Gene therapy compositions and treatment of arrhythmogenic right ventricular cardiomyopathy

Abstract

This invention provides a method for treating or preventing human cardiomyopathy. [Solution] A method for treating or preventing cardiomyopathy in a human subject, comprising delivering a therapeutic dose of a gene therapy vector to cardiomyocytes of the human subject, wherein the gene therapy vector comprises a nucleic acid sequence encoding placofilin-2 (PKP2) or a functional variant thereof.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 903,103, filed on September 20, 2019, the disclosure of which is hereby incorporated by reference in its entirety into this specification.

[0002] The present invention relates to the treatment of heart diseases (e.g., cardiomyopathy), and more particularly, to methods and pharmaceutical compositions for gene therapy for the treatment of cardiomyopathy.

Background Art

[0003] Despite pharmacological advances in the treatment of various heart conditions, such as heart failure, mortality and morbidity remain unacceptably high. Furthermore, certain treatment approaches are not suitable for many patients (e.g., patients with advanced heart failure conditions associated with other co - morbidities). Alternative approaches, such as gene therapy and cell therapy, are increasingly attracting attention due to their potential to be specifically tailored to the target and to address the underlying causes of the pathogenesis of many heart diseases.

Summary of the Invention

Problems to be Solved by the Invention

[0004] It is an object of the present invention to provide a method for delivering a therapeutic polynucleotide sequence to cardiomyocytes of a human subject.

[0005] Vectorizing a polynucleotide sequence encoding a plakophilin - 2 (PKP2) protein in a viral vector such as an adeno - associated virus is a further object of certain embodiments of the present invention.

[0006] Gene therapy methods to correct haploinsufficiency in PKP2 mutant cardiomyocytes Utilizing this is a further objective of certain embodiments of the present invention. [Means for solving the problem]

[0007] The above objectives and others are intended to treat cardiomyopathy in human subjects in certain embodiments. This invention is directed toward methods for treatment or prevention. The methods include, for example, therapeutic The process involves delivering a certain amount of gene therapy vector to human target cardiomyocytes, and the gene therapy vector — contains the nucleic acid sequence that encodes PKP2.

[0008] In some embodiments, gene therapy vectors include viral vectors. Morphologically, viral vectors include AAV1, AAV2, AAV3, AAV4, and AAV 5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, This includes one or more of these variations and combinations thereof. In this embodiment, the viral vector includes AAV6 or AAV9.

[0009] In some embodiments, the therapeutic dose is measured by human cardiomyocytes to PKP2 protein Treating or preventing arrhythmogenic right ventricular cardiomyopathy (ARVC) by causing the production of [unclear]. It is effective for that purpose.

[0010] Certain other embodiments are adapted for expressing nucleic acid sequences in human cardiomyocytes. It is intended for gene therapy vectors. The nucleic acid sequence is, for example, the PKP2 protein Includes a first sequence that encodes and a second sequence that includes a promoter. In some embodiments In some embodiments, gene therapy vectors include viral vectors. The sub-vectors are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV 7, AAV8, AAV9, AAV10, AAV11, AAV12, these variations This includes n, and one or more combinations thereof. In some embodiments, IllusVector includes AAV6 or AAV9.

[0011] In some embodiments, the promoter includes TNNT2. [Modes for carrying out the invention]

[0012] definition As used herein, the singular forms "a," "an," and "the" are used in sentences. Unless the pulse clearly points elsewhere, it includes references to multiple things. For example, "drugs" ( A reference to a drug refers to a mixture of two or more different drugs in addition to a single drug. The reference to "viral vector" implies a single viral vector. This could also include a mixture of two or more different viral vectors, for example.

[0013] Furthermore, as used herein, "approximately" is used in relation to the quantity being measured. In such cases, the level of attention required for the measurement, the purpose of the measurement, and the accuracy of the measuring instrument must be appropriate. This refers to the normal variation in the quantity being measured, as expected by a person skilled in the art. In a particular embodiment, the term “about” includes ±10% of the stated number. As a result, "approximately 10" includes 9-11.

[0014] Furthermore, as used herein, "polynucleotide" means in the art. It has its usual and customary meaning and refers to any polymeric nucleic acid, such as DNA or R In addition to NA molecules, it contains chemical derivatives known to those skilled in the art. Polynucleotides are used in therapeutic drugs. This includes not only those that encode proteins, but also those that use known techniques in the art. Sequences that may be used to reduce the expression of targeted nucleic acid sequences (e.g., antisensory sequences) Polynucleotides include (nucleic acids, interference, or small molecule interference). Polynucleotides are also found in the cells of the cardiovascular system. Initiation or increase of the expression of targeted nucleic acid sequences or the production of targeted proteins within the target protein. It can be used to add. Targeted nucleic acids and proteins are targeted Nucleic acids and proteins that are normally found in tissues, such naturally occurring nucleic acids or These are protein derivatives, naturally occurring nucleic acids not normally found in targeted tissues, and Examples include, but are not limited to, proteins, or synthetic nucleic acids or proteins. i. One or more polynucleotides are one or more targeted nucleic acid sequences or These are used in combination to increase and / or decrease protein, simultaneously and / Alternatively, it may be administered sequentially.

[0015] Furthermore, as used herein, “exogenous” nucleic acids or genes refer to nucleic acid transfer. In vectors used for this purpose, there are substances that do not exist in nature, for example, viral vectors with odors. Although not found in nature, the term refers to substances that are naturally present in the patient or host. It is not intended to exclude nucleic acids that encode proteins or polypeptides.

[0016] Furthermore, as used herein, "cardiac cells" refers to the maintenance or function of the structure of the heart. Any cells of the heart involved in providing function, such as cardiomyocytes, cardiovascular cells, or heart cells. This includes cells present in the valves. Cardiac cells include cardiomyocytes (both normal and abnormal electrical activity). (possessing specific characteristics), epithelial cells, endothelial cells, fibroblasts, cells of conductive tissue, cardiac pacemakers Examples include Kerr cells and neurons.

[0017] Furthermore, as used herein, "adeno-associated virus" or "AAV" means In addition to all subtypes, serotypes, and pseudotypes, naturally occurring and recombinant It encompasses various forms. Various AAV serotypes and strains are known in the art. Available from suppliers, e.g., ATCC and academic or commercial suppliers. Alternative AAV serotypes and strains that have been published and / or are available from various databases. These sequences may be synthesized using known techniques.

[0018] Furthermore, as used herein, "serotype" refers to a capsulated antiserum with a defined antiserum. Identified by protein reactivity and distinguished from other AAVs based on said reactivity. , refers to AAV. Including AAV1 to AAV12, at least 12 known human AAVs While there are existing serotypes, additional serotypes continue to be discovered, and the use of newly discovered serotypes is increasing. It is expected.

