WO2017136533A1 - Méthodes pour traiter, diagnostiquer, et surveiller le traitement des mucopolysaccharidoses - Google Patents

Méthodes pour traiter, diagnostiquer, et surveiller le traitement des mucopolysaccharidoses Download PDF

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WO2017136533A1
WO2017136533A1 PCT/US2017/016188 US2017016188W WO2017136533A1 WO 2017136533 A1 WO2017136533 A1 WO 2017136533A1 US 2017016188 W US2017016188 W US 2017016188W WO 2017136533 A1 WO2017136533 A1 WO 2017136533A1
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spermine
patient
sample
mps
alpha
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James M. Wilson
Christian HINDERER
Nathan Katz
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The Trustees Of The University Of Pennsylvania
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/132—Amines having two or more amino groups, e.g. spermidine, putrescine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • MPSs mucopolysaccharidoses
  • GAGs glycosaminoglycans
  • GAGs glycosaminoglycans
  • etiology of cognitive dysfunction is not known, although MPS neurons have been shown to exhibit aberrant overgrowth of neurites and upregulation of GAP43, a key regulator of neurite growth, which may lead to defects in neural development and plasticity.
  • Mucopolysaccharidosis type I is a recessive disorder caused by mutations in the gene encoding a-L-iduronidase (IDUA), a lysosomal enzyme responsible for the breakdown of complex polysaccharides called glycosaminoglycans (GAGs).
  • IDUA a-L-iduronidase
  • GAGs glycosaminoglycans
  • MPS I is characterized by systemic accumulation of heparan and dermatan sulfate, two GAGs that are normally degraded by IDUA.
  • MPS I patients present with a constellation of clinical findings, many of which are directly linked to mechanical consequences of GAG storage, such as cardiac valve thickening, liver and spleen enlargement, corneal clouding, and airway infiltration E. F. Neufeld, J.
  • ganglioside accumulation in neurons occurs to a similar degree in MPS VI, a disease without cognitive impairment, indicating that elevated brain gangliosides are unlikely to be related to CNS pathogenesis in MPS I, II, III, and VII [S. U. Walkley et al, Abnormal neuronal metabolism and storage in mucopolysaccharidosis type VI (Maroteaux-Lamy) disease. Neuropathology and applied neurobiology 31, 536-544 (2005); published online EpubOct (10.1111/j.1365- 2990.2005.00675.x).].
  • Murine models of MPS III show progressive overexpression of GAP43 in the brain, and cultured neurons from MPS mice likewise overexpress GAP43 and elaborate longer neurites with more complex branching patterns than normal neurons [M. Hocquemiller, et al repeal J. M. Heard, GAP43 overexpression and enhanced neurite outgrowth in
  • CSF cerebrospinal fluid
  • MPS mucopolysaccharidosis
  • threshold spermine levels refers to the spermine concentration as assessed in a suitable biological sample from a patient (e.g., cerebrospinal fluid (CSF)), which levels (i.e., concentrations) are at a predetermined value. Values above the threshold levels are indicative of a disease state. Such a predetermined value may be a value for a patient subpopulation (e.g., an MPS type or subtype, an age group, and/or a gender-based subgrouping) or may be determined on an individual basis to facilitate personalized treatment. In certain embodiments, these levels are determined in a quantitative manner, e.g., by measuring in a mass spectral apparatus.
  • CSF cerebrospinal fluid
  • spermine concentrations in CSF of at least about 2 ng/mL, or greater than about 3 ng/mL are threshold values indicative of MPS disease.
  • spermine concentrations below 3 ng/mL e.g., below about 2 ng/mL, or about 0. 1 ng/mL to about 2 ng/mL, are considered normal levels.
  • threshold spermine levels may be determined by values relative to normal subjects using other apparatus or methods.
  • spermine levels up to 30 fold higher were observed in the CSF of animals having MPSI or MPSVII to normal levels. Similar trends are observed in the human population.
  • spermine levels e.g., a 2 to 3 fold decrease in spermine concentrations
  • This significant reduction of spermine post- treatment is indicative of the ability of the method of invention to be used in monitoring patient treatment for the neurodegenerative component of MPS.
  • Similar trends may be observed in other biological samples, such that spermine levels can be used to monitor the efficacy of systemic treatments.
  • Such other samples may include, e.g., plasma, fibroblasts or urine.
  • a method for determining if a patient has a mucopolysaccharidosis disorder selected from type I, type II, type III or type VII is provided.
  • the method involves (a) obtaining a sample from a patient; and (b) detecting whether spermine is present in said sample above a threshold concentration, whereby these elevated concentrations (used interchangeably with the term "level") correlate with a patient having an MPS disorder.
  • the method may further involve determining the type of MPS disorder the patient has. This may involve obtaining a sample from the patient diagnosed with an MPS disorder as provided herein and screening the sample for a deficiency in one or more of a human enzyme selected from: a-L-iduronidase (IDUA), human iduronate sulfates (hIDS), and/or beta-glucoronidase.
  • IDUA a-L-iduronidase
  • hIDS human iduronate sulfates
  • beta-glucoronidase a human enzyme selected from: a-L-iduronidase (IDUA), human iduronate sulfates (hIDS), and/or beta-glucoronidas
  • the sample is preferably obtained from the cerebrospinal fluid.
  • the systemic effects of an MPS condition may be monitored using plasma, urine, or another suitable sample.
  • a method for monitoring a patient having a prior MPS diagnosis involves (a) obtaining a sample from a patient; and (b) detecting whether spermine above a threshold level is present in said sample, whereby these elevated levels of spermine correlate to a patient having an MPS disorder.
  • efficacy of an MPS treatment can be monitored using the level of spermine, which when above a threshold level allows for a decision to be made regarding the timing and dosing of further therapy in the patient, which may be enzyme replacement therapy (ERT), ERT co-therapy in a patient who is a gene therapy patient or candidate, and/or bone marrow transplant (BMT).
  • ERT enzyme replacement therapy
  • BMT bone marrow transplant
  • a viral vector expressing at least one enzyme selected from alpha-L-Iduronidase, iduronate sulfatase, heparan N-sulfatase, alpha-N-acetylglucosaminidase, Acetyl CoA: alpha-glycosaminide acetyltransferase, N- acetylglucosamine 6-sulfatase, or beta-glucuronidase in treating a mucopolysaccharidosis in a patient having spermine levels above the threshold level.
