Muscle-specific regulatory cassette

Abstract

The present disclosure relates to nucleic acid regulatory cassettes useful for the expression of transgene payloads in specific muscle tissues. Also included in the present disclosure are vectors, including adeno-associated virus (AAV) vectors, comprising the regulatory cassettes of the invention, as well as compositions and methods for treating genetic diseases or genetic disorders, also comprising the regulatory cassettes.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority under 35 U.S.C.§119(e) to U.S. Provisional Patent Application No. 63 / 390,886, filed on July 20, 2022, which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application is filed via the Patent Center in XML format and includes a sequence listing that is hereby incorporated by reference in its entirety. The XML file was created on July 19, 2023, named "369055_7028WO1_00100_SequenceListingST_26.xml", and has a size of 4,096 bytes.

Background Art

[0003] Background of the Invention AAV - mediated gene therapy for myopathies requires regulatory cassettes that confer high levels of skeletal muscle - specific activity. However, cardiotoxicity caused by transgene expression in the heart has emerged as a major concern in clinical trials for Duchenne muscular dystrophy and X - linked myotubular myopathy. Furthermore, the size of certain therapeutic cargoes requires regulatory cassettes to be even smaller than current established versions. Therefore, there is a clear need in the art for highly minimized regulatory cassettes that retain high activity in skeletal muscle and have either low or no activity in the myocardium. The present invention addresses this need.

Summary of the Invention

[0004] As described herein, the present disclosure relates to a nucleic acid encoding a regulatory cassette useful for the expression of a transgene payload in a specific muscle tissue. Also included are vectors, including AAV vectors, that contain the regulatory cassette of the invention, as well as compositions and methods for treating genetic diseases or disorders that also contain the regulatory cassette.

[0005] Accordingly, in one aspect, the invention includes a polynucleotide encoding a muscle-specific regulatory cassette that includes at least three modified enhancer sequences located upstream of and operably linked to a promoter, wherein the three modified enhancer sequences include a Trex sequence, an AT-rich sequence, a right E-box sequence, and an MEF2 sequence, the right E-box sequence is repeated at least once, and the promoter is a -80 to +50 promoter that contains a consensus Inr sequence.

[0006] In certain embodiments, the cassette has high expression activity in skeletal muscle tissue.

[0007] In certain embodiments, the skeletal muscle tissue is fast-twitch muscle tissue.

[0008] In certain embodiments, the cassette has very low expression in cardiac tissue compared to skeletal muscle tissue.

[0009] In certain preferred embodiments, the cassette is encoded by a nucleic acid that includes the sequence shown in SEQ ID NO:1.

[0010] In certain embodiments, the cassette is encoded by a nucleic acid that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, and at least 99% sequence identity to the sequence shown in SEQ ID NO:1.

[0011] In another aspect, the invention includes a polynucleotide encoding a muscle-specific regulatory cassette comprising at least three modified enhancer arrays located upstream of and operably linked to a promoter, wherein the at least three modified enhancer arrays include a Trex array, an AT-rich array, a left E-box array, a right E-box array, and an MEF2 array, and the promoter is an -80 to +50 promoter containing a consensus Inr sequence.

[0012] In certain embodiments, the cassette has high expression activity in skeletal muscle tissue.

[0013] In certain embodiments, the cassette has low expression activity in heart tissue compared to skeletal muscle tissue.

[0014] In certain preferred embodiments, the cassette is encoded by a nucleic acid comprising the sequence shown in SEQ ID NO:2.

[0015] In certain embodiments, the cassette is encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, and at least 99% sequence identity to the sequence shown in SEQ ID NO:2.

[0016] In another aspect, the invention includes an AAV vector comprising a muscle-specific regulatory cassette comprising a polynucleotide of any one of the above aspects for the embodiment or any other aspect or embodiment disclosed herein.

[0017] In another aspect, the invention includes an AAV vector comprising a muscle-specific regulatory cassette comprising a polynucleotide of any one of any one of the above aspects for the embodiment or any other aspect or embodiment disclosed herein.

[0018] In certain embodiments, the AAV vector comprises an AAV capsid of a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, AAV-B1, AAV-DJ, AAV-Retro, AAVrh8, AAVrh10, AAVrh25, Anc80L65, LK03, AAVrh18, rAAVrh74, AAVrh32.33, AAVrh39, AAVrh43, MyoAAV2, Oligo001, PHP-B, and Spark100.

[0019] In certain preferred embodiments, the capsid is of the rAAVrh74 serotype.

[0020] In certain preferred embodiments, the capsid is of the AAV9 serotype.

[0021] In certain preferred embodiments, the capsid is of the MyoAAV2 serotype.

[0022] In certain embodiments, the capsid has specificity for muscle tissue.

[0023] In another aspect, the invention includes a composition comprising an AAV vector particle comprising a polynucleotide of any one of the above aspects or embodiments or any aspect or embodiment disclosed herein, and a pharmaceutically acceptable carrier or excipient.

[0024] In another aspect, the invention includes administering to a subject in need thereof an effective amount of an AAV vector comprising a muscle-specific regulatory cassette comprising at least three modified enhancer sequences located upstream of and operably linked to a promoter, thereby treating a genetic disease or disorder, the method of treating a genetic disease or disorder in a subject in need thereof, the three modified enhancer sequences comprising a Trex sequence, an AT-rich sequence, a right E-box sequence, and a MEF2 sequence, the right E-box sequence being repeated at least once, and the promoter being a -80 to +50 promoter containing a consensus Inr sequence.

[0025] In certain preferred embodiments, the cassette is encoded by a nucleic acid comprising the sequence shown in SEQ ID NO:1.

[0026] In another aspect, the invention includes a method of treating a genetic disease or disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of an AAV vector comprising a muscle-specific regulatory cassette comprising at least three modified enhancer sequences located upstream of and operably linked to a promoter, the at least three modified enhancer sequences comprising a Trex sequence, an AT-rich sequence, a left E-box sequence, a right E-box sequence, and a MEF2 sequence, and the promoter being a -80 to +50 promoter containing a consensus Inr sequence.

[0027] In certain preferred embodiments, the cassette is encoded by a nucleic acid comprising the sequence shown in SEQ ID NO:2.

[0028] In certain embodiments, the AAV vector comprises an AAV capsid of a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, AAV-B1, AAV-DJ, AAV-Retro, AAVrh8, AAVrh10, AAVrh25, Anc80L65, LK03, AAVrh18, rAAVrh74, AAVrh32.33, AAVrh39, AAVrh43, MyoAAV2, Oligo001, PHP-B, and Spark100.

[0029] In certain embodiments, the capsid is of the rAAVrh74 serotype.

[0030] In certain embodiments, the capsid is of the AAV9 serotype.

[0031] In certain embodiments, the capsid is of the MyoAAV2 serotype.

[0032] In certain embodiments, the AAV vector has specificity for muscle tissue.

[0033] In certain embodiments, the genetic disease or genetic disorder is a muscle-related genetic disease or genetic disorder.

[0034] In certain embodiments, the muscle-related genetic disease or genetic disorder is selected from the list consisting of facioscapulohumeral muscular dystrophy (FSHD), X-linked myotubular myopathy (XLMTM), central core myopathy, inclusion body myositis, nemaline myopathy, distal myopathy, centronuclear myopathy, oculopharyngeal muscular dystrophy, dysferlinopathy, limb-girdle muscular dystrophy (LGMD), and Duchenne muscular dystrophy (DMD).

[0035] In another aspect, the present invention includes a method of introducing a transgene into a cell, the method comprising contacting the target cell with a polynucleotide encoding a muscle-specific regulatory cassette comprising at least three modified enhancer sequences located upstream of and operably linked to a promoter, wherein the three modified enhancer sequences include a Trex sequence, an AT-rich sequence, a right E-box sequence, and an MEF2 sequence, the right E-box sequence is repeated at least once, and the promoter is an -80 to +50 promoter containing a consensus Inr sequence.

[0036] In certain embodiments, the cassette has high expression activity in skeletal muscle tissue.

[0037] In certain embodiments, the skeletal muscle tissue is fast-twitch muscle tissue.

[0038] In certain embodiments, the cassette has very low expression in cardiac tissue compared to skeletal muscle tissue.

[0039] In certain preferred embodiments, the cassette is encoded by a nucleic acid comprising the sequence shown in SEQ ID NO:1.

[0040] In certain embodiments, the cassette is encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, and at least 99% sequence identity to the sequence shown in SEQ ID NO:1.

[0041] In another aspect, the present invention includes a method of introducing a transgene into a cell, the method comprising contacting the target cell with a polynucleotide encoding a muscle-specific regulatory cassette comprising at least three modified enhancer sequences located upstream of and operably linked to a promoter, wherein the at least three modified enhancer sequences include a Trex sequence, an AT-rich sequence, a left E-box sequence, a right E-box sequence, and an MEF2 sequence, and the promoter is an -80 to +50 promoter containing a consensus Inr sequence.

[0042] In certain embodiments, the cassette has high expression activity in skeletal muscle tissue.

[0043] In certain embodiments, the cassette has low expression activity in heart tissue compared to skeletal muscle tissue.

[0044] In certain preferred embodiments, the cassette is encoded by a nucleic acid comprising the sequence shown in SEQ ID NO:2.

[0045] In certain embodiments, the cassette is encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, and at least 99% sequence identity to the sequence shown in SEQ ID NO:2. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The following detailed description of the preferred embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. For purposes of illustration of the present invention, preferred drawing embodiments are shown herein. However, it will be understood that the present invention is not limited to the exact arrangements and instrumentalities of the embodiments shown in the drawings.

[0047]

Figure 1

Figure 2A

Figure 2B

Figure 2C

Figure 3-1

Figure 3-2

Figure 4-1

Figure 4-2

Figure 5

Figure 6-1

Figure 6-2

Figure 7-1

Figure 7-2

Figure 8-1

Figure 8-2

Figure 9-1

Figure 9-2

Mode for Carrying Out the Invention

[0048] Detailed Description Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing the present invention, the preferred materials and methods are described herein. The following technical terms are used in the description and claims of the present invention.

[0049] It should also be understood that the technical terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0050] As used herein, the articles "a" and "an" are used to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. By way of example, "an element" means one element or more than one element.

