MXPA99002458A - THERAPY FOR&agr;-GALACTOSIDASE A DEFICIENCY - Google Patents

Abstract

A therapeutic method whereby an individual suspected of having an&agr;-galactosidase A deficiency, such as Fabry disease, is treated either with (1) human cells that have been genetically modified to overexpress and secrete human&agr;-gal A, or (2) purified human&agr;-gal A obtained from cultured, genetically modified human cells.

Description

THERAPY FOR -GALACTOSIDASE A DEFICIENCY BACKGROUND OF THE INVENTION This invention relates to α-galactosidase A and to treatment for a-galactosidase A deficiency. Fabry disease is a hereditary lysosomal storage disease linked to X characterized by symptoms such as severe kidney damage, angiokeratomas and abnormalities cardiovascular events, including ventricular enlargement and mitral valve insufficiency. The disease also affects the peripheral nervous system, causing episodes of burning, dying pain in the extremities. Fabry disease is caused by a deficiency in the enzyme a-galactosidase A (a-gal A), which results in a blockade of the catabolism of neutral glycosphingolipids and the accumulation of the substrate of the enzyme, ceramide trihexoside, within the cells and in the bloodstream. Due to the inheritance pattern linked to X of the disease, essentially all patients with Fabry disease are men. Although a few severely affected female heterozygotes have been observed, female heterozygotes are generally asymptomatic or have relatively moderate symptoms largely limited to a characteristic opacity of the cornea. An atypical variant of Fabry disease, exhibiting low residual a-gal A activity and still very mild symptoms and apparently without other characteristic symptoms in Fabry disease, correlates with left ventricular hypertrophy and heart disease (Nakano et al. others, New Engl. J. Med. 333: 288-293, 1995). It has been speculated that the reduction in a-gal A may be the cause of such cardiac abnormalities. The cDNA and the gene encoding human a-gal A have been isolated and sequenced (Bishop et al, Proc Nati, Acad Sci US 83: 4859, 1986, Kornreich et al, Nuc Acids Res. 3301, 1988; Oeltjen et al., Mammalian Genome 6: 335-338, 1995). Human a-gal is expressed as a polypeptide of 429 amino acids, of which 31 N-terminal amino acids constitute a signal peptide. The human enzyme has been expressed in Chinese Hamster Ovary (OHC) cells (Desnick, U.S. Patent No. 5,356,804, loannou et al., J. Cell Biol. 119: 1137, 1992); insect cells (Calhoun et al., U.S. Patent No. 5,179,023); and COS cells (Tsuji et al., Eur. J. Biochem. 165: 275, 1987). Pilot trials of a-gal A replacement therapies have been reported using proteins derived from human tissues (Mapes et al., Science 169: 987 1970; Brady et al., N. Engl. J. Med. 289: 9, 1973; Desnick et al., Proc. Nati, Acad. Sci. USA 76: 5326, 1979), but currently there is no effective treatment for Fabry disease. SUMMARY OF THE INVENTION It has been found that the expression of human α-gal A encoding DNA in cultured human cells produces a peptide that is appropriately glycosylated, so that it is not only enzymatically active but capable of acting on the glycosphingolipid substrate that is they accumulate in Fabry's disease, but they are also efficiently interned by the cells via the cell surface receptors that are directed exactly where they are needed in this disease: the lysosomal behavior of the affected cells, particularly the endothelial cells that cover the blood spleens of the patient. This discovery, which is discussed in more detail below, means that an individual suspected of having a deficiency of a-gal A, such as Fabry disease, can be treated with either (1) human cells that have been genetically modified to over-express and secrete human a-gal A, or (2) purified human a-gal A obtained from human, genetically modified, cultured cells. The therapy via the first route, i.e., with the cells modified by themselves, involves the genetic manipulation of human cells (e.g., primary cells, secondary cells, or immortalized cells) in vitro or ex vivo to induce them for express and secrete high levels of human a-gal A, followed by implanting the cells in the patient, as generally described in Selden et al., WO 93/09222 (incorporated herein by reference). When the cells are genetically modified, for the purposes of Fabry disease treatment, by gene therapy or enzyme replacement therapy, a DNA molecule containing an a-gal A cDNA or genomic DNA sequence, can obtained within an expression construct and introduced into primary or secondary human cells (e.g., fibroblasts, epithelial cells including mammary and intestinal epithelial cells, endothelial cells, elements formed from the blood including lymphocytes and cells from the bone marrow, cells of glia, hepatocytes, keratinocytes, muscle cells, neural cells or the precursors of these cell types) by normal methods of transfection including, but not limited to, liposome-mediated transfection, polybrennes, or DEAE dextran, eiectroporation, precipitation of calcium phosphate, microinjection or speed-driven microprojectiles ("biolistics").

Alternatively, a system that delivers DNA by viral vector could be used. Viruses known to be useful for gene transfer include adenoviruses, associated adenoviruses, herpes viruses, mumps virus, poliovirus, retroviruses, Sindbis virus and vaccinia viruses such as pox virus of canaries. Although primary and secondary cell cultures are preferred for the therapy methods of the invention, immortalized human cells can also be used. Examples of immortalized human cell lines useful in the present method include, but are not limited to, Bowes Melanoma cells (ATCC accession No. CCL 9607), Daudi cells (Accession of ATCC No. CCL 213), cells of HeLa and HeLa cell derivatives (Accessions of ATCC Nos. CCL 2, CCL 2.1 and CCL 2.2), cells of HL-60 (Access of ATCC No. CCL 240), cells of HT1080 (Access of ATCC No. CCL 121 ), cells Jurkat (ATCC Access No. TIB 152), KB carcinoma cells (Accession of ATCC No. CCL 17), leukemia cells of K-562 (ATCC Accession No. CCL 243), breast cancer cells MCF-7 (ATCC Access No. 1582 BTH 22), MOLT-4 cells (Access from ATCC No. 1582), Namalwa cells (ATCC Access No. CRL 1432), Raji cells (Accession of ATCC No. CCL 86), cells of RPMI 8226 (ATCC access No. CCL 155), U-937 cells (Accession of ATCC No. CRL 1593), cells of the 2R4 subline of Wl-38VA13 (Accession of ATCC No. CLL 75.1) and ovarian carcinoma cells 2780AD (Van der Blick et al., Cancer Res. 48: 5927-5932 , 1988) as well as heterohybridoma cells produced by fusion of human cells and cells of other species. Secondary human fibroblast strains, such as Wl-38 (ATCC Access No. CCL 75) and MRC-5 (ATCC Access No.

CCL 171). Following the genetic engineering of human cells with a DNA molecule encoding a-gal A (or following another appropriate genetic modification, as described below) to produce a cell that overexpresses and secretes α-gal A, a strain of clonal cells consisting essentially of a plurality of genetically identical identical primary human cells, or, where the cells are immortalized, a clonal cell line consisting essentially of a plurality of genetically identical immortalized human cells can be generated. Preferably, the cells of the clonal cell strain or the clonal cell line are fibroblasts. Genetically modified cells can be prepared and introduced into the patient by appropriate methods, e.g., as described in Selden et al., WO 93/09222. The gene therapy according to the invention has a number of advantages over enzyme replacement therapy with enzyme derived from human or animal tissues. For example, the method of the invention does not depend on the possibly inconsistent availability of appropriate tissue sources, and thus it is a commercially viable medium for treating a-gal A deficiency. Relatively there is no risk compared to the therapy of enzyme replacement with the enzyme derived from human tissues, from what it could be if it is infected with known or unknown viruses and other infectious agents. In addition, gene therapy according to the invention has a number of advantages over enzyme replacement therapy in general. For example, the method of the invention (1) provides the benefits of a long-term treatment strategy that eliminates the need for daily injections; (2) eliminates extreme fluctuations in serum and tissue concentrations of the therapeutic protein, which normally accompanies conventional drug delivery; and (3) it is probably less expensive than the enzyme replacement climb because the production and purification of the protein for frequent administration are unnecessary.

As described above, individuals with deficiencies of x-gal A can also be treated with purified a-gal A (ie, enzyme replacement therapy). Human primary, secondary or immortalized cells genetically engineered to overexpress human α-gal A will also be useful for the production of proteins in vitro, in order to produce protein that can be purified for enzyme replacement therapy. Secondary or immortalized human cells can be chosen from those described above and can be genetically modified by the transfection or transduction methods also described above. After genetic modification, the cells are cultured under conditions that allow the overexpression and secretion of α-gal A. The protein is isolated from the cultured cells by collecting the medium in which the cells develop and / or the cells are lysed for release its content and then apply normal protein purification techniques. One such technique involves passing the culture medium or any sample containing human α-gal A onto a hydrophobic interaction resin such as Butyl Sepharose® or another resin having a functional portion that includes a butyl group. Passing the sample on said resin may constitute the first step of chromatography. If further purification is required, the α-gal A containing material eluted from the hydrophobic interaction resin, can be passed over a column containing a second resin, such as an immobilized heparin resin such as Heparin Sepharose®, hydroxyapatite, a anion exchange resin such as Q Sepharose®, or a size exclusion resin such as Superdex® 200. Preferably, the purification protocol could include the use of each of the above types of resins. Alternatively, one of the last resins could be used before, or instead, of the hydrophobic interaction resin. The previous methods for the preparation of a-gal A with relatively high purity, depends on the use of affinity chromatography, using a combination of lectin affinity chromatography (concanavalin A (Con A) Sepharose) and affinity chromatography based on the binding of α-gal A to the analogous substrate N-6-aminohexanoyl-aD-galactosylamine coupled to a Sepharose matrix (Bishop et al., J. Biol. Chem. 256: 1307-1316, 1981). The use of lectin protein affinity resins and analogous resins of the substrate is usually associated with the continuous leaching of the solid support affinity agent (see Marikar et al., Anal. Biochem. 201: 306-310, 1992), as a result the contamination of the purified product with the affinity agent either free in solution or bound to eluted protein. Such contaminants render the product unsuitable for use in pharmaceutical preparations. The analogues of the bound substrate and the lectins can also have substantially negative effects on the enzymatic, functional and structural properties of the proteins. In addition, said affinity resins are usually of expensive preparation, making the use of such resins more suitable for production on a commercial scale than most conventional chromatography resins. Therefore, the development of a purification protocol using conventional chromatography resins, which are readily available with suppliers and have adequate quality for commercial use on a large scale, is a significant advantage of the present invention. An individual suspected of having a deficiency of a-gal A can be treated by the administration of purified human pharmaceutically acceptable α-gal A by any normal method, including but not limited to intravenous, subcutaneous or intramuscular injection, or as a solid implant The purified protein can be formulated into a therapeutic composition consisting of an aqueous solution containing a physiologically acceptable excipient, e.g., a carrier such as human serum albumin, at a pH of 6.5 or less. The present invention, therefore, provides a means to obtain large amounts of appropriately glycosylated human a-gal A, and therefore, therapeutically useful. This makes commercially viable enzyme replacement therapy for a-gal deficiency as well as relatively risk-free, compared to enzyme therapy derived from human or animal tissues.

