WO2022031759A1 - Expression de mecp2 induite par décitabine et ses utilisations - Google Patents

Expression de mecp2 induite par décitabine et ses utilisations Download PDF

Info

Publication number
WO2022031759A1
WO2022031759A1 PCT/US2021/044415 US2021044415W WO2022031759A1 WO 2022031759 A1 WO2022031759 A1 WO 2022031759A1 US 2021044415 W US2021044415 W US 2021044415W WO 2022031759 A1 WO2022031759 A1 WO 2022031759A1
Authority
WO
WIPO (PCT)
Prior art keywords
mecp2
subject
chromosome
optionally substituted
alkyl
Prior art date
Application number
PCT/US2021/044415
Other languages
English (en)
Inventor
Luigi ENRIQUEZ
Ryan Thomas JONES
Chia-Yao LEE
Pavan Ramkumar
Kevan SHAH
Gaia SKIBINSKI
Zhixiang TONG
Original Assignee
Herophilus, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Herophilus, Inc. filed Critical Herophilus, Inc.
Publication of WO2022031759A1 publication Critical patent/WO2022031759A1/fr

Links

Classifications

    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system

Definitions

  • Rett syndrome is a progressive neurological disorder that affects 1 in 10,000-15,000 female births.
  • Loss of function mutations in methyl-CpG-binding-binding protein (MeCP2, locus located on the X-chromosome) is the major cause of RTT.
  • Random X chromosome inactivation causes cells to express either mutant or wild-type MeCP2, leading to a mosaic pattern of mutant MeCP2 protein expression throughout the body.
  • Mouse models of RTT have shown that an increase of 5 to 10% in wild-type MeCP2 is sufficient to improve RTT phenotypes and delay premature death. Thus, new methods that increase MeCP2 protein levels are needed to treat RTT.
  • the present disclosure provides a method of treating an X-chromosome- linked disease which comprise: identifying a subject with an X-chromosome genetic mutation, wherein the X-chromosome genetic mutation is outside of the MeCP2 Exon 2 region on the X- chromosome in a subject; and administering a DNA methyltransferase inhibitor to the subject, wherein the DNA methyltransferase inhibitor activates an inactive X-chromosome.
  • the genetic mutation is a missense, non-sense, frameshift, insertion, deletion or a duplication mutation.
  • the MeCP2 Exon 2 region contains no genetic mutations.
  • the MeCP2 Exon 2 region contains mutations selected from: reference SNP rs267608409.
  • the X-chromosome-linked disease is selected from CDKL5 deficiency disorder, fragile x syndrome, Alport syndrome, X-linked Charcot-Mari e- tooth disease, X-linked dominant porphyria, Vitamin D resistant rickets, Incontinentia pigmenti, CLCN4-related disorder, and facioscapulohumeral muscular dystrophy.
  • the X-chromosome-linked disease is Rett Syndrome.
  • administering the DNA methyltransferase inhibitor induces wild-type MeCP2 expression.
  • administering the DNA methyltransferase inhibitor induces wild-type MeCP2 in human neural tissue.
  • the present disclosure provides a method of treating an X-chromosome- linked disease which further comprises the DNA methyltransferase inhibitor activating an inactive X-chromosome in the following in vitro assay: (a) reprogramming X-chromosome linked disease fibroblasts into induced pluripotent stem cells; (b) differentiating the induced pluripotent stem cells into brain organoids; (c) contacting said brain organoids in a vessel with a test DNA methyltransferase inhibitor, wherein the test DNA methyltransferase inhibitor is administered every other day for two weeks; and (d) detecting MeCP2 level in said vessel following step (c) and comparing the MeCP2 level to a MeCP2 level prior to said contacting in step (c), wherein when the MeCP2 levels are elevated by at least 1% and the test DNA methyltransferase inhibitor is a DNA methyltransferase inhibitor.
  • the X-chromosome linked disease which further comprises the DNA methyltransfera
  • the DNA methyltransferase is decitabine or a salt thereof.
  • the DNA methyltransferase inhibitor is a compound of
  • R 1 and R 2 are each independently selected from hydrogen and -Si(R 3 )(R 4 )(R 5 ) ;
  • R 3 , R 4 , and R 5 are each independently selected from:
  • Ci-6 alkyl optionally substituted with one or more substituents selected from halogen, -OR 6 , -NO2, -CN, and C3-8 carbocycle optionally substituted with one more substituents selected from halogen, -OR 6 , -NO2, -CN, Ci-6 alkyl and Ci-e haloalkyl; and
  • C3-10 carbocycle optionally substituted with one or more substituents selected from halogen, -OR 6 , -NO2, CN and Ci-6 alkyl optionally substituted with one or more substituents selected from halogen, -OR 6 , -NO2, and CN; and
  • R 6 is independently selected at each occurrence from hydrogen, Ci-6 alkyl, and Ci-6 haloalkyl.
  • the DNA methyltransferase inhibitor is a compound of Formula (I- A): pharmaceutically acceptable salt thereof.
  • the present disclosure provides a method of reactivating inactive X- chromosomes, the method comprising: identifying a subject with an X-chromosome genetic mutation, wherein the X-chromosome genetic mutation is outside of the MeCP2 Exon 2 region on the X-chromosome in a subject; and administering a DNA methyltransferase inhibitor to the subject, wherein the DNA methyltransferase inhibitor activates an inactive X-chromosome.
  • methylation patterns in the subject’s blood are decreased by at least 5% for two weeks or more.
  • the decitabine or salt thereof is administered daily, every other day or once weekly.
  • the subject has Rett Syndrome.
  • the present disclosure provides a method of treating an X-chromosome- linked disease, comprises administering decitabine or a salt thereof to a subject in need thereof over a period of two weeks or more, wherein the subject does not have cancer.
  • methylation patterns in the subject’s blood is decreased by at least 5% for two weeks or more.
  • the decitabine or salt thereof is administered daily, every other day or once weekly.
  • the subject has Rett Syndrome.
  • the present disclosure provides a method of reactivating an inactive X- chromosomes, comprising administering decitabine to a subject in need thereof in an amount sufficient to increase cerebrospinal fluid levels of KCC2 by at least 10% for two weeks or more.
  • methylation patterns in the subject’s blood is decreased by at least 5% for two weeks or more.
  • the decitabine or salt thereof is administered daily, every other day or once weekly.
  • the subject has Rett Syndrome.
  • the present disclosure provides a method of reactivating an inactive X- chromosomes, comprising administering decitabine to a subject in need thereof in an amount sufficient to increase cerebrospinal fluid levels of brain-derived neurotrophic factor by at least 10% for two weeks or more.
  • methylation patterns in the subject’s blood is decreased by at least 5% for two weeks or more.
  • the decitabine or salt thereof is administered daily, every other day or once weekly.
  • the subject has Rett Syndrome.
  • the present disclosure provides a method of reactivating an inactive X- chromosomes, comprising administering decitabine to a subject in need thereof in an amount sufficient to reduce oxidative stress biomarkers by at least 10% for two weeks or more.
  • methylation patterns in the subject’s blood is decreased by at least 5% for two weeks or more.
  • the decitabine or salt thereof is administered daily, every other day or once weekly.
  • the subject has Rett Syndrome.
  • the present disclosure provides a method of reactivating inactive X- chromosomes, comprising administering decitabine to a subject in need thereof in an amount sufficient to reduce DNA methylation patterns in the subject’s blood by at least 1% for two weeks or more.
  • DNA methylation patterns in the subject’s blood is decreased by at least 5% for two weeks or more.
  • the decitabine or salt thereof is administered daily, every other day or once weekly.
  • the subject has Rett Syndrome.
  • the present disclosure provides a method of treating an X chromosome-linked disease, the method comprising: administering a DNA methyltransferase inhibitor to the subject, wherein the DNA methyltransferase inhibitor is a compound of Formula (II): or a pharmaceutically acceptable sale thereof wherein:
  • R 11 and R 12 are each independently selected from hydrogen or -Si(R 3 )(R 4 )(R 5 ) and at least one of R 11 and R 12 is -Si(R 3 )(R 4 )(R 5 );
  • R 3 , R 4 , and R 5 are each independently selected from: Ci-6 alkyl optionally substituted with one or more substituents selected from halogen, -OR 6 , -NO2, -CN, and C3-8 carbocycle optionally substituted with one more substituents selected from halogen, -OR 6 , -NO2, -CN, Ci-6 alkyl and Ci-e haloalkyl; and
