Gene therapy compositions and treatments for cardiomyopathy associated with muscular dystrophin

Abstract

A composition and method for treating or preventing cardiomyopathy in a human subject is disclosed. In one embodiment, a method comprises delivering a gene therapy drug to cardiac muscle tissue of the human subject. The gene therapy drug comprises a vector comprising a polynucleotide coding sequence of a micro muscular dystrophin.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 300,167, filed on January 17, 2022, the disclosure of which is hereby incorporated by reference in its entirety. Technical Field

[0003] The present invention relates to the treatment of cardiac diseases (eg, cardiomyopathies), and more particularly, to gene therapy methods and pharmaceutical compositions for treating dystrophin-associated cardiomyopathies. Background of the Invention

[0005] Despite advances in pharmacology for the treatment of various cardiac disorders such as heart failure, mortality and morbidity remain unacceptably high. In addition, certain treatments are not suitable for many patients (e.g., patients with advanced heart failure and other comorbidities). Alternative approaches, such as gene therapy and cell therapy, have attracted considerable attention because they have the potential to be uniquely targeted and effective in addressing the underlying pathogenic mechanisms of many heart diseases. Summary of the invention

[0006] It is an object of certain embodiments of the present invention to provide methods for delivering therapeutic polynucleotide sequences to cardiomyocytes of human subjects.

[0007] It is another object of certain embodiments of the present invention to utilize gene therapy approaches for treating dystrophin-related cardiomyopathies.

[0008] It is another object of certain embodiments of the present invention to vectorize the polynucleotide sequence encoding the micro-dystrophin construct.

[0009] Another object of certain embodiments of the present invention is to deliver vectored micro-dystrophin constructs to cardiac tissue in human patients.

[0010] The above objectives and other objectives are met by the present invention, wherein certain embodiments relate to a gene therapy drug for treating or preventing cardiomyopathy in a human subject, the gene therapy drug comprising: a vector comprising a polynucleotide sequence encoding a micro-dystrophin protein, wherein the polynucleotide encoding sequence encodes a subset of repeating units R1-R24 of the dystrophin protein, and wherein the repeating units of the subset are selected from the group consisting of: R1, R2, R24, portions thereof, and combinations thereof, with the proviso that R1 and R24 or portions thereof are present in the subset.

[0011] In at least one embodiment, the polynucleotide coding sequence encodes only hinge 1, hinge 3 and hinge 4 of the hinge domain of the dystrophin protein.

[0012] In at least one embodiment, the polynucleotide coding sequence encodes only hinge 1 and hinge 4 of the hinge domain of the dystrophin protein.

[0013] In at least one embodiment, the repeating unit of the subset consists of R1 and R24 or a portion thereof. In at least one embodiment, the polynucleotide coding sequence encodes the entire CT domain of the dystrophin protein. In at least one embodiment, the polynucleotide coding sequence encodes about 65% to about 85% of R1 and about 20% to about 40% of R24. In at least one embodiment, the polynucleotide coding sequence encodes about 65% to about 85% of R1 and about 50% to about 70% of R24, provided that the polynucleotide sequence completely encodes exon 60 of the dystrophin gene. In at least one embodiment, the polynucleotide coding sequence encodes about 95% or more of R1 and about 60% or less of R24, provided that the polynucleotide sequence completely encodes exon 60 of the dystrophin gene.

[0014] In at least one embodiment, the polynucleotide sequence encodes only exons 70-75 of the cysteine-rich domain of the dystrophin protein or a portion thereof. In at least one embodiment, the polynucleotide sequence further encodes at least a portion of the repeating unit R2. In at least one embodiment, the polynucleotide sequence encodes only hinge 1, hinge 3 and hinge 4 of the hinge domain of the dystrophin protein, and wherein the repeating units of the subset consist of R1, R2 and R24 or a portion thereof.

[0015] In at least one embodiment, the gene therapy drug comprises a polynucleotide sequence comprising a micro-dystrophin coding sequence that is at least 90% identical to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

[0016] In at least one embodiment, the polynucleotide sequence further encodes a myocardium-specific promoter. In at least one embodiment, the myocardium-specific promoter is a TNNT2 promoter.

[0017] In at least one embodiment, the vector comprises a viral vector. In at least one embodiment, the viral vector is an AAV vector. In at least one embodiment, the AAV vector comprises AAV9.

[0018] In at least one embodiment, the expression product of the polynucleotide coding sequence in a human subject is capable of restoring syntrophin binding and localization in dystrophin-deficient myocardium.

[0019] In at least one embodiment, a method of treating or preventing a dystrophin-related cardiomyopathy in a human subject comprises delivering any of the aforementioned gene therapy drugs to cardiac tissue of the human subject. In at least one embodiment, the dystrophin-related cardiomyopathy is associated with Duchenne muscular dystrophy or Becker muscular dystrophy.

[0020] At least one embodiment relates to a functional micro-dystrophin protein encoded by SEQ ID NO:2.

[0021] At least one embodiment relates to a functional micro-dystrophin protein encoded by SEQ ID NO:3.

[0022] At least one embodiment relates to a functional micro-dystrophin protein encoded by SEQ ID NO:4.

[0023] At least one embodiment relates to a functional micro-dystrophin protein encoded by SEQ ID NO:5. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The above and other features of the present disclosure, its nature and various advantages will become more apparent upon consideration of the following detailed description taken in conjunction with the accompanying drawings, in which:

[0025] Figure 1 shows an exemplary micro-dystrophin construct according to an embodiment of the present disclosure;

[0026] Figure 2A is a plot of vector copy number analysis in mouse heart 4 weeks after delivery of a micro-dystrophin construct according to an embodiment of the present disclosure;

[0027] Figure 2B is a plot of vector copy number analysis in mouse heart 12 weeks after delivery of a micro-dystrophin construct according to an embodiment of the present disclosure;

[0028] Figure 3 is a graph of vector copy number analysis (mean / group) in mouse hearts at 4 and 12 weeks after delivery of a micro-dystrophin construct according to an embodiment of the present disclosure;

[0029] Figure 4 is a plot of the relative quantification of micro-dystrophin mRNA in the heart tissue of each experimental group;

[0030] Figure 5is a plot of relative quantification of micro-dystrophin mRNA in cardiac tissue (mean / group);

[0031] Fig. 6A Western Blot assay, indicating the end of the CT domain expressing micro-dystrophin;

[0032] Figure 6B is a Western blot assay, which indicates the expression of the R1 domain of micro-dystrophin;

[0033] Figure 7 For mdx 4CV Immunofluorescence images of micro-dystrophin expression in mouse heart;

[0034] Figure 8 Describes normal syntrophin expression, mdx in normal mouse heart according to embodiments of the present disclosure 4CV Aberrant syntrophin expression in mouse hearts and restoration of syntrophin expression following gene therapy with micro-dystrophins 1, 2, and 4;

[0035] Fig.9A Plot showing heart rate, QRS width, and QT interval obtained by electrocardiographic measurements three months after injection of a micro-dystrophin construct in newborn mice;

[0036] Fig. 9B Plot showing heart rate, QRS width, and QT interval obtained from electrocardiographic measurements six months after injection of a micro-dystrophin construct in newborn mice;

[0037] Fig. 10A shows structural remodeling of the left ventricle three months after injection of a micro-dystrophin construct in newborn mice;

[0038] Fig. 10B showed structural remodeling of the left ventricle six months after injection of a micro-dystrophin construct in newborn mice;

[0039] Fig.11A Plots showing systolic and diastolic function three months after injection of a micro-dystrophin construct in newborn mice;

[0040] Fig. 11B Plots showing systolic and diastolic function six months after injection of a micro-dystrophin construct in newborn mice;

[0041] Fig.12 shows the biodistribution profiles measured in different regions of the heart and liver three months after injection of micro-dystrophin constructs in newborn mice;

[0042] Fig.13Western blot images showing evaluation of micro-dystrophin expression in left ventricular tissue three months after injection;

[0043] Fig.14 Shows the expression level of micro-dystrophin in the septal ventricle of the heart;

[0044] Fig.15 showed the beating rate and force produced by post-treatment micro-dystrophin expression in engineered cardiac tissue;

[0045] Fig.16 showed arrhythmia scores after micro-dystrophin expression in engineered cardiac tissue;

[0046] Fig.17 A vector showing an exemplary micro-dystrophin construct according to the first embodiment;

[0047] Fig.18 A vector showing an exemplary micro-dystrophin construct according to a second embodiment;

[0048] Fig.19 A vector showing an exemplary micro-dystrophin construct according to the third embodiment; and

[0049] Fig. 20 A vector of an exemplary micro-dystrophin construct according to the fourth embodiment is shown.

