PT1740204T

PT1740204T - Medicinal use of alpha-mannosidase

Info

Publication number: PT1740204T
Application number: PT57151466T
Authority: PT
Inventors: Andersson Claes; Fogh Jens; Weigelt Cecilia; Prieto Roces Diego; Von Figura Kurt; Irani Meher
Original assignee: Chiesi Farm Spa
Priority date: 2004-04-01
Filing date: 2005-04-01
Publication date: 2018-06-20
Also published as: HUE037787T2; LT1740204T; TR201808258T4; ES2672646T3

Abstract

Uso de una alfa-manosidasa lisosómica para la preparación de un medicamento para el tratamiento de alfamanosidosis, en el que dicho medicamento debe administrarse por administración intravenosa.Use of a lysosomal alpha-mannosidase for the preparation of a medicament for the treatment of alpha-mannosis, wherein said medicament must be administered by intravenous administration.

Description

DESCRIPTION "ALPHA-MANOSIDASE MEDICAL USE"

FIELD OF THE INVENTION The present invention provides means and strategies for treating lysosomal storage disorder alpha-mannosidosis by enzyme replacement therapy. In particular, the invention relates to the reduction of neutral mannose-rich oligosaccharides stored in cells within the central nervous system.

BACKGROUND OF THE INVENTION

Alpha-mannosidosis Alpha-mannosidosis is an autosomal recessive disease occurring on a worldwide scale between 1/1 000 000 and 1/500 000. Mannosidosis is found in all ethnic groups in Europe, America, Africa, and also in Asia. It is detected in all countries with a good diagnostic service for lysosomal storage disorders at a similar frequency. They are born apparently healthy, yet the symptoms of disease are progressive. Alpha-mannosidosis presents clinical heterogeneity, ranging from very severe to very mild. Typical clinical symptoms are: mental retardation, skeletal changes, impaired immune system resulting in recurrent infections, hearing loss, and often the disease is associated with atypical facial features such as a coarse face, prominent forehead, a flattened nasal bridge, a small nose and a wide mouth. In very severe cases (type I mannosidosis) children suffer from hepatosplenomegaly, and they die during the first years of life. Probably, this early death is caused by serious infections due to immunodeficiency caused by the disease. In milder cases (type 2 mannosidosis) patients usually reach adulthood. Skeletal weaknesses of patients result in the need for wheelchairs at 20 to 40 years of age. The disease causes diffuse dysfunction of the brain that often results in poor mental performance that excludes everything except the most basic skills of simple reading and writing. These problems associated with hearing impairments and other clinical manifestations prevent the patient from living independently and the consequence is the need for lifelong care.

Lysosomal alpha-mannosidase Alpha-mannosidosis results from a deficient lysosomal alpha-mannosidase activity (LAMAN, EC3.2.1.24). The disease is characterized by the massive intracellular accumulation of mannose-rich oligosaccharides, i.e., oligosaccharides having al, 2-, al, 3- and Î ±, 6-mannosyl residues at their non-reducing ends. These oligosaccharides result mainly from the intralysossomic degradation of glycoproteins with N-linked oligosaccharides. However, some result from the catabolism of dolicol-bound oligosaccharides and misfolded glycoproteins redirected into the cytosol for degradation by the proteasome (4,5). Lysosomal storage is observed in a wide range of cell and tissue types, including neurons in all regions of the brain. LAMAN is an exoglycosidase which hydrolyzes these terminal non-reducing alpha-D-mannosis residues in alpha-D-mannosides from the non-reducing end during orderly degradation of the N-linked glycoproteins (Aronson and Kuranda FASEB J 3: 2615-2622. 1989). The human enzyme is synthesized as a single 1011 amino acid polypeptide with a putative 49 residue signal peptide which is processed into three major 15, 42 and 70 kD glycopeptides. (Nilssen, et al., Hum. Mol.Genet, 6, 717-726, 1997). The lysosomal alpha-mannosidase gene The gene encoding LAMAN (MANB) is located on chromosome 19 (19cen-q12), (Kaneda et al., Chromosome 95: 8-12, 1987). The MANB consists of 24 exons, which covers 21.5 kb (GenBank Accession Nos. U60885-U60899; Riise et al., Genomics 42: 200-207, 1997). The LAMAN transcript is 3 500 nucleotides (nts) and contains an open reading frame encoding 1011 amino acids (GenBank U60266.1). Cloning and sequencing of the human cDNA encoding LAMAN have been published in three papers (Nilssen et al., Hum.Mol.Genet.6, 717-726 1997, Liao et al., J. Biol. Chem., 271, 28348-28358 1996, Nebes et al., Biochem.Biophys.Res.Commun 200, 239-245, 1994). Curiously, the three sequences are not identical. When compared to the sequence of Nilssen et al. (accession # U60266.1), Liao et al. and Nebes et al. observed a modification of TA to AT at positions 1670 and 1671 which resulted in a substitution of valine for aspartic acid.

Mutations

In 1997 Nilssen et al. (Hum Mol Genet 6: 717-726) described the first disease-causing mutation in the fibroblasts of two siblings with mannosidosis in a highly inbred Palestinian family. In these two patients, the cause of the disease is a point mutation in the gene, with the nucleotide Adenine substituted by Thymidine. Since then, several genetic causes have been found for the disease in humans. Nowadays mutations that cause alpha-mannosidosis have been characterized in more than 60 patients, mainly in Europeans. These mutations vary in type, encompassing nucleotide substitutions, deletions, and additions, as they span most of the 24 gene exons (Berg et al., Am J Genet 64: 77-88, 1999).

The consequences of mutations at the protein level also range from amino acid substitutions affecting both the active site and folding, to early grating deviations and premature termination codons, which result in a completely inactive product. The amino acid sequence and structure of the enzyme in a number of different species. When comparing species, many of the observed human and bovine mutations are located in a highly "conserved" mannosidase polypeptide region for various species such as humans, cattle, cat, whale, birds, cod and mucilaginous background, indicating that these regions are very important for the function of the enzyme.

Despite the many different types of mutations, it appears that all mutations cause the complete complete loss of enzymatic activity in cells. Thus, apparently, variations relative to clinical severity can not be explained by variations in enzyme activity. Instead, these variations are brought about by other factors, which are attributed to the body's general ability to reduce the harmful effects of the oligomannosides that accumulate, and by the means to prevent and treat infections and other clinical symptoms in patients.

Diagnosis The diagnosis of alpha-mannosidosis is currently based on clinical evaluation, detection of mannose-rich oligosaccharides in urine, and direct measurements of alpha-mannosidase activity in various cell types, such as leukocytes, fibroblasts, and amniocytes (Chester et al. , In: Durand P, O'Brian J (eds) Genetic errors of glycoprotein metabolism Edi-Ermes, Milan, pp. 89-120.

Beaudet. In: Scriver CR, Beaudet AL, Sly WA, Valle D (eds) The metabolic and molecular bases of inherited disease. Vol 5. McGraw-Hill, New York, pp 2529-2562. 1995).

Since symptoms are often mild initially and biochemical diagnosis is difficult, diagnosis is often made late in the course of the disease. It is obvious that patients and their families would benefit substantially from an early diagnosis.

Animal models Alpha-mannosidosis was described in cattle (Hocking et al., Biochem J 128: 69-78, 1972), cats (Walkley et al., Proc. Nat. Acad. Sci., 91: 2970-2974, 1994) and guinea pigs (Crawley et al., Pediatr Res 46: 501-509, 1999). A mouse model was recently generated by directed disruption of the alpha-mannosidase gene (Stinchi et al., Hum Mol Genet 8: 1366-72, 1999).

As in humans, alpha-mannosidosis appears to be elicited by specific mutations in the gene encoding lysosomal alpha-mannosidase. Berg et al. (Biochem. J. 328: 863-870, 1997) reported the purification of feline liver lysosomal alpha-mannosidase and determination of its cDNA sequence. The active enzyme consists of 3 polypeptides, with molecular weights of 72, 41 and 12 kD. Analogously to the human enzyme it has been shown that the feline enzyme is synthesized as a single-stranded precursor with a putative signal peptide of 50 amino acids followed by a polypeptide chain of 957 amino acids which is dissociated in the 3 polypeptides of the mature enzyme. The deduced amino acid sequence was 81.1% and 83.2% identical to the human and bovine sequences, respectively. A 4 bp deletion was identified in an affected Persian cat; suppression resulted in a shift of the codon 583 grid and premature termination at codon 645. It was not possible to detect enzymatic activity in the cat's liver. A domestic long-haired cat expressing a lighter phenotype had an enzymatic activity that was 2% of normal; this cat did not have suppression of 4 bp. Tollersrud et al. (Eur J Biochem 246: 410-419, 1997) purified the bovine kidney enzyme to homogeneity and cloned the gene. The gene was organized into 24 exons spanning 16 kb. Based on the gene sequence identified two mutations in cattle.

At the present time the mice with inactivated α-mannosidase gene provide the best pharmacological model available for the investigational treatment of α-mannosidosis to support regulatory compliance. Currently, there are no acceptable models that do not use live animals.

Medical necessity of therapy for alpha-mannosidosis In light of the serious clinical manifestations resulting from the accumulation of mannose-rich oligosaccharides, the lack of effective treatment for alpha-mannosidosis is well recognized. At present, the main therapeutic options for the treatment of the disease are bone marrow transplantation and enzyme replacement therapy.

Bone marrow transplant

In 1996 Walkley et al. (Proc Nat Nat. Acad Sci 1991, 91: 2970-294, 1994) published an article on 3 mannosidose kittens that were treated with bone marrow transplantation (BMT) in 1991. In the 2 animals that were sacrificed, not only in the body, but more importantly also in the brain. The third cat was well after 6 years. Normally, an untreated cat dies with 3-6 months. In 1987 a child with mannosidosis was treated with BMT (Will et al., Arch Dis Child 1987 Oct; 62 (10): 1044-9). He died after 18 weeks due to complications related to the procedure. In the brain, little enzymatic activity was found. This disappointing result could be explained by heavy immunosuppressive treatment before death, or by the fact that time is required for there to be an increase in the enzymatic activity in the brain after BMT. The donor was the mother (who as carrier would be expected to have less than 50% enzymatic activity) or it may be that BMT in humans has no effect on the function of the enzyme in the brain. In spite of having variable results, the few bone marrow transplantation attempts thus indicate that a successful graft can correct the clinical manifestations of alpha-mannosidosis, at least in part. However, the challenge to reduce serious complications related to the procedure when applying bone marrow transplantation in human therapy remains unresolved.

Enzyme replacement therapy

When lysosomal storage diseases were discovered, hopes were raised that they could be treated by enzyme replacement. Enzyme replacement therapy has been shown to be effective in Gaucher disease. When exogenous lysosomal glucocerebrosidase is injected into the patient, this enzyme is taken up by enzyme deficient cells (Barton et al., N Engl J Med 324: 1464-1470). Such uptake is regulated by certain receptors on the cell surface such as, for example, the mannose-6-phosphate receptor, which is almost ubiquitous on the surface of cells and other receptors such as the asialoglycoprotein receptor and the mannose receptor, which restricted to certain cell types such as monocyte / macrophage cell line cells and hepatocytes. Cell uptake of the enzyme is therefore strongly dependent on its glycosylation profile. If properly designed, the deficient enzyme could be replaced by regular injections of an exogenous enzyme in the same way that diabetic patients receive insulin. In vitro studies with purified active lysosomal alpha-mannosidase, added to enzyme-deficient fibroblast media showed a correction of lysosomal substrate accumulation. In vivo treatment, on the other hand, has been prevented, in part, by the problem of producing sufficient amount of enzymes, and by complications resulting from immunological reactions against the exogenous enzyme. However, more importantly, special considerations apply to lysosomal storage diseases with a major neurological component, such as alpha-mannosidosis, in which clinical manifestations are associated with increased lysosomal storage within the central nervous system. Thus, enzyme replacement therapy has not been shown to be effective against the acute neuronopathic variant of Gaucher disease (Prows et al., Am J Med Genet 71: 16-21). The administration of therapeutic enzymes to the brain is hindered by the lack of transport of these large molecules through the blood-brain barrier. From the general notion that the blood-brain barrier must be bypassed to observe an effect of therapeutic agents on the brain, the use of a wide variety of delivery systems has been envisaged. These include invasive techniques such as the osmotic opening of the blood-brain barrier with, for example, mannitol and non-invasive techniques such as chimeric enzyme receptor mediated endocytosis. Since enzyme replacement is expected to require administration of the enzyme on a regular basis, the use of invasive techniques should be avoided. The use of non-invasive techniques, on the other hand, did not provide promising results. It was considered that reduced storage in visceral organs and meninges could reduce the amount of oligosaccharides that is transported to the brain. However, it is considered that these considerations do not apply to lysosomal disorders in which neurological damage is primary and severe (Neufeld, EF Enzyme replacement therapy, in Lysosomal disorders of the brain (Platt, FM Walkley, S. V: (WO 02/099092 (Fogh et al., 2002) and Berg et al., Molecular Genetics and Metabolism, vol 73 , No. 1, April 2001, pages 18-29, discloses the use of a lysosomal alpha-mannosidase (LAMAN) for the preparation of a medicament for reducing the intracellular levels of mannose-rich oligosaccharides within the CNS by administering LAMAN "on cell membranes ".

Fogh et al., 2002, discusses enzyme replacement therapy and discloses that if appropriately designed the missing enzyme could be injected regularly as the diabetic individual injects insulin. Furthermore, Fogh et al. , 2002 and Berg et al. , 2001 disclose the expression and purification of human LAMAN from CHO cells and demonstrate in vitro the uptake of LAMAN in fibroblasts from a mannosidose patient. However, Fogh et al., 2002, and Berg et al., 2001, do not disclose administration by repeated intravenous injection at a specific frequency.

SUMMARY OF THE INVENTION

A first aspect of the present invention provides the use of a lysosomal alpha-mannosidase for the preparation of a medicament for the treatment of alpha-mannosidosis, wherein said medicament is to be administered by intravenous administration.

An embodiment of the present invention relates to the use, wherein the medicament is to be administered by infusion, or repeated intravenous injection at a rate corresponding to 1 to 5 injections daily, 1 to 5 injections per week, 1 to 5 injections every 2 weeks or 1 to 5 injections every month.

A second embodiment of the present invention relates to the use, wherein said medicament does not comprise a component with a known ability to target cells within the central nervous system.

A third embodiment of the present invention relates to the use, wherein the lysosomal alpha-mannosidase comprises the amino acid sequence of SEQ ID NO: 1.

Another embodiment of the present invention relates to the use, wherein said medicament does not comprise a component for targeting cells within the central nervous system and does not comprise a component capable of acting as a vehicle for passage through the blood-brain barrier.

Another embodiment of the present invention relates to the use, wherein said medicament is administered in an amount, which is within the range of 5 to 300 mU lysosomal alpha-mannosidase per gram of body weight of the subject to be treated.

Yet another embodiment of the present invention relates to the use, wherein a fraction of said lysosomal alpha-mannosidase consists of lysosomal alpha-mannosidase having one or more N-linked oligosaccharides carrying mannose-6-phosphate residues.

A preferred embodiment of the present invention relates to the use, wherein said lysosomal alpha-mannosidase is a recombinant enzyme.

DEFINITIONS AND TERMS

By "component" we mean broadly an element that is part of the whole. More specifically, the term refers to a substance present in a mixture. Such a substance may be a compound defined as a material consisting of elements chemically combined in defined mass proportions. Examples of inorganic compounds such as small molecular species, organic compounds such as carbohydrates including simple sugars and complex polysaccharide polymers, peptides, proteins, peptidomimetics, nucleic acids and antibodies. The term "central nervous system" refers to the brain, spinal cord, and spinal nerves. The central nervous system does not include the peripheral nerves in the arms, legs, muscles, and organs.

In the present context the term "brain" refers to that portion of the central nervous system which is located inside the skull including its two halves (right and left) referred to as hemispheres.

Also in the present context, extraneuronal cells such as cells in the endothelium and choroid plexus epithelia are not to be considered as part of the central nervous system and brain. Similarly, the brain and central nervous system do not understand the meninges. The "choroid plexus" is a vascular proliferation or fringe of the choroidal screen in the third and fourth ventricles and in the lateral cerebral ventricle; secretes the cerebrospinal fluid, thereby regulating intraventricular pressure to a certain extent. The term "meninges" refers to the three membranes covering the brain and spinal cord. The outer meninge is called the dura mater, and is the most resilient of the three. The middle layer is the pia mater, and the innermost thin layer is the arachnoid.

By "complex" or "conjugate" is meant a molecule or a group of molecules which are held together by van der Waals and / or electrostatic and / or covalent bonds. The term "exogenous" refers to a component that is introduced from the outside or produced outside the organism or system; specifically, the term refers to components that are not synthesized within the organism or system. The term "permeant" refers to a component that is capable of passing through a particular semipermeable membrane. Such a semipermeable membrane may be the blood-brain barrier and / or the maternal-fetal barrier.

The terms "blood-brain barrier" and "blood-brain barrier" refer to the barrier system that separates blood from the parenchyma of the central nervous system. The term "directed to" refers to the process of the process of having proteins with certain signals such that the proteins are targeted specifically to certain cell locations, for example the lysosome.

