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EP2198027A1 - Die verbesserung enzymatischer aktivität fördende, durch kurzes polypeptid vermittelte konformationsänderungen - Google Patents

Die verbesserung enzymatischer aktivität fördende, durch kurzes polypeptid vermittelte konformationsänderungen

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Publication number
EP2198027A1
EP2198027A1 EP08804083A EP08804083A EP2198027A1 EP 2198027 A1 EP2198027 A1 EP 2198027A1 EP 08804083 A EP08804083 A EP 08804083A EP 08804083 A EP08804083 A EP 08804083A EP 2198027 A1 EP2198027 A1 EP 2198027A1
Authority
EP
European Patent Office
Prior art keywords
isolated
purified
protein
dimeric protein
galns
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Withdrawn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
EP08804083A
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English (en)
French (fr)
Inventor
Julia Hepp
Christoph Heinzen
Francois Forcellino
Iris Kornelia Fux
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.)
Vivendy Therapeutics Ltd
Original Assignee
Vivendy Therapeutics Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Vivendy Therapeutics Ltd filed Critical Vivendy Therapeutics Ltd
Publication of EP2198027A1 publication Critical patent/EP2198027A1/de
Withdrawn legal-status Critical Current

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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention is in the field of molecular biology, biochemistry and clinical medicine.
  • the present invention features pharmaceutical compositions and methods for treating lysosomal storage diseases. More specifically the invention provides methods and compositions for treating sulfatase deficiency diseases.
  • the invention relates to methods and compositions for treating suifatase deficiency disease caused all or in part by deficiencies in human N-acetylgalactosamine- ⁇ - sulfate sulfatase (also known as N ⁇ acetylgalactosamine-6 ⁇ suifatase or GAINS) including mucopolysaccharidosis IV A disease (MPS iVA).
  • the present invention relates to an isolated and purified tagged dimeric protein having improved stability and targeting ability and improved plasma half life as well as the production and purification thereof,
  • MPS IV A disease type SV A mucopolysaccharidosis
  • mucopoiy- saccharidosis which forms a group of lysosomal diseases caused by a deficiency of enzymes necessary for the metabolism of glycosaminoglycan (hereinafter referred to as GAG)
  • GAG glycosaminoglycan
  • its clinical symptoms vary among individual patients, its common characteristic pathologies include swelling of cells caused by the accumulation of GAG in lysosomes, hypertrophy of organs, destruction of tissues and failing organs.
  • MPS IV A disease is an autosomal recessive genetic disease caused by an anomaly in the gene for a lysosomal enzyme, N-acetylgalactosamine-6-sulfate sulfatase (hereinafter referred to as GALNS) and is classified as type IV A mucopolysaccharidosis.
  • GALNS is an enzyme that hydroiyses the sulfate groups of chondroitin-6-suffate and keratan sulfate, which are species of GAG, and the deficiency of the enzyme causes intra-tissue deposition of GAG and its increased excretion in the urine.
  • MPS IV A disease One of the cfinicai characteristics of MPS IV A disease is bone dysplasia, and thus short statute, scoli ⁇ ' kyphasis, brevicollis, coxa vaiga, and articular hyperextension have been reported to occur. Also reported are corneal opacity, deafness and cardiac vaivuiar disorders.
  • the effective therapies are not yet available for MPS IV A disease.
  • the bone marrow transplantation provides no more than a marginal improvement of osteopathy.
  • most of current treatments are addressed to symptomatic therapy or control of symptoms, like orthopaedic treatment to prevent dislocation in upper cervical vertebrae.
  • polyethylene glycol is coupled to proteins (EP 0 671 905, Amgen, inc.).
  • dimers of two EPO molecules linked either by chemical cross- linking or by a polypeptide exhibit enhanced in vivo activities and a prolonged half- life 3AS (DaKe 1 2001 ; Kocbendoerfer, 2003; Sytkowski, 1998).
  • the enhanced activity may be due to the more efficient binding of the EPO dimer to one receptor, and the prolonged in vivo half-life due to the larger size of the dimer protein.
  • the chemical cross-linking process is not efficient and is difficult to control.
  • the linkage peptide in the dimer of EPO may alter the three-dimensional structure of EPO moiecule and the peptide itself may stimulate immunogenic responses in vivo.
  • the main problem to overcome is the instability of enzymatic activity over time in vivo. It has been shown that human GALNS has a relatively short plasma half-life and, hence, rapid loss of enzymatic activity in blood is observed. In the present invention it is demonstrated that a short-polypeptide (D6) induced conformational change leads to GALNS species with a larger molecular size (dimerization) that exhibits an increased plasma half-life and an enhanced biological activity in vivo.
  • D6 short-polypeptide
  • the present invention provides an isolated and purified tagged dimeric protein wherein a tagged monomer is non-covalently bound to another tagged monomer.
  • the isolated and purified tagged dimeric protein of the invention is characterized in that said tagged monomer is a fusion protein comprising a physiologically active protein and a short peptide comprising 4 - 15 acidic amino acids linked to said physiologically active protein on the N-terminal side thereof.
  • the isolated and purified tagged dimeric protein of the invention is characterized in that said physiologically active protein is an enzyme, particularly a N-acetylgalactosamine -6-sulfate sulfatase and variants thereof.
  • the isolated and purified tagged dimeric protein of the invention is characterized in that said short peptide consists of 4 - 12 acidic amino acids. particularly of 4 - 8 acidic amino acids, particularly of 6 acidic amino acids.
  • the isolated and purified tagged dimeric protein of the invention is characterized in that said acidic amino acids are giutamic acid or aspartic acid or a combination thereof, but particularly giutamic acid.
  • the isolated and purified tagged dimeric protein of the invention is characterized in that said short peptide is attached to the N-terminus of the physiologically active protein via a linker peptide, particularly a small peptide of 1 to 15 amino acids.
  • the isolated and purified tagged dimeric protein of the invention is characterized in that the amino acid sequence of said tagged monomer is SEQ [D No 2, a biologically active fragment thereof and/or biologically active variants thereof.
