Treatment of diseases associated with ENPP1 or ENPP3 deficiency

Abstract

The present disclosure provides, among other things, vectors for the in vivo expression of ENPP1 or ENPP3 and methods for the treatment of mineralization and ossification disorders in a subject.

Description

[Technical Field]

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

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

[0003] ENPP1 (also known as PC-1) is a type 2 extracellular membrane-bound glycoprotein located in mineral-depositing matrix vesicles of osteoblasts and chondrocytes. It hydrolyzes extracellular nucleotides (primarily ATP) to adenosine monophosphate (AMP) and inorganic pyrophosphate (PPi). PPi functions as a potent inhibitor of ectopic 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-nonspecific 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 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 in various molecules, including deoxynucleotides, NAD, and nucleotide sugars. ENPP1 has been shown to be effective in treating certain diseases of ectopic tissue calcification, such as reducing systemic arterial calcification in a mouse model of GACI (generalized arterial calcification of infancy), a severe disease that occurs in infants and involves widespread 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 viral vectors 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, a herpes simplex vector, an alphavirus vector, or a lentivirus 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 present disclosure provides an adeno-associated virus 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 polypeptide 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 is capable of infecting mammalian cells, such as human cells (e.g., human hepatocytes or HEK cells, HeLa or A549 or hepatocytes). In some embodiments, the viral vector is capable of infecting, entering, and / or fusing with mammalian cells, such as human cells. In some embodiments, all or a functional portion of the polynucleotide of the viral vector (e.g., capable of expressing a polypeptide described herein) integrates or is integrated into the genome of a cell contacted by a viral vector described herein. In some embodiments, all or a functional portion of the polynucleotide of the viral vector can persist extrachromosomally without being integrated into the genome of a mammalian cell contacted by a viral vector described herein.

[0019] In some embodiments, the recombinant polynucleotide comprises a vector or plasmid encoding viral proteins and / or human ENPP1. In some embodiments, the recombinant polynucleotide comprises a vector or plasmid encoding viral proteins and / or human ENPP3. In some embodiments, the vector or said plasmid is capable of expressing an encoded polypeptide comprising an 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) and is non-secreted, 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 the 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, forming, from amino terminus to carboxy terminus, azurocidin signal peptide-ENPP1-Fc or azurocidin signal peptide-ENPP3-Fc, respectively.

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

[0028] In some embodiments, the Fc or albumin sequence is fused directly 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 an albumin promoter, a phosphoenolpyruvate carboxykinase (PEPCK) promoter, and an 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 an 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 has a serotype 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 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, from amino terminus to carboxy terminus, azurocidin signal peptide-ENPP1-Fc or azurocidin signal peptide-ENPP3-Fc, respectively.

[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 an ENPP1 or 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.

[0034] In yet another aspect, the disclosure provides a cell (e.g., a mammalian cell, e.g., 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 steps of: i. providing a cell comprising a polynucleotide of the invention; ii. maintaining the cells under conditions suitable for virus assembly; and iii. Purifying the viral vector produced by the cells.

[0036] In another aspect, the present disclosure provides a method for producing a recombinant viral vector, the method comprising the steps of: i. providing a cell or population of cells comprising a polynucleotide described herein, wherein the cells express viral proteins 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 assembly of the packaging of said 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 has been maintained.

[0038] In some embodiments, the cell is a mammalian cell, eg, a rodent cell (eg, a rat cell, a mouse cell, a hamster cell), a non-human primate cell, or a human cell (eg, 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), e.g., infecting the cell or population of cells with a helper virus containing such recombinant nucleic acid, transfecting the cell or population of cells with a helper plasmid containing such recombinant nucleic acid, etc.

[0040] In some embodiments, the viral vector is capable of expressing one or more polypeptides described herein upon infection in a target cell.

[0041] In some embodiments, the present disclosure provides pharmaceutical compositions comprising the purified viral vectors described herein. In some embodiments, the present disclosure provides sterile pharmaceutical compositions comprising the sterile / endotoxin-free purified viral vectors 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 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 invention provides a method of providing ENPP1 or ENPP3 to a mammal, the method comprising administering to the mammal a viral vector of the invention.

[0045] In certain embodiments, the present disclosure provides methods for expressing ENPP1 or ENPP3 in a mammal (e.g., a human, e.g., a human in need of such expression), the method comprising administering any of the viral vectors described herein to the mammal. Prior to, concurrently 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, activity level of ENPP1 and / or ENPP3, and / or level or concentration of pyrophosphate. In some embodiments, the one or more parameters are detected or measured within 1 week, 1-2 weeks, and / or 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 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 provides a method of preventing or reducing the progression of a condition or disease in a mammal in need thereof, comprising administering to said mammal a therapeutically effective amount of a composition according to the present invention, wherein the condition or disease includes, but is not limited to, one or more of the following: NPP1 deficiency, low levels of PPi, a progressive disorder characterized by accumulation of calcium and other mineral deposits in arteries and / or connective tissue, ectopic calcification of soft tissue, arterial or venous calcification, calcification of cardiac tissue, e.g., aortic tissue and coronary vascular tissue, pseudoxanthoma elasticum (PXE), 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), whereby said disease in said mammal is prevented or its progression is reduced.

[0048] In another aspect, the disclosure provides methods of treating, preventing, and / or ameliorating a pathological calcification or pathological ossification disease or disorder 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 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. In one aspect of the invention, the viral vector encodes a human ENPP1 or human ENPP3 polypeptide.

[0050] In another embodiment, the subject has a disease or disorder associated with a loss-of-function mutation in the subject's NPP1 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 in an amount of 1 x 10 12 ~1×10 15 vg / kg, preferably 1 × 10 13 ~1×10 14 The drug is administered to the subject at a dosage of 1000 mg / 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 5×10 11 ~5×10 15 The drug is administered to the subject at a dosage of 1000 mg / 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 x 10 per subject is administered for delivery and expression of the ENPP1-Fc polypeptide. 12 ~1×10 15 vg / kg is administered.

[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 in an amount of 1 x 10 12 ~1×10 15 vg / kg, preferably 1 × 10 13 ~1×10 14 The drug is administered to the subject at a dosage of 1000 mg / 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 5×1011 ~5×10 15 The drug is administered to the subject at a dosage of 1000 mg / 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 approximately 1 x 10 per subject is administered for delivery and expression of the ENPP3-Fc polypeptide. 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 the subject.

[0058] Prior to, concurrently with, and / or after administration of the viral vector to the mammal, any of the methods described herein can further include 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, activity level of ENPP1 and / or ENPP3, and / or level or concentration of pyrophosphate. In some embodiments, the one or more parameters are detected or measured within 1 week, 1-2 weeks, and / or 1 month after administration of the viral vector to the mammal.

[0059] In yet another aspect, the present disclosure provides a method of treating or preventing a pathological calcification or pathological ossification disease or disorder in a subject in need thereof, the method comprising administering to said 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 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 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 viral vector or a herpes simplex vector or an alphavirus vector or a lentivirus vector.

[0070] In some embodiments of any of the methods described herein, 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.

[0071] In some embodiments of any of the methods described herein, 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.

[0072] In some embodiments of any of the methods described herein, the AAV vector encoding the ENPP1-Fc fusion polypeptide is 1 x 10 12 ~1×10 15 The drug is administered to the subject at a dosage of 1000 mg / 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 5×10 11 ~5×10 15 The drug is administered to the subject at a dosage of 1000 mg / kg.

[0075] In some embodiments of any of the methods described herein, the vector is an AAV vector encoding ENPP1-Fc, and is 1 x 10 12 ~1×10 15 The drug is administered to the subject at a dosage of 1000 mg / kg.

[0076] In some embodiments of any of the aforementioned methods, 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.

[0077] In another aspect, the disclosure features a viral vector that includes 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 a 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 of SEQ ID NO:1 (Pro Ser Cys-Gln Glu Asp).

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

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

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

[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 an albumin promoter, a phosphoenolpyruvate carboxykinase (PEPCK) promoter, and an 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 an azurocidin signal peptide.

[0095] In some embodiments of any of the viral vectors described herein, the viral vector is an adeno-associated viral (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, from amino terminus to carboxy terminus, azurocidin signal peptide-ENPP1-Fc or azurocidin signal peptide-ENPP3-Fc, respectively.

[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 to form, from amino terminus to carboxy terminus, azurocidin signal peptide-ENPP1-albumin or azurocidin signal peptide-ENPP3-albumin, respectively.

[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 extending domain.

[0100] In some embodiments of any of the viral vectors described herein, the half-life prolonging domain is an IgG Fc domain or a functional fragment of an IgG Fc 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 IgG Fc domain or functional fragment thereof.

[0101] In some embodiments of any of the viral vectors described herein, the half-life prolonging domain is an albumin domain or a functional fragment of an 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.

[0102] In some embodiments of any of the viral vectors described herein, the half-life prolonging 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, a linker sequence connects the ENPP1 or ENPP3 protein and the half-life extending domain of the fusion protein.

[0108] In some embodiments of any of the viral vectors described herein, the polypeptide comprises the amino acid sequence 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 steps of: i. providing a cell or a population of cells comprising a polynucleotide encoding a polypeptide comprising the catalytic domain of an ENPP1 or ENPP3 protein, wherein the cells express viral proteins 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 suitable for assembly of the packaging of said recombinant viral vector containing 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 disclosure features a recombinant viral vector purified from the methods for producing and / or purifying a 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 the progression of a disease in a mammal in need thereof, comprising administering to said 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 pathological calcification or pathological ossification disease or disorder in a subject in need thereof, 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 said disease or disorder.

[0119] In another aspect, the disclosure features a method of treating a subject having a deficiency in ENPP1 protein, the method including 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 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 comprises 1 x 10 12 ~1×10 15 The dosage is administered in vg / kg of subject or mammal.

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

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

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

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

[0126] In some embodiments, any of the aforementioned methods may 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 enzymatic activity of ENPP1 or ENPP3.

[0127] In some embodiments of any of the methods described herein, the detecting or measuring is performed before administering the viral vector or pharmaceutical composition. [Brief explanation of the drawings]

[0128] [Figure 1] FIG. 1 is a schematic diagram showing AAV constructs. [Figure 2] FIG. 1 shows an increase in the expression level of ENPP1 when the azurocidin signal sequence is used compared to the NPP2 and NPP7 signal sequences. [Figure 3] Plasmid map of the vector expressing ENPP1-Fc fusion [Figure 4] FIG. 1 is a schematic diagram showing administration of viral particles containing an ENPP1 construct to a mouse model. [Figure 5] FIG. 1 shows the dose-dependent increase in ENPP1 activity in plasma samples obtained from control, low-dose, and high-dose mouse cohorts collected on days 7, 28, and 56 after administration of the viral vector. [Figure 6] FIG. 1 shows the dose-dependent increase in ENPP1 concentrations in plasma samples obtained from control, low-dose, and high-dose mouse cohorts collected on days 7, 28, and 56 after administration of the viral vector. [Figure 7] FIG. 1 shows a dose-dependent increase in plasma PPi concentrations in plasma samples obtained from control, low-dose, and high-dose mouse cohorts collected on days 7, 28, and 56 after administration of the viral vector. [Figure 8] FIG. 1 shows sustained expression of ENPP1 for up to 112 days after viral vector administration. [Figure 9] FIG. 1 shows the dose-dependent increase in ENPP1 activity in plasma samples obtained from control, low-dose, and high-dose mouse cohorts collected on days 7, 28, 56, and 112 after administration of the viral vector. DETAILED DESCRIPTION OF THE INVENTION

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

[0130] definition Unless otherwise defined, 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, 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 to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0132] The following notation applies to this disclosure for clarity. Regardless, any teachings herein that do not follow this convention are still part of this disclosure and can be fully understood given the context in which the teachings are disclosed. Protein symbols are disclosed in non-italicized capital letters. As a non-limiting example, "ENPP1" refers to the protein. In certain embodiments, if the protein is a human protein, an "h" is used before the protein symbol. In other embodiments, if the protein is a mouse protein, an "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 capitalized and the remaining letters lowercase; further, mouse gene symbols are italicized. As a non-limiting example, the mouse gene that produces the protein mEnpp1 is Enpp1. Notations for gene mutations are 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 using non-invasive methods such as X-ray, micro-CT and MRI. Reduction of calcification also refers to 99m Tc-pyrophosphate ( 99mThe presence of calcification in mice is assessed via micro-computed tomography (CT) scans and necropsy on tissue sections obtained from the heart, aorta, and kidneys using dyes such as hematoxylin and eosin (H&E) and alizarin red, according to a 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.

[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 enzymatic rate of NPP1 is determined using ATP as a substrate. HPLC analysis of the enzymatic reaction can demonstrate that NPP1 cleaves ATP, and the identity of the substrate and product 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 ATP substrate, and the data are curve-fitted to derive the enzymatic rate constant. At physiological pH, the kinetic rate constants for NPP1 are Km = 144 μM and kcat = 1. t =7.8s -1 is.

[0137] ATP hydrolysis activity of NPP3 The enzymatic activity of NPP3 was measured using pNP-TMP or ATP as a substrate. NPP3 protein was incubated at 37°C in the presence of 100 mM Tris-HCl at pH 8.9 and either 5 mM pNP-TMP or 50 μM [γ-32P]ATP. Hydrolysis of pNP-TMP was stopped by 10-fold dilution in 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 colorimetrically at 405 nm. ATP hydrolysis was stopped by adding 100 mM EDTA. 1 μl of the reaction mixture was analyzed by thin-layer chromatography on polyethyleneimine cellulose plates (Merck). Nucleotides and degradation products were separated by ascending chromatography in 750 mM KH2PO4 at pH 3.0. Radioactive spots were visualized by autoradiography. Nucleotidylated intermediates formed during the hydrolysis of 50 μM [α-32P]ATP were trapped according to the method of Blytt et al. (H.J.Blytt, J.E.Brotherton, L.Butler Anal.Biochem.147(1985), pp.517-520) with minor modifications (R.Gijsbers, H.Ceulemans, W.Stalmans, M.Bollen J.Biol.Chem.276(2001), pp.1361-1368). After SDS-PAGE, the trapped intermediates were visualized by autoradiography. Bis-pNPP and pNPP were also tested as substrates for NPP3. NPP3 isoforms were incubated in 100 mM Tris-HCl, pH 8.9, and 5 mM of either bis-pNPP or pNPP for 2.5 h at 37°C. The formed pNP was subsequently quantified colorimetrically 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 exhibited a pH of approximately 2.59 (±0.04) s, similar to that of ENPP1. -1 and Km (<8 μM) values (WO2017 / 087936).

[0138] HPLC Protocol The HPLC protocol used to measure ATP cleavage by NPP1 and identify products is adapted from the literature (Stocchi et al., 1985, Anal. Biochem. 146:118-124). Reactions containing various concentrations of ATP in a 50 mM Tris pH 8.0, 140 mM NaCl, 5 mM KCl, 1 mM MgCl, and 1 mM CaCl buffer are initiated by the addition of 0.2–1 μM NPP1. The reactions are quenched at various times with an equivalent volume of 3 M formic acid or 0.5 N KOH and reacidified to pH 6 with glacial acetic acid. The quenched reaction solutions are serially diluted and loaded onto an HPLC system (Waters, Milford Mass.), where substrate and product are monitored by UV absorbance at 254 or 259 nm. Separate the substrate and product on a C18, 5um 250 x 4.6mm HPLC column (Higgins Analytical, Mountain View, Calif.) using a 0% to 10% (or 20%) methanol gradient in 15mM ammonium acetate pH 6.0 solution. Identify the product and substrate by the integrals of their corresponding peaks and the formula:

number

[0139] "Pathological calcification": As used herein, this term refers to the abnormal deposition of calcium salts in the soft tissues, secretory, and excretory ducts of the body, hardening them. There are two types: dystrophic calcification, which occurs in dying and dead tissue, and metastatic calcification (hypercalcemia), which occurs when the extracellular level of calcium is elevated beyond the homeostatic capacity of cells and tissues. Calcification can involve cells as well as extracellular matrix components, such as collagen in the basement membrane and elastic fibers in the arterial wall. Some examples of tissues prone to calcification include the gastric mucosa—the inner epithelial lining 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 develops in tissues that are not part of the skeletal system and in connective tissues that do not normally exhibit osteogenic properties. Ossification is classified into three types depending on the nature of the affected tissue or organ: endochondral ossification is ossification that develops in and replaces cartilage; intramembranous ossification is ossification of bone that develops in and replaces connective tissue; modified ossification, the development of bone in normally soft body structures, is also called heterotrophic ossification.

[0141] NPP1 "deficiency" refers to a condition in which a subject has less than 5%-10% of the normal level of NPP1 in their plasma. Normal levels of NPP1 in healthy human subjects are approximately 10-30 ng / ml (Am J Pathol. 2001 Feb;158(2):543-554).

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

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

[0144] "Ectopic soft tissue calcification" refers to inappropriate biomineralization, typically composed of calcium phosphate, hydroxyapatite, calcium oxalate, and octacalcium phosphate, occurring in soft tissues and leading to loss of soft tissue hardening. "Arterial calcification" refers to ectopic calcification occurring in arteries and heart valves, leading to hardening and / or narrowing of the arteries. 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 ectopic calcification occurring in veins that reduces the elasticity of veins and restricts blood flow, which in turn can lead to elevated blood pressure and coronary vascular defects.

[0146] "Vascular calcification" refers to the pathological deposition of minerals in the vascular system. It has various forms, including intimal and medial calcification, but can also be found in the valves of the heart. Vascular calcification is associated with atherosclerosis, diabetes, certain genetic conditions, and kidney disease, particularly CKD. Patients with vascular calcification are at higher risk for 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] "Cerebral calcification" (BC) refers to a nonspecific neuropathological condition in which deposition of calcium and other minerals occurs in blood vessel walls and parenchymal tissue, leading to neuronal death and gliosis. Cerebral calcification is often associated with a variety of 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 cardiac tissue refers to the accumulation of calcium (and possibly other minerals) deposits in cardiac tissue, such as aortic and coronary tissue.

[0149] As used herein, "chronic kidney disease (CKD)" refers to abnormalities in kidney structure or function that persist for more than three months and affect health. Generally, in most chronic kidney diseases, excretory, endocrine, and metabolic functions simultaneously decline. 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. The prevalence of vascular calcification increases with declining kidney function, and calcification occurs many years earlier in CKD patients than in the general population. Preventing, reducing, and / or reversing vascular calcification can increase the survival time of patients with CKD.

