Compositions and methods for treatment of homocystinuria
Provided herein are improved compositions and methods for enzyme replacement therapy using modified human cystathionine beta synthase (CBS) in the treatment of homocystinuria and related diseases and disorders.
1 . An isolated polypeptide dimer comprising two monomeric human truncated cystathionine β-synthase (htCBS) mutant polypeptide subunits, wherein each monomeric htCBS mutant polypeptide subunit independently comprises:
an amino acid sequence sharing an amino acid sequence identity of at least 80%, at least 90%, at least 95%, or at least 99% compared with SEQ ID NO:2, wherein the amino acid sequence of the htCBS mutant polypeptide further comprises
a carboyxyl-terminal truncation, wherein the truncation comprises residues 383-551, 397-551, 414-551, 442-551, 489-551, 497-551, 524-551, 534-551, or 544-551 of SEQ ID NO: 2; and
a mutation of a cysteine to a serine at amino acid position 15 (C15S) compared to a cystathionine β-synthase (CBS) protein comprising the amino acid sequence of SEQ ID NO: 2;
wherein each htCBS mutant polypeptide subunit is independently covalently linked to at least one polyethylene glycol (PEG) molecule, wherein the at least one PEG molecule is an NHS ester-activated PEG molecule or has a molecular weight greater than or equal to 20 kDa.
2 . The isolated polypeptide dimer of claim 1 , wherein each monomeric htCBS mutant polypeptide subunit independently comprises an amino acid sequence sharing an amino acid sequence identity of at least 99% compared with SEQ ID NO: 2.
3 . The isolated polypeptide dimer of claim 1 , wherein the carboxyl-terminated truncation of each monomeric htCBS mutant polypeptide subunit comprises residues 414-551 of SEQ ID NO: 2.
4 . The isolated polypeptide dimer of claim 1 , wherein each monomeric htCBS mutant polypeptide subunit is independently covalently linked to a plurality of PEG molecules, wherein each PEG molecule is an NHS ester-activated PEG molecule having a molecular weight greater than or equal to 20 kDa.
5 . The isolated polypeptide dimer of claim 1 , wherein each PEG molecule covalently linked to each htCBS mutant polypeptide subunit is independently an unbranched PEG molecule or a branched PEG molecule.
6 . An isolated polypeptide dimer comprising two monomeric human truncated cystathionine β-synthase (htCBS) mutant polypeptide subunits, wherein each monomeric htCBS mutant polypeptide subunit independently comprises an amino acid sequence sharing an amino acid sequence identity of at least 99% compared with SEQ ID NO: 13, wherein each htCBS mutant polypeptide subunit is independently covalently linked to at least one 20 kDa NHS ester-actived PEG molecule.
7 . The isolated polypeptide dimer of claim 6 , wherein each monomeric htCBS mutant polypeptide subunit independently comprises amino acids 2-413 of the amino acid sequence of SEQ ID NO: 13.
8 . The isolated polypeptide dimer of claim 7 , wherein each monomeric htCBS mutant polypeptide subunit is independently covalently linked to a plurality of independent 20 kDa NHS ester-actived PEG molecules.
9 . The isolated polypeptide dimer of claim 8 , wherein the polypeptide dimer is purified from other protein components such that the polypeptide dimer comprises at least about 80% weight/weight of the total protein in a given composition, at least about 85% weight/weight of the total protein in a given composition, at least about 90% weight/weight of the total protein in a given composition, at least about 91% weight/weight of the total protein in a given composition, at least about 92% weight/weight of the total protein in a given composition, at least about 93% weight/weight of the total protein in a given composition, at least about 94% weight/weight of the total protein in a given composition, at least about 95% weight/weight of the total protein in a given composition, at least about 96% weight/weight of the total protein in a given composition, at least about 97% weight/weight of the total protein in a given composition, at least about 98% weight/weight of the total protein in a given composition, or at least about 99 weight/weight of the total protein in a given composition.
10 . The isolated polypeptide dimer of claim 9 , wherein the isolated polypeptide dimer is non-heme binding and remains catalytically active.
11 . A pharmaceutical composition comprising:
a therapeutically effective amount of the isolated polypeptide dimer of claim 8 ; and
a pharmaceutically acceptable carrier, diluent, or excipient.
12 . A method for treating or ameliorating a disease, disorder, or condition associated with elevated homocysteine in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the isolated polypeptide dimer of claim 8 or a pharmaceutical composition of claim 11 .
13 . A method for increasing the amount of a metabolite in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the isolated polypeptide dimer of claim 8 or a pharmaceutical composition of claim 11 , wherein the metabolite is cystathionine or cysteine.
14 . A method for decreasing the amount of a metabolite in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the isolated polypeptide dimer of claim 8 or a pharmaceutical composition of claim 11 , wherein the metabolite is homocysteine, methionine, or S-adenosyl homocysteine.
15 . A method for treating liver disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the isolated polypeptide dimer of claim 8 or a pharmaceutical composition of claim 11 .
16 . A method for treating homocystinuria or homocysteine remethylation disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the isolated polypeptide dimer of claim 8 or a pharmaceutical composition of claim 11 .
17 . The method of claim 16 , wherein the homocystinuria or homocysteine remethylation disorder is mental retardation, osteoporosis, ectopia lentis, kyphoscoliosis, stroke, myocardial infarction, or pulmonary embolism.
18 . The method of claim 16 , wherein the subject in need is also administered one or more of an anti-coagulant, a statin, a relaxed protein restricted diet, anethole dithiolethione, or betaine.
19 . The method of claim 18 , wherein betaine is administered twice daily.
20 . The method of claim 18 , wherein the individual is on a relaxed protein restricted diet.
21 . The method of claim 16 , wherein the isolated polypeptide dimer is administered to the subject in need thereof by an implanted osmotic pump, intravenous administration, subcutaneous administration, or intraperitoneal administration.