Producing active highly phosphorylated human lysosomal sulphatase enzymes and use thereof

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Disclosed is a purified preparation of recombinant human N-acetylgalactosamine-6-sulfatase (GALNS) enzyme, where said enzyme includes an amino acid sequence which is at least 95% identical to amino acids 27-522 SEQ ID NO:4, which is suitable for treating a subject suffering from a lysosomal storage disease associated with GALNS, where: (a) said GALNS enzyme preparation has purity of at least about 95% as determined by Coomassie Blue staining when subjected to SDS-PAGE under non-reducing conditions; and (b) the cysteine residue at position 79 of at least 50% of molecules of the GALNS enzyme in said GALNS enzyme preparation is converted to Cα-formylglycine (FGly); where said GALNS enzyme is N-linked glycosylated at the asparagine residues at positions 204 and 423, wherein at least about 50% of the oligomannose chains attached to the asparagine residue at position 204 are bis-phosphorylated. Disclosed is a method of treating a subject suffering from mucopolysaccharidosis type IVa (MPS IVa), Morquio A syndrome or multiple sulfatase deficiency (MSD), which involves administering a therapeutically effective amount of said purified preparation of recombinant human GALNS to the subject.

EFFECT: invention enables to obtain a pharmaceutical preparation of recombinant highly phosphorylated human GALNS, having a high content of molecules with a cysteine residue at position 79 converted to Cα-formylglycine, owing to which it is highly absorbed through the mannose-6-phosphate receptor (MPR) and has high activity.

29 cl, 13 dwg, 15 tbl, 11 ex

 

This application claims the priority of provisional patent application U.S. No. 61/022179, filed January 18, 2008, according to provisional patent application U.S. No. 61/099373, filed September 23, 2008, and provisional patent application U.S. No. 61/110246, filed October 31, 2008, the descriptions of which are incorporated herein as references.

The SCOPE of the INVENTION

The invention relates to the technical field of cell and molecular biology and medicine, in particular to the manufacture of active vysokotsentralizovannym lysosomal enzymes sulfates person and to their use for management for lysosomal diseases accumulation associated with deficiency of lysosomal enzymes sulfates. In particular, the present invention relates to the manufacture of active vysokovostrebovannye recombinant N-atsetilgalaktozamin-6-sulfatase (GALNS) human rights and its application for control over mucopolysaccharidosis IVa (MPS Iva, or syndrome Morquio A) and other lysosomal diseases accumulation associated with deficiency of GALNS.

The prior art TO WHICH the INVENTION RELATES

Lysosomal storage disorders (LSD) are a consequence of deficiency of specific lysosomal enzymes in the cell that are necessary for the degradation of cellular waste in lysosome. The shortage of such lysosomal what's enzymes leads to accumulation in lysosome non-degraded material accumulation", which causes swelling and impaired function of the lysosomes, and, ultimately, to the damage of cells and tissues. A large number of lysosomal enzymes identified and correlated with their associated diseases. After identification of a missing enzyme treatment can be reduced to a single issue of effective delivery of the substitution of the enzyme in the affected tissue of patients.

One of the ways of treatment of lysosomal diseases, accumulation of the intravenous enzyme replacement therapy (ERT) (Kakkis, Expert Opin. Investig. Drugs 11 (5): 675-685, 2002). In ERT vessels are used for delivery of the enzyme from one area introduction in most tissues. After the enzyme is widely distributed, it must be captured by the cells. The basis for engagement in cells is a unique characteristic of lysosomal enzymes. Lysosomal enzymes constitute a separate class of glycoproteins defined by phosphate in the 6 position terminal mannose residues. Mannose-6-phosphate binds with high affinity and specificity to a receptor located on the surface of most cells (Munier-Lehmann et al., Biochem. Soc. Trans. 24(1): 133-136, 1996; Marnell et al., J. Cell. Biol. 99(6): 1907-1916, 1984). The receptor for mannose-6-phosphate (MPR), which has two binding mannose-6-phosphate site on the polypeptide chain (Tong et al., J. Biol Chem. 264:7962-7969, 1989), captures farms the NTA from the blood into the tissues, and then mediates intracellular delivery to lysosome.

Large-scale production of lysosomal enzymes involves the expression in cell lines mammals. Its aim is the predominant secretion of the recombinant enzyme in the environment for growth for subsequent collection and processing. In the ideal system for large-scale production of lysosomal enzymes, enzyme effectively fosfauriliruetsa, and then is directed mainly to the cell surface (i.e. for secretion), instead of the direction, first of all, in lysosome. As described above, this separation of phosphorylated lysosomal enzymes completely the opposite of what occurs in normal cells. Production of cell lines used for production of lysosomal enzymes, focused on maximizing the level of mannose-6-phosphate per mole of enzyme, but it is characterized by low specific productivity. Attempts productsin vitrolysosomal enzymes containing high levels of groups of mannose-6-phosphate,had variable success (Canfield et al., U.S. patent No. 6537785). Enzymein vitroit has high levels of mannose-6-phosphate, as well as high levels of unmodified terminal mannose. Competition between mannose-6-phosphate and mannose receptors for the lysosomal enzyme drive the t to the need of high doses of the enzyme for effectiveness and may lead to higher immunogenicity harmful for the subject being treated.

Sulfatase represent a unique subclass of lysosomal enzymes. Sulfatase otscheplaut sulfate esters from various substrates, including, for example, steroids, carbohydrates, proteoglycans and glycolipids. All known eukaryotic sulfatase contain a cysteine residue in their catalytic center. Activity sulfatase requires posttranslational modification of the cysteine residue in the Cα-formylglycine (FGly). Post-translational activation of the enzyme by modification of cysteine to FGly occurs in the endoplasmic reticulum on nevernude sulfatase immediately after the broadcast, before sending sulfates in lysosome (Dierks et al., Proc. Natl. Acad. Sci. USA 94:11963-11968, 1997). Forming formylglycine the enzyme that catalyzes this reaction is modifying sulfatase factor 1 (SUMF1). The importance of this unique posttranslational modification indicates that SUMF1 mutations in which lead to the violation of FGly formation in lysosomal enzymes sulfatase lead to multiple sulfatase deficiency (MSD) in humans (Diez-Ruiz et al., Annu. Rev. Genomics Hum. Genet. 6:355-379, 2005).

Thus, therapeutic effectiveness of lysosomal enzyme sulfatase depends on the level of mannose-6-phosphate and from the presence of the active enzyme in this drug.

Thus, in this area there is a need for an efficient and productive system for large-scale production of therapeutically effective active vysokotsentralizovannym lysosomal enzymes sulfates to control for lysosomal diseases accumulation caused by deficiency of such lysosomal enzymes sulfates or associated.

The INVENTION

The present invention relates to the discovery that when a derived cell line CHO-K1 (denoted G71), which is defective in endosomal acidification, design so that it is expressed recombinant modifying sulfatase factor 1 (SUMF1) man modified G71 cells produce high output active vysokovostrebovannye recombinant lysosomal enzymes sulfatase partly by preventing the loss of material in lysosomal Department producing cell lines. In one embodiment, the invention relates to a cell line group complementaly END3, which coexpressed recombinant human SUMF1 and recombinant N-atsetilgalaktozamin-6-sulfatase (GALNS) person providing high outputs are active vysokopostavlennogo enzyme. Illustrative cell lines are G71, G71S and their derivatives, is the quiet retain the desired property G71, i.e. the ability to produce high yield of active vysokovostrebovannye recombinant lysosomal enzymes sulfatase. This application of the modified cell line CHO-K1 group complementaly END3, coexpression recombinant human SUMF1 and recombinant lysosomal enzyme sulfatase, may be particularly suitable for the manufacture of active vysokotsentralizovannym lysosomal enzymes sulfates to use in order to control for lysosomal diseases accumulation by enzyme-replacement therapy (ERT).

In the first aspect, the present invention relates to a new method of production of active vysokotsentralizovannym recombinant lysosomal enzymes sulfates person or their biologically active fragments, mutants, variants or derivatives in the cell CHO group complementaly END3 or its derivative in amounts that provide their therapeutic application. In a broad embodiment, the method comprises the stage of: (a) culturing originating from CHO cells group complementaly END3 or its derivative; (b) receiving the first expressing vector mammals, able to Express the active vysokovostrebovannye recombinant lysosomal enzyme sulfatase person or its biologically active fragments which, mutant, variant or derivative in originating from CHO cell group complementaly END3 or its derivative; (c) receiving the second expressing vector mammals, able to Express the recombinant modifying sulfatase factor 1 (SUMF1) person or its biologically active fragment, mutant, variant or derivative in originating from CHO cell group complementaly END3 or its derivative; (d) transfection originating from CHO cells group complementaly END3 or its first derivative and the second expressing vectors; (e) selection and cloning of transfectant originating from CHO cells group complementaly END3 or its derivative, which expresses active vysokovostrebovannye recombinant lysosomal enzyme sulfatase person or its biologically active fragment, mutant, variant or derivative; and (f) optimization method of culturing cells for the manufacture of vysokopostavlennogo recombinant lysosomal enzyme sulfatase person or its biologically active fragment, mutant, variant or derivative. Recombinant lysosomal enzyme sulfatase person selected from the group consisting of Ukrainian A (ARSA), Ukrainian B (ARSB), iduronate-2-sulfatase (IDS), sulfamidate/heparin-N-sulfatase (SGSH), N-acetylglucosamine-sulfatase (G6S) and N-and acylgalactosamine-6-sulfatase (GALNS).

The method involves phase transfection with cDNA encoding the entire lysosomal enzyme sulfatase or part thereof, and cDNA encoding the entire human SUMF1 or part thereof, originating from CHO cell group complementaly END3 or its derivative. In some embodiments, the implementation, the first and the second expressing vectors that can Express the cDNA encoding the active vysokovostrebovannye recombinant lysosomal enzyme sulfatase human and human SUMF1, respectively, transferout simultaneously originating from CHO cell group complementaly END3 or its derivative. In some embodiments, the implementation, the first and the second expressing vectors transferout in originating from CHO cell group complementaly END3 or its derived series. In some embodiments, implementation, use of cDNA encoding full-lysosomal enzyme sulfatase person, while in other embodiments, implementation of the use of cDNA encoding its biologically active fragment, mutant, variant or derivative. In some embodiments, implementation, use of cDNA encoding a full-sized human SUMF1, while in other embodiments, implementation of the use of cDNA encoding its biologically active fragment, mutant, variant or derivative. In some embodiments, the OS is enforced, for simultaneous transfer of cDNA lysosomal enzyme sulfatase person and SUMF1 or consistently originating from CHO cell group complementaly END3 or its derivative use several expressing vectors. In some embodiments, implement, for simultaneous transfer of cDNA lysosomal enzyme sulfatase and SUMF1 person originating from CHO cell group complementaly END3 or its derivative use one expressing vector. In a preferred embodiment, derived from CHO cell group complementaly END3 or its derivative is a cell line G71, cell line G71S or derived G71 or G71S.

In a preferred embodiment, the method includes the production of active vysokopostavlennogo recombinant lysosomal enzyme sulfatase person, for example Ukrainian A (ARSA), Ukrainian B (ARSB), iduronate-2-sulfatase (IDS), sulfamidate/heparin-N-sulfatase (SGSH), N-acetylglucosamine-sulfatase (G6S) or N-atsetilgalaktozamin-6-sulfatase (GALNS), the cell line CHO group complementaly END3 or its derivative. In a particularly preferred embodiment, the method includes the production of active vysokovostrebovannye recombinant N-atsetilgalaktozamin-6-sulfatase person (GALNS) cell line CHO group of the complement is AI END3 or its derivative. Cell line group complementaly END3 is any modified cell line CHO, which retains the properties of the cell line groups complementaly END3, such as defective endosomal acidification. In a preferred embodiment, derived from CHO cell group complementaly END3 or its derivative is a cell line G71, cell line G71S or derived G71 or G71S.

In the second aspect, the present invention relates to deficient endosomal acidification cell line, characterized by its ability to produce active vysokovostrebovannye recombinant lysosomal enzymes sulfatase man in quantities which give the possibility of therapeutic application of lysosomal enzyme sulfatase. In preferred embodiments, the implementation, the invention relates to the events of the CHO-K1 cell lines group complementaly END3, denoted by G71, G71S, or their derivatives, which are capable of producing high output active vysokovostrebovannye recombinant lysosomal enzymes sulfatase person, thereby allowing large-scale production of such therapeutic lysosomal enzymes sulfates. In more preferred versions of the implementation, the cell line expresses and secreter the em recombinant lysosomal enzyme sulfatase person in the amount of at least about 0.5, preferably at least about 0.75, and more preferably at least about 1.0, and even more preferably at least about 1.25 PG/cell/day.

Cell line group complementaly END3 is any modified cell line CHO, which retains the properties of the cell line groups complementaly END3, such as defective endosomal acidification. In one embodiment, the cell line CHO group complementaly END3 formed from G71 or its derivative and contains (a) expressing a vector for recombinant modifying sulfatase factor 1 (SUMF1) human and (b) expressing a vector for recombinant lysosomal enzyme sulfatase human recombinant lysosomal enzyme sulfatase person selected from the group consisting of Ukrainian A (ARSA), Ukrainian B (ARSB), iduronate-2-sulfatase (IDS), sulfamidate/heparin-N-sulfatase (SGSH), N-acetylglucosamine-sulfatase (G6S) and N-atsetilgalaktozamin-6-sulfatase (GALNS). In a preferred embodiment, the cell line CHO group complementaly END3 contains expressing vector for recombinant N-atsetilgalaktozamin-6-sulfatase (GALNS) person. In a more preferred embodiment, the cell line CHO group complementaly END3 expressio the t and secretes a recombinant human GALNS. In another preferred embodiment, the cell line CHO group complementaly END3 selected from the group consisting of clone 4, clone 5, clone C6, clone C2, clone C5, clone C7 clone C10, clone C11 and clone C30. In a more preferred embodiment, the cell line CHO group complementaly END3 is a clone of C2. In another preferred embodiment, the cell line CHO group complementaly END3 adapted for growth in suspension.

In the third aspect, the invention relates to recombinant lysosomal enzymes to sulfatases man, produced by methods of the present invention and, thus, present in quantities that make it possible therapeutic application of lysosomal enzymes sulfates. Lysosomal enzymes sulfatase can be a full-sized proteins or their fragments, mutants, variants or derivatives. In some embodiments, implementation, lysosomal enzyme sulfatase or a fragment, mutant, variant or derivative according to this invention can, if desired, be modified to enhance their stability or pharmacokinetic properties (e.g., tahilramani, mutagenesis, fusion, conjugation). In preferred embodiments, the implementation, the enzyme is a lysosomal enzyme sulfatase person, ragment lysosomal enzyme sulfatase person, having the biological activity of the native enzyme sulfatase, or polypeptide, which has significant amino acid sequence homology with lysosomal enzyme by sulfatases person. In some embodiments, implementation, lysosomal enzyme sulfatase is a protein source or origin of the sequence which is the human or mammal. In other embodiments, implementation of lysosomal enzyme sulfatase is such that its deficiency leads to the disease in humans, such as metachroma leukodystrophy, or MLD (i.e. arylsulfatase A (ARSA)), the syndrome Maroto-Lamy, or MPS VI (i.e. arylsulfatase B (ARSB)), hunter's syndrome, or MPS II (i.e. iduronate-2-sulfatase (IDS)), syndrome Sanfilippo's title A, or MPS IIIa (i.e. sulfamidate/heparin-N-sulfatase (SGSH)), syndrome Sanfilippo's title D, or MPS IIId (i.e. N-acetylglucosamine-sulfatase (G6S)), and the syndrome Morquio A, or MPS IVa (i.e. N-atsetilgalaktozamin-6-sulfatase (GALNS)). In a particularly preferred embodiment, the lysosomal enzyme sulfatase is such that its deficiency causes the syndrome Morquio A or MPS IVa (i.e. N-atsetilgalaktozamin-6-sulfatase (GALNS)). In another particularly preferred embodiment, the lysosomal enzyme sulfatase is such that its deficiency is associated with human disease, such as Dagestana Solfatara failure or MSD (i.e. N-atsetilgalaktozamin-6-sulfatase (GALNS)).

The source or origin of a sequence of lysosomal enzyme sulfatase can also be a person or a mammal. In other embodiments of the invention, in each of its aspects, the lysosomal enzyme sulfatase is identical in amino acid sequence corresponding to part of the amino acid sequence of lysosomal enzyme sulfatase human or mammal. In other embodiments, implementation, polypeptide portion is a native lysosomal enzyme sulfatase from human or mammal. In other embodiments, implementation, polypeptide lysosomal enzyme sulfatase is essentially homologous (i.e. at least approximately 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical in amino acid sequence) for at least about 25, 50, 100, 150 or 200 amino acids or the full length polypeptide amino acid sequence of native lysosomal enzyme sulfatase human or mammal. In other embodiments, implementation of the subject, which enter the lysosomal enzyme sulfatase, is the man.

In preferred embodiments, implementation, lysosomal enzyme sulfatase is vysokovostrebovannye R is combinatii lysosomal enzyme sulfatase person, produced deficient endosomal acidification cell line, for example originating from a CHO cell line group complementaly END3. Cell line group complementaly END3 is any modified cell line CHO, which retains the properties of the cell line groups complementaly END3, such as defective endosomal acidification. In a preferred embodiment, derived from CHO cell group complementaly END3 or are derived cell line G71, cell line G71S or derived G71 or G71S.

In more preferred embodiments, implementation, recombinant lysosomal enzyme sulfatase person has a high level of phosphorylated oligosaccharides (i.e. more than approximately 0.25, preferably greater than 0.5 and more preferably more than approximately 0,75 bis-phosphorylated oligomannose chains on the protein chain). In more preferred versions of the implementation, the enzyme is vysokovostrebovannye recombinant N-atsetilgalaktozamin-6-sulfatase (GALNS) person.

In more preferred embodiments, implementation, recombinant lysosomal enzyme sulfatase person has a high percentage (i.e. at least about 50%, preferably at least approx is Ino 70%, more preferably at least about 90%, more preferably at least about 95%) of the cysteine residue of the active site, converted to Cα-formylglycine (FGly). In even more preferred embodiments, the implementation, the enzyme is an active recombinant N-atsetilgalaktozamin-6-sulfatase (GALNS) person.

In more preferred embodiments, implementation, recombinant lysosomal enzyme sulfatase person has a high level of phosphorylated oligosaccharides (i.e. more than approximately 0.25, preferably greater than 0.5 and more preferably more than approximately 0,75 bis-phosphorylated oligomannose chains on the protein chain) and a high percentage (i.e. at least about 50%, preferably at least about 70%, more preferably at least about 90%, more preferably at least about 95%) of the cysteine residue of the active site, converted to Cα-formylglycine (FGly). In the most preferred options for implementation, the enzyme is an active vysokovostrebovannye recombinant N-atsetilgalaktozamin-6-sulfatase person (GALNS).

In the fourth aspect, the invention relates to a method of purification of recombinant lysosomal enzymes sulfates people the century, produced by methods of the present invention. In a preferred embodiment, the lysosomal enzymes sulfatase cleaned using the method of two-column (chromatography dye-ligand, such as Blue-Sepharose, anion-exchange chromatography, for example SE Hi-Cap), including at least five stages purification: (1) filtering the collected material, i.e. culture medium from cell lines CHO group complementaly END3 or its derivative, which expresses modifying sulfatase factor 1 (SUMF1) and recombinant human lysosomal enzyme sulfatase person; (2) bringing the pH of the filtered collected material to a pH of 4.5 (for induction deposition of pollutants proteins); (3) applying the filtered collected material with increased pH on the column dye-ligand, for example a column of Blue-Sepharose, washing the column and elution of lysosomal enzyme sulfatase from the column; (4) applying the eluate from the column dye-ligand in aminobenzo column, for example column SE Hi-Cap, washing the column and elution of lysosomal enzyme sulfatase from the column; (5) ultrafiltration and diafiltration of the eluate from the anion exchange column. Optionally, the filtered collected material stage (1) concentrated 10-20 fold by ultrafiltration before bringing the pH. Optional, ultrafi trevanny and definitavely lysosomal enzyme sulfatase stage (5) prepare buffer for manufacturing. In a particularly preferred embodiment, the lysosomal enzyme is a recombinant N-atsetilgalaktozamin-6-sulfatase (GALNS) person.

In another preferred embodiment, the lysosomal enzymes sulfatase cleaned using the method with three columns (chromatography with carbon capture, such as anion-exchange SE Hi-Cap; interim chromatography, for example, dye-ligand Capto BlueZinc, Chelating Sepharose FF or Capto Adhere; and chromatography purification, such as ToyoPearl Butyl 650M, Phenyl Sepharose Hi-Sub or Phenyl Sepharose Low-Sub), including at least five stages purification: (1) ultrafiltration of the collected material, i.e. culture medium of the cell line CHO group complementaly END3 or its derivative, which Express a modifying factor sulfatase 1 (SUMF1) and recombinant human lysosomal enzyme sulfatase person, for example, using Sartoon Cassettes (10 kDa, Hydrosart); (2) bringing the pH of the filtered collected material to a pH of 4.5 (to induce precipitation of contaminating proteins); (3) applying the filtered collected material with increased pH in a capture column, such as anion-exchange column of Fractogel EMD SE Hi-CAP (M), washing the column and elution of lysosomal enzyme sulfatase from the column; (4) applying the eluate from the capture column at an intermediate column, for example column is recital-ligand Capto BlueZinc, Chelating Sepharose FF or Capto Adhere, washing the column and elution of lysosomal enzyme sulfatase from the column; (5) applying the eluate to column purification, for example, ToyoPearl Butyl 650M, Phenyl Sepharose Hi-Sub or Phenyl Sepharose Low-Sub, washing the column and elution of lysosomal enzyme from the column. Suirvey lysosomal enzyme stage (5) prepare buffer for manufacturing. Optional, suirvey lysosomal enzyme sulfatase stage (5) is subjected to ultrafiltration, and then prepared in the buffer for manufacturing. Optional, lysosomal enzyme sulfatase column of stage (4) is exposed to pH 3.5 for inactivation of viruses low pH before applying to column purification stage (5). In a particularly preferred embodiment, the lysosomal enzyme is a recombinant N-atsetilgalaktozamin-6-sulfatase (GALNS) person.

In the fifth aspect, the invention relates to purified active vysokovostrebovannye recombinant N-atsetilgalaktozamin-6-sulfatase (GALNS) of a person or a biologically active mutant, variant or derivative, suitable for treatment of a subject suffering from lysosomal disease accumulation, which is caused (e.g., mucopolysaccharidosis type IVa (MPS IVa), or the syndrome Morquio A) or associated with (e.g., multiple sulfatase deficiency (MSD) deficiency GALNS enzyme. In a preferred embodiment, the purified active vysokovostrebovannye recombinant human GALNS: (a) has a purity of at least about 90% when determining the staining of Kumasi blue when SDS-PAGE in non conditions; (b) has at least about 90% of the cysteine residue in position 53, converted into aα-formylglycine (FGly); and (c) is glycosylated N-linked glycosylation at asparagine residues at positions 178 and 397, where at least about 50% oligomannose circuits associated with the asparagine residue at position 178, are bis-phosphorylated. Purified active vysokovostrebovannye recombinant human GALNS has a major band at approximately 55-60 kDa (i.e. predecessor GALNS person, of at least about 75%, preferably at least about 85%, more preferably at least about 90% and more preferably at least approximately 95% of the visible proteins) and a minor band at ~39 kDa and ~19 kDa (i.e. Mature or protestirovanny GALNS man, which constitutes less than about 25%, preferably less than about 15%, more preferably less than about 10% and even more preferably less than about 5% visible the proteins in SDS-PAGE in reducing conditions. In a particularly preferred embodiment, the purified active vysokovostrebovannye recombinant human GALNS has essentially a single band at approximately 55-60 kDa (i.e. the predecessor of human GALNS) by SDS-PAGE in reducing conditions. In one embodiment, the purified active vysokovostrebovannye recombinant human GALNS suitable for the treatment of MPS IVa, or syndrome Morquio A. In one embodiment, the purified active vysokovostrebovannye recombinant human GALNS suitable for the treatment of MSD.

In the sixth aspect, the invention relates to a method of treating diseases caused wholly or partly by a deficiency of lysosomal enzyme sulfatase or associated with its deficiency. The method includes the introduction of therapeutic recombinant lysosomal enzyme sulfatase man, produced by methods of the present invention, where lysosomal enzyme sulfatase binds to the receptor MPR and transported through the cell membrane, enters the cell and delivered to the lysosomes in the cell.

In one embodiment, the method includes treating a subject suffering from a deficiency of the lysosomal enzyme sulfatase, including introduction to the subject in need, a therapeutically effective amount specified Liz is Smolnogo the enzyme sulfatase, where specified lysosomal enzyme sulfatase is a recombinant lysosomal enzyme sulfatase person or its biologically active fragment, mutant, variant or derivative produced originating from CHO cell group complementaly END3 or its derivative. In some embodiments, the implementation, the method includes the introduction of therapeutic recombinant lysosomal enzyme sulfatase person or its biologically active fragment, mutant, variant or derivative alone or in combination with a pharmaceutically acceptable carrier, diluent or excipient. Preferred embodiments of include optimizing the dosage in accordance with the needs of the subjects being treated, preferably mammals and most preferably humans, for the most effective mitigation deficiency of lysosomal enzyme sulfatase.

Such therapeutic lysosomal enzymes sulfatase particularly suitable, for example, for the treatment of patients suffering from lysosomal diseases accumulation caused by deficiency of lysosomal enzyme sulfatase, such as patients suffering from the metachromatic leukodystrophy, or MLD, mucopolysaccharidosis type VI (MPS VI), or syndrome Maroto-Lamy, mucopolysaccharidosis type II (MPS II), or syndrome Hante is a, the mucopolysaccharidosis type IIIa (MPS IIIa), or syndrome, Sanfilippo's title a, mucopolysaccharidosis type IIId (MPS IIId), or syndrome, Sanfilippo's title D, and mucopolysaccharidosis type IVa (MPS IVa), or syndrome Morquio A. In a particularly preferred embodiment, the lysosomal disease accumulation represents MPS Iva, or syndrome Morquio A, and lysosomal enzyme sulfatase is a recombinant N-atsetilgalaktozamin-6-sulfatase (GALNS) person. In other embodiments, the implementation, the invention also relates to pharmaceutical compositions containing the defective lysosomal enzyme sulfatase causing lysosomal disease accumulation, and a pharmaceutically acceptable carrier, diluent or excipient.

In another embodiment, the method includes treating a subject suffering from lysosomal disease accumulation, which is associated with a deficiency of one or more lysosomal enzymes sulfates, including introduction to the subject in need, a therapeutically effective amount of lysosomal enzyme sulfatase where specified lysosomal enzyme sulfatase is a recombinant N-atsetilgalaktozamin-6-sulfatase (GALNS) person or its biologically active fragment, mutant, variant or derivative produced originating from CHO cell group complem is ncacii END3 or its derivative. In some embodiments, the implementation, the method includes the introduction of therapeutic recombinant GALNS enzyme of the person or its biologically active fragment, mutant, variant or derivative alone or in combination with a pharmaceutically acceptable carrier, diluent or excipient. In a particularly preferred embodiment, the lysosomal disease accumulation is a multiple sulfatase deficiency (MSD).

In particularly preferred embodiments, implementation, originating from CHO cell group complementaly END3 or its derivative is a cell line G71, cell line G71S or derived G71 or G71S.

In another embodiment, the present invention relates to a method enzyme-replacement therapy by introducing a therapeutically effective amount of lysosomal enzyme sulfatase to a subject in need of enzyme-replacement therapy, where cells of the patient have a complementary mechanism, which contain an insufficient number of lysosomal enzyme sulfatase to prevent or reduce damage to the cells, where complementary mechanism penetrate a sufficient number of lysosomal enzyme sulfatase to prevent or reduce damage to the cells. Cells may reside in the Central nervous system or outside of it or not, capture the shape of the blood through the capillary walls, endothelial cells are tightly closed to diffusion of the active substance through the close contacts.

