Enpp1 polypeptides and methods of using same

Abstract

The present disclosure includes ENPP1 mutant polypeptides with improved in vivo half-lives.

1 . An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide fused to a Fc region of an immunoglobulin, wherein the ENPP1 polypeptide comprises the mutation I256T as relating to SEQ ID NO:7. 
     
     
         2 . The polypeptide fusion of  claim 1 , wherein the Fc region comprises at least one mutation selected from the group consisting of M883Y, S885N, S885T, T887E, H1064K, and N1065F as relating to SEQ ID NO:7. 
     
     
         3 . (canceled) 
     
     
         4 . The polypeptide fusion of  claim 1 , wherein the ENPP1 polypeptide further comprises at least one mutation selected from the group consisting of C 25 N, K27T, and V29N as relating to SEQ ID NO:7. 
     
     
         5 . (canceled) 
     
     
         6 . The polypeptide fusion of  claim 1 , wherein the ENPP1 polypeptide further comprises at least one mutation selected from the group consisting of K369N, and I371T as relating to SEQ ID NO:7. 
     
     
         7 . (canceled) 
     
     
         8 . The polypeptide fusion of  claim 1 , wherein the ENPP1 polypeptide further comprises at least one mutation selected from the group consisting of P534N, V536T, R545T, P554L, E592N, R741D, and S766N as relating to SEQ ID NO:7. 
     
     
         9 . (canceled) 
     
     
         10 . The polypeptide fusion of  claim 1 , wherein the ENPP1 polypeptide further comprises at least one mutation selected from the group consisting of E864N and L866T as relating to SEQ ID NO:7. 
     
     
         11 . The polypeptide fusion of  claim 10 , wherein the ENPP1 polypeptide comprises at least one of the following:
 (a) at least one mutation selected from the group consisting of S885N, M883Y, M883Y/S885T/T887E, and H1064K/N1065F as relating to SEQ ID NO:7;   (b) at least one mutation selected from the group consisting of C 25 N/K27T and V29N as relating to SEQ ID NO:7;   (c) the mutation K369N/I371T as relating to SEQ ID NO:7;   (d) at least one mutation selected from the group consisting of at least one mutation selected from the group consisting of P534N/V536T, P554L/R545T, E592N, E592N/R741D, and S766N as relating to SEQ ID NO:7;   (e) at least the mutation E864N/L866T as relating to SEQ ID NO:7.   
     
     
         12 . The polypeptide fusion of  claim 1 , comprising at least one mutation selected from the group consisting of C 25 N, K27T, V29N, C 25 N/K27T, K369N, I371T, K369N/I371T, P534N, V536T, R545T, P554L, E592N, R741D, S766N, P534N/V536T, P554L/R545T, E592N/R741D, E864N, L866T, E864N/L866T, M883Y, S885N, S885T, T887E, H1064K, N1065F, M883Y/S885T/T887E, H1064K/N1065F as relating to SEQ ID NO:7. 
     
     
         13 . The polypeptide fusion of  claim 1 , wherein the Fc region is of an IgG. 
     
     
         14 . The polypeptide fusion of  claim 1 , comprising at least one mutation selected from the group consisting of P534N, V536T, R545T, P554L, S766N, and E592N as relating to SEQ ID NO:7. 
     
     
         15 . The polypeptide fusion of  claim 1 , comprising at least one of the following:
 (a) at least one mutation selected from the group consisting of S766N, P534N/Y536T, P554L/R545T, and E592N as relating to SEQ ID NO:7;   (b) at least one mutation selected from the group consisting of S885N, S766N, M883Y/S885T/T887E, E864N/L866T, P534N/V536T/H1064K/N1065F, P554L/R545T, S766N/H1064K/N1065F, E592N/H1064K/N1065F, and P534N/V536T/M883Y/S885T/T887E as relating to SEQ ID NO:7.   
     
     
         16 . (canceled) 
     
     
         17 . An ENPP1 polypeptide fusion comprising an ENPP1 polypeptide and a Fc region of an immunoglobulin, the polypeptide fusion comprising at least one of the following:
 (a) mutations I256T, M883Y, S885T, and T887E as relating to SEQ ID NO:7;   (b) mutations I256T, P534N, V536T, M883Y, S885T, and T887E as relating to SEQ ID NO:7;   (c) mutations I256T, E592N, H1064K, and N1065F as relating to SEQ ID NO:7.   
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . An ENPP1 mutant polypeptide comprising amino acids 23-849 of SEQ ID NO:7, wherein the mutant polypeptide comprises mutation I256T and further comprises a mutation selected from the group consisting of S766N, P534N, V536T, P554L, R545T, and E592N as relating to SEQ ID NO:7. 
     