[0019] Furthermore, as used herein, "pseudotype" AAV refers to one serotype Capsid proteins from different or heterologous serotypes, as well as the 5' and 3' reversed ends of different or heterologous serotypes. This refers to AAV containing a viral genome including ITR (intracraviolet retrieval). Pseudotype recombinant AA V(rAAV) is a gene that matches the cell surface binding properties of the capsid serotype and the ITR serotype. It is expected to have genetic characteristics. Pseudotype rAAV is a capsid protein. As long as it is a heterogeneous serotype for the ITR, VP1, VP2, and VP3 caps AAV capsid proteins, including sid proteins, and AAV1-AAV12 It may include any primate AAV serotype and ITR from any serotype AAV. In pseudotype rAAV, the 5' and 3' ITRs are either the same or different. This is also acceptable. Pseudotype rAAV uses standard techniques described in the relevant art. It is manufactured using [a specific method / technology].

[0020] Furthermore, as used herein, the “chimera” rAAV vector is a heterocapsulated vector. The rAAV vector contains the cytoprotein, meaning that the rAAV vector contains its caps The side proteins VP1, VP2, and VP3 may be chimeric, and as a result VP1, VP2, and VP3 are not all from the same serotype AAV. Chimeric AAV When used herein, this includes, but is not limited to, AAV1 and A Including the capsid protein from AV2, the capsid proteins VP1, VP2, and VP3 encompasses different AAVs in serotype; other parvovirus capsid proteins A mixture of, or other viral proteins or other proteins, for example, desired Chimeric rAAV includes proteins that target AAV delivery to cells or tissues. Furthermore, when used herein, rAA containing chimeric 5' and 3' ITRs may also be used. Includes V.

[0021] Furthermore, as used herein, “pharmaceutically acceptable excipients or carriers” means This refers to any inactive component in a composition that can be combined with the active agent in the formulation. It is pharmaceutically acceptable. Excipients include carbohydrates (e.g., glucose, sucrose, or dextran), Antioxidants (e.g., ascorbic acid or glutathione), chelating agents, low molecular weight proteins Examples include high molecular weight polymers, gel-forming agents, or other stabilizers and additives. These are not the only examples of pharmaceutically acceptable carriers. Other examples of pharmaceutically acceptable carriers include wetting agents and emulsifiers. These include preservatives, dispersants, or antimicrobial agents, the latter of which prevent the growth or action of microorganisms. It is particularly useful for this purpose. Various preservatives are well known, for example, phenol and ascorbic acid. Examples include nic acid. Examples of carriers, stabilizers, or adjuvants can be found in Remington's Pharmaceutical Sciences. Find it in ences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985). It is possible.

[0022] Furthermore, as used herein, “patient” means “not a patient who has any indication of need for treatment.” Or, a condition that presents with clinical signs of multiple specific symptoms, is treated preventively, Or a subject, especially a human (but including non-humans), who has been diagnosed with a condition that requires treatment. It refers to obtaining something.

[0023] Furthermore, as used herein, “subject” encompasses the definition of the term “patient.” This includes healthy individuals, and does not exclude healthy individuals.

[0024] Furthermore, as used herein, "treatment" and "to treat" mean a state, for example This includes the administration of drugs intended to reduce the severity of heart disease or prevent such a condition. nothing.

[0025] Furthermore, as used herein, "prevention" and "to prevent" mean a state, for example This includes avoiding the onset of heart disease.

[0026] Furthermore, as used herein, the term "condition" or "con" may be used. Dictions are medical conditions that can be treated, alleviated, or prevented by administering an effective amount of a drug to a subject. It refers to a specific condition, such as heart disease.

[0027] Furthermore, as used herein, "effective amount" means the amount that produces a beneficial or desired effect. At a level easily detectable by methods commonly used to detect such effects This refers to a sufficient amount of drug to produce the effect. In some embodiments, such an effect is Even if the result is a change of at least 10% from the baseline level when no drug is administered, To reduce. In other embodiments, the change is at least 20%, 50%, and 8% from the baseline level. 0% or a higher percentage. The effective dose depends on the age of the subject, their overall health status, the severity of the condition being treated, and the amount administered. It can vary from person to person depending on the specific drug or other substance used. The "effectiveness" can be determined by referring to relevant texts and literature and / or by routine practice. This can be determined by those skilled in the art through the use of trials.

[0028] Furthermore, as used herein, “activator” means “for the purpose of a government agency.” Therefore, regardless of whether it is approved or not, therapeutic, preventive, or other intended effects This refers to any material that is intended to produce a certain effect.

[0029] The ranges of values ​​described herein, unless otherwise indicated herein, are defined as ranges. It is simply intended to serve as a simplified way to refer to each separate value that goes inside individually. Each separate value is incorporated herein as if it were individually described herein. All methods described herein, unless otherwise indicated herein, Alternatively, it may be done in any preferred order, as long as it does not clearly contradict the context otherwise. Any and all examples or illustrative expressions provided in (for example, "For example") The use of "etc." is simply intended to shed light on certain materials and methods, and within a range of... No limitations are imposed on this. No expression in this specification shall apply to the implementation of the disclosed materials and methods. It should be interpreted as referring to any unclaimed element that is essential for it. do not have.

[0030] Detailed explanation Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a major myocardial disorder, and is often seen in young individuals. It is the primary cause of spontaneous cardiac death (SCD). This is due to myocardial degeneration and myocardial fibrofatty replacement. Characterized by these, they are located in the right and / or left ventricles and cause progressive cardiac failure. It can ultimately lead to all of the following. Clinical cardiac phenotypes include the presence of typical electrocardiogram abnormalities and ventricular dysfunction. Increased load on the regular heart rate and large-scale myocardial scarring on cardiac magnetic resonance imaging Characterized.

[0031] ARVC is familial in approximately 50% of cases and is usually an autosomal dominant trait. It is hereditary. Approximately 30% of patients of Caucasian descent have a dominant mutation in the PKP2 gene. They differ. Most mutations result in haploinsufficiency, insertions-deletions, and other mutations. Sense, or an abnormal or severed result resulting from a splice site mutation. This results in the production of proteins.

[0032] ARVCs are desmodendrocytes, which are dense electron structures that provide mechanical connections between cardiomyocytes. It is thought to be a disease of the desmosome. PKP2 forms a part of the desmosome protein complex. It is one of several genes in which mutations leading to ARVC have been identified. PKP2 protein deficiency through haploinsufficiency destabilizes the desmosome protein complex. This process involves mechanical and signal transduction consequences.