[0050] definition

[0051] As used herein, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a drug" includes a single drug as well as a mixture of two or more different drugs; and reference to "a viral vector" includes a single viral vector as well as a mixture of two or more different viral vectors, and so forth.

[0052] Also as used herein, when used in conjunction with a measured amount, "about" refers to the normal variation of the measured amount as expected by one of ordinary skill in the art to match the measurement and operation with the objective of the measurement and the precision of the measuring equipment at the level of interest. In certain embodiments, the term "about" includes the stated value ± 10%, such that "about 10" would include 9 to 11.

[0053] As used herein, "polynucleotide" has its common and customary meaning in the art and includes any polymeric nucleic acid such as DNA or RNA molecules, and chemical derivatives known to those skilled in the art. Polynucleotides include not only those polynucleotides encoding therapeutic proteins, but also sequences (e.g., antisense, interference or less interfering nucleic acids) that can be used to reduce the expression of target nucleic acid sequences using techniques known in the art. Polynucleotides can also be used to start or increase the expression of target nucleic acid sequences or the production of target proteins in cells of the cardiovascular system. Target nucleic acids and proteins include, but are not limited to, nucleic acids and proteins commonly found in target tissues, derivatives of such naturally occurring nucleic acids or proteins, naturally occurring nucleic acids or proteins that are not commonly found in target tissues, or synthetic nucleic acids or proteins. One or more polynucleotides can be used in combination, administered simultaneously and / or sequentially, to increase and / or reduce one or more target nucleic acid sequences or proteins.

[0054] Also as used herein, a "foreign" nucleic acid or gene is a nucleic acid that does not occur naturally in the vector used for nucleic acid transfer; for example, a nucleic acid that does not occur naturally in a viral vector, but the term is not intended to exclude nucleic acids encoding proteins or polypeptides that occur naturally in a patient or host.

[0055] As also used herein, "cardiac cells" include any cardiac cell that participates in maintaining the structure or providing cardiac function, such as cardiomyocytes, cells of the cardiac vasculature, or cells present in heart valves. Cardiac cells include cardiomyocytes (with both normal and abnormal electrical properties), epithelial cells, endothelial cells, fibroblasts, cells of conductive tissue, cardiac pacemaker cells, and neurons.

[0056] Also as used herein, "adeno-associated virus" or "AAV" encompasses all subtypes, serotypes, and pseudotypes, as well as naturally occurring and recombinant forms. Various AAV serotypes and strains are known in the art and are publicly available from sources such as the ATCC, and academic or commercial sources. Alternatively, sequences from AAV serotypes and strains that are publicly available and / or purchased from various databases can be synthesized using known techniques.

[0057] Also as used herein, "serotype" refers to an AAV that is identified and distinguished from other AAVs based on capsid protein reactivity with defined antisera. There are at least twelve known serotypes of human AAV, including AAV1 to AAV12, however, additional serotypes continue to be discovered, and newly discovered serotypes are contemplated for use.

[0058] As also used herein, "pseudotyped" AAV refers to an AAV containing capsid proteins from one serotype and a viral genome that includes 5' and 3' inverted terminal repeats (ITRs) of a different or heterologous serotype. Pseudotyped recombinant AAV (rAAV) will be expected to have the cell surface binding properties of the capsid serotype and genetic properties consistent with the ITR serotype. Pseudotyped rAAV may comprise AAV capsid proteins, including VP1, VP2 and VP3 capsid proteins, and ITRs from any serotype of AAV, including any primate AAV serotype from AAV1 to AAV12, as long as the capsid proteins have a serotype that is heterologous to the serotype of the ITR. In pseudotyped rAAV, 5' and 3' ITRs may be identical or heterologous. Pseudotyped rAAV is produced using standard techniques described in the art.

[0059] As also used herein, a "chimeric" rAAV vector encompasses an AAV vector comprising heterologous capsid proteins; that is, the rAAV vector may be chimeric with respect to its capsid proteins VP1, VP2, and VP3, such that VP1, VP2, and VP3 are not all of the same serotype AAV. Chimeric AAV as used herein encompasses AAVs in which the capsid proteins VP1, VP2, and VP3 differ in serotype, including, for example, but not limited to, capsid proteins from AAV1 and AAV2; a mixture of other parvovirus capsid proteins or comprising other viral proteins or other proteins, such as proteins that target AAV delivery to desired cells or tissues. Chimeric rAAV as used herein also encompasses rAAVs comprising chimeric 5' and 3' ITRs.

[0060] As used herein, "pharmaceutically acceptable excipient or carrier" refers to any inert ingredient in a composition that is combined with an active agent in a formulation. Pharmaceutically acceptable excipients may include, but are not limited to, carbohydrates (e.g., glucose, sucrose, or dextran), antioxidants (e.g., ascorbic acid or glutathione), chelating agents, low molecular weight proteins, high molecular weight polymers, gelling agents or other stabilizers and additives. Examples of other pharmaceutically acceptable carriers include wetting agents, emulsifiers, dispersants, or preservatives that are particularly suitable for preventing microbial growth or activity. Various preservatives are well known and include, for example, phenol and ascorbic acid. Examples of carriers, stabilizers, or adjuvants can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th edition. (1985).

[0061] Also as used herein, "patient" refers to a subject, particularly a human (although non-humans are also contemplated), who has clinical manifestations of one or more specific symptoms indicating a need for treatment, who is being treated to prevent or treat a disorder, or who has been diagnosed with a disorder to be treated.

[0062] Also as used herein, "subject" encompasses the definition of the term "patient" and does not exclude otherwise healthy individuals.

[0063] Also as used herein, "treatment of" and "treating" include the administration of drugs intended to lessen the severity of a condition, such as heart disease, or to prevent the condition.

[0064] Also as used herein, "prevention of" and "preventing" include avoiding the onset of a disorder such as a heart attack.

[0065] Also as used herein, "condition" or "conditions" refer to those medical conditions, such as heart disease, that can be treated, alleviated or prevented by administering an effective amount of a drug to a subject.

[0066] As also used herein, "effective amount" refers to the amount of a drug that is sufficient to produce a beneficial or desired effect at a level that can be easily detected by methods commonly used to detect such effects. In some embodiments, such an effect causes a change of at least 10% from the value of the basal level of the drug that has not been administered. In other embodiments, the change is at least 20%, 50%, 80% or even higher percentages compared to the basal level. As will be described below, the effective amount of the drug may vary with each subject, depending on age, the general condition of the subject, the severity of the illness being treated, the specific drug being administered, etc. The appropriate "effective" amount in any individual case may be determined by a person of ordinary skill in the art with reference to relevant texts and literature and / or by using routine experiments.

[0067] Also as used herein, "active agent" means any substance intended to produce a therapeutic, prophylactic, or other desired effect, whether or not it is approved by a governmental agency for that purpose.

[0068] Unless otherwise indicated, the enumeration of the value range herein is intended only to serve as a shorthand method for individually referring to each independent value within the range, and each independent value is incorporated into this specification as if individually listed herein. Unless otherwise indicated herein or clearly contradictory to the content, all methods described herein can be carried out in any suitable order. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to illustrate certain materials and methods and is not intended to limit the scope. The language of this specification should not be interpreted as indicating that any non-claimed element is essential for the practice of the disclosed materials and methods. DETAILED DESCRIPTION

[0069] The present invention relates to a micro-dystrophin construct for treating cardiomyopathy associated with dystrophin. Dystrophin is a cytoplasmic protein that is part of a protein complex that connects the cytoskeleton of a muscle fiber to the extracellular matrix via a cell membrane. The DMD gene encoding dystrophin is about 2.3 megabases in length and is located at locus Xp21. SEQ ID NO: 1 is a muscle transcript of 79 exons of the DMD gene, which includes 11,058 base pairs (bp). The complete dystrophin includes the following regions from N-terminus to C-terminus: an actin binding domain, hinge 1 (H1), repeat units 1-3, hinge 2 (H2), repeat units 4-19, hinge 3 (H3), repeat units 20-24, hinge 4 (H4), a cysteine-rich (CR) domain and a C-terminal region that overlap with the H4 portion. Mutations in the dystrophin gene (i.e., spontaneous mutations or inherited mutations) can lead to different forms of muscular dystrophy. The most common conditions caused by dystrophin defects are Duchenne muscular dystrophy and Becker muscular dystrophy.