  • C3-10 carbocycle optionally substituted with one or more substituents selected from halogen, -OR 6 , -NO2, CN and Ci-6 alkyl optionally substituted with one or more substituents selected from halogen, -OR 6 , -NO2, and CN; and
  • R 6 is independently selected at each occurrence from hydrogen, Ci-6 alkyl, and Ci-6 haloalkyl, wherein the DNA methyltransferase inhibitor activates an inactive X-chromosome.
  • the compound of Formula (II) is represented by Formula (II- A):
  • the X-chromosome-linked disease is selected from CDKL5 deficiency disorder, fragile x syndrome, Alport syndrome, X-linked Charcot-Marie-tooth disease, X-linked dominant porphyria, Vitamin D resistant rickets, Incontinentia pigmenti, CLCN4-related disorder, and facioscapulohumeral muscular dystrophy.
  • the X-chromosome-linked disease is Rett Syndrome.
  • the administration of a compound or salt of Formula (II) or (II- A) induces wild-type MeCP2 expression.
  • the administration of a compound or salt of Formula (II) or (II- A) induces wildtype MeCP2 expression in human neural tissue.
  • FIG. 1A-C illustrates a wild type MeCP2 protein expression assay design.
  • FIG. 2 illustrates decitabine induced MeCP2 protein expression in a dose-dependent manner following 2 week treatment.
  • FIG. 3 illustrates decitabine induced MeCP2 protein expression in a dose-dependent manner following 2 week treatment.
  • FIG. 4 illustrates decitabine induced MeCP2 protein expression in a dose-dependent manner following 1 week treatment.
  • FIG. 5 illustrates decitabine induced MeCP2 protein expression in a dose-dependent manner following 1 week treatment.
  • FIG. 6 illustrates quantified decitabine toxicity after treatment for 1 week and 2 weeks with DAPI immunostaining.
  • FIG. 7 illustrates quantified decitabine toxicity after treatment for 1 week and 2 weeks with MAP2 immunostaining.
  • FIG. 8A-B illustrates immunolabeling images of decitabine induced MeCP2 expression.
  • FIG. 9 illustrates 5 ’-O-tri ethylsilyl 5-aza-2'-deoxycytidine induced MeCP2 protein expression in a dose-dependent manner following 2 week treatment.
  • FIG. 10 illustrates 5 ’-O-tri ethylsilyl 5-aza-2'-deoxycytidine induced MeCP2 protein expression in a dose-dependent manner following 2 week treatment.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • a sample includes a plurality of samples, including mixtures thereof.
  • C x -y when used in conjunction with a chemical moiety, such as alkyl, is meant to include groups that contain from x to y carbons in the chain.
  • Ci-ealkyl refers to saturated hydrocarbon groups, including straight-chain alkyl and branched-chain alkyl groups that contain from 1 to 6 carbons.
  • -Ci-6 alkyl- may be selected from methyl, ethyl, propyl, butyl, pentyl, and hexyl, any one of which is optionally substituted.
  • Alkyl refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, and preferably having from one to fifteen carbon atoms (i.e., C1-C15 alkyl).
  • an alkyl comprises one to thirteen carbon atoms (i.e., C1-C13 alkyl).
  • an alkyl comprises one to eight carbon atoms (i.e., Ci-Cs alkyl).
  • an alkyl comprises one to five carbon atoms (i.e., C1-C5 alkyl).
  • an alkyl comprises one to four carbon atoms (i.e., C1-C4 alkyl). In other embodiments, an alkyl comprises one to three carbon atoms (i.e., C1-C3 alkyl). In other embodiments, an alkyl comprises one to two carbon atoms (i.e., C1-C2 alkyl). In other embodiments, an alkyl comprises one carbon atom (z.e., Ci alkyl). In other embodiments, an alkyl comprises five to fifteen carbon atoms (z.e., C5-C15 alkyl). In other embodiments, an alkyl comprises five to eight carbon atoms (z.e., Cs-Cs alkyl).
  • an alkyl comprises two to five carbon atoms (z.e., C2-C5 alkyl). In other embodiments, an alkyl comprises three to five carbon atoms (z.e., C3-C5 alkyl).
  • the alkyl group is selected from methyl, ethyl, 1 -propyl (zz-propyl), 1 -methylethyl (z.w-propyl), 1 -butyl (zz-butyl), 1 -methylpropyl (.scc-butyl), 2- methylpropyl (z.w-butyl), 1,1 -dimethylethyl (tert-butyl), 1 -pentyl (zz-pentyl).
  • the alkyl is attached to the rest of the molecule by a single bond.
  • Halo or "halogen” as used herein refers to halogen substituents such as bromo, chloro, fluoro and iodo substituents.
  • Haloalkyl refers to an alkyl radical, as defined above, that is substituted by one or more halogen radicals, for example, trifluoromethyl, dichloromethyl, bromomethyl, 2,2,2- trifluoroethyl, l-fluoromethyl-2-fluoroethyl, and the like.
  • halogen substituted alkanes include halomethane (e.g., chloromethane, bromomethane, fluoromethane, iodomethane), di-and trihalomethane (e.g., trichloromethane, tribromomethane, trifluoromethane, triiodomethane), 1-haloethane, 2-haloethane, 1,2-dihaloethane, and any other suitable combinations of alkanes (or substituted alkanes) and halogens.
  • each halogen may be independently selected, for example 1 -chloro, 2- bromoethane.
  • Carbocycle refers to a saturated, unsaturated or aromatic ring in which each atom of the ring is carbon.
  • Carbocycle may include 3- to 10-membered monocyclic rings, 6- to 12-membered bicyclic rings, and 6- to 12-membered bridged rings.
  • Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated, and aromatic rings.
  • the carbocycle is an aryl.
  • the carbocycle is a cycloalkyl.
  • the carbocycle is a cycloalkenyl.
  • an aromatic ring e.g., phenyl
  • a saturated or unsaturated ring e.g., cyclohexane, cyclopentane, or cyclohexene.
  • Exemplary carbocycles include cyclopentyl, cyclohexyl, cyclohexenyl, adamantyl, phenyl, indanyl, and naphthyl.
  • Carbocycle may be optionally substituted by one or more substituents such as those substituents described herein.
  • Bicyclic carbocycles may be fused, bridged or spiro-ring systems.
  • Carbocycle-alkyl refers to a radical alkylene bound to a carbocyclic group.
  • An exemplary carbocycle-alkyl includes benzyl, cyclopropyl-methyl and phenethyl.
  • Aryl refers to a radical derived from an aromatic monocyclic or aromatic multicyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom.
  • the aromatic monocyclic or aromatic multicyclic hydrocarbon ring system contains only hydrogen and carbon and from five to eighteen carbon atoms, where at least one of the rings in the ring system is aromatic, /. ⁇ ., it contains a cyclic, delocalized (4n+2) ⁇ -electron system in accordance with the Hiickel theory.
  • the ring system from which aryl groups are derived include, but are not limited to, groups such as benzene, fluorene, indane, indene, tetralin and naphthalene.
  • Aryl-alkyl refers to radical alkylene bound to an aryl ring, e.g., benzyl, phenethyl, and phenpropyl.
  • Substituted refers to moieties having substituents replacing a hydrogen on one or more carbons or substitutable heteroatoms, e.g., an NH or NH2 of a compound. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, /. ⁇ ., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
  • substituted refers to moieties having substituents replacing two hydrogen atoms on the same carbon atom, such as substituting the two hydrogen atoms on a single carbon with an oxo, imino or thioxo group.
  • substituted is contemplated to include all permissible substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • a pharmaceutically acceptable salt also refers to any salt which may form in vivo as a result of administration of an acid, another salt, or a prodrug which is converted into an acid or salt.
  • a salt comprises one or more ionic forms of the compound, such as a conjugate acid or base, associated with one or more corresponding counterions. Salts can form from or incorporate one or more deprotonated acidic groups (e.g. carboxylic acids), one or more protonated basic groups (e.g. amines ), or both (e.g. zwitterions).
  • salt or “pharmaceutically acceptable salt” refers to salts derived from a variety of organic and inorganic counter ions well known in the art.
  • Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids.
  • Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
  • Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, -toluenesulfonic acid, salicylic acid, and the like.
  • Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.
  • Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like.
  • Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
  • the pharmaceutically acceptable base addition salt is chosen from ammonium, potassium, sodium, calcium, and magnesium salts.