  • an enzyme selected from one or more of: alpha-L-Iduronidase, iduronate sulfatase, heparan N-sulfatase, alpha-N- acetylglucosaminidase, Acetyl CoA: alpha-glycosaminide acetyltransferase, N- acetylglucosamine 6-sulfatase, or beta-Glucuronidase, for use in treating a mucopolysaccharidosis in a patient, comprising: assaying a patient sample spermine levels above a threshold concentration; assaying a sample from the patient having spermine levels exceeding a threshold concentration for a deficiency in one or more of the enzymes: and administering a therapeutically effective amount of the enzyme in which the patient is deficient.
  • compositions comprising at least one viral vector expressing at least one enzyme selected from one or more of: alpha-L- Iduronidase, iduronate sulfatase, heparan N-sulfatase, alpha-N-acetylglucosaminidase, Acetyl CoA: alpha-glycosaminide acetyltransferase, N-acetylglucosamine 6-sulfatase, or beta-Glucuronidase for use in treating a mucopolysaccharidosis in a patient, comprising: assaying a sample from a patient for spermine levels above a threshold concentration; assaying a sample from the patient having spermine levels elevated above the threshold level for a deficiency in one or more of the enzymes: and administering a therapeutically effective amount of the composition expressing the enzyme in which the patient is deficient.
  • alpha-L- Iduronidase iduronate sulfat
  • a method of treating MPSI, II, III, or VII in a patient comprises administering an effective amount of a spermine or spermine synthase inhibitor to the patient.
  • the patient is a human.
  • a method of treating patient with a spermine synthase inhibitor is provided, wherein the inhibitor is an anti-spermine synthase antibody.
  • a method of treating a patient by delivering with a cell surface proteoglycan that blocks binding of spermine to heparin sulfate is provided.
  • a method of treating a patient involves delivering a selective spermine synthase inhibitor selected from N-(3-aminopropyl)cyclohexylamine (APCHA) and methylthiopropolyamine (MTPA) .
  • a patient having MPSI, MPSII, III or VII is treated by delivering an an oligonucleotide inhibitor specific for spermine synthase.
  • a method of treating cognitive dysfunction in a patient having MPSI, II, III, or VII in a patient comprises administering an effective amount of an inhibitor of the activity of spermine synthase to the patient.
  • FIGS 1A-1B illustrate elevated CSF spermine in MPS I.
  • FIG 1A shows a heatmap of the top 100 differentially detected metabolites (ANOVA) is shown. The youngest animal in the MPS I cohort (28 days of age) is indicated by an asterisk.
  • FIG IB is a graph showing spermine concentration was measured by a quantitative isotope dilution LC/MS assay in CSF samples from 6 infants with MPS I and 2 normal infants.
  • FIGS 2A-2F illustrate spermine dependent aberrant neurite growth in MPS I neurons.
  • Cortical neurons harvested from E18 wild-type or MPS I mouse embryos were treated with spermine (50 ng/mL) or the spermine synthase inhibitor APCHA 24 hours after plating. Neurite number, length and branching were quantified for 45-65 randomly selected neurons from duplicate cultures per treatment condition by a blinded reviewer.
  • FIG 2A is a bar chart providing neurites for MPSI, MPSI + APCHA, or
  • FIG 2B is a bar chart providing branch points for MPSI, MPSI + APCHA, or MPSI+APCHA+spermine, as compared to a wild-type.
  • FIG 2C is a bar chart providing arbor length for MPSI, MPSI + APCHA, or MPSI+APCHA+spermine, as compared to a wild-type *** p ⁇ 0.0001 (ANOVA followed by Dunnett's test).
  • FIG 2D is a bar chart comparing neurites/cell for wild-type treated with spermine as compared to wild-type.
  • FIG 2E is a bar chart comparing branch points/cell for wild-type treated with spermine as compared to wild-type.
  • FIG 2F is a bar chart comparing arbor length /cell for wild-type treated with spermine as compared to wild-type.
  • FIGS 3A - 3C illustrate normalization of CSF spermine levels and brain GAP43 expression in MPS I dogs following gene therapy.
  • Five MPS I dogs were treated with an intrathecal injection of an AAV9 vector expressing canine IDUA at one month of age.
  • Two of the dogs (1-549, 1-552) were tolerized to IDUA by liver directed gene therapy on postnatal day 1 in order to prevent the antibody response that is elicited to IDUA in some MPS I dogs.
  • FIG 3A is a bar chart showing the results of IDUA activity measured in brain tissue six months after intrathecal vector injection.
  • FIGS 3B and 3C are graphs showing results following measurement of GAP43 in cortical brain samples quantified relative to ⁇ -actin by densitometry.
  • FIGS 4A-4B are graphs which illustrate the use of spermine as a CSF biomarker for evaluation of CNS directed gene therapy in MPS I.
  • FIG 4A provides results following measurement of CSF spermine levels measured six months after treatment.
  • Three MPS I cats were treated with intrathecal AAV9 expressing feline IDUA (1012 GC/kg).
  • FIG 5 illustrates the mean decrease accuracy for metabolites identified by random forest analysis.
  • FIG 6 illustrates the expression of enzymes in the polyamine synthetic pathway in MPS I dog brain samples.
  • FIG 7 illustrates spermine concentration in MPS VII dog CSF.
  • FIG 8 illustrates that there is no impact of APCHA treatment on WT neuron growth.
  • FIG 9 illustrates spermine levels in MPSI patients receiving: 1 intrathecal dose enzyme replacement therapy (ERT) + one dose iv ERT (no bone marrow transplant
  • BMT (second point); 2 IT ERT and is 3 months post BMT (third point), and 3 IT ERT and is 6-months post-BMT (last point).
  • the present invention provides methods for treatment of the neurodegenerative and systemic symptoms and pathology of MPSI, MPSII, MPSIII and MPSVIII.
  • spermine accumulation is associated with mucopolysaccharidoses (MPS) type I, as well as other types of MPS associated with heparin accumulation, such as MPSII, MPSIII, and MPSVIII.
  • MPS mucopolysaccharidoses
  • MPSIII mucopolysaccharidoses
  • the potential for impaired HS metabolism to trigger accumulation of a metabolite that alters neuron growth could point to a novel connection between enzyme deficiencies and the abnormal neurite growth phenotype in MPS, which may explain the cognitive dysfunction associated with these disorders.
  • Prevention of spermine accumulation may be useful in preventing or alleviating these cognitive issues.
  • Spermine is a polyamine.
  • the sequence of human spermine synthase is provided in UniProtKB - P52788 (SPSY_Human) jitti //www.umprot.orgA.miprot, P52788.
  • SEQ ID NO: 1 Isoform 2 of spermine synthase is reproduced in SEQ ID NO: 2.
