WO2014127038A1 - Procédés et compositions associés à l'homéostase du calcium et la maladie de fabry - Google Patents

Procédés et compositions associés à l'homéostase du calcium et la maladie de fabry Download PDF

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WO2014127038A1
WO2014127038A1 PCT/US2014/016094 US2014016094W WO2014127038A1 WO 2014127038 A1 WO2014127038 A1 WO 2014127038A1 US 2014016094 W US2014016094 W US 2014016094W WO 2014127038 A1 WO2014127038 A1 WO 2014127038A1
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calcium
modulator
cycling
nucleic acid
calcium cycling
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PCT/US2014/016094
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English (en)
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Xingli Meng
Jinsong Shen
Raphael Schiffmann
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Baylor Research Institute
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    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/554—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one sulfur as ring hetero atoms, e.g. clothiapine, diltiazem
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
    • 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
    • A61K31/275—Nitriles; Isonitriles
    • A61K31/277—Nitriles; Isonitriles having a ring, e.g. verapamil
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/06—Antiarrhythmics

Definitions

  • the present invention relates generally to the field of medicine. More particularly, it concerns the use of calcium cycling modulators to treat arrhythmias and related heart conditions in Fabry disease patients.
  • Fabry disease is an X-linked lysosomal storage disorder, caused by insufficient activity of a-galactosidase A (oc-Gal A).
  • oc-Gal A a-galactosidase A
  • GSLs glycosphingolipids
  • Gb3 globotriaosylceramide
  • Arrhythmias are one of the most prominent cardiac manifestations of Fabry disease, which can cause sudden death and precipitate heart failure in the patients.
  • ERT enzyme replace therapy
  • ERT in Fabry disease has very limited effect, especially in preventing and treating cardiac arrhythmias and other cardiac complications.
  • Another treatment called pharmacological chaperon is in development for Fabry disease patients who have amendable mutations. Both the ERT and chaperon studied are based on the concept of the supplement of the deficient enzyme to the patient or stabilizing the mutant enzyme to increase enzyme activity.
  • Methods and compositions are provided for treating arrhythmia or treating a patient with or at risk for Fabry disease. Additional methods and compositions can be used for treating patients with suspected of having arrhythmia or brachycardia or having symptoms of arrhythmia or brachycardia, or patients at risk for arrhythmia or brachycardia. Methods and compositions concern regulating calcium homeostasis in cardiac cells. In certain embodiments, calcium homeostasis is restored in cardiac cells, such as cardiomyocytes.
  • Some embodiments concern a pharmaceutical composition comprising a calcium cycling modulator, wherein the calcium cycling modulator restores calcium homeostasis in the cardiac cells.
  • the calcium cycling modulator is an L-type calcium channel antisense nucleic acid.
  • the L-type calcium channel antisense nucleic acid is 8 to 50 nucleotides in length.
  • Further embodiments include an L- type calcium channel antisense nucleic acid that is partly or fully double stranded.
  • Some embodiments involve an antisense nucleic acid that comprises DNA. It is contemplated that the nucleic acid may include one or more modified nucleic acid residues.
  • the calcium cycling modulator is a CASQ2 polypeptide or nucleic acid encoding a CASQ2 polypeptide.
  • the nucleic acid is an expression vector or expression construct.
  • the expression vector is a viral vector.
  • Other embodiments include a number of different methods that may or may not use the pharmaceutical compositions described above or elsewhere.
  • there are methods of treating a patient with Fabry disease comprising administering to the patient an effective amount of a composition comprising a calcium cycling modulator, wherein the calcium cycling modulator restores calcium homeostasis in cardiac cells of the patient.
  • methods of treating a patient with Fabry disease comprising administering to the patient an effective amount of a composition comprising a calcium cycling modulator, wherein the calcium cycling modulator restores calcium homeostasis in other systems (apart from the heart) of the patient.
  • Other methods include treating arrhythmia in a patient with Fabry disease comprising administering to the patient an effective amount of a composition comprising a calcium cycling modulator, wherein the calcium cycling modulator restores calcium homeostasis in the cardiac cells.
  • Further embodiments include methods of treating brachycardia in a Fabry disease patient comprising administering to the patient an effective amount of a composition comprising a calcium cycling modulator.
  • Other embodiments include methods of decreasing Ryanodine receptor 2 hyperphosphorylation (e.g., by use of a phosphatase) in a Fabry disease patient, comprising administering to the patient an effective amount of a composition comprising a calcium cycling modulator.
  • Additional embodiments include methods of increasing phospholamban in a Fabry disease patient comprising administering to the patient an effective amount of a composition comprising a calcium cycling modulator.
  • the calcium cycling modulator is a phospholamban polypeptide or nucleic acid encoding a phospholamban polypeptide.
  • the nucleic acid is an expression vector or expression construct.
  • the expression vector is a viral vector.
  • Other embodiments include methods of restoring calcium homeostasis in a patient in need thereof comprising administering a calcium cycling modulator.
  • Patients may have been diagnosed or identified as in need of treatment in some embodiments.
  • a patient is determined to be in need of restoration of calcium homeostasis.
  • the calcium cycling modulator restores calcium homeostasis by preventing calcium overload and/or reducing sarcoplasmic reticulum (SR) calcium leak.
  • the calcium cycling modulator inhibits calcium channel activity or expression.
  • the calcium cycling modulator selectively inhibits calcium channel activity or expression.
  • the calcium cycling modulator inhibits L-type calcium channel activity or expression. It is contemplated that the calcium cycling modulator is a non-dihydropyridine in some embodiments. Additional embodiments concern a calcium cycling modulator that inhibits L-type calcium channel activity.