  • phrases “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • the terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.
  • zzz vivo is used to describe an event that takes place in a subject’s body.
  • ex vivo is used to describe an event that takes place outside of a subject’s body.
  • An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject.
  • An example of an ex vivo assay performed on a sample is an “zzz vitro" assay.
  • zzz vitro is used to describe an event that takes place in a container for holding laboratory reagent such that it is separated from the biological source from which the material is obtained.
  • In vitro assays can encompass cell-based assays in which living or dead cells are employed.
  • In vitro assays can also encompass a cell-free assay in which no intact cells are employed.
  • the term “about” a number refers to that number plus or minus 10% of that number.
  • the term “about” a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value.
  • a “subject” can be a biological entity containing expressed genetic materials.
  • the biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa.
  • the subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro.
  • the subject can be a mammal.
  • the mammal can be a human.
  • the subject may be diagnosed or suspected of being at high risk for a disease.
  • the subject can be a human (e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other health worker in a hospital, as an outpatient, or other clinical context.
  • the subject may not be under the care or prescription of a physician or other health worker.
  • the subject is not necessarily diagnosed or suspected of being at high risk for the disease.
  • a subject in need thereof' refers to a subject, as described infra, that suffers from, or is at risk for, a pathology to be prophylactically or therapeutically treated with a compound or salt described herein.
  • administer are defined as providing a composition to a subject via a route known in the art, including but not limited to intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, or intraperitoneal routes of administration.
  • oral routes of administering a composition can be used.
  • administer should be understood to mean providing a compound of the invention or a prodrug of a compound of the invention to the individual in need.
  • treatment or “treating” are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient.
  • beneficial or desired results include but are not limited to a therapeutic benefit and/or a prophylactic benefit.
  • a therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated.
  • a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder.
  • a prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.
  • a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.
  • DNA methyltransferase inhibitors are defined as molecules or compositions that inhibit the catalyzed transfer of a methyl group to DNA. Such molecules or compositions can be synthetically produced, naturally derived, or semi-synthetically produced. Such molecules and compositions can directly or indirectly inhibit the catalyzed transfer of a methyl group to DNA. [0054] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.
  • the present disclosure provides a method of treating an X-chromosome- linked disease which comprise: identifying a subject with an X-chromosome genetic mutation, wherein the X-chromosome genetic mutation is outside of the MeCP2 Exon 2 region on the X- chromosome in a subject; and administering a DNA methyltransferase inhibitor to the subject, wherein the DNA methyltransferase inhibitor activates an inactive X-chromosome.
  • the genetic mutation outside of the MeCP2 Exon 2 region is at least one of a missense, non-sense, frameshift, insertion, deletion or a duplication mutation, or any combination thereof.
  • the genetic mutation is a missense, non-sense, frameshift, insertion, deletion or a duplication mutation.
  • the genetic mutation is a missense mutation outside of the MeCP2 Exon 2 region.
  • the genetic mutation is a non-sense mutation outside of the MeCP2 Exon 2 region.
  • the genetic mutation is a frameshift mutation outside of the MeCP2 Exon 2 region.
  • the genetic mutation is an insertion mutation outside of the MeCP2 Exon 2 region.
  • the genetic mutation is a deletion mutation outside of the MeCP2 Exon 2 region. In some embodiments, the genetic mutation is a duplication mutation outside of the MeCP2 Exon 2 region. In some embodiments, the genetic mutation outside of SEQ. ID NO. 1 region is at least one of a missense mutation, non-sense mutation, frameshift, insertion, deletion or duplication mutation, or any combination thereof. In some embodiments, the genetic mutation is a missense, non-sense, frameshift, insertion, deletion or duplication mutation. In some embodiments, the genetic mutation is a missense mutation outside of the SEQ. ID NO. 1 region. In some embodiments, the genetic mutation is a non-sense mutation outside of the SEQ. ID NO. 1 region.
  • the genetic mutation is a frameshift mutation outside of the SEQ. ID NO. 1 region. In some embodiments, the genetic mutation is an insertion mutation outside of the SEQ. ID NO. 1 region. In some embodiments, the genetic mutation is a deletion mutation outside of the SEQ. ID NO. 1 region. In some embodiments, the genetic mutation is a duplication mutation outside of the SEQ. ID NO. 1 region. In some embodiments, the MeCP2 Exon 2 region contains no genetic mutations. In some embodiments, the SEQ. ID NO. 1 region contains no genetic mutations. In some embodiments, the MeCP2 Exon 2 region contains mutations selected from: reference SNP rs267608409. In some embodiments, the SEQ. ID NO.
  • the X-chromosome-linked disease is selected from CDKL5 deficiency disorder, fragile x syndrome, Rett syndrome, Alport syndrome, X-linked Charcot-Marie- tooth disease, X-linked dominant porphyria, Vitamin D resistant rickets, Incontinentia pigmenti, CLCN4-related disorder, and facioscapulohumeral muscular dystrophy.
  • the X-chromosome-linked disease is selected from CDKL5 deficiency disorder, fragile x syndrome, Rett syndrome, Alport syndrome, X-linked Charcot-Marie-tooth disease, X-linked dominant porphyria, and Vitamin D resistant rickets.
  • the X-chromosome-linked disease is selected from fragile x syndrome and Rett syndrome.
  • the X-chromosome-linked disease is Rett syndrome.
  • administering the DNA methyltransferase inhibitor induces wildtype MeCP2 expression. In some embodiments, administering the DNA methyltransferase inhibitor induces the reactivation of wild-type MeCP2 expression. In some embodiments, administering the DNA methyltransferase inhibitor induces wild-type MeCP2 expression in human neural tissue. In some embodiments, administering the DNA methyltransferase inhibitor induces the reactivation of wild-type MeCP2 expression in human neural tissue.
  • the DNA methyltransferase inhibitor is a compound of Formula (I): or a pharmaceutically acceptable salt thereof, wherein:
  • R 1 and R 2 are each independently selected from hydrogen and -Si(R 3 )(R 4 )(R 5 ) ;
  • R 3 , R 4 , and R 5 are each independently selected from:
  • Ci-6 alkyl optionally substituted with one or more substituents selected from halogen, -OR 6 , -NO2, -CN, and C3-8 carbocycle optionally substituted with one more substituents selected from halogen, -OR 6 , -NO2, -CN, Ci-6 alkyl and Ci-e haloalkyl; and
  • C3-10 carbocycle optionally substituted with one or more substituents selected from halogen, -OR 6 , -NO2, CN and Ci-6 alkyl optionally substituted with one or more substituents selected from halogen, -OR 6 , -NO2, and CN; and R 6 is independently selected at each occurrence from hydrogen, Ci-6 alkyl, and Ci-6 haloalkyl; wherein the DNA methyltransferase inhibitor activates an inactive X-chromosome.
  • R 3 , R 4 , and R 5 are each independently selected from Ci-6 alkyl optionally substituted with one or more substituents selected from a saturated C3-8 carbocycle and phenyl each of which is optionally substituted with one more substituents selected from halogen, -OR 6 , -NO2, -CN, Ci-6 alkyl and Ci-6 haloalkyl.
  • R 3 , R 4 , and R 5 are each independently selected from Ci-6 alkyl optionally substituted with one or more substituents selected from a saturated C3-8 carbocycle and phenyl.
  • R 3 , R 4 , and R 5 are each independently selected from Ci-6 alkyl optionally substituted with one or more substituents independently selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl each of which is optionally substituted.
