LYSOSOMAL STORAGE DISEASE ENZYMES
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.