Acta1 expression control element

Abstract

This disclosure relates to the fields of molecular biology and nucleic acid technology. This disclosure also relates to therapies and prevention of diseases.

Description

Technical Field

[0001] This disclosure relates to the fields of molecular biology and nucleic acid technology. This disclosure also relates to therapies and prevention of diseases. Background Technology

[0002] Muscle tissue represents the largest tissue category in the human body and is divided into three main muscle types: skeletal muscle, cardiac muscle, and smooth muscle. Skeletal and cardiac muscles are grouped together as striated muscle, characterized by its ability to produce contraction, force, and movement (Mukund & Subramaniam, 2020). Several gene mutations cause devastating muscle diseases, including muscular dystrophys such as DMD, Becker's muscular dystrophy, or limb-girdle muscular dystrophy, as well as hereditary myopathy such as X-linked myotube myopathy. As with many other disease areas, rAAV vectors have shown promising results in clinical trials treating these previously incurable and devastating muscle diseases (Braun et al., 2014; D. Wang et al., 2014).

[0003] ACTA1 encodes α-skeletal actin, which is essential for muscle contraction. Mutations in ACTA1 can lead to abnormalities in the protein's structure / level and disruption of muscle cell structure / function, manifesting as muscle weakness, such as rare forms of congenital myopathy.

[0004] Congenital myopathy is a rare, inherited muscle disorder with genetic, histological, and clinical heterogeneity. Clinical features include, among other things, decreased muscle tone and weakness due to affected skeletal muscle fibers. The disease ranges in severity from a severe neonatal form to a milder childhood form, all caused by gene mutations.

[0005] Mutations in ACTA1 result in overlapping congenital myopathy, such as, for example, linear myopathy, congenital myofibril heterogeneity myopathy, and myopathy with nucleoid regions (see, for example, Wang, CH, et al., J. Child. Neurol. 27 (2012) 363-382; Cassandrini, D., et al., Ital. J. Pediatr. 43 (2017) 101).

[0006] ACTA1 encodes skeletal muscle α-actin, the major actin isoform in skeletal muscle, and forms the core of filaments that interact with myosin during muscle contraction. To date, more than 200 pathogenic mutations in ACTA1 have been identified (see, for example, Sparrow, JC, et al., Neuromusc. Disord. 13 (2003) 519-531; Feng, JJ. and Marston, S., Neuromuscul. Doisord. 19 (2009) 6-16; Laing, NG, et al., Hum. Mutat. 30 (2009) 1267-1277; Laitila, J. and Wallgren-Petterson, C. Neuromusc. Disord. 31 (2021) 955-967).

[0007] Approximately 90% of the mutations are dominant (most commonly de novo, although some are hereditary) and cause severe linear myopathy primarily due to the negative effects of dominance. No significant mutational hotspots have been identified in the genes or proteins (see, for example, Feng, JJ. and Marston, S., Neuromuscul. Doisord. 19 (2009) 6–16; Laing, NG, et al., Hum. Mutat. 30 (2009) 1267–1277).

[0008] However, over 50% of patients with severe linear myopathy have ACTA1 mutations (see, for example, Labasse, C., et al., Acta Neuropathol. Commun. 10 (2022) 101; Neuhaus, SB, et al., Neuromusc. Disord. 30 (2020) 866-875). The severe phenotype presents with infantile hypotonia, bradykinesia, medullary and respiratory weakness, leading to early death. A small percentage of affected patients develop hypertrophic cardiomyopathy.

[0009] Restoration of actin function improves the pathogenic phenotype. Rare cases have been observed in children with homozygous null mutations in ACTA1 (see, e.g., Nowak, KJ, et al., Ann. Neurol. 61 (2007) 175-184; O'Grady, GL, et al., Eur. J. Hum. Genet. 23 (2015) 883-886; Nowak, KJ, et al., 2013), in which cardiac actin (ACTC1) substitution compensates for the loss of skeletal actin expression, as the ACTC1 gene, normally expressed during fetal skeletal muscle development, continues to be expressed. Patients with increased cardiac actin expression have longer lifespans due to the high homology between the two actin isoforms (difference of 4 amino acids) (see, e.g., Labasse, C., et al., Acta Neuropathol. Commun. 10 (2022) 101).

[0010] Evidence comes from disease models. ACTC1 overexpression was observed in ACTA1-ineffective mice and a disease model (ACTA1...). D286G The loss of function was compensated and their lifespan was significantly extended (see, for example, Nowak, KJ, et al., J. Cell Biol. 185 (2009) 903-915; Ravencroft, G., et al., Hum. Mol. Genet. 22 (2013) 3987-3997).

[0011] However, in another mouse model (ACTA1) H40Y In this study, overexpression of ACTC1 did not improve lifespan or phenotype (see, for example, Ravencroft, G., et al., Hum. Mol. Genet. 22 (2013) 3987-3997).

[0012] Therefore, relying on actin mutation restoration without removing the mutated form may not be sufficient for effective treatment. Summary of the Invention

[0013] This article presents an AAV-based gene therapy that uses skeletal muscle-specific transgenic regulation and optionally a muscle-loving capsid to restore functional skeletal muscle actin while inactivating mutant actin.

[0014] More generally, this article presents expression control elements suitable for restoring skeletal actin function in patients with ACTA1 mutations. Without being bound by this theory, it is hypothesized that optimal patient benefit can be achieved using disease-modifying potential when the mutated actin is inactivated and skeletal actin is replaced; that is, a therapeutic modality encompassing the restoration of functional skeletal actin while simultaneously inactivating the mutated actin. This is achieved through selective miRNA silencing of mutated ACTA1 expression.

[0015] In one aspect, a regulatory polynucleotide for inhibiting ACTA1 expression is provided, wherein the regulatory polynucleotide encodes a miRNA scaffold, wherein the miRNA scaffold comprises a repressive RNA (RNAi) molecule, wherein the RNAi molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 82 and SEQ ID NO: 84, and wherein the antisense strand and the sense strand share a length of at least 20, at least 21, at least 22, at least 23, or at least 24. Or at least 25 complementary regions of nucleotides, wherein the antisense strand sequence differs from the reverse complementary nucleotide sequence of the sense strand sequence by no more than 4 nucleotides.

[0016] On the one hand, regulatory polynucleotides inhibit the activity of the ACTA1 gene.

[0017] On one hand, regulatory polynucleotides are: single-stranded DNA, double-stranded DNA, DNA as a mixture of single-stranded and double-stranded regions, single-stranded RNA, double-stranded RNA, RNA as a mixture of single-stranded and double-stranded regions, single-stranded molecules containing DNA and RNA, double-stranded molecules containing DNA and RNA, or molecules containing DNA and RNA having a mixture of single-stranded and double-stranded regions, particularly wherein the regulatory polynucleotide is single-stranded DNA.

[0018] On the one hand, the ACTA1 gene is an endogenous gene with mutations in cells or subjects.

[0019] On one hand, the ACTA1 gene is the wild-type ACTA1 gene.

[0020] On the one hand, the ACTA1 gene is a mutated ACTA1 gene with at least one pathogenic mutation.

[0021] On the one hand, regulatory polynucleotides inhibit the activity of both the wild-type ACTA1 gene and the mutated ACTA1 gene with at least one pathogenic mutation.

[0022] On the one hand, regulatory polynucleotides do not inhibit the activity of codon-optimized ACTA1 genes.

[0023] On one hand, the regulatory polynucleotide contains the sequence of SEQ ID NO: 93 or a sequence having at least 85% sequence identity with SEQ ID NO: 93.

[0024] On one hand, the regulatory polynucleotide comprises a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the group consisting of: SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, and SEQ ID NO: 124.

[0025] In one aspect, an expression system is provided comprising a regulatory polynucleotide as described above, wherein the regulatory polynucleotide is operatively linked to a promoter, and optionally wherein the expression system further comprises a codon-optimized polynucleotide encoding ACTA1.

[0026] In one aspect, an adeno-associated virus (AAV) vector is provided, comprising a regulatory polynucleotide as described above, wherein the regulatory polynucleotide is operatively linked to a promoter, and optionally wherein the AAV vector further comprises a codon-optimized polynucleotide encoding ACTA1.

[0027] In one aspect, the expression system or vector comprises: a first expression cassette containing a polynucleotide encoding a codon-optimized polynucleotide encoding ACTA1; and a second expression cassette containing a regulatory polynucleotide as described above.

[0028] On one hand, the first expression cassette contains an intron that contains a second expression cassette encoding a regulatory polynucleotide.

[0029] In one aspect, an adeno-associated virus (AAV) vector is provided, comprising a vector genome, wherein the vector genome comprises, in a 5' to 3' sequence: (i) a 5' inverted terminal repeat (ITR) sequence; (ii) a promoter; (iii) optionally, a 5' UTR sequence; (iv) a polynucleotide encoding the ACTA1 gene, or a codon-optimized variant thereof; (v) a 3' UTR sequence; and (vi) a 3' inverted terminal repeat (ITR) sequence, wherein the vector genome comprises the regulatory polynucleotides as described above.

[0030] On the one hand, the vector genome is suitable for being packaged into adeno-associated virus particles.

[0031] In one aspect, a pharmaceutical composition is provided comprising an expression system as described above, or an AAV carrier as described above, and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0032] In one aspect, there are provided expression systems as described above, or AAV vectors as described above, or pharmaceutical compositions as described above, for use in the treatment or prevention of diseases or conditions from which therapeutic or preventive benefits are derived by an increase in the expression level of ACTA1 in skeletal muscle cells.

[0033] In one aspect, the use of the expression system as described above, or the AAV carrier as described above, or the pharmaceutical composition as described above in the manufacture of a medicament for the treatment or prevention of a disease or condition from which therapeutic or preventive benefits would be derived by an increase in the expression level of ACTA1.

[0034] In one aspect, a method is provided for treating or preventing a disease or symptom from which therapeutic or preventive benefits are derived by an increase in the expression level of ACTA1, the method comprising administering to a subject an expression system as described above, or an AAV carrier as described above, or a pharmaceutical composition as described above.

[0035] On one hand, an expression system, vector, AAV vector, or pharmaceutical composition as described above, or a method of treatment as described above, is provided, wherein the disease is a congenital myopathy, particularly wherein the disease is selected from the group consisting of: linear myopathy, intranuclear rod myopathy, actin filament aggregation myopathy, congenital myofiber heterogeneity, and myopathy with nucleoid regions.

[0036] Other technical features will be apparent to those skilled in the art from the following figures, description and claims. Detailed Implementation

[0037] The present invention is based, at least in part, on the identification of highly specific expression control elements (particularly inactivated constructs).

[0038] Lowering endogenous ACTA1 expression is essential for clinical efficacy. More than 200 mutations are known to be distributed throughout the gene. Therefore, targeting endogenous ACTA1 must be performed at a location where no known mutations are found, and high specificity is also required, as ACTA1 and cardiac actin (ACTC1) are highly conserved.

[0039] The present invention is based, at least in part, on the identification of highly specific target sequences that allow for the successful rescue of disease phenotypes.

[0040] Using the sequence according to the invention, efficient knockdown of wtACTA1 can be achieved, for example, expression reduction of more than 75%.

[0041] The negative impact of the sequences according to the invention on therapeutic transgenic expression (i.e., codon-optimized or modified ACTA1 (coACTA1)) is limited or even eliminated due to the achievement of high splicing efficiency.

[0042] According to the sequences of the present invention, only limited toxicity has been observed, such as off-target effects, RNAi pathway interference, etc.

[0043] Small non-coding RNA molecules (containing approximately 22 nucleotides) found in different species are referred to as microRNAs (abbreviated as miRNAs). These miRNAs have post-transcriptional regulatory functions in RNA silencing and gene expression. The mode of action of miRNAs is to pair with complementary sequence bases within the target mRNA molecule, thereby silencing the transcription of the target mRNA.

[0044] “microRNA” or “miRNA” is a small non-coding RNA molecule capable of mediating post-transcriptional or post-translational gene silencing. Typically, miRNAs are transcribed into hairpin or stem-loop (e.g., with a self-complementary, single-stranded backbone) double-stranded structure called primary miRNA (pri-miRNA), which is then enzymatically processed (e.g., by Drosha, DGCR8, Pasha, etc.) into pre-miRNA. The length of pri-miRNA can vary. In some embodiments, the length of pri-miRNA ranges from about 100 to about 5000 base pairs (e.g., about 100, about 200, about 500, about 1000, about 1200, about 1500, about 1800, or about 2000 base pairs). In some embodiments, the length of pri-miRNA is greater than 200 base pairs, for example, 2500, 5000, 7000, 9000, or more base pairs.

[0045] The structural feature of miRNA is that it folds itself, thus forming a short hairpin.

[0046] The nomenclature for miRNAs is as follows: a stem “mir / R” followed by a dash and a number. The uppercase “R” in the stem indicates the mature form of the miRNA, while the smaller letter “r” indicates the pre-miRNA and pri-miRNA forms. miRNAs with only one or two nucleotide differences in their sequence contain an additional lowercase letter.

[0047] The three-letter prefix in the name indicates the original species of the miRNA; for example, hsa-miR-33 is human (Homo sapiens) miRNA-33.

[0048] Equal amounts of miRNAs from opposite arms of the same pre-miRNA are indicated by the suffix -3p or -5p, or by "s" for the sense strand and "as" for the antisense strand. Typically, miRNAs from one arm of the hairpin are found to be much more abundant than those from the other arm. This is indicated by the use of an asterisk (*) after the name to signify a lower level of the species.

[0049] Pre-miRNAs are also characterized by a hairpin or stem-loop double-stranded structure, and their length can vary. In some embodiments, the length of a pre-miRNA ranges from about 40 base pairs to about 500 base pairs. In some embodiments, the length of a pre-miRNA ranges from about 50 to 100 base pairs. In some embodiments, the length of a pre-miRNA ranges from about 50 to about 90 base pairs (e.g., lengths of about 50, about 52, about 54, about 56, about 58, about 60, about 62, about 64, about 66, about 68, about 70, about 72, about 74, about 76, about 78, about 80, about 82, about 84, about 86, about 88, or about 90 base pairs).

[0050] Typically, pre-miRNA is exported into the cytoplasm and enzymatically treated with Dicer to first produce an imperfect miRNA / miRNA* duplex, and then to produce a single-stranded mature miRNA molecule, which is subsequently loaded into an RNA-induced silencing complex (RISC). The mature miRNA molecule is typically between about 19 and 30 base pairs in length. In some embodiments, the mature miRNA molecule is about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 base pairs in length.

[0051] miRNA scaffolds are structured nucleic acid molecules, typically RNA, that serve as a platform for assembling and processing microRNA (miRNA) molecules. These scaffolds provide a structural framework for the formation of functional miRNA complexes, allowing for precise regulation of gene expression. miRNA scaffolds promote the maturation and stabilization of miRNAs, enabling them to function in post-transcriptional gene regulation.

[0052] The present invention provides, in some aspects, a regulatory polynucleotide sequence that interferes with the expression of a target gene and / or the production of a target protein and a method of using the same, particularly wherein the regulatory polynucleotide sequence is integrated into a miRNA-33 scaffold.

[0053] "Regulatory polynucleotides" refer to synthetic genetic material designed to influence or interfere with the expression of specific target genes. For example, in some aspects of the present invention, small interfering RNAs (siRNAs) are provided, enabling precise regulation of gene expression at the posttranscriptional level. In some aspects of the present invention, repressive RNA (RNAi) molecules are embedded in miRNA scaffolds. Preferably, the present invention provides regulatory polynucleotides encoding miRNA scaffolds containing repressive RNA (RNAi) molecules. A preferred example of such regulatory polynucleotides encoding miRNA scaffolds containing RNAi molecules is a single-stranded DNA (e.g., packaged in a viral vector) encoding a miRNA scaffold containing an RNAi molecule according to the present invention. By targeting specific mRNA sequences corresponding to genes of interest, regulatory polynucleotides can effectively regulate gene expression. Regulatory polynucleotides as described herein can be described by RNA sequences (e.g., exemplary sequences of SEQ ID NO 45 to 86). Alternatively, regulatory polynucleotides can be described by DNA sequences encoding relevant RNA sequences forming RNAi molecules (e.g., exemplary sequences of SEQ ID NO 109 to 124).

[0054] This invention is based at least in part on the discovery that the miRNA scaffold miR33 is beneficial for suppressing endogenous gene products (especially genes such as ACTA1), particularly when used in recombinant adeno-associated virus particles.

[0055] Table 1: Examples of miRNA scaffolds

[0056]

[0057] In recent years, microRNAs have become important regulators of cellular function in both normal and diseased cells. miRNAs coordinate the regulation of a large number of target genes through mRNA degradation and / or translational repression. In animals, the mRNA target specificity of miRNAs is primarily encoded within a 7-nt “seed region” located at positions 2 to 8 of the 5' end of the molecule. Results indicate that even a single nucleotide substitution within the seed region can alter the spectrum of mRNA targets by >50%. This high functional cost, even for single nucleotide changes within the seed region, is consistent with the high sequence conservation among miRNA families within and between species, suggesting a process that may form the basis of evolutionary miRNA regulatory control (see, e.g., Hill, CG, et al., PloS One 9 (2014) e115241).

[0058] Regulatory polynucleotides

[0059] This article discloses a novel regulatory polynucleotide.

