Treatment of diseases involving deficiency of ENPP1 or ENPP3

Abstract

To provide a recombinant polynucleotide for treating a particular disease of ectopic tissue calcification, and to provide a viral vector containing the recombinant polynucleotide.SOLUTION: Provided is a recombinant polynucleotide encoding a precursor polypeptide comprising an Azurocidin signal peptide fused to ectonucleotide pyrophosphatase / phosphodiesterase- 1 (ENPP1) or to ectonucleotide pyrophosphatase / phosphodiesterase-3 (ENPP3), the precursor polypeptide being proteolytically cleaved to produce soluble ENPP1 or soluble ENPP3 which is active to reduce ectopic calcification of soft tissue, upon expression of the polynucleotide in mammalian cells.SELECTED DRAWING: Figure 4

Description

Technical Field

[0001] Cross-reference This application claims priority to U.S. Application No. 62 / 794,450, filed on January 18, 2019 (01 / 18 / 2019), U.S. Application No. 62 / 821,692, filed on March 21, 2019 (03 / 21 / 2019), and U.S. Application No. 62 / 877,044, filed on July 22, 2019 (07 / 22 / 2019), the contents of each of which are hereby incorporated by reference in their entirety.

[0002] The present invention generally relates to the treatment of diseases associated with a deficiency of ENPP1 or ENPP3 by providing a nucleic acid encoding ENPP1 or ENPP3 to a mammal.

Background Art

[0003] ENPP1 (also known as PC-1) is a type 2 extracellular membrane-bound glycoprotein located in the mineral deposition matrix vesicles of osteoblasts and chondrocytes, and hydrolyzes extracellular nucleotides (primarily ATP) to adenosine monophosphate (AMP) and inorganic pyrophosphate (PPi). PPi functions as a potent inhibitor of heterotopic tissue mineralization by binding to nascent hydroxyapatite (HA) crystals, thereby preventing the future growth of these crystals. ENPP1 generates PPi by hydrolysis of nucleotide triphosphate (NTP), Progressive Ankylosis Protein (ANK) transports intracellular PPi to the extracellular space, and tissue non-specific alkaline phosphatase (TNAP) removes PPi via direct hydrolysis of PPi to Pi. WO2011 / 113027 - Quinn et al., WO2012 / 125182 - Quinn et al., WO2016 / 100803 - Quinn et al., and WO2017 / 218786 - Yan et al. describe NPP1.

[0004] Like ENPP1, ENPP3 also belongs to the phosphodiesterase I / nucleotide pyrophosphatase enzyme family. These enzymes are type II transmembrane proteins that catalyze the cleavage of phosphodiester and phosphosulfate bonds of various molecules, including deoxynucleotides, NAD, and nucleotide sugars. ENPP1 has been shown to be effective in reducing systemic arterial calcification in a mouse model of a specific disease with ectopic tissue calcification, for example, GACI (generalized arterial calcification of infancy), a severe disease that occurs in infants and is involved in extensive arterial calcification (Albright, et al., 2015, Nature Comm. 10006).

[0005] Summary of the Invention In one aspect, the present disclosure provides a recombinant polynucleotide encoding a recombinant polypeptide comprising ectonucleotide pyrophosphatase / phosphodiesterase-1 (ENPP1) or ectonucleotide pyrophosphatase / phosphodiesterase-3 (ENPP3).

[0006] In another aspect, the present disclosure provides a viral vector comprising any of the recombinant polynucleotides described herein.

[0007] In some embodiments, the recombinant polynucleotide encodes a human ENPP1 or human ENPP3 polypeptide. Accordingly, the present disclosure also provides a viral vector comprising a recombinant polynucleotide encoding a recombinant polypeptide comprising ectonucleotide pyrophosphatase / phosphodiesterase-1 (ENPP1) or ectonucleotide pyrophosphatase / phosphodiesterase-3 (ENPP3).

[0008] In some embodiments of any of the polynucleotides or viral vectors described herein, the recombinant polypeptide is an ENPP1 fusion polypeptide.

[0009] In some embodiments of any of the polynucleotides or viral vectors described herein, the recombinant polypeptide is an ENPP3 fusion polypeptide.

[0010] In some embodiments of any of the polynucleotides or viral vectors described herein, the ENPP1 fusion polypeptide is an ENPP1-Fc fusion polypeptide or an ENPP1-albumin fusion polypeptide.

[0011] In some embodiments of any of the polynucleotides or viral vectors described herein, the ENPP3 fusion polypeptide is an ENPP3-Fc fusion polypeptide or an ENPP3-albumin fusion polypeptide.

[0012] In some embodiments of any of the polynucleotides or viral vectors described herein, the recombinant polypeptide comprises a signal peptide fused to ENPP1 or ENPP3.

[0013] In some embodiments of any of the polynucleotides or viral vectors described herein, the signal peptide is an azurocidin signal peptide or an NPP2 signal peptide or an NPP7 signal peptide.

[0014] In some embodiments of any of the polynucleotides or viral vectors described herein, the viral vector is an adeno-associated viral vector or a herpes simplex vector or an alphavirus vector or a lentiviral vector. In one aspect of the invention, the serotype of the adeno-associated viral vector (AAV) is AAV1 or AAV2 or AAV3 or AAV4 or AAV5 or AAV6 or AAV7 or AAV8 or AAV9 or AAV-rh74.

[0015] In yet another aspect, the disclosure provides an adeno-associated viral vector comprising a recombinant polypeptide encoding an ENPP1-Fc fusion polypeptide.

[0016] In yet another aspect, the present disclosure provides an adeno-associated virus vector comprising a recombinant polynucleotide encoding a recombinant polypeptide comprising an azurocidin signal peptide fused to an ENPP1-Fc fusion polypeptide.

[0017] In some embodiments, the viral vector is not an insect viral vector such as a baculovirus vector.

[0018] In some embodiments, the viral vector can infect mammalian cells, such as human cells (e.g., human hepatocytes or HEK cells, HeLa or A549 or hepatocytes). In some embodiments, the viral vector can infect, enter, and / or fuse with mammalian cells such as human cells. In some embodiments, all or a functional portion of the polynucleotide of the viral vector integrates or is integrated into the genome of the cells contacted by the viral vector described herein. In some embodiments, all or a functional portion of the polynucleotide of the viral vector can persist in an episomal state without integrating into the genome of the mammalian cells contacted by the viral vector described herein.

[0019] In some embodiments, the recombinant polynucleotide comprises a vector or plasmid encoding a viral protein and / or human ENPP1. In some embodiments, the recombinant polynucleotide comprises a vector or plasmid encoding a viral protein and / or human ENPP3. In some embodiments, the vector or the plasmid can express a polypeptide encoded by the azurocidin signal peptide fused to ectonucleotide pyrophosphatase / phosphodiesterase-1 (ENPP1) or to ectonucleotide pyrophosphatase / phosphodiesterase-3 (ENPP3).

[0020] In some embodiments, the encoded polypeptide comprises an azurocidin signal peptide fused to ectonucleotide pyrophosphatase / phosphodiesterase-1 (ENPP1), and comprises a transmembrane domain, a somatomedin domain, a catalytic domain, and a nuclease domain.

[0021] In some embodiments, the encoded polypeptide comprises an azurocidin signal peptide fused to ectonucleotide pyrophosphatase / phosphodiesterase-1 (ENPP1) and is secreted into the cytosol.

[0022] In some embodiments, the recombinant polynucleotide encoding the polypeptide comprises a transmembrane domain fused to ectonucleotide pyrophosphatase / phosphodiesterase-1 (ENPP1), is not secreted, and is membrane-bound.

[0023] In some embodiments, the present disclosure provides a recombinant polynucleotide encoding a polypeptide comprising ectonucleotide pyrophosphatase / phosphodiesterase-1 (ENPP1). In some embodiments, the polypeptide comprising ectonucleotide pyrophosphatase / phosphodiesterase-1 (ENPP1) comprises the amino acid residues of SEQ ID NO: 1.

[0024] In some embodiments, the encoded polypeptide comprises an azurocidin signal peptide fused to ectonucleotide pyrophosphatase / phosphodiesterase-1 (ENPP1).

[0025] In some embodiments, the encoded polypeptide comprising an azurocidin signal peptide fused to ectonucleotide pyrophosphatase / phosphodiesterase-1 (ENPP1) lacks a polyaspartic acid domain or a negatively charged bone targeting domain.

[0026] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, any of the polynucleotides described herein encodes an azurocidin signal peptide fused to ENPP1, or an azurocidin signal peptide fused to ENPP3 and ENPP1 or ENPP3 fused to an Fc polypeptide, to form azurocidin signal peptide-ENPP1-Fc or azurocidin signal peptide-ENPP3-Fc, respectively, in order from amino terminus to carboxy terminus.

[0027] In some embodiments, the recombinant polynucleotide encodes an azurocidin signal peptide fused to ENPP1 or an azurocidin signal peptide fused to ENPP3 and ENPP1 or ENPP3 fused to human serum albumin, to form azurocidin signal peptide-ENPP1-albumin or azurocidin signal peptide-ENPP3-albumin, respectively, in order from amino terminus to carboxy terminus.

[0028] In some embodiments, the Fc or albumin sequence is directly fused to the C-terminus of the ENPP1 or ENPP3 protein. In some embodiments, the Fc or albumin sequence is fused to the C-terminus of the ENPP1 or ENPP3 protein via a linker such as a flexible linker. In some embodiments, the linker is selected from SEQ ID NOs: 57-88.

[0029] In some embodiments, the viral vector comprises a nucleic acid sequence encoding a signal peptide fused to the N-terminus of ENPP1 or ENPP3 and is capable of expressing the same. In some embodiments of the viral vector, the vector comprises a promoter. In some embodiments of the viral vector, the promoter is a liver-specific promoter.

[0030] In some embodiments of the viral vector, the liver-specific promoter is selected from the group consisting of the albumin promoter, the phosphoenolpyruvate carboxykinase (PEPCK) promoter, and the alpha-1-antitrypsin promoter. In some embodiments of the viral vector, the vector comprises a sequence encoding a polyadenylation signal.

[0031] In some embodiments of the viral vector, the signal peptide is the azurocidin signal peptide. In some embodiments of the viral vector, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments of the viral vector, the AAV vector having a serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV-rh74.

[0032] In some embodiments of the viral vector, the polynucleotide of the present invention encodes an azurocidin signal peptide fused to ENPP1 or an azurocidin signal peptide fused to ENPP3 and ENPP1 or ENPP3 fused to an Fc polypeptide, forming azurocidin signal peptide-ENPP1-Fc or azurocidin signal peptide-ENPP3-Fc, respectively, in order from the amino terminus to the carboxy terminus.

[0033] In some embodiments of the viral vector, the polynucleotide encodes an azurocidin signal peptide fused to ENPP1 or an azurocidin signal peptide fused to ENPP3 and ENPP1 or ENPP3 fused to human serum albumin, forming azurocidin signal peptide-ENPP1-albumin or azurocidin signal peptide-ENPP3-albumin, respectively, in order from the amino terminus to the carboxy terminus.

[0034] In yet another aspect, the present disclosure provides a cell (e.g., a mammalian cell, such as a rodent cell, a non-human primate cell, or a human cell) comprising any of the polynucleotides described herein.

[0035] In some embodiments, the present invention also provides a method for obtaining a recombinant viral vector, comprising the following steps: i. providing a cell comprising a polynucleotide of the present invention; ii. maintaining the cell under conditions suitable for viral assembly; and iii. purifying the viral vector produced by the cell.

[0036] In another aspect, the present disclosure provides a method for producing a recombinant viral vector. The method comprises the following steps: i. providing a cell or population of cells comprising a polynucleotide described herein, wherein the cell expresses a viral protein essential for packaging or assembly of the polynucleotide into a recombinant viral vector; and ii. maintaining the cell or population of cells under conditions suitable for packaging or assembly of the recombinant viral vector.

[0037] In some embodiments, the method comprises purifying the viral vector from the cell or population of cells, or from the medium in which the cell or population of cells was maintained.

[0038] In some embodiments, the cell is a mammalian cell, such as a rodent cell (e.g., a rat cell, a mouse cell, a hamster cell), a non-human primate cell, or a human cell (e.g., HEK293, HeLa, or A549).

[0039] In some embodiments, the method further comprises introducing into a cell or population of cells a recombinant nucleic acid encoding one or more viral proteins (e.g., those essential for packaging or assembly of a viral vector), such as infecting the cell or population of cells with a helper virus comprising such recombinant nucleic acid, transfecting the cell or population of cells with a helper plasmid comprising such recombinant nucleic acid, and the like.

[0040] In some embodiments, the viral vector can express one or more polypeptides described herein upon infection of a target cell.

[0041] In some embodiments, the present disclosure provides a pharmaceutical composition comprising the purified viral vector described herein. In some embodiments, the present disclosure provides a sterile pharmaceutical composition comprising the sterile / endotoxin-free purified viral vector described herein.

[0042] In another aspect, the present disclosure provides a purified viral vector obtained by any of the methods described herein.

[0043] In another aspect, the present disclosure provides a pharmaceutical composition comprising any of the purified viral vectors obtained by any of the methods described herein.

[0044] In certain embodiments, the present invention provides a method of providing ENPP1 or ENPP3 to a mammal, the method comprising administering to the mammal a viral vector of the present invention.

[0045] In certain embodiments, the present disclosure provides a method of expressing ENPP1 or ENPP3 in a mammal (e.g., a human, e.g., a human in need of such expression), the method comprising administering to the mammal any of the viral vectors described herein. Prior to, simultaneously with, and / or after administration of the viral vector to the mammal, the method can further comprise detecting and / or measuring one or more of the following parameters in a biological sample obtained from the mammal: expression of ENPP1 and / or ENPP3, the activity level of ENPP1 and / or ENPP3, and / or the level or concentration of pyrophosphate. In some embodiments, the one or more parameters are detected or measured within 1 week, within 1-2 weeks, and / or within 1 month after administration of the viral vector to the mammal. In some embodiments, the mammal (e.g., a human) has a deficiency of ENPP1 or ABCC6.

[0046] In another aspect, the present disclosure provides a pharmaceutical composition comprising any of the viral vectors described herein and a physiologically compatible carrier.

[0047] In some embodiments, the present disclosure is a method of preventing or reducing the progression of a condition or disease in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of a composition according to the present invention, wherein the condition or disease includes, but is not limited to, the following: deficiency of NPP1, low levels of PPi, progressive disorders characterized by the accumulation of calcium and other mineral deposits in arteries and / or connective tissues, ectopic calcification of soft tissues, arterial or venous calcification, calcification of heart tissue, such as aortic tissue and coronary vascular tissue, pseudoxanthoma elasticum (PXE), X-linked hypophosphatemia (XLH), chronic kidney disease (CKD), mineral bone disorder (MBD), vascular calcification, pathologic calcification of soft tissues, pathologic ossification of soft tissues, generalized arterial calcification of infancy (GACI), and ossification of the posterior longitudinal ligament (OPLL), one or more of which thereby provides a method of preventing the disease in the mammal or reducing its progression.

[0048] In another aspect, the present disclosure is a method of treating, preventing and / or ameliorating a disease or disorder of pathologic calcification or pathologic ossification in a subject in need thereof, comprising administering a therapeutically effective amount of any of the viral vectors described herein, thereby treating, preventing or ameliorating the disease or disorder. In some embodiments, the viral vector comprises a polynucleotide encoding a human ENPP1 or human ENPP3 polypeptide.

[0049] In another aspect, the present disclosure is a method of treating a subject having a deficiency of ENPP1 protein, comprising administering to the subject a therapeutically effective amount of a viral vector encoding a recombinant ENPP1 or ENPP3 polypeptide, thereby treating the subject. In one aspect of the invention, the viral vector encodes a human ENPP1 or human ENPP3 polypeptide.

[0050] In another aspect, the subject has a disease or disorder associated with a loss-of-function mutation in the subject's ENPP1 gene or a loss-of-function mutation in the subject's ABCC6 gene or a deficiency in ENPP1 protein.

[0051] In some embodiments of any of the methods described herein, the viral vector is an AAV vector encoding an ENPP1-Fc fusion polypeptide, and the vector is administered to the subject at a dosage of 1×10 12 ~1×10 15 vg / kg, preferably 1×10 13 ~1×10 14 vg / kg.

[0052] In some embodiments of any of the methods described herein, the viral vector is an AAV vector encoding an ENPP1-Fc fusion polypeptide, and the vector is administered to the subject at a dosage of 5×10 11 ~5×10 15 vg / kg.

[0053] In some embodiments of any of the methods described herein, the viral vector is an AAV vector encoding an ENPP1-Fc fusion polypeptide, and approximately 1×10 12 ~1×10 15 vg / kg is administered per subject for delivery and expression of the ENPP1-Fc polypeptide.

[0054] In some embodiments of any of the methods described herein, the viral vector is an AAV vector encoding an ENPP3-Fc fusion polypeptide, and the vector is administered to the subject at a dosage of 1×10 12 ~1×10 15 vg / kg, preferably 1×10 13 ~1×10 14 vg / kg.

[0055] In some embodiments of any of the methods described herein, the viral vector is an AAV vector encoding an ENPP3-Fc fusion polypeptide, and the vector is administered to the subject at a dosage of 5×1011 ~5×10 15 is administered to the subject at a dosage of vg / kg.

[0056] In some embodiments of any of the methods described herein, the viral vector is an AAV vector encoding an ENPP3-Fc fusion polypeptide, and for delivery and expression of the ENPP3-Fc polypeptide, approximately 1×10 12 ~1×10 15 vg / kg is administered.

[0057] In some embodiments of any of the methods described herein, administration of an AAV vector encoding an ENPP1-Fc polypeptide to a subject results in a dose-dependent increase in plasma pyrophosphate (PPi) and a dose-dependent increase in plasma ENPP1 concentration in said subject.

[0058] Prior to, simultaneously with, and / or after administration of the viral vector to a mammal, any of the methods described herein can further comprise detecting and / or measuring in a biological sample obtained from the mammal one or more of the following parameters: expression of ENPP1 and / or ENPP3, activity levels of ENPP1 and / or ENPP3, and / or levels or concentrations of pyrophosphate. In some embodiments, the one or more parameters are detected or measured 1 week, within 1-2 weeks, and / or within 1 month after administration of the viral vector to the mammal.

[0059] In yet another aspect, the disclosure provides a method of treating or preventing a disease or disorder of pathologic calcification or pathologic ossification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a viral vector encoding a recombinant ENPP1 or ENPP3 polypeptide, thereby treating or preventing said disease or disorder.

[0060] In another aspect, the present disclosure provides a method of treating a subject having a deficiency of ENPP1 protein, the method comprising administering to the subject a therapeutically effective amount of a viral vector encoding a recombinant ENPP1 or ENPP3 polypeptide, thereby treating the subject.

[0061] In some embodiments of any of the methods described herein, the disease or disorder or the deficiency of the ENPP1 protein is associated with a loss-of-function mutation in the NPP1 gene or a loss-of-function mutation in the ABCC6 gene in the subject.

[0062] In some embodiments of any of the methods described herein, the viral vector encodes a recombinant ENPP1 polypeptide.

[0063] In some embodiments of any of the methods described herein, the viral vector encodes a recombinant ENPP3 polypeptide.

[0064] In some embodiments of any of the methods described herein, the viral vector encodes a recombinant ENPP1-Fc fusion polypeptide or a recombinant ENPP1-albumin fusion polypeptide.

[0065] In some embodiments of any of the methods described herein, the viral vector encodes a recombinant ENPP3-Fc fusion polypeptide or a recombinant ENPP3-albumin fusion polypeptide.

[0066] In some embodiments of any of the methods described herein, the viral vector encodes a recombinant polypeptide comprising a signal peptide fused to ENPP1 or ENPP3.

[0067] In some embodiments of any of the methods described herein, the vector encodes ENPP1-Fc or ENPP1-albumin.

[0068] In some embodiments of any of the methods described herein, the signal peptide is an azurocidin signal peptide, an NPP2 signal peptide, or an NPP7 signal peptide.

[0069] In some embodiments of any of the methods described herein, the viral vector is an adeno-associated virus vector, a herpes simplex vector, an alphavirus vector, or a lentivirus vector.

[0070] In some embodiments of any of the methods described herein, the serotype of the adeno-associated virus vector (AAV) is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV-rh74.

[0071] In some embodiments of any of the methods described herein, the viral vector is an adeno-associated virus (AAV) vector encoding a recombinant polypeptide comprising an azurocidin signal peptide fused to an ENPP1-Fc fusion polypeptide.

[0072] In some embodiments of any of the methods described herein, the AAV vector encoding the ENPP1-Fc fusion polypeptide is administered to a subject at a dosage of 1×10 12 ~1×10 15 vg / kg.

[0073] In some embodiments of any of the methods described herein, the dosage is 1×10 13 ~1×10 14 vg / kg.

[0074] In some embodiments of any of the methods described herein, the AAV vector is administered to a subject at a dosage of 5×10 11 ~5×10 15 vg / kg.

[0075] In some embodiments of any of the methods described herein, the vector is an AAV vector encoding ENPP1-Fc, at 1×10 12 ~1×10 15 vg / kg and is administered to a subject at a dosage of vg / kg.

[0076] In some embodiments of any of the aforementioned methods, administration of the AAV vector encoding the ENPP1-Fc polypeptide to a subject results in a dose-dependent increase in plasma pyrophosphate (PPi) and a dose-dependent increase in plasma ENPP1 concentration in the subject.

[0077] In another aspect, the present disclosure features a viral vector comprising a polynucleotide sequence encoding a polypeptide comprising the catalytic domain of an ENPP1 or ENPP3 protein.

[0078] In some embodiments of any of the viral vectors described herein, the polypeptide sequence comprises the extracellular domain of an ENPP1 or ENPP3 protein.

[0079] In some embodiments of any of the viral vectors described herein, the polypeptide comprises the transmembrane domain of an ENPP1 or ENPP3 protein.

[0080] In some embodiments of any of the viral vectors described herein, the polypeptide comprises the nuclease domain of an ENPP1 or ENPP3 protein.

[0081] In some embodiments of any of the viral vectors described herein, the polypeptide comprises residues 99-925 (Pro Ser Cys~Gln Glu Asp) of SEQ ID NO: 1.

[0082] In some embodiments of any of the viral vectors described herein, the polypeptide comprises residues 31-875 (Leu Leu Val~Thr Thr Ile) of SEQ ID NO: 7.

[0083] In some embodiments of any of the viral vectors described herein, the polypeptide comprises residues 191 - 591 (Val Glu Glu - Gly Ser Leu) of SEQ ID NO: 1.

[0084] In some embodiments of any of the viral vectors described herein, the polypeptide comprises residues 140 - 510 (Leu Glu Glu - Glu Val Glu) of SEQ ID NO: 7.

[0085] In some embodiments of any of the viral vectors described herein, the polypeptide comprises residues 1 - 827 (Pro Ser Cys - Gln Glu Asp) of SEQ ID NO: 92.

[0086] In some embodiments of any of the viral vectors described herein, the polypeptide comprises residues 1 - 833 (Phe Thr Ala - Gln Glu Asp) of SEQ ID NO: 89 or residues 1 - 830 (Gly Leu Lys - Gln Glu Asp) of SEQ ID NO: 91.

[0087] In some embodiments of any of the viral vectors described herein, the viral vector is not an insect viral vector.

[0088] In some embodiments of any of the viral vectors described herein, the viral vector infects, or is capable of infecting, mammalian cells.

[0089] In some embodiments of any of the viral vectors described herein, the polynucleotide sequence encodes a promoter sequence.

[0090] In some embodiments of any of the viral vectors described herein, the promoter is a liver - specific promoter.

[0091] In some embodiments of any of the viral vectors described herein, the liver-specific promoter is selected from the group consisting of the albumin promoter, the phosphoenolpyruvate carboxykinase (PEPCK) promoter, and the alpha-1-antitrypsin promoter.

[0092] In some embodiments of any of the viral vectors described herein, the polynucleotide sequence comprises a nucleotide sequence encoding a polyadenylation signal.

[0093] In some embodiments of any of the viral vectors described herein, the polynucleotide encodes a signal peptide that is amino-terminal to the nucleotide sequence encoding the ENPP1 or ENPP3 protein.

[0094] In some embodiments of any of the viral vectors described herein, the signal peptide is the azurocidin signal peptide.

[0095] In some embodiments of any of the viral vectors described herein, the viral vector is an adeno-associated virus (AAV) vector.

[0096] In some embodiments of any of the viral vectors described herein, the AAV vector has a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV-rh74.

[0097] In some embodiments of any of the viral vectors described herein, the polynucleotide sequence encodes the azurocidin signal peptide fused to the ENPP1 or the azurocidin signal peptide fused to the ENPP3 and the ENPP1 or the ENPP3 fused to an Fc polypeptide, forming azurocidin signal peptide-ENPP1-Fc or azurocidin signal peptide-ENPP3-Fc, respectively, in the order from the amino terminus to the carboxy terminus.

[0098] In some embodiments of any of the viral vectors described herein, the polynucleotide sequence encodes the azurocidin signal peptide fused to the ENPP1 or the azurocidin signal peptide fused to the ENPP3 and the ENPP1 or the ENPP3 fused to human serum albumin, forming azurocidin signal peptide-ENPP1-albumin or azurocidin signal peptide-ENPP3-albumin, respectively, in the order from the amino terminus to the carboxy terminus.

[0099] In some embodiments of any of the viral vectors described herein, the polypeptide is a fusion protein comprising (i) an ENPP1 protein or an ENPP3 protein and (ii) a half-life extension domain.

[0100] In some embodiments of any of the viral vectors described herein, the half-life extension domain is an IgG Fc domain or a functional fragment thereof that can extend the half-life of the polypeptide in a mammal as compared to the half-life of the polypeptide in the absence of the IgG Fc domain or its functional fragment.

[0101] In some embodiments of any of the viral vectors described herein, the half-life extension domain is an albumin domain or a functional fragment thereof that can extend the half-life of a polypeptide in a mammal as compared to the half-life of the polypeptide in the absence of the albumin domain or its functional fragment.

[0102] In some embodiments of any of the viral vectors described herein, the half-life extension domain is carboxy-terminal to the NPP1 or ENPP3 protein in the E fusion protein.

[0103] In some embodiments of any of the viral vectors described herein, the IgG Fc domain comprises the amino acid sequence set forth in SEQ ID NO: 34.

[0104] In some embodiments of any of the viral vectors described herein, the albumin domain comprises the amino acid sequence set forth in SEQ ID NO: 35.

[0105] In some embodiments of any of the viral vectors described herein, the polynucleotide encodes a linker sequence.

[0106] In some embodiments of any of the viral vectors described herein, the linker sequence is selected from the group consisting of SEQ ID NOs: 57-88.

[0107] In some embodiments of any of the viral vectors described herein, the linker sequence connects the ENPP1 or ENPP3 protein of the fusion protein to the half-life extension domain.

[0108] In some embodiments of any of the viral vectors described herein, the polypeptide comprises the amino acid sequences set forth in SEQ ID NOs: 89, 91, 92, and 93.

[0109] In another aspect, the present disclosure provides a method for producing a recombinant viral vector, comprising the following steps: i. providing a cell or population of cells comprising a polynucleotide encoding a polypeptide comprising the catalytic domain of an ENPP1 or ENPP3 protein, wherein the cell expresses a viral protein essential for packaging and / or assembly of the polynucleotide into a recombinant viral vector; and ii. maintaining the cell or population of cells under conditions appropriate for packaging and assembly of the recombinant viral vector comprising the polynucleotide.

[0110] In some embodiments of any of the methods described herein, the mammalian cell is a rodent cell or a human cell.

[0111] In some embodiments of any of the methods described herein, the viral vector is any one of the viral vectors described herein.

[0112] In some embodiments, any of the methods described herein can further comprise purifying the recombinant viral vector from the cell or population of cells or from the medium in which the cell or population of cells was maintained.

[0113] In another aspect, the present disclosure features a recombinant viral vector purified from a method for producing and / or purifying the recombinant viral vector described herein.

[0114] In another aspect, the present disclosure provides a pharmaceutical composition comprising any one of the viral vectors or recombinant viral vectors described herein and a pharmaceutically acceptable carrier.

[0115] In yet another aspect, the present disclosure provides a method of preventing or reducing disease progression in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of any one of the pharmaceutical compositions described herein, thereby preventing or reducing the progression of the disease or disorder.

[0116] In some embodiments of any of the methods described herein, the mammal is a human.

[0117] In some embodiments of any of the methods described herein, the disease is selected from the group consisting of X-linked hypophosphatemia (XLH), chronic kidney disease (CKD), mineral bone disorder (MBD), vascular calcification, pathologic calcification of soft tissue, pathologic ossification of soft tissue, PXE, generalized arterial calcification of infancy (GACI), and ossification of the posterior longitudinal ligament (OPLL).

[0118] In another aspect, the present disclosure provides a method of treating or preventing a disease or disorder of pathologic calcification or pathologic ossification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any one of the viral vectors or pharmaceutical compositions described herein, thereby treating or preventing the disease or disorder.

[0119] In another aspect, the present disclosure features a method of treating a subject having a deficiency of ENPP1 protein, the method comprising administering to the subject a therapeutically effective amount of any one of the viral vectors or pharmaceutical compositions described herein, thereby treating the subject.

[0120] In some embodiments of any of the methods described herein, the disease or disorder or the deficiency of the ENPP1 protein is associated with a loss-of-function mutation in the NPP1 gene or a loss-of-function mutation in the ABCC6 gene in the subject.

[0121] In some embodiments of any of the methods described herein, the viral vector or pharmaceutical composition is administered at a dosage of 1×10 12 ~1×10 15 vg / subject or kg of the mammal.

[0122] In some embodiments of any of the methods described herein, the viral vector or pharmaceutical composition is administered at a dosage of 1×10 13 ~1×10 14 vg / subject or kg of the mammal.

[0123] In some embodiments of any of the methods described herein, the viral vector or pharmaceutical composition is administered at a dosage of 5×10 11 ~5×10 15 vg / subject or kg of the mammal.

[0124] In some embodiments of any of the methods described herein, the viral vector or pharmaceutical composition is administered at a dosage of 1×10 12 ~1×10 15 vg / subject or kg of the mammal.

[0125] In some embodiments of any of the methods described herein, the administration of the viral vector or pharmaceutical composition to the subject or mammal increases plasma pyrophosphate (PPi) and / or plasma ENPP1 or ENPP3 concentration in the subject or mammal.

[0126] In some embodiments, any of the aforementioned methods can further include detecting or measuring one or more of the following parameters in a biological sample obtained from the subject or mammal: (i) the concentration of pyrophosphate, (ii) the expression level of ENPP1 or ENPP3, and (iii) the enzyme activity of ENPP1 or ENPP3.

