Engineered nucleic acid regulatory elements and methods of use thereof

Abstract

The present invention relates to nucleic acid expression cassettes engineered to enhance gene expression. Vectors and methods are provided that utilize expression cassettes containing novel chimeric regulatory elements. The present invention is particularly useful for delivering transgenes to target cells, conferring desirable properties for muscle gene therapy. Furthermore, the present invention relates to gene therapy methods for delivering therapeutic agents to treat various disorders.

Description

[Technical Field]

[0001] Sequence Listing Reference The Sequence Listing submitted as an xml file named 38013_0032P1_SEQLIST.xml, created on October 11, 2023, and having a size of 105,583 bytes, is incorporated herein by reference pursuant to 37 CFR §§ 1.831-1.835.

[0002] 1. Field of the Invention The present invention relates to engineered nucleic acid regulatory elements that enhance gene expression, methods for using the regulatory elements, and their uses. The use of engineered regulatory elements upstream of a transgene delivered to a target cell can confer desired properties, in some cases desirable for gene therapy. In particular, the present invention provides nucleic acid regulatory elements operably linked to a heterologous gene (transgene) inserted into an expression cassette such that the regulatory element drives transgene expression in specific cells. Accordingly, the present invention also provides methods for tissue targeting, particularly in muscle, where expression cassettes containing the engineered regulatory elements improve transgene expression and systemically deliver therapeutic agents for the treatment of various disorders. [Background technology]

[0003] 2.Background The use of regulatory elements to drive gene expression is highly complex. Both naturally occurring and synthetic regulatory elements, such as enhancers and promoters, have been described in the art. It is unknown whether multiple elements engineered for heterologous gene expression will produce individual aberrant, unstable, and / or competing transcripts in a given tissue environment.

[0004] Highly productive and stable gene expression vectors may be suitable for gene therapy. Transgenes delivered by AAV or other viral vectors aim to provide long-term gene expression, thereby increasing the systemic expression level or serum half-life of biopharmaceutical transgenes. Therefore, improved gene expression systems for gene therapy would be of great benefit to patients compared with direct injection of biopharmaceuticals, for example, in enzyme replacement therapy. Although AAV capsid proteins carrying genomic DNA can confer specific tissue tropism for delivering DNA to target cells, due to their low immunogenicity, it is desirable to express genes of interest in greater amounts in the liver (Pastore, et al. Human Gene Therapy Vol. 10(11):1773-81, July 1999).

[0005] Therefore, good expression in muscle is desirable for systemic delivery of biopharmaceuticals. Muscle is a frequent target for gene therapy. However, muscle diseases vary significantly in terms of etiology and specific affected tissues. Therefore, expanding the toolbox of available muscle promoters with unique properties is desirable to improve the efficacy of current-generation muscle-directed gene therapies. Notably, because muscle-targeted gene therapy typically utilizes high AAV doses, stronger muscle promoters with low off-target activity would be highly valuable as a means to reduce the clinically effective dose. There remains a need for highly productive tissue-targeted gene expression and vectors in skeletal muscle. Summary of the Invention

[0006] 3. Summary of the Invention Provided are recombinant expression cassettes comprising composite nucleic acid regulatory elements for enhancing or regulating gene expression in the liver and in skeletal muscle, or in skeletal muscle or cardiac muscle. In embodiments, liver and / or cardiac tissues are detargeted. The regulatory element is a complex comprising at least one enhancer and at least one promoter operably linked to a transgene.

[0007] Provided are recombinant expression cassettes comprising composite nucleic acid regulatory elements, the elements comprising one or more promoters arranged in tandem, including, in embodiments, two or more promoters (i.e., composite promoters), where the composite promoters are muscle-specific promoters, including those in Table 1. In embodiments, the composite nucleic acid regulatory element comprises a CK promoter. In embodiments, the composite nucleic acid regulatory element comprises an ACTA core promoter in Table 1 (any one of the nucleotide sequences of SEQ ID NOS: 10-13).

[0008] The composite nucleic acid regulatory element comprises one or more enhancer elements, and in embodiments, two or more enhancer elements. At least one of the enhancer elements may also be muscle-specific. In embodiments, the composite nucleic acid regulatory element comprises one or two muscle-specific cis-regulatory elements (CREs) or Mus CREs. In embodiments, the Mus CRE may be Mus022 (SEQ ID NO: 10), Mus007 (SEQ ID NO: 11), Mus011 (SEQ ID NO: 12), or Mus035 (SEQ ID NO: 13) (see Table 1). In embodiments, the composite nucleic acid regulatory element comprises one or two muscle-specific regulatory elements or enhancers comprising or consisting of the nucleotide sequence of SEQ ID NO: 4, 5, 6, or 7.

[0009] In embodiments, the composite nucleic acid regulatory element comprises or consists of Mus CRE and the CK promoter, e.g., Mus CRE is 5' to the CK promoter. In embodiments, the composite regulatory element is any one of the nucleotide sequences comprising or consisting of SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NOs: 27-54. In embodiments, the composite nucleic acid regulatory element comprises or consists of any one of the nucleotide sequences of SEQ ID NOs: 1, 9, 27-54, 68, or 69.

[0010] In some embodiments, provided is an expression cassette comprising the composite nucleic acid regulatory element operably linked to a transgene. The transgene can be any one of the genes or nucleic acids encoding a therapeutic protein, including, but not limited to, those listed in Tables 4A, 4B, and 4C. In certain embodiments, the transgene encodes a therapeutic antibody having full-length heavy and light chains, or an antigen-binding fragment, e.g., a Fab fragment or scFv. In embodiments, the expression cassette is flanked by AAV ITR sequences and can be present in a cis-plasmid construct for AAV particle production or in an artificial genome within an AAV capsid.

[0011] In some embodiments, provided is a vector comprising a transgene operably linked to a composite nucleic acid regulatory element comprising or consisting of a nucleic acid sequence having a muscle CRE (e.g., see Table 1) 5' to a muscle-specific promoter or two tandem promoters. In embodiments, the muscle-specific promoter is a CK promoter, mSyn100 promoter, SPc5.12 promoter, or a variant thereof, such as SPc5v2 (see WO 2023 / 178053), or an ACTA core promoter. In embodiments, the composite nucleic acid regulatory element is Mus022.CK, including an element having a nucleotide sequence comprising or consisting of SEQ ID NO:9, and the transgene is expressed in skeletal muscle. In certain embodiments, the transgene is expressed at a lower level in cardiac or cardiac tissue compared to skeletal muscle. In certain embodiments, the transgene is expressed at a higher level in cardiac or cardiac tissue compared to skeletal muscle. In other embodiments, the transgene is expressed at a lower level in liver compared to muscle, including skeletal muscle.

[0012] Provided are methods for enhancing transgene expression, the methods comprising delivery of a viral vector containing a nucleic acid construct comprising or consisting of a recombinant expression cassette provided herein. Also provided are methods for enhancing transgene expression, the methods comprising delivery of a viral vector containing a nucleic acid construct having the following components: (1) AAV inverted terminal repeats (ITRs) on either side of the expression cassette, (2) control elements comprising: a) one or more regulatory elements in combination with one or more promoters, including embodiments in which the control elements comprise or consist of at least one or more enhancers of any one of SEQ ID NOS: 10-13, b) a polyA signal, and c) optionally an intron, and 3) a transgene providing (e.g., encoding) one or more RNA or protein products of interest.

[0013] Provided are methods of using nucleic acid constructs comprising or consisting of the recombinant expression cassettes provided herein. Also provided are methods of using nucleic acid constructs having the following components: (1) AAV inverted terminal repeats (ITRs) on either side of the expression cassette, (2) control elements comprising: a) one or more regulatory elements comprising at least one enhancer of any one of SEQ ID NOS: 10-13 in combination with a CK promoter, b) a polyA signal, and c) optionally an intron, and 3) a transgene providing (e.g., encoding) one or more RNA or protein products of interest, e.g., those in Tables 4A, 4B, and 4C. Also provided are methods of using nucleic acid constructs having the following components: (1) AAV inverted terminal repeats (ITRs) on either side of the expression cassette, (2) control elements including: a) one or more regulatory elements including the Mus022 enhancer (SEQ ID NO: 10) in combination with the CK promoter (SEQ ID NO: 8), b) a polyA signal, and c) optionally an intron, and 3) a transgene providing (e.g., encoding) one or more RNA or protein products of interest, e.g., those in Tables 4A, 4B, and 4C.

[0014] Also provided are methods of using nucleic acid constructs having the following components: (1) AAV inverted terminal repeats (ITRs) on either side of the expression cassette; (2) control elements including: a) one or more regulatory elements including one or more promoters comprising or consisting of any one of SEQ ID NOS: 14-26, alone or in combination with one or more enhancers; b) a polyA signal; and c) optionally an intron; and 3) a transgene providing (e.g., encoding) one or more RNA or protein products of interest. Also provided are methods of using nucleic acid constructs having the following components: (1) AAV inverted terminal repeats (ITRs) on either side of the expression cassette; (2) control elements including: a) one or more regulatory elements including at least one of the ACTA core promoters of any one of SEQ ID NOS: 14-26, alone or in combination with one or more enhancers of any one of SEQ ID NOS: 4-7; b) a polyA signal; and c) optionally an intron; and 3) a transgene providing (e.g., encoding) one or more RNA or protein products of interest, e.g., those in Tables 4A, 4B, and 4C.

[0015] In some embodiments, provided are viral vectors incorporating the recombinant expression cassettes described herein, including recombinant AAV (rAAV).

[0016] In another aspect, methods of treatment by delivery of an rAAV containing a nucleic acid expression cassette described herein are also provided. Provided are methods of treating a disease or disorder, including but not limited to those outlined in Tables 4A and 4B, in a subject in need thereof, the method comprising administering recombinant AAV particles containing an expression cassette comprising the composite nucleic acid regulatory sequence of any one of SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:68, SEQ ID NO:69, or SEQ ID NOs:27-54.

[0017] Also provided is a method for producing a recombinant AAV vector, the method comprising: 1) culturing in culture host cells a) an artificial genome comprising an expression cassette comprising any one of the composite regulatory elements of Table 1, e.g., SEQ ID NO: 9, or SEQ ID NOs: 27-54, or SEQ ID NOs: 68-69, operably linked to a transgene, flanked by AAV ITRs, and b) a trans expression cassette lacking AAV ITRs, encoding AAV rep and AAV capsid proteins operably linked to expression control elements that drive expression of and provide AAV rep and AAV capsid proteins in trans, and c) sufficient adenovirus helper functions to enable replication and packaging of the artificial genome by the AAV capsid proteins; and 2) recovering from the cell culture recombinant AAV encapsidated with the artificial genome. Also provided is a host cell for producing a recombinant AAV comprising an artificial genome comprising an expression cassette described herein.

[0018] The present invention is illustrated below by examples describing the structure and function of gene cassettes engineered with composite regulatory elements designed based on several liver-specific enhancers and promoters in tandem, including muscle-specific enhancers and promoters, with downstream elements modified at their translation start sites.

[0019] 3.1 Implementation Embodiment 1. A recombinant expression cassette comprising a) a muscle cis-regulatory element (CRE), and b) a composite nucleic acid regulatory element comprising a CK promoter, an Spc5-12 promoter, or a variant thereof, or an ACTA core promoter, operably linked to a transgene.

[0020] Embodiment 2. The recombinant expression cassette of embodiment 1, wherein the muscle CRE is Mus022 (SEQ ID NO: 10), Mus077 (SEQ ID NO: 11), Mus011 (SEQ ID NO: 12), or Mus035 (SEQ ID NO: 13).

[0021] Embodiment 3. The recombinant expression cassette of embodiment 2, wherein said muscle CRE is Mus022 (SEQ ID NO: 10).

[0022] Embodiment 4. The recombinant expression cassette of any one of embodiments 1 to 3, wherein the composite nucleic acid regulatory element is Mus022.CK (SEQ ID NO: 9).

[0023] Embodiment 5. The recombinant expression cassette of embodiment 1, wherein the muscle CRE is eMCK (SEQ ID NO: 4), seMCK (SEQ ID NO: 5), mSYN100E (SEQ ID NO: 6), or SPc5v2 (SEQ ID NO: 7).

[0024] Embodiment 6. The recombinant expression cassette of any one of embodiments 1 to 3 or 5, wherein the ACTA promoter is mmACTA1 (SEQ ID NO: 14), mmACTA2 (SEQ ID NO: 15), mmACTA3 (SEQ ID NO: 16), mmACTA4 (SEQ ID NO: 17), huACTA1 (SEQ ID NO: 18), huACTA2 (SEQ ID NO: 19), huACTA3 (SEQ ID NO: 20), mmACTA2-shortUTR (SEQ ID NO: 21), mmACTA2-midUTR (SEQ ID NO: 22), huACTA2-shortUTR (SEQ ID NO: 23), huACTA2-midUTR (SEQ ID NO: 24), mmACTA3-shortUTR (SEQ ID NO: 25), or huACTA3-midUTR (SEQ ID NO: 26).

[0025] Embodiment 7. A recombinant expression cassette comprising a composite nucleic acid regulatory element comprising or consisting of a nucleic acid sequence that a) has 99%, 95%, 90%, 85%, or 80% sequence identity to any one of SEQ ID NOs: 9, 27-54, 68, and 69, and / or b) has 1 to 10 nucleotide substitutions compared to any one of SEQ ID NOs: 9, 27-54, 68, and 69, wherein the composite nucleic acid regulatory element retains the biological activity of any one of SEQ ID NOs: 9, 27-54, 68, and 69.

[0026] Embodiment 8. The recombinant expression cassette of embodiment 7, wherein the composite nucleic acid regulatory element comprises any one of the nucleotide sequences of SEQ ID NOs: 9, 27-54, 68, or 69.

[0027] Embodiment 9. The recombinant expression cassette of embodiment 8, wherein the composite nucleic acid regulatory element consists of any one of the nucleotide sequences of SEQ ID NOs: 9, 27-54, 68, or 69.

[0028] Embodiment 10. The recombinant expression cassette of any one of embodiments 1 to 9, further comprising an intron sequence between the composite nucleic acid regulatory element and the transgene.