  • the present invention provides for an isolated and purified DNA molecule, having a nucleotide sequence encoding a tagged monomer as disclosed herein, particularly an isolated and purified DNA molecule comprising the sequence SEQ ID No 1 , a biologically active fragment thereof and/or biologically active variants thereof.
  • the present invention provides for an expression vector, comprising said isolated and purified DNA molecule of the invention and as described herein, and a host cell comprising said isolated and purified DNA molecule and/or at least one copy of said expression vector, particularly a host cell selected from the group consisting of CHO, CHO-K1, HE ⁇ 193T, HEK293, COS, PC12, HiB5, RN33b, BHK cells, but especially a Chinese hamster ovary (CHO) cell.
  • CHO Chinese hamster ovary
  • the present invention provides a pharmaceutical composition comprising a pharmaceutically effective amount of the isolated and purified tagged dimeric protein according to the invention and as described herein, optionally in combination with one or more pharmaceutically acceptable carriers.
  • the present invention provides a process for producing and purifying the isolated and purified tagged dimeric protein in a suitable host cell comprising the steps of:
  • DNA molecule of the invention as described herein,;
  • the present invention provides the use of the isolated and purified tagged dimeric protein as a medicament.
  • the isolated and purified tagged dimeric protein is used in the preparation of a medicament for the treatment of sulfatase deficiency diseases, and more particularly, for the treatment of N-acetylgalactosamine-6-sulfat ⁇ suifatas ⁇ deficiency diseases, but especially for mucopolysaccharidosis IVA.
  • the present invention provides a method for the treatment of sulfatase deficiency diseases, comprising administering to a subject an effective amount of the isolated and purified tagged dimeric protein, as described herein, and/or the pharmaceutical composition of the present invention.
  • the invention particularly provides a method for the treatment of N-acetylgalactosamine- 6-suifate sulfatase deficiency diseases, but especially a method for the treatment of mucopolysaccharidosis !VA.
  • the present invention provides a method of using the isolated and purified tagged dimeric protein and/or the pharmaceutical composition of the invention and as described herein, for the preparation of a medicament for use in the treatment of suifatase deficiency diseases, particularly for use in the treatment of N- acetylgalactosamine- ⁇ -suifate suffatase deficiency diseases, but especially for use in the treatment of mucopolysaccharidosis IVA.
  • the present invention provides a pharmaceutical composition for use as a medicament, particularly for use in the treatment of a sulfatase deficiency disease, particularly for use in the treatment of a N-acetylgalactosamine-6-sulfate sulfatase deficiency disease, characterized in that it comprises a pharmaceutically effective amount of the isolated and purified tagged dimeric protein of the invention and as described herein, optionaiiy in combination with one or more pharmaceutically acceptable carriers.
  • the present invention provides a pharmaceutical composition for use in the treatment of mucopolysaccharidosis IVA.
  • FIG. 1 Chromatogram of the direct capture of D6-GALNS from cel ⁇ culture fluid by anion exchange chromatography on DEAE sepharose FF matrix.
  • the pooled harvest (960 mL D6-GALNS supernatant, S018) was 1 :2 diluted in 20 mM Tris pH 7.0 buffer and (A) ioaded onto the column with a flow rate of 5 mL/min.
  • the column was washed after loading with (B) 120 mM NaC! in 20 mM Tris, pH 7.0 buffer for 20 CV.
  • FIG. 2 SDS-PAGE analysis followed by silver staining of direct capture of D6-GALNS from ceil culture fluid by anion exchange chromatography on DEAE sepharose FF matrix. The molecular weight and running position of the marker proteins (M) are indicated on the left.
  • the samples are run in different lanes and labeled accordingly: marker (M); positive control of rhGALNS (+); lane (1) pooled harvest of S018; lane (2) pooled harvest of S018 1 :2 diluted in 20 mM Tris, pH 7; lane (3) ffowthrough of anion exchange chromatography run; lane (4) fractions of 120 mM NaCI wash collected early; iane (5) fractions of 120 mM NaCl wash coliected later; iane (6) eluted fractions at 600 mM NaCI.
  • marker (M) positive control of rhGALNS (+)
  • lane (3) ffowthrough of anion exchange chromatography run lane (4) fractions of 120 mM NaCI wash collected early; iane (5) fractions of 120 mM NaCl wash
  • FIG. 3 Analytical size exclusion chromatogram of D6-GALNS preparation with indicated retention volumes.
  • Retention volumes are marking the maximum absorption peaks of the UV 280 ⁇ m trace.
  • FIG. 4 MALDI mass spectrometry analysis of D6-GALNS.
  • the experiment is performed on an UltraFlexTOF/TOF MALDi tandem time-of-flight (TOF/TOF) mass spectrometer (Bruker Daitonics Inc.).
  • the applied voltage from two sources was 25 kV and 23.45 kV respectively with a linear detector voltage of 1.607 kV and a laser repletion rate of 100 Hz.
  • the mass/charge (m/z) ration of the D6-GALNS protein sample is recorded and plotted against the intensity measured in absorption units (a. u.). The corresponding apparent molecular weight is indicated above the respective peaks and assigned to (M+H)+ (60 kDa), (M+2H)2+, and (M+3H ⁇ 3+.
  • FIG. 5 Demonstration of in Vivo Enhanced Enzymatic Effectiveness of Dimeric D6-GALNS.
  • Accumulative enzymatic activity of the monomeric GALNS and dimeric D6-GALNS enzymes in the blood circulation of homozygous GALNS " ' " mice is determined after an intravenous single injection of 250 U/g body weight enzyme. Blood is taken after 2 min, 5 min, 10 min, 15 min, 20 min, 30 min, 1 h and 2 h of the intravenous injection.
  • the enzymatic activity of GALNS (open squares D) in the blood circulation of GALNS "A mice shows a biphasic kinetic with two distinct half lives of 1.84 min and 13.3 min.
  • the enzymatic activity of dimeric D6-GALNS displays a prolonged monophasic kinetic with a half life of 15.6 min.
  • the D6 polypeptide mediated conformational change in D6-GALNS leads to an unexpectedly prolonged half life that results in an increase enzymatic exposure. Plotting the enzymatic activity in vivo over time one can determine the area under the curve (AUC) that is an indirect measure for the enzymatic exposure.
