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WO1990011353A1 - Alpha-galactosidase de recombinaison, une therapie contre la maladie de fabry - Google Patents

Alpha-galactosidase de recombinaison, une therapie contre la maladie de fabry Download PDF

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Publication number
WO1990011353A1
WO1990011353A1 PCT/US1990/001571 US9001571W WO9011353A1 WO 1990011353 A1 WO1990011353 A1 WO 1990011353A1 US 9001571 W US9001571 W US 9001571W WO 9011353 A1 WO9011353 A1 WO 9011353A1
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gly
ala
asp
ser
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PCT/US1990/001571
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English (en)
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David H. Calhoun
George Coppola
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Research Corporation Technologies, Inc.
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Publication of WO1990011353A1 publication Critical patent/WO1990011353A1/fr

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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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)
    • C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • Fabry disease results from an X-linked deficiency in the enzyme ⁇ -galactosidase A.
  • the present invention is directed to recombinant human ⁇ -galactosidase A and provides baculovirus expression vectors and recombinant virus that provide stable expression of extracellular and intracellular levels of this enzyme in an insect cell culture.
  • the recombinant-derived enzyme can be used in enzyme replacement therapy to treat Fabry patients.
  • Compositions useful in therapeutic administration of ⁇ -galactosidase A are also provided.
  • Fabry disease is an X-linked inborn error of metabolism resulting from a deficiency of the lysosomal enzyme, ⁇ -galactosidase A.
  • Deficiency of ⁇ -galactosidase A results in the accumulation of its major glycosphingolipid substrate, globotriaosylceramide and related glycolipids with terminal ⁇ -galactosidic linkages.
  • Fabry diseases is one of approximately 30 lysosomal storage diseases known to affect humans. Each of these diseases result from an inherited trait which affects the levels of enzymes in the lyosome. Tay-Sach's disease and Gauucher disease are members of this group of diseases. Since specific pathways for the uptake of these other lysosomal enzymes also exist, enzyme replacement therapy is expected to be effective in Fabry disease and could logically be expected to be successful in these other diseases as well. 1 Although these diseases are individually rare, (e.g., several thousand patients with Fabry disease are known to occur world wide, i.e., 1 to 40,000), as a group this class of diseases t- accounts for a significant fraction of all inherited diseases.
  • the ⁇ -galactosidase A enzyme is a lysosomal enzyme which hydrolyzes globotriaosylceramide and related glycolipids which have terminal ⁇ -galactosidase linkages. It is a 45 kDa N-glycosylated protein encoded on the long arm of the X chromosome.
  • the partial cDNA clone was used to construct an E. coli expression vector by placing the ⁇ -galactosidase A coding sequence under control of the trp promoter
  • a full length cDNA of human ⁇ -galactosidase A is needed which can be incorporated into an expression vector under control of a strong promoter.
  • this vector r- should provide stable expression of the cDNA and use a host system in which the processing and glycosylation may occur.
  • One such expression vector is provided by the baculovirus expression system of the present invention.
  • the early baculovirus expression vectors employ a strong promoter for a nonessential gene, the polyhedrin gene.
  • the polyhedrin gene To facilitate cloning, a DNA sequence encoding several restriction endonuclease sites had been inserted into the polyhedrin
  • the present invention is directed to replicable expression vectors that express the human ⁇ -galactosidase A enzyme.
  • These vectors are baculovirus derivatives which have c been genetically engineered to contain a full length cDNA or recombinant DNA encoding the precursor form of ⁇ -galactosidase A.
  • the cDNA for example, is operably linked to -the baculovirus polyhedrin promoter so that the promoter directs expression of biologically active, human 0 ⁇ -galactosidase A under the appropriate conditions.
  • This promoter can be either a genetically engineered polyhedrin promoter with an insertion of DNA encoding several restriction endonuclease sites or the wild type polyhedrin promoter.
  • Transformant microorganisms or cell cultures j c; carrying these vectors are also provided by the present invention.