[0051] As used herein, "about", when referring to measurable values such as amounts, temporal periods, and the like, is intended to encompass variations of ±20% or ±10% from a particular value, more preferably ±5%, still more preferably ±1%, and yet still more preferably ±0.1%, such that the variations are suitable in practicing the disclosed methods.

[0052] As used herein, the term "AAV vector" refers to a polynucleotide vector containing one or more genes of interest (or transgenes) adjacent to AAV terminal repeats (ITRs). An AAV vector is generated when one or more helper plasmids encoding and expressing the rep and cap proteins and one or more proteins derived from the adenovirus open reading frame E4orf6 are transfected into a host cell and can be packaged into infectious virus particles. The AAV vector may be operably linked to promoter and enhancer sequences that can regulate the expression of the proteins encoded by the AAV vector.

[0053] As used herein, the term "AAV virion" or "AAV virus particle" or "AAV vector particle" refers to a virus particle composed of capsid proteins derived from at least one AAV serotype surrounding the polynucleotide AAV vector. If the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to mammalian cells), it is typically referred to as an "AAV vector particle" or simply an "AAV vector". Therefore, the production of AAV vector particles necessarily involves the production of AAV vectors, and thus the vector is contained within the AAV vector particle.

[0054] As used herein, the term "packaging" refers to the intracellular process by which virus virions or particles (e.g., AAV virions or particles), particularly virus vector particles or virions, are assembled in a host cell. "Packaging" cells contain the polynucleotide (e.g., helper plasmid) and protein components necessary to assemble functional virus virions.

[0055] An "agent" is intended to be any nucleic acid molecule, small molecule chemical compound, antibody, or polypeptide, or fragment thereof.

[0056] "Change" or "variation" is intended to mean an increase or a decrease. The change may be at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or 40%, 50%, 60%, or even up to 70%, 75%, 80%, 90%, or 100%.

[0057] As used herein, the term "self" refers to any material being obtained from the same individual from which it will be re-introduced later.

[0058] "Allogeneic" refers to the graft being obtained from different animals of the same species.

[0059] "Xenogenicity" refers to the graft being obtained from animals of different species.

[0060] "Biological sample" is intended to mean any tissue, cell, body fluid, or other material obtained from a living organism.

[0061] As used herein, the term "cassette" or "expression cassette" or "regulatory cassette" refers to a discrete nucleic acid vector comprising a payload sequence (e.g., encoding a transgene or RNA) and regulatory sequences that control its expression (i.e., promoter, enhancer, terminator, and the like). Upon successful insertion into a host cell, the regulatory sequences enable transcription and translation of the payload transgene.

[0062] As used herein, the term "conservative sequence modification" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. The modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. Such families include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids having beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody can be replaced with other amino acid residues from the same side chain family, and the altered antibody can be tested for its ability to bind an antigen using the functional assays described herein.

[0063] A "disease" is a health condition of an animal in which the animal is unable to maintain homeostasis and, if the disease is not alleviated, the health of the animal will deteriorate. In contrast, a "disorder" in an animal is a health condition in which the animal can maintain homeostasis, but the health condition of the animal is not as good as it would be assumed in the absence of the disorder. Without treatment, a disorder does not necessarily cause further decline in the health condition of the animal.

[0064] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to the amount of a compound, formulation, material, or composition described herein that is effective to achieve a particular biological result or to provide a therapeutic or prophylactic benefit. Such results may include, but are not limited to, antitumor activity determined by any suitable means in the art.

[0065] "Encoding" refers to the unique property of a specific sequence of nucleotides in a polynucleotide such as a gene, cDNA, or mRNA to serve as a template for the synthesis of other polymers and macromolecules in biological processes, either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties arising therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene results in the production of the protein in a cell or other biological system. A nucleotide sequence that is identical to the mRNA sequence and both the coding strand, which is usually provided in the sequence listing, and the non-coding strand used as a template for transcription of the gene or cDNA can be said to encode the protein or other product of that gene or cDNA.

[0066] As used herein, "endogenous" refers to any material that is derived from or produced inside a living organism, cell, tissue, or system.

[0067] As used herein, the term "exogenous" refers to any material that is introduced from outside or produced outside a living organism, cell, tissue, or system.

[0068] The term "expression" as used herein is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0069] "Expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains cis-acting elements sufficient for expression, and other elements for expression can be supplied by the host cell or can be supplied in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) into which the recombinant polynucleotide has been incorporated, and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus).

[0070] "Homologous", as used herein, refers to subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. Subunit positions in both of the two molecules are occupied by the same monomeric subunit, e.g., if the positions in each of two DNA molecules are occupied by adenine, then they are homologous at that position. The homology between two sequences is a linear function of the number of matching or homologous positions, e.g., if half of the positions in two sequences (e.g., 5 positions in a polymer of 10 subunits in length) are homologous, then the two sequences are 50% homologous, and if 90% of the positions (e.g., 9 out of 10) match or are homologous, then the two sequences are 90% homologous.

[0071] "Identity", as used herein, refers to subunit sequence identity between two polymer molecules, specifically, between two amino acid molecules such as between two polypeptide molecules. Two amino acid sequences are identical at a position when they have the same residue at the same position, for example, if the positions in each of two polypeptide molecules are occupied by arginine, then they are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a linear function of the number of match positions or identical positions. For example, if half of the positions in two sequences (e.g., 5 positions in a polymer of 10 amino acids in length) are identical, then the two sequences are 50% identical, and if 90% of the positions (e.g., 9 out of 10) match or are identical, then the two amino acid sequences are 90% identical.

[0072] As used herein, "teaching material" includes publications, records, charts, or any other medium of expression that can be used to convey the usefulness of the compositions and methods of the present invention. The teaching material of the kit of the present invention may, for example, be attached to a container containing the nucleic acid, peptide, and / or composition of the present invention, or be delivered together with the container containing the nucleic acid, peptide, and / or composition. Alternatively, the teaching material may be delivered separately from the container with the intention that the teaching material and the compound be used together by the recipient.

[0073] "Isolated" means changed or removed from its natural context. For example, a nucleic acid or peptide that naturally exists in a living animal is not "isolated", but the same nucleic acid or peptide that is partially or completely separated from its coexisting materials in its natural context is "isolated". An isolated nucleic acid or protein may exist in a substantially purified form or may exist in a non-natural environment such as, for example, a host cell.

[0074] As used herein, the term "modified" is intended to mean that the molecular or cellular context or structure of the invention has been changed. A molecule may be modified in many ways, including chemically, structurally, and functionally. A cell may be modified through the introduction of nucleic acid.

[0075] As used herein, the term "modulating" is intended to mean mediating a detectable increase or decrease in the level of a response in a subject as compared to the level of the response in the subject in the absence of treatment or compound and / or as compared to the level of the response in the same subject except for not having received treatment, which includes perturbing and / or affecting a natural signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0076] In the context of the present invention, the following abbreviations are used for commonly occurring nucleobases. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0077] Unless otherwise defined, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of one another and encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may include introns to the extent that a nucleotide sequence encoding a protein may contain some versions of intron(s).

[0078] The term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is arranged in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, are in the same reading frame.

[0079] "Parenteral" administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.

[0080] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Further, a nucleic acid is a polymer of nucleotides. Thus, the nucleic acids and polynucleotides used herein are interchangeable. One of ordinary skill in the art has the general knowledge that a nucleic acid is a polynucleotide that can be hydrolyzed to monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed to nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any available means in the art, including, but not limited to, recombinant means using conventional cloning techniques and PCR (trademark) and the like, i.e., cloning of nucleic acid sequences from recombinant libraries or cell genomes, as well as synthetic means.

[0081] As used herein, the terms "peptide", "polypeptide", and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limitation on the maximum number of amino acids that can be included in the sequence of the protein or peptide. Polypeptides include any peptide or protein containing two or more amino acids joined together by peptide bonds. As used herein, the term refers to both short chains, which are also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers, and the many types of longer chains, which are commonly referred to in the art as proteins. "Polypeptide" includes, for example, among others, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers of polypeptides, heterodimers, variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0082] As used herein, the term "promoter" is defined as a DNA sequence recognized by the cell's synthetic machinery or introduced synthetic machinery that is required to initiate specific transcription of a polynucleotide sequence.

[0083] As used herein, the term "promoter / regulatory sequence" means a nucleic acid sequence required for the expression of a gene product that is operably linked to the promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other instances, this sequence may include enhancer sequences and other regulatory elements required for the expression of the gene product. The promoter / regulatory sequence may, for example, express the gene product in a tissue-specific manner.

[0084] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0085] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell substantially only when the corresponding inducer of the promoter is present in the cell.

[0086] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes the gene product to be produced in a cell substantially only when the cell is of the tissue type corresponding to the promoter.

[0087] The term "epigenetic", as used herein, refers to a heritable influence on gene expression without a change in the DNA nucleotide sequence. Epigenetic regulation can enhance or inhibit the expression of the affected gene and can involve chemical modification of the deoxyribose backbone of DNA or the association of DNA / histone protein complexes or both.

[0088] The term "epigenetic regulator", as used herein, refers to a factor, enzyme, compound, or composition that acts to change the epigenetic state of a particular DNA locus. An epigenetic regulator can induce or catalyze the modification of DNA-related proteins or the chemical structure of DNA itself.

[0089] As used interchangeably herein, the terms "epigenetic tag" or "epigenetic marker" or "epigenetic mark" describe specific chemical modifications made to DNA or DNA-related proteins that result in epigenetic regulation of gene expression. Examples of epigenetic marks or tags include, but are not limited to, the addition or removal of methyl or acetyl groups from CpG dinucleotides and histone proteins. The number and density of epigenetic tags or epigenetic marks can correlate with the degree of epigenetic regulation of a particular DNA locus.

[0090] "Signaling pathway" refers to the biochemical relationships among various signaling molecules that play a role in the transmission of signals from one part of a cell to another part of the cell. The phrase "cell surface receptor" includes molecules or molecular complexes that can receive signals and transmit them across the plasma membrane of the cell.