[0127] In some embodiments of any of the methods described herein, the detection or measurement is performed before administering the viral vector or pharmaceutical composition.

Brief Description of the Drawings

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Figure 9

Modes for Carrying Out the Invention

[0129] The present invention relates to the delivery of a nucleic acid encoding mammalian ENPP1 or mammalian ENPP3 to a mammal having a deficiency of ENPP1 or ENPP3.

[0130] Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the exemplary methods and materials are described. As used herein, each of the following terms has the meaning associated with it in this section.

[0131] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. By way of example, "an element" means one element or more than one element.

[0132] The following notations are applied to this disclosure for clarity. In any case, any teaching in this specification that does not conform to this convention remains part of this disclosure and can be fully understood in consideration of the context in which the teaching is disclosed. Protein symbols are disclosed in non-italicized capital letters. As a non-limiting example, "ENPP1" refers to a protein. In certain embodiments, when the protein is a human protein, "h" is used before the protein symbol. In other embodiments, when the protein is a mouse protein, "m" is used before the symbol. Human ENPP1 is referred to as "hENPP1", and mouse ENPP1 is referred to as "mENPP1". Human gene symbols are disclosed in italicized capital letters. As a non-limiting example, the human gene corresponding to the protein hENPP1 is ENPP1. Mouse gene symbols are disclosed with the first letter in capital and the remaining letters in lowercase; further, mouse gene symbols are italicized. As a non-limiting example, the mouse gene that produces the protein mEnpp1 is Enpp1. The notation for gene mutations is shown in capital text. "Human ENPP1": Human NPP1 (NCBI accession NP_006199 / Uniprot-Swissprot P22413) "Soluble human ENPP1": Residues 96 - 925 of NCBI accession NP_006199 "Human ENPP3": Human NPP3 (UniProtKB / Swiss-Prot: O14638.2) "Soluble human ENPP3": Residues 49 - 875 of UniProtKB / Swiss-Prot: O14638.2

[0133] "Reduction of calcification": As used herein, reduction of calcification is observed by using non-invasive methods such as X-ray, micro-CT and MRI. Reduction of calcification is also 99m Tc-pyrophosphate ( 99mIt is inferred by using radioactive imaging with the uptake of PYP). The presence of calcification in mice was evaluated by necropsy of tissue sections obtained from the heart, aorta, and kidneys using micro-computed tomography (CT) scans and dyes such as hematoxylin-eosin (H&E) and alizarin red according to the protocol established by Braddock et al. (Nature Communications volume 6, Article number: 10006 (2015)).

[0134] "Enzymatically active" for ENPP1 or ENPP3 is defined as having ATP hydrolysis activity to AMP and PPi and / or AP3a hydrolysis to ATP. It has substrate binding activity.

[0135] ATP hydrolysis activity can be determined as follows.

[0136] ATP hydrolysis activity of NPP1 NPP1 readily hydrolyzes ATP to AMP and PPi. The steady-state Michaelis-Menten enzyme constants of NPP1 are determined using ATP as the substrate. HPLC analysis of the enzyme reaction can show that NPP1 cleaves ATP, and the identity of the substrates and products of the reaction is confirmed by using ATP, AMP, and ADP standards. The ATP substrate degrades over time in the presence of NPP1 with the accumulation of the enzymatic product AMP. Initial rate velocities for NPP1 in the presence of ATP are derived using various concentrations of the ATP substrate, and the data are fitted to a curve to derive the enzyme rate constants. At physiological pH, the kinetic rate constants of NPP1 are Km = 144 μM and kcat t = 7.8 s -1 is.

[0137] ATP hydrolysis activity of NPP3 The enzymatic activity of NPP3 was measured using pNP-TMP or ATP as substrates. The NPP3 protein was incubated at 37 °C in the presence of either 100 mM Tris-HCl at pH 8.9 and 5 mM pNP-TMP or 50 μM [γ-32P]ATP. Hydrolysis of pNP-TMP was stopped by 10-fold dilution into 3% (w / v) trichloroacetic acid. Subsequently, the reaction mixture was neutralized with 60 μl of 5 N NaOH, and the formed p-nitrophenol (pNP) was quantified by colorimetric measurement at 405 nm. Hydrolysis of ATP was stopped by adding 100 mM EDTA. 1 μl of the reaction mixture was analyzed by thin layer chromatography on a polyethyleneimine cellulose plate (Merck). Nucleotides and degradation products were separated by ascending chromatography in 750 mM KH2PO4 at pH 3.0. Radioactive spots were visualized by autoradiography. According to Blytt et al. (H.J. Blytt, J.E. Brotherton, L. Butler Anal. Biochem. 147 (1985), pp. 517-520), with some modifications (R. Gijsbers, H. Ceulemans, W. Stalmans, M. Bollen J. Biol. Chem., 276 (2001), pp. 1361-1368), the nucleotidylated intermediates formed during hydrolysis of 50 μM [α-32P]ATP were trapped. After SDS-PAGE, the trapped intermediates were visualized by autoradiography. Bis-pNPP and pNPP were also tested as substrates for NPP3. The NPP3 isoform was incubated at 37 °C for 2.5 h in either 100 mM Tris-HCl at pH 8.9 and 5 mM of bis-pNPP or pNPP. Subsequently, the formed pNP was quantified by colorimetric measurement at 405 nm (Gijsbers R1, Aoki J, Arai H, Bollen M, FEBS Lett. 2003 Mar 13;538(1-3):60-4.). At physiological pH, NPP3 has a kcat value of approximately 2.59 (±0.04) s -1 and a Km (<8 μM) value similar to ENPP1 (WO2017 / 087936).

[0138] HPLC protocol The HPLC protocol used to measure ATP cleavage by NPP1 and for product identification is modified from the literature (Stocchi et al., 1985, Anal. Biochem. 146:118 - 124). 50 mM Tris pH 8.0, 140 mM NaCl, 5 mM KCl, 1 mM MgCl 2 and 1 mM CaCl 2 Reactions containing various concentrations of ATP in buffer are initiated by adding 0.2 - 1 μM NPP1, and the reaction is quenched at various time points with an equivalent of 3 M formic acid or 0.5 N KOH and re - acidified to pH 6 with glacial acetic acid. The quenched reaction solutions are systematically diluted and loaded onto an HPLC system (Waters, Milford Mass.), and the substrate and product are monitored by UV absorbance at 254 or 259 nm. Using a 0% - 10% (or 20%) methanol gradient, with a 15 mM ammonium acetate pH 6.0 solution, the substrate and product are separated on a C18, 5 μm 250×4.6 mm HPLC column (Higgins Analytical, Mountain View, Calif.). The product and substrate are quantified by integrating their corresponding peak and the formula:

Number

[0139] "Pathological calcification": As used herein, this term refers to the abnormal deposition of calcium salts that hardens soft tissues of the body, secretory and excretory ducts. There are two types: dystrophic calcification that occurs in dying and dead tissues, and metastatic calcification (hypercalcemia) that raises extracellular levels of calcium beyond the homeostatic capacity of cells and tissues. Calcification can involve cells as well as extracellular matrix components such as collagen of the basement membrane and elastic fibers of the arterial wall. Some examples of tissues prone to calcification include the gastric mucosa - the inner epithelial layer of the stomach, the kidneys and lungs, the cornea, systemic arteries and pulmonary veins.

[0140] "Pathological ossification": As used herein, this term refers to a pathological condition in which bone forms in tissues that are not part of the skeletal system and connective tissues that do not normally manifest osteogenic properties. Ossification is classified into three types according to the nature of the affected tissue or organ. Endochondral ossification occurs in cartilage and is the ossification that replaces cartilage. Intramembranous ossification occurs in connective tissue and is the ossification of bone that replaces connective tissue. Heterotopic ossification, usually the formation of bone in soft body structures, is also called secondary dystrophic ossification.

[0141] "Deficiency" of NPP1 refers to a state in which a subject has only 5% - 10% or less of the normal level of NPP1 in plasma. The normal level of NPP1 in healthy human subjects is approximately between 10 - 30 ng / ml (Am J Pathol. 2001 Feb;158(2):543 - 554.).

[0142] "Low" level of PPi refers to a state in which a subject has only 2% - 5% or less of the normal level of plasma pyrophosphate (PPi). The normal level of plasma PPi in healthy human subjects is approximately 1.8 - 2.6 μM (Arthritis and Rheumatism, Vol. 22, No. 8 (August 1979)).

[0143] "Heterotopic calcification" refers to a condition characterized by the pathological deposition of calcium salts in tissues or bone growth in soft tissues.

[0144] "Soft tissue heterotopic calcification" refers to inappropriate biomineralization that occurs in soft tissue and leads to the loss of soft tissue compliance, typically composed of calcium phosphate, hydroxyapatite, calcium oxalate, and octacalcium phosphate. "Arterial calcification" refers to heterotopic calcification that occurs in arteries and heart valves and leads to arterial stiffening and / or narrowing. Arterial calcification is associated with increased atherosclerotic plaque burden and risk of myocardial infarction, increased ischemic episodes in peripheral vascular disease, and increased risk of dissection after angioplasty.

[0145] "Venous calcification" refers to heterotopic calcification that occurs in veins, reducing venous elasticity, restricting blood flow, and potentially leading to increased blood pressure and coronary vascular deficits.

[0146] "Vascular calcification" refers to the pathological deposition of minerals in the vascular system. It has various forms, including intimal and medial calcification, and can also be found in heart valves. Vascular calcification is associated with atherosclerosis, diabetes, certain genetic conditions, and kidney disease, particularly CKD. Patients with vascular calcification are at higher risk of adverse cardiovascular events. Vascular calcification affects a wide variety of patients. Idiopathic infantile arterial calcification is a rare form of vascular calcification in which the arteries of newborns become calcified.

[0147] "Brain calcification" (BC) refers to a non-specific neuropathology in which deposits of calcium and other minerals occur in the blood vessel walls and parenchymal tissue, leading to neuron death and gliosis. Brain calcification is often associated with various chronic and acute brain disorders, including Down syndrome, Lewy body disease, Alzheimer's disease, Parkinson's disease, vascular dementia, brain tumors, and various endocrinological conditions.

[0148] Calcification of heart tissue refers to the accumulation of calcium (possibly including other minerals) deposits in heart tissue, such as aorta tissue and coronary vascular tissue.

[0149] "Chronic kidney disease (CKD)", as used herein, refers to an abnormal structure or function of the kidneys that persists for more than three months and affects health. Generally, in most chronic kidney diseases, the excretory, endocrine, and metabolic functions decline simultaneously. Cardiovascular disease is the most common cause of death in patients with chronic kidney disease (CKD), and vascular calcification is one of the strongest predictors of cardiovascular risk. As kidney function declines, the prevalence of vascular calcification increases, and calcification occurs several years earlier in CKD patients compared to the general population. By preventing, reducing, and / or reversing vascular calcification, the survival time of patients with CKD can be increased.

[0150] Clinical symptoms of chronic kidney disease include itching, muscle cramps, nausea, loss of appetite, swelling of the feet and ankles, insomnia, and shortness of breath on exertion. Chronic kidney disease tends to progress to end-stage renal disease (ESRD) if left untreated. General symptoms of ESRD include anuria, fatigue, lethargy, weight loss, bone pain, changes in skin color, frequent formation of bruises, and edema of the outer extremities such as fingers, toes, hands, and feet. Calciphylaxis or uremic arteriolopathy (CUA) is a condition in which calcium accumulates inside the blood vessels of fat and skin. A subset of patients with ESRD may also develop calciphylaxis. General symptoms of calciphylaxis include large purple net-like patterns on the skin, open sores with non-healing dark brown crusts, ulcerated deep painful masses, skin lesions on the lower extremities or areas with higher fat content, such as the thighs, breasts, buttocks, and abdomen. People with calciphylaxis may have higher blood calcium (hypercalcemia) and phosphate (hyperphosphatemia) levels than normal. They may also have symptoms of hyperparathyroidism. Hyperparathyroidism occurs when the parathyroid glands produce excessive parathyroid hormone (PTH). Reduced plasma pyrophosphate (PPi) levels are also present in vascular calcification associated with end-stage renal disease (ESRD).

[0151] Vascular calcification associated with ESRD contributes to poor outcomes by increasing pulse pressure, causing or worsening hypertension, and inducing or enhancing myocardial infarction and stroke. Most patients with ESRD do not die of renal failure but die of cardiovascular complications of ESRD, and it is important to note that many very young patients with ESRD on dialysis have coronary artery calcification. The histological subtype of vascular calcification associated with CKD is known as Monckeberg sclerosis, which is a form of arteriosclerosis in which calcium deposition is seen in the muscular layer of the medial vascular wall. This form of calcification is histologically different from the intimal or neointimal vascular wall calcification commonly observed in atherosclerosis but is identical to the vascular calcification observed in human CKD patients and rodent models of the diseases described herein.

[0152] "Generalized arterial calcification of infancy (GACI)" (also known as IACI), as used herein, refers to a disorder affecting the circulatory system that becomes apparent prenatally or within the first few months of life. It is characterized by abnormal accumulation (calcification) of mineral calcium in the walls of the blood vessels (arteries) that carry blood from the heart to the rest of the body. Calcification often occurs along with thickening of the inner layer (intima) of the arterial wall. These changes lead to narrowing (stenosis) and stiffening of the arteries, which causes the heart to work harder to pump blood. As a result, heart failure can occur in affected individuals, along with symptoms and signs including shortness of breath, fluid accumulation (edema) in the limbs, a bluish tint to the skin or lips (cyanosis), severe high blood pressure (hypertension), and an enlarged heart (cardiomegaly). People with GACI may also have calcification in other organs and tissues, particularly around the joints. In addition, they may have hearing loss or softening and weakening of the bones, known as osteomalacia or rickets.

[0153] Generalized arterial calcification (GACI) or idiopathic infantile arterial calcification (IIAC) is characterized by abnormal accumulation (calcification) of mineral calcium in the walls of blood vessels (arteries) that carry blood from the heart to other parts of the body. Calcification often occurs along with thickening of the inner layer (intima) of the arterial wall. These changes lead to narrowing (stenosis) and stiffening of the arteries, which causes the heart to work harder to pump blood. As a result, symptoms and signs including shortness of breath, fluid accumulation (edema) in the limbs, a bluish appearance of the skin or lips (cyanosis), severe high blood pressure (hypertension), and an enlarged heart (cardiomegaly) may occur in affected individuals, and heart failure may develop.

[0154] "Arterial calcification" or "vascular calcification" or "arterial sclerosis", as used herein, refers to a process characterized by thickening of the muscular arterial wall and loss of elasticity. The thickening and loss of elasticity occur at two different sites, namely, in the intima and media layers of the vascular structure (medial vascular calcification). Intimal calcification is associated with atherosclerotic plaques, and medial calcification is characteristic of vascular stiffness and arteriosclerosis. This reduces the elasticity of the arteries and increases the incidence and mortality trends due to dysfunction of the hemodynamics of the cardiovascular system.

[0155] "Mineral bone disorder (MBD)", as used herein, refers to a disorder characterized by abnormal hormone levels that destabilize calcium and phosphorus levels in a person's blood. Mineral and bone disorders generally occur in people with CKD and affect most people with end-stage renal disease undergoing dialysis.

[0156] Osteopenia is a bone condition characterized by a decrease in bone density that leads to weakened bones and an increased risk of fractures. Osteomalacia is a bone disorder characterized by a decrease in the mineralization of newly formed bone. Osteomalacia is caused by severe vitamin D deficiency (which can be nutritional or caused by a genetic syndrome) and conditions that cause very low blood phosphate levels. Both osteomalacia and osteopenia increase the risk of bone fractures. Symptoms of osteomalacia include bone pain and muscle weakness, bone tenderness, difficulty walking, and muscle spasms.

[0157] "Age-related osteopenia," as used herein, refers to a condition in which bone mineral content is lower than normal. Generally, patients with osteopenia have a bone mineral content T-score of -1.0 to -2.5. Osteopenia, if left untreated, progresses to osteoporosis, in which bones become extremely brittle and prone to breaking.

[0158] "Ossification of the posterior longitudinal ligament (OPLL)," as used herein, refers to a state of excessive ossification (excessive bone growth) that results in heterotopic calcification of the posterior longitudinal ligament. The posterior longitudinal ligament connects and stabilizes the bones of the spinal column. Hypertrophied or calcified ligaments can compress the spinal cord and result in myelopathy. Symptoms of myelopathy include difficulty walking and difficulty controlling the bowel and bladder. OPLL can also cause radiculopathy or compression of the nerve roots. Symptoms of cervical radiculopathy include pain, tingling, or numbness in the neck, shoulder, arm, or hand.

[0159] The clinical symptoms and signs caused by OPLL are classified as follows: (1) myelopathy or spinal cord lesions with motor and sensory disturbances in the upper and lower extremities, spasticity, and bladder dysfunction; (2) cervical radiculopathy with pain and sensory disturbances in the upper extremities; and (3) axial discomfort with pain and stiffness around the neck. The most common symptoms of early OPLL include abnormal sensations and tingling in the hands, as well as clumsiness. As the neuropathy progresses, symptoms in the lower extremities, such as gait disturbances, may appear. OPLL is detected by lateral plain radiographs, and the diagnosis and morphological details of cervical OPLL are clearly shown by magnetic resonance imaging (MRI) and computed tomography (CT).

[0160] "Pseudoxanthoma elasticum (PXE)" as used herein refers to a progressive disorder characterized by the accumulation (mineralization) of calcium and other mineral deposits in elastic fibers. Elastic fibers are components of connective tissue that give structure strength and mobility throughout the body. In PXE, mineralization can affect elastic fibers in the skin, eyes, and blood vessels, as well as in other areas such as the gastrointestinal tract, although to a lesser extent. People with PXE may have yellowish raised bumps called papules in areas of the skin that are touched when the neck, underarms, and joints are bent. Mineralization of the blood vessels (arteries) that carry blood from the heart to the rest of the body can cause other signs and symptoms of PXE. For example, people with this condition may develop a condition called claudication, which is characterized by narrowing of the arteries (arteriosclerosis) or muscle cramps and pain during exercise due to a decrease in blood flow to the arms and legs.

[0161] Pseudoxanthoma elasticum (PXE), also known as Grönblad-Strandberg syndrome, is a hereditary disorder that causes fragmentation and mineralization of elastic fibers in several tissues. The most common problems occur in the skin and eyes, and later in the blood vessels in the form of premature atherosclerotic disease. PXE is caused by autosomal recessive mutations in the ABCC6 gene on the short arm of chromosome 16 (16p13.1). In some cases, some infants survive through GACI, grow into adults, and eventually develop pseudoxanthoma elasticum (PXE). PXE is characterized by the accumulation of calcium and other minerals (mineralization) in elastic fibers, which are components of connective tissue. Connective tissue gives structure strength and mobility throughout the body. Distinctive features specific to PXE, which also occurs in GACI, include yellowish raised bumps called papules in the armpits and other areas of the skin that are touched when the joints bend (flexor areas); arterial stenosis, and an abnormality called retinal pigment streaks (retinal hemorrhage) that affects the tissue behind the eye and is detected during an eye examination.

[0162] "End-stage renal disease (ESRD): As used herein, this term refers to an advanced stage of chronic kidney disease in which the patient's kidneys are no longer functional. Common symptoms include fatigue associated with anemia (low blood iron), loss of appetite, nausea, vomiting, abnormal clinical test values including elevated potassium, abnormal hormones related to bone health, elevated phosphorus and / or decreased calcium, high blood pressure (hypertension), swelling in the hands / feet / eyes / lumbar region (sacrum), and shortness of breath.

[0163] "Uremic arteriosclerosis (CUA)" or "calciphylaxis," as used herein, refers to a condition with high morbidity and mortality rates seen in patients with kidney disease, particularly those with end-stage renal disease (ESRD). This is characterized by the calcification of small blood vessels located deeper within adipose tissue and the skin, which leads to blood clots and skin cell death due to reduced blood flow caused by excessive calcification.

[0164] "Hypophosphatemic rickets", as used herein, refers to a disorder in which bones become soft and bend easily due to low levels of phosphate in the blood. Symptoms usually begin in early childhood and can range in severity from knock knees, bone deformities; bone pain; joint pain; insufficient bone growth; and short stature.

[0165] "Hereditary hypophosphatemic rickets", as used herein, refers to a disorder (hypophosphatemia) associated with low levels of phosphate in the blood. Phosphate is a mineral essential for the normal formation of bones and teeth. Most commonly, this is caused by mutations in the PHEX gene. Other genes involved in this condition include the CLCN5, DMP1, ENPP1, FGF23, and SLC34A3 genes. Other signs and symptoms of hereditary hypophosphatemic rickets can include premature fusion of the skull (craniosynostosis) and dental abnormalities. This disorder can also cause abnormal bone growth (enthesopathy) of the ligaments and tendons attached to the joints. In adults, hypophosphatemia is characterized by softening of the bones, known as osteomalacia. Another rare form of this disorder is known as hereditary hypophosphatemic rickets with hypercalciuria (HHRH), which, in addition to hypophosphatemia, is characterized by high levels of calcium excretion in the urine (hypercalciuria).

[0166] "X-linked hypophosphatemia (XLH)", as used herein, is a term, also known as X-linked dominant hypophosphatemic rickets or X-linked vitamin D-resistant rickets, that refers to the X-linked dominant form of rickets (or osteomalacia), which differs from most cases of rickets in that vitamin D supplementation does not cure it. This can cause bone deformities including short stature and knock knees (genu valgum). This is associated with a mutated sequence of the PHEX gene (Xp.22) and subsequent inactivation of the PHEX protein.

[0167] "Autosomal recessive hypophosphatemic rickets type 2 (ARHR2)", as used herein, refers to a hereditary renal phosphate wasting disorder hypophosphatemia characterized by rickets and / or osteomalacia and growth retardation. Autosomal recessive hypophosphatemic rickets type 2 (ARHR2) is caused by homozygous loss-of-function mutations in the ENPP1 gene.

[0168] "Autosomal dominant hypophosphatemic rickets (ADHR)", as used herein, refers to a rare genetic disorder in which excessive loss of phosphate in the urine leads to poorly formed bones (rickets), bone pain, and dental abscesses. ADHR is caused by mutations in fibroblast growth factor 23 (FGF23). ADHR is characterized by impaired bone mineralization, rickets and / or osteomalacia, suppression of the levels of calcitriol (1,25-dihydroxyvitamin D3), renal phosphate wasting, and hypophosphatemia. Mutations in FGF23 make the protein more stable and impossible to be cleaved by proteases, resulting in enhanced biological activity of FGF23. The enhanced activity of the FGF23 mutant reduces the expression of the sodium-phosphate cotransporters, NPT2a and NPT2c, at the apical surface of proximal renal tubular cells, leading to renal phosphate wasting.

[0169] Hypophosphatemic rickets (formerly called vitamin D-resistant rickets) is a disorder in which low levels of phosphate in the blood cause bones to become so soft and painful that they easily bend. Symptoms can include bowing of the legs and other bone deformities; bone pain; joint pain; inadequate bone growth; and short stature. Some affected infants develop premature closure of the spaces between the skull bones, leading to craniosynostosis. Most patients show abnormalities in calcium-phosphate metabolism, dental enamel, delayed tooth eruption, and an elongated head (dolichocephaly).

[0170] As used interchangeably herein, the terms "adeno-associated virus vector", "AAV vector", "adeno-associated virus", "AAV virus", "AAV virion", "AAV virus particle", and "AAV particle" refer to virus particles composed of at least one AAV capsid protein (preferably, all of the capsid proteins of a particular AAV serotype) and a recombinant viral genome encapsulated by the capsid. The particles contain a recombinant viral genome having a heterologous polynucleotide that includes a sequence encoding a transcriptional control region that includes at least a human ENPP1 or human ENPP3 or a functionally equivalent variant thereof and a promoter adjacent to the AAV inverted terminal repeat. The particles are typically referred to as "AAV vector particles" or "AAV vectors".

[0171] As used herein, the term "vector" means a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, the vector is a plasmid, i.e., a circular double-stranded DNA loop into which additional DNA segments can be ligated. In some embodiments, the vector is a viral vector into which additional nucleotide sequences can be ligated in the viral genome. In some embodiments, the vector is capable of autonomous replication in the introduced host cell (e.g., a bacterial vector having a bacterial origin of replication and an episomal mammalian vector). In other embodiments, the vector (e.g., a non-episomal mammalian vector) is integrated into the genome of the host cell upon introduction into the host cell and is thereby replicated with the host genome. Further, a particular vector (expression vector) can direct the expression of a gene operably linked thereto.

[0172] As used herein, the term "recombinant host cell" (or simply "host cell") means a cell into which an exogenous nucleic acid and / or recombinant vector has been introduced as used herein. It should be understood that "recombinant host cell" and "host cell" mean not only a particular target cell but also the progeny of such a cell. Such progeny may not actually be identical to the parent cell because a particular modification may occur in later generations due to either mutation or environmental influences, but are still included within the scope of the term "host cell" as used herein.

[0173] As used herein, the term "recombinant viral genome" refers to an AAV genome into which at least one foreign expression cassette polynucleotide has been inserted into the naturally occurring AAV genome. The AAV genome according to the present invention typically includes cis-acting 5' and 3' inverted terminal repeat sequences (ITRs) and an expression cassette.

[0174] As used herein, the term "expression cassette" refers to a recombinant or synthetically produced nucleic acid construct having a series of specific nucleic acid elements that enable transcription of a particular nucleic acid in a target cell. The expression cassette of the recombinant viral genome of the AAV vector according to the present invention includes a transcriptional control region operably linked to a nucleotide sequence encoding ENPP1 or ENPP3 or a functionally equivalent variant thereof.

[0175] As used herein, the term "transcriptional control region" refers to a nucleic acid fragment capable of regulating the expression of one or more genes. The transcriptional control region according to the present invention includes a promoter and optionally an enhancer.

[0176] As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more polynucleotides located upstream of a polynucleotide sequence(s), and that is structurally defined by the presence of a binding site for DNA-dependent RNA polymerase, a transcription start site, and any other DNA sequences, such as, but not limited to, transcription factor binding sites, repressors, and activator protein binding sites, and any other sequence of nucleotides known in the art that directly or indirectly acts to regulate the amount of transcription from the promoter. Any type of promoter, including inducible promoters, constitutive promoters, and tissue-specific promoters, can be used in the present invention.

[0177] As used herein, the term "enhancer" refers to a DNA sequence element to which a transcription factor binds to increase gene transcription. Examples of enhancers can include, but are not limited to, the RSV enhancer, the CMV enhancer, the HCR enhancer, etc. In another embodiment, the enhancer is a liver-specific enhancer, more preferably a hepatic control region enhancer (HCR).

[0178] As used herein, the term "operably linked" refers to the functional relationship and position of a promoter sequence relative to a polynucleotide of interest (e.g., a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence). Generally, an operably linked promoter is in proximity to the sequence of interest. However, an enhancer need not be in proximity to the sequence of interest to control its expression. In another embodiment, a nucleotide sequence encoding a promoter and ENPP1 or ENPP3 or a functionally equivalent variant thereof.

[0179] The term "therapeutically effective amount" refers to a non-toxic but sufficient amount of a viral vector encoding ENPP1 or ENPP3 to provide the desired biological result. The result can be a reduction and / or alleviation of the signs, symptoms or causes of a disease, or any other desired change in a biological system. For example, a therapeutically effective amount of an AAV vector according to the invention is an amount sufficient to produce.

[0180] The term "Cap protein", as used herein, refers to a polypeptide having at least one functional activity of a native AAV Cap protein (e.g., VP, VP2, VP3). Examples of the functional activities of the Cap protein include the ability to induce capsid formation, facilitate the accumulation of single-stranded DNA, facilitate the packaging of AAV DNA into the capsid (i.e., capsid formation), bind to cell receptors, and facilitate the entry of virions into host cells. In principle, any Cap protein can be used in the present invention.

[0181] The term "capsid", as used herein, refers to the structure in which the viral genome is packaged. The capsid consists of several oligomeric structural subunits made of protein. For example, AAV has an icosahedral capsid formed by the interaction of three capsid proteins: VP1, VP2 and VP3.

[0182] As used herein, the term "Rep protein" refers to a polypeptide having at least one functional activity of a native AAV Rep protein (e.g., Rep40, 52, 68, 78). "Functional activity" of a Rep protein is any activity associated with the physiological function of the protein, such as facilitating DNA replication through recognition, binding, and nicking of the AAV origin of DNA replication, as well as DNA helicase activity. Further functions include modulation of transcription from an AAV (or other heterologous) promoter and site-specific integration of AAV DNA into the host chromosome. In certain embodiments, the AAV rep gene is derived from AAV serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAVrh10; more preferably, from an AAV serotype selected from the group consisting of AAV2, AAV5, AAV7, AAV8, AAV9, AAV10, and AAVrh10.

[0183] As used herein, the expression "viral protein on which AAV depends for replication" refers to a polypeptide that performs a function on which AAV depends for replication (i.e., a "helper function"). Helper functions include, but are not limited to, those functions required for AAV replication, including activation of transcription of AAV genes, stage-specific AAV mRNA splicing, replication of AAV DNA, synthesis of cap expression products, and portions involved in assembly of the AAV capsid. The virus-based accessory function is derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type 1), and vaccinia virus. Helper functions include, but are not limited to, adenovirus E1, E2a, VA, and E4 or herpesvirus UL5, ULB, UL52, and UL29, as well as the herpesvirus polymerase. In another embodiment, the protein on which AAV depends for replication is derived from adenovirus.

[0184] The term "adeno-associated virus ITR" or "AAV ITR", as used herein, refers to the inverted terminal repeats present at both ends of the DNA strands of the adeno-associated virus genome. The ITR sequences are required for efficient propagation of the AAV genome. Another property of these sequences is their ability to form hairpins. This feature contributes to their self-priming, which enables primer-independent synthesis of the second DNA strand. Procedures for modifying these ITR sequences are known in the art (Brown T, "Gene Cloning", Chapman & Hall, London, GB, 1995; Watson R, et al., "Recombinant DNA", 2 nd Ed. Scientific American Books, New York, N.Y., US, 1992; Alberts B, et al., "Molecular Biology of the Cell", Garland Publishing Inc., New York, N.Y., US, 2008; Innis M, et al., Eds., "PCR Protocols. A Guide to Methods and Applications", Academic Press Inc., San Diego, Calif., US, 1990; and Schleef M, Ed., "Plasmid for Therapy and Vaccination", Wiley-VCH Verlag GmbH, Weinheim, Del., 2001).