In a specific embodiment, the invention relates to compositions and pharmaceutical compositions containing the active recombinant lysosomal enzyme sulfatase person that has a biological activity, which is reduced, defective, or not in lysosome target, and which is administered to the subject. Preferred active lysosomal enzymes sulfatase person include, but are not limited to, arylsulfatase arylsulfatase B, iduronate-2-sulfatase, sulfamidate/heparan-N-sulfatase, N-acetylglucosamine-6-sulfatase and N-atsetilgalaktozamin-6-sulfatase. In a preferred embodiment, N-atsetilgalaktozamin-6-sulfatase is an active recombinant lysosomal enzyme sulfatase person.

In a preferred embodiment, the invention relates to a method of treatment of a subject suffering from MPS Iva, or syndrome Morquio A, or MSD, by introducing to the subject a therapeutically effective amount of recombinant N-atsetilgalaktozamin-6-sulfatase (GALNS) human recombinant GALNS person has a high level of conversion of the cysteine residue in the active center in the Cα-formylglycine (FGly) (i.e. at least approx the positive 50%, preferably at least about 70%, more preferably at least about 90%, more preferably at least about 95% conversion) and high levels of phosphorylation (i.e. more than approximately 0.25, preferably more than about 0.5 and more preferably more than approximately 0,75 bis-phosphorylated oligomannose chains on the protein chain).

In a more preferred embodiment, the invention relates to a method of treatment of a subject suffering from MPS Iva, or syndrome Morquio A, or MSD, by introducing to the subject a therapeutically effective amount of recombinant N-atsetilgalaktozamin-6-sulfatase (GALNS) man, produced by cells of the group of complementaly END3, where recombinant human GALNS has a high level of conversion of cysteine residues in the active site in the Cα-formylglycine (FGly) (i.e. at least about 50%, preferably at least about 70%, more preferably at least about 90%, more preferably at least about 95% conversion) and high levels of phosphorylation (i.e. more than approximately 0.25, preferably greater than 0.5 and more preferably more than approximately 0,75 bis-phosphorylated oligomannose chains on the protein price is b).

In a particularly preferred embodiment, the invention relates to a method of treatment of a subject suffering from MPS Iva, or syndrome Morquio A, or MSD, by introducing to the subject a therapeutically effective amount of purified active vysokovostrebovannye recombinant human GALNS, which: (a) has a purity of at least about 90% when determining the staining of Kumasi blue when SDS-PAGE in non conditions; (b) has at least approximately 90% of the cysteine residues at position 53, converted into aα-formylglycine (FGly); and (c) is glycosylated N-linked glycosylation at asparagine residues at positions 178 and 397, where at least about 50% oligomannose circuits associated with the asparagine residue at position 178, are bis-phosphorylated. Purified active vysokovostrebovannye recombinant human GALNS has a major band at approximately 55-60 kDa (i.e. predecessor GALNS person, of at least about 75%, preferably at least about 85%, more preferably at least about 90% and more preferably at least approximately 95% of the visible proteins) and a minor band at ~39 kDa and ~19 kDa (i.e. Mature or protestirovanny GALNS human component IU is it than approximately 25%, preferably less than about 15%, more preferably less than about 10% and even more preferably less than about 5% of the visible proteins in SDS-PAGE in reducing conditions. In a particularly preferred embodiment, the purified active vysokovostrebovannye recombinant human GALNS has essentially a single band at approximately 55-60 kDa (i.e. the predecessor of human GALNS) by SDS-PAGE in reducing conditions.

In some embodiments, the implementation, the subject suffers from MPS Iva, or syndrome Morquio A. In some embodiments, the implementation, the subject suffers from MSD.

Also provides for the appropriate use of active vysokotsentralizovannym lysosomal enzymes sulfates according to the invention are preferably produced by the methods according to the invention, for manufacturing a medicinal product for the treatment of lysosomal diseases of accumulation described above.

In the seventh aspect, the present invention relates to pharmaceutical compositions containing the active vysokovostrebovannye recombinant lysosomal enzyme sulfatase person, as described herein above, which is suitable for treatment of diseases caused wholly or partly by the lack of lysosomal enzyme sulfatase or associated the data with him, and one or more pharmaceutically acceptable carriers, diluents or excipients. In a preferred embodiment, the pharmaceutical composition contains an active vysokovostrebovannye recombinant N-atsetilgalaktozamin-6-sulfatase (GALNS) person or its biologically active fragment, mutant, variant or derivative produced by methods according to the invention, and one or more pharmaceutically acceptable carriers, diluents or excipients. Such pharmaceutical compositions may be suitable for introduction in various ways, such as intrathecal, parenteral, local, intranasal, inhalation or oral administration. In a preferred embodiment, pharmaceutical compositions suitable for parenteral administration. In the scope of this aspect are variants of implementation related to the sequences of nucleic acids encoding a full-sized lysosomal enzymes sulfatase or their fragments, mutants, variants or derivatives, you can enterin vivoin cells affected by deficiency of lysosomal enzymes.

In another aspect, the invention relates to a method of detecting activity of the lysosomal enzyme sulfatase, comprising (a) culturing cells of chondrocytes from a patient suffering from a deficit Liz is Smolnogo the enzyme sulfatase, for example a patient suffering from a syndrome Morquio, in conditions which ensure the maintenance of differentiation of chondrocytes; (b) contacting chondrocytes with the lysosomal enzyme sulfatase that destroys keratinolytic; and (c) detection levels createsurface in cells, where a reduced level of keratomalacia in cells that were subjected to contacting with the lysosomal enzyme sulfatase, compared with cells that were not subjected to contact with the lysosomal enzyme sulfatase indicates the activity of the lysosomal enzyme sulfatase. In some embodiments, implementation, lysosomal enzyme sulfatase is an N-atsetilgalaktozamin-6-sulfatase (GALNS). In some embodiments, execution, cultivation is carried out in a medium containing the growth factor insulin 1 (IGF1), transforming growth factor-beta (TGF-β), transferrin, insulin and ascorbic acid. In some embodiments, implementation, detection of karacasogut performed using confocal microscopy or by binding with the antibody against createsurface. The method can be performed with any lysosomal enzyme by sulfatases, including naturally occurring or recombinant human enzyme, or its fragments or variants, including variants containing amino acid sequence is, at least 80%, 85%, 90%, 95% or 100% identical to the precursor enzyme of man, without the signal sequence, or its Mature form.

In another aspect, the invention relates to cell analysis for measuring the activity of recombinant lysosomal enzyme in the degradation of natural substrates. The method comprises (a) culturing the selected cells of a person with deficiency of lysosomal enzyme under conditions in which are accumulated the natural substrates of lysosomal enzyme; (b) contacting the cells with lysosomal enzyme; (c) lysis of the cells; (d) adding to the cell lysate enzyme, which (i) is specific to natural substrates, (ii) it is a small oligosaccharides from natural substrates; (e) labelling of small oligosaccharides amenable to detection by the group; (f) optional separation of labeled small oligosaccharides; (g) detection of labeled small oligosaccharides; (h) determining the activity lysosomal enzyme in the degradation of natural substrates by comparing (i) the number of labeled small oligosaccharide from cells that had been subjected to contact with lysosomal enzyme, and (ii) the number of labeled small oligosaccharides from cells that were not subjected to contacting lysosomal enzyme, where the decrease (h)(i) compared to(h)(ii) indicates the activity of the lysosomal enzyme in the degradation of natural substrates. In one embodiment, a small oligosaccharide is a mono-, di - or trisaccharide. In related embodiments, implementation, small oligosaccharide is a disaccharide. In some embodiments, the implementation, the lysosomal enzyme is selected from the group consisting of Ukrainian B (ARSB), iduronate-2-sulfatase (IDS), sulfamidate/heparin-N-sulfatase (SGSH), N-acetylglucosamine-sulfatase (G6S) and N-atsetilgalaktozamin-6-sulfatase (GALNS). In some embodiments, implementation, lysosomal enzyme is an α-L-iduronidase (IDU). In some embodiments, implementation, lysosomal enzyme is an acid α-glucosidase (GAA). In some embodiments, the implementation, the lysosomal enzyme is a β-glucuronidase (GUSB). In some embodiments, the implementation, the lysosomal enzyme is a β-galactosidase (GLB1).

Suitable human cells that can be used in cellular analysis, include any cell that is deficient in the lysosomal enzyme to be checked, so that it could accumulate natural substrates for lysosomal enzyme. For example, you can use cells naturally expressing full (100%) or partial deficiency of activity, for example 30%, 50%, 70%, 80%, 90%, 95% the reduction of activity or more. Can the use of cells, expressing mutant enzyme with reduced activity, or cells from patients suffering from lysosomal disease accumulation, such as mucopolysaccharidosis. You can use cells, recombinante modified for knockout or reduce the activity of the lysosomal enzyme, for example, by introducing mutations in the encoding gene, or the promoter or other regulatory region. You can use cells, treated to reduce the activity of the lysosomal enzyme, such as processed antisense RNA or RNAi for the induction of expression of the enzyme.

Specialists in this field can choose suitable enzymes that otscheplaut (hydrolyzing) small oligosaccharides from carbohydrates and which are "specific" (i.e., predominantly hydrolyzing) natural substrates lysosomal enzyme. For example, for detecting activity GALNS or GLB1 (enzymes that destroy keratinolytic) enzyme stage (d) can represent keratinase II or any enzyme that mainly affects keratinolytic. As another example, for detection IDU, ARSB, IDS or GUSB (enzymes that destroy dermatologit) enzyme stage (d) can be chondroitinase ABC or any enzyme that acts predominantly on dermatologit. As another example, for detection IDU,IDS, SGHS, G6S or GUSB (enzymes that destroy heparansulfate) enzyme stage (d) can represent heparinase I, or heparinase II, or both of them. As another example, for the detection of GAA (the enzyme that breaks down glycogen) enzyme stage (d) may consist of α-amylase or any enzyme that mainly affects glycogen.

This cellular method is capable of high sensitivity to determine the activity of the lysosomal enzyme. In some embodiments, implementation, activity of lysosomal enzyme is determined when the concentration of the lysosomal enzyme is only about 10 nm, or about 5 nm or about 1 nm, or approximately 0,75 nm, or about 0.5 nm, or approximately 0.25 nm, or approximately 0.1 nm, or about 0.05 nm, or about 0.01 nm, or approximately 0,005 nm, or about 1 PM, or about 0.5 PM.

Other characteristics and advantages of the invention will be evident from the below detailed description. However, it should be understood that the detailed description and specific examples, though, and indicate the preferred embodiments of the invention, given for illustration only, since various changes and modifications relating to the nature and scope of the invention will be apparent specifications the leaves in this area from this detailed description.

BRIEF DESCRIPTION of DRAWINGS

The figure 1 presents the nucleotide sequence of modifying sulfatase factor 1 (SUMF1) human (SEQ ID NO:1).

The figure 2 presents the amino acid sequence of modifying sulfatase factor 1 (SUMF1) human (SEQ ID NO:2).

The figure 3 presents the nucleotide sequence of N-atsetilgalaktozamin-6-sulfatase (GALNS) human (SEQ ID NO:3).

The figure 4 presents the amino acid sequence of the N-atsetilgalaktozamin-6-sulfatase (GALNS) human (SEQ ID NO:4). In protestirovanny GALNS lacks a signal peptide of 26 amino acids at N-end.

The figure 5 presents the structure and characteristics protestirovanny N-atsetilgalaktozamin-6-sulfatase (GALNS) human (SEQ ID NO: 5).

The figure 6 presents the expression of N-atsetilgalaktozamin-6-sulfatase (GALNS) human cells G71S, cotransfection expressing vectors for modifying sulfatase factor 1 (SUMF1) human rights and human GALNS. (A) Screening of clone G71S against active GALNS in 96-well plates. (B) the Productivity of clone G71S in respect of GALNS in pilgrammage per cell per day.

The figure 7 presents a diagram of the bioreactor controller WAVE used for large-scale production of cells G71S expressing N-atsetilgalaktozamin-6-sulfatase (GALNS) of man and its options.

On Phi is ur 8 presents the stability of the purified enzyme N-atsetilgalaktozamin-6-sulfatase (GALNS) person when stored at 4°C (diamonds) or at -70°C (triangles).

The figure 9 presents the purification of N-atsetilgalaktozamin-6-sulfatase (GALNS) with (A) chromatography with a Blue Sepharose 6 Fast Flow followed (B) chromatography with Fractogel SE Hi-CAP. Purity is determined by staining Kumasi blue in SDS-PAGE (left) and by Western blotting using antibodies against GALNS (IVA) (right).

The figure 10 presents the purification of N-atsetilgalaktozamin-6-sulfatase (GALNS) person ultrafiltration/diafiltration (UF/DF), chromatography with Fractogel SE Hi-Cap, chromatography with Zn-chelating Sepharose and chromatography ToyoPearl Butyl 650M. Purity is determined by staining Kumasi blue in SDS-PAGE (top left) and by Western blotting using antibodies against GALNS (top right), antibodies against cathepsin L (bottom left) and antibodies against CHOP (cell proteins of Chinese hamster ovary) (bottom right).

The figure 11 shows that in the treated IDU cells GM01391 observed a dose-dependent decrease in the number of substrate dermatosurgery.

The figure 12 shows that in the treated ARSB cells GM00519 observed a dose-dependent decrease in the number of substrate dermatosurgery.

In the figure 13 presents the capture of N-atsetilgalaktozamin-6-sulfatase (GALNS) person or unlabeled (circles), or conjugated with A488 (squares) or A555 (triangles), cultured synoviocytes.

A DETAILED DESCRIPTION of IZABERETE THE OIA

The present invention relates to the discovery of the method that satisfies the need for large-scale production of recombinant lysosomal enzymes sulfates requiring product in the form of active vysokopostavlennogo lysosomal enzyme sulfatase, which is effective for targeting complementary mechanism and, thus, is therapeutically effective.

therapeutic effectiveness of lysosomal enzyme depends on the level of mannose-6-phosphate in the product. Phosphate is added to the glycoprotein target by posttranslational modification in the endoplasmic reticulum and early Golgi apparatus. Rolled lysosomal enzymes have a unique tertiary structure determinants, which is recognized by the enzyme modification of the oligosaccharide. Determinant consists of a series in a certain way lysine residues located, and it is found on most lysosomal enzymes despite the lack of primary sequence homology. The modifying enzyme, UDP-GlcNAc of phosphotransferase, binds to a protein determinant and adds GlcNAc-1-phosphate to 6-position terminal mannose residues on the oligosaccharide near the binding site; then a second enzyme, phosphodiester-α-GlcNAcase, cleaves GlcNAc-phosphate communication with the formation of mannose-6-fosfat the th end of the oligosaccharide (Canfield et al., U.S. patent No. 6537785). The purpose of modification of mannose-6-phosphate is the deviation of lysosomal enzymes from the secretory cascade lysosomal cascade in the cell. The enzyme carrier mannose-6-phosphate, MPR associates in the TRANS-Golgi apparatus and is directed in lysosome instead of the cell surface.

In addition to the presence of mannose-6-phosphate marker on the oligosaccharides of lysosomal enzymes, enzymes in complementary mechanism depends on acidification transportiruyushchie endosomes, leaving the end of the pile TRANS-Golgi apparatus. Chemical quenching acidic environment in these endosomes main diffusing molecules leads to output the contents of the vesicles, including lysosomal enzymes in the extracellular environment (Braulke et al., Eur. J. Cell Biol. 43(3): 316-321, 1987). Acidification requires specific using Atraz, pokruženy in the membrane of endosome (Nishi et al., Nat. Rev. Mol. Cell Biol. 3(2): 94-103, 2002). The lack of this Atraz, as expected, can increase the secretion of lysosomal enzymes instead of sending complementary mechanism. It can be expected that the production of cell lines, which have defects in using Atrise, can prevent unproductive deviation phosphorylated recombinant enzyme in intracellular lysosomal compartment.

In 1984 were obtained and characterized mutants of cells of the Chinese hamster ovary (CHO) defective in endosomal acidification (Park et al., Somat. Cell Mol. Genet. 17(2): 137-150, 1991). Cells CHO-K1 was subjected to chemical mutagenesis and selected for survival at elevated temperatures in the presence of toxins. These toxins demanded endosomal acidification for the full manifestation of their mortality (Marnell et al., J. Cell. Biol. 99(6): 1907-1916, 1984). In the previous study, chose a combination of two toxins with different mechanisms of action to avoid toxin-specific sustainability. The principle is that although the probability of random mutations that lead to resistance to one particular toxin is very small, the probability of two simultaneous random mutations that are specific to two completely different toxins, is nonexistent. Selection was carried out at an elevated temperature in order to allow temperature-sensitive mutations. This genetic screening led to two mutants, one of which was designated as G.7.1 (G71), which were resistant to toxins at high temperatures. Damage in the G71 was not the result of capture or mechanism of action of the two toxins, and was a result of the failure of clone zakislate of endosome at elevated temperatures. This failure was also evident at the permissive temperature (34°C), although to a lesser degree. It was also revealed that the G71 cells are auxotrophic in respect to the research Institute for iron at elevated temperatures despite normal capture transferrin from the environment (Timchak et al., J. Biol. Chem. 261(30): 14154-14159, 1986). Because iron is released from transferrin only at low pH, auxotroph iron despite normal capture transferrin indicated no endosomal acidification. In another study it was shown that the defect in the acidification was manifested mainly in endosomes, but not in lysosomes (Stone et al., J. Biol. Chem. 262(20): 9883-9886, 1987). Data concerning G71, consistent with the conclusion that the mutation destabilized using Atraz responsible for endosomal acidification. Destabilization was most evident at elevated temperatures (39.5°C), but was partially manifested even at lower temperatures (34°C). Study of transport of the two endogenous lysosomal enzymes, cathepsin D and alpha-glucosidase, G71 cells (Park et al., Somat. Cell Mol. Genet. 17(2): 137-150, 1991) showed that both enzymes in detectable quantities secretarials at elevated temperatures, and glycosylation enzymes has not been changed. Secretion of phosphorylated acid alpha-glucosidase was significantly increased when nopermission temperatures.

therapeutic effectiveness of lysosomal enzyme sulfatase depends not only on the level of mannose-6-phosphate, but it also depends on the presence of active enzyme in this preparation. All known sulfatase contain the remainder C is steina in their catalytic center. this cysteine residue excision of modified Cα-formylglycine (FGly) to activate the enzyme. This post-translational activation of the enzyme, which is catalyzed by modifying sulfatase factor 1 (SUMF1), occurs in the endoplasmic reticulum on nevernude sulfatase immediately after the broadcast, before targeting sulfates in lysosome (Dierks et al., Proc. Natl. Acad. Sci. USA 94:11963-11968, 1997). The importance of this unique posttranslational modification indicates that SUMF1 mutations in which lead to the violation of FGly formation in lysosomal enzymes sulfatase lead to multiple sulfatase deficiency (MSD) in humans (Diez-Ruiz et al., Annu. Rev. Genomics Hum. Genet. 6:355-379, 2005).

Thus, the ability of cells G71 - mutant CHO cells that are defective in endosomal acidification, coexpression recombinant modifying sulfatase enzyme (SUMF1) man and lysosomal enzyme sulfatase person provides a mechanism for large-scale production of active vysokotsentralizovannym recombinant lysosomal enzymes sulfates person suitable for management for lysosomal diseases accumulation, caused by deficiency of such lysosomal enzymes sulfates or associated.

I DEFINE

If not defined otherwise, all technical and scientific the terms used in this document have the same value, which usually means a specialist in the field to which this invention relates. The following links are provided for experts in the field of General definitions of many terms used for this invention: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988); THE GLOSSARY OF GENETICS, 5TH ED., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale &Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY (1991).

Each publication, patent application, patent and other reference cited herein, are incorporated herein as references in full to the extent that they do not contradict this description.

It should be noted that, as used in this description and the attached claims, the singular includes the plural, unless the context clearly indicates otherwise.

As used herein, the following terms shall have ascribed to them the values, unless otherwise indicated.

"Allelic variant" refers to any of two or more polymorphic forms of a gene occupying the same genetic locus. Allelic variants arise in nature by mutation and can lead to phenotypic polymorphism in populations. Mutations in genes can be silent (that is. without changes in the encoded polypeptide), or they may encode polypeptides having altered amino acid sequences. "Allelic variants" also belong to the cDNA derived from mRNA transcripts genetic allelic variants, as well as to proteins encoded by them.

"Amplification" refers to any method by which the polynucleotide sequence is copied and thus multiplies in more polynucleotide molecules, e.g., by reverse transcription, polymerase chain reaction and ligase chain reaction.

The first sequence is an "antisense sequence" in relation to a second sequence if polynucleotide, the sequence is a first sequence-specific hybridized with polynucleotide, the sequence of which represents the second sequence.

"cDNA" refers to a DNA that is complementary or identical to an mRNA or single-stranded or double-stranded form.

For a description of the polynucleotide sequences in this document uses the standard notation: the left end of the single-stranded polynucleotide sequence is a 5'-end; the left-hand direction of double-stranded polynucleotide sequence is lnasty referred to as the 5'-direction. The direction of adding nucleotides to the resulting RNA transcripts from the 5' to 3' is called the direction of transcription. The DNA strand having the same sequence as the mRNA, called "coding circuit"; sequences on the DNA strand having the same sequence as mRNA, transcribed from this DNA, and located on the 5'-side from the 5'-end of the RNA transcript, referred to as "upstream sequences"; sequence on the DNA strand having the same sequence as the RNA and located on the 3'side of the 3'-end coding RNA transcript, referred to as "downstream sequences".

"Complementary" refers to the topological compatibility or matching of the interacting surfaces of the two polynucleotides. Thus, two molecules can be described as complementary, and what is more, the property of their contact surfaces is that they are complementary to each other. First polynucleotide complementary second polynucleotide, if the nucleotide sequence of the first polynucleotide identical to the nucleotide sequence of polynucleotide-binding partner of the second polynucleotide. Thus, polynucleotide with the sequence 5'-TATAC-3' complementary to polynucleotide with the sequence 5'-GTATA-3'. Nucleotide sequence", there is the complementary reference nucleotide sequence, if the sequence considered complementary nucleotide sequence that is essentially identical to a reference nucleotide sequence.

"Conservative substitution" refers to substitution in the polypeptide amino acid functionally similar amino acid. Each of the following six groups contain amino acids that are conservative substitutions for one another:

1) alanine (A), serine (S), threonine (T);

2) aspartic acid (D), glutamic acid (E);

3) asparagine (N), glutamine (Q);

4) arginine (R), lysine (K);

5) isoleucine (I), leucine (L), methionine (M), valine (V);

6) phenylalanine (F), tyrosine (Y), tryptophan (W).

The term "fragment"when used in relation to polypeptides, refers to polypeptides that are shorter than a full-sized polypeptide, due to the shortening or N-end or at the C-end of the protein or on both of them and/or because of internal deletions of part or region of the protein. Fragments of the polypeptide can be obtained by methods known in this field.

The term "mutant", when used in relation to polypeptides, refers to polypeptides in which one or more amino acids of the protein replaced by a different amino acid. Amino acid replacement can be a conservative substitution, as defined above, or it can p is establet a non-conservative substitution. Mutant polypeptides can be obtained by methods known in this field.

The term "derivative"when used in relation to polypeptides, refers to polypeptides chemically modified by such methods as, for example, but not limited to, ubiquitinylation, tagging (e.g., with radionuclides or various enzymes), covalent joining of the polymer, such as tahilramani (i.e. the formation of a derivative with polyethylene glycol) and the embedding or substitution by chemical synthesis of amino acids such as ornithine, which do not normally found in proteins person. Derivatives of polypeptides can be obtained by methods known in this field.

The term "derivative"when used in relation to cell lines, refers to cell lines that are descendants of the parent cell line; for example, the term includes cells which have passively or subclinically from the parent cell and which retain the desired property of the descendants of the parent cell lines, which were subjected to mutation and selected for preservation of the desired properties, and descendants of the parent cell lines that have been modified to contain different expressing vectors or different exogenously added nucleic acids.

"Detection" refers to ODA the division availability, absence or quantity of the analyzed component in the sample, and it may include determination of the number of analyzed component in the sample or a cell sample.

"Amenable to detection group" or "label" refers to compositions amenable to detection by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, suitable labels include32P,35S, fluorescent dyes, electroplate reagents, enzymes (as commonly used in an ELISA), Biotin-streptavidin, digoxigenin, haptens and proteins for which antisera or monoclonal antibodies, or nucleic acid molecule with a sequence complementary to the target. Amenable to detection group often gives a measurable signal, such as radioactive, chromogenic or fluorescent signal, which can be used to determine the number associated amenable to detection group in the sample. Amenable to detection group can be included in the primer or probe or attach to them, either covalently, or through ionic, van der Waals or hydrogen bonds, for example, you can include nucleotides or biotinylating nucleotides that are recognized by streptavidin. Amenable to detection group can yield detection directly or nepra is O. Indirect detection can involve linking amenable to detection by the second group amenable to direct or indirect detection of the group. For example, amenable to detection group can be a ligand of a binding partner, such as Biotin, which is a partner binding to streptavidin, or a nucleotide sequence that is a partner binding to complementary sequences with which it may be specific to gibridizatsiya. Partner binding itself can be a direct detection, for example, the antibody itself can be observed with a fluorescent molecule. The binding partner may also indirect succumb detection, for example, a nucleic acid having a complementary nucleotide sequence may be part of a branched DNA molecules, which, in turn, lends itself to detection by hybridization with other labeled molecules of nucleic acids (see, for example, Fahrlander et al., Bio/Technology 6:1165, 1988). Quantification of the signal is carried out, for example, by scintillation counting, densitometry or flow cytometry.

"Diagnosis" means the identification of the presence or determining the nature of the pathological condition. Diagnostic methods differ in their specificity and selectivity. Although specific diag is eticheski method may not provide a definitive diagnosis of the condition, it is sufficient that the method provides a positive indication that contributes to the diagnosis.

The term "effective amount" means a dosage sufficient to provide the desired effect on health, pathology and disease of the subject or for diagnostic purposes. The desired result may include subjective or objective improvement in the recipient of the dosage. "Therapeutically effective amount" refers to an amount effective to provide certain favorable effect on health.

"Encoding" refers to the property that specific sequences of nucleotides in polynucleotide, such as a gene, cDNA, or an mRNA, to serve as a matrix for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e. rRNA, tRNA and mRNA)or a defined sequence of amino acids and the biological properties resulting from them. Thus, the gene encodes a protein, if the transcription and translation of mRNA produced from this gene leads to the formation of a protein in a cell or other biological system. As the coding circuit, a nucleotide sequence which is identical to the mRNA sequence and is usually provided in sequence listings and nicodemous the I circuit, used as the template for transcription of a gene or cDNA, can be specified as encoding a protein or other product of this gene or cDNA. If there are no other instructions, "the nucleotide sequence encoding amino acid sequence"includes all nucleotide sequences that are degenerate variants of each other and which encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA can include introns.

"Dose equivalent" refers to a dose that contains an equal number of active funds.

Sequence controls expression" refers to a nucleotide sequence in polynucleotide, which regulates the expression (transcription and/or translation) of the nucleotide sequence is functionally associated with it. "Functionally linked" refers to functional relationships between the two parts, in which the activity of one part (for example, the ability to regulate transcription) leads to an effect on another part (for example, transcription of the sequence). Sequences control expression can include, for example, without limitation, the sequences of promoters (e.g., inducible or constitutive), enhancers, terminators, transcripti is, the initiating codon (i.e. ATG), splicing signals for introns and stop codons.

"Expressing vector" refers to a vector containing a recombinant polynucleotide containing sequences control the expression of functionally associated with nucleotide sequence of subject expression. Expressing the vector contains a sufficient number of CIS-regulatory elements for expression; other elements for expression can be provided by the host-cell or expressing systemin vitro. Expressing vectors include all vectors known in this field, such as Comedy, plasmids (e.g., simple or in liposomes and viruses, which include recombinant polynucleotide.