Skilled artisans will recognize that the DNA sequence of human a-gal A (either cDNA or genomic DNA), or sequences that differ from it due to silent codon changes, or to the codon changes that produce amino acid substitutions Conservatives can be used to genetically modify cultured human cells so that they will overexpress and secrete the enzyme. It is also possible that certain mutations in the DNA sequence of a-gal A encode polypeptides that retain or exhibit improved a-gal A enzymatic activity (as would be evident by expressing the mutant DNA molecule in cultured cells, by purifying the encoded polypeptide and mediating catalytic activity, as described herein). For example, conservative amino acid substitutions could be expected to have little or no effect on biological activity, particularly if they represent less than 10% of the total number of residues in the protein. Conservative substitutions usually include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine, aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arge; and phenylalanine, tyrosine. The production of a-gal A by the cells can be maximized by certain genetic manipulations. For example, the DNA molecule encoding a-gal A can also encode a heterologous signal peptide, such as the human growth hormone (hGH) signal peptide, eptropoietin, HIV factor, Factor IX, glucagon, the receptor for Low density hpoprotein (LBD) or a hsosomal enzyme other than α-gal A Preferably, the signal peptide is the hGH signal peptide (SEQ ID NO 21) and is at the N-terminus of the encoded protein The DNA sequence encoding the signal peptide may contain an intron such as the first mtron of the hGH gene, resulting in a DNA sequence such as SEQ ID NO 27 (see also FIG. 10). In addition, the DNA molecule may also contain a sequence 3 'untranslated (UTS) that is at least 6 nucieotides in length (in contrast to the mRNA of a-gal A found in humans, which do not have UTS 3', the site of polyadenylation required being within the sequence coding sequence) The UTS are placed immediately at 3 'for the codon of termination of the coding sequence and include a polyadenylation site. They preferably have at least 6 nucleotides in length, more preferably at least 12 and even more preferably at least minus 30 and in all cases contain the AATAAA sequence or a related sequence which serves to promote polyadenylation. A DNA molecule as described is that it encodes a hGH signal peptide linked to a-gal A and that contains a UTS 3 'which includes a polyadenylation site and preferably includes an expression control sequence is also within the invention. Also within the scope of the invention is a DNA molecule that encodes a protein that includes the hGH signal peptide linked to a -gal A or any other heterologous polypeptide (i.e., any polypeptide other than hGH or an hGH analog). The heterologous polypeptide is usually a mammalian protein, e.g., any medically convenient human polypeptide. Other aspects and advantages of the invention will be apparent from the following detailed description and from the claims.

The term "genetically modified", as used herein with reference to cells, is understood to encompass cells that express a particular gene product following the DNA molecule encoding the product of genes and / or regulatory elements that control the expression of a coding sequence. The introduction of the DNA molecule can be achieved by gene targeting (ie, introduction of a DNA molecule to a particular genomic site); in addition homologous recombination allows the replacement of the defective gene itself (the defective a-gal A gene or a portion thereof could be replaced in the cells of a patient with Fabry disease with the entire gene or a portion thereof). The term "α-gal A", as used herein, means an α-gal A without a signal peptide, ie, SEQ ID NO: 26 (Fig. 9). There is some indication that residues 371 to 398 or 373 to 398 of SEQ ID NO: 26 (Fig. 9) can be removed in the lysosome; however, it is not thought that the removal of this putative propeptide affects the activity of the enzyme. This suggests that any portion of the putative propeptide could be suppressed without affecting the activity. Therefore, the term "α-gal A" as used herein, also covers a protein having a sequence corresponding to SEQ ID NO: 26 except that it lacks 28 residues at the C-terminus of that sequence. By "a-gal A deficiency" is meant any deficiency in the amount or activity of this enzyme in a patient. The deficiency can induce severe symptoms as is normally observed in men suffering from Fabry disease, or it can only be partial or induce relatively mild symptoms as can be observed in heterozygous female carriers of the defective gene. As used herein, the term "primary cell" includes cells present in a suspension of cells isolated from a vertebrate tissue source (before they are plated, i.e., bound to the tissue culture substrate. such as a plate or flask), cells present in a tissue-derived explant, both of the previous types of cells seeded for the first time, and suspensions of cells derived from these cells plated. "Secondary cells" refers to the cells in all subsequent steps of the culture. That is, the first time that a primary cell seeded in plates is removed from the culture substrate, it is replanted in plates (it is passed), it refers to a secondary cell, like all the cells in the subsequent passages. A "cell strain" consists of secondary cells, which have been passed one or more times; they exhibit a finite number of doublets of the average population in culture; exhibit anchor-dependent growth properties inhibited by contact (except for cells propagated in suspension culture); and they are not immortalized. By "immortalized cell" is meant a cell of an established cell line that exhibits a life extension apparently not limited in the culture. By "signal peptide" is meant a peptide sequence that is directed to a newly synthesized polypeptide to which the endoplasmic reticulum is attached for further post-translational processing and / or distribution. The term "heterologous signal peptide", as used herein in the context of α-gal A, means a signal peptide that is not the signal peptide of α-gal A (ie, which is encoded by the nucleotides 36-128 of SEQ ID NO: 18). It is usually the signal peptide of some mammalian protein other than α-gal A. The term "first step chromatography" refers to the first application of a sample to a chromatography column (all the steps associated with the preparation of the sample are excluded) Brief Description of the Drawings Figure 1 is a representation of the 210 bp probe that was used to isolate a-gal A from a fibroblast cDNA library (SEQ ID NO: 19). The sequence is from exon 7 of the a-gal A gene. The probe was isolated from human genomic DNA by the polymerase chain reaction (PCR). The regions underlined in the figure correspond to the sequences of the amplification primers. Figure 2 is a representation of the sequence of the DNA fragment that completes the 5 'end of a-gal A cDNA (SEQ ID NO: 20). This fragment was amplified from human genomic DNA by PCR. The underlined regions correspond to the sequences of the amplification primers. The positions of the restriction endonuclease sites Ncol and Sacll, which are used to subclone as described in Example IA, are also shown. Figure 3 is a representation of the a-gal A cDNA sequence, including the sequence encoding the signal peptide (SEQ ID NO: 18). Figure 4 is a schematic map of pXAG-16, an a-gal A expression construct that includes the CMV (cytomegalovirus) and intron promoter, the sequence encoding the hGH signal peptide and the first intron, the CDNA for a-gal A (ie lacking the signal peptide sequence of α-gal A) and the UTS of hGH 3 'Figure 5 is a schematic map of pXAG-28, an expression construct of α-gal A which includes the collagen Ia2 promoter, a β-actin intron, the hGH signal peptide encoding the sequence and the first intron, the cDNA for α-gal A (ie lacking the sequence of the α-gal signal peptide A) and the 3 'UTS of hGH. Figure 6 is a chromatogram of the purification step of a-gal A using the Butyl Sepharose® resin. The absorbance at 280 nm (whole line) and the α-gal A activity (dotted line) of the selected fractions are shown. Figure 7 is a line graph describing the internalization of Fabry fibroblasts of human a-gal A prepared according to the invention. The intracellular α-gal A activity and the total protein concentration were measured after incubating the cells with increasing concentrations of human α-gal A prepared according to the invention. The effects of the internalization inhibitor potential of mannose-6-phosphate (M6P; open diamonds) and tomorrow (open circles). Figure 8 is a schematic diagram of the experimental paradigm designed to examine Fabry fibroblasts after internalization of a-gal A. The a-gal A activity of Fabry cells was measured after exposure to normal human fibroblasts o overexpressing a-gal A cultured in Transwe? TM 'M6P' inserts = mannose-6 phosphate; "Notrf HF" = untransfected human fibroblasts; "BRS11" = a strain of fibroblasts overexpressing transfected a-gal A. 9, is a representation of the amino acid sequence of human a-gal A (SEQ ID NO: 26). Figure 10 is a representation of the DNA sequence encoding the hGH signal peptide and containing the first intron of hGH (underlined) (SEQ ID NO: 27) Figure 11 is a representation of the DNA sequence encoding the hGH signal peptide without the intron (SEQ ID NO: 22) Figure 12 is a representation of the amino acid sequence of the signal peptide of hGH (SEQ ID NO: 21) Figure 13 is a representation of the cDNA sequence encoding human α-gal A (without the signal peptide) (SEQ ID NO: 25). Detailed Description Lysosomal enzymes such as a-gal A are directed to the lysosomal compartment of a cell through interaction with the mannose-6-phosphate receptor (M6P), which binds to M6P residues present in the oligosaccharide moieties of the enzymes destined for the lysosomal compartment (Kornfeld, S. and Mellman, I. Ann. Rev. Cell Biol. 5: 483-525, 1989). The primary interaction occurs in the Golgi apparatus, where the enzymes bound to the Golgi M6P receptors are secreted by the transport of lysosomes. A secondary type of interaction is ght to take place between the extracellular a-gal and M6P receptors on the surface of the cells. The extracellular substances internalized by the cells are transported through the cytoplasm into endocytic vesicles, which fuse with primary lysosomes and empty their contents into the iisosomes. In this process, M6P receptors on the cell surface are also incorporated into the endocytic vesicles and transported to the lysosomes. Any α-gal A present in the extracellular part, if it has M6P residues, can bind to the M6P receptors on the cell surface and thus be transported in the lysosomal compartment together with the receptors. Once in the lysosomal compartment, as a result of this eliminator route, the enzyme can carry out its proper function. Therefore, even if a cell is genetically deficient to produce its own agal A, there is a mechanism for it to absorb the exogenously produced enzyme, as long as (a) the enzyme is glycosylated properly and (b) the cell deficient have M6P receivers.

In Fabry disease, endothelial cells of the kidney and heart have been shown to exhibit severe histopaogical abnormalities and contribute to the clinical paogy of the disease; these cells, which carry the M6P receptors, are a particular target of the invention currently claimed. The α-gal A produced according to the invention can be delivered either locally or systemically to cells affected by gene therapy (ie, by genetically modified cells that express and secrete the glycosylated enzyme within the patient), or by routes conventional pharmacological administration. An α-gal A in which M6P is present in the N-linked oligosaccharides is therefore of greater importance for the therapy according to the invention. In addition, the degree to which the N-linked oligosaccharides of α-gal A are modified by sialylation is also of great importance. In the absence of appropriate sialylation, a-gal A will be rapidly removed from the circulation due to binding by hepatic asialoglycoprotein receptors, followed by internalization and degradation by hepatocytes (Ashwell and Harford, Ann.Rev. Biochem. 51: 531-554 , 1982). This decreases the amount of a-gal A available in the circulation for binding to M6P receptors on cells that contribute to the clinical paogy of Fabry disease, such as vascular endothelial cells of the kidney and heart. Surprisingly, applications have found that α-gal A secreted by transfected human cells has glycosylation properties that are suitable for the treatment of Fabry disease by gene therapy or by conventional pharmaceutical administration of the purified secreted protein. This is in contrast to the situation with the best-studied lysosomal enzyme, glucocerebrosidase, in which delivery of purified enzyme from human placenta or secreted from CHO cells transfected to clinically relevant cells in the body, requires complex enzymatic modification of the enzyme after purification (see Beutler, New Engl. J. Med. 325: 1354-1360, 1991). The therapy of the invention can be carried out in two general ways: by introducing into the patient a therapeutically effective amount of purified human α-gal A from cultured human cells genetically modified to overexpress and secrete the enzyme, or by introducing the cell itself Overexpression in the patient. The techniques to achieve the necessary genetic modifications are discussed below, as well as the mes of purification, formulation and treatment. Example. Preparation and Use of Constructs Designed to Supply and Express a-gal A Two expression plasmids, pXAG-16 and pXAG-28, were constructed. These plasmids contain human a-gal A cDNA encoding 398 amino acids of the enzyme a-gal A (wit the signal peptide of α-gal A); the genomic DNA sequence of human growth hormone (hHG), which is interrupted by the first intron of the hGH gene; and the 3 'untranslated sequence (UTS) of the hGH gene, which contains a signal for polyadenylation. The plasmid pXAG-16 has the immediate early promoter of human cytomegalovirus (CMV IE) and the first intron (flanked by exon sequences without coding), while pXAG-28 is driven by the collagen Ia2 promoter and also contains UTS 5 'of the ß-actin gene, which contains the first ß-actin nucleon.

