Synthetic regulatory cassettes for muscle-specific gene expression

Abstract

We describe a library of muscle-specific artificial expression cassettes (MSECs) for muscle-specific gene expression. Different transcription levels can be obtained in different types of muscle cells by selecting different members of the MSEC library for different research or therapeutic purposes. MSECs contained in the MSEC library can be used to develop treatments for muscle-related diseases.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 173,295, filed April 9, 2021, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] The present disclosure relates to the fields of molecular biology, medicine, and genetics, and more particularly to a regulatory cassette that can induce expression of a protein or RNA product at various levels in skeletal muscle cells and cardiac muscle cells, which expresses the protein or RNA product at low levels in cell types other than skeletal muscle cells and cardiac muscle cells. The muscle-specific artificial expression cassettes (MSECs) of the present invention can be used to develop treatments for virtually any type of muscle-related disease in vertebrates, and can also be used to develop treatments for many other diseases in humans and vertebrates that can utilize skeletal muscle tissue for the production of beneficial secretory products, such as hormones, clotting factors, antibodies, and other beneficial proteins or metabolites. Summary of the Invention [Means for solving the problem]

[0003] The muscle-specific expression cassette (MSEC) of the present invention is an artificial expression construct comprising DNA that can be utilized for selective expression of an operably linked coding sequence in skeletal and cardiac muscle cells, and the artificial expression construct does not substantially express the coding sequence in cells other than muscle cells. MSECs can be linked to cDNAs encoding proteins or RNAs, which can be any protein or RNA, and the MSECs are inserted (transduced) into cardiac or skeletal muscle cells to synthesize the protein or RNA products encoded by the cDNA. By varying the design of MSECs, the expression of protein or RNA products from individual MSECs can be varied over a concentration range of more than 1000-fold. Thus, by using MSECs contained within the library disclosed herein, selected proteins or RNAs can be selectively expressed over a very wide concentration range in cardiac and / or skeletal muscle cells. The performance of this MSEC library can also be utilized to develop gene therapy for neuromuscular diseases, cardiac diseases, cancer cachexia, and aging diseases, as well as other diseases in which factors produced and secreted by muscle cells have a beneficial effect. The beneficial factors produced and secreted by muscle cells range from secreted polypeptide hormones and cytokines to extracellular matrix proteins, enzymes, clotting factors, antibodies, and metabolites. Furthermore, MSECs can be used for immunization against virtually any type of antigen, for a variety of veterinary and livestock agricultural purposes, and for the production of cell-cultured meat. [Brief description of the drawings]

[0004] Some of the drawings may be more easily understood in color, and applicants hereby contemplate that color versions of these drawings are part of the original application and reserve the right to submit color versions of the drawings in later proceedings.

[0005] [Figure 1]Representative embodiments of muscle-specific expression cassettes (MSECs) are shown. MSECs can be used to induce expression of cDNA products. A combination of MSECs and cDNA products can be inserted into a delivery vector and delivered to a patient, which allows expression of the cDNA products in skeletal and cardiac muscles, but not in tissues other than muscle. In some embodiments, the delivery vector is a viral vector, such as AAV. In some embodiments, differential expression can be induced in cardiac muscle and skeletal muscle by selecting MSECs with differential expression activity in skeletal and cardiac muscles. The MSEC library of the present invention also allows the expression level of the transgene to be specifically adjusted, and expression can be enhanced or suppressed in the target tissue. Due to the small size of MSECs, they can be used in a variety of vectors, including delivery vectors that are limited in the size of the base pairs that can be inserted, such as viral vectors such as AAV.

[0006] [Diagram 2] Various types of representative muscle therapeutic gene constructs are shown, which are constructed with skeletal muscle control elements, cardiac muscle control elements, transcriptionally permissive promoters, and combinations thereof. A specific combination of skeletal muscle control elements and cardiac muscle control elements is ligated to a transcriptionally permissive promoter. This basic form of MSEC can then be ligated to the cDNA of the transcript. Specific MSECs can be selected to induce differential expression in various target tissues. For example, in some embodiments, MSECs containing a combination of skeletal muscle control elements and cardiac muscle control elements can induce differential expression in skeletal muscle and cardiac muscle. In certain embodiments, MSECs can increase, decrease, or be similar to the expression of the transgene in skeletal muscle compared to cardiac muscle. By designing various MSECs with different control elements as components, the amount of cDNA expression in the target muscle tissue can be adjusted.

[0007] [Diagram 3] In some embodiments, MSECs are constructed based on various promoter and enhancer regions of muscle-specific genes, such as the M-type creatine kinase (CKM) gene. Native CKM is highly expressed in both skeletal and cardiac muscles and is activated only during muscle differentiation. The present disclosure is based on the findings of the 5'-enhancer, intron 1 enhancer and proximal promoter regions of CKM and its binding to E-boxes and MyoD, and the finding that these control elements play an essential role in the muscle-specific expression of CKM as well as in the expression of many other muscle genes. By utilizing quantitative proteomics, previously unknown transcription factors containing novel DNA binding sites were identified in the enhancer and promoter of CKM that constitute key control elements (Six4 / 5, MAZ and KLF3) for muscle gene expression. Depending on the size that can be packaged in the vector, the utilization of regulatory elements via CKM allows expression of cDNA-encoded gene products in differentiated skeletal muscle cells and differentiated cardiomyocytes, but with limited expression in non-muscle cells. However, when used with vectors such as adeno-associated virus (AAV), which have limited packageable size, the native CKM regulatory region and cDNAs larger than about 4 kb in size must be packaged efficiently into the vector by miniaturizing the CKM regulatory region. Miniaturization of the regulatory region can be achieved by removing non-conserved genomic sequence regions based on sequence alignments between various mammalian and / or vertebrate species (see Figures 8-10). Such miniaturization of the regulatory region can have the additional effect of increasing or decreasing the transcriptional activity of the miniaturized regulatory element (see Figures 5 and 7).

[0008] [Figure 4]A standard cell culture assay for evaluating MSEC expression activity in differentiated skeletal muscle cells is shown. A mouse skeletal muscle cell line (MM14) derived from a permanent cell clone is seeded in culture wells in the presence of fibroblast growth factor (FGF), which stimulates proliferation and inhibits differentiation. The MSEC test plasmid and the internal standard plasmid are added to transfect the cultured cells. The MSEC test plasmid contains MSEC ligated to luciferase cDNA. The internal standard plasmid contains native CKM and is referred to herein as MSEC-1263a ligated to Renilla cDNA. The internal standard plasmid serves as an assay standardization protocol to correct each assay for the number and percentage of transfected cells in each culture plate and the degree of differentiation, by calculating the ratio of the expression amount of luciferase cDNA to the expression amount of Renilla cDNA. After 4 hours of culture, the culture supernatant is aspirated, the cells are rinsed, and replaced with low serum-containing differentiation medium without FGF. After 48 hours of incubation, the cultures were harvested and frozen to analyze the relative expression of luciferase and Renilla activity in the extracts. As shown in the graph, some MSECs (t, u, v, w, x) expressed higher amounts of luciferase product than the MSEC-1263a control (c), while the remaining MSECs (y and z) expressed less luciferase product. In a typical experiment, four culture dishes were tested for each MSEC, and the average relative expression of luciferase to Renilla cDNA expression was calculated and compared with previous experimental data. The average expression of the same MSEC was considered as the same MSEC used in repeated assays.