[0029] Embodiment 11. The recombinant expression cassette of any one of Embodiments 1-10, wherein the transgene is a gene or nucleic acid encoding a therapeutic agent listed in Tables 4A-4C.

[0030] Embodiment 12. The recombinant expression cassette of embodiment 11, wherein the transgene is a muscle-derived protein.

[0031] Embodiment 13. The recombinant expression cassette of embodiment 11, wherein the transgene is a mini-dystrophin gene or a micro-dystrophin gene.

[0032] Embodiment 14. The recombinant expression cassette of embodiment 13, wherein the microdystrophin is Dys1 (SEQ ID NO: 73), Dys3 (SEQ ID NO: 74), Dys5 (SEQ ID NO: 75), human MD1(R4-R23 / ΔCT) (SEQ ID NO: 76), human microdystrophin (SEQ ID NO: 77), Dys3978 (SEQ ID NO: 78), human MD3 (SEQ ID NO: 79), or human MD4 (SEQ ID NO: 80).

[0033] Embodiment 15. The recombinant expression cassette of any one of embodiments 1 to 10, wherein the transgene is a gene or nucleic acid encoding a therapeutic antibody, or antigen-binding fragment thereof, listed in Table 4B.

[0034] Embodiment 16. A vector comprising the recombinant expression cassette according to any one of embodiments 1 to 15.

[0035] Embodiment 17. The vector of embodiment 16, further comprising AAV ITRs on both sides of the expression cassette.

[0036] Embodiment 18. The vector of embodiment 16 or embodiment 17, wherein the recombinant expression cassette is suitable for packaging into an ssAAV or scAAV vector.

[0037] Embodiment 19. The vector according to any one of embodiments 16 to 18, and AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, AAV-16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV. rAAV particles comprising capsid proteins derived from an AAV capsid serotype selected from AV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or a derivative, modification, or pseudotype thereof.

[0038] Embodiment 20. A method of enhancing expression of a transgene in a subject, comprising delivering a viral vector comprising a recombinant expression cassette comprising a composite nucleic acid regulatory element comprising, in 5' to 3' configuration: a) a Mus022 sequence; b) at least one muscle-specific promoter; c) a transgene; and d) a polyadenylation signal sequence.

[0039] Embodiment 21. The method of embodiment 20, wherein the muscle-specific promoter is the CK promoter (SEQ ID NO: 8).

[0040] Embodiment 22 The method of embodiment 20 or embodiment 21, wherein the viral vector is administered intravenously or intramuscularly.

[0041] Embodiment 23. The method of any one of embodiments 20 to 22, wherein expression of the transgene is enhanced in the blood circulation or systemically.

[0042] Embodiment 24. The method of any one of embodiments 20 to 23, wherein expression of the transgene is enhanced in the liver or skeletal muscle.

[0043] Embodiment 25. A method of treatment comprising delivering an rAAV comprising a recombinant expression cassette described in any one of embodiments 1 to 15, or a vector described in any one of embodiments 16 to 18, or delivering an rAAV described in embodiment 19. [Brief explanation of the drawings]

[0044] 4. Brief description of the drawings [Figure 1] Arranged from 5' to 3' are: 5'-ITR, muscle CRE (Mus CRE), CK promoter, optionally an intron, gene of interest, polyadenylation (polyA) sequence, and a depiction of the AAV genome cassette of 5'-ITR. [Figure 2A] Arranged from 5' to 3' are: 5'-ITR, muscle CRE (Mus CRE), CK promoter, intron, eGFP transgene, barcode sequence, polyadenylation (polyA) sequence, and depiction of the reporter gene cassette in 3'-ITR. [Figure 2B] Representative photomicrographs of each CRE candidate cassette transfected into differentiated C2C12 cells showing eGFP expression (fluorescence). [Figure 2C]Data graph showing fold change in CRE activity for each plasmid normalized to a control plasmid (CK promoter not containing an upstream CRE). [Figure 2D] Relative promoter activity, e.g., the relative abundance of transgene RNA transcripts versus the relative abundance of DNA in C2C12 cells, is shown. [Figure 3] Normalized promoter activity is shown in mouse tissues: liver, tibialis anterior (TA), gastrocnemius (GAS), and heart. [Figure 4A] Copies of micro-dystrophin transcripts per copy of TBP in mouse tissues: gastrocnemius muscle (GAS), tibialis anterior muscle (TA), liver, and heart. [Figure 4B] Representative DNA biodistribution of CK7 compared to Mus022.CK vector. [Figure 4C] Representative immunofluorescence showing transgene expression in cardiac tissue. [Figure 4D] Representative immunofluorescence showing transgene expression in GAS tissues. [Figure 5] Table 1. Representative AAV genomic cassettes showing various composite nucleic acid regulatory element (enhancer / promoter) combinations. [Figure 6] Table 1. Representative AAV genomic cassettes showing various composite nucleic acid regulatory element (enhancer / promoter) combinations and their approximate promoter lengths in base pairs (bp). [Figure 7] Table 1. Representative AAV genomic cassettes showing various composite nucleic acid regulatory element (enhancer / promoter) combinations. [Figure 8] Representative bar graphs showing promoter activity of the eMCK.mmACTA2shortUTR(EMP1), SPC5v2.mmACTA2shortUTR(EMP2), and eMCK.SPC5v2.mmACTA2shortUTR(EMP3) promoters compared to CK7 and spc5-12 (A) or data normalized compared to CK7 (B and C). [Figure 9A]Representative bar graph showing promoter activity in gastrocnemius muscle (GAS) of mouse tissues identified by relative abundance of RNA versus DNA for promoters listed in Table 6. [Figure 9B] Representative bar graph showing promoter activity in quadriceps (Quad) muscle of mouse tissues identified by relative abundance of RNA vs. DNA for promoters listed in Table 6. [Figure 9C] Representative bar graph showing promoter activity in mouse tissues identified by relative abundance of RNA versus DNA for promoters listed in Table 6, tibialis anterior (TA). [Figure 9D] Representative bar graph showing promoter activity in mouse tissue diaphragm (DIA) identified by relative abundance of RNA vs. DNA for promoters listed in Table 6. [Figure 9E] Representative bar graph showing promoter activity in the heart of mouse tissues identified by relative abundance of RNA vs. DNA for promoters listed in Table 6. [Figure 9F] Representative bar graph showing promoter activity in liver of mouse tissues identified by relative abundance of RNA vs. DNA for promoters listed in Table 6. [Figure 10A] Representative bar graph showing promoter activity in monkey tissue gastrocnemius (GAS) identified by relative abundance of RNA versus DNA for promoters listed in Table 6. [Figure 10B] Representative bar graph showing promoter activity in monkey tissue quadriceps (Quad) identified by relative abundance of RNA vs. DNA for promoters listed in Table 6. [Figure 10C] Representative bar graph showing promoter activity in monkey tissue tibialis anterior (TA) muscle, identified by relative abundance of RNA versus DNA for promoters listed in Table 6. [Figure 10D] Representative bar graph showing promoter activity in monkey tissue diaphragm (DIA) identified by relative abundance of RNA vs. DNA for promoters listed in Table 6. [Figure 10E]Representative bar graph showing promoter activity in monkey tissue heart, identified by relative abundance of RNA vs. DNA, for promoters listed in Table 6. [Figure 10F] Representative bar graph showing promoter activity in liver of monkey tissues identified by relative abundance of RNA vs. DNA for promoters listed in Table 6. DETAILED DESCRIPTION OF THE INVENTION

[0045] 5. Detailed Description Provided are unique combinations of promoter and enhancer sequences in expression cassettes suitable for improving transgene expression while maintaining or conferring tissue specificity. Provided are vectors, e.g., viral vectors, incorporating the recombinant expression cassettes described herein, including rAAV for therapeutic use, as well as methods and host cells for producing the same. Novel regulatory element nucleic acids have been generated to improve and target transgene expression. Ultimately, these designs may improve the therapeutic efficacy of gene transfer by providing more robust levels of transgene expression, improved stability / persistence, and reduced expression in undesirable tissues, which may allow for lower doses. Provided are composite regulatory elements that promote expression in skeletal muscle with minimal expression in cardiac or myocardial tissue or liver tissue, or, in embodiments, high expression in myocardial tissue.

[0046] 5.1.Definition The term "regulatory element" or "nucleic acid regulatory element" refers to a non-coding nucleic acid sequence that controls transcription of adjacent genes. Cis-regulatory elements typically regulate gene transcription by binding transcription factors. This includes "composite nucleic acid regulatory elements" that contain multiple enhancer or promoter elements as described herein.

[0047] The term "expression cassette" or "nucleic acid expression cassette" refers to a nucleic acid molecule comprising one or more transcriptional control elements, including, but not limited to, promoters, enhancers and / or regulatory elements, introns, and polyadenylation sequences. The enhancers and promoters typically function to direct (trans)gene expression in one or more desired cell types, tissues, or organs.

[0048] The terms "operably linked" and "operably linked to" refer to linked, usually contiguous or substantially contiguous, nucleic acid sequences that connect two protein-coding regions, optionally contiguous and in reading frame. However, because enhancers generally function when separated by several kilobases from the promoter and because intron sequences can be of variable length, some polynucleotide elements can still function when operably linked and not immediately adjacent to the downstream promoter and transgene.

[0049] The term "AAV" or "adeno-associated virus" refers to a Dependoparvovirus within the Parvoviridae genus of viruses. The AAV may be derived from a naturally occurring "wild-type" virus, or may be derived from a rAAV genome packaged in a capsid containing capsid proteins encoded by a naturally occurring cap gene and / or from a rAAV genome packaged in a capsid containing capsid proteins encoded by a non-naturally occurring capsid cap gene. Examples of the latter include rAAVs having capsid proteins containing peptide insertions within or modifications of the amino acid sequence of the naturally occurring capsid.

[0050] The term "rAAV" refers to "recombinant AAV." In some embodiments, a recombinant AAV has an AAV genome in which some or all of the rep and cap genes have been replaced with heterologous sequences.

[0051] The term "rep-cap helper plasmid" refers to a plasmid that provides viral rep and cap gene functions and assists in the production of AAV from rAAV genomes that lack functional rep and / or cap gene sequences.

[0052] The term "cap gene" refers to a nucleic acid sequence that encodes a capsid protein that forms or helps form the capsid coat of a virus. In the case of AAV, the capsid protein can be VP1, VP2, or VP3.

[0053] The term "rep gene" refers to a nucleic acid sequence that encodes a nonstructural protein necessary for viral replication and production.

[0054] The terms "nucleic acid" and "nucleotide sequence" include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), combinations of DNA and RNA molecules or hybrid DNA / RNA molecules, and analogs of DNA or RNA molecules. Such analogs can be generated using nucleotide analogs, including, but not limited to, inosine or tritylated bases. Such analogs can also include DNA or RNA molecules containing modified backbones that confer beneficial attributes on the molecule, such as, for example, increased nuclease resistance or ability to cross cell membranes. The nucleic acid or nucleotide sequence can be single-stranded or double-stranded, and can contain both single-stranded and double-stranded portions, or can contain triple-stranded portions, but is preferably double-stranded DNA.

[0055] The terms "subject," "host," and "patient" are used interchangeably. As used herein, a subject is preferably a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans), most preferably a human.

[0056] The term "therapeutic agent" or "biopharmaceutical agent" refers to any agent that can be used in treating, managing, or ameliorating symptoms associated with a disease or disorder, where the disease or disorder is related to the function provided by the transgene. As used herein, a "therapeutically effective amount" refers to an amount of agent (e.g., the amount of product expressed by the transgene) that provides at least one therapeutic effect in the treatment or management of a target disease or disorder when administered to a subject suffering from the disease or disorder. Furthermore, a therapeutically effective amount with respect to an agent of the present invention means that the amount of agent, alone or in combination with other therapies, provides at least one therapeutic effect in the treatment or management of a disease or disorder.

[0057] The terms "muscle-specific" or "muscle-tropic" refer to nucleic acid elements that are adapted for their activity in muscle cells or tissues due to the interaction of the nucleic acid element with the intracellular environment of the muscle cell. Muscle cells include skeletal and cardiac muscles. Secretion of transgene products into muscle and / or the bloodstream can also be enhanced due to intramuscular expression in the presence of a muscle-specific promoter after various routes of administration, e.g., intravenous or intramuscular administration. Various therapeutic agents benefit from muscle-specific expression of a transgene, or from both muscle- and liver-specific expression of a transgene. Muscle production of a biopharmaceutical (e.g., produced by a delivered transgene) can also result in increased immune tolerance to the drug in the host compared to an equivalent amount of protein drug injected directly into the host.

[0058] Regulatory Elements One embodiment relates to nucleic acid regulatory elements that are chimeric with respect to the arrangement of tandem elements in an expression cassette. Regulatory elements generally have multiple functions: initiating or regulating transcription, coordinating with cell-specific machinery to drive expression upon signal transduction, and as recognition sites to enhance expression of downstream genes.

[0059] Provided are combined arrangements of nucleic acid regulatory elements that promote transgene expression in muscle (including skeletal muscle) tissue. In particular, certain elements are arranged with one or more copies of individual enhancer and promoter elements arranged in tandem and operably linked to a transgene to promote expression, particularly tissue-specific expression. Exemplary nucleic acid sequences of individual promoter and enhancer elements are provided in Table 1. Also provided in Table 1 are exemplary composite nucleic acid regulatory elements comprising promoter and enhancer elements, including one or two promoters and / or one or two enhancer elements. In certain embodiments, the downstream promoter is the ACTA core promoter (e.g., SEQ ID NOs: 14-26) or the CK promoter (SEQ ID NO: 8).

[0060] Thus, for muscle-specific expression, provided are nucleic acid regulatory elements that include or consist of a promoter and other nucleic acid elements, such as enhancers. In embodiments, the enhancers for muscle-specific expression include the muscle CRE, Mus022 (SEQ ID NO: 10), and also include Mus007 (SEQ ID NO: 11), Mus011 (SEQ ID NO: 12), and Mus035 (SEQ ID NO: 13). These may be included as a single copy or in tandem as two or more copies (or two different promoters).

