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WO1998031817A2 - Sialidase lysosomiale humaine et utilisations therapeutiques de celle-ci - Google Patents

Sialidase lysosomiale humaine et utilisations therapeutiques de celle-ci Download PDF

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Publication number
WO1998031817A2
WO1998031817A2 PCT/CA1998/000026 CA9800026W WO9831817A2 WO 1998031817 A2 WO1998031817 A2 WO 1998031817A2 CA 9800026 W CA9800026 W CA 9800026W WO 9831817 A2 WO9831817 A2 WO 9831817A2
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sialidase
patients
sialidosis
dna
human
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PCT/CA1998/000026
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WO1998031817A3 (fr
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Michel Potier
Alexey V. Pshezhetsky
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Hopital Sainte-Justine
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Priority to AU55468/98A priority Critical patent/AU5546898A/en
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Publication of WO1998031817A3 publication Critical patent/WO1998031817A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01018—Exo-alpha-sialidase (3.2.1.18), i.e. trans-sialidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to the identification of a complete cDNA coding for human lysosomal sialidase, its cloning, sequencing and expression, and to the identification of mutations found in sialidosis patients and chromosomal mapping of the sialidase gene.
  • Sialidosis is an autosomal recessive disease caused by genetic deficiency of a lysosomal sialidase (EC 3.2.1.18, neuraminidase ) which catalyzes the hydrolysis of the terminal sialic acid residues of glycoconjugates .
  • Sialidase acetylneuraminyl hydrolase or neuraminidase, EC 3.2.1.18 catalyzes the hydrolysis of terminal sialic acid residues of oligosaccharides, glycoproteins and glycolipids.
  • Sialidase has been well studied in viruses and bacteria where it destroys the sialic acid receptors on host cells, and mobilize bacterial nutrients.
  • sialidases In mammals, three types of sialidases, localized to the lysosome, plasma membrane and cytosol, have been described. They have different substrate specificities and biochemical properties and are probably encoded by different genes.
  • the lysosomal acid-sialidase is associated with cathepsin A (also named "protective protein"), and ⁇ -galactosidase in a 1.27 MDa multienzyme complex.
  • This complex is essential for the expression of sialidase activity and for the stabilisation of ⁇ -galactosidase in the lysosome as demonstrated by the existence of an autosomal recessive disease, galactosialidosis, characterized by combined deficiency of ⁇ -galactosidase and sialidase activities secondary to cathepsin A deficiency.
  • the genetic deficiency of lysosomal sialidase in humans causes an autosomal recessive lysosomal storage disease, sialidosis, associated with tissue accumulation and urinary excretion of sialylated oligosaccharides and glycolipids.
  • Sialidosis includes two main clinical variants with different ages of onset and severity.
  • Sialidosis type I is the late-onset form characterized by bilateral macular cherry-red spots and myoclonus.
  • Sialidosis type II is the infantile-onset form which is also associated with skeletal dysplasia, Hurler-like phenotype, mental retardation and hepatosplenomegaly.
  • a severe form of the disease also manifests prenataly and is associated with ascites and hydrops fetalis.
  • the molecular defects in sialidosis have not been characterized since the lysosomal sialidase has never been purified.
  • One aim of the present invention is to provide the identification of a complete cDNA coding for human lysosomal sialidase, its cloning, sequencing and expression.
  • Another aim of the present invention is to provide the identification of mutations found in sialidosis patients and chromosomal mapping of the sialidase gene.
  • a human lysosomal sialidase encoded by the DNA sequence set forth in Fig. 1 and having the amino acid sequence depicted in Fig. 1.
  • a method of mutations analysis in patients affected with sialidosis or similar diseases which comprises the steps of: a) isolating DNA from a biological sample of said patients; b) comparing the DNA of step a) with the DNA of the non-mutated protein to determine the presence of any mutation, whereby the presence of a mutation is indicative of sialidosis or similar diseases.
  • a method of treatment of lysosomal storage disorders in patients which comprises administering to said patients recombinant human lysosomal sialidase.