DETAILED DESCRIPTION The hallmark of α-mannosidosis is the storage of neutral oligosaccharides in a wide variety of tissues (see Fig. 2, lane 1 and lane 2). These oligosaccharides contain 2-9 mannose residues and a N-acetylglucosamine residue at their reducing end and thus result from the action of an endoglucosaminidase. The strong involvement of the central nervous system is reflected in the clinical phenotype of alpha-mannosidosis ranging from severe infantile forms to lighter juvenile forms with only moderate mental retardation. The rationale behind the present invention emerges from the surprising observation that repeated intravenous administration of recombinant alpha-mannosidase results in a marked reduction in the levels of neutral mannose-rich oligosaccharides stored in the brain.

In its broadest aspect, the present disclosure relates to a recombinant LAMAN polypeptide and to a nucleotide sequence encoding the LAMAN polypeptide, to an expression system capable of expressing the polypeptide as well as to pharmaceutical compositions comprising the polypeptide or part thereof and to the use of the polypeptide to prevent or treat the development of alpha-mannosidosis-related symptoms caused by a deficiency of the enzyme lysosomal alpha-mannosidase (LAMAN) in an individual.

In one main aspect the invention provides the use of a lysosomal alpha-mannosidase for the preparation of a medicament for reducing intracellular levels of neutral mannose-rich oligosaccharides in cells within one or more regions of the central nervous system, such as one or more regions within the brain. Neutral mannose-rich oligosaccharides contain two to nine mannose residues attached to a single residue of N-acetylglucosamine.

Another major aspect of the invention relates to a lysosomal alpha-mannosidase formulation for use as a medicament for reducing intracellular levels of neutral mannose-rich oligosaccharides in cells within one or more regions of the central nervous system.

For both major aspects of the invention it is even more preferred that the lysosomal alpha-mannosidase is selected from the group consisting of (a) the amino acid sequence of SEQ ID NO: 1; (b) a portion of the sequence in (a) which is enzymatically equivalent to recombinant human lysosomal alpha-mannosidase (c) an analogous amino acid sequence having at least 75% sequence identity to any of the sequences in (a) or (b) and which is at the same time enzymatically equivalent to recombinant human lysosomal alpha-mannosidase.

It may be preferred that the degree of sequence identity between the nucleic acid sequence comprised within the cell according to the invention and SEQ ID NO: 1 is at least 80%, such as at least 85%, at least 90% , at least 95%, at least 97%, at least 98% or at least 99%.

In the present context, an analogous amino acid sequence or a portion of an amino acid sequence that is enzymatically equivalent to recombinant human lysosomal alpha-mannosidase, is a polypeptide, which has a specific activity of at least 5 U / mg when present in a purity of at least 90%. Purity is conveniently determined using standard techniques such as SDS-PAGE. The specific activity can be determined in an enzyme assay as described in Example 2 of the present application. Briefly, the purified polypeptide is incubated with the substrate, p-nitrophenyl-α-mannopyranoside at 37 ° C for an appropriate amount of time before the reaction is stopped by the addition of 0.4 M glycine / NaOH pH 10.4. The amount of the converted substrate can be determined by changing the absorbance at 405 nm (s = 18500 M -1 cm -1).

In some embodiments the lysosomal alpha-mannosidase is formulated for systemic administration. Formulations for systemic administration may comprise a variety of pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. "Pharmaceutically acceptable carriers, excipients or stabilizers" are nontoxic to the recipients at the dosages and concentrations used, and for the present application are also present at concentrations which have no effect on the permeability of the blood-brain barrier. Buffers such as phosphate, citrate and other organic acids are included; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride: phenol, butyl or benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m- cresol); low molecular weight polypeptide (less than about 10 residues); proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; counterions forming salts such as sodium; metal complexes (e.g., Zn protein complexes); and / or nonionic surfactants such as TWEEN, PLURONICST or polyethylene glycol (PEG). Successful alpha-mannosidosis therapy should effectively address the challenges posed by the central nervous system and the blood-brain barrier, in particular, as it prevents the entry of lysosomal enzymes into the brain. The anatomical component of the blood-brain barrier consists of single endothelial cells in the cerebral capillaries, which have hermetic junctions without fenestrations and with few microvilli and few vesicles to transport fluids. Its physiological component consists in part of enzymes unique to brain endothelia and active transport through carrier proteins.

Additional barriers are present in addition to the blood-brain barrier. Perivascular spaces are patrolled by phagocytes that can sequester foreign compounds and, in addition, layers of basal laminae, macromolecular protein meshes are located between the vasculature and the parenchyma of the central nervous system. After this, glia lamintan, a coating consisting of the terminal legs of astroglia and microglia form another physical barrier. Finally, the solutes after the endothelial barrier are also subjected to a flow of massive liquid out through contiguous spaces. The interstitial fluid of the parenchyma of the central nervous system travels partially to the ventricular system and perivascular spaces, and subsequently the solutes are transported to the subarachnoid spaces from which the soluble constituents return to the bloodstream. Thus, there are multiple barriers that limit and regulate the entry of bloodstream elements into the brain.

It has been considered that the blood-brain barrier problem can be solved with the development of drug targeting technology for the brain. This technology relies on the delivery of therapeutic proteins to the brain through access in endogenous transport systems located within the brain's capillary endothelium, which forms the blood-brain barrier in vivo. Non-limiting examples of components with a capability to direct or mediate targeting to the central nervous system and / or with a known capacity to act as vehicles for administration by blood-brain barrier and / or cell membranes are given below. 1) Peptides and proteins as vehicles for the passage of LAMAN to target cells through the passage of cell membranes and / or BBB:

A number of animal studies have shown that certain proteins and / or peptides can act as vehicles for BBB passage. For example, proteins modified by the insulin fragment (Fukuta et al., Phaomacol Res 11: 1681-1688) or the antibodies against the transferrin receptor (Friden et al., Proc Natl Acad Sci USA 88: 4771-4775) blood-brain barrier. Likewise, proteins modified by polyamine coupling (Poduslo and Curran, J Neurochem 66: 1599-1606) have also been reported to pass the blood-brain barrier. In addition, peptidomimetic (MAb) monoclonal antibodies undergo receptor-mediated transthyme (RMT) through BBB in vivo by transport at the endogenous BBB (TfR) or insulin receptor (IR) transferrin receptor. The therapeutic proteins may be conjugated to the MAb transport vectors with avidin-biotin technology or as fusion proteins. 2) Toxins as vehicles for the passage of LAMAN to target cells by passage of cell membranes and / or BBB:

Different bacteria, plants and animals produce toxins. Toxins have many different targets such as the intestine (enterotoxins), the nerves or synapses (neurotoxins). Toxins may cross cell membranes through receptor-mediated processes and the embodiment of the present invention is to utilize toxins as vehicles for the passage of rhLAMAN to target cells through cell membranes and / or BBB. Preferred target cells are cells in the CNS and / or the peripheral nervous system. It is to be understood that only the part of the toxin which is responsible for association and translocation across the cell membranes is used. An example of a toxin used as a carrier is a bacterial toxin such as Diphtheria Toxin (DT) from Corynebacterium Diptheriae. Bacterial toxins exhibit a wide range of toxicities and are distributed in groups by structure and function. The toxin binds to a target cell and enters the cell through a receptor, and is reduced to separate the fragments. The processed toxin can be divided into the following 3 domains: the catalytic domain (C), the (R) receptor domain and the translocation domain (T). The catalytic fragment and the toxin receptor fragment or fragments thereof are replaced by LAMAN. This fusion protein may cross cell membranes and / or BBB and thereby administer LAMAN to target cells. An example of the manipulation of a hybrid molecule could be by recombinant technology

Other examples of bacterial toxins to be used as carriers are Clostridium Botulinum, Pseudomonas Exotoxin A produced by Psedomonas aeruginosa, Cholera Toxin produced by Vibrio cholerae, and Convulsive Cough Toxin produced by Bordetella pertussis.

Other examples of toxins used as carriers are the plant toxins selected from the list of the following plant toxins: cholinesterase inhibitors, protease inhibitors, amylase inhibitors, tannins, cyanogenic glycosides, goitrogenic compounds, lectin proteins, and lactogenic compounds, pyrrozidine alkaloids .

Still other examples of toxins used as vehicles are the toxins of molluscs and crustaceans (saxitoxin) and snakes (alfa-bungarotoxin). The skilled person may add further examples in light of the details and features of the invention. 3) Proteins and / or peptides isolated from bacteria or viruses as vehicles for the passage of rhLAMAN to target cells by passage of cell membranes and / or BBB: the transaction element and / or transaction protein of bacteria or viruses may be used in conjunction with rhLAMAN to cross the BBB and / or cell membranes.

Examples of bacteria are selected from the following list: Nos. meningitidis, S. pneumoniae, Hemophilus influenzae, Staphylococcus species, Proteus species, Pseudomonas species, E. coli, Listeria monocytogenes, M. tuberculosis, Neurolues, Spirochetes Borrelia burgdorferi of Iodex ricinus.

Virus examples are selected from the following list of virus families: Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxviridae, Picornaviridae, Reoviridae, Togaviridae, Arenaviridae, Coronaviridae, Retroviridae, Bunyaviridae, Orthomyxoviridae, Paramyxoviridae and Rhabdoviridae. Relevant examples of viruses selected from the list mentioned above are, for example, Measles viruses, Papova viruses and JC viruses. 4) Cell-mediated delivery systems The cell-mediated approach to therapy can solve storage disorders in more than one fundamental way. The first is to replace or compensate deficient cell populations with normal equivalents. A second way to obtain a therapeutic benefit may be through cross-correction. Appropriate cell types provide the ability not only to cross the endothelial barrier from the blood but also to migrate actively into the parenchyma and settle in the midst of diseased cells. The preferred human cell is selected from human monocytes, human fibroblasts and human lymphocytes. 5) Disturbance of the blood-brain barrier

Another invasive strategy to increase drug delivery to the CNS involves systemic drug administration in conjunction with the transient BBB rupture (BBBD). Theoretically, with weakened BBB, systemically administered drugs may suffer higher rates of extravasation in the cerebral endothelium, which leads to an increase in parenchymal drug concentrations. We investigated a variety of techniques that transiently break the BBB; however, while physiologically interesting, many are unacceptably toxic and therefore are not clinically useful. These include the infusion of solvents such as dimethylsulfoxide or ethanol and metals such as aluminum; irradiation with X-rays; and the induction of pathological conditions including hypertension, hypercapnia, hypoxia or ischemia.

A somewhat safer technique involves the systemic administration of the convulsant drug, metrazol, which transiently increases the permeability of BBB while causing seizures. Simultaneous administration of anticonvulsive pentobarbital blocks seizures while allowing BBBD to remain. BBB may also be compromised by the systemic administration of various antineoplastic agents including VP-16, cisplatin, hydroxylurea, flurouracil, and etoposide.

Hematoencephalic Barrier Osmotic Routing Intracarotid injection of an inert hypertonic solution such as mannitol or arabinose was used to initiate contraction of endothelial cells and the opening of the hermetic junctions of BBB over a period of a few hours, and this allows the administration of antineoplastic agents to the brain. Osmotic breakdown has been tested as a strategy for the administration of macromolecular drugs such as monoclonal antibodies, nanoparticles and viruses. However, the procedure destroys the brain's self-defense mechanism and makes it vulnerable to damage or infection by any chemicals or circulating toxins. Risk factors include plasma protein passage, altered glucose uptake, heat shock protein expression, microembolism, or abnormal neuronal function. Thus, although a large number of techniques have been developed to facilitate administration of compounds through the blood-brain barrier many of them exhibit limited success in approaching the other barriers mentioned above. Therefore, its applicability in the treatment of alpha-mannosidosis is limited.

On the contrary, a very important aspect of the invention relates, however, to a scheme for reducing the intralysossomic accumulation of neutral mannose rich oligosaccharides within the central nervous system based on a technique, which is not dependent on the means to mediate transport through and / or penetrate and / or rupture the blood-brain barrier. In preferred embodiments of the invention the approaches described above for accessing the central nervous system are therefore disclosed. In a preferred embodiment of the invention, the medicament according to the invention does not comprise a component with a known ability to direct or mediate targeting into cells within the central nervous system and / or a component with a known ability to act as a carrier for passage through the blood-brain barrier.

Expressed in more exact terms, said medicament or formulation does not comprise a component with a known ability to target neuronal and / or glial cells of the brain.

Preferred embodiments thus relate to the use of lysosomal alpha-mannosidase, wherein said medicament does not comprise lysosomal alpha-mannosidase in a form which is capable of permeating the central nervous system / blood-brain barrier. Therefore, it is also preferred that lysosomal alpha-mannosidase is not modified to provide carrier-mediated transport across the blood-brain barrier and that lysosomal alpha-mannosidase is not formulated in order to obtain rupture of the blood-brain barrier.

In equally preferred embodiments the medicament comprises a lysosomal alpha-mannosidase and does not comprise a) a vehicle or vector, such as a peptide or polypeptide, for the administration of lysosomal alpha-mannosidase in the central nervous system, and b) a component with a known capacity to cause the opening or rupture of the blood-brain barrier, and c) an intact cell

More specifically, it is an object of the present invention to provide the use of a lysosomal alpha-mannosidase for the preparation of a medicament, wherein said medicament does not comprise a vehicle selected from the group consisting of exogenous peptides and proteins, including the insulin fragment , and peptides and proteins derived from viruses or bacteria, polyamines, lipophilic units including phospholipids, antibodies or antibody fragments such as antibodies to the transferrin receptor and toxins.

Also, the invention relates to the use of lysosomal alpha-mannosidase wherein said preparation of a medicament does not comprise the modification of said lysosomal alpha-mannosidase by the attachment of polyamines. The medicament according to the invention may comprise or consist of a lysosomal alpha-mannosidase, which is formulated in an isotonic solution. The term "isotonic solution" conventionally refers to a solution having the same osmotic pressure as the blood. In the context of the present invention, the solution may be, but is not limited to, a human isotonic solution. Presently, formulation buffers comprising or consisting of 3.10-3.50 mM Na2 HPO4, 0.4-0.6 mM NaH2 PO4, 25-30 mM Glycine, 220-250 mM Mannitol, and water for injectable preparation are preferred. Also preferred are formulation buffers which comprise or consist of 4.0-5.0 mM Sodium Citrate, 0.3-0.8 mM Citric Acid, 200-250 mM Mannitol, 3.0-5 Tween 80 , 0 mM, and water for injectable preparation. Alternatively, lysosomal alpha-mannosidase can be formulated in phosphate buffered saline (0.01 M phosphate buffer + 0.15 M NaCl, pH = 7.2).

As mentioned above, the use of intact cells may provide a means for the administration of substances through the blood-brain barrier. The present invention, however, relates to a medicament which does not comprise an intact cell.

Accordingly, an even more preferred embodiment of the invention relates to the use of lysosomal alpha-mannosidase, wherein said preparation of a medicament does not involve the use of a cell mediated delivery system. In particular, the present invention is not based on the use of an intact cell selected from the group consisting of a transduced autologous cell, such as a transduced fibroblast or peripheral blood lymphocyte, and polymer encapsulated cell line capable of secreting alpha-mannosidase for cerebrospinal fluid.

The administration of lysosomal alpha-mannosidase is predicted to result in less accumulation of neutral oligosaccharides in a wide variety of cells within the central nervous system. However, in preferred embodiments of the invention, said cells within one or more regions of the central nervous system comprise neuronal cells and / or glial cells. In addition, said cells may comprise extraneuronal cells present within the central nervous system. The brain comprises four major parts, the brain, the diencephalon, the brainstem and the cerebellum. It is to be understood that the administration of alpha-mannosidase affects the storage of oligosaccharides in one or more of these parts. In even more preferred embodiments of the invention, said one or more regions of the central nervous system comprise one or more regions within the brain, diencephalon, brainstem and / or cerebellum. The reduction in the accumulation of neutral mannose-rich oligosaccharides within certain specific regions of the above-mentioned four brain parts is likely to be of particular relevance to the reduction of the clinical manifestations of alpha-mannosidosis. In more particular embodiments of the invention, the accumulation of oligosaccharides is reduced within one or more regions within the brain which are selected from the group comprising one or more regions within the cerebral cortex, one or more regions within the subcortical nuclei, and one or more regions within the limbic system.

A number of sensory and / or motor functions were attributed to several specific regions within the cerebral cortex. Within the Frontal Lobe, which is generally motor in terms of function, the Precentral Circumvolution (Primary Motor Cortex) is responsible for fine movement control, the Motor Association Cortex is responsible for integrating information for complex movements, the Cortex Motor of Discourse is involved in the production of speech; (Brocas area), and the Pre Frontal Cortex is involved in decision making; direction of attention.

Within the Parietal Lobe, which is usually sensory in terms of function, the Postcentral Circumvolution (Primary Somatosensory Cortex) is involved in the perception of sensation from the skin (Cutaneous) and muscle (propriocetic), the Sensory Association Cortex is involved in the integration of sensory signals; direction of sensory attention, the Left Association Cortex is involved in the production of meaning for language; (Wernicke area), and the Right Association Cortex is involved in producing meaning for spatial relationships

Within the Occipital Lobe, which is usually sensory in terms of function, the Visual Cortex is involved in the perception of visual sensation, the Visual Association Cortex is involved in the perception of content and visual meaning

Within the Temporal Lobe, which is usually sensory in terms of function, the Primary Auditory Cortex is involved in the perception of auditory sensation, the Auditory Association Cortex is involved in the perception of auditory content and meaning; it extends to the parietal cortex Gustatory and Olfactory Cortex that are involved in the perception of gustatory and olfactory sensation, the Insulating Cortex located in the deep extension of the lateral sulcus is involved in providing knowledge about the outcome of events.

