WO2026011009A1

WO2026011009A1 - Compositions and methods for muscle disorders

Info

Publication number: WO2026011009A1
Application number: PCT/US2025/036162
Authority: WO
Inventors: Saurav SESHADRI; Sharif TABEBORDBAR; Shayan TABEBORDBAR
Original assignee: Kate Therapeutics Inc
Current assignee: Kate Therapeutics Inc
Priority date: 2024-07-02
Filing date: 2025-07-01
Publication date: 2026-01-08
Anticipated expiration: 2027-01-02

Abstract

The present invention provides recombinant nucleic acids comprising a primary microRNA (pri-miRNA) scaffold comprising a scaffold sequence of miR-138 or miR-139 together with a guide sequence that targets a gene transcript. Methods of use of such recombinant nucleic acids for inhibiting expression of a gene or gene product are also disclosed herein.

Description

COMPOSITIONS AND METHODS FOR MUSCLE DISORDERS

CLAIM OF PRIORITY

This application claims the benefit of U.S. Provisional Patent Application No.

63/666.905, filed July 2, 2024, which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The invention relates to miRNA molecules.

BACKGROUND

Genetic disorders are a major source of disease burden, and many of them have few or no medical or curative treatments. For example, genetic defects in muscle can result in myopathies and muscular dystrophies due to mutations in genes encoding muscle proteins. Many of these genes encode proteins that provide structural stability or bolster membrane integrity, while others are involved in protein turnover, trafficking, and electrical excitability. When mutations arise, they can lead to devastating consequences for skeletal muscle, leading to impaired ambulation, compromised breathing, and early death in the most severe conditions. microRNAs (miRNAs) are small RNA molecules that can modulate the expression of genes. Synthetic miRNAs have been proposed as a treatment for genetic disorders that involve the expression of a harmful copies of a gene, by modulating that harmful gene expression and reestablishing tissue health.

However, target specificity has remained a hurdle for miRNA-based therapies. Any one miRNA may target many genes, resulting in off-target effects which need to be addressed. Off-target gene silencing can lead to neuro and immunotoxicity and may reduce its therapeutic effects. Thus, there is a need for engineered miRNAs that target a gene of interest with high specificity while resulting in minimal off- target effects.

SUMMARY

The present invention provides composition and methods comprising microRNA (miRNA) scaffold molecules comprising the scaffold miR-138 and miR-139 together with a miRNA that targets a gene transcript, thereby reducing the transgene rnRNA levels and interfering with translation of the transcript. By the present invention, it was discovered that miR-138 and miR-139 scaffolds can be highly expressed and efficiently processed from primary miRs (pri-miRNAs) into mature miR transcripts in muscle tissue, for example, skeletal muscle tissue and cardiac tissue. This allows the present invention to provide for the expression of a mature microRNAs (miRs) comprising a nucleic acid guide targeting a transcript within skeletal and cardiac muscle, resulting in interference/silencing of targeted gene expression. Advantageously, without being bound by a mechanism of action, the miR- 138 and miR-139 scaffolds provide a construct that protects the guide sequence (for example, within the cytoplasm of cells) and allows for safe processing of the guide sequence into a mature miR.

Accordingly, the present invention provides a nucleic acid molecule comprising a pri- miR scaffold having the scaffold sequence of miR-138 or miR-139 and a guide sequence within the scaffold that targets a gene transcript. pri-miR scaffolds together with their guide and passenger sequence may form a hairpin loop structure. The hairpin loop structure may be greater than 250 nucleotides in length (for example, between 250-270 nucleotides in length). The pri-miR may have the structure, in order, of a first (“upstream”) scaffold sequence , the guide sequence, a hairpin loop, a sequence complimentary to the guide sequence (called a “passenger” sequence), and second (“downstream”) scaffold sequence. The guide sequence and passenger sequence form a double stranded RNA (dsRNA), with the first and second scaffold sequence being single stranded RNA (sRNA) on either end of the double stranded molecule (referred to as “arms”). The passenger strand may be fully complementary to the guide sequence or may have one or more mismatched nucleotides (e.g, 1 mismatched nucleotide, 2 mismatched nucleotides, 3 mismatched nucleotides, 4 mismatched nucleotides, 5 or more mismatched nucleotides) to the guide strand. For example, mismatches or additional nucleotides may result in ’’bulges” in the pri-miRNA or the pre-miRNA, while maintaining overall hybridization between the guide strand and passenger strand. The hairpin loop is found at the opposite end of the dsRNA from the first and second scaffold sequences, connecting the 5' and 3' end of the guide and passenger.

The hairpin structure may be processed by cleaving near the junction betw een dsRNA and ssRNA arms. After processing, which may result in substantially cleaving of the ssRNA arms, the pn-miRNAs loop structure is generally 60-100 nucleotides long, and is referred to as a precursor miRNA (pre-miRNA).

The pre-miRNA may be exported to the cytoplasm and further processed by an enzyme, for example (Dicer). The enzyme may process the pre-miRNA at the 5’ and 3' ends of the hairpin by cutting away the loop joining the 3' and 5' arms, resulting in an miRNA(guide):miRNA (passenger) duplex about 21 nucleotides in length. The duplexed miRNA may then by processed to form a precursor to an RNA-induced silencing complex (RISC). The complex may unwind the duplex and the passenger RNA strand may then be discarded, leaving behind a mature RISC carrying the mature, single stranded guide miRNA.

In aspects of the invention, the pri-miR scaffold comprises the scaffold sequence of miR-138.

For example, the nucleic acid molecule (e.g. pri-miR) may comprising in order: a first scaffold sequence of SEQ ID NO: 2; the guide sequence; a loop scaffold sequence of SEQ ID NO: 3; a passenger sequence to the guide sequence; and a second scaffold sequence of SEQ ID NO: 4.

Together, the nucleic acid molecule (e.g. pri-miRNA) may comprise a sequence having at least 95% sequence identity with the defined nucleic acids in SEQ ID NO: 1, where N is any nucleic acid and indicates the position of the guide and the passenger strands within the scaffold. In such instances, the first series of Ns can be a sequence of a guide strand and the second series of Ns can be a sequence of a passenger strand, or vice versa.

In aspects of the invention, the pri-miR scaffold comprises the scaffold sequence of miR-139.

For example, the nucleic acid molecule (e.g. pri-miR) may comprising in order: a first scaffold sequence of SEQ ID NO: 6; the guide sequence; a loop scaffold sequence of SEQ ID NO: 7; a passenger sequence to the guide sequence; and a second scaffold sequence of SEQ ID NO: 8.

Together, the nucleic acid molecule (e.g. pri-miRNA) may comprise a sequence having at least 95% sequence identity with the defined nucleic acids in SEQ ID NO: 5, where N is any nucleic acid and indicates the position of the guide and the passenger strands within the scaffold. In such instances, the first series of Ns can be a sequence of a guide strand and the second series of Ns can be a sequence of a passenger strand, or vice versa.

Guide sequences of the invention may be any guide sequence targeting a gene transcript, e.g. any mRNA transcript for which RNA/translational interference is needed.

Typically, guide sequences are approximately 21-22 nucleotides in length. In aspects of the invention, the guide sequence may be 21 nucleotides in length.

Advantageously, the guide sequence may be a guide sequence targeting a disordered gene transcript expressed in muscle, for example skeletal and cardiac muscle. The guide sequence may target a gene transcript for a disordered muscle protein, for example, double homeobox 4 (DUX4) or troponin (e.g. troponin T (TnT) or troponin (TnC).

Aspects of the invention also provide a nucleic acid molecule encoding pri-miRNAs of the invention. Advantageously, by the present invention it was discovered that when provided to a muscle cells, pri-miRNAs of the invention are expressed (and processed) in muscle cells to form mature miRs.

Encoding nucleic acid molecules of the invention may further comprise a promoter, for example a muscle specific promoter to further driver pri-miR expression. The promoter may be a U6 promoter sequence, MHCK7 promoter sequence, CK6 promoter sequence, tMCK promoter sequence. CK5 promoter sequence, MCK promoter sequence, HAS promoter sequence, MPZ promoter sequence, desmin promoter sequence, APOA2 promoter sequence, hAAT promoter sequence, INS promoter sequence, IRS2 promoter sequence, MYH6 promoter sequence, MYL2 promoter sequence, TNNI3 promoter sequence, SYN1 promoter sequence, GFAP promoter sequence, NES promoter sequence, MBP promoter sequence, or TH promoter sequence.

The promoter may be a cardiac specific promoter. The promoter may be a TNNT2 promoter, MHCK9 promoter, CB A (Chicken P-Actin) promoter, CMV or mini CMV promoter, a Desmin promoter, MYBPC3 promoter, HSPB7 promoter, XIRP1 promoter, ACTA1 promoter, LDB3 promoter, CSRP3 promoter, COX6A2 promoter, HRC promoter, CRY AB promoter, or NMRK2 promoter.

Nucleic acid molecules of the invention may be delivered to cells by any know n method, for example, nucleic acid molecules of the invention may be delivered by lipid nanoparticles (LNP) or viral vectors. The viral vector may be any viral vector, for example an adeno-associated virus (AAV) vector.

Accordingly, aspects of the invention provide an AAV vector comprising a promoter sequence, an encoding nucleic acid molecule of the invention (encoding a miR scaffold and a miR guide sequence that targets a gene transcript), and a capsid protein.

The viral vector may comprise at least one modification that results in reduced livertropism of the AAV vector and/or preferential targeting of the AAV vector to muscle tissue. The capsid protein may comprise at least one modification that is an insertion between any two contiguous amino acids between amino acids 262-269, 327-332, 382-386, 452-460, 488- 505, 527-539, 545-558, 581-593, 704-714, or any combination thereof in an AAV9 capsid polypeptide or in an analogous position in an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, AAV rh. 10 capsid polypeptide.

Aspects of the invention also provide methods for inhibiting expression of a gene or gene product in a cell, the method comprising administering to a subject a composition that results in expression within the cell of a nucleic acid molecules of the invention (comprising a miR scaffold and a miR guide sequence that targets a gene transcript).

In methods of the invention, nucleic acid molecules of the invention may be delivered to cells by any known method. In preferred aspects, the nucleic acid molecules may be delivered by AAV vectors. Advantageously, the AAV vectors may comprise at least one modification results in reduced liver-tropism of the AAV vector and/or preferential targeting of the AAV vector to muscle tissue.

The methods of the invention may result in treatment of myopathy. The myopathy may be a cardiomyopathy, for example hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM). Aspects of the invention may treat a muscular dystrophy. The muscular dystrophy may be facioscapulohumeral muscular dystrophy or myotonic dystrophy type 1. The treatment may comprise arresting the muscular effects of the myopathy or muscular dystrophy. The treatment may comprise reversing the symptoms of the cardiomyopathy or muscular dystrophy.

Accordingly, the present invention provides methods of inhibiting expression of a gene or gene product in a cell, the method comprising providing to a subject a composition comprising a nucleic acid molecule comprising, a pri-miR scaffold having the scaffold sequence of miR-138 or miR-139, and a guide sequence within the scaffold that targets a gene transcript.

Aspects of the present disclosure provide a recombinant nucleic acid comprising a primary microRNA (pri-miRNA) scaffold comprising a scaffold sequence of miR-138 or miR-139; and a guide sequence that targets a gene of interest, wherein the guide sequence is positioned within the scaffold sequence.

In some embodiments, the pri-miRNA scaffold comprises the scaffold sequence of miR-138. In some embodiments, the recombinant nucleic acid comprises, from 5' to 3', a first scaffold sequence comprising or consisting of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 2; the guide sequence; a loop scaffold sequence comprising or consisting of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 3; a passenger sequence that is at least partially complementary to the guide sequence; and a second scaffold sequence comprising or consisting of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 4. In some embodiments, the first scaffold sequence comprises or consists of SEQ ID NO: 2; the loop scaffold sequence comprises or consists of SEQ ID NO: 3; and the second scaffold sequence comprises or consists of SEQ ID NO: 4.

