AU2914192A - Synthetic alpha-l-iduronidase and genetic sequences encoding same - Google Patents

Description

SYNTHETIC α-L-IDURONIDASE AND GENETIC SEQUENCES

ENCODING SAME

The present invention relates generally to α-L-iduronidase and to genetic sequences encoding same and to the use of these in the investigation, diagnosi and treatment of subjects suspected of or suffering from α-L-iduronidase deficiency.

The lysomal enzyme α-L-iduronidase (IDUA; glycosaminoglycan α-L- iduronohydrolase, EC 3.2.1.76) hydrolyzes the nonreducing terminal α-L-iduro glycosidic bonds in the glycosaminoglycans heparan sulfate and dermatan sulfa (1,2). IDUA has served as a model for process and maturation events underg by lysosomal enzymes (3-8). A deficiency of IDUA in humans results in the lysosomal storage disorder mucopolysaccharidosis type I (MPS-I; cp-onyms, Hurler, Hurler/Scheic, and Scheic syndromes), which is inherited as an autoso recessive disease and shows wide variation of clinical presentation. Severely affected patients have mental retardation, somatic tissue complications and a reduced life span, while mildly affected patients may have only mild somatic complications and a normal life span. Multiple different mutant alleles at the IDUA locus are thought to be responsible for the spectrum of clinical phenoty (1,9), but biochemical characterisation of the residual IDUA activity has enabl discrimination only between the extremes of clinical phenotypes (10-12). In w leading up to the present invention, the isolation of the IDUA gene was undertaken to provide a DNA probe for molecular analysis of mutations in M patients and for use in enzyme and gene therapy experiments in the canine m Accordingly, the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides which encodes, or are complementary to a sequence which encodes, a mammalian αrL-iduronidase (IDUA) or fragment or derivative thereof or its like molecule.

Preferably, the mammal is a human, livestock animal, companion animal, wild animal or laboratory test animal (e.g. rabbit, rat, mouse or guinea pig). Most preferably, the mammal is a human. Conveniently, the IDUA is isolatable from the liver. However, the present invention extends to all mammalian IDUA enzymes and from any anatomical or cellular source and/or any biological fluid source, such as but not limited to plasma, serum, cell extract or lymph fluid.

Although a preferred embodiment of the present invention contemplates the use of human IDUA or genomic or recombinant genetic sequences encoding same in the investigation, diagnosis and/or treatment of human subjects (i.e. homologous system), one skilled in the art will appreciate that the enzyme or genetic sequences encoding same from a non-human animal may also be useful. Such a heterologous system is encompassed by the present invention.

The "nucleic acid molecule" of the present invention may be RNA or DNA (eg. cDNA), single or double stranded and linear or covalently closed. The nucleic acid molecule may also be genomic DNA corresponding to the entire gene or a substantial portion thereof or to fragments and derivatives thereof. The nucleotide sequence may correspond to the nautrally occurring nucleotide sequence or may contain single or multiple nucleotide substitutions, deletions and/or additions. All such modifications encode the IDUA-like molecules contemplated by the present invention. The length of the nucleotide sequence may vary from a few bases, such as in nucleic acid probes or primers, to a full length sequence. The nucleic acid molecule of the present invention may constitute solely the nucleotide sequence encoding IDUA or like molecule or may be part of a lar nucleic acid molecule and extends to the genomic clone of IDUA. The non- IDUA encoding sequences in a larger nucleic acid molecule may include vecto promoter, terminator, enhancer, replication or signal sequences or non-coding regions of the genomic clone.

The present invention is particularly directed to the nucleic acid in cDNA for and particularly when inserted in an expression vector. The expression vector may be replicable in a eukaryotic or prokaryotic cell and may either produce mRNA or the mRNA may be subsequently translated into IDUA or like molecule. Particularly preferred eukaryotic cells include CHO cells but may b any other suitable mammalian cells or cell lines or non-mammalian cells such yeast or insect cells.

The present invention is further directed to synthetic IDUA or like molecule. The term "synthetic" includes recombinant forms and molecules produced by t sequential addition of amino acid residues, or groups of amino acid residues, i defined order. In a most preferred embodiment, the invention relates to recombinant IDUA or like molecule encoded by or expressed from the nucleic acid molecules as hereinbefore described.

The synthetic or recombinant IDUA may comprise an amino acid sequence corresponding to the naturally occurring amino acid sequence or may contain single or multiple amino acid substitutions, deletions and/or additions. The length of the amino acid sequence may range from a few residues to a full len molecule. Accordingly, this aspect of the present invention contemplates a proteinaceous molecule comprising an amino acid sequence corresponding to t full length mammalian IDUA enzyme or to a like molecule. The like molecul therefore, comprises parts, derivatives and/or portions of the IDUA enzyme whether functional or not. Preferably, the mammal is human but may be of n human origin as contemplated above.

Advantageously, the recombinant IDUA is a biologically pure preparation meaning that it has undergone some purification away for other proteins and/or non-proteinacous material. The purity of the preparation may be represented as at least 40% of the enzyme, preferably at least 60%, more preferably at least 75%, even more preferably at least 85% and still more preferably at least 95% relative to non-IDUA material as determined by weight, activity, amino acid homology or similarity, antibody reactivity or other convenient means.

Amino acid insertional derivatives of IDUA of the present invention include amino and/or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterised by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Typical substitutions are those made in accordance with the following Table 1:

TABLE 1 Suitable residues for amino acid substitutions

10

15

20

Where the enzyme is derivatised by amino acid substitution, the amino acids are generally replaced by other amino acids having like properties such as hydrophobicity, hydrophUicity, electronegativity, bulky side chains and the like. Amino acid substitutions are typically of single residues. Amino acid insertions will usually be in the order of about 1-10 amino acid residues and deletions will range from about 1-20 residues. Preferably, deletions or insertions are made in adjacent pairs, i.e. a deletion of two residues or insertion of two residues.