Other regions include the Para-hippocampal Circumvolution, which overlaps the hippocampus, the Hippocampus, which is involved in the formation of long-term memory and the Amygdala, which is involved in the feeling of emotion.

In even more particular embodiments of the invention, the levels of oligosaccharides are reduced in one or more regions within the cerebral cortex which are selected from the group comprising the frontal lobe, parietal lobe, temporal lobe and occipital lobe.

In further particular embodiments of the invention, the levels of oligosaccharides are reduced in one or more regions within the diencephalon which are selected from the group comprising the thalamus and the hypothalamus. The thalamus is a component of the diencephalon deeply buried in the nucleus of the brain. Tightly and reciprocally bound with cortical structures, the thalamic nuclei function both to retransmit and modulate the transmission of information throughout the brain. The thalamus comprises the following major parts: the anterior nuclear group, the midline nuclear group, the medial-dorsal nucleus, the intralaminar nuclear group, the lateral nuclear group, the ventral nuclear group, the geniculate bodies, the posterior nuclear complex, the thalamic reticular nucleus, the thalamic fiber segments, and the zonal stratum of the thalamus. The limbic system is a group of brain structures that are involved in various emotions such as violence, fear, pleasure and also memory formation. The limbic system also affects the endocrine system and the autonomic nervous system. It consists of several structures located around the thalamus, immediately below the brain: the hippocampus, which is involved in the formation of long-term memory, the amygdala, which is involved in violence and fear, the cingulate gyrus, the fornication , the paleocortex and the hypothalamus, which controls the autonomic nervous system and regulates blood pressure, heart rate, appetite, thirst and sexual arousal.

It attaches to the pituitary gland and thereby regulates the endocrine system.

Neurological functions located in the brainstem include those necessary for survival (breathing, digestion, heart rate, blood pressure) and on awakening (being awake and alert). Most cranial nerves come from the brainstem. The brainstem is the pathway for all fiber segments that rise and descend from the peripheral nerves and spinal cord to the upper parts of the brain. In further particular embodiments of the invention the levels of oligosaccharides are reduced in one or more regions within the brainstem which are selected from the group comprising the mesencephalon, the varolus bridge and the medulla oblongata. The medulla oblongata acts mainly as a relay station for the crossing of motorways between the spinal cord and the brain. It also contains the respiratory, vasomotor and cardiac centers, as well as many mechanisms to control reflex activities such as coughing, choking, swallowing and vomiting. The midbrain serves as the nerve pathway of the cerebral hemispheres and contains centers of auditory and visual reflexes. The Valorio bridge is a bridge-like structure that connects different parts of the brain and serves as a relay station for the spinal cord to the upper cortical structures of the brain. Contains respiratory center. The function of the cerebellum, which includes the cerebellar cortex, is to facilitate the performance of movements by coordinating the action of the various groups of muscles involved. In other particular embodiments of the invention, the levels of oligosaccharides are reduced in one or more regions within the cerebellum comprising the cerebellar cortex.

A primary advantage of the use of recombinant lysosomal alpha-mannosidase according to the present invention relates to the fact that the treatment effects observed in the brain can combine with the effects on cells which are not part of the central nervous system. Thus, the administration of exogenous lysosomal alpha-mannosidase is likely to correct most, if not all, of the clinical manifestations of alpha-mannosidosis. In preferred embodiments, the invention thus relates to the use of a medicament which further reduces levels of neutral mannose-rich oligosaccharides in cells of neurological tissues that are not within the central nervous system. In this embodiment of the invention, said medicament further comprises a component, which is capable of targeting cells of neurological tissues, which are not within the central nervous system. In particular, the use of this medicament results in the correction of lysosomal storage in cells not within the central nervous system, which comprise the most prominent cells of the peripheral nervous system. These comprise neuronal cells and / or Schwann cells.

Clinical manifestations of alpha-mannosidosis may be partially alleviated by the isolated effects of alpha-mannosidase deficiency on visceral tissues. However, a central part of the concept, which forms the basis of the present invention, relates to the fact that the beneficial effects of therapy on lysosomal storage in the central nervous system is secondary and / or dependent on a reduction in lysosomal storage in tissues outside the central nervous system. Accordingly, it is an important feature of the invention that said medicament is also used to obtain a reduction in the levels of neutral mannose rich oligosaccharides in cells within non-neurological tissues and that the medicament comprises a component capable of targeting cells within such tissues .

In this context, the effects of exogenous alpha-mannosidase on visceral tissues are judged to be of primary importance. According to a preferred embodiment of the invention, the medicament for this purpose comprises a component, which is capable of targeting cells of neurological and / or non-neurological tissues outside the central nervous system. In an even more preferred embodiment, said neurological and / or non-neurological tissues outside the central nervous system comprise visceral tissues selected from the group comprising liver, spleen, kidney and heart.

It is implicit that exogenous alpha-mannosidase can be administered to target cells in relevant tissues outside the central nervous system through the use of, for example, receptor-mediated endocytosis. Preferably, alpha-mannosidase is administered to cell membranes of target cells such as those that take advantage of a mannose or mannose-P-6 receptor mediated uptake. Therefore, the above-mentioned factor having targeting capabilities is preferably mannose-6-phosphate.

Any mammalian alpha-mannosidase is believed to be used to replace the deficient enzyme in a subject. However, where the subject is a human the use of a human lysosomal alpha-mannosidase will be preferred in order, for example, to minimize the occurrence of antigenic reactions. Accordingly, preferred embodiments of the invention relate to the use of a lysosomal alpha-mannosidase, which is a human lysosomal alpha-mannosidase.

In a most preferred embodiment, the lysosomal alpha-mannosidase comprises the amino acid sequence of SEQ ID NO: 1.

For practical and economic reasons, it is preferred that alpha-mannosidase is produced recombinantly. In addition, it is envisaged that for a purified tissue lysosomal hydrolase, that enzyme has a low mannose-6-phosphate content. Thus, through the recombinant production it will also be possible to obtain a preparation of the enzyme in which a large fraction contains mannose-6-phosphate. Recombinant production can be achieved after transfection of a cell using a nucleic acid sequence comprising the sequence of SEQ ID NO: 2. Alpha-mannosidase is preferably prepared in a mammalian cell system to provide a glycosylation profile, which ensures an efficient receptor-mediated uptake in cells, for example, of the body's visceral organs. In particular, it has been determined that the production of the enzyme in CHO, COS or BHK cells ensures a suitable post-translational modification of the enzyme by the addition of mannose-6-phosphate residues. In addition a correct sialylation profile is obtained. It is known that correct sialylation is important to prevent liver uptake due to the exposed galactose residues.

In still more preferred embodiments, the mammalian cell system is therefore selected from the group comprising CHO, COS cells or BHK cells (Stein et al., J Biol Chem 1989, 264, 1252-1259). It may further be preferred that the mammalian cell system is a human fibroblast cell line.

In a most preferred embodiment, the mammalian cell system is a CHO cell line.

Preferred embodiments of the invention are also concerned with the use of a lysosomal alpha-mannosidase preparation wherein a fraction of said preparation consists of lysosomal alpha-mannosidase having one or more N-linked oligosaccharides carrying groups of mannose-6-phosphate. It is even more preferred that a fraction of a preparation of said lysosomal alpha-mannosidase is capable of binding to mannose-6-phosphate receptors. The ability of the enzyme to bind to mannose-6-phosphate receptors can be determined in an in vitro assay as described in Example 1 of the present application. Here, the binding of the enzyme to an MPR 300 Affinity Matrix provides a measure of its ability to bind to mannose-6-phosphate receptors. In a preferred embodiment of the invention, the binding of the enzyme to mannose-6-phosphate receptors occurs in vitro.

In more preferred embodiments of the invention, this fraction corresponds to from 1 to 75% of the activity of a lysosomal alpha-mannosidase preparation, such as from 2 to 70%, such as from 5 to 60%, such as from 10 to 50%, such as from 15 to 45%, such as from 20 to 40%, such as from 30 to 35%.

Accordingly, it is preferred that lysosomal alpha-mannosidase has a mannose-6-phosphate residues content that allows mannose-6-phosphate-dependent binding from 2 to 100%, 5 to 95%, 10 to 90%, 20 to 80%, 30 to 70% or 40 to 60% of the amount of enzyme to a matrix of Man-6-P receptors. Presently, the degree of phosphorylation has been analyzed in several batches of enzyme, and typically, from 30 to 45% of the enzyme is phosphorylated and binds to the affinity matrix. It is still more preferred that a fraction consisting of from 2 - 100%, 5-90%, 10-80%, 20-75%, 30-70%, 35-65% or 40-60% of the amount of said alpha- mannosidase binds to the Man-6-P receptor with high affinity. Theoretically, two mannose-6-phosphate groups should be placed close together so that the enzyme binds to a Man-6-P receptor with high affinity. Recent observations suggest that the distance between the phosphorylated mannose residues should be 40 Âμm or less to give high affinity binding. In human lysosomal alpha-mannosidase according to SEQ ID NO: 1 the two mannose-6-phosphate residues may be located in the asparagine residues at positions 367 and 766. It is therefore preferred that the medicament according to present invention comprises lysosomal alpha-mannosidase, a fraction of which has mannose-6-phosphate groups in both these asparagine residues.

It will be understood that various different means of administration may be used for the administration of the enzyme. These include parenteral infusion or infusion including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular. Since administration is likely to be repeated on a regular basis, the use of simple techniques for administration is preferred. In a currently highly preferred embodiment of the invention, alpha-mannosidase is used for the preparation of a medicament for intravenous administration. In particular, administration by injection directly into the central nervous system is considered to be inadequate, since administration through this route can not be performed by a lay person.

It will of course be possible to coadminister the medicament which is prepared by the use of the alpha-mannosidase according to the invention with a composition comprising a component with a known ability to direct or mediate the targeting of the central nervous system. In a most preferred embodiment of the present invention such coadministration is neither necessary nor desired and is not to be included in the treatment schedule. This embodiment relates to the use according to the invention, wherein said medicament is to be administered without the supplemental administration of a composition comprising a component of known ability to target the central nervous system.

Apparently the medicament is to be administered in doses, wherein the amount of lysosomal alpha-mannosidase is sufficient to correct defects in lysosomal storage. It appears from previous studies on related lysosomal disorders that a complete replacement of the deficient enzyme may not be necessary in order to achieve levels corresponding to those observed in healthy individuals. In order to minimize the cost of therapy and probably also reduce putative adverse effects the goal should be to utilize as small amounts of the enzyme as possible. In addition, it appears possible to "adjust" the treatment including the amounts administered and the frequency of administration according to the particular individual's profile. Accordingly, in preferred embodiments of the present invention, the medicament is administered in an amount, which is within the range of 1 to 400 mU lysosomal alpha-mannosidase per gram of body weight of the subject to be treated, such as within the range from 2 to 350, 5 to 300, 7.5 to 200, 10 to 200, 15 to 150, 20 to 100, 25 to 75, 30 to 60, 35 to 50 or 40 to 45 mU of lysosomal alpha-mannosidase per gram of body weight,

In a currently more preferred embodiment of the invention the medicament is administered in an amount of 250 mU lysosomal alpha-mannosidase per gram of body weight. In an equally preferred embodiment of the invention, the medicament is administered in an amount of 50 mU lysosomal alpha-mannosidase per gram of body weight. The specific activity of lysosomal alpha-mannosidase may be a crucial factor influencing ability, wherein said lysosomal alpha-mannosidase has a specific activity of 5-100 U / mg, such as from 10-90 U / mg, 80 U / mg, 20-70 U / mg, 25-65 U / mg, 30-60 U / mg or 35-55 U / mg.

In the enzyme preparation used in the present invention, a fraction of the amount of lysosomal alpha-mannosidase is present in the form of a 260 kDa dimer comprised of 130 kDa monomers of an amino acid sequence that is at least 75%, 80%, 85% , 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1. However, when analyzed under reducing conditions, a variable fraction of the amount of enzyme appears to have been processed into the forms of 70 and 55 kDa. This indicates that a fraction of the amount of lysosomal alpha-mannosidase has been partially dissociated, but is still held together in the form of 260 kDa by disulfide bridges. Both the 130 kDa form and the processed forms are active, but it is preferred that a large fraction of the amount of enzyme is present as dimers of the 130 kDa precursor form when administered to the subject. Preferably, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% is present as a dimer of the 130 kDa form. When the enzyme is subsequently taken up in the lysosomes it is processed into the 15, 42 and 70 kDa peptides. The 70 kDa peptide is further processed into three smaller peptides which are further held together by disulfide bridges.

Since enzyme replacement therapy is expected to only generate transient effects, it appears evident that the medicament can be administered at a frequency ranging from several daily injections to injections once or twice a week or month. In preferred embodiments, the medicament can be administered at a frequency corresponding to 1 to 5 injections daily, 1 to 5 injections weekly, 1 to 5 injections every 2 weeks or 1 to 5 injections every month. Alternatively, the frequency may correspond to an injection at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.

In equally preferred embodiments, the medicament is administered by repeated intravenous injection on a daily basis, by repeated intravenous injection every 2, 3, 4, 5 or 6 days, or by repeated intravenous injection on a weekly, biweekly or monthly basis.

In a currently preferred embodiment of the invention, the amount of lysosomal alpha-mannosidase is administered once every 3 to 4 days. Administration of the medicament according to the present invention by intravenous injection only leads to insignificant increases in the amount of lysosomal alpha-mannosidase present within the central nervous system. Although not intended to be limited by this theory, it appears that reduced levels of neutral oligosaccharides may be secondary and a direct or indirect result of increased enzyme levels in the peripheral tissue and visceral organs. Since lysosomal alpha-mannosidase appears to be rapidly eliminated from circulation, it seems critical that the amounts of enzyme administered are such that circulating enzyme levels remain adequately increased for an amount of time that allows for the cellular uptake of appropriate amounts of the enzyme through the Man-6-P receptor or probably also through other presently unknown transport mechanisms. In addition, enzyme levels within the visceral organs and the peripheral tissue must remain adequately increased for a sufficient amount of time to cause a reduction in the neutral sulfatídeos not only within the organs or tissue but also within the central nervous system. Accordingly, it is preferred that the amount of lysosomal alpha-mannosidase administered is such that: a) effective levels of the enzyme are maintained circulating for not less than 5-30 minutes, such as not less than 5, 10, 15, 20, 25 or 30 minutes, and / or b) effective levels of the enzyme are maintained in the visceral organs, including the kidney, spleen and heart, for not less than 1,2,3,4,5,6,7,8, 9, 10, 12 or 24 hours, and / or c) effective levels of the enzyme are maintained in the liver for not less than 6, 12 or 24 hours or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days after the injection, such as intravenous injection, of said medicament.

In the present context the term "effective levels" refers to levels of lysosomal alpha-mannosidase that are increased by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold or at least 50-fold compared to enzyme levels observed in serum or organ extracts from untreated individuals when analyzed in an enzyme assay as described in Example 2 of the present application.

Preferably, the enzyme once administered to said subject will lead to a reduction in intralysosomal oligosaccharide levels which corresponds preferably to 20%, more preferably 30%, still more preferably 40%, still more preferably 50%, still more preferably 60%, very much preferably more than 70%.

In particular, the amount of lysosomal alpha-mannosidase administered is preferably such that: a) the amount of neutral oligosaccharides in the visceral organs including the liver, spleen, kidney and / or heart is reduced by 10-80%, 15-85% , 20-90%, 25-95% or 30-100%, such as at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% over 2, 4, 6, 8, 10, 12 or 24 hours compared to pretreatment levels or levels observed in untreated individuals, and / or b) the amount of neutral oligosaccharides in the visceral organs including the liver, spleen kidney and / or heart is reduced by 40-80%, 45-85%, 50-90%, 55-95% or 60-100%, such as at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% over a period of 1-4, 2-6 or 4-8 days, and / or c) storage of oligosaccharides neutrophils in the liver is reduced over a period of 1 to 12, 2-10 or 4-8 days as determined by a reduced presence of storage vacuoles within the sinus endothelial cells, Kupffer cells and hepatocytes subsequent to the injection, such as intravenous injection, of said medicament.

Essential for the present invention is the observation that a reduction in neutral oligosaccharide levels in the central nervous system is only observed after repeated administration of lysosomal alpha-mannosidase. Therefore, it may be preferred that the amount of lysosomal alpha-mannosidase administered is such that: a) effective levels of the circulating enzyme are maintained, and / or b) effective levels of the enzyme are maintained in the visceral organs, including the kidney, spleen, and heart and / or c) effective levels of the enzyme in the liver are maintained by repeated injection such as intravenous injection of the medicament on a daily basis by repeated injection such as intravenous injection every 2, 5 or 6 days, or by repeated injection, such as intravenous injection, on a weekly, bi-weekly or monthly basis.

