Gene therapy for fibroblast growth factor 23-related hypophosphatemia

Abstract

The present invention relates to nucleic acid constructs for use in the gene therapy of FGF-23-related hypophosphatemia, in particular for the gene therapy of muscle, liver or hematopoietic tissue, more particularly liver tissue. The invention also relates to vectors comprising the nucleic acid constructs and their use in the treatment of FGF-23 associated hypophosphatemia, in particular XLH, by gene therapy.

Description

Technical Field

[0001] This invention relates to the field of gene therapy for FGF-23-related hypophosphatemia, particularly X-linked hypophosphatemia (XLH). The invention specifically relates to nucleic acid constructs for gene therapy of FGF-23-related hypophosphatemia, particularly targeting muscle, liver, or hematopoietic tissue, and more particularly liver tissue. The invention also relates to vectors comprising nucleic acid constructs, and their use in treating FGF-23-related hypophosphatemia, particularly XLH, via gene therapy. Background Technology

[0002] Fibroblast growth factor 23 (FGF-23 or FGF23) is a phosphorylated hormone produced by bone that exerts its effects by binding to the Klotho-FGF receptor complex. Overactivity of FGF23 can lead to hypophosphatemia, 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 disease (CKD-MBD) (reviewed in Seiji Fukumoto, Calcif. Tissue Int., 2016, 98, 334-340).

[0003] X-linked hypophosphatemia (XLH, OMIM # 307800) presents clinically with a range of symptoms from isolated hypophosphatemia to severe lower limb curvature. The disease typically manifests within the first two years of life, presenting with lower limb curvature. In adults, tendinopathy (tendon calcification) associated with joint pain, spontaneous dental abscesses, and sensorineural hearing loss has been reported. XLH is caused by mutations in the gene for phosphate-regulated neutral endopeptidase (PHEX), which induces increased circulating FGF23 levels. Increased FGF23 function leads to downregulation of sodium phosphate cotransporters in the kidneys. Cotransporters, located in the proximal tubules of the kidneys, mediate the reabsorption of phosphate in urine. Their downregulation results in impaired phosphate reabsorption and decreased serum phosphate levels. Furthermore, increased FGF23 is associated with impaired synthesis and increased degradation of 1,25(OH)2 vitamin D. Decreased serum phosphorus levels and low vitamin D levels contribute to bone mineralization defects and fractures.

[0004] Classic XLH treatment involves oral phosphate and high-dose calcitriol (the active form of vitamin D). However, the response to intravenous phosphate therapy is sometimes unpredictable, and complications include overshooting of hyperphosphatemia, hypocalcemia, and metastatic calcification; parenteral regimens are also impractical for chronic cases. Oral therapy requires high doses, which often leads to diarrhea or gastric irritation, and replacement therapy alone is insufficient when renal phosphate is heavily depleted. Therefore, novel strategies for treating FGF23-related hypophosphatemia are needed.

[0005] An animal model of the disease, the HypDuk model, exists, derived from a natural deletion of the PHEX gene. This model summarizes most of the disease manifestations, including low blood phosphate levels and impaired bone growth. Different treatment strategies have been tested in HypDuk mice. One approach uses anti-FGF23 neutralizing antibodies. Recently, a monoclonal antibody against FGF23 has been approved for the treatment of pediatric XLH (Crysvita®, Ultragenyx). Another strategy involves using a truncated form of human FGF23 that binds to the FGF23 receptor without inducing an intracellular activation cascade caused by the functional interaction between the receptor and FGF23. This truncated FGF23 can be used as a competitor to reduce the increased FGF23 function observed in XLH (Goetz R. et al., PNAS, 2009, 107, 407-412).

[0006] To date, there are no reports of gene therapy for FGF-23-related hypophosphatemia (such as XLH). Therefore, gene therapy is needed to treat FGF-23-related hypophosphatemia, especially XLH. Invention Overview The inventors have designed a nucleic acid construct and a derived AAV vector for gene therapy of FGF-23-related hypophosphatemia. Following a single injection of this AAV vector into HypDuk mice, normalized increases in body weight, body size, tail length, and circulating phosphate were observed in the treated animals. The disease correction observed at biochemical, macroscopic, and functional levels after a single injection of this AAV vector confirms the enhanced efficacy of gene therapy methods based on this nucleic acid construct and derived vector, particularly the AAV vector, for treating FGF-23-related hypophosphatemia.

[0007] Therefore, the present invention relates to nucleic acid constructs for gene therapy of FGF-23-related hypophosphatemia, encoding an FGF23 fusion protein, said fusion protein comprising: (a) Signal peptide, (b) The FGF23 C-terminal peptide bound to the FGFR / klotho complex, (c) Cuttable connectors, and (d) The stable portion of the protein, The signal peptide is located at the N-terminus of the fusion protein, and the FGF23 C-terminal peptide and the stable portion of the protein are separated by a cleavable linker.

[0008] In some embodiments, the FGF23 C-terminal peptide comprises a sequence from any one of positions 175-189 to positions 203-251 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 with said sequence.

[0009] In some embodiments, the FGF23 C-terminal peptide contains the RXXR motif at positions 176-179 of SEQ ID NO: 1.

[0010] In some preferred embodiments, the FGF23 C-terminal peptide comprises the sequence 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 with said sequence.

[0011] In some embodiments, the signal peptide comprises a sequence selected from SEQ ID NO: 3-8; preferably SEQ ID NO: 7.

[0012] In some embodiments, the protein stabilizing portion is human serum albumin, preferably containing the sequence SEQ ID NO:9.

[0013] In some implementations, the cuttable connector includes the sequence SEQ ID NO: 10.

[0014] In some preferred embodiments, the nucleic acid construct encodes the FGF23 protein, which comprises the sequence 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 with any of the said sequences.

[0015] In some implementations, the nucleic acid constructs are codons optimized for expression in humans.

[0016] In some preferred embodiments, the nucleic acid construct comprises a sequence 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 with any of the said sequences.

[0017] In some embodiments, the nucleic acid construct comprises an expression cassette in which the coding sequence is operatively linked to at least one promoter that functions in a target cell or tissue of an individual, particularly muscle, liver, or hematopoietic cells or tissue. In some specific embodiments, the promoter is a liver-specific promoter, preferably a human α-1 antitrypsin promoter.

[0018] In some embodiments, the nucleic acid construct further comprises one or more control elements selected from: a promoter-associated enhancer, preferably a human ApoE control region; an intron positioned 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 a bovine growth hormone polyadenylation signal.

[0019] In some implementations, the nucleic acid construct contains or is composed of DNA.

[0020] In some implementations, the nucleic acid construct contains or is composed of RNA.

[0021] The present invention also relates to vectors for gene therapy, said vectors comprising nucleic acid constructs according to the present invention.

[0022] In some implementations, the vector is a viral vector, particularly an AAV or lentiviral vector, preferably an AAV vector containing a capsid selected from the following: AAV1, AAV2, AAV5, AAV8, AAV2i8, AAV9, AAVrh10, AAVrh39, AAVrh43, AAVrh74, AAV-LK03, AAV2G9, AAV.PHP, AAV-Anc80, AAV3B capsids and their chimeric capsids, particularly AAV8, AAV9 or AAVrh74 capsids, such as AAV8 or AAV9 capsids, more preferably AAV8 capsids.

[0023] In some other embodiments, the carrier is a particle or vesicle, particularly lipid-based micro or nano vesicles or particles.

[0024] This invention relates to cells genetically modified by nucleic acid constructs or vectors according to the invention, preferably liver, muscle or hematopoietic cells, more preferably liver cells.

[0025] The present invention further relates to a pharmaceutical composition comprising at least one nucleic acid construct selected from the present invention, a carrier according to the present invention, or a cell active agent and a pharmaceutically acceptable carrier according to the present invention.

[0026] This invention relates to pharmaceutical compositions according to the invention for treating FGF-23-related hypophosphatemia via gene therapy or cell therapy.

[0027] In some embodiments of the described use, FGF-23-related hypophosphatemia is selected from the following genetic disorders: X-linked hypophosphatemia (XLH), autosomal dominant hypophosphatemic rickets (ADHR), autosomal recessive hypophosphatemic rickets 1 (ADHR1), autosomal recessive hypophosphatemic rickets 2 (ADHR2), osteoglophonic dysplasia, Jansen type metaphyseal dysplasia, hypophosphatemia, dental abnormalities and ectopic calcification, McCune-Albright syndrome / fibrous dysplasia and hypophosphatemia, skin and bone lesions, or acquired diseases selected from the following: tumor-induced osteomalacia, hypophosphatemic osteomalacia, complications of kidney transplantation or parenteral iron therapy, chronic kidney disease and its complications such as hyperparathyroidism; preferably X-linked hypophosphatemia. Invention Details Nucleic acid constructs This invention provides a nucleic acid construct for gene therapy of FGF-23-related hypophosphatemia.

[0029] The nucleic acid construct of the present invention encodes an FGF23 fusion protein, the fusion protein comprising... (a) Signal peptide, (b) The FGF23 C-terminal peptide bound to the FGFR / klotho complex, (c) Cuttable connectors, and (d) The stable portion of the protein, The signal peptide is located at the N-terminus of the fusion protein, and the FGF23 C-terminal peptide and the stable portion of the protein are separated by a cleavable linker.