In some embodiments, the pri-miRNA scaffold comprises the scaffold sequence of miR-139. In some embodiments, the recombinant nucleic acid comprises, from 5' to 3', a first scaffold sequence comprising or consisting of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 6; the guide sequence; a loop scaffold sequence comprising or consisting of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 7; a passenger sequence that is at least partially complementary to the guide sequence; and a second scaffold sequence comprising or consisting of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 8.

In some embodiments, the first scaffold sequence comprises or consists of SEQ ID NO: 6; the loop scaffold sequence comprises or consists of SEQ ID NO: 7; and the second scaffold sequence comprises or consists of SEQ ID NO: 8.

In some embodiments, the guide sequence is 21 nucleotides in length.

In some embodiments, the recombinant nucleic acid further comprises a tissue specific promoter. In some embodiments, when provided to a muscle cell, the recombinant nucleic acid molecule comprising the pri-miRNA scaffold and the guide sequence is expressed in muscle cells.

In some embodiments, the recombinant nucleic acid is comprised in a vector, optionally wherein the vector is a viral vector. In some embodiments, the vector is an adeno- associated viral (AAV) vector.

Aspects of the present disclosure provide an adeno-associated virus (AAV) particle comprising a capsid protein, and any one of the recombinant nucleic acids described herein.

In some embodiments, the capsid protein comprises at least one modification that results in reduced liver-tropism of the AAV particle and/or preferential targeting of the AAV particle to muscle tissue. In some embodiments, the capsid protein comprises at least one modification that is an insertion between any two contiguous amino acids between amino acids 262-269, 327-332, 382-386, 452-460, 488-505, 527-539, 545-558, 581-593, 704-714, or any combination thereof in an AAV9 capsid polypeptide or in an analogous position in an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, AAV rh.10 capsid polypeptide.

Aspects of the present disclosure provide a method of inhibiting expression of a gene or gene product in a cell, the method comprising providing to a subject a composition comprising any one of the recombinant nucleic acids described herein.

In some embodiments, providing the recombinant nucleic acid to the subject results in expression of the recombinant nucleic acid molecule in muscle cells of the subject.

In some embodiments, inhibiting expression of the gene or gene product results in treatment of a myopathy or a muscular dystrophy.

In some embodiments, the gene or gene product is a gene or gene product associated with a muscular dystrophy.

In some embodiments, the cell is a muscle cell. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph of pri-miRNA expression using miR-138, miR-139, and miR- 33 scaffolds described herein. The miR-33 scaffold was included as a positive control.

FIG. 2 shows a graph of mature miRNA expression using miR-138, miR-139, and miR-33 scaffolds described herein. The miR-33 scaffold was included as a positive control.

FIGs. 3A-3B show graphs of DUX4 (FIG. 3A) and DUX4 target gene (FIG. 3B) expression in FSHD patient myotubes following DUX4 targeting miR expression using miR- 138, miR-139, and miR-33 scaffolds. The miR-33 scaffold was included as a positive control.

FIG. 4 shows a graph of endogenous troponin T (TNNT2) knockdown in mice using an engineered pri-miRNA including a guide strand targeting TNNT2 positioned within the miR-139 scaffold.

FIG. 5 shows a graph of endogenous troponin C type 1 (TNNC1) knockdown in mice using an engineered pri-miRNA including a guide strand targeting TNNC1 positioned within the miR-139 scaffold.

FIG. 6 shows a graph of endogenous myotonic dystrophy protein kinase (DMPK) knockdown in healthy human myotubes using engineered pri-miRNAs including a DMPK targeting miRNA positioned within the miR-139 scaffold. Healthy human myotubes were transduced using 2E4 or 6E4 vg/cell. Each engineered miRNA represents a unique DMPK targeting miRNA.

DETAILED DESCRIPTION

The present invention provides recombinant nucleic acids comprising a rimary microRNA (pri-miRNA) scaffold comprising a scaffold sequence of miR-138 or miR-139 together with a guide that targets a gene transcript. Methods of use of such recombinant nucleic acids for inhibiting expression of a gene or gene product are also disclosed herein.

Recombinant Nucleic Acids

A “scaffold sequence of miR-138’' refers to the sequence of a naturally occurring miR-138 (e.g, mouse miR-138) outside of the mature guide-passenger strand duplex. A “scaffold sequence of miR-139” refers to the sequence of a naturally occurring miR-139 (e.g, mouse miR-139) outside of the mature guide-passenger strand duplex. A scaffold sequence can comprise a first scaffold sequence, a loop sequence, and a second scaffold sequence.

Recombinant nucleic acids described herein can comprise a pri-miRNA scaffold comprising a scaffold sequence of miR-138. In some embodiments, the scaffold sequence of miR-138 comprises a first scaffold sequence, a loop sequence, and a second scaffold sequence. In some embodiments, the scaffold sequence of miR-138 comprises a first scaffold sequence comprising or consisting of a nucleic acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to of SEQ ID NO: 2; a loop scaffold sequence comprising or consisting of a nucleic acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to of SEQ ID NO: 3; a passenger sequence that is at least partially complementary to the guide sequence: and a second scaffold sequence comprising or consisting of a nucleic acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4. In some embodiments, the scaffold sequence of miR-138 comprises a first scaffold sequence comprising or consisting of SEQ ID NO: 2; a loop scaffold sequence comprising or consisting of SEQ ID NO: 3; and a second scaffold sequence comprising or consisting of SEQ ID NO: 4.

Recombinant nucleic acids described herein can comprise a pri-miRNA scaffold comprising a scaffold sequence of miR-139. In some embodiments, the scaffold sequence of miR-139 comprises a first scaffold sequence, a loop sequence, and a second scaffold sequence. In some embodiments, the scaffold sequence of miR-139 comprises a first scaffold sequence comprising or consisting of a nucleic acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to of SEQ ID NO: 6; a loop scaffold sequence comprising or consisting of a nucleic acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to of SEQ ID NO: 7; a passenger sequence that is at least partially complementary to the guide sequence; and a second scaffold sequence comprising or consisting of a nucleic acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 8. In some embodiments, the scaffold sequence of miR-139 comprises a first scaffold sequence comprising or consisting of SEQ ID NO: 6; a loop scaffold sequence comprising or consisting of SEQ ID NO: 7; and a second scaffold sequence comprising or consisting of SEQ ID NO: 8 Recombinant nucleic acids described herein can comprise a guide sequence that is any nucleotides in length, e.g, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25. 26. 27. 28. 29, or 30 nucleotides in length.

Recombinant nucleic acids described herein can comprise a guide sequence that is at least partially complementary to any target gene or gene product of interest. In some embodiments, the guide sequence is at least partially complementary to a target gene or gene product of interest that is associated with a muscular dystrophy (e.g., facioscapulohumeral muscular dystrophy (FSHD), Duchenne muscular dystrophy (DMD), Limb-girdle muscular dystrophy (LGMD), myotonic dystrophy type 1 (DM1)) or a cardiomyopathy (e.g., cardiomyopathy associated with a genetic mutation in TNNT2 and/or TNNC1).

Recombinant nucleic acids described herein can comprise one or more additional elements, e.g., a promoter, a PolyA signal, an intron, a 5' inverted terminal repeat (ITR) and a 3' ITR, and any combination thereof.

MicroRNA

MicroRNA (miRNA) are small, single-stranded, non-coding RNA molecules. miRNAs base-pair to complementary sequences in mRNA molecules, thereby silencing post- transcriptional regulation of gene expression. Typically, miRNA molecules silence mRNA translation by cleavage of mRNA strand into two pieces or destabilization of the mRNA byshortening its poly(A) tail. miRNAs resemble small interfering RNAs (siRNAs), however miRNAs derive from regions of RNA transcripts that fold back on themselves to form short hairpins.

A miRNA in its final form is a non-coding RNA molecule ~22 nucleotides in length. However, it is initially transcribed as part of one arm of an RNA stem-loop that in turn forms part of a several hundred nucleotide-long miRNA precursor termed a primary miRNA transcript (pri-miRNA). Pri-miRNAs have hairpin structures that are processed by the Drosha enzy me (as part of the microprocessor complex). The microprocessor complex functions byrecognizing and cleaving near the junction between hairpin structure and ssRNA. After Drosha processing, the pri-miRNAs are only 60-100 nucleotides long, and are called precursor miRNAs (pre-miRNAs).

At this point, the pre-miRNA is exported to the cytoplasm, where it typically- encounters the RNAase enzy me Dicer. Dicer interacts with 5’ and 3’ ends of the hairpin and cuts away the loop joining the 3'' and 5’ arms, resulting in an miRNA:miRNA duplex about 22 nucleotides in length. After processing, the duplexed miRNA strands are loaded onto an Argonaute (AGO) protein to form a precursor to an RNA-induced silencing complex (RISC). The complex causes the duplex to unwind and the passenger RNA strand is discarded, leaving behind a mature RISC earn ing the mature, single stranded miRNA. The miRNA remains part of the RISC as it silences the expression of its target genes. Overall hairpin length and loop size influence the efficiency of Dicer processing. Although either strand of the duplex may potentially act as a functional miRNA, only one strand is generally incorporated into the RNA-induced silencing complex (RISC) where the miRNA and its mRNA target interact.

A variety of other pathways have been discovered, including Drosha-independent pathways (such as the mirtron pathway, snoRNA-derived pathway, and shRNA-derived pathway) and Dicer-independent pathways (such as one that relies on AGO for cleavage, and another which is dependent on tRNaseZ)

Advantageously, artificial miRNA molecules may be engineered to comprise the scaffold of endogenous miRNA and a targeting sequence to a gene of interest. Aspects of the invention comprise miRNA molecules that target gene transcripts using the scaffold of miR- 138 or miR-139. miR-138 is a family of microRNA precursors found in animals, including humans. miR-138 is transcribed as a ~70 nucleotide precursor and subsequently processed by the Dicer enzyme to give a ~22 nucleotide product. The excised region or, mature product, of the miR-138 precursor is the microRNA mir-138.

Endogenously, although miR-138 precursor is expressed ubiquitously, the mature product is found only in certain cell types. In adult mice, endogenously miR-138 is only expressed in brain tissue. Its expression is not uniform throughout the brain but restricted to distinct neuronal populations. In the zebrafish, miR-138 is expressed in specific domains in the heart and is required to establish appropriate chamber-specific gene expression patterns. miRNA-based therapies, including miRNA inhibition and miRNA replacement, may be used to treat many diseases such as hepatitis C viral infection, muscular dystrophies, neurodegenerative diseases, peripheral neuropathies, chronic heart failure and post- myocardial infarction remodeling and cancers. In addition, miRNA directed regulation of gene expression may improve traditional gene therapy approaches in which the vector payload is a protein coding gene. microRNA sequences are described in U.S. Patent Publication Nos. 2020-0248179, 2019-0300903. 2019-0136235, 2019-0024083, 2017-0029849, and 2014-0322169. the contents of each of which are incorporated by reference herein. Myopathies and Muscular Dystrophies

A muscle cell, also known as a myocyte, is a mature contractile cell in the muscle of an animal. In humans and other vertebrates there are three types: skeletal, smooth, and cardiac.

Skeletal muscle cells are large and multinucleated cells containing highly organized contractile proteins that interact with each other to generate force and allow movement of the body. Skeletal muscle contains multiple bundles of muscle fibers, with each muscle fibers composed of myofibrils. The myofibrils are composed of actin and myosin filaments called myofilaments, repeated in units called sarcomeres, which are the basic functional, contractile units of the muscle fiber necessary for muscle contraction. Each muscle cell is innerv ated from a single synapse of the motor neuron, with the contact point termed the neuromuscular junction, which is the site where muscle contraction is initiated.