[0060] "Polynucleotide" refers to a polymer chain of multiple nucleotide monomers linked by bonds between monomers, typically phosphodiester bonds (e.g., in the case of polynucleotides formed from naturally occurring nucleotide monomers). Polynucleotides include oligonucleotides, which typically contain ≤50 nucleotides. Polynucleotides can be single-stranded or double-stranded (i.e., can contain a double strand formed by hydrogen bonds between complementary nucleotides). Polynucleotides according to this disclosure can comprise or consist of: single-stranded DNA, double-stranded DNA, DNA being a mixture of single-stranded and double-stranded regions, single-stranded RNA, double-stranded RNA, RNA being a mixture of single-stranded and double-stranded regions, single-stranded molecules containing DNA and RNA, double-stranded molecules containing DNA and RNA, and molecules containing a mixture of single-stranded and double-stranded regions of DNA and RNA.

[0061] In some aspects, a polynucleotide comprises or is composed of DNA. In some embodiments, a polynucleotide is a polydeoxyribonucleotide. In some embodiments, a polynucleotide comprises or is composed of RNA. In some embodiments, a polynucleotide is a polynucleotide.

[0062] In the polynucleotides of this disclosure defined by reference to a given nucleotide sequence and in aspects where the given nucleotide sequence comprises or is composed of and / or is a polynucleotide, it should be understood that instances of “T” representing thymidine in such sequences are replaced by “U” representing uracil.

[0063] This disclosure also contemplates polynucleotides comprising modified nucleotides, such as deoxyribonucleotides or ribonucleotides whose phosphate esters and / or ribose and / or bases are chemically modified. Nucleotide modifications contemplated according to this disclosure include those described in Hu et al., Sig. Transduc. Tar. Ther. (2020) 5(101), which is hereby incorporated herein by reference in its entirety.

[0064] Phosphate modification can be selected from thiophosphates (e.g., Rp isomers, Sp isomers), dithiophosphates, methylphosphonates, methoxypropylphosphonates, 5'-(E)-vinylphosphonates, 5'-methylphosphonates, (S)-5'-C-methyl phosphates, 5'-thiophosphates, and peptide nucleic acid modifications. Ribose modification can be selected from 2'-O-methyl, 2'-O-methoxyethyl, 2'-fluorine, 2'-deoxy-2'-fluorine, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxy, 2'-arabinose-fluorine, 2'-O-benzyl, 2'-O-methyl-4-pyridine, locked nucleic acids, (S)-cEt-BNA, tricyclic DNA, PMO, non-locked nucleic acids, hexitol nucleic acids, and ethylene glycol nucleic acid modifications. The base modification can be selected from pseudouridine, 2'-thiouridine, N6'-methyladenosine, 5'-methylcytidine, 5'-fluoro-2'-deoxyuridine, adenine modified with N-ethylpiperidine 7'-EAA triazole, adenine modified with N-ethylpiperidine 6'-triazole, 6'-phenylpyrrolo-cytosine, 2',4'-difluorotolyl ribonucleoside, and 5'-nitroindole.

[0065] In some respects, the modified nucleotide may be selected from 2'-O-methyluridine-3'-phosphate, 2'-O-methyladenosine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methyluridine-3'-thiophosphate, 2'-O-methyladenosine-3'-thiophosphate, and 2'-O-methylguanosine-3'-thiophosphate. Ester, 2'-O-methylcytidine-3'-thiophosphate, 2'-fluorouridine-3'-phosphate, 2'-fluoroadenosine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluorocytidine-3'-thiophosphate, 2'-fluoroguanosine-3'-thiophosphate, 2'-fluoroadenosine-3'-thiophosphate and 2'-fluorouridine-3'-thiophosphate.

[0066] In some respects, regulatory polynucleotides inhibit the activity of target genes. "Inhibition of target gene activity" refers to the prevention or reduction of the expression or function of a specific gene (target gene). Various methods and compounds can result in the inhibition of target gene activity, such as gene silencing or gene knockdown. Methods for measuring the expression or activity of target genes are known in the art and are also described herein, for example, in the Examples and Functional Properties of Polynucleotides sections.

[0067] In some respects, target genes are mutated endogenous genes in cells or subjects. Mutated endogenous genes are genes that are naturally present in an organism (endogenous), but whose DNA sequence has been altered (mutated). These mutations can occur naturally due to DNA replication errors or environmental factors such as radiation or certain chemicals. Depending on their location and nature, these mutations can lead to changes in gene function, which may result in disease or ailment.

[0068] In some respects, regulatory polynucleotides are isolated regulatory polynucleotides. Isolated regulatory polynucleotides are a type of regulatory polynucleotide that has been isolated from components in its natural environment (if it exists in nature).

[0069] In some respects, regulatory polynucleotides contain or encode repressive RNAs (RNAi molecules). "Repressive RNA" refers to a class of RNA molecules that typically participate in regulating gene expression by silencing or inhibiting the activity of specific genes. The most common types of repressive RNAs are small interfering RNAs (siRNAs) and microRNAs (miRNAs). These molecules can bind to messenger RNA (mRNA) and prevent them from being translated into proteins, thereby effectively reducing or inhibiting the expression of target genes. This mechanism is used by cells to regulate gene expression and can also be used for research or therapeutic purposes. In some respects, the activity of the target gene is its expression. In some respects, regulatory polynucleotides silence the target gene. Gene silencing, or gene silencing, refers to the mechanism of reducing, shutting down (or silencing) gene expression. Gene silencing can occur during transcription or translation. In some respects, regulatory polynucleotides reduce or inhibit the expression of target genes.

[0070] In some respects, regulatory polynucleotide sequences contain or encode “sense strand” and “antisense strand” sequences. The sense strand, also known as the coding strand, has the same or highly similar sequence as a portion of the target gene mRNA. The antisense strand is complementary to the sense strand, meaning its sequence is the same as or highly similar to the complementary sequence of the sense strand. Sense and antisense strands are the typical nomenclature for siRNA molecules. For miRNA molecules, the antisense strand and sense strand are usually referred to as the guide strand and lagging strand, respectively. siRNA antisense strands and miRNA guide strands function in targeting and downregulating gene expression by guiding the RNA-induced silencing complex (RISC) to the complementary mRNA sequence used for cleavage or repression. Conversely, the siRNA sense strand, similar to the miRNA lagging strand, typically functions through degradation, ensuring the specificity and efficiency of the gene silencing process. In Table 3, sequences labeled as antisense (strand) sequences also refer to the corresponding guide strand sequences (e.g., SEQ ID NOs 45 to 88). Similarly, sequences labeled as positive (chain) sequences also refer to corresponding follower chain sequences (e.g., SEQ ID NO 128 to 171). Furthermore, in the specific embodiments described below, references to antisense chain sequences also include corresponding references to the lead chain sequence. And references to positive chain sequences also include corresponding references to the follower chain sequences.

[0071] The term "complementary nucleotide sequence" refers to a DNA or RNA sequence of bases that can form specific base pairs with another nucleotide sequence by following the rules of base pairing. In DNA, adenine (A) pairs with thymine (T), and cytosine (C) pairs with guanine (G), while in RNA, uracil (U) replaces thymine. Therefore, a complementary nucleotide sequence will have bases that can form hydrogen bonds with their corresponding bases in another sequence, allowing the two sequences to pair together and form a double-stranded structure.

[0072] In some aspects, the sense strand sequence comprises at least 20 consecutive nucleotides, and the antisense strand sequence comprises at least 20 consecutive nucleotides. In a preferred aspect, the sense strand sequence comprises at least 21 consecutive nucleotides, and the antisense strand sequence comprises at least 21 consecutive nucleotides. In some aspects, the sense strand sequence comprises at least 22 consecutive nucleotides, and the antisense strand sequence comprises at least 22 consecutive nucleotides. In some aspects, the sense strand sequence comprises at least 23 consecutive nucleotides, and the antisense strand sequence comprises at least 23 consecutive nucleotides. In some aspects, the sense strand sequence comprises at least 24 consecutive nucleotides, and the antisense strand sequence comprises at least 24 consecutive nucleotides. In some aspects, the sense strand sequence comprises at least 23 consecutive nucleotides, and the antisense strand sequence comprises at least 23 consecutive nucleotides. In some aspects, the sense strand sequence comprises at least 25 consecutive nucleotides, and the antisense strand sequence comprises at least 25 consecutive nucleotides.

[0073] In some respects, the target gene is the actin gene. An "actin gene" refers to any gene that encodes actin (a class of globular, multifunctional proteins that form microfilaments). Actin is one of the most highly conserved proteins and plays a crucial role in various types of cell movement, structure, and integrity. In humans, there are six actin isoforms, each encoded by a different gene. These include four α-actin (found in muscle tissue) and two β / γ cytoplasmic actin. Mutations in actin genes can lead to a variety of diseases, including a group of conditions known as actinopathy.

[0074] In some respects, the target gene is ACTA1. In some respects, the target gene is human ACTA1 (exemplary references: HGNC:129, NM_001100). In some respects, regulatory polynucleotides repress or inhibit the expression of the ACTA1 gene. In some respects, the ACTA1 gene is the wild-type ACTA1 gene, a mutated ACTA1 gene with at least one pathogenic mutation, or both. In some respects, the ACTA1 gene is a mutated ACTA1 gene with at least one pathogenic mutation. A "pathogenic mutation" is a change in the DNA sequence that causes disease or symptoms. For example, a mutation can alter the function of a gene, causing the protein to malfunction or not be produced at a non-pathogenic level.

[0075] In some respects, the antisense strand (or guide strand) sequence differs from the nucleotide sequence selected from the group consisting of the following: SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85 and SEQ ID NO: 86.

[0076] In a preferred embodiment, the antisense strand (or guide strand) sequence differs from the nucleotide sequence selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 82 and SEQ ID NO: 84.

[0077] In a more preferred aspect, the antisense strand (or guide strand) sequence differs from the nucleotide sequence selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 68, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 79 and SEQ ID NO: 82.

[0078] In some respects, the sequence of the positive strand (or lagging strand) differs from the nucleotide sequence of the reverse complementary nucleotide sequence selected from the group consisting of the following by no more than 4 nucleotides: SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85 and SEQ ID NO: 86, wherein the sense and antisense sequences share a complementary region of at least four nucleotides in length.

[0079] In a preferred aspect, the positive (or negative) strand sequence differs from the antisense strand sequence of a nucleotide sequence selected from the group consisting of no more than 4 nucleotides by a length not exceeding 4 nucleotides: SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 82 and SEQ ID NO: 84, and wherein the positive and negative strand sequences share a complementary region of at least four nucleotides in length.

[0080] In a more preferred aspect, the nucleotide sequence of the sense strand (or lagging strand) differs from the nucleotide sequence of the antisense strand selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 68, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 79 and SEQ ID NO: 82 by no more than 4 nucleotides, and wherein the sense strand sequence and the antisense strand sequence share a complementary region of at least four nucleotides in length.

[0081] In some respects, the sequence of the positive strand (or lagging strand) differs from the nucleotide sequence of the group consisting of the following: SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152. SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168 and SEQ ID NO: 169.

[0082] In a preferred embodiment, the sequence of the positive strand (or lagging strand) differs from the nucleotide sequence of the group consisting of the following: SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 162, SEQ ID NO: 165, and SEQ ID NO: 167.

[0083] In a more preferred aspect, the sequence of the positive strand (or lagging strand) differs from the nucleotide sequence of the group consisting of the following: SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 139, SEQ ID NO: 151, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 162 and SEQ ID NO: 165.

[0084] In some respects, regulatory polynucleotides are siRNAs. siRNA stands for small interfering RNA. These are double-stranded RNA molecules, typically 20 to 25 base pairs in length, that play a crucial role in the RNA interference (RNAi) pathway, interfering with the expression of specific genes. The siRNA molecule binds to the RNA-induced silencing complex (RISC), directing the complex to degrade messenger RNA (mRNA) that matches the siRNA sequence, or to inhibit its translation. This effectively silences the expression of the target gene. In some respects, siRNAs are 20 to 25 nucleotides in length.

[0085] On the one hand, a siRNA molecule that inhibits the expression of ACTA1 is provided. In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69. SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85 and SEQ ID NO: 86.

[0086] In a preferred aspect, a siRNA molecule for inhibiting the expression of ACTA1 is provided. In some aspects, a siRNA molecule for inhibiting the expression of ACTA1 is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 82 and SEQ ID NO: 84.

[0087] In a more preferred aspect, a siRNA molecule for inhibiting the expression of ACTA1 is provided. In some aspects, a siRNA molecule for inhibiting the expression of ACTA1 is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 68, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 79 and SEQ ID NO: 82.

[0088] In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85 and SEQ ID NO: 86, wherein the sense strand shares a complementary region of at least 20, at least 21, at least 22, at least 23, at least 24 or at least 25 nucleotides in length with the sense strand, and wherein the antisense strand sequence differs from the sense strand nucleotide sequence by no more than 4 nucleotides.In a preferred aspect, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85 and SEQ ID NO: 86, wherein the antisense strand and the sense strand share a complementary region of length 21, and wherein the nucleotide sequence of the antisense strand differs from that of the sense strand by no more than 4 nucleotides.

[0089] In a preferred aspect, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 82 and SEQ ID NO: 84, and wherein the sense strand and the antisense strand share a complementary region of at least 20, at least 21, at least 22, at least 23, at least 24 or at least 25 nucleotides in length, and wherein the nucleotide sequence of the antisense strand differs from that of the sense strand by no more than 4 nucleotides. In a preferred aspect, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 82 and SEQ ID NO: 84, and wherein the sense strand shares a complementary region of length 21, and wherein the nucleotide sequence of the antisense strand differs from that of the sense strand by no more than 4 nucleotides.

[0090] In a more preferred aspect, a siRNA molecule for inhibiting the expression of ACTA1 is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 68, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 79 and SEQ ID NO: 82, and wherein the sense strand and the antisense strand share a complementary region of at least 20, at least 21, at least 22, at least 23, at least 24 or at least 25 nucleotides in length, and wherein the nucleotide sequence of the antisense strand differs from that of the sense strand by no more than 4 nucleotides. In a preferred aspect, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 68, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 79 and SEQ ID NO: 82, and wherein the sense strand shares a complementary region of length 21, and wherein the sequence of the antisense strand differs from the nucleotide sequence of the sense strand by no more than 4 nucleotides.

[0091] In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 45, and wherein the antisense strand shares a complementary region of at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length with the sense strand, and wherein the sequence of the antisense strand differs from the nucleotide sequence of the sense strand by no more than 4 nucleotides. In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 45, and wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 128.

[0092] In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 46, and wherein the antisense strand shares a complementary region of at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length with the sense strand. In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 46, and wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 129.

[0093] In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 47, and wherein the antisense strand shares a complementary region of at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length with the sense strand. In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 47, and wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 130.

[0094] In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 51, and wherein the antisense strand shares a complementary region of at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length with the sense strand. In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 51, and wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 134.

[0095] In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 52, and wherein the antisense strand shares a complementary region of at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length with the sense strand. In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 52, and wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 135.

[0096] In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 53, and wherein the antisense strand shares a complementary region of at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length with the sense strand. In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 53, and wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 136.

[0097] In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 56, and wherein the antisense strand shares a complementary region of at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length with the sense strand. In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 56, and wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 139.

[0098] In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 58, and wherein the antisense strand shares a complementary region of at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length with the sense strand. In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 58, and wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 141.

[0099] In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 63, and wherein the antisense strand shares a complementary region of at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length with the sense strand. In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 63, and wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 146.

[0100] In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 65, and wherein the antisense strand shares a complementary region of at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length with the sense strand. In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 65, and wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 148.

[0101] In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 66, and wherein the antisense strand shares a complementary region of at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length with the sense strand. In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 66, and wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 149.

[0102] In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 68, and wherein the antisense strand shares a complementary region of at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length with the sense strand. In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 68, and wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 151.

[0103] In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 74, and wherein the antisense strand shares a complementary region of at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length with the sense strand. In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 74, and wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 157.

[0104] In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 76, and wherein the antisense strand shares a complementary region of at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length with the sense strand. In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 76, and wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 159.

[0105] In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 79, and wherein the antisense strand shares a complementary region of at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length with the sense strand. In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 79, and wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 162.

[0106] In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 82, and wherein the antisense strand shares a complementary region of at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length with the sense strand. In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 82, and wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 165.

[0107] In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 84, and wherein the antisense strand shares a complementary region of at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length with the sense strand. In some aspects, a siRNA molecule for inhibiting ACTA1 expression is provided, wherein the siRNA molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO: 84, and wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 167.

[0108] In some aspects, an shRNA molecule that inhibits the expression of ACTA1 is provided. shRNA stands for short hairpin RNA. It is an RNA sequence that allows a tight hairpin to rotate and can be used to silence target gene expression. Once inside the cell, shRNA is cleaved into siRNA (small interfering RNA) by the enzyme Dicer. These siRNAs can then be incorporated into an RNA-induced silencing complex (RISC), which can degrade messenger RNA or inhibit its translation, thereby effectively reducing or suppressing the expression of the target gene. In some aspects, an shRNA molecule that inhibits the expression of ACTA1 is provided, wherein the shRNA molecule comprises the siRNA molecule as described above. In some aspects, a regulatory polynucleotide is provided that comprises an shRNA molecule that inhibits the expression of ACTA1.

[0109] In some aspects, a miRNA scaffold comprising the regulatory polynucleotides described above is provided. In other aspects, the regulatory polynucleotides are embedded (integrated) into the miRNA scaffold. For example, a portion of the complementary strand of the miRNA scaffold is replaced by a corresponding sequence of the regulatory polynucleotide (e.g., siRNA molecule). Embedding the regulatory polynucleotides into the miRNA scaffold improves its stability and transcriptional efficiency.