The amino acid variants referred to above may readily be made using peptide synd etic techniques well known in the art, such as solid phase peptide synthesis (Merrifield synthesis) and the like, or by recombinant DNA manipulations. Techniques for making substitution mutations at predetermined sites in DNA having known or partially known sequence are well known and include, for example, M13 mutagenesis. The manipulation of DNA sequence to produce variant proteins which manifest as substitutional, insertional or deletional variants are conveniently elsewhere described such as Sambrook et al, 1989 Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratories, Cold Spring Harbor, NY.

The derivatives or like molecules include single or multiple substitutions, deletions and/ or additions of any components) naturally or artificially associated with the IDUA enzyme such as carbohydrate, lipid and/or other proteinaceous moieties. For example, the present invention extends to glycosylated and non- glycosylated forms of the molecule. All such molecules are encompassed by d e expression "mutants", "derivatives", "fragments", "portions" and "like" molecules. These molecules may be active or non-active and may contain specific regions, such as a catalytic region. Particularly, preferred derivative molecules include those with altered glycosylation patterns relative to the naturally occurring molecule. Even more particularly, the recombinant molecule is more highly glycosylated than the naturally occurring molecule. Such higly glycosylated derivatives may have improved take-up properties and enhanced half-lives. The present invention also extends to synthetic IDUA or like molecules when fused to other proteinaceous molecules. The latter may include another enzy reporter molecule, purification site or an amino acid sequence which facilitate transport of the molecule out of a cell, such as a signal sequence.

In a most preferred embodiment, the present invention has an amino acid or corresponding IDUA cDNA nucleotide sequence substantially as setforth in Figure 2 or genomic nucleotide sequence substantially as set forth in Figure 4 and 4B or having at least 40% similarity, preferably at least 60% similarity thereto or more preferably at least 80% or 85-90% similarity thereto.

The present invention further contemplates antibodies to synthetic IDUA or li molecule. The antibodies may be polyclonal or monoclonal, naturally occurri or synthetic (including recombinant, fragment or fusion forms). Such antibodi will be useful in developing immunoassays for IDUA.

A further aspect of the present invention contemplates a method of screening abberations in the IDUA gene. Such a method may be accomplished in a number of ways including isolating a source of DNA to be tested or mRNA therefrom and hybridising thereto a nucleic acid molecule as hereinbefore described. Generally, the nucleic acid is probe or primer size and polymerase chain reaction is a convenient means by which to analyse the RNA or DNA. Other suitable assays include the ligation chain reaction and the strand displacement amplification methods. The IDUA sequence can also be determined and compared to the naturally occurring sequence. Such methods may be useful in adults and children and may be adapted for a pre-natal test. The DNA to be tested includes a genomic sample carrying the IDUA gene, a cDNA clone and/or amplification product.

In accordance with this aspect of the present invention there is provided a method for screening for abberations in the IDUA gene including the absence such a gene or a portion or a substantial portion thereof comprising isolating sample of DNA or mRNA corresponding to a region of said DNA and contacting same with an oligonucleotide probe capable of hybridising to one or more complementary sequences within the IDUA gene and then detecting the hybridisation, the extent of hybridisation or the absence of hybridisation. Alternatively, the probe is a primer and capable of directing amplification of one or more regions of said IDUA gene and die amplification products and/or profile of amplification products is compared to an individual carrying the full gene or to a reference date base. Conveniently, the amplification products are sequenced to determine the presence or absence of d e full gene.

The present invention further extends to a method of treating patients suffering from IDUA deficiency, such as in MPS-I, said method comprising administering to said patient an effective amount of IDUA or active like form thereof. Preferably, the IDUA is in recombinant form. Such a method is referred to as "enzyme therapy". Alternatively, gene therapy can be employed including introducing an active gene (i.e. a nucleic acid molecule as hereinbefore described) or to parts of the gene or other sequences which facilitate expression of a naturally occurring IDUA gene.

Administration of the IDUA for enzyme therapy may be by oral, intravenous, suppository, intraperitoneal, intramuscular, intranasal, intradermal or subcutaneous administration or by infusion or implantation. The IDUA is preferably as hereinbefore described including active mutants or derivatives thereof and glycosylation variants thereof. Administration may also be by way of gene therapy including expression of the gene by inclusion of the gene in viral vectors which are introduced into the animal (e.g. human) host to be treated. Alternatively, the gene may be expressed in a bacterial host which is then introduced and becomes part of the bacterial flora in the animal to be tested.

Still yet another aspect of the present invention is directed to a pharmaceutical composition comprising synthetic (e.g. recombinant) IDUA or like molecule, including active derivatives and fragments thereof, alone or in combination with other active molecules. Such other molecules may act synergistically with the enzyme or facilitates its entry to a target cell. The composition will also conta one or more pharmaceutically acceptable carriers and/or diluents. The composition may alternatively comprise a genetic component useful in gene therapy.