[0150] Clinical symptoms of chronic kidney disease include itching, muscle spasms, nausea, loss of appetite, swelling of the feet and ankles, insomnia, and labored breathing. If left untreated, chronic kidney disease tends to progress to end-stage renal disease (ESRD). Common symptoms of ESRD include inability to urinate, fatigue, lethargy, weight loss, bone pain, changes in skin color, frequent bruising, and swelling of the outer extremities, such as the fingers, toes, hands, and feet. Calciphylaxis, or calcific uremic arteriolopathy (CUA), is a condition that causes calcium to accumulate inside the blood vessels in the fat and skin. A subpopulation of patients with ESRD may also develop calciphylaxis. Common symptoms of calciphylaxis include large, purple, web-like patterns on the skin, deep, painful lumps that ulcerate and create open wounds with non-healing, dark-brown crusts, and skin lesions on the lower extremities or areas with higher fat content, such as the thighs, breasts, buttocks, and abdomen. Individuals with calciphylaxis may have higher than normal levels of calcium (hypercalcemia) and phosphate (hyperphosphatemia) in the blood. They may also have symptoms of hyperparathyroidism. Hyperparathyroidism occurs when the parathyroid glands produce excess parathyroid hormone (PTH). Decreased 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 potentiating myocardial infarction and stroke. It is important to note that while most patients with ESRD do not die of renal failure, many very young patients with ESRD who die from cardiovascular complications of ESRD and are on dialysis have coronary artery calcification. A histological subtype of vascular calcification associated with CKD is known as Mönckeberg sclerosis, a form of vascular sclerosis characterized by calcium deposits in the muscular layer of the medial vascular wall. This form of calcification is histologically distinct from the calcification of the intimal or neointimal vascular wall commonly observed in atherosclerosis, but is identical to the vascular calcification observed in human CKD patients and the rodent models of the disease 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 before birth or within the first few months of life. It is characterized by an abnormal buildup of the mineral calcium (calcification) in the walls of 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, affected individuals may develop heart failure, accompanied by signs and symptoms including difficulty breathing, fluid accumulation in the extremities (edema), a bluish discoloration of 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, especially around the joints. In addition, they may have hearing loss or softening and weakening of the bones, known as rickets.

[0153] Common arterial calcification (GACI) or idiopathic infantile arterial calcification (IIAC) is characterized by an abnormal buildup of the mineral calcium (calcification) in the walls of 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, affected individuals may develop heart failure, with signs and symptoms including difficulty breathing, accumulation of fluid in the extremities (edema), a bluish discoloration of the skin or lips (cyanosis), severe high blood pressure (hypertension), and an enlarged heart (cardiomegaly).

[0154] As used herein, "arterial calcification" or "vascular calcification" or "arterial hardening" refers to a process characterized by thickening and loss of elasticity of the muscular arterial wall. Thickening and loss of elasticity occur in two different locations, namely, the intima and medial layers of the vascular structure (medial vascular calcification). Intimal calcification is associated with atherosclerotic plaque, while medial calcification is characterized by vascular stiffness and arteriosclerosis. This reduces arterial elasticity and increases the tendency for morbidity and mortality due to hemodynamic dysfunction of the cardiovascular system.

[0155] "Mineral bone disorder (MBD)," as used herein, refers to a disorder characterized by abnormal hormone levels that destabilizes calcium and phosphorus levels in a person's blood. Mineral and bone disorders commonly occur in people with CKD and affect most people with kidney failure who are undergoing dialysis.

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

[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. If left untreated, osteopenia progresses to osteoporosis, an extreme condition in which bones become brittle and prone to fracture.

[0158] As used herein, "ossification of the posterior longitudinal ligament (OPLL)" refers to a condition of hyperossification (excessive bone growth) that results in ectopic calcification of the posterior longitudinal ligament. The posterior longitudinal ligament connects and stabilizes the bones of the spine. A thickened or calcified ligament can compress the spinal cord, resulting in myelopathy. Symptoms of myelopathy include difficulty walking and difficulty with bowel and bladder control. OPLL can also cause radiculopathy, or compression of nerve roots. Symptoms of cervical radiculopathy include pain, tingling, or numbness in the neck, shoulders, arms, or hands.

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

[0160] As used herein, "pseudoxanthoma elasticum (PXE)" refers to a progressive disorder characterized by the accumulation of calcium and other mineral deposits (mineralization) in elastic fibers. Elastic fibers are components of connective tissue that provide strength and mobility to structures throughout the body. In PXE, mineralization can affect elastic fibers in the skin, eyes, and blood vessels, and less commonly in other areas such as the gastrointestinal tract. People with PXE may have yellowish bumps called papules on the neck, armpits, and other areas of the skin that are touched when bending joints. 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 muscle cramps and pain during exercise due to narrowing of the arteries (arteriosclerosis) or reduced blood flow to the arms and legs.

[0161] Pseudoxanthoma elasticum (PXE), also known as Glennblad-Strandberg syndrome, is a genetic disorder that causes fragmentation and mineralization of elastic fibers in several tissues. The most common problems occur in the skin and eyes, followed by blood vessels in the form of premature atherosclerosis. PXE is caused by an autosomal recessive mutation in the ABCC6 gene, located on the short arm of chromosome 16 (16p13.1). In some cases, some infants survive GACI and eventually develop pseudoxanthoma elasticum (PXE) as they grow into adulthood. PXE is characterized by the accumulation of calcium and other minerals (mineralization) in elastic fibers, a component of connective tissue. Connective tissue provides strength and mobility to structures throughout the body. Features specific to PXE, which also occurs in GACI, include yellowish bumps called papules in the armpits and other areas of the skin that come into contact when joints bend (flexor areas); arterial narrowing, and abnormalities called angioid streaks (retinal hemorrhages) that affect the tissues behind the eyes, detected during an eye exam.

[0162] "End Stage Renal Disease (ESRD): As used herein, this term refers to an advanced stage of chronic kidney disease in which a patient's kidneys are no longer functional. Common symptoms include fatigue associated with anemia (low blood iron), loss of appetite, nausea, vomiting, abnormal laboratory tests including elevated potassium, hormonal abnormalities related to bone health, elevated phosphorus and / or low calcium, high blood pressure (hypertension), swelling of the hands / feet / eyes / lower back (sacrum), and shortness of breath.

[0163] "Uremic arterial calcification (CUA)" or "calciphylaxis," as used herein, refers to a condition associated with high morbidity and mortality seen in patients with kidney disease, particularly those with end-stage renal disease (ESRD). It is characterized by calcification of small blood vessels located in fatty tissue and deeper layers of the skin, leading 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 easily bent due to low levels of phosphate in the blood. Symptoms usually begin in early childhood and can range in severity from bowing of the feet, bone deformities; bone pain; joint pain; poor bone growth; and short stature.

[0165] "Hereditary hypophosphatemic rickets," as used herein, refers to a disorder associated with low levels of phosphate in the blood (hypophosphatemia). Phosphate is a mineral essential for the normal formation of bones and teeth. Most commonly, it 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 bones (craniosynostosis) and dental abnormalities. This disorder can also cause abnormal bone growth of ligaments and tendons that attach to joints (enthesopathy). 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); in addition to hypophosphatemia, this condition is characterized by the excretion of high levels of calcium in the urine (hypercalciuria).

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

[0167] "Autosomal recessive hypophosphatemic rickets type 2 (ARHR2)," as used herein, refers to the inherited renal phosphate-wasting disorder hypophosphatemia, characterized by rickets and / or osteomalacia and slow growth. 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 urinary phosphate loss 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, suppressed calcitriol (1,25-dihydroxyvitamin D3) levels, renal phosphate wasting, and low serum phosphate. FGF23 mutations render the protein more stable and incapable of being cleaved by proteases, resulting in enhanced biological activity of FGF23. The enhanced activity of FGF23 mutants reduces the expression of sodium-phosphate cotransporters, NPT2a and NPT2c, at the apical surface of proximal renal tubule cells, resulting in 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 painfully soft and easily bent. Symptoms can include bowing of the feet and other bone deformities; bone pain; joint pain; poor bone growth; and short stature. In some affected babies, the spaces between the skull bones close too early, leading to craniosynostosis. Most patients exhibit abnormalities of calcium-phosphate metabolism, abnormalities of tooth enamel, delayed tooth eruption, and an elongated head (dolichocephaly).

[0170] The terms "adeno-associated viral vector," "AAV vector," "adeno-associated virus," "AAV virus," "AAV virion," "AAV viral particle," and "AAV particle," used interchangeably herein, refer to a viral particle composed of at least one AAV capsid protein (preferably all of the capsid proteins of a particular AAV serotype) and an encapsidated recombinant viral genome. The particle contains a recombinant viral genome carrying a heterologous polynucleotide comprising a sequence encoding human ENPP1 or human ENPP3 or a functionally equivalent variant thereof and a transcriptional regulatory region comprising at least a promoter flanked by AAV inverted terminal repeats. The particle is typically referred to as an "AAV vector particle" or "AAV vector."

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

[0172] As used herein, the term "recombinant host cell" (or simply "host cell"), as used herein, refers to a cell into which an exogenous nucleic acid and / or recombinant vector has been introduced. It should be understood that "recombinant host cell" and "host cell" refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations, either due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

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

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

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

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

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

[0178] The term "operably linked" as used herein 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 adjacent to a sequence of interest. However, an enhancer does not need to be adjacent to a sequence of interest to control its expression. In another embodiment, a promoter and a nucleotide sequence encoding 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 a desired biological result. The result may be a reduction and / or alleviation of 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 present invention is an amount sufficient to produce a therapeutically effective amount.

[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 functional activities of a Cap protein include the ability to induce capsid formation, facilitate the accumulation of single-stranded DNA, facilitate packaging of AAV DNA into capsids (i.e., encapsidation), bind to a cellular receptor, and facilitate virion entry 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. Capsid is composed of several oligomeric structural subunits made of proteins. For example, AAV has an icosahedral capsid formed by the interaction of three capsid proteins: VP1, VP2 and VP3.

[0182] The term "Rep protein," as used herein, refers to a polypeptide having at least one functional activity of a native AAV Rep protein (e.g., Rep40, 52, 68, 78). A "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 AAV origins of DNA replication, and DNA helicase activity. Additional functions include modulation of transcription from an AAV (or other heterologous) promoter and site-specific integration of AAV DNA into a host chromosome. In certain embodiments, the AAV rep gene is derived from an AAV serotype selected from the group consisting of serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAVrhlO; more preferably, AAV2, AAV5, AAV7, AAV8, AAV9, AAV10, and AAVrhlO.

[0183] The phrase "viral proteins on which AAV depends for replication" as used herein refers to polypeptides that perform functions on which AAV depends for replication (i.e., "helper functions"). Helper functions include those functions required for AAV replication, including, but not limited to, those involved in activating AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and assembly of AAV capsids. Viral-based accessory functions are 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, and herpesvirus polymerase. In another embodiment, the proteins on which AAV depends for replication are 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 strand of the genome of an adeno-associated virus. 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, allowing primase-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", 2002). nd and Schleef M, Ed., “Plasmid for Therapy and Vaccination”, Wiley-VCH Verlag GmbH, Weinheim, Del., 2001).

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

[0186] The term "skeletal muscle-specific promoter," as used herein, refers to a nucleic acid sequence that acts as a promoter (i.e., regulates the expression of a selected nucleic acid sequence operably linked to the promoter) and promotes the expression of a 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] The term "liver-specific promoter," as used herein, refers to a nucleic acid sequence that acts as a promoter (i.e., regulates the expression of a selected nucleic acid sequence operably linked to the promoter) and promotes the expression of a 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]l-antitrypsin (AAT) promoter, the thyroid hormone-binding globulin promoter, the alpha-fetoprotein promoter, the alcohol dehydrogenase promoter, the factor VIII (FVIII) promoter, the HBV basic core promoter (BCP) and PreS2 promoter, the albumin promoter, the -460 to 73 bp phosphoenolpyruvate carboxykinase (PEPCK) promoter, the thyroxine-binding globulin (TBG) promoter, the hepatic control region (HCR)-ApoCII hybrid promoter, the HCR-hAAT hybrid promoter, the AAT promoter combined with the mouse albumin gene enhancer (Ealb) element, and the apolipoprotein B12 promoter. Examples of tissue-specific promoters include the protein E promoter, low-density lipoprotein promoter, pyruvate kinase promoter, lecithin-cholesterol acyltransferase (LCAT) promoter, apolipoprotein H (ApoH) promoter, transferrin promoter, transthyretin promoter, alpha-fibrinogen and beta-fibrinogen promoters, alpha1-antichymotrypsin promoter, alpha2-HS glycoprotein promoter, haptoglobin promoter, ceruloplasmin promoter, plasminogen promoter, promoters of complement proteins (CIq, CIr, C2, C3, C4, C5, C6, C8, C9, complement factor I and factor H), C3 complement activator, and [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 alpha 1-antitrypsin promoter, more preferably an alpha 1-antitrypsin promoter, and even more preferably a human alpha 1-antitrypsin promoter.

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

[0189] The term "constitutive promoter," as used herein, 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 consideration of cellular environmental conditions. In another embodiment, the transcriptional control region allows for constitutive expression of ENPP1. Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), cytomegalovirus (CMV) promoter (optionally with a CMV enhancer), SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter, and EF1a promoter (Boshart M, et al., Cell 1985;41:521-530). Preferably, a constitutive promoter is suitable for expression of ENPP1 in the liver, including, but not limited to, the promoter of hypoxanthine phosphoribosyltransferase (HPTR), the promoter of adenosine deaminase, the promoter of pyruvate kinase, the promoter of β-actin, the promoter of elongation factor 1 alpha (EF1), the promoter of phosphoglycerate kinase (PGK), the promoter of ubiquitin (Ubc), the promoter of albumin, 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. USA 78:1441-1445).

[0190] The term "polyadenylation signal" as used herein 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, SV40 early polyadenylation signal, SV40 late polyadenylation signal, HSV thymidine kinase polyadenylation signal, protamine gene polyadenylation signal, adenovirus 5 EIb polyadenylation signal, bovine growth hormone polyadenylation signal, human mutant growth hormone polyadenylation signal, etc.

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

[0192] The term "signal peptide," as used herein, refers to a sequence of amino acid residues (ranging from 10 to 30 residues in length) that is attached 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 cleaved by signal peptidase after transport in the endoplasmic reticulum (Lodish et al., 2000, Molecular Cell Biology, 4th edition).

[0193] The term "subject," as used herein, refers to an individual mammal, such as a human, a non-human primate (e.g., chimpanzee and other ape and monkey species), livestock (e.g., birds, fish, cattle, sheep, pigs, goats, and horses), domestic mammals (e.g., dogs and cats), or laboratory animals (e.g., rodents, such as mice, rats, and guinea pigs). 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 "alleviated" if the severity of the symptoms of the disease or disorder, the frequency with which such symptoms are experienced by the patient, or both, is reduced.

[0195] As used herein, the terms "alteration," "deletion," "mutation," or "mutation" refer to a mutation in 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 in an animal in which the animal is unable to maintain homeostasis and if the disease is not ameliorated, the animal's health will continue to deteriorate.

[0197] An animal "disorder" is a health condition in which the animal is able to maintain homeostasis, but the animal's health condition is not better than it would be in the absence of the disorder. If left untreated, the disorder does not necessarily cause a further deterioration in the animal's health condition.

[0198] As used herein, the term "immune response" or "immune reaction" refers to the host's immune system responding to an antigen in a pathogenic organism or to the introduction or expression of a foreign protein. The immune response is generally humoral and localized, and antibodies produced by B cells bind to the antigen in an antigen-antibody complex to inactivate or neutralize the antigen. Immune responses are often observed when human proteins are injected into mouse model systems. Generally, mouse model systems are made immune tolerant by injecting immune suppressors before the introduction of foreign antigens to ensure better survival rates.

[0199] As used herein, the term "immunosuppression" refers to the deliberate reduction in 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) antibodies, 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 incorporated herein by reference in its entirety.

[0202] Mammalian ENPP1 and ENPP3 polypeptides, mutants or mutant fragments thereof have been previously disclosed in International PCT Application Publication Nos. 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 incorporated herein by reference in their entireties.

[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 No. WO / 2017 / 087936 to Braddock et al., which is incorporated herein 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. During proteolysis, the signal sequence is cleaved from ENPP1 to yield the ENPP1 protein. Signal peptide sequences useful within the present invention include, but are not limited to, the albumin signal sequence, the azurocidin signal sequence, the ENPP1 signal peptide sequence, the ENPP2 signal peptide sequence, the ENPP7 signal peptide sequence, and / or the 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. During proteolysis, the signal sequence is cleaved from ENPP3 to yield the ENPP3 protein. Signal peptide sequences useful within the present invention include, but are not limited to, an albumin signal peptide sequence, an azurocidin signal peptide sequence, an ENPP1 signal peptide sequence, an ENPP2 signal peptide sequence, an ENPP7 signal peptide sequence, and / or an 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 azurosin signal peptide (MTRLTVLALLAGLLASSRA) is fused to the nucleotide sequence of the NPP1 or NPP3 gene, which, when encoded, produces the ENPP1 or 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 recombinantly fused and / or chemically conjugated (including both covalent and non-covalent conjugation) to 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 recombinantly fused and / or chemically conjugated (including both covalent and non-covalent conjugation) to 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 human IgG (immunoglobulin) Fc domain. 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" refers to the portion of an IgG molecule associated with the crystallizable fragment obtained by papain digestion of the IgG molecule. The Fc region comprises the C-terminal halves of the two heavy chains of an IgG molecule, which are linked by disulfide bonds. It does not have antigen-binding activity but contains carbohydrate moieties and binding sites for complement and Fc receptors, such as the FcRn receptor. The Fc fragment contains 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 term "IgG hinge-Fc region" or "hinge-Fc fragment" refers to the region of an 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 than the other portions of the immunoglobulin, i.e., the variable domains, which contain the antigen-binding site. The 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" as applied to nucleic acids 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" as 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 changed or removed from the natural state. For example, a nucleic acid or polypeptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or polypeptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0213] An "oligonucleotide" or "polynucleotide" is a nucleic acid ranging in length from at least 2, and in certain embodiments at least 8, 15, or 25 nucleotides, but may be up to 50, 100, 1000, or 5000 nucleotides in length, or 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 administration of the compound to a patient. Multiple techniques for administering a compound exist 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, e.g., 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 undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained, 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, cows, and horses. Due to its 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 a modified enzymatic assay using 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, from 1 to 2 μM. Subjects with incomplete ENPP1 expression tend to have low PPi levels, which are at least 10% below normal, at least 20% below normal, at least 30% below normal, at least 40% below normal, at least 50% below normal, at least 60% below normal, at least 70% below normal, at least 80% below normal, and combinations thereof. In patients with GACI, PPi levels have been found to be less than 1 μm, and in some cases, below 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, related naturally occurring structural variants and synthetic non-naturally occurring analogues thereof, linked through peptide bonds.

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

[0220] As used herein, the term "prevent" or "prevention" means that a disorder or disease will not occur if none occurred, or that a further disorder or disease will not occur if a disorder or disease has already occurred. The ability to prevent some or all of the symptoms associated with a disorder or disease is also considered.

[0221] "Sample" or "biological sample," as used herein, refers to biological material isolated from a subject. A biological sample can include any biological material suitable for detecting mRNA, polypeptides, or other markers of physiological or pathological processes in a subject, and can include bodily fluids, tissues, cellular and / or non-cellular material obtained from an individual.

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

[0223] As used herein, the term "treatment" or "treating" is defined as the application or administration of a therapeutic agent, i.e., a compound useful within the present invention (alone or in combination with another pharmaceutical agent), to a patient, or the application or administration of a therapeutic agent to tissue or cell lines isolated from a patient having a disease or disorder, a symptom of a disease or disorder, or the likelihood of developing a disease or disorder (e.g., for diagnostic or ex vivo applications), for the purpose of curing, curing, alleviating, palliating, altering, relieving, ameliorating, reversing, or affecting the disease or disorder, the symptoms of a disease or disorder, or the likelihood of developing a disease or disorder.