  • Another aspect of the present invention provides recombinant baculoviruses having a gene sequence for the precursor form of a human ⁇ -galactosidase A enzyme and which
  • a further aspect of this invention provides homogenous recombinant human ⁇ -galactosidase A and antibodies directed against this enzyme or directed against any active fragment or derivative of the enzyme.
  • a still further aspect of the present invention provides pharmaceutical compositions comprising an effective amount of recombinant human ⁇ -galactosidase A, or an active derivative thereof, and a pharmaceutically acceptable carrier. These compositions are useful in treating diseases go associated with a deficiency of this enzyme, especially Fabry disease.
  • Fig. 1 depicts the human ⁇ -galactosidase cDNA features encoded on M13.L21.1 (top), the restriction enzyme recognition sites in the polylinker region of pSPRl (middle) t - and a partial restriction map and some of the features of pAc373, a baculovirus transfer vector (bottom) .
  • Fig. 2 depicts the differences in DNA sequence between transfer vectors pCC4 (top) and pCC14 (bottom).
  • Elimination of 93 bp from the promoter sequences of pCC4 0 (underlined) and replacement with 7bp (underlined) in pCC14 increases ⁇ -galactosidase A protein expression at least 2-10 fold.
  • the ATG start codon (box) is indicated in both sequences.
  • the present invention is directed to replicable p expression vectors which are used to express human ⁇ -galactosidase A.
  • These vectors are recombinant baculoviruses having an ⁇ -galactosidase A cDNA under control of the Autographa californica nuclear polyhedrosis virus (AcMNPV) polyhedrin promoter or any other strong baculovirus o promoter capable of expressing foreign genes.
  • An insect cell culture for example Spodoptera frugiperda line Sf9, infected with this recombinant virus expresses ⁇ -galactosidase A both intracellularly and extracellularly at unexpected levels.
  • Baculovirus expression vectors and their use are reviewed in Luckow et al.
  • the baculoviruses have double-stranded circular DNA genomes approximately 130 kb in length. These viruses have a wide host range infecting about 30 species of Lepidopterm insects.
  • AcMNPV infection of Spodoptera frugiperda cell cultures produce very o high levels of a protein known as polyhedrin which may account for 50% of the cellular protein.
  • Polyhedrin is a protein known as polyhedrin which may account for 50% of the cellular protein.
  • the recombinant viruses are r- then used to infect a S . frugiperda cell culture to produce the desired protein.
  • a particular advantage of this system is its similarity to higher eukaryotes with regard to protein modification, processing and transport. Thus, recombinant- derived eukaryotic proteins will be processed and 0 glycosylated in a manner important for biological activity.
  • To produce a recombinant baculovirus which expresses a foreign gene that gene is inserted into a transfer vector downstream of the polyhedrin promoter.
  • the transfer vector is a plasmid which has been genetically
  • engineered polyhedrin promoter can be mainpulated, e.g. its sequences changed to
  • the transfer vector and viral DNA are used to co-transfect insect cell cultures. Homologous recombination will occur between the transfer vector and viral DNA during transfection and the
  • Occ ⁇ occlusion negative virus
  • the recombinant baculovirus expression vectors of the present invention are made by inserting a cDNA or a recombinant DNA for the precursor form of human ⁇ -galactosidase A downstream of the polyhedrin promoter in an baculovirus transfer vector, for example the AcMNPV transfer vector pAc373.
  • the precursor form consists of the signal peptide and the mature ⁇ -galactosidase A. This transfer vector and many other suitable vectors are described in
  • the pSPRl polylinker is composed of two smaller polylinkers cloned head to head at a unique EcoRI site, it provides a convenient way to introduce a variety of restriction enzyme sites on the ends of any EcoRI fragment.
  • 25 ⁇ -galactosidase A gene is then cloned into the unique BamHI site in pAc373.
  • the resulting transfer vector, designated pCC4 contains the complete ⁇ -galactosidase A cDNA in the proper orientation relative to the polyhedrin promoter.