  • the disorders treated include, without limitation, Sialidosis, Tay-Sachs and Sandhof diseases.
  • the use of the human lysosomal sialidase of the present invention for screening pharmaceutical agents against viral sialidase without side effects to the human, wherein said pharmaceutical agents are useful in the treatment of viral infections.
  • the human lysosomal sialidase of the present invention as poly-His and GST fusion proteins for therapeutic protein expression.
  • the use of the human lysosomal sialidase of the present invention for the digestion of sialylated oligosaccharides and glycolipids in milk.
  • an human lysosomal sialidase of the present invention inactivated as an anti-viral agent, wherein the inactivated sialidase can bind with high affinity to surface sialic acid residues thereby preventing the viral protein from binding with the cells .
  • the disorders include, without limitation, Sialidosis, Tay-Sachs and Sandhof diseases.
  • Fig. 1 illustrates the cDNA (SEQ ID N0:1) and deduced amino acid (SEQ ID NO: 2) sequences of human lysosomal sialidase;
  • Fig. 2 illustrates the amino acid sequence alignment of human lysosomal (hum) (SEQ ID NO: 18) and Salmonella typhimurium (salty) (SEQ ID NO: 19) siali- dases by the Lipman-Pearson algorithm;
  • Fig. 3 illustrates an insertion frameshift and two missense mutations identified in sialidosis patients
  • Fig. 4 illustrates the detection of identified mutations in genomic DNA
  • Fig. 5 illustrates the allele-specific amplification of DNA from sialidosis patient GM01718A
  • Fig. 6 illustrates the Northern blot analysis of
  • Fig. 7 illustrates FISH mapping of human lysosomal sialidase on chromosome 6 (6p21.3). DETAILED DESCRIPTION OF THE INVENTION
  • lysosomal sialidase is necessary for the understanding of the molecular bases of two severe human genetic diseases, sialidosis and galactosialidosis.
  • the identification and sequencing of sialidase has been hampered by low tissue content, instability and complete inactivation of the enzyme after dissociation of the 1.27 MDa complex.
  • the identity of the clone was confirmed by expression of sialidase activity, structural analysis of the deduced amino acid sequence, the characterization of mutations in sialidosis patients and chromosomal mapping.
  • the human lysosomal sialidase cDNA was identified, cloned and sequenced.
  • the deduced amino acid sequence of human sialidase is homologous to those of bacterial sialidases, suggesting that the human enzyme shares the same " ⁇ -propeller" fold.
  • Expression of the cloned cDNA in sialidosis fibroblasts produced a 15-fold increase of intracellular sialidase activity.
  • Three mutations, one frameshift insertion and two missense, were identified in six patients affected with the neurodegenerative disease.
  • the gene coding for lysosomal sialidase was mapped to human chromosome 6 (6p21.3), which is consistent with the previous chromosomal assignment of the sialidase gene in proximity to the HLA locus.
  • RNA isolation, analyses and probes Human skin fibroblasts from sialidosis patients were obtained from NIGMS Human Genetic Mutant Cell Repository (GM1718A, GM11604, GM02685, GM02837, GM02921) and the Montreal Children's Hospital Cell Repository (WG544). Cells were cultured to confluency in Eagle's Minimal Essential Medium (Mediatech, Washington DC), supplemented with 10% (v/v) fetal calf serum (MultiCell) and antibiotics. Total RNA or DNA were isolated as described (Maniatis,T. , Fritsch,E.F. and Sambrook,J. (eds) (1982) Molecular cloning: a laboratory manual . Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
  • the human sialidase cDNA was obtained by reverse transcription of total human fibroblast RNA using the Marathon cDNA amplification Kit (Clonetech) according to the manufacturer's protocol, followed by cDNA amplification using 5 ' -CCCAAGCTTAGATCTTGGAGTCTAGCTGCC AGGGT-3' (SEQ ID NO: 3) and 5 ' -AGTTCCCTGAGTTCACATTG-3 ' ( SEQ ID NO : 4 ) primers complementary to the cDNA sequence of the I.M.A.G.E. Consortium clones 31259 and HIBBL61, respectively.