[0030] As used herein, the term fibroblast growth factor 23 (FGF-23 or FGF23) (also known as phosphorylated protein or tumor-derived hypophosphatemia-inducing factor) refers to the protein encoded by the FGF23 gene in the mammalian genome. Human FGF23 has a 251-amino acid sequence (UniProtKB / Swiss-Prot accession number Q9GZV9.1 or NCBI accession number NP_065689, SEQ ID NO: 1). FGF23 is expressed as a precursor containing an N-terminal signal peptide (24 amino acids), which is cleaved to produce the mature protein (FGF23). To exert its phosphate-urinary activity, FGF23 requires a binary FGF receptor (FGFR)-Klotho complex. Furthermore, the activity of FGF23 is mediated through… 176 RXXR 179 The motif is regulated by proteolytic cleavage at the boundary between the FGF core homology domain and the 72-residue long C-terminal tail of FGF23. Proteolytic cleavage yields an inactive N-terminal fragment (Y25-R179), the 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 to bind to the FGFR-Klotho complex and block FGF23 signaling. This indicates that the FGF23 C-terminal fragment (180-251) antagonizes the phosphate-urinary activity of FGF23 in vivo. The smaller C-terminal fragment (FGF 180-205) also exhibits FGF23 antagonist activity (Goetz et al. PNAS, 2010, 107, 407-410). Residues 189-203 of the mature 251-residue FGF23 are essential for FGF23 activity, while residue 203 of the 3'-amino acid FGF23 is not essential for initiating FGF23-dependent intracellular signaling (Garringer et al., Am. J. Physiol. Endocrinol. Metab., 2008, 295, E929-E937).

[0031] In the following description, residues are named by standard single-letter amino acid codes, and the indicated positions are determined by comparison with SEQ ID NO: 1.

[0032] Unless the context clearly indicates otherwise, “a,” “an,” and “the” include plural indicators. Therefore, the terms “a” (or “an”), “one or more,” or “at least one” are used interchangeably in this document; unless otherwise stated, “or” means “and / or.”

[0033] Nucleic acid constructs may contain or consist of DNA, RNA, or synthetic or semi-synthetic nucleic acids that can be expressed in the target cells or tissues of an individual.

[0034] The FGF23 C-terminal peptide, generated by cleaving the FGF23 fusion protein in vivo, binds to the FGFR / klotho complex. Mature FGF23 fusion proteins (without their signal peptide) can also bind to the FGFR / klotho complex. This binding inhibits FGF23 signaling via the FGFR-klotho complex. The binding activity of the FGF23 fusion protein and its derived C-terminal peptide to the FGFR / klotho complex, and the inhibition of FGF23 signaling via the FGFR-klotho complex, can be verified by standard assays well known in the art and disclosed, for example, in Goetz et al. PNAS, 2010, 107, 407-410. The FGF23 C-terminal peptide, generated by cleaving the FGF23 fusion protein according to the invention in vivo, is a specific inhibitor or antagonist of the FGFR-klotho-dependent function of FGF23. The FGF23 fusion protein according to the 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 possible fusion proteins to neutralize the Klotho-dependent function of FGF23, the FGF23 fusion protein of the present invention can be used as a therapeutic agent for treating FGF23-related hypophosphatemia.

[0035] The FGF23 C-terminal peptide comprises or consists of the sequence of any one of positions 175-189 to 203-251 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 with said sequence, said sequence binding to the FGFR / klotho complex. The FGF23 C-terminal peptide may comprise positions 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, or 189 of SEQ ID NO: 1. Positions 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, 229, 230, 231, 232, 233, 234, 235, 23 The sequence at positions 6, 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 with said sequence. Preferably, the FGF23 C-terminal peptide comprises or consists of: any one of positions 175-180 to 205-251 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 with said sequence, said sequence binding to the FGFR / klotho complex.

[0036] In some specific embodiments, the FGF23 C-terminal peptide of the present invention comprises 176 RXXR 179The motif (positions 176-179 of SEQ ID NO: 1). In some specific embodiments, the FGF23 C-terminal peptide of the present invention terminates at position 203 of SEQ ID NO: 1. In some other specific embodiments, the FGF23 C-terminal peptide of the present invention terminates at position 204 or more of SEQ ID NO: 1, for example, 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) or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with said sequence, said sequence binding to the FGFR / klotho complex. In some embodiments, the FGF23 C-terminal peptide of the present invention contains a mutation, particularly a mutation that increases its binding affinity to the FGFR / klotho complex.

[0037] The FGF23 C-terminal peptide according to the present invention comprises or is composed of a C-terminal fragment of FGF23 consisting of 15-77 amino acids. Therefore, the FGF23 C-terminal peptide differs from the full-length FGF23 protein and does not contain any sequence from the FGF23 N-terminal region (positions 25-174 of SEQ ID NO: 1). In some embodiments, the FGF23 C-terminal peptide according to the present invention comprises or is composed of a C-terminal fragment of FGF23 consisting of at least 20, 25, 30, or more amino acids.

[0038] The percentage of amino acid or nucleotide sequence identity is defined as the percentage of amino acid residues or nucleotides in the comparison sequence that are identical to those in the reference sequence after alignment and, where necessary, the introduction of vacancies, to achieve maximum sequence identity without regard to any conserved substitutions in the amino acid sequence that are part of the sequence identity. Sequence identity is calculated over the entire length of the reference sequence. Alignments used to determine the percentage of amino acid sequence identity can be implemented 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, such as vacancy penalties and expansion penalties, are preferred. The BLASTP program defaults to a word length of 3 (W) and an expected value of 10 (E).

[0039] The FGF23 fusion protein contains a signal peptide at its N-terminus. The signal peptide (SP) is a short peptide sequence located at the secretory N-terminus and used to target secreted proteins. Instead of a strictly shared sequence, the signal peptide has a tri-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). Many signal peptides are known in the art and are publicly available (see the signal peptide website and the SPdb sequence database; Puzzo et al., Sci. Transl. Med., 2017, 9(418): doi:10.1126). Furthermore, methods for selecting suitable SP sequences for efficient protein secretion are known in the art (see Stern et al., BMC Proc., 2011, 5 (suppl 8):013).

[0040] The signal peptide can be an endogenous or natural FGF23 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 is 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 this invention include, but are not limited to: α-1 antitrypsin (SEQ ID NO: 4); synthetic mut1 (SEQ ID NO: 5); synthetic mut3 (SEQ ID NO: 6); chymotrypsinogen B2 (CTRB2) (Uniprot accession number Q6GPI1 or NCBI accession number NP_001020371 or positions 1-18 of SEQ ID NO: 7) and plasma protease inhibitor C1 (Uniprot accession number P05155 or positions 1-22 of SEQ ID NO: 7).

[0041] In some embodiments, the signal peptide is a heterologous signal peptide, preferably the chymotrypsinogen B2 signal peptide (SEQ ID NO: 7).

[0042] The FGF23 C-terminal peptide is linked to a 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 linked and is suitable for therapeutic applications. Various protein stabilizing moieties that have been used to stabilize therapeutic proteins are known in the art (see, for example, Sven Berger, Peter Lowe & Michael Tesar (2015) Fusion protein technologies forbiopharmaceuticals: Applications and challenge, mAbs, 7:3, 456-460, DOI:10.1080 / 19420862.2015.1019788). Examples of protein stabilizing moieties that can be used in this invention include, but are not limited to: serum albumin, particularly human serum albumin; immunoglobulin Fc fragments; the carboxyl-terminal peptide (CTP) of human chorionic gonadotropin; receptors fused with their ligands (GHR fused with GH); and latency-related peptides of TGF-β (linked to cleavage sites of metalloproteinases).

[0043] In some implementations, the protein stabilizing portion differs from the immunoglobulin Fc fragment.

[0044] In some embodiments, the protein stabilizing portion is derived from serum transporters. Serum transporters include, but are not limited to, albumin family proteins and evolution-associated serum transporters, 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 transporters can be derived from any vertebrate, including mammals, birds, fish, etc. This invention includes functional variants, such as naturally occurring polymorphic variants, as well as functional fragments of serum transporters. A functional fragment or variant of a serum transporter refers to a variant or fragment capable of increasing the half-life or duration of action of the therapeutic protein / peptide to which it is linked and suitable for therapeutic application.

[0045] In some specific embodiments, the protein stabilizing portion is albumin, including its functional fragments or variants as defined above. Albumin can be derived from any vertebrate, especially any mammal, such as human, cattle, sheep, or pig. Non-mammalian albumins include, but are not limited to, hens and salmon. The albumin portion of the albumin-linked polypeptide can be derived from an animal different from the therapeutic polypeptide portion. In particular, the albumin fusion protein of the present invention can include polymorphic variants of naturally occurring human albumin (HA) and fragments of human albumin. The albumin portion of the albumin fusion protein can contain the full-length HA sequence (NCBI Registry No. NP_000468), preferably containing human serum albumin without a signal peptide (NCBI Registry No. NP_000468 or positions 25-609 of SEQ ID NO: 9), or can include one or more fragments thereof capable of stabilizing or prolonging therapeutic activity. Such fragments can be 10 or more amino acids in length, or can include about 15, 20, 25, 30, 50, 70 or more consecutive amino acids from the HA sequence, or can include some or all of the HA-specific domains.

[0046] In some preferred embodiments, the protein stabilizing portion is human serum albumin (NCBI accession number NP_000468), preferably comprising human serum albumin without the signal peptide (NCBI accession number NP_000468 or positions 25-609 of SEQ ID NO: 9).

[0047] A cleavable linker is any peptide linker that can be cleaved in vivo. Various cleavable peptide linkers that have been used in therapeutic protein constructs are known in the art. Examples of cleavable peptide linkers that can be used in this invention include, but are not limited to: coagulation factor activating sequences, particularly FIX activating sequences (NCBI accession number NP_000124, aa 182-200 or 182-203): SEQ ID NO: 10 or SEQ ID NO: 11.

[0048] In some preferred embodiments, the cuttable connector comprises or consists of the sequence SEQ ID NO: 10.

