Gene therapy for fibroblast growth factor 23-associated hypophosphatemic disorders

Abstract

The present invention relates to nucleic acid constructs for gene therapy of FGF-23-associated hypophosphatemic disorders, particularly gene therapy directed to muscle, liver, or hematopoietic tissue, more particularly liver tissue. The present invention also relates to vectors containing the nucleic acid constructs and their use for the treatment of FGF-23-associated hypophosphatemic disorders, particularly XLH, by gene therapy.

Description

[Technical field]

[0001] The present invention is in the field of gene therapy for FGF-23-related hypophosphatemic disorders, particularly X-linked hypophosphatemia (XLH). The present invention relates to a nucleic acid construct for gene therapy for FGF-23-related hypophosphatemic disorders, particularly gene therapy directed to muscle, liver, or hematopoietic tissue, more particularly liver tissue. The present invention also relates to vectors comprising the nucleic acid constructs and their uses for the treatment of FGF-23-related hypophosphatemic disorders, particularly XLH, by gene therapy. [Background technology]

[0002] Fibroblast growth factor 23 (FGF-23 or FGF23) is a phosphaturic hormone produced by bone, which acts by binding to the Klotho-FGF receptor complex. Excessive activity of FGF23 leads to hypophosphatemic diseases, including various genetic disorders such as X-linked hypophosphatemia (XLH) and autosomal dominant or recessive hypophosphatemic rickets (ADHR, ADHR1, ADHR2), as well as acquired disorders such as tumor-induced osteomalacia (TIO) and chronic kidney disease-mineral and bone disorder (CKD-MBD) (reviewed in Seiji Fukumoto, Calcif. Tissue Int., 2016, 98, pp. 334-340).

[0003] Clinical signs of X-linked hypophosphatemia (XLH, OMIM #307800) range from isolated hypophosphatemia to severe lower limb deformities. The disease manifests within the first 2 years of life with lower limb deformities. In adults, enthesopathy (tendon calcification) with joint pain, spontaneous dental abscesses, and sensorineural hearing loss have been reported. XLH is caused by mutations in the phosphate-regulating neutral endopeptidase (PHEX) gene that induce increased circulating levels of FGF23. Increased FGF23 function leads to downregulation of sodium phosphate cotransporters in the kidney. The cotransporters, located in the renal proximal tubules, mediate re-adsorption of phosphate from urine. Its downregulation leads to insufficient reabsorption of phosphate and lower phosphate levels in the blood. In addition, increased FGF23 is associated with deficient 1,25(OH)2 vitamin D synthesis and increased degradation. Decreased blood phosphate levels and low vitamin D levels lead to defective bone mineralization and fractures.

[0004] Classical XLH treatment consists of oral phosphate and high doses of calcitriol, the active form of vitamin D. In practice, the response to iv phosphate therapy can be unpredictable, complications include "excessive" hyperphosphatemia, hypocalcemia, and metastatic calcification, and parenteral regimens are impractical for chronic disorders. However, oral therapy requires high doses that frequently lead to diarrhea or gastric irritation, and replacement therapy alone is never sufficient in the presence of significant renal phosphate wasting. Therefore, novel strategies for the treatment of FGF23-associated hypophosphatemia are needed.

[0005] There is an animal model of the disease resulting from a natural deletion of the PHEX gene, the HypDuk model. This model reproduces most of the disease symptoms, with lower phosphate levels in the blood and defective bone growth. Various therapeutic strategies have been tested in HypDuk mice. One approach uses anti-FGF23 neutralizing antibodies. Recently, a monoclonal antibody anti-FGF23 was approved for the treatment of pediatric forms of XLH (Crysvita®, Ultragenyx). Another strategy consists of the use of a truncated form of human FGF23 that can bind to the FGF23 receptor without inducing the intracellular cascade of activation caused by the functional interaction between the receptor and FGF23. This truncated FGF23 can be used as a competitive factor to reduce the increase in FGF23 function observed in XLH (Goetz R. et al., PNAS, 2009, 107, 407-412).

[0006] No gene therapy for FGF-23-related hypophosphatemic disorders, such as XLH, has been reported so far. Therefore, there is a need for a gene therapy approach for the treatment of FGF-23-related hypophosphatemic disorders, especially XLH. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] United States Patent Publication No. US 2003 / 0157064 [Patent Document 2] WO2015110449 [Patent Document 3] WO2009130208 [Patent Document 4] WO2017 / 182607 [Patent Document 5] WO2015013313 [Patent Document 6] WO2005 / 118792 [Non-patent literature]

[0008] [Non-Patent Document 1] Seiji Fukumoto, Calcif. Tissue Int., 2016, 98, pp. 334 - 340 [Non-Patent Document 2] Goetz R. et al., PNAS, 2009, 107, pp. 407 - 412 [Non-Patent Document 3] Goetz et al., PNAS, 2010, 107, pp. 407 - 410 [Non-Patent Document 4] Garringer et al., Am. J. Physiol. Endocrinol. Metab., 2008, 295, pp. E929 - E937 [Non-Patent Document 5] Altschul et al., J. Mol. Biol., 1990, 215, pp. 403 - [Non-Patent Document 6] Puzzo et al., Sci. Transl. Med., 2017, 9(418): doi:10.1126 [Non-Patent Document 7] Stern et al., BMC Proc., 2011, 5 (suppl 8):013 [Non-Patent Document 8] Sven Berger, Peter Lowe, and Michael Tesar (2015) Fusion protein technologies for biopharmaceuticals: Applications and challenge, mAbs, 7:3, pp. 456 - 460, DOI:10.1080 / 19420862.2015.1019788 [Non-Patent Document 9] Beattie and Dugaiczyk, Gene 1982, 20, pp. 415 - 422 [Non-Patent Document 10] Lichenstein et al., J. Biol. Chem., 1994, 269, pp. 18149 - 18154 [Non-Patent Document 11] Cooke and David, J. Clin. Invest., 1985, 76, pp. 2420 - 2424

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[0009] The present inventors have engineered a nucleic acid construct and derived AAV vector for gene therapy of FGF-23-associated hypophosphatemic disease. After a single injection of this AAV vector in HypDuk mice, normalized increases in body weight, body size, tail length, and circulating phosphate were observed in treated animals. The correction of the disease at biochemical, macroscopic, and functional levels observed after a single injection of this AAV vector demonstrates the enhanced efficacy of gene therapy approaches based on this nucleic acid construct and derived vector, particularly AAV vector, for the treatment of FGF-23-associated hypophosphatemic disease.

[0010] Therefore, the present invention relates to a method for producing a (a) a signal peptide, (b) FGF23 C-terminal peptide that binds to the FGFR / klotho complex, (c) a cleavable linker, and (d) Protein stabilizing moiety wherein the signal peptide is at the N-terminus of the fusion protein, and the FGF23 C-terminal peptide and the protein stabilizing moiety are separated by a cleavable linker.

[0011] In some embodiments, the FGF23 C-terminal peptide comprises a sequence from any one of positions 175 to 189 to any one of positions 203 to 251 of SEQ ID NO:1, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to said sequence.

[0012] In some embodiments, the FGF23 C-terminal peptide comprises an RXXR motif at positions 176-179 of SEQ ID NO:1.

[0013] In some preferred embodiments, the FGF23 C-terminal peptide comprises the sequence of SEQ ID NO:2, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0014] In some embodiments, the signal peptide comprises a sequence selected from the group consisting of SEQ ID NO:3-8, preferably SEQ ID NO:7.

[0015] In some embodiments, the protein stabilizing moiety is human serum albumin, preferably comprising the sequence of SEQ ID NO:9.

[0016] In some embodiments, the cleavable linker comprises the sequence of SEQ ID NO:10.

[0017] In some preferred embodiments, the nucleic acid construct encodes an FGF23 protein comprising the sequence of SEQ ID NO: 12 or 52, or a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of the foregoing sequences.

[0018] In some embodiments, the nucleic acid construct is codon optimized for expression in humans.

[0019] In some preferred embodiments, the nucleic acid construct comprises the sequence of SEQ ID NO: 13, 51, or 57, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of said sequences.

[0020] In some embodiments, the nucleic acid construct comprises an expression cassette in which a coding sequence is operably linked to at least one promoter that is functional in a target cell or tissue of an individual, particularly a muscle, liver, or hematopoietic cell or tissue. In some particular embodiments, the promoter is a liver-specific promoter, preferably the human alpha-1 antitrypsin promoter.

[0021] In some embodiments, the nucleic acid construct further comprises one or more control elements selected from the group consisting of an enhancer associated with the promoter, preferably the human ApoE regulatory region; an intron located between the promoter and the coding sequence, preferably the modified HBB2 intron of SEQ ID NO: 17 or the modified FIX intron of SEQ ID NO: 19; and a transcription termination signal, preferably the bovine growth hormone polyadenylation signal.

[0022] In some embodiments, the nucleic acid construct comprises or consists of DNA.

[0023] In other embodiments, the nucleic acid construct comprises or consists of RNA.

[0024] The present invention also relates to a vector for gene therapy comprising a nucleic acid construct according to the invention.

[0025] In some embodiments, the vector is a viral vector, particularly an AAV or lentiviral vector, preferably an AAV vector comprising a capsid selected from the group consisting of AAV1, AAV2, AAV5, AAV8, AAV2i8, AAV9, AAVrh10, AAVrh39, AAVrh43, AAVrh74, AAV-LK03, AAV2G9, AAV.PHP, AAV-Anc80, AAV3B capsids, and chimeric capsids thereof, particularly an AAV8, AAV9, or AAVrh74 capsid, such as an AAV8 or AAV9 capsid, more preferably an AAV8 capsid.

[0026] In some other embodiments, the vector is a particle or vesicle, particularly a lipid-based micro- or nano-vesicle or particle.

[0027] The present invention relates to a cell genetically modified by a nucleic acid construct according to the invention or by a vector according to the invention, which is preferably a liver, muscle or hematopoietic cell, more preferably a liver cell.

[0028] The present invention further relates to a pharmaceutical composition comprising at least one active agent selected from a nucleic acid construct according to the invention, a vector according to the invention or a cell according to the invention, and a pharma- ceutically acceptable carrier.

[0029] The present invention relates to a pharmaceutical composition according to the invention for use in the treatment of FGF-23-associated hypophosphatemic disorders by gene therapy or cell therapy.