[0009] [Diagram 5]The transcriptional activity of 59 MSECs in mouse skeletal muscle cultures was plotted and corrected by setting the value of MSEC-1263a, which is the region from -1256 to +7 of native mouse CKM, as 1. The data shown in the graph show MSECs containing recombinant mouse CKM enhancer and recombinant mouse CKM promoter regions, MSECs containing recombinant mouse CKM regions with multimerized small intronic enhancers (SIEs), MSECs containing recombinant human CKM regulatory regions, and MSECs containing a fully synthetic enhancer ligated to a miniaturized CKM or human ACTA1 promoter. Data from a ubiquitously active (non-muscle-specific) cytomegalovirus enhancer / promoter cassette (CMV) are shown as a control. These MSECs showed transcription rates that showed a stepwise increase in activity over a range of about 20-fold higher than that of native MCK, the second most active skeletal muscle gene. Many of the human MSECs had activities similar to their analog mouse CKM. The synthetic regulatory cassette contained a variety of optimized control elements derived from diverse striated muscle genes (CK7=MSEC-571, CK8=MSEC-438a, MHCK7=MSEC-770, native mouse=native CKM=MSEC-1263a).

[0010] [Figure 6] The mRNA TPMs (transcript levels per million) of various genes in human adult skeletal muscle are shown (Gtex portal at the Broad Institute). Human diseases associated with mutations in these genes are shown in bold. Referring to this table without considering unidentified parameters such as mRNA and protein half-life, the diseases shown in the table have 5000-fold higher mRNA expression than normal, suggesting that optimal gene replacement therapy for the diseases shown in the table will require MSECs with broad transcriptional activity to optimize expression of the therapeutic gene product (see Figure 7).

[0011] [Figure 7]1 shows a logarithmic scale plot showing that the transcriptional activity of the MSECs of the present invention spans a 1000-fold range. This range of activity allows for easy selection of MSECs that may be suitable for different gene therapy targets depending on vector dose and transduction efficiency.

[0012] [Figure 8] Sequence conservation of the 5'-enhancer region of the CKM gene in six mammalian sequences aligned with the mouse sequence (-1256 to -1051) and the sequence compaction strategy are shown. Highly conserved sequences are boxed and named. X indicates non-conserved regions that were assessed for whether complete or partial deletion significantly reduces transcriptional activity. MSECs with enhanced, reduced, and unchanged transcriptional activity were included in the total MSEC sequence library.

[0013] [Figure 9]The sequence conservation of the proximal promoter region of the CKM gene in five mammalian sequences aligned with the mouse sequence (-358 to +7) and the miniaturization method of MSEC are shown. The highly conserved sequences identified are boxed and named, while other highly conserved unknown sequences are not boxed. Deletion tests of the unboxed conserved regions were performed, and transcriptional activity was reduced. X indicates a non-conserved region that was evaluated for whether complete or partial deletion significantly reduces transcriptional activity. Replacement of parts of these deleted sequences with alternative regulatory elements was also tested. Furthermore, a long conserved region was found by aligning with the sequence of the exon 1 region of the CKM gene. Deletion tests of this conserved region showed that the MSEC containing the most miniaturized CKM enhancer + promoter showed higher transcriptional activity when combined with the +1 to +50 region of a particular MSEC than when combined with the +1 to +7 region, but showed better transcriptional activity when combined with the +1 to +12 region than when combined with either exon 1 region. MSECs with enhanced, decreased and unchanged transcriptional activity were included in the total MSEC sequence library (CK7=MSEC-571, CK8=MSEC-538a, CK9=MSEC-336c).

[0014] [Figure 10]Sequence alignment of mouse and human cardiac troponin T (TNNT2) genomic sequences in the constructed 5' end portions of mouse and human cardiac troponin T genes. Identified regulatory elements are boxed, and unknown conserved regions are underlined. A truncated design was tested in which a portion of the enhancer-promoter region of human TNNT2 was deleted, creating a truncated 130 bp cardiac-specific enhancer located between the two triangles and a truncated 170 bp proximal promoter. MSEC-320 and MSEC-455c, which contain this truncated promoter and one or two truncated enhancers, show very high cardiac-specific transcriptional activity. Furthermore, this 130 bp enhancer shows even higher transcriptional activity in skeletal and cardiac muscle when ligated to CKM-based MSECs (e.g., MSEC-571a) (e.g., MSEC-725a and MSEC-875). See also FIG. 16.

[0015] [Figure 11] The activity of MSECs can be increased stepwise by the addition of one or more MCK small intron enhancers (SIEs). In the experiment shown, multimerized SIEs were ligated to the 5' end of MSEC-571 and expression activity in skeletal muscle cultures was tested using a dual luciferase reporter assay (Figure 4). Conclusion: Multimerized SIEs allow stepwise increase in MSEC activity, achieving expression activity levels 15-fold higher than wild-type MSECs and 7-fold higher than a universally active regulatory cassette containing a strong CMV enhancer / promoter. The base pair lengths of the individual MSECs are shown below the x-axis.

[0016] [Figure 12] A representative pGL3 plasmid map of MSEC-285 is shown, which contains a complete synthetic enhancer with various regulatory elements as shown, ligated to a truncated 99 bp promoter from human skeletal α-actin (ACTA1) followed by a luciferase reporter cDNA.

[0017] [Figure 13] A representative pGL3 plasmid map of MSEC-429a is shown. This MSEC contains various regulatory elements and a synthetic enhancer whose linear order and spacing differ from the MSEC-285 enhancer (Figure 12). The synthetic enhancer is ligated to a truncated 129 bp promoter from human skeletal alpha actin (ACTA1) followed by a luciferase reporter cDNA.

[0018] [Figure 14] A representative pGL3 plasmid map of MSEC-393b is shown. This MSEC contains various regulatory elements and a synthetic enhancer whose linear order and spacing differ from the MSEC-285 and MSEC-429a enhancers (Figures 12 and 13). The synthetic enhancer has been ligated to a truncated 134 bp basal promoter + exon 1 region (-84 to +50) from mouse CKM followed by a luciferase reporter cDNA.

[0019] [Figure 15]DNA linkers of various lengths and sequences were included in various MSECs as insertion sequences between enhancer and promoter regions, between enhancers, and between multimerized miRNA target sites. Examples are shown to show that short and long linker sequences included in various MSECs have various lengths and sequences. The insertion of linkers into MSEC sequences has three basic aspects: (1.) the linker introduces an artificial sequence between the ligated components; (2.) the linker introduces various spatial distances between the ligated components; and (3.) both of these two factors can affect the transcriptional activity of each MSEC, regardless of the intrinsic activity of the enhancer and promoter sequences of the MSEC. Thus, the linker sequence can be considered as a functional component in each MSEC, related to a specific transcription level in muscle cells. These linkers do not affect the selectivity of expression in muscle cells and cell types other than muscle cells.

[0020] [Figure 16]Figure 1 shows the transcriptional activity of five MSECs in different tissues of adult mice after systemic administration of the five MSECs to adult mice with serotype 6 adeno-associated virus (AAV6) at a dose of 2 x 1012 vector genomes per kg body weight. Each MSEC was ligated to human placental alkaline phosphatase (hPAP) cDNA, and each cDNA contained the same polyA sequence ligated to its 3' end. Additionally, AAV LTR sequences were ligated to the 5' and 3' ends of each construct. Adult mice were injected retro-orbitally (N=6) and euthanized 5 weeks later. Various tissues were harvested, frozen, and then analyzed for hPAP activity levels per mg of protein. The transcriptional activity of each MSEC was compared by correcting all hPAP activity levels to the hPAP activity levels in the tibialis anterior muscle of mice injected with MSEC-571a. Overall the data supported the following eight conclusions: (1.) Each MSEC is expressed at a higher level in muscle tissue than in nonmuscle tissue. (2.) Each MSEC is expressed at a different level in various muscles according to anatomical classification. (3.) MSEC-438a (CK8e) is more active in all skeletal muscles than other MSECs. (4.) MSEC-455a is active in cardiac muscle but is essentially inactive in skeletal muscle. (5.) MSEC-725a, which was generated by adding a truncated 144-bp hTNNT2 enhancer in the positive orientation to MSEC-571a, has increased transcriptional activity not only in all skeletal muscles but also in cardiac muscle (this 144-bp sequence is the 144-bp sequence most 5'-terminal of the sequence designated MSEC-725a in the MSEC library). (6.) A second hTNNT2 enhancer was ligated in the positive orientation to the 5' end of the first enhancer, but a deletion of the most 5' base pairs of the first enhancer did not result in any additional increase in activity. (7.) Both MSECs containing an additional hTNNT2 enhancer showed the highest activity in cardiac muscle.(8.) In tissues other than muscle tissue, the expression levels of all MSECs are very low, thus avoiding the deleterious effects of expressing the gene products in unintended cell types and avoiding stimulation of immune system dendritic cell responses to the gene products.

