WO2006052911A2 - Expression de laforine pour diagnostiquer et traiter un cancer et d'autres maladies - Google Patents

Expression de laforine pour diagnostiquer et traiter un cancer et d'autres maladies Download PDF

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WO2006052911A2
WO2006052911A2 PCT/US2005/040338 US2005040338W WO2006052911A2 WO 2006052911 A2 WO2006052911 A2 WO 2006052911A2 US 2005040338 W US2005040338 W US 2005040338W WO 2006052911 A2 WO2006052911 A2 WO 2006052911A2
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laforin
epm2a
person
cancer
gene
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PCT/US2005/040338
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WO2006052911A3 (fr
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Pan Zheng
Yang Liu
Yin Wang
Yan Liu
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The Ohio State University Research Foundation
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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11026—Tau-protein kinase (2.7.11.26)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03016—Phosphoprotein phosphatase (3.1.3.16), i.e. calcineurin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • This invention relates to diagnosis and treatment of cancer and other diseases.
  • it relates to diseases that involve abnormal expression of the EPM2A gene, its product, laforin and/or its substrate GSK-3.
  • this invention relates to methods of characterizing an etiology of a cancer by testing at least one cancer cell for the presence of at least one of a) methylation of the EPM2A gene; b) reduced expression of the EPM2A gene; and c) mutation of the EPM2A gene.
  • the invention relates to methods of characterizing an etiology of a cancer by testing at least one cancer cell for the presence of lafora bodies.
  • the invention relates to methods of treating a cancer in a person identified as having a cancer associated with methylated EPM2A gene(s), by administering to the person an effective amount of a drug that demethylates DNA.
  • the invention relates to methods of treating a person identified as having a disease associated with reduced expression or mutation of the EPM2A gene, by activating EPM2A gene(s) in cells of the person's body.
  • the disease can be chosen, for example, from Lafora disease and cancer.
  • the invention relates to methods of treating a person identified as having a disease associated with reduced expression or mutation of the EPM2A gene, by administering laforin to the person.
  • the disease can be chosen from, for example, Lafora disease and cancer.
  • administering the laforin involves administering the laforin protein to the person.
  • administering the laforin involves administering a laforin-expressing genetic construct to the person.
  • the invention involves methods of treating a person identified as having a disease associated with reduced expression or mutation of the EPM2A gene, by administering a genetically engineered GSK3 ⁇ that cannot be phosphorylated at the Serine 9 position.
  • the GSK3/? that cannot be phosphorylated at the Serine 9 position includes an amino acid mutation at Serine 9 with Alanine or any other amino acids.
  • the invention relates to methods of treating a person identified as having a disease associated with reduced expression or mutation of EPM2A gene, by administering to the person at least one compound that restores laforin's ability to phosphorylate GSK3y?.
  • the invention also relates to methods of identifying compounds that modify laforin's ability to phosphorylate GSK3/? at Serine 9, wherein the methods include a step of testing laforin's ability to phosphorylate GSK3/? in the presence and absence of the test compounds.
  • the laforin tested is wild-type laforin and the compounds identified aje either inhibitors or enhancers of laforin's ability to phosphorylate GSK3/?.
  • the laforin tested is mutated laforin and the compounds identified restore laforin's ability to phosphorylate GSK3 ⁇ .
  • the invention also provides methods of treating Alzheimer's disease or other diseases with over-activated GSK3 by administering to a person in need of treatment a compound identified according to the aforementioned methods.
  • the invention provides methods of determining the type of cancer in a person, by assaying a tissue sample from the person for the presence of at least one of a) methylation of EPM2A gene; b) reduced expression of EPM2A gene; and c) mutation of EPM2A gene.
  • the person is a blood relative of a person having Lafora disease.
  • the invention is directed to methods of reducing the likelihood of cancer or other diseases in a person identified as positive for the presence of at least one of a) reduced expression of EPM2A, b) methylation of EPM2A gene, or c) mutation of EPM2A gene, comprising modifying the person's glucose metabolism.
  • the person's glucose metabolism may be modified through diet modification.
  • the person's glucose metabolism is modified by administration of a. drug such as 2- deoxyglucose and/or metforin.
  • the invention is directed to methods of reducing the likelihood of diseases in a person identified as having over-activated GSK3, such as Alzheimer's disease, by administering drugs that reduce expression of EPM2a gene or its products.
  • One of the compounds can be SiRNA using sequence information from Laforin cDNA.
  • FIG. 1 A transgenic mouse (TG-B) line with high penetrance.
  • transgenes the re-arranged ⁇ and ⁇ chain genes
  • mice were obtained from a CD8 + T cell clone with specificity for SV40 large T antigen. Since its generation, the transgenic mice have been backcrossed to B10.BR background for more than 30 generations.
  • A. The survival distribution function among B10.BR, TG-B and F1 (TG-B x C57BL/6) mice. The incidence of lymphoma is significantly higher among TG-B in comparison to WT (P ⁇ 0.0001) and F1 (TG-B x B6) mice (PO.0001). F1 (TG-B x B6) mice developed lymphoma at a significant rate in comparison to WT mice (PO.0001).