  • the amplification conditions were: denaturation at 94 °C for 6 min, 30 cycles each consisting of 1 min at 94°C, 45 sec at 58°C and 2 min at 72°C followed by 8 min at 72°C.
  • the amplified sialidase cDNA was directly cloned into pCRII vector using the TA cloning kit (InVitrogen) according to the manufacturer's protocol. Nucleotide sequencing
  • a complete cDNA was obtained by RT-PCR amplification of total RNA from human fibroblast and placenta using primers complementary to the sequences of two overlapping clones, 31259 and HIBBL61, cloned into a pCRII vector and sequenced.
  • the resulting cDNA includes 1245 bp of open reading frame and a polyadenylation site within 3' untranslated region (Fig. 1).
  • the predicted signal peptide is underlined, lysosomal C-terminal targeting motif is double- underlined. Potential glycosylation sites are marked with asterisks.
  • Asp-boxes are boxed.
  • the deduced amino acid sequence contains four Asp-boxes (Fig.
  • lysosomal sialidase The analysis of the deduced amino acid sequence of lysosomal sialidase revealed the presence of a possible signal peptide, three glycosylation sites and a Gly-Tyr-X-X-hydrophobic-residue lysosomal-targeting signal, common to several lysosomal integral membrane proteins such as glucocerebrosidase, LAMP-1, LAMP-2, and LGP-85. All these proteins are transported to the lysosome by association of their C-terminus with cytoplasmic proteins called adapters.
  • lysosomal acid phosphatase which is synthesized as a transmembrane protein but is cleaved inside the lysosome into a soluble form.
  • the dual localization of sialidase in the lysosomal membrane and in matrix and the demonstration that the intralysosomal processing of sialidase is necessary for its activation suggest that it may be also transported by a similar mechanism.
  • the Neu-1 gene which controls lysosomal sialidase activity in liver, was identified and mapped within the major histocompatibility gene complex in H-2 locus of mouse and RT-1 locus of rat.
  • the gene causing sialidosis cosegregates with a specific HLA haplotype, also indicating the close linkage of lysosomal sialidase gene locus to HLA at 6p21.3.
  • RNA isolated from patients ' fibroblasts were reverse transcribed after denaturation for 3 min at 65 °C, using 140 ng of oligonucleotide primer dt2o * 20 ⁇ l of IX H-RT buffer (Gibco BRL), containing 1 mM dNTP, 39 units of rRNasin (Promega, Madison), 1 mM DTT and 200 units of Superscript RT. The mixture was incubated 30 min at 42 °C followed by 5 min at 95°C.
  • cDNA was amplified as above and entirely sequenced, instead of using SSCP analysis.
  • the insertion frameshift mutation of patient GM11604 was confirmed by heteroduplex analysis.
  • 50 ng of genomic DNA from patient (identified as GM11604) containing the ACTG ⁇ -11 insertion and from control were PCR amplified using the following primers: 5 ' -CCCAAGCTTAGATCTTGGAGTCTAGCTGCCAGGGT-3 ' (SEQ ID NO: 3) and 5 ' -GCGGCCCCCAGCGTCTGTCC-3 ' (SEQ ID NO: 10).
  • PCR conditions were as for the amplification of cDNA except the annealing temperature was 62°C.
  • 5 ⁇ l of PCR reaction mix was subjected to PAGE (12% (w/v) of polyacrylamide) .
  • 10 ⁇ l of PCR products from control and patient were mixed, heated at 94 °C for 6 min and slowly cooled to room temperature prior to PAGE.
  • a fragment containing the mutation was PCR-amplified from 100 ng of genomic DNA from GM01718A and controls as described above using the forward primer, 5 ' -CGCTACGGAAGTGGGGTCAG-3 ' (SEQ ID NO:8), and reverse primer, 5 ' -TCGGCAGTGGCAGTGGTAGT-3 ' (SEQ ID NO: 12).