It may also be preferred that the amount of lysosomal alpha-mannosidase administered is such that a) the storage of neutral oligosaccharides in the kidney and heart is fully corrected or fully corrected at least over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days, and / or b) the amount of neutral oligosaccharides in the spleen is reduced by at least 10, 20, 30, 40, 70, 80, 90 or 100% or reduced thereby at least over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days , and / or c) the storage of neutral oligosaccharides in the liver and kidney is reduced or reduced over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 13 or 14 days as determined by a reduced presence of storage vacuoles within sinus endothelial cells, Kupffer cells and liver hepatocytes, and tubular epithelia of the kidney by repeated injection, such as intravenous injection, of the med on a daily basis, by repeated injection, such as intravenous injection, every 2, 3, 4, 5 or 6 days, or by repeated injection, such as intravenous injection, on a weekly, biweekly or monthly basis. The reduction of stored oligosaccharides in the brain can result, at least in part, from the clearance of material stored from the cerebral endothelium and the meninges, which may improve microcirculation and cerebrospinal fluid flow. A most preferred embodiment of the present invention thus relates to the use of lysosomal alpha-mannosidase for the preparation of a medicament for reducing intracellular levels of neutral mannose-rich oligosaccharides in cells within one or more regions of the cerebral and / or the meninges.

It is to be understood that the medicament may have the characteristics described above. Thus, in a preferred embodiment of this aspect of the invention, said medicament does not comprise a component with a known ability to direct or mediate targeting to the central nervous system and / or a component with a known ability to act as a vehicle for passage through the central nervous system. blood-brain barrier.

Again, it is preferred that the method comprises administering said alpha-mannosidase medicament via a route other than intracerebroventricular, spinal, intrathecal or intracranial administration. The administration of the medicament by intravenous or subcutaneous injection is particularly preferred. Coadministration of the medicament according to the present invention with a component with a known ability to direct or mediate central nervous system targeting and / or a component with known ability to act as a vehicle for blood-brain barrier passage is neither necessary nor and is not to be included in the treatment schedule. Thus, it is preferred that the administration of said medicament is not supplemented by administering a composition comprising a component of known ability to target the central nervous system.

According to a recent publication, intrathecal injection (i.e., injection directly into the cerebrospinal fluid) of recombinant human alpha-L-iduronidase (rhIDU) may reduce the storage of carbohydrates in brain tissue in a canine model of mucopolysaccharidosis ( MPL) (Kakkis, 2003). While intravenous administration of the medicament according to the present invention is by far preferred, Kakkis' observations suggest that such administration may be, at least in some cases, combined with other treatments, including other routes of administration. The invention may thus provide the use of a lysosomal alpha-mannosidase for the preparation of a medicament for intravenous administration, as well as for intrathecal or spinal injection administration, wherein the combined intravenous and intrathecal administration results in a reduction in the levels of galactosylsulphate into target cells within the peripheral nervous system and into target cells within the central nervous system in a subject.

In addition, the invention may provide the use of a lysosomal alpha-mannosidase for the preparation of a medicament for intravenous administration, wherein the intravenous administration of the medicament results in a reduction in the levels of galactosylsulfatide in target cells within the peripheral nervous system and in target cells within the central nervous system.

In yet another aspect, there is provided a method for the preparation of recombinant human alpha-mannosidase, wherein the method comprises a) introducing, in a suitable vector, a nucleic acid fragment comprising a DNA fragment encoding the amino acid sequence that is synthesized. shown in SEQ ID NO: 1; b) transforming a cell with the vector obtained in step a); c) culturing the transformed host cell under conditions facilitating expression of the nucleic acid sequence; d) recovering the culture expression product. The method may further comprise a series of one or more purification steps.

In particular, there is provided a method, wherein the nucleic acid fragment comprises the 30 66 base pair EcoRI-Xbal fragment of the DNA fragment shown in SEQ ID NO 2. The production of recombinant alpha-mannosidase, which is mannose-6-P comprises one or more or all of the following general steps (one sketch): A. Synthesis of rhLAMAN Cloning of specific rhLAMAN B. Transformation 2-10 æg of hybrid rhLAMAN vector DNA is used for transfection by methodology of precipitated phosphate / glycerol shock, in mammalian cells (for example CHO, COS cells or BHK cells). The transfer can also be carried out with an electric shock methodology.

C. Expression of rhLAMAN The synthesis of the active protein and the mannose-6-P modification of said protein is effected during expression in the mammalian cell system.

D. Purification of rhLAMAN Recombinant human alpha-mannosidase is purified using a 6-step procedure - see example 1.

E. Test system for mannose-6-PA receptor-mediated uptake ability of the recombinant alpha-mannosidase produced to be active in a mannose-6-P receptor-mediated uptake is assayed by incubating the alpha-mannosidase with normal fibroblasts or fibroblasts of patients with alpha-mannosidosis. Cell uptake is analyzed by increased alpha-mannosidase activity.

In a currently preferred embodiment, the alpha-mannosidase is produced in a mammalian cell system selected from the group comprising BHK, COS and CHO cells. One of the critical parameters of the method is the glycosylation profile of the alpha-mannosidase in the final preparation. The glycosylation profile is an important determinant of the enzyme's corrective activity in vivo through its effects on receptor-mediated endocytosis and clearance of the enzyme from the bloodstream. When the above-mentioned preferred mammalian cell systems have been used, it has been found that alpha-mannosidosis with an appropriate glycosylation profile is produced; in particular, the use of CHO cells leads to the production of alpha-mannosidase with an adequate mannose-6-phosphate content. In the examples of the present application, the ability of alpha-mannosidase to bind an MPR 300 column provides a measure of its mannose-6-phosphate content. Mannose-6-phosphate containing alpha-mannosidase is secreted into the medium and purification of the enzyme can be facilitated by the use of ammonium salts (NH 4 Cl) in the fermentation step.

One factor, which must be taken into account when isolating and purifying the enzyme, is the occurrence of dephosphorylation during isolation. Although the preparation of human alpha-mannosidase in which a fraction corresponding to 40% or more of the enzymatic activity was able to bind to the MRP 300 column, this fraction may be as low as a small percentage. In order to reduce dephosphorylation during the processing steps, the presence of one or more phosphatase inhibitors appears to be crucial. Such phosphatase inhibitors are available from commercial sources.

An important aspect of the invention relates to the use of a recombinant alpha-mannosidase for the preparation of a medicament for the treatment of alpha-mannosidosis. In particular, it is preferred that alpha-mannosidase is a recombinant human alpha-mannosidase. The disease which is the target of the inventive method is alpha-mannosidosis and therefore the catalyst is the alpha-mannosidase or an enzymatically equivalent part or analog thereof. It is most preferred that the catalyst is a recombinant form of the human alpha-mannosidase enzyme or the enzymatically equivalent part or analog thereof, since recombinant production will allow large-scale production which, with the presently available means, does not appear feasible if the enzyme had to be purified from a native source.

Preferably, alpha-mannosidosis is prepared by recombinant techniques. In another embodiment, alpha-mannosidosis is human (hLAMAN) and, even more preferred, mature human alpha-mannosidase (mhLAMAN) or a fragment thereof. The fragment may be modified, however, the active sites of the enzyme must be preserved.

It is envisaged that in the alpha-mannosidosis preparations according to the present invention a fraction of the enzyme is represented by its precursor form, while other fractions represent proteolytically processed forms of approximately 55 and 70 kDa. In general, the target cell is a cell in which enzymatic activity, such as alpha-mannosidase activity, is insufficient for optimal cell functioning. Insufficient alpha-mannosidase activity may be measured by one or more of the parameters selected from monitoring the increased levels of mannose-rich oligosaccharides in the urine, analyzing alpha-mannosidase activity in an individual material such as leukocytes and / or skin fibroblasts, presence of clinical symptoms, or increased rate of development of clinical symptoms of alpha-mannosidosis.

A significant feature of insufficient alpha-mannosidase activity is a cell in which a massive intracellular accumulation of mannose-rich oligosaccharides is present. Naturally, such a cell is a target cell in accordance with the present invention. The target cell may also be a cell of the nervous system. In addition, target cells for administration of the enzyme also include one or more cell types selected from human monocytes, human fibroblasts, human lymphocytes, human macrophages.

An increased alpha-mannosidase activity may be used as a parameter to evaluate the success of a treatment plan. The activity can be measured by one or more of the selected monitoring parameters of increased mannose rich oligosaccharide levels in the urine, analysis of alpha-mannosidase activity in patient material such as leukocytes and / or skin fibroblasts, presence of clinical symptoms or increased rate of development of clinical symptoms of alpha-mannosidosis. It is a very important aspect of the invention to carry out the treatment of a possible enzymatic defect in the prenatal form. In a further aspect of the invention, the cell membrane is the maternal-fetal barrier (placenta). As is the case for the blood-brain barrier, it may be an important feature of the present invention that the effective administration of the enzyme over the maternal-fetal barrier or rupture of the maternal-fetal barrier is unnecessary and undesirable.

With respect to the foregoing description of the various aspects of the present invention and the specific embodiments of these aspects it is to be understood that any particularity and feature described or mentioned above with respect to an aspect and / or an embodiment of an aspect of the invention applies, by analogy, to any or all other aspects and / or embodiments of the disclosed invention.

Where, in the present application, an object according to the present invention or one of its particularities or characteristics is referred to in the singular, this also refers to the object or its particularities or features in the plural. As an example, when referring to "a cell" it is understood that reference is made to one or more cells.

Throughout the present description, the word "comprise", or variants such as "comprises" or "comprising," will be understood to imply the inclusion of an element, integer or pitch, or group of elements, integers or steps specified, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

PICTURE'S DESCRIPTION

Fig. 1: Standard of bovine, rat and human LAMAN polypeptide 10æg of bovine kidney purified LAMAN or the secretions of cells which overexpress rat or human LAMAN were separated by SDS-PAGE and developed with Coomassie Blue. The 130 kDa polypeptides observed in rat and human LAMAN correspond to precursors and polypeptides ranging from 11 to 70 kDa to proteolytically processed forms of LAMAN. The 46-48 kDa polypeptide in bovine LAMAN represents partially processed intermediates.

Fig. 2: Thin layer chromatography of neutral oligosaccharides in the liver, spleen, kidney and heart. The fraction of neutral oligosaccharides of equal amounts of tissues was separated by thin layer chromatography. Lane 1 contains the sample of control rats, lane 2 of sham-injected LAMAN rats, lanes 3, 4 and 5 the samples of α-mannosidosis mice injected with 100 mU rat LAMAN per g body weight and died after 2, 4 and 10 h, respectively (see also Table 1). The oligosaccharides were detected with orcinol / sulfuric acid. Mass spectrometry and a-mannosidase sensitivity of madagascar bean demonstrated that M2 to M9 oligosaccharides contain 2 to 9 mannose residues and a single residue of N-acetylglucosamine at the reducing end. The oligosaccharides were quantified by densitometry. For storage calculation, neutral oligosaccharide values in α-mannosidosis mice were corrected for the neutral oligosaccharides in control mice and expressed as the percentage of those in simulated injected α-mannosidosis mice. Storage in mice with α-mannosidosis injected with LAMAN was expressed as the percentage of that in mice with simulated injected a-mannosidosis. Mass spectrometry and α-mannosidase sensitivity of madagascar bean showed that M2 to M9 oligosaccharides contain 2 to 9 mannose residues and a single residue of N-acetylglucosamine at the reducing end.

Fig. 3: Neutral oligosaccharides in tissue extracts of α-mannosidosis mice injected with 100 mU rat LAMAN per g body weight.

The mice were killed 4, 8 and 16 days after injection and the neutral oligosaccharides in the liver, spleen, kidney and heart extracts were quantified by densitometry after separation by thin layer chromatography (see legend of Fig. 2).

Fig. 4: Depuration of LAMAN from bovine ()), murine ()) or human (origem) serum, 50 mU / g body weight, was injected into the tail vein of α-mannosidosis with 8 weeks of age. Blood was collected from the retroorbital plexus 5 to 65 min after injection. Each value represents the average of two animals. The range is indicated by the bars where it exceeds the size of the symbols.

Corrective effect of bovine, rat and human LAMAN on storage of neutral oligosaccharides Rats received 50 mU LAMAN per g body weight (see Fig. 3) 2 days prior to tissue analysis. Neutral oligosaccharides were quantified as in Figures 2 and 3. The bars indicate the range observed in two independent mice.

Fig. 6: Neutral oligosaccharides in rat tissue extracts with α-mannosidosis after injection of a single dose of 250 mU of human α-mannosidase per g body weight. Rats were killed 1, 3, 6 and 12 days post-injection. Neutral oligosaccharides in liver, spleen, kidney and heart tissue extracts were quantified by thin layer chromatography and densitometry as in Figures 1 and 2.

Fig. 7: Disappearance and reappearance of storage vacuoles in liver A, C, E, optical microscopy (semifinished sections); B, D, F, Kupffer cell ultrastructure; A, B, simulated injected arabinosidosis mouse (14 weeks of age). The hepatocytes show transparent vacuoles along the bile ducts (A, arrows). Strongly vacuolized breast wall cells are not clearly distinguished at the level of optical microscopy (A) because of the extremely narrow cytoplasmic bridges between the vacuoles as seen in (B). C, D, mouse with a-mannosidosis, 1 day after injection of 250 mU of human LAMAN per g of body weight. Vacuoles disappeared from hepatocytes (C). Very few vacuolized sinus wall cells (C, arrow) are observed. The vacuoles in the Kupffer cells are smaller than in the treated animal in a simulated fashion and may contain a dense matrix in electrons (D). E, F mice with a-mannosidosis 12 days after injection of 250 mU of human LAMAN per g body weight. The vacuoles reappeared in the cells of the sinus wall (E, arrows) and less in the hepatocytes. In the Kupffer (F) cells, the vacuoles resemble qualitatively those in simulated injected rats. The dense inclusions in the hepatocytes shown in A, C and E represent lipid droplets, which are also present in the corresponding wild type mice (not shown). The bars represent 20 μg and 2 μμ respectively.

Fig. 8: Neutral oligosaccharides in tissue extracts of α-mannosidosis mice injected twice with 250 mU of human LAMAN per g body weight

Control rats (lane 1) and α-mannosidosis (lane 2 and 3) were injected with PBS (lane 1 and 2) or 250 mU human LAMAN per g body weight (lane 3) 7 and 3.5 days before of the analysis. Neutral oligosaccharides were prepared and quantified as in Figs. 1 and 2. Migration of oligosaccharides consisting of 2 to 9 mannose residues and a single residue of N-acetylglucosamine (M 2 to M 9) is indicated. The arrows mark positive material for orcinol, insensitive to LAMAN. Results in a duplicate set of rats (not shown) varied less than 15%.

Fig. 9: Optical microscopy of the kidney in arabinosidose rats Rats with α-mannosidosis were injected with PBS (A) or 250 mU of human LAMAN per g of body weight (B) 7 and 3.5 days before death. The outer band of the outer medulla is shown. Numerous clear vacuoles are seen in the thicker raised limb (TAL) of the simulated injected mouse Henle loop (A, arrows) in contrast to the LAMAN (B) treated mouse. The proximal straight tubules (PST) are free of pathological vacuoles in any of the animals. The bars represent 20 and m.

Fig. 10: HPLC separation of neutral oligosaccharides from the brain derivatized with 2-anthranilamide

Neutral brain oligosaccharides from PBS (top scan) injected arabinosidosis mice or 250 mU of human LAMAN per g body weight (bottom scan) 7 and 3.5 days before death were mixed with 220 pmol of GlcNAc4Man3Gal3 as an internal standard, derivatized with 2-anthranilamide and separated by HPLC. The position of the internal standard and the oligosaccharides consisting of 2 to 9 mannose residues and a single residue of N-acetylglucosamine (M 2 to M 9) are indicated.

EXAMPLES

Example 1: Production and characterization of LAMAN Experimental procedures

Expression and purification of recombinant human LAMAN in CHO cells

The human LAMAN cDNA isolated from the HepG2 cDNA library and subcloned into an expression vector carrying a dihydrofolate reductase gene and the LAMAN cDNA under the control of the human CMV promoter was expressed in Ovary cells from Chinese Hamster (CHO) deficient in dihydrofolate reductase. CHO cells were cultured in a two-chamber CELLine flask (Integra Biosciences Inc.) in serum-free ExCell 302 medium (JRH Biosciences) supplemented with 20 nM methotrexate at 37øC in a humidified atmosphere containing 5% CO2. The medium was diafiltered using a Pellicon Biomax polysulfone filter with a 100 kDa cut against 4 volumes of 0.02 M Tris-HCl, pH 7.6. Ion exchange chromatography was performed on DEAE-Sepharose FF (Amersham Pharmacia Biotech AB) using a NaCl gradient in 0.02 M Tris-HCl, pH 7.6. Active fractions were concentrated using a 30 kDa Amicon Centricon Plus-80 centrifugal filter and subjected to gel filtration on a HiPrep 26/60 Sephacryl Hi-Res column (Amersham Pharmacia Biotech AB) in 0.02 Tris-HCl M, pH 7.6, containing 0.15 M NaCl. After concentration, the final preparation had a specific activity of 9-15 U / mg.