[0070] Micro-dystrophin gene therapy is a method of using a shorter but functional variant of dystrophin (referred to as "microdystrophin protein" or "microdystrophin") to reduce muscle damage in patients with muscular dystrophy. Embodiments of the present application relate to various micro-dystrophin constructs targeted for the treatment of cardiomyopathy. For example, such constructs can be expressed using a cardiac-specific promoter (e.g., the TNNT promoter). Such constructs can be used to treat Duchenne muscular dystrophy, Becker muscular dystrophy, and other X-linked disorders caused by abnormal dystrophin. In certain embodiments, the micro-dystrophin constructs described herein contain a complete CR domain and a complete or nearly complete C-terminal region (e.g., up to exon 75, which includes a syntrophin binding domain that facilitates binding to ion channels in the myocardium).

[0071] Currently, the only curative treatment for all genetic diseases and heart failure is heart transplantation. Cardiac gene therapy based on AAV vectors has great promise for treating cardiomyopathies associated with dystrophin. AAV vectors are non-pathogenic, cannot replicate on their own, are maintained in the host cell nucleus in an extrachromosomal form, and can be delivered by intramyocardial or intracoronary or systemic injection.

[0072] Certain embodiments of the present disclosure relate to different methods of combining one or more AAVs related to AAV-mediated expression of a micro-dystrophin gene in cardiomyocytes (e.g., hiPSC-derived cardiomyocytes). In some embodiments, each box is packaged into a suitable AAV. For example, the boxes can each be packaged in rAAV2 / 9, which is a particularly efficient serotype for cardiomyocyte transduction.

[0073] Certain embodiments of the present disclosure relate to the expression of a functional micro-dystrophin containing about 25-40% of the total amino acids of the complete dystrophin protein (e.g., encoded by a nucleic acid sequence having a total length of 3000 bp to 4000 bp). Exemplary micro-dystrophin constructs are shown in Figure 1 In the dystrophin protein domains described above, only a subset of the dystrophin regions (or portions thereof) may be encoded. These include:

[0074] the N-terminal (NT) region encoded by SEQ ID NO:7;

[0075] the C-terminal (CT) region encoded by SEQ ID NO: 11;

[0076] the hinge 1 region encoded by SEQ ID NO:8;

[0077] the hinge 3 region encoded by SEQ ID NO:20;

[0078] the hinge 4 region encoded by SEQ ID NO:9;

[0079] the repeat sequence 1 (R1) region encoded by SEQ ID NO: 13;

[0080] the repeat 2 (R2) region encoded by SEQ ID NO: 19;

[0081] The repeat 24 (R24) region encoded by SEQ ID NO: 17; and

[0082] a cysteine-rich (CR) domain encoded by SEQ ID NO: 10,

[0083] wherein any given sequence within any micro-dystrophin construct may be at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the corresponding sequence above.

[0084] In at least one embodiment, one or more hinge regions may be present in the micro-dystrophin protein. For example, the micro-dystrophin protein construct may encode one or more of hinge 1, hinge 3, or hinge 4 (omit hinge 2). In at least one embodiment, the micro-dystrophin protein construct may encode hinge 1, hinge 4, and optionally hinge 3.

[0085] In at least one embodiment, the micro-dystrophin construct encoding the R1 region may encode less than the full length of the R1 region, such as less than about 80%, less than about 70%, less than about 60%, or less than about 50% (e.g., about 50% to about 85% or about 70% to about 80%). Exemplary sequences encoding a portion of R1 include SEQ ID NO: 12.

[0086] In at least one embodiment, the micro-dystrophin construct encoding the R2 region may encode less than the full length of the R2 region, such as less than about 10% (eg, about 4% to about 8%). An exemplary sequence encoding a portion of R2 includes SEQ ID NO:18.

[0087] In at least one embodiment, the micro-dystrophin construct encoding the R24 region may encode less than the full length of the R24 region, such as less than about 80%, less than about 70%, less than about 60%, less than about 50%, or less than about 40% (e.g., about 50% to about 70% or about 20% to about 40%). Exemplary sequences encoding portions of R24 include SEQ ID NO: 15 and SEQ ID NO: 16.

[0088] In at least one embodiment, the micro-dystrophin construct encoding the CT region may encode less than the full length of the CT region, such as less than about 70%, less than about 60%, less than about 50%, or less than about 40% (e.g., about 50% to about 60%). An exemplary sequence encoding a portion of the CT region is included as SEQ ID NO: 14.

[0089] In at least one embodiment, the TNNT2 promoter may correspond to the nucleotide sequence encoded by SEQ ID NO:6.

[0090] In at least one embodiment, the micro-dystrophin construct nucleic acid sequence is at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 2. In at least one embodiment, the micro-dystrophin may have an amino acid sequence that is at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 25. For example, Figure 1 Exemplary construct 1 (μDys1) of encodes the following dystrophin regions: NT region, hinge 1, about 65-85% of R1, about 20-40% of R24, hinge 4, CR domain, and CT region.

[0091] In at least one embodiment, the micro-dystrophin construct nucleic acid sequence is at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:3. In at least one embodiment, the micro-dystrophin may have an amino acid sequence that is at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:26. For example, Figure 1 Exemplary construct 2 (μDys2) of encodes the following dystrophin regions: NT region, hinge 1, about 65-85% of R1, about 50-70% of R24, hinge 4, CR domain, and CT region.

[0092] In at least one embodiment, the micro-dystrophin construct nucleic acid sequence is at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 4. In at least one embodiment, the micro-dystrophin may have an amino acid sequence that is at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 27. For example, Figure 1 Exemplary construct 3 (μDys3) of encodes the following dystrophin regions: NT region, hinge 1, R1, about 4-10% of R2, about 50-70% of R24 (entire exon 60), hinge 4, CR domain, and CT region.

[0093] In at least one embodiment, the micro-dystrophin construct nucleic acid sequence is at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 5. In at least one embodiment, the micro-dystrophin may have an amino acid sequence that is at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 28. For example, Figure 1 Exemplary construct 4 (μDys4) of encodes the following regions of dystrophin: NT region, hinge 1, R1, R2, hinge 3, R24, hinge 4, CR domain, and part of the CT region (exons 70-75).

[0094] Although many embodiments herein are described with respect to micro-dystrophin, it should be understood that the expression of other proteins (e.g., myofibrillar proteins) is encompassed. Other exemplary proteins include, but are not limited to, MYH7, PKP2, SERCA2, MYBPC3, MYL3, MYL2, ACTC1, TPM1, TNNT2, TNNI3, TTN, FHL1, ALPK3, FKRP, dystrophin-related proteins, one or more of their variants or combinations thereof. The one or more proteins used may also be functional variants of the proteins mentioned herein and may exhibit significant amino acid sequence identity compared to the original protein. For example, the amino acid identity can add up to 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%, at least about 97%, at least about 98%, or at least about 99%. In this context, the term "functional variant" means that a variant of a protein is able to partially or completely fulfill the function of the naturally occurring corresponding protein. Functional variants of a protein can include proteins that differ from their naturally occurring counterparts, for example, by one or more amino acid substitutions, deletions, or additions.

[0095] Amino acid substitutions can be conservative or non-conservative. Preferably, substitutions are conservative substitutions, i.e., amino acid residues are substituted with amino acids of similar polarity that serve as functional equivalents. Preferably, the amino acid residue used as a substitute is selected from the same amino acid group as the amino acid residue to be substituted. For example, a hydrophobic residue can be substituted by another hydrophobic residue, or a polar residue can be substituted by another polar residue with the same charge. Functionally homologous amino acids that can be used for conservative substitutions include, for example, non-polar amino acids such as glycine, valine, alanine, isoleucine, leucine, methionine, proline, phenylalanine and tryptophan. Examples of non-charged polar amino acids include serine, threonine, glutamine, asparagine, tyrosine and cysteine. Examples of charged polar (basic) amino acids include histidine, arginine and lysine. Examples of charged polar (acidic) amino acids include aspartic acid and glutamic acid.

