Viral vectors encoding parathyroid hormone fusions and their use in treating hypoparathyroidism - Patents.com

Abstract

Compositions and methods for treating hypoparathyroidism in a subject are provided. A viral vector is provided that includes a nucleic acid molecule that includes a sequence encoding a PTH fusion protein and a regulatory sequence that directs its expression.

Description

[Technical field]

[0001] Electronic Sequence Listing Reference The contents of the electronic sequence listing (22-10017.PCT_Seq-Listing.xml, size: 304kb, and creation date: March 1, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0002] Parathyroid hormone (PTH) is an 84 amino acid hormone produced by the parathyroid gland. PTH regulates serum calcium concentrations through its effects on bone, kidney, and intestine. Hypoparathyroidism (HP) is a rare condition in which PTH is not secreted by the parathyroid gland. HP causes hypocalcemia, which leads to further symptoms including weakness, muscle spasms, headache, and / or uncontrollable muscle twitching and seizures. Recombinant human PTH (rhPTH(1-84)) has been approved by the FDA as a treatment for chronic HP in adults. Because PTH has a short serum half-life, rhPTH(1-84) needs to be injected subcutaneously daily. Although rhPTH(1-84) has shown a good safety profile in clinical trials, exogenous PTH increased the risk of osteosarcoma in rats, suggesting safety concerns for the long-term continuous use of rhPTH(1-84).

[0003] What is needed is an improved therapy for hypoparathyroidism. Summary of the Invention

[0004] Provided herein are viral vectors encoding parathyroid hormone (PTH) fusion protein constructs. These viral vectors, in some embodiments, can achieve sustained expression and / or increased circulating half-life of PTH fusions in a subject compared to vector-mediated delivery of PTH fusions without a fusion partner. Methods of making and using such viral vectors are also provided.

[0005] In one aspect, a viral vector is provided that includes a nucleic acid comprising a polynucleotide sequence encoding a fusion protein. The fusion protein includes (a) a leader sequence including a secretory signal peptide and a propeptide, (b) a parathyroid hormone (PTH) fusion, and (c) a fusion domain that includes either (i) an 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.

[0006] In one embodiment, (i) the secretory signal peptide of the leader sequence comprises a thrombin signal peptide, (ii) the leader sequence comprises a thrombin propeptide, and / or (iii) the leader sequence comprises a thrombin leader sequence. In another embodiment, the leader sequence comprises an IL-2 leader sequence in combination with a PTH propeptide or a thrombin propeptide. In one embodiment, the PTH fusion is selected from 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, and SEQ ID NO:48, and functional variants thereof.

[0007] In one embodiment, the fusion domain is a 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 a 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 an embodiment, the fusion domain is a rhesus 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 a 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.

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

[0009] In another aspect, a pharmaceutical composition suitable for use in 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.

[0010] In yet another aspect, the use of the viral vectors described herein is provided for the manufacture of a medicament for treating a subject having hypoparathyroidism, and optionally, diabetes.

[0011] In another aspect, a method of treating a subject having hypoparathyroidism is provided, comprising administering to the subject an effective amount of a viral vector or composition described herein.

[0012] Other aspects and advantages of the present invention will become readily apparent from the following detailed description of the invention. [Brief description of the drawings]

[0013] [Figure 1A] FIG. 1 is a schematic diagram outlining the PTH constructs described herein that utilize a heterologous leader (signal peptide and propeptide) in combination with mature PTH. [Figure 1B] 1 shows a table showing the experimental outline for the experiment described in Example 3. [Figure 1C] The results of the experiment in Figure 1B are shown. Briefly, three groups of RagKO mice were treated with vectors utilizing the native leader or IL2 signal (in combination with the PTH propeptide), or vehicle. Plasma PTH levels (day 14) and serum calcium levels (day 28) were comparable for both vectors. [Figure 2A]FIG. 1 shows a schematic diagram of a PTH construct described herein that utilizes a fusion partner in combination with truncated PTH. [Figure 2B] FIG. 1 shows a schematic diagram of a PTH construct described herein that utilizes a fusion partner in combination with truncated PTH. [Figure 2C] FIG. 2B shows in vitro expression in HEK293 cells of the constructs described in FIG. 2B. [Figure 3A] 1 is a gel showing stained PTH Fc fusion protein produced from the indicated constructs and purified using Protein A column purification, with fractions 3 and 4 produced under reducing (R) and non-reducing (NR) conditions indicated by the ladder. [Figure 3B] 3B is a graph showing the results of a potency assay from the purified proteins of FIG 3 A. Purified PTH73 Fc fusions from fractions 3 and 4 showed similar potency to PTH34 (the active portion of the protein). [Figure 3C] FIG. 1 is a graph showing hPTH73 Fc fusion levels produced from inducible constructs in vitro upon induction with 40 nM rapamycin. [Figure 4A] 1 shows a table showing the experimental outline for the experiment described in Example 3. [Figure 4B] The results of the experiment in Figure 4A are shown. Briefly, three groups of RagKO mice were treated with vehicle, vector utilizing the native leader (hPTH(N)), or native leader utilizing an Fc fusion (hPTH73.P.Fc). Plasma hPTH potency was determined from bleeds on days 14 and 28. [Figure 5A] 1 shows a table depicting the experimental outline for the experiment described in Example 3. Briefly, three groups of RagKO mice were treated with vehicle, an inducible vector utilizing the native leader along with an Fc fusion. Rapamycin was administered on day 12 for groups 2 and 3, and again on day 38 for group 3. [Figure 5B] Plasma hPTH potency determined from bleeds on days 14 and 38 is shown. [Figure 5C] Serum calcium levels are shown. [Figure 6] The full length sequence of human PTH is provided in SEQ ID NO:1. [Figure 7A] 1 shows the study design of the experiment described in Example 4. [Figure 7B] Preliminary data including NAB potency of five different AAV capsids are presented. [Figure 8A] 1 shows the results of the experiment described in Example 5. The body weights of animals treated with the indicated vectors and dosages are shown. [Figure 8B] 1 shows the results of the experiment described in Example 5. Serum calcium in animals treated with the indicated vectors and doses is shown. [Figure 8C] 1 shows the results of the experiment described in Example 5. Plasma hPTH levels of animals treated with the indicated vectors and dosages are shown. [Figure 9A] 1 shows the results of the experiment described in Example 6. Plasma hPTH levels are shown for animals treated with the indicated vectors and dosages. [Figure 8B] 1 shows the results of the experiment described in Example 6. Serum calcium in animals treated with the indicated vectors and doses is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Described herein is an adeno-associated virus (AAV) vector expressing PTH fusion protein for treating hypoparathyroidism in patients with a single intramuscular vector administration.Described is the delivery of these constructs to subjects in need of delivery via multiple routes, particularly by in vivo expression mediated by recombinant vectors such as rAAV vectors.Also provided is a method of using these constructs in a regimen in a subject in need of treating HP to increase the half-life of PTH in the subject.In addition, provided is a method for enhancing the activity of PTH in a subject.

[0015] PTH fusion protein Hypoparathyroidism (HP) is characterized by low serum calcium levels caused by insufficient secretion of parathyroid hormone (PTH). Despite normalization of serum calcium levels by treatment with activated vitamin D protein and calcium supplementation, patients suffer from a reduced quality of life and an increased risk of several comorbidities. Several recent studies have investigated replacement therapy with recombinant human PTH (rhPTH(1-84)) and therapy with an N-terminal PTH fragment (rhPTH(1-34)). Both drugs have been shown to normalize serum calcium while reducing the need for activated vitamin D and calcium supplements. However, once-daily injections cause large fluctuations in serum calcium. Twice-daily injections reduce the fluctuations but do not restore normal physiological function of calcium homeostasis.

[0016] Parathyroid hormone (PTH) is a single polypeptide chain of 115 amino acids with the following sequence: MIPAKDMAKV MIVMLAICFL TKSDGKSVKK RSVSEIQLMH NLGKHLNSMERVEWLRKKLQ DVHNFVALGA PLAPRDAGSQ RPRKKEDNVL VESHEKSLGEADKADVNVLT KAKSQ (SEQ ID NO: 1)

[0017] The full-length sequence includes the signal peptide (amino acids 1-25) and the propeptide (amino acids The mature protein consists of amino acids 32 to 115 of SEQ ID NO:1 - SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ (SEQ ID NO:2).

[0018] The present disclosure provides fusion proteins comprising 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 sequences encompassing full-length pre-proparathyroid hormone (including signal sequence and propeptide), propeptide and mature peptide (e.g., about 26-115 amino acids), mature peptide (e.g., about 32-115 amino acids), peptides with C-terminal or N-terminal truncations. In particular, 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 may be combined with the native PTH propeptide and signal peptide, or with heterologous propeptides and / or signal peptides. Several constructs embodying various combinations of these sequences are described herein. As used herein, references to PTH and the following numbers (e.g., PTHX, PTH34, PTH73) refer to truncated PTH peptides that include the first X amino acids of the peptide starting at the N-terminus of SEQ ID NO: 2. For example, PTH34 includes the first 34 amino acids of the mature peptide, while PTH73 includes the first 73 amino acids of the mature peptide.

[0019] As used herein, the term "leader sequence" refers to any N-terminal sequence of a PTH polypeptide. In some embodiments, the leader includes a secretory signal peptide, a propeptide, or both.

[0020] In some embodiments, the fusion protein comprises a polynucleotide sequence encoding a fusion protein comprising (a) a 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 functional variant thereof. In another embodiment, the fusion protein comprises a thrombin leader, a PTH protein, and an albumin or functional variant thereof. In another embodiment, the PTH fusion comprises a PTH leader sequence, a PTH protein, and an IgG Fc or functional variant thereof. In another embodiment, the fusion protein comprises a PTH leader, a PTH protein, and an albumin or 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 functional variant thereof. In another embodiment, the fusion protein comprises an IL2 signal sequence, a PTH propeptide, a PTH protein, and an IgG Fc or functional variant thereof. In another embodiment, the fusion protein comprises an IL2 signal sequence, a PTH propeptide, a PTH protein, and an albumin or functional variant thereof.

[0021] In some embodiments, the PTH fusions include variants that retain the function of the wild-type sequence and may include up to about 10% variation from the PTH nucleic acid or amino acid sequences described herein or known in the art. As used herein, "retaining function" means that the nucleic acid or amino acid functions in the same manner as the wild-type sequence, although not necessarily at the same level of expression or activity. For example, in one embodiment, the functional variant has increased expression or activity compared to the wild-type sequence. In another embodiment, the functional variant has decreased expression or activity compared to the wild-type sequence. In one embodiment, the functional variant has a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more increase or decrease in expression or activity compared to the wild-type sequence. In some embodiments, the functional variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes compared to the wild-type sequence.

[0022] In one embodiment, the fusion comprises a PTH protein combined with 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 up to 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 human. For example, PTH may be derived from non-human primates, dogs, cats, mice, rats, sheep, cows, horses, etc. In some embodiments, it is desirable to modify the native PTH sequence to optimize one or more characteristics thereof. In one embodiment, the PTH protein has a sequence that includes or consists 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.

[0023] The leader sequence may be derived from the same species as that for which administration is ultimately intended, e.g., human. As used herein, the term "derived" or "derived from" means that the sequence or protein is sourced from or shares the same sequence as a protein or sequence sourced from a particular subject species. For example, a leader sequence "derived" from a human shares the same sequence (or a variant thereof as defined herein) as the same leader sequence expressed in a human. However, the specified nucleic acid or amino acid need not actually be sourced from a human. A variety of techniques are known in the art that can produce a desired sequence, including mutagenesis of a similar protein (e.g., a homologue), or artificial production of a nucleic acid or amino acid sequence. A "derived" nucleic acid or amino acid retains the function of the same nucleic acid or amino acid in the species from which it is "derived", regardless of the actual source of the derived sequence.

[0024] The term "amino acid substitution" and its synonyms are intended to encompass the modification of an amino acid sequence by replacing an amino acid with another alternative amino acid. The substitution may be a conservative substitution. It may be a non-conservative substitution. The term conservative, when referring to two amino acids, is intended to mean that the amino acids share a common property recognized by those of skill in the art. For example, amino acids with hydrophobic non-acidic side chains, amino acids with hydrophobic acidic side chains, amino acids with hydrophilic non-acidic side chains, amino acids with hydrophilic acidic side chains, and amino acids with hydrophilic basic side chains. The common property may also be amino acids with hydrophobic side chains, amino acids with aliphatic hydrophobic side chains, amino acids with aromatic hydrophobic side chains, amino acids with polar neutral side chains, amino acids with charged side chains, amino acids with charged acidic side chains, and amino acids with charged basic side chains. Both naturally occurring and non-naturally occurring amino acids are known in the art and may be used as substitute amino acids in embodiments. Methods for substituting amino acids are well known to those of skill in the art and include, but are not limited to, mutations in the nucleotide sequence encoding the amino acid sequence. References herein to "one or more" are intended to encompass, for example, one, two, three, four, five, six or more individual embodiments.

[0025] In one embodiment, the leader is a human thrombin (Factor II) sequence. In one embodiment, the thrombin leader is SEQ ID NO:3: MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRR, or a functional variant thereof having up to 1, 2, or 3 amino acid substitutions.