[0061] The recombinant expression cassettes provided herein include: i) a) a Mus CRE, including a muscle-specific enhancer region, e.g., Mus022 (SEQ ID NO: 10); b) a muscle-specific promoter, including the CK promoter (SEQ ID NO: 8); and c) a composite nucleic acid regulatory element, optionally including an intron; and ii) a transgene to which the composite nucleic acid regulatory element is operably linked, as well as other regulatory elements, e.g., a polyadenylation signal. In some embodiments, the composite nucleic acid regulatory element comprises mus022.CK (SEQ ID NO: 9) in Table 1. In some embodiments, the composite nucleic acid regulatory element is operably linked to a transgene. The transgene can be any one of the genes or nucleic acids encoding a therapeutic protein, including, but not limited to, those listed in Tables 4A, 4B, and 4C. The transgene can also encode a therapeutic antibody, including a full-length antibody or an antigen-binding fragment, e.g., a Fab fragment.

[0062] Also provided is a nucleic acid regulatory element comprising a muscle CRE (comprising MusO22 (SEQ ID NO:10), MusO07 (SEQ ID NO:11), MusOl 1 (SEQ ID NO:12), or MusO35 (SEQ ID NO:13)) upstream (5') of the CK promoter or any other muscle-specific promoter (see Table 1), including, for example, Spc5-12, which, in embodiments, is operably linked to a transgene.

[0063] Provided are composite regulatory elements that enhance gene expression in skeletal and / or cardiac muscle and have 99%, 95%, 90%, 85%, or 80% sequence identity to SEQ ID NO:9 (Mus022.CK) and / or contain 1 to 10 nucleic acid substitutions while retaining the biological activity of the composite regulatory element. Also provided are composite regulatory elements that enhance gene expression in skeletal and / or cardiac muscle and comprise or consist of SEQ ID NO:9.

[0064] Provided are composite regulatory elements that enhance gene expression in skeletal and / or cardiac muscle and have 99%, 95%, 90%, 85%, or 80% sequence identity to any one of SEQ ID NOS: 27-54, 68, and 69 (composite enhancer / ACTA core promoter) and / or contain 1 to 10 nucleic acid substitutions while retaining the biological activity of the composite regulatory element. Also provided are composite regulatory elements that enhance gene expression in skeletal and / or cardiac muscle and comprise or consist of any one of SEQ ID NOS: 27-54, 69, and 69.

[0065] In the context of nucleic acid sequences, the terms "sequence identity," "percent sequence identity," or "percent identical" refer to the residues in two sequences that are the same when aligned for maximum correspondence. The length of sequence identity comparison can be over the entire length of a gene sequence or component shown or claimed in the sequence listing, e.g., an enhancer or promoter or composite promoter sequence, or over a fragment or portion thereof, e.g., a nucleotide sequence encoding a composite enhancer / ACTA core promoter. However, identity between smaller fragments, e.g., at least about 10 nucleotides, usually at least about 20-24 nucleotides, at least about 28-32 nucleotides, at least about 36 or more nucleotides, may also be desired. Similarly, "percent sequence identity" can be readily determined for amino acid sequences over the entire length of a protein, or a fragment or portion thereof, e.g., a transgene protein product or capsid protein.

[0066] Alignment is carried out using any of a variety of publicly or commercially available multiple sequence alignment programs, for example, Clustal W, which can be accessed via a web server on the Internet. There are several algorithms known in the art that can be used to measure nucleotide sequence identity, including those included in the programs described herein. Similar programs are also available for amino acid sequences, such as the Clustal X program (Soding, J. (2005) Bioinformatics 21,951-960; Thompson JD et al., (1994) Nucleic Acids Res.22(22):4673-4680; Larkin, MA et al. (2007) Bioinformatics,23,2947-2948). Generally, any of these programs can be used with default settings, but those skilled in the art can change these settings as needed. Alternatively, one skilled in the art can utilize another algorithm or computer program that provides a level of identity or alignment at least that provided by the referenced algorithms and programs (Higgins DG et al., (2005) PNAS USA; 102(30):10411-10412; Raghava and Barton (2006) BMC Bioinformatics, 7:415).

[0067] The terms "substantial homology" or "substantial similarity," when referring to a nucleic acid or a fragment thereof, indicate that when optimally aligned with another nucleic acid (or its complementary strand) by appropriate nucleotide insertion or deletion, there is at least about 95-99% nucleotide sequence identity in the aligned sequence. In some instances, the homology spans the full-length sequence or its open reading frame, e.g., promoter, enhancer, cap sequence, rep sequence, transgene, or another suitable fragment at least 15 nucleotides in length. Examples of suitable fragments are described herein.

[0068] The terms "substantial homology" or "substantial similarity," when referring to an amino acid or fragment thereof, indicate that when optimally aligned with another amino acid (or its complementary strand) by appropriate amino acid insertion or deletion, there is at least about 95-99% amino acid sequence identity in the aligned sequence. In some instances, the homology is over the full-length sequence, or over a protein thereof, e.g., a cap protein, a rep protein, a therapeutic protein (transgene product), or a fragment or portion thereof that is at least 8 amino acids, or at least 15 amino acids in length. Examples of suitable fragments are described herein.

[0069] In embodiments of the present invention, various regulatory elements and element combinations were used to design and generate nucleic acid expression cassettes, which are listed in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] [Table 1-15]

[0070] 5.2.1 Enhancers The present inventors have surprisingly discovered that multiple enhancers are suitable for tandem arrangement and simultaneously operably linked to one or more promoters, which enhancers, when arranged in tandem and operably linked to a promoter and a transgene, promote tissue-specific expression of the transgene.

[0071] Thus, provided are muscle creatine kinase (MCK) enhancers, particularly the eMCK enhancer (modified MCK enhancer) or seMCK enhancer (short modified MCK enhancer) of SEQ ID NO: 4 or 5. Also provided is mSYN100E (SEQ ID NO: 6). Also provided are muscle-specific enhancers, e.g., Mus022 (SEQ ID NO: 10). In embodiments, enhancers enhance muscle-specific expression of muscle CREs, e.g., Mus022 (SEQ ID NO: 10), which also includes Mus007 (SEQ ID NO: 11), Mus011 (SEQ ID NO: 12), and Mus035 (SEQ ID NO: 13).

[0072] In embodiments, the enhancer comprises the nucleic acid sequence of SEQ ID NO: 4, or a sequence that is 99%, 95%, 90%, 85%, or 80% identical to SEQ ID NO: 4 and / or contains 1 to 10 nucleic acid substitutions while retaining the biological activity of the enhancer. In embodiments, the enhancer comprises the nucleic acid sequence of SEQ ID NO: 5, or a sequence that is 99%, 95%, 90%, 85%, or 80% identical to SEQ ID NO: 5 and / or contains 1 to 10 nucleic acid substitutions while retaining the biological activity of the enhancer. In embodiments, the enhancer comprises the nucleic acid sequence of SEQ ID NO: 6, or a sequence that is 99%, 95%, 90%, 85%, or 80% identical to SEQ ID NO: 6 and / or contains 1 to 10 nucleic acid substitutions while retaining the biological activity of the enhancer. In embodiments, the enhancer comprises the nucleic acid sequence of SEQ ID NO: 10, or a sequence that is 99%, 95%, 90%, 85%, or 80% identical to SEQ ID NO: 10 and / or contains 1 to 10 nucleic acid substitutions while retaining the biological activity of the enhancer. In embodiments, the enhancer comprises the nucleic acid sequence of SEQ ID NO: 11, or a sequence that is 99%, 95%, 90%, 85%, or 80% identical to SEQ ID NO: 11 and / or contains 1 to 10 nucleic acid substitutions while retaining the biological activity of the enhancer. In embodiments, the enhancer comprises the nucleic acid sequence of SEQ ID NO: 12, or a sequence that is 99%, 95%, 90%, 85%, or 80% identical to SEQ ID NO: 12 and / or contains 1 to 10 nucleic acid substitutions while retaining the biological activity of the enhancer. In embodiments, the enhancer comprises the nucleic acid sequence of SEQ ID NO: 13, or a sequence that is 99%, 95%, 90%, 85%, or 80% identical to SEQ ID NO: 13 and / or contains 1 to 10 nucleic acid substitutions while retaining the biological activity of the enhancer.

[0073] In embodiments, the enhancer element comprises or consists of one or more of SEQ ID NOs: 4, 5, 6, 10, 11, 12, or 13.

[0074] Other enhancers are known to those of skill in the art.

[0075] 5.2.2 Promoters Another aspect of the present invention relates to nucleic acid expression cassettes containing chimeric regulatory elements designed to confer or enhance liver-specific and muscle-specific expression (including skeletal muscle-specific expression). The invention involves engineering tandem regulatory elements, including promoter elements, enhancer elements, and, optionally, introns. Examples include, but are not limited to, Spc5-12, or variants thereof, such as SPc5v2 (see WO 2023 / 178053, incorporated herein by reference in its entirety), or the syn100 promoter (e.g., SEQ ID NOS: 2, 3, 7, and 61), the CK promoter (SEQ ID NOS: 8), and the ACTA core promoter (SEQ ID NOS: 14-26).

[0076] Provided are promoter elements that enhance gene expression in skeletal muscle and have 99%, 95%, 90%, 85%, or 80% sequence identity to any one of SEQ ID NOs: 2, 3, 7, 14-26, and 61, and / or contain 1-10 nucleic acid substitutions while retaining the biological activity of the promoter. In embodiments, the promoter element comprises or consists of one or more of SEQ ID NOs: 2, 3, 7, 14-26, and 61.

[0077] The unique combinations of promoter and enhancer sequences provided herein improve transgene expression while maintaining tissue specificity. Novel regulatory element nucleic acids were generated using methods that improve transgene expression from tandem enhancer / promoter combinations while reducing expression in undesired cells or tissues and reducing the size of the promoter in the gene cassette. Ultimately, these designs aim to improve the therapeutic efficacy of gene transfer by providing more robust levels of transgene expression, improved stability / persistence, and lower doses via AAV delivery.

[0078] The CAG promoter (SEQ ID NO: 17) refers to a chimeric promoter constructed from the following sequences: cytomegalovirus (CMV) early enhancer element (C), chicken beta-actin promoter (the first exon and first intron of the chicken beta-actin gene) (A), and splice acceptor of rabbit beta-globin gene (G). The CAG promoter is frequently used in the art to drive high-level expression in mammalian cells, and is not tissue-specific and therefore is generally used as a general-purpose promoter. Other promoters that can be used in combination with the regulatory elements herein are well known to those skilled in the art.

[0079] 5.2.3 Introns Another aspect of the present invention relates to nucleic acid expression cassettes containing an intron within the regulatory cassette. In some embodiments, the intron nucleic acid is a chimeric intron derived from human β-globin and Ig heavy chain (also known as the β-globin splice donor / immunoglobulin heavy chain splice acceptor intron, or the β-globin / IgG chimeric intron; Reed, R., et al., Genes and Development, 1989). The use of an intron can further induce efficient splicing in eukaryotic cells. While the use of an intron may not result in increased expression relative to an already strong promoter, the presence of an intron can increase the expression level of the transgene and may also extend the duration of expression in vivo.

[0080] In some embodiments, the intron is a VH4 intron. The VH4 intron nucleic acid may comprise SEQ ID NO: 64, as shown in Table 2 below. A VH4 intron 5' of the coding sequence may enhance proper splicing and thus expression of the transgene. Thus, in some embodiments, the intron is attached to the 5' end of the transgene sequence. In other embodiments, the intron is less than 100 nucleotides in length. [Table 2]

[0081] In other embodiments, the intron is a chimeric intron derived from human β-globin and Ig heavy chain (also known as a β-globin splice donor / immunoglobulin heavy chain splice acceptor intron, or a β-globin / IgG chimeric intron) (Table 2, SEQ ID NO: 63). Other introns known to those skilled in the art may also be used, such as the chicken β-actin intron, minute virus of mice (MVM) intron, human factor IX intron (e.g., FIX split intron 1), β-globin splice donor / immunoglobulin heavy chain splice acceptor intron, adenovirus splice donor / immunoglobulin splice acceptor intron, and SV40 late splice donor / splice acceptor (19S / 16S) intron (Table 2, SEQ ID NO: 65).

[0082] Other introns known to those skilled in the art may also be used.

[0083] 5.2.4 Other regulatory elements 5.2.4.1 PolyA Another aspect of the present disclosure relates to an expression cassette comprising a polyadenylation (polyA) site downstream of the transgene coding region. Any polyA site that signals the end of transcription and directs the synthesis of a polyA tail is suitable for use in the AAV vectors of the present disclosure. Exemplary polyA signals include, but are not limited to, those derived from the SV40 late gene, rabbit β-globin gene (SEQ ID NO: 67), bovine growth hormone (BPH) gene, human growth hormone (hGH) gene, synthetic polyA (SPA) site, and bovine growth hormone (bGH) gene. See, for example, Powell and Rivera-Soto, 2015, Discov. Med., 19(102):49-57. In one embodiment, the polyA signal comprises SEQ ID NO: 66, as shown in Table 3. [Table 3]

[0084] 5.3. Gene Delivery Vectors Another aspect of the present invention relates to the genetic engineering of tandem nucleic acid regulatory elements and the incorporation of these nucleic acid sequences into vector expression systems. In one embodiment, the vector is a recombinant adeno-associated virus (rAAV) vector (e.g., Gao G., et al. 2003 Proc. Natl. Acad. Sci. USA 100(10):6081-6086), a lentiviral vector (e.g., Matrai, J, et al. 2011, Hepatology 53, 1696-707), a retroviral vector (e.g., Axelrod, JH, et al. 1990. Proc Natl Acad Sci USA; 87, 5173-7), an adenoviral vector (e.g., Brown et al., 2004 Blood 103, 804-10), a herpes simplex virus vector (Marconi, P. et al. Proc Natl Acad Sci USA. 1996 93(21):11319-11320, Baez, MV, et al. Chapter 19-Using Herpes Simplex Virus Type 1-Based Amplicon Vectors for Neuroscience Research and Gene Therapy of Neurologic Diseases, Ed.: Robert T. Gerlai, Molecular-Genetic and Statistical Techniques for Behavioral and Neural Research, Academic Press, 2018: Pages 445-477) and retrotransposon-based vector systems (e.g., Soifer, 2004, Current Gene Therapy 4(4): 373-384). In another embodiment, the vector is a non-viral vector. rAAV vectors have a limited packaging capacity (i.e., approximately 4.7 kb) of vector particles, which limits the size of the transgene expression cassette required to obtain a functional vector (Jiang et al., 2006 Blood. 108: 107-15).The length of the transgene and the length of the regulatory nucleic acid sequence, including the tandem enhancer(s) and promoter(s), are considered when selecting an appropriate regulatory region for a particular transgene and target tissue.