The expression "vysokovostrebovannye", "high level phosphorylation" and "high level of phosphorylated oligosaccharides" refers to preparations of lysosomal enzymes sulfates, in which at least 50% of the lysosomal enzyme sulfatase associated with the cation-independent receptor for mannose-6-phosphate via phosphorylated oligosaccharides. Linking, in addition, characterized by the exposure to competition with mannose-6-phosphate. Vysokovostrebovannye lysosomal enzyme sulfatase can also refer to the lysosomal enzyme sulfatase with men is her least 0,25, preferably at least 0.5 and more preferably at least 0,75 bis-phosphorylated oligomannose chains on the protein chain.

The term "bis-phosphorylated oligomannose chain", as used herein, refers to containing mannose oligosaccharide chains, which are N-linked to asparagine residues in lysosomal enzymes sulfatase and contain two remaining mannose-6-phosphate. As a rule, bis-phosphorylated oligomannose chains have 7 residues of mannose, i.e. bis-fosfatados 7 (BPM7)that are associated with two GlcNAc residues, which, in turn, linked to the asparagine residue in the lysosomal enzyme sulfatase.

The terms "active", "activated" and "high level of activation" refers to preparations of lysosomal enzymes sulfates, in which at least 50%, preferably at least 70%, more preferably at least 90% and even more preferably at least 95% of the cysteine residue in the active center of the protein excision of modified Cα-formylglycine (FGly).

"Active vysokovostrebovannye" refers to preparations of lysosomal enzymes sulfates, in which at least 50%, preferably at least 70%, more preferably at least 90% and even more preferably at least 95% of the residue C is Stein active centre of the protein excision of modified C α-formylglycine (FGly) and with at least a 0.25, preferably at least 0.5 and more preferably at least 0,75 bis-phosphorylated oligomannose chains on the protein chain.

The term "biologically active" refers to the polypeptide (i.e., the enzyme) fragments, mutants, variants or derivatives, that retain at least a significant amount (e.g., at least about 50%, preferably at least about 70% and more preferably at least about 90%) of one or more types of biological activity of the full-length polypeptide. When this term is used in relation to lysosomal enzyme sulfatase, its biologically active fragment, mutant, variant or derivative retains at least a significant level of activity sulfatase (i.e. the removal of sulfate esters from their substrates-targets). When used in relation to modifying sulfatase factor 1 (SUMF1), its active fragment, mutant, variant or derivative retains at least a significant level of activity education formylglycine (i.e. modification of the cysteine residue in the active center of lysosomal enzyme sulfatase in Cα-formylglycine (FGly)).

The term "clean" or "pure", when used against the AI polypeptides, refers to the amount of the polypeptide being analysed, compared with any pollutants that can be detected using a specific method. For recombinant lysosomal enzymes sulfates according to the invention, the "purity" can be defined, subjecting the product of the enzyme sulfatase electrophoretic separation by SDS-PAGE in reducing or non conditions with subsequent staining of Kumasi blue or silver, or by chromatographic separation by HPLC (for example, reversed-phase (RP) C4), or through any other chromatographic separation, for example gel filtration (SEC), etc. using these methods, purified recombinant lysosomal enzymes sulfatase according to the invention have a purity of at least about 80%, preferably at least about 90%, more preferably at least about 95% and even more preferably at least about 98%, or 99%.

The term "predecessor" or "form predecessor" refers to the shape of recombinant lysosomal enzyme sulfatase, which is secreted from mammalian cells, i.e. without the signal sequence, but without certain modifications, such as internal cleavage Bel is s, which normally occurs in lysosome. The terms "Mature", "Mature form", "processionary" or "protestirovanny form" refers to the form of recombinant lysosomal enzyme sulfatase, which in norm exists in lysosome. For recombinant lysosomal enzymes sulfates according to the invention, the relative content of the "predecessor" or "forms predecessor" and "Mature", "Mature form", "processioning" or "protestirovanny forms of the enzyme can be determined by exposing the preparation of enzyme sulfatase electrophoretic separation by SDS-PAGE in reducing conditions followed by staining of Kumasi blue or silver, or by chromatographic separation by HPLC (for example, reversed-phase (RP) C4) or by any other chromatographic separation, for example gel filtration (SEC), etc. using these methods, peeled recombinant lysosomal enzymes sulfatase according to the invention comprise at least about 75%, preferably at least about 85%, more preferably at least about 90% and even more preferably at least approximately 95% of the "predecessor" or "forms predecessor". An alternative to these methods, purified recombinant lysosomal farm what you sulfatase according to the invention contain less than about 25%, preferably less than about 15%, more preferably less than about 10% and even more preferably less than about 5% "Mature", "Mature form", "processioning" or "protestirovanny form of the enzyme. In some embodiments, implementation, reveals only the "predecessor" or "form of predecessor (i.e. the product of the enzyme sulfatase essentially consists of one amenable to detection band in SDS-PAGE in reducing conditions, or a single peak in the HPLC analysis).

The terms "identical" or percent "identity" in the context of two or more polynucleotide or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a certain percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, when measured using one of the following algorithms for comparing sequences or visual examination.

"Linker" refers to a molecule that binds two other molecule either covalently, or through ionic, baths der Waals or hydrogen bonds, for example a molecule of nucleic acid, which hybridizes with one of the complementary sequence of the 5'-end and with the other complementary sequence on the 3'-end, thus, combining two complementary sequence.

"Low level phosphorylation" or "low phosphorylation" refers to the preparation of lysosomal enzymes sulfates, in which half-maximal concentration for engagement in fibroblastlike cells is less than 10 nm or fraction of lysosomal enzymes sulfates, which binds the column with receptor mannose-6-phosphate is less than about 25%.

The term "naturally occurring"applied to the object, refers to the fact that the object can be found in nature. For example, the sequence of the polypeptide or polynucleotide, which is present in an organism (including viruses)that can be isolated from a source in nature and which is not intentionally modified by man in the laboratory is naturally occurring.

"Pharmaceutical composition" refers to compositions suitable for pharmaceutical use in an animal subject, including humans and mammals. The pharmaceutical composition comprises a pharmacologically effective amount of therapeutic lysosomal enzyme sulfatase, and it contains one or more pharmaceutically acceptable carriers, diluents or excipients. The pharmaceutical composition comprises the composition, with the holding of the active ingredient(s) and the inert ingredient(s), which are a carrier, diluent or excipient, as well as any product that is the result, directly or indirectly, combination, complex formation or aggregation of any two or more ingredients, or dissociation of one or more ingredients, or other types of reactions or interactions of one or more of the ingredients. Thus, the pharmaceutical compositions of the present invention include any composition made by mixing lysosomal enzyme sulfatase of the present invention and one or more pharmaceutically acceptable carriers, diluents or excipients.

The expression "pharmaceutically acceptable carrier, diluent or excipient" refers to any of the standard pharmaceutical carriers, diluents, buffers and excipients, such as, for example, is not limited to, phosphate-saline buffer, 5% aqueous solution of dextrose, and emulsions, such as emulsion type oil/water or water/oil, and various types of wetting agents and/or adjuvants. Suitable pharmaceutical carriers, diluents or excipients and formulations described in Remington''s Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton, 1995). Preferred pharmaceutical carriers, diluents or excipients depend on the method of introduction of the active substance. Typical is diversified methods of introduction include, for example, not limited to, enteral (e.g. oral) or parenteral (e.g. subcutaneous, intramuscular, intravenous or intraperitoneal) injection or local, transdermal or transmucosal introduction.

"Pharmaceutically acceptable salt" is a salt that can produce lysosomal enzyme sulfatase for pharmaceutical applications, including, for example, metal salts (sodium, potassium, magnesium, calcium, etc. and salts with ammonia or organic amines.

"Polynucleotide" refers to a polymer composed of nucleotide elements. Polynucleotide include naturally occurring nucleic acids such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), and analogs of nucleic acids. Analogues of nucleic acids include analogs that contain non-naturally occurring bases, nucleotides, which have connections with other nucleotides that differ from naturally occurring phosphodiester bond, or which contain a base connected through a connection other than fosfolipidnyh links. Thus, analogs of nucleotides include, for example, without limitation, phosphorothioate, phosphorodithioate, postretrieval, phosphoramidate, boranophosphate, methylphosphonate, chiral methylphosphonate, 2-O-methylribonucleotide peptide nucleic acid (PNA), etc. Such polynucleotide can be synthesized, for example, using automated devices for DNA synthesis. The term "nucleic acid", mainly refers to the large polynucleotides. The term "oligonucleotide", mainly refers to short polynucleotides, usually not more than about 50 nucleotides. It will be understood that when a nucleotide sequence is a DNA sequence (i.e. A, T, G, C), it also includes an RNA sequence (i.e. A, U, G, C)in which "U" replaces "T".

"Polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic is not naturally occurring analogues, linked through peptide bonds. Synthetic polypeptides can be synthesized, for example, using an automated device for synthesis of polypeptides. The term "protein", mainly refers to large polypeptides. The term "peptide", mainly refers to short polypeptides. Herein to describe a polypeptide sequence using the conventional designation: the left end of the polypeptide sequence is an N-end; the right end of the polypeptide sequence is a With-end.

"Primer" from OSISA to polynucleotide, which is capable of specific to gibridizatsiya with a specific polynucleotide matrix and provide a point of initiation of synthesis of complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is a condition in which induced the synthesis, i.e. in the presence of nucleotides, complementary to the polynucleotide matrix, and means for polymerization such as DNA polymerase. Primer, usually a single-stranded, but may be double-stranded. Primers usually are deoxyribonucleic acid, however, for many applications fit a wide variety of synthetic and naturally occurring primers. Primer complementary matrix for hybridization with which it is designed to serve as the site for initiation of synthesis, however it should not reflect the exact sequence of the matrix. In this case, specific hybridization of the primer with the matrix depends on the stringency of the hybridization conditions. The primers can be labeled, e.g., chromogenic, radioactive or fluorescent groups, and you can use them as amenable to detection groups.

"Probe", when used in respect of polynucleotide, refers to polynucleotide, which is capable of specific to gibridizatsiya with a certain sequence, etc is different polynucleotide. The probe specific hybridized with complementary polynucleotide target, but it should not reflect the exact complementary sequence of the matrix. In this case, specific hybridization of the probe with the matrix depends on the stringency of the hybridization conditions. The probes may be in the state of, for example, chromogenic, radioactive or fluorescent groups, and you can use them as amenable to detection groups.

"Prophylactic" treatment is a treatment for a subject, who has no symptoms or only early signs, to reduce the risk of developing pathology. Compounds according to the invention can be entered as a prophylactic treatment to reduce the likelihood of development of a pathology or to minimize the severity of the pathology, if it develops.

"Recombinant polynucleotide" refers to polynucleotide having a sequence that is not associated in nature with each other. Amplificatory or assembled recombinant polynucleotide can be included in a suitable vector, and the vector can be used to transform a suitable host cell. Cell host, which contains recombinant polynucleotide, referred to as "recombinant cell host. Then gene is expressed in the recombinant cell host with education, for example, recom is anantnag polypeptide". Recombinant polynucleotide can also perform non-coding function (e.g., promoter, origin of replication, the binding site of the ribosome).

The expression "specific hybridization", "specific hybridization" or "selectively gibridizatsiya with" refers to the binding, the formation of duplexes or hybridization of nucleic acid molecules, preferably with a specific nucleotide sequence in stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.

The term "stringent conditions" refers to conditions under which a probe hybridizes preferably with its sequence target and to a lesser extent hybridized, or not hybridized, with other sequences. "Stringent hybridization" and "stringent washing conditions during hybridization" in the context of experiments on hybridization of nucleic acids, such as southern and Northern hybridization, are dependent on the sequence and are different under different environmental parameters. A detailed guide to the hybridization of nucleic acids can be found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes part I chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York. Typically, hybridization conditions of high stringency and washing the choice is up so, so they were approximately 5°C below the annealing temperature (Tm) for the specific sequence at a defined ionic strength and pH. Tm is the temperature (under defined ionic strength and pH)at which 50% of the target sequence hybridizes with exactly the same probe. Conditions of very high stringency chosen so that they were equal to the Tm for a particular probe.

An example of hybridization conditions of high stringency for hybridization of complementary nucleic acids that have more than 100 complementary residues on a filter in a southern or Northern band is a 50% formalin with 1 mg of heparin at 42°C, and hybridization carried out during the night. An example of the washing conditions of high stringency are 0.15 M NaCl at 72°C for approximately 15 minutes. An example of stringent wash is of 0.2× SSC at 65°C for 15 minutes (see Sambrook et al. for a description of SSC buffer). Often washing high severity preceded by the washing of low stringency to remove background probe signal. An example of a washing cycle, the average severity for duplex, for example, more than 100 nucleotides, is 1× SSC at 45°C for 15 minutes. Example flush low severity for duplex, for example, more than 100 nucleotides, is 4-6× SSC at 40°C for 15 minutes. Typically, the ratio of signal to noise, stood the determinant of 2× (or more) regarding attitude, observed for an unrelated probe in the particular hybridization analysis indicates detection of a specific hybridization.

A "subject" of diagnosis or treatment is a human or non-human animal, including a mammal, or a Primate.

The expression "essentially homologous" or "substantially identical" in the context of two nucleic acids or polypeptides, generally refers to two or more sequences or subsequences that have at least 40%, 60%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity of nucleotide or amino acid residues, when compared and aligned for maximum correspondence and determining, using one of the following algorithms for comparing sequences or visual examination. Preferably, the substantial identity exists over a region of sequence length of at least about 50 residues, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues. In the most preferred embodiment, the sequences are substantially identical over the entire length and any and compare both biopolymers.

To compare sequences, typically one sequence acts as a reference sequence, with which compare the test sequence. When using the algorithm of sequence comparison, test and reference sequences are introduced into the computer, denote the coordinates of suppositionally, if necessary, and specify the parameters of the program with the algorithm of sequence comparison. The algorithm then compare sequences calculates the percent sequence identity for the test sequence(s) relative to a reference sequence based on the specified parameters.

Optimal alignment of sequences for comparison can be conducted, for example, using the algorithm of the local homology Smith & Waterman, Adv. Appl. Math. 2:482, 1981, using the algorithm of alignment homology Needleman &Wunsch, J. Mol. Biol. 48:443, 1970, by way of finding similarities Pearson &Lipman, Proc. Natl. Acad. ScL USA 85:2444, 1988, by using a computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI) or visual examination.

One example of a suitable algorithm is PILEUP. A PILEUP performs multiple sequence alignment from a group of related members is of telestai using progressive pairwise alignment to show the relationship and the percent identity of the sequences. He also builds a tree or dendrogram showing cluster correlation, used to facilitate alignment. In a PILEUP uses a simplification of the method of progressive alignment Feng &Doolittle, J. Mol. Evol., 35:351-360, 1987. The method used is similar to the method described by Higgins &Sharp, CABIOS 5:151-153, 1989). The program can align up to 300 sequences, the maximum length of each of which is 5000 nucleotides or amino acids. Process multiple alignment begins with a pairwise alignment of the two most similar sequences, providing a cluster of two aligned sequences. Then this cluster is aligned with the next most similar sequence or cluster of aligned sequences. Two clusters of sequences aligned by a simple extension of the pairwise alignment of two separate sequences. The final alignment is performed using a series of progressive pairwise alignments. The program perform, specifying certain sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and indicating the parameters of the program. For example, the reference sequence can srawniwa the ü with other test sequences to determine the ratio of the percentage identities of the sequences using the following parameters: the penalty for making a pass default (3,00), the penalty for continuing to pass by default (0,10) and the estimated limit passes. Another algorithm that is suitable for holding multiple sequence alignment is a Clustal W (Thompson et al., Nucleic Acids Research 22: 4673-4680, 1994).

Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, described in Altschul, et al., J. Mol. Biol., 215: 403-410, 1990. Software for performing BLAST analyses is freely available through the National Center for Biotechnology Information. This algorithm includes the initial identification of pairs of sequences with the maximum similarity (HSP) by identifying short words of length W in the interest of the sequence, which either match or satisfy some positive evaluation threshold value T when aligned with a word of the same length in the sequence from the database. T is defined as the threshold value for the neighboring words (Altschul, et al., J. Mol. Biol., 215: 403-410, 1990). These initial matches neighboring words act as precursors for the start of the search in order to detect longer HSP containing them. The matching words propagates in both directions along each sequence for as long, until the cumulative value to stabilize the air traffic management may increase. The total value is calculated using, in the case of nucleotide sequences, the parameters M (the compensation value for a pair of matching residues, always >0) and N (the size of the penalty for not matching residues, always <0). In the case of amino acid sequences to calculate the total values using the evaluation matrix. The continuation of matching words in each direction is stopped if: the total value for alignment decreases the value of X relative to its maximum achieved value; the total value is reduced to zero or below, due to the accumulation of one or more aligned residues with a negative value; reached the end of any sequence. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, the expected value (E) of 10, M=5, N=-4 and a comparison of both circuits. In the case of amino acid sequences, the BLASTP program uses as defaults a word length 3 and the expected values (E) 10 and estimate the BLOSUM62 matrix (see Henikoff &Henikoff, Proc. Natl. Acad. Sci. USA 89:of 10,915, 1989).

In addition to calculating percent sequence identity, the BLAST algorithm also performs statisticheskiy similarity between two sequences (see, for example, Karlin and Altschul, Proc. NAT'l. Acad. Sci. USA 90:5873-5787, 1993). One of the parameters of the similarity obtained using the BLAST algorithm is the smallest sum probability (P(N)), which involves specifying the probability with which a match between two nucleotide or amino acid sequences can occur randomly. For example, a nucleic acid is considered similar to the nucleic acid reference sequence if the smallest sum probability in a comparison of the analyzed nucleic acid to the reference nucleic acid is less than about 0.1, less than about 0.01, or less than about 0.001 in.

As an indication that two nucleic acid sequences or two polypeptide are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reacts with the polypeptide encoded by the second nucleic acid, as described below. Thus, the polypeptide, as a rule, is essentially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other in a strict service is established, as described in this document.

"Essentially pure" or "isolated" means that the component in question is predominantly present component (i.e. on a molar basis it is more common than any other individual macromolecular component in the composition), and substantially purified fraction is a composition, where this component contains at least about 50% (on a molar basis) of all present macromolecular products. As a rule, essentially pure composition means that approximately 80% to 90% or more of the macromolecular constituents present in the composition, are of interest purified component. This component purified essentially to a homogeneous state (contaminants cannot be detected in the composition of conventional detection methods), if the composition consists essentially of a single macromolecular product. For this definition, various solvents, low molecular weight compounds (<500 daltons), stabilizers (e.g., BSA) and particles of elemental ions do not consider macromolecular components. In some embodiments, implementation of lysosomal enzymes sulfatase according to the invention are essentially pure or selected. In some the older versions of the implementation, lysosomal enzymes sulfatase according to the invention are essentially pure or allocated in respect of the macromolecular starting materials used for their synthesis. In some embodiments, implementation of the pharmaceutical composition according to the invention contains essentially cleared or selected therapeutic lysosomal enzyme sulfatase mixed with one or more pharmaceutically acceptable carriers, diluents or excipients.

"Therapeutic" treatment is a treatment for a subject, which has signs or symptoms of pathology, to reduce or eliminate these signs or symptoms. Signs or symptoms may be biochemical, cellular, histological, functional, subjective or objective. Lysosomal enzymes sulfatase according to the invention it is possible to enter as medical treatment or diagnosis.

"Therapeutic index" refers to the range of dosages (quantitative and/or temporary) above the minimum therapeutic quantity, and below unacceptable toxic amounts.

"Treatment" refers to prophylactic treatment or therapeutic treatment or diagnostic treatment.

The term "unit dosage form"as used herein refers to eticheski discrete units, suitable as single dosages for the subjects-people or animals, where each element contains a given number of lysosomal enzyme sulfatase of the present invention, calculated to be sufficient to provide the desired effect, together with one or more pharmaceutically acceptable carriers, diluents or excipients. Characteristics of the new unit dosage forms of the present invention depend on the specific lysosomal enzyme sulfatase and effect to be achieved, and the pharmacodynamics associated with each of the lysosomal enzyme sulfatase the owner.

II PRODUCTION of LYSOSOMAL ENZYMES SULFATES

In one aspect the present invention relates to a new method of production of active vysokotsentralizovannym lysosomal enzymes sulfates in amounts that allow the use of such enzymes. Generally, the method comprises transforming a suitable cell line cDNA coding modifying sulfatase factor 1 (SUMF1) person or its biologically active fragment, mutant, variant or derivative, and cDNA that encodes a full-sized lysosomal enzyme sulfatase or its biologically active fragment, mutant, variant or derivative. Specialists in this area who can get expressing designs, other than structures specifically described herein, for the optimum production of such lysosomal enzymes sulfates suitable transfected their cell lines. Moreover, professionals can easily construct the cDNA fragments encoding biologically active fragments, variants, mutants or derivatives of naturally occurring SUMF1 or lysosomal enzyme sulfatase, which have the same biological activity as the naturally occurring full-length enzymes, or similar.

Cell-hosts

Cell owners used for the production of recombinant lysosomal enzymes sulfates, are deficient in endosomal acidification cell lines characterized by their ability to produce such lysosomal enzymes sulfatase in amounts that allow therapeutic use of the enzyme. The invention relates to the events of the CHO-K1 cell line group complementaly END3, denoted G71. The invention also relates to a cell line G71, which is adapted for growth in serum-free suspension culture, denoted G71S. The invention also relates to derivatives G71 and cell lines G71S, which further subcloned or which contain different ex is rezerwuj plasmids.

Cells that contain and Express DNA or RNA, coding for a recombinant protein, referred to herein genetically modified cells. Mammalian cells that contain and Express DNA or RNA, coding for a recombinant protein, referred to as genetically modified mammalian cells. The introduction of DNA or RNA in cells is conducted in a known manner transfection, such as, but not limited to, electroporation, microinjection, bombardment microkeratome, precipitation with calcium phosphate, modified precipitation with calcium phosphate, processing cationic lipid, photoporation, methods of fusion, mediated by receptors transfer or deposition with polybrene. Alternative DNA or RNA can be entered by infection with a viral vector. How products for cells, including mammalian cells, which Express DNA or RNA, coding for a recombinant protein described in simultaneously considering patent applications U.S. serial No. 08/334797, entitled "In Vivo Protien Production and Delivery System for Gene Therapy", Richard F. Selden, Douglas A. Treco, Michael W. Heartlein (filed November 4, 1994); in the application U.S. serial number No. 08/334455, entitled "In Vivo Production and Delivery of Erythropoetin or Insulinotropin for Gene Therapy", Richard F. Selden, Douglas A. Treco and Michael W. Heartlein (filed November 4, 1994), and in the application U.S. serial No 08/231439, called "Targeted Introduction of DNA Into Primary or Secondary Cells and Their Use for Gene Therapy", Douglas A. Treco, Michael W. Heartlein and Richard F. Selden (filed April 20, 1994). Specify all of these applications are incorporated herein as references in full.

In preferred variants of implementation, a host cell used for the production of recombinant lysosomal enzymes sulfates, is a cell line deficient in endosomal acidification, which is characterized by its ability to produce such lysosomal enzymes sulfatase in amounts that allow therapeutic use of the enzyme. In preferred embodiments, the implementation, the invention relates to the events of the CHO-K1 cell line group complementaly END3, denoted by G71, and cell line G71, which is adapted for growth in serum-free suspension culture, denoted G71S, which coexpression modifying sulfatase factor 1 (SUMF1) and recombinant human lysosomal enzyme sulfatase and, thus, capable of producing high output active vysokovostrebovannye lysosomal enzymes sulfatase, as defined in section "DEFINITIONS", thereby allowing large-scale production of therapeutic lysosomal enzymes sulfates. In the most preferred embodiments implementing the tvline, cell line G71 or G71S or its derivative expresses and secretes a recombinant lysosomal enzymes sulfatase in amounts of at least about 0.5, preferably at least about 0.75, and more preferably at least about 1.0, and even more preferably at least about 1.25 PG/cell/day.

Vectors and structures of nucleic acids

The design of nucleic acid used for the expression of recombinant protein, or modifying sulfatase factor 1 person (SUMFl)or lysosomal enzyme sulfatase, or both of them may be a structure which is expressed extrachromosomal (episome) in transtitional cell of a mammal, or design, which is embedded, either randomly or in a pre-selected area of the target, using homologous recombination in the genome of the recipient cells. Design, which is expressed Negroamaro, contains, in addition to encoding the recombinant protein sequences, sequences sufficient for expression of the protein in cells and, optionally, to replicate the design. It typically includes a promoter, encoding a recombinant protein DNA and the polyadenylation site. DNA encoding a recombinant the first protein, located in the design so that its expression was under the control of the promoter. Optionally, the construct may contain additional components such as one or more of the following: plot splicing, enhancer sequence, gene selective marker under the control of an appropriate promoter, amplifiers marker gene under the control of an appropriate promoter and the region of attachment to the matrix (MAR) or other elements known in this field, which enhance the expression region where it is built.

In the variants of implementation, in which the design DNA integrates into the host cell genome, it necessarily includes only coding recombinant protein nucleic acid sequences. Optionally, it may include promoter and enhancer sequence, a parcel or parcels of polyadenylation, a parcel or parcels of splicing, nucleic acid sequences that encode a selective marker or markers, nucleic acid sequences that encode amplificatory marker, the area of attachment to the matrix (MAR) or other elements known in this field, which enhance the expression of the area in which it is embedded, and/or DNA homologous to genomic DNA in the recipient cell, for the directional striven what I DNA in the selected region of the genome (for targeting DNA or DNA sequence).

Methods cell culture

Mammalian cells containing encoding a recombinant protein, DNA or RNA, cultured under conditions suitable for cell growth and expression of DNA or RNA. Cells that Express a recombinant protein, can be identified using well known methods and methods described herein, the recombinant protein can be extracted and purified by using known methods and methods described herein, or by increased production of recombinant protein, or without it. Identification can be performed, for example, by screening of genetically modified mammalian cells that have the phenotype, indicating the presence of DNA or RNA that encodes a recombinant protein, such as screening by PCR screening southern-blot analysis, or screening for expression of the recombinant protein. The selection of cells that contain included in each encoding a recombinant protein DNA can be performed by incorporating a selective marker in the design DNA with subsequent cultivation of transfected or infected cells containing the gene selective marker, under conditions suitable for the survival of only those cells that Express the gene selective marker. In addition, the amplification time viennoiserie DNA can be enhanced by cultivation of genetically modified mammalian cells under appropriate conditions (for example, the cultivation of genetically modified mammalian cells containing amplificatory marker gene in the presence of concentrations of drugs, which can survive only cells containing multiple copies amplifierarava marker gene).

Genetically modified mammalian cells expressing the recombinant protein, can be identified as described herein, by detecting expressing the product. For example, mammalian cells expressing active vysokovostrebovannye lysosomal enzymes sulfatase, it is possible to identify immunological sandwich enzyme-analysis. Antibodies may be directed to the portion of the active tool.

Variants of lysosomal enzymes sulfates

In certain embodiments implemented, it is possible to obtain mutants or variants active vysokopostavlennogo lysosomal enzyme sulfatase, and they can be used in various applications in which it is possible to use active vysokovostrebovannye lysosomal enzymes sulfatase. Mutants or variants of the amino acid sequence of the polypeptide can be a mutant or variants with substitutions, insertions or deletions. Mutants or variants with deletions are deprived of one or more OS Atkov native protein, which are not essential for function or immunogenic activity. Common type of mutant or variant with a deletion is a mutant or variant devoid of secretory signal sequences or signal sequences directing the protein to bind to a specific part of the cell. Mutants or variants with insertions, usually involve adding material in reconcavo point of the polypeptide. This may include the insertion of immunologically reactive epitope or just one of balance. End of add, also called fused proteins, discussed below.

