MXPA00011137A - Recombinant (alpha)-l-iduronidase, methods for producing and purifying the same and methods for treating diseases caused by deficiencies thereof - Google Patents

Abstract

The present invention provides a recombinant&agr;-L-iduronidase and biologically active fragments and mutants thereof, methods to produce and purify this enzyme as well as methods to treat certain genetic disorders including&agr;-L-iduronidase deficiency and mucopolysaccharidosis I (MPS I).

Description

g-L-IDURONIDASA RECOMBINES YOU, METHODS TO PRODUCE AND PURIFY THE SAME AND METHODS TO TREAT DISEASES CAUSED BY DEFICIENCIES OF THE SAME FIELD OF THE INVENTION The present invention is in the field of molecular biology, enzymology, biochemistry and clinical medicine. In particular, the present invention provides a recombinant a-L-iduronidase, methods for producing and purifying this enzyme, as well as methods for treating certain genetic disorders including deficiency of a-L-iduronidase and mucopolysaccharidosis I (MPS I).

BACKGROUND OF THE INVENTION Carbohydrates play a number of important roles in the functioning of living organisms. In addition to their metabolic roles, carbohydrates are structural components of the human body that covalently bind to many other entities, such as proteins and lipids (called glycoconjugates). For example, human connective tissues and cell membranes comprise proteins, carbohydrates and a proteoglycan matrix. The carbohydrate portion of this proteoglycan matrix provides important properties to the structure of the body. A genetic deficiency of carbohydrate dissociation, the a-L-iduronidase of the lysosomal enzyme causes a lysosomal storage disorder which is known as mucopolysaccharidosis I (MPS I) (Neufeld, E.F., and Muenzer, J. (1989)). Mucopolysaccharidosis in 'The Metabolic Basis of Inherited Disease' (Scriver, CR, Beaudet, AL, Sly, .S., And Valle, D., Eds.), Pp. 1565-1587, McGraw-Hill, New York). In a severe form, MPS I is commonly referred to as Hurler's syndrome and is associated with multiple problems such as mental retardation, clouding of the cornea, thickened facial features, heart disease, respiratory disease, enlargement of the liver and spleen, hernias and joint stiffness Patients suffering from Hurler's syndrome usually die before the age of 10. In an intermediate form known as Hurler-Scheie syndrome, mental function is generally not severely affected, but physical problems can lead to death by adolescence or when they are in their twenties.Science syndrome is the lightest form of MPS I. It is compatible with a normal life span, but hardening of joints s, clouding of the cornea and heart valve disease cause significant problems. It is estimated that the frequency of MPS I is 1: 100,000 according to a survey in British Columbia in all newborns (Lowry et al., Human Genetics 8_5, 389-390 (1990)) and 1: 70,000 According to an Irish study (Nelson, Human Genetics 101: 355-348 (1990)), it seems that there is no ethnic predilection for this disease, it is probable that on a world scale the disease is being under-diagnosed either because the patient dies of a complication before a diagnosis is made, or because the lighter forms of arthritis syndrome are misinterpreted or completely overlooked. Effective screening of newborns by MPS I would very likely find some patients who do not previously detected by bone marrow transplantation, there are no significant therapies available for MPS I. Bone marrow transplants can be effective in the treatment of some of the symptoms of l disorder, but they have high morbidity and mortality in MPS I and are usually not available to patients due to a lack of adequate donors. An alternative therapy available to all affected patients would be to provide an important discovery to treat and manage this disease. Enzyme replacement therapy has long been considered as a potential therapy for MPS I, after the discovery that a-L-iduronidase can correct the enzyme defect in Hurler cells in culture. In this corrective process, the enzyme containing a mannose-6-phosphate residue is taken into the cells through a receptor-mediated endocytosis and transported to the lysosomes where it cleans the stored substrates, heparan sulfate and dermatan sulfate. The application of this therapy to humans has not been previously possible due to inadequate sources of a-L-iduronidase in tissues. The concept of enzyme replacement was first applied effectively in Gaucher patients in a modified placental glucocerebrosidase. The administration and effective uptake of glucocerebrosidase in a Gaucher patient demonstrated that the enzyme could be taken in vivo in sufficient amounts to provide effective therapy. For the therapy of the enzyme a-L-iduronidase in MPS I, a recombinant source of the enzyme has been needed in order to obtain therapeutically sufficient supplies of the enzyme. The mammalian enzyme was cloned in 1990 (Stolzfus et al., J. Biol. Chem. 267: 6570-6575 (1992), and the human enzyme was cloned in 1991 (Moskowitz et al., FASEB J 6: A77 (1992)) .

DESCRIPTION OF THE FIGURES Figure 1 represents the nucleotide and deduced amino acid sequences of the cDNA encoding a-L-iduronidase. Nucleotides 1 to 6200 are provided. Amino acids are provided starting with the first methionine in the open reading frame. Figure 2 depicts the results an SDS-PAGE course of the eluate that was obtained in accordance with the procedure set forth in Example 1. Line 1 was blank. Line 2 contained high molecular weight standards. Line 3 was blank. Line 4 contained bovine serum albumin in a concentration of 50 μg. Lines 5 to 10 represent the eluate containing the a-L-iduronidase produced recombinantly in amounts of 1 μg, 2 μg, 5 μg, 5 μg, 5 μg and 5 μg, respectively. Figure 3 reveals urinary GAG levels in 16 patients with MPS I in relation to normal excretion values. There is a wide range of GAG values for urine in MPS I patients without treatment. A reduction of more than 50 percent in the excretion of GAGs without degradation after therapy with the recombinant a-L-iduronidase is a valid means of measuring an individual's response to therapy. Figure 4 demonstrates the activity of leukocyte iduronidase before and after enzyme treatment in patients with MPS I. Figure 5 demonstrates the activity of oral iduronidase before and after treatment with the enzyme. Figure 6 demonstrates in three patients that substantial shrinkage of the liver and spleen was associated with significant clinical improvement in the joint and storage of soft tissue, with a greater 65 percent reduction in GAG without degrading after only 8 weeks of treatment with the recombinant enzyme. Figure 7 demonstrates that there is a substantial normalization of livers and spleens in patients who were treated with the recombinant enzyme after only 12 weeks of therapy. Figure 8 demonstrates a precipitous drop in urinary GAG excretion during 22 weeks of recombinant enzyme therapy in 6 patients.

BRIEF SUMMARY OF THE INVENTION In one aspect, the present invention presents a method for producing α-L-iduronidase in amounts that allow the use of the enzyme in a therapeutic manner. In a broad embodiment, the method comprises the step of transfecting a cDNA encoding all or a portion of the a-L-iduronidase into a suitable cell for expression thereof. In some embodiments, a cDNA encoding a complete α-L-iduronidase, preferably a human α-L-iduronidase, is used. However, in other embodiments, a cDNA encoding a biologically active fragment or mutant thereof can be used. Specifically, one or more amino acid substitutions can be made, while the biological activity of the enzyme is preserved or improved. In other preferred embodiments, an expression vector is used to transfer the cDNA into a suitable cell cell line for expression thereof. In a particularly preferred embodiment, the cDNA is transfected into a Chinese hamster ovary to create the 2.131 cell line. In yet another preferred embodiment, the production process has one or more of the following characteristics, which have shown particularly high production levels: (a) the pH of the cell growth culture can be decreased to about 6.5 to 7.0, Preference of approximately 6.7 - 6.8 during the production process, (b) Approximately 2/3 to 3/4 of the medium can be changed approximately every 12 hours, (c) oxygen saturation can be optimized to approximately 80 percent, using intermittent splash of pure oxygen, (d) initially microcarriers with approximately 10 percent serum can be used ", to produce the cell mass followed by a change of fast washing to protein-free medium for production, (e) a protein-free or low-protein medium such as a PF-CHO product from JRH Biosciences can be optimized to include complementary amounts of one or more ingredients that are selected from the group consisting of glutamate, aspartate, glycine, ribonucleosides and deoxyribonucleosides, (f) an infusion rod such as a warp perfusion stick can be used in a frequent batch feeding process, rather than a standard intended perfusion process, and (g) can be used a process of induction of light sodium butyrate, to induce the expression of the increased aL-iduronidase. In a second aspect, the present invention provides a transfected cell line, which has the ability to produce a-L-iduronidase in amounts that allow the use of the enzyme in a therapeutic manner. In preferred embodiments, the present invention features a recombinant Chinese hamster ovary cell line, such as cell line 2.131 which reliably produces amounts of α-L-iduronidase that allow the use of the enzyme in a therapeutic manner. In some preferred embodiments, the cell line may contain at least about 10 copies of an expression construct. In even more preferred embodiments, the cell line expresses the recombinant a-L-iduronidase in amounts of at least 20-40 micrograms per cell at 10 7 per day. In a third aspect, the present invention provides novel vectors suitable for producing a-L-iduronidase in amounts that facilitate the use of the enzyme in a therapeutic manner. In preferred embodiments, the present invention features an expression vector comprising a cytomegalovirus promoter / enhancer element, a 5 'intron consisting of a murine Ca intron, a cDNA encoding all or a fragment or mutant of an aL- iduronidase, and a polyadenylation site of growth hormone. Also, preferably the cDNA encoding all or a fragment or mutant of an α-L-iduronidase is about 2.2. kb in length This expression vector can be transfected in, for example, a ratio of 50 to 1 with any appropriate common selection vector such as, for example, pSV2NE0, to improve the insertions of multiple copies. Alternatively, gene amplification can be used to induce the insertions of multiple copies. In a fourth aspect, the present invention provides the a-L-iduronidase that was produced in accordance with the methods of the present invention and present thereby in amounts that allow the use of the enzyme in a therapeutic manner. The specific activity of a-L-iduronidase according to the present invention is in excess of 200,000 units per milligram of protein. Preferably, it is in excess of about 240,000 units per milligram of protein. The molecular weight of the a-L-iduronidase of the present invention is about 82,000 daltons, with about 70,000 daltons amino acids and about 12,000 daltons being carbohydrates. In a fifth aspect, the present invention presents a novel method for purifying an α-L-iduronidase. According to a first embodiment, a cell mass in serum can be cultured at approximately 10 percent containing the medium, followed by switching to a production medium free of modified protein, without any significant adaptation, to produce a starting material of high specific activity. Preferably, a concentration / diafiltration scheme is used that allows removal of exogenous materials that may be required for recombinant production thereof such as, for example, Pluronics F-68, a supernatant that is commonly used to protect damage cells by splash. These exogenous materials should normally be separated from the raw mass to avoid contaminating the columns. In another preferred modality, a first loading of the column is acidified to minimize the competitive inhibition effect of the uronic acids that were found in the protein free medium formulations. "Also preferably, a purification scheme of heparin, phenyl and of classification by size, to produce the pure enzyme using the steps that can be automated and the means that can be validated In another preferred embodiment, the heparin and phenyl column steps are used to eliminate the less desirable aL-iduronidase In another preferred embodiment, an acidic pH treatment step is used to deactivate potential viruses without damage to the enzyme In a sixth aspect, the present invention presents novel methods for treating diseases caused in whole or in part due to a deficiency of aL-iduronidase In one embodiment, this method presents the administration of an aL-iduronidase recomb or a biologically active fragment or mutant thereof alone and in combination with a pharmaceutically suitable carrier. In another embodiment, this method presents the transfer of a nucleic acid encoding all or part of an a-L-iduronidase within one or more host cells in vivo. Preferred embodiments include optimizing the dose for the needs of the organism to be treated, preferably mammals or humans, to effectively improve the symptoms of the disease. In the preferred embodiments, the disease is mucopolysaccharidosis I (MPS I), Hurler syndrome, Hurler-Scheie syndrome, Scheie syndrome. In a seventh aspect, the present invention features novel pharmaceutical compositions comprising a-L-iduronidase useful for treating a disease caused wholly or in part by a deficiency in a-L-iduronidase. These compositions may be suitable for administration in a number of ways, such as parenteral, topical, intranasal, inhalation or oral administration. Within the scope of this aspect are the embodiments that present nucleic acid sequences that encode all or part of an α-L-iduronidase, which can be administered in vivo within affected cells with a deficiency of α-L-iduronidase.

