Title of Invention

A PHARMACEUTICAL COMPOSITION COMPRISING A HIGH MANNOSE GLUCOCEREBROSIDASE (hmGCB)

Abstract

A method of producing a high mannose glucocerebrosidase (hmGCB), comprising:providing a cell which is capable of expressing glucocerebrosidase (GCB) and which is maintained in culture;contacting the cell with kifunensine such that the removal of at least one mannose residue distal to the pentasaccharide core of the precursor oligosaccharide of GCB is prevented; and allowing the cell to produce hmGCB, to thereby produce an hmGCB preparation.

Full Text

FORM 2 THE PATENTS ACT 1970 [39 OF 1970] & THE PATENTS RULES, 2003 COMPLETE SPECIFICATION [See Section 10; rule 13] "HIGH MANNOSE PROTEINS AND METHODS OF MAKING HIGH MANNOSE PROTEINS" SHIRE HUMAN GENETIC THERAPIES INC. of 195 Albany Street, Cambridge, Massachusetts 02139, United States of America, The following specification particularly describes and ascertains the nature of this invention and the manner in which it is to be performed: WO 02/15927 PCT/US01/25882 HIGH MANNOSE PROTEINS AND METHODS OF MAKING HIGH MANNOSE PROTEINS Background of the Invention Gaucher disease is an autosomal recessive lysosomal storage disorder characterized by a deficiency in the lysosomal enzyme, glucocerebrosidase (GCB). GCB hydrolyzes the glycolipid glucocerebroside that is formed after degradation of glycosphingolipids in the membranes of white blood cells and red blood cells. The deficiency in this enzyme causes glucocerebroside to accumulate in large quantities in the lysosomes of phagocytic cells located in the liver, spleen and bone marrow of Gaucher patients. Accumulation of these molecules causes a range of clinical manifestations including splenomegaly, hepatomegaly, skeletal disorder, thrombocytopeniaand anemia- (Beutler et al. Gaucher disease; In: The Metabolic and Molecular Bases of Inherited Disease (McGraw-Hill, Inc, New York, 1995) pp.2625-2639) Treatments for patients suffering from this disease include administration of analgesics for relief of bone pain, blood and platelet transfusions and, in some cases, splenectomy. Joint replacement is sometimes necessary for patients who experience bone erosion. Enzyme replacement therapy with GCB has been used as a treatment for Gaucher disease. Current treatment of patients with Gaucher disease includes administration of a carbohydrate remodeled GCB derived from human placenta or Chinese hamster ovary (CHO) cells transfected with a GCB expression construct and known as alglucerase or imiglucerase, respectively. The treatment is extremely expensive in part because of the cost of removing sugars from GCB to expose the trimannosyl core of complex glycans in order to target the enzyme to mannose receptors on cells of reticuloendothelial origin. The scarcity of the human placental tissue (in the case of alglucerase), complex purification protocols, and the relatively large amounts of the carbohydrate remodeled GCB required all contribute to the cost of the treatment. WO 02/15927 PCTfUSGl/25882 Summary of the Invention The invention is based, in part on the discovery that by preventing removal of one or - more masnose residues distal from the pentasaccharide core of a precursor oligosaccharide chain of a protein, e.g., a lysosomal storage en2yme, a high mannose protein such as high 5 mannose glucocerebrosidase (hmGCB) can be obtained These high mannose proteins can be used to target the protein to cells "which express mannose receptors. Such cells can include cells of reticuloendothelial origin including macrophages, Kupffer cells and histiocytes. Thus, these high mannose proteins can be used, for example, to target delivery by receptor mediated endocytosis to lysosomes to treat various lysosomal storage diseases. 10 In particular, hmGCB has been found to efficiently target mannose receptors. Mannose receptors are present on macrophages and other cells, e.g.* dendritic cells, cardiomyocytes and glial cells, and are instrumental in receptor-mediated endocytosis. The absence of GCB in patients with Gaucher disease leads to accumulation of glucocerebroside, primarily in cells of reticuloendothelial origin including macrophages, Kupffer cells and 15 histiocytes. Because these cells express mannose receptors on their surface, hmGCB can be used to effectively target delivery of a corrective enzyme to the lysosomes through receptor-mediated endocytosis, thereby treating Gaucher disease. Surprisingly, it was found that hmGCB uptake by macrophages was increased as compared to uptake of GCB secreted from cells. 20 Accordingly, in one aspect, the invention features a method of producing a preparation of high mannose glucocerebrosidase (hmGCB). The method includes: providing a cell which is capable of expressing GCB; and allowing production of GCB having a precursor oligosaccharide under conditions which prevent the removal of at least one maimose residue distal to the pentasacchande core 25 of the precursor ohgosaccharide of GCB, to thereby produce an hmGCB preparation. In a preferred embodiment, the GCB is human GCB. In a preferred embodiment, the cell is a human cell. In a preferred embodiment, the removal of: one or more a 1,2 mannose residue(s) distal to the pentasaccharide core is prevented; an a 1,3 mannose residue distal to the 30 pentasaccharide core is prevented; and/or an a 1,6 mannose residue distal to the WO 02/1593 PCT/US01/25882 pentassccnHii core is ^s^ented. Preferably, the removal of one or more a I_2 mannose ..: residue(s; oisrai to Trie pentasaccharide cere is prevented. In a preferred embodiment, the method can include contacting the cell with a substance which prevents the removal of at least one mannose residue distal to the 5 pentasaccharide core of the precursor oligosaccharide ofGCB^ e.g., prevents removal of one or more a 1.2 mannose residue(s) distal to the pentasaccharide core, an a 1,3 mannose residue distal to the pentasaccharide core and/or an a 1,6 mannose residue distal to the pentasaccharide core. Preferably, the removal of one or more a 1.2 mannose(s) residue distal to the pentasaccharide core is prevented. 10 In a preferred embodiment, the method includes contacting the cell with a substance which prevents the removal of at least one mannose residue distal to the pentasaccharide core of the precursor oligosaccharide of GCB, wherein the substance is a mannosidase inhibitor. The mannosidase inhibitor can be a class 1 processing mannosidase inhibitor, a class 2 processing mannosidase inhibitor or both. The class 1 processing mannosidase inhibitor can 15 be one or more of: kifunensine, deoxymannojirimycin, or a similar inhibitor. Preferably, the class 1 processing mannosidase inhibitor is kifunensine. Useful class 2 processing mannosidase inhibitors can include one or more of: swainsonine, mannostatin, 6-deoxy-l, 4-dideoxy-1, 4-imino-D-mannitol (6-deoxy-DIM), and 6-deoxy-6-fmoro-l, 4-dideoxy-l, 4-imino-D--ma^mitol (6-deoxy-6-fluoro-DIM). Preferably, the class 2 processing mannosidase 20 inhibitor is swainsonine. In a preferred embodiment, a mannosidase inhibitor is present at a concentration between about 0.025 to 20.0 M-g/ml, 0.05 to 10 jig/ml, 0.05 to 5 ug/ml, preferably between about 0.1 to 2.0 u.g/ml. In a preferred embodiment, the method further includes contacting the cell with a 25 class I processing mannosidase inhibitor and a class 2 processing mannosidase inhibitor. In a preferred embodiment, the class 1 processing mannosidase inhibitor is present at a concentration between about 0.025 to 20.0 ug/ml, 0.05 to 10 U-g/ml, 0.05 to 5 ug/ml, preferably between about 0.1 to 2.0 ug/ml; the class 2 processing mannosidase inhibitor is present at a concentration between about 0.025 to 20.0 ug/ml, 0.05 to 10 ug/ml, 0.05 to 5 30 ug/ml, preferably between about 0.1 to 2.0 pg/ml; each of the class 1 processing and class 2 WO 02/15927 PCT/US01/25882 processing mannosidase inidbiKKS are Tsressr-s a concentration between about 0.025 toJZQ.-O ug/mh 0.05 to 10 ug/rnl. 0.05 to 5 u^mL ps&abry between about 0.1 to 2.0 ug/ml: the total concentration of the class 1 processing and class 2 processing mannosidase inhibitors present is between about 0.025 to 40.0 tig/ml 0.05 to 20 ug/ml, 0.05 to 10 ug/ml, preferably between about 0.1 to 4.0 ug/ml. In a preferred embodiment, the cell carries a mutation for, e.g., a knockout for, at least one Golgi processing mannosidase. The mutation can he one which reduces the expression of the gene, reduces protein or activity levels, or alters the distribution or other post translational modifications of the mannosidase, e.g., the processing of the carbohydrate chains. The mutation can be one which reduces the level of the Golgi processing mannosidase activity, e.g., one which reduces gene expression, e.g., a null mutation, e.g., a deletion, a frameshift or an insertion, ha a preferred embodiment the mutation is a knockout, e.g., in the mannosidase gene. The mutation can affect the structure (and activity of the protein), and can, e.g., be a temperature sensitive mutation or a truncation. In a preferred embodiment the cell carries a mutation, e.g., a knockout, for: a class 1 processing mannosidase; a class 2 processing mannosidase; a class 1 processing mannosidase and a class 2 processing mannosidase. In a preferred embodiment, the class 1 processing mannosidase is: Golgi mannosidase IA; Golgi mannosidase IB; Golgi mannosidase IC; or combinations thereof. In a preferred embodiment, the class 2 processing mannosidase is: Golgi mannosidase II. In a preferred embodiment, the cell includes a nucleic acid sequence, such as an antisense molecule or ribozyme, which can bind to or inactivate a cellular mannosidase nucleic acid sequence, e.g., mRNA. and inhibit expression of the protein. In a preferred embodiment, the nucleic acid sequence is: a class 1 processing mannosidase antisense molecule; a class 2 processing mannosidase antisense molecule; both a class 1 processing mannosidase antisense molecule and a class 2 processing mannosidase antisense molecule. In a preferred embodiment, the class 1 processing mannosidase is: Golgi mannosidase IA; Golgi mannosidase IB; Golgi mannosidase IC; and combinations thereof. In a preferred embodiment, the class 2 processing mannosidase is: Golgi mannosidase H. WO 02/15927 PCT/US01/25882 si arsTle^red embodiment the cell includes a molecule, e.g., an exogenously . soppliec moiscale, which binds and inhibits a mannosidase. The molecule can be, e.g., a single cham antibody, an intracellular protein or a competitive or non-competitive inhibitor. In a preferred embodiment, the hmGCB molecule includes a carbohydrate chain having at least four mannose residues. For example, the hmGCB molecule has at least one carbohydrate chain having five mannose residues, the hmGCB molecule has at least one carbohydrate chain having six mannose residues, the hmGCB molecule has at least one carbohydrate chain having seven mannose residues, the hmGCB molecule has at least one carbohydrate chain having eight mannose residues, the hmGCB molecule has at least one carbohydrate chain having nine mannose residues. Preferably, the hmGCB molecule has at least one carbohydrate chain having five, eight or nine mannose residues. In a preferred embodiment, the hmGCB produced (either one or more hmGCB molecules or the preparation as a whole) has a ratio of mannose residues to GIcNAc residues which is greater than 3 mannose residues to 2 GIcNAc residues, preferably the ratio of mannose to GIcNAc is 4:2, 5:2, 6:2, 7:2, 8:2, 9:2, more preferably the ratio of mannose to GIcNAc is 5:2, 8:2 or 9:2. In a preferred embodiment, the removal of one or more mannose residues distal to the pentasaccharide core is prevented on one, two, three or four of the carbohydrate chains of an hmGCB molecule. In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or all of the hmGCB molecules of the preparation have at least one, and preferably two, three or four carbohydrate chains in which the removal of one or more mannose residues distal to the pentasaccharide core has been prevented. In a preferred embodiment, the hmGCB preparation is a relatively heterogeneous preparation. Preferably, less than 80%, 70%, 60%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 1% of the carbohydrate chains in the hmGCB preparation have the same number of mannose residues in addition to the pentasaccharide core. For example, the ratio of carbohydrate chains having the same number of mannose resides in addition to the pentasaccharide core to carbohydrate chains having a different number of mannose residues WO 02/15927 PCT/US01/25882 can be about: 60% 40% 50%:50%: 4Q%:60%; 30%:70%: 25%:75%; 20%:SD%; 15%:85%; 10%:90%;5%or lessar 95EA-ar3BEze. In a preferred embodiment, activity of Golgi mannosidase IA and/or IB and/or IC is inhibited and at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the carbohydrate chains in the hmGCB preparation have five or more mannose residues, e.o., five, six, seven, eight and/or nine mannose residues. Tn a preferred embodiment, activity of Golgi mannosidase I is inhibited and the ratio of carbohydrate chains having five or more mannose residues to carbohydrate chains having four or less mannose residues is about 60%:40%; 70%:30%; 75%:25%; 80%:20%; 85%:15%; 90%:10%; 95%:5%; 99%:1%; or 100%:0%. In a preferred embodiment activity of Golgi mannosidase II is inhibited and at least about 60%, 70%, 75%, 80%s 85%, 90%, 95%, 98%, 99% or 100% of the carbohydrate chains in the hmGCB preparation have five or more mannose residues, e.g., five, six, seven, eight and/or nine mannose .residues. In a preferred embodiment, activity of Golgi mannosidase IE is inhibited and the ratio of carbohydrate chains having five or more mannose residues to • carbohydrate chains having four or less mannose residues is about 60%:40%; 70%:30%; 75%:25%; 80%:20%; 85%:15%; 90%:10%; 95%:5%; 99%:1%; or 100%:0%. In a preferred embodiment, the cell includes an exogenous nucleic acid sequence which includes a GCB coding region. In a preferred embodiment, the cell further includes a regulatory sequence, an endogenous or exogenous regulatory sequences which functions to regulate expression of the exogenous GCB coding region. In a preferred embodiment, the cell includes an exogenous regulatory sequence which functions to regulate expression of an endogenous GCB coding sequence, e.g., the regulatory sequence is integrated into the genome of the cell such that it regulates expression of an endogenous GCB coding sequence. In a preferred embodiment, the regulatory sequence includes one or more of: a promoter, an enhancer, an upstream activating sequence (UAS), a scaffold-attachment region or a transcription factor-binding site. In a preferred embodiment, the regulatory sequence. includes: a regulatory sequence from a metallothionein-I gene, e.g., a mouse merallothionein-I gene, a regulatory sequence from an SV-40 gene, a regulatory sequence from a WO 02/15927 PCT/USO1/25882 cytomegalovirus gene, a regulatory seqassceiiK" acsllagea gene, a regulatory sequence irom an acttn gene, a regulatory seqn^oe £vm £n mnmmcglabulm gens. 2 regulatory sequence from the HMG-CoA reductase gene, or a regulatory sequence from the EF-la gene. In a preferred embodiment the ceil is: a eukaryotic cell. In a preferred embodiment, 5 the cell is of fungal, plant or animal origin, e.g., vertebrate origin. In a preferred embodiment, the cell is: a mammalian ceil, e.g., a primary or secondary mammalian cell, e.g., a fibroblast, a hematopoietic stem cell, a myoblast, a keratrnocyte, an epithelial cell, an endothelial cell, a glial cell, a neural cell, a cell comprising a formed element of the blood, a muscle cell and precursors of these somatic cells; a transformed or immortalized cell line, to Preferably, the cell is a human cell. Examples of immortalized human cell lines useful in the present method include, but are not limited to: a Bowes Melanoma cell (ATCC Accession No. CRL 9607), a Daudi cell (ATCC Accession No. CCL 213), a HeLa cell and a derivative of 3 HeLa cell (ATCC Accession Nos. CCL2 CCL2. J and CCL 2.2), a BL-6D cell (ATCC Accession No. CCL 240), anHT-1080 ceil (ATCC Accession No. CCL 121), a Jurkat ceil 15 (ATCC Accession No. TIB 152), a KB carcinoma ceil (ATCC Accession No. CCL i 7), a K-562 leukemia cell (ATCC Accession No. CCL 243), a MCF-7 breast cancer cell (ATCC Accession No. BTH 22), a MOLT-4 cell (ATCC Accession No. 1582), a Namalwa cell (ATCC Accession No. CRL 1432), aRaji cell (ATCC Accession No. CCL 86), aRPMI 8226 cell (ATCC Accession No. CCL 155), a U-937 cell (ATCC Accession No. 1593), WI-20 28VA13 sub line 2R4 cells (ATCC Accession No. CLL 155), a CCRF-CEM cell (ATCC Accession No. CCL 119) and a 2780AD ovarian carcinoma cell (Van Der Blick et al., Cancer Res. 48:5927-5932,1988), as well as hsgrobybndaTna. cells produced by fusion of human cells and cells of another species. In another embodiment, the immortalized cell line can be cell line other than a human cell line, e.g.;, a CHO cell Ihie, a COS cell line. In another 25 embodiment, the cell can be a from a clonal cell strain or clonal cell line. In a preferred embodiment, a population of cells which are capable of expressing hmGCB is provided, and at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%o, 99% or all of the cells, produce hmGCB molecules with at least one carbohydrate chain, and preferably two,-three, or four carbohydrate chains, having the specified number of 30 mannose residues. WO 02/15927 PCT/US01/25882 In a preferred embodiment, the cell is cultured in caimr= medium which includes at"" . least one mannosidase inhibitor. In a preferred emborr^ag. the msdiod further includes obtaining the hmGCB from the medium in which the ceil is cultured. 5 -In another aspect, the invention features a method of producing a preparation of hmGCB.' The method includes: providing a cell which is capable of expressing GCB; and allowing production of GCB having a precursor oligosaccharide under conditions which inhibit class 1 processing mannosidase activity and class 2 processing mannosidase 10 activity such that the removal of at least one mannose residue distal to the pentasaccharide core of the precursor oligosaccharide of GCB is prevented, to thereby produce an hmGCB preparation. In a preferred embodiment, the GCB is human GCB. In a preferred embodiment, the cell is a human ceil. 15 In a preferred embodiment, the removal of: one or more al^ mannose residue(s) distal to the pentasaccharide core is prevented; an a 1,3 mannose residue distal to the pentasaccharide core is prevented; and/or an a 1,6 mannose residue distal to the pentasaccharide core is prevented. Preferably, the removal of one or more a 1,2 mannose ' residue(s) distal to the pentasaccharide core is prevented. 20 In a preferred embodiment, the method can include: contacting the cell with a substance which inhibits a class 1 processing mannosidase activity and a substance which inhibits a class 2 processing mannosidase activity thereby p:evenirag the removal of at least one mannose residue distal to the pentasaccharide core of the precursor ohgosaccharide of GCB. In a preferred embodiment, the substances prevent removal of one or more a 1,2 25 mannose residue distal to the pentasaccharide core. In a preferred embodiment, the method includes contacting the cell with a substance which inhibits a class 1 processing mannosidase activity and a substance which inhibits a class 2 processing mannosidase activity, wherein the substances are a class 1 processing mannosidase inhibitor and a class 2 processing mannosidase inhibitor. In a preferred 30 embodiment, the class 1 processing mannosidase inhibitor can be one or more of: WO 02/15927 PCT/US01/25882 3 gnTTTr?