     
         21 . The mutant polypeptide of  claim 20 , which comprises the amino acid sequence of SEQ ID NO:7. 
     
     
         22 . The mutant polypeptide of  claim 20 , which lacks a signal peptide sequence. 
     
     
         23 . The mutant polypeptide of  claim 20 , wherein the mutant polypeptide comprises at least one mutation selected from the group consisting of S766N, P534N/V536T, P554L/R545T, and E592N as relating to SEQ ID NO:7. 
     
     
         24 . The mutant polypeptide of  claim 21 , comprising mutations selected from the group consisting of: S885N, S766N, M883Y/S885T/T887E, P534N/V536T/H1064K/N1065F, P554L/R545T, S766N/H1064K/N1065F, E592N/H1064K/N1065F, and P534N/V536T/M883Y/S885T/T887E as relating to SEQ ID NO:7. 
     
     
         25 . The mutant polypeptide of  claim 21 , comprising a S885N mutation as relating to SEQ ID NO:7. 
     
     
         26 . The mutant polypeptide of  claim 20 , comprising a S766N mutation as relating to SEQ ID NO:7. 
     
     
         27 . The mutant polypeptide of  claim 21 , comprising at least one of the following:
 (a) mutations M883Y, S885T, and T887E as relating to SEQ ID NO:7,   (b) mutations P534N, V536T, H1064K, and N1065F as relating to SEQ ID NO:7;   (c) mutations S766N, H1064K, and N1065F as relating to SEQ ID NO:7;   (d) mutations E592N, H1064K, and N1065F as relating to SEQ ID NO:7;   (e) mutations P534N, V536T, M883Y, S885T, and T887E as relating to SEQ ID NO:7.   
     
     
         28 . (canceled) 
     
     
         29 . The mutant polypeptide of  claim 20 , comprising mutations P554L and R545T as relating to SEQ ID NO:7. 
     
     
         30 - 32 . (canceled) 
     
     
         33 . The polypeptide fusion of  claim 1 , which is expressed from a CHO cell line stably transfected with human ST6 beta-galactoside alpha-2,6-sialyltransferase (also known as ST6GAL1). 
     
     
         34 . The polypeptide fusion of  claim 1 , which is grown in a cell culture supplemented with at least one of sialic acid and/er N-acetylmannosamine (also known as 1,3,4-O-Bu 3 ManNAc). 
     
     
         35 . A method of reducing or preventing progression of pathological calcification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion of  claim 1 . 
     
     
         36 . A method of reducing or preventing progression of pathological ossification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion of  claim 1 . 
     
     
         37 . A method of reducing or preventing progression of ectopic calcification of soft tissue in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion of  claim 1 . 
     
     
         38 . A method of treating, reversing, or preventing progression of ossification of the posterior longitudinal ligament (OPLL) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion of  claim 1 . 
     
     
         39 . A method of treating, reverting, or preventing progression of hypophosphatemic rickets in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion of  claim 1 . 
     
     
         40 . A method of reducing or preventing progression of at least one disease selected from the group consisting of chronic kidney disease (CKD), end stage renal disease (ESRD), calcific uremic arteriolopathy (CUA), calciphylaxis, ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, aging related hardening of arteries, idiopathic infantile arterial calcification (IIAC), Generalized Arterial Calcification of Infancy (GACI), and calcification of atherosclerotic plaques in a subject diagnosed with the at least one disease, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion of  claim 1 . 
     
     
         41 . A method of reducing or preventing progression of aging related hardening of arteries in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusion of  claim 1 . 
     
     
         42 . The method of  claim 35 , wherein the pathological calcification is selected from the group consisting of idiopathic infantile arterial calcification (IIAC) and calcification of atherosclerotic plaques. 
     
     
         43 . The method of  claim 36 , wherein the pathological ossification is selected from the group consisting of ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, and osteoarthritis. 
     
     
         44 . The method of  claim 37 , wherein the soft tissue calcification is selected from the group consisting of IIAC and osteoarthritis. 
     
     
         45 . The method of  claim 37 , wherein the soft tissue is selected from the group consisting of atherosclerotic plaques, muscular arteries, joint, spine, articular cartilage, vertebral disk cartilage, vessels, and connective tissue. 
     