[0033] Mechanical components are found under several extracellular matrix genes, for example, different collagens. Regulatory and fibrillating collagen, fibronectin, and other fibrosis-promoting compounds Carr, for example, PKP2 protein under mechanical stress conditions accompanied by strong upward regulation of TIMP1 Abnormal gene expression patterns caused by a lack of quality result in odor in vitro. This is emphasized. In preclinical and clinical settings, this is in the PKP2-mouse model. Exercise-induced exacerbation of ARVC and phenotypic effects in humans, such as athletes This is reflected in the harmful effects of exercise. At the level of signaling, the deficiency of pracophyllin This causes the translocation of placoglobin to the nucleus, which is canonical Wnt / β-cateni This leads to a reduction in signal transduction and an increase in the expression of genes for fibrosis and adipogenesis.

[0034] The two main forms of PKP2 are PKP2 isoform 2a (SEQ ID NO: 2) and PKP2 isoform 2b (SEQ ID NO: 4) is an example. The protein-coding portion of the PKP2 gene for a is a 2764bp cDNA sequence. (GenBank:BC126199.1; Sequence ID No. 1) is contained in this invention It can be vectorized during AAV. When used herein, "PKP2" The term "PKP2 protein" is used unless otherwise stated or implied by the context. For example, including PKP2 isoform 2a and PKP2 isoform 2b, PKP2 It should be interpreted as encompassing the isoforms of [the original form].

[0035] In certain embodiments, positive gene transfer occurs via AAV9-TNNT2-PKP2-mediated gene transfer. Haploinsufficiency of the PKP2 protein can be corrected by substituting a common allele. In a specific embodiment, the composition and method of the present invention include, for example, (1) PKP2 protein (2) Precisely localizing the quality to desmosomes; and (2) PKP2 mutant human artificial In pluripotent stem cell-derived cardiomyocytes (iPSC-CM), haploinsufficiency is corrected, and as a result, It may be possible to correct the smosome protein complex. In certain embodiments, Furthermore, in trans iPSC-CMs with two pathogenic mutations, complete or complete This is expected to result in a PKP2 deficiency similar to that of cardiomyocytes. Non-limiting exemplary embodiments for testing the delivery of creotide include (1) TNNT2 Using a lomotor to vectorize PKP2 during AAV9 and / or AAV6 and; PKP2 mutation (either one mutation or two mutations in trans) To produce iPSC-CMs possessing 2D PKP2 mutant heart cells in vitro. AAV6-PKP2 or AAV in muscle cell cultures (with one or two mutations) Transduce 9-PKP2 and test its intracellular localization into desmosomes; cell size Test molecular and physiological data, including dysplasia, contractility, and transcriptome analysis. This includes doing so.

[0036] Many embodiments described herein relate to the PKP2 protein, but additional It should be understood that protein expression (e.g., sarcomere proteins) is assumed to occur. In addition to PKP2, exemplary proteins include, non-limitingly, SERCA2 and MYBP. C3, MYH7, MYL3, MYL2, ACTC1, TPM1, TNNT2, TNNI3 , TTN, FHL1, ALPK3, dystrophin, FKRP, these variants, Or it may include one or more of these combinations. The protein is also a functional variant of the protein referred to herein. It is acceptable for it to exhibit remarkable amino acid sequence identity compared to the original protein. For example, amino acid identity is at least about 30%, at least about 35%, at least about 40% %, at least about 45%, at least about 50%, at least about 55%, at least about 60% %, at least about 65%, at least about 70%, at least about 75%, at least about 80% %, at least about 85%, at least about 90%, at least about 95%, at least about 96% %, or at least about 97%, at least about 98%, or at least about 99% In this context, the term "functional variant" refers to a variant of a protein. However, it is possible to partially or completely perform the function of the corresponding naturally occurring protein. This means that a functional variant of a protein is, for example, one or more amino acid substitutions. , containing proteins that differ from their naturally occurring counterparts due to deletion or addition. That's good too.

[0037] Amino acid substitutions can be conservative or non-conservative. A substitution is a conservative substitution. That is, amino acid residues with similar polarity that act as functional equivalents. Preferably, the substitution is of the amino acid residue used as a substitute. Preferably, the amino acid residue used as a substitute is of the same type. The replacement amino acid is selected from the same group of amino acids as the amino acid residue being replaced. For example, a hydrophobic residue is replaced with another hydrophobic amino acid. It can be substituted with an aqueous residue, or a polar residue can be replaced with another polar residue having the same charge. They can be substituted. Functionally homogeneous amino acids may be used for conservative substitution. For example, nonpolar amino acids such as glycine, valine, alanine, isoleucine, leucine Contains methionine, proline, phenylalanine, and tryptophan. Uncharged. Examples of polar amino acids include serine, threonine, glutamine, asparagine, tyrosine, and Contains cysteine. Examples of charged polar (basic) amino acids include histidine, arginine, and It also contains lysine. Examples of charged polar (acidic) amino acids are aspartic acid and glutamic acid. Contains nic acid.

[0038] One or more (for example, 2, 3, 4, 5, 10, or 15) additional amino acids Proteins that differ from their naturally occurring counterparts are also considered variants. The additional amino acids are present within the original protein's amino acid sequence (i.e., as insertions). They may be attached to one or both ends of the protein. Basically, the addition of amino acids is the mechanism of naturally occurring proteins in the target being treated. Insertion can occur at any position as long as it does not impair the polypeptide's ability to perform its function. Yes, it is possible. Furthermore, the protein variant is also one less than the original polypeptide. This includes proteins that are missing multiple amino acids. Such deletions affect the protein. Any amino acid position may be affected, as long as it does not impair the ability to perform normal functions.

[0039] Finally, variants of cardiac sarcomere proteins (e.g., PKP2) are also structurally Modification, such as modified amino acids, refers to proteins that differ from naturally occurring proteins. Modified amino acids are the result of natural processes, such as processing or post-translational modification, or the same These are amino acids modified by any of the chemical modification processes known in the technical field. Typical amino acid modifications include phosphorylation, glycosylation, acetylation, and O-linked N-acetylcholine. Glucosamine formation, glutathione formation, acylation, branching, ADP-ribose formation, cross-linking, disulfide formation Fidobridge formation, formylation, hydroxylation, carboxylation, methylation, demethylation Additive, amidative, cyclization, and / or phosphatidylinositol, flavin derivatives, This includes lipoteichoic acid, fatty acids, or covalent or non-covalent bonds to lipids.

[0040] Therapeutic polynucleotide sequences encoding target proteins are used in gene therapy vectors. In other words, other sequences required to provide expression of exogenous nucleic acids, such as promoters, Near the kozak sequence and the polyA signal, including the translation and stop codons, It may be administered to the target to be treated in the form of a nucleic acid construct containing a coding sequence. .