Cardiac muscle cells form the cardiac muscle in the walls of the heart chambers and have a single central nucleus. Cardiac muscle cells are joined to neighboring cells byintercalated discs, and when joined in a visible unit they are described as a cardiac muscle fiber.

Skeletal muscle cells and cardiac muscle cells both contain myofibrils and sarcomeres and form a striated muscle tissue. Neuromuscular junction signals are transduced to the contractile apparatus via a process called excitation contraction coupling (ECC), which is mediated through a specialized structure called the triad.

Muscle diseases are collectively referred to as myopathies and/or muscular dystrophies (MD), depending on the underlying genetic cause and the morphological appearance of the abnormal muscle on biopsy. Defects can arise at essentially all points of the contractile process, from the neuromuscular junction, to the triad, to the contractile apparatus itself, and to the specialized matrix-membrane contacts that maintain and preserve membrane integrity.

Muscular dystrophies are characterized by progressive weakness and degeneration of the skeletal muscles that control movement or breathing. Some forms of MD develop in infancy or childhood, while others may not appear until middle age or later. The disorders differ in terms of the distribution and extent of muscle weakness (some forms of MD also affect cardiac muscle), the age of onset, the rate of progression, and the pattern of inheritance. Facioscapulohumeral Muscular Dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is a complex autosomal dominant disorder characterized by progressive and asymmetric weakness of facial, shoulder and limb muscles. Symptoms typically arise in adulthood with most patients showing clinical features before age thirty. About five percent of patients develop symptoms as infants or juveniles and these are generally more severely affected. Clinical presentation can vary from mild (e.g., some limited muscle weakness) to severe (e.g., wheelchair dependence). Historically, FSHD was classified as the third most common MD, affecting one in 20,000 individuals worldwide. However, recent data indicate FSHD is the most common MD in Europe, suggesting its worldwide incidence could be as high as 1 in 8,333 individuals.

There are two main types of FSHD, which are FSHD type 1 (FSHD1) and FSHD type 2 (FSHD2). Typical FSHD cases (also known as FSHD1A) are linked to heterozygous chromosomal deletions that decrease the copy number of 3.3 kilobase (kb) D4Z4 repeats on human chromosome 4q35. Simplistically, normal individuals have 11-100 tandemly-repeated D4Z4 copies on both 4q35 alleles, while patients with FSHD have one normal and one contracted allele containing 1-10 repeats. In addition, FSHD-associated D4Z4 contractions must occur on specific disease-permissive chromosome 4q35 backgrounds (called 4qA). Importantly, no genes are completely lost or structurally mutated as a result of FSHD- associated deletions. Instead, genetic changes associated with FSHD give rise to expression of the toxic DUX4 gene, which is damaging to muscle. FSHD2 (also known as FSHD IB) is phenotypically identical to FSHD1, is associated with DUX4 expression, and requires the 4qA chromosomal background. FSHD2 is not associated with D4Z4 repeat contraction, but is instead caused by mutation in the SMCHD1 gene, which is a chromatin regulator normally involved in repressing the DUX4 locus at 4qA. Mutated SMCHD1 proteins fail to participate in adding heterochromatin to the 4qA DUX4 allele, thereby allowing DUX4 gene expression.

In the leading FSHD pathogenesis model, D4Z4 contractions are proposed to cause epigenetic changes that permit expression of the DUX4 gene. As a result, the aberrant overexpression of otherwise silent or near-silent DUX4 gene, and the genes it regulates, may ultimately cause FSHD. This model is consistent with data showing normal 4q35 D4Z4 repeats have heterochromatin characteristics, while FSHD-linked D4Z4 repeats contain marks more indicative of actively transcribed euchromatin. These transcription-permissive epigenetic changes, coupled with the observation that complete monosomic D4Z4 deletions (z.e., zero repeats) do not cause FSHD, support the hypothesis that D4Z4 repeats harbor potentially myopathic open reading frames (ORFs), which are abnormally expressed in FSHD muscles. This notion was initially considered in 1994, when a D4Z4-localized ORF, called DUX4, was first identified. However, the locus had some characteristics of an unexpressed pseudogene and DUX4 was therefore summarily dismissed as an FSHD candidate. For many years thereafter, the search for FSHD-related genes was mainly focused outside the D4Z4 repeats, and although some intriguing candidates emerged from these studies, no single gene had been conclusively linked to FSHD development. This slow progress led to the re-emergence of DUX4 as an FSHD candidate in 2007.

The role of DUX4 in FSHD pathogenesis can be explained as follows. First, D4Z4 repeats contain identical DUX4 coding regions, and D4Z4 repeats also harbor smaller sense and antisense transcripts, including some resembling microRNAs. Over-expressed DUX4 transcripts and an about 50 kDa full-length DUX4 protein are found in biopsies and cell lines from FSHD patients. These data are consistent with a transcriptional de-repression model of FSHD pathogenesis. In addition, unlike pseudogenes, D4Z4 repeats and DUX4 likely have functional importance, since tandemly-arrayed D4Z4 repeats are conserved in at least eleven different placental mammalian species (non-placental animals lack D4Z4 repeats), with the greatest sequence conservation occurring within the DUX4 ORF. Second, over-expressed DUX4 is toxic to tissue culture cells and embryonic progenitors of developing lower organisms in vivo. This toxicity occurs at least partly through a pro-apoptotic mechanism, indicated by caspase-3 activation in DUX4 transfected cells, and presence of TUNEL- positive nuclei in developmentally arrested Xenopus embryos injected with DUX4 mRNA at the two-cell stage. These findings are consistent with studies showing some pro-apoptotic proteins, including caspase-3, are present in FSHD patient muscles. In addition to stimulating apoptosis, DUX4 may negatively regulate myogenesis. Human DUX4 inhibits differentiation of mouse C2C12 myoblasts in vitro, potentially by interfering with PAX3 and/or PAX7, and causes developmental arrest and reduced staining of some muscle markers when delivered to progenitor cells of zebrafish or Xenopus embryos. Finally, aberrant DUX4 function is directly associated with potentially important molecular changes seen in FSHD patient muscles. Specifically, full-length human DUX4 encodes an approximately 50 kDa double homeodomain transcription factor, and DUX4 targets can be found at elevated levels in FSHD patient muscles. These data support that DUX4 catalyzes numerous downstream molecular changes that are incompatible with maintaining normal muscle integrity. Cardiomyopathies

Cardiomyopathies are a class of disease of heart muscle that adversely impacts the heart’s ability to circulate blood through the cardiovascular system. Various types of cardiomyopathies exist, including hypertrophic cardiomyopathy (HCM), a condition characterized by thickening (hypertrophy) of the cardiac muscle, dilated cardiomyopathy (DCM), a condition characterized by enlargement and thinning of the left ventricle, or restrictive cardiomyopathy (RCM), a condition characterized by stiffening of cardiac muscle.

Troponin is a central regulatory protein of striated muscle contraction, and together with tropomyosin, is located on the actin fdament. Troponin consists of 3 subunits: inhibitor troponin I (Tnl), which is the inhibitor of actomyosin ATPase; troponin T (TnT); and troponin C (TnC).

Cardiac troponin T (cTnT) in cardiac muscle cells binds tropomyosin to regulate heart contractions and is encoded by the TNNT2 gene. Mutations in the TNNT2 gene can cause familial HCM, DCM, and RCM. Most TNNT2 gene mutations in familial hypertrophic cardiomyopathy or dilated cardiomyopathy change single amino acids in the cTnT protein. The altered protein is incorporated into the troponin complex, resulting in possible dysfunction.

Troponin C (TnC) is found in the troponin complex on actin thin filaments of striated muscle (cardiac, fast-twitch skeletal, or slow-twitch skeletal), and is responsible for binding calcium to activate muscle contraction. Troponin C is encoded by the TNNC1 gene in humans for both cardiac and slow skeletal muscle. TnC works in concert with inhibitor troponin I (Tnl) and TnT, which provides a direct link to tropomyosin and assists in transducing the contractile signal to the rest of the thin filament. Recently, cTnC mutations have been identified as associated with causing both HCM and DCM.

Adeno Associated Virus Vector

Any of the recombinant nucleic acids described herein can be included in a recombinant expression vector (e.g., a viral vector (e.g.. an AAV vector (e.g., a rAAV vector))).

AAVs are particularly appropriate viral vectors for delivery of genetic material into mammalian cells. AAVs are not known to cause disease in mammals and cause a very mild immune response. Additionally, AAVs are able to infect cells in multiple stages whether at rest or in a phase of the cell replication cycle. Advantageously, AAV DNA is not regularly inserted into the host’s genome at random sites, reducing the oncogenic properties of this vector.

AAVs have been engineered to deliver a variety of treatments, especially for genetic disorders caused by single nucleotide polymorphisms (“SNP”). Genetic diseases that have been studied in conjunction with AAV vectors include Cystic fibrosis, hemophilia, arthritis, macular degeneration, muscular dystrophy, Parkinson’s disease, congestive heart failure, and Alzheimer’s disease. The AAV can be used as a vector to deliver engineered nucleic acid to a host and utilize the host’s own ribosomes to transcribe that nucleic acid into the desired proteins. See, e.g, West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); and Muzyczka, J. Clin. Invest. 94: 1351 (1994). AAVs have some deficiency in their replication and/or pathogenicity and thus can be safer that adenoviral vectors. In some embodiments, the AAV can integrate into a specific site on chromosome 19 of a human cell with no observable side effects. In some embodiments, the capacity of the AAV vector, system thereof, and/or AAV particles can be up to about 4.7 kb. The AAV vector or system thereof can include one or more engineered capsid polynucleotides described herein.

AAVs are small, replication-defective, nonenveloped viruses that infect humans and other primate species and have a linear single-stranded DNA genome. Naturally occurring AAV serotypes exhibit liver tropism. As a result, transfection of non-liver tissue with traditional AAV vectors is impeded by the virus’s natural liver tropism. Moreover, because the liver acts to break down substances delivered to a subject, transfection of non-liver tissue with unmodified AAV vectors requires higher dosing to provide sufficient viral load to overcome the liver and reach non-liver tissue. More than 30 naturally occurring serotypes of AAV are available. Many natural variants in the AAV capsid exist. AAV serotypes include, but are not limited to, AAV serotypes AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAV13. AAVs may be engineered using conventional molecular biology techniques, making it possible to optimize these particles, for example, for cell specific delivery, for minimizing immunogenicity, for tuning stability and particle lifetime, for efficient degradation, for accurate delivery to the nucleus. AAV vectors can be specifically targeted to one or more types of cells by choosing the appropriate combination of AAV serotype, promoter, and delivery method.

Previous approaches to identify AAV sequences correlated with tropism have relied upon the comparison of highly related extant serotypes with distinct characteristics, random domain swaps between unrelated serotypes, or consideration of higher-order structure, to identify motifs that define liver tropism. For example, mapping determinants of AAV tropism have been carried out by comparing highly related serotypes. One such example is the singleamino acid change (E531K) between AAV1 and AAV6 that improves murine liver transduction in AAV1. See, e.g., Wu et al. (2006) J. Virol., 80(22): 11393-7, incorporated by reference herein. Another example is a reciprocal domain swap between AAV2 and AAV8 that alters tropism, but fails to define any robust specific tissue-targeting motifs. See. e.g., Raupp et al. (201) J. Virol., 86(17):9396-408, incorporated by reference herein. Further, global consideration of structure has only highlighted gross differences between better- or worse-liver-transducers that are more observational than useful in practice. Nam et al (2007) J. Virol., 81(22): 12260-71.