[0085] Another aspect of the invention relates to a viral vector comprising an expression cassette comprising a nucleic acid regulatory element described herein operably linked to a transgene. In some embodiments, the expression cassette comprises a nucleic acid regulatory element comprising the nucleic acid sequence of SEQ ID NO: 9, or a sequence that is 99%, 95%, 90%, 85%, or 80% identical to SEQ ID NO: 9, and that enhances expression of the transgene in skeletal muscle (in embodiments, with minimal or reduced expression in cardiac tissue).

[0086] In another aspect, the expression cassette is suitable for packaging into an AAV capsid, and thus the cassette comprises (1) AAV inverted terminal repeats (ITRs) on either side of the expression cassette, (2) regulatory control elements: a) a promoter / enhancer, e.g., any one of the promoters listed in Table 1, b) a polyA signal, and c) optionally an intron, and (3) a transgene that provides (e.g., encodes) one or more RNA or protein products of interest. In certain embodiments, the transgene is one of Tables 4A, 4B, or 4C. In a further aspect, the expression cassette is suitable for packaging as an ssAAV vector or scAAV vector.

[0087] 5.3.1 AAV Another aspect of the present invention relates to an expression cassette suitable for packaging into an AAV capsid, such that the cassette comprises: (1) AAV inverted terminal repeats (ITRs) on either side of the expression cassette; (2) regulatory control elements consisting essentially of one or more enhancers and one or more promoters, particularly the muscle-specific regulatory elements provided herein, including the regulatory elements of Table 1 or composite enhancer / promoters; d) a polyA signal; and e) optionally, an intron; and (3) a transgene providing (e.g., encoding) one or more RNA or protein products of interest.

[0088] The provided nucleic acids and methods are suitable for use in producing any isolated recombinant AAV particle, in producing a composition comprising any isolated recombinant AAV particle, or in a method of treating a disease or disorder in a subject in need thereof, comprising administering any isolated recombinant AAV particle. Accordingly, the rAAV can be of any serotype, modification, or derivative known in the art, or any combination thereof known in the art (e.g., a population of rAAV particles comprising two or more serotypes, e.g., comprising two or more rAAV2, rAAV8, and rAAV9 particles). In some embodiments, the rAAV particles include AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV -16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV. PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or other rAAV particle, or a combination of two or more thereof.

[0089] In some embodiments, the rAAV particles include AAV1, AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-1. 6, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AA and having a capsid protein derived from an AAV serotype selected from V2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or a derivative, modification, or pseudotype thereof.In some embodiments, the rAAV particles are AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AA V10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, rAAV.Anc80L65, A AV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3 , AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, and / or AAV.HSC17.

[0090] In some embodiments, the rAAV particles include AAV1, AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16. , AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2 and a capsid protein derived from an AAV capsid serotype selected from AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or a derivative, modification, or pseudotype thereof.In some embodiments, the rAAV particles include AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16, AAV.rh8, AAV.rh10, AAV.rh20. , AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, A AV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3 , AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, and / or AAV.HSC17.

[0091] In some embodiments, the rAAV particles comprise a capsid of Anc80 or Anc80L65, as described in Zinn et al., 2015, Cell Rep. 12(6):1056-1068, which is incorporated by reference in its entirety. In certain embodiments, the rAAV particles comprise a capsid with one of the following amino acid insertions: LGETTRP (SEQ ID NO: 71) or LALGETTRP (SEQ ID NO: 72), as described in U.S. Patent Nos. 9,193,956, 9,458,517, and 9,587,282 and U.S. Patent Application Publication No. 2016 / 0376323, each of which is incorporated by reference in its entirety. In some embodiments, the rAAV particles comprise the AAV.7m8 capsid described in U.S. Patent Nos. 9,193,956, 9,458,517, and 9,587,282 and U.S. Patent Application Publication No. 2016 / 0376323, each of which is incorporated herein by reference in its entirety. In some embodiments, the rAAV particles comprise any AAV capsid disclosed in U.S. Patent No. 9,585,971, such as AAV-PHP.B. In some embodiments, the rAAV particles comprise any AAV capsid disclosed in U.S. Patent No. 9,840,719 and WO2015 / 013313, each of which is incorporated herein by reference in its entirety, such as AAV.Rh74 and RHM4-1. In some embodiments, the rAAV particles comprise any AAV capsid disclosed in WO 2014 / 172669, which is incorporated herein by reference in its entirety, e.g., AAVrh.74. In some embodiments, the rAAV particles comprise an AAV2 / 5 capsid described in Georgiadis et al., 2016, Gene Therapy 23:857-862 and Georgiadis et al., 2018, Gene Therapy 25:450, each of which is incorporated herein by reference in its entirety. In some embodiments, the rAAV particles comprise any AAV capsid disclosed in WO 2017 / 070491, which is incorporated herein by reference in its entirety, e.g., AAV2tYF.In some embodiments, the rAAV particles comprise the AAVLK03 or AAV3B capsid described in Puzzo et al., 2017, Sci. Transl. Med. 29(9):418, which is incorporated by reference in its entirety. In some embodiments, the rAAV particles comprise any AAV capsid disclosed in U.S. Patent No. 8,628,966, U.S. Patent No. 8,927,514, U.S. Patent No. 9,923,120, and WO2016 / 049230, each of which is incorporated by reference in its entirety, such as HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16.

[0092] In some embodiments, the rAAV particles comprise an AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: U.S. Pat. Nos. 7,282,199, 7,906,111, 8,524,446, 8,999,678, 8,628,966, 8,927,514, 8,734,809, US Pat. Nos. 9,284,357, 9,409,953, and . Nos. 9,169,299, 9,193,956, 9458517, and 9,587,282, U.S. Patent Application Publication Nos. 2015 / 0374803, 2015 / 0126588, 2017 / 0067908, 2013 / 0224836, 2016 / 0215024, 2017 / 0051257, and International Patent Application Nos. PCT / US2015 / 034799 and PCT / EP2015 / 053335. In some embodiments, the rAAV particles have capsid proteins that are at least 80% or more identical, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical, to the VP1, VP2, and / or VP3 sequences of the AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: U.S. Pat. Nos. 7,282,199, 7,906,111, 8,524,446, and 8,999. ,678, 8,628,966, 8,927,514, 8,734,809, US9,284,357, 9,409,953, 9,169,299, 9,193,956, 9458517, and 9,587,282, US Patent Application Publication No. 2015 / 0 374803, 2015 / 0126588, 2017 / 0067908, 2013 / 0224836, 2016 / 0215024, 2017 / 0051257, and International Patent Applications Nos. PCT / US2015 / 034799 and PCT / EP2015 / 053335.

[0093] In some embodiments, the rAAV particles are prepared using techniques described in International Application Publication Nos. WO2003 / 052051 (see, e.g., SEQ ID NO: 2 of the '051 publication), WO2005 / 033321 (see, e.g., SEQ ID NOs: 123 and 88 of the '321 publication), WO03 / 042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97 of the '397 publication), WO2006 / 068888 (see, e.g., SEQ ID NOs: 1 and 3-6 of the '888 publication), WO2006 / 11068 9 (see, e.g., SEQ ID NOS: 5-38 of the '689 publication), WO2009 / 104964 (see, e.g., SEQ ID NOS: 1-5, 7, 9, 20, 22, 24, and 31 of the '964 publication), WO2010 / 127097 (see, e.g., SEQ ID NOS: 5-38 of the '097 publication), and WO2015 / 191508 (see, e.g., SEQ ID NOS: 80-294 of the '508 publication), and U.S. Application Publication No. 20150023924 (see, e.g., SEQ ID NOS: 1, 5-10 of the '924 publication).In some embodiments, the rAAV particles are prepared using the methods described in International Application Publication Nos. WO2003 / 052051 (see, e.g., SEQ ID NO: 2 of the '051 publication), WO2005 / 033321 (see, e.g., SEQ ID NOs: 123 and 88 of the '321 publication), WO03 / 042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97 of the '397 publication), WO2006 / 068888 (see, e.g., SEQ ID NOs: 1 and 3-6 of the '888 publication), WO2006 / 110689 (see, e.g., SEQ ID NOs: 5-38 of the '689 publication), WO2009 / 104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24, and 31 of the '964 publication), The capsid proteins are at least 80% or more identical, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical, to the VP1, VP2, and / or VP3 sequences of the AAV capsid disclosed in WO2010 / 127097 (see, e.g., SEQ ID NOS: 5-38 of the '097 publication), and WO2015 / 191508 (see, e.g., SEQ ID NOS: 80-294 of the '508 publication), and U.S. Application Publication No. 20150023924 (see, e.g., SEQ ID NOS: 1, 5-10 of the '924 publication).

[0094] Nucleic acid sequences of AAV-based viral vectors and methods for producing recombinant AAV and AAV capsids are described in, for example, U.S. Patent Nos. 7,282,199, 7,906,111, 8,524,446, 8,999,678, 8,628,966, 8,927,514, 8,734,809, US Pat. Nos. 9,284,357, 9,409,953, 9,169,299, 9,193,956, 9458517, and 9,587,282; U.S. Patent Application Publication Nos. 2015 / 0374803, 2015 / 0126588, 2017 / 0 067908, 2013 / 0224836, 2016 / 0215024, 2017 / 0051257, International Patent Application Nos. PCT / US2015 / 034799, PCT / EP2015 / 053335, WO2003 / 052051, WO2005 / 033321, WO03 / 042397, WO2006 / 068888, WO2006 / 110689, WO2009 / 104964, WO2010 / 127097, and WO2015 / 191508, and U.S. Publication No. 20150023924.

[0095] The provided methods are suitable for use in generating recombinant AAV encoding a transgene. In some embodiments, provided herein is a rAAV viral vector encoding an anti-VEGF Fab. In some embodiments, provided herein is a rAAV8-based viral vector encoding an anti-VEGF Fab. In further embodiments, provided herein is a rAAV8-based viral vector encoding ranibizumab. In some embodiments, provided herein is a rAAV viral vector encoding iduronidase (IDUA). In some embodiments, provided herein is a rAAV9-based viral vector encoding IDUA. In some embodiments, provided herein is a rAAV viral vector encoding iduronate 2-sulfatase (IDS). In some embodiments, provided herein is a rAAV9-based viral vector encoding an IDS. In some embodiments, provided herein is a rAAV viral vector encoding low density lipoprotein receptor (LDLR). In some embodiments, provided herein is a rAAV8-based viral vector encoding LDLR. In some embodiments, provided herein are rAAV viral vectors encoding tripeptidyl peptidase 1 (TPP1) protein. In some embodiments, provided herein are rAAV9-based viral vectors encoding TPP. In some embodiments, provided herein are rAAV viral vectors encoding anti-kallikrein (anti-pKal) antibodies. In some embodiments, provided herein are rAAV8- or rAAV9-based viral vectors encoding Fab or full-length antibodies of pKal antibodies.

[0096] In further embodiments, the rAAV particles comprise pseudotyped AAV capsids. In some embodiments, the pseudotyped AAV capsids are rAAV2 / 8 or rAAV2 / 9 pseudotyped AAV capsids. Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081 (2001)).

[0097] In further embodiments, the rAAV particles comprise capsids that comprise capsid proteins that are chimeric of two or more AAV capsid serotypes, hi some embodiments, the capsid proteins are AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, and AAV-16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV. and a chimera of two or more AAV capsid proteins derived from an AAV serotype selected from PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.

[0098] In certain embodiments, single-stranded AAV (ssAAV) may be used. In certain embodiments, self-complementary vectors, such as scAAV, may be used (see, for example, Wu, 2007, Human Gene Therapy, 18(2):171-82; McCarty, DM, et al, 2001, Gene Therapy, Vol. 8, Number 16, Pages 1248-1254, and U.S. Patent Nos. 6,596,535, 7,125,717, and 7,456,683, each of which is incorporated herein by reference in its entirety).

[0099] "Self-complementary AAV" refers to a plasmid or vector containing an expression cassette designed so that the coding region carried by the recombinant AAV nucleic acid sequence forms an intramolecular double-stranded DNA template. Unlike ssDNA genomes, scAAV genomes are not affected by host cell DNA polymerases and do not require synthesis of a complementary strand. After infection, rather than waiting for cell-mediated synthesis of a second strand, the two complementary halves of the scAAV associate to form a single double-stranded DNA (dsDNA) unit ready for immediate replication and transcription. See, e.g., McCarty, DM, et al., 2001, supra. Self-complementary AAVs are described, for example, in U.S. Patent Nos. 6,596,535, 7,125,717, and 7,456,683 (each of which is incorporated herein by reference in its entirety). Genomes of 2500 kb or less in size may benefit from packaging into self-complementary AAV vectors.

[0100] Single-stranded AAV (ssAAV) vectors, in which the coding sequence and complementary sequence of a transgene expression cassette are located on separate strands, are packaged into separate viral capsids. In the case of ssAAV, after transduction occurs and the genome enters the nucleus, the DNA is converted from single-stranded to double-stranded via intermolecular annealing or second-strand synthesis. In certain embodiments, single-stranded AAV (ssAAV) may be used. In the case of self-complementary AAV (scAAV) vectors, both the coding sequence and complementary sequence of the transgene expression cassette are present on each plus- and minus-strand genome. In contrast, scAAV vectors, which are half the size of the ssAAV genome, have mutations in the terminal separation sites (TRSs) to form vector genomes with wild-type ITRs at both ends and mutant ITRs at the center of symmetry. After uncoating in the target cell nucleus, this DNA structure can easily fold into a transcriptionally active double-stranded form via intramolecular annealing. In certain embodiments, self-complementary vectors, such as scAAV, may be used (see, e.g., Wu, 2007, supra; McCarty et al., 2001, supra). In some embodiments, the expression cassette contains one ITR (5'-) mutated to form a self-complementary AAV (scAAV) genome, or two wild-type ITRs (5'- and 3'-) to form a single-stranded AAV (ssAAV) genome. Alternative ITR sequences are known in the art.