The active ingredients of the pharmaceutical composition comprising the synth or recombinant IDUA or mutants or fragments or derivatives thereof are contemplated to exhibit excellent activity in treating patients with a deficiency the enzyme when administered in an amount which depends on the particular case. The variation depends, for example, on the patient and the IDUA used. For example, from about 0.5 ug to about 20 mg of enzyme per animal body or, depending on the animal and other factors, per kilogram of body weight may b administered. Dosage regima may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or in other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation. Accordingly, alternative dosages in the order of 1.0 μg to 15 mg, 2.0 μg to 10 or lOμg to 5mg may be administered in a single or as part of multiple doses. active compound may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intramuscular, subcutaneous, intranasa intradermal or suppository routes or implanting (eg using slow release molecul Depending on the route of administration, the active ingredients which compris a synthetic (e.g. recombinant) IDUA or fragments, derivatives or mutants there may be required to be coated in a material to protect same from the action of enzymes, acids and other natural conditions which may inactivate said ingredie For example, the low lipophilicity of IDUA will allow it to be destroyed in the gastrointestinal tract by enzymes capable of cleaving peptide bonds and in the stomach by acid hydrolysis. In order to administer the vaccine by other than parenteral administration, the enzyme will be coated by, or administered with, material to prevent its inactivation. For example, the enzyme may be administered in an adjuvant, co-administered with enzyme inhibitors or in liposomes. Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon. Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl edier and n- hexadecyl polyethylene ether. Conveniently, the adjuvant is Freund's Complete or Incomplete Adjuvant. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol. Liposomes include water-in-oil-in- water CGF emulsions as well as conventional liposomes.

The active compound may also be administered in dispersions prepared in glycerol, liquid polyethylene glycols, and/or mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compoun the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized act ingredient(s) into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case sterile powders for the preparation of sterile injectable solutions, the preferre methods of preparation are vacuum drying and the freeze-drying technique w yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

When the IDUA of the present invention is suitably protected as described ab the composition may be orally administered, for example, with an inert diluen with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporate directly with the food of the diet. For oral therapeutic administration, the acti compound may be incorporated with excipients and used in the form of ingest tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, an the like. Such compositions and preparations should contain at least 1% by w of active compound. The percentage of the compositions and preparations ma course, be varied and may conveniently be between about 5 to about 80% of t weight of the unit. The amount of active compound in the vaccine compositio such diat a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared,so that an oral dosage unit form contains between about 0.5 ug and 20 mg of active compoun

The tablets, troches, pills, capsules and the like may also contain the followin a binder such as gum gragacanth, acacia, corn starch or gelatin; excipients suc dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavo agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings o to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release reparations and formulations.

As used herein "pharmaceutically acceptable carriers and/or diluents" include any and all solvents, dispersion media, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the an. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the pharmaceutical compositions is contemplated. Supplementary active ingredients can also be incorporated into die compositions.

The present invention further relates to the use of IDUA or active fragment, mutant or derivative thereof in the manufacture of a medicament for the treatment of patients suffering from a deficiency in the naturally occurring enzyme (e.g. MPS-1).

The present invention is further described with reference to the following non- limiting figures and examples. With reference to the figures:

FIGURE 1 is a schematic representation showing a model to connect the sev major polypeptides in immune purified human liver IDUA present after

SDS/PAGE as shown on the left, with the polypeptide sizes indicated in kDa The three amino-terminal sequences present are represented by the letters A or C next to the polypeptides. The proteolytic sites cleaved to produce the s polypeptides from the 74-kDa polypeptide are numbered 1, 2 and 3.

FIGURE 2 is a representation of a compiled nucleotide sequence for IDUA cDNA and the deduced amino acid sequence of the protein. The amino acid sequence is shown in single letter code above the cDNA sequence. Nucleoti and amino acid numbers are in the right margin. The probable site of signal peptide peptidase cleavage is shown by a large arrow, and small arrows indic exon junctions. Exons II and IV, which are alternatively spliced in some RN transcripts, are boxed. Amino acids colinear with either amino-terminal pept data or tryptic peptides are underlined and named above the sequence. Pote N-glycosylation sites are asterisked. Oligonucleotides used in this study are underlined below the nucleotide sequence with the arrows indicating either s (-») or antisense (<-). The cDNA clone λRPCI extended from base 541 to ba 1269 and λE8A extended from base 391 to the 3' end of the sequence shown.

FIGURE 3 is a representation of reverse-transcribed normal fibroblast RNA showing the alternative splicinng of exons II and IV. Lane 1, PCR between 1 and 1D57, howing a major 225-bp product and a minor 84-bp product: lane pUC19 Hpa II markers: lane 3, PCR between IDNT and ID39, showing a ma 222-bp product and a minor 114-bp product. Partial sequences of the two mi products and their encoded amino acid are at the left and right of the figures The position of the missing exon is indicated by the arrow labelled "Exon junction". FIGURE 4 shows the sequence of the human genomic IDUA gene. Primers were made every 200 to 400 bp to completely sequence areas of interest in both directions. The coding region of the exons are in uppercase letters; untranslated sequence and introns are in lowercase letters. (A) Exons I and II of the human IDUA gene are shown in the 1.8 kb segment. The Alu repeat sequence and the four best potential OC boxes in the promoter region of IDUA are boxed. Potential transcription start sites are underlined. (B) Exons III to XIV of the human IDUA gene are shown in this 4.5 kb segment. Potential polyadenylation signals are underlined.

EXAMPLE 1. MATERIALS AND METHODS

Polypeptide Isolation and Sequencing. All seven major polypeptides of IDUA (7) were directiy sequenced from their amino termini as previously described (17). Tryptic peptide sequences from 150μg of purified human liver IDUA were generated as previously described (18).