Expression and purification of recombinant mouse LAMAN The cDNA encoding rat LAMAN (16) was subcloned into the expression vector pMPSVEH (17). A polyhistidine tail (6 residues) had been added to the C-terminal part of the enzyme. Small and large mannose-6-phosphate (mpr - / - MEF) (18) receptor embryonic fibroblasts were transfected and stably expressing clones were selected with 50æL / mL hygromycin. For the production of recombinant rat LAMAN, the cells were cultured in medium supplemented with 10% FCS in a humidified atmosphere containing 5% CO2. The secreted recombinant rat LAMAN was purified from conditioned medium using a three step procedure. In the first step the medium was dialysed against 20 mM sodium phosphate buffer, pH 7.8 which contained 500 mM sodium chloride and then loaded onto a Probond column (Invitrogen). The retained enzyme was eluted with a gradient of 0 to 0.35 M imidazole (total volume 80 mL) in 20 mM sodium phosphate buffer, pH 6.0 containing 500 mM sodium chloride. Fractions containing LAMAN were dialyzed against 10 mM sodium phosphate, pH 6.0 and loaded onto a DEAE-cellulose. The enzyme was eluted in a 0 to 0.25 M sodium chloride gradient (total volume 80 mL) in 10 mM sodium phosphate buffer. Finally, rat LAMAN was adsorbed on ConA-Sepharose (20 mM Tris-HCl loading buffer, pH 7.4, containing 1 mM MgCl 2, 1 mM MnCl 2, 1 mM CaCl 2 and 0.5 M NaCl), and eluted with α-mannopyranoside (0.0-1.0 M) in the same buffer. The final preparation had a specific activity of 17-25 U / mg.

Purification of bovine LAMAN Bovine LAMAN was purified from kidney as described (19). The final preparation had a specific activity of 10 U / mg.

Purification of human LAMAN LAMAN was expressed on Chinese Hamster Ovary (CHO-) cells which were cultured in a two compartment CelLine flask at 37øC in a humidified atmosphere with 5% CO2. The enzyme obtained from the CHO cells was purified according to a purification procedure consisting of 4 steps:

Diafiltration The diafiltration was based on the TFF model, using a polysulfone Pellicon Biomax filter with a cut of 100 kDa. The sample was diafiltered against approximately 3 x volume of the sample with 0.02 M Tris-HCl, pH 7.6.

Ion Exchange Chromatography Ion exchange chromatography was performed using flow filtration with DEAE Sepharose. The buffer was 0.02 M Tris-HCl pH 7.6. The gel was packaged on a 16/20 XK column from Amersham Pharmacia Biotech AB. Elution was performed with a NaCl gradient.

Sample concentration

Prior to gel filtration, the sample was concentrated to a protein concentration of ~6 mg / mL and a final volume of 10 mL. This was performed using an Amicon Centricon Plus-80 centrifugal filter with a 30 kDa cut.

Gel filtration Gel filtration was performed on a HiPrep 26/60 Sephacryl Hi-Res column from Amersham Pharmacia Biotech AB. The sample was eluted with 0.02 M Tris-HCl supplied with 0.15 M NaCl, pH 7.6.

An example of a LAMAN purification is summarized in the table below. Usually the yield is in the range of 30-50% and the specific activity is 9-15 U / mg.

Summary of LAMAN Purification

Purified LAMAN is stored at -80 ° C.

In addition, a purification process for rhLAMAN at a scale of 20-200 mL is developed for scaling up for large-scale production. The quality and purity of the final product (rhLAMAN) are very high and in compliance with the specifications (approved for clinical trials). The process includes a capture step, 1-2 intermediate purification steps, 1 finishing step, 1-2 virus removal steps and 1 formulation step. Also included are 1 or more buffer exchange steps (diafiltration). The small-scale production process is transferred to intermediate scale and finally large-scale production.

Experimental design: Several different chromatography gels are tested and the performance of the different steps will be analyzed by a battery of analytical methods which is briefly described below:

Enzyme activity: Alpha-mannosidase assay

Total Protein Concentration: BCA Analysis

RhLAMAN concentration: rhLAMAN ELISA

Purity: HPLC and SDS-PAGE

Identity: HPLC

HCP proteins; ELISA Endotoxin level; Requested to subcontracted laboratory

Outline of the scale purification process of a 20 mL column

Step 1: Concentration / Diafiltration

The medium produced in T-500 flasks (0.06U / ml) with expressed rhLAMAN is concentrated approximately 10% * using

Tangential flow filtration (TFF) against a 100 kDa Pellicon Biomax polysulfone filter.

Example: 0.5-1.0 liter of rotary flask media is concentrated using TFF against a 50 cm 2 Biomax 100 kDa filter to 50-100 ml. The transmembrane pressure (TMP) is 25 (Pentade = 30 psi; Pose = 20 psi).

Thereafter diafiltration is applied against 7 volumes of 20 mM Tris-HCl, pH 7.6 with TMP of 25. The specific activity of the concentrated sample is 0.5-1.5 U / mg.

Yield 70 - 90%

Step 2: Capture step - DEAE sepharose FF

The concentrated sample from step 1 is applied to 20 ml of DEAE sepharose packed in a 16 mm diameter column (Pharmacia XK 16) equilibrated with 20 mM Tris-HCl, pH 7.6 (referred to as: standard buffer). Flow rate is 3 mL / min. The DEAE gel-bound protein is then washed with 2-4 column volumes (VC) of standard buffer followed by 2-4 VCs of 30 mM NaCl in standard buffer. RhLAMAN is eluted with a linear gradient of 30-300 mM NaCl in standard buffer for 20 minutes. Alternatively, for large-scale production: elution is achieved by applying 75-150 mM NaCl in standard buffer. The DEAE gel is washed with 2-4 VCs of 0.5 M NaCl in standard buffer, followed by 2.4 VCs of 1.0 M NaOH (contact time 40-60 minutes). Fractions containing rhLAMAN activity are pooled (specific activity: 2-5 U / mg) and used for further purification. Yield 70-90%.

Step 3: Intermediate 1 1. Hydrophobic interaction chromatography: butyl-, phenyl- or octyl-sepharose FF. The pooled sample from step 2 is mixed 1: 1 with 2.0 M Na2SO4 and applied on a 20 ml HIC (butyl-, phenyl- or octyl-sepharose) column packed in a balanced 16 mm diameter (Pharmacia XK 16) column with standard buffer + 1.0 Na2 SO4. The flow rate is 3-4 mL / min. The column is washed with 2-4 VCs of the same buffer. The rhLAMAN is eluted with standard buffer and the fractions containing activity are pooled and used for further purification. 2. Macroprep, Type I or II ceramic hydroxyapatite.

Brief description: Balancing column (gel volume = 20 mL) with 4-6 VCs of 10 mM Sodium Phosphate, H 7.6 (Buffer A). Flow rate 2-5 mL / min. Change the sample buffer from step 3 with TFF to Buffer A. Charge the sample from step 3 to the column. Wash with 2-4 VCs of Buffer A. Elute with buffer A containing 100 mM NaCl. Collect the peak containing rhLAMAN activity.

Step 4: Intermediate 2 1. Macro prep, Type I or II ceramic hydroxyapatite, 40æm. Brief description: see Step 3 2. Source 15 Q - anion exchanger

Balance the column with 4 VCs of 200 mM Tris-HCl pH 7.6. Change to 5 VCs of 20 mM Tris-HCl, pH7.6 (standard buffer). Flow rate: 2.5 mL / min. Load the sample from step 3 (it must be in standard buffer before application) into the column. Wash with 2-4 VCs of standard buffer. Apply a mild gradient of 0 to 100% 1M NaCl in standard buffer (flow 2 mL / min, gradient time 50 minutes). Collect fractions that contain rhLAMAN activity. 3. Source 15 S - cat ion exchanger

It should run at acidic pH. Balancing Buffer = 20 mM Sodium Acetate, pH 4.5. Elute with a gradient of NaCl (increasing salt concentration) or pH (increasing pH).

Step 5: Finishing Step 1. Macro prep, Ceramic Hydroxyapatite Type II, 40æm. Description of the parameters: see Step 3. 2. Source 15 Q - anion exchanger Description of the parameters: see Step 4. 3. Source 15 S - cation exchanger Description of parameters: see Step 4. 4. Affinity chromatography

Step 6: Diafiltration / Formulation Step Tangential flow filtration (TFF) is run against a 100 kDa Pellicon Biomax polysulfone filter against 5-10 volumes of formulation buffer. Two formulation buffers are tested:

Formulation buffer 1.

Water for Injection (WFI)

Formulation buffer 2.

Water for Injection Preparation (WFI) The pH and osmolality in both Formulation Buffer are equilibrated at 7.5 ± 0.2 and 300 ± 50 mOsm / kg, respectively. The final protein concentration is according to the description (> 5 mg / ml).

Step 7: Formulation, Filling and Freeze-drying Formulation and dosage form

In the development of the dosage form the stability of rhLAMAN is focused. The development process begins with an aqueous solution and will most likely end up as a freeze-dried product. Two different formulations are tested: Formulation Buffer 1 and Formulation Buffer 6, see Step 6.

Both of these formulations are known to stabilize proteins in aqueous solutions, as well as in freeze-dried powders. The pH and the osmolality in both Formulation Buffer will be equilibrated at 7.5 ± 0.2 and 300 ± 50 mOsm / kg, respectively. The final protein concentration should be according to the specification and in the range 4-10 mg / mL.

A freeze-dried product of rhLAMAN will be produced in a production facility in accordance with GMP EU practice. Filling and freeze-drying shall be performed in a room classified as Class A. During production, the filling zone is monitored with particulate counting plates and deposition plates. Personnel are trained regularly according to US GMP and monitored after each production with glove prints. The sterility of the equipment and materials is ensured by validated sterilization procedures.

Filling The pharmacological bulk substance of rhLAMAN is filled aseptically into sterile type I glass vials.

Freeze-drying

The vials are freeze-dried with freeze-drying cycles developed specifically for rhLAMAN in the two different formulations described above. Nitrogen gas is introduced into the flasks at the end of the cycle and eventually closed with stoppers and capped. The batch is finally analyzed and released according to the description.

Determination of LAMAN phosphorylation LAMAN was incubated overnight with an MPR 300 affinity matrix as described (20). LAMAN activity was determined on unbound fraction and fractions were eluted with 5 mM 6-phosphate glucose and 5 mM mannose-6-phosphate.

Results Purified LAMAN from three different species was used for enzymatic replacement in mice with aplasidosis (Fig. 1). Bovine kidney LAMAN is represented by a mixture of polypeptides (11-38 kDa) generated by limited proteolysis of a common precursor (19). As expected for a purified tissue lysosomal hydrolase and which has thus been exposed to endosomal / lysosomal phosphatase activity, the enzyme has a low Man6P recognition marker content. When incubated with a Man6P receptor affinity matrix, 6.4% of the LAMAN activity bound to the matrix in a Man6P-dependent manner. Recombinant rat and human LAMAN were isolated from secretions of Man6P receptor deficient mouse fibroblasts and CHO cells, respectively. Enzymes were largely represented by their precursor forms, but contained a variable fraction (5-35%) of proteolytically processed forms of 55 and 70 kDa (Fig. 1). Rat LAMAN had a higher content of Man6P recognition marker, which mediated the binding of 73.6% of the activity to the affinity matrix. Of the only 4.2% human LAMAN bound in a Man6P-dependent manner to the Man6P receptor affinity matrix, which indicates that the content of Man6P recognition marker is as low as for bovine LAMAN. Several batches were subsequently analyzed, for example: batch lot 2003-09 - 2004-02: 40% batch phosphorylation 2004-02-02 - 06: 30% batch phosphorylation batch 2003-01-21 - 04-16 42% phosphorylation

In general, approximately 40% of the amount of enzyme binds to the affinity matrix. The low degree of phosphorylation observed for human and bovine LAMAN is believed to result from prolonged storage of the enzymes.

Example 2: Effects of in vivo administration of LAMAN on stored oligosaccharide levels

Experimental procedures

Rat Injection LAMAN was injected into the tail vein of mice with 8-14 week old aosinoseose (final volume up to 5.3 æl / g body weight). Simulated injected rats received the same volume of 10 mM phosphate, pH 7.4 in 0.15 M NaCl (PBS). In a single experiment, rats from up to three litters did not differ in age. 5 min after injection, blood was collected from the retroorbital plexus to control the amount of enzyme injected. The serum was prepared and stored at -20 ° C.

Preparation of organ extracts

The rats were anesthetized with 20 μl of a solution of Ketavet 10 mg / ml (Parke Davis) and 2 mg / ml Rompun (Bayer) in 0.15 M NaCl and perfused with PBS. The organs (liver, spleen, kidney, heart and brain) were collected and stored at -78 ° C. About 50-70 mg of each organ were homogenized at 4 ° C in 9 volumes (by weight) of 10 mM Tris / HCl, 150 mM NaCl, 1 mM PMSF (in isopropanol), 1 mM iodoacetamide, 5 mM EDTA. Triton X-100 was added to a final concentration of 0.5% w / v (1% to the liver). After incubation for 30 minutes on ice, the samples were sonicated and then centrifuged for 15 minutes at 13000 g. The supernatant was stored at -20 ° C.

Enzymatic assays

For determination of LAMAN activity in organ and serum extracts, 10-50 μl of enzyme sample were incubated in 0.2 ml of 0.2 M sodium citrate, pH 4.6, 0.08% NaN 3 , 0.4% BSA, 0.15% NaCl, and 10 mM p-nitrophenyl-α-mannopyranoside as substrate for 0.5-5 h at 37øC. 1 mL of 0.4 M glycine / NaOH, pH 10.4 was added to stop the reaction. The absorbance was read at 405 nm (ε = 18500 M -1 cm -1). All determinations were performed in duplicate and with appropriate whites.

Western transfer

For Western blotting of human LAMAN, 20-40æg of protein was separated on a 10% SDS-polyacrylamide gel. After SDS-PAGE electrophoresis, the proteins were transferred to PVDF membranes using a semi-transparent transfer system. The transfer efficiency was verified by revelation with Ponceau. The membranes were subsequently blocked with 5% skimmed milk powder and incubated with the primary antibody at a 1:50,000 or 1: 100,000 dilution. After washing in PBS, 0.1% Tween 20, the blots were incubated with horseradish peroxidase-coupled secondary antibodies (HRP) (1:20,000). The signals were visualized using the ECL Detection System (Amersham, Freiburg, Germany).

Isolation of neutral oligosaccharides

Tissue samples (50-60 mg) were cut into small pieces and homogenized with 0.6 mL H2 O (HPLC grade) at 4 ° C. After freezing (-20 ° C) and thawing twice and ultrasonic treatment, the proteins were precipitated by the addition of 4 vol of methanol and extracted by the addition of 4 vol of chloroform / iO (1: 3) (21). The supernatant was

desalted by incubation for 1 h at 4 ° C with mixed bed ion exchange resin (AG 501-X8, 20-50 mesh). The unbound material was lyophilized and resuspended in water (1 mL per mg tissue).

Separation of mannose oligosaccharides by thin layer chromatography (TLC)

Neutral oligosaccharides extracted from equal aliquots of tissue were loaded onto TLC plates (20x20 Silica gel F60, Merck). After drying the plates at room temperature for ~ 1 h, the oligosaccharides were separated by overnight development with n-butanol / acetic acid / H 2 O (100: 50: 50). After drying (~1 h at room temperature and then 5 min at 110 ° C), the plates were grown for 4 h in n-propanol / nitromethane / H 2 O (100: 80: 60). For development, the plates were sprayed with 0.2% orcinol in H2 SO4 (20% in water), and heated to 110 ° C. The size of oligosaccharides was determined by MALDI-TOF (see below).

Digestion of liver oligosaccharides with α-glycosidase or madagascar bean α-mannosidase In order to avoid interference with glycogen-derived oligosaccharides, 20 μl of liver and heart oligosaccharide extracts were incubated overnight another at 37 ° C with 40 U / ml Bacillus stearothermophilus α-glycosidase (Sigma) in 20 mM phosphate, pH 6.8. The incubation mixture was heated to 96 ° C to denature the proteins. After centrifugation for 10 min at 13000 g, the supernatant was desalted by incubation for 1 h at 4øC with an ion exchange resin (AG 501-X8, 20-50 mesh), lyophilized and resuspended in 20 ul of water and then separated by TLC. In order to verify the nature of the oligosaccharides, 20 æl of the oligosaccharide extracts were incubated overnight at 37øC with 30 U / ml madagascar bean α-mannosidase (Sigma) in 0.1 M sodium, pH 5.0, containing 2 mM ZnCl 2. After incubation, the oligosaccharides were prepared as described for the α-glycosidase digested samples and separated by TLC.

Quantitative analysis of neutral oligosaccharides in organs

The oligosaccharides (0.3æl) were mixed with 220 pmol of a decassaccharide which served as an internal standard and had the composition GlcNAc4Man3Gal3. The mixture was lyophilized and resuspended in 5 æl DMSO / acetic acid (7: 3) containing 0.34 M 2-anthranilamide (Aldrich) and 1 M NaBH 3 CN (Fluka). After incubation for 2 h at 65 ° C, the samples were purified by paper chromatography (developed with ethyl acetate). The oligosaccharides, located at the starting point, were extracted by subjecting the paper to lyophilized waterborne ultrasound, resuspended in 300 μl acetonitrile / 80 mM ammonium formate, pH 4.4 (65:35), loaded onto a Gluco-Sepharose column (Ludger) and eluted at a flow rate of 0.4 mL / min with acetonitrile / ammonium formate buffer. Fluorescence (excitation 350 nm, emission 450 nm) was recorded (Shimadzu, RF-10A XL) and the oligosaccharide masses determined by MALDI-TOF.