[0185] The term "tissue-specific" promoter is active only in a particular type of differentiated cell or tissue. Typically, the downstream gene of a tissue-specific promoter is much more highly active in the tissue(s) for which it is specific than in any other tissue. In this case, in any tissue other than the tissue(s) for which it is specific, the promoter may have little or substantially no activity.

[0186] As used herein, the term "skeletal muscle-specific promoter" refers to a nucleic acid sequence that functions as a promoter (i.e., regulates the expression of a selected nucleic acid sequence operably linked to the promoter) and promotes the expression of the selected nucleic acid sequence in specific tissue cells of skeletal muscle. Examples of skeletal muscle-specific promoters include, but are not limited to, the myosin light chain promoter (MLC) and the muscle creatine kinase promoter (MCK).

[0187] As used herein, the term "liver-specific promoter" refers to a nucleic acid sequence that functions as a promoter (i.e., regulates the expression of a selected nucleic acid sequence operably linked thereto) and promotes the expression of the selected nucleic acid sequence in hepatocytes. Typically, a liver-specific promoter is more active in the liver compared to its activity in any other tissue in the body. A liver-specific promoter can be constitutive or inducible. Suitable liver-specific promoters include, but are not limited to, the [alpha]1-antitrypsin (AAT) promoter, the thyroxine-binding globulin promoter, the alpha-fetoprotein promoter, the alcohol dehydrogenase promoter, the factor VIII (FVIII) promoter, the HBV basal core promoter (BCP) and the PreS2 promoter, the albumin promoter, the -460 to 73 bp phosphoenolpyruvate carboxykinase (PEPCK) promoter, the thyroxine-binding globulin (TBG) promoter, the liver control region (HCR)-ApoCII hybrid promoter, the HCR-hAAT hybrid promoter, the AAT promoter combined with the mouse albumin gene enhancer (Ealb) element, the apolipoprotein E promoter, the low density lipoprotein promoter, the pyruvate kinase promoter, the lecithin-cholesterol acyltransferase (LCAT) promoter, the apolipoprotein H (ApoH) promoter, the transferrin promoter, the transthyretin promoter, the alpha-fibrinogen and beta-fibrinogen promoters, the alpha1-antichymotrypsin promoter, the alpha2-HS glycoprotein promoter, the haptoglobin promoter, the ceruloplasmin promoter, the plasminogen promoter, the promoters of complement proteins (CIq, CIr, C2, C3, C4, C5, C6, C8, C9, complement factor I and factor H), the C3 complement activator, and the [alpha]-acid glycoprotein promoter. Additional tissue-specific promoters can be found in the Tissue-Specific Promoter Database, TiProD (Nucleic Acids Research, J4:D104 - D107 (2006)).In another embodiment, the liver-specific promoter is selected from the group consisting of an albumin promoter, a phosphoenolpyruvate carboxykinase (PEPCK) promoter, and an alpha1-antitrypsin promoter, more preferably the alpha1-antitrypsin promoter, and even more preferably the human alpha1-antitrypsin promoter.

[0188] As used herein, the term "inducible promoter" refers to a promoter that is physiologically or developmentally regulated, for example, by the application of a chemical inducer. For example, this can be a tetracycline-inducible promoter, a mifepristone (RU-486)-inducible promoter, and the like.

[0189] As used herein, the term "constitutive promoter" refers to a promoter whose activity is maintained at a relatively constant level in all cells of an organism or during most developmental stages, with little or no regard for cell environmental conditions. In another embodiment, the transcriptional control region enables constitutive expression of ENPP1. Examples of constitutive promoters include, but are not limited to, the Rous sarcoma virus (RSV) LTR promoter of retrovirus (optionally with an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerate kinase (PGK) promoter, and the EF1a promoter (Boshart M, et al., Cell 1985;41:521-530). Preferably, the constitutive promoter is suitable for the expression of ENPP1 in the liver, and examples of constitutive promoters include, but are not limited to, the promoter of hypoxanthine phosphoribosyl transferase (HPTR), the promoter of adenosine deaminase, the promoter of pyruvate kinase, the promoter of β-actin, the elongation factor 1 alpha (EF1) promoter, the phosphoglycerate kinase (PGK) promoter, the ubiquitin (Ubc) promoter, the albumin promoter, and other constitutive promoters. Exemplary viral promoters that function constitutively in cells include, for example, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), or the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445).

[0190] As used herein, the term "polyadenylation signal" refers to a nucleic acid sequence that mediates the attachment of a polyadenine stretch to the 3' end of mRNA. Suitable polyadenylation signals include, but are not limited to, the SV40 early polyadenylation signal, the SV40 late polyadenylation signal, the HSV thymidine kinase polyadenylation signal, the protamine gene polyadenylation signal, the adenovirus 5 E1b polyadenylation signal, the bovine growth hormone polyadenylation signal, the human mutant growth hormone polyadenylation signal, and the like.

[0191] As used herein, the term "nucleotide or nucleic acid sequence" is used interchangeably with "polynucleotide" and refers to any polymeric form of nucleotides of any length. The nucleotide sequence encodes a signal peptide and the ENPP1 protein or a functionally equivalent variant thereof.

[0192] As used herein, the term "signal peptide" refers to a sequence of amino acid residues (ranging in length from 10 to 30 residues) that binds to the amino terminus of a nascent protein of interest during protein translation. The signal peptide is recognized by the signal recognition particle (SRP) and is cleaved by signal peptidase after transport to the endoplasmic reticulum (Lodish et al., 2000, Molecular Cell Biology, 4th edition).

[0193] As used herein, the term "subject" refers to an individual mammal, such as a human, a non-human primate (e.g., chimpanzee and other ape and monkey species), a domestic animal (e.g., bird, fish, cow, sheep, pig, goat, and horse), a domesticated mammal (e.g., dog and cat), or a laboratory animal (e.g., rodent, e.g., mouse, rat, and guinea pig). The term includes subjects of any age or sex. In another embodiment, the subject is a mammal, preferably a human.

[0194] A disease or disorder is "reduced" when the severity of the symptoms of the disease or disorder, the frequency with which such symptoms are experienced by a patient, or both are reduced.

[0195] As used herein, the terms "change", "defect", "variant" or "mutation" refer to a mutation of a gene in a cell that affects the function, activity, expression (transcription or translation) or conformation of the polypeptide it encodes, including missense and nonsense mutations, insertions, deletions, frameshifts and premature terminations.

[0196] A "disease" is a state of health of an animal in which the animal is unable to maintain homeostasis and, if the disease is not improved, the health of the animal continues to deteriorate.

[0197] A "disorder" of an animal is a state of health in which the animal is able to maintain homeostasis, but the health of the animal is not as good as it would be in the absence of the disorder. If left untreated, a disorder does not necessarily cause a further decline in the health of the animal.

[0198] As used herein, the terms "immune response" or "immune reaction" refer to the host immune system in response to an antigen in a pathogenic organism or to the introduction or expression of a foreign protein. The immune response is generally humoral and local, and antibodies produced by B cells bind to antigens in antigen-antibody complexes to inactivate or neutralize the antigens. The immune response is often observed when human proteins are injected into a mouse model system. Generally, the mouse model system is made tolerant to the immune system by injecting an immunosuppressor prior to the introduction of the foreign antigen to ensure better survival.

[0199] As used herein, the term "immunosuppression" is the planned reduction of the activation or effectiveness of the host immune system using immunosuppressive drugs to promote immune tolerance to foreign antigens such as foreign proteins, organ transplants, bone marrow, and tissue transplants. Non-limiting examples of immunosuppressive drugs include anti-CD4 (GK1.5) antibody, cyclophosphamide, azathioprine (Imuran), mycophenolate mofetil (CellCept), cyclosporine (Neoral, Sandimmune, Gengraf), methotrexate (Rheumatrex), leflunomide (Arava), cyclophosphamide (Cytoxan), and chlorambucil (Leukeran).

[0200] As used herein, the term "ENPP" or "NPP" refers to ectonucleotide pyrophosphatase / phosphodiesterase.

[0201] As used herein, the term "ENPP1 protein" or "ENPP1 polypeptide" refers to the ectonucleotide pyrophosphatase / phosphodiesterase-1 protein encoded by the ENPP1 gene. The encoded protein is a type II transmembrane glycoprotein that cleaves various substrates, including phosphodiester bonds between nucleotides and nucleotide sugars and pyrophosphate bonds between nucleotides and nucleotide sugars. The ENPP1 protein has a transmembrane domain and a soluble extracellular domain. The extracellular domain is further subdivided into a somatomedin B domain, a catalytic domain, and a nuclease domain. The sequence and structure of wild-type ENPP1 are described in detail in PCT Application Publication No. WO2014 / 126965 to Braddock et al., which is hereby incorporated by reference in its entirety.

[0202] Mammalian ENPP1 and ENPP3 polypeptides, mutants or mutant fragments thereof have been previously disclosed in International PCT Application Publications WO / 2014 / 126965 - Braddock et al., WO / 2016 / 187408 - Braddock et al., WO / 2017 / 087936 - Braddock et al., and WO2018 / 027024 - Braddock et al., all of which are hereby incorporated by reference in their entirety.

[0203] As used herein, the term "ENPP3 protein" or "ENPP3 polypeptide" refers to the ectonucleotide pyrophosphatase / phosphodiesterase - 3 protein encoded by the ENPP3 gene. The encoded protein is a type II transmembrane glycoprotein that cleaves various substrates including phosphodiester bonds between nucleotides and nucleotide sugars and pyrophosphate bonds between nucleotides and nucleotide sugars. The ENPP3 protein has a transmembrane domain and a soluble extracellular domain. The sequence and structure of wild - type ENPP3 are described in detail in PCT Application Publication WO / 2017 / 087936 to Braddock et al., which is hereby incorporated by reference in its entirety.

[0204] As used herein, the term "ENPP1 precursor protein" refers to ENPP1 having its signal peptide sequence at the N - terminus of ENPP1. Upon proteolysis, the signal sequence is cleaved from ENPP1, resulting in the ENPP1 protein. Signal peptide sequences useful within the present invention include, but are not limited to, albumin signal sequence, azurocidin signal sequence, ENPP1 signal peptide sequence, ENPP2 signal peptide sequence, ENPP7 signal peptide sequence, and / or ENPP5 signal peptide sequence.

[0205] As used herein, the term "ENPP3 precursor protein" refers to ENPP3 having its signal peptide sequence at the N-terminus of ENPP3. Upon proteolysis, the signal sequence is cleaved from ENPP3, resulting in the ENPP3 protein. Signal peptide sequences useful within the present invention include, but are not limited to, albumin signal peptide sequence, azurocidin signal peptide sequence, ENPP1 signal peptide sequence, ENPP2 signal peptide sequence, ENPP7 signal peptide sequence, and / or ENPP5 signal peptide sequence.

[0206] As used herein, the term "azurocidin signal peptide sequence" refers to a signal peptide derived from human azurocidin. Azurocidin, also known as cationic antimicrobial protein CAP37 or heparin-binding protein (HBP), is a protein encoded by the AZU1 gene in humans. The nucleotide sequence encoding the azurocidin signal peptide (MTRLTVLALLAGLLASSRA) is fused to the nucleotide sequence of the NPP1 or NPP3 gene such that when encoded, it produces the ENPP1 precursor protein or the ENPP3 precursor protein. (Optimized signal peptides for the development of high expressing CHO cell lines, Kober et al., Biotechnol Bioeng. 2013 Apr;110(4):1164-73).

[0207] As used herein, the term "ENPP1-Fc construct" refers to ENPP1 that is recombinantly fused to and / or chemically conjugated to (including both covalent conjugation and non-covalent conjugation) the FcR-binding domain of an IgG molecule (preferably, human IgG). In certain embodiments, the C-terminus of ENPP1 is fused or conjugated to the N-terminus of the FcR-binding domain.

[0208] As used herein, the term "ENPP3-Fc construct" refers to ENPP3 that is recombinantly fused to and / or chemically conjugated to (including both covalent and non-covalent conjugation) the FcR-binding domain of an IgG molecule (preferably, human IgG). In certain embodiments, the C-terminus of ENPP1 is fused or conjugated to the N-terminus of the FcR-binding domain.

[0209] As used herein, the term "Fc" refers to the Fc domain of human IgG (immunoglobulin). Subtypes of IgG, such as IgG1, IgG2, IgG3, and IgG4, are contemplated for use as the Fc domain.

[0210] As used herein, "Fc region or Fc polypeptide" is a portion of an IgG molecule that is related to the crystalline fragment obtained by papain digestion of the IgG molecule. The Fc region includes the C-terminal halves of the two heavy chains of the IgG molecule, which are linked by disulfide bonds. This has no antigen-binding activity but includes carbohydrate moieties and binding sites for complement and Fc receptors, such as the FcRn receptor. The Fc fragment includes the complete second constant domain CH2 (residues 231-340 of human IgG1 according to the Kabat numbering system) and the third constant domain CH3 (residues 341-447). The terms "IgG hinge-Fc region" or "hinge-Fc fragment" refer to the region of the IgG molecule consisting of the Fc region (residues 231-447) and the hinge region (residues 216-230) extending from the N-terminus of the Fc region. The term "constant domain" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to the other portions of the immunoglobulin, i.e., the variable domains that include the antigen-binding sites. Constant domains include the CH1, CH2, and CH3 domains of the heavy chain and the CHL domain of the light chain.

[0211] As used herein, the term "fragment" when applied to a nucleic acid refers to a subsequence of a larger nucleic acid. A "fragment" of a nucleic acid can be at least about 15, 50 - 100, 100 - 500, 500 - 1000, 1000 - 1500 nucleotides, 1500 - 2500 or 2500 nucleotides (and any integer value therebetween). As used herein, the term "fragment" when applied to a protein or peptide refers to a subsequence of a larger protein or peptide and can be at least about 20, 50, 100, 200, 300 or 400 amino acids in length (and any integer value therebetween).

[0212] "Isolated" means altered or removed from its natural state. For example, a nucleic acid or polypeptide that occurs naturally in a living animal is not "isolated", but the same nucleic acid or polypeptide that is partially or completely separated from the coexisting materials in its natural state is "isolated". An isolated nucleic acid or protein can exist in a substantially purified form or, for example, in a non-natural environment such as a host cell.

[0213] "Oligonucleotide" or "polynucleotide" refers to a nucleic acid having a length of at least 2, and in certain embodiments at least 8, 15 or 25 nucleotides, but can be up to 50, 100, 1000 or 5000 nucleotides in length, or can be a compound that specifically hybridizes to a polynucleotide.

[0214] As used herein, the terms "patient", "individual" or "subject" refer to a human.

[0215] As used herein, the term "pharmaceutical composition" or "composition" refers to a mixture of at least one compound useful within the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates the administration of the compound to a patient. There are a plurality of techniques for administering the compound in the art, including, but not limited to, subcutaneous, intravenous, oral, aerosol, inhalation, rectal, vaginal, transdermal, intranasal, buccal, sublingual, parenteral, intrathecal, intragastric, ocular, pulmonary, and topical administration.

[0216] As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, that does not interfere with the biological activity or properties of the compound and is relatively non-toxic, i.e., the material can be administered to an individual without causing undesirable biological effects or interacting in a harmful manner with any of the components of the composition in which it is included, e.g., phosphate buffered saline (PBS).

[0217] As used herein, the term "plasma pyrophosphate (PPi) level" refers to the amount of pyrophosphate present in the plasma of an animal. In certain embodiments, animals include rats, mice, cats, dogs, humans, female cows, and horses. Due to release from platelets, it is necessary to measure PPi in plasma rather than serum. There are several methods for measuring PPi, one of which is by an enzyme assay using modified uridine diphosphoglucose (UDPG) pyrophosphorylase (Lust & Seegmiller, 1976, Clin. Chim. Acta 66:241-249; Cheung & Suhadolnik, 1977, Anal. Biochem. 83:61-63). Typically, normal PPi levels in healthy subjects range from about 1 μM to about 3 μM, and in some cases 1-2 μM. Subjects with incomplete ENPP1 expression tend to show low PPi levels, which are at least 10% lower than normal levels, at least 20% lower than normal levels, at least 30% lower than normal levels, at least 40% lower than normal levels, at least 50% lower than normal levels, at least 60% lower than normal levels, at least 70% lower than normal levels, at least 80% lower than normal levels, and combinations thereof. In patients with GACI, PPi levels are found to be less than 1 μM, and in some cases lower than the detection level. In patients with PXE, PPi levels are less than 0.5 μM (Arterioscler Thromb Vasc Biol. 2014 Sep;34(9):1985-9; Braddock et al., Nat Commun. 2015;6:10006.).

[0218] As used herein, the term "polypeptide" refers to a polymer composed of amino acid residues, their related naturally occurring structural variants, and synthetic non-naturally occurring analogs linked via peptide bonds.

[0219] As used herein, the term "PPi" refers to pyrophosphate.

[0220] As used herein, the terms "prevent" or "prevention" mean that, when nothing has occurred, a disorder or disease does not occur, or when a disorder or disease has already occurred, no further disorder or disease occurs. The ability to prevent some or all of the symptoms associated with a disorder or disease is also considered.

[0221] As used herein, "sample" or "biological sample" means a biological substance isolated from a subject. A biological sample can include any biological substance suitable for detecting mRNA, polypeptide, or other markers of physiological or pathological processes in a subject, and can include body fluids, tissues, cellular and / or acellular substances obtained from an individual.

[0222] As used herein, "substantially purified" refers to essentially being free of other components. For example, a substantially purified polypeptide is a polypeptide that has been separated from other components to which it is normally bound in its native state. Non-limiting embodiments include 95% purity, 99% purity, 99.5% purity, 99.9% purity, and 100% purity.

[0223] As used herein, the terms "treat" or "treatment" are directed to the application or administration of a therapeutic agent, i.e., a compound useful within the present invention (alone or in combination with another pharmaceutical), to a patient for the purpose of curing, treating, alleviating, soothing, altering, salvaging, improving, ameliorating, or affecting a disease or disorder, the symptoms of a disease or disorder, or the likelihood of developing a disease or disorder, or the application or administration of a therapeutic agent (e.g., for diagnostic or ex vivo applications) to a tissue or cell line isolated from a patient having a disease or disorder, the symptoms of a disease or disorder, or the likelihood of developing a disease or disorder.

[0224] As used herein, the terms "prevent," "preventing," and "prevention" refer to inhibiting the onset of a disease or reducing the incidence of a disease in a subject. Prevention may be complete (e.g., the complete absence of diseased cells in the subject) or partial. Prevention also refers to a reduction in susceptibility to a clinical condition.

[0225] As used herein, the term "wild-type" refers to a gene or gene product isolated from a naturally occurring source. Wild-type genes are the most frequently observed within a population and are thus arbitrarily referred to as the "normal" or "wild-type" form of the human NPP1 or NPP3 gene. In contrast, the term "functionally equivalent" refers to an NPP1 or NPP3 gene or gene product that exhibits a modification in sequence and / or functional properties (i.e., altered characteristics) as compared to the wild-type gene or gene product. Naturally occurring mutants can be isolated and are identified by the fact that they have altered characteristics (including altered nucleic acid sequences) as compared to the wild-type gene or gene product.

[0226] As used herein, the term "functionally equivalent variant" relates to a polypeptide that is substantially homologous to the ENPP1 or ENPP3 sequence (as defined above) and retains the enzymatic and biological activities of ENPP1 or ENPP3, respectively. Methods for determining whether a variant retains the biological activity of native ENPP1 or ENPP3 are well known to those of skill in the art and include any of the assays used in the experimental portion of the foregoing application. In particular, functionally equivalent variants of ENPP1 or ENPP3 delivered by viral vectors are encompassed by the present invention.

[0227] Functionally equivalent variants of ENPP1 or ENPP3 are polypeptides that are substantially homologous to native ENPP1 or ENPP3, respectively. The expression "substantially homologous" relates to protein sequences where the protein sequences have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the ENPP1 or ENPP3 sequences described above, respectively.

[0228] The identity between two polypeptides is determined using computer algorithms and methods well known to those skilled in the art. The identity between two amino acid sequences is preferably determined by using the BLASTP algorithm (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990)), although other similar algorithms can also be used. BLAST and BLAST 2.0 are used with the parameters described herein to determine the percent sequence identity. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information.

[0229] "Functionally equivalent variants" of ENPP1 or ENPP3 can be obtained by replacing nucleotides within the polynucleotide that account for codon preference in the host cell used to produce ENPP1 or ENPP3, respectively. Such "codon optimization" can be determined via computer algorithms that incorporate codon frequency tables such as "Human high.cod" for codon preference provided by the University of Wisconsin Package Version 9.0, Genetics Computer Group, Madison, Wis.

[0230] As used herein, "about" when referring to a measurable value such as an amount, a temporal duration, etc., means within ±20%, or ±10% in certain embodiments, ±5% in certain embodiments, ±1% in certain embodiments, or ±0.1% in certain embodiments of a particular value, because such variations are appropriate for carrying out the disclosed methods.

[0231] The present disclosure provides representative examples of the protein and nucleic acid sequences of the invention. The described protein sequences can be converted into nucleic acid sequences by reverse translation and codon optimization. There are several tools available in the art, such as Expasy (https: / / www.expasy.org / ) and bioinformatics servers (http: / / www.bioinformatics.org), that enable such conversions.

[0232] Ranges: Throughout this disclosure, various aspects of the invention may be presented in range format. The description in range format is merely for convenience and brevity and should not be construed as an immutable limitation on the scope of the invention. Accordingly, a description of a range should be considered to have specifically disclosed all the possible sub-ranges and individual numerical values within that range. For example, a range description such as 1-6 should be considered to have specifically disclosed sub-ranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., and individual numbers within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0233] Viral vectors for in vivo expression of ENPP1 and ENPP3 To compensate for the lack of ENPP1 or ENPP3 polypeptide, genetic material such as a polypeptide containing the NPP1 or NPP3 sequence can be inserted into a mammal.

[0234] Certain modified viruses are often used as vectors for carrying coding sequences because, after administration to a mammal, the virus infects cells and expresses the encoded protein. Modified viruses useful according to the present invention are derived from viruses including, for example: parvovirus, picornavirus, pseudorabies virus, hepatitis virus A, B or C, papillomavirus, papovavirus (e.g., polyoma and SV40) or herpes virus (e.g., Epstein - Barr virus, varicella - zoster virus, cytomegalovirus, herpes zoster virus and herpes simplex virus types 1 and 2), RNA virus or retrovirus, for example, Moloney murine leukemia virus or lentivirus (i.e., derived from human immunodeficiency virus, feline immunodeficiency virus, equine infectious anemia virus, etc.). Among the DNA viruses useful according to the present invention are: adeno - associated virus, adenovirus, alphavirus and lentivirus.

[0235] Viral vectors are generally administered by intravenous injection (IV), usually directly into the body or directly into a specific tissue where it is taken up by individual cells. Alternatively, a viral vector can be administered by contacting the viral vector ex vivo with a sample of the patient's cells, thereby infecting the cells with the viral vector, and then the cells containing the vector are returned to the patient. Once the viral vector is delivered, the coding sequence is expressed, resulting in a functional protein. Generally, infection and transduction of cells by viral vectors occur by the following series of consecutive events: interaction of the viral capsid with receptors on the surface of the target cell, internalization by endocytosis, intracellular transport through the endocytosis / proteasome compartment, endosomal escape, nuclear translocation, uncoating of the virion, and viral DNA double - strand conversion leading to transcription and expression of the recombinant coding sequence of interest (Colella et al., Mol Ther Methods Clin Dev. 2017 Dec 1;8:87 - 104.).

[0236] Adeno-associated virus vector according to the present invention AAV refers to a virus belonging to the genus Dependovirus of the Parvoviridae family. The AAV genome is approximately 4.7 kilobases in length and consists of linear single-stranded deoxyribonucleic acid (ssDNA) that can be either plus or minus sense. The genome contains inverted terminal repeats (ITRs) at both ends of the DNA strand as well as two open reading frames (ORFs): rep and cap. The rep frame is composed of four overlapping genes that encode non-structural replication (Rep) proteins required for the life cycle of AAV. The cap frame contains overlapping nucleotide sequences of the structural VP capsid proteins: VP1, VP2, and VP3 that interact together to form an icosahedral symmetric capsid.

[0237] The 145 nucleotides at the ends are self-complementary and are organized so that an energetically stable intramolecular double-strand that forms a T-shaped hairpin can be formed. These hairpin structures function as starting points for viral DNA replication and act as primers for the cellular DNA polymerase complex. After infection with wild-type AAV in mammalian cells, the rep genes (i.e., Rep78 and Rep52) are expressed from the P5 promoter and the P19 promoter, respectively, and both Rep proteins have functions in viral genome replication. Splicing events of the rep ORF actually result in the expression of four Rep proteins (i.e., Rep78, Rep68, Rep52, and Rep40). However, unspliced mRNAs encoding the Rep78 and Rep52 proteins in mammalian cells have been shown to be sufficient for AAV vector production. In insect cells as well, the Rep78 and Rep52 proteins are sufficient for AAV vector production.

[0238] AAV vectors typically lack the rep and cap frames. Such AAV vectors are present in host cells transfected with a vector that encodes and expresses the rep and cap gene products (i.e., AAV Rep and Cap proteins), and can replicate and be packaged into infectious virus particles when the host cells are transfected with a vector that encodes and expresses a protein derived from the adenoviral open reading frame E4orf6.

[0239] In one embodiment, the present invention relates to an adeno-associated virus (AAV) expression vector comprising a sequence encoding mammalian ENPP1 or mammalian ENPP3, wherein upon administration to a mammal, the vector expresses an ENPP1 or ENPP3 precursor in cells, and the precursor comprises an azurocidin signal peptide fused to the amino terminus of ENPP1 or ENPP3 at its carboxy terminus. The ENPP1 or ENPP3 precursor can comprise a stabilizing domain, such as an IgG Fc region or human albumin. Upon secretion of the precursor from the cell, the signal peptide is cleaved, resulting in an enzymatically active soluble mammalian ENPP1 or ENPP3 being brought extracellularly.

[0240] The AAV expression vector can comprise an expression cassette, the expression cassette comprising a nucleotide sequence operably linked to a transcription control region, the nucleotide sequence encoding a polypeptide comprising an azurocidin signal peptide sequence and an ectonucleotide pyrophosphatase / phosphodiesterase (ENPP1) polypeptide sequence operably linked to a transcription control region.

[0241] In some embodiments, the expression cassette comprises a promoter and enhancer, a Kozak sequence GCCACCATGG, a nucleotide sequence encoding a mammalian NPP1 protein or a nucleotide sequence encoding a mammalian NPP3 protein, other suitable regulatory elements, and a polyadenylation signal.

[0242] In some embodiments, the AAV recombinant genome of the AAV vector according to the present invention lacks the rep open reading frame and / or the cap open reading frame.

[0243] The AAV vector according to the present invention includes a capsid derived from any serotype. Generally, AAV serotypes have genomic sequences with significant homology at the amino acid and nucleic acid levels, provide the same set of genetic functions, and replicate and assemble through substantially the same mechanism. In particular, the AAV of the present invention may belong to AAV serotype 1 (AAV1), AAV2, AAV3 (including 3A and 3B types), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV.

[0244] Examples of the sequences of the genomes of various AAV serotypes can be found in the literature or in public databases such as GenBank. For example, GENBANK accession numbers NC_001401.2 (AAV2), NC_001829.1 (AAV4), NC_006152.1 (AAV5), AF028704.1 (AAV6), NC_006260.1 (AAV7), NC_006261.1 (AAV8), AX753250.1 (AAV9) and AX753362.1 (AAV10).

[0245] In some embodiments, the adeno-associated virus vector according to the present invention includes a capsid derived from a serotype selected from the group consisting of AAV2, AAV5, AAV7, AAV8, AAV9, AAV10 and AAVrh10 serotypes. In another embodiment, the serotype of AAV is AAV8. When the viral vector includes a sequence encoding a capsid protein, these can be modified to include foreign sequences for directing the AAV to specific cell type(s), or for enhancing the efficiency of delivery of the targeted vector to cells, or for facilitating the purification or detection of the AAV, or for reducing the host response.

[0246] The published application US2017 / 0290926 - Smith et al., the contents of which are hereby incorporated by reference in their entirety, describes in detail the process by which AAV vectors are generated, delivered, and administered.

[0247] Adenoviral vectors useful according to the present invention Adenoviruses can be engineered to encode and express a desired gene product (e.g., ENPP1 or ENPP3) and at the same time be inactivated with respect to their ability to replicate in the normal lytic viral life cycle. Further, adenoviruses have a natural tropism for airway epithelium. The virus can infect quiescent cells as seen in the airway, providing a significant advantage over retroviruses. Adenoviral expression is achieved without integration of viral DNA into the host cell chromosome, thereby reducing concerns about insertional mutagenesis. Additionally, adenoviruses have been used for many years as live intestinal vaccines with an excellent safety profile (Schwartz, A.R. et al. (1974) Am. Rev. Respir. Dis. 109:233 - 238). Finally, adenovirus - mediated gene transfer has been demonstrated in several examples, including the transfer of alpha - 1 - antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, M.A. et al. (1991) Science 252:431 - 434; Rosenfeld et al., (1992) Cell 68:143 - 155). Further, extensive studies attempting to establish adenoviruses as causative agents in human cancer have been uniformly negative (Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA 76:6606).

[0248] Pseudoadenoviral vectors (PAVs) contain the adenoviral inverted terminal repeats and the minimal adenoviral 5' sequences required for helper virus-dependent replication and packaging of the vector. These vectors do not contain potentially harmful viral genes, have a theoretical capacity of approximately 36 kb for foreign substances, can result in moderately high titers, and maintain the tropism of the parental virus for both dividing and non-dividing human target cell types. PAV vectors can be maintained either as plasmid-derived constructs or as infectious virus particles. As plasmid constructs, PAVs are composed of the minimal sequences derived from wild-type adenovirus type 2 that are required for efficient replication and packaging of these sequences by either wild-type or incomplete helper viruses, as well as any desired additional foreign genetic material.

[0249] U.S. Patent Publication US7,318,919 - Gregory et al. describes in detail the process by which adenoviral vectors are produced and delivered, and their corresponding use for the treatment of diseases, the content of which is hereby incorporated by reference in its entirety. The present invention contemplates the use of adenoviral vectors for delivering nucleotides encoding ENPP1 or ENPP3 to subjects in need thereof, and methods of treatment using the same.

