Recombinant alpha-L-iduronidase, methods for producing and purifying the same and methods for treating diseases caused by deficiencies thereof

Abstract

The present invention provides a recombinant α-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—α-L-iduronidase deficiency and mucopolysaccharidosis I (MPS 1).

Description

[0001] This application is a continuation application of U.S. patent application Ser. No. 09 / 439,923, filed Nov. 12, 1999, now U.S. Pat. No. 6,426,208, issued Jul. 30, 2002, which is incorporated herein by reference.FIELD OF THE INVENTION [0002] The present invention is in the field of molecular biology, enzymology, biochemistry and clinical medicine. In particular, the present invention provides a recombinant α-L-iduronidase, methods to produce and purify this enzyme as well as methods to treat certain genetic disorders including α-L-iduronidase deficiency and mucopolysaccharidosis I (MPS I). BACKGROUND OF THE INVENTION [0003] 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 covalently attached to numerous other entities such as proteins and lipids (called glycoconjugates). For example, human connective tissues and cell membranes comprise proteins, c...

AI Technical Summary

Benefits of technology

[0017] In one aspect, the present invention features a method to produce α-L-iduronidase in amounts which enable using the enzyme therapeutically. In a broad embodiment, the method comprises the step of transfecting a cDNA encoding for all or a part of an α-L-iduronidase into a cell suitable for the expression thereof. In some embodiments, a cDNA encoding for a complete α-L-iduronidase is used, preferably a human α-L-iduronidase. However, in other embodiments, a cDNA encoding for a biologically active fragment or mutant thereof may be used. Specifically, one or more amino acid substitutions may be made while preserving or enhancing the biological activity of the enzyme. In other preferred embodiments, an expression vector is used to transfer the cDNA into a suitable cell or cell line for expression thereof. In one particularly preferred embodiment, the cDNA is transfected into a Chinese hamster ovary cell to create cell line 2.131. In yet other preferred embodiments, the production procedure features one or more of the following characteristics which have demonstrated particularly high production levels: (a) the pH of the cell growth culture may be lowered to about 6.5 to 7.0, preferably to about 6.7-6.8 during the production process, (b) about ⅔ to ¾ of the medium may be changed approximately every 12 hours, (c) oxygen saturation may be optimized at about 80% using intermittent pure oxygen sparging, (d) microcarriers with about 10% serum initially may be used to produce cell mass followed by a rapid washout shift to protein-free medium for production, (e) a protein-free or low protein medium such as a JRH Biosciences PF-CHO product may be optimized to include supplemental amounts of one or more ingredients selected from the group consisting of glutamate, aspartate, glycine, ribonucleosides and deoxyribonucleosides, (f) a perfusion wand such as a Bellco perfusion wand may be used in a frequent batch-feed process rather than a standard intended perfusion process, and (g) a mild sodium butyrate induction process may be used to induce increased α-L-iduronidase expression.

[0021] In a fifth aspect, the present invention features a novel method to purify α-L-iduronidase. According to a first embodiment, a cell mass may be grown in about 10% serum containing medium followed by a switch to a modified protein-free production medium without any significant adaptation to produce a high specific activity starting material for purification. Preferably, a concentration / diafiltration scheme is employed that allows for the removal of exogenous materials that may be required for recombinant production of the same such as, for example, Pluronics F-68, a commonly used surfactant for protecting cells from sparging damage. Such exogenous materials should normally be separated from the crude bulk to prevent fouling of the columns. In another preferred embodiment, a first column load is acidified to minimize the competitive inhibition effect of uronic acids found in protein-free medium formulations. In other preferred embodiments, a heparin, phenyl and sizing column purification scheme is used to produce pure enzyme using automatable steps and validatable media. In other preferred embodiments, the heparin and phenyl column steps are used to eliminate less desirable α-L-iduronidase that is nicked or degraded. In yet other preferred embodiments, a three step column chromatography may be used to purify the enzyme. Such a three step column chromatography may include using a blue sepharose FF, a Cu++ chelating sepharose chromatography and a phenyl sepharose HP chromatography. In another preferred embodiment, an acid pH treatment step is used to inactivate potential viruses without harming the enzyme.

Problems solved by technology

In an intermediate form known as Hurler-Scheie syndrome, mental function is generally not severely affected, but physical problems may lead to death by the teens or twenties.

Scheie syndrome is the mildest form of MPS I. It is compatible with a normal life span, but joint stiffness, corneal clouding and heart valve disease cause significant problems.

It is likely that worldwide the disease is underdiagnosed either because the patient dies of a complication before the diagnosis is made or because the milder forms of the syndrome may be mistaken for arthritis or missed entirely.

Except for bone marrow transplantation, there are no significant therapies available for MPS I. Bone marrow transplants can be effective in treating some of the symptoms of the disorder but have high morbidity and mortality in MPS I and often are not available to patients because of a lack of suitable donors.

Application of this therapy to humans has previously not been possible due to inadequate sources of α-L-iduronidase in tissues.

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