A. Cloning of the complete α-gal A cDNA and Construction of α-gal A Expression Plasmid pXAG-16 The human a-gal cDNA was cloned from a fibroblast cDNA library that was constructed in the following manner. Poly-A + mRNA was isolated from total RNA and cDNA synthesis was carried out using reagents for the Zapll® system according to the manufacturer's instructions (Stratagene Ine, La Jolla, CA). Briefly, the "first strand" DNA was generated by reverse transcription in the presence of an oligo-dT primer containing an internal Xhol restriction endonuclease site. After treatment with RNase H, the cDNA was translated by insertion with DNA I polymerase to generate double stranded cDNA. This cDNA was blunt-ended with T4 DNA polymerase and ligated to EcoRl adapters. The products of this ligation were treated with T4 DNA kinase and were digested by Xhol. The cDNA was fractionated by Sephacryl ~ 400X chromatography. The large and medium size fractions were combined and the cDNAs were ligated to EcoRl and Zapll Lambda limbs digested with XhoI. The products of this ligature were packed and titled. The primary bank had a titre of 1.2 x 107 pfu / ml and an average insert size of 925 bp. A 210 bp probe of exon 7 of the human a-gal A gene (Figure 1, SEQ ID NO: 19) was used to isolate the cDNA. The probe itself was isolated from the genomic DNA by the polymerase chain reaction (PCR) using the following oligonucleotides: 5'-CTGGGCTGTAGCTATGATAAAC-3 '(Oligo 1, SEQ ID NO 1) and 5'-TCTAGCTGAAGCAAAACAGTG-3' (Oligo 2 , SEQ ID NO 2) The PCR product was then used to screen the cDNA library of the fibroblasts and the positive clones were isolated and further characterized, a positive clone, phage 3A, was subjected to the excision protocol of the Zapll® system (Stratagene, Ine, La Jolla, CA), according to the manufacturer's instructions This procedure yielded the plasmid pBSAG3A, which contains the a-gal A cDNA sequence in the base structure of the plasmid pBIuescpptSK ™ DNA sequencing revealed that this plasmid does not contain the complete 5 'end of the cDNA sequence Therefore, the 5' end was reconstructed using an amplified PCR fragment of human genomic DNA To achieve this, a genomic DNA fragment (Figure 2, SEQ ID NO 20) s and amplified using the following oligonucleotides 5'-ATTGGTCCCGCCCCTGAGGT-3 ' (Ohgo 3, SEQ ID NO 3) and S'-TGATGCAGGAATCTGGCTCT-S '(Oligo 4, SEQ ID NO 4) This fragment was subcloned into a "TA" cloning plasmid (Invitrogen Corp, San Diego CA) to generate the plasmid pTAAGEl Plasmid pBSAG3A, which contains the majority of the cDNA sequence of a-gal A and pTTAGEI, which contains a 5 end of the a-gal A cDNA, were each digested with Sacll and Ncol. Relevant Sacll and Ncol sites within the amplified DNA fragment are shown in Figure 2 The 02 kb Sacll-Ncol fragment of PTTAGEl was isolated and ligated to equivalently digested pBSAG3A This plasmid, pAGAL, contains the cDNA sequence of a- A complete gal including the sequence encoding the a-gal A signal peptide. The cDNA was completely sequenced (shown in Figure 3 including the signal peptide of α-gal A; SEQ ID NO: 18) and found to be is identical to the sequence published for the human a-gal A cDNA (Genbank sequence HUMGALA). Plasmid pXAG-16 was constructed via several intermediates, as follows. First, pAGAL was digested with Sacll and Xhol and made blunt end. Second, the complete a-gal A cDNA ends were ligated to the Xbal links and subcloned into digested pEF-BOS from Xbal (Mizushima et al., Nucí Acids Res. 18: 5322, 1990) creating pXAG-1. This construct contains the stimulation factor of granulocyte (G-CSF) 3 'UTS colonies and the human elongation factor 1a promoter (EF-1a) flanking the cDNA encoding a-gal A plus the signal peptide of a- gal A, so that the 5 'end of the α-gal A cDNA is fused with the EF-1a promoter. To create a construct with the CMV IE promoter and the first intron, the a-gal A cDNA and the 3 'UTS of G-CSF were removed from pXAG-1 as a two kb Xbal-BamHI fragment. The fragment had a blunt end, was ligated to the BamHI linkages and inserted into digested BamHl from pCMVfINeo (which was constructed as described below). The orientation was such that the 5 'end of the α-gal A cDNA was fused to the CMV IE promoter region.

PCMVfINeo was created from the following: A fragment of CMV IE gene promoter was amplified by PCR using CMV genomic DNA as an primer and the oligonucleotides: 5'-TTTTGGATCCCTCGAGGACATTGATTATTGATAG-3 '(SEQ ID NO: 23) and d' -TTTTGGATCCCGTGTCAAGGACGGTGAC-S '(SEQ ID NO: 24). The resulting product (a 1.6 kb fragment) was digested with BamHI, giving a fragment containing the CMV promoter with digested BamHl cohesive ends. The new expression unit was isolated from the pMCIneopA plasmid (Stratagene Ine, La Jolla, CA) as a 1.1 kb Xhol-BamHI fragment. Fragments containing the CMV promoter and new were inserted into a plasmid digested with BamHI, XhoI (pUC12). Notably pCMVMpNeo contains the CMV IE promoter region, starting at nucleotide 546 and ending at nucleotide 2105 (Genbank sequence HS5MIEP) and the neomycin resistance gene driven by the herpes simplex virus thymidine kinase promoter (HSV) , for its acronym in English) (the TKneo gene) immediately at 5 'for the CMV IE promoter fragment. The transcription direction of the new gene is the same as that for the CMV promoter fragment. This construction of intermediaries was called pXAG-4. To add 3 'UTS of hGH, the 3' UTS of GCSF was removed from pXAG-4 as a Xbal-Smal fragment and the ends of pXAG-4 were blunt-formed. The 3 'UTS of hGH was removed from pXGH5 (Selden et al., Mol Cellular Biol. 6: 3173-3179, 1986) as a 0.6 kb fragment. After finishing this fragment, it was ligated into pXAG-4 immediately after the blunt-ended Xbal site of pXAG-4. This intermediary was called pXAG-7. The TKneo fragment was removed from this plasmid as a fragment of Hí nd! I! -Cla 1 and the plasmid ends were enromoted to "fill" with the Klenow fragment of DNA polymerase I. A neomycin resistance gene driven by the SV40 early promoter was ligated as a Clal-BsmBI fragment from a PCDNeo digestion (Chen et al., Mol Cellular Biol. 7: 2745-2752, 1987), placing the new transcript in the same orientation as the a-gal A transcription unit. This intermediate was called pXAG-13. To complete pXAG-16, which has the 26 amino acid hGH signal peptide encoding the sequence and the first intron of the hGH gene, a 2.0 kb EcoR1-BamHI fragment of pXAG-13 was first removed. This fragment included a-gal A cDNA and the 3 'UTS of hGH. This large fragment was replaced with three fragments. The first fragment consisted of a 0.3 kb PCR product from pXGH5, which contains the hGH signal peptide coding sequence and includes the first hGH introns sequence from a synthetic BamHl site located just upstream of the sequence consensual of Kozak to the end of the hGH signal peptide coding sequence. The following oligonucleotides were used to amplify this fragment (Fragment 1): 5'-TTTTGGATCCACCATGGCTA-3 '(Oligo HGH101; SEQ ID NO: 5) and 5'-TTTTGCCGGCACTGCCCTCTTGAA-3' (Oligo HGH102- SEQ ID NO: 6). The second fragment consisted of a 0.27 kb PCR product containing sequences corresponding to the start of the cDNA encoding the a-gal A enzyme of 398 amino acids (ie lacking the signal peptide a-gal A) to the Nhel site . The following oligonucleotides were used to amplify this fragment (Fragment 2): 5'- TTTTCAGCTGGACAATGGATTGGC-3 '(Oligo AG10; SEQ ID NO: 7) and d'-TTTTGCTAGCTGGCGAATCC-S' (Oligo AG11; SEQ ID NO: 8). The third segment consisted of the Nhel-EcoRI fragment of pXAG-7 containing the remaining a-gal A sequence as well as the 3 'UTS of hGH (Fragment 3). Fragment 1 (digested with BamHl and Nael), Fragment 2 (digested with Pvull and Nhel) and Fragment 3 were mixed in the 6.5 kb BamHI-EcoRI fragment of pXAG-13 containing the new gene and the CMV IE promoter and ligated to generate the plasmid pXAG-16 (Figure 4) . B. Construction of α-gal A Expression Plasmid pXAG-28 The collagen Ia2 promoter was isolated for use in the construction of pXAG-28 expression of α-gal A in the following manner. A 480 bp PCR fragment of human genomic DNA containing part of the human collagen Ia2 promoter was isolated using the following oligonucleotides: 5'- TTTTGGATCCGTGTCCCATAGTGTTTCCAA-3 '(Oligo 72; SEQ ID NO: 9) and d'-TTTTGGATCCGCAGTCGTGGCCAGTACC- S '(Oligo 73; SEQ ID NOX0).