     
         46 . A method of raising pyrophosphate (PPi) levels in a subject having PPi level lower than PPi normal level, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of the polypeptide fusion of  claim 1 , whereby upon the administration the level of the PPi in the subject is elevated to a normal level of at least 2 μM and is maintained at approximately the same level. 
     
     
         47 . A method of reducing or preventing the progression of pathological calcification or ossification in a subject having pyrophosphate (PPi) level lower than PPi normal level, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion of  claim 1 , whereby pathological calcification or ossification in the subject is reduced or progression of pathological calcification or ossification in the subject is prevented. 
     
     
         48 . A method of treating ENPP1 deficiency manifested by a reduction of extracellular pyrophosphate (PPi) concentration in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide fusion of  claim 1 , whereby the level of the PPi in the subject is elevated. 
     
     
         49 . The method of  claim 35 , wherein the polypeptide fusion is a secreted product of a ENPP1 precursor protein expressed in a mammalian cell, wherein the ENPP1 precursor protein comprises a signal peptide sequence and an ENPP1 polypeptide, wherein the ENPP1 precursor protein undergoes proteolytic processing to yield the ENPP1 polypeptide. 
     
     
         50 . The method of  claim 49 , wherein in the ENPP1 precursor protein the signal peptide sequence is conjugated to the N-terminus of the ENPP1 polypeptide. 
     
     
         51 . The method of  claim 49 , wherein the signal peptide sequence is selected from the group consisting of ENPP1 signal peptide sequence, ENPP2 signal peptide sequence, ENPP7 signal peptide sequence, and ENPP5 signal peptide sequence. 
     
     
         52 . The method of  claim 35 , wherein the polypeptide fusion is administered acutely or chronically to the subject. 
     
     
         53 . The method of  claim 35 , wherein the polypeptide fusion is administered locally, regionally, parenterally, or systemically to the subject. 
     
     
         54 . The method of  claim 35 , wherein the polypeptide fusion is administered to the subject by at least one route selected from the group consisting of subcutaneous, oral, aerosol, inhalational, rectal, vaginal, transdermal, subcutaneous, intranasal, buccal, sublingual, parenteral, intrathecal, intragastrical, ophthalmic, pulmonary, and topical. 
     
     
         55 . The method of  claim 35 , wherein the polypeptide fusion is administered to the subject as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier. 
     
     
         56 . The method of  claim 35 , wherein the subject is a mammal. 
     
     
         57 . The method of  claim 56 , wherein the mammal is human. 
     
     
         58 . An ENPP1 mutant polypeptide comprising one or more amino acid substitutions as relating to SEQ ID NO:7, wherein the polypeptide comprises an amino acid substitution at position 256 relative to SEQ ID NO:7. 
     
     
         59 . The ENPP1 mutant polypeptide of  claim 58 , wherein the ENPP1 mutant polypeptide amino acid sequence is at least 90% identical to amino acids 23-849 of SEQ ID NO:7. 
     
     
         60 . An ENPP1 mutant polypeptide comprising amino acids 23-849 of SEQ ID NO:7,
 wherein no more than ten (10) amino acid substitutions relative to amino acids 23-849 of SEQ ID NO:7 are present, and   wherein the ENPP1 mutant polypeptide comprises an amino acid substitution at position 256 relative to SEQ ID NO:7.   
     
     
         61 . The ENPP1 mutant polypeptide of  claim 58 , wherein the amino acid substitution is the substitution of isoleucine (I) for threonine (T) at position 256 relative to SEQ ID NO:7. 
     
     
         62 . The ENPP1 mutant polypeptide of  claim 58 , wherein the amino acid substitution is the substitution of isoleucine (I) for serine (S) at position 256 relative to SEQ ID NO:7. 
     
     
         63 . An ENPP1 mutant polypeptide comprising an amino acid sequence that is at least 90% identical to amino acids 23-849 of SEQ ID NO:7,
 wherein the mutant polypeptide comprises mutation I256T as relating to SEQ ID NO:7, and   wherein the mutant polypeptide further comprises a mutation selected from the group consisting of S766N, P534N, V536T, P554L, R545T, and E592N as relating to SEQ ID NO:7.   
     
     
         64 . The ENPP1 mutant polypeptide of  claim 63 , wherein the mutant polypeptide comprises at least one amino acid substitution selected from the group consisting of S766N, P534N/V536T, P554L/R545T, and E592N as relating to SEQ ID NO:7. 
     