DETAILED DESCRIPTION OF THE INVENTION In one aspect, the present invention provides a method for producing an α-L-iduronidase in amounts that allow the use of the enzyme in a therapeutic manner. In general, the method presents the transformation of a suitable cell line with the cDNA encoding all of the a-L-iduronidase or a biologically active fragment or mutant thereof. Those of skill in the art can prepare expression constructs in addition to those expressly described herein for the optimal production of an α-L-iduronidase in the appropriate cell lines that are transfected therewith. In addition, skilled artisans can easily design fragment of the cDNA encoding the biologically active fragments and mutants of the aL-iduronidase that occurs naturally, which possesses the same or a similar biological activity as the full-length enzyme that occurs in a manner natural. To create a recombinant source for an α-L-iduronidase, a long series of expression vectors can be constructed and tested for the expression of an α-L-iduronidase cDNA. Based on transient transfection experiments, as well as stable transfections, an expression construct can be identified that provides particularly high level expressions. In one embodiment of the present invention, a Chinese hamster cell line 2113 that was developed by transfection of the expression construct of a-L-iduronidase and selection for a high expression clone provides particularly high level expression. This Chinese hamster cell line according to this embodiment of the present invention can secrete approximately 5,000 to 7,000 times more a-L-iduronidase than normal. The aL-iduronidase which was produced by the same, can be processed in an appropriate manner, which are taken into cells with high affinity and is corrective for cells deficient in aL-iduronidase, such as those of patients suffering from Hurler's Syndrome . The method for producing a-L-iduronidase in amounts that allow the use of the enzyme in a therapeutic manner, presents a production process that was designed specifically to produce the enzyme in high amounts. In accordance with the preferred embodiments of this process, the microcarriers are used as a low cost scalable surface on which to grow the adherent cells. In accordance with other preferred embodiments of the method for producing a-L-iduronidase according to the present invention, a culture system is optimized. In a preferred embodiment, the pH of the culture is decreased to about 6.5 to 7.0, preferably to about 6.7-6.8 during the production process. An advantage of this pH is that it improves the accumulation of lysosomal enzymes that are more stable at an acidic pH. In a second mode, approximately 2/3 to 3/4 of the medium is changed approximately every 12 hours. An advantage of this method is to improve the secretion rate of the recombinant aL-iduronidase and capture the most active enzyme, in a third mode, the oxygen saturation is optimized to approximately 80 percent, using the intermittent pure oxygen spray, rather than the continuous dew. In a fourth embodiment, Citodex 2 microcarriers are initially used with approximately 10 percent serum to produce a cell mass followed by a rapid wash change to a protein-free medium for production. In a fifth embodiment, a culture medium such as the PF-CHO product from JRH Biosciences can be optimized to include complementary amounts of one or more of the ingredients that were selected from the group consisting of glutamate, aspartate, glycine, ribonucleosides and deoxyribonucleosides. In a sixth embodiment, a perfusion rod such as a warp perfusion rod can be used in a frequent batch feeding process, rather than a standard purported perfusion process. In a seventh embodiment, a sodium butyrate induction process can be used to induce the expression of increased α-L-iduronidase, without a substantial effect on carbohydrate processing and cellular uptake of the enzyme. This induction process can provide an approximation of a double increase in production, without significantly altering post-translational processing. Particularly preferred embodiments of the method for producing a-L-iduronidase according to the present invention have one, more than one. or all the optimizations described herein. The production method of the present invention can therefore provide a production culture process having the following characteristics: 1. Preferably a microcarrier-based culture using Cytodex 2 globules or an equivalent thereof in flasks was used. large-scale culture with upper rod shaking using a warp infusion rod, or an equivalent thereof. Adherence to these beads can be achieved by culturing in a 10 percent fetal bovine serum medium in 1: 1 DME / F12, which was modified with ingredients that include ribonucleosides, deoxyribonucleosides, pyruvate, non-essential amino acids, and HEPES and at a pH of about 6.7-6.9. After 3 days in this medium, a washing procedure was started in which the protein-free medium replaced approximately 2/3 of the medium approximately every 12 hours, for a total of approximately 3-4 washes. Subsequently and throughout the remaining culture period, the cells were cultured in the protein-free medium. 2. Preferably the culture conditions were maintained at a dissolved oxygen of 80 percent air saturation at a pH of about 6.7 and at a temperature of about 37 ° C. This can be achieved by using a control tower, the service unit and the appropriate probes such as those produced by Wheaton. However, skilled technicians will readily appreciate that this can be easily achieved by equivalent control systems produced by other manufacturers. An air saturation of about 80 percent results in an improved secretion of a-L-iduronidase over that of 40 percent and 60 percent air saturation. However, 90 percent air saturation does not provide significantly improved secretion over 80 percent air saturation. Dissolved oxygen can be supplied by spraying pure intermittent oxygen using a 5 micron stainless steel sprayer or equivalent thereof. A pH of about 6.7 is optimal for the accumulation of the enzyme a-L-iduronidase. The enzyme is particularly unstable at a pH above about 7.0. Below a pH of about 6.7, the rate of secretion could decrease, particularly below about 6.5. Therefore, the culture is optimally maintained between a pH of about 6.6 to 6.8. 3. The production culture medium could be a modified form of the patented medium commercially available with JRH Biosciences, called Excell PF CHO. This medium supports secretion levels equivalent to those of serum using a cell line such as cell line 2.131. It could be modified preferably to include an acidic pH of about 6.7 (+/- 0.1), and it could be regulated with pH with HEPES at 7.5 mM. The medium could contain 0.05 to 0.1 percent Pluronics F-68 (BASF), a nonionic supernatant or an equivalent thereof that has the advantage of protecting the cells from the shear forces associated with watering. The medium could also contain a patented supplement that proves that it is important in increasing the productivity of the medium, on other protein-free media that are currently available. Those skilled in the art will readily understand that the selection of the culture medium can be optimized continuously, in accordance with the particular commercial modalities available at the particular time points. These changes include no more than routine experimentation and are intended to be within the scope of the present invention. 4. The production medium can be analyzed using an amino acid analyzer that compares the medium that was spent with the starting medium. These analyzes have shown that the 2.131 cell line reduces a standard PF CHO medium of glycine, glutamate and aspartate to a level of about 10 percent of the start-up concentration. Supplementation of these amino acids at higher levels could result in improved culture density and productivity that could lead to 2 to 3 times higher production than in the baseline. Experienced technicians will appreciate that other cell lines within the scope of the present invention could be equally useful for producing a-L-iduronidase according to the present method. Therefore, more or less supplementary nutrients may be required to optimize the medium. It is intended that these optimizations are within the scope of the present invention and that they can be practiced without unnecessary experimentation. 5. The medium can be supplemented with ribonucleosides and deoxyribonucleosides to support the 2,131 cell line deficient in dihydrofolate reductase. The skilled artisan will appreciate that other cell lines within the scope of the present invention could be equally useful for producing the α-L-iduronidase according to the present method. Therefore, more or less ribonucleosides and deoxyribonucleosides could be required to optimize the medium. It is intended that these optimizations are within the scope of the present invention and that they can be practiced without unnecessary experimentation. 6. After reaching the confluence in approximately 3-4 days of culture, approximately 2/3 of the medium can be changed every 12 hours. The change of the medium can be achieved using, for example, a warp perfusion rod, which is a shaker with a hollow center and a filter wired at its tip. By means of pumping out the medium through the hollow interior of the rod through the 40 micron wiring, the microcarriers with the cell mass 2,131 are separated from the supernatant containing the enzyme. 7. Productivity studies have shown that rapid and frequent changes in the environment result in an improved total collection of the enzyme from cell culture. Less frequent changes result in a smaller total accumulation of the enzyme. Studies of the enzyme's rate of secretion during a 12-hour culture cycle showed that the cells are active secretory enzymes for most of the culture period. It is unlikely that the most frequent changes produce substantially more enzyme. The method of this modality has proven to be superior to the perfusion culture and much better than the strict batch culture or the daily batch feeding strategies or every third day. Using the change approximately every 12 hours, the cells can be maintained in excellent conditions with high degrees of viability and a high level of productivity. 8. The production of a-L-iduronidase can be improved by the use of sodium butyrate induction of gene expression. Systematic studies of the 2.131 cell line demonstrated that approximately 2 mM of butyrate can be applied - and result in approximately a two-fold or greater induction of enzyme production with minimal effects on carbohydrate processing. It has not been shown to induce lower levels of butyrate as well, and substantially higher levels could result in higher induction but diminished affinity of the enzyme that occurred for cells suffering from a deficiency of a-L-iduronidase. The results suggest that a 2-fold or greater induction results in less carbohydrate processing and less phosphate addition to the enzyme, as well as increased toxicity. One method that is particularly preferred uses 2 mM addition of butyrate every 48 hours to the culture systems. This modality results in a roughly double induction of enzyme production, using this method without a significant effect on the enzyme uptake affinity, (uptake of K of less than 30 U / ml or 2 mM). Using the modalities of the present method having all the above modifications and induction, a 15 liter culture system can produce approximately 25 milligrams per liter of culture per day, or more at a peak culture density.

