WO2012017105A1 - Microrna that can be used for treating arrhythmogenic channelopathies - Google Patents

Microrna that can be used for treating arrhythmogenic channelopathies Download PDF

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Publication number
WO2012017105A1
WO2012017105A1 PCT/ES2011/000248 ES2011000248W WO2012017105A1 WO 2012017105 A1 WO2012017105 A1 WO 2012017105A1 ES 2011000248 W ES2011000248 W ES 2011000248W WO 2012017105 A1 WO2012017105 A1 WO 2012017105A1
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seq
arrhythmogenic
polynucleotide
sequence
mir
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PCT/ES2011/000248
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Spanish (es)
French (fr)
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Diego Franco Jaime
Amelia Eva Aranega Jimenez
Houría DAIMI
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Universidad De Jaen
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical

Definitions

  • the present invention falls within the field of gene therapy and cardiology and specifically refers to the use of the hsa-mir-219-5p microRNA, of SEQ ID NO: 1, of the modified polynucleotides derived therefrom, SEQ ID NO: 3 or SEQ ID NO: 5, or of its precursors, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, for the preparation of useful medicines for the treatment of arrhythmogenic canalopathies, preferably those that are due to alterations in the function of the cardiac sodium channel, more preferably of type 3 long QT syndrome and Brugada syndrome.
  • Ventricular arrhythmias are the most frequent cause of sudden death in developed societies and are usually related to the existence of ischemic heart disease, of which they may be the first manifestation in hitherto asymptomatic subjects. Less frequently it occurs in subjects without structural heart disease and younger. Advances in molecular biology in the last decade have allowed the identification of several clinical entities that share their origin in mutations in genes that code for subunits of ion channels, which are responsible for generating the potential for action of heart cells. This set of inherited diseases are called arrhythmogenic canalopathies. In recent years, much progress has been made in the knowledge of the genetic and functional basis of different arrhythmogenic syndromes, including Brugada syndrome and long QT syndrome.
  • Brugada syndrome is a canalopathy associated with abnormalities in the sodium channels, specifically mutations in the SCN5A gene that codes for the transmembrane protein Nav1 .5, which in all cases cause a lack of function of this channel and usually occur associated with polymorphic ventricular arrhythmias in subjects without heart disease. These subjects they have a characteristic electrocardiogram: ST segment elevation in the right precordial leads and blocking pattern of the right bundle of the His bundle, usually with normal QT.
  • microRNAs are small-sized single-stranded RNA molecules (approximately 22 nucleotides) and non-coding, which bind to specific sequences of the non-coding regions (mainly the 3 ' UTRs) of the target messenger RNAs, inhibiting their translation and / or destabilizing them, which also causes a decrease in the total amount of protein.
  • target transcripts encoding cardiac ion channel proteins for example, microRNA-195 and microRNA-1 act on the expression of the SCN5A gene (Baofeng Yang, et al., 2008, Cardiovascular Research, Vol. 79: 571-580).
  • the present invention proposes the use of the hsa-mir-219-5p microRNA, of SEQ ID NO: 1, of the modified polynucleotides derived therefrom, SEQ ID NO: 3 or SEQ ID NO: 5, or of its precursors, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, for the preparation of medicines useful for the treatment of arrhythmogenic canalopathies, preferably those that are due to alterations in the function of the cardiac sodium channel, more preferably of syndrome of long QT type 3 and Brugada syndrome.
  • the small size of the microRNAs their easy manipulation, the bioavailability of their precursors (pre-miR or pre-microRNA) and their high thermodynamic stability, make them molecules with a high therapeutic potential.
  • the conjugation of the polynucleotides of the invention with, for example, but not limited to lipid residues, allows their bioavailability in the organism of the patient to which they are administered.
  • polynucleotide sequences of the invention or polynucleotides of the invention allow modulating the function of the cardiac sodium channel through its binding to the mRNA encoding the Nav1.5 protein, or in the case of precursors, providing the polynucleotide that binds to this mRNA, which gives the possibility of increase the function of the sodium channel in patients with, for example, but not limited to, Brugada syndrome, or decrease it in patients with, for example, but not limited to, LQT3 syndrome, and thus reverse the altered biological function in these pathologies cardiac, as opposed to current biomechanical or pharmacological methods that only partially mitigate the symptoms of these diseases.
  • the inventors demonstrate that the hsa-mir-219-5p microRNA, or miR-219a, of SEQ ID NO: 1, is capable of reducing the expression of the SCN5A gene, which codes for the Nav1.5 transmembrane protein responsible for the potential of action in cardiac muscle cells.
  • SCN5A gene which codes for the Nav1.5 transmembrane protein responsible for the potential of action in cardiac muscle cells.
  • In vitro analysis of this microRNA shows that its overexpression decreases the expression of the SCN5A gene up to 50% in atrial cardiac muscle cells ( Figure 3), thus reducing its capacity and contractile rhythm.
  • the present invention proposes the use of this microRNA for the preparation of medicaments for the treatment of arrhythmogenic canalopathies, preferably of those arrhythmogenic canalopathies whose cause is alterations in the sodium channels, more preferably when the alterations in said channels are due to function gain mutations in the SCN5A gene, such as, but not limited to, the LQT3 syndrome.
  • the three nucleotides (CAA) adjacent to the 3 " end of the GAUUGUC sequence or complementary sequence to the mRNA of the SCN5A gene comprised in SEQ ID NO: 1, have been replaced by three other nucleotides (AGC), of so that SEQ ID NO: 3 has been obtained, a polynucleotide that has at least 85% identity with SEQ ID NO: 1 and that comprises three complementary sites more than this sequence with the mRNA of the SCN5A gene, so it has a greater binding capacity to it than the sequence of the native microRNA or SEQ ID NO: 1.
  • SEQ ID NO: 3 is also useful for the preparation of medicaments for the treatment of arrhythmogenic canalopathies, preferably of those arrhythmogenic canalopathies whose cause they are alterations in the sodium channels, more preferably when the alterations in said channels are due to function gain mutations in this gene, such as, but not limited to, the sin drome of LQT3.
  • a first aspect of the invention relates to the use of an isolated polynucleotide, hereinafter "polynucleotide of the invention", which comprises a nucleotide sequence with at least 60%, preferably with at least 75%, more preferably with at least 80% and even more preferably with at least 85% identity with respect to the full length of the sequence SEQ ID NO: 1 for the preparation of a medicament, or alternatively, to an isolated polynucleotide comprising a nucleotide sequence with at least 60%, preferably with at least 75%, more preferably with at least 80% and even more preferably with at least 85% identity with respect to the full length of the sequence SEQ ID NO: 1 for use as a medicine.
  • polynucleotide of the invention which comprises a nucleotide sequence with at least 60%, preferably with at least 75%, more preferably with at least 80% and even more preferably with at least 85% identity with respect to the full length of the sequence SEQ ID NO: 1 for use as
  • nucleotide sequence refers to a polymeric form of nucleotides of any length that may or may not be, chemically or biochemically modified. Thus, they refer to any polyiribonucleotide or polydeoxyribonucleotide , both single chain and double strand.
  • the polynucleotide of the invention can be obtained artificially by conventional cloning and selection methods, or by sequencing.
  • the polynucleotide of the invention comprises the nucleotide sequence SEQ ID NO: 3, which has at least 85% identity with respect to the full length of SEQ ID NO: 1.
  • the polynucleotide of the invention is SEQ ID NO: 4, a precursor nucleotide sequence of SEQ ID NO: 3 comprising this sequence.
  • SEQ ID NO: 5 a polynucleotide, SEQ ID NO: 5, which has at least 85% identity with SEQ ID NO: 1 and comprising three complementary sites less than this sequence with the mRNA of the SCN5A gene, so it has a lower binding capacity than the sequence of the native microRNA or SEQ ID NO: 1.
  • the SEQ ID NO: 5 is also useful for the preparation of drugs for the treatment of arrhythmogenic canalopathies, preferably of those arrhythmogenic canalopathies whose cause is alterations in the sodium channels, more preferably when the Alterations in these channels are due to function gain mutations in this gene, such as, but not limited to, LQT3 syndrome, since, although this sequence has fewer complementary sites than SEQ ID NO: 1 with the target mRNA It is also able to bind to said mRNA and reduce its function;
  • These medications may be useful, for example, but not limited to, in patients with LQT3 syndrome in which there is a deficiency in the expression of the native microRNA.
  • the polynucleotide of the invention comprises the nucleotide sequence SEQ ID NO: 5, which has at least 85% identity with respect to the full length of SEQ ID NO: 1.
  • the polynucleotide of the invention is SEQ ID NO: 6, a precursor nucleotide sequence of SEQ ID NO: 5 comprising this sequence.
  • the polynucleotide of the invention comprises a nucleotide sequence with at least 90% identity with respect to the full length of the sequence SEQ ID NO: 1.
  • the polynucleotide of the invention comprises a nucleotide sequence with at least 95% of identity with respect to the full length of the sequence SEQ ID NO: 1.
  • the polynucleotide of the invention comprises a nucleotide sequence with at least 98% identity with respect to the full length of the sequence SEQ ID NO: 1.
  • the polynucleotide of the invention comprises the nucleotide sequence SEQ ID NO: 1.
  • the polynucleotide of the invention is SEQ ID NO: 2, a precursor nucleotide sequence of SEQ ID NO: 1 comprising this sequence. Also within the scope of this invention are precursor nucleotide sequences or precursors.
  • precursor or “precursor sequence” as used herein includes any nucleotide sequence that when processed by, for example, but not limited to, enzymatic cleavage, is capable of providing a polynucleotide with at least 60%, preferably with at least 65%, more preferably with at least 70%, with at least 75%, with at least 80%, with at least 85%, with at least 90%, with at least 95% or with at least 98% identity with respect to the full length of SEQ ID NO: 1, or that it is capable of providing a sequence polynucleotide of SEQ ID NO: 1; or that is capable of providing a sequence polynucleotide SEQ ID NO: 3; or that is capable of providing a polynucleotide of sequence SEQ ID NO: 5.
  • said precursor is a nucleotide sequence that increases the bioavailability of the polynucleotides that it provides when administered to an individual or that enhances their release in a compartment biological.
  • the precursor nucleotide sequences of the invention are SEQ ID NO: 2, a sequence that will also be referred to as pre-miR-219a and that is a precursor to SEQ ID NO: 1; SEQ ID NO: 4, precursor sequence of SEQ ID NO: 3; and SEQ ID NO: 6, precursor sequence of SEQ ID NO: 5. All of these precursor nucleotide sequences comprise polynucleotides SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, respectively.
  • the medicaments and pharmaceutical compositions of the invention are useful for the treatment of arrhythmogenic canalopathies, preferably, for the treatment of arrhythmogenic canalopathies that are due to alterations in the sodium channels, since the polynucleotide of the invention is capable of reducing the expression of the SCN5A gene that codes for a transmembrane protein of sodium channels, so it can be used in those arrhythmogenic canalopathies due to alterations in function gain in these channels.
  • the polynucleotide of the invention comprising the nucleotide sequence SEQ ID NO: 5 or that of SEQ ID NO: 6, has a lower complementarity with the mRNA of this gene than the native microRNA, so competing with the latter for said Binding is able to increase the levels of mRNAs of this gene, compared to the native microRNA, so it is useful for the treatment of arrhythmogenic canalopathies due to impaired loss of function in the sodium channels.
  • another aspect of the invention relates to the use of the polynucleotide of the invention for the preparation of a medicament for the treatment of arrhythmogenic canalopathies, or alternatively, to the polynucleotide of the invention for use as a medicament for the treatment of arrhythmogenic canalopathies.
  • arrhythmogenic canalopathies are due to alterations in the sodium channels.
  • the arrhythmogenic canalopathy that is due to alterations in the sodium channels is the long QT syndrome type 3.
  • Another aspect of the invention relates to the use of the polynucleotide of the invention comprising the nucleotide sequence SEQ ID.
  • medication refers to any substance used for the prevention, diagnosis, relief, treatment or cure of diseases in man and women. animals. In the context of the present invention it refers to a preparation comprising at least the polynucleotide of the invention.
  • the polynucleotide of the invention is formulated in an appropriate pharmaceutical composition, in the therapeutically effective amount, preferably together with one or more pharmaceutically acceptable carriers, adjuvants or excipients.
  • treatment is to combat the effects caused as a consequence of arrhythmogenic canalopathies, preferably, of arrhythmogenic canalopathies that are due to alterations in the sodium channels, more preferably of the QT syndrome long type 3 or Brugada syndrome, to stabilize the condition of individuals or prevent further damage.
  • prevention is to prevent the occurrence of damage whose cause are arrhythmogenic canalopathies, preferably, arrhythmogenic canalopathies that are due to alterations in the sodium channels, more preferably the syndrome of Long QT type 3 or Brugada syndrome.
