ES2374244B1 - MicroRNA useful for the treatment of arrhythmogenic canalopathies. - Google Patents

Abstract

MocroRNA useful for the treatment of arrhythmogenic canalopathies. # 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 function of the cardiac sodium channel, more preferably of type 3 long QT syndrome and Brugada syndrome. These polynucleotides are capable of modulating the function of the cardiac sodium channel through their binding to the mRNA of the SCN5A gene.

Description

MicroRNA useful for the treatment of arrhythmogenic canalopathies.

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.

Prior art

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 us to identify various 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 cardiac cell action. 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 have a characteristic electrocardiogram: ST segment elevation in the right precordial leads and block 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. Saf fi tz, 2005, Circulation, 112: 36723674).

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): 10471049) and also exert a systemic effect on it, aimed at alleviating but not restoring cardiac pathophysiology.

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).

Another possibility for the treatment of arrhythmogenic canalopathies related to dysfunctions in the sodium channels is the use of polypeptides encoded by polynucleotides comprising the sequence of the SCN5A gene with mutations that are present in healthy individuals (US2008214457 A1).

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 found in alterations of function gain in the channels sodium (G. Alex Papadatos, et al., 2002, PNAS, Vol. 99 (9): 6210-6215).

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'UTR) 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 merely limit the symptomatology of the disease. On the other hand, these new therapeutic strategies should avoid the complications of current drug or cardioverter therapies.

Description of the invention

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 development of useful medicines for the treatment of arrhythmogenic canalopathies,

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preferably of 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.

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.

The polynucleotide sequences of the invention or polynucleotides of the invention, preferably, SEQ ID NO: 1, SEQ ID NO: 2, SEQ 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 that codes for 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. 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, LQT3 syndrome.

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 those arrhythmogenic canalopathies whose cause 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 syndrome rome of LQT3.

Therefore, a first aspect of the invention refers 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.

The terms "nucleotide sequence", "nucleotide sequence", "nucleic acid", "oligonucleotide" and "polynucleotide" are used interchangeably herein and refer to a polymeric form of nucleotides of any length that may or may not be chemical or biochemically modified. They refer, therefore, to any polyiribonucleotide or polydeoxyribonucleotide, both single-stranded and double-stranded.

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.

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.

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 native microRNA sequence

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o SEQ ID NO: 1. Therefore, SEQ ID NO: 5 is also useful for the preparation of medicines for the treatment of arrhythmogenic canalopathies, preferably those arrhythmogenic canalopathies whose cause is alterations in the sodium channels, more preferably when 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 whom 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.

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.

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% 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 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: 5oeldeSEQ 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 capable of 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 alterations of loss of function in the sodium channels.

Therefore, another aspect of the invention refers 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 due to alterations in the sodium channels is the long QT syndrome type 3.

Another aspect of the invention refers to the use of the polynucleotide of the invention comprising the nucleotide sequence SEQ ID NO: 5 or the polynucleotide of the invention of sequence SEQ ID NO: 6 for the preparation of a medicament for the treatment of syndrome of Brugada

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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 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.

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 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 (of 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 which it is possible to make a potential diagnosis of the altered currents (Antzelevitch et al. , 2005a, 2005b, Circulation, 111: 659-670); or by PCR of the genes that code for the sodium channels, preferably of the SCN5A gene, and subsequent sequencing, which allows detecting 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).

The “type 3 long QT syndrome” or “LQT3” is characterized by an alteration in ventricular repolarization translated in the electrocardiogram by an elongation in the corrected QT interval (QTc) by Bazzet's formula

√

(QTc = QT / R) ≥ 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).

The "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, seven types of Brugada syndrome have been identified, the most frequent being, but not limited to, Brugada syndrome type 1 or Brugada syndrome type 2.

Another aspect of the invention refers to a pharmaceutical composition, hereinafter, "first pharmaceutical composition of the invention", comprising 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 less than 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: 3odeSEQ ID NO: 4.

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.

Another aspect of the invention refers to a pharmaceutical composition, hereinafter, "second pharmaceutical composition of the invention", which comprises the polynucleotide of the invention comprising the nucleotide sequence SEQ ID NO: 5odeSEQ ID NO: 6.

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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.