In a second aspect, the present invention provides a transfected cell line that possesses the unique ability to produce a-L-iduronidase in amounts that allow the use of the enzyme in a therapeutic manner. In preferred embodiments, the present invention features a Chinese hamster ovary cell line such as cell line 2,131 which stably and reliably produces amounts of α-L-iduronidase. In preferred embodiments, the cell line could contain at least about 10 copies of an expression construct comprising a CMV promoter, a Ca intron, a human aL-iduronidase cDNA, and a bovine growth hormone polyadenylation sequence. . In the most preferred embodiments, the cell line expresses aL-iduronidase in amounts of at least about 20-40 microgramqs per 10 7 cells per day, in a high, appropriately processed form of uptake, which is suitable for the therapy of enzyme replacement. In accordance with the above, for the preferred embodiments of this aspect of the invention, the transfected cell line that was adapted to produce the aL-iduronidase in amounts that facilitate and use the enzyme in a therapeutic manner, possesses one or more of the following characteristics: 1. The cell line of the preferred modalities is derived from a parent cell line where the cells are evacuated in the culture until they have acquired a smaller size and a faster growth rate and until they bind rapidly to the substrates. 2. The cell line of the preferred embodiments is transfected with an expression vector containing both 2 and 3, a human cDNA of approximately 2.2 kb in length, and a 3 'bovine growth hormone cytomegalovirus promoter / enhancer element, a 5 'intron consisting of the murine Ca intron between the polyadenylation site of exons. This expression vector can be transfected in, for example, a ratio of 50 to 1 with an appropriate common selection vector, such as pSV2NEO. In turn, the selection vector pSV2NEO confers resistance G418 on the cells that were successfully transfected. In the modalities that are particularly preferred, a ratio of approximately 50 to 1 is used, since this ratio improves the acquisition of multi-copy number inserts. In accordance with one embodiment wherein the Chinese hamster ovary cell line 2113 is provided, there are approximately 10 copies of the expression vector for α-L-iduronidase. This cell line has demonstrated the ability to produce large amounts of human a-L-iduronidase (minimum 20 micrograms per 10 million cells per day) ^.

Particularly preferred embodiments, such as the 2.131 cell line, possess the ability to produce an appropriately processed enzyme containing the N-linked oligosaccharides containing high-chain phosphate-modified chains at position 6, in an amount sufficient to produce an enzyme with high affinity (uptake of K of less than 3 nM). 3. The enzyme that was produced from the cell lines of the present invention, such as the Chinese hamster ovary cell line 2.131, is rapidly assimilated into cells, eliminates glyco-saminoglycan storage and has a half-life of 5 days in the cells of patients suffering from a deficiency of aL-iduronidase. 4. The cell line of preferred embodiments, such as a cell line 2.131, is adapted to large-scale culture and produces human a-L-iduronidase stably under these conditions. Cells of the preferred embodiments can breed and secrete a-L-iduronidase at an acidic pH of about 6.6 to 6.8 in which the enhanced accumulation of a-L-iduronidase can occur. 5. Particularly preferred embodiments of the cell line according to the invention, such as cell line 2.131, can secrete human a-L-iduronidase at levels exceeding 2000 units per milliliter. (8 micrograms per milliliter) twice a day, using a specially formulated protein-free medium.

In a third aspect, the present invention provides novel vectors suitable for producing a-L-iduronidase in amounts that allow the use of the enzyme in a therapeutic manner. The production of the appropriate amounts of the recombinant a-L-iduronidase is a critical prerequisite for studies on the structure of the enzyme, as well as for enzyme replacement therapy. The cell lines according to the present invention allow the production of significant quantities of recombinant α-L-iduronidase which are processed in an appropriate manner for uptake. Overexpression of Chinese hamster ovary cells (CHO) have been described for three other lysosomal enzymes, α-galactosidase (Ioannou et al., J. Cell, Biol. 119: 1137-1150 (1992)), 2- iduronate sulphatase (Bielicki et al., Biochem. J. 289: 241-246 (1993)), and N-acetylgalactose-mine 4-sulphatase (Anson et al., Biochem. J. 284: 789-794 (1992)). ), using a variety of promoters and, in one case, amplification. The present invention features a CHO cell line deficient in dihydrofolate reductase, but in accordance with the preferred embodiments of the invention, amplification is unnecessary. Additionally, the present invention provides a high level of expression of human a-L-iduronidase, using the promoter / enhancer of the CMV immediate early gene.

The present invention features, in preferred embodiments, an expression vector comprising a cytomegalovirus promoter / enhancer element, a 5 'intron consisting of the murine Ca intron which is derived from the long chain murine immunoglobulin Ca gene between exons 2 and 3, a human cDNA approximately 2.2 kb in length, and a polyadenylation site of bovine growth hormone 3 '. This expression vector can be transfected in, for example, a ratio of 50 to 1 with an appropriate common selection vector such as, for example, pSV2NEO. In turn, the selection vector pSV2NEO confers resistance G418 on the cells that were successfully transfected. In the modalities that are particularly preferred, a ratio of about 50 to 1 of expression vector is used with the selection vector, since this ratio improves the acquisition of multiple copy number inserts. According to a modality wherein the Chinese hamster ovary cell line 2113 is provided, there are approximately 10 copies of the expression vector for a-L-iduronidase. This cell line has demonstrated the ability to produce large amounts of human α-L-iduronidase (minimum 20 micrograms per 10 million cells per day) in a suitable cell line such as, for example, a 2,113 cell line of Chinese hamster ovary.