  • pCC4 DNA was cut with EcoRV and BssHII restriction endonucleases and the 213 bp EcoRV/BssHII fragment was discarded. This deletes 178 bp c upstream- and 35 bp downstream of the ⁇ -galactosidase A ATG start codon of pCC4 (See Figure 2).
  • 4 overlapping oligonucleotides were synthesized, annealed together and
  • 20 can increase up to 1000-fold for ⁇ -galactosidase A, relative to expression from pCC4.
  • a transfer vector carrying that DNA for example pCC4 or pCC14,
  • -y e - and AcMNPV DNA are used to cotransfect an insect cell culture line, preferably £. frugiperda cell line Sf9.
  • the transfer vector and viral DNA undergo recombination between the homologous baculovirus sequences which flank the ⁇ -galactosidase A gene and thereby produce the desired go recombinant baculovirus.
  • the viral progeny from the transfection are
  • CAC ATC AGC CCT CAA GCC AAA GCT CTC CTT CAG GAT AAG GAC GTA 855 His He Ser Pro Gin Ala Lys Ala Leu Leu Gin Asp Lys Asp Val 285
  • the recombinant baculoviruses of the present invention provide high level, stable expression of biologically active human ⁇ -galactosidase A.
  • the stable expression of high levels of biologically active human is a stable expression of biologically active human ⁇ -galactosidase A.
  • 1 ⁇ -galactosidase A is unique to the present invention. Such expression and activity is particularly high in insect cell cultures infected with these recombinant baculoviruses in
  • Recombinant ⁇ -galactosidase A activity is present in the culture medium as well as the cells, with the 'majority of the activity found in the culture medium. Extracellular, recombinant enzyme activity is thus achieved by the present
  • the expression system of the present invention provides glycosylated ⁇ -galactosidase A which is biologically active.
  • the ⁇ -gaiactosidase A activity can be measured using a fluorescent substrate, 4-methylumbelliferyl- ⁇ -D-galactopyranoside, for example.
  • Another aspect of the present invention provides homogeneous recombinant human ⁇ -galactosidase A.
  • Homogeneous preparations are particularly useful for enzyme replacement r- therapy of Fabry disease and any other disease resulting from a deficiency of ⁇ -galactosidase A.
  • Recombinant production of ⁇ -galactosidase A provides a plentiful source of active enzyme which was heretofore unavailable from natural sources or other recombinant sources.
  • the increased ⁇ n activity relative to the E. coli expression system already described is due to glycosylation and processing of the enzyme neither of which occurred when the ⁇ -galactosidase was expressed in E. coli.
  • Purification of recombinant ⁇ -galactosidase A from l -,p r- the culture medium or the intact cells, if desired, is achieved by conventional purification means such is ammonium sulfate precipitation, column chromatography and the like by following the enzymatic activity of the recombinant ⁇ -galactosidase A by the assay described herein.
  • One preferred scheme to purify recombinant ⁇ -galactosidase A produced by a baculovirus expression vector of the present invention involves harvesting the culture supernatent when the ⁇ -galactosidase A activity is at a peak, typically about 48 to 72 hours after viral infection of Sf9 cells.
  • the proteins in the supernatent are precipitated by ammonium sulfate, dialyzed into an appropriate buffer and applied to a Concanavalin A-Sepharose (Pharmacia trademark) chromatography resin.
  • the resin is eluted with 0.1 M ⁇ -meth ⁇ lglucoside to remove contaminants and then with 1 M Q ⁇ -methylglucoside to release the bound ⁇ -galactosidase A activity. After concentrating and dialyzing the eluate which
  • the present invention also provides polyclonal and monoclonal antibodies to recombinant ⁇ -galactosidase A,
  • Monoclonal antibodies are conveniently prepared by immunizing mice with homogeneous or partially purified recombinant ⁇ -galactosidase A.
  • fragments or n active derivatives of ⁇ -galactosidase A may be used for immunization. These fragments may be made by proteolytic digestion and purified by conventional means.