[0021] [Figure 17] We demonstrate the use of microRNA (miR) target sites to restrict expression of a transcript in cells where the transcript is harmful or where it may have a deleterious effect (e.g., cardiac muscle). Although many of the MSECs exhibit variable transcriptional activity in different muscle types and much higher activity in muscle than in non-muscle cells (e.g., FIG. 16), some of the therapeutic gene products may be deleterious if expressed above a certain expression level in one or more tissues when delivered systemically using a vector. This problem can be ameliorated by inserting binding site sequences complementary to specific miRs known to be present in one or more cell types where the expressed gene product is undesirable. In this study, three tandem miR208a target sites (see miR208aTS×3 on page 1 of Figure 27) were ligated to the 3' end of luciferase cDNA, and the targeted cDNA and a control cDNA were ligated to the MSEC-438a construct (shown as CK8e in the figure), and each construct was transiently transfected into differentiation-competent MM14 skeletal muscle cultures or neonatal rat cardiomyocyte cultures, and culture extracts were harvested after 48 hours. The presence of the miR208a target sites resulted in 95% suppression of luciferase product expression in cardiomyocytes, whereas the presence of the miR208a target sites did not suppress luciferase product expression in differentiated skeletal muscle cells (both cultures were transfected with MSEC-1263a as a positive control to indicate increased transcriptional activity of MSEC-438a).

[0022] [Figure 18]The use of microRNA (miR) target sites to restrict the expression of transcripts in skeletal muscle is shown. Three tandem miR206 target sites (see miR206TS-3Gx3 on page 1 of FIG. 27) were ligated to the 3' end of luciferase cDNA, and the targeted cDNA and control cDNA were ligated to MSEC-438a construct (shown as CK8e in the figure) or MSEC-725a construct (shown as h-cTnt(E)-CK7 in the figure), respectively, and each construct was electroporated into intact mouse flexor digitorum brevis myofibers isolated and cultured directly from adult mice to mimic the differentiated in vivo myofiber environment, or transiently transfected into neonatal rat cardiomyocyte cultures (shown as right bar in each group), and culture extracts were harvested after 48 hours. Both MSEC constructs contained the miR206 target site, which suppressed the expression of luciferase product by 90% in skeletal muscle fibers, but did not suppress the expression of luciferase product in differentiated cardiomyocytes, even though the miR206 target site was present.

[0023] [Figure 19]By extracting data from the Jaspar 2020 transcription factor database (http: / / jaspar.genereg.net / search?q=TATA+box&collection=CORE&tax_group=vertebrates&tax_id=all&type=all&class=all&family=all&version=all), we present a plot of the frequency of each base position in the TATA box components identified from all vertebrate genes. We compare this plot with the mouse CKM TATA box sequence listed at the bottom of the graph. This graph reveals three concepts: (1.) The most frequent TATA box core sequence is TATAAAA. (2.) TATA box sequences are highly variable. (3.) The core sequence of the TATA box of mouse CKM differs from the vertebrate "consensus" TATA sequence by the deletion of two 3'-terminal A residues, whereas the TATA box of the most highly transcribed human skeletal muscle gene, ACTA1 (Figure 6), is a perfect match for the vertebrate consensus TATA sequence (see the sequence of MSEC-239). These comparisons are consistent with the hypothesis that the TATA box sequence is a critical determinant of the transcriptional activity of MSEC.

[0024] [Figure 20] TATA box variants of native adenovirus major late promoters show a 20-fold difference in activity. Similar to the sequence variations and transcriptional activity differences seen in adenovirus TATA boxes, in some embodiments, the TATA of a selected MSEC can be modified to decrease or increase activity with or without affecting the response of the MSEC to the binding of other transcription factors (TFs) to its regulatory elements.

[0025] [Figure 21]N-box regulatory elements are present in the promoters and enhancers of muscle genes that are highly transcribed in muscle fiber nuclei near the neuromuscular junction (NMJ). This figure shows that native N-box elements are present in a wide variety of locations, and that these N-boxes function within the proximal promoter region close to the transcription start site (TSS) or within enhancer regions 5' or 3' to the transcription start site. This suggests that N-boxes can be inserted into various locations within MSECs and still exert their function. These N-boxes can be miniaturized N-box-containing enhancers isolated from muscle genes such as the acetylcholinesterase (ACHE) gene or the acetylcholine receptor subunit (CHRM1, CHRND, CHRNG) genes, or they can be multimerized N-box clusters such as the 43 bp sequence of three clustered N-boxes shown in this figure. In vitro assays have demonstrated that such N-box-containing MSECs exert their functions in differentiated skeletal muscle cells in response to the addition of specific ligands, such as agrin and neuregulin, to the culture medium (PMID:11498047; and references in the figure). Based on this, we determine the optimal location of the N-box within MSECs by quantitatively comparing the expression levels of N-box-containing MSECs in response to exogenous neurogenic factors that stimulate N-box-binding transcription factors, such as GABP, that can bind to the inserted N-box (PMID:11498047). Therefore, by combining N-box functions with various MSECs ligated to therapeutic cDNA, it is expected that therapeutic gene products can be delivered to the NMJ region, with only a small amount of therapeutic gene product being delivered to regions other than the NMJ region of skeletal muscle fibers.

[0026] [Figure 22]We present an MSEC design scheme for miniaturization of the α-MyHC enhancer to engineer the compact MSEC-770. The length of the human α-MyHC enhancer can be shortened from 190 bp to 90 bp by stepwise deletion of unannotated and non-conserved regions. Combining the smallest miniaturized α-MyHC enhancer with MSEC-438a results in a new muscle-specific cassette with a shortened length of 526 bp (compared to MSEC-770) and increased activity. Reducing the length of MSECs facilitates the combination packaging of MSECs with larger cDNAs into viral vectors or other gene delivery systems that have limited space for inserting nucleic acid cargo.

[0027] [Figure 23] Using the enhancer and promoter regions of human cardiac troponin T (TNNT2) (see Figure 10), various MSECs were engineered and showed graded expression upregulation in differentiated cardiomyocytes in vitro, including MSEC-455a, which contains two truncated TNNT2 enhancers and shows high levels of cardiomyocyte-specific activity in vivo (see data for MSEC-455a in Figure 16).

[0028] [Figure 24] Striated muscle-specific synthetic enhancers are designed by combining, spaced in 5'→3' order, various muscle gene control elements that confer various degrees of qualitative and quantitative transcriptional activity to various types of striated muscle. Small or large enhancers can be easily designed because any number of control elements can be linked at various intervals. Synergistic interactions of transcription factors can also be promoted by placing control elements known to bind synergistic transcription factors adjacent to each other. The resulting synthetic enhancers are then linked to promoters and cDNAs of various muscle or universal genes to obtain the desired expression levels of the gene products.