  • Spermine has the molecular formula: CioH 2 6N 4 and is characterized by the structure:
  • threshold spermine levels refers to the spermine concentration as assessed in a suitable biological sample from a patient (e.g., cerebrospinal fluid (CSF)), which levels (i.e., concentrations) are at a predetermined value. Values above the threshold levels are indicative of a disease state. Such a predetermined value may be a value for a patient subpopulation (e.g., an MPS type or subtype, an age group, and/or a gender-based subgrouping) or may be determined on an individual basis to facilitate personalized treatment. In certain embodiments, these levels are determined in a quantitative manner, e.g., by measuring in a mass spectral apparatus.
  • CSF cerebrospinal fluid
  • spermine concentrations in CSF of at least about 2 ng/mL, or greater than about 3 ng/mL are threshold values indicative of MPS disease.
  • spermine concentrations below 3 ng/mL e.g., below about 2 ng/mL, or about 0. 1 ng/mL to about 2 ng/mL, are considered normal levels.
  • threshold spermine levels may be determined by values relative to normal subjects using other apparatus or methods.
  • spermine levels up to 30 fold higher were observed in the CSF of animals having MPSI or MPSVII to normal levels. Similar trends are observed in the human population.
  • spermine levels e.g., a 2 to 3 fold decrease in spermine concentrations
  • This significant reduction of spermine post- treatment is indicative of the ability of the method of invention to be used in monitoring patient treatment for the neurodegenerative component of MPS.
  • Similar trends may be observed in other biological samples, such that spermine levels can be used to monitor the efficacy of systemic treatments.
  • Such other samples may include, e.g., plasma, fibroblasts or urine.
  • spermine concentrations in cerebrospinal fluid are used to monitor treatment or the disease progression of a patient having a neurodegenerative disorder, including one or more MPS disorder.
  • personalized spermine concentrations for a selected patient may be used to monitor treatment post- diagnosis. Such diagnosis may be the methods described herein, or by conventional diagnostic methods.
  • an initial CSF spermine concentration may be obtained from a sample pre-treatment and one or more follow-up CSF spermine concentration may be obtained from a sample post-treatment.
  • Such treatment may be enzyme replacement therapy, bone marrow transplant, or gene therapy, or a regimen involving a combination of one or more of these.
  • the threshold value is not restricted to the values defined above, as the subject has an existing diagnosis and to allow for the patient-specific spermine concentration levels.
  • spermine levels in plasma may range from about 1 ng/mL to about 32 ng/mL; and are more typically in the range of about 10 ng/mL to about 12 ng/mL, and values within these ranges.
  • a "normal subject” refers to a subject (e.g., a human) who does not have a neurodegenerative disorder associated with storage diseases, e.g., an MPSI, MPSII, an MPSIII, or an MPS VII disorder.
  • a "spermine inhibitor” is any moiety which inhibits the activity of spermine, including its ability to bind to heparin sulfate.
  • specific (or selective) spermine inhibitors are selected. Such selective spermine inhibitors are characterized by inhibiting spermine activity, but not substantially inhibiting the activity of spermidine or other polyamines.
  • an "inhibitor of spermine synthase” is a moiety which inhibits the ability of spermine synthase to catalyze the production of spermine from spermidine and decarboxylated S-adenosylmethionine (dcSAM).
  • specific spermine synthase inhibitors are selected. Such spermine synthase inhibitors are characterized by inhibiting spermine synthesis, but not substantially inhibiting the activity of spermidine synthase or other polyamine synthetases.
  • Inhibitors may be selected from a variety of classes of compounds, including, e.g., polynucleotide sequences (e.g., antisense oligonucleotides, R A), antibodies, peptides, proteins, or small molecules.
  • suitable oligonucucleotides may be those which prevent transcription of spermine synthase, e.g., antisense oligonucleotides, including antisense RNA, RNAi, and/or siRNA.
  • the inhibitor is an anti-spermine antibody.
  • the inhibitor may be another protein or a peptide that blocks binding of spermine to heparin sulfate, such as, e.g., a cell surface proteoglycan, e.g., a member of the syndecan family.
  • the inhibitor is a selective spermine synthase inhibitor.
  • One examples of a selected synthase inhibitor is N-(3-aminopropyl)cyclohexylamine (APCHA).
  • an inhibitor of spermidine/spermidine synthase may be targeted, in order to prevent conversion to spermine.
  • spermidine synthase inhibitors are adenosyl spermidine, Ado DATO, DCHA, trans-4-methylcyclohexylamine (4MCHA), cyclohexylamine, methylglyoxal bis-( cyclopentylamidinohydrazone) (MGMBP), 2-mercaptopropylamine, N-chlorosulfonyldicyclohexylamine, '-((3- aminopropyl) amino)-5'-deoxy adenosine, l-aminooxyl-3-aminopropane, 5-(isobutylthio) adenosine, 5'-(methylthio) adenosine and any functional homologs and analogs thereof.
  • inhibitors may be non-specific for spermine and additional inhibitors for related polyamine pathways may be required for combination with these inhibitors.
  • inhibitor means to reduce spermine levels and/or spermine activity to below the elevated levels associated with mucopolysaccharidosis. In certain embodiments, levels are reduced by 5%, reduced by 10%, reduced by 15%, or reduced to within 10% of normal spermine levels in plasma and/or cerebrospinal fluid (CSF). In certain embodiments, levels are reduced to less than 2 ng/mL or less than 1 ng/mL in the CSF, or less than 10 to 12% ng/mL in plasma.
  • Spermine concentration may be determined using a suitable assay.
  • a suitable assay For example the assay described in J Sanchez-Lopez, et al, "Underivatives polyamine analsyis is plant samples by ion pair liquid chromatography coupled with electrospray tandem mass spectrometry," Plant Physiology and Biochemistry, 47 (2009): 592-598, avail online 28 Feb 2009; MR Hakkinen et al, "Analysis of underivatized poly amines by reversed phase liquid chromatography with electrospray tandem mass spectrometry", J Pharm Biomec Analysis, 44 (2007): 625-634, quantitative isotope dilution liquid chromatography (LC)/mass spectrometry (MS) assay.
  • LC liquid chromatography
  • MS mass spectrometry
  • a method of diagnosing mucopolysaccharidoses (MPS) I is provided.
  • the method involves obtaining a cerebrospinal fluid or plasma sample in a human patient; detecting spermine concentration levels in the sample; and diagnosing the patient with a mucopolysaccharidosis selected from MPS I, II, III or VII in a patient having spermine concentrations in excess of normal levels.