[0101] In some embodiments, the rAAV particles comprise capsid proteins derived from an AAV capsid serotype selected from AAV-8 or AAV-9. In some embodiments, the rAAV particles have an AAV-1 or a derivative, modified, or pseudotyped AAV capsid serotype thereof. In some embodiments, the rAAV particles have an AAV-4 or a derivative, modified, or pseudotyped AAV capsid serotype thereof. In some embodiments, the rAAV particles have an AAV-5 or a derivative, modified, or pseudotyped AAV capsid serotype thereof. In some embodiments, the rAAV particles have an AAV-8 or a derivative, modified, or pseudotyped AAV capsid serotype thereof. In some embodiments, the rAAV particles have an AAV-9 or a derivative, modified, or pseudotyped AAV capsid serotype thereof.

[0102] In some embodiments, the rAAV particles comprise capsid proteins that are derivatives, modifications, or pseudotypes of AAV-8 or AAV-9 capsid proteins. In some embodiments, the rAAV particles comprise capsid proteins that have at least 80% or more identity, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., to the VP1, VP2, and / or VP3 sequences of the AAV-8 capsid protein, i.e., up to 100% identity to the AAV-8 capsid protein.

[0103] In some embodiments, the rAAV particles comprise capsid proteins that are derivatives, modifications, or pseudotypes of the AAV-9 capsid protein. In some embodiments, the rAAV particles in the purified supply comprise capsid proteins that have at least 80% or more identity, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., to the VP1, VP2, and / or VP3 sequences of the AAV-9 capsid protein, i.e., up to 100% identity to the AAV-8 capsid protein.

[0104] In further embodiments, the rAAV particles comprise a mosaic capsid. Mosaic AAV particles are composed of a mixture of viral capsid proteins from different AAV serotypes. In some embodiments, the rAAV particles comprise any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, and AAV-16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV. and a mosaic capsid comprising capsid proteins of a serotype selected from PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16.

[0105] In some embodiments, the rAAV particles comprise a mosaic capsid comprising capsid proteins of a serotype selected from AAV-1, AAV-2, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAVrh.8, and AAVrh.10. In further embodiments, the rAAV particles comprise pseudotyped rAAV particles. In some embodiments, the pseudotyped rAAV particles comprise (a) a nucleic acid vector comprising the ITRs of an AAV, and (b) a capsid composed of capsid proteins derived from an AAVx (e.g., AAV-1, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, and AAV-16). In further embodiments, the rAAV particles are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, and pseudotyped rAAV particles comprised of capsid proteins of an AAV serotype selected from AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In further embodiments, the rAAV particles comprise pseudotyped rAAV particles comprised of AAV-8 capsid proteins. In further embodiments, the rAAV particles comprise pseudotyped rAAV particles comprised of AAV-9 capsid proteins. In some embodiments, the pseudotyped AAV8 or AAV9 particles are rAAV2 / 8 or rAAV2 / 9 pseudotyped particles.Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001)).

[0106] In further embodiments, the rAAV particles comprise capsids that include capsid proteins that are chimeric of two or more AAV capsid serotypes, including AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.C, AAV.PHP.D, AAV.PHP.F, AAV.PHP.H ... A chimera of two or more AAV capsid proteins from an AAV serotype selected from AAV2.5, AAV2tYF, AAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In a further embodiment, the capsid protein is a chimera of two or more AAV capsid proteins from an AAV serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.8, and AAVrh.10.

[0107] In some embodiments, the rAAV particles contain AAV-8 capsid proteins as well as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP. and an AAV capsid protein that is a chimera of one or more AAV capsid proteins from an AAV serotype selected from AAV.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the rAAV particles comprise AAV-8 capsid proteins and AAV capsid proteins that are chimeras of one or more AAV capsid proteins from an AAV serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV9, AAV10, AAVrh.8, and AAVrh.10.

[0108] In some embodiments, the rAAV particles contain AAV-9 capsid proteins as well as any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP and AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16.

[0109] In some embodiments, the rAAV particles comprise AAV-9 capsid proteins and AAV capsid proteins that are chimeras of capsid proteins of one or more AAV capsid serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, and AAVrh.10.

[0110] In embodiments, rAAVs containing a recombinant expression cassette comprising a composite nucleic acid regulatory element comprising or consisting of Mus022.CK and Mus035.CK exhibit reduced activity in cardiac muscle compared to the CK7 and Spc5-12 promoters, despite having similar skeletal muscle-specific activity. rAAVs containing a recombinant expression cassette comprising a composite nucleic acid regulatory element comprising or consisting of Mus022.CK and Mus035.CK exhibit significantly lower RNA expression in the heart, resulting in an advantageous gene expression profile for operably linking the Mus022.CK promoter to a therapeutic gene that is preferentially delivered to skeletal muscle compared to cardiac tissue.

[0111] Method for producing rAAV vectors Another aspect of the invention involves producing the molecules disclosed herein. In some embodiments, the molecules of the invention are produced by providing nucleotides comprising a nucleic acid sequence encoding an AAV capsid protein and using a packaging cell line to prepare rAAV particles having a capsid coat composed of the corresponding capsid protein. In some embodiments, the nucleic acid sequence encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, or 95%, 96%, 97%, 98%, 99%, or 99.9% identity to the sequence of a capsid protein molecule described herein, and retains (or substantially retains) the biological function of the capsid protein and an inserted peptide derived from a heterologous protein or domain thereof. In some embodiments, the nucleic acid encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, or 95%, 96%, 97%, 98%, 99% or 99.9% identity to a particular sequence of the AAV capsid protein while retaining (or substantially retaining) the biological function of the AAV capsid protein.

[0112] The capsid proteins, coats, and rAAV particles can be produced by techniques known in the art. In some embodiments, the viral genome comprises at least one inverted terminal end sequence to enable packaging into a vector. In some embodiments, the viral genome further comprises a cap gene and / or a rep gene for expression and splicing of the cap gene. In certain embodiments, the cap and rep genes are provided by the packaging cell and are not present in the viral genome.

[0113] In some embodiments, the nucleic acid encoding the capsid protein is cloned into an AAV Rep-Cap helper plasmid in place of the existing capsid gene. When introduced into a host cell together, this plasmid helps package the rAAV genome into the capsid protein as the capsid coat. Packaging cells can be any cell type that has the genes necessary to promote AAV genome replication, capsid assembly, and packaging. Non-limiting examples include 293 cells or their derivatives, HELA cells, or insect cells.

[0114] Standard techniques for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection) may be used. Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications, as commonly practiced in the art, or as described herein. The foregoing techniques and procedures may generally be performed according to conventional methods known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference for all purposes. Unless detailed definitions are provided, the nomenclature used in connection with, and the laboratory methods and techniques for, analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. Nucleic acid sequences of AAV-based viral vectors and methods for producing recombinant AAV and AAV capsids are taught, for example, in US 7,282,199, US 7,790,449, US 8,318,480, US 8,962,332, and PCT / EP2014 / 076466, each of which is incorporated herein by reference in its entirety.

[0115] In preferred embodiments, the rAAV provides a transgene delivery vector that can be used in therapeutic and prophylactic applications, as discussed in more detail below. The rAAV vector also contains the regulatory control elements described above that affect expression of the RNA and / or protein product encoded by the nucleic acid (transgene) in target cells of a subject.

[0116] In certain embodiments, provided are AAV vectors comprising a viral genome comprising an expression cassette flanked by ITRs for expression of the transgene under the control of the regulatory elements, and an engineered viral capsid described herein or an engineered viral capsid that is at least 95%, 96%, 97%, 98%, 99%, or 99.9% identical to the amino acid sequence of the AAV capsid protein.

[0117] The recombinant adenovirus may be a first-generation vector with E1 deletion, E3 deletion or non-deletion, and the expression cassette inserted into either of the deleted regions. The recombinant adenovirus may be a second-generation vector with complete or partial deletion of the E2 and E4 regions. Helper-dependent adenoviruses retain only the adenovirus inverted terminal end sequences and packaging signal (phi). The transgene is usually inserted between the packaging signal and the 3' ITR, with or without a stuffer sequence, maintaining a genome close to the wild-type size of approximately 36 kb. Exemplary protocols for generating adenovirus vectors can be found in Alba et al., 2005, "Gutless adenovirus: last generation adenovirus for gene therapy," Gene Therapy 12:S18-S27, incorporated herein by reference in its entirety.

[0118] The rAAV vector for delivering the transgene to the target tissue, cell, or organ may also have tropism for that particular target tissue, cell, or organ, e.g., liver and / or muscle, in conjunction with the use of tissue-specific promoters described herein. The construct can further include additional expression control elements, such as introns that enhance expression of the transgene (e.g., introns such as chicken β-actin intron, minute virus of mouse (MVM) intron, human factor IX intron (e.g., FIX split intron 1), β-globin splice donor / immunoglobulin heavy chain splice acceptor intron, adenovirus splice donor / immunoglobulin splice acceptor intron, SV40 late splice donor / splice acceptor (19S / 16S) intron, and hybrid adenovirus splice donor / IgG splice acceptor intron, as well as polyA signals, e.g., rabbit β-globin polyA signal, human growth hormone (hGH) polyA signal, SV40 late polyA signal, synthetic polyA (SPA) signal, and bovine growth hormone (bGH) polyA signal. See, e.g., Powell and Rivera-Soto, 2015, Discov. See Med., 19(102):49-57.

[0119] In certain embodiments, the nucleic acid sequences disclosed herein may be codon optimized, for example, using any codon optimization technique known to those of skill in the art (see, e.g., review by Quax et al., 2015, Mol Cell 59:149-161).

[0120] In one embodiment, the constructs described herein comprise the following components: (1) AAV inverted terminal repeats (ITRs) on either side of the expression cassette, (2) control elements comprising: a) one or more regulatory elements comprising at least one or more enhancers of any one of SEQ ID NOS: 10-13, alone or in combination with one or more promoters, b) a polyA signal, and c) optionally an intron, 3) a transgene providing (e.g., encoding) one or more RNA or protein products of interest. In another embodiment, the constructs described herein comprise the following components: (1) AAV inverted terminal repeats (ITRs) on either side of the expression cassette, (2) control elements comprising: a) one or more regulatory elements comprising at least one or more enhancers of any one of SEQ ID NOS: 10-13, alone or in combination with one or more CK promoters, b) a polyA signal, and c) optionally an intron, 3) a transgene providing (e.g., encoding) one or more RNA or protein products of interest. In yet another embodiment, the constructs described herein comprise the following components: (1) AAV inverted terminal repeats (ITRs) on either side of the expression cassette; (2) control elements comprising: a) one or more regulatory elements including at least the Mus022 enhancer (SEQ ID NO: 10) in combination with the CK promoter (SEQ ID NO: 8); b) a polyA signal; and c) optionally an intron; and 3) a transgene providing (e.g., encoding) one or more RNA or protein products of interest, e.g., those in Table 4A, 4B, or 4C.

[0121] In certain embodiments, the constructs described herein comprise the following components: (1) AAV inverted terminal repeats (ITRs) on either side of the expression cassette; (2) control elements comprising: a) one or more regulatory elements comprising at least one of the promoters of any one of SEQ ID NOs: 14-26, alone or in combination with one or more enhancers; b) a polyA signal; and c) optionally an intron; and 3) a transgene providing (e.g., encoding) one or more RNA or protein products of interest. In another embodiment, the constructs described herein comprise the following components: (1) AAV inverted terminal repeats (ITRs) on either side of the expression cassette; (2) control elements comprising: a) one or more regulatory elements comprising at least one of the ACTA core promoters of any one of SEQ ID NOS: 14-26, alone or in combination with one or more enhancers of any one of SEQ ID NOS: 4-7; b) a polyA signal; and c) optionally an intron; and 3) a transgene providing (e.g., encoding) one or more RNA or protein products of interest, e.g., those in Table 4A, 4B, or 4C.

[0122] The viral vectors provided herein can be produced using mammalian host cells, including host cells derived from humans, monkeys, mice, rats, rabbits, or hamsters. Non-limiting examples include A549, WEHI, 10T1 / 2, BHK, MDCK, COS1, COS7, BSC1, BSC40, BMT10, VERO, W138, HeLa, 293, Saos, C2C12, L, HT1080, HepG2, primary fibroblasts, hepatocytes, and myoblasts. Typically, the host cells are stably transformed with sequences encoding the transgene and related elements (i.e., the vector genome), as well as genetic components for producing virus in the host cells, such as replication and capsid genes (e.g., AAV rep and cap genes). For methods of generating recombinant AAV vectors with AAV8 capsids, see Section IV of the detailed description in U.S. Patent No. 7,282,199 B2, incorporated herein by reference in its entirety. The titer of genome copies of the vector can be determined, for example, by TAQMAN® analysis. Virions can be collected, for example, by CsCl2 precipitation. Alternatively, a baculovirus expression system in insect cells can be used to produce AAV vectors. For a review, see Aponte-Ubillus et al., 2018, Appl. Microbiol. Biotechnol. 102:1045-1054, which is incorporated herein by reference in its entirety for production techniques.

[0123] In vitro assays, such as cell culture assays, can be used to measure transgene expression from the vectors described herein and thus, for example, demonstrate vector efficacy. For example, the PER.C6® cell line (Lonza), a cell line derived from human embryonic retinal cells, or retinal pigment epithelial cells, such as the retinal pigment epithelial cell line hTERT RPE-1 (available from ATCC®), can be used to assess transgene expression. Alternatively, cell lines derived from liver or muscle or other cell types, such as, but not limited to, HuH-7, HEK293, fibrosarcoma HT-1080, HKB-11, C2C12 myoblasts, and CAP cells, can be used. After expression, the expression product (transgenic product) can be characterized, including serum half-life, protein functional activity (e.g., enzymatic activity or target binding), glycosylation and tyrosine sulfation pattern determination, and other assays known in the art for characterizing proteins.