[0029] [Diagram 25] All MSEC designs were tested for expression in non-muscle cell cultures to determine muscle-specific expression. In Figure 25, a typical MSEC showed very low activity in 3T3 mouse fibroblast cultures and human foreskin fibroblast cultures, while two designs combining CMV enhancer and promoter showed high activity in both cell types. Further examples of universal enhancer and universal promoter combinations compared to MSEC in various cell types other than muscle cells are published in PMID:17235310.

[0030] [Figure 26] In vivo comparison data between two MSECs with different lengths of CKM exon 1 components and regulatory elements are shown (see comparison of library sequences for MSEC-411 and MSEC-438a). Four weeks after injection, mice were euthanized and tissues were harvested, frozen, extracted, and analyzed as described in FIG. 26. The average hPAP activity (×106 enzyme units) per mg of extracted protein was measured for each tissue, and the relative expression in each tissue was calculated by determining the ratio of activity in MSEC-411 to activity in MSEC-438a. These data show that MSEC-411 has approximately twice the activity of MSEC-438a in all skeletal muscles, but that MSEC-411 and MSEC-438a have similar activity in cardiac muscle, and that these MSECs have very low expression in non-muscle tissues (liver).

[0031] [Figure 27]A library of representative MSEC sequences supporting the present disclosure is shown. All sequences are listed from left to right in the 5' to 3' orientation. Linker sequences are underlined in most sequences. In some representative MSEC sequences, intronic and / or 3' untranslated region sequences are also shown. The sequences of the miRNA target sites are listed first in the list, and these miRNA target site sequences are inserted 3' into the cDNA that is ligated to the MSEC. Sequences are listed from shortest to longest. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Skeletal and cardiac muscles are affected by hundreds of genetic diseases and can undergo various types of physical damage, leading to a gradual decline in their function due to disuse and aging. In addition, skeletal muscles undergo exhaustive catabolic degradation associated with cancer. Since all skeletal muscle fibers are innervated and the function of the myocyte synaptic regions where muscle contraction is stimulated by neuronal axons depends on nerve interactions, muscle cells also exhibit various neuromuscular junction (NMJ) disorders. Furthermore, muscle plays a key role in the normal function of neurons innervating skeletal muscle fibers, since their maintenance depends in part on muscle-mediated signals. The muscle-specific expression cassettes (MSECs) described herein can play an important role in the development of therapies to combat all of these medical challenges and similar challenges in the veterinary field. Importantly, two previously developed MSECs have shown promising results in some of the ongoing clinical trials for Duchenne muscular dystrophy.

[0033] The DNA sequences described herein were determined by an iterative design and testing strategy based on data from 40 years of basic research studies focused exclusively on understanding skeletal and cardiac muscle gene regulation. These basic research studies identified the regulatory region of the mouse M-type creatine kinase (MCK / CKM) gene by investigating its regulatory region. However, many of the basic research papers summarized below do not address the application of the fundamental findings of the CKM gene regulatory region to the development of gene therapy or other commercial applications. A transgenic mouse paper published in 1993 (Cox, GA et al.; PMID:8355788) finally demonstrated the possibility of treating Duchenne muscular dystrophy (DMD) by gene replacement therapy, but the 6,300 bp regulatory sequence and 14,000 bp dystrophin cDNA sequence used in this study were not available for practical therapeutic strategies at the time. Furthermore, although this study and many subsequent basic research studies published a vast amount of information about the regulatory elements of the mouse CKM gene, for many years no researchers applied this research data for therapeutic purposes, and it was not until 2000 that it was announced that a miniaturized MSEC ("CK6") could be used for therapeutic purposes (Hauser, MA et al., 2000; PMID:10899824).

[0034] An example of the discussion of the use of MSECs disclosed herein is given in relation to striated muscle, including a comparison between skeletal and cardiac striated muscle. Striated muscle is the largest tissue mass in any vertebrate. Humans and many vertebrates have over 600 anatomically distinct skeletal muscle types, with a wide variety of cardiac muscle types. Striated muscle is composed of fibers and myocytes that have specific gene expression for each type. Human skeletal muscle is a specialized anatomical variety that contains tens to thousands of multinucleated myofibers, with varying proportions of each of the three fiber types and mixed fibers. Each fiber ranges in length from 1 mm in the inner ear to 60 cm in the thigh, with approximately 50 myonuclei per mm. Thus, the longest muscle fibers in tall individuals contain as many as 30,000 myonuclei per fiber, and muscles throughout the body contain well over 10 million myonuclei. With a few exceptions, all myonuclei in each skeletal muscle fiber express the same genes, with expression levels assumed to be appropriate for each gene. Based on quantitative analysis of mRNA abundance in all skeletal muscles, the steady-state relative gene expression rates vary by more than 5000-fold between the most and least active genes. In contrast, human ventricular, atrial, and several other types of cardiac muscle contain only one to three myonuclei per cardiomyocyte and display a wide range of transcription rates for the various types of genes.

[0035] Multinucleation may be beneficial for skeletal gene therapy, but may be less beneficial for cardiac gene therapy. Ideally, in striated muscle gene therapy, the same number of vectors would be transduced into each myocyte nucleus, so that all nuclei produce the same amount of therapeutic gene product. This goal has not yet been achieved with viral therapy, and current protocols, which are being tested using the highest FDA-approved human vector doses translated into mouse body weight doses, have shown that not all skeletal and cardiac myonuclei are transduced. Furthermore, the number of vectors contained in transduced nuclei appears to vary. This has a significant impact on the efficacy of gene therapy. The reason for this is that if too few myonuclei are transduced, some regions of long skeletal muscle fibers may have suboptimal expression of the therapeutic gene product, and some myonuclei of cardiomyocytes may not be transduced at all, resulting in no direct therapeutic benefit. Most of the variability in transduction efficiency in skeletal muscle myonuclei can be overcome by utilizing muscle-specific expression cassettes (MSECs) with very high transcriptional activity, where the gene product is highly expressed by transduced myonuclei and can diffuse laterally to non-transduced fiber regions, thus compensating for these non-transduced regions. On the other hand, human cardiomyocytes usually have only 1-3 nuclei, so at least one nucleus in each cardiomyocyte must be transduced to obtain the benefits of transduction. However, a very large number of vectors may be randomly transduced into myonuclei, which may result in excessive amounts of gene product being produced in myonuclei transduced with a large number of vectors, resulting in local toxicity. In the treatment of some muscle diseases, a partial benefit may be obtained even for adjacent fibers and cardiomyocytes that do not express sufficient therapeutic gene product, but optimal treatment requires that a non-toxic but sufficient amount of therapeutic gene product is obtained.

[0036] The above-mentioned differences in fiber types are crucial in designing MSECs for muscle gene therapy. The reason is that different fiber types have different transcription factors (TFs), which may result in excessive expression of the therapeutic gene product in some fibers and insufficient expression in other fibers, even when the same MSECs are used. It would therefore be advantageous to have MSECs that show similar, high, medium or low transcript expression in all fiber types, or MSECs that show fast, medium, slow or "off" transcription rates depending on the fiber type (e.g., type I has fast transcription rates, type IIa has medium transcription rates, and type IIx has slow or "off" transcription). Similar changes in transcription factors result in different gene expression levels in atrial, ventricular and conduction system cardiomyocytes of the heart. Additionally, by varying the type, sequence, spacing, and flanking regulatory elements, we were able to generate MSECs that respond differently to a wide variety of physiological signals associated with different muscle types and varying workloads, resulting in the wide range of MSEC transcriptional activity seen in the MSEC libraries of the present disclosure. As described below, each therapeutic gene product has unique properties, and pathological differences among various diseases will result in different optimal MSECs for each therapeutic goal.