  • Spermine concentrations may be determined in a quantitative manner, e.g., by measuring in a mass specification apparatus.
  • threshold values are about 3 ng/mL or in certain embodiments, about 2 ng/mL.
  • untreated patients having an MPS disorder as descried herein have spermine concentrations above about 3 ng/mL, or in some embodiments, above 2 ng/mL are above threshold spermine values, wherein normal subjects, having values generally below Ing/mL to 2 ng/mL, and up to but not including 3 ng/mL.
  • significantly elevated spermine levels may be determined by values relative to normal subjects using other apparatus or methods.
  • the sample is a cerebrospinal fluid sample. In other embodiments, the sample is a plasma sample.
  • the spermine concentration levels are measured by any suitable method, e.g., an isotype dilution liquid chromatography - mass spectrometry. Suitable instruments are commercially available. See, e.g., AB Sciex QTrap 5500 mass spectrometer system [Metabolon]; Waters TQ-S Triple Quad mass spectrometer system; Thermo Scientific Q Exactive HF mass spectrometer system. Still other apparatus may be selected by one of skill in the art provided with the information in this specification.
  • MPS mucopolysaccharidoses
  • hIDUA human alpha-L-iduronidase
  • the hIDUA is delivered via a viral vector.
  • the hIDUA is delivered intrathecally.
  • the hIDUA is delivered to the liver.
  • the hIDUA is delivered via enzyme replacement therapy (ERT).
  • ERT enzyme replacement therapy
  • Such ERT may involve administering a fusion protein comprising the hIDUA.
  • a method of diagnosing and/or treating MPSI in a patient, or monitoring treatment involves obtaining a suitable sample from a human patient suspected of having MPSI; detecting spermine concentration levels in the sample; diagnosing the patient with a mucopolysaccharidosis selected from MPS I in the patient having significantly elevated spermine concentrations; and delivering an effective amount of human IDUA to the diagnosed patient as provided herein.
  • the amino acid sequence of human IDUA is reproduced in SEQ ID NO: 10.
  • This enzyme may be used to deliver functional IDUA to a patient for enzyme replacement therapy.
  • the coding sequence for this enzyme may be used to construct a viral vector expressing this enzyme.
  • An example of a suitable enzyme may include laronidase (available as AldurazymeTM (Genzyme)).
  • the method involves monitoring and adjusting MPSII therapy. Such method involves obtaining a sample from a human patient undergoing therapy for MPSII; detecting spermine concentration levels in the sample by performing a mass spectral analysis; adjusting dosing levels of the MPSII therapeutic if spermine concentrations above the threshold level are detected.
  • patients having untreated MPSII may have spermine concentration levels of greater than 2 ng/mL, more commonly above 3 ng/mL and up to about 100 ng/mL. If a patient has levels approaching normal levels, dosing may be lowered. Conversely, if a patient has higher than desired MPSII levels, higher doses, or an additional therapy, e.g., ERT may be provided to the patient.
  • Human IDS includes the protein encoded by the published gene coding sequence of
  • the expression cassette contains a hIDS gene characterized by having the nucleotide sequence at least about 80% identical to SEQ ID NO: 8 and encodes a functional human iduronate-2-sulfatase.
  • the sequence is at least about 85% identity to SEQ ID NO: 8 or at least about 90% identical to SEQ ID NO: 8 and encodes a functional human iduronate-2- sulfatase.
  • a functional human iduronate-2-sulfatase may include a synthetic amino acid sequence in which all or a portion of the first 25 amino acids of the preproprotein SEQ ID NO: 7, which correspond to the leader (signal) peptide, are replaced with a heterologous leader peptide.
  • This leader peptide e.g., such as the leader peptides from interleukin-2 (IL-2) or oncostatin, can improve transport of the enzyme out of the cell through its secretory pathway into the circulation and consequently facilitate reuptake by untransduced cells in addition to normal trafficking of the enzyme to the intracellular lysosomal compartment.
  • Suitable leader peptides are preferably, although not necessarily of human original.
  • Suitable leader peptides may be chosen from http://proline.bic.nus.edu.sg/spdb/zhang270.htm , which is incorporated by reference herein, or may be determined using a variety of computational programs for determining the leader (signal) peptide in a selected protein. Although not limited, such sequences may be from about 15 to about 50 amino acids in length, or about 19 to about 28 amino acids in length, or may be larger or smaller as required. In addition, at least one in vitro assay has been described as being useful to assess the enzymatic activity of an IDS enzyme [see, e.g., Dean et al, Clinical Chemistry, 2006 Apr; 52(4): 643-649].
  • all or a portion of the proprotein may be removed and optionally replaced with a heterologous mature peptide.
  • human IDS variant 2 [SEQ ID NO: 9], or a sequence encoding same, may be used to express the protein.
  • the hIDS is delivered via a viral vector.
  • the hIDS is delivered intrathecally.
  • the hIDS is delivered to the liver. Suitable vector constructs and methods have been described, e.g., US Published
  • the hIDS is delivered via enzyme replacement therapy (ERT) (e.g., using Elaprase® or another suitable composition).
  • ERT enzyme replacement therapy
  • Such ERT may involve administering a fusion protein comprising the hIDS.
  • the enzyme is delivered intrathecally.
  • the enzyme is delivered to the liver.
  • a method of diagnosing and treating a MPS III in a patient comprises: obtaining a sample from a human patient; detecting spermine concentration levels in the sample; diagnosing the patient with a mucopolysaccharidosis selected from a MPS III in a patient having spermine concentrations above the threshold level; and (d) delivering an effective amount of a therapeutic gene to the diagnosed patient.
  • the patient may be diagnosed with Sanfilippo Syndrome, of which there are four types: (A): heparin N- sulfatase (gene location 17q25.3), MPSIII-B (N-acetyl-alpha-D-glucosaminadase) (gene location 17q21), MPIII-C (acetyl-CoA; alpha-glucosaminide acetyltransferase (gene location 8pl l-ql3); MPSIII-D: N-acetylglucosamine-G-sulfate sulfatase (gnee location 12ql4).
  • the different subtypes of MPS III are treated using different enzymes.
  • MPS IIIA may be treated using N-sulfoglucosamine sulfohydrolase (SGSH) [UniProtKB - P51688; signal peptide (aa 1-20) and mature chain (aa 21-502), SEQ ID NO: 3] or heparan N-sulfatase (Sanfilippo syndrome); MPS IIIB may be treated using N- acetyl-alpha-D-glucosaminadase [UniProtKB - P54802; signal peptide (aa 1-23); 82kDa chain (aa 24-743); 77 kDa chain (aa 59-743); SEQ ID NO:4]; MPS IIIC may be treated using acetyl-CoA:alpha-glucosaminidase [UniProtKB - Q68CP4; SEQ ID NO:5; and MPS HID may be treated using N-acetylglucosamine-6-sulfate s
  • the enzyme may be expressed from a viral vector delivered according to gene therapy methods.