  • R 3 , R 4 , and R 5 are each independently selected from Ci-6 alkyl optionally substituted with one or more phenyls. In some embodiments, R 3 , R 4 , and R 5 are each independently selected from methyl, ethyl, propyl, and butyl each of which is unsubstituted. In some embodiments, R 3 , R 4 , and R 5 are each ethyl.
  • both R 1 and R 2 are selected from -Si(R 3 )(R 4 )(R 5 ). In some embodiments, both R 1 and R 2 are -Si(R 3 )(R 4 )(R 5 ) and R 3 , R 4 , and R 5 are each ethyl. In some embodiments, at least one of R 1 and R 2 are -Si(R 3 )(R 4 )(R 5 ) and R 3 , R 4 , and R 5 are each ethyl. In some embodiments, the DNA methyltransferase inhibitor is 5’-O- triethylsilyl 5-aza-2'-deoxycytidine.
  • the compound or salt of Formula (I) is represented by Formula (I-
  • administering decitabine or a salt thereof induces wild-type
  • administering decitabine or a salt thereof induces the reactivation of wild-type MeCP2 expression. In some embodiments, administering decitabine or a salt thereof induces wild-type MeCP2 expression in human neural tissue. In some embodiments, administering decitabine or a salt thereof induces the reactivation of wild-type MeCP2 expression in human neural tissue.
  • administering a compound or salt of Formula (I) or (I-A) induces wild-type MeCP2 expression. In some embodiments, administering a compound or salt of Formula (I) or (I-A) induces the reactivation of wild-type MeCP2 expression. In some embodiments, administering a compound or salt of Formula (I) or (I-A) induces wild-type MeCP2 expression in human neural tissue. In some embodiments, administering a compound or salt of Formula (I) or (I- A) induces the reactivation of wild-type MeCP2 expression in human neural tissue.
  • the method of treating an X-chromosome-linked disease comprises a DNA methyltransferase inhibitor, wherein the DNA methyltransferase inhibitor activates an inactive X-chromosome in the following in vitro assay: (a) reprogramming X- chromosome linked disease fibroblasts into induced pluripotent stem cells; (b) differentiating the induced pluripotent stem cells into brain organoids; (c) contacting said brain organoids in a vessel with a test DNA methyltransferase inhibitor, wherein the test DNA methyltransferase inhibitor is administered every other day for two weeks; and (d) detecting MeCP2 level in said vessel following step (c) and comparing the MeCP2 level to a MeCP2 level prior to said contacting in step (c), wherein when the MeCP2 levels are elevated by at least 1% and the test DNA methyltransferase inhibitor is a DNA methyltransferase inhibitor.
  • the X-chromosome-linked disease fibroblasts are Rett Syndrome fibroblasts.
  • the DNA methyltransferase inhibitor is decitabine or a salt thereof.
  • the DNA methyltransferase inhibitor is a compound or salt of Formula (I) or (I-A).
  • the DNA methyltransferase inhibitor is 5 ’-O-tri ethylsilyl 5-aza-2'-deoxycytidine.
  • the method of treating an X-chromosome-linked disease comprises a DNA methyltransferase inhibitor, wherein the DNA methyltransferase inhibitor induces wild-type MeCP2 in the following in vitro assay: (a) reprogramming X-chromosome linked disease fibroblasts into induced pluripotent stem cells; (b) differentiating the induced pluripotent stem cells into brain organoids; (c) contacting said brain organoids in a vessel with a test DNA methyltransferase inhibitor, wherein the test DNA methyltransferase inhibitor is administered every other day for two weeks; and (d) detecting MeCP2 level in said vessel following step (c) and comparing the MeCP2 level to a MeCP2 level prior to said contacting in step (c), wherein when the MeCP2 levels are elevated by at least 1% and the test DNA methyltransferase inhibitor is a DNA methyltransferase inhibitor.
  • the X-chromosome-linked disease fibroblasts are Rett Syndrome fibroblasts.
  • the DNA methyltransferase inhibitor is decitabine or a salt thereof.
  • the DNA methyltransferase inhibitor is a compound or salt of Formula (I) or (I-A).
  • the DNA methyltransferase inhibitor is 5’-O- triethylsilyl 5-aza-2'-deoxycytidine.
  • the assay provides an increased level of MeCP2 expression compared to the level of MeCP2 expression prior to the administration of the DNA methyltransferase inhibitor.
  • the level of expression is increased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.
  • the level of expression is increased by 5% to about 50%.
  • the DNA methyltransferase inhibitor is administered to a subject in need thereof for a period to induce MeCP2 expression. In some embodiments, the subject in need thereof does not have cancer. In some embodiments, the DNA methyltransferase inhibitor is administered to a subject in need thereof for a period of at least 7 days, at least 10 days, at least 14 days, at least 20 days, at least 30 days, at least 90 days, at least 200 days, at least 350 days or at least 500 days. In some embodiments, the method of treating an X-chromosome-linked disease, comprises administering DNA methyltransferase inhibitor to a subject in need thereof over a period of two weeks or more, wherein the subject does not have cancer.
  • a compound or salt of Formula (I) or (I-A) is administered to a subject in need thereof for a period to induce MeCP2 expression. In some embodiments, the subject in need thereof does not have cancer. In some embodiments, a compound or salt of Formula (I) or (I- A) is administered to a subject in need thereof for a period of at least 7 days, at least 10 days, at least 14 days, at least 20 days, at least 30 days, at least 90 days, at least 200 days, at least 350 days or at least 500 days.
  • the method of treating an X-chromosome-linked disease comprises administering a compound or salt of Formula (I) or (I-A) to a subject in need thereof over a period of two weeks or more, wherein the subj ect does not have cancer.
  • decitabine or a salt thereof is administered to a subject in need thereof for a period to induce MeCP2 expression. In some embodiments, the subject in need thereof does not have cancer. In some embodiments, decitabine or a salt thereof is administered to a subject in need thereof for a period of at least 7 days, at least 10 days, at least 14 days, at least 20 days, at least 30 days, at least 90 days, at least 200 days, at least 350 days or at least 500 days. In some embodiments, the method of treating an X-chromosome-linked disease, comprises administering decitabine or a salt thereof to a subject in need thereof over a period of two weeks or more, wherein the subj ect does not have cancer.
  • the present disclosure provides a method of treating an X-chromosome- linked disease, the method comprising: administering a DNA methyltransferase inhibitor to the subject, wherein the DNA methyltransferase inhibitor is a compound of Formula (II):
  • R 11 and R 12 are each independently selected from hydrogen or -Si(R 3 )(R 4 )(R 5 ) and at least one of R 11 and R 12 is -Si(R 3 )(R 4 )(R 5 );
  • R 3 , R 4 , and R 5 are each independently selected from:
  • Ci-6 alkyl optionally substituted with one or more substituents selected from halogen, -OR 6 , -NO2, -CN, and C3-8 carbocycle optionally substituted with one more substituents selected from halogen, -OR 6 , -NO2, -CN, Ci-6 alkyl and Ci-e haloalkyl; and
  • C3-10 carbocycle optionally substituted with one or more substituents selected from halogen, -OR 6 , -NO2, CN and Ci-6 alkyl optionally substituted with one or more substituents selected from halogen, -OR 6 , -NO2, and CN; and
  • R 6 is independently selected at each occurrence from hydrogen, Ci-6 alkyl, and Ci-6 haloalkyl; wherein the DNA methyltransferase inhibitor activates an inactive X-chromosome.
  • R 3 , R 4 , and R 5 are each independently selected from Ci-6 alkyl optionally substituted with one or more substituents selected from a saturated C3-8 carbocycle and phenyl each of which is optionally substituted with one more substituents selected from halogen, -OR 6 , -NO2, -CN, Ci-6 alkyl and Ci-e haloalkyl.
  • R 3 , R 4 , and R 5 are each independently selected from Ci-6 alkyl optionally substituted with one or more substituents selected from a saturated C3-8 carbocycle and phenyl.
  • R 3 , R 4 , and R 5 are each independently selected from Ci-6 alkyl, optionally substituted C3-8 carbocycle and optionally substituted C3-8 carbocycle-alkyl.
  • R 3 , R 4 , and R 5 are each independently selected from alkyl, optionally substituted aryl, and optionally substituted aryl-alkyl.