[0110] In some aspects, the miRNA scaffold is selected from the group consisting of miRNA-517a, miR30a, miR155, miR450b, mmu-pri-miR-33, miR16-2(3G), and miR802. In a preferred aspect, the miRNA scaffold is a miRNA-33 scaffold (e.g., mmu-pri-miR-33). Exemplary miRNA-33 scaffold sequences are included below and in the appended examples and exemplary sequences. In a particular aspect, the miRNA scaffold (without regulatory polynucleotides, i.e., prior to the embedding of regulatory polynucleotides into the miRNA scaffold) comprises or is composed of the polynucleotide sequence of SEQ ID NO: 93. In a preferred embodiment, the miRNA-33 scaffold comprises the sequence of SEQ ID NO: 93 or a sequence having at least 90% sequence identity with SEQ ID NO: 93.

[0111] In some respects, the regulatory polynucleotide comprises a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a nucleotide sequence selected from the group consisting of: SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, and SEQ ID NO: 124.

[0112] In a preferred aspect, a regulatory polynucleotide is provided comprising a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a nucleotide sequence selected from the group consisting of: SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123.

[0113] In a most preferred aspect, a regulatory polynucleotide is provided comprising a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a nucleotide sequence selected from the group consisting of: SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 122, and SEQ ID NO: 123. In a particular such most preferred aspect, the regulatory polynucleotide is selected from the group consisting of: SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 122, and SEQ ID NO: 123.

[0114] In some aspects, the regulatory polynucleotide (including the miRNA-33 scaffold containing the regulatory polynucleotide) described above is operatively linked to a promoter element. In some aspects, the promoter element is a promoter element selected from a list consisting of: ACTA1 promoter (NCBI gene ID 58, actin α1), CACNG1 promoter (NCBI gene ID 786, voltage-dependent calcium channel γ-1 subunit), MYBPC2 promoter (NCBI gene ID 4606, myosin-binding protein C, fast type), MYH2 promoter (NCBI gene ID 4620, myosin-2), MYLPF promoter (NCBI gene ID 29895, and myosin regulatory light chain 2, skeletal muscle isotype), MYBC1 promoter, TTN promoter (NCBI gene ID 7273, titin), or a fragment thereof that retains tissue-specific expression (specifically, skeletal muscle-specific expression).

[0115] In the aspects described above, the promoter and / or promoter element preferably exhibit the functional characteristics described below.

[0116] Functional properties of polynucleotides

[0117] In aspects and embodiments of this disclosure, the regulatory polynucleotides of this disclosure can be characterized by reference to one or more functional properties. Some very important functional properties of the provided regulatory polynucleotides comprising a miRNA-33 scaffold containing a regulatory polynucleotide are scaffold stability, transcriptional efficiency, and knockdown of target genes, such as endogenous ACTA1 (e.g., a mutated ACTA1 gene with at least one pathogenic mutation).

[0118] In some embodiments, regulatory polynucleotides inhibit the expression of ACTA1. In the context of RNA interference, the term "repressing gene expression" refers to the process of inhibiting or reducing the activity or production of a specific gene's protein product by introducing small interfering RNA (siRNA) or microRNA (miRNA) molecules. These RNA molecules selectively bind to complementary messenger RNA (mRNA) molecules, leading to their degradation or inhibiting their translation into proteins. This targeted interference at the RNA level effectively reduces the expression of the corresponding gene, providing a precise mechanism for regulating gene activity in various biological processes and applications, including gene function research and therapeutic interventions.

[0119] In some embodiments, skeletal muscle cells containing the regulatory polynucleotide of the present invention have reduced or absent expression of endogenous ACTA1 (e.g., a mutated ACTA1 gene with at least one pathogenic mutation). In a preferred embodiment, skeletal muscle cells containing the regulatory polynucleotide of the present disclosure substantially do not express endogenous ACTA1 (e.g., a mutated ACTA1 gene with at least one pathogenic mutation).

[0120] In some embodiments, cells that substantially do not express endogenous ACTA1 may exhibit expression levels less than 0.2 times that of cells expressing endogenous ACTA1, for example, ≤0.1 times, ≤0.09 times, ≤0.08 times, ≤0.07 times, ≤0.06 times, ≤0.05 times, ≤0.04 times, ≤0.03 times, ≤0.02 times, or ≤0.01 times. In some embodiments, cells “expressing” endogenous ACTA1 may exhibit expression levels greater than 5 times (e.g., ≥10 times, ≥20 times, ≥50 times, ≥100 times, ≥1000 times, ≥5000 times, or ≥10000 times) that of cells that substantially do not express endogenous ACTA1.

[0121] The expression of a (target) gene can be assessed using any suitable technique for detecting and / or quantifying the relevant polypeptide encoded by the gene. Such techniques include, for example, antibody-based methods such as immunocytochemistry (e.g., Western blotting, ELISA), fluorescence microscopy, and flow cytometry. The expression of the target gene can also be measured at the mRNA level. Methods for measuring mRNA levels include techniques such as reverse transcription polymerase chain reaction (RT-PCR), quantitative PCR (qPCR) or real-time PCR, RNA sequencing (RNA-Seq), Northern blotting, and microarrays. In some embodiments, the expression of the polypeptide of interest can be assessed as described in the experimental examples of this disclosure.

[0122] For example, the expression level of endogenous ACTA1 can be measured in target cells (e.g., skeletal muscle cells or cell lines), and its expression in target cells can be compared to that in the same cell type or cell line containing nucleic acids (including the miRNA-33 scaffold and regulatory polynucleotides as described above). The ratio of endogenous ACTA1 expression in cells without nucleic acids (containing the miRNA-33 scaffold containing regulatory polynucleotides) to that in cells with nucleic acids (containing the miRNA-33 scaffold containing regulatory polynucleotides) can be calculated to illustrate the knockdown efficacy.

[0123] In some respects, i) after 5 days of AAV transduction with serotype 2 containing a miRNA-33 scaffold (containing a regulatory polynucleotide operablely linked to a promoter) at an MOI of 20,000, the expression level of endogenous ACTA1 was compared with ii) after 5 days of AAV transduction with the same MOI of 20,000, at a MOI of 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, 0.1 or less, 0.01 or less, and 0.001 or less. In some respects, the expression of endogenous ACTA1 was determined by qPCR.

[0124] An exemplary miRNA-33 scaffold containing a regulatory polynucleotide exhibiting such functional properties comprises a polynucleotide having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the group consisting of: SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 122, and SEQ ID NO: 123.

[0125] It should be understood that the functional properties described herein were assessed using similar (or identical) experimental conditions on cells containing different regulatory polynucleotides. However, some experimental conditions may need to be adjusted, for example, depending on the cell type. Adjustments to experimental conditions for specific cell types can be made according to methods known in the art and as described in the experimental section.

[0126] Expression System

[0127] In some embodiments, an expression system is provided comprising a regulatory polynucleotide as described above. In some embodiments, the regulatory polynucleotide comprises a miRNA-33 scaffold containing the regulatory polynucleotide as described above. In some embodiments, the regulatory polynucleotide is operatively linked to a polynucleotide encoding a polypeptide of interest.

[0128] The term "operably linked" can include situations where a nucleic acid sequence (e.g., a polynucleotide sequence containing a regulatory polynucleotide or encoding a polypeptide of interest according to this disclosure) and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked in such a manner to place the expression of the regulatory polynucleotide under the influence or control of the regulatory nucleic acid sequence (thereby forming an expression cassette). Thus, if the regulatory sequence is capable of influencing the transcription of a selected nucleic acid sequence, the regulatory sequence is operably linked to that nucleic acid sequence. The resulting transcript can then be a regulatory polynucleotide, or the transcript can be translated into the desired polypeptide of interest.

[0129] In some embodiments, the target peptide is a peptide suitable for the treatment or prevention of a disease / symptom. In some embodiments, the peptide of interest is the actin gene. In some embodiments, the peptide of interest is ACTA1. In some embodiments, the peptide of interest replaces a mutated ACTA1 (such as, for example, in gene replacement therapy). In some embodiments, the mutated ACTA1 is associated with a disease or symptom. In some embodiments, the mutated ACTA1 is associated with congenital myopathy, such as, for example, filamentous myopathy, intranuclear rod myopathy, actin filament aggregation myopathy, congenital myofiber heterogeneity, and myopathy with nucleoid regions.

[0130] In some embodiments, the polynucleotide encoding the polypeptide of interest (e.g., ACTA1) is codon-optimized. "Codon optimization" refers to the process of altering the DNA sequence of a gene to improve its expression in a host organism without changing the protein it encodes. This is achieved by changing the codons (three-letter sequences in mRNA that specify a particular amino acid in the protein being produced) to the most commonly used codons in the host organism. This can increase the rate and amount of protein production. In the context of polynucleotides, "codon-optimized" refers to a polynucleotide sequence altered in this way to improve its expression.

[0131] In some respects, the polypeptide of interest comprises or consists of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a sequence selected from Column E of Table 2.

[0132] In addition to regulatory polynucleotides and polynucleotides encoding polypeptides of interest, the regulatory polynucleotides of the present invention may also contain additional nucleotide sequences and / or sequence features.

[0133] The expression system disclosed herein may include a start codon at the 5' position (i.e., upstream of the polynucleotide sequence in the context of the polynucleotide sequence) of a polynucleotide sequence encoding a polypeptide of interest (e.g., ACTA1). The start codon is preferably a trinucleotide 'ATG'.

[0134] In some embodiments, the expression system of the present invention further comprises a Kozak sequence. In a preferred embodiment, the Kozak sequence is provided upstream of the start codon used to initiate the translation of the polypeptide of interest (e.g., ACTA1).

[0135] In some embodiments, the expression system of the present invention further comprises one or more promoter sequences. The one or more promoter sequences are preferably polynucleotides encoding a polypeptide of interest (e.g., ACTA1) and / or the 5' end of a regulatory polynucleotide comprising a miRNA-33 scaffold according to the present disclosure.

[0136] In some embodiments, the expression system of the present invention comprises a 5'UTR. The term 5'UTR stands for 5' untranslated region. This region is transcribed but not translated into protein. The 5'UTR may contain regulatory elements and may regulate the stability of the mRNA, its localization, and the rate of protein synthesis. In some embodiments, the 5'UTR is operatively linked to a synthetic skeletal muscle-specific promoter of the present disclosure.

[0137] In some embodiments, the 5'UTR contains introns. In a preferred embodiment, the intron consists of fewer nucleotides than the number of nucleotides in a known (e.g., naturally occurring) intron. In some aspects, the 5'UTR does not contain a complete, naturally occurring promoter intron. In some aspects, the intron is a truncated natural or synthetic intron. In some aspects, the 5'UTR does not contain an intron.

[0138] In some embodiments, the 5'UTR comprises, or consists of, a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from columns B, C, and / or D of Table 2.

[0139] In some embodiments, the expression system of the present invention further includes a stop codon. The stop codon is preferably provided at the 3' of the trinucleotide immediately following the terminal amino acid encoding the polypeptide of interest (e.g., ACTA1) (i.e., downstream of it in the context of a polynucleotide sequence).

[0140] In some embodiments, the expression system of the present invention further comprises a polyadenylation signal sequence. In a preferred embodiment, the polyadenylation signal sequence is provided at the 3' position of the nucleotide sequence encoding the polypeptide of interest (e.g., ACTA1) (i.e., downstream of the nucleotide sequence of the polynucleotide). In some embodiments, the expression system of the present invention further comprises a terminator sequence. The terminator sequence is preferably located at the 3' position of the nucleotide sequence encoding the polypeptide of interest (and at the 3' position of the polyadenylation signal sequence, if present).

[0141] In some embodiments, the expression system of the present invention includes a 3'UTR. The term 3'UTR stands for 3' untranslated region. This region is transcribed but not translated into protein. The 3'UTR refers to the polynucleotide sequence following the stop codon and typically includes a polyadenylation signal.

[0142] In some embodiments, the 3'UTR comprises, or consists of, a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from columns H and / or I of Table 2.

[0143] In a preferred embodiment, the constituent nucleotide sequences of the expression system of the present invention are provided adjacent to each other. However, in some embodiments, the polynucleotide is further included among one or more adapter nucleotide sequences in one or more of the constituent nucleotide sequences of the expression system of the present invention. The adapter nucleotide sequence may comprise or consist of 1 to 10 nucleotides (e.g., one of 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 nucleotides). When the expression system of the present invention according to this disclosure includes one or more adapter nucleotide sequences, the adapter sequences are preferably selected such that they do not alter the amino acid sequence of the polypeptide encoded by the polynucleotide. In a preferred embodiment, when the expression system of the present invention according to this disclosure includes one or more adapter sequences, the expression system of the present invention encodes the same polypeptide as an equivalent expression system of the present invention lacking the adapter nucleotide sequence.

[0144] In some embodiments, the expression system of the present invention further comprises an inverted terminal repeat (ITR) sequence.

[0145] In some embodiments, the polynucleotide is contained in the ITR at the 5' of the promoter and / or enhancer sequence (if present). In some embodiments, the polynucleotide is contained in the ITR at the 3' of the nucleotide sequence encoding the polypeptide of interest (and at the 3' of the stop codon, polyadenylation signal sequence, and / or terminator sequence (if present).

[0146] In some embodiments, the 5'ITR comprises, or consists of, a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from Column A of Table 2.

[0147] In some embodiments, the 3'ITR comprises, or consists of, a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from column J of Table 2.

[0148] In some embodiments, the expression system of this disclosure has a size that allows it to be delivered as a gene therapy (i.e., in a suitable vector). In some embodiments, the vector consists of a nucleotide sequence having a size within the packaging limits of a vector for delivering polynucleotides.

[0149] In some embodiments, the expression system has a size within the packaging limits of the AAV vector. In some embodiments, the expression system has a size within the packaging limits of the AAV vector of one of the following serotypes: AAV9, AAV9.45, AAV-PHP.eB, AAV1, AAV2, AAV2i8, AAV5, AAV6, AAV8, AAV10, or AAVrh74.

[0150] In some embodiments, the expression system of this disclosure consists of less than 6,000 nucleotides (e.g., one of ≤5,000, ≤4,750, ≤4,500, ≤4,250, ≤4,000, ≤3,750, ≤3,500, ≤3,250, ≤3,000, ≤2,750, ≤2,500, ≤2,250, ≤2,000, ≤1,750, ≤1,500, ≤1,250, or ≤1,000 nucleotides).

[0151] In some embodiments, the expression system includes AAV padding sequences. For example, such padding sequences may be needed to accommodate promoter elements of different sizes or polynucleotides encoding the protein of interest.

[0152] In some embodiments, the expression system of this disclosure includes one or more nucleotide sequences encoding selection markers to facilitate the identification and / or selection of cells containing / expressing the polypeptide of interest. Selection markers include proteins conferring resistance to antibiotics or other toxins (e.g., blast fungicide, ampicillin, neomycin, methotrexate, or tetracycline) and proteins that supplement nutritional deficiencies.

[0153] In some embodiments, the expression system of this disclosure comprises a nucleotide sequence encoding an internal ribosome entry site (IRES). In some embodiments, the expression system of this disclosure comprises a nucleotide sequence that allows two or more polypeptides to be translated separately from a single polynucleotide.

[0154] The expression system disclosed herein can be provided in purified or isolated form, i.e., isolated from other nucleic acids or naturally occurring biological materials.

[0155] In some embodiments, the expression system of this disclosure is a vector. As used herein, "vector" refers to a polynucleotide used as a medium for transferring exogenous nucleic acids into cells. A vector can be a carrier for expressing nucleic acids in cells (i.e., a vector can be an expression vector). Such a vector may include a promoter sequence operatively linked to a nucleotide sequence to be expressed. The vector may also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon can be used for vectors according to this disclosure.

[0156] Vectors contemplated in this disclosure include DNA vectors, RNA vectors, plasmids (e.g., conjugating plasmids (e.g., F plasmids), non-conjugating plasmids, R plasmids, col plasmids, episomes), viral vectors (e.g., retroviral vectors, such as gamma retroviral vectors (e.g., vectors derived from murine leukemia virus (MLV), such as SFG vectors), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, baculovirus vectors, and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes), such as those described in Maus et al., Annu Rev Immunol (2014) 32:189-225 and Morgan and Boyerinas, Biomedicines (2016) 4:9, both of which are hereby incorporated herein by reference in their entirety.

[0157] In some embodiments, the vector may be a eukaryotic vector, i.e., a vector containing elements necessary for protein expression from a vector in a eukaryotic cell. In some embodiments, the vector may be a mammalian vector, which, for example, contains a cytomegalovirus (CMV) or SV40 promoter to drive protein expression. In some embodiments, the vector contains a CMV (e.g., mCMV), SV40, RSV, or PGK promoter.

[0158] In some embodiments, the vector is selected based on the tropism of the cell type / tissue / organ to which the expression system according to this disclosure is to be delivered. In some embodiments, the vector is selected based on the tropism of the cell type / tissue / organ in which the polypeptide of interest is to be expressed. For example, it may be desirable to deliver the expression system to a cell type / tissue / organ affected by a disease / symptom to be treated / prevented according to this disclosure (e.g., a cell type / tissue / organ exhibiting symptoms of the disease / symptom), and / or to express the polypeptide of interest in that cell type / tissue / organ.

[0159] For example, it may be desirable to deliver the expression system of this disclosure encoding a polypeptide of interest (e.g., ACTA1) to skeletal muscle tissue, and in such cases a vector that is tropism for such cells / tissues may be employed.

