GENERATION OF MANNOSE-6-PHOSPHATE CONTAINING RECOMBINANT ALPHA-N-ACETYL GLUCOSAMINIDASE

1 . A method of producing mannose-6-phosphate (M6P)-containing recombinant alpha-N-acetyl-glucosaminidase (Naglu), comprising the steps of:

providing a high mannose containing recombinant Naglu protein; and

contacting the high mannose containing recombinant Naglu protein with N-acetylglucosamine-1-phosphotransferase (GNPT) under conditions that permit phosphorylation of one or more mannose residues on the recombinant Naglu protein, thereby generating M6P-containing recombinant Naglu.

2 . The method of claim 1 , wherein the high mannose containing recombinant Naglu protein comprises substantially Man9(GlcNAc)2, Man8(GlcNAc)2, Man7(GlcNAc)2, Man6(GlcNAc)2, and/or Man5(GlcNAc)2 N-linked oligosaccharides.

3 . The method of claim 1 or 2 , wherein the high mannose containing recombinant Naglu protein comprises substantially Man9(GlcNAc)2 N-linked oligosaccharides.

4 . The method of any one of the preceding claims, wherein the high mannose containing recombinant Naglu protein is produced from cells cultured in the presence of an alpha-mannosidase I inhibitor.

5 . The method of claim 4 , wherein the alpha-mannosidase I inhibitor is selected from the group consisting of deoxymannojirimycin, kifunensine, D-mannonolactam amidrazone, N-butyl-dexoymannojirimycin, and combinations thereof.

6 . The method of claim 4 or 5 , wherein the alpha-mannosidase I inhibitor is kifunensine.

7 . The method of any one of claims 4 - 6 , wherein the cells are mammalian cells.

8 . The method of any one of claims 4 - 7 , wherein the cells are human cells.

9 . The method of any one of claims 4 - 7 , wherein the cells are CHO cells.

10 . The method of any one of claims 4 - 9 , wherein the method further comprises a step of purifying the high mannose containing recombinant Naglu protein from the cells.

11 . The method of any one of the preceding claims, wherein contacting the high mannose containing recombinant Naglu protein with the GNPT is carried out in vitro.

12 . The method of any one of the preceding claims, wherein contacting the high mannose containing recombinant Naglu protein with the GNPT is carried out in a reaction vessel.

13 . The method of any one of the preceding claims, wherein the conditions that permit phosphorylation comprise providing the GNPT in an amount greater than the recombinant Naglu protein.

14 . The method of any one of the preceding claims, wherein the conditions that permit phosphorylation comprise incubating the high mannose containing recombinant Naglu protein with the GNPT in the presence of UDP-GlcNac.

15 . The method of any one of the preceding claims, wherein the conditions that permit phosphorylation comprise incubating the high mannose containing recombinant Naglu protein with the GNPT at about 20° C.

16 . The method of any one of the preceding claims, wherein the method further comprises a step of incubating the M6P-containing recombinant Naglu with N-acetylglucosamine 1-phosphodiester α-N-acetylglucosaminidase.

17 . The method of any one of the preceding claims, wherein the recombinant Naglu protein comprises an amino acid sequence of SEQ ID NO:1 (corresponding to the naturally occurring wild-type Naglu sequence).

18 . The method of any one of the preceding claims, wherein the recombinant Naglu protein is not fused or conjugated to a peptide.

19 . A mannose-6-phosphate (M6P)-containing recombinant alpha-N-acetyl-glucosaminidase (Naglu) protein produced by a method of any one of the preceding claims.

20 . A composition comprising recombinant alpha-N-acetyl-glucosaminidase (Naglu) protein characterized by mannose-6-phosphate (M6P) containing glycans.

21 . The composition of claim 20 , wherein the M6P containing glycans comprise capped-M6P, 1M6P and/or 2M6P containing glycans.

22 . The composition of claim 20 or 21 , wherein the recombinant Naglu protein is further characterized by high mannose containing glycans and/or sialic acid containing glycans.

23 . The composition of claim 22 , wherein the sialic acid containing glycans comprise one, two, three, and/or four sialic acid containing glycans.

24 . A composition comprising recombinant alpha-N-acetyl-glucosaminidase (Naglu) protein characterized by substantially high mannose containing glycans.

25 . The composition of claim 24 , wherein the high mannose containing glycans comprise Man9(GlcNAc)2, Man8(GlcNAc)2, Man7(GlcNAc)2, Man6(GlcNAc)2, and/or Man5(GlcNAc)2 N-linked oligosaccharides.

26 . The composition of claim 24 or 25 , wherein the high mannose containing glycans comprises substantially Man9(GlcNAc)2 N-linked oligosaccharides.

27 . The composition of any one of claims 24 - 26 , wherein the recombinant Naglu protein is produced from mammalian cells.

28 . The composition of claim 27 , wherein the mammalian cells are human cells.

29 . The composition of claim 27 , wherein the mammalian cells are CHO cells.

30 . The composition of any one of claims 20 - 29 , wherein the recombinant Naglu protein comprises an amino acid sequence of SEQ ID NO:1 (corresponding to the naturally occurring wild-type Naglu sequence).

31 . The composition of any one of claims 20 - 30 , wherein the recombinant Naglu protein is not fused or conjugated to a peptide.

32 . The composition of any one of claims 20 - 23 , 30 and 31 , wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

33 . A method of treating Sanfilippo Syndrome Type B (MPS IIIB) comprising administering to a subject in need of treatment a therapeutically effective amount of the composition of any one of claims 20 to 32 .