AAVs exhibiting modified tissue tropism that may be used with the present invention are described in U.S. Patent No. 9,695,220, U.S. Patent No. 9,719,070; U.S. Patent No. 10,119,125; U.S. Patent No. 10,526,584; U.S. Patent Application Publication No. 2018- 0369414; U.S. Patent Application Publication No. 2020-0123504; U.S. Patent Application Publication No. 2020-0318082; PCT International Patent Application Publication No. WO 2015/054653; PCT International Patent Application Publication No. WO 2016/179496; PCT International Patent Application Publication No. WO 2017/100791 ; and PCT International Patent Application Publication No. WO 2019/217911, the entirety of the contents of each of which are incorporated by reference herein.

The AAV vector or system thereof may include one or more regulatory molecules, such as promoters, enhancers, repressors and the like. In some embodiments, the AAV vector or system thereof can include one or more polynucleotides that can encode one or more regulatory' proteins. In some embodiments, the one or more regulatory' proteins can be selected from Rep78, Rep68, Rep52, Rep40, variants thereof, and combinations thereof. In some embodiments, the muscle specific promoter can drive expression of an engineered AAV capsid polynucleotide.

The AAV vector or system thereof can include one or more polynucleotides that can encode one or more capsid proteins, such as the engineered AAV capsid proteins described elsewhere herein. The engineered capsid proteins can be capable of assembling into a protein shell (an engineered capsid) of the AAV virus particle. The engineered capsid can have a cell-, tissue-, and/or organ-specific tropism.

The AAV vector or system thereof can be configured to produce AAV particles having a specific serotype. In some embodiments, the serotype can be AAV-1, AAV -2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, AAV-9 or any combinations thereof. In some embodiments, the AAV can be AAV1, AAV-2, AAV-5, AAV-9 or any combination thereof. One can select the AAV of the AAV with regard to the cells to be targeted; e.g., one can select AAV serotypes 1, 2, 5, 9 or a hybrid capsid AAV-1, AAV-2, AAV-5, AAV-9 or any combination thereof for targeting brain and/or neuronal cells; and one can select AAV-4 for targeting cardiac tissue; and one can select AAV-8 for delivery to the liver. Thus, in some embodiments, an AAV vector or system thereof capable of producing AAV particles capable of targeting the brain and/or neuronal cells can be configured to generate AAV particles having serotypes 1, 2, 5 or a hybrid capsid AAV-1, AAV-2, AAV-5 or any combination thereof. In some embodiments, an AAV vector or system thereof capable of producing AAV particles capable of targeting cardiac tissue can be configured to generate an AAV particle having an AAV-4 serotype. In some embodiments, an AAV vector or system thereof capable of producing AAV particles capable of targeting the liver can be configured to generate an AAV having an AAV-8 serotype. See also Srivastava. 2017. Curr. Opin. Virol. 21 :75-80.

It will be appreciated that while the different serotypes can provide some level of cell, tissue, and/or organ specificity, each serotype still is multi-tropic and thus can result in tissuetoxicity if using that serotype to target a tissue that the serotype is less efficient in transducing. Thus, in addition to achieving some tissue targeting capacity’ via selecting an AAV of a particular serotype, it will be appreciated that the tropism of the AAV serotype can be modified by an engineered AAV capsid described herein. As described elsewhere herein, variants of wild-type AAV of any serotype can be generated via a method described herein and determined to have a particular cell-specific tropism, which can be the same or different as that of the reference wild-type AAV serotype. In some embodiments, the cell, tissue, and/or specificity of the wild-type serotype can be enhanced (e.g., made more selective or specific for a particular cell type that the serotype is already biased towards). For example, wild-type AAV-9 is biased towards muscle and brain in humans (see, e.g., Srivastava. 2017. Curr. Opin. Virol. 21 :75-80.) By including an engineered AAV capsid and/or capsid protein variant of wild-type AAV-9 as described herein, the tropism for nervous cells might be reduced or eliminated and/or the muscle specificity increased such that the nervous specificity appears reduced in comparison, thus enhancing the specificity for muscle as compared to the wild-type AAV-9. As previously mentioned, inclusion of an engineered capsid and/or capsid protein variant of a wild-type AAV serotype can have a different tropism than the wild-type reference AAV serotype. For example, an engineered AAV capsid and/or capsid protein variant of AAV-9 can have specificity for a tissue other than muscle or brain in humans. In some embodiments, the AAV vector is a hybrid AAV vector or system thereof. Hybrid AAVs are AAVs that include genomes with elements from one serotype that are packaged into a capsid derived from at least one different serotype. For example, if it is the rAAV2/5 that is to be produced, and if the production method is based on the helper-free, transient transfection method discussed above, the 1st plasmid and the 3rd plasmid (the adeno helper plasmid) will be the same as discussed for rAAV2 production. However, the 2nd plasmid, the pRepCap will be different. In this plasmid, called pRep2/Cap5, the Rep gene is still derived from AAV2, while the Cap gene is derived from AAV5. The production scheme is the same as the above-mentioned approach for AAV2 production. The resulting rAAV is called rAAV2/5, in which the genome is based on recombinant AAV2, while the capsid is based on AAV5. It is assumed the cell or tissue-tropism displayed by this AAV2/5 hybrid virus should be the same as that of AAV5. It will be appreciated that wild-type hybrid AAV particles suffer the same specificity issues as with the non-hybrid wild-tj pe serotypes previously discussed.

Advantages achieved by the wild-type based hybrid AAV systems can be combined with the increased and customizable cell-specificity that can be achieved with the engineered AAV capsids can be combined by generating a hybrid AAV that can include an engineered AAV capsid described elsewhere herein. It will be appreciated that hybrid AAVs can contain an engineered AAV capsid containing a genome with elements from a different serotype than the reference wild-type serotype that the engineered AAV capsid is a variant of. For example, a hybrid AAV can be produced that includes an engineered AAV capsid that is a variant of an AAV-9 serotype that is used to package a genome that contains components (e.g, rep elements) from an AAV -2 serotype. As with wild-type based hybrid AAVs previously discussed, the tropism of the resulting AAV particle will be that of the engineered AAV capsid.

In some embodiments, the AAV vector or system thereof is configured as a “gutless” vector, similar to that described in connection with a retroviral vector. In some embodiments, the “gutless” AAV vector or system thereof can have the cis-acting viral DNA elements involved in genome amplification and packaging in linkage with the heterologous sequences of interest (e.g, the engineered AAV capsid polynucleotide(s)).

The vectors described herein can be constructed using any suitable process or technique. In some embodiments, one or more suitable recombination and/or cloning methods or techniques can be used to the vector(s) described herein. Suitable recombination and/or cloning techniques and/or methods can include, but not limited to, those described in U.S. Application publication No. US 2004-0171156 Al. Other suitable methods and techniques are described elsewhere herein.

Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81 :6466-6470 (1984); and Samulski et al.. J. Virol. 63:03822-3828 (1989). Any of the techniques and/or methods can be used and/or adapted for constructing an AAV or other vector described herein. AAV vectors are discussed elsewhere herein.

In some embodiments, the vector can have one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a “cloning site'’). In some embodiments, one or more insertion sites (e. , about or more than about 1. 2, 3, 4. 5, 6, 7, 8. 9, 10, or more insertion sites) are located upstream and/or downstream of one or more sequence elements of one or more vectors.

Delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for expression of one or more elements of a engineered AAV capsid system described herein are as used in the foregoing documents, such as International Patent Application Publications WO 2021/050974, WO 2021/077000 and WO 2022/020616, the contents of which are incorporated by reference herein.

Additional AAV vectors are described in International Patent Application Publication WO 2019/2071632, the contents of which are incorporated by reference herein.

Further AAV vectors are described in International Patent Application Publications WO 2020/086881 and WO 2020/235543, the contents of each of which are incorporated by reference herein.

Further AAV vectors are described in International Patent Application Publications WO 2005/033321; WO 2006/110689; WO 2007/127264; WO 2008/027084; WO 2009/073103; WO 2009/073104; WO 2009/105084; WO 2009/134681; WO 2009/136977; WO 2010/051367; WO 2010/138675; WO 2001/038187; WO 2012/112832; WO 2015/054653; WO 2016/179496; WO 2017/100791; WO 2017/019994; WO 2018/209154; WO 2019/067982; WO 2019/195701; WO 2019/217911; WO 2020/041498; WO 2020/210839; U.S. Patent No. 7,906,1 11; U.S. Patent No. 9,737,618; U.S. Patent No.10,265,417; U.S. Patent No. 10,485,883; U.S. Patent No. 10,695,441; U.S. Patent No. 10,722.598; U.S. Patent No. 8.999,678; U.S. Patent No. 10,301,648; U.S. Patent No. 10,626.415; U.S. Patent No. 9.198,984; U.S. Patent No. 10.155,931; U.S. Patent No. 8,524,219; U.S. Patent No. 9,206,238; U.S. Patent No. 8,685,387; U.S. Patent No. 9,359,618; U.S. Patent No. 8,231,880; U.S. Patent No. 8,470.310; U.S. Patent No. 9,597,363; U.S. Patent No. 8.940,290; U.S. Patent No. 9,593,346; U.S. Patent No. 10.501,757; U.S. Patent No. 10,786,568; U.S. Patent No. 10,973,928; U.S. Patent No. 10,519,198; U.S. Patent No. 8,846,031; U.S. Patent No. 9,617,561; U.S. Patent No. 9,884,071; U.S. Patent No. 10,406,173; U.S. Patent No. 9,596,220; U.S. Patent No. 9,719,010; U.S. Patent No. 10,117.125; U.S. Patent No. 10,526,584; U.S. Patent No. 10,881,548; U.S. Patent No. 10.738.087; U.S. Patent Publication No. 2011-023353; U.S. Patent Publication No. 2019- 0015527; U.S. Patent Publication No. 2020-155704; U.S. Patent Publication No 2017- 0191079; U.S. Patent Publication No. 2019-0218574; U.S. Patent Publication No. 2020- 0208176; U.S. Patent Publication No. 2020-0325491; U.S. Patent Publication No. 2019- 0055523; U.S. Patent Publication No. 2020-0385689; U.S. Patent Publication No. 2009- 0317417; U.S. Patent Publication No. 2016-0051603; U.S. Patent Publication No. 2016- 00244783; U.S. Patent Publication No. 2017-0183636; U.S. Patent Publication No. 2020- 0263201; U.S. Patent Publication No. 2020-0101099; U.S. Patent Publication No. 2020- 0318082; U.S. Patent Publication No. 2018-0369414; U.S. Patent Publication No. 2019- 0330278; U.S. Patent Publication No. 2020-0231986, the contents of each of which are incorporated by reference herein.

Promoters

Recombinant nucleic acids comprising a pri-miRNA described herein may also include a muscle specific promoter or another promoter. The promoter may be linked to the nucleic acid sequence so that the transcription preferably occurs within myocytes. Promoter regions enable the host cells to replicate the AAV delivered nucleic acid only in those cell types and tissues or organs in which the desired protein should be created. Here, the muscle specific promoter is included because it is principally desired that the proteins only be translated in myocytes. Specificity of the cell type into which the nucleic acid is delivered and thus the proteins translated is desired because of the adverse effects that may ensue from delivering the nucleic acid and having it translated in cells in which that nucleic acid and thus protein is not needed.

The myocyte specific promoter may be coupled or otherwise associated with a truncated DUX4 sequence. In some embodiments, the promoter may be directly attached, and in others there may be a linker molecule or another indirect coupling method to attach to the truncated DUX4 sequence. In some embodiments, there may be an associated polypeptide or other particle that is coupled to the truncated DUX4 sequence. In some embodiments, the muscle specific promoter yields increased muscle cell potency, muscle cell specificity, reduced immunogenicity, or any combination thereof. As used herein the terms “muscle-specific”, “muscle cell specificity”, “muscle cell potency,” “myocyte specific” and the like, refer to the increased specificity, selectivity, or potency, of the muscle-specific targeting moieties and compositions incorporating said muscle-specific targeting moieties of the present invention for myocytes relative to non-muscle cells. In some embodiments, the cell specificity, or selectivity, or potency, or a combination thereof of a muscle-specific targeting moiety or composition incorporating a muscle-specific targeting moiety⁷ described herein is at least 2 to at least 500 times more specific, selective, and/or potent for/in a muscle cell relative to a non-muscle cell.