Options can be essentially homologous or essentially identical remotefilename the lysosomal enzyme sulfatase, as described above. Preferred variants are variants of the polypeptide active vysokopostavlennogo lysosomal enzyme sulfatase, which retain at least some biological activity, for example sulfatase activity, lysosomal enzyme sulfatase. Other preferred variants include polypeptide N-atsetilgalaktozamin-6-sulfatase person that retains at least part sulfatase activity of N-atsetilgalaktozamin-6-sulfatase person.

In mutants or variants with substitutions, as a rule, one amino acid poly is eptide wild-type replaced by a different amino acid at one or several sites of the protein, and they can be designed to modelirovanie one or more properties of the polypeptide, such as, for example, is not limited to, resistance to proteolytic cleavage, without the loss of other functions or properties. Replacement of this type preferably are conservative, i.e. one amino acid is replaced by an amino acid with similar shape and charge. Conservative substitutions are well known in this field and include, for example, replacement of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to Proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.

One aspect of the present invention provides for producing mutants or variants of the glycosylation site, which is a mutant plot of O - or N-linked glycosylation of lysosomal enzyme sulfatase. Such mutants or variants can provide important information regarding the biological activity, the physical is the first structure and capacity with respect to the binding of substrate at the active vysokopostavlennogo lysosomal enzyme sulfatase. In specific aspects, provides that you can get other mutants or variants of the peptide active vysokopostavlennogo lysosomal enzyme sulfatase that retain biological activity but have increased or decreased activity of the binding of the substrate. Essentially, specifically provides for the mutation of the active site or catalytic region for the formation of mutants or variants of a protein with an altered activity of the binding of the substrate. In such scenarios, the implementation, the sequence of active vysokopostavlennogo lysosomal enzyme sulfatase compared with the sequence of other related enzymes and selected residues are subjected to certain mutations.

The numbering of amino acid Mature protein with the intended N-end as amino acid number 1, illustrative mutations that may be suitable include, for example, replacement of all or some of the potentially glycosylated asparagine residues, including provisions 397 178 and recombinant N-atsetilgalaktozamin-6-sulfatase (GALNS) of the person (see figure 5).

The binding of a substrate can be modified by mutations in the active centre/near the active centre of the lysosomal enzyme sulfatase. Taking these mutations as illustrative, the experts in this area and will understand what can make other mutation sequence of the enzyme to obtain structural and functional information about the protein and its activity.

For constructing mutants or variants, such as mutants or variants described above, the person skilled in the art can use standard, well-known methods. Specifically provided N-terminal deletions, C-terminal deletions, internal deletions, as well as random and targeted mutagenesis.

N-terminal and C-terminal deletions represent a form of deletion mutagenesis using, for example, the availability of suitable single restriction site near the end of the C - or N-terminal region. DNA is cleaved at this site, and split ends are broken down by nucleases, such as BAL31, exonuclease III, Tnkase I and S1 nuclease. The connection of the two ends of the leads to a series of DNA with deletions of various sizes in the area of the site restriction. Proteins expressed from such mutants can be analyzed in respect of the relevant biological functions, such as enzymatic activity, using methods standard in the field and described in the description. For mutants with an internal deletion is possible to apply similar methods using two suitable manner placed restriction sites, thereby ensuring that the conduct is part of a certain deletions, and re-ligating the ends, as described above.

Also provides mutants with partial splitting. In such cases, the person skilled in the art may use "frequent splitter", which cleaves DNA at different places, depending on the duration of the reaction time. Thus, by varying the reaction conditions, it is possible to obtain a series of mutants of various sizes, which can then be subjected to screening for activity.

You can also make random insertional mutation, cutting the DNA sequence of Dnazol I, for example, by embedding the plot of nucleotides that encode 3, 6, 9, 12, etc., amino acids and re-ligera the end. After making mutations, mutants can be screened for various kinds of activity, which manifests protein wild-type.

You can also use point mutagenesis to accurately identify which amino acid residues important for specific types of activity associated with the biological activity of lysosomal enzyme sulfatase. Thus, the person skilled in the art can carry out the replacement of individual bases in the DNA chain, to obtain the modified codon or missense mutation.

The specific amino acids of the protein can be modified to create an equivalent, or even superior, the molecules of the second generation is possible. Such changes include the replacement of this amino acid protein without significant loss of binding ability of interaction structures, such as antigennegative region of antibodies or binding sites on molecules of substrates or receptors. Because the ability to interact and nature define this protein's biological functional activity of the protein, the protein sequence can to make certain amino acid substitutions, as well as in the underlying encoding DNA sequence, and nevertheless obtain a protein with similar properties. Thus, in the DNA sequence of genes is possible to make various modifications without appreciable loss of their biological applicability or activity, as discussed below.

In carrying out such changes, you can consider the hydrophobicity index of amino acids. It is generally accepted that the relative hydrophobicity of amino acids provides the secondary structure of the final protein, which, in turn, determines the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Each amino acid is assigned the index of hydrophobicity on the basis of its characteristics, hydrophobicity and charge (Kyte &Doolittle, J. Mol. Biol., 157(1): 105-132, 1982, included in this the document is t as a reference). Typically, amino acids can be substituted by other amino acids, which have a similar index or indicator of hydrophobicity and, nevertheless, lead to a protein with similar biological activity, i.e, however, provide equivalent from the point of view of biological function of the protein.

In addition, the substitution of similar amino acids can be effectively carried out on the basis of hydrophilicity. In U.S. patent 4554101, incorporated herein by reference, States that the most high local average hydrophilicity of a protein, determined by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. Essentially, the amino acid can be replaced with another amino acid having a similar hydrophilicity value and, nevertheless, to obtain a biologically equivalent, and immunologically equivalent protein.

Illustrative amino acid substitutions that can be used in the context of the invention, include, but are not limited to, the replacement between arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; valine, leucine and isoleucine. Other such changes, which take into consideration the need to preserve part or all of the biological activity when changing the secondary structure of the protein, are well known in the art given in the second field.

Another type of option, which provides for obtaining polypeptides according to the invention is the use of peptide mimetics. Mimetics are containing peptide molecules which mimic the secondary structure elements of the protein. See, for example, Johnson et al., "Pepride Turn Mimetics", BIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Eds., Chapman and Hall, New York (1993). The rationale for the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to Orient the side chains of amino acids so that was simplified molecular interactions such as the interaction between antibody and antigen. A peptide mimetic according to expectations, may permit molecular interactions similar to the natural molecule. These principles can be used in conjunction with the principles described above, to construct molecules of the second generation, with many of the natural properties of lysosomal enzymes sulfates, but with changed or even improved characteristics.

Modified glycosylation of lysosomal enzymes sulfates

You can also get options active vysokopostavlennogo lysosomal enzyme sulfatase, which have a modified glycosylation pattern relative to the original polypeptide, for example, a deletion of one or several is gcih carbohydrate groups and/or by adding one or more glycosylation sites, which are not present in the native polypeptide.

Glycosylation, as a rule, is either N-linked or O-linked. N-linked glycosylation refers to the attachment of a carbohydrate group to the side chain of an asparagine residue. Tripeptide sequence asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except Proline, are sequences that are recognized for enzymatic joining of carbohydrate groups to the side chain of asparagine. The presence of any of these Tripeptide sequences in the polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars, which represents the N-atsetilgalaktozamin, galactose, or xylose to hydroxynicotinate, usually serine or threonine, although you can also use 5-hydroxyproline or 5-hydroxylysine. The sites of O-linked glycosylation can be added by embedding or replacing one or more residues of serine or threonine in the sequence of the original polypeptide.

Switching domains

Different parts of the protein lysosomal enzymes sulfates possess significant homology sequences. In the polypeptides of lysosomal enzyme sulfatase can identificirovat the mutation, which change their function. These studies are potentially important at least for two reasons. First, they provide a reasonable expectation that in related species, such as rat, rabbit, monkey, Gibbon, chimp, the APE, baboon, cow, pig, horse and cat, there may be other homologues, allelic variants and mutants of this gene. When the allocation of these homologues, variants and mutants and in conjunction with other tests can identify some active or functional domains. Secondly, it can provide a starting point for further mutational analysis of the molecule, as described above. One of the ways in which you can use for this information is "switching domains.

Switching domains involves the preparation of recombinant molecules using different, but related polypeptides. For example, by comparing sequence lysosomal enzyme sulfatase, for example N-atsetilgalaktozamin-6-sulfatase, with a sequence similar lysosomal enzyme sulfatase from another source and mutants and allelic variants of these polypeptides can predict functionally important region of these molecules. Then you can switch related domains of these molecules to determine the importance of these is areas for enzyme function and effects of lysosomal diseases of accumulation. These molecules may have more value, because these "chimeras" may differ from the natural molecules, with the possible implementation of the same or even enhanced functions.

On the basis of many lysosomal enzymes sulfates identified at the present time, additional analysis of mutations and their predicted effect on the secondary structure will complement this understanding. Provided that the mutants, in which the switched domains between lysosomal enzymes by sulfatase, can provide suitable information about the relationships of the structures/functions of these molecules and polypeptides that interact with them.

Slit proteins

In addition to the mutations described above, the present invention also provides for a specialized variant type with insertion, known as protein. This molecule generally has all or a substantial part of natural molecules, linked at the N - or C-end with all the second polypeptide or part thereof. For example, when merging, generally used leader sequences from other species to ensure recombinant expression of a protein in a heterologous host. Other suitable fusion involves adding immunologically active domain, such as the epitope antibodies, to facilitate purification of fused protein. Included is their cleavage site at the confluence or near it can facilitate the removal of unnecessary polypeptide after purification. Other suitable merger include linking functional domains, such as active centers of enzymes, glycosylation domains, cell targeting signals or transmembrane region.

There are various commercially available system for expressing fused proteins, which can be used in the present invention. Especially suitable systems include, but are not limited to, a system of glutathione-S-transferase (GST) (Pharmacia, Piscataway, NJ), maltose binding protein (NEB, Beverley, MA), system FLAG (IBI, New Haven, CT), a 6×His (Qiagen, Chatsworth, CA). These systems are capable of producing recombinant polypeptides having only a small number of additional amino acids, which will probably not affect the antigenic capacity of the recombinant polypeptide. For example, systems like FLAG and 6×His add only short sequences, both of which are known to be poorly antigenic not have any adverse effect on the folding of the polypeptide in its native conformation. Other N-terminal fusion, which is considered suitable, is the merger of the dipeptide Met-Lys N-terminal region of the protein or peptides. Such a merger may provide a favorable increase the expression or activity of the protein.

Particularly suitable design for merging can be design in which the second polypeptide active vysokopostavlennogo lysosomal enzyme sulfatase or its fragment are merged with the hapten to enhance the immunogenicity of the slit design lysosomal enzyme sulfatase. This may be suitable for the production of antibodies to the active vysokodetalizirovannom the lysosomal enzyme sulfatase to ensure detection of the protein. In other embodiments, the implementation can be obtained fused design, which can enhance the targeting associated with the lysosomal enzyme sulfatase compositions in a specific area or cell.

Other fused designs, including metrologichna peptide with the desired properties, such as motive, can target the lysosomal enzyme sulfatase in a specific organ, tissue or cell type. In a preferred embodiment, the slit design, including target bone peptide, for example 6 aspartic acid residues (6×Asp or 6D), merged with the lysosomal enzyme sulfatase can target the enzyme to specific areas of the bone.

Also provided by other merged configuration including a heterologous polypeptide with the required properties, such as constant plot Ig for longer half-life in serum or antibody or its fragment to target. Other fused systems form a polypeptide hybrids where it is desirable cleavage partner to merge from the desired polypeptide. In one embodiment, the partner merger bound by the recombinant polypeptide active vysokofosforistoy the tion of lysosomal enzyme sulfatase using peptide sequence, contains the specific sequence for recognition by the protease. Examples of suitable sequences are sequence recognized by the protease of the virus engraving tobacco (Life Technologies, Gaithersburg, MD) or factor Xa (New England Biolabs, Beverley, MA).

Derivatives

As indicated above, the derivative refers to polypeptides chemically modified by such methods as, for example, without limitation, ubiquitinylation, tagging (e.g., with radionuclides or various enzymes), covalent joining of the polymer, such as tahilramani (transformation by polyethylene glycol) and the embedding or substitution by chemical synthesis of amino acids such as ornithine. Derived lysosomal enzyme sulfatase also suitable as pharmaceuticals, and can be obtained by methods according to the invention.

Polyethylene glycol (PEG) may be attached to the lysosomal enzyme sulfatase produced by methods according to the invention, to provide a longer half-life ofin vivo. The PEG group may have any suitable molecular weight and may be linear or branched. The average molecular weight of the PEG is preferably in the range from about 2 kilodaltons ("KD") to about 100 kDa, more preferably from about 5 kDa to arr is siteline 50 kDa, most preferably from about 5 kDa to about 10 kDa. Group PEG, mainly, can be associated with lysosomal enzymes by sulfatase according to the invention via acylation or reductive alkylation through a reactive group on the PEG group (e.g., aldehyde, amino, Tilney or ester group and a reactive group on the group of the protein (e.g., aldehyde, amino, Tilney or ester group). Adding groups of PEG to interest the polypeptides can be made using methods well known in the field. See, for example, international publication No. WO 96/11953 and U.S. patent No. 4179337.

Ligation of the polypeptide of lysosomal enzyme sulfatase with PEG is usually carried out in aqueous phase, and it can easily be monitored using a reversed-phase analytical HPLC. Pegylated peptides can be easily cleaned preparative HPLC and oharakterizovat analytical HPLC, amino acids analysis and mass spectrometry laser desorption.

Label

In some embodiments, implementation of therapeutic lysosomal enzyme sulfatase mark to facilitate its detection. "Label" or "amenable to detection group" is a composition amenable to detection by spectroscopic, photochemical,biochemical, immunochemical, chemical, or other physical methods. For example, labels that are suitable for use in the present invention include, but are not limited to, radioactive labels (e.g.,32P), fluorophores (such as fluorescein), electron-dense reagents, enzymes (as commonly used in an ELISA), Biotin, digoxigenin or haptens and proteins which can be made amenable to detection, for example through the incorporation of radioactive label in the hapten or peptide, or which can be used for detection of antibodies specific reactive to the hapten or peptide.

Examples of labels suitable for use in the present invention include, but are not limited to, fluorescent dyes (e.g., fluoresceinisothiocyanate, Texas red, rhodamine, and the like), radioactive labels (e.g.,3H,125I35S14C or32P), enzymes (such as horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold, colored glass or plastic pellets (e.g., polystyrene, polypropylene, latex etc).

A label can be attached directly or indirectly to the desired component of the lysosomal enzyme sulfatase in accordance with methods well known in the field. Site is preferably, in one embodiment, the label is covalently linked to the lysosomal enzyme sulfatase using reagent-based isocyanate to active conjugation means according to the invention. In one aspect of the invention, conjugation of the label with the lysosomal enzyme sulfatase you can use bifunctional reagents based on isocyanate according to the invention with the formation of a conjugate of a label and lysosomal enzyme sulfatase without active tool associated with it. The conjugate of a label and lysosomal enzyme sulfatase can be used as intermediate compounds for the synthesis of labeled conjugate according to the invention or it can be used for detection conjugate lysosomal enzyme sulfatase. As stated above, you can use a wide variety of labels, and the label selection depends on the required sensitivity, ease of conjugation with a required component of the lysosomal enzyme sulfatase, sustainability requirements, available equipment and software removal. Non-radioactive labels often associated indirect ways. Generally, a ligand molecule (e.g., Biotin) is covalently associated with the lysosomal enzyme sulfatase. Then the ligand bind to another molecule (e.g., streptavidin), which is either inherently amenable to detection, or covalent what about connected with alarm system such as amenable to the detection of an enzyme, a fluorescent compound or a chemiluminescent compound.

Lysosomal enzymes sulfatase according to the invention can also be konjugierte right from creating the signal connections, for example, by conjugation with an enzyme or fluorophore. Enzymes suitable for use as labels include, but are not limited to, hydrolases, in particular phosphatase, esterase and glycosidase or oxidase, particularly peroxidases. Fluorescent compounds, i.e. fluorophores suitable for use as labels include, but are not limited to, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone etc. the Following examples of suitable fluorophores include, but are not limited to, eosin, TRITC-amine, quinine, fluorescein W, acridine yellow, lissamine-rhodamine B, sulphonylchloride-erythrocin, ruthenium (Tris, bipyridine), Texas red, nicotinamide adenine dinucleotide, flavine adenine dinucleotide, etc. Chemiluminescent compounds suitable for use as labels include, but are not limited to, luciferin and 2,3-dihydropteridine, such as luminal. For a review of various systems for marking or formation of a signal that can be used in the methods of the present invention, see U.S. patent No. 4391904.

Means for detecti the label is well known to specialists in this field. Thus, for example, when the label is radioactive, means for detection include a scintillation counter or photographic film, for example, when autoradiography. When the label is a fluorescent label, its detection can be performed by excitation of fluorochrome the corresponding wavelength and detecting the resulting fluorescence. The fluorescence detection can be performed visually, with the use of electronic detectors such as charge-coupled devices (CCD) or photomultipliers and the like, Similarly, the detection of enzyme labels can be performed by providing appropriate substrates for the enzyme and detecting the resulting product of the reaction. Detection colorimetric or chemiluminescent labels can be performed simply by observing the color associated with the label. Other systems for labeling and detection, suitable for use in the methods of the present invention, will be apparent to experts in this field. Such labeled modulators and ligands can be used in diagnostics of the diseases or health.

In a preferred embodiment, the method includes a step of production of active vysokotsentralizovannym lysosomal enzymes sulfates of cell lines with defects in endosomal transport. In a particularly preferred variants of the e implementation the method includes a step of production of active vysokovostrebovannye recombinant N-atsetilgalaktozamin-6-sulfatase (GALNS) person from the cell line CHO G71 or its derivative. Production of lysosomal enzymes sulfates, such as, for example, without limitation, GALNS, involves the following stages: (a) development derived cell lines or G71 G71, which coexpressed recombinant lysosomal enzyme sulfatase person, for example N-atsetilgalaktozamin-6-sulfatase (GALNS), and recombinant modifying sulfatase factor 1 (SUMF1) person; (b) culturing cell lines, coexpression lysosomal enzyme sulfatase and SUMF1 person; and (c) zoom in cell lines, coexpression lysosomal enzyme sulfatase person and SUMF1 to a bioreactor for the production of lysosomal enzymes sulfates. In preferred embodiments, implementation, cDNA lysosomal enzyme sulfatase person, for example N-atsetilgalaktozamin-6-sulfatase (GALNS) and SUMF1 person, subcloning in expressing vectors mammals, essentially as described herein below.

To develop a cell line G71 or G71S, clone G71 adapted for growth in serum-free suspension culture, were cotranslational vector mammals expressing human GALNS, vector mammals, expre siraudin SUMF1 person, gene and the selective marker and selected stable transformants. After the first round of sublimirovanny stable transfectants, cell lines were subjected to selection using the fluorescent substrate and in a certain way meant. Cell line G71 or G71S analyzed in relation to the cell-specific productivity (PG product/cage) in torque devices Mironosetsky or in suspension culture, respectively. The best producers of GALNS person identified and has scaled up to a bioreactor for the production of pre-clinical material.

In another embodiment, the invention relates to cell analysis for measuring the activity of recombinant lysosomal enzyme for degradation of natural substrates. The method comprises (a) culturing the selected cell lines of human, deficient in the lysosomal enzyme, under conditions which accumulate natural substrates for lysosomal enzyme; (b) contacting the cells with lysosomal enzyme; (c) lysis of the cells; (d) adding to the cell lysate enzyme, which (i) is specific for the natural substrates, (ii) it is a small oligosaccharides from natural substrates; (e) the tagging of small oligosaccharides with amenable to detection by the group; (f) optionally separating uchenykh small oligosaccharides; (g) detection of labeled small oligosaccharides; (h) determining the activity of the lysosomal enzyme in the degradation of natural substrates by comparing (i) the number of labeled small oligosaccharide from cells that had been subjected to contact with lysosomal enzyme with (ii) the number of labeled small oligosaccharides from cells that were not subjected to contact with lysosomal enzyme, where the decrease (h)(i) compared to (h)(ii) indicates the activity of the lysosomal enzyme in the degradation of natural substrates. In one embodiment, a small oligosaccharide is a mono-, di - or trisaccharide. In related embodiments, implementation, small oligosaccharide is a disaccharide.

In some embodiments, the implementation, the lysosomal enzyme is selected from the group consisting of Ukrainian B (ARSB), iduronate-2-sulfatase (IDS), sulfamidate/heparin-N-sulfatase (SGSH), N-acetylglucosaminidase (G6S) and N-atsetilgalaktozamin-6-sulfatase (GALNS). In some embodiments, implementation, lysosomal enzyme is an α-L-iduronidase (IDU). In some embodiments, implementation, lysosomal enzyme is an acid α-glucosidase (GAA). In some embodiments, the implementation, the lysosomal enzyme is a β-glucuronidase (GUSB). In nektarinukiniai implementation lysosomal enzyme is a β-galactosidase (GLB1).

Suitable human cells that can be used in cellular analysis, include any cell that is deficient in the lysosomal enzyme subjected to testing so that it could accumulate natural substrates for lysosomal enzyme. For example, you can use the cells in nature with full (100%) or partial deficiency of activity, for example 30%, 50%, 70%, 80%, 90%, 95% decreased activity or more. You can use cells expressing mutant enzyme with reduced activity, or cells derived from patients suffering from lysosomal disease accumulation, such as mucopolysaccharidosis. You can use cells, recombinante modified for knockout or reduce the activity of the lysosomal enzyme, for example, by introducing mutations in the encoding gene or its promoter or other regulatory region. You can use cells, treated to reduce the activity of the lysosomal enzyme, such as processed antisense RNA or RNAi to reduce the expression of the enzyme.

Suitable enzymes that cut (otscheplaut) small oligosaccharides from carbohydrates and which are "specific" (i.e. mainly split) natural substrates lysosomal enzyme is, can choose the experts in this field. For example, for detecting activity GALNS or GLB1 (enzymes that carry out the degradation createsurface) enzyme stage (d) can represent keratinase II or any enzyme that acts mainly on keratinolytic. As another example, for detection IDU, ARSB, IDS or GUSB (enzymes that carry out the degradation dermatosurgery), the enzyme stage (d) can be chondroitinase ABC or any enzyme that acts mainly on dermatologit. As another example, for detection IDU, IDS, SGHS, G6S or GUSB (enzymes that carry out the degradation of heparan sulfate), an enzyme stage (d) can represent heparinase I, or heparinase II, or both of them. As another example, for the detection of GAA (the enzyme that carries out the degradation of glycogen), the enzyme stage (d) may consist of α-amylase or any enzyme that acts primarily on glycogen.

This cell may have a high sensitivity in terms of detection of the activity of the lysosomal enzyme. In some embodiments, implementation, activity of lysosomal enzyme amenable to detection, when the concentration of lysosomal enzyme is only about 10 nm, or about 5 nm, or about 1 n is, or approximately 0,75 nm, or about 0.5 nm, or approximately 0.25 nm, or approximately 0.1 nm, or about 0.05 nm, or about 0.01 nm, or approximately 0,005 nm, or about 1 PM, or about 0.5 PM.

III PURIFICATION of LYSOSOMAL ENZYMES SULFATES

Material bioreactor containing recombinant human GALNS, subjected to sterile filtration through the 0.2-μm filter and kept at 4°C. the Material of the bioreactor or put in a capture column directly, or concentrated from 10 to 20-fold by ultrafiltration before applying in a capture column. The material of the bioreactor or concentrated material bioreactor pH was brought to pH 4.5, and then put it on a column of Blue-Sepharose, washed successively 20 mm with a mixture of acetate/phosphate, 50 mm NaCl, pH 4.5, and 20 mm with a mixture of acetate/phosphate, 50 mm NaCl, pH to 6.0, and suirable 20 mm with a mixture of acetate/phosphate, 100 mm NaCl, pH 7.0. Then the eluate of the column Blue-Sepharose was applied to a Fractogel SE Hi-Cap, washed successively 20 mm with a mixture of acetate/phosphate, 50 mm NaCl, pH 5.0, and 20 mm with a mixture of acetate/phosphate, 50 mm NaCl, pH 5.5, and suirable 20 mm with a mixture of acetate/phosphate, 50-350 mm gradient of NaCl, pH 5.5. The eluate Fractogel SE Hi-Cap were made in 10 mm NaOAc, 1 mm NaH2PO4, of 0.005% Tween-80, pH 5.5.

Alternatively, the material of the bioreactor containing recombinant human GALNS, concentrated 20-fold by ultrafiltration before NAS is senjem in a capture column. In the concentrated material bioreactor pH was brought to pH 4.5, filtered, and then applied on a column of Fractogel SE Hi-Cap, washed sequentially with 10 mm mixture of acetate/phosphate, 50 mm NaCl, pH 4.5 and 10 mm mixture of acetate/phosphate, 50 mm NaCl, pH 5.0, and suirable 10 mm mixture of acetate/phosphate, 140 mm NaCl, pH 5.0. Then the eluate column of Fractogel SE Hi-Cap had brought, 500 mm NaCl, pH 7.0, and applied to a column of Zn-chelating Sepharose (Zn-IMAC), washed with 10 mm mixture of acetate/phosphate, 125 mm NaCl, 10 mm imidazole, pH 7.0, and was suirable 10 mm mixture of acetate/phosphate, 125 mm NaCl, 90 mm imidazole, pH 7.0. The eluate of the column with Zn-chelating Sepharose (Zn-IMAC) was brought to pH 3.5 for inactivation of the virus to low pH was brought to 10 mm mixture of acetate/phosphate, 2M NaCl, pH 5.0, and then put in a column of ToyoPearl Butyl 650M, washed with 10 mm mixture of acetate/phosphate, 2M NaCl, pH 5.0, and suirable 10 mm mixture of acetate/phosphate, and 0.7 M NaCl, pH 5.0. The eluate ToyoPearl Butyl 650M subjected ultrafiltration and diafiltration in 20 mm acetate, 1 mm phosphate, 150 mm NaCl, pH 5.5, and then prepared in 20 mm acetate, 1 mm phosphate, 150 mm NaCl, 0.01% of Tween-20, pH 5.5.

Purification of recombinant human GALNS described in detail below, and purification of recombinant human GALNS according to methods modified from the above Protocol, described in detail below.

Recombinant GALNS enzyme human expressed in cells G71S, as described in example III, and was purified as described in example V. the Purified recombinant GALS man according to the invention can be compared with other described drugs GALNS. In Masue et al., J. Biochem. 110:965-970, 1991, describes the purification and karakterizacija GALNS from human placenta. It was found that the purified enzyme has a molecular mass of 120 kDa and consists of the polypeptide with a mass of 40 kDa and 15 kDa, the latter of which, as shown, is a glycoprotein. Thus, Masue et al. the GALNS enzyme, appears to be consistent with protestirovanny form presented on figure 5. In Bielicki et al., Biochem. J. 279:515-520, 1991, describes the purification and karakterizacija GALNS from human liver. Upon analysis by SDS-PAGE, enzyme had a molecular mass of 70 kDa in non conditions and the molecular weight of 57 kDa, 39 kDa and 19 kDa in reducing conditions. In Bielicki et al., Biochem j 311: 333-339, 1995, describes the purification and chracterization recombinant human GALNS from ovary cells Chinese hamster. When SDS-PAGE revealed that the purified enzyme has a molecular mass of 58-60 kDa in non conditions and molecular weight 55-57 kDa, 39 kDa and 38 kDa in reducing conditions. Thus, Bielicki et al. the GALNS enzyme, apparently, correspond to a mixture of repressirovannoy form (predecessor) enzyme and protestirovanny forms presented on figure 5. In contrast, recombinant GALNS enzyme of man according to the invention consists almost entirely from the form of the precursor enzyme (see figure 9), or predominantly (i.e. at the ore approximately 85%) from the form of the precursor enzyme (see figure 10).

