EP0578790A1 - Alpha-l iduronidase synthetique et sequences genetiques la codant - Google Patents

Alpha-l iduronidase synthetique et sequences genetiques la codant

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Publication number
EP0578790A1
EP0578790A1 EP92923074A EP92923074A EP0578790A1 EP 0578790 A1 EP0578790 A1 EP 0578790A1 EP 92923074 A EP92923074 A EP 92923074A EP 92923074 A EP92923074 A EP 92923074A EP 0578790 A1 EP0578790 A1 EP 0578790A1
Authority
EP
European Patent Office
Prior art keywords
idua
mammalian
molecule
nucleic acid
isolated nucleic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP92923074A
Other languages
German (de)
English (en)
Other versions
EP0578790A4 (fr
Inventor
Hamish Steel Scott
Donald Stewart Anson
Annette Marie Orsborn
Paul Victor Nelson
Peter Roy Clements
Charles Phillip Morris
John Joseph Hopwood
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Womens and Childrens Hospital Adelaide
Original Assignee
Womens and Childrens Hospital Adelaide
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Filing date
Publication date
Application filed by Womens and Childrens Hospital Adelaide filed Critical Womens and Childrens Hospital Adelaide
Publication of EP0578790A1 publication Critical patent/EP0578790A1/fr
Publication of EP0578790A4 publication Critical patent/EP0578790A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01076—L-Iduronidase (3.2.1.76)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to ⁇ -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 ⁇ -L-iduronidase deficiency.
  • the lysomal enzyme ⁇ -L-iduronidase (IDUA; glycosaminoglycan ⁇ -L- iduronohydrolase, EC 3.2.1.76) hydrolyzes the nonreducing terminal ⁇ -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).
  • IDUA 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).
  • 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
  • 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 ⁇ rL-iduronidase (IDUA) or fragment or derivative thereof or its like molecule.
  • IDUA mammalian ⁇ rL-iduronidase
  • 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.
  • the IDUA is isolatable from the liver.
  • 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.
  • 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.
  • 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.
  • synthetic includes recombinant forms and molecules produced by t sequential addition of amino acid residues, or groups of amino acid residues, i defined order.
  • 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.
  • 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.
  • the mammal is human but may be of n human origin as contemplated above.
  • 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:
  • 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.
  • deletions or insertions are made in adjacent pairs, i.e. a deletion of two residues or insertion of two residues.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • IDUA is in recombinant form.
  • Enzyme therapy Such a method is referred to as "enzyme therapy”.
  • 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
  • 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.
  • Dosage procedures 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.
  • 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.
  • 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
  • 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.
  • the enzyme will be coated by, or administered with, material to prevent its inactivation.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions and preparations 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.
  • a binder such as gum gragacanth, acacia, corn starch or gelatin
  • excipients suc dicalcium phosphate such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or a flavo agent such as peppermint, oil of wintergreen, or cherry flavour
  • 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.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compound may be incorporated into sustained-release reparations and formulations.
  • 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).
  • a deficiency in the naturally occurring enzyme e.g. MPS-1
  • FIGURE 1 is a schematic representation showing a model to connect the sev major polypeptides in immune purified human liver IDUA present after
  • 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 ⁇ RPCI extended from base 541 to ba 1269 and ⁇ E8A 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.
  • 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.
  • Exons III to XIV of the human IDUA gene are shown in this 4.5 kb segment. Potential polyadenylation signals are underlined.
  • AH oligonucleotides were synthesised on an Applied Biosystems 391 DNA synthesiser. ED47, 5'-
  • AACTTCGAGACCTGGAACGAGCCCGACCAGCACGACTTCGACAACGT- 3' was used for initial library screening.
  • IDUA-specific primers used for PCR from cDNA were IDNT, ID39, ID56, ID57, ID58, ID60 and ID61 (see Figure 2). Library Screening.
  • 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.
  • 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 ⁇ g of total RNA and 10 and 40 ⁇ g of poly (a)+ RNA as described (17).
  • RNA (3 ⁇ g) 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 ⁇ l.
  • Incubation at 37°C fo m was followed by hydrolysis of the RNA by the addition of 5 ⁇ l of 3 M NaOH and further incubation at 37° for 30 min. The NaOH was neutralised by the addition of 1.25 ⁇ l of 10.3 M HC1, and the cDNA was precipitated and resuspended in 50 ⁇ l of water.
  • Each PCR used 5 ⁇ l of cDNA.
  • PCR reagents were as described by Saiki et al (26) except that the final concentrations of deoxynucleotides were 400 ⁇ M 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.
  • FMC Nusieve GTG agarose
  • IDUA 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 100 bp 3' of a unique Kpri. restriction iste (bases 818-823, see Figure 2). Utilizing the Hir ⁇ lH and the Kpri.
  • 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 ⁇ 8A.
  • aU 48 clones were analysed and only one was found to be correct (fuU length).
  • This insert was excised with Hir ⁇ lll 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.
  • 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 ⁇ g/m streptomycin sulfate at 100 ⁇ g/ml, and kanamycin sulfate at 120 ⁇ g/ml at 37 ° a 5% v/v C0 2 atmosphere.
  • CHO ceUs (1.2 x 10 7 ) were electroporated at 0 °C using a BRL Cell-Porator at a pulse of 330 ⁇ F and 275 V in the presence 15 ⁇ 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 ⁇ g/ml.
  • G418 sulfa Geneticin; GIBCO
  • a bulk culture of resistant cells was extracted (14) and assayed for IDUA activity with the fluorogenic substrate 4- methylumbeUiferyl ⁇ -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).
  • ID47 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 ( ⁇ RPCl, bases 541-1269; see Figure 2) from the ⁇ gtlO 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 ⁇ RPCI insert was then used to screen a ⁇ gtll human endothelial cDNA library. Twenty clones were isolated, and the insert of the longest clone, ⁇ E8A, 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.
  • peptide Z' secondary tryptic peptide
  • 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 using the oligonucleotide pairs ID56 to ID57 IDNT to ID39 produced two products per reaction.
  • the smaUer products were 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.
  • the alternatively spliced mRNA species individuaUy missin exons II and IV would produce peptide products of 606 and 617 amino acids, respectively.
  • 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.
  • 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 ⁇ M) 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).
  • IDUA activity relative to IDUA protein.
  • 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').
  • 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.
  • the origi expression plasmid was also modified such that the RSV-LTR promoter elem was replaced with the human elongation factor 1 ⁇ 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).
  • 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.
  • 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
  • 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.
  • 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.
  • 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.