--^~TTrtrg- Prcisrahfy, Idfunensine, deoxymarmojirimyciri, or a In a preferred embodiment, a class 1 processing mannosidase inhibitor is present at a concentration between about 0.025 to 20.0 ug/ml, 0.05 to 10 y.g/ml,0.05 to 5 jig/ml, preferably between about 0.1 to 2.0 fig/rnl; a class 2 processing mannosidase inhibitor is present at a concentration between about 0.025 to 20.0 p.g/ml, 0.05 to 10 fig/ml, 0.05 to 5 10 M-g/nil, preferably between about 0.1 to 2.0 ug/ml; each of the class 1 processing and class 2 processing mannosidase inhibitors are present at a concentration between about 0.025 to 20.0 ug/ml, 0.05 to 10 jig/ml, 0.05 to 5 ug/ml, preferably between about 0.1 to 2.0 |ig/ml; the total concentration of the class 1 processing and class 2 processing mannosidase inhibitors present is between about 0.025 to 40.0 ug/ml, 0.05 to 20 (J-g/ml, 0.05 to 10 p.g/ml, preferably between 15 about 0.1 to 4.0 Ug/ml. In a preferred embodiment, the cell carries a mutation for, e.g., a knockout for, a class 1 mannosidase and a class 2 mannosidase. The mutation can be one which reduces the expression of the gene, reduces protein or activity levels, or alters the distribution or other post translation! modifications of the mannosidase, e.g., the processing of the carbohydrate 20 chains. The mutation can be one which reduces the level of a class 1 processing mannosidase and/or a class 2 processing mannosidase activity, e.g., one which reduces gene expression, e.g., a null mutation, e.g., a deletion, a irameshrrt, or an insertion. In a preferred embodiment, the mutation is a knockout in the mannosidase gene. The mutation can affect the structure (and activity of the protein), and can, e.g., be a temperature sensitive mutant. In 25 a preferred embodiment, the class 1 processing mannosidase is: Golgi mannosidase IA; Golgi mannosidase IB; Golgi mannosidase IC; combinations thereof. In a preferred embodiment, the class 2 processing mannosidase is: Golgi mannosidase H. In a preferred embodiment, the cell includes both a class 1 processing mannosidase antisense molecule and a class 2 processing mannosidase antisense molecule. In a preferred 30 embodiment, the class 1 processing mannosidase is: Golgi mannosidase IA; Golgi WO 02/15927 PCT/US01/25882 mannosidase IB; Golgi mannosidase IC; combinations thereof m. a prefered sz^odiznent the class 2 processing mannosidase is: Golgi mannosidase IL In a preferred embodiment, the cell includes a molecule, e.g.? an exogenously supplied molecule, which binds and inhibits a mannosidase. The molecule can be, e.g., a single chain antibody, an intracellular protein or a competitive or non-competitive inhibitor. In a preferred embodiment, the class 1 processing mannosidase activity and the class 2 mannosidase activity can be inhibited by different mechanisms. For example, a class 1 processing mannosidase activity can be inhibited by contacting the cell with a substrate which inhibits a class 1 processing mannosidase, e.g., a class 1 mannosidase inhibitor, and the class 2 processing mannosidase can be inhibited by using a cell which is aicnockout of a class 2 mannosidase and/or includes a class 2 mannosidase antrseiise molecule. In another preferred embodiment, a class 2 processing mannosidase activity can be inhibited ~by contacting the cell with a substrate which inhibits a class 2 processing mannosidase, e.g., a class 2 mannosidase inhibitor, and the class 1 processing mannosidase can be inhibited by using a cell which is a knockout of a class 1 mannosidase and/or includes a class 1 mannosidase antisense molecule. In a preferred embodiment, the hmGCB molecule includes a carbohydrate chain having at least four mannose residues. For example, the hmGCB molecule has at least one carbohydrate chain having five mannose residues, the hmGCB molecule has at least one carbohydrate chain having six mannose residues, the hmGCB molecule has at least one carbohydrate chain having seven mannose residues, the hmGCB molecule has at least one carbohydrate chain having eight mannose residues, the hmGCB molecule has at least one carbohydrate chain having nine mannose residues. Preferably, the hmGCB molecule has at least one carbohydrate chain having five, eight or nine mannose residues. In a preferred embodiment, the hmGCB produced (either one or more hmGCB molecules or the preparation as a whole) has a ratio of mannose residues to GlcNAc residues which is greater than 3 mannose residues to 2 GlcNAc residues, preferably the ratio of mannose to GlcNAc is 4:2, 5:2, 6:2, 7:2,. 8:2, 9:2, more preferably the ratio of mannose to GlcNAc is 8:2 or 9:2. WO 02/15927 PCT/U30L'25S82 In a preferred embodiment, the removal of one or more mannose residnas dissltD fhs . psntasscciiEride core is prevented on one, two. three or four of the carbohydr^ie ^hsiris of"n±e hmGCB molecule. . In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, S5°/% 90%, 95%, 98%, 99% or all of the hmGCB molecules of the preparation have at least one, and preferably two, three or four carbohydrate chains in which the removal of one or more mannose residues distal to the pentasaccharide core has been prevented. : In a preferred embodiment, the hmGCB preparation is a relatively heterogeneous preparation. Preferably, less than 80%, 70%, 60%, 50%, 45%, 40%, 35%, 30%, 25%, 20%o, 15%, 10%, 5% or 1% of the carbohydrate chains in the hmGCB preparation have the same number of mannose residues in addition to the pentasaccharide core. For example, tie ratio of carbohydrate chains having the same number of mannose resides in addition to the pentasaccharide core to carbohydrate chains having a different number of mannose residues can be about: 60%;40%; 50%:50%; 40%:60%; 30%:70%; 25%:75%; 20%:80%; 15%:85%; 10%:90%; 5% or less:95% or more. In a preferred embodiment, activity of a class 1 processing mannosidase, e.g., Golgi mannosidase IA and/or Golgi mannosidase IB and/or Golgi mannosidase IC, and activity of a class 2 processing mannosidase, e.g., Golgi mannosidase H, are inhibited and at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100%, of the carbohydrate chains in the hmGCB preparation have five or more mannose residues, e.g., five, six, seven, eight and/or nine mannose residues. In a preferred embodiment, activity of a class 1 processing mannosidase, e.g., Golgi mannosidase IA and/or Golgi mannosidase IB and/or Golgi mannosidase IC, and activity of a class 2 processing mannosidase, e.g., Golgi mannosidase II, are inhibited and the ratio of carbohydrate chains having five or more mannose residues to carbohydrate chains having four or less mannose residues, respectively, is about 60%:40%; 70%:30%; 75%:25%; 80%:20%; 85%:15%; 90%:10%; 95%:5%; 99%: 1%; or 100%:0%. In a preferred embodiment, the cell includes an exogenous nucleic acid sequence which includes a GCB coding region.. In apreferred embodiment, the cell further includes a regulatory sequence, an endogenous or exogenous regulatory sequence, which functions to regulate expression of the exogenous GCB coding region. WO 02/15927 PCTAJSOl/25882 In a preferred embodiment the cell includes an exogenous regulatory sequence which functions to regulate expression of an endogenous GCB coding sequence, e.g.s the regulatory sequence is integrated into the genome of the cell such that it regulates expression of an endogenous GCB coding sequence. In a preferred embodiment, the regulatory sequent includes one or more of: a promoter, an enhancer, an upstream activating sequence (UAS), a scaffold-attachment region or a transcription factor-binding site. In a preferred embodiment, the regulatory sequence includes: a regulatory sequence from a metallothionein-I ^Q> e.g., a mouse metallothionem-I gene, a regulatory sequence from an SY-40 gene, a regulatory sequence from a cytomegalovirus gene, a regulatory sequence from a collagen gene, a regulatory sequence from an actin gene, a regulatory sequence from an imrnun°gl°buu*n genej a regulatory sequence from the HMG-CoA reductase gene, or a regulatory sequence from the EF-la gene. In a preferred embodiment., the cell is: a eukaryotiP cell. In a preferred embodiment, the cell is of fungal, plant or animal origin, e.g., vertebrate origin. In a preferred embodiment, the cell is: a mammalian cell, e.g., a primary or secondary mammalian cell, e.g., a fibroblast, a hematopoietic stem cell, a myoblast, a keratinocyte, an epithelial cell, an endothelial cell, a glial cell, a neural cell, a cell comprising a formed element of the blood, a muscle cell and precursors of these somatic cells; a transfPnried or immortalized cell line. Preferably, the cell is a human cell. Examples nfimmnrtalized human cell lines useful in the present method include, but are not limited to: a Bowes Melanoma cell (ATCC Accession No. CRL 9607), a Daudi cell (ATCC Accession No. CCL 213), a HeLa cell and a derivative of a HeLa cell (ATCC Accession Nos. CCL2 CCL2.1 and CCL 2.2), a HL-60 cell (ATCC Accession No. CCL 240), an HT-2080 cell (ATCC Accession No. CCL 121), a Juricat cell (ATCC Accession No. TIB 152), aKB carcinoma cell (ATCC Accession NO. CCL 17), al-562 leukemia cell (ATCC Accession No. CCL 243), a MCF-7 breast cancer cell (ATCC Accession No. BTH 22), a MOLT-4 cell (ATCC Accession No. 1582), a Namalwa cell (ATCC Accession No. CRL 1432), a Raji cell (ATCC Accession No. CCL 86), a RPMI 8226 cell (ATCC Accession No. CCL 155), a U-937 cell (ATCC Accession No. 1593), WI-28VA13 sub line 2R4 cells (ATCC Accession No. CLL 1^5), a CCRF-CEM cell (ATCC Accession No. CCL 119) and a 2780AD ovarian carcinoma cell (Van Der BHck et al., Cancer Res. 48:5927-5932, 1988), as well as heterohybridoma cells produced by fusion of human WO 02/15927 PCT/USO1/25882 ceils and cells of another species. In snother embodiment, the immortalized cell line can be cell line other than a human cell fine, e.g., a CEO cell line, a COS cell line. In another embodiment, the cell can be from a clonal cell strain or clonal cell line. In a preferred embodiment, a population of cells which are capable of expressing 5 hmGCB is provided, and at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or all of the cells produce hmGCB with at least one carbohydrate chain, preferably two, three, or four carbohydrate chains, having the specified number of mannose residues. In a preferred embodiment, the cell is cultured in a culture medium which includes at least one class 1 processing mannosidase inhibitor and at least one class 2 processing 10 mannosidase inhibitor. In a preferred embodiment, the method further includes obtaining the hmGCB from the medium in which the cell is .cultured. In another aspect, the invention features a method of producing a preparation of hmGCB. The method includes: 15 providing a cell into which a'nucleic acid sequence comprising an exogenous regulatory sequence has been introduced such that the regulatory sequence regulates the expression of an endogenous GCB coding region; and allowing production of GCB having a precursor oligosaccharide under conditions which prevent the removal of at least one mannose residue distal to the pentasaccharide core 20 of the precursor oligosaccharide of GCB, to thereby produce an hmGCB preparation. In a preferred embodiment, the GCB is human GCB. In a preferred embodiment, the removal of: one or more a 1,2 mannose residue(s) distal to the pentasaccharide core is prevented; an a 2,3 mannose residue distal to the pentasaccharide core is prevented; and/or an a 1,6 mannose residue distal to the 25 pentasaccharide core is prevented. Preferably, the removal of one or more a 1,2 mannose residue(s) distal to the pentasaccharide core is prevented. In a preferred embodiment, the method can include contacting the cell with a substance which prevents the removal of at least one mannose residue distal to the pentasaccharide core of the precursor oligosaccharide of GCB, e.g., prevents removal of one 30 or more a 1,2 mannose residue(s) distal to the pentasaccharide core, an a 1,3 mannose "WO 02/35927 PCT/US01/258S2 residue distal to the pentasaccharide core and/or an c 1.5"iaannose residue distal to the pentasaccharide core. Preferably, the removal of one cr uxors a 12 maiiao3e(s} residue distal to the pentasaccharide core is prevented. In a preferred embodiment, the method includes contacting the cell with a substance 5 which prevents the removal of at feast one mannose residue distal to the pentasaccharide core of the precursor oligosaccharide of GCB, and the substance is a mannosidase inhibitor. The mannosidase inhibitor can be a class 1 processing mannosidase inhibitor, a class 2 processing mannosidase inhibitor or both. The class 1 processing mannosidase inhibitor can be one or more of: kifunensine, deoxymannojirimycin, or a similar inhibitor. Preferably, the class 1 10 processing mannosidase inhibitor is kifunensine. Useful class 2 processing mannosidase inhibitors can include one or more of: swainsonine, mannostatux, 6-deoxy-DIM, 6-deoxy-6-fluoro-DIM. Preferably, the class 2 processing mannosidase inhibitor is swainsonine. In a preferred embodiment, a mannosidase inhibitor is present at a concentration between about 0.025 to 20.0 ug/ml, 0.05 to 10 ug/ml, 0.05 to 5 ug/ml, preferably between 15 about 0.1 to 2.0 fig/ml. In a preferred embodiment, the method further includes contacting the cell with a class 1 processing mannosidase inhibitor and a class 2 processing mannosidase inhibitor. In a preferred embodiment, a class 1 processing mannosidase inhibitor is present at a concentration between about 0.025 to 20.0 ug/ml, 0.05 to 10 ug/ml, 0.05 to 5 ug/ml, 20 preferably between about 0.1 to 2^0 ug/ml; a class 2 processing mannosidase inhibitor is present at a concentration between about 0.025 to 20.0 ug/ml, 0.05 to 10 ug/ml, 0.05 to 5 ug/ml, preferably between about 0.1 to 2.0 ug/ml; each of the class 1 processing and class 2 processing mannosidase inhibitors are present at a concentration between about 0.025 to 20.0 ug/ml, 0.05 to 10 ug/ml, 0.05 to 5 ug/ml, preferably between about 0.1 to 2.0 ug/ml; the total 25 concentration of the class 1 processing and class 2 processing mannosidase inhibitors present is between about 0.025 to 40.0 Ug/ml, 0.05 to 20 Ug/ml, 0.05 to 10 Ug/ml, preferably between about 0.1 to 4.0 ug/ml. In a preferred embodiment, the cell carries a mutation for, e.g., a knockout for, at least one mannosidase. The mutation can be one which reduces the expression the gene, 30 reduces protein or activity levels, or alters the distribution or other post translational WO 02/15927 PCT/TJS01/25882 modifications of the mannosidase, e.g.. the processing of the carbohydrate chains. The mutation can be one which reduces the level of the Golgi processing mauiiosiGass activity, e.g., one which reduces gene expression, e.g., a null mutation, e.g., a deletion, a fzameshiit or an insertion. In a preferred embodiment the mutation is a knockout, e.g., in the mannosidase gene. The mutation can affect the structure (and activity of the protein), and can, e.g., be a temperature sensitive mutation or a truncation. In a preferred embodiment, the cell carries a mutation, e.g., a knockout, for: a class 1 processing mannosidase; a class 2 processing mannosidase; a mutant, e.g., a knockout, for a class 1 processing mannosidase and a class 2 processing mannosidase. In a preferred embodiment, the class 1 processing mannosidase is: Golgi mannosidase IA; Golgi mannosidase IB; Golgi mannosidase IC; or combinations thereof. In a preferred embodiment, the class 2 processing mannosidase is: Golgi mannosidase II. In a preferred embodiment, the cell includes a nucleic acid sequence, such as an antisense molecule or ribozyme, which can bind to or inactivate a cellular mannosidase nucleic acid sequence, e.g., niRNA, and inhibit expression of the protein. In a preferred embodiment, the nucleic acid sequence is: a class 1 processing mannosidase antisense molecule; a class 2 processing mannosidase antisense molecule; both a class 1 processing mannosidase antisense molecule and a class 2 processing mannosidase antisense molecule. In a preferred embodiment, the class 1 processing mannosidase is: Golgi mannosidase IA; Golgi mannosidase IB; Golgi mannosidase IC; combinations thereof. In a preferred embodiment, the class 2 processing mannosidase is: Golgi mannosidase n. In a preferred embodiment, the cell includes a molecule, e.g.. an exogenousfy supplied molecule, which binds and inhibits a mannosidase. The molecule can be, e.g., a single chain antibody, an intracellular protein or a competitive or non-competitive inhibitor. In a preferred embodiment, the hmGCB molecule includes a carbohydrate chain having at least four mannose residues. For example, the hmGCB molecule has at least one carbohydrate chain having five mannose residues, the hmGCB molecule has at least one carbohydrate chain having six mannose residues, the hmGCB molecule has at least one carbohydrate chain having seven mannose residues, the hmGCB molecule has at least one carbohydrate chain having eight mannose residues, the hmGCB molecule has at least one WO 02/15927 PCT/USO1725882 carbohydrate chain having nine mannose residues. Preferably, the hmGCB molecule has at least one carbohydrate chain having five, eight or nine mannose residues. In a preferred embodiment, the hmGCB produced (either one or more hmGCB molecules or the preparation as a whole) has a ratio of mannose residues to GlcNAc residues 5 which is greater than 3 mannose residues to 2 GlcNAc residues, preferably the ratio of mannose to GlcNAc is 4:2, 5:2, 6:2, 7:2, 8:2, 9:2. more preferably the ratio of mannose to GlcNAc is 5:2, 8:2 or 9:2. In a preferred embodiment, the removal of one or more mannose residues distal to the pentasaccharide core is prevented on one, two, three or four of the carbohydrate chains of the 10 hmGCB molecule. In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or all of the hmGCB molecules of the preparation have at least one, and preferably two, three or four carbohydrate chains in which the removal of one or more mannose residues distal to the pentasaccharide core has been prevented. 15 In a preferred embodiment, the hmGCB preparation is a relatively heterogeneous preparation. Preferably, less than 80%, 70%, 60%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 1% of the carbohydrate chains in ihe hmGCB preparation have the same number of mannose residues in addition to ihs pentasaccharide core. For example, the ratio of carbohydrate chains having the same number of mannose resides in addition to the 20 pentasaccharide core to carbohydrate chains having a different number of mannose residues can be about: 60%:40%; 50%:50%; 40%;60%; 30%:70%; 25%:75%; 20%:80%; 15%:85%; 10%:90%; 5% or less:95% or more. In a preferred embodiment, activity of Golgi mannosidase IA and/or IB and/or IC is inhibited and at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of 25 the carbohydrate chains in the hmGCB preparation have five or more mannose residues, e.g., five, six, seven, eight, and/or nine mannose residues. In a preferred embodiment, activity of Golgi mannosidase I is inhibited and the ratio of carbohydrate chains having rive or more mannose residues to carbohydrate chains having four or less mannose residues is about 60%:40%; 70%:30%; 75%:25%; 80%:20%; 85%:15%; 90%:10%; 95%:5%; 99%:1%; or 30 100%:0%. WO 02/15927 PCT/US01/25882 In a preferred embodiment, activity of Golgi mannosidase H is inhibited and at least about 60% 70% 75% 80% 85% 90%, 95% 98% 99% or 100% of the carbohydrate chains in the hmGCB preparation have five or more mannose residues. In a preferred embodiment, activity of Golgi mannosidase II is inhibited and the ratio of carbohydrate chains having five or more mannose residues to carbohydrate chains having four or less mannose residues is about 60%;40%; 70%:30%; 75%:25%; 80%:20%; 85%:15%; 90%:10%; 95%:5%; 99%:1%; or 100%:Q%. In a preferred embodiment the regulatory sequence includes one or-more of: a promoter,.an enhancer, an upstream activating sequence (UAS), a scaffold-attachment region or a transcription factor-binding site. In a preferred embodiment, the regulatory sequence includes: a regulatory sequence from a metallothionein-I gene, e.g., a mouse metallothionein-I gene, a regulatory sequence from an SV-40 gene, a regulatory sequence from a cytomegalovirus gene, a regulatory sequence, from a collagen gene, a regulatory sequence from an actin gene, a regulatory sequence from an immunoglobulin gene, a regulatory sequence from the HMG-CoA reductase gene, or a regulatory sequence from the BF-la gene. In a preferred embodiment, the cell is: a eukaryoric cell. In a preferred embodiment, the cell is of fungal, plant or animal origin, e.g., vertebrate origin. In a preferred embodiment, the cell is: a mammalian cell, e.g., a primary or secondary mammalian cell, e.g., a fibroblast, a hematopoietic stem cell, a myoblast, a keratinocyte, an epithelial cell, an endothelial cell, a glial cell, a neural cell, a cell comprising a formed element of the blood, a muscle cell and precursors of these somatic cells; a transformed or immortalized cell line. Preferably, the cell is a human cell. Examples of immortalized human cell lines useful in the present method include, but are not limited to; a Bowes Melanoma cell (ATCC Accession No. CRL 9607), a Daudi cell (ATCC Accession No. CCL 213), a HeLa cell and a derivative of a HeLa cell (ATCC Accession Nos. CCL2, CCL2.1 and CCL 2.2), a HL-60 cell (ATCC Accession No. CCL 240), an HT-1080 cell (ATCC Accession No. CCL 121), a Jurkat cell (ATCC Accession No. TIB 152), a KB carcinoma cell (ATCC Accession No. CCL 17), a K-562 leukemia cell (ATCC Accession No. CCL 243), a MCF-7 breast cancer cell (ATCC Accession No. BTH 22), a MOLT-4 cell (ATCC Accession No. 1582), a Namalwa cell (ATCC Accession No. CRL 1432), a Raji cell (ATCC Accession No. CCL 86), a RPMI 8226 cell (ATCC Accession No. CCL 155), a U-937 cell (ATCC Accession No. 1593), WI- WO 02/15927 PCT/US01/25882 28VA13-sub line 2R4 cells (ATCC Accession No. CIX 155% - CCRF-CEM cell (ATCC Accession No. CCL 119) and a 2780AD ovarian carcinoma csE (Van :DerBlick et si., Cancer Res. 48:5927-5932. 