  • R 3 , R 4 , and R 5 are each independently selected from Ci-6 alkyl optionally substituted with one or more substituents independently selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl each of which is optionally substituted.
  • R 3 , R 4 , and R 5 are each independently selected Ci-6 alkyl optionally substituted with one or more substituents selected from -OR 6 , and C3-8 carbocycle, wherein the C3-8 carbocycle is optionally substituted with one more substituents selected from -OR 6 , and Ci-6 alkyl and Ci-6 haloalkyl.
  • R 3 , R 4 , and R 5 are each independently selected from Ci-6 alkyl optionally substituted with one or more substituents selected from a saturated C3-8 carbocycle and phenyl. In some embodiments, R 3 , R 4 , and R 5 are each independently selected from methyl, ethyl, propyl, and butyl each of which is unsubstituted. In some embodiments, R 3 , R 4 , and R 5 are each ethyl.
  • both R 11 and R 12 are selected from -Si(R 3 )(R 4 )(R 5 ). In some embodiments, both R 11 and R 12 are -Si(R 3 )(R 4 )(R 5 ) and R 3 , R 4 , and R 5 are each ethyl. In some embodiments, at least one of R 11 and R 12 are -Si(R 3 )(R 4 )(R 5 ) and R 3 , R 4 , and R 5 are each ethyl. In some embodiments, the DNA methyltransferase inhibitor is 5’-O- triethylsilyl 5-aza-2'-deoxycytidine.
  • the X-chromosome-linked disease is selected from CDKL5 deficiency disorder, fragile x syndrome, Rett syndrome, Alport syndrome, X-linked Charcot-Marie- tooth disease, X-linked dominant porphyria, Vitamin D resistant rickets, Incontinentia pigmenti, CLCN4-related disorder, and facioscapulohumeral muscular dystrophy.
  • the X-chromosome-linked disease is selected from CDKL5 deficiency disorder, fragile x syndrome, Rett syndrome, Alport syndrome, X-linked Charcot-Marie-tooth disease, X-linked dominant porphyria, and Vitamin D resistant rickets.
  • the X-chromosome-linked disease is selected from fragile x syndrome and Rett syndrome.
  • the X-chromosome-linked disease is Rett syndrome.
  • administering the DNA methyltransferase inhibitor induces wildtype MeCP2 expression. In some embodiments, administering the DNA methyltransferase inhibitor induces the reactivation of wild-type MeCP2 expression. In some embodiments, administering the DNA methyltransferase inhibitor induces wild-type MeCP2 expression in human neural tissue. In some embodiments, administering the DNA methyltransferase inhibitor induces the reactivation of wild-type MeCP2 expression in human neural tissue.
  • administering a compound or salt of Formula (II) or (II-A) induces wild-type MeCP2 expression. In some embodiments, administering a compound or salt of Formula (II) or (II-A) induces the reactivation of wild-type MeCP2 expression. In some embodiments, administering a compound or salt of Formula (II) or (II-A) induces wild-type MeCP2 expression in human neural tissue. In some embodiments, administering a compound or salt of Formula (II) or (II-A) induces the reactivation of wild-type MeCP2 expression in human neural tissue.
  • a compound or salt of Formula (II) or (II-A) is administered to a subject in need thereof for a period to induce MeCP2 expression. In some embodiments, the subject in need thereof does not have cancer. In some embodiments, a compound or salt of Formula (II) or (II-A) is administered to a subject in need thereof for a period of at least 7 days, at least 10 days, at least 14 days, at least 20 days, at least 30 days, at least 90 days, at least 200 days, at least 350 days or at least 500 days.
  • the method of treating an X-chromosome-linked disease comprises administering a compound or salt of Formula (II) or (II-A) to a subject in need thereof over a period of two weeks or more, wherein the subject does not have cancer.
  • the present disclosure provides a method of reactivating inactive X- chromosomes, the method comprising: identifying a subject with an X-chromosome genetic mutation, wherein the X-chromosome genetic mutation is outside of the MeCP2 Exon 2 region on the X-chromosome in a subject; and administering a DNA methyltransferase inhibitor to the subject, wherein the DNA methyltransferase inhibitor activates an inactive X-chromosome.
  • the genetic mutation outside of the MeCP2 Exon 2 region is at least one of a missense, non-sense, frameshift, insertion, deletion or a duplication mutation, or any combination thereof.
  • the genetic mutation is a missense, non-sense, frameshift, insertion, deletion or a duplication mutation. In some embodiments, the genetic mutation is a missense mutation outside of the MeCP2 Exon 2 region. In some embodiments, the genetic mutation is a nonsense mutation outside of the MeCP2 Exon 2 region. In some embodiments, the genetic mutation is a frameshift mutation outside of the MeCP2 Exon 2 region. In some embodiments, the genetic mutation is an insertion mutation outside of the MeCP2 Exon 2 region. In some embodiments, the genetic mutation is a deletion mutation outside of the MeCP2 Exon 2 region. In some embodiments, the genetic mutation is a duplication mutation outside of the MeCP2 Exon 2 region.
  • the genetic mutation outside of SEQ. ID NO. 1 region is at least one of a missense mutation, non-sense mutation, frameshift, insertion, deletion or a duplication mutation, or any combination thereof.
  • the genetic mutation is a missense, non-sense, frameshift, insertion, deletion or a duplication mutation.
  • the genetic mutation is a missense mutation outside of the SEQ. ID NO. 1 region.
  • the genetic mutation is a non-sense mutation outside of the SEQ. ID NO. 1 region.
  • the genetic mutation is a frameshift mutation outside of the SEQ. ID NO. 1 region.
  • the genetic mutation is an insertion mutation outside of the SEQ. ID NO. 1 region.
  • the genetic mutation is a deletion mutation outside of the SEQ. ID NO. 1 region. In some embodiments, the genetic mutation is a nonsense mutation outside of the SEQ. ID NO. 1 region. In some embodiments, the MeCP2 Exon 2 region contains no genetic mutations. In some embodiments, the SEQ. ID NO. 1 region contains no genetic mutations. In some embodiments, the MeCP2 Exon 2 region contains mutations selected from: reference SNP rs267608409. In some embodiments, the SEQ. ID NO. 1 region contains mutations selected from: reference SNP rs267608409.
  • the method of reactivating an inactive X-chromosome comprises administering a DNA methyltransferase inhibitor to a subject in need thereof in an amount sufficient to increase at least one biomarker selected from: KCC2, brain-derived neurotrophic factor, oxidative stress biomarkers, and DNA methylation patterns for at least 1 week or more.
  • the biomarker is increased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.
  • the biomarker is increased by 5% to about 50%.
  • the biomarker is decreased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, the biomarkers is decreased by 5% to about 50%. In some embodiments, the biomarker is increased for a period of at least 7 days, at least 10 days, at least 14 days, at least 20 days, at least 30 days, at least 90 days, at least 200 days, at least 350 days or at least 500 days. In some cases, the subject in need thereof is administered decitabine or a salt thereof in an amount sufficient to increase at least one biomarker selected from: KCC2, and brain-derived neurotrophic factor, for at least 1 week or more.
  • the subject in need thereof is administered decitabine or a salt thereof in an amount sufficient to decrease at least one biomarker selected from: oxidative stress biomarkers and DNA methylation patterns, for at least 1 week or more.
  • the subject in need thereof is administered a compound or salt of Formula (I), (I- A), (II), or (II- A) in an amount sufficient to decrease at least one biomarker selected from: oxidative stress biomarkers and DNA methylation patterns, for at least 1 week or more.
  • the present disclosure provides a method of reactivating an inactive X- chromosomes, comprising administering decitabine or a salt thereof to a subject in need thereof in an amount sufficient to increase cerebrospinal fluid levels of KCC2 by at least 10% for two weeks or more.
  • the cerebrospinal fluid levels of KCC2 is increased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.
  • the cerebrospinal fluid levels of KCC2 is increased by 5% to about 50%.
  • KCC2 levels can be measured using any number of methods known to those skilled in the art.
  • the present disclosure provides a method of reactivating an inactive X- chromosomes, comprising administering decitabine or a salt thereof to a subject in need thereof in an amount sufficient to increase cerebrospinal fluid levels of brain-derived neurotrophic factor by at least 10% for two weeks or more.