[0030] In some embodiments of said use, the FGF-23 related hypophosphatemic disorder is a genetic disorder selected from the group comprising X-linked hypophosphatemia (XLH), autosomal dominant hypophosphatemic rickets (ADHR), autosomal recessive hypophosphatemic rickets 1 (ADHR1), autosomal recessive hypophosphatemic rickets 2 (ADHR2), cavitary skeletal dysplasia, Janssen type metaphyseal chondrodysplasia, hypophosphatemia, dental abnormalities and focal calcifications, McCune-Albright syndrome / fibrous dysplasia, and hypophosphatemia, skin and bone lesions, or an acquired disorder selected from the group comprising tumor-induced osteomalacia, hypophosphatemic osteomalacia, complications from kidney transplantation or parenteral iron therapy, chronic kidney disease and hyperparathyroidism; preferably X-linked hypophosphatemic rickets. [Brief description of the drawings]

[0031] [Figure 1] Schematic diagram of the nucleic acid construct and AAV expression vector for a truncated FGF23 fusion protein according to the invention. A. Nucleic acid construct for a truncated FGF23 fusion protein according to the invention. The C-terminal amino acids of human FGF protein (aa 175-251, cFGF) were fused to human serum albumin without a signal peptide (aa 25-609) through a cleavable linker derived from amino acids 182-200 of human coagulation factor IX (hFIX). Finally, a signal peptide derived from the first 18 amino acids of chymotrypsinogen B2 was fused at the N-terminus of the construct (CTRB2). B. AAV expression vector for a truncated FGF23 fusion protein according to the invention. The nucleic acid construct was inserted into an AAV expression vector containing a liver-specific promoter (apolipoprotein E (ApoE) enhancer-human alpha 1 antitrypsin (hAAT) promoter), a modified HBB2 intron, and a bovine growth hormone (bGH) polyadenylation signal. [Diagram 2]FIG. 1. Stabilization of FGF23 c-terminal tail in vitro by albumin fusion. Human liver hepatoma cells (Huh-7) were transfected with plasmids expressing various forms of human FGF23 as listed in Table 1. 48 hours after transfection, media was collected and analyzed by Western blot using anti-FGF23 antibody. (A) Comparison of all constructs showing relative expression of the various constructs and higher expression levels achieved with fusion proteins of FGF23-C and albumin moiety. The locations of FGF23, FGF23-C, or fusion protein of FGF23-C and human albumin (FGF-23-albumin) are indicated on the left. (B) Comparison of expression levels achieved after transfection with triplicates of plasmids expressing FGF23-C albumin fusion proteins in Huh-7 cells. [Diagram 3] FIG. 1. Stabilization of the c-terminal part of FGF23 in vivo by albumin fusion. Wild-type C57BL6 / J mice were injected with 1×1012 vg / mouse of AAV8 vectors expressing a codon-optimized version of the c-terminal part of FGF23 (coFGF23-C, amino acids 175-251) containing the RRHTR motif (constructs 7, 9, and 12 only), the sp7 signal peptide, and fused (constructs 9, 11, and 12) or not fused to an albumin moiety (construct 7). Construct 12 contained a linker composed of amino acids 182-200 of human coagulation factor IX, whereas constructs 9 and 11 had no linker. One month after vector injection, mice were sacrificed and plasma and liver were harvested to measure the levels of phosphorylation and expression achieved with the different AAV vectors. (A) Phosphorylation measured in blood. (B) Vector genome copy number per diploid genome (VGCN) measured in liver. (C) Western blot analysis performed on plasma using anti-FGF23 antibody. (D) Histogram showing relative intensity of bands depicted in panel C. Statistical analysis was performed by t-test (*P<0.05 vs. PBS-treated mice, n=4-5 per group). [Figure 4-1]AAV-mediated rescue of growth retardation in Hypduk mice. One-month-old Hypduk mice were treated with either PBS (KO, PBS) or 1×1012 vg / mouse of an AAV vector expressing FGF23-C fused to sp7 and albumin through an hFIX linker and containing an RRHTR motif (KO, AAV). PBS-treated wild-type mice were used as controls (WT, PBS). A-C. Mice were measured 1, 2, and 3 months after vector injection. They showed increases over the 3-month period in (A) body weight, (B) tail length, and (C) body size. Statistical analysis was performed by ANOVA (*KO, P<0.05 vs. PBS; #WT, P<0.05 vs. PBS; n=9-11 per group). [Figure 4-2] AAV-mediated rescue of growth retardation in Hypduk mice. One-month-old Hypduk mice were treated with either PBS (KO, PBS) or 1×1012 vg / mouse of an AAV vector expressing FGF23-C fused to sp7 and albumin through an hFIX linker and containing an RRHTR motif (KO, AAV). PBS-treated wild-type mice were used as controls (WT, PBS). A-C. Mice were measured 1, 2, and 3 months after vector injection. They showed increases over the 3-month period in (A) body weight, (B) tail length, and (C) body size. Statistical analysis was performed by ANOVA (*KO, P<0.05 vs. PBS; #WT, P<0.05 vs. PBS; n=9-11 per group). [Diagram 5] AAV-mediated rescue of hypophosphatemia in Hypduk mice. One-month-old Hypduk mice were treated with either PBS (KO, PBS) or 1x1012vg / mouse of AAV vector expressing FGF23-C fused to sp7 and albumin through a hFIX linker and containing the RRHTR motif (KO, AAV). PBS-treated wild-type mice were used as controls (WT, PBS). Phosphate levels in blood were measured 3 months after vector injection. Statistical analysis was performed by ANOVA (*=P<0.05, NS not significant as indicated). [Figure 6]AAV-mediated rescue of muscle function in Hypduk mice. One-month-old Hypduk mice were treated with either PBS (KO, PBS) or 1x1012vg / mouse of AAV vector expressing FGF23-C fused to sp7 and albumin through a hFIX linker and containing a RRHTR motif (KO, AAV). PBS-treated wild-type mice were used as controls (WT, PBS). Inverted grid performance was measured 3 months after vector injection. Statistical analysis was performed by ANOVA (*=P<0.05, NS not significant as indicated). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] nucleic acid construct The present invention provides nucleic acid constructs for gene therapy of FGF-23-associated hypophosphatemic disorders.

[0033] The nucleic acid construct of the present invention comprises at least (a) a signal peptide, (b) FGF23 C-terminal peptide that binds to the FGFR / klotho complex, (c) a cleavable linker, and (d) Protein stabilizing moiety wherein the signal peptide is at the N-terminus of the fusion protein, and the FGF23 C-terminal peptide and the protein stabilizing moiety are separated by a cleavable linker.

[0034] As used herein, the term fibroblast growth factor 23 (FGF-23 or FGF23), also known as phosphatonin or tumor-derived hypophosphatemia-inducing factor, refers to the protein encoded by the FGF23 gene in the mammalian genome. Human FGF23 has the UniProtKB / Swiss-Prot accession number Q9GZV9.1 or NCBI accession number NP_065689 (SEQ ID NO: 1) of a 251 amino acid sequence. FGF23 is expressed as a precursor containing an N-terminal signal peptide (24 amino acids), which is cleaved to yield the mature protein (FGF23). To exert its phosphaturic activity, FGF23 requires a two-component FGF receptor (FGFR)-Klotho complex. In addition, FGF23 activity is located at the interface between the FGF core homology domain and the 72-residue long C-terminal tail of FGF23. 176 RXXR 179 It is regulated by proteolytic cleavage at the motif. Proteolytic cleavage generates an inactive N-terminal fragment (Y25-R179), an FGF core homology domain, and a C-terminal fragment (S180-I251). The FGF23 C-terminal fragment is an endogenous inhibitor or antagonist of FGF23, competing with the full-length ligand for binding to the FGFR-Klotho complex and blocking FGF23 signaling. The FGF23 C-terminal fragment (180-251) has been shown to antagonize the phosphaturic activity of FGF23 in vivo. A smaller C-terminal fragment (FGF 180-205) has also been shown to exhibit FGF23 antagonist activity (Goetz et al., PNAS, 2010, pp. 107, 407-410). Residues 189-203 in the mature 251 residue FGF23 are required for FGF23 activity, whereas FGF23 amino acid 3' to residue 203 is dispensable for initiating FGF23-dependent intracellular signaling (Garringer et al., Am. J. Physiol. Endocrinol. Metab., 2008, 295, E929-E937).

[0035] In the following description, residues are designated by the standard single letter amino acid code, with the positions indicated being determined by alignment with SEQ ID NO:1.

[0036] Unless the context clearly indicates otherwise, "a," "an," and "the" include plural referents. Thus, the terms "a" (or "an"), "one or more," or "at least one" can be used interchangeably herein; unless otherwise specified, "or" means "and / or."

[0037] The nucleic acid construct may comprise or consist of DNA, RNA, or synthetic or semi-synthetic nucleic acid that is expressible in a target cell or tissue of an individual.

[0038] The FGF23 C-terminal peptide produced by in vivo cleavage of the FGF23 fusion protein binds to the FGFR / klotho complex. The mature FGF23 fusion protein (without its signal peptide) can also bind to the FGFR / klotho complex. This binding inhibits FGF23 signaling through the FGFR-klotho complex. The binding activity of FGF23 fusion proteins and derived C-terminal peptides to the FGFR / klotho complex, as well as the inhibition of FGF23 signaling through the FGFR-klotho complex, are well known in the art and can be verified by standard assays, for example those disclosed in Goetz et al., PNAS, 2010, 107, 407-410. The FGF23 C-terminal peptide produced by in vivo cleavage of the FGF23 fusion protein according to the present invention is a specific inhibitor or antagonist of the FGFR-klotho-dependent function of FGF23. The FGF23 fusion protein according to the present invention can also be a specific inhibitor or antagonist of the FGFR-klotho-dependent function of FGF23. Due to the ability of the FGF23 C-terminal peptide and optionally the ability of the fusion protein to neutralize the Klotho-dependent function of FGF23, the FGF23 fusion protein of the present invention is useful as a therapeutic agent for treating FGF23-related hypophosphatemic diseases.

[0039] The FGF23 C-terminal peptide comprises or consists of a sequence from any one of positions 175 to 189 to any one of positions 203 to 251 of SEQ ID NO: 1, which binds to the FGFR / klotho complex, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the above sequence. FGF23 The C-terminal peptide is selected from the group consisting of positions 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, and 189 to 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, and 229 of SEQ ID NO: 1. The nucleic acid sequence may comprise a sequence up to position 9, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, or 251, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. Preferably, the FGF23 C-terminal peptide comprises or consists of a sequence from any one of positions 175 to 180 to any one of positions 205 to 251 of SEQ ID NO: 1, which binds to the FGFR / klotho complex, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to said sequence.