IV LYSOSOMAL ENZYMES SULFATASE AND LYSOSOMAL storage disorders

Lysosomal enzyme sulfatase is a full sized enzyme or fragment, mutant, variant or derivative, which retain at least at a high level (for example, at least about 50%, preferably at least about 75% and more preferably at least about 90%), essentially all, or all therapeutic or biological activity (e.g., sulfatase activity) of the enzyme.

In some embodiments, implementation, lysosomal enzyme sulfatase is an enzyme that, if it is not expressed or is not produced or if its expression or products being reduced, can lead to disease, including, but not limited to, lysosomal storage disorders. In some embodiments, implementation, lysosomal enzyme sulfatase is an enzyme that, if it is not expressed or is not produced or if its expression or production is essentially reduced, may not cause disease, but the absence or reduced expression or the production of which is associated with a disease, including, but not limited to, lysosomal storage disorders. Predpochtite is) lysosomal enzyme sulfatase occurs or obtained from a person.

Preferably, in the treatment of lysosomal diseases accumulation of lysosomal enzyme sulfatase is an enzyme found in the cell and which, if it is not expressed or is not produced or if its expression or products being reduced, can lead to lysosomal storage disorders. Alternative in the treatment of lysosomal diseases accumulation of lysosomal enzyme sulfatase is an enzyme, the absence or a substantial reduction in the expression or production of which is associated with the disease, although the absence of or essentially reduced expression or the products themselves may not lead to disease. Preferably, the lysosomal enzyme sulfatase occurs or obtained from a person.

Preferably, the enzyme is a lysosomal enzyme sulfatase, such as arylsulfatase A (ARSA) (registration number Genbank No. NPJ300478 (isoform a), registration number Genbank No. NP_001078897 (isoform b) and other variants), arylsulfatase B/N-acetylglucosamine-4-sulfatase (ARSB) (registration number Genbank No. P15848), iduronate-2-sulfatase (IDS) (registration number Genbank No. NP_000193 (isoform a), registration number Genbank No. NP_006114 (isoform b)), sulfamidate/heparin-N-sulfatase (SGSH) registration number Genbank No. NP_000190), N-acetylglucosamine-sulfatase (G6S) (registration number Genbank No. NP_002067), galactose-6-sulfatase/N-atsetilgalaktozamin-6-sulfatase (GALNS) (registration number Genbank No. NP_000503). The table below lysosomal diseases of accumulation and lysosomal enzymes sulfates, deficient in them, which are suitable as drugs:

Lysosomal disease accumulationDeficiency of lysosomal sulfatase
Mucopolysaccharidosis type II, hunter's syndromeIduronate-2-sulfatase
Mucopolysaccharidosis type IIIA, syndrome, Sanfilippo's titleSulfamidate/heparin-N-sulfatase
Mucopolysaccharidosis type IIID syndrome Sanfilippo's titleN-acetylglucosamine-6-sulfatase
Mucopolysaccharidosis type IVA syndrome MorquioN-acetylglucosamine-6-sulfatase
Mucopolysaccharidosis type VIN-acetylglucosamine-4-sulfatase
Metachromatic leukodystrophy (MLD)Arylsulfatase A
Multiple sulfatase failureSeveral sulfates

In preferred embodiments, implementation, lysosomal enzyme sulfatase is a recombinant lysosomal enzyme sulfatase man produced deficient endosomal acidification cell line. In more preferred embodiments, implementation, recombinant lysosomal enzyme sulfatase person is active and has a high level of phosphorylated oligosaccharides, as described in the section "DEFINITIONS". In the most preferred options for implementation, lysosomal enzyme sulfatase is an active vysokovostrebovannye recombinant N-atsetilgalaktozamin-6-sulfatase (GALNS) person.

Thus, lysosomal storage disorders that can be treated or prevented using the methods of the present invention, include, but are not limited to, metachronous the leukodystrophy, or MLD, the syndrome Maroto-Lamy, or MPS VI, hunter's syndrome, or MPS II syndrome Sanfilippo's title A, or MPS IIIa syndrome Sanfilippo's title D, or MPS IIId, and the syndrome Morquio A, or MPS IVa. In a particularly preferred embodiment, the lysosomal enzyme sulfatase is such that its deficiency causes the syndrome Morquio A, or MPS IVa. Another person is but a preferred embodiment, lysosomal enzyme sulfatase is such that its deficiency is associated with lysosomal disease accumulation of man, such as multiple sulfatase failure or MSD.

Thus, according to the above table, for each disease lysosomal enzyme sulfatase preferably includes specific active lysosomal enzyme sulfatase, deficient disease. For example, for methods involving MPS II, the preferred enzyme is iduronate-2-sulfatase. For methods involving MPS IIIA, the preferred enzyme is sulfamidate/heparin-N-sulfatase. For methods involving MPS IIID, the preferred enzyme is N-acetylglucosamine-6-sulfatase. For methods involving MPS IVA, the preferred enzyme is galactose-6-sulfatase/N-atsetilgalaktozamin-6-sulfatase. For methods involving MPS VI, the preferred enzyme is N-atsetilgalaktozamin-4-sulfatase. For methods involving metachromatism the leukodystrophy (MLD), the preferred enzyme is arylsulfatase A. For methods involving multiple sulfatase deficiency (MSD), the enzyme may represent arylsulfatase arylsulfatase B/N-acetylglucosamine-4-sulfatase, iduronate-2-sulfatase, sulfamidate/heparin-N-sulfatase, N-acetyl is cosamin-sulfatase or galactose-6-sulfatase/N-atsetilgalaktozamin-6-sulfatase, and the preferred enzyme is galactose-6-sulfatase/N-atsetilgalaktozamin-6-sulfatase.

V MUCOPOLYSACCHARIDOSIS TYPE IVA (SYNDROME MORQUIO, MPS IVA)

Mucopolysaccharidosis type IVA (syndrome Morquio, MPS IVa) is a congenital autosomal recessive disorder belonging to the group of diseases is the accumulation of mucopolysaccharides. The syndrome Morquio is caused by deficiency of lysosomal enzyme required for the degradation of two glycosaminoglycans (GAG): createsurface (KS) and chondroitin-6-sulfate (C6S). Specifically, MPS IVa charaterizes the lack of the enzyme N-atsetilgalaktozamin-6-sulfatase (GALNS) and KS excretion in the urine. The lack of GALNS leads to abnormally large amounts of mucopolysaccharides in hyaline cartilage, the main component of skeletal tissue. All patients have a systemic skeletal dysplasia. Other symptoms vary in severity from patient to patient and may include hearing loss, cataracts, spinal instability, disease of the heart valves and respiratory problems, among others.

GALNS hydrolyzes sulfate ester bonds in galactose-6-sulfate from KS and N-atsetilgalaktozamin-6-sulfate from C6S. GALNS person is expressed as a protein precursor mass of 55-60 kDa with only 2 potential associated with asparagine glycosylation sites. Mannose-6-phosphate (M6P) is frequent the Yu oligosaccharides, present on the molecule GALNS. M6P is recognized by a receptor on the surface of lysosomal cells and, therefore, is important for effective capture of GALNS.

Like all sulfatases, GALNS should processionals formylglycine-activating enzyme (FGE), encoded by the genome modifier to sulfatase factor 1 (SUMF1), to increase activity. Due to this stage of activation, involving post-translational modification of cysteine residue of the active site in the Cα-formylglycine (FGly), overexpression of recombinant sulfates can lead to the production of the enzymes sulfates as low specific activity (i.e. a mixture of activated and unactivated sulfatase enzymes), and low-titer production (i.e. degradation and/or lack of secretion of the unactivated sulfates).

The task of this invention is the provision of active vysokopostavlennogo enzyme N-atsetilgalaktozamin-6-sulfatase person suitable for the treatment of syndrome Morquio and other diseases, for example multiple sulfatase deficiency (MSD), which are caused by deficiency of the enzyme N-atsetilgalaktozamin-6-sulfatase or associated with. Such active vysokovostrebovannye the enzyme N-atsetilgalaktozamin-6-sulfatase person has the ability to be localized in the tissues in which building up the scarfing KS and C6S, has sufficient levels of M6P to effectively capture, has a fairly high percentage of FGly for activity of the enzyme and has relatively high levels of production.

It should be understood that the methods according to the invention, described herein, are applicable to products of other lysosomal enzymes sulfates, for example Ukrainian A (ARSA), Ukrainian (B/N-acetylglucosamine-4-sulfatase (ARSB), iduronate-2-sulfatase (IDS), sulfamidate/heparin-N-sulfatase (SGSH) and N-acetylglucosamine-sulfatase (G6S), suitable for the treatment of lysosomal diseases of accumulation, which is caused or characterized by a deficit.

VI PHARMACEUTICAL COMPOSITION AND INTRODUCTION

Lysosomal enzymes sulfatase according to the invention can be administered in a number of ways. For oral preparations, lysosomal enzymes sulfatase can be used alone or in combination with appropriate additives for the manufacture of tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, gum Arabic, corn starch or gelatin; (C dezinfeciruyuhimi substances such as corn starch, potato starch or carboximetilzellulozu is sodium; with lubricating agents such as talc or magnesium stearate; and if desired, with diluents, buffering means, moisturizing agents, preservatives and flavors.

Lysosomal enzymes sulfatase according to the invention can be produced in the form of preparations for injections by dissolving, suspension or emulsification them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acids glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives, such as solubilization providing isotonicity substances suspendresume substances, emulsifiers, stabilizers and preservatives.

Lysosomal enzymes sulfatase according to the invention can be used in aerosol composition for administration by inhalation. Lysosomal enzymes sulfatase according to the invention can be produced in an acceptable propellants under pressure, such as DICHLORODIFLUOROMETHANE, propane, nitrogen, etc.

Moreover, lysosomal enzymes sulfatase according to the invention can be produced in the form of suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. Lysosomal enzymes sulfatase according to the invention it is possible to enter rectal use soup is ository. The suppository can include carriers such as cocoa butter, carbowax and glycols, which melt at body temperature and are solid at room temperature.

Can be provided a single dosage forms of lysosomal enzymes sulfates according to the invention for oral or rectal administration, such as syrups, elixirs and suspensions, where each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a given number of lysosomal enzyme sulfatase containing the active substance. Similarly, unit dosage forms for injection or intravenous administration may contain lysosomal enzyme sulfatase as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.

In practice, lysosomal enzymes sulfatase according to the invention can be combined as the active ingredient in a homogeneous mixture with one or more pharmaceutically acceptable carriers, diluents or excipients according to the conventional manufacturing methods. The carrier, diluent or excipient can take various forms, depending on the preferred form of the desired drug for administration, for example oral, or parenteral (in the including intravenous) form. Upon receipt of the compositions of lysosomal enzyme sulfatase for oral dosage forms, you can use any of the usual pharmaceutical media, such as water, glycols, oils, alcohols, flavoring, preservatives, dyes, etc. in the case of oral liquid preparations such as suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating tools, lubricants, binders, dezintegriruetsja substances, etc. in the case of oral solid preparations such as powders, hard and soft capsules and tablets, with the solid oral drugs are preferred relative to liquid preparations.

In relation to transdermal routes of administration, methods of transdermal administration of drugs described in Remington's Pharmaceutical Sciences, 17th Edition (Gennaro et al., Eds. Mack Publishing Co., 1985). Dermal or skin patches are the preferred means for transdermal delivery of lysosomal enzymes sulfates according to the invention. The patches are preferably provided power suction, such as DMSO, to improve the absorption of lysosomal enzymes sulfates. Other methods of transdermal drug delivery is described in U.S. patent No. 5962012, 6261595 and 6261595, all of which is clucene in this document as a reference in full.

Pharmaceutically acceptable excipients, such as carriers, adjuvants, carriers, or diluents, are commercially available. Moreover, are also commercially available pharmaceutically acceptable excipients, such as adjustment of pH and buffer means, corrective toychest tools, stabilizers, wetting agents, etc.

In each of these aspects, the compositions of lysosomal enzymes sulfates include, but are not limited to, compositions suitable for oral, rectal, local, parenteral (including subcutaneous, intramuscular and intravenous), pulmonary (nasal or buccal inhalation), or nasal introduction, although the most suitable way in any given case depends partly on the nature and severity of the conditions being treated and on the nature of the active ingredient. Illustrative ways of administration are oral and intravenous route. Composition of lysosomal enzyme sulfatase may conveniently be presented in unit dosage form and received by any of the methods well known in the pharmaceutical field.

Due to ease of administration, tablets and capsules represent the most advantageous oral unit dosage form, in which case typically use solid is farmacevticheskie media. If desired, tablets may be coated standard aqueous or nonaqueous techniques. The percentage of active lysosomal enzyme sulfatase in these compositions may, of course, vary, and conveniently, so that he was between approximately 2% and approximately 60 percent of the weight of the unit.

Composition of lysosomal enzyme sulfatase according to the invention it is possible to enter encapsulated in viral membranes or vesicles, or attached thereto or incorporated into the cells. Vesicles are micellar particles that are generally spherical and which are often lipid. Liposomes are vesicles formed from a bilayer membranes. Suitable vesicles include, but are not limited to, single-layer vesicles and multilamellar lipid vesicles or liposomes. Such vesicles and liposomes can be made from a wide range of lipid or phospholipid compounds such as phosphatidylcholine, phosphatidic acid, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, glycolipids, ganglioside etc. using standard methods, such as described, for example, in U.S. patent No. 4394448. Such vesicles or liposomes can be used for the introduction of lysosomal enzymes sulfates and intracellular delivery of lysosomal enzymes sulf the pelvis in target organs. Using encapsulation, you can achieve a controlled release of interest lysosomal enzyme sulfatase (see, for example, U.S. patent No. 5186941).

You can use any method of administration, which residetial composition of lysosomal enzyme sulfatase in the bloodstream or preferably at least outside the blood-brain barrier. Preferably the composition of the lysosomal enzyme sulfatase injected at the periphery, most preferably intravenously or through a cardiac catheter. Also suitable vnutrennie and vnutrikvartalnye injection. Composition of lysosomal enzyme sulfatase you can enter local or regional, for example intraperitoneally, subcutaneously or intramuscularly. In one aspect the composition of lysosomal enzyme sulfatase injected with one or more pharmaceutically acceptable carriers, diluents or excipients.

Specialists in this field will appreciate that dose levels can vary depending on the specific lysosomal enzyme sulfatase, symptom severity and sensitivity of the subject to side effects. Preferred dosages for a given lysosomal enzyme sulfatase can easily identify experts in this field in various ways, including, but not limited to the United as them assessment of response to dose and pharmacokinetic evaluation of patients in the tested animals andin vitro.

Dosages that need to be instituted, may also depend on individual needs, from the desired effect, from specific lysosomal enzyme sulfatase and the selected route of administration. Dosage lysosomal enzyme sulfatase range from about 0.2 pmol/kg to about 20 nmol/kg, preferred dosages range from 2 pmol/kg to 2 nmol/kg, and particularly preferred dosage range from 2 pmol/kg to 200 pmol/kg Alternative dosage lysosomal enzyme sulfatase can be in the range from 0.01 to 1000 mg/kg, preferred dosages may be in the range of from 0.1 to 100 mg/kg, and particularly preferred dosage range from 0.1 to 10 mg/kg At these dosages may be affected, for example, not limited to their specific lysosomal enzyme sulfatase, the form of pharmaceutical compositions, method of administration, and the scope of a specific lysosomal enzyme sulfatase.

Lysosomal enzymes sulfatase according to the invention are suitable for therapeutic and diagnostic interventions in animals, particularly in humans. Lysosomal enzymes sulfatase can demonstrate the preferred accumulation in spiral is different tissues. Preferred medical indications for diagnostic applications include, for example, any condition associated with interest target organ (e.g. lung, liver, kidney, spleen).

The considered methods are applicable for the treatment of many different illnesses. In certain embodiments of the implementation of special interest is the application of techniques discussed in painful conditions, under which was previously identified lysosomal enzyme sulfatase having the desired activity, but in which the lysosomal enzyme sulfatase not delivered properly in the plot-target area target or compartment target to ensure a fully satisfactory therapeutic result. In the case of such lysosomal enzymes sulfates, the methods of production of active vysokotsentralizovannym lysosomal enzymes sulfates can be used to improve therapeutic efficacy and therapeutic index of lysosomal enzyme sulfatase.

Imply that treatment covers any favorable outcome for a subject, associated with the introduction of the lysosomal enzyme sulfatase, including reducing the likelihood of disease, prevention of disease, slowing, remains the internals or reversal of disease progression or alleviation of symptoms, associated with a disease state that occurs in the host, where the terms "mitigation" or "benefit" is used in a broad sense to refer to at least reduce the value of the parameter, e.g. symptom, associated with the pathological condition being treated, such as inflammation and its associated pain. Essentially, the treatment also includes cases where the pathological condition, or at least symptoms associated with it, are completely inhibited, for example, are prevented or stopped, e.g. terminated, so that the host no longer suffers from a pathological condition, or at least does not suffer more from symptoms that characterize the pathological condition.

Many owners or entities can be treated under consideration ways. Typically, these masters are "mammals" or "mammalian," where these terms are widely used to describe organisms that belong to the class of mammals, including groups of predators (e.g. cats and dogs), rodents (such as mice, Guinea pigs and rats), and primates (e.g., humans, chimpanzees, and monkeys). In many variants of implementation, the master is the man.

After describing, in General, the invention will be described more clearly with the help of the following examples, in which PR is delivered illustrative protocols for the production and purification of active vysokotsentralizovannym lysosomal enzymes sulfates and their use for the treatment of lysosomal diseases of accumulation. Examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy in terms of numbers (e.g., amounts, temperatures, etc), but of course, you should avoid some experimental error and deviation.

EXAMPLES

EXAMPLE I

EXPRESSING the VECTORS MAMMALS FOR MODIFYING SULFATASE FACTOR 1 (SUMF1) PERSON AND N-ATSETILGALAKTOZAMIN-6-SULFATASE (GALNS) PERSON

The task was to construct expressing vectors mammals, suitable for production in stably transfected cells active lysosomal enzyme sulfatase in sufficient quantities to elevated levels of phosphorylation.

cDNA full-modifying sulfatase factor 1 (SUMF1) of the person (see patent application U.S. No. US 20005/0123949, with a publication date June 9, 2005, and US 2004/0229250, with a publication date November 8, 2004, both of which are incorporated herein as reference in full), which encodes the polypeptide of 374 amino acids, was cloned in expressing vector mammals cDNA4 (Invitrogen, Carlsbad, CA), which contains the enhancer-promoter, CMV person and polylinker. Efficient termination of transcription was provided by the presence of a sequence is eljnosti polyadenylation bovine growth hormone. Selective marker was a gene for resistance to zeocin under the control of the promoter EM-7 and the early sequence of the SV40 polyadenylation. The obtained plasmid was designated as pcDNA4 SUMF1. Polynucleotide (SEQ ID NO:1) and polypeptide (SEQ ID NO:2) sequences of human SUMF1 presented on figure 1 and figure 2, respectively.

cDNA full-sized N-atsetilgalaktozamin-6-sulfatase (GALNS) of the person (see Tomatsu et al., Biochem. Biophys. Res. Commun. 181(2):677-683, 1991), which encodes the polypeptide of 522 amino acids, including a signal peptide of 26 amino acids, was cloned in expressing vector mammals pCIN (BioMarin), which contains the enhancer-promoter, CMV person associated with the intron of the β-globin IVS2 rabbit, and polylinker. Efficient termination of transcription was provided by the presence of a polyadenylation sequence of bovine growth hormone. Selective marker represented gene phosphotransferase neomycin, which carries a point mutation that reduces the efficiency of the enzyme. Weakened marker further weakened weak promoter, HSV-tk. The obtained plasmid was designated as pCIN 4A. Polynucleotide (SEQ ID NO:3) and polypeptide (SEQ ID NO:4) sequences of human GALNS presented on figure 3 and figure 4, respectively.

To increase levels of expression of SUMF1 and GALNS, the elements of the frame/field attachment to the matrix (MAR) (see Mermd et al., U.S. patent No. 7129062) cloned in expressing plasmids for SUMF1 and GALNS.

BMAR SUMF1 was obtained by splitting P<1_68 X_X NcoI filled MAR (Selexis) via BamHI and HincII, and then embedding the released fragment MAR pcDNA4 SUMF1, split BgIII and NruI.

PMAR SUMF1 was obtained by splitting P<1_68 NcoI filled (MAR) EGFP SV40 (Selexis) via HindIII and XbaI to remove the EGFP gene, and then embed SUMF1 gene, which was freed from pcDNA4 SUMF1 by splitting HindIII and XbaI.

BMAR 4A was obtained by splitting BMAR SUMF1 by PmeI and SpeI to remove the SUMF1 gene, and then embed GALNS gene, which was freed from pCIN 4A by splitting PmeI and SpeI.

PMAR 4A was obtained by splitting P<1_68 NcoI filled (MAR) EGFP SV40 (Selexis) via HindIII and XbaI to remove the EGFP gene, and then embed GALNS gene, which was freed from pCIN 4A splitting HindIII and XbaI.

Full-GALNS cDNA person also cloned in expressing vector mammals pcDNA4 (Invitrogen, Carlsbad, CA). pcDNA4 SUMF1 were digested by HindIII and XbaI to remove cDNA SUMF1 and pCIN 4A were digested HindIII and XbaI to highlight GALNS cDNA. The cDNA fragment GALNS HindIII/XbaT ligated into the vector fragment pcDNA4 HindIII/XbaI. The obtained plasmid was designated pcDNA4-4A.

The integrity of the GALNS gene in expressing vectors pCIN 4A, BMAR and pcDNA4-4A was confirmed by restriction mapping using enzymes obtained from New England Biolabs. Expressing the vector PMAR 4A is not mapped.

the Structure completely protestirovanny form N-atsetilgalaktozamin-6-sulfatase (GALNS) of a person are shown on figure 5. GALNS is expressed as a polypeptide of 522 amino acid sequence of the signal peptide of 26 amino acids. Polypeptide GALNS of 496 amino acids secreted as a pre-protestirovanny form (predecessor) enzyme having a molecular weight of approximately 55-60 kDa. In the active GALNS, the cysteine residue at position 53 of the predecessor or fully processioning polypeptide GALNS (corresponding to position 79 full-length polypeptide GALNS) turned into Cα-formylglycine (FGly) through modifying sulfatase factor 1 (SUMF1). In lysosome GALNS is cleaved after provisions 325 fully processioning polypeptide GALNS, forming peptide fragments GALNS weight of approximately 40 kDa and 19 kDa. These peptides GALNS are linked by a disulfide bridge between cysteine residues (C) at positions 282 and 393 completely processioning polypeptide GALNS. There are two canonical site of N-linked glycosylation at positions 178 and 397 completely processioning polypeptide GALNS. Bis-phosphorylated mannose 7 (BPM7)containing residues 2 mannose-6-phosphate was detected at N178, but not on N397.

EXAMPLE II

CELL LINES G71S, COEXPRESSION MODIFYING SULFATASE HUMAN FACTOR 1 (SUMF1) AND N-ATSETILGALAKTOZAMIN-6-SULFATASE (GALNS) PERSON

The challenge was to develop a cell if the s, capable of producing active lysosomal enzymes sulfatase with elevated levels of phosphorylation.

Cells G71 (Rockford K. Draper) were obtained directly from CHO-K1 (ATCC CCL-61). Cell line G71 is a temperature-sensitive mutant of CHO-K1 in relation to acidification of the endosome, which, as shown, carries out different General secretion protein and phosphorylation on residues mannose several enzymes at elevated temperatures (Park et al., Somat. Cell Mol. Genet. 17(2): 137-150, 1991; Marnell et al., J. Cell. Biol. 99(6): 1907-1916, 1984).

The G71 cells were maintained at 34°C in the environment BioWhittaker UltraCHO, supplemented with 2.5% fetal calf serum, 2 mm glutamine, gentamicin, and amphotericin.

For easier application of cell lines for production of the protein is attached G71 cells pre-adapted to serum-free medium for growth using the Protocol for the adaptation-dependent attachment-dependent serum cell lines mammals to high-density serum-free suspension culture (Sinacore et al., Mol. Biotechnol. 15(3):249-257, 2000), getting adapted to serum-free suspension culture cell line G71S. Alternative attached G71 cells after stable transfection, as described below, can be adapted to serum-free medium for growth, as described in Sinacore e al.

Paired combinations expressing vectors for human SUMF1 and human GALNS (example I): or pcDNA4 SUMF1 together with pCIN4 4A or BMAR SUMF1 together with BMAR 4A or PMAR SUMF1 together PMAR 4A, transfusional in accordance with the Protocol MARtech II, as described Selexis, cells G71S growing in culture medium supplemented with antibiotic-antimycotic solution (100 IU penicillin, 10 mg streptomycin, and 25 μg amphotericin B, Cellgro). Pools of transfectants were grown in UltraCHO medium (Cambrex), supplemented with 5% γ-irradiated fetal calf serum (FBS, JRH), 200 μg/ml G418 (AG Scientific) and 200 μg/ml of zeocin (Invitrogen), and cloned by the method of limiting dilutions in 96-well tablets in the same environment for growth. Monitoring of the growth of clones was performed by visualization of Cell Screen (Innovatis). All clones were subjected to screening using ELISA with activity capture enzyme active GALNS (see example IV). Cell productivity was calculated by dividing the GALNS activity in ELISA with activity capture enzyme on the cell growth (Vi-Cell, Beckman Coulter) per day for 4 days.

Received 202 clone G71S, and they were subjected to screening for active GALNS: 86 clones, cotransfection pcDNA4 SUMF1 together with pCIN 4A, 65 clones, cotransfection BMAR SUMF1 together with BMAR 4A, and 51 clones, cotransfection PMAR SUMF1 together PMAR 4A. First, the clones were subjected to selection on the basis of high levels of GALNS 96-l the night of tablets for the cultivation of tissues (figure 6A). The GALNS activity was measured using ELISA with activity capture enzyme and presented in ng/ml (y-axis). On the x-axis presents three conditions cotransfection used for the expression of SUMF1 and GALNS: the hCMV promoter without the MAR, the hCMV promoter with MAR and the SV40 promoter with MAR. Each column corresponds to a separate clone of the target population. In this screening of clones from 96-hole did not account for cell density, and this figure is presented not all of cotransfection clones G71S.

The most active producing GALNS clones G71S chose to analyze productivity (figure 6B). Cell productivity per day expressed in PG/cell/day and was obtained by dividing the GALNS activity at the cellular density on these days. This figure presents the fourth day (96 hours) after sowing in an amount of 5×105cells/vial. The clones were analyzed in relation to GALNS using ELISA with activity capture enzyme in PG/cell/day (y-axis). Positive controls consisted of BHK expressing GALNS, and CHO clones (BioMarin). Each vertical column corresponds to a separate clone. Active GALNS was produced clones pCIN 4A, but only slightly above background levels in the analysis.

Analysis of clones by the screening of 96 wells and analysis of productivity for 4 days showed that cotransfection expressing vectors with elements of MAR raised products is Yunosti clones G71S compared with cotransfection expressing vectors without items MAR. Cotransfection clones BMAR 4A + BMAR SUMF1 showed the rapid formation of a pool, the rapid growth of the clones and the ability to produce more than 2 times more active GALNS compared to most vysokoplodorodnye clones PMAR 4A and up to 10-fold increase relative to the CHO clones 4A and BHK 4A deprived of MAR elements.

Expressing GALNS clones G71S adapted to serum-free medium for growth using the Protocol described in Sinacore et al., Mol. Biotechnol. 15(3):249-257, 2000. Full adaptation was performed in the presence of both tools for breeding (zeocin at a concentration of 200 μg/ml neomycin at a concentration of 200 μg/ml). Expressing GALNS clones G71, cultured in T-flasks were divided as follows: (1) in 125-ml flasks for shaking with the environment Cambrex UltraCHO and 5% FBS (lot # 8L2242); (2) in 125-ml flasks for shaking with the environment JRH 302M environment (for products) and 5% FBS; (3) in T-flasks as duplication (UltraCHO, 5% FBS). After the establishment of suspension culture, the attached cells were removed and started removing FBS. When the growth rate was back up >0,5 (l/day) for 3 passages and viability was >95%, the FBS concentration was reduced by 50%. The cells were left for a given concentration of FBS for at least 3 passages. After adaptation to growth in 2.5% FBS, the cells were collected directly in serum-free medium. The cells were kept in fresh from the food with 10% (vol./about.) DMSO. Tested test defrost in order to ensure that the cells survive the process of freezing. For two expressing GALNS clones G71S after transfection BMAR 4A + BMAR SUMF1, clones 4 and 5, adapted to serum-free suspension culture took about 15 passages. Also identified clone C6 expressing GALNS after transfection pcDNA4 SUMF1 together with pCIN 4A, and adapted it to a serum-free culture.