  • B The survival distribution function among B10.BR, TG-B and F1 mice. The incidence of lymphoma is significantly higher among TG-B in comparison to WT (P ⁇ 0.0001) and F1 (TG-B x B6) mice (PO.0001). F1 (TG-B x B6) mice developed lymphoma at a significant rate in comparison to WT mice (PO.0001).
  • probe used is the constant region of the TCR ⁇ chain. The location of the
  • transgene is indicated by an arrow in the left panel, the positions of chromosomes 10 and 14 are marked in the right panel.
  • chromosome 14 reflect the location of the endogenous TCR ⁇ loci.
  • TCR gene sequence The junction sequence of TCR Ca and chromosome 10
  • FIG. 3 Defective expression of laforin in lymphoma.
  • A Defective expression of EPM2A mRNA in transgenic mice with lymphoma (Tg- Tu), but not in the non-transgenic littermates (Ntg), or in young transgenic mice that have not yet developed lymphoma (Tg). Total RNA was isolated from thymi. The cDNA was prepared by reverse transcription and amplified using primers that amplify the nearly entire coding sequence of Epm2a.
  • B Absence of the laforin protein expression in Tg-Tu cells, as analyzed by Western blot using a polyclonal anti-laforin antibody.
  • C Defective expression of laforin in lymphoma.
  • T, B cell lineages Down-regulation of the laforin protein in multiple murine malignancies of T, B cell lineages.
  • the thymocytes from non-transgenic littermates (Ntg) and two transgenic mice with lymphoma (Tg-Tu) were examined as control.
  • the malignant cell lines used were T cell lymphoma EL4, YAC-1 , BW5147 and RMA-S 1 B cell lymphoma A20, RAW8.1 , CH27, and myeloma S194. Data shown are representative of three independent experiments.
  • Epm2a is hvpermethylated in lymphoma cells.
  • A Diagram of 5'-CpG island and the location of the 4 primer sets used to amplify the bisulfite treated DNA to examine the methylation status of the Epm2a gene.
  • the 5'-CpG island is 1225 bp in length with 59.2% GC content.
  • the ratio of observed CpG : expected CpG is 0.948.
  • the length of PCR products and the number of CpG di-nucleotides (in parentheses) within each PCR products are labelled.
  • B Summary of the results of bisulfite sequencing of PCR products with LDM3 which contained 45 CpG di-nucleotides.
  • the cell lines used were T cell lymphoma EL4, YAC-1 and BW5147, mastocytoma P815, colon cancer MC38 and fibrosarcoma Meth A.
  • D DNA methylation in lymphoma cell lines. Genomic DNA from the cell lines were isolated and subjected to digested or undigested with methylation sensitive enzyme Sacll overnight. The 280 bp promoter was amplified by real-time PCR.
  • FIG. 5 Laforin suppresses the growth of tumor cells.
  • A. Laforin induces apoptosis in lymphoma cells.
  • V5 tagged Epm2a lentiviral expression construct was transduced into murine T lymphoma cell lines, EL-4, YAC-1 , RMA- S, and BW5147. Forty-eight hours after transduction, the cells were stained with FITC conjugated anti-V5 antibody and 7-amino-actinomycin (7-AAD) to measure DNA contents. Cells were gated for V5 negative (upper panels) and V5 positive cells (Epm2a transduced cells) (lower panels). DNA contents corresponding to 2C and 4C are indicated by arrows.
  • Laforin suppresses the growth of murine T cell lymphoma BW5147.
  • BW5147 cells were transduced with either vector or Epm2a cDNA lentivirus.
  • Polyclonal stable transfectants were injected subcutaneously into the RAG-2(-/-) BALB/c mice (3 x 10 5 cells/mouse). The growth kinetics are presented.
  • C. Laforin suppresses the growth of P185neu- transformed NIH3T3 cell line B104-1-1.
  • B104-1-1 cells were transfected with either vector or Epm2a cDNA.
  • RNAi silencing of laforin expression in fibroblast cell line EEF8 increases its tumorigenicity. EEF8 were transduced with either Epm2a-siRNA or its control lentivirus as detailed in methods.
  • the insert shows the laforin protein levels of representative clones from each group.
  • the control and silenced clones of EEF8 cells (1x10 6 /mouse) were injected subcutaneously into RAG-2(-/-) mice. The incidence and growth kinetics were recorded over a two-month period. Data shown represent two independent experiments. In combination, tumor was detected in all 8 mice that received the silenced clone, but in none of the 8 mice that received control clone.
  • Laforin is a phosphatase of GSK-36 at Ser 9. A.
  • Iaforin-V5 transfected NIH3T3 cells were stimulated with PDGF for given periods of time.
  • laforin-transfected NIH3T3 cells were stimulated with PDGF for 30 minutes and
  • the immunoprecipitates were used for kinase assay to determine the
  • GSK-3 ⁇ kinase activity Recombinant Tau protein and 32 P- ⁇ -ATP were used as substrates. Specific phosphorylation of Tau was determined by 32 P
  • the upper panel shows the efficiency of endogenous Epm2a suppression in three stable clones C1 , C2 and
  • GSK-3 ⁇ was the protein loading control.