Oligonucleotides and Primers. AH oligonucleotides were synthesised on an Applied Biosystems 391 DNA synthesiser. ED47, 5'-

AACTTCGAGACCTGGAACGAGCCCGACCAGCACGACTTCGACAACGT- 3', designed from residue 2 to residue 17 of peptide 8 (see Figure 2), was used for initial library screening. ID13, 5'-GCCCGGGCGGCA/GTCCACC/TTG-3' (a mixture of four sequences; nucleotides separated by / are options at the same position), designed from residue 13 to residue 7 of the 74/13-kDa amino-terminal amino acid sequence (see Figure 2), was used to screen Southern blots of the cosmid clone A157.1 (15). IDUA-specific primers used for PCR from cDNA were IDNT, ID39, ID56, ID57, ID58, ID60 and ID61 (see Figure 2). Library Screening.

All libraries screened were of human origin and were purchased from Clontech They were a leukocyte genomic DNA in EMGL3 (catalogue number HL1006) and the following cDNA libraries: colon (random primed, HL1034a), unbUical endothelial (HL1024b), umbilical endothelial 5' stretch (HL1070b), and T-ceU stretch (HL1068b). All libraries were plated at a density of between 40,000 an 55,000 plaques per 140 mm plate. The host cells used for each library were NM538 for the EMBL3 genomic library, C600 for the λgtll cDNA libraries. Probes were either labeUed at the 5' end (19) or labeUed by primer extension random oligonucleotide primers (20) and the Colony/Plaquescreen filters (DuPont/NEN) were prehybridised, hybridised, and washed according to the manufacturer's instructions.

Sequencing.

Specific oligonucleotides were made every 200-400 base pairs (bp) to fuUy sequence fragments in both directions (21). Compressed areas of G + C-rich sequence were resolved by using 7-deazaguanosine (22). Direct PCR sequenci was by the linear PCR method (23).

RNA Isolation and Northern Blot Analysis.

Total RNA was isolated from normal human placental, liver and kidney tissue cultured normal human fibroblasts as previously described (24). Poly (A) + R was obtained (25) from placental RNA and Northern blotting was carried out 40 μg of total RNA and 10 and 40 μg of poly (a)+ RNA as described (17).

cDNA Synthesis.

Total RNA (3 μg) from normal fibroblasts was added to a reaction mix containing lx Moloney murine leukaemia virus (Mo-MLV) reverse transcriptas buffer (BRL), 40 units of RNAsin (Promega), 500 ng of random octamers, 0.5 mM deoxynucleotides (Boehringer Mannheim), and 200 units of Mo-MLV reve transcriptase (BRL) to a final reaction volume of 50 μl. Incubation at 37°C fo m was followed by hydrolysis of the RNA by the addition of 5μl of 3 M NaOH and further incubation at 37° for 30 min. The NaOH was neutralised by the addition of 1.25 μl of 10.3 M HC1, and the cDNA was precipitated and resuspended in 50 μl of water. Each PCR used 5μl of cDNA.

PCR

PCR reagents were as described by Saiki et al (26) except that the final concentrations of deoxynucleotides were 400 μM and 10% v/v dimethyl sulfoxide was present in the reaction mix. Forty cycles of denaturation at 94 °C for 45 s, annealing at 58 °C for 43 s, and elongation at 72 °C for 2 min were carried out. PCR products were analysed on 4% w/v Nusieve GTG agarose (FMC) gels.

Construction of Full-Length IDUA cDNA. cDNA from a mixture of normal human fibroblast ceU lines was used for PCR as described, using the primers ID60 and ID6L. ID60 spans the initiating ATG codon and has a Ht/rilll restriction site with a 4bp GC clamp on the 5' end. ID61 is = 100 bp 3' of a unique Kpri. restriction iste (bases 818-823, see Figure 2). Utilizing the HirύlH and the Kpri. sites, the PCR product was directionaUy cloned in a pTZ19 vector that contained the rest of the IDUA coding sequence from the KpriL site to the EccRI cloning site of the clone λΕ8A. In aU, 48 clones were analysed and only one was found to be correct (fuU length). This insert was excised with Hirύlll and EccRI and was directionaUy cloned in the expression vector pRSVN.07 (which drives expression of the insert from the Rous sarcoma virus long terminal repeat) to give pPSVNID7I. This fuU length IDUA cDNA insert was also subcloned in M13 and sequenced between the tiilll and Kpri. restriction sites, using IDUA-specific oligonucleotide primers to determine if any errors were present in the sequence. Expression of IDUA

CHO (Chinese hamster ovary) ceUs (strain DKI) were grown in Ham's F12 medium (GIBCO), 10% v/v fetal calf serum (GOBCO), penicUlin at 100 μg/m streptomycin sulfate at 100 μg/ml, and kanamycin sulfate at 120 μg/ml at 37 ° a 5% v/v C0₂ atmosphere. CHO ceUs (1.2 x 10⁷) were electroporated at 0 °C using a BRL Cell-Porator at a pulse of 330 μF and 275 V in the presence 15 μ of pRSVNID21. CeUs were grown in nonselective medium for 48 hr and then 1:20 and 1:100 dUutions of the electroporated ceUs were selected in G418 sulfa (Geneticin; GIBCO) at 750 μg/ml. A bulk culture of resistant cells was extracted (14) and assayed for IDUA activity with the fluorogenic substrate 4- methylumbeUiferyl α-L-iduronide (Calbiochem) (6). The Bio-Rad protein assa was used to quantitate the amount of protein in each sample according to the manufacturer's instructions. The monoclonal antibody IdlA was used for immunocapture (14) and immunoquantification in conjunction with a polyclona antibody (12) to assay the specific activity of the expressed HDUA (7).

2. RESULTS

All seven polypeptides of IDUA were subjected to direct amino-terminal sequencing, and tiiree different amino-terminal sequences were found to be present. The 65-, 60-, and 18-kDa species have a common amino-terminal ami acid sequence, the 49- and 44-kDa another, and the 74- and 13-kDa species another. Assuming that all seven species represent part of a single IDUA polypeptide, a model (Figure 1), is proposed showing three sites of proteolytic processing of the 74-kDa polypeptide to produce the seven major species of IDUA.