[0026] In another embodiment, the leader is a human IL2 sequence. In one embodiment, the IL2 leader has the sequence set forth in SEQ ID NO: 4: MYKMQLLSCIALTLVLVANS, or a functional variant thereof having up to 1, 2, or 3 amino acid substitutions. In certain embodiments, the leader includes a propeptide (e.g., derived from human PTH, human thrombin, or other sequences) in addition to the IL2 signal peptide.

[0027] In another embodiment, the leader is a human PTH sequence. In one embodiment, the PTH leader has the sequence set forth in SEQ ID NO: 5: MIPAKDMAKVMIVMLAICFLTKSDGKSVKKR, or a functional variant thereof having up to 1, 2, or 3 amino acid substitutions.

[0028] In one embodiment, functional variants of desired leaders include variants that retain the function of the wild-type sequence and may contain up to about 10% variation from the leader nucleic acid or amino acid sequences described herein or known in the art.

[0029] In some embodiments, the coding regions for both the leader and the PTH peptide are incorporated into a single nucleic acid sequence with no linker between the leader and PTH coding sequences.

[0030] In certain embodiments, the fusion protein further comprises a fusion domain. The fusion domain is, in one embodiment, a human IgG Fc fragment or a functional variant thereof. Immunoglobulins typically have a long circulating half-life in vivo. By fusing the PTH protein (and leader) to IgG Fc, the circulation time of the fusion protein is extended while maintaining the function of PTH. In another embodiment, the fusion domain is a rhesus IgG Fc fragment or a functional variant thereof.

[0031] As used herein, the Fc portion of an immunoglobulin has the meaning commonly given to the term in the field of immunology. Specifically, the term refers to an antibody fragment that does not contain the two antigen-binding regions (Fab fragments) from the antibody. The Fc portion consists of the constant regions of the antibody from both heavy chains, which are associated by non-covalent interactions and disulfide bonds. The Fc portion includes the hinge region and may extend through the CH2 and CH3 domains to the c-terminus of the antibody. The Fc portion may further include one or more glycosylation sites. In one embodiment, the fusion domain is human IgG Fc. The four highly conserved subclasses, IgG1, IgG2, IgG3, and IgG4, differ in their constant regions, especially their hinge and upper CH2 domains. See Vidarsson et al, IgG Subclasses and Allotypes: From Structure to Effector Functions, Front Immunol. Oct. 2014; 5:520, which is incorporated herein by reference. The Fc domain can be derived from any human IgG, including human IgG1, human IgG2, human IgG3, or human IgG4. In one embodiment, the human IgG Fc is an IgG4 Fc. In one embodiment, the human IgG Fc is SEQ ID NO:6: AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG. In another embodiment, the human IgG Fc shares at least 90% identity, at least 95% identity, at least 99% identity, or at least 100% identity to SEQ ID NO:6.

[0032] In one embodiment, the human IgG Fc is an IgG1 Fc. In one embodiment, the human IgG Fc is SEQ ID NO: 7: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG. In another embodiment, the human IgG Fc shares at least 90% identity, at least 95% identity, at least 99% identity, or at least 100% identity to SEQ ID NO: 7.

[0033] In another embodiment, the fusion domain is a Rhesus IgG Fc. The Fc domain can be derived from any Rhesus IgG, including Rhesus IgG1, Rhesus IgG2, Rhesus IgG3, or Rhesus IgG4. In one embodiment, the Rhesus IgG Fc is an IgG4 Fc. In one embodiment, the Rhesus IgG Fc is SEQ ID NO:8: PPCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAQTKPRE RQFNSTYRVV SVLTVTHQDW LNGKEYTCKV SNKGLPAPIE KTISKAKGQP REPQVYILPP PQEELTKNQV SLTCLVTGFY PSDIAVEWES NGQPENTYKT TPPVLDSDGS YLLYSKLTVN KSRWQPGNIF TCSVMHEALH NHYTQKSLSV SPGK. In another embodiment, the rhesus IgG Fc shares at least 90% identity, at least 95% identity, at least 99% identity, or at least 100% identity to SEQ ID NO: 8. In one embodiment, the rhesus IgG further comprises a hinge sequence.

[0034] In another embodiment, the fusion domain is human albumin or a functional variant thereof. In one embodiment, the human albumin is SEQ ID NO:9: In another embodiment, the human albumin shares at least 90% identity, at least 95% identity, at least 99% identity, or at least 100% identity to SEQ ID NO:9.

[0035] The in vivo function and stability of the fusion proteins of the present disclosure may be potentially undesirable, for example. To prevent domain interaction or for other reasons, it may be optimized by adding a small amount of peptide linker. Furthermore, the glycine-rich linker may provide some structural flexibility so that the PTH protein portion can productively interact with the PTH receptor on target cells, such as pancreatic beta cells. Thus, 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. In one embodiment, the linker comprises 1, 1.5, or 2 repeats of a G-rich peptide linker having GGGSGGGGSGGGGS (SEQ ID NO: 10) or a similar sequence.

[0036] In one embodiment, the fusion protein comprises (a) a human thrombin leader, (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:X, or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. SEQ ID NO: 11MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRRSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0037] 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. Sequence number 12: ATGGCTCAcGTTCGAGGACTGCAGCTGCCTGGGATGTCTGGCTCTGGCCGCTTGTGTAGCCTGTGCACAGCCAGCAGAGCAcGTGTTTCTGGCTCCTCAGCAaGCCAGATCACTGCTGCAGCGCGTCAGAAGAAAGCGTCAGCGAGATCCAGCTGATGCACAACCTGGGCAAGCACCTGAACAGCATGGAACGCGTGGAAgTGGCTGCGGAAGAAACTGCAGGACGTGCAAAcGGTGGCGGCGGAAGCGGTGGCGGAAGGATCAGGCGGTGGATCTGGCGAGTCTGCCGGATCCTGAGCTCCGcTGTCCTGCCCcGAAGCTGCTGGCGGCCCATCCGTGTTCTGTCCCCCCAAAGCCTAAGGACACCCTGATGGACACCCTGATGATGACCTGCGTGGTGGTGGACGGTTCCCCAAAGGAGGATCCTGAGTCCTGAGGTCAGTTCAATTGGTAACTGGACGGGGT GGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCAAGAGAACCCCAGGTGTACACACTGCCTCCAAGCCAAGAGGAAAATGACCAAGAACCA GGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTCGAGTGGGAGAGCAAcGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCcggCTGACCGTGGACAAGAGCAGgTGGCAAAGAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCCACAACCACTACACCAGAAGTCTCTGAG CCTGAGCCTGGGCTGATGA

[0038] In one embodiment, the fusion protein comprises (a) a human thrombin leader, (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: 13, or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. SEQ ID NO: 13 MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRRSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG**

[0039] 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.

[0040] In one embodiment, the fusion protein comprises: (a) a human thrombin leader; (b) a PTH( In one embodiment, the fusion protein comprises (a) a human thrombin leader, (b) PTH(1-74), a linker, and (c) a human IgG Fc. In one embodiment, the fusion protein comprises (a) a human thrombin leader, (b) PTH(1-84), a linker, and (c) a human IgG Fc. In one embodiment, the fusion protein comprises (a) a human thrombin leader, (b) PTH(1-84), a linker, and (c) a human IgG Fc. In one embodiment, the fusion protein has a sequence of SEQ ID NO: 15, or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. SEQ ID NO: 15 MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRRSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0041] 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.

[0042] In one embodiment, the fusion protein comprises (a) a human PTH leader, (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 at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. SEQ ID NO: 17 MIPAKDMAKVMIVMLAICFLTKSDGKSVKKRSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*

[0043] 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. Sequence number 18: ATGATTCCCGCCAAGACATGGCCAAAGTGATGATCGTGATGCTGGCCATCTGCTTTCTGACCAAGAGCGACGGCAAGAGCGTGAAGAAAAAGCGTGTCCGAGATCCAGCTGATGCACAACCTGGGCAAGCACCTGAACAGCATGGAACGCGTGGAGTGGCTGCGGGAAGAAACTGCAGGACGTGCACAACTTCGACAAGACCCACACCTGTCTCTCCTGCTCCCGAAGCTGCGGCCCTTCGTGTTCCTCCCAAAGCCTAAGGACACCCTGATGGACCTGCGTGTGGTGGTGGATGTGTCCCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTA CAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAACCATCAGCAAGGCCAAGGGCCAGCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGACAAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTTTACC CCAGCGACATTGCCGTCGAGTGGGAGCTAACGGCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGAGCCGGTGGCAGCAGGGCAATGTGTTCAGCTGTAGCGTGATGCACGAGGCCCTGCACAACCACTACACCAGAAGTCCCTGAGCCTGTCTCCTGGCTGA

[0044] In one embodiment, the fusion protein comprises (a) a human PTH leader, (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 at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. SEQ ID NO: 19 MIPAKDMAKVMIVMLAICFLTKSDGKSVKKRSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*

[0045] 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. Sequence number 20: ATGATTCCCGCCAAGGACATGGCCAAAGTGATGATCGTGATGCTGGCCATCTGCTTTCTGACCAAGAGCGACGGCAAGAGCGTGAAGAAAAGAAGCGTGTCCGAGATCCAGCTGATGCACAACCTGGGCAAGCACCTGAACAGCATGGAACGCGTGGAGTGGCTGCGGAAGAAACTGCAGGACGTGCACAACTTCGTGGCCCTGGGAGCACCTCTGGCTCCTAGGGACGCCGGCAGCCAGCGGCCTAGAAAGAAAGAGGATAACGTCCTGGTCGAGAGCCACGAGAAGTCTCTGGGCGAAGCCGACAAGGCCGACAAAACACACACCTGTCCTCCCTGTCCTGCTCCCGAAGCTGCTGGCGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTCCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCTAACAAGGCCCTGCCTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACACTCCCTCCAAGCAGGGACGAGCTGACAAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTCGAGTGGGAGTCCAACGGCCAGCCAGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGAGCCGGTGGCAGCAGGGCAATGTGTTCAGCTGTAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGCTGA。

[0046] In one embodiment, the fusion protein comprises (a) a human PTH leader, (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 at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. SEQ ID NO:21 MIPAKDMAKVMIVMLAICFLTKSDGKSVKKRSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*

[0047] In one embodiment, the sequence encoding the fusion protein is SEQ ID NO: 22, or at least 75%, at least 85%, at least 90%, at least 95%, at least Both sequences are 98% or at least 99% identical.

[0048] In one embodiment, the fusion protein comprises (a) a human PTH leader (including the propeptide), (b) PTH(1-75), a linker, and (c) a human IgG Fc. In one embodiment, the fusion protein has a sequence of SEQ ID NO: 23, or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. SEQ ID NO: 23 MIPAKDMAKVMIVMLAICFLTKSDGKSVKKRSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0049] In one embodiment, the sequence encoding the fusion protein is SEQ ID NO:24, or a sequence at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. SEQ ID NO: 24: ATGATTCCCGCCAAGGACATGGCCAAAGTGATGATCGTGATGCTGGCCATCTGCTTTCTGACCAAGAGCGACGGCAAGAGCGTGAAGAAAAGAAGCGTGTCCGAGATCCAGCTGATGCACAACCTGGGCAAGCACCTGAACAGCATGGAACGC GTGGAGTGGCTGCGGAAGAAACTGCAGGACGTGCACAACTTCGTGGCCCTGGGAGCACCTCTGGCTCCTAGAGACGCCGGCAGCCAGCGGCCTAGAAAGAAAGAGGATAACGTCCTGGTCGAGAGCCACGAGAAGTCTCTGGGCGAAGCCGACAAGGCCGACGTGGACAAGACACACACCTGTCCTCCCTGTCCTGCTCCCGAAGCTGCTGGCGGCCCTTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGACAAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTCGAGTGGGAGTCCAACGGCCAGCCAGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGAGCCGGTGGCAGCAGGGCAATGTGTTCAGCTGTAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGCTGA

[0050] In one embodiment, the fusion protein comprises (a) a human PTH leader, (b) PTH(1-84), a linker, and (c) a human IgG Fc.

[0051] In one embodiment, the fusion protein comprises (a) a human IL2 leader (including a PTH or thrombin propeptide), (b) PTH(1-34), a linker, and (c) a human IgG In one embodiment, the fusion protein comprises (a) a human IL2 leader (comprising PTH or thrombin propeptide), (b) PTH(1-73), a linker, and (c) a human IgG Fc. In one embodiment, the fusion protein comprises (a) a human IL2 leader (comprising PTH or thrombin propeptide), (b) PTH(1-74), a linker, and (c) a human IgG Fc. In one embodiment, the fusion protein comprises (a) a human IL2 leader (comprising PTH or thrombin propeptide), (b) PTH(1-75), a linker, and (c) a human IgG Fc. In one embodiment, the fusion protein comprises (a) a human IL2 leader (comprising PTH or thrombin propeptide), (b) PTH(1-84), a linker, and (c) a human IgG Fc.