In some embodiments, the promoter comprises a cardiac specific promoter. In some embodiments, the promoter is TNNT2. In some embodiments, the promoter is MHCK9. In some embodiments, the promoter is MHCK.7. In some embodiments, the promoter is CBA (chicken P-actin). In some embodiments, the promoter is CMV or mini CMV. In some embodiments, the promoter is a desmin promoter.

In some embodiments, the myocyte-selective promoter utilized is MHCK.7. MHCK7is a 770 base pair length promoter that is small enough to be included in an AAV vector. MHCK7 directs expression in fast and slow skeletal and cardiac muscle, with low expression in the liver, lung, and spleen. It is less active in smooth muscle. The MHCK7 promoter is associated with high levels of expression in skeletal muscles, including the diaphragm, and includes an enhancer to especially drive expression in the heart, whereas expression in off- target tissues is minimal.

In some embodiments, the promoters described herein are inserted into an AAV protein (e.g., an AAV capsid protein) that has reduced specificity (or no detectable, measurable, or clinically relevant interaction) for one or more non-muscle cell types. Exemplary non-muscle cell types include, but are not limited to, liver, kidney, lung, heart, spleen, central or peripheral nervous system cells, bone, immune, stomach, intestine, eye, skin cells and the like. In some embodiments, the non-muscle cells are liver cells.

The term “operably linked” refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other.

Further exemplary tissue specific promoters include U6 promoter sequence, MHCK7 promoter sequence, CK6 promoter sequence, tMCK promoter sequence, CK5 promoter sequence, MCK promoter sequence, HAS promoter sequence, MPZ promoter sequence, desmin promoter sequence, APOA2 promoter sequence, hAAT promoter sequence, INS promoter sequence, IRS2 promoter sequence, MYH6 promoter sequence, MYL2 promoter sequence, TNNI3 promoter sequence, SYN1 promoter sequence, GFAP promoter sequence, NES promoter sequence, MBP promoter sequence, or TH promoter sequence.

Muscle specific promoters are described in International Patent Application Publications WO 2020/006458 and WO 2021/126880, the contents of each of which are incorporated by reference herein.

Further muscle specific promoters are described in U.S. Patent No. 9,133,482; U.S. Patent No. 10,105,453; U.S. Patent No. 10,301,367; U.S. Patent Publication No. 2020- 0360534: PCT International Patent Publication Nos. WO 2020/006458; WO 2021/035120: WO 2021/053124; and WO 2021/077000. the contents of each of which are incorporated by reference herein.

It may be convenient to use an RNA polymerase II or III promoter; these are known to the person skilled in the art and reviewed in, e.g., Kornberg 1999. However, transcripts from an RNA II polymerase often have complex transcription terminators and transcripts are polyadenylated; this may hamper with the requirements of the miRNA strand which because both its 5' and 3' ends need to be precisely defined in order to achieve the required secondary structure to produce a functional molecule. These drawbacks can how ever be circumvented. In case an RNA polymerase II or III promoter is used, the polynucleotide encoding the miRNA strand may also encode self-processing ribozymes and may be operably linked to an RNA polymerase II or III promoter; as such the polynucleotide encodes a pre- miRNA strand comprising the miRNA strand and self-processing ribozymes, wherein, when transcribed, the miRNA strand is released by the self-processing ribozymes from the pre- miRNA strand de transcript.

Preferably, in a composition according to the present invention the AAV vector is comprised of an RNA polymerase II promoter or III promoter, and encodes a pre- miRNA strand comprising the miRNA strand and self-processing ribozy mes, wherein, when transcribed, the miRNA strand is released by the self-processing ribozymes from the pre- miRNA strand transcript. Conveniently, multiple pre-miRNA strands and multiple selfprocessing ribozymes may be encoded by a single polynucleotide, operably linked to one or more RNA polymerase II promoters.

RNA polymerase II or III promoters that are inducible and/or tissue-specific have been previously described. RNA polymerase promoters are known in the art and further described in U.S. Patent Publication 11,149,288, the contents of which is incorporated by reference herein.

Viral Particles and Capsid Proteins

Any of the recombinant nucleic acids or recombinant expression vectors (e.g, recombinant expression vector (e.g.. viral vector (e.g, AAV (e.g, rAAV))) described herein can be packaged or encapsulated in a viral particle (e.g, AAV particle). Viral particles for use in compositions and methods described herein can include any viral capsid protein (e.g, AAV capsid protein or variant thereof) known in the art or described herein.

The capsid protein is the shell or coating of the virus that enables its delivery into the host. Without the protein, the nucleic acids would be destroyed by the host without entering into the host cells and beginning transcription and translation. The capsid protein may be in the natural conformation of a naturally occurring AAV, or it may be modified.

In certain example embodiments, the AAV capsid protein is an engineered AAV capsid protein having reduced or eliminated uptake in a non-muscle cell as compared to a corresponding wild-type AAV capsid polypeptide.

In some embodiments, the engineered AAV capsid encoding polynucleotide can be included in a polynucleotide that is configured to be an AAV genome donor in an AAV vector system that can be used to generate engineered AAV particles described elsewhere herein. In some embodiments, the engineered AAV capsid encoding polynucleotide can be operably coupled to a poly adenylation tail. In some embodiments, the poly adenylation tail can be an SV40 poly adenylation tail. In some embodiments, the AAV capsid encoding polynucleotide can be operably coupled to a promoter. In some embodiments, the promoter can be a tissue specific promoter. In some embodiments, the tissue specific promoter is specific for muscle (e.g., cardiac, skeletal, and/or smooth muscle), neurons and supporting cells (e.g., astrocytes, glial cells, Schwann cells, etc.), fat, spleen, liver, kidney, immune cells, spinal fluid cells, synovial fluid cells, skin cells, cartilage, tendons, connective tissue, bone, pancreas, adrenal gland, blood cell, bone marrow cells, placenta, endothelial cells, and combinations thereof. In some embodiments, the promoter can be a constitutive promoter. Suitable tissue specific promoters and constitutive promoters are discussed elsewhere herein and are generally known in the art and can be commercially available. Suitable muscle specific promoters include, but are not limited to CK8, MHCK7, Myoglobin promoter (Mb). Desmin promoter, muscle creatine kinase promoter (MCK) and variants thereof, and SPc5-12 synthetic promoter. Described herein are various embodiments of engineered viral capsids, such as adeno- associated virus (AAV) capsids, that can be engineered to confer cell-specific tropism, such as muscle specific tropism, to an engineered viral particle. Engineered viral capsids can be lentiviral, retroviral, adenoviral, or AAV capsids. The engineered capsids can be included in an engineered vims particle (e.g., an engineered lentiviral, retroviral, adenoviral, or AAV virus particle), and can confer cell-specific tropism, reduced immunogenicity, or both to the engineered viral particle. The engineered viral capsids described herein can include one or more engineered viral capsid proteins described herein. The engineered viral capsids described herein can include one or more engineered viral capsid proteins described herein that can contain a muscle-specific targeting moiety containing or composed of an n-mer motif described elsewhere herein.

The engineered viral capsid and/or capsid proteins can be encoded by one or more engineered viral capsid polynucleotides. In some embodiments, the engineered viral capsid polynucleotide is an engineered AAV capsid polynucleotide, engineered lentiviral capsid polynucleotide, engineered retroviral capsid polynucleotide, or engineered adenovirus capsid polynucleotide. In some embodiments, an engineered viral capsid polynucleotide e.g., an engineered AAV capsid polynucleotide, engineered lentiviral capsid polynucleotide, engineered retroviral capsid polynucleotide, or engineered adenovirus capsid polynucleotide) can include a 3' poly adenylation signal. The polyadenylation signal can be an SV40 polyadenylation signal.

The engineered viral capsids can be variants of wild-type viral capsid. For example, in some embodiments, the engineered AAV capsids can be variants of wild-type AAV capsids. In some embodiments, the wild-type AAV capsids can be composed of VP1, VP2, VP3 capsid proteins or a combination thereof. In other words, the engineered AAV capsids can include one or more variants of a wild-type VP1, wild-type VP2, and/or wild-type VP3 capsid proteins. In some embodiments, the serotype of the reference wild-type AAV capsid can be AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, AAV-9 or any combination thereof. In some embodiments, the serotype of the wild-type AAV capsid can be AAV-9. The engineered AAV capsids can have a different tropism than that of the reference wild-type AAV capsid.

The engineered viral capsid can contain 1-60 engineered capsid proteins. In some embodiments, the engineered viral capsids can contain 1, 2, 3, 4, 5, 6, 7, 8, 9. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26. 27. 28. 29. 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 engineered capsid proteins. In some embodiments, the engineered viral capsid can contain 0- 59 wild-type viral capsid proteins. In some embodiments, the engineered viral capsid can contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 wild-ty pe viral capsid proteins.

In some embodiments, the engineered AAV capsid can contain 1 -60 engineered capsid proteins. In some embodiments, the engineered AAV capsids can contain 1. 2, 3, 4. 5,

6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 engineered capsid proteins. In some embodiments, the engineered AAV capsid can contain 0-59 wild-type AAV capsid proteins. In some embodiments, the engineered AAV capsid can contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 wild-type AAV capsid proteins.

In some embodiments, the engineered viral capsid protein can have an n-mer amino acid motif, where n can be at least 3 amino acids. In some embodiments, n can be 3, 4, 5, 6,

7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In some embodiments, an engineered AAV capsid can have a 6-mer or 7-mer amino acid motif. In some embodiments, the n-mer amino acid motif can be inserted between two amino acids in the wild-type viral protein (VP) (or capsid protein). In some embodiments, the n-mer motif can be inserted between two amino acids in a variable amino acid region in a viral capsid protein.

In some embodiments, the n-mer motif can be inserted between two amino acids in a variable amino acid region in an AAV capsid protein. The core of each wild-type AAV viral protein contains an eight-stranded beta-barrel motif (betaB to betal) and an alpha-helix (alphaA) that are conserved in autonomous parv ovirus capsids (see, e.g., DiMattia et al. 2012. J. Virol. 86(12):6947-6958). Structural variable regions (VRs) occur in the surface loops that connect the beta-strands, which cluster to produce local variations in the capsid surface. AAVs have 12 variable regions (also referred to as hypervariable regions) (see e.g., Weitzman and Linden. 2011. “Adeno- Associated Virus Biology.” In Snyder, R.O., Moullier, P. (eds.) Totowa, NJ: Humana Press). In some embodiments, one or more n-mer motifs can be inserted between two amino acids in one or more of the 12 variable regions in the wildtype AVV capsid proteins. In some embodiments, the one or more n-mer motifs can be each be inserted between two amino acids in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-III, VR-IX, VR-X. VR-XI, VR-XII, or a combination thereof. In some embodiments, the rt-mer can be inserted between two amino acids in the VR-III of a capsid protein. In some embodiments, the engineered capsid can have an /?-mer inserted between any two contiguous amino acids between amino acids 262 and 269, between any two contiguous amino acids between amino acids 327 and 332, between any two contiguous amino acids between amino acids 382 and 386, between any two contiguous amino acids between amino acids 452 and 460, between any two contiguous amino acids between amino acids 488 and 505, between any two contiguous amino acids between amino acids 545 and 558, between any two contiguous amino acids between amino acids 581 and 593, between any two contiguous amino acids between amino acids 704 and 714 of an AAV9 viral protein. In some embodiments, the engineered capsid can have an /7-mer inserted between amino acids 588 and 589 of an AAV9 viral protein. In some embodiments, the engineered capsid can have a 7- mer motif inserted between amino acids 588 and 589 of an AAV9 viral protein. In other embodiments, the motif inserted is a 10-mer motif, with replacement of amino acids 586-88 and an insertion before 589. It will be appreciated that w-mers can be inserted in analogous positions in AAV viral proteins of other serotypes. In some embodiments as previously discussed, the n-mer(s) can be inserted betw een any two contiguous amino acids within the AAV viral protein and in some embodiments the insertion is made in a variable region.