[0049] In some implementations, the signal peptide, the FGF23 C-terminal peptide, the cleavable linker, and the protein stabilizing moiety are derived from the N-to-C-terminus of the FGF23 fusion protein, which means that the protein stabilizing moiety is fused to the C-terminus of the FGF23 C-terminal peptide.

[0050] In some implementations, the nucleic acid construct contains or is composed of DNA.

[0051] In some other implementations, the nucleic acid construct contains RNA, particularly mRNA, or is composed of it.

[0052] 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 described in the embodiments of this application and Figure 1 As shown in A, corresponding to constructor n°12 in Table 1, - A nucleic acid construct encoding the FGF23 protein, comprising the sequence SEQ ID NO: 52, as shown in the embodiments of this application, corresponding to construct n°10 in Table 1, and - A nucleic acid construct encoding the FGF23 protein, comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the sequences 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 with any of the sequences; more preferably a sequence having at least 95%, 96%, 97%, 98%, or 99% identity with any of the sequences.

[0053] SEQ ID NO: 12 comprises, from its N-terminus to its C-terminus: chymotrypsinogen B2 signal peptide (SEQ ID NO: 7), the FGF23 C-terminal peptide of SEQ ID NO: 2, the cleavable linker of SEQ ID NO: 10, and human serum albumin of SEQ ID NO: 9. SEQ ID NO: 52 comprises, from its N-terminus to its C-terminus: chymotrypsinogen B2 signal peptide (SEQ ID NO: 7), the FGF23 C-terminal peptide consisting of positions 180-251 of SEQ ID NO: 1, the cleavable linker of SEQ ID NO: 10, and human serum albumin of SEQ ID NO: 9.

[0054] In some implementations, the nucleic acid construct comprises a sequence codon-optimized for expression in individuals treated with gene therapy, preferably human individuals. Suitable software for codon optimization in the desired individuals 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 ).

[0055] In some preferred embodiments, the nucleic acid construct comprises the nucleotide sequence SEQ ID NO: 13 or SEQ ID NO: 57, a codon-optimized sequence for expression in humans, encoding the FGF23 fusion protein of SEQ ID NO: 12; the nucleotide sequence SEQ ID NO: 51, 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 of the sequences; preferably a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the sequences; more preferably a sequence having at least 95%, 96%, 97%, 98%, or 99% identity with any of the sequences. The sequences are advantageously codon-optimized sequences for expression in humans.

[0056] In some embodiments, the nucleic acid construct comprises an expression cassette in which a coding sequence is operatively linked to a suitable regulatory sequence to express a transgene in target cells or tissues of an individual. In some specific embodiments, the target tissue is muscle or hepatocytes or tissues or hematopoietic cells, more particularly hepatocytes or tissues. Such sequences well known in the art particularly include promoters and other regulatory sequences capable of further controlling transgene expression, such as, but not limited to, enhancers, terminators, introns, silencers, particularly tissue-specific silencers and microRNAs.

[0057] Promoters can be tissue-specific, ubiquitous, constitutive, or inducible promoters that function in the target cells or tissues of an individual, particularly muscle, liver, or hematopoietic cells or tissues, and more particularly hepatocytes or tissues. Examples of constitutive promoters that can be used in this invention include, but are not limited to: phosphoglycerate kinase promoters (PGK), elongation factor-1α (EF-1α) promoters, including the short form (EFS) of said promoters, viral promoters such as cytomegalovirus (CMV) immediate early enhancers and promoters, CMV enhancer / chicken β-actin (CAG) promoters, SV40 early promoters, and retroviral 5' and 3' LTR promoters, including hybrid LTR promoters. A preferred ubiquitous promoter is the CAG promoter. Examples of inducible promoters that can be used in this invention include tetracycline-regulated promoters. Advantageously, promoters are human promoters, i.e., promoters derived from human cells or human viruses. Such promoters are well known in the art, and their sequences are available in public sequence databases.

[0058] In some specific embodiments, the promoter is a liver-specific promoter. Non-limiting examples of liver-specific promoters that can be used in this invention include the human α-1 antitrypsin promoter (hAAT) (SEQ ID NO: 14), transthyretin promoter, albumin promoter, thyroxine-binding globulin (TBG) promoter, LSP promoter (containing a thyroxine-binding globulin promoter sequence, two copies of an α1-microglobulin / bikunin enhancer sequence and a leader sequence; Charles R. et al., Blood Coag. Fibrinol, 1997, 8: S23–S30), etc. Other useful liver-specific promoters are known in the art, for example, from the liver-specific gene promoter database compiled at Cold Spring Harbor Laboratory (…). http: / / rulai.cshl.edu / LSPD / Those listed in (). In the context of this invention, the preferred liver-specific promoter is the hAAT promoter.

[0059] In other specific embodiments, the promoter is a muscle-specific promoter. Non-limiting examples of muscle-specific promoters include the muscle creatine kinase (MCK) promoter. Suitable non-limiting examples of muscle creatine kinase promoters are the human muscle creatine kinase promoter and the 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 1158 (representative GenBank accession number NC_000019.9, accessed December 26, 2012). Other examples of muscle-specific promoters include the synthetic promoter C5.12 (spC5.12, alternatively referred to herein as “C5.12”), such as spC5.12 or the spC5.12 promoter (published in Wang et al, Gene Therapy, 2008, 15, 1489-99); the MHCK7 promoter (Salva et al. Mol... Ther. Feb. 2007; 15(2):320-9); myosin light chain (MLC) promoters, such as MLC2 (gene ID 4633; GenBank accession number NG_007554.1, accessed December 26, 2012); myosin heavy chain (MHC) promoters, such as α-MHC (gene ID 4624; GenBank accession number NG_023444.1, accessed December 26, 2012); desmin promoter (gene ID 1674; GenBank accession number NG_008043.1, accessed December 26, 2012); cardiac troponin C promoter (gene ID 7134; GenBank accession number NG_008963.1, accessed December 26, 2012); troponin I promoter (gene ID 1 Gene IDs 7135, 7136, and 7137 (representative GenBank accessions NG_016649.1, NG_011621.1, and NG_007866.2, accessed December 26, 2012); myoD gene family promoters (Weintraub et al., Science, 251, 761 (1991); gene ID 4654; representative GenBank accession NM_002478, accessed December 26, 2012); α-actin promoters (gene IDs 58, 59, and 70; representative GenBank accessions NG_006672.1, NG_011541.1, and NG_007553.1, accessed December 26, 2012); β-actin promoters (gene ID 60; representative GenBank accession NG_007992).1. Accessed on December 26, 2012; γ-actin promoters (gene IDs 71 and 72; representative GenBank accessions NG_011433.1 and NM_001199893, accessed on December 26, 2012); muscle-specific promoters located in intron 1 of ocular Pitx3 (gene ID 5309) (Coulon et al.; muscle-selective promoters corresponding to residues 11219-11527 of representative GenBank accession NG_008147, accessed on December 26, 2012); and promoters described in US Patent Publication US 2003 / 0157064, and 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 disclosed in (Gene Therapy, 2008, 15, 1489–99), which comprises a combination of an MCK-derived enhancer and the spC5.12 promoter. In a specific embodiment of the invention, the muscle-specific promoter is selected from the group consisting of: 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, α-actin promoter, β-actin promoter, γ-actin promoter, muscle-specific promoter located in intron 1 of the eye-shaped Pitx3, and CK6 promoter. In a specific embodiment, the muscle-specific promoter is selected from the group consisting of: spC5.12, desmin, and MCK promoters. In another embodiment, the muscle-specific promoter is selected from the group consisting of: spC5.12 and MCK promoters. In a particular implementation, the muscle-specific promoter is the spC5.12 promoter.

[0060] In some other specific embodiments, the promoter is a ubiquitous promoter. Representative ubiquitous promoters include the cytomegalovirus enhancer / chicken β-actin (CAG) promoter, the cytomegalovirus enhancer / promoter (CMV) (optionally having a CMV enhancer) [see, for example, Boshart et al., Cell, 41:521-530 (1985)], the PGK promoter, the SV40 early promoter, the retroviral sarcomavirus (RSV) LTR promoter (optionally having an RSV enhancer), the dihydrofolate reductase promoter, the β-actin promoter, the glycerol phosphokinase (PGK) promoter, and the EF1α promoter.

[0061] In some other specific embodiments, the promoter is an α-globin or β-globin promoter. The β-globin promoter is expressed only in erythrocyte-like cells.

[0062] In other specific implementations, the promoter is an endogenous promoter, such as the albumin promoter or the GDE (glycogen debranching enzyme) promoter. GDE is amylase-1,6-glucosidase-4-α-glucan transferase or AGL, corresponding to human gene ID: 178 (representative GenBank accession number NG_012865, accessed on September 16, 2018).