  • TCAGCCCAAGCAGGAAAATGATTT-3 ' SEQ ID NO: 14
  • 5'- TCAGCCCAAGCAGGAAAATGATTA-3 SEQ ID NO: 15
  • Two reverse primers 5 ' -GCCACTGGGGCCTGGCCATA-3 ' (SEQ ID NO: 16) and 5 ' -GCCACTGGGGCCTGGCCATG-3 ' (SEQ ID NO: 17) corresponded to normal sequence and to ⁇ 88-to-C mutation.
  • the PCR conditions were as described above except that annealing temperature was 66 °C. 7 ⁇ l of the resulting PCR products were analyzed by electrophoresis in 1% (w/v) agarose gel.
  • Sialidase cDNA was PCR-amplified as three overlaping fragments from reverse-transcribed total fibroblast RNA of six sialidosis patients.
  • SSCP analysis of the first fragment revealed a band with altered migration for cell line GM11604, as compared to other cell lines.
  • the ⁇ - strands localized in the Salmonella typhimurium sialidase X-ray structure (Crennell, S.J. et al., (1993) Proc . Natl . Acad. Sci . USA, 90:9852-9856) are underlined and indicated.
  • N indicate N-acetyl-binding residues
  • C carboxylate- binding residues
  • Y anomeric cycle-binding residue. Sequencing of the variant band demonstrated that the patient was homozygous for a frameshift mutation, an ACTG duplication after nucleotide 7 (+ACTG8-11, Fig. 3A, middle panel). To confirm the mutation, the surrounding sequence was amplified from the genomic DNA of GM11604 and of a control- The PCR products were mixed, heated to 94°C, and analyzed by PAGE to test for heteroduplex formation.
  • the mutation T- j - j y-to-A does not create or remove any restriction site. Therefore we amplified the fragment containing this mutation using a reverse primer with a single mismatch which itself creates Dral site only in amplification products from normal genomic DNA.
  • the Tyyg-to-A mutation removes the Dral site in one allele of GM01718A (Fig. 4C, lower panel).
  • T- j - j q-to-A and T ⁇ o88- to-A mutations were not found in genomic DNA of 28 normal unrelated individuals when tested by the above methods .
  • Fig. 5A Allele-specific PCR on genomic DNA was used to determine whether the mutations are present on the same or different alleles in GM01718A. Fig. 5A, the allele-specific forward and reverse primers terminate in a nucleotide mismatches relative to ⁇ q- to-A and T ⁇ o88 ⁇ to-C mutations.
  • Fig. 6 10 ⁇ g of total RNA from sialidosis fibroblasts and 5 ⁇ g from control were applied on gel. The membranes were hybridized with 32 P-labelled sialidase (A) and tubulin (B) cDNA probes as described above. The size of sialidase mRNA (1.9 kb) is consistent with that of its cDNA, indicating that the cloned cDNA is probably full-length.
  • Fig. 6 (Top) shows SSCP patterns of PCR- amplified cDNA fragments from four sialidosis patients and from control (Figs. 3A, fragment 1; 3B, fragment 2; 3C, fragment 3). Patient GM11604 shows a lower mobility band corresponding to fragment 1 indicating the homozygous insertion; patient GM01718A shows duplication of bands corresponding to fragments 2 and 3 indicating two heterozygous nucleotide changes.
  • Fig. 6 shows the nucleotide sequences of cDNA fragments amplified from patients (upper panels) and controls (lower panels).
  • Patient GM11604 is homozygous for ACTGg-n insertion; patient GM01718A is heterozygous for two mutations: a T'A transversion at nucleotide 779 in fragment 2 and a T*C transition at nucleotide 1088 in fragment 3.
  • the sialidosis patients with identified exonic mutations belong to the severe early-onset clinical form, sialidosis type II.
  • sialidase activity in sialidosis cultured skin fibroblasts
  • T- - j g- -o-A substitution Phe260 to Tyr
  • Tj ⁇ gg-to-C substitution Leu3g3 to Pro
  • Leu3 ⁇ 3 to Pro is likely to induce the structural change which affects the sialidase activity or stability.
  • the ability to genotype patients with sialidosis will permit DNA-based diagnosis and genotype-phenotype correlations .