  • NIH3T3 cells were stimulated with PDGF for 30 min and cell lysates were
  • the lower panel shows association
  • FIG. 7 Laforin modulates Wnt signaling pathway.
  • V Vector
  • D2, D7, D14 mutant
  • C265S mutant-transfected NIH3T3 cells
  • the cell lysates were analyzed for phosphorylation of GSK-3 ⁇ at Ser
  • pTOPFLASH or pFOPFLASH TCF luciferase reporter vector was co-transfected with pRL-SV40 Ranilla control luciferase reporter plasmid into Vector (V), laforin (D14) or mutant laforin (C265S) stably transfected NIH3T3 cells. Twenty four hours after transfection, the cells were stimulated with Wnt-3A conditional medium or control medium overnight. Cell lysate supematants were evaluated for luciferase activity by Dual-luciferase reporter assay system. The results are shown after normalized to corresponding Renilla
  • siRNA (C3) transfected NIH3T3 cells were cultured in the presence of 0.5 ⁇ M
  • thymocytes from transgenic mice with lymphoma (Tg-Tu), but not the normal mice (Ntg) and the transgenic mice yet to develop lymphoma (Tg).
  • Tg-Tu lymphoma
  • Ntg normal mice
  • Tg lymphoma
  • Freshly isolated thymi were lysed and nuclear and cytoplasmic proteins were analyzed
  • FIG. 1 Figure S1. Over-expression of genes adjacent to TCR transqene integration site does not lead to tumorigenesis.
  • c Summary of observation period of 8 transgenic founder mice that express mCG16603 under the control of the proximal lck promoter to induce high expression in the thymus.
  • NIH3T3 cells were stimulated with PDGF for given periods of time.
  • FIG. S4 A preliminary study identifying a 333 bp fragment with most methylated CpG.
  • Top diagram illustrates the CpG contents of the 5' region of the mouse EPM2a gene, with the CpG island shaded. The numbers underneath the black/white are those of the methylated clones over the number of clones analyzed. The average numbers of methylated per clone are given in the parentheses.
  • the sequence of the 5' region of the gene is provided in the bottom. The sequence in exon 1 are shown in upper cases, with remaining sequence in lower case.
  • Fig. S5. The CpG island of human EPM2a gene. Top diagram illustrates the CpG contents of the 5' region of the mouse EPM2a gene, with the CpG island shaded. The sequence of the 5 J region of the gene is provided in the bottom. The sequence in exon 1 are shown in upper cases, with remaining sequence in lower case.
  • Fig. S6 Treatment of 2-d ⁇ prolong survival of TqB mice.
  • TgB mice (20 mice/group) between 2-4 month of age were treated with either PBS or 2-dG (20 mg/kg) every 3 days for 6 weeks. After 4 weeks of interval, all mice were again treated for another cycle. Mice that were moribund were sacrificed, and the presence of lymphoma was confirmed by necropsy.
  • this invention relates to methods of characterizing an etiology of a cancer by testing at least one cancer cell for the presence of at least one of a) methylation of the EPM2A gene; b) reduced expression of the EPM2A gene; and c) mutation of the EPM2A gene.
  • the invention also provides methods of determining an increased likelihood of cancer in a person, by assaying a tissue sample from the person for the presence of at least one of a) methylation of EPM2A gene; b) reduced expression of EPM2A gene; and c) mutation of EPM2A gene.
  • Methylation of the EPM2A gene can be tested using the bisulfite sequencing method, which generally involves converting non-methylated cytosines into thymines, while methylated cytosines remain unchanged.
  • the putative CpG island of the human EPM2a gene is illustrated in Fig. S5.
  • the modified DNA can then be analyzed using a variety of methods, examples of which are known in the art. Examples of methods for determining the level of DNA expression are also well known in the art, and include but are not limited to, assaying mRNA or protein.
  • mutation of the EPM2A gene can be determined with a variety of well known methods, including but not limited to, those involving T4 endonuclease VII, which recognizes structural differences in heteroduplex DNA, rather than the sequence itself. Many readily available assays can be used in accordance with this aspect of the invention.
  • the invention relates to methods of characterizing an etiology of a cancer by testing at least one cancer cell for the presence of lafora bodies.
  • These stored polysaccharide bodies are pale, well- defined masses, round or kidney-shaped, that generally displace the nuclei and reside primarily in periportal hepatocytes. They are weakly positive with diastase- PAS, stain with methenamine silver and colloidal iron stains, and, by electron microscopy, are composed of a nonmembrane-bound combination of interwoven fibrils (6 to 10 nm diameter), aggregates of smooth endoplasmic reticulum, and granular material with glycogen rosettes.
  • the invention also relates to methods of identifying compounds that modify laforin's ability to phosphorylate GSK3/? at Serine 9, wherein the methods include a step of testing laforin's ability to phosphorylate GSK3/? in the presence and absence of the test compounds.
  • the laforin tested is wild-type laforin and the compounds identified are inhibitors of laforin's ability to phosphorylate GSK3/?.