  • the gene is delivered via enzyme replacement therapy (ERT).
  • ERT enzyme replacement therapy
  • Such ERT may involve administering a fusion protein comprising the enzyme.
  • the enzyme is delivered intrathecally.
  • the enzyme is delivered to the liver.
  • the method involves monitoring and adjusting MPSIII therapy.
  • Such method involves obtaining a sample from a human patient undergoing therapy for MPSIII; detecting spermine concentration levels in the sample by performing a mass spectral analysis; adjusting dosing levels of the MPS III therapeutic if spermine concentrations above a threshold value are detected.
  • certain patients having an untreated MPSIII may have spermine concentration levels of greater than 2 ng/mL, more generally 3 ng/mL and up to about 100 ng/mL.
  • dosing may be lowered.
  • higher doses, or an additional therapy e.g., ERT may be provided to the patient.
  • a method of diagnosing and treating a MPS VII patient comprises: obtaining a cerebrospinal fluid or plasma sample in a human patient; detecting spermine concentration levels in the sample by performing a mass spectral analysis; diagnosing the patient with a MPS VII patient (Sly syndrome)
  • mucopolysaccharidosis selected from MPS VII in a patient having spermine
  • the gene is delivered intrathecally.
  • the gene is delivered to the liver.
  • the gene is delivered via enzyme replacement therapy (ERT).
  • ERT may involve administering a fusion protein comprising the enzyme.
  • the treatment involves delivering the enzyme ⁇ -glucuronidase (GUS).
  • GUS ⁇ -glucuronidase
  • the enzyme may be expressed from a viral vector delivered according to gene therapy methods.
  • ERT is used.
  • An example of a suitable enzyme for therapy is ABO- 102 [Abeona Therapeutics] .
  • the method involves monitoring and adjusting MPS VII (Sly syndrome) therapy.
  • MPS VII Sly syndrome
  • Such method involves obtaining a sample from a human patient undergoing therapy for MPS VII; detecting spermine concentration levels in the sample by performing a mass spectral analysis; adjusting dosing levels of the MPS VII therapeutic if spermine levels above a threshold level are detected.
  • patients having untreated MPS VII may have spermine
  • concentration levels of greater than 2 ng/mL may be from 3 ng/mL and up to about 100 ng/mL. If a patient has levels approaching normal levels, dosing may be lowered. Conversely, if a patient has higher than desired MPS VII levels, higher doses, or an additional therapy, e.g., ERT may be provided to the patient.
  • the amino acid sequence of human GUSB variant 1 is reproduced in SEQ ID NO: 11.
  • This enzyme may be used to deliver functional GUSB A to a patient for enzyme replacement therapy.
  • the coding sequence for this enzyme may be used to construct a viral vector expressing this enzyme.
  • An example of a suitable enzyme includes Naglazyme® (galsulfase; BioMarin) and UX003 (Ultragenyz Pharmaceutical),
  • a variety of vectors may be selected for use in delivering one or more enzymes useful in treating an MPS disorder.
  • the viral vector is an adeno- associated viral (AAV) vector.
  • AAV adeno- associated viral
  • One desirable AAV is a replication-defective AAV having an AAV9 capsid.
  • AAV9 capsid refers to the AAV9 having the amino acid sequence of GenBank accession: AAS99264, which is incorporated by reference herein. The sequence is reproduced in SEQ ID NO: 12.
  • AAV may include, e.g., natural isolates (e.g., hu31 or hu32), or variants of AAV 9 having amino acid substitutions, deletions or additions, e.g., including but not limited to amino acid substitutions selected from alternate residues "recruited” from the corresponding position in any other AAV capsid aligned with the AAV9 capsid; e.g., such as described in US 9,102,949, US 8,927,514, US2015/349911; and WO 2016/04923 OA 1.
  • Some variation from this encoded sequence is encompassed by certain embodiments, which may include sequences having about 99% identity to the referenced amino acid sequence in GenBank accession:AAS99264 and US7906111 (also WO 2005/033321) (i.e., less than about 1% variation from the referenced sequence.
  • other variants of AAV9, or AAV9 capsids having at least about 95% identity to the above-referenced sequences may be selected. See, e.g., US 2015/0079038. Methods of generating the capsid, coding sequences therefore, and methods for production of rAAV viral vectors have been described. See, e.g., Gao, et al, Proc. Natl.
  • AAV9 intermediate or "AAV9 vector intermediate” refers to an assembled rAAV capsid which lacks the desired genomic sequences packaged therein. These may also be termed an "empty" capsid. Such a capsid may contain no detectable genomic sequences of an expression cassette, or only partially packaged genomic sequences which are insufficient to achieve expression of the gene product. These empty capsids are non-functional to transfer the gene of interest to a host cell.
  • AAV capsids may be selected or generated.
  • sequences of AAV 8 and methods of generating vectors based on the AAV8 capsid are described in US Patent 7,282,199 B2, US 7,790,449, and US 8,318,480, which are incorporated herein by reference.
  • the sequences of a number of such AAV are provided in the above-cited US Patent 7,282, 199 B2, US 7,790,449, US 8,318,480, and US Patent 7,906,111, and/or are available from GenBank.
  • the sequences of any of the AAV capsids can be readily generated synthetically or using a variety of molecular biology and genetic engineering techniques. Suitable production techniques are well known to those of skill in the art. See, e.g.
  • oligonucleotides encoding peptides e.g., CDRs
  • the peptides themselves can generated synthetically, e.g., by the well-known solid phase peptide synthesis methods (Merrifield, (1962) J. Am. Chem. Soc , 85:2149; Stewart and Young, Solid Phase Peptide Synthesis (Freeman, San Francisco, 1969) pp. 27-62).
  • solid phase peptide synthesis methods (Merrifield, (1962) J. Am. Chem. Soc , 85:2149; Stewart and Young, Solid Phase Peptide Synthesis (Freeman, San Francisco, 1969) pp. 27-62).
  • Replication-defective recombinant AAV may be generated using techniques which are known. See, e.g., WO 2003/042397; WO 2005/033321, WO 2006/110689; US 7588772 B2.
  • Such a method involves culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid; a functional rep gene; an expression cassette composed of, at a minimum, AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the expression cassette into the AAV capsid protein.