  • the calcium cycling modulator is verapamil or diltiazem, or a salt or prodrug thereof.
  • the calcium cycling modulator is not diltiazem. In other embodiments, the calcium cycling modulator is mibefradil, bepridil, fluspirilene, or fendiline, or a salt or prodrug thereof. In some embodiments, the calcium cycling modulator is an L-type calcium channel antisense nucleic acid, as described herein. [0011 J Methods include a calcium cycling modulator stabilizes Ryr2 channels or increases calsequestrin 2 (CASQ2) activity or expression. In some cases, the calcium cycling modulator increases CASQ2 activity or expression. In some cases, the calcium cycling modulator increases CASQ2 expression. In some cases, the calcium cycling modulator increases phospholamban activity or expression.
  • CASQ2 calsequestrin 2
  • Certain embodiments involve a calcium cycling modulator that is 1 ,4- benzothiazepine analogue JTV519 ( 201), JTV519 analogue (S I 07), RyR2 blocking agent tetracaine or CaMKII inhibitor K.N-93.
  • the calcium cycling modulator is BAPTA or EDTA.
  • Some methods involve a patient who has previously been administered enzyme replacement therapy. Other methods comprise treating the patient with enzyme replacement therapy.
  • the calcium cycling modulator is administered multiple times. It may be administered multiple times per day. It may be administered intravenously, intraarterial ly, intraperitoneally, intradermal ly, intramuscularly, subcutaneously, intranasally, intratracheally, intraspinally, intracranial ly, orally, or by infusion.
  • Methods may include evaluating the patient for cardiac arrhythmia or for Fabry's disease or for brachycardia. A patient may be further monitored for brachycardia or cardiac arrhythmia following treatment.
  • compositions described herein for the treatment of arrhythmia in a patient with Fabry disease.
  • a dose may be administered on an as needed basis or every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 18, or 24 hours (or any range derivable therein) or 1 , 2, 3, 4, 5, 6, 7, 8, 9, or times per day (or any range derivable therein).
  • a dose may be first administered before or after signs of arrhythmia are exhibited or felt by a patient or after a clinician evaluates the patient for an arrhythmia.
  • the patient is administered a first dose of a regimen 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 hours (or any range derivable therein) or 1 , 2, 3, 4, or 5 days after the patient experiences or exhibits signs or symptoms of an arrhythmia (or any range derivable therein).
  • the patient may be treated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days (or any range derivable therein) or until symptoms of an arrhythmia have disappeared or been reduced or after 6, 12, 18, or 24 hours or 1 , 2, 3, 4, or 5 days after symptoms of arrhythmia have disappeared or been reduced.
  • 0017) As used herein the specification, "a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words "a” or “an” may mean one or more than one.
  • FIG. 1A-1G Generation of human iPSC from Fabry patients and healthy controls. Dermal fibroblasts (A) from Fabry patients and healthy controls are used for generation of iPSC (B,C). The established iPSC lines are positive for TRA- 1 -60 (D), SSEA-4 € and Nanog (F). The mutations in the GLA gene of Fabry patients iPSC are confirmed by DNA sequencing (G).
  • FIG. 2A-2G Cardiac differentiation. Cardiac cells (A) differentiated from Fabry patient and control iPSC express cardiac cell-specific markers. Troponin T, GATA-4 (B) and sarcomeric a-Actinin (C-F). Quantitative mRNA expression analysis (G) show cardiomyocytes from Fabry and healthy controls express similar level of TNNT2, troponin T type 2 (cardiac); MYH6, myosin, heavy chain 6, cardiac muscle, alpha and NKX2.5, NK2 homeobox 5.
  • FIG. 3A-3B GSLs accumulation. Gb 3 immunostaining in untreated (A) and a-Gal A treated (B) Fabry patient iPSC-derived cardiomyocytes.
  • FIG. 4A-4B "Bradycardia" in cardiomyocytes differentiated from Fabry patient iPSC. Representative differentiated beating clusters of cardiomyocytes are shown in A. Spontaneous beating rates of the beating clusters from Fabry patients are significantly slower than those from the healthy controls (B). *P ⁇ 0.01. [0026] FIG. 5: Arrhythmia in donor Fabry disease patient. E G recording of
  • Fabry patient showed abnormal EKG: marked sinus bradycardia with 1 st degree A-V block, right bundle branch block, left anterior fascicular block, incomplete trifascicular block, lateral infact. Vent rate: 48 BPM; PR interval: 232 ms; QRS duration 180 ms; QT/QTc: 478/427 ms.
  • FIG. 6 Quantitative RT-PCR analysis of the expression levels of calcium regulatory proteins in Fabry patient and control iPSC-derived cardiomyocytes. *p ⁇
  • FIG. 7A-7C Ca2+ imaging study in Fabry patients and healthy control cardiomyocytes.
  • A Ventricular cardiomyocytes were identified by Tomato red (arrow).
  • B Ca2+ transients were recorded.
  • C The spontaneous firing rates, amplitude, upstroke and decay velocities of the Ca2+ transient in Fabry patient and healthy control cardiomyocytes were measured.
  • FIG. 8A-8B Western blot analysis of RyR2 and PLN.
  • A The phosphorylated and total RyR2 and PLN were measured in the Fabry patient iPSC-derived cardiomyocyte clusters by Western blot.
  • B Western blot analysis of RyR2 and PLN in the Fabry mouse heart tissues.