[0040] In certain embodiments, the FGF23 C-terminal peptide of the present invention comprises: 176 RXXR 179In some particular embodiments, the FGF23 C-terminal peptide of the present invention terminates at position 203 of SEQ ID NO:1. In some other particular embodiments, the FGF23 C-terminal peptide of the present invention terminates at position 204 or later of SEQ ID NO:1, such as at position 232 or 251 of SEQ ID NO:1. In some preferred embodiments, the FGF23 C-terminal peptide of the present invention comprises or consists of the sequence of SEQ ID NO:2 (positions 175-251 of SEQ ID NO:1), which binds to the FGFR / klotho complex, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the FGF23 C-terminal peptide of the present invention comprises a mutation, in particular a mutation that increases its binding affinity to the FGFR / klotho complex.

[0041] The FGF23 C-terminal peptide according to the present invention comprises or consists of a C-terminal fragment of FGF23 of 15 to 77 amino acids. Thus, unlike the full-length FGF23 protein, the FGF23 C-terminal peptide does not contain any sequence derived from the N-terminal region of FGF23 (positions 25 to 174 of SEQ ID NO: 1). In some embodiments, the FGF23 C-terminal peptide according to the present invention comprises or consists of a C-terminal fragment of FGF23 of at least 20, 25, 30, or more amino acids.

[0042] Percent amino acid or nucleotide sequence identity is defined as the percentage of amino acid residues or nucleotides in a comparison sequence that are identical to a reference sequence after aligning the sequences to achieve maximum sequence identity and introducing gaps if necessary, and any conservative substitutions for amino acid sequences are not considered as part of the sequence identity. Sequence identity is calculated over the entire length of the reference sequence. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways known to those skilled in the art, for example, using publicly available computer software such as BLAST (Altschul et al., J. Mol. Biol., 1990, 215, 403-). When using such software, default parameters are preferably used, for example, for gap penalty and extension penalty. The BLASTP program uses a word length (W) of 3 and an expectation (E) of 10 as default settings.

[0043] FGF23 fusion proteins contain a signal peptide at their N-terminus. A signal peptide (SP) is a short peptide sequence present at the N-terminus of a secretory and used to target a protein for secretion. Signal peptides do not consist of a strict consensus sequence, but have a three-region design consisting of a positively charged N-terminal region (N-region, 1-5 residues), a hydrophobic central region (H-region, 7-15 residues), and a neutral polar C-terminal region (C-region, 3-5 residues). Numerous signal peptides are known in the art and publicly available (see, inter alia, the Signal Peptide Website and the SPdb sequence database; Puzzo et al., Sci. Transl. Med., 2017, 9(418): doi:10.1126). In addition, methods for selecting appropriate SP sequences for efficient protein secretion are known in the art (see, inter alia, Stern et al., BMC Proc., 2011, 5 (suppl 8):013).

[0044] The signal peptide can be the FGF23 endogenous or native signal peptide (SEQ ID NO:3; positions 1-24 of SEQ ID NO:1) or a heterologous signal peptide. As used herein, a heterologous signal peptide refers to a signal peptide that is different from the FGF23 signal peptide, particularly the human FGF23 signal peptide. Examples of heterologous signal peptides that can be used in the present invention include, but are not limited to, alpha-1 antitrypsin (SEQ ID NO:4); synthetic mut1 (SEQ ID NO:5); synthetic mut3 (SEQ ID NO:6); chymotrypsinogen B2 (CTRB2), (positions 1-18 of Uniprot Accession No. Q6GPI1 or NCBI Accession No. NP_001020371, or SEQ ID NO:7), and plasma protease inhibitor C1 (positions 1-22 of Uniprot Accession No. P05155, or SEQ ID NO:8).

[0045] In some embodiments, the signal peptide is a heterologous signal peptide, preferably chymotrypsinogen B2 signal peptide (SEQ ID NO: 7).

[0046] The FGF23 C-terminal peptide is linked to the protein stabilizing moiety via a cleavable linker. A protein stabilizing moiety is any protein moiety that increases the half-life or duration of action of the therapeutic protein / peptide to which it is attached and is suitable for therapeutic applications. A variety of protein stabilizing moieties that have been used to stabilize therapeutic proteins are known in the art (see, for example, Sven Berger, Peter Lowe, and Michael Tesar (2015) Fusion protein technologies for biopharmaceuticals: Applications and challenge, mAbs, 7:3, pp. 456-460, DOI:10.1080 / 19420862.2015.1019788). Examples of protein stabilizing moieties that can be used in the present invention include, but are not limited to, serum albumin, particularly human serum albumin; immunoglobulin Fc fragments; human chorionic gonadotropin carboxy-terminal peptide (CTP); a receptor fused to its ligand (GHR fused to GH); and the latency-associated peptide of TGF-beta (linked to a cleavage site for a metalloprotease).

[0047] In some embodiments, the protein stabilizing moiety is different from the immunoglobulin Fc fragment.

[0048] In some embodiments, the protein stabilizing moiety is derived from a serum transport protein. Serum transport proteins include, but are not limited to, the albumin family of proteins and evolutionarily related serum transport proteins, such as albumin, alpha-fetoprotein (AFP; Beattie and Dugaiczyk, Gene 1982, 20, 415-422), afamin (AFM; Lichenstein et al., J. Biol. Chem., 1994, 269, 18149-18154), and vitamin D binding protein (DBP; Cooke and David, J. Clin. Invest., 1985, 76, 2420-2424). Serum transport proteins can be from any vertebrate, including mammals, birds, fish, and others. The present invention encompasses functional variants, such as naturally occurring polymorphic variants, of serum transport proteins, as well as functional fragments. A functional fragment or variant of a serum transport protein refers to a variant or fragment that is capable of increasing the half-life or duration of action of a therapeutic protein / peptide to which it is attached and is suitable for therapeutic applications.

[0049] In certain embodiments, the protein stabilizing moiety is albumin, including functional fragments or variants thereof as defined above. Albumin may be from any vertebrate, particularly any mammal, such as human, bovine, ovine, or porcine. Non-mammalian albumins include, but are not limited to, hen and salmon. The albumin portion of the albumin-linked polypeptide may be from a different animal than the therapeutic polypeptide portion. In particular, the albumin fusion proteins of the invention may include naturally occurring polymorphic variants of human albumin (HA) and fragments of human albumin. The albumin portion of the albumin fusion protein may include the full length HA sequence (NCBI Accession No. NP_000468), preferably including human serum albumin without the signal peptide (positions 25-609 of NCBI Accession No. NP_000468, or SEQ ID NO: 9), or may include one or more fragments thereof that may stabilize or prolong therapeutic activity. Such fragments may be 10 or more amino acids in length, or may contain about 15, 20, 25, 30, 50, 70 or more contiguous amino acids from the HA sequence, or may contain some or all of a specific domain of HA.

[0050] In some preferred embodiments, the protein stabilizing moiety is human serum albumin (NCBI Accession No. NP_000468), preferably including human serum albumin without the signal peptide (positions 25-609 of NCBI Accession No. NP_000468, or SEQ ID NO: 9).

[0051] A cleavable linker is any peptide linker that is cleavable in vivo. A variety of cleavable peptide linkers that have been used in therapeutic protein constructs are known in the art. Examples of cleavable peptide linkers that may be used in the present invention include, but are not limited to, coagulation factor activation sequences, particularly FIX activation sequences (aa 182-200 or 182-203 of NCBI Accession No. NP_000124): SEQ ID NO: 10 or SEQ ID NO: 11.

[0052] In some preferred embodiments, the cleavable linker comprises or consists of the sequence of SEQ ID NO:10.

[0053] In some embodiments, the signal peptide, the FGF23 C-terminal peptide, the cleavable linker, and the protein stabilizing moiety are N- to C-terminal of the FGF23 fusion protein, meaning that the protein stabilizing moiety is fused to the C-terminus of the FGF23 C-terminal peptide.

[0054] In some embodiments, the nucleic acid construct comprises or consists of DNA.

[0055] In some other embodiments, the nucleic acid construct comprises or consists of RNA, in particular mRNA.

[0056] Examples of preferred nucleic acid constructs of the present invention include: - a nucleic acid construct encoding the FGF23 protein comprising the sequence SEQ ID NO: 12 as shown in the examples of the present application and in FIG. 1A, which corresponds to construct number 12 in Table 1; - a nucleic acid construct encoding an FGF23 protein comprising the sequence SEQ ID NO: 52 as shown in the examples of the present application, which corresponds to construct number 10 in Table 1, and - a nucleic acid construct encoding an FGF23 protein comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of the sequences of SEQ ID NO: 12 or 52; preferably a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of said sequences; more preferably a sequence having at least 95%, 96%, 97%, 98% or 99% identity to any one of said sequences. Includes:

[0057] SEQ ID NO: 12 contains, from its N to C terminus, a chymotrypsinogen B2 signal peptide (SEQ ID NO: 7), an FGF23 C-terminal peptide of SEQ ID NO: 2, a cleavable linker of SEQ ID NO: 10, and human serum albumin of SEQ ID NO: 9. SEQ ID NO: 52 contains, from its N to C terminus, a chymotrypsinogen B2 signal peptide (SEQ ID NO: 7), an FGF23 C-terminal peptide consisting of a sequence derived from positions 180 to 251 of SEQ ID NO: 1, a cleavable linker of SEQ ID NO: 10, and human serum albumin of SEQ ID NO: 9.

[0058] In some embodiments, the nucleic acid construct comprises a sequence that is codon-optimized for expression in an individual, preferably a human individual, to be treated by gene therapy. Suitable software for codon optimization in a desired individual is well known in the art and publicly available (see, for example, http: / / www.genscript;com / cgi-bin / rare_codon_analysis; or https: / / eu.idtdna.com / site / account / login?returnurl=%2FCodonOpt).

[0059] In some preferred embodiments, the nucleic acid construct comprises the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 57, which is a codon-optimized sequence for expression in humans, encoding the FGF23 fusion protein of SEQ ID NO: 12, the nucleotide sequence of SEQ ID NO: 51, which is a codon-optimized sequence for expression in humans, encoding the FGF23 fusion protein of SEQ ID NO: 52, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any one of the above sequences; preferably, a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any one of the above sequences; more preferably, a sequence having at least 95%, 96%, 97%, 98%, or 99% identity with any one of the above sequences. The above sequences are advantageously codon-optimized sequences for expression in humans.