[0160] In a preferred embodiment, the vector is an adeno-associated virus (AAV) vector. AAV vectors and their applications in vector gene therapy are reviewed, for example, in Wang et al., Nat. Rev. Drug Discov. (2019) 18: 358-378 and Li and Samulski, Nat. Rev. Genet. (2020) 12: 255-272, both of which are hereby incorporated by reference in their entirety. In some embodiments, the vector may be the adeno-associated virus vector described in Wang et al., Nat. Rev. Drug Discov. (2019) 18: 358-378. In some embodiments, the vector may be the adeno-associated virus vector described in Li and Samulski, Nat. Rev. Genet. (2020) 12: 255-272.

[0161] In some embodiments, the vector is a self-complementary adeno-associated virus (scAAV) vector. Self-complementary adeno-associated virus vectors are described, for example, in McCarty, Mol Ther. (2008) 16 (10): 1648-56, which is hereby incorporated in its entirety by reference. Conventional AAVs have a single-stranded DNA genome and rely on the DNA replication mechanism of the transduced cell to synthesize the complementary strand, thereby delaying transgene expression. In contrast, scAAVs contain a complementary sequence that anneals spontaneously upon infection, thus eliminating the need for DNA synthesis in the transduced host cell. Compared to classic single-stranded AAV vectors, scAAV vectors have been shown to provide accelerated transgene expression and increased transgene expression levels.

[0162] In some embodiments, the vector may be an adeno-associated virus vector of one of the following serotypes: AAV9 (including AAV9 variants AAV-PHP.eB and AAV9.45), AAV1, AAV2 (including AAV2 variant AAV2i8), AAV5, AAV6, AAV8, AAV10, or AAVrh74. In some embodiments, the vector is an AAV9 vector.

[0163] In some embodiments, the vector contains modifications to increase binding and / or transduction with the cell type of interest (i.e., compared to the binding / transduction level of an unmodified vector). In some embodiments, the modification is a modification of the capsid protein.

[0164] In some embodiments, the carrier comprises a capsid protein containing a cell-targeting peptide. In some embodiments, the cell-targeting peptide is the cell-targeting peptide described herein by reference to Büning and Srivastava, Molecular Therapy: Methods & Clinical Development (2019) 12: 248-265, which are incorporated herein by reference, such as the cell-targeting peptides shown in Tables 1, 2, 3 or 4 thereon.

[0165] In some embodiments, the carrier comprises a capsid protein containing substitutions for one or more tyrosine residues, such as one or more surface-exposed tyrosine residues. In some embodiments, one or more tyrosine residues of the capsid protein are substituted with phenylalanine. In some embodiments, the carrier comprises a capsid protein wherein one or more tyrosine residues are substituted with another amino acid, as described in Iida et al., Biomed Res Int. (2013) 2013: 974819, which is hereby cited in its entirety.

[0166] In some embodiments, the vector may be the adeno-associated virus vector described above by Büning and Srivastava. In some embodiments, the vector may be the adeno-associated virus vector described above by Iida et al.

[0167] In some embodiments, the carrier includes a control element for inducible expression of the expression system of this disclosure.

[0168] In some embodiments, an adeno-associated virus (AAV) comprising a vector genome is provided, wherein the vector genome comprises, in a 5' to 3' sequence:

[0169] (i) 5' inverted terminal repeat (ITR) sequence;

[0170] (ii) Promoter;

[0171] (iii) Optionally, a 5' UTR sequence;

[0172] (iv) Codon-optimized polynucleotides encoding ACTA1;

[0173] (v) 3' UTR sequence; and

[0174] (vi) 3' Inverted Terminal Repeat (ITR) sequence,

[0175] The vector genome contains a regulatory polynucleotide that inhibits the expression of both the wild-type ACTA1 gene and the mutated ACTA1 gene with at least one pathogenic mutation, wherein the regulatory polynucleotide contains a polynucleotide sequence having at least 85% sequence identity with a sequence selected from the group consisting of: SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122 and SEQ ID NO: 123.

[0176] In some embodiments, an adeno-associated virus (AAV) comprising a vector genome is provided, wherein the vector genome comprises, in a 5' to 3' sequence:

[0177] (i) 5' inverted terminal repeat (ITR) sequence;

[0178] (ii) Promoter;

[0179] (iii) Optionally, a 5' UTR sequence;

[0180] (iv) Codon-optimized polynucleotides encoding ACTA1;

[0181] (v) 3' UTR sequence; and

[0182] (vi) 3' Inverted Terminal Repeat (ITR) sequence,

[0183] The vector genome contains polynucleotides selected from the group consisting of: SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122 and SEQ ID NO: 123.

[0184] In some embodiments, an adeno-associated virus (AAV) comprising a vector genome is provided, wherein the vector genome comprises, in a 5' to 3' sequence:

[0185] (i) 5' inverted terminal repeat (ITR) sequence;

[0186] (ii) Promoter;

[0187] (iii) A 5' UTR sequence comprising a regulatory polynucleotide that inhibits the expression of both the wild-type ACTA1 gene and a mutated ACTA1 gene having at least one pathogenic mutation, wherein the regulatory polynucleotide comprises a polynucleotide sequence having at least 85% sequence identity with a sequence selected from the group consisting of: SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122 and SEQ ID NO: 123;

[0188] (iv) Codon-optimized polynucleotides encoding ACTA1;

[0189] (v) 3' UTR sequence; and

[0190] (vi) 3' Inverted terminal repeat (ITR) sequence.

[0191] In some embodiments, an adeno-associated virus (AAV) comprising a vector genome is provided, wherein the vector genome comprises, in a 5' to 3' sequence:

[0192] (i) 5' inverted terminal repeat (ITR) sequence;

[0193] (ii) Promoter;

[0194] (iii) A 5' UTR sequence comprising a polynucleotide sequence selected from the group consisting of: SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122 and SEQ ID NO:123;

[0195] (iv) Codon-optimized polynucleotides encoding ACTA1;

[0196] (v) 3' UTR sequence; and

[0197] (vi) 3' Inverted terminal repeat (ITR) sequence.

[0198] In some embodiments, an adeno-associated virus (AAV) comprising a vector genome is provided, wherein the vector genome comprises, in a 5' to 3' sequence:

[0199] (i) 5' inverted terminal repeat (ITR) sequence;

[0200] (ii) Promoter;

[0201] (iii) Optional 5' UTR;

[0202] (iv) A codon-optimized polynucleotide encoding ACTA1, wherein the polynucleotide encoding the codon-optimized polynucleotide further comprises a regulatory polynucleotide that inhibits the expression of both the wild-type ACTA1 gene and the mutated ACTA1 gene having at least one pathogenic mutation, wherein the regulatory polynucleotide comprises a polynucleotide sequence having at least 85% sequence identity with a sequence selected from the group consisting of: SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122 and SEQ ID NO: 123;

[0203] (v) 3' UTR sequence; and

[0204] (vi) 3' Inverted terminal repeat (ITR) sequence.

[0205] In some embodiments, an adeno-associated virus (AAV) comprising a vector genome is provided, wherein the vector genome comprises, in a 5' to 3' sequence:

[0206] (i) 5' inverted terminal repeat (ITR) sequence;

[0207] (ii) Promoter;

[0208] (iii) Optional 5' UTR;

[0209] (iv) A codon-optimized polynucleotide encoding ACTA1, wherein the polynucleotide encoding the codon-optimized polynucleotide further comprises a regulatory polynucleotide that inhibits the expression of both the wild-type ACTA1 gene and the mutated ACTA1 gene having at least one pathogenic mutation, wherein the regulatory polynucleotide comprises a sequence selected from the group consisting of: SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122 and SEQ ID NO: 123;

[0210] (v) 3' UTR sequence; and

[0211] (vi) 3' Inverted terminal repeat (ITR) sequence.

[0212] In any of the AAVs provided above, the regulatory polynucleotide does not inhibit the expression of the codon-optimized polynucleotide encoding ACTA1.

[0213] In some embodiments, the AAV comprises (i) a 5' ITR sequence comprising or consisting of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from Column A of Table 2.

[0214] In some embodiments, the AAV includes (ii) a promoter comprising or consisting of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from column B of Table 2. Preferably, the promoter comprises or consists of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 99.

[0215] In some embodiments, the AAV includes (iii) a 5'UTR that contains or consists of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from columns B, C, and / or D of Table 2.

[0216] In some embodiments, the AAV comprises (iv) a polynucleotide encoding a protein of interest, the polynucleotide comprising or consisting of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from column E of Table 2.

[0217] In some embodiments, the AAV includes (v) a 3'UTR, which contains or is composed of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from columns H and / or I of Table 2.

[0218] In some embodiments, the AAV includes (vi) a 3'ITR, which comprises or consists of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from column J of Table 2.

[0219] In some embodiments, the AAV further comprises a polynucleotide comprising or consisting of a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from Column F of Table 2.

[0220] In some embodiments, an adeno-associated virus (AAV) is provided that comprises sequences A to G from one row selected from Table 2 from 5' to 3', these sequences optionally linked by a linker polynucleotide sequence.

[0221] cell

[0222] This disclosure also provides a cell comprising a regulatory polynucleotide according to the invention. A cell comprising an expression system (such as one or more vectors) according to the invention is also provided.

[0223] The cell can be a eukaryotic cell, such as a mammalian cell. The mammal can be a primate (rhesus monkey, cynomolgus monkey, non-human primate, or human) or a non-human mammal (e.g., rabbit, guinea pig, rat, mouse, hamster, or other rodent (including any animal in the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, such as dairy cows, or any animal in the genus Bos), horse (including any animal in the family Equidae), donkey, or non-human primate). In a preferred embodiment, the cell is a human cell.

[0224] This disclosure also provides a method for producing cells comprising an expression system / vector according to this disclosure, the method comprising introducing the expression system / vector of this disclosure into the cells. In some embodiments, introducing the expression system / vector according to this disclosure into the cells comprises transformation, transfection, electroporation, or transduction (e.g., adeno-associated virus transduction). In some embodiments, the expression system / vector is introduced into cells in vivo, for example by administering a vector according to this disclosure (e.g., a viral vector, such as an adeno-associated virus vector) to a subject. In some embodiments, the expression system / vector is introduced into cells cultured in vitro or in vitro.

[0225] Cells according to this disclosure can be produced using any suitable method. Such methods may include nucleic acid transfer for permanent (i.e., stable) or transient expression of polynucleotides according to this disclosure. In some embodiments, after introduction into the cell, the polynucleotide may be integrated into the cell's genomic DNA or form part of the cell's genomic DNA. In some embodiments, after introduction into the cell, the expression system / vector may be maintained extrachromosomally.

[0226] Any suitable genetic engineering platform can be used, including gamma retroviral vectors, lentiviral vectors, adenoviral vectors, DNA transfection, transposon-based gene delivery, and RNA transfection, such as those described in Maus et al., AnnuRev Immunol (2014) 32:189-225, which is hereby incorporated by reference in its entirety. Methods also include those described, for example, in Wang and Rivière Mol Ther Oncolytics. (2016) 3:16015, the entire contents of which are incorporated herein by reference. Suitable methods for introducing nucleic acids / vectors into cells include transduction, transfection, and electroporation.

[0227] In some embodiments, the method further includes maintaining the cells under conditions suitable for the expression of the cell vector / peptide of interest.

[0228] This disclosure also provides cells that can be obtained or acquired by the methods according to this disclosure.

[0229] Composition

[0230] This disclosure also provides compositions comprising the regulatory polynucleotides, expression systems, vectors, and cells described herein. Specifically, this disclosure provides pharmaceutical compositions and pharmaceuticals comprising the regulatory polynucleotides, expression systems, vectors, and cells of this disclosure.

[0231] Such compositions may comprise related articles (i.e., regulatory polynucleotides / expression systems / vectors / cells) suitable for clinical use in formulations. This disclosure specifically relates to pharmaceutical compositions / medications comprising a vector according to this disclosure.

[0232] The pharmaceutical compositions / medications disclosed herein may comprise one or more pharmaceutically acceptable carriers (e.g., liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g., starch, cellulose, cellulose derivatives, polyols, glucose, maltodextrin, magnesium stearate), excipients, fillers, buffers, preservatives (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methylparaben, propylparaben), antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), lubricants (e.g., magnesium stearate, talc, silica, stearic acid, vegetable stearin), binders (e.g., sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, or colorants (e.g., titanium dioxide).

[0233] As used herein, the term "pharmaceutical acceptable" refers to compounds, ingredients, materials, compositions, dosage forms, etc., that, to the extent of reasonable medical judgment, are suitable for contact with the tissues of a relevant subject (e.g., a human subject) without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, antioxidant, lubricant, binder, stabilizer, solubilizer, surfactant, masking agent, colorant, flavoring agent, or sweetener in the compositions according to this disclosure must also be "acceptable" in the sense of compatibility with the other components of the formulation. Suitable carriers, diluents, excipients, auxiliaries, fillers, buffers, preservatives, antioxidants, lubricants, binders, stabilizers, solubilizers, surfactants, masking agents, colorants, flavorings, or sweeteners can be found in standard pharmacy textbooks (e.g., Remington's "The Science and Practice of Pharmacy" ((A)Adejare, ed.), 23rd edition (2020), Academic Press).

[0234] The pharmaceutical compositions / medications according to this disclosure can be formulated for administration to a subject, for example, via a route of administration as appropriate, depending on the nature of the therapeutic agent and the disease to be treated / prevented. In some embodiments, the pharmaceutical compositions / medications can be formulated for parenteral, systemic, local, intracavitary, intravascular, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, oral, or transdermal administration. In some embodiments, the pharmaceutical compositions / medications can be formulated for administration by injection or infusion, or by ingestion.

[0235] Drugs and drug compositions can be formulated for administration to blood vessels or to target tissues / organs (e.g., tissues / organs affected by disease / symptoms, such as tissues / organs where symptoms of disease / symptoms are manifest).

[0236] The pharmaceutical composition / drug may comprise a regulatory polynucleotide / expression system / vector / cell in a sterile or isotonic medium. The pharmaceutical composition / drug may be provided in a fluid form, including a gel. Fluid formulations may be formulated for administration to a selected area of ​​a blood vessel or human or animal body via injection or infusion (e.g., via a catheter). The pharmaceutical composition / drug may be provided in a solid form, such as a lyophilized form.

[0237] This disclosure also provides methods for producing pharmaceutical compositions / medications according to this disclosure. Such methods may include mixing the regulatory polynucleotide / expression system / vector / cell described herein with a pharmaceutically acceptable load, diluent, excipient, adjuvant, filler, buffer, preservative, antioxidant, lubricant, binder, stabilizer, solubilizer, surfactant, masking agent, colorant, flavoring agent, or sweetener. Such methods typically involve the step of associating the regulatory polynucleotide / expression system / vector / cell with a load constituting one or more adjuvant components. Generally, compositions are prepared by uniformly and tightly associating the active compound with a load (e.g., a liquid load, a finely dispersed solid load, etc.) and then shaping the product if necessary.

[0238] The regulatory polynucleotide / expression systems, vectors, cells, and compositions disclosed herein can be modified and / or formulated to facilitate delivery to and / or uptake by cell types / tissues / organs of interest (e.g., cell types / tissues / organs exhibiting symptoms of disease / symptoms).

[0239] Strategies for targeted delivery of polynucleotides are reviewed, for example, in Li et al., Int. J. Mol. Sci. (2015) 16:19518-19536 and Fu et al., Bioconjug Chem. (2014) 25(9): 1602-1608, which are hereby incorporated in their entirety by reference.

[0240] In some embodiments, the articles of this disclosure may be encapsulated in nanoparticles or liposomes. In some embodiments, the articles of this disclosure may be associated (covalently or non-covalently) with cell-penetrating peptides (e.g., protein transduction domains, trojan peptides, arginine-rich peptides, vectocell peptides), cationic polymers, cationic lipids, or viral vectors.

[0241] Nanoparticles can be organic, such as micelles, liposomes, proteins, solid lipid particles, solid polymer particles, dendritic molecules, and polymeric therapeutic agents. Nanoparticles can also be inorganic, such as nanotubes or metal particles, optionally with the addition of organic molecules. In some embodiments, nanoparticles are those described in Chen et al., Mol Ther Methods ClinDev. (2016) 3:16023, the entire contents of which are incorporated herein by reference. In some embodiments, nanoparticles are PLGA, peptides, poly(β-amino esters), DOPE, β-cyclodextrin-containing polycationic, linear PEI, PAMAM dendritic macromolecules, branched PEI, chitosan, or polyphosphate nanoparticles.

[0242] In some embodiments, the expression systems and vectors according to this disclosure include modifications to incorporate one or more portions to facilitate delivery to and / or uptake by a cell type, organ, or tissue of interest (e.g., a cell type / tissue / organ exhibiting symptoms of disease / symptoms). In some embodiments, a polynucleotide or vector according to this disclosure is linked (e.g., chemically conjugated) to one or more portions to facilitate delivery to and / or uptake by a cell type, tissue, or organ of interest.

[0243] Therapeutic / Preventive Application

[0244] The regulatory polynucleotides, expression systems, vectors and cells disclosed herein, as well as the compositions thereof, can be used in treatment and prevention.

[0245] Therefore, this disclosure provides the regulatory polynucleotides, expression systems, vectors, cells, or compositions described herein for use in methods of medical treatment or prevention. It also provides the regulatory polynucleotides, expression systems, vectors, cells, or compositions described herein for use in methods of treating or preventing the diseases / symptoms described herein. Furthermore, it provides the use of the regulatory polynucleotides, expression systems, vectors, cells, or compositions described herein in the manufacture of a medicament for treating or preventing the diseases or symptoms described herein. Finally, it provides a method of treating or preventing the diseases or symptoms described herein, comprising administering to a subject a therapeutically or preventively effective amount of the regulatory polynucleotides, expression systems, vectors, cells, or compositions described herein.