  • the enzyme derivatives may be made by chemical modification or site-directed mutagenesis of the cloned ⁇ -galactosidase A
  • any of these ⁇ -galactosidase A preparation are used to prepare polyclonal antibodies in rabbits or other animals such as goat, sheep, rats or the like. Methods of identifying the desired antibody include ELISA assay using any purified ⁇ -galactosidase A as the test
  • the antibodies are useful to affinity purify large quantities of ⁇ -galactosidase A, including the recombinant enzyme.
  • the present invention also provides
  • ⁇ -galactosidase A to homologous human proteins including, but not limited to, albumin, insulin and apoprotein B. Fusions may be constructed to specific fragments of these genes that stabilize the ⁇ -galactosidase A and retain specific receptors for endocytosis.
  • This technique also contemplates the genetically altered versions of the proteins employed where, for example, it may be advantageous to eliminate the biological activity of the protein, e.g., insulin, while
  • Another aspect of the present invention provides recombinant human ⁇ -galactosidase A as a valuable therapeutic agent for treating diseases resulting from deficiencies of this enzyme, especially Fabry disease, in a mammal by commandeing to said mammal an effective amount of recombinant ⁇ -galactosidase A or an active derivative or fragment thereof for a time and under conditions sufficient to treat the deficiency by increasing enzyme level.
  • ⁇ -galactosidase A may range from about 50 to about 10,000 units enzyme activity per kg body weight per day.
  • a unit of ⁇ -galactosidase A activity is as defined in Calhoun et al. , with one unit corresponding to one nanomole of
  • the subject invention contemplates treating Fabry disease or other diseases resulting from a deficiency of ⁇ -galactosidase A in mammals by administering a r- pharmaceutical composition containing a pharmaceutically effective amount of recombinant ⁇ -galactosidase A or an active fragment or derivative thereof.
  • a method for treating Fabry disease (or other diseases characterized by this enzyme deficiency) in a mammal is P contemplated in which a nucleic acid molecule encoding ⁇ -galactosidase A is introduced into a cell in such a manner that said nucleic acid molecule is expressed intracellularly.
  • active fragments any part of the enzyme which is derived from the intact whole enzyme and still retains ⁇ c - biological activity.
  • derivatives of ⁇ -galactosidase mean enzymes which have been chemically modified or genetically engineered to effect minor changes, for example amino acid substitutions, which maintain biological activity. Such expression may be extrachromosomal
  • the nucleic acid molecule may be carried to the cell and transferred into said cell by a second nucleic acid molecule (e.g., various viruses).
  • the first nucleic acid molecule is manipulated such that it contains the appropriate signals for expression in the target cell. That is, in accordance with the present invention, a method of treating Fabry disease in a mammal is contemplated by administering a first nucleic acid molecule encoding go ⁇ -galactosidase A.
  • This nucleic acid is contained in a pharmacologically acceptable second nucleic acid carrier such
  • said first nucleic acid enters a target cell and is either maintained extrachromosomally or integrated into the genome of said target.
  • expression of the first nucleic acid produces an ' effective amount of human ⁇ -galactosidase A.
  • the active ingredients of the pharmaceutical compositions comprising recombinant ⁇ -galactosidase A are contemplated to exhibit excellent and effective therapeutic activity in replacing the enzymatic deficiency found in Fabry disease or other conditions resulting from this deficiency.
  • the active ingredients of the therapeutic compositions including recombinant ⁇ -galactosidase A exhibit enzymatic activity when administered in therapeutic amounts from about
  • the dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • a decided practical advantage is that the active compound may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intra ⁇ muscular, intravenous, intranasal, intradermal, subcutaneous, or suppository routes.
  • the active ingredients of a recombinant ⁇ -galactosidase A-containing pharmaceutical composition may be required to be coated in a material to protect said ingredients from the action of enzymes, acids or other natural conditions.
  • the active compounds may also be administered parenterally or intraperitoneally.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of - 18- 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 carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and 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 surfactants.
  • microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • 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.