[0224] The terms "prevent," "preventing," and "prevention," as used herein, refer to inhibiting the onset of disease or reducing the occurrence of disease in a subject. Prevention may be complete (e.g., the subject is completely free of pathological cells) or partial. Prevention also refers to reducing 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. A wild-type gene is the one most frequently observed in a population and is therefore arbitrarily referred to as the "normal" or "wild-type" form of a human NPP1 or NPP3 gene. In contrast, the term "functionally equivalent" refers to an NPP1 or NPP3 gene or gene product that exhibits altered sequence and / or functional properties (i.e., altered characteristics) compared to a 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) compared to a wild-type gene or gene product.

[0226] The term "functionally equivalent variant," as used herein, refers to a polypeptide that is substantially homologous to the sequence of ENPP1 or ENPP3 (as defined above) and retains the enzymatic and biological activity of ENPP1 or ENPP3, respectively. Methods for determining whether a variant retains the biological activity of native ENPP1 or ENPP3 are widely known to those skilled in the art and include any of the assays used in the experimental part of the 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 a protein sequence when said protein sequence has a degree of identity of 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% to the above-mentioned ENPP1 or ENPP3 sequences, respectively.

[0228] The degree of identity between two polypeptides is determined using computer algorithms and methods that are 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)), but other similar algorithms can also be used.To determine percent sequence identity, BLAST and BLAST 2.0 are used with the parameters described herein.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 substituting nucleotides within a polynucleotide that account for the codon preferences in the host cell used to produce ENPP1 or ENPP3, respectively. Such "codon optimization" can be determined via a computer algorithm that incorporates a codon frequency table, such as "Human high.cod," for codon preferences 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, e.g., an amount, a temporal duration, etc., is meant to encompass a variation of ±20% or ±10%, in certain embodiments ±5%, in certain embodiments ±1%, and in certain embodiments ±0.1% from the particular value, as such variations are appropriate for practicing the disclosed methods.

[0231] This disclosure provides representative examples of protein and nucleic acid sequences of the present 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 that enable such conversion, such as Expasy (https: / / www.expasy.org / ) and bioinformatics servers (http: / / www.bioinformatics.org).

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

[0233] Viral vectors for in vivo expression of ENPP1 and ENPP3 To compensate for a deficiency of an ENPP1 or ENPP3 polypeptide, genetic material, such as a polypeptide comprising an 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, parvoviruses, picornaviruses, pseudorabies virus, hepatitis virus A, B, or C, papillomaviruses, papovaviruses (e.g., polyoma and SV40), or herpesviruses (e.g., Epstein-Barr virus, varicella-zoster virus, cytomegalovirus, varicella-zoster virus, and herpes simplex virus types 1 and 2), RNA viruses, or retroviruses, such as Moloney murine leukemia virus or lentiviruses (i.e., derived from human immunodeficiency virus, feline immunodeficiency virus, equine infectious anemia virus, etc.). Among DNA viruses useful according to the present invention are adeno-associated viruses, adenoviruses, alphaviruses, and lentiviruses.

[0235] Viral vectors are generally administered directly into the body or into specific tissues where they are taken up by individual cells, most often by intravenous injection (IV). Alternatively, viral vectors can be administered by ex vivo contacting the viral vector with a sample of patient cells, thereby infecting the cells with the viral vector, and then the vector-containing cells are returned to the patient. Once the viral vector is delivered, the coding sequence is expressed, resulting in a functional protein. In general, viral vector infection and transduction of cells occurs through a series of consecutive events: interaction of the viral capsid with a receptor on the surface of the target cell, internalization by endocytosis, intracellular transport through the endocytic / proteasome compartment, endosomal escape, nuclear import, virion uncoating, 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 viruses belonging to the Dependovirus genus of the Parvoviridae family. The AAV genome is approximately 4.7 kilobases long and is composed of linear, single-stranded deoxyribonucleic acid (ssDNA) that can be either positive- or negative-sense. The genome contains inverted terminal repeats (ITRs) at both ends of the DNA strand and two open reading frames (ORFs): rep and cap. The rep frame consists of four overlapping genes encoding nonstructural replication (Rep) proteins required for the AAV life cycle. The cap frame contains overlapping nucleotide sequences of the structural VP capsid proteins: VP1, VP2, and VP3, which interact together to form a capsid with icosahedral symmetry.

[0237] The terminal 145 nucleotides are self-complementary and organized to allow the formation of an energetically stable intramolecular duplex that forms a T-shaped hairpin. These hairpin structures serve as initiation sites for viral DNA replication and as primers for the cellular DNA polymerase complex. After wild-type AAV infection in mammalian cells, the rep genes (i.e., Rep78 and Rep52) are expressed from the P5 and P19 promoters, respectively, and both Rep proteins function in viral genome replication. Splicing events in the rep ORF actually result in the expression of four Rep proteins (i.e., Rep78, Rep68, Rep52, and Rep40). However, it has been shown that unspliced mRNAs encoding the Rep78 and Rep52 proteins are sufficient for AAV vector production in mammalian cells. In insect cells, the Rep78 and Rep52 proteins are also sufficient for AAV vector production.

[0238] AAV vectors typically lack the rep and cap frames and can replicate and be packaged into infectious viral particles when present in a host cell transfected with a vector encoding and expressing the rep and cap gene products (i.e., AAV Rep and Cap proteins) and the host cell is transfected with a vector encoding and expressing proteins 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 a mammalian ENPP1 or a mammalian ENPP3. Upon administration to a mammal, the vector expresses an ENPP1 or ENPP3 precursor in cells, the precursor comprising an azurocidin signal peptide fused at its carboxy terminus to the amino terminus of ENPP1 or ENPP3. The ENPP1 or ENPP3 precursor may comprise a stabilizing domain, e.g., an IgG Fc region or human albumin. Upon secretion of the precursor from the cell, the signal peptide is cleaved, resulting in enzymatically active, soluble mammalian ENPP1 or ENPP3 outside the cell.

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

[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 an AAV vector according to the invention lacks the rep open reading frame and / or the cap open reading frame.

[0243] The AAV vector of the present invention comprises capsids from any serotype.Generally, AAV serotypes have significant homology in genome sequence at the amino acid and nucleic acid level, provide the same set of genetic functions, and replicate and assemble through virtually the same mechanism.In particular, the AAV of the present invention can belong to AAV serotype 1 (AAV1), AAV2, AAV3 (including type 3A and type 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhlO, AAV11, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV.

[0244] Examples of genome sequences for various AAV serotypes can be found in the literature or in public databases such as GenBank, e.g., 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 viral vector according to the present invention comprises a capsid derived from a serotype selected from the group consisting of AAV2, AAV5, AAV7, AAV8, AAV9, AAV10, and AAVrhlO serotypes. In another embodiment, the AAV serotype is AAV8. When the viral vector comprises a sequence encoding a capsid protein, it can be modified to contain an exogenous sequence to direct the AAV to a specific cell type(s), to increase the efficiency of delivery of the targeted vector to cells, to facilitate purification or detection of the AAV, or to reduce host responses.

[0246] Published application US2017 / 0290926-Smith et al., the contents of which are incorporated herein 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 Invention Adenoviruses can be engineered to encode and express a desired gene product (e.g., ENPP1 or ENPP3) while simultaneously being inactivated with respect to their ability to replicate in the normal lytic viral life cycle. Furthermore, adenoviruses have a natural tropism for respiratory epithelia. The virus can infect quiescent cells, such as those found in the respiratory tract, offering a significant advantage over retroviruses. Adenoviral expression is achieved without integration of viral DNA into host cell chromosomes, thereby mitigating concerns about insertional mutagenesis. Furthermore, adenoviruses have been used for many years as live enteric vaccines with an excellent safety profile (Schwartz, AR et al. (1974) Am. Rev. Respir. Dis. 109:233-238). Finally, adenovirus-mediated gene transfer has been demonstrated in several instances, including the transfer of alpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, MA et al. (1991) Science 252:431-434; Rosenfeld et al., (1992) Cell 68:143-155). Furthermore, extensive studies attempting to establish adenovirus as a causative agent in human cancer have been uniformly negative (Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA 76:6606).

[0248] Pseudoadenoviral vectors (PAVs)—PAVs contain adenoviral inverted terminal repeats and minimal adenoviral 5' sequences required for helper virus-dependent replication and packaging of the vector. These vectors are free of potentially harmful viral genes, have a theoretical carrying capacity of approximately 36 kb for exogenous material, can produce reasonably high titers, and maintain the tropism of the parent virus for dividing and non-dividing human target cell types. PAV vectors can be maintained either as plasmid-derived constructs or as infectious viral particles. As plasmid constructs, PAVs consist of the minimal sequences derived from wild-type adenovirus type 2 required for efficient replication and packaging of these sequences by either wild-type or defective helper viruses, as well as any desired additional exogenous genetic material.

[0249] U.S. Patent Publication US 7,318,919 - Gregory et al., describes in detail the process by which adenoviral vectors are generated and delivered, and their corresponding uses for the treatment of disease, the contents of which are incorporated herein by reference in their entirety. The present invention contemplates the use of adenoviral vectors to deliver 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 Invention Herpes simplex vectors (HSV-based viral vectors) are suitable for use as vectors for introducing nucleic acid sequences into numerous cell types. Mature HSV virions consist of an enveloped icosahedral capsid with 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, HSV-based viral vectors deficient in at least one essential HSV gene are replication-deficient. Most replication-deficient HSV vectors contain deletions to remove one or more intermediate-early, early, or late HSV genes to prevent replication. For example, HSV vectors may be deficient in an immediate-early gene selected from the group consisting of ICP4, ICP22, ICP27, ICP47, and combinations thereof. Advantages of HSV vectors are their ability to enter a latency phase, which can result in long-term DNA expression, and their large viral DNA genome, which can accommodate foreign DNA inserts up to 25 kb.

[0251] HSV-based vectors are described, for example, in U.S. Patent No. 5,837,532 (Preston et al.), U.S. Patent No. 5,846,782 (Wickham et al.), and U.S. Patent No. 5,804,413 (Deluca et al.), and International Patent Application 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. HSV vectors may lack the essential gene functions for replication of only the 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 early and late regions of the HSV genome. The generation of HSV vectors involves the use of standard molecular biology techniques well known in the art.

[0252] Replication-deficient HSV vectors are typically produced in complementation cell lines that provide gene functions not present in the replication-deficient HSV vector but required for viral propagation at levels appropriate for generating high-titer viral vector stocks. Expression of the nucleic acid sequence encoding the 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 promotes, enhances, or controls the expression (typically, and preferably, transcription) of another nucleic acid sequence.

[0253] Suitable expression control sequences include constitutive promoters, inducible promoters, repressible 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 human cytomegalovirus (HCMV) promoters, such as the HCMV immediate early promoter (HCMV IEp), promoters derived from 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 agent is particularly useful in pain management applications. For example, the promoter may be a pharmaceutical-inducible promoter (e.g., tetracycline-responsive). The promoter can be inserted into the genome of the vector by methods known in the art, for example, by introducing a unique restriction site into a given region of the genome.

[0255] U.S. Patent Publication US 7,531,167 - Glorioso et al., the contents of which are incorporated herein by reference in their entirety, describes in detail the processes by which herpes simplex vectors are produced and delivered, and their corresponding uses for the treatment of disease. The present invention contemplates the use of herpes simplex vectors to deliver nucleotides encoding ENPP1 or ENPP3 to a subject in need thereof, and methods of treatment using the same.

[0256] Alphavirus Vectors Useful According to the 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. Alphavirus replicons contain an open reading frame at their 5' end encoding the viral replicase (Rep), which is translated when viral RNA is transfected into cells. Rep is expressed as a polyprotein, which is subsequently processed into four subunits (nsps 1-4). Unprocessed Rep can copy the RNA vector into negative-strand RNA, a process that occurs during the first 3-4 hours after transfection or infection. Once processed, Rep uses the negative-strand RNA as a template to synthesize more replicon molecules. Processed Rep can also recognize internal sequences in the negative-strand RNA, or subgenomic promoters, from which it synthesizes subgenomic positive-strand RNA corresponding to the 3' end of the replicon. This subgenomic RNA is translated to produce large amounts of the heterologous protein.

[0257] A noncytopathic mutant isolated from SIN (SIN P726L vector in nsp2) containing a single amino acid change (P for L) at position 726 of 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 noncytopathic SIN vector has been widely used in vitro because it can provide long-term, persistent transgene expression with good stability and an expression level approximately 4% of that obtained with the original SIN vector (Agapov et al., 1998, Proc. Natl. Acad. Sci. USA 95:12989-94). Similarly, patent application WO2008065225 - Smerdou et al. describes a non-cytopathic SFV vector carrying the mutations R649H / P718T in the replicase nsp2 subunit. This vector makes it possible to obtain cell lines capable of constitutively and stably expressing a gene of interest by culturing them in the presence of an antibiotic resistance gene integrated into the alphavirus vector (Casales et al. 2008. Virology. 376:242-51).

[0258] The present invention contemplates designing a vector containing a DNA sequence complementary to an alphavirus replicon incorporating the sequence of a gene of interest, such as NPP1 or NPP3, along with a recognition sequence for site-specific recombination. This vector allows the alphavirus replicon containing the sequence of the gene of interest to be integrated into the cellular genome, thereby enabling the production and selection of cells that stably express the ENPP1 or ENPP3 polypeptide. The present invention also contemplates generating expression vectors in which the alphavirus replicon is under the control of an inducible promoter. This vector, when integrated into a cell further modified by integration of an expression cassette encoding a transcriptional activator in the presence of a given ligand capable of positively regulating the activity of the promoter that controls transcription of the alphavirus replicon, is further contemplated.

[0259] U.S. Patent Publication US 10,011,847 - Aranda et al., the contents of which are incorporated herein by reference in their entirety, describes in detail the process by which alphavirus vectors are generated and delivered, and their corresponding uses for the treatment of disease. The present invention contemplates the use of alphavirus vectors to deliver nucleotides encoding ENPP1 or ENPP3 to subjects in need thereof, and methods of treatment using the same.

[0260] Lentiviral Vectors Useful According to the Invention Lentiviruses belong to the Retroviridae family of viruses and are characterized by a long incubation period. Lentiviruses possess a unique ability among retroviruses: they can deliver significant amounts of viral RNA into the host cell's DNA and infect non-dividing cells. Lentiviral vectors, particularly those derived from HIV-1, have been extensively studied and are frequently used vectors. The evolution of the lentiviral vector backbone and the virus's ability to deliver recombinant DNA molecules (transgenes) to target cells has led to their use in gene therapy to restore functional genes and in vitro recombinant protein production.

[0261] The present invention contemplates lentiviral vectors containing a promoter and transgene suitable for expressing a protein of interest, such as ENPP1 or ENPP3. Typically, the vector backbone is derived from a 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 encompasses methods for producing lentiviral vectors, compositions containing lentiviral vectors expressing a gene of interest, and their use in gene therapy to express ENPP1 or ENPP3 proteins for the treatment of calcification or ossification diseases. Lentiviral vectors according to the present invention can also be used in gene therapy methods to promote the secretion of therapeutic proteins. As a further example, the present invention provides for the secretion of therapeutic proteins into the respiratory lumen or circulatory system. Thus, administration of a vector according to the present invention and its uptake by airway cells may enable the use of the lungs (or nose or airways) as a "factory" for producing therapeutic proteins that are then secreted and enter the systemic circulation at therapeutic levels, where they can migrate to cells / tissues of interest and induce a therapeutic effect. In contrast to intracellular or membrane proteins, the production of such secreted proteins does not depend on specific disease target cells to be transduced, which is an important advantage, achieving high levels of protein expression. Therefore, lentiviral vectors can also be used to treat diseases other than respiratory diseases, such as cardiovascular diseases and blood disorders. Lentiviral vectors, such as those according to the present invention, can be integrated into the genome of transduced cells, leading to long-term, persistent expression, making them suitable for transducing stem / progenitor cells.

[0262] U.S. Patent Application Publication US2017 / 0096684 - Alton et al., the contents of which are incorporated herein by reference in their entirety, describes in detail the process by which lentiviral vectors are generated and delivered and their corresponding uses for the treatment of disease. The present invention contemplates the use of lentiviral vectors to deliver nucleotides encoding ENPP1 or ENPP3 to a subject in need thereof, and methods of treatment using the same.

[0263] array 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 contains the cytoplasmic domain, transmembrane domain, SMB1 domain, SMB2 domain, phosphodiesterase / catalytic domain, linker domain, and nuclease domain. The SMB1 domain, SMB2 domain, catalytic domain, linker domain, and nuclease domain are collectively 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 connecting 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. Residue numbering and domain classification are based on the human NPP1 sequence (NCBI accession NP_006199 / Uniprot-Swissprot P22413).

[0264] SEQ ID NO:2-azurocidin-ENPP1-FC [Table 2-1] [Table 2-2] Single underline - azurocidin signal sequence, double underline - start and end of ENPP1 sequence, bold residues - Fc sequence, ** The cleavage point of the signal sequence is indicated.

[0265] SEQ ID NO:3-azurocidin-ENPP1-Alb [Table 3] Single underline - azurocidin signal sequence, double underline - start and end of ENPP1 sequence, bold residues - albumin sequence, ** The cleavage point of the signal sequence is indicated.

[0266] SEQ ID NO:4-Azurocidin-ENPP1 [Table 4-1] [Table 4-2] Single underline - azurocidin signal sequence, double underline - start and end of ENPP1 sequence, ** The cleavage point of the signal sequence is indicated.

[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] The NPP3 amino acid sequence shown above contains the cytoplasmic domain, transmembrane domain, phosphodiesterase / catalytic domain, and nuclease domain. The catalytic domain and nuclease domain together are referred to as the extracellular domain. Residues 1-11 (Met Glu Ser-Ala Thr Glu) correspond to the cytoplasmic domain. Residues 12-30 (Gln Pro Val-Leu Leu Ala) correspond to the transmembrane domain. Residues 31-875 (Leu Leu Val-Thr Thr Ile) correspond to the extracellular domain. Residues 140-510 (Leu Glu Glu-Glu Val Glu) correspond to the catalytic / phosphodiesterase domain. Residues 605-875 (Lys Val Asn-Thr Thr Ile) correspond to the nuclease domain. Residue numbering and domain classification are based on the human NPP3 sequence (UniProtKB / Swiss-Prot: O14638.2).

[0270] SEQ ID NO:8-azurocidin-ENPP3-FC [Table 8] Single underline - azurocidin signal sequence, double underline - start and end of ENPP3 sequence, bold residues - Fc sequence, ** indicates the cleavage point of the signal sequence.

[0271] SEQ ID NO:9-azurocidin-ENPP3-albumin [Table 9-1] [Table 9-2] Single underline - azurocidin signal sequence, double underline - start and end of ENPP3 sequence, bold residues - albumin sequence, ** indicates the cleavage point of the signal sequence.

[0272] SEQ ID 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] SEQ ID NO:11 - ENPP4 amino acid sequence - wild type [Table 11-1] [Table 11-2]

[0274] SEQ ID NO: 12 - ENPP51 amino acid sequence [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4] Single underlined: signal peptide sequence; double underlined: start and end of NPP1; ** = Cleavage site of the signal peptide sequence

[0275] SEQ ID NO:13 - ENPP51-ALB amino acid sequence: [Table 13-1] [Table 13-2] [Table 13-3] [Table 13-4] Single underlined: signal peptide sequence; double underlined: start and end of NPP1; ** = cleavage position of the signal peptide sequence; bold residues indicate the albumin sequence.

[0276] SEQ ID NO: 14 - ENPP5-NPP3-Fc sequence [Table 14-1] [Table 14-2] [Table 14-3] Single underlined: signal peptide sequence; double underlined: start and end of NPP33; ** = cleavage position of the signal peptide sequence; bold residues indicate the albumin sequence.