  • Arrhythmogenic canalopathy is understood as any genetic cardiomyopathy (or impaired myocardial function) that results from mutations in the genes responsible for the proper functioning of the ion channels that generate the potential for transmembrane action, which leads to defects in function ( gain or loss) of said channels, physiological alterations of the duration of the potential for transmembrane action and / or predisposition to develop ventricular arrhythmias in the absence of structural heart disease.
  • arrhythmogenic canalopathies include, but are not limited to, Brugada syndrome, long QT syndrome, short QT syndrome or catecholaminergic polymorphic ventricular tachycardia.
  • Arrhythmogenic canalopathies can be diagnosed, for example, but not limited to, as described in Cabrera Ortega M., et al., 2009, Revista Cubana Pediatr ⁇ a, 81 (4).
  • "Arrhythmogenic canalopathies due to alterations in sodium channels” are those canalopathies whose genetic basis is found in mutations (loss or gain of function) in at least one of the genes that code for said channels, preferably, in the SCN5A gene of SEQ ID NO: 7.
  • the detection of this type of canalopathies can be performed by, for example, but not limited to, an electrocardiogram, by means of which it is possible to make a potential diagnosis of the altered currents (Antzelevitch et al.
  • Brugada syndrome is characterized by the probability of presenting syncopal episodes or cardiac arrest caused by polymorphic ventricular tachycardia or ventricular fibrillation during rest or sleep, with an electrocardiographic pattern of apparent right bundle branch block and supra-level ST segment that falls slowly and ends on a negative T wave in V1, V2 and V3, without depression of the opposite leads. Genetically they have identified seven types of Brugada syndrome, the most frequent are, but not limited to, Brugada syndrome type 1 or Brugada syndrome type 2.
  • first pharmaceutical composition of the invention which comprises the polynucleotide of the invention comprising a nucleotide sequence with at least 60%, preferably with at least 65%, more preferably with at least 70%, with at least 75% , with at least 80%, with at least 85%, with at least 90%, with at least 95% or with at least 98% identity with respect to the full length of the sequence SEQ ID NO: 1, or comprising the nucleotide sequence SEQ ID NO: 1, or of SEQ ID NO: 2, or comprising the nucleotide sequence SEQ ID NO: 3 or of SEQ ID NO: 4.
  • the first pharmaceutical composition of the invention further comprises another active ingredient.
  • the first pharmaceutical composition of the invention further comprises a pharmaceutically acceptable carrier.
  • second pharmaceutical composition of the invention comprising the polynucleotide of the invention comprising the nucleotide sequence SEQ ID NO: 5 or SEQ ID NO: 6.
  • the second pharmaceutical composition of the invention further comprises another active ingredient.
  • the second pharmaceutical composition of the invention further comprises a pharmaceutically acceptable carrier.
  • active substance As used herein, the term “active substance”, “active substance”, “pharmaceutically active substance”, “active ingredient” or “ingredient pharmaceutically active” “means any component that potentially provides a pharmacological activity or other effect different in the diagnosis, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals.
  • Adjuvants and “pharmaceutically acceptable carriers” refer to those substances, or combination of substances, known in the pharmaceutical sector, used in the preparation of pharmaceutical forms of administration and include, but are not limited to, solids, liquids, solvents or surfactants. .
  • Pharmaceutically acceptable carriers that can be used in the present invention are vehicles known in the state of the art, such as, but not limited to, lipid residues.
  • compositions and medicaments of the present invention can be used in a method of treatment or prevention in isolation or in conjunction with other pharmaceutical compounds intended for the treatment or prevention of arrhythmogenic canalopathies, preferably, of arrhythmogenic canalopathies that are due to alterations in the sodium channels, more preferably, of type 3 long QT syndrome or Brugada syndrome.
  • the pharmaceutical compositions of the present invention can be formulated for administration in a variety of ways known in the state of the art.
  • compositions and / or their formulations can be administered to an animal, including a mammal and, therefore, to man, in a variety of ways, including, but not limited to, parenteral, intraperitoneal, intravenous, intradermal, intraarticular, intrasynovial, intralesional, intraarterial, intracardiac, intramuscular, intranasal, subcutaneous, intracapsular, topical, by transdermal patches, by percutaneous administration, surgical implant, internal surgical painting, infusion pump or catheter.
  • the dosage to obtain a therapeutically effective amount depends on a variety of factors, such as the age, weight, sex or tolerance of the individual.
  • the term "therapeutically effective amount” refers to the amount of the pharmaceutical compositions of the invention that produces the desired effect and, in general, will be determined, among other causes, by the characteristics of said pharmaceutical compositions and the therapeutic effect to be achieved.
  • the medicament is for the treatment of arrhythmogenic canalopathies.
  • arrhythmogenic canalopathies are due to alterations in the sodium channels.
  • arrhythmogenic canalopathy due to alterations in sodium channels is the long QT syndrome type 3.
  • Another aspect of the invention relates to the use of the second pharmaceutical composition of the invention for the preparation of a medicament for the treatment of Brugada syndrome, or alternatively, to the second pharmaceutical composition of the invention for use as a medicament for the treatment. of Brugada syndrome.
  • Fig. 1 Shows the complementarity scheme of hsa-mir-219-5p (SEQ ID NO: 1) with the 3 UTR region of the SCN5A gene messenger.
  • Upper sequence 3 ' UTR region of the messenger of the SCN5A gene (position 1 .416-1 .422, 5 X - 3 " ).
  • Lower sequence sequence of hsa-mir-219-5p (SEQ ID NO: 1, 3 " - 5 " ) with the sequence complementary to the messenger of the SCN5A gene (GAUUGUC) marked in white.
  • Black bars complementary nucleotides.
  • Fig. 2. Shows the sequence of the mature hsa-mir-219-5p and its precursor.
  • Fig. 4 Shows the results of immunohistochemical tests against the expression product of the SCN5A gene in control cells (panel left) and in cells that were transfected with hsa-mir-219-5p (right panel).
  • the left panel shows the normal protein expression of Nav1 .5 (SCN5A) in the cardiomyocytic cell.
  • Nav1 .5 appears to be sequestered in the Golgi apparatus, and therefore is very poorly represented in the cytoplasmic membrane, where it normally exerts its sodium ion transport function.
  • Fig. 5 It shows an illustrative scheme of the strategy for the treatment of long QT syndrome type 3 where there is gain of function of the SCN5A gene.
  • A. Upper sequence: 3 ' UTR region of the messenger of the SCN5A gene (position 1 .416-1 .422, 5 " - 3 " ).
  • the box in the lower sequence shows the three nucleotides that have been replaced by the nucleotides shown in the lower part to give rise to SEQ ID NO: 3. The complementarity between both upper and lower sequences (bars) is shown.
  • the gray bars represent the new complementary sites created to give rise to SEQ ID NO: 3.
  • B Shows the precursor sequence of SEQ ID NO: 1, pre-miR-219a, of SEQ ID NO: 2, where the mature sequence of hsa-mir-219-5p (SEQ ID NO: 1) stands out in gray in the two complementary strands of the pre-miR-219a, in a box the three modified nucleotides are indicated to design SEQ ID NO: 3
  • C Shows SEQ ID NO: 3 (in bold) included in its precursor sequence or SEQ ID NO: 4. In gray, the modified nucleotides are highlighted.
  • Fig. 6. It shows an illustrative scheme of the strategy for the treatment of Brugada syndrome where there is loss of function of the SCN5A gene. TO.
  • Upper sequence 3 ' UTR region of the messenger of the SCN5A gene (position 1 .416-1 .422, 5 N - 3 " ).
  • Lower sequence sequence of hsa-mir-219-5p (SEQ ID NO: 1, 3 ' - 5 ' ) with the sequence complementary to the messenger of the SCN5A gene (GAUUGUC) marked in white.
  • the boxes in the lower sequence show the three nucleotides that have been replaced by the nucleotides that are shown at the bottom to give rise to SEQ ID NO: 5. The complementarity between both upper and lower sequences (bars) is shown.
  • FIG. 7 Shows the expression of the SCN5A gene in atrial myocardial cells (HL-1) transfected with SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 for 12 hours.
  • Control cells where the precursor of endogenous hsa-mir-219-5p or miR-219a (SEQ ID NO: 2) was transfected.
  • miR-219a- Cells transfected with the precursor of miR-219a- (SEQ ID NO: 4), designed to treat arrhythmogenic canalopathies due to a gain in function in the SCN5A gene.
  • miR-219a + Cells transfected with the precursor of miR-219 + (SEQ ID NO: 6), designed to treat arrhythmogenic canalopathies due to a loss of function in the SCN5A gene.
  • the figure shows that HL-1 cells transfected with SEQ ID NO: 4 show a decrease in SCN5A expression compared to cells transfected with the endogenous precursor of miR-219a (SEQ ID NO: 2), while those Myocardial cells transfected with SEQ ID NO: 6 show increased expression of SCN5A compared to cells transfected with the endogenous precursor of miR-219a (SEQ ID NO: 2).
  • EXAMPLE 1 Effect of the expression of hsa-mir-219-5p on the expression of the SCN5A gene in atrial myocardial cells.
  • the hsa-mir-219-5p (SEQ ID NO: 1) is capable of binding to the 3 ' UTR region of the mRNA of the SCN5A gene as shown in Figure 1, in particular by the region corresponding to the GAUUGUC sequence included in SEQ ID NO: 1 (marked in bold in Figure 2A).
  • This hsa-mir-219-5p is included in the sequence of its precursor or pre-miR-219a (SEQ ID NO: 2) as seen in Figures 2B and 2C.
  • hsa-mir-219-5p conditioned the contractile capacity of cardiomyocytes in culture.
  • contractions of control cardiomyocytes and cardiomyocytes transfected with hsa-mir-219-5p were counted, and it was observed that the rate was decreased by approximately 30% in the latter, and that the contraction pattern was asynchronous (Table 1). Therefore, these data revealed that hsa-mir-219-5p can regulate the function of the sodium channel, which is very important since it implies a molecular mechanism of easy manipulation and high accessibility that allows to correct the lack or gain of function of the Cardiac sodium channel underlying arrhythmogenic syndromes such as Brugada or long QT, respectively.

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Abstract

The present invention proposes the use of hsa-mir-219-5ρ microRNA, of SEQ ID NO: 1, of the modified polynucleotides derived therefrom, SEQ ID NO: 3 or SEQ ID NO: 5, or of the precursors thereof, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, for producing drugs that can be used to treat arrhythmogenic channelopathies, preferably those caused by changes in cardiac sodium channel function, more preferably long QT syndrome type 3 and Brugada syndrome. These polynucleotides are capable of modulating cardiac sodium channel function by means of the binding thereof to the mRNA of the SCN5A gene.

Description

MicroRNA útil para el tratamiento de canalopatías arritmogénicas  MicroRNA useful for the treatment of arrhythmogenic canalopathies
La presente invención se encuadra en el campo de la terapia génica y la cardiología y específicamente se refiere al uso del microRNA hsa-mir-219-5p, de SEQ ID NO: 1 , de los polinucleótidos modificados derivados de él, SEQ ID NO: 3 o SEQ ID NO: 5, o de sus precursores, SEQ ID NO: 2, SEQ ID NO: 4 o SEQ ID NO: 6, para la elaboración de medicamentos útiles para el tratamiento de canalopatías arritmogénicas, preferiblemente de aquellas que se deben a alteraciones en la función del canal de sodio cardíaco, más preferiblemente de síndrome de QT largo tipo 3 y de síndrome de Brugada. The present invention falls within the field of gene therapy and cardiology and specifically refers to the use of the hsa-mir-219-5p microRNA, of SEQ ID NO: 1, of the modified polynucleotides derived therefrom, SEQ ID NO: 3 or SEQ ID NO: 5, or of its precursors, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, for the preparation of useful medicines for the treatment of arrhythmogenic canalopathies, preferably those that are due to alterations in the function of the cardiac sodium channel, more preferably of type 3 long QT syndrome and Brugada syndrome.