After tryptic digestion and separation by HPLC (18) of immunopurified IDUA, nine major peptides were sequenced. One tryptic peptide was the same as the 65/60/ 18-kDa amino-terminal sequence, and one of the two tryptic peptide species present in part 3 were contained within the 49 /44-kDa amino-terminal sequence. Incorporating choices based on human codon usage and assuming that the undetermined amino acid at position 16 of peptide 8 was a glycosylated asparagine residue (see Figure 2) the sequence was used to design a 74-mer oligonucleotide (ED47) for library screening.

Using ID47 as a probe, 500,000 clones were screened of the EMBL3 human genomic library and obtained 8 clones. A genomic clone, ID-475, was purified and an ID47-positive 1.6 kilobase (kb) PsfL fragment was subcloned in pUC19 to produce pID89 (14). This 1.6-kb insert was then used to screen a number of cDNA libraries, this screening yielded only 1 clone, which contained an insert of 729 bp (λRPCl, bases 541-1269; see Figure 2) from the λgtlO random-promed human colon cDNA library. The sequence of this clone was colinear with six peptide sequences, including the 49 /44-kDa amino-terminal sequence, but the clone ended within peptide 9.

The λRPCI insert was then used to screen a λgtll human endothelial cDNA library. Twenty clones were isolated, and the insert of the longest clone, λE8A, was fully sequenced. The 11765-bp insert contained an open reading frame starting just before the position of the 65/60/18-kDa amino terminus (base 391 in Figure 2) to a stop codon (base 2048). Six further tryptic peptides were matched to the translated DNA sequence but, significantly, the sequence of the 74/13-kDa amino terminus, a secondary tryptic peptide (peptide Z'), a signal peptide, and an initiating methronine were not present in this clone. Of the other clones, 7 ended at the same base at the 5' end, whUe all the others were shorter. A 5' probe derived from λE8A was used to screen another seven cDNA libraries. No clones were obtained from the screening of five of these cDNA libraries. Screening of two 5' "stretch" cDNA libraries (umbUϊcal endothelial and T cell) resulted in a further 38 clones. PCR analysis of these clones showed that aU ended at the same 5' base as λE8A. Major secondary structures present in the IDUA mRNA may be responsible for the premature termination of these clones at their 5' ends. Using the polypeptide model for IDUA (Figure 1) it was hypothesised that the 74/13-kDa amino-terminal peptide sequence lay at the 5' end of the IDUA mRNA. A mixed oligonucleotide, ID13, made to the 74/13-kDa amino-termin sequence was used to probe Southern blots of the cosmid A157.1, which spans area of the IDUA gene (15). A 2.8 kb Baπύrll fragment was isolated and partially sequenced. The sequence contained an initiating methionine, a signal peptide, 74/13 kDa amino terminus, and the start of the last unmatched trypti peptide (peptide 2' in figure 2). A number of oligonucleotides were made to t exon and PCR used to amplify normal fibroblast cDNA. A major PCR produ was obtained between ID58 and ID61, and the oligonucleotides ID56 and ID5 was directly sequenced (23). The collated DNA sequence (Figure 2) encodes protein containing all amino-terminal and tryptic peptide sequences obtained from purified IDUA and is consistent with the model for EDUA (Figure 1).

PCR of normal fibroblast cDNA at the 5' end of the IDUA mRNA, using the oligonucleotides ID58 and ID61, produced a major product representing the sequence described (Figure 2) and several minor products that also hybridised an internal oligonucleotide, ID56. This indicates that the minor products were representative of alternative mRNA species from the IDUA gene, as has been reported for a number of other genes, including lysosomal hydrolases (27-29).

PCR of normal fibroblast cDNA using the oligonucleotide pairs ID56 to ID57 IDNT to ID39 produced two products per reaction. The smaUer products wer isolated and directly sequenced; they showed alternative splicing of exons II a IV of IDUA (Figure 3). The polypeptides from these alternatively spliced ID mRNA species would maintain the translation frame for the IDUA protein (se Figure 3) leaving the primary sequence of the translated peptide identical to t of the deduced IDUA peptide except for the omission of 47 and 36 amino aci respectively. Thus, the alternatively spliced mRNA species individuaUy missin exons II and IV would produce peptide products of 606 and 617 amino acids, respectively. Using the insert of λESA as a probe against total placental RNA and poly(A) + RNA, a single 2.3 kb band only was detected when 40 μg of poly(A)+ RNA was loaded fn a single track. The strength of the signal also indicated that the mRN for IDUA has a considerably lower abundance than the iduronate-2-sulfatase mRNA in placental RNA (16). Multiple PCR products of the same relative intensity were observed when reverse-transcribed liver, kidney, or placental RNA was used as template, indicating that this splicing does not appear to be tissue specific and that these products may be minor mRNA species not detectable by Northern blot analysis. The alternative splicing of exon II introduces a tryptophan residue into the amino acid sequence at the splice junction, and the alternative splicing of exons II and IV both interrupt reported peptide sequences (peptide 2' and the 65/60/18 kDa amino terminus of IDUA, respectively, see Figure 2). Thus, it was thought that the major PCR product was most likely to represent the fuU-length mRNA encoding IDUA. Expression of this putative fuU- length mRNA would establish that the nucleotide sequence presented here in Figure 2 encodes enzymicaUy active IDUA.