[0277] SEQ ID NO: 15 - ENPP5-NPP3-Albumin sequence [Table 15-1] [Table 15-2] [Table 15-3] [Table 15-4] [Table 15-5] Single underlined: signal peptide sequence; double underlined: start and end of NPP3; ** = cleavage position of the signal peptide sequence; bold residues indicate the albumin sequence.

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

[0279] SEQ ID NO: 17-ENPP5-1-Fc [Table 17-1] [Table 17-2] [Table 17-3] [Table 17-4] Single underlined: signal peptide sequence; double underlined: start and end of NPP3; ** = cleavage position of signal peptide sequence; bolded residues indicate Fc sequence.

[0280] SEQ ID NO: 18 - ENPP7-1-Fc amino acid sequence [Table 18-1] [Table 18-2] [Table 18-3] [Table 18-4] Single underlined: signal peptide sequence; double underlined: start and end of NPP1; ** = cleavage position of signal peptide sequence; bolded residues indicate Fc sequence.

[0281] SEQ ID NO:19 - ENPP71 (NPP1 lacking N-terminal GLK) amino acid sequence: [Table 19-1] [Table 19-2] [Table 19-3] Single underlined: signal peptide sequence; double underlined: start and end of NPP3; ** = cleavage position of the signal peptide sequence.

[0282] SEQ ID NO:20 - ENPP71 (NPP1 lacking N-terminal GLK) - Fc amino acid sequence: [Table 20-1] [Table 20-2] [Table 20-3] [Table 20-4] Single underlined: signal peptide sequence; double underlined: start and end of NPP1; ** = cleavage position of signal peptide sequence; bolded residues indicate Fc sequence.

[0283] SEQ ID NO:21 - ENPP7-1 (lacking the N-terminal GLK of NPP1)-ALB amino acid sequence [Table 21-1] [Table 21-2] [Table 21-3] [Table 21-4] [Table 21-5] Single underlined: signal peptide sequence; double underlined: start and end of NPP1; ** = cleavage position of the signal peptide sequence; bold residues indicate the albumin sequence.

[0284] SEQ ID NO:22 - ENPP7-NPP3-Fc sequence: [Table 22-1] [Table 22-2] [Table 22-3] Single underlined: signal peptide sequence; double underlined: start and end of NPP3; ** = cleavage position of signal peptide sequence; bolded residues indicate Fc sequence.

[0285] SEQ ID NO:23-ENPP7-1-Albumin [Table 23-1] [Table 23-2] [Table 23-3] [Table 23-4] Single underlined: signal peptide sequence; double underlined: start and end of NPP3; ** = cleavage position of signal peptide sequence; bolded residues indicate Fc sequence.

[0286] SEQ ID NO:24-ENPP7-NPP3-Albumin [Table 24-1] [Table 24-2] [Table 24-3] [Table 24-4] [Table 24-5] Single underlined: signal peptide sequence; double underlined: start and end of NPP3; ** = cleavage position of the signal peptide sequence; bold residues indicate the albumin sequence.

[0287] SEQ ID NO:25-ENPP7-ENPP3-Albumin [Table 25-1] [Table 25-2] [Table 25-3] [Table 25-4] [Table 25-5] Single underlined: signal peptide sequence; double underlined: start and end of NPP3; ** = cleavage position of the signal peptide sequence; bold residues indicate the albumin sequence.

[0288] SEQ ID NO: 26 - ENPP71-GLK amino acid sequence [Table 26-1] [Table 26-2] [Table 26-3] Single underlined: signal peptide sequence; double underlined: start and end of NPP1; ** = cleavage position of the signal peptide sequence.

[0289] SEQ ID NO: 27 - ENPP121 amino acid sequence [Table 27-1] [Table 27-2] [Table 27-3] Single underlined: signal peptide sequence; double underlined: start and end of NPP1; ** = cleavage position of the signal peptide sequence.

[0290] SEQ ID NO: 28 - ENPP121-Fc amino acid sequence [Table 28-1] [Table 28-2] [Table 28-3] Single underlined: signal peptide sequence; double underlined: start and end of NPP1; ** = cleavage position of signal peptide sequence; bolded residues indicate Fc sequence.

[0291] SEQ ID NO:29 - ENPP121-ALB amino acid sequence: [Table 29-1] [Table 29-2] [Table 29-3] [Table 29-4] Single underlined: signal peptide sequence; double underlined: start and end of NPP1; ** = cleavage position of the signal peptide sequence; bold residues indicate the albumin sequence.

[0292] SEQ ID NO: 30 - ENPP121-NPP3-Fc sequence [Table 30-1] [Table 30-2] [Table 30-3] Single underlined: signal peptide sequence; double underlined: start and end of NPP1; ** = cleavage position of signal peptide sequence; bolded residues indicate Fc sequence.

[0293] SEQ ID NO: 31 - ENPP121-NPP3-Albumin sequence [Table 31-1] [Table 31-2] [Table 31-3] [Table 31-4] Single underlined: signal peptide sequence; double underlined: start and end of NPP3; ** = cleavage position of the signal peptide sequence; bold residues indicate the albumin sequence.

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

[0295] SEQ ID NO: 33 - Albumin sequence [Table 33-1] [Table 33-2] [Table 33-3]

[0296] SEQ ID NO: 34 - Human IgG Fc domain, Fc [Table 34]

[0297] SEQ ID NO: 35 - Albumin sequence [Table 35-1] [Table 35-2] [Table 35-3]

[0298] SEQ ID NO:36 - ENPP2 signal peptide [Table 36]

[0299] SEQ ID NO: 37 - Signal sequence ENPP7 [Table 37]

[0300] SEQ ID NO: 38 - Signal sequence ENPP7 [Table 38]

[0301] SEQ ID NO: 39 - Signal sequence ENPP1-2-1 [Table 39-1] [Table 39-2]

[0302] SEQ ID NO: 40-exENPP3 [Table 40]

[0303] SEQ ID NO: 41 - Signal sequence ENPP5: [Table 41]

[0304] SEQ ID NO:42 - Azurocidin-ENPP1-FC nucleotide sequence [Table 42-1] [Table 42-2] Legend: Blue = restriction sites; bold = start / stop codons; green = Kozak sequence; underlined = nucleotide sequence of signal peptide.

[0305] SEQ ID NO:43 - Azurocidin-ENPP1-albumin nucleotide sequence [Table 43-1] [Table 43-2]

[0306] SEQ ID NO:44 - Azurocidin-ENPP1 nucleotide sequence [Table 44-1] [Table 44-2]

[0307] SEQ ID NO:45 - Azurocidin-ENPP3-FC nucleotide sequence [Table 45-1] [Table 45-2]

[0308] SEQ ID NO: 46 - Azurocidin-ENPP3-Albumin nucleotide sequence [Table 46-1] [Table 46-2]

[0309] SEQ ID NO: 47 - Azurocidin-ENPP3 - Nucleotide sequence [Table 47-1] [Table 47-2]

[0310] SEQ ID NO:48 - ENPP7-1-Fc nucleotide sequence [Table 48-1] [Table 48-2] [Table 48-3]

[0311] SEQ ID NO:49 - ENPP7-NPP1 albumin nucleotide sequence: [Table 49-1] [Table 49-2] [Table 49-3]

[0312] SEQ ID NO: 50 - Nucleotide sequence of NPP121-NPP3-Fc [Table 50-1] [Table 50-2] [Table 50-3]

[0313] SEQ ID NO: 51 - Nucleotide sequence of NPP121-NPP3-Fc [Table 51-1] [Table 51-2] [Table 51-3] [Table 51-4]

[0314] SEQ ID NO: 52—Nucleotide sequence of hNPP3-hFc-pcDNA3 [Table 52-1] [Table 52-2] [Table 52-3] [Table 52-4] [Table 52-5] [Table 52-6]

[0315] SEQ ID NO: 53 - ENPP121-Fc - nucleotide sequence [Table 53-1] [Table 53-2]

[0316] SEQ ID NO:54 - ENPP121 - Albumin nucleotide sequence [Table 54-1] [Table 54-2] [Table 54-3] [Table 54-4]

[0317] SEQ ID NO:55 - ENPP3 nucleotide sequence [Table 55-1] [Table 55-2]

[0318] SEQ ID NO:56 - ENPP1 nucleotide sequence: [Table 56-1] [Table 56-2]

[0319] SEQ ID NO:57 - Linker [Table 57]

[0320] SEQ ID NO:58 - Linker [Table 58]

[0321] SEQ ID NO:59 - Linker [Table 59]

[0322] SEQ ID NO: 60 - Linker [Table 60]

[0323] SEQ ID NO: 61 - Linker [Table 61]

[0324] SEQ ID NO: 62 - Linker [Table 62]

[0325] SEQ ID NO: 63 - Linker [Table 63]

[0326] SEQ ID NO: 64 - Linker [Table 64]

[0327] SEQ ID NO: 65 - Linker [Table 65]

[0328] SEQ ID NO: 66 - Linker [Table 66]

[0329] SEQ ID NO: 67 - Linker [Table 67]

[0330] SEQ ID NO: 68 - Linker [Table 68]

[0331] SEQ ID NO: 69 - Linker [Table 69]

[0332] SEQ ID NO: 70 - Linker [Table 70]

[0333] SEQ ID NO:71 - Linker [Table 71]

[0334] SEQ ID NO:72 - Linker [Table 72]

[0335] SEQ ID NO:73 - Linker [Table 73]

[0336] SEQ ID NO:74 - Linker [Table 74]

[0337] SEQ ID NO: 75 - Linker [Table 75]

[0338] SEQ ID NO:76 - Linker [Table 76]

[0339] SEQ ID NO:77 - Linker [Table 77]

[0340] SEQ ID NO:78 - Linker [Table 78]

[0341] SEQ ID NO:79 - Linker [Table 79]

[0342] SEQ ID NO:80 - Linker [Table 80]

[0343] SEQ ID NO:81 - Linker [Table 81]

[0344] SEQ ID NO:82 - Linker [Table 82]

[0345] SEQ ID NO:83 - Linker [Table 83]

[0346] SEQ ID NO:84 - Linker [Table 84]

[0347] SEQ ID NO:85 - Linker [Table 85]

[0348] SEQ ID NO:86 - Linker [Table 86]

[0349] SEQ ID NO:87 - Linker [Table 87]

[0350] SEQ ID NO:88 - Linker [Table 88]

[0351] SEQ ID NO:89 - Soluble NPP1-Fc fusion protein sequence [Table 89-1] [Table 89-2] Double underlined: start and end of NPP1; bold residues indicate Fc sequence.

[0352] SEQ ID NO: 90 - Nucleotide sequence of soluble NPP1-Fc [Table 90-1] [Table 90-2] [Table 90-3]

[0353] SEQ ID NO:91 - Soluble NPP1-(GLK)-Fc fusion protein sequence [Table 91-1] [Table 91-2] Double underlined: start and end of NPP1; bold residues indicate Fc sequence.

[0354] SEQ ID NO: 92 - Soluble NPP1-Fc fusion protein sequence [Table 92-1] [Table 92-2] [Table 92-3] Double underlined: start and end of NPP1; bold residues indicate Fc sequence.

[0355] SEQ ID NO: 93 - Soluble NPP1-Fc fusion protein sequence [Table 93-1] [Table 93-2] Double underlined: start and end of NPP1; bold residues indicate Fc sequence.

[0356] SEQ ID NO:94 - Linker [Table 94]

[0357] Pharmaceutical compositions 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 the formulation of said vector into a pharmaceutical composition. In one embodiment, the present invention relates to a pharmaceutical composition (hereinafter referred to as "the pharmaceutical composition according to the present invention") comprising an AAV vector comprising a recombinant viral genome, wherein said 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.

[0358] All embodiments disclosed in connection with the adeno-associated viral vectors, herpes simplex vectors, adenoviral vectors, alphaviral vectors and lentiviral vectors according to the invention are also applicable to the pharmaceutical compositions according to the invention.

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

[0360] In some embodiments, a 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, a 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, a 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 an amount of an AAV vector according to the present invention calculated to produce a desired effect, and is generally determined by, among other factors, the specific characteristics of the viral vector according to the present invention and the therapeutic effect to be obtained. The amount of the viral vector according to the present invention that is effective in treating a disease can be determined by standard clinical techniques described herein or known in the art. Furthermore, in vitro tests can optionally be used to help identify optimal dosage ranges. The exact dose to be used in the formulation will depend on the route of administration and the severity of the condition, and should be determined at the discretion of the physician and according to the circumstances of each patient.

[0364] promoter Vectors used in gene therapy require an expression cassette. An expression cassette consists of three key 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 is active only in certain cell types. The use of a tissue-specific promoter in an expression cassette can limit unwanted transgene expression and facilitate sustained transgene expression. Commonly used promoters 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 LTR promoters of other retroviruses. Eukaryotic promoters can also be used for gene therapy; common examples of eukaryotic promoters include the human a1-antitrypsin (hAAT) and mouse 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 AI, ApoE and a1-antitrypsin (hAAT) allow tissue-specific expression of target proteins 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 transcription targeting using eukaryotic promoters in gene therapy, all of which are fully incorporated herein by reference.

[0365] Dosage and Mode of Administration AAV titers are expressed as "physical" titers of vector or viral genomes per ml (vg / ml) or vector or viral genomes per kilogram dosage (vg / kg). Titers can be determined using QPCR of purified vector particles. One method for titering AAV VG count is as follows: purified AAV vector samples are first treated with DNase to remove non-encapsidated AAV genomic DNA or contaminating plasmid DNA from the production process. DNase-resistant particles are then heat-treated to release the genome from the capsid. The released genomes are quantified by real-time PCR using primer / probe sets targeting specific regions of the viral genome.

[0366] The viral composition is administered to a human patient at a dose of approximately 1.0 x 10 9 vg / kg ~ approx. 1.0×10 15 vg / kg, preferably 1.0 × 10 12 vg / kg~1.0×10 14 The dosage unit can be formulated to contain an amount of viral vector in the range of 1.0 x 10 vg / kg. Preferably, the dose of virus in the formulation is 1.0 x 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, the dose administered to a mammal, particularly a human, in the context of the present invention will vary depending on the particular viral vector, the composition comprising the vector and a carrier therefor (discussed above), and the mode of administration. The dose is sufficient to produce a desired response, e.g., a therapeutic or prophylactic response, within a desired time frame. In terms of viral vectors, the dose is 1×10 15 vg / kg.

[0368] The vectors of the present invention enable long-term gene expression, resulting in a long-term effect of a therapeutic protein. The phrases "long-term expression," "continuous expression," and "sustained expression" are used interchangeably. Long-term expression according to the present invention preferably refers to expression of a therapeutic gene and / or protein at therapeutic levels 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 refers to 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 doses (if possible) or by a single dose.

[0369] Repeat doses can be administered twice daily, once daily, twice weekly, weekly, monthly, every two months, every three months, every four months, every six months, yearly, every two years, or more. Dosing can continue for as long as necessary, for example, 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 compositions of the present invention can be administered locally or systemically, intramuscularly, intravenously, and parenterally. Delivery of the therapeutic compositions of the present invention can be directed to the central nervous system, cardiac system, and pulmonary system. A common delivery strategy is direct intramuscular injection. As a non-limiting example, skeletal muscle has been shown to be an efficiently transduced target tissue type. Once transduced, muscle cells serve as a production site for protein production, which can be acted on locally or systemically by many AAV variants.

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

[0372] By way of non-limiting example, systemic administration includes systemic injection of an AAV vector according to the invention, for example, 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, an AAV vector according to the 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 by methods known in the art, for example, but not limited to, intravenous administration, intraportal administration, intrabiliary administration, intraarterial administration and direct injection into the liver parenchyma.In another embodiment, the pharmaceutical composition is administered intravenously.

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

[0375] formulation The preparation may also contain buffer salts. Alternatively, the composition may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use. If necessary, the composition may also contain a local anesthetic such as lidocaine to ease pain at the injection site. If the composition is to be administered by infiltration, it can be dispensed in an infiltration bottle containing pharmaceutical-grade water or saline. If the composition is to be administered by injection, a water for injection or sterile saline vial can be provided so that the ingredients can be mixed prior to administration. Preferably, the pharmaceutically acceptable carrier is saline and a surfactant such as Pluronic®.

[0376] Compositions according to the invention can be formulated for delivery to animals for veterinary purposes (e.g., livestock (cows, pigs, etc.) and other non-human mammalian subjects), as well as to human subjects. AAV vectors can be formulated with a physiologically acceptable carrier for use in gene transfer and gene therapy applications. Non-limiting examples include the use of adjuvants in combination with or mixed with AAV vectors according to the invention. Contemplated adjuvants include, but are not limited to, mineral salt adjuvants or mineral salt gel adjuvants, particulate adjuvants, microparticulate adjuvants, and mucosal adjuvants. Adjuvants can be administered to a subject as a mixture with AAV vectors according to the invention or 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 formulation. Pharmaceutically acceptable carriers are essentially non-toxic to recipients at the dosages and concentrations used and are compatible with the other ingredients of the formulation. The number and nature of the pharmaceutically acceptable carriers depend on the desired form of administration. 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, SA, Madrid, ES, 1993; Gennaro A, Ed., "Remington: The Science and Practice of Pharmacy," 20th ed. Lippincott Williams & Wilkins, Philadelphia, Pa., US, 2003).

[0378] As a 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 virus compositions can take forms such as suspensions, solutions, or emulsions in oily or aqueous vehicles 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-hydroxybenzoate or sorbic acid).

[0379] Preparations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the preparation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives.The preparations can be provided in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) state, requiring only the addition of a sterile liquid excipient, such as water, for injection immediately before use.Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the type described above.

[0380] In addition, the composition may contain additional therapeutic or biologically active agents. For example, therapeutic agents useful in the treatment of specific indications may be present. Inflammation-controlling agents, such as ibuprofen or steroids, may be part of the composition to reduce swelling and inflammation and physiological distress associated with in vivo administration of the vector. Immune system suppressors may be administered in the composition to reduce any immune response against the vector itself or associated with the disorder. Administration of immunosuppressive pharmaceuticals or immunosuppressants is the primary method of deliberately induced immunosuppression; in optimal situations, the immunosuppressive drug targets only any hyperactive components of the immune system.

[0381] Immunosuppressants, or anti-rejection medications, are drugs that inhibit or prevent the activity of the immune system. These include glucocorticoids, cytostatics, antibodies, and drugs acting on immunophilins. Glucocorticoids, such as prednisone, dexamethasone, and hydrocortisone, are used in pharmacological (supraphysiological) doses to suppress various allergic and inflammatory reactions. Cytostatics include purine analogs, alkylating agents, such as nitrogen mustard (cyclophosphamide), nitrosoureas, and platinum compounds. Cyclophosphamide (Baxter's Cytoxan) is perhaps the most potent immunosuppressant. Antimetabolites include 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, are the most important, are used in kidney transplants. Other cytotoxic antibiotics are anthracyclines, mitomycin C, bleomycin, and mithramycin. Antibodies may also be used as rapid and potent immunosuppressive therapy to prevent acute rejection (e.g., anti-CD20 monoclonal).

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

[0383] Antibiotics, ie, bactericides and fungicides, may be present to reduce the risk of infection associated with the gene transfer procedure and other disorders.