[0060] In some embodiments, the nucleic acid construct comprises an expression cassette in which the coding sequence is operably linked to a suitable regulatory sequence for the expression of the transgene in a target cell or tissue of an individual. In some particular embodiments, the target tissue is a muscle or liver cell or tissue, or a hematopoietic cell, more particularly a liver cell or tissue. Such sequences, which are well known in the art, include in particular promoters, as well as further regulatory sequences that can further control the expression of the transgene, such as, but not limited to, enhancers, terminators, introns, silencers, in particular tissue-specific silencers, and microRNAs.

[0061] The promoter may be a tissue-specific, universal, constitutive, or inducible promoter that is functional in the target cells or tissues of an individual, particularly in muscle, liver, or hematopoietic cells or tissues, more particularly in liver cells or tissues. Examples of constitutive promoters that may be used in the present invention include, but are not limited to, phosphoglycerate kinase promoter (PGK), elongation factor-1 alpha (EF-1 alpha) promoter, including the short form (EFS) of said promoter, viral promoters such as cytomegalovirus (CMV) immediate early enhancer and promoter, cytomegalovirus enhancer / chicken beta actin (CAG) promoter, SV40 early promoter, and retroviral 5' and 3' LTR promoters, including hybrid LTR promoters. A preferred universal promoter is the CAG promoter. Examples of inducible promoters that may be used in the present invention include tetracycline-regulated promoters. The promoter is advantageously a human promoter, i.e. a promoter derived from a human cell or a human virus. Such promoters are well known in the art and their sequences are available in public sequence databases.

[0062] In some particular embodiments, the promoter is a liver-specific promoter. Non-limiting examples of liver-specific promoters that can be used in the present invention include human alpha-1 antitrypsin promoter (hAAT) (SEQ ID NO: 14), transthyretin promoter, albumin promoter, thyroxine-binding globulin (TBG) promoter, LSP promoter (containing thyroid hormone-binding globulin promoter sequence, two copies of alpha 1-microglobulin / bikunin enhancer sequence, and leader sequence; Charles R. et al., Blood Coag. Fibrinol, 1997, 8: S23-S30), and others. Other useful liver-specific promoters are known in the art, such as those listed in the Liver Specific Gene Promoter Database compiled by Cold Spring Harbor Laboratory (http: / / rulai.cshl.edu / LSPD / ). A preferred liver-specific promoter in the context of the present invention is the hAAT promoter.

[0063] In other specific embodiments, the promoter is a muscle-specific promoter. Non-limiting examples of muscle-specific promoters include muscle creatine kinase (MCK) promoter. Non-limiting examples of suitable muscle creatine kinase promoters are human muscle creatine kinase promoter and truncated mouse muscle creatine kinase [(tMCK) promoter] (Wang et al., Gene Therapy, 2008, 15, 1489-99); (representative GenBank accession number AF188002). Human muscle creatine kinase has Gene ID number 1158 (representative GenBank accession number NC_000019.9, accessed December 26, 2012). Other examples of muscle-specific promoters include synthetic promoters C5.12 (spC5.12, or referred to herein as "C5.12"), such as spC5.12 or spC5.12 promoter (disclosed in Wang et al., Gene Therapy, 2008, 15, 1489-99); MHCK7 promoter (Salva et al., Mol Ther., 2007, 15, 320-9); myosin light chain (MLC) promoters, such as MLC2 (Gene ID number 4633; representative GenBank accession number NG_007554.1, accessed December 26, 2012); myosin heavy chain (MHC) promoters, such as alpha-MHC (Gene ID number 4624; representative GenBank accession number NG_023444.1, accessed December 26, 2012); desmin promoter (Gene ID number 4624; representative GenBank accession number NG_023444.1, accessed December 26, 2012); ID no. 1674; accessed 26 December 2012, representative GenBank accession no. NG_008043.1); cardiac troponin C promoter (Gene ID no. 7134; accessed 26 December 2012, representative GenBank accession no. NG_008963.1); troponin I promoter (Gene ID nos. 7135, 7136, and 7137; accessed 26 December 2012, representative GenBank accession nos. NG_016649.1, NG_011621.1, and NG_007866.2); myoD gene family promoters (Weintraub et al., Science, 251, 761 (1991); Gene ID number 4654; representative GenBank accession number NM_002478, accessed December 26, 2012); alpha actin promoters (Gene ID numbers 58, 59, and 70; representative GenBank accession numbers NG_006672.1, NG_011541.1, and NG_007553.1, accessed December 26, 2012); beta actin promoters (Gene ID number 60; representative GenBank accession number NG_007992.1, accessed December 26, 2012); gamma actin promoters (Gene ID number Representative GenBank Accession Nos. NG_011433.1 and NM_001199893, accessed December 26, 2012; a muscle-specific promoter within intron 1 of the ocular form of Pitx3 (Gene ID No. 5309) (Coulon et al.; a muscle-selective promoter corresponding to residues 11219-11527 of Representative GenBank Accession No. NG_008147, accessed December 26, 2012); and promoters described in U.S. Patent Publication No. US 2003 / 0157064, as well as the CK6 promoter (Wang et al., 2008 doi: 10.1038 / gt.2008.104). In another specific embodiment, the muscle-specific promoter is the E-Syn promoter described in Wang et al. (Gene Therapy, 2008, 15, pp. 1489-99), which comprises a combination of the MCK-derived enhancer and the spC5.12 promoter. In a specific embodiment of the invention, the muscle-specific promoter is spC5.12 promoter, MHCK7 promoter, E-syn promoter, muscle creatine kinase myosin light chain (MLC) promoter, myosin heavy chain (MHC) promoter, cardiac troponin C promoter, troponin I promoter, myoD gene family promoter, alpha actin promoter, beta actin promoter, gamma actin promoter, muscle specific promoter within intron 1 of the eye form of Pitx3, and CK6 promoter. In certain embodiments, the muscle specific promoter is selected from the group consisting of spC5.12, desmin, and MCK promoters. In further embodiments, the muscle specific promoter is selected from the group consisting of spC5.12 and MCK promoters. In certain embodiments, the muscle specific promoter is spC5.12 promoter.

[0064] In some other specific embodiments, the promoter is a universal promoter.Exemplary universal promoters include cytomegalovirus enhancer / chicken beta actin (CAG) promoter, cytomegalovirus enhancer / promoter (CMV) (optionally with CMV enhancer) [see, for example, Boshart et al., Cell, 41:521-530 (1985)], PGK promoter, SV40 early promoter, retrovirus Rous sarcoma virus (RSV) LTR promoter (optionally with RSV enhancer), dihydrofolate reductase promoter, beta-actin promoter, phosphoglycerol kinase (PGK) promoter, and EF1 alpha promoter.

[0065] In certain other embodiments, the promoter is an alpha-globin or beta-globin promoter. The beta-globin promoter is expressed only in erythroid cells.

[0066] In yet other specific embodiments, the promoter is an endogenous promoter, such as the albumin promoter or the GDE (glycogen debranching enzyme) promoter, where GDE is amylo-1,6-glucosidase 4-alpha-glucanotransferase or AGL, which corresponds to human Gene ID: 178 (representative GenBank accession number NG_012865, accessed September 16, 2018).

[0067] In certain embodiments, the promoter is associated with an enhancer sequence, such as a cis-regulatory module (CRM) or an artificial enhancer sequence. CRMs useful in the practice of the invention include those described in Rincon et al., Mol Ther., 2015, 23, 43-52, Chuah et al., Mol Ther. 2014, 22, 1605-13, or Nair et al., Blood, 2014 123, 20, 3195-9. Other regulatory elements that may enhance, in particular, muscle-specific expression of genes, in particular expression in cardiac and / or skeletal muscles, are those disclosed in WO2015110449. Particular examples of nucleic acid regulatory elements, including artificial sequences, include regulatory elements obtained by rearranging transcription factor binding sites (TFBS) present in the sequences disclosed in WO2015110449. The rearrangement may involve changing the order of the TFBSs and / or changing the location of one or more TFBSs relative to other TFBSs and / or changing the copy number of one or more of the TFBSs. For example, a nucleic acid regulatory element for enhancing muscle-specific gene expression, particularly cardiac and skeletal muscle-specific gene expression, can contain binding sites for E2A, HNH1, NF1, C / EBP, LRF, MyoD, and SREBP; or for E2A, NF1, p53, C / EBP, LRF, and SREBP; or for E2A, HNH1, HNF3a, HNF3b, NF1, C / EBP, LRF, MyoD, and SREBP; or for E2A, HNF3a, NF1, C / EBP, LRF, MyoD, and SREBP; or for E2A, HNF3a, NF1, CEBP, LRF, MyoD, and SREBP; or for HNF4, NF1, RSRFC4, C / EBP, LRF, and MyoD, or for NF1, PPAR, p53, C / EBP, LRF, and MyoD.For example, nucleic acid regulatory elements for enhancing muscle-specific gene expression, particularly skeletal muscle-specific gene expression, may also contain binding sites for E2A, NF1, SRFC, p53, C / EBP, LRF, and MyoD; or for E2A, NF1, C / EBP, LRF, MyoD, and SREBP; or for E2A, HNF3a, C / EBP, LRF, MyoD, SEREBP, and Tal1_b; or for E2A, SRF, p53, C / EBP, LRF, MyoD, and SREBP; or for HNF4, NF1, RSRFC4, C / EBP, LRF, and SREBP; or for E2A, HNF3a, HNF3b, NF1, SRF, C / EBP, LRF, MyoD, and SREBP; or for E2A, CEBP, and MyoD. In further examples, these nucleic acid regulatory elements include at least two, such as two, three, four or more copies of one or more of the TFBSs listed above. In particular, other regulatory elements that may enhance liver-specific expression of genes are those disclosed in WO2009130208. Further examples of enhancers that may be used in the present invention include the ApoE regulatory region, in particular the human ApoE regulatory region (or human apolipoprotein E / CI locus, hepatic control region HCR-1; Genbank accession number U32510, SEQ ID NO: 15). In some more specific embodiments, enhancer sequences such as the ApoE regulatory region, preferably the human ApoE regulatory region, are associated with liver-specific promoters such as those listed above, in particular the hAAT promoter.

