WO2016083625A1 - Acide nucléique codant pour la triadine destinée à la prévention et/ou au traitement d'une insuffisance cardiaque - Google Patents

Acide nucléique codant pour la triadine destinée à la prévention et/ou au traitement d'une insuffisance cardiaque Download PDF

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WO2016083625A1
WO2016083625A1 PCT/EP2015/078109 EP2015078109W WO2016083625A1 WO 2016083625 A1 WO2016083625 A1 WO 2016083625A1 EP 2015078109 W EP2015078109 W EP 2015078109W WO 2016083625 A1 WO2016083625 A1 WO 2016083625A1
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Prior art keywords
triadin
sequence
vector
nucleic acid
sequence seq
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PCT/EP2015/078109
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English (en)
Inventor
Christophe HEYMES
Isabelle Marty
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Institut National De La Sante Et De La Recherche Medicale (Inserm)
Universite Grenoble Alpes
Universite Paul Sabatier Toulouse Iii
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Publication of WO2016083625A1 publication Critical patent/WO2016083625A1/fr

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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
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/06—Antiarrhythmics
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to a method for preventing and/or treating heart failure, in particular using gene therapy.
  • Heart failure is a serious public health issue with a growing prevalence in industrialized countries. Heart failure occurs when the heart is unable to pump sufficiently to maintain a blood flow that meets the needs of the body. Symptoms generally include shortness of breath, excessive tiredness and leg swelling. Heart failure often results from a coronary artery disease (including myocardial infarction), high blood pressure or cardiomyopathy. Heart failure generally begins with an adaptive myocardial remodeling leading to a Left Ventricular Hypertrophy (LVH) and is later associated with arrhythmias.
  • LHL Left Ventricular Hypertrophy
  • Heart failure is characterized by multiple defects in Ca 2+ -handling proteins involved in excitation-contraction coupling.
  • contractility indeed depends on calcium release from sarcoplasmic reticulum. This release is controlled by a macro-molecular complex, composed by ryanodine receptor (RyR2) and its associated regulatory protein junctin (JCN), calsequestrin (CASQ2) and triadin. Alterations of this complex, as evidenced by disturbed calcium release outside the sarcoplasmic reticulum, is often observed during the development of heart failure, resulting in a LVH associated with contractile dysfunction and fatal ventricular arrhythmias.
  • RyR2 ryanodine receptor
  • JCN regulatory protein junctin
  • CASQ2 calsequestrin
  • Roux-Buisson et al. disclose the identification of three mutations in the triadin gene, in a panel of 97 patients suffering from CPVT (Catecholaminergic Polymorphism Ventricular Tachycardia) not involving a mutation of the genes RyR2 and CASQ2. These mutations lead to the absence of expression of triadin or to an unstable triadin that is destroyed in the body.
  • CPVT is a rare inherited heart rhythm disturbance.
  • Treatments of heart failure usually include administration of an angiotensin converting enzyme (ACE) inhibitor, angiotensin receptor blocker, beta adrenergic blocker and/or diuretic.
  • ACE angiotensin converting enzyme
  • angiotensin receptor blocker angiotensin receptor blocker
  • beta adrenergic blocker angiotensin receptor blocker
  • diuretic adrenergic blocker
  • the Inventors have surprisingly found that the use of triadin in gene therapy enables to prevent and/or treat heart failure.
  • triadin accelerates the transition towards heart failure, acting on left ventricular hypertrophy (LVH) and contractile dysfunction, in three cardiac pathological experimental models on mice with triadin gene invalidation: 1 . a pressure overload with transversal aorta constriction (TAC), 2. a chronic infusion of catecholamines (Isoproterenol, Iso) with osmotic minipumps and 3. a chronic HF with myocardial infarction (Ml). More particularly, in response to TAC or a chronic infusion of catecholamines, triadin knock-out mice develop a greater LVH compared to wild-type mice. Also with TAC, triadin knock-out mice show an eccentric LVH associated with a severe cardiac dysfunction, as compared to wild-type mice. Besides, the lack of triadin increases the occurrence of post-ischemic cardiac arrhythmias.
  • TAC transversal aorta constriction
  • Ml myocardial infarction
  • the re-expression of triadin in the heart by gene therapy for example using an AAV9 vector, in the TAC model on mice with triadin gene invalidation, enables to reduce cardiac hypertrophy and pulmonary edema, as well as to prevent cardiac dysfunction, 28 days after TAC surgery. Delivery of a nucleic acid encoding triadin in triadin knock-out mice therefore prevents adverse remodeling and the associated cardiac dysfunction.
  • the therapeutic effect of triadin expression by gene therapy was also assessed in an animal model of heart failure further to a myocardial infarction in wild type mice.
  • the animal models used in the present invention are very interesting, since they represent more than 85% of the cases of heart failure in humans.
  • triadin in heart is essential, as shown in the CPVT cases involving mutations in the triadin gene, the fact that the solely expression of triadin would allow preventing and/or treating heart failure was unexpected.
  • the use of gene therapy is particularly advantageous for the prevention and/or treatment of heart failure, since one single injection of a nucleic acid encoding triadin is sufficient to provide an efficient treatment for several months or years.
  • the present invention thus relates to a nucleic acid encoding triadin for use in the prevention and/or treatment of heart failure.
  • Heart failure is preferably characterized by cardiac arrhythmia, cardiac hypertrophy, myocardial fibrosis and/or cardiac post-ischemia contractile dysfunction.
  • the triadin encoded by the nucleic acid for use according to the invention comprises or consists of a sequence selected in the group consisting of sequence SEQ ID NO: 2, a sequence at least 80% identical to sequence SEQ ID NO: 2, sequence SEQ ID NO: 6, a sequence at least 80% identical to sequence SEQ ID NO: 6, sequence SEQ ID NO: 8 and a sequence at least 80% identical to sequence SEQ ID NO: 6.
  • the nucleic acid encoding triadin for use according to the invention comprises or consists of a sequence selected in the group consisting of sequence SEQ ID NO: 1 , a sequence at least 80% identical to sequence SEQ ID NO: 1 , sequence SEQ ID NO: 5, a sequence at least 80% identical to sequence SEQ ID NO: 5, sequence SEQ ID NO: 7 and a sequence at least 80% identical to sequence SEQ ID NO: 7.
  • the nucleic acid encoding triadin for use according to the invention is preferably provided in the form of a vector comprising said nucleic acid.
  • Said vector is preferably cardiotropic.
  • said vector is a non-viral vector or a viral vector, said viral vector being preferably selected in the group consisting of a retrovirus vector, a lentivirus vector, an adenovirus vector and an adeno-associated virus vector.
  • a preferred vector for use according to the invention is an adeno-associated virus selected in the group consisting of AAV1 , AAV2, AAV6, AAV8, AAV9 and a hybrid AAV vector thereof.