  • ITRs AAV inverted terminal repeats
  • identity or “percent sequence identity” may be readily determined for amino acid sequences, over the full-length of a protein, a subunit, or a fragment thereof.
  • a fragment is at least about 8 amino acids in length, and may be up to about 700 amino acids. Examples of suitable fragments are described herein.
  • identity is determined in reference to “aligned” sequences.
  • Alignments refer to multiple nucleic acid sequences or protein (amino acids) sequences, often containing corrections for missing or additional bases or amino acids as compared to a reference sequence. Alignments are performed using any of a variety of publicly or commercially available Multiple
  • Sequence Alignment Programs examples include, “Clustal W”, “CAP Sequence Assembly”, “MAP”, and “MEME”, which are accessible through Web Servers on the internet. Other sources for such programs are known to those of skill in the art. Alternatively, Vector NTI utilities are also used. There are also a number of algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the programs described above. As another example, polynucleotide sequences can be compared using FastaTM, a program in GCG Version 6.1. FastaTM provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences.
  • percent sequence identity between nucleic acid sequences can be determined using FastaTM with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) as provided in GCG Version 6.1, herein incorporated by reference.
  • Multiple sequence alignment programs are also available for amino acid sequences, e.g. , the "Clustal X”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box" programs.
  • any of these programs are used at default settings, although one of skill in the art can alter these settings as needed.
  • one of skill in the art can utilize another algorithm or computer program which provides at least the level of identity or alignment as that provided by the referenced algorithms and programs. See, e.g., J. D. Thomson et al, Nucl. Acids Res., "A comprehensive comparison of multiple sequence alignments", 27(13):2682-2690 (1999).
  • compositions which provides a combination or co-therapy useful in treating a patient having an MPS disorder.
  • Such compositions may comprise one or more viral vector stocks, each stock expressing at least one functional enzyme and optionally, a second active component.
  • a patient having an MPS disorder may have more than one of MPSI, MPSII, MPSIII, or MPSVII deficiencies, or may have a second condition requiring therapy.
  • active component may include enzyme replacement therapy which is the same or different from the enzyme expressed from the viral vector.
  • another component may include an immune modulator (e.g., an immunosuppressant), or another active component.
  • a virus particle includes one or more virus particles.
  • the terms “comprise”, “comprising”, “contain”, “containing”, and their variants are open claim language, i.e., are permissive of additional elements.
  • the terms “consists”, “consisting”, and its variants are closed claim language, i.e., exclusive additional elements.
  • a method for determining if a patient has a mucopolysaccharidosis disorder selected from type I, type II, type III or type VII involves (a) obtaining a plasma or CSF sample from a patient; and (b) detecting whether spermine is present in said sample above a threshold value, whereby an amount of spermine in the sample above the threshold correlates to a patient having an MPS disorder.
  • the method may further involve determining the type of MPS disorder the patient has.
  • This may involve obtaining a sample from the patient diagnosed with an MPS disorder as provided herein and screening the sample for a deficiency in one or more of a human enzyme selected from: alpha-L-Iduronidase, iduronate sulfatase, heparan N-sulfatase, alpha-N-acetylglucosaminidase, Acetyl CoA: alpha-glycosaminide acetyltransferase, N-acetylglucosamine 6-sulfatase, or beta-glucuronidase.
  • a human enzyme selected from: alpha-L-Iduronidase, iduronate sulfatase, heparan N-sulfatase, alpha-N-acetylglucosaminidase, Acetyl CoA: alpha-glycosaminide acetyltransferase, N-acetylglucosamine
  • a method for monitoring a patient having a prior MPS diagnosis involves (a) obtaining a plasma or CSF sample from a patient; and (b) detecting whether spermine is present above the threshold amount in said sample.
  • An evaluation of the level of spermine allows for a decision to be made regarding the timing and dosing of further therapy in the patient, which may be enzyme replacement therapy (ERT), or ERT co-therapy in a patient who is a gene therapy patient or candidate.
  • a viral vector expressing at least one enzyme selected from alpha-L-Iduronidase, iduronate sulfatase, heparan N-sulfatase, alpha-N-acetylglucosaminidase, Acetyl CoA: alpha-glycosaminide acetyltransferase, N- acetylglucosamine 6-sulfatase, or beta-glucuronidase in treating a mucopolysaccharidosis in a patient having significantly elevated spermine levels.
  • an enzyme selected from one or more of: alpha-L-Iduronidase, iduronate sulfatase, heparan N-sulfatase, alpha-N- acetylglucosaminidase, Acetyl CoA: alpha-glycosaminide acetyltransferase, N- acetylglucosamine 6-sulfatase, or beta-Glucuronidase, for use in treating a
  • mucopolysaccharidosis in a patient comprising: assaying a plasma or CSF sample in a patient for the presence of spermine above a threshold amount; assaying a sample from the patient having spermine above the threshold for a deficiency in one or more of the enzymes: and administering a therapeutically effective amount of the enzyme in which the patient is deficient.
  • compositions comprising at least one viral vector expressing at least one enzyme selected from one or more of: alpha-L- Iduronidase, iduronate sulfatase, heparan N-sulfatase, alpha-N-acetylglucosaminidase, Acetyl CoA: alpha-glycosaminide acetyltransferase, N-acetylglucosamine 6-sulfatase, or beta-Glucuronidase for use in treating a mucopolysaccharidosis in a patient, comprising: assaying a CSF sample in a patient for the presence of spermine in an amount in excess of a threshold value; assaying a sample from the patient having spermine in excess of the threshold value in CSF for a deficiency in one or more of the enzymes: and administering a therapeutically effective amount of the composition expressing the enzyme in which the patient is deficient
  • a method of treating MPSI, II, III, or VII in a patient comprises administering an effective amount of a spermine or spermine synthase inhibitor to the patient.
  • the patient is a human.
  • a method of treating patient with a spermine synthase inhibitor is provided, wherein the inhibitor is an anti-spermine antibody.
  • a method of treating a patient by delivering with a cell surface is provided, wherein the inhibitor is an anti-spermine antibody.
  • a method of treating a patient which involves delivering a selective spermine synthase inhibitor selected from N-(3-aminopropyl)cyclohexylamine (APCHA) and methylthiopropolyamine (MTPA).
  • APCHA N-(3-aminopropyl)cyclohexylamine
  • MTPA methylthiopropolyamine
  • a patient having MPSI, MPSII, III or VII is treated by delivering an oligonucleotide inhibitor specific for spermine synthase.