[0091] As used herein, the term "tropism" refers to the ability of a cell type or tissue type to be productively infected by a particular virus or virus-based vector (e.g., an AAV vector). Although not necessarily excluding others, tropism is often expressed as "preference" or "specificity" of a virus or virus vector for a particular cell type or tissue type. Since it is a parasitic entity, cell tropism or tissue tropism is one of the main characteristics of certain viruses that often require insertion into a particular cell type to maintain a good infectious cycle in the target cell. When referring to a virus vector (e.g., an AAV vector), tissue tropism or cell tropism or specificity or preference can be used to direct a therapeutic payload to a particular tissue type or cell type.

[0092] As used herein with respect to an antibody, the term "specifically binds" is intended to mean that the antibody recognizes a particular antigen, but substantially does not recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. However, such cross-reactivity by itself does not change the classification of the antibody as being specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity by itself does not change the classification of the antibody as being specific. In some cases, the terms "specific binding" or "specifically binds" are used with respect to the interaction of an antibody, protein, or peptide with a second chemical species, and may mean that the interaction depends on the presence of a particular structure on the chemical species (e.g., an antigenic determinant or epitope). For example, an antibody generally recognizes and binds to a particular protein structure rather than to the protein per se. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free unlabeled A) is thought to reduce the amount of labeled A bound to the antibody in a reaction containing labeled "A" and the antibody.

[0093] The term "subject" is intended to include a living organism (e.g., a mammal) in which an immune response can be elicited. A "subject" or "patient", as used herein, can be a human or non-human mammal. Non-human mammals include, for example, livestock and pets such as sheep, cows, pigs, dogs, cats, and mouse mammals. Preferably, the subject is a human.

[0094] A "target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0095] As used herein, the term "therapeutic" means treatment and / or prevention. A therapeutic effect is obtained by suppression, alleviation, or eradication of a disease condition.

[0096] As used herein, the terms "transfected", "transformed", or "transduced" refer to the process by which exogenous nucleic acid is introduced or transferred into a host cell. A "transfected", "transformed", or "transduced" cell has been transfected, transformed, or transduced with exogenous nucleic acid. Cells include primary subject cells and their progeny.

[0097] The term "transgene" refers to genetic material that has been or is to be artificially inserted into the genome of mammalian cells of an animal, specifically a mammal, more specifically a living animal.

[0098] The term "transgenic animal" refers to a non-human animal, usually a mammal, having a non-endogenous (i.e., heterologous) nucleic acid sequence that is present as an extrachromosomal element in some of its cells or stably integrated into its germline DNA (i.e., in most or all of its genomic sequences), e.g., a transgenic mouse. The heterologous nucleic acid is introduced into the germline of such a transgenic animal, for example, by genetic manipulation of an embryo or embryonic stem cell of the host animal.

[0099] The term "knockout mouse" refers to a mouse having an inactivated existing gene (i.e., a "knockout"). In some embodiments, the gene is inactivated by homologous recombination. In some embodiments, the gene is inactivated by replacement or disruption with an artificial nucleic acid sequence.

[0100] "Treating" a disease, as the term is used herein, means reducing the frequency or severity of at least one sign or symptom of a disease or disorder that a subject has.

[0101] As used herein, the phrases "under transcriptional control" or "functionally linked" mean that the promoter is in the correct position and orientation in relation to the polynucleotide so as to control the initiation of transcription by RNA polymerase and the expression of the polynucleotide.

[0102] "Vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid inside a cell. A number of vectors are known, including, but not limited to, linear polynucleotides, polynucleotides complexed with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes self-replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the entry of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and the like.

[0103] Range: Throughout this disclosure, various aspects of the invention may be presented in range format. It should be understood that descriptions in range format are merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges and individual numerical values within that range. For example, a description of a range such as 1-6 should be considered to have specifically disclosed sub-ranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., and individual numbers such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6 within that range. This applies regardless of the width of the range.

[0104] Explanation The present invention is based on the unexpected finding that in certain muscle tissues, a muscle-specific regulatory cassette can be constructed that significantly and preferentially expresses a payload gene contained within the cassette. In certain embodiments, the muscle-specific regulatory cassette of the present invention efficiently expresses the payload gene in skeletal muscle tissue but not in heart tissue. Also included in the present invention are muscle-specific regulatory cassettes that have similarly robust payload gene expression in skeletal muscle tissue but, although reduced, still have a detectable level of expression in heart tissue.

[0105] The regulatory cassettes of the present invention are also minimized with respect to size, such that they are smaller than other muscle-specific regulatory cassettes known in the art. Thus, such cassettes maximize the size of the payload gene that can be packaged in cell transduction systems such as AAV vector systems and the like. Accordingly, in certain embodiments, the regulatory cassettes of the present invention can be included within an AAV vector system that efficiently transfers the regulatory cassette and its associated transgene into mammalian cells and tissues.

[0106] Cardiotoxicity is an issue for good gene therapy Gene therapy approaches in preclinical models of muscle diseases can result in cardiotoxicity caused by the expression of the transgene in the heart. This toxicity can be prevented by using a muscle-specific regulatory cassette in combination with miRNA target derepression sequences, while such elements together add a significant size to the therapeutic cassette, which is already limited by the tight packaging capacity of the recombinant AAV (rAAV) vector. A particular major concern is the severe adverse events reported in recent gene therapy trials for two muscle diseases. The death in the ASPIRO X-linked myotubular myopathy (XLMTM) clinical trial was due to liver failure, although some patients had laboratory findings indicative of myocarditis, and two instances of myocarditis were reported in the Pfizer trial for Duchenne muscular dystrophy (DMD). Therefore, there is a clinical need for a minimized gene regulatory cassette that has high activity in skeletal muscle but low or no activity in the heart.

[0107] Regulatory regions of the muscle creatine kinase (Ckm) gene that mediate transcription have been found in previous studies to be useful in driving highly tissue-specific expression in skeletal and cardiac muscle tissues (Salva et al. (2007) Mol Ther, 2007. 15(2): p. 320-9.). Regulatory cassettes containing sequences obtained from such regions have been demonstrated to drive robust tissue-specific transgene expression.

[0108] In certain embodiments, the invention includes muscle creatine kinase (CKM)-based regulatory cassettes that have been modified in multiple ways to alter tissue specificity beyond currently available cassettes: (1) a No Heart (NH) cassette that is highly active in most fast skeletal muscles but essentially inactive in the soleus muscle (slow muscle), diaphragm, and heart, and (2) a Have a Little Heart (HLH) cassette that exhibits extremely high activity in fast muscles, moderate activity in the soleus muscle and diaphragm, and low activity in the heart. Thus, the NH cassette is thought to be useful for skeletal muscle indications where cardiac expression needs to be absent or tolerated, while the HLH cassette is thought to be useful for providing transgene expression in all striated muscles while avoiding cardiotoxicity associated with high transgene expression. Collectively, these cassettes are applicable to a variety of gene therapy strategies targeting a wide range of myopathies.

[0109] AAV vector AAV is a relatively small non-enveloped virus with a genome of approximately 4 kb adjacent to inverted terminal repeat sequences (ITRs). The genome contains two open reading frames, one of which provides the proteins necessary for replication and the other provides the components necessary for the construction of the viral capsid. Since the helper proteins essential for the packaging of the AAV genome into virions are provided by adenovirus, wild-type AAV is typically found in the presence of adenovirus. For this reason, AAV production exploits co-infection with adenovirus and relies on the following three key elements: the ITR-adjacent genome, the open reading frame, and the adenovirus helper gene. Due to its non-pathogenic ability to easily infect human cells, AAV has been well studied as a vector for gene delivery. AAV is readily obtainable and its use as a vector for gene delivery is described, for example, in Muzyczka, 1992, U.S. Patent No. 4,797,368, and PCT Publication WO 91 / 18088. The construction of AAV vectors is described in several publications, including Lebkowski et al., 1988, Tratschin et al., 1985, and Hermonat and Muzyczka, 1984.

[0110] AAV-based vector systems typically separate the viral AAV genes, the helper genes derived from adenovirus, and the transgene payload onto two or three separate plasmids. The three-plasmid system consists of an AAV helper plasmid containing the rep (replication) gene and the cap (capsid) gene, an adenovirus helper plasmid containing at least the E2a gene, the E4 gene, and VA (virus-associated) RNA, and a payload plasmid containing the transgene adjacent to the ITR sequence and the associated promoter and enhancer. The helper plasmid(s) do not contain the ITRs to prevent the packaging of a functional infectious viral genome.

[0111] Two plasmid systems simplify viral vector production by integrating the AAV rep and cap genes and the adenovirus helper genes onto a single plasmid and reducing the number of plasmids to be transfected. Often, a dedicated packaging cell line engineered to express the AAV / helper genes prior to introduction of the payload plasmid is used.

[0112] Good gene therapy requires efficient infection of target tissues and establishment of long-term gene expression. AAV vectors have been demonstrated in previous studies to be able to infect and transduce well into a wide variety of cell types and tissue types, such as the brain, liver, and muscle, and to have the ability to infect both dividing and resting cells. Furthermore, AAV-mediated transduction of tissues has been demonstrated to result in long-term transgene expression exceeding 1.5 years in animal models including dogs, mice, and hamsters.

[0113] Although the tissue tropism of AAV vector particles is affected by the serotype of the capsid protein, the receptors and co-receptors to which the capsid protein binds are often poorly understood and can be expressed by multiple tissue types. For example, AAV2, one of the most well-studied serotypes, mainly has a binding affinity for heparan sulfate proteoglycan (HSPG) and thus has tropism for the eye, brain, lung, liver, muscle, and connective tissues in humans. Similarly, AAV1, 4, 5, and 6 mainly have a binding affinity for sialic acid and tropism for neural tissues, while AAV5 and 8 share tropism for skeletal muscle. Thus, the serotype of the AAV capsid protein can be selected to target the payload nucleic acid (e.g., regulatory cassette) of the AAV vector to a specific tissue type or cell type. Alteration or modification of the capsid protein structure can also change the tissue tropism and affinity or cell tropism and affinity of the resulting AAV vector particles.

[0114] In certain preferred embodiments, the invention includes an AAV vector comprising a capsid protein obtained from AAV9 and variants thereof. AAV9 and AAV9-based capsid proteins transduce muscle, liver, and lung tissues more than about 100-fold more efficiently than AAV2, while also being able to cross the blood-brain barrier.