[0041] For example, the gene therapy vector may be a portion of the mammalian expression system. Expression systems and expression constructs are commercially available. Additionally, several mammalian expression systems are available. They are supplied by different manufacturers and can be used in this invention, for example, Plasmid or viral vector-based systems, e.g., LENTI-Smart (Trademark) (InvivoGen), GenScript (Trademark) Expression vectors, pAdVAntage(TM) (Promega), ViraPow er(trademark) Lentiviral, Adenoviral Expression Systems (Invitrogen), and adeno-associated virus expression system (Cell Biolabs)

[0042] The gene therapy vector for expressing the exogenous therapeutic polynucleotide sequence of the present invention is For example, an exogenous therapeutic polynucleotide sequence is introduced into a cell, and the nucleic acid encodes a tan Suitable viral or nonviral expression vectors for subsequent expression of the protein It can be an expression vector. The expression vector is an episomal vector, i.e., an intracellular expression vector. A vector that can autonomously self-replicate, or an embedded vector, i.e., stable in the cell genome. It can be incorporated constitutively. Expression in host cells can be constitutive or regulatory. It can be (for example, inductive).

[0043] In a particular embodiment, the gene therapy vector is a viral expression vector. The viral vector for use in the invention does not destroy the infectivity of the virus. In order to introduce a species polynucleotide, a portion of the native sequence of the viral gene is deleted. It may contain mu. Due to specific interactions between viral components and host cell receptors. Viral vectors are highly suitable for the efficient transfer of genes into target cells. Suitable viral vectors for promoting gene transfer into mammalian cells are different types of viral vectors. Rus, for example, AAV, adenovirus, retrovirus, herpes simplex virus, bovine Papillomavirus, lentivirus, vaccinia virus, polyomavirus, sen Divirus, Orthomyxovirus, Paramyxovirus, Papovavirus, Picorna Suitable for use with viruses, poxviruses, alphaviruses, or gene therapy. Other virus shuttles, variations of these, and combinations of these originate from It is possible.

[0044] "Adenovirus expression vector" or "adenovirus" is (a) a therapeutic polynucleo To support the packaging of rheotide sequence constructs, and / or (b) its To ultimately express the cloned tissue and / or cell-specific constructs within This means that the construct contains a sufficient adenovirus sequence. One of the present inventions In this embodiment, the expression vector comprises a genetically modified form of adenovirus. The genetics of adenoviruses, which are linear double-stranded DNA viruses with 36 kilobases (kb). This knowledge makes it possible to replace large portions of adenovirus DNA with foreign sequences up to 7kb. Let's assume that.

[0045] The replication and manipulation of adenoviruses are known to those skilled in the art, in vit They exhibit a broad host range both in vivo and in vivo. Viruses in this group have high titers, for example... For example, 10 per 1 mL 9 ~10 11 It can be obtained as a plaque-forming unit, and is highly infectious It is a sex. The life cycle of adenoviruses does not require integration into the host cell genome. The foreign gene delivered by the adenovirus vector is episomal, therefore It has low genotoxicity to host cells. Vaccination with wild-type adenovirus No side effects have been reported in the study, and as an in vivo gene transfer vector, Their safety and / or therapeutic potential has been demonstrated.

[0046] Retroviruses (also called "retroviral vectors") carry those genes. It integrates into the main genome, introduces large amounts of foreign genetic material, infects a wide range of species and cell types, Furthermore, due to their ability to be packaged in specific cell lines, gene delivery vectors - May be selected as such.

[0047] The retroviral genome contains three genes, gag, pol, and env. These are the capsid protein, polymerase enzyme, and envelope component, respectively. The sequence found upstream of the gag gene is used for packaging the genome into virions. It contains signals for . Two long-chain terminal repeat (LTR) sequences are 5 of the viral genome. They are located at the ' and 3' ends. These contain strong promoter and enhancer sequences. These are also required for integration into the host cell genome.

[0048] To construct a retroviral vector, the nucleic acid encoding the gene of interest is A specific viral sequence is inserted into the viral genome at a particular location, resulting in the production of a virus with replication defects. To produce virions, the gag, pol, and / or env genes are used. Packaging cell systems that contain but do not have LTR and / or packaging components It is constructed. It contains cDNA along with retroviral LTRs and packaging sequences. When recombinant plasmids are introduced into this cell line (for example, by calcium phosphate precipitation) The packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles. It is then sizing and secreted into the culture medium. The medium containing recombinant retrovirus is The retroviruses are then collected, selectively enriched, and used for gene transfer. The vector can infect a wide variety of cell types. However, integration and safety Severe expression requires host cell division.

[0049] Retroviruses can originate from any subfamily. For example, mouse sarcoma Virus, bovine leukemia, Roussarcoma virus, mouse leukemia virus, mink Vectors from follicular lesion-inducing viruses, reticuloendotheliosis viruses, or avian leukemia viruses. Those skilled in the art can use parts derived from different retroviruses, for example, LT Recombinant retrograde tRNA by combining R, tRNA binding site, and packaging signal. Rus can be obtained. These retroviruses are then usually competent at transduction. Used to produce retroviral vector particles. For this purpose, vector It is introduced into a suitable packaging cell line. Retroviruses are also chimeric integra By incorporating the -ase enzyme into retroviral particles, site-specific association with host cell DNA is achieved. It can be built for inclusion.

[0050] Because herpes simplex virus (HSV) is neurotropic, it is used in the treatment of nervous system disorders. It has attracted considerable interest. Furthermore, it can be incorporated into host cell chromosomes, as well as other host cells. HS establishes latent infection in non-dividing neuronal cells without altering cell metabolism. V's ability, along with the presence of an active promoter during the latency period, makes HSV an attractive vector. While much attention is focused on the neurotropic applications of HSV, this vector also... Given its wide host range, it can be utilized for other tissues.

[0051] Another factor that makes HSV an attractive vector is the size and organization of the genome. Because it is large, the incorporation of multiple genes or expression cassettes is possible with other smaller viruses. It is less problematic than in the stem. Additionally, it has various properties (time, strength, etc.). Due to the availability of different viral control sequences, it is more efficient than in other systems. Expression can be controlled to a certain extent. Splicin contains relatively little virus. It also has the benefit of having a message that can be manipulated, making gene manipulation even easier. It is a point.

[0052] HSVs are also relatively easy to manipulate and can be grown to high titers. Therefore, the volume required to achieve a sufficient multiple infection degree (MOI), and From the perspective of both the reduced need for repeated medication and the less problematic delivery, delivery becomes less of an issue. Non-pathogenic variants of HSV have been developed for use in the context of gene therapy. It is readily available.

[0053] Lentiviruses are complex retroviruses, and they share a common retroviral gene, gag. In addition to pol and env, it contains other genes with regulatory or structural functions. The higher complexity lies in the fact that the virus undergoes its life cycle, such as during the course of latent infection. It enables modulation. Some examples of lentiviruses include human immunodeficiency virus (HVM). Examples include viruses (HIV-1, HIV-2) and primate immunodeficiency virus (SIV). By doubling, the HIV pathogenicity gene is weakened, allowing the lentiviral vector to... For example, the genes env, vif, vpr, vpu, and nef are deleted. Therefore, the vectors are considered biologically safe.