  • AUC area under the curve
  • FIG. 6 SEQ ID No 1 : Partial nucleotide sequence of ⁇ CXN-p97-D ⁇ -GALNS containing D6-GALNS cDNA. Nt 1-57 p97 signal sequence, nt 61-78: polynucleotide encoding the D6 polypeptide, nt 79-96: linker sequence, nt 97-1587: gains cDNA nucleotide sequence without signal sequence,
  • Figure 7 SEQ ID No 2: Amino acid sequence of D6-GALNS with ⁇ 97 signal sequence. AA 1-19: p97 signal sequence, AA 21-26: D6, AA 27-32: linker sequence, AA 33-528: GALNS without signal sequence.
  • a cell includes a plurality of ce ⁇ s, including mixtures thereof.
  • a protein includes a plurality of proteins.
  • peptide As used herein, the terms “peptide”, “protein”, “polypeptide”, “polypeptide” and “peptidic” are used interchangeably to designate a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
  • enzymes are bioiogical catalysts that speed up the rate of reaction between substances without themselves being consumed in the reaction.
  • the dimeric enzyme of the present invention is intended to encompass any protein consisting of two monomers and possessing enzymatic properties.
  • Tagged protein or “tagged monomer” refers to a protein or a monomer which has a “tag” usually located on its N-terminal.
  • a “tag” is used to indicate a removable molecuie, preferably a short peptide, added to the protein during or after synthesis thereof.
  • This "tag” can selectively interact with celiuiar receptors or other cell surface proteins, or can selectively interact with certain components of the environment, or can selectively interact with the protein either intramolecular or intermolecular, either free or bound to a surface.
  • the "tag” can act as address for sending "tagged protein” to a specific celiuiar compartment,
  • “Dimeric protein” refers to dimerization of two identical subunits, herein two monomers, which are bound together via non-covalent bonds.
  • “Bound” can be defined as connected, attached, linked or put into contact.
  • “Non-covale ⁇ t bond” or “no ⁇ -covending!y bound” refers herein to a type of chemical bond, typically between rnacromolecules, such as proteins, that does not involve the sharing of pairs of electrons. Examples of non-covalent bonds are hydrogen bonding, ionic interactions, Van der Waais interactions, hydrophobic bonds or binding interactions which hold proteins in a particular three-dimensional conformation.
  • the conformation of the isolated and purified tagged dimeric protein under non- denaturing physioiogicai conditions was investigated by analytical size exciusion chromatography as described in the following examples.
  • the obtained results have demonstrated that the D6-GALNS (tagged monomer) from CHO cell cultivation displays under non-denaturing physioiogicai conditions a dimeric conformation in solution.
  • D6-GALNS displays a globular monomeric conformation under the reducing and denaturing conditions of the SDS-PAGE analysis but under the non-denaturing physiological conditions of the size exclusion chromatography the D ⁇ -GALNS protein construct appears to adapt a stable dimeric conformation.
  • the D6-GALNS protein sample contained a single well defined macro molecule with the apparent mass calculated from the m/z ratio of 60 kDa.
  • This apparent mass of 60 kDa determined by MALD! TOF MS corresponds well to the calculated theoretical molecular weight of D6-GALNS based on the primary amino acid sequence (57 kDa).
  • the conformations! dimer that was observed in the analytical size exciusion chromatography is a non-covalent association of two monomeric D6-GALNS proteins that can be separated under the denaturing conditions of the applied TCA precipitation and ionization of MALDi mass spectrometry (as well as under the denaturing and reducing conditions of the SDS-PAGE analysis).
  • said tagged monomer is usually a fusion protein comprising a physiologically active protein and a short peptide comprising 4 - 15 acidic amino acids linked to said physiologically active protein on the N-terminal side thereof.
  • said physiologically active protein is an enzyme and more preferably N- acetySgaiactosamine -6-sulfate suifatase and variants thereof.
  • variant refers to a peptide having an amino acid sequence that differ to some extent from a native sequence peptide, that is an amino acid sequence that vary from the native sequence by conservative amino acid substitutions, whereby one or more amino acids are substituted by another with same characteristics and conformational roles.
  • the amino acid sequence variants possess substitutions, deletions, side-chain modifications and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence. Conservative amino acid substitutions are herein defined as exchanges within one of the following five groups:
  • Lys residues may be substituted by ornithine, homoarginine, nor-Lys, N-methyl-Lys, N, M-dimethyl-Lys and N, N, N- trimethyl-Lys.
  • Lys residues can aiso be replaced with synthetic basic amino acids including, but not limited to, N-1- (2-pyrazolinyl)-Arg, 2- (4-piperiny!)-Gly, 2- (4- piperinyl)-Ala, 2- [3- (2S) pyrrolininyI]-Gly and2- [3- (2S) pyrolininyl]-A!a.
  • Tyr residues may be substituted with 4-methoxy tyrosine (MeY), meta-Tyr,ortho-Tyr, nor- Tyr,1251-Tyr, mono-halo-Tyr, di-haio ⁇ Tyr, O-sulpho-Tyr, O-phospho-Tyr, and nitro- Tyr.
  • Tyr residues may also be substituted with the 3-hydroxyl or 2-hydroxyl isomers (meta-Tyr or ortho-Tyr, respectively) and corresponding O-suipho-and O-phospho derivatives. Tyr residues can also be replaced with synthetic hydroxy!
  • amino acids including, but not limited to4-hydroxymethyi-Phe, 4-hydroxypheny!- GIy, 2, 6-dimethyi-Tyr and 5-amino-Tyr.
  • Aliphatic amino acids may be substituted by synthetic derivatives bearing non-natural aliphatic branched or linear side chains CnH2n+2 where n is a number from 1 up to and including 8. Examples of suitable conservative substitutions by non-conventiona! amino acids are given in WO 02/064740.
  • Insertions encompass the addition of one or more naturally occurring or non conventional amino acid residues. Deletion encompasses the deletion of one or more amino acid residues.
  • the physiological active protein of the invention may be prepared in order to include D-forms and/or "retro-in verso isomers" of the peptide.