  • the laforin tested is mutated laforin and the compounds identified restore laforin's ability to phosphorylate GSK3/?.
  • These assays can be performed in a variety of ways, examples of which are well known in the art.
  • the invention also provides methods of treating Alzheimer's disease by administering to a person in need of treatment a compound identified according to the aforementioned methods.
  • the invention also relates to methods of treating cancer in a person identified as having a cancer associated with methylated EPM2A gene(s), by administering to the person an effective amount of a drug that demethylates DNA.
  • the invention relates to methods of treating a person identified as having a disease associated with reduced expression or mutation of the EPM2A gene, by activating EPM2A ge ⁇ e(s) in cells of the person's body.
  • the invention relates to methods of treating a person identified as having a disease associated with reduced expression or mutation of the EPM2A gene, by administering laforin to the person.
  • the invention involves methods of treating a person identified as having a disease associated with reduced expression or mutation of the EPM2A gene, by administering a genetically engineered GSK3/? that cannot be phosphorylated at the Serine 9 position. In some embodiments, the invention relates to methods of treating a person identified as having a disease associated with reduced expression or mutation of EPM2A gene, by administering to the person at least one compound that restores laforin's ability to phosphorylate GSK3/?.
  • an expression construct comprising the oligonucleotide for treatment may be entrapped in a liposome.
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They can form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat (1991) Targeting of liposomes to hepatocytes. Targeted Diagn. Ther 4: 87-103). Also contemplated are lipofectamine-DNA complexes.
  • an eleven-amino-acid sequence the "protein transduction domain" (PTD), from the human immunodeficiency virus TAT protein (Green and Loewenstein, 1988, Cell, 55:1179-88.; Frankel and Pabo, 1988, Cell, 55:1189-93.) is fused to the protein to be delivered, e.g., laforin.
  • the purified protein can then be put in contact with the surface of the tumor cells and the cells take up the protein, which functions to inhibit or suppress growth of that cell.
  • the protein can be administered to the human by a variety of methods.
  • the protein is administered by intratumoral or intralesional injection. Methods of administration are provided in more detail below.
  • Laforin proteins that are fused to PTD can be made by fusing the DNA sequence encoding the laforin protein with the DNA sequence encoding the PTD.
  • the resulting laforin-PTD fusion gene can be incorporated into a vector, for example a plasmid or viral vector, that facilitates introduction of the fusion gene into a organism and expression of the gene at high levels in the organism such that large amounts of the fusion protein are made therein.
  • a vector for example a plasmid or viral vector
  • One such organism in which the vector containing the fusion gene can be expressed is a bacterium, such as Escherichia coli. Other organisms are also commonly used by those skilled in the art.
  • the fusion protein is purified from the organism using protein purification techniques well known to those skilled in the art.
  • the methods of the invention can be practiced orally, parenterally (IV, IM, depot-lM, SQ, and depot-SQ), sublingually, intranasally (inhalation), intrathecal ⁇ , topically, or rectally. Dosage forms known to those of skill in the art are suitable for delivery of the compounds employed in the methods of the invention.
  • compositions are provided that contain therapeutically effective amounts of the compounds employed in the methods of the invention.
  • the compounds can be formulated into suitable pharmaceutical preparations such as tablets, capsules, or elixirs for oral administration or in sterile solutions or suspensions for parenteral administration.
  • suitable pharmaceutical preparations such as tablets, capsules, or elixirs for oral administration or in sterile solutions or suspensions for parenteral administration.
  • the compounds can be formulated into pharmaceutical compositions using techniques and procedures well known in the art.
  • compositions can be formulated in a unit dosage form, each dosage containing from about 1 to about 500 mg, or about 10 to about 100 mg of the active ingredient.
  • unit dosage from refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
  • compositions one or more compounds employed in the methods of the invention are mixed with a suitable pharmaceutically acceptable carrier.
  • a suitable pharmaceutically acceptable carrier Upon mixing or addition of the compound(s), the resulting mixture may be a solution, suspension, emulsion, or the like. Liposomal suspensions may also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient for lessening or ameliorating at least one symptom of the disease, disorder, or condition treated and may be empirically determined.
  • compositions suitable for administration of the compounds in the methods provided herein include any such carriers suitable for the particular mode of administration.
  • the active materials can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, or have another action.
  • the compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.
  • solubilizing may be used. Such methods are known and include, but are not limited to, using co-solvents such as dimethylsulfoxide (DMSO), using surfactants such as TWEEN, and dissolution in aqueous sodium bicarbonate. Derivatives of the compounds, such as salts or prodrugs, may also be used in formulating effective pharmaceutical compositions.
  • DMSO dimethylsulfoxide
  • surfactants such as TWEEN
  • dissolution in aqueous sodium bicarbonate such as sodium bicarbonate
  • Derivatives of the compounds, such as salts or prodrugs may also be used in formulating effective pharmaceutical compositions.
  • the compounds - be it polynucleotides or polypeptides - can be complexed or tagged with targeting moieties to improve their delivery.
  • the concentration of the compound is effective for delivery of an amount upon administration that lessens or ameliorates at least one symptom of the disorder for which the compound is administered.