As used herein, the term "active ingredient", "active substance", "pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient" means any component that potentially provides a pharmacological activity or other different effect on 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 includes those components that promote a chemical change in the elaboration of the drug and are present therein in a planned modified manner that provides the specific activity or effect.

The "adjuvants" and "pharmaceutically acceptable carriers" refer to those substances, or combination of substances, known in the pharmaceutical sector, used in the preparation of pharmaceutical administration forms 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.

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 long Q3 syndrome type 3 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.

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 paint, infusion pump or catheter route.

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.

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.

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 those skilled in the art, other objects, advantages and characteristics of the invention will be derived partly from the description and partly 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.

Description of the fi gures

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: region 3’UTR of the messenger of the SCN5A gene (position 1.416-1.422, 5’-3 ’). Lower sequence: sequence of hsa-mir-219-5p (SEQ ID NO: 1, 3’-5 ’) with the complementary sequence to the SCN5A gene messenger (GAUUGUC) marked in white. Black bars: complementary nucleotides.

Fig. 2. Shows the sequence of the mature hsa-mir-219-5p and its precursor. A. Shows the sequence of mature hsa-mir219-5p (SEQ ID NO: 1). In bold, the sequence that shows complementarity with the 3’UTR region of the SCN5A gene messenger is highlighted, that is, the GAUUGUC sequence. B. Shows the sequence of pre-miR-219a (SEQ ID NO: 2). In bold, the region corresponding to the mature hsa-mir-219-5p stands out (SEQ ID NO: 1). C. Shows the outline of the fork structure of the pre-miR-219a. Gray highlights the mature sequence of hsamir-219-5p (SEQ ID NO: 1) in the two complementary strands of pre-miR-219a (SEQ ID NO: 2).

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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-21 9-5p 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. It shows the results of immunohistochemical tests against the expression product of the SCN5A gene in control cells (left panel) and in cells that were transfected by 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. 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: region 3’UTR of the messenger of the SCN5A gene (position 1.416-1.422, 5’-3 ’). Lower sequence: sequence of hsa-mir-219-5p (SEQ ID NO: 1, 3’-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 to design SEQ ID NO: 3 are indicated. C. Shows SEQ ID NO: 3 (in bold) included in its precursor sequence or SEQ ID NO: 4. Gray modi fi ed nucleotides stand out.

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. A. Upper sequence: region 3’UTR of the messenger of the SCN5A gene (position 1.416-1.422, 5’-3 ’). Lower sequence: sequence of hsa-mir-219-5p (SEQ ID NO: 1, 3’-5 ’) with the complementary sequence to the SCN5A gene messenger (GAUUGUC) marked in white. The boxes in the lower sequence show the three nucleotides that have been replaced by the nucleotides shown in the lower part 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 HL1 cells transfected with SEQ ID NO: 4 show a decrease in SCN5A expression compared with cells transfected with the endogenous precursor of miR-219a (SEQ ID NO: 2), while myocardial cells Transfected with SEQ ID NO: 6 show increased expression of SCN5A compared to cells transfected with the endogenous miR-219a precursor (SEQ ID NO: 2).

Examples

The invention will now be illustrated by tests carried out by the inventors, which demonstrate 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 the present invention. These examples are included for illustrative purposes only and should not be construed as limitations to the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application.

Example 1

Effect of hsa-mir-219-5p expression on SCN5A gene expression 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.

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 levels were assessed

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of expression of the SCN5A gene therein by qRT-PCR. Analysis of the expression of SCN5A at 6 h posttransfection 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).

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 it was retained in the endoplasmic reticulum and / or Golgi apparatus, and the cytoplasmic and / or cell membrane location was severely diminished (Figure 4). Therefore, these data were consistent with the data obtained by qRT-PCR.

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.

TABLE 1

Contractile capacity of atrial myocardial cells (HL-1) control and transfected with hsa-mir-219-5p

Example 2

Effect of expression of the 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

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 (figures 5 and 6) was partially modified, to increase or decrease, respectively, its ability to bind to the 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

Three nucleotides were modified in the native pre-miR-219a according to the sequence of the 3'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

On the other hand, three nucleotides comprised in the GAUUGUC sequence of hsa-mir-219-5p contained in the pre-miR-219a precursor were modified according to the sequence of the 3'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 binding sites, and also dispersed, to the target mRNA instead of the 9 native ones. 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).