In a fourth aspect, the present invention provides the a-L-iduronidase which was produced according to the methods of the present invention and which is present therein in amounts that allow the use of the enzyme in a therapeutic manner. The methods of the present invention produce a substantially pure α-L-iduronidase which is processed in an appropriate manner and in a high uptake form appropriate for enzyme replacement therapy and which is effective in in vivo therapy. The specific activity of a-L-iduronidase according to the present invention is in excess of about 200,000 units per milligram of protein. Preferably, it is in excess of about 240, 0OO units per milligram of protein. The molecular weight of the full-length a-L-iduronidase of the present invention is about 82.00 daltons, with about 70,000 daltons amino acids and about 12,000 daltons being carbohydrates. The recombinant enzyme of the present invention can be subjected to endocytosis even more efficiently than previously reported for a partially purified preparation of the urinary enzyme. The recombinant enzyme according to the present invention, is effective in reducing the accumulation of GAG labeled with "S radioactive in the fibroblasts deficient in aL-iduronidase, indicating that it is transported to the lysosomes, the storage site of GAG.The surprisingly low concentration of aL-iduronidase What is needed for this correction (mean maximum correction at 0.7 pM) could be very important for the success of enzyme replacement therapy The cDNA of an aL-iduronidase predicts a protein of 653 amino acids and an expected molecular weight of 70,000 daltons After the dissociation of the signal peptide, the amino acid sequencing reveals alanine 26 at the N terminus which gives an expected protein of 629 amino acids.The recombinant human aL-iduronidase has a histidine at the 8-position of the mature protein. sequence of the predicted protein comprises six potential sites of modification of oligosaccharides linked by N. All these can be modified in the recombinant protein. It has been shown that the third and sixth sites contain one or more residues of mannose 6-phosphate responsible for the uptake of high affinity within the cells. The following peptide corresponds to Amino Acids 26-45 of Human Recombinant a-L-iduronidase with an alanine of N-terminus and the following sequence: ala-glu-ala-pro-his-leu-val-his-val-asp-ala-ala-arg-ala-leu-trp-pro-leu-arg-arg Overexpression of the aL-iduronidase of the present invention it does not result in the generalized secretion of other lysosomal enzymes that are dependent on the goal of mannose-6-P. The secreted recombinant a-L-iduronidase is similar to the normal secreted enzyme in many respects. If molecular size, which is found to be 77, 82, 84, and 89 kDa in different determinations, is comparable to the 87 kDa, which is found for the urinary corrective factor (Barton et al., J. Bi ol. Chem. 246 : 7773-7779 (1971)), and with the 76 kDa and 82 kDa, which are found for the enzyme that secreted cultured human fibroblasts (Myerowitz et al., J. Biol. Chem. 256: 3044-3048 (1991); Taylor et al. Biochem J. 274: 263-268 (1991)). The differences within and between the studies are attributed to the imprecision of the measures. The pattern of intracellular processing of the recombinant enzyme - a slight decrease in molecular size and the eventual appearance of a smaller additional band by 9 kDa, is the same as for the human fibroblast enzyme. This faster band arises through the proteolytic dissociation of the 80 N-terminal amino acids. In a fifth aspect, the present invention presents a novel method for purifying a-L-iduronidase. In preferred embodiments, the present invention features a method for purifying recombinant a-L-iduronidase that has been optimized to produce a rapid and efficient purification with validatable chromatography resins and a single charge, levigated wash operation. The method for purifying an α-L-iduronidase of the present invention includes a series of column chromatography steps, which allow the high-throughput purification of the enzyme from the protein-free production medium. In accordance with a first embodiment, the cell mass is cultured in approximately 10 percent serum containing the medium, followed by switching to a modified protein-free production medium, without any significant adaptation to produce an activity starting material. specific high for purification. In a second mode, a concentration / diafiltration scheme is used that allows the removal of exogenous materials such as Pluronics F-68 from the raw mass, to avoid contamination of the columns. In a third embodiment, a first column charge is acidified to minimize the competitive inhibition effect of compounds such as the uronic acids found in the protein free medium formulations. In a fourth embodiment, a heparin, phenyl, and sizing column purification scheme is used to produce the pure enzyme using the steps that can be automated. In a fifth embodiment, the steps of the heparin and phenyl column are used to remove the less desirable a-L-iduronidase that is nicked or degraded. In a sixth embodiment, an acid pH treatment step is used to deactivate the potential viruses without damage to the enzyme. Particularly preferred methods of the method for purifying the α-L-iduronidase according to the present invention have more than one or all of the optimizations according to the following particular embodiments. The purification method of the present invention can therefore provide a purified a-L-iduronidase having the characteristics described herein. 1. Concentration / diafiltration: The crude supernatant is processed with a hollow fiber concentrator (A / G Technologies, 30K cut) to reduce the fluid volume by approximately 75 percent and then diafiltered with a charge pH regulator of heparin (10 mM NaP04, pH 5.3, 200 mM NaCl). Diafiltration is an important step that eliminates undesirable compounds such as the Pluronics F-68, of the supernatant, a supernatant that is needed in many cell cultures of the present invention that can contaminate the columns. Diafiltration can also partially remove competitor inhibitors that may prevent heparin fixation to the column. These inhibitors can be found in the PF-CHO medium and are believed to be uronic acids that are derived from a soybean hydrolyzate present in this particular medium. 2. Heparin Column: The load can be adjusted to a pH of about 5.0, before loading onto Heparin Sepharose CL-6B. other types of heparin columns such as an FF heparin (Pharmacia) have different linkages and do not bind a-L-iduronidase as efficiently. A lower pH neutralizes uronic acids to a certain degree, which decreases their competitive effect. Without diafiltration and pH adjustment, heparin columns can not be run using the PF-CHO medium without having a substantial enzyme influx. The column can be washed with a pH regulator with a pH of about 5.3 and then levigated in 0.6 M NaCl. The adjusted range of fixation and levigation of the salt concentration leads to an efficient purification step and the enzyme that is frequently greater than 90 percent pure after One step. 3. Heparin Column: You can use Phenyl Phenyl Sepharose BP (Pharmacia) in the next step. The heparin eluate can be adjusted to approximately 1.5 M NaCl and loaded onto the column. The selection of the resin is important, as is the concentration of salt to ensure that the enzyme is completely fixed (no influx) and is still easily and completely levigated with approximately 0.15 M NaCl. The eluate obtained is almost pure a-L-iduronidase. 4. A pH inactivation can be performed to provide a strong step for the removal of potential viruses. The phenyl group is adjusted to a pH of about 3.3. using Citrate with a pH of 3.9 and kept at room temperature for about 4 hours. Then the enzyme can be neutralized. It has been shown that the modalities that present this step eliminate viruses to a minimum of approximately 5 load units. The step does not deactivate or substantially affect the activity of the enzyme. 5. Then the enzyme can be concentrated and injected over a Sephacryl S-200 column and the peak of the enzyme collected. It has been shown that the enzyme that was purified in this manner contains mannose-6-phosphate residues in sufficient quantity at positions 3 and 6 of the N-linked sugars, to give the enzyme uptake affinity of less than 30 units per milliliter (less than 2 nM) of enzyme. The enzyme is substantially corrective for glycosaminoglycan storage disorders and has a half-life within cells of about 5 days. In a sixth aspect, the present invention presents novel methods for treating diseases caused all or in part by a deficiency of a-L-iduronidase. The recombinant a-L-iduronidase provides enzyme replacement therapy in a canine model of MPS I. This canine model is deficient in a-L-iduronidase due to a genetic mutation and is similar to human MPS I. Purified a-L-iduronidase, appropriately processed intravenously, was administered to 11 dogs. In those dogs that were treated with weekly doses of 25,000 to 125,000 units per kilogram, for 3, 6 or 13 months, the enzyme was captured in a variety of tissues and decreased lysosomal storage in many tissues. Long-term treatment of the disease was associated with clinical improvement in behavior, joint stiffness, fur and growth. The highest doses of therapy (125,000 units per kilogram per week), resulted in better efficacy and included normalization of urinary GAG excretion in addition to faster clinical improvement in behavior, hardening of the joints and fur. Enzyme therapy even at small doses of 25,000 units (0.1 milligram / kilogram / week) resulted in significant enzyme distribution in some tissues and decreases in GAG storage. If it continues for 1 year, the significant clinical effects of the therapy in terms of activity, mobility, growth and general health will be evident. The therapy at these doses did not improve other tissues that are important sites for the disease in this entity, such as cartilage and brain. The highest doses of 125,000 units (0.5 milligrams / kilogram) that were given 5 times during two weeks, showed that improved tissue penetration can be achieved, and a tissue-level therapeutic effect was achieved in as few as 2 weeks. Studies in this increased dose have been conducted in two dogs. These dogs with MPS I showed significant clinical improvement and substantial decreases in urinary GAG excretion within the normal range. Apart from an immune reaction controlled by altered administration techniques, enzyme therapy has not shown significant clinical or biochemical toxicity. The enzyme therapy in its highest weekly doses is effective to improve some clinical characteristics of MPS I and to decrease storage without significant toxicity. In a seventh aspect, the present invention features novel pharmaceutical compositions comprising human a-L-iduronidase useful for the treatment of a deficiency of a-L-iduronidase. The recombinant enzyme can be administered in a number of ways, such as parenteral, topical, intranasal, inhalation, or oral administration. Another aspect of the invention is to provide for the administration of the enzyme by means of formulating it with a pharmaceutically acceptable carrier which can be solid, semi-solid, or liquid or an ingestible capsule. Examples of the pharmaceutical compositions include tablets, drops such as nasal drops, compositions for topical application such as ointments, jellies, creams and suspensions, aerosols for inhalation, nasal sprays, and liposomes. Usually, the recombinant enzyme comprises between 0.05 and 99 percent or between 0.5 and 99 percent by weight of the composition, for example, between 0.5 and 20 percent for compositions that are presented for injection and between 0.1 and 50 percent for the compositions that are desired for oral administration. To produce the pharmaceutical compositions in this form of dosage units for oral application containing a therapeutic enzyme, the enzyme can be mixed with a solid, powdery carrier, for example lactose, sucrose, sorbitol, mannitol, a starch such as starch of potato, corn starch, amylopectin, laminaria powder or citrus pulp powder, a cellulose derivative or gelatin and may also include lubricants such as magnesium or calcium stearate or a carbowax or other polyethylene glycol waxes and compress to form tablets or cores for dragees. If dragees are required, the cores may be coated, for example, with concentrated sugar solutions which may contain gum arabic, talc and / or titanium dioxide, or alternatively with a film-forming agent that dissolves in organic solvents easily. volatile or mixtures of organic solvents. Dyes can be added to these coatings, for example, to distinguish between the different contents of the active substance. For the composition of soft gelatine capsules consisting of gelatin and, for example, glycerol as a plasticizer, or similar closed capsules, you can mix the active substance with a Carbowax® or a suitable oil such as, for example, sesame oil, olive oil, or arachis oil. Hard gelatine capsules may contain granules of the active substance with solid, powdery carriers, such as lactose *, sucrose, sorbitol, mannitol, starches such as potato starch, corn starch, or amylopectin, cellulose derivatives or gelatin, and also they may include magnesium stearate or stearic acid as lubricants. The therapeutic enzymes of the subject invention can also be administered parenterally, such as by subcutaneous, intramuscular or intravenous injection or by a sustained release subcutaneous implant. In subcutaneous, intramuscular or intravenous injection, the therapeutic enzyme (the active ingredient) can be dissolved or dispersed in a liquid carrier vehicle. For parenteral administration, the active material can be suitably mixed with an acceptable carrier, preferably from the variety of vegetable oils, such as peanut oil, cottonseed oil, or the like. Other parenteral vehicles such as compositions using solketal, glycerol, formal, and aqueous parenteral formulations may also be used. For parenteral application by injection, the compositions may comprise an aqueous solution of a pharmaceutically acceptable salt soluble in water of the active acids, according to the invention, desirably at a concentration of 0.5-10 percent, and optionally also a stabilizing agent and / or pH regulating substances in aqueous solution. The dosing units of the solution can be conveniently enclosed in ampoules. When the therapeutic enzymes are administered in the form of a subcutaneous implant, the compound is suspended or dissolved in a slowly dispersed material known to those skilled in the art, or administered in a device that slowly releases the active material through the use of a constant driving force, such as an osmotic pump. In these cases, administration is possible for an extended period of time. For topical application, the pharmaceutical compositions are suitably in the form of an ointment, gel, suspension, cream or the like. The amount of the active substance may vary, for example, between 0.05 - 20 percent by weight of the active substance. These pharmaceutical compositions for topical application can be prepared in a known manner by mixing the active substance with the known carrier materials, such as isopropanol, glycerol, paraffin, stearyl alcohol, polyethylene glycol, and the like. The pharmaceutically acceptable carrier can also include a known chemical absorption promoter. Examples of absorption promoters are, for example, dimethylacetamide (U.S. Patent No. 3,472,931), trichloroethanol or trifluoroethanol (U.S. Patent No. 3,891,757), certain alcohols and mixtures thereof (British Patent Number 1,001,949). A carrier material for topical application to a cracked skin is also described in British Patent Specification No. 1,464,975, which describes a carrier material consisting of a solvent comprising 40-70 percent (v / v) isopropanol and -60 percent (v / v) glycerol, the balance, if any, being an inert constituent of a diluent that does not exceed 40 percent of the total volume of the solvent. The dose at which the therapeutic enzyme containing the pharmaceutical compositions is administered can vary within a wide range and will depend on different factors such as, for example, the severity of the disease, the age of the patient, etc., and could also be have to adjust individually. As a possible range for the amount of therapeutic enzyme that can be administered per day, we can mention give from approximately 0.1 milligrams to approximately 2000 milligrams or from approximately 1 milligram to approximately 2000 milligrams. Pharmaceutical compositions containing the therapeutic enzyme can be formulated suitably, so as to provide doses within these ranges either as single-dose units or as multiple-dose units. In addition to containing a therapeutic enzyme (or therapeutic enzymes), the subject formulations may contain one or more substrates or cofactors for the reaction that was catalyzed by the therapeutic enzyme in the compositions.The therapeutic enzyme containing the compositions may also contain more The recombinant enzyme that is used in the subject methods and compositions can also be administered by transforming the patient's cells with nucleic acids encoding the aL-iduronidase.The nucleic acid sequence encoding in this manner is they can be incorporated into a vector for transformation within the cells of the subject to be treated.The preferred embodiments of these vectors are described herein.The vector can be designed so as to be integrated into the subject's chromosomes, by example, retroviral vectors, or replicate autonomously in the host cells s The vectors containing the nucleotide sequences encoding a-L-iduronidase can be designed to provide continuous or regulated expression of the enzyme. Additionally, the genetic vector encoding the enzyme can be designed so that it is stably integrated into the genome of the cell or only transiently present. The general methodology of conventional gene therapy can be applied to the polynucleotide sequences encoding a-L-iduronidase. Reviews of conventional gene therapy techniques can be found in Friedman, Science 244: 1275-1281 (1989); Ledley, J. Inheri t. Metab. Dis. 13: 587-616 (1990); and Toltoshev et al., Curr Opinions Bictech. _1: 55-61 (1990). A method that is particularly preferred for administering the recombinant enzyme is intravenously. A particularly preferred composition comprises the recombinant α-L-iduronidase, normal saline, phosphate pH regulator to maintain the pH at about 5.8, and human albumin These ingredients can be provided in the following amounts:aL-iduronidase 0.05-0.2 mg / mL or 12,500- 50,000 units per mL 150 mM sodium chloride solution in an IV bag, 50-250 cc total volume Phosphate pH Regulator 10-50 mM, pH 5.8 sodium Human albumin 1 mg / mL Having described the invention, the following examples are offered to illustrate the invention by way of illustration, not by way of limitation.