[0052] In one embodiment, the fusion protein comprises (a) a human thrombin leader, (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 at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. SEQ ID NO: 25 MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRRSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNGGGGGGSGGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLS QRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYK FQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALG

[0053] 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. GAACCTGATCAAGCAGAACTGCGAGCTGTTCGAGCAGCTGGGCGAGTACAAGTTCCAGAACGCCCTGCTCGTGCGGTACACCAAGAAGGTGCCCCAGGTTTCCACACCTACACTGGTTGAGGTGTCCCGGAATCTGGGCAAAGTGGGCAGC AAGTGCTGCAAGCACCCTGAGGCCAAGAGAATGCCTTGCGCCGAGGATTACCTGAGCGTGGTGCTGAATCAGCTGTGCGTGCTGCACGAGAAAACCCCTGTGTCCGACAGAGTGACCAAGTGCTGTACCGAGAGCCTGGTCAACAGACGGC CTTGCTTTAGCGCCCTCGAGGTGGACGAGACATACGTGCCCAAAGAGTTCAACGCCGAGACATTCACCTTCCACGCCGACATCTGTACCCTGAGCGAGAAAGAGCGGCAGATCAAGAAACAGACTGCCCTGGTGGAACTGGTCAAGCACAA GCCCAAGGCCACCAAAGAACAGCTGAAGGCCGTGATGGACGACTTCGCCGCCTTCGTGGAAAAATGCTGCAAGGCCGACGACAAAGAGACATGCTTCGCCGAAGAGGGCAAGAAACTGGTGGCCGCTTCTCAGGCTGCCCTGGGATGATAA

[0054] In one embodiment, the fusion protein comprises (a) a human thrombin leader, (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 at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. SEQ ID NO: 27 MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRRSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNGGGGGGSGGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTEC CHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKK VPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALG

[0055] 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. Sequence number 28:ATGGCTCAcGTTCGAGGACTGCAGCTGCCTGGGATGTCTGGCTCTTGCCGCTTGTGTAGCCTGTGCACAGCCAGCAGCAcGTGTTTCTGGCTCCTCAGCAaGCCAGATCACTGCTGCAGCGCGTCAGAAGAAGCGTCAGCGAATCCAGCTGATGCACAACCTGGGCAAGCACCTGAACAGCATGGAACGCGTGGAAgTGGCTGCGGAAGAAACTGCAGGACGTGCACAAcGGTGGCGGCGGAAGCGGTGGCGGAGGGATCAGGCGGTGGT

[0056] In one embodiment, the fusion protein comprises (a) a human thrombin leader, (b) PTH(1-74), a linker, and (c) human albumin. In one embodiment, the fusion protein comprises (a) a human thrombin leader, (b) PTH(1-75), a linker, and (c) human albumin. In one embodiment, the fusion protein comprises (a) a human thrombin leader, (b) PTH(1-75), a linker, and (c) human albumin. a leader, (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 at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. SEQ ID NO: 29 MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRRSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQGGGGSGGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLS QRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYK FQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALG

[0057] 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. Sequence number 30: ATGGCTCAcGTTCGAGGACTGCAGCTGCCTGGGATGTCTGGCTCTGGCCTTGTGTAGCCTGTGCACAGCCAGCAcGTGTTTCTGGCTCCTCAgCAaGCCAGATCACTGCTGCAGCGCGTCAGAAGAAAGCGTCAGCGAGATCCAGCTGATGCACAACCTGGGCAAGCACCTGAACAGCATGGAACGCGTGGAAgTGGCTGCGGAAGAAACTGCAGGACGTGCACAACTTCGTGGCCCTGGGAGCCACCTCTGGCTCCAAGAGAcGCGGCAGCCAACGGCCCTAGAAAGAAAGGACAACGTCTGGTCGGAGAGCCAGAAGTCTCTGGGCGGAAGCCGATAAGGCCGAGGTGAACGTGCTGACCAAGGCCAAGTCTCAAGGTGCGGAGGCGGAGGATCTGGTGGTGGGATCTGGCGGCGGAGGTTCTAcGCCCACAAATCTGAAGTG GCCCACCGGTTCAAGGACCTGGGAGAAGAGAACTTCAAGGCCCTGGTGCTGATCGCCTTCGCTCAGTACCTGCAGCAgTGCCCCTTCGAGGACCACGTGAAGCTGGTCAACGAAGTGACCGAGTTCGCCAAAGACCTGCGTGGCCGAcGAGAGCGCGAGAACTGTGATAAGAGCCTGCACACCCTTGTGGCGACAAGCTGTGTACAGTGGCCACACTGAGAGAAACCTA CGGCGAGATGGCCGACTGCTGCGCCAAGCAAGAGCCCGAGAGAAACGAGTGCTTCCTGCAGCACAAGGACGACAACCCCAACCTGCCTAGACTCGTGCGGCCTGAAGTGGACGTGATGTGCACGCCTTCACACGACAACGAGGAACCTTCCTGAAAGAAGTACCTGTACGAGATCGCCAGACGGCACCCCTACTTTTACGCCCCTGAGCTGCTGTTCTTCGCCAAGCGGT

[0058] In one embodiment, the fusion protein comprises (a) a human PTH leader (including the propeptide), (b) PTH(1-34), a linker, and (c) human albumin. In one embodiment, the fusion protein comprises (a) a human PTH leader (including the propeptide), (b) PTH(1-73), a linker, and (c) human albumin. In one embodiment, the fusion protein comprises (a) a human PTH leader (including the propeptide), (b) PTH(1-74), a linker, and (c) human albumin. In one embodiment, the fusion protein comprises (a) a human PTH leader (including the propeptide), (b) PTH(1-75), a linker, and (c) human albumin. In one embodiment, the fusion protein comprises (a) a human PTH leader (including the propeptide), (b) PTH(1-84), a linker, and (c) human albumin.

[0059] In one embodiment, the fusion protein comprises (a) a human IL2 leader (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 (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 (comprising PTH or thrombin propeptide), (b) PTH(1-74), a linker, and (c) human albumin. and (c) human albumin. In one embodiment, the fusion protein comprises (a) a human IL2 leader (including 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 (including PTH or thrombin propeptide), (b) PTH(1-84), a linker, and (c) human albumin.

[0060] If variants or fragments of the leader sequence, PTH protein, or fusion domain are desired, the coding sequences of these peptides can be generated using site-directed mutagenesis of the wild-type nucleic acid sequence. Alternatively or additionally, web-based or commercially available computer programs, as well as service-based companies, can be used to back-translate the amino acid sequence into a nucleic acid coding sequence, including both RNA and / or cDNA. See, for example, backtranseq by EMBOSS, ebi.ac.uk / Tools / st / ; Gene Infinity (geneinfinity.org / sms- / sms_backtranslation.html); ExPasy (expasy.org / tools / ). In one embodiment, the RNA coding sequence and / or the cDNA coding sequence are designed for optimal expression in the subject species to which administration is ultimately intended, e.g., humans.

[0061] Coding sequences can be designed for optimal expression using codon optimization. Codon-optimized coding regions can be designed by a variety of different methods. This optimization can be done using methods available online, published methods, or companies that provide codon optimization services. One codon optimization method is described, for example, in International Patent Application Publication No. 2015 / 012924, which is incorporated herein by reference. Briefly, a nucleic acid sequence that encodes a product is modified with synonymous codon sequences. Preferably, the entire length of the product's open reading frame (ORF) is modified. However, in some embodiments, only a fragment of the ORF may be modified. By using one of these methods, a frequency can be applied to any given polypeptide sequence to produce a nucleic acid fragment of a codon-optimized coding region that encodes a polypeptide.

[0062] In addition to the leader sequences, PTH proteins, fusion domains, and fusion proteins provided herein, nucleic acid sequences encoding these polypeptides are provided. In one embodiment, a nucleic acid sequence encoding a PTH peptide as described herein is provided. In some embodiments, this may include any nucleic acid sequence that encodes the PTH sequence of SEQ ID NO:1. In another embodiment, this includes any nucleic acid that includes the PTH sequence of SEQ ID NO:2.

[0063] In one embodiment, a nucleic acid sequence encoding a PTH fusion protein as described herein is provided, which in another embodiment includes any nucleic acid sequence encoding 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.

[0064] Expression cassette In another aspect, provided herein is an expression cassette comprising a nucleic acid encoding a PTH fusion protein as described herein. As used herein, "expression cassette" refers to a nucleic acid molecule comprising a biologically useful nucleic acid sequence and a regulatory sequence operably linked thereto that directs or regulates the transcription, translation, and / or expression of the nucleic acid sequence (e.g., a gene cDNA encoding a protein, enzyme, or other useful gene product, mRNA, etc.) and its gene product. As used herein, "operably linked" sequences include both regulatory sequences (also referred to as elements) that are contiguous or non-contiguous with the nucleic acid sequence and regulatory sequences that act in trans or cis with the nucleic acid sequence. Such regulatory sequences typically include, for example, promoters, enhancers, transcription factors, transcription terminators, introns, sequences that enhance translation efficiency (i.e., Kozak consensus sequences), slicing and polyadenylation sequences, etc. The expression cassette may include one or more of the following: an efficient RNA processing signal of the ribosomal RNA ... In another embodiment, the term "transgene" refers to one or more DNA sequences from an exogenous source that are inserted into a target cell.

[0065] In one embodiment, an expression cassette refers to a nucleic acid molecule that includes a PTH construct coding sequence (e.g., a coding sequence for a PTH fusion protein), a promoter, and may include other regulatory sequences therefor, which may be engineered into a genetic element and / or packaged into a viral vector capsid (e.g., a viral particle). Typically, such an expression cassette for producing a viral vector includes a PTH construct sequence as described herein adjacent to a packaging signal in the viral genome (and referred to as the "vector genome"), and other expression control sequences, such as those described herein. Any of the expression control sequences may be optimized for a particular species using techniques known in the art, including codon optimization, for example, as described herein.

[0066] In certain embodiments, the expression cassette comprises a constitutive promoter. In another embodiment, CB7 is used. CB7 is a chicken beta actin promoter with a cytomegalovirus enhancer element. In some embodiments, the CB7 promoter has the nucleic acid sequence of SEQ ID NO: 31:

[0067] In one embodiment, the promoter is a CMV promoter. In some embodiments, the CMV promoter is represented by SEQ ID NO: 32: cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggt aaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtgg atagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgt It has a nucleic acid sequence of aacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatc.

[0068] In another embodiment, tissue-specific promoter is used.Alternatively, other liver-specific promoters can be used, for example, those listed in the Liver Specific Gene Promoter Database, Cold Spring Harbor, (rulai.schl.edu / LSPD), including but not limited to alpha 1 antitrypsin (A1AT), human albumin (Miyatake et al., J.Virol., 71:5124 32(1997)), humAlb; Hepatitis B virus core promoter (Sandig et al., Gene Ther., 3:1002 9(1996)), TTR minimal enhancer / promoter, alpha-antitrypsin promoter, liver-specific promoter (LSP) (Wu et al.Mol Ther.16:280-289(2008)), TBG liver-specific promoter. Other promoters, such as viral promoters, constitutive promoters, regulatable promoters (see, e.g., WO2011 / 126808 and WO2013 / 049493), or promoters that respond to physiological cues, may be used and utilized in the vectors described herein.

[0069] In one embodiment, the promoter is included in an inducible gene expression system. The inducible gene regulation / expression system includes at least the following components: a promoter (also referred to as a regulatable promoter) operably linked to a transgene encoding a PTH fusion protein described herein, an activation domain, a DNA binding domain, and a zinc finger homeodomain binding site(s). In other embodiments, additional components may be included in the expression system, as further described herein.

[0070] 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 as shown in SEQ ID NO: 32. In one embodiment, the promoter is a ubiquitous inducible promoter Z12I, which contains 12 repeated copies of binding sites for ZFHD1 and an IL2 minimal promoter. See, for example, Chen et al, Hum Gene Ther Methods. 2013 Aug; 24(4): 270-278, which is incorporated herein.

[0071] The expression system comprises an activation domain, which is preferably located upstream of the DNA binding domain. In one embodiment, the activation domain is a fusion of the carboxy terminus from the p65 subunit of NF-kappa B with the FKBP12-rapamycin binding (FRB) domain of FKBP12-rapamycin related protein (FRAP). In one embodiment, the activation domain is the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin related protein (FRAP) fused to the carboxy terminus from the p65 subunit of NF-kappa B from human origin.

[0072] In one embodiment, there is a linker between the transactivation domain and the DNA binding domain. In one embodiment, the linker can be F2A or IRES. In one embodiment, the linker is selected from an IRES or a 2A peptide. In one embodiment, the linker is a cleavable 2A peptide. In one embodiment, the 2A peptide is selected to increase packaging limits to allow for a single vector system.

[0073] The DNA-binding domain consists of a DNA-binding fusion of zinc finger homeodomain 1 (ZFHD1) joined to up to three copies of FK506-binding protein (FKBP). In the presence of an inducer, e.g., a rapalog such as rapamycin, the DNA-binding and activation domains dimerize through the interaction of their FKBP and FRB domains, resulting in transcriptional activation of the transgene. In some embodiments, ZFHD1 is included in frame with GT2A or an IRES.

[0074] The expression system is designed to have one, two, or three copies of the FKBP sequence. These are referred to herein as FKBP subunits. In one embodiment, the subunits are designed to express the same protein, but with different nucleic acids from each other to minimize recombination. For example, SEQ ID NO: 33 provides three "wobbled" coding sequences for FKBP, each of which encodes the sequence shown in SEQ ID NO: 34: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE.

[0075] The expression system further comprises a zinc finger homeodomain binding site. The nucleic acid molecule comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 binding sites for a ZFHD. In one embodiment, the expression system comprises 12 zinc finger homeodomain binding sites (binding partners) (12XZFHD). In another embodiment, the expression system comprises 8 zinc finger homeodomain binding sites (binding partners) (12XZFHD). However, the invention encompasses expression systems having from 2 to about 12 copies of a zinc finger binding site. An example of a single copy of a ZFHD binding site is AATGATGGGGCGCTCGAGT (SEQ ID NO: 35).