This fragment was used to screen a human leukocyte bank in EMBL3 (Clontech Ine, Palo Alto, CA). A positive clone (phage 7H) containing an EcoRI fragment of 3.8 Kb was isolated and cloned into pBSIISK + (Stratagene Ine, La Jolla, CA) at the EcoRI site (creating pBS / 7H.2). an Avrll site was introduced into pBSIISK + by digesting with Spel, which is separated within the pBSIISK + polylinker, "filling" with the Klenow fragment of DNA polymerase I by inserting the oligonucleotide 5'-CTAGTCCTAGG-3 '(SEQ ID NO: 11 ). This variant of pBSIISK + was digested with BamHl and Avrll and ligated to the 121 bp BamHI-Avrll fragment of the original 408 bp collagen Ia2 promoter RCP fragment described above, creating PBS / 121COL.6. Plasmid pBS / 121COL.6 was digested with Xbal, which is separated within the pBSIISK + polyslinker sequence, "filled" with the Klenow fragment of DNA polymerase I, and digested with Avrll. The 3.8 kb BamHI-AvrlI fragment of pBS / 7H.2 was isolated and the BamH1 site was blunt-ended by treatment with the Klenow enzyme. The fragment was then run with Avrll and ligated to the digested vector of Avrll, thus creating plasmid pBS / 121 pbCOL7H.18 of collagen promoter. Then, the collagen promoter was fused to the 5 'UTS of the human β-actin gene, which contains the first intron of the human β-actin gene. To isolate this sequence, a 2 kb PCR fragment of human genomic DNA was isolated using the following oligonucleotides: 5'-TTTTGAGCACAGAGCCTCGCCT-3 '(Oiigo BA1; SEQ ID NO: 12) and 5'-TTTTGGATCCGGTGAGCTGCGAGAATAGCC-3 '(Oligo BA1; SEQ ID NO: 13). This fragment was digested with BamHl and BsiHKAI to release a 0.8 kb fragment containing the 5 'UTS of β-actin and the intron. A fragment of Sal1-Srfl of 3.6 kb was isolated after the collagen promoter plasmid pBS / 121 pbCOL7H.18 in the following manner: pBS / 121 bpCOL7H.18 was partially digested with BamHl (the BamHl site lies at the extreme 'of the collagen Ia2 promoter fragment), made with the blunt end by the Klenow treatment and ligated to a SalI linker (5'-GGTCGACC-3'), thus placing a Sali site upstream of the Ia2 promoter of collagen. This plasmid was then digested with Sali and Srfl (the Srfl site is 110 bp upstream of the CAP site of the collagen Ia2 promoter) and the 3.6 kb fragment was isolated. Fragments of 0.8 and 3.6 kb were combined with pSIISK digested with SaM and BamHl (Stratagene Ine, La Jolla, CA) and a fragment composed of the following four oligonucleotides was annealed (forming the fragment with a blunt end and one end of BsiHKAI) : 5'-GGGCCCCCAGCCCCAGCCCTCCCATTGGTGGAGGCCCTTTTGGA- GGCACCCTAGGGCCAGGAAACTTTTGCCGTAT-3 '(Oligo COL-1; SEQ ID NOX4), 5'-AAATAGGGCAGATCCGGGCTTTATTATTTTAGCA CCACGGCCGCCGAGACCGCGTCCGCCCCGCGAGCA-3 '(Oligo COL-2; SEQ ID NOX5), 5'-GCCC TGCCCTATTTATACGGCAAAAGTTTCCTGGCCCT AGGGTGCCTCCAAAAGGGCTCCACCAATGGGAGGGCTGGGGCTGGG-3' (Oligo COL-3; SEQ ID NO: 16) and 5'- CGCGGGGCGGACGCGGTCTCGGCGGCCGTGGTGCTAAAATAATAAAG CCCGGATC-3 '(Oligo COL- 4; SEQ ID NO: 17). These four oligonucleotides, when annealed, corresponded to the region that starts at the Srfl site of collagen promoter and continue through the BsiHKAI site of the β-actin promoter. The resulting plasmid was designated pCOL / β-actin. To complete the construction of pXAG-28, the fragment of Sal1-BamHI from pCOL / β-actin, which contains the Ia2 promoter of collagen and 5 'UTS of β-actin, was isolated, this fragment was ligated to two fragments of pXAG-16 (see Example 1A and Figure 4): ( 1) the 6.0 kb BamHl fragment (containing the new gene, structure of the plasmid base, and the cDNA encoding the a-gal A enzyme 398 amino acids, and the 3 'UTS of hGH); and (2) the 0.3 kb BamHI-XhoI fragment (which contains the SV40 poly A sequence of pcDneo). PXAG-28 Contains the human collagen Ia2 promoter fused to the 5 'UTS of human β-actin, the hGH signal peptide (which was interrupted by the first intron of hGH). The cDNA encoding the a-gal A enzyme and the 3 'UTS of hGH. A map of the complete expression construction pXAG-28 is shown in Figure . C. Transfection and Selection of Electrophored Fibroblasts with the α-gal A Expression Plasmids In order to express α-gal A in fibroblasts, secondary fibroblasts were cultured and transfected according to published procedures (Selden et al., WO 93/09222). Plasmids pXAG-13, pXAG-16 and pXAG-28 were transfected by electroporation into human anterior skin fibroblasts to generate strains of stably transfected cloned cells and the resulting a-gal A expression levels were monitored as described in Example ID. The secretion of a-gal A by normal anterior skin fibroblasts is in the range of 2-10 units / 106 cells / 24 hours. In contrast, the transfected fibroblasts exhibit medium expression levels as shown in Table 1: Table 1 Mean expression levels of a-gal A (+ / - normal deviation) pXAG-13: 420 +/- 344 U / 106 cells / day N = 26 clonal strains (scale 3 - 1133 U / 106 cells / day) ? XAG-16: 2.051 +/- 1253 U / 106 cells / day N = 24 clonal strains (scale 422 - 5200 U / 106 cells / day) pXAG-28: 141 +/- 131 U / 106 cells / day N = 38 clonal strains (scale 20 - 616 U / 106 cells / day) These data show that the three expression constructs are capable of increasing the expression of α-gal A many times than non-transfected fibroblasts. The expression of fibroblasts stably transfected with pXAG-13, which codes for a-gal A bound to the signal peptide of a-gal A, was substantially lower than expression by fibroblasts transfected with pXAG-16, which differs only in that the peptide of the signal is the peptide of the hGH signal, the coding sequence of which is interrupted by the first intron of the hGH gene. Each time the transfected cells were passaged, the secreted a-gal A activity was determined, the cells were counted and the cell density was calculated. Based on the number of cells cultured and the time allowed for the secretion of α-gal A, the specific expression regimen of α-gal A was determined as reported in Tables 2 and 3 as secreted units (from α-gal A ) by 106 cells over a period of 24 hours. Strains of cells desirable for gene therapy or for use in the generation of material for purification of a-gal A should exhibit growth and stable expression for several steps. The data of the cell strains shown in Tables 2 and 3, which were stably transfected with the expression construct of α-gal A pXAG-16, illustrate the fact that the expression of α-gal A is stably maintained during the step in series.

Table 2: Growth and Expression of BRS-11 Cells Containing the Expression Construction of a-gal A pXAG-16 Table 3: Growth and Expression of HF503-242 Cells Containing Construction of Expression a-Gal A pXAG-16 Quantification of a-gal A Expression Activity activity of a-gal A was measured using the water-soluble 4-methylumbelliferyl-aD-galactopyranoside substrate (4-MUF-gal, Research Products, Inc.) by a modification of the protocol described by loannou et al. (J. Cell Biol. 119: 1137-1150, 1992). The substrate was dissolved in substrate regulating solution (0.1 M citrate-phosphate pH 4.6) at a concentration of 1.69 mg / ml (5 mM). Typically, 10 μl of culture supernatant was added to 75 μl of the substrate solution. The tubes were covered and allowed to incubate in a 37 ° C water bath for 60 minutes. At the end of the incubation period, 2 ml of a glycine buffer (130 mM glycine, 83 mM sodium carbonate at pH 10.6) was used to stop the reaction. The relative fluorescence of each sample was measured using a fluorometer model TK0100 (Hoefer Scientific Instruments) which has a fixed excitation wavelength of 365 nm and detects a fixed emission wavelength of 460 nm. The readings of the samples were compared with normal prepared from a 1μM stock solution of methylumbelliferone (Sigma Chemical Co.), and the amount of hydrolysed substrate was calculated. The activity of a-gal A was expressed in units; a unit of activity of a-gal A is equivalent to a nanomole of hydrolyzed substrate per hour at 37 ° C. Cell expression data were generally expressed as units of secreted a-gal A activity / 10 6 cells / 24 hours. This analysis was also used to measure the amount of α-gal A in cell lysates and in samples of several a-gal purification steps, as discussed above Example II. Purification of a-gal A from Conditioned Medium from Strains of Stably Transfected Human Cells Examples IIA-IIE illustrate that a-gal A can be purified to near homogeneity from the conditioned medium of strains of cultured human cells that have been stably transfected to produce the enzyme. A. Use of Butyl Sepharose® Chromatography as a primer Step in the Purification of a-gal A The cold conditioned medium (1.34 liters) was clarified by centrifugation and filtered through a 0.45 μm cellulose acetate filter using glass fiber prefilters. While stirring, the pH of the cold filtered medium was adjusted to 5.6 by the dropwise addition of 1 N HCl and ammonium sulfate was added to a final concentration of 0.66 M by the dropwise addition of a stock solution (room temperature). ) of 3.9 M ultrapure ammonium sulfate. The medium was stirred for an additional 5 minutes at 4 ° C, filtered as above and applied to a Butyl Sepharose® Rapid Flow column (column volume 81 ml, 2.5 x 16.5 cm). Pharmacia, Uppsala, Sweden) which was equilibrated in 10 mM MES-Tris, pH 5.6, containing 0.66 M ammonium sulfate (buffer A). Chromatography was carried out at 4 ° C in a Gradi-Frac ™ system (Pharmacia, Uppsaia, Sweden) equipped with in-line UV (280 nM) and conductivity monitors to evaluate total protein and salt concentration, respectively, after application of the sample at a flow rate of 10 ml / min, the column was washed with 10 column volumes of buffer solution A. The a-gal A was eluted from the column Butyl Sepharose® with gradient line! of volume of column 14 of a buffer solution A * (containing ammonium sulfate) at 10 mM MES-Tris, pH 5.6 (without ammonium sulfate.) The fractions were analyzed for a-gal A activity by the analysis of 4- MUF-gal and those containing the appreciable enzyme activity were combined.As seen in Figure 6 and the purification summary (Table 3), this step removes approximately 99% of the contamination protein (pre-column sample). = 8.14 g of total protein, sample of posterior to coiumna = 0.0638 g of total protein).

Table 4: Purification of a-Gal A from Conditioned Medium of Human Fibroblasts Stably Transfected B. Use of Heparin Chromatography of Sepharose® as a Purification Step of a-gal A The peak fractions of the Sepharose® Butyl Column were dialyzed at 4 ° C against (4 liters) of 10 mM MES-Tris, pH 5.6 (changed once). The conductivity of the diaiisate was adjusted to 1.0 mMHO at 4 ° C by the addition of H20 or NaCl as necessary. Then, the sample was amplified to a Sepharose® Heparin fast-flow column (Pharmacia, Uppsala, Sweden, 29 ml column volume, 2.5 x 6 cm) which was equilibrated in 10 mM MES-Tris, pH 5.6, containing 9 mM NaCl (buffer B). This was done at 4 ° C at a flow rate of 10 ml / min. Online UV (280 nm) and conductivity monitors measured total protein and salt concentration. After the sample was applied, the column was washed with 10 volumes of column columns of buffer B followed by a linear gradient of 3 column volumes at 8% buffer solution C / 92% buffer B (when the buffer C is 10 mM MES-Tris, pH 5.6, containing 250 mM NaCl) and a 10-volume wash of a column with 8% Regulatory solution C. This was followed by elution of a-gal A with a linear gradient of 1.5 volumes of a column at 29% of buffer C and a linear gradient 10 volumes of the column subsequent to buffer C of 35%. The fractions were analyzed for a-gal A activity and those containing the appreciable activity were combined. C. Use of Hydroxyapatite Chromatography as a Step for For Purification of a-gal A Heparin was filtered and applied directly to a column of Ceramic Hydroxyapatite HC (40 μm, American International Chemical, Natick, MA, 12 column volumes, 1.5 x 6.8 cm) equilibrated in 1 mM sodium phosphate, pH 6.0 (buffer D). Chromatography was carried out at room temperature in a Gradi-Frac ™ / FPLC® Hybrid System (Pharmacia, Uppsala, Sweden) equipped with in-line UV monitors (280 nm) and conductivity. After it was applied to the sample (5 ml / min), the column was washed with 10 column volumes of D. buffer. The a-gal A was eluted with a linear gradient of 7 column volumes at 42% buffer solution E / 58% buffer solution D (where the solution regulator E is 250 mM sodium phosphate, pH 6.0) followed by a gradient of 10 column volumes at 52% buffer E. The fractions were analyzed for a-gal A activity and the fractions containing appreciable activity were combined . D. Use of Q Sepharose® Anion Exchange Chromatography as a Step for Purification of α-gal A The hydroxyapatite combination was diluted approximately 1.5 times with H2O at a final conductivity of 3.4-3.6 mMHO at room temperature. After filtering, the sample was applied to a column of Q Sepharose® HP (Pharmacia, Uppsala, Sweden; column volume 5.1 ml, 1.5 x 2.9 cm) balanced in 10% buffer G / 90% buffer F, when buffer F is 25 M sodium phosphate, pH 6.0 and buffer G is 25 mM sodium phosphate, pH 6.0, 250 mM NaCl. Chromatography was carried out at room temperature in the Gradi-Frac ™ System / FPLC15 Hybrid (Pharmacia, Uppsala, Sweden) and the total protein and salt concentrations were monitored by the monitors online. The sample was applied at a flow rate of 5 ml / min, then the following steps were carried out: (1) a wash with 5 column volumes at 10% buffer G, (2) a wash with 7 volumes column at 12% buffer G, (3) a linear gradient with 3 column volumes at 50% buffer G, (4) a linear gradient with 10 column volumes at 53% buffer G, (5) ) a gradient of 3 column volumes at 100% buffer G, and (6) a wash with 10 column volumes at 100% buffer G. a-gal A was eluted mainly during steps 3 and 4. fractions containing appreciable activity were combined (the "Q combination"). E. Use of SuperdexJ Gel Filtration Chromatography as a Step for the Purification of a-gal A The combination Q was concentrated to approximately 5 times using Centriprep®-10 Centrifuge Concentrator units (Amicon, Beverly, MA), and applied a column of Superdex® 200 (Pharmacia, Uppsala, Sweden, column volume 189 ml, 1.6 x 94 cm). The column was equilibrated and eluted with 25 mM sodium phosphate, pH 6.0, containing 150 mM NaCl. Chromatography was carried out in a FOLC® system (Pharmacia, Uppsala, Sweden) at room temperature using the in-line UV monitor (280 nm) to follow the elution of the protein. The volume of the sample applied to the column was 2 ml, the flow rate was 0.5 ml / min and the fraction size was 2 ml. Multiple column operations were carried out; the fractions were analyzed for a-gal A activity and the fractions containing appreciable activity were combined. The combined fractions of the Superdex® 200 column were concentrated using Centriprep-10 units, aliquots were taken, frozen under pressure and stored at -80 ° C for short times. A summary of this example of purification of a-gal A is shown in Table 3. The final yield of a-gal A was 59% of the activity of the starting material and the specific activity of the purified product was 2.92 x 106 units / mg of protein. The resulting product showed a higher level of purity after electrophoresis under reduced conditions on an SDS-prolylacrylamide gel 4-15% which subsequently was stained with silver. Example III Formulation and Storage of highly purified a-gal A purified a-gal is not stable for extended periods when it is shown as a diluted solution of purified protein (< mg protein / ml). Therefore, a formulation was provided to improve stability during prolonged storage, i.e., storage that takes from several weeks to at least several months. The purified enzyme was concentrated to at least 1 mg / ml using a centrifuge concentrator (in enzyme buffer containing 25 mM sodium phosphate (pH 6. 0) and 150 mM NaCl). Human serum albumin was added (HSA; BuminateX Baxter-Hyland) to the final concentration of 2.5 mg / ml. The protein solution was filtered with sterilization using a 0.2 μm cellulose acetate filter (Schleicher and Schuell) attached to a syringe. A solution of a-gal A was dispensed in sterile, pyrogen-free glass vials sealed with a cap of Teflon, frozen under pressure and stored at -20 ° C. The stability of the activity of a-gal A was evaluated over a period of three months using the 4-MUF-gal analysis. The data presented in Table 5 demonstrate that there was no loss of enzyme activity during the test period. The acidic pH of the formulation (< 6.5) is critical for the stability of the highly purified enzyme.