  • a method of treating cognitive dysfunction in a patient having MPSI, II, III, or VII in a patient comprises administering an effective amount of an inhibitor of the activity of spermine synthase/spermine synthase to the patient.
  • Cell surface proteoglycans such as glypican-1 can bind spermine through their HS moieties, and after endocytosis of the glypican protein, intracellular cleavage of the HS chain releases bound spermine into the cell [H. H. Li, et al.,. Journal of neuroscience research 69, 30-38 (2002); C. Hinderer, et al., Molecular Genetics and Metabolism, ht p://dx.doi.org. ⁇ lO.1016/j.ymgme.2016.06.006)] .
  • intact HS recycling is essential for spermine uptake.
  • spermine accumulation is not the sole mediator contributing to neurite outgrowth in MPS I.
  • many neurotrophic factors bind through HS modified receptors, and interactions with HS in extracellular matrix can influence neurite growth [D. Georgiev, et al, A critical importance of polyamine site in NMDA receptors for neurite outgrowth and fasciculation at early stages of P19 neuronal differentiation.
  • Spermine accumulation may therefore be one of several factors promoting abnormal neurite growth in MPS I. Of the 15 MPS I dog CSF samples screened, only one fell within the normal range of spermine concentration.
  • CSF spermine is a useful as a noninvasive biomarker for assessing pharmacodynamics of novel CNS-directed therapies for MPS.
  • Samples were stored at -80 ° C until processing. Samples were prepared using the MicroLab STAR® system (Hamilton Company). A recovery standard was added prior to the first step in the extraction process for QC purposes. Proteins were precipitated with methanol under vigorous shaking for 2 min followed by centrifugation.
  • the resulting extract was divided into five fractions: one for analysis by reverse phase (RP)UPLC- MS/MS with positive ion mode electrospray ionization, one for analysis by RP/UPLC- MS/MS with negative ion mode electrospray ionization, one for analysis by hydrophilic interaction chromatography (HILIC)/UPLC-MS/MS with negative ion mode electrospray ionization, one for analysis by GC- MS, and one sample was reserved for backup. Samples were placed briefly on a TurboVap® (Zymark) to remove the organic solvent. For LC, the samples were stored overnight under nitrogen before preparation for analysis. For GC, each sample was dried under vacuum overnight before preparation for analysis.
  • RP reverse phase
  • HILIC hydrophilic interaction chromatography
  • GC- MS hydrophilic interaction chromatography
  • the LC/MS portion of the platform was based on a Waters ACQUITY ultra- performance liquid chromatography (UPLC) and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution.
  • the sample extract was dried then reconstituted in solvents compatible to each of the LC/MS methods.
  • Each reconstitution solvent contained a series of standards at fixed
  • the third aliquot was analyzed via negative ionization following elution from a HILIC column (Waters UPLC BEH Amide 2.1x150 mm, 1.7 um) using a gradient consisting of water and acetonitrile with lOmM ammonium formate.
  • the MS analysis alternated between MS and data-dependent MSn scans using dynamic exclusion. The scan range varied slightly between methods but covered 80-1000 m/z.
  • the samples destined for analysis by GC-MS were dried under vacuum for a minimum of 18 h prior to being derivatized under dried nitrogen using bistrimethyl- silyltrifluoroacetamide.
  • Derivatized samples were separated on a 5% diphenyl / 95% dimethyl polysiloxane fused silica column (20 m x 0.18 mm ID; 0.18 um film thickness) with helium as carrier gas and a temperature ramp from 60° to 340°C in a 17.5 min period.
  • Samples were analyzed on a Thermo- Finnigan Trace DSQ fast-scanning single- quadrupole mass spectrometer using electron impact ionization (EI) and operated at unit mass resolving power. The scan range was from 50-750 m/z.
  • Instrument variability was determined by calculating the median relative standard deviation (RSD) for the standards that were added to each sample prior to injection into the mass spectrometers. Overall process variability was determined by calculating the median RSD for all endogenous metabolites (i.e., non-instrument standards) present in 100% of the pooled matrix samples. Experimental samples were randomized across the platform run with QC samples spaced evenly among the injections.
  • RSS median relative standard deviation
  • Metabolites were identified by automated comparison of the ion features in the experimental samples to a reference library of chemical standard entries that included retention time, molecular weight (m z), preferred adducts, and in-source fragments as well as associated MS spectra and curated by visual inspection for quality control using software developed at Metabolon. Identification of known chemical entities was based on comparison to metabolomics library entries of purified standards. Peaks were quantified using area-under-the-curve measurements. Raw area counts for each metabolite in each sample were normalized to correct for variation resulting from instrument inter-day tuning differences by the median value for each run-day, therefore, setting the medians to 1.0 for each run. This preserved variation between samples but allowed metabolites of widely different raw peak areas to be compared on a similar graphical scale. Missing values were imputed with the observed minimum after normalization.
  • Quantitative MS assay CSF samples (50 ⁇ ,) were mixed with a spermine-d8 internal standard (IsoSciences). Samples were deproteinized by mixing with a 4-fold excess of methanol and centrifuging at 12,000 x g at 4°C. The supernatant was dried under a stream of nitrogen, and then resuspended in 50 ⁇ ⁇ of water. An aliquot of 5 ⁇ ⁇ was subjected to LC-MS analysis. The LC separations were carried out using a Waters ACQUITY UPLC system (Waters Corp., Milford, MA, USA) equipped with an
  • Xbridge® C18 column (3.5 ⁇ , 150 ⁇ 2.1 mm). The flow- rate was 0.15 mL/min, solvent A was 0.1% formic acid and solvent B was 98/2 acetonitrile/H20 (v/v) with 0.1% formic acid.
  • the elution conditions were as follows: 2% B at 0 min, 2% B at 2 min, 60% B at 5 min, 80% B at 10 min, 98% B at 11 min, 98% B at 16 min, 2% B at 17 min, 2% B at 22 min, with the column temperature being 35 °C.
  • a Finnigan TSQ Quantum Ultra spectrometer (Thermo Fisher, San Jose, CA) was used to conduct MS/MS analysis in positive ion mode with the following parameters: spray voltage at 4000 V, capillary temperature at 270 °C, sheath gas pressure at 35 arbitrary units, ion sweep gas pressure at 2 arbitrary units, auxiliary gas pressure at 10 arbitrary units, vaporizer temperature at 200 °C, tube lens offset at 50, capillary offset at 35 and skimmer offset at 0. The following transitions were monitored: 203.1/112.1 (spermine); 211.1/120.1 (spermine-d8) with scan width of 0.002 m/z, and scan time being 0.15 s.