[0037] Alternatively, the expression of gene products is controlled by various microRNAs (miRNAs) expressed in proliferative skeletal myoblasts, differentiated fibers, and differentiated cardiomyocytes. In general, miRNAs affect the amount of a particular mRNA translated into protein by increasing the rate of degradation of the mRNA to which the miRNA binds. The selectivity of mRNA degradation is determined by the qualitative and concentration differences of miRNAs produced by each type of cell, and also by the binding affinity of miRNAs to various miR target site (miRTS) sequences that are present in the mRNAs of various muscle genes, which are slightly different. In light of this, by inserting a variable number of miRNA target sites (miRTs) with low to high binding affinity into the non-coding region of the cDNA of the desired gene product, the expression of the gene product in a specific muscle type can be regulated with even greater precision than transcriptional regulation alone. Thus, even finer tuning of the expression of gene products among various muscle types can be achieved by selecting the appropriate MSECs and miRTSs for the treatment of each disease.

[0038] As described herein, skeletal muscles contain a mixture of fast and slow twitch fibers whose biochemical and physiological properties are heavily influenced by the embryonic cell line from which they are derived, as well as by their subsequent innervation and workload. The relative number and spatial distribution of fast and slow twitch fibers vary between and within individual anatomical muscle subregions. Slow twitch fibers (type I) contract over a relatively long period of time, utilize aerobic metabolism, and express type I myosin heavy chain. Fast twitch fibers, on the other hand, contract and relax rapidly, rely on highly active glycolytic metabolism, and express two or three myosin heavy chain genes. Type IIa and IIx fibers are found in humans, dogs, sheep, and cattle, while types IIa, IIx, and IIb fibers are found in pigs, cats, and rodents, and similar fiber types are found in poultry. Each type of fiber also contains a unique subset of contractile proteins and other proteins that are specialized for slow-twitch or fast-twitch function.

[0039] Muscle diseases often vary in severity depending on the type of striated muscle, and also between skeletal and cardiac muscles. Age-related sarcopenia, cancer cachexia, and spinal cord injury tend to affect type II fibers more than type I fibers. Duchenne muscular dystrophy (DMD) and facioscapulohumeral muscular dystrophy (FSHD) also frequently affect type II fibers, but FKRP-mediated dystroglycanopathies (MDDGA5, MDDGB5, and MDDGC5) result in a relative increase in type I fibers, possibly due to a gradual conversion of type II to type I fibers. In contrast, myotonic dystrophies and some types of limb-girdle muscular dystrophies (e.g., limb-girdle muscular dystrophy type 2A due to calpain 3 deficiency) are associated with a decrease in type I fibers. Neuromuscular junction (NMJ) diseases also cause conversion of skeletal muscle fiber types. For example, infants with the most severe forms of spinal muscular atrophy (SMA) have a very low number of type II fibers, with a corresponding increase in type I fibers. Patients with advanced amyotrophic lateral sclerosis (ALS) have a conversion of type II fibers to type I fibers. Some striated muscle diseases, such as Duchenne muscular dystrophy (DMD), affect both skeletal and cardiac muscles, whereas other striated muscle diseases primarily affect skeletal or cardiac muscles, while some cardiac diseases most significantly affect the ventricles, atria, or conduction system. These disease-specific changes in muscle types highlight the importance of developing MSECs that can be optimized for a variety of diseases, with the aim of targeting gene therapy to the most affected muscle and fiber types.

[0040] Challenges of achieving product-level control in the treatment of neuromuscular diseases The natural gene expression levels of all striated muscle proteins and striated muscle RNAs in various muscle types were varied to obtain optimal levels of expression. A crucial effect is under- or over-expression of therapeutic gene products at least at suboptimal levels. Over-expression can be detrimental due to dominant-negative protein-protein interaction effects or inappropriate activity of therapeutic proteins in certain muscle types (e.g., systemic administration of calpain 3 protease via AAV under the control of the relatively highly active desmin gene promoter is beneficial in skeletal muscle of a mouse model of limb-girdle muscular dystrophy type 2A, but calpain 3 toxicity is shown in the ventricular muscle of these mice due to calpain 3 over-expression).

[0041] Normal expression levels of each muscle protein and regulatory RNA species is achieved through a combination of transcriptional, post-transcriptional, and translational mechanisms. Transcriptional regulation occurs through the interaction of universal or tissue-specific transcription factors and control element complexes (proximal promoter and one or more enhancers) within the regulatory regions of each gene. Post-transcriptional regulation occurs through miRNAs and their target sites, and through protein-mRNA interactions. Translational regulation occurs through the mRNA splice machinery. Control of gene product abundance is further regulated by mechanisms that affect protein half-life, such as the ubiquitin-mediated degradation pathway. Standard methods for determining optimal MSEC and vector doses are described elsewhere herein.

[0042] Relationship between vector dose and MSEC activity It is important to realize that the expression levels of therapeutic gene products obtained with various MSECs vary depending on the vector dose and the inherent differences in transduction efficiency of each vector type, so that a sufficient amount of vector must be transduced into each muscle fiber or cardiomyocyte to obtain at least a minimally beneficial expression level of the therapeutic gene product. In various limb-girdle muscular dystrophies in which genes encoding enzymes are affected, the expression level of the gene product required may be significantly lower than that required for gene products encoding abundant contractile proteins such as actin. This is demonstrated by quantitative analyses of relative transcript levels of mRNA available on public websites such as the Broad Institute's gtexportal.org site (e.g., skeletal actin ACTA1 mRNA expression level in adult skeletal muscle is 5,000-fold higher than that of dystroglycan glycosylase (GMPPB)). Thus, while it may seem that MDDGC14 (GMPPB enzyme myopathy) can be treated simply by using 5,000-fold less of the same highly expressing MSEC vector used to treat nemaline myopathy (ACTA1 myopathy), this is incorrect because simply reducing the vector dose would inevitably result in some muscle fibers receiving no vector at all or too little vector to receive any therapeutic benefit. Also, the relative mRNA expression levels listed on the gtexportal.org site are derived from older subjects who died primarily of cardiac disease, so the numbers obtained from this site may not be as accurate for younger patients with various disease phenotypes.

[0043] Features of MSEC Basic research has identified various enhancer and promoter regions of skeletal and cardiac muscle genes, their control elements, and the interactions of these DNA components with transcription factors. The growing knowledge of these components, the signaling pathways that affect them, the identification of muscle type-specific miRNAs, and the various miRNA binding sites on mRNAs has provided a basic method for selecting MSECs that show different qualitative and quantitative expression levels of desired gene products depending on the muscle type.

[0044] In this disclosure, we describe over 300 MSECs that can be used to express protein- or RNA-encoding cDNAs in differentiated skeletal muscle cells and differentiated cardiomyocytes. These MSECs show background levels of expression in non-myocytes. We tested the transcriptional activity of all these regulatory cassettes in differentiated skeletal muscle cultures, and many of them were also evaluated in neonatal rat cardiomyocyte and non-myocyte cultures. Additionally, we tested a select subset for in vivo expression in transgenic mice or by systemic delivery of AAV vectors via blood vessels.

[0045] Overall findings from in vivo studies are that the relative transcriptional activities of various MSECs shown in in vitro studies are suggestive but cannot fully predict the relative activities of these MSECs in vivo.

[0046] (1) All in vitro evaluations of MSECs involve stepwise increased expression of desired protein or RNA products over a 1000-fold concentration range in differentiated skeletal muscle cell cultures and differentiated cardiac muscle cell cultures.

[0047] (2) The lack of expression of MSEC in non-muscle cell cultures correlates extremely well with the lack of expression of MSEC in any in vivo tissues, including striated muscle.

[0048] (3) The differences in MSEC expression levels observed in differentiated skeletal muscle cell cultures and differentiated cardiomyocyte cultures suggest that there may be similar relative differences in MSEC expression levels in vivo, which may be useful as a starting point for identifying optimal MSECs for specific in vivo therapeutic purposes.

