Variant, recombinant beta-glucocerebrosidase proteins with increased stability and increased retained catalytic activity
View Patent ↗Described herein are variant, recombinant β-glucocerebrosidase proteins characterized as having increased stability relative to recombinant wild-type β-glucocerebrosidase. Also provided herein are variant, recombinant β-glucocerebrosidase proteins characterized as retaining more catalytic activity relative to recombinant wild-type β-glucocerebrosidase. Further described herein are variant, recombinant β-glucocerebrosidase proteins that can have amino acid variations at one or more of the following positions: 316, 317, 321 and 145. Methods of making the variant, recombinant β-glucocerebrosidase proteins are also described as well as methods of treating patients having lysosomal storage diseases.
1. A composition comprising:
a variant, recombinant human β-glucocerebrosidase protein, wherein the protein has at least one substitution relative to SEQ ID NO: 1, and wherein the substitution is selected from the group consisting of
F316A relative to SEQ ID NO: 21,
L317F relative to SEQ ID NO: 21,
K321V relative to SEQ ID NO: 21,
K321A relative to SEQ ID NO: 21,
H145L relative to SEQ ID NO: 17,
H145F relative to SEQ ID NO: 17,
and combinations thereof; and
isofagomine.
2. The composition of claim 1 , wherein the protein has increased stability relative to human wild-type, recombinant β-glucocerebrosidase.
3. The composition of claim 1 , wherein the substitution is selected from the group consisting of F316A, L317F, K321V, K321A, and combinations thereof relative to SEQ ID NO: 21.
4. The composition of claim 1 , wherein the substitution is selected from the group consisting of H145L and H145F relative to SEQ ID NO: 17.
5. The composition of claim 1 , wherein the protein retains more catalytic activity relative to human wild-type, recombinant β-glucocerebrosidase.
6. The composition of claim 1 , wherein catalytic activity is retained at conditions of about neutral pH and about 37° C.
7. The composition of claim 1 , wherein the composition has a pH of about 7.5.
8. A method for treating a lysosomal storage disease, comprising administering to a human subject in need thereof the composition of claim 1 .
9. The method of claim 8 , wherein the lysosomal storage disease is Gaucher disease.
10. A variant, recombinant human β-glucocerebrosidase protein, wherein the protein has a variation relative to SEQ ID NO: 1, the variation consisting essentially of a substitution at a position selected from the group consisting of 316, 317, 321, 145, and combinations thereof.
11. The protein of claim 10 , wherein the protein has increased stability relative to human wild-type, recombinant β-glucocerebrosidase.
12. The protein of claim 11 , wherein the increased stability is monitored at conditions of about neutral pH and about 37° C. over a period of about three hours.
13. The protein of claim 10 , wherein the position is 316, 317 or 321.
14. The protein of claim 10 , wherein the position is 145.
15. A composition comprising the protein of claim 10 and isofagomine.
16. A method for treating a lysosomal storage disease, comprising administering to a human subject in need thereof the protein of claim 10 .
17. The method of claim 16 , wherein the lysosomal storage disease is Gaucher disease.
18. A method for treating a lysosomal storage disease, comprising administering to a human subject in need thereof the composition of claim 15 .
19. The method of claim 18 , wherein the lysosomal storage disease is Gaucher disease.
20. A composition comprising:
isofagomine; and
a variant, recombinant β-glucocerebrosidase protein characterized as having any one of the following amino acid sequences: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.