[0054] Lentiviral vectors are plasmid-based or virus-based, and use foreign nucleic acids. It has essential sequences for incorporation, selection, and transfer of nucleic acids into host cells. It is configured in such a way that the gag, pol, and env genes of the vector of interest are also included. It is publicly known in the relevant technical field. Therefore, the relevant genes are selected for the vector. It is cloned and then used to transform target cells of interest.

[0055] Vaccinia virus vectors are easy to construct and produce relatively high levels of expression. It is used on a large scale due to its broad host range and large capacity for holding DNA. Senior has a linear double-stranded DNA genome of approximately 186kb that exhibits a marked preference for "AT". It contains approximately 10.5 kb of reverse-terminal repeats adjacent to the genome. The majority of essential genes are It appears to map to the most highly conserved central region among poxviruses. The estimated number of open reading frames in vaccinia virus is 150-20. It is 0. Both chains are coding, but the reading frame has a large overhead Wrapping is not common.

[0056] At least 25kb can be inserted into the vaccinia virus genome. The xinia vector is inserted into the viral thymidine kinase gene via homologous recombination. Contains a transgene. The vector is selected based on the tk phenotype. Encephalomyocarditis virus Including the non-translating reader sequence results in higher expression levels than with conventional vectors. As a result, the introduced gene brings about 10% or more of the protein in infected cells within 24 hours. It accumulates in a certain place.

[0057] Papovaviruses, such as the empty capsid of mouse polyomavirus, are used for gene transfer. It is attracting attention as a possible vector. The use of empty polyomas is polyoma DNA and The first description was that when purified empty capsids were incubated in a cell-free system, The DNA of the new particles was protected from the action of pancreatic DNase. The reconstituted particles were It was used to transfer transformable polyoma DNA fragments into rat FIII cells. The capsid and reconstituted particles contain polyomacapsid antigens VP1, VP2, and V It consists of all three P3 components.

[0058] AAV is a parvovirus belonging to the genus Dependovirus. They are small, non- It is an enveloped, single-stranded DNA virus that requires helper viruses to replicate. To form a functionally complete AAV virion, helper viruses (e.g., Aden) A co-infection with a novirus, herpesvirus, or vacciniavirus is required. In vitro, in the absence of co-infection with helper viruses, AAV is in a latent state. Once established, the viral genome exists in episomal form during the latent state, but infectious viruses Lyon is not produced. Subsequent infection by the helper virus "rescues" the genome. And so the genome is replicated and packaged into a viral capsid, and so Infectious virions are reconstituted. Both wild-type AAV and recombinant AAV are reconstituted in vivo. Recent data indicate that it mainly exists as a large episomal concatemer. In one embodiment, the gene therapy vector used herein is AAV It is a vector. AAV vectors are purified, replicated, incompetent, pseudotypes. rAAV particles may also be used.

[0059] AAV is not associated with any known human disease and is generally not considered pathogenic. It does not appear to modify the physiological characteristics of host cells through integration. AAV is used in non-dividing cells. It can infect a wide range of host cells, including cells, and can infect cells from different species. This can be achieved. Rapid clearance or inactivation occurs through both cellular and humoral responses. In contrast to some vectors, AAV vectors exhibit various behaviors in vivo. It has been shown to induce sustained transgene expression in tissues. The persistence of recombinant AAV-mediated transgenes in non-dividing cells is linked to the native AAV virus. The lack of the S gene and the ITR-related ability of vectors to form episomal concatemers They can be sent back.

[0060] AAV has high persistence as an episomal concatemer and contains cardiomyocytes. Furthermore, because it can infect non-dividing cells, for example, in tissue culture and in vivo Because it is useful for gene delivery to mammalian cells in the present invention, the cell shape It is an attractive vector system for use in quality introduction.

[0061] Typically, rAAV uses two AAV terminal repeats to create adjacent genes of interest. Plasmids containing and / or wild-type AAV coding sequences without terminal repeats It can be produced by co-transfecting with an expression plasmid containing it, such as pIM45. The cells also possess the adenovirus genes required for AAV helper function. Infection and / or transfection using adenovirus and / or plasmids The rAAV stock produced in this manner is contaminated with adenovirus. , (for example, by cesium chloride density centrifugation or column chromatography) It must be physically separated from the rAAV particles. Alternatively, the AAV coding region Adenovirus vectors containing the region and / or AAV coding region and / Alternatively, a cell line containing some or all of the adenovirus helper genes may be used. It can be used. Cell systems that have rAAV DNA as an incorporated provirus can also be used. It can be used.

[0062] Multiple serotypes of AAV exist naturally, with at least 12 serotypes (AAV1 to AAV1) 2) Despite a high degree of homology, different serotypes are different for different tissues. It has pism. In transfection, AAV is (if any) minor in the host. It only induces an immune response. Therefore, AAV is highly suitable for gene therapy approaches. Suitable.

[0063] This disclosure, in some embodiments, refers to AAV1, AAV2, AAV3, AAV4, AA V5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 ANC AAV, chimeric AAV derived from these, variations of these, and This is even more suitable for highly efficient trait introduction within the organization of interest. Directed to drugs containing AAV vectors that are one or more of the combinations of these. It is possible. In a particular embodiment, the gene therapy vector is a vector for AAV serotype 1. —In a particular embodiment, the gene therapy vector is a vector for AAV serotype 2. —In a particular embodiment, the gene therapy vector is a vector for AAV serotype 3. —In a particular embodiment, the gene therapy vector is a vector for AAV serotype 4. —In a particular embodiment, the gene therapy vector is a vector for AAV serotype 5. —In a particular embodiment, the gene therapy vector is a vector for AAV serotype 6. —In a particular embodiment, the gene therapy vector is a vector for AAV serotype 7. —In a particular embodiment, the gene therapy vector is a vector for AAV serotype 8. - is. In certain embodiments, the gene therapy vector is a vector of AAV serotype 9 - is. In certain embodiments, the gene therapy vector is a vector of AAV serotype 10 - is. In certain embodiments, the gene therapy vector is a vector of AAV serotype 11 - is. In certain embodiments, the gene therapy vector is a vector of AAV serotype 12 - is a vector.