EXAMPLE 1 Production of Recombinant Iduronidase Standard techniques such as those described by Sambrook et al. (1987) can be used.

Molecular Cloning: A Laboratory Manual, 2 edition, Cold Springs Harbor Laboratory, Cold Spring Harbor, NY, to clone the cDNA encoding human aL-iduronidase The cDNA of the aL-iduronidase that had been previously cloned was subcloned, in PRCCMV (in Vitrogen) as a HindIII-Xbal fragment from a KS subclone of cyanocopy.An intron cartridge was constructed that is derived from the murine immunoglobulin Cot intron between exons 2 and 3, using the amplification of the polymerase chain reaction of bases 788-1372 (Tucker et al., Prcc Nati Acad Sci USA USA 78: 7684-7688 (1991) clone pRIR14.5 (Kakkis et al., Nucleic Acids Res. 16: 7796 (1988).) The cartridge included 136 bp from the 3 'end of exon 2 and 242 bp from the 5' end of exon 3, which would remain in the cDNA that was appropriately divided.There were no ATG sequences present in the coding. , the intron cartridge region, the cartridge was cloned of the intron at the 5 'HindIII site of the a-L-iduronidase cDNA. The new gene was deleted by Xhol digestion followed by recircularization of the vector to make pCMVhldu. A flask was thawed from the master cell bank and placed in three T150 flasks in DME / F12 plus the supplements, plus 10 percent FBS and 500 llg / ml G418. After 3-4 days, the cells are passaged using trypsin-EDTA to 6 cylinders of high capacity cylinder in the same medium. The 2 x 109 cell inoculum was added to a Wheaton microcarrier matra, z containing 60 grams of Cytodex 2 microcarriers, and DME / F12 plus supplements, 10 percent FBS and 500 llg / ml of G418 to a final volume of 13 liters. The flask was agitated by a superior warp pulse with an infusion rod stirrer. The culture is monitored by temperature, OD and pH probes and controlled using the Wheaton mini-plant control system with a PC interface (BioPro software). The parameters are controlled at set points, 37 ° C, 80 percent air saturation, and pH 6.7, using a heating blanket, oxygen sprayer and base pump. The culture was incubated for 3-4 days, at which time the culture is leaving the growth of the loading phase at 1-3 x 106 cells per milliliter. After this, at 12 hour intervals, the medium was changed with PF-CHO medium (with the customary modifications, JRH Biosciences). The first 2 collections were separated as 'washed out.' The third harvest is the start of the production series, sodium butyrate was added at 2 mM final, every 48 hours to induce an increase in the expression of iduronidase. with medium changes of 10 liters every 12 hours and the collections were filtered through a 1 micron filter to eliminate the free cells and the waste.The culture was monitored by 1 temperature, pH and OD on a continuous basis. Twice daily, the culture was sampled before the medium change and tested for the condition of the cells and microorganisms, by phase contrast microscopy, the content of the glucose using a portable glucometer, the activity of the iduronidase using a fluorescent substrate assay The cell mass was tested several times during the run, using a total cell protein assay. Lular reaches 107 cells per milliliter. The production medium that was collected containing the iduronidase was then concentrated five times., using a hollow fiber molecular filter from A / G Technology with a molecular weight limitation of 30,000. After diafiltrating the concentrate with a volume of three times minimum of 0.2 M NaCl in 10 mM NaP04, pH 5.8 for a period of 8 hours. This step removes the Pluronics F68 and the uronic acids from the concentrate. These molecules can inhibit the function of the heparin column. The concentrate was adjusted to a pH of 5.0, filtered through 1.0 and 0.2 micron filters and then loaded onto a Heparin-Sepharose CL-6B column. The column was washed with 10 column volumes of 0.2 M NaCl, 10 mM NaP04, pH 5.3, and the enzyme was levigated with 0.6 MI, 10 mM NaP04, pH 5.8. The eluate was adjusted to 1.5 M NaCl, filtered through a 1 micron filter and loaded onto a HP Phenyl-Sepharose column. The column was washed with 10 column volumes of 1.5 M NaCl, 10 mM NaP04, pH 5.8 and the enzyme was levigated with 0.15 M NaCl, 10 mM NaP04, pH 5.8. Viral deactivation was performed by acidifying the enzyme fraction to a pH of 3.3, using 1 M citric acid with a pH of 2.9 and incubating the enzyme at a pH of 3.3 at room temperature for 4 hours and readjusting pH to 5.8. , using 1 M of phosphate pH regulator. It has been shown that this step removes 5 charges or better from a retrovirus in the drilling experiments. The deactivated enzyme was filtered through a 0.2 μ filter, concentrated on a hollow fiber concentrator apparatus from A / G Technologies (molecular weight limitation of 30,000) and injected in cycles on a Sephacryl S200 gel filtration column. and the peaks were collected. The pooled peaks were filtered through a 0.2 μ filter, formulated for 0.1 M NaP04, pH 5.8 and passed through the vial. A set of studies can be carried out to evaluate the quality, purity, and potency of the enzyme. In Figure 2 the results of an SDS-PAGE analysis of the eluate are provided. A recombinant human a-L-iduronidase obtained from this procedure demonstrated a potency of 100,000 units per milliliter and had a total protein concentration of 0.313 milligrams / milliliter.

EXAMPLE 2 Therapy with the α-Iduronidase Recombinan e is Effective Short-term intravenous administration of purified recombinant human of-L-iduronidase to 9 dogs with MPS I and 6 cats with MPS I demonstrated significant uptake of the enzyme in a variety of tissues with an estimated 50 percent or more recovery in tissues, 24 hours after a single dose. Although the liver and spleen capture the greatest amount of the enzyme, and have the best pathological progress, improvements in pathology and glycosaminoglycan content have been observed in many, but not all, tissues. In particular, the heart's cartilage, brain and valve did not improve significantly. Clinical improvement was observed in a single dog in a long-term treatment for 13 months, but other studies have been limited to 6 months or less. All dogs, and most cats, that received the recombinant human enzyme developed antibodies to the human product. The IgG antibodies are of the complement activating type (probable canine IgG). This phenomenon was also observed in at least 13 percent of Gaucher patients treated with alglucerase. Proteinuria was observed in a dog, which can be related to the disease of the immune complex. No other effect of the antibodies was observed in the other animals that were treated. No specific toxicity was observed and clinical laboratory studies (Complete blood counts, electrolytes, BLJN / creatinine, liver enzymes, uranization) have been normal in every other way. Enzyme therapy even at small doses of 25,000 units (0.1 milligrams / kilogram / week) resulted in significant enzyme distribution to some tissues and decreases in GAG storage. If it continues for more than 1 year, the significant clinical effects of the therapy in terms of activity, mobility, growth and general health will be evident. The therapy at this dose did not improve other tissues that are important sites for the disease in this entity, such as cartilage and brain. The highest doses of 125,000 units (0.5 milligrams / kilogram) that were given 5 times during two weeks, showed that improved tissue penetration can be achieved, and a tissue-level therapeutic effect was achieved in as few as 2 weeks. Studies in this increased dose are currently being conducted in two dogs. These dogs with MPS I show significant clinical improvement and substantial decreases in urinary GAG excretion within the normal range. Apart from an immune reaction controlled by altered administration techniques, enzyme therapy has not shown significant clinical or biochemical toxicity. The enzyme therapy in its highest weekly doses is effective to improve some clinical characteristics of MPS I and to decrease storage without significant toxicity. The results of these different studies in dogs with MPS I and a study in cats with MPS I show that recombinant human a-L-iduronidase is safe. These same results also provide a major reason that this recombinant enzyme should be effective in treating a-L-iduronidase deficiency.