[0096] The protein that is also considered as a variant is a protein that differs from its naturally occurring counterpart by one or more (e.g., 2, 3, 4, 5, 10 or 15) additional amino acids. These additional amino acids may be present in the amino acid sequence of the original protein (i.e., as an insertion), or they may be added to one or both ends of the protein. Basically, if the addition of amino acids does not weaken the ability of the polypeptide to achieve the function of the naturally occurring protein in the treated subject, the insertion may be performed at any position. In addition, the variant of the protein also includes proteins that lack one or more amino acids compared to the original polypeptide. Such deletions may affect any amino acid position, provided that it does not weaken the ability to achieve the normal function of the protein.

[0097] Finally, variants of cardiac myofibrillar proteins also refer to proteins that differ from naturally occurring proteins by structural modifications, such as modified amino acids. Modified amino acids are amino acids that have been modified by natural processes, such as processing or post-translational modifications, or by chemical modification methods known in the art. Typical amino acid modifications include phosphorylation; glycosylation; acetylation; O-linked N-acetylglucosaminylation; glutathionylation; acylation; branching; ADP ribosylation; cross-linking; disulfide bridge formation; formylation; hydroxylation; carboxylation; methylation; demethylation; amidation; cyclization and / or covalent or non-covalent bonding with phosphatidylinositol, flavin derivatives, lipoteichoic acid, fatty acids or lipids.

[0098] The therapeutic polynucleotide sequence encoding the target protein can be administered to the subject to be treated in the form of a gene therapy vector, i.e., a nucleic acid construct comprising a coding sequence adjacent to other sequences required for providing expression of the exogenous nucleic acid, such as a promoter, a kozak sequence, a polyA signal, etc., including translation and termination codons.

[0099] For example, the gene therapy vector can be part of a mammalian expression system. Useful mammalian expression systems and expression constructs are commercially available. In addition, several mammalian expression systems are sold by different manufacturers and can be used in the present invention, such as systems based on plasmids or viral vectors, such as LENTI-Smart TM (InvivoGen), GenScript TM Expression vector, pAdVAntage TM (Promega), ViraPower TM Lentivirus, adenovirus expression system (Invitrogen) and adeno-associated virus expression system (Cell Biolabs).

[0100] The gene therapy vector used to express the exogenous therapeutic polynucleotide sequence of the present invention can be, for example, a viral or non-viral expression vector, which is suitable for introducing the exogenous therapeutic polynucleotide sequence into the cell for subsequent expression of the protein encoded by the nucleic acid. The expression vector can be an episomal vector, i.e., a vector that can replicate autonomously in the host cell; or an integrating vector, i.e., a vector that is stably incorporated into the cell genome. Expression in the host cell can be constitutive or regulated (e.g., inducible).

[0101] In a certain embodiment, the gene therapy vector is a viral expression vector. The viral vector used in the present invention may comprise a viral genome in which a portion of the native sequence has been deleted in order to introduce a heterogeneous polynucleotide without destroying the infectivity of the virus. Due to the specific interaction between the viral components and the host cell receptors, the viral vector is highly suitable for effectively transferring genes into the target cells. The viral vector suitable for facilitating gene transfer into mammalian cells can be derived from different types of viruses, such as AAV, adenovirus, retrovirus, herpes simplex virus, bovine papilloma virus, lentivirus, vaccinia virus, polyoma virus, Sendai virus, orthomyxovirus, paramyxovirus, papovavirus, small RNA virus, poxvirus, alpha virus or any other viral shuttle suitable for gene therapy, its variants and combinations thereof.

[0102] "Adenoviral expression vector" or "adenovirus" is intended to include those constructs containing adenoviral sequences sufficient to (a) support packaging of therapeutic polynucleotide sequence constructs, and / or (b) ultimately express tissue and / or cell specific constructs cloned therein. In one embodiment of the invention, the expression vector comprises a genetically engineered form of adenovirus. Knowing the genetic organization of adenovirus, the 36 kilobase (kb) linear double-stranded DNA virus can allow up to 7 kb of exogenous sequence to replace large fragments of adenoviral DNA.

[0103] Adenovirus growth and manipulation are known to those skilled in the art and exhibit a broad host range in vitro and in vivo. This group of viruses can be grown at high titers, e.g., 10 9 Up to 10 11 Plaque forming units / ml are obtained, and it is highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and therefore have low genotoxicity to host cells. No side effects were reported in studies of vaccination with wild-type adenovirus, indicating its safety and / or therapeutic potential as an in vivo gene transfer vector.

[0104] Retroviruses (also called "retroviral vectors") may be chosen as gene delivery vehicles due to their ability to integrate their genes into the host genome, transfer large amounts of foreign genetic material, infect a wide range of species and cell types, and for packaging in specialized cell lines.

[0105] The retroviral genome contains three genes gag, pol and env that encode capsid protein, polymerase and envelope components respectively. The sequence found upstream of the gag gene contains a signal for packaging the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These sequences contain strong promoter and enhancer sequences and are also required for integration into the host cell genome.

[0106] In order to construct retroviral vectors, nucleic acid encoding a gene of interest is inserted into the viral genome in place of some viral sequences to produce a virus that can replicate defectively. In order to produce virions, a packaging cell line containing gag, pol and / or env genes but without LTR and / or encapsulation components is constructed. When the recombinant plasmid containing cDNA and the retroviral LTR and encapsulation sequence are introduced into this cell line (e.g., by calcium phosphate precipitation), the encapsulation sequence encapsulates the RNA transcript of the recombinant plasmid in the viral particle, which is then secreted in the culture medium. The culture medium containing the recombinant retrovirus is then collected, optionally concentrated and used for gene transfer. Retroviral vectors can infect a wide variety of cell types. However, integration and stable expression require split host cells.

[0107] Retrovirus can be derived from any one of the subfamily. For example, vectors from murine sarcoma virus, bovine leukemia, virus Rous Sarcoma Virus, murine leukemia virus, mink cell focus-initiating virus, reticuloendotheliosis virus or avian leukemia virus can be used. Those skilled in the art will be able to combine parts derived from different retroviruses, such as LTR, tRNA binding sites and packaging signals to provide recombinant retroviruses. These retroviruses are then generally used to produce transduction competent retroviral vector particles. For this purpose, the vector is introduced into a suitable packaging cell line. Retroviruses can also be constructed so that site-specific integration into the DNA of the host cell is carried out by incorporating a chimeric integrase into the retroviral particles.

[0108] Because herpes simplex virus (HSV) is neurotropic, it has generated a lot of attention in the treatment of nervous system disorders. In addition, the ability of HSV to establish latent infection in non-dividing neuronal cells without integrating into the host cell chromosome or otherwise altering the metabolism of the host cell, as well as the presence of promoters that are active during the latent period make HSV an attractive vector. And although much attention has been focused on the neurological applications of HSV, given its broad host range, this vector can also be used in other tissues.

[0109] Another factor that makes HSV an attractive vector is the size and organization of the genome. Because HSV is so large, incorporation of multiple genes or expression cassettes is less of a problem than in other smaller viral systems. In addition, the availability of different viral control sequences with different potencies (time, strength, etc.) makes it possible to control expression to a greater extent than in other systems. There is also the advantage that the virus has relatively little splicing information, further facilitating genetic manipulation.

[0110] HSV is also relatively easy to manipulate and can be grown to high titers. Therefore, delivery is not a problem in terms of the volume required to obtain sufficient infection multiplicity (MOI) and in terms of reducing the need for repeated dosing. Non-toxic variants of HSV have been developed and can be easily used in gene therapy settings.

[0111] Lentiviruses are complex retroviruses that contain other genes with regulatory or structural functions in addition to the common retroviral genes gag, pol and env. The higher complexity enables the virus to regulate its life cycle, such as during latent infection. Some examples of lentiviruses include human immunodeficiency virus (HIV-1, HIV-2) and simian immunodeficiency virus (SIV). Lentivirus vectors have been produced by multiple mitigation of HIV virulence genes, such as deletion of genes env, vif, vpr, vpu and nef to make the vector biosafe.

