Lysosomal storage disease enzymes

Abstract

The present invention provides compositions of recombinant human lysosomal acid lipase having particular glycosylation patterns for internalization into target cells, a vector containing the nucleic acid encoding human lysosomal acid lipase, a host cell transformed with the vector, pharmaceutical compositions comprising the recombinant human lysosomal acid lipase and method of treating conditions associated with lysosomal acid lipase deficiency.

1 .- 72 . (canceled) 
     
     
         73 . A composition comprising an isolated human recombinant lysosomal acid lipase (rhLAL) comprising one or more N-glycan structures, wherein the rhLAL is N-linked glycosylated at Asn 15 , Asn 80 , Asn 140 , Asn 252  and Asn 300  of SEQ ID NO:2. 
     
     
         74 . The composition of  claim 73 , wherein the LAL comprises a N-glycan structure comprising a phosphorylated mannose at Asn 80 , Asn 140  or Asn 252  of SEQ ID NO:2. 
     
     
         75 . The composition of  claim 74 , wherein the LAL comprises an N-glycan structure comprising a phosphorylated mannose at Asn 80 . 
     
     
         76 . The composition of  claim 75 , wherein at least 30% of N-glycan structures associated with Asn 80  have a phosphorylated mannose. 
     
     
         77 . The composition of  claim 74 , wherein the LAL comprises an N-glycan structure comprising a bisphosphorylated mannose at Asn 80 . 
     
     
         78 . The composition of  claim 74 , wherein the LAL comprises an N-glycan structure comprising a phosphorylated mannose (M6P) at Asn 140 . 
     
     
         79 . The composition of  claim 78 , wherein between about 10% and about 50% of N-glycan structures associated with Asn 140  comprise a M6P. 
     
     
         80 . The composition of  claim 74 , wherein the LAL comprises an N-glycan structure comprising a phosphorylated mannose (M6P) at Asn 252 . 
     
     
         81 . The composition of  claim 80 , wherein at least 50% of the N-glycan structures at Asn 252  comprise a M6P. 
     
     
         82 . The composition of  claim 73 , wherein the LAL comprises an N-glycan structure comprising a high-mannose group at Asn 80  or Asn 252 . 
     
     
         83 . The composition of  claim 82 , wherein the high mannose-group comprises 6, 7, 8, 9 or 10 mannose. 
     
     
         84 . The composition of  claim 83 , wherein the LAL comprises an N-glycan structure comprising 7, 8 or 9 mannose at Asn 80 . 
     
     
         85 . The composition of  claim 83 , wherein the LAL comprises an N-glycan structure comprising 7, 8 or 9 mannose at Asn 252 . 
     
     
         86 . The composition of  claim 73 , wherein the LAL comprises an N-glycan structure comprising a terminal galactose at Asn 15 , Asn 140  or Asn 300 . 
     
     
         87 . The composition of  claim 86 , wherein about 2% to about 10% of N-glycan structures associated with Asn 15  comprise a terminal galactose. 
     
     
         88 . The composition of  claim 87 , wherein less than 5% of N-glycan structures associated with Asn 140  comprise a terminal galactose. 
     
     
         89 . The composition of  claim 88 , wherein the LAL comprises a terminal galactose at Asn 300 . 
     
     
         90 . The composition of  claim 73 , wherein Asn 51  of the LAL is unglycosylated. 
     
     
         91 . The composition of  claim 73 , wherein the LAL comprises N-glycan structures having no xylose and less than 15% of the N-glycan structures contain sialic acid. 
     
     
         92 . The composition of  claim 91 , wherein the LAL comprises N-glycan structures having no xylose and less than 10% of the N-glycan structures contain sialic acid. 
     
     
         93 . The composition of  claim 92 , wherein the LAL comprises N-glycan structures having no xylose and less than 5% of the N-glycan structures contain sialic acid. 
     
     
         94 . The composition of  claim 73 , wherein the LAL contains no sialic acid and no xylose. 
     
     
         95 . The composition of  claim 73 , wherein the LAL contains no fucose. 
     
     
         96 . The composition of  claim 73 , wherein the LAL comprises a N-linked glycosylation profile as follows:
 a) at Asn 15 , GlcNAc4Man3GlcNAc2, or
 Gal1GlcNAc4Man3GlcNAc2; 
   b) at Asn 80 , Phos2Man7GlcNAc2;   c) at Asn 140 , Phos1Man6GlcNAc2,
 GlcNAc1Phos1Man6GlcNAc2; 
 Man3GlcNAc2; 
 GlcNAc2Man3GlcNAc2; 
 GlcNAc3Man3GlcNAc2; 
 GlcNAc4Man3GlcNAc2, or 
 Gal1GlcNAc4Man3GlcNAc2; 
   d) at Asn 252 , Man7GlcNAc2,
 Man8GlcNAc2, 
 Man9GlcNAc2, 
 Phos1Man8GlcNAc2, or 
 Phos1Man9GlcNAc2; and 
   e) at Asn 300 , GlcNAc2Man3GlcNAc2,
 GlcNAc3Man3GlcNAc2, 
 GlcNAc4Man3GlcNAc2, 
 Gal1GlcNAc4Man3GlcNAc2, 
 GlcNAc5Man3GlcNAc2, 
 Gal1GlcNAc5Man3GlcNAc2, 
 GlcNAc6Man3GlcNAc2, or 
 Gal1GlcNAc6Man3GlcNAc2, 
   wherein Man=mannose,
 GlcNAc=N-Acetyl Glucosamine 
 Phos=phosphate 
 Gal=galactose. 
   
     
     
         97 . The composition of  claim 73 , wherein the LAL is produced in a germ-line transgenic avian. 
     
     
         98 . The composition of  claim 97 , wherein the LAL is produced from an oviduct cell of the germ-line transgenic avian. 
     
     
         99 . The composition of  claim 98 , wherein the avian is a chicken. 
     
     
         100 . A composition comprising a mixture of recombinant human lysosomal acid lipase (LAL), the mixture comprising at least two human LAL selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:19. 
     
     
         101 . The composition of  claim 100 , wherein the mixture of human LAL comprises N-glycans selected from the following structures: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         102 . A method of producing glycosylated human lysosomal acid lipase (LAL), comprising creating a germ-line transgenic avian which contains a transgene encoding human LAL operably linked to a promoter and expresses the human LAL in an oviduct cell, wherein the LAL is glycosylated in the oviduct cell and deposited into a hard shell egg laid by the transgenic avian; and obtaining the LAL from egg white of the egg. 
     
     
         103 . A transgenic avian which produces glycosylated human lysosomal acid lipase (LAL) according to  claim 73  or  claim 100 . 
     
     
         104 . An egg laid by a transgenic avian according to  claim 103 . 
     
     
         105 . A pharmaceutical formulation comprising a composition according to  claim 73  or  claim 100  in combination with a pharmaceutically acceptable carrier, diluent or excipient. 
     
     
         106 . The pharmaceutical formulation of  claim 105 , wherein the formulation comprises at least one agent selected from the group consisting of trisodium citrate dehydrate, citric acid and human serum albumin. 
     
     
         107 . The pharmaceutical formulation of  claim 105 , wherein the formulation is provided in an aqueous solution maintained at pH between about 5.6 and about 6.2.