Dephosphorylated lysosomal storage disease proteins and methods of use thereof

Abstract

Provided are substantially dephosphorylated forms of lysosomal storage disease (LSD) proteins, including dephosphorylated forms of iduronate-2-sulfatase (IDS, or 12D) and iduronidase (IDU), having increased ability to traverse or penetrate the blood brain barrier (BBB) relative to phosphorylated forms of the protein, and p97 conjugates thereof. Also provided are compositions comprising such dephosphorylated LSD proteins and p97 conjugates, and methods of use thereof, for instance, to treat any one or more lysosomal storage diseases, such as Hunter Syndrome (or MPS Type II).

1 - 50 . (canceled) 
     
     
         51 . An isolated human lysosomal storage disease (LSD) polypeptide produced in a human cell that is at least 75% dephosphorylated and which has substantially the same degree of glycosylation, or number of glycans, relative to an LSD polypeptide produced in a human cell, wherein the LSD polypeptide is α-galactosidase A. 
     
     
         52 . The isolated LSD polypeptide of  claim 51 , wherein the LSD polypeptide is substantially free of mannose-6-phospate (M6P) residues relative to an LSD polypeptide produced in a HT-1080 fibrosarcoma cell. 
     
     
         53 . The isolated LSD polypeptide of  claim 51 , wherein the LSD polypeptide is dephosphorylated by enzymatic digestion with an acid phosphatase or an alkaline phosphatase. 
     
     
         54 . A conjugate comprising a p97 polypeptide that is covalently or operatively linked to an LSD polypeptide as defined in  claim 51 . 
     
     
         55 . A composition comprising the isolated LSD polypeptide of  claim 51 . 
     
     
         56 . A composition comprising the conjugate of  claim 54 . 
     
     
         57 . A conjugate comprising:
 (a) a p97 polypeptide that is covalently or operatively linked to (b) a recombinant human lysosomal storage disease (LSD) polypeptide,   wherein the recombinant human LSD polypeptide is α-galactosidase A, wherein the LSD polypeptide is substantially free of mannose-6-phosphate (M6P) residues, and wherein the LSD polypeptide has oligomannose glycans at all 8 of the N-linked glycosylation sites.   
     
     
         58 . The conjugate of  claim 57 , wherein the recombinant human LSD polypeptide has increased ability to transfer across the blood brain barrier relative to the normally phosphorylated human LSD polypeptide. 
     
     
         59 . A composition comprising the conjugate of  claim 57 , and a pharmaceutically acceptable carrier, where the conjugate is at least 80% pure. 
     
     
         60 . The composition of  claim 59 , wherein the human LSD polypeptide is dephosphorylated by enzymatic digestion with an acid phosphatase or an alkaline phosphatase. 
     
     
         61 . The composition of  claim 59 , wherein the LSD polypeptide has a M6P content of less than 0.5 pmol M6P/pmol LSD polypeptide. 
     
     
         62 . A method of treating a lysosomal storage disease (LSD) in a subject in need thereof comprising administering to the subject in need thereof a therapeutically effective amount of:
 a) an isolated human lysosomal storage disease (LSD) polypeptide produced in a human cell that is at least 75% dephosphorylated and which has substantially the same degree of glycosylation, or number of glycans, relative to an LSD polypeptide produced in a human cell, wherein the LSD polypeptide is α-galactosidase A;   b) a conjugate comprising a p97 polypeptide that is covalently or operatively linked to an isolated human lysosomal storage disease (LSD) polypeptide produced in a human cell that is at least 75% dephosphorylated and which has substantially the same degree of glycosylation, or number of glycans, relative to an LSD polypeptide produced in a human cell, wherein the LSD polypeptide is α-galactosidase A; or   c) A conjugate comprising: a p97 polypeptide that is covalently or operatively linked to a recombinant human lysosomal storage disease (LSD) polypeptide, wherein the recombinant human LSD polypeptide is α-galactosidase A, wherein the LSD polypeptide is substantially free of mannose-6-phosphate (M6P) residues, and wherein the LSD polypeptide has oligomannose glycans at all 8 of the N-linked glycosylation sites.   
     