ESTADO DE LA TÉCNICA ANTERIOR STATE OF THE PREVIOUS TECHNIQUE
Las arritmias ventriculares constituyen la causa más frecuente de muerte súbita en las sociedades desarrolladas y suelen relacionarse con la existencia de cardiopatía isquémica, de la que pueden ser la primera manifestación en sujetos hasta entonces asintomáticos. Con menor frecuencia ocurre en sujetos sin cardiopatía estructural y más jóvenes. Los avances de la biología molecular en la última década han permitido identificar diversas entidades clínicas que comparten su origen en mutaciones en genes que codifican para subunidades de canales iónicos, los cuales son responsables de la generación del potencial de acción de las células cardíacas. Este conjunto de enfermedades hereditarias se denominan canalopatías arritmogénicas. En los últimos años, se ha avanzado mucho en el conocimiento de la base genética y funcional de distintos síndromes arritmogénicos, entre los cuales cabe destacar el síndrome de Brugada y el síndrome de QT largo. Ventricular arrhythmias are the most frequent cause of sudden death in developed societies and are usually related to the existence of ischemic heart disease, of which they may be the first manifestation in hitherto asymptomatic subjects. Less frequently it occurs in subjects without structural heart disease and younger. Advances in molecular biology in the last decade have allowed the identification of several clinical entities that share their origin in mutations in genes that code for subunits of ion channels, which are responsible for generating the potential for action of heart cells. This set of inherited diseases are called arrhythmogenic canalopathies. In recent years, much progress has been made in the knowledge of the genetic and functional basis of different arrhythmogenic syndromes, including Brugada syndrome and long QT syndrome.
El síndrome de Brugada es una canalopatía asociada a anomalías en los canales de sodio, concretamente a mutaciones en el gen SCN5A que codifica para la proteína transmembrana Nav1 .5, las cuales provocan en todos los casos una falta de función de dicho canal y suele presentarse asociada a arritmias ventriculares polimórficas en sujetos sin cardiopatía. Estos sujetos presentan un electrocardiograma característico: elevación del segmento ST en las derivaciones precordiales derechas y patrón de bloqueo de la rama derecha del haz de His, generalmente con QT normal. Por otro lado, la base genética del síndrome de QT largo tipo 3 ó LQT3 es más heterogénea, aunque hay que resaltar que esta patología también presenta alteraciones moleculares en los canales de sodio debidas a mutaciones en el gen SCN5A, que provocan en este caso una ganancia de función de dicho canal (Jeffrey E. Saffitz, 2005, Circulation, 1 12:3672-3674). En la actualidad existen tratamientos farmacológicos, como los agentes beta- bloqueantes (Riera A. R., et al., 2007, Cardiology Journal, 14(1 ):97-106), que permiten modular este tipo de patologías arritmogénicas, si bien tienen una alta variabilidad en su efectividad en función del paciente tratado (Michowitz Y., et al., 2009, Heart Rhythm, 6(7): 1047-1049) y además ejercen un efecto sistémico en él, encaminado a paliar pero no a restituir la fisiopatología cardíaca. Brugada syndrome is a canalopathy associated with abnormalities in the sodium channels, specifically mutations in the SCN5A gene that codes for the transmembrane protein Nav1 .5, which in all cases cause a lack of function of this channel and usually occur associated with polymorphic ventricular arrhythmias in subjects without heart disease. These subjects they have a characteristic electrocardiogram: ST segment elevation in the right precordial leads and blocking pattern of the right bundle of the His bundle, usually with normal QT. On the other hand, the genetic basis of long QT syndrome type 3 or LQT3 is more heterogeneous, although it should be noted that this pathology also presents molecular alterations in sodium channels due to mutations in the SCN5A gene, which in this case cause gain of function of said channel (Jeffrey E. Saffitz, 2005, Circulation, 1 12: 3672-3674). Currently there are pharmacological treatments, such as beta-blocking agents (Riera AR, et al., 2007, Cardiology Journal, 14 (1): 97-106), which allow modulating this type of arrhythmogenic pathologies, although they have a high variability in its effectiveness depending on the patient treated (Michowitz Y., et al., 2009, Heart Rhythm, 6 (7): 1047-1049) and also exert a systemic effect on it, aimed at alleviating but not restoring the pathophysiology cardiac
Una alterativa a los tratamientos farmacológicos es la implantación de un cardiovertor (ICD), sin embargo, este mecanismo, además de suponer un elevado coste médico, está contraindicado en pacientes con alto riesgo de sufrir episodios de muerte súbita (Sherrid M. V., et al., 2008, Progress in Cardiovascular Diseases, 51 (3):237-63). An alternative to pharmacological treatments is the implantation of a cardioverter (ICD), however, this mechanism, in addition to assuming a high medical cost, is contraindicated in patients at high risk of sudden death episodes (Sherrid MV, et al. , 2008, Progress in Cardiovascular Diseases, 51 (3): 237-63).
Otra posibilidad para el tratamiento de canalopatías arritmogénicas relacionadas con disfunciones en los canales de sodio es el uso de polipéptidos codificados por polinucleótidos que comprenden la secuencia del gen SCN5A con mutaciones que están presentes en los individuos sanos (US2008214457 A1 ). Another possibility for the treatment of arrhythmogenic canalopathies related to dysfunctions in the sodium channels is the use of polypeptides encoded by polynucleotides that comprise the sequence of the SCN5A gene with mutations that are present in healthy individuals (US2008214457 A1).
Se sabe que algunas mutaciones en el gen SCN5A conducen a una disminución en la conductancia del canal de sodio, por lo que esta aproximación se plantea como otra posibilidad para controlar las arritmias cardíacas cuya base molecular se encuentre en alteraciones de ganancia de función en los canales de sodio (G. Alex Papadatos, et al., 2002, PNAS, Vol. 99(9): 6210-6215). It is known that some mutations in the SCN5A gene lead to a decrease in the conductance of the sodium channel, so this approach is considered as another possibility to control cardiac arrhythmias whose molecular basis is in alterations of gain of function in sodium channels (G. Alex Papadatos, et al., 2002, PNAS, Vol. 99 (9): 6210-6215).
Por otro lado, los microRNAs son moléculas de RNA monocatenario de pequeño tamaño (22 nucleótidos aproximadamente) y no codificantes, que se unen a secuencias especificas de las regiones no codificantes (principalmente las 3'UTR) de los RNA mensajeros diana, inhibiendo su traducción y/o desestabilizándolos, lo que provoca igualmente una disminución en la cantidad total de proteína. Existen diversos microRNAs que tienen como diana transcritos que codifican para proteínas de los canales iónicos cardíacos, por ejemplo, el microRNA-195 y el microRNA-1 actúan sobre la expresión del gen SCN5A (Baofeng Yang, et al., 2008, Cardiovascular Research, Vol. 79: 571- 580). En resumen, es necesario diseñar nuevas estrategias terapéuticas de modulación de la función del canal de sodio para tratar arritmias cardíacas, las cuales restauren la deficiencia biológica del paciente y no se limiten únicamente a paliar la sintomatología de la enfermedad. Por otro lado, estas nuevas estrategias terapéuticas deberían evitar las complicaciones de las actuales terapias con drogas o con el cardiovertor. On the other hand, microRNAs are small-sized single-stranded RNA molecules (approximately 22 nucleotides) and non-coding, which bind to specific sequences of the non-coding regions (mainly the 3 ' UTRs) of the target messenger RNAs, inhibiting their translation and / or destabilizing them, which also causes a decrease in the total amount of protein. There are several microRNAs that target transcripts encoding cardiac ion channel proteins, for example, microRNA-195 and microRNA-1 act on the expression of the SCN5A gene (Baofeng Yang, et al., 2008, Cardiovascular Research, Vol. 79: 571-580). In summary, it is necessary to design new therapeutic strategies for modulating the function of the sodium channel to treat cardiac arrhythmias, which will restore the patient's biological deficiency and not only limit the symptomatology of the disease. On the other hand, these new therapeutic strategies should avoid the complications of current drug or cardioverter therapies.
DESCRIPCIÓN DE LA INVENCIÓN DESCRIPTION OF THE INVENTION
La presente invención propone el uso del microRNA hsa-mir-219-5p, de SEQ ID NO: 1 , de los polinucleótidos modificados derivados de él, SEQ ID NO: 3 o SEQ ID NO: 5, o de sus precursores, SEQ ID NO: 2, SEQ ID NO: 4 o SEQ ID NO: 6, para la elaboración de medicamentos útiles para el tratamiento de canalopatías arritmogénicas, preferiblemente de aquellas que se deben a alteraciones en la función del canal de sodio cardíaco, más preferiblemente de síndrome de QT largo tipo 3 y de síndrome de Brugada. El pequeño tamaño de los microRNAs, su fácil manipulación, la biodisponibilidad de sus precursores (pre-miR o pre-microRNA) y su elevada estabilidad termodinámica, hacen de ellos moléculas con un alto potencial terapéutico. Además, la conjugación de los polinucleótidos de la invención con, por ejemplo, aunque sin limitarnos, residuos lipidíeos, permite su biodisponibilidad en el organismo del paciente al que le son administrados. The present invention proposes the use of the hsa-mir-219-5p microRNA, of SEQ ID NO: 1, of the modified polynucleotides derived therefrom, SEQ ID NO: 3 or SEQ ID NO: 5, or of its precursors, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, for the preparation of medicines useful for the treatment of arrhythmogenic canalopathies, preferably those that are due to alterations in the function of the cardiac sodium channel, more preferably of syndrome of long QT type 3 and Brugada syndrome. The small size of the microRNAs, their easy manipulation, the bioavailability of their precursors (pre-miR or pre-microRNA) and their high thermodynamic stability, make them molecules with a high therapeutic potential. In addition, the conjugation of the polynucleotides of the invention with, for example, but not limited to lipid residues, allows their bioavailability in the organism of the patient to which they are administered.
Las secuencias polinucleotídicas de la invención o polinucleótidos de la invención, preferiblemente, las SEQ ID NO: 1 , SEQ ID NO: 2, SEO ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 o SEQ ID NO: 6, permiten modular la función del canal de sodio cardíaco a través de su unión al mRNA que codifica para la proteína Nav1.5, o en el caso de los precursores, proporcionando el polinucleótido que se une a este mRNA, lo que arroja la posibilidad de incrementar la función del canal de sodio en pacientes con, por ejemplo, aunque sin limitarnos, síndrome de Brugada, o disminuirla en pacientes con, por ejemplo, aunque sin limitarnos, síndrome LQT3, y de este modo revertir la función biológica alterada en dichas patologías cardíacas, en contraposición con los actuales métodos biomecánicos o farmacológicos que solo permiten mitigar parcialmente la sintomatología de estas enfermedades. The polynucleotide sequences of the invention or polynucleotides of the invention, preferably, SEQ ID NO: 1, SEQ ID NO: 2, SEO ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 , allow modulating the function of the cardiac sodium channel through its binding to the mRNA encoding the Nav1.5 protein, or in the case of precursors, providing the polynucleotide that binds to this mRNA, which gives the possibility of increase the function of the sodium channel in patients with, for example, but not limited to, Brugada syndrome, or decrease it in patients with, for example, but not limited to, LQT3 syndrome, and thus reverse the altered biological function in these pathologies cardiac, as opposed to current biomechanical or pharmacological methods that only partially mitigate the symptoms of these diseases.
Los inventores demuestran que el microRNA hsa-mir-219-5p, o miR-219a, de SEQ ID NO: 1 , es capaz de reducir la expresión del gen SCN5A, el cual codifica para la proteína transmembrana Nav1.5 responsable del potencial de acción en las células musculares cardíacas. El análisis in vitro de este microRNA muestra que su sobreexpresión disminuye la expresión del gen SCN5A hasta un 50% en las células musculares cardíacas auriculares (Figura 3), reduciendo así su capacidad y ritmo contráctil. Por ello, en la presente invención se propone el uso de este microRNA para la elaboración de medicamentos destinados al tratamiento de canalopatías arritmogénicas, preferiblemente de aquellas canalopatías arritmogénicas cuya causa sean alteraciones en los canales de sodio, más preferiblemente cuando las alteraciones en dichos canales se deban a mutaciones de ganancia de función en el gen SCN5A, como por ejemplo, aunque sin limitarnos, el síndrome de LQT3. The inventors demonstrate that the hsa-mir-219-5p microRNA, or miR-219a, of SEQ ID NO: 1, is capable of reducing the expression of the SCN5A gene, which codes for the Nav1.5 transmembrane protein responsible for the potential of action in cardiac muscle cells. In vitro analysis of this microRNA shows that its overexpression decreases the expression of the SCN5A gene up to 50% in atrial cardiac muscle cells (Figure 3), thus reducing its capacity and contractile rhythm. Therefore, the present invention proposes the use of this microRNA for the preparation of medicaments for the treatment of arrhythmogenic canalopathies, preferably of those arrhythmogenic canalopathies whose cause is alterations in the sodium channels, more preferably when the alterations in said channels are due to function gain mutations in the SCN5A gene, such as, but not limited to, the LQT3 syndrome.