[0384] A pharmaceutical composition can be formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous or intramuscular administration to human beings.

[0385] Treatment methods 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 mineralization or ossification.

[0386] In one aspect, the present invention relates to an adeno-associated viral vector comprising a recombinant viral genome, wherein said 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 a disease of pathological calcification or ossification.

[0387] In another aspect, the present invention relates to the use of an adeno-associated viral vector comprising a recombinant viral genome, wherein said 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 the manufacture of a medicament for the treatment and / or prevention of a disease of pathological calcification or ossification.

[0388] In another aspect, the present invention provides a method for the treatment and / or prevention of a pathological calcification or ossification disease in a subject in need thereof, comprising administering to said subject an adeno-associated viral vector comprising a recombinant viral genome, said recombinant viral genome comprising 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 embodiment, the pathological calcification or ossification disease treated by the compositions and methods of the present invention 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).

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

[0391] In one embodiment, a polynucleotide according to the present invention comprises a transcriptional 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 the albumin promoter, the phosphoenolpyruvate carboxykinase (PEPCK) promoter, and the alpha-1-antitrypsin promoter (most preferably the human alpha-1-antitrypsin promoter). In another embodiment, the transcriptional control region of a polynucleotide according to the present invention further comprises an enhancer, preferably a liver-specific enhancer, more preferably a liver control region enhancer (HCR), operably linked to the promoter.

[0392] In another embodiment, the expression cassette of the polynucleotide according to the present invention further comprises a polyadenylation signal, more preferably SV40 poly A. 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 a polynucleotide according to the present invention. In a specific embodiment, 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 are not present in the viral vector, as they can be provided in trans by another vector, e.g., a plasmid, or by stable integration of the sequences into a packaging cell line.

[0395] Methods for Obtaining AAV According to the Invention The present invention also relates to a method for obtaining a viral vector according to the present invention, including, but not limited to, an AAV vector. The AAV vector can be obtained by introducing a polynucleotide according to the present invention into a cell that constitutively expresses Rep and Cap proteins or 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 viral vector, comprising the steps of: (i) providing a cell containing a polynucleotide according to the invention, an AAV Cap protein, an AAV Rep protein, and optionally viral proteins on which AAV depends for replication; (ii) maintaining the cells under conditions suitable for assembly of AAV; and (iii) purifying the adeno-associated virus vector produced by the cells.

[0396] The generation of recombinant AAV (rAAV) for vectoring 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 and insect cells.

[0397] In one embodiment, a producer cell line is transiently transfected with a polynucleotide according to the invention (comprising an expression cassette flanked by ITRs) and construct(s) encoding Rep and Cap proteins and providing helper functions. In another embodiment, a cell line stably supplies helper functions and is transiently transfected with a polynucleotide according to the invention (comprising an expression cassette flanked by ITRs) and construct(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 the 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. Methods for producing and using these and other AAV systems have been 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 providing 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 capacity of AAV can be increased by providing two genomic AAV ITRs that can anneal to form head-to-tail concatemers. Generally, upon AAV entry into a host cell, single-stranded DNA containing the transgene is converted to double-stranded DNA by the host cell's DNA polymerase complex, and the ITRs then assist in concatemer formation in the nucleus. Alternatively, AAV can be engineered to be self-complementary (sc) AAV, which allows the viral vector to bypass the second-strand synthesis step upon entry into target cells, providing a scAAV viral vector with faster and potentially higher (e.g., up to 100-fold) transgene expression.

[0401] For example, AAVs can be engineered to have a genome comprising two linked single-stranded DNAs, each encoding a transgene unit and its complement, which can snap together after delivery into a target cell to produce a double-stranded DNA encoding the desired transgene unit. Self-complementary AAVs have been described in the art (Carter B, U.S. Pat. No. 6,596,535; Carter B, U.S. Pat. No. 7,125,717; and Takano H et al., U.S. Pat. No. 7,456,683).

[0402] Preferably, all structural and non-structural coding sequences (Cap and Rep proteins) are not present in the AAV vector, as they can be provided in trans by a vector, such as a plasmid. Cap proteins have been reported to affect the host tropism, cell, tissue, or organ specificity, receptor usage, infection efficiency, and immunogenicity of AAV viruses. Therefore, the AAV Cap for use in rAAV can be selected taking into account, for example, the target species (e.g., human or non-human), the target's immunological status, the target's suitability for long-term or short-term treatment, or a specific therapeutic application (e.g., treatment of a specific disease or disorder, or delivery to specific cells, tissues, or organs).

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

[0404] In some embodiments, an AAV Cap for use in a method according to the invention can be generated by mutagenesis (i.e., by insertion, deletion, or substitution) of one of the aforementioned AAV Caps or their encoding nucleic acids, hi 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 comprising 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 comprises more than one of the aforementioned Caps.

[0406] In some embodiments, the AAV Cap for use in the rAAV composition is engineered to contain 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 so that the surface of the rAAV is pegylated (i.e., PEGylated), which can facilitate immune evasion. The Cap protein can also be mutagenized (e.g., to eliminate its natural receptor binding or to mask immunogenic epitopes).

[0407] In some embodiments, AAV Rep proteins for use in methods according to the invention can be generated by mutagenesis (i.e., by insertion, deletion, or substitution) of one of the aforementioned AAV Reps or their encoding nucleic acids, hi 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 AAVrhlO.

[0409] In some embodiments, a viral protein on which AAV depends for replication for use in a method according to the invention can be generated by mutagenesis (i.e., by insertion, deletion, or substitution) of one of the aforementioned viral proteins or their encoding nucleic acids, hi 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 function of Cap proteins, Rep proteins, and viral proteins on which AAV depends for replication are well known in the art. The genes AAV rep, AAV cap, and genes providing helper functions can be inserted into cells by incorporating the genes into a vector, such as a plasmid, and introducing the vector into cells. The genes can be incorporated into the same or different plasmids. In another embodiment, the AAV rep and cap genes are incorporated into one plasmid, and the genes providing helper functions are incorporated into another plasmid. Examples of plasmids containing AAV rep and cap genes suitable for use with the methods of the present invention include pHLP19 and pRep6cap6 vectors (Colisi P, U.S. Pat. No. 6,001,650 and Russell D et al., U.S. Pat. No. 6,156,303).

[0411] The polynucleotides of the present invention and the polynucleotides containing the AAV rep and cap genes or genes providing helper functions can be inserted into cells by any suitable method 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 certain embodiments, transfection is carried out by co-precipitation with calcium phosphate. If a cell lacks expression of either the AAV rep and cap genes or the genes providing adenoviral helper functions, the genes can be inserted into the cell simultaneously with the polynucleotides of the present invention.

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

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

[0414] The present invention encompasses methods that involve maintaining cells under conditions suitable for AAV assembly. Exemplary conditions for culturing packaging cells and promoting AAV vector particle release, such as producing cell lysates, can be performed as described in the Examples herein. Producer cells are grown for an appropriate period of time to promote AAV assembly and viral vector release 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, or up to about 10 days. After about 10 days (or sooner, depending on the culture conditions and the specific producer cells used), the level of production generally declines significantly. Generally, the time of culture 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 suitable 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 the onset of virus production).

[0415] The present invention encompasses a method for purifying adeno-associated viral vectors produced by cells. AAV according to the present invention can be obtained from both i) cells transfected with a polynucleotide according to the present invention and ii) the culture medium of the cells after a certain period of time, preferably 72 hours, after transfection. Any method for purifying AAV from the cells or culture medium can be used to obtain AAV according to the present invention. In a specific embodiment, AAV according to the present invention is purified according to an optimized method 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 viral genome titer can be determined by quantitative PCR according to the protocol described for the AAV2 reference standard (Lock M, et al., Hum. Gene Ther. 2010;21:1273-1285), using linearized plasmid DNA as a standard curve.

[0416] In another embodiment, purification is further carried out by a polyethylene glycol precipitation step or cesium chloride gradient fractionation. In some embodiments, the method further comprises a purification step, such as treating the cell lysate with benzonase, purifying the cell lysate through a CsCl gradient, or purifying the cell lysate using heparin sulfate chromatography (Halbert C, et al., Methods Mol. Biol. 2004;246:201-212).

[0417] Various naturally occurring and recombinant AAVs, their encoding nucleic acids, AAV Cap and Rep proteins and their sequences, and methods for isolating or producing, propagating, and purifying such AAVs, particularly their capsids suitable for use in AAV production, 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 in generalized arterial calcification of infancy (GACI) asj / asj Model; Li, et al., 2013, Disease Models & Mech. 6(5): 1227-35. 2. Enpp1 in generalized arterial calcification of infancy (GACI) asj / asj Model;Li,et al,2014,PloS one 9(12):el 13542. 3. ABCC6 in Pseudoxanthoma Elasticum (PXE) - / - Mouse model; Jiang, et al., 2007, J. Invest. Derm. 127(6):1392-4102. 4. HYP mouse model of X-linked hypophosphatasia (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 l04(6):492-7) and osteoarthritis (Bertrand, et al, 2012, Annals Rheum. Diseases 71(7):1249-53). 7. Dietary adenine-induced chronic kidney disease (CKD) in rat models; Schibler, et al., 1968, Clin. Sci. 35(2):363-72; O'Neill, et al., 2011, Kidney Int. 79(5):512-7. 8. Dietary adenine-induced chronic kidney disease (CKD) in a mouse model; 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 osteopenia; Mackenzie, et al, 2012, PloS one 7(2):e32177.

[0419] The animal models described above can be used to test for changes in soft tissue mineralization and ossification upon administration of a vector 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 (diet is a high calcium, low magnesium diet (e.g., Teklad Labs diet TD.00042, Harlan Labs, Madison, WI)).

[0420] Npt2a - / - Mice exhibit kidney stone formation when fed with normal chow from weaning age and persist for at least 10 weeks of age. Conversely, double mutant Npt2a - / - / Enpp1 asj / asj Mice exhibit twice the level of kidney stone formation when compared to Npt2a- / - mice fed a normal chow diet. - / - Mouse and Npt2a - / - / Enpp1 asj / asj Mice are commercially available from the Jackson laboratory, ME. Double mutant mice (Npt2a - / - / Enpp1 asj / asj) was purified by Npt2a following standard protocols known in the art (Jackson Laboratory Recourse Manual, (2007, 1-29)). - / - Mice and Enpp1 asj / asj It is produced by cross-breeding mice. Npt2a for kidney stone-related diseases - / - or Npt2a - / - / Enpp1 asj / asj The double mutant mouse model can be used to test the efficacy of treatments according to the invention (Khan & Canales, 2011, J. Urol. 186(3):1107-13; Wu, 2015, Urolithiasis 43(Suppl 1):65-76). Rodent models of oxalate stone formation, 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 are no rodent models in the literature that recapitulate 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] Experimental details regarding ENPP1 enzymatic activity, ENPP3 enzymatic activity, plasma PPi quantification, micro-CT scanning, and plasma PPi uptake quantification are described in detail in patent applications and publications 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 incorporated herein in their entirety.

[0423] The present invention is further illustrated by the following examples, which should not be construed as further limiting in any way. The contents of all cited references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference. [Example]

[0424] Example 1 - Cloning of NPP1 sequences 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 from AAV2. The AAV2 genome can be pseudotyped with AAV8. The expression cassette can have the following elements from the 5' to 3' direction: a liver-specific enhancer liver control region (HCR), a liver-specific promoter human alpha antitrypsin (hAAT), an intron, a polypeptide containing an N-terminal azurocidin signal sequence, an NPP1 cDNA, a C-terminal Fc sequence, and an SV40 polyadenylation signal. The expression cassette is flanked by the 5' and 3' ITRs from AAV2. The resulting construct is shown in the schematic diagram in 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), (b) residues 1-22 of ENPP7, (c) residues 1-24 of ENPP5, (d) human serum albumin, or (e) human azurocidin.

[0426] SEQ ID NOs: 1-4, 6-15, 17-31, and 42-56 show several ENPP1-Fc and ENPP3-Fc constructs, all of which can be used for cloning ENPP1 or ENPP3 sequences into the AAV system and generating constructs for AAV infection.

[0427] The modified NPP1 sequence was cloned into a plasmid using standard molecular biology protocols. A non-coding plasmid carrying the same components of the construct but lacking the NPP1 cDNA and containing a multiple cloning site was used to generate null particles as a control.

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

[0429] After three freeze / thaw cycles, the lysate is centrifuged at 2500 g for 30 minutes. The supernatant from this centrifugation is added to the medium, and vector particles are precipitated by incubation with 8% PEG8000 (Sigma) for 15 hours and pelleted at 2500 g for 30 minutes. The vector-containing pellet from the cells and medium is thoroughly reconstituted in TBS, treated with benzonase (Merck) for 30 minutes at 37°C, and centrifuged at 10,000 g for 10 minutes. The supernatant is loaded into a 37.5 ml UltraClear tube (Beckman) containing a 1.3-1.5 g / ml CsCl density step gradient and centrifuged at 28,000 rpm for 17 hours in an SW28 rotor (Beckman). The virus band was collected using a 10 ml syringe and an 18-gauge needle and transferred to a new 12.5 ml UltraClear tube filled with 1.379 g / ml CsCl solution to generate a continuous gradient. The tube was centrifuged at 38,000 rpm for 48 hours in an SW40Ti rotor (Beckman). Finally, the intact particle band was collected, dialyzed in 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 reduced empty AAV capsids and DNA and protein impurities from the virus stock, thus increasing AAV purity and ultimately resulting in higher in vivo transduction. The same protocol was used to generate infectious AAV particles carrying a "null" vector that does not encode any ENPP proteins.

[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., a sequence disclosed elsewhere herein) is placed into a vector suitable for large-scale protein production. A variety of such vectors are available from commercial sources.

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

[0432] Single stably transfected cell clones are then established and screened for high-expression clones of the desired fusion protein. Screening of single cell clones for ENPP1 protein expression is performed in a high-throughput manner in 96-well plates 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] Once high-expressing clones are identified through screening, protein production is carried out in shake flasks or using bioreactors, as previously described for ENPP1 (Albright, et al., 2015, Nat Commun. 6:10006). ENPP1 is purified 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) is surprisingly 5-fold higher than that produced using the NPP2 (127 mg / liter) or NPP7 (136 mg / liter) signal sequences. The ENPP1 protein produced in this manner can be further purified using additional techniques and / or chromatography steps, as described above, to reach substantially higher purities, such as about 99% purity.

[0435] The enzymatic activity of the ENPP1 thus produced was measured by determining the steady-state hydrolysis of ATP by human NPP1 using HPLC. Briefly, the enzymatic reaction was initiated by adding 10 nM ENPP1 to various concentrations of ATP in a reaction buffer containing 20 mM Tris, pH 7.4, 150 mM NaCl, 4.5 nM KCl, 14 μM ZnCl2, 1 mM MgCl2, and 1 mM CaCl2. At various time points, 50 μl of the reaction solution was removed and quenched with an equivalent volume of 3 M formic acid. The quenched reaction solution was loaded onto a C-18 (5 μm, 250 × 4.6 mm) column (Higgins Analytical) equilibrated in 5 mM ammonium acetate (pH 6.0) and eluted with a 0% to 20% methanol gradient. The substrate and product were monitored by UV absorbance at 259 nm and quantified by integrating their corresponding peaks and using a calibration curve. Thus, the ENPP1 protein is characterized according to the protocols discussed herein and elsewhere in 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 viral particles encoding ENPP1-Fc into mice and measurement of weight gain, bone density, bone strength and bone mass Mouse models, e.g., Enpp1 asj / asj Mouse model, ABCC6 - / - Mouse models, including the HYP mouse model, the ttw mouse model, a mouse model of chronic kidney disease (CKD), or a 5 / 6th nephrectomy rat model of CKD, are used to test the efficacy of delivery of vectors capable of encoding and expressing NPP1 or NPP3. As a non-limiting example, the following experiments use Enpp1 as a mouse model. asj / asj The mouse model uses the azurocidin-NPP1-Fc construct as the polynucleotide delivered to the mouse, and delivery is carried out in vivo by using AAV particles (prepared as described in Example 1) encoding the ENPP1-Fc protein.

[0437] Those skilled in the art will recognize that the same experiments can be repeated by using alternative mouse models, alternative polynucleotide constructs comprising alternative signal sequences (such as NPP2, NPP5, NPP7, albumin or azurosidi) encoding different ENPP1 fusion proteins disclosed in the present invention (such as ENPP1-albumin, or ENPP1-Fc, or an ENPP1 functional equivalent, 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 an ENPP3 functional equivalent) to test the efficacy of gene therapy for treating diseases of pathological calcification or ossification. The azurocidin-NPP1-Fc construct utilized in the experiments encodes the human ENPP1-Fc protein as a proof of concept, and the same experiments can be repeated using the azurocidin-NPP3-Fc construct encoding human ENPP3-Fc.

[0438] Four sets of mice were used in this experiment, with each set having at least five mice (6–8 weeks old). Prior to injection of AAV particles, all sets of mice were tolerized by intraperitoneal injection of Titer GK1.5CD4 antibody at a concentration of 1000 μg / ml (final dose of 25–40 μg / animal) to reduce the immune response of mice to the human protein produced by the AAV construct, and ENPP1 served as a control group. wt The first cohort of mice was injected with AAV particles containing a null vector, ENPP1, to serve as a control group. asj / asj A second cohort of mice was injected with AAV particles containing the null vector, ENPP1, to serve as the study group. wt A third cohort of mice was injected with AAV particles containing a polynucleotide encoding the ENPP1-Fc protein, which served as the test group. asj / asjThe fourth cohort will be injected with AAV particles containing a polynucleotide encoding the ENPP1-Fc protein. Tolerization injections will be repeated weekly after AAV injection for each cohort (i.e., on days 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, and 105 after AAV administration).

[0439] Experimental mice were fed either a phosphorus-enriched, magnesium-reduced acceleration diet (Harlan Teklad, Rodent diet TD.00442, Madison, WI) or regular chow (Laboratory Autoclavable Rodent Diet 5010; PMI Nutritional International, Brentwood, MO). After 6–8 weeks of age, mice were fed approximately 1 × 10 Fecal pellets in PBS pH 7.4. 12 ~1×10 15 vg / kg , preferably 1 x 10 13 ~1×10 14 vg / kgAll mice will receive retro-orbital or tail vein injections of AAV. The injected vector will either be empty "null" (control group) or carry the NPP1 gene (study group). Body weight will be measured once daily, and any gain or loss in body weight after AAV injection will be recorded. Blood, urine, bone, and tissue samples will be collected from the mice and analyzed 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, the entire contents of which are incorporated herein by reference in their entirety. At the end of the study (days 7, 28, and 112), all mice were euthanized following retro-orbital bleeding under deep isoflurane anesthesia, and vital organs were removed 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 The animals were retro-orbitally bled using a heparinized micropipette, and the blood was dispensed into heparinized Eppendorf tubes, which were placed on ice. The samples were spun at 4,000 rpm for 5 minutes in a pre-cooled microcentrifuge at 4°C, and the plasma was collected and diluted in one volume of 50 mM Tris-acetate pH = 8.0. The collected plasma was filtered through a 300 KDa membrane by ultracentrifugation (NanoSep 300K, Pall Corp., Ann Arbor, MI) and frozen at -80°C. Pyrophosphate was converted to uridine 5'-diphospho[ 14The reaction product, i.e., uridine 5'-diphospho[C], was quantified using a standard three-step enzymatic assay using [C] glucose. 14 [C]gluconate was recorded (Analysis of inorganic pyrophosphate at the picomole level. Cheung CP, Suhadolnik RJ, Anal Biochem. 1977 Nov;83(1):61-3). Briefly, 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 A reaction mixture (100 μl) containing [C] glucose, 0.25 units of uridine 5'-diphosphoglucose pyrophosphorylase, 0.25 units of phosphoglucose mutase, 0.5 units of glucose 6-phosphate dehydrogenase, and inorganic pyrophosphate (50–200 pmol) was incubated at 37°C for 30 min. The reaction was terminated by adding 200 μl of 2% charcoal thoroughly suspended in water. A 200 μl aliquot of the supernatant was then counted in scintillation solution.