In some embodiments, the first 1, 2, 3, or 4 amino acids of an n-mer motif can replace 1, 2, 3, or 4 amino acids of a polypeptide into which it is inserted and preceding the insertion site. In some embodiments, the amino acids of the n-mer motif that replace 1 or more amino acids of the polypeptide into which the n-mer motif is inserted come before or immediately before an “RGD"’ in an n-mer motif. The first three amino acids can replace 1 -3 amino acids into a polypeptide to which they may be inserted. Using an AAV as another non-limiting example, one or more of the n-mer motifs can be inserted into, e. , and AAV9 capsid prolylpeptide between amino acids 588 and 589 and the insert can replace amino acids 586, 587, and 588 such that the amino acid immediately preceding the n-mer motif after insertion is residue 585. It will be appreciated that this principle can apply in any other insertion context and is not necessarily limited to insertion between residues 588 and 589 of an AAV9 capsid or equivalent position in another AAV capsid. It will further be appreciated that in some embodiments, no amino acids in the polypeptide into which the n-mer motif is inserted are replaced by the n-mer motif.

In some embodiments, the AAV capsids or other viral capsids or compositions can be muscle-specific. In some embodiments, muscle-specificity of the engineered AAV or other

T1 viral capsid or other composition is conferred by a muscle specific n-mer motif incorporated in the engineered AAV or other viral capsid or other composition described herein. While not intending to be bound by theory, it is believed that the n-mer motif confers a 3D structure to or within a domain or region of the engineered AAV capsid or other viral capsid or other composition such that the interaction of the viral particle or other composition containing the engineered AAV capsid or other viral capsid or other composition described herein has increased or improved interactions (e.g.. increased affinity) with a cell surface receptor and/or other molecule on the surface of a muscle cell. In some embodiments, the cell surface receptor is AAV receptor (AAVR). In some embodiments, the cell surface receptor is a muscle cell specific AAV receptor. In some embodiments, the cell surface receptor or other molecule is a cell surface receptor or other molecule selectively expressed on the surface of a muscle cell. In some embodiments, the cell surface receptor or molecule is an integrin or dimer thereof. In some embodiments, the cell surface receptor or molecule is an Vb6 integrin heterodimer.

In some embodiments, a muscle specific engineered viral particle or other composition described herein containing the muscle-specific capsid, n-mer motif, or musclespecific targeting moiety described herein can have an increased uptake, delivery rate, transduction rate, efficiency, amount, or a combination thereof in a muscle cell as compared to other cells types and/or other virus particles (including but not limited to AAVs) and other compositions that do not contain the muscle-specific n-mer motif of the present invention.

First- and second-generation muscle specific AAV capsids were developed using a muscle specific promoter and the resulting capsid libraries were screened in mice and nonhuman primates as described elsewhere herein and/or in, e.g., U.S. Provisional Application Serial Nos. 62/899,453, 62/916,207. 63/018,454, and 63/242,008. First and second generation myoAAV capsids were further optimized in mice and non-human primates as previously described to generate enhanced myoAAV capsids.

Pharmaceutical Compositions

Any of the recombinant nucleic acids, the expression vectors (e.g., AAV vectors), or the AAV particles described herein can be included in a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

Some embodiments of the invention may include any acceptable form of providing the AAV vector to a subject. For example, the AAV vector may be provided to the subject in the form of a composition or formulation comprising the AAV vector. The expression vector (e.g, AAV vector) of this invention can be formulated and administered to treat a variety of disease states by any means that produces contact of the active ingredient with the agent's site of action in the body of the subject. The compositions, polynucleotides, polypeptides, particles, cells, vector systems and combinations thereof described herein can be contained in a formulation, such as a pharmaceutical formulation. In some embodiments, the formulations can be used to generate polypeptides and other particles that include one or more musclespecific targeting moieties described herein. In some embodiments, the formulations can be delivered to a subject in need thereof. In some embodiments, component(s) of the engineered AAV capsid system, engineered cells, engineered AAV capsid particles, and/or combinations thereof described herein can be included in a formulation that can be delivered to a subject or a cell. In some embodiments, the formulation is a pharmaceutical formulation. One or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be provided to a subject in need thereof or a cell alone or as an active ingredient, such as in a pharmaceutical formulation. As such, also described herein are pharmaceutical formulations containing an amount of one or more of the polypeptides, polynucleotides, vectors, cells, or combinations thereof described herein. In some embodiments, the pharmaceutical formulation can contain an effective amount of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein. The pharmaceutical formulations described herein can be administered to a subject in need thereof or a cell.

In some embodiments, the amount of the one or more of the polypeptides, polynucleotides, vectors, cells, vims particles, nanoparticles, other delivery' particles, and combinations thereof described herein contained in the pharmaceutical formulation can range from about 1 pg/kg to about 10 mg/kg based upon the body weight of the subject in need thereof or average body weight of the specific patient population to which the pharmaceutical formulation can be administered. The amount of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein in the pharmaceutical formulation can range from about 1 pg to about 10 g, from about 10 nL to about 10 ml. In embodiments where the pharmaceutical formulation contains one or more cells, the amount can range from about 1 cell to 1 x 10², 1 x 10³, 1 x 10⁴, 1 x 10⁵, 1 x 10⁶, 1 x 10⁷, 1 x 10⁸, 1 x 10⁹, 1 x IO¹⁰ or more cells. In embodiments where the pharmaceutical formulation contains one or more cells, the amount can range from about 1 cell to I x 10², 1 x 10³, 1 x 10⁴, 1 x 10⁵. 1 x 10⁶, 1 x 10⁷, 1 x 10⁸, 1 x 10⁹. 1 x IO¹⁰ or more cells per nL, pL, mL. or L. In embodiments, where engineered AAV capsid particles are included in the formulation, the formulation can contain 1 to 1 x 10², 1 x 10³. 1 x 10⁴, 1 x 10⁵, 1 x 10⁶. 1 x 10⁷, 1 x 10⁸, 1 x 10⁹, 1 x IO¹⁰, 1 x 10¹¹, 1 x IO¹², 1 x 10¹³, 1 x 10¹⁴, 1 x 10¹⁵, 1 x 10¹⁶, 1 x 10¹⁷, 1 x IO¹⁸, 1 x 10¹⁹, or 1 x IO²⁰ transducing units (TU)/mL of the engineered AAV capsid particles. In some embodiments, the formulation can be 0. 1 to 100 mL in volume and can contain 1 to 1 x 10², 1 x 10³, 1 x 10⁴, 1 x 10⁵, 1 x 10⁶, 1 x 10⁷, 1 x 10⁸, 1 x 10⁹, 1 x IO¹⁰, 1 x IO¹¹, 1 x 10¹², 1 x IO¹³, 1 x 10¹⁴. 1 x IO¹⁵, 1 x 10¹⁶, 1 x 10¹⁷. 1 x IO¹⁸. 1 x 10¹⁹, or 1 x IO²⁰ transducing units (TU)/mL of the engineered AAV capsid particles.

Pharmaceutically Acceptable Carriers and Auxiliary Ingredients and Agents

In embodiments, the pharmaceutical formulation containing an amount of one or more of the polypeptides, polynucleotides, vectors, cells, virus particles, nanoparticles, other delivery particles, and combinations thereof described herein can further include a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxy methylcellulose, and polyvinyl pyrrolidone, which do not deleteriously react with the active composition.

The pharmaceutical formulations can be sterilized, and if desired, mixed with auxiliary agents, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances, and the like which do not deleteriously react with the active composition.

In some embodiments, the pharmaceutical formulations described herein may be in a dosage form. The dosage forms can be adapted for administration by any appropriate route. Appropriate routes include, but are not limited to, oral (including buccal or sublingual), rectal, epidural, intracranial, intraocular, inhaled, intranasal, topical (including buccal, sublingual, or transdermal), vaginal, intraurethral, parenteral, intracranial, subcutaneous, intramuscular, intravenous, intraperitoneal, intradermal, intraosseous, intracardiac, intraarticular, intracavemous, intrathecal, intravitreal, intracerebral, gingival, subgingival, intracerebroventricular, and intradermal. Such formulations may be prepared by any method known in the art.

Dosage forms adapted for oral administration can be discrete dosage units such as capsules, pellets or tablets, powders or granules, solutions, or suspensions in aqueous or non- aqueous liquids; edible foams or whips, or in oil-in-water liquid emulsions or water-in-oil liquid emulsions. In some embodiments, the pharmaceutical formulations adapted for oral administration also include one or more agents which flavor, preserve, color, or help disperse the pharmaceutical formulation. Dosage forms prepared for oral administration can also be in the form of a liquid solution that can be delivered as foam, spray, or liquid solution. In some embodiments, the oral dosage form can contain about 1 ng to 1000 g of a pharmaceutical formulation containing a therapeutically effective amount or an appropriate fraction thereof of the targeted effector fusion protein and/or complex thereof or composition containing the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein. The oral dosage form can be administered to a subject in need thereof.

Where appropriate, the dosage forms described herein can be microencapsulated. The dosage form can also be prepared to prolong or sustain the release of any ingredient. In some embodiments, the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be the ingredient whose release is delayed. In other embodiments, the release of an optionally included auxiliary ingredient is delayed. Suitable methods for delaying the release of an ingredient include, but are not limited to, coating or embedding the ingredients in material in polymers, wax, gels, and the like. Delayed release dosage formulations can be prepared as described in standard references such as “Pharmaceutical dosage form tablets,'’ eds. Liberman et. al. (New York, Marcel Dekker, Inc.. 1989), “Remington - The science and practice of pharmacy”, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and “Pharmaceutical dosage forms and drug delivery systems”, 6th Edition, Ansel et al., (Media, PA: Williams and Wilkins, 1995). These references provide information on excipients, materials, equipment, and processes for preparing tablets and capsules and delayed release dosage forms of tablets and pellets, capsules, and granules. The delayed release can be anywhere from about an hour to about 3 months or more.

Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacry lic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt. Germany), zein, shellac, and polysaccharides.

Coatings may be formed with a different ratio of water-soluble polymer, water insoluble polymers, and/or pH dependent polymers, with or without water insoluble/water soluble non-polymeric excipient, to produce the desired release profile. The coating is either performed on the dosage form (matrix or simple) which includes, but is not limited to, tablets (compressed with or without coated beads), capsules (with or without coated beads), beads, particle and compositions, formulated as, but not limited to, suspension form or as a sprinkle dosage form.

Dosage forms adapted for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils. In some embodiments for treatments of the eye or other external tissues, for example the mouth or the skin, the pharmaceutical formulations are applied as a topical ointment or cream. When formulated in an ointment, the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be formulated with a paraffinic or water- miscible ointment base. In some embodiments, the active ingredient can be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Dosage forms adapted for topical administration in the mouth include lozenges, pastilles, and mouth washes.