1988), as well as heterohybridoma cells produced by fusion of human cells and cells of another species. In another embodiment, the immortalized cell line can be 5 cell line other than a human cell line, e.g., a CHO cell line, a COS cell line. In another embodiment, the cell can be from a clonal cell strain or clonal cell line. In a preferred embodiment, a population of cells which are capable of expressing hmGCB is provided, and at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or all of the cells produce hmGCB with at least one carbohydrate chain, preferably 10 two, three, or four carbohydrate chains, having the specified number of mannose residues. In a preferred embodiment, the cell is cultured in culture medium which includes at least one mannosidase inhibitor. In a preferred embodiment, the method further includes obtaining the hmGCB from the medium in which the cell is cultured. 15 In another aspect, the invention features an hmGCB molecule, e.g., an hmGCB molecule described herein, e.g., a human hmGCB, produced by any of the methods described herein. Preferably, the hmGCB molecule includes at least one carbohydrate chain, preferably two, three, or four carbohydrate chains, having at least four mannose residues of a precursor oligosaccharide chain. 20 In another aspect, the invention features an hmGCB preparation which includes a portion of hmGCB molecules which include at least one carbohydrate cnsin, preferably two, three, or four carbohydrate chains, having at least four mannose residues of a precursor oligosaccharide chain. Preferably, the hmGCB preparation is produced by any of the 25 methods described herein. - In a preferred embodiment, the hmGCB is human hmGCB. In a preferred embodiment, the hmGCB molecule can have: at least one carbohydrate chain having five mannose residues; at least one carbohydrate chain having six mannose residues; at least one carbohydrate chain having seven mannose residues; at least one 30 carbohydrate chain having eight mannose residues; at least one carbohydrate chain having nine mannose residues. WO 02/15927 PCTAJS01/25882 In z preferred embodkosit the hmGCB produced (either one or more hmGCB molecules or the pr^s^fei as a ■seholc) has at least one carbohydrate chain having a ratio of mannose residues io GIcNAc residues which is greater than 3 mannose residues to 2 GlcNAc residues, preferably the ratio of mannose to GlcNAc is 4:2, 5:2, 6:2, 7:2, 8:2, 9:2, more 5 preferably the ratio of mannose to GlcNAc is 5:2, 8:2 or 9:2. In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or ail of the hmGCB of the preparation have at least one, preferably, two, three or four carbohydrate chains in which the removal of one or more mannose residues distal to the pentasaccharide core has been prevented. 10 In another aspect the invention features a cell having at least one mannosidase activity inhibited and which includes a nucleic acid sequence comprising an ex o geno us regulatory sequence which has been introduced such that the regulatory sequence regulates the expression of an endogenous GCB coding region, wherein the cell produces GCB in 15 which the removal of at least one mannose residue distal to the pentasaccharide core of a precursor oh go saccharide of GCB is prevented. In a preferred embodiment, the cell produces an hmGCB preparation, e.g., a human hmGCB preparation, in which the removal of: one or more a 1,2 mannose residue(s) distal to the pentasaccharide core is prevented; an a 1,3 mannose residue distal to the pentasaccharide 20 core is prevented; and/or an a 1,6 mannose residue distal to the pentasaccharide core is prevented. Preferably, the removal of one or more a 1,2 mannose residue(s) distal to the pentasaccharide core is prevented. In a preferred embodiment, at least one mannosidase activity in the cell has been inhibited by contacting the cell with a substance which inhibits a mannosidase. In a preferred 25 embodiment, the substance is a mannosidase inhibitor. The mannosidase inhibitor can be a class 1 processing mannosidase inhibitor, a class 2 processing mannosidase inhibitor or both. In a preferred embodiment, the class 1 processing mannosidase inhibitor can be one or more of: kifunensine and deoxymannojirimycin. Preferably, the class 1 processing mannosidase inhibitor is kifunensine. In a preferred embodiment, the class. 2 processing mannosidase 30 inhibitor can be one or more of: swainsonine, mannostatin, 6-deoxy-DIM, and 6-deoxy-6-fluoro-DIM. Preferably, the class 2 processing mannosidase inhibitor is swainsonine. WO 02/15927 PCT/US01/25882 In a preferred embodimsr; ih^ cdi carries £ mutation for. e.g., a knockout for. at least one Golgi processing msszicadiSe. The msns&on can be one which reduces the expression of the gene, reduces protein or activity levels, or alters the distribution or other post translational modifications of the mannosidase, e.g., the processing of a carbohydrate chain. The mutant can be one which reduces the level of Golgi processing mannosidase activity, e.g., one which reduces gene expression, e.g., a null mutation, e.g., a deletion, a frarneshift, or an insertion. In a preferred embodiment, the mutation is a knockout in the mannosidase gene. The mutation can affect the structure (and activity of the protein), and can, e.g., be a temperature sensitive mutation. In a preferred embodiment, the ceil is a mutant, e.g., a knockout, for a class 1 processing mannosidase; a class 2 processing mannosidase; a class 1 processing marmosidase and a class 2 processing mannosidase. In a preferred embodiment, the class I processing mannosidase is: Golgi mannosidase IA; Goigi mannosidase IB; Golgi mannosidase IC; combinations thereof. In a preferred embodiment, the class 2 processing mannosidase is: Golgi mannosidase II. In a preferred embodiment, the cell further includes a nucleic acid sequence, such as an antisense molecule or ribozyme, which can bind to or inactivate a cellular mannosidase nucleic acid sequence, e.g., mRNA, and inhibit expression of the protein. In a preferred embodiment, the nucleic acid sequence is: a class I processing mannosidase antisense molecule; a class 2 processing mannosidase antisense molecule; both a class 1 processing mannosidase antisense molecule and a class 2 processing mannosidase antisense molecule. In a preferred embodiment, the class 1 processing mannosidase is: Golgi mannosidase IA; Golgi mannosidase IB; Golgi mannosidase IC; combinations thereof. In a preferred embodiment, the class 2 processing mannosidase is: Golgi mannosidase H. In a preferred embodiment, the cell includes a molecule, e.g., an exogenously supplied molecule, which binds and inhibits a mannosidase. The molecule can be, e.g., a single chain antibody, an intracellular protein or a competitive or non-competitive inhibitor. In a preferred embodiment, the hmGCB molecule produced by the cell has a ratio of mannose residues to GlcNAc residues which is greater than 3 mannose residues to 2 GlcNAc residues, preferably the ratio of mannose to GlcNAc is 4:2, 5:2, 6:2, 7:2, 8:2, 9:2, more preferably the ratio of mannose to GlcNAc is 5:2, 8:2 or 9:2. "R70 02-15927 PCT/US01/25882 rz a preferred embodiment lie cell is unable to remove of one or more mannose " res-±i2S drsrsl ID me pentasaccharide core on one. two. three or four of the carbohydrate C5SZLS OI ■ u.LJVJV^iJ. M a preferred embodiment, at least 30%, 40% 50%, 60%, 70%, 75%, 80%, 85%, 90%: 95%, 98%, 99% or all of the hmGCB molecules produced by the cell have at least one, preferably, two, three or four carbohydrate chains in which the removal of one or more mannose residues distal to the pentasaccharide core has been prevented. In a preferred embodiment, the regulatory sequence includes one or more of: a promoter, an enhancer, an upstream activating sequence (UAS), a scaffold-attachment region or a transcription factor-binding site. In a preferred embodiment, the regulatory sequence includes: a regulatory sequence from a metallothionein-I gene, e.g., a mouse metallothionein-I gene, a regulatory sequence from an SV-40 gene, a regulatory sequence from a cytomegalovirus gene, a regulatory sequence from a collagen gene, a regulatory sequence from an actin gene, a regulatory sequence from an immunoglobulin gene, a regulatory sequence from the HMG-CoA reductase gene, or a regulatory sequence from the EF-1 a gene. In a preferred embodiment, the cell is: a eukaryotic cell. In a preferred embodiment, the cell is of fungal, plant or animal origin, e.g., vertebrate origin. In a preferred embodiment, the cell is: a mammalian cell, e.g., a primary or secondary mammalian cell, e.g., a fibroblast, a hematopoietic stem cell, a myoblast, a keratinocyte, an epithelial cell, an endothelial cell, a glial cell, a neural cell, a cell comprising a formed element of the blood, a muscle cell and precursors of these somatic cells; a transformed or immortalized cell. line. Preferably, lis cell is a human cell. Examples of immortalized human cell lines useful in the present method include, but are not limited to: a Bowes Melanoma cell (ATCC Accession No. CRL 9607), a Daudi cell (ATCC Accession No. CCL 213), a HeLa cell and a derivative of aHeLacell (ATCC Accession Nos. CCL2, CCL2.1, and CCL 2.2), aHL-60 cell (ATCC Accession No. CCL 240), an HT-1080 cell (ATCC Accession No. CCL 121), a Jurkat cell (ATCC Accession No. TIB 152), a KB carcinoma cell (ATCC Accession No. CCL 17), a K-562 leukemia cell (ATCC Accession No. CCL 243), a MCF-7 breast cancer cell (ATCC Accession No. BTH 22), a MOLT-4 cell (ATCC Accession No. 1582), a Namalwa cell (ATCC Axcession No. CRL 1432), a Raji cell (ATCC Accession No. CCL 86), a RPMI 8226 cell (ATCC Accession No. CCL 155), a U-937 cell (ATCC Accession No. 1593), WI- WO 02/15927 PCT/USG1/25882 2SVA13 nsb irse 2R4 cells (ATCC Accession No. CLL 155), a CCRF-CEM cell (ATCC Accs3E.No. CCL 119) and a 2780AD ovarian carcinoma cell (Van Der Blick et al, Cancer Res. 48:5927-5932. 1988), as well as heterohyhridoma cells produced by fusion of human cells and ceils of another species, m another embodiment, the immortalized cell line can be 5 cell line other than a human cell line, e.g., a CHO cell line, a COS cell line. In another embodiment, the cell can be from a clonal cell strain or clonal cell line. In another aspect, the invention features a pharmaceutical composition which includes an hmGCB molecule, e.g., a human hmGCB, which includes at least one 10 carbohydrate chain, preferably two, three, or four carbohydrate chains, having at least four mannose residues of a precursor oligosaccharide chain, in an amount suitable for treating Gaucher disease. In a preferred embodiment, the pharmaceutical composition further includes a pharmaceutically acceptable carrier or diluent. 15 Another aspect of the invention features a method of treating a subject having Gaucher disease. The method includes administering to a subject having Gaucher disease an hmGCB preparation, e.g., a human hmGCB preparation, which includes at least one carbohydrate chain, preferably two, three, or four carbohydrate chains, having at least four 20 mannose residues of a precursor oligosaccharide chain, in an amount suitable for treating Gaucher disease. In another aspect, the invention features a method of purifying hmGCB from a sample. The method includes: providing a harvested hmGCB product; and subjecting the 25 hmGCB product to hydrophobic charge induction chromatography (HCIC) and/or hydrophobic interaction chromatography (HIC), thereby obtaining purified hmGCB. In a preferred embodiment, MEP Hypercel® is used for HCIC. In another preferred embodiment, MacroPrep Methyl® is used for HIC. In another preferred embodiment, the method further includes subjecting the hmGCB 30 product to ion exchange chromatography. The hmGCB product can be subjected to HCIC and/or HIC prior to ion exchange chromatography or the hmGCB product can be subjected to WO 02/15927 PCTYUS01/25882 ion excnznss -j^-'- r'-f^y pdor to HCIC and/orHIC. Preferably, the hmGCB product is subjected tc ^ncicirisi c-neiGn exchange chromatography step. The ion exchange chromatography can be: anion exchange chromatography, cation exchange chromatography or both. 5 In a preferred embodiment anion exchange chromatography is performed using one or more of: Q Sepharose Fast Plow®, MacroPrep High Q Support®, DEAE Sepharose Fast Flow®, and Macro-Prep DEAE®. In a preferred embodiment, cation exchange chromatography is performed using one or more of SP Sepharose Fast Flow®, Source 30S®, CM Sepharose Fast Flow®, Macro-Prep CM Support®, and Macro-Prep High S 10 Support®. In a preferred embodiment, the method further includes subjecting the hmGCB product to size exclusion chroiaatography. Preferably, the size exclusion chromatography is performed using one or more of: Superdex 200®, Sephacryl S-200 HR® and Bio-Gel A 1.5m®. 15 In another aspect, the invention features a method of purifying hmGCB. The method includes: providing a harvested hmGCB product; subjecting the hmGCB product to hydrophobic charge induction chromatography (HCIC) and/or hydrophobic interaction chromatography (HIC); and subjecting the hmGCB product to one or more of anion 20 exchange chromatography, cation exchange chromatography, and size exclusion chromatography, to thereby obtain purified hmGCB. In a preferred embodiment, MEP Hypercel® is used for HCIC. In another preferred embodiment, MacroPrep Methyl® is used for HIC. 25 In a preferred embodiment, the method includes using anion exchange chromatography. Preferably, anion exchange chromatography is performed using one or more of: Q Sepharose Fast Flow®, MacroPrep High Q Support®, DEAE Sepharose Fast Flow®, and Macro-Prep DEAE®. In a preferred embodiment, the method includes using cation exchange 30 chromatography. Preferably, cation exchange chromatography is performed using one or WO 02*15927 PCT7US01/25882 —-err of S? Sepharose Fsst FlawS, Source 50S®, CM Sepharose Fast Flow®, Macro-Prep CM Scppon®, and Macro-Prep High S Support®. In a preferred embodiment, the method includes using size exclusion chromatography. Preferably, the size exclusion chromatography is performed using one or 5 more of: Superdex 200®, Sephacryl S-200 HR® and Bio-Gel A 1.5m®. In a preferred embodiment, the hmGCB is subjected to (in any order): anion exchange chromatography and cation exchange chromatography; anion exchange chromatography and size exclusion chromatography; cation exchange chromatography and size exclusion chromatography; anion exchange chromatography, cation exchange chromatography and size 10 exclusion chromatography. Preferably, the hmGrCB is subjected to all three of these chromatography steps in the following order: anion exchange chromatography, cation exchange chromatography and size exclusion chromatography. In another aspect, the invention features a method of purifying hmGCB. The method 15 includes: providing a harvested hmGCB product; subjecting the hmGCB product to hydrophobic charge induction chromatography (HCIC) and/or hydrophobic interaction . chromatography (HIC); subjecting the HCIC and/or HIC purified hmGCB product to anion exchange chromatography; subjecting the anion exchange purified hmGCB to cation exchange chromatography; and, subjecting the cation exchange purified hmGCB to size 20 exclusion chromatography, to thereby obtain purified hmGCB. In a preferred embodiment, MEP Hypercel® is used for HCIC. In another preferred embodiment MacroPrep Methyl® is used for HIC. In a preferred embodiment, anion exchange chromatography is performed using one or more of: Q Sepharose Fast Flow®, MacroPrep High Q Support®, DEAE Sepharose Fast 25 Flow®, and Macro-Prep DEAE®. In a preferred embodiment, cation exchange chromatography is performed using one or more of: SP Sepharose Fast Flow®, Source 30S®, CM Sepharose Fast Flow®, Macro¬Prep CM Support®, and Macro-Prep High S Support®. In a preferred embodiment, size exclusion chromatography is performed using one or 30 more of: Superdex 200®, Sephacryl S-200 HR® and Bio-Gel A 1.5m®. WO 02/15927 PCT/US01/25882 The tss "H^f- n^n^rsa gliicocarebro-sidsss (hraGCS)" as used herein refers to glucocerehrosiazsc raving at least one carbohydrate chain having four or more marmose residues -from a precursor oUgosacchaiide. Preferably, the hmGCB has five, six, seven, eight or nine mannose residues from ihe precursor oligosaccharide chain. Most preferably, the 5 hmGCB has five, eight or nine mannose residues from the precursor oligosaccharide chain. The term :1imGCB preparation5' refers to two or more hmGCB molecules. The term "primary cell" includes cells present in a suspension of cells isolated from a vertebrate tissue source (prior to their being plated i.e., attached to a tissue culture substrate 10 such as a dish or flask), cells present in an explant derived from tissue, both of the previous types of cells plated for the first tim^ and cell suspensions derived from these plated cells. The term secondary cell or cell strain refers to cells at all subsequent' steps in ciilturmg. That is, the first time a plated primary cell is removed from the culture substrate and replated (passaged), it is referred to herein as a secondary cell, as are all cells in subsequent passages. 15 Secondary cells are cell strains which consist of secondary cells which have been passaged one or more times. A cell strain consists of secondary cells that: 1) have been passaged one or more times; 2) exhibit a finite number of mean population doublings in -culture; 3) exhibit the properties of contact-inhibited, anchorage dependent growth (anchorage-dependence does not apply to cells that are propagated in suspension culture); and 4) are not immortalized. A 20 "clonal cell strain" is defined as a cell strain that is derived from a single founder cell. A "heterogenous cell strain" is defined as a cell strain that is derived from two or more founder cells. 