[0250] Herpes simplex vectors useful according to the present invention A simplex herpes vector (HSV-based viral vector) is suitable for use as a vector for introducing nucleic acid sequences into a number of cell types. A mature HSV virion consists of an enveloped icosahedral capsid having a viral genome consisting of a 152 kb linear double-stranded DNA molecule. In another embodiment, the HSV-based viral vector is deficient in at least one essential HSV gene. In some embodiments, the HSV-based viral vector deficient in at least one essential HSV gene is replication-deficient. Most replication-deficient HSV vectors contain deletions for removing one or more immediate-early, early or late HSV genes to prevent replication. For example, the HSV vector may be deficient in immediate-early genes selected from the group consisting of ICP4, ICP22, ICP27, ICP47 and combinations thereof. Advantages of the HSV vector are its ability to enter a latent state that can result in long-term DNA expression, and its large viral DNA genome that can accommodate exogenous DNA inserts up to 25 kb.

[0251] HSV-based vectors are described, for example, in U.S. Patent Nos. 5,837,532 - Preston et al., 5,846,782 - Wickham et al., and 5,804,413 - DeLuca et al., and International Patent Applications WO91 / 02788 - Preston et al., WO96 / 04394 - Preston et al., WO98 / 15637 - DeLuca et al., and WO99 / 06583 - Glorioso et al., which are incorporated herein by reference. The HSV vector may be deficient in gene functions essential for replication of only the immediate-early region of the HSV genome, only the immediate-early region of the HSV genome, only the late region of the HSV genome, or both the immediate-early and late regions of the HSV genome. Generation of the HSV vector involves the use of standard molecular biological techniques well known in the art.

[0252] Replication-deficient HSV vectors are typically produced in complementing cell lines that provide gene functions necessary for viral growth but are not present in the replication-deficient HSV vector, at appropriate levels to generate high-titer viral vector stocks. Expression of a nucleic acid sequence encoding a protein is controlled by an appropriate expression control sequence operably linked to the nucleic acid sequence. An "expression control sequence" is any nucleic acid sequence that facilitates, enhances, or controls the expression (typically, and preferably transcription) of another nucleic acid sequence.

[0253] Suitable expression control sequences include constitutive promoters, inducible promoters, repressive promoters, and enhancers. The nucleic acid sequence encoding a protein in the vector can be regulated by its endogenous promoter or, preferably, by a non-native promoter sequence. Examples of suitable non-native promoters include the human cytomegalovirus (HCMV) promoter, such as the HCMV immediate early promoter (HCMV IEp), promoters derived from the human immunodeficiency virus (HIV), such as the HIV long terminal repeat promoter, the phosphoglycerate kinase (PGK) promoter, the Rous sarcoma virus (RSV) promoter, such as the RSV long terminal repeat, the mouse mammary tumor virus (MMTV) promoter, the Lap2 promoter, or the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci., 78, 1444-1445 (1981)), promoters derived from SV40 or Epstein-Barr virus, and the like. In another embodiment, the promoter is HCMV IEp.

[0254] The promoter may also be an inducible promoter, i.e., a promoter that is up- and / or down-regulated in response to an appropriate signal. For example, an expression control sequence that is up-regulated by a pharmaceutical is particularly useful in pain management applications. For example, the promoter can be a pharmaceutically inducible promoter (e.g., tetracycline-responsive). The promoter can be inserted into the genome of the vector by methods known in the art, e.g., by introducing unique restriction sites into a given region of the genome.

[0255] U.S. Patent Publication US7,531,167 - Glorioso et al. describes in detail the process by which herpes simplex vectors are generated and delivered, and their corresponding use for the treatment of diseases, the contents of which are hereby fully incorporated by reference. The present invention contemplates the use of herpes simplex vectors for delivering nucleotides encoding ENPP1 or ENPP3 to subjects in need thereof, and methods of treatment using the same.

[0256] Alpha virus vectors useful according to the present invention Alphavirus expression vectors have been developed from different types of alphaviruses, including Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan equine encephalitis (VEE) virus. The alphavirus replicon contains an open reading frame encoding the viral replicase (Rep) at its 5’ end, which is translated when the viral RNA is transfected into cells. Rep is expressed as a polyprotein, which is subsequently processed into four subunits (nsps1-4). Unprocessed Rep can copy the RNA vector into minus-strand RNA, a process that occurs during the first 3-4 hours after transfection or infection. Once processed, Rep uses the minus-strand RNA as a template to synthesize more replicon molecules. Processed Rep can also recognize internal sequences of the minus-strand RNA or the subgenomic promoter, from which it synthesizes the subgenomic plus-strand RNA corresponding to the 3’ end of the replicon. This subgenomic RNA is translated to produce large amounts of heterologous protein.

[0257] A non-cytopathic mutant isolated from SIN containing a single amino acid change at position 726 of nsp2 (P for L (P726L vector of SIN in nsp2)) showed Rep hyperprocessing (Frolov et al., 1999, J. Virol. 73: 3854 - 65). This mutant was able to efficiently establish continuous replication in BHK cells. This non-cytopathic SIN vector is widely used in vitro because it can provide long-term persistent transgene expression at a good stability level and an expression level of about 4% of that obtained using the original SIN vector (Agapov et al., 1998, Proc. Natl. Acad. Sci. USA. 95: 12989 - 94). Similarly, patent application WO2008065225 - Smerdou et al. describes that a non-cytopathic SFV vector has mutations R649H / P718T in the replicase nsp2 subunit. The aforementioned vector enables the obtaining of cell lines that can constitutively and stably express the gene of interest by culturing in the presence of an antibiotic resistance gene incorporated into the alphavirus vector (Casales et al. 2008. Virology. 376: 242 - 51).

[0258] The present invention contemplates designing a vector comprising a DNA sequence complementary to an alphavirus replicon into which the sequence of a gene of interest, such as NPP1 or NPP3, is incorporated together with a recognition sequence for site-specific recombination. By means of the said vector, it is possible to obtain and select cells in which an alphavirus replicon containing the sequence of the gene of interest is incorporated into the cell genome, with the result that the cells stably express the ENPP1 or ENPP3 polypeptide. The present invention also contemplates generating an expression vector in which the alphavirus replicon is under the control of an inducible promoter. The said vector when incorporated into a cell further modified by incorporating an expression cassette encoding a transcriptional activator in the presence of a given ligand that can positively regulate the activity of a promoter that regulates the transcription of the alphavirus replicon.

[0259] U.S. Patent Publication US10,011,847 - Aranda et al. describes in detail the process by which alphavirus vectors are generated and delivered, and their corresponding use for the treatment of diseases, the content of which is hereby fully incorporated by reference. The present invention contemplates the use of alphavirus vectors for delivering nucleotides encoding ENPP1 or ENPP3 to a subject in need thereof, and a method of treatment using the same.

[0260] Lentiviral vectors useful according to the present invention Lentiviruses belong to the genus of viruses in the Retroviridae family and are characterized by a long incubation period. Lentiviruses have the unique ability among retroviruses to deliver a significant amount of viral RNA into the DNA of host cells and to infect non - dividing cells. Lentiviral vectors, particularly those derived from HIV - 1, have been widely studied and are frequently used vectors. The evolution of the lentiviral vector backbone and the ability of the virus to deliver recombinant DNA molecules (transgenes) to target cells have led to its use in the repair of functional genes in gene therapy and in recombinant protein production in vitro.

[0261] The present invention contemplates a lentiviral vector containing a promoter and a transgene suitable for expressing a protein of interest, such as ENPP1 or ENPP3. Typically, the vector backbone is derived from simian immunodeficiency virus (SIV), such as SIV1 or African green monkey SIV (SIV-AGM). In one embodiment, the promoter is preferably a hybrid human CMV enhancer / EF1a (hCEF) promoter. The present invention includes a method for producing a lentiviral vector, a composition containing a lentiviral vector that expresses a gene of interest, and use in gene therapy for expressing ENPP1 or ENPP3 protein for the purpose of treating diseases of calcification or ossification. The lentiviral vector according to the present invention can also be used in a method of gene therapy for promoting the secretion of a therapeutic protein. As a further example, the present invention provides for the secretion of a therapeutic protein into the respiratory lumen or the circulatory system. Thus, administration of the vector according to the present invention and its uptake by airway cells can then be secreted, enter the systemic circulation at therapeutic levels, where it can move to the target cells / tissues of interest and enable the use of the lung (or nose or airway) as a "factory" for producing a therapeutic protein that can induce a therapeutic effect. In contrast to intracellular or membrane proteins, the production of such secreted proteins does not rely on the specific disease target cells being transduced, which is an important advantage and achieves high levels of protein expression. Thus, other diseases not of the respiratory tract, such as cardiovascular diseases and blood disorders, can also be treated with a lentiviral vector, such as that according to the present invention. A lentiviral vector, such as that according to the present invention, can be integrated into the genome of the transduced cells, leading to long-term persistent expression and making it suitable for transduction of stem / progenitor cells.

[0262] U.S. Patent Application Publication US2017 / 0096684 - Alton et al. describes in detail the process by which lentiviral vectors are generated and delivered and their corresponding use for the treatment of diseases, the content of which is hereby incorporated by reference in its entirety. The present invention contemplates the use of lentiviral vectors for delivering nucleotides encoding ENPP1 or ENPP3 to subjects in need thereof, and methods of treatment using the same.

[0263] Sequence SEQ ID NO:1 - ENPP1 Amino Acid Sequence - Wild - type [Table 1 - 1] [Table 1 - 2] [Table 1 - 3] The NPP1 amino acid sequence shown above includes a cytoplasmic domain, a transmembrane domain, an SMB1 domain, an SMB2 domain, a phosphodiesterase / catalytic domain, a linker domain, and a nuclease domain. The SMB1 domain, SMB2 domain, catalytic domain, linker domain, and nuclease domain together are referred to as the extracellular domain. Residues 1-76 (Met Glu Arg~Thr Tyr Lys) correspond to the cytoplasmic domain. Residues 77-97 (Val Leu Ser~Phe Gly Leu) correspond to the transmembrane domain. Residues 99-925 (Pro Ser Cys~Gln Glu Asp) correspond to the extracellular domain. Residues 104-144 (Glu Val Lys~Glu Pro Glu) correspond to the SMB1 domain, and residues 145-189 (His Ile Trp~Glu Lys Ser) correspond to the SMB2 domain. Residues 597-647 correspond to the linker domain that binds the catalytic and nuclease domains. Residues 191-591 (Val Glu Glu~Gly Ser Leu) correspond to the catalytic / phosphodiesterase domain. Residues 654-925 (His Glu Thr~Gln Glu Asp) correspond to the nuclease domain. The residue numbering and domain classification are based on the human NPP1 sequence (NCBI accession NP_006199 / Uniprot-Swissprot P22413).

[0264] SEQ ID NO: 2 - Azlosidine-ENPP1-Fc

Table 2-1

Table 2-2

[0265] SEQ ID NO: 3 - Azlosidine-ENPP1-Alb

Table 3

[0266] SEQ ID NO: 4 - Azurocidin - ENPP1

Table 4 - 1

Table 4 - 2

[0267] SEQ ID NO: 5 - ENPP2 amino acid sequence - wild type

Table 5 - 1

Table 5 - 2

Table 5 - 3

[0268] SEQ ID NO: 6 - Extracellular domain of ENPP3:

Table 6 - 1

Table 6 - 2

Table 6 - 3

[0269] SEQ ID NO: 7 - NPP3 amino acid sequence:

Table 7 - 1

Table 7 - 2

Table 7-3

[0270] SEQ ID NO: 8-Azurosidine-ENPP3-Fc

Table 8

[0271] SEQ ID NO: 9-Azurosidine-ENPP3-albumin

Table 9-1

Table 9-2

[0272] Accession No. 10 - Azurocidin - ENPP3 [Table 10] Single underline - Azurocidin signal sequence, double underline - start and end of ENPP3 sequence, ** indicates the cleavage point of the signal sequence.

[0273] Accession No. 11 - ENPP4 Amino Acid Sequence - Wild Type [Table 11 - 1] [Table 11 - 2]

[0274] Accession No. 12 - ENPP51 Amino Acid Sequence [Table 12 - 1] [Table 12 - 2] [Table 12 - 3] [Table 12 - 4] Underlined once: signal peptide sequence; underlined twice: start and end of NPP1; ** = cleavage position of the signal peptide sequence

[0275] Accession No. 13 - ENPP51 - ALB Amino Acid Sequence: [Table 13 - 1] [Table 13 - 2] [Table 13 - 3]

Table 13-4

[0276] SEQ ID NO: 14 - ENPP5 - NPP3 - Fc sequence

Table 14-1

Table 14-2

Table 14-3

[0277] SEQ ID NO: 15 - ENPP5 - NPP3 - albumin sequence

Table 15-1

Table 15-2

Table 15-3

Table 15-4

Table 15-5

[0278] SEQ ID NO: 16 - ENPP5 protein export signal sequence

Table 16

[0279] Accession No. 17 - ENPP5 - 1 - Fc

Table 17 - 1

Table 17 - 2

Table 17 - 3

Table 17 - 4

[0280] Accession No. 18 - ENPP7 - 1 - Fc Amino Acid Sequence

Table 18 - 1

Table 18 - 2

Table 18 - 3

Table 18 - 4

[0281] Accession No. 19 - ENPP71 (lacking N - terminal GLK of NPP1) Amino Acid Sequence:

Table 19 - 1

Table 19-2

Table 19-3

[0282] SEQ ID NO: 20 - ENPP71 (lacking N-terminal GLK of NPP1) - Fc amino acid sequence:

Table 20-1

Table 20-2

Table 20-3

Table 20-4

[0283] SEQ ID NO: 21 - ENPP7-1 (lacking N-terminal GLK of NPP1) - ALB amino acid sequence

Table 21-1

Table 21-2

Table 21-3

Table 21-4

Table 21-5

[0284] SEQ ID NO: 22 - ENPP7 - NPP3 - Fc sequence:

Table 22 - 1

Table 22 - 2

Table 22 - 3

[0285] SEQ ID NO: 23 - ENPP7 - 1 - albumin

Table 23 - 1

Table 23 - 2

Table 23 - 3

Table 23 - 4

[0286] SEQ ID NO: 24 - ENPP7 - NPP3 - albumin

Table 24 - 1

Table 24 - 2

Table 24-3

Table 24-4

Table 24-5

[0287] SEQ ID NO: 25 - ENPP7 - ENPP3 - Albumin

Table 25-1

Table 25-2

Table 25-3

Table 25-4

Table 25-5

[0288] SEQ ID NO: 26 - ENPP71 - GLK Amino Acid Sequence

Table 26-1

Table 26-2

Table 26-3

[0289] SEQ ID NO: 27 - ENPP1 21 - amino acid sequence

Table 27 - 1

Table 27 - 2

Table 27 - 3

[0290] SEQ ID NO: 28 - ENPP1 21 - Fc amino acid sequence

Table 28 - 1

Table 28 - 2

Table 28 - 3

[0291] SEQ ID NO: 29 - ENPP1 21 - ALB amino acid sequence:

Table 29 - 1

Table 29 - 2

Table 29 - 3

Table 29 - 4

[0292] SEQ ID NO: 30 - ENPP121 - NPP3 - Fc sequence

Table 30 - 1

Table 30 - 2

Table 30 - 3

[0293] SEQ ID NO: 31 - ENPP121 - NPP3 - albumin sequence

Table 31 - 1

Table 31 - 2

Table 31 - 3

Table 31 - 4

[0294] SEQ ID NO: 32 - ENPP121GLK protein export signal sequence

Table 32

[0295] Sequence number 33 - Albumin sequence

Table 33 - 1

Table 33 - 2

Table 33 - 3

[0296] Sequence number 34 - Human IgG Fc domain, Fc

Table 34

[0297] Sequence number 35 - Albumin sequence

Table 35 - 1

Table 35 - 2

Table 35 - 3

[0298] Sequence number 36 - ENPP2 signal peptide

Table 36

[0299] Sequence number 37 - Signal sequence ENPP7

Table 37

[0300] Sequence number 38 - Signal sequence ENPP7

Table 38

[0301] Array No. 39 - Signal sequence ENPP1 - 2 - 1

Table 39 - 1

Table 39 - 2

[0302] Array No. 40 - exENPP3

Table 40

[0303] Array No. 41 - Signal sequence ENPP5:

Table 41

[0304] Array No. 42 - Azurocidin - ENPP1 - FC nucleotide sequence

Table 42 - 1

Table 42 - 2

[0305] Array No. 43 - Azurocidin - ENPP1 - albumin nucleotide sequence

Table 43 - 1

Table 43 - 2

[0306] Array No. 44 - Azurocidin - ENPP1 nucleotide sequence

Table 44 - 1

Table 44-2

[0307] Array No. 45 - Azurosidine - ENPP3 - FC Nucleotide Sequence

Table 45-1

Table 45-2

[0308] Array No.: 46 - Azurosidine - ENPP3 - Albumin Nucleotide Sequence

Table 46-1

Table 46-2

[0309] Array No. 47 - Azurosidine - ENPP3 - Nucleotide Sequence

Table 47-1

Table 47-2

[0310] Array No. 48 - ENPP7-1 - FC Nucleotide Sequence

Table 48-1

Table 48-2

Table 48-3

[0311] Array No. 49 - ENPP7 - NPP1 Albumin Nucleotide Sequence:

Table 49 - 1

Table 49 - 2

Table 49 - 3

[0312] Nucleotide Sequence of Array No. 50 - NPP121 - NPP3 - Fc

Table 50 - 1

Table 50 - 2

Table 50 - 3

[0313] Nucleotide Sequence of Array No. 51 - NPP121 - NPP3 - Fc

Table 51 - 1

Table 51 - 2

Table 51 - 3

Table 51 - 4

[0314] Nucleotide Sequence of Array No. 52 - hNPP3 - hFc - pcDNA3

Table 52 - 1

Table 52 - 2

Table 52-3

Table 52-4

Table 52-5

Table 52-6

[0315] Array number 53 - ENPP121 - Fc - nucleotide sequence

Table 53-1

Table 53-2

[0316] Array number 54 - ENPP121 - albumin nucleotide sequence

Table 54-1

Table 54-2

Table 54-3

Table 54-4

[0317] Array number 55 - ENPP3 nucleotide sequence

Table 55-1

Table 55-2

[0318] Array number 56 - ENPP1 nucleotide sequence:

Table 56-1

Table 56-2

[0319] Array number 57 - Linker

Table 57

[0320] Array number 58 - Linker

Table 58

[0321] Array number 59 - Linker

Table 59

[0322] Array number 60 - Linker

Table 60

[0323] Array number 61 - Linker

Table 61

[0324] Array number 62 - Linker

Table 62

[0325] Array number 63 - Linker

Table 63

[0326] Array No. 64 - Linker [Table 64]

[0327] Array No. 65 - Linker [Table 65]

[0328] Array No. 66 - Linker [Table 66]

[0329] Array No. 67 - Linker [Table 67]

[0330] Array No. 68 - Linker [Table 68]

[0331] Array No. 69 - Linker [Table 69]

[0332] Array No. 70 - Linker [Table 70]

[0333] Array No. 71 - Linker [Table 71]

[0334] Array No. 72 - Linker

Table 72

[0335] Array number 73 - Linker

Table 73

[0336] Array number 74 - Linker

Table 74

[0337] Array number 75 - Linker

Table 75

[0338] Array number 76 - Linker

Table 76

[0339] Array number 77 - Linker

Table 77

[0340] Array number 78 - Linker

Table 78

[0341] Array number 79 - Linker

Table 79

[0342] Array number 80 - Linker

Table 80

[0343] Array No. 81 - Linker

Table 81

[0344] Array No. 82 - Linker

Table 82

[0345] Array No. 83 - Linker

Table 83

[0346] Array No. 84 - Linker

Table 84

[0347] Array No. 85 - Linker

Table 85

[0348] Array No. 86 - Linker

Table 86

[0349] Array No. 87 - Linker

Table 87

[0350] Array No. 88 - Linker

Table 88

[0351] Accession No. 89 - Soluble NPP1 - Fc Fusion Protein Sequence [Table 89 - 1] [Table 89 - 2] Underlined twice: Start and end of NPP1; Bold residues indicate the Fc sequence.

[0352] Accession No. 90 - Nucleotide Sequence of Soluble NPP1 - Fc [Table 90 - 1] [Table 90 - 2] [Table 90 - 3]

[0353] Accession No. 91 - Soluble NPP1 - (GLK) - Fc Fusion Protein Sequence [Table 91 - 1] [Table 91 - 2] Underlined twice: Start and end of NPP1; Bold residues indicate the Fc sequence.

[0354] Accession No. 92 - Soluble NPP1 - Fc Fusion Protein Sequence [Table 92 - 1] [Table 92 - 2] [Table 92 - 3] Underlined twice: Start and end of NPP1; Bold residues indicate the Fc sequence.

[0355] Array No. 93 - Soluble NPP1 - Fc fusion protein sequence

Table 93 - 1

Table 93 - 2

[0356] Array No. 94 - Linker

Table 94

[0357] Pharmaceutical composition according to the present invention The AAV vector according to the present invention can be administered to the human or animal body by conventional methods, which requires formulation of the vector into a pharmaceutical composition. In one embodiment, the present invention relates to a pharmaceutical composition comprising an AAV vector comprising a recombinant viral genome (hereinafter referred to as "the pharmaceutical composition according to the present invention"), wherein the recombinant viral genome comprises an expression cassette operably linked to a nucleotide sequence encoding ENPP1 or ENPP3 or a functionally equivalent variant thereof and comprising a transcriptional control region.

[0358] All embodiments disclosed in connection with the adeno - associated virus vector, herpes simplex vector, adenovirus vector, alphavirus vector and lentivirus vector according to the present invention are also applicable to the pharmaceutical composition according to the present invention.

[0359] In some embodiments, the pharmaceutical composition can comprise a therapeutically effective amount of the AAV vector according to the present invention and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition can comprise a therapeutically effective amount of the adenovirus vector according to the present invention and a pharmaceutically acceptable carrier.

[0360] In some embodiments, the pharmaceutical composition can comprise a therapeutically effective amount of a lentiviral vector according to the present invention and a pharmaceutically acceptable carrier.

[0361] In some embodiments, the pharmaceutical composition can comprise a therapeutically effective amount of an alphavirus vector according to the present invention and a pharmaceutically acceptable carrier.

[0362] In some embodiments, the pharmaceutical composition can comprise a therapeutically effective amount of a herpes simplex virus vector according to the present invention and a pharmaceutically acceptable carrier.

[0363] The term "therapeutically effective amount" refers to the amount of the AAV vector according to the present invention calculated to produce the desired effect, and is generally determined by, among other reasons, the particular characteristics of the viral vector according to the present invention and the resulting therapeutic effect. The amount of the viral vector according to the present invention that is effective for treating a disease can be determined by standard clinical techniques described herein or known in the art. Additionally, in vitro tests can optionally be used to assist in identifying the optimal dosage range. The exact dosage to be used in the formulation depends on the route of administration and the severity of the condition, and should be determined by a physician's judgment and according to the circumstances of each patient.

[0364] Promoter Vectors used in gene therapy require an expression cassette. The expression cassette consists of three important components: a promoter, a therapeutic gene, and a polyadenylation signal. The promoter is essential for controlling the expression of the therapeutic gene. A tissue-specific promoter is a promoter that has activity only in a specific cell type. The use of a tissue-specific promoter in an expression cassette can limit unwanted transgene expression and facilitate sustained transgene expression. Promoters commonly used for gene therapy include the cytomegalovirus immediate early (CMV-IE) promoter, the Rous sarcoma virus long terminal repeat (RSV-LTR), the Moloney murine leukemia virus (MoMLV) LTR, and the LTR promoters of other retroviruses. Eukaryotic promoters can be used for gene therapy, and general examples of eukaryotic promoters include the human α1-antitrypsin (hAAT) and murine RNA polymerase II (large subunit) promoters. Non-tissue-specific promoters such as the small nuclear RNA U1b promoter, the EF1α promoter, and the PGK1 promoter are also available for use in gene therapy. Tissue-specific promoters such as Apo A-I, ApoE, and α1-antitrypsin (hAAT) enable tissue-specific expression of the protein of interest in gene therapy. Table I of Papadakis et al. (Promoters and Control Elements: Designing Expression Cassettes for Gene Therapy, Current Gene Therapy, 2004, 4, 89-113) lists examples of transcriptional targeting using eukaryotic promoters in gene therapy, all of which are hereby incorporated by reference in their entirety.

[0365] Dosage and administration mode The AAV titer is expressed as the "physical" titer of vector or viral genome per milliliter (vg / ml) or per kilogram dosage of vector or viral genome (vg / kg). The titer can be determined using QPCR of purified vector particles. One method for measuring the titer of AAV VG number is as follows: The purified AAV vector sample is first treated with DNase to remove AAV genomic DNA not encapsulated by the capsid or contaminating plasmid DNA from the production process. The DNase-resistant particles are then subjected to heat treatment to release the genome from the capsid. The released genome is quantified by real-time PCR using a primer / probe set that targets a specific region of the viral genome.

[0366] The viral composition can be formulated in dosage units to contain an amount of viral vector in the range of about 1.0×10 9 vg / kg to about 1.0×10 15 vg / kg, preferably 1.0×10 12 vg / kg to 1.0×10 14 vg / kg for human patients. Preferably, the dosage of the virus in the formulation is 1.0×10 9 vg / kg, 5.0×10 9 vg / kg, 1.0×10 10 vg / kg, 5.0×10 10 vg / kg, 1.0×10 11 vg / kg, 5.0×10 11 vg / kg, 1.0×10 12 vg / kg, 5.0×10 12 vg / kg, or 1.0×10 13 vg / kg, 5.0×10 13 vg / kg, 1.0×10 14 vg / kg, 5.0×10 14 vg / kg, or 1.0×10 15 vg / kg, or 5.0×10 15 vg / kg.

[0367] In some embodiments, in the context of the present invention, the dosage administered to a mammal, particularly a human, will vary depending on the particular viral vector, vector and composition therefor (described above), as well as the mode of administration. The dosage is sufficient to produce a desired response, e.g., a therapeutic or prophylactic response, within a desired time frame. In terms of the viral vector, the dosage can be up to 1×10 15 vg / kg.

[0368] The vectors of the present invention enable long-term gene expression, and as a result, provide a long-term effect of the therapeutic protein. The phrases "long-term expression", "continuous expression" and "sustained expression" are used interchangeably. Long-term expression according to the present invention preferably means expression of a therapeutic gene and / or protein at a therapeutic level for at least 45 days, at least 60 days, at least 90 days, at least 120 days, at least 180 days, at least 250 days, at least 360 days, at least 450 days, at least 730 days or more. Preferably, long-term expression means expression for at least 90 days, at least 120 days, at least 180 days, at least 250 days, at least 360 days, at least 450 days, at least 720 days or more, more preferably, at least 360 days, at least 450 days, at least 720 days or more. This long-term expression can be achieved by repeated dosages (if possible) or by a single dosage.

[0369] Repeated dosages can be administered twice a day, once a day, twice a week, once a week, once a month, every two months, every three months, every four months, every six months, once a year, every two years, or more. The dosing can continue for as long as necessary, e.g., for at least six months, at least one year, two years, three years, four years, five years, ten years, fifteen years, twenty years or more, up to the lifetime of the patient being treated.

[0370] The pharmaceutical composition according to the present invention can be administered locally or systemically, intramuscularly, intravenously, and parenterally. Delivery of the therapeutic composition according to the present invention can be directed to the central nervous system, the cardiovascular system, and the pulmonary system. A common delivery strategy is direct intramuscular injection. As a non-limiting example, skeletal muscle has been shown to be a target tissue type that is efficiently transduced. Once transduced, muscle cells serve as a site of production of protein production that can act locally or systemically by many AAV variants.

[0371] In one embodiment, the pharmaceutical composition is administered near the tissue or organ whose cells are to be transduced. In certain embodiments, the pharmaceutical composition according to the present invention is administered locally to the liver by injection into the liver parenchyma. In another embodiment, the pharmaceutical composition according to the present invention is administered systemically.

[0372] As a non-limiting example, systemic administration includes systemic injection of the AAV vector according to the present invention, such as intramuscular (im), intravascular (ie), intraarterial (ia), intravenous (iv), intraperitoneal (ip), or subcutaneous injection. Preferably, systemic administration is via im, ip, is, or iv injection. In some embodiments, the AAV vector according to the present invention is administered via intravenous injection.

[0373] In another embodiment, the pharmaceutical composition according to the present invention is delivered to the liver of a subject. Administration to the liver is achieved using methods known in the art, such as, but not limited to, intravenous administration, portal vein administration, intra-biliary administration, intra-arterial administration, and direct injection into the liver parenchyma. In another embodiment, the pharmaceutical composition is administered intravenously.

[0374] The pharmaceutical composition according to the present invention can be administered in a single dose, or in certain embodiments according to the present invention, a plurality of doses (e.g., 2, 3, 4 or more administrations) can be used to achieve a therapeutic effect. Preferably, the AAV vectors contained in the pharmaceutical composition according to the present invention are derived from different serotypes when multiple doses are required to overcome the effects of neutralizing antibodies.

[0375] Formulation The preparation can also contain a buffer salt. Alternatively, the composition may be in powder form for constitution with a suitable vehicle (e.g., sterile water free of pyrogens) before use. If necessary, the composition can also contain a local anesthetic such as lidocaine to relieve pain at the injection site. If the composition is intended to be administered by infiltration, this can be dispensed in an infiltration bottle containing pharmaceutical quality water or saline. If the composition is to be administered by injection, a water vial for injection or sterile saline can be provided so that the components can be mixed before administration. Preferably, the pharmaceutically acceptable carrier is saline and a surfactant such as Pluronic®.

[0376] The compositions according to the invention can be formulated for delivery to animals for veterinary purposes (e.g., livestock (such as cows, pigs, etc.) and other non-human mammals), as well as to human subjects. The AAV vectors can be formulated with a physiologically acceptable carrier for use in gene transfer and gene therapy applications. By way of non-limiting example, the use of adjuvants in combination with, or mixed with, the AAV vectors according to the invention is also encompassed. Intended adjuvants include, but are not limited to, inorganic salt adjuvants or inorganic salt gels, particulate adjuvants, microparticulate adjuvants, mucosal adjuvants. The adjuvant can be administered to the subject as a mixture with the AAV vectors according to the invention or can be used in combination with said AAV vectors.

[0377] The terms "pharmaceutically acceptable carrier", "pharmaceutically acceptable diluent", "pharmaceutically acceptable excipient", or "pharmaceutically acceptable vehicle" are used interchangeably herein and refer to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, or any conventional type of auxiliary preparation. A pharmaceutically acceptable carrier is essentially non-toxic to the recipient at the dosages and concentrations employed and is compatible with the other ingredients of the formulation. The number and nature of pharmaceutically acceptable carriers depend on the desired dosage form. Pharmaceutically acceptable carriers are known and can be prepared by methods well known in the art (Fauli i Trillo C, "Tratado de Farmacia Galenica". Ed. Luzan 5, S.A., Madrid, ES, 1993; Gennaro A, Ed., "Remington: The Science and Practice of Pharmacy" 20th ed. Lippincott Williams & Wilkins, Philadelphia, Pa., US, 2003).