Paired combinations expressing vectors for human SUMF1 and human GALNS (example I), pcDNA4 SUMF1 together with pcDNA4-4A, was transfusional cells G71S, mainly, as described above, except that selection was used at 200 µg/ml zeocin (Invitrogen). Six clones expressing GALNS, C2, C5, C7, C10, C11 and C30, were isolated and adapted to serum-free suspensino culture, mainly, as described above.

EXAMPLE III

Large-SCALE CULTURE CELL LINES G71S EXPRESSING N-ATSETILGALAKTOZAMIN-6-SULFATASE (GALNS) PERSON

The task was to measure the production of enzyme from clones G71S expressing N-atsetilgalaktozamin-6-sulfatase (GALNS) person. Adapted to serum-free suspension culture cell line G71S, coexpression SUMF1 human GALNS and man, were cultivated on a large scale and were assessed in relation to the production of active enzyme GALNS.

Pascalc the adaptation to serum-free suspension culture was relatively quick for cell line host G71S, it was decided that production can be carried out in the WAVE bioreactor operating in the mode of perfusion. The WAVE bioreactor allows greater flexibility in terms of the volume of inoculum, since the scaling can be performed directly in the bag, reducing the risk of contamination and accelerating the production of the material. The figure 7 presents a diagram of the system of the WAVE bioreactor. The graph shows, in the mode of perfusion that voltage sensor monitors the volume of medium in the bag by determining the mass of the bag and levels adjustment filing and collection to maintain the desired volume. In bag of 10 l, pH is also controlled to the desired preset value of the probe that is placed in the bag.

The material of expressing GALNS clones G71S 4 and 5 received on a scale of 1 L. In these analyses the pH of the culture is not controlled. Operational limitation bag WAVE is 3 volume production capacity per day (VV/day). To prevent any inactivation of the material, the specific rate of perfusion of the target cells (CSPR) was 0.3 nl/cell/day, providing an average residence time for GALNS enzyme, comprising eight hours. Thus, the density of cells in the bag was maintained at a level of approximately 10-12×106cells/ml the Rate of growth of expressing GALNS clones G71S 4 and 5 amounted to 0.16 and 0.20, respectively. Merging is supported for the I density of target cells was carried out directly from the bag.

the pH of the collected liquid was brought to a pH between 5.5 and 6.5 for the maintenance of enzymatic activity, as was previously shown that GALNS is stable at pH 6. It spent a bolus addition of schedule 5% by volume pH 4,0 citrate-sodium buffer, homogeneously mixed with the collected liquid leaving the reactor. Brought collected fluid was stored at 4°C before further processing. Two expressing GALNS clone G71S 4 and 5 had average titers of approximately 4.2 mg/l associated with a specific productivity of approximately 1.25 PG/cell/day.

Expressing GALNS clones G71S, C2, C5, C6, C7, C10, C11 and C30 similarly cultivated on a large scale and were assessed in relation to the production of active enzyme GALNS.

EXAMPLE IV

The MEASUREMENT of the CONCENTRATION AND activity of N-ATSETILGALAKTOZAMIN-6-SULFATASE (GALNS) PERSON

Enzyme-linked immunosorbent assay (ELISA) was developed for measuring the concentration and activity of the GALNS enzyme clones G71S, coexpressing human SUMF1 and N-atsetilgalaktozamin-6-sulfatase (GALNS) person.

ELISA activity capture enzyme

In ELISA with activity capture enzyme is measured by the GALNS enzyme activity in the solid phase, with subsequent removal of specific antibody against GALNS associated with the tablet for ELISA.

Buffers. Buffer A(carbonate buffer): dissolve to 3.09 grams of Na 2CO3and 5,88 grams of NaHCO3in 900 ml of deionized (DI) H2O, then add DI H2O to a final volume of 1000 ml to Check that the pH is between about 9.4 and 9.6, then sterilisation by filtration. For the application completely in one 96-well microplate, 100 μl per well, diluted with 19 μl of antibodies against GALNS in a single vial (12 ml). Buffer B (blocking buffer for ELISA and buffer for serial dilution): 1× acidic PBS, 0.05% of Tween-20 and 2% BSA, brought to pH 6.5 with acetic acid. Buffer Bw(the buffer for washing): 100 mm NaOAc and 0.05% Tween-20, brought to pH 6.5 with acetic acid. Buffer C (buffer substrate): 25 mm sodium acetate, 1 mm NaCl, 0.5 mg/ml demineralized BSA and 0.01% sodium azide, brought to a pH of 4.0 glacial acetic acid. Buffer D (buffer for β-galactosidase): 300 mm dibasic sodium phosphate, 0.1 mg/ml BSA, 0.01% sodium azide and 0.01% Tween-20, brought to a pH of 7.2 with phosphoric acid. Buffer E (Buffer to stop): 350 mm glycine and 440 mm carbonate buffer, brought to a pH of 10.7 using 6 M NaOH.

Reagents. Antibody IgG against GALNS: polyclonal rabbit antibodies are purified by protein G from whey. In D-PBS, total protein = 3,17 mg/ml (BCA). Aliquots (19 μl) and stored at -20°C, each for a single use. The substrate 4MU-Gal-6-S (solid; 440 MM): 100 mm original solution was obtained in DI water and stored at 4°C. β-galactosidase (Sigma G-4155): dilute to 12 ug/ml per puff is re D before use.

Protocol. To bind the antibody against GALNS tablet: tablet Nunc MaxiSorp ELISA (Nalge/Nunc International, Fisher # 12-565-135) coated with antibody against GALNS at a final concentration of 5 µg protein/ml in buffer A. For the preparation of this solution to unfreeze one 19-ál aliquot, ottsentrirovat quickly (10) in microcentrifuge for collecting fluid. To move all of 19 ál 12 ml of buffer A. Mix vigorously by inversion, then pour into a container and then put in the tablet (100 μl per well) using a multichannel pipette. To close the tablet and incubate at 4°C over night. To remove unbound antibody against GALNS: wash the tablet filling buffer Bwthree times. Block: to block the plate with buffer B (320 μl per well), then close the tablet and incubate at 37°C for 1 h to Prepare a series of dilutions of purified standard GALNS and test samples (unknown) during stage block: standard diluted in buffer B to the upper linear range of the assay (128 ng/ml in row A), then serially diluted (2-fold) in rows B-G 96-well plate. The number H is an empty buffer (i.e. without GALNS enzyme). First prepare 500 ál with a concentration of 128 ng/ml in buffer B. Then serially diluted 2 times in buffer B (250 ál 250 ál) to achieve 2 ng/ml to Delete buffer for blocking: after stage blocks the Finance buffer B are removed. To link the standard of GALNS enzyme and the test sample with the antibody against GALNS: apply to the tablet with 100 μl/well of serially diluted standard and test samples (in duplicate). To close the tablet and incubate at 37°C for 1 h to Remove inhibitors GALNS: wash the tablet filling buffer Bwthree times. Add substrate GALNS (first reaction): to prepare a sufficient quantity of the finished substrate solution for the application of 100 μl per well (prepare no more than 1 hour before use). Dilute the original solution 4MU-Gal-6-S (100 mm) to 1 mm in buffer C. Apply 100 ál per well. To close the tablet and incubate at 37°C for 30 minutes Add β-galactosidase (the second reaction): add 50 ál of β-galactosidase at a concentration of 12 μg/ml in buffer D to each well. To close the tablet and incubate at 37°C for 15 min, the Reaction stops: add 100 ál buffer E (buffer stop) to each well for ionization 4MU released. To move the tablet to determine fluorescence: move (8 holes at once) 200 ál 250 ál in each well of the tablet for ELISA in black raw titration microplate with flat bottom (Fluoroplate, Costar #3915). Read fluorescence: read the tablet in the reader Gemini (Molecular Devices Corporation) using SOFTmax PRO (excitation 366 nm, the emission of 446 nm, the frontier - 435 nm).

ELISA for GALNS

In ELISA for GALNS determine the concentration of the GALNS enzyme is in the air-conditioned cell culture medium or other process samples using immunological sandwich-analysis.

The buffers.Buffer A (carbonate buffer): dissolve to 3.09 grams of Na2CO3and 5,88 grams of NaHCO3in 900 ml of deionized (DI) H2O, then add DI H2O to a final volume of 1000 ml to Check that the pH is between about 9.4 and 9.6, then sterilisation by filtration. For full coverage of one 96-well microplate, 100 μl per well, diluted with 19 μl of antibodies against GALNS in a single vial (12 ml). Buffer B (blocking buffer for ELISA and buffer for serial dilution): 1× acidic PBS, 0.05% of Tween-20 and 2% BSA, brought to pH 6.5 with acetic acid. Buffer Bw(the buffer for washing): 100 mm NaOAc and 0.05% Tween-20, brought to pH 6.5 with acetic acid. Buffer F (buffer to stop): 2 N H2SO4: just for 600 ml, add 100 ml of 12 N H2SO4and 500 ml of MilliQ water.

Reagents. Antibody IgG against GALNS: polyclonal rabbit antibodies are purified by protein G from whey. In D-PBS, total protein = 3,17 mg/ml (BCA). Aliquots (19 μl) and stored at -20°C, each for a single use. Conjugated with HRP antibody for detection (RIVAH): end-conjugated antibody diluted 1:100 in D-PBS/1% BSA and stored in 20-µl aliquot at -20°C for a single use. The set of substrate EIA TMB Substrate Kit (BioRad #172-1067).

Protocol. To bind the antibody against GALNS tablet: tablet Nunc MaxiSorp ELISA (Nalge/Nunc International, Fisher # 12-565-135) coated with antibody against GALNS at a final concentration of 5 µg protein/ml in buffer A. For the preparation of this solution, thaw one 19-ál aliquot, ottsentrirovat quickly (10) in microcentrifuge for collecting fluid. To move all of 19 ál 12 ml of buffer A. Mix vigorously by inversion, then pour into a container and then put in the tablet (100 μl per well) using a multichannel pipette. To close the tablet and incubate at 37°C (convection incubator) for two hours. Do not use hot block. To remove unbound antibody against GALNS: wash the tablet filling buffer Bwthree times. Block: to block the plate with buffer B (320 μl per well), then close the tablet and incubate at 37°C for 1 h to Prepare a series of dilutions of purified standard GALNS and test samples (unknown) during stage block: standard diluted in buffer B to the upper linear range of the assay (128 ng/ml in row A), then serially diluted (2-fold) in rows B-G 96-well plate. The number H is an empty buffer (i.e. without GALNS enzyme). First prepare 500 ál with a concentration of 40 ng/ml in buffer B. Then serially times ablate 2 times in buffer B (250 ál 250 ál) to the achievement of 0.625 ng/ml Remove the buffer to block: after the stage of blocking buffer B are removed. To link the standard of GALNS enzyme and the test sample with the antibody against GALNS: apply to the tablet with 100 μl/well of serially diluted standard and test samples (in duplicate). To close the tablet and incubate at 37°C for 1 h Wash: wash the tablet filling buffer Bwthree times. To bind the conjugate antibodies for detection: thaw one aliquot (120 ál) antibody RIVAH, briefly ottsentrirovat (10) in microcentrifuge for collecting fluid. Dilute all 120 ál of 11.9 ml of buffer B and vigorously to turn the tube to mix. Pour into a container and add 100 ál per well using a multichannel pipette. To close the tablet and incubate at 37°C for 30 minutes Rinse: rinse the tablet filling buffer Bwthree times. The TMB substrate: prepare final substrate solution by mixing 1.2 ml of solution B from 10.8 ml of solution A. to Pour into a container and add 100 ál per well using a multichannel pipette. To close the tablet and incubate at 37°C for 15 minutes the Solution to stop: pipette 12 ml of 2 N H2SO4the stop solution in the tank and add 100 ál per well using a multichannel pipette. To gently tap for mixing. Consider A450: to consider a tablet device for cityuniversity.

The analysis of the specific activity GALNS

In the analysis of the specific activity GALNS measure the enzymatic activity of GALNS in solution using a specific substrate for GALNS.

Buffers. For all buffers use MilliQ H2O. the dilution Buffer (DB): for 1 l DB dissolving of 1.74 ml of acetic acid, 0.75 g of sodium acetate, 233,6 mg NaCl, 2 ml of 50% Tween-20 and 10 ml of 1% sodium azide in MilliQ H2O and to bring the pH to 4.0 +/- 0,5 using 0.1 M NaOH, if the pH is less than 3,95, and using 0.1 M acetic acid, if the pH exceeds 4,05. The final concentrations are: 19,5 mm acetic acid, 5.5 mm sodium acetate, 1 mm NaCl, 0.1% Tween-20 and 0.01% sodium azide. Phosphate buffer (PB): for 1 l of PB, dissolving of 13.9 g of NaH2PO4-H2O and 55 g NaHPO4-7H2O in MilliQ H2O and to bring the pH to 7.2. The final concentration of 300 mm NaPi. The buffer stop (SB): for 1 l of SB, and dilute to 26.2 g of glycine and 46.6 g of sodium carbonate in MilliQ H2O and to bring the pH to 10.6 using NaOH. Buffer for analysis (AB): dilute the original solution 4MU-Gal-6S 1:50 DB (final concentration 2 mm). Buffer for β-galactosidase (βGB): 25 μl/ml β-galactosidase 300 mm NaPi, pH 7,2.

Reagents. 4MU-Gal-6S: 100 mm, H2O (Toronto Research Chemicals, catalog # M334480). β-galactosidase: Sigma G-4155. 4-methylumbelliferone (standard 4MU): Sigma M-1381 (10 mm initial solution in DMSO).

Protocol. To perform a serial dilution of the GALNS enzyme. For purified is Oh and cooked GALNS (~1.5 mg/ml), dilute samples 1:10,000 in microcentrifuge tubes with low adhesion protein (USA Scientific, catalog #1415-2600)containing the DB, before serial dilutions 1:1. Place 100 ál DB 96-well plate. In the first row of pipette 100 ál of the sample GALNS. Continue to carry out serial dilutions (1:1) in the tablet (A-G 96-hole tablets). In the hole H (empty) sample is added. The linear range of this assay is 1-75 ng/ml to Use the same procedure to obtain a standard curve 4MU. Dilute 10 mm initial solution 4MU in DMSO 1:100 in DB. To start a standard curve 4MU by adding 50 μl of 50 μm 4MU in the first hole, and then to a serial dilution. Add 50 μl of substrate diluted in AB (2 mm 4MU-galactose-6S in DB) in a 96-well plate to determine the fluorescence. Pre-incubate the substrate for 10 min at 37°C. Add 50 μl of the 100 μl of serial dilutions GALNS and standards 4MU in 50 μl of substrate in AB. Incubate at 37°C for 30 min (this first reaction removes sulfate from the substrate), quenching the first reaction and start the second reaction by adding 50 ál of β-galactosidase (to dilute the original solution of β-galactosidase to 25 µg/ml in βGB). Phosphate inhibits GALNS, and an increase in pH also stops the reaction GALNS. After that the final pH is in the optimum range of pH for β-galactosidases. Incubate this second reaction mixture at t the value of 15 min at 37°C. To ionize 4MU released by addition of 100 μl of SB. To read Ex355, Em460 on the reader 96-well plates to determine the fluorescence. Calculation of enzyme activity (at 37°C in buffer with a pH of 4.0): 1 unit = number µmol released 4MU/min; activity = number µmol 4MU/min/ml; specific activity = µmol 4MU/min/mg. calculation of the protein concentration: use the extinction coefficient GALNS (1 mg/ml = 1,708 units absorption at 280 nm).

EXAMPLE V

PURIFICATION of N-ATSETILGALAKTOZAMIN-6-SULFATASE PERSON (GALNS)

The task consisted in the production of large quantities of recombinant N-atsetilgalaktozamin-6-sulfatase person (GALNS). Stable transfetsirovannyh cells G71, coexpression SUMF1 human GALNS and man, were grown in culture conditions in the bioreactor and cell environment was purified active enzyme GALNS.

Device for liquid chromatography.System Amersham Pharmacia Biotech AKTA explorer 900 using software Unicorn control.

Methods for protein analysis.For SDS-PAGE, staining Kumasi blue (B101-02-COOM), Western blotting and Bradford protein analysis followed standard methods. Purification was assessed by the release of activity and purity of the product GALNS was assessed visually using SDS-PAGE. The presence protestirovannyx impurities were detected by Western-blotting using the antibodies against the GALNS. The protein concentration was measured using Bradford protein analysis. The concentration of the final purified GALNS protein was measured by measuring A280using the extinction coefficient 1,708.

Chromatographic resin.Blue Sepharose 6 FF (GE Healthcare, party #306346) and Fractogel SE Hi-Cap (Merck KgaA, FC040894449).

Determination of enzyme activity GALNS. The specific activity of GALNS was determined using small fluorescent substrate 4-methylumbelliferyl-6-S-GAL (4-MU-6-S-GAL). The analysis of the specific activity GALNS involves a two-step reaction, where necessary adding β-galactosidase activity after incubation with GALNS substrate within a certain period of time to release the fluorescent label. The measurements were carried out using a fluorescent reader tablets.

Column for 10DG desalting (Bio-RAD) was balanced buffer for equilibration (EQB, 50 mm NaOAc, 10 mm NaCl, pH 5.8). For all buffers used MilliQ H2A. Three (3) ml of purified GALNS (0.5-2 mg/ml) was applied on the column for desalting, suirable and collected in 4-ml aliquot in a separate test tubes using the EQB. The protein concentration was calculated using an extinction coefficient GALNS (1 mg/ml = 1,708 units absorption at 280 nm).

Desalted samples GALNS were subjected to serial dilution (1:1) in the buffer for cultivation (DB, 50 mm NaOAc, 1 mm NaC, pH of 4.0+0.5 mg/ml BSA). The original solution of BSA was absoluely before use by applying a source of BSA solution at a concentration of 50 mg/ml (not more than 5% CV) on a G25 column, pre-equilibrated milliQ H2O. 100 ál of desalted sample GALNS was pietravalle in the first row of the 96-well plate with low-binding protein, and serially diluted samples GALNS was transferred by pipette into the tablet (rows A-G in 96-well tablets). 100 μl of DB pietravalle in the last hole (H). The upper end of the linear range of this assay is 200 ng/ml and the linear range is 3-200 ng/ml, The same method was carried out to obtain a standard curve (4-methylumbelliferone (4MU) (Sigma M-1381, 10 mm initial solution in DMSO). 50 μl of the 100 μl of serial dilutions GALNS and 4MU was transferred into a new 96-well plate to determine fluorescence (tablet black bottom). 50 μl of 2 mm 4MU-galactose-6S (in milliQ H2O) was added to the samples subject to analysis, and incubated at 37°C for 30 minutes. This first reaction is extinguished and started the second reaction by adding 50 ál of β-galactosidase (Sigma G-4155, the original solution is diluted to 12 µg/ml in 300 mm NaPi, pH of 7.2), and incubated at 37°C for 15 minutes. Released 4MU ionized by addition of 100 μl of buffer stop (glycine/sodium carbonate, pH to 10.6). Spent reading tablets, the fluorescent reader 96 well the tablets (excitation 355 nm, the emission 460 nm). 1 unit is defined as 1 µmol released 4MU/min, the enzyme activity is given in µmol 4MU/min/ml and the specific activity is given in µmol 4MU/min/mg, all at 37°C in buffer with a pH of 4.0.

The first cleaning process.The first cleaning process included stage ultrafiltration (UF) followed by a cleaning process on 2 columns.

1. Filtering the collected liquid (HF): material bioreactor was subjected to sterile filtration through the 0.2-μm filter.

2. Ultrafiltration (UF): material bioreactor concentrated 10-20× ultrafiltration through a 30 kDa membrane Sartocon.

3. Bringing the pH to 4.5: concentrated material bioreactor (UF (20×)) brought to pH 4.5 buffer to bring the pH (1,75 M NaOAc, pH 4.0) is at room temperature before application to the column of Blue Sepharose was subjected to sterile filtration.

4. Blue Sepharose 6 Fast Flow (FF): UF with increased pH to 4.5 (20×) was applied to a column of Blue Sepharose and protein GALNS was suirable, as shown in table 1 and on figure 9A.

Table 1
Chromatography with Blue Sepharose 6 Fast Flow
StageCV*The buffer
Trim5 20 mm acetate/phosphate, 50 mm NaCl, pH 4.5
ApplicationProduct UF, brought to pH 4.5, filtered
Washing 1420 mm acetate/phosphate, 50 mm NaCl, pH 4.5
Washed 2820 mm acetate/phosphate, 50 mm NaCl, pH 6,0
Elution820 mm acetate/phosphate, 100 mm NaCl, pH 7.0
Desorption520 mm acetate/phosphate, 1 M NaCl, pH 7.0
Clean4of 0.1 N NaOH, 0.5 hours
Regeneration5H2O
Storage320% ETOH
* CV: the volume of the column. Flow rate = 92 cm·h-1

5. Fractogel SE Hi-Cap: the eluate from the column with a Blue Sepharose was brought to pH 4.3 and put in a column of Fractogel SE Hi-Cap and GALNS protein was suirable, as presented in table 2 and on figure 9B.

Table 2
Chromatography with Fractogel SE Hi-Cap
StageCV*The buffer
Trim520 mm acetate/phosphate, 50 mm NaCl, pH 4,3
ApplicationThe eluate Blue Sepharose, brought to pH 4.3 and diluted 1:1 with water MQ
Washing 1520 mm acetate/phosphate, 50 mm NaCl, pH 5.0
Washed 2520 mm acetate/phosphate, 50 mm NaCl, pH 5.5
Elution2020 mm acetate/phosphate, 50-350 mm gradient of NaCl, pH 5.5
Regeneration 1520 mm acetate/phosphate, 500 mm NaCl, pH 5.5
Regeneration 2520 mm acetate/phosphate, 50 mm NaCl, pH 4,3
Clean50.5 N NaOH, 0.5 hours
Regeneration 34H2O
Storage320% ETOH
* CV: the volume of the column. Flow rate = 150 cm·h-1

Protein GALNS in the eluate was collected by fractionation, removing preliminay initial fraction and posterwerundu final fraction.

6. The final UF/HF: the eluate from the column of Fractogel SE Hi-CAP was concentrated by ultrafiltration and subjected to sterile filtration, as described above.

Composition. Purified GALNS protein were prepared in 10 mm NaOAc, 1 mm NaH2PO4, of 0.005% Tween-80, pH 5.5.

Stability studies. Monitoring the stability of the final cooked purified GALNS was carried out at 4°C and -70°C as a function of time by storing small aliquot samples GALNS at appropriate temperatures. At certain time points, aliquots of the frozen samples were rapidly thawed in a water bath at 37°C before activity measurements. The figure 8 shows that purified GALNS was stable in buffer for manufacturing at 4°C and -70°C for up to at least 79 days.

The results of the first cleaning. Table 3 presents the outputs after cleaning three drugs GALNS protein produced by clone 4 G71S in Biore store for suspension culture. Purity was assessed visually, by SDS-PAGE at the 95% level in all cases.

Table 3
Output N-atsetilgalaktozamin-6-sulfatase (GALNS) from clone 4 G71S after cleaning of the reactor WAVE
Output
StageThe drug 1The product 2Drug 3AverageThe standard deviation
UFN/A1001001000
Blue Sepharose 6 FF93103101995,3
SE Hi-CAP908790891,7

The figure 9 shows the SDS-PAGE protein GALNS separated (A) chromatography with Blu Sepharose 6 Fast Flow followed (B) chromatography with Fractogel SE Hi-CAP. Gels were stained with Kumasi blue (left) or antibody against GALNS (right). For Western blots, the antibody against GALNS rabbit diluted 1:5000, and the secondary antibody was an antibody rabbit against alkaline phosphatase. Protein GALNS had an apparent molecular mass of ~55-60 kDa in SDS-PAGE, consistent with the expected size of the secreted pre protestirovanny form (predecessor) of the enzyme lacking the signal peptide of 26 amino acid residues, and also without splitting after position 325.

Karakterizacija N-end.N-end purified GALNS protein was determined by LC/MS. The sequence of the N-end was a APQPPN, which corresponds to the predicted N-end of the secreted form of GALNS, devoid of the signal peptide of 26 amino acid residues (compare: polypeptide sequence of human GALNS in the figure 4 and figure 5).

The second cleaning process.The second cleaning process included stage ultrafiltration/diafiltration (UF/DF) with the subsequent cleaning process on 3 columns.

1. Ultrafiltration (UF/DF): material bioreactor was concentrated 20× ultrafiltration/diafiltration through a 30 kDa membrane Sartocon at pH 5,5.

2. Bringing the pH to 4.5: concentrated material bioreactor (UF/DF (20×)) brought to pH 4.5 using a buffer to bring the pH (1,75 M NaOAc, pH 4.0) is at room temperature the e and before applying on a column of Fractogel EMD SE Hi-Cap was subjected to sterile filtration.

3. Fractogel EMD SE Hi-Cap: UF/DF (20×), brought to pH 4.5, was applied on a column of Fractogel EMD SE Hi-Cap, washed sequentially with 10 mm mixture of acetate/phosphate, 50 mm NaCl, pH 4.5 and 10 mm mixture of acetate/phosphate, 50 mm NaCl, pH 5.0, and GALNS protein was suirable 10 mm mixture of acetate/phosphate, 140 mm NaCl, pH 5.0.

5. Zn-chelating Sepharose FF: the eluate from the column with Fractogel EMD SE Hi-Cap had brought, 500 mm NaCl, pH 7.0, and applied to a column of Zn-chelating Sepharose FF (Zn-OMAS), washed with 10 mm mixture of acetate/phosphate, 125 mm NaCl, 10 mm imidazole, pH 7.0, and GALNS protein was suirable 10 mm mixture of acetate/phosphate, 125 mm NaCl, 90 mm imidazole, pH 7.0.

6. Bring to pH 3.5: the eluate from the Zn-chelating Sepharose column FF containing protein GALNS, brought to pH 3.5 for inactivation of the virus to low pH, and then brought up to 10 mm mixture of acetate/phosphate, 2 M NaCl, pH 5.0.

7. ToyoPearl Butyl 650M: the eluate with brought to the low pH values of the Zn-chelating Sepharose column FF, put in a column of ToyoPearl Butyl 650M, washed with 10 mm mixture of acetate/phosphate, 2 M NaCl, pH 5.0, and GALNS protein was suirable 10 mm mixture of acetate/phosphate, and 0.7 M NaCl, pH 5.0.

8. The final UF/HF: the eluate from the ToyoPearl Butyl 650M was subjected to ultrafiltration and diafiltration in 20 mm acetate, 1 mm phosphate, 150 mm NaCl, pH 5.5.

Preparation.Purified GALNS protein were prepared in 10 mm NaOAc/HOAc, 1 mm NaH2PO4, 150 mm NaCl, 0.01% of Tween-20, pH 5.5.

The results of the second cleaning process.Table 4 presents the output GALNS protein produced from clone C2 G71S in bio is auctore with the suspension culture using a second cleaning process. The purity of the prepared GALNS enzyme (i.e. the form of precursor and Mature or protestirovany forms together) amounted to approximately 98% when using C3 RP-HPLC. The percentage of forms predecessor of the GALNS enzyme was approximately 85% in the determination SDS-capillary gel electrophoresis.