'Tmmortalized cells1*, as used herein, are cell lines (as opposed to cell strains with the designation "strain" reserved for primary and secondary cells), a critical feature of which is 25 that they exhibit an apparently unlimited lifespan in culture. The term "transfected cell" refers to a cell into which an exogenous synthetic nucleic acid sequence, e.g., a sequence which encodes a protein, is introduced. Once in the cell, the . synthetic nucleic acid sequence can integrate into the recipients cells chromosomal DNA or ' ' can exist episomalfy. Standard transfection methods can be used to introduce the synthetic 30 nucleic acid sequence into a cell, e.g., transfection mediated by liposome, polybrene, DEAE dextran-mediated transfection, electroporation, calcium phosphate precipitation or WO 02/15927 PCT/US01/25882 uncroiajectiorL The term "transfection" does notinclude delivery of DNA orRNAinto a cell by a virus . The term "infected cell" or "transduced cell" refers to a cell into which an exogenous synthetic nucleic acid sequence, e.g., a sequence which encodes a protein, is introduced by a virus. Viruses known to be useful for gene transfer include an adenovirus, an adeno-associated virus, a herpes virus, a mumps virus, apoliovirus, a retrovirus, a Sindbis virus, a lentivirus and a vaccinia virus such as a canary pox virus. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. Brief Description of the Drawing Figure J is a diagram showing the trimming of N-linked glycans as it occurs in the endoplasmic reticulum, the intermediate compartment and in the Golgi apparatus. The enzymes are numbered as follows: (1) a-glucosidase I; (2) a-glucosidase IT; (3) ER mannosidase I; (4) ER mannosidase II; (5) ER glucosyl transferase; (6) endomannosidase; (7) Golgi mannosidase LA, IB and IC; (8) GlcNAc transferase I; (9) Golgi mannosidase n. A, Glucose; D, GlcNAc; 0, Mannose. Enzymes (3) and (7) are inhibited by kifunensine; enzyme (9) is inhibited by swainsonine. Detailed Description of the Invention The invention is based, in part, on the discovery that inhibition of the removal of one or more mannose residues distal from the pentasaccharide core of a precursor oligosaccharide chain of glucocerebrosidase (GCB), results in high mannose glucocerebrosidase (hmGCB) that is efficiently targeted to mannose receptors. The removal of a mannose residue from the pentasaccharide core of a precursor oligosaccharide chain can be prevented by inhibiting or reducing the activity of one or more mannosidase enzymes, e.g., one or more class 1 processing maimosidase(s) and/or class 2 processing mannosidase(s). By preventing or inhibiting the removal of one or more mannose residues, hmGCB having at least one "WO 0X15927 FCT/US0LO58S2 cErbozryGTHS chzni with four or more mannose residues -from the precursor oligosaccharide Gaucher disease is caused by a deficiency of GCB. GCB is required for degradation of glycosphingoiipid giucocerebroside. In the absence of GCB, the giucocerebroside 5 accumulates primarily in phagocytic cells, e.g., macrophages, and, ultimately, builds up in the liver, spleen and bone marrow. Macrophages have mannose receptors. These receptors play a role in receptor-mediated endocytosis by these cells. hrnGCB efficiently targets the mairaose receptors on . macrophages and improves the uptake of GCB (in the form of hmGCB) into these cells. By 10 directing GCB (in the form of hmGCB) to the cells in which giucocerebroside accumulates, hmGCB can be used to hydiolyze giucocerebroside in the macrophages, thereby reducing the subsequent accumulation of this glycolipid in the liver, spleen and bone marrow of patients having Gaucher disease. GIuco cerebrosi dase 15 Nucleotide sequence information is available for genes encoding glucocerebrosidase from various species. (See Horowitz et al. (1989) Genomics 4(l):87-96; Beutler et ai. (1992) Genomics 12(4):795-800), Mature human GCB has five potential N-lmked glycosylation sites at Asn-I9, Asn-59, Asn-146, Asu-270, and Asn-462. Glycosylation occurs at four of the five sites in human 20 tissue derived GCB (Erickson et al. (1985) /. Biol Chem. 260:14319-14324). Studies employing site-directed mutagenesis have demonstrated that the site at Asn-462 is never occupied (Berg-Fussman et al. (1993) I Biol. Chem. 268:14861-14866). Approximately 20% ofths released glycan chains from human placental GCB were shown to be of the high mannose type containing up to seven mannose residues, whereas the majority of the glycan 25 chains were of the complex type with sialylated biantennary and triantennary structures. (Takasaki etal. (1984)/. Biol Chem. 259:10112-10117) The first event in GCB N-glycosylate on is the co-translational transfer in the lumen of the endoplasmic reticulum (ER) of Glc3Man9GlcNAc2 from oligosaccharide-PP-dolichol to nascent peptide. The presence of the three glucose residues on the donor oligosaccharide 30 allows for efficient transfer to an acceptor asparagine by oligosaccharyl transferase. WO 02/15927 PCT/USOl/25882 FollowingN-slyco5vistE3Zi. ^ gnz^ese residues are rapitfly removed from GCB during the folding process by BR gri^xjsidsses I and H Two different ER manncsidases are each capable of hydrolyzing a single mannase residue from Maxi9GIcNAc2 to form two different isomers of MangGlcNAci (see Fig. 1). Accessible glycaris are then further processed in the 5 Golgi to Man5GlcNAc2 by the removal of up to four al ,2-linked mannose residues by Golgi mannosidase I. There are af least three different human genes encoding related Golgi mannosidase I isoforms (IA, IB, and IC) with shghtly different substrate specificities and tissue expression but all are capable of trimming four maP&ose residues from Man$GlcNAc2 glycans to form Man5GIcNAc2 (Tremblay et al. (July 27, 2000) /. Biol Chem. [epub ahead of 10 print]). They are located on chromosomes 6q22, lp!3, afld lp35-36 and their cDNA sequences are obtainable from GenBank as X74837, AF027156, and AF261655, respectively. The final stage of processing that commits a glycan to the biosynthetic pathway for complex glycans requires the initial conversion of Man5Gric'jSiAc2 to GlcNAcMansGiclSiAc2 15 by the action of GlcNAc transferase I, after which Golgi inannosidase U can catalyse the removal of two further mannose residues to yield GIcNA^Man3GlcNAc2. This is the substrate for glycan elongation by glycosyl transferases located in the trans Golgi and the trans Golgi network to form complex type chains. If the high mannose chains transferred to GCB in the initial N-glycosylati on step can 20 be prevented from being processed to complex chains in tbe Golgi, then GCB with high mannose chains (hmGCB) will effectively target the mannose receptors on reticuloendothelial cells. Cells 25 Primary and secondary cells to be transfected or infected can be obtained from a variety of tissues and include cell types which can be maintained and propagated in culture. For example, primary and secondary cells which can be transfected or infected include fibroblasts, keratinccytes, epithelial cells (e.g., mammary epithelial cells, intestinal epithelial cells), endothelial cells, glial cells, neural cells, formed elements of the blood (e.g., 30 lymphocytes, bone marrow cells), muscle cells and precursors of these somatic cell types. Primary cells are preferably obtained from the individual to whom the transfected or infected WO 02/15927 PCT/USOl/25882 primary or secondary cells 'are admisster (Le~ £2 autologous cell). However.primary cells may be obtained from £ donor (other nzsn the recipient) of the same species (i.e.. an allogeneic cell) or another species (ie.; a xenogeneic cell) (e.g., mouse, rat, rabbit, cat, dog, pig, cow, bird, sheep, goat, horse: monkey, baboon). Primary or secondary ceils of vertebrate, particularly mammalian, origin can be transfected or infected with an exogenous DNA sequence, e.g., an exogenous DNA sequence encoding a therapeutic protein, and produce an encoded therapeutic protein stably and reproducibly, both in vitro and in vivo, over extended periods of time. In addition, the transfected or infected primary and secondary cells can express the encoded product in vivo at physiologically relevant levels, cells can be recovered after implantation and, upon reculturing, to grow and display their preimplantaiion properties. Cells can be modified to reduce cell surface histo compatibility, complex or foreign carbohydrate moieties to reduce immunogenecity, e.g., a universal donor cell. Alternatively, primary or secondary cells of vertebrate, particularly mammalian, origin can be transfected or infected with an exogenous DNA sequence which includes a regulatory sequence. Examples of such regulatory sequences include one or more of: a promoter, an enhancer, an UAS, a scaffold attachment region or a transcription binding site. The targeting event can result in the insertion of the regulatory sequence of the DNA sequence, placing a targeted endogenous gene under their control (for example, by insertion of either a promoter or an enhancer, or both, upstream of the endogenous gene or regulatory region). Optionally, the targeting event can simultaneously result in the deletion of an endogenous regulatory sequence, such as the deletion of a tissue-specific negative regulatory sequence, of a gene. The targeting event can replace an existing regulatory sequence; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the endogenous elements, or displays a pattern of regulation or induction that is different from the corresponding nontransfected or nonmfected cell. In this regard, the endogenous sequences are deleted and new sequences are added. Alternatively, the endogenous regulatory sequences are not removed or replaced but are disrupted or disabled by the targeting event, such as by targeting the exogenous sequences within the endogenous regulatory elements. Introduction of a regulatory sequence by homologous recombination can result in primary or secondary cells expressing a therapeutic WO 02/15927 PCT7US01/25882 protein which it does not normally express, ha af-fttor-. :srgs=d inm^daciion-oi a regulatory sequence can be used for cells which make or contain the thsrspenizc protein hnt in lower quantities than norma] (in quantities less than the physiologically sornral lower level) or in defective form, and for cells which make the therapeutic protein at physiologically normal 5 levels, but are to be augmented or enhanced in their content or production. Methods of activating an endogenous coding sequence are described in U.S. Patent No.: 5,641,670, U.S. Patent No.: 5,733,761 and U.S. Patent No.: 5,968,502, the contents of which are incorporated herein by reference. The transfected or infected primary or secondary cells may also include a DNA 10 sequence encoding a selectable marker which confers a selectable phenotype upon them, facilitating their identification and isolation. Methods for producing transfected primary or secondary cells which stably express the DNA sequence, clonal cell strains and heterogenous cell strains of such transfected cells, methods of producing the clonal and heterogenous cell strains, are known and described, for example, in U.S. Patent No.: 5,641,670, U.S. Patent 15 No.: 5,733,761 and U.S. Patent No.: 5,968,502, the contents of which are incorporated herein by reference. Transfected primary or secondary cells, can be made by electroporation. Electrop oration is carried out at appropriate voltage and capacitance (and corresponding time constant) to result in entry of the DNA construct(s) into the primary or secondary cells. 20 Electroporation can be carried out over a wide range of voltages (e.g., 50 to 2000 volts) and corresponding capacitance. Total DNA of approximately 0.1 to 500 u.g is generally used. Alternatively, known methods such as calcium phosphate precipitation, microinjection, modified calcium phosphate precipitation and porybrsne precipitation, liposome fusion and receptor-mediated gene delivery can be used to transfect cells. 25 Processing of Glucocerebrosidase Oligosaccharide assembly in cells which have not been treated to prevent removal of mannose residues usually proceeds as discussed below: The oligosaccharide chains of GCB are attached to the polypeptide backbone by N-30 glycosidic linkages. N-linked glycans have an amide bond that connects the anomeric carbon WO 02/15927 PCT/T501/25882 (Crl) of a Teducrng-terminal N-acetylglut^samme (GIcHAc) resist cc ~he oE^Ds-cebsdas and a nitrogen of an asparagine (A 5m) residue of the polypeptide. Initiation of N-linked oligosaccharide assembly does not occur directry on me Asn residues of the GCB protein, but rather involves preass&nhly of a lipid-linlceG 14 sugar 5 precursor oligosaccharide which is then transferred to the protein in the ER during or very soon after its translation from mRNA. A "precursor ohgosacchande" as used herein refers to the oligosaccharide chain involved in the initial steps in biosynthesis of carbohydrate chains. A "precursor oligosaccharide" can be an oligosaccharide structure which includes at least the following sugars: Man9GlcNAc2, for example, a precursor oligosaccharide can have the 1 o following structure: Glc3MangGlcNAc2, as shown in Figure 1. The precursor ohgosacchande is synthesized while attached via a pyrophosphate bridge to a polyisoprenoid carrier lipid, a dolichoh This assembly involves at least six distinct membrane-bound giyc^syirransfexases. Some of these enzymes transfer monosaccharides from nucleotide sugars, while others utilize dolichol-iinked monosaccharides as sugar donors. After assembly of the hpid-hnked 15 precursor is complete, another membrane-bound enzyme transfers it to sterically accessible Asn residues which occur as part of the sequence -Asn-X-Ser/Thr-. Glycosylated Asn residues of newly-synthesized GCB transiently carry , Glc3Man9GIcNAc2, also referred to herein as an "unprocessed carbohydrate chain". The processmg of N-linked oligosaccharides is accomplished by the sequential action 20 of a number of membrane-bound enzymes and begins immediately after transfer of the precursor oligosaccharide Glc3Man9GlcNAc2 to the protein. The terms "processing", "trimming" and "modifying" are used interchangeably herein. N-linked oligosaccharide processing can be divided into three stages: removal of the three glucose residues, removal of a variable number of marrnose residues, and addition of 25 various sugar residues to the resulting trimmed core. The removal of the glucose residues in the first stage of processing involves removal of all three glucose residues to generate N-linked Man9GlcNAc2. This structure is also referred to herein as: Manal-2Manal-2Manal-3[Manal-2Manal-3(Manal-2Manal- 6)Manal~6]Man£l-4GlcNAc(3l-4GlcNAc (See Figure 1, structure 9'). Processmg normally 30 continues to the second stage with removal of marrnose residues. WO 02/1=927 PCT/OS01/25SS? Four of the mannose residues of the Man5GIcNA(>= moiety are bound by a 1.2 linkages. Up to four of these a 1:2-Iinked mannose residues can be removed by mariDcsidss^ LA IB and IC to generate N-linked Man5.gGlcNAc2. Protein-linked Man5G3cNAc2 can then serve as a substrate for GlcNAc transferase I, 5 which transfers a p 1 J2-linked GlcNAc residue from UDP-GlcNAc to the core a 1,3-linked mannose residue to form GlcNAcMan5GlcNAc2- Mannosidase H can then complete the triinming phase of the processing pathway by removing two mannose residues to generate a protein-linked oligosaccharide which contains within it a Man3GlcNAc2, the "pentasaccharide core". The structure GIcNAcMan3G]cNAc2 is then a substrate for GlcNAc 10 transferase II, which can transfer a p ls2-linked GlcNAc residue to the a 1,6-linked mannose residue. After the trimming phase, monosaccharides are sequentially added to the growing oligosaccharide chain by a series of membrane-bound Golgi glycosyltransferases, each of which is highly specific with respect to the acceptor oligosaccharide, the donor sugar, and the 15 type of linkage formed between the sugars. These can include distinct GlcNAc transferases (producing p 1,2; p 1, 4; or p 1,6 linkages); galactosyltransferases (producing p 1, 4; p 1,3; and a 1,3 linkages); sialyltransferases (one producing a 2, 3 and another, a 2, 6 linkages); fiicosyltransferases (producing a 1,2; a 1,3; a 1, 4 or a 1,6 linkages); and a growing list of other enzymes responsible for a variety of unusual linkages. The cooperative action of these 20 glycosyltransferases produces a diverse family of structures collectively referred to as "complex" oligosaccharides. These may contain two, three or four outer branches ("antennae") attached to the invariant core pentasaccharide, Mar^GlcNAc^. These structures are referred to in terms of the number of their outer branches: biantennary (two branches), triantennary (three branches) or tetraantennary (four branches). The size of these complex 25 glycans can vary. Processing of High Mannose Glucocerebrosidase hmGCB can be produced by reducing or preventing cellular carbohydrate modification (i.e., processing) of GCB. Carbohydrate modification can be prevented by 30 allowing production of GCB under conditions which prevent the removal of at least one mannose residue distal to the pentasaccharide core of a precursor oligosaccharide chain of WO 02/15927 PCT/US01/25882 GCB. Per example, one or more of the 'trimming" stages durmg me removal of mannose residues from a precursor oligosaccharide can be prevented. Cellular mannosidases fall into two broad classes: class 1 processing enzymes, which include ER mannosidase I. Golgi mannosidase IA, IB and IC and which hydrolyze a.1,2- 5 linked marmose residues, and require Ca2+ for activity; and class 2 processing enzymes, which include ER mannosidase II, Golgi mannosidase II, cytosolic a-mannosidase, and lysosomal a-mannosidase and which have a broader substrate specificity and do not require Ca2+ for activity. The trimming of mannose residues from the precursor oligosaccharide involves at 10 least the following mannosidase enzymes: Golgi mannosidase IA, IB and IC, and Golgi mannosidase II. By inhibiting one or more of these mannosidases during N-linked oligosaccharide assembly in a cell, GCB can he produced which has at least one carbohydrate chain with one or more mannose residues in addition to the pentasaccharide core. For example, inhibition of both ER mannosidase I and Golgi mannosidase I can 15 produce hmGCB with at least one carbohydrate chain (and preferably all chains) having at least eight mannose residues from the precursor oligosaccharide; inhibition of Golgi mannosidase II can produce hmGCB with at least one carbohydrate chain (and preferably all chains) having at least five mannose residues from the precursor oligosaccharide. Trirnming by a mannosidase can be inhibited, for example, by contacting the cell with 20 a substance which prevents the removal of one or more mannose residues from a precursor oligosaccharide of GCB or by producing GCB in a cell which does not produce or produces at deficient levels at least one mannosidase, or in a cell which produces a mutated and/or inactive mannosidase. For example, the cell can be a knockout for at least one mannosidase, can express at least one antisense mannosidase molecule or can be dominant negative for at 25 least one mannosidase. Substances Which Prevent Removal of Mannose Residues A substance which prevents the removal of one or more mannose residues from a precursor oligosaccharide of GCB can be used to produce an hmGCB preparation. For 30 example, a cell which expresses GCB can be contacted with a substance which prevents the removal of one or more a 1,2 mannose residues of a precursor oligosaccharide of GCB, WO 02/15927 PCT/US01/25882 and/or removal of an alp mannose residue of a precursor oligosaccharide of GCB, and/or removal of ana 1.6 mannose residue of a precursor oligosaccharide of GCB. Preferably; the substance is a mannosidase inhibitor, e.g., a class 1 processing mannosidase inhibitor or a class 2 processing mannosidase inhibitor. Cellular mannosidases fall into two broad classes on the basis of protein sequence homologies (Moremen et al. (1994) Glycobiology 4:113-125). These two classes are mechanistically different Class 1 enzymes, which include ER mannosidase I and Golgi mannosidase I isoforms, have a mass of about 63-73 kDa, hydrolyze al,2-Unked mannose residues and require Ca2+for activity. Class 1 processing mannosidases can be blocked, for example, by treatment with a substrate mimic, e^g., a pyranose analog of mannose'. For example, class 1 processing mannosidases can be blocked by treatment with one or more of the following enzymatic inhibitors: kifunensine, deoxymannojirimycin, or a combination thereof. Class 2 enzymes, which include ER mannosidase I, Golgi mannosidase H, cystolic a-mannosidase, and lysosomal a-mannosidase, have a greater mass of about 107-136 kDa, do not require Ca2+ for activity and have a broader substrate specificity. Class 2 processing mannosidases can be blocked, for example, by treatment with furanose transition state analogues of the mannosyl cation (Daniels et al. (1994) GlycoBiol. 