  • the nucleic acid or the vector for use according to the invention is administered by the arterial, venous, intramyocardial, intrapericardial and/or intraperitoneal route.
  • the present invention also relates to an adeno-associated virus vector comprising a nucleic acid encoding triadin, wherein said nucleic acid comprises or consists of a sequence selected in the group consisting of sequence SEQ ID NO: 1 , a sequence at least 80% identical to sequence SEQ ID NO: 1 , sequence SEQ ID NO: 5, a sequence at least 80% identical to sequence SEQ ID NO: 5, sequence SEQ ID NO: 7 and a sequence at least 80% identical to sequence SEQ ID NO: 7.
  • Triadin is a transmembrane protein.
  • the human triadin gene is located on chromosome 6 and comprises 41 exons.
  • the sequence of the human triadin gene is for example the sequence of access number MIM#603283 or NG_030438.1 (NCBI database), as available on November 27, 2014.
  • TRDN gene there are four triadin isoforms resulting from an alternative splicing of a single triadin gene, called TRDN gene.
  • isoforms of triadin are tissue specific. Isoforms Trisk 95 and Trisk 51 are indeed exclusively expressed on skeletal muscle, whereas isoform Trisk 32, also called
  • CT1 is mainly expressed in cardiac muscle.
  • the isoform Trisk 32 (Cardiac Triadin Isoform 1 ) is the shortest isoform of a molecular weight of 35 kDa (non glycosylated form) or 40 kDa (glycosylated form).
  • triadin associates with the ryanodine receptor (RyR2), calsequestrin
  • CASQ2 CASQ2
  • junctin a macromolecular Ca 2+ signaling complex in the junctional sarcoplasmic reticulum.
  • triadin is the isoform Trisk 32.
  • the triadin for use according to the invention is a functional triadin.
  • triadin or "triadin with a functional activity” it is herein meant that triadin is expressed in the triad/dyade junction, colocalised and associated to RyR2.
  • triadin The functional activity of triadin may be assessed by any method well-known by the skilled person, for example, by immunofluorescent labelling and immunoprecipitation.
  • Triadin is preferably of mammalian origin.
  • triadin of mammalian origin includes a mammalian triadin or a variant thereof.
  • a variant of a mammalian triadin is a functional triadin that has a sequence at least 80% identical to said mammalian triadin, preferably at least 85% identical to said mammalian triadin, more preferably at least 90% identical to said mammalian triadin, still more preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to said mammalian triadin.
  • mammalian includes human and non-human mammalian.
  • non-human mammalian for example includes rat, murine, pig, cat, dog, rabbit or primate.
  • the human triadin trisk 32 may be of sequence SEQ ID NO: 6 or a sequence at least 80% identical to sequence SEQ ID NO: 6, preferably at least 85% identical to sequence SEQ ID NO: 6, more preferably at least 90% identical to sequence SEQ ID NO: 6, still more preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to sequence SEQ ID NO: 6.
  • said human triadin trisk 32 comprises the amino acids 217 to 231 of sequence SEQ ID NO: 6.
  • Sequence SEQ ID NO: 6 corresponds to sequence of access number JN900469 in the Gen Bank database, as available on November 27, 2014.
  • the human triadin trisk 32 may be of sequence SEQ ID NO: 16 or a sequence at least 80% identical to sequence SEQ ID NO: 16, preferably at least 85% identical to sequence SEQ ID NO: 16, more preferably at least 90% identical to sequence SEQ ID NO: 16, still more preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to sequence SEQ ID NO: 16.
  • said human triadin trisk 32 comprises the amino acids 217 to 231 of sequence SEQ ID NO: 16.
  • the rat triadin trisk 32 may be of sequence SEQ ID NO: 2 or a sequence at least 80% identical to sequence SEQ ID NO: 2, preferably at least 85% identical to sequence SEQ ID NO: 2, more preferably at least 90% identical to sequence SEQ ID NO: 2, still more preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to sequence SEQ ID NO: 2.
  • Sequence SEQ ID NO: 2 corresponds to the sequence of access number AJ812276 in the GenBank database, as available on November 27, 2014.
  • the rat triadin trisk 32 may be of sequence SEQ ID NO: 14 or a sequence at least 80% identical to sequence SEQ ID NO: 14, preferably at least 85% identical to sequence SEQ ID NO: 14, more preferably at least 90% identical to sequence SEQ ID NO: 14, still more preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to sequence SEQ ID NO: 14.
  • Sequence SEQ ID NO: 14 corresponds to the sequence of access number AF220558.1 in the GenBank database, as available on November 27, 2014.
  • the murine triadin trisk 32 may be of sequence SEQ ID NO: 8 or a sequence at least 80% identical to sequence SEQ ID NO: 8, preferably at least 85% identical to sequence SEQ ID NO: 8, more preferably at least 90% identical to sequence SEQ ID NO: 8, still more preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to sequence SEQ ID NO: 8.
  • Sequence SEQ ID NO: 8 corresponds to the sequence of access number AF223415 in the Gen Bank database, as available on November 27, 2014.
  • the triadin comprises or consists of a sequence selected in the group consisting of sequence SEQ ID NO: 2, a sequence at least 80% identical to sequence SEQ ID NO: 2, sequence SEQ ID NO: 6, a sequence at least 80% identical to sequence SEQ ID NO: 6, sequence SEQ ID NO: 8 and a sequence at least 80% identical to sequence SEQ ID NO: 8.
  • the triadin comprises or consists of a sequence selected in the group consisting of sequence SEQ ID NO: 2, sequence SEQ ID NO: 14, sequence SEQ ID NO: 6, sequence SEQ ID NO: 16 and sequence SEQ ID NO: 8.
  • an amino acid sequence "at least 80%, 85%, 90%, 95%, 96%,
  • 97%, 98% or 99% identical to a reference sequence may comprise mutations, such as deletions, insertions and/or substitutions compared to the reference sequence.
  • substitutions preferably corresponds to a conservative substitution as indicated in the Table 1 below.
  • sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence only differs from the reference sequence by conservative substitutions.
  • amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence corresponds to a naturally-occurring allelic variant of the reference sequence.
  • the amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence corresponds to a homologous sequence derived from another mammalian species than the reference sequence.
  • the amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence differs from the reference sequence by conservative substitutions and/or corresponds to a homologous sequence derived from another mammalian species than the reference sequence.
  • sequence at least x% identical to a reference sequence it is intended that the sequence is identical to the reference sequence or differ from the reference sequence by up to 100-x amino acid alterations per each 100 amino acids of the reference sequence.
  • the present invention particularly relates to a nucleic acid encoding triadin.
  • Triadin is preferably as defined above in the section "Triadin”.