  • Coatings for ⁇ -galactosidase A preparations are useful to reduce degradation of the enzyme when administered as a therapeutic agent. Coatings also reduce the - 19-
  • -- is polyethylene glycol
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the reguired other ingredients from those enumerated above.
  • the active compound may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed c - into tablets, or it may be incorporated directly with the food of the diet.
  • the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • compositions and preparation should contain at least 1% of active compound. The percentage of the compositions and preparation should contain at least 1% of active compound.
  • 1 preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit.
  • compositions is such that a suitable dosage is obtained.
  • D Preferred compositions or preparations according to the present invention are prepared so that an oral unit dosage form contains between about 10 ug and 1000 ug of active compound.
  • the tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum agragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant
  • the dosage form when it is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other p materials may be present as coatings or to otherwise modify the physical form of the unit dosage. 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 pj . 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
  • _ used herein refers to physically discrete units suited as D unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the principal active ingredient is compounded for convenient n and effective administration in pharmaceutically effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed.
  • a unit dosage form can, for example, contain the principal active compound in amounts ranging from 10 ug to about 1000 ug. Expressed in r _ proportions, the active compound is generally present in from about 10 ug to about 1000 ug/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and
  • Supplementary active ingredients can also be incorporated into the compositions.
  • Human ⁇ -Galactosidase A The clone that contains the complete coding region for the precursor form of human ⁇ -galactosidase A, M13.L21.1 (Hanztopoulos) was digested with
  • recombinants were selected for ampicillin resistance, and restriction mapped to identify two clones having the pSPRl-derived BamHI fragment inserted into the BamHI site of pAc373 in opposite orientations.
  • These clones are the transfer vectors pCC4 which has the ⁇ -galactosidase A cDNA in the orientation properly aligned the polyhedrin promoter and pCC5 which has the cDNA in the opposite orientation.
  • the vector pCC5 is a control plasmid.
  • Transfer vector pCC4 or pCC5 were co-transfected with AcMNPV DNA into S. frugiperda Sf9 cells by the methods described in Summers et al. About 3% of the plaques were Occ ⁇ . Several recombinant viral clones were plaque purified -24- three times from the Occ clones. The resulting recombinant baculoviruses from transfer vector pCC4 are designated AcCCl and AcCC2. The control recombinant virus from transfer vector pCC5 is designated AcCC3.
  • Fig. 1 Restriction maps of M13.L21.1 and pAc373 are shown in Fig. 1 (top and bottom). The center portion of Fig. 1 depicts the polylinker region of pSRPl.
  • D used to infect Sf9 cells. Cells were cultured and infections carried out as described in Summers et al. Exponential phase infected cells were harvested, separated from the culture medium, and both the cells and the culture medium tested for
  • ⁇ -galactosidase A activity The wild type virus, AcMNPV and mock infected cells were included as controls.
  • the activity of ⁇ -galactosidase A was measured using the fluorescent substrate, 4-methylumbelliferyl- ⁇ -D-galactopyrassoside and is expressed as units (nanomoles per hour at 37°C) present in
  • Table 1 indicates that recombinant viruses AcCCl and AcCC2 express high levels of active recombinant human ⁇ -galactosidase A in the culture medium and significant
  • control viruses AcCC3, and AcMNPV as well as mock infections have relatively little enzyme activity.
  • the recombinant viruses AcCCl and AcCC2 express about 1000-fold higher levels of enzyme activity than the recombinant control virus, AcCC3.
  • EXAMPLE 3 Construction and Expression of Human ⁇ -Galactosidase A in a Modified Baculovirus Vector: Transfer vector pCC4, containing the human ⁇ -galactosidase A cDNA cloned in proper orientation to the modified polyhedrin promoter derived from pAc373, was digested with EcoRV and BssHII (enzymes and used as recommended by the manufacturer, New England Biolabs). The 213 bp EcoRV/BssHII fragment was discarded.
  • SF9 cells as described in Example 2, virally infected cloned cells were tested for ⁇ -galactosidase A activity.