  • FIG. 9A-9B Effect of verapamil on the beating rates of Fabry patient iPSC-derived cardiac clusters.
  • A MEA tracing of diseased cardiac clusters treated with low dose verapamil.
  • B Beating rates of diseased and normal control cardiac clusters treated with verapamil. Data were presented as mean ⁇ s.e.m. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
  • compositions and methods of using these compositions can treat a subject (e.g., Fabry disease subject with calcium homeostasis pathology) having, suspected of having, or at risk of developing cardiac manifestations of Fabry disease.
  • a subject e.g., Fabry disease subject with calcium homeostasis pathology
  • Fabry disease also known as Fabry's disease, Anderson-Fabry disease, angiokeratoma corporis diffusum and alpha-galactosidase
  • a deficiency is a rare genetic lysosomal storage disease, inherited in an X-linked manner.
  • Fabry disease can cause a wide range of systemic symptoms, including pain, renal involvement, cardiac manifestations, dermatological manifestations, ocular manifestations, Fatigue, neuropathy (in particular, burning extremity pain), cerebrovascular effects leading to an increased risk of stroke, tinnitus, vertigo, nausea, inability to gain weight, chemical imbalances, and diarrhea.
  • Fabry disease is a form of sphingolipidosis, as it involves dysfunctional metabolism of sphingolipids.
  • a deficiency of the enzyme alpha galactosidase A (a-GAL A, encoded by GLA) due to mutation causes a glycolipid known as globotriaosylceramide (abbreviated as Gb3, GL-3, or ceramide trihexoside) to accumulate within the blood vessels, other tissues, and organs. This accumulation leads to an impairment of their proper function.
  • a-GAL A alpha galactosidase A
  • Gb3, GL-3 globotriaosylceramide
  • ceramide trihexoside glycolipid known as globotriaosylceramide
  • Cardiac complications occur in Fabry disease subjects when glycolipids build up in different heart cells. Heart related effects worsen with age and may lead to increased risk of heart disease. Hypertension (high blood pressure) and cardiomyopathy are commonly observed.
  • cardiac cells of a Fabry disease subject are treated by administering a composition to affect calcium modulation in cardiac cells and restore calcium homeostasis.
  • the composition to affect calcium modulation comprises a calcium cycling modulator.
  • a calcium cycling modulator may operate in specific embodiments by preventing calcium overload or reducing calcium leak from the sarcoplasmic reticulum.
  • a calcium cycling modulator inhibits calcium channel activity.
  • calcium channel activity is selectively inhibited.
  • the calcium channel is an L-type calcium channel.
  • the calcium modulator that inhibits an L-type calcium channel is a non- dihydropyridine.
  • a dihydropyridine belongs to one of the phenylalkylamines or benzothiazepine groups.
  • the calcium cycling modulator is Verapamil, Gallopamil, Fendiline or Diltiazem.
  • the calcium cycling modulator is mibefradil, bepridil, fluspirilene, or fendiline.
  • Administration of drugs such as Verapamil, Gallopamil, Fendiline, Diltiazem, mibefradil, bepridil, fluspirilene, or fendiline may be at dosages, concentrations or schedules to achieve blood or plasma concentrations in a range of 1 -100, or 50-500, or 100-1000 ⁇ g/L in persons on therapy with the drug.
  • it will be desirable to have multiple administrations of the composition e.g., 2, 3, 4, 5, 6 or more administrations.
  • the administrations can be at 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9 ,10, 1 1 , 12 twelve day intervals, including all ranges there between.
  • a calcium cycling modulator inhibits calcium channel expression. Inhibition of calcium channel expression may be achieved by methods known to those of skill in the art. Particularly, RNA interference, antisense RNA, siRNA, gene editing are contemplated as methods to inhibit calcium channel expression. In still other aspects, a calcium cycling modulator inhibits calcium channel stability and leads to increased calcium channel turnover or degradation. In other aspects a calcium cycling modulator decreases the open probability of a calcium channel.
  • nucleic acid molecules identical or complementary to all or part of any L-type calcium channel, ryanodine 2 receptor or calsequestrin 2 may be employed in diagnostic, prognostic and therapeutic methods and compositions described herein.
  • nucleic acids can be labeled, used as probes, in array analysis, or employed in other diagnostic or prognostic applications, particularly those related to detecting L-type calcium channel, ryanodine 2 receptor overexpression or calsequestrin 2 underexpression and/or related Fabry disease cardiac symptoms or manifestations.
  • the expression of genes associated with Fabry disease cardiac symptoms or manifestations may be assayed or detected by methods used to detect and/or measure nucleic acid expression described below.
  • nucleic acids can be used as antisense or siR A molecules targeted at a L-type calcium channel or ryanodine 2 receptor gene for use in reducing expression of that gene.
  • reduction of expression provides inhibition of L-type calcium channel or ryanodine 2 receptor and accordingly activity of an L-type calcium channel or ryanodine 2 receptor.
  • These therapeutic nucleic acids may be modified to enhance their stability in storage or in vivo, bioavailability, activity, or localization.
  • the nucleic acids may have been endogenously produced by a cell, or been synthesized or produced chemically or recombinantly. They may be isolated and/or purified. Nucleic acids used in methods and compositions disclosed herein may have regions of identity or complementarity to another nucleic acid, such as a L-type calcium channel or ryanodine 2 receptor.
  • the region of complementarity or identity can be at least 5 contiguous residues, though it is specifically contemplated that the region is, is at least, or is at most 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 1 10, 120, 130
  • complementarity within a gene, gene transcript or between a gene target and a nucleic acid are such lengths.
