Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Reexamination Certificate
1998-10-21
2001-04-03
Prouty, Rebecca E. (Department: 1652)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
C435S200000, C435S208000, C530S350000
Reexamination Certificate
active
06210666
ABSTRACT:
BACKGROUND OF THE INVENTION
Fabry disease is an X-linked disorder of glycosphingolipid metabolism caused by a deficiency of &agr;-galactosidase A (&agr;-Gal A). Human &agr;-Gal A is a lysosomal enzyme that catalyzes the hydrolysis of &agr;-galactosidic linkages of glycoconjugates. An X-linked inborn deficiency of this hydrolase, which is the cause of Fabry disease, leads to the accumulation of neutral glycosphingolipids, predominantly ceramide trihexoxide, primarily in the plasma and in the lysosomes of the vascular endothelium. Affected hemizygous males show various clinical symptoms, such as acroparesthesias, angiokeratoma, hypohidrosis, corneal and lenticular opacities, and progressive vascular disease of the kidney, heart, and brain, leading to death in early adulthood.
Several lines of evidence suggest that enzyme replacement therapy may be beneficial for patients with Fabry disease. For example, it has been demonstrated in cell cultures of fibroblasts obtained from patients with this disease that &agr;-Gal A present in the culture medium is specifically transported to lysosomes. Moreover, the enzyme has been administered to patients with Fabry disease using infusions of normal plasma (Mapes et al.,
Science,
169, 987 (1970)); &agr;-Gal A purified from placenta (Brady et al.,
New Eng. J. Med.,
279, 1163 (1973)); and &agr;-Gal A purified from spleen or plasma (Desnick et al.,
Processor. Natl. Acad. Sci. USA,
76, 5326 (1979)). In one study (Desnick et al., supra), intravenous injection of purified enzyme resulted in a transient reduction in the plasma levels of the substrate globtriaosylceramide. However, insufficient quantities of the purified human enzyme were available for further study.
The structure of the &agr;-Gal A gene has been determined. The 14 kb &agr;-Gal A genomic sequence contains 7 exons encoding a 429 amino acid polypeptide, including an NH
2
-terminal 31-residue signal peptide. Studies on unrelated families with Fabry disease have shown a variety of molecular changes in the &agr;-Gal A gene in affected individuals. More than 70 mutations in the coding region of the &agr;-Gal A gene have been reported, the majority of which result in a classical phenotype with no &agr;-Gal A activity. However, a few mutations that result in single amino acid substitutions in the carboxy (C)-terminal region of &agr;-Gal A lead to an atypical variant of Fabry disease in males, with manifestations limited to the heart (H. Sakuraba et al.,
Am. J. Hum. Genet.,
47, 784 (1990); W. V. Scheidt et al.,
New Engl. J. of Med.,
324, 394 (1991); Y. Nagao et al.,
Clin. Genet.,
39, 233 (1991); S. Nakao et al.,
New Engl. J of Med.,
333, 288 (1995)). It has been suggested that this variant type might be more common than previously believed, occurring in male patients with unexplained left ventricular hypertrophy (S. Nakao et al.,
New Engl. J. of Med.,
333, 288 (1995)).
Several mutations of the &agr;-Gal A gene that result in the introduction of premature stop codons have also been described, including a mutant &agr;-Gal A gene termed E398X which encodes a polypeptide that lacks 32 amino acid residues of the C-terninus of &agr;-Gal A. All hemizygous patients with these mutations are reported to manifest a classical phenotype (C. Meaney et al.,
Hum. Mol. Genet.,
3, 1019 (1994); M. E. Christine et al.,
Hum. Mol. Genet.,
3, 1795 (1994)). While some investigators have speculated that 26 or 28 amino acid residues from the C-terminus of &agr;-Gal A might be proteolytically cleaved to generate the final polypeptide product present in cells (Quinn et al.,
Gene,
58, 177 (1987) and Bishop et al.,
Proc. Natl. Acad. Sci. USA,
85, 3903 (1988)), it remains uncertain whether the C-terminus of &agr;-Gal A is necessary for enzymatic activity.
Thus, a continuing need exists for agents which have increased enzymatic activity relative to wild type &agr;-Gal A.