[0115] In certain preferred embodiments, the invention includes an AAV vector comprising a capsid protein obtained from AAVrh74 and variants thereof. AAVrh74 and AAVrh74-based capsid proteins have tropism for skeletal and cardiac tissues and are useful for delivering therapeutic nucleic acids to such tissues.

[0116] In certain preferred embodiments, the invention includes an AAV vector comprising a capsid protein obtained from a MyoAAV capsid and variants thereof. MyoAAV is a family of capsid proteins that share a common arginine-glycine-aspartic acid (RGD) motif and was generated through evolution such that high specificity and expression in muscle tissue were selected, particularly as compared to AAV9. MyoAAV capsids known in the art include, but are not limited to, MyoAAV 1A, MyoAAV 2 or 2A, so-called second-generation capsids, MyoAAV 3A, MyoAAV 4A, MyoAAV 4C, and MyoAAV 4E. Given the tissue specificity of MyoAAV family capsids for muscle tissue, it is contemplated that any MyoAAV capsid may be used with the therapeutic cassettes, vectors, compositions, and methods of the invention. In certain preferred embodiments, the AAV vector of the invention comprises a capsid protein obtained from MyoAAV2 or a variant thereof.

[0117] In particular, it is contemplated that the regulatory cassette of the present invention can be used with any naturally occurring AAV capsid protein, modified AAV capsid protein, hybrid AAV capsid protein, or engineered AAV capsid protein that confers a desired tissue tropism, including but not limited to AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, AAV-B1, AAV-DJ, AAV-Retro, AAVrh8, AAVrh10, AAVrh25, Anc80L65, LK03, AAVrh18, AAVrh74, AAVrh32.33, AAVrh39, AAVrh43, MyoAAV2, Oligo001, PHP-B, and Spark100 or variants thereof. One of ordinary skill in the art will be able to select a capsid protein suitable for use with the present invention based on the desired target tissue type or cell type.

[0118] Nucleic acid and vector The present disclosure provides an isolated polynucleotide encoding a muscle-specific regulatory cassette. In certain embodiments, the muscle-specific regulatory cassette comprises at least three modified enhancer sequences located upstream of and operably linked to a promoter, the three modified enhancer sequences comprising a Trex sequence, an AT-rich sequence, a right E-box sequence, and a MEF2 sequence, the right E-box sequence repeating at least once, and the promoter being an -80 to +50 promoter containing a consensus Inr sequence.

[0119] In certain embodiments, the muscle-specific regulatory cassette is encoded by a nucleic acid comprising a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to SEQ ID NO:1.

[0120] In certain embodiments, the muscle-specific regulatory cassette is encoded by a nucleic acid comprising the polynucleotide sequence set forth in SEQ ID NO:1.

[0121] In certain embodiments, the muscle-specific regulatory cassette is encoded by a nucleic acid consisting of the polynucleotide sequence set forth in SEQ ID NO:1.

[0122] In certain embodiments, the muscle-specific regulatory cassette comprises at least three modified enhancer sequences located upstream of and operably linked to a promoter, the at least three modified enhancer sequences comprising a Trex sequence, an AT-rich sequence, a left E-box sequence, a right E-box sequence, and a MEF2 sequence, and the promoter is a -80 to +50 promoter containing a consensus Inr sequence.

[0123] In certain embodiments, the muscle-specific regulatory cassette is encoded by a nucleic acid comprising a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to SEQ ID NO:2.

[0124] In certain embodiments, the muscle-specific regulatory cassette is encoded by a nucleic acid comprising the polynucleotide sequence set forth in SEQ ID NO:2.

[0125] In certain embodiments, the muscle-specific regulatory cassette is encoded by a nucleic acid consisting of the polynucleotide sequence set forth in SEQ ID NO:2.

[0126] Gene transfer system and adeno-associated virus (AAV) Gene transfer systems, such as those described in the present invention, rely on vectors or vector systems for freely transporting genetic constructs into target cells. Methods for introducing nucleic acids into hematopoietic stem cells or progenitor cells include physical, biological, and chemical methods. Physical methods for introducing polynucleotides, such as RNA, into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. RNA can be introduced into target cells using commercially available methods including electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany)). RNA can also be introduced into cells using cationic liposome-mediated transfection using lipofection, using polymer encapsulation, using peptide-mediated transfection, or using a biolistic particle delivery system such as a "gene gun" (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001).

[0127] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including water-in-oil emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0128] Suitable lipids can be obtained from commercial sources. For example, dimyristoyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO. Dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY). Cholesterol ("Choi") can be obtained from Calbiochem-Behring. Dimyristoyl phosphatidylglycerol ("DMPG") and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Since chloroform evaporates more readily than methanol, it is used as the sole solvent. "Liposome" is a general term encompassing various single-layer and multi-layer lipid mediators formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure containing a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. They are formed spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-reorganization before the formation of the closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., (1991) Glycobiology 5: 505-10). However, compositions having solution structures different from normal vesicular structures are also included. For example, the lipids may assume a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0129] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be obtained from lentiviruses, poxviruses, herpes simplex virus type I, adenoviruses, and adeno-associated viruses, and the like. See, e.g., U.S. Pat. Nos. 5,350,674 and 5,585,362. Currently, the most efficient and effective manner of achieving the transfer of genetic constructs into living cells is via the use of vector systems based on replication-defective viruses. Some of the most effective vectors known in the art are those based on adeno-associated virus (AAV). AAV is a parvovirus that is an attractive vector for gene transfer in that it is replication-defective, is not known to cause any human diseases, elicits only a very mild immune response, can infect both actively dividing cells and resting cells, and persists stably in an episomal situation without integration into the genome of the target cell. In certain embodiments, the disclosure provides AAV vectors comprising the dCas9-based CRISPRi system of the invention.

[0130] Regardless of the method used to introduce a nucleic acid into a cell, various assays may be performed to confirm the presence of the nucleic acid in the cell. Such assays include, for example, "molecular biology" assays well known to those of skill in the art, such as Southern and Northern blots, RT-PCR, and PCR, "biochemical" assays that detect the presence or absence of a particular peptide, e.g., by immunological means (ELISA and Western blot), or assays described herein for identifying agents within the scope of the invention.

[0131] Methods of treatment and use In certain embodiments, the invention comprises administering to a subject in need thereof an effective amount of an AAV vector comprising a muscle-specific regulatory cassette comprising at least three modified enhancer sequences located upstream of and operably linked to a promoter, thereby treating a genetic disease or disorder, said method of treating a genetic disease or disorder in a subject in need thereof comprising said step, wherein the three modified enhancer sequences comprise a Trex sequence, an AT-rich sequence, a right E-box sequence, and an MEF2 sequence, the right E-box sequence is repeated at least once, and the promoter is a -80 to +50 promoter containing a consensus Inr sequence. Thus, in certain embodiments, the AAV vector has specificity or tropism for muscle tissue. In certain embodiments, the regulatory cassette is highly active in fast skeletal muscle, with relatively low expression activity in soleus muscle and diaphragm muscle and no expression activity in heart tissue. Thus, methods comprising such regulatory cassettes are ideally suited for the treatment of genetic diseases or disorders such as skeletal muscle myopathies that do not affect the myocardium (e.g., FSHD, XLMTM, central core myopathy, inclusion body myositis, nemaline myopathy, distal myopathy, centronuclear myopathy, and most cases of oculopharyngeal muscular dystrophy, as well as certain subtypes of LGMD in patients without cardiac lesions).

[0132] In certain aspects, the invention of the present disclosure includes a method of treating a genetic disease or genetic disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of an AAV vector comprising a muscle-specific regulatory cassette encoding a polynucleotide comprising at least three modified enhancer sequences located upstream of and operably linked to a promoter, wherein the at least three modified enhancer sequences include a Trex sequence, an AT-rich sequence, a left E-box sequence, a right E-box sequence, and an MEF2 sequence, and the promoter is a -80 to +50 promoter containing a consensus Inr sequence. Thus, in certain aspects, the AAV vector has specificity or tropism for muscle tissue. In certain aspects, the regulatory cassette exhibits moderate to high activity in all skeletal muscles and low expression activity in heart tissue. Thus, such cassettes provide moderate to high levels of expression across all skeletal muscles with low-level expression in the heart, thereby affecting all striated muscles, but are well-suited for the treatment of genetic diseases or genetic disorders in which high expression of the therapeutic transgene is toxic in cardiomyocytes. Examples of such include, but are not limited to, many subtypes of DMD, XLMTM, and LGMD. For such diseases or disorders, effective treatment requires some therapeutic transgene expression in cardiomyocytes, but low expression levels will prevent myocarditis.

[0133] In certain aspects, the genetic disease or genetic disorder treated by the methods of the invention is a muscle-related genetic disease or genetic disorder. Non-limiting examples of muscle-related genetic diseases or genetic disorders that can be treated by the methods of the invention include facioscapulohumeral muscular dystrophy (FSHD), X-linked myotubular myopathy (XLMTM), central core myopathy, inclusion body myositis, nemaline myopathy, distal myopathy, centronuclear myopathy, oculopharyngeal muscular dystrophy, dysferlinopathy, limb-girdle muscular dystrophy (LGMD), and Duchenne muscular dystrophy (DMD), but are not limited thereto.

[0134] In certain embodiments, the present invention includes a method of introducing a transgene into a target cell, the method comprising contacting the target cell with a polynucleotide encoding a muscle-specific regulatory cassette comprising at least three modified enhancer sequences located upstream of and functionally linked to a promoter, the three modified enhancer sequences comprising a Trex sequence, an AT-rich sequence, a right E-box sequence, and a MEF2 sequence, the right E-box sequence being repeated at least once, and the promoter being an -80 to +50 promoter containing a consensus Inr sequence. In certain embodiments, the target cell is a mammalian muscle cell. In certain embodiments, the cassette has high expression activity in skeletal muscle cells. In certain preferred embodiments, the skeletal muscle tissue is fast-twitch muscle tissue. As used herein, the term "fast-twitch muscle tissue" refers to type II muscle tissue specialized for short, powerful contractions, as contrasted with "slow-twitch" or type I muscle tissue, which can maintain contraction over a long period of time. In certain embodiments, the cassette has very low expression in heart tissue compared to skeletal muscle tissue.