  • the recombinant virus which has incorporated pCC14 sequences is called AcCC4 and produce at least 2-10 fold more -27- ⁇ -galactosidase A activity than does the pCC4 containing virus AcCCl after on round of plague purification.
  • the ⁇ -galactosidase A levels can increase up to about 1000 fold relative to AcCCl-derived enzyme levels.

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Abstract

La maladie de Fabry est le résultat d'une insuffisance de liaison X, se produisant dans l'enzyme α-galactosidase A. L'invention concerne une α-galactosidase A humaine de recombinaison, et fournit des vecteurs d'expression de baculovirus ainsi qu'un virus de recombinaison produisant une expression stable de niveau extracellulaire et intracellulaire de cette enzyme, dans une culture cellulaire d'insecte. On peut utiliser l'enzyme dérivée par recombinaison, dans une thérapie de remplacement d'enzyme destinée à traiter des patients atteints de la maladie de Fabry. L'invention concerne également une composition utile dans l'administration thérapeutique d'une α-galactosidase A.
PCT/US1990/001571 1989-03-24 1990-03-23 Alpha-galactosidase de recombinaison, une therapie contre la maladie de fabry WO1990011353A1 (fr)

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US32842189A 1989-03-24 1989-03-24
US328,421 1989-03-24

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Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GR900100374A (en) * 1989-05-16 1991-10-10 Aek Dev Corp Composition and method for reducing gastro-intestinal distress due to alpha-d-galactoside-linked/containing sugars
EP0529160A1 (fr) * 1990-07-16 1993-03-03 The Regents Of The University Of California Produits de gène contenant des proteines et méthodes de thérapie cellulaire
EP0554385A4 (en) * 1990-10-24 1994-08-17 Sinai School Medicine Cloning and expression of biologically active alpha-n-acetylgalactosaminidase
US5401650A (en) * 1990-10-24 1995-03-28 The Mount Sinai School Of Medicine Of The City University Of New York Cloning and expression of biologically active α-galactosidase A
WO1998011206A3 (fr) * 1996-09-13 1998-08-13 Transkaryotic Therapies Inc TRAITEMENT POUR CARENCE EN α-GALACTOSIDASE A
US6083725A (en) * 1996-09-13 2000-07-04 Transkaryotic Therapies, Inc. Tranfected human cells expressing human α-galactosidase A protein
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US6458574B1 (en) 1996-09-12 2002-10-01 Transkaryotic Therapies, Inc. Treatment of a α-galactosidase a deficiency
WO2003090695A2 (fr) 2002-04-25 2003-11-06 Transkaryotic Therapies, Inc. Traitement d'un deficit en alpha-galactosidase a
AU2003220717B2 (en) * 1996-09-13 2007-10-18 Shire Human Genetic Therapies, Inc. Therapy for alpha-galactosidase a deficiency
US20120220632A1 (en) * 2006-05-16 2012-08-30 Amicus Therapeutics, Inc. Methods For Treatment of Fabry Disease
WO2014017088A1 (fr) 2012-07-26 2014-01-30 Jcr Pharmaceuticals Co., Ltd. Procédé de production d'alpha-galactosidase a humaine recombinante
WO2014152673A1 (fr) 2013-03-14 2014-09-25 Shire Human Genetic Therapies, Inc. Évaluation quantitative de l'efficacité de coiffage de l'arn messager
WO2014152659A1 (fr) 2013-03-14 2014-09-25 Shire Human Genetic Therapies, Inc. Évaluation quantitative pour l'efficacité de coiffage de l'arn messager
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WO2016117341A1 (fr) 2015-01-22 2016-07-28 Jcr Pharmaceuticals Co., Ltd. Procédé de purification d'alpha-galactosidase a humaine de recombinaison à partir d'une matière contenant des protéines de cellules hôtes polluantes