     
         63 . The method according to  claim 62 , wherein the LSD is selected from the group consisting of: mucopolysaccharidosis type II (Hunter Syndrome), mucopolysaccharidosis type I (Hurler Syndrome), aspartylglucosaminuria, cholesterol ester storage disease, Wolman disease, cystinosis, Danon disease, Fabry disease, Farber lipogranulomatosis, Farber disease, fucosidosis, galactosialidosis types I/II, Gaucher disease types I/II/III, Gaucher disease, globoid cell leucodystrophy, Krabbe disease, glycogen storage disease II, Pompe disease, GMI-gangliosidosis types I/II/III, GM2-gangliosidosis type I, Tay Sachs disease, GM2-gangliosidosis type II, Sandhoff disease, GM2-gangliosidosis, α-mannosidosis types I/II, mannosidosis, metachromatic leucodystrophy, mucolipidosis type I, sialidosis types I/II, mucolipidosis types II/IIII-cell disease, mucolipidosis type IIIC, pseudo-Hurler polydystrophy, mucopolysaccharidosis type IIIA, Sanfilippo syndrome, mucopolysaccharidosis type IIIB, mucopolysaccharidosis type IIIC, mucopolysaccharidosis type IIID, mucopolysaccharidosis type IVA, Morquio syndrome, mucopolysaccharidosis type IVB, mucopolysaccharidosis type VI, mucopolysaccharidosis type VII, Sly syndrome, mucopolysaccharidosis type IX, multiple sulfatase deficiency, neuronal ceroid lipofuscinosis, CLNI Batten disease, Niemann-Pick disease type A, Niemann-Pick disease B, Niemann-Pick disease, Niemann-Pick disease type CI, Niemann-Pick disease type C2, pycnodysostosis, Schindler disease types I/II, Schindler disease, sialic acid storage disease, and any combination thereof. 
     
     
         64 . The method according to  claim 63 , wherein the LSD is Fabry disease 
     
     
         65 . The method according to  claim 62 , wherein the LSD has central nervous system (CNS) involvement, or the subject is at risk for developing CNS involvement of the LSD. 
     
     
         66 . An isolated human lysosomal storage disease (LSD) polypeptide produced in a human cell that is at least 75% dephosphorylated and which has substantially the same degree of glycosylation, or number of glycans, relative to an LSD polypeptide produced in a human cell, wherein the LSD polypeptide is α-L-iduronidase. 
     
     
         67 . The isolated LSD polypeptide of  claim 66 , wherein the LSD polypeptide is substantially free of mannose-6-phospate (M6P) residues relative to an LSD polypeptide produced in a HT-1080 fibrosarcoma cell. 
     
     
         68 . The isolated LSD polypeptide of  claim 66 , wherein the LSD polypeptide is dephosphorylated by enzymatic digestion with an acid phosphatase or an alkaline phosphatase. 
     
     
         69 . A conjugate comprising a p97 polypeptide that is covalently or operatively linked to an LSD polypeptide as defined in  claim 66 . 
     
     
         70 . A composition comprising the isolated LSD polypeptide of  claim 66 . 
     
     
         71 . A composition comprising the conjugate of  claim 69 . 
     
     
         72 . A conjugate comprising:
 (a) a p97 polypeptide that is covalently or operatively linked to (b) a recombinant human lysosomal storage disease (LSD) polypeptide,   wherein the recombinant human LSD polypeptide is α-L-iduronidase, wherein the LSD polypeptide is substantially free of mannose-6-phosphate (M6P) residues, and wherein the LSD polypeptide has oligomannose glycans at all 8 of the N-linked glycosylation sites.   
     
     
         73 . The conjugate of  claim 72 , wherein the recombinant human LSD polypeptide has increased ability to transfer across the blood brain barrier relative to the normally phosphorylated human LSD polypeptide. 
     