Además, en la presente invención se han sustituido los tres nucleótidos (CAA) adyacentes al extremo 3" de la secuencia GAUUGUC o secuencia complementaria al mRNA del gen SCN5A comprendida en la SEQ ID NO: 1 , por otros tres nucleótidos (AGC), de manera que se ha obtenido la SEQ ID NO: 3, polinucleótido que presenta al menos un 85% de identidad con la SEQ ID NO: 1 y que comprende tres sitios complementarios más que esta secuencia con el mRNA del gen SCN5A, por lo que posee una mayor capacidad de unión al mismo que la secuencia del microRNA nativo o SEQ ID NO: 1 . Por ello, la SEQ ID NO: 3 también es útil para la elaboración de medicamentos destinados al tratamiento de canalopatías arritmogénicas, preferiblemente de aquellas canalopatías arritmogénicas cuya causa sean alteraciones en los canales de sodio, más preferiblemente cuando las alteraciones en dichos canales se deban a mutaciones de ganancia de función en este gen, como por ejemplo, aunque sin limitarnos, el síndrome de LQT3. In addition, in the present invention the three nucleotides (CAA) adjacent to the 3 " end of the GAUUGUC sequence or complementary sequence to the mRNA of the SCN5A gene comprised in SEQ ID NO: 1, have been replaced by three other nucleotides (AGC), of so that SEQ ID NO: 3 has been obtained, a polynucleotide that has at least 85% identity with SEQ ID NO: 1 and that comprises three complementary sites more than this sequence with the mRNA of the SCN5A gene, so it has a greater binding capacity to it than the sequence of the native microRNA or SEQ ID NO: 1. Therefore, SEQ ID NO: 3 is also useful for the preparation of medicaments for the treatment of arrhythmogenic canalopathies, preferably of those arrhythmogenic canalopathies whose cause they are alterations in the sodium channels, more preferably when the alterations in said channels are due to function gain mutations in this gene, such as, but not limited to, the sin drome of LQT3.
Por tanto, un primer aspecto de la invención se refiere al uso de un polinucleótido aislado, de ahora en adelante "polinucleótido de la invención", que comprende una secuencia de nucleótidos con al menos un 60%, preferiblemente con al menos un 75%, más preferiblemente con al menos un 80% y aun más preferiblemente con al menos un 85% de identidad con respecto a la longitud completa de la secuencia SEQ ID NO: 1 para la elaboración de un medicamento, o alternativamente, a un polinucleótido aislado que comprende una secuencia de nucleótidos con al menos un 60%, preferiblemente con al menos un 75%, más preferiblemente con al menos un 80% y aun más preferiblemente con al menos un 85% de identidad con respecto a la longitud completa de la secuencia SEQ ID NO: 1 para su uso como medicamento. Thus, a first aspect of the invention relates to the use of an isolated polynucleotide, hereinafter "polynucleotide of the invention", which comprises a nucleotide sequence with at least 60%, preferably with at least 75%, more preferably with at least 80% and even more preferably with at least 85% identity with respect to the full length of the sequence SEQ ID NO: 1 for the preparation of a medicament, or alternatively, to an isolated polynucleotide comprising a nucleotide sequence with at least 60%, preferably with at least 75%, more preferably with at least 80% and even more preferably with at least 85% identity with respect to the full length of the sequence SEQ ID NO: 1 for use as a medicine.
Los términos "secuencia nucleotídica", "secuencia de nucleótidos", "ácido nucleico", "oligonucleótido" y "polinucleotido" se usan aquí de manera intercambiable y se refieren a una forma polimérica de nucleótidos de cualquier longitud que pueden estar o no, química o bioquímicamente modificados. Se refieren, por tanto, a cualquier polirribonucleótido o polidesoxirribonucleótido, tanto de cadena sencilla como de doble hebra. The terms "nucleotide sequence", "nucleotide sequence", "acid nucleic "," oligonucleotide "and" polynucleotide "are used interchangeably herein and refer to a polymeric form of nucleotides of any length that may or may not be, chemically or biochemically modified. Thus, they refer to any polyiribonucleotide or polydeoxyribonucleotide , both single chain and double strand.
El polinucleotido de la invención puede obtenerse de manera artificial mediante métodos de clonación y selección convencional, o mediante secuenciación. En una realización preferida, el polinucleótido de la invención comprende la secuencia de nucleótidos SEQ ID NO: 3, la cual presenta al menos un 85% de identidad con respecto a la longitud completa de la SEQ ID NO: 1. En una realización más preferida, el polinucleótido de la invención es SEQ ID NO: 4, secuencia nucleotídica precursora de la SEQ ID NO: 3 que comprende esta secuencia. The polynucleotide of the invention can be obtained artificially by conventional cloning and selection methods, or by sequencing. In a preferred embodiment, the polynucleotide of the invention comprises the nucleotide sequence SEQ ID NO: 3, which has at least 85% identity with respect to the full length of SEQ ID NO: 1. In a more preferred embodiment , the polynucleotide of the invention is SEQ ID NO: 4, a precursor nucleotide sequence of SEQ ID NO: 3 comprising this sequence.
Otro aspecto de la invención se refiere a un polinucleótido aislado que comprende la secuencia de nucleótidos SEQ ID NO: 3. Otro aspecto de la invención se refiere a un polinucleótido aislado que comprende la secuencia de nucleótidos SEQ ID NO: 4. Another aspect of the invention relates to an isolated polynucleotide comprising the nucleotide sequence SEQ ID NO: 3. Another aspect of the invention relates to an isolated polynucleotide comprising the nucleotide sequence SEQ ID NO: 4.
Por otro lado, en la presente invención se han sustituido tres de los nucleótidos de la secuencia GAUUGUC comprendida en la SEQ ID NO: 1 para obtener un polinucleótido, SEQ ID NO: 5, que presenta al menos un 85% de identidad con la SEQ ID NO: 1 y que comprende tres sitios complementarios menos que esta secuencia con el mRNA del gen SCN5A, por lo que posee una menor capacidad de unión al mismo que la secuencia del microRNA nativo o SEQ ID NO: 1. Por ello, la SEQ ID NO: 5 también es útil para la elaboración de medicamentos destinados al tratamiento de canalopatías arritmogénicas, preferiblemente de aquellas canalopatías arritmogénicas cuya causa sean alteraciones en los canales de sodio, más preferiblemente cuando las alteraciones en dichos canales se deban a mutaciones de ganancia de función en este gen, como por ejemplo, aunque sin limitarnos, el síndrome de LQT3, puesto que, aunque esta secuencia posea menos sitios complementarios que la SEQ ID NO: 1 con el mRNA diana también es capaz de unirse a dicho mRNA y reducir su función; estos medicamentos podrían ser útiles, por ejemplo, aunque sin limitarnos, en pacientes de síndrome de LQT3 en los que exista una deficiencia en la expresión del microRNA nativo. La SEQ ID NO: 5 también es útil para la elaboración de medicamentos destinados al tratamiento de canalopatías arritmogénicas cuya causa sean mutaciones de pérdida de función en este gen, como por ejemplo, aunque sin limitarnos, el síndrome de Brugada, ya que esta secuencia compite con el microRNA nativo presente en las células por la unión al mRNA diana reduciendo la capacidad de este último de inhibir la función de dicho gen. Por ello, en otra realización preferida del primer aspecto de la invención, el polinucleótido de la invención comprende la secuencia de nucleótidos SEQ ID NO: 5, la cual presenta al menos un 85% de identidad con respecto a la longitud completa de la SEQ ID NO: 1. En una realización más preferida, el polinucleótido de la invención es SEQ ID NO: 6, secuencia nucleotídica precursora de la SEQ ID NO: 5 que comprende esta secuencia. On the other hand, three of the nucleotides of the GAUUGUC sequence comprised in SEQ ID NO: 1 have been substituted in the present invention to obtain a polynucleotide, SEQ ID NO: 5, which has at least 85% identity with SEQ ID NO: 1 and comprising three complementary sites less than this sequence with the mRNA of the SCN5A gene, so it has a lower binding capacity than the sequence of the native microRNA or SEQ ID NO: 1. Therefore, the SEQ ID NO: 5 is also useful for the preparation of drugs for the treatment of arrhythmogenic canalopathies, preferably of those arrhythmogenic canalopathies whose cause is alterations in the sodium channels, more preferably when the Alterations in these channels are due to function gain mutations in this gene, such as, but not limited to, LQT3 syndrome, since, although this sequence has fewer complementary sites than SEQ ID NO: 1 with the target mRNA It is also able to bind to said mRNA and reduce its function; These medications may be useful, for example, but not limited to, in patients with LQT3 syndrome in which there is a deficiency in the expression of the native microRNA. SEQ ID NO: 5 is also useful for the preparation of drugs for the treatment of arrhythmogenic canalopathies whose cause is mutations of loss of function in this gene, such as, but not limited to, Brugada syndrome, since this sequence competes with the native microRNA present in the cells by binding to the target mRNA reducing the latter's ability to inhibit the function of said gene. Therefore, in another preferred embodiment of the first aspect of the invention, the polynucleotide of the invention comprises the nucleotide sequence SEQ ID NO: 5, which has at least 85% identity with respect to the full length of SEQ ID NO: 1. In a more preferred embodiment, the polynucleotide of the invention is SEQ ID NO: 6, a precursor nucleotide sequence of SEQ ID NO: 5 comprising this sequence.
Otro aspecto de la invención se refiere a un polinucleótido aislado que comprende la secuencia de nucleótidos SEQ ID NO: 5. Otro aspecto de la invención se refiere a un polinucleótido aislado que comprende la secuencia de nucleótidos SEQ ID NO: 6. Another aspect of the invention relates to an isolated polynucleotide comprising the nucleotide sequence SEQ ID NO: 5. Another aspect of the invention relates to an isolated polynucleotide comprising the nucleotide sequence SEQ ID NO: 6.