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US9937246B2 (en) 2013-03-14 2018-04-10 Icahn School Of Medicine At Mount Sinai Therapeutic acid ceramidase compositions and methods of making and using them
US9999618B2 (en) 2007-04-26 2018-06-19 Amicus Therapeutics, Inc. Dosing regimens for the treatment of lysosomal storage diseases using pharmacological chaperones
US10350277B2 (en) 2011-09-07 2019-07-16 Icahn School Of Medicine At Mount Sinai Ceramidase and cell differentiation
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EP0529160A1 (fr) * 1990-07-16 1993-03-03 The Regents Of The University Of California Produits de gène contenant des proteines et méthodes de thérapie cellulaire
EP0554385A4 (en) * 1990-10-24 1994-08-17 Sinai School Medicine Cloning and expression of biologically active alpha-n-acetylgalactosaminidase
US5401650A (en) * 1990-10-24 1995-03-28 The Mount Sinai School Of Medicine Of The City University Of New York Cloning and expression of biologically active α-galactosidase A
US5580757A (en) * 1990-10-24 1996-12-03 The Mount Sinai School Of Medicine Of The City University Of New York Cloning and expression of biologically active α-galactosidase A as a fusion protein
EP1375665A1 (fr) * 1990-10-24 2004-01-02 The Mount Sinai School Of Medicine Of The City University Of New York Compositions d'enzymes lysosomales
EP0670896A4 (fr) * 1992-11-30 1997-08-20 Sinai School Medicine Clonage et expression d'alpha-galactosidase a biologiquement active.
EP1942189A3 (fr) * 1992-11-30 2008-09-10 The Mount Sinai School of Medicine of New York University Procédé de production de protéines secrétées
US6329191B1 (en) 1993-08-30 2001-12-11 Hawaii Biotechnology Group, Inc. DNA encoding recombinant coffee bean alpha-galactosidase
US6458574B1 (en) 1996-09-12 2002-10-01 Transkaryotic Therapies, Inc. Treatment of a α-galactosidase a deficiency
US6566099B1 (en) 1996-09-13 2003-05-20 Transkaryotic Therapies, Inc. Nucleic acid encoding a chimeric polypeptide
EP2327775A3 (fr) * 1996-09-13 2012-10-24 Shire Human Genetic Therapies, Inc. Production de l'alpha-galactosidase A humaine
US6395884B1 (en) 1996-09-13 2002-05-28 Transkaryotic Therapies, Inc. Therapy for α-galactosidase a deficiency
EP0935651B1 (fr) * 1996-09-13 2004-12-29 Transkaryotic Therapies, Inc. TRAITEMENT POUR CARENCE EN alpha-GALACTOSIDASE A
US7122354B2 (en) 1996-09-13 2006-10-17 Transkaryotic Therapies, Inc. Nucleic acid encoding a chimeric polypeptide
EP1538202A3 (fr) * 1996-09-13 2007-05-30 Transkaryotic Therapies, Inc. Production de l'alpha-galactosidase A humaine
AU2003220717B2 (en) * 1996-09-13 2007-10-18 Shire Human Genetic Therapies, Inc. Therapy for alpha-galactosidase a deficiency
US6083725A (en) * 1996-09-13 2000-07-04 Transkaryotic Therapies, Inc. Tranfected human cells expressing human α-galactosidase A protein
WO1998011206A3 (fr) * 1996-09-13 1998-08-13 Transkaryotic Therapies Inc TRAITEMENT POUR CARENCE EN α-GALACTOSIDASE A
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US7833742B2 (en) 2002-04-25 2010-11-16 Shire Human Genetic Therapies, Inc. Treatment of α-galactosidase A deficiency
US9267166B2 (en) 2002-04-25 2016-02-23 Shire Human Genetic Therapies, Inc. Treatment of α-galactosidase A deficiency
WO2003090695A2 (fr) 2002-04-25 2003-11-06 Transkaryotic Therapies, Inc. Traitement d'un deficit en alpha-galactosidase a
EP2361631A1 (fr) 2002-04-25 2011-08-31 Shire Human Genetic Therapies, Inc. TRAITEMENT POUR CARENCE EN alpha-GALACTOSIDASE A