  • the complementarity may be expressed as a percentage, meaning that the complementarity between a probe and its target is 90% or greater over the length of the probe.
  • complementarity is or is at least 90%, 95% or 100%, or any range derivable therein.
  • lengths may be applied to any nucleic acid comprising a nucleic acid sequence identified.
  • the commonly used name of the genes or gene targets is given throughout the application.
  • the sequence of a gene can be used to design the sequence of any probe, primer or siRNA molecule that is complementary or identical to a target L-type calcium channel or ryanodine 2 receptor gene identified herein.
  • nucleic acid may be derived from genomic sequences or a gene.
  • gene is used for simplicity to refer to the genomic sequence encoding the transcript for a given amino acid sequence.
  • embodiments may involve genomic sequences of a gene that are involved in its expression, such as a promoter or other regulatory sequences.
  • nucleic acid is well known in the art.
  • a “nucleic acid” as used herein will generally refer to a molecule (one or more strands) of DNA, RNA or a derivative or analog thereof, comprising a nucleobase.
  • a nucleobase includes, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., an adenine "A,” a guanine “G,” a thymine “T” or a cytosine “C”) or RNA (e.g., an A, a G, an uracil “U” or a C).
  • DNA e.g., an adenine "A,” a guanine "G,” a thymine “T” or a cytosine "C”
  • RNA e.g., an A, a G, an uracil "U” or a C.
  • nucleic acid encompasses the terms “oligonucleotide” and “polynucleotide,” each as a subgenus of the term “nucleic acid.”
  • hybridization As used herein, “hybridization”, “hybridizes” or “capable of hybridizing” is understood to mean the forming of a double or triple stranded molecule or a molecule with partial double or triple stranded nature.
  • anneal is synonymous with “hybridize.”
  • hybridization “hybridize(s)” or “capable of hybridizing” encompasses the terms “stringent condition(s)” or “high stringency” and the terms “low stringency” or “low stringency condition(s).”
  • stringent condition(s) or “high stringency” are those conditions that allow hybridization between or within one or more nucleic acid strand(s) containing complementary sequence(s), but preclude hybridization of random sequences. Stringent conditions tolerate little, if any, mismatch between a nucleic acid and a target strand. Such conditions are well known to those of ordinary skill in the art, and are preferred for applications requiring high selectivity. Non-limiting applications include isolating a nucleic acid, such as a gene or a nucleic acid segment thereof, or detecting at least one specific mRNA transcript or a nucleic acid segment thereof, and the like.
  • Stringent conditions may comprise low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.5 M NaCl at temperatures of about 42°C to about 70°C. It is understood that the temperature and ionic strength of a desired stringency are determined in part by the length of the particular nucleic acid(s), the length and nucleobase content of the target sequence(s), the charge composition of the nucleic acid(s), and to the presence or concentration of formamide, tetramethylammonium chloride or other solvent(s) in a hybridization mixture.
  • a nucleic acid may comprise, or be composed entirely of, a derivative or analog of a nucleobase, a nucleobase linker moiety and/or backbone moiety that may be present in a naturally occurring nucleic acid.
  • RNA with nucleic acid analogs may also be labeled according to methods disclosed herein.
  • a "derivative” refers to a chemically modified or altered form of a naturally occurring molecule, while the terms “mimic” or “analog” refer to a molecule that may or may not structurally resemble a naturally occurring molecule or moiety, but possesses similar functions.
  • a “moiety” generally refers to a smaller chemical or molecular component of a larger chemical or molecular structure. Nucleobase, nucleoside, and nucleotide analogs or derivatives are well known in the art, and have been described (see for example, Scheit, 1980, incorporated herein by reference).
  • nucleic acid molecules comprise at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, or at least 85% sequence complementarity to a target region within the target nucleic acid. In other embodiments, the molecules comprise at least 90% sequence complementarity to a target region within the target nucleic acid. In other embodiments, the molecules comprise at least 95% or at least 99% sequence complementarity to a target region within the target nucleic acid. For example, a nucleic acid molecule in which 18 of 20 nucleobases of it are complementary to a target sequence would represent 90 percent complementarity.
  • the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases.
  • an oligomeric compound that is 18 nucleobases in length having 4 noncomplementary nucleobases that are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid.
  • Percent complementarity of an oligomeric compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
  • a nucleic acid molecule is "targeted" to a molecule (that is a nucleic acid) in some embodiments. This means there is sufficient sequence complementarity to achieve a level of hybridization that accomplishes a particular goal with respect to the nucleic acid molecule, such as in the context of being a probe, a primer or an siRNA. In some embodiments, expression or function is to be modulated.
  • the targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the interaction to occur such that the desired effect, e.g., modulation of levels, expression or function, will result.
  • region is defined as a portion of the target nucleic acid having at least one identifiable sequence, structure, function, or characteristic.
  • segments Within regions of target nucleic acids are segments.
  • Segments are defined as smaller or sub- portions of regions within a target nucleic acid.
  • Sites as used in the present invention, are defined as specific positions within a target nucleic acid.
  • region, segment, and site can also be used to describe an oligomeric compound of the invention such as for example a gapped oligomeric compound having three separate segments.
  • Targets of the nucleic acid molecules described in embodiments include both coding and non-coding nucleic acid sequences.
  • the translation initiation codon is typically 5 -AUG (in transcribed mRNA molecules; 5 -ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the "AUG codon,” the “start codon” or the "AUG start codon.”