MALDI-TOF

Samples of the HPLC fractions were lyophilized and dissolved in 2-3 μl of water. The aqueous extracts from the TLC plates were derivatized with 1-phenyl-3-methyl-5-pyrazolone and dissolved in 2-3 μl of water. 2,5-Dihydroxybenzoic acid (DHB, 5 mg / mL in water) was used as the matrix. 0.5 μl of DHB and 1 μl of sample were applied to the Anchorchip target (Bruker Daltonik) and dried at room temperature. Mass spectrometric analysis was performed on a MALDI-TOF Reflex III (Bruker Daltonik) with a 337 nm UV laser.

Histological examinations

For electron microscopy, small sections of liver and kidney collected at time of death were dipped in 0.1 M phosphate, 6% glutardialdehyde pH 7.4. The tissue samples were post-fixed with 2% osmium tetroxide, dehydrated and embedded in Araldite. The semifinished sections were developed with toluidine blue. The ultrafine sections were processed according to standard techniques. For histochemical investigations, the sections were immersed with Bouin's solution diluted 1: 4 in 10 mM phosphate, pH 7.4, 0.15 NaCl. Incorporation was carried out in low melting point paraffin (Wolff, Wetzlar, Germany). Successive sections (7 pm) were cut and mounted on Biobond coated glass slides (British Biocell, London, UK). The central sections of each series were developed with hematoxylin and eosin for standard optical microscopy.

Results

Corrective effect of a single intravenous injection of rat LAMAN In order to study the short- and long-term effect of a single dose of LAMAN on storage of neutral oligosaccharides, mice with 9 week old α-mannosidosis received 100 mU of LAMAN of rat per g of body weight intravenously. To control the amount of enzyme injected and its clearance from the circulation, blood was collected at 5, 30 and 60 min after injection. At 5 min after injection the LAMAN activity varied less than ± 6%, indicating that the rats received comparable amounts of enzyme. The enzyme was cleared from the circulation with a half-life of less than 20 min. Rats were killed 2, 4 and 10 h after injection and organ extracts were prepared to determine the activity of LAMAN and the neutral oligosaccharides. The maximal LAMAN activity in the organs was observed 2-4 h after injection (Table 1). In liver the activity was 6-7 times higher than in control rats, but also in the spleen and heart the peak values exceeded the controls. In the kidney a maximum of one quarter of the activity was achieved in control mice. It is likely that the small activity observed in the brain is of the LAMAN present in the vascular system. Between 4 and 10 h after injection, enzymatic activity rapidly decreased in the liver, spleen and kidney with an apparent half-life of about 3 h. Western blotting demonstrated that the internalized precursor of LAMAN was rapidly processed in mature forms (not shown). The hallmark of α-mannosidosis is the storage of neutral oligosaccharides in a wide variety of tissues (see Fig. 2, lane 1 and lane 2). These oligosaccharides contain 2-9 mannose residues and an N-acetylglucosamine residue at their reducing end and thus result from the action of an endoglucosaminidase. The amount of neutral oligosaccharides in the liver and spleen progressively decreased over time to 15% and 7% of simulated injected animals, whereas in the kidney and heart the storage reduced only to about 49% and 74% (Figure 2) . TABLE 1: LAMAN activity in tissue extracts of control and α-mannosidosis mice before and after injection of 100 mU rat LAMAN per g body weight. * + / + refers to control mice, - / - mice with a-mannosidosis, and to the number of animals investigated. + All values were corrected for the mean serum a-mannosidase activity at tzero (2350 mU / ml serum). The correction factors were 0.98, 1.07 and 0.96 for the 2, 4 and 10 h, respectively.

To determine how long the corrective effect of a single dose of LAMAN remained, rats were examined 4, 8 and 16 days after the injection of 100 mU rat LAMAN per g body weight. At all points in time LAMAN activity in the organs (spleen, kidney, heart, brain) was in the range of untreated rats or mice with α-mannosidosis injected in a simulated fashion, except for the liver, where 4 days after injection LAMAN activity (15.1 mU / g wet weight) was still about 5-fold higher than in simulated injected rats. Storage of neutral oligosaccharides in the liver, spleen and kidney 4 days after injection was in the range observed 10 h after injection (compare Fig. 2 and Fig. 3). At heart, storage decreased from 74% after 10 h to 42% after 4 days. This indicates that the corrective effect observed after 10 h remains for about 4 days, although little or no LAMAN activity is detectable 10 h after injection into organs such as kidney or heart. After 4 days, the storage of oligosaccharides clearly began to increase again. The increase observed between day 4 and day 16 after injection corresponded to 20-40% of the storage observed in simulated injected α-mannosidosis rats (Fig. 3).

Comparison of clearance and corrective effect of bovine, rat and human LAMAN

To compare the corrective effect of the LAMAN preparations from the three sources, the mice were injected with a dose of LAMAN which was expected to produce a partial repair. Consequently, we injected 50 mU of LAMAN per g of body weight. Bovine and human LAMAN were rapidly cleared from the circulation with half-lives of 4 min and 8 min, respectively (Fig. 4). The clearance of highly phosphorylated rat LAMAN was slower and at least biphasic. About 85% of the enzyme was purified with an apparent shelf life of 12 min, while apparent clearance of the remaining fraction was about 47 min (Fig. 4).

Rats were killed 2 days after injection and liver, spleen, kidney and heart extracts were examined for neutral oligosaccharides (Fig. 5). With the exception of the liver, the corrective effect was higher for human LAMAN and lower for bovine LAMAN. The corrective effect of the rat enzyme was intermediate. Only in the liver, the corrective effect of bovine enzyme (remained 4% of storage) was more pronounced than that of the human enzyme (remained 14% of storage). In liver, the corrective effect of rat LAMAN was the weakest (remained 23% of storage).

Corrective effect of human LAMAN at high dose Comparison of bovine, rat and human LAMAN indicated that poorly phosphorylated human LAMAN had a relatively higher corrective potential in the kidney and heart, two organs that are more resistant to metabolic correction than the liver and the spleen. In order to evaluate the corrective potential of the human enzyme we injected a single dose of 250 mU of LAMAN and analyzed the mice 1 to 12 days after injection. In the liver, storage was fully corrected 1 and 3 days after injection. After 6 and 12 days, the neutral oligosaccharides began to accumulate again, but only reached about 30% of the storage level before treatment (Fig. 6). Optic microscopy examination of the liver revealed the near-complete disappearance of storage vacuoles, which are prominent in sinus endothelial cells, Kupffer cells and hepatocytes of non-α-mannosidosis rats and reappear 12 days after injection (Figure 7) . In the spleen and kidney, the storage of neutral oligosaccharides decreased to 12 and 18%, respectively. It is noteworthy that in the spleen and kidney the maximum correction was observed after 3 and 6 days, respectively. In both organs, the neutral oligosaccharides began to accumulate again 3 and 6 days after the injection (see Fig. 5). Neutral oligosaccharides in the brain of α-mannosidosis rats were not affected by the treatment (not shown).

The activities of human LAMAN recovered 24 h after injection were much higher than expected from rat LAMAN experiments. In the liver, the activity of human LAMAN was 6 times higher than in the control liver. In the spleen and kidney it was responsible for 10-15% of that in the control. To follow the uptake and stability of the human enzyme, LAMAN activity was determined in prepared tissue extracts 4, 16 and 24 h after injection of 250 mU of human LAMAN per g body weight. Less than 20% of the injected LAMAN was recovered after 4 h in the tissues examined (liver 18%, kidney 0.4%, spleen 0.12% and heart 0.04%). When compared to the rat LAMAN uptake and taking into account that a 2.5-fold higher amount of human LAMAN was injected, the activity recovered in the liver, kidney and heart 4 h after injection was comparable for both enzyme preparations ( compare Table 1 and Table 2). Spleen uptake appeared to be 2-3 times less efficient for the human enzyme. The major difference between the human and rat enzyme was the greater stability of human LAMAN internalized by the liver, kidney and spleen. While human LAMAN activity decreased in these organs after 24 h to 17-33% of the value at 4 h (see Table 2), that of the rat enzyme decreased to 20-26% of the value at 4 h, shortly after 10 h see Table 1). The corrective effect of rat LAMAN (see Fig. 3) and human LAMAN was only transient. The neutral oligosaccharides began to accumulate again 3 to 6 days after injection. Increasing the amount of enzyme injected would be a means to retard re-accumulation. The corrective effect of a given dose of LAMAN is predicted to be higher when administered as two half-doses separated by an appropriate range than as a single dose. Instead of increasing the amount of LAMAN to 500 mU / g body weight we twice administered 250 mU of human LAMAN per g of body weight each on day 7 and 3.5 before analysis. This resulted in a complete correction of deposition in the kidney and heart (Fig. 8). Optical microscopy showed the absence of storage vacuoles in the liver (not shown) and in the tubular epithelia of the kidney (Fig. 9). Residual spleen storage was less than 20% of that in simulated injected α-mannosidosis mice. Most especially in the brain, the level of neutral oligosaccharides as quantified by TLC was only half that in the brain of simulated-injected a-mannosidosis mice (Fig. 8). TABLE 2: Activity of LAMAN in rat tissue extracts with a-mannosidosis 4-24 h after injection of 250 mU of human α-mannosidase per g of body weight. "1" All values were corrected for the mean activity of LAMAN in serum at zero (5012 mU / mL serum). The correction factors ranged from 0.903 to 1.222. * For LAMAN activity in tissue extracts from control rats and uninjected rats see

Table 1.

In order to quantify the neutral oligosaccharides on a molar basis, the neutral oligosaccharides extracted from the brain of simulated-injected α-mannosidosis mice and LAMAN were reacted with 2-anthranilamide, which introduces a fluorescent label at the oligosaccharide reducing ends. The fluorescently labeled oligosaccharides were separated by HPLC and quantitated by comparison with an internal standard oligosaccharide added prior to derivatization with 2-anthranilamide (Fig. 10). The major oligosaccharide species in the brains of α-mannosidosis mice contains 2, 3 or 4 mannose residues. These are present at concentrations of 319, 87 and 164 pmol / g tissue, respectively, and account for 61% of all neutral oligosaccharides in the brain. In the brains of treated α-mannosidose rats the concentration of neutral oligosaccharides decreased to 26% of the injected mice in a simulated fashion. The corrective effect was observed for each of the oligosaccharide species, but was relatively higher for the species with 9, 7 and 4 mannose residues (see Fig. 10).

In the present study, purified LAMAN from bovine kidney was used. This enzyme preparation consisted of mature polypeptides with a low mannose-6-phosphate content. Recombinant human LAMAN isolated from the secretion of CHO cells consisted mainly of the precursor form, but was also poorly phosphorylated. The low mannose-6-phosphate content of this preparation is likely to be due to dephosphorylation during isolation. Occasionally, preparations of human LAMAN of which up to 40% were bound to the MRP 300 column (C. Andersson, DP Roces, unpublished observation) were obtained. Recombinant mouse LAMAN purified from secretions of rat embryonic fibroblasts lacking mannose-6-phosphate receptors was highly phosphorylated and represented by the precursor form. The clearance of the circulation was faster for the two less phosphorylated LAMAN preparations of bovine kidney and CHO cells than for the highly phosphorylated rat LAMAN. The uptake of the different preparations of LAMAN by the organs was not directly compared. However, comparison of data from different experiments suggests that similar fractions of the poorly phosphorylated human LAMAN and highly phosphorylated rat LAMAN are captured by the liver, kidney and heart. In the spleen only the uptake was different and facilitated by the higher phosphorylation. In a related study on the uptake of phosphorylated and non-phosphorylated β-glucuronidase in rats, no difference was observed for uptake into the liver and spleen, whereas uptake into the kidney and heart was higher for the phosphorylated enzyme (26). For a-galactosidase preparations differing in glycosylation and phosphorylation a similar uptake was observed by the liver, spleen and kidney (27). This indicates that the effect of glycosylation and phosphorylation on the uptake of lysosomal enzymes to the different organs depends on the enzyme.

A major difference between LAMAN preparations from different sources is their stability. Assuming a first-order kinetics, rat LAMAN (see Table 1) and bovine (not shown) LAMAN activity decreased in the liver, kidney and spleen with an apparent half-life of 3 h or less, while the apparent half-life of human LAMAN was higher 3 times longer. This may explain the greater corrective efficacy of human LAMAN on the storage of oligosaccharides in the spleen, kidney and heart (see Fig. 5).

A single intravenous administration of LAMAN led to a rapid and marked decrease in lysosomal storage of neutral oligosaccharides regardless of the source of the enzyme. The corrective effect was very pronounced in the liver. This is also the tissue where the increase in LAMAN activity relative to that in the wild type mice was the highest. Histological examination revealed a correction of storage in both parenchymal and non-parenchymal liver cells. This was true for highly phosphorylated rat LAMAN (not shown) and for poorly phosphorylated human LAMAN (see Fig. 7). This was unexpected since an earlier study with β-glucuronidase showed that non-phosphorylated forms of enzymes were located almost exclusively in non-parenchymal liver cells, whereas phosphorylated forms were located in both parenchymal and non-parenchymal liver cells (26). The corrective effect of LAMAN was transient in all tissues. The neutral oligosaccharides began to accumulate again 2 to 6 days after the injection.

A remarkable correction of storage was observed when injection of 250 mU of human LAMAN per g body weight, which reduces spleen and kidney deposition by 80-90% was repeated after 3.5 days. In less than a week, the storage disappeared not only from the liver but also from the kidney and heart. Even more importantly, the concentration of neutral oligosaccharides also decreased in the brain to about a quarter. The latter can not be attributed to an uptake of LAMAN to nerve cells. In rats the blood-brain barrier matures within the first two weeks of life, and lysosomal enzymes administered intravenously after this period do not cross the blood-brain barrier (28-31). According to these observations, we observed trace amounts of LAMAN in the brain homogenates of treated rats (see Table 1 and 2). These activities are attributed to extraneuronal cells, such as endothelium, choroid plexus epithelium and incompletely removed meninges. Since the administered LAMAN is unlikely to gain access to the oligosaccharides deposited in neuronal and glial cells, alternative mechanisms must be considered. Cleansing of oligosaccharides through the bloodstream or through an improved flow of cerebrospinal fluid may contribute to the decrease of stores of neutral oligosaccharides in the brain.

Example 3: rhLAMAN - Efficacy of repeated dose replacement therapy in arabosidose rats The aim of the study is

Demonstrate a total correction of mannose oligosaccharide storage in all peripheral tissues, and check the correction of storage in the brain.

To determine whether the injection of a repeated dose of rhLAMAN also has an effect on the LAMAN activities measured in the different peripheral tissues.

Follow the development of antibodies after repeated injections and check if they precipitate the enzyme. Determine whether, as soon as the mice develop antibodies, these have an effect on the clearance of the enzyme.

Determine whether an increased level of antibodies has an effect on blood brain barrier (BBB) permeability.

To compare gene expression in inactivated, inactivated and wild type inactivated rats after 1 and 4 weeks of treatment. A microarray experiment with liver RNA will be performed. The mice used for this experiment are selected from heterozygous pups (see below).

Justification of the test system Test item The test item is supplied as a solution at 5.4 mg.mL-1 (8.2 U / mg, lot 041202) and is used as supplied. The test item is stored frozen (ca -20 ° C) in the dispensary when not in use.

Animals and management

For the experiment 5 different groups of rats are used: (- / -) rats with C57 / B6 (+ / +) a-mannosidosis (wild type rats) (+/-) heterozygous rats that are offspring of a cross between rats (+/-) mice (+/-) (- / -) mice with heterozygous crosses (+/-) (+/-) rats with heterozygous crosses (+/- mice)

Age: 8 weeks at baseline. Number of animals: 64 rats (male and / or female)

Health Status: Animals suspected of being ill are eliminated from the study. If the significant number of animals is improper, the entire batch of animals is rejected and a new batch is obtained. Acclimatization: Animals are allowed to acclimatize to accommodation for a minimum of 3 days prior to initiation of administration.

Allocation to Dosage Groups: All animals are weighed and the required number of animals is randomly assigned to the 5 dose groups. No stratification by weight and / or sex is required as the dose is adjusted for weight and to date no gender-specific differences have been found. Only males are used for the Micromatrix experiment (groups 2 *, 4 * and 5 *, see below), since in gene expression studies, sex has often been described as a relevant factor.

Room Environment: Animals are housed 4 / cage or 8 / cage in a polypropylene cage with stainless steel mesh tops and solid bottoms with wood chips as bedding material. The cages (267 x 207 x 104 mm or 425 x 266 x 188 mm) are changed, as required.

The cages are provided with a polycarbonate water bottle (capacity 250 mL) with a stainless steel spout and Duretano cap. Bottles of water are filled as needed and changed / washed at least once a week. There is automatic temperature and humidity control, the target ranges are 20 ° C ± 2 ° C and 50% ± 15%, respectively, with an airflow in the room to give a minimum of 15 air changes per hour. The temperature and humidity ranges are monitored and recorded daily. There is an automatic control of the light cycle; the hours of light are normally from 0600-1800.