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US11241422B2 (en) 2006-05-16 2022-02-08 Amicus Therapeutics, Inc. Methods for treatment of Fabry disease
US10383864B2 (en) 2006-05-16 2019-08-20 Amicus Therapeutics, Inc. Methods for treatment of Fabry disease
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US9987263B2 (en) 2006-05-16 2018-06-05 Amicus Therapeutics, Inc. Methods for treatment of Fabry disease
US9000011B2 (en) * 2006-05-16 2015-04-07 Amicus Therapeutics, Inc. Methods for treatment of Fabry disease
US10406143B2 (en) 2006-05-16 2019-09-10 Amicus Therapeutics, Inc. Methods for treatment of fabry disease
US20120220632A1 (en) * 2006-05-16 2012-08-30 Amicus Therapeutics, Inc. Methods For Treatment of Fabry Disease
US9999618B2 (en) 2007-04-26 2018-06-19 Amicus Therapeutics, Inc. Dosing regimens for the treatment of lysosomal storage diseases using pharmacological chaperones
US10525045B2 (en) 2007-04-26 2020-01-07 Amicus Therapeutics, Inc. Dosing regimens for the treatment of lysosomal storage diseases using pharmacological chaperones
US11033538B2 (en) 2007-04-26 2021-06-15 Amicus Therapeutics, Inc. Dosing regimens for the treatment of lysosomal storage diseases using pharmacological chaperones
US10925866B2 (en) 2007-04-26 2021-02-23 Amicus Therapeutics, Inc. Dosing regimens for the treatment of lysosomal storage diseases using pharmacological chaperones
US10350277B2 (en) 2011-09-07 2019-07-16 Icahn School Of Medicine At Mount Sinai Ceramidase and cell differentiation
US9492514B2 (en) 2012-06-01 2016-11-15 Icahn School Of Medicine At Mount Sinai Ceramide levels in the treatment and prevention of infections
US10159724B2 (en) 2012-06-01 2018-12-25 Icahn School Of Medicine At Mount Sinai Ceramide levels in the treatment and prevention of infections
WO2014017088A1 (fr) 2012-07-26 2014-01-30 Jcr Pharmaceuticals Co., Ltd. Procédé de production d'alpha-galactosidase a humaine recombinante
EP3495505A1 (fr) 2013-03-14 2019-06-12 Translate Bio, Inc. Évaluation quantitative de l'efficacité de coiffe d'arn messager
WO2014152659A1 (fr) 2013-03-14 2014-09-25 Shire Human Genetic Therapies, Inc. Évaluation quantitative pour l'efficacité de coiffage de l'arn messager
US10918702B2 (en) 2013-03-14 2021-02-16 Icahn School Of Medicine At Mount Sinai Therapeutic acid ceramidase compositions and methods of making and using them
US9937246B2 (en) 2013-03-14 2018-04-10 Icahn School Of Medicine At Mount Sinai Therapeutic acid ceramidase compositions and methods of making and using them
WO2014152673A1 (fr) 2013-03-14 2014-09-25 Shire Human Genetic Therapies, Inc. Évaluation quantitative de l'efficacité de coiffage de l'arn messager
US10238721B2 (en) 2013-03-14 2019-03-26 Icahn School Of Medicine At Mount Sinai Therapeutic acid ceramidase compositions and methods of making and using them
US10385325B2 (en) 2015-01-22 2019-08-20 Jcr Pharmaceuticals Co., Ltd. Method for production of recombinant human alpha-galactosidase A from material containing contaminant host cell proteins by chromatography
WO2016117341A1 (fr) 2015-01-22 2016-07-28 Jcr Pharmaceuticals Co., Ltd. Procédé de purification d'alpha-galactosidase a humaine de recombinaison à partir d'une matière contenant des protéines de cellules hôtes polluantes
US12070453B2 (en) 2016-07-19 2024-08-27 Amicus Therapeutics, Inc. Treatment of Fabry disease in ERT-naïve and ERT-experienced patients

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