En otra realización preferida del primer aspecto de la invención, el polinucleótido de la invención comprende una secuencia de nucleótidos con al menos un 90% de identidad con respecto a la longitud completa de la secuencia SEQ ID NO: 1. En una realización más preferida, el polinucleótido de la invención comprende una secuencia de nucleótidos con al menos un 95% de identidad con respecto a la longitud completa de la secuencia SEQ ID NO: 1. En una realización más preferida, el polinucleotido de la invención comprende una secuencia de nucleótidos con al menos un 98% de identidad con respecto a la longitud completa de la secuencia SEQ ID NO: 1. En una realización más preferida, el polinucleotido de la invención comprende la secuencia de nucleótidos SEQ ID NO: 1 . En una realización aun más preferida, el polinucleotido de la invención es SEQ ID NO: 2, secuencia nucleotídica precursora de la SEQ ID NO: 1 que comprende esta secuencia. Dentro del alcance de esta invención se encuentran también los precursores o secuencias nucleotídicas precursoras. El término "precursor" o "secuencia precursora" tal como aquí se utiliza incluye a cualquier secuencia nucleotídica que cuando se procesa mediante, por ejemplo, aunque sin limitarnos, corte enzimático, es capaz de proporcionar un polinucleotido con al menos un 60%, preferiblemente con al menos un 65%, más preferiblemente con al menos un 70%, con al menos un 75%, con al menos un 80%, con al menos un 85%, con al menos un 90%, con al menos un 95% o con al menos un 98% de identidad con respecto a la longitud completa de la SEQ ID NO: 1 , o bien que es capaz de proporcionar un polinucleotido de secuencia SEQ ID NO: 1 ; o que es capaz de proporcionar un polinucleotido de secuencia SEQ ID NO: 3; o que es capaz de proporcionar un polinucleotido de secuencia SEQ ID NO: 5. Ventajosamente, dicho precursor es una secuencia nucleotídica que aumenta la biodisponibilidad de los polinucleótidos que proporciona cuando se administra a un individuo o que potencia la liberación de los mismos en un compartimento biológico. Preferiblemente, las secuencias nucleotídicas precursoras de la invención son la SEQ ID NO: 2, secuencia a la que también se hará referencia como pre-miR-219a y que es precursora de la SEQ ID NO: 1 ; la SEQ ID NO: 4, secuencia precursora de la SEQ ID NO: 3; y la SEQ ID NO: 6, secuencia precursora de la SEQ ID NO: 5. Todas estas secuencias nucleotídicas precursoras comprenden los polinucleótidos SEQ ID NO: 1 , SEQ ID NO: 3 y SEQ ID NO: 5, respectivamente. Los medicamentos y composiciones farmacéuticas de la invención son útiles para el tratamiento de canalopatías arritmogénicas, preferiblemente, para el tratamiento de canalopatías arritmogénicas que se deben a alteraciones en los canales de sodio, ya que el polinucleotido de la invención es capaz de reducir la expresión del gen SCN5A que codifica para una proteína transmembrana de los canales de sodio, por lo que puede ser empleado en aquellas canalopatías arritmogénicas debidas a alteraciones de ganancia de función en estos canales. El polinucleótido de la invención que comprende la secuencia de nucleótidos SEQ ID NO: 5 o el de SEQ ID NO: 6, presenta una menor complementariedad con el mRNA de este gen que el microRNA nativo, por lo que al competir con este último por dicha unión es capaz de incrementar los niveles de mRNAs de este gen, en comparación con el microRNA nativo, por lo que es útil para el tratamiento de canalopatías arritmogénicas debidas a alteraciones de pérdida de función en los canales de sodio. In another preferred embodiment of the first aspect of the invention, the polynucleotide of the invention comprises a nucleotide sequence with at least 90% identity with respect to the full length of the sequence SEQ ID NO: 1. In a more preferred embodiment, the polynucleotide of the invention comprises a nucleotide sequence with at least 95% of identity with respect to the full length of the sequence SEQ ID NO: 1. In a more preferred embodiment, the polynucleotide of the invention comprises a nucleotide sequence with at least 98% identity with respect to the full length of the sequence SEQ ID NO: 1. In a more preferred embodiment, the polynucleotide of the invention comprises the nucleotide sequence SEQ ID NO: 1. In an even more preferred embodiment, the polynucleotide of the invention is SEQ ID NO: 2, a precursor nucleotide sequence of SEQ ID NO: 1 comprising this sequence. Also within the scope of this invention are precursor nucleotide sequences or precursors. The term "precursor" or "precursor sequence" as used herein includes any nucleotide sequence that when processed by, for example, but not limited to, enzymatic cleavage, is capable of providing a polynucleotide with at least 60%, preferably with at least 65%, more preferably with at least 70%, with at least 75%, with at least 80%, with at least 85%, with at least 90%, with at least 95% or with at least 98% identity with respect to the full length of SEQ ID NO: 1, or that it is capable of providing a sequence polynucleotide of SEQ ID NO: 1; or that is capable of providing a sequence polynucleotide SEQ ID NO: 3; or that is capable of providing a polynucleotide of sequence SEQ ID NO: 5. Advantageously, said precursor is a nucleotide sequence that increases the bioavailability of the polynucleotides that it provides when administered to an individual or that enhances their release in a compartment biological. Preferably, the precursor nucleotide sequences of the invention are SEQ ID NO: 2, a sequence that will also be referred to as pre-miR-219a and that is a precursor to SEQ ID NO: 1; SEQ ID NO: 4, precursor sequence of SEQ ID NO: 3; and SEQ ID NO: 6, precursor sequence of SEQ ID NO: 5. All of these precursor nucleotide sequences comprise polynucleotides SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, respectively. The medicaments and pharmaceutical compositions of the invention are useful for the treatment of arrhythmogenic canalopathies, preferably, for the treatment of arrhythmogenic canalopathies that are due to alterations in the sodium channels, since the polynucleotide of the invention is capable of reducing the expression of the SCN5A gene that codes for a transmembrane protein of sodium channels, so it can be used in those arrhythmogenic canalopathies due to alterations in function gain in these channels. The polynucleotide of the invention comprising the nucleotide sequence SEQ ID NO: 5 or that of SEQ ID NO: 6, has a lower complementarity with the mRNA of this gene than the native microRNA, so competing with the latter for said Binding is able to increase the levels of mRNAs of this gene, compared to the native microRNA, so it is useful for the treatment of arrhythmogenic canalopathies due to impaired loss of function in the sodium channels.
Por tanto, otro aspecto de la invención se refiere al uso del polinucleótido de la invención para la elaboración de un medicamento para el tratamiento de canalopatías arritmogénicas, o alternativamente, al polinucleótido de la invención para su uso como medicamento para el tratamiento de canalopatías arritmogénicas. En una realización preferida, las canalopatías arritmogénicas se deben a alteraciones en los canales de sodio. En una realización más preferida, la canalopatía arritmogénica que se debe a alteraciones en los canales de sodio es el síndrome de QT largo tipo 3. Otro aspecto de la invención se refiere al uso del polinucleótido de la invención que comprende la secuencia de nucleótidos SEQ ID NO: 5 o del polinucleótido de la invención de secuencia SEQ ID NO: 6 para la elaboración de un medicamento para el tratamiento de síndrome de Brugada. El término "medicamento", tal y como se usa en esta descripción, hace referencia a cualquier sustancia usada para la prevención, el diagnóstico, el alivio, el tratamiento o la curación de enfermedades en el hombre y los animales. En el contexto de la presente invención se refiere a una preparación que comprenda, al menos, el polinucleótido de la invención. Therefore, another aspect of the invention relates to the use of the polynucleotide of the invention for the preparation of a medicament for the treatment of arrhythmogenic canalopathies, or alternatively, to the polynucleotide of the invention for use as a medicament for the treatment of arrhythmogenic canalopathies. In a preferred embodiment, arrhythmogenic canalopathies are due to alterations in the sodium channels. In a more preferred embodiment, the arrhythmogenic canalopathy that is due to alterations in the sodium channels is the long QT syndrome type 3. Another aspect of the invention relates to the use of the polynucleotide of the invention comprising the nucleotide sequence SEQ ID. NO: 5 or of the polynucleotide of the invention of sequence SEQ ID NO: 6 for the preparation of a medicament for the treatment of Brugada syndrome. The term "medication", as used in this description, refers to any substance used for the prevention, diagnosis, relief, treatment or cure of diseases in man and women. animals. In the context of the present invention it refers to a preparation comprising at least the polynucleotide of the invention.
El polinucleótido de la invención se formula en una composición farmacéutica apropiada, en la cantidad terapéuticamente efectiva, preferiblemente junto con uno o más vehículos, adyuvantes o excipientes farmacéuticamente aceptables. The polynucleotide of the invention is formulated in an appropriate pharmaceutical composition, in the therapeutically effective amount, preferably together with one or more pharmaceutically acceptable carriers, adjuvants or excipients.
El término "tratamiento", tal como se entiende en la presente invención, supone combatir los efectos causados como consecuencia de las canalopatías arritmogénicas, preferiblemente, de las canalopatías arritmogénicas que se deben a alteraciones en los canales de sodio, más preferiblemente del síndrome de QT largo tipo 3 o del síndrome de Brugada, para estabilizar el estado de los individuos o prevenir daños posteriores. El término "prevención", tal como se entiende en la presente invención, consiste en evitar la aparición de daños cuya causa sean las canalopatías arritmogénicas, preferiblemente, las canalopatías arritmogénicas que se deben a alteraciones en los canales de sodio, más preferiblemente el síndrome de QT largo tipo 3 o el síndrome de Brugada. Se entiende por "canalopatía arritmogénica" cualquier cardiomiopatía (o alteración de la función del miocardio) genética que resulta de mutaciones en los genes responsables del correcto funcionamiento de los canales iónicos que generan el potencial de acción transmembrana, lo cual conlleva defectos en la función (ganancia o pérdida) de dichos canales, alteraciones fisiológicas de la duración del potencial de acción transmembrana y/o predisposición a desarrollar arritmias ventriculares en ausencia de cardiopatía estructural. Ejemplos de canalopatías arritmogénicas incluyen, aunque sin limitarnos, síndrome de Brugada, síndrome de QT largo, síndrome de QT corto o taquicardia ventricular polimórfica catecolaminérgica. Las canalopatías arritmogénicas pueden ser diagnosticadas, por ejemplo, aunque sin limitarnos, como se describe en Cabrera Ortega M., et al., 2009, Revista Cubana Pediatría, 81 (4). Las "canalopatías arritmogénicas que se deben a alteraciones en los canales de sodio" son aquellas canalopatías cuya base genética se encuentra en mutaciones (de pérdida o de ganancia de función) en al menos uno de los genes que codifican para dichos canales, preferiblemente, en el gen SCN5A de SEQ ID NO: 7. La detección de este tipo de canalopatías se puede realizar mediante, por ejemplo, aunque sin limitarnos, un electrocardiograma, mediante el cual es posible hacer un diagnóstico potencial de las corrientes alteradas (Antzelevitch et al., 2005a, 2005b, Circulation, 1 1 1 :659-670); o bien mediante PCR de los genes que codifican para los canales de sodio, preferiblemente del gen SCN5A, y posterior secuenciación, lo cual permite detectar alteraciones en el gen que puedan ser las causantes de la canalopatía (Ashley & Niebauer, 2004, J. Cardiology explained; Kapplinger et al., 2010, Heart Rhythm, 7(1 ):33- 46). The term "treatment", as understood in the present invention, is to combat the effects caused as a consequence of arrhythmogenic canalopathies, preferably, of arrhythmogenic canalopathies that are due to alterations in the sodium channels, more preferably of the QT syndrome long type 3 or Brugada syndrome, to stabilize the condition of individuals or prevent further damage. The term "prevention", as understood in the present invention, is to prevent the occurrence of damage whose cause are arrhythmogenic canalopathies, preferably, arrhythmogenic canalopathies that are due to alterations in the sodium channels, more preferably the syndrome of Long QT type 3 or Brugada syndrome. "Arrhythmogenic canalopathy" is understood as any genetic cardiomyopathy (or impaired myocardial function) that results from mutations in the genes responsible for the proper functioning of the ion channels that generate the potential for transmembrane action, which leads to defects in function ( gain or loss) of said channels, physiological alterations of the duration of the potential for transmembrane action and / or predisposition to develop ventricular arrhythmias in the absence of structural heart disease. Examples of arrhythmogenic canalopathies include, but are not limited to, Brugada syndrome, long QT syndrome, short QT syndrome or catecholaminergic polymorphic ventricular tachycardia. Arrhythmogenic canalopathies can be diagnosed, for example, but not limited to, as described in Cabrera Ortega M., et al., 2009, Revista Cubana Pediatría, 81 (4). "Arrhythmogenic canalopathies due to alterations in sodium channels" are those canalopathies whose genetic basis is found in mutations (loss or gain of function) in at least one of the genes that code for said channels, preferably, in the SCN5A gene of SEQ ID NO: 7. The detection of this type of canalopathies can be performed by, for example, but not limited to, an electrocardiogram, by means of which it is possible to make a potential diagnosis of the altered currents (Antzelevitch et al. , 2005a, 2005b, Circulation, 1 1 1: 659-670); or by PCR of the genes that code for the sodium channels, preferably of the SCN5A gene, and subsequent sequencing, which allows to detect alterations in the gene that may be the cause of the canalopathy (Ashley & Niebauer, 2004, J. Cardiology explained; Kapplinger et al., 2010, Heart Rhythm, 7 (1): 33-46).
El "síndrome de QT largo tipo 3" o "LQT3" se caracteriza por una alteración en la repolarización ventricular traducida en el electrocardiograma por un alargamiento en el intervalo QT corregido (QTc) por fórmula de Bazzet (QTc=QT/VR)≥ 440 ms, que predispone a arritmia de puntas torcidas (torsade de pointe) y muerte súbita. Puede diagnosticarse, por tanto, mediante por ejemplo, aunque sin limitarnos, electrocardiograma o registro Holter. Otros criterios diagnósticos de este síndrome son el síncope inducido por estrés y/o alternancia eléctrica de la onda T. Este síndrome se debe a mutaciones en el gen SCN5A que originan anomalías en el canal de sodio. Este gen se localiza en el brazo corto del cromosoma 3 (3p21 -24). "Long QT syndrome type 3" or "LQT3" is characterized by an alteration in ventricular repolarization translated on the electrocardiogram by an elongation in the corrected QT interval (QTc) by Bazzet's formula (QTc = QT / VR) ≥ 440 ms, which predisposes to arrhythmia of crooked tips (torsade de pointe) and sudden death. It can be diagnosed, therefore, by means of, for example, but not limited to, electrocardiogram or Holter recording. Other diagnostic criteria for this syndrome are stress-induced syncope and / or electrical alternation of the T wave. This syndrome is due to mutations in the SCN5A gene that cause sodium channel abnormalities. This gene is located in the short arm of chromosome 3 (3p21-24).