[0063] In certain embodiments, the promoter is linked to an enhancer sequence, such as a cis-regulatory module (CRM) or an artificial enhancer sequence. CRMs usable in this practice 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, 2014123, 20, 3195-9. In particular, other regulatory elements capable of enhancing muscle-specific expression of genes (especially in cardiac and / or skeletal muscle) are those disclosed in WO2015110449. Specific examples of nucleic acid regulatory elements comprising artificial sequences include regulatory elements obtained by rearranging transcription factor binding sites (TFBSs) present in the sequences disclosed in WO2015110449. Such rearrangements may encompass altering the order of TFBSs and / or altering the position of one or more TFBSs relative to other TFBSs and / or altering the copy number of one or more TFBSs. For example, nucleic acid regulatory elements for enhancing muscle-specific gene expression (especially cardiac and skeletal muscle-specific gene expression) may include binding sites for E2A, HNH1, NF1, C / EBP, LRF, MyoD, and SREBP; or binding sites for E2A, NF1, p53, C / EBP, LRF, and SREBP; or binding sites for E2A, HNH1, HNF3a, HNF3b, NF1, C / EBP, LRF, MyoD, and SREBP; or binding sites for E2A, HNF3a, NF1, C / EBP, LRF, MyoD, and SREBP; or binding sites for E2A, HNF3a, NF1, CEBP, LRF, MyoD, and SREBP; or binding sites for HNF4, NF1, RSRFC4, C / EBP, LRF, and MyoD; or binding sites for NF1, PPAR, p53, C / EBP, LRF, and MyoD.For example, nucleic acid regulatory elements used to enhance the expression of muscle-specific genes (especially skeletal muscle-specific genes) may also include binding sites for E2A, NF1, SRFC, p53, C / EBP, LRF, and MyoD; or binding sites for E2A, NF1, C / EBP, LRF, MyoD, and SREBP; or binding sites for E2A, HNF3a, C / EBP, LRF, MyoD, SEREBP, and Tal1_b; or binding sites for E2A, SRF, p53, C / EBP, LRF, MyoD, and SREBP; or binding sites for HNF4, NF1, RSRFC4, C / EBP, LRF, and SREBP; or binding sites for E2A, HNF3a, HNF3b, NF1, SRF, C / EBP, LRF, MyoD, and SREBP; or binding sites for E2A, CEBP, and MyoD. In other examples, these nucleic acid regulatory elements comprise at least two (e.g., 2, 3, 4) or more copies of one or more of the aforementioned TFBS. In particular, other regulatory elements capable of enhancing liver-specific expression of genes are those disclosed in WO2009130208. Other embodiments of enhancers that can be used in this invention include ApoE control regions, particularly human ApoE control regions (or human apolipoprotein E / CI locus, liver control region HCR-1; Genbank accession number U32510, SEQ ID NO: 15). In some more specific embodiments, enhancer sequences (such as ApoE control regions, preferably human ApoE control regions) are associated with liver-specific promoters (such as those listed above, particularly, for example, the hAAT promoter).

[0064] In certain embodiments, the nucleic acid construct contains introns, particularly introns positioned between the promoter and the coding sequence. Introducing introns increases mRNA stability and protein yield. Furthermore, modified introns designed to reduce or even completely remove the number of alternative open reading frames (ARFs) found in said introns can significantly improve transgene expression. Additionally, by reducing the number of ARFs within the introns contained in the constructs of the present invention, the immunogenicity of the construct is believed to be reduced as well. Preferably, ARFs with a length span exceeding 50 bp and containing a stop codon within a start codon frame are removed. ARFs can be removed by nucleotide substitution, insertion, or deletion, preferably by nucleotide substitution. For example, within the sequence of the target intron, ATG or GTG (which is not a start codon) can be replaced by CTG. Examples of introns that can be used in this invention include human β-globin b2 (or HBB2; SEQ ID NO:16) introns, modified HBB2 introns (SEQ ID NO:17), coagulation factor IX (FIX) introns, particularly those derived from the first intron (SEQ ID NO:18) and its modified introns (SEQ ID NO:19), chicken β-globin introns (SEQ ID NO:20) and their modified introns (SEQ ID NO:21), and SV40 introns. Preferred introns are modified HBB2 introns (SEQ ID NO:17) and modified FIX introns (SEQ ID NO:19).

[0065] In a particular embodiment, the nucleic acid construct further includes a transcription termination signal (polyadenylation signal) operatively linked to the coding sequence (i.e., at the 3' end of the coding sequence). Examples of polyA that can be used in this invention include bovine growth hormone (bGH) polyA (SEQ ID NO: 22).

[0066] In some preferred embodiments, the expression cassette includes a liver-specific promoter, preferably the hAAT promoter, in the 5' to 3' orientation; a coding sequence; and a polyadenylation signal (e.g., (bGH) polyA (SEQ ID NO: 22)). In some more preferred embodiments, the expression cassette further includes one or more other regulatory elements selected from enhancers, preferably the human ApoE control region (SEQ ID NO: 15), and introns, preferably modified HBB2 introns (SEQ ID NO: 17). In the examples and... Figure 1Examples of preferred expression cassettes disclosed in B include sequence SEQ ID NO: 23, which comprises, in the 5' to 3' direction: an ApoE control region (SEQ ID NO: 15), an α-1 antitrypsin promoter (hAAT) (SEQ ID NO: 14), a modified HBB2 intron (SEQ ID NO: 17), a coding sequence (SEQ ID NO: 13), and (bGH) polyA (SEQ ID NO: 22). Another example of a preferred expression cassette disclosed in the embodiments comprises, in the 5' to 3' direction: an ApoE control region (SEQ ID NO: 15), an α-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 comprises sequence SEQ ID NO: 23.

[0067] carrier The present invention also relates to a vector comprising the above-described nucleic acid construct.

[0068] This invention can use any vector suitable for delivering and expressing nucleic acids into individual cells, particularly suitable for gene therapy, and even more particularly for targeted gene therapy to target tissues or cells of an individual. Such vectors well known in the art include viral and nonviral vectors, wherein the vectors may be integrated or non-integrated; replicating or non-replicating. In some specific embodiments, gene therapy targets muscle, liver, or hematopoietic cells or tissues, more particularly liver cells or tissues.

[0069] As used herein, the term "individual" or "patient" refers to a mammal. Preferably, the patient or individual according to the invention is a human being. "Individual" or "patient" includes adults, children, infants, and the elderly.

[0070] Non-viral vectors include a variety of (non-viral) agents commonly used to introduce or maintain nucleic acids into individual cells. Agents used to introduce nucleic acids into individual cells via various mechanisms particularly include polymer-based, particle-based, lipid-based, peptide-based delivery vectors, or combinations thereof, such as, but not limited to, cationic polymers, dendritic polymers, micelles, liposomes, exosomes, microparticles, and nanoparticles, including lipid nanoparticles (LNPs); and cell-penetrating peptides (CPPs). CPPs are particularly cationic peptides, such as poly-L-lysine (PLL), oligo-arginine, Tat peptides, Penetratin, or Transportan peptides and their derivatives, such as Pip. Agents used to maintain nucleic acids within individual cells (integrated into chromosomes or in extrachromosomal form) particularly include naked nucleic acid vectors, such as plasmids, transposons, and small loops, as well as gene editing and RNA editing systems. Transposons particularly include the Sleeping Beauty Hyperactive (SB100X) transposon system (Mates et al. 2009). Gene editing and RNA editing systems can utilize any site-specific endonuclease, such as Cas nucleases, TALENs, broad-spectrum nucleases, zinc finger nucleases, etc. Furthermore, these methods can be advantageously combined to introduce and maintain the nucleic acids of the present invention into individual cells.

[0071] According to a process called viral transduction, viral vectors are essentially able to penetrate cells and deliver target nucleic acids into the cells.

[0072] As used herein, the term "viral vector" refers to a non-replicating, non-pathogenic virus engineered to deliver genetic material into cells. In a viral vector, the viral genes necessary for replication and virulence are replaced by expression cassettes of the target transgene. Thus, the viral vector genome contains transgene expression cassettes flanking the viral sequences required for viral vector production.

[0073] As used herein, the term "recombinant virus" refers to a virus, particularly a viral vector, produced using standard recombinant DNA techniques known in the art.

[0074] As used herein, the term “viral particle” or “particle of a virus” is intended to refer to the extracellular form of nonpathogenic viruses, particularly viral vectors, which consist of genetic material made of DNA or RNA surrounded by a protein coat (called a capsid), and in some cases, the envelope is derived from a portion of the host cell membrane, including viral glycoproteins.

[0075] As used in this article, viral vector refers to viral vector particles.

[0076] Preferred vectors for delivering the nucleic acids (nucleic acid constructs) of the present invention are viral vectors, particularly suitable for gene therapy, and even more particularly for gene therapy targeting target tissues or cells (e.g., muscle, liver, or hematopoietic cells or tissues, especially hepatocytes or tissues) in an individual. Specifically, viral vectors can be derived from non-pathogenic parvoviruses (such as adeno-associated virus (AAV)), retroviruses (such as gamma retroviruses), foamy viruses and lentiviruses, adenoviruses, poxviruses, and herpesviruses. Viral vectors are preferably integrative vectors, such as AAV or lentiviral vectors, with AAV vectors being preferred. Lentiviral vectors can be pseudotyped with envelope glycoproteins from another virus to target target cells / tissues, such as muscle cells, hepatocytes, or hematopoietic cells. In some embodiments, such as those disclosed in WO 2017 / 182607, lentiviruses are pseudotyped with syncytial.

[0077] The vector contains the viral sequence required for viral vector production, such as a lentiviral LTR sequence or an AAV ITR sequence located on the flanking side of the expression cassette.

[0078] In certain embodiments, the carrier is a particle or vesicle, particularly lipid-based micro or nanovesicles or particles, such as liposomes or lipid nanoparticles (LNPs). In more specific embodiments, the nucleic acid is RNA and the carrier is a particle or vesicle as described above.

[0079] In another specific implementation, the vector is a lentiviral vector, particularly a pseudo-lentiviral vector as described above.

[0080] In another specific implementation, the vector is an AAV vector. Human parvovirus adeno-associated virus (AAV) is a naturally occurring replication-defective dependent virus capable of integrating into the genome of infected cells to establish latent infection. This last characteristic appears to be unique among mammalian viruses because integration occurs at a specific site in the human genome located on chromosome 19 (19q13.3 qter) called AAVS1. Therefore, AAV vectors have gained considerable interest as potential vectors for human gene therapy. Advantageous properties of the virus include its lack of association with any human disease, its ability to infect both dividing and non-dividing cells, and the ability to infect a wide range of cell lines derived from different tissues.