[0076] In some embodiments, there is a minimal IL2 promoter downstream of the zinc finger homeodomain binding site. An exemplary IL2 promoter is shown in SEQ ID NO:36.

[0077] Such induction systems are known in the art and are described, for example, in Rivera et al, A humanized system for pharmacologic control of gene expression, Nature Medicine volume 2, pages 1028-1032 (September 1996) and Rivera et al, Long-term pharmacologically regulated expression of erythropoietin in primates following AAV-mediated gene transfer, Blood, 15 February 2005, volume 1 105, number 4, both of which are incorporated herein by reference. In one embodiment, the inducible gene expression system comprises a CMV promoter and the activation domain is the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin related protein (FRAP) fused to the carboxy terminus from the p65 subunit of NF-kappa B of human origin, a GT2A peptide, a ZFHD1 DNA binding domain, three FKBP subunits, hGH polyA, 12XZFHD, and a minimal sIL2 promoter. These sequences are added to the coding sequence for the PTH fusion protein, and optionally other regulatory sequences.

[0078] In addition to the promoter, the expression cassette and / or vector may include suitable transcription initiation, termination, and enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals, sequences that stabilize cytoplasmic mRNA, sequences that increase translation efficiency (i.e., Kozak consensus sequences), sequences that enhance protein stability, and, if necessary, sequences that enhance secretion of the encoded product. Examples of suitable polyA sequences include, for example, SV40, bovine growth hormone (bGH), human growth hormone (hGH), SV40, rabbit β-globin (also called rabbit globin polyA; RGB), modified RGB (mRGB), and TK polyA. Examples of suitable enhancers include, for example, alpha fetoprotein enhancer, TTR minimal promoter / enhancer, LSP (TH binding globulin promoter / alpha 1-microglobulin / bikunin enhancer), among others. In one embodiment, the polyA is rabbit globin polyA.

[0079] 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 with a gene of interest, and expression control sequences that act in trans or at a distance to control the gene of interest.

[0080] In one embodiment, a rAAV is provided that comprises a 5' ITR, a CB7 promoter, a chicken beta-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, a rabbit globin polyA, and a 3' ITR. In another embodiment, the rAAV comprises a polynucleotide comprising a CMV promoter, an activation domain comprising the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin related protein (FRAP) fused to the carboxy terminus from the p65 subunit of NF-kappa B from human, an IRES, a ZFHD1 DNA binding domain, three FKBP subunits, hGH polyA, 8XZFHD, a minimal sIL2 promoter, 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.

[0081] In another embodiment, a two-vector induction system is provided. The first rAAV comprises 12XZFHD, a minimal IL2 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. A stuffer sequence can be included to increase the package size of the vector. The second rAAV comprises a polynucleotide comprising a CMV promoter, an intron, and the activation domain is the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin-related protein (FRAP) fused to the carboxy terminus from the p65 subunit of NF-kappa B from human, an IRES, a ZFHD1 DNA binding domain, and polyA.

[0082] In one embodiment, an expression cassette is provided comprising a polynucleotide comprising the CB7 promoter, a chicken beta-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 a rabbit globin polyA.

[0083] In another embodiment, an expression cassette is provided comprising a polynucleotide comprising the CB7 promoter, a chicken beta-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 a rabbit globin polyA.

[0084] Viral Vectors In another aspect, a viral vector is provided that comprises the expression cassette described herein. In certain embodiments of the viral vector described herein, the viral vector is an adeno-associated virus (AAV) viral vector or a recombinant AAV (rAAV). The term "recombinant AAV" or "rAAV" as used herein refers to naturally occurring adeno-associated viruses, adeno-associated viruses available to those skilled in the art and / or available in the context of the composition(s) and method(s) described herein, as well as artificial AAV. An adeno-associated virus (AAV) viral vector is an AAV DNase-resistant particle that has an AAV protein capsid, in which the expression cassette packaged is flanked by AAV inverted terminal repeats (ITRs) for delivery to target cells (collectively referred to as "vector genome"). The AAV capsid is composed of 60 capsid protein subunits, VP1, VP2, and VP3, arranged in icosahedral symmetry in a ratio of approximately 1:1:10 to 1:1:20, depending on the AAV selected. Various AAVs can be selected as the source of capsids for the AAV viral vectors identified above. In one embodiment, the AAV capsid is the AAVrh91 capsid or a variant thereof. In certain embodiments, the capsid proteins are designated by a number or a combination of numbers and letters following the term "AAV" in the name of the rAAV vector. Unless otherwise indicated, the AAV capsids, ITRs, and other selected AAV components described herein may be readily selected from among any AAV, including, but not limited to, the AAVs identified as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAVhu37, AAVrh32.33, AAVAnc80, AAV10, AAV11, AAV12, AAVrh8, AAVrh74, AAV-DJ8, AAV-DJ, AAVhu.37, AAVrh.64R1, and AAVhu68. See, e.g., U.S. Patent Application Publication No. 2007-0036760-A1, U.S. Patent Application Publication No. 2009-0197338-A1, and EP 1310571.See also WO2003 / 042397 (AAV7 and other simian AAVs), U.S. Pat. No. 7,790,449 and U.S. Pat. No. 7,282,199 (AAV8), WO2005 / 033321 and US Pat. No. 7,906,111 (AAV9), as well as WO2006 / 110689, and WO2003 / 042397 (rh.10), WO2005 / 033321, WO2018 / 160582 (AAVhu68), which are incorporated by reference herein. Other suitable AAVs may include, but are not limited to, AAVrh90 [filed April 28, 2020, PCT / US20 / 30273], AAVrh91 [filed April 28, 2020, PCT / US20 / 030266, current publication WO2020 / 223231 published November 5, 2020] AAVrh92, AAVrh93, AAVrh91.93 [filed April 28, 2020, PCT / US20 / 30281], which are incorporated herein by reference. Other suitable AAVs include the AAV3B variants described in U.S. Provisional Patent Application No. 62 / 924,112, filed October 21, 2019, and U.S. Provisional Patent Application No. 63 / 025,753, filed May 15, 2020, including AAV3B.AR2.01, AAV3B.AR2.02, AAV3B.AR2.03, AAV3B.AR2.04, AAV3B.AR2.05, AAV3B.AR2.06, AAV3B.AR2.07, AAV3B.AR2.09, AAV3B.AR2.10, AAV3B.AR2.11, AAV3B.AR2.12, AAV3B.AR2.13, AAV3B.AR2.14, AAV3B.AR2.15, AAV3B.AR2.16, AAV3B.AR2.17, AAV3B.AR2.18, AAV3B.AR2.19, AAV3B.AR2.20, AAV3B.AR2.21, AAV3B.AR2.22, AAV3B.AR2.23, AAV3B.AR2.24, AAV3B.AR2.25, AAV3B.AR2.26, AAV3B.AR2.27, AAV3B.AR2.28, AAV3B.AR2.29, AAV3B.AR2.30, AAV3B.AR2.31, AAV3B.AR2.32, AAV3B.AR2.33, AAV3B.AR2.34, AAV3B.AR2.35, AAV3B.AR2.36, AAV3B.AR2.37, AAV3B.AR2.38, AAV3B.AR2.39, AAV3B.AR , AAV3B.AR2.06, AAV3B.AR2.07, AAV3B.AR2.08, AAV3B.AR2.10, AAV3B.AR2.11, AAV3B.AR2.12, AAV3B.AR2.13, AAV3B.AR2.14, AAV3B.AR2.15, AAV3B.AR2.16, or AAV3B.AR2.17, which are incorporated herein by reference. See also International Patent Application No. PCT / US21 / 45945, filed August 13, 2021, U.S. Provisional Patent Application No. 63 / 065,616, filed August 14, 2020, and U.S. Provisional Patent Application No. 63 / 109,734, filed November 4, 2020, all of which are incorporated herein by reference in their entireties. These documents also describe other AAV capsids that can be selected to generate rAAV, and are incorporated by reference. Among the well-characterized AAVs isolated or engineered from non-human primates (NHPs), human AAV2 was the first AAV developed as a gene transfer vector and has been widely used for efficient gene transfer experiments in various target tissues and animal models.

[0085] As used herein, with respect to AAV, the term "variant" refers to any AAV sequence derived from a known AAV sequence, including AAV sequences with conservative amino acid substitutions and AAV sequences that share at least 90%, at least 95%, at least 97%, at least 99% or more sequence identity across the amino acid or nucleic acid sequence. In another embodiment, the AAV capsid includes a variant that may include up to about 10% variation from any described or known AAV capsid sequence. That is, the AAV capsid shares about 90% identity to about 99.9% identity, about 95% to about 99% identity, or about 97% to about 98% identity with an AAV capsid provided herein and / or known in the art. In one embodiment, the AAV capsid shares at least 95% identity with an AAV capsid. When determining the percent identity of an AAV capsid, the comparison can be made across any of the variable proteins (e.g., vp1, vp2, or vp3).

[0086] In one embodiment, the viral vector is a rAAV having a capsid of AAV8 or a functional variant thereof. In one embodiment, the viral vector is a rAAV having a capsid of AAVrh91 or a functional variant thereof. In one embodiment, the viral vector is a rAAV having a capsid of AAV3.AR.2.12 or a functional variant thereof. In one embodiment, the viral vector is a rAAV having a capsid selected from AAV9, AAVrh64R1, AAVhu37, or AAVrh10. In one embodiment, the viral vector is a rAAV having a capsid of AAVhu68 or a functional variant thereof.

[0087] In certain embodiments, the viral vector has an AAVrh91 capsid. A nucleic acid sequence encoding the AAVrh91 capsid is provided in SEQ ID NO: 37, and the encoded amino acid sequence is provided in SEQ ID NO: 38. Provided herein is an rAAV 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 the AAVrh91 amino acid sequence is provided in SEQ ID NO: 19, and the 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 an N-terminal and / or C-terminal truncation (e.g., a truncation of about 1 to about 10 amino acids).

[0088] In certain embodiments, the AAVrh91 capsid is characterized by one or more of the following: (1) a heterologous collection of AAVrh91 vp1 proteins selected from a vp1 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of 1-736 of SEQ ID NO:38, a vp1 protein produced from SEQ ID NO:37, or a vp1 protein produced from a nucleic acid sequence that is at least 70% identical to SEQ ID NO:37 encoding the predicted amino acid sequence of 1-736 of SEQ ID NO:38; a vp2 protein produced by expression from a nucleic acid sequence encoding at least about 138-736 amino acids of SEQ ID NO:38; a vp2 protein produced from a sequence that includes at least nucleotides 412-2208 of SEQ ID NO:37; or a sequence a heterologous collection of AAVrh91 vp2 proteins selected from vp2 proteins produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 412-2208 of SEQ ID NO:37, which encodes a predicted amino acid sequence of at least about 138-736 amino acids of SEQ ID NO:38; a vp3 protein produced from expression from a nucleic acid sequence that encodes a predicted amino acid sequence of at least about 203-736 amino acids of SEQ ID NO:38; a vp3 protein produced from a sequence that includes at least nucleotides 607-2208 of SEQ ID NO:37; or a vp3 protein produced from a nucleic acid sequence that encodes a predicted amino acid sequence of at least about 203-736 amino acids of SEQ ID NO:38. and / or (2) a heterogeneous collection of vp1 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of at least about amino acids 138-736 of SEQ ID NO:38, a heterogeneous collection of vp2 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:38, wherein the vp1 proteins, vp2 proteins, and vp3 proteins are the product of a nucleic acid sequence encoding an asparagine-glycosylated amino acid sequence of SEQ ID NO:38. a heterogeneous collection of vp1 proteins, a heterogeneous collection of vp2 proteins, a heterogeneous collection of vp3 proteins, each of which comprises a subpopulation having an amino acid modification comprising at least two highly deamidated asparagines (N) in a syn pair, and optionally further comprising a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change, and (B) a vector genome in an AAVrh91 capsid, the vector genome comprising a nucleic acid molecule comprising an AAV inverted terminal repeat and a non-AAV nucleic acid sequence encoding a product operably linked to a sequence that directs expression of the product in a host cell.

[0089] In certain embodiments, the AAVrh91 capsid is characterized by one or more of the following: (1) a heterologous collection of AAVrh91 vp1 proteins selected from a vp1 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of 1-736 of SEQ ID NO:38, a vp1 protein produced from SEQ ID NO:19, or a vp1 protein produced from a nucleic acid sequence that encodes the predicted amino acid sequence of 1-736 of SEQ ID NO:38; a vp2 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about 138-736 amino acids of SEQ ID NO:38, a vp2 protein produced from a sequence including at least nucleotides 412-2208 of SEQ ID NO:19, or a vp2 protein produced from a nucleic acid sequence that encodes the predicted amino acid sequence of at least about 138-736 amino acids of SEQ ID NO:38; a heterologous collection of vp2 proteins, vp3 proteins produced from expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:38, vp3 proteins produced from a sequence comprising at least nucleotides 607-2208 of SEQ ID NO:19, or vp3 proteins produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 607-2208 of SEQ ID NO:19 encoding the predicted amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:38.and / or (2) a heterogeneous collection of vp1 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of at least about amino acids 138-736 of SEQ ID NO:38, a heterogeneous collection of vp2 proteins that are the product of a nucleic acid sequence encoding an amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:38, wherein the vp1 proteins, vp2 proteins, and vp3 proteins comprise a subpopulation having an amino acid modification comprising at least two highly deamidated asparagines (N) in the asparagine-glycine pair of SEQ ID NO:38, and optionally further subpopulations comprising other deamidated amino acids, wherein the deamidation results in an amino acid change. a heterologous collection of proteins, a heterologous collection of vp2 proteins, a heterologous collection of vp3 proteins, and (B) a vector genome in an AAVrh91 capsid, the vector genome comprising a nucleic acid molecule comprising an AAV inverted terminal repeat and a non-AAV nucleic acid sequence encoding a product operably linked to a sequence that directs expression of the product in a host cell.