SUMMARY OF THE INVENTION
The present invention provides an isolated and purified &agr;-galactosidase A (&agr;-Gal A) polypeptide, or a variant thereof, which has a carboxy-terminal deletion. Preferably, the truncated &agr;-Gal A polypeptide is biologically active, and can be used to treat Fabry disease, as discussed above. As described hereinbelow, carboxy-terminal deletions of about 2 to about 10 amino acid residues of human &agr;-Gal A unexpectedly resulted in an increase in enzyme activity relative to wild type &agr;-Gal A polypeptide. These results indicate that the C-terminal domain of &agr;-Gal A is important in the regulation of the activity of &agr;-Gal A. In contrast, deletions of 12 or more amino acid residues resulted in a complete loss of enzyme activity.
A “carboxy-terminal” or “C-terminal” truncated &agr;-Gal A polypeptide preferably refers to a polypeptide that lacks about 12, preferably about 10, and more preferably about 8 peptidyl residues, but more than 1 residue, relative to the residues at the C-terminus of native or wild type &agr;-Gal A (SEQ ID NO:1). For example, preferred C-terminal truncated &agr;-Gal A polypeptides include polypeptides corresponding to SEQ ID NO:3 (“&Dgr;4”), SEQ ID NO:2 (“&Dgr;2”), SEQ ID NO:4 (“&Dgr;5”), SEQ ID NO:5 (“&Dgr;6”), SEQ ID NO:6 (“&Dgr;7”), SEQ ID NO:7 (“&Dgr;8”), SEQ ID NO:8 (“&Dgr;9”), and SEQ ID NO:9 (“&Dgr;10”). Preferably, the polypeptides of the invention are biologically active.
As used herein, a “biologically active” polypeptide means that the polypeptide has an activity that is similar to or greater than, e.g., about 1.5-fold, preferably 3-fold, more preferably 4-fold, and even more preferably 6-fold, the activity of wild type &agr;-Gal A. Methods to determine &agr;-Gal A activity are well known to the art, see, for example, Beutler et al.,
Am. J. Hum. Genet.,
24, 235 (1972) and Mayes et al.,
Clin. Chim. Acta,
112, 247 (1981).
An isolated “variant” of a C-terminal truncated &agr;-Gal A polypeptide is a polypeptide that has at least about 50%, preferably at least about 80%, and more preferably at least about 90%, but less than 100%, amino acid sequence homology or identity to the amino acid sequence of the corresponding wild type, preferably carboxy-terminal truncated, &agr;-Gal A polypeptide. Thus, a variant &agr;-Gal A polypeptide of the invention may include amino acid residues not present in the corresponding wild type &agr;-Gal A polypeptide, and may include internal and/or amino-terminal deletions, as well as C-terminal deletions, relative to the corresponding wild type polypeptide. Variants of the invention include polypeptides having at least one D-amino acid. Preferably, the variant polypeptides of the invention are biologically active.
&agr;-Gal A polypeptides and variants thereof which are subjected to chemical modifications, such as esterification, amidation, reduction, protection and the like, are referred to as “derivatives.”
Also provided is an isolated nucleic acid molecule which encodes an &agr;-Gal A polypeptide, or a variant thereof, which has a carboxy-terminal deletion. Preferably the isolated nucleic acid molecule further comprises a transcriptional control unit which is operably linked to the nucleic acid sequence so to provide an expression cassette.
Further provided is a therapeutic method which comprises administering to a mammal, e.g., a human, at risk of, or afflicted with, a condition associated with an &agr;-Gal A deficiency an effective amount of a carboxy-terminal truncated form of &agr;-Gal A or a variant thereof, which is biologically active. Preferably, the condition is Fabry disease. Also provided is a therapeutic method comprising administering to a mammal at risk of, or afflicted with, Fabry disease an effective amount of an isolated and purified nucleic acid molecule comprising a nucleic acid sequence encoding a carboxy-terminal truncated &agr;-Gal A polypeptide.
The invention also provides an isolated and purified nucleic acid molecule comprising an &agr;-Gal A gene having a stop codon at amino acid position 365 in &agr;- Gal A. As described below, such a nucleic acid molecule was isolated from a family with Fabry disease. The
Hutson Richard
Orphan Medical, Inc.
Prouty Rebecca E.
Schwegman Lundberg Woessner & Kluth P.A.
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