  • nucleic acid refers to the phosphate ester polymeric form of ribonucleosides (also called “RNA molecule”), deoxyribonucleosides (also called “DNA molecule”) or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form or a double- stranded form.
  • nucleic acid encoding triadin is thereafter also referred to as "nucleic acid”.
  • the nucleic acid encoding triadin is preferably an isolated nucleic acid.
  • isolated in reference to a biological component (such as a nucleic acid, a vector or a protein) refers to a biological component that has been substantially separated or purified away from other biological components in the cell of the organism, or the organism itself, in which the component naturally occurs, such as other chromosomal and extra-chromosomal DNA and RNA, proteins, cells, and organelles.
  • the sequence of the nucleic acid encoding triadin is preferably the same as those of the cDNA corresponding to the mature mRNA encoding triadin obtained after splicing of the triadin pre-mRNA, or a sequence at least 80% identical to said cDNA sequence, preferably at least 85%, more preferably at least 90%, still more preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to said cDNA sequence.
  • the triadin pre-mRNA is obtained by transcription of the triadin gene.
  • sequence of the nucleic acid encoding human triadin trisk 32 is, for example, sequence SEQ ID NO: 5 or a sequence at least 80% identical to sequence SEQ ID NO: 5, preferably at least 85% identical to sequence SEQ ID NO: 5, more preferably at least 90% identical to sequence SEQ ID NO: 5, still more preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to sequence SEQ ID NO: 5.
  • sequence of the nucleic acid encoding human triadin trisk 32 may also be sequence SEQ ID NO: 15 or a sequence at least 80% identical to sequence SEQ ID NO: 15, preferably at least 85% identical to sequence SEQ ID NO: 15, more preferably at least 90% identical to sequence SEQ ID NO: 15, still more preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to sequence SEQ ID NO: 15.
  • sequence of the nucleic acid encoding rat triadin trisk 32 may, for example, be encoded by sequence SEQ ID NO: 1 or a sequence at least 80% identical to sequence SEQ ID NO: 1 , preferably at least 85% identical to sequence SEQ ID NO: 1 , more preferably at least 90% identical to sequence SEQ ID NO: 1 , still more preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to sequence SEQ ID NO: 1 .
  • sequence of the nucleic acid encoding rat triadin trisk 32 may, for example, be encoded by sequence SEQ ID NO: 13 or a sequence at least 80% identical to sequence SEQ ID NO: 13, preferably at least 85% identical to sequence SEQ ID NO: 13, more preferably at least 90% identical to sequence SEQ ID NO: 13, still more preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to sequence SEQ ID NO: 13.
  • sequence of the nucleic acid encoding murine triadin trisk 32 may, for example, be encoded by sequence SEQ ID NO: 7 or a sequence at least 80% identical to sequence SEQ ID NO: 7, preferably at least 85% identical to sequence SEQ ID NO: 7, more preferably at least 90% identical to sequence SEQ ID NO: 7, still more preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to sequence SEQ ID NO: 7.
  • the nucleic acid encoding triadin comprises or consists of a sequence selected in the group consisting of sequence SEQ ID NO: 1 , a sequence at least 80% identical to sequence SEQ ID NO: 1 , sequence SEQ ID NO: 5, a sequence at least 80% identical to sequence SEQ ID NO: 5, sequence SEQ ID NO: 7 and a sequence at least 80% identical to sequence SEQ ID NO: 7.
  • the nucleic acid encoding triadin comprises or consists of a sequence selected in the group consisting of sequence SEQ ID NO: 1 , sequence SEQ ID NO: 13, sequence SEQ ID NO: 5, sequence SEQ ID NO: 15 and sequence SEQ ID NO: 7 .
  • a nucleic sequence "at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical" to a reference sequence may comprise mutations, such as deletions, insertions and/or substitutions compared to the reference sequence.
  • substitutions preferably corresponds to a silent substitution or a substitution leading to a conservative substitution in the translated amino acid sequence, by comparison to the reference sequence, for example as indicated in the Table 1 above.
  • the nucleic sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence only differs from the reference sequence by silent substitution(s) and/or substitution(s) leading to a conservative amino- acid substitution.
  • the nucleic acid encoding triadin is preferably cloned into a vector.
  • Vector comprising a nucleic acid encoding triadin
  • the nucleic acid encoding triadin is preferably provided in the form of a vector comprising said nucleic acid.
  • Triadin and "the nucleic acid encoding triadin” are particularly as defined above in the sections of the same name.
  • the present invention thus also relates to a vector comprising a nucleic acid encoding triadin.
  • the "vector comprising a nucleic acid encoding triadin” is thereafter also referred to as "vector".
  • the vector is preferably an isolated vector.
  • the vector comprising a nucleic acid encoding triadin is preferably cardiotropic.
  • cardiotropic vector means a vector that is preferentially expressed in heart by comparison to the other parts of a body, in particular after intravenous and/or intra-arterial administration.
  • the cardiotropism may result from an intrinsic tropism, for example in the case of a viral vector and/or may result from the use of a tissue specific promoter and/or from the use of a heart targeting molecule, in particularly in the case of a non-viral vector.
  • the vector preferably comprises a triadin expression cassette, i.e. a nucleic acid encoding triadin placed under the control of at least one expression signal allowing its expression.
  • the expression signal is particularly selected among a promoter, a terminator, an enhancer and their combinations.
  • Suitable promoters, terminators and enhancers are well-known by the skilled person.
  • the promoter is the CMV promoter.
  • the terminator is the SV 40 late polyadenylation signal.
  • Said promoter may be a heart-specific promoter.
  • An example of heart-specific promoter is the a-myosin heavy chain promoter.
  • the enhancer is the CMV immediate early enhancer.
  • a preferred triadin expression cassette comprises the CMV immediate early enhancer, the CMV promoter, a nucleic acid encoding triadin and the SV40 late polyadenylation signal.
  • the vector comprising a nucleic acid encoding triadin is preferably a non-viral vector or a viral vector.
  • a non-viral vector may be selected in the group consisting of a plasmid, a liposomal nucleic acid complex and a carrier-associated nucleic acid.
  • plasmid it is herein meant a double-stranded circular DNA.
  • the plasmid may include a marker gene enabling to select the cells comprising said plasmid, an origin of replication to allow the cell to replicate the plasmid and/or a multiple cloning site allowing the insertion of a DNA fragment, in particular the nucleic acid encoding triadin according to the invention.
  • Non-limitative examples of carrier-associated nucleic acid are polymer-carried DNA and cationic lipids.
  • liposomes used in liposomal DNA complexes are well-known in the art. Said liposomes may be cationic, anionic or neutral liposomes.
  • Cationic lipids are also known in the art and are commonly used for gene delivery.