[0090] In certain embodiments, the vp1, vp2, and vp3 proteins of AAVrh91 comprise subpopulations having amino acid modifications that include at least two highly deamidated asparagines (N) in the asparagine-glycine pair of SEQ ID NO: 38, and optionally further comprise subpopulations that include other deamidated amino acids, where deamidation results in an amino acid change. High levels of deamidation are observed at NG pairs N57, N383, and / or N512, relative to the numbers in SEQ ID NO: 38. Deamidation has been observed at other residues. In certain embodiments, AAVrh91 may have other residues that are deamidated, e.g., typically less than 10%, and / or may have other modifications including phosphorylation (e.g., in a range of about 2 to about 30%, or about 2 to about 20%, or about 2 to about 10%, if present) (e.g., at S149), or oxidation (e.g., at one or more of about W22, about M211, W247, M403, M435, M471, W478, W503, about M537, about M541, about M559, about M599, M635, and / or W695). Optionally, W may be oxidized to kynurenine. [Table 1]

[0091] In certain embodiments, the AAVrh91 capsid is modified at one or more of the positions identified in the table above, within the ranges provided below, as determined using mass spectrometry with trypsin enzyme. In certain embodiments, one or more positions, or the glycine following the N, is modified as described herein. Residue numbers are based on the AAVrh91 sequence provided herein. See SEQ ID NO:38.

[0092] In certain embodiments, the AAVrh91 capsid comprises a heterogeneous collection of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:38, a heterogeneous collection of vp2 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence at least about amino acids 138-736 of SEQ ID NO:38, and a heterogeneous collection of vp3 proteins that are the product of a nucleic acid sequence encoding at least amino acids 203-736 of SEQ ID NO:38.

[0093] In certain embodiments, modified AAVrh91 nucleic acid sequences can be used to generate mutant rAAVs having capsids that contain less deamidation than the native AAVrh91 capsid. Such mutant rAAVs can have reduced immunogenicity and / or increased stability upon storage, particularly in suspension form.

[0094] In one aspect, a recombinant AAV (rAAV) is provided that comprises an AAV capsid derived from the adeno-associated virus rh91 and a vector genome packaged within the AAV capsid, the vector genome comprising the AAV inverted terminal repeats (ITRs), a coding sequence for 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 that directs expression of the PTH fusion.

[0095] In certain embodiments, the AAVhu68 capsid is further characterized by one or more of the following: AAVhu68 capsid protein includes 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, vp1 protein produced from SEQ ID NO:98 or 99, or vp1 protein produced from a nucleic acid sequence that is at least 70% identical to SEQ ID NO:98, which encodes the predicted amino acid sequence of 1 to 736 of SEQ ID NO:100; AAVhu68 vp2 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:100, vp2 protein produced from a sequence that includes at least nucleotides 412 to 2211 of SEQ ID NO:98 or 99, or 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, which encodes the predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:100, and / or AAVhu68 vp2 protein produced by expression from a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID NO:100. vp3 protein, a vp3 protein produced from a sequence including at least nucleotides 607-2211 of SEQ ID NO:98, or a vp3 protein produced from a nucleic acid sequence that is at least 70% identical to at least nucleotides 607-2211 of SEQ ID NO:98 or 99 that encodes a predicted amino acid sequence of at least about amino acids 203-736 of SEQ ID NO:100.

[0096] Additionally or alternatively, optionally, an AAV capsid is provided that comprises a heterogeneous population of vp1 proteins that comprise a valine at position 157, optionally a heterogeneous population of vp2 proteins that comprise a valine at position 157, and a heterogeneous population of vp3 proteins, wherein at least a subpopulation of vp1 and vp2 proteins comprise a valine at position 157 and optionally further comprise a glutamic acid at position 67, based on the numbering of the vp1 capsid in SEQ ID NO:100. Additionally or alternatively, there is provided an AAVhu68 capsid comprising a heterogeneous population of vp1 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:100, a heterogeneous population of vp2 proteins that are the product of a nucleic acid sequence encoding the amino acid sequence of at least about amino acids 138-736 of SEQ ID NO:100, and a heterogeneous population of vp3 proteins that are the product of a nucleic acid sequence encoding at least amino acids 203-736 of SEQ ID NO:100, wherein the vp1 proteins, vp2 proteins, and vp3 proteins include subpopulations having amino acid modifications.

[0097] The AAVhu68 vp1, vp2, and vp3 proteins are typically expressed as alternative splice variants encoded by the same nucleic acid sequence that encodes the full-length vp1 amino acid sequence (amino acids 1-736) of SEQ ID NO: 100. Optionally, the vp1 coding sequence is used alone to express the vp1, vp2, and vp3 proteins. Alternatively, the sequence may be expressed using a sequence that is distinct from the vp1 unique region (from about aa1 to about aa137) and / or the vp2 unique region (from about aa1 to about aa202). or a complementary strand thereto, the corresponding mRNA or tRNA (from 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 that encodes aa 203 to 736 of SEQ ID NO: 100. Additionally or alternatively, the vp1 and / or vp2 coding sequences may be co-expressed with a nucleic acid sequence encoding the AAVhu68 vp2 amino acid sequence of SEQ ID NO:100 (about aa 1 to about 137) without the vp1 unique region (about aa 1 to about 137), or a complementary strand thereto, the corresponding mRNA or tRNA (e.g., nt 412 to 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 about aa 138 to 736 of SEQ ID NO:100.

[0098] 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, additional nucleic acid sequences, such as sequences encoding a vp3 protein that does not include the vp1 unique region and / or the vp2 unique region. rAAVhu68 resulting from production using a single nucleic acid sequence vp1 produces a heterogeneous population of vp1, vp2, and vp3 proteins. More specifically, AAVhu68 capsids contain subpopulations within the vp1, vp2, and vp3 proteins that have modifications from the predicted amino acid residues of SEQ ID NO: 100. These subpopulations contain, at a minimum, deamidated asparagine (N or Asn) residues. For example, the asparagine in an asparagine-glycine pair is highly deamidated.

[0099] In one embodiment, the AAVhu68 vp1 nucleic acid sequence has the sequence of SEQ ID NO: 98 or 99, or a complementary strand thereto, e.g., the corresponding mRNA or tRNA. In certain embodiments, the vp2 and / or vp3 proteins may additionally or alternatively be expressed from a nucleic acid sequence different from vp1, e.g., to alter the ratio of vp proteins in a selected expression system. In certain embodiments, a nucleic acid sequence encoding the AAVhu68 vp3 amino acid sequence of SEQ ID NO: 100 (about aa 203-736) is also provided that does not include the vp1 unique region (about aa 1 to about aa 137) and / or the vp2 unique region (about aa 1 to about aa 202), or a complementary strand thereto, the corresponding mRNA or tRNA (about nt 607 to about nt 2211 of SEQ ID NO: 98 or 99). In certain embodiments, a nucleic acid sequence encoding the AAVhu68 vp2 amino acid sequence of SEQ ID NO: 100 (about aa 138-736), without the vp1 unique region (about aa 1 to about 137), or the complementary strand thereto, the corresponding mRNA or tRNA (nt 412-2211 of SEQ ID NO: 98 or 99), is also provided.

[0100] However, other nucleic acid sequences encoding the amino acid sequence of SEQ ID NO: 100 may be selected for use in producing rAAVhu68 capsid. In certain embodiments, the nucleic acid sequence has a sequence at least 70% to 99% identical, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to the nucleic acid sequence of SEQ ID NO: 98 or 99, or a sequence encoding SEQ ID NO: 100. In certain embodiments, the nucleic acid sequence has a sequence at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identical to the nucleic acid sequence of SEQ ID NO: 98 or 99, or a sequence from about nt 412 to about nt 2211 of SEQ ID NO: 98 or 99, and encodes 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 from about nt 607 to about nt 2211 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 SEQ ID NO: 98 or 99, and encodes the vp3 capsid protein of SEQ ID NO: 100 (about aa 203 to 736). [Table 2-1] [Table 2-2]

[0101] In certain embodiments, an AAVhu68 capsid is characterized in having a capsid protein in which at least 45% of the N residues are deamidated at at least one of positions N57, N329, N452, and / or N512, based on the numbering of the amino acid sequence of SEQ ID NO: 100. In certain embodiments, at least about 60%, at least about 70%, at least about 80%, or at least 90% of the N residues are deamidated at one or more of 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). In these and other embodiments, the AAVhu68 capsid is characterized in having a population of proteins in which about 1% to about 20% of N residues have deamidation at one or more of positions N94, N253, N270, N304, N409, N477, and / or Q599, based on the numbering of the amino acid sequence of SEQ ID NO:100. In certain embodiments, AAVhu68 comprises at least a subpopulation of vp1, vp2, and / or vp3 proteins that are deamidated at one or more of positions N35, N57, N66, N94, N113, N252, N253, Q259, N270, N303, N304, N305, N319, N328, N329, N336, N409, N410, N452, N477, N515, N598, Q599, N628, N651, N663, N709, N735, or combinations thereof, based on the numbering of the amino acid sequence of SEQ ID NO: 100. In certain embodiments, the capsid protein may have one or more amidated amino acids.

[0102] In one aspect, a recombinant AAV (rAAV) is provided that comprises an AAV capsid derived from the adeno-associated virus hu68 and a vector genome packaged within the AAV capsid, the vector genome comprising the AAV inverted terminal repeats (ITRs), a coding sequence for 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 regulatory sequences that direct expression of the PTH fusion.

[0103] In another embodiment, a recombinant adeno-associated virus (rAAV) having an AAVhu68 capsid and a vector genome is provided, wherein (a) the AAVhu68 capsid comprises a heterogeneous population of AAVhu68 vp1 proteins, a heterogeneous population of AAVhu68 vp2 proteins, and a heterogeneous population of AAVhu68 vp3 proteins, and the heterogeneous AAVhu68 vp1, AAVhu68 vp2, and AAVhu68 vp3 proteins are identified using mass spectrometry. and optionally further comprising a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change such that the deamidated asparagine is deamidated to aspartic acid, isoaspartic acid, an interconverting aspartic acid / isoaspartic acid pair, or a combination thereof, and the AAVhu68 capsid comprises a subpopulation having an amino acid modification comprising 50% to 100% deamidation of at least two asparagines (N) in an asparagine-glycine pair at two or more of N57, N329, N452, N512 of SEQ ID NO: 100, and optionally further comprising a subpopulation comprising other deamidated amino acids, wherein the deamidation results in an amino acid change such that the deamidated asparagine is deamidated to aspartic acid, isoaspartic acid, an interconverting aspartic acid / isoaspartic acid pair, or a combination thereof, and the AAVhu68 capsid comprises (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; (b) at least 75% of the N in the asparagine-glycine pair at position N329 of the vp1 protein, v2 protein, and vp3 protein are deamidated, based on the residue numbering of the amino acid sequence of SEQ ID NO:100; (c) at least 50% of the N in the asparagine-glycine pair at position N452 of the vp1 protein, v2 protein, and vp3 protein are deamidated, based on the residue numbering of the amino acid sequence of SEQ ID NO: 100; and / or (d) at least 75% of the N in the asparagine-glycine pair at position N512 of the vp1 protein, v2 protein, and vp3 protein are deamidated, based on the residue numbering of the amino acid sequence of SEQ ID NO:100; The vector genome further comprises an AAVhu68 capsid, the vector genome comprising an AAV inverted terminal repeat sequence and a non-AAV nucleic acid sequence encoding a PTH fusion described herein operably linked to a sequence that directs expression of the PTH fusion in a target cell.

[0104] Still other modifications are observed, most of which do not involve conversion of an amino acid to 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 proteins have one or more phosphorylations. For example, a particular vp1 capsid protein may be phosphorylated at position 149.

[0105] In one embodiment, the rAAV is a scAAV. The abbreviation "sc" refers to self-complementary. "Self-complementary AAV" refers to a plasmid or vector with an expression cassette in which the coding region carried by the recombinant AAV nucleic acid sequence is designed to form an intramolecular double-stranded DNA template. Upon infection, rather than waiting for cell-mediated synthesis of the second strand, the two complementary halves of the scAAV will associate to form one double-stranded DNA (dsDNA) unit ready for immediate replication and transcription. See, for example, DM McCarty et al, "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis", Gene Therapy, (August 2001), Vol 8, Number 16, Pages 1248-1254. Self-complementary AAVs are described, for example, in U.S. Pat. Nos. 6,596,535, 7,125,717, and 7,456,683. Nos. 5,393,925, 5,393 ... each of which is incorporated herein by reference in its entirety.