ES 2 374 244 A1

Claims (25)

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.

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.

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.

Four.

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.

Use of an isolated polynucleotide according to claim 4 wherein the polynucleotide comprises the nucleotide sequence SEQ ID NO: 1.

6. Use of an isolated polynucleotide according to claim 5 wherein the polynucleotide is SEQ ID NO: 2.

7.

Use of an isolated polynucleotide according to claim 1 wherein the polynucleotide comprises the nucleotide sequence SEQ ID NO: 3.

8. Use of an isolated polynucleotide according to claim 7 wherein the polynucleotide is SEQ ID NO: 4.

9.

Use of an isolated polynucleotide according to claim 1 wherein the polynucleotide comprises the nucleotide sequence SEQ ID NO: 5.

10. Use of an isolated polynucleotide according to claim 9 wherein the polynucleotide is SEQ ID NO: 6.

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.

Use of an isolated polynucleotide according to claim 11 wherein the arrhythmogenic canalopathies are due to alterations in the sodium channels.

13.

Use of an isolated polynucleotide according to claim 12 wherein the arrhythmogenic canalopathy is type 3 long QT syndrome.

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.

fifteen.

Pharmaceutical composition comprising an isolated polynucleotide as defined in any of claims 1-8.

16.

Pharmaceutical composition according to claim 15 further comprising a pharmaceutically acceptable carrier.

17.

Pharmaceutical composition comprising an isolated polynucleotide as defined in any of claims 9 or 10.

18.

Pharmaceutical composition according to claim 17 further comprising a pharmaceutically acceptable carrier.

19.

Use of the pharmaceutical composition according to any of claims 15 to 18 for the preparation of a medicament.

twenty.

Use of the pharmaceutical composition according to claim 19 wherein the medicament is for the treatment of arrhythmogenic canalopathies.

twenty-one.

Use of the pharmaceutical composition according to claim 20 wherein the arrhythmogenic canalopathies are due to alterations in the sodium channels.

22

Use of the pharmaceutical composition according to claim 21 wherein the arrhythmogenic canalopathy is the long QT syndrome type 3.

2. 3.

Use of the pharmaceutical composition according to any of claims 17 or 18 for the preparation of a medicament for the treatment of Brugada syndrome.

ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 SEQUENCE LIST <110> University of Jaén <120> "microRNA useful for the treatment of arrhythmogenic canalopathies" <130> ES1881.6 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 21 <212> RNA <213> Homo sapiens <400> 1 <210> 2 <211> 110 <212> RNA <213> Homo sapiens <400> 2 <210> 3 <211> 21 <212> RNA <213> Artificial sequence <220> <223> Polynucleotide comprising 3 complementary sites more than native hsa-mir-219-5p (SEQ ID NO: 1) with the mRNA of the SCN5A gene <400> 3 <210> 4 <211> 110 <212> RNA <213> Artificial sequence <220> <223> Precursor of SEQ ID NO: 3 ES 2 374 244 A1 <400> 4 <210> 5 <211> 21 <212> RNA <213> Artificial sequence <220> <223> Polynucleotide comprising 3 complementary sites less than the native hsa-mir-219-5p (SEQ ID NO: 1) with the mRNA of the SCN5A gene <400> 5 <210> 6 <211> 110 <212> RNA <213> Artificial sequence <220> <223> Precursor of SEQ ID NO: 5 <400> 6 <210> 7 <211> 102817 <212> DNA <213> Homo sapiens ES 2 374 244 A1 <400> 7 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 ES 2 374 244 A1 SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 201031205 SPAIN Date of submission of the application: 02.08.2010 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: See Additional Sheet RELEVANT DOCUMENTS

Category

56 Documents cited Claims Affected

X

US 20100010073 (JULIUS-MAXIMILIANS-UNIVERSITÄT WÜRZBURG) 14.01.2010, the whole document. 1-5,15,16,19

TO

XIAOBIN LUO et al., "Regulation of human cardiac ion channel genes by microRNAs: theoretical perspective and pathophysiological implications" Cellular Physiology and Biochemistry (May 18, 2010) vol 25: No. 6; pages 571-586; DOI: 10.1159 / 000315076; whole document. 1-23