  • Such lipids include Lipofectin Tm also known as DOTMA (N- [I- (2, 3-dioleyloxy) propyls N, N, N-trimethylammonium chloride), DOTAP (1 , 2-bis (oleyloxy)-3 (trimethylammonio) propane), DDAB (dimethyldioctadecyl- ammonium bromide), DOGS (dioctadecylamidologlycyl spermine) and cholesterol derivatives such as DC-Chol (3 beta- (N- ( ⁇ ', ⁇ '- dimethyl aminomethane)-carbamoyl) cholesterol).
  • Cationic lipids for gene delivery are preferably used in association with a neutral lipid such as DOPE (dioleyl phosphatidylethanolamine).
  • DOPE dioleyl phosphatidylethanolamine
  • the vector comprising a nucleic acid encoding triadin is a viral vector.
  • viral vector it is herein meant a recombinant viral vector.
  • the viral vector is preferably selected in the group consisting of a retrovirus vector, a lentivirus vector, an adenovirus vector and an adeno-associated virus vector.
  • a retrovirus vector is a single-stranded positive-sense RNA that encodes a transcriptase enabling to generate double-stranded DNA.
  • a lentivirus vector is a single-stranded RNA that integrates in the genome of the host, thereby allowing long term expression of the nucleic acid.
  • An adenovirus vector is a double-stranded DNA that does not integrate in the genome of the host.
  • An adeno-associated virus vector also called AAV, is a single-stranded DNA virus.
  • the vector is an adeno-associated virus.
  • An adeno-associated virus vector is preferably selected in the group consisting of an AAV1 vector, AAV2 vector, AAV6 vector, AAV8 vector, AAV9 vector and a hybrid AAV vector.
  • a “hybrid AAV vector” is a vector comprising the rep gene of one AAV vector and the cap gene of another AAV vector.
  • Non-limitative examples of hybrid AAV vector are an AAV1/2 vector, i.e. a vector comprising AAV1 rep gene and AAV2 cap gene, or an AAV2/9 vector, i.e. a vector comprising with AAV2 rep gene and AAV9 cap gene.
  • An example of AAV2/9 vector is pZac2.1 (for example from Penn Vector Core, Upenn).
  • Preferred viral vectors are AAV1 , AAV6, AAV8, AAV9 vectors and hybrid AAV vectors thereof, because they are highly cardiotropic.
  • the vector comprising a nucleic acid encoding triadin is the AAV1 or AAV9 vector, in particular for cardiac human gene therapy.
  • the vector is an AAV2/9 vector comprising a triadin expression cassette, said triadin expression cassette comprising the CMV immediate early enhancer, the CMV promoter, a nucleic acid encoding triadin and a SV40 polyadenylation signal.
  • Said vector thus corresponds to an AAV9 vector comprising a triadin expression cassette flanked by inverted terminal repeats derived from AAV serotype 2, said triadin expression cassette comprising the CMV immediate early enhancer, the CMV promoter, a nucleic acid encoding triadin and a SV40 polyadenylation signal.
  • the vector is an adeno- associated virus vector comprising a nucleic acid encoding triadin, wherein said nucleic acid comprises or consists of a sequence selected in the group consisting of sequence SEQ ID NO: 1 , a sequence at least 80% identical to sequence SEQ ID NO: 1 (for example sequence SEQ ID NO 13), sequence SEQ ID NO: 5, a sequence at least 80% identical to sequence SEQ ID NO: 5 (for example sequence SEQ ID NO: 15), sequence SEQ ID NO: 7 and a sequence at least 80% identical to sequence SEQ ID NO: 7.
  • the vector is an AAV1/2 vector comprising a triadin expression cassette, wherein said triadin expression cassette comprises the CMV immediate early enhancer, the CMV promoter, a nucleic acid encoding triadin and a SV40 polyadenylation signal and wherein said nucleic acid comprises or consists of a sequence selected in the group consisting of sequence SEQ ID NO: 1 , a sequence at least 80% identical to sequence SEQ ID NO: 1 (for example sequence SEQ ID NO 13), sequence SEQ ID NO: 5, a sequence at least 80% identical to sequence SEQ ID NO: 5 (for example sequence SEQ ID NO: 15), sequence SEQ ID NO: 7 and a sequence at least 80% identical to sequence SEQ ID NO: 7.
  • the vector comprises or consists of sequence SEQ ID NO: 3.
  • the vector comprises or consists of sequence SEQ ID NO: 4.
  • the nucleic acid encoding triadin or the vector comprising said nucleic acid may be formulated into a pharmaceutical composition.
  • the invention contemplates a pharmaceutical composition comprising a nucleic acid encoding triadin or a vector comprising a nucleic acid encoding triadin, and a pharmaceutically acceptable vehicle.
  • nucleic acid encoding triadin and the vector comprising said nucleic acid are as defined above in the sections of the same names.
  • compositions comprising a nucleic acid encoding triadin or a vector comprising a nucleic acid encoding triadin include all compositions, wherein said nucleic acid or said vector is contained in an amount effective to achieve the intended purpose.
  • pharmaceutically acceptable is meant to encompass any carrier, which does not interfere with the effectiveness of the biological activity of the active ingredient and that is preferably not toxic to the host to which is administered.
  • Pharmaceutically acceptable vehicles can be prepared by any method known by those skilled in the art.
  • Suitable pharmaceutically acceptable vehicles may comprise excipients and auxiliaries, which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Suitable pharmaceutically acceptable vehicles are described for example in Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton, USA, 1985), which is a standard reference text in this field. Pharmaceutically acceptable vehicles can be routinely selected in accordance with the mode of administration, solubility and stability of nucleic acid or vector. For example, formulations for intravenous administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. The use of biomaterials and other polymers for drug delivery, as well the different techniques and models to validate a specific mode of administration, are disclosed in literature.
  • the nucleic acid or vector according to the invention is preferably formulated as liquid (e.g., solutions, suspensions).
  • the amount of nucleic acid or vector to be used in a pharmaceutical composition depends, for example, on the strength of the promoter used in the DNA construct, the immunogenicity, the condition of the mammal intended for administration (e.g., weight, age, sex, health, concurrent treatment, if any, and frequency of treatment), the mode of administration and the type of formulation.
  • the pharmaceutical composition may comprise from 1 ⁇ 9 to 8 mg, preferably from 1 ⁇ 9 to 1 mg, preferably from 10 ⁇ g to 800 ⁇ g, more preferably from 25 ⁇ g to 250 ⁇ g, of the nucleic acid or vector, in particular for administration to human adults and/or when using a non-viral vector.