El "síndrome de Brugada" se caracteriza por la probabilidad de presentar episodios sincópales o parada cardíaca causados por taquicardia ventricular polimórfica o fibrilación ventricular durante el reposo o el sueño, con un patrón electrocardiográfico de aparente bloqueo de rama derecha y supradesnivel del segmento ST que cae con lentitud y finaliza en una onda T negativa en V1 , V2 y V3, sin depresión de las derivaciones opuestas. Genéticamente se han identificado siete tipos de síndrome de Brugada, los más frecuentes son, aunque sin limitarnos, síndrome de Brugada de tipo 1 o síndrome de Brugada de tipo 2. Otro aspecto de la invención se refiere a una composición farmacéutica, de ahora en adelante, "primera composición farmacéutica de la invención", que comprende el polinucleotido de la invención que comprende una secuencia de nucleótidos con al menos un 60%, preferiblemente con al menos un 65%, más preferiblemente con al menos un 70%, con al menos un 75%, con al menos un 80%, con al menos un 85%, con al menos un 90%, con al menos un 95% o con al menos un 98% de identidad con respecto a la longitud completa de la secuencia SEQ ID NO: 1 , o que comprende la secuencia de nucleótidos SEQ ID NO: 1 , o de SEQ ID NO: 2, o que comprende la secuencia de nucleótidos SEQ ID NO: 3 o de SEQ ID NO: 4. "Brugada syndrome" is characterized by the probability of presenting syncopal episodes or cardiac arrest caused by polymorphic ventricular tachycardia or ventricular fibrillation during rest or sleep, with an electrocardiographic pattern of apparent right bundle branch block and supra-level ST segment that falls slowly and ends on a negative T wave in V1, V2 and V3, without depression of the opposite leads. Genetically they have identified seven types of Brugada syndrome, the most frequent are, but not limited to, Brugada syndrome type 1 or Brugada syndrome type 2. Another aspect of the invention relates to a pharmaceutical composition, hereafter, "first pharmaceutical composition of the invention ", which comprises the polynucleotide of the invention comprising a nucleotide sequence with at least 60%, preferably with at least 65%, more preferably with at least 70%, with at least 75% , with at least 80%, with at least 85%, with at least 90%, with at least 95% or with at least 98% identity with respect to the full length of the sequence SEQ ID NO: 1, or comprising the nucleotide sequence SEQ ID NO: 1, or of SEQ ID NO: 2, or comprising the nucleotide sequence SEQ ID NO: 3 or of SEQ ID NO: 4.
En una realización preferida, la primera composición farmacéutica de la invención además comprende otro principio activo. En una realización más preferida, la primera composición farmacéutica de la invención además comprende un vehículo farmacéuticamente aceptable. In a preferred embodiment, the first pharmaceutical composition of the invention further comprises another active ingredient. In a more preferred embodiment, the first pharmaceutical composition of the invention further comprises a pharmaceutically acceptable carrier.
Otro aspecto de la invención se refiere a una composición farmacéutica, de ahora en adelante, "segunda composición farmacéutica de la invención", que comprende el polinucleotido de la invención que comprende la secuencia de nucleótidos SEQ ID NO: 5 o de SEQ ID NO: 6. Another aspect of the invention relates to a pharmaceutical composition, hereinafter, "second pharmaceutical composition of the invention", comprising the polynucleotide of the invention comprising the nucleotide sequence SEQ ID NO: 5 or SEQ ID NO: 6.
En una realización preferida, la segunda composición farmacéutica de la invención además comprende otro principio activo. En una realización más preferida, la segunda composición farmacéutica de la invención además comprende un vehículo farmacéuticamente aceptable. In a preferred embodiment, the second pharmaceutical composition of the invention further comprises another active ingredient. In a more preferred embodiment, the second pharmaceutical composition of the invention further comprises a pharmaceutically acceptable carrier.
Como se emplea aquí, el término "principio activo", "sustancia activa", "sustancia farmacéuticamente activa", "ingrediente activo" ó "ingrediente farmacéuticamente activo" significa cualquier componente que potencialmente proporcione una actividad farmacológica u otro efecto diferente en el diagnóstico, cura, mitigación, tratamiento, o prevención de una enfermedad, o que afecta a la estructura o función del cuerpo del hombre u otros animales. El término incluye aquellos componentes que promueven un cambio químico en la elaboración del fármaco y están presentes en el mismo de una forma modificada prevista que proporciona la actividad específica o el efecto. As used herein, the term "active substance", "active substance", "pharmaceutically active substance", "active ingredient" or "ingredient pharmaceutically active "means any component that potentially provides a pharmacological activity or other effect different in the diagnosis, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals. The term It includes those components that promote a chemical change in the preparation of the drug and are present therein in a modified form intended to provide the specific activity or effect.
Los "adyuvantes" y "vehículos farmacéuticamente aceptables" se refieren a aquellas sustancias, o combinación de sustancias, conocidas en el sector farmacéutico, utilizadas en la elaboración de formas farmacéuticas de administración e incluyen, pero sin limitarse, sólidos, líquidos, disolventes o tensioactivos. Los vehículos farmacéuticamente aceptables que pueden ser utilizados en la presente invención son los vehículos conocidos en el estado de la técnica, como por ejemplo, aunque sin limitarnos, los residuos lipidíeos. "Adjuvants" and "pharmaceutically acceptable carriers" refer to those substances, or combination of substances, known in the pharmaceutical sector, used in the preparation of pharmaceutical forms of administration and include, but are not limited to, solids, liquids, solvents or surfactants. . Pharmaceutically acceptable carriers that can be used in the present invention are vehicles known in the state of the art, such as, but not limited to, lipid residues.
Las composiciones farmacéuticas y medicamentos de la presente invención pueden utilizarse en un método de tratamiento o de prevención de forma aislada o conjuntamente con otros compuestos farmacéuticos destinados al tratamiento o prevención de las canalopatías arritmogénicas, preferiblemente, de las canalopatías arritmogénicas que se deben a alteraciones en los canales de sodio, más preferiblemente, de síndrome de QT largo tipo 3 o de síndrome de Brugada. Las composiciones farmacéuticas de la presente invención pueden formularse para su administración en una variedad de formas conocidas en el estado de la técnica. The pharmaceutical compositions and medicaments of the present invention can be used in a method of treatment or prevention in isolation or in conjunction with other pharmaceutical compounds intended for the treatment or prevention of arrhythmogenic canalopathies, preferably, of arrhythmogenic canalopathies that are due to alterations in the sodium channels, more preferably, of type 3 long QT syndrome or Brugada syndrome. The pharmaceutical compositions of the present invention can be formulated for administration in a variety of ways known in the state of the art.
Tales composiciones y/o sus formulaciones pueden administrarse a un animal, incluyendo un mamífero y, por tanto, al hombre, en una variedad de formas, incluyendo, pero sin limitarse, parenteral, intraperitoneal, intravenosa, intradérmica, intraarticular, intrasinovial, intralesional, intraarterial, intracardíaca, intramuscular, intranasal, subcutánea, intracapsular, tópica, mediante parches transdérmicos, mediante administración percutánea, implante quirúrgico, pintura quirúrgica interna, bomba de infusión o vía catéter. La dosificación para obtener una cantidad terapéuticamente efectiva depende de una variedad de factores, como por ejemplo, la edad, peso, sexo o tolerancia del individuo. En el sentido utilizado en esta descripción, la expresión "cantidad terapéuticamente efectiva" se refiere a la cantidad de las composiciones farmacéuticas de la invención que produzca el efecto deseado y, en general, vendrá determinada, entre otras causas, por las características propias de dichas composiciones farmacéuticas y el efecto terapéutico a conseguir. Such compositions and / or their formulations can be administered to an animal, including a mammal and, therefore, to man, in a variety of ways, including, but not limited to, parenteral, intraperitoneal, intravenous, intradermal, intraarticular, intrasynovial, intralesional, intraarterial, intracardiac, intramuscular, intranasal, subcutaneous, intracapsular, topical, by transdermal patches, by percutaneous administration, surgical implant, internal surgical painting, infusion pump or catheter. The dosage to obtain a therapeutically effective amount depends on a variety of factors, such as the age, weight, sex or tolerance of the individual. In the sense used in this description, the term "therapeutically effective amount" refers to the amount of the pharmaceutical compositions of the invention that produces the desired effect and, in general, will be determined, among other causes, by the characteristics of said pharmaceutical compositions and the therapeutic effect to be achieved.
Otro aspecto de la invención se refiere al uso de la primera y de la segunda composición farmacéutica de la invención para la elaboración de un medicamento, o alternativamente, a la primera y segunda composición farmacéutica de la invención para su uso como medicamento. En una realización preferida, el medicamento es para el tratamiento de canalopatías arritmogénicas. En una realización más preferida, las canalopatías arritmogénicas se deben a alteraciones en los canales de sodio. En una realización aun más preferida, la canalopatía arritmogénica que se debe a alteraciones en los canales de sodio es el síndrome de QT largo tipo 3. Another aspect of the invention relates to the use of the first and second pharmaceutical composition of the invention for the preparation of a medicament, or alternatively, the first and second pharmaceutical composition of the invention for use as a medicament. In a preferred embodiment, the medicament is for the treatment of arrhythmogenic canalopathies. In a more preferred embodiment, arrhythmogenic canalopathies are due to alterations in the sodium channels. In an even more preferred embodiment, arrhythmogenic canalopathy due to alterations in sodium channels is the long QT syndrome type 3.
Otro aspecto de la invención se refiere al uso de la segunda composición farmacéutica de la invención para la elaboración de un medicamento para el tratamiento de síndrome de Brugada, o alternativamente, a la segunda composición farmacéutica de la invención para su uso como medicamento para el tratamiento de síndrome de Brugada. A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos o componentes. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Los siguientes ejemplos y dibujos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. Another aspect of the invention relates to the use of the second pharmaceutical composition of the invention for the preparation of a medicament for the treatment of Brugada syndrome, or alternatively, to the second pharmaceutical composition of the invention for use as a medicament for the treatment. of Brugada syndrome. Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives or components. For experts in the field, other objects, advantages and Characteristics of the invention will be apparent in part from the description and in part from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.
DESCRIPCIÓN DE LAS FIGURAS DESCRIPTION OF THE FIGURES
Fig. 1. Muestra el esquema de complementariedad del hsa-mir-219-5p (SEQ ID NO: 1) con la región 3 UTR del mensajero del gen SCN5A. Secuencia superior: región 3' UTR del mensajero del gen SCN5A (posición 1 .416-1 .422, 5X- 3"). Secuencia inferior: secuencia del hsa-mir-219-5p (SEQ ID NO: 1 , 3"- 5") con la secuencia complementaria al mensajero del gen SCN5A (GAUUGUC) marcada en blanco. Barras negras: nucleótidos complementarios. Fig. 2. Muestra la secuencia del hsa-mir-219-5p maduro y de su precursor.Fig. 1. Shows the complementarity scheme of hsa-mir-219-5p (SEQ ID NO: 1) with the 3 UTR region of the SCN5A gene messenger. Upper sequence: 3 ' UTR region of the messenger of the SCN5A gene (position 1 .416-1 .422, 5 X - 3 " ). Lower sequence: sequence of hsa-mir-219-5p (SEQ ID NO: 1, 3 " - 5 " ) with the sequence complementary to the messenger of the SCN5A gene (GAUUGUC) marked in white. Black bars: complementary nucleotides. Fig. 2. Shows the sequence of the mature hsa-mir-219-5p and its precursor.
A. Muestra la secuencia del hsa-mir-219-5p maduro (SEQ ID NO: 1 ). En negrita se destaca la secuencia que presenta complementariedad con la región 3" UTR del mensajero del gen SCN5A, es decir, la secuencia GAUUGUC. B. Muestra la secuencia del pre-miR-219a (SEQ ID NO: 2). En negrita se destaca la región correspondiente al hsa-mir-219-5p maduro (SEQ ID NO: 1 ). C. Muestra el esquema de la estructura en horquilla del pre-miR-219a. En gris se destaca la secuencia madura del hsa-mir-219-5p (SEQ ID NO: 1 ) en las dos hebras complementarias del pre-miR-219a (SEQ ID NO: 2). Fig. 3. Muestra el análisis por qRT-PCR de la expresión del gen SCN5A en células miocárdicas atriales (HL-1) 6 horas tras la transfección con el hsa- mir-219-5p de SEQ ID NO: 1. Barras negras: células control, donde no se transfectó el hsa-mir-219-5p. Barras blancas: células donde se transfectó el hsa-mir-219-5p. A. Shows the sequence of mature hsa-mir-219-5p (SEQ ID NO: 1). In bold, the sequence that shows complementarity with the 3 " UTR region of the messenger of the SCN5A gene is highlighted, ie the GAUUGUC sequence. B. It shows the sequence of the pre-miR-219a (SEQ ID NO: 2). the region corresponding to the mature hsa-mir-219-5p stands out (SEQ ID NO: 1) C. It shows the outline of the hairpin structure of the pre-miR-219a, in gray the mature sequence of the hsa-mir- stands out 219-5p (SEQ ID NO: 1) in the two complementary strands of pre-miR-219a (SEQ ID NO: 2) Fig. 3. Shows the qRT-PCR analysis of the expression of the SCN5A gene in atrial myocardial cells (HL-1) 6 hours after transfection with the hsa-mir-219-5p of SEQ ID NO: 1. Black bars: control cells, where hsa-mir-219-5p was not transfected White bars: cells where hsa-mir-219-5p was transfected.
