Variant forms of urate oxidase and use thereof

Abstract

Genetically modified proteins with uricolytic activity are described. Proteins comprising truncated urate oxidases and methods for producing them, including PEGylated proteins comprising truncated urate oxidase are described.

1 . An isolated truncated mammalian uricase comprising a mammalian uricase amino acid sequence truncated at the amino terminus or the carboxy terminus or both the amino and carboxy termini by about 1-13 amino acids and further comprising an amino acid substitution at about position 46. 
     
     
         2 . The uricase of  claim 1 , further comprising an amino terminal amino acid, wherein the amino terminal amino acid is alanine, glycine, proline, serine, or threonine. 
     
     
         3 . (canceled) 
     
     
         4 . The uricase of  claim 1 , wherein the substitution is with threonine or alanine. 
     
     
         5 . (canceled) 
     
     
         6 . The uricase of  claim 4 , comprising the amino acid sequence of SEQ ID No: 8. 
     
     
         7 . The uricase of  claim 1 , wherein the uricase is conjugated with a polymer. 
     
     
         8 . A polyethylene glycol-uricase conjugate comprising the uricase of  claim 1 . 
     
     
         9 . The conjugate of  claim 8 , comprising 2 to 12 polyethylene glycol molecules on each uricase subunit. 
     
     
         10 . (canceled) 
     
     
         11 . The conjugate of  claim 8 , wherein each polyethylene glycol molecule has a molecular weight between about 1 kD and 100 kD. 
     
     
         12 - 14 . (canceled) 
     
     
         15 . A pharmaceutical composition comprising a uricase of  claim 1 . 
     
     
         16 . A pharmaceutical composition comprising a conjugate of  claim 8 . 
     
     
         17 - 18 . (canceled) 
     
     
         19 . A method of reducing uric acid levels in a biological fluid of a subject in need thereof, comprising administering the composition of  claim 15  to the subject. 
     
     
         20 . A method of reducing uric acid levels in a biological fluid of a subject in need thereof, comprising administering the composition of  claim 16  to the subject. 
     
     
         21 . The method of  claim 19 , wherein the biological fluid is blood. 
     
     
         22 . The method of  claim 20 , wherein the biological fluid is blood. 
     
     
         23 . (canceled) 
     
     
         24 . The uricase of  claim 1 , wherein the N terminal amino acid is methionine. 
     
     
         25 . The uricase of  claim 24 , comprising the amino acid sequence of SEQ ID NO: 7. 
     
     
         26 . An isolated nucleic acid comprising a nucleic acid sequence which encodes the uricase of  claim 1 . 
     
     
         27 . The isolated nucleic acid of  claim 26 , wherein the nucleic acid sequence is operatively linked to a heterologous promoter. 
     
     
         28 . The nucleic acid of  claim 27 , wherein the promoter is the osmB promoter. 
     
     
         29 . A nucleic acid vector comprising the nucleic acid of  claim 27 . 
     
     
         30 . A host cell comprising the vector of  claim 29 . 
     
     
         31 . An isolated nucleic acid comprising a nucleic acid sequence encoding a uricase comprising the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8. 
     
     
         32 . The isolated nucleic acid of  claim 31 , wherein the nucleic acid sequence comprises SEQ ID NO: 9 or SEQ ID NO: 10. 
     
     
         33 - 36 . (canceled) 
     
     
         37 . A method for producing a uricase comprising the steps of culturing a host cell of  claim 30  under conditions such that the nucleic acid sequence is expressed by the host cell and isolating the expressed uricase.