Diet and Water Supply Food and water are available for animals ad libitum. It is considered that the food and water used do not contain any additional substances in sufficient concentration to have any influence on the outcome of the study.

Treatment

Dose Groups / Dose Level

Group 1 (for biochemical investigations, 16 LAMAN rats). Rats are treated twice a week intravenously with 250 mU / g body weight rhLAMAN, and groups of 4 mice are killed after 1, 2 and 4 weeks of treatment.

Group 2 * (for microarray investigations, 8 heterozygous cross-over LAMAN rats), are treated twice weekly with 250 mU / g body weight rhLAMAN, and groups of 3 mice are killed after 1 and 4 weeks of treatment.

Group 3 (for blood brain barrier, antibody and plasma clearance investigations, 12 LAMAN rats), are treated once weekly with 250 mU / g body weight of rhLAMAN. Both groups of rats, the 4 rats used for BBB investigations, and the 4 rats used for investigational clearance, are killed after 4 weeks of treatment.

Group 4 (Rats treated in a simulated manner, 12 LAMAN rats) are treated once / twice weekly with rhLAMAN vehicle at the same dose volume as Group 3 / Group 1 treated animals. A group of 4 mice injected one once a week are killed after 4 weeks of treatment (to be compared with the mice in group 3), and groups of 2 mice are killed after 1, 2 and 4 weeks of treatment (to be compared with group 1)

Group 4 * (Rats treated in a simulated manner, 8 heterozygous crossing LAMAN rats) and Group 5 * (8 heterozygous wild type crossing) are treated twice a week with vehicle for rhLAMAN at the same dose volume as the animals treated in the Group 2*. Groups of 3 rats are killed after 1 and 4 weeks. * Rats with α-mannosidosis of heterozygous (+/-) pups, descended from a reproduction between rats with wild-type (+/-) and wild-type A-mannosidosis (- / -) and C57 / B6. TABLE 3: Allocation of animals to dose groups:

TABLE 3: Allocation of animals to dose groups (continued)

Route, Duration and Method of Administration The test item is administered through an intravenous bolus injection in one of the lateral tail veins at the target dose levels. The volume of dose required is calculated according to the body weight of the most recently registered animal.

Prior to administration, the rats are anesthetized (intraperitoneally) with 75 æl per 100 g body weight of a solution of 10 mg / ml Ketavet (Parke Davis) and 2 mg / ml Rompun (Bayer) and are maintained at about 37 ° C until recovery from anesthesia.

Comments

Viability

All animals are checked every day for viability, early in the morning and as late as possible. Any animal that shows signs of weakness or severe intoxication and is considered to be in a dying state is killed. Any animal killed in extremis or found dead is, whenever possible, subjected to a macroscopic post mortem examination on the day of death. If macroscopic post-mortem examination is not possible on the day of death (eg late afternoon or on weekends), the carcass is placed in a polyethylene bag and kept in a refrigerator (4 ° C) until examination on day Following.

Clinical Signs

All animals are observed on days of administration for reaction to treatment. The beginning, intensity and duration of any observed signals are recorded.

Body weight

Prior to administration all animals are weighed weekly and recorded and classified.

Laboratory investigations

Treatment verification

Five minutes after injection, blood from the retroorbital plexus is collected to measure the plasma levels of rhLAMAN (according to internal procedure 2). If an animal has not received the full dose, the missing amount in% is calculated and given to the animal on the same day.

Kinetics

Of the 4 mice in group 3, blood is collected from the retroorbital plexus one day prior to injection, and also 5, 15 and 30 minutes after each injection, to investigate a possible Ab effect on enzyme clearance.

If enzyme is still available at the end of the experiment (4 weeks), treatment of these mice will continue until 8 weeks.

Determination of antibodies

Prior to administration, blood from the retroorbital plexus of the animals of group 2, as well as from all other animals at the end, is collected to measure antibody titres.

Terminal studies Method of death

Rats are anesthetized with 600æL of a 10 mg / mL solution of Ketavet (Parke Davis) and 2 mg / mL Rompun (Bayer) in 0.15 M NaCl, perfused with PBS and killed by bleeding.

For all groups except group 3, death occurs three and a half days after the last treatment. For group 3, the rats are killed 1 week after the last treatment (see table 3).

Histopathology

The following organs are harvested and fixed with 0.1 mM NaPi / 6% buffered Gluteraldehyde at pH 7.4: liver, kidney, brain Small sections are cut with a razor blade and the fixed slides are subjected to analysis by electron microscopy .

Concentrations of LAMAN in tissues

Liver, spleen, kidney, heart, TBS / PI (Protease Inhibitors) homogenates are measured for LAMAN activity using p-nitrophenyl-a-mannopyranoside as a substrate and 0.2 M sodium citrate, pH 4, 6, 0.08% NaN3, 0.4% BSA as buffer. LAMAN processing is followed by Western blotting (~ 1 mU required).

Oligosaccharides in Tissues From the organs described in 10.3 a preparation of neutral oligosaccharide extracts and quantified by densitometry is prepared to study the normalization of storage in organs by TLC according to procedure 4 and 5.

Neutral oligosaccharides from the liver and heart will be digested with α-glycosidase to prevent glycogen interference.

Statistical analysis of results

Unless otherwise noted, all statistical tests shall be bilateral and performed at a significance level of 5% using laboratory software. The following paired comparisons are performed:

Vehicle Control Group 4A vs Group IA

Vehicle Control Group 4B vs Group 1B

Vehicle Control Group 4C vs. Group 1C

Vehicle Control Group 4D vs. Group 3A

Vehicle Control Group 5A *, 5B * vs Group 4E *, 4F *

Vehicle Control Group 5A *, 5B * vs Group 2A *, 2B *

Vehicle Control Group 4E *, 4F * vs Group 2A *, 2B *

Data are analyzed for normality of distribution using the Shapiro-Wilk test and quantil-quantil graphs. When a normal distribution can be assumed, the differences between groups are tested using linear models (ANOVA) with or without the homoscedasticity assumption. If the data (or data transformed by log) do not have a normal distribution, a Kruskal-Wallis test is used. Additional data analysis steps may be performed depending on the data. (* heterozygous pups, see below)

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Purification of normal and mutant lysosomal alpha-mannosidase from human liver. Life Sci 37 (25): 2365-2371 60. Joss V, Rogers TR, Hugh-Jones K, Beilby B, Joshi R, Williamson S, Foroozanfar N, Riches P, Turner M, Benson PD, Hobbs JR. 1982 Exp Hematol 10, 52 61. Kelly BM, Yu CZ, Chang PL. 1989. Presence of a lysosomal enzyme, arylsulfatase-A, in the pre-isolated endosome compartments of human cultured fibroblasts. Eur J Cell Biol 48 (1): 71-78 62. Klein MA, Frigg R, Flechsig E, Raeber AJ, Kalinke U, Bluethmann H, Bootz F, Suter M, Zinkernagel RM, Aguzzi A. 1997. The crucial role for B cells in neuroinvasive scrapie. Nature 390 (6661): 687-690 63. Kreysing J, Polten A, Hess B, von Figure K, Menz K, Steiner F, Gieselmann V. 1994. Structure of the lysosomal mouse alpha-mannosidase gene and cDNA. Genomics 19 (2): 249-256 64. Kreysing J, von Figure K, Gieselmann V. 1990. Structure of the lysosomal alpha-mannosidase gene. Eur J Biochem 1990. Eur J Biochem. 191 (3): 627-631 65. Krivit W, Shapiro E, Kennedy W, Lipton M, Lockman L, Smith S, Summers CG, Wenger DA, Tsai MY, Ramsay NK, et al. 1990. Treatment of late infantile metachromatic leukodystrophy by bone marrow transplantation. N Engl J Med 322: 28-32 66. Krivit W, Shapiro E, Hoogerbrugge PM, Moser HW. 1992. State of the art review. Bone marrow transplantation treatment for storage diseases. Keystone. January 23, 1992. Bone Marrow Transplant 10 Suppl 1: 87-96 67. Laidler PM, Waheed A, Van Etten RL. 1985. Structural and immunochemical characterization of human urine lysosomal alpha-mannosidase purified by affinity chromatography. Biochim Biophys Acta 827 (1): 73-83 68. Lee GD, van Etten RL. 1975. Evidence of an essential histidine residue in rabbit liver aryl sulfatase A. Arch Biochem Biophys 171 (2): 424-434 69. Luijten JAFM, Van Der Heijden MCM, Rijksen G, Staal GEJ. 1978. Purification and characterization of lysosomal alpha-mannosidase from human urine. J Mol Med. 1978, 3, 213 70. Manowitz P, Fine LV, Nora R, Chokroverty S, Nathan PE, Fazzaro JM. 1988. A new electrophoretic variant of lysosomal alpha-mannosidase. Biochem Med Metab Biol 39 (1): 117-120 71. Mold DL, Dawson AM, Rennie JC. 1970. Very early replication of scrapie in lymphocytic tissue. Nature 228 (273): 779-780 72. Ohashi T, Watabe K, Sato Y, Saito I, Barranger JA, Matalon R, Eto Y. 1996. Gene therapy for metachromatic leukodystrophy. Acta Paediatr Jpn 38 (2): 193-201 73. Oya Y, Nakayasu H, Fujita N, Suzuki K, Suzuki K. 1998. Pathological study of mice with total deficiency of sphingolipid activator proteins (SAP knockout mice). Acta Neuropathol (Berl) 96 (1): 29-40 74. Poretz RD, Yang RS, Canas B, Lackland H, Stein S, Manowitz P. 1992. The structural basis for the electrophoretic isoforms of normal and variant human platelet arylsulphatase A Biochem J 287 (Pt 3): 979-983 75. Porter MT, Fluharty AL, Kihara H. 1971. Correction of abnormal cerebroside sulfate metabolism in cultured metachromatic leukodystrophy fibroblasts. Science 1971, 172, 1263-1265 76. Sangalli A, Taveggia C, Salviati A, Wrabetz L, Bordignon C, Severini GM. 1998. Transduced fibroblasts and metachromatic leukodystrophy lymphocytes transfer lysosomal alpha-mannosidase to myelinating glia and deficient cells in vitro. Hum Gene Ther 9 (14): 2111-2119 77. Sarafian TA, Tsay KK, Jackson WE, Fluharty AL, Kihara H. 1985. Studies on the charge isomers of lysosomal alpha-mannosidase. Biochem Med 33 (3): 372-380 78. Schmidt B, Selmer T, Ingendoh A, von Figure K. 1995. A novel amino acid modification in sulfates that is defective in multiple sulfatase deficiency. Cell 82 (2): 271-278 79. Selmer T, Hallmann A, Schmidt B, Sumper M, von Figure K. 1996. The evolutionary conservation of a novel protein modification, the conversion of cysteine to serinesemialdehyde in arylsulfatase from Volvox carteri. Eur J Biochem 238: 341-345 80. Shapira E, Nadler HL. 1975. Purification and some properties of soluble human liver arylsulfatases. Arch Biochem Biophys 170 (1): 179-187 81. Shapiro EG, Lockman LA, Balthazor M, Krivit W. 1995. Neuropsychological outcomes of several storage diseases with and without bone marrow transplantation. J Inherit Metab Dis 18: 413-429 82. Stein C, Gieselmann V, Kreysing J, Schmidt B, Pohlmann R, Waheed A, Meyer HE, O'Brien JS, von Figure K. 1989. Cloning and expression of human lysosomal alpha -mannosidase. J Biol Chem 264: 1252-1259 83. Stevens RL, Fluharty AL, Skokut MH, Kihara H. 1975. Purification and properties of arylsulfatase. A from human urine. J Biol Chem 250 (7): 2495-2501 84. Stevens RL, Fluharty AL, Killgrove AR, Kihara H. 1976. Microheterogeneity of lysosomal alpha-mannosidase from human tissues. Biochim Biophys Acta 445 (3): 661-671 85. von Figure K, Steckel F, Conary J, Hasilik A, Shaw E. 1986. Heterogeneity in late-onset metachromatic leukodystrophy. Effect of inhibitors of cysteine proteinases. Am J Hum Genet, 39, 371-382 86. Waheed A, Hasilik A, von Figure K. 1982. Synthesis and processing of lysosomal alpha-mannosidase in human skin fibroblasts. Hoppe Seylers Z Physiol Chem 363 (4): 425-430 87. Waheed A, van Etten RL. 1985. Phosphorylation and sulfation of lysosomal alpha-mannosidase accompanies biosynthesis of the enzyme in normal and carcinoma cell lines. Biochim Biophys Acta 847 (1): 53-61 88. WO 02/99092 A (Fogh Jens; Hemebiotech AS (DK); Andersson Claes (SE); Weigelt Cecilia) December 12, 2002 89. Berg Thomas et al .: "Purification and characterization of recombinant human lysosomal alpha-mannosidase", Molecular Genetics and Metabolism, vol. 73, no. 1, April 2001, pages 18-29

LIST OF SEQUENCES <110> Jens Fogh Claes Anderson Cecilia Weigelt Diego Prieto Roces Kurt von Figure Meher Irani

<120> Use of alpha-mannosidase for the correction of abnormal lysosomal storage in the CNS <130> 36564PC01 <150> PA200400528 <151> 2004-04-01 <150> US 60 / 558,108 <151> 2004-04-01 <160> 2 <170> FastSEQ for Windows Version 4.0

<210> 1 <211> 1011 <212> PRT <213> Homo Sapiens <4 0 0> 1

Met Gly Ala Tyr Ala Arg Ala Ser Gly Val Cys Ala Arg Gly Cys Leu 15 10 15

Asp Ser Wing Gly Pro Trp Thr Met Ser Arg Ala Leu Arg Pro Pro Leu 20 25 30

Pro Pro Leu Cys Phe Phe Leu Leu Leu Leu Ala Ala Ala Gly Ala Arg 35 40 45

Ala Gly Gly Tyr Glu Thr Cys Pro Thr Val Gin Pro Asn Met Leu Asn 50 55 60

Val His Leu Leu Pro His Thr His Asp Asp Val Gly Trp Leu Lys Thr. 65 70 75 80

Val Asp Gin Tyr Phe Tyr Gly Ile Lys Asn Asp Ile His His Gly 85 90 95

Go Gin Tyr lie Leu Asp Ser Val lie Ser Lea Leu Leu Ala Asp Pro 100 105 110

Thr Arg Arg Phe Ile Tyr Val Glu Ile Ala Phe Phe Ser Arg Trp Trp 115 120 125

His Gin Gin Thr Asn Ala Thr Gin Glu Val Val Arg Asp Leu Val Arg 130 135 140

Gin Gly Arg Leu Glu Phe Ala Asn Gly Gly Trp Val Met Asn Asp Glu 145 150 155 160

Wing Wing Thr His Tyr Gly Ala He Val Asp Gin Met Thr Leu Gly Leu 165 170 175

Arg Phe Leu Glu Asp Thr Phe Gly Asn Asp Gly Arg Pro Arg Val Ala 180 185 190

Trp His Ile Asp Pro Phe Gly His Ser · Arg Glu Gin Ala Ser Leu Phe 195 200 205

Ala Gin Met Gly Phe Asp Gly Phe Phe Phe Gly Arg Leu Asp Tyr Gin 210 215 220

Asp Lys Trp Val Arg Met Gin Lys Leu Glu Met Glu Gin Val Trp Arg 225 230 235 240

Ala Ser Thr Ser Leu Lys Pro Thr Ala Asp Leu Phe Thr Gly Val 245 250 255

Leu Pro Asn Gly Tyr Asn Pro Pro Arg Asn Leu Cys Trp Asp Val Leu 260 265 270

Cys Val Asp Gin Pro Leu Val Glu Asp Pro Arg Ser Pro Glu Tyr Asn 275 280 285

Ala Lys Glu Leu Val Asp Tyr Phe Leu Asn Val Ala Thr Ala Gin Gly 290 295 300

Arg Tyr Tyr Arg Thr Asn His Thr Val Met Thr Met Gly Ser Asp Phe 305 310 315 320

Gin Tyr Glu Asn Ala Asn Met Trp Phe Lys Asn Leu Asp Lys Leu lie 325 330 335

Arg Leu Val Asn Wing Gin Gin Wing Lys Gly Ser Ser Val His Val Leu 340 345 350

Tyr Ser Thr Pro Ala Cys Tyr Leu Trp Glu Leu Asn Lys Ala Asn Leu 355 360 365

Thr Trp Ser Val Lys His Asp Asp Phe Phe Pro Tyr Ala Asp Gly Pro 370 375 380

His Gin Phe Trp Thr Gly Tyr Phe Ser Ser Arg Pro Ala Leu Lys Arg 385 390 395 400

Tyr Glu Arg Leu Ser Tyr Asn Phe Leu Gin Val Cys Asn Gin Leu Glu 405 410 415

Ala Leu Val Gly Leu Ala Ala Asn Val Gly Pro Tyr Gly Ser Gly Asp 420 425 430

Be Ala Pro Leu Asn Glu Ala Met Ala Val Leu Gin His His Asp Ala 435 440 445

Val Ser Gly Thr Ser Arg Gin His Val Ala Asn Asp Tyr Ala Arg Gin 450 455 460

Leu Wing Wing Gly Trp Gly Pro Cys Glu Val Leu Leu Ser Asn Wing Leu 465 470 475 480