  • a minority of genes have a translation initiation codon having the RNA sequence 5'-GUG, 5 -UUG or 5'-CUG, and 5'- AUA, 5'-ACG and 5'-CUG have been shown to function in vivo.
  • translation initiation codon and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA transcribed from a gene encoding a nucleic acid target, regardless of the sequence(s) of such codons.
  • a translation termination codon (or "stop codon") of a gene may have one of three sequences, i.e., 5'-UAA, 5'-UAG and 5 -UGA (the corresponding DNA sequences are 5'- TAA, 5'-TAG and 5'-TGA, respectively).
  • a nucleic acid may be made by any technique known to one of ordinary skill in the art, such as for example, chemical synthesis, enzymatic production, or biological production. It is specifically contemplated that nucleic acid probes are chemically synthesized.
  • nucleic acids are recovered or isolated from a biological sample. The nucleic acids may be recombinant or it may be natural or endogenous to the cell (produced from the cell's genome). It is contemplated that a biological sample may be treated in a way so as to enhance the recovery of small RNA molecules such as mRNA or miR As.
  • U.S. Patent Application Serial No. 10/667,126 describes such methods and is specifically incorporated herein by reference.
  • nucleic acid synthesis is performed according to standard methods. See, for example, Itakura and Riggs (1980). Additionally, U.S. Patents 4,704,362, 5,221 ,619, and 5,583,013 each describe various methods of preparing synthetic nucleic acids.
  • Non-limiting examples of a synthetic nucleic acid include a nucleic acid made by in vitro chemical synthesis using phosphotriester, phosphite, or phosphoramidite chemistry and solid phase techniques such as described in EP 266,032, incorporated herein by reference, or via deoxynucleoside H-phosphonate intermediates as described by Froehler et al., 1986 and U.S. Patent 5,705,629, each incorporated herein by reference.
  • one or more oligonucleotide may be used.
  • Various different mechanisms of oligonucleotide synthesis have been disclosed in for example, U.S.
  • a non-limiting example of an enzymatically produced nucleic acid include one produced by enzymes in amplification reactions such as PCRTM (see for example, U.S. Patents 4,683,202 and 4,682,195, each incorporated herein by reference), or the synthesis of an oligonucleotide as described in U.S. Patent 5,645,897, incorporated herein by reference.
  • a non-limiting example of a biologically produced nucleic acid includes a recombinant nucleic acid produced (i.e., replicated) in a living cell, such as a recombinant DNA vector replicated in bacteria (see for example, Sambrook et al., 2001, incorporated herein by reference).
  • Oligonucleotide synthesis is well known to those of skill in the art. Various different mechanisms of oligonucleotide synthesis have been disclosed in for example, U.S. Patents 4,659,774, 4,816,571 , 5,141 ,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which is incorporated herein by reference.
  • chemical synthesis can be achieved by the diester method, the triester method, polynucleotide phosphorylase method, and by solid-phase chemistry.
  • the diester method was the first to be developed to a usable state, primarily by horana and co- workers. ( horana, 1979).
  • the basic step is the joining of two suitably protected deoxynucleotides to form a dideoxynucleotide containing a phosphodiester bond.
  • Polynucleotide phosphorylase method is an enzymatic method of DNA synthesis that can be used to synthesize many useful oligonucleotides (Gillam et al., 1978; Gillam et al., 1979). Under controlled conditions, polynucleotide phosphorylase adds predominantly a single nucleotide to a short oligonucleotide. Chromatographic purification allows the desired single adduct to be obtained. At least a trimer is required to start the procedure, and this primer must be obtained by some other method. The polynucleotide phosphorylase method works and has the advantage that the procedures involved are familiar to most biochemists.
  • Phosphoramidite chemistry (Beaucage and Lyer, 1992) has become the most widely used coupling chemistry for the synthesis of oligonucleotides.
  • Phosphoramidite synthesis of oligonucleotides involves activation of nucleoside phosphoramidite monomer precursors by reaction with an activating agent to form activated intermediates, followed by sequential addition of the activated intermediates to the growing oligonucleotide chain (generally anchored at one end to a suitable solid support) to form the oligonucleotide product.
  • Recombinant methods for producing nucleic acids in a cell are well known to those of skill in the art. These include the use of vectors (viral and non-viral), plasmids, cosmids, and other vehicles for delivering a nucleic acid to a cell, which may be the target cell (e.g., a cardiac cell) or simply a host cell (to produce large quantities of the desired RNA molecule). Alternatively, such vehicles can be used in the context of a cell free system so long as the reagents for generating the RNA molecule are present. Such methods include those described in Sambrook, 2003, Sambrook, 2001 and Sambrook, 1989, which are hereby incorporated by reference.
  • nucleic acid molecules are not synthetic.
  • the nucleic acid molecule has a chemical structure of a naturally occurring nucleic acid and a sequence of a naturally occurring nucleic acid.
  • non-synthetic nucleic acids may be generated chemically, such as by employing technology used for creating oligonucleotides.
  • Nucleic acids may be isolated using techniques well known to those of skill in the art, though in particular embodiments, methods for isolating small nucleic acid molecules, and/or isolating mRNA molecules can be employed. Chromatography is a process often used to separate or isolate nucleic acids from protein or from other nucleic acids. Such methods can involve electrophoresis with a gel matrix, filter columns, alcohol precipitation, and/or other chromatography.
  • methods generally involve lysing the cells with a chaotropic (e.g., guanidinium isothiocyanate) and/or detergent (e.g., N-lauroyl sarcosine) prior to implementing processes for isolating particular populations of RNA.