[0081] AAV viruses can be engineered using conventional molecular biology techniques, enabling the optimization of these particles for cell-specific delivery of nucleic acid sequences, minimization of immunogenicity, regulation of stability and particle lifespan, efficient degradation, and precise delivery to the cell nucleus.

[0082] As is known in the art, additional suitable sequences can be introduced into the nucleic acid constructs of the present invention to obtain functional viral vectors. Suitable sequences include AAV ITRs. Desired AAV fragments for assembly into the vector 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 readily used in a variety of vector systems and host cells. AAV-based recombinant vectors lacking Rep proteins integrate into the host genome inefficiently, existing primarily as stable circular appendages (which can persist in target cells for years).

[0083] In the context of this invention, the AAV vector comprises an AAV capsid capable of transducing target cells, particularly muscle, liver, or hematopoietic cells or tissues, and more particularly hepatocytes or tissues. The AAV capsid may be derived from one or more natural or artificial AAV serotypes.

[0084] Among the AAV serotypes isolated from and well characterized from humans or non-human primates (NHP), human serotype 2 was the first AAV to be developed as a gene transfer vector. Other currently used AAV serotypes include AAV-1 and AAV-2 variants (such as the quadruple mutant capsid-optimized AAV-2, which contains an engineered capsid with the Y44+500+730F+T491V variation, disclosed in Ling et al., 2016 Jul 18, Hum Gene Ther Methods); -3 and AAV-3 variants (such as the AAV3-ST variant, which contains an engineered AAV3 capsid with the two amino acid variations S663V+T492V, 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 (such as the AAV6 variant containing a triple mutant AAV6 capsid in the form Y731F / Y705F / T492V, disclosed in Rosario et al., 2016, Mol Ther Methods). Clin Dev. 3, p.16026); -7, -8, -9, -2G9; -10 such as cy10 and rh10, rh39, -rh43; -rh74; -dj; Anc80; LK03; AAV.PHP; AAV2i8; porcine AAV serotypes such as AAVpo4 and AAVpo6; and tyrosine, lysine and serine capsid mutants of AAV serotypes, etc.

[0085] As an alternative to using natural AAV serotypes, artificial AAV serotypes, i.e., those having non-naturally occurring capsid proteins, can be used in the context of this invention, including but not limited to chimeric AAV capsids, recombinant AAV capsids, or “humanized AAV capsids.” Such artificial capsids can be generated by any suitable technique using a combination of selected AAV sequences (e.g., fragments of the vpl capsid protein) and heterologous sequences obtainable from different selected AAV serotypes, discontinuous portions of AAV, or the same AAV serotype from non-AAV or non-viral sources. Modified capsids can also be derived from capsid modifications via error-prone PCR and / or peptide insertion (e.g., as described in Bartel et al., 2011). Furthermore, capsid variants can include single amino acid changes such as tyrosine mutants (e.g., as described in Zhong et al., 2008). In the context of this invention, a “modified capsid” can be a chimeric capsid or a capsid containing one or more variant VP capsid proteins derived from one or more wild-type AAV VP capsid proteins.

[0086] In some embodiments, the AAV vector is a chimeric vector, i.e., its capsid contains a VP capsid protein derived from at least two different AAV serotypes, or contains at least one chimeric VP protein combined with VP protein regions or domains derived from at least two AAV serotypes. Examples of such chimeric AAV vectors that can be used to transduce hepatocytes are described in Shen et al., Molecular Therapy, 2007 and Tenney et al., Virology, 2014. For example, a chimeric AAV vector may be derived from a combination of an AAV8 capsid sequence with a sequence of an AAV serotype other than AAV8 (such as any of those specifically mentioned above). In another embodiment, the capsid of the AAV vector contains one or more variant VP capsid proteins, such as those described in WO2015013313, particularly RHM4 1, RHM15 1, RHM15 2, RHM15 3 / RHM15 5, RHM154, and RHM15 6 capsid variants that exhibit high hepaticity.

[0087] In a further embodiment, the AAV vector is a pseudotype vector, meaning its genome and capsid are derived from different serotypes of AAV, such as the AAV serotypes mentioned above. Furthermore, the genome of the AAV vector can be single-stranded or self-complementary double-stranded (McCarty et al., Gene Therapy, 2003). Self-complementary double-stranded AAV vectors are generated by deleting the end resolution site from one of the terminal repeat sequences of the AAV. These modified vectors (whose replicated genome is half the length of the wild-type AAV genome) tend to package DNA dimers.

[0088] In some embodiments, the AAV vector is suitable for gene therapy targeting target tissues or cells in an individual, particularly muscle, liver, or hematopoietic cells or tissues, and more particularly hepatocytes or tissues. In specific 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 capsids, and chimeric capsids thereof. In some preferred embodiments, the AAV vector comprises an AAV8, AAV9, AAVrh74, or AAV2i8 capsid, particularly an AAV8, AAV9, or AAVrh74 capsid, such as an AAV8 or AAV9 capsid, and more particularly an AAV8 capsid. The genome of the AAV vector can be derived from different serotypes (pseudotype vectors) and is advantageously single-stranded.

[0089] The present invention also relates to an isolated cell, particularly a cell derived from an individual, which has been genetically modified or transformed by the nucleic acid or vector of the present invention. The individual is advantageously a patient to be treated. In some embodiments, the cell is a hepatocyte, preferably a patient's hepatocyte.

[0090] As used in this article, “hepatocytes” include primary hepatocytes, such as those derived from adult or fetal livers; hepatocytes matured in vitro, hepatocyte 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.

[0091] As used herein, the term "hematopoietic cell" refers to cells differentiated from hematopoietic stem cells (HSCs or HSCs). Hematopoietic cells include HSCs, pluripotent and lineage-directed 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 microglia.

[0092] As used in this article, the term "hematopoietic stem cell (HSC)" refers to self-renewing cells capable of regenerating hematopoiesis in the short or long term after transplantation.

[0093] As used herein, the term “genetic modification” refers to the insertion, deletion, and / or substitution of one or more nucleotides into a genome sequence.

[0094] As used in this article, muscle tissue specifically includes cardiac and skeletal muscle tissue.

[0095] As used in this article, the term "muscle cell" refers to myocytes, myotubes, myoblasts, and / or satellite cells.

[0096] Pharmaceutical Compositions and Therapeutic Uses Another aspect of the present invention is a pharmaceutical composition comprising at least one nucleic acid selected from the present invention, the carrier of the present invention, or the cell activator of the present invention, and a pharmaceutically acceptable carrier.

[0097] The nucleic acid, vector, and derived pharmaceutical compositions of the present invention can be used to treat diseases through gene therapy, particularly targeted gene therapy of muscle, liver, or hematopoietic cells or tissues, and more particularly hepatocytes or tissues. The cell and derived pharmaceutical compositions of the present invention can be used to treat diseases through cell therapy, particularly cell therapy of muscle, liver, or hematopoietic cells, preferably cell therapy of the liver.

[0098] As used in this article, "gene therapy" refers to treatment involving the delivery of a target nucleic acid into an individual's cells for the purpose of treating a disease. Nucleic acid delivery is typically achieved using a delivery vector (also called a vector). Viral and non-viral vectors can be used to deliver genes into a patient's cells.

[0099] As used herein, “cell therapy” refers to a method in which cells modified with the nucleic acid or vector of the present invention are delivered to an individual in need by any suitable means, such as by intravenous injection (infusion) or injection into a target tissue (implantation or transplantation). In a particular embodiment, cell therapy includes collecting cells from an individual, modifying the individual’s cells with the nucleic acid or vector of the present invention, and administering the modified cells back to the patient. As used herein, “cell” means isolated cells, natural or artificial cell aggregates, bioartificial cell scaffolds, and bioartificial organs or tissues.

[0100] A "pharmaceutically acceptable carrier" is a carrier in which a therapeutic agent is administered, and which, when properly administered to mammals, particularly humans, does not produce adverse, allergic, or other adverse reactions. A pharmaceutically acceptable carrier refers to any type of non-toxic solid or liquid filler, diluent, adjuvant, excipient, encapsulating material, or formulation aid. Pharmaceutical compositions formulated according to standard procedures can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, implants, etc.

[0101] The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of a nucleic acid, a carrier, or a cell therapeutic agent, preferably in a purified form, and an appropriate amount of carrier to provide a form suitable for administration to a subject.

[0102] In the context of this invention, a therapeutically effective dose means a dose sufficient to reverse, alleviate or inhibit the development of the disease or condition to which the term applies, or to reverse, alleviate or inhibit the development of one or more symptoms of the disease or condition to which the term applies.

[0103] The determination and adjustment of the effective dose depends on a variety of factors, such as the composition used, the route of administration, the physical characteristics of the individual considered (e.g., sex, age, and weight), concomitant medications, and other factors that a medical professional would recognize. The effective dose can be determined using standard clinical techniques. Additionally, in vivo and / or in vitro assays may be optionally used to help predict the optimal dose range. In cases involving treatments that include administration of a viral vector (e.g., an AAV vector) to the subject, the typical dose of the vector is at least 1 x 10⁻⁶. 8 One vector genome / kg body weight (vg / kg), for example at least 1x10 9 vg / kg, at least 1x10 10 vg / kg, at least 1x10 11 vg / kg, at least 1x10 12 vg / kg, at least 1x10 13 vg / kg, at least 1x10 14 vg / kg.