     
         65 . The ENPP1 mutant polypeptide of  claim 63 , wherein the mutant polypeptide comprises the amino acid substitution V29N. 
     
     
         66 . The ENPP1 mutant polypeptide of  claim 58 , wherein the mutant polypeptide comprises the amino acid sequence of SEQ ID NO:11. 
     
     
         67 . An ENPP1 mutant polypeptide fusion comprising the ENPP1 mutant polypeptide of  claim 58  and a heterologous protein. 
     
     
         68 . The ENPP1 mutant polypeptide fusion of  claim 67 , wherein the heterologous protein is an FcRn binding domain. 
     
     
         69 . The ENPP1 mutant polypeptide fusion of  claim 67 , wherein the heterologous protein is carboxy-terminal to the ENPP1 mutant polypeptide of the fusion. 
     
     
         70 . The ENPP1 mutant polypeptide fusion of  claim 67 , wherein the heterologous protein is amino-terminal to the ENPP1 mutant polypeptide of the fusion. 
     
     
         71 . The ENPP1 mutant polypeptide fusion of  claim 68 , wherein the FcRn binding domain is an albumin polypeptide. 
     
     
         72 . The ENPP1 mutant polypeptide fusion of  claim 68 , wherein the FcRn binding domain is a Fc portion of an immunoglobulin molecule. 
     
     
         73 . The ENPP1 mutant polypeptide fusion of  claim 72 , wherein the immunoglobulin molecule is an IgG1. 
     
     
         74 . The ENPP1 mutant polypeptide fusion of  claim 68 , wherein the FcRn binding domain comprises one more amino acid substitutions relative to a wild type FcRn binding domain. 
     
     
         75 . The ENPP1 mutant polypeptide fusion of  claim 68 , and, wherein the FcRn binding domain is the Fc portion of a human IgG1 molecule and comprises the following amino acid substitutions: M883Y, S885T, and T887E, each relative to SEQ ID NO:7. 
     
     
         76 . The ENPP1 mutant polypeptide fusion of  claim 68 , and, wherein the ENPP1 mutant polypeptide fusion comprises one or more of the following substitutions: S885N, S766N, M883Y/S885T/T887E, P534N/V536T/H1064K/N1065F, P554L/R545T, S766N/H1064K/N1065F, E592N/H1064K/N1065F, or P534N/V536T/M883Y/S885T/T887E, each as relating to SEQ ID NO:7. 
     
     
         77 . The ENPP1 mutant polypeptide fusion of  claim 68 , and, wherein the ENPP1 mutant polypeptide fusion comprises the S885N mutation as relating to SEQ ID NO:7. 
     
     
         78 . The ENPP1 mutant polypeptide fusion of  claim 68 , and, wherein the ENPP1 mutant polypeptide fusion comprises at least one of the following:
 (a) the S766N mutation as relating to SEQ ID NO:7;   (b) mutations M883Y, S885T, and T887E as relating to SEQ ID NO:7;   (c) mutations P534N, V536T, H1064K, and N1065F as relating to SEQ ID NO:7;   (d) mutations P554L and R545T as relating to SEQ ID NO:7;   (e) mutations S766N, H1064K, and N1065F as relating to SEQ ID NO:7;   (f) mutations E592N, H1064K, and N1065F as relating to SEQ ID NO:7;   (g) mutations P534N, V536T, M883Y, S885T, and T887E as relating to SEQ ID NO:7.   
     
     
         79 - 84 . (canceled) 
     
     
         85 . The ENPP1 mutant polypeptide fusion of  claim 68 , wherein the Fc region comprises at least one mutation selected from the group consisting of M883Y, S885N, S885T, T887E, H1064K, and N1065F as relating to SEQ ID NO:7. 
     
     
         86 . The ENPP1 mutant polypeptide fusion of  claim 68 , wherein the Fc region comprises at least one mutation selected from the group consisting of S885N, M883Y, M883Y/S885T/T887E, and H1064K/N1065F as relating to SEQ ID NO:7. 
     
     
         87 . The ENPP1 mutant polypeptide fusion of  claim 68 , wherein the ENPP1 mutant polypeptide fusion comprises at least one mutation selected from the group consisting of C 25 N, K27T, and V29N as relating to SEQ ID NO:7. 
     
     
         88 . (canceled) 
     
     
         89 . The ENPP1 mutant polypeptide fusion of  claim 68 , wherein the ENPP1 mutant polypeptide fusion comprises one mutation selected from the group consisting of K369N and I371T as relating to SEQ ID NO:7. 
     