[0246] The interventions described in the preceding paragraph can effectively reduce the development or progression of a disease / symptom, alleviate the symptoms of a disease / symptom, or reduce the pathology of a disease / symptom. Interventions can effectively prevent the progression of a disease / symptom, such as preventing its worsening or slowing its rate of development. In some embodiments, interventions can lead to improvement in the disease / symptom, such as a reduction in the symptoms of the disease / symptom or a reduction in some other associated severity / activity of the disease / symptom. In some embodiments, interventions can prevent the progression / development of the disease / symptom in later stages (e.g., the chronic phase).

[0247] It should be understood that the regulatory polynucleotides, expression systems, vectors, cells, and compositions described herein can be used to treat / prevent any disease / symptom from which therapeutic or preventive benefits will be derived by an increase in the level of the polypeptide of interest (i.e., the polypeptide of interest encoded by the polynucleotide).

[0248] For example, a disease / symptom can be associated with and / or characterized by the deficiency / insufficiency of the target peptide. The deficiency / insufficiency of the target peptide may be positively correlated with the onset, development, or progression of the disease / symptom, and / or with the severity of one or more symptoms of the disease / symptom. The deficiency / insufficiency of the target peptide may be a risk factor for the onset, development, or progression of the disease / symptom. A disease / symptom is characterized by a reduced level of expression or activity of the target peptide, for example, compared to the expression / activity level in the absence of the disease / symptom. In some embodiments, a disease / symptom may be characterized by a reduced number / proportion / activity of cells expressing the target peptide, for example, compared to the level / number / proportion / activity in the absence of the disease / symptom (e.g., in healthy subjects or equivalent non-disease tissues).

[0249] For example, in some embodiments, the polypeptide of interest may be ACTA1, and the disease / symptom to be treated / prevented according to this disclosure may be a disease / symptom caused by a deficiency / insufficiency of ACTA1, such as congenital myopathy, such as linear myopathy, intranuclear rod myopathy, actin filament aggregation myopathy, congenital fiber type imbalance and myopathy with core-like regions (Laing et al., Hum Mutat. Sep 2009; 30(9): 1267-1277).

[0250] As a further example, in embodiments where the regulatory polynucleotide can inhibit the expression and / or activity of the target antigen of interest, the disease / symptom can be a disease / symptom in which the target antigen is pathologically involved or cells containing / expressing the target antigen are present, for example, a disease / symptom in which an increase in the level / activity of the target antigen or an increase in the number / proportion / activity of cells containing / expressing the target antigen is positively correlated with the onset, development, or progression of the disease / symptom and / or the severity of one or more symptoms of the disease / symptom. In some embodiments, an increase in the level / activity of the target antigen, or an increase in the number / proportion / activity of cells containing / expressing the target antigen, can be a risk factor for the onset, development, or progression of the disease / symptom. The disease / symptom can be characterized by an increase in the expression or activity level of the target antigen, for example, compared to the expression / activity level in the absence of the disease / symptom. In some embodiments, the disease / symptom can be characterized by an increase in the number / proportion / activity of cells expressing the target antigen, for example, compared to the level / number / proportion / activity in the absence of the disease / symptom (e.g., in healthy subjects or equivalent non-disease tissues). The therapeutic / preventive interventions according to this disclosure can achieve one or more of the following in subjects (compared to equivalent untreated subjects or subjects treated with appropriate controls): a reduction in the level of (mutated) target antigen; a reduction in the activity of the target antigen; and / or a reduction in the number / proportion / activity of cells containing / expressing the target antigen.

[0251] This disclosure provides products of the disclosure for use, uses of products of the disclosure, and methods of administering a regulatory polynucleotide, carrier, cell, or composition according to the disclosure to a subject (e.g., a subject requiring treatment).

[0252] The application of the articles disclosed herein is preferably in a "therapeuticly effective" or "preventively effective" amount, sufficient to demonstrate a therapeutic or preventative benefit to the subject. The actual dosage, as well as the rate and timing of administration, will depend on the nature and severity of the disease / symptom and the specific article being applied. Prescribing treatment, such as determining the dosage, is within the responsibility of a general practitioner and other physicians, and generally takes into account the disease / symptom to be treated, the individual subject's condition, the site of delivery, the method of administration, and other factors known to the physician. Examples of the above techniques and protocols can be found in Remington's "The Science and Practice of Pharmacy" (ed. (A) Adejare), 23rd edition (2020), Academic Press.

[0253] The items disclosed herein can be administered parenterally, systemically, intravenously, intra-arterially, intramuscularly, intracavitarily, intrathecally, intraocularly, intravitreally, intraconjunctivally, subretinally, suprachoroidally, subcutaneously, intradermally, or orally, nasally, topically, or percutaneously. Administration can be performed by injection or infusion.

[0254] Multiple doses of the article disclosed herein may be provided. The multiple doses may be separated by a predetermined time interval, which may be selected as 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 or 31 days, or one of 1, 2, 3, 4, 5 or 6 months.

[0255] Other methods

[0256] This disclosure also provides a method for modifying cells to express a protein of interest according to this disclosure, the method comprising introducing an expression system or vector according to this disclosure into the cells.

[0257] In some embodiments, the introduction of an expression system or vector according to the present disclosure into cells includes transformation, transfection, electroporation, or transduction (e.g., retroviral transduction).

[0258] Transfection involves the introduction of nucleic acids into cells using methods other than viral infection, and is therefore a non-viral approach. Transfection can be performed using physical / mechanical methods (including electroporation, acoustic perforation, magnetic transfection, gene microinjection, and laser irradiation) or chemical methods (liposome-based or non-liposome-based). Liposome-based transfection reagents are chemicals that enable the formation of positively charged lipid aggregates, which can then fuse with the cellular phospholipid bilayer to facilitate the entry of foreign genetic material. Examples of liposome-based transfection reagents include, but are not limited to, Oligofectamine®, Lipofectamine®, and DharmaFECT®. Non-liposome-based transfection reagents include, but are not limited to, calcium phosphate, nanoparticles, polymers, dendritic molecules, and non-liposomal lipids. One example of a non-liposome-based transfection reagent is polyethyleneimine (PEI).

[0259] Electroporation can be performed, for example as described in Koh et al., Molecular Therapy – Nucleic Acids (2013) 2, e114, which is hereby incorporated herein by reference in its entirety.

[0260] Transduction is the process of introducing nucleic acids into cells via viruses or viral vectors. Therefore, in some embodiments, polynucleotides are or are contained in a viral vector, or the vector is a viral vector. Transduction of immune cells with viral vectors is described, for example, in Simmons and Albertola-Ila, Methods Mol Biol. (2016) 1323:99-108, the entire contents of which are incorporated herein by reference. Reagents may be used in the methods of this disclosure to enhance transduction efficiency. Hexadimethrine bromide (polygluconine) is a cationic polymer commonly used to improve transduction by neutralizing the charge repulsion between viral particles and sialic acid residues expressed on the cell surface. Other reagents commonly used to enhance transduction include, for example, poloxamer-based reagents such as LentiBOOST (Sirion Biotech), Retrolectin (Takara), Vectofusin (Miltenyi Biotech), and SureENTRY (Qiagen) and ViraDuctin (Cell Biolabs).

[0261] In some embodiments, the method includes centrifuging (referred to in the art as “spinfection”) cells into which the expression system or vector according to the present disclosure is desired to be introduced in the presence of a cell culture medium containing a viral vector containing an expression system.

[0262] In some embodiments, the method includes culturing cells under conditions suitable for the expression of the polypeptide of interest. In some embodiments, the method includes culturing cells under conditions suitable for transcription of polynucleotides. In some embodiments, the method includes culturing cells under conditions suitable for post-transcriptional processing (e.g., splicing) of polynucleotides. In some embodiments, the method includes culturing cells under conditions suitable for translation of polypeptides from polynucleotides.

[0263] Methods for in vitro / in vitro culture (including generation and / or expansion) of cell populations—including suitable culture conditions (i.e., cell culture medium, additives, stimulation, temperature, gaseous atmosphere), cell numbers, culture cycles, etc.—are well known to those skilled in the art. Conveniently, cell cultures according to this disclosure can be maintained at 37°C in a humid atmosphere containing 5% CO2.

[0264] This disclosure also provides a method for modifying cells to express a polypeptide of interest, the method comprising introducing an expression system or vector according to this disclosure into the cell. When the polynucleotide is an OFF-switch or wherein the vector contains / encodes an OFF-switch, the cell may express the polypeptide of interest after the expression system / vector is introduced into the cell.

[0265] Subjects

[0266] According to various aspects of this disclosure, the subject can be any animal or human. Therapeutic and prophylactic applications can be in humans or animals (for veterinary use).

[0267] The subject to be administered the articles of this disclosure (e.g., according to a therapeutic or preventative intervention) may be a subject in need of such intervention. The subject is preferably a mammal, more preferably a human. The subject may be a non-human mammal, but more preferably a human. The subject may be male or female. In one embodiment, the subject is female. The subject may be a patient.

[0268] Subjects may have (e.g., may have been diagnosed with) the diseases or conditions described herein, may be suspected of having such diseases / conditions, or may be at risk of developing / contracting such diseases / conditions. In embodiments according to this disclosure, subjects may be selected for treatment based on a method of characterizing one or more markers of such diseases / conditions.

[0269] Sequence identity

[0270] The “sequence identity” between a given nucleotide sequence (e.g., the nucleotide sequence of a polynucleotide) and a reference nucleotide sequence is calculated by determining the percentage of nucleotides in the given nucleotide sequence that are identical to those in the reference nucleotide sequence after aligning the sequences and, if necessary, introducing gaps to achieve the maximum percentage sequence identity between the two sequences. Similarly, the “sequence identity” between a given amino acid sequence (e.g., the amino acid sequence of a polypeptide) and a reference amino acid sequence is calculated by determining the percentage of amino acids in the given amino acid sequence that are identical to those in the reference amino acid sequence after aligning the sequences and, if necessary, introducing gaps to achieve the maximum percentage sequence identity between the two sequences.

[0271] Pairwise and multiple sequence alignments performed for the purpose of assessing the percentage of sequence identity between two or more amino acid or nucleic acid sequences can be performed in various ways known to those skilled in the art, for example, using publicly available computer software such as ClustalOmega (Söding, J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)), and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772–780). When using such software, it is preferable to use default parameters, such as those for vacancy penalties and extension penalties.

[0272] Table 2: Exemplary Constructs

[0273]

[0274] Table 3: Exemplary Sequences

[0275]

[0276] In Table 3 above, sequences marked as antisense (chain) sequences also refer to the corresponding lead chain sequences (e.g., SEQ ID NO 45 to 88). Similarly, sequences marked as positive (chain) sequences also refer to the corresponding follower chain sequences (e.g., SEQ ID NO 128 to 171).

[0277] Numbering statements

[0278] The following numbered paragraphs describe specific aspects and embodiments of this disclosure:

[0279] 1. A regulatory polynucleotide for inhibiting ACTA1 expression, wherein the regulatory polynucleotide encodes a miRNA scaffold, wherein the miRNA scaffold is selected from the group consisting of miR30a scaffold, miR33 scaffold, and miR155 scaffold, wherein the miRNA scaffold comprises a repressive RNA (RNAi) molecule, wherein the RNAi molecule comprises a sense strand and an antisense strand forming a double-stranded region, and wherein the antisense strand shares a complementary region of at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length with the sense strand, and wherein the antisense strand sequence differs from the inverse complementary nucleotide sequence of the sense strand sequence by no more than 4 nucleotides.

[0280] 2. The regulatory polynucleotide as described in paragraph 1, wherein the miRNA scaffold is selected from the group consisting of miRNA30a scaffold, miRNA33 scaffold and miRNA155 scaffold.

[0281] 3. The regulatory polynucleotide as described in paragraph 1 or 2, wherein the miRNA scaffold is miRNA-33.

[0282] 4. The regulatory polynucleotide according to any one of paragraphs 1 to 3, wherein the antisense strand (or guide strand) comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 82 and SEQ ID NO: 84.

[0283] 5. The regulatory polynucleotide according to any one of the preceding paragraphs, wherein the regulatory polynucleotide inhibits the activity of the ACTA1 gene.

[0284] 6. The regulatory polynucleotide according to any one of the preceding paragraphs, wherein the regulatory polynucleotide inhibits or silences the ACTA1 gene.

[0285] 7. The regulatory polynucleotide according to any one of the preceding paragraphs, wherein the regulatory polynucleotide reduces the expression of the ACTA1 gene.

[0286] 8. A regulatory polynucleotide according to any one of paragraphs 5 to 7, wherein the regulatory polynucleotide is: single-stranded DNA, double-stranded DNA, DNA as a mixture of single-stranded and double-stranded regions, single-stranded RNA, double-stranded RNA, RNA as a mixture of single-stranded and double-stranded regions, a single-stranded molecule containing DNA and RNA, a double-stranded molecule containing DNA and RNA, or a molecule containing DNA and RNA having a mixture of single-stranded and double-stranded regions, particularly wherein the regulatory polynucleotide is single-stranded DNA.

[0287] 9. A regulatory polynucleotide according to any one of paragraphs 5 to 8, wherein the ACTA1 gene is a wild-type ACTA1 gene or a mutated ACTA1 gene.

[0288] 10. The regulatory polynucleotide according to any one of paragraphs 5 to 9, wherein the ACTA1 gene is a mutated ACTA1 gene having at least one pathogenic mutation.

[0289] 11. The regulatory polynucleotide according to any one of the preceding paragraphs, wherein the regulatory polynucleotide is an isolated regulatory polynucleotide.

[0290] 12. The regulatory polynucleotide according to any one of the preceding paragraphs, wherein the regulatory polynucleotide inhibits the activity of both the wild-type ACTA1 gene and the mutated ACTA1 gene having at least one pathogenic mutation.

[0291] 13. The regulatory polynucleotide according to any one of the preceding paragraphs, wherein the regulatory polynucleotide does not inhibit the activity of the codon-optimized ACTA1 gene.

[0292] 14. The regulatory polynucleotide according to any one of the preceding paragraphs, wherein the sense strand sequence comprises at least 20 consecutive nucleotides, and wherein the antisense strand sequence comprises at least 20 consecutive nucleotides.

[0293] 15. The regulatory polynucleotide according to any one of the preceding paragraphs, wherein the sense strand sequence comprises at least 21 consecutive nucleotides, and wherein the antisense strand sequence comprises at least 21 consecutive nucleotides.

[0294] 16. The regulatory polynucleotide according to any one of the preceding paragraphs, wherein the sense strand sequence comprises at least 22 consecutive nucleotides, and wherein the antisense strand sequence comprises at least 22 consecutive nucleotides.

[0295] 17. The regulatory polynucleotide according to any one of the preceding paragraphs, wherein the sense strand sequence comprises at least 23 consecutive nucleotides, and wherein the antisense strand sequence comprises at least 23 consecutive nucleotides.

[0296] 18. The regulatory polynucleotide according to any one of the preceding paragraphs, wherein the sense strand sequence comprises at least 24 consecutive nucleotides, and wherein the antisense strand sequence comprises at least 24 consecutive nucleotides.

[0297] 19. The regulatory polynucleotide according to any one of the preceding paragraphs, wherein the sense strand sequence comprises at least 25 consecutive nucleotides, and wherein the antisense strand sequence comprises at least 25 consecutive nucleotides.

[0298] 20. The regulatory polynucleotide according to any one of the preceding paragraphs, wherein the regulatory polynucleotide comprises an siRNA molecule.

[0299] 21. The regulatory polynucleotide according to any one of the preceding paragraphs, wherein the regulatory polynucleotide comprises an shRNA molecule.

[0300] 22. The regulatory polynucleotide according to any one of the preceding paragraphs, wherein the antisense strand (or guide strand) sequence differs from the nucleotide sequence selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 68, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 79 and SEQ ID NO: 82.

[0301] 23. The regulatory polynucleotide according to any one of the preceding paragraphs, wherein the sequence of the sense strand (or lagging strand) differs from the sequence of a nucleotide complementary to a nucleotide sequence selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 68, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 79 and SEQ ID NO: 82 by no more than 4 nucleotides.

[0302] 24. The regulatory polynucleotide according to any one of the preceding paragraphs, wherein the antisense strand (or guide strand) sequence differs from the nucleotide sequence selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 68, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 79 and SEQ ID NO: 82, wherein the sense strand (or lagging strand) sequence differs from the nucleotide sequence selected from the group consisting of: SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 139, SEQ ID NO: 151, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 162 and SEQ ID NO: 165.

[0303] 25. The regulatory polynucleotide according to any one of the preceding paragraphs, wherein the regulatory polynucleotide comprises the sequence of SEQ ID NO: 93 or a sequence having at least 85% sequence identity with SEQ ID NO: 93.

[0304] 26. The regulatory polynucleotide according to any one of the preceding paragraphs, wherein the regulatory polynucleotide is capable of forming a double-stranded region with the sequence of the ACTA1 gene.

[0305] 27. The regulatory polynucleotide according to paragraph 23, wherein the length of the double-stranded region relative to the sense strand is at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides, and wherein the nucleotide sequence of the double-stranded region differs from that of the sense strand by no more than 4 nucleotides.

[0306] 28. The regulatory polynucleotide according to any one of the preceding paragraphs, wherein the regulatory polynucleotide comprises a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the group consisting of: SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, and SEQ ID NO: 124, particularly, wherein the regulatory polynucleotide comprises a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the group consisting of: SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123.

