Treatment of alpha-galactosidase a deficiency

Abstract

The invention provides highly purified α-Gal A, and various methods for purifying it; α-Gal A preparations with altered charge and methods for making those preparations; α-Gal A preparations that have an extended circulating half-life in a mammalian host, and methods for making same; and methods and dosages for administering an α-Gal A preparation to a subject.

What is claimed is:  
     
         1 . A composition comprising a human α-Gal A preparation, purified to at least 98% homogeneity, as measured by SDS-PAGE or reverse phase HPLC.  
     
     
         2 . A composition comprising a human α-Gal A preparation, having a specific activity of at least 2.0×10 6  units/mg protein.  
     
     
         3 . A method for producing an α-Gal A preparation comprising various a Gal A glycoforms and purified to at least 98% homogeneity, comprising 
 separating the all A glycoforms from other components on a hydrophobic interaction resin,  
 wherein the α-Gal A preparation is purified to at least 98% homogeneity and wherein the purification does not include a lectin chromatography step.  
 
     
     
         4 . An α-Gal A preparation comprising various α-Gal A glycoforms, purified to at least 98% homogeneity, produced by the method of any one of  claim 3 .  
     
     
         5 . A method for producing an α-Gal A preparation comprising various α-Gal A glycoforms, purified to at least 98% homogeneity, comprising: 
 (a) binding the α-Gal A glycoforms to a cation exchange resin at acidic pH in an equilibration buffer,  
 (b) washing the resin with the equilibration buffer to elute the unbound material, and  
 (c) eluting the α-Gal A glycoforms using an elution solution selected from the group consisting of a salt solution of 10-100 mM, a buffered solution of pH 45, and a combination thereof wherein the α-Gal A preparation is purified to at least 98% homogeneity.  
 
     
     
         6 . An α-Gal A preparation comprising various α-Gal A glycoforms, purified to at least 98% homogeneity, produced by the method of  claim 5 .  
     
     
         7 . A method for producing an α-Gal A preparation comprising various α-Gal A glycoforms, purified to at least 98% homogeneity, comprising 
 separating the α-Gal A glycoforms in a sample from the other components in the sample using a purification procedure comprising a step selected from the group consisting of chromatofocusing chromatography, metal chelate affinity chromatography and immunoaffinity chromatography, wherein the α-Gal A is purified to at least 98% homogeneity.  
 
     
     
         8 . An α-Gal A preparation comprising various α-Gal A glycoforms, purified to at least 98% homogeneity, produced by the method of  claim 7 .  
     
     
         9 . A human glycosylated α-Gal A preparation, wherein at least 35% of the oligosaccharides are charged.  
     
     
         10 . A human glycosylated α-Gal A preparation, wherein the preparation includes multiple glycoforms, comprising at least 20% complex glycans with 2-4 sialic acid residues.  
     
     
         11 . A human glycosylated α-Gal A preparation, wherein the oligosaccharide charge, as measured by the Z number, is greater than 100.  
     
     
         12 . A human glycosylated α-Gal A preparation, wherein the preparation includes multiple glycoforms, said glycoforms being at least on average between 25-50% phosphorylated.  
     
     
         13 . A human glycosylated α-Gal A preparation, wherein the preparation includes multiple glycoforms, and, wherein between 50-75% of the total glycans are sialylated.  
     
     
         14 . A method for producing a glycosylated a4Gal A preparation having an increased oligosaccharide charge, comprising: 
 (a) introducing a polynucleotide which on expression codes for GlcNAc transferase III (GnT-III) into an α-Gal A producing-cell or introducing a regulatory sequence by homologous recombination that regulates expression of an endogenous GnT-III gene;    (b) culturing the α-Gal A production cell under culture conditions which results in expression of α-Gal A and GnT-III; and    (c) isolating the a Gal A preparation, wherein the α-Gal A preparation has increased oligosaccharide charge as compared to α-Gal A from an α-Gal A producing-cell that lacks the polynucleotide in step (a).    
     
     
         15 . The method of  claim 14 , wherein at least 35% of the oligosaccharides are charged.  
     
     
         16 . The method of  claim 14 , wherein the preparation includes multiple glycoforms, comprising at least 20% complex glycans with 2-4 sialic acid residues.  
     
     
         17 . The method of  claim 14 , wherein the oligosaccharide charge, as measured by the Z number, is greater than 100.  
     
     
         18 . The method of  claim 14 , wherein the preparation includes multiple glycoforms, said glycoforms being at least on average between 25-50%/ phosphorylated.  
     
     
         19 . A glycosylated α-Gal A preparation having an increased oligosaccharide charge produced by the method of any one of claims  14 - 18 .  
     
     
         20 . A method for producing a glycosylated α-Gal A preparation with increased oligosaccharide charge, comprising: 
 (a) introducing a polynucleotide which on expression codes for sialyl transferase into an α-Gal A producing-cell or introducing a regulatory sequence by homologous recombination that regulates expression of an endogenous sialyl transferase;  
 (b) culturing the α-Gal A production cell under culture conditions which results in expression of α-Gal A and sialyl transferase; and  
 (c) isolating the α-Gal A preparation, wherein the α-Gal A preparation has increased oligosaccharide charge as compared to α-Gal A from an α-Gal A producing-cell that lacks the polynucleotide in step (a).  
 
     
     
         21 . The method for producing a glycosylated α-Gal A preparation of  claim 20 , further comprising: 
 (d) selecting for α-Gal A glycoforms with increased size or increased charge by fractionation or purification of the preparations of step (c).  
 