[0068] In certain embodiments, the nucleic acid construct comprises an intron, particularly an intron located between the promoter and the coding sequence. The intron is introduced to increase mRNA stability and protein production. In addition, modified introns designed to reduce or even completely remove the number of alternative open reading frames (ARFs) found in said introns can significantly improve the expression of the transgene. Furthermore, by reducing the number of ARFs in the introns contained in the constructs of the present invention, it is believed that the immunogenicity of the constructs is also reduced. Preferably, ARFs that are more than 50 bp in length and have a stop codon in frame with the start codon are removed. ARFs can be removed by nucleotide substitution, insertion, or deletion, preferably by nucleotide substitution. For example, ATG or GTG that is not a start codon in the sequence of the intron of interest can be replaced by CTG. Examples of introns that can be used in the present invention include the human beta globin b2 (or HBB2; SEQ ID NO: 16) intron, the modified HBB2 intron (SEQ ID NO: 17), the coagulation factor IX (FIX) intron (SEQ ID NO: 18) and its modified intron (SEQ ID NO: 19), especially derived from the first intron, the chicken beta-globin intron (SEQ ID NO: 20) and its modified intron (SEQ ID NO: 21), and the SV40 intron. Preferred introns are the modified HBB2 intron (SEQ ID NO: 17) and the modified FIX intron (SEQ ID NO: 19).

[0069] In certain embodiments, the nucleic acid construct further comprises a transcription termination signal (polyadenylation signal) operably linked to the coding sequence (i.e., to the 3' end of the coding sequence). An example of a polyA that can be used in the present invention includes bovine growth hormone (bGH) polyA (SEQ ID NO:22).

[0070] In some preferred embodiments, the expression cassette comprises, in the 5' to 3' orientation, a liver-specific promoter, preferably the hAAT promoter; a coding sequence; and a polyadenylation signal, such as (bGH) polyA (SEQ ID NO:22). In some more preferred embodiments, the expression cassette further comprises one or more additional regulatory elements selected from an enhancer, preferably the human ApoE regulatory region (SEQ ID NO:15), and an intron, preferably the modified HBB2 intron (SEQ ID NO:17). An example of a preferred expression cassette is disclosed in the Examples and in FIG. 1B and comprises, in the 5' to 3' orientation, the sequence of SEQ ID NO:23, which comprises the ApoE regulatory region (SEQ ID NO:15), the alpha-1 antitrypsin promoter (hAAT) (SEQ ID NO:14), the modified HBB2 intron (SEQ ID NO:17), the coding sequence (SEQ ID NO:13), and the (bGH) polyA (SEQ ID NO:22). Another example of a preferred expression cassette disclosed in the Examples contains, in the 5' to 3' orientation, an ApoE regulatory region (SEQ ID NO: 15), an alpha-1 antitrypsin promoter (hAAT) (SEQ ID NO: 14), a modified HBB2 intron (SEQ ID NO: 17), a coding sequence (SEQ ID NO: 51), and (bGH) polyA (SEQ ID NO: 22). A more preferred expression cassette contains the sequence of SEQ ID NO: 23.

[0071] vector The present invention also relates to a vector comprising the nucleic acid construct described above.

[0072] The present invention may use any vector suitable for delivery and expression of nucleic acid into cells of an individual, particularly suitable for gene therapy, more particularly targeted gene therapy directed to target tissues or cells in an individual. Such vectors, which are well known in the art, include viral and non-viral vectors, and the vectors may be integrative or non-integrative; replicating or non-replicating. In some specific embodiments, gene therapy is directed to muscle, liver, or hematopoietic cells or tissues, more particularly liver cells or tissues.

[0073] As used herein, the term "individual" or "patient" refers to a mammal. Preferably, a patient or individual according to the present invention is a human. An "individual" or "patient" includes adults, children, infants, and the elderly.

[0074] Non-viral vectors include various (non-viral) agents that are commonly used to introduce or maintain nucleic acid in the cells of an individual. Agents that are used to introduce nucleic acid into the cells of an individual by various means include, but are not limited to, nanoparticles, including cationic polymers, dendrimers, micelles, liposomes, exosomes, microparticles, and lipid nanoparticles (LNPs); and cell-penetrating peptides (CPPs), among others, polymer-based, particle-based, lipid-based, peptide-based delivery vehicles, or combinations thereof. CPPs are particularly cationic peptides, such as poly-L-lysine (PLL), oligo-arginine, Tat peptide, Penetratin or Transportan peptide, and its derivatives, such as Pip. Agents that are used to maintain nucleic acid in the cells of an individual (integrated into chromosomes or otherwise in extrachromosomal form) include, among others, naked nucleic acid vectors, such as plasmids, transposons, and minicircles, as well as gene editing and RNA editing systems. Transposons include, in particular, the hyperactive Sleeping Beauty (SB100X) transposon system (Mates et al., 2009). Gene editing and RNA editing systems can use any site-specific endonuclease, such as Cas nuclease, TALEN, meganuclease, zinc finger nuclease, etc. In addition, these methods can be advantageously combined to introduce and maintain the nucleic acid of the present invention in the cells of an individual.

[0075] Viral vectors are naturally capable of penetrating cells and delivering a nucleic acid of interest into the cell through a process termed viral transduction.

[0076] As used herein, the term "viral vector" refers to a non-replicating, non-pathogenic virus engineered for the delivery of genetic material into cells. In viral vectors, viral genes essential for replication and virulence are replaced with an expression cassette for a transgene of interest. Thus, the viral vector genome contains a transgene expression cassette flanked by viral sequences required for viral vector production.

[0077] As used herein, the term "recombinant virus" refers to viruses, particularly viral vectors, produced by standard recombinant DNA technology techniques known in the art.

[0078] As used herein, the term "virus particle" or "viral particle" is intended to mean the extracellular form of a non-pathogenic virus, in particular a viral vector, that is composed of genetic material made of DNA or RNA surrounded by a protein coat called a capsid, in some cases by an envelope derived from a portion of the host cell membrane and that contains viral glycoproteins.

[0079] As used herein, a viral vector refers to a viral vector particle.

[0080] A preferred vector for delivering the nucleic acid (nucleic acid construct) of the present invention is a viral vector, particularly suitable for gene therapy, more particularly directed gene therapy to target tissues or cells in an individual, such as muscle, liver, or hematopoietic cells or tissues, more particularly liver cells or tissues. In particular, the viral vector can be derived from non-pathogenic parvoviruses, such as adeno-associated viruses (AAV), retroviruses, such as gammaretroviruses, spumaviruses, and lentiviruses, adenoviruses, poxviruses, and herpesviruses. The viral vector is preferably an integrating vector, such as an AAV or lentiviral vector, preferably an AAV vector. The lentiviral vector can be pseudotyped with envelope glycoproteins from another virus to target cells / tissues of interest, such as muscle cells, liver cells, or hematopoietic cells. In some embodiments, the lentivirus is pseudotyped with syncytin, as disclosed in WO2017 / 182607.

[0081] The vector contains viral sequences required for viral vector production, such as lentiviral LTR sequences or AAV ITR sequences flanking the expression cassette.

[0082] In a particular embodiment, the vector is a particle or vesicle, in particular a lipid-based micro- or nano-vesicle or particle, such as a liposome or lipid nanoparticle (LNP). In a more particular embodiment, the nucleic acid is RNA and the vector is a particle or vesicle as described above.

[0083] In another particular embodiment, the vector is a lentiviral vector, in particular a pseudotyped lentiviral vector as described above.

[0084] In another specific embodiment, the vector is an AAV vector. The human parvovirus adeno-associated virus (AAV) is a naturally replication-defective dependent virus that can integrate into the genome of infected cells to establish latent infection. The last property appears to be unique among mammalian viruses, since integration occurs at a specific site in the human genome, called AAVS1, located on chromosome 19 (19q13.3-qter). Therefore, AAV vectors have attracted great interest as vectors for human gene therapy. Among the desirable properties of the virus are its lack of association with any human disease, its ability to infect both dividing and non-dividing cells, and the wide range of cell lines derived from various tissues that can be infected.

[0085] AAV viruses can be engineered using conventional molecular biology techniques, allowing these particles to be optimized for cell-specific delivery of nucleic acid sequences, to minimize immunogenicity, to tailor stability and particle longevity, for efficient degradation, and for precise delivery to the nucleus.

[0086] As known in the art, additional suitable sequences can be introduced into the nucleic acid construct of the present invention to obtain a functional viral vector. Suitable sequences include AAV ITRs. Desirable AAV fragments for assembly into vectors include cap proteins, including vp1, vp2, vp3, and hypervariable regions, rep proteins, including rep 78, rep 68, rep 52, and rep 40, and sequences encoding these proteins. These fragments can be easily utilized in a variety of vector systems and host cells. AAV-based recombinant vectors that lack Rep proteins integrate with low efficiency into the genome of the host and exist primarily as stable circular episomes that can persist in target cells for years.

[0087] In the context of the present invention, an AAV vector comprises an AAV capsid capable of transducing a target cell of interest, in particular a muscle, liver, or hematopoietic cell or tissue, more particularly a liver cell or tissue. The AAV capsid may be derived from one or more AAV natural or artificial serotypes.

[0088] Among the well-characterized AAV serotypes isolated from humans or non-human primates (NHPs), human serotype 2 was the first AAV to be developed as a gene transfer vector. Other AAV serotypes currently in use include AAV-1, AAV-2 variants (such as the quadruple mutant capsid-optimized AAV-2, which contains an engineered capsid with Y44+500+730F+T491V changes, as disclosed in Ling et al., July 18, 2016, Hum Gene Ther Methods.), -3 and AAV-3 variants (such as the AAV3-ST variant, which contains an engineered AAV3 capsid with two amino acid changes S663V+T492V, as disclosed in Vercauteren et al., 2016, Mol. Ther. Vol. 24(6), p. 1042), -3B and AAV-3B variants, -4, -5, -6 and AAV-6 variants (Rosario et al., 2016, Mol Ther Methods Clin Dev. 3, p. 16026), porcine AAV serotypes such as -10, rh39, -rh43, -rh74, -dj, Anc80, LK03, AAV.PHP, AAV2i8, AAVpo4 and AAVpo6, as well as tyrosine, lysine and serine capsid mutants of AAV serotypes.

[0089] Instead of using AAV natural serotypes, artificial AAV serotypes with artificial, i.e. non-naturally occurring, capsid proteins can be used in the context of the present invention, including, but not limited to, chimeric AAV capsids, recombinant AAV capsids, or "humanized" AAV capsids. Such artificial capsids can be created by any suitable technique using selected AAV sequences (e.g., fragments of vp1 capsid protein) in combination with heterologous sequences that can be obtained from different selected AAV serotypes, non-contiguous parts of the same AAV serotype, from non-AAV viral sources, or from non-viral sources. Modified capsids can also result from capsid modifications inserted by error-prone PCR and / or peptide insertion (e.g., as described in Bartel et al., 2011). In addition, capsid variants can include single amino acid changes, such as tyrosine mutations (e.g., as described in Zhong et al., 2008). In the context of the present invention, a "modified capsid" can be a chimeric capsid or a capsid that comprises one or more variant VP capsid proteins derived from one or more wild-type AAV VP capsid proteins.