[0307] 29. A regulatory polynucleotide for inhibiting ACTA1 expression, comprising a polynucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the group consisting of: SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 123, and SEQ ID NO: 124, particularly wherein the regulatory polynucleotide comprises a ...10, SEQ ID NO: 120, SEQ ID NO: 120, SEQ ID NO: 122 NO: 109, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 122 and SEQ ID NO: 123.

[0308] 30. An expression system comprising a regulatory polynucleotide according to any one of paragraphs 1 to 29, wherein the regulatory polynucleotide is operatively linked to a promoter, and optionally wherein the expression system further comprises a codon-optimized polynucleotide encoding a polypeptide of interest, particularly wherein the polypeptide of interest is ACTA1.

[0309] 31. A vector comprising a regulatory polynucleotide according to any one of paragraphs 1 to 29, wherein the regulatory polynucleotide is operatively linked to a promoter, and optionally wherein the vector further comprises a codon-optimized polynucleotide encoding a polypeptide of interest, particularly wherein the polypeptide of interest is ACTA1.

[0310] 32. The expression system according to paragraph 30 or the vector according to paragraph 31, comprising a start codon at the 5' position of a polynucleotide encoding a polypeptide of interest.

[0311] 33. The expression system according to paragraph 30 or 32, or the vector according to paragraph 31 or 32, further comprising a polyadenylated sequence at the 3' of a nucleotide sequence encoding a polypeptide of interest.

[0312] 34. The expression system according to any one of paragraphs 30 or 32 to 33, or the vector according to any one of paragraphs 30 to 32, further comprising an inverted terminal repeat (ITR) sequence at its 5' end and an ITR sequence at its 3' end.

[0313] 35. An expression system according to any one of paragraphs 30 or 32 to 34, or a vector according to any one of paragraphs 28 to 31, wherein the expression system or vector comprises: a first expression cassette containing a polynucleotide encoding a polypeptide of interest; and a second expression cassette containing a regulatory polynucleotide according to any one of paragraphs 1 to 25.

[0314] 36. An expression system according to any one of paragraphs 30 or 32 to 35, or a vector according to any one of paragraphs 31 to 35, wherein the first expression cassette contains an intron that contains a second expression cassette encoding a regulatory polynucleotide.

[0315] 37. An expression system according to any one of paragraphs 30 or 32 to 35, or a vector according to any one of paragraphs 31 to 36, wherein a regulatory polynucleotide is integrated into a polynucleotide encoding a polypeptide of interest, optionally wherein the regulatory polynucleotide is integrated into an intron contained in the polynucleotide encoding the polypeptide of interest.

[0316] 38. The expression system according to any one of paragraphs 30 or 32 to 37, or the vector according to any one of paragraphs 31 to 37, wherein the second expression cassette encoding the regulatory polynucleotide comprises a promoter operatively linked to the regulatory polynucleotide.

[0317] 39. An expression system according to any one of paragraphs 30 or 32 to 38, or a vector according to any one of paragraphs 31 to 38, wherein the promoter of the second expression cassette is a constitutive promoter, a cell type-specific promoter, or an inducible promoter.

[0318] 40. The expression system according to any one of paragraphs 30 or 32 to 39, or the vector according to any one of paragraphs 31 to 39, wherein the promoter of the second expression box is a pol III promoter or a U6 promoter.

[0319] 41. An expression system according to any one of paragraphs 30 or 32 to 40, or a vector according to any one of paragraphs 31 to 40, wherein the regulatory polynucleotide cannot be operatively linked to a promoter.

[0320] 42. The expression system according to any one of paragraphs 30 or 32 to 41, or the vector according to any one of paragraphs 31 to 41, wherein the first expression cassette contains a promoter operatively linked to a polynucleotide encoding a polypeptide of interest.

[0321] 43. An expression system according to any one of paragraphs 30 or 32 to 42, or a vector according to any one of paragraphs 31 to 42, wherein the promoter of the first expression cassette is a constitutive promoter, a cell type-specific promoter, or an inducible promoter.

[0322] 44. The expression system according to any one of paragraphs 30 or 32 to 43, or the carrier according to any one of paragraphs 31 to 43, wherein the first and / or second expression box contains an enhancing sub-element.

[0323] 45. An expression system according to any one of paragraphs 30 or 32 to 44, or a vector according to any one of paragraphs 31 to 44, wherein the first and / or second expression cassette comprises introns, filling polynucleotide sequences, polyadenylation signal sequences, or combinations thereof.

[0324] 46. ​​The expression system according to any one of paragraphs 30 or 32 to 45, or the vector according to any one of paragraphs 31 to 45, wherein the expression of the regulatory polynucleotide repressor gene in the cell or subject is about 20% to about 80% relative to the expression level prior to administration of the expression vector containing the regulatory polynucleotide according to any one of Examples 1 to 29 to the cell or subject.

[0325] 47. An expression system according to any one of paragraphs 30 or 32 to 46, or a vector according to any one of paragraphs 31 to 46, wherein the inhibition or suppression of gene expression is less than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% relative to the expression level prior to administration of the expression vector comprising the regulatory polynucleotide according to any one of paragraphs 1 to 29 to a cell or subject.

[0326] 48. The vector according to any one of paragraphs 31 to 47, wherein the vector is an adeno-associated virus (AAV) vector.

[0327] 49. An adeno-associated virus (AAV) vector comprising a vector genome, wherein the vector genome comprises, in a 5' to 3' sequence:

[0328] (i) 5' inverted terminal repeat (ITR) sequence;

[0329] (ii) Promoter;

[0330] (iii) Optionally, a 5' UTR sequence;

[0331] (iv) Polynucleotides encoding the protein of interest;

[0332] (v) 3' UTR sequence; and

[0333] (vi) 3' Inverted Terminal Repeat (ITR) sequence,

[0334] The vector genome contains regulatory polynucleotides according to any one of paragraphs 1 to 29.

[0335] 50. The AAV vector according to paragraph 49, wherein the regulatory polynucleotide is included in (iii) the 5' UTR sequence.

[0336] 51. The AAV vector according to paragraph 49, wherein the regulatory polynucleotide is included in (iv) the polynucleotide encoding the protein of interest.

[0337] 52. The AAV vector according to any one of paragraphs 49 to 51, wherein the 5' ITR sequence comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequence according to SEQ ID NO: 126.

[0338] 53. The AAV vector according to any one of paragraphs 49 to 52, wherein the 5' UTR sequence comprises:

[0339] (i) A sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 99;

[0340] (ii) A sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a nucleotide sequence selected from the group consisting of: SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 122, and SEQ ID NO: 123; and / or

[0341] (iii) A sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 100.

[0342] 54. The AAV vector according to any one of paragraphs 49 to 53, wherein the polynucleotide sequence encoding the protein of interest is at the 3' position of the polynucleotide sequence containing the regulatory polynucleotide.

[0343] 55. The AAV vector according to any one of paragraphs 49 to 54, wherein the polynucleotide sequence encoding the protein of interest is a cDNA sequence.

[0344] 56. The AAV vector according to any one of paragraphs 49 to 55, wherein the polypeptide of interest is a functional variant of a target gene of a regulatory polynucleotide.

[0345] 57. The AAV vector according to any one of paragraphs 49 to 56, wherein the polypeptide of interest is the ACTA1 gene or a codon-optimized variant thereof.

[0346] 58. The AAV vector according to any one of paragraphs 49 to 57, wherein the polynucleotide encoding the protein of interest comprises or consists of a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequence according to SEQ ID NO: 101.

[0347] 59. The AAV vector according to any one of paragraphs 49 to 58, wherein the 3' UTR sequence comprises a polyadenylated sequence.

[0348] 60. The AAV vector according to any one of paragraphs 49 to 59, wherein the 3' UTR sequence comprises:

[0349] (i) a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 104; and / or

[0350] (ii) A sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 105.

[0351] 61. The AAV vector according to any one of paragraphs 49 to 60, wherein the 3' ITR sequence comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequence according to SEQ ID NO: 127.

[0352] 62. The AAV vector according to any one of paragraphs 49 to 61, wherein the vector genome is suitable for packaging into adeno-associated virus particles.

[0353] 63. A cell comprising an expression system according to any one of paragraphs 30 or 32 to 47, or a vector according to any one of paragraphs 31 to 48, or an AAV vector according to any one of paragraphs 49 to 62.

[0354] 64. A pharmaceutical composition comprising an expression system according to any one of paragraphs 30 or 32 to 47, or a carrier according to any one of paragraphs 31 to 48, or an AAV carrier according to any one of paragraphs 49 to 62, and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0355] 65. An expression system according to any one of paragraphs 30 or 32 to 47, or a vector according to any one of paragraphs 31 to 48, or an AAV vector according to any one of paragraphs 49 to 62, or a pharmaceutical composition according to paragraph 64, used for the treatment or prevention of a disease or condition from which therapeutic or preventive benefits are derived by an increase in the expression level of ACTA1 in skeletal muscle cells.

[0356] 66. The use of an expression system according to any one of paragraphs 30 or 32 to 47, or a vector according to any one of paragraphs 31 to 48, or an AAV vector according to any one of paragraphs 49 to 62, or a pharmaceutical composition according to paragraph 64 in the manufacture of a medicament for the treatment or prevention of a disease or condition from which a therapeutic or preventive benefit would be derived by an increase in the expression level of ACTA1.

[0357] 67. A method for treating or preventing a disease or symptom from which therapeutic or preventive benefits are derived by an increase in the expression level of ACTA1, the method comprising administering to a subject an expression system according to any one of paragraphs 30 or 32 to 47, or a carrier according to any one of paragraphs 31 to 48, or an AAV carrier according to any one of paragraphs 49 to 62, or a pharmaceutical composition according to paragraph 64.

[0358] 68. The expression system, vector, AAV vector, or pharmaceutical composition used according to paragraph 65 or 66, or the treatment method according to paragraph 67, wherein the disease is a congenital myopathy, particularly wherein the disease is selected from the group consisting of: linear myopathy, intranuclear rod myopathy, actin filament aggregation myopathy, congenital myofiber heterogeneity, and myopathy with nucleoid regions.

[0359] 69. The present invention as described above and with reference to the accompanying drawings and examples.

[0360] ***

[0361] This disclosure includes combinations of the described aspects and preferred features, unless such combination is obviously not permitted or explicitly avoided.

[0362] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described.

[0363] Aspects and embodiments of this disclosure will now be illustrated by way of example with reference to the accompanying drawings. Other aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated by reference.

[0364] Throughout the specification (including the following claims), unless the context otherwise requires, the word “comprising” and variations such as “including” and “containing” should be understood to imply inclusion of the stated integer or step or group of integers or steps, but not to exclude any other integer or step or group of integers or steps.

[0365] As used herein, a "peptide" is a chain of two or more amino acid monomers linked by peptide bonds. Peptides typically have a length of approximately 2 to 50 amino acids. A "polypeptide" is a polymer chain of two or more peptides. Polypeptides typically have a length greater than approximately 50 amino acids. The peptides, polypeptides, and proteins mentioned herein also include glycopeptides / glycopeptides / glycoproteins, lipopeptides / lipopeptides / lipoproteins, nucleopeptides / nucleopeptides / nucleoproteins, etc.

[0366] As used herein, an amino acid sequence or peptide region “corresponding to” a specified reference amino acid sequence or peptide region has at least 60% (e.g., at least ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) sequence identity with that amino acid sequence / region / position. The amino acid sequence / region / position of the peptide / amino acid sequence “corresponding to” a specified reference amino acid sequence / region / position can be identified by sequence alignment of the test sequence with a reference sequence, for example using sequence alignment software such as ClustalOmega (Söding, J. 2005, Bioinformatics 21, 951-960).

[0367] Similarly, a nucleotide sequence or polynucleotide region “corresponding” to a specified reference nucleotide sequence or polynucleotide region has at least 60% (e.g., at least ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) sequence identity with the amino acid sequence of that nucleotide sequence / polynucleotide / region. The polynucleotide / polynucleotide / region / position “corresponding” to the specified reference nucleotide sequence / region / position can be identified by sequence alignment of the test sequence with the reference sequence, for example using sequence alignment software such as ClustalOmega (Söding, J.2005, Bioinformatics 21, 951-960).

[0368] As used herein, an amino acid sequence “derived from” a reference amino acid sequence (e.g., an amino acid sequence of a reference peptide / peptide / domain / region) comprises, or is composed of, an amino acid sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the reference amino acid sequence. Similarly, a nucleotide sequence “derived from” a reference nucleotide sequence (e.g., a nucleotide sequence of a polynucleotide) comprises, or is composed of, a nucleotide sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the reference nucleotide sequence.

[0369] It should be noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context explicitly specifies otherwise. A range herein may be expressed as “about” a particular value and / or to “about” another particular value. When expressing such a range, another embodiment includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation, the use of the antecedent “about” will be understood to form another embodiment of a particular value.

[0370] When this paper discloses the nucleic acid sequence, its reverse complementary sequence is also explicitly considered.

[0371] The methods described herein are preferably performed in vitro. The term "in vitro" is intended to cover procedures performed in cultures using cells, while the term "in vivo" is intended to cover procedures performed using / on intact multicellular organisms. Attached Figure Description

[0372] Embodiments and experiments illustrating the principles of this disclosure will now be discussed with reference to the accompanying drawings.

[0373] Figure 1: An example of rAAV encoding for an intronic artificial miRNA scaffold capable of simultaneously knocking down endogenous gene and transgene expression.

[0374] Figure 2: miRNA knockdown screening system in CHO targeted integration cell lines. The front plasmid encodes a gene expression cassette with a CMV-driven intronic artificial miRNA scaffold encoding a miRNA sequence targeting the gene of interest. It also encodes coACTA1-T2A-mCherry expression to monitor the splicing efficiency of the intronic miRNA scaffold. The back plasmid encodes two expression cassettes: (i) a targeted reporter expression regulated by the artificial miRNA; and (ii) a normalized reporter transgene expression cassette.

[0375] Figure 3: Normalized measurements of GFP / iRFP in a miRNA knockdown selection system in CHO-targeted integration cell lines encoding different artificial miRNA scaffolds and introns. CHO-TI cells were transfected and selected for stable integration of Front and Back plasmids, and GFP and iRFP expression were measured by flow cytometry. A design using a minI-miR33a scaffold encoding a non-target siRNA (Luci) was employed. Values ​​were normalized to introns without an artificial miRNA scaffold.

[0376] Figure 4: Raw flow cytometry data of the miRNA knockdown screening system in the CHO-targeted integration cell line, which stably encodes artificial miR33 scaffolds with different ACTA1-targeting siRNAs. CHO-TI cells were transfected and selected to stably integrate Front and Back plasmids, and GFP, mCherry, and iRFP expression was measured by flow cytometry. Performance differences in CMV-driven expression with the minI-intron artificial miR33 scaffold encoding ACTA1-targeting siRNA and in the GFP miRNA knockdown efficiency of the control were shown. A control design (minI) without an artificial miRNA scaffold in the intron and a minI-miR33a scaffold encoding non-target siRNAs (shGFP, shLuc) were also shown.

[0377] Figure 5: Raw flow cytometry data of the miRNA knockdown selection system in the CHO-targeted integration cell line encoding artificial miR33 scaffolds with different ACTA1-targeting siRNAs, comparing two different promoters. CHO-TI cells were transfected and selected to stably integrate Front and Back plasmids, and GFP, mCherry, and iRFP expression was measured by flow cytometry. Comparison of miRNA knockdown efficiency with CMV-driven and minCMV-driven artificial miR33 scaffolds encoding ACTA1-targeting siRNAs, and controls. As controls, designs without artificial miRNAs in the introns (minI) and designs with minI-miR33a scaffolds encoding non-target siRNAs (shGFP) and no fluorescence (negative control) were used.

[0378] Figure 6: Raw flow cytometry data of rAAV-delivered artificial miRNA-mediated GFP knockdown in the HEK293-GFP high-cell line. Different MOIs encoding CMV-driven mCherry-expressing rAAV were used to transduce the HEK293-GFP high-cell line with stable GFP expression, which has minimal introns but no miRNA scaffold. rAAV transduction was measured by gene knockdown mediated by mCherry expression and miRNAs with GFP expression. The HEK293 cell line without GFP was used as a comparison.

[0379] Figure 7: Raw flow cytometry data of rAAV-delivered artificial miRNA-mediated GFP knockdown in the HEK293-GFP high-growth cell line. Different MOIs encoding a CMV-driven minI-intron miR33a-GFP scaffold and mCherry-expressing rAAV were used to transduce the HEK293-GFP high-growth cell line with stable GFP expression. rAAV transduction was measured by gene knockdown mediated by mCherry expression and miRNAs with GFP expression. The HEK293 cell line without GFP was used as a comparison.

[0380] Figure 8: Performance of rAAV-delivered artificial miRNA-mediated ACTA-1 knockdown in HEK293 cell lines stably expressing ACTA1 or ACTC1. Transduction efficiency of rAAV encoding CMV-driven GFP expression, with a minI-intron miR33a scaffold carrying a different ACTA1-targeting siRNA, was measured by flow cytometry after 5 days. HEK293 cell lines without GFP were used as a comparison.

[0381] Figure 9: Performance of rAAV-delivered artificial miRNA-mediated ACTA-1 knockdown in HEK293 cell lines stably expressing ACTA1 or ACTC1. ACTA1 or ACTC1 knockdown was measured by qPCR after 5 days. Relative quantification (RQ) of different rAAVs encoding CMV-driven GFP expression is shown, including controls (no AAV, no miRNA scaffold) with a minI-intron miR33a scaffold carrying a different ACTA1-targeting siRNA. GUSB expression levels were used for RQ calculation.