[0049] (4) Because many MSECs differ in only one or two very few variables, in vivo data from one member of a related MSEC population is very likely to be predictive of most other members of that population.

[0050] The MSECs in the library of the present disclosure have a wide range of transcriptional activity. Importantly, several MSECs have been tested in vivo and shown to have different relative expression levels in various anatomical muscle types and muscle fibers. Since many MSECs have been designed using regulatory elements of the M-type creatine kinase gene (CKM), and the native CKM gene shows higher transcriptional activity in skeletal muscle than cardiac muscle and in fast muscle fibers than slow muscle fibers, it was expected that MSECs derived from the CKM gene would also show such properties. This has been demonstrated, for example, by the relative expression of one of the more active MSECs, "MSEC-438a," in mouse tibialis anterior and soleus muscle fibers in the order of type IIb>type IIx>type IIa>type I, in a ratio of approximately 8:4:2:1. Because humans do not have type IIb fibers, the relative expression levels of therapeutic gene products expressed through MSEC-438a in humans are predicted to be in the following ratio, although this has not been verified: type IIx fibers:type IIa fibers:type I fibers = approximately 4:2:1.

[0051] An important inference from the differences in transcriptional activity of these fiber types is that when MSEC-438a is used to treat Duchenne muscular dystrophy using microdystrophin, the ratio of microdystrophin produced in patients is estimated to be 4:2:1 between type IIx fibers, type IIa fibers, and type I fibers. Previous mouse Duchenne muscular dystrophy studies have suggested that higher than normal amounts of dystrophin are non-toxic, and it has been reported that the amount of microdystrophin in type IIx fibers can be four times higher than type I fibers and twice as high as type IIa fibers in Duchenne muscular dystrophy patients, regardless of vector dose. Biopsy data from ongoing human clinical trials using MSEC-438a should show the same results, and physiological studies examining the distribution of various muscle fiber types in anatomical muscle classifications should show whether functionality is achieved.

[0052] However, if type IIx fibers show barely sufficient microdystrophin for a given vector dose, then the amount of microdystrophin in type IIa and type I fibers will likely be significantly less than that in type IIx fibers. In this case, type IIx fibers will be considered "cured" and the amount of microdystrophin in type IIa and type I fibers will be 2-4 times lower than in type IIx fibers. It would therefore be highly beneficial to obtain sufficient gene product expression in all muscle fibers, and novel MSECs containing various combinations of striated muscle control elements in various configurations could provide these properties.

[0053] MSEC in clinical trials Two previously developed MSECs (MSEC-770 and MSEC-438a) are performing well in ongoing clinical trials for Duchenne muscular dystrophy (DMD) by Serepta and Solid Biosciences. For example, biopsy data from the Serepta clinical trial indicate that MSEC-770 likely expresses microdystrophin in all fiber types (relative expression levels not published), and plasma creatine kinase levels in treated boys were significantly reduced, mitigating systemic muscle fiber damage caused by muscular dystrophy. Based on these findings, these same MSECs were used to perform pilot studies of gene therapy for the treatment of other neuromuscular diseases. However, as detailed data from ongoing clinical trials will soon become clear, there is a more or less clear indication that more or less transcriptional activity than in this clinical trial is beneficial for the treatment of Duchenne muscular dystrophy, and therefore a different MSEC should be selected for the next clinical trial. Moreover, protein products involved in different neuromuscular diseases have different functions and require different concentrations than microdystrophin used to treat Duchenne muscular dystrophy, so it is very likely that different MSECs are suitable for treating different neuromuscular diseases. Identifying MSECs beneficial for each of the various neuromuscular diseases requires several in vitro preliminary tests followed by in vivo preliminary tests to identify the optimal MSECs. The availability of hundreds of MSECs with transcriptional activity ranging over 1000-fold makes this process very easy. An example of such a sequential approach to rapidly identify optimal MSECs for various neuromuscular diseases is described below.

[0054] The assessment of vector dose ranges for a particular application can be based on previously established safety studies of vector doses, transduction efficiency of viral vectors, knowledge of post-transcriptional diffusion / transport, and half-life parameters of mRNAs and proteins involved in neuromuscular diseases. The "ideal vector dose" may envision transducing all myonuclei of skeletal muscle cells and myonuclei of cardiomyocytes with only one transcriptionally active therapeutic vector genome capable of producing the same concentration of neuromuscular disease-related products as those produced by normal myonuclei, but this simple goal remains unattainable. At a therapeutically safe vector dose, the number of vectors contained in some myonuclei is always greater than the number of vectors contained in other myonuclei, and some myonuclei are vector-free. Thus, at very high vector doses, some myonuclei may produce concentrations of therapeutic gene products that are locally toxic, and at low vector doses, some myonuclei are unable to produce the therapeutic gene products. Therefore, current AAV doses are best determined based on practical experience associated with specific serotypes, delivery routes, and infusion rate parameters, as well as knowledge of transduction efficiency in various anatomical muscle categories.

[0055] Because there are virtually no data on transduction efficiency in humans, vector doses are usually determined based on extrapolation from mouse and other animal studies. As a specific example, a clinical trial for the treatment of Duchenne muscular dystrophy genes in humans used 2 × 10 14 It has been suggested that beneficial systemic delivery of micro-dystrophin could be achieved by delivering a dose of AAVrh74 of 1 × 10 vg / kg (Asher et al., 2020. http: / / doi.org / 10.1080 / 14712598.2020.1725469). This dose is at least one-third the AAV dose in mice and non-human primates known to be tolerated without overt vector toxicity. Because lower vector doses are preferable for cost and safety reasons, a reasonable two-way AAV dose for initial MSEC selection studies in mice is 2 × 1014 vg / kg and 2 × 10 13 vg / kg, and 7 × 10 13 vg / kg (3.5 × 10 per 20 g body weight of a 5-week-old male mouse) 12 Alternatively, 1 × 10 10 We were able to obtain preliminary data on the expression of therapeutic gene products via MSECs by intramuscular injection of .vg. Before generating AAV vectors, we always test MSEC therapeutic cDNA constructs in cost-effective and time-effective skeletal and cardiac muscle cultures as well as non-muscle cell cultures to confirm that muscle-specific products are expressed at reasonable levels. This avoids problems due to cloning errors and unexpected positive or negative effects when the cDNA sequence is transcribed.

[0056] Identification of the regulatory region of the CKM gene The mouse CKM gene was first identified by screening a lambda phage mouse genomic DNA library with a plasmid containing a portion of the mouse CKM cDNA (Chamberlain, JS, et al., 1985; PMID: 3990682; Jaynes, JB, et al., 1986). This method identified a 20 kb mouse genomic DNA fragment that contained what was thought to be the "entire" mouse CKM gene (Figure 3). This fragment contained eight different exons in an 11 kb region, with 3.3 kb of genomic DNA 5' to exon 1 and 6 kb of genomic DNA 3' to exon 8. The CKM transcription start site (TSS) was then identified using this genomic DNA fragment, which contained almost the full-length cDNA information, and a typical TATA box sequence (TATATAA) was identified 5' to the transcription start site. The fact that this genomic region contained CKM regulatory sequences was confirmed by two mutually complementary methods. In the first method, a 21 bp unique "mRNA tagging sequence" was inserted into exon 7 within a 16 kb genomic sequence in the region from -3,300 bp to +12,700 bp, or into a putative "promoter-less" genomic sequence lacking the region from -3,300 to +7, and transfected into proliferative MM14 mouse myoblasts or differentiated MM14 mouse muscle cells. RNA analysis showed that labeled mRNA was detected only in differentiated cultures transfected with the entire 16 kb region. In the second method, chloramphenicol acetyltransferase (CAT) cDNA was ligated into the region from -3,300 to +7 and transfected into proliferative MM14 myoblasts or differentiated muscle cells. Subsequent analysis showed that CAT expression was extremely high only in differentiated cultures (Figure 4 and another publication by Stephen D. Hauschka). Importantly, these two studies established that the mouse CKM gene region from −3,300 to +7 contains regulatory information that displays selective transcriptional activity in differentiated muscle cultures.Since the enhancer region contained in this region from -3,300 to +7 had not been reported before, we named this mouse CKM gene region from -3,300 to +7 "MCK gene promoter" based on the currently used common terminology. In addition, this natural mouse genomic DNA sequence is shown in the MSEC sequence library in Figure 27 under the name "MSEC-3363". In this study, we successfully analyzed this CAT reporter gene cDNA, which showed that this 3.3 kb CKM promoter can selectively express heterologous proteins in differentiated muscle cells. Based on this finding, we investigated the use of CKM gene regulatory sequences that can express virtually any protein of interest (Figures 1 and 2).