EXAMPLE 3 Recombinant a-L-Iduronidase Therapy is Effective in Humans The human cDNA of a-L-iduronidase predicts a protein of 653 amino acids and an epserated molecular weight of 70,000 daltons after dissociation of the signal peptide. The amino acid sequencing reveals alanine 26 in the N-terminus that gives an expected protein of 629 amino acids. Recombinant human a-L-iduronidase has a histidine at position 8 of the mature protein. The sequence of the predicted protein comprises six potential sites of modification of N-linked oligosaccharides. All of these can be modified in the recombinant protein. It has been shown that the third and sixth sites contain one or more residues of mannose 6-phosphate responsible for the uptake of high affinity within the cells. This peptide corresponds to amino acids 26-45 of Human Recombinant a-L-iduronidase with an alanine of N-terminus and the following sequence: ala-glu-ala-pro-his-leu-val-his-val-asp-ala-ala-arg-ala-leu-trp-pro-leu-arg-arg The recombinant enzyme has an apparent molecular weight of 82,000 daltons on SDS-PAGE due to modifications of the carbohydrate. The recombinant human recombinant a-L-iduronidase has been sequenced by the UCLA Protein Sequencing facility. It is preferred to administer the recombinant enzyme intravenously. The recombinant human a-L-iduronidase was delivered in 10 mL polypropylene vials at a concentration of 0.05-0.2 milligrams / milliliter (12.500-50,000 units per milliliter). The final dosage form of the enzyme includes the recombinant α-L-iduronidase, normal saline, phosphate pH regulator at a pH of 5.8, and human albumin at 1 milligram / milliliter. These were prepared in a bag of normal saline solution.