  • the compositions are formulated for single dosage administration.
  • the compounds employed in the methods of the invention may be prepared with carriers that protect them against rapid elimination from the body, such as time-release formulations or coatings.
  • Such carriers include controlled release formulations, such as, but not limited to, microencapsulated delivery systems.
  • the active compound can be included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated.
  • the therapeutically effective concentration may be determined empirically by testing the compounds in known in vitro and in vivo model systems for the treated disorder.
  • kits for example, including component parts that can be assembled for use.
  • a compound in lyophilized form and a suitable diluent may be provided as separated components for combination prior to use.
  • a kit may include a compound and a second therapeutic agent for co-administration.
  • the compound and second therapeutic agent may be provided as separate component parts.
  • a kit may include a plurality of containers, each container holding one or more unit dose of the inventive compound employed in the method of the invention.
  • the containers can be adapted for the desired mode of administration, including, but not limited to tablets, gel capsules, sustained-release capsules, and the like for oral administration; depot products, pre-filled syringes, ampoules, vials, and the like for parenteral administration; and patches, medipads, creams, and the like for topical administration.
  • concentration of active compound in the drug composition will depend on absorption, inactivation, and excretion rates of the active compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.
  • the active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.
  • the compound can be provided in a composition that protects it from the acidic environment of the stomach.
  • the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine.
  • the composition may also be formulated in combination with an antacid or other such ingredient.
  • Oral compositions can include an inert diluent or an edible carrier and may be compressed into tablets or enclosed in gelatin capsules.
  • the active compound or compounds can be incorporated with excipients and used in the form of tablets, capsules, or troches.
  • Pharmaceutically compatible binding agents and adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches, and the like can contain any of the following ingredients or compounds of a similar nature: a binder such as, but not limited to, gum tragacanth, acacia, corn starch, or gelatin; an excipient such as microcrystalline cellulose, starch, or lactose; a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate; a glidant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and a flavoring agent such as peppermint, methyl salicylate, or fruit flavoring.
  • a binder such as, but not limited to, gum tragacanth, acacia, corn starch, or gelatin
  • an excipient such as microcrystalline cellulose, starch, or lactose
  • a disintegrating agent such as, but not limited to, algin
  • the dosage unit form when it is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil.
  • dosage unit forms can contain various other materials, which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents.
  • the compounds can also be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like.
  • a syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings, and flavors.
  • the active materials can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action.
  • the compounds can be used, for example, in combination with an antitumor agent, a hormone, a steroid, or a retinoid.
  • the antitumor agent may be one of numerous chemotherapy agents such as an demethylating agent, an antimetabolite, a hormonal agent, an antibiotic, colchicine, a vinca alkaloid, L- asparaginase, procarbazine, hydroxyurea, mitotane, nitrosoureas, or an imidazole carboxamide.
  • Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent such as water for injection, saline solution, fixed oil, a naturally occurring vegetable oil such as sesame oil, coconut oil, peanut oil, cottonseed oil, and the like, or a synthetic fatty vehicle such as ethyl oleate, and the like, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvent; antimicrobial agents such as benzyl alcohol and methyl parabens; antioxidants such as ascorbic acid and sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, and phosphates; and agents for the adjustment of tonicity such as sodium chloride and dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oil, a naturally occurring vegetable oil
  • suitable carriers include, but are not limited to, physiological saline, phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, polypropyleneglycol, and mixtures thereof.
  • PBS phosphate buffered saline
  • suitable carriers include, but are not limited to, physiological saline, phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, polypropyleneglycol, and mixtures thereof.
  • Liposomal suspensions including tissue-targeted liposomes may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known in the art.
  • the compounds may be prepared with carriers that protect the compound against rapid elimination from the body, such as time-release formulations or coatings.
  • carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and the like. Methods for preparation of such formulations are known to those skilled in the art.
  • Compounds employed in the methods of the invention may be administered enterally or parenterally.
  • compounds employed in the methods of the invention can be administered in usual dosage forms for oral administration as is well known to those skilled in the art.
  • These dosage forms include the usual solid unit dosage forms of tablets and capsules as well as liquid dosage forms such as solutions, suspensions, and elixirs.
  • the solid dosage forms can be of the sustained release type so that the compounds employed in the methods of the invention need to be administered only once or twice daily.
  • the oral dosage forms can be administered to the patient 1 , 2, 3, or 4 times daily.
  • the compounds employed in the methods of the invention can be administered either three or fewer times, or even once or twice daily.
  • the compounds employed in the methods of the invention be administered in oral dosage form.
  • Whatever oral dosage form is used they can be designed so as to protect the compounds employed in the methods of the invention from the acidic environment of the stomach. Enteric coated tablets are well known to those skilled in the art.
  • capsules filled with small spheres each coated to protect from the acidic stomach are also well known to those skilled in the art.
  • the compounds and methods can be used to inhibit neoplastic cell proliferation in an animal.
  • the methods comprise administering to an animal having at least one neoplastic cell present in its body a therapeutically effective amount of at least one of the compounds, in compositions as described above.
  • the animal can be a mammal, including a domesticated mammal.