  • the pharmaceutical composition comprises a quantity of a viral vector according to the invention, in particular an adeno-associated virus vector, ranging from 5.10 7 DRP to 5.10 16 DRP, preferably from 10 8 DRP to 10 16 DRP, more preferably from 5.10 8 DRP to 5.10 15 DRP, more preferably from 10 9 DRP to 10 15 DRP.
  • a viral vector according to the invention in particular an adeno-associated virus vector, ranging from 5.10 7 DRP to 5.10 16 DRP, preferably from 10 8 DRP to 10 16 DRP, more preferably from 5.10 8 DRP to 5.10 15 DRP, more preferably from 10 9 DRP to 10 15 DRP.
  • the pharmaceutical composition comprises a quantity of a viral vector according to the invention, in particular an adeno-associated virus vector, ranging from 10 11 DRP to 10 15 DRP, preferably from 5.10 11 DRP to 5.10 14 DRP, more preferably from 10 12 DRP to 10 14 DRP, for example 1 ,4.10 12 DRP, 6.10 12 DRP, 3.10 13 DRP or 10 14 DRP, in particular for a systemic administration of said pharmaceutical composition (for example by the arterial, venous and/or intraperitoneal route).
  • a viral vector according to the invention in particular an adeno-associated virus vector, ranging from 10 11 DRP to 10 15 DRP, preferably from 5.10 11 DRP to 5.10 14 DRP, more preferably from 10 12 DRP to 10 14 DRP, for example 1 ,4.10 12 DRP, 6.10 12 DRP, 3.10 13 DRP or 10 14 DRP, in particular for a systemic administration of said pharmaceutical composition (for example by the arterial, venous and/or intraperitoneal route).
  • the pharmaceutical composition comprises a quantity of a viral vector according to the invention, in particular an adeno-associated virus vector, ranging from 10 10 DRP to 10 14 DRP, preferably from 5.10 10 DRP to 5.10 13 DRP, more preferably from 10 11 DRP to 10 13 DRP, for example 1 .4.10 11 DRP, 6.10 11 DRP, 3.10 12 DRP or 10 13 DRP, in particular for an intramyocardial and/or intrapericardial administration of said pharmaceutical composition.
  • a viral vector according to the invention in particular an adeno-associated virus vector, ranging from 10 10 DRP to 10 14 DRP, preferably from 5.10 10 DRP to 5.10 13 DRP, more preferably from 10 11 DRP to 10 13 DRP, for example 1 .4.10 11 DRP, 6.10 11 DRP, 3.10 12 DRP or 10 13 DRP, in particular for an intramyocardial and/or intrapericardial administration of said pharmaceutical composition.
  • the pharmaceutical composition comprises a quantity of a viral vector according to the invention, in particular an adeno-associated virus vector, ranging from 10 9 DRP to 10 13 DRP, preferably from 5.10 9 DRP to 5.10 12 DRP, more preferably from 10 10 DRP to 10 12 DRP, for example 1 ,4.10 10 DRP, 6.10 10 DRP, 3.10 11 DRP or 10 12 DRP, in particular for a paediatric use of said pharmaceutical composition.
  • a viral vector according to the invention in particular an adeno-associated virus vector, ranging from 10 9 DRP to 10 13 DRP, preferably from 5.10 9 DRP to 5.10 12 DRP, more preferably from 10 10 DRP to 10 12 DRP, for example 1 ,4.10 10 DRP, 6.10 10 DRP, 3.10 11 DRP or 10 12 DRP, in particular for a paediatric use of said pharmaceutical composition.
  • DNase-resistant particles By “DRP” or “DNase-resistant particles”, it is herein meant the number of packaged vector genomes.
  • the administration may be achieved in a single dose or several doses of the pharmaceutical composition according to the invention, said several doses being preferably injected at the same time.
  • the pharmaceutical compositions are presented in unit dosage forms to facilitate accurate dosing.
  • unit dosage forms refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a pre-determined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
  • Typical unit dosage forms include pre-filled, pre-measured ampoules or syringes of the liquid compositions.
  • the nucleic acid or vector is usually a minor component, with the remainder being various vehicles or carriers and processing aids helpful for forming the desired dosing form.
  • kits comprising a pharmaceutical composition comprising a nucleic acid or vector according to the invention and instructions regarding the mode of administration. These instructions may e.g. indicate the medical indication, the route of administration, the dosage and/or the group of patients to be treated.
  • a subject in need of a treatment for preventing and/or treating heart failure may be a mammal, for example a human being or a non-human mammal.
  • a human being is also referred to as an "individual” or a "patient”.
  • Said human being may be of any age, for example an infant, child, adolescent, adult, elderly people, and of any sex.
  • a non-human mammal is preferably a mouse, rat, cat, dog, rabbit or primate.
  • the subject to be treated may suffer from heart failure or may be likely to be affected by heart failure.
  • CPVT Choaminergic Polymorphism Ventricular Tachycardia
  • cardiac ryanodine receptor gene the cardiac calsequestrin gene and/or the triadin gene.
  • said mutation leads to the absence of expression of triadin or to an unstable triadin that is destroyed.
  • the subject to be treated has a CPVT involving a mutation in the triadin gene.
  • the subject to be treated does not have a CPVT involving a mutation in the triadin gene, or more generally, does not have a CPVT.
  • the subject to be treated has a gene encoding a functional triadin.
  • the subject to be treated thus does not suffer or may be likely to be affected by heart failure resulting from mutation(s) in the triadin gene.
  • the subject to be treated has a gene encoding a functional junctin, a gene encoding a functional RyR2 gene and/or a gene encoding a functional CASQ2 gene.
  • the subject to be treated does not suffer or may be likely to be affected by heart failure resulting from mutation(s) in the junctin gene, in the RyR2 gene and/or in the CASQ2 gene, in particular wherein said mutation(s) lead to the absence of expression of the protein or to a non-functional and/or unstable protein.
  • Failing hearts are characterized by an increased occurrence of cardiac arrhythmia, cardiac hypertrophy, myocardial fibrosis and/or cardiac post-ischemia contractile dysfunction.
  • Heart failure is generally associated a high rate of mortality.
  • the present failure may relate to the two types of heart failure: heart failure due to left ventricular dysfunction and heart failure with normal ejection fraction.
  • the present invention preferably relates to heart failure due to left ventricular dysfunction.
  • Heart failure is particularly characterized by a contractile dysfunction.
  • the heart failure may be selected in the group consisting of acute heart failure, chronic heart failure, end-stage heart failure, congestive heart failure, left heart failure, Class I, II, III, or IV heart failure as defined by New York Heart Association Functional Classification, systolic heart failure, diastolic heart failure, low-output heart failure and high-output heart failure.
  • heart failure is characterized by at least one of cardiac arrhythmias, cardiac hypertrophy, myocardial fibrosis, cardiac post-ischemia or contractile dysfunction.