  • a chaotropic e.g., guanidinium isothiocyanate
  • detergent e.g., N-lauroyl sarcosine
  • a gel matrix is prepared using polyacrylamide, though agarose can also be used.
  • the gels may be graded by concentration or they may be uniform. Plates or tubing can be used to hold the gel matrix for electrophoresis. Usually one-dimensional electrophoresis is employed for the separation of nucleic acids. Plates are used to prepare a slab gel, while the tubing (glass or rubber, typically) can be used to prepare a tube gel.
  • the phrase "tube electrophoresis” refers to the use of a tube or tubing, instead of plates, to form the gel. Materials for implementing tube electrophoresis can be readily prepared by a person of skill in the art or purchased.
  • Methods may involve the use of organic solvents and/or alcohol to isolate nucleic acids, particularly RNA used in methods and compositions disclosed herein. Some embodiments are described in U.S. Patent Application Serial No. 10/667,126, which is hereby incorporated by reference. Generally, this disclosure provides methods for efficiently isolating small RNA molecules from cells comprising: adding an alcohol solution to a cell lysate and applying the alcohol/lysate mixture to a solid support before eluting the RNA molecules from the solid support. In some embodiments, the amount of alcohol added to a cell lysate achieves an alcohol concentration of about 55% to 60%. While different alcohols can be employed, ethanol works well.
  • RNA isolation processes include: a) lysing cells in the sample with a lysing solution comprising guanidinium, wherein a lysate with a concentration of at least about 1 M guanidinium is produced; b) extracting RNA molecules from the lysate with an extraction solution comprising phenol; c) adding to the lysate an alcohol solution for forming a lysate/alcohol mixture, wherein the concentration of alcohol in the mixture is between about 35% to about 70%; d) applying the lysate/alcohol mixture to a solid support; e) eluting the RNA molecules from the solid support with an ionic solution; and, f) capturing the RNA molecules.
  • the sample is dried down and resuspended in
  • siRNA Small interfering RNA
  • siRNAs are double-stranded RNA molecules, 20-25 base pairs in length.
  • siRNAs form part of the RNA interference (RNAi) pathway, where they interfere with the expression of specific genes with complementary nucleotide sequence. Any of the embodiments discussed above regarding nucleic acids may be implemented with respect to siRNA molecules.
  • Therapeutic siRNA may be administered to a patient to modulate the expression of one or more of the genes identified as involved in calcium homeostasis in cardiac cells related to Fabry disease.
  • siRNA may also target the expression of the spectrum of genes/gene products related to calcium homeostasis (e.g., L-type calcium channels, ryanodine 2 receptor).
  • the design of siRNA to target expression of a gene is a process well known in the art (Nat Biotechnol. 2004 Mar;22(3):326-30. Rational siRNA design for RNA interference. Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, hvorova A.).
  • the therapeutic siRNA modulates an L-type calcium channel gene selected from CACNA1 C, CACH2, CACN2, CACNL1 A1 , CCHL1A1 , CACNA1C, CACH2, CACN2, CACNL1 A1 , CCHL1 A1 , CACNA1F, CACNAF1, CACNA1 S, CACH1, CACN1 , CACNL1A3, CACNA1 D, CACH3, CACN4, CACNL1 A2, CCHL1A2, CACNB1 CACNLB1 , CACNB2, CACNLB2, MYSB, CACNB3, CACNLB3, CACNB4, CACNLB4, CACNA1 D, CACNA1D, CACNA1 S, CACNA1 C, CACNA1C, CACNA1C, CACNA1C, CACNA1C, CACNB4, CACNA1D, CACNB2, CACNA1 C, CACNB3, CACNB4, CACNB4, CACNB3, CACNB4, CACN
  • siRNA molecules may, but is not limited to, methodology described in paragraphs above.
  • nucleic acid molecules described above may be employed as siRNA molecules targeting a L-type calcium channel or ryanodine receptor such as ryanodine receptor 2.
  • a calcium cycling modulator targets a ryanodine receptor.
  • a calcium cycling modulator targets ryanodine receptor 2.
  • Ryanodine receptor 2 expression or activity may be targeted by therapeutic siRNA or therapeutic molecules that direct the knockdown of ryanodine receptor 2 through the RNA interference pathway.
  • Ryanodine receptor 2 activity may be therapeutically modulated by altering its phosphorylation state by drugs and phosphatases that generally or specifically target ryanodine receptor 2.
  • drugs or pharmacological modulators that decrease ryanodine receptor 2 open probability may be used to therapeutically inhibit ryanodine receptor 2 activity.
  • Pharmacological modulators may either stimulate or inhibit Ca2+ release, depending on concentration or incubation time, such concentration and exposure times are known to those of ordinary skill in the art.
  • Ryanodine receptor 2 pharmacological modulators include but are not limited to ryanoids, purine derivatives and related compounds, methylxanthines, carboline derivatives and carbazole derivatives, sulmazole, anthraquinones, digitalis glycosides, milrinone and other bipyridine derivatives, suramin, halogenated hydrocarbons and phenols, volatile anesthetics, phenol derivatives, hexachlorocyclohexane.
  • macrocyclic compounds immunosuppressant macrolides, bastadins, quinolidomicin al, heparin polyamines, ruthenium red, aminoglycosides, fla365, dantrolene, local anesthetics and phenylalkylamines.
  • Certain embodiments involve a calcium cycling modulator that is 1 ,4- benzothiazepine analogue JTV519 (K201), JTV519 analogue (SI 07), RyR2 blocking agent tetracaine or CaMK.II inhibitor KN-93.