[0112] Lentiviral vectors are plasmid-based or virus-based and are configured to carry the necessary sequences for incorporating foreign nucleic acids, for selection, and for transferring nucleic acids into host cells. The gag, pol, and env genes of the vector of interest are also known in the art. Therefore, the relevant gene is cloned into the selected vector and then used to transform the target cell of interest.

[0113] Vaccinia virus vectors have been widely used because of their ease of construction, relatively high expression levels obtained, wide host range, and large capacity for carrying DNA. Vaccinia contains a linear double-stranded DNA genome of approximately 186 kb, which exhibits a significant "AT" preference. Approximately 10.5 kb of inverted terminal repeats flank the genome. Most essential genes appear to be located in the central region, which is the most highly conserved among poxviruses. The number of open reading frames of vaccinia virus is estimated to be 150 to 200. Despite encoding two strands, extensive overlap of reading frames is not common.

[0114] At least 25 kb can be inserted into the vaccinia virus genome. The prototype vaccinia vector contains a transgene inserted into the viral thymidine kinase gene via homologous recombination. The vector is selected based on the tk phenotype. The inclusion of an untranslated leader sequence from encephalomyocarditis virus produces higher expression levels than conventional vectors, with the transgene accumulating in 24 hours at 10% or more of the protein in infected cells.

[0115] For example, the empty capsids of the papovavirus of mouse polyomavirus have been noted as possible vectors for gene transfer. The use of polyomavirus was first described when polyomavirus DNA and purified empty capsids were incubated in a cell-free system. The DNA of the new particles is protected from the action of pancreatic DNA enzymes. The reconstructed particles are used to transfer the transformed polyomavirus DNA fragments to rat FIII cells. The empty capsids and reconstructed particles are composed of all three polyomavirus capsid antigens VP1, VP2 and VP3.

[0116] AAV is a parvovirus belonging to the genus Dependaviridae. It is a small non-enveloped single-stranded DNA virus that requires a helper virus for replication. It is necessary to co-infect with a helper virus (e.g., adenovirus, herpes virus, or vaccinia virus) to form functionally complete AAV virions. In vitro, in the absence of co-infection with a helper virus, AAV establishes a latent state in which the viral genome exists in an episomal form but does not produce infectious virions. The genome is then "rescued" by infection with a helper virus, allowing it to replicate and be encapsulated in a viral capsid, thereby reconstructing infectious virions. The latest data indicate that both wild-type AAV and recombinant AAV in vivo exist mainly in the form of large free-type concatemers. In one embodiment, the gene therapy vector used herein is an AAV vector. The AAV vector may be a purified replication-incompetent pseudotype rAAV particle.

[0117] AAV is not associated with any known human disease, is not generally considered pathogenic, and does not appear to alter the physiological properties of host cells when integrated. AAV can infect a wide range of host cells, including non-dividing cells, and can infect cells from different species. Compared to some vectors that are rapidly cleared or inactivated by both cellular and humoral responses, AAV vectors have been shown to induce persistent transgene expression in various tissues in vivo. The persistence of recombinant AAV-mediated transgenes in non-dividing cells in vivo can be attributed to the lack of the ability of natural AAV viral genes and vectors to form episomal concatemers associated with ITRs.

[0118] AAV is an attractive vector system for cell transduction in the present invention because it has high frequency persistence as an episomal concatemer and it can infect non-dividing cells, including cardiomyocytes, thus making it suitable for gene delivery into mammalian cells, for example in tissue culture and in vivo.

[0119] Typically, rAAV is manufactured by co-transfection of a plasmid containing two AAV terminal repeats flanking the gene of interest and / or an expression plasmid containing a wild-type AAV coding sequence without terminal repeats (e.g., pIM45). Cells are also infected and / or transfected with a plasmid containing adenovirus and / or adenovirus genes required for AAV auxiliary functions. The stock solution of rAAV manufactured in this way is mixed with adenovirus, which must be physically separated from rAAV particles (e.g., by cesium chloride density centrifugation or column chromatography). Alternatively, adenovirus vectors containing AAV coding regions and / or cell lines containing AAV coding regions and / or some or all adenovirus auxiliary genes can be used. Cell lines carrying rAAV DNA as integrated proviruses can also be used.

[0120] A variety of AAV serotypes exist in nature, with at least twelve serotypes (AAV1-AAV12). Despite their high homology, different serotypes have tropism for different tissues. After transfection, AAV only triggers a minor immune response in the host (if any). Therefore, AAV is highly suitable for gene therapy methods.

[0121] In some embodiments, the present disclosure may be directed to a medicament comprising an AAV vector, wherein the AAV vector is one or more of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, ANC AAV, chimeric AAVs derived therefrom, variants thereof, and combinations thereof, which will be even better suited for efficient transduction in tissues of interest. In certain embodiments, the gene therapy vector is an AAV serotype 1 vector. In certain embodiments, the gene therapy vector is an AAV serotype 2 vector. In certain embodiments, the gene therapy vector is an AAV serotype 3 vector. In certain embodiments, the gene therapy vector is an AAV serotype 4 vector. In certain embodiments, the gene therapy vector is an AAV serotype 5 vector. In certain embodiments, the gene therapy vector is an AAV serotype 6 vector. In certain embodiments, the gene therapy vector is an AAV serotype 7 vector. In certain embodiments, the gene therapy vector is an AAV serotype 8 vector. In certain embodiments, the gene therapy vector is an AAV serotype 9 vector. In certain embodiments, the gene therapy vector is an AAV serotype 10 vector. In certain embodiments, the gene therapy vector is an AAV serotype 11 vector. In certain embodiments, the gene therapy vector is an AAV serotype 12 vector.

[0122] In some embodiments, the gene therapy vector may be an AAV serotype having one or more of the capsid proteins disclosed in U.S. Pat. Nos. 7,198,951 and 7,906,111, the disclosures of which are incorporated herein by reference in their entireties.

[0123] A suitable dose of AAV for use in humans can be about 1×10 8 Vector genome / kg body weight (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 about 1×10 14 The total amount of viral particles or DRP is, is about, is at least, is at least about, does not exceed, or does not exceed about 5×10 15 vg / kg, 4×10 15 vg / kg, 3×10 15 vg / kg, 2×10 15 vg / kg, 1×1015 vg / kg、9×10 14 vg / kg、8×10 14 vg / kg、7×10 14 vg / 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、9×10 13 vg / kg、8×10 13 vg / kg、7×10 13 vg / kg、6×10 13 vg / 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、8×10 12 vg / kg、7×10 12 vg / kg、6×10 12 vg / kg、5×10 12 vg / 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、7×10 11 vg / kg、6×10 11 vg / kg、5×10 11 vg / kg、4×10 11 vg / 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、6×10 10 vg / kg、5×10 10 vg / kg、4×10 10 vg / kg、3×10 10vg / kg, 2×10 10 vg / kg, 1×10 10 vg / kg, 9×10 9 vg / kg, 8×10 9 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, 1×10 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 vg / kg, 2×10 8 vg / kg or 1×10 8 vg / kg or within the range defined by any two of these values. The doses listed above are in units of vg / kg heart tissue.

[0124] In addition to viral vectors, non-viral expression constructs can also be used to introduce genes encoding target proteins or functional variants or fragments thereof into patient cells. Non-viral expression vectors that allow in vivo expression of proteins in target cells include, for example, plasmids, modified RNA, cDNA, antisense oligomers, DNA-lipid complexes, nanoparticles, exosomes, any other non-viral shuttles suitable for gene therapy, variants thereof, and combinations thereof.

[0125] In addition to viral vectors and non-viral expression vectors, nuclease systems can also be used in conjunction with vectors and / or electroporation systems to enter the patient's cells and introduce genes encoding target proteins or functional variants or fragments thereof therein. Exemplary nuclease systems may include, but are not limited to, clustered regularly interspaced short palindromic repeats (CRISPR), DNA cutting enzymes (e.g., Cas9), large-range nucleases, TALENs, zinc finger nucleases, any other nuclease systems suitable for gene therapy, variants thereof, and combinations thereof. For example, in one embodiment, a viral vector (e.g., AAV) can be used for a nuclease (e.g., CRISPR) and another viral vector (e.g., AAV) can be used for a DNA cutting enzyme (e.g., Cas9) to introduce both (nucleases and DNA cutting enzymes) into the target cell.