PCRs were performed with reverse-transcribed fibroblast RNA as template and the primers ID60 and ID61. The 840 bp PCR product was subcloned in the pTZ19 vector to produce a "fuU-length" IDUA cDNA clone. Sequence analysis of this full-length insert found four nucleotides that were different from the previously determined sequence. The differences, numbered as in Figure 2, were A to C (base 276), G to A (base 402), T to C (base 440), and T to C (base 631). The first two differences alter the amino acid residues coded for by the cDNA from Gin to Pro (amino acid 63) and Arg to Gin (amino acid 105), respectively. The T to C (base 440) is a silent change that alters a Leu (amino acid 118) codon from TTG to CTG and introduces a second Kpri site into the cDNA. Thus, the cloned PCR product presumably resulted from partial digestion with Kpri or the Iigation of three fragments. The last change T to C (base 631) is a sUent change in the third base of an Asn (amino acid 181) codon. All of these differences may be polymorphic, but as two change amino acids, they may be transcription errors introduced by Tag DNA polymerase during PCR in the presence of high concentrations of dNTPs (400 μM) for 40 cycles (30). However, these condi were essential to produce enough PCR product to conduct the experiment.

This full-length cDNA construct was subcloned in the expression vector pRSVN.07 to produce the construct pRSVNID2L CHO cells were electropo in the in the presence of pRSVNID21, and G418-resistant colonies were sele and grown as a mass culture. CeUular extracts from control CHO cells, mix normal human skin fibroblasts, and pRSVNID21 transfected ceUs were assay for total IDUA activity by using the IDUA-specific fluorogenic substrate. C cell extract contained a low level of IDUA activity. CeUular extract from C cells transfected with pRSVNID21 gave a total activity 160-fold greater than control normal human fibroblast activity (Table 2). To compare the specific activities of the recombinant and fibroblast IDUA serial dilutions of the ceU extracts were assayed in parallel, using human IDUA-specific IDIA monoclo antibody based immunocapture (14) and ELISA assays (12). The CHO ceU extract gave sero background in both assays. The ELISA result was normali to the normal fibroblast extract and showed a 12.7 fold higher expression of human IDUA in the pRSVNID21 transfected CHO cells. The immunocaptu assay showed that this results in an almost proportional increase in EDUA ac in the transfected CHO ceUs, demonstrating that the normal and recombina enzymes have similar specific activities (Table 2). These results prove that t IDUA sequence used in this experiment codes for a protein that has a specif activity similar to the IDUA activity present in normal cultured human skin fibroblasts.

TABLE 2 Expression of IDUA

IDUA activity¹ Relative IDUA Relative IDUA

CeU Type Total Captured⁴ protein² specific activity³

CHO 1 ND ND

CHO with pRSVNID21 160 152 12.7 12.0

Normal human fibroblasts 16 12.6 1 12.6

ND none detected i Activity is in pmol x 10^'2 per min per mg of ceU protein

The amount of human IDUA protein captured in the ELISA assay per mg of ceU protein normalised against human fibroblasts.

Expressed as IDUA activity relative to IDUA protein.

IDUA activity captured in the immunocapture assay.

A further expression construct was made such that the normal 5' non-coding sequence of the IDUA mRNA, was found in the full length cDNA clone described, was replaced with 30 bp of the 5' non-coding sequence of the rat preproinsulin mRNA (5'- AACCATCAGCAAGCAGGTCATTGTTCCAACGCGTGGCC-3'). At the same time, the four nucleotide differences noted in the PCR-produced 840 bp portion of the original cDNA used for expression (A→C, bp 276; G→A, bp 402 T→C bp 440; T→C bp 631) were corrected. This ensures efficient mRNA translation (34) and has been shown to lead to high-level expression of other lysosomal enzymes in CHO cell expression systems (32,33). This modification also led to greatly enhanced expression of IDUA in CHO-K1 cells. The origi expression plasmid was also modified such that the RSV-LTR promoter elem was replaced with the human elongation factor 1 α gene promoter from pEFB (35). This promoter is 5 times more efficient in CHO-K1 cells than the RSV- LTR.

The total coding sequence, therefore, for IDUA has an open reading frame of 1959 bp encoding a peptide of 653 amino acids. A signal peptide of 26 amino acids with a consensus cleavage site (31) was present immediately adjacent to mature amino terminus of the protein (74/13 kDa amino terminus). Thus, th mature human IDUA protein of 627 amino acids has a molecular mass of 70,0 Da, which is consistent with the previous estimates of IDUA size after aUowin for post-translational modifications (5-8). AU major peptide species sequences are present in the translation of the open reading frame, totalling 234 amino a (42%) of the 627 amino acids of the mature IDUA. This includes several peptides that were present as minor sequences in peptide peaks (secondary peptides, e.g. peptide 7'). The presence of all three amino-terminal sequences from purified human liver IDUA in the peptide sequence presented in Figure supports the hypothesised model of proteolytic processing of the 74 kDa IDU polypeptide (Figure 1). Of six potential stes in the 65/60/18 kDa amino-termi sequence and peptide 8 was not detected in sequencing and may, therefore, b glycosylated. The potential glycosylation site at the very end of peptide 9 was also not defected, but this may be due to a weak signal towards the end of the sequence rather than a glycosylated residue. No significant homology was found between the human IDUA amino acid sequence and proteins in the GenBank, National Biomedical Research Foundation, or Swiss-Prot data bases (aU releases of May, 1991).

Having determined the cDNA sequence, tiie genomic sequence was then sought. The IDUA genomic sequence is valuable for defining mutations in MPS-1 patients, for defining diagnostically useful polymorphisms for MPS-1 and

Huntington's disease and for refining the genetic and physical map of the IDUA gene. The genomic sequence is shown in Figure 4A and B as two segments.