  • the animal can be a human.
  • neoplastic cell is used to denote a cell that shows aberrant cell growth.
  • the aberrant cell growth of a neoplastic cell includes increased cell growth.
  • a neoplastic cell may be, for example, a hyperplastic cell, a cell that shows a lack of contact inhibition of growth in vitro, a benign tumor cell that is incapable of metastasis in vivo, or a cancer cell that is capable of metastases in vivo and that may recur after attempted removal.
  • tumorgenesis is used to denote the induction of cell proliferation that leads to the development of a neoplastic growth.
  • therapeutically effective amount and "therapeutically effective period of time” are used to denote treatments at dosages and for periods effective to reduce neoplastic cell growth.
  • administration can be parenteral, oral, sublingual, transdermal, topical, intranasal, or intrarectal.
  • the therapeutic composition can be administered at a sufficient dosage to attain a blood level of the compounds of from about 0.1 ⁇ M to about 100 mM.
  • concentrations for localized administration, much lower concentrations than this can be effective, and much higher concentrations may be tolerated.
  • concentrations for localized administration, much lower concentrations than this can be effective, and much higher concentrations may be tolerated.
  • therapeutic effect resulting in a lower effective concentration of the compound may vary considerably depending on the tissue, organ, or the particular animal or patient to be treated according to the invention. It is also understood that while a patient may be started at one dose, that dose may be varied over time as the patient's condition changes.
  • cell proliferative disease or condition is meant to refer to any condition characterized by aberrant cell growth, preferably abnormally increased cellular proliferation.
  • Examples of such cell proliferative diseases or conditions include, but are not limited to, cancer, restenosis, and psoriasis.
  • Cancers treatable according to the invention include, but are not limited to, prostate cancer, lung cancer, acute leukemia, multiple myeloma, bladder carcinoma, renal carcinoma, breast carcinoma, colorectal carcinoma, neuroblastoma, or melanoma.
  • PDGF phobol myristate acetate
  • PMA phobol myristate acetate
  • FISH fluorescence in situ hybridization
  • TCR transgenic mouse genomic DNA was digested by restrictive enzyme Pst ⁇ and dephosphorylated to allow ligation with the
  • Dephosphorylated A-tailed DNA was ligated with TOPO ® Linker and further amplified by PCR.
  • the PCR products were cloned into a TA-TOPO cloning vector (Invitrogen) and sequenced. The sequences were searched with the Celera Discovery System (Rockville, MD).
  • PCR products were purified by Gel Extraction kit (Qiagen), subcloned into the TA-TOPO vector system (Invitrogen) and sequenced by an ABI/PE 3700 capillary sequencer. 8-10 clones were sequenced for each lymphoma or normal thymus.
  • Genomic DNA from three Epm2a high cell lines (P815, Meth A and MC38) and three Epm2a low lymphoma cell lines (EL-4, BW5147 and YAC-1) were subjected to methylation-sensitive Sac Il digestion. Digested or undigested genomic DNA were used as templates for real time PCR.
  • Anti-laforin polyclonal antibody The antibody was produced by Genemed Synthesis, Inc. (San Francisco, CA). Rabbits were immunized with the synthetic peptide of 16 amino acid residues (YKFLQREPGGELHWEG, residues 85-100 in laforin protein, Accession No. AAD26336) coupled with keyhole limpet hemocyanin (KLH) in complete Freund's adjuvant. The antiserum was purified using peptide conjugated Affigel column.
  • Epm2a siRNA constructs Oligonucleotides encoding siRNA directed against Epm2a at C terminal region of 934 to 954 nucleotides (5'- AAGGTGCAGTACTTCATCATG-3') (C) or polyA region of 1212 to 1233 nucleotides ( ⁇ '-AAGGAAAACTGCATGCCACAT-S 1 ) (E) were cloned into pSilencer 1.0-U6 siRNA expression vector (Ambion, Austin, TX) to generate C- siRNA according to manufacturer's protocol. NIH3T3 cells were co-transfected with C- or E-siRNA vector and pcDNA4/V5-His (Invitrogen) using Fugene 6 and
  • polybrene for 16 hours.
  • the fresh medium was replaced and continued to culture for another 24 hours before flow cytometry analysis.
  • Epm2a full length cDNA was cloned into the BamH ⁇ and Xho ⁇ sites of pGEX-6P- 1(Amersham) to create GST-Epm2a fusion protein construct and transformed into E.coli strain BL21.
  • the soluble GST-fused protein was purified by glutathione-agarose beads, and eluted with 5OmM reduced glutathione.
  • the total cell lysates and nuclear fractions were separated in 12% SDS-PAGE and probed with different antibodies after transfer to nitrocellulose membranes.
  • TG-B mice had a high incidence of lymphoma regardless of sex.
  • the maximal survival time for TG-B mice was 9 months with a median survival time of 6 months (Fig. 1A).
  • the lymphoma in TG-B mice was first seen in lymphoid organs, including the thymus, spleen and lymph nodes (Fig. 1B).
  • the tumor cells infiltrated other organs such as liver, kidney (Fig. 1C), lung, pancreas, intestines and salivary glands (data not shown).