[0126] Other vector delivery systems that can be used to deliver therapeutic polynucleotide sequences encoding therapeutic genes into cells are receptor-mediated delivery vehicles. These utilize the selective uptake of macromolecules in nearly all eukaryotic cells through receptor-mediated endocytosis. Due to the cell type-specific distribution of various receptors, delivery can be highly specific. Receptor-mediated gene targeting vehicles can include two components: a cell receptor-specific ligand and a DNA binder.

[0127] Suitable methods for transferring non-viral vectors into target cells are, for example, liposome transfection, calcium phosphate coprecipitation, DEAE-dextran, and direct DNA introduction using glass cannula, ultrasound, electroporation, etc. Prior to the introduction of the vector, cardiomyocytes may be treated with a permeabilizing agent such as phosphatidylcholine, streptolysin, sodium decanoate, decanoylcarnitine, tartaric acid, lysolecithin, Triton X-100, etc. Exosomes can also be used to transfer naked DNA or AAV encapsidated DNA.

[0128] The gene therapy vector of the present invention may include a promoter functionally connected to a nucleic acid sequence encoding a target protein. The promoter sequence must be tight and ensure strong expression. Preferably, the promoter provides expression of the target protein in the myocardium of a patient treated with a gene therapy vector. In some embodiments, the gene therapy vector comprises a myocardial-specific promoter operably connected to a nucleic acid sequence encoding a target protein. As used herein, a "cardiac-specific promoter" refers to a promoter whose activity in cardiac cells is at least 2 times higher than that in any other non-cardiac cell type. Preferably, the cardiac-specific promoter suitable for use in the vector of the present invention has an activity in cardiac cells that is at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, or at least 50 times higher than that in non-cardiac cell types.

[0129] The cardiac-specific promoter may be a selected human promoter, or a promoter comprising a functionally equivalent sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the selected human promoter. An exemplary non-limiting promoter that can be used is the troponin T promoter (TNNT2). Other non-limiting examples of promoters include alpha myosin heavy chain promoter, myosin light chain 2v promoter, alpha myosin heavy chain promoter, alpha-cardiac actin promoter, alpha-actin promoter, cardiac troponin C promoter, cardiac troponin I promoter, cardiac myosin-binding protein C promoter, and sarcoplasmic reticulum / endoplasmic reticulum Ca promoter. 2+ - ATPase (SERCA) promoter (eg isoform 2 of this promoter (SERCA2)).

[0130] Vectors suitable for use in the present invention may have different transduction efficiencies. Thus, a viral vector or non-viral vector transduces more than, equal to, or at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% of the cells at the target vascular site. More than one vector (viral or non-viral or a combination thereof) may be used simultaneously or sequentially. This can be used to transfer more than one polynucleotide, and / or target more than one cell type. When multiple vectors or multiple agents are used, more than one transduction / transfection efficiency may be produced.

[0131] The pharmaceutical composition containing the gene therapy vector can be prepared as a liquid solution or suspension. The pharmaceutical composition of the present invention may include a commonly used pharmaceutically acceptable excipient, such as a diluent and a carrier. In particular, the composition comprises a pharmaceutically acceptable carrier, such as water, saline, Ringer's solution or dextrose solution. In addition to the carrier, the pharmaceutical composition may also contain an emulsifier, a pH buffer, a stabilizer, a dye, etc.

[0132] In certain embodiments, the pharmaceutical composition will comprise a therapeutically effective gene dose, which is a dose that is capable of preventing or treating a cardiomyopathy in a subject without being toxic to the subject. Prevention or treatment of a cardiomyopathy can be assessed as a change in a phenotypic characteristic associated with a cardiomyopathy, wherein the change is effective in preventing or treating the cardiomyopathy. Thus, a therapeutically effective gene dose is generally a gene dose that is sufficient to ameliorate or prevent a pathogenic cardiac phenotype in a treated subject when administered in a physiologically tolerable composition.

[0133] In certain embodiments, the gene therapy vector can be transduced into a subject via several different methods, including intravenous delivery, intra-arterial delivery, or intraperitoneal delivery. In some embodiments, the gene therapy vector can be directly administered to cardiac tissue, for example, by intracoronary administration. In some embodiments, tissue transduction of the myocardium can be achieved by catheter-mediated intramyocardial delivery or by retrograde intravenous injection into the cardiac vein via the coronary sinus, which can be used to transfer vector-free cDNA linked or unlinked to the transduction enhancement vector into the myocardium.

[0134] In certain embodiments, the medicament will comprise a therapeutically effective gene dose. A therapeutically effective gene dose is a gene dose that is capable of preventing or treating a particular cardiac disorder in a patient without being toxic to the patient.

[0135] Cardiac disorders that can be treated by the methods disclosed herein can include, but are not limited to, one or more of the following: hereditary cardiomyopathy (e.g., hereditary cardiomyopathy), arrhythmic heart disease, heart failure, ischemia, arrhythmia, myocardial infarction, congestive heart failure, transplant rejection, abnormal heart contraction, non-ischemic cardiomyopathy, mitral regurgitation, aortic stenosis or regurgitation, abnormal Ca 2+ Metabolic, congenital heart disease, primary or secondary myocardial tumors and combinations thereof.

[0136] Exemplary Embodiments

[0137] The following examples are set forth to aid in understanding the present disclosure and should not, of course, be construed as specifically limiting the embodiments described and claimed herein. Such variations of the embodiments, including substitution of all equivalents currently known or subsequently developed that will be within the capabilities of those skilled in the art, as well as changes in formulation or minor changes in experimental design, will be considered to fall within the scope of the embodiments incorporated herein.

[0138] Example 1 describes four different micro-dystrophin constructs that were used in the following various studies.

[0139] Examples 2-6 are described in mdx 4CV Tolerance, gene expression, and protein expression studies at 4 and 12 weeks after intravenous injection of AAV9-hTNNT-micro-dystrophin vectors in a mouse model.

[0140] Example 7 describes the treatment of DMD mdx Gene therapy efficacy studies in neonatal rats.

[0141] Example 8 describes AAV9-micro-dystrophin transduction in engineered cardiac tissue.

[0142] Each vector used in the study included a micro-dystrophin construct with a sequence length below the approximately 4.7 kb packaging limit of AAV.

[0143] Example 1: Micro-dystrophin constructs

[0144] Preparation of four different rAAV9 vectors encoding human micro-dystrophin under the control of the hTNNT2 promoter for IV injection into mdx 4CV In mice. Figure 1 The micro-dystrophin constructs used in the examples are shown. Each vector includes a construct encoding micro-dystrophin (NT to CT) and also includes a hTNNT2 promoter sequence (SEQ ID NO: 6) and a bovine growth hormone polyadenylation (bGH PolyA) coding sequence after each CT.

[0145] Each construct included the NT domain coding sequence (SEQ ID NO: 7) and the H1 coding sequence (SEQ ID NO: 8).

[0146] Each construct sequence is additionally flanked by inverted terminal repeat (ITR) sequences.

[0147] Construct 1 (μDys1, SEQ ID NO:2) also included sequences encoding approximately 75% of R1 up to amino acid (aa) 419 (SEQ ID NO:12), approximately 30% of R24 (SEQ ID NO:15), H4 (SEQ ID NO:9), the CR domain (SEQ ID NO:10), and the CT domain (SEQ ID NO:11).

[0148] Construct 2 (μDys2, SEQ ID NO:3) also includes sequences encoding approximately 75% of R1 until amino acid (aa) 419 (SEQ ID NO:12), approximately 60% of R24 including the entire exon 60 (SEQ ID NO:16), H4 (SEQ ID NO:9), the CR domain (SEQ ID NO:10), and the CT domain (SEQ ID NO:11).

[0149] Construct 3 (μDys3, SEQ ID NO:4) also includes sequences encoding R1 (SEQ ID NO:13), an R2 linker sequence (SEQ ID NO:18), approximately 60% of R24 including the entire exon 60 (SEQ ID NO:16), H4 (SEQ ID NO:9), a CR domain (SEQ ID NO:10), and a CT domain (SEQ ID NO:11).