[0135] In certain embodiments, the present invention includes a method of introducing a transgene into a target cell, the method comprising contacting the target cell with a polynucleotide encoding a muscle-specific regulatory cassette comprising at least three modified enhancer sequences located upstream of and functionally linked to a promoter, the at least three modified enhancer sequences comprising a Trex sequence, an AT-rich sequence, a left E-box sequence, a right E-box sequence, and a MEF2 sequence, and the promoter being an -80 to +50 promoter containing a consensus Inr sequence. In certain embodiments, the target cell is a mammalian muscle cell. In certain embodiments, the cassette has high expression activity in skeletal muscle tissue achieved by expression that is at a low level but still present in heart tissue.

[0136] Pharmaceutical composition The pharmaceutical composition of the present invention may comprise those described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, adjuvants, or excipients. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline, and the like, carbohydrates such as glucose, mannose, sucrose or dextran, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0137] The pharmaceutical compositions of the present disclosure may comprise the AAV vector particles described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline (PBS), and the like, carbohydrates such as glucose, mannose, sucrose or dextran, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives. The compositions of the present disclosure are preferably formulated for several routes of administration including oral, inhalation, nasal, spray, intravenous injection, intramuscular injection, intrathecal injection, intrapleural injection, intracisternal injection, subcutaneous injection, and / or transdermal injection. The pharmaceutical compositions of the present disclosure may be administered in a manner suitable for the disease to be treated (or prevented). The dosage and frequency of administration are determined by factors such as the patient's condition, the type and severity of the patient's disease, and the type and functional nature of the patient's immune response to the phage particles, but appropriate dosages may be determined by clinical trials.

[0138] The AAV vector particles of the present disclosure can be administered in dosages, routes, and frequencies to be determined in appropriate preclinical and clinical experiments and trials. Administration of the AAV vector particles of the present disclosure may be combined with other methods useful in treating the desired disease or condition as determined by those skilled in the art.

[0139] In certain embodiments, the effective dosage range is measured in units known to those of ordinary skill in the art suitable for the description of the AAV vector particle dosage. In some embodiments, the effective dosage range of the vaccines or therapeutic compounds of the present disclosure is measured by transduction units (TU) / kg / dosage or genomic copies (GC) / kg / dosage or particles / kg / dosage. In some embodiments, the dosage administered to a patient is about 10 6 ~10 14 TU / kg. In some embodiments, the dosage administered to a patient is about 10 6 ~10 14 GC / kg. In some embodiments, the effective dosage range is measured by colony forming units (CFU), 50% tissue culture infective dose (TCID 50 ), and combinations thereof.

[0140] The actual dosage level of the active ingredient in the pharmaceutical compositions of the present disclosure can be varied so that an amount of the active ingredient effective to achieve the desired therapeutic response in a particular patient, composition, and mode of administration is obtained without causing toxicity to the patient.

[0141] The therapeutically effective amount or therapeutically effective dosage of the compounds of the present disclosure depends on the age, sex, and weight of the patient, the current medical condition of the patient, and the progression of the disease or disorder contemplated in the present disclosure.

[0142] A physician having ordinary skill in the art, e.g., a medical doctor or veterinarian, can readily determine and prescribe the effective amount of the required pharmaceutical composition. For example, a medical doctor or veterinarian can start with a dosage of the compounds of the present disclosure utilized in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0143] In certain embodiments, the compositions of the present disclosure are administered to a patient at a dosage range of 1 to 5 times per day or more. In other embodiments, the compositions of the present disclosure are administered to a patient at dosage ranges including, but not limited to, once a day, once every two days, once every three days to once a week, and once every two weeks. The frequency of administration of the various combination compositions of the present disclosure will vary from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, general health, and other factors. Thus, the present disclosure should not be construed as limited to any particular dosage regimen, and the exact dosage and composition to be administered to any patient will be determined by the attending physician taking into account all other factors regarding the patient.

[0144] The dosage size can be adjusted according to the weight, age, and disease stage of the subject being treated. The AAV vector particles may be administered multiple times at such dosages. The AAV vector particles can be administered by using infusion techniques generally known in the technical fields of immunotherapy or vaccinology. The optimal dosage and treatment regimen for a particular patient can be readily determined by a person skilled in the art by monitoring the patient for signs of the disease and adjusting the treatment accordingly.

[0145] Administration of the AAV vector particle compositions of the present disclosure may be carried out in any convenient manner known to those skilled in the art. The AAV vector particles of the present disclosure may be administered to a subject by aerosol inhalation, injection, oral ingestion, transfusion, implantation, or transplantation. The compositions described herein may be administered to a subject or patient via arterial, subcutaneous, intranasal, intradermal, intratumoral, intra-articular, intramedullary, intramuscular, intravenous, or intraperitoneal routes. In other examples, the AAV vector particles of the present disclosure are directly injected into the site of inflammation in a subject, a local disease site in a subject, LNs, organs, tumors, and the like. It should be understood that the methods and compositions that may be useful in the present disclosure are not limited to the specific formulations shown in the examples.

[0146] In certain embodiments, the compositions of the present disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the present disclosure comprise a therapeutically effective amount of a compound of the present disclosure and a pharmaceutically acceptable carrier.

[0147] The carrier can be, for example, a solvent or dispersion medium containing saline, buffered saline, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Suitable fluidity can be maintained, for example, by the use of coatings such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents such as, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is advisable to include in the composition isotonic agents such as, for example, sugars, sodium chloride, or polyhydric alcohols such as mannitol and sorbitol.

[0148] The formulations can be utilized in admixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances, suitable for any appropriate mode of administration known in the art. The pharmaceutical preparations are sterilized and, if desired, can be mixed with adjuvants such as, for example, lubricants, preservatives, stabilizers, wetting agents, emulsifying agents, salts for influencing osmotic pressure buffers, coloring substances, flavoring substances, and / or aromatic substances, and the like. They can also be combined, if desired, with other active agents such as, for example, analgesics.

[0149] In the practice of the present disclosure, unless otherwise specified, conventional techniques in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology that are well within the purview of those of ordinary skill in the art are utilized. Such techniques are fully described in the literature such as “Molecular Cloning: A Laboratory Manual”, fourth edition (Sambrook, 2012), “Oligonucleotide Synthesis” (Gait, 1984), “Culture of Animal Cells” (Freshney, 2010), “Methods in Enzymology”, “Handbook of Experimental Immunology” (Weir, 1997), “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987), “Short Protocols in Molecular Biology” (Ausubel, 2002), “Polymerase Chain Reaction: Principles, Applications and Troubleshooting”, (Babar, 2011), “Current Protocols in Immunology” (Coligan, 2002). Such techniques are applicable to the production of the polynucleotides and AAV particles of the present disclosure and are thus contemplated in the practice and implementation of the present disclosure.

[0150] It should be understood that the methods and compositions that may be useful in the present disclosure are not limited to the specific formulations shown in the examples. The following examples are provided to fully disclose and describe to those of ordinary skill in the art how to implement and use cell, growth, and culture methods, as well as the therapeutic methods of the present disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure.

Examples

[0151] Experimental Examples The present invention will be described in more detail by reference to the following experimental examples. Such examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should not be construed as being limited to the following examples in any way, but rather should be construed as encompassing any and all variations that become apparent as a result of the teachings provided herein.

[0152] Even without further explanation, those skilled in the art will be able to make and use the compounds of the present invention and carry out the claimed methods using the foregoing description and the following illustrative examples. For this reason, the following practical examples are specific illustrations of preferred embodiments of the present invention and should not be construed as limiting the remainder of the disclosure in any way.

[0153] Materials and methods are described herein.

[0154] Plasmid The NH cassette (Himeda et al. (2021) Mol Ther Methods Clin Dev, 2021. 20: p. 298-311) consists of three tandem copies of a modified Ckm enhancer located upstream of the modified Ckm promoter. The main features are as follows: (1) a mutation from the left E-box to the right E-box (Nguyen et al., (2003) J Biol Chem. 278(47): p. 46494-505 and Hauser et al. (2000) Mol Ther. 2(1): p. 16-25), (2) removal of the enhancer CArG site and AP2 site, (3) removal of 63 bp between the right E-box and the MEF2 site (Salva et al., (2007) Mol Ther. 15(2): p320-9), (4) minimization of the sequences between transcription factor binding motifs, (5) use of the -80 (Donoviel et al., (1996) Mol Cell Biol. 16(4): p1649-58) to +50 (Salva et al., (2007) Mol Ther. 15(2): p320-9) promoter sequence, and (6) addition of a consensus initiator element (Inr) (Salva et al. (2007) Mol Ther. 15(2): p. 320-9). The HLH cassette is based on the NH cassette and has the following modifications: the additional right E-box was replaced with the original left E-box from the Ckm enhancer. For both cassettes, an mCherry reporter was inserted downstream of the transcription start site. The reporter cassette was synthesized and fully sequenced by GENEWIZ, LLC (South Plainfield, NJ). For the evaluation of cassette activity in vivo, each regulatory cassette driving the mCherry reporter was cloned between the AAV2 ITRs (using MluI and RsrII) of the pAAV-CA plasmid (Menegas et al., (2015) Elife 4: pe10032), a gift from Naoshige Uchida (Addgene plasmid # 69616; RRID:Addgene_69616).The recombinant AAVrh74 particles were produced by Vector Biolabs (Malvern, PA). The vector sequences are disclosed in Table 1.

[0155] Animal All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Nevada, Reno. Prior to retro-orbital injection (ROI) of rAAVrh74 (2.6×10 14 GC / kg) containing either the NH cassette or the HLH cassette that regulates the expression of mCherry, 3.5-week-old male wild-type mice (C57BL / 6J) were anesthetized with 3.5% isoflurane.