Component Composition aL-iduronidase 0.05-0.2 mg / mL or 12,500- 50,000 units per L 150 mM sodium chloride solution in an IV bag, 50-250 cc total volume 10-50 mM Phosphate pH Regulator, pH 5.8 sodium Human albumin 1 mg / mL The study included human patients who manifest a clinical phenotype of MPS I disorder with a level of a-L-iduronidase less than 1 percent of normal in leukocytes and fibroblasts. All patients showed some clinical evidence of visceral glycosaminoglycan and soft tissue accumulation with varying degrees of functional impairment. Efficacy was determined by measuring the percentage reduction in urinary GAG excretion over time. Figure 3 reveals the levels of urinary GAG in 16 patients with MPS I, in relation to normal excretion values. There is a wide range of urine GAG values in patients with untreated MPS I. A reduction of more than 50 percent in the excretion of GAG without degradation after therapy with the recombinant a-L-iduronidase is a valid means of measuring an individual's response to therapy. Figure 4 demonstrates the activity of leukocyte iduronidase before and after enzyme therapy in patients with MPS I. The activity of oral iduronidase before and after therapy is described in Figure 5. Figure 6 shows in three patients that a substantial shrinkage of the liver and spleen was associated, together with a significant clinical improvement in the storage of the joints and soft tissue, with a reduction of more than 65 percent in the GAG without degrading , only 8 weeks of treatment with the recombinant enzyme. Figure 7 demonstrates that there is a substantial normalization of livers and spleens in patients who were treated with the recombinant enzyme, after only 12 weeks of therapy with the recombinant enzyme. Figure 8 demonstrates a precipitous drop in urinary GAG excretion during 22 weeks of recombinant enzyme therapy in 11 patients. Clinical evaluation of the size of the liver and spleen has been the most accepted element to evaluate treatment with successful bone marrow transplantation in patients with MPS I (Hoogerbrugge et al., Lancet 345: 1398 (1995)). These measurements correlate greatly with decreased visceral storage of GAGs in patients with MPS I. Although the invention has been described with reference to the embodiments referred to herein, it should be understood that various modifications can be made, without departing from the spirit of the invention. In accordance with the foregoing, the invention is limited only by the following claims LIST OF SEQUENCES < 110 > Harbor - UCLA < 120 > Recombinant Alpha-L-Iduronidase, Methods to Produce and Purify the Same and Methods to Treat the Diseases Caused by the Deficiency of the same. < 130 > 08000007PC00 < 140 > PCT / US99 / 10102 < 141 > 1999-05-07 < 160 > 2 < 170 > FastSEQ for Windows Version 3.0 < 210 > 1 < 211 > 6200 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (1558) (3516) 400 > 1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 gacgtcaata cccgcccatt atgacgtatg ttcccatagt aacgccaata gggactttcc 20 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 catgacctta atgcccagta tgggactttc ctacttggca gtattagtca gtacatctac 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 ggactttcca aaaatcaacg aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttaact ggcttatc ga aattaatacg actcactata gggagaccca agcttcgcag 900 aattcctgcg gctgctacag tgtgtccagc gtcctgcctg gctgtgctga gcgctggaac 960 agtggcgcat cattcaagtg cacagttacc catcctgagt ctggcacctt aactggcaca 1020 attgccaaag tcacaggtga gctcagatgc ataccaggac attgtatgac gttccctgct 1080 cacatgcctg ctttcttcct ataatacaga tggtcaacta actgctcatg tccttatatc 1140 attggagcta acagagggaa tctgaggaac tgcccagaag ggaagggcag aggggtcttg 1200 ctgagccata ctctccttgt ctaccttcca actcttcttt tcccacccca gtgaacacct 1260 ggtccacctg ctaccgccgc cgtcggagga gctggccctg aatgagctct tgtccctgac 1320 cgagctttca atgcctggtg accctaaaga agtgctggtg cgatggctgc atggaaatga 1380 ggagctgtcc ccagaaagct acctagtgtt tgagccccta aaggagccag gcgagggagc 1440 ctggtgacaa caccacctac gcgtgttgcg tgtatcagct gaaagcttga tatcgaattc 1500 cggaggcgga accggcagtg cagcccgaag ccccgcagtc cccgagcacg cgtggcc atg 1560 Met 1 cgc ccc ctg cgc ccc cgc gcc gcg ctg ctg gcg etc ctg gcc teg etc 1608 Arg Pro Leu Arg Pro Arg Ala Ala Leu Leu Ala Leu Leu Ala Ser Leu 5 10 15 ctg gcc gcg ccc ceg gtg gcc ceg gcc gag gcc ceg cae ctg gtg cat 1656 Leu Ala Ala Pro Pro Val Ala Pro Ala Glu Ala Pro His Leu Val His 20 25 30 gtg gac gcg gcc cgc gcg ctg tgg ccc ctg cgg cgc ttc tgg agg age 1704 Val Asp Ala Ala Arg Ala Leu Trp Pro Leu Arg Arg Phe Trp Arg Ser 35 or 45 here ggc ttc tgc ccc ceg ctg cea falls age cag gct gac cag tac gtc 1752 Thr Gly Phe Cys Pro Pro Leu Pro His Ser Gln Wing Asp Gln Tyr Val 5 ° 55 60 65 etc age tgg gac cag cag etc aac etc gcc tat gtg ggc gcc gtc cct 1800 Leu Ser Trp Asp Gln Gln Leu Asn Leu Ala Tyr Val Gly Ala Val Pro 70 75 80 fall cgc ggc ate aag cag gtc cgg acc fall tgg ctg ctg gag ctt gtc 1848 His Arg Gly He Lys Gln Val Arg Thr His Trp Leu Leu Glu Leu Val 85 90 95 acc action agg ggg tec act gga cgg ggc ctg age tac aac ttc acc falls 1896 Thr Thr Arg Gly Ser Thr Gly Arg Gly Leu Ser Tyr Asn Phe Thr His 100 105 110 ctg gac ggg tac ctg gac ctt etc agg gag aac cag etc etc cea ggg 1944 Leu Asp Gly Tyr Leu Asp Leu Leu Arg Glu Asn Gln Leu Leu Pro Gly 115 120 125 ttt gag ctg atg ggc age gcc tcg ggc falls ttc act gac ttt gag gac 1992 Phe Glu Leu Met Gly Ser Wing Ser Gly His Phe Thr Asp Phe Glu Asp 130 135 140 145 aag cag gtg ttt gag tgg aag gac ttg gtc tec age ctg gcc agg 2040 Lys Gln Gln Val Phe Glu Trp Lys Asp Leu Val Ser Ser Leu Ala Arg 150 155 160 aga tac ate ggt agg tac gga ctg gcg cat gtt tec-aag tgg aac ttc 2088 Arg Tyr He Gly Arg • Tyr Gly Leu Wing His Val Ser Lys Trp Asn Phe 165 170 J75 gag acg tgg aat gag cea gac cae falls gac ttt gac aac gtc tec atg 2136 Glu Thr Trp Asn Glu Pro Asp His His Asp Phe Asp Asn Val Ser Met 180 185 190. acc atg ca ggc ttc ctg aac tac tac gat gcc tgc tcg gag ggt ctg 2184 Thr Met Gln Gly Phe Leu Asn Tyr Tyr Asp Wing Cys Ser Glu Gly Leu 195 200 205 cgc gcc gcc age ccc gcc ctg cgg ggg cg ggc ccc ggc gac tec ttc 2232 Arg Wing Wing Pro Pro Wing Leu Arg Leu Gly Gly Pro Gly Asp Ser Phe 210 215 220 225 cae agg cea ceg cga tec ceg ctg age tgg ggc etc ctg cgc falls tgc 2280 Hrs Arg Pro Pro Arg Ser Pro Leu Ser Trp Gly Leu Leu Arg His Cys 230 235 240 falls gac ggt acc aac ttc ttc act ggg gag gcg ggc gtg cgg ctg'gac 2328 His Asp Gly Thr Asn Phe Phe Thr Gly Glu Wing Gly Val Arg Leu Asp 245 250 255 tac ate tec etc drops agg aag ggt gcg cgc age tec ate tec ctg! 376 Tyr lie Ser Leu His Arg Lys Gly Ala Arg Ser Ser He Be lie Leu 260 265 270 gag cag gag aag gtc gtc gcg cag cag ate. cgg cag etc ttc ccc aag 2424 Glu Gln Glu Lys Val Val Ala Gln Gln He Arg Gln Leu Phe Pro Lys 275 280 285 ttc gcg gac acc ccc att tac aac gac gac gcg gac ceg ctg gtg ggc 2472 Phe Wing Asp Thr Pro He Tyr Asn Asp Glu Wing Asp Pro Leu Val Gly 290 295 300 305 tgg tec ctg cea cag ceg tgg agg gcg gac gtg acc tac gcg gcc atg 2520 Trp Ser Leu Pro Gln Pro Trp Arg Wing Asp Val Thr Tyr Wing Ala Met 310 315 320 gtg gtg aag gtc ate gcg cag cat cag aac ctg cta ctg gcc aac acc 2568 Val Val Lys Val He Wing Gln His Gln Asn Leu Leu Leu Wing Asn Thr 325 330 335 acc tec gcc ttc ccc tac gcg etc ctg age aac gac aat gcc ttc ctg 2616 Thr Ser Wing Phe Pro Tyr Wing Leu Leu Ser Asn Asp Asn Wing Phe Leu 340 345 350 age tac falls ceg falls ccc ttc gcg cag cgc acg etc acc gcg cgc ttc 2664 Ser Tyr His Pro His Pro Phe Wing Gln Arg Thr Leu Thr Ala Arg Phe 355 • 360 365 cag gtc aac aac a cc cc cc cc cc cc cc cc gc tc cgc aag ceg 2712 Gln Val Asn Asn Thr Arg Pro Pro His Val Gln Leu Leu Arg Lys Pro 370 375 380 385 gtg etc acg gcc atg ggg ctg ctg gcg ctg ctg gat gag gag cag etc 2760 Val Leu Thr Ala Met Gly Leu Leu Ala Leu Leu Asp. Glu Glu Gln Leu 390 395 400 tgg gcc gaa gtg cag gcc ggg acc gtc ctg gac age aac falls acg 2808 Trp Wing Glu Val Ser Gln Wing Gly Thr Val Leu Asp Ser Asn His Thr 405 410 415 gtg ggc gtc ctg gcc age gcc falls cgc ccc cag ggc ceg gcc gac gcc 2856 Val Gly Val Leu Wing Ser Wing His Arg Pro Gln Gly Pro Wing Asp Wing 420 425 430 tgg cgc gcc gcg gtg ctg ate tac gcg age gac gac acc cgc gcc drops 2904 Trp Arg Ala Ala Val Leu He Tyr Wing Ser Asp Asp Thr Arg Wing His 435 440 445 ccc aac cgc age gtc gcg gtg acc ctg cgg cg cgc ggg gtg ccc ccc 2952 Pro Asn Arg Ser Val Wing Val Thr Leu Arg Leu Arg Gly Val Pro Pro 450 455 460 465 ggc ceg ggc ctg gtc tac gtc acg cgc tac ctg gac aac ggg etc tgc 3000 Gly Pro Gly Leu Val Tyr Val Thr Arg Tyr Leu Asp Asn Gly Leu Cys 470 475 480 age ccc gac ggc gag tgg cgg cgc cgc ggg cgg gcc ccc gcc tc ccc acg 3048 Pro Pro Asp Gly Glu Trp Arg Arg Leu Gly Arg Pro Val Phe Pro Thr 485 490 495 gca gag cag ttc cgg cgc atg cgc gcg gct gag gac ceg gtg gcc gcg 3096 Wing Glu Gln Phe Arg Arg Wing Arg Wing Wing Glu Asp Pro Wing Wing 500 500 505 510 gcg ccc cgc ccc tta ccc gcc ggc ggc cgc ctg acg ctg cgc ccc gcg 3144 Wing Pro Arg Pro Leu Pro Wing Gly Gly Arg Leu Thr Leu Arg Pro Wing 515 520 525 ctg cgg cg cg ctg ctt ttg ctg gtg gtg gtg gcg cgc ccc gag 3192 Leu Arg Leu Pro Ser Leu Leu Leu Val His Val Cys Ala Arg Pro Glu 530 535 540 545 aag ceg ccc ggg cag gtc acg cgg etc cgc gcc ctg ccc ctg acc at ca 3240 Lys Pro Pro Gly Gln Val Thr Arg Leu Arg Ala Leu Pro Leu Thr Gln 550 555 560 ggg cag ctg gtt ctg gtc tgg tcg gat gaa falls gtg ggc tec aag tgc 3288 Gly Gln Leu Val Leu Val Trp Ser Asp Glu His Val Gly Ser Lys Cys 565 570 575 ctg tgg here tac gag ate cag ttc tet cag gac ggt aag gcg tac acc 3336 Leu Trp Thr Tyr Glu He Gln Phe Ser Gln Asp Gly Lys Wing Tyr Thr 580 585 590 ceg gtc age agg aag cea tcg acc ttc aac etc ttt gtg ttc age cea 3384 Pro Val Ser Arg Lys Pro Ser Thr Phe Asn Leu Phe Val Phe Ser Pro 595 600 605 gac here ggt gct gtc tet ggc tec tac cga gtt cga gcc ctg gac tac 3432 Asp Thr Gly Ala Val Ser Gly Ser Tyr Arg Val Arg Ala Leu Asp Tyr 610 615 620. 625 tgg gcc cga ce ggc ccc tcc tcg gac cct gtg ceg tac ctg gag gtc 3480 trp Wing Arg Pro Gly Pro Phe Ser Asp Pro Val Val Tyr Leu Glu Val 630 635 640 cct gtg cea aga ggg ccc ce ce tec ggc aat cea tgagcctgtg 3526 Pro Val Pro Arg Gly Pro Pro Ser Pro Gly Asn Pro 645,650 gtgggttgca ctgagcccca cctccaccgg cagtcagcga gctggggctg cactgtgccc 3586 atgctgccct cccatcaccc cctttgcaat atatttttat attttaaaaa aaaaaaaaaa 3646 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaagaattec 3706 tgcagcccgg gggatccact agttctagag ggcccgttta aacccgctga tcagcctcga 3766 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 3826 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 3886 tgagtaggtg teattetatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 3946 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa 4006 gggctcgaga gaaccagctg gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa 4066 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg 4126 gggtgcctaa tgagtgagct aaetcacatt aattgcgttg cgctcactgc ccgctttcca 4186 gtcgggaaac ctgtcgtgcc agetgeatta atgaateggc caacgcgcgg ggagaggcgg 4246 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4306 gctgcggcga gcggtatcag ggcggtaata ctcactcaaa cggttatcca cagaatcagg 4366 ggaaagaaca ggataaegea tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4426 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gaegageate acaaaaatcg 4486 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4546 tggaagctcc ctggtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4606 ctttctccct tcgggaagcg tggcgctttc tcaatgetca cgctgtaggt atetcagtte 4666 gttcgctcca ggtgtaggtc agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4726 ctgcg cctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4786 gccactggta actggcagca acaggattag cagagcgagg tatgtaggcg gtgetacaga 4846 tggtggccta gttcttgaag actacggcta cactagaagg acagtatttg gtatctgcgc 4906 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4966 caccgctggt agcggtggtt tttttgtttg caageageag attacgcgca gaaaaaaagg 5026 gatcctttga atctcaagaa tcttttctac ggggtctgac gctcagtgga acgaaaactc 5086 acgttaaggg attttggtca aaaaaggatc tgagattatc tteaectaga tccttttaaa 5146 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 5206 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catecatagt 5266 tgcctgactc cccgtcgtgt agataactac gatacgggag ggettaccat ctggccccag 5326 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 5386 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 5446 tattaattgt tgccgggaag ctagagtaag tagttegeca gttaatagtt tgcgcaacgt 5506 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 5566 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 5626 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcaetcat 5686 gcactgcata ggttatggca tgtcatgcca attctgttac tccgtaagat gcttttctgt 5746 tactcaacca gactggtgag agtcattctg agaatagtgt atgcggcgac cgagttgctc 5806 ttgcccggcg tcaatacggg ataatacege gccacatagc agaactttaa aagtgctcat 5866 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 5926 ttcgatgtaa cccactcgtg cacccaactg atetteagea tcttttactt tcaccagcgt 5986 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 6046 gaaatgttga ataetcatac tcttcctttt tcaatattat tgaageattt atcagggtta 6106 ttgtctcatg ageggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 6166 gcgcacattt ccccgaaaag tgccacctga cgtc 6200 < 210 > 2 < 211 > 653 < 212 > PRT < 213 > Homo sapiens > 2 Met Arg Pro Leu Arg Pro Arg Ala Ala Leu Leu Ala Leu Leu Ala Ser 5 10 15 Leu Leu Ala Ala Pro Pro Val Ala Pro Ala Glu Ala Pro His Leu Val 25 30 His Val Asp Ala Ala Arg Ala Leu Trp Pro Leu Arg Arg Phe Trp Arg 35 40 45 Ser Thr Gly Phe Cys Pro Pro Leu Pro His Ser Gln Wing Asp Gln Tyr 50 55 60 Val Leu Ser Trp Asp Gln Gln Leu Asn Leu Wing Tyr Val Gly Wing Val 65 70 75 80 Pro His Arg Gly He Lys Gln Val Arg Thr His Trp Leu Leu Glu Leu 85 90 95 Val Thr Thr Arg Gly Ser Thr Gly Arg Gly Leu Ser Tyr Asn Phe Thr 100 105 110 His Leu Asp Gly Tyr Leu Asp Leu Leu Arg Glu Asn Gln Leu Leu Pro 115 120 125 Gly Phe Glu Leu Met Gly Ser Wing Ser Gly His Phe Thr Asp Phe Glu 130 135 140 Asp Lys Gln Gln Val Phe Glu Trp Lys Asp Leu Val Ser Ser Leu Ala 145 150 155 160 Arg Arg Tyr He Gly Arg Tyr Gly Leu Wing His Val Ser Lys Trp Asn 165 170 175 Phe Glu Thr Trp Asn Glu Pro Asp His His Asp Phe Asp Asn Val Ser 180 185 190 Met Thr Met Gln Gly Phe Leu Asn Tyr Tyr Asp Wing Cys Ser Glu Gly 195 200 205.Leu Arg Wing Wing Pro Pro Wing Leu Arg Leu Gly Gly Pro Gly Asp Ser 210 215 220.Phe His Arg Pro Pro Arg Ser Pro Leu Ser Trp Gly Leu Leu Arg His 225 230 235 240 Cys His Asp Gly Thr Asn Phe Phe Thr Gly Glu Wing Cly Val Arg Leu 245 250 255 Asp Tyr Be Ser Leu His Arg Lys Gly Ala Arg Be Ser Be He Be 260 265 270 Leu Glu Gln Glu Lys Val Val Wing Gln Gln He Arg Gln Leu Phe Pro 275 280 285 Lye Phe Wing Asp Thr Pro He Tyr Asn Asp Glu Wing Asp Pro Leu Val 290 295 300 Gly Trp Ser Leu Pro Gln Pro Trp Arg Wing Asp Val Thr Tyr Wing Ala 305 310 315 320 Met Val Val Lys Val He Wing Gln His Gln Asn Leu Leu Leu Wing Asn 325 330, 335 Thr Thr Ser Wing Phe Pro Tyr Wing Leu Leu Ser Aen Asp Asn Wing Phe 340 345 350 Leu Ser Tyr His Pro His Pro Phe Wing Gln Arg Thr Leu Thr Wing Arg 355 360 365 Phe Gln Val Asn Asn Thr Arg Pro Pro His Val Gln Leu Leu Arg Lys 370 375 380 Pro Val Leu Thr Ala Met Gly Leu Leu Ala Leu Leu Asp Glu Glu Gln 385 390 395 400 Leu Trp Wing Glu Val Ser Gln Wing Gly Thr Val Leu Asp Ser Asn His 405 410 415 Thr Val Gly Val Leu Wing Ser Wing His Arg Pro Gln Gly Pro Wing Asp 420 425 430 Wing Trp Arg Wing Wing Val Leu He Tyr Wing Ser Asp Asp Thr Arg Wing 435 440 445 His Pro Asn Arg Ser Val Wing Val Thr Leu Arg Leu Arg Gly Val Pro 450 455 460 Pro Gly Pro Gly Leu Val Tyr Val Thr Arg Tyr Leu Asp Asn Gly Leu 465 470 475 480 Cys Ser Pro Asp Gly Glu Trp Arg Arg Leu Gly Arg Pro Val Phe Pro 485 490 495 Thr Ala Glu Gln Phe Arg Arg Arg Wing Ala Glu Asp Pro Val Wing 500 505 510 Wing Wing Pro Arg Pro Leu Pro Wing Gly Gly Arg Leu Thr Leu Arg Pro 515 520 525 Wing Leu Arg Leu Pro Ser Leu Leu Val Val Val Cys Ala Arg Pro 530 535 540 Glu Lys Pro Pro Gly Gln Val Thr Arg Leu Arg Ala Leu Pro Leu Thr 545 550 555 5so Gln Gly Gln Leu Val Leu Val Trp Ser Asp Glu His Val Gly Ser Lys 565 570 575, Cys Leu Trp Thr Tyr Glu He Gln Phe Ser Gln Asp Gly Lys Ala Tyr 580 585 590 Thr Pro Val Ser Arg Lys Pro Ser Thr Phe Asn Leu Phe Val Phe Ser 595 600 605 Pro Asp Thr Gly Wing Val Ser Gly Ser Tyr Arg Val Arg Ala Leu Asp 610 615 620 Tyr Trp Wing Arg Pro Gly Pro Phe Ser Asp Pro Val Pro Tyr Leu Glu 625 630 635 6 0 Val Pro Pro Pro Arg Gly Pro Pro Pro Pro Gly Asn Pro 645 650