  • the cerebrospinal fluid levels of brain- derived neurotrophic factor is increased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.
  • the brain-derived neurotrophic factor is increased by 5% to about 50%. Brain-derived neurotrophic factor levels can be measured using any number of methods known to those skilled in the art.
  • the present disclosure provides a method of reactivating an inactive X- chromosomes, comprising administering decitabine or a salt thereof to a subject in need thereof in an amount sufficient to reduce oxidative stress biomarkers by at least 10% for two weeks or more.
  • the oxidative stress biomarkers are decreased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.
  • the oxidative stress biomarkers are decreased by 5% to about 50%. Oxidative stress biomarkers levels can be measured using any number of methods known to those skilled in the art.
  • the present disclosure provides a method of reactivating inactive X- chromosomes, comprising administering decitabine or a salt thereof to a subject in need thereof in an amount sufficient to reduce DNA methylation patterns in the subject’s blood by at least 1% for two weeks or more.
  • DNA methylation patterns are decreased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.
  • DNA methylation patterns are decreased by 5% to about 50%.
  • DNA methylation patterns levels can be measured using any number of methods known to those skilled in the art.
  • DNA methylation patterns in the subject’s blood are decreased by at least 5% for two weeks or more.
  • the subject has Rett Syndrome.
  • the decitabine or salt thereof is administered daily, every other day or once weekly.
  • the method comprises, chronically administering decitabine or a salt thereof daily, every other day, every third day, once a week, or once a month.
  • the method comprises, chronically administering decitabine or a salt thereof daily, every other day, every third day, once a week, or once a month.
  • the method comprises, chronically administering decitabine or a salt thereof at least daily, every other day, every third day, once a week, or once a month.
  • the method comprises chronically administering decitabine or a salt thereof at least one time a week, two times a week, three times a week, four times a week, five times a week, six times a week, seven times a week, eight times a week, nine times a week, ten times a week, or more.
  • chronically administrating decitabine or a salt thereof the compound over the course of at least about 7 days, 10 days, 14 days, 21 days, 30 days, 60 days, 90 days, or more.
  • the present disclosure provides a method of reactivating an inactive X- chromosomes, comprising administering a compound or salt of Formula (I), (I- A), (II), or (II- A) to a subject in need thereof in an amount sufficient to increase cerebrospinal fluid levels of KCC2 by at least 10% for two weeks or more.
  • the cerebrospinal fluid levels of KCC2 is increased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.
  • the cerebrospinal fluid levels of KCC2 is increased by 5% to about 50%.
  • KCC2 levels can be measured using any number of methods known to those skilled in the art.
  • the present disclosure provides a method of reactivating an inactive X- chromosomes, comprising administering a compound or salt of Formula (I), (I- A), (II), or (II- A) to a subject in need thereof in an amount sufficient to increase cerebrospinal fluid levels of brain-derived neurotrophic factor by at least 10% for two weeks or more.
  • the cerebrospinal fluid levels of brain-derived neurotrophic factor is increased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.
  • the brain- derived neurotrophic factor is increased by 5% to about 50%. Brain-derived neurotrophic factor levels can be measured using any number of methods known to those skilled in the art.
  • the present disclosure provides a method of reactivating an inactive X- chromosomes, comprising administering a compound or salt of Formula (I), (I- A), (II), or (II- A) to a subject in need thereof in an amount sufficient to reduce oxidative stress biomarkers by at least 10% for two weeks or more.
  • the oxidative stress biomarkers are decreased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.
  • the oxidative stress biomarkers are decreased by 5% to about 50%. Oxidative stress biomarkers levels can be measured using any number of methods known to those skilled in the art.
  • the present disclosure provides a method of reactivating inactive X- chromosomes, comprising administering a compound or salt of Formula (I), (I- A), (II), or (II- A) to a subject in need thereof in an amount sufficient to reduce DNA methylation patterns in the subject’s blood by at least 1% for two weeks or more.
  • DNA methylation patterns are decreased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.
  • DNA methylation patterns are decreased by 5% to about 50%.
  • DNA methylation patterns levels can be measured using any number of methods known to those skilled in the art.
  • DNA methylation patterns in the subject’s blood are decreased by at least 5% for two weeks or more.
  • the subject has Rett Syndrome.
  • a compound or salt of Formula (I), (I- A), (II), or (II- A) is administered daily, every other day or once weekly.
  • the method comprises, chronically administering a compound or salt of Formula (I), (I- A), (II), or (II- A) daily, every other day, every third day, once a week, or once a month. In some embodiments, the method comprises, chronically administering a compound or salt of Formula (I), (I- A), (II), or (II- A) daily, every other day, every third day, once a week, or once a month. In some embodiments, the method comprises, chronically administering a compound or salt of Formula (I), (I- A), (II), or (II- A) at least daily, every other day, every third day, once a week, or once a month.
  • the method comprises chronically administering a compound or salt of Formula (I), (I- A), (II), or (II- A) at least one time a week, two times a week, three times a week, four times a week, five times a week, six times a week, seven times a week, eight times a week, nine times a week, ten times a week, or more.
  • FIG. 1A-C shows a wild type MeCP2 protein expression assay design.
  • FIG. 1A shows isogenic control patient derived organoid (PDO) models are generated from Rett patient iPSC lines in which the wild type MeCP2 gene (WT MeCP2) on the active X chromosome (Xa) is expressed. The mutated MeCP2 gene is on the inactive X chromosome (Xi) and not expressed. Immunolabeling with a C-terminal targeting MeCP2 antibody results in detectable MeCP2 expression in cells.
  • FIG. 1A shows isogenic control patient derived organoid (PDO) models are generated from Rett patient iPSC lines in which the wild type MeCP2 gene (WT MeCP2) on the active X chromosome (Xa) is expressed. The mutated MeCP2 gene is on the inactive X chromosome (Xi) and not expressed. Immunolabeling with a C-terminal targeting MeCP2 antibody results in detectable Me
  • IB shows PDO models of Rett syndrome generated from iPSC lines expressing mutated MeCP2 protein with premature truncation on the Xa and wild type MeCP2 on Xi. C-terminal targeting MeCP2 immunolabeling results in no detectable MeCP2 protein.
  • FIG.1C shows a drug compound inducing re-activation of wild type MeCP2 from the inactivated X chromosome (Xi*) that results in measurable C-terminal immunolabeling enabling quantification of percentage MeCP2 reactivation.
  • Patient fibroblasts with mutation R270X, were obtained from Rett Syndrome Research Trust.
  • Patient fibroblasts were reprogrammed into induced pluripotent stem cells (iPSCs) using nonintegrating Sendai Virus (Cytotune 2.0).
  • the iPSCs were maintained in StemFlex medium on matrigel-coated plates.
  • iPSCs were seeded at 10K cells/well in ultra-low attachment 96 well plates and differentiated into brain organoids using methods and procedures according to Velasco et al., Nature 2019, 570, 523-529, relevant portions of which are incorporated herein by reference in its entirety. Following neural induction, organoids were transferred to low-attachment 24 well plates and cultured in neuron culture media.
  • organoids were dissociated into single cell suspension with TrypLE/DNasel at 37C for 30 mins. Dissociated cells were plated at 25K cells/well in neuron culture medium onto matrigel-coated 96 well Cellvis plates.