     
     
         22 . A glycosylated α-Gal A preparation with increased oligosaccharide charge produced by the method of any one of claims  20 - 21 .  
     
     
         23 . A method for producing a glycosylated α-Gal A preparation with increased sialylation, comprising contacting an α-Gal A production cell with a culture medium having an ammonium concentration below 10 mM.  
     
     
         24 . The method of  claim 23 , wherein the contacting step comprises continuously or intermittently perfusing the α-Gal A production cell with flesh culture medium to maintain the ammonium concentration below 10 mM.  
     
     
         25 . A method for producing a glycosylated α-Gal A preparation having increased phosphorylation, comprising: 
 (a) introducing a polynucleotide which on expression codes for phosphoryl transferase into an α-Gal A producing-cell or introducing a regulatory sequence by homologous recombination that regulates expression of an endogenous phosphoryl transferase;  
 (b) culturing the α-Gal A production cell under culture conditions which result in expression of α-Gal A and phosphoryl transferase; and  
 (c) isolating the α-Gal A, wherein the isolated α-Gal A has increased phosphorylation as compared to the α-Gal A produced in a cell without the polynucleotide.  
 
     
     
         26 . A glycosylated α-Gal A preparation having increased phosphorylation produced by the method of  claim 25 .  
     
     
         27 . A human glycosylated α-Gal A preparation with an extended circulating half-life when administered to a patient, wherein the preparation includes multiple glycoforms, comprising at least 20% complex glycans with 2-4 sialic acid residues.  
     
     
         28 . A human glycosylated α-Gal A preparation with an extended circulating half-life when administered to a patient, wherein the preparation includes multiple glycoforms, said glycoforms being at least on average between 25-50/o phosphorylated.  
     
     
         29 . A human glycosylated α-Gal A preparation with an extended circulating half-life when administered to a patient, wherein the preparation includes multiple glycoforms, and, wherein between 50-75% of the total glycans are sialylated.  
     
     
         30 . A method for producing a glycosylated α-Gal A preparation with a reduced number of sialic acid and terminal galactose residues on the oligosaccharide chains, comprising: 
 (a) contacting α-Gal A with neuraminidase (sialidase) to remove sialic acid residues, leaving the terminal galactose moieties exposed; and  
 (b) contacting the desialylated α-Gal A of step (a) with β-galactosidase to remove terminal galactose residues, 
 such that the desialylated, degalactosylated α-Gal A product of step (b) has a reduced number of terminal sialic acid or galactose residues on the oligosaccharide chains compared to α-Gal A from uncontacted α-Gal A.  
 
 
     
     
         31 . A method for producing a glycosylated α-Gal A with a reduced number of terminal galactose residues on the oligosaccharide chains, comprising: 
 contacting α-Gal A with β-galactosidase to remove terminal galactose residues, such that the product has a reduced number of terminal galactose residues on the oligosaccharide chains compared to α-Gal A from uncontacted α-Gal A.  
 
     
     
         32 . A degalactosylated α-Gal A preparation produced according to the method of  claim 31 .  
     
     
         33 . A formulation comprising an α-Gal A preparation that is substantially free of proteins other than α-Gal A.  
     
     
         34 . A formulation comprising an α-Gal A preparation that is substantially free of albumin.  
     
     
         35 . A method for administering an α-Gal A preparation to a subject, comprising administering a dose of between 0.05-5.0 mg of the α-Gal A preparation weekly or biweekly.  
     
     
         36 . The method of  claim 35 , wherein the dose is about 0.2 mg per kg body weight biweekly.  
     
     
         37 . The method of any one of claims  35 - 36 , wherein the dose is administered intramuscularly, orally, rectally, subcutaneously, intra-arterially, intraperitoneally, intracerebrally, intranasally, intrathecally, transmucosally, transdermally, or via inhalation.  
     
     
         38 . A method for delivering α-Gal A preparation to a subject, comprising subcutaneously administering a dose ranging between 0.01-10 mg of the α-Gal A preparation per kg body weight biweekly or weekly.  
     
     
         39 . The method of any of claims  35 - 36 , wherein the α-Gal A preparation is administered using a delivery system selected from the group consisting of pump delivery, encapsulated cell delivery, liposomal delivery, needle-delivered injection, needle-less injection, nebulizer, aeorosolizer, electroporation, and transdermal patch.  
     
     
         40 . A method of treating a patient with Fabry disease, comprising administering an α-Gal A preparation to the patient in a dose of between 0.05-5.0 mg of the α-Gal A preparation per kg body weight weekly or biweekly.  
     
     
         41 . The method of  claim 40 , wherein the dose is about 0.2 mg per kg body weight biweekly.  
     
     
         42 . A method for treating a patient with Fabry disease comprising subcutaneously administering an α-Gal A preparation to the patient in a dose ranging between 0.01-10 mg of the α-Gal A preparation per kg body weight biweekly or weekly.  
     
     
         43 . A method of treating a patient with atypical variant of Fabry disease, comprising administering to the patient an α-Gal A preparation at a dose of between 0.05-5.0 mg of the α-Gal A preparation per kg body weight weekly or biweekly.  
     
     
         44 . The method of  claim 43 , wherein the patient suffers from a cardiovascular abnormality.  
     
     
         45 . The method of  claim 44 , wherein the cardiovascular abnormality is left ventricular hypertrophy (LVH).