The gene for IDUA is split into 14 exons spaning approximately 19 kb. The first 2 exons are separated by a 566 bp intron and the last 12 exons are separated by a 566 bp intron and the last 12 exons are clustered in a 4.2 kb region. Two variant polyadenylation signals consistent with a 2.3 kb mRNA transcript are underlined in Figure 4B. From the position of the proposed polyadenylation signals, the mRNA produced would be 2203 and 2285 bp with an additional 20-30 prior to the poly(A) tail.

Accordingly, the potential promoter for IDUA is bounded by an Alu repeat sequence and has only GC box type concensus sequences (Figure 4A).

The fuU length cDNA and genomic sequence described herein for human IDUA makes it possible to characterise MPS-I mutations and to determine how much of the clinical variability reflects different mutations and how much reflects other genetic or environmental influeneces. Furthermore, large-scale expression of IDUA wiU provide enzyme for evaluation of enzyme therapy, for example in the dog model for MPS-I and the cDNA in the appropriate vectors may be used for experimental gene therapy in the same model. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically descri It is to be understood that the invention includes all such variations and modifications. The invention also includes aU of the steps, features, composit and compounds referred to or indicated in this specification, individuaUy or collectively, and any and all combinations of any two or more of said steps or features.

REFERENCES:

1. Neufeld, EF & Muenzer, J, (1989) The Metabolic Basis of Inherited Diseas pp 1565-1587.

2. Hopwood, JJ, (1989) Heparin: Chemical and Biological Properties, Clinical Applications, pp 190-229.

3. Shapiro, LI, Hall, CW, Leder U, & Neufeld, EF, (1976) Arch. Biochem. Biophys. 172: 156-161.

4. Rome, IH, Garvin, A, Neufeld, EF, (1978) Arch. Biochem. Biophys. 189: 344-353.

5. Myerowitz, R, & Neufeld EF, (1981) /. Biol. Chem. 256: 3044-3048.

6. Clements, PR, Brooks, DA, Saccone, GTP, & Hopwood, JJ, (1985) Ew, J. Biochem. 152:21-28.

7. Clements, PR, Brooks, DA, McCourt, PAG, & Hopwood, JJ, (1989) Biochem. J. 259: 199-208.

8. Taylor, JA, Gibson, GJ, Brooks, DA & Hopwood, JJ, (1991) Biochem. J. 274:263-268.

9. Hopwood, JJ, & Morris, CP, (1990) Mol. Biol. Med. 7: 381-404.

10. Hopwood, JJ, & Muller, V, (1979) Clin. Sci. 57: 265-272.

11. Muller, V, & Hopwood, JJ, (1984) Clin. Genet. 26: 414-421.

12. Ashton, LJ, Brooks, DA, McCourt, PAG, Clements, PR, & Hopwood, JJ, (1991) Am. J. Humn. Genet, in press

13. Spellacy, E, ShuU, RM, Constantopoulos, G, & Neufled, EF (1983) Proc. Natl. Acad. Sci. USA 80: 6091-6095.

14. Scott, HS, Ashton, LJ, Eyre, HJ, Baker, E, Brooks, DA, CaUen, DF, Sutherland, GR, Morris, CP & Hopwood, JJ, (1990) Am. J. Hum. Genet. 47: 802-807.

15. MacDonald, ME, Scott, HS, Whaley, WL, Phol, T, Wasmuth, JJ, Lehrach, H, Morris, CP, Frischuaf, AM, Hopwood, JJ, & Gusella, JF (1991) Somatic Cell Mol. Genet. 17: 421-425. 16. Stoltzfus, LY, Uhrhammer, N, Sosa-Pineda, B, Teplow, DB, & Neufeld EF, (1990) Am. J. Hum. Genet. 47: A147 (abstract 655).

17. Wilson, PJ, Morris, CP, Anson, DS, Occhiodoro, T, Bielicki, J, Clement PR & Hopwood, JJ, (1990) Proc. Nad. Acad. Sci. USA 57: 8531-8535.

18. Robertson, DA, Freeman, C, Nelson, PV, Morris, CP, & Hopwood, JJ, (1988) Biochem. Biophys. Res. Commun. 157: 218-224.

19. Chaconas, G, & van de Sande, JH, (1980) Methods Enzymol. 65: 75-88.

20. Feinberg, AP, & Vogelstein, B, (1983) Anal. Biochem. 132: 6-13.

21. Sanger, F, Nicklen, S, & Coulson, AR, (1977) Proc. Natl. Acad. Sci. US 74: 5463-5467.

22. Mizusawa, S, Nishimura, S, & Seela, F, (1986) Nucleic Acids Res. 14: 1 1324.

23. Murray, V, (1989) Nucleic Acids Res. 17: 8889.

24. Chomezynski, P, & Sacchi, N, (1987) And. Biochem. 162: 156-159.

25. Kingston, RE, (1987) Current Protocols in Molecular Biology pp 4.5.1-4.5

26. Saiki, RK, Gelfand, DH, Stoffel, S, Scharf, SJ, Higuchi, R, Horn, GT, Mullis, KB, & Erlich, HA, (1988) Science 239: 481-491.

27. Oshima, A, Kyle, JW, MiUer, RD, Hoffman, JW, Powell, P, Grubb, JH, Sly, WS, Tropak, M, Guise, S, & Gravel, RA (1987) Proc. Natl. Acad. USA 54: 685-689.

28. Morreau, H, Galjart, NJ, Gillemans, N, WiUemsen, R, van der Horts, & d'Azzo, A, (1989) J. Biol. Chem. 264: 20655-20663.