[0382] Figure 10: Performance of rAAV-delivered artificial miRNA-mediated ACTA-1 knockdown in primary human skeletal muscle myotubes (pHSMM). Transduction efficiency of rAAV encoding CMV-driven GFP expression, 3 days after delivery, was measured by flow cytometry. This rAAV possesses a minI-intron miR33a scaffold with a different ACTA1-targeting siRNA. No AAV treatment was used as a control.

[0383] Figure 11: Performance of rAAV-delivered artificial miRNA-mediated ACTA-1 knockdown in primary human skeletal muscle myotubes (pHSMM). Measurements of ACTA1 or ACTC1 knockdown by qPCR after 3 days at MOI=6250 are shown. Normalized relative quantification (RQ) of different rAAVs encoding CMV-driven GFP expression to AAV-free treatment is shown, including controls (AAV-free, miRNA-free scaffold) with a minI-intron miR33a scaffold carrying different ACTA1-targeting siRNAs. GUSB expression levels were used for RQ calculation.

[0384] Figure 12: Aggregate phenotype (“linear rods”) of HEK293 cell lines stably overexpressing ACTA1 H40Y but not the D286G mutation. A) Confocal micrographs of HEK293 cells overexpressing the ACTA1 H40Y mutation and different immunofluorescence staining: DAPI (nucleus), phalloidin (actin filaments), and overlap. B) Confocal micrographs of HEK293 cells overexpressing the ACTA1 D286G mutation and different immunofluorescence staining: DAPI (nucleus), phalloidin (actin filaments), and overlap of all three stainings.

[0385] Figure 13: The clustering phenotype (“linear rods”) of the HEK293 cell line stably overexpressing the ACTA1 H40Y mutation can be reversed by expression of an ACTA1-targeting miR33 scaffold delivered by rAAV. A. Confocal micrographs and different immunofluorescence stainings of HEK293 cells overexpressing the ACTA1 H40Y mutation: DAPI (nuclei), phalloidin (actin filaments). Overlap of two stainings before and after rAAV treatment encoding miR33-ACTA1 is shown. For the control without rAAV treatment (negctr) and for rAAV treatment of two miR33 scaffolds expressing two different ACTA1-targeting siRNAs (166 and 169), the total linear rod intensity per nucleus is shown. B. Description of the methods used for measuring total rod intensity and normalizing to cell counts (nuclei).

[0386] Figure 14: Performance of rAAV-delivered artificial miRNA-mediated gene knockdown in primary human skeletal muscle myotubes (pHSMMs) as measured by DRUG-seq and its effects on different genes. Different MOIs encoding CMV-driven minI-intron miR33-ACTA1 scaffolds and rAAV expressed in coACTA1-T2A-GFP or GFP were used for pHSMM transduction, illustrating different expression levels of specific genes: 14A) Wild-type ACTA1 gene expression normalized to the non-transduction control. 14B) Wild-type ACTA1 gene expression. As controls, designs using scaffolds without artificial miRNA in the introns (210_minI+GFP and 146_MinI+coA+GFP) and designs using minI-miR33 scaffolds encoding non-target siRNA (shLuc+GFP) and cells without fluorescence (negcon (no AAV)) were used.

[0387] Figure 15: Performance of rAAV-delivered artificial miRNA-mediated gene knockdown in primary human skeletal muscle myotubes (pHSMMs) as measured by DRUG-seq and its effects on different genes. Different MOIs encoding CMV-driven minI-intron miR33-ACTA1 scaffolds and rAAV expressed in coACTA1-T2A-GFP or GFP are used for pHSMM transduction, illustrating different gene expression levels: 15A) GFP gene expression. 15B) coACTA1 gene expression. As controls, designs using scaffolds without artificial miRNA in introns (210_minI+GFP and 146_MinI+coA+GFP) and designs using minI-miR33 scaffolds encoding non-target siRNA (shLuc+GFP) and cells without fluorescence (negcon (no AAV)) are also used.

[0388] Figure 16: Performance of rAAV-delivered artificial miRNA-mediated gene knockdown in primary human skeletal muscle myotubes (pHSMMs) as measured by DRUG-seq and its effects on different genes. Different MOIs encoding CMV-driven minI-intron miR33-ACTA1 scaffolds and rAAV expressed in coACTA1-T2A-GFP or GFP were used for pHSMM transduction, illustrating different expression levels of specific genes: 16A) ACTC1 gene expression; 16B) BAX gene expression. As controls, designs using scaffolds without artificial miRNA in introns (210_minI+GFP and 146_MinI+coA+GFP) and designs using minI-miR33 scaffolds encoding non-target siRNA (shLuc+GFP) and cells without fluorescence (negcon (no AAV)) were used.

[0389] Figure 17: Performance of rAAV-delivered artificial miRNA-mediated gene knockdown in primary human skeletal muscle myotubes (pHSMMs) as measured by DRUG-seq and its effects on different genes. Different MOIs encoding CMV-driven minI-intron miR33-ACTA1 scaffolds and rAAV expressed in coACTA1-T2A-GFP or GFP were used for pHSMM transduction, illustrating different expression levels of specific genes: 17A) CASP3 gene expression. 17B) TP53 gene expression. As controls, designs using scaffolds without artificial miRNA in introns (210_minI+GFP and 146_MinI+coA+GFP) and designs using minI-miR33 scaffolds encoding non-target siRNA (shLuc+GFP) and cells without fluorescence (negcon (no AAV)) were used.

[0390] Figure 18: Performance of rAAV-delivered artificial miRNA-mediated gene knockdown in primary human skeletal muscle myotubes (pHSMMs) as measured by DRUG-seq and its effect on the expression of different genes. A volcano plot showing the different gene expression of rAAVs encoded by the CMV-driven minI-intron miR33-ACTA1 scaffold and coACTA1-T2A-GFP (18B) or GFP (18A) expressed in pHSMMs, compared to untreated cells. C. Differential gene expression analysis of different rAAVs encoded by the CMV-driven minI-intron miR33-ACTA1 scaffold and coACTA1-T2A-GFP (18B) or GFP (18A) expressed in pHSMMs. As a control, designs using introns without artificial miRNA scaffolds (introns) and designs using minI-miR33 scaffolds encoding non-target siRNAs (miLuc) were used.

[0391] Example

[0392] Example 1: General Method

[0393] 1.1 Recombinant DNA Technology

[0394] DNA was manipulated using standard methods, as described in Sambrook, J. et al., *Molecular Cloning: A Laboratory Manual*; Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturer's instructions. The desired gene fragment was synthesized from synthetic oligonucleotides and PCR products using automated gene synthesis at Geneart AG (Regensburg, Germany). The gene fragment, flanked by a single restriction endonuclease cleavage site, was cloned into a standard cloning / sequencing plasmid. The plasmid DNA was purified from transformed bacteria, and its concentration was determined by UV spectroscopy. The DNA sequence of the cloned gene fragment was confirmed by DNA sequencing. The gene fragment was designed with appropriate restriction sites to allow transfer into the corresponding reporter vector. The miRNA scaffold was cloned into different introns of a CMV-driven gene expression cassette that encodes either a single transgene or two transgenes linked via a T2A cleavage site (Figure 1). mmu-pri-miR-33 was engineered by substituting the miRNA and miRNA* sequences, as described by Xie et al., Molecular Therapy (2020). Other scaffolds were derived from the miRBase database and engineered by substituting the miRNA and miRNA* sequences. The genetic elements used are described in Table 2.

[0395] 1.2 Production of rAAV via triple transfection

[0396] As recommended by the manufacturer, recombinant AAV was produced by triple transfection of Expi293F (Thermo-Fisher Scientific) cells using PEI MAX (Polysciences) as the transfection reagent. In short, Expi293F cells were grown to 3 x 10⁻⁶ cells in 500 mL shake flasks and 125 mL culture volumes. 6 A density of cells / mL was used. For transfection, equimolar amounts of the first plasmid (pAAV2 rep / cap) carrying the AAV2 rep-cap gene, the second plasmid (pHelper) carrying the adenovirus helper genes E4, E2a, and VA, and the third plasmid (pTransgene) carrying the reporter gene of interest flanked by AAV2ITR were combined and diluted in 2.5 mL of Expi293 expression medium. Per 10... 6A total of 0.6 µg of DNA was used per cell. PEI MAX was mixed separately with 2.5 mL of Expi293 expression medium and then combined with the previously prepared DNA mixture at a ratio of 1:2.5 (DNA:PEI). After incubation, the DNA-PEI complex was added dropwise to the cells. Finally, the transfected cell cultures were incubated at 37 °C, 125 rpm, and 8% CO2 for 72 hours.

[0397] To harvest recombinant AAV, cells were centrifuged at 300 g for 5 min and chemically lysed using a Triton CG-110-based lysis buffer containing 50 U / mLDENARASE (c-LEcta). For complete lysis, cells were incubated at 37 °C and 180 rpm with agitation for 1.5 h. To remove cell debris after lysis, the suspension was centrifuged at 4,000 x g for 60 min. Finally, the supernatant was passed through a 0.8 / 0.2 µm double filter and collected. The sterile and clear cell lysate was stored at -80 °C until iodixanol gradient purification was performed.

[0398] 1.3 Purification of rAAV by gradient ultracentrifugation with iodixanol

[0399] To purify and isolate the desired intact rAAV capsid, density gradient ultracentrifugation with iodixanol was performed, using layers of 60%, 40%, 25%, and 15% iodixanol. The filtered viral lysate was thawed and transferred to ultracentrifuge tubes. Subsequently, a gradient of different iodixanol concentrations was added to the top, the tubes were equilibrated, sealed, and centrifuged at 230,000 xg for 2 hours at 4°C in a Beckman 50.2Ti rotor. After centrifugation, the tubes were firmly secured and punctured at the top with a 21G needle to allow air inflow. A second 21G needle, connected to a syringe, was carefully inserted below the 60–40% interface, and 1.0 to 2.5 mL was collected from the 40% phase. The resulting vector stock solution was stored at -80°C or further processed to exchange iodixanol with PBS and increase viral concentration. This was achieved through ultrafiltration using an Amicon Ultra-15 centrifuge filter. Store the concentrated sample at -80°C.

[0400] 1.4 Analysis of rAAV transduction and reporter gene expression

[0401] Primary human skeletal muscle myoblasts (HSMMs) were used as a cell model to measure promoter activity in human skeletal muscle. For HSMM transduction, cells were cultured at 3 to 4.5 x 10⁻⁶ cells per hectare. 4 cells / cm 2 Cells are typically seeded in 96-well format in growth medium (Lonza SkBM-2 or PromoCell skeletal muscle growth medium with appropriate supplements provided by the supplier) and checked for correct morphology. The next day, cells are transduced using constructs diluted in MEM (L-glutamine, Pen / Strep, 10 µM etoposide, phenol red-free). Four to five hours after transduction, cells are supplemented with fetal bovine serum (FBS). f =2.5% v / v). On day 3, etoposide was removed by changing the growth medium. On day 5, the medium was changed to differentiation medium (Lonza DMEM / BioWhittaker F-12 [1:1], L-glutamine, heat-inactivated horse serum plus Pen / Strep). Between days 7 and 9, the expression of fluorescent proteins (e.g., eGFP, mCherry) in the cells was usually analyzed on Incucyte (Sartorius) or by flow cytometry. For flow cytometry, the cells were washed twice with PBS and separated with 0.05% trypsin / EDTA. Separation was stopped by adding growth medium or PBS / 10% FBS. The cells were then centrifuged, washed, and co-stained for viability (7AAD) for 15 minutes.

[0402] Transduction and knockdown efficiencies were tested using adherent Hek293 cells and four different Hek293-based ACTA1 / ACTC1 cell lines. For transduction, cells were cultured at 3 to 3.5 x 10⁻⁶ cells per cell line. 4 cells / cm 2 Cells were typically seeded (in 96-well format) in growth medium (DMEM high glucose (PAN biotech)) containing fetal bovine serum (c = 10% v / v) and L-glutamine (4 mM). Two hours later, cells were transduced using constructs diluted in DMEM medium. DMEM medium (0.25 v / v) was added the next day. On day 5, cell knockdown efficiency was analyzed by qRT-PCR, and fluorescent protein expression (e.g., eGFP) was analyzed by flow cytometry. For flow cytometry, cells were isolated with 0.05% Accutase. Isolation was stopped by adding PBS / 2.5% FBS. Cells were then centrifuged, washed, and measured using FACS CantoII.

[0403] Example 2: CHO TI Generation and FACS Analysis

[0404] Based on the targeted integration technology in CHO cells, a novel artificial miRNA gene knockdown screening method was developed. This targeted integration technology simultaneously utilizes the stable integration of two plasmids (Front and Back) mediated by RMCE (Figure 2).

[0405] TI host cells were propagated in disposable 125 ml aerated shake flasks in proprietary DMEM / F12-based medium at a constant stirring rate of 150 rpm under standard humidified conditions (95% rH, 37°C, and 5% CO2). Cells were seeded every 3 to 4 days in a chemically defined medium containing effective concentrations of blast fungicide S (selection marker 1) and hygromycin B (selection marker 2). Culture density and viability were measured using a Cedex HiRes cell counter (F. Hoffmann-La Roche Ltd, Basel, Switzerland).

[0406] For stable transfection, TI host cells were seeded in fresh medium at a density of 4 x 10⁵ cells / ml two days prior to transfection. Transfection was performed using a MaxCyte STX electroporator (MaxCyte Inc., Gaithersburg) with an OC-400 electroporation kit, following the manufacturer's protocol. A total of 30 µg of plasmid was used to transfect 3 x 10⁷ cells per transfection, with a plasmid ratio of 2.5:2.5:1 (front-, back-, Cre- recombinase plasmids). After transfection, cells were seeded in 30 ml of medium without selectants.

[0407] On day 5 post-inoculation, cells were centrifuged (1200 rpm, 5 min) and transferred at 6 x 10⁵ cells / mL to 80 mL of chemically defined culture medium containing effective concentrations of puromycin (selector 1) and 1-(2'-deoxy-2'-fluoro-1-β-D-arabinofuranyl-5-iodo)uracil (FIAU; selector 2) for selecting recombinant cells. From this day onward, cells were incubated at 37°C, 150 rpm, 5% CO₂, and 85% humidity without division or passage. Cell density and viability of the culture were monitored periodically. When culture viability began to increase again, the concentrations of selectors 1 and 2 were reduced to half of the previously used amounts. More specifically, to facilitate cell recovery, the selection pressure was reduced if viability > 40% and viable cell density (VCD) > 0.5 x 10⁶ cells / mL. Therefore, 4 x 10⁵ cells / ml were centrifuged and resuspended in 40 ml of Selective Medium II (chemically defined medium, ½ selector marker 1 and ½ selector marker 2). Cells were incubated under the same conditions as before and continued to divide and be monitored periodically. Pool formation was considered complete once viability > 90%.

[0408] Cre-mediated cassette exchange success was examined by measuring the expression of both fluorescent reporter GFP (used to assess miRNA knockdown efficiency) and mCherry (used to measure protein expression of the CMV-intron-miRNA-mCherry cassette) using flow cytometry. Flow cytometry was performed using a BD FACSCelesta™ flow cytometer (BD, Heidelberg, Germany). Ten thousand events were measured for each sample. Live cells were gated in forward scatter (FSC) versus side scatter (SSC) plots. Live cell gates were defined by untransfected TI host cells and applied to all samples using FlowJo 10.8.1 EN software (Ashland, OR 97520, USA). GFP fluorescence was quantified in the FITC channel (excited at 488 nm, detected at 530 nm). mCherry was measured in the PE-CF594 channel (excited at 561 nm, detected at 610 nm). Parental CHO cells, those used to generate TI host cells, were used as a negative control for GFP and mCherry expression.

[0409] We cloned different introns (see Table 2) containing different miRNA scaffolds (see Table 2) into the Front vector. These miRNA scaffolds encoded siRNAs targeting either GFP (target) or luciferase (off-target control). After stable CHO-TI pooling was generated, GFP, mCherry, and iRFP were measured to quantify gene knockdown efficiency. Different GFP knockdown efficiencies were measured based on the intron and the miRNA scaffold (Figure 3). For example, the miR450b scaffold did not result in GFP gene knockdown, while miR30a, miR33, and miR155 (eSIBR) resulted in highly efficient GFP knockdown independent of the intron environment.

[0410] We cloned different siRNAs targeting ACTA-1 into the minI-miR33 scaffold of the Front vector to identify candidates with high ACTA-1 gene knockdown efficiency. Therefore, the ACTA-1 sequence was codon-optimized in a manner as different as possible from the wild-type ACTA-1 sequence (named coACTA-1) to avoid transcripts of miR-ACTA1 candidates that self-target themselves (Figure 2). Stable CHO-TI pools were generated for all candidates and for GFP, mCherry, and iRPF measured by flow cytometry (Figure 4). Interestingly, the ACTA-1 gene knockdown efficiency varied for different miR33-ACTA1 variants, allowing for candidate selection based on this measured gene knockdown performance. Importantly, all CHO-TI cells carrying both the miRNA scaffold and the corresponding targeting transgenes exhibited narrow and clear GFP intensity peaks due to stable integration, thereby improving the differentiation of gene knockdown performance mediated by different miR-ACTA1 variants. Replacing the CMV promoter driving miRNA scaffold expression with a minimal promoter (minCMV) significantly reduced the dynamic range of gene knockdown, such as the ACTA1 gene knockdown efficiency among different miR-ACTA1 candidates (Figure 5).