     
         90 . (canceled) 
     
     
         91 . The ENPP1 mutant polypeptide fusion of any one of  claims 68 - 70  and  72 - 90  or the ENPP1 mutant polypeptide of any one of  claims 58 - 65  and  87 - 90 , wherein the ENPP1 mutant polypeptide fusion or ENPP1 mutant polypeptide comprises at least one mutation selected from the group consisting of P534N, V536T, R545T, P554L, E592N, R741D, and S766N as relating to SEQ ID NO:7. 
     
     
         92 . The ENPP1 mutant polypeptide fusion of  claim 68 , wherein the ENPP1 mutant polypeptide fusion comprises at least one of the following:
 (a) at least one mutation selected from the group consisting of C 25 N/K27T and V29N as relating to SEQ ID NO:7;   (b) the mutation K369N/I371T as relating to SEQ ID NO:7;   (c) at least one mutation selected from the group consisting of P534N/V536T, P554L/R545T, E592N, E592N/R741D, and S766N as relating to SEQ ID NO:7.   
     
     
         93 . The ENPP1 mutant polypeptide fusion of  claim 68 , wherein the ENPP1 mutant polypeptide fusion or ENPP1 mutant polypeptide comprises at least one mutation selected from the group consisting of C 25 N, K27T, V29N, C 25 N/K27T, K369N, I371T, K369N/I371T, P534N, V536T, R545T, P554L, E592N, R741D, S766N, P534N/V536T, P554L/R545T, E592N/R741D, E864N, L866T, E864N/L866T, M883Y, S885N, S885T, T887E, H1064K, N1065F, M883Y/S885T/T887E, H1064K/N1065F as relating to SEQ ID NO:7. 
     
     
         94 . The ENPP1 mutant polypeptide fusion of  claim 68 , wherein the ENPP1 mutant polypeptide fusion comprises at least one mutation selected from the group consisting of P534N, V536T, R545T, P554L, S766N, and E592N as relating to SEQ ID NO:7. 
     
     
         95 . The ENPP1 mutant polypeptide fusion of  claim 68 , wherein the ENPP1 mutant polypeptide fusion comprises at least one mutation selected from the group consisting of S766N, P534N/Y536T, P554L/R545T, and E592N as relating to SEQ ID NO:7. 
     
     
         96 . The ENPP1 mutant polypeptide fusion of  claim 68 , wherein the ENPP1 mutant polypeptide fusion comprises at least one mutation selected from the group consisting of S885N, S766N, M883Y/S885T/T887E, E864N/L866T, P534N/V536T/H1064K/N1065F, P554L/R545T, S766N/H1064K/N1065F, E592N/H1064K/N1065F, and P534N/V536T/M883Y/S885T/T887E as relating to SEQ ID NO:7. 
     
     
         97 . The ENPP1 mutant polypeptide fusion of  claim 68 , wherein the ENPP1 mutant polypeptide fusion comprises at least one of the following:
 (a) mutations I256T, M883Y, S885T, and T887E as relating to SEQ ID NO:7,   (b) mutations I256T, P534N, V536T, M883Y, S885T, and T887E as relating to SEQ ID NO:7;   (c) mutations I256T, E592N, H1064K, and N1065F as relating to SEQ ID NO:7.   
     
     
         98 - 99 . (canceled) 
     
     
         100 . The fusion of  claim 67 , comprising a linker amino acid sequence. 
     
     
         101 . The fusion of  claim 100 , wherein the linker amino acid sequence connects the ENPP1 mutant polypeptide portion of the fusion and the heterologous protein. 
     
     
         102 . The fusion of  claim 100 , wherein the linker amino acid sequence comprises SEQ ID NO:8 or SEQ ID NO:9. 
     
     
         103 . A nucleic acid encoding the ENPP1 mutant polypeptide of  claim 58 . 
     
     
         104 . A vector or an expression vector comprising the nucleic acid of  claim 103 . 
     
     
         105 . (canceled) 
     
     
         106 . A cell or plurality of cells, each comprising the nucleic acid of  claim 103 . 
     
     
         107 . The cell or plurality of cells of  claim 106 , wherein the cell(s) is/are a CHO cell(s) or an NS0 cell(s). 
     
     
         108 . The cell or plurality of cells of  claim 107 , wherein the CHO cell is stably transfected with human ST6 beta-galactoside alpha-2,6-sialyltransferase. 
     