  • Full-length human sialidase cDNA was obtained as a Xmalll fragment (1.9 kb) from pCRII vector and cloned into the NotI site of a pCMV expression vector (MacGregor,G.R. and Caskey,C.T. (1989) Nucleic Acids Res, 17:2365).
  • Skin fibroblasts of sialidosis type II, WG544 cell line were cultured as described, trypsinized, suspended at a density of 6 X 10° cells per ml in OPTI- MEM medium (BRL) supplemented with 5% (v/v) of fetal calf serum and electroporated twice using a Gene Zapper device (IBI). 24 h after the transfection, sialidase and hexosaminidase activities in cellular homogenates were assayed using the corresponding fluorogenic 4-methylumbelliferyl-glycoside substrates according to published methods (Potier,M. et al. (1979) Anal . Biochem. 94:287-296).
  • One unit of enzyme activity (U) is defined as the conversion of 1 ⁇ mol of substrate per min.
  • the WG544 fibroblast cell line which showed the reduced sialidase mRNA level and negligible sialidase activity was used for the expression of the cloned cDNA.
  • the full-length cDNA was inserted into a pCMV vector containing the CMV immediate-early promoter (MacGregor,G.R. and Caskey,C.T. (1989) Nucleic Acids Res, 17:2365).
  • the resulting construct was electroporated into fibroblasts and, 24 h after transfection, sialidase and ⁇ -hexosaminidase (control) activities were assayed in fibroblast homogenates .
  • the cells transfected with pCMV-SIAL showed a 15-fold increase of sialidase activity (45 ⁇ 15 pmol/min ⁇ g of protein) as compared with untransfected cells (4 ⁇ 0.3 pmol/min ⁇ g of protein) or with pCMV-transfected cells (3 ⁇ 0.1 pmol/min ⁇ g of protein).
  • Heteroduplexes (shown by arrows, Fig. 4A) were identified when the PCR products amplified from GM11604 and a control genomic DNA were mixed, heated at 94 °C for 6 min, slowly cooled to room temperature and analyzed by electrophoresis in 12% polyacrylamide gel. Lane 1-control, lane 2 - GM11604, lane 3 - control plus GM11604. The Ncol restriction site created in one of two alleles by T'C transition was found in PCR product amplified from genomic DNA of GM01718A but not of the control (Fig. 4B) . Lanes 1 and 2 - non-digested control and GMD1718A, lanes 3 and 4 - control and GM01718A, digested with 10 U of Ncol.
  • the Dral restriction site created in DNA fragments amplified from control genomic DNA using the reverse primer with a single nucleotide T for G mismatch was not found in one allele of DNA amplified from GM01718A. Lanes 1 and 2 - non-digested control and GM01718A, lanes 3 and 4 - control and GM01718A, digested with 20 U of Dral.
  • Sialidase cDNA subcloned in pCRII vector was nick-translated with biotin-16-dUTP (Bionick, BRL), and hybridized with lymphocyte chromosomes overnight at 37 °C according to Lichter and Cremer (Lichter, P. and Cremer, T. (1992) In : Rooney,D. E. , and Czepulkowski ,B. H. (eds) Human cytogenetics . A practical approach, Oxford University Press, N.Y. vol. 1:157-192).
  • the biotinylated DNA was detected using rabbit anti-biotin antibodies (Enzo Diagnostic), biotinylated goat anti-rabbit antibodies (Sigma) and streptavidin- FITC (BRL) as recommended by manufacturer.

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Abstract

La présente invention concerne l'identification d'un ADNc complet codant pour la sialidase lysosomiale humaine, le clonage, le séquençage et l'expression de cet ADNc, l'identification de mutations trouvées dans des patients présentant une sialidose, ainsi que la cartographie chromosomique du gène de la sialidase. On décrit notamment une sialidase lysosomiale humaine, codée par la séquence d'ADN développée dans la figure (1) (SEQ ID NO:1) et possédant la séquence d'acides aminés illustrée dans cette figure (1) (SEQ ID NO:2). L'invention concerne également un procédé d'analyse de mutations chez des patients atteints de sialidose ou de maladies similaires, ce procédé consistant: a) à isoler l'ADN à partir d'un échantillon biologique prélevé sur de tels patients, et b) à comparer l'ADN de l'étape a) avec l'ADN de la présente invention, afin de déterminer la présence d'une quelconque mutation, laquelle présence est révélatrice d'une sialidose ou de maladies similaires.