Dosage forms adapted for nasal or inhalation administration include aerosols, solutions, suspension drops, gels, or dry powders. In some embodiments, the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein is contained in a dosage form adapted for inhalation is in a particle-size-reduced form that is obtained or obtainable by micronization. In some embodiments, the particle size of the size reduced (e.g, micronized) compound or salt or solvate thereof, is defined by a D50 value of about 0.5 to about 10 microns as measured by an appropriate method known in the art. Dosage forms adapted for administration by inhalation also include particle dusts or mists. Suitable dosage forms wherein the carrier or excipient is a liquid for administration as a nasal spray or drops include aqueous or oil solutions/suspensions of an active ingredient (e.g., the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein and/or auxiliary active agent), which may be generated by various types of metered dose pressurized aerosols, nebulizers, or insufflators.

In some embodiments, the dosage forms can be aerosol formulations suitable for administration by inhalation. In some of these embodiments, the aerosol formulation can contain a solution or fine suspension of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein and a pharmaceutically acceptable aqueous or non-aqueous solvent. Aerosol formulations can be presented in single or multidose quantities in sterile form in a sealed container. For some of these embodiments, the sealed container is a single dose or multi-dose nasal, or an aerosol dispenser fitted with a metering valve (e.g., metered dose inhaler), which is intended for disposal once the contents of the container have been exhausted.

Where the aerosol dosage form is contained in an aerosol dispenser, the dispenser contains a suitable propellant under pressure, such as compressed air, carbon dioxide, or an organic propellant, including but not limited to a hydrofluorocarbon. The aerosol formulation dosage forms in other embodiments are contained in a pump-atomizer. The pressurized aerosol formulation can also contain a solution or a suspension of one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein. In further embodiments, the aerosol formulation can also contain co-solvents and/or modifiers incorporated to improve, for example, the stability and/or taste and/or fine particle mass characteristics (amount and/or profile) of the formulation. Administration of the aerosol formulation can be once daily or several times daily, for example, 2, 3, 4, or 8 times daily, in which 1 , 2, or 3 doses are delivered each time.

For some dosage forms suitable and/or adapted for inhaled administration, the pharmaceutical formulation is a dry powder inhalable formulation. In addition to the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein, an auxiliary active ingredient, and/or pharmaceutically acceptable salt thereof, such a dosage form can contain a powder base such as lactose, glucose, trehalose, mannitol, and/or starch. In some of these embodiments, the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein is in a particle-size reduced form. In further embodiments, a performance modifier, such as L-leucine or another amino acid, cellobiose octaacetate, and/or metals salts of stearic acid, such as magnesium or calcium stearate.

In some embodiments, the aerosol dosage forms can be arranged so that each metered dose of aerosol contains a predetermined amount of an active ingredient, such as the one or more of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein.

Dosage forms adapted for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulations. Dosage forms adapted for rectal administration include suppositories or enemas.

Dosage forms adapted for parenteral administration and/or adapted for any type of injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, intradermal, intraosseous, epidural, intracardiac, intraarticular, intracavemous. gingival, subgingival, intrathecal, intravitreal, intracerebral, and intracerebroventricular) can include aqueous and/or non-aqueous sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, solutes that render the composition isotonic with the blood of the subject, and aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents. The dosage forms adapted for parenteral administration can be presented in a single- unit dose or multi-unit dose containers, including but not limited to sealed ampoules or vials. The doses can be lyophilized and resuspended in a sterile carrier to reconstitute the dose prior to administration. Extemporaneous injection solutions and suspensions can be prepared in some embodiments, from sterile powders, granules, and tablets.

Dosage forms adapted for ocular administration can include aqueous and/or nonaqueous sterile solutions that can optionally be adapted for injection, and which can optionally contain anti-oxidants, buffers, bacteriostats, solutes that render the composition isotonic with the eye or fluid contained therein or around the eye of the subject, and aqueous and nonaqueous sterile suspensions, which can include suspending agents and thickening agents.

For some embodiments, the dosage form contains a predetermined amount of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein per unit dose. In some embodiments, the predetermined amount of the Such unit doses may therefore be administered once or more than once a day. Such pharmaceutical formulations may be prepared by any of the methods well known in the art.

Methods

Also disclosed herein are methods of inhibiting a gene or gene product in a cell of a subject comprising administrating of therapeutically effective amount of one or more compositions (e.g., recombinant nucleic acid, rAAV particle, AAV vector, pharmaceutical composition) disclosed herein to the subject.

Methods described herein encompass inhibiting any gene or gene product using the recombinant nucleic acids described herein. In such instances, the recombinant nucleic acid comprises a guide strand that is at least partially complementary to the gene or gene product of interest (e.g., DUX4, TNNT2, TNNC1, DMPK). In some embodiments, the gene or gene product is associated with a muscular dystrophy (e.g., facioscapulohumeral muscular dystrophy (FSHD), Duchenne muscular dystrophy (DMD), Limb-girdle muscular dystrophy (LGMD), myotonic dystrophy type 1 (DM1)) or a cardiomyopathy (e.g., cardiomyopathy associated with a genetic mutation in TNNT2 and/or TNNC1). Methods described herein encompass inhibiting any gene or gene product in any cell using the recombinant nucleic acids described herein. In some embodiments, the gene or gene product comprises DUX4, TNNT2, TNNC1, or DMPK. In some embodiments, the cell is a muscle cell (e.g., a skeletal muscle cell, a heart muscle cell).

Other Embodiments

Without further elaboration, it is believed that one skilled in the art can. based on the above description, utilize the present disclosure to its fullest extent. The specific embodiments herein are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or sub-combination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

In any of the aspects and embodiments described herein, the nucleic acid sequences contemplated can be DNA, RNA, or modified versions thereof. Modified nucleic acids can be distinguished from naturally occurring nucleic acids by modifications to the backbone of the polynucleotide chain, for example, peptide nucleic acids (PNA), morpholinos, locked nucleic acids (LNA). glycol nucleic acids (GNA) and threose nucleic acid (TNA). Modified nucleic acids can also include analogs with modifications to the four nucleobases. In some embodiments, the nucleic acids are PNAs. In some embodiments, the nucleic acids are LNAs. In some embodiments, the nucleic acids are morpholinos. In some embodiments, the nucleic acids are in a single-stranded form. In some embodiments, the nucleic acids are in doublestranded form. In some embodiments, the nucleic acids are linear. In some embodiments, the nucleic acids are circular. In some embodiments, the nucleic acids are plasmids.

Non-limiting embodiments of the present disclosure include:

Embodiment 1 is a nucleic acid molecule comprising a primary microRNA (pri- miRNA) scaffold having the scaffold sequence of miR-138 or miR-139; and a guide sequence within the scaffold that targets a gene transcript.

Embodiment 2 is the nucleic acid of Embodiment 1, wherein the pri-miRNA scaffold comprises the scaffold sequence of miR-138. Embodiment 3 is the nucleic acid of Embodiment 2, wherein the nucleic acid comprises, in order, a first scaffold sequence of SEQ ID NO: 2; the guide sequence; a loop scaffold sequence of SEQ ID NO: 3; a passenger sequence to the guide sequence; and a second scaffold sequence of SEQ ID NO: 4. Embodiment 4 is the nucleic acid of Embodiment 3, wherein the nucleic acid molecule comprises a sequence having at least 95% sequence identity with the sequence of SEQ ID NO: 1.

Embodiment 5 is the nucleic acid of Embodiment 1, wherein the pri-miRNA scaffold comprises the scaffold sequence of miR-139. Embodiment 6 is the nucleic acid of Embodiment 5, wherein the nucleic acid molecule comprises, in order, a first scaffold sequence of SEQ ID NO: 6; the guide sequence; a loop scaffold sequence of SEQ ID NO: 7; a passenger sequence to the guide sequence: and a second scaffold sequence of SEQ ID NO: 8. Embodiment 7 is the nucleic acid of Embodiment 6, wherein the nucleic acid molecule comprises a sequence having at least 95% sequence identity with the sequence of SEQ ID NO: 5.

Embodiment 8 is the nucleic acid of Embodiment 1, wherein the guide sequence is 21 nucleotides in length.

Embodiment 9 is an encoding nucleic acid molecule encoding the nucleic acid molecule of Embodiment 1. Embodiment 10 is the encoding nucleic acid of Embodiment 9, wherein when provided to a muscle cells the nucleic acid molecule comprising the pri- miRNA scaffold and the guide sequence is expressed in muscle cells.

Embodiment 11 is an adeno-associated virus (AAV) vector comprising a capsid protein, and a nucleic acid molecule encoding a promoter sequence; a pri-miRNA scaffold having the scaffold sequence of miR-138 or miR-139; and a guide sequence within the scaffold that targets a gene transcript.

Embodiment 12 is the AAV vector of Embodiment 11, wherein the pri-miRNA scaffold comprises the scaffold sequence of miR-138. Embodiment 13 is the AAV vector of Embodiment 12, wherein the AAV vector comprises, in order, a first scaffold sequence of SEQ ID NO: 2; the guide sequence; a loop scaffold sequence of SEQ ID NO: 3; a passenger sequence to the guide sequence; and a second scaffold sequence of SEQ ID NO: 4. Embodiment 14 is the AAV vector of Embodiment 13, wherein the nucleic acid molecule comprises a sequence having at least 95% sequence identity with the sequence of SEQ ID NO: 1.

Embodiment 15 is the AAV vector of Embodiment 11, wherein the pri-miRNA scaffold comprises the scaffold sequence of miR-139. Embodiment 16 is the AAV vector of Embodiment 15, wherein the AAV vector comprises, in order, a first scaffold sequence of SEQ ID NO: 6; the guide sequence; a loop scaffold sequence of SEQ ID NO: 7; a passenger sequence to the guide sequence; and a second scaffold sequence of SEQ ID NO: 8. Embodiment 17 is the AAV vector of Embodiment 16, wherein the nucleic acid molecule comprises a sequence having at least 95% sequence identity with the sequence of SEQ ID NO: 5.

Embodiment 18 is the AAV vector of Embodiment 11, wherein the guide sequence is 21 nucleotides in length.

Embodiment 19 is the AAV vector of Embodiment 11 , further comprising a promoter sequence.

Embodiment 20 is the AAV vector of Embodiment 11, wherein when provided to muscle cells the nucleic acid molecule is expressed in muscle cells.

Embodiment 21 is the AAV vector of Embodiment 11, wherein the capsid protein comprises at least one modification that results in reduced liver-tropism of the AAV vector and/or preferential targeting of the AAV vector to muscle tissue.

Embodiment 22 is the AAV vector of Embodiment 11, wherein the capsid protein comprises at least one modification that is an insertion between any two contiguous amino acids between amino acids 262-269. 327-332, 382-386, 452-460, 488-505, 527-539, 545-558, 581-593, 704-714, or any combination thereof in an AAV9 capsid polypeptide or in an analogous position in an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, AAV rh.10 capsid polypeptide.

Embodiment 23 is a method of inhibiting expression of a gene or gene product in a cell, the method comprising providing to a subject a composition comprising a nucleic acid molecule comprising a pri-miRNA scaffold having the scaffold sequence of miR-138 or miR- 139; and a guide sequence within the scaffold that targets a double homeobox 4 (DUX4) transcript.

Embodiment 24 is the method of Embodiment 23. wherein the pri-miRNA scaffold comprises the scaffold sequence of miR-138. Embodiment 25 is the method of Embodiment 24, wherein the nucleic acid molecule comprises, in order, a first scaffold sequence of SEQ ID NO: 2; the guide sequence; a loop scaffold sequence of SEQ ID NO: 3; a passenger sequence; and a second scaffold sequence of SEQ ID NO: 4. Embodiment 26 is the method of Embodiment 25, wherein the nucleic acid molecule comprises a sequence having at least 95% sequence identity with the sequence of SEQ ID NO: 1 .

Embodiment 27 is the method of Embodiment 23, wherein the pri-miRNA scaffold comprises the scaffold sequence of miR-139. Embodiment 28 is the method of Embodiment 27, wherein the nucleic acid molecule comprises, in order, a first scaffold sequence of SEQ ID NO: 6; the guide sequence; a loop scaffold sequence of SEQ ID NO: 7; a passenger sequence ; and a second scaffold sequence of SEQ ID NO: 8.