[0090] In some embodiments, the AAV vector is a chimeric vector, i.e., its capsid comprises VP capsid proteins from at least two different AAV serotypes, or comprises at least one chimeric VP protein that combines VP protein regions or domains from at least two AAV serotypes. Examples of such chimeric AAV vectors useful for transducing liver cells are described in Shen et al., Molecular Therapy, 2007 and Tenney et al., Virology, 2014. For example, a chimeric AAV vector can be derived from a combination of AAV8 capsid sequences and sequences of an AAV serotype different from the AAV8 serotype, such as any of those specifically mentioned above. In other embodiments, the capsid of the AAV vector comprises one or more variant VP capsid proteins, such as those described in WO2015013313, particularly the RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, and RHM15-6 capsid variants, which exhibit enhanced liver tropism.

[0091] In further embodiments, the AAV vector is a pseudotype vector, i.e., its genome and capsid are derived from different serotypes of AAV, such as the AAV serotypes mentioned above. In addition, the genome of the AAV vector can be a single-stranded or self-complementary double-stranded genome (McCarty et al., Gene Therapy, 2003). A self-complementary double-stranded AAV vector is created by deleting the terminal resolution site from one of the AAV terminal repeats. These modified vectors, whose replicating genome is half the length of the wild-type AAV genome, have a tendency to package DNA dimers.

[0092] In some embodiments, the AAV vector is suitable for gene therapy directed to target tissue or cells in an individual, particularly muscle, liver, or hematopoietic cells or tissues, more particularly liver cells or tissues.In certain embodiments, the AAV vector comprises a capsid selected from the group consisting of AAV1, AAV2, AAV2i8, AAV5, AAV8, AAV9, AAVrh10, AAVrh39, AAVrh43, AAVrh74, AAV-LK03, AAV2G9, AAV.PHP, AAV-Anc80, AAV3B capsid, and chimeric capsids thereof.In some preferred embodiments, the AAV vector comprises an AAV8, AAV9, or AAVrh74 capsid, such as an AAV8, AAV9, AAVrh74, or AAV2i8 capsid, particularly an AAV8 or AAV9 capsid, more particularly an AAV8 capsid. The genome of the AAV vector can be derived from different serotypes (pseudotype vectors) and is advantageously single stranded.

[0093] The present invention also relates to isolated cells, in particular cells from an individual, which have been genetically modified or transformed with a nucleic acid or vector of the invention. The individual is advantageously the patient to be treated. In some embodiments, the cells are liver cells, preferably liver cells of the patient.

[0094] As used herein, "liver cells" includes primary hepatocytes, such as those derived from adult or fetal liver; in vitro matured hepatocytes, hepatocyte cell lines; hepatic progenitor cells; or pluripotent stem cells, such as induced pluripotent stem cells (iPS cells), embryonic stem cells, fetal stem cells, and adult stem cells.

[0095] As used herein, the term "hematopoietic cells" refers to cells produced by differentiation of hematopoietic stem cells (HSC(s). Hematopoietic cells include HSCs, multipotent and lineage committed progenitor cells, precursor cells, and mature cells. Mature hematopoietic cells include, but are not limited to, lymphocytes (B, T), NK cells, monocytes, macrophages, granulocytes, erythrocytes, platelets, plasmacytoid and myeloid dendritic cells, and microglial cells.

[0096] As used herein, the term "hematopoietic stem cell (HSC)" refers to a self-renewing cell that can reconstitute short-term or long-term hematopoiesis after transplantation.

[0097] As used herein, the term "genetic modification" refers to the insertion, deletion, and / or substitution of one or more nucleotides in a genomic sequence.

[0098] As used herein, muscle tissue specifically includes cardiac and skeletal muscle tissue.

[0099] As used herein, the term "muscle cells" refers to muscle cells, myotubes, myoblasts, and / or satellite cells.

[0100] Pharmaceutical Compositions and Therapeutic Uses Another aspect of the invention is a pharmaceutical composition comprising at least one active agent selected from a nucleic acid of the invention, a vector of the invention, or a cell of the invention, and a pharma- ceutically acceptable carrier.

[0101] The nucleic acids, vectors and derived pharmaceutical compositions of the invention may be used to treat diseases by gene therapy, in particular targeted gene therapy directed to muscle, liver or hematopoietic cells or tissue, more particularly liver cells or tissue. The cells and derived pharmaceutical compositions of the invention may be used to treat diseases by cell therapy, in particular cell therapy directed to muscle, liver or hematopoietic cells, preferably liver directed cell therapy.

[0102] As used herein, "gene therapy" refers to the treatment of an individual with the delivery of a nucleic acid of interest into the cells of the individual for the purpose of treating a disease. The delivery of the nucleic acid is generally achieved using a delivery vehicle, also known as a vector. Viral and non-viral vectors can be employed to deliver genes to the cells of a patient.

[0103] As used herein, "cell therapy" refers to the process of delivering cells modified with a nucleic acid or vector of the invention to an individual in need thereof by any suitable means, such as, for example, by intravenous injection (infusion) or injection in a tissue of interest (implantation or transplantation). In certain embodiments, cell therapy includes the steps of recovering cells from an individual, modifying the individual's cells with a nucleic acid or vector of the invention, and administering the modified cells to the patient. As used herein, "cells" refers to isolated cells, natural or artificial cell aggregates, bioartificial cell scaffolds, and bioartificial organs or tissues.

[0104] A "pharmaceutical acceptable carrier" refers to a vehicle in which a therapeutic is administered and which, when administered to a mammal, particularly a human, as appropriate, does not produce adverse, allergic, or other untoward reactions. A pharmaceutical acceptable carrier refers to a non-toxic solid or liquid filler, diluent, adjuvant, excipient, encapsulating material, or formulation auxiliary of any type. Pharmaceutical compositions formulated according to standard procedures can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, implants, and other forms.

[0105] Pharmaceutical compositions of the present invention will contain a therapeutically effective amount of the nucleic acid, vector, or cell therapy, preferably in purified form, together with a suitable amount of carrier to provide the form for proper administration to a subject.

[0106] In the context of this invention, a therapeutically effective amount refers to a dose sufficient to reverse, alleviate, or inhibit the progression of the disorder or condition to which such term applies, or to reverse, alleviate, or inhibit the progression of one or more symptoms of the disorder or condition to which such term applies.

[0107] The effective dose will be determined and adjusted depending on factors such as the composition used, the route of administration, the physical characteristics of the individual under consideration, such as sex, age, and weight, concomitant medications, and other factors that one skilled in the art of medicine would recognize. The effective dose may be determined by standard clinical techniques. In addition, in vivo and / or in vitro assays may optionally be employed to help predict optimal dosage ranges. For treatments involving administering a viral vector, such as an AAV vector, to a subject, a typical dose of the vector is at least 1×10 8 Vector genomes per kilogram body weight (vg / kg), e.g., at least 1 × 10 9 vg / kg, at least 1 × 10 10 vg / kg, at least 1 × 10 11 vg / kg, at least 1 × 10 12 vg / kg, at least 1 × 10 13 vg / kg, at least 1 × 10 14 vg / kg.

[0108] In some embodiments, the pharmaceutical compositions contain a pharma- ceutically acceptable vehicle, especially for formulations that can be injected into human individuals. These include, in particular, sterile isotonic aqueous solutions or suspensions, such as saline (monosodium or disodium phosphate, sodium, potassium, calcium, or magnesium chloride, or mixtures of such salts), or dry, especially lyophilized, compositions that allow the constitution of an injectable solution upon the addition of sterile water or physiological saline, as the case may be. The solution or suspension may contain additives that are compatible with the nucleic acid and viral vector and do not prevent the entry of the nucleic acid or viral vector particles into the target cells. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Examples of suitable solutions are buffers, such as phosphate-buffered saline (PBS) or lactated Ringer's.

[0109] In some specific embodiments, the nucleic acid, vector, or cell of the invention is formulated in a composition comprising phosphate buffered saline and supplemented with 0.25% human serum albumin. In other specific embodiments, the nucleic acid, vector, or cell of the invention is formulated in a composition comprising lactated Ringer's and a non-ionic surfactant such as Pluronic F68 at a final concentration of 0.01-0.0001%, such as a concentration of 0.001% by weight of the total composition. The formulation may further comprise serum albumin, particularly human serum albumin, such as 0.25% human serum albumin. Other suitable formulations for either storage or administration are known in the art, particularly from WO2005 / 118792 or Allay et al., 2011, Hum. Gene Ther, May 2011; 22(5):595-604.

[0110] In some other embodiments, the pharmaceutical composition is formulated as an implant. The implant can be of porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. The implant is useful for localized administration of the pharmaceutical composition of the present invention to areas requiring treatment, particularly the liver.

[0111] In yet other embodiments, the pharmaceutical composition is formulated as a controlled release system.

[0112] The pharmaceutical compositions may also include additional therapeutic agents, particularly agents useful in the treatment of FGF-23-related disorders, particularly FGF-23-related hypophosphatemic disorders, such as calcitriol, among others.

[0113] Another aspect of the invention relates to the nucleic acids, vectors, cells, pharmaceutical compositions of the invention for use as medicaments.

[0114] A further aspect of the invention relates to the nucleic acids, vectors, cells, pharmaceutical compositions of the invention for use in the treatment of FGF-23-related disorders, in particular FGF-23-related hypophosphatemic disorders, by gene therapy or cell therapy as described above, preferably liver-directed gene therapy or cell therapy.

[0115] Yet another aspect of the invention relates to the use of the nucleic acids, vectors, cells, pharmaceutical compositions of the invention in the manufacture of a medicament for the treatment of FGF-23-related disorders, in particular FGF-23-related hypophosphatemic disorders, by gene therapy or cell therapy as described above, preferably liver-directed gene therapy or cell therapy.

[0116] The nucleic acid constructs, vectors and compositions according to the present invention are used to treat diseases caused by excessive action of FGF23, particularly diseases mediated by the interaction of FGF23 with the FGF23-FGF receptor (FGFR) / klotho complex, which can be treated by inhibiting the formation of the FGFR-Klotho complex.

[0117] Diseases that may be treated are particularly FGF-23-associated hypophosphatemic diseases, which may be diagnosed by elevated FGF23 levels in the presence of hypophosphatemia, since FGF23 levels are low in patients with hypophosphatemia due to other causes.