[0106] In one embodiment, the nucleic acid sequence encoding the PTH construct described herein is engineered into any suitable genetic element, e.g., naked DNA, phage, transposon, cosmid, RNA molecule (e.g., mRNA), episome, etc., which introduces the PTH sequence carried thereon into a host cell, e.g., to generate nanoparticles carrying DNA or RNA, viral vectors in a packaging host cell, and / or for delivery to a host cell in a subject. In one embodiment, the genetic element is a plasmid. The selected genetic element can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. Methods used to generate such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0107] As used herein, the term "host cell" may refer to a packaging cell line in which a vector (e.g., recombinant AAV or rAAV) is produced from a production plasmid. Alternatively, the term "host cell" may refer to any target cell in which expression of a gene product described herein is desired. Thus, "host cell" refers to a prokaryotic or eukaryotic cell (e.g., bacterial, human, or insect cell) that contains exogenous or heterologous DNA introduced into the cell by any means, such as electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. In certain embodiments herein, the term "host cell" refers to cultures of cells of various mammalian species for in vitro evaluation of the compositions described herein. In other embodiments herein, the term "host cell" refers to cells used to produce and package viral vectors or recombinant viruses. In further embodiments, the term "host cell" is an intestinal cell, a small intestinal cell, a pancreatic cell, or a liver cell.

[0108] As used herein, the term "target cell" refers to any target cell in which expression of a heterologous nucleic acid sequence or protein is desired. In certain embodiments, the target cell is a liver cell. In other embodiments, the target cell is a muscle cell.

[0109] In one embodiment, an rAAV is provided that includes a vector genome that includes an expression cassette, the expression cassette including a CMV promoter and an activation domain including the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin related protein (FRAP) fused to the carboxy terminus from the p65 subunit of NF-kappa B of human origin, a GT2A_V1 peptide, a ZFHD1 DNA binding domain, three FKBP subunits, hGH polyA, 12XZFHD, a minimal sIL2 promoter, a coding sequence for a PTH fusion protein of SEQ ID NO:14, and a rabbit beta globin polyA. In another embodiment, an rAAV is provided that includes a vector genome that includes an expression cassette, the expression cassette including a CMV promoter and an activation domain that is the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin related protein (FRAP) fused to the carboxy terminus from the p65 subunit of NF-kappa B of human origin, a GT2A_V2 peptide, a ZFHD1 DNA binding domain, three FKBP subunits, hGH polyA, 12XZFHD, a minimal sIL2 promoter, a coding sequence for a PTH fusion protein of SEQ ID NO: 14, and a rabbit beta globin polyA.

[0110] In one embodiment, an rAAV is provided that comprises a vector genome comprising an expression cassette, the expression cassette comprising a CB7 promoter, a chicken beta-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, a rabbit globin polyA, and a 3' ITR. In another embodiment, an rAAV is provided that includes a vector genome that includes an expression cassette, the expression cassette including a CMV promoter and an activation domain is the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin related protein (FRAP) fused to the carboxy terminus from the p65 subunit of NF-kappa B of human origin, an IRES, a ZFHD1 DNA binding domain, three FKBP subunits, hGH polyA, 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 a rabbit beta globin polyA.

[0111] In another embodiment, a two vector induction system is provided. In one embodiment, a first rAAV is provided that includes a vector genome that includes an expression cassette, the expression cassette includes 12XZFHD, a minimal IL2 promoter, a coding sequence for a 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 can be included to increase the package size of the vector. A second rAAV includes a vector genome that includes an expression cassette, the expression cassette includes a polynucleotide that includes a CMV promoter, an intron, and an activation domain is the FKBP12-rapamycin binding (FRB) domain of human FKBP12-rapamycin related protein (FRAP) fused to the carboxy terminus from the p65 subunit of NF-kappa B from human, an IRES, a ZFHD1 DNA binding domain, and polyA.

[0112] In one embodiment, the rAAV comprises a vector genome comprising an expression cassette comprising a polynucleotide comprising a CB7 promoter, a chicken beta-actin intron, a coding sequence for a fusion protein of SEQ ID NO: 1, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 39, 42, 44, 46, or 48, and a rabbit globin polyA.

[0113] The minimal sequences required to package an expression cassette into an AAV viral particle are the AAV 5' and 3' ITRs, which may be of the same AAV origin as the capsid or of a different AAV origin (to generate AAV pseudotypes). In one embodiment, the ITR sequences from AAV2, or a deleted version thereof (ΔITR), are used for convenience and to speed regulatory approval. However, ITRs from other AAV sources may be selected. Preferably, the source of the ITRs is the same as the source of the Rep protein provided in trans for production. Typically, an expression cassette for an AAV vector comprises the AAV 5' ITR, the PTH fusion protein coding sequence, and any regulatory sequences, as well as the AAV 3' ITR. However, other configurations of these elements may be suitable. A shortened version of the 5' ITR, in which the D sequence and the terminal separation site (trs) are deleted (designated ΔITR), has been described. In another embodiment, the full-length AAV The 5' and 3' ITRs are used.

[0114] The ITRs are the only AAV components required in cis in the same construct as the genes for packaging the expression cassette into virions. In one embodiment, the replication (rep) and / or capsid (cap) coding sequences are removed from the AAV genome and supplied in trans or by a packaging cell line to generate the AAV vector. For example, as described above, pseudotyped AAV may contain ITRs from a source different from the source of the AAV capsid. In one embodiment, a chimeric AAV capsid may be utilized. Still other AAV components may be selected. Sources of such AAV sequences are described herein and may be obtained from academic, commercial, or public sources (e.g., American Type AAV sequences may be isolated or obtained from the National AAV Culture Collection, Manassas, VA. AAV sequences may be obtained synthetically or through other suitable means by reference to published sequences such as those available in the literature or databases (e.g., GenBank®, PubMed®, etc.).

[0115] Methods for generating and isolating AAV viral vectors suitable for delivery to subjects are known in the art.See, for example, US Patent No. 7,790,449, US Patent No. 7,282,199, WO2003 / 042397, WO2005 / 033321, WO2006 / 110689, and US7,588,772 B2. In one system, producer cell lines are transiently transfected with construct(s) encoding transgenes flanked by ITRs, and constructs encoding rep and cap. In the second system, packaging cell lines that stably supply rep and cap are transiently transfected with constructs encoding transgenes flanked by ITRs. In each of these systems, AAV virions are produced in response to infection with helper adenovirus or herpesvirus, requiring the separation of rAAV from contaminating viruses. More recently, systems have been developed that do not require infection with a helper virus to restore AAV, and the necessary helper functions (i.e., adenovirus E1, E2a, VA, and E4, or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase) are also supplied by the system in trans. In these newer systems, the helper functions can be supplied by transient transfection of cells with constructs encoding the required helper functions, or the cells can be engineered to stably contain genes encoding helper functions, the expression of which can be controlled at the transcriptional or post-transcriptional level. In yet another system, the transgene flanked by ITRs and the rep / cap genes are introduced into insect cells by infection with a baculovirus-derived vector. For a review of these production systems generally, see, e.g., Zhang et al., 2009, "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, the contents of each of which are incorporated herein by reference in their entireties.Methods of making and using these and other AAV production systems are also described in the following U.S. patents, the contents of each of which are incorporated herein by reference in their entirety: 5,139,941, 5,741,683, 6,057,152, 6,204,059, 6,268,213, 6,491,907, 6,660,514, 6,951,753, 7,094,604, 7,172,893, 7,201,898, 7,229,823, and 7,439,065. See generally, for example, Grieger & Samulski, 2005, "Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications," Adv. Biochem. Engin / Biotechnol. 99:119-145, Buning. et al., 2008, “Recent developments in adeno-associated virus vector technology,” J. Gene Med. 10:717-733, and the references cited below, each of which is incorporated herein by reference in its entirety. The methods used to construct any embodiment of the present invention are known to those skilled in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Methods for producing AV virions are well known, and the selection of a suitable method is not a limitation of the present invention, see, e.g., K. Fisher et al., (1993) J. Virol., 70:520-532 and U.S. Patent No. 5,478,745.

[0116] 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 regulatory sequences that direct insertion of the polynucleotide sequence encoding the fusion protein into the genome of a host cell.

[0117] As used herein, "vector genome" refers to a nucleic acid sequence packaged inside a parvovirus (e.g., rAAV) capsid that forms a viral particle. Such a nucleic acid sequence includes AAV inverted terminal repeats (ITRs). In the example herein, the vector genome includes at least, from 5' to 3', an AAV 5'ITR, a coding sequence(s) (i.e., transgene(s)), and an AAV 3'ITR. ITRs from AAV2, a source AAV different from the capsid, or other than full-length ITRs can be selected. In certain embodiments, the ITRs are from the same AAV source as the AAV that provides the rep function or trans-complementing AAV during production. Additionally, other ITRs can be used, such as self-complementary (scAAV) ITRs. Both single-stranded and self-complementary (sc) AAVs are included in rAAV. A transgene is a nucleic acid coding sequence heterologous to the vector sequence that encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor), or other gene product of interest. The nucleic acid coding sequence is operably linked to regulatory elements in a manner that allows transcription, translation, and / or expression of the transgene in cells of the target tissue. Suitable components of a vector genome are discussed in more detail herein. In one example, a "vector genome" includes at least, from 5' to 3', a vector-specific sequence and a nucleic acid sequence encoding a PTH construct operably linked to a regulatory control sequence (that directs its expression in the target sequence), and the vector-specific sequence can be a terminal repeat sequence that specifically packages the vector genome into a viral vector capsid or envelope protein. For example, AAV inverted terminal repeats are utilized for packaging into AAV and certain other parvovirus capsids.

[0118] The AAV sequences of the vector typically contain cis-acting 5' and 3' inverted terminal repeat sequences (see, e.g., BJ Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155 168 (1990)). The ITR sequences are approximately 145 bp in length. Preferably, substantially complete sequences encoding the ITRs are used in the molecule, although some minimal modification of these sequences is tolerated. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., Sambrook et al, "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J. Virol., 70:520 532 (1996)). One example of such a molecule used in the present invention is a "cis-acting" plasmid containing a transgene, where the selected transgene sequence and associated regulatory elements are flanked by 5' and 3' AAV ITR sequences. In one embodiment, the ITRs are from a different AAV than the one supplying the capsid. In one embodiment, the ITR sequences are from AAV2. However, ITRs from other AAV sources may be selected. A shortened version of the 5'ITR (designated ΔITR) has been described, in which the D sequence and terminal separation sites (trs) are deleted. In one particular embodiment, the vector genome comprises a 130 base pair truncated AAV2 ITR, in which the external A elements are deleted. Without wishing to be bound by theory, it is believed that the shortened ITRs facilitate the amplification of vector DNA using the internal (A') elements as a template. It is believed that the ITRs are reverted to the wild-type length of 145 base pairs in width. In other embodiments, full-length AAV 5' and 3' ITRs are used. If the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be referred to as pseudotyped. However, other configurations of these elements may also be suitable.

[0119] Optionally, the PTH constructs described herein can be delivered via a viral vector other than rAAV. Such other viral vectors can include any virus suitable for gene therapy, including but not limited to adenovirus, herpesvirus, lentivirus, retrovirus, etc. Preferably, when one of these other vectors is produced, it is produced as a replication-defective viral vector.

[0120] "Replication-defective virus" or "viral vector" refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, and any viral genomic sequences also packaged in the viral capsid or envelope are replication-defective, i.e., they are unable to generate progeny virions, but retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome can be engineered to be "gutless", containing only the transgene of interest flanked by signals required for amplification and packaging of the artificial genome), although these genes can be supplied during production. It is therefore considered safe for use in gene therapy, since replication and infection by progeny virions cannot occur except in the presence of viral enzymes required for replication.

[0121] Compositions comprising the viral vector constructs described herein are also provided. The pharmaceutical compositions described herein are designed to be delivered to a subject in need thereof by any suitable route or combination of different routes. Direct delivery to the liver (optionally intravenously, via the hepatic artery, or by transplantation), oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral routes of administration. The viral vectors described herein may be delivered in a single composition or multiple compositions. Optionally, two or more different AAVs, or multiple viruses, may be delivered [see, for example, WO2011 / 126808 and WO2013 / 049493]. In another embodiment, the multiple viruses may include different replication-deficient viruses (e.g., AAV and adenovirus). In one embodiment, administration is intramuscular. In another embodiment, administration is intravenous.

[0122] Replication-defective viruses can be formulated with physiologically acceptable carriers for use in gene transfer and gene therapy applications. In the case of AAV viral vectors, quantification of genome copies ("GC") can be used as a measure of the dose contained in the formulation. Any method known in the art can be used to determine the genome copy (GC) number of the replication-defective virus composition of the present invention. One method for performing GC number titration of AAV is as follows. Purified AAV vector samples are first treated with DNase to eliminate non-encapsidated 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 that targets a specific region of the viral genome (usually the polyA signal). Another suitable method for determining genome copies is quantitative PCR (qPCR), in particular optimized qPCR or digital droplet PCR [Lock Martin, et al, Human Gene Therapy Methods. April 2014, 25(2):115-125. doi:10.1089 / hgtb.2013.131, published online December 13, 2013 ahead of edit].