[0057] Overview of the 5'-enhancer and promoter regions of the CKM gene In a basic research study, we identified the 5'-enhancer and proximal promoter regions of the native mouse CKM gene. In this basic research study, we removed the 5'-end portion of the CKM region from -3,300 to +7 by stepwise truncation from the 5'-end, ligated each remaining segment to CAT cDNA, and tested the transcriptional activity of each segment in differentiated and proliferative MM14 mouse muscle cultures (Figure 1 in Jaynes, JB et al., 1988 (PMID:3336366)). In this study, we found that 100% of the activity exhibited by the -3,300 to +7 region was maintained by a short genomic fragment from -1256 to +7. This native mouse genomic DNA sequence is shown in Figure 27 under the name "MSEC-1263a," which indicates the total number of base pairs in MSEC, including the +7 base pair on the 3' side of the transcription start site, and is also referred to as the "1256-bp mouse MCK gene promoter" based on the currently used common terminology. On the other hand, when MSEC-1020 (-1020 to +7 region), which is an additional 243-bp deleted from the 5'-DNA, was created and tested, the transcription activity of MSEC-1020 in differentiated skeletal muscle cultures was reduced by more than 95%, and when multiple fragments with further deletions of the 5'-DNA were tested, the transcription activity was further reduced to a few percent (Figure 1 in PMID:3336366). These findings suggest that the skeletal muscle gene enhancer may be located within the deleted 243-bp genomic DNA region, and further suggest that the "proximal promoter" region, MSEC-783 (-776 to +7), contains additional muscle gene regulatory elements. However, when this proximal promoter region was truncated to a fragment from -80 to +7 (termed "MSEC-87"), which contains the "basal promoter" and the TATA box, it showed only background levels of activity in differentiated muscle cultures (Figure 2 in PMID:3336366).Furthermore, we evaluated enhancer-containing genomic fragments, and found that a 206 bp fragment spanning the region from -1256 to -1050, i.e., MSEC-206a, showed strong transcriptional activity in differentiated muscle cultures when ligated to the proximal or basal promoter fragments (referred to as "MSEC-1263a" and "MSEC-297a," respectively), but not in undifferentiated myoblasts or non-muscle cells. Furthermore, when ligated in the reverse 3'→5' orientation, the 206 bp fragment was highly active, even when ligated to the 3' end of a CAT cDNA reporter (Figure 2 in PMID:3336366). The 206 bp fragment was able to function regardless of the distance from the transcription start site to the 5' end or the distance from the 3' end to the 5' end, or the orientation, proving that this DNA sequence is an enhancer. This interpretation was supported by the fact that the 206 bp fragment was responsive to muscle differentiation signals even when ligated to a basal promoter from a heterologous gene, such as a promoter from thymidine kinase or SV40 virus (PMID:3336366, Figure 4). These studies were the first to identify muscle-specific expression enhancer sequences.

[0058] In vivo analysis of the 5'-enhancer and promoter regions of the CKM gene: To investigate whether the mouse CKM sequence is active in vivo, whether the 206bp region exhibits enhancer properties, and whether the various genomic fragments described in PMID:3336366 show similar transcriptional activity in cardiac muscle and are inactive in tissues other than striated muscle, we tested the genomic fragments in transgenic mice (Fig. 1 in PMID:2796990). These studies revealed several important facts for the design of MSECs in the future: (1.) The CKM gene genomic fragment containing the 5'-enhancer (207bp in this study) is transcriptionally active in both skeletal and cardiac muscle and shows background levels of activity in tissues other than striated muscle (Tables 1 and 2 in PMID:2796990). (2.) These enhancer-mediated activities occur both in combination with the proximal promoter fragment of the mouse CKM gene and in combination with the basic promoter fragment of the mouse CKM gene, but are enhanced when combined with larger proximal promoter fragments, e.g., MSEC-1263a shows stronger activity than MSEC-335a (Table 2 in PMID:2796990). (3.) The proximal promoter region surrounded by the region from -723 to +7 of the mouse CKM gene (MSEC-730) was shown to show low transcriptional activity in skeletal and cardiac muscles in the absence of a 5'-enhancer (Table 2 in PMID:2796990). (4.) Another region from +738 to 1599 of the mouse CKM gene (E2) shows enhancer-like activity in skeletal and cardiac muscles, but not in other types of tissues (Table 2 in PMID:2796990). Furthermore, MSEC containing the E2 region shows higher transcriptional activity when combined with a large proximal promoter fragment such as the -776 to +7 region of the CKM gene (e.g., MSEC-1676) than when ligated to the -80 to +7 proximal promoter fragment (e.g., MSEC-974). Thus, the mouse CKM gene contains at least two enhancers.In subsequent studies, the E2 enhancer was renamed "regulatory region 1 (MR1)" and further analyzed to define the core enhancer region as a 95 bp fragment termed the "small intronic enhancer (SIE)" (see Figures 3, 5 and 11 below). In several types of MSECs, this SIE or smaller fragments are used alone or in multimerized form.

[0059] Identification of the 5'-enhancer regulatory element of the CKM gene In the next basic study, we analyzed the 5'-enhancer of mouse CKM gene to identify its control elements (Fig. 3). Since all vertebrates are known to have CKM gene, we hypothesized that CKM genes of other species would have enhancers with very similar DNA sequences, since the basic gene regulatory mechanisms are similar in all vertebrates and have undergone evolutionary processes. Sequence searches of CKM genes of other vertebrates confirmed this hypothesis, and we aligned the putative enhancer sequences from various kinds of CKM genes to maximize similarity, and found that they contained several blocks of highly conserved sequences that were likely to be control elements (Fig. 8 and Fig. 9). To test this hypothesis, in the mouse CKM gene, two types of mutations were introduced into the putative control element region contained in the enhancer region in the genomic fragment from -1256 to +7 and into the putative control element region contained in the enhancer region in the genomic fragment from -1256 to -1048 (i.e., a fragment of 208 bp in this experiment), and then ligated in the positive or negative direction to the basic promoter in the region from -80 to +7, and the effect of each mutation on the transcriptional activity in differentiation-competent skeletal muscle and differentiation-competent cardiomyocytes was examined (PMID: 8474439, Figures 3, 4, and 5).