4:551-566). For example, class 2 processing mannosidases can by blocked by treatment with one or more of the following inhibitors:, swainsonine, 6-deoxy-DIM, 6-deoxy-6-fluoro-DIMJ mannostatin A, or combinations thereof. Kifunensine can be used as an inhibitor of the endoplasmic reticulum mannosidase I and/or Golgi mannosidase IA and/or IB and/or 1C; deoxymannojirimycin can be used as an inhibitor of ER maimosidase I, ER mannosidase EE and/or of Golgi mannosidase IA and/or IB and/or 1C; swainsonine can be used an inhibitor of Golgi mannosidase H; and mannostatin A can be used as an inhibitor of Golgi mannosidase U. Use of a mannosidase inhibitor can inhibit the processing of a carbohydrate chain of GCB past a certain stage of mannose residue trimming during oligosaccharide assembly. For example, contacting a cell with kifunensine can inhibit triinrning of any, or one, two, three, or four of the mannose residues of a precursor oligosaccharide. Processing a-mannosidases can be blocked by treatment of cells with one or more of the following enzyme inhibitors: WO 02/15927 PCTYUSOi/25882 • Kifuneiisme, an inhibitor of ihe endoplasmic reticulum I and Golgi mannosidase I enzymes (Weng and Spiro (1993) J. Biol Qiern 268:25656-25663; Elbein et ai. (1990) J. Biol Chem 265:15599-15605). 5 • Swainsonine, an inhibitor of the Golgi mannosidase U enzyme (Tulsiani et al. (1982) /,' Biol Chem 257:7936^7939). • Deoxymannojirimycin, an inhibitor of both endoplasmic reticulum mannosidases I and II and of Golgi mannosidase I (Weng and Spiro (1993) /. Biol Chem 268:25656-25663; Tremblay and Herscovics (2000) J. Biol. Chem. Jul 27; [epub ahead of print]) 10 . DIM (l,4-dideoxy-l,4-imino-D-mannitol), an inhibitor of Golgi mannosidase H (Palamarzyk et al. (1985) Arch. Biochem. Biojjhys. 243:35-45). • 6-Deoxy-DIM and 6-deoxy-6-fLuoro-DIM, inhibitors of Golgi mannosidase II (Winchester et al. (1993) Biochem ]. 290:743-749). • Mannostatin A, an inhibitor of Golgi mannosidase H (Tropea et al. (1990) Biochemistry 15 29:10062-10069). Various mannosidase inhibitors can be selected by their ability to penetrate particular cell types as well as by the inhibitory potency of the mannosidase inhibitor. Fox example, swainsonine is rapidly internalized by cultured fibroblasts in a time- and concentratzon- 20 dependent manner. Swainsonine is also a potent inhibitor of a class 2 mannosidase, e.g., Golgi mannosidase U. Thus, swainsonine can be used to produce hmGCB in cultured fibroblasts, e.g., hmGCB having at least one carbohydrate chain which has at least four or five mannose residues of the precursor oligosaccharide. In addition, Hfunensine is readily taken up by cultured fibroblasts and is a potent inhibitor of class 1 mannosidases, e.g., ER 25 mannosidase I and Golgi mannosidase I. Thus, kifunensine can be used to produce hmGCB in cultured fibroblasts, e.g., hmGCB having at least one carbohydrate chain which has at least four, five, six, seven, eight or nine mannose residues of the precursor oligosaccharide. Preferably, the mannosidase inhibitor is present at a concentration of 0.025 to 20.0 ug/ml, 0.05 to 10 ug/ml, 0.05 to 5 tig/ml, preferably between about 0.1 to 2.0 jig/ml. For 30 " example, a class 1 processing mannosidase inhibitor can be present at a concentration between about 0.Q25 to 20.0 ug/ml, 0.05 to 10 ug/ml, 0.05 to 5 ug/ml, preferably between about 0.1 to 2.0 ug/ml; a class 2 processing mannosidase inhibitor can be present at a concentration between about 0.025 to 20.0 Jig/ml, 0.05 to 10 ug/ml, 0.05 to 5 u.g/ml, preferably between about 0.1 to 2.0 ug/ml; each of the class 1 processing and class 2 35 processing mannosidase inhibitors can be present at a concentration between about 0.025 to WO 02/15927 PC17US01/25882 20.0 ug/in].-0.05 to ibp-g/ml, 0.05 to 5 ug/mL preferably between, about 0.1 to 2.0 ptg/ml; or the total concentration of the class 1 processing and class 2 processing mannosidase inhibitors present can be between about 0.025 to 40.0 ug/rni, 0.05 to 20 U-g/ml, 0.05 to 10 ug/ml, preferably between about 0.1 to 5.0 ug/ml. 5 The cell can be contacted with a mannosidase inhibitor by, for example, culturing the cell on medium which includes at least one mannosidase inhibitor. Mannosidase Mutant Cell Mannosidase Knockout Cell 10 Permanent or regulated inactivation of mannosidase gene expression can be achieved by targeting to a mannosidase locus with a transfected plasmid DNA construct or a synthetic oligonucleotide. The plasmid construct or oligonucleotide can be designed to several forms. These include the following: 1) insertion of selectable marker genes or other sequences within an exon of a mannosidase gene; 2) insertion of exogenous sequences in regulatory 15 regions of non-coding sequence; 3) deletion or replacement of regulatory and/or coding sequences; and, 4) alteration of a protein coding sequence by site specific mutagenesis. In the case of insertion of a selectable marker gene into coding sequence, it is possible to create an in-frame fusion of an endogenous mannosidase exon with the mannosidase exon engineered to contain, for example, a selectable marker gene. In this way following 2fl successful targeting, the endogenous mannosidase gene expresses a fusion mRNA (mannosidase sequence "plus selectable marker sequence). Moreover, the fusion mRNA would be unable to produce a functional mannosidase translation product. In the case of insertion of DNA sequences into regulatory regions, the transcription of a mannosidase gene can be silenced by disrupting the endogenous promoter region or any 25 other regions in the 5' untranslated region (5* UTR) that is needed for transcription. Such regions include, for example, translational control regions and splice donors of introns. Secondly, a new regulatory sequence can be inserted upstream of the mannosidase gene that would render the mannosidase gene subject to the control of extracellular factors. It would thus be possible to down-regulate or extinguish mannosidase gene expression as desired for 30 optimal hmGCB production. Moreover, a sequence which includes a selectable marker and a promoter can be used to disrupt expression of the endogenous sequence. Finally, all or part WO OZ'15927 PCT/USOl/25882 c-ftht endogenous mannosidase gene could be. deleted by appropriate design of targeting In order to create a cell which includes a knockout of at least one chromosomal copy of the human Golgi mannosidase IA, IB or IC gene, the genomic DNA comprising at least the 5' portion of the gene (including regulatory sequences, 5' UTR, coding sequence) is isolated. For example, the GenBank sequence, Accession No.: NM0059Q7 (human), can be used to generate a probe for Golgi mannosidase IA or Accession Nos.: AAF97058 can be used to generate a probe for Golgi mannosidase IB or IC using polymerase chain reaction (PCR). Oligonucleotides for PCR can be designated based upon the GenBank sequence. The resulting probe can hybridize to the single copy Golgi mannosidase IA, IB or IC gene. This probe can then be used to screen a commercially available recombinant phage library (e.g., a library made from human genomic DNA) to isolate a clone comprising all or part of the mannosidase I structural genes. Once a recombinant clone comprising a mannosidase regulatory and/or coding sequence is isolated, specific targeting plasmids designed to achieve the inactivation of mannosidase gene expression can then be constructed. Inactivation of mannosidase activity results from the insertion of exogenous DNA into regulatory or coding sequences to disrupt the translational reading frame. Inactivation of the enzyme can also be the result of disruption of mRNA transcription or rnRNA processing, or by deletion of endogenous mannosidase regulatory or coding sequences. The nucleic acid sequence of other class 1 and class 2 processing mannosidase are also available, for example, in GenBank. Using the methods described above for Golgi mannosidase IA, IB or IC, a knockout cell for other class 1 and/or class 2 processing -mannosidases can be produced. A mannosidase knockout cell can be used, for example, in gene therapy. A knockout cell can be administered to a subject, e.g., a subject having Gaucher disease, such that the cell produces hmGCB in vivo. Antisense Mannosidase Nucleic Acid Sequences Nucleic acid molecules which are antisense to a nucleotide encoding a mannosidase, e.g., a class 1 processing or class 2 processing mannosidase, can be used as an inactivating WO 02/15527 PCT/US01/25882" sgcr^ uracil inhibit expression of a mannosidase. For example, Golgi mannosidase 1A_ Gebd maniiosiGESs IB. Golgi mannosidase IC. and/or Golgi mannosidase II explosion can be isinbiteG by an sniisense nucleic acid molecule. An "antisense" nucleic acid i^.Jndes a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a mannosidase, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can form hydrogen bonds with a sense nucleic acid. The antisense nucleic acid can be complementary to an entire mannosidase coding strand, or to only a portion thereof. For example, an . antisense nucleic acid molecule which antisense to the "coding region" of the coding strand of a nucleotide sequence encoding a mannosidase can be used. . As the coding strand sequences encoding various mannosidases are disclosed in, for . example, Bause (1993) Eur. J. Biochem. 217(2):535-540; Gonzalez et al. (1999) J. Biol Chem. 274(30):21375-21386; Misago et al. (1995) Proc. Nail Acad. Set USA 92(25):11766-11770; Tremblay et al. (1998) Glycobiology 8(6):585-595, Tremblay et al. (2000) J. Biol Chem. Jul 27:[epub ahead of print], antisense nucleic acids can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can comprise sequence complementary to the entire coding region of a mannosidase mRNA, but more preferably is an oligonucleotide which is complementary to only a portion of the coding or noncoding region of a mannosidase mRNA For example, the antisense oligonucleotide can comprise sequence complementary to the region surrounding the translation start site of a mannosidase mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35,40, 45, 50, or more nucleotides in length. An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracii, 5-bromouraciI, 5-chlorouracil, 5-iodouracii, hypoxanthine, xanthine, 4-acetylcvtosine, 5-(carboxyhydroxylmethyl)uraciI, 5-carboxymethylaminomethyl-2- WO 02/15927 PCT/USO1725882 tnioundine, S-caruci^i drjisdsEsetrhciL dmydrouracil, beta-D-galactosylqueosine: inosine, N6-isopenisiryl£deEizi&. l-iu&hylgusniiic. 1 -methyiinosine, 2,2-dimemyiguanine, 2-rnethyladenine, 2-methyIguanine. S-rnethylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, S-memylammomemyiuracil. 5-methoxyammornethyl-2-thiouracil, beta-D-mannosylqueosiDe, i'-memoxycarboxymethyluracil, 5-methoxyuraciI, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), ybutoxosine, pseudouracil, queosine, 2-* thiocytosine, 5-memyl-2-thioi2racil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester. uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-arnino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RKA transcribed from the inserted nucleic acid "will be of an antisense orientation relative to a target nucleic acid of interest. Purification of hrnGCB The term "purified" hmGCB, as used herein, refers to hmGCB that is substantially free of cellular material when produced by a cell which expresses GCB. The language "substantially free of cellular material" includes preparations of hmGCB in which the protein , is separated from cellular components of the cells in which it is produced. In one embodiment, the language "substantially free of cellular material" includes preparations of hmGCB having less than about 30% (by dry weight) of non-GCB protein (also referred to herein as a "protein impurity" or "contaminating protein17), more preferably less than about 20% of non-GCB protein, still more preferably less than about 10% of non-GCB protein, and most preferably less than about 5% non-GCB protein. When the hmGCB is obtained (i.e., harvested) from culture media, it is also preferably substantially free of a component of the culture medium, i.e., components of the culture medium represent less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the dry weight of the protein preparation. Various methods can be used to harvest hmGCB from culture media. The term "harvested hmGCB" as used herein refers to hmGCB obtained from culture media or from a cell. For example, one of the following alternatives can be used'to prepare the harvested hmGCB prior to a purification procedure. These can include: 1) filtering the fresh harvest; 2) filtering the fresh harvest and freezing, e.g., at about-20°C to ~-80°C, the filtered product WO 02/13927 PCT/US01/25882 . until ready for processing (at which tins t sn be xh^red end. optionally, filtered); 3) filtering the fresh harvest concentrHtnrg- nhssd prc-ds^i (e.g., by about 8 to 10 "fold), and then, optionally, filtering again; 4) filtering the fresh harvest, concentrating filtered product (e.g., by about 8 to 10 fold), optionally, filtering again, and then freezing, e.g., at about -20°C to -80°C, until ready for processing (at which time it can be thawed and, optionally, filtered). Variations of these alternatives can also be performed. For example, when the harvested product or concentrated harvested product is frozen, different harvests can be pooled after thawing and filtered. In addition, for harvested or concentrated harvested product, the product can be held at a cooling temperature, e.g., about 2°C to 8°C, for short periods of time, e.g., about 1 to 3 days, preferably 1 day, prior to purification. The harvested product held at the cooling temperature can be pooled prior to purification. When a concentration of harvest is performed, an ultrafiltration membrane with a 5,000 to 50,000 mw cutoff; preferably a 10,000 to 30,000 mw cutoff, can be employed. Filter clarification will typically employ a 1.2 um/0.5 urn prefilter, followed by a 0.2 urn final filter. HmGCB can be purified by the following purification techniques. For example, hydrophobic charge induction chromatography (HCIC) can be used to purify the hmGCB preparation. Alternatively, hydrophobic interaction chromatography (HIC) can be used to purify the hmGCB preparation. Both HCIC and HIC are described below. HCIC or HIC can be used alone or in combination with one or more ion exchange steps. Ion exchange steps that can be used in combination with an HCIC or HIC step (either before.or after KCIC or HIC) include the use of anion exchange and/or cation exchange chromatography. Generally known commercially available anion exchange supports used in the purification of proteins bear quaternary ammonium functional groups. Preferred matrices for use in the present process are agarose or cellulose based matrices such as microcrystalline cellulose or cross-linked agaroses. Also particularly preferred are those matrices bearing diethyl aminoethyl, triethyl aminomethyl, or trimethyi aminomethyl functional groups. A particularly preferred anion exchange matrix is trimethyi aminomethyl crosslinked agarose, which is commercially available, e.g., Q-Sepharose Fast Flow® (Pharmacia). Generally known commercially available cation exchange supports that may be used in the purification of proteins bear acidic functionalities, including carboxy and sulfonic acids. Matrices WO 02/15927 PCT/USO1/258 82 - containing the cation functionalities include various fonzs of ceHnksss snd polystyrene based matrices. For example, weak cation exchangers krmrz. inisan xrjc^Kie. but are not limited to, Carboxym ethyl-Sepharose® and Carboxymethyl-CeMose®. Strong canon exchangers known in the art include, but are not limited to. sulfonated polystyrenes (AG 5.0W®, Bio-Rex 70®), sulfonated celluloses (SP~Sephadex®), and sulfonated Sepharoses (S-Sepharose®). A particularly preferred cation exchange matrix is S-Sephaiose Fast Flow® (Pharmacia). The chromatographic step involving these matrices is most preferably conducted as a column chromatography step or in alternative a batch absorptive technique, which optionally can be performed at a temperature between 25°C to 40°C. Preferably, a salt is added to a washing or eluting buffer to increase the ionic strength of the buffer. Any of the salts conventionally used may be employed for this purpose as can be readily determined by one skilled in the art, with NaCl being one of the most frequently and conveniently used salts. A conventional gel filtration step can also be used in combination with the HCIC or HIC chromatography process step. Representative examples of these matrices are polydexrrans cross Jinked with acrylamides, such as composite hydrophilic gels prepared by covalently cross linking allyl dextran with N, N'-methylene bisacrylamide and crosslinked cellulose or agarose gels. Commercially available crosslinked dextran-acrylamides are known under the trade name Sephacryl® and are available from Pharmacia. Commercially available crosslinked dextran-agarose resins are known under the trade name Superdex®, available from Pharmacia. A preferred Superdex® gel is Superdex 200®. Examples of crosslinked cellulose gels are those commercially available cross linking porous cellulose gels, e.g., GLC 300® or GLC 1,000® that are available from Amicon Inc. Silica based resins such as TSK-Gel SW®, available from TosoHaas can be utilized. Polymer based resins such as TSK-Gel PW®, TSK Alpha Series®, Toyopearl HW packings® (copolymerization of ethylene glycol and methyl acrylate polymers) are also available from TosoHaas. Preferably, HCIC or HIC can be combined with one or more of these ion exchange steps. When a combination of HCIC or HIC and various ion exchange or ge] filtration steps are used, they can be performed in any order. For example, as described below a four step procedure can be followed which includes HCIC using MEP Hypercel® chromatography or WO 02/15927 VCT.VSQV25881 HIC using MacroPrep Methyl® chromatography,- then Q Sepharose Fast Flow 5? Sepharose Fast Flow1© and lastly Superdex 200®. Several of these procedure are set form in more detail below. 5 MEP Hypercel Chromatography MEP (mercaptoethylpyridine) Hypercel®.(BioSepra, Life Technologies) can be used for HCIC. It is a resin consisting of NEP linked to a regenerated cellulose bead of high porosity (80-100 microns). The functional group (MEP), consisting of a hydrophobic tail and an ionizable head group, is uncharged at neutral pH and can bind certain protein ligands 10 based on hydrophobic interaction at a physiological ionic strength: Elution is accomplished by decreasing pH to 4 to 5, at which MEP is positively charged, and the protein elates from the column due to electrostatic repulsion. For example, prepared harvest or harvest concentrate can be applied directly to the MEP column equilibrated with 25 mM so, :um phosphate, pH 6.8, containing 180 mM sodium chloride and 2 mM DTT. Optionally, the 15 column can then be washed with equilibration buffer containing 25 mM sodium caprylate until the absorbance at 280 nm (A280) stabilizes. The hmGCB can.be eluted from the column with 50mM sodium acetate, 2 mM DTT, pH 4.7, and the peak as monitored at 280 nm can be collected. 