[0156] Optical imaging in vivo Prior to imaging, the fur in the ventral abdominal region was removed using depilatory cream. Fluorescent images of mCherry in live mice under anesthesia (2% isoflurane) were acquired weekly from the ROI for 10 weeks using an IVIS Lumina III and Living Image software version 4.3.1 (PerkinElmer) with a λex = 580 nm / λem = 620 nm filter, an exposure time of 1 second, a 12.5×12.5 cm FOV, and an f / stop of 4. All images were equally processed in Living Image using a trans-illumination fluorescence scale with a minimum = 7×10 8 and a maximum = 3.2×10 10 , smoothing = 5×5, and binning = 2. Total light flux (photons / second) and maximum luminance (photons / second / cm 2 / steradian) were measured using the Living Image software.

[0157] Optical imaging in vitro At 12 weeks after injection, all mice were euthanized, tissues were dissected, and mCherry signals were acquired using a Leica THUNDER / DFC-7000T fluorescence imaging system and Leica LAS X software with the same exposure time unless otherwise indicated in the figure. Images were constructed using Adobe Photoshop 24.1.0 and the exposures were adjusted equally. The fluorescence signal in the selected tissues was further measured using an IVIS Lumina III and Living Image software with a filter λex = 580 nm / λem = 620 nm, exposure time 0.5 s, FOV 4×4-cm, f / stop 8. Using the Living Image software, the total light flux (photons / sec) and maximum luminance (photons / sec / cm 2 / steradian) were measured.

[0158] Quantification of viral transduction After in vitro imaging, genomic DNA was isolated from each tissue for evaluation of rAAV infection. The viral genome was quantified by qPCR (100 ng of genomic DNA) using primers against the bovine growth hormone polyadenylation signal (bGH PAS) present in the transgene construct sequence and normalized to the endogenous single-copy Rosa26 locus. Each tissue had 3 biological replicates. The oligonucleotide primer sequences were as reported (Himeda et al., (2021) Mol Ther Methods Clin Dev. 20: p. 298-311 and Himeda et al., (2016) Mol Ther. 24(3): p. 527-35).

[0159] (Table 1) Gene expression regulatory cassette TIFF2025523952000001.tif91164

[0160] The experimental results are described below.

[0161] Example 1: No Heart (NH): A muscle-specific cassette with high activity in fast muscle and no or low expression in myocardium The goal of the research of the present disclosure was to design a highly minimized skeletal muscle-specific regulatory cassette for use in a CRISPR inhibition approach against facioscapulohumeral muscular dystrophy (FSHD). It was necessary to minimize the current regulatory cassette in order to accommodate all therapeutic CRISPR components within a single rAAV vector.

[0162] Based on previous work (Salva, M.Z., et al., Mol Ther, 2007. 15(2): p. 320-9 and Himeda, C.L., et al., Methods Mol Biol, 2011. 709: p. 3-19), a skeletal muscle regulatory cassette was designed to drive the expression of larger therapeutic components, taking into account that the myocardium is not involved in FSHD. Several muscle-specific regulatory cassettes optimized for expression in striated muscle have been developed (PCT application No. WO2022216988A2) and are actively used in both academic and clinical settings. However, the lack of consistent nomenclature among users is a major problem, and researchers often refer to different cassettes by the same name (for example, in the "tMCK" cassette in Addgene plasmid #105556, three copies of the wild-type Ckm enhancer are used, while in "tMCK" in Addgene plasmid #149410, three copies of a modified Ckm enhancer are used). Although the importance of these differences is unclear, they can certainly complicate the comparison of efficacy and specificity between studies.

[0163] Starting with the well-established and widely used CK8 cassette, which is a modified version of three CKM enhancers located upstream of the CKM promoter, additional sequences between the elements were removed, and CarG and AP2 sites unnecessary for expression in skeletal muscle were deleted (Figure 1). The left E-box sequence was also mutated to create an additional right E-box, a change reported to increase activity in skeletal muscle cells (Nguyen et al., (2003) J Biol Chem. 278(47): p46494-505 and Hauser et al. (2000) Mol Ther. 2(1): p16-25), and the promoter was shortened to an 80-bp basal sequence (Donoviel et al., (1996) Mol Cell Biol. 16(4): p1649-58). This cassette was highly active in all skeletal muscles tested except for soleus, extensor digitorum longus (EDL), and diaphragm, and was virtually inactive in heart and non-muscle tissues (Himeda et al. (2021) Mol Ther Methods Clin Dev. 20: p298-311). Therefore, the inventors named this regulatory cassette no-heart (NH). Importantly, NH is active in most skeletal muscles, which are a very important target tissue for FSHD and also the host of other skeletal muscle myopathies, and the absence of cardiac expression is not important for FSHD because the heart is not pathologically affected (Galetta et al. (2005) Neuromuscul Disord. 15(6): p403-8, van Dijk et al. (2014) Funct Neurol. 29(3): p159-65, Ducharme-Smith et al. (2021) Front Neurol. 12: p668180).

[0164] Example 2: Hub A Little Heart (HLH): A muscle-specific cassette with moderate to high activity in all anatomical skeletal muscles and low activity in the heart NH is highly active in most fast-twitch muscles, while one weakness is that it is extremely lowly active in soleus muscle, EDL, and diaphragm (Himeda et al. (2021) Mol Ther Methods Clin Dev. 20: p298-311). To address this, we redesigned NH such that the additional right E-box replaces the original left E-box from the Ckm enhancer (Himeda, C.L., et al., Methods Mol Biol, 2011. 709: p. 3-19) (Figure 1). After the analysis described below, this cassette was named Hub·A·Little·Heart (HLH). Using systemic rAAVrh74-mediated transgene delivery into wild-type mice, the in vivo activities of the two cassettes were directly compared. Virus particles were delivered by retro-orbital injection (2.6×10 14 genome copies [GC] / kg body weight), and mCherry reporter activity (evaluated as epifluorescence in abdominal and thoracic muscles) was monitored over time. Over 10 weeks of monitoring, HLH showed significantly higher skeletal muscle activity than NH at all time points (Figure 2). Most notably, HLH achieved rapid high expression, and within just 2 weeks after delivery, the total light flux of HLH (mean 1.21×10 11 ) was already 95% of the peak total light flux of NH (mean 1.27×10 11 ) observed 8 weeks after delivery, and reached 485% at 3 weeks (mean 6.16×10 11 ) (Figure 2A). Total light flux is the integrated epifluorescence signal (brightness) over the area where the signal was detected and can vary between animals. However, even by maximum brightness measurement (the highest brightness for pixels in the field of view), HLH exceeded NH (Figure 2B). HLH showed 81% of the peak NH maximum brightness by the first week after ROI (mean 3.74×10 9 compared to 4.64×10 9 of NH at 6 weeks), increased to 165% at 2 weeks (mean 7.63×10 9 ), and reached 606% at 3 weeks (mean 2.81×10 10)。Overall, HLH exhibited 5- to 6-fold higher activity than NH in the abdominal / thoracic muscles.

[0165] To further evaluate both cassettes in other muscles and tissues, mice were sacrificed at 12 weeks post-injection for epifluorescence quantification and fluorescence imaging in vitro. As previously reported (Pozsgai et al. (2017) Mol Ther. 25(4): p855-869, Potter et al. (2021) Hum Gene Ther. 32(7-8): p375-389), quantitative PCR (qPCR) for viral genomes present across tissues showed that rAAVrh74 transduced the liver strongly and transduced the heart, kidney, and skeletal muscle moderately (Figure 5). Nevertheless, both cassettes maintained high activity across striated muscles, with HLH showing 7.4- to 36.5-fold higher activity than NH and showing no activity in non-muscle tissues (Figures 2-4, 6-9).

[0166] Mice injected with NH-mCherry in rAAVrh74 showed strong expression in the tibialis anterior muscle (TA), gastrocnemius muscle (GA), and quadriceps muscle (QUA), as well as in the pectoralis, abdominal, dorsal, and facial muscles (Figures 3-4, 6). Expression was virtually undetectable in the soleus muscle (SOL), EDL, diaphragm, and heart (Figures 3-4), remained virtually undetectable even with increased exposure (Figure 6), and was also virtually undetectable in non-muscle tissues (Figure 7). This reproduced the expression pattern observed by the inventors with this cassette delivered in AAV9 virions (Himeda et al. (2021) Mol Ther Methods Clin Dev. 20: p298-311), confirming that the low activity in certain skeletal muscles and the virtually undetectable activity in the heart are inherent to NH and not due to differences in the transduction ability of the vector.

[0167] The return of the left E-box in HLH (Figure 1) dramatically restored reporter expression in all fast muscles, conferred low expression in the heart, and most importantly, enabled moderate expression in SOL, EDL, and the diaphragm (Figures 3-4, 8). Whole-beam measurements showed mCherry expression 8.6-fold, 12.3-fold, 20-fold, and 36.5-fold higher in HLH than in NH in the QUA, GA, TA, and EDL muscles (Figure 3A), and this high expression even turned the muscles pink under visible light (Figure 4). In contrast, the low mCherry signal in the heart, despite its size and higher AAV transduction ability than skeletal muscle, indicates that this cassette has low activity in the heart. To exclude the influence of tissue size on signal measurement, we compared the mCherry signals in skeletal muscle and the heart using the maximum luminance data from in vitro optical imaging analysis (Figure 3B) and identified high-to-low HLH activities as follows: TA = GA = QUA (high: about 2.5-fold) > EDL = abdomen (moderate: 1-fold) > heart = SOL (low: about 0.3-fold). The maximum luminance of the HLH mCherry signal in the abdominal muscle was 2.5×10 11 which was 5.12-fold greater than that of NH (4.96×10 10 ), so the comparative HLH activity relative to NH (abdominal activity = 1) was TA = GA = QUA (high: about 12.8-fold) > EDL = abdomen (moderate: 5.1-fold) > heart = SOL (low: about 1.5-fold). We conclude that HLH exhibits extremely high activity in most skeletal muscles and no detectable expression in non-muscle tissues (Figure 9).