Fig. 4. Muestra los resultados de los ensayos inmunohistoquímicos contra el producto de expresión del gen SCN5A en células control (panel izquierdo) y en células a las que se les transfectó el hsa-mir-219-5p (panel derecho). El panel izquierdo muestra la expresión proteica normal de Nav1 .5 (SCN5A) en la célula cardiomiocítica. Tras la transfección con miR-219-5p (panel derecho) Nav1 .5 parece quedar secuestrado en el aparato de Golgi, y por tanto está muy poco representado en la membrana citoplasmática, donde normalmente ejerce su función de transporte de iones sodio. Fig. 4. Shows the results of immunohistochemical tests against the expression product of the SCN5A gene in control cells (panel left) and in cells that were transfected with hsa-mir-219-5p (right panel). The left panel shows the normal protein expression of Nav1 .5 (SCN5A) in the cardiomyocytic cell. After transfection with miR-219-5p (right panel) Nav1 .5 appears to be sequestered in the Golgi apparatus, and therefore is very poorly represented in the cytoplasmic membrane, where it normally exerts its sodium ion transport function.
Fig. 5. Muestra un esquema ilustrativo de la estrategia para el tratamiento de síndrome de QT largo tipo 3 donde hay ganancia de función del gen SCN5A. A. Secuencia superior: región 3'UTR del mensajero del gen SCN5A (posición 1 .416-1 .422, 5"- 3"). Secuencia inferior: secuencia del hsa-mir-219-5p (SEQ ID NO: 1 , 3X- 5') con la secuencia complementaria al mensajero del gen SCN5A (GAUUGUC) marcada en blanco. El recuadro de la secuencia inferior muestra los tres nucleótidos que se han sustituido por los nucleótidos que se muestran en la parte inferior para dar lugar a la SEQ ID NO: 3. Se muestra la complementariedad entre ambas secuencias, superior e inferior (barras). Las barras grises representan los nuevos sitios complementarios creados para dar lugar a la SEQ ID NO: 3. B. Muestra la secuencia del precursor de la SEQ ID NO: 1 , pre-miR-219a, de SEQ ID NO: 2, donde se destaca en gris la secuencia madura del hsa-mir-219-5p (SEQ ID NO: 1 ) en las dos hebras complementarias del pre-miR-219a, en un recuadro se señalan los tres nucleótidos modificados para diseñar la SEQ ID NO: 3. C. Muestra la SEQ ID NO: 3 (en negrita) comprendida en su secuencia precursora o SEQ ID NO: 4. En gris se destacan los nucleótidos modificados. Fig. 5. It shows an illustrative scheme of the strategy for the treatment of long QT syndrome type 3 where there is gain of function of the SCN5A gene. A. Upper sequence: 3 ' UTR region of the messenger of the SCN5A gene (position 1 .416-1 .422, 5 " - 3 " ). Lower sequence: hsa-mir-219-5p sequence (SEQ ID NO: 1, 3 X - 5 ' ) with the complementary sequence to the SCN5A gene messenger (GAUUGUC) marked in white. The box in the lower sequence shows the three nucleotides that have been replaced by the nucleotides shown in the lower part to give rise to SEQ ID NO: 3. The complementarity between both upper and lower sequences (bars) is shown. The gray bars represent the new complementary sites created to give rise to SEQ ID NO: 3. B. Shows the precursor sequence of SEQ ID NO: 1, pre-miR-219a, of SEQ ID NO: 2, where the mature sequence of hsa-mir-219-5p (SEQ ID NO: 1) stands out in gray in the two complementary strands of the pre-miR-219a, in a box the three modified nucleotides are indicated to design SEQ ID NO: 3 C. Shows SEQ ID NO: 3 (in bold) included in its precursor sequence or SEQ ID NO: 4. In gray, the modified nucleotides are highlighted.
Fig. 6. Muestra un esquema ilustrativo de la estrategia para el tratamiento de síndrome de Brugada donde hay pérdida de función del gen SCN5A. A.Fig. 6. It shows an illustrative scheme of the strategy for the treatment of Brugada syndrome where there is loss of function of the SCN5A gene. TO.
Secuencia superior: región 3' UTR del mensajero del gen SCN5A (posición 1 .416-1 .422, 5N- 3"). Secuencia inferior: secuencia del hsa-mir-219-5p (SEQ ID NO: 1 , 3'- 5') con la secuencia complementaria al mensajero del gen SCN5A (GAUUGUC) marcada en blanco. Los recuadros de la secuencia inferior muestran los tres nucleótidos que se han sustituido por los nucleótidos que se muestran en la parte inferior para dar lugar a la SEQ ID NO: 5. Se muestra la complementariedad entre ambas secuencias, superior e inferior (barras). B. Muestra la secuencia del precursor de la SEQ ID NO: 1 pre-miR-219a, de SEQ ID NO: 2, donde se destaca en gris la secuencia madura del hsa-mir-219-5p (SEQ ID NO: 1 ) en las dos hebras complementarias del pre-miR-219a, en un recuadro se señalan los tres nucleótidos modificados para diseñar la SEQ ID NO: 5. C. Muestra la SEQ ID NO: 5 (en negrita) comprendida en su secuencia precursora o SEQ ID NO: 6. En gris se destacan los nucleótidos modificados. FIG. 7. Muestra la expresión del gen SCN5A en células miocárdicas atriales (HL-1) transfectadas con la SEQ ID NO: 2, SEQ ID NO: 4 o SEQ ID NO: 6 durante 12 horas. Células control (wt), donde se transfectó el precursor de hsa-mir-219-5p o miR-219a endógeno (SEQ ID NO: 2). miR-219a-: Células transfectadas con el precursor de miR-219a-(SEQ ID NO: 4), diseñado para tratar canalopatías arritmogénicas debidas a una ganancia de función en el gen SCN5A. miR-219a+: Células transfectadas con el precursor de miR-219+ (SEQ ID NO: 6), diseñado para tratar canalopatías arritmogénicas debidas a una pérdida de función en el gen SCN5A. La figura muestra que las células HL-1 transfectadas con la SEQ ID NO: 4 muestran una disminución en la expresión de SCN5A en comparación con las células transfectadas con el precursor endógeno de miR-219a (SEQ ID NO: 2), mientras que las células miocárdicas transfectadas con la SEQ ID NO: 6 muestran incremento en la expresión de SCN5A en comparación con las células transfectadas con el precursor endógeno de miR-219a (SEQ ID NO: 2). Upper sequence: 3 ' UTR region of the messenger of the SCN5A gene (position 1 .416-1 .422, 5 N - 3 " ). Lower sequence: sequence of hsa-mir-219-5p (SEQ ID NO: 1, 3 ' - 5 ' ) with the sequence complementary to the messenger of the SCN5A gene (GAUUGUC) marked in white.The boxes in the lower sequence show the three nucleotides that have been replaced by the nucleotides that are shown at the bottom to give rise to SEQ ID NO: 5. The complementarity between both upper and lower sequences (bars) is shown. B. Shows the sequence of the precursor of SEQ ID NO: 1 pre-miR-219a, of SEQ ID NO: 2, where the mature sequence of hsa-mir-219-5p is highlighted in gray (SEQ ID NO: 1) in the two complementary strands of the pre-miR-219a, a box indicates the three modified nucleotides to design SEQ ID NO: 5. C. Shows SEQ ID NO: 5 (in bold) included in its precursor sequence or SEQ ID NO: 6. Gray shows the modified nucleotides. FIG. 7. Shows the expression of the SCN5A gene in atrial myocardial cells (HL-1) transfected with SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 for 12 hours. Control cells (wt), where the precursor of endogenous hsa-mir-219-5p or miR-219a (SEQ ID NO: 2) was transfected. miR-219a-: Cells transfected with the precursor of miR-219a- (SEQ ID NO: 4), designed to treat arrhythmogenic canalopathies due to a gain in function in the SCN5A gene. miR-219a +: Cells transfected with the precursor of miR-219 + (SEQ ID NO: 6), designed to treat arrhythmogenic canalopathies due to a loss of function in the SCN5A gene. The figure shows that HL-1 cells transfected with SEQ ID NO: 4 show a decrease in SCN5A expression compared to cells transfected with the endogenous precursor of miR-219a (SEQ ID NO: 2), while those Myocardial cells transfected with SEQ ID NO: 6 show increased expression of SCN5A compared to cells transfected with the endogenous precursor of miR-219a (SEQ ID NO: 2).
EJEMPLOS EXAMPLES
A continuación se ilustrará la invención mediante unos ensayos realizados por los inventores, que ponen de manifiesto la especificidad y la efectividad de las secuencias polinucleotídicas de la invención en la modulación de la función del gen SCN5A. Los siguientes ejemplos específicos que se proporcionan en este documento de patente sirven para ilustrar la naturaleza de la presente invención. Estos ejemplos se incluyen solamente con fines ilustrativos y no han de ser interpretados como limitaciones a la invención que aquí se reivindica. Por tanto, los ejemplos descritos más adelante ilustran la invención sin limitar el campo de aplicación de la misma. The invention will now be illustrated by tests carried out by the inventors, which show the specificity and effectiveness of the polynucleotide sequences of the invention in modulating the function of the SCN5A gene. The following specific examples provided in this patent document serve to illustrate the nature of this invention. These examples are included for illustrative purposes only and should not be construed as limitations on the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application.
EJEMPLO 1. Efecto de la expresión de hsa-mir-219-5p en la expresión del gen SCN5A en células miocárdicas atriales. EXAMPLE 1. Effect of the expression of hsa-mir-219-5p on the expression of the SCN5A gene in atrial myocardial cells.
El hsa-mir-219-5p (SEQ ID NO: 1 ) es capaz de unirse a la región 3'UTR del mRNA del gen SCN5A como se muestra en la figura 1 , en concreto mediante la región correspondiente a la secuencia GAUUGUC incluida en la SEQ ID NO: 1 (marcada en negrita en la figura 2A). Este hsa-mir-219-5p se encuentra incluido en la secuencia de su precursor o pre-miR-219a (SEQ ID NO: 2) como se observa en las figuras 2B y 2C. The hsa-mir-219-5p (SEQ ID NO: 1) is capable of binding to the 3 ' UTR region of the mRNA of the SCN5A gene as shown in Figure 1, in particular by the region corresponding to the GAUUGUC sequence included in SEQ ID NO: 1 (marked in bold in Figure 2A). This hsa-mir-219-5p is included in the sequence of its precursor or pre-miR-219a (SEQ ID NO: 2) as seen in Figures 2B and 2C.
En la presente invención, se exploró in vitro el efecto de la sobreexpresión de hsa-mir-219-5p en la actividad del gen Scn5a. Para ello se transfectaron células miocárdicas atriales (HL-1 ) con hsa-mir-219-5p, y se valoraron los niveles de expresión del gen SCN5A en las mismas mediante qRT-PCR. El análisis de la expresión de SCN5A a 6h post-transfección y normalizado con dos controles internos distintos (beta-actina y gapdh) mostró una severa disminución de la expresión del canal de sodio mediada por SCN5A (Figura 3). In the present invention, the effect of overexpression of hsa-mir-219-5p on the activity of the Scn5a gene was explored in vitro. For this, atrial myocardial cells (HL-1) were transfected with hsa-mir-219-5p, and the expression levels of the SCN5A gene were evaluated in them by qRT-PCR. The analysis of the expression of SCN5A at 6h post-transfection and normalized with two different internal controls (beta-actin and gapdh) showed a severe decrease in the expression of the sodium channel mediated by SCN5A (Figure 3).
Para constatar estos datos de forma independiente, se realizaron también ensayos inmunohistoquímicos contra el producto de expresión de SCN5A, en los cuales se observó que existía un cambio sustancial en la localización del canal de sodio codificado por SCN5A tras la transfección con hsa-mir-219-5p. En las células control, la localización del producto de expresión de SCN5A era principalmente citoplasmática y, de forma global, se encontraba localizado en la membrana plasmática, mientras que en las células transfectadas con hsa-mir- 219-5p, el canal de sodio se encontraba retenido en el retículo endoplasmático y/o aparato de Golgi, y la localización citoplasmática y/o de membrana celular se encontraba seriamente disminuida (Figura 4). Por tanto, estos datos estaban en consonancia con los datos obtenidos mediante qRT-PCR. To verify these data independently, immunohistochemical tests were also performed against the expression product of SCN5A, in which it was observed that there was a substantial change in the location of the sodium channel encoded by SCN5A after transfection with hsa-mir-219 -5 p. In the control cells, the location of the SCN5A expression product was mainly cytoplasmic and, overall, was located in the plasma membrane, while in the cells transfected with hsa-mir-219-5p, the sodium channel was was retained in the endoplasmic reticulum and / or Golgi apparatus, and the cytoplasmic and / or cell membrane location It was seriously diminished (Figure 4). Therefore, these data were consistent with the data obtained by qRT-PCR.
