Glycosylated lysosomal proteins, method of production and uses

1. A lysosomal protein composition comprising a plurality of lysosomal proteins that are glycosylated according to a glycosylation pattern, wherein said glycosylation pattern has at least 60% paucimannosidic N-glycans (molar %), wherein the paucimannosidic N-glycan comprises the structure of formula 1:

wherein a square represents N-Acetylglucosamine (GlcNAc), a circle represents mannose (Man), and a circle with a T represents a terminal mannose, wherein one or more of the GlcNAc or Man subunits may be α1,3-fucosylated, α1,6-fucosylated and/or β1,2-xylosylated.

2. The lysosomal protein composition according to claim 1 wherein the lysosomal protein comprises at least one of α-Galactosidase; α-Mannosidase; Aspartylglucosaminidase; β-Mannosidase; Acid Ceremidase; α-Fucosidase; β-Galactosidase, β-Hexosaminidase activator protein; Galactocerebrosidaseb; Galactoceramidase; lysosomal acid lipase (LAL); α-Iduronidase; Iduronate-2-sulfatase; Glucosamine-N-sulfatase; Heparansulfatsulfamidase (SGSH); α-N-acetyl-glucosaminidase (NAGLU); α-glucosaminide-N-acetyltransferase; N-Acetygalactosamine-6-sulfatase; β-Galactosidase; N-Acetygalactosamine-4-sulfatase; β-Glucoronidase; Neuraminidase; Sphingomyelinase; Sphingomyelin phosphodiesterase; Acid alpha-1,4-glucosidase; β-Hexosaminidase, or its a subunit; Alpha-N-acetylgalactosaminidase (NAGA); α-Galactosaminidase; β-Hexosaminidase A; Galactose-6-sulfate sulfatase; and Hyaluronidase.

3. The lysosomal protein composition according to claim 1 , wherein one or more of the GlcNAc or Man subunits may be α1,6-fucosylated.

4. The lysosomal protein composition according to claim 1 wherein the glycosylation pattern has at least 1% N-glycans of the formula GlcNAc 2 -Hex 2 -methyl-Hex; and/or wherein the glycosylation pattern comprises the following N-glycans:

0% to 35% -GlcNAc 2 -(Man 2 methyl-Hex);

30% to 80% -GlcNAc 2 -Man 3 ;

0% to 30% -GlcNAc 2 -Man 3 -GlcNAc;

0% to 15% -GlcNAc 2 -Man 3 -GlcNAc 2 ;

0% to 5% -GlcNAc 2 -Man 3 -Hex 2 ,

0% to 11% -GlcNAc 2 -Man 3 -Hex 3 ;

0% to 10% -GlcNAc 2 -Man 3 -Hex 4 ;

0% to 10% -GlcNAc 2 -Man 3 -Hex 5 ;

wherein all of these compounds together amount to 100% or less than 100%, wherein GlcNAc is a N-Acetylglucosamine subunit, Man is a mannose subunit, Hex is a hexose subunit, methyl-Hex is a methylated hexose subunit; with the proviso that -GlcNAc 2 -(Man 2 methyl-Hex) and -GlcNAc 2 -Man 3 together amount to at least 45%, (all % are molar %); wherein the GlcNAc at the reducing end of the glycan is not fucosylated in any one of the above N-glycans; wherein a Man at a branching point, is not xylosylated in any one of the above N-glycans.

5. The lysosomal protein composition according to claim 1 comprising non-phosphorylated lysosomal proteins.

6. An in vitro method of processing a lysosomal protein comprising a complex N-glycan, said method comprising providing the lysosomal protein of claim 1 in a sample and contacting the sample with a bryophyte HEXO, whereby the bryophyte HEXO enzyme cleaves terminal GlcNAc residues from the lysosomal protein thereby producing a paucimannosidic N-glycan.

7. The method of treatment of a lysosomal storage disease comprising administering a lysosomal protein composition according to claim 1 .

8. The lysosomal protein composition according to claim 2 , wherein the α-Galactosidase is selected from α-Galactosidase A (GLA); ß-Glucoceramidase, and β-glucosidase (glucocerebrosidase).

9. The lysosomal protein composition according to claim 4 , wherein the glycosylation pattern further comprises at least one of the following N-glycans:

1% to 30% -GlcNAc 2 -(Man 2 methyl-Hex);

40% to 70% -GlcNAc 2 -Man 3 ;

4% to 22%, -GlcNAc 2 -Man 3 -GlcNAc;

2% to 12% -GlcNAc 2 -Man 3 -GlcNAc 2 ;

0% to 3% -GlcNAc 2 -Man 3 -Hex 2 ,

1% to 8% -GlcNAc 2 -Man 3 -Hex 3 ;

1% to 7% -GlcNAc 2 -Man 3 -Hex 4 ;

1% to 7% -GlcNAc 2 -Man 3 -Hex 5 .

10. The lysosomal protein composition according to claim 4 , wherein methyl-Hex is 2-0 methyl hexose.

11. The lysosomal protein composition according to claim 4 , wherein Hex is Man.

12. The lysosomal protein composition according to claim 1 , wherein the glycosylation pattern has at least 45% paucimannosidic N-glycans (molar %) that are not α1,3-fucosylated and that are not β1,2-xylosylated.

13. The in vitro method of processing a lysosomal protein comprising a complex N-glycan according to claim 6 , wherein said bryophyte HEXO is an HEXO3 enzyme.

14. The method of treatment of a lysosomal storage disease according to claim 7 wherein the disease and lysosomal protein are selected from the following table:

Disease

lysosomal protein

Fabry Disease

α-Galactosidase A (GLA)

Gaucher's Disease

β-Glucoceramidase, β-glucosidase

(glucocerebrosidase)

Alpha-Mannosidosis

α-Mannosidase

Aspartylglucosaminuria

Aspartylglucosaminidase

Beta-Mannosidosis

β-Mannosidase

Farber Disease

Acid Ceremidase

Fucosidosis

α-Fucosidase

GM1-Gangliosidosis

β-Galactosidase, β-Hexosaminidase

activator protein

Krabbe Disease

Galactocerebrosidase; Galactoceramidase

Lysosomal Acid Lipase

lysosomal acid lipase (LAL)

(LAL) Deficiency

Mucopolysaccharidoses

α-Iduronidase

Iduronate-2-sulfatase

Glucosamine-N-sulfatase;

Heparansulfatsulfamidase (SGSH)

α-N-acetyl-glucosaminidase (NAGLU)

α-glucosaminide-N-acetyltransferase

N-Acetygalactosamine-6-sulfatase

Galactose-6-sulfate sulfatase

β-Galactosidase

N-Acetygalactosamine-4-sulfatase

β-Glucoronidase

Hyaluronidase

Niemann Pick Disease

Sphingomyelinase

Pompe Disease

(Acid) alpha-1,4-glucosidase

Sandhoff Disease

β-Hexosaminidase, or its α subunit

Schindler Disease

Alpha-N-acetylgalactosaminidase (NAGA);

α-Galactosaminidase

Tay-Sachs Syndrome

β-Hexosaminidase A

Sialidosis

Neuraminidase.

15. The lysosomal protein composition according to claim 1 , wherein one or more of the mannose is methylated.