[0378] By way of non-limiting example, AAV vectors can be formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Injectable formulations can be provided in unit dosage forms (e.g., ampoules or multi-dose containers) with added preservatives. The viral composition can take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and can contain formulating agents such as suspending, stabilizing or dispersing agents. Liquid preparations of AAV formulations can be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifying agents (e.g., lecithin or gum arabic), non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils), and preservatives (e.g., methyl or propyl-p-hydroxybenzoic acid or sorbic acid).

[0379] Formulations suitable for parenteral administration can include aqueous and non-aqueous isotonic sterile injection solutions which can contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of a given recipient, as well as aqueous and non-aqueous sterile suspensions which can contain suspending agents, solubilizing agents, thickening agents, stabilizers and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and can be stored in a freeze-dried (lyophilized) state which requires only the addition of sterile liquid excipients, for example water, immediately prior to use for injection. Immediate injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the above-described types.

[0380] Furthermore, the composition can include additional therapeutic or biologically active agents. For example, there can be present therapeutic factors useful in the treatment of specific indications. Factors which control inflammation, for example, ibuprofen or steroids, can be part of the composition to reduce swelling and inflammation and physical pain associated with in vivo administration of the vector. Immunosuppressors can be administered in the composition in a manner to reduce any immune response against the vector itself or associated with the disorder. Administration of immunosuppressive pharmaceuticals or immunosuppressive agents is the primary method of immunosuppression induced in a planned manner and, in an optimal situation, the immunosuppressive drug targets only any hyperactive component of the immune system.

[0381] Immunosuppressive drugs, immunosuppressants, or antirejection drugs are drugs that inhibit or prevent the activity of the immune system. Such drugs include glucocorticoids, cell division inhibitors, antibodies, and drugs that act on immunophilins. At pharmacological (supraphysiological) doses, glucocorticoids such as prednisone, dexamethasone, and hydrocortisone are used to suppress various allergic and inflammatory reactions. Cell division inhibitors such as purine analogs, alkylating agents such as nitrogen mustard (cyclophosphamide), nitrosoureas, platinum compounds, etc. Cyclophosphamide (Baxter's Cytoxan) is perhaps the most potent immunosuppressive compound. Metabolic antagonists such as folic acid analogs such as methotrexate, purine analogs such as azathioprine and mercaptopurine, pyrimidine analogs such as fluorouracil, and protein synthesis inhibitors. Cytotoxic antibiotics, among which dactinomycin is the most important. This is used in kidney transplantation. Other cytotoxic antibiotics are anthracyclines, mitomycin C, bleomycin, mitramycin. Antibodies may also be used as a rapid and potent immunosuppressive therapy to prevent acute rejection reactions (e.g., anti-CD20 monoclonal).

[0382] Alternatively, immunostimulants can be included in the composition to upregulate the body's natural defenses against the disease.

[0383] Antibiotics, i.e., bactericidal and fungicidal drugs, may be present to reduce the risk of infections associated with gene transfer procedures and other complications.

[0384] The pharmaceutical composition can be formulated according to routine procedures as a pharmaceutical composition suitable for intravenous, subcutaneous, or intramuscular administration to humans.

[0385] The treatment method according to the present invention As a non-limiting example, a viral vector encoding human ENPP1 or ENPP3 is administered to a mammal, resulting in delivery of DNA encoding ENPP1 or ENPP3 and expression of the protein in the mammal, thereby restoring the levels of ENPP1 or ENPP3 required to reduce soft tissue calcification or ossification.

[0386] In one aspect, the invention relates to an adeno-associated viral vector comprising a recombinant viral genome, wherein the recombinant viral genome comprises an expression cassette comprising a transcriptional control region operably linked to a nucleotide sequence encoding ENPP1 or ENPP3 or a functionally equivalent variant thereof, or a pharmaceutical composition comprising said viral vector for use in the treatment and / or prevention of diseases of pathological calcification or ossification.

[0387] In another aspect, the invention relates to an adeno-associated viral vector comprising a recombinant viral genome for use in the manufacture of a medicament for the treatment and / or prevention of diseases of pathological calcification or ossification, wherein the recombinant viral genome comprises an expression cassette comprising a transcriptional control region operably linked to a nucleotide sequence encoding ENPP1 or ENPP3 or a functionally equivalent variant thereof, or the use of a pharmaceutical composition comprising said viral vector.

[0388] In another aspect, the invention provides a method for the treatment and / or prevention of diseases of pathological calcification or ossification in a subject in need thereof, which comprises administering to the subject an adeno-associated viral vector comprising a recombinant viral genome, wherein the recombinant viral genome comprises an expression cassette comprising a transcriptional control region operably linked to a nucleotide sequence encoding ENPP1 or ENPP3 or a functionally equivalent variant thereof, or a pharmaceutical composition comprising said viral vector.

[0389] In another aspect, the diseases of pathological calcification or ossification treated by the compositions and methods of the present invention are selected from the group consisting of X-linked hypophosphatemia (XLH), chronic kidney disease (CKD), mineral bone disorder (MBD), vascular calcification, pathological calcification of soft tissue, pathological ossification of soft tissue, generalized arterial calcification in infants (GACI), and ossification of the posterior longitudinal ligament (OPLL).

[0390] Polynucleotides, vectors and plasmids according to the present invention The present invention also relates to polynucleotides useful for generating viral vectors, such as AAV vectors according to the present invention. In one embodiment, the present invention relates to a polynucleotide comprising an expression cassette adjacent to an adeno-associated virus ITR (a "polynucleotide according to the present invention"), wherein the expression cassette comprises a transcription control region operably linked to a nucleotide sequence encoding ENPP1 or ENPP3 or a functionally equivalent variant thereof.

[0391] In one embodiment, the polynucleotide according to the present invention comprises a transcription control region comprising a promoter, preferably a constitutive promoter, more preferably a liver-specific promoter, more preferably a liver-specific promoter selected from the group consisting of albumin promoter, phosphoenolpyruvate carboxykinase (PEPCK) promoter and alpha1-antitrypsin promoter (most preferably the human alpha1-antitrypsin promoter). In another embodiment, the transcription control region of the polynucleotide according to the present invention further comprises an enhancer operably linked to the promoter, preferably a liver-specific enhancer, more preferably a liver control region enhancer (HCR).

[0392] In another embodiment, the expression cassette of the polynucleotide according to the present invention further comprises a polyadenylation signal, more preferably SV40 polyA. In another embodiment, the ENPP1 encoded by the polynucleotide according to the present invention is selected from the group consisting of human ENPP1 and human ENPP3.

[0393] The polynucleotide according to the present invention can be incorporated into a vector such as a plasmid. Thus, in another aspect, the present invention relates to a vector or plasmid comprising the polynucleotide according to the present invention. In certain embodiments, the polynucleotide according to the present invention is incorporated into an adeno-associated virus vector or plasmid.

[0394] Preferably, all other structural and non-structural coding sequences necessary for the production of adeno-associated virus can be provided in trans by another vector, such as a plasmid, or by stably integrating the sequences into a packaging cell line, so that they are not present in the viral vector.

[0395] Method for obtaining AAV according to the present invention The present invention also relates to a method for obtaining a viral vector according to the present invention, such as an AAV vector by way of non-limiting example. The AAV vector can be obtained by constitutively expressing the Rep and Cap proteins, or by introducing the polynucleotide according to the present invention into a cell in which the Rep and Cap coding sequences are provided in a plasmid or vector. Thus, in another aspect, the present invention relates to a method for obtaining an adeno-associated virus vector, comprising the following steps: (i) providing a cell comprising the polynucleotide according to the present invention, an AAV Cap protein, an AAV Rep protein, and optionally a viral protein on which AAV depends for replication; (ii) maintaining the cell under conditions appropriate for AAV assembly; and (iii) purifying the adeno-associated virus vector produced by the cell.

[0396] The generation of recombinant AAV (rAAV) for vectorizing transgenes has been previously described (Ayuso E, et al., Curr. Gene Ther. 2010, 10:423-436; Okada T, et al., Hum. Gene Ther. 2009, 20:1013-1021; Zhang H, et al., Hum. Gene Ther. 2009, 20:922-929; and Virag T, et al., Hum. Gene Ther. 2009, 20:807-817). These protocols can be used or adapted to generate AAV according to the present invention. Any cell capable of producing adeno-associated virus vectors can be used in the present invention, including mammalian cells and insect cells.

[0397] In one embodiment, the producer cell line is transiently transfected with a polynucleotide according to the present invention (including an expression cassette adjacent to the ITR) and constructs (s) encoding Rep and Cap proteins and providing helper functions. In another embodiment, the cell line stably supplies helper functions and is transiently transfected with a polynucleotide according to the present invention (including an expression cassette adjacent to the ITR) and constructs (s) encoding Rep and Cap proteins.

[0398] In another embodiment, the cell line stably supplies Rep and Cap proteins and helper functions and is transiently transfected with a polynucleotide according to the present invention. In another embodiment, the cell line stably supplies Rep and Cap proteins and is transiently transfected with a polynucleotide according to the present invention and a polynucleotide encoding helper functions. In yet another embodiment, the cell line stably supplies a polynucleotide according to the present invention, Rep and Cap proteins, and helper functions. These and other methods of manufacturing and using AAV systems are described in the art.

[0399] In another embodiment, the producer cell line is an insect cell line (typically Sf9 cells) infected with a baculovirus expression vector that provides the Rep and Cap proteins. This system does not require adenovirus helper genes (Ayuso E, et al., Curr. Gene Ther. 2010, 10:423-436).

[0400] In another embodiment, the transgene delivery ability of AAV can be increased by providing two genomic AAV ITRs that can anneal to form head-to-tail concatemers. Generally, upon entry of AAV into a host cell, the single-stranded DNA containing the transgene is converted to double-stranded DNA by the host cell's DNA polymerase complex, and then the ITRs assist in concatemer formation in the nucleus. As an alternative, AAV can be engineered to be self-complementary (sc) AAV, which allows the viral vector to bypass the step of second-strand synthesis upon entry into the target cell, providing an scAAV viral vector with faster and potentially higher (e.g., up to 100-fold) transgene expression.

[0401] For example, AAV can be engineered to have a genome containing two joined single-stranded DNAs that each encode a transgene unit and its complement, and these single-stranded DNAs can snap together after delivery into the target cell to yield a double-stranded DNA encoding the transgene unit of interest. Self-complementary AAVs are described in the art (Carter B, U.S. Patent No. 6,596,535, Carter B, U.S. Patent No. 7,125,717, and Takano H et al., U.S. Patent No. 7,456,683).

[0402] Preferably, since all structural and non-structural coding sequences (Cap protein and Rep protein) can be provided in trans by a vector, such as a plasmid, they are not present in the AAV vector. The Cap protein has been reported to affect the host tropism, cell, tissue or organ specificity, receptor usage, infection efficiency and immunogenicity of AAV virus. Thus, AAV Cap for use in rAAV can be selected, for example, considering the species of the subject (e.g., human or non-human), the immunological status of the subject, the suitability of the subject for long-term or short-term treatment, or a specific therapeutic use (e.g., treatment of a specific disease or disorder, or delivery to a specific cell, tissue or organ).

[0403] In another embodiment, the Cap protein is derived from an AAV of the group consisting of AAV2, AAV5, AAV7, AAV8, AAV9, AAV10 and AAVrh10 serotypes. In another embodiment, the Cap protein is derived from AAV8.

[0404] In some embodiments, the AAV Cap for use in the method according to the invention can be generated by mutagenesis of one of the aforementioned AAV Caps or its coding nucleic acid (i.e., by insertion, deletion or substitution). In some embodiments, the AAV Cap is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% or more similar to one or more of the aforementioned AAV Caps.

[0405] In some embodiments, the AAV Cap is a chimera containing domains from two, three, four or more of the aforementioned AAV Caps. In some embodiments, the AAV Cap is a mosaic of VP1, VP2 and VP3 monomers from two or three different AAVs or recombinant AAVs. In some embodiments, the rAAV composition contains more than one of the aforementioned Caps.

[0406] In some embodiments, the AAV Cap for use in rAAV compositions is engineered to include heterologous sequences or other modifications. For example, peptide or protein sequences that confer selective targeting or immune evasion can be engineered into the Cap protein. Alternatively, or in addition, the Cap can be chemically modified such that the surface of the rAAV is polyethylene glycolated (i.e., pegylated), which can facilitate immune evasion. The Cap protein can also be mutagenized (e.g., to remove its native receptor binding or to mask immunogenic epitopes).

[0407] In some embodiments, the AAV Rep protein for use in the methods according to the invention can be generated by mutagenesis of one of the aforementioned AAV Reps or its coding nucleic acid (i.e., by insertion, deletion, or substitution). In some embodiments, the AAV Rep is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% or more similar to one or more of the aforementioned AAV Reps.

[0408] In another embodiment, the AAV Rep and Cap proteins are derived from an AAV serotype selected from the group consisting of AAV2, AAV5, AAV7, AAV8, AAV9, AAV10, and AAVrh10.

[0409] In some embodiments, the viral protein on which AAV depends for replication for use in the methods according to the invention can be generated by mutagenesis of one of the aforementioned viral proteins or its coding nucleic acid (i.e., by insertion, deletion, or substitution). In some embodiments, the viral protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% or more similar to one or more of the aforementioned viral proteins.

[0410] Methods for assaying the functions of the Cap protein, Rep protein, and viral proteins on which AAV depends for replication are well known in the art. The genes providing the AAV rep, AAV cap, and helper functions can be inserted into cells by incorporating the genes into a vector, such as a plasmid, and introducing the vector into the cells. The genes can be incorporated into the same plasmid or different plasmids. In another embodiment, the AAV rep and cap genes are incorporated into one plasmid, and the genes providing the helper functions are incorporated into another plasmid. Examples of plasmids containing the AAV rep and cap genes suitable for use with the method according to the present invention include the pHLP19 and pRep6cap6 vectors (Colisi P, US Patent No. 6,001,650 and Russell D et al., US Patent No. 6,156,303).

[0411] The polynucleotides according to the present invention, as well as polynucleotides containing the AAV rep and cap genes or genes providing helper functions, can be inserted into cells by any suitable method well known in the art. Examples of transfection methods include, but are not limited to, co-precipitation with calcium phosphate, DEAE-dextran, polybrene, electroporation, microinjection, liposome-mediated fusion, lipofection, retroviral infection, and biolistic transfection. In a particular embodiment, transfection is performed by co-precipitation with calcium phosphate. If the cells lack the expression of any of the AAV rep and cap genes and the genes providing the helper functions of adenovirus, the genes can be inserted into the cells simultaneously with the polynucleotides according to the present invention.

[0412] Alternatively, the genes can be inserted into the cells before or after the introduction of the polynucleotides according to the present invention. In a particular embodiment, the cells are transfected simultaneously with the following three plasmids: 1) A plasmid containing the polynucleotide according to the present invention 2) A plasmid containing the AAV rep and cap genes 3) A plasmid containing a gene that provides helper functions.

[0413] Alternatively, the AAV rep and cap genes and the gene that provides helper functions can be episomally and / or integrated into the genome of the packaging cell and carried by the packaging cell.

[0414] The present invention encompasses methods that include maintaining cells under conditions appropriate for AAV assembly. Methods of culturing packaging cells and exemplary conditions that promote the release of AAV vector particles, such as the generation of cell lysates, can be carried out as described in the examples herein. Producer cells are grown for an appropriate period to promote AAV assembly and the release of viral vectors into the medium. Generally, cells can be grown for about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, up to about 10 days maximum. After about 10 days (or earlier depending on the culture conditions used and the particular producer cells), the level of production generally decreases significantly. Generally, the culture time is measured from the time of virus production. For example, in the case of AAV, virus production generally begins upon supplying helper virus functions in appropriate producer cells as described herein. Generally, cells are harvested about 48 to about 100, preferably about 48 to about 96, preferably about 72 to about 96, preferably about 68 to about 72 hours after infection with the helper virus (or after virus production has started).

[0415] The present invention encompasses a method for purifying adeno-associated virus vectors produced by cells. The AAV according to the present invention can be obtained from both i) cells transfected with the polynucleotide according to the present invention and ii) the culture medium of said cells at a certain period after transfection, preferably 72 hours after transfection. Any method for purifying AAV from said cells or said culture medium can be used to obtain the AAV according to the present invention. In a particular embodiment, the AAV according to the present invention is purified according to a method optimized based on a polyethylene glycol precipitation step and two consecutive cesium chloride (CsCl) gradients. The purified AAV according to the present invention can be dialyzed against PBS, filtered, and stored at -80°C. The titer of the viral genome can be determined by quantitative PCR according to the protocol described for the AAV2 reference standard using linearized plasmid DNA as a standard curve (Lock M, et al., Hum. Gene Ther. 2010;21:1273-1285).

[0416] In another embodiment, purification is further performed by a polyethylene glycol precipitation step or cesium chloride gradient fractionation. In some embodiments, this method further includes purification steps, such as treatment of cell lysates with benzonase, purification of cell lysates by CsCl gradient, or purification of cell lysates using heparin sulfate chromatography (Halbert C, et al., Methods Mol. Biol. 2004;246:201-212).

[0417] Methods for isolating or generating, propagating, and purifying various naturally occurring and recombinant AAVs, their coding nucleic acids, AAV Cap and Rep proteins and their sequences, and such AAVs, particularly their capsids suitable for use in the production of AAV, are known in the art.

[0418] Animal models The following are non-limiting animal models that can be used to test the effectiveness of administering ENPP1 or ENPP3 to prevent or reduce the progression of pathological ossification or calcification. 1. Enpp1 of Generalized Arterial Calcification in Infancy (GACI); asj / asj Model; Li, et al., 2013, Disease Models & Mech. 6(5):1227-35. 2. Enpp1 of Generalized Arterial Calcification in Infancy (GACI); asj / asj Model; Li, et al, 2014, PloS one 9(12):e113542. 3. ABCC6 of Pseudoxanthoma Elasticum (PXE); - / - Mouse model; Jiang, et al., 2007, J.Invest.Derm. 127(6):1392-4102. 4. HYP mouse model of X-linked Hypophosphatemia (XLH); Liang, et al., 2009, Calcif.Tissue Int. 85(3):235-46. 5. LmnaG609G / + mouse model of Hutchinson-Gilford Progeria Syndrome; Villa-Bellosta, et al, 2013, Circulation 127(24):2442-51. 6. Tip toe walking (ttw) mouse models of Ossification of the Posterior Longitudinal Ligament (OPLL) (Okawa, et al, 1998, Nature Genetics 19(3):271-3; Nakamura, et al, 1999, Human Genetics 104(6):492-7) and Osteoarthritis (Bertrand, et al, 2012, Annals Rheum.Diseases 71(7):1249-53). 7. Rat model of Chronic Kidney Disease (CKD) by adenine diet; Schibler, et al., 1968, Clin.Sci. 35(2):363-72; O’Neill, et al, 2011, Kidney Int. 79(5):512-7. 8. Mouse model of chronic kidney disease (CKD) by adenine diet; Jia, et al., 2013, BMC Nephrol. 14:116. 9. 5 / 6 of CKD th Nephrectomized rat model; Morrison, 1962, Lab Invest. 11:321 - 32; Shimamura & Morrison, 1975, Am. J. Pathol. 79(1):95 - 106. 10. ENPP1 knockout mouse model of GACI and osteoporosis; Mackenzie, et al, 2012, PloS one 7(2):e32177.

[0419] Animal models as described above are used to test for changes in soft tissue calcification and ossification upon administration of vectors encoding ENPP1 or ENPP3 according to the present invention. For example, the following mouse models: (a) Npt2a - / - (b) Double mutant Npt2a - / - / Enpp1 asj / asj and (c) C57BL / 6 mice (Jackson Labs) subjected to diet - induced kidney stone formation (the diet is a high - calcium, low - magnesium diet (e.g., Teklad Labs diet TD.00042, Harlan Labs, Madison, WI)).

[0420] Npt2a - / - Mice show kidney stone formation when fed a normal solid diet from weaning age and maintained for at least 10 weeks of age. Conversely, double mutant Npt2a - / - / Enpp1 asj / asj mice exhibit a two - fold higher level of kidney stone formation compared to Npt2a - / - mice when fed a normal solid diet. Npt2a - / - mice and Npt2a - / - / Enpp1 asj / asj mice are commercially available from Jackson laboratory, ME. Double mutant mice (Npt2a - / - / Enpp1 asj / asj) was prepared by cross-breeding Npt2a - / - mice with Enpp1 asj / asj mice according to standard protocols known in the art (Jackson Laboratory Recourse Manual, (2007, 1-29)). Npt2a for kidney stone-related diseases - / - or Npt2a - / - / Enpp1 asj / asj double mutant mouse models can be used to test the effectiveness of the treatment according to the present invention (Khan & Canales, 2011, J.Urol. 186(3):1107-13; Wu, 2015, Urolithiasis 43(Suppl 1):65-76). Oxalate stone-forming rodent models, i.e., mice or rats given ethylene glycol, hydroxyl purine, or intraperitoneal injection of sodium oxalate in mice and rats (Khan & Glenton, J.Urology 184:1189-1196), uric acid stone formation (Wu, et al., 1994, Proc.Natl.Acad.Sci.USA 91(2):742-6), and cystinuria mouse models (Zee, et al., 2017, Nat.Med. 23(3):288-290; Sahota, et al., 2014, Urology 84(5):1249 e9-15) can also be tested.

[0421] In certain embodiments, there is no rodent model in the literature that recapitulates the adult form of the human disease GACI, also known as autosomal recessive hypophosphatemic rickets type 2 (ARHR2) (Levy-Litan, et al, 2010, Am.J.Human Gen. 86(2):273-8.).

[0422] Details of experiments regarding the enzymatic activity of ENPP1, the enzymatic activity of ENPP3, the quantification of plasma PPi, micro-CT scans, and the quantification of plasma PPi uptake are described in detail in the patent applications and publications of PCT / US2016 / 33236 - Braddock et al., WO2014 / 126965 - Braddock et al., WO2017 / 087936 - Braddock et al., and US2015 / 0359858 - Braddock et al., all of which are hereby incorporated by reference in their entirety.

[0423] The present invention is further illustrated by the following examples, which should in no way be construed as limiting. The contents of all cited reference documents (including references, registered patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference into this specification.

Examples

[0424] Example: 1 - Cloning of the NPP1 sequence into the AAV system, generation of constructs for AAV infection, production, and purification of AAV The AAV plasmid used in this example contains an expression cassette flanked by two ITRs derived from AAV2. The genome of AAV2 can be pseudotyped with AAV8. The expression cassette can have the following elements in the 5' to 3' direction: a liver-specific enhancer, a liver control region (HCR), a liver-specific promoter, human alpha-1 antitrypsin (hAAT), an intron, a polypeptide containing an N-terminal azurocidin signal sequence, NPP1 cDNA, a C-terminal Fc sequence, and an SV40 polyadenylation signal. The expression cassette is flanked by the 5' ITR and 3' ITR derived from AAV2. The generated construct is shown in the schematic diagram of Figure 1.

[0425] The ENPP1 protein is a transmembrane protein that localizes to the cell surface using a unique intramembrane domain. The ENPP1 protein was made soluble by removing the transmembrane domain. Human NPP1 (NCBI accession NP_006199) was modified to express a soluble recombinant protein by replacing its transmembrane region (e.g., residues 77 - 98 of ENPP1, NCBI accession NP_006199) with an appropriate signal peptide sequence selected from the group consisting of (a) residues 12 - 30 of human NPP2 (NCBI accession NP_001124335), or (b) residues 1 - 22 of ENPP7, or (c) residues 1 - 24 of ENPP5, or (d) human serum albumin, or (e) human azurocidin.

[0426] Sequence numbers (1 - 4, 6 - 15, 17 - 31 and 42 - 56) represent several ENPP1 - Fc and ENPP3 - Fc constructs, all of which can be used for cloning of the ENPP1 or ENPP3 sequences into the AAV system and for generation of constructs for AAV infection.

[0427] Using standard molecular biology protocols, the modified NPP1 sequence was cloned into a plasmid. Null particles were produced as a control using a non - coding plasmid that carried the same components of the construct but lacked the NPP1 cDNA and had a multiple cloning site.

[0428] Infectious AAV vector particles were generated in Hek293 cells cultured in roller bottles by co-transfecting each roller bottle with 125 μg of a vector plasmid (containing ITRs and an expression cassette), 125 μg of a rep / cap plasmid (expressing the capsid protein of the AAV particle and the proteins necessary for viral replication), and 150 μg of a helper plasmid expressing the helper functions of adenovirus by calcium phosphate co-precipitation. A total of 10 roller bottles were used for each vector preparation. Approximately 3 days after transfection, the cells were harvested and centrifuged at 2500 g for 10 minutes. The cell pellet and the medium were then processed separately. The cell pellet was completely reconstituted in TBS (50 mM TrisHCl, 150 mM NaCl, 2 mM MgCl2, pH 8.0).

[0429] After 3 freeze / thaw cycles, the solubilized solution is centrifuged at 2500 g for 30 minutes. The supernatant from this centrifugation is added to the medium and the vector particles are precipitated by incubation with 8% PEG8000 (Sigma) for 15 hours and pelleted at 2500 g for 30 minutes. The pellet containing the vector from the cells and medium is completely reconstituted in TBS, treated with Benzonase (Merck) at 37 °C for 30 minutes and centrifuged at 10,000 g for 10 minutes. The supernatant is loaded into a 37.5 ml UltraClear tube (Beckman) containing a CsCl density step gradient of 1.3 - 1.5 g / ml and centrifuged at 28,000 rpm for 17 hours in an SW28 rotor (Beckman). The virus band is collected using a 10 ml syringe and an 18 gauge needle and transferred to a new 12.5 ml UltraClear tube which is filled with a CsCl solution of 1.379 g / ml to generate a continuous gradient. The tube is centrifuged at 38,000 rpm for 48 hours in an SW40Ti rotor (Beckman). Finally, the band of complete particles is collected, dialyzed against PBS using a 10 KDa membrane (Slide-A-Lyzer Dialysis Products, Pierce) and filtered through a 0.45 μm Millipore filter. This PEG and CsCl-based purification protocol dramatically reduces empty AAV capsids as well as DNA and protein impurities from the virus stock, thus enhancing AAV purity and ultimately resulting in higher transduction in vivo. The same protocol is used to generate infectious AAV particles carrying a "null" vector that does not encode any ENPP protein.

[0430] Example - 2 - Expression of ENPP1 using different signal sequences ENPP1 is produced by establishing stable transfection in CHO or HEK293 mammalian cells. To establish a stable cell line, a nucleic acid sequence encoding an ENPP1 fusion protein (e.g., the sequences disclosed elsewhere in this specification) is placed into a vector suitable for large-scale protein production. There are various such vectors available from commercial suppliers.

[0431] For example, Figure 3 shows NPP2 シグナル -NPP1-Fc cloned into the pcDNA3 plasmid using appropriate endonuclease restriction sites, NPP7 シグナル -NPP1-Fc cloned into the pcDNA3 plasmid, and azurocidin シグナル -NPP1-Fc plasmid maps. The pcDNA3 plasmid containing the desired protein construct is stably transfected into an expression plasmid using established techniques such as electroporation or lipofectamine, and the cells are grown under antibiotic selection to expand the stably transfected cells.

[0432] Next, clones of single stably transfected cells are established and high-expressing clones of the desired fusion protein are screened. Screening of single cell clones for ENPP1 protein expression is performed in 96-well plates in high-throughput using the synthetic enzyme substrate pNP-TMP, as previously described for ENPP1 (Saunders, et al., 2008, Mol. Cancer Ther. 7(10):3352-62; Albright, et al., 2015, Nat Commun. 6:10006).

[0433] Upon identifying high-expressing clones through screening, protein production is carried out in shake flasks or using a bioreactor as previously described for ENPP1 (Albright, et al., 2015, Nat Commun. 6:10006). Purification of ENPP1 is performed using a combination of standard purification techniques known in the art.

[0434] As shown in Figure 2, the construct containing the azurocidin signal sequence produces the highest amount of NPP1 protein. The amount of ENPP1 protein produced using the azurocidin signal sequence (731 mg / liter) was surprisingly 5-fold higher compared to the ENPP1 protein produced using NPP2 (127 mg / liter) or the NPP7 (136 mg / liter) signal sequence. The ENPP1 protein thus produced is further purified using additional techniques and / or chromatographic steps as described above to reach a substantially higher purity, such as about 99% purity.

[0435] The enzymatic activity of ENPP1 thus produced is measured by determining the steady-state hydrolysis of ATP by human NPP1 using HPLC. Briefly, 20 mM Tris, pH 7.4, 150 mM NaCl, 4.5 nM KCl, 14 μM ZnCl 2 , 1 mM MgCl 2 and 1 mM CaCl 2The enzyme reaction is initiated by adding 10 nM ENPP1 to various concentrations of ATP in a reaction buffer containing []. At various time points, 50 μl of the reaction solution is removed and quenched with an equivalent amount of 3 M formic acid. The quenched reaction solution is loaded onto a C-18 (5 μm, 250 × 4.6 mm) column (Higgins Analytical) equilibrated in a 5 mM ammonium acetate (pH 6.0) solution and eluted with a 0% to 20% methanol gradient. The substrate and product are monitored by UV absorbance at 259 nm and quantified by integration of their corresponding peaks and a calibration curve. Thus, the ENPP1 protein is characterized according to the protocol discussed herein and in other places such as PCT / 2014 / 015945 - Braddock et al.; PCT / 2016 / 033236 - Braddock et al., and PCT / 2016 / 063034 - Braddock et al.

[0436] Example - 3 - Injection of AAV virus particles encoding ENPP1 - Fc into mice and measurement of body weight gain, bone density, bone strength, and bone mass Mouse models, for example, Enpp1 asj / asj Mouse models, ABCC6 - / - Using a mouse model, such as an Enpp1 mouse model, an ABCC6 mouse model, a HYP mouse model, a ttw mouse model, a mouse model of chronic kidney disease (CKD), or a 5 / 6th nephrectomized rat model of CKD, test the effectiveness of delivery of a vector capable of encoding and expressing NPP1 or NPP3. As a non - limiting example, the following experiments use Enpp1 as a mouse model asj / asj Mice are used with an azurocidin - NPP1 - Fc construct as the polynucleotide to be delivered to the mouse model, and delivery is performed in vivo by using AAV particles (prepared as shown in Example 1) encoding the ENPP1 - Fc protein.