  • the calcium cycling modulator is BAPTA or EDTA.
  • calcium homeostasis is treated in cardiac cell of a Fabry disease subject by increasing calcium buffering.
  • activity of calsequestrin-2 is increased or potentiated.
  • compositions are administered to a subject. Different aspects may involve administering an effective amount of a composition to a subject.
  • a composition comprising a calcium cycling modulator may be administered to the patient to treat Fabry disease. More specifically, regulation or homeostatsis of calcium treats cardiac manifestations associated with Fabry disease, including arrhythmias, cardiac hypertrophy and bradychardia.
  • an expression vector encoding one or more such nucleic acids, polypeptides or peptides may be given to a patient as a treatment.
  • such compositions can be administered in combination with traditional cardiac therapies to treat arrhythmias, cardiac hypertrophy and bradychardia.
  • Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • phrases "pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • the active compounds can be formulated for parenteral administration, e.g., formulated for injection via the mucosal, intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes.
  • parenteral administration e.g., formulated for injection via the mucosal, intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes.
  • such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • a proteinaceous compositions may be formulated into a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • a pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization or an equivalent procedure.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum- drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Administration of the compositions will typically be via any common route.
  • compositions that include physiologically acceptable carriers, buffers or other excipients.
  • unit dose refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and regimen.
  • the quantity to be administered both according to number of treatments and unit dose, depends on the protection desired.
  • compositions also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. [0089 J Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.
  • Administration of drugs such as Verapamil, Gallopamil, Fendiline, Diltiazem, mibefradil, bepridil, fluspirilene, or fendiline may be at dosages, concentrations or schedules to achieve blood or plasma concentrations in a range of 1-100, or 50-500, or 100-1000 ⁇ g/L in persons on therapy with the drug.
  • administrations of the composition e.g., 2, 3, 4, 5, 6 or more administrations.
  • the administrations can be at 1 , 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9 ,10, 1 1 , 12 twelve day intervals, including all ranges there between. Additionally, the administrations can be at 1 , 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9 ,10, 1 1 , 12 twelve week intervals, including all ranges there between.
  • compositions and related methods particularly administration of a therapy to restore calcium homeostasis that comprises inhibitors of L-type calcium channel or ryanodine receptor 2 expression or activity, a potentiator of PLN or CASQ2 activity, a RyR2 specific or general phosphatase, Ryr2 stabilizing agent may also be used in combination with the administration of Fabry disease enzyme replacement therapy (alpha-galctosidase A therapy or an enzyme that mimics alpha-galactosidase A activity such as agalsidase beta, also known as Fabryzme ®) or traditional therapies or drugs to treat arrhythmias, bradychardia, cardiac hypertrophy or other cardiac manifestations of Fabry disease.
  • Fabry disease enzyme replacement therapy alpha-galctosidase A therapy or an enzyme that mimics alpha-galactosidase A activity such as agalsidase beta, also known as Fabryzme ®
  • traditional therapies or drugs to treat arrhythm
  • An arrhythmia therapy may comprise any form of ablation, defibrillation or a device to treat arrhythmia.
  • ablation forms include radiofrequency ablation, transcatheter ablation or catheter ablation.
  • Devices contemplated to treat arrhythmias include implantable cardioverter defibrillators and pacemakers.
  • a treatment of cardiac hypertrophy may specifically treat ventricular hypertrophy, left ventricular hypertrophy, right ventricular hypertrophy or associate hypertension.
  • Examples of medications that may be used to treat ventricular hypertrophy an associated hypertension include but are not limited to Cozaar®(losartan potassium) and Hyzaar®(Losartan- hydrochlorothiazide).
  • Treatments for bradychardia may include epinephrine, dopamine, atropine, hyoscyamine sulfate, or Levsin®.
  • a therapy to restore calcium homeostasis includes inhibitors of L-type calcium channel or ryanodine receptor 2 expression or activity, potentiator of PLN or CASQ2 activity, RyR2 specific or general phosphatase, Ryr2 stabilizing agent that are used in conjunction with Fabry disease enzyme replacement therapy (alpha-galctosidase A therapy or an enzyme that mimics alpha-galactosidase A activity such as agalsidase beta, also known as Fabryzme ®) or traditional therapies or drugs to treat arrhythmias, bradychardia, cardiac hypertrophy or other cardiac manifestations of Fabry disease.
  • Fabry disease enzyme replacement therapy alpha-galctosidase A therapy or an enzyme that mimics alpha-galactosidase A activity such as agalsidase beta, also known as Fabryzme ®
  • the therapy may precede or follow the other agent treatment by intervals ranging from minutes to weeks.
  • the other agents, drugs and/or a proteins or polynucleotides are administered separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the therapeutic composition would still be able to exert an advantageously combined effect on the subject.
  • Various combinations of therapy may be employed, for example inhibitors of L-type calcium channel or RyR2 expression or activity, potentiator of PLN or CASQ2 activity, RyR2 specific or general phosphatase, Ryr2 stabilizing agent is "A” and traditional therapies or drugs to treat arrhythmias, bradychardia, cardiac hypertrophy or other cardiac manifestations of Fabry disease is "B":
  • Fabry disease is an X-linked lyosomal storage disorder, caused by insufficient activity of a-galactosidase A.
  • glycosphingolipids with terminal a-D-galactosyl moieties primarily globotriaosylceramide, accumulate in multiple organs.
  • Arrhythmias and cardiac hypertrophy are the most prominent cardiac manifestations of Fabry disease, which can cause sudden death and precipitate heart failure in the patients. The pathogenesis of arrhythmias in Fabry disease remains unclear.