Table 5: Stability of a-Gal A Formulated at -20 ° C Example IV. The a-gal A Produced by Human Cell Strains is Suitable for the Treatment of a-gal A Deficiency The structural and functional properties of purified human a-gal A prepared according to the invention, has been investigated in order to demonstrate that the DNA molecules described herein and the corresponding expressed glycoproteins produced by strains of transfected human cells can be used in gene or enzyme replacement therapies, respectively. A. Size of α-gal A Produced by Human Cells Stable Transfected in Culture The molecular mass of a-gal A was calculated by MALDI-TOF mass spectrometry. These results show that the molecular mass of the dimer is 102.353 Da, while that of the monomer is 51.002 Da. The expected molecular mass of the monomer, based on the amino acid composition, is 45,400 Da. Therefore, it can be inferred that the carbohydrate content of the enzyme accounts for up to 5,600 Da of molecular weight. The results of normal amino acid analysis performed on the purified protein are consistent with the conclusion that the protein produced by transfected human cells is identical to the purified protein of human tissues at the amino acid level. B. N-Terminal Processing of α-gal A Produced by Stably Transfected Human Cells The nucleotide sequence of human a-gal A cDNA encodes 429 amino acids. The N-terminal 31 amino acids constitute a signal peptide sequence, which is separated as the nascent protein transits the endoplasmic reticulum membrane (LeDonne et al., Arch. Biochem. Biophys., 224: 186, 1983; Lemansky et al. J. Biol. Chem. 262: 2062, 1987). In order to confirm that α-gal A is properly processed when associated with a heterologous signal peptide sequence (e.g., the human growth hormone signal sequence) and expressed in transfected human fibroblasts, ten microsequences were sequenced N-terminal amino acids of the secreted protein. Samples were electrophoresed by SDS-PAGE and transferred to ProBlotX (ABI, Foster City, CA) using 10 mM CPAS (pH 11.0), 10% methanol regulator system. The protein in ProBlott® was visualized by Coomassie tinsion and an approximate size band (50 kDa) was excised. The N-terminal sequence was obtained using an impulse phase amino acid sequencer from an Applied Biosystems liquid that performs automatic Edman degradation. The obtained N-terminal sequence, LDNGLARTPT (SEQ ID NO: 28), agrees with the appropriate separation of the signal peptide and is matched to the N-terminal sequence predicted by the secreted protein. C. C-Terminal A-amino acid A-gal A Produced by Stably Transfected Human Cells The C-terminal amino acid residue of secreted a-gal A produced in accordance with the invention was identified using an automatic Hewlett Packard C-terminal sequencer. The results indicated a leucine residue at the C-terminus, which matches the C-terminal amino acid predicted by the DNA sequence. D. Modification of α-gal A Carbohydrates Produced by Stably Transfected Human Cells The glycosylation pattern of α-gal A produced according to the invention was also evaluated. Proper glycosylation is important for the optimal in vivo activity of a-gal A; α-gal A expressed in systems without glycosylation is inactive or unstable (Hantzopolous et al., Gene 57: 159, 1987). Glycosylation is also important to internalize a-gal A in the desired target cells, and affects the circulating half-life of the enzyme in vivo. In each subunit of a-gal A there are four sites available for the addition of asparagine-linked carbohydrate chains, of which only three are occupied (Desnick et al., In The Metabolic and Molecular Bases of Inherited Disease, pp. 2741-2780 , MacGraw Hill, New York, 1995). A sample of α-gal A produced by stably transfected cells was treated with neuraminidase, which was isolated from A. urafaciens, (Boehringer-Mannheim, Indianapolis, IN) to remove sialic acid. This reaction was carried out by treating 5 μg of a-gal A overnight with 10 mU of neuraminidase at room temperature in a total volume of 10 μl of acetate buffered saline (ABS, 20 mM sodium acetate, pH 5.2 , 150 mM NaCl). The purified a-gal A produced by stably transfected cells was also dephosphorylated using alkaline phosphatase (calf intestinal alkaline phosphatase, Boehringer-Mannheim, Indianapolis, IN), treating 5 μg of a-gal A overnight at room temperature with 15 U of alkaline phosphatase in ABS (rinse pH at 7.5 with 1 M Tris). The samples were analyzed by Western blots with an antibody specific for a-gal A. The antibody used was a polyclonal rabbit anti-peptide antibody, which was produced using a peptide representing amino acids 68-81 of a-gal A as an immunogen. After transfer of the PVDF protein (Millipore, Bedford, MA), the membrane was tested with a 1: 2000 dilution of the anti-serum in 2.5% dryer (dry milk without fat in 20 mM Tris-HCl, pH 7.5, 0.05% Tween-20). This was followed by detection of goat anti-rabbit IgG conjugated with horseradish peroxidase (Organon Teknika / Cappel, Durham, NC, dilution at 1: 5000) and chemiluminescent ECL reagents (Amersham, Arlington Heights, IN). Treatment of a-gal A with neuraminidase results in a change in molecular mass (approximately 1500-2000 Da or 4-6 sialic acids / monomer), suggesting that there is extensive modification of a-gal A with sialic acid. By reference, the plasma form of a-gal A has 5-6 sialic acid residues per monomer and the placental form has O.5-1.0 sialic acid residues per monomer (Bishop et al., J. Biol. Chem. 256: 1307, 1981). Another method used to examine sialic acid and modifications of mannose-6-phosphate from a-gal A was the isoelectric focusing (EIE), where the samples are separated based on their isoelectric point (pl) or net charge. Therefore, it could be expected that the removal of charged residues such as sialic acid or a-gal A phosphate will alter the mobility of the protein in the EIE system. To perform the EIE experiment, the samples of a-gal A produced according to the invention, were treated with neuraminidase and alkaline phosphatase, mixed at 1: 1 with 2x Novex sample buffer solution (with 8 M urea, pH 3.0-7.0) and loaded on an EIE gel of 6 M urea (5.5% polyacrylamide) made using Pharmalyte® (Pharmacia, Uppsala, Sweden, pH 3.0-6.5, Pharmalyte® 4-6.5 and 2.5-5.5, 0.25 ml each per gel). Isoelectric point standards (Bio-Rad) were also included). After electrophoresis, the gel was transferred to PVDF and the Western blot analysis was performed as described above. The α-gal A produced by stably transfected human fibroblasts consisted of three main isoforms with a pl scale of approximately 4.4-4.65. These values are similar to plasma pl and splenic forms of α-gal A (Bishop et al, J. Bioi, Chem. 256: 1307, 1981). Neuraminidase treatment of the enzyme increased the pl of all three isoforms, indicating that they were modified to some extent by sialic acid. These data suggest that α-gal A produced by stably transfected human cells, should have a convenient plasma half-life, indicating that this material is indicated for pharmacological use. In addition, the treatment of a-gal A treated with neuraminidase with alkaline phosphatase further increased the pl of a portion of the protein to about 5.0-5.1, indicating that the enzyme has one or more mannose-6-phosphate residues. This modification is significant in that it is required for the efficient internalization of a-gal A by the target cells. E. Specific Activity of Purified a-gal A from Stably Transfected Fibroblasts The potency or specific activity of purified a-gal A was calculated by measuring the catalytic activity of the enzyme (with 4-MUF-gal analysis) and protein concentration. The protein concentration can be determined by any normal method, such as with the BCA system (Pierce) or by measuring the absrobancy at 280 nm and using the extinction coefficient in mg / ml of 2.3 (determined from the amino acid analysis) to calculate the value. Using these techniques, the specific activity of a-gal A purified from the conditioned medium of transfected human fibroblasts is 2.2-2.9 x 106 units / mg protein, which is comparable with the specific activity of a-gal A which is purified from human tissues (Bishop et al., J. Biol. Chem. 256: 1301, 1981). F. Internalization mediated by Mannose or Mannose-6-a-gal Phosphate A In order that a-gal A produced by stably transfected cells is an effective therapeutic agent for α-gal A deficiencies, the enzyme must be internalized by the cells affected, a-gal A is not active at physiological pH levels and is not likely to be effective in the. interstitial blood fluids. Metabolizes optimally accumulated lipid substrates only when internalized in the acid environment of the lysosome. This internalization is measured by the binding of α-gal A to the mannose-6-phosphate (M6P) receptors, which is expressed on the surface of the cells and supplies the enzyme to the lysosome via the endocytic pathway. The M6P receptor was ubiquitously expressed; most somatic cells express it to some degree. The mannose receptor, which is specific for residues of mannose exposed on glycoproteins, prevails less. The last receptors are usually found alone on macrophages and macrophage-like cells and provide an additional means of entry of a-gal A into these cell types. In order to demonstrate the M6P-mediated internalization of a-gai A, the fibroblasts of the skin of a patient with Fabry (Deposit of Human Genetic Mutant Cells NIGMS) were grown overnight in the presence of increasing concentrations of purified a-gal A of the invention. Some of the samples contained 5 mM soluble M6P, which competitively inhibits the binding to, and as a result of, internalization by the mannose-6-phosphate receptor. Other samples contained 30 μg / ml of morning, which inhibits binding to, and as a result of, internalization by the mannose receptor. After incubation, the cells were washed and cultured by scraping in the lysis regulatory solution (10 mM Tris, pH 7.2, 100 mM NaCl, 5 mM EDTA, 2 mM Pefabloc ™, (Boehringer-Mannheim, Indianapolis , IN) and 1% of NP-40). The samples used were then analyzed for protein concentration and a-gal A activity. The results were expressed as units of activity a-gal A / mg of cell proteins. The Fabry cells inter-nalized a-gal A in a manner that depends on the dose (Figure 7). This internalization was inhibited by mannose-6-phosphate, but there was no inhibition with morning. Therefore, the internalization of a-gal A in Fabry fibroblasts was mediated by the mannose-6-phosphate receptor, but not by the mannose receptor. α-gal A was also internalized in vitro by endothelial cells, target cells important for the treatment of Fabry disease. Human umbilical vein endothelial cells (HUVECs) were grown overnight with 7500 units of a-gal A; some of the wells contained M6P. After the incubation period the cells were recovered and dialysed for a-gai A as described above. Cells incubated with a-gal A only had enzyme levels almost 10 times higher than those of control cells (without incubation with a-gal A). M6P inhibited the intracellular accumulation of a-gal A, suggesting that the internalization of a-gal A by HUVECs is mediated by the M6P receptor. Therefore, the human a-gal A of the invention was internalized by clinically relevant cells. Few lines of cultured human cells are known to express the mannose receptor. However, a cell line similar to mouse macrophages (J774.E-) having mannose receptors but few if mannose-6-phosphate receptors can be used to determine whether the purified a-gal A of the invention it is internalized or not via the mannose receptor (Diment et al., J. Leukocyte Biol. 42: 485-490, 1987). J774.E cells were cultured during the. overnight in the presence of 10,000 units / ml of a-gal A. The selected samples also contained 2 mM of M6P and others contained 100 μg / ml of morning. The cells were washed and recovered as described above, and the total protein and a-gal A activity of each sample were determined. The results are shown in Table 5. M6P does not inhibit the uptake of a-gal A by these cells, while tomorrow it reduces the levels of a-gal A accumulated to 75%. Therefore, a-gal A of the invention can be internalized by the mannose receptor in the cell types that express this particular cell surface receptor. Table 6. Internalization of a-Gal A by Cells J774.E. Activity of a-Gal (units / mg of total protein) Sin + a-gal A + a-gal A, + a-gal A, additions + M6P + tomorrow J774.E 409 + 25 6444 + 554 6297 + 674 1654 + 323 [manana] = 100 μg / ml [M6P] = 2 mM or 660 μg / ml These experiments demonstrate that a-gal A produced by stably transfected human cells can be internalized by cells via the mannose or mannose-6-phosphate receptor . G. Correction of Fabrv Fibroblasts by Human Fibroblasts Expressing a-gal A For gene therapy, an implant of autologous cells producing a-gal A should produce the enzyme in a modified form appropriately to "correct" the deficiency of α-gal A in white cells. To assess the effect of a-gal A production by transfected human fibroblasts on Fabry cells, fibroblasts recovered from patients with Fabry disease (Deposit of Human Genetic Mutant Cells NIGMS) were co-cultured with a strain of a-gal A producing cells (BRS-11) in Transwells ^ (Costar, Cambridge, MA). The experimental scheme is described in Figure 8. Fabry cells were grown in 12-well tissue culture plates, some of which contained inserts (Transwells®, 0.4 μm pore size) having a surface on which they can develop the cells. The growth matrix of the insert is porous and allows the macromolecules to pass from the top into the lower environment. A group of inserts contained the fibroblasts of normal human anterior skin (HF), which secrete minimal levels of a-gal A, while that another group contained the strain of stably transfected human fibroblasts, BRS-11, which secretes large amounts of a-gal A. Wells co-cultured with cells that produce a-gal A, a-gal A can enter the bathing medium Fabry cells and can potentially be internalized by Fabry cells. The data in Table 7 shows that the Fabry cells internalized the secreted a-gal A. The intracellular levels of a-gal A were monitored for three days. Cells grown alone (without insert) or in the presence of non-transfected anterior skin fibroblasts (PH insert) had very low intracellular levels of a-gal A activity. Fabry cells cultured with cells that produce a-gal A (insert of BRS-11), however they exhibited enzyme levels similar to those of normal cells at the end of Day 2 (normal fibroblasts contain 25-80 units of a-gal A / mg protein). That the correction is attributed to α-gal A absorbed via the M6P receptor is demonstrated by its inhibition with mannose-6-phosphate (insert BRS-11 + M6P). Table 7. Correction of Fabry Fibroblasts by Human Fibroblasts that Express a-Gal A Activity of a-Gal A (units / mg of total protein) H. Utility of Other Types of Cells Other types of cells can be used in the method described herein. The cells can be obtained from a variety of tissues and include all types of cells that can be maintained in the culture. For example, primary and secondary cells that can be transfected by the present method include human fibroblasts, keratinocytes, epithelial cells (e.g., mammary or intestinal epithelial cells), endothelial cells, glia cells, neural cells, formed elements of blood (e.g., lymphocytes and bone marrow cells), muscle cells and precursors of these types of somatic cells. The fibroblasts are of particular interest. The primary cells are preferably obtained from the individual to whom the primary or secondary cells transfected are to be administered so that they will not be rejected by the patient's immune system. However, if appropriate attention is paid to avoid or suppress immuno-rejection (as described below), cells from a human donor different from the patient may also be used. This could allow the use of cells from a line of stably transfected, stable and normalized cells in all patients. I. Administration of Cells Expressing a-gal A The cells described above can be introduced into an individual, through several normalized routes, so that they will reside in, for example, the renal subcapsule, a subcutaneous compartment, the central nervous system. , the intrathecal space, the liver, the intraperitoneal cavity, or within a muscle, the cells can also be injected intravenously or intra-arterially so that they circulate within the individual's bloodstream. Once implanted in the individual, the transfected cells will produce and secrete the therapeutic product, glycosylated human a-gal A. The number of genetically modified cells that will be introduced into the individual will vary, but can be determined by the experts. The age, weight, sex and general physical condition of each patient, as well as the volume of distribution, the half-life and bioavailability of the enzyme and the in vivo productivity of the genetically modified cells, will be among the main considerations to determine the dose and administration route. Normally, between one million and one billion cells will be used, with expression levels ranging from 100-100,000 units per 106 cells per day. If necessary, the procedure may be repeated or modified until the desired result is achieved, for example, relief of symptoms associated with Fabry disease. As described above, the cells used will generally be specific to the patient, ie, obtained from the individual to whom the transfected primary or secondary cells are to be administered, so that they will not be rejected by the patient's immune system. However, if this is not possible or convenient, the genetically modified cells can be obtained from another individual as described herein and implanted in the patient who suffers from a-gal A deficiency. The use of an individual's cells Different from the recipient may require the administration of an inmusupressor, alteration of histocompatibility antigens or the use of a barrier device to prevent rejection of implanted cells. The barrier device is made of a material (e.g., a membrane such as XM-50 from Amicon, Beverly, MA) that allows the secreted product to pass into the recipient's circulation or tissues, but avoids contact between the implanted cells and the immuno-receptor system and therefore prevents an immune response from (and possible rejection of cells by the recipient.) For further guidance regarding gene therapy, refer to Selden et al. (WO 93/09222) The cells can alternatively be embedded in a matrix or gel material, as described in the co-ownership of U.S.S.N. 08/548/002, which describes the use of hybrid matrix implants, or in Jain et al. (PCT Application WO 95/19430), which describes macroencapsulation of secretory cells in a hydrophilic gel material (each of which is incorporated here by reference). J. Pharmaceutical Formulation for the Conventional Administration of α-gal A Protein The α-gal A protein that was expressed and secreted by human cells stably transfected (or somehow genetically modified) and purified as described herein, they can be administered to individuals who produce insufficient or defective a-gal A protein. The protein can be administered in a pharmaceutically acceptable carrier, at a pH below 6.5, e.g., in a formulation as described in Example III. Examples of excipients that may be included with the formulation are regulatory solutions such as citrate buffer solutions, phosphate buffer, acetate buffer and bicarbonate buffer, amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins, such as serum albumin and gelatin, EDTA, sodium chloride, liposomes, polyvinylpyrrolidone, mannitol, sorbitol, glycerol propylene glycol and polyethylene glycol (e.g., PEG-4000, PEG-6000). The administration route, for example, can be intravenous, intraarterial. subcutaneous, intraperitoneal, intraperitoneal, intramuscular, intrapulmonal or transmucosal. The route of administration and amount of proteins delivered will be determined by factors that are well within the capacity of the experts. In addition, skilled artisans will be aware that the route of administration and the dose of a therapeutic protein may vary for a given patient until a therapeutic dose level is obtained. Normally, doses of a-gal A of 0.01-100 mg / kg of body weight will be administered. It is expected that regularly repeated doses of the protein will be necessary for the patient's life. K. Treatment of Other Conditions Caused by Enzyme Deficiencies It is likely that other conditions caused by deficiencies in lysosomal storage enzymes other than α-gal A are subject to treatment by methods comparable to those described herein. In these cases, the DNA encoding a functional form of the deficient enzyme could be replaced by the DNA encoding α-gal A in the expression constructs described herein. Examples of enzyme deficiency syndromes that have been identified and that can be treated as described herein are shown in Table 8. The information in this Table is taken from E. Neufeld (Ann.Rev. Biochem. 60: 257-280, 1991), which is incorporated herein by reference.