29. Quintern, LE, Schuchman, EH, Levran, O, Suchi, M, Ferlinz, K, Reink H, Sandhoff, K, & Desnick, RJ, (1989) EMBO J. 5: 2469-2473.

30. Eckert, KA, & Kunkel, TA (1990) Nucleic Acids Res. 18: 3739-3744.

31. von Heijne, G, (1986) Nucleic Acids Res. 74: 4683-4690.

32. Anson, DS, et al (1992) Biochem. J. 284: 789-794.

33. Bielicki, J, et al (1992) Biochem. J. (in press).

34. Cullen, BJ, (1988) DNA 7: 645-650.

35. Mizishima, S, & Nagata, S, (1990) BAR 18: 5322.

Claims (43)

CLAIMS:

1. An isolated nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a sequence which encodes a mammalian crL- iduronidase (IDUA) or fragment or derivative thereof.

2. The isolated nucleic acid molecule according to claim 1 wherein the nucleotides are deox ribonucleotides.

3. The isolated nucleic acid molecule according to claim 2 wherein said molecule is cDNA.

4. The isolated nucleic acid molecule according to claim 2 wherein said molecule is a genomic DNA molecule.

5. The isolated nucleic acid according to any one of claims 1 to 4 wherein the mammal is a human animal.

6. The isolated nucleic acid according to claim 5 wherein the IDUA is of liver origin.

7. The isolated nucleic acid molecule according to claim 3 or 4 wherein said molecule is carried by a vector capable of replication in a eukaryotic ceU and/or a prokaryotic ceU.

8. The isolated nucleic acid molecule according to claim 7 wherein the vector is an expression vector.

9. The isolated nucleic acid molecule according to claim 1 having a nucleotide sequence substantially as set forth in Figure 2 or having at least 40% similarity to a region thereof.

10. The isolated nucleic acid molecule according to claim 9 wherein the percentage simUarity is at least 60%.

11. The isolated nucleic acid molecule according to claim 10 wherein the percentage homology is at least 80%.

12. The isolated nucleic acid molecule according to claim 8 when expressed eukaryotic cells.

13. The isolated nucleic acid molecule according to claim 1 when expressed mammalian cells.

14. A recombinant mammalian α-L-iduronidase (IDUA) or fragment or derivative thereof.

15. The recombinant mammalian IDUA according to claim 14 in substantia pure form.

16. The recombinant mammalian IDUA according to claim 14 or 15 when expressed in mammalian, yeast or insect cells.

17. The recombinant mammalian IDUA according to claim 16 when express in mammalian ceUs.

18. The recombinant mammalian IDUA according to claim 17 having an altered glycosylation pattern compared to the naturally occurring molecu

19. The recombinant mammalian IDUA according to claim 18 being more highly glycosylated compared to the naturally occurring molecule.

20. The recombinant mammalian IDUA according to claim 14 wherein the mammalian IDUA is of human origin.

21. The recombinant mammalian IDUA according to claim 14 when fused to another proteinaceous molecule.

22. The recombinant mammalian IDUA according to claim 21 wherein said other proteinaceous molecule is an enzyme, reporter molecule, purification site and/or a signal sequence.

23. The recombinant mammalian IDUA according to claim 14 having an amino acid sequence substantially as set forth in Figure 2 or having at least 40% similarity to a region thereof.

24. A method for treating a patient suffering from α-L-iduronidase (IDUA) deficiency said method comprising administering to said patient an effective amount of recombinant mammalian IDUA or active fragment or derivative thereof.

25. The method according to claim 24 wherein the mammalian IDUA is of human origin.

26. The method according to claim 24 wherein the patient is suffering from mucopolysaccharidosis type I.

27. The method according to claim 24 or 25 wherein the recombinant IDUA is expressed in mammalian cells.

28. The method according to claim 27 wherein the recombinant IDUA comprises an altered glycosylation pattern compared to the naturaUy occurring enzyme.

29. The method according to claim 28 wherein the IDUA is more highly glycosylated than the naturally occurring molecule.

30. The method according to any one of claims 24 to 28 wherein administration of the IDUA is by oral, intravenous, suppository, intraperetoneal, intramuscular, intranasal, intradermal or subcutaneous administration by infusion or implantation or by gene therapy.

31. The method according to claim 30 wherein the method of administration by intravenous injection or by gene therapy.

32. A pharmaceutical composition comprising recombinant mammalian α-L- iduronidase (IDUA) or an active fragment or derivative thereof and one more pharmaceutically acceptable carriers and/or dUuents.

33. The pharmaceutical composition according to claim 32 wherein the mammalian IDUA is of human origin.

34. The pharmaceutical composition according to claim 32 or 33 when expressed in a mammalian cell.

35. The pharmaceutical composition according to claim 34 wherein the IDU has an altered glycosylation pattern compared to the naturaUy occurring molecule.

36. The pharmaceutical composition according to claim 35 wherein the IDU is more highly glycosylated compared to the naturally occurring molecule.

37. A pharmaceutical composition comprising recombinant mammalian IDU when used in the method according to claim 24.

38. Use of recombinant mammalian α-L-iduronidase (IDUA) or an active fragment or derivative thereof in the manufacture of a medicament for t treatment of IDUA deficiency is a patient.

39. The use according to claim 38 wherein the mammalian IDUA is of human origin.

40. The use according to claim 38 or 39 wherein the patient is suffering from mucopolysaceharidosis type I.

41. The use according to claim 40 wherein the IDUA is expressed in mammalian ceUs.

42. The use according to claim 41 wherein the IDUA has an altered glycosylation pattern compared to the naturaUy occurring molecule.

43. The use according to claim 42 wherein the IDUA is more highly glycosylated compared to the naturally occurring molecule.