  • retro-inverso isomers of short parts, variants or combinations of the physiological active protein of the invention are prepared.
  • Retro-inverso peptides are prepared for peptides of known sequence as described for example in SeIa and Zisman, (1997).
  • retro-inverso isomer is meant an isomer of a linear peptide in which the direction of the sequence is reversed and the chirality of each amino acid residue is inverted; thus, there can be no end-group complementarity.
  • the invention also includes analogs in which one or more peptide bonds have been replaced with an alternative type of covalent bond (a "peptide mimetic") which is not susceptible to cleavage by peptidases.
  • a peptide mimetic an alternative type of covalent bond
  • proteolytic degradation of the peptides following injection into the subject is a problem
  • replacement of a particularly sensitive peptide bond with a noncleavable peptide mimetic will make the resulting peptide more stable and thus more useful as an active substance.
  • mimetics, and methods of incorporating them into peptides are well known in the art.
  • the short peptide consists preferably of 4-12 acidic amino acids, more preferably of 4-8 acidic amino acids, and still more preferably of 6 acidic amino acids.
  • acidic amino acid means glutamic acid or aspartic acid or a combination thereof, more preferably aspartic acid, most preferably 6 aspartic acids (hereinafter referred to as D6 or, bound to a polypeptide, as D6 polypeptide, iike D6-GALNS). These acidic amino acids are used for the preparation of said "short peptide”. Acidic amino acids may be used in any arbitrary combination including a simple use of one or the other alone for the preparation of a short peptide.
  • the short peptide consisting of acidic amino acids is directly attached to the N-terminus of a physiologically active protein via a peptide bond or via a linker peptide, more preferably via a linker peptide.
  • a linker peptide is not an indispensable component. It is optionally used for attaching a short peptide consisting of acidic amino acids to the N-terminus of a physiologically active protein. If used, a linker peptide is a small peptide consisting preferably of 1 to 15, more preferably of 1 to 10, and still more preferably of 1 to 6 amino acids. A linker peptide may be also a single amino acid molecule which can bind the short peptide to the physiologically active protein via peptide bonds. A linker peptide may be made of any amino acid as desired.
  • the method for binding a short peptide to a physiologically active protein it is of advantage to form and use a transfected cell expressing the fusion protein consisting of the short peptide and the physiologically active protein.
  • the present invention relates to an isolated and purified tagged dimeric protein wherein the amino acid sequence of the tagged monomer is SEQ ID No 2, a biologically active fragment thereof and/or biologically active variants thereof.
  • Biologically active means affecting any physical or biochemical properties of a living organism or biological process.
  • Biologically Active Substance refers to any molecule or mixture or complex of molecules that exerts a biological effect in vitro and/or in vivo, including pharmaceuticals, drugs, proteins, peptides, polypeptides, hormones, vitamins, steroids, polyanions, nucleosides, nucleotides, nucleic acids (e.g. DNA or RNA), nucleotides, polynucleotides, etc.
  • “Fragments” refer to sequences sharing at least 40% amino acids in length with the respective sequence of the substrate active site. These sequences can be used as long as they exhibit the same biological properties as the native sequence from which they derive. Preferably these sequences share more than 70%, preferably more than 80%, in particular more than 90% amino acids in length with the respective sequence from which it derives. These fragments can be prepared by a variety of methods and techniques known in the art such as for example chemical synthesis.
  • the present invention also relates to an isolated and purified DNA molecule having a nucleotide sequence encoding the tagged monomer of the present invention.
  • the isolated and purified DNA molecule sequence comprises the SEQ ID No 1 , a biologically active fragment thereof, and/or biologically active variants thereof.
  • isolated requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide present in a living animal is not isolated, but the same polynucleotide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • purified does not require absolute purity; rather, it is intended as a relative definition.
  • DNA molecule or sequence refers to the state in which the nucleic acid molecule is free or substantially free of material with which it is naturally associated such as other polypeptides or nucleic acids with which it is found in its natural environment, or the environment in which it is prepared (e. g. eel! culture) when such preparation is by recombinant nucleic acid technology practiced in vitro or in vivo.
  • DNA which can be used herein is any polydeoxynuciotide sequence, including, e.g. double-stranded DNA, singie-stranded DNA, doubie-stranded DNA wherein one or both strands are composed of two or more fragments, doubie-stranded DNA wherein one or both strands have an uninterrupted phosphodiester backbone, DNA containing one or more singie-stranded portion(s) and one or more double-stranded portion(s), double-stranded DNA wherein the DNA strands are fully complementary, double-stranded DNA wherein the DNA strands are only partially complementary, circular DNA, covalently- closed DNA, linear DNA.
  • covaSently cross-linked DNA cDNA, chemicaliy- synthesized DNA, semi-synthetic DNA 1 biosynthetic DNA, naturally-isolated DNA, enzyme-digested DNA, sheared DNA, labeled DNA, such as radiolabeied DNA and fiuorochrome-iabeled DNA, DNA containing one or more n ⁇ n- natura ⁇ y occurring species of nucleic acid.
  • RNA polyribonucleotide
  • RNA RNA
  • RNA polyribonucleotide
  • RNA including, e.g., single-stranded RNA, double- stranded RNA, double-stranded RNA wherein one or both strands are composed of two or more fragments, double-stranded RNA wherein one or both strands have an uninterrupted phosphodiester backbone, RNA containing one or more single- stranded portton(s) and one or more double-stranded portion(s), double-stranded RNA wherein the RNA strands are fully complementary, doubie-stranded RNA wherein the RNA strands are only partially compiementary, covalently crossl ⁇ nked RNA, enzyme-digested RNA, sheared RNA, mRNA, chemically-synthesized RNA, semi-synthetic RNA, biosynthetic RNA, naturally-isolated RNA, labeled RNA, such
  • the isolated and purified nucleic acid sequence also comprises an isolated and purified nucleic acid sequence having substantial sequence identity or homology to a nucleic acid sequence encoding the monomer of the invention.
  • the nucleic acid will have substantial sequence identity for example at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% nucleic acid identity; more preferably 90% nucleic acid identity; and most preferably at ieast 95%, 96%, 97%, 98%, or 99% sequence identity.