  • CSF samples were collected 6-8 months after vector administration.
  • primary cortical neuron cultures were prepared from El 8 IDUA-/- or IDUA+/+ embryos.
  • GAP43 western Samples of frontal cortex were homogenized in 0.2% triton X- 100 using a Qiagen Tissuelyser at 30 Hz for 5 min. Samples were clarified by centrifugation at 4° C. Protein concentration was determined in supernatants by BCA assay. Samples were incubated in NuPAGE LDS buffer with DTT (Thermo Fisher Scientific) at 70° C for 1 hr and separated on a Bis-Tris 4-12% polyacrylamide gel in MOPS buffer. Protein was transferred to a PVDF membrane, and blocked for 1 hr in 5% nonfat dry milk.
  • the membrane was probed with rabbit polyclonal anti-GAP43 antibody (Abeam) diluted to 1 ⁇ g/mL in 5% nonfat dry milk followed by an HRP conjugated polyclonal anti-rabbit antibody (Thermo Fisher Scientific) diluted 1: 10,000 in 5% nonfat dry milk. Bands were detected using SuperSignal West Pico substrate (Thermo Fisher Scientific). Densitometry was performed using Image Lab 5.1 (Bio-Rad).
  • Neurite growth assay Day 18 embryonic cortical neurons were harvested as described above, and plated at a concentration of 100,000 cells / mL on chamber slides (Sigma S6815) or poly-L-lysine (Sigma) coated tissue culture plates in serum-free Neurobasal medium (Gibco) supplemented by B27 (Gibco). Treatments were applied to duplicate wells 24 hours after plating (day 1). Phase-contrast images for quantification were taken on a Nikon Eclipse Ti at 20X using a 600 ms manual exposure and 1.70x gain on high contrast. An individual blind to treatment conditions captured 10-20 images per well and coded them. Images were converted to 8-bit format in ImageJ (NIH) and traced in NeuronJ [E. Meijering, et al. complicat Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images. Cytometry. Part A : the journal of the journal of the journal of the journal of the journal of the journal of the journal of the journal of the journal of the
  • RNA 500 ng was reverse transcribed using the High Capacity cDNA Synthesis Kit (Applied Biosystems) with random hexamer primers. Transcripts for arginase, ornithine decarboxylase, spermine synthase, spermidine synthase, spermine- spermidine acetyltransferase and glyceraldehyde phosphate dehydrogenase were quantified by Sybr green PCR using an Applied Biosystems 7500.
  • CSF samples were collected from 15 normal dogs and 15 MPS I dogs. CSF samples were evaluated for relative quantities of metabolites by LC and GC-MS. A total of 281 metabolites could be positively identified in CSF samples by mass spectrometry. Of these, 47 (17%) were significantly elevated in MPS I dogs relative to controls, and 88 (31%) were decreased relative to controls A heat map of the 50 metabolites most different between groups is shown in Figure 1. Metabolite profiling identified marked differences in polyamine, sphingolipid, acetylated amino acid, and nucleotide metabolism between MPS I and normal dogs.
  • Random forest clustering analysis identified the polyamine spermine as the largest contributor to the metabolite differences between MPS I and normal dogs (FIG 5). On average spermine was more than 30-fold elevated in MPS I dogs, with the exception of one MPS I dog that was under 1 month of age at the time of sample collection.
  • a stable isotope dilution (SID)-LC-MS/MS assay was developed to quantitatively measure spermine in CSF. Samples were screened from 6 children with Hurler syndrome (ages 6-26 months), as well as 2 healthy controls (ages 36 and 48 months).
  • spermine elevation was a general property of heparan sulfate storage diseases or specific to MPS I, spermine was measured in a CSF sample from a canine model of MPS VII, which exhibited a similar elevation (FIG 7).
  • GAP43 a central regulator of neurite growth
  • MPS III mouse neurons both in vitro and in vivo, suggesting that the same neurite growth pathway aberrantly activated in neuron cultures is also active in vivo
  • MPS I dogs can develop antibodies to the normal IDUA enzyme, so two of the dogs were pre-treated as newborns with hepatic IDUA gene transfer to induce immunological tolerance to the protein.
  • Both tolerized dogs exhibited brain IDUA activity well above normal following AAV9 treatment.
  • the three non-tolerized dogs exhibited varying levels of expression, with one animal reaching levels greater than normal and the other two exhibiting expression near normal ( Figure 3A).
  • CSF spermine reduction was inversely proportional to brain IDUA activity, with a 3 -fold reduction relative to untreated animals in the two dogs with the lowest IDUA expression, and more than 20-fold reduction in the animal with the highest expression (Figure 3).
  • GAP43 was upregulated in frontal cortex of MPS I dogs, and expression was normalized in all vector treated animals (Figure 3).
  • NMDA signaling induces neurite outgrowth, and the spermine sensitive subunit of the receptor is highly expressed during development [D. Georgiev, et al, Experimental cell research 314, 2603-2617 (2008); published online EpubAug 15 (10.1016/j.yexcr.2008.06.009); R. G. Kalb, Regulation of motor neuron dendrite growth by NMDA receptor activation. Development 120, 3063-3071 (1994); J. Zhong, et al, Expression of
  • CSF spermine may be useful as a noninvasive biomarker for assessing pharmacodynamics of novel CNS-directed therapies for MPS.
  • sequence Listing Free Text The following information is provided for sequences containing free text under numeric identifier ⁇ 223>.

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Abstract

L'invention concerne des méthodes permettant de diagnostiquer et de traiter la mucopolysaccharidose au moyen de concentrations de spermine. Les méthodes de traitement comprennent l'utilisation d'inhibiteurs de polyamine spécifiques.
PCT/US2017/016188 2016-02-03 2017-02-02 Méthodes pour traiter, diagnostiquer, et surveiller le traitement des mucopolysaccharidoses WO2017136533A1 (fr)

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WO2022165313A1 (fr) 2021-02-01 2022-08-04 Regenxbio Inc. Thérapie génique de céroïdes-lipofuscinoses neuronales
CN115032377A (zh) * 2022-08-11 2022-09-09 裕菁科技(上海)有限公司 一种粘多糖贮积症生物标记物及应用
US11555206B2 (en) 2017-11-30 2023-01-17 The Trustees Of The University Of Pennsylvania Gene therapy for mucopolysaccharidosis IIIA
US11723989B2 (en) 2017-11-30 2023-08-15 The Trustees Of The University Of Pennsylvania Gene therapy for mucopolysaccharidosis IIIB

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