[0441] In vivo 99m PYP Imaging Bone imaging can be performed if desired. The bone imaging agent 99mTc-pyrophosphate (Pharmalucence, Inc.) is evaluated in a cohort of animals using a preclinical microSPECT / CT hybrid imaging system with a dual 1 mm pinhole collimator (X-SPECT, Gamma Medica-Ideas). 2–5 mCi of radiolabeled tracer is injected intraperitoneally into each animal, and the animal is imaged 1–1.5 h after injection. CT scans (512 projections at 50 kVp, 800 μA, and a magnification factor of 1.25) are obtained for anatomical colocalization with SPECT images. SPECT imaging is obtained with a counterclockwise rotation of 180° per collimator head, 32 projections, a 7.0 cm ROR, an 8.95 cm FOV, and an energy window of 140 keV ± 20 s per projection. CT images are reconstructed with FLEX XO CT software (Gamma Medica-Ideas) using filtered backprojection. SPECT images will be reconstructed using FLEX SPECT software (5 repetitions, 4 subsets) and subsequently fused with CT images and analyzed using AMIRA software.

[0442] 99 Quantification of mPYP uptake 99 For mPYP mouse scans, 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 delineated around the head (target organ) and whole body of each animal. Percent injected activity (PIA), often referred to as the "percent injected dose," is calculated by comparing the ratio of head counts to whole body counts and expressed as a percent injected dose, providing a measure of the affinity of radiotracer uptake by the region of interest (head). The total counts for each scan are considered a whole-body measure of the injected dose.

[0443] Blood and urine parameters Biochemical analysis can also be performed using blood samples (obtained by retro-orbital bleeding) and spot urine collected after an overnight fast at the same time between 10:00 AM and 2:00 PM. Following deproteinization of heparinized plasma by filtration (NanoSep 300 K, Pall Corp., Ann Arbor, MI), plasma and urinary total pyrophosphate (PPi) concentrations are determined using a fluorescent probe (AB112155, ABCAM, Cambridge, MA). Urinary PPi is corrected for urinary creatinine measured by LC-MS / MS or by ELISA using appropriate controls to adjust for inter-assay variability.

[0444] Kidney histology The left kidney was fixed in 4% formalin / PBS for 12 hours at 4°C, then dehydrated in increasing concentrations of ethanol and xylene, followed by paraffin embedding. Mineral deposition was measured on 10 μm von Kossa-stained sections counterstained with 1% methyl green. Hematoxylin / eosin was used as the counterstain for morphological assessment. Histomorphometric evaluation of sagittal kidney sections, including the cortex, medulla, and pelvis, was performed blinded by two independent observers using an Osteomeasure system (Osteometrics, Atlanta, GA). Percent mineralization area was determined using the formula: % calculated area = 100 * mineralization area / total area (including the cortex, medulla, and pelvic lumen) and was dependent on the number of areas observed per section. Mineralization size was determined using the formula: calculated size = mineralization area / number of calcifications observed per section.

[0445] For transmission electron microscopy, 1 mm of the left kidney was 3The blocks were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in phosphate-buffered saline for 2 hours, followed by post-fixation in 1% osmium for 2 hours. Dehydration was performed using a series of ethanol concentrations (50%–100%). The kidney tissue was embedded in epoxy resin and polymerized overnight at 60°C. Thin sections (50 nm) were prepared, and the tissue was double-stained with uranium and lead and visualized using a Tecnai Biotwin (LaB6, 80 kV) (FEI, Thermo Fisher, Hillsboro, OR).

[0446] Histology, histomorphometry and microCT The tibiae and femurs of mice were stripped of soft tissue, 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 were performed on a fixed area just below the growth plate, corresponding to the primary cancellous bone (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)). Analysis was performed using Osteomeas software (Osteometrics, Atlanta, GA). Bones were scanned using a Scanco μCT-35 (Scanco, Brutissellen, Switzerland) to analyze multiple structural parameters in both the proximal tibia and distal femur just below the growth plate (trabecular bone) and the midshaft of the tibia or femur (cortical bone).

[0447] Bone Biomechanical Testing Femurs from mice receiving the enhanced diet were loaded to failure in three-point bending, while femurs from mice receiving a normal chow diet were loaded to failure in four-point bending. All whole-bone tests were performed by loading the femur in a posterior-to-anterior direction, with the anterior quadrant under tension. The widths of the lower and upper supports of the four-point bending apparatus were 7 mm and 3 mm, respectively. Tests were performed using a servohydraulic testing machine (Instron Model 8874; Instron Corp., Norwood, MA, USA) at a deflection rate of 0.05 mm / s. Load and midspan deflection were acquired directly at a sampling frequency of 200 Hz. Load-deflection curves were analyzed for stiffness, maximum load, and work to failure. Yield was defined as a 10% reduction in secant stiffness (load range normalized to deflection range) compared to the initial tangent stiffness. Femurs were tested at room temperature and kept moist with phosphate-buffered saline (PBS). The post-yield deflection, defined as the deflection at failure minus the deflection at yield, is also measured.

[0448] Example 4 - Treatment of chronic kidney disease using viral vectors 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 CKD. For illustrative purposes, ENPP1-Fc and ENPP3-Fc are used in these examples, and similar results can be obtained using other ENPP1 or ENPP3 fusions of the invention.

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

[0450] Control cohort: In this experiment, a first cohort of ENPP1 wt mice, which serve as a control group, will be injected with AAV particles containing a null vector, and a second cohort of CKD mice, which serve as a control group, will be injected with AAV particles containing a null vector.

[0451] Cohort of ENPP1-treated mice:ENPP1 wt A third cohort of mice will be injected with AAV particles engineered to express the ENPP1-Fc protein, and a fourth cohort of CKD mice will be injected with AAV particles engineered to express the ENPP1-Fc protein.

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

[0453] Adenine diet: CKD mice were maintained on an adenine diet, while wild-type mice were maintained on a regular chow diet (Laboratory Autoclavable Rodent Diet 5010; PMI Nutritional International, Brentwood, MO). To provide the adenine-containing chow for CKD mice, adenine was mixed with a casein-based diet to mask odor and taste. Adenine was purchased from Sigma-Aldrich (MO, USA), and powdered casein-based diet was purchased from Special Diets Services (SDS, UK) (reference number 824522). Other components of the diet were 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 x 1012 ~1×10 15 vg / kg , preferably 1 x 10 13 ~1×10 14 vg / kg All mice were given a retro-orbital or tail vein injection of either an empty "null" vector (control group) or carrying the NPP1 or NPP3 gene (study group).

[0455] Assays: Renal histology, 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 creatine levels, and plasma pyrophosphate (PPi), will be analyzed for each cohort as described in Example 3. Urine will be collected as a spot urine sample after spontaneous voiding. Serum and urinary calcium, phosphorus, creatinine, and urea levels will be measured using a Konelab 20XTi (Thermo Scientific, Finland). Creatinine concentrations will be confirmed using a colorimetric assay (BioChain, CA, USA). PTH will be measured using a mouse intact PTH ELISA kit (Immutopics, CA, USA), FGF23 levels will be measured using an intact FGF23 ELISA (Kainos, Japan), and vitamin D will be measured using an EIA kit (Immunodiagnostic Systems, UK). Details of the experiment will be 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 decreased urinary urea / serum urea and urinary creatinine / serum creatinine ratios. In contrast, CKD mice treated with AAV expressing ENPP1 or ENPP3 proteins are expected to show increased weight, approaching the weight range 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 carrying ENPP1 or ENPP3) CKD mice are expected to show improved kidney function, manifested by decreased serum urea levels and increased urinary urea levels, leading to higher urinary urea / serum urea ratios.

[0457] Renal histology analysis of kidney tissue from CKD mice is expected to show deposition of crystalline structures in areas such as tubular lumens, microabscesses, and dilated tubules, Periodic Acid-Schiff (PAS) staining showing dilated Bowman's spaces, the presence of atrophic tubules with protein casts ("thyroidization") and tubular atrophy with thickening of the tubular basement membrane, the presence of mild interstitial fibrosis visible through Ladewig staining, and the appearance of extensive calcification of tubular structures visible through von Kossa staining. In contrast, CKD mice treated according to the present invention with ENPP1 or ENPP3 are expected to show reduced or absent renal mineral deposition in tubular lumens and soft tissue vasculature, with a histology similar to that of healthy wild-type mice.

[0458] Untreated CKD mice are expected to exhibit significantly elevated serum inorganic phosphorus (Pi), elevated PTH and FGF23 levels, but reduced 1,25(OH)2-vitamin D levels, and lower PPi levels (approximately 0.5 μM) when compared to healthy wild-type mice (normal levels of PPi are approximately 2-4 μM; PTH is approximately 10-65 ng / L; median FGF23 levels are 13 RU / mL, normal FGF23 levels range from 5-210 RU / mL; normal vitamin D levels are 20 ng / mL-50 ng / mL). In contrast, treated CKD mice are expected to exhibit elevated PPi levels (approximately 4-5 μM), which are higher than the PPi levels (approximately 0.5 μM) seen in untreated CKD mice. Thus, by observing one or more factors such as a reduction in calcification of renal soft tissue and coronary arteries visualized through histological analysis (25%, 50%, 70%, 90%, or 100% reduction), an increase in serum PPi levels from blood analysis, normalization of vitamin D levels, a reduction in FGF23 levels to the normal range, normalization of PTH levels, an increase in survival time, and improved kidney function observed by an increase in urinary urea and creatine along with increased weight gain, one skilled in the art can determine the therapeutic efficacy of vector-based ENPP1 or ENPP3 in treating chronic kidney disease.

[0459] Treatment of human subjects: Approximately 5 x 10 cells per subject in 1 x PBS, pH 7.4, can be used to deliver and express ENPP1 or ENPP3. 11 ~5×10 15 vg / kg and in some embodiments, approximately 1×10 in 1×PBS at pH 7.4 12 ~1×10 15 vg / kgHuman patients with CKD are treated by intravenous injection containing a compound containing ENPP1 or ENPP3. Successful CKD treatment is observed by monitoring one or more of the aforementioned parameters through regular blood and urine tests, as described for the mouse model. Rather than histological analysis, which requires staining of kidney slices or arterial tissue, which cannot be performed in living patients, non-invasive visualization techniques commonly known in the art, such as CT scans, ultrasound, or intravenous pyelography, are used to visualize the presence of calcification and its reduction in response to vector-based ENPP1 or ENPP3 delivery and expression in patients with CKD. Intravenous pyelography is an X-ray examination that uses a contrast agent, which acts as a dye to visualize the urinary tract and detect the presence of nephrocalcinosis. 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. Nephrocalcinosis is visualized by CT scans. CT scans collect x-ray images from various angles around the body to produce detailed cross-sectional images and three-dimensional images of the body's internal structures and organs. CT scans can also be used on arteries to detect the presence of calcification and subsequent reduction of calcification after treatment. A computer analyzes the radiation transmitted through the body to reconstruct images of internal structures and organs.

[0460] Physicians skilled in visualizing soft tissue calcification, cardiac calcification, and myocardial infarction attempt to treat subjects with CKD by administering AAV virions expressing human ENPP1 or human ENPP3. Physicians administer viral particles that deliver constructs of hENPP1 or hENPP3 and express the corresponding proteins under the control of an inducible promoter. Thus, physicians have the option to control the dosage (the amount of hENPP1 or hENPP3 expressed) based on the rate and extent of symptom improvement. Successful treatment is defined as improved kidney function, improved urinary creatine levels (normal creatine levels in urine are 40-278 mg / dL for men and 29-226 mg / dL for women) and urinary urea levels (normal urea levels in urine for adults are 26-43 g / 24 hours), normal serum creatine levels (normal serum creatinine range is 0.6-1.1 mg / dL for women and 0.7-1.3 mg / dL for men), and normal vitamin D levels (20 ng / ml-50 ng / ml is considered adequate for healthy people). The diagnosis is observed by a medical professional in the field by observing one or more positive symptoms such as: a vitamin D deficiency (levels below 12 ng / mL indicate vitamin D deficiency), normal blood urea nitrogen levels (BUN levels for a healthy adult are 7-20 mg / dL), weight gain, an increase in serum PPi levels (at least about 4-5 μm), a reduction in calcification of arterial tissue (a 25%, 50%, 70%, 90%, or 100% reduction), and / or a reduction in calcification of renal tubules visualized by non-invasive techniques such as CT or ultrasound scans.

[0461] Example 5 - Treatment of GACI using viral vectors 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 these examples, and similar results can be obtained using other ENPP1 or ENPP3 fusions of the invention.

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

[0463] Control cohort: In this experiment, the first cohort of ENPP1 wt mice, which served as the control group, was injected with AAV particles containing a null vector, and the second cohort of ENPP1 wt mice, which served as the control group, was injected with AAV particles containing a null vector. asj / asj A second cohort of mice is injected with AAV particles containing the null vector.

[0464] Cohort of ENPP1-treated mice:ENPP1 wt A third cohort of mice was injected with AAV particles engineered to express the ENPP1-Fc protein, resulting in the expression of ENPP1. asj / asj A fourth cohort of mice will be injected with AAV particles engineered to express the ENPP1-Fc protein.

[0465] Cohort of ENPP3-treated mice:ENPP1 wt A fifth cohort of mice was injected with AAV particles engineered to express the ENPP3-Fc protein, and ENPP1 asj / asj A sixth cohort of mice will be injected with AAV particles engineered to express the ENPP3-Fc protein. Wild-type mice will be maintained on a normal chow diet and will be injected with Enpp1. asj / asj Mice are fed a high phosphate Teklad diet.

[0466] Vector injection: After 2 weeks of age, approximately 1 x 10 12 ~1×10 15 vg / kg , preferably 1 x 10 13 ~1×10 14 vg / kgAll mice were injected retro-orbitally or by tail vein with either an empty "null" vector (control group) or carrying the NPP1 or NPP3 gene (study group).

[0467] Assays: Kidney histology, PPi levels and hematological parameters such as FGF-23 levels, vitamin D, parathyroid hormone (PTH) levels, serum / blood urea levels, blood urea nitrogen (BUN) levels, serum / blood creatine levels and plasma pyrophosphate (PPi) will be analyzed for each cohort as described in Examples 3 and 4.

[0468] Results: Untreated Enpp1 asj / asj Mice generally exhibit a loss of body weight and increased mortality. In contrast, mice treated with AAV expressing ENPP1 or ENPP3 proteins showed a significant decrease in the body weight and increased mortality. asj / asj Mice are expected to exhibit weight gain approaching the weight range of normal WT mice.

[0469] Enpp1 treated with null vector asj / asj Mice are expected to show calcification in the heart, aorta, and coronary arteries, as well as histological evidence of myocardial infarction in the free wall of the right ventricle, calcification in the coronary arteries, heart, ascending aorta, and descending aorta, myocardial cellular necrosis, and myocardial fibrosis in myocardial tissue adjacent to areas of coronary artery calcification. asj / asj Animals are expected to show no cardiac, arterial, or aortic calcification on histology or postmortem micro-CT. asj / asj Mice also exhibited calcification centered in the renal medulla, with intense and widespread calcification centered in the outer medulla and extending into the renal cortex. asj / asj The mice are expected to show reduced or absent renal mineral deposition in the tubular lumen and soft tissue vasculature, with a histology similar to that of healthy wild-type mice.

[0470] In addition to survival time, daily animal weights, and end-of-life histology, evaluate treatment response by postmortem high-resolution micro-CT scans for imaging vascular calcification, plasma PPi concentrations, and 99mTc PPi (99mPYP) uptake in untreated (null vector) Enpp1 mice. asj / asj In contrast to the dramatic calcification expected in the aorta, coronary arteries, and hearts of our cohort, WT and treated (vector expressing ENPP1 or ENPP3) Enpp1 asj / asj None of the mice were expected to have any vascular calcification via microCT. Furthermore, the mice treated with Enpp1 (vectors expressing ENPP1 or ENPP3) asj / asj The animals' serum PPi concentrations (5.2 μM) are expected to increase to WT levels (4.4 μM), significantly exceeding those 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, making it sensitive to areas of abnormally high bone remodeling activity. asj / asj Weekly serial imaging of animals showed that Enpp1 treated asj / asj It is expected that GACI mice will show greater uptake of 99mPYP in the head compared to control animals. Measurements will be performed 30-35 days and 50-65 days after administration of viral particles containing a null vector or a vector expressing ENPP1. Comparison of these experimental groups is expected to show that ENPP1-Fc or ENPP3-Fc treatment restored 99mPYP uptake in GACI mice to WT levels, indicating that ENPP1-Fc or ENPP3-Fc treatment increases extracellular PPi concentrations, thereby inhibiting ENPP1. asj / asj These observations suggest that uncontrolled mineralization of tissues, sinus hairs, and skull can be prevented in mice. These observations support the conclusion that viral particles containing vectors expressing ENPP1-Fc or ENPP3-Fc can be administered to mice.asj / asj It is expected that the mice will show no vascular calcification and have normal plasma PPi concentrations.

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

[0473] Treatment of Human Subjects Approximately 5 x 10 cells per subject in 1 x PBS at pH 7.4 can be delivered and expressed 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 / kgHuman patients suffering from GACI are treated by injecting a compound containing GACI. Successful treatment of GACI is observed by monitoring one or more of the aforementioned parameters through periodic blood and urine tests as described for the mouse model. Rather than histological analysis, which requires staining of kidney slices or arterial tissue, which cannot be performed in living patients, non-invasive visualization techniques are used instead, as described in Example 4.

[0474] Physicians skilled in visualizing soft tissue calcification, cardiac calcification, and myocardial infarction attempt to treat subjects with GACI by administering AAV virions expressing hENPP1 or hENPP3. Physicians administer viral particles delivering constructs encoding hENPP1 or hENPP3, and the vectors express the ENPP protein under the control of an inducible promoter. Physicians can adjust the dosage (the amount of hENPP1 or hENPP3 expressed) based on the rate and extent of symptomatic improvement. Successful treatment is observed by a medical professional in the field by observing one or more positive symptoms such as normal vitamin D levels (20 ng / ml to 50 ng / ml is considered adequate for healthy people; levels below 12 ng / ml indicate vitamin D deficiency), normal blood urea nitrogen levels (BUN levels for healthy adults are 7 to 20 mg / dL), weight gain, increased serum PPi levels (at least about 4 to 5 μm), reduced calcification of arterial tissue (25%, 50%, 70%, 90%, or 100% reduction), and / or reduced calcification of renal tubules visualized by non-invasive techniques such as CT or ultrasound scans.

[0475] Example 6 - Treatment of PXE using viral vectors 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 PXE. For illustrative purposes, ENPP1-Fc and ENPP3-Fc are used in these examples, and similar results can be obtained using other ENPP1 or ENPP3 fusions of the invention.