[0104] In some embodiments, the pharmaceutical composition comprises excipients that are pharmaceutically acceptable for formulations that can be injected, particularly into a human individual. These include, in particular, sterile isotonic aqueous solutions or suspensions, such as saline solutions (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or mixtures of these salts), or dry, especially lyophilized compositions, which, as appropriate, are prepared as injectable solutions using sterile water or physiological saline upon addition. The solution or suspension may contain additives compatible with nucleic acids and viral vectors and that do not prevent nucleic acid or viral vector particles from entering target cells. In all cases, the form must be sterile and must be fluid to the extent that a syringe can easily inject it. It must be stable under the conditions of manufacture and storage and must be protected against contamination by microorganisms, such as bacteria and fungi. Examples of suitable solutions are buffers, such as phosphate-buffered saline (PBS) or Ringer's lactate.

[0105] In some specific embodiments, the nucleic acids, vectors, or cells of the present invention are formulated in a composition comprising phosphate-buffered saline supplemented with 0.25% human serum albumin. In other specific embodiments, the nucleic acids, vectors, or cells of the present invention are formulated to a final concentration of 0.01-0.0001% by weight of the total composition, for example, a concentration of 0.001%, comprising Ringer's lactate and a nonionic surfactant (e.g., Prönkel F68). The formulation may also contain serum albumin, particularly human serum albumin, for example, 0.25% human serum albumin. Other suitable formulations for storage or administration are known in the art, particularly from WO 2005 / 118792 or Alley et al., 2011, Hum. Gene Ther, 2011 May; 22(5):595-604.

[0106] In some other embodiments, the pharmaceutical composition is formulated as an implant. The implant may be a porous, non-porous, or gel-like material, including membranes such as sialic acid membranes or fibers. The implant can be used to locally apply the pharmaceutical composition of the present invention to the area requiring treatment, namely the liver.

[0107] In other embodiments, the pharmaceutical composition is formulated into a controlled-release system.

[0108] The pharmaceutical composition may also contain additional therapeutic agents, particularly those for treating FGF-23-related diseases, especially FGF-23-related hypophosphatemia, such as calcitriol.

[0109] Another aspect of the present invention relates to the nucleic acids, vectors, cells, and pharmaceutical compositions of the present invention, which are used as pharmaceuticals.

[0110] Another aspect of the present invention relates to the nucleic acids, vectors, cells, and pharmaceutical compositions of the present invention for treating FGF-23-related diseases, particularly FGF-2-related hypophosphatemia, by gene therapy or cell therapy as described above, preferably liver-directed gene therapy or cell therapy.

[0111] Another aspect of the invention relates to the use of the nucleic acid, vector, cell, and pharmaceutical composition of the invention in the preparation of a medicament for treating FGF-23-related diseases, particularly FGF-2-related hypophosphatemia, by gene therapy or cell therapy as described above, preferably liver-directed gene therapy or cell therapy.

[0112] The nucleic acid constructs, vectors, and compositions according to the present invention are used to treat diseases that can be treated by inhibiting the formation of the FGF23-FGF receptor (FGFR)-Klotho complex, such as diseases caused by the overactivity of FGF23, and especially diseases mediated by the interaction between FGF23 and the FGFR / klotho complex.

[0113] Treatable conditions include, in particular, FGF-2-related hypophosphatemia. These conditions can be diagnosed by the presence of high FGF23 levels in hypophosphatemia, as patients with hypophosphatemia due to other causes have low FGF23 levels.

[0114] Examples of treatable FGF-23-related hypophosphatemia 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; osteoglophonic dysplasia caused by mutations in the FRFR1 gene; Jansen-type metaphyseal dysplasia caused by mutations in the PTH1R gene; hypophosphatemia, dental abnormalities, and ectopic 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.

[0115] Other treatable examples of FGF-23-related hypophosphatemia include acquired diseases such as tumor-induced osteomalacia (TIO), hypophosphatemic osteomalacia caused by glycosylated iron oxide or polymaltose iron, complications of kidney transplantation or parenteral iron therapy, chronic kidney disease and its complications such as hyperparathyroidism.

[0116] In some preferred embodiments, the FGF-23-related hypophosphatemia is a genetic disorder, preferably XLH. The disorder is preferably treated with targeted gene therapy, particularly targeting muscle, liver, or hematopoietic cells or tissues, more particularly hepatocytes or tissues, or with cell therapy, more preferably using AAV or lentiviral vectors, particularly AAV8 vectors.

[0117] Another aspect of the present invention relates to a method for treating the aforementioned FGF-23-related hypophosphatemia, comprising: administering to a patient a therapeutically effective amount of the aforementioned nucleic acid, carrier, cell, or pharmaceutical composition.

[0118] In the context of this invention, as used herein, the term "treatment" means reversing, alleviating or inhibiting the development of a disease or condition to which the term applies, or reversing, alleviating or inhibiting the development of one or more symptoms of a disease or condition to which the term applies.

[0119] The nucleic acid, carrier, cell, or pharmaceutical composition of the present invention is typically administered according to known procedures at doses and time periods that effectively induce therapeutic effects in patients. The nucleic acids of the present invention, whether or not carrier-based, can be administered via any convenient route, such as by infusion or bolus in a non-limiting manner, or by absorption through the epithelial or mucosal skin lining (e.g., oral mucosa, rectal and intestinal mucosa). Administration can be systemic or local; systemic administration includes parenteral and oral administration, and local administration includes local and regional administration. Parenteral administration is advantageously via injection or infusion such as subcutaneous (SC), intramuscular (IM), intravascular such as intravenous (IV), intraarterial, intraperitoneal (IP), intradermal (ID), intranasal, epidural, or other methods. Furthermore, it may be necessary to introduce the pharmaceutical composition of the present invention into the liver of the subject via any suitable route.

[0120] The nucleic acid, carrier, cell, or pharmaceutical composition of the present invention can be used in combination with other bioactive agents, wherein the combination is performed by simultaneous, separate, or sequential administration.

[0121] Unless otherwise stated, the practice of this invention will take place using conventional techniques within the scope of the art. These techniques are fully explained in the literature.

[0122] The invention will now be described with reference to the accompanying drawings and the following non-limiting embodiments, in which: Attached Figure Description

[0123] Figure 1 A schematic diagram of the nucleic acid construct and AAV expression vector of the truncated FGF23 fusion protein according to the present invention.

[0124] A. A nucleic acid construct of the truncated FGF23 fusion protein according to the present invention. The C-terminal amino acids (aa 175-251, cFGF) of human FGF protein are fused to human serum albumin (aa 25-609) without a signal peptide via a cleavable linker derived from human coagulation factor IX (hFIX) amino acids 182-200. Finally, a signal peptide derived from the first 18 amino acids of chymotrypsinogen B2 is fused to the N-terminus of the construct (CTRB2).

[0125] B. The AAV expression vector of the truncated FGF23 fusion protein according to the present invention. The nucleic acid construct was inserted into an AAV expression vector containing a liver-specific promoter (apolipoprotein E (ApoE) enhancer-human α1 antitrypsin (hAAT) promoter), a modified HBB2 intron, and a bovine growth hormone (bGH) polyadenylation signal.

[0126] Figure 2The C-terminal portion of FGF23 was stabilized by in vitro albumin fusion. As shown in Table 1, human hepatocellular carcinoma cells (Huh-7) were transfected with plasmids expressing different versions of human FGF23. Forty-eight hours after transfection, the culture medium was harvested and analyzed by Western blot using an anti-FGF23 antibody. (A) Comparison of all constructs, showing the relative expression of different constructs and the higher expression levels achieved using fusion proteins of FGF23-C and the albumin portion. The positions of FGF23, FGF23-C, or the fusion protein of FGF23-C with human albumin (FGF-23-albumin) are shown on the left. (B) Comparison of expression levels obtained after transfection of triplicate plasmids expressing the FGF23-C albumin fusion protein in Huh-7 cells.

[0127] Figure 3 The C-terminal portion of FGF23 is stabilized through in vivo albumin fusion. Wild-type C57BL6 / J mice were injected with 1x10... 12 AAV8 vectors were used in vg / mouse to express a codon-optimized version (coFGF23-C, amino acids 175-251) of the C-terminal portion of FGF23 containing the RRHTR motif (constructors 7, 9, and 12 only), the sp7 signal peptide fused to the albumin portion (constructors 9, 11, and 12), or not fused (constructor 7). Construct 12 included a linker consisting of amino acids 182-200 of human coagulation factor IX, while constructs 9 and 11 lacked a linker. One month after vector injection, mice were sacrificed and plasma and liver were collected to measure phosphate levels and expression levels achieved with different AAV vectors. (A) Phosphate levels measured in blood. (B) Vector genome copy number (VGCN) per diploid genome measured in liver. (C) Western blot analysis in plasma using anti-FGF23 antibody. (D) Bar chart showing the relative intensities of the bands described in inset C. Statistical analysis was performed using a t-test. P<0.05 vs. PBS-treated mice, n=4-5 mice / group).

[0128] Figure 4 AAV-mediated relief of growth retardation in Hypduk mice Use PBS (KO, PBS) or 1x10 12 One-month-old Hypduk mice were treated with an AAV vector expressing FGF23-C fused to sp7 and albumin via an hFIX linker and containing the RRHTR motif (KO, AAV). Wild-type mice treated with PBS served as controls (WT, PBS). AC. Mice were measured at one, two, and three months post-vector injection. The increases in (A) body weight, (B) tail length, and (C) body size over three months are shown. Statistical analysis was performed by ANOVA. P<0.05 vs. KO, PBS; # P<0.05 vs. WT, PBS; n=9-11 animals / group).