  • Identity is a relationship between two or more amino acid sequences or two or more nucleic acid sequences, as determined by comparing the sequences, it also refers to the degree of sequence reiatedness between amino acid or nucieic acid sequences, as the case may be, as determined by the match between strings of such sequences.
  • Identity and similarity are well known terms to skilled artisans and they can be calculated by conventional methods (for example see Computational Molecular Biology, Lesk. A, M. ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W. ed,, Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M. and Griffin, H.
  • the present invention also includes variants of the afore-mentioned sequence SEQ ID NO: 1
  • variants or “variants of a sequence” is meant a nucleic acid sequence that vary form the reference sequence by conservative nucleic acid substitutions, whereby one or more nucleic acids are substituted by another with same characteristics, Variants encompass as well degenerated sequences, sequences with deletions and insertions, as long as such modified sequences exhibit the same function (functionally equivalent) as the reference sequence.
  • the invention also encompasses allelic variants of the disclosed purified and isolated nucieic sequence; that is, naturally-occurring alternative forms of the isolated and purified nucleic acid that also encode peptides that are identical, homologous or related to that encoded by the purified and isolated nucleic sequences.
  • allelic variants may be produced by mutagenesis techniques or by direct synthesis.
  • Molecular chimera of SEQ ID No 1 are also considered in the present invention.
  • molecular chimera is intended a nucleotide sequence that may inciude a functional portion of the isolated DNA molecule according to the invention and that will be obtained by molecular biology methods known by those skilled in the art.
  • DNA molecules or fragments or sub- portions thereof are also considered in the present invention.
  • fragments can be prepared by a variety of methods known in the art. These methods include, but are not limited to, digestion with restriction enzymes and recovery of the fragments, chemical synthesis or polymerase chain reactions (PCR).
  • mutant and mutant refer to nucleic acid or protein sequences which are not found in nature
  • truncated refers to nucleic acid or protein sequences that are shorter than those found in nature.
  • Another concern of the present invention is to provide an expression vector comprising the isolated and purified DNA molecule having a nucleotide sequence encoding the tagged monomer of the present invention.
  • the expression vector of the present invention comprises as well the sequence SEQ ID No 1 , a biologically active fragment thereof and/or biologically active variants thereof,
  • vector As used herein, "vector”, "plasmid” and “expression vector” are used interchangeably, as the piasmid is the most commonly used vector form.
  • an expression vector depends directly, as it is well known in the art, on the desired functional properties, e.g., peptide expression and the host cell to be transformed or transfected.
  • the expression vector may further comprise a promoter operabiy linked to the isolated DNA sequence.
  • a promoter operabiy linked to the isolated DNA sequence This means that the linked isolated DNA sequence encoding the monomer of the present invention is under control of a suitable regulatory sequence which allows expression, i.e. transcription and translation of the inserted isolated DNA sequence.
  • a wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention.
  • Useful expression vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences.
  • Suitable vectors include derivatives of SV40 and known bacteria! plasmids, e. g., E. coii plasmids col El 1 pCRI, pCXN, pBR322, pcDNA3, pMB9 and their derivatives, pfasmids such as RP4; phage DNAs, e. g., the numerous derivatives of phage X, e. g., NM989, and other phage DNA, e.
  • yeast plasmids such as the 2 ⁇ piasmid or derivatives thereof
  • vectors useful in eukaryotic cells such as vectors useful in insect or mammalian cells
  • vectors derived from combinations of plasmids and phage DNAs such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.
  • the expression vector is pCXN.
  • Another object of the present invention Is to provide a host cell comprising the isolated and purified DNA molecule of the invention and/or at least one copy of the expression vector described herein.
  • a wide variety of host ceils are useful in expressing the DNA sequences of this invention.
  • the host eel! of the present invention are selected from the group consisting of CHO, CHO-K1 , HES193T,
  • the host cell is Chinese hamster ovary (CHO) cell.
  • compositions comprising a pharmaceutically effective amount of the isolated and purified tagged dimeric protein of the present invention, optionally in combination with one or more pharmaceutically acceptable carriers.
  • a pharmaceutically effective amount refers to a chemica! material or compound which, when administered to a human or animai organism induces a detectable pharmacologic and/or physiologic effect.
  • the respective pharmaceuticaiiy effect amount can depend on the specific patient to be treated, on the disease to be treated and on the method of administration. Further, the pharmaceuticaiiy effective amount depends on the specific protein used, especially if the protein additionally contains a drug as described or not.
  • the treatment usually comprises a multiple administration of the pharmaceutical composition, usually in intervals of several hours, days or weeks.
  • the pharmaceutically effective amount of a dosage unit of the polypeptide usually is in the range of 0.01 mg to 10 mg per kg of body weight of the patient to be treated.
  • phrases “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • An isolated and purified tagged dimeric protein of the present invention in particular a protein comprising the enzyme N-acetylgalaclosamine-6-suifate sulfatase and a short peptide, may be prepared in the form of a pharmaceutical composition containing the isolated and purified tagged dimeric protein dissolved or dispersed in a pharmaceutically acceptable carrier well known to those skilled in the art, for parenteral administration by, e. g., intravenous, subcutaneous or intramuscular injection or by intravenous drip infusion.
  • any conventional additives may be used such as excipients, adjuvants, binders, dtsintegrants, dispersing agents, lubricants, diluents, absorption enhancers, buffering agents, surfactants, solubiSizi ⁇ g agents, preservatives, emulsifters, isotonizers, stabilizers, solubilizers for injection, pH adjusting agents, etc.
  • Acceptable carriers, diluents and adjuvants which facilitates processing of the active compounds into preparation which can be used pharmaceuticaiiy are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyidimethyibenzyl ammonium chloride; hexamethonium chloride; benzaikonium chloride, benz ⁇ thonium chioride; phenoi, butyl orbenzyi alcohol; aikyl parabens such as methyi or propy!
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid and methionine
  • preservatives such as octadecyidimethyibenzyl ammonium chloride; hexamethonium chloride; benzai
  • paraben catechol; resorcinol; cyclohexano ⁇ ; 3-pentanoi; and m-cresol); low molecular weight ⁇ less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophiSic polymers such as polyvinylpyrrolidone; amino acids such as glycine, giutamine, asparagine.