20 MacroPrep Methyl Chromatography An alternative to MEP Hypercel® is MacroPrep Methyl®, which is a hydrophobic interaction chromatography (HIC) resin. This resin consists of a methyl functional group attached to a bead composition of macroporous co-polymerized glycol methacryiaie and diethylene glycol dirnethacrylate. For example, MacroPrep Methyl® (BioRad) 25 chromatography can be performed as follows. The pH of the harvest or harvest concentrate is adjusted to 5.6, and ammonium sulfate is added to 0.70 M final concentration. The prepared harvest can be applied to the MacroPrep Methyl® column, which has been equilibrated in 0.70 M ammonium sulfate, 10 mM MES, pH 5.6. After application of the load, the column is washed with equilibrated buffer until the A280 returns to baseline. The 30 hmGCB can be eluted with 10 mM MES, pH 5.6. The eluted hmGCB can be ultrafiltered WO 02/15927 PCT/US01/25882 and/or dianltered in preparation for steps such as an ion exchange step such as Q Senirsi^se chromatography, gp Sepharose chromatography and/or Superdex 200 Crtromatograprry. QjjgBfairose Chromatography Q Sepharose Fast Flow® (Amersham 'Pharmacia) is a relati vely strong anion exchange chromatography resin. The functional substituted is a quaternary amine group, which is positively charged over the working pH range of 2 to 12. Proteins with a net negative charge at the working pH will tend to bind to the resin at a relatively low ionic strength and can he eluted at higher ionic strength or lower pH. HmGCB does not bind to Q Sepharose at approximately pH 6 and low ionic strength, but impurities do bind, thereby purifying the sample. For example, the following protocol can be used to purify hmGCB in the sample by Q Sepharose Fast Flow® chromatography. Under appropriate conditions. hmGCB flows through this column, so the product is found in the flowthrough/wash fraction, Sodium phosphate (250 mM, pH 6) is added to the MEP elution pool prepared as described above to a final concentration of 25 mM, and the pH of the pool is adjusted to pH 6 with NaOH (and HC1 if necessary). The conductivity is adjusted to 2.5 +0.1 mS/cm by dilution with water or by ultrafiltration/diafiltration using 25 mM sodium phosphate, 2 mM DTT, at approximately pH 6. The material is then filtered and applied to a column of Q Sepharose Fast Flow® which has been equilibrated in 25 mM sodium phosphate, 2 mM DTT, pH 6.0. After application of the load, the column is washed with equilibration buffer until the A280 reaches baseline. The flowthrough/wash fraction can then be processed through another column., e.g.j SP Sepharose Fast Flow® column, shortly thereafter, e.g.^ within 24 hours, or frozen and stored at about -20°C to -S0°C prior to further processing. Other strong anion exchange resins, such as Macro-Prep High Q Support® (BioRad) can be used in place of Q Sepharose. A weaker anion exchange resin such as DEAE Sepharose Fast Flow® (Pharmacia) or Macro-Prep DEAE® (BioRad) can also be used. The column is equilibrated in buffer, e.g., 25 mM sodium phosphate, pH 6. The pH of the sample is adjusted to pH 6 and the conductivity is adjusted by dilution or diafiltration to a relatively low ionic strength, which allows impurities to bind to the column and hmGCB to flow through. The sample is applied and the column is washed with equilibration buffer. Impurities are still bound to the column, and can be eluted with application of salt, e.g., WO 02/15927 PCT7US01/25882 sodium chloride or potassium chloride, or application of a lower pH buffer, or a combination of increased salt and lower pH; The hmGCB can also be allowed to bind the anion exchange column during loading by decreasing the salt concentration in the load or by running the column at a higher pH, or 5 by a combination of both decreased salt and higher pH. SP Sepharose Chromatography SP Sepharose Fast Flow® (Amersham Pharmacia) is a relatively strong cation exchange chromatography resin. The functional substltutent is a charged sulfonic acid group, 10 which is negatively charged over a working pH range of 2 to 12. Proteins with a net positive charge at the working pH will tend to bind to the resin at a relatively low ionic strength and can be eluted at higher ionic strength or higher pH.' HmGCB binds to SP Sepharose at approximately pH 6 and intermediate ionic strength (e.g., 6.5 mSJcm) and can be eluted at higher ionic strength (e.g., 10.7 mS/cm). Impurity proteins remain bound to SP Sepharose 15 under conditions of hmGCB elution, thereby purifying the hmGCB in the sample. For example, the following protocol can be used to purify hmGCB by SP Sepharose Fast Flow® chromatography. Sodium chloride (2.0 M stock) is added to the Q Sepharose® flowthrough/wash until the conductivity is 6.3 mS/cm, The pH is checked and readjusted to pH 6.0 if necessary. Then, addition of sodium chloride stock is continued until the 20 conductivity is 6.5 mS/cm. The material is filtered and applied to a column of SP Sepharose Fast Flow®, which has been equilibrated with 25 mM sodium phosphate, 44 mM sodium chloride, pH 6.0. After application of the load, the column is washed with equilibration buffer until the baseline is reached and eluted with 25 mM sodium phosphate, 84 mM sodium chloride, pH 6.0. HmGCB is found in the elution fraction. 25 Another cation exchange resin, e.g., Source 30S® (Pharmacia), CM Sepharose Fast Flow® (Pharmacia), Macro-Prep CM Support® (BioRad) or Macro-Prep High S Support® (Bioliad), can be used as an alternative to SP Sepharose. The hmGCB can bind to the column at approximately pH 6 and low to intermediate ionic strength, such as 4 to 7 mS/cm. A buffer, e.g., 10 mM sodium citrate, pH 6.0, 10 mM MES, pH 6.0, 25 mM sodium 30 phosphate, pH 6.0, or other buffer with adequate buffering capacity at pH 6.0 can be used to equilibrate the column. The ionic strength of the sample is adjusted by dilution or WO 02/15927 PCTYUS01/25882 diafiltratioii-to a level which will accommodate binding to the column. The sample'is applied to the column sd the cohsss is washed after the load to remove unbound material. A salt, e.g., sodium chloride or potassium chloride, can be used to elute the hmGCB from the column. Alternatively, the hmGCB" can be eluted from the column with a buffer of higher 5 pH or a combination of higher sah concentration and higher pH. The hmGCB can also be made to flow through the cation exchange column during loading by increasing the salt concentration in the equilibration buffer and in the sample load, by running the column at a higher pH or by a combination of both increased salt and higher pH. 10 Superdex 200 Chromatography Superdex 200 prep grade® (Amersham Pharmacia) is used for size exclusion chromatography of hmGCB, whereby molecules are separated by size, molecular mass, strokes radius or hydrodynamic volume. Superdex 200 is composed of dextran covalently 15 cross linked to agarose and has a fractionation range of 10,000 to 60,000 molecular weight for globular proteins. For example, the following protocol can be used to purify hmGCB by Superdex 200® chromatography. The S~P elution pool is concentrated by ultrafiltration using a 10,000 niw cutoff membrane. The concentrated pool is filtered, then applied to a Superdex 200 prep grade® column which has been equilibrated in 50 mM sodium citrate, pH 6.0. The 20 A280 of the column effluent in the initial fractions is collected and, for example, an 8 to 16% SDS poly aery lamide gel is run to determine pooling of fractions. Pooling may be decided based on visual inspection of the silver-stained gel. Other size exclusion chromatography resins such as Sephacryl S-200 HR®, Bio-Gel A 1.5m®, or TosoHaas TSK Gel resins can also be used to purify hmGCB. The buffer used 25 for size exclusion chromatography of hmGCB is 50 mM sodium citrate, pH 6.0. Other buffers can also be used such as 25 mM sodium phosphate, pH 6.0 containing 0.15 M sodium chloride. The pH of the buffer can be between pH 5 and pH 7 and should have sufficient ionic strength to inininiize ionic interactions with the column. W'6 02/15927'" PCT/US01/25882 Variations of pH. buffer and/or salt concentration in any of the purification protocols described above can be performed by rc-vrrfne medaods to achieve the desired purified product. Assays For Determining Macrophage Uptake and Cellular Targeting of hmGCB 5 The uptake efficiency of hmGCB by macrophages can be determined by assaying, e.g., protein levels and/or enzyme activity in macrophages. For example, as described in the Examples below and in Diment et al. (1987) /. Leukocyte Biol 42:485-490, an in vitro assay using a macrophage cell line can be used to determine absolute and mannose receptor specific uptake of hmGCB. 10 In addition, in vivo comparison of uptake of hmGCB and GCB by liver cells can be determined as described, for example, in Friedman et al. (1999) Blood 93:2807-2816. Briefly a mouse model can be injected'with hmGCB or GCB, and then sacrificed shortly thereafter. The liver of the animal can then be used to prepare a suspension of liver cells, e.g., parenchymal cells, KupfTer cells, endothelial cells and hepatocytes. The cells can then be 15 separated, identified by morphology and the protein levels and/or enzymatic activity of hmGCB and GCB in the various liver cell types can be determined. Alternatively, immunohistochemical detection maybe be used to localize hmGCB to a specific cell or cell type in tissue of treated animals. 20 Pharmaceutical Compositions High mannose glucocerebrosidase (hmGCB) can be incorporated into pharmaceutical compositions suitable for administration to a subject, e.g., a human. The composition can include a sufficient dosage of hmGCB to treat a subject having Gaucher disease. As used herein the language "pharmaceuticaily acceptable carrier" is intended to include any and all 25 solvents, excipients, dispersion media, coatings, antibacterial and antifungal agents, isotonic and adsorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceuticaily active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the invention. Supplementary 30 active compounds can also be incorporated into the compositions. WO 02/15927 PCTYUS01/25882 A pharmaceutical composition of-fee invention is formulated to be compatible with its intended route of adrmrdstratioiL Examples of routes of administration include parenteral e.g., intravenous, intradermal, and subcutaneous administration. Preferably, the route of adniimstration is intravenous. Solutions or suspensions used for parenteral application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as emylenedlaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders, e.g., lyophilized preparations, for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged stability of the injectable . compositions can be brought about by including in the composition an agent which delays adsorption, for example, aluminum monostearate, human serum albumin and gelatin. \V0 02/15927 PCT/0SO1/25882 Sterile injectable solutions can be prepared by incorporating the hmGCB in the reqrfred amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic 5 dispersion medium and the required other ingredients from those enumerated above, in the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, e.g., lyophilization, which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. 10 In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyandrydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of 15 such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutical^ acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in 20 U.S. Patent No. 4,522,811. Treatment of Gaucher Disease HmGCB, e.g., any hmGCB molecule or preparation described herein, can be used to treat a subject having Gaucher disease. Alternatively, any mannosidase knockout cell 25 described herein, can be introduced into a subject having Gaucher disease to deliver hmGCB to the subject. Various routed of administration and various sites can be used. Once implanted in individual, the knockout cell can produce hmGCB. Preferably, the knockout cells used will generally be patient-specific genetically engineered cells. It is possible, however, to obtain cells form another individual of the same 30 species or form a different species. Use of such cells might require administration of an WO GZ--"L5?2" PCT7US01/2S882 irrrrt-^xycrggassanL HiuaiiiJoD of histocompatibility antigens, or use of a barrier device to Gaucher disease is an autosomal recessive lysosomal storage disorder characterized by a deficiency in the lysosomal enzyme, glucocerebrosidase (GCB). GCB hydrolyzes the 5 glycolipid glucocerebroside that is formed after degradation of glycospliingolipids in the membranes of white blood cells and red blood cells. If GCB hydrolysis is insufficient then glucocerebroside can accumulate in macrophages (Gaucher cells), causing anemia, thrombocytopenia, organomegaly and major bone problems. There are several types of Gaucher disease including Gaucher type 1, type 2 and type 10 3, which can arise due to various mutations in the GCB gene. A "therapeutically effective amount" of hmGCB. i.e.. a dosage of hmGCB sufficient to treat Gaucher disease, can be given to a subject having this disorder. The term "treat" as used herein refers to reducing or inhibiting one or more symptoms of Gaucher disease. Symptoms of Gaucher disease type I include: skeletal complications such as bone pain, bone lesions, osteopenia, osteonecrosis, 15 avascular necrosis and pathological fractures; anemia; hepatosplenomegaly; splenic nodules and liver dysfunction; thrombocytopenia; and/or delayed growth and pubertal development. Symptoms of Gaucher disease type n include the symptoms of Gaucher type I as well as neck rigidity, apathy, catatonia, strabismus, increased deep reflex and laryngeal spasm. Symptoms of Gaucher disease type HI are similar to Gaucher type II except milder and later 20 in onset. A therapeutically effective amount of hmGCB can be determined on an individual basis and will be based, at least in part, on consideration of the size of the patient, the agent used, the type of delivery system used, the time of administration relative to the severity of the disease, and whether a single, multiple, or a controlled release dose regimen is employed. 25 Preferably, the dosage of hmGCB sufficient to treat Gaucher disease is less than the dosage of human tissue derived or human placenta derived GCB, or GCB produced by cells in vitro and then trimmed to expose core marmose residues. Treatment of Other Lysosomal Storage Diseases 30 Generally, the invention described herein can be used to produce proteins for targeting any cells that express mannose receptors on their surface. Thus, the invention WO 02/15927 . - PCT/US01/25882 described herein can be nsd H: T7=Et airy disorder in which it is desirable to target a protein for treatment to a mannese recepic^-zxpressing cell For example, the invention described herein can also be applied to other lysosomal storage enzymes and other lysosomal storage diseases in which cells, e.g., the cells of reticuloendothelial origin, accumulate undigested substrate. Reticuloendothelial cells include macrophages, Kupffer cells in the liver and histiocytes in the spleen. Such lysosomal storage diseases include, but are not limited to, Farber disease and Neimann-Pick disease. Farber disease is an autosomal recessive lysosomal storage disorder characterized by a deficiency in acid ceramidase. Ceramidases are enzymes responsible for degradation of ceramide. If ceramide degradation is insufficient then ceramide accumulates leading to granuloma formation and histiocytic response. (Moser, H.W. Ceramidase deficiency: Farber lipogranulomatosis; In: The Metabolic and Molecular Basis of Inherited Disease (C.R, Scriver, AX. Beaudet, W.S. Sly and D. Valle, Eds.), Seventh edition, pp. 2589-2599, McGraw-Hill Inc., New York (1995)) There are several types of Farber disease including Farber type 1, type 2, type 3, type 4, and type 5 which differ in severity and sites of major tissue involvement. There is also type 6 and type 7 Farber disease. High mannose acid ceramidase can be given to a subject having Farber disease to treat, i.e., alleviate or reduce at least one symptom, of the disease. Symptoms of Farber disease type 1 include: swelling of the joints (particularly the interphalangeal, metacarpal, ankle, wrist, knee and elbow), palpable nodules in relation to the affected joints and over pressure points, a hoarse cry that may progress to aphonia, feeding and respiratory difficulty, poor weight gain and intermittent fever. The symptoms usually occur between ages two weeks and ibis: months. Symptoms of Farber type 2 and type 3 include: subcutaneous nodulaes, joint deformities, and laryngeal involvement. These subjects survive longei than subjects having Farber type 1. Farber disease type 5 symptoms include psychomotor deterioration beginning at one to two and half years of age. Neimann-Pick disease type A and type B are an autosomal recessive lysosomal storage disorder characterized by a deficiency acid sphingomyelinase. Acid sphingomyelinase is an enzyme responsible for degradation of sphingomyelin. If sphingomyelinase is deficient, sphingomyelin and other lipids can accumulate in the WO 02/15927 PCT/USO1/25882 ' monocyte-macrophage system. (schmen . r K and Desnck, RJ. Neiinann-Pick Disease types A and B: "acid sphingomyelinase rHvrieigies: In; Th= Metabolic and Molecular Basis of Inherited Disease (C.R. Scriver. AX,. Beandet, W.S. Sly and D. Valle, Eds.), Seventh edition, pp. 2589-2599, McGraw-Hill Inc., New York (1995)) 5 There are several types of Neimann-Pick disease including type A and type B. High ■mannose acid sphingomyelinase can he given to a subject havmg Neimann-Pick disease to treat, i.e., alleviate or reduce at least one symptom, of the disease. Symptoms of Neimann-Pick disease type A include: enlargement of the spleen and liver, lymphadenopathy, microcytic anemia, decreased platelet count, hypotonia, muscular weakness, psychomotor 10 retardation. Symptoms of Neimann-Pick type B include; enlargement of the liver and/or spleen, heptoslenomegaly; pulmonary compromise. Thus, high mannose lysosomal storage enzymes such as high mannose acid ceramidase or high mannose acid spingomyelinase can be produced by the methods described herein in order to target these proteins to mannose receptor-expressing cells. 15 Examples In experiments with HT-1080 cells expressing Gene-Activated"™ GCB (GA-GCB), the cells were treated with either Hfunensine or swainsonine at concentrations ranging from 0.1 to2ug/mL. 20 Effect of Kifunensine or Swainsonine on GA-GCB Glycofprms HT-1080 cells producing GA-GCB were plated in duplicate 6-weII plates and the Production Medium adjusted to the following of kifunensine or swainsonine: 0 (no drug), 0.1, 0.25, 0.5, 1, and 2 u-g/mL. The medium was harvested and the cells refed every 24 hours for three days. The samples from the third day were subjected to isoelectric 25 focusing (IEF) analysis. The effect of kifunensine and swainsonme on the molecular charge of GA-GCB is shown by the IEF analysis. With both drugs, a concentration dependent increase in the apparent isoelectric point (pi) was observed, with kifunensine causing a much - larger shift in pi than swainsonine at the highest concentration tested (2 u.g/mL). 30 WO 02/15927 PCT/US01/25882 Effect of Kifunensine or Swainsonine on GA-GCB 'PiDdnakzi Ten roller bottles (surface area, 1700 cm2 each) were seeded in Growth Medium (DMEM with 10% calf serum) with HT-1080 cells producing GA-GCB. Following two weeks of growth, the medium was aspirated and 200 mL of fresh Production Medium 5 (DMEM/F12, 0% calf serum) was added to three sets of roller bottles. Two sets of 4 roller bottles were treated with 1 ug/mL of either kifunensine or swainsonine. The third group of two roller bottles received no drug treatment. After approximately 24 hours, the medium from each roller bottle was harvested, pooled and a sample taken for GA-GCB enzymatic activity analysis. This procedure was repeated for seven days. Stable production of GA- 10 GCB was observed for all roller bottles throughout the seven daily harvests (Table 1). Absolute levels of the enzyme, however, varied according to drug treatment group with the following average GA-GCB production levels observed across the seven harvests: 38.3±3.5 mg/L (control, no drug treatment), 24.5±4.0 mg/L (swainsonine, 1 pg/mL), and 21.3±2,8 mg/L (kifunensine, 1 pg/mL). Both drugs, therefore, resulted in stable, but lower production 15 levels with the largest decrease seen for kifunensme (44% reduction relative to control). Table 1. Roller Bottle Production of Glucocerebrosidase in Cells Treated with Mannosidase Inhibitors Treatment Glucocerebrosidasea' Activity (D) mg / Liter) Harvest 1 Harvest. 