     
         109 . A method of producing an ENPP1 mutant polypeptide or fusion, the method comprising culturing the cell or plurality of cells of  claim 106 , under conditions suitable for expression of the ENPP1 mutant polypeptide or fusion by the cell or cells. 
     
     
         110 . The method of  claim 109 , wherein the cells are cultured in a medium supplemented with sialic acid and/or N-acetylmannosamine. 
     
     
         111 . The method of  claim 109 , further comprising purifying the ENPP1 mutant polypeptide or fusion from the cell, plurality of cells, or the media in which the cell or plurality of cells were cultured. 
     
     
         112 . An ENPP1 mutant polypeptide or fusion purified by the method of  claim 111 . 
     
     
         113 . A conjugate comprising (i) the ENPP1 mutant polypeptide of  claim 58  and (ii) a heterologous moiety. 
     
     
         114 . The conjugate of  claim 113 , wherein the heterologous moiety is polyethylene glycol. 
     
     
         115 . A pharmaceutical composition comprising the ENPP1 mutant polypeptide of  claim 58  and a pharmaceutically acceptable carrier. 
     
     
         116 . A method of reducing or preventing progression of pathological calcification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of
 the ENPP1 mutant polypeptide of  claim 58     to thereby reduce or prevent progression of pathological calcification in the subject.   
     
     
         117 . A method of reducing or preventing progression of pathological ossification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of
 the ENPP1 mutant polypeptide of  claim 58     to thereby reduce or prevent progression of pathological ossification in the subject.   
     
     
         118 . A method of reducing or preventing progression of ectopic calcification of soft tissue in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of
 the ENPP1 mutant polypeptide of  claim 58     to thereby reduce or prevent progression of ectopic calcification of soft tissue in the subject.   
     
     
         119 . A method of treating, reversing, or preventing progression of ossification of the posterior longitudinal ligament (OPLL) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of
 the ENPP1 mutant polypeptide of  claim 58     to thereby reduce, reverse, or prevent ossification of the posterior longitudinal ligament (OPLL) in the subject.   
     
     
         120 . A method of treating, reverting, or preventing progression of hypophosphatemic rickets in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of
 the ENPP1 mutant polypeptide of  claim 58  to thereby reduce, reverse, or prevent progression of hypophosphatemic rickets in the subject.   
     
     
         121 . A method of reducing or preventing progression of at least one disease selected from the group consisting of chronic kidney disease (CKD), end stage renal disease (ESRD), calcific uremic arteriolopathy (CUA), calciphylaxis, ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, aging related hardening of arteries, idiopathic infantile arterial calcification (IIAC), Generalized Arterial Calcification of Infancy (GACI), and calcification of atherosclerotic plaques in a subject diagnosed with the at least one disease, the method comprising administering to the subject a therapeutically effective amount of
 ENPP1 mutant polypeptide of  claim 58     to thereby reduce or prevent progression of the disease.   
     
     
         122 . A method of reducing or preventing progression of aging related hardening of arteries in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of
 the ENPP1 mutant polypeptide of  claim 58     to thereby reduce or prevent progression of aging related hardening of arteries in the subject.   
     
     
         123 . A method of raising pyrophosphate (PPi) levels in a subject having PPi level lower than PPi normal level, the method comprising administering to the subject a therapeutically effective amount of
 the ENPP1 mutant polypeptide of  claim 58     whereby upon the administration the level of the PPi in the subject is elevated to a normal level of at least 2 μM and is maintained at approximately the same level.   
     
     
         124 . A method of reducing or preventing the progression of pathological calcification or ossification in a subject having pyrophosphate (PPi) level lower than PPi normal level, the method comprising administering to the subject a therapeutically effective amount of
 the ENPP1 mutant polypeptide of  claim 58     whereby pathological calcification or ossification in the subject is reduced or progression of pathological calcification or ossification in the subject is prevented.   
     
     
         125 . A method of treating ENPP1 deficiency manifested by a reduction of extracellular pyrophosphate (PPi) concentration in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of:
 the ENPP1 mutant polypeptide of  claim 58     whereby the level of the PPi in the subject is elevated.   
     
     
         126 . The method of  claim 116 , wherein the mutant polypeptide is administered acutely or chronically to the subject. 
     
     
         127 . The method of  claim 116 , wherein the mutant polypeptide is administered locally, regionally, parenterally, or systemically to the subject. 
     
     
         128 . The method of  claim 116 , wherein the subject is human.