  • TCR ⁇ and TCR ⁇ transgenes were cloned and sequenced.
  • transgene was inserted into the intron 1 of Epm2a, which encodes laforin, a dual specific protein phosphatase (Fig. 2B)(Minassian et al., 1998; Serratosa et al., 1999b). Further PCR analysis using primers corresponding to the 3' terminus of Epm2a
  • Fig. 1 A the high tumor incidence and the lack of laforin documented in Fig. 1 A were observed in heterozygous mice. Since the wild type allele was not deleted in the tumor cells lacking laforin expression (data not shown), it may have been silenced by epigenetic mechanisms.
  • the Epm2a gene contains a 1.2 kb 5'-CpG island that spans the promoter region, exon 1 and part of intron 1 (Fig. 4A).
  • DNA hypermethylation is an epigenetic mechanism for laforin gene down-regulation in TG-B lymphoma.
  • Fig. S4 Our preliminary analyses of the tumor samples indicated that a 333-bp PCR fragment was the most frequently methylated area (Fig. S4). We therefore focused on this region and examined six TG-B mice that developed lymphoma and six TG-B mice that had not developed tumors. Based on the methylation-mediated protection of OT conversion by the sodium bisulfite, we identified the methylated CpG di-nucleotides. As illustrated in Fig. 4B, extensive hypermethylation was found in the Epm2a gene of all six lymphoma samples. In contrast, none of the normal thyrni had methylation in this region. Our survey of multiple mouse lymphoma cell lines revealed that depression in the Epm2a gene was wide-spread among lymphomas (Fig.
  • Epm2a expression was determined by quantitative real-time PCR based on susceptibility to a methylation-sensitive restriction enzyme. As shown in Fig.
  • Epm2a was constitutively expressed at high levels in mastocytoma cell line P815, colon cancer cell line MC38 and fibrosarcoma cell line Meth A. Treatment with 5-aza- 2'-deoxycytidine had little effect on Epm2a expression in these three cell lines. Since the methylation-sensitive Sac Il cuts more than 99% of the Epm2a promoter, lack of PCR product indicated that Epm2a gene in these three tumor cell lines was not methylated. In contrast, in three T lym phoma cell lines tested, the Epm2a promoter region is completely resistant to Sac Il digestion.
  • Eprn2a substantially induced Epm2a expression among the three T lymphoma cell lines.
  • the transgene expression may potentially activate genes in the area. There are two genes within 300kb to the transgene integration site. The proximal gene is 56 kb away from the integration site and encodes a protein that contains an F-box domain (mCG16603), while the other gene is 135 kb from the integration site (mCG140768) (Fig. S1A). We performed RNase protection assay to determine the mRNA expression of these two genes. The expression level of the distal gene (mCG140768) was not affected by transgene or lymphoma development, while the expression of the proximal gene (mCG16603) was up- regulated by the transgene regardless of lymphoma formation (Fig. S1 b).
  • proximal gene Although the lack of further elevation of the proximal gene was inconsistent with its role in lymphoma development, we tested this possibility by producing 8 independent founders of transgenic mice that express mCG 16603 under the control of a proximal lck promoter, which resulted in high levels of transgene expression in the thymus. None of the mice developed lymphoma during the over more than 1 year period (Fig. S1c). Thus, the lymphoma development in TG-B mice is unlikely due to the abnormal expression of other genes near the integration site.
  • the DNA contents in the majority of the V5 negative cells were between DNA content 2C and 4C, which indicated live cells at different stages of cell cycle.
  • 50-70% of the Epm2a- ⁇ /5 positive cells had DNA contents less than 2C, indicating very active DNA degradation and cell death. This result demonstrates that laforin expression induces apoptosis of tumor cells.
  • Laforin is a phosphatase for GSK-3 ⁇
  • SiRNA specific for another region of the Epm2a gene partially reduced laforin expression and lead to quantitative changes
  • GSK-3 ⁇ but not control IgGI , brought down both GSK-3 ⁇ and V5-tagged laforin
  • GSK-3 ⁇ and laforin are associated in the NIH3T3 cells.
  • GSK-3 ⁇ associate with endogenous laforin protein, we precipitated the GSK-3 ⁇ in
  • Laforin is an important modulator for Wnt signaling
  • GSK-3 ⁇ is a major component in the Wnt signalling pathway (He
  • Laforin is a tumor suppressor in the mouse
  • GSK-3 ⁇ and modulator for Wnt signaling provide a mechanism by which laforin
  • mice with targeted mutation have been reported to survive to 12 months of age, although it has not been examined as to whether the mice are more susceptible to lymphoma (Ganesh et al., 2002). Therefore targeted mutation of Epm2a in the mixed 129Svj x C57BL/6 background (H-2 b ) does not seem to form early lethal lymphoma as observed in the TG-B mice. This apparent inconsistency can be reconciled in two ways. The C57BL/6 (H-2 b ) mice are less sensitive to lymphoma in B10.BR, as we showed that the offspring of TG-B(BIO.
  • BR, H-2 k developed lymphoma substantially faster than the C57BL/6 x TG-B F1 mice.