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

[0477] Control cohort: In this experiment, the first cohort of ENPP1 wt mice, which served as the control group, was injected with AAV particles containing a null vector, and the second cohort of ABCC6 mice, which served as the control group, was injected with AAV particles containing a null vector. - / - A second cohort of mice is injected with AAV particles containing the null vector.

[0478] Cohort of ENPP1-treated mice:ENPP1 wt A third cohort of mice was injected with AAV particles engineered to express the ENPP1-Fc protein, ABCC6 - / - A fourth cohort of mice will be injected with AAV particles engineered to express the ENPP1-Fc protein.

[0479] Cohort of ENPP3-treated mice:ENPP1 wt A fifth cohort of mice was injected with AAV particles engineered to express the ENPP3-Fc protein, ABCC6 - / - A sixth cohort of mice will be injected with AAV particles engineered to express the ENPP3-Fc protein. Wild-type mice will be maintained on a normal chow diet and will be bred to express ABCC6. - / - Mice are fed a high phosphate Teklad diet.

[0480] Vector injection: Approximately 1 x 10 in PBS pH 7.4 per mouse after 2 weeks of age. 12 ~1×10 15 vg / kg , preferably 1 x 10 13 ~1×10 14 vg / kg All mice were injected retro-orbitally or by tail vein with either an empty "null" vector (control group) or carrying the NPP1 or NPP3 gene (study group).

[0481] Assays: Kidney histology, PPi levels and hematological parameters such as FGF-23 levels, vitamin D, parathyroid hormone (PTH) levels, serum / blood urea levels, blood urea nitrogen (BUN) levels, serum / blood creatine levels and plasma pyrophosphate (PPi) will be analyzed for each cohort as described in Examples 3 and 4.

[0482] Results: Untreated ABCC6- / - mice are generally expected to exhibit reduced body weight and increased mortality. In contrast, ABCC6- / - mice treated with AAVs expressing ENPP1 or ENPP3 proteins are expected to exhibit increased body weight approaching the weight range of normal WT mice. ABCC6- / - mice treated with null vectors are expected to exhibit calcification in the heart, aorta, and coronary arteries, as well as histological evidence of myocardial infarction in the free wall of the right ventricle, calcification in the coronary arteries, heart, ascending aorta, and descending aorta, myocardial cellular necrosis, and myocardial fibrosis in myocardial tissue adjacent to areas of coronary artery calcification. In contrast, ABCC6- / - animals treated with vectors expressing ENPP1-Fc or ENPP3-Fc are expected to demonstrate an absence of calcification in the heart, arteries, or aorta on histology or postmortem microCT. Enpp1 treated with null vectors is expected to exhibit increased cardiac, aortic, and aortic calcification. asj / asj Mice also exhibited intense and widespread calcification centered in the renal medulla with extension into the renal cortex. In contrast, mice treated with viral vector-based expression of ENPP1 or ENPP3 showed no significant changes in the renal medulla. asj / asjThe mice are expected to show reduced or absent renal mineral deposition in the tubular lumen and soft tissue vasculature, with a histology similar to that of healthy wild-type mice.

[0483] Assess the treatment response by postmortem high-resolution micro-CT scans to image vascular calcification and plasma PPi concentrations, in addition to survival time, daily animal weights, and end-of-life histology in WT and treated (vector expressing ENPP1) ABCC6 mice. - / - None of these were untreated (null vector) ABCC6 - / - In contrast to the dramatic calcification expected to be seen in the aorta, coronary arteries, and heart of this cohort, mice were not expected to have any vascular calcification via microCT. Furthermore, mice treated with ABCC6 (vector expressing ENPP1) - / - Serum PPi concentrations in treated animals (5.2 μM) increased to WT levels (4.4 μM), whereas those in untreated ABCC6 - / - It is expected that the level of ATP will be significantly higher than that of ATP (0.5 μM).

[0484] Untreated ABCC6 - / - Mice exhibit significant increases in serum inorganic phosphorus (pI), PTH, and FGF23 levels when compared to those of healthy wild-type mice (normal levels of PP are approximately 2-4 μM; PTH is approximately 10-65 ng / L; median FGF23 level is 13 RU / ml, normal FGF23 levels range from 5-210 RU / ml; normal vitamin D levels are 20 ng / ml-50 ng / ml), but also reduced 1,25(OH)2-vitamin D levels, and lower PPi levels (approximately 0.5 μM) are expected. In contrast, treated ABCC6 mice exhibited significantly increased serum inorganic phosphorus (pI), increased PTH, and FGF23 levels compared to healthy wild-type mice (normal levels of PP are approximately 2-4 μM; PTH is approximately 10-65 ng / L; median FGF23 level is 13 RU / ml, normal FGF23 levels range from 5-210 RU / ml; normal vitamin D levels are 20 ng / ml-50 ng / ml). - / - Mice are expected to show elevated PPi levels (approximately 4-5 μM), which is consistent with untreated ABCC6 - / -This is expected to be higher than the PPi level (about 0.5 μM) seen in mice. Therefore, one skilled in the art can determine the therapeutic efficacy of vector-based ENPP1 or ENPP3 in treating PXE by observing one or more factors such as a reduction in renal soft tissue and coronary artery calcification visualized through histological analysis (25%, 50%, 70%, 90%, or 100% reduction), an increase in serum PPi levels from blood analysis, normalization of vitamin D levels, a reduction in FGF23 levels and normalization of PTH levels to the normal range, an increase in survival time, and improved kidney function observed by an increase in urinary urea and creatine along with increased body weight gain.

[0485] Treatment of human subjects: Approximately 5 x 10 cells per subject in 1 x PBS, pH 7.4, can be used to deliver and express ENPP1 or ENPP3. 11 ~5×10 15 vg / kg In some embodiments, approximately 1×10 in 1×PBS at pH 7.4 12 ~1×10 15 vg / kg Human patients suffering from PXE are treated by intravenous injection containing PXE. Successful treatment of PXE is observed by monitoring one or more of the aforementioned parameters through periodic blood and urine tests as described for the mouse model. Rather than histological analysis, which requires staining of kidney slices or arterial tissue, which cannot be performed in living patients, non-invasive visualization techniques are used instead, as described in Example 4.

[0486] Physicians skilled in visualizing soft tissue calcification, cardiac calcification, and myocardial infarction can attempt to treat subjects with PXE by administering AAV virions expressing ENPP1 or ENPP3. Physicians can also use viral particles that deliver ENPP1 or ENPP3 constructs and express the corresponding proteins under the control of an inducible promoter. Thus, physicians have the option to control the dosage (the amount of ENPP1 or ENPP3 expressed) based on the rate and extent of symptom improvement. Successful treatment and appropriate dosage are readily inferred by medical professionals in the field by observing one or more positive symptoms, such as normal vitamin D levels (20 ng / ml to 50 ng / mL is considered adequate for healthy people; levels below 12 ng / mL indicate vitamin D deficiency), disappearance or reduction in the size and / or number of pigmented streaks, reduction or absence of retinal hemorrhages, normal blood urea nitrogen levels (BUN levels for healthy adults are 7 to 20 mg / dL), weight gain, increased serum PPi levels (at least about 4 to 5 μm), reduction in calcification of arterial tissue, connective tissue (25%, 50%, 70%, 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 viral vectors expressing human ENPP1 or ENPP3 The following examples provide AAVs expressing human ENPP1 or ENPP3 that 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 these examples, and similar results can be obtained using other ENPP1 or ENPP3 fusions of the invention.

[0488] AAV virions expressing ENPP1-Fc protein or ENPP3-Fc protein are produced according to Example 1 and administered to tiptoe 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 104(6):492-7). Six sets of mice are used for treatment with ENPP1 and ENPP3.

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

[0490] Cohort of ENPP1-treated mice:ENPP1 wt A third cohort of mice will be injected with AAV particles engineered to express the ENPP1-Fc protein, and a fourth cohort of ttw mice will be injected with AAV particles engineered to express the ENPP1-Fc protein.

[0491] Cohort of ENPP3-treated mice:ENPP1 wt A fifth cohort of mice will be injected with AAV particles engineered to express the ENPP3-Fc protein, and a sixth cohort of ttw mice will be injected with AAV particles engineered to express the ENPP3-Fc protein. Wild-type mice will be maintained on a normal chow diet, while ttw mice will be fed a high-phosphate Teklad diet.

[0492] Vector injection: After 2 weeks of age, approximately 1 x 10 12 ~1×10 15 vg / kg , preferably 1 x 10 13 ~1×10 14 vg / kgAll mice were injected retro-orbitally or by tail vein with either an empty "null" vector (control group) or carrying the NPP1 or NPP3 gene (study group).

[0493] Assays: Kidney histology, PPi levels and hematological parameters such as FGF-23 levels, vitamin D, parathyroid hormone (PTH) levels, serum / blood urea levels, blood urea nitrogen (BUN) levels, serum / blood creatine levels and plasma pyrophosphate (PPi) will be analyzed for each cohort as described in Examples 3 and 4.

[0494] Results: Untreated ttw mice generally exhibit reduced body weight, spinal thickening, lethargy, and increased mortality. In contrast, ttw mice treated with AAVs expressing ENPP1 or ENPP3 proteins are expected to exhibit increased body weight approaching the weight range of normal WT mice, normal alertness, and reduced spinal thickness approaching that of wild-type mice. ttw mice treated with null vectors are expected to exhibit calcification in the heart, aorta, and coronary arteries, as well as histological evidence of myocardial infarction in the free wall of the right ventricle, calcification in the coronary arteries, heart, ascending aorta, and descending aorta, myocardial cellular necrosis, and myocardial fibrosis in myocardial tissue adjacent to areas of coronary artery calcification. In contrast, ttw animals treated with vectors expressing ENPP1-Fc or ENPP3-Fc are expected to demonstrate an absence of calcification in the heart, arteries, or aorta on histology or postmortem microCT scans. Null vector-treated ttw mice will also exhibit calcification centered in the renal medulla, with intense and widespread calcification centered in the outer medulla and extending into the renal cortex. In contrast, ttw mice treated with viral vector-based expression of ENPP1 or ENPP3 are expected to exhibit reduced or absent renal mineral deposition in the tubular lumen and reduced calcification of the spine and soft tissue vasculature, with a histology similar to that of healthy wild-type mice.

[0495] In addition to survival time, daily animal weights, and terminal histology, treatment response will be assessed by postmortem high-resolution micro-CT scans to image vascular calcification and plasma PPi concentrations. Neither WT nor treated (ENPP1-expressing vector) ttw mice 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) ttw cohort. Furthermore, treated (ENPP1-expressing vector) ttw mice are expected to have any vascular calcification via micro-CT. - The animals' serum PPi concentrations (5.2 μM) are expected to rise to WT levels (4.4 μM), significantly above those in untreated ttw (0.5 μM).

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

[0497] Treatment of human subjects: Approximately 5 x 10 cells per subject in 1 x PBS at pH 7.4 can be delivered and expressed 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 / kg Human patients suffering from OPLL are treated by intravenous injection containing OPLL. Successful treatment of OPLL is observed by monitoring one or more of the aforementioned parameters through regular blood and urine tests as described for the mouse model. Rather than histological analysis, which requires staining of kidney slices or arterial tissue, which cannot be performed in living patients, non-invasive visualization techniques are used instead, as described in Example 4.

[0498] Physicians skilled in visualizing soft tissue calcification, cardiac calcification, and myocardial infarction can attempt to treat subjects suffering from OPLL by administering AAV virions expressing hENPP1 or hENPP3. In some embodiments, physicians use viral particles that deliver constructs of hENPP1 or hENPP3 and express the corresponding proteins under the control of an inducible promoter. Thus, physicians have the option to control the dosage (the amount of hENPP1 or hENPP3 expressed) based on the rate and extent of symptomatic improvement. Successful treatment and appropriate dosage are readily inferred by medical professionals in the field by observing one or more positive symptoms, such as normal vitamin D levels (20 ng / ml to 50 ng / ml is considered adequate for healthy people; levels below 12 ng / ml indicate vitamin D deficiency), normal blood urea nitrogen levels (BUN levels for healthy adults are 7 to 20 mg / dL), weight gain, increased serum PPi levels (at least about 4 to 5 μm), reduced calcification of arterial tissue (25%, 50%, 70%, 90%, or 100% reduction), reduced spinal thickness and pain sensation, and reduced spinal canal narrowing 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 viral vectors expressing ENPP1 or ENPP3 The following examples provide AAVs expressing ENPP1 or ENPP3 that are expected to be effective in treating conditions associated with osteopenia and / or osteomalacia. For illustrative purposes, ENPP1-Fc and ENPP3-Fc are used in these examples, and similar results can be obtained using other ENPP1 or ENPP3 fusions of the invention.

[0500] AAV virions expressing ENPP1-Fc or ENPP3-Fc proteins are produced according to Example 1 and administered to tiptoe walking (ttw) mice, a mouse model of osteoarthritis (Bertrand, et al., 2012, Annals Rheum. Diseases 71(7):1249-53). Six sets of mice are used for treatment with ENPP1 and ENPP3. ENPP1 knockout mice (ENPP1), which also serve as a model of osteopenia in addition to GACI (Mackenzie, et al., 2012, PloS one 7(2):e32177), are used. KO ) and repeat the same experiment.

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

[0502] Cohort of ENPP1-treated mice:ENPP1 wt A third cohort of mice was injected with AAV particles engineered to express the ENPP1-Fc protein, designated ttw mice (or ENPP1 KO A fourth cohort of mice will be injected with AAV particles engineered to express the ENPP1-Fc protein.

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

[0504] Vector injection: After 2 weeks of age, approximately 1 x 10 12 ~1×10 15 vg / kg , preferably 1 x 10 13 ~1×10 14 vg / kg All mice were injected retro-orbitally or by tail vein with either an empty "null" vector (control group) or carrying the NPP1 or NPP3 gene (study group).

[0505] Assays: Kidney histology, PPi levels and hematological parameters such as FGF-23 levels, vitamin D, parathyroid hormone (PTH) levels, serum / blood urea levels, blood urea nitrogen (BUN) levels, serum / blood creatine levels and plasma pyrophosphate (PPi) will be analyzed for each cohort as described in Examples 3 and 4.

[0506] Histology, histomorphometry and microCT: Bone analysis is performed according to the protocol described in Example 3.

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

[0508] Results: Untreated ttw (or ENPP1 KO ) mice generally exhibit reduced body weight, lethargy, decreased cortical and trabecular bone mass, mineralization of cartilage and ligaments, reduced bone density in the femur and tibia, and increased mortality compared to wild-type mice. In contrast, ttw (or ENPP1) mice treated with AAVs expressing ENPP1 or ENPP3 proteins exhibited significantly reduced body weight, lethargy, decreased cortical and trabecular bone mass, reduced cartilage and ligament mineralization, and decreased bone density in the femur and tibia. KO ) mice are expected to exhibit increased body weight approaching the weight range of normal WT mice, normal alertness, increased bone mineral content, improved cortical and trabecular bone mass, increased bone strength and flexibility. KO) mice are expected to show calcification in the heart, aorta, and coronary arteries, as well as histological evidence of myocardial infarction in the free wall of the right ventricle, calcification in the coronary arteries, heart, ascending aorta, and descending aorta, myocardial cellular necrosis, and myocardial fibrosis in myocardial tissue adjacent to areas of coronary artery calcification. In contrast, ttw (or ENPP1) mice treated with vectors expressing ENPP1-Fc or ENPP3-Fc are expected to show histological evidence of myocardial infarction in the free wall of the right ventricle, calcification in the coronary arteries, heart, ascending aorta, and descending aorta, myocardial necrosis, and myocardial fibrosis in myocardial tissue adjacent to areas of coronary artery calcification. KO ) Animals are expected to show no cardiac, arterial, or aortic calcification on histology or postmortem micro-CT. KO ) mice show intense and widespread calcification centered in the renal medulla with extension into the renal cortex. In contrast, ttw (or ENPP1) mice treated with viral vector-based expression of ENPP1 or ENPP3 show calcification centered in the renal medulla. KO ) mice are expected to show a histology similar to that of healthy wild-type mice, with reduced or absent renal mineral deposits in the tubular lumen and reduced calcification of the spinal and soft tissue vasculature.

[0509] In addition to survival time, daily animal weight, and end-of-life histology, treatment response is assessed by postmortem high-resolution micro-CT scanning to image vascular calcification and plasma PPi concentrations. WT or treated (vector expressing ENPP1) ttw (or ENPP1) mice are evaluated. KO ) were not treated with untreated (null vector) ttw (or ENPP1 KO ) cohort, are not expected to have any vascular calcification via microCT, in contrast to the dramatic calcification expected to be seen in the aorta, coronary arteries, and heart. Furthermore, treated (vector expressing ENPP1) ttw (or ENPP1 KO ) animals, serum PPi concentrations (5.2 μM) increased to WT levels (4.4 μM), compared with untreated ttw (or ENPP1 KO ) level (0.5 μM).

[0510] Untreated ttw (or ENPP1 KO) mice show significantly increased serum inorganic phosphorus (pI), increased PTH and FGF23 levels when compared to healthy wild-type mice (normal levels of PP are approximately 2-4 μM; for PTH, approximately 10-65 ng / L; median FGF23 level is 13 RU / ml, normal FGF23 levels range from 5-210 RU / ml; normal vitamin D levels are 20 ng / ml-50 ng / ml), but reduced 1,25(OH)2-vitamin D levels, and lower PPi levels (approximately 0.5 μM) are also expected. In contrast, treated ttw (or ENPP1) mice show significantly increased serum inorganic phosphorus (pI), increased PTH and FGF23 levels when compared to healthy wild-type mice (normal levels of PP are approximately 2-4 μM; for PTH, approximately 10-65 ng / L; median FGF23 level is 13 RU / ml, normal FGF23 levels range from 5-210 RU / ml; normal vitamin D levels are 20 ng / ml-50 ng / ml). KO ) mice are expected to show elevated PPi levels (approximately 4-5 μM), which is consistent with untreated ttw (or ENPP1 KO ) mice (approximately 0.5 μM). Thus, by observing one or more factors such as a reduction in renal soft tissue and coronary artery calcification visualized through histological analysis (25%, 50%, 70%, 90%, or 100% reduction), an increase in serum PPi levels from blood analysis, normalization of vitamin D levels, a reduction in FGF23 levels and normalization of PTH levels to the normal range, improved long bone strength, increased bone mineral density, improved cortical bone thickness and trabecular bone mass, increased survival time, and improved kidney function observed by increased urinary urea and creatine along with increased body weight gain, one skilled in the art can determine the therapeutic efficacy of vector-based ENPP1 or ENPP3 in treating osteopenia or osteomalacia or osteoarthritis.

[0511] Treatment of human subjects: Approximately 5 x 10 cells per subject in 1 x PBS at pH 7.4 can be delivered and expressed 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 / kgHuman patients suffering from osteopenia, osteomalacia, or osteoarthritis are treated by intravenous injection containing the compound. Successful treatment of osteopenia, osteomalacia, or osteoarthritis is observed by monitoring one or more of the above parameters through periodic blood and urine tests for bone strength and bone density, as described for the mouse model. Rather than histological analysis, which requires staining of kidney slices or arterial tissue, which cannot be performed in living patients, non-invasive visualization techniques are used instead, as described in Example 4.