[0118] Examples of FGF-23-related hypophosphatemic disorders that may be treated include genetic disorders such as X-linked hypophosphatemia (XLH) caused by mutations in the PHEX gene; autosomal dominant hypophosphatemic rickets (ADHR) caused by mutations in the FGF23 gene; autosomal recessive hypophosphatemic rickets 1 (ADHR1) caused by mutations in the DMP1 gene; autosomal recessive hypophosphatemic rickets 2 (ADHR2) caused by mutations in the ENPP1 gene; cavitary dysplasia caused by mutations in the FRFR1 gene; Jansen type metaphyseal chondrodysplasia caused by mutations in the PTH1R gene; hypophosphatemia, dental abnormalities, and focal calcification caused by mutations in the FAM20C gene; McCune-Albright syndrome / fibrous dysplasia caused by mutations in the GNAS1 gene; and hypophosphatemia, skin and bone lesions caused by mutations in the HRAS or NRAS genes.

[0119] Other examples of FGF-23-related hypophosphatemic disorders that can be treated are acquired disorders such as tumor-induced osteomalacia (TIO), hypophosphatemic osteomalacia due to ferric oxide or ferric polymaltose, complications from kidney transplantation or parenteral iron therapy, chronic kidney disease and its complications such as hyperparathyroidism.

[0120] In some preferred embodiments, the FGF-23-associated hypophosphatemic disorder is a genetic disorder, preferably XLH, and the disorder is preferably treated by targeted gene therapy, particularly directed to muscle, liver, or hematopoietic cells or tissues, more particularly liver cells or tissues, or by cell therapy, more preferably using an AAV or lentiviral vector, particularly an AAV8 vector.

[0121] Another aspect of the invention relates to a method of treating an FGF-23-associated hypophosphatemic disorder as described above, comprising administering to a patient a therapeutically effective amount of a nucleic acid, vector, cell, or pharmaceutical composition as described above.

[0122] In the context of the present invention, the term "treating" or "treatment" as used herein means reversing, alleviating, or inhibiting the progression of the disorder or condition to which such term applies, or reversing, alleviating, or inhibiting the progression of one or more symptoms of the disorder or condition to which such term applies.

[0123] The nucleic acid, vector, cell, or pharmaceutical composition of the present invention is generally administered according to known procedures in a dosage and for a period of time effective to induce a therapeutic effect in a patient. The nucleic acid of the present invention, whether vectorized or not, can be administered by any convenient route, including but not limited to, by infusion or bolus injection, by absorption through epithelial or mucocutaneous layers (e.g., oral mucosa, rectal mucosa, intestinal mucosa, etc.). Administration can be systemic or local; systemic includes parenteral and oral, and local includes local and locoregional. Parenteral administration is advantageously by injection or perfusion, such as subcutaneous (SC), intramuscular (IM), intravenous (IV), intravascular, such as intraarterial, intraperitoneal (IP), intradermal (ID), intranasal, epidural, or other. In addition, it may be desirable to introduce the pharmaceutical composition of the present invention into the liver of a subject by any suitable route.

[0124] The nucleic acids, vectors, cells or pharmaceutical compositions of the invention may be used in combination with other biologically active agents, the combined use being by simultaneous, separate or sequential administration.

[0125] The practice of the present invention will employ, unless otherwise indicated, conventional techniques that are within the skill of the art, and such techniques are fully explained in the literature.

[0126] The invention will now be illustrated by way of the following non-limiting examples, with reference to the accompanying drawings. EXAMPLES

[0127] Materials and Methods Construction of nucleic acids encoding truncated FGF23 A nucleic acid construct encoding a truncated FGF23 was created by fusing amino acids 175-251 of the human FGF23 protein (aa 175-251 of NP_065689; SEQ ID NO:2), which contains an RXXR motif (amino acids 175-179 of the human FGF23 protein) preceded by R175 (RRHTR motif; amino acids 175-179 of the human FGF23 protein), or amino acids 180-251 of the human FGF protein of SEQ ID NO:1 (which does not contain said RXXR motif), to human serum albumin without a signal peptide (aa 25-609 of NP_000468 or SEQ ID NO:9) via a cleavable linker derived from human coagulation factor IX (cFIX; aa 182-200 of NP_000124; SEQ ID NO:10). Finally, a signal peptide derived from the first 18 amino acids of chymotrypsinogen B2 (sp7; aa 1-18 of NP_001020371 or SEQ ID NO: 7) was inserted at the N-terminus of the construct to mediate efficient secretion (SEQ ID NO: 12 (Figure 1A); SEQ ID NO: 52). These (Thes) sequences were codon-optimized by a commercial algorithm. The resulting sequences are SEQ ID NO: 57 (construct no. 12 in Table 1) containing the CDS of SEQ ID NO: 13 encoding the fusion protein of SEQ ID NO: 12 flanked in 5' with MluI / Kozak; and SEQ ID NO: 51 (construct no. 10 in Table 1) containing the CDS of SEQ ID NO: 52 encoding the fusion protein of SEQ ID NO: 52 flanked in 5' with MluI / Kozak. The codon-optimized sequences were cloned into a transgene expression cassette optimized for liver expression (Figure 1B).

[0128] For comparison, other constructs were created (Table 1). Nucleic acid constructs encoding native human FGF23 (SEQ ID NO: 1) were created by cloning wild-type or codon-optimized human FGF23 sequences into a transgene expression cassette optimized for liver expression (FIG. 1B).

[0129] Truncated C-terminal FGF23 constructs (FGF23-C) were created by fusing FGF23-C (amino acids 175-251 or 180-251 of the human FGF23 protein) containing or not containing the RXXR motif with the native FGF23 or sp7 signal peptide. These sequences were codon-optimized by a commercial algorithm and cloned into a transgene expression cassette optimized for liver expression.

[0130] Chimeric proteins were created by fusing FGF23-C without the RXXR motif with human serum albumin (without signal peptide) without a cleavable linker derived from human coagulation factor IX, or by fusing FGF23-C with the RXXR motif with human serum albumin (without signal peptide) without a cleavable linker derived from human coagulation factor IX. A signal peptide (sp7) derived from the first 18 amino acids of chymotrypsinogen B2 was inserted at the N-terminus of the construct to mediate efficient secretion. These sequences were codon-optimized by a commercial algorithm and cloned into a transgene expression cassette optimized for liver expression (Figure 1B).

[0131] AAV vector production AAV vectors were produced using an adenovirus-free transient transfection method (Matsushita et al., Gene Therapy, 1998, 5, 938-945) and purified as previously described (Ayuso et al., Gene Therapy, 2010, 17, 503-510). Titers of AAV vector stocks were determined using quantitative real-time PCR (qPCR) and confirmed by SDS-PAGE followed by SYPRO® Ruby protein gel staining and band densitometry.

[0132] In vitro studies Huh-7 cells at 70-80% confluence were transfected by lipofectamine with the constructs shown in Table 1. Two days after transfection, medium was collected and the levels of FGF23 were analyzed by Western blot.

[0133] Western blot Huh-7 medium after transfection and serum from mice injected with various AAV vectors were loaded onto 4-15% gradient polyacrylamide gels and subjected to SDS-PAGE. After transfer onto nitrocellulose, the membranes were blocked and incubated with anti-FGF23 antibody. The membranes were incubated with appropriate secondary antibodies and visualized by the Odyssey imaging system.

[0134] Mouse Study AAV vectors were administered intravenously via the tail vein to 6-week-old C57BL6 / J mice. One month after vector injection, circulating FGF23-C levels were analyzed by Western blot. Livers were harvested and vector genome copy numbers were measured by qPCR.

[0135] AAV vectors were administered intravenously via the tail vein to 1-month-old male Hypduk mice. PBS-injected wild-type and Hypduk littermates served as controls. Mice were weighed and measured monthly for 3 months after vector injection to assess correction of disease at the macroscopic level. Measurement of blood phosphatemia and functional assessment of muscle strength were performed 3 months after vector injection.

[0136] Phosphatemia To measure circulating phosphate levels, mice were bled 3 months after vector injection. After centrifugation at 10,000×g for 10 min, serum inorganic phosphate content was measured using a standard commercial kit.

[0137] Reversed Grating Test Mice were placed on the grid and allowed to acclimate for 3-5 s before the grid was inverted and kept at least 35 cm above a mouse cage containing 5-7 cm of soft bedding. The number of falls was measured over a 3 min period and reported as falls per minute.

[0138] result The C-terminal part of human FGF23 (FGF23-C) competes with native FGF23 and reduces intracellular signaling following receptor binding.

[0139] One important limitation in the use of FGF23-C as a therapy for XLH is that this peptide is highly unstable in the circulation. To secrete this peptide from the liver and improve its stability, a gene therapy approach was developed. Amino acids 175-251 (SEQ ID NO:2) of human FGF protein containing an RXXR motif preceded by R175 (RRHTR motif; amino acids 175-179 of human FGF23 protein) or amino acids 180-251 (without said RXXR motif) of human FGF protein SEQ ID NO:1 were fused to human serum albumin without a signal peptide (SEQ ID NO:9) through a cleavable linker (SEQ ID NO:10) derived from human coagulation factor IX. Finally, a signal peptide (SEQ ID NO:7) derived from the first 18 amino acids of chymotrypsinogen B2 was inserted at the N-terminus of the construct to mediate efficient secretion (SEQ ID NO:12 (Figure 1A); SEQ ID NO:52). These sequences were codon-optimized by a commercial algorithm. The resulting sequences are SEQ ID NO:57 (construct no. 12 in Table 1) containing the CDS of SEQ ID NO:13 encoding the fusion protein of SEQ ID NO:12 flanked at 5' with MluI / Kozak; and SEQ ID NO:51 (construct no. 10 in Table 1) containing the CDS of SEQ ID NO:52 encoding the fusion protein flanked at 5' with MluI / Kozak. The codon-optimized sequences were cloned into transgene expression cassettes optimized for liver expression (Figure 1B). The transgene expression cassette containing SEQ ID NO:13 contained in SEQ ID NO:57 had the sequence of SEQ ID NO:23. The AAV transfer vector containing the expression cassette of SEQ ID NO:23 flanked by AAV ITRs had the sequence of SEQ ID NO:24.

[0140] To confirm the low stability of FGF23-C and to test strategies to improve its secretion and stability, we transfected human hepatic hepatoma cells with various constructs expressing both native FGF23 and FGF23-C under the transcriptional control of the hAAT promoter (Table 1 and FIG. 1).