  • H-2 region has been shown to contain a major modifier for susceptibility to lymphoma (Kamoto et al., 1996).
  • TCR transgenic mice are immune-deficient because the majority of their T cells are directed to a single CTL epitope and therefore unable to recognize tumor antigen. In combination, these two factors may explain the lack of report on lymphoma in the Epm2a-deficient mice.
  • EPM2A gene may be a tumor suppressor in human.
  • Lafora's disease due to EPM2A mutations is an extremely rare genetic disease with early lethality. The patients usually die before they reached 20 years of age. It is therefore not possible to evaluate the direct impact of this mutation on cancer incidence. Nevertheless, we have observed high incidence of loss of heterozygosity (LOH) in human cancer (data not shown).
  • LHO heterozygosity
  • the EPM2A is located in 6q24 of human genome where deletions or LOH were reported in cancers from a large number of different organs including carcinomas of the breast (Devilee et al., 1991 ; Sheng et al., 1996), endometrium (Tibiletti et al., 1997; Tibiletti et al., 1999), prostate (Cooney et al., 1996), lymphomas and leukemias (Gerard et al., 1997; Zhang et al., 1997), and melanomas (Walker et al., 1994).
  • Laforin is a phosphatase for GSK-3 ⁇
  • GSK-3 ⁇ is a key modulator for several important signal
  • GSK-3 ⁇ is inactivated by phosphorylation at the
  • AKT substrates such as FKHR and AFX.
  • knockdown of laforin in either NIH3T3 cells or in mouse thymus substantially increased phosphorylation
  • GSK-3 ⁇ important in a variety of biological processes including cellular
  • Wnt signalling may reduce GSK activity of Ser9>A mutant protein, as suggested
  • Laforin is a key regulator for the function of GSK3.
  • GSK3 plays a critical role in a wide spectrum of human diseases, including the Alzheimer's disease (Phiel et al., 2003). Since the expression of Laforin is essential for optimal GSK3 function, it may be possible to inhibit GSK3 function by either down regulating the expression of EPM2a or by inhibition of the Laforin phophatase activity.
  • Axin a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3beta and beta-catenin and promotes GSK-3beta-dependent phosphorylation of beta-catenin. Embo J 17, 1371-1384.
  • Minassian, B. A. (2001). Lafora's disease: towards a clinical, pathologic, and molecular synthesis. Pediatr Neurol 25, 21-29.
  • TCR/CD3 ligation of a TCR-transgenic T lymphoma blocks its proliferation in vitro but does not affect its growth in vivo.
  • Exp Clin lmmunogenet 11 197-208.
  • NF-kappaB is a target of AKT in anti-apoptotic PDGF signalling. Nature 401 , 86-90.
  • Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423, 448-452.

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Abstract

La présente invention concerne des procédés pour caractériser l'étiologie d'un cancer et d'autres maladies humaines, qui consistent à tester au moins une cellule à propos de la présence d'au moins un des paramètres suivants a) une méthylation du gène EPM2A, b) une expression réduite du gène EPM2A et c) une mutation du gène EPM2A. Cette invention concerne également des procédés pour traiter un cancer et d'autres maladies.
PCT/US2005/040338 2004-11-08 2005-11-07 Expression de laforine pour diagnostiquer et traiter un cancer et d'autres maladies WO2006052911A2 (fr)

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WO2009050321A1 (fr) * 2007-10-19 2009-04-23 Consejo Superior De Investigaciones Científicas (66%) Méthode de diagnostic et de traitement du cancer basée sur la laforine, éléments biologiques permettant la mise en oeuvre de ladite méthode et applications
EP3154570A4 (fr) * 2014-06-13 2017-12-27 Valerion Therapeutics, LLC Procédés et compositions pour le traitement de glycogénoses et de troubles du métabolisme du glycogène

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WANG Y. ET AL.: 'Epm2a suppresses tumor growth in an immunocompromised host by inhibiting Wnt signaling' CANCER CELL vol. 10, September 2006, pages 179 - 190, XP003011158 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009050321A1 (fr) * 2007-10-19 2009-04-23 Consejo Superior De Investigaciones Científicas (66%) Méthode de diagnostic et de traitement du cancer basée sur la laforine, éléments biologiques permettant la mise en oeuvre de ladite méthode et applications
ES2326014A1 (es) * 2007-10-19 2009-09-28 Consejo Superior De Investigaciones Cientificas (66%) Procedimiento de diagnostico y tratamiento del cancer basado en la laforina, elementos biologicos para llevarlos a cabo y sus aplicaciones.
EP3154570A4 (fr) * 2014-06-13 2017-12-27 Valerion Therapeutics, LLC Procédés et compositions pour le traitement de glycogénoses et de troubles du métabolisme du glycogène
US10202591B2 (en) 2014-06-13 2019-02-12 Valerion Therapeutics, Llc Methods and compositions for treatment of glycogen storage diseases and glycogen metabolism disorders
US10781434B2 (en) 2014-06-13 2020-09-22 Valerion Therapeutics, Llc Methods and compositions for treatment of glycogen storage diseases and glycogen metabolism disorders

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