[0124] Provided are methods for producing a recombinant AAV, which involve culturing a host cell capable of producing a recombinant AAV described herein under conditions suitable for producing the recombinant AAV, which comprises an artificial genome comprising an expression cassette comprising a synthetic promoter operably linked to a transgene. In particular, the method provides (1) culturing (i) an artificial genome comprising an AAV ITR flanked by a recombinant cis expression cassette comprising nucleic acid regulatory elements, the nucleic acid regulatory elements comprising a composite nucleic acid regulatory element disclosed herein, operably linked to a transgene; (ii) a trans expression cassette lacking the AAV ITRs, encoding AAV rep and AAV capsid proteins operably linked to expression control elements that drive expression of and provide AAV rep and AAV capsid proteins in trans in host cells in culture; and (iii) culturing the host cells containing sufficient adenovirus helper functions to allow replication and packaging of the artificial genome by the AAV capsid proteins; and (2) recovering from the cell culture a recombinant AAV encapsidated with the artificial genome. Also provided is an artificial genome comprising (i) an AAV ITR flanking a recombinant cis expression cassette comprising a composite nucleic acid regulatory element disclosed herein operably linked to a transgene; (ii) a trans expression cassette lacking the AAV ITRs encoding AAV rep and AAV capsid proteins operably linked to expression control elements that drive expression of the AAV rep and AAV capsid proteins in a host cell in culture and provide the AAV rep and AAV capsid proteins in trans; and, optionally, (iii) a host cell comprising sufficient adenovirus helper functions to enable replication and packaging of the artificial genome by the AAV capsid proteins. In certain embodiments, the composite nucleic acid regulatory element is Mus022.CK or SEQ ID NO:9. In certain embodiments, the artificial genome comprises a transgene encoding one of the therapeutic agents listed in Tables 4A, 4B, and 4C.

[0125] 5.4. Therapeutic and prophylactic uses Another aspect relates to a treatment for delaying, preventing, treating, and / or managing a disease or disorder and / or ameliorating one or more symptoms associated therewith, comprising administering a transgene to a subject in need thereof via the rAAV vector of the present invention. The subject in need thereof includes a subject suffering from the disease or disorder or a subject predisposed thereto, for example, a subject at risk of developing the disease or disorder or experiencing a recurrence thereof. Typically, a rAAV carrying a specific transgene is utilized for a given disease or disorder in a subject in which the subject's native gene corresponding to the transgene is defective in providing the correct gene product or the correct amount of gene product. The transgene can then provide a copy of the defective gene in the subject.

[0126] Typically, the transgene comprises a cDNA that restores protein function to a subject with a genetic mutation(s) in the corresponding native gene. In some embodiments, the cDNA comprises the relevant RNA for genome engineering, for example, genome editing via homologous recombination. In some embodiments, the transgene encodes a therapeutic RNA, for example, an shRNA, an artificial miRNA, or an element that affects splicing.

[0127] In some aspects, the therapeutic agent encoded by one or more of the transgenes of the present disclosure may be encoded by microdystrophin. Micro-dystrophins include those having the amino acid sequence of micro-dystrophins consisting of dystrophin domains arranged from the amino terminus to the carboxy terminus as follows: ABD-H1-R1-R2-R3-H3-R24-H4-CR-CT, where ABD is the actin-binding domain of dystrophin, H1 is the hinge 1 region of dystrophin, R1 is the spectrin 1 region of dystrophin, R2 is the spectrin 2 region of dystrophin, R3 is the spectrin 3 region of dystrophin, H3 is the hinge 3 region of dystrophin, R24 is the spectrin 24 region of dystrophin, H4 is the hinge 4 region of dystrophin, CR is the cysteine-rich region of dystrophin, and CT is the C-terminal domain (including at least a portion of the CT domain containing the α1-syntrophin binding site), such as the micro-dystrophin of RGX-202. The amino acid sequences of the dystrophin components used to form micro- or mini-dystrophins are described in the full-length human DMD protein UniProtDB-11532, which is incorporated herein by reference. Further embodiments are disclosed in International Application No. PCT / US2020 / 062484, filed November 27, 2020, which is incorporated herein by reference in its entirety.

[0128] Provided is the method for treating any muscular dystrophy disease that can be treated by providing functional dystrophin to human subject.In some embodiments, the functional dystrophin is one or more of the micro-dystrophins disclosed herein.DMD is the most common muscular dystrophy disease, but other diseases can be treated, such as but not limited to Becker muscular dystrophy (BMD), myotonic muscular dystrophy (Steinert's disease), facioscapulohumeral muscular dystrophy (FSHD), limb-girdle muscular dystrophy, X-linked dilated cardiomyopathy or oculopharyngeal muscular dystrophy.

[0129] Tables 4A, 4B, and 4C below provide lists of transgenes that can be used in any of the recombinant expression cassettes described herein, preferably to treat or prevent the disease associated with the transgene, also described in Tables 4A-4B. Table 4A includes, but is not limited to, several transgenes known to restore muscle cells or muscle activity in a patient when delivered to the patient via muscle secretion, via the blood circulation, and / or via the CNS at levels determined to be effective. Some transgenes are muscle-derived proteins. As described herein, the AAV vectors can be engineered as described herein to target appropriate tissues for transgene delivery for therapeutic or prophylactic use. Appropriate AAV serotypes can be selected to optimize the tissue tropism and transduction of the vector. [Table 4A] [Table 4B-1] [Table 4B-2] [Table 4B-3]

[0130] In some embodiments, provided is a recombinant expression cassette comprising a mini-dystrophin or micro-dystrophin transgene. In embodiments, the micro-dystrophin comprises a dystrophin domain, or a portion thereof, arranged from amino-terminus to carboxy-terminus as follows: ABD-H1-R1-R2-R3-H3-R24-H4-CR-CT, where ABD is the actin-binding domain of dystrophin, H1 is the hinge 1 region of dystrophin, R1 is the spectrin 1 region of dystrophin, R2 is the spectrin 2 region of dystrophin, R3 is the spectrin 3 region of dystrophin, H3 is the hinge 3 region of dystrophin, R24 is the spectrin 24 region of dystrophin, H4 is the hinge 4 region of dystrophin, CR is the cysteine-rich region of dystrophin, and CT is the C-terminal domain.

[0131] The present disclosure contemplates variants of microdystrophin, including those that substantially maintain the therapeutic effects of microdystrophin, including functional activities such as (1) binding to one, a combination, or all of actin, β-dystoglycan, α1-syntrophin, α-dystrobrevin, and nNOS, (2) improving muscle function in an animal model (e.g., the mdx mouse model) or a human subject, and / or (3) cardioprotection or improved myocardial function in an animal model or a human patient.

[0132] Table 4C shows the amino acid sequences of embodiments of microdystrophins according to the present disclosure. In certain embodiments, the microdystrophins have the amino acid sequence of SEQ ID NO: 73 (DYS1), 74 (DYS3), or 75 (DYS5). In other embodiments, the microdystrophins have the amino acid sequence of SEQ ID NO: 76 (human MD1 (R4-R23 / ΔCT), SEQ ID NO: 77 (microdystrophin), SEQ ID NO: 78 (Dys3978), SEQ ID NO: 79 (MD3), or SEQ ID NO: 80 (MD4). Other embodiments are also contemplated as substitution variants of microdystrophins defined by SEQ ID NO: 73 (DYS1), 74 (DYS3), or 75 (DYS5).

[0133] In embodiments, micro-dystrophin may have at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 73, 74, or 75, and may maintain the functional activity of micro-dystrophin as determined, for example, by one or more in vitro assays or in vivo assays in animal models, for example, as disclosed in WO 2021 / 108755. [Table 4C-1] [Table 4C-2] [Table 4C-3] [Table 4C-4] [Table 4C-5] [Table 4C-6] [Table 4C-7] [Table 4C-8]

[0134] In another example, rAAV containing a transgene encoding an anti-kallikrein antibody, e.g., lanadelumab, is used to treat / prevent / manage hereditary angioedema (HAE). In yet another example, rAAV containing a transgene encoding a lysosomal enzyme is used to treat / prevent / manage mucopolysaccharidoses. Generally, the rAAV vector is administered systemically, and after transduction, production of the protein product by the vector is enhanced by an expression cassette using engineered muscle-specific nucleic acid regulatory elements. For example, the rAAV vector can be administered intravenously, intramuscularly, and / or intraperitoneally for secretion of the protein of interest (encoded by the transgene) from muscle as a reservoir.

[0135] For the therapeutic antibodies of Table 4B, an expression cassette comprising regulatory sequences operably linked to a transgene encoding the therapeutic antibody can be packaged into an rAAV for delivery having an AAV8 capsid, an AAV9 capsid, or an AAVrh74 capsid for targeting to or expression in muscle cells.

[0136] In some embodiments, the rAAV of the present invention is used to deliver to a target tissue associated with the disorder or disease to be treated / prevented. A disease or disorder associated with a specific tissue or cell type is one that affects the specific tissue or cell type more significantly than other tissues or cell types in the body, or the effects or symptoms of the disorder are manifested in the specific tissue or cell type. A method for delivering a transgene to a target tissue in a subject in need thereof comprises administering to the subject an rAAV, wherein the expression cassette comprises a nucleic acid regulatory element operably linked to the transgene.

[0137] Following transduction of target cells, expression of the protein product is enhanced by using such liver-specific and muscle-specific expression cassettes. Such enhancement can be measured by the following non-limiting list of measurements: 1) protein titer using assays known to those skilled in the art, including but not limited to sandwich ELISA, Western blot, histological staining, and liquid chromatography-tandem mass spectrometry (LC-MS / MS); 2) protein activity using assays such as binding assays, functional assays, enzymatic assays, and / or substrate detection assays; and / or 3) serum half-life or long-term expression. Enhanced transgene expression can be identified as effective and suitable for human therapy (Hintze, JP et al., Biomarker Insights 2011:6 69-78). Evaluation of quantitative and functional properties of transgenes using such in vitro and in vivo cell, blood, and tissue assays has been shown to correlate with the efficacy of certain treatments (Hintze, JP et al., 2011, supra) and is used to evaluate response to transgene gene therapy using the vectors described herein.

[0138] The rAAV vectors of the present invention can also facilitate the delivery, particularly targeted delivery, of transgenes operably linked to the chimeric regulatory sequences described herein, including, but not limited to, oligonucleotides, drugs, contrast agents, inorganic nanoparticles, liposomes, and antibodies, to target cells or tissues. The rAAV vectors can also facilitate the delivery, particularly targeted delivery, of non-coding DNA, RNA, or oligonucleotides to target tissues.

[0139] The agents can be provided in pharmaceutically acceptable compositions known in the art and / or described herein. In some embodiments, the rAAV molecule can be administered alone or in combination with other prophylactic and / or therapeutic agents.

[0140] The dosage amounts and administration frequencies provided herein are encompassed by the terms therapeutically effective and prophylactically effective. The dosage amount and frequency will typically vary depending on patient-specific factors, such as the particular therapeutic or prophylactic agent being administered, the severity and type of disease, the route of administration, and the patient's age, weight, response, and medical history, and should be determined according to the judgment of the physician and each patient's circumstances. Appropriate regimens will be determined by considering such factors and will be discussed in detail in, for example, the literature and in the Physician's Desk Reference (56 th The dosages can be selected by one skilled in the art according to the dosages recommended in the "Prophylactic and / or Therapeutic Agents" section of ...

[0141] The amount of an agent of the present invention that will be effective can be determined by standard clinical techniques. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. For any agent used in the methods of the present invention, the therapeutically effective dose can be initially estimated from cell culture assays. Doses can be determined based on the IC20 determined in cell culture. 50Animal models can be developed to provide a circulating plasma concentration range that includes the concentration of the test compound that achieves a half-maximal inhibition of symptoms (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

[0142] Prophylactic and / or therapeutic agents, and combinations thereof, can be tested in appropriate animal model systems prior to use in humans. Such animal model systems include, but are not limited to, rats, mice, chickens, cows, monkeys, pigs, dogs, rabbits, etc. Any animal system known in the art can be used. Such model systems are widely used and well known to those skilled in the art. In some preferred embodiments, animal model systems for CNS conditions based on rats, mice, or other small mammals other than primates are used.

[0143] After the prophylactic and / or therapeutic agents of the present invention are tested in animal models, they can be tested in clinical trials to establish their efficacy. The establishment of clinical trials can be carried out according to general methods known to those skilled in the art, and the optimal dosage and administration route as well as the toxicity profile of the agents of the present invention can be established. For example, clinical trials can be designed to test the efficacy and toxicity of the rAAV molecules of the present invention in human patients.

[0144] Toxicity and efficacy of prophylactic and / or therapeutic agents of the invention can be measured, for example, by LD 50 The LD50 (lethal dose in 50% of the population) and ED50 (therapeutically effective dose in 50% of the population) can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutic effects is the therapeutic index, which is defined as the LD 50 / ED 50 The therapeutic index can be expressed as a ratio. Prophylactic and / or therapeutic agents that exhibit large therapeutic indices are preferred. While prophylactic and / or therapeutic agents that exhibit toxic side effects may also be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells, thereby reducing side effects.

[0145] The rAAV molecules of the invention are generally administered for a time and in an amount effective to achieve the desired therapeutic and / or prophylactic effect. Data obtained from the cell culture assays and animal studies can be used in formulating dosage ranges and / or schedules of prophylactic and / or therapeutic agents for use in humans. The dosage of such agents preferably is within the ED range with little or no toxicity. 50 The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

[0146] The therapeutically effective dosage of rAAV vectors for patients is typically at a concentration of approximately 1 x 10 9 ~Approx. 1×10 16 genomic rAAV vector, or approximately 1 x 10 10 ~Approx. 1×10 15 , about 1×10 12 ~Approx. 1×10 16 , or approximately 1 × 10 14 ~Approx. 1×10 16 It is a solution of about 0.1 ml to about 100 ml containing the AAV genome. The level of expression of the transgene can be observed to determine / adjust dosage, frequency, scheduling, etc.