  • FIG. 2 shows decitabine induced MeCP2 protein expression in a dose-dependent manner following 2 week treatment, with 0.1-0.3 uM inducing the greatest MeCP2 protein expression by MeCP2 reactivation quantification.
  • MeCP2 reactivation rate quantified as the number of MeCP2 positive cells per 100 DAPI positive cells, n.s., not significant; *, p ⁇ 0.01; **, p ⁇ 0.001; ***, p ⁇ 0.0001 : one-sided unpaired / test with respect to untreated and DMSO negative controls.
  • Each dot represents a single fluorescence microscopy image of a well plated with 2-D dissociated PDOs, then compound treated, fixed and immunolabeled prior to imaging.
  • FIG. 4 shows decitabine treatment for 1 week is insufficient to induce MeCP2 protein expression at all evaluated concentrations evaluated by MeCP2 reactivation quantification.
  • Each dot represents a single fluorescence microscopy image of a well plated with 2-D dissociated PDOs, then compound treated, fixed and immunolabeled prior to imaging.
  • FIG. 8A-B shows immunolabeling images of decitabine induced MeCP2 expression.
  • FIG. 8A shows representative immunolabeling images from Rett PDOs treated with DMSO. Immunolabeling with C-terminal MeCP2 antibody shows no detectable signal. Additional counter stains shown for the nucleus (DAPI) and neurites (MAP2).
  • FIG. 8B shows representative immunolabeling images from Rett PDOs treated with 0.1 uM decitabine for 2 weeks showing qualitative MeCP2 protein reactivation. Each column is a different imaging field used for MeCP2 reativation quantification.
  • FIG. 9 shows 5 ’-O-triethylsilyl 5-aza-2'-deoxycytidine induced MeCP2 protein expression in a dose-dependent manner following 2 week treatment with, 0.1 uM inducing the greatest MeCP2 protein expression by MeCP2 reactivation quantification.
  • Negative control data generated using Rett brain organoids expressing mutated MeCP2 and either untreated, or treated with DMSO vehicle.
  • MeCP2 reactivation rate quantified as the number of MeCP2 positive cells per 100 DAPI positive cells, n.s.
  • Each dot represents a single fluorescence microscopy image of a well plated with 2-D dissociated brain organoids, then compound treated, fixed and immunolabeled prior to imaging.
  • DAPI channels DNA-stained image channels
  • Identified segments were post-hoc artifact-corrected for potential debris, background staining or imaging artifacts using thresholds on DAPI intensity and size, as well as brightfield image intensity.
  • the number of DAPI positive nuclei were counted in each image.
  • Therapeutic efficacy and toxicity quantification metrics were computed for each image using DAPI positive nuclei as follows:
  • Efficacy was quantified using two different metrics.
  • the MECP2 reactivation rate was computed by first applying the DAPI segmented nuclei as a mask on the MECP2 channel. Second, thresholding the MECP2 channel intensity with suitably derived fixed intensity thresholds. Third, counting the number of MECP2 positive cells and normalizing by DAPI positive cells expressed as a percent.
  • tail score metrics were also computed to detect low effect sizes. Using DAPI segmented nuclei as a mask on the MECP2 channel, the distribution of mean MECP2 intensity within masks was estimated. Then quantification of the extent to which this distribution was right-shifted with respect to a normal distribution, and labeled as the tail score. The tail score is simply computed as the ratio of the difference between the 99th percentile and the 75th percentile, with the difference between the 99th and 50th percentile for the distribution of interest; this ratio is then normalized by the same ratio for a Gaussian distribution.
  • the tail score for a Gaussian distribution is 1
  • the tail score for a left- shifted distribution is ⁇ 1
  • the tail score for a right-skewed distribution is > 1.
  • FIG. 3 shows decitabine induced MeCP2 protein expression in a dose-dependent manner following 2 week treatment, with 0.1-0.3 uM inducing significant MeCP2 protein expression by tail score quantification.
  • MeCP2 intensity tail score quantified as a measure of tail weight in the distribution of MeCP2 intensity over all DAPI positive cells, n.s., not significant; *, p ⁇ 0.01; **, p ⁇ 0.001; ***, p ⁇ 0.0001 : one-sided unpaired / test with respect to untreated and DMSO negative controls.
  • Each dot represents a single fluorescence microscopy image of a well plated with 2-D dissociated PDOs, then compound treated, fixed and immunolabeled prior to imaging.
  • FIG. 5 shows decitabine treatment for 1 week at 0.1 and 0.3 uM and induces subtle MeCP2 expression by histogram tail score quantification. Other concentrations are unable to induce MeCP2 expression.
  • MeCP2 intensity tail score quantified as a measure of tail weight in the distribution of MeCP2 intensity over all DAPI positive cells, n.s., not significant; *, p ⁇ 0.01; **, p ⁇ 0.001; ***, p ⁇ 0.0001 : one-sided unpaired / test with respect to untreated and DMSO negative controls.
  • Each dot represents a single fluorescence microscopy image of a well plated with 2-D dissociated PDOs, then compound treated, fixed and immunolabeled prior to imaging.
  • Toxicity was quantified using two different metrics, both based on estimates of percent cell survival in the treatment group relative to a negative control group (untreated or DMSO vehicle treated samples).
  • the first metric calculated from the ratio of DAPI positive cell counts from treatment and control groups, and expressed as a percent.
  • the second metric calculated from the ratio of the overall background-removed MAP2 channel intensity between treatment and control groups, and expressed as a percent.
  • Empirically calculated MAP2 estimates of toxicity were less variable across technical replicates than DAPI estimates, although both estimates suggest no toxicity at the optimally efficacious dose.
  • FIG. 6 shows the quantified toxicity of decitabine treatment for 1 week and 2 weeks using percent survival relative to untreated or DMSO vehicle treated control using DAPI staining. No toxicity at 1 week incubation, statistically significant toxicity observed only at 0.3 uM at 2 weeks incubation.
  • Negative control data generated using Rett brain organoids expressing mutated MeCP2 and either untreated or treated with DMSO vehicle. Percent survival quantified as percent of DAPI positive cells in treatment groups relative to negative control group; *, p ⁇ 0.01; **, p ⁇ 0.001; ***, p ⁇ 0.0001 : one-sided unpaired t test with respect to untreated and DMSO negative controls.
  • FIG. 7 shows the quantified toxicity of decitabine treatment for 1 week and 2 weeks using percent survival relative to untreated or DMSO vehicle treated control using MAP2 immunostaining. No toxicity at 1 week incubation, statistically significant toxicity observed only at 0.3, 1.0, 3.0 and 10.0 uM at 2 weeks incubation. Negative control data generated using Rett brain organoids expressing mutated MeCP2 and either untreated or treated with DMSO vehicle.
  • Each dot represents a single fluorescence microscopy image of a well plated with 2-D dissociated PDOs, then compound treated, fixed and immunolabeled prior to imaging.
  • FIG. 10 shows 5 ’-O-triethylsilyl 5-aza-2'-deoxycytidine induced MeCP2 protein expression in a dose-dependent manner following 2 week treatment with, 0.1 uM inducing the greatest MeCP2 protein expression by tail score quantification.
  • Negative control data generated using Rett brain organoids expressing mutated MeCP2 and either untreated, or treated with DMSO vehicle.
  • MeCP2 intensity tail score quantified as a measure of tail weight in the distribution of MeCP2 intensity of all DAPI positive cells, n.s.
  • Each dot represents a single fluorescence microscopy image of a well plated with 2-D dissociated brain organoids, then compound treated, fixed and immunolabeled prior to imaging.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Neurosurgery (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Neurology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Epidemiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne des méthodes d'induction de l'expression et/ou de la réactivation de MECP2 par l'intermédiaire de la décitabine ou de ses sels, et des composés de formules (I), (I-A), (II), (II-A) ou de leurs sels.
PCT/US2021/044415 2020-08-04 2021-08-03 Expression de mecp2 induite par décitabine et ses utilisations WO2022031759A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202063061075P 2020-08-04 2020-08-04
US63/061,075 2020-08-04
US202063077444P 2020-09-11 2020-09-11
US63/077,444 2020-09-11