[0437] To test the effectiveness of gene therapy for treating diseases of calcification or ossification, one of ordinary skill in the art will recognize that the same experiment can be repeated by using alternative mouse models, different ENPP1 fusion proteins disclosed in the present invention (such as ENPP1-albumin, or ENPP1-Fc, or ENPP1 functional equivalents, or ENPP1 lacking the Fc or albumin domain), or different ENPP3 fusion proteins (such as ENPP3-Fc, or ENPP3-albumin, or ENPP3 lacking the Fc or albumin domain, or ENPP3 functional equivalents), and alternative polynucleotide constructs containing alternative signal sequences (such as NPP2, NPP5, NPP7, albumin, or azurocidin). The azurocidin-NPP1-Fc construct used in the experiment encodes the human ENPP1-Fc protein as a proof of concept, and the same experiment can be repeated using the azurocidin-NPP3-Fc construct encoding human ENPP3-Fc.

[0438] In this experiment, four sets of mice were used, each set having at least five mice (6-8 weeks old). Before injection of AAV particles, the entire set of mice was tolerized by intraperitoneal injection of the Titer GK1.5 CD4 antibody at a concentration of 1000 μg / ml (final dose of 25-40 μg / animal) to reduce the immune response of the mice to the human proteins produced by the AAV construct, and ENPP1 served as a control group. wt AAV particles containing a null vector were injected into the first cohort of mice, and ENPP1 served as a control group. asj / asj AAV particles containing a null vector were injected into the second cohort of mice, and ENPP1 served as a study group. wt AAV particles containing a polynucleotide encoding the ENPP1-Fc protein were injected into the third cohort of mice, and ENPP1 served as a test group. asj / asjInject AAV particles containing a polynucleotide encoding the ENPP1-Fc protein into the fourth cohort. The tolerizing injections are repeated once a week (i.e., on days 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, and 105 after AAV administration) after the AAV injection into each cohort.

[0439] Give the experimental mice either an acceleration diet (Harlan Teklad, Rodent diet TD.00442, Madison, WI) in which phosphorus is enhanced and magnesium content is reduced or a normal solid diet (Laboratory Autoclavable Rodent Diet 5010; PMI Nutritional International, Brentwood, MO). After 6 - 8 weeks of age, about 1×10 12 ~1×10 15 vg / kg and preferably 1×10 13 ~1×10 14 vg / kgPerform retro-orbital injection or tail vein injection on all mice. The vector to be injected is either an empty "null" (control group) or one carrying the NPP1 gene (study group). Measure body weight once a day and record any increase or decrease in body weight after AAV injection. Collect blood, urine, bone, and tissue samples from the mice and analyze them as follows. The experimental protocol is described in detail in Albright et al., Nat Commun. 2015 Dec 1;6:10006, and Caballero et al., PLoS One. 2017;12(7):e0180098, and all of their contents are hereby incorporated by reference in their entirety. At the end of the study (on days 7, 28, and 112), euthanize all mice following retro-orbital blood collection under deep anesthesia with isoflurane and remove the organs necessary for life support as described in the art (Impaired urinary osteopontin excretion in Npt2a- / - mice., Caballero et al., Am J Physiol Renal Physiol. 2017 Jan 1;312(1):F77-F83; Response of Npt2a knockout mice to dietary calcium and phosphorus, Li Y et al., PLoS One. 2017;12(4):e0176232.).

[0440] Quantification of plasma PPi Use a heparinized micropipette to cause retro-orbital bleeding in the animals, dispense the blood into a heparinized Eppendorf tube, and place this on ice. Spin the sample at 4,000 r.p.m. for 5 minutes in a pre-cooled microcentrifuge at 4 °C, collect the plasma, and dilute it in 1 volume of 50 mM Tris-acetate pH = 8.0. Filter the collected plasma through a 300 KDa membrane by ultrafiltration (NanoSep 300K, Pall Corp., Ann Arbor, MI) and freeze it at -80 °C. Pyrophosphate is uridine 5'diphospho 14Quantify using a standard three-step enzymatic assay using glucose to record the reaction product, namely uridine 5'-diphospho 14 C] gluconic acid (Analysis of inorganic pyrophosphate at the picomole level. Cheung CP, Suhadolnik RJ, Anal Biochem. 1977 Nov;83(1):61-3). Briefly, a reaction mixture (100 μl) containing 5 mM MgCl2, 90 mM KCl, 63 mM Tris-HCl (pH 7.6), 1 nmol NADP+, 2 nmol glucose 1,6-diphosphate, 400 pmol uridine 5'-diphosphoglucose, 0.02 μCi uridine 5'-diphospho 14 C] glucose, 0.25 unit of uridine 5'-diphosphoglucose pyrophosphorylase, 0.25 unit of phosphoglucose mutase, 0.5 unit of glucose 6-phosphate dehydrogenase and inorganic pyrophosphate (50 - 200 pmol) was incubated at 37 °C for 30 minutes. The reaction was terminated by adding 200 μl of 2% charcoal thoroughly suspended in water. Then, a 200 μl aliquot of the supernatant was counted in a scintillation solution.

[0441] In vivo 99m PYP imaging Bone imaging can be performed as needed. The bone imaging agent 99mTc-pyrophosphate (Pharmalucence, Inc.) is evaluated in a cohort of animals using a preclinical micro SPECT / CT hybrid imaging system with a dual 1 mm pinhole collimator (X-SPECT, Gamma Medica-Ideas) 38. Each animal is intraperitoneally injected with 2 - 5 mCi of the radiolabeled tracer, and the animal is imaged 1 - 1.5 hours after injection. For anatomical co-registration with the SPECT images, a CT scan (512 projections at 50 kVp, 800 uA, and a magnification factor of 1.25) is obtained. SPECT imaging is obtained at 60 seconds per projection at 180° counterclockwise per collimator head, 32 projections, a ROR of 7.0 cm, a FOV of 8.95 cm, and an energy window of 140 keV ± 20. The CT images are reconstructed with FLEX X-O CT software (Gamma Medica-Ideas) using filtered backprojection. The SPECT images are reconstructed using FLEX SPECT software (5 iterations, 4 subsets), then fused with the CT images and analyzed using AMIRA software.

[0442] 99 Quantification of mPYP uptake 99 For scans of mice with mPYP, the animals are imaged within 7 days of injection. The resulting SPECT scans are imported into NIH's ImageJ image processing software, and regions of interest are drawn surrounding the head (target organ) and the whole body of each animal. The percent injected activity (PIA), often referred to as "percent injected dose", is calculated by comparing the ratio of head counts to whole body counts and expressed as percent injected dose to obtain a measure of the affinity by which the radioactive tracer is taken up by the region of interest (head). The total counts of each scan are considered as the whole body measure of the injected dose.

[0443] Blood and urine parameters Biochemical analyses can also be performed simultaneously at the same time between 10:00 am and 2:00 pm, using blood samples (obtained by orbital blood sampling) and spot urine collected after an overnight fast. Following protein removal from heparinized plasma by filtration (NanoSep 300 K, Pall Corp., Ann Arbor, MI), fluorescence probes (AB112155, ABCAM, Cambridge, MA) are used to determine the total pyrophosphate (PPi) concentrations in plasma and urine. Urine PPi is corrected for urinary creatinine measured by LC-MS / MS or by ELISA using appropriate controls to adjust for variability between assays.

[0444] Renal tissue images The left kidney is fixed in 4% formalin / PBS at 4 °C for 12 h, then dehydrated by increasing the concentrations of ethanol and xylene, followed by paraffin embedding. Mineral deposition is measured in 10-μm von Kossa-stained sections counterstained with 1% methyl green. As a morphological evaluation, hematoxylin / eosin is used as a counterstain. The histomorphometric evaluation of sagittal kidney sections containing the cortex, medulla, and pelvis is performed blindly by two independent observers using an Osteomeasure system (Osteometrics, Atlanta, GA). The percent calcified area is determined using the formula: % calculated area = 100 * calcified area / total area (including cortex, medulla, and pelvic lumen) and depends on the number of observed areas per section. The size of mineralization is determined using the formula: calculated size = calcified area / number of observed calcifications per section.

[0445] For transmission electron microscopy, 1 mm of the left kidney 3The blocks are fixed in phosphate buffered saline containing 2.5% glutaraldehyde and 2% paraformaldehyde for 2 hours, followed by post-fixation in 1% osmium solution for 2 hours. Dehydration is carried out using a series of ethanol concentrations (50% - 100%). The kidney tissues are embedded in epoxy resin and polymerized overnight at 60°C. After preparing thin sections (50 nm), the tissues are double-stained with uranium and lead and observed using Tecnai Biotwin (LaB6, 80 kV) (FEI, Thermo Fisher, Hillsboro, OR).

[0446] Tissue imaging, histomorphometry and micro-CT The tibiae and femurs of mice are dissected free of soft tissues, fixed in 70% ethanol, dehydrated, embedded in methyl methacrylate, sectioned, and stained with toluidine blue (C.B. Ware et al., Targeted disruption of the low-affinity leukemia inhibitory factor receptor gene causes placental, skeletal, neural and metabolic defects and results in perinatal death. Development 121, 1283 - 1299 (1995)). Histomorphometric measurements are made in the fixed region immediately below the growth plate corresponding to the primary spongiosa (A.M. Parfitt et al., Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res 2, 595 - 610 (1987)) and analyzed using OsteoMeasure software (Osteometrics, Atlanta, GA). The bones are scanned using Scanco μCT-35 (Scanco, Brutissellen, Switzerland), and a number of structural parameters are analyzed in both the proximal tibia and distal femur (trabecular bone) immediately below the growth plate and the central shaft (cortical bone) of the tibia or femur.

[0447] Biomechanical Tests of Bone The femurs of mice on a high - protein diet are loaded until fracture by three - point bending, while the femurs of mice on a normal chow diet are loaded until fracture by four - point bending. All whole - bone tests are performed by loading the femur in the posterior - to - anterior direction such that the anterior one - quarter segment is under tensile load. The widths of the lower and upper supports of the four - point bending apparatus are 7 mm and 3 mm, respectively. The tests are performed using a servo - hydraulic testing machine (Instron model 8874; Instron Corp., Norwood, MA, USA) at a deflection rate of 0.05 mm / sec. Load and mid - span deflection are obtained directly at a sampling frequency of 200 Hz. The load - deflection curve is analyzed for stiffness, maximum load, and work to fracture. Yield is defined as a 10% reduction in secant stiffness (load range normalized to the deflection range) compared to the initial tangent stiffness. The femurs are tested at room temperature and kept moist with phosphate - buffered saline (PBS). The post - yield deflection, defined as the deflection at fracture minus the deflection at yield, is also measured.

[0448] Treatment of Chronic Kidney Disease Using a Viral Vector Expressing ENPP1 or ENPP3 The following examples provide AAVs expressing ENPP1 or ENPP3, which are expected to be effective in the treatment of vascular calcification and symptoms associated with CKD. For illustrative purposes, ENPP1 - Fc and ENPP3 - Fc are used in this example, and similar results can be obtained using other ENPP1 or ENPP3 fusions of the present invention.

[0449] AAV virions expressing ENPP1 - Fc and ENPP3 - Fc proteins are prepared according to Example 1 and administered to CKD mice (which are a model of chronic kidney disease (CKD) (BMC Nephrology, 2013, 14:116)). Six sets of mice are used for the treatment with ENPP1 and ENPP3.

[0450] Control cohort: In this experiment, AAV particles containing a null vector are injected into the first cohort of ENPP1 wt mice serving as a control group, and AAV particles containing a null vector are injected into the second cohort of CKD mice serving as a control group.

[0451] Cohort of ENPP1-treated mice: ENPP1 wt AAV particles engineered to express ENPP1-Fc protein are injected into the third cohort of mice, and AAV particles engineered to express ENPP1-Fc protein are injected into the fourth cohort of CKD mice.

[0452] Cohort of ENPP3-treated mice: ENPP1 wt AAV particles engineered to express ENPP3-Fc protein are injected into the fifth cohort of mice, and AAV particles engineered to express ENPP3-Fc protein are injected into the sixth cohort of CKD mice.

[0453] Adenine diet: CKD mice are maintained on an adenine diet, while wild-type mice are maintained on a normal solid diet (Laboratory Autoclavable Rodent Diet 5010; PMI Nutritional International, Brentwood, MO). To provide the adenine-containing solid diet that CKD mice consume, adenine is mixed with a casein-based diet that dulls the smell and taste. Adenine is purchased from Sigma Aldrich (MO, USA), and the powdered casein-based diet is purchased from Special Diets Services (SDS, UK) (reference number 824522). The other components of the diet are corn starch (39.3%), casein (20.0%), maltodextrin (14.0%), sucrose (9.2%), corn / corn oil (5%), cellulose (5%), vitamin mixture (1.0%), DL-methionine (0.3%), and choline bitartrate (0.2%).

[0454] Vector injection: After 2 weeks of age, approximately 1 × 10 per mouse in PBS pH 7.412 ~1×10 15 vg / kg 、 preferably, 1×10 13 ~1×10 14 vg / kg Posterior orbital injection or tail vein injection is performed on all mice. The vector to be injected is either an empty "null" (control group) or one that carried the NPP1 or NPP3 gene (study group).

[0455] Assay: Analyze the kidney histology, PPi levels, and blood and urine parameters, such as FGF-23 levels, vitamin D, parathyroid hormone (PTH) levels, serum / blood urea levels, blood urea nitrogen (BUN) levels, serum / blood creatinine levels, and plasma pyrophosphate (PPi) for each cohort as described in Example 3. Urine is collected as a spot urine sample after spontaneous urination. Serum and urine calcium, phosphorus, creatinine, and urea levels are measured with a Konelab 20XTi (Thermo Scientific, Finland). Creatinine concentration is confirmed by a colorimetric assay (BioChain, CA, USA). PTH is measured by a mouse intact PTH ELISA kit (Immutopics, CA, USA), FGF23 levels are measured by an intact FGF23 ELISA (Kainos, Japan), and vitamin D is measured by an EIA kit (Immunodiagnostic Systems, UK). Details of the experiments are published in BMC Nephrology, 2013, 14:116, and PLoS One. 2017 Jul 13;12(7).

[0456] Results: Untreated CKD mice generally show weight loss and signs of declining kidney function, such as a decrease in the ratios of urinary urea / serum urea and urinary creatinine / serum creatinine. In contrast, CKD mice treated with AAV expressing ENPP1 or ENPP3 protein are expected to show an increase in weight approaching that of normal WT mice. Generally, serum urea levels in the range of 80 - 100 mg / dL are considered optimal. Urea levels above 100 mg / dL are associated with increased morbidity along with weight loss and reduced physical activity. Treated (AAV with ENPP1 or ENPP3) CKD mice are expected to show improvement in kidney function manifested by a decrease in serum urea levels leading to a higher urinary urea / serum urea ratio and an increase in urinary urea levels.

[0457] Renal histopathological analysis of the kidney tissue of CKD mice shows deposition of crystal structures in areas such as the tubular lumen, microabscesses, and dilated tubules, periodic acid Schiff (PAS) staining showing dilated Bowman's space, the presence of atrophic tubules with proteinaceous casts ("thyroidization") and tubular atrophy with thickening of the tubular basement membrane, the presence of mild interstitial fibrosis seen through Ladewig staining, and the appearance of extensive calcification of the tubular structures seen through von Kossa staining. In contrast, CKD mice treated according to the invention with ENPP1 or ENPP3 are expected to show a tissue image similar to that of healthy wild-type mice, showing a reduction or absence of renal mineral deposits in the tubular lumen and soft tissue vascular structures.

[0458] Untreated CKD mice show a significant increase in serum inorganic phosphorus (pi), increases in PTH and FGF23 levels when compared to those of healthy wild-type mice (normal levels of PPi are about 2 - 4 μM; for PTH are about 10 - 65 ng / L; the median FGF23 level is 13 RU / ml, and normal FGF23 levels are in the range of 5 - 210 RU / ml; normal vitamin D levels are 20 ng / mL - 50 ng / mL), but 1,25(OH) 2- A decrease in vitamin D levels and lower PPi levels (about 0.5 μM) are expected. In contrast, treated CKD mice are expected to show an increase in PPi levels (about 4 - 5 μM), which is expected to be higher than the PPi levels (about 0.5 μM) seen in untreated CKD mice. Thus, through histological analysis, a reduction in soft tissue and coronary artery calcification of the kidney (a 25% or 50% or 70% or 90% or 100% reduction) visualized, an increase in serum PPi levels from blood analysis, normalization of vitamin D levels, reduction of FGF23 levels to the normal range, normalization of PTH levels, increase in survival time, increase in weight gain, and increase in urine urea and creatinine, resulting in improvement of kidney function, such as the observation of one or more factors, a person skilled in the art can determine the therapeutic efficacy of vector - based ENPP1 or ENPP3 in the treatment of chronic kidney disease.

[0459] Treatment of human subjects: Approximately 5×10 11 ~5×10 15 vg / kg , in some embodiments approximately 1×10 in 1×PBS at pH 7.4 12 ~1×10 15 vg / kgTreat human patients suffering from CKD by performing an intravenous injection comprising. The success of the treatment of CKD is observed by monitoring one or more of the aforementioned parameters through regular blood and urine tests as described for the mouse model. Instead of histological analysis that requires staining of kidney slices or arterial tissue, which is not possible to perform on living patients, non-invasive visualization techniques commonly known in the art, such as CT scans, ultrasounds, or intravenous pyelograms, are used to visualize the presence of calcification and the reduction of calcification in patients suffering from CKD in response to vector-based delivery and expression of ENPP1 or ENPP3. Intravenous pyelogram is an X-ray examination that uses a contrast agent that functions as a dye to visualize the urinary tract and assist in detecting the presence of renal calcification. Computed tomography is a non-invasive imaging technique that uses X-ray technology to depict internal structures of the body, such as the urinary tract. Renal calcification is visualized by a CT scan. A CT scan collects X-ray images from various angles around the body to generate detailed cross-sectional images as well as three-dimensional images of the internal structures and organs of the body. A CT scan can also be used in arteries to detect the presence of post-treatment calcification and subsequent reduction of calcification. The computer analyzes the radiation transmitted through the body to reconstruct images of the internal structures and organs.

[0460] A physician having the skill to visualize soft tissue calcification, cardiac calcification, and myocardial infarction attempts to treat a subject suffering from CKD by administering AAV virions expressing human ENPP1 or human ENPP3. The physician delivers a construct of hENPP1 or hENPP3 and administers viral particles that express the corresponding protein under the control of an inducible promoter. Thus, the physician has the option to control the dosage (the amount of hENPP1 or hENPP3 expressed) based on the rate and extent of symptom improvement. The success of the treatment is observed by medical experts in the field by observing one or more positive symptoms such as improvement in kidney function, improvement in urinary creatinine levels (normal urinary creatinine levels are 40 - 278 mg / dL for men and 29 - 226 mg / dL for women), and improvement in urinary urea levels (normal urinary urea levels for adults are 26 - 43 g / 24 hours), normal serum creatinine levels (normal serum creatinine range is 0.6 - 1.1 mg / dL for women and 0.7 - 1.3 mg / dL for men), normal vitamin D levels (levels of 20 ng / ml - 50 ng / mL are considered appropriate for healthy people. Levels below 12 ng / mL indicate vitamin D deficiency), normal blood urea nitrogen levels (BUN levels for healthy adults are 7 - 20 mg / dL), weight gain, increase in serum PPi levels (at least about 4 - 5 μm), reduction in arterial tissue calcification (25% or 50% or 70% or 90% or 100% reduction), and reduction in kidney tubule calcification visualized by non-invasive techniques such as CT or ultrasound scans.

[0461] Example 5 - Treatment of GACI Using a Viral Vector Expressing ENPP1 or ENPP3 The following examples provide AAVs expressing ENPP1 or ENPP3 that are expected to be effective in treating vascular calcification and symptoms associated with GACI. For illustrative purposes, ENPP1-Fc and ENPP3-Fc are used in this example, and similar results can be obtained using other ENPP1 or ENPP3 fusions of the present invention.

[0462] AAV virions expressing ENPP1-Fc and ENPP3-Fc proteins were produced according to Example 1, and Enpp1 asj / asj mice (which are a model of infantile systemic arterial calcification (Li, et al., 2013, Disease Models & Mech. 6(5):1227-35)) are administered. For treatment with ENPP1 and ENPP3, 6 sets of mice are used.

[0463] Control cohort: In this experiment, AAV particles containing a null vector are injected into the first cohort of ENPP1 wt mice that serve as a control group, and ENPP1 asj / asj AAV particles containing a null vector are injected into the second cohort of mice that serve as a control group.

[0464] ENPP1-treated mouse cohort: ENPP1 wt AAV particles engineered to express ENPP1-Fc protein are injected into the third cohort of mice, and ENPP1 asj / asj AAV particles engineered to express ENPP1-Fc protein are injected into the fourth cohort of mice.

[0465] ENPP3-treated mouse cohort: ENPP1 wt AAV particles engineered to express ENPP3-Fc protein are injected into the fifth cohort of mice, and ENPP1 asj / asj AAV particles engineered to express ENPP3-Fc protein are injected into the sixth cohort of mice. Wild-type mice are maintained on a normal chow diet, and Enpp1 asj / asj mice are given a high-phosphate Teklad diet.

[0466] Vector injection: After 2 weeks of age, per mouse, about 1×10 12 ~1×10 15 vg / kg , preferably 1×10 13 ~1×10 14 vg / kgPerform retro-orbital injection or tail vein injection on all mice. The vector to be injected is either an empty "null" (control group) or one carrying the NPP1 or NPP3 gene (study group).

[0467] Assay: Analyze kidney histology, PPi levels, and blood and urine parameters, such as FGF-23 levels, vitamin D, parathyroid hormone (PTH) levels, serum / blood urea levels, blood urea nitrogen (BUN) levels, serum / blood creatinine levels, and plasma pyrophosphate (PPi) for each cohort as described in Examples 3 and 4.

[0468] Results: Untreated Enpp1 asj / asj mice generally show weight loss and increased mortality. In contrast, Enpp1 treated with AAV expressing ENPP1 protein or ENPP3 protein asj / asj mice are expected to show weight gain approaching that of normal WT mice.

[0469] Enpp1 treated with a null vector asj / asj mice are expected to show calcification of the heart, aorta, and coronary arteries, as well as histological evidence of myocardial infarction in the free wall of the right ventricle, calcification of the coronary arteries, heart, ascending aorta, and descending aorta, myocardial cell necrosis, and myocardial fibrosis in the myocardial tissue adjacent to the area of coronary artery calcification. In contrast, Enpp1 treated with AAV expressing ENPP1-Fc or ENPP3-Fc asj / asj animals are expected to show no calcification of the heart, arteries, or aorta by histology or postmortem micro-CT. Enpp1 treated with a null vector asj / asj mice show intense extensive calcification centered in the outer medulla and extending into the renal cortex, as well as calcification centered in the renal medulla. In contrast, Enpp1 treated according to the present invention with ENPP1 or ENPP3 asj / asj mice are expected to show a tissue image similar to that of healthy wild-type mice, with reduced or no renal mineral deposition in the tubular lumen and soft tissue vascular structures.

[0470] In addition to survival time, daily animal body weight, and terminal tissue images, treatment response is evaluated by postmortem high-resolution micro-CT scans for imaging vascular calcification, plasma PPi concentration, and uptake of 99mTc PPi (99mPYP). Untreated (null vector) Enpp1 asj / asj In contrast to the dramatic calcification expected in the aorta, coronary arteries, and heart of the cohort, WT and treated (vector expressing ENPP1 or ENPP3) Enpp1 asj / asj are both expected to have no vascular calcification via micro-CT. Furthermore, the serum PPi concentration (5.2 μM) of treated (vector expressing ENPP1 or ENPP3) Enpp1 asj / asj animals is expected to increase to WT levels (4.4 μM) and significantly exceed the levels of untreated enpp1 asj / asj (0.5 μM).

[0471] 99mPYP is an imaging agent typically used in cardiac imaging and bone remodeling. It localizes to the surface of hydroxyapatite and can then be taken up by osteoclasts, so it is sensitive to regions of abnormally high bone remodeling activity. Untreated Enpp1 asj / asj weekly serial imaging of animals is expected to show greater uptake of 99mPYP in the head compared to treated Enpp1 asj / asj animals. Measurements are taken on days 30 - 35 and 50 - 65 after administration of viral particles containing the null vector or vector expressing ENPP1. Comparison of these experimental groups is expected to show that treatment with ENPP1-Fc or ENPP3-Fc returned 99mPYP uptake to WT levels in GACI mice, which suggests that treatment with ENPP1-Fc or ENPP3-Fc can prevent uncontrolled tissue, trabecular, and cranial mineralization in Enpp1 asj / asj mice. These observations are in Enpp1 animals administered viral particles expressing a vector for ENPP1-Fc or ENPP3-Fcasj / asj The mouse is expected to show no vascular calcification and have normal plasma PPi concentration.

[0472] Untreated Enpp1 asj / asj The mouse shows a significant increase in serum inorganic phosphorus (pi), an increase in PTH and FGF23 levels when compared to that of healthy wild-type mice (normal levels of PP are about 2 - 4 μM; for PTH are about 10 - 65 ng / L; the median FGF23 level is 13 RU / ml, and normal FGF23 levels range from 5 - 210 RU / ml; normal vitamin D levels are 20 ng / mL - 50 ng / mL), but a decrease in 1,25(OH) 2 -vitamin D levels, and a lower PPi level (about 0.5 μM) are also expected. In contrast, treated Enpp1 asj / asj The mouse is expected to show an increase in PPi level (about 4 - 5 μM), which is expected to be higher than the PPi level (about 0.5 μM) seen in untreated CKD mice. Thus, through histological analysis, one or more factors such as a reduction in renal soft tissue and coronary artery calcification (a 25% or 50% or 70% or 90% or 100% reduction) visualized, an increase in serum PPi level from blood analysis, normalization of vitamin D levels, reduction of FGF23 levels towards the normal range and normalization of PTH levels, an increase in survival time, an increase in weight gain along with an increase in urinary urea and creatinine, indicating an improvement in renal function, can be observed by those skilled in the art to determine the therapeutic efficacy of vector-based ENPP1 or ENPP3 in the treatment of GACI.

[0473] Treatment of human subjects Approximately 5×10 per subject, in 1×PBS at pH 7.4, that can deliver and express hENPP1 or hENPP3 11 ~5×10 15 vg / kg In some embodiments, approximately 1×10 in 1×PBS at pH 7.4 12 ~1×10 15 vg / kgBy administering an injection comprising [the relevant substance], a human patient suffering from GACI is treated. The success of the treatment of GACI is observed by monitoring one or more of the aforementioned parameters through regular blood and urine tests as described for the mouse model. Instead of histological analysis that requires staining of kidney slices or arterial tissue, which is not possible to perform on living patients, non-invasive visualization techniques as described in Example 4 are used.

[0474] A physician with the skill to visualize soft tissue calcification, heart calcification, and myocardial infarction attempts to treat a subject suffering from GACI by administering AAV virions expressing hENPP1 or hENPP3. The physician administers viral particles that deliver a construct encoding hENPP1 or hENPP3, and the vector expresses the ENPP protein under the control of an inducible promoter. The physician can control the dosage (the amount of hENPP1 or hENPP3 expressed) based on the rate and extent of symptom improvement. The success of the treatment is observed by medical experts in the field by observing one or more positive symptoms such as normal vitamin D levels (levels between 20 ng / ml and 50 ng / mL are considered appropriate for healthy people. Levels below 12 ng / mL indicate vitamin D deficiency), normal blood urea nitrogen levels (the BUN level for healthy adults is 7 - 20 mg / dL), weight gain, an increase in serum PPi levels (at least about 4 - 5 μm), a reduction in arterial tissue calcification (a 25% or 50% or 70% or 90% or 100% reduction), and / or a reduction in kidney tubule calcification visualized by non-invasive techniques such as CT or ultrasound scans.

[0475] Example 6 - Treatment of PXE using a viral vector expressing ENPP1 or ENPP3 The following examples provide AAVs that express ENPP1 or ENPP3, which are expected to be effective in treating vascular calcification and symptoms associated with PXE. For purposes of illustration, ENPP1-Fc and ENPP3-Fc are used in this example, and similar results can be obtained using other ENPP1 or ENPP3 fusions of the invention.

[0476] AAV virions expressing ENPP1-Fc protein and ENPP3-Fc protein were produced according to Example 1 and administered to ABCC6 - / - mice (which are a model of pseudoxanthoma elasticum; Jiang, et al., 2007, J. Invest. Derm. 127(6):1392 - 4102). For treatment with ENPP1 and ENPP3, six sets of mice are used.

[0477] Control cohort: In this experiment, AAV particles containing a null vector are injected into a first cohort of ENPP1 wt mice that serve as a control group, and AAV particles containing a null vector are injected into a second cohort of ABCC6 - / - mice that serve as a control group.

[0478] ENPP1 - treated mouse cohort: AAV particles engineered to express ENPP1 wt are injected into a third cohort of mice, and AAV particles engineered to express ENPP1 - / - are injected into a fourth cohort of ABCC6 mice.

[0479] ENPP3 - treated mouse cohort: AAV particles engineered to express ENPP1 wt are injected into a fifth cohort of mice, and AAV particles engineered to express ENPP1 - / - are injected into a sixth cohort of mice. Wild - type mice are maintained on a normal chow diet, and ABCC6 - / - mice are fed a high - phosphate Teklad diet.

[0480] Vector injection: After 2 weeks of age, approximately 1×10 12 ~1×10 15 vg / kg , preferably 1×10 13 ~1×10 14 vg / kg of retro-orbital injection or tail vein injection is performed on all mice. The vector to be injected is either an empty "null" (control group) or one that carried the NPP1 or NPP3 gene (study group).

[0481] Assay: Tissue images of the kidneys, PPi levels and blood urine parameters, such as FGF-23 levels, vitamin D, parathyroid hormone (PTH) levels, serum / blood urea levels, blood urea nitrogen (BUN) levels, serum / blood creatinine levels and plasma pyrophosphate (PPi) are analyzed for each cohort as described in Examples 3 and 4.