Ala Arg Leu Arg Gly Phe Lys Asp His Phe Thr Phe Cys Gin Gin Leu 485 490 495

Asn lie Ser Cys Pro Leu Ser Gin Thr Ala Ala Arg Phe Gin Val 500 505 510 lie Val Tyr Asn Pro Leu Gly Arg Lys Val Asn Trp Met Val Arg Leu 515 520 525

Pro Val Ser Glu Gly Val Phe Val Val Lys Asp Pro Asn Gly Arg Thr 530 535 540

Val Pro Ser Asp Val Val phe Pro Ser Ser Asp Ser Gin Ala His 545 550 555 560

Pro Pro Glu Leu Leu Phe Ser Ala Ser Leu Pro Ala Leu Gly Phe Ser 565 570 575

Thr Tyr Ser Val Ala Gin Val Pro Arg Trp Lys Pro Gin Ala Arg Ala 580 585 590

Pro Gin Pro Ile Pro Arg Arg Ser Trp Ser Pro Ala Leu Thr Ile Glu 595 600 605

Asn Glu His Ile Arg Ala Thr Phe Asp Pro Asp Thr Gly Leu Leu Met 610 615 620

Glu Ile Met Asn Met Asn Gin Gin Leu Leu Leu Pro Val Arg Gin Thr 625 630 635 640

Phe Phe Trp Tyr Asn Ala Ser is Gly Asp Asn Glu Ser Asp Gin Ala 645 650 655

Ser Gly Ala Tyr lie Phe Arg Pro Asn Gin Gin Lys Pro Leu Pro Val 660 665 670

Ser Arg Trp Ala Gin lie His Leu Val Lys Thr Pro Leu Val Gin Glu 675 680 685

Val His Gin Asn Phe Ser Ala Trp Cys Ser Gin Val Val Arg Leu Tyr. 690 695 700

Pro Gly Gin Arg His Leu Glu Leu Glu Trp Ser Gly Pro Pro 705 710 715 720

Val Gly Asp Thr Trp Gly Lys Glu Val Ile Ser Arg Phe Asp Thr Pro 725 730 735

Leu Glu Thr Lys Gly Arg Phe Tyr Thr Asp Ser Asn Gly Arg Glu Ile 740 745 750

Leu Glu Arg Arg Arg Asp Tyr Arg Pro Thr Trp Lys Leu Asn Gin Thr 755 760 765

Glu Pro Val Ala Gly Asn Tyr Tyr Pro Val Asn Thr Arg Ile Tyr Ile 770 775 780

Thr Asp Gly Asn Met Gin Leu Thr Val Leu Thr Asp Arg Ser Gin Gly 785 790 795 800

Gly Ser Ser Leu Arg Asp Gly Ser Leu Glu Leu Met Val His Arg Arg 805 810 815

Leu Leu Lys Asp Asp Gly Arg Gly Val Ser Glu Pro Leu Met Glu Asn 820 825 830

Gly Ser Gly Ala Trp Val Arg Gly Arg His Leu Val Leu Leu Asp Thr 835 840 845

Ala Gin Wing Wing Wing Wing Gly His Arg Leu Wing Wing Glu Gin Glu Val 850 855 860

Leu Wing Pro Gin Val Val Leu Wing Pro Gly Gly Gly Wing Wing Tyr Asn_ 865 870 875 880

Leu Gly Ala Pro Pro Arg Thr Gin Phe Ser Gly Leu Arg Arg Asp Leu 885 890 895

Pro Pro Ser Val His Leu Leu Thr Leu Ala Ser Trp Gly Pro Glu Met 900 905 910

Val Leu Leu Arg Leu Glu His Gin Phe Ala Val Gly Glu Asp Ser Gly 915 920 925

Arg Asn Leu Ser Ala Pro Val Thr Leu Asn Leu Arg Asp Leu Phe Ser 930 935 940

Thr Phe Thr Ile Thr Arg Leu Gin Glu Thr Thr Leu Val Ala Asn Gin 945 950 955 960

Leu Arg Glu Ala Ala Ser Arg Leu Lys Trp Thr Thr Asn Thr Gly Pro 965 970 975

Thr Pro His Gin Thr Pro Tyr Gin Leu Asp Pro Ala Asn lie Thr Leu 980 985 990

Glu Pro Met Glu Ile Arg Thr Phe Leu Ala Ser Val Gin Trp Lys Glu 995 1000 1005

Val Asp Gly 1010

<210> 2 <211> 8079 <212> DNA <213> Artificial Sequence <22 0><223> Expression Plasmid pLamanExpl <400> 2 agiatcttoáa iattggcoa t: tagecafcatt art oat: iggt tataiagaa ·: aaa least.at. 60 tggctattgg ocattgeata cgttgtatct at the teat AT: 120 atgtacatfcfc atatcggctc atgcccaata tgaccgccat gfctggcattg attattgact agttattaaft agtaafccsac 180 taoggggtca ttagtfccata gcccatatat ggagttccgc gttacataac ttacggtaaa 240 tggeecgcet ggctgaccgc ccaacgaccc cc.gocc.at.tg aegtcaataa fcgacgtatgt 300 tcccatagta acgccaatag ggactttcca ttgacgteaa tgggfcggagt atfctacggta 360 aaotgcccac ttggcagtac atcaagtgta tcatatgcca agtccgcccc ctattgacgt 420 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttac gggactttcc 480 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca 540 gtacaccaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat 600 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa 660 caactgcgat cgcccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc 720 tatataagca gagctcgttt agtgaaccgt cagatcacta gaagctttat tgcggtagtt 780 tatcacagtt aaattgctaa cgcagtcagt gcttctgaca caacagtctc gaacttaagc 840 tgcagtgact ctcttaaggt agccttgcag aagttggtcg tgaggcactg ggcaggtaag 900 tatcaaggtt acaagacagg tttaaggaga ccaatagaaa ctgggcttgt cgagacagag 960 aagactcttg cgtttctgat aggcacctat tggtcttact gacatccact ttgcctttct 1020 ctccacaggt gtccactccc agttcaatta cagctcttaa ggctagagta cttaatacga 1080 ctcactatag gctagcctcg agaattcgcc gccatgggcg cctacgcgcg ggcttcgggg 1140 gaggctgcct ggactcagca ggcccctgga gtctgcgctc ccatgtcccg cgccctgcgg 1200 ccaccgctcc cgcctctctg ctttttcctt ttgttgctgg cggctgccgg tgctcgggcc 1260 gggggatacg agacatgccc cacagtgcag ccgaacatgc tgaacgtgca cctgctgcct 1320 cacacacatg atgacgtggg ctggctcaaa accgtggacc agtactttta tggaatcaag 1380 aatgacatcc agcacgccgg tgtgcagtac atcctggact cggtcatctc tgccttgctg 1440 gcagatccca cccgtcgctt catttacgtg gagattgcct tcttctcccg ttggtggcac 1500 cagcagacaa atgccacaca ggaagtcgtg cgagaccttg tgcgccaggg gcgcctggag 1560 ttcgccaatg gtggctgggt gatgaacgat gaggcagcca cccactacgg tgccatcgtg 1620 gaccagatga cacttgggct gcgctttctg gaggacacat ttggcaatga tgggcgaccc 1680 cgtgtggcct ggcacattga ccccttcggc cactctcggg agcaggcctc gctgtttgcg 1740 cagatgg gct tcgacggctt cttctttggg cgccttgatt atcaagataa gtgggtacgg 1800 atgcagaagc tggagatgga gcaggtgtgg cgggccagca ccagcctgaa gcccccgacc 1860 gcggacctct tcactggtgt gcttcccaat ggttacaacc cgccaaggaa tctgtgctgg 1920 gatgtgctgt gtgtcgatca gccgctggtg gaggaccctc gcagccccga gtacaacgcc 1980 aaggagctgg tcgattactt cctaaatgtg gccactgccc agggccggta ttaccgcacc 2040 aaccacactg tgatgaccat gggctcggac ttccaatatg agaatgccaa catgtggttc 2100 aagaaccttg acaagctcat ccggctggta aatgcgcagc aggcaaaagg aagcagtgtc 2160 catgttctct actccacccc cgcttgttac ctctgggagc tgaacaaggc caacctcacc 2220 aacatgacga cttcttccct tggtcagtga tacgcggatg gcccccacca gttctggacc 2280 ccagtcggcc ggccctcaaa ggttactttt cgctacgagc gcctcagcta caacttcctg 2340 caggtgtgca accagctgga ggcgctggtg ggcctggcgg ccaacgtggg accctatggc 2400 tccggagaca gtgcacccct caatgaggcg atggctgtgc tccagcatca cgacgccgtc 2460 agcggcacct cccgccagca cgtggccaac gactacgcgc gccagcttgc ggcaggctgg 2520 gggccttgcg aggttcttct gagcaacgcg ctggcgcggc tcagaggctt caaagatcac 2580 ttcacctttt o caacagct aaacatcagc atctgcccgc tcagccagac ggcggcgcgc 2640 ttccaggtca tcqtttataa tcccctgggg cggaaggtga attqqatggt acggctgccg 2700 gtcagcgaag gcgttttcgt tgtgaaggac cccaatggca ggacagtgcc cagcgatgtg 2760 gtaatatttc ccagctcaga cagccaggcg caccctccgg agctgctgtt ctcagcctca 2820 ctgcccgccc tgggcttcag cacctattca gtagcccagg tgcctcgctg gaagccccag 2880 gcccgcgcac cacagcccat ccccagaaga tcctggtccc ctgctttaac catcgaaaat 2940 gagcacatcc gggcaacgtt tgatcctgac acagggctgt tgatggagat tatgaacatg 3000 aatcagcaac tcctgctgcc tgttcgccag accttcttct ggtacaacgc cagtataggt 3060 gtgaccaggc gacaacgaaa ctcaggtgcc tacatcttca gacccaacca acagaaaccg 3120 ctgcctgtga gccgctgggc tcagatccac ctggtgaaga cacccttggt gcaggaggtg 3180 caccagaact tctcagcttg gtgttcccag gtggttcgcc tgtacccagg acagcggcac 3240 ctggagctag agtggtcggt ggggccgata cctgtgggcg acacctgggg gaaggaggtc 3300 atcagccgtt ttgacacacc gctggagaca aagggacgct tctacacaga cagcaatggc 3360 cgggagatcc tggagaggag gcgggattat cgacccacct ggaaactgaa ccagacggag 3420 cccgtggcag gaaactac rt tccagtcaac acccggattt acatcacgga tggaaacatg 3480 cagctgactg tgctgactga ccgctcccag gggggcagca gcctgagaga tggctcgctg 3540 gagctcatgg tgcaccgaag gctgctgaag gacgatggac gcggagtatc ggagccacta 3600 atggagaacg ggtcgggggc gtgggtgcga gggcgccacc tggtgctgct ggacacagcc 3660 caggctgcag ccgccggaca ccggctcctg gcggagcagg aggtcctggc ccctcaggtg 3720 gtgctggccc cgggtggcgg cgccgcctac aatctcgggg ctcctccgcg cacgcagttc 3780 tcagggctgc gcagggacct gccgccctcg gtgcacctgc tcacgctggc cagctggggc 3840 cccgaaatgg tgctgctgcg cttggagcac cagtttgccg taggagagga ttccggacgt 3900 aacctgagcg cccccgttac cttgaacttg agggacctgt tctccacctt caccatcacc 3960 cgcctgcagg agaccacgct ggtggccaac cagctccgcg aggcagcctc caggctcaag 4020 tggacaacaa acacaggccc cacaccccac caaactccgt accagctgga cccggccaac 4080 atcacgctgg aacccatgga aatccgcact ttcctggcct cagttcaatg gaaggaggtg 4140 gatggttagg tctgctggga tgggccctct agagtcgacc cgggcggccg cttcccttta 4200 gtgagggtta atgcttcgag cagacatgat aagatacatt gatgagtttg gacaaaccac 4260 aactagaatg cagtgaaaaa aat gctttat ttgtgaaatt tgtgatgcta ttgctttatt 4320 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt 4380 tcaggttcag ggggagatgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg 4440 taaaatccga taaggatcga tccgggctgg cgtaatagcg aagaggcccg caccgatcgc 4500 ccttcccaac agttgcgcag cctgaatggc gaatggacgc gccctgtagc ggcgcattaa 4560 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 4620 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 4680 ctctaaatcg ggggctccct ttagggttcc gatttagagc tttacggcac ctcgaccgca 4740 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 4800 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 4860 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgggg atttcggcct 4920 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt 4980 gtgtcagtta gggtgtggaa agtccccagg ctccccaggc aggcagaagt atgcaaagca 5040 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa 5100 gtatgcaaag catgcatctc aattagtca g caaccatagt cccgccccta actccgccca 5160 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt 5220 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag 5280 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg atggttcgac cattgaactg 5340 catcgtcgcc gtgtcccaaa atatggggat tggcaagaac ggagacctac cctggcctcc 5400 gctcaggaac gagttcaagt acttccaaag aatgaccaca acctcttcag tggaaggtaa 5460 acagaatctg gtgattatgg gtaggaaaac ctggttctcc attcctgaga agaatcgacc 5520 tttaaaggac agaattaata tagttctcag tagagaactc aaagaaccac cacgaggagc 5580 tcattttctt gccaaaagtt tggatgatgc cttaagactt attgaacaac cggaattggc 5640 aagtaaagta gacatggttt ggatagtcgg aggcagttct gtttaccagg aagccatgaa 5700 tcaaccaggc caccttagac tctttgtgac aaggatcatg caggaatttg aaagtgacac 5760 gtttttccca gaaattgatt tggggaaata taaacttctc ccagaatacc caggcgtcct 5820 ctctgaggtc caggaggaaa aaggcatcaa gtataagttt gaagtctacg agaagaaaga 5880 ctaattcgaa atgaccgacc aagcgacgcc caacctgcca tcacgatggc cgcaataaaa 5940 tatctttatt ttcattacat ctgtgtgttg gttt tttgtg tgaatcgata gcgataagga 6000 tccgcgtatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc 6060 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 6120 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 6180 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga 6240 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaceccta 6300 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat 6360 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc 6420 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga 6480 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca 6540 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt 6600 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg 6660 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc 6720 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata 6780 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt 6840 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag 6900 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca 6960 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg 7020 aggcggataa agttgcagga ccacttctgc gctoggccct tccggctggc tggtttattg 7080 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag 7140 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg 7200 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag 7260 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga 7320 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt 7380 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc 7440 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 7500 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 7560 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac 7620 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctg ccagt ggcgataagt 7680 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct 7740 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat 7800 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt 7860 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 7920 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt 7980 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt 8040 ttgctggcct tttgctcaca tggctcgac tcctggcctt 8079

Claims

Use of a lysosomal alpha-mannosidase for the preparation of a medicament for the treatment of alpha-mannosidosis, wherein said medicament is to be administered by intravenous administration.

Use according to claim 1, wherein the medicament is to be administered by infusion, or repeated intravenous injection at a rate corresponding to 1 to 5 injections daily, 1 to 5 injections per week, 1 to 5 injections every 2 weeks or 1 to 5 injections every month.

Use according to claim 1 or claim 2, wherein said medicament does not comprise a component with a known ability to target cells within the central nervous system.

Use according to any one of claims 1 - 3, wherein the lysosomal alpha-mannosidase comprises the amino acid sequence of SEQ ID NO: 1.

Use according to any one of claims 1-4, wherein said medicament is to be administered at a frequency corresponding to 1-5 injections per week.

Use according to any one of claims 1-5, wherein said medicament does not comprise a component for targeting cells within the central nervous system and does not comprise a component capable of acting as a vehicle for passage through the blood-brain barrier.

Use according to any one of the preceding claims wherein said medicament is administered in an amount that is within the range of 5 to 300 mU of lysosomal alpha-mannosidase per gram of body weight of the subject to be treated.

Use according to any one of the preceding claims, wherein said medicament is administered in an amount that is within the range of from 20 to 100 mU lysosomal alpha-mannosidase per gram of body weight of the subject to be treated.

Use according to any one of the preceding claims, wherein a fraction of said lysosomal alpha-mannosidase consists of lysosomal alpha-mannosidase having one or more N-linked oligosaccharides carrying mannose-6-phosphate residues.

Use according to any one of the preceding claims, wherein a fraction corresponding to from 1 to 75% of the activity of a lysosomal alpha-mannosidase preparation binds to the mannose-6-phosphate receptor.

Use according to claim 10, wherein said fraction corresponds to from 10 to 50% of the activity of a lysosomal alpha-mannosidase preparation.

Use according to claim 10, wherein said fraction corresponds to from 20 to 40% of the activity of a lysosomal alpha-mannosidase preparation.

Use according to any one of the preceding claims, wherein said lysosomal alpha-mannosidase is a recombinant enzyme.

Use according to any one of the preceding claims, wherein said lysosomal alpha-mannosidase is produced in a mammalian cell system.

Use according to claim 14, wherein said mammalian cell system is a CHO cell line.