Table 4
Output N-atsetilgalaktozamin-6-sulfatase (GALNS) person to clone C2 G71S
Stage of the processOutput (%)
Bringing pH96
Column with Fractogel SE Hi-CAP98
Column with Zn-IMAC89
Inactivation of the virus to low pH89
Column of ToyoPearl Butyl 650M99
Cooking99
Total70

The figure 10 shows SDS-PAGE of the GALNS enzyme, separated by ultrafiltration/diafiltration (UF/DF), chromatography with Fractogel SE Hi-CAP, chromatography with Zn-chelating Sepharos FF and chromatography with ToyoPearl Butyl 650M. Gels were stained with Kumasi blue (top left), antibody against GALNS (top right), antibody against cathepsin L (bottom left) and an antibody against the protein CHO (CHOP, bottom right). For Western blots, polyclonal antibody rabbit against GALNS was diluted to 1:5000, and the secondary antibody was a conjugate antibodies against rabbit antibodies and AP; polyclonal antibody goat against cathepsin L was diluted to 1:1000 and secondary antibody was a conjugate antibodies against antibodies goat-HRP; and polyclonal antibody rabbit against CHOP was diluted to 1:1000 and secondary antibody was a conjugate antibodies against rabbit antibodies and HRP. The predecessor of the GALNS enzyme has an apparent molecular weight of ~55-60 kDa in SDS-PAGE, and Mature or protestirovanny form of GALNS enzyme have apparent molecular weight ~39 kDa and ~19 kDa in SDS-PAGE.

The generalization of the first cleaning process.The GALNS enzyme was purified using a series of cleanings, which was a modified standard series (see table 5). The material collected from the bioreactor were subjected to sterile filtration through the 0.2-μm filter and kept at 4°C before application to the column for catching Blue-Sepharose. The filtered material bioreactor or caused directly or concentrated up to 15× ultrafiltration. Modification of series PTS is a runoff was necessary, because the subsequent stage of purification, chromatography with SP Sepharose followed by chromatography with Phenyl Sepharose did not lead to enough clean GALNS. Using chromatography with SE Hi-Cap as a replacement for the subsequent two columns for purification in the purification process with 2 columns, the purity of the target material is substantially increased, and the overall yield GALNS significantly increased from ~22% to ~80%. The purity of the GALNS enzyme (consisting essentially of the form predecessor, see figure 9), with the C4 definition OF chromatography, was approximately estimated as >95%, and purified GALNS enzyme was stable in buffer for manufacturing for more than 79 days as at 4°C and -70°C.

Table 5
The first series of cleanings N-atsetilgalaktozamin-6-sulfatase (GALNS) person
StageThe usual processThe modified process
1HF (1×)HF (1×)
2*UF (5×)UF (15×)
3Bring to pH 4.5Bringing the pH to 4.5
4Blue-Sepharose 6 FFBlue-Sepharose 6 FF
5SP SepharoseSE Hi-Cap
6Phenyl Sepharose Hi-SubThe final UF/DF
7The final UF/DF
* This step is optional

The generalization of the second cleaning process. The GALNS enzyme was also purified using the second series of cleanings (see table 6). The total yield of GALNS was approximately 70%, and the purity of the GALNS enzyme (including both the predecessor and Mature or protestirovanny form, see figure 10), with the C4 definition OF chromatography, was roughly estimated as approximately 97%.

Table 6
The second series of cleanings N-atsetilgalaktozamin-6-sulfatase (GALNS) person
StageProcess
1HF (1×)
2UF/DF (20×)
3Bring to pH 4.5
4SE Hi-Cap
5Zn-chelating Sepharose
6Bring to pH 3.5
7ToyoPearl Butyl 650M
8The final UF/DF

These analyses indicate that the protocols described above for the preparation of recombinant lysosomal enzymes sulfates ensure effective production of large quantities of highly purified enzyme, in particular secreted pre protestirovanny form (predecessor) N-atsetilgalaktozamin-6-sulfatase (GALNS) person.

EXAMPLE VI

KARAKTERIZACIJA PURIFIED N-ATSETILGALAKTOZAMIN-6-SULFATASE (GALNS) PERSON

Cell line G71 produce proteins (e.g., lysosomal enzymes) with higher levels of phosphorylation of oligosaccharides with a high content of mannose than observed in the average mammalian cell lines, and respectively a lower level nefosfaurilirovanna oligosaccharides with a high content of mannose. Lysosomal enzyme sulfatase (e.g., recombinant N-atsetilgalaktozamin-6-sulfatase (GALNS) che is owaka), having a high level of bis-phosphorylated oligosaccharides with a high content of mannose, as defined in this document, compared to molecules obtained according to Canfield et al., U.S. patent 6537785 that do not contain complex oligosaccharides and have only oligosaccharides with a high content of mannose.

To determine the levels nefosfaurilirovanna oligosaccharides with a high content of mannose lysosomal enzyme sulfatase, the person skilled in the art can use sequencing using ectoparasites released oligosaccharides ("sequencing FACE"), to identify the percent nefosfaurilirovanna polisaharidnykh circuits with a high content of mannose. Normal profileru.exe gel FACE in reduced volume nefosfaurilirovanna oligosaccharides with a high content of mannose migrate together with the specific complex oligosaccharides (for example, oligomannose 6 and fully suliranin complex of the two branches). Then nefosfaurilirovanna oligosaccharides with a high content of mannose differentiate from other oligosaccharides using an enzymatic sequencing.

To determine whether purified lysosomal enzyme sulfatase (e.g., recombinant N-atsetilgalaktozamin-6-sulfatase (GALNS) man), expressed in cells G71S, increasing the military phosphorylation, to determine the level of mannose-6-phosphate (M6P) on the lysosomal enzyme sulfatase, as well as the ability of the enzyme to contact the M6P receptor (MPR).

Recombinant GALNS enzyme of the person, expressed in cells G71S and purified, and analyzed by electrophoresis hydrocarbons using fluorescence (FACE) and chromatography on resin MPR-Sepharose. In the system of the FACE is used polyacrylamide gel electrophoresis to separate, quantify and identify the sequence of oligosaccharides released from glycoproteins. The relative intensity of the band oligomannose-7-bis-phosphate (O7P) at the FACE (Hague et al., Electrophoresis 19(15): 2612-20, 1998) and the percentage of the active substance is retained on the column MPR (Cacia et al., Biochemistry 37(43): 15154-61, 1998), provide reliable indicators of the level of phosphorylation per mole of protein.

Specific activity. The specific activity of recombinant N-atsetilgalaktozamin-6-sulfatase (GALNS) of a person was determined using small fluorescent substrate 4-methylumbelliferyl-6-S-GAL (4MU-Gal-6S) at 37°C. using this assay, the specific activity of purified GALNS was 165 µmol/min/mg (0,165 U/mg).

Stability in human serum.Determined the stability of GALNS in serumex vivo. The human serum (Sigma H-4522) was subjected to sterile filtration through the 0.2-μm filter PES and 4 ml of sterilized F. what letricia human serum pre-incubated in bottles for culturing cells in T-25 for 1 hour at 37°C in an atmosphere of 10% CO 2(the pH at this point is 7.4 ± 0,1). To pre-incubated serum was added 0.4 ml of desalted purified GALNS (2 mg/ml purified GALNS was absoluely in PBS using columns Bio-RAD 10DG) or control in the form of PBS containing 0.5 mg/l BSA. A 100-µl samples were collected at specified time points (for example, 0, 1, and 3.5, 7.5 and 26 hours) and added to 900 ml of a buffer to absorb (QB, 50 mm NaOAc, pH 5,6 + 150 mm NaCl + 0.5 mg/ml BSA + of 0.001% Tween-80). The samples were stored at 4°C before measuring the activity of the GALNS enzyme.

The GALNS enzyme activity was measured using ELISA with activity capture enzyme. Extrapolating the exponential decaying curve % residual activity of the GALNS enzyme was estimated that the half-life of purified GALNSex vivoserum is 217 hours.

Capture synoviocyte (chondrocytes).Determined the ability of GALNS be captured by synoviocyte (chondrocytes).

The chondrocytes (number ATCC CRL-1832) were cultured in the medium for growth (F12 ham + 10% FBS) at 37°C in 5% CO212-hole cups. The analysis of trapping in three samples requires 4× 12-hole of the tablet. The cleaned samples GALNS and reference GALNS was diluted to 1 μm in acPBS/BSA (acidic PBS + 200 μg/ml BSA). From 1 μm initial solutions were obtained curves capture for dilution of samples GALNS and reference: a 50.5 μl (1 μm rhASB) in 5 ml of diluent for analysis of capture (UAD, DMEM + 2 mm L-glutamine + 0.5 mg/ml BSA), which led to 10Nm samples GALNS and reference which then serially diluted to 5, 2,5, 1,25, 0,62, at 0.31 and 0.16 nm serial twofold dilutions in UAD. Environment for the growth of the 12-hole cups with a monolayer of chondrocytes aspirated, the wells were added to 1 ml or UAD (blank)or serial dilutions of samples GALNS or standards, and incubated for 4 hours at 37°C in an incubator with 10% CO2. Environment for capturing aspirated, tilting each Cup completely, and each well was washed with 1 ml PBS. PBS aspirated and the chondrocytes were detached by addition of 0.5 ml of a mixture of trypsin/EDTA (0.25% trypsin/ 0.1% EDTA (Mediatech 25-053-CI, party 25053025)) per well. After release from the tablet, the chondrocytes were divided into aliquots in pre-chilled on ice Eppendorf tubes (30 tubes). Trypsinization chondrocytes was cooled, and then besieged at low speeds in microcentrifuge (4000 rpm for 3 minutes). Trypsin fully aspirated, the cell sediment was washed with 1 ml PBS, repeating once stage in microcentrifuge and aspirirovanna. To each tube was added 200 μl of buffer for lysis of cells (CLB, 50 mm sodium acetate, pH 5,6 + 0,1% Triton X-100). Cellular precipitation resuspendable pulse by shaking three times. After resuspendable mixture for lysis of the cells were stored overnight at -80°C or directly analyzed.

Cell lysates were thawed at room temperature and after time is oriane transferred on ice. Cell lysates were shaken for resuspendable any visible solid material, and then centrifugally in microcentrifuge at 14 thousand rpm for 10 minutes at 4°C to precipitate insoluble material. Supernatant was transferred into a fresh set of tubes and the precipitate was removed. Then there was the analysis of GALNS activity in supernatant. Usually get the curve cultivation of the seven points (serial two-fold dilutions, ranging from 10 nm to 0.16 nm), which covers the expected Kgripfairly evenly on both sides. The both molarity of the original sample is calculated using a molecular weight of only protein.

Purified GALNS had Kd capture synoviocyte based on binding of a ligand at one site, part of 4.9 nm.

The analysis of binding with the tablet with the receptor, mannose-6-phosphate (M6P).The ability GALNS contact with the receptor, mannose-6-phosphate (M6P) was determined in the analysis of binding with the tablet. On tablets FluoroNunc with high binding inflicted 4 μg/ml of M6P receptor. Coated tablets were washed two times with 250 μl/well of buffer to wash (WB, TBS + 0.05% of Tween 20) and nonspecific binding was blocked with 200 μl/well of buffer to block and breeding (BDB, Pierce SuperBlock buffer, party #CA46485). The plates were incubated for 1 hour at room temperature (RT). During this stage, blocking the untreated samples GALNS (0.5-2 mg/ml, stored at 4°C for 2 weeks) was diluted to 10 nm in BDB, and then serially diluted with buffer for cultivation (DB, 50 mm NaOAc, 1 mm NaCl, pH 4,0 + 0.5 mg/ml BSA) (250 ál + 250 μl) to 5, 2,5, 1,25, 0,62, at 0.31 and 0.16 nm. Blocked tablets washed WB, as described above, and diluted samples GALNS was distributed into the wells in duplicate in the amount of 100 μl/well and incubated for 1 hour at RT. During this stage of incubation, received a 2 mm substrate for determining the activity of breeding 0.1 ml of 100 mm initial solution 6S-galactose-4MU (stored in the H2O, -20°C) in 5 ml of DB and pre-heated in a water bath at 37°C. After incubation, the tablets were washed twice WB, as described above, was added 100 μl of diluted substrate and defined specific activity GALNS.

When applying this analysis, the purified GALNS had a Kd for binding to the M6P receptor from binding in the same area, component of 2.4 nm.

The binding column to a receptor mannose-6-phosphate (M6P).The ability GALNS contact with the receptor, mannose-6-phosphate (M6P) was determined in the analysis of the binding column. Column M6P receptor was obtained according to the manufacturer's instructions. The M6P receptor was from the laboratory of Peter Lobel, the resin column was represented by the activated resin NHS (Bio-RAD Affi-Gel 15), and the column size was 0.7 ml Column with M6P receptor balanced 10 volume and column (CV) of buffer for equilibration (EQ, acidic PBS, pH of 6.0, containing 5 mm β-glycerol, 0.05% of Tween 20, 5 mm glucose-1-phosphate and 0.02% NaN3) with a flow rate of 0.25 ml/min 6 μg of purified GALNS (200 μl) was applied on the column with the M6P receptor a flow rate of 0.1 ml/min Unbound GALNS was washed from the column with 10 CV EQ a flow rate of 0.25 ml/min Associated GALNS was suirable with a column using 0-100% gradient of buffer for elution (EL, acidic PBS, pH of 6.0, containing 5 mm β-glycerol, 0.05% of Tween 20, 5 mm mannose-6-phosphate and 0.02% NaN3) (10 CV), and then 2 CV 100% EL. The column was re-balanced with 3 CV of EQ.

Using ELISA for GALNS, it was determined that the percentage of purified GALNS, which is associated with the M6P receptor, was 56%.

Analysis of total oligosaccharides by capillary electrophoresis (CE).To determine the level of phosphorylation of mannose-6 on GALNS, determined the profile of N-linked carbohydrates on the total oligosaccharides on GALNS capillary electrophoresis (CE)as described in Ma et al., Anal. Chem. 71(22):5185-5192, 1999. In the method used PNGase F cleavage of N-linked to asparagine oligosaccharides. Derived oligosaccharides were isolated, received them derived from a fluorescent dye and put on a rotating G10 column to remove excess dye. Purified fluorescently labeled oligosaccharides were separated by electrophoresis, and then defined peaks use the education software MDQ-CE (32 Karat Ver. 7.0).

Using this analysis, the number of oligosaccharides containing bis-phosphorylated mannose 7 (BPM7), mono-phosphorylated mannose 6 (MPM6) and sialic acid, purified GALNS was of 0.58 mol/mol of enzyme, 0.08 mol/mol of enzyme and was not determined, respectively. It is estimated that the percentage of GALNS protein containing BPM7 amounted to 29%.

Karakterizacija bis-7 oligosaccharide.Determined the localization of oligosaccharides with bis-phosphorylated mannose 7 (BPM7) on GALNS. The residue asparagine (Asn) at position 178 was glycosylated N-linked glycosylation via BPM7. The Asn residue at position 397 was not glycosylated N-linked glycosylation via BPM7, however, it was revealed that he mainly glycosylated sugar type oligomannose.

Affinity for hydroxyapatite.A model was developed bonein vitroto determine whether GALNS capacity for targeting bone. A suspension of hydroxyapatite at a concentration of 4 mg/ml class HTP-DNA (Bio-RAD) was received and balanced in DBS + 50 μg/ml BSA, pH of 7.4. Purified GALNS, after adding 50 μg/ml BSA, absoluely in DBS, a pH of 7.4. Desalted GALNS at a final concentration of approximately 2 mg/ml was subjected to serial dilution in DBS + 50 μg/ml BSA, pH 7,4, in 96-well pad. 50 μl of serially diluted GALNS was transferred into a 96-well plate for filter (Milipore #MSGVN2210, hydrophilic PVDF, low binding of the protein, the pore size of 22 microns). 50 μl of the suspension of hydroxyapatite was added to the wells to filter containing serial dilution of GALNS, and incubated for 1 hour at 37°C under mild shaking. The tablet was subjected to vacuum filtration.

Supernatant with vacuum filter was analyzed either by HPLC or by activity of the GALNS enzyme, as described above. Purified GALNS had Kd for hydroxyapatite 3-4,0 μm.

Cell line G71S expressing modifying sulfatase factor 1 (SUMF1) the person produces lysosomal enzymes sulfatase with higher levels of activation (i.e. conversion of the cysteine residue of the active site in the Cα-formylglycine (FGly)).

To determine, is there a purified recombinant lysosomal enzyme sulfatase (for example, N-acetyl galactosamine-6-sulfatase (GALNS) man), coexpression with SUMF1 in cells G71S, increased activation was determined by the amount of conversion of the cysteine residue of the active site in FGly on purified lysosomal enzyme sulfatase.

Activation of GALNS.Percentage activation, i.e. the percentage conversion of the cysteine residue (Cys) active site in the Cα-formylglycine (FGly), GALNS was determined by LC/MS (TFA). TIC/1000 for Cys, FGly and Gly was 39, 1840 and 183, respectively, indicating that p is blithedale 90% purified GALNS is an active form (i.e. FGly).

The compilation.Table 7 presents the generalization to characterizatio recombinant GALNS, expressed in cells of clone 4 G71S. Table 8 presents the generalization to characterizatio recombinant GALNS, expressed in cells C2 clone.

Table 7
Karakterizacija N-atsetilgalaktozamin-6-sulfatase (GALNS) man produced from clone 4 G71S
Category analysisGALNS
Specific activity: Activity/Antigen in ELISA0,165 U/mg
Specific activity: Activity/Protein7.7 u/mg
Purity at C4-PF>95% (6 of tested parties)
Size in SEQ115 kDa (glycosilated)
Stability in serum at 37°C217 hours
Capture: the Chondrocytesof 4.9 nm
Capture: Fibroblasts5,0 nm
Capture: Osteoblasts 7,8 nm
Productivity1,3 PG/cell/day
Title4,2 mg/l
Linking with the tablet with the M6P receptor2,4 nm
Binding to the column with M6P receptor: % associated56%
The content of M6P when CE: % of total carbohydrates29%
The M6P content: mol M6P/mol GALNS0,58
The content of sialic acid when CE1%
Affinity for hydroxyapatite4 µm
Activation: % FGly90%

Table 8
Karakterizacija N-atsetilgalaktozamin-6-sulfatase (GALNS) man produced from clone C2 G71S
Category analysisGALNS
Specific activity: Activity/Protein6,4 U/mg
Cleanliness at the 4-PF >97%
Size in SEQ115 kDa (glycosilated)
Capture: Fibroblasts3.4 nm
Title6.4 mg/l (4 tested party)
Linking with the tablet with the M6P receptor5,7 nm
The content of M6P when CE: % of total carbohydrates34,5%
The M6P content: mol M6P/mol GALNS0,69

These results demonstrate that purified recombinant human GALNS has a high level of activation and high levels of phosphorylation of mannose-6-phosphate. Thus, cells G71S, coexpression SUMF1 and lysosomal enzyme sulfatase (i.e. GALNS), effectively produce active vysokovostrebovannye lysosomal enzyme sulfatase. Elevated levels of residues with a high content of mannose on such lysosomal enzymes sulfatase leads to increased capture using MPR on the cells.

EXAMPLE VII

Engagement AND ACTIVITY of RECOMBINANT N-ATSETILGALAKTOZAMIN-6-SULFATASE (GALNS) HUMAN CHONDROCYTES SYNDROME, MORQUIOIN VITRO

Estimated capture of recombinant N-and acylgalactosamine-6-sulfatase (GALNS) human lysosomes chondrocytes syndrome, Morquio and ability GALNS destroy keratinolytic (KS) in vitro.

The chondrocytes from patients with mucopolysaccharidosis type IVa (MPS IVa syndrome Morquio) have reduced activity of GALNS and show accumulation in lysosome KS. Created model MPS IVain vitrousing chondrocytes isolated from biopsies of the iliac crest of the patient with MPS IVa. However, in the culture of primary chondrocytes lose their differentiation and lose their characteristics of chondrocytes. Thus, have established such culturing conditions to induce differentiation of chondrocytesin vitro.

Chondrocytes isolated from a patient with MPS IVa, denoted MQCH, were cultured in alginate beads in the presence of IGF-1, TGF-β, transferrin, insulin and ascorbic acid (Chondrocyte Growth Medium, Lonza #CC-3225). The culture medium was replaced twice a week during the experiments within 6 to 15 weeks. These culturing conditions induced the expression of the phenotype of chondrocytes and differentiation. These MQCH cells expressed markers of chondrocytes, including determining floor region Y-box 9 (Sox 9), collagen II, collagen X, oligomeric matrix protein cartilage and mRNA aggrecan, when measuring the quantitative analysis OF RT-PCR using RNA isolated from cell cultures MQCH. These cultured cells MQCH also produced extracellular matrix.

To confirm that the MQCH cells accumulate KS, conducted to the focal microscopy. The MQCH cells in 8-week culture was trimensional, besieged by cytocentrifugation on glass slides were fixed in acetone and frozen until use. After thawing the cells rehydratable and stained using, as primary and secondary antibodies, monoclonal antibodies against KS (Chemicon) and conjugated with Alexa-488 (green) antibodies goat antibodies against rabbit, respectively. Cells MQ-CH point showed intracellular staining, which is consistent with the accumulation of KS in the lysosomes.

To determine whether purified recombinant human GALNS be captured MQCH cells in a complementary mechanism to carry out the degradation KS, 6-week culture MQCH cells were incubated with 10 nm recombinant human GALNS twice a week for 9 weeks. Capture GALNS and excretion KS was determined by confocal microscopy. Used primary antibodies were: (a) polyclonal antibody rabbit against GALNS and monoclonal antibody against associated with lysosomes membrane protein 1 (LAMP-1) or (b) a monoclonal antibody against KS and polyclonal antibody against LAMP-1. Used secondary antibodies were: conjugated with Alexa-488 (green) antibodies for detection of antibodies against GALNS or against KS or conjugated with Alexa-555 or -594 (red) antibodies DL is the detection of antibodies against LAMP-1. Drugs MQCH cells were prepared for microscopic examination in histology environment DAPI, which stains the nucleus.

In MQCH cells treated with GALNS, watched expressed colocalization GALNS enzyme and KS with the lysosomal marker LAMP-1. When exposed to MQCH recombinant human GALNS, the number of intracellular KS declined.

Also measured the capture GALNS using ELISA for detecting GALNS enzyme and ELISA for the specific activity GALNS, both of which are described in example IV above. Normal human chondrocytes (NHKC), which Express GALNS was used as a positive control. As shown in tables 9 and 10, the raw MQCH cells had no measurable detection GALNS enzyme or activity, where cells MCQH processed within 9 weeks 10 nm GALNS, had a significant amount of enzyme and the activity of GALNS.

Table 9
ELISA for the removal of the GALNS enzyme using MQCH cells
Cells MQCHNHKC
No processingN.D.a0,12b
10 nm GALNS for 9 h is del 3,990,88
aNot detected;bng antigen GALNS/µg total protein

Table 10
The analysis of the specific activity GALNS using ELISA using MQCH cells
Cells MQCHNHKC
No processingN.D.awas 2.76b
10 nm GALNS for 9 weeks3,685,15
aNot detected;bthe GALNS activity/ng antigen

These results demonstrate that purified recombinant human GALNS is captured by chondrocytes syndrome, Morquio in complementary mechanism and can lysosomal degradation KSin vitro. These chondrocytes syndrome, Morquio suitable as a model of efficiencyin vitrofor testing lysosomal enzymes sulfates, such as GALNS, which are degradation KS.

EXAMPLE VIII

The ACTIVITY of RECOMBINANT LYSOSOMAL ENZYMES PERSON WHO compared the degradation of NATURAL SUBSTRATES IN CELL ANALYSIS IN VITRO

Cellular analysesin vitrowas developed for measuring the activity of recombinant lysosomal enzymes person, such as lysosomal enzymes sulfates, degradation of natural substrates.

Enzymatic activity of recombinant lysosomal enzymes person, such as lysosomal enzymes sulfates, usually measured in cell-free analysis ofin vitrousing artificial fluorogenic substrate (see example 4 for GALNS). However, the measured activity of the enzyme depends on the size of the artificial substrate, i.e. on the number of monosaccharide components. In addition, the enzyme activity is measured in an environment that does not reflect the situationin vivo. Thus, cell-free analysis ofin vitrodoes not take into account neither the ability of lysosomal enzymes to break down natural, nor their ability to capture the target cells and localized in the lysosomes.

Was developed cell analysisin vitrofor measuring the activity of recombinant lysosomal enzymes person: alpha-L-iduronidase (IDU) and Ukrainian B (ARSB), degradation of their natural substrates, i.e. intracellular containing dermatologit (DS) substrates. DS contains variable way sulfated disaccharide glycosides elements Euronova acid-β-(1-3)-N-atsetilgalaktozamin β(1-4).

<> Deficient ARSB fibroblastlike cells GM00519 person or deficient IDU fibroblastlike cells GM01391 human cultivation to a monolayer of 12-hole tablets and culture after closing monolayer supported within 3-6 weeks to ensure accumulation of intracellular DS.

Then cells GM00519 or GM01391 after closing monolayer was affected by saturating doses of recombinant human ARSB (10 nm) or recombinant IDU man (25 nm), respectively, within 4-5 days. Raw and processed by lysosomal enzyme sulfatase cells collected were subjected to lysis and centrifuged.

The activity of lysosomal enzymes in cell lysates was measured by determining the content of residual DS cells by: (1) lysis of the cells; (2) specific cleavage containing DS substrates on disaccharides using chondroitin-ABC-LiAZ (EC 4.2.2.4) in the cell lysate; (3) labeling of DS disaccharides fluorescent dye (for example, 2-aminoacridone, AMAC); (4) separation of DS disaccharides (e.g., capillary zone electrophoresis, CZE); (5) detection of labeled DS disaccharides (e.g., laser-induced fluorescence, LIF). Such methods are described, for example, in Zinellu et al., Electrophoresis 2:2439-2447, 2007, and Lamari et al., J. Chromatogr. B 730:129-133, 1999 (review Volpi et al., Electrophoresis 29:3095-3106, 2008).

Table 11 presents the percentage degrades what I DS using cells GM00519, processed ARSB, when determining by measuring the amount of disaccharide containing N-atsetilgalaktozamin-4-sulfate (disaccharide 4S), which is the predominant disaccharide DS. Similar results were obtained using cells GM01391 treated with IDU.

Table 11
Depletion DS by ARSB in cell analysisin vitro
The age of the cells (weeks)Cells GM00519 (% degradation)a
386
492
592
689
aThe percentage degradation was calculated by measuring the area under the curve of the disaccharide 4S identified by scanning CZE-LIF in the treated ARSB lysates compared with untreated cells

The above analysis showed that the target cell capture recombinant ARSB and IDU person, which are then localized in the lysosomes, where they carry out the degradation of their natural substrate, intracellular DS.

The experiment to detect on the PS was performed to determine the concentration, in which IDU becomes rate-limiting in this cell analysis. Cells GM01391 were cultured in 12-hole tablets. 4 weeks after the closing of the monolayer, the cells were subjected to different concentrations of IDU, from 0.8 nm to 25 nm, for 6 or 26 hours. Cell lysates were obtained and processed as described above. Has determined that the IDU does not become the rate-limiting below 1 nm.

In the second experiment, to identify the dose, the cells GM01391 3 weeks after the closing of the monolayer was subjected to various concentrations of IDU, from 0.01 to 0.2 nm, for 2 days. Cell lysates were obtained and processed as described above. In this experiment, cell lysates were added to a known amount of internal standard monosaccharide, GlcNAc-6S, exit control during processing. As shown in figure 11, in the treated IDU cells GM01391 observed a dose-dependent decrease in the number of substrate DS.

In a similar experiment to identify the dose, the cells GM00519 3 weeks after the closing of the monolayer was subjected to various concentrations of ARSB, from 0.001 to 0.06 nm, for 5 days. Cell lysates were obtained and processed as described above. In this experiment, cell lysates were added to a known amount of internal standard monosaccharide, GlcNAc-6S, exit control during processing. As before the tableno in figure 12, in the treated ARSB cells GM00519 observed a dose-dependent decrease in the number of substrate DS.