2 Harvest 3 Harvest 4 Harvest 5 Harvest 6 Harvest 7 Average + Standard Deviation No drug added 35.8 36.6 44.9 4QS 34.6 3 S3 372 383 ±3.5 Swainsonine (1 ug/mi) 28.6 0 17.4 2S.5 27.0 22.9 25.0 223 245 ±4M Kifunensine (1 Pg/ml) 26.0 22.9 17.7 21.2 18.4 21.0 22.0 21.3+2.8 Assay performed as follows: test article is mixed with the enzyme substrate (4-melhylumbelliferyI-p-D-glucopyranoside) and incubated for 1 hour at 37°C. The reaction is stopped by the addition of NaOH/GIycine buffer. Fluorescence is quantified by the use of a fluorescence spectrophotometer. Specific activity: 2,500 Units/mg. One unit is defined as the conversion of 1 uMole of substrate in 1 hour at 25 37aC. WO 02/15927 PCT-USOL'25882 Effect of Kifaneasine or Swainsonine on GA-GCB Uptake into Macroohsss GA-GCB produced in HT-1080 cells was used in an in vitro assay re -issr~23s uptake efficiency in a mouse macrophage cell line. The specific objective of the experiment was to determine the absolute and mannose receptor-specific uptake of GA-GCB in mouse 5 J774E ceils. One day prior to assay, J774B cells were plated at 50,000 cells/cm1 in 12 well plates in Growth Medium. For the assay, 0.5 mL of Production Medium (DMEM/FI2), 0% calf serum) containing 50 nM vitamin D3 (1,2-5, Dihydroxy vitamin D3) was added to the cells; Unpurified GA-GCB (from harvest 4, Table 1) was added to the test wells at a final concentration of 10 pg/mL in the presence or absence of 2 pg/mL mannan (a competitor for 10 the mannose receptor). Three different forms of GA-GCB were used: GA-GCB from cells treated with kiiunensine (1 ug/mL), GA-GCB from cells treated with swainsonine (1 pg/mL), and GA-GCB (1 pg/mL) from untreated cells. Control wells received no GA-GCB. The wells were incubated for 4 hours at 37°C, then washed extensively in buffered saline, scraped into GA-GCB enzyme reaction buffer, passed through 2 freeze/thaw cycles, arid clarified by 15 centrifugation. The supernantant was then quantitatively tested for enzyme activity and total protein. Internalization of GA-GCB into mouse J744E cells is shown in Table 2 and is reported as Units/mg of cell lysate. These results demonstrated that uptake of GA-GCB from kifunensine treated cells was 14-fold over background and 73% inhibitable by mannan and that uptake of GA-GCB from swamsonine treated cells was 7-fold over background and 67% 20 inhibitable by mannan. In addition, they also demonstrate that uptake of GA-GCB from untreated cells was approximately 3-fold over background and 53% inhibitable by mannam Thus, the inhibition of intracellular rnannosidases by either kifunensine or sw'airisomne results in GA-GCB that can be transported into cells efficiently via the mannose receptor, with kifunensine causing an approximately 2-fold greater uptake than swainsonine. 25 Improvement in targeting of GA-GCB to cells via mannose receptors can therefore be optimized by production of GA-GCB in the presence of kifunensine or swainsonine. 30 WO 02/15927 PCT/DS01/25882 Table 2. Internalization of Glucocerebrosidase Into J774E Cells. Glucocerebrosidase Produced from Cells Treated with Mannosidase Inhibitors D> Sample aj Glucocerebrosidase Activity (Units/mg cell lysate) Inhibition (%) Absolute Background Corrected Background (no GA-GCB added) 655 0 - GA-GCB from untreated cells -fMannan 2816 2161 - 1678 1023 53 GA-GCB from kifunensine treated cells + Mannan 9185 8530 - 2977 2322 73 GA-GCB from swainsonine treated cells +'Mannan 4787 4132 - 2036 1381 67 5 a* Assay performed as follows: sample is mixed with the enzyme substrate (^methylumbelliferyl-p-D- glucopyranoside) and incubated for 1 hour at 37°C. The reaction is stopped by the addition of NaOH/Glycine buffer. Fluorescence is quantified by the use of a fluorescence spectrophotometer. Total protein determined in freeze/thaw cell lysates by bicinchoninic acid (BCA). Activity reported as nnits/mg total protein. One 10 Unit is defined as the conversion of 1 uMole of substrate in 1 hourat37°C. ' Cells treated with drug received 1 ug/mL of either Kifunensine or Swainsonine in the presence or absence of mannan (2 \isjmL). Purification and Characterization of hmGCB 15 HmGCB was purified from the culture medium of human fibroblasts grown in the presence of kifunensine at a concentration of 2 U-g/ml The four N-linked glycans present on hmGCB were released by peptide N-glycosidaseF and purified using a Sep-pakC18 WO 02/15927 PC17US01/25882 cartridge. Oligosaccharides eluting in the 5% acetic acid fraction were perrnethylated using sodium hydroxide and methyl iodide, dissolved in methanohwater (80:20), and pcmons of me p&rmethyiaied glycan mixture were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (MALDI-TOF-MS). The sample was analyzed on a Voyager STR. Biospectrometry Research Station laser-desorption mass spectrometer coupled with Delayed Extraction using a matrix of 2, 5-dihydroxybenzoic acid. A pattern of pseudomolecular ions is seen in the range m/z 1500-2500, indicating the presence of high-mannose glycans ranging from Man5GlcNAc2 to Man9GlcHAc2. Table 3. Summary of Data Obtained from MALDI-TOF-MS Analysis of N-GJycans from hmGCB from Kifunensine-Treated Cells M/Z Peak Assignment Approximate % of Total Glycans 15S0 Man5GlcNAc2 13 1730 1752 1784 MarifiGlcNAcz 11.2 1934 1957 1988 Man7GlcNAc2 23.3 2139 2161 2192 MangGIcNAcz 32.0 2343 2365 2397 Man9GlcNAc2 31.2 2969 Biantennary complex 1.0 The most abundant high mannose glycans present are Man9GlcNAc2 and ManSGicKAcj, with decreasing abundances of Man7GIcNAc2, Man^GlcNAoz, and Man5GlcNAc2. A trace amount of a hicosylated biantennary complex glycan containing two sialic acid residues was observed. An approximate indication of the relative abundancy of each crjycan is obtained by measuring the peak heights. See Table 3. A more accurate assessment of the average chain length of the high mannose glycans was obtained by MALDI-TOF-MS analysis of the intact glycoprotein. A sharp peak was obtained at m/z WO 02/15927 PCT7US01/25882 62:4S3.1 due to the homogeneity of the glyean chains. The mass of the mature peptide calculated from the amino acid seaueuce is 55;577.6: indicating the four N-linked glycan chains contribute 6905.5 to the total mass of hmGCB. Froni this number, it can be calculated that the average glycan length is 8.15 mannose residues. All patents and references cited herein are incorporated in their entirety by reference. Other embodiments are within the following claims. Claims to be filed in national phase of 10278-017WO1 1. A method of producing a high mannose glucocerebrosidase (hmGCB), comprising: providing a cell which is capable of expressing glucocerebrosidase (GCB); contacting the cell with kifunensine such that the removal of at least one mannose, residue distal to the pentasaccharide core of the precursor oligosaccharide of GCB is prevented; and allowing the cell to produce hmGCB, to thereby produce an hmGCB preparation. 2. The method of claim 1, wherein removal of one or more a 1,2 mannose residue(s) distal to the pentasaccharide core is prevented. 3- The method of claim \, wherein removal of one a 1,3 mannose residue distal to the pentasaccharide core is prevented. 4. The method of claim I, wherein removal of one a 1,6 mannose residue distal to the pentasaccharide core is prevented. . 5. The method of claim 1, wherein the ldfunensine is present at a concentration between about 0.05 to 20.0 p-g/ml. 6. The method of claim 1, wherein the kifunensine is present at a concentration between about 0.1 to 2.0 jig/ml 7. The method of claim 1, further comprising contacting the cell with a class 2 processing mannosidase inhibitor. 8. The method of claim 7, wherein the class 2 processing mannosidase inhibitor is selected from the group consisting of: swainsonine, mannostatin, 6-deoxy DIM, 6-deoxy-6-fluoro-DIM and combinations thereof. 9. The method of claim 7, wherein the class 2 processing mannosidase inhibitor is swainsonine. 10. The method of claim 7, wherein the class 2 processing mannosidase inhibitor is present at a concentration between 0.05 to 20.0 ug/ml. 11. The method of claim 1, wherein the hmGCB has at least one carbohydrate chain having five mannose residues. 12. The method of claim 1, wherein the hmGCB has at least one carbohydrate chain having eight mannose residues. 13. The method of claim 1, wherein the hmGCB has at least one carbohydrate chain having nine mannose residues. 14. The method of claim 1, wherein the removal of one or more mannose residues distal to the pentasaccharide core is prevented on at least two carbohydrate chains of hmGCB. 35. The method of claim 1, wherein at least 60% of the hmGCB of the preparation have one or more carbohydrate chains in which the removal of one or more mannose residues distal to the pentasaccharide core has been prevented. 16. The method of claim 15, wherein the removal of three or more mannose residues distal to the pentasaccharide core has been prevented. 17. The method of claim 1, wherein at least about 20% of the hmGCB of the preparation have one or more carbohydrate chains having at least eight mannose residues. 18. The method of claim 17, wherein at least about 40% of the hmGCB of the preparation have one or more carbohydrate chains having at least eight mannose residues. 19. me method of claim IS, wherein at least about 60% of the hmGCB of the preparanon have one or more carbohydrate chains having at least eight mannose residues. 20. The method of claim 1, wherein at least about 80% or more of the carbohydrate chains of the hmGCB preparation have six or more mannose residues. 21. The method of claim 1, wherein the cell is a knockout for at least one class 2 processing mannosidase. 22. The method of claim 1, wherein the cell comprises a class 2 processing mannosidase antisense molecule. 23. The method of claim 1, wherein the cell comprises an exogenous nucleic acid sequence comprising a GCB coding region. 24. The method of claim 23, wherein the cell further comprises an exogenous regulatory sequence which functions to regulate expression of the GCB coding region. 25. The method of claim 1, wherein the cell comprises an exogenous regulatory sequence which fiinctions to regulate expression of an endogenous GCB coding sequence. 26. The method of claim 1, wherein the cell is a primary cell. 27. The method of claim 1, wherein the cell is a secondary cell. 28. The method of claim 1, wherein the cell is a mammalian cell 29. The method of claim 28, wherein the cell is a human cell. 30. The method of claim 29, wherein the cell is a fibroblast or a myoblast. 31 The method of claim 29, wherein the cell is an immortalized cell. 32. The method of claim 31, wherein the cell is anHT-1080 cell. 33. The method of claim 1, wherein the cell is contacted with kifunensine in ' culture media. 34. The method of claim 33, wherein the hmGCB is obtained from the media in which the cell is cultured. 35. A method of purifying hmGCB from a sample, comprising: providing a harvested hmGCB product; and subjecting thehmGCB product to hydrophobic charge induction chromatography (HCIC) or hydrophobic interaction chromatography (HIC), thereby obtaining purified hmGCB. 36. The method of claim 35, further comprising subjecting the hmGCB product to ion exchange chromatography. 37. The method of claim 36, wherein the hmGCB product is subjected to HCIC or HIC prior to ion exchange chromatography. 38. The method of claim 36, wherein the hmGCB product is subjected to ion exchange chromatography prior to HCIC or HIC. 39. The method of claim 36, wherein the hmGCB product is subjected to more than one ion exchange chromatography step. 40. The method of claim 36, wherein the ion exchange chromatography is selected from the group consisting of anion exchange chromatography and cation exchange chromatography. 41. ire -method c-f claim 36, further comprising subjecting the hniGCB product to size esclixsioL: chztsnistography. 42. The method of claim 35, further comprising subjecting the hmGCB product to one or more of anion exchange chromatography, cation exchange chromatography, and size exclusion chromatography. 43. The method of claim 35, further comprising: subjecting the HCIC or HIC purified hmGCB product to anion exchange chromatography; subjecting the anion exchange purified hmGCB to cation exchange chromatography; and, subjecting the cation exchange purified hmGCB to size exclusion chromatography. 44. The method of claim 35, 42 or 43, wherein MEP Hypercel® is used for HCIC. 45. The method of claim 35, 42 or 43, wherein MacroPrep Methyl® is used for HIC. 46. The method of claim 35, 42 or 43, wherein anion exchange chromatography is performed using one or more of: Q Sepharose Fast Flow®, MacroPrep High Q Support®, DEAE Sepharose Fast Flow®, and Macro-Prep DEAE®. 47. The method of claim 35, 42 or 43, wherein cation exchange chromatography is performed using one or more of: SV Sepharose Fast Flow®, Source 30S®, CM Sepharose Fast Flow®, Macro-Prep CM Support®, and Macro-Prep High S Support®. 48. The method of claim 41 or 43, wherein the size exclusion chromato graph y is performed using one or more of: Superdex 200®, Sephacryl S-200 HR® and Bio-Gel A 1.5m®. 49. ' Amethod ofprodixingliighiriannoseglucocerebrosid^se (hmGCB): comprising: providing a cell into which a nucleic acid sequence comprising an exogenous regulatory sequence has been introduced such that the regulatory sequence regulates the expression of an endogenous GCB coding region; contacting the cell with a substance which prevents the removal of at 1 east one mannose residue distal to the pentasaccharide core of a precursor oligosaccharide of GCB; and allowing the cell to produce hmGCB, to thereby produce an hmGCB preparation. 50. The method of claim 49, wherein removal of one or more a 1,2 mannose residue(s) distal to the pentasaccharide core is prevented. 51. The method of claim 49, wherein removal of one a 1,3 mannose residue distal to the pentasaccharide core is prevented. 52. The method of claim 49. wherein removal of one a 1,6 mannose residue distal to the pentasaccharide core is prevented. 53. The method of claim 49, wherein the substance is a class 1 processing mannosidase inhibitor. 54. The method of claim 53, wherein the class 1 processing mannosidase inhibitor is kifunensine. 55. The method of claim 54, wherein the kifunensine is present at a concentration between about 0.05 to 20-0 U-g/mh 56. The method of claim 55, wherein the kifunensine is present at a concentration between about 0.1 to 2.0 ug/ml. 57. The method of claim 54, further ecmBrisiiig contacting the cell with a class 2 processing mannosidase inhibitor. 58. The method of claim 57, wherein the class 2 processing mannosidase inhibitor is selected from the group consisting of: swamsonine. mannosratin, 6-deoxy DIM, 6-deoxy-6-fluoro-DhM and combinations thereof 59. The method of claim 57, wherein the class 2 processing mannosidase inhibitor is swainsonine. 60. The method of claim 57, wherein the class 2 processing mannosidase inhibitor is present at a concentration between 0.05 to 20.0 ug/ml. 61. The method of claim 49, wherein the cell is a knockout for at least one class 2 processing mannosidase. 62. The method of claim 49, wherein the cell comprises a class 2 processing mannosidase antisense molecule. 63. The method of claim 49, wherein the hmGCB has at least one carbohydrate chain having six mannose residues of the precursor oligosaccharide. 64. The method of claim 49, wherein the hmGCB has at least one carbohydrate chain having eight mannose residues of the precursor oligosaccharide. 65. The method of claim 49, wherein the hmGCB has at least one carbohydrate chain having nine mannose residues of the precursor oligosaccharide. 66. The method of claim 49, wherein the substance nrevsrs rsnscvsl of at least three mannose residues distal to the pentasaccharide core of the prec^irsGr oligosaccharide of GCB. 67. The method of claim 49, wherein the removal of one or more mannose residues distal to the pentasaccharide core is prevented on at least two of the carbohydrate chains of hmGCB. 68. The method of claim 49, wherein at least 60% of the hmGCB of the preparation have one or more carbohydrate chains in which the removal of three or more mannose residues distal to the pentasaccharide core has been prevented. 69. The method of claim 49, wherein at least 20% of the hmGCB of the preparation have one or more carbohydrate chains having at least eight mannose residues. 70. The method of claim 69, wherein at least 40% of the hmGCB of the preparation have one or more carbohydrate chains having at least eight mannose residues. 71. The method of claim 70, wherein at least 60% of the hmGCB of the preparation have one or more carbohydrate chains having at least eight-mannose residues. 72. The method of claim 49, wherein at least about 80% or more of the carbohydrate chains of the hmGCB preparation have six or more mannose residues. 73. The method of claim 49, wherein the cell is a primary cell. 74. . The method of claim 49, wherein the cell is a secondary cell. 75. The method of claim 49, wherein the cell is a mammalian cell 76. The method of claim 75, wherein the cell is a human cell. 77. The method of claim 76, wherein the cell is a fibroblast or a myoblast. 78. The method of claim 16, -wherein the ceU is an immortahzed ceil 79. The method of claim 78, wherein the cell is an HT-1080 cell. 80. The method of claim 54, wherein the ceil is conta.ctQd with kifunensine in culture media. 81. The method of claim 80, wherein the hmGCB is obtained from the media in which the cell is cultured. 82. A high mannose glucocerebrosidase (hmGCB) preparation, wherein each hmGCB has four carbohydrate chains and wherein at least about 10% of the carbohydrate chains in the hmGCB preparation have six or more mannose residues of a precursor oligosaccharide. 83. The hmGCB preparation of claim 82, wherein at least about 30% of the carbohydrate chains in the hmGCB preparation have six or more mannose residues of a precursor oligosaccharide. 84. The hmGCB preparation of claim 83, wherein at least about 60% of the carbohydrate chains in the hmGCB preparation have six or more maimosidase residues of a precursor oligosaccharide. 85. The hmGCB preparation of claim 82, wherein at least about 10% of The carbohydrate chains in the hmGCB preparation have eight or more mannose residues of a precursor oligosaccharide. 86. The hmGCB preparation of claim 85, wherein at least about 30% of the carbohydrate chains in the hmGCB preparation have eight or more mannose residues of a precursor oligosaccharide. 87. The hmGCB preparation of claim 86, wherein at least about 60% of the . carbohydrate chains in the hmGCB preparation have eight or more mannose residues of a precursor oligosaccharide. 88. A high mannose glucocerebrosidase (hmGCB) comprising at least one carbohydrate chain having six or more mannose residues of a precursor oligosaccharide. 89. The hmGCB of claim 88, wherein at least two carbohydrate chains having six or more mannose residues of a precursor oligosaccharide. 90. The hmGCB of claim 89, wherein the carbohydrate chain has eight or more mannose residues of a precursor ohgosaccharide. 91. A pharmaceutical composition, comprising: the hmGCB preparation of any of claims 82-90, in an amount suitable for treating Gaucher disease. 92. The composition of claim 91, further comprising a pharmaceutically acceptable carrier or diluent. 93. A method of treating a subject having Gaucher disease, comprising: administering to the subject the composition of claim 91, to thereby treat Gaucher disease.