[0129] Figure 5 AAV-mediated relief of hypophosphatemia in Hypduk mice Use PBS (KO, PBS) or 1x10 12 One-month-old Hypduk mice were treated with an AAV vector from vg / mouse, which expressed FGF23-C fused to sp7 and albumin via an hFIX linker and contained the RRHTR motif (KO, AAV). Wild-type mice treated with PBS served as controls (WT, PBS). Serum phosphate levels were measured three months after vector injection. Statistical analysis was performed using ANOVA. As shown, NS is not significant.

[0130] Figure 6 AAV-mediated relief of muscle function in Hypduk mice Use PBS (KO, PBS) or 1x10 12 One-month-old Hypduk mice were treated with an AAV vector from vg / mouse, which expressed FGF23-C fused to sp7 and albumin via an hFIX linker and contained the RRHTR motif (KO, AAV). Wild-type mice treated with PBS served as controls (WT, PBS). Inverted grid performance was measured three months after vector injection. Statistical analysis was performed by ANOVA. As shown, NS is not significant. Detailed Implementation

[0131] Materials and methods Construction of nucleic acid encoding truncated FGF23 A nucleic acid construct encoding truncated FGF23 was generated by fusing the following amino acids to human FGF23 protein (amino acids 175-179 of human FGF23 protein, previously R175 (RRHTR motif; amino acids 175-179 of human FGF23 protein) (aa 175-251 of NP_065689; SEQ ID NO:2) or amino acids 180-251 of human FGF protein (excluding the RXXR motif) of SEQ ID NO:1 with human serum albumin (aa 25-609 of NP_000468 or SEQ ID NO:9) without the signal peptide via a cleavable linker derived from human coagulation factor IX (cFIX; amino acids 182-200 of NP_000124; SEQ ID NO:10). Finally, a signal peptide derived from the first 18 amino acids of chymotrypsinogen B2 (sp7; amino acids 1-18 of NP_001020371 or SEQ ID NO: 7) was inserted into the N-terminus of the construct to mediate effective secretion (SEQ ID NO: 12). Figure 1 A); SEQ ID NO: 52). These sequences were codon-optimized using a commercial algorithm. The resulting sequence was SEQ ID NO: 57, which contained the CDS of SEQ ID NO: 13 encoding the fusion protein of SEQ ID NO: 12, with MluI / Kozak flanking at 5' (construction n°12 in Table 1); and SEQ ID NO: 51, which contained the CDS encoding the fusion protein of SEQ ID NO: 52, with MluI / Kozak flanking at 5' (construction n°10 in Table 1). The codon-optimized sequences were cloned into a transgenic expression cassette optimized for liver expression ( Figure 1 B).

[0132] Other constructs were generated for comparison (Table 1). Nucleic acid constructs encoding natural human FGF23 (SEQ ID NO: 1) were generated by cloning wild-type or codon-optimized human FGF23 sequences in transgenic expression cassettes optimized for liver expression. Figure 1 B).

[0133] A truncated C-terminal FGF23 construct (FGF23-C) was generated by fusing FGF23-C, with or without the RXXR motif (amino acids 175-251 or 180-251 of the human FGF23 protein), to native FGF23 or the sp7 signal peptide. These sequences were codon-optimized using commercial algorithms and cloned into transgenic expression cassettes optimized for liver expression.

[0134] Chimeric proteins were generated by fusing FGF23-C without the RXXR motif to human serum albumin (without the signal peptide) in the absence of a cleavable linker derived from human coagulation factor IX, or by fusing FGF23-C containing the RXXR motif to human serum albumin (without the signal peptide) in the absence of a cleavable linker derived from human coagulation factor IX. A signal peptide derived from the first 18 amino acids of chymotrypsinogen B2 (sp7) was inserted into the N-terminus of the construct to mediate efficient secretion. These sequences were codon-optimized using commercial algorithms and cloned into transgenic expression cassettes optimized for liver expression. Figure 1 B).

[0135] AAV vector production The AAV vector was generated using a transient transfection method without adenovirus (Matsushita et al., Gene Therapy, 1998, 5, 938-945) and purified as previously described (Ayuso et al., Gene Therapy, 2010, 17, 503-510). The titer of the AAV vector stock solution was determined by real-time quantitative PCR (qPCR) and confirmed by SDS-PAGE, followed by SYPRO® Ruby protein gel staining and band density assay.

[0136] In vitro studies 70-80% confluent Huh-7 cells were transfected via liposomes using the constructs shown in Table 1. Two days after transfection, the culture medium was harvested and FGF23 levels were analyzed by Western blotting.

[0137] Protein blot Transfected Huh-7 medium and serum from mice injected with different AAV vectors were loaded onto 4–15% gradient polyacrylamide gels for SDS-PAGE. After transfer onto nitrocellulose membranes, the membranes were blocked and incubated with anti-FGF23 antibody. The membranes were then incubated with appropriate secondary antibodies and visualized using an Odyssey imaging system.

[0138] Mouse studies AAV vector was injected intravenously into six-week-old C57BL6 / J mice via the tail vein. One month post-injection, the level of circulating FGF23-C was analyzed by Western blotting. Liver samples were harvested, and the vector genome copy number was measured by qPCR.

[0139] One-month-old male Hypduk mice were intravenously injected with the AAV vector via the tail vein. Wild-type and Hypduk littermates injected with PBS served as controls. Mice were weighed and measured monthly for three months post-injection to assess disease correction at a macroscopic level. Serum phosphate levels and muscle strength were measured three months post-injection for functional assessment.

[0140] Phosphateemia To measure circulating phosphate levels, mice were bled three months after injection of the vector. Serum inorganic phosphate levels were measured using a standard commercial kit after centrifugation at 10000 xg for 10 minutes.

[0141] Inverted Mesh Test Place the mouse on the grid and allow it to settle for 3-5 seconds. Then invert the grid and hold it at least 35 cm above the mouse cage containing a 5-7 cm pad. Measure the number of falls over a three-minute period and report it as falls per minute.

[0142] result The C-terminal portion of human FGF23 (FGF23-C) competes with natural FGF23 and reduces intracellular signal transduction after receptor binding.

[0143] A significant limitation of using FGF23-C as an XLH therapy is the peptide's high instability in circulation. Gene therapy methods were developed to secrete this peptide from the liver and improve its stability. Amino acids 175-251 of human FGF protein (SEQ ID NO: 2), containing the RXXR motif followed by R175 (RRHTR motif; amino acids 175-179 of human FGF23 protein), or amino acids 180-251 of human FGF protein (without the RXXR motif) from SEQ ID NO: 1, were fused to human serum albumin (SEQ ID NO: 9) without a signal peptide via a cleavable linker derived from human coagulation factor IX (SEQ ID NO: 10). Finally, a signal peptide, consisting of the first 18 amino acids of chymotrypsinogen B2 (SEQ ID NO: 7), was inserted into 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 using a commercial algorithm. The resulting sequences were SEQ ID NO: 57, which contained the CDS of SEQ ID NO: 13 encoding the fusion protein of SEQ ID NO: 12, with MluI / Kozak flanking at 5' (construction n°12 in Table 1); and SEQ ID NO: 51, which contained the CDS of the fusion protein of SEQ ID NO: 52, with MluI / Kozak flanking at 5' (construction n°10 in Table 1). The codon-optimized sequences were cloned into a transgenic expression cassette optimized for liver expression ( Figure 1 B). The transgenic expression cassette containing SEQ ID NO: 13, included in SEQ ID NO: 57, has the sequence SEQ ID NO: 23. The AAV transfer vector containing the AAV ITR-side-attached expression cassette of SEQ ID NO: 23 has the sequence SEQ ID NO: 24.

[0144] To confirm the low stability of FGF23-C and test strategies to improve its secretion and stability, the inventors transfected human hepatocellular carcinoma cells with different constructs expressing native FGF23 and FGF23-C under the transcriptional control of the hAAT promoter (Table 1 and 2000). Figure 1 ).

[0145] Table 1

[0146] Table 1. Description of the tested constructs. FGF23, fibroblast growth factor 23; FGF23-C, the C-terminal portion of FGF23 (amino acids 175-251); spFGF23, native FGF23 signal peptide; sp7, chymotrypsinogen B2 signal peptide; RXXRR, amino acids 176-179 of FGF23-C; wt, native FGF23 sequence; co, codon optimized; cFIX, amino acids 182-200 of human coagulation factor IX; albumin, amino acids 25-609 of human serum albumin sequence.

[0147] -Construction n°1 (nucleotide (nt) sequence (SEQ ID NO: 25); corresponding amino acid (aa) sequence (SEQ ID NO: 26, 1 or 27).

[0148] -Constructor n°2 (nucleotide (nt) sequence (SEQ ID NO: 28); corresponding amino acid (aa) sequence (SEQ ID NO: 29, 1, 26 or 27).

[0149] -Construction n°3 (nucleotide (nt) sequence (SEQ ID NO: 30); corresponding amino acid (aa) sequence (SEQ ID NO: 31 or 32).

[0150] -Constructor n°4 (nucleotide (nt) sequence (SEQ ID NO: 33); corresponding amino acid (aa) sequence (SEQ ID NO: 34 or 35).

[0151] - Construct n°5 (nucleotide (nt) sequence (SEQ ID NO: 36); corresponding amino acid (aa) sequence (SEQ ID NO: 37 or 38).

[0152] -Constructor n°6 (nucleotide (nt) sequence (SEQ ID NO: 39); corresponding amino acid (aa) sequence (SEQ ID NO: 40 or 41).

[0153] -Constructor n°7 (nucleotide (nt) sequence (SEQ ID NO: 42); corresponding amino acid (aa) sequence (SEQ ID NO: 43 or 44).

[0154] -Constructor n°8 (nucleotide (nt) sequence (SEQ ID NO: 45); corresponding amino acid (aa) sequence (SEQ ID NO: 46 or 47).