Table 8. Lysosomal Storage Disorder Summary Main Deficiency [secondary deficiency] Sphingolipid Degradation Disorders Fabry a-galactosidase disease Farber ceramidase disease Gaucher disease glucocerebrosidase Gangliosidosis of G? β-galactosidase Gangliosidosis of GM2 β-hexosaminidase, subunit-a Tay-Sachs disease [hexosaminidase A] Disease of β-hexosaminidase, Sandhoff subunit-β [hexosaminidase A and B] Activator deficiency activator of Krabbe disease galactosylceramidase GM2 Activator of deficient form of leukodystrophy enzyme metachromasulfatide of arylsulfatase A tica-activator of deficient form / saposin Mucolipidosis IV unknown major defect [ganglioside sialidase] Multiple sulfatase deficiency unknown major defect [deficiency of all sulphatases] Niemann-Pick disease sphingomyelinase Schindler's disease ar -? / - acetylgalactosaminidase Disorders of glycoprotein degradation Aspa rtilglicosaminu ria aspa rti Iglicosaminidase Protein a-L-fucosidase fucosidosis Galactosialidosis [ß-galactosidase and sialidase] protein / cathepsin a-Mannosidosis a-mannosidase ß-Mannosidosis ß-mannosidase Sialidosis sialidase Disorders of glycosaminoglycan degradation Hunter syndrome sulfatase of iduronate Syndromes Hurler and Scheie aL-idouronidase Maroteaux-Lamy syndrome GalNAc 4- sulfatase / ari I sulfatase Morquio syndrome subtype A Gal 6-sulfatase subtype B ß-galactosidase Sanfilippo syndrome subtype A? / - heparan sulfatase subtype B a -? / - acetyl glucosaminidsase subtype C AcetylCoA: glucosamine? / - acetyltransferase subtype D GIcNAc 6-sulfatase Sly Syndrome ß-glucuronidase Other Single Enzyme Deficiency Disorders Pompe disease (glycogenosis α-glucosidase Wolman's disease lipase acid Lysosomal enzyme biosynthesis disorders I-Cell and pseudoHurler disease 6-phospho -? - acetylglucosamine transferase Lysosomal polydystrophy [misplacement of many enzymes] Lysosomal membrane transport disorders Cystinosis Cystine transport Sialic storage and transport of Salic diseasesl acid. V. Other Modes The invention described herein has been exemplified in part by methods of treatment employing cells that express a particular gene product after transfection, i.e., after the introduction of a construct encoding the product of gene and that has regulatory elements that control the expression of the coding sequence. These methods can also be carried out using cells that have been genetically modified by other methods, including targeting a gene and activating the gene (see Treco et al., WO 95/31560, incorporated herein by reference; see also Selden et al., WO 93/09222). The hGH signal peptide can be used with heterologous proteins other than α-gal A, to increase the level of expression and secretion of heterologous protein. Examples of such proteins include α-1 antitrysone, antithrombin III, apolipoprotein E, apolipoprotein A-1, blood coagulation factors V, VII, VII, IX, X and XIII, bone growth factor-2, growth factor. of bones-7, calcitonin, catalytic antibodies, DNAse, erythropoietin, FSH-ß, globins, glucagon, glucocerebrosidase, G-CSF, GM-CSF, growth hormone, immune response modifiers, immunoglobulins, insulin, insulinotropin, growth factors insulin-like, interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-6, interleukin-11, interleukin-12, IL-2 receptor, IL-1 receptor antagonists, low lipoprotein receptor density, M-CSF, parathyroid hormone, protein kinase C, soluble CD4, superoxide dismutase, tissue plasminogen activator, TGF-β, tumor necrosis factor, TSHβ, tyrosine hydroxylase and urokinase. Other embodiments are within the following claims.