Publications (1)

Publication Number Publication Date
WO2022031759A1 true WO2022031759A1 (fr) 2022-02-10

Family

ID=77448146

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2021/044415 WO2022031759A1 (fr) 2020-08-04 2021-08-03 Expression de mecp2 induite par décitabine et ses utilisations

Country Status (1)

Country Link
WO (1) WO2022031759A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023192629A1 (fr) * 2022-04-01 2023-10-05 Herophilus, Inc. Induction de l'expression de mecp2 par des inhibiteurs de l'adn méthyl transférase

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3207932A1 (fr) * 2016-02-19 2017-08-23 Universität Stuttgart Inhibiteurs de méthyltransférase d'adn pour la thérapie du syndrome de rett
EP3252067A1 (fr) * 2016-04-21 2017-12-06 Ohara Pharmaceutical Co., Ltd. Dérivé d'éther de silyle à fragment de sucre de 5-azacytidine

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3207932A1 (fr) * 2016-02-19 2017-08-23 Universität Stuttgart Inhibiteurs de méthyltransférase d'adn pour la thérapie du syndrome de rett
EP3252067A1 (fr) * 2016-04-21 2017-12-06 Ohara Pharmaceutical Co., Ltd. Dérivé d'éther de silyle à fragment de sucre de 5-azacytidine

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
AMIR R E ET AL: "Mutations in exon 1 of MECP2 are a rare cause of Rett syndrome", JOURNAL OF MEDICAL GENETICS, vol. 42, no. 2, 1 February 2005 (2005-02-01), pages e15 - e15, XP055857854, Retrieved from the Internet <URL:https://jmg.bmj.com/content/jmedgenet/42/2/e15.full.pdf> DOI: 10.1136/jmg.2004.026161 *
HATTORI NAOKO ET AL: "Novel prodrugs of decitabine with greater metabolic stability and less toxicity", CLINICAL EPIGENETICS, vol. 11, no. 1, 1 August 2019 (2019-08-01), London, UK, pages 111, XP055809414, ISSN: 1868-7075, Retrieved from the Internet <URL:http://link.springer.com/article/10.1186/s13148-019-0709-y/fulltext.html> DOI: 10.1186/s13148-019-0709-y *
VELASCO ET AL., NATURE, vol. 570, 2019, pages 523 - 529

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023192629A1 (fr) * 2022-04-01 2023-10-05 Herophilus, Inc. Induction de l'expression de mecp2 par des inhibiteurs de l'adn méthyl transférase

Similar Documents

Publication Publication Date Title
Rodenburg Mitochondrial complex I-linked disease
Taubes et al. Experimental and real-world evidence supporting the computational repurposing of bumetanide for APOE4-related Alzheimer’s disease
Hua et al. Sex and age differences in atrophic rates: an ADNI study with n= 1368 MRI scans
Seshadri et al. Genetic correlates of brain aging on MRI and cognitive test measures: a genome-wide association and linkage analysis in the Framingham Study
Sweet et al. Reduced dendritic spine density in auditory cortex of subjects with schizophrenia
Williams et al. UBQLN2/ubiquilin 2 mutation and pathology in familial amyotrophic lateral sclerosis
Wilcock et al. The differential involvement of subcortical nuclei in senile dementia of Alzheimer's type.
Hachon et al. Behavioural and cognitive phenotypes in children with neurofibromatosis type 1 (NF1): the link with the neurobiological level
Lista et al. Imaging epigenetics in Alzheimer's disease
US20150045306A1 (en) Determination of single nucleotide polymorphisms useful to predict response for glatiramer acetate
MXPA06014611A (es) Evaluacion de un tratamiento para disminuir el riesgo de un desorden cerebral progresivo o para frenar el envejecimiento del cerebro.
WO2019236750A2 (fr) Traitement ciblé d&#39;un trouble du spectre autistique et d&#39;autres troubles neurologiques ou psychiatriques
Nagy et al. Schöpf-Schulz-Passarge syndrome resulting from a homozygous nonsense mutation in WNT10A
WO2022031759A1 (fr) Expression de mecp2 induite par décitabine et ses utilisations
Choudhary et al. Current progress in understanding schizophrenia using genomics and pluripotent stem cells: A meta-analytical overview
WO2023192629A1 (fr) Induction de l&#39;expression de mecp2 par des inhibiteurs de l&#39;adn méthyl transférase
Liu et al. Decreased DNA methylation of RGMA is associated with intracranial hypertension after severe traumatic brain injury: an exploratory epigenome-wide association study
Chirichigno et al. Stress-induced mitochondrial depolarization and oxidative damage in PSP cybrids
Jiang et al. Identification of novel quantitative traits-associated susceptibility loci for APOE ε 4 non-carriers of Alzheimer’s disease
Auranen Molecular genetics of autism spectrum disorders in the Finnish population
Guo et al. Genome-wide association study of white matter hyperintensity volume in elderly persons without dementia
Hill et al. Can a herpes simplex virus type 1 neuroinvasive score be correlated to other risk factors in Alzheimer’s disease?
Wang et al. Interaction of COMT and KIBRA modulates the association between hippocampal structure and episodic memory performance in healthy young adults
Highley et al. Motor neuron disorders
Beckers et al. Specific suppression of microgliosis cannot circumvent the severe neuropathology in peroxisomal β-oxidation-deficient mice

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21758972

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21758972

Country of ref document: EP

Kind code of ref document: A1