[0123] In addition, the replication-deficient virus composition contains approximately 1.0×10 9 GC~approx. 1.0×10 15 The dosage unit can be formulated to contain an amount of replication-deficient virus in the range of GC. In another embodiment, this amount of viral genome can be delivered in divided doses. In one embodiment, the dose is about 1.0×10 for an average human subject of about 70 kg. 10 GC~approx.3.0×10 14 In another embodiment, the dose is about 1×10 9 For example, the dose of AAV virus is about 1×10 10 GC, 1×10 11 GC, approx. 5×10 11 GC, approx. 1×10 12 GC, approx. 5×10 12GC, or approximately 1 × 10 13 In another embodiment, the dosage is about 1.0×10 9 GC / kg ~ approx. 3.0×10 14 In another embodiment, the dose is about 1×10 9 For example, the dose of AAV virus is about 1×10 10 GC / kg, 1×10 11 GC / kg, approx. 5×10 11 GC / kg, approx. 1×10 12 GC / kg, approx. 5×10 12 GC / kg, or approximately 1 x 10 13 GC / kg. In one embodiment, the construct may be delivered in a volume of 1 μL to about 100 mL. As used herein, the term "dose" or "amount" may refer to the total dose or amount delivered to a subject over the course of treatment, or the dose or amount delivered in a single unit (or multiple units or split doses).

[0124] The above-mentioned recombinant vector can be delivered to a host cell according to published methods. Preferably, rAAV suspended in a physiologically compatible carrier can be administered to a desired subject, including humans. A suitable carrier can be easily selected by those skilled in the art in view of the indication for which the introduced virus is intended. For example, one suitable carrier includes saline, which can be formulated with various buffer solutions (e.g., phosphate-buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present invention.

[0125] In another embodiment, the composition comprises a carrier, diluent, excipient and / or adjuvant. In certain embodiments, for administration to a human patient, the rAAV is suitably suspended in an aqueous solution containing saline, a surfactant, and a pharma- ceutically and / or physiologically compatible salt or mixture of salts. Suitably, the formulation is adjusted to a physiologically acceptable pH, for example, in the range of pH 6-9, or pH 6.0-7.5, or pH 6.2-7.7, or pH 6.5-7.5, pH 7.0-7.7, or pH 7.2-7.8, or about pH 7.0. In certain embodiments, the formulation is adjusted to a pH of about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, or about 7.8. In certain embodiments, a pH of about 7.28 to about 7.32, about 6.0 to about 7.5, about 6.2 to about 7.7, about 7.5 to about 7.8, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, or about 7.8 may be desired. In certain embodiments, for intravenous delivery, a pH of about 6.8 to about 7.2 may be desired. However, other pHs in the broader range, and subranges thereof, may be selected for other delivery routes.

[0126] Optionally, the compositions of the invention may contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to the rAAV and / or variant and carrier(s). Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0127] As used herein, "carrier" refers to any and all solvents, dispersion media, vehicles, These include coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharma- ceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmacologically 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 compositions of the present invention into suitable host cells. In particular, the rAAV vector-delivered transgene can be formulated for delivery either encapsulated in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles, etc.

[0128] In one embodiment, the composition comprises a final formulation suitable for delivery to a subject, for example, an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration.Optionally, one or more surfactants are present in the formulation.In another embodiment, the composition can be delivered as a concentrate that is diluted for administration to a subject.In other embodiments, the composition can be lyophilized and reconstituted at the time of administration.

[0129] Suitable surfactants or combinations of surfactants may be selected from non-toxic non-ionic surfactants. In one embodiment, a primary hydroxyl terminated bifunctional block copolymer surfactant is selected, such as Pluronic® F68 [BASF], also known as Poloxamer 188, with neutral pH and average molecular weight of 8400. Other surfactants and other poloxamers may be selected, i.e., non-ionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (macrogol-15 hydroxystearate), LABRASOL (polyoxycaprylic acid glyceride), polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol, and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are generally named with the letter "P" (for poloxamer) followed by three digits, the first two digits x 100 giving the approximate molecular mass of the polyoxypropylene core and the last digit x 10 giving the percentage of polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005% to about 0.001% of the suspension.

[0130] The dosage of a viral vector depends primarily on factors such as the condition being treated, the age, weight, and health of the patient, and may therefore vary between patients. For example, a therapeutically effective human dosage of a viral vector is generally about 1×10 (to treat an average subject weighing 70 kg) in the range of about 25 to about 1000 microliters to about 100 mL. 9 ~1×10 16 The concentration of the genomic viral vector (including all integers or fractions therein, preferably 1.0×10 for human patients) 12 GC~1.0×10 13GC). The compositions of the present invention may be delivered in a volume of about 0.1 μL to about 10 mL, including all values ​​within the range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method. In one embodiment, the volume is about 50 μL. In another embodiment, the volume is about 70 μL. In another embodiment, the volume is about 100 μL. In another embodiment, the volume is about 125 μL. In another embodiment, the volume is about 150 μL. In another embodiment, the volume is about 175 μL. In yet another embodiment, the volume is about 200 μL. In another embodiment, the volume is about 250 μL. In another embodiment, the volume is about 300 μL. In another embodiment, the volume is about 450 μL. In another embodiment, the volume is about 500 μL. In another embodiment, the volume is about 600 μL. In another embodiment, the volume is about 750 μL. In another embodiment, the volume is about 850 μL. In another embodiment, the volume is 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.

[0131] In some embodiments, the concentration of the recombinant adeno-associated virus having a nucleic acid sequence encoding a desired transgene under the control of a regulatory sequence in the composition is desirably about 10 per milliliter. 7 ~10 14 The range is in genome copies (GC / mL).

[0132] In one embodiment, the dosage of rAAV in the composition is about 1.0×10 9 GC / kg ~ approx. 1.5×10 13 In one embodiment, the dosage is about 1.0×10 10 In one embodiment, the dosage is about 1.0×10 11 In one embodiment, the dosage is about 1.0×10 12 In one embodiment, the dosage is about 5.0×10 12 In one embodiment, the dosage is about 1.0×10 13 GC / kg. All ranges stated herein are inclusive of the endpoints.

[0133] In one embodiment, the effective dose (total genome copies delivered) is about 10 7 ~10 13 In one embodiment, the total administered dose is about 10 8 In one embodiment, the total administered dose is about 10 9 In one embodiment, the total administered dose is about 10 10 In one embodiment, the total administered dose is about 10 11 In one embodiment, the total administered dose is about 10 12 In one embodiment, the total administered dose is about 10 13 In one embodiment, the total administered dose is about 10 14 In one embodiment, the total administered dose is about 10 15 It is a genome copy.

[0134] It is desirable to utilize the lowest effective concentration of the virus to reduce the risk of undesirable effects such as toxicity. Further dosages and dosage volumes within these ranges can be selected by the attending physician, taking into account the physical condition of the subject, preferably a human, being treated, the age of the subject, the particular disorder, and, if progressive, the degree of progression of the disorder.

[0135] In certain embodiments, the composition comprises a rAAV comprising an inducible PTH agonist construct. In certain embodiments, the inducing agent or molecule is rapamycin or a rapalog. In certain embodiments, the inducing agent is rapamycin and is administered at least once or more, at least twice or more, or at least three times or more after the composition comprising the rAAV. In some embodiments, the rapamycin is administered at a dose of at least about 4 to at least about 40 nM. In certain embodiments, the inducing agent (i.e., rapamycin) is administered at a dose of at least about 0.1 mg / kg to at least about 3.0 mg / kg. In certain embodiments, the inducing agent (i.e., rapamycin) is administered at a dose of at least about 0.5 mg / kg to at least about 2.0 mg / kg.

[0136] The viral vectors and other constructs described herein may be used in preparing medicaments for delivering a PTH fusion protein construct to a subject in need thereof, for providing a subject with increased half-life PTH, and / or for treating HP in a subject. Thus, in another aspect, a method of treating HP is provided. The method comprises administering a composition described herein to a subject in need thereof. In one embodiment, the composition comprises administering a viral vector comprising an expression cassette for a PTH fusion protein described herein. include.

[0137] As used herein, the term "treatment" or "treating" is defined to include administering to a subject one or more compounds or compositions described herein for the purpose of alleviating one or more symptoms of short bowel syndrome. Thus, "treatment" can include one or more of the following in a given subject: reducing the progression of HP, reducing the severity of symptoms, eliminating disease symptoms, slowing disease progression, or increasing the effectiveness of therapy.

[0138] 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 described herein. In one embodiment, the subject is a human.

[0139] A course of treatment may optionally include repeated administration of the same viral vector (e.g., AAVrh91 vector) or different viral vectors (e.g., AAVrh91 and AAV3B.AR2.12). Still other combinations may be selected using the viral vectors described herein. Optionally, the compositions described herein may be combined in a regimen with nutritional therapy (enteral or parenteral nutrition), drugs, such as drugs used to control gastric acid, drugs used to reduce diarrhea or improve intestinal absorption, or PTH protein, or surgery. In certain embodiments, the AAV vector and the combination therapy are administered essentially simultaneously. In other embodiments, the AAV vector is administered first. In other embodiments, the combination therapy is administered first.

[0140] In one embodiment, the composition is administered in combination 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, and recombinant PTH (rhPTH1-84).In another embodiment, the composition is administered in combination with an effective amount of calcium carbonate or vitamin D.

[0141] In some embodiments, the combination of rAAV and PTH protein described herein reduces the PTH protein dosage requirement in a subject compared to before treatment with a viral vector. Such dosage requirement 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 PTH protein that the subject requires. For example, the subject can be treated with PTH protein or other therapy that the treating physician can continue upon administration of the AAV vector. Such PTH protein or other combination therapy can then be continued, reduced, or discontinued as needed.

[0142] In one embodiment, the expression cassette, vector genome, rAAV-containing composition described herein for gene therapy, or other composition is delivered as a single dose per patient. In one embodiment, the subject is delivered a therapeutically effective amount of the composition described herein. As used herein, "therapeutically effective amount" refers to the amount of expression cassette or vector, or combination thereof, that delivers and expresses a sufficient amount of PTH fusion protein to target cells to achieve a therapeutic goal. The therapeutically effective amount can be selected by the treating physician or can be guided based on previously determined guidelines. For example, teriparatide can be provided subcutaneously at an initial dose of 20 μg twice daily. rAAV can be delivered to the subject and then supplemented with oral or subcutaneous teriparatide, or other drugs as needed, to reach the equivalent of the desired dose of 20 μg twice daily.

[0143] In certain embodiments, the therapeutic goal is to alleviate or treat one or more of the symptoms of HP. Therapeutically effective amounts may be determined based on animal models rather than human patients.

[0144] As used herein, the term "heterologous" or any grammatical variation thereof when used to refer to vp capsid proteins refers to a population of non-identical members, e.g., having vp1, vp2, or vp3 monomers (proteins) with different modified amino acid sequences. SEQ ID NO: 38 provides the encoded amino acid sequence of the AAVrh91 vp1 protein. The term "heterologous" as used in reference to vp1, vp2, and vp3 proteins (alternatively referred to as isoforms) refers to differences in the amino acid sequences of the vp1, vp2, and vp3 proteins within the capsid. AAV capsids contain subpopulations within the vp1, vp2, and vp3 proteins with predicted amino acid residue modifications. These subpopulations contain, at a minimum, certain deamidated asparagine (N or Asn) residues. For example, certain subpopulations contain at least one, two, three, or four highly deamidated asparagine (N) positions in asparagine-glycine pairs, and optionally further contain other deamidated amino acids, where deamidation results in an amino acid change and other optional modifications.

[0145] As used herein, a "subpopulation" of vp proteins refers to a group of vp proteins that have at least one defined common feature and that consists of at least one group member and fewer than all members of the reference group, unless otherwise specified. For example, a "subpopulation" of vp1 proteins is at least one vp1 protein and fewer than all vp1 proteins in an assembled AAV capsid, unless otherwise specified. A "subpopulation" of vp3 can be one vp3 protein and fewer than all vp3 proteins in an assembled AAV capsid, unless otherwise specified. For example, in an assembled AAV capsid, vp1 protein can be a subpopulation of vp proteins, vp2 protein can be another subpopulation of vp proteins, and vp3 is yet another subpopulation of vp proteins. In another example, the vp1, vp2, and vp3 proteins can include subpopulations having, e.g., at least one, two, three, or four highly deamidated asparagines, e.g., different modifications at asparagine-glycine pairs.

[0146] As used herein, a "stock" of rAAV refers to a population of rAAV. Despite the heterogeneity of capsid proteins due to deamidation, rAAV within a stock are expected to share five identical vector genomes. A stock may contain, for example, rAAV with capsids having selected AAV capsid proteins and heterogeneous deamidation patterns characteristic of a selected production system. A stock may be produced from a single production system or may be pooled from multiple runs of a production system. A variety of production systems may be selected, including but not limited to those described herein. As used herein, the terms "PTH construct", "PTH expression construct" and synonyms include the PTH sequence described herein in combination with the leader and fusion domain. The terms "PTH construct", "PTH expression construct" and synonyms may be used to refer to a nucleic acid sequence encoding a PTH fusion protein or its expression product.