LIST OF SEQUENCES (1) GENERAL INFORMATION (I) APPLICANT: Transkaryotic Therapies, Inc. (i) TITLE OF THE INVENTION: THERAPY FOR DEFICIENCY OF a-GALACTOSIDASE A (iii) NUMBER OF SEQUENCES: 28 (iv) DIRECTION OF CORRESPONDENCE ( A) RECIPIENT: Fish ¿S Richardson, PC (B) STREET: 225 Franklin Street (C) CITY: Boston (D) STATE: MA (E) COUNTRY: E.U.A. (F) ZP: 02110-2804 (v) COMPUTER LEADABLE FORM: (A) TYPE OF MEDIUM: Hard Disk (B) COMPUTER: compatible with IBM PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Reléase # 1.0, Version # 1.30 (vi) CURRENT REQUEST DATA: (A) APPLICATION NUMBER: (B) DATE OF SUBMISSION: (C) CLASSIFICATION: (vii) PREVIOUS APPLICATION DATA- (A) APPLICATION NUMBER: 08 / 712,614 (B) DATE OF PRESENTATION. 13-SEPTEMBER-1996 (C) CLASSIFICATION: (viii) EMPLOYEE / AGENT INFORMATION: (A) NAME: Fraser, Janis K. (B) REGISTRATION NUMBER: 34,819 (C) REFERENCE / CASE NUMBER: 07236 / 003WO1 ( ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 617 / 542-5070 (B) TELEFAX: 617 / 542-8906 (C) TELEX: 200154 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CTGGGCTGTA GTATGATAA AC 22 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 2: TCTAGCTGAA GCAAAACAGT G 21 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) FORM OF THREAD: simple (D) TOPOLOGY: line al (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: ATTGGTCCGC CCCTGAGGT 19 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: acid nucleic (C) THREAD FORM: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: TGATGCAGGA ATCTGGCTCT 20 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: ( A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: TTTTGGATCC ACCATGGCTA 20 (2) INFORMATION FOR SEQ ID NO: 6 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: TTTTGCCGGC ACTGCCCTCT TGAA 24 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY : linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: TTTTCAGCTG GACAATGGAT TGGC 24 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (xi) ) SEQUENCE DESCRIPTION: SEQ ID NO: 8: TTTTGCTAGC TGGCGAATCC 20 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) ) THREAD FORM: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: TTTTGGATCC GTGTCCCATA GTGTTTCCAA 30 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: TTTTGGATCC GCAGTCGTGG CCAGTACC 28 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF SECU ENCIA: SEQ ID NO: 11 CTAGTCCTAG GA 12 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH. 22 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: TTTTGAGCAC AGAGCCTCGC CT 22TION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (xi) ) SEQUENCE DESCRIPTION: SEQ ID NO: 13: TTTTGGATCC GGTGAGCTGC GAGAATAGCC 30 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 76 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (xi) ) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GGGCCCCCAG CCCCAGCCCT CCCATTGGTG GAGGCCCTTT TGGAGGCACC 50 CTAGGGCCCC GAAATTTTG CCGTAT 76 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH 69 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: AAATAGGGCA GATCCGGGCT TTATTATTTT AGCACCACGG CCGCCGAGAC 50 CGCGTCCGCC CCGCGAGCA 69 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (xi) ) SEQUENCE DESCRIPTION: SEQ ID NO: 16: TGCCCTATTT ATACGGCAAA AGTTTCCTGG CCCTAGGGTG CTCCAAAAG 50 GGCCTCCACC AATGGGAGGG CTGGGGCTGG GGGCCC 86 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 55 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (xi) ) SEQUENCE DESCRIPTION: SEQ ID NO: 17: CGCGGGGCG ACGCGGTCTC GGCGGCCGTG GTGCTAAAAT AATAAAGCC 50 GGATC 55 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1343 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: CCGCGGGAAA TTT? TGCTGT CCGGTCACCG TGACAATGCA GCTGAGGAAC CCAGAACTAC 60 ATCTGGGCTG CGCGCTTGCG CTTCGCTTCC TGGCCCTCGT TTCCTGGGAC ATCCCTGGGG 120 CTAGAGCACT GGACAATGGA TTGGCAAGGA CGCCTACCAT GGGCTGGCTG CACTGGGAGC 180 GCTTCATGTG CAACCTTGAC TGCCAGGAAG AGCCAGATTC CTGCATCAGT GAGAAGCTCT 240 TCATGGAGAT GGCAGAGCTC ATGGTCTCAG AAGGCTGGAA GGATGCAGGT TATGAGTACC 300 TCTGCATTGA TGACTGTTGG ATGGCTCCCC AAAGAGATTC AGAAGGCAG? CTTCAGGCAG 360 ACCCTCAGCG CTTTCCTCAT GGGATTCGCC AGCTAGCTAA TTATGTTCAC AGCAAAGGAC 420 TGAAGCTAGG GATTTATGCA GATGTTGGAA ATAAAACCTG CGCAGGCTTC CCTCGGAGTT 480 TTGGATACTA CGACATTGAT GCCCAGACCT TTGCTGACTG GGGAGTAGAT CTGCTAAAAT 540 TTGATGGTTG TTACTGTGAC AGTTTGGAAA ATTTGGCAGA TGGTTATAAG CACATGTCCT 600 TGGCCCTGAA TAGGACTGGC AGAAGCATTG TGTACTCCTG TGAGTGGCCT CTTTATATGT 660 GGCCCTTTCA AAAGCCCAAT TATACAGAAA TCCGACAGTA CTGCAATCAC TGGCGAAATT 720 TTGCTGACAT TGATGATTCC TGGAAAAGTA TAAAGAGTAT CTTGGACTGG ACATCTTTTA 780 ACCAGGAGAG AATTGTTGAT GTTGCTGGAC CAGGGGGTTG GAATGACCCA GATATGTTAG_840_TGATTGGCAA CTTTGGCCTC AGCTGGAATC AGCAAGTAAC TCAGATGGCC CTCTGGGCTA 900 TCATGGCTGC TCCTTTATTC ATGTCTAATG ACCTCCGACA CATCAGCCCT CAAGCCAAAG 960 CTCTCCTTC? GGATAACGAC GTAATTGCCA TCAATCAGGA CCCCTTGGCC AAGCAAGGGT 1020 ACCAGCTTAG ACAGGGAGAC AACTTTGAAG TGTGGGAACG ACCTCTCTCA GGCTTAGCCT 1080 GGGCTGTAGC TATGATAAAC CGGCAGGAGA TTGGTGGACC TCGCTCTTAT ACCATCGCAG 1140 TTGCTTCCCT GGGTAAAGGA GTGGCCTGTA ATCCTGCCTG CTTCATCACA CAGCTCCTCC 1200 CTGTGAAAAG GAAGCTAGGG TTCTATGAAT GGACTTCAAG GTTAAGAAGT CACATAAATC 1260 CCACAGGCAC TGTTTTGCTT CAGCTAGAAA ATACAATGCA GATGTCATTA AAAGACTTAC 1320 TTTAAAAAAA AAAAAAACTC GAG 1343 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 210 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY : linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: CTGGGCTGTA GCTATGATAA ACCGGCAGGA GATTGGTGG? CCTCGCTCTT ATACCATCGC 60 AGTTGCTTCC CTCGGTAAAG GAGTGGCCTG TAATCCTGCC TGCTTCATCA CACAGCTCCT 120 CCCTGTGAAA AGCAAGCTAG GGTTCTATGA ATGGACTTCA AGGTTAAGAA GTCACATAAA 180 TCCCACAGGC ACTGTTTTGC TTCAGCTAGA 210 (2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 268 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY: linear (xi) ) SEQUENCE DESCRIPTION: SEQ ID NO: 20:CCCTGAGGTT AATCTTAAAA GCCCAGGTTA CCCGCGGAAA TTTATGCTGT 60 CCGGTCACCG TGACAATGCA GCTGAGGA? C CCAGAACTAC ATCTGGGCTG CGCGCTTGCG 120 CTTCGCTTCC TGGCCCTCGT TTCCTGGGAC ATCCCTGGGG CTAGAGCACT GGACAATGGA 180 TTGGCAAGGA CGCCTACCAT GGGCTGGCTG CACTGGGAGC GCTTCATGTG CAACCTTGAC 240 TGCCAGGAAG AGCCAGATTC CTGCATCA 268 (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 amino acids (B) TYPE: amino acid (C) THREAD FORM: not relevant (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21 Hßt Ala Thr Gly Sßr? Rg Thr Ser Lßu Lßu Lßu Ala Phß Gly Lßu Lßu 1 5 10 15 Cya Lßu Pro Trp Lßu Gln Glu ßly Sßr? La 20 25 (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 78 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (xi) ) SEQUENCE DESCRIPTION: SEQ ID NO: 22: ATGGCTACAG GCTCCCGGAC GTCCCTGCTC CTGGCTTTTG GCCTGCTCTG 50 CCTGCCCTGG CTTCAAGAGG GCAGTGCC 78 (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) ) TYPE nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: TTTTGGATCC CTCGAGGACA TTGATTATTG ACTAG_35_(2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: TTTTGGATCC CGTGTCAAGG ACGGTGAC 28 (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1197 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (xi) ) SEQUENCE DESCRIPTION: SEQ ID NO: 25: CTGGACAATE GATTGGCAAG GACGCCTACC ATGGGCTGGC TGCACTGGGA GCGCTTCATO 60 TGCAACCTTG ACTGCCAGGA AGAGCCAGAT TCCTGCATCA GTGAGAAGCT CTTCATGGAG 120 ATGGCAGAGC TCATGGTCTC AGAAGGCTGG AAGGATGCAG GTTATGAGTA CCTCTGCATT 180 GATG? CTGTT GGATGGCTCC CCAAAGAGAT TC? G ?? GGC? OACTTC? ßGC AGACCCTCAG 240 CGCTTTCCTC ATGGGATTCG CCAGCTAGCT AATTATGTTC ACAGCAAAGG ACTGAAGCTA 300 GGGATTTATG CAG? TGTTGG AAATAAAACC TGCGC? GGCT TCCCTGGGAG TTTTUGAT? C 360 TACGACATTG ATGCCCAGAC CTTTGCTGAC TGGGGAGTAG ATCTGCTAAA ATTTGATGGT 420 TGTTACTGTG ACAGTTTGGA AAATTTGGCA GATGGTTATA AGCACATGTC CTTGGCCCTG 480 AATAGGACTG GCAGAAGCAT TGTGTACTCC TGTGAGTGGC CTCTTTATAT GTGGCCCTTT 540 CA? AAGCCCA ATTATAC? G? AATCCGACAG TACTGCAATC ACTGGCGAAA TTTTGCTGAC 600 ATTGATGATT CCTGGAAAAG TATAAAGAGT ATCTTGGACT GGACATCTTT TAACCAGGAO 660 AGAATTGTTG ATGTTGCTGG ACCAGGGGGT TGGAATGACC CAGATATGTT AGTGATTGGC 720 AACTTTGGCC TCAGCTGGAA TCAGCAAGTA ACTCAGATGG CCCTCTGGGC TATCATGGCT 780 GCTCCTTTAT TCATGTCTAA TGACCTCCGA CACATCAGCC CTCAAGCCAA AGCTCTCCTT 840 CAGGATAAGG ACGTAATTGC CATCAATCAG GACCCCTTGG GCAAGCAAGG GTACCAGCTT 900 AGACAGGGAG ACAACTTTGA AGTGTGGGAA CGACCTCTCT CAGGCTTAGC CTGGGCTGTA 960 GCTATGATAA ACCGGCAGGA GATTGGTGGA CCTCGCTCTT ATACCATCGC AGTTGCTTCC 1020 CTGGGTAAAG GAGTGGCCTG TAATCCTGCC TGCTTCATCA CACAGCTCCT CCCTGTGAAA 1080 AGGAAGCTAG GGTTCTATGA ATGGACTTCA AGGTTAAGAA GTCACATAAA TCCCACAGGC 1140 ACTGTTTTGC TTCAGCT? G? AAATACAATG CAGATGTCAT TA ?? AGACTT ACTTTAA 1197 (2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 398 amino acids (B) TYPE amino acids (C) THREAD FORM: not relevant (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide x? SEQUENCE DESCRIPTION: SEQ ID NO: 26: Lßu Aßp Aßn Gly Lßu Wing Arg Thr Pro Thr Mßt Gly Trp Lßu Hiß Trp 1 5 10 15 Glu Arg Phß Mßt Cyß? ßn Lßu? ßp Cyß ßln Glu ßlu Pro? ßp Sßr Cyß 20 25 30 Ilß Sßr Glu Lyß Lßu Phß Mßt ßlu Mßt Ala ßlu Lßu Mßt Val Sßr ßlu 35 40 45 ßly Trp Lyß Aßp? The Gly Tyr ßlu Tyr Lßu Cyß llβ? ßp? ßp Cyß Trp 50 55 60 Mßt? The Pro ßln? Rg? ßp Sßr ßlu ßly? Rg Lßu ßln? La? ßp Pro ßln 65 70 75 80? Rg Phß Pro Hiß ßly He? Rg ßln Lßu ? the? ßn Tyr Val Hia Sßr Lyß 85 90 95 Gly Lßu Lyß Lßu Gly Ilß Tyr? The Aßp Val Gly? ßn Lyß Thr Cyß? The 100 105 110 Gly Phß Pro ßly Sßr Phß ßly and Tyr Tyr? ßp He? ßp? The ßln Thr Pbe 115 120 125 Ala Aßp Trp ßly and Val Aßp Lßu Lßu Lyß Phß? ßp ßly Cyß Tyr Cyß? ßp 130 135 140 Sßr Leu Glu? ßn Leu? The Aßp Gly Tyr Lyß Hiß Mßt Sßr Leu? The Lßu 145 150 155 160 Aßn Arg Thr Gly Arg Ser He Val Tyr Ser Cyß Glu Trp Pro Lßu Tyr 165 170 175 Mßt Trp Pro Phß Gln Lyß Pro Aßn Tyr Thr ßlu Ilß Arg Gln Tyr Cyß 180 185 190 Aßn Hls Trp Arg? An Phe Ala Aap He Aβp Aβp Ser Trp Lyß Ser Xlβ 195 200 205 Lyß Sßr lie Leu Aβp Trp Thr Ser Phe Aβn Gln ßlu Arg He Val Aßp 210 215 220 Val Wing Gly Pro Gly Cly Trp? ßn? Sp Pro? ßp Mßt Leu Val He ßly 225 230 235 240 Asn Phe Gly Leu Ser Trp? ßn Gln Gln Val Thr Gln Met? The Leu Trp 245 250 255 Ala llß Met Ala? The Pro Leu Phe Met Ser? An? ßp Leu? Rg Hiß He 260 265 270 Sßr Pro Gln? The Lyß Ala Leu Leu Gln? ßp Lyß? ßp Val Hß? The He 275 280 285 Aßn Gln Aßp Pro Leu Gly Lyß ßln Gly Tyr Gln Leu Arg ßln Gly? ßp 290 295 300 Aßn Phe Glu Val Trp ßlu Arg Pro Leu Ser ßly Leu? The Trp? Val 305 310 315 320? The Met He? ßn? Rg ßln ßlu He ßly ßly Pro? rg Ser Tyr Thr? le 325 330 335 Wing Val Wing Ser Leu ßly Lyß ßly Val Ala Cyß? ßn Pro? Cyß Phe 340 345 350 He Thr ßln Leu Leu Pro Val Lyß? rg Lyß Leu ßly Phe Tyr ßlu Trp 355 360 365 Thr Ser Arg Leu? Rg Ser Hiß He? An Pro Thr ßly Thr val Leu Leu 370 375 380 ßln Leu ßlu? ßn Thr Met ßln Met Ser Leu Lyß? Sp Leu Leu 385 390 395 (2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 338 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 27: TGGCTACAG GT ?? GCGCCC CTAA? ATCCC TTTGGGCAC? ? TGTGTCCTG AGGGGAGAGG 60 CAGCGACCTG TAG? TGGGAC GßGGGC? CT? ACCCTC? GGT TTßß GCTTC TGAATGTGAG 120 TATCGCCATG TAAGCCCAGT ATTTGGCCAA TCTCAGAAAG CTCCTGGTCC CTGGAGGGAT 180 GGAGAGAGAA AAACAAACAG CTCCTGGAGC AGGGAGAGTG CTGGCCTCTT GCTCTCCGGC 240 TCCCTCTGTT GCCCTCTGGT TTCTCCCCAG GCTCCCGGAC GTCCCTGCTC CTCGCTTTTG 300 GCCTGCTCTG CCTGCCCTGG CTTCAAGAGG GCAGTGCC 338 (2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) THREAD FORM: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: Leu Asp Asn Gly Leu Ala Arg Thr Pro Thr 1 5 10