     
         74 . A composition comprising the conjugate of  claim 72 , and a pharmaceutically acceptable carrier, where the conjugate is at least 80% pure. 
     
     
         75 . The composition of  claim 74 , wherein the human LSD polypeptide is dephosphorylated by enzymatic digestion with an acid phosphatase or an alkaline phosphatase. 
     
     
         76 . The composition of  claim 59 , wherein the LSD polypeptide has a M6P content of less than 0.5 pmol M6P/pmol LSD polypeptide. 
     
     
         77 . A method of treating a lysosomal storage disease (LSD) in a subject in need thereof comprising administering to the subject in need thereof a therapeutically effective amount of:
 a) an isolated human lysosomal storage disease (LSD) polypeptide produced in a human cell that is at least 75% dephosphorylated and which has substantially the same degree of glycosylation, or number of glycans, relative to an LSD polypeptide produced in a human cell, wherein the LSD polypeptide is α-L-iduronidase;   b) a conjugate comprising a p97 polypeptide that is covalently or operatively linked to an isolated human lysosomal storage disease (LSD) polypeptide produced in a human cell that is at least 75% dephosphorylated and which has substantially the same degree of glycosylation, or number of glycans, relative to an LSD polypeptide produced in a human cell, wherein the LSD polypeptide is α-L-iduronidase; or   c) A conjugate comprising: a p97 polypeptide that is covalently or operatively linked to a recombinant human lysosomal storage disease (LSD) polypeptide, wherein the recombinant human LSD polypeptide is α-L-iduronidase, wherein the LSD polypeptide is substantially free of mannose-6-phosphate (M6P) residues, and wherein the LSD polypeptide has oligomannose glycans at all 8 of the N-linked glycosylation sites.   
     
     
         78 . The method according to  claim 77 , wherein the LSD is selected from the group consisting of: mucopolysaccharidosis type II (Hunter Syndrome), mucopolysaccharidosis type I (Hurler Syndrome), aspartylglucosaminuria, cholesterol ester storage disease, Wolman disease, cystinosis, Danon disease, Fabry disease, Farber lipogranulomatosis, Farber disease, fucosidosis, galactosialidosis types I/II, Gaucher disease types I/II/III, Gaucher disease, globoid cell leucodystrophy, Krabbe disease, glycogen storage disease II, Pompe disease, GMI-gangliosidosis types I/II/III, GM2-gangliosidosis type I, Tay Sachs disease, GM2-gangliosidosis type II, Sandhoff disease, GM2-gangliosidosis, α-mannosidosis types I/II, mannosidosis, metachromatic leucodystrophy, mucolipidosis type I, sialidosis types I/II, mucolipidosis types II/IIII-cell disease, mucolipidosis type IIIC, pseudo-Hurler polydystrophy, mucopolysaccharidosis type IIIA, Sanfilippo syndrome, mucopolysaccharidosis type IIIB, mucopolysaccharidosis type IIIC, mucopolysaccharidosis type IIID, mucopolysaccharidosis type IVA, Morquio syndrome, mucopolysaccharidosis type IVB, mucopolysaccharidosis type VI, mucopolysaccharidosis type VII, Sly syndrome, mucopolysaccharidosis type IX, multiple sulfatase deficiency, neuronal ceroid lipofuscinosis, CLNI Batten disease, Niemann-Pick disease type A, Niemann-Pick disease B, Niemann-Pick disease, Niemann-Pick disease type CI, Niemann-Pick disease type C2, pycnodysostosis, Schindler disease types I/II, Schindler disease, sialic acid storage disease, and any combination thereof. 
     
     
         79 . The method according to  claim 78 , wherein the LSD is mucopolysaccharidosis type I (Hurler Syndrome). 
     
     
         80 . The method according to  claim 77 , wherein the LSD has central nervous system (CNS) involvement, or the subject is at risk for developing CNS involvement of the LSD.