[0064] A suitable dose of AAV for humans is about 1×10 8 vg / kg to about 3×10 14 vg / kg, about 1×10 8 vg / kg, about 1×10 9 vg / kg, about 1×10 10 vg / kg, about 1×10 11 vg / kg, about 1×10 12 vg / kg, about 1×10 13 vg / kg, or it may be in the range of about 1×10 14 vg / kg. The total amount of viral particles or DRP is , about, at least, at least about, at most, or at most about, 5×10 15 vg / kg , 4×10 15 vg / kg, 3×10 15 vg / kg, 2×10 15 vg / kg, 1×1 0 15 vg / kg, 9×10 14 vg / kg, 8×10 14 vg / kg, 7×10 14 v g / kg, 6×10 14 vg / kg, 5×10 14 vg / kg, 4×10 14 vg / kg , 3×10 14 vg / kg, 2×10 14 vg / kg, 1×10 14 vg / kg, 9×1 013 vg / kg、8×10 13 vg / kg、7×10 13 vg / kg、6×10 13 v g / kg、5×10 13 vg / kg、4×10 13 vg / kg、3×10 13 vg / kg 、2×10 13 vg / kg、1×10 13 vg / kg、9×10 12 vg / kg、8×1 0 12 vg / kg、7×10 12 vg / kg、6×10 12 vg / kg、5×10 12 v g / kg、4×10 12 vg / kg、3×10 12 vg / kg、2×10 12 vg / kg 、1×10 12 vg / kg、9×10 11 vg / kg、8×10 11 vg / kg、7×1 0 11 vg / kg、6×10 11 vg / kg、5×10 11 vg / kg、4×10 11 v g / kg、3×10 11 vg / kg、2×10 11 vg / kg、1×10 11 vg / kg 、9×10 10 vg / kg、8×10 10 vg / kg、7×10 10 vg / kg、6×1 0 10 vg / kg、5×10 10 vg / kg、4×10 10 vg / kg、3×10 10 v g / kg、2×10 10 vg / kg、1×10 10 vg / kg、9×10 9 vg / kg、 8×109 vg / kg, 7×10 9 vg / kg, 6×10 9 vg / kg, 5×10 9 vg / kg, 4×10 9 vg / kg, 3 × 10 9 vg / kg, 2 × 10 9 vg / kg, 1×1 0 9 vg / kg, 9×10 8 vg / kg, 8×10 8 vg / kg, 7×10 8 vg / kg , 6×10 8 vg / kg, 5×10 8 vg / kg, 4×10 8 vg / kg, 3 × 10 8 v g / kg, 2 × 10 8 vg / kg, or 1 × 10⁻⁶ 8 It is vg / kg, or these The listed dosages above fall within the range defined by any two of the values ​​of the cardiac tissue. This is the unit vg per 1kg.

[0065] In addition to viral vectors, non-viral expression constructs can also target proteins in patient cells. Used to introduce genes that encode functional variants or fragments thereof. This may also be done. Nonviral expression beds that allow in vivo expression of proteins in target cells. Examples of subjects include plasmids, modified RNA, cDNA, and antisense oligomers. DNA-lipid complexes, nanoparticles, exosomes, and any other suitable for gene therapy. Examples include non-viral shuttles, their variations, and combinations thereof.

[0066] In addition to viral vectors and non-viral expression vectors, nuclease systems also It enters the patient's cells and encodes the target protein or its functional variant or fragment. To introduce the gene there, a vector and / or electroporation system It may be used in combination with a stem. An example of a nuclease system is a non Limited, clustered regularly interspaced sho rt palindromic repeats (CRISPR), DNA cutting enzymes (examples) For example, Cas9, meganuclease, TALEN, zinc finger nuclease, Any other nuclease system, variations thereof, suitable for genetic therapy. And combinations of these can be listed. For example, in one embodiment, one U Illus vectors (e.g., AAV) are used for nucleases (e.g., CRISPR). It may be used, and another viral vector (e.g., AAV) may be used with a DNA-cutting enzyme (e.g., It may also be used for Cas9, thereby enabling both (nuclease and DNA cleavage). The enzyme is introduced into the target cells.

[0067] Used to deliver therapeutic polynucleotide sequences encoding therapeutic genes to cells. Other possible vector delivery systems are receptor-mediated delivery media. These are almost all This utilizes the selective uptake of macromolecules by receptor-mediated endocytosis in eukaryotic cells. Due to the cell-type-specific distribution of various receptors, delivery can be highly specific. The gene-mediated targeting medium consists of two components: a cell receptor-specific ligand and a DNA binding agent. It may contain an agent.

[0068] A preferred method for transferring nonviral vectors into target cells is, for example, lipofecti The ion method, calcium phosphate coprecipitation method, DEAE dextran method, and microglass method. Direct DNA delivery methods using tubes, ultrasound, and electroporation Yes. Before vector introduction, cardiomyocytes are treated with a permeabilizing agent, such as phosphatidylcholine. Streptolysine, sodium caprate, decanoyl carnitine, tartaric acid, lysolecithin They may also be treated with , and Triton X-100, etc. Exosomes are also , It may be used to transfer Ikid DNA or AAV capsid-encapsulated DNA.

[0069] The gene therapy vector of the present invention is functionally linked to a nucleic acid sequence encoding a target protein. The promoter sequence may include a compact and strong expression sequence. It must be effective. Preferably, the promoter is the gene therapy vector It provides the expression of the target protein in the myocardium of patients treated with it. Some embodiments In this context, the gene therapy vector is operably linked to the nucleic acid sequence encoding the target protein. Contains a linked cardiac-specific promoter. As used herein, "cardiac-specific" The "promoter" is such that its activity in cardiac cells is more than in any other non-cardiac cell type. It also refers to a promoter that is at least twice as high. Preferably used in the vector of the present invention. A suitable cardiac-specific promoter for this purpose has less activity compared to its activity in non-cardiac cell types. At least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 2 It exhibits five times, or at least 50 times, higher activity in cardiac cells.

[0070] Cardiac-specific promoters are selected human promoters, or selected human promoters. At least approximately 80%, at least approximately 90%, at least approximately 95%, and less than the motor. At least 96%, at least 97%, at least 98%, or at least 99% A promoter containing a functionally equivalent sequence with % sequence identity may also be used. An example of an unspecified promoter that may be used is the cardiac troponin T promoter (TNNT). 2) Another non-limiting example of a promoter is alpha-myosin heavy chain promoter. Term, myosin light chain 2v promoter, alpha myosin heavy chain promoter, alpha- Cardiac actin promoter, alpha-tropomyosin promoter, cardiac troponin C Promoter, cardiac troponin I promoter, cardiac myosin-binding protein C promoter Ca, and sarcoplasmic reticulum / endoplasmic reticulum Ca 2+ -ATPase (SERCA) promoter ( For example, this promoter isoform 2 (SERCA2) is one such example.

[0071] The vectors useful in this invention may have various transduction efficiencies. As a result, Lus or non-viral vectors penetrate approximately 10% of cells in the targeted vascular territory, approximately 2%. 0%, approximately 30%, approximately 40%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 7 5%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 99%, or higher than 100%, Transduction is performed using an equal or greater number of vectors (including viruses). (either viruses, non-viruses, or combinations thereof) can be used simultaneously or sequentially. This can be used to import one or more polynucleotides, and / or one It can target a wider variety of cells. Multiple vectors or multiple agents can be used. If so, it results in one or more transfection efficiencies. It is possible.