Developed cell analysisin vitrofor measuring the activity of recombinant lysosomal enzyme sulfatase human GALNS, degradation of its natural substrate, i.e. intracellular containing keratinolytic (KS) substrates.

Scarce on GALNS MQCH cells were cultured as described in example 7 above, and treated with recombinant human GALNS in quantities of 1 or 10 nm. After treatment, cell lysates MQCH received and digested with certanty II (EC 3.2.1), which destroys the larger oligosaccharides KS to disaccharides KS. Disaccharides KS noted AMAC, separated by CZE and subjected to detection by LIF, as described above, for DS disaccharides. GlcNAc-6S, a monosaccharide KS, was added to the cell lysates as an internal control for output during processing. Measured quantities of the two characteristic disaccharides KS, Gal6S-GlcNAc6S and Gal-GlcNAc6S, and the obtained data were corrected for the number of allocated GlcNAc6S. Table 12 presents the percentage degradation of KS using MQCH cells treated with GALNS, when determining by measuring the amount of two characteristic disaccharides KS.

Table 12
Depletion KS through GALNS in cell analysisin vitro
Gal6S-GlcNAc6SGal-GlcNAc6S
1 nm GALNS85,7a78,5b
10 nm GALNS88,681,5
a,bThe percentage degradation was calculated by measuring the area under the curve for Gal6S-GlcNAc6S and Gal-GlcNAc6S identified when scanning CZE-LIF in lysates treated GALNS compared with untreated cells MQCH, and correction by area under the curve for added control GlcNAc6S

The above analysis showed that the target cell capture recombinant human GALNS, which is then localized in the lysosomes, where GALNS was carried out by the degradation of its natural substrate, intracellular KS.

In General, these results demonstrated that the activity of recombinant lysosomal enzymes person, ARSB, IDU and GALNS, against the degradation of their natural substrates can be measured and quantified in cell analysisin vitro. It is clear that this cell analysisin vitroyou can easily modify to measure and quantify the activity of other lysosomal the x of the enzymes sulfates, along with a wide variety of recombinant lysosomal enzymes.

EXAMPLE IX

DELIVERY of RECOMBINANT N-ATSETILGALAKTOZAMIN-6-SULFATASE (GALNS) person IN a PARTICULAR TISSUE

Evaluated the ability of recombinant N-atsetilgalaktozamin-6-sulfatase (GALNS) of a person, expressed in cells G71 and purified, to be delivered to a particular tissue affected by deficit GALNS or associate with him, when administered to mice.

Highly specific distribution of createsurface provides highly characteristic phenotype of mucopolysaccharidosis type IVa (MPS IVA), or syndrome Morquio. Keratinolytic mainly found in cartilage (connection plates of bone, heart valves, larynx and nasal septum) and in the cornea, and it is in these tissues accumulation of keratomalacia in patients with MPS IVA. Thus, for an N-atsetilgalaktozamin-6-sulfatase (GALNS), which is deficient in MPS IVA, or syndrome Morquio, it is important to show delivery of the GALNS enzyme in the growth plate of long bones, heart valves, corneas, larynx and nose. To study these specific tissues, which are poorly vascularization target, investigated the delivery of fluorescent GALNS in mice.

Mice were tested in two ways immunohistochemical staining: (1) human GALNS, anywhereman the Alexa 488, and (2) unconjugated human GALNS. Conjugation GALNS person with Alexa 488 was performed using a kit for labelling maleinimide Molecular Probes Alexa Fluor 488 C5maleimid labeling kit (A-10254). The conjugation reaction with maleinimide resulted in the ratio of 1:1 between the label and the protein.

To confirm that the fluorescent label does not prevent the capture of GALNS, conducted immunocytochemically experiment using cultured synoviocytes (ATCC # CRL-1832). For comparison unconjugated GALNS conjugated with GALNS (GALNS-A488 or GALNS-A555) used the standard analysis of capture. Cells were incubated with GALNS enzyme for 4 hours followed by displacement of α-L-iduronidase (IDU) for 2 hours. The result showed that the conjugation with Alexa 488 not prevented the capture of cells. In the figure 13 presents the estimated Kd for GALNS, GALNS-A488 and GALNS-A555. The capture was measured using ELISA with antigen in the cell lysate, and not by the activity of the enzyme due to tagging inactivated enzyme. Kd unconjugated and conjugated GALNS enzyme was defined as approximately equal.

To determine the stability of the fluorescent label after turning on the GALNS enzyme in the cell, were immune staining for unconjugated and conjugated GALNS. Used for staining, the primary antibody was a purified with protein G antibodies what about the rabbit against GALNS at a concentration of 1 µg/ml All images were obtained on wide-field EPI-fluorescence microscope (Leica IRE2 using the software MetaMorph. To measure colocalization in these images was required deconvolution of multiple images due to the presence of neprosteno light. Reverse convolution was performed using the program for visualization AutoQuant/AutoDeblur using theoretical functions of a point of deployment (blind algorithm).

Immune staining showed good overlap with the amplified signal on the material GALNS-A488. The observed increase in sensitivity was due to the fact that both primary and secondary antibodies were polyclonal.

To determine whether the GALNS enzyme is aimed at lysosome, were immune staining of cultured synoviocytes using Molecular Probes Lysotracker or another enzyme that is localized in lysosome. It turned out that Lysotracker showed some overlap with the GALNS enzyme-488; however, the staining was not uniform. Eviction within 2 h of recombinant N-acetylgalactosamine-4-sulfatase (rhASB) human lysosomal enzyme did not show any colocalization with GALNS.

The above experiments showed that the GALNS enzyme-A488 red cells and is localized in lysosome, and you can use the La definition bearsdley in vivo.

Conducted two surveysin vivo. The first pilot study was performed with bolus injection of a single dose (10 mg/kg) into the tail vein of normal Balb/c mice, followed by a second study with multiple (5) injections of 10 mg/kg every two days into the tail vein of normal Balb/c mice. In table 13 and table 14 describes the experiments for the first and second studies, respectively.

Table 13
The experimental design of the first pilot studies
GroupOnlyTime 2 hoursTime 24 hours
Control in the form of PBS422
GALNS-A488422
Unlabeled GALNS422
Unlabeled ASB110

Table 14
Diagram of the second experiment research
GroupOnlyTime 2 hoursTime : 4 hoursTime 8 hours
Control in the form of PBS2101
Control in the form of PBS/Cys4202
GALNS-A4889333
Unlabeled GALNS6303
Unlabeled ASB3201

In the first pilot study, heart, liver and joint leg/thigh was received at time 2 hours and 24 hours. In the second study received heart is, kidney, liver and bone with the quadriceps muscle and the soleus muscle at time 2 hours, 4 hours and 8 hours. For both studies, heart, kidney and liver were fixed by immersion in 4% paraformaldehyde (PFA) for 4 days, immersed in paraffin, then make a slice thickness of 7 μm. Bone, including the muscle in the second study, were fixed by immersion in 4% PFA for 8 days, decalzinirute, was dipped in paraffin and made them a slice thickness of 7 μm.

Images of mice that were injected with GALNS-A488 received on a laser scanning confocal microscope Zeiss. For the analysis in the first pilot study received one confocal set on the sample valve of the heart and liver and used it for volumetric analysis. For the record growth received two confocal set/sample and used for volumetric analysis. In the second study received one confocal set/sample valve of the heart, kidneys and liver and used for volumetric analysis; two confocal set/sample received for record growth and the rest of the zone of cartilage (zrc) and used for volumetric analysis.

Studies on the visualization of confocal microscopy has made the following conclusions: (1) it was possible to detect fluorescent GALNSin vivo; (2) the signal was specific (no background) and localisations who was lysosomal; (3) the presence of GALNS was shown in the sinusoidal cell in the liver; (5) in the heart, the GALNS enzyme was present in the partition and the atrium, but most importantly, he was clearly visible at the level of the heart valves, where he was more deeply divided after repeated injections; (6) in the joint of the femur/tibia, the GALNS enzyme was in the mineralized part of the bone (epiphysis), as well as in the bone marrow. GALNS was present in the growth plate. More specifically, GALNS was distributed in the chondrocytes of the resting zone (or zone of reserve cartilage), was present at the beginning of the proliferative zone and reappeared in large numbers in the zone of ossification at the end of the growth plate. Although it is difficult to quantify the cumulative effect of multiple injections, the second study, as it turned out, showed a wider distribution. Table 15 presents a summary of research visualization by confocal microscopy.

Table 15
The biodistribution GALNS in mice
ClothLocalization
Bone (femur)
The mineralized area Yes
Bone marrowYes
The growth plateYes
Heart
Heart valveYes
AtriumYes
PartitionYes
Liver
Hepatocytesno
Sinusoidal cellsYes

For secondary staining, the initial stage was the optimization of the primary antibodies GALNS. Various tissues were stained with dilutions from 1:100 to 1:400 using purified by protein G antibody rabbit against GALNS. Results in the first pilot study showed that a dilution of 1:100 was optimal for high signal-to-noise. This result was confirmed in a second study. The remaining slides were treated with a dilution of primary antibody 1:100 dilution of secondary antibody 1:1000.

The signal for Balb/c mice, which were injected GALNS was higher than the control signal (i.e., for mice, which were injected PS-Cys) staining of purified with protein G antibody against GALNS. To confirm that the GALNS enzyme was localized in lysosome, sections were stained with antibody against LAMP1. LAMP1 is a marker of lysosomes. The image showed overlap between antibodies against LAMP1 and against GALNS, indicating that the GALNS enzyme was localized in lysosome.

In General, two studiesin vivoindicate that the biodistribution GALNS associated with vascularity, i.e. more vascularity tissue have a greater fluorescent signal. More importantly, studies have shown the presence of GALNS in the areas of accumulation of createsurface syndrome, Morquio, even if these areas are poorly vascularsurgery.

EXAMPLE X

EFFECTS of RECOMBINANT N-ATSETILGALAKTOZAMIN-6-SULFATASE (GALNS) of HUMANS AND OTHER LYSOSOMAL ENZYMES SULFATES IN MICE DEFICIENT activity of the LYSOSOMAL ENZYME SULFATASE

Evaluated the effects of active vysokotsentralizovannym lysosomal enzymes sulfates man according to the invention, for example, recombinant N-atsetilgalaktozamin-6-sulfatase (GALNS) human, mice deficient activity of the lysosomal enzyme sulfatase.

Recombinant protein of human GALNS expressed in cells G71S and cleansed. Other recombinant lysosomal enzymes sulfatase person can Express and clear, essentially according to the methods described is herein or known in this field.

There are several models in mice for deficiency of lysosomal enzyme sulfatase of human rights, including: the metachromatic leukodystrophy (MLD) (deficit Ukrainian (A), (Hess et al., Proc. Natl. Acad. Sci. USA 93:14821-14826, 1996), mucopolysaccharidosis type VI (MPS VI), or the syndrome Maroto-Lamy (deficit Ukrainian (B) (Evers et al., Proc. Natl. Acad. Sci. USA 93:8214-8219, 1996), mucopolysaccharidosis type II (MPS II or hunter syndrome (deficit iduronate-2-sulfatase) (Muenzer et al., Acta Paediatr. Suppl. 91(439): 98-99, 2002; Cardone et al., Hum. Mol. Genet. 15:1225-1236, 2006), mucopolysacharides type IIIa (MPS IIIa), or a syndrome, Sanfilippo's title A (deficit sulfamidate/heparan-N-sulfatase) (Bhaumik et al., Glycohiology 9(12): 1389-1396, 1999), mucopolysaccharidosis type IVa (MPS IVa), or the syndrome Morquio A (deficiency of N-atsetilgalaktozamin-6-sulfatase) (Tomatsu et al., Hum. Mol. Genet. 12:3349-3358, 2003), and multiple sulfatase deficiency (MSD) (deficit modifying sulfatase factor 1) (Settembre et al., Proc. Natl. Acad. ScL USA 104:4506-4511, 2007). The model in mice for mucopolysaccharidosis type IIId (MPS IIId), or syndrome, Sanfilippo's title D (deficiency of N-acetylglucosamine-6-sulfatase), yet to be described.

Model in mice for deficiency of lysosomal enzyme sulfatase person can be used to assess the possibility of enzyme replacement therapy (ERT) as a method for the treatment of lysosomal diseases of accumulation. For example, mice with knockout of MPS IVa (GALNS-/-; Tomatsu et al., Hum. Mol. Genet. 12:3349-3358, 2003) do not have is Asia detection of enzyme activity GALNS and have elevated levels of glycosaminoglycans (GAG) in the urine, i.e. createsurface and chondroitin-6-sulfate, and GAG accumulation in many tissues and organs, such as liver, kidney, spleen, heart, brain and cartilage. However, mouse GALNS-/-do not have skeletal anomalies associated with human disease. Was developed another model in mice MPS IVa, which is expressed inactive GALNS human and mutant inactive endogenous GALNS mouse (mouse GALNStm(hC79S.mC76S)slu; Tomatsu et al., Hum. Mol. Genet. 14:3321 - 3335, 2005). In mice GALNStm(hC79S.mC76S)sluthat are not amenable to detection of enzyme activity GALNS, GAG excretion in the urine is increased, GAG accumulate in many tissues, including internal organs, brain, cornea, bone, ligament, and bone marrow, the accumulation in the lysosomes is expressed in many tissues, and accumulation in the bones is obvious. Pathological changes in mice GALNStm(hC79S.mC76S)sludifferent from the pathological changes observed in mice GAINS-/-. However, like mice GAINS-/-mouse GALNStm(hC79S.mC76S)sludo not have disorders of the skeleton associated with human disease. Thus, mice GAINS-/-or GALNStm(hC79S.mC76S)slucan be used to study the effect of the introduction of recombinant human GALNS at elevated GAG in the urine and accumulation of GAG in the tissues.

Mice GAINS-/-, GALNStm(hC79S.mC76S)sluor wild the IPA at the age of four weeks weekly intravenous injections (n = at least 6 or 8 per group) of different doses of recombinant human GALNS (for example, 0,1, 0,3, 1, 3, 10 mg/kg) or control in the form of media until the age of 16-20 weeks, and then kill them for histological study. The urine of the mice collect and define the GAG excretion in the urine, as described (Tomatsu et al., Hum. Mol. Genet. 12: 3349-3358, 2003). Pathological examination of various tissues were carried out as described (Tomatsu et al., Hum. Mol. Genet. 12:3349-3358, 2003).

When using mice GAINS-/-or GALNStm(hC79S.mC76S)slu, it is expected that recombinant human GALNS will demonstrate the ability of the invention to reduce: (1) excretion of GAG with urine; (2) the accumulation of GAG in many tissues, such as visceral organs, brain, cornea, bone, ligaments, and bone marrow; (3) lysosomal accumulation in many tissues; (4) accumulation in the bones.

Effect of recombinant human GALNS explore the model in mice for multiple sulfatase deficiency (MSD) (mouse SUMF-/-; Settembre et al., Proc. Natl. Acad. Sci USA 104: 4506-4511, 2007). Because mouse SUMF1-/-show frequent mortality at early life stages, injection of these mice with recombinant human GALNS started more early than described for mice GALNS-/-.

Following the methods known in this field, the effects of other recombinant lysosomal enzymes sulfates person, i.e. a Ukrainian, Ukrainian B, iduronate-2-sulfatase, sulfamidate/heparin-N-sulfatase and N-acetylglucosamine-6-sulf the basins, explore models in mice for MLD (mouseASA-/-; Hess et al., Proc. Natl. Acad. Sci. USA 93: 14821-14826, 1996), MPS VI (mouseAsl-s-/-; Evers et al., Proc. Natl. Acad. Sci. USA 93:8214-8219, 1996), MPS II (mouseidsy/-; Cardone et al., Hum. Mol. Genet. 15: 1225-1236, 2006), MPS IIIa (Bhaumik et al., Glycobiology 9(12): 1389-1396, 1999) and MSD (mouse SUMF1-/-; Settembre et al., Proc. Natl. Acad. Sci. USA 104:4506-4511, 2007).

EXAMPLE XI

TREATMENT of PATIENTS-PEOPLE WITH MUCOPOLYSACCHARIDOSIS TYPE IVA (OR SYNDROME MORQUIO) OR OTHER DEFICIENCIES of LYSOSOMAL ENZYMES SULFATES RECOMBINANT N-ATSETILGALAKTOZAMIN-6-SULFATASE (GALNS) of HUMAN AND OTHER SULFATASE ENZYMES

It is assumed that enzyme replacement therapy with recombinant enzyme, i.e. N-atsetilgalaktozamin-6-sulfatase (GALNS) person, subject to patients-people with clinical manifestations of the phenotype deficiency of lysosomal enzyme sulfatase, such as patients with a diagnosis of mucopolysaccharidosis type IVA (MPS IVa, or syndrome Morquio). All patients suffering from deficiency of lysosomal enzyme sulfatase, there are some manifestations of clinical signs of excessive or harmful accumulation in the internal organs and soft tissues of the stored material in their lysosomes, as manifested by varying degrees of functional impairment or deterioration of health associated with specific lysosomal disease accumulation. All the patient is with MPS IVa are manifestations of some clinical signs of deformation of the bones, low growth and disturbed gait and/or accumulation of glycosaminoglycans and collagen (GAG) in blood or urine with different degrees of functional impairment.

Preferably, in a patient suffering from a deficiency of the lysosomal enzyme sulfatase, monitor levels of enzymes to confirm there is no activity or reduced activity of the lysosomal enzyme sulfatase in their tissues. Patients with the activity of the lysosomal enzyme is less than 10%, preferably less than 5%, more preferably less than 2% and even more preferably less than 1%, the rest being healthy subjects, are appropriate candidates for treatment relevant lysosomal enzyme by sulfatases. When determining the activity of the lysosomal enzyme sulfatase the patient can receive data before, during and after therapy.

Efficiency is determined by measuring the percentage reduction in the excretion of urine substrate, i.e. fractions (GAG), lysosomal enzyme sulfatase over time. The levels of GAG in the urine of patients suffering from a deficiency of the lysosomal enzyme sulfatase, compared with normal levels of excretion, and/or levels in patients without treatment, suffering from deficiency of the same lysosomal enzyme sulfatase, and/or levels of the same patient before therapy of lysosomal enzyme sulfate is Oh. Reliable indicators for measuring the individual response to therapy are more than 25% reduction, preferably greater than 50% decrease excretion of non-degraded GAG after therapy of lysosomal enzyme by sulfatases.

Efficiency can be defined to reduce signs and symptoms and pathology associated with lysosomal disease accumulation. Efficiency can be determined by tissue biopsy and analysis of cells and/or lysosomes to determine the extent to which GAG decrease in the lysosomes, cells or tissues. Efficiency can be defined using a functional assessment, which may be objective or subjective (e.g., reduced pain or difficulty functioning, increased muscle strength or endurance, increased cardiac output, endurance during physical activity, changes in body weight, growth or appearance etc).

The pharmaceutical composition comprises a recombinant human GALNS, expressed in cells G71S and purified, manufactured by methods known in this field. Preferred is the introduction of a pharmaceutical composition according to the invention intravenously.

The basic structure of the original clinical trial to study the effect of the introduction of recombinant human GALNS patients with MPS IVa involves open study the security-related/efficacy with increasing dose at different doses, when different doses of the enzyme is administered to patients intravenously with fixed intervals, for example, without limitation, injection of enzymes once a week.

For patients with MPS IVa, efficiency is determined by measuring, for example, reduced GAG in the blood or urine, which can probably occur when ERT for weeks, increased stamina during tests of cardiac, pulmonary and/or motor function, which may be observed during ERT in the months and/or changes of the skeleton and/or growth of the organism, which are likely to occur when ERT during the years.

Measurement of GAG in the urine is suitable for establishing an appropriate dosing regimen and to determine the effectiveness, by measuring the percentage reduction in GAG excretion in urine over time.

You can use a variety of endurance tests, including, for example, is not limited to, tests of distance (distance traveled for 6 or 12 minutes), climbing stairs (steps per minute) and pulmonary/respiratory function, including cardiac function (ECG, echocardiogram), pulmonary function (high-flow, FEV1maximum expiratory flow).

For younger patients being treated for extended periods of time, it is possible to measure the growth (height).

Lysosomal storage disorders, associated what's with the lack of activity of the lysosomal enzyme sulfatase, which can be treated or prevented using the methods of the present invention, are: the metachromatic leukodystrophy (MLD), mucopolysaccharidosis type VI (MPS VI), or the syndrome Maroto-Lamy, mucopolysaccharidosis type II (MPS II or hunter syndrome, mucopolysacharides type IIIa (MPS IIIa), or a syndrome, Sanfilippo's title A, mucopolysaccharidosis type IIId (MPS IIId), or syndrome, Sanfilippo's title D, mucopolysaccharidosis type IVa (MPS TVa), or the syndrome Morquio A, or multiple sulfatase deficiency (MSD). For each of lysosomal storage disorders, recombinant lysosomal enzyme sulfatase may include specific lysosomal enzyme sulfatase.

For methods involving MLD, preferred lysosomal enzyme by sulfatases is arylsulfatase A. For methods involving MPS VI, preferred lysosomal enzyme by sulfatases is arylsulfatase B. For methods involving MPS II, preferred lysosomal enzyme by sulfatases is iduronate-2-sulfatase. For methods involving MPS IIIA, preferred lysosomal enzyme by sulfatases is sulfamidate/heparan-N-sulfatase. For methods involving MPS IIID, preferred lysosomal enzyme by sulfatases is N-acetylglucosamine-β-sulfatase. For methods involving MPS IVA, preferred lysosomal what enzyme sulfatase is N-atsetilgalaktozamin-6-sulfatase. For methods involving MSD, preferred lysosomal enzyme by sulfatases is N-atsetilgalaktozamin-6-sulfatase.

It is expected that the experts can devise various modifications and variations of the invention specified in the illustrated examples. Therefore, the invention should be subject only to such limitations as are specified in the attached claims.

1. The purified recombinant enzyme N-atsetilgalaktozamin-6-sulfatase (GALNS), where the specified enzyme comprises the amino acid sequence at least 95% identical to amino acids 27-522 SEQ ID NO:4, suitable for treatment of a subject suffering from lysosomal disease accumulation, which is caused by deficiency of the specified GALNS or associated with him, where
(a) the specified drug GALNS enzyme has a purity of at least approximately 95% when determining the staining of Kumasi blue when SDS-PAGE in non conditions; and
(b) a cysteine residue at position 79 of at least about 50% of the molecules of the GALNS enzyme is in the specified drug GALNS enzyme is converted to Cα-formylglycine (FGly);
where specified GALNS enzyme
(c) is glycosylated N-linked glycosylation at asparagine residues at positions 204 and 423, where at least about 50% by oligom nosnik circuits, attached to the asparagine residue at position 204, are bis-phosphorylated.

2. The drug GALNS according to claim 1, where lysosomal disease accumulation is a mucopolysaccharidosis type IVa (MPS IVa), or the syndrome Morquio A.

3. The drug GALNS according to claim 1, where lysosomal disease accumulation is a multiple sulfatase failure.

4. The drug GALNS according to claim 1, where the GALNS enzyme has a major band at approximately 55-60 kDa, which is at least approximately 75% of the visible proteins when determining the staining of Kumasi blue when SDS-PAGE in reducing conditions.

5. The drug GALNS according to claim 1, where the GALNS enzyme has a major band at approximately 55-60 kDa, which is at least approximately 85% of the visible proteins when determining the staining of Kumasi blue when SDS-PAGE in reducing conditions.

6. The drug GALNS according to claim 1, where the GALNS enzyme has a major band at approximately 55-60 kDa, which is at least approximately 90% of the visible proteins when determining the staining of Kumasi blue when SDS-PAGE in reducing conditions.

7. The drug GALNS according to claim 1, where the cysteine residue at position 79 of at least approximately 70% of the molecules of the GALNS enzyme is in the specified drug GALNS enzyme is converted to Cα-formylglycine (FGly).

8. The drug GALNS at p., where the cysteine residue at position 79 of at least approximately 90% of the molecules of the GALNS enzyme is in the specified drug GALNS enzyme is converted to Cα-formylglycine (FGly).

9. The drug GALNS according to claim 1, where the GALNS enzyme is a protein that contains a cellular targeting signal located at the N - or C-end of the GALNS enzyme.

10. The drug GALNS according to claim 9, where the cellular targeting signal contains the target bone peptide.

11. The drug GALNS of claim 10, where the target bone peptide contains six residues aspartic acid.

12. The method of treatment of a subject suffering from mucopolysaccharidosis type IVa (MPS IVa), or syndrome Morquio A, or multiple sulfatase deficiency (MSD), including introduction to the subject a therapeutically effective amount of purified recombinant enzyme N-atsetilgalaktozamin-6-sulfatase (GALNS), where the specified enzyme comprises the amino acid sequence at least 95% identical to amino acids 27-522 SEQ ID NO:4, where
(a) the specified drug GALNS enzyme has a purity of at least approximately 95% when determining the staining of Kumasi blue when SDS-PAGE in non conditions; and
(b) a cysteine residue at position 79 of at least about 50% of the molecules of the GALNS enzyme is in the specified drug GALNS enzyme is converted to Cαwhere specified GALNS enzyme
(c) is glycosylated N-linked glycosylation at asparagine residues at positions 204 and 423, where at least about 50% oligomannose chains attached to the asparagine residue at position 204, are bis-phosphorylated.

13. The method according to item 12, where the subject is suffering from MPS Iva, or syndrome Morquio A.

14. The method according to item 12, where the subject is suffering from MSD.

15. The method according to item 12, where the GALNS enzyme has a major band at approximately 55-60 kDa, which is at least approximately 75% of the visible proteins when determining the staining of Kumasi blue when SDS-PAGE in reducing conditions.

16. The method according to item 12, where the GALNS enzyme has a major band at approximately 55-60 kDa, which is at least approximately 85% of the visible proteins when determining the staining of Kumasi blue when SDS-PAGE in reducing conditions.

17. The method according to item 12, where the GALNS enzyme has a major band at approximately 55-60 kDa, which is at least approximately 90% of the visible proteins when determining the staining of Kumasi blue when SDS-PAGE in reducing conditions.

18. The method according to item 12, where the cysteine residue at position 79 of at least approximately 70% of the molecules of the GALNS enzyme is in the specified drug GALNS enzyme is converted to Cα-formylglycine the (FGly).

19. The method according to item 12, where the cysteine residue at position 79 of at least approximately 90% of the molecules of the GALNS enzyme is in the specified drug GALNS enzyme is converted to Cα-formylglycine (FGly).

20. The method according to item 13, where the effectiveness of treatment is determined by measuring urinary excretion of createsurface (KS) for a subject, where the KS levels in the urine of a subject suffering from mucopolysaccharidosis type IVa (MPS IVa), or syndrome Morquio A, compared to the KS levels in the urine of normal subjects and/or subject not receiving treatment, suffering from mucopolysaccharidosis
type IVa (MPS IVa), or syndrome Morquio A, and/or from the same subject prior to treatment with GALNS enzyme.

21. The method according to claim 20, where, after treatment of the GALNS enzyme is more than 25% decrease of KS in urine.

22. The method according to claim 20, where, after treatment of the GALNS enzyme is more than 50% decrease of KS in urine.

23. The method according to item 13, where the effectiveness of treatment is determined by the functional assessments of the subject by measuring endurance tests of walking, climbing stairs and pulmonary/respiratory function.

24. The method according to item 23, where in the test distance measure distance traveled for 6 or 12 minutes.

25. The method according to item 23, where in the test of stairs measure the number of steps traveled per minute.

26. The method according to item 23, where pulmonary/respiratory function measured by measuring heart f is NCLI (echocardiogram) or by measurement of lung function (high-flow, FEV1or peak expiratory flow rate).

27. The method according to item 12, where the GALNS enzyme is a protein that contains a cellular targeting signal located at the N - or C-end of the GALNS enzyme.

28. The method according to item 27, where cellular targeting signal contains the target bone peptide.

29. The method according to p, where the target bone peptide contains six residues of aspartic acid.



 

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3 ex

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2 ex

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1 cl, 9 ex, 3 tbl

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