PCT/CA1998/000026 1997-01-14 1998-01-13 Sialidase lysosomiale humaine et utilisations therapeutiques de celle-ci WO1998031817A2 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003052104A1 (fr) * 2001-12-14 2003-06-26 Provost, Fellows And Scholars Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth Near Dublin Gene rapporteur
WO2004047735A2 (fr) 2002-11-22 2004-06-10 Mang Yu Therapies et prophylaxie anti-virus a large spectre
US7645448B2 (en) 2002-11-22 2010-01-12 Nexbio, Inc. Class of therapeutic protein based molecules
US8722869B2 (en) 2002-11-22 2014-05-13 Ansun Biopharma, Inc. Class of therapeutic protein based molecules
JP2014526904A (ja) * 2011-08-31 2014-10-09 セント ジュード チルドレンズ リサーチ ホスピタル リソソームエキソサイトーシス活性のレベルを検出する方法及び組成物並びに使用方法
US20220364101A1 (en) * 2019-07-05 2022-11-17 Tokushima University Modified neuraminidase

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EP0349666B1 (fr) * 1988-07-07 1992-12-23 Milupa Aktiengesellschaft Procédé pour la production enzymatique de denrées alimentaires bifidogéniques diététiques pour les nourrissons
GB9410817D0 (en) * 1994-05-28 1994-07-20 Glaxo Group Ltd Medicaments

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WO2003052104A1 (fr) * 2001-12-14 2003-06-26 Provost, Fellows And Scholars Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth Near Dublin Gene rapporteur
US8084036B2 (en) 2002-11-22 2011-12-27 Nexbio, Inc. Broad spectrum anti-viral therapeutics and prophylaxis
US10525109B2 (en) 2002-11-22 2020-01-07 Ansun Biopharma, Inc. Class of therapeutic protein based molecules
EP1567185A4 (fr) * 2002-11-22 2009-04-22 Mang Yu Therapies et prophylaxie anti-virus a large spectre
US7645448B2 (en) 2002-11-22 2010-01-12 Nexbio, Inc. Class of therapeutic protein based molecules
US7807174B2 (en) 2002-11-22 2010-10-05 Nexbio, Inc. Class of therapeutic protein based molecules
WO2004047735A2 (fr) 2002-11-22 2004-06-10 Mang Yu Therapies et prophylaxie anti-virus a large spectre
US8722869B2 (en) 2002-11-22 2014-05-13 Ansun Biopharma, Inc. Class of therapeutic protein based molecules
JP2006508193A (ja) * 2002-11-22 2006-03-09 ユ，マン 広域抗ウイルス治療および予防薬
US9212353B2 (en) 2002-11-22 2015-12-15 Ansun Biopharma, Inc. Class of therapeutic protein based molecules
US9764007B2 (en) 2002-11-22 2017-09-19 Ansun Biopharma, Inc. Class of therapeutic protein based molecules
JP2014526904A (ja) * 2011-08-31 2014-10-09 セント ジュード チルドレンズ リサーチ ホスピタル リソソームエキソサイトーシス活性のレベルを検出する方法及び組成物並びに使用方法
US9840727B2 (en) 2011-08-31 2017-12-12 St. Jude Children's Research Hospital Methods and compositions to detect the level of lysosomal exocytosis activity and methods of use
US10533208B2 (en) 2011-08-31 2020-01-14 St. Jude Children's Research Hospital Methods of treating dementia associated with Alzheimer's disease with protective protein/cathepsin A (PPCA)
US20220364101A1 (en) * 2019-07-05 2022-11-17 Tokushima University Modified neuraminidase

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WO1998000551A2 (fr) Gene codant la synthase hyaluronan

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