  • chelating agents such as EDTA
  • sugars such as sucrose, mannitot, trehalose or sorbitol
  • salt-forming counter-ions such as sodium
  • metal complexes e.g. Zn-protein complexes
  • non-ionic surfactants such as TWEEN®, PLURON1CS® or polyethylene glycol (PEG).
  • administration of the pharmaceutical composition may be systemic or topical.
  • administration of such a composition may be various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, buccal routes or via an implanted device, and may also be delivered by peristaltic means.
  • composition comprising an isolated and purified tagged dimeric protein, as described herein, as an active agent may also be incorporated or impregnated into a bioabsorbable matrix, with the matrix being administered in the form of a suspension of matrix, a ge! or a solid support.
  • the matrix may be comprised of a biopolymer.
  • Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semi permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, po!y(2-hydroxyethy!-m ⁇ thacryiate), or poly(vinyialcohol)), polyiactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and [gamma] ethyi-L-glutar ⁇ ate non-degradable ethylene-vinyl acetate
  • degradable lactic acid- glycoiic acid copolymers such as the LUPRON DEPOT(TM) (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolJde acetate), and poiy-D- (-)-3-hydroxybutyric acid.
  • the formulations to be used for in vivo administration must be sterile. This is readily accomplished for example by filtration through sterile filtration membranes.
  • the suitable dosage of a peptide of the present invention will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any and the nature of the effect desired.
  • the appropriate dosage form wili depend on the disease, the protein, and the mode of administration; possibilities include tablets, capsules, Sozenges, dental pastes, suppositories, inhalants, solutions, ointments and parenteral depots.
  • Another concern of the present invention is a process for producing and purifying the isolated and purified tagged dimeric protein of the present invention in a suitable host cell comprising the steps of;
  • the host cell is Chinese hamster ovary (CHO) eel!.
  • Suitable culture conditions are those conventionally used for in vitro cultivation of host cells as described e.g. in WO 96/39488.
  • the process of the present invention comprises a purification process performed with one single anion exchange column with appropriate buffers and is more preferably monitored by recording the UV absorption at 280 nm.
  • the singie anion exchange column is an DEAE sepharose FF column and the appropriate buffers have a pH 7.0.
  • Transformation or transfection of suitable host cells with an expression vector comprising an isolated DNA sequence according to the invention is accomplished by well known methods that typically depend on the type of vector used. With regard to these methods, see for example, Sambrook et ai. Molecular Cloning a Laboratory Manual, Second Edition, Cold Spring Habour Laboratory Press, 1989.
  • the generation of the expression vector pCXN-GALNS has been already described in detail 7 ' 8 (Tomatsu, 2003; T ⁇ matsu, 1991 ).
  • the generation of the modified expression vector pCXN-p97 ⁇ D6-GALNS was performed analogous to the previously described construction of the expression vector pCXN-p97-NBT-GALNS (US2005/0276796 A1 ).
  • pCXN-GALNS refers to the expression vector pCXN comprising the DNA sequence of the native human N-acetylgaiactosamine-6-sulfate sulfatase.
  • pCXN-p97-D ⁇ -GALNS refers to the expression vector of the monomer of the present invention.
  • cell transfected or "ceil transformed” or “transfected/transformed cell” means the ceil into which the extracellular DNA has been introduced and thus harbors the extracellular DNA.
  • the DNA might be introduced into the cell so that the nucleic acid is repiicable either as a chromosomal integrant or as an extra chromosomal element.
  • Further concern of the present invention is an isolated and purified tagged dimeric protein, as herein described, obtainable by the process of the present invention.
  • the Applicant has surprisingly characterized an isolated and purified tagged dimeric protein that has improved targeting ability and stability over time in vivo, thus with increased plasma half-life and enhanced biological activity in vivo.
  • improved refers to the capacity of an isolated and purified tagged dimeric protein to bind to the bone tissue and to increase the plasma half life, thus enhancing the biological activity in vivo. This capacity can be measured by, for example, measuring the enzymatic activity and by measuring in vivo the enzymatic effectiveness (see Examples of the present invention).
  • the present invention relates to compositions, methods and use of the isolated and purified tagged dimeric protein, as described herein, for the treatment of lysosomal storage diseases. More specifically the invention provides methods and compositions for treating sulfatase deficiency. In particular, the invention relates to methods and compositions for treating suifatase deficiency disease caused ail or in part by deficiencies in human N-acetylgalactosamine-6-sulfate sulfatase, including mucopolysaccharidosis IVA.
  • Yet another concern of the present invention is the use of the isolated and purified tagged dimeric protein, herein described, as a medicament.
  • Also encompassed by the present invention is the use of the isolated and purified tagged dimeric protein, as described herein, in the preparation of a medicament for the treatment of suifatase deficiency diseases and more preferably for the treatment of N-acetylgalactosamine-6-suSfate sulfatase deficiency diseases, most preferably mucopolysaccharidosis IVA disease.
  • An isolated and purified tagged dimeric protein of the present invention in particular an isolated and purified tagged dimeric protein comprising enzyme N- acetylgalactosamine-6-suifate sulfatase and a short peptide, may be used advantageously in place of the conventional native human enzyme in a substitution therapy for the treatment of mucopolysaccharidosis IVA disease .
  • a pharmaceutically effective amount of the isolated and purified tagged dimeric protein may be administered intravenously, subcutaneously or intramuscularly.
  • the respective pharmaceutically effect amount can depend on the specific patient to be treated, on the disease to be treated and on the method of administration. Further, the pharmaceutically effective amount depends on the specific peptide used, especially if the peptide additio ⁇ aity contains a drug as described or not.
  • the treatment usually comprises a multiple administration of the pharmaceutical composition, usually in intervals of several hours, days or weeks.
  • the pharmaceutically effective amount of a dosage unit of the polypeptide usua ⁇ y is in the range of 0.001 ng to 100 ⁇ g per kg of body weight of the patient to be treated.
  • subject or “patient” are weii-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human.
  • the subject is a subject in need of treatment.
  • the subject can be a normal subject.