Embodiment 29 is the method of Embodiment 28, wherein the nucleic acid molecule comprises a sequence having at least 95% sequence identity with the sequence of SEQ ID NO: 5.

Embodiment 30 is the method of Embodiment 23, wherein the guide sequence is 21 nucleotides in length.

Embodiment 31 is the method of Embodiment 23, wherein the method comprises providing to a subject an encoding nucleic acid molecule encoding the nucleic acid molecule comprising a pri-miRNA scaffold having the scaffold sequence and the guide sequence. Embodiment 32 is the method of Embodiment 31. wherein the step of providing to the subject the encoding nucleic acid molecule results in expression of the nucleic acid molecule comprising a pri-miRNA scaffold having the scaffold sequence and the guide sequence in skeletal muscle cells of the subject.

Embodiment 33 is the method of Embodiment 23. wherein the composition comprises an adeno-associated virus (AAV) vector comprising a capsid protein, and a nucleic acid molecule comprising a promoter sequence; a pri-miRNA scaffold having the scaffold sequence of miR-138 or miR-139; and a guide sequence within the scaffold that targets a gene transcript.

Embodiment 34 is the method of Embodiment 33. wherein the capsid protein comprises at least one modification that results in reduced liver-tropism of the AAV vector and/or preferential targeting of the AAV vector to muscle tissue.

Embodiment 35 is the method of Embodiment 34, wherein the AAV vector comprises at least one modification that is an insertion between any two contiguous amino acids between ammo acids 262-269, 327-332, 382-386, 452-460, 488-505, 527-539, 545-558, 581- 593, 704-714, or any combination thereof in an AAV9 capsid polypeptide or in an analogous position in an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, AAV rh. 10 capsid polypeptide.

Embodiment 36 is the method of Embodiment 23. wherein inhibition of the gene or gene product results in treatment of a myopathy or muscular dystrophy. Embodiment 37 is the method of Embodiment 36, wherein the muscular dystrophy is facioscapulohumeral muscular dystrophy. Embodiment 38 is the method of Embodiment 36, wherein the myopathy is a cardiomyopathy. Embodiment 39 is the method of Embodiment 36, wherein the treatment may comprise arresting the muscular effects of muscular dystrophy. Embodiment 40 is the method of Embodiment 36. wherein the treatment may comprise reversing the muscular effects of the muscular dystrophy.

EXPERIMENTAL EXAMPLES

In order that the invention described may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the methods and compositions provided herein and are not to be construed in any way as limiting their scope.

Example 1: Efficient Processing of Engineered Pri-miRNAs Produces Mature Guide Strand With Minimal Passenger Strand Production

To determine whether the miR-138 and miR-139 scaffolds could be used to deliver a guide strand, an engineered pri-miRNA comprising a guide strand targeting DUX4 and a passenger strand was inserted into the mouse pri-miR-139 and pri-miR-138 scaffolds (SEQ ID NO: 1 and SEQ ID NO: 5, respectively). The guide strand targeting DUX4 and the passenger strand was also inserted into the pri-miR-33 scaffold as a positive control. Constructs were transduced into cells and expression of the pri-miRNA and the mature miRNA was determined.

FIG. 1 shows a graph of pri-miRNA expression using miR-138, miR-139, and miR- 33 scaffolds. As shown in FIG. 1. mouse miR-139 and miR-138 scaffolds enabled high levels of expression of the engineered pri-miRNAs, with dose dependent expression.

FIG. 2 shows a graph of mature miRNA expression using miR-138, miR-139, and miR-33 scaffolds. As shown in FIG. 2, miR-138 and miR-139 scaffolds also showed high levels of processing into mature miRs (e.g. via RISC).

Taken together, these results demonstrate that engineered pri-miRNA comprising either the miR-138 or the miR-139 scaffold and a DUX4 targeting guide strand can be highly expressed in cells in a dose dependent manner. These results also demonstrate that the miR- 138 and the miR-139 scaffolds can provide efficient processing of the mature guide strand with minimal passenger strand production. This has the advantage of reducing potential off- target effects of the passenger strand. Example 2: Engineered Pri-miRNAs Targeting DUX4 Can Knockdown DUX4 and DUX4 Downstream Genes in FSHD Patient Myotubes

Expression of DUX4 and DUX4 downstream genes (also referred to as FSHD composite gene expression) was measured in FSHD patient myotubes transduced with the engineered pri-miRNAs described in Example 1.

FIGs. 3A-3B show a graph of DUX4 and FSHD composite gene expression (which includes DUX4 downstream targets hCCNAl, hKHDCl. hMBD3L2. hRFPL2, hTRIM43. and hZSCAN4) in FSHD patient myotubes following DUX4 targeting pri-miRNA expression.

Expression of the engineered pri-miRNAs of the invention targeting DUX4 resulted in knockdown of DUX4 and downregulation of DUX4 target genes in FSHD patient myotubes across all doses, with a dose dependent response (FIGs. 3A-3B).

These results also demonstrated that the DUX4 targeted miRNA in the context of the miR-139 scaffold had greater knockdown compared to the DUX4 targeted miRNA in context of the miR-33 scaffold, across all doses.

Taken together, these results demonstrate that the DUX4 targeting miRNA in the context of the miRNA-138 scaffold or the miRNA- 139 scaffold can effectively decrease expression of DUX4 and DUX4 downstream target genes.

Example 3: Engineered Pri-miRNA Targeting TNNT2 or TNNC1 Can Knockdown TNNT2 or TNNC1 in Mice

Engineered pri-miRNAs comprising TNNT2 or TNNC1 targeting guide sequences inserted into the mouse miR-139 scaffold were expressed in mice using an engineered AAV vector. A scrambled sequence was inserted into the mouse miR-139 scaffold as a control. Tissues were harvested 4 weeks after injection (2E+13 vg/kg) and in vivo knockdown of endogenous mTNNT2 and mTNNC 1 was quantified by qPCR.

FIG. 4 shows a graph of endogenous TNNT2 knockdow n in mice using an engineered pri-miRNA targeting TNNT2 comprising a TNNT2 miRNA inserted into a miR- 139 scaffold.

FIG. 5 shows a graph of endogenous TNNC1 knockdown in mice using an engineered pri-miRNA targeting TNNC1 comprising a TNNC1 miRNA inserted into a miR- 139 scaffold.

Expression of the engineered pri-miRNAs of the invention targeting TNNT2 or TNNC1 resulted in nearly complete knockdown of TNNT2 and TNNC1. These results demonstrate that miRNAs targeting TNNT2 or TNNC1 in the context of the miRNA-139 scaffold can effectively decrease target gene expression.

Example 4: Engineered Pri-miRNAs Targeting DMPK Can Knockdown DMPK in Healthy Human Myotubes

Engineered pri-miRNAs comprising a DMPK targeting guide and passenger sequence inserted into the mouse miR-139 scaffold were expressed in healthy human myotubes using an engineered AAV vector. Each engineered pri-miRNA included a unique DMPK targeting miRNA inserted into the mouse miR-139 scaffold. Eleven engineered pri-miRNAs were tested (pri-miRNA #1-11). Three scramble miRNA sequences in the miR-139 scaffold were used as controls. Healthy human myotubes were transduced 3 days after differentiation and then harvested 4 days after transduction. DMPK mRNA was quantified using qPCR.

FIG. 6 shows a graph of DMPK mRNA expression in healthy human myotubes transduced with an engineered DMPK targeting pri-miRNA. Expression of the engineered pri-miRNAs targeting DMPK resulted in significantly greater knockdown of DMPK mRNA compared to scrambled control sequences. These results demonstrated that the DMPK targeted miRNA in the context of the miR-139 scaffold can effectively knockdown DMPK.

Taken together, the experimental data described herein demonstrates that the miR-139 scaffold can be used to effectively deliver miRNA sequences targeting different genes including DUX4, TNNT2, TNNC1, and DMPK.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

EQUIVALENTS

Various modifications of the invention and many further embodiments thereof, in addition to those show n and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

CLAIMS What Is Claimed Is:

1. A recombinant nucleic acid comprising: a primary microRNA (pri-miRNA) scaffold comprising a scaffold sequence of miR-

138 or miR-139; and a guide sequence that targets a gene of interest, wherein the guide sequence is positioned within the scaffold sequence.

2. The recombinant nucleic acid of claim 1, wherein the pri-miRNA scaffold comprises the scaffold sequence of miR-138.

3. The recombinant nucleic acid of claim 2, wherein the recombinant nucleic acid comprises from 5' to 3': a first scaffold sequence comprising or consisting of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 2; the guide sequence; a loop scaffold sequence comprising or consisting of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 3; a passenger sequence that is at least partially complementary to the guide sequence; and a second scaffold sequence comprising or consisting of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 4.

4. The recombinant nucleic acid of claim 3, wherein: the first scaffold sequence comprises or consists of SEQ ID NO: 2; the loop scaffold sequence comprises or consists of SEQ ID NO: 3; and the second scaffold sequence comprises or consists of SEQ ID NO: 4.

5. The recombinant nucleic acid of claim 1, wherein the pri-miRNA scaffold comprises the scaffold sequence of miR-139.

6. The recombinant nucleic acid of claim 5, wherein the recombinant nucleic acid comprises from 5' to 3': a first scaffold sequence comprising or consisting of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 6; the guide sequence; a loop scaffold sequence comprising or consisting of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 7; a passenger sequence that is at least partially complementary to the guide sequence; and a second scaffold sequence comprising or consisting of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 8.

7. The recombinant nucleic acid of claim 6, wherein: the first scaffold sequence comprises or consists of SEQ ID NO: 6; the loop scaffold sequence comprises or consists of SEQ ID NO: 7; and the second scaffold sequence comprises or consists of SEQ ID NO: 8.

8. The recombinant nucleic acid of claim 1, wherein the guide sequence is 21 nucleotides in length.

9. The recombinant nucleic acid of claim 1 , further comprising a tissue specific promoter.

10. The recombinant nucleic acid of claim 1, wherein, when provided to a muscle cell, the recombinant nucleic acid molecule comprising the pri-miRNA scaffold and the guide sequence is expressed in muscle cells.

11. The recombinant nucleic acid of claim 1, wherein the recombinant nucleic acid is comprised in a vector, optionally wherein the vector is a viral vector.

12. The recombinant nucleic acid of claim 11, wherein the vector is an adeno-associated viral (AAV) vector.

13. An adeno-associated virus (AAV) particle comprising: a capsid protein, and the recombinant nucleic acid of claim 1.

14. The AAV particle of claim 13, wherein the capsid protein comprises at least one modification that results in reduced liver-tropism of the AAV particle and/or preferential targeting of the AAV particle to muscle tissue.

15. The AAV particle of claim 13, wherein the capsid protein comprises at least one modification that is an insertion between any two contiguous amino acids between amino acids 262-269, 327-332, 382-386, 452-460, 488-505, 527-539, 545-558, 581-593, 704-714, or any combination thereof in an AAV9 capsid polypeptide or in an analogous position in an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh.74, AAV rh.10 capsid polypeptide.

16. A method of inhibiting expression of a gene or gene product in a cell, the method comprising providing to a subject a composition comprising the recombinant nucleic acid of claim 1.

17. The method of claim 16. wherein providing the recombinant nucleic acid to the subject results in expression of the recombinant nucleic acid molecule in muscle cells of the subject.

18. The method of claim 16, wherein inhibiting expression of the gene or gene product results in treatment of a myopathy or a muscular dystrophy.

19. The method of claim 16, wherein the gene or gene product is a gene or gene product associated with a muscular dystrophy.

20. The method of claim 16, wherein the cell is a muscle cell.