[0147] Treatment of a subject with a therapeutically or prophylactically effective amount of the agent of the present invention can include a single treatment or a series of treatments. For example, a pharmaceutical composition containing the agent of the present invention can be administered once daily, twice daily, or three times daily. In some embodiments, the agent can be administered once daily, every other day, once weekly, twice weekly, once every two weeks, once monthly, once every six weeks, once every two months, twice yearly, or once yearly. It is also understood that the effective dosage of a particular agent, for example, the effective dosage of an agent containing a dual antigen-binding molecule of the present invention, can increase or decrease during the course of treatment.

[0148] Methods of administering agents of the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous, including infusion or bolus injection), epidural, and by absorption through epithelial or mucocutaneous or mucosal linings (e.g., intranasal, oral, rectal, and intestinal mucosa, etc.). In certain embodiments, the transgene is administered intravenously, even if it is intended to be expressed in the CNS, for example, by forming a depot in the liver where the transgene is expressed and secreted into the bloodstream.

[0149] In certain embodiments, the agents of the present invention are administered intravenously or intramuscularly and may be administered together with other biologically active agents.

[0150] In another specific embodiment, the agents of the present invention can be delivered in a sustained-release formulation, for example, where the formulation provides sustained release and thus extends the half-life of the administered agent. Suitable controlled-release systems include, but are not limited to, diffusion-controlled, solvent-controlled, and chemical-controlled systems. Diffusion-controlled systems include, for example, reservoir devices in which the molecules of the present invention are encapsulated within the device so that release of the molecules is controlled by permeation through a diffusion barrier. Common reservoir devices include, for example, membranes, capsules, microcapsules, liposomes, and hollow fibers. Monolithic (matrix) devices are another type of diffusion-controlled system, in which the dual antigen-binding molecules are dispersed or dissolved in a rate-controlling matrix (e.g., a polymer matrix). The agents of the present invention can be homogeneously dispersed throughout the rate-controlling matrix, and the rate of release is controlled by diffusion through the matrix. Suitable polymers for use in monolithic matrix devices include naturally occurring polymers, synthetic polymers, and synthetically modified natural polymers, as well as polymer derivatives.

[0151] Any technique known to those skilled in the art can be used to produce sustained release formulations containing one or more of the agents described herein. See, for example, U.S. Pat. No. 4,526,938, PCT Publication No. WO 91 / 05548, PCT Publication No. WO 96 / 20698, Ning et al., "Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel," Radiotherapy & Oncology, 39:179-189, 1996, Song et al., "Antibody Mediated Lung Targeting of Long-Circulating Emulsions," PDA Journal of Pharmaceutical Science & Technology, 50:372-397, 1995, Cleek et al., "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application," Proc. Intl. Symp. Control. Rel. Bioact. Mater., 24:853-854, 1997, and Lam et al., "Microencapsulation of Recombinant Humanized See, "Monoclonal Antibody for Local Delivery," Proc. Int'l. Symp. Control Rel. Bioact. Mater., 24:759-760, 1997, each of which is incorporated herein by reference in its entirety. In one embodiment, a pump can be used in a controlled release system (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng., 14:20, 1987; Buchwald et al., Surgery, 88:507, 1980; and Saudek et al., N. Engl. J. Med., 321:574, 1989).In another embodiment, polymeric materials can be used to achieve controlled release of drugs comprising the bivalent antigen-binding molecule, or antigen-binding fragments thereof (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NY (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem., 23:61, 1983; also, Levy et al., Science, 228:190, 1985; During et al., Ann. Neurol., 25:351, 1989; Howard et al., J. Neurosurg., 7 1:105, 1989), U.S. Pat. No. 5,679,377, U.S. Pat. No. 5,916,597, U.S. Pat. No. 5,912,015, U.S. Pat. No. 5,989,463, U.S. Pat. No. 5,128,326, PCT Publication No. WO 99 / 15154, and PCT Publication No. WO 99 / 20253. In yet another embodiment, a controlled-release system can be placed in proximity to the therapeutic target (e.g., an affected joint), thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled-release systems are discussed in the review by Langer, Science, 249:1527-1533, 1990.

[0152] Additionally, the rAAV can be used for in vivo delivery of transgenes for scientific research, such as gene knockdown with miRNA, delivery of recombinases for conditional gene deletion, gene editing with CRISPR, etc.

[0153] 5.5. Pharmaceutical Compositions and KitsThe present invention further provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an agent of the invention, the agent comprising an rAAV molecule of the invention comprising a transgene cassette, wherein expression of the transgene is driven by a chimeric regulatory element described herein. In a preferred embodiment, the pharmaceutical composition comprises an rAAV in combination with a pharmaceutically acceptable carrier for administration to a subject. In certain embodiments, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias for use in animals, more specifically in humans. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's complete and incomplete adjuvant), excipient, or vehicle with which the agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, including, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a common carrier when the pharmaceutical composition is administered intravenously or intramuscularly. Physiological saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc.Further examples of pharmaceutically acceptable carriers, excipients, and stabilizers include, but are not limited to, buffers such as phosphate, citric acid, and other organic acids, antioxidants including ascorbic acid, low molecular weight polypeptides, proteins such as serum albumin and gelatin, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming counterions such as sodium, and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONIC™, which are known in the art. In addition to the above ingredients, the pharmaceutical compositions of the present invention may also contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, and preservatives. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.

[0154] In certain embodiments of the invention, pharmaceutical compositions are provided for use in accordance with the methods of the invention, the pharmaceutical compositions comprising a therapeutically and / or prophylactically effective amount of an agent of the invention together with a pharmaceutically acceptable carrier.

[0155] In a preferred embodiment, the agent of the present invention is substantially pure (i.e., substantially free from substances that limit its effectiveness or cause undesirable side effects). In certain embodiments, the host or subject is an animal, preferably a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) and a primate (e.g., monkey, e.g., cynomolgus monkey, and human). In a preferred embodiment, the host is a human.

[0156] The present invention further provides a kit that can be used in the above-mentioned method. In one embodiment, the kit includes, for example, one or more agents of the present invention in one or more containers. In another embodiment, the kit further includes, in one or more containers, one or more other prophylactic or therapeutic agents useful for treating the condition.

[0157] The present invention also provides agents of the present invention packaged in a sealed container, e.g., an ampoule or sachet, indicating the quantity of agent or active agent. In one embodiment, the agent is supplied as a dry, sterile, lyophilized powder or water-free concentrate in a sealed container, which can be reconstituted, e.g., with water or saline, to a concentration suitable for administration to a subject. Typically, the agent is supplied as a dry, sterile, lyophilized powder in a sealed container in a unit dosage of at least 5 mg, more often at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg. Lyophilized agents should be stored in their original container at 2-8°C, and the agent should be administered within 12 hours, usually within 6 hours, 5 hours, 3 hours, or 1 hour, after reconstitution. In an alternative embodiment, agents of the present invention are supplied in liquid form in a sealed container indicating the quantity and concentration of agent or active agent. Typically, the liquid form of the agent is supplied in a sealed container at at least 1 mg / ml, at least 2.5 mg / ml, at least 5 mg / ml, at least 8 mg / ml, at least 10 mg / ml, at least 15 mg / kg, or at least 25 mg / ml.

[0158] Compositions of the present invention include bulk drug compositions (e.g., impure or non-sterile compositions) useful in the manufacture of pharmaceutical compositions and pharmaceutical compositions (i.e., compositions suitable for administration to a subject or patient). Bulk drug compositions can be used, for example, to prepare unit dosage forms containing a prophylactically or therapeutically effective amount of an agent disclosed herein, or a combination of such an agent and a pharmaceutically acceptable carrier.

[0159] The present invention further provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the agents of the present invention. One or more other prophylactic or therapeutic agents useful for treating the target disease or disorder may also be included in the pharmaceutical pack or kit. The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical composition of the present invention. Optionally associated with such container(s) may be a notice in a format prescribed by a governmental authority regulating the manufacture, use, or sale of drugs or biopharmaceuticals, which notice reflects approval by the authority for manufacture, use, or sale for human administration.

[0160] Usually, the components of the composition of the present invention are supplied separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or a water-free concentrate in a sealed container, for example, an ampoule or a sachet, indicating the amount of drug or active agent.When the composition is administered by injection, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline.When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration. [Example]

[0161] 6. Working Example 6.1. Example 1 - Discovery of cis-regulatory elements (CREs) for muscle-specific transgene expression Candidate cis-regulatory element (CRE) sequences derived from the proximal regions of genes specifically or significantly enriched in skeletal or cardiac muscle were identified in the ENCODE database (Davis, C. et al., 2018 Nucleic Acids Res. The Encyclopedia of DNA elements (ENCODE): data portal update; 46(D1):D794-D801. doi:10.1093 / nar / gkx1081). Compact CRE sequences were cloned upstream of the muscle-specific CK promoter in cis-reporter plasmids individually barcoded between the eGFP coding sequence and the RBG polyA tail (Figure 2A). The cis-plasmids were mixed to generate a single vector preparation, or individual preparations could be made and then combined.

[0162] An AAV "promoter" library was generated and then screened for enhancer specificity in vitro by characterizing transgene expression in C2C12 myotubes compared to the CK promoter alone. The enhancer activity of each CRE was semiquantified by calculating the fold change in transgene expression (eGFP fluorescence) normalized to a control vector (containing the CK promoter without an upstream CRE) (Figures 2B and 2C). Cis-regulatory modules (CRMs), Mus007, Mus011, Mus022, and Mus035 (also known as composite enhancer / promoters Mus007.CK, Mus011.CK, Mus022.CK, and Mus035.CK, respectively), were identified as showing a greater than two-fold increase in transgene expression normalized to the control plasmid (CK promoter without an upstream CRE). Mus022 and Mus035 showed significantly increased transgene expression and were selected for further evaluation. The promoter activity of Mus022 was further evaluated in C2C12 myoblasts and expressed as the RNA RA divided by the DNA RA for each barcode within each sample (Figure 2D), demonstrating that Mus022 exhibits higher relative activity in differentiated C2C12 cells compared to the CK7 and Spc5-12 promoters.

[0163] 6.2. Example 2 - Evaluation of Engineered Muscle-Specific Promoters in Mice The selected engineered promoter cassette, containing a novel muscle CRE upstream of the CK promoter region, was further evaluated in mice. Using the same barcoded AAV library (as described in Example 1 above), cis-regulatory modules (CRMs) that confer unique expression profiles to muscle promoters were evaluated in different tissues after injection of vector pools into mice. Briefly, AAV vector preparations were generated according to a 2L-scale version of the protocol in suspension-cultured HEK293 cells triple-transfected with a helper plasmid, a rep / cap plasmid (AAV2 / 9), and a transgene (cis) plasmid. Packaged AAV9 vectors were purified from the supernatant and clarified cell lysates using iodixanol gradient ultracentrifugation and formulated. Vector titers were measured by ddPCR using appropriate transgene-specific primers.

[0164] C57 / BL6 mice (n=5) were administered systemically at 5e13GC / kg via tail vein injection. Promoter activity (RNA RA divided by DNA RA for each barcode within each sample) was calculated and normalized. Normalized promoter activity was calculated by dividing normalized RNA RA by normalized DNA RA. Normalized RNA is the RNA RA (≥1) per barcode multiplied by total RNA copies per TATA box-binding protein (TBP) copy, measured by ddPCR using an eGFP primer probe in cDNA. Normalized DNA is the DNA RA (≥1) per barcode multiplied by the total AAV genome per diploid genome, measured by ddPCR.

[0165] As shown in Table 5 and Figure 3, except for the unexpectedly low activity in mouse heart tissue, the Mus022.CK CRM and Mus035.CK CRM showed similar muscle-specific activity compared to the CK7 and Spc5-12 promoters. Mus022 CRM activity was approximately 5-fold lower in the heart (Figure 3), indicating the effect of combining the Mus022 CRE with the CK7 promoter to "detarget" the CK7-active promoter in the mouse heart. [Table 5]

[0166] 6.3. Example 3: Single Vector Evaluation of CK7 Against a Cardiac "Detargeted" Mus022 Promoter in mdx Mice Individual vector preparations of AAV9 packaging CK7.μDys and Mus022.μDys were generated similarly to the method described above. This cis-plasmid encoded the microdystrophin (μDys) protein. C57BL / 10ScSn-Dmdmdx / J (mdx) mice (n=5) were systemically administered with 5e13GC / kg of each vector. Tissues were harvested 5 weeks after administration. Briefly, 10 mdx mice, 5 mice per group, were administered as follows: Group 1 = AAV9.CK7.μDys and Group 2 = AAV9.Mus022.CK.μDys. Mice were systemically administered via tail vein injection at 5e13GC / kg per group and necropsied 5 weeks after injection. Tissues were harvested and flash-frozen in an isopentane / LN2 double bath. RNA was extracted using a KingFisher Apex kit, and DNA was extracted using a Qiagen DNEASY kit. cDNA was then synthesized using the MAXIMA RT kit with dsDNA SEI, and normalized RNA and DNA levels were assessed using a ddPCR duplex assay. Hearts were sectioned and stained for DysB and laminin.

[0167] A. Quantification of μDys protein The cDNA copies per TBP copy were analyzed via ddPCR in gastrocnemius muscle (GAS), tibialis anterior muscle (TA), quadriceps muscle (Quad), liver, and heart, as depicted in Figure 4A.

[0168] B. Quantification of AAV genomes per diploid genome in tissues DNA biodistribution in GAS, TA, Quad, liver, and heart is depicted in Figure 4B.

[0169] C. Immunofluorescence (IF) staining of human dystrophin in the heart IF staining of human dystrophin reveals similar Mus022 detargeting in mouse hearts at the protein level in cardiac tissue sections comparing mice treated with CK7.μDys and Mus022.μDys, as depicted in Figure 4C .

[0170] D. IF staining of human dystrophin in gastrocnemius muscle (GAS) IF staining of human dystrophin in GAS sections from mice treated with CK7.μDys and Mus022.μDys shows similar protein levels, as depicted in Figure 4D.