Claims (41)

1. A method for producing α-L-iduronidase, comprising the step of transforming a suitable cell line with a cDNA encoding the whole or the biologically active fragment or mutant of α-L-iduronidase.

2. The method of claim 1, wherein the appropriate cell line is a 2.131 Chinese hamster ovary cell line.

3. The method of claim 2, wherein the Chinese hamster cell line secretes about 5,000 to 7,000 times more aL-iduronidase than it secreted before introducing the cDNA encoding all aL-iduronidase or a biologically active fragment Of the same.

4. The method of claim 1, wherein the transfected cells are cultured on microcarriers.

The method of claim 1, wherein a culture system is optimized so that the culture pH is reduced to approximately 6.7-6.8 during the production process.

6. The method of claim 1, wherein about 2/3 to 3/4 of a growth medium of the culture system is changed approximately every 12 hours.

The method of claim 1, wherein the oxygen saturation of the culture system is optimized to approximately 80 percent.

The method of claim 7, wherein the oxygen saturation of the culture system is optimized to approximately 80 percent, using pure intermittent oxygen spray.

The method of claim 1, wherein the microcarriers having serum at about 10 percent, are initially used to produce a mass of cells for a culture system.

10. The method of claim 1, characterized in that it also comprises the passage of a washing change to the protein-free medium for production.

The method of claim 1, wherein a culture system comprising a growth medium PF-CHO of JRH Biosciences is used.

The method of claim 11, wherein the growth medium is optimized to include complementary amounts of one or more ingredients that are selected from the group consisting of glutamate, aspartate, glycine, ribonucleosides and deoxyribonucleosides.

The method of claim 1, wherein a batch feed process is performed by an infusion rod.

The method of claim 1, wherein sodium butyrate is added to a culture system.

15. A transfected cell line that has the ability to produce a-L-iduronidase.

16. A transfected cell line according to claim 15, wherein the transfected cell line is a recombinant Chinese hamster ovary cell line.

17. A transfected cell line according to claim 15, wherein the transfected cell line is a 2.131 cell line of recombinant Chinese hamster ovary.

18. A transfected cell line according to claim 15, wherein the transfected cell line contains at least about 10 copies of an expression construct comprising a CMV promoter, an intron of Ca, a cDNA of aL-iduronidase, and a polyadenylation sequence of bovine growth hormone.

19. A transfected cell line according to claim 15, wherein the transfected cell line expresses a-L-iduronidase in amounts of at least about 20-40 micrograms per 10 7 cells per day.

20. A vector adapted to produce human a-L-iduronidase in a transfected cell.

21. The vector according to claim 20, which is adapted to produce human a-L-iduronidase in the cells of a Chinese hamster ovary (CHO).

22. The vector according to claim 20, characterized in that it comprises a promoter / enhancer of the immediate early gene of CMV.

23. The vector according to claim 20, characterized in that it comprises a prcmotor / enhancer element of the cyto egalovirus, a 5 'intron consisting of an intron of murine Ca between exons 2 and 3, a cDNA encoding all or a biologically active fragment of a-L-iduronidase and a polyadenylation site of bovine growth hormone 3.

24. A recombinant aL-iduronidase that is produced according to the method of claim 1.

25. A recombinant aL-iduronidase that is produced according to the method of claim 1 having a specific activity of at least about 200,000 units per milligram.

26. An a-L-iduronidase according to claim 25, characterized in that it has a specific activity of at least about 240,000 units per milligram.

27. A method for purifying a-L-iduronidase, comprising the steps of: (a) performing a concentration / filtration procedure to remove one or more undesirable compounds from a sample; (b) acidifying the sample from step (a); (c) running the sample from step (b) on a heparin column; (d) running the sample from step (c) on a phenyl column; (e) running the sample from step (d) on a Sephacryl column; and (f) running the substantially purified a-L-iduronidase.

28. A method for treating a disease caused in whole or in part by a deficiency in a-L-iduronidase, comprising the step of administering a recombinant a-L-iduronidase.

29. A method for treating a disease caused in whole or in part in a human being by a deficiency in a-L-iduronidase, comprising the step of administering a recombinant human a-L-iduronidase.

30. The method of the. Claim 28, wherein the disease is mucopolysaccharidosis.

31. The method of claim 28, wherein the disease is MPS I.

32. The method of claim 28, wherein the disease is selected from the group consisting of Hurler's disease, Scheie's syndrome, and Hurler-Scheie syndrome.

The method of claim 28, wherein a patient suffering from the disease demonstrates approximately 1 percent or less of a normal a-L-iduronidase activity.

34. The method of claim 28, wherein at least about 25,000 units or 0.1 milligrams / kilogram of a recombinant a-L-iduronidase are administered weekly to a patient suffering from a deficiency thereof.

35. The method of claim 28, wherein at least about 125,000 units or 0.5 milligrams / kilogram of a recombinant a-L-iduronidase are administered weekly to a patient suffering from a deficiency thereof.

36. A pharmaceutical composition comprising the recombinant a-L-iduronidase and a pharmaceutically acceptable carrier.

37. The pharmaceutical composition of claim 36, characterized in that it further comprises a solution of sodium chloride, a pH regulator and a human albumin.

38. The pharmaceutical composition of claim 36, wherein the recombinant a-L-iduronidase is present at a concentration of about 0.05 0.20 milligrams / milliliter or about 12,500 about 50,000 units per milliliter.

39. The pharmaceutical composition of claim 36, wherein the human albumin is present at a concentration of at least about milligram / milliliter.

40. The pharmaceutical composition of claim 36, wherein the pH regulator is a pH regulator of sodium phosphate at a concentration of about 10-50 mM.

41. The pharmaceutical composition of claim 36, wherein the pH of the composition is maintained at about 5.8.