[0482] Results: Untreated ABCC6− / − mice generally show a reduction in body weight and an increase in mortality. In contrast, ABCC6− / − mice treated with AAV expressing ENPP1 or ENPP3 protein are expected to show an increase in body weight approaching that of normal WT mice. ABCC6− / − mice treated with a null vector are expected to show calcification of the heart, aorta and coronary arteries, as well as histological evidence of myocardial infarction in the free wall of the right ventricle, calcification of the coronary arteries, heart, ascending aorta and descending aorta, necrosis of myocardial cells, and myocardial fibrosis in the myocardial tissue adjacent to the areas of coronary artery calcification. In contrast, ABCC6− / − animals treated with a vector expressing ENPP1-Fc or ENPP3-Fc are expected to show no calcification of the heart, arteries or aorta by histological examination or postmortem micro-CT. Enpp1 asj / asj mice treated with a null vector show intense extensive calcification centered on the outer medulla and extending into the renal cortex, as well as calcification centered on the renal medulla. In contrast, Enpp1 treated with viral vector-based expression of ENPP1 or ENPP3 asj / asjThe mouse is expected to show a tissue image similar to that of a healthy wild-type mouse, with a reduction or absence of renal mineral deposits in the renal tubule lumen and soft tissue vascular structures.

[0483] In addition to survival time, daily animal body weight, and final tissue images, the treatment response is evaluated by postmortem high-resolution micro-CT scans for imaging vascular calcification and plasma PPi concentration. WT and treated (vector expressing ENPP1) ABCC6 - / - None of the untreated (null vector) ABCC6 - / - cohorts are expected to have any vascular calcification via micro-CT, in contrast to the dramatic calcification expected to be seen in the aorta, coronary arteries, and heart of the untreated (null vector) ABCC6 - / - animals. Furthermore, the serum PPi concentration of treated (vector expressing ENPP1) ABCC6 - / - animals (5.2 μM) is expected to increase to WT levels (4.4 μM) and significantly exceed the levels of untreated ABCC6

[0484] untreated ABCC6 - / - mice show a significant increase in serum inorganic phosphorus (pi), an increase in PTH and FGF23 levels, a decrease in 1,25(OH) 2 -vitamin D levels, and lower PPi levels (about 0.5 μM) when compared to those of healthy wild-type mice (normal levels of PP are about 2 - 4 μM; for PTH it is about 10 - 65 ng / L; the median FGF23 level is 13 RU / ml and normal FGF23 levels are in the range of 5 - 210 RU / ml; normal vitamin D levels are 20 ng / mL - 50 ng / mL). In contrast, treated ABCC6 - / - mice are expected to show an increase in PPi levels (about 4 - 5 μM), which is higher than that of untreated ABCC6 - / -It is expected to be higher than the PPi level seen in mice (about 0.5 μM). Thus, through histological analysis, reduction of soft tissue of the kidney and calcification of coronary arteries visualized (25% or 50% or 70% or 90% or 100% reduction), increase in serum PPi levels from blood analysis, normalization of vitamin D levels, reduction of FGF23 levels and normalization of PTH levels relative to the normal range, increase in survival time, and improvement of kidney function observed by an increase in urinary urea and creatinine along with an increase in body weight gain, one or more factors such as these allow those skilled in the art to determine the therapeutic efficacy of vector-based ENPP1 or ENPP3 in the treatment of PXE.

[0485] Treatment of human subjects: Treat human patients suffering from PXE by intravenous injection, per subject, containing approximately 5×10 11 ~5×10 15 vg / kg , and in some embodiments, approximately 1×10 12 ~1×10 15 vg / kg in 1×PBS at pH 7.4. The success of the treatment of PXE is observed by monitoring one or more of the aforementioned parameters through regular blood and urine tests as described for the mouse model. Instead of histological analysis that requires staining of kidney slices or arterial tissue, which is not possible to perform on living patients, use non-invasive visualization techniques as described in Example 4.

[0486] A physician having the skill to visualize soft tissue calcification, heart calcification, and myocardial infarction can attempt to treat a subject suffering from PXE by administering AAV virions that express ENPP1 or ENPP3. The physician can also use viral particles that deliver a construct of ENPP1 or ENPP3 and express the corresponding protein under the control of an inducible promoter. Thus, the physician has the option to control the dosage (the amount of ENPP1 or ENPP3 expressed) based on the rate and extent of symptom improvement. The success of the treatment and the appropriate dosage can be readily inferred by medical experts in the field by observing one or more positive symptoms such as normal vitamin D levels (levels of 20 ng / ml to 50 ng / mL are considered appropriate for healthy people; levels less than 12 ng / mL indicate vitamin D deficiency), disappearance or reduction in size and / or number of retinal pigment streaks, reduction or absence of retinal hemorrhage, normal blood urea nitrogen levels (BUN levels for healthy adults are 7 to 20 mg / dL), weight gain, increase in serum PPi levels (at least about 4 to 5 μm), reduction in calcification of arterial tissue and connective tissue (25% or 50% or 70% or 90% or 100% reduction), and / or reduction in calcification of renal tubules visualized by non-invasive techniques such as CT or ultrasound scans.

[0487] Example 7 - Treatment of OPLL Using a Viral Vector Expressing Human ENPP1 or ENPP3 The following examples provide AAVs that express human ENPP1 or ENPP3, which are expected to be effective in treating vascular calcification and symptoms associated with PXE. For illustrative purposes, ENPP1-Fc and ENPP3-Fc fusions are used in this example, and similar results can be obtained using other ENPP1 or ENPP3 fusions of the present invention.

[0488] AAV virions expressing ENPP1-Fc protein or ENPP3-Fc protein were prepared according to Example 1 and administered to toe tip walking (ttw) mice, which are a model of ossification of the posterior longitudinal ligament; (Okawa, et al, 1998, Nature Genetics 19(3):271-3; Nakamura, et al, 1999, Human Genetics l04(6):492-7). For treatment with ENPP1 and ENPP3, 6 sets of mice are used.

[0489] Control cohort: In this experiment, AAV particles containing a null vector are injected into the first cohort of ENPP1 wt mice that serve as a control group, and AAV particles containing a null vector are injected into the second cohort of ttw mice that serve as a control group.

[0490] Cohort of ENPP1-treated mice: ENPP1 wt AAV particles engineered to express ENPP1-Fc protein are injected into the third cohort of mice, and AAV particles engineered to express ENPP1-Fc protein are injected into the fourth cohort of ttw mice.

[0491] Cohort of ENPP3-treated mice: ENPP1 wt AAV particles engineered to express ENPP3-Fc protein are injected into the fifth cohort of mice, and AAV particles engineered to express ENPP3-Fc protein are injected into the sixth cohort of ttw mice. Wild-type mice are maintained on a normal solid diet, and ttw mice are given a high-phosphate Teklad diet.

[0492] Vector injection: After 2 weeks of age, per mouse, about 1×10 12 ~1×10 15 vg / kg Preferably 1×10 13 ~1×10 14 vg / kgPosterior orbital injection or tail vein injection is performed on all mice. The vector to be injected is either an empty "null" (control group) or one that carried the NPP1 or NPP3 gene (study group).

[0493] Assay: Tissue images of the kidney, PPi levels and blood urine parameters such as FGF-23 levels, vitamin D, parathyroid hormone (PTH) levels, serum / blood urea levels, blood urea nitrogen (BUN) levels, serum / blood creatinine levels and plasma pyrophosphate (PPi) are analyzed for each cohort as described in Examples 3 and 4.

[0494] Results: Untreated ttw mice generally show weight loss, spinal hypertrophy, lethargy and increased mortality. In contrast, ttw mice treated with AAV expressing ENPP1 protein or ENPP3 protein are expected to show weight gain approaching that of normal WT mice, normal wakefulness, and reduced spinal thickness approaching that of wild-type mice. ttw mice treated with a null vector are expected to show calcification of the heart, aorta and coronary arteries, as well as histological evidence of myocardial infarction in the free wall of the right ventricle, calcification of the coronary arteries, heart, ascending aorta and descending aorta, myocardial cell necrosis, and myocardial fibrosis in the myocardial tissue adjacent to the area of coronary artery calcification. In contrast, ttw animals treated with a vector expressing ENPP1-Fc or ENPP3-Fc are expected to show no calcification of the heart, arteries or aorta by tissue imaging or postmortem micro-CT. ttw mice treated with a null vector show intense widespread calcification centered on the outer medulla and extending into the renal cortex, as well as calcification centered on the renal medulla. In contrast, ttw mice treated with viral vector-based expression of ENPP1 or ENPP3 are expected to show tissue images similar to those of healthy wild-type mice, reduced or no renal mineral deposits in the lumens of renal tubules, and reduced calcification of the spine and soft tissue vascular structures.

[0495] In addition to survival time, daily animal body weight, and terminal tissue images, treatment response is evaluated by post - mortem high - resolution micro - CT scans for imaging vascular calcification and plasma PPi concentration. Neither WT nor treated (vector expressing ENPP1) ttw is expected to have any vascular calcification via micro - CT, in contrast to the dramatic calcification expected to be seen in the aorta, coronary arteries, and heart of the untreated (null vector) ttw cohort. Furthermore, treated (vector expressing ENPP1) ttw - The serum PPi concentration of the animals (5.2 μM) is expected to increase to WT levels (4.4 μM) and significantly exceed the levels of untreated ttw (0.5 μM).

[0496] Untreated ttw mice show a significant increase in serum inorganic phosphorus (pi), increases in PTH and FGF23 levels when compared to those of healthy wild - type mice (normal levels of PP are about 2 - 4 μM; for PTH are about 10 - 65 ng / L; the median FGF23 level is 13 RU / ml and normal FGF23 levels are in the range of 5 - 210 RU / ml; normal vitamin D levels are 20 ng / mL - 50 ng / mL), but also a decrease in 1,25(OH) 2 - vitamin D levels, and lower PPi levels (about 0.5 μM) are also expected. In contrast, treated ttw mice are expected to show an increase in PPi levels (about 4 - 5 μM), which is expected to be higher than the PPi levels (about 0.5 μM) seen in untreated ttw mice. Thus, one or more factors such as a reduction in renal soft - tissue and coronary artery calcification (25% or 50% or 70% or 90% or 100% reduction) visualized through histological analysis, an increase in serum PPi levels from blood analysis, normalization of vitamin D levels, reduction of FGF23 levels to the normal range and normalization of PTH levels, an increase in survival time, and an increase in body weight gain along with an increase in urinary urea and creatinine indicating improved renal function observed by those skilled in the art can determine the therapeutic efficacy of vector - based ENPP1 or ENPP3 in the treatment of OPLL.

[0497] Treatment of human subjects: Administering an intravenous injection containing approximately 5×10 11 ~5×10 15 vg / kg in 1×PBS at pH 7.4 per subject, which can deliver and express hENPP1 or hENPP3. In some embodiments, approximately 1×10 12 ~1×10 15 vg / kg Treat human patients suffering from OPLL by intravenous injection. The success of OPLL treatment is observed by monitoring one or more of the aforementioned parameters through regular blood and urine tests as described for the mouse model. Instead of histological analysis requiring staining of kidney slices or arterial tissue, which is not possible to perform on living patients, non-invasive visualization techniques as described in Example 4 are used.

[0498] A physician having the skill to visualize soft tissue calcification, cardiac calcification, and myocardial infarction can attempt to treat a subject suffering from OPLL by administering AAV virions expressing hENPP1 or hENPP3. In some embodiments, the physician uses viral particles that deliver a construct of hENPP1 or hENPP3 and express the corresponding protein under the control of an inducible promoter. Thus, the physician has the option to control the dosage (the amount of hENPP1 or hENPP3 expressed) based on the rate and extent of symptom improvement. The success of the treatment and the appropriate dosage can be readily inferred by medical professionals in the field by observing one or more positive symptoms such as normal vitamin D levels (levels of 20 ng / ml to 50 ng / mL are considered appropriate for healthy people; levels below 12 ng / mL indicate vitamin D deficiency), normal blood urea nitrogen levels (the BUN level for healthy adults is 7 to 20 mg / dL), weight gain, an increase in serum PPi levels (at least about 4 to 5 μm), a reduction in arterial tissue calcification (a 25% or 50% or 70% or 90% or 100% reduction), a reduction in spinal thickness and pain sensation, and a reduction in spinal canal stenosis visualized by non-invasive techniques such as CT, magnetic resonance imaging (MRI), or ultrasound scans.

[0499] Example 8 - Treatment of Osteopenia and / or Osteomalacia Using a Viral Vector Expressing ENPP1 or ENPP3 The following examples provide AAVs expressing ENPP1 or ENPP3 that are expected to be effective in treating symptoms associated with osteopenia and / or osteomalacia. For purposes of illustration, ENPP1-Fc and ENPP3-Fc are used in this example, and similar results can be obtained using other ENPP1 or ENPP3 fusions of the present invention.

[0500] AAV virions expressing ENPP1-Fc protein or ENPP3-Fc protein are prepared according to Example 1 and administered to toe tip walking (ttw) mice, which are a mouse model of osteoarthritis (Bertrand, et al, 2012, Annals Rheum. Diseases 71(7):1249-53). For treatment with ENPP1 and ENPP3, six sets of mice are used. In addition to GACI, which also serves as a model of osteoporosis (Mackenzie, et al, 2012, PloS one 7(2):e32177), ENPP1 knockout mice (ENPP1 KO ) are used to repeat the same experiment.

[0501] Control cohort: In this experiment, AAV particles containing a null vector are injected into the first cohort of ENPP1 wt mice, which serve as a control group, and AAV particles containing a null vector are injected into the second cohort of ttw (or ENPP1 KO ) mice, which serve as a control group.

[0502] Cohort of ENPP1-treated mice: AAV particles engineered to express ENPP1-Fc protein are injected into the third cohort of ENPP1 wt mice, and AAV particles engineered to express ENPP1-Fc protein are injected into the fourth cohort of ttw mice (or ENPP1 KO ).

[0503] Cohort of ENPP3-treated mice: AAV particles engineered to express ENPP3-Fc protein are injected into the fifth cohort of ENPP1 wt mice, and AAV particles engineered to express ENPP3-Fc protein are injected into the sixth cohort of ttw (or ENPP1 KO ) mice. Wild-type mice are maintained on a normal chow diet, and ttw mice (or ENPP1 KO ) are given a high-phosphate Teklad diet.

[0504] Vector injection: After 2 weeks of age, approximately 1×10 12 ~1×10 15 vg / kg , preferably 1×10 13 ~1×10 14 vg / kg of retro-orbital injection or tail vein injection per mouse is performed on all mice. The vector to be injected is either an empty "null" (control group) or one that carried the NPP1 or NPP3 gene (study group).

[0505] Assay: Tissue images of the kidneys, PPi levels and blood and urine parameters, such as FGF-23 levels, vitamin D, parathyroid hormone (PTH) levels, serum / blood urea levels, blood urea nitrogen (BUN) levels, serum / blood creatinine levels and plasma pyrophosphate (PPi), are analyzed for each cohort as described in Examples 3 and 4.

[0506] Tissue imaging, histomorphometry and micro-CT: Bone analysis is performed according to the protocol described in Example 3.

[0507] Biomechanical testing of bone: Bone analysis is performed according to the protocol described in Example 3.

[0508] Results: Untreated ttw (or ENPP1 KO ) mice generally show a decrease in body weight, lethargy, a decrease in cortical bone thickness and trabecular bone mass, calcification of cartilage and ligaments, a decrease in bone density such as in the femur and tibia, and an increase in mortality compared to wild type. In contrast, ttw (or ENPP1 KO ) mice treated with AAV expressing ENPP1 protein or ENPP3 protein are expected to show an increase in body weight approaching the normal WT mouse body weight range, normal wakefulness, an increase in bone mineral content, improvement in cortical bone thickness and trabecular bone mass, and an increase in bone strength and bone flexibility. ttw (or ENPP1 KO)The mouse is expected to show calcification of the heart, aorta and coronary arteries, as well as histological evidence of myocardial infarction in the free wall of the right ventricle, calcification of the coronary arteries, heart, ascending aorta and descending aorta, myocardial cell necrosis, and myocardial fibrosis in the myocardial tissue adjacent to the area of coronary artery calcification. In contrast, ttw (or ENPP1) treated with a vector expressing ENPP1-Fc or ENPP3-Fc KO )animals are expected to show no calcification of the heart, arteries or aorta by histological examination or postmortem micro-CT. ttw (or ENPP1) treated with a null vector KO )mice show intense extensive calcification centered on the outer medulla and extending into the renal cortex, as well as calcification centered on the renal medulla. In contrast, ttw (or ENPP1) treated with viral vector-based expression of ENPP1 or ENPP3 KO )mice are expected to show a tissue appearance similar to that of healthy wild-type mice, with reduced or absent renal mineral deposits in the tubular lumen and reduced calcification of the spine and soft tissue vascular structures.

[0509] In addition to survival time, daily animal body weight, and terminal tissue appearance, the treatment response is evaluated by postmortem high-resolution micro-CT scans for imaging vascular calcification and plasma PPi concentration. Neither WT nor treated (vector expressing ENPP1) ttw (or ENPP1 KO )is expected to have any vascular calcification via micro-CT, in contrast to the dramatic calcification seen in the aorta, coronary arteries and heart of the untreated (null vector) ttw (or ENPP1 KO )cohort. Furthermore, the serum PPi concentration (5.2 μM) of treated (vector expressing ENPP1) ttw (or ENPP1 KO )animals is expected to increase to WT levels (4.4 μM) and significantly exceed the levels (0.5 μM) of untreated ttw (or ENPP1 KO ).

[0510] Untreated ttw (or ENPP1 KO)The mouse shows a significant increase in serum inorganic phosphorus (pi), an increase in PTH and FGF23 levels, a decrease in 1,25(OH) 2 -vitamin D levels, and a lower PPi level (about 0.5 μM) when compared to that of a healthy wild-type mouse (the normal level of PP is about 2 - 4 μM; for PTH it is about 10 - 65 ng / L; the median FGF23 level is 13 RU / ml, and the normal FGF23 level ranges from 5 - 210 RU / ml; the normal vitamin D level is 20 ng / mL - 50 ng / mL). In contrast, the treated ttw (or ENPP1 KO ) mouse is expected to show an increase in PPi level (about 4 - 5 μM), which is expected to be higher than the PPi level (about 0.5 μM) seen in the untreated ttw (or ENPP1 KO ) mouse. Thus, through histological analysis, a reduction in renal soft tissue and coronary artery calcification (25% or 50% or 70% or 90% or 100% reduction) visualized, an increase in serum PPi level from blood analysis, normalization of vitamin D level, reduction of FGF23 level towards the normal range and normalization of PTH level, improvement of long bone strength, increase in bone density, improvement of cortical bone thickness and trabecular bone mass, increase in survival time, and improvement of renal function observed as an increase in urinary urea and creatinine along with an increase in body weight gain, one or more factors such as these allow one of ordinary skill in the art to determine the therapeutic efficacy of vector-based ENPP1 or ENPP3 in the treatment of osteopenia or osteomalacia or osteoarthritis.

[0511] Treatment of human subjects: Approximately 5×10 11 ~5×10 15 vg / kg , and in some embodiments, approximately 1×10 12 ~1×10 15 vg / kgBy administering an intravenous injection comprising [the compound], a human patient suffering from osteopenia, osteomalacia, or osteoarthritis is treated. The success of the treatment of osteopenia, osteomalacia, or osteoarthritis is observed by monitoring one or more of the aforementioned parameters through regular blood and urine tests of bone strength and bone density as described for the mouse model. Instead of histological analysis requiring staining of kidney slices or arterial tissue, which is not feasible in living patients, non-invasive visualization techniques as described in Example 4 are used.

[0512] Similarly, the patient is subjected to regular bone density measurements using dual energy X-ray absorptiometry (DXA), peripheral dual energy X-ray absorptiometry (pDXA), quantitative ultrasound (QUS), or peripheral quantitative computed tomography (pQCT). The bone density score obtained from one of these methods provides an indication of the state and progression obtained after treatment. A T-score of 1.0 or higher is considered normal bone density, a T-score between -1.0 and -2.5 indicates the presence of osteopenia, while a T-score of -2.5 or lower indicates the presence of osteoporosis. In patients treated with ENPP1 or ENPP3 of the present invention, a stepwise improvement in the T-score is expected.

[0513] A physician having the skill to visualize soft tissue calcification, heart calcification, and bone density visualization attempts to treat a subject suffering from osteoporosis or osteoarthritis by administering AAV virions expressing hENPP1 or hENPP3. In some embodiments, the physician delivers a construct of hENPP1 or hENPP3 and uses viral particles that express the corresponding protein under the control of an inducible promoter. Thus, the physician has the option to control the dosage (the amount of hENPP1 or hENPP3 expressed) based on the rate and extent of symptom improvement. The success of the treatment and the appropriate dosage can be readily inferred by medical professionals in the field by observing one or more positive symptoms such as normal vitamin D levels (levels of 20 ng / ml to 50 ng / mL are considered appropriate for healthy people; levels below 12 ng / mL indicate vitamin D deficiency), normal bone density (T-score of ≧ -1), normal blood urea nitrogen levels (BUN levels for healthy adults are 7 to 20 mg / dL), weight gain, an increase in serum PPi levels (at least about 4 to 5 μm), a reduction in arterial tissue calcification (a reduction of 25% or 50% or 70% or 90% or 100%), and an improvement in bone strength visualized by non-invasive techniques such as CT, magnetic resonance imaging (MRI), or ultrasound scans.

[0514] Example 9 - Treatment of ADHR-2 or ARHR-2 and / or XLH Using a Viral Vector Expressing ENPP1 or ENPP3 The following examples provide AAVs expressing ENPP1 or ENPP3 that are expected to be effective in treating symptoms associated with ADHR-2 or ARHR-2 or XLH. For illustrative purposes, ENPP1-Fc and ENPP3-Fc are used in this example, and similar results can be obtained using other ENPP1 or ENPP3 fusions of the present invention.

[0515] AAV virions expressing ENPP1-Fc protein or ENPP3-Fc protein are prepared according to Example 1 and administered to a HYP mouse model of X-linked hypophosphatemia (XLH) (Liang, et al., 2009, Calcif. Tissue Int. 85(3):235-46). For the treatment with ENPP1 and ENPP3, six sets of mice are used. In addition to GACI, it also acts as a model of ARHR-2 (Am J Hum Genet. 2010 Feb 12;86(2):273-278.) ENPP1 age stiffened joint mouse (ENPP1 asj / asj ) is used to repeat the same experiment.

[0516] Control cohort: In this experiment, AAV particles containing a null vector are injected into the first cohort of ENPP1 wt mice that serve as a control group, and AAV particles containing a null vector are injected into the second cohort of HYP (or ENPP1 asj / asj ) mice that serve as a control group.

[0517] Cohort of ENPP1-treated mice: AAV particles engineered to express ENPP1-Fc protein are injected into the third cohort of ENPP1 wt mice, and AAV particles engineered to express ENPP1-Fc protein are injected into the fourth cohort of HYP (or ENPP1 asj / asj ) mice.

[0518] Cohort of ENPP3-treated mice: AAV particles engineered to express ENPP3-Fc protein are injected into the fifth cohort of ENPP1 wt mice, and AAV particles engineered to express ENPP3-Fc protein are injected into the sixth cohort of HYP (or ENPP1 asj / asj ) mice. Wild-type mice are maintained on a normal solid diet, and HYP (or ENPP1 asj / asj ) mice are given a high-phosphate Teklad diet.

[0519] Vector injection: After 2 weeks of age, approximately 1×10 12 ~1×101 5 vg / kg , preferably 1×10 13 ~1×10 14 vg / kg of retro-orbital or tail vein injection per mouse is performed on all mice. The vector to be injected is either an empty "null" (control group) or one carrying the NPP1 or NPP3 gene (study group).

[0520] Assay: For each cohort, histological images of the kidneys, PPi levels and blood and urine parameters such as FGF-23 levels, vitamin D, parathyroid hormone (PTH) levels, serum / blood urea levels, blood urea nitrogen (BUN) levels, serum / blood creatinine levels and plasma pyrophosphate (PPi) are analyzed as described in Examples 3 and 4.

[0521] Histological images, histomorphometry and micro-CT: Bone analysis is performed according to the protocol described in Example 3.

[0522] Biomechanical testing of bone: Bone analysis is performed according to the protocol described in Example 3.

[0523] Results: Untreated HYP (or ENPP1 asj / asj ) mice generally show weight loss, lethargy, decreased cortical bone thickness and trabecular bone mass, calcification of cartilage and ligaments, decreased bone density such as in the femur and tibia, and increased mortality compared to wild type. In contrast, HYP (or ENPP1 asj / asj ) mice treated with AAV expressing ENPP1 protein or ENPP3 protein are expected to show weight gain approaching the weight range of normal WT mice, normal wakefulness, increased bone mineral content, improved cortical bone thickness and trabecular bone mass, and increased bone strength and bone flexibility. HYP (or ENPP1 asj / asj)The mouse is expected to show calcification of the heart, aorta and coronary arteries, as well as histological evidence of myocardial infarction in the free wall of the right ventricle, calcification of the coronary arteries, heart, ascending aorta and descending aorta, myocardial cell necrosis, and myocardial fibrosis in the myocardial tissue adjacent to the area of coronary artery calcification. In contrast, HYP (or ENPP1) mice treated with a vector expressing ENPP1-Fc or ENPP3-Fc are expected to show no calcification of the heart, arteries or aorta by histological examination or post-mortem micro-CT. asj / asj )HYP (or ENPP1) mice treated with a null vector are expected to show severe extensive calcification centered on the outer medulla and extending into the renal cortex, as well as calcification centered on the renal medulla. In contrast, HYP (or ENPP1) mice treated with viral vector-based expression of ENPP1 or ENPP3 are expected to show a tissue appearance similar to that of healthy wild-type mice, with reduced or absent renal mineral deposits in the tubule lumen and reduced calcification of the spine and soft tissue vascular structures. asj / asj )HYP (or ENPP1) mice treated with a null vector are expected to show severe extensive calcification centered on the outer medulla and extending into the renal cortex, as well as calcification centered on the renal medulla. In contrast, HYP (or ENPP1) mice treated with viral vector-based expression of ENPP1 or ENPP3 are expected to show a tissue appearance similar to that of healthy wild-type mice, with reduced or absent renal mineral deposits in the tubule lumen and reduced calcification of the spine and soft tissue vascular structures. asj / asj )HYP (or ENPP1) mice treated with viral vector-based expression of ENPP1 or ENPP3 are expected to show a tissue appearance similar to that of healthy wild-type mice, with reduced or absent renal mineral deposits in the tubule lumen and reduced calcification of the spine and soft tissue vascular structures.

[0524] In addition to survival time, daily animal body weight, and final tissue appearance, the treatment response is evaluated by post-mortem high-resolution micro-CT scans for imaging vascular calcification and plasma PPi concentration. None of the WT or treated (vector expressing ENPP1) HYP (or ENPP1) mice are expected to have any vascular calcification via micro-CT, in contrast to the dramatic calcification seen in the aorta, coronary arteries and heart of the untreated (null vector) HYP (or ENPP1) cohort. Furthermore, the serum PPi concentration (5.2 μM) of treated (vector expressing ENPP1) HYP (or ENPP1) mice is expected to increase to WT levels (4.4 μM) and significantly exceed the level (0.5 μM) of untreated HYP (or ENPP1). asj / as j) None of the mice, whether WT or treated (vector expressing ENPP1) HYP (or ENPP1), are expected to have any vascular calcification via micro-CT, in contrast to the dramatic calcification seen in the aorta, coronary arteries and heart of the untreated (null vector) HYP (or ENPP1) cohort. asj / asj )Furthermore, the serum PPi concentration (5.2 μM) of treated (vector expressing ENPP1) HYP (or ENPP1) mice is expected to increase to WT levels (4.4 μM) and significantly exceed the level (0.5 μM) of untreated HYP (or ENPP1). asj / asj )Furthermore, the serum PPi concentration (5.2 μM) of treated (vector expressing ENPP1) HYP (or ENPP1) mice is expected to increase to WT levels (4.4 μM) and significantly exceed the level (0.5 μM) of untreated HYP (or ENPP1). asj / asj )Furthermore, the serum PPi concentration (5.2 μM) of treated (vector expressing ENPP1) HYP (or ENPP1) mice is expected to increase to WT levels (4.4 μM) and significantly exceed the level (0.5 μM) of untreated HYP (or ENPP1).

[0525] Untreated HYP (or ENPP1)asj / asj ) The mouse shows a significant increase in serum inorganic phosphorus (pi), increases in PTH and FGF23 levels when compared to that of healthy wild-type mice (the normal level of PP is about 2 - 4 μM; for PTH it is about 10 - 65 ng / L; the median FGF23 level is 13 RU / ml and the normal FGF23 level ranges from 5 - 210 RU / ml; the normal vitamin D level is 20 ng / mL - 50 ng / mL), but a decrease in 1,25(OH) 2 -vitamin D level, and a lower PPi level (about 0.5 μM) are also expected. In contrast, the treated HYP (or ENPP1 asj / asj ) mouse is expected to show an increase in PPi level (about 4 - 5 μM), which is expected to be higher than the PPi level (about 0.5 μM) seen in untreated HYP (or ENPP1 asj / asj ) mice. Thus, through histological analysis, a reduction in soft tissue of the kidney and coronary artery calcification (25% or 50% or 70% or 90% or 100% reduction) visualized, an increase in serum PPi level from blood analysis, normalization of vitamin D level, reduction of FGF23 level towards the normal range and normalization of PTH level, improvement in long bone strength, increase in bone density, improvement in cortical bone thickness and trabecular bone mass, increase in survival time, and improvement in kidney function observed as an increase in urinary urea and creatinine along with an increase in body weight gain, one or more factors such as these allow one of ordinary skill in the art to determine the therapeutic efficacy of vector-based ENPP1 or ENPP3 in the treatment of ADHR-2 or ARHR-2 or XLH.

[0526] Treatment of human subjects: Approximately 5×10 per subject, in 1×PBS at pH 7.4, that can deliver and express hENPP1 or hENPP3 11 ~5×10 15 vg / kg , in some embodiments, approximately 1×10 in 1×PBS at pH 7.4 12 ~1×10 15vg / kgBy performing an intravenous injection comprising, treat a human patient suffering from ADHR-2 or ARHR-2 or XLH. The success of the treatment of ADHR-2 or ARHR-2 or XLH is observed by monitoring one or more of the aforementioned parameters through regular bone strength, blood and urine tests for bone density as described for the mouse model. Instead of histological analysis requiring staining of kidney slices or arterial tissue, which is not possible to perform on living patients, use non-invasive visualization techniques as described in Example 4.

[0527] Similarly, subject the patient to regular bone density measurements using dual energy X-ray absorptiometry (DXA) or peripheral dual energy X-ray absorptiometry (pDXA) or quantitative ultrasound (QUS) or peripheral quantitative computed tomography (pQCT). The bone density score obtained from one of these methods provides an indication of the state and progression obtained after treatment. A T-score of 1.0 or greater is considered normal bone density, a T-score between -1.0 and -2.5 indicates the presence of osteopenia, while a T-score of -2.5 or less indicates the presence of osteoporosis. In patients treated with ENPP1 or ENPP3 of the present invention, a stepwise improvement in the T-score is expected.