[0171] The entire Mus022.CK is a muscle-specific promoter of approximately 800 bp consisting of a 347-bp CRE (SEQ ID NO: 10) and the mouse creatine kinase core promoter. The upstream Mus022 CRE was isolated from the most recent human genome assembly (GRCh38) and is derived from the MYLPF gene (also known as MYL11 or MLC2B), which encodes myosin light chain, a phosphorylatable fast skeletal muscle protein. Mus022.CK showed slightly weaker RNA expression relative to CK7 in mdx skeletal muscle but significantly lower RNA expression in mdx hearts, providing a favorable gene expression profile for operably linking the Mus022.CK promoter to therapeutic genes preferentially delivered to skeletal muscle. IF staining confirmed significant differences in microdystrophin expression between the Mus022.CK and CK7 promoters in mdx hearts.

[0172] 6.4. Example 4: Small Muscle-Specific Promoter Another AAV library constructed similarly to the one described above to generate the vector pool was utilized to evaluate various hybrid promoters driving fluorescent marker transgenes (Figure 5). The muscle promoter pool was evaluated in mdx mice (n = 5) at a systemic dose of 8e13GC / kg. The engineered muscle promoter (EMP) is a novel muscle promoter designed using bioinformatics and rational engineering. Briefly, a library of cis-regulatory element (CRE) sequences was obtained from genes specific to or significantly enriched in skeletal or cardiac muscle, e.g., the proximal region of the alpha (α)-actin (ACTA) gene. Sequence databases such as ENCODE and TRANSFAC were utilized to aid in the identification of key genetic features related to muscle-specific transcriptional activity, such as positive and negative regulators of transcription and transcription factor binding sites. Novel promoters were created by engineering each CRE within a composite sequence containing an upstream enhancer, e.g., eMCK, with or without untranslated regions (UTRs). These were then cloned into AAV vector transgene cassettes with unique barcodes to enable NGS-based evaluation of transgene expression. (Figures 5-7).

[0173] Several iterations of EMPs with novel enhancer regions were designed to further improve transcriptional activity in muscle. These include tandem promoters, such as SPC5v2 upstream of the ACTA2 CRE, and a distinct untranslated region between the TSS and the start codon of the ACTA2 gene (the UTR within the CRE does not contain an ATG, which could aberrantly initiate early transgene transcription). Some EMPs improve muscle expression while increasing or decreasing transcriptional activity in the heart. These EMPs are also compact in size, e.g., less than approximately 600 nucleotide bases, compared to the CAG promoter, which is over 1,600 bases long. Compact promoters are important in gene therapy, allowing the size of gene cassettes (limited to approximately 4.7 kb in AAV) to be maximized with other regulatory elements, RNAi components, RNA stabilization elements, or large transgenes.

[0174] Comparison of the CK7, SPC5-12, and eMCK.mmACTA2shortUTR muscle promoters using NGS was performed in skeletal muscle (GAS, quad, TA), heart, diaphragm, liver, and pancreas, and activity of SPC5-12 and eMCK.mmACTA2shortUTR in each tissue was normalized to CK7 (Figure 8A).

[0175] We also compared the second-generation engineered muscle promoters, SPC5v2.mmACTA2shortUTR and eMCK.SPC5v2.mmACTA2shortUTR, with eMCK.mmACTA2shortUTR and CK7. Using next-generation sequencing (NGS), activity was normalized to CK7 (Figure 8B). Normalized promoter activity was calculated using RNA copies / TBP copies and AAV genomes per diploid genome (Figure 8C). These engineered muscle promoters (EMPs) showed enhanced activity in mouse muscle compared to CK7. The small size (approximately 280 bp) of these promoters, particularly eMCK.mmACTA2shortUTR, allows for the expression of large transgenes via rAAV vectors, which is highly useful due to the limited genome size that can be packaged by AAV. These new promoters may result in strong transcriptional activity in human muscle, ultimately reducing the effective dose level of muscle gene therapy.

[0176] 6.5. Example 5: In vivo studies measuring promoter activity of small muscle-specific promoters driving expression of muscle proteins Eleven muscle-specific promoters (Table 1) selected from previous studies were cloned into barcoded microdystrophin AAV transgene plasmids, similar to Figures 5–7, except that the transgene downstream of the promoter encoded the microdystrophin protein. Each microdystrophin plasmid contained a barcode in the 3' untranslated region (UTR) and between the transgene and poly(A) sequence. Microdystrophin is an engineered protein intended to mimic the function of dystrophin, an essential structural muscle protein with important functions, including maintaining muscle membrane integrity. For each barcoded muscle promoter transgene plasmid, individual AAV vector preparations (packaged in AAVhu32 capsids) were generated by triple transfection and purified by iodixanol gradient. The AAV preparations were pooled with equal contributions to create a single AAV muscle promoter pool consisting of these 11 unique muscle promoters driving the expression of barcoded microdystrophin mRNA. [Table 6]

[0177] Five 6-week-old mice were injected with 1 × 10 AAV muscle promoter pool per mouse. 14The mice were administered at a dose of gc / kg. Tissues were collected 4 weeks after administration, and DNA / RNA was extracted from the collected tissues. The relative abundance (RA) of barcodes in each sample (both RNA and DNA) was determined by next-generation sequencing (NGS) of the barcode amplicons using the Illumina MiSeq platform. Relative promoter activity per muscle promoter in each tissue was determined by dividing the relative abundance (RA) of RNA by the RA of DNA for each promoter. Normalized promoter activity was calculated by dividing normalized RNA by normalized DNA. Normalized RNA was the RA (≥1) of RNA per barcode multiplied by the total RNA copies per TATA box-binding protein (TBP) copy, measured by ddPCR using an eGFP primer probe in cDNA. Normalized DNA was calculated similarly, but using the total AAV genome per diploid genome, measured by ddPCR.

[0178] Two NHPs were also injected with the AAV muscle promoter pool at 1 × 10 14 The mice were administered at a dose of gc / kg, and tissue samples were collected 3 months after administration.Promoter activity was analyzed in the same manner as in the mouse study.

[0179] result

[0180] The muscle promoter was evaluated for activity in different tissues against two controls, CK7 and SPC5v2. In this study, the novel hybrid promoters were confirmed to drive increased transgene expression in skeletal muscle and diaphragm, but either low expression or high expression in cardiac tissue. Depending on the transgene delivered via gene therapy, promoters with low or high expression in the heart, particularly relatively small promoters (less than approximately 1000 bp), may be therapeutically useful. NGS analysis of various mouse tissues revealed that the eMCK.SPC5v2.MMACTA2shortUTR, SPC5v2.MMACTA4, and eMCK.SPC5v2.HuACTA2midUTR promoters drove the strongest expression in skeletal muscle and diaphragm (Figure 9A-D), with expression levels ranging from 2- to 3-fold higher than that of the control muscle promoter, CK7. In the heart, the eMCK.SPC5v2.MMACTA2shortUTR promoter drove the highest expression, up to fourfold higher than CK7 (Figure 9E). Other promoters, such as SPC5v2.MMACTA4 and eMCK.SPC5v2.HuACTA2midUTR, were still more potent than CK7, with nearly twofold higher activity. NGS results from liver samples revealed that all promoters in this pool had similar activity to CK7 and SPC5v2, which are known to be muscle-specific and liver-detargeted promoters (Figure 9F).

[0181] The results of the NHP study were similar, showing that the same three promoters, eMCK.SPC5v2.MMACTA2shortUTR, SPC5v2.MMACTA4, and eMCK.SPC5v2.HuACTA2midUTR, remained the best promoters in skeletal muscle for promoter activity. These promoters were nearly two-fold better than CK7 in the gastrocnemius (GAS), quadriceps (Quad), and tibialis anterior (TA) muscles (Figure 10A-C). Interestingly, the most active promoters in the NHP heart were different from those in the mouse heart; eMCK.HUACTA3 and mSYN100E.HuACTA3 were the strongest cardiac promoters in this study (Figure 10E). In the diaphragm, the best promoter was eMCK.MMACTA2shortUTR, slightly outperforming CK7 (Figure 10D). Finally, all promoters in this study had similar activity in the liver compared to CK7, indicating lower promoter activity in the liver of NHPs for all promoters tested (Figure 10F).

[0182] equivalent Although the present invention has been described in detail with reference to specific embodiments thereof, it will be understood that functionally equivalent variants are within the scope of the present invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0183] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0184] The discussion herein provides a better understanding of the nature of the problems facing the art and should not be construed as an admission of prior art in any way, nor should the citation of any reference herein be construed as an admission that such reference constitutes "prior art" to the instant application.

[0185] All references, including patent applications and publications, cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Many modifications and variations of the present invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are provided by way of example only, and the present invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

1. A recombinant expression cassette comprising a composite nucleic acid regulatory element comprising: a) a muscle cis-regulatory element (CRE); and b) a CK promoter, an Spc5-12 promoter, or a variant thereof, or an ACTA core promoter, operably linked to a transgene.

2. 2. The recombinant expression cassette of claim 1, wherein the muscle CRE is Mus022 (SEQ ID NO: 10), Mus077 (SEQ ID NO: 11), Mus011 (SEQ ID NO: 12), or Mus035 (SEQ ID NO: 13).

3. 3. The recombinant expression cassette of claim 2, wherein the muscle CRE is Mus022 (SEQ ID NO: 10).

4. 4. The recombinant expression cassette of claim 1, wherein the composite nucleic acid regulatory element is Mus022.CK (SEQ ID NO: 9).

5. 2. The recombinant expression cassette of claim 1, wherein the muscle CRE is eMCK (SEQ ID NO: 4), seMCK (SEQ ID NO: 5), mSYN100E (SEQ ID NO: 6), or SPc5v2 (SEQ ID NO: 7).

6. 6. The recombinant expression cassette of any one of claims 1 to 3 or 5, wherein the ACTA promoter is mmACTA1 (SEQ ID NO: 14), mmACTA2 (SEQ ID NO: 15), mmACTA3 (SEQ ID NO: 16), mmACTA4 (SEQ ID NO: 17), huACTA1 (SEQ ID NO: 18), huACTA2 (SEQ ID NO: 19), huACTA3 (SEQ ID NO: 20), mmACTA2-shortUTR (SEQ ID NO: 21), mmACTA2-midUTR (SEQ ID NO: 22), huACTA2-shortUTR (SEQ ID NO: 23), huACTA2-midUTR (SEQ ID NO: 24), mmACTA3-shortUTR (SEQ ID NO: 25), or huACTA3-midUTR (SEQ ID NO: 26).

7. 1. A recombinant expression cassette comprising a composite nucleic acid regulatory element comprising or consisting of a nucleic acid sequence that a) has 99%, 95%, 90%, 85%, or 80% sequence identity to any one of SEQ ID NOs: 9, 27-54, 68, and 69, and / or b) has 1 to 10 nucleotide substitutions compared to any one of SEQ ID NOs: 9, 27-54, 68, and 69, wherein the composite nucleic acid regulatory element retains the biological activity of any one of SEQ ID NOs: 9, 27-54, 68, and 69.

8. 8. The recombinant expression cassette of claim 7, wherein the composite nucleic acid regulatory element comprises any one of the nucleotide sequences of SEQ ID NOs: 9, 27-54, 68, or 69.

9. 9. The recombinant expression cassette of claim 8, wherein the composite nucleic acid regulatory element consists of any one of the nucleotide sequences of SEQ ID NOs: 9, 27-54, 68, or 69.

10. The recombinant expression cassette of any one of claims 1 to 9, further comprising an intron sequence between the composite nucleic acid regulatory element and the transgene.

11. 11. The recombinant expression cassette of any one of claims 1-10, wherein the transgene is a gene or nucleic acid encoding a therapeutic agent listed in Tables 4A-4C.

12. 12. The recombinant expression cassette of claim 11, wherein the transgene is a muscle-derived protein.

13. 12. The recombinant expression cassette of claim 11, wherein the transgene is a mini-dystrophin gene or a micro-dystrophin gene.

14. 14. The recombinant expression cassette of claim 13, wherein the microdystrophin is Dys1 (SEQ ID NO: 73), Dys3 (SEQ ID NO: 74), Dys5 (SEQ ID NO: 75), human MD1 (R4-R23 / ΔCT) (SEQ ID NO: 76), human microdystrophin (SEQ ID NO: 77), Dys3978 (SEQ ID NO: 78), human MD3 (SEQ ID NO: 79), or human MD4 (SEQ ID NO: 80).

15. 11. The recombinant expression cassette of any one of claims 1 to 10, wherein the transgene is a gene or nucleic acid encoding a therapeutic antibody listed in Table 4B, or an antigen-binding fragment thereof.

16. A vector comprising the recombinant expression cassette of any one of claims 1 to 15.

17. The vector of claim 16, further comprising an AAV ITR on either side of the expression cassette.

18. 18. The vector of claim 16 or claim 17, wherein the recombinant expression cassette is suitable for packaging into an ssAAV or scAAV vector.

19. The vector according to any one of claims 16 to 18, and AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, AAV-16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP. and rAAV particles comprising capsid proteins derived from an AAV capsid serotype selected from AAV.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or derivatives, modifications, or pseudotypes thereof.

20. 1. A method for enhancing expression of a transgene in a subject, comprising delivering a viral vector comprising a recombinant expression cassette comprising a composite nucleic acid regulatory element comprising, in 5' to 3' arrangement: a) a Mus022 sequence; b) at least one muscle-specific promoter; c) a transgene; and d) a polyadenylation signal sequence.

21. 21. The method of claim 20, wherein the muscle-specific promoter is the CK promoter (SEQ ID NO: 8).

22. 22. The method of claim 20 or claim 21, wherein the viral vector is administered intravenously or intramuscularly.

23. The method according to any one of claims 20 to 22, wherein expression of the transgene is enhanced in the blood circulation or systemically.

24. The method of any one of claims 20 to 23, wherein expression of the transgene is enhanced in the liver or skeletal muscle.

25. A method of treatment comprising delivering an rAAV comprising the recombinant expression cassette of any one of claims 1 to 15, or the vector of any one of claims 16 to 18, or delivering the rAAV of claim 19.

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