  • “Mamma! for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, monkeys etc.
  • the mamma! is human.
  • Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. Hence, the mamma! to be treated herein may have been diagnosed as having the disorder or may be predisposed or susceptible to the disorder.
  • Disease refers to a pathoiogical condition of a part, organ, or system of an organism resulting from various causes, such as infection, genetic defect, or environmental stress, and characterized by an identifiable group of signs or symptoms.
  • the isolated and purified tagged dimeric protein of the invention will generally be used in an amount to achieve the intended purpose.
  • the isolated and purified tagged dimeric protein or the pharmaceutical compositions thereof is administered or applied in a therapeutically effective amount.
  • a "therapeutically effective amount” is an amount effective to ameliorate or prevent the symptoms, or alleviate osteopathy of the subject being treated. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • administering refers to contact of the isolated and purified tagged dimeric protein or the pharmaceutical compositions to the subject, preferably a human.
  • a therapeutically effective amount or dose can be estimated initially from In vitro assays.
  • a dose can be formulated in animal modeis to achieve a circulating concentration range that includes the 1C50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
  • initial doses can also be estimated from in vivo data, e.g. animal modeis, using techniques that are well known in the art.
  • in vivo data e.g. animal modeis
  • One ordinarily skill in the art could readily optimise administration to humans based on animal data and will, of course, depend on the subject being treated, on the subject's weight, the severity of the disorder, the manner of administration and the judgement of the prescribing physician,
  • sulfatase deficiency diseases are N-acetyigalactosamine-6-sulfate sulfatase deficiency diseases. More preferably the disease is mucopolysaccharidosis !VA.
  • the generation of the expression vector pCXN-GALNS has been described in detail previously (Tomatsu et al. 1991 ).
  • the generation of the modified expression vector pCXN-p97-D6-GALNS was performed analogous to the previously described construction of the expression vector pCXN-p97-NBT-GALNS (US2005/0276796 A1 ).
  • an expression vector encoding the human GALNS with an W-terminaliy attached short polypetide (D6) connected via a linker peptide was constructed starting from the pCXN-GALNS expression vector.
  • Chinese hamster ovary ceils of the a single selected D6-GALNS clone are grown in T-175 flasks in HP1 media supplemented with 20% FBS, 34.5 Qg/mL Prolin, 400 Dg/mL G418 and 10 Dg/mL puromycin until celis are confluent.
  • the supernatant is coliected and centrifuged by 200 g at RT for 3 minutes.
  • the supernatant is pooled and centrifuged a second time at 7000 g at 4 "C for 20 minutes.
  • the cell pellet from the first centrifugation is resuspended in Ex-Cell Media (without FBS 1 with 34.5 Dg/mL Prolin) and cells are seeded back in the origina! T-Flask.
  • the doubly centrifuged supernatant, containing D6-GALNS is stored and frozen at -20 °C Over the duration of 9 days, ali harvests are collected as describe above and frozen at -20 0 C.
  • a DEAE sepharose FF column (GE healthcare, 17-0709-10) with a column volume (CV) of 5 mL (16 mm diameter x 2.5 cm bed height) is run with a flow speed of 5 mL/min at a temperature of 6 ⁇ 2 0 C.
  • the column is equilibrated with 20 mM Tris pH 7.0 buffer with an equivalent of 10 CV.
  • the pooled harvest (960 mL D ⁇ -GALNS supernatant) are 1 :2 diluted in 20 mM Tris pH 7.0 buffer prior to loading onto the column with a flow rate of 5 mL/min (see Figure 1 ).
  • the column is washed after loading with 120 rriM NaCl in 20 mM Tris, pH 7.0 buffer for 20 CV, Complete eiution of bound protein is achieved with a step-wise eiution profile consisting of 600 and 1000 mM NaCI in 20 mM Tris, pH 7.0 buffer for 20 CV each.
  • Protein samples are prepared for SDS-PAGE analysis under denaturing and reducing conditions by adding equivalent amounts of reducing Laemli loading buffer 2 x to the sample. After boiling 3-10 min at 95 0 C the samples are cooled on ice and centrifuged at 7000 g for 10 sec. A denaturing 8 % SDS acrylamide gel is prepared and the samples are run at constant current of 200 V until complete separation is reached as judged by position of the running front of sample dye. SDS-PAGE analysis of the DEAE chromatography run of D6-GALNS supernatant suggests that D6-GALNS protein elutes mainly during the wash step with 120 mM NaCl ( Figure 3).
  • the dominant protein band in lane 4 runs at an apparent molecular weight equivalent to 67 kDa corresponding well to the theoretical MW of D6-GALNS of 57 kDa.
  • a small amount of D6-GALNS is also eiuted during the elution step with 600 mM NaCl. This is probably due to the fact that a 120 mM NaCI concentration might not be high enough to completely elute the protein.
  • the purity achieved in the main D6-GALNS containing pool is high. Under denaturing and reducing conditions the anion exchange chromatography purified D6-GALNS runs as a well separated single band at the expected molecular weight.
  • the apparent molecular weight of 67 kDa on the denaturing SDS-PAGE gel corresponds weli to an assumed doubly glycosylated D6-GALNS protein with a theoretical molecular weight of 57 kDa (based on primary amino acid sequence on!y).
  • DNA quantification is performed according to Picogreen assay kit of invitrogen (cat. Nr. P11496). Approximately 37 ⁇ fo!d more DNA is eluted in a pool of fractions eluted at 600 mm NaCi than at 120 mM NaCI. A pool of fractions eluted at 120 mM NaCI and containing most GALNS activity still contains approximately 900 ng DNA/ml (data not shown). These findings explain the chromatograrn data recorded by UV absorption at 280 nm ( Figure 1 , (C)).
  • GALNS enzymatic activity assay is performed as described in detail previously (Tomatsu et al. 2003; Hum MoI Genet. 12(24):3349-58).
  • the substrate 4- Methyiumbeiliferyl- ⁇ -galactosid-6- sulfat is convert by the enzyme GALNS to 4-Methylumbel ⁇ iferylgalactosid.