[0528] A physician having the skill to visualize soft tissue calcification, heart calcification, and bone density visualization attempts to treat a subject suffering from ADHR-2 or ARHR-2 or XLH by administering AAV virions expressing hENPP1 or hENPP3. In some embodiments, the physician delivers a construct of hENPP1 or hENPP3 and uses viral particles that express the corresponding protein under the control of an inducible promoter. Thus, the physician has the option to control the dosage (the amount of hENPP1 or hENPP3 expressed) based on the rate and extent of symptom improvement. The success of the treatment and the appropriate dosage are easily inferred by medical experts in the field by observing one or more positive symptoms such as normal vitamin D levels (levels of 20 ng / ml to 50 ng / mL are considered appropriate for healthy people. Levels below 12 ng / mL indicate vitamin D deficiency), normal bone density (T-score of ≧ -1), normal blood urea nitrogen levels (the BUN level for healthy adults is 7 - 20 mg / dL), weight gain, an increase in serum PPi levels (at least about 4 - 5 μm), a reduction in arterial tissue calcification (a reduction of 25% or 50% or 70% or 90% or 100%), and an improvement in bone strength visualized by non-invasive techniques such as CT, magnetic resonance imaging (MRI), or ultrasound scans.

[0529] Example 10 - Analysis of Plasma PPi Levels, ENPP1 Concentration, and Activity Levels in Model Mice after Viral Administration Three cohorts of normal mice were used in this experiment. Each cohort included five adult mice. The first cohort was used as the "control group" and saline was injected into the control group. The second cohort was used as the "low-dose group" and an AAV vector at a concentration of 1e 13 vg / kg was injected into the low-dose group. The third cohort used the "high-dose group" and an AAV vector at a concentration of 1e 14Mice in the high-dose group were injected with an AAV vector at a concentration of vg / kg. The process of generating virus particles from the AAV construct and injecting the recombinant AAV virus particles containing the ENPP1 fusion protein into normal mice is schematically shown in Figure 4. Blood was collected from the mice in all cohorts on days 7, 28, and 56 after injection, and plasma and serum were collected.

[0530] Blood was collected into heparinized tubes. Plasma was isolated and platelets were removed by filtration through a Nanosep 30 kDa Omega centrifugal filter (Pall, OD030C35). The samples were centrifuged at the maximum speed (about 20 kg) for 20 minutes at 4 °C. The flow-through was collected and the samples were snap-frozen on dry ice. The samples were stored at -80 °C for later use in assays.

[0531] First, the collected samples were assayed to determine the activity level of ENPP1 using the colorimetric substrate, p-nitrophenyl thymidine 5'-monophosphate (Sigma). Plasma samples were incubated for 1 hour with 1 mg / ml p-nitrophenyl thymidine 5'-monophosphate in 1% Triton, 200 mM Tris, pH 8.0 buffer. After 1 hour, 100 mM NaOH was added to stop the reaction and the absorbance was measured at 405 nm. The specific activity was determined according to the following assay protocol disclosed by R&D Systems for recombinant human ENPP-1; catalog number: 6136-WN.

Number

[0532] The results of the ENPP1 activity assay are shown in Figure 5, which shows a dose-dependent increase in ENPP1 activity after injection. Plasma from normal mice was used as a reference standard to normalize ENPP1 activity levels, and one-way ANOVA was used for statistical analysis. Figure 5 shows that the ENPP1 activity levels were higher in the low-dose group when compared to those of the control group. Similarly, the ENPP1 activity levels were higher in the high-dose group when compared to those of the low-dose and control groups. Among the low-dose and high-dose cohorts, ENPP1 activity was stable in plasma samples from 7 to 56 days in the high-dose group, but there was a slight decrease in ENPP1 activity from 28 to 56 days in the low-dose group.

[0533] Samples were then assayed to determine the concentration of ENPP1 using a sandwich ELISA assay with an ENPP1 polyclonal antibody obtained from Sigma (SAB1400199). 96-well clear flat-bottom polystyrene high-binding microplates (Corning catalog number 9018), BSA (Sigma #7906), 10× Dulbecco's phosphate buffered saline (DPBS) (Quality Biological catalog number 119-068-101), Tween-20 (Sigma catalog number P2287), anti-ENPP1 antibody produced in mice (Sigma-Aldrich catalog number SAB1400199), Sure Blue TMB microwell peroxidase substrate (1-component) (KPL product number 52-00-01), 2N sulfuric acid (BDH product number BDH7500-1), MilliQ water, C57BL / 6 mouse plasma NaHep pooled gender (BioIVT catalog number MSE01PLNHPNN), mouse serum (BIO IVT elevating Science catalog number MSE01SRMPNN) were used in the ELISA assay.

[0534] A calibration curve for the ENPP1-Fc protein is created by the following standard procedure known in the art. Briefly, serial dilutions of the ENPP1-Fc protein in the range of 2 mg / ml to 30 ng / ml were made. A 96-well plate was first coated with an overnight coating solution containing the ENPP1 capture antibody in 1×PBS at 1 μg / 1 mL. The wells were then incubated with 5% BSA in PBS for 1 hour and then washed with a post-block solution. The ENPP1 dilution samples were added to the coated 96-well plate and incubated for 1.5 hours. After incubation, the wells were washed 4 times with 300 μl of 0.05 T% PBST. The washed wells were then treated with 100 μL / well of the detection HRP antibody conjugate and incubated for 1 hour. After incubation with the HRP antibody conjugate, the wells were washed 4 times with 300 μl of 0.05 T% PBST. The washed wells were then treated with 100 μl / well of the TMB microplate peroxidase substrate and incubated in the dark for 30 minutes. The wells were then washed 4 times with 300 μl of 0.05 T% PBST and the reaction was stopped using 2N sulfuric acid. The absorbance of the wells was read using a microplate reader at a wavelength of 450 nm. A calibration curve was created using the absorbance readings and the corresponding concentrations of the ENPP1 serial dilution samples.

[0535] Subsequently, the assays were repeated using plasma samples obtained from the control, low-dose, and high-dose cohorts on days 7, 28, and 56 after virus injection. The absorbance generated with each plasma sample was correlated with the calibration curve of ENPP1-Fc to determine the concentration of ENPP1-Fc in the plasma samples. The results of the ENPP1 concentration assay are shown in Figure 6, which shows a dose-dependent increase in ENPP1 concentration after injection of the viral vector. The ENPP1 concentration levels were normalized using plasma from normal mice as a reference standard, and one-way ANOVA was used for statistical analysis. Figure 6 shows that the ENPP1 concentration was higher in the low-dose group compared to that of the control group. Similarly, the ENPP1 activity levels were higher in the high-dose group compared to those of the low-dose and control groups. Among the low-dose and high-dose cohorts, the ENPP1 levels were stable in the high-dose group in samples from days 7 to 56, but there was a slight decrease in ENPP1 levels in the low-dose group from days 28 to 56.

[0536] Samples were also assayed to determine the concentration of plasma PPi using a sulfirylase assay. ATP sulfirylase (NEB-M0394L, lot number: 10028529), adenosine 5'-phosphosulfate (APS; Santa Cruz, sc-214506)), PPi: 100 uM stock, HEPES pH 7.4 buffer (Boston Bioproducts BB2076), 1 M magnesium sulfate (MgSO4) solution, 1 M calcium chloride (CaCl2) solution, BactiterGlo (Promega G8231), plates (Costar 3915, black flat bottom), and a plate reader (Molecular Devices Spectramax I3x) were used for the PPi-sulfirylase assay. Serial dilutions were used to prepare PPi standards (0.125 - 4 μM) in water. In the presence of excess adenosine 5'-phosphosulfate (APS), PPi in the PPi standards and filtered plasma samples was converted to ATP by ATP sulfirylase. 8 mM CaCl 2The sample (15 μl) was treated with 5 μl of a mixture containing 2 mM MgSO4, 40 mM HEPES pH 7.4, 80 μM APS (Santa Cruz, sc-214506), and 0.1 U / ml ATP sulfurylase (NEB-M0394L). The mixture was incubated at 37 °C for 40 minutes and then the ATP sulfurylase was inactivated by incubating at 90 °C for 10 minutes. The generated ATP was measured using BactiterGlo (Promega G8231) by mixing 20 μl of the treated sample or standard with 20 μl of BactiterGlo reagent. Subsequently, bioluminescence was measured with a microplate reader, and then the amount of PPi generated in each sample was determined from the calibration curve.

[0537] The results of the plasma PPi assay are shown in Figure 7. The results show a dose-dependent increase in plasma PPi after injection of the viral vector. Plasma PPi concentration levels were normalized using plasma from normal mice as a reference standard, and one-way ANOVA was used for statistical analysis. Figure 7 shows that the plasma PPi concentration was slightly higher in the low-dose group when compared to that of the control group. Similarly, the plasma PPi concentration was higher in the high-dose group when compared to that of the low-dose and control groups. Among the low-dose and high-dose cohorts, the ENPP1 levels were stable in plasma samples from the high-dose group up to 7 - 56 days, while a slight decrease in ENPP1 levels was observed in the low-dose group from days 28 - 56.

[0538] In related experiments, a single dose of 1e14 vg / kg of AAV viral vector or vehicle control (without AAV vector) was intravenously administered to 5- to 6-week-old C57 / Bl male mice. GK1.5 (40 μg / mouse, 1 day before administration of the viral vector or vehicle, and then 25 μg / mouse every 7 days until the study was completed) was administered to the animals. The AAV viral vector was engineered to express a fusion protein of ENPP1 and IgG Fc, which was similar to the polypeptide described in Example 10, except that the ENPP portion and the IgG Fc portion of the fusion protein were connected by the following linker amino acid sequence: GGGGS. Mice administered the AAV viral vector showed higher levels of ENPP1 enzyme activity than the vehicle-only control when measured over a period of approximately 40 days.

[0539] Example 11 - Analysis of the Concentration and Activity Levels of ENPP1 in Model Mouse Viruses 112 Days after Virus Administration Three cohorts of normal mice were used in this experiment. Each cohort included 5 adult mice. The first cohort was used as the "control group" and saline was injected into the control group. The second cohort was used as the "low-dose group" and an AAV vector at a concentration of 1e 13 vg / kg was injected into the low-dose group. The third cohort was used as the "high-dose group" and an AAV vector at a concentration of 1e 14 vg / kg was injected into the high-dose group. The process of generating virus particles from the AAV construct and injecting recombinant AAV virus particles containing the ENPP1 fusion protein into normal mice is schematically shown in Figure 4. Blood was collected from the mice in all cohorts on days 7, 28, 56, and 112 after injection to collect plasma and serum.

[0540] Blood was collected into heparinized tubes. The samples were centrifuged at a maximum speed (about 20 kg) for 20 minutes at 4°C. The flow-through was collected and the samples were snap-frozen on dry ice. The samples were stored at -80°C for later use in assays.

[0541] First, as described in Example 10, the collected samples were assayed to determine the activity level of ENPP1 using the colorimetric substrate, p-nitrophenyl thymidine 5'-monophosphate (Sigma). The results of the ENPP1 activity assay are shown in Figure 9, which shows a dose-dependent increase in ENPP1 activity after injection. Normal mouse plasma was used as a reference standard to normalize the ENPP1 activity levels, and one-way ANOVA was used for statistical analysis. Figure 9 shows that the ENPP1 activity levels were higher in the low-dose group when compared to those of the control group. Similarly, the ENPP1 activity levels were higher in the high-dose group when compared to those of the low-dose and control groups.

[0542] Next, the samples were assayed to determine the concentration of ENPP1 using a sandwich ELISA assay with an ENPP1 polyclonal antibody obtained from Sigma (SAB1400199) according to the protocol taught in Example 10. The assay was then repeated using plasma samples obtained from the control, low-dose, and high-dose cohorts at 7, 28, 56, and 112 days post-viral injection. The absorbance generated for each plasma sample was correlated with a calibration curve of ENPP1-Fc to determine the concentration of ENPP1-Fc in the plasma sample.

[0543] The results of the ENPP1 concentration assay are shown in Figure 8, which shows a dose-dependent increase in ENPP1 concentration after injection of the viral vector. Normal mouse plasma was used as a reference standard to normalize the ENPP1 concentration levels, and one-way ANOVA was used for statistical analysis. Figure 8 shows that the ENPP1 concentration was higher in the low-dose group when compared to those of the control group. Similarly, the ENPP1 levels were higher in the high-dose group when compared to those of the low-dose and control groups.

[0544] Other embodiments From the foregoing description, for treating any disease characterized by the presence of pathological calcification or ossification, various uses and conditions can be adopted, including the use of different signal sequences for expressing functional variants of ENPP1 or ENPP3 or combinations thereof in different viral vectors or different cell types having different promoters or enhancers known in the art. It will be apparent that modifications and variations to the invention described herein can be made within the scope of the invention. Other embodiments according to the invention are within the scope of the following claims.

[0545] The recitation of a list of elements in any definition of a variable herein includes the definition of that variable as any single element or combination (or sub-combination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portions thereof.

[0546] All publications and patent applications mentioned herein are indicative of the level of skill of those of ordinary skill in the art to which this invention pertains. All publications and patent applications are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0547] Other embodiments are within the scope of the following claims.

Claims

1. A recombinant polynucleotide encoding a precursor polypeptide comprising an azurocidin signal peptide fused to ectonucleotide pyrophosphatase / phosphodiesterase-1 (ENPP1) or ectonucleotide pyrophosphatase / phosphodiesterase-3 (ENPP3), wherein upon expression of the polynucleotide in a mammalian cell, the precursor polypeptide is proteolytically cleaved to generate soluble ENPP1 or soluble ENPP3 that is active in reducing ectopic calcification of soft tissue.

2. The recombinant polynucleotide of claim 1 , comprising a vector or a plasmid.

3. 2. The recombinant polynucleotide of claim 1, wherein the vector or plasmid is capable of expressing the encoded polypeptide.

4. The recombinant polynucleotide of claim 3 , wherein the vector is a viral vector.

5. The recombinant polynucleotide of claim 4, wherein the viral vector is an adeno-associated viral (AAV) vector.

6. The recombinant polynucleotide of any one of claims 1 to 5, wherein the polynucleotide encodes the azurocidin signal peptide fused to the ENPP1 or the azurocidin signal peptide fused to the ENPP3, and the ENPP1 or the ENPP3 fused to an Fc polypeptide, forming, from amino terminus to carboxy terminus, azurocidin signal peptide-ENPP1-Fc or azurocidin signal peptide-ENPP3-Fc, respectively.

7. The recombinant polynucleotide of any one of claims 1 to 5, wherein the polynucleotide encodes the azurocidin signal peptide fused to the ENPP1 or the azurocidin signal peptide fused to the ENPP3, and the ENPP1 or the ENPP3 fused to human serum albumin to form, in order from amino terminus to carboxy terminus, azurocidin signal peptide-ENPP1-albumin or azurocidin signal peptide-ENPP3-albumin, respectively.

8. A viral vector comprising a nucleic acid sequence encoding a signal peptide fused to the N-terminus of ENPP1 or ENPP3 and capable of expressing the same.

9. The viral vector of claim 8 , which comprises a promoter.

10. The viral vector of claim 9 , wherein the promoter is a liver-specific promoter.

11. The viral vector of claim 10, wherein the liver-specific promoter is selected from the group consisting of an albumin promoter, a phosphoenolpyruvate carboxykinase (PEPCK) promoter, and an alpha-1-antitrypsin promoter.

12. A viral vector according to any one of claims 8 to 11, comprising a sequence encoding a polyadenylation signal.

13. The viral vector according to any one of claims 8 to 12, wherein the signal peptide is an azurocidin signal peptide.

14. The viral vector according to any one of claims 8 to 13, which is an adeno-associated viral (AAV) vector.

15. The viral vector of claim 14, wherein the AAV vector has a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and AAV-rh74.

16. The viral vector of any one of claims 13 to 15, wherein the polynucleotide encodes the azurocidin signal peptide fused to the ENPP1 or the azurocidin signal peptide fused to the ENPP3, and the ENPP1 or the ENPP3 fused to an Fc polypeptide, forming, from amino terminus to carboxy terminus, azurocidin signal peptide-ENPP1-Fc or azurocidin signal peptide-ENPP3-Fc, respectively.

17. The viral vector of any one of claims 13 to 15, wherein the polynucleotide encodes the azurocidin signal peptide fused to the ENPP1 or the azurocidin signal peptide fused to the ENPP3, and the ENPP1 or the ENPP3 fused to human serum albumin, forming, from amino terminus to carboxy terminus, azurocidin signal peptide-ENPP1-albumin or azurocidin signal peptide-ENPP3-albumin, respectively.

18. A method for obtaining a recombinant viral vector according to any one of claims 8 to 17, comprising the steps of: i. Providing a cell comprising the polynucleotide of any one of claims 1 to 7; ii. maintaining the cells under conditions suitable for assembly of the virus; and iii. Purifying the viral vector produced by the cells. A method comprising:

19. 1. A method of providing an ENPP1 or ENPP3 protein to a mammal, comprising: A method comprising administering to the mammal a viral vector according to any one of claims 8 to 17.

20. A pharmaceutical composition comprising a viral vector according to any one of claims 8 to 17 and a physiologically compatible carrier.

21. 21. A method of preventing or reducing the progression of a disease in a mammal in need thereof, comprising administering to said mammal a therapeutically effective amount of the pharmaceutical composition of claim 20, wherein said disease is selected from the group consisting of X-linked hypophosphatemia (XLH), chronic kidney disease (CKD), mineral bone disorder (MBD), vascular calcification, pathological calcification of soft tissue, pathological ossification of soft tissue, generalized arterial calcification of infancy (GACI) and ossification of the posterior longitudinal ligament (OPLL), thereby preventing or reducing the progression of said disease in said mammal.

22. A cell comprising the polynucleotide according to any one of claims 1 to 7.

23. A method for treating or preventing a pathological calcification or pathological ossification disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a viral vector encoding a recombinant ENPP1 or ENPP3 polypeptide, thereby treating or preventing the disease or disorder.

24. A method for treating a subject having a deficiency of ENPP1 protein, comprising administering to the subject a therapeutically effective amount of a viral vector encoding a recombinant ENPP1 or ENPP3 polypeptide, thereby treating the subject.

25. The method of claim 23 or 24, wherein the disease or disorder or deficiency of the ENPP1 protein is associated with a loss-of-function mutation in the NPP1 gene or a loss-of-function mutation in the ABCC6 gene in the subject.

26. The method of claims 23 to 25, wherein the viral vector encodes a recombinant ENPP1 polypeptide.

27. The method of claims 23 to 25, wherein the viral vector encodes a recombinant ENPP3 polypeptide.

28. The method of claims 23 to 26, wherein the viral vector encodes a recombinant ENPP1-Fc fusion polypeptide or a recombinant ENPP1-albumin fusion polypeptide.

29. The method of claim 27, wherein the viral vector encodes a recombinant ENPP3-Fc fusion polypeptide or a recombinant ENPP3-albumin fusion polypeptide.

30. The method of claims 23 to 29, wherein the viral vector encodes a recombinant polypeptide comprising a signal peptide fused to ENPP1 or ENPP3.

31. The method according to claims 23 to 30, wherein the vector encodes ENPP1-Fc or ENPP1-albumin.

32. The method according to claims 23 to 30, wherein the signal peptide is an azurocidin signal peptide, an NPP2 signal peptide or an NPP7 signal peptide.

33. The method according to claims 23 to 30, wherein the viral vector is an adeno-associated viral vector or a herpes simplex vector or an alphavirus vector or a lentivirus vector.

34. The method of claim 33, wherein the serotype of the adeno-associated viral vector (AAV) is AAV1 or AAV2 or AAV3 or AAV4 or AAV5 or AAV6 or AAV7 or AAV8 or AAV9 or AAV-rh74.

35. The method according to claims 23 to 32, wherein the viral vector is an adeno-associated viral (AAV) vector encoding a recombinant polypeptide comprising an azurocidin signal peptide fused to an ENPP1-Fc fusion polypeptide.

36. The AAV vector encoding the ENPP1-Fc fusion polypeptide is 1×10 １２ ~1×10 １５ 36. The method of claim 35, wherein the subject is administered a dosage of 100 mg / kg.

37. The dosage is 1×10 １３ ~1×10 １４ 36. The method of claim 35, wherein the dose is 100 mg / kg.

38. The AAV vector is 5×10 １１ ~5×10 １５ 36. The method of claim 35, wherein the subject is administered a dosage of 100 mg / kg.

39. The vector is an AAV vector encoding ENPP1-Fc, １２ ~1×10 １５ 36. The method of claim 35, wherein the subject is administered a dosage of 100 mg / kg.

40. The method of claim 35, wherein administration of the AAV vector encoding an ENPP1-Fc polypeptide to a subject results in a dose-dependent increase in plasma pyrophosphate (PPi) and a dose-dependent increase in plasma ENPP1 concentration in the subject.

41. A viral vector comprising a polynucleotide sequence encoding a polypeptide comprising the catalytic domain of an ENPP1 or ENPP3 protein.

42. The viral vector of claim 41 , wherein the polypeptide sequence comprises the extracellular domain of an ENPP1 or ENPP3 protein.

43. 43. The viral vector of claim 41 or 42, wherein the polypeptide comprises the transmembrane domain of an ENPP1 or ENPP3 protein.

44. A viral vector according to any one of claims 41 to 43, wherein the polypeptide comprises a nuclease domain of an ENPP1 or ENPP3 protein.

45. 45. The viral vector of any one of claims 41 to 44, wherein the polypeptide comprises residues 99 to 925 (Pro Ser Cys to Gln Glu Asp) of SEQ ID NO:

1.

46. 45. The viral vector of any one of claims 41 to 44, wherein the polypeptide comprises residues 31 to 875 (Leu Leu Val to Thr Thr Ile) of SEQ ID NO:

7.

47. 45. The viral vector of any one of claims 41 to 44, wherein the polypeptide comprises residues 191 to 591 (Val Glu Glu to Gly Ser Leu) of SEQ ID NO:

1.

48. 45. The viral vector of any one of claims 41 to 44, wherein the polypeptide comprises residues 140 to 510 (Leu Glu Glu to Glu Val Glu) of SEQ ID NO:

7.

49. 45. The viral vector of any one of claims 41 to 44, wherein the polypeptide comprises residues 1 to 827 (Pro Ser Cys to Gln Glu Asp) of SEQ ID NO:

92.

50. 45. The viral vector of any one of claims 41 to 44, wherein the polypeptide comprises residues 1 to 833 (Phe Thr Ala to Gln Glu Asp) of SEQ ID NO: 89 or residues 1 to 830 (Gly Leu Lys to Gln Glu Asp) of SEQ ID NO:

91.

51. The viral vector according to any one of claims 41 to 50, which is not an insect viral vector.

52. The viral vector according to any one of claims 41 to 51, which infects a mammalian cell.

53. The viral vector of any one of claims 41 to 52, wherein the polynucleotide sequence encodes a promoter sequence.

54. The viral vector of claim 53, wherein the promoter is a liver-specific promoter.

55. 55. The viral vector of claim 54, wherein the liver-specific promoter is selected from the group consisting of an albumin promoter, a phosphoenolpyruvate carboxykinase (PEPCK) promoter, and an alpha-1-antitrypsin promoter.

56. The viral vector of any one of claims 41 to 55, wherein the polynucleotide sequence comprises a nucleotide sequence encoding a polyadenylation signal.

57. The viral vector of any one of claims 41 to 55, wherein the polynucleotide encodes a signal peptide that is amino-terminal to the nucleotide sequence encoding the ENPP1 or ENPP3 protein.

58. 58. The viral vector of claim 57, wherein the signal peptide is an azurocidin signal peptide.

59. The viral vector of any one of claims 41 to 58, which is an adeno-associated viral (AAV) vector.

60. 60. The viral vector of claim 59, wherein the AAV vector has a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and AAV-rh74.

61. The viral vector of any one of claims 41 to 60, wherein the polynucleotide sequence encodes the azurocidin signal peptide fused to the ENPP1 or the azurocidin signal peptide fused to the ENPP3, and the ENPP1 or the ENPP3 fused to an Fc polypeptide, forming, in order from amino terminus to carboxy terminus, azurocidin signal peptide-ENPP1-Fc or azurocidin signal peptide-ENPP3-Fc, respectively.

62. The viral vector of any one of claims 41 to 60, wherein the polynucleotide sequence encodes the azurocidin signal peptide fused to the ENPP1 or the azurocidin signal peptide fused to the ENPP3 and the ENPP1 or the ENPP3 fused to human serum albumin to form, in order from amino terminus to carboxy terminus, azurocidin signal peptide-ENPP1-albumin or azurocidin signal peptide-ENPP3-albumin, respectively.

63. The viral vector of any one of claims 41 to 62, wherein the polypeptide is a fusion protein comprising (i) an ENPP1 protein or an ENPP3 protein and (ii) a half-life prolonging domain.

64. 64. The viral vector of claim 63, wherein the half-life prolonging domain is an IgG Fc domain or a functional fragment of the IgG Fc domain capable of extending the half-life of the polypeptide in a mammal compared to the half-life of the polypeptide in the absence of the IgG Fc domain or functional fragment thereof.

65. The viral vector of claim 63, wherein the half-life prolonging domain is an albumin domain or a functional fragment of the albumin domain that is capable of extending the half-life of the polypeptide in a mammal compared to the half-life of the polypeptide in the absence of the albumin domain or functional fragment thereof.

66. The viral vector of any one of claims 63 to 65, wherein the half-life prolonging domain is carboxy-terminal to the ENPP1 or ENPP3 protein in the fusion protein.

67. The viral vector of claim 64 or 66, wherein the IgG Fc domain comprises the amino acid sequence set forth in SEQ ID NO:

34.

68. The viral vector of claim 65 or 66, wherein the albumin domain comprises the amino acid sequence set forth in SEQ ID NO:

35.

69. The viral vector of any one of claims 41 to 68, wherein the polynucleotide encodes a linker sequence.

70. The viral vector of claim 69, wherein the linker sequence is selected from the group consisting of SEQ ID NOs: 57-88 and 94.

71. The viral vector of any one of claims 63 to 70, wherein the linker sequence connects the ENPP1 or ENPP3 protein and the half-life prolonging domain of the fusion protein.

72. The viral vector of any one of claims 41 to 64, 66 or 67, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NOs: 89, 91, 92 and 93.

73. 1. A method for producing a recombinant viral vector, comprising: i. providing a cell or a population of cells comprising a polynucleotide encoding a polypeptide comprising a catalytic domain of an ENPP1 or ENPP3 protein, said cell expressing a viral protein essential for packaging and / or assembly of said polynucleotide into a recombinant viral vector; and ii. maintaining said cell or population of cells under conditions suitable for said assembly of said packaging of said recombinant viral vector comprising said polynucleotide. A method comprising:

74. 74. The method of claim 73, wherein the cell is a mammalian cell.

75. 75. The method of claim 74, wherein the mammalian cell is a rodent cell or a human cell.

76. The method according to any one of claims 73 to 75, wherein the viral vector is a vector according to any one of claims 41 to 72.

77. 77. The method of any one of claims 73 to 76, further comprising purifying said recombinant viral vector from said cell or population of cells or from the medium in which said cell or population of cells was maintained.

78. 78. A recombinant viral vector purified from the method of claim 77.

79. A pharmaceutical composition comprising a viral vector according to any one of claims 41 to 72 or a recombinant viral vector according to claim 78 and a pharma- ceutically acceptable carrier.

80. 80. A method of preventing or reducing the progression of a disease in a mammal in need thereof, comprising administering to said mammal a therapeutically effective amount of the pharmaceutical composition of claim 79, thereby preventing or reducing the progression of said disease or disorder.

81. 81. The method of claim 80, wherein the mammal is a human.

82. 83. The method of claim 81 or 82, wherein the disease is selected from the group consisting of X-linked hypophosphatemia (XLH), chronic kidney disease (CKD), mineral bone disorder (MBD), vascular calcification, pathological calcification of soft tissue, pathological ossification of soft tissue, PXE, generalized arterial calcification of infancy (GACI) and ossification of the posterior longitudinal ligament (OPLL).

83. A method for treating or preventing a pathological calcification or pathological ossification disease or disorder in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a viral vector according to any one of claims 41 to 72 or a pharmaceutical composition according to claim 79, thereby treating or preventing said disease or disorder.

84. A method for treating a subject having a deficiency of ENPP1 protein, comprising administering to the subject a therapeutically effective amount of a viral vector described in any one of claims 41 to 72 or a pharmaceutical composition described in claim 79, thereby treating the subject.

85. 85. The method of claim 84, wherein the disease or disorder or deficiency of the ENPP1 protein is associated with a loss-of-function mutation in the NPP1 gene or a loss-of-function mutation in the ABCC6 gene in the subject.

86. The viral vector or pharmaceutical composition is 1×10 １２ ~1×10 １５ 86. The method of any one of claims 80-85, administered in a dosage of vg / kg of subject or mammal.

87. The viral vector or pharmaceutical composition is 1×10 １３ ~1×10 １４ 86. The method of any one of claims 80-85, administered in a dosage of vg / kg of subject or mammal.

88. The viral vector or pharmaceutical composition is 5×10 １１ ~5×10 １５ 86. The method of any one of claims 80-85, administered in a dosage of vg / kg of subject or mammal.

89. The viral vector or pharmaceutical composition is 1×10 １２ ~1×10 １５ 86. The method of any one of claims 80-85, administered in a dosage of vg / kg of subject or mammal.

90. A method according to any one of claims 80 to 89, wherein administration of the viral vector or pharmaceutical composition to the subject or mammal increases plasma pyrophosphate (PPi) and / or plasma ENPP1 or ENPP3 concentrations in the subject or mammal.

91. The method of any one of claims 80 to 89, further comprising detecting or measuring one or more of the following parameters in a biological sample obtained from the subject or mammal: (i) pyrophosphate concentration, (ii) expression level of ENPP1 or ENPP3, and (iii) enzymatic activity of ENPP1 or ENPP3.

92. 92. The method of claim 91, wherein the detection or measurement is performed prior to administering the viral vector or pharmaceutical composition.

93. 93. The method of claim 91 or 92, wherein the detection or measuring is performed simultaneously or nearly simultaneously with administration of the viral vector or pharmaceutical composition.

94. The method of any one of claims 91 to 93, wherein the detection or measurement is performed after administration of the viral vector or pharmaceutical composition.

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