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Abstract

La présente invention concerne généralement l'alpha-L-iduronidase et les séquences génétiques la codant. Plus particulièrement, la présente invention concerne une molécule d'acide nucléique isolée comprenant une séquence de nucléotides qui codent ou qui sont complémentaires à une séquence codant l'alpha-L-iduronidase des mammifères ou un fragment ou un dérivé de celle-ci et à une enzyme recombinante codée par ce moyen. Ces molécules sont utiles dans l'investigation, le diagnostic et le traitement de sujets suspectés de souffrir d'une insuffisance d'alpha-L-iduronidase.
EP9292923074A 1991-11-14 1992-11-12 Alpha-l iduronidase synthetique et sequences genetiques la codant. Withdrawn EP0578790A4 (fr)

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BR9910323A (pt) * 1998-05-13 2001-01-30 Harbor Ucla Alfa-l-iduronidase recombinante, métodos para produção e purificação da mesma e métodos para tratamento de doenças ocasionadas deficiência da mesma
US6426208B1 (en) 1999-11-12 2002-07-30 Harbor-Ucla Research And Education Institute Recombinant α-L-iduronidase, methods for producing and purifying the same and methods for treating diseases caused by deficiencies thereof
US6569661B1 (en) 1999-11-12 2003-05-27 Biomarin Pharmaceutical Inc. Recombinant α-L-iduronidase, methods for producing and purifying the same and methods for treating diseases caused by deficiencies thereof
US6585971B1 (en) 1999-11-12 2003-07-01 Harbor-Ucla Research And Education Institute Recombinant α-L-iduronidase, methods for producing and purifying the same and methods for treating disease caused by deficiencies thereof
GB0606190D0 (en) 2006-03-28 2006-05-10 Isis Innovation Construct
US8679478B2 (en) 2010-10-04 2014-03-25 Duke University Methods of lysosomal storage disease therapy
CA2815212A1 (fr) 2010-10-22 2012-04-26 Curna, Inc. Traitement de maladies associees a l'alpha-l-iduronidase (idua) par inhibition du transcrit antisens endogene de idua
WO2017049157A1 (fr) 2015-09-18 2017-03-23 Duke University Méthodes et compositions pour le traitement de troubles associés à une stéatose
IL268076B2 (en) * 2017-01-31 2024-10-01 Regenxbio Inc Treatment of mucopolysaccharidoses I with human iduronidase L (IDUA) and full human glycosylations
EP3676385A1 (fr) 2017-07-06 2020-07-08 The Trustees of The University of Pennsylvania Thérapie génique médiée par aav9 pour traiter la mucopolysaccharidose de type i
EP3684938A1 (fr) 2017-09-22 2020-07-29 The Trustees of the University of Pennsylvania Thérapie génique pour le traitement de la mucopolysaccharidose de type ii

Non-Patent Citations (5)

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Title
EUROPEAN JOURNAL OF BIOCHEMISTRY vol. 152, no. 1 , October 1985 , BERLIN, DE pages 21 - 28 CLEMENTS, P. ET AL. 'Human alpha-L-iduronidase. 1. Purification, monoclonal antibody production, native and subunit molecular mass' *
GENOMICS vol. 13, no. 4 , August 1992 SCOTT, H. ET AL. 'Structure and sequence of the human alpha-L-iduronidase gene' *
HUMAN GENETICS vol. 65, no. 3 , 1984 pages 268 - 272 FUJIBAYASHI, S. ET AL. 'Properties of alpha-L-iduronidase in cultured skin fibroblasts from alpha-L-iduronidase-deficient patients' *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA vol. 88, no. 21 , 1 November 1991 , WASHINGTON US pages 9695 - 9699 SCOTT, H. ET AL. 'Human alpha-L-iduronidase: cDNA isolation and expression' *
See also references of WO9310244A1 *

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