  • Arrhythmia corresponds to an irregular heartbeat. Arrhythmia can occur with a normal, slow or rapid heart rate.
  • Cardiac hypertrophy is an adaptive response to pressure or volume stress, mutations of sarcomeric or other proteins, or loss of contractile mass from prior infarction, in order to maintain a normal pump functioning. Cardiac hypertrophy results from a cardiac remodelling.
  • Myocardial fibrosis is defined as a diffuse replacement or invasion of the myocardium by fibrous connective tissue, which results in a reduced elasticity of the heart.
  • Cardiac post-ischemia dysfunction is a cardiac dysfunction resulting from the cardiac remodeling that occurs in response to a cardiac ischemia.
  • Heart failure may be associated with CPVT.
  • heart failure is not associated with a CPVT involving at least one mutation of the triadin gene or, more generally, is not associated with a CPVT. In one embodiment, heart failure does not result from mutation(s) in the junctin gene, the RyR2 gene, the CASQ2 gene and/or the triadin gene.
  • heart failure does not result from mutation(s) in a given gene
  • said gene encodes a functional protein, although the expression of said functional protein may be altered or not during heart failure.
  • CPVT is as defined above in the section "subject to be treated”.
  • treatment of heart failure it is herein meant to eliminate or reduce ventricular hypertrophy, adverse remodelling, myocardial fibrosis and/or arrhythmias, to eliminate or improve contractile dysfunction, and/or to stop or slow down the progression of ventricular hypertrophy, adverse remodelling, myocardial fibrosis, contractile dysfunction and/or arrhythmias, in particular in a subject suffering from heart failure.
  • prevention of heart failure it is herein meant to prevent, at least partially, contractile dysfunction, ventricular hypertrophy, adverse remodelling, myocardial fibrosis and/or arrhythmias, in particular in a subject likely to be affected by heart failure.
  • a subject likely to be affected by heart failure is for example a subject that has suffered from a myocardial infarction.
  • Contractile function may be assessed by any method well known by the skilled person. For example, contractile function may be assessed by echocardiography. Ventricular hypertrophy may be assessed by any method well known by the skilled person. For example, ventricular hypertrophy may be assessed by a measure of the thickness of the heart muscle by echocardiography.
  • Adverse remodelling may be assessed by any method well known by the skilled person. For example, adverse remodelling may be assessed by echocardiography.
  • Myocardial fibrosis may be assessed by any method well known by the skilled person. For example, myocardial fibrosis may be assessed by Masson's Trichrome staining.
  • Arrhythmia may be assessed by any method well known by the skilled person. For example, arrhythmia may be assessed by recording electrical heart's activity using an electrocardiogram (ECG) recordings.
  • ECG electrocardiogram
  • Acid nucleic encoding triadin for use in the prevention and/or treatment of heart failure and method of prevention and/or treatment of heart failure
  • the present invention also relates to a nucleic acid encoding triadin for use in the prevention and/or treatment of heart failure, in particular in a subject in need thereof.
  • the present invention is also directed to a method for preventing and/or treating heart failure in a subject in need thereof, said method comprising a step of administering an effective amount of a nucleic acid encoding triadin to said subject.
  • the nucleic acid encoding triadin is preferably provided in the form of a vector.
  • the present invention thus also relates to a vector comprising a nucleic acid encoding triadin for use in the prevention and/or treatment of heart failure, in particular in a subject in need thereof.
  • the present invention also relates to a method for preventing and/or treating heart failure in a subject in need thereof, said method comprising a step of administering an effective amount of a vector comprising a nucleic acid encoding triadin to said subject.
  • the vector may be provided in the form of a pharmaceutical composition.
  • the subject in need thereof is as defined above in the section "subject to be treated”.
  • an effective amount or “therapeutically effective amount” it is herein meant an amount sufficient to achieve a triadin expression, which is capable of preventing and/or treating heart failure.
  • effective amounts can be routinely determined by those of skilled in the art.
  • the amount of the compound actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, sex, weight, and response of the individual patient, the severity of the patient's symptoms, and the like. It will also be appreciated by those of skilled in the art that the dosage may be dependent on the stability of the administered nucleic acid or vector.
  • the effective amount may also vary according to the drug or prodrug with which the nucleic acid or vector may be co-administered.
  • a therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the triadin are outweighed by the therapeutically beneficial effects.
  • the nucleic acid, vector or pharmaceutical composition is preferably used or administered by arterial, venous, intramyocardial, intraperitoneal and/or intrapericardial route.
  • the intramyocardial route includes epicardial and/or endocardial route.
  • the nucleic acid, vector or pharmaceutical composition is used or administered by arterial route, for example in the left coronary artery and/or in the right coronary artery.
  • the nucleic acid, vector or pharmaceutical composition is administered as a single dose, said single dose being for example efficient for the prevention and/or treatment of heart failure for several months or years.
  • one part of said single dose is administered in the anterolateral myocardium, and one part is administered in the posterolateral myocardium.
  • two-thirds of said single dose is administered in the anterolateral myocardium and one-third is administered in the posterolateral myocardium.
  • a combination of at least two vectors may be used.
  • Said at least two vectors may be used or administered simultaneously, separately or sequentially, in any order.
  • the combination of at least two vectors may comprise non-viral vector(s) and/or viral vector(s), each of said vectors comprising a nucleic acid encoding triadin.
  • the nucleic acid encoding triadin may be identical or different in said at least two vectors.
  • the vector is a non-viral vector, such as a plasmid
  • said vector may be used or administered in a naked form, i.e. free from any delivery vehicles.
  • the nucleic acid may be simply diluted in a physiologically acceptable solution, such as sterile saline or sterile buffered saline, with or without a carrier.
  • the carrier is preferably isotonic, hypotonic, or weakly hypertonic, and has a relatively low ionic strength, such as provided by a sucrose solution, e. g., a solution containing 20% sucrose.
  • a sucrose solution e. g., a solution containing 20% sucrose.
  • the non-viral vector may be administered in association with agents that assist in cellular uptake, for example chemicals that modify cellular permeability, such as bupivacaine.
  • a viral vector in particular an adeno-associated virus vector, may be used or administered in a quantity ranging from 10 7 DRP/kg to 10 14 DRP/kg, preferably from 5.10 7 DRP/kg to 5.10 13 DRP/kg, more preferably from 10 8 DRP/kg to 10 13 DRP/kg.
  • a viral vector in particular an adeno-associated virus vector, is preferably used or administered in a quantity ranging from 10 8 DRP/kg to 10 12 DRP/kg, preferably from 5.10 8 DRP/kg to 5.10 11 DRP/kg, more preferably from 10 9 DRP/kg to 2.5.10 11 DRP/kg, for example 2.10 9 DRP/kg, 8,6.10 9 DRP/kg, 4,3.10 10 DRP/kg or 1 ,4.10 11 DRP/kg of the subject to be treated, in particular for intramyocardial and/or intrapericardial administration (for example by the arterial, venous and/or intraperitoneal route).