[0150] Construct 4 (μDys4, SEQ ID NO:5) also included sequences encoding R1 (SEQ ID NO:13), R2 (SEQ ID NO:19), H3 (SEQ ID NO:20), R24 (SEQ ID NO:17), H4 (SEQ ID NO:9), the CR domain (SEQ ID NO:10), and the CT domain containing only exons 70-75 ending at aa 3540 (SEQ ID NO:14).

[0151] Construct 5 (rAAV9-MCKE-μDMD-PC) was used as a positive control.

[0152] An exemplary AAV-vector sequence that was used to bind various micro-dystrophin constructs and was used in the studies is as follows: SEQ ID NO: 21 ( Fig.17 ); SEQ ID NO:22( Fig.18 ); SEQ ID NO:23( Fig.19 ); and SEQ ID NO:24 ( Fig. 20 ).

[0153] Example 2: Experimental Design

[0154] Animals were injected between 11 and 14 weeks of age and randomly assigned to different groups. The experimental parameters for each animal group are summarized in Table 1 below.

[0155] Table 1: Experimental parameters for treatment with micro-dystrophin constructs and controls

[0156] Group deal with dose Duration of follow-up after infection Number of animals 1A μDys1 <![CDATA[10 14 vg / kg]]> About 4 weeks (28 days ± 3 days) 5 2A μDys2 <![CDATA[10 14 vg / kg]]> About 4 weeks (28 days ± 3 days) 5 3A μDys3 <![CDATA[10 14 vg / kg]]> About 4 weeks (28 days ± 3 days) 5 4A μDys4 <![CDATA[10 14 vg / kg]]> About 4 weeks (28 days ± 3 days) 5 PC Positive Control <![CDATA[10 14 vg / kg]]> About 4 weeks (28 days ± 3 days) 2 Veh Vehicle control N / A About 4 weeks (28 days ± 3 days) 5 1B μDys1 <![CDATA[10 14 vg / kg]]> About 12 weeks (84 days ± 6 days) 5 2B μDys2 <![CDATA[10 14 vg / kg]]> About 12 weeks (84 days ± 6 days) 5 3B μDys3 <![CDATA[10 14 vg / kg]]> About 12 weeks (84 days ± 6 days) 5 4B μDys4 <![CDATA[10 14 vg / kg]]> About 12 weeks (84 days ± 6 days) 5 PC Positive Control <![CDATA[10 14 vg / kg]]> About 12 weeks (84 days ± 6 days) 2 Veh Vehicle control N / A About 12 weeks (84 days ± 6 days) 5

[0157] Example 3: Carrier Quantification

[0158] Vector quantification was performed using qPCR on gDNA from cardiac issues, including vector copy number analysis and vector biodistribution analysis in the heart (one sample per mouse, using the upper half of the heart with the left and right ventricles).

[0159] gDNA extraction was performed using the Gentra Puregene kit and Tissue Lyser II (both from Qiagen).

[0160] Simplex qPCR analysis was performed in duplicate on 50 ng of gDNA: the micro-dystrophin sequence (target sequence, the same assay was used for all constructs and targeted to the 5' portion of the CR domain); and the mAlbumin gene (murine endogenous gene).

[0161] Figure 2A and Figure 2B Vector copy number analysis at 4 and 12 weeks is shown, respectively, and Figure 3 This data is shown based on the mean / group. This data demonstrates the biodistribution of constructs 1-4 comparable to construct 5 (positive control). Data are expressed as vector genome copies per diploid genome (vg / dg).

[0162] Example 4: Quantification of mRNA

[0163] Quantification of micro-dystrophin mRNA was performed using qPCR on cDNA from cardiac problems (one sample per mouse, using the upper half of the heart with left and right ventricles). Initially, total RNA was extracted from cardiac tissue using Trizol. 500 ng of total RNA was DNase I treated using ezDNase. 500 ng of DNase I treated RNA was reverse transcribed using SuperScript IV VILO master mix and then diluted (1 / 40) to obtain cDNA. Simplex Sybr qPCR analysis was performed in duplicate for each of the following: micro-dystrophin sequence (target sequence); and mHPRT1 gene (murine endogenous gene).

[0164] Figure 4 and Figure 5 Relative quantification of micro-dystrophin mRNA in individual heart tissues and mean values ​​per group are shown, respectively, demonstrating increased expression of constructs 1-4 relative to the positive control (construct 5).

[0165] Example 5: Micro-dystrophin expression

[0166] Western blot and immunofluorescence (IF) assays were used to analyze micro-dystrophin expression in the heart after four weeks, as Fig. 6A and Figure 6B (Western blot) As shown. For Western blot assay, 50 μg of total protein lysate (extracted with RIPA buffer) was loaded on 3-8% triacetate gel (exposure time 10 min).

[0167] Example 6: Immunofluorescence

[0168] Figure 7 For mdx 4CV Immunofluorescence images of micro-dystrophin expression in mouse heart.

[0169] Figure 8 Describes normal syntrophin expression, mdx in normal mouse heart according to embodiments of the present disclosure 4CV Aberrant syntrophin expression in mouse heart and restoration of syntrophin expression following gene therapy with micro-dystrophin constructs 1, 2, and 4. Figure 8 Restoration of the dystrophin-dystroglycan complex after gene therapy with micro-dystrophin constructs 1, 2, and 4 is further depicted, with increased expression of α-dystroglycan, β-dystroglycan, α-sarcoglycan, and γ-sarcoglycan.

[0170] Example 7: DMD mdx Studying the efficacy of gene therapy in neonatal rats

[0171] The efficacy of gene therapy was studied in 80 Sprague Dawley male rats, including those carrying DMD. mdx Rats with mutant dystrophin gene and wild-type dystrophin gene. Table 2 shows the experimental parameters of this study. Rats were injected with vectors encoding micro-dystrophin constructs (AAV9-TNNT2-μDys2 or AAV9-TNNT2-μDys4), empty vector or vehicle at 3-7 days of age. The number of animals in each group was 8. At 3 and 6 months after injection, cardiac function, biodistribution, RNA expression, protein expression and localization, and immunohistochemistry were evaluated.

[0172] Table 2: DMD research mdx Experimental parameters of freshman ratio

[0173] Group genotype Test items dose Follow-up 1A <![CDATA[DMD mdx ]]> μDys2 <![CDATA[2×10 14 vg / kg]]> 3 months 1B <![CDATA[DMD mdx ]]> μDys2 <![CDATA[2×10 14 vg / kg]]> 6 months 2A <![CDATA[DMD mdx ]]> μDys4 <![CDATA[2×10 14 vg / kg]]> 3 months 2B <![CDATA[DMD mdx ]]> μDys4 <![CDATA[2×10 14 vg / kg]]> 6 months 3A <![CDATA[DMD mdx ]]> Empty vector <![CDATA[2×10 14 vg / kg]]> 3 months 3B <![CDATA[DMD mdx ]]> Empty vector <![CDATA[2×10 14 vg / kg]]> 6 months 4A <![CDATA[DMD mdx ]]> vehicle - 3 months 4B <![CDATA[DMD mdx ]]> vehicle - 6 months 5A wild type vehicle - 3 months 5B wild type vehicle - 6 months

[0174] Fig.9A and Fig. 9B Plots showing heart rate, QRS width, and QT interval obtained from electrocardiographic measurements at three months (9A) and six months (9B) after injection revealing treated DMD mdx Trends in the therapeutic effect for reduction of QRS and QT intervals in rats.

[0175] Fig. 10A and Fig. 10B Structural remodeling of the left ventricle (LV) is shown at three months (10A) and six months (10B) after injection, including plots of LV diameter and LV thickness. Fig.11A and Fig. 11B Plots showing systolic function (ejection fraction) and diastolic function (E / A ratio) at three months (11A) and six months (11B) post-injection. Data reveal DMD treated with micro-dystrophin mdx The rats tended to be more similar to wild-type rats, with ejection fraction significantly altered at three months of age.

[0176] Fig.12 Shown are biodistribution plots (vg / dg) of μDys2 and μDys4 vectors measured in different regions of the heart and liver three months after injection, showing that both vectors resulted in a higher degree of localization and transduction in the ventricles than in the atria. The vg / dl measured in the liver may be a result of the delivery method (intraperitoneal injection).