[0168] Example 3: Selective consideration The studies presented herein disclose two highly minimized muscle-specific cassettes that have utility for different gene therapy strategies across a broad range of muscle disorders. Due to their small size, both cassettes can be used in diverse strategies including gene replacement, exon skipping, and CRISPR-based gene editing, activation, or inhibition approaches. Since clinically relevant gene therapy using rAAV requires a therapeutic cassette contained within a single vector, it is not straightforward to allow for an increase in transgene size downstream of tissue-specific gene regulation (Hareendran et al. (2013) Rev Med Virol. 23(6): p399-413). Eliminating the need for more than one vector is extremely important in increasing delivery efficiency, reducing the high cost of therapy, and reducing the immunotoxicity associated with high viral doses. This is particularly a challenge for some CRISPR-based approaches that often utilize multiple vectors to accommodate all therapeutic components, and for gene replacement approaches for DMD where the ability to increase cargo size by even hundreds of base pairs can be critically important for efficacy. Importantly, the minimized regulatory cassettes of the present invention enable a single vector platform. As a non-limiting example, at 378 bp, they can be accommodated within the 4.4 kb packaging limit of an rAAV vector into an all-in-one therapeutic cassette containing dSaCas9 fused to a minimized effector domain and all sgRNA components. Potential therapeutic applications are further expanded when combined with engineered miniCas9 proteins or more recently reported, smaller Cas9 orthologs that can allow for multiple sgRNA cassettes or larger effector domain fusions.

[0169] The NH cassette of the present invention confers high-level expression in most fast skeletal muscles and essentially no expression in the heart. Therefore, it is ideally suited for the treatment of skeletal muscle myopathies that do not affect the myocardium, including, but not limited to, most cases of facioscapulohumeral muscular dystrophy (FSHD), X-linked myotubular myopathy (XLMTM), central core myopathy, inclusion body myositis, nemaline myopathy, distal myopathy, centronuclear myopathy, and oculopharyngeal muscular dystrophy, as well as certain subtypes of limb-girdle muscular dystrophy (LGMD) in which the patient does not exhibit cardiac lesions. Examples of the latter include LGMD2A / R1 (calpainopathy), one of the most common LGMD subtypes, and most dysferlinopathies.

[0170] In contrast to the NH cassette, the HLH cassette confers moderate to very high levels of expression across all skeletal muscles with low-level expression in the heart, thus affecting all striated muscles and being well-suited for the treatment of disorders where high expression of the therapeutic product in the myocardium can be detrimental. Such disorders include, but are not limited to, Duchenne muscular dystrophy (DMD) as well as some subtypes of LGMD and EDMD. For such diseases, effective treatment requires some therapeutic expression in the myocardium, but results from clinical trials suggest that reducing the expression level in the heart prevents the occurrence of myocarditis. Since AAV capsids highly directed to skeletal muscle are also highly directed to the myocardium, the best mode for selectively reducing transgene expression in the heart is through transcriptional regulation. Therefore, the HLH cassette of the present invention can provide optimal regulation for the therapeutic transgene in such indications. Furthermore, since HLH was highly active in all skeletal muscles (including soleus and EDL where NH was almost inactive), the HLH cassette would be well-suited for an approach where high skeletal muscle transgene expression is desired and low cardiac transgene expression is sufficiently tolerated. In FSHD, for example, the soleus muscle is often severely affected and would be a desirable target muscle for therapeutic delivery. Therefore, despite the absence of clinical heart pathology in FSHD, the HLH cassette can be a better overall therapeutic option for this myopathy and potentially other myopathies as well.

[0171] The research disclosed herein describes two novel variants of the CK8 regulatory cassette that are widely used in gene therapy approaches for muscular dystrophy. These minimized cassettes provide a significantly increased space for the transgene cargo while maintaining high activity and specificity to skeletal muscle in combination with no (NH) activity or low (HLH) activity in the heart. Importantly, neither cassette has any detectable activity in non-muscle tissues, regardless of the viral transduction level. Without wishing to be bound by theory, these new cassettes are thought to be valuable tools for enhancing the safety of gene therapy approaches for many myopathies.

[0172] Enumerated aspect The following enumerated aspects are provided, but their numbering should not be construed as specifying an order of importance. Aspect 1 provides the following: A polynucleotide encoding a muscle-specific regulatory cassette comprising at least three modified enhancer sequences located upstream of and functionally linked to a promoter, wherein said three modified enhancer sequences include a Trex sequence, an AT-rich sequence, a right E-box sequence, and a MEF2 sequence, wherein said right E-box sequence is repeated at least once, and wherein said promoter is an -80 to +50 promoter containing a consensus Inr sequence, said polynucleotide. Aspect 2 provides the following: The polynucleotide according to Aspect 1, wherein said cassette has high expression activity in skeletal muscle tissue. Aspect 3 provides the following: The polynucleotide according to Aspect 2, wherein the skeletal muscle tissue is fast-twitch muscle tissue. Aspect 4 provides the following: The polynucleotide according to Aspect 1, wherein said cassette has very low expression in heart tissue compared to skeletal muscle tissue. Aspect 5 provides the following: The polynucleotide according to embodiment 1, wherein the cassette is encoded by a nucleic acid comprising the sequence shown in SEQ ID NO:1. Embodiment 6 provides the following: The polynucleotide according to embodiment 1, wherein the cassette is encoded by a nucleic acid comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, and at least 99% sequence identity with the sequence shown in SEQ ID NO:1. Embodiment 7 provides the following: A polynucleotide encoding a muscle-specific regulatory cassette comprising at least three modified enhancer sequences located upstream of and functionally linked to a promoter, wherein the at least three modified enhancer sequences comprise a Trex sequence, an AT-rich sequence, a left E-box sequence, a right E-box sequence, and a MEF2 sequence, and wherein the promoter is an -80 to +50 promoter containing a consensus Inr sequence. Embodiment 8 provides the following: The polynucleotide according to embodiment 7, wherein the cassette has high expression activity in skeletal muscle tissue. Embodiment 9 provides the following: The polynucleotide according to embodiment 7, wherein the cassette has low expression activity in heart tissue compared to skeletal muscle tissue. Embodiment 10 provides the following: The polynucleotide according to embodiment 7, wherein the cassette is encoded by a nucleic acid comprising the sequence shown in SEQ ID NO:2. Embodiment 11 provides the following: The polynucleotide according to embodiment 7, wherein the cassette is encoded by a nucleic acid comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, and at least 99% sequence identity with the sequence shown in SEQ ID NO:2. Embodiment 12 provides the following: An AAV vector comprising a muscle-specific regulatory cassette comprising any one of the polynucleotides of Aspects 1 to 6. Aspect 13 provides the following: An AAV vector comprising a muscle-specific regulatory cassette comprising any one of the polynucleotides of Aspects 7 to 11. Aspect 14 provides the following: An AAV vector of any one of Aspects 12 to 13, comprising an AAV capsid that is a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, AAV-B1, AAV-DJ, AAV-Retro, AAVrh8, AAVrh10, AAVrh25, Anc80L65, LK03, AAVrh18, rAAVrh74, AAVrh32.33, AAVrh39, AAVrh43, MyoAAV2, Oligo001, PHP-B, and Spark100. Aspect 15 provides the following: The AAV vector according to Aspect 14, wherein the capsid is of the rAAVrh74 serotype. Aspect 16 provides the following: The AAV vector according to Aspect 14, wherein the capsid is of the AAV9 serotype. Aspect 17 provides the following: The AAV vector according to Aspect 14, wherein the capsid is of the MyoAAV2 serotype. Aspect 18 provides the following: The AAV vector according to Aspect 14, wherein the capsid has specificity for muscle tissue. Aspect 19 provides the following: A composition comprising AAV vector particles comprising any one of the polynucleotides of Aspects 1 to 11 and a pharmaceutically acceptable carrier or excipient. Aspect 20 provides the following: A method of treating a genetic disease or genetic disorder in a subject in need thereof, Administering to the subject an effective amount of an AAV vector comprising a muscle-specific regulatory cassette comprising at least three modified enhancer arrays located upstream of and operably linked to a promoter, thereby treating the genetic disease or the genetic disorder comprising wherein the three modified enhancer arrays comprise a Trex array, an AT-rich array, a right E-box array, and a MEF2 array wherein the right E-box array is repeated at least once wherein the promoter is a -80 to +50 promoter containing a consensus Inr sequence the method Aspect 21 provides the following: The method according to aspect 20, wherein the cassette is encoded by a nucleic acid comprising the sequence shown in SEQ ID NO:1 Aspect 22 provides the following: A method of treating a genetic disease or genetic disorder in a subject in need thereof comprising administering to the subject an effective amount of an AAV vector comprising a polynucleotide encoding a muscle-specific regulatory cassette comprising at least three modified enhancer arrays located upstream of and operably linked to a promoter comprising wherein the at least three modified enhancer arrays comprise a Trex array, an AT-rich array, a left E-box array, a right E-box array, and a MEF2 array wherein the promoter is a -80 to +50 promoter containing a consensus Inr sequence the method Aspect 23 provides the following: The method according to aspect 22, wherein the cassette is encoded by a nucleic acid comprising the sequence shown in SEQ ID NO:2 Aspect 24 provides the following: The method according to any one of aspects 20 to 23, wherein the AAV vector comprises an AAV capsid of a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, AAV-B1, AAV-DJ, AAV-Retro, AAVrh8, AAVrh10, AAVrh25, Anc80L65, LK03, AAVrh18, rAAVrh74, AAVrh32.33, AAVrh39, AAVrh43, MyoAAV2, Oligo001, PHP-B, and Spark100. Aspect 25 provides the following: The method according to aspect 24, wherein the capsid is of the rAAVrh74 serotype. Aspect 25 provides the following: The method according to aspect 24, wherein the capsid is of the AAV9 serotype. Aspect 26 provides the following: The method according to aspect 24, wherein the capsid is of the MyoAAV2 serotype. Aspect 27 provides the following: The method according to any one of aspects 20 to 23, wherein the AAV vector has specificity for muscle tissue. Aspect 28 provides the following: The method according to any one of aspects 20 to 26, wherein the genetic disease or genetic disorder is a muscle-related genetic disease or genetic disorder. Aspect 29 provides the following: The method according to aspect 28, wherein the muscle-related genetic disease or genetic disorder is selected from the list consisting of facioscapulohumeral muscular dystrophy (FSHD), X-linked myotubular myopathy (XLMTM), central core myopathy, inclusion body myositis, nemaline myopathy, distal myopathy, centronuclear myopathy, oculopharyngeal muscular dystrophy, dysferlinopathy, limb-girdle muscular dystrophy (LGMD), and Duchenne muscular dystrophy (DMD). Aspect 30 provides the following: A method of introducing a transgene into a target cell, comprising: contacting the cells with a polynucleotide encoding a muscle-specific regulatory cassette comprising at least three modified enhancer sequences located upstream of and operably linked to a promoter comprising wherein the three modified enhancer sequences comprise a Trex sequence, an AT-rich sequence, a right E-box sequence, and an MEF2 sequence wherein the right E-box sequence is repeated at least once wherein the promoter is a -80 to +50 promoter containing a consensus Inr sequence the method Aspect 31 provides the following: The method according to aspect 30, wherein the cassette has high expression activity in skeletal muscle tissue Aspect 32 provides the following: The method according to aspect 30, wherein the skeletal muscle tissue is fast muscle tissue Aspect 33 provides the following: The method according to aspect 30, wherein the cassette has very low expression in heart tissue compared to skeletal muscle tissue Aspect 34 provides the following: The method according to aspect 30, wherein the cassette is encoded by a nucleic acid comprising the sequence shown in SEQ ID NO:1 Aspect 35 provides the following: The method according to aspect 30, wherein the cassette is encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, and at least 99% sequence identity to the sequence shown in SEQ ID NO:1 Aspect 36 provides the following: A method of introducing a transgene into a target cell, comprising contacting the cells with a polynucleotide encoding a muscle-specific regulatory cassette comprising at least three modified enhancer sequences located upstream of and operably linked to a promoter comprising The at least three modified enhancer arrays include a Trex array, an AT-rich array, a left E-box array, a right E-box array, and an MEF2 array, The promoter is a -80 to +50 promoter containing a consensus Inr array, The method. Aspect 37 provides the following: The method according to aspect 36, wherein the cassette has high expression activity in skeletal muscle tissue. Aspect 38 provides the following: The method according to aspect 36, wherein the cassette has low expression activity in heart tissue compared to skeletal muscle tissue. Aspect 39 provides the following: The method according to aspect 36, wherein the cassette is encoded by a nucleic acid comprising the sequence shown in SEQ ID NO:2. Aspect 40 provides the following: The method according to aspect 36, wherein the cassette is encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, and at least 99% sequence identity to the sequence shown in SEQ ID NO:2.