[0155] -Constructor n°9 (nucleotide (nt) sequence (SEQ ID NO: 48); corresponding amino acid (aa) sequence (SEQ ID NO: 49 or 50) -Constructor n°10 (nucleotide (nt) sequence (SEQ ID NO: 51); corresponding amino acid (aa) sequence (SEQ ID NO: 52, which is the same as SEQ ID NO: 53).

[0156] -Constructor n°11 (nucleotide (nt) sequence (SEQ ID NO: 54); corresponding amino acid (aa) sequence (SEQ ID NO: 55 or 56).

[0157] -Constructor n°12 (nucleotide (nt) sequence (SEQ ID NO: 57); corresponding amino acid (aa) sequence SEQ ID NO: 58, which is identical to SEQ ID NO: 12).

[0158] Wild-type and codon-optimized versions of the FGF-23 and FGF23-C genes were tested together with versions fused with a heterologous signal peptide of native FGF23 and prochymotrypsinogen B2 (SEQ ID NO: 7, sp7). The different sequences tested included or excluded the RRHTR motif, which is present in human FGF23 and responsible for cleaving it into the FGF23 N-terminal and C-terminal peptides.

[0159] Natural FGF23 in transfected Huh-7 cells and its codon-optimized versions (constructors n° 1 and 2, Figure 2 Efficient production and secretion in culture medium A). Except for the sp7 fusion of FGF23-C (structure n° 8) without the RHTR motif. Figure 2 A) Apart from this, no FGF23-C variants were produced in the culture medium. Interestingly, fusion with the albumin moiety (SEQ ID NO: 9) enhanced FGF23-C secretion, regardless of the chimeric protein (constructors n° 9-12). Figure 2 A) What is its composition? Compared to other chimeric proteins containing albumin motifs, the fusion of FGF23-C with albumin in the absence of the RHTR motif and a cleavable linker derived from human coagulation factor IX (construct n° 11) results in higher in vitro secretion levels (constructions n° 9, n° 10, and n° 12). Figure 2 B).

[0160] AAV8 vectors expressing constructs n° 7, n° 9, n° 11, and n° 12 were prepared: i) FGF23-C fused to sp7 and containing the RRHTR motif (constructor n° 7), ii) FGF23-C fused to sp7 and albumin and containing the RRHTR motif (constructor n° 9), iii) FGF23-C fused to sp7 and albumin without the RRHTR motif (constructor n° 11), and iv) FGF23-C fused to sp7 and albumin via an hFIX linker and containing the RRHTR motif (constructor n° 12, Table 1 and 1). Figure 1 A). The AAV8 carrier uses a 1x10 12 The dose of vg / mouse was generated and injected into six-week-old C57BL6 / J mice. One month after vector injection, mice were exsanguinated to measure phosphate and FGF23-C levels by Western blotting. Importantly, increased blood phosphorus levels were observed only in mice injected with the AAV8 vector expressing construct n°12. Figure 3 A), although other constructs achieved similar levels of liver transduction ( Figure 3 B), and higher expression levels were achieved in vitro using the construct n° 11.

[0161] The band corresponding to the molecular weight of the FGF23-C-albumin chimeric protein was only observed in mice injected with the AAV8 vector, which specifically expressed constructs n°9 and n°12 in the liver. Conversely, no band compatible with FGF23-C, i.e., FGF23-C fused with sp7, was detected in mice injected with the AAV8 vector, confirming the in vivo instability of FGF23-C. Figure 3 C). Importantly, with the construct n° 9 (no joints; Figure 3 Compared to D), mice injected with construct n° 12 containing the cleavable linker (hFIX; SEQ ID NO: 10) showed significantly higher levels of circulating FGF-23-C fusion protein, despite a lower vector genome copy number per cell ( Figure 3 The higher expression levels achieved in vitro by B) and construct n° 11 demonstrate similar hepatic transduction. These data indicate that the C-terminal portion of FGF23 (FGF23-C) can only be expressed by the liver in a fusion form with the albumin moiety. They also show that when FGF23-C fuses with the albumin moiety via a cleavable linker (specifically derived from human coagulation factor IX (SEQ ID NO:10)), the composition of the fusion protein affects its stability and function in circulation.

[0162] To verify the composition of FGF23-C and the application of the hFIX-derived connector according to the present invention (construction n° 12, Table 1 and... Figure 1 A) Whether the chimeric protein fused with sp7 and albumin is active in vivo was determined by administering 1 x 10⁻⁶ doses to one-month-old Hypduk mice. 12 vg / mice (KO, AAV) were injected with the AAV8 vector expressing this construct. Hypduk (KO, PBS) and wild-type littermates (WT, PBS) were used as controls. Phenotypic correction was assessed by monthly measurements of body weight, body length, and tail length starting one month after vector injection. Figure 4 Typically, Hypduk mice treated with PBS have lower body weight, body length, and tail length compared to wild-type animals. Figure 4 AC). Hypduk mice treated with the AAV8 vector expressing the FGF23-C chimeric protein according to the invention showed increases in body weight, body length, and tail length over a 3-month period compared to PBS-treated Hypduk mice, and were similar to those observed in wild-type animals. Figure 4 AC). Although a complete relief of body length increase was observed in AAV-treated animals ( Figure 4 B), but weight and tail length were only partially compensated ( Figure 4A, C). After three 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 indistinguishable from those in wild-type animals. Figure 5 Impaired bone growth and tendon calcification may lead to decreased motor function and muscle strength. Functional assessment of the Hypduk mouse phenotype was performed using an inverted grid test. We observed an increased number of falls per minute in Hypduk mice compared to wild-type mice that were fully relieved by AAV gene therapy. Figure 6 ).

Claims

1. A nucleic acid construct for gene therapy of FGF-23-related hypophosphatemia, encoding an FGF23 fusion protein, said fusion protein comprising: (a) Signal peptide, (b) The FGF23 C-terminal peptide bound to the FGFR / klotho complex, (c) Cuttable connectors, and (d) The stable portion of the protein, The signal peptide is located at the N-terminus of the fusion protein, and the FGF23 C-terminal peptide and the protein stabilizing portion are separated by the cleavable linker.

2. The nucleic acid construct according to claim 1, wherein the FGF23 C-terminal peptide comprises a sequence from any one of positions 175-189 to positions 203-251 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 with said sequence.

3. The nucleic acid construct according to claim 1 or 2, wherein the FGF23 C-terminal peptide comprises the RXXR motif at positions 176-179 of SEQ ID NO:

1.

4. The nucleic acid construct according to any one of claims 1-3, wherein the FGF23 C-terminal peptide comprises the sequence 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 with the sequence.

5. The nucleic acid construct according to any one of claims 1-4, wherein the signal peptide comprises a sequence selected from the group consisting of: SEQ ID NO: 3-8; preferably SEQ ID NO: 7, and / or the cleavable adapter comprises the sequence SEQ ID NO:

10.

6. The nucleic acid construct according to any one of claims 1-5, wherein the protein stabilizing portion is human serum albumin, preferably comprising the sequence SEQ ID NO:

9.

7. The nucleic acid construct according to any one of claims 1-6, encoding an FGF23 protein, said FGF23 protein comprising the sequence 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 with any one of said sequences.

8. The nucleic acid construct according to any one of claims 1-7, wherein the codons are optimized for expression in humans.

9. The nucleic acid construct according to claim 7 or 8, comprising a sequence 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 with any of the said sequences.

10. The nucleic acid construct according to any one of claims 1-9, comprising an expression cassette, wherein the coding sequence is operatively linked to a promoter that is functional in at least muscle, liver, or hematopoietic cells or tissues, particularly a liver-specific promoter, preferably a human α-1 antitrypsin promoter.

11. The nucleic acid construct of claim 10, further comprising one or more control elements selected from the group consisting of: a promoter-associated enhancer, preferably a human ApoE control 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 a bovine growth hormone polyadenylation signal.

12. The nucleic acid construct according to any one of claims 1-11, wherein it is DNA or RNA.

13. A vector for gene therapy, said vector comprising the nucleic acid construct according to any one of claims 1-12.

14. The vector according to claim 13, wherein it 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 their chimeric capsids, more preferably an AAV8 capsid.

15. The carrier of claim 13, wherein it is a particle or vesicle, particularly a lipid-based micro or nano vesicle or particle, preferably comprising the RNA construct of claim 12.

16. Cells, genetically modified from nucleic acid constructs according to any one of claims 1-12 or vectors according to any one of claims 13-15, particularly muscle, liver or hematopoietic cells, preferably hepatocytes.

17. A pharmaceutical composition comprising at least one nucleic acid construct selected from any one of claims 1-12, a carrier selected from any one of claims 13-15, or a cell activator selected from claim 16, and a pharmaceutically acceptable carrier.

18. The pharmaceutical composition of claim 17, for treating FGF-23-related hypophosphatemia by gene therapy or cell therapy, wherein the FGF-23-related hypophosphatemia is preferably a genetic disease selected from the group consisting of: X-linked hypophosphatemia, autosomal dominant hypophosphatemic rickets, autosomal recessive hypophosphatemic rickets 1, autosomal recessive hypophosphatemic rickets 2, osteoglophonic dysplasia, Jansen type metaphyseal dysplasia, hypophosphatemia, dental abnormalities and ectopic calcification, McCune-Albright syndrome / fibrous dysplasia and hypophosphatemia, skin and bone lesions, or an acquired disease selected from the group consisting of: tumor-induced osteomalacia, hypophosphatemic osteomalacia, complications of kidney transplantation or parenteral iron therapy, chronic kidney disease and its complications such as hyperparathyroidism; preferably X-linked hypophosphatemia.

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