[0177] Fig.13 Western blot images evaluating micro-dystrophin expression in left ventricular tissue three months after injection are shown. Fig.14The expression levels of micro-dystrophin in the septal ventricle of the heart are shown, which reveals uniform expression of μDys4 in the ventricle. Efficient transduction of the heart was observed for both vectors. Similar transgene expression was observed in the ventricle and atrium, but more μDys4 transcripts were observed in the septum.

[0178] Example 8: AAV9-micro-dystrophin transduction in engineered cardiac tissue

[0179] Both constructs (Constructs 2 and 4 correspond to μDys2 and μDys4, respectively) were evaluated in engineered heart tissue (EHT). EHT were cast with hiPSC-CMs (cell-attached cpHet7% cTnT positive cardiomyocytes) and transduced with AAV9 constructs. Relaxation kinetics were measured in spontaneously contracting EHT cultured in complete medium. In addition, several relaxation parameters were evaluated under electrical stimulation at 1 Hz at 1 and 2 weeks after transduction. Spontaneous beat frequency was analyzed over time in complete medium and serum-free DMEM medium 2 weeks after transduction.

[0180] Some EHT cardiomyocytes were collected 14 days after transduction for protein extraction and further analysis. Exogenous protein levels were measured by western blot using an antibody against micro-dystrophin. Cardiac TnT protein levels were used as a reference.

[0181] EHT function was analyzed in the absence of electrical stimulation using an EHT test system equipped with CTMV.10 software, which provides standardized and robust video-optical evaluation of contractility in a 24-well format as described by Hansen et al. (Development of a Drug Screening Platform Based on Engineered Heart Tissue, Circulation Research, Vol. 107, No. 1, 2010, pp. 35-44). EHT was then measured under spontaneous beating conditions in complete medium after 4 weeks of culture.

[0182] EHT measurement data were collected using CTMV.10 software and graphs were generated using GraphPad Prism software. Fig.15 The resulting beating rate and force of treatments with μDys2 and μDys4, non-transduced (NT) and control samples (non-diseased isogenic control) are shown, revealing that micro-dystrophin expression in transduced DMD EHTs stabilizes beating rate and improves contractile force. Fig.16 Arrhythmia scores are shown, revealing that micro-dystrophin expression in transduced DMD EHTs protects against arrhythmic events in response to adrenergic stimulation.

[0183] In the foregoing description, numerous specific details such as specific materials, dimensions, process parameters, etc. are described to provide a thorough understanding of the present invention. In one or more embodiments, specific features, structures, materials or characteristics can be combined in any suitable manner. The words "embodiment" or "exemplary" are used herein to mean serving as examples, examples or illustrations. It is not necessary to understand any aspect or design described herein as "embodiment" or "exemplary" as being superior or advantageous to other aspects or designs. In fact, the use of the words "embodiment" or "exemplary" is intended to present concepts in a specific way. As used in this application, the term "or" is intended to mean inclusive "or" rather than exclusive "or". That is, unless otherwise specified, or it is obvious from the context, "X includes A or B" is intended to mean any one of the natural inclusive substitutions. That is, if X includes A; X includes B; or X includes both A and B, then "X includes A or B" is satisfied in any of the foregoing situations. "One embodiment", "certain embodiments" or "one embodiment" mentioned throughout this specification means that the specific features, structures or features described in conjunction with the embodiment are included in at least one embodiment. Thus, the appearances of the phrases "one embodiment," "some embodiments," or "an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment.

[0184] The present invention has been described with reference to specific exemplary embodiments thereof. Therefore, the description and drawings should be viewed in an illustrative sense rather than a restrictive sense. Various modifications of the present invention, in addition to those shown and described herein, will become apparent to those skilled in the art and are intended to fall within the scope of the appended claims.

Claims

1. A gene therapy drug for treating or preventing cardiomyopathy in a human subject, the gene therapy drug comprising: A vector comprising a polynucleotide sequence encoding a micro-dystrophin protein, wherein the polynucleotide encoding sequence encodes a subset of repeating units R1-R24 of the dystrophin protein, and wherein the repeating units of the subset are selected from the group consisting of: R1, R2, R24, a portion thereof, and a combination thereof, with the proviso that R1 and R24 or a portion thereof are present in the subset.

2. The gene therapy drug of claim 1, wherein the polynucleotide coding sequence encodes only hinge 1, hinge 3 and hinge 4 of the hinge domain of the dystrophin protein.

3. The gene therapy drug of claim 1, wherein the polynucleotide coding sequence encodes only hinge 1 and hinge 4 of the hinge domain of the dystrophin protein.

4. The gene therapy drug of claim 1, wherein the repeating units of the subset consist of R1 and R24 or a portion thereof.

5. The gene therapy drug of claim 4, wherein the polynucleotide coding sequence encodes the entire CT domain of the dystrophin protein.

6. The gene therapy drug of claim 4, wherein the polynucleotide coding sequence encodes about 65% to about 85% of R1 and about 20% to about 40% of R24.

7. The gene therapy drug of claim 4, wherein the polynucleotide coding sequence encodes about 65% to about 85% of R1 and about 50% to about 70% of R24, provided that the polynucleotide sequence completely encodes exon 60 of the dystrophin gene.

8. The gene therapy drug of claim 4, wherein the polynucleotide coding sequence encodes about 95% or more of R1 and about 60% or less of R24, provided that the polynucleotide sequence completely encodes exon 60 of the dystrophin gene.

9. The gene therapy drug of claim 1, wherein the polynucleotide sequence encodes only exons 70-75 or a portion thereof of the cysteine-rich domain of the dystrophin protein.

10. The gene therapy drug of claim 9, wherein the polynucleotide sequence further encodes at least a portion of the repeating unit R2.

11. The gene therapy drug of claim 9, wherein the polynucleotide sequence encodes only hinge 1, hinge 3, and hinge 4 of the hinge domain of the dystrophin protein, and wherein the repeating units of the subset consist of R1, R2, and R24 or portions thereof.

12. A gene therapy drug comprising a polynucleotide sequence comprising a micro-dystrophin coding sequence that is at least 90% identical to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:

5.

13. The gene therapy drug according to any one of claims 1 to 12, wherein the polynucleotide sequence further encodes a myocardial-specific promoter. The gene therapy drug according to claim 13 , wherein the myocardium-specific promoter is the TNNT2 promoter.

15. The gene therapy drug of any one of claims 1-14, wherein the vector comprises a viral vector.

16. The gene therapy drug of claim 15, wherein the viral vector is an AAV vector.

17. The gene therapy drug of claim 16, wherein the AAV vector comprises AAV9.

18. The gene therapy drug of any one of claims 1-17, wherein the expression product of the polynucleotide coding sequence in the human subject is capable of restoring syntrophin binding and localization in dystrophin-deficient myocardium.

19. A functional micro-dystrophin protein encoded by SEQ ID NO: 2 or a variant thereof having at least about 85% identity thereto, or having the amino acid sequence of SEQ ID NO: 25 or a variant thereof having at least about 85% identity thereto.

20. A functional micro-dystrophin protein encoded by SEQ ID NO: 3 or a variant thereof having at least about 85% identity thereto, or having an amino acid sequence of SEQ ID NO: 26 or a variant thereof having at least about 85% identity thereto.

21. A functional micro-dystrophin protein encoded by SEQ ID NO:4 or a variant thereof having at least about 85% identity thereto, or having the amino acid sequence of SEQ ID NO:27 or a variant thereof having at least about 85% identity thereto.

22. A functional micro-dystrophin protein encoded by SEQ ID NO: 5 or a variant thereof having at least about 85% identity thereto, or having the amino acid sequence of SEQ ID NO: 28 or a variant thereof having at least about 85% identity thereto.

23. A gene therapy drug for expressing the functional micro-dystrophin protein of any one of claims 19 to 22 in cardiac tissue of a human subject.

24. A method of treating or preventing a dystrophin-related cardiomyopathy in a human subject, the method comprising: Delivering the gene therapy drug of any one of claims 1-18 or 23 to the cardiac tissue of the human subject.

25. The method of claim 24, wherein the dystrophin-related cardiomyopathy is associated with Duchenne muscular dystrophy or Becker muscular dystrophy.

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