[0173] Other aspects The recitation of a list of elements in any definition of a variable term herein includes that definition of the variable term as any single element or combination (or partial combination) of the listed elements. The recitation of an aspect herein includes that aspect as any single aspect or in combination with any other aspect or part thereof.

[0174] The disclosure of each and every patent, patent application, and publication cited herein is hereby incorporated by reference in its entirety. Although the invention has been disclosed with respect to specific embodiments, it is apparent that other embodiments and variations of the invention may be devised by those skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

1. A polynucleotide encoding a muscle-specific regulatory cassette comprising at least three modified enhancer arrays located upstream of and functionally linked to a promoter, wherein the three modified enhancer arrays include a Trex array, an AT-rich array, a right E-box array, and a MEF2 array, the right E-box array repeats at least once, the promoter is an -80 to +50 promoter containing a consensus Inr sequence, the polynucleotide.

2. The polynucleotide according to claim 1, wherein the cassette has high expression activity in skeletal muscle tissue.

3. The polynucleotide according to claim 2, wherein the skeletal muscle tissue is fast muscle tissue.

4. The polynucleotide according to claim 1, wherein the cassette has very low expression in heart tissue compared to skeletal muscle tissue.

5. The polynucleotide according to claim 1, wherein the cassette is encoded by a nucleic acid comprising the sequence shown in SEQ ID NO:

1.

6. The polynucleotide according to claim 1, wherein the cassette is encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, and at least 99% sequence identity with the sequence shown in SEQ ID NO:

1.

7. A polynucleotide encoding a muscle-specific regulatory cassette comprising at least three modified enhancer arrays located upstream of and functionally linked to a promoter, wherein the at least three modified enhancer arrays include a Trex array, an AT-rich array, a left E-box array, a right E-box array, and a MEF2 array, the promoter is an -80 to +50 promoter containing a consensus Inr sequence, the polynucleotide.

8. The polynucleotide according to claim 7, wherein the cassette has high expression activity in skeletal muscle tissue.

9. The polynucleotide according to claim 7, wherein the cassette has low expression activity in heart tissue compared to skeletal muscle tissue.

10. The polynucleotide according to claim 7, wherein the cassette is encoded by a nucleic acid comprising the sequence shown in SEQ ID NO:

2.

11. The polynucleotide according to claim 7, wherein the cassette is encoded by a nucleic acid comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, and at least 99% sequence identity with the sequence shown in SEQ ID NO:

2.

12. An AAV vector comprising a muscle-specific regulatory cassette, comprising the polynucleotide according to any one of claims 1 to 6.

13. An AAV vector comprising a muscle-specific regulatory cassette, comprising the polynucleotide according to any one of claims 7 to 11.

14. The AAV vector according to any one of claims 12 to 13, comprising an AAV capsid that is a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, AAV-B1, AAV-DJ, AAV-Retro, AAVrh8, AAVrh10, AAVrh25, Anc80L65, LK03, AAVrh18, rAAVrh74, AAVrh32.33, AAVrh39, AAVrh43, MyoAAV2, Oligo001, PHP-B, and Spark100.

15. The AAV vector according to claim 14, wherein the capsid is of the rAAVrh74 serotype.

16. The AAV vector according to claim 14, wherein the capsid is of the AAV9 serotype.

17. The AAV vector according to claim 14, wherein the capsid is of the MyoAAV2 serotype.

18. The AAV vector according to claim 14, wherein the capsid has specificity for muscle tissue.

19. A composition comprising AAV vector particles comprising the polynucleotide according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier or excipient.

20. A method of treating a genetic disease or genetic disorder in a subject in need thereof, administering to the subject an effective amount of an AAV vector comprising a muscle-specific regulatory cassette comprising at least three modified enhancer sequences located upstream of and functionally linked to a promoter, thereby treating the genetic disease or the genetic disorder. comprising the three modified enhancer sequences comprising a Trex sequence, an AT-rich sequence, a right E-box sequence, and a MEF2 sequence, the right E-box sequence repeating at least once The promoter is a -80 to +50 promoter containing a consensus Inr sequence. The method.

21. The method according to claim 20, wherein the cassette is encoded by a nucleic acid comprising the sequence shown in SEQ ID NO:

1.

22. A method for treating a genetic disease or genetic disorder in a subject in need thereof, administering to the subject an effective amount of an AAV vector comprising a muscle-specific regulatory cassette comprising at least three modified enhancer sequences located upstream of and operably linked to a promoter comprising the at least three modified enhancer sequences comprising a Trex sequence, an AT-rich sequence, a left E-box sequence, a right E-box sequence, and a MEF2 sequence, the promoter being a -80 to +50 promoter containing a consensus Inr sequence. The method.

23. The method according to claim 22, wherein the cassette is encoded by a nucleic acid comprising the sequence shown in SEQ ID NO:

2.

24. The method according to any one of claims 20 to 23, wherein the AAV vector comprises an AAV capsid of a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, AAV-B1, AAV-DJ, AAV-Retro, AAVrh8, AAVrh10, AAVrh25, Anc80L65, LK03, AAVrh18, rAAVrh74, AAVrh32.33, AAVrh39, AAVrh43, MyoAAV2, Oligo001, PHP-B, and Spark100.

25. The method according to claim 24, wherein the capsid is of the rAAVrh74 serotype.

26. The method according to claim 24, wherein the capsid is of the AAV9 serotype.

27. The method according to claim 24, wherein the capsid is of the MyoAAV2 serotype.

28. The method according to any one of claims 20 to 27, wherein the AAV vector has specificity for muscle tissue.

29. The method according to any one of claims 20 to 27, wherein the genetic disease or genetic disorder is a muscle-related genetic disease or genetic disorder.

30. The method according to claim 29, wherein the muscle-related genetic disease or genetic disorder is selected from the list consisting of facioscapulohumeral muscular dystrophy (FSHD), X-linked myotubular myopathy (XLMTM), central core myopathy, inclusion body myositis, nemaline myopathy, distal myopathy, centronuclear myopathy, oculopharyngeal muscular dystrophy, dysferlinopathy, limb-girdle muscular dystrophy (LGMD), and Duchenne muscular dystrophy (DMD).

31. A method for introducing a transgene into a target cell, comprising: contacting the cell with a polynucleotide encoding a muscle-specific regulatory cassette comprising at least three modified enhancer sequences located upstream of and functionally linked to a promoter wherein: the three modified enhancer sequences include a Trex sequence, an AT-rich sequence, a right E-box sequence, and a MEF2 sequence; the right E-box sequence is repeated at least once; the promoter is a -80 to +50 promoter containing a consensus Inr sequence; the method.

32. The method according to claim 31, wherein the cassette has high expression activity in skeletal muscle tissue.

33. The method according to claim 32, wherein the skeletal muscle tissue is fast muscle tissue.

34. The method according to claim 31, wherein the cassette has very low expression in cardiac tissue compared to skeletal muscle tissue.

35. The method according to claim 31, wherein the cassette is encoded by a nucleic acid comprising the sequence shown in SEQ ID NO:

1.

36. The method according to claim 31, wherein the cassette is encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, and at least 99% sequence identity to the sequence shown in SEQ ID NO:

1.

37. A method for introducing a transgene into a target cell, comprising: contacting the cell with a polynucleotide encoding a muscle-specific regulatory cassette comprising at least three modified enhancer sequences located upstream of and functionally linked to a promoter wherein: the at least three modified enhancer sequences include a Trex sequence, an AT-rich sequence, a left E-box sequence, a right E-box sequence, and a MEF2 sequence; The promoter is a -80 to +50 promoter containing a consensus Inr sequence. The method. Claim 38 The method according to claim 37, wherein the cassette has high expression activity in skeletal muscle tissue. Claim 39 The method according to claim 37, wherein the cassette has low expression activity in heart tissue as compared to skeletal muscle tissue. Claim 40 The method according to claim 37, wherein the cassette is encoded by a nucleic acid comprising the sequence shown in SEQ ID NO:

2. Claim 41 The method according to claim 37, wherein the cassette is encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, and at least 99% sequence identity with the sequence shown in SEQ ID NO: 2.

Images