[0147] The terms "percent identity", "sequence identity", "percent sequence identity" or "percent identical" in the context of nucleic acid sequences refer to bases in two sequences that are the same when aligned for correspondence. The length of sequence identity comparison can span the entire length of a genome, the entire length of a gene coding sequence, or a fragment of at least about 100-150 nucleotides, or as desired. However, for example, at least about 9 nucleotides, usually at least about 20-24 nucleotides, at least Identity between smaller fragments of about 28-32 nucleotides, at least about 36 or more nucleotides, may also be desired. Multiple sequence alignment programs are also available for nucleic acid sequences. Examples of such programs include "Clustal W", "CAP Sequence Assembly", "BLAST", "MAP", and "MEME", which are accessible through web servers on the Internet. Other sources of such programs are known to those of skill in the art. Alternatively, the Vector NTI utility is also used. There are also several algorithms known in the art that can be used to measure nucleotide sequence identity, including those included in the programs described above. As another example, polynucleotide sequences can be compared using Fasta™, a program in GCG version 6.1. Fasta™ provides alignments and percent sequence identity of the best overlapping regions between the query and search sequences. For example, percent sequence identity between nucleic acid sequences can be determined using Fasta™ using its default parameters (word size 6 and NOPAM factor for the scoring matrix) provided in GCG version 6.1, which is incorporated herein by reference.

[0148] The term "highly conserved" means at least 80% identity, preferably at least 90% identity, more preferably more than 97% identity. Identity is easily determined by those skilled in the art by resorting to algorithms and computer programs known to those skilled in the art.

[0149] Unless otherwise stated in the upper range, the percentage of identity will be understood to be a minimum level of identity and includes all higher levels of identity up to 100% identity to the reference sequence. Unless otherwise stated, the percentage of identity will be understood to be a minimum level of identity and includes all higher levels of identity up to 100% identity to the reference sequence. For example, "95% identity" and "at least 95% identity" can be used interchangeably, including 95%, 96%, 97%, 98%, 99%, up to 100% identity to the reference sequence, and all fractions therebetween.

[0150] The terms "percent identity (%)", "sequence identity", "percent sequence identity", or "percent identity" in the context of amino acid sequences refer to residues in two sequences that are the same when aligned so that they correspond. Percent identity can be readily determined for the corresponding nucleic acid sequence encoding a full-length polypeptide, polypeptide, about 70 amino acids to about 100 amino acids, or peptide fragments thereof, or sequencers of a protein. Suitable amino acid fragments can be at least about 8 amino acids in length and can be up to about 150 amino acids in length. In general, when referring to "identity", "homology", or "similarity" between two different sequences, the "identity", "homology", or "similarity" is determined with reference to the "aligned" sequences. An "aligned" sequence or "alignment" refers to multiple nucleic acid or protein (amino acid) sequences, often including corrections for missing or additional bases or amino acids, as compared to a reference sequence. Alignment is performed using any of a variety of publicly or commercially available multiple sequence alignment programs. Sequence alignment programs are available for amino acid sequences, including, for example, the "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME", and "Match-Box" programs. Typically, one of these programs is used with default settings, but one of skill in the art may modify these settings as necessary. Alternatively, one of skill in the art may utilize another algorithm or computer program that provides at least the same level of identity or alignment as that provided by the referenced algorithms and programs. See, for example, J.D. Thomson et al. al, Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments", 27(13):2682-2690 (1999).

[0151] It should be noted that the terms "a" or "an" refer to one or more. Thus, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0152] The terms "comprise", "comprises", and "comprising" should be interpreted inclusively rather than exclusively. The terms "consist", "consisting", and variations thereof should be interpreted exclusively rather than inclusively. Although various embodiments herein are set forth using the word "comprising", it is also intended that in other circumstances the relevant embodiment should be construed and described using the word "consisting of" or "consisting essentially of".

[0153] As used herein, "patient" or "subject" refers to a mammal, meaning humans, veterinary or agricultural animals, domestic or pet animals, and animals typically used in clinical research. In one embodiment, the subject of these methods and compositions is a human. In another embodiment, the subject is not a feline.

[0154] As used herein, the term "about" means a variability of 10% (±10%, e.g., ±1, ±2, ±3, ±4, ±5, ±6, ±7, ±8, ±9, ±10, or any value therebetween) from a given reference, unless otherwise specified.

[0155] In certain cases, the term "E+#" or "e+#" is used to refer to the exponent. For example, "5E10" or "5e10" refers to 5 × 10 10 These terms may be used interchangeably.

[0156] The term "modulate" or variations thereof as used herein refers to the ability of a composition to inhibit one or more components of a biological pathway.

[0157] As used herein, "disease," "disorder," and "condition" are used interchangeably to refer to an abnormal condition in a subject.

[0158] Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and by reference to published documents which provide general guidance to those of ordinary skill in the art for many of the terms used herein.

[0159] References to "one embodiment" or "another embodiment" when describing an embodiment do not imply that the referenced embodiment is mutually exclusive with another embodiment (e.g., an embodiment described before the referenced embodiment), unless expressly specified otherwise. EXAMPLES

[0160] The following examples are provided to illustrate various embodiments of the invention and are not intended to limit the invention in any manner.

[0161] Example 1 - Construction of PTH Vector A pharmacologically regulated gene expression system was reported in which transgene expression can be controlled by orally available drugs such as rapamycin and its proteins (Rapalogs). This gene circuit can be delivered to skeletal muscle by AAV vectors and provide sustained regulated transgene expression for more than 5 years in non-human primates (Rivera et al., 2005). Applying this technology to PTH could be a solution for HP patients to avoid daily injections of PTH protein and be safe when patients require treatment. To create a potent AAV vector that can provide therapeutic levels of PTH expression with lower vector doses and less frequent oral inducers, various versions of the PTH transgene were developed for favorable kinetics of PTH expression relative to rapamycin, with interest in the level and duration of induced expression. Figure 2B.

[0162] All transgenes were codon optimized for universal human codon usage. For better expression by skeletal muscle cells, various secretory sequences were also introduced (Figure 1A). For longer serum half-life, several N-terminal truncated forms of PTH were fused to the Fc domain from human IgG1 and IgG4 or to serum albumin (Figure 2A).

[0163] More specifically, vectors were constructed with a leader sequence placed upstream of one of several PTH amino acid sequences, followed by a fusion domain (Figure 2B). Sequences were generated and cloned into an expression vector containing a CMV promoter under the control of an inducible expression system. The expression construct was flanked by AAV2 ITRs. In other constructs, sequences were generated and cloned into an expression vector containing a CB7 constitutive promoter.

[0164] The following constructs were packaged into the AAVrh91 vector by triple transfection and iodixanol gradient purification as previously described.

[0165] Example 2 - In vitro expression PTH fusions were assayed for inducible human PTH-Fc in the culture supernatant of plasmid-transfected HEK293 cells. PTH-Fc was quantified by staining as shown in FIG. 3A. Fractions 3 and 4 under reducing (R) and non-reducing (NR) conditions are shown. PTH cannot form dimers under reduced conditions. The potency of these purified proteins was tested. The two tested fusions were found to have potencies similar to PTH(1-34). FIG. 3B

[0166] The inducible construct was tested with 0 or 40 nM rapamycin, and was expressed in slightly less amount compared to the constitutive construct (FIG. 3C).

[0167] Example 3 - Pilot expression in Rag1KO mice As shown in Figure 1B, Rag1KO female mice were administered the vector AAVrh91.CB7.CI.hPTH(N).rBG (1 × 10 11 GC / mouse) (with native leader) or AAVrh91.CB7.CI.hPTH(IL2).rBG (1 × 10 11 GC / mice) (with IL2 leader). Plasma on day 14 showed similar PTH levels, with the IL2 leader providing slightly higher secreted PTH. Figure 1C. Serum calcium on day 28 showed similar results. Figure 1C.

[0168] Rag1KO female mice were administered the vector AAVrh91.CB7.CI.hPTH(N).rBG (1 × 10 11 GC / mouse) or AAVrh91.CB7.CI.hPTH73.P.Fc.rBG (1 × 10 11 GC / mouse). Figure 4A. Plasma was isolated on days 14 and 28 and tested for hPTH potency. CB7.hPTH73.P.Fc demonstrated superior potency compared to the construct lacking the fusion. Figure 4B.

[0169] As shown in Figure 5A, Rag1KO female mice were administered the vector AAVrh91.CMV.TF1Nc.3.hGH (1 × 10 11 GC / mouse) and AAVrh91.CB7.CI.hPTH73.P.Fc.rBG (1 × 10 11 GC / mouse). On day 12 (in groups 2-3) rapamycin was administered at a dose of 10 mg / kg and on day 38 (in group 3) rapamycin was administered at a dose of 3 mg / kg. Groups 1 and 2 were bled and necropsied on day 14, and group 3 was bled (necropsied) on days 14, 21, 28, and 40. Plasma was isolated and tested for potency (Figure 5B). Groups 2 and 3 showed comparable potency on day 14, and a second rapamycin induction resulted in increased potency in group 3. Serum calcium is shown in Figure 5C.

[0170] Example 4 - Constitutive PTH-Fc vector in AFG monkeys As shown in Figure 7, female African green monkeys were administered the vector AAVrh91.CB7.CI.hPTH73.P.Fc.rBG (1 x 10 12 GC / kg) injections. These monkeys demonstrated NAB titers, as shown in Figure 7B.

[0171] Example 5 - TPTX model in Rag2KO rats Rag2KO rats were subjected to thyroparathyroidectomy (TPTX) to validate the TPTX model. Surgical resection of the parathyroid gland was performed on 8-week-old Sprague Dawley / Rag2KO rats using 5-aminolevulinic acid fluorescence identification technique. 5-ALA, 500 mg / kg, was administered intraperitoneally before surgery, resulting in phototoxicity. Therefore, the rats were placed under soft light for 2 hours before surgery. A vertical incision was made in the midline of the neck, and the red fluorescent PTH gland was observed under white-blue light and UV filter illumination. The gland was excised with a cold knife. Postoperative complications were low calcium levels, which caused uncontrollable muscle spasms and contractions. Without calcium supplementation provided in water or custom-formulated diet (AIN-93G 0.5% Ca), death occurred within 4 days.

[0172] TPTX rats were administered vehicle, inducible hPTH73.P.Fc (1 × 10 12 GC / kg or 1×10 13 Rapamycin was administered IM with 10 mg / kg rapamycin per 100 mg / kg of rapamycin per day on day 14 and the second dose (3 mg / kg per 100 mg / kg per day) on day 40. Clinical signs are shown in the table below. [Table 3]

[0173] The results are shown in Figures 8A-8C. The two rats that received the vehicle injections underwent incomplete parathyroidectomy. The rats became severely ill for unknown reasons.

[0174] Example 6 - Constitutive PTH-Fc vector in Rag2KO rats Rag2KO rats (3 rats / group) were injected with AAVrh91.CB7.CI.hPTH73.P.Fc.rBG (1 × 10 13 GC / kg or 3×10 13 GC / kg) was injected IM. 13 One rat from the group receiving GC / kg exhibited hind limb paralysis and had to be euthanized, presumably due to pathological fracture of the spinal cord. Plasma PTH (Figure 9A) and serum calcium (Figure 9B) were measured.

[0175] All documents cited herein are incorporated herein by reference. U.S. Provisional Patent Application No. 63 / 316,215, filed March 3, 2022, is incorporated herein by reference. Although the present invention has been described with reference to certain embodiments, it will be understood that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. 1. A composition comprising a nucleic acid comprising a sequence encoding a fusion protein comprising a parathyroid hormone (PTH) protein and an IgG4 Fc, wherein the fusion protein has the 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. 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 a sequence sharing at least 75% identity thereto.

3. A composition comprising a viral vector, (a) an adeno-associated virus (AAV) capsid; (b) a vector genome packaged within the AAV capsid, the vector genome comprising AAV inverted terminal repeats (ITRs), a coding sequence for a fusion protein, and a regulatory sequence that directs 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 of any one of claims 1 to 4, wherein the fusion protein is under the control of an inducible gene expression system.

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

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

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

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

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

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

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

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

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

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

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

17. 17. The composition of any one of claims 5 to 16, comprising a regulatable promoter, an activation domain comprising a p65 transactivation domain and the FKBP12-rapamycin binding (FRB) domain of FKBP12-rapamycin related protein (FRAP), a DNA binding domain comprising a zinc finger homeodomain (ZFHD) and three FK506 binding protein domain (FKBP) subunit genes, 12 copies of binding sites for said ZFHD, and a PTH protein of any of SEQ ID NOs: 1, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 39, 42, 44, 46, or 48, and a regulatory sequence directing expression of said fusion protein.

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

19. 20. A composition according to any one of claims 1 to 18 for use in a method for treating a subject with hypoparathyroidism.

20. 20. Use of a composition according to any one of claims 1 to 18 in the manufacture of a medicament for treating a subject with hypoparathyroidism.

21. The composition is 1×10 ９ GC / kg ~ 5 x 10 １３ 19. The composition of any one of claims 1 to 18, formulated to administer a dose of GC / kg of the rAAV.

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

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

24. 1. A method of treating a subject having hypoparathyroidism, comprising delivering to the subject an rAAV having an AAV capsid derived from AAVrh91 or AAVhu68, and a vector genome packaged in the AAV capsid, wherein the vector genome comprises AAV inverted terminal repeats (ITRs), a sequence encoding a fusion protein comprising a PTH protein of any of SEQ ID NOs: 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 patient is administered a composition according to any one of claims 1 to 18.

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

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

28. A composition according to any one of claims 1 to 18 for treating hypoparathyroidism in humans.

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