Documents:

70-MUMNP-2009-ABSTRACT(23-8-2012).pdf

70-mumnp-2009-abstract.doc

70-mumnp-2009-abstract.pdf

70-MUMNP-2009-CLAIMS(AMENDED)-(23-8-2012).pdf

70-MUMNP-2009-CLAIMS(AMENDED)-(5-3-2013).pdf

70-MUMNP-2009-CLAIMS(MARKED COPY)-(5-3-2013).pdf

70-mumnp-2009-claims.doc

70-mumnp-2009-claims.pdf

70-MUMNP-2009-CORRESPONDENCE(14-5-2009).pdf

70-MUMNP-2009-CORRESPONDENCE(2-7-2009).pdf

70-MUMNP-2009-CORRESPONDENCE(28-1-2013).pdf

70-MUMNP-2009-CORRESPONDENCE(31-1-2013).pdf

70-MUMNP-2009-CORRESPONDENCE(4-9-2013).pdf

70-mumnp-2009-correspondence.pdf

70-mumnp-2009-description(complete).doc

70-mumnp-2009-description(complete).pdf

70-MUMNP-2009-DRAWING(23-8-2012).pdf

70-mumnp-2009-drawing.pdf

70-MUMNP-2009-FORM 1(23-8-2012).pdf

70-MUMNP-2009-FORM 1(5-3-2013).pdf

70-mumnp-2009-form 1.pdf

70-mumnp-2009-form 13(14-5-2009).pdf

70-MUMNP-2009-FORM 18(2-7-2009).pdf

70-MUMNP-2009-FORM 2(TITLE PAGE)-(23-8-2012).pdf

70-mumnp-2009-form 2(title page).pdf

70-mumnp-2009-form 2.doc

70-mumnp-2009-form 2.pdf

70-MUMNP-2009-FORM 3(23-8-2012).pdf

70-MUMNP-2009-FORM 3(4-9-2013).pdf

70-MUMNP-2009-FORM 3(5-3-2013).pdf

70-mumnp-2009-form 3.pdf

70-mumnp-2009-form 5.pdf

70-MUMNP-2009-OTHER DOCUMENT(23-8-2012).pdf

70-MUMNP-2009-OTHER DOCUMENT(5-3-2013).pdf

70-MUMNP-2009-PETITION UNDER RULE 137(23-8-2012).pdf

70-MUMNP-2009-POWER OF AUTHORITY(2-7-2009).pdf

70-MUMNP-2009-POWER OF AUTHORITY(23-8-2012).pdf

70-MUMNP-2009-POWER OF AUTHORITY(5-3-2013).pdf

70-MUMNP-2009-REPLY TO EXAMINATION REPORT(23-8-2012).pdf

70-MUMNP-2009-REPLY TO HEARING(5-3-2013).pdf

abstract1.jpg