Finalmente se evaluó si la transfección de hsa-mir-219-5p condicionaba la capacidad contráctil de los cardiomiocitos en cultivo. De este modo, se contaron las contracciones de los cardiomiocitos controles y de los cardiomiocitos transfectados con hsa-mir-219-5p, y se observó que el ritmo estaba disminuido en un 30% aproximadamente en estos últimos, y que el patrón de contracción era asincrónico (Tabla 1 ). Por tanto, estos datos revelaron que hsa-mir-219-5p puede regular la función del canal de sodio, lo cual es muy importante puesto que supone un mecanismo molecular de fácil manipulación y alta accesibilidad que permite corregir la falta o ganancia de función del canal de sodio cardíaco que subyace a síndromes arritmogénicos como Brugada o QT largo, respectivamente. Finally, it was evaluated whether the transfection of hsa-mir-219-5p conditioned the contractile capacity of cardiomyocytes in culture. Thus, contractions of control cardiomyocytes and cardiomyocytes transfected with hsa-mir-219-5p were counted, and it was observed that the rate was decreased by approximately 30% in the latter, and that the contraction pattern was asynchronous (Table 1). Therefore, these data revealed that hsa-mir-219-5p can regulate the function of the sodium channel, which is very important since it implies a molecular mechanism of easy manipulation and high accessibility that allows to correct the lack or gain of function of the Cardiac sodium channel underlying arrhythmogenic syndromes such as Brugada or long QT, respectively.
Figure imgf000020_0001
Figure imgf000020_0001
Tabla 1. Capacidad contráctil de las células miocárdicas atriales (HL-1 ) control y transfectadas con hsa-mir-219-5p. EJEMPLO 2. Efecto de la expresión de los hsa-mir-219-5p modificados de SEQ ID NO: 3 y de SEQ ID NO: 5 en la expresión del gen SCN5A en células miocárdicas atriales. Table 1. Contractile capacity of atrial myocardial cells (HL-1) control and transfected with hsa-mir-219-5p. EXAMPLE 2. Effect of the expression of modified hsa-mir-219-5p of SEQ ID NO: 3 and SEQ ID NO: 5 on the expression of the SCN5A gene in atrial myocardial cells.
Respecto a la generación de los microRNAs modificados, de SEQ ID NO: 3 y SEQ ID NO: 5, se modificó parcialmente la estructura primaria del precursor pre-miR-219a (figuras 5 y 6), para incrementar o disminuir, respectivamente, su capacidad de unión al RNA mensajero de SCN5A respecto al hsa-mir-219-5p, y de esta forma aumentar (en pacientes con síndrome de Brugada) o disminuir (en pacientes con síndrome de QT largo tipo 3) la función del canal de sodio cardíaco. 2.1. Polinucleótido para el tratamiento de síndrome de QT largo tipo 3. Regarding the generation of the modified microRNAs, of SEQ ID NO: 3 and SEQ ID NO: 5, the primary structure of the pre-miR-219a precursor was partially modified (Figures 5 and 6), to increase or decrease, respectively, its ability to bind to messenger RNA of SCN5A with respect to hsa-mir-219-5p, and thus increase (in patients with long QT syndrome type 3) the sodium channel function cardiac. 2.1. Polynucleotide for the treatment of long QT syndrome type 3.
Se modificaron tres nucleotidos en el pre-miR-219a nativo de acuerdo con la secuencia de la región 3XUTR del mensajero del gen SCN5A, como se muestra en la figura 5A, 5B y 5C, diseñándose así una hebra madura que tenía 12 sitios de unión complementarios al mRNA diana en lugar de los 9 nativos. Por tanto, el precursor de este microRNA, de SEQ ID NO: 4, así diseñado incrementó la eficacia en la reducción de la expresión del gen SCN5A (Figura 7). 2.2. Polinucleótido para el tratamiento de síndrome de Brugada. Three nucleotides were modified in the native pre-miR-219a according to the sequence of the 3 X UTR region of the messenger of the SCN5A gene, as shown in Figure 5A, 5B and 5C, thus designing a mature strand that had 12 sites of binding complementary to the target mRNA instead of the 9 natives. Therefore, the precursor of this microRNA, of SEQ ID NO: 4, thus designed increased the efficiency in reducing the expression of the SCN5A gene (Figure 7). 2.2. Polynucleotide for the treatment of Brugada syndrome.
Por otro lado, se modificaron tres nucleotidos comprendidos en la secuencia GAUUGUC del hsa-mir-219-5p contenido en el precursor pre-miR-219a de acuerdo con la secuencia de la región 3XUTR del mensajero del gen SCN5A, como se muestra en la figura 6A, 6B y 6C, diseñándose así una hebra madura que tenía únicamente 6 sitios de unión complementarios, y además dispersos, al mRNA diana en lugar de los 9 nativos. Por tanto, el precursor de este microRNA, de SEQ ID NO: 6, así diseñado incrementó la expresión del gen SCN5A respecto al microRNA-219a nativo (Figura 7). On the other hand, three nucleotides comprised in the GAUUGUC sequence of the hsa-mir-219-5p contained in the pre-miR-219a precursor were modified according to the sequence of the 3 X UTR region of the SCN5A gene messenger, as shown in Figure 6A, 6B and 6C, thus designing a mature strand that had only 6 complementary, and also dispersed, binding sites to the target mRNA instead of the native 9. Therefore, the precursor of this microRNA, of SEQ ID NO: 6, thus designed increased the expression of the SCN5A gene with respect to the native microRNA-219a (Figure 7).

Claims

REIVINDICACIONES
1 . Uso de un polinucleótido aislado que comprende una secuencia de nucleótidos con al menos un 85% de identidad con respecto a la longitud completa de la secuencia SEQ ID NO: 1 para la elaboración de un medicamento. one . Use of an isolated polynucleotide comprising a nucleotide sequence with at least 85% identity with respect to the full length of the sequence SEQ ID NO: 1 for the preparation of a medicament.
2. Uso de un polinucleótido aislado según la reivindicación 1 donde el polinucleótido comprende una secuencia de nucleótidos con al menos un 90% de identidad con respecto a la longitud completa de la secuencia SEQ ID NO: 1 . 2. Use of an isolated polynucleotide according to claim 1 wherein the polynucleotide comprises a nucleotide sequence with at least 90% identity with respect to the full length of the sequence SEQ ID NO: 1.
3. Uso de un polinucleótido aislado según la reivindicación 2 donde el polinucleótido comprende una secuencia de nucleótidos con al menos un 95% de identidad con respecto a la longitud completa de la secuencia SEQ ID NO: 1 . 3. Use of an isolated polynucleotide according to claim 2 wherein the polynucleotide comprises a nucleotide sequence with at least 95% identity with respect to the full length of the sequence SEQ ID NO: 1.
4. Uso de un polinucleótido aislado según la reivindicación 3 donde el polinucleótido comprende una secuencia de nucleótidos con al menos un 98% de identidad con respecto a la longitud completa de la secuencia SEQ ID NO: 1 . 4. Use of an isolated polynucleotide according to claim 3 wherein the polynucleotide comprises a nucleotide sequence with at least 98% identity with respect to the full length of the sequence SEQ ID NO: 1.
5. Uso de un polinucleótido aislado según la reivindicación 4 donde el polinucleótido comprende la secuencia de nucleótidos SEQ ID NO: 1 . 5. Use of an isolated polynucleotide according to claim 4 wherein the polynucleotide comprises the nucleotide sequence SEQ ID NO: 1.
6. Uso de un polinucleótido aislado según la reivindicación 5 donde el polinucleótido es SEQ ID NO: 2. 6. Use of an isolated polynucleotide according to claim 5 wherein the polynucleotide is SEQ ID NO: 2.
7. Uso de un polinucleótido aislado según la reivindicación 1 donde el polinucleótido comprende la secuencia de nucleótidos SEQ ID NO: 3. 7. Use of an isolated polynucleotide according to claim 1 wherein the polynucleotide comprises the nucleotide sequence SEQ ID NO: 3.
8. Uso de un poiinucleotido aislado según la reivindicación 7 donde el poiinucleotido es SEQ ID NO: 4. 8. Use of an isolated polynucleotide according to claim 7 wherein the polynucleotide is SEQ ID NO: 4.
9. Uso de un poiinucleotido aislado según la reivindicación 1 donde el poiinucleotido comprende la secuencia de nucleótidos SEQ ID NO: 5. 9. Use of an isolated polynucleotide according to claim 1 wherein the polynucleotide comprises the nucleotide sequence SEQ ID NO: 5.
10. Uso de un poiinucleotido aislado según la reivindicación 9 donde el poiinucleotido es SEQ ID NO: 6. 10. Use of an isolated polynucleotide according to claim 9 wherein the polynucleotide is SEQ ID NO: 6.
1 1 . Uso de un poiinucleotido aislado como se define en cualquiera de las reivindicaciones 1 a 10 para la elaboración de un medicamento para el tratamiento de canalopatías arritmogénicas. eleven . Use of an isolated polynucleotide as defined in any one of claims 1 to 10 for the preparation of a medicament for the treatment of arrhythmogenic canalopathies.
12. Uso de un poiinucleotido aislado según la reivindicación 1 1 donde las canalopatías arritmogénicas se deben a alteraciones en los canales de sodio. 12. Use of an isolated polynucleotide according to claim 1, wherein the arrhythmogenic canalopathies are due to alterations in the sodium channels.
13. Uso de un poiinucleotido aislado según la reivindicación 12 donde la canalopatía arritmogénica es el síndrome de QT largo tipo 3. 13. Use of an isolated polynucleotide according to claim 12 wherein the arrhythmogenic canalopathy is the long QT syndrome type 3.
14. Uso de un poiinucleotido aislado como se define en cualquiera de las reivindicaciones 9 ó 10 para la elaboración de un medicamento para el tratamiento de síndrome de Brugada. 14. Use of an isolated polynucleotide as defined in any of claims 9 or 10 for the preparation of a medicament for the treatment of Brugada syndrome.
15. Composición farmacéutica que comprende un poiinucleotido aislado como se define en cualquiera de las reivindicaciones 1 a 8. 15. Pharmaceutical composition comprising an isolated polynucleotide as defined in any one of claims 1 to 8.
16. Composición farmacéutica según la reivindicación 15 que además comprende otro principio activo. 16. Pharmaceutical composition according to claim 15 further comprising another active ingredient.
17. Composición farmacéutica según cualquiera de las reivindicaciones 15 ó 16 que además comprende un vehículo farmacéuticamente aceptable. 17. Pharmaceutical composition according to any of claims 15 or 16 further comprising a pharmaceutically acceptable carrier.
18. Composición farmacéutica que comprende un polinucleótido aislado como se define en cualquiera de las reivindicaciones 9 ó 10. 18. Pharmaceutical composition comprising an isolated polynucleotide as defined in any of claims 9 or 10.
19. Composición farmacéutica según la reivindicación 18 que además comprende otro principio activo. 19. Pharmaceutical composition according to claim 18 further comprising another active ingredient.
20. Composición farmacéutica según cualquiera de las reivindicaciones 18 ó 19 que además comprende un vehículo farmacéuticamente aceptable. 20. Pharmaceutical composition according to any of claims 18 or 19 further comprising a pharmaceutically acceptable carrier.
21. Uso de la composición farmacéutica según cualquiera de las reivindicaciones 15 a 20 para la elaboración de un medicamento. 21. Use of the pharmaceutical composition according to any of claims 15 to 20 for the preparation of a medicament.
22. Uso de la composición farmacéutica según la reivindicación 21 donde el medicamento es para el tratamiento de canalopatías arritmogénicas. 22. Use of the pharmaceutical composition according to claim 21 wherein the medicament is for the treatment of arrhythmogenic canalopathies.
23. Uso de la composición farmacéutica según la reivindicación 22 donde las canalopatías arritmogénicas se deben a alteraciones en los canales de sodio. 23. Use of the pharmaceutical composition according to claim 22 wherein the arrhythmogenic canalopathies are due to alterations in the sodium channels.
24. Uso de la composición farmacéutica según la reivindicación 23 donde la canalopatía arritmogénica es el síndrome de QT largo tipo 3. 24. Use of the pharmaceutical composition according to claim 23 wherein the arrhythmogenic canalopathy is the long QT syndrome type 3.
25. Uso de la composición farmacéutica según cualquiera de las reivindicaciones 18 a 20 para la elaboración de un medicamento para el tratamiento de síndrome de Brugada. 25. Use of the pharmaceutical composition according to any of claims 18 to 20 for the preparation of a medicament for the treatment of Brugada syndrome.
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