TO

BLANCHE SCHOROEN et al., "MicroRNAs and beyond: the heart reveals its treasures" Hypertension (October 19, 2009) vol. 54; pages 1189-1194; DOI: 10.1161 / HYPERTENSIONAHA.109.133942; ISSN 0194-911X (online ISSN 1524-4563); whole document. 1-23

TO

HEATHER S. DUFFY “The ever-shrinking world of cardiac ion channel remodeling: the role of microRNAs in heart disease” Heart Rhythm Society (2009) Vol. 6; No. 12; pages 1810-1811; DOI: 10.1016 / j.hrthm. 2009.09.021; ISSN 1547-5271; whole document. 1-23

TO

STANLEY et al., "Ion-channel mRNA-expression profiling: insights into cardiac remodeling and arrhythmic substrates" Journal of Molecular and Cellular Cardiology (July 23, 2009) Vol. 48; pages 96-105; DOI: 10.1016 / j.yjmcc.2009.07.016; whole document. 1-23

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 13.01.2012

Examiner M. M. García Coca Page 1/4

REPORT OF THE STATE OF THE TECHNIQUE Application number: 201031205 CLASSIFICATION OBJECT OF THE APPLICATION A61K48 / 00 (2006.01) C07H21 / 02 (2006.01) A61P9 / 00 (2006.01) Minimum documentation sought (classification system followed by classification symbols) A61K, C07H, A61P Electronic databases consulted during the search (name of the database and, if possible, terms of search used) INVENTIONS, EPODOC State of the Art Report Page 2/4  WRITTEN OPINION Application number: 201031205 Date of Written Opinion: 13.01.2012 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims Claims 6-23 1-5 IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims Claims 6-14, 17, 18, 20-23 1-5, 15, 16, 19 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/4  WRITTEN OPINION Application number: 201031205 1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

US 2010/0010073 (JULIUS-MAXIMILIANS-UNIVERSITÄT WÜRZBURG) 01/14/2010

2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The object of the invention, as set forth in claims 1-23, relates to the use of the hsa-mir-219-5p microRNA, of the modified polynucleotides derived therefrom, or of its precursors, for the preparation of a medicament for the treatment of arrhythmogenic canalopathies that are due to alterations in the sodium channels, more specifically, for the treatment of long QT syndrome type 3 and for Brugada syndrome (reiv. 1-14). The subject of the invention is also a pharmaceutical composition containing the hsa-mir-219-5p microRNA, of the modified polynucleotides derived therefrom, or of its precursors (reiv. 15-18) and their use for the preparation of a medicament for the treatment of arrhythmogenic canalopathies that are due to alterations in the sodium channels, more specifically, for the treatment of long QT syndrome type 3 and for Brugada syndrome (reiv. 19-23). 1. Novelty (art. 6.1 of Law 11/1986 on Patents). SEQ ID NO: 1 is disclosed in various documents, such as document D01, where SEQ ID NO: 1 of the application corresponds to SEQ ID NO: 26 of document D01. This document discloses the use of microRNAs (miRNAs) for the diagnosis, prophylaxis and treatment of heart disease. In particular, the use of such microRNAs for the preparation of a medicament for both the treatment and for the diagnosis of such diseases is disclosed (see abstract, page 1 paragraphs [0001] and [0002], page 2 paragraphs [0028] and [0029] and claims 1 and 2). Therefore, in view of what is disclosed in document D01, the object of the invention, as set out in claims 1-5, is novel in the sense of art. 6.1 of Law 11/1986 on Patents and therefore on inventive activity as established in art. 8.1 of Law 11/1986 on Patents. 2. Inventive Activity (art. 8.1 of Law 11/1986 on Patents). Document D01 discloses the use of microRNAs (miRNAs) for the diagnosis, prophylaxis and treatment of heart disease. In particular, the use of such microRNAs for the preparation of a medicament for both the treatment and for the diagnosis of such diseases is disclosed (see abstract, page 1 paragraphs [0001] and [0002], page 2 paragraphs [0028] and [0029] and claims 1 and 2). It would be obvious to one skilled in the art to obtain a pharmaceutical composition with the microRNA of the invention and a pharmaceutically acceptable excipient for making a medicament. Therefore, in view of what is disclosed in document D01, the object of the invention, as set out in claims 15, 16 and 19 lacks inventive activity as set forth in art. 8.1 of Law 11/1986 on Patents. State of the Art Report Page 4/4