  • a viral vector in particular an adeno-associated virus vector, is preferably used or administered in a quantity ranging from 10 9 DRP/kg to 10 13 DRP/kg, preferably from 5.10 9 DRP/kg to 5.10 12 DRP/kg, more preferably from 10 10 DRP/kg to 2.5.10 13 DRP/kg, for example 2.10 10 DRP/kg, 8,6.10 10 DRP/kg, 4,3.10 11 DRP/kg or 1 ,4.10 12 DRP/kg of the subject to be treated, in particular for an systemic administration .
  • the nucleic acid encoding triadin may be used or administered in combination with at least another compound useful for the prevention and/or treatment of heart failure.
  • angiotensin converting enzyme (ACE) inhibitor such as alacepril, benazepril, benazeprilat, captopril, ceronapril, cilazapril, delapril, enalapril, enaprilat, fosinopril, imidapril, lisinopril, moveltopril, perindopril, quinapril, ramipril, spirapril, temocapril, and trandolapril, angiotensin receptor blocker such as valsartan, losartan, candesartan, telmisartan, eprosartan, olmesartan, irbesartan and tasosartan, beta adrenergic blocker such as bisoprolol, metoprolol, carvedilo
  • ACE angiotensin converting enzyme
  • the nucleic acid according to the invention is preferably used or administered in combination with at least one compound increasing and/or restoring the expression of junctin, RyR2 and/or CASQ2, respectively.
  • ESV End-systolic volume
  • EDV end-diastolic volume
  • C genotypes
  • Figure 4 LV catheterization. Two-way ANOVA was performed to analyze differences by treatment and genotype, including a Bonferroni post hoc analysis when significant interaction occurred.
  • FIG. 5 Increased Triadin expression blocks the cardiac hypertrophic response.
  • A Immunoblot analysis of Triadin was performed on left ventricular (LV) homogenates from WT mice, Triadin KO mice, and Triadin KO mice injected with AAV9-Triadin.
  • B LV weight(LVW)/TL ratios and
  • C lung weight of WT, Triadin KO mice, and AAV9-Triadin treated mice after 4 weeks TAC surgery. *** P ⁇ 0.001 versus WT; % P ⁇ 0.05 versus KO TAC; %% P ⁇ 0.01 versus KO TAC.
  • FIG. 6 Summary data from M-mode echocardiography in WT, Triadin KO mice, and AAV9-Triadin treated mice demonstrating that injection of the AAV9-Triadin reduced cardiac dilation and improved cardiac function. *** P ⁇ 0.001 versus WT TAC; %%% P ⁇ 0.001 versus Triadin KO TAC.
  • FIG. 7 LV catheterization data demonstrating that AAV9-Triadin treated KO mice are characterized by a better cardiac function.
  • A Left ventricular end-diastolic pressure (LVendP),
  • B left ventricular developed pressure (LVdevP),
  • C dP/dt max (rate of rise of LV pressure),
  • D dP/dt min (rate of fall of LV pressure). ** P ⁇ 0.01 versus WT TAC; *** P ⁇ 0.001 versus WT TAC; % P ⁇ 0.05 versus KO TAC; %%% P ⁇ 0.001 versus KO TAC.
  • FIG. 8 (A) Data from echocardiography demonstrating significant decreased ejection fraction (FE) in Triadin KO after 28 days of myocardial infarction (Ml), as compared to WT. * P ⁇ 0.05 versus Sham; ## P ⁇ 0.01 versus WT. (B) ECG analysis demonstrating an increased corrected QT interval (QT) in KO Ml after isoproterenol (1 mg/kg) infusion ## P ⁇ 0.01 versus WT. (C) Arrhythmic events. Ventricular tachycardias were counted in WT and Triadin KO mice 28 days following Ml. *** P ⁇ 0.001 versus WT Ml. (D) Dot plot demonstrating that sparks duration (a reflect of leaky RyR receptors during diastole) was significantly increased in KO Ml group. ## P ⁇ 0.01 versus WT.
  • SEQ ID NO: 1 corresponds to the coding sequence of a rat triadin.
  • SEQ ID NO: 2 corresponds to the amino acid sequence of the rat triadin encoded by sequence SEQ ID NO: 1 .
  • SEQ ID NO: 3 comprises sequence SEQ ID NO: 1 and corresponds to a sequence to be integrated in a vector, for example in the adeno-associated virus vector pZac2.1 .
  • SEQ ID NO: 4 corresponds to plasmid pZac2.1 comprising sequence SEQ ID NO: 13.
  • SEQ ID NO: 5 corresponds to the coding sequence of a human triadin.
  • SEQ ID NO: 6 corresponds to the amino acid sequence of the human triadin encoded by sequence SEQ ID NO: 5.
  • SEQ ID NO: 7 corresponds to the coding sequence of a murine triadin.
  • SEQ ID NO: 8 corresponds to the amino acid sequence of the murine triadin encoded by sequence SEQ ID NO: 7.
  • SEQ ID NO 9 corresponds to a forward primer of rat trisk 32.
  • SEQ ID NO 10 corresponds to a reverse primer of rat trisk 32.
  • SEQ ID NO 1 1 corresponds to a forward primer of mouse GAPDH.
  • SEQ ID NO 12 corresponds to a reverse primer of mouse GAPDH.
  • SEQ ID NO 13 corresponds to the coding sequence of a rat triadin.
  • SEQ ID NO 14 corresponds to the amino acid sequence of the rat triadin encoded by sequence SEQ ID NO: 13.
  • SEQ ID NO: 15 corresponds to the coding sequence of a human triadin.
  • SEQ ID NO: 16 corresponds to the amino acid sequence of the human triadin encoded by sequence SEQ ID NO: 15.
  • TAC Transverse thoracic aortic constriction
  • mice 8 week-old male WT littermate and Triadin KO mice (a kind gift of Dr. I. Marty).
  • male 8 week-old mice were anesthetized by an intraperitoneal (i.p.) injection of a cocktail of ketamine (100mg/kg) and xylazine (10mg/kg), intubated, and connected to a mouse ventilator (MiniVent, Harvard Apparatus, Holliston, MA).
  • a double blunted 26-gauge needle was tied to the aorta between the innominate and left common carotid arteries using 6/0 silk suture. The needle was then removed and the chest and skin were sutured closed.
  • the sham group (without ligation of the aorta) was set up as the control group.
  • mice were anesthetized in order to produce a near physiologic heart rate of 500 beats/min.
  • the mice were intubated and ventilated with a Harvard ventilator set at 200-400 ⁇ .