[0141] [Table 1A]

[0142] [Table 1B]

[0143] Wild-type and codon-optimized versions of the FGF-23 and FGF23-C genes were tested, along with native FGF23 and versions fused to the heterologous signal peptide of chymotrypsinogen B2 (SEQ ID NO: 7, sp7). The various sequences tested either included or did not include the RRHTR motif that is present in human FGF23 and is involved in its cleavage into the FGF23 N- and C-terminal peptides.

[0144] Native FGF23, as well as its codon-optimized form, were efficiently produced and secreted in the medium of transfected Huh-7 cells (constructs no. 1 and 2, FIG. 2A). None of the FGF23-C variants were produced in the medium, except for sp7-fused FGF23-C without the RHTR motif (construct no. 8, FIG. 2A). Interestingly, fusion with an albumin moiety (SEQ ID NO: 9) enhanced the secretion of FGF23-C, regardless of the composition of the chimeric protein (constructs no. 9-12, FIG. 2A). Fusion of FGF23-C with albumin in the absence of the RHTR motif and the cleavable linker derived from human coagulation factor IX (construct no. 11) led to a higher level of secretion in vitro when compared to other chimeric proteins containing the albumin moiety (constructs no. 9, no. 10, and no. 12, FIG. 2B).

[0145] AAV8 vectors were prepared expressing constructs no. 7, no. 9, no. 11, and no. 12: i) FGF23-C fused to sp7 and containing an RRHTR motif (construct no. 7), ii) FGF23-C fused to sp7 and albumin and containing an RRHTR motif (construct no. 9), iii) FGF23-C fused to sp7 and albumin without an RRHTR motif (construct no. 11), and iv) FGF23-C fused to sp7 and albumin through a hFIX linker and containing an RRHTR motif (construct no. 12, Table 1 and FIG. 1A). AAV8 vectors were produced and injected into 6-week-old C57BL6 / J mice at 1×10 12 vg / mouse. One month after vector injection, mice were bled to measure phosphorus levels and FGF23-C levels by Western blot. Importantly, increased phosphorus levels in the blood were observed only in mice injected with AAV8 vectors expressing construct no. 12 (Figure 3A), despite similar levels of liver transduction achieved with the other constructs (Figure 3B) and higher expression levels achieved in vitro with construct no. 11.

[0146] A band corresponding to the molecular weight of FGF23-C-albumin chimeric protein was only evident in mice injected with AAV8 vectors expressing constructs #9 and #12 specifically in the liver. In contrast, a band with a size compatible with FGF23-C was undetectable in mice injected with construct #7, an AAV8 vector expressing FGF23-C fused to sp7, confirming the instability of FGF23-C in vivo (Figure 3C). Importantly, mice injected with construct #12 containing a cleavable linker (hFIX; SEQ ID NO:10) showed significantly higher levels of circulating FGF-23-C fusion protein when compared to construct #9 (no linker; Figure 3D), despite comparable liver transduction as demonstrated by vector genome copy number per cell (Figure 3B) and higher expression levels achieved in vitro with construct #11. These data indicate that the C-terminal part of FGF23 (FGF23-C) can be expressed by the liver only as a fusion with the albumin moiety. They also suggest that the composition of the fusion protein affects its stability and function in the circulation, which is increased when FGF23-C is fused to the albumin moiety, especially through a cleavable linker derived from human coagulation factor IX (SEQ ID NO: 10).

[0147] To verify whether the chimeric protein according to the present invention, composed of FGF23-C and fused to sp7 and albumin through a linker derived from hFIX (construct no. 12, Table 1 and FIG. 1A), is active in vivo, an AAV8 vector expressing this construct was injected into 1-month-old Hypduk mice at 1×10 12vg / mouse (KO, AAV). PBS-injected Hypduk (KO, PBS) and wild-type littermates (WT, PBS) were used as controls. Phenotypic correction was assessed by monthly measurements of body weight, height, and tail length starting one month after vector injection (Figure 4). In general, PBS-treated Hypduk mice had lower body weight, height, and tail length when compared to wild-type animals (Figures 4A-C). Hypduk mice treated with AAV8 vectors expressing FGF23-C chimeric proteins according to the present invention showed an increase in body weight, height, and tail length over a 3-month period that was higher than that of PBS-treated Hypduk mice and comparable to that observed in wild-type animals (Figures 4A-C). Although a complete rescue of height increase was observed in animals treated with AAV (Figure 4B), body weight and tail length were only partially rescued (Figure 4A, Figure 4C). After 3 months of treatment, serum phosphate levels were measured. In AAV-treated Hypduk mice, phosphate levels were significantly different from those measured in PBS-treated Hypduk mice and were indistinguishable from those in wild-type animals (Figure 5). Reduced bone growth and tendon mineralization may result in reduced motor function and muscle strength. Functional evaluation of the phenotype in Hypduk mice was performed by the inverted grid test. We observed an increase in the number of falls per minute in Hypduk mice compared to wild-type, which was completely rescued by AAV gene therapy (Figure 6).

Claims

1. expressible in a target cell or tissue of the individual; (a) a signal peptide, (b) FGF23 C-terminal peptide that binds to the FGFR / klotho complex, (c) a cleavable linker that is cleavable in vivo, and (d) Protein stabilizing moiety 1. A nucleic acid construct having a nucleotide sequence encoding an FGF23 fusion protein comprising:

2. A pharmaceutical composition for gene therapy of FGF-23-associated hypophosphatemic diseases, comprising:

2. The pharmaceutical composition of claim 1, wherein the FGF23 C-terminal peptide comprises the RRHTR motif at positions 175 to 179 of SEQ ID NO:

1.

3. The pharmaceutical composition according to claim 1 or 2, wherein the FGF23 C-terminal peptide comprises the sequence of SEQ ID NO: 2 or a sequence having at least 90% identity thereto.

4. 4. The pharmaceutical composition according to claim 1, wherein the signal peptide comprises a sequence selected from the group consisting of SEQ ID NO: 3 to 8 and / or the cleavable linker comprises the sequence of SEQ ID NO:

10.

5. 5. The pharmaceutical composition of claim 1, wherein the protein stabilizing moiety is human serum albumin.

6. 6. The pharmaceutical composition of claim 5, wherein the human serum albumin comprises the sequence of SEQ ID NO:

9.

7. 7. The pharmaceutical composition of any one of claims 1 to 6, wherein the nucleic acid construct encodes an FGF23 fusion protein comprising the sequence of SEQ ID NO: 12 or 52, or a sequence having at least 90% identity to any one of the sequences.

8. 8. The pharmaceutical composition of claim 1, wherein the nucleic acid construct is codon-optimized for expression in humans.

9. 9. The pharmaceutical composition of claim 7 or 8, wherein the nucleic acid construct comprises the sequence of SEQ ID NO: 13, 51, or 57, or a sequence having at least 90% identity to any one of said sequences.

10. 10. The pharmaceutical composition of any one of claims 1 to 9, wherein the nucleic acid construct comprises an expression cassette in which a coding sequence is operably linked to at least one promoter that is functional in muscle, liver, or hematopoietic cells or tissues.

11. The pharmaceutical composition of claim 10, wherein the promoter is a liver-specific promoter.

12. The pharmaceutical composition of claim 11, wherein the promoter is a human alpha-1 antitrypsin promoter.

13. 13. The pharmaceutical composition of any one of claims 10 to 12, wherein the nucleic acid construct further comprises one or more control elements selected from the group consisting of an enhancer associated with the promoter; an intron located between the promoter and the coding sequence; and a transcription termination signal.

14. 14. The pharmaceutical composition of claim 13, wherein the one or more control elements are selected from the human ApoE control region, a modified HBB2 intron of SEQ ID NO: 17, a modified FIX intron of SEQ ID NO: 19, and a bovine growth hormone polyadenylation signal.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the nucleic acid construct is DNA or RNA.

16. 16. A pharmaceutical composition for gene therapy comprising a vector comprising a nucleic acid construct as defined in any one of claims 1 to 15.

17. The pharmaceutical composition of claim 16, wherein the vector is a viral vector.

18. The pharmaceutical composition of claim 17, wherein the vector is an AAV or lentiviral vector.

19. 19. The pharmaceutical composition of claim 18, wherein the vector is an AAV vector comprising a capsid selected from the group consisting of an AAV1 capsid, an AAV2 capsid, an AAV5 capsid, an AAV8 capsid, an AAV2i8 capsid, an AAV9 capsid, an AAVrh10 capsid, an AAVrh39 capsid, an AAVrh43 capsid, an AAVrh74 capsid, an AAV-LK03 capsid, an AAV2G9 capsid, an AAV.PHP capsid, an AAV-Anc80 capsid, and an AAV3B capsid.

20. The vector comprising VP capsid proteins from at least two different AAV serotypes, or at least one chimeric VP capsid protein combining VP capsid protein regions or domains from at least two AAV serotypes. The pharmaceutical composition of claim 18, which is an AAV vector comprising a chimeric AAV capsid.

21. 17. The pharmaceutical composition of claim 16, wherein the vector is a lipid-based micro- or nano-vesicle or particle.

22. A cell genetically modified with the pharmaceutical composition of any one of claims 1 to 21.

23. 22. The pharmaceutical composition of any one of claims 1 to 21, further comprising a pharma- ceutically acceptable carrier.

24. 1. A pharmaceutical composition for use in the treatment of an FGF-23-associated hypophosphatemic disorder by gene therapy or cell therapy, said FGF-23-associated hypophosphatemic disorder comprising: a genetic disease selected from the group consisting of X-linked hypophosphatemia caused by a mutation in the PHEX gene, autosomal dominant hypophosphatemic rickets caused by a mutation in the FGF23 gene, autosomal recessive hypophosphatemic rickets 1 caused by a mutation in the DMP1 gene, autosomal recessive hypophosphatemic rickets 2 caused by a mutation in the ENPP1 gene, cavitary dysplasia caused by a mutation in the FRFR1 gene, Jansen metaphyseal chondrodysplasia caused by a mutation in the PTH1R gene, hypophosphatemia, dental abnormalities and focal calcifications caused by a mutation in the FAM20C gene, McCune-Albright syndrome / fibrous dysplasia caused by a mutation in the GNAS1 gene, and hypophosphatemia, skin lesions and bone lesions caused by a mutation in the HRAS or NRAS gene; or an acquired disease selected from the group consisting of tumor-induced osteomalacia, hypophosphatemic osteomalacia, complications from kidney transplantation or parenteral iron therapy, chronic kidney disease, and hyperparathyroidism; 24. The pharmaceutical composition of claim 23.

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