  • iPSC induced pluripotent stem cells
  • iPSC Induced pluripotent stem cells
  • Fabry cardiomyocytes have slower beating rates than healthy controls, mimicking the clinical bradycardia that is present in these donor Fabry patients.
  • Treatment of Fabry cardiomyocytes with recombinant a-Gal A significantly increased the beating rates compared with mock-treated counterparts. This suggested that arrhythmia in Fabry disease is likely caused by intrinsic dysfunction of cardiomyocytes that iPSC-derived cardiomyocytes are a useful model for studying disease mechanism of arrhythmia in Fabry disease.
  • LTCC L-type calcium channel
  • CASQ2 calsequenstrin 2
  • Established iPSC clones are pluripotent by the assessment of their expression of pluripotent stem cell markers (Fig. 1) and teratoma formation assay (data not shown).
  • Fabry patient and healthy control iPSC were differentiated into spontaneously contracting cardiomyocytes in vitro by embryoid body (EB)-based cardiac differentiation method [4].
  • the spontaneous beating rates of cardiac clusters were measured and compared between Fabry patients and healthy controls at day 30 of differentiation.
  • Fig. 4 Abnormal expression of calcium handling proteins
  • LTCC Up-regulated L-type calcium channel
  • the electrical activities (action potential) of cardiomyocytes are mediated by the ion flows across the cell membranes.
  • Ion channels are proteins responsible for the gating of these ion flows.
  • the expression and function of these channel proteins is important for maintaining the normal electrical activities of cardiomyocytes.
  • the inventors studied the expression levels of major ion channels in iPSC-derived cardiomyocytes by quantitative RT-PCR and compared between Fabry patients and healthy controls. The inventors found the expression of LTCC, the cell membrane sensor the calcium cycling (Wehrens, et al, 2004), is significantly up- regulated in Fabry disease patient cardiomyocytes ( Figure 6).
  • the inventors further studied whether there is abnormal expression of other calcium handling proteins.
  • the inventors found significant down-regulated expression of CASQ2 at mRNA level, the most abundant calcium buffering protein in cardiomyocytes [6].
  • Treatment of arrhythmias Calcium handling abnormalities play a central role in the pathogenesis of a variety of heart conditions including arrhythmias, cardiomyopathies and heart failure (Yano, et al., 2008; Tomaselli, 2012). Targeting the molecules/pathways in the calcium cycling can be a novel effective treatment strategies for the arrhythmia in Fabry disease.
  • LTCC inhibitors such as Diltiazem, verapamil.
  • the use may be before cardiac arrhythmias occur.
  • Target molecules that prevent/reduce calcium leak in Fabry cardiac cells include RyR2 stabilizing molecules such as JTV-519 (Aetas Pharma Ltd. Japan) and/or over-expression of calcium buffering protein or drug of the sarcoplasmic reticulum such as CASQ2 by gene transfer.
  • RyR2 stabilizing molecules such as JTV-519 (Aetas Pharma Ltd. Japan) and/or over-expression of calcium buffering protein or drug of the sarcoplasmic reticulum such as CASQ2 by gene transfer.
  • RyR2 and SERCA2a receptors regulate SR Ca2+ release and uptake.
  • the inventors studied the protein expression levels of RyR2 receptor, SERCA2a and PLN in Fabry patient and healthy control iPSC-derived cardiac clusters by Western blot.
  • LTCC is the main entrance for Ca2+ influx in cardiac cells and determines the activity of the whole heart. Changes in Ca2+ influx balance in the cardiac cells are directly related to human and animal cardiac diseases, including arrhythmia and cardiac hypertrophy (Mukherjee et al.).
  • the inventors observed increased mRNA expression of LTCC in Fabry patient-derived cardiac cells and heart tissues of Fabry mice, treated Fabry patients' cardiac clusters with a LTCC inhibitor, verapamil, and evaluated its effect on decreasing beating rates. At higher doses verapamil decreased beating rates in both Fabry patients' and healthy controls' cells (Fig. 9b). However, at a low dose (10e-9 M) the inhibitor significantly increased the beating rates of Fabry cardiomyocytes. The beating rate of normal control cells remained unchanged at these low verapamil concentrations. (Fig. 9).
  • the inventors identified abnormal SR Ca2+ release and uptake, upregulated LTCC expression, hyperphosphorylation of RyR2, decreased total PLN in Fabry patient iPSC-derived cardiomyocytes.
  • abnormal Ca2+ handling underlies the pathogenesis of Fabry heart disease and is a therapeutic target for treatment of arrhythmia in Fabry disease.
  • LTCC inhibitor verapamil improved beating rates of Fabry patient-derived cardiac clusters, suggesting that targeting Ca2+ cycling is effective in improving arrhythmia in Fabry disease and possibly other Fabry cardiac manifestations such as hypertrophic cardiomyopathy and vascular disease.

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Abstract

La présente invention concerne des procédés et des compositions pour traiter une arythmie chez des patients atteints de la maladie de Fabry en utilisant des modulateurs de cyclisation du calcium. La maladie de Fabry est un trouble de surcharge lysosomale liée à l'X, causée par une activité insuffisante d'une β-galactosidase A (a-Gal A). En conséquence de la déficience enzymatique, des glycosphingolipides (GSL) avec des fragments a-D-galactosyle terminaux, principalement globotriaosylcéramide (Gb3), s'accumulent dans des organes multiples (Brady, et al., 1967).
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