Claims (35)

1. CLAIMS 1. A method of treatment comprising identifying a patient suspected of having a-galactosidase A deficiency by introducing a genetically modified human cell into the patient to overexpress and secrete human a-gal A. The method of claim 1, wherein the cells were transfected in vitro with a DNA molecule having a coding sequence encoding a polypeptide comprising human α-gal A (SEQ ID NO: 26). 3. The method of claim 2, wherein the polypeptide further comprises a heterologous signal peptide. The method of claim 3, wherein the heterologous signal peptide is the signal peptide of human growth hormone (hGH) (SEQ ID NO: 21). The method of claim 3, wherein the DNA molecule comprises an intron within the sequence encoding the signal peptide. The method of claim 3, wherein the DNA molecule comprising a non-translated sequence of at least 6 nucleotides that are immediately 3 'of the stop codon of the coding sequence, the untranslated sequence comprising a site of polyadenylation. 7. A DNA molecule comprising a first sequence encoding the hGH signal peptide (SEQ ID NO: 21), said first sequence comprising an intron; and linked to the 3 'end of the first sequence, a second sequence encoding a polypeptide comprising human α-gal A. 8. The DNA molecule of claim 7, further comprising a 3 'untranslated sequence comprising a polyadeniiation site. 9. An expression construct comprising the DNA molecule of claim 7 and is suitable for expression in a human cell. 10. A cultured human cell that contains a molecule of DNA that (a) encodes a polypeptide comprising human α-gal A linked to a heterologous signal peptide, and (b) allows expression of the polypeptide in the cell. 11. The cell of claim 10, said cell being a fibroblast. The cell of claim 10, wherein the cell is selected from the group consisting of an epithelial cell, an endothelial cell, a bone marrow cell, a glia cell, a hepatocyte, a keratinocyte, a myocyte, a neuron or a precursor of these types of cells. The cell of claim 10, wherein the signal peptide is the hGH signal peptide (SEQ ID NO: 21). 14. A clonal cell strain of cultured human cells transfected with a DNA molecule that (a) encodes a polypeptide comprising human α-gal A linked to a heterologous signal peptide, and (b) allows overexpression of the polypeptide at the cells 15. The clonal cell strain of claim 14, the cells being fibroblasts. 16. A line of clonal cells of immortalized human cells transfected with a DNA molecule that (a) encodes a polypeptide comprising human a-gal A linked to a heterologous signal peptide, and (b) allows overexpression of the polypeptide at the cells 17. The clonal cell line of claim 16, wherein the cell is selected from the group consisting of a Bowes melanoma cell, a Daudi cell, a HeLa cell, an HL-60 cell, an HT1080 cell , a Jurkat cell, a KB carcinoma cell, a K-562 leukemia cell, a MCF-7 breast cancer cell, a Molt-4 cell, a Namalwa cell. a Raji cell, an RPMI 8226 cell, a U-937 cell, a WI-38VA13 cell (2R4 subline) and an ovarian carcinoma cell 2780AD. 18. A protein comprising (a) the hGH signal peptide (SEQ ID NO: 21) ligated by a peptide linked to (b) a-gal A human. 19. A process for forming glycosylated human a-gal A, which comprises culturing the cell of claim 11 under conditions that allows the expression of human α-gal A of the DNA molecule and the secretion of glycosylated human α-gal A into the cell culture medium; Y . isolate glycosylated human a-gal A from the culture medium. 20. A purified human glycosylated a-gal A made by the process of claim 19. 21. A process for purifying human a-gal A from a sample, comprising a first step of chromatography wherein the sample is passed over a resin of hydrophobic interaction. 22. The process of claim 21, wherein the functional portion on the resin comprises a butyl group. 23. A process for forming glycosylated human α-gal A, comprising culturing the cell of claim 16 under conditions that allow the expression of human a-gal A of said DNA molecule and the secretion of glycosylated human α-gal A in the cell culture medium; and isolating glycosylated human a-gal A from the culture medium. The process of claim 21, further comprising the step of passing the sample over a second resin selected from the group consisting of an immobilized heparin resin, hydroxyapatite, an anion exchange resin, and size exclusion resin. 25. A method of treatment comprising identifying a patient suspected of having a-gal A deficiency and administering to the patient the purified glycosylated human a-gal A of claim 20. 26. A therapeutic composition comprising a-gal A human pharmaceutically acceptable salt having N-linked mannose-6-phosphate in a pharmaceutically acceptable carrier. 27. A therapeutic composition comprising the purified human a-gal A of claim 20 and a pharmaceutically acceptable excipient. 28. The therapeutic composition of claim 27, formulated at pH 6.5 or lower. 29. The therapeutic composition of claim 28, wherein the pharmaceutically acceptable excipient is human serum albumin. 30. A DNA molecule encoding a protein comprising the hGH signal peptide (SEQ ID: 21) linked to a polypeptide other than hGH. 31. The DNA molecules of claim 30, wherein the DNA molecule comprises an intron within the sequence encoding the signal peptide. 32. A cultured human cell transfected with a DNA molecule that (a) encodes a polypeptide comprising human α-gal A, and (b) allows overexpression of the polypeptide in the cell. 33. A process for forming glycosylated human α-gal A, which comprises culturing a human cell containing a DNA molecule encoding the polypeptide under conditions that allow the expression of α-gal A, and a heterologous signal peptide under conditions that allow the expression of human a-gal A of said DNA molecule. 34. The therapeutic composition of claim 26, further comprising a pharmaceutically acceptable excipient. 35. A process for purifying human α-gal A from a sample comprising passing the sample onto a hydrophobic interaction resin comprising a butyl group as a functional moiety.