[0411] Example 3: Generation of adherent HEK293-ACTA1 stable cell lines

[0412] To evaluate the activity of different ACTA1 shRNA candidates in in vitro cell models, adherent HEK293 cell lines were genetically engineered to express wild-type ACTA1, the ACTA1_H40Y mutant, the ACTA1_D286G mutant, or as an off-target ACTC1. For modification, a transposon vector system consisting of two plasmids was used: a transposon vector containing two inverted / direct repeats (IR / DR) flanking the gene of interest, and a vector encoding the Sleeping Beauty transposase SB100x. The full-length wild-type cDNA encoding human ACTA1, ACTA1_H40Y, ACTA1_D286G, or ACTC1, along with its 3' UTR region, was subcloned into the transposon vector, which carried puromycin resistance. Expression in the final plasmid was controlled by the CMV promoter. Following the manufacturer's protocol, the transposon and transposase vector were co-transfected into adherent HEK293 cells using the 293Fectin reagent (Gibco, #12347019). The adherent HEK293 cells were maintained in DMEM medium (PAN, #P04-03609) supplemented with 10% FCS (Gibco, #10500) and 2 mM L-glutamine, with 2.5 µg / mL puromycin (Gibco, #A11138-03) added after stable transfection. Adherent HEK293 cells stably expressing human ACTA1, ACTA1 mutants, or ACTC1 were isolated by single-cell sorting using a BD FACSAria III cell sorter (BD Biosciences) and cultured to establish stable cell clones. Stable cell clones were screened for ACTA1 or ACTC1 expression and amplification. Cells were prepared using 4% PFA and methanol, and ACTA1 cell clones were stained with mIgG1 mouse anti-actin clone 3B3 (NovusBio) and anti-mouse IgG-PE (Jackson Immunoresearch) within 4 weeks, or with anti-actin antibody [ERP8484] (Abcam) and anti-rabbit IgG-PE (Jackson Immunoresearch) after staining. The expression levels and stability were then confirmed by flow cytometry analysis.

[0413] To evaluate the gene knockdown efficiency of the rAAV-delivered miRNA scaffold, HEK293 cells stably expressing GFP were used as a cell model. rAAVs encoding either the CMV-driven minI-intron miR33-GFP scaffold (targeted) or the absence of the miR33 scaffold (control, no miR) also encoded mCherry as a transgene. After rAAV transduction using different MOIs, MOI-dependent transduction efficiency as measured by mCherry expression levels and MOI-dependent GFP gene knockdown efficiency as measured by GFP expression levels were observed for miR33-GFP rAAV (Fig. 7), but not for control rAAV (Fig. 6). Importantly, GFP gene knockdown efficiency increased with increasing transduction efficiency. At a maximum MOI of 250 kJ, the GFP intensity level was comparable to that of the HEK293 cell line without GFP expression (Fig. 7).

[0414] To evaluate the performance of different miR33-ACTA1 candidates in a cellular setting, two different cell models were used. In the first experiment, HEK293 cells stably overexpressing either ACTA1 or ACTC1 were used. rAAVs encoding either a CMV-driven minI-intron miR33-ACTA1 scaffold (targeted) or no miR33 scaffold (control, no miR) encoding different ACTA1 siRNAs also encoded co-ACTA1-T2A-GFP as a transgene. Five days after rAAV transduction, rAAVs efficiently transduced cells by flow cytometry-based GFP intensity (Figure 8), and no significant differences were observed between miR33-ACTA1 candidates or between the two stable cell lines. Five days later, the knockdown efficiency of ACTA1 or ACTC1 genes was measured by qPCR (Figure 9), showing strong knockdown of the miR33-ACTA1 candidates and no effect on control rAAVs. ACTC1 expression was only slightly affected by some miR33-ACTA1 candidates.

[0415] Furthermore, primary human skeletal muscle myotube (pHSMM) cells were transduced in a dose-dependent manner with the same rAAV (using different MOIs). Here, well-defined dose-dependent transduction efficiencies were measured by flow cytometry (Fig. 10), demonstrating comparable transduction efficiencies across the rAAV variants. ACTA1 gene knockdown was highly efficient with the miR33-ACTA1 candidate (Fig. 11), and no ACTA1 gene knockdown was observed in the control rAAV (without miR). ACTC1 expression was moderately reduced with the miR-ACTA1 candidate.

[0416] A stable HEK293 cell line overexpressing ACTA1 protein has been established, containing two clinically observed mutations in patients that have been further characterized (xxx references!), namely H40Y and D286G. Interestingly, the HEK293-ACTA1-H40Y cell line exhibits a large number of aggregate-like phenotypes, resembling rods in the cytoplasm and nucleus (described in the literature), while the HEK293-ACTA1-D286G cell line does not exhibit this phenotype (Figure 12). Treatment of HEK293-ACTA1-H40Y with either a minI-miR33-ACTA1 scaffold containing ACTA1 or rAAV without a miRNA scaffold demonstrated rod removal after three days with the minI-miR33-ACTA1 variant (Figure 13). This indicates that the rods observed in the HEK293-ACTA1-H40Y cell line are triggered by ACTA1-H40Y overexpression, and that this phenotype can be reversed by gene knockdown of artificial ACTA1 miRNA delivered by rAAV.

[0417] Example 4: Rescuing HEK cells expressing mutant ACTA1

[0418] Cell culture and confocal immunofluorescence analysis. HEK cells (H40Y with a D268G mutation stably expressing ACTA1) were grown on calibrated glass coverslips at 80% confluence. Cells were then fixed with 4% freshly prepared paraformaldehyde, washed, and incubated with Alexa Fluor™ 488 phalloidin (1:100; Thermo) at room temperature for 1 h followed by DAPI incubation for 5 min. Alexa Fluor™ 488 phalloidin possesses conformational epitopes located in grooves within actin filaments composed of at least three actin monomers. We found that phalloidin strongly labeled both cytoplasmic and nuclear rods in H40Y cells, but not in D268G mutant cells, indicating that the H40Y structural binding site is specific for filamentous actin. Imaging was performed using a hybrid detector (HyD) on a Leica Falcon SP8 confocal microscope. Imaging conditions were as follows: 63× / 1.40 NA oil immersion microscope, with sequential acquisition of each channel using a 405 nm laser diode and white light laser excitation (Ex), at 405 nm / emission (Em) 413–455 nm and Ex 488 / Em 495–537 nm, respectively. Images were recorded sequentially. Images were gated to exclude filamentous stress fibers from image analysis. Total Alexa Fluor™ 488 phalloidin fluorescence in H40Y filamentous rods was then measured using MetaMorph imaging software (Molecular Devices) and normalized to the cell number determined by DAPI staining.

[0419] Interestingly, the HEK293-ACTA1-H40Y cell line exhibited a large number of cluster-like phenotypes, resembling rods in the cytoplasm and nucleus, while the HEK293-ACTA1-D286G cell line did not exhibit this phenotype (Fig. 12). Treatment of HEK293-ACTA1-H40Y with either a minI-miR33-ACTA1 scaffold containing ACTA1 or an rAAV without a miRNA scaffold demonstrated that the rods were removed three days after treatment with the minI-miR33-ACTA1 variant (Fig. 13). This suggests that the rods observed in the HEK293-ACTA1-H40Y cell line are triggered by ACTA1-H40Y overexpression, and that this phenotype can be reversed by gene knockdown of artificial ACTA1 miRNA delivered by rAAV.

[0420] Example 5: RNA quantification and DRUG-SEQ

[0421] Human skeletal muscle myoblasts (HSMMs) (Lonza CC-2580, batch number 20TL293904, donor 5) were seeded at a density of 20,000 cells per well in two 96-well plates in growth medium (Lonza SkbM-2 CC-3244 plus provided supplements). 24 hours post-seeding, the medium was replaced with differentiation medium (500 ml DMEM: F12 Thermo Fisher 11330-032, 10 ml horse serum, Thermo Fisher 26050070, 5 ml Pen / Strep Thermo Fisher 15140122), and simultaneously transduced in triplicate with AAV at MOIs of 500, 1000, 6000, and 20000. The culture medium was changed every 2 to 3 days, and HSMM cultures were treated 8 days post-transduction using the DRUG-Seq (Alithea Genomics 10841) RNA sequencing library preparation protocol, following the manufacturer's instructions. Briefly, cells were washed with PBS and incubated on ice with lysis buffer for 15 minutes to promote cytoplasmic lysis. After centrifugation (300x g, 5 min, +4 C), the lysates were used directly for reverse transcription (RT) using oligomeric dT primers with pore-specific barcodes and unique molecular identifiers (UMIs), allowing all samples to be pooled in a single tube after RT incubation. Standard RNA library preparation steps were then performed, including second-strand synthesis, labeling, library amplification, and multiple rounds of solid-phase reversible immobilization (SPRI) bead purification. The final libraries were sequenced on a NovaSeq 6000 SP1 flow cell (Illumina) with a target of at least 5 million reads per sample.

[0422] The obtained FASTQ file was aligned with the human genome (GRCh38.p14, Ensembl May 2023), and GFP and codon-optimized ACTA1 sequences were amplified. The spliced ​​transcript was aligned to the reference (STAR, Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, ​​Chaisson M, Gingeras TR. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. January 1, 2013; 29(1):15-21. doi: 10.1093 / bioinformatics / bts635. Epub October 25, 2012. PMID: 23104886; PMCID: PMC3530905.) with the aligner in “solo” mode, generating a UMI-corrected counting matrix for each 96-well plate. Of the 192 samples, 181 met the quality threshold of at least 6,000 reads per sample, and the average number of genes per sample was 15,000. The count matrix was analyzed using the Seurat package in Rstudio (version 2023.06.0+421) (Butler A, Hoffman P, Smibert P, Papalexi E, Satija R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol. 2018 Jun; 36(5):411-420. doi: 10.1038 / nbt.4096. Epub 2018 Apr. PMID: 29608179; PMCID: PMC6700744.), and transcript levels were compared using counts per million (CPM) normalization.For differential gene expression analysis, the likelihood ratio test implemented by Seurat was used to compare transduced samples with untransduced controls (McDavid A, Finak G, Chattopadyay PK, Dominguez M, Lamoreaux L, Ma SS, Roederer M, Gottardo R. Data exploration, quality control and testing in single-cell qPCR-based gene expression experiments. Bioinformatics. 2013 Feb 15; 29(4):461-7. doi: 10.1093 / bioinformatics / bts714.Epub 2012 Dec 24.PMID: 23267174; PMCID:PMC3570210.). Genes with an adjusted p-value below 0.05 and a logarithmic change (LFC) less than -0.32 or greater than 0.32 (indicating at least 25% change in expression) are considered differentially expressed and are considered potential off-targets.

[0423] DRUG-seq was performed on pHSMM cells to validate qPCR data and to obtain differential gene expression data from the rAAV-delivered minI-miR33-ACTA1 variant and controls (Figs. 14–18). For the experiments, different MOIs encoding minI-miR33-ACTA1 with coACTA1-T2A-GFP or GFP only were transduced in pHSMM cells. DRUG-seq was performed after 8 days. For the minI-miR33-ACTA1 candidate, wild-type ACTA1 expression was knocked down to varying degrees in a dose-dependent manner, while control rAAV had no strong effect on wild-type ACTA1 expression (Fig. 14). For all rAAV constructs encoding the corresponding transgenes, GFP and coACTA1 expression increased with increasing MOI (Fig. 15). Other genes, such as ACTC1, CASP3, and T53, were unaffected by rAAV transduction (Figs. 16, 17A). However, under high MOI conditions in different constructs, the apoptosis marker BAX was often expressed at higher levels, which may represent the negative impact of increased AAV titers on cell viability (Fig. 17B). A comparison of two rAAV designs with the same minI-miR33-ACTA1 variant (shSA013) but two different transgenes (coACTA-T2A-GFP or GFP only) showed the effect of coACTA1 on the expression of several genes (Fig. 18). The volcano plot of minI-miR33-ACTA1 candidates was able to select candidates with low off-target characteristics (examples are shown in Fig. 18).

Claims

1. A regulatory polynucleotide for inhibiting ACTA1 expression, wherein the regulatory polynucleotide encodes a miRNA-33 scaffold, wherein the miRNA-33 scaffold comprises a repressive RNA (RNAi) molecule, wherein the RNAi molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 82 and SEQ ID NO: 84, and wherein the antisense strand shares a length with the sense strand of at least 20, at least 21, at least 22, at least 23, at least 24 or at least 25. The complementary region of 1 nucleotide, wherein the antisense strand sequence differs from the reverse complementary nucleotide sequence of the sense strand sequence by no more than 4 nucleotides.

2. The regulatory polynucleotide according to claim 1, wherein the regulatory polynucleotide is: single-stranded DNA, double-stranded DNA, DNA as a mixture of single-stranded and double-stranded regions, single-stranded RNA, double-stranded RNA, RNA as a mixture of single-stranded and double-stranded regions, a single-stranded molecule containing DNA and RNA, a double-stranded molecule containing DNA and RNA, or a molecule containing DNA and RNA having a mixture of single-stranded and double-stranded regions, particularly wherein the regulatory polynucleotide is single-stranded DNA.

3. The regulatory polynucleotide according to claim 1 or 2, wherein the regulatory polynucleotide inhibits the activity of the ACTA1 gene.

4. The regulatory polynucleotide according to claim 3, wherein the ACTA1 gene is a wild-type ACTA1 gene or a mutated ACTA1 gene.

5. The regulatory polynucleotide according to claim 3 or 4, wherein the ACTA1 gene is a mutated ACTA1 gene having at least one pathogenic mutation.

6. The regulatory polynucleotide according to any one of claims 1 to 5, wherein the regulatory polynucleotide inhibits the activity of both the wild-type ACTA1 gene and the mutated ACTA1 gene having at least one pathogenic mutation.

7. The regulatory polynucleotide according to any one of claims 1 to 6, wherein the regulatory polynucleotide does not inhibit the activity of the codon-optimized ACTA1 gene.

8. The regulatory polynucleotide according to any one of claims 1 to 7, wherein the regulatory polynucleotide comprises the sequence of SEQ ID NO: 93 or a sequence having at least 85% sequence identity with SEQ ID NO:

93.

9. The regulatory polynucleotide according to any one of claims 1 to 8, wherein the regulatory polynucleotide comprises a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the group consisting of: SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, and SEQ ID NO:

124.

10. An expression system comprising a regulatory polynucleotide according to any one of claims 1 to 9, wherein the regulatory polynucleotide is operatively linked to a promoter, and optionally wherein the expression system further comprises a codon-optimized polynucleotide encoding ACTA1.

11. An adeno-associated virus (AAV) vector comprising a regulatory polynucleotide according to any one of claims 1 to 9, wherein the regulatory polynucleotide is operatively linked to a promoter, and optionally wherein the AAV vector further comprises a codon-optimized polynucleotide encoding ACTA1.

12. The expression system of claim 10 or the AAV vector of claim 11, wherein the expression system or vector comprises: a first expression cassette comprising a polynucleotide encoding a codon-optimized polynucleotide encoding ACTA1; and a second expression cassette comprising a regulatory polynucleotide of any one of claims 1 to 9.

13. The expression system of claim 10 or 12 or the AAV vector of claim 11, wherein the first expression cassette comprises an intron, the intron comprising a second expression cassette encoding the regulatory polynucleotide.

14. An adeno-associated virus (AAV) vector comprising a vector genome, wherein the vector genome comprises, in a 5' to 3' sequence: (i) 5' inverted terminal repeat (ITR) sequence; (ii) Promoter; (iii) Optionally, a 5' UTR sequence; (iv) Codon-optimized polynucleotides encoding ACTA1; (v) 3' UTR sequence; and (vi) 3' Inverted Terminal Repeat (ITR) sequence, The vector genome contains the regulatory polynucleotide according to any one of claims 1 to 9.

15. The AAV vector of claim 14, wherein the vector genome is suitable for packaging into adeno-associated virus particles.

16. A pharmaceutical composition comprising: an expression system according to any one of claims 10, 12 or 13, or an AAV carrier according to any one of claims 11, 14 or 15; and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

17. The expression system according to any one of claims 10, 12 or 13, or the AAV vector according to any one of claims 11, 14 or 15, or the pharmaceutical composition according to claim 16, used for the treatment or prevention of a disease or condition from which therapeutic or preventive benefits are derived by an increase in the expression level of ACTA1 in skeletal muscle cells.

18. Use in the manufacture of a medicament of an expression system according to any one of claims 10, 12 or 13, or an AAV vector according to any one of claims 11, 14 or 15, or a pharmaceutical composition according to claim 16, for the treatment or prevention of a disease or condition from which therapeutic or preventive benefits will be derived by an increase in the expression level of ACTA1.

19. A method for treating or preventing a disease or symptom from which therapeutic or preventive benefits are derived by an increase in the expression level of ACTA1, the method comprising administering to a subject an expression system according to any one of claims 10, 12, or 13, or an AAV carrier according to any one of claims 11, 14, or 15, or a pharmaceutical composition according to claim 16.

20. The expression system, vector, AAV vector, or pharmaceutical composition used according to claim 18, or the method of treatment according to claim 19, wherein the disease is a congenital myopathy, and particularly wherein the disease is selected from the group consisting of: linear myopathy, intranuclear rod myopathy, actin filament aggregation myopathy, congenital myofiber heterogeneity, and myopathy with nucleoid regions.

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