  • the bilateral carotid arteries were identified in the region of the trachea and the right carotid was cannulated with with a 1 .0 French mouse pressure catheter (Millar, SPR-1000).
  • the catheter was advanced retrogradely through the aortic valve into the left ventricle to assess pressure volume loops.
  • LV systolic and diastolic pressures, the derivative of LV pressure (dP/dT), and tau were recorded and analyzed with Emka analysis software (iox2).
  • mice were anesthetized using isoflurane inhalation (0.8-1 .0% volume in oxygen) and surface ECGs were recorded and analyzed using iox2 acquisition system.
  • ECGs were measured continuously before and after i.p. administration of the b-agonist, isoproterenol (1 mg/kg bodyweight). QTc intervals, and arrhythmias were analysed (ECG-auto, EMKA Technologies).
  • RT-PCR mPtNA expression levels of Triadin were determined by quantitative RT-PCR analysis. Total mRNA was extracted using TriPure reagent (Roche, Basel, Switzerland). PCRs were performed using an AbiPrism 7900 Sequence Detection System instrument and software (Applied Biosystems, Foster City, CA, USA).
  • Primer sequences were as follows: rat Trisk 32: forward, 5'-AGCCAAAGAAACTCCGAAAA-3' (sequence SEQ ID NO: 9); reverse, 5'-TCCAGTGGC CGTATGTACTTC-3' (sequence SEQ ID NO: 10); mouse GAPDH: forward, 5'-CGTGCCGCCTGGAGAAAC-3' (sequence SEQ ID NO: 1 1 ); reverse, 5'-TGGGAGTTGCTGTTGAAGTCG-3' (sequence SEQ ID NO: 12).
  • Full-length cDNA coding for rat Trisk 32 (accession number: EMBL AJ812276) was inserted into pZac2.1 for AAV development.
  • the sequence of the obtained recombinant vector is shown in sequence SEQ ID NO: 4.
  • the virus was engineered and produced by the Penn Vector Core (Philadelphia, PA, USA). 5 week-old male KO mice (4 weeks before cardiac surgery) were anesthetized with xylazine (10 ⁇ g/g) and ketamine (100 ⁇ g g), and injected with 2 ⁇ 10 11 viral genome copy diluted in 100 ⁇ of 150 mM NaCI, by a single injection in retro-orbital sinus.
  • Results are expressed as means ⁇ SEM. Data were analyzed using ANOVA followed by Bonferroni post-test when the data were available using Graphpad Prism 6.0 (San Diego, CA). Differences between the groups were considered statistically significant when the p value was under 0.05.
  • Triadin protein expression in the adult heart we obtained samples from the left ventricle of control donor hearts (NF) and of patients in end-stage heart failure at the time of cardiac transplantation (DCM). Immunoblot analysis revealed a significant decrease in Triadin expression in dilated human hearts compared to control hearts ( Figure 1, A 1 and A2). These patients in end-stage heart failure were not suffering from CPVT. A similar result was obtained in left ventricular homogenates from mice subjected to 28 days of TAC, as compared to sham mice ( Figure 1, B1 and B2). WT and Triadin _/" mice were subjected to TAC or sham operation. Animals were excluded from analysis if perioperative death occurred within the first 24 hours after surgery.
  • mice were included in the study, and their mortality was followed up for 28 days after surgery. After hemodynamic measurements were obtained, mice were killed. TAC resulted in a significant increase in mortality in Triadin _/" mice compared with the WT group ( Figure 2A). Furthermore, the 30-day survival was significantly lower in Triadin _/" mice (23 of 65) compared with WT mice (6 of 63, Figure 2A). Triadin knockout animals showed an excessive increase of left ventricle-weight to tibia-length ratios as compared to wild-type mice subjected to aortic constriction (Figure 2B). Moreover, Triadin-null mice revealed increased pulmonary congestion after TAC, as shown by higher lung-weight (Figure 2C). A similar increase in cardiomyocyte cross- sectional areas was also confirmed by morphometric analysis of LV sections ( Figure 2D).
  • Cardiovascular performance was also assessed in anesthetized WT and KO mice using in vivo catheterization. Under both basal conditions, the results showed no change in in systolic left ventricular pressure (LVsysP, Figure 4A), left ventricular end-diastolic pressure (LVendP, Figure 4B), left ventricular developped pressure (LVdevP, Figure 4C), the maximal rate of left ventricular pressure development (dP/dtmax, Figure 4D), a measure of contractility, and the maximal rate of left ventricular pressure decay (dP/dtmin, Figure 4E), which is a measure of relaxation.
  • LVsysP left ventricular end-diastolic pressure
  • LdevP left ventricular developped pressure
  • dP/dtmax maximal rate of left ventricular pressure development
  • dP/dtmin a measure of contractility
  • mice The therapeutic effect of a nucleic acid encoding triadin was also checked on an animal model of heart failure further to a myocardial infarction in wild type mice.
  • Myocardial infarction (Ml) was performed on 8 week-old male C57BL/6J.
  • male 8 week-old mice were anesthetized by an intraperitoneal (i.p.) injection of a cocktail of ketamine (100mg/kg) and xylazine (10mg/kg), intubated, and connected to a mouse ventilator (MiniVent, Harvard Apparatus, Holliston, MA). Permanent ligation of the left anterior descending artery was blocked using a segment of saline 9-0 prolene.
  • the sham group (without ligation of the left anterior descending artery) was set up as the control group. All surgical procedures were performed under sterile conditions. Successful cardiac infarction was confirmed by apparent S-T segment elevation. One day post- surgery, physiological serum solution (100 ⁇ L) containing 3 ⁇ 10 11 genome copies per 1 mL AAV9-Triadin was injected in the retro-orbital sinus. 8 weeks post-surgery, echocardiography and left ventricular catheter were performed; subsequently, cardiac tissues from different regions were harvested for further analysis.

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Abstract

La présente invention concerne un acide nucléique codant pour la triadine destinée à être utilisée dans la prévention et/ou le traitement de l'insuffisance cardiaque, en particulier l'insuffisance cardiaque caractérisée par une arythmie, une hypertrophie cardiaque, une fibrose du myocarde et/ou un dysfonctionnement post-ischémique cardiaque. L'acide nucléique peut être compris dans un vecteur viral ou non viral. La présente invention concerne également une méthode destinée à prévenir et/ou à traiter l'insuffisance cardiaque chez un sujet qui en a besoin, ladite méthode comprenant une étape d'administration audit sujet d'une quantité efficace d'un acide nucléique codant pour la triadine.
PCT/EP2015/078109 2014-11-28 2015-11-30 Acide nucléique codant pour la triadine destinée à la prévention et/ou au traitement d'une insuffisance cardiaque WO2016083625A1 (fr)

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Cited By (1)

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