[0072] A pharmaceutical composition containing a gene therapy vector may be expressed as either a liquid solution or a suspension. The pharmaceutical compositions of the present invention may be prepared using commonly used pharmaceutically acceptable excipients. The composition may include agents, such as diluents and carriers. In particular, the composition may be pharmaceutically acceptable. A carrier, for example, contains water, saline solution, Ringer's solution, or dextrose solution. Furthermore, the pharmaceutical composition also contains emulsifiers, pH buffering agents, stabilizers, and dyes, etc. That's good too.

[0073] In a particular embodiment, the pharmaceutical composition comprises a therapeutically effective gene dose, This allows for the prevention or treatment of cardiomyopathy in the subject without being toxic to the subject. This is a possible dosage. Prevention or treatment of cardiomyopathy is based on the phenotypic features associated with cardiomyopathy. These changes may be evaluated as changes in cardiomyopathy, and such changes may be used to prevent or treat cardiomyopathy. Therefore, the therapeutically effective gene dose is typically physiological. When administered in a composition acceptable to the patient, the pathogenic cardiac phenotype in the patient being treated It is sufficient to improve or prevent it.

[0074] In certain embodiments, the gene therapy vector is delivered intravenously, intra-arterially, and The subject may be transduced through several different methods, including intraperitoneal delivery. In one embodiment, the gene therapy vector is directly delivered to cardiac tissue, for example, by intracoronary administration. It may be administered via catheter. In some embodiments, myocardial tissue transduction is performed via catheter. This may also be achieved by mediated intramyocardial delivery, which is carried out while linked to a transduction-enhancing carrier. Alternatively, it may be used to transfer unlinked vector-free cDNA into the cardiomyocyte. .

[0075] In certain embodiments, the drug contains a therapeutically effective gene dose. The effective gene dose is one that does not cause toxicity to the patient and predicts a specific cardiac condition in the patient. It is something that can be prevented or treated.

[0076] Cardiac conditions that can be treated by the methods disclosed herein are, without limitation, genetically determined. Determined heart disease (e.g., genetically determined cardiomyopathy), arrhythmia heart disease, heart failure, Ischemia, arrhythmia, myocardial infarction, congestive heart failure, graft rejection, abnormal cardiac contraction, non-ischemic cardiomyopathy mitral valve regurgitation, aortic stenosis or regurgitation, abnormal calcium levels 2+ Metabolism, congenital heart disease, original This may include primary or secondary cardiac tumors, and one or more combinations thereof. stomach. [Examples]

[0077] Practical and prophetic examples The following embodiments are provided to aid in understanding the present disclosure and, naturally, in this application This should not be construed as particularly limiting the embodiments described and claimed. Including the replacement of all publicly known or subsequently developed equivalents within the scope of the contractor's knowledge. Such variations of the embodiments, and changes or experimental designs in formulations Such minor modifications should be considered to fall within the scope of the embodiments incorporated herein. ru.

[0078] In a practical example of an in vitro system, PKP2 isoform 2a cDNA distribution The column (2764bp cDNA, GenBank:BC126199.1; Sequence ID 1) Using AAV2 internal terminal repeats (ITRs), the cardiac-specific TNNT2 promoter (sequence) Number 5) Cloned below: ITR-TNNT2-PKP2cDNA-ITR. As an example of this, the nucleic acid sequence encoding PKP2 is PKP2 isoform 2b ( This is a codon-optimized version (sequence number 3) of the PKP2 gene that encodes sequence number 4. It is also acceptable for the construct to be vectorized during AAV, for example, AAV6 and AAV9. Good. Identify the protein after transfection with anti-Flag and use it as an endogenous agent. A construct with a flag added to distinguish it from sex proteins (Flag-PKP2) A solution may be prepared. Sequence ID 6 expresses, for example, PKP2 isoform 2b. The following is an example construct sequence. PKP2 expression in vitro is PKP2- The cell membrane and dense plaque may also be observed using immunofluorescence microscopy with the following antibody, thereby revealing the cell membrane and dense plaque. The localization of PKP2 in Lark is revealed.

[0079] AAV6-TNNT2-PKP2 is a normal cardiomyocyte; one (from an ARVC patient) Cardiomyocytes with heterozygous PKP2 mutations; and trans with two PKP2 mutations. Transfection of iPSC-CM in 2D cell cultures containing cardiomyocytes with different characteristics It is used for [purpose].

[0080] Successful transfection and characteristic development at the PKP2 RNA and protein levels. After marking, a comparison was made between normal CM, PKP2-deficient CM, and PKP2-corrected CM, based on cell size. Furthermore, genes whose expression is known to be altered in PKP2 deficiency (MYL2, SCN) Correction of 5A (whose protein product is NaV1.5), GJA1, and TTN This is done for numerous leadouts, including [mention specific examples].

[0081] Similar methodologies include ex vivo treatment in human 3D culture models, as well as PKP2 It is assumed that this could be adapted for in vivo treatment in mutant mouse models. ru.

[0082] When the (Flag-)PKP2 protein is expressed, it has precise intracellular localization ( (Smosomes) Also, transfection occurs in PKP2 haploinsufficiency or complete absence It is thought to correct damaged cells at the RNA and protein levels. In P2-deficient cells, PKP2 transfection also affects the desmosomes. Restoration of the desmosome protein complex, particularly the reduction in PKP2 levels. It is believed that lacoglobin can be restored.

[0083] To express PKP2 isoform 2a, PKP2 isoform 2b, or both It is further hypothesized that the gene therapy vector may be delivered to human cardiac tissue. For example, a gene therapy vector is one or more gene therapy vectors and pharmaceuticals The formulation may be formulated into a therapeutic preparation containing a suitably acceptable excipient or carrier. The preparation may be administered intravenously. Human-to-human pairings can be delivered through several different methods, including intra-arterial delivery, intraperitoneal delivery, or intraperitoneal delivery. It may be introduced into elephants. The gene therapy vector can be directly delivered to cardiac tissue, for example, by intracoronary administration. It may be administered via intracardiac delivery. Gene therapy vectors may also be delivered via catheter-mediated intramyocardial delivery. It may be delivered in this manner.

[0084] Gene therapy vectors, for example, isolate the target coronary circulation from the target systemic circulation and close the gap. Forming a complex circuit and targeting fluids (e.g., formulations including gene therapy vectors). By perfusing the isolated coronary circulation, it is administered locally to the target cardiac tissue. It is also anticipated that this may be beneficial. Perfusion is effective in the non-stopping, beating heart of the subject. This may be done by: The closed circuit is, for example, a first drug delivery system located in the patient's right coronary artery. A catheter, a second drug delivery catheter placed in the left main coronary artery of the patient, into the coronary sinus. Between the placed drug collection catheter, coronary arteries, coronary venous system, and venous and arterial branches It may be formed using an interlocked external membrane oxygenator. Such local delivery is 20 International application PCT / IB2020 / 000692 filed on August 26, 2020 ( Even if the disclosure is made as described in relation to (the entire disclosure is incorporated herein by reference) good.