  • the results of the GALNS enzymatic activity assay confirm that the majority of functional D6-GALNS protein eluted at 120 mM NaCl, 20 mM Tris, pH 7.0.
  • the protein purification of cell culture supernatant yielded D6-GALNS enzyme with 80 % protein purity as judged by SDS-PAGE analysis.
  • the protein displays the expected molecular weight on a denaturing SDS-PAGE gei and shows corresponding enzymatic activity.
  • Analytical Size Exclusion Chromatography To analyze the conformation of the purified D6-GALNS under non-denaturing physiological conditions the protein preparation consisting of D6-GALNS eluted at 120 mM NaCl is investigated by analytical size exclusion chromatography as described in detail below.
  • a Superdex 75 10/300 GL (GE healthcare, 17-5174-01 ) column with a CV of 23.5 mL is run with 20 rnM Tris, 150 mM NaCl, pH 7.0 buffer at a flow of 0.5 mL/min at 6 ⁇ 2 0 C. Calibration of the Superdex 75 column for globular protein molecular weight separation is performed with 35 micro litres of Biorad get filtration standard (cat. Nr.
  • the separation of the D6-GALNS protein sample on analytical size exclusion chromatography column is monitored by recoding of UV at 280 mm ( Figure 3, blue line, scale left Y-axis) and conductivity ( Figure 3, brown line, scale right Y-axis).
  • Two major peaks are observed with 9.51 mL and 13.64 mL retention volume respectively.
  • the retention volume of 9.51 mL of the first peak corresponds to an apparent moiecuiar weight of 112 kD
  • the second peak with maximum absorption at a retention volume of 13.64 mL might be due to DNA fragment that are present in the solution (see DNA Quantification).
  • the first peak with maximum absorption at a retention volume of 9.51 mL contains the D6-GALNS protein and shows enzymatic activity in the GALNS activity assay.
  • the D6-GALNS protein sample eiuted of the DEAE column at 120 mM NaCi in 20 mM Tris, pH 7.0 buffer is subjected to trichloroacetic acid (TCA) protein precipitation prior to mass spectrometry analysis.
  • TCA trichloroacetic acid
  • the protein content of the DEAE-coJumn eiuted fraction is precipitated by adding 2 volumes of ice cold 20 % TCA, vortex, and incubation from 10 mins to several hours on ice. Ideally a final concentration of 15 % TCA is achieved.
  • the precipitate is centrifuged in a micro centrifuge at 7000 g for 30 sec, the supernatant is discharged and the peliet is resuspended in equi-voiume of ice-cold etha ⁇ ohether (1 :1 v/v).
  • a second centrifugation step is performed under same conditions and the resulting pellet is dried and resuspended in reduced volume.
  • TOF mass spectrometers operate on the principle that when a temporally and spatially wel! defined group of ions of differing mass/charge (m/z) ratios are subjected to the same applied eSectric field and allowed to drift in a region of constant electric field, they wil! traverse this region in a time which depends upon their m/z ratios.
  • the experiment is performed on an UitraFlexTOF/TOF MALDI tandem time-of-flight (TOF/TOF) mass spectrometer (Bruker Daltonics Inc.).
  • the applied diharmonic pressure from two sources was 25 kV and 23.45 kV respectively with a linear detector voltage of 1.607 kV and a laser repletion rate of 100 Hz.
  • the mass/charge (m/z) ration of the D6-GALNS protein sample is recorded and plotted against the intensity measured in absorption units (a.u.) ( Figure 4).
  • the D6-GALNS protein sample contained a single wel! defined macromolecule with the apparent mass calculated from the m/z ratio of 60 kDa.
  • This apparent mass of 60 kDa determined by MALDI TOF MS corresponds welt to the calculated theoretical molecular weight of D6-GALNS based on the primary amino acid sequence (57 kDa).
  • the conformational dimer that was observed in the analytical seize exclusion chromatography is a non ⁇ covalent association of two mon ⁇ meric D6-GALNS proteins that can be separated under the denaturing conditions of the applied TCA precipitation and ionization of MALDI mass spectrometry (as wel! as under the denaturing and reducing conditions of the SDS-PAGE analysis).
  • the aim of this experiment is to asses the in vivo enzymatic effectiveness of conformational dimeric D ⁇ -GALNS by observing the accumulative enzymatic activity of D6-GALNS over time in the circulation of 3-month-old homozygous mutant MPS i VA knockout mice.
  • D6-GALNS is diluted in PBS and injected intravenously through the lateral tail vein.
  • Homozygous knockout mutant (Gains ' ' 1' ) mice are obtained from the MPS IVA mouse colony (Tomatsu et al. 2003). All mice are identified at birth as normal or mutants by obtaining genomic DNA from tissue obtained by a toe dip and ampfifying with primer.
  • a dose of 250 units/g body weight of GALNS-D6 is injected into the tail vein of 3-month-oid mice and blood samples are collected by retro-orbital puncture at different intervals after the infusion.
  • Treated mice are examined by assaying the GALNS activity (U/mi) at 0, 2, 5, 10, 20, 30, 60, 120, and 180 minutes post infusion to determine cumulative enzymatic activity of enzyme in the blood stream ( Figure 5),
  • the enzymatic activity of GALNS in the blood circulation of GALNS + mice shows a biphasic kinetic with two distinct half lives of 1.84 min and 13.3 min.
  • the enzymatic activity of dimeric D6-GALNS displays a prolonged monophasic kinetic with a half life of 15.6 min.
  • the D6 polypeptide mediated conformational change in D6-GALNS leads to an unpredictable prolonged half fife that results in a beneficial increase of enzymatic exposure in vivo. Plotting the enzymatic activity in vivo over time one can determine the area under the curve (AUC) that is an indirect measure for the enzymatic exposure in vivo.
  • AUC area under the curve
  • the area under the enzymatic activity (U/mL) per time (min) curve is increased dramatically by four fold (approximately 5'400 1 OOO U/mL*min "1 for D6- GALNS versus 1 '350 1 OOO U/mL * rnin '1 for GALNS).
  • the content of the present invention is the novei D ⁇ polypetide-mediated conformational change of GALNS leading to a beneficial enhancement of cumulative overal! effective enzymatic activity in vivo.

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