[0512] Similarly, patients undergo periodic bone mineral 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 mineral density score obtained from one of these methods provides an indication of the condition and progression achieved after treatment. A T-score of 1.0 or greater is considered normal bone mineral 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. A gradual improvement in T-score is expected in patients treated with ENPP1 or ENPP3 of the present invention.

[0513] Physicians skilled in visualizing soft tissue calcification, cardiac calcification, and bone density visualization attempt to treat subjects suffering from osteopenia or osteoarthritis by administering AAV virions expressing hENPP1 or hENPP3. In some embodiments, physicians use viral particles that deliver constructs of hENPP1 or hENPP3 and express the corresponding proteins under the control of an inducible promoter. Thus, physicians have the option to control the dosage (the amount of hENPP1 or hENPP3 expressed) based on the speed and extent of symptom improvement. Successful treatment and appropriate dosage are readily inferred by medical professionals in the field by observing one or more positive symptoms, such as normal vitamin D levels (20 ng / ml to 50 ng / ml is considered adequate for healthy people; levels below 12 ng / ml indicate vitamin D deficiency), normal bone mineral density (T-score of ≥ -1), normal blood urea nitrogen levels (BUN levels for healthy adults are 7 to 20 mg / dL), weight gain, increased serum PPi levels (at least about 4 to 5 μm), reduced calcification of arterial tissue (25%, 50%, 70%, 90%, or 100% reduction), and improved 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 viral vectors expressing ENPP1 or ENPP3 The following examples provide AAVs expressing ENPP1 or ENPP3 that are expected to be effective in treating conditions associated with ADHR-2 or ARHR-2 or XLH. For illustrative purposes, ENPP1-Fc and ENPP3-Fc are used in these examples, and similar results can be obtained using other ENPP1 or ENPP3 fusions of the invention.

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

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

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

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

[0519] Vector injection: Approximately 1 x 10 in PBS pH 7.4 per mouse after 2 weeks of age. 12 ~1×101 5 vg / kg , preferably 1 x 10 13 ~1×10 14 vg / kg All mice were injected retro-orbitally or by tail vein with either an empty "null" vector (control group) or carrying the NPP1 or NPP3 gene (study group).

[0520] Assays: Kidney histology, PPi levels and hematological parameters such as FGF-23 levels, vitamin D, parathyroid hormone (PTH) levels, serum / blood urea levels, blood urea nitrogen (BUN) levels, serum / blood creatine levels and plasma pyrophosphate (PPi) will be analyzed for each cohort as described in Examples 3 and 4.

[0521] Histology, histomorphometry and microCT: Bone analysis is performed according to the protocol described in Example 3.

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

[0523] Results: Untreated HYP (or ENPP1 asj / asj ) mice generally exhibit reduced body weight, lethargy, reduced cortical and trabecular bone mass, mineralization of cartilage and ligaments, reduced bone density in the femur and tibia, and increased mortality compared to wild-type mice. In contrast, HYP (or ENPP1) mice treated with AAVs expressing ENPP1 or ENPP3 proteins exhibited significantly reduced body weight, lethargy, reduced cortical and trabecular bone mass, reduced cartilage and ligament mineralization, reduced bone density in the femur and tibia, and increased mortality. asj / asj ) mice are expected to exhibit increased body weight approaching the weight range of normal WT mice, normal alertness, increased bone mineral content, improved cortical and trabecular bone mass, increased bone strength and flexibility. asj / asj) mice are expected to show calcification in the heart, aorta, and coronary arteries, as well as histological evidence of myocardial infarction in the free wall of the right ventricle, calcification in the coronary arteries, heart, ascending aorta, and descending aorta, myocardial cellular necrosis, and myocardial fibrosis in myocardial tissue adjacent to areas of coronary artery calcification. In contrast, HYP (or ENPP1) mice treated with vectors expressing ENPP1-Fc or ENPP3-Fc are expected to show histological evidence of myocardial infarction in the free wall of the right ventricle, calcification in the coronary arteries, heart, ascending aorta, and descending aorta, myocardial necrosis, and myocardial fibrosis in myocardial tissue adjacent to areas of coronary artery calcification. asj / asj ) mice are expected to show no cardiac, arterial, or aortic calcification in histology or postmortem micro-CT scans. asj / asj ) mice show intense and widespread calcification centered in the renal medulla with extension into the renal cortex. In contrast, HYP (or ENPP1) mice treated with viral vector-based expression of ENPP1 or ENPP3 show calcification centered in the renal medulla. asj / asj ) mice are expected to show a histology similar to that of healthy wild-type mice, with reduced or absent renal mineral deposits in the tubular lumen and reduced calcification of the spinal and soft tissue vasculature.

[0524] In addition to survival time, daily animal weight, and end-of-life histology, treatment response is assessed by postmortem high-resolution micro-CT scanning to image vascular calcification and plasma PPi concentrations. WT or treated (vector expressing ENPP1) HYP (or ENPP1) mice are evaluated. asj / as j) None of the mice were treated with untreated (null vector) HYP (or ENPP1 asj / asj ) cohort, are not expected to have any vascular calcification via microCT, in contrast to the dramatic calcification expected to be seen in the aorta, coronary arteries, and heart. Furthermore, treated (vector expressing ENPP1) HYP (or ENPP1 asj / asj ) mice, serum PPi concentrations (5.2 μM) increased to WT levels (4.4 μM), whereas untreated HYP (or ENPP1 asj / asj ) level (0.5 μM).

[0525] Untreated HYP (or ENPP1asj / asj ) mice show significantly increased serum inorganic phosphorus (pI), increased PTH and FGF23 levels when compared to healthy wild-type mice (normal levels of PP are approximately 2-4 μM; for PTH, approximately 10-65 ng / L; median FGF23 level is 13 RU / ml, normal FGF23 levels range from 5-210 RU / ml; normal vitamin D levels are 20 ng / ml-50 ng / ml), but reduced 1,25(OH)2-vitamin D levels and lower PPi levels (approximately 0.5 μM) are also expected. In contrast, treated HYP (or ENPP1) mice show significantly increased serum inorganic phosphorus (pI), increased PTH and FGF23 levels when compared to healthy wild-type mice (normal levels of PP are approximately 2-4 μM; for PTH, approximately 10-65 ng / L; median FGF23 level is 13 RU / ml, normal FGF23 levels range from 5-210 RU / ml; normal vitamin D levels are 20 ng / ml-50 ng / ml). asj / asj ) mice are expected to show elevated PPi levels (approximately 4-5 μM), which is consistent with untreated HYP (or ENPP1 asj / asj ) mice, is expected to be higher than the PPi levels (about 0.5 μM) seen in mice. Thus, one skilled in the art can determine the therapeutic efficacy of vector-based ENPP1 or ENPP3 in treating ADHR-2 or ARHR-2 or XLH by observing one or more factors such as reduced calcification in renal soft tissue and coronary arteries visualized through histological analysis (25%, 50%, 70%, 90%, or 100% reduction), increased serum PPi levels from blood analysis, normalization of vitamin D levels, reduced FGF23 levels and normalization of PTH levels to the normal range, improved long bone strength, increased bone mineral density, improved cortical bone thickness and trabecular bone mass, increased survival time, and improved kidney function observed by increased urinary urea and creatine along with increased body weight gain.

[0526] Treatment of human subjects: Approximately 5 x 10 cells per subject in 1 x PBS at pH 7.4 can be delivered and expressed 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 / kgHuman patients suffering from ADHR-2, ARHR-2, or XLH are treated by intravenous injection containing a compound containing 2-aminopropyl methylcellulose (MAMM). Successful treatment of ADHR-2, ARHR-2, or XLH is observed by monitoring one or more of the aforementioned parameters through periodic blood and urine tests for bone strength and bone density, as described for the mouse model. Rather than histological analysis, which requires staining of kidney slices or arterial tissue, which cannot be performed in living patients, non-invasive visualization techniques, as described in Example 4, are used instead.

[0527] Similarly, patients undergo periodic bone mineral 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 mineral density score obtained from one of these methods provides an indication of the condition and progression achieved after treatment. A T-score of 1.0 or greater is considered normal bone mineral 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. A gradual improvement in T-score is expected in patients treated with ENPP1 or ENPP3 of the present invention.

[0528] Physicians skilled in visualizing soft tissue calcification, cardiac calcification, and bone density visualization attempt to treat subjects suffering from ADHR-2, ARHR-2, or XLH by administering AAV virions expressing hENPP1 or hENPP3. In some embodiments, physicians use viral particles that deliver constructs of hENPP1 or hENPP3 and express the corresponding proteins under the control of an inducible promoter. Thus, physicians have the option to control the dosage (the amount of hENPP1 or hENPP3 expressed) based on the speed and extent of symptomatic improvement. Successful treatment and appropriate dosage are readily inferred by medical professionals in the field by observing one or more positive symptoms, such as normal vitamin D levels (20 ng / ml to 50 ng / ml is considered adequate for healthy people; levels below 12 ng / ml indicate vitamin D deficiency), normal bone mineral density (T-score of ≥ -1), normal blood urea nitrogen levels (BUN levels for healthy adults are 7 to 20 mg / dL), weight gain, increased serum PPi levels (at least about 4 to 5 μm), reduced calcification of arterial tissue (25%, 50%, 70%, 90%, or 100% reduction), and improved 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 contained five adult mice. The first cohort served as the "control group" and was injected with saline. The second cohort served as the "low-dose group" and was injected with 1e 13 The low-dose group was injected with AAV vectors at a concentration of 1e vg / kg. The third cohort was the "high-dose group," with 1e 14The high-dose group was injected with an AAV vector at a concentration of 0.05 mg / kg. The process of generating viral particles from the AAV construct and injecting recombinant AAV viral particles containing the ENPP1 fusion protein into normal mice is shown schematically in Figure 4. Blood was collected from all cohorts of mice on days 7, 28, and 56 post-injection to collect plasma and serum.

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

[0531] First, collected samples were assayed to determine the activity level of ENPP1 using the colorimetric substrate, p-nitrophenylthymidine 5'-monophosphate (Sigma). Plasma samples were incubated with 1 mg / ml p-nitrophenylthymidine 5'-monophosphate in 1% Triton, 200 mM Tris, pH 8.0 buffer for 1 hour. After 1 hour, 100 mM NaOH was added to stop the reaction, and absorbance was measured at 405 nm. Specific activity was determined according to the following assay protocol published 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 demonstrate a dose-dependent increase in ENPP1 activity after injection. Normal mouse plasma was used as a reference standard to normalize ENPP1 activity levels, and one-way ANOVA was used for statistical analysis. Figure 5 shows that ENPP1 activity levels were higher in the low-dose group compared to those in the control group. Similarly, ENPP1 activity levels were higher in the high-dose group compared to those in the low-dose and control groups. Among the low- and high-dose cohorts, ENPP1 activity was stable in plasma samples from days 7 to 56 in the high-dose group, whereas there was a slight decrease in ENPP1 activity from days 28 to 56 in the low-dose group.

[0533] Samples were then assayed to determine the concentration of ENPP1 using a sandwich ELISA assay with ENPP1 polyclonal antibody obtained from Sigma (SAB1400199). 96-well clear flat-bottom polystyrene high-binding microplates (Corning catalog no. 9018), BSA (Sigma #7906), 10x Dulbecco's phosphate-buffered saline (DPBS) (Quality Biological catalog no. 119-068-101), Tween-20 (Sigma catalog no. P2287), anti-ENPP1 antibody produced in mouse (Sigma-Aldrich catalog no. SAB1400199), Sure Blue TMB Microwell Peroxidase Substrate (1-component) (KPL product no. 52-00-01), 2N sulfuric acid (BDH product no. BDH7500-1), MilliQ water, C57BL / 6 mouse plasma NaHep Pooled Gender (BioIVT catalog no. MSE01PLNHPNN), and mouse serum (BIO IVT elevating Science catalog no. MSE01SRMPNN) were used for the ELISA assay.

[0534] A standard curve for ENPP1-Fc protein was prepared by the following standard procedures known in the art. Briefly, serial dilutions of ENPP1-Fc protein ranging from 2 mg / ml to 30 ng / ml were prepared. A 96-well plate was first coated with an overnight coating solution containing 1 μg / 1 mL of ENPP1 capture antibody in 1x PBS. The wells were then incubated with 5% BSA in PBS for 1 hour and then washed with post-blocking solution. ENPP1 diluted samples were added to the coated 96-well plate, which was then incubated for 1.5 hours. After incubation, the wells were washed four times with 300 μl of 0.05% PBST. The washed wells were then treated with 100 μL / well of detection HRP antibody conjugate and incubated for 1 hour. After incubation with the HRP antibody conjugate, the wells were washed four times with 300 μl of 0.05% PBST. The washed wells were then treated with 100 μl of TMB microwell peroxidase substrate per well and incubated in the dark for 30 minutes. The wells were then washed four times with 300 μl of 0.05% 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 constructed using the absorbance readings and the corresponding concentrations of the ENPP serially diluted samples.

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

[0536] Samples were also assayed to determine plasma PPi concentrations using a sulfurylase assay. ATP sulfurylase (NEB-M0394L, lot number: 10028529), adenosine 5'-phosphosulfate (APS; Santa Cruz, sc-214506), PPi: 100 μM 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-sulfurylase assay. PPi standards (0.125–4 μM) were prepared in water using serial dilutions. PPi in PPi standards and filtered plasma samples was converted to ATP by ATP sulfurylase in the presence of excess adenosine 5'-phosphosulfate (APS). Samples (15 μl) were treated with 5 μl of a mixture containing 8 mM CaCl2, 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, followed by inactivation of ATP sulfurylase at 90°C for 10 minutes. ATP generated was measured using BactiterGlo (Promega G8231) by mixing 20 μl of treated sample or standard with 20 μl of BactiterGlo reagent. Bioluminescence was then measured using a microplate reader, and the amount of PPi generated in each sample was subsequently determined from a calibration curve.

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

[0538] In a related experiment, a single dose of 1e14 vg / kg of AAV viral vector or vehicle control (no AAV vector) was administered intravenously to 5-6 week-old C57 / Bl male mice. Animals were administered GK1.5 (40 μg / mouse 1 day prior to viral vector or vehicle administration, then 25 μg / mouse every 7 days thereafter until study completion). The AAV viral vector was engineered to express a fusion protein of ENPP1 and IgG Fc, similar to the polypeptide described in Example 10, except that the ENPP and IgG Fc portions of the fusion protein were connected by the following linker amino acid sequence: GGGGS. Mice administered with the AAV viral vector exhibited higher levels of ENPP1 enzymatic activity than vehicle-only controls when measured over a period of approximately 40 days.

[0539] Example 11 - Analysis of ENPP1 concentration and activity levels in a mouse virus model 112 days after viral administration Three cohorts of normal mice were used in this experiment. Each cohort contained five adult mice. The first cohort served as the "control group" and was injected with saline. The second cohort served as the "low-dose group" and was injected with 1e 13 The low-dose group was injected with AAV vectors at a concentration of 1e vg / kg. The third cohort was the "high-dose group," with 1e 14The high-dose group was injected with AAV vectors at a concentration of 1000 mg / kg. The process of generating viral particles from the AAV construct and injecting recombinant AAV viral particles containing the ENPP1 fusion protein into normal mice is shown schematically in Figure 4. Blood was collected from all cohorts of mice on days 7, 28, 56, and 112 post-injection to collect plasma and serum.

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

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

[0542] The samples were then assayed to determine the concentration of ENPP1 using a sandwich ELISA assay with 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 on days 7, 28, 56, and 112 post-virus injection. The absorbance generated in each plasma sample was correlated with a standard curve of ENPP1-Fc to determine the concentration of ENPP1-Fc in the plasma samples.

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

[0544] Other embodiments From the foregoing, it will be apparent that it is within the scope of the present invention that variations and modifications can be made to the invention described herein to adapt it to a variety of uses and conditions, including the use of different viral vectors with different promoters or enhancers or different signal sequences to express functional variants of ENPP1 or ENPP3 or combinations thereof in different cell types, as known in the art, to treat any disease characterized by the presence of pathological calcification or ossification. Other embodiments of the present 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 that definition of that variable as any single element or combination (or subcombination) 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 portion thereof.

[0546] All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated 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 pharmaceutical composition for treating a disease of pathological calcification by increasing ectonucleotide pyrophosphatase / phosphodiesterase-1 (ENPP1) levels and reducing pathological calcification in a subject in need thereof, the pharmaceutical composition comprising a therapeutically effective amount of an adeno-associated virus (AAV) vector encoding a recombinant ENPP1-Fc fusion polypeptide, the AAV vector comprising a liver-specific promoter and having the AAV8 serotype.

2. The pharmaceutical composition of claim 1, wherein the disease is a disease caused by a deficiency of ENPP1 protein associated with a loss-of-function mutation in the NPP1 gene, or the disease is a disease caused by a loss-of-function mutation in the ABCC6 gene in the subject.

3. The AAV vector is 1×10 １２ ~1 x 10 １５ vg / kg or 1 x 10 １３ ~1 x 10 １４ can be administered to a subject at a dosage of 1000 mg / kg; The AAV vector is 5×10 １１ ~5 x 10 １５ can be administered to a subject at a dosage of 100 mg / kg or The AAV vector is 1×10 １２ ~1 x 10 １５ can be administered to a subject at a dosage of 1000 mg / kg; The pharmaceutical composition of claim 1 or 2, wherein administration of the AAV vector 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.

4. the ENPP1-Fc fusion polypeptide comprises an ENPP1 polypeptide fused to Fc, and the ENPP1 polypeptide comprises residues 99 to 925 (Pro Ser Cys to Gln Glu Asp) of SEQ ID NO: 1; the ENPP1 polypeptide comprises residues 1 to 827 (Pro Ser Cys to Gln Glu Asp) of SEQ ID NO: 92; or 4. The pharmaceutical composition of claim 1, wherein the ENPP1 polypeptide comprises residues 1 to 833 (Phe Thr 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.

5. 5. The pharmaceutical composition of claim 1, 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.

6. 6. The pharmaceutical composition of claim 1, further comprising a pharmaceutically acceptable carrier.

7. 7. The pharmaceutical composition of claim 6, 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).

8. 7. The pharmaceutical composition of claim 6 for treating a subject having a deficiency of the ENPP1 protein.

9. The pharmaceutical composition of claim 6 for treating a subject having a loss-of-function mutation in the ABCC6 gene in said subject.

10. The pharmaceutical composition is 1×10 １２ ~1 x 10 １５ can be administered at a dosage of vg / kg of subject; The pharmaceutical composition is 1×10 １３ ~1 x 10 １４ can be administered at a dosage of vg / kg of subject; The pharmaceutical composition is 5×10 １１ ~5 x 10 １５ can be administered at a dosage of vg / kg of subject, or The pharmaceutical composition is 1×10 １２ ~1 x 10 １５ vg / kg of subject; The pharmaceutical composition of any one of claims 6 to 9, wherein administration of the pharmaceutical composition to the subject increases plasma pyrophosphate (PPi) and / or plasma ENPP1 concentrations in the subject.

11. The pharmaceutical composition of claim 10, wherein the plasma pyrophosphate (PPi) and / or plasma ENPP1 concentration is indicated by detecting or measuring one or more of the following parameters in a biological sample obtained from the subject: (i) pyrophosphate concentration, (ii) ENPP1 expression level, and (iii) ENPP1 enzyme activity, and the detection or measurement is performed before administering the pharmaceutical composition, simultaneously or nearly simultaneously with administering the pharmaceutical composition, or after administering the pharmaceutical composition.

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