[0085] In the above description, in order to provide a thorough understanding of the present invention, numerous specific details and examples have been provided. For example, specific materials, dimensions, process parameters, etc. are described. Specific configuration, structure, materials The materials or features are combined in any preferred manner in one or more embodiments. It is also acceptable to use the words "example" or "exemplary" to mean an example, case, or example. As used herein, "example" or "exemplary" is used to mean the same thing. Any aspect or design described in the details is preferable or better than any other aspect or design. It is not necessarily construed as being advantageous. Rather, the use of the words "example" or "exemplary" is simply intended to present the concept in a specific manner. As used in this application in the case, the term "or" is meant to be an inclusive "or" rather than an exclusive "or" is intended. That is, unless otherwise specified or clear from the context is not, "X includes A or B" is meant to mean any of the natural inclusive permutations is intended. That is, if X includes A, or X includes B, or X includes both A and B "X includes A or B" is satisfied under any of the above cases . Throughout this specification, references to "embodiments", "certain embodiments", or "one embodiment" mean that a particular configuration, structure, or feature described in connection with the embodiment is included in at least one embodiment. Therefore, the appearances of the phrases "embodiments", "certain embodiments", or "one embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment. Throughout this specification, references to "embodiments", "certain embodiments", or "one embodiment" do not necessarily all refer to the same embodiment.

[0086] The present invention has been described with reference to its particular exemplary embodiments. This specification and the drawings are to be regarded in an illustrative rather than a restrictive sense. Various modifications of the invention will become apparent to those skilled in the art in addition to what is shown and described herein, and these are intended to fall within the scope of the appended claims. Therefore, they should be considered in an illustrative rather than a restrictive sense. In addition to what is shown and described in this specification , various modifications of the present invention will become apparent to those skilled in the art, and these are intended to fall within the scope of the appended claims.

[0087] The following SEQ ID NO: 1 is a cDNA copy of the mRNA sequence containing the protein coding sequence of PKP2 isoform 2a (GenBank: BC126199.1):

[0088] ​​ [Chemistry]

[0089] [Chemistry]

[0090] The following SEQ ID NO: 2 is the amino acid sequence of PKP2 isoform 2a:

[0091] [Chemistry]

[0092] The following SEQ ID NO: 3 is the codon-optimized cDNA encoding PKP2 isoform 2b:

[0093] [Chemistry]

[0094] [Chemistry]

[0095] The following SEQ ID NO: 4 is the amino acid sequence of PKP2 isoform 2b:

[0096] [Chemistry]

[0097] The following SEQ ID NO: 5 is the nucleic acid sequence encoding the TNNT2 promoter:

[0098] [Chemistry]

[0099] The following Sequence ID 6 exemplifies the expression of PKP2 isoform 2b in cardiomyocytes. It is a vector construct:

[0100] [ka]

[0101] [ka]

[0102] [ka]

[0103] [ka]

[0104] [ka]

Claims

1. A method for treating or preventing cardiomyopathy in human subjects, wherein the therapeutic dose of gene therapy The process includes delivering the gene therapy vector to the human target cardiomyocytes, wherein the gene therapy vector is Contains nucleic acid sequences encoding lacofilin-2 (PKP2) or its functional variant. Hmm, a method.

2. The method according to claim 1, wherein the gene therapy vector comprises a viral vector.

3. The aforementioned viral vectors are AAV1, AAV2, AAV3, AAV4, AAV5, AA V6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, these Claim 2 includes one or more of the variations and combinations thereof. The method.

4. The method according to claim 2, wherein the viral vector comprises AAV6 or AAV9.

5. The method according to claim 2, wherein the viral vector comprises AAV6.

6. Any one of claims 1 to 5, wherein the nucleic acid sequence further encodes a heart-specific promoter. The method described in item 1.

7. The therapeutic dose is the PKP2 or its functional effect on the cardiomyocytes of the human subject. Treating arrhythmogenic right ventricular cardiomyopathy (ARVC) by inducing the production of variants The method according to any one of claims 1 to 6, which is effective for prevention.

8. The method according to any one of claims 1 to 7, wherein the delivery of the therapeutic dose is performed intravenously. Law.

9. A gene therapy vector adapted for expressing nucleic acid sequences in human cardiomyocytes. The nucleic acid sequence is, A first sequence encoding PKP2 or a functional variant thereof, and Second sequence containing a cardiac-specific promoter Gene therapy vectors, including those mentioned above.

10. The gene therapy vector according to claim 9, wherein the gene therapy vector comprises a viral vector. Tar.

11. The aforementioned viral vectors are AAV1, AAV2, AAV3, AAV4, AAV5, AA V6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, these The variations and one or more combinations thereof as described in claim 10. Gene therapy vectors.

12. The gene therapy according to claim 10, wherein the viral vector comprises AAV6 or AAV9. Law vector.

13. The cardiac-specific promoter comprises TNNT2, as described in any one of claims 9 to 12. Gene therapy vectors.

14. A therapeutic formulation for treating or preventing cardiomyopathy in human subjects, pharmaceutically acceptable excipients or carriers, Viral vectors containing nucleic acid sequences encoding PKP2 or its functional variants. A therapeutic formulation containing [the active ingredient].

15. One or more non-PKP2 sarcomere proteins or their functional variants One or more additional viral vectors, each containing a nucleic acid sequence to be routed. The therapeutic formulation according to claim 14, further comprising the above.

16. Genetically modify PKP2 mutant cardiomyocytes to express non-mutant PKP2. It is a method, The nucleic acid sequence encoding the non-mutant PKP2 is introduced into the PKP2 mutant cardiomyocytes. Transfecting Methods that include...

17. The nucleic acid sequence is delivered via a viral vector containing AAV6 or AAV9. The method according to claim 16.

18. The method according to claim 17, wherein the viral vector comprises AAV6.

19. Any of claims 16 to 18, wherein the nucleic acid sequence further encodes a heart-specific promoter The method described in any one of the items.

20. The method according to claim 19, wherein the cardiac-specific promoter includes TNNT2.

21. The PKP2 is PKP2 isoform 2a, according to claims 1 to 8 or 16 to 20. The method described in any one of the items.

22. The PKP2 is PKP2 isoform 2b, according to claims 1 to 8 or 16 to 20. The method described in any one of the items.

23. The PKP2 is PKP2 isoform 2a, according to any one of claims 9 to 15. The gene therapy vector or therapeutic formulation described herein.

24. The PKP2 is PKP2 isoform 2b, according to any one of claims 9 to 15. The gene therapy vector or therapeutic formulation described herein.

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