[0060] The results of this study demonstrated that at least six types of control elements exist within the 5'-enhancer region of the mouse CKM gene, and by inference based on sequence homology, the same six types of control elements exist in the 5'-enhancers of CKM genes from various other vertebrates. As shown in the data in Figure 5 of PMID:8474439, each mutation confers various transcriptional activity to the entire DNA construct, and it was revealed that mutations in the five control element regions of CArG, A / T, left E box, MEF1 (right E box), and MEF2, as well as mutations in the E boxes on both sides, reduced transcriptional activity, and mutations in the AP2 control element increased transcriptional activity. Subsequent basic research identified a sixth positively acting control element (Six4) within the 5'-enhancer region of the CKM gene (see below) (PMID:8617727; PMID:14966291). The differential activity of CKM 5'-enhancer fragments containing mutations in each of the regulatory elements was then shown to provide a basis for designing MSECs with graded levels of transcriptional activity that may be useful for gene therapy and other muscle-specific expression applications (see representative embodiments). Furthermore, both AP2 mutations increased transcriptional activity, indicating that these modifications can be used to construct design strategies for increasing MSEC activity in both skeletal and cardiac muscles. The wild-type and mutant sequences of this 206 bp enhancer sequence are shown in Figure 27 as MSEC-297a-297L and MSEC-1263a-1263v. Another study described herein discloses that a 110 bp genomic segment lacking three of the seven regulatory elements (CArG, AP2, and MEF2) within the 206 bp enhancer region described above exhibits skeletal muscle-specific enhancer activity when combined with the basal promoter region of the -80 to +7 region of the CKM gene, but not cardiac muscle (Figure 3 in PMID:8474439). This construct (MSEC-206b) may be applicable to gene therapy where expression in skeletal muscle is desirable but not cardiac muscle (see representative embodiments).

[0061] Identification of transcription factors associated with unknown CKM gene regulatory elements As shown in Figure 8, the 5'-enhancer regions of the CKM genes from various vertebrate species contain many highly conserved continuous base sequence regions. Most of these regions have sequences identified as matching consensus DNA binding sites of known transcription factors (PMID:8474439). However, some of the conserved sequence regions did not match any known consensus binding sites, nor did they show similarity to the binding sites of several transcription factors (PMID:8617727; PMID:14966291). To identify specific transcription factors that bind to unknown wild-type conserved sequences in differentiated skeletal muscle cells, mutations were introduced into each sequence and the transcriptional activity of the 5'-enhancer for the native and mutant sequences was compared. The introduction of mutations showed a significant decrease in activity, indicating that the conserved regions bind to one or more transcription factors and induce positive transcriptional activity (PMID:8617727). We then performed differential quantitative proteomics to distinguish between transcription factors that can bind only to wild-type DNA sequences and those that can bind to both wild-type and mutant sequences. As an example, this method identified Six / 4 (called TREX in earlier studies) as a major transcription factor that binds to conserved sequence regions in skeletal muscle cells, and Six / 4 was found to be located between the AP-2 and A / T-rich regulatory elements in the 5'-enhancer of mouse CKM (PMID:14966291). Using the same regulatory element discovery method, two other regulatory elements were identified within the proximal promoter of the mouse CKM gene (PMID:18710939; PMID:20404088). An important point regarding the design of MSECs is that the Six / 4 regulatory element in the 5'-enhancer of the CKM gene was found to be important for obtaining maximal transcriptional activity of the mouse native CKM genomic fragment (-1256 to +7) (MSEC-1263a) and the 5'-enhancer region ligated to the proximal promoter (-80 to +7) (MSEC-297a) in differentiated skeletal muscle cells, but not in cardiomyocytes (PMID:8617727).This suggests that a synthetic enhancer containing multiple Six / 4 regulatory elements can increase expression in skeletal muscle without increasing expression in cardiac muscle (see representative embodiments).

[0062] Identification of regulatory elements in the CKM gene promoter Similar to the cluster of conserved sequences found in the 5'-enhancer of the mouse CKM gene, a cluster of highly conserved sequences was also found in basic research on the proximal promoter of the mouse CKM gene. These conserved sequences are present in the region from -356 to +7 (Figure 9), and as in the case of the 5'-enhancer, several conserved sequences could be identified based on their similarity to consensus sequences of known transcription factors, such as E-box sequences, CArG / SRF sequences, SP1 sequences, AP2 sequences, and TATA box sequences. Among these, mutational analysis of the conserved proximal promoter E-boxes was particularly important because the E-boxes had high transcriptional activity in the 5'-enhancer of the CKM gene. In transgenic mice, introduction of mutations in the E-box of the proximal promoter within the entire region from -1256 to +7 (MSEC-1263c) or deletion of the E-box region within the region from -358 to +7 (MSEC-466) ligated to the previously described 206 bp 5'-enhancer significantly reduced transcriptional activity (see Figure 1 and Table 1 in PMID:8756664). Furthermore, a study investigating a deletion of the proximal promoter region containing the conserved CArG / SRF regulatory element found that transcriptional activity was reduced when the proximal promoter containing this deletion was ligated to the previously described 206 bp enhancer (MSEC-476) (see Figure 1 and Table 2 in PMID:8756664).

[0063] Several other highly conserved sequence regions found within the proximal promoter region of the mouse CKM gene from -358 to +7 did not match DNA binding sites of known transcription factors found in muscle tissue (Figure 9). Based on this result, we investigated these conserved regions using the same mutagenesis and differential quantitative proteomics methods that successfully identified the 5'-enhancer regulatory element Six / 4 and its transcription factors (PMID:14966291). This study identified MAZ and KLF regulatory elements that bind the transcription factors MAZ and KLF3, respectively (PMID:18710939; PMID:20404088). The MAZ regulatory element of the mouse CKM gene is located in the region from -81 to -67, and mutation of this MAZ regulatory element reduces transcriptional activity by more than a factor of two in both skeletal and cardiac muscle cultures (PMID:18710939, Figure 1). In addition, the mouse CKM gene contains two KLF3 regulatory elements in the -136 to -130 and -50 to -44 regions, and mutation or deletion of these elements reduces transcriptional activity by 2- to 3-fold in skeletal muscle cultures (Figure 3 in PMID:20404088).

[0064] Putative regulatory elements of the CKM gene Although the highly conserved 5'-enhancer and promoter regions of the CKM gene were identified to bind transcription factors, these 5'-enhancer and 5'-promoter regions contain several highly conserved sequences for which binding factors have not been identified. This 5'-enhancer region contains a 25-bp region with 80% sequence conservation between the right E-box and the 3' MEF2 control element, which made it highly likely that this 25-bp region confers one or more control functions (Figure 8). However, deletion of this entire region in vitro or in vivo under physiological conditions had little or no effect, but further testing was performed on the expression level of MSEC. The proximal promoter of the CKM gene in the region from -358 to +7 contained seven unknown regions with very high sequence conservation that did not contain known binding factors (Figure 9), but the introduction of mutations or deletions in one of these putative control elements did not reduce transcriptional activity significantly (see the paragraph below under the heading "Miniaturization of MSEC").

[0065] Generalization of the CKM gene regulatory element to similar elements in other muscle genes An important aspect of the identification of all the control elements in the mouse CKM gene as described above is that sequence searches for similar control elements in the control regions of other skeletal and cardiac muscle genes have shown that these control elements are also present in many other muscle genes, albeit with minor sequence variations. Thus, it is predicted that mutating or multimerizing these control elements in the enhancers and promoters of muscle genes in other vertebrates could have a similar effect on the transcriptional activity of these control regions (see representative embodiments). Furthermore, the finding that the same control elements were present in different skeletal enhancers and different skeletal promoters suggests that completely synthetic muscle regulatory regions could be designed by combining libraries of muscle gene control elements in various linear orders (see the "Synthetic MSEC" section below and representative embodiments).

[0066] Minimization of the 5'-enhancer and promoter sequences of the CKM gene to facilitate packaging into adeno-associated viruses and other small vectors Sequence alignment of the enhancer and promoter regions of the CKM gene of various vertebrates has revealed regions with very low sequence conservation between the identified control elements. Although some of these regions may create significant spatial distance between consecutive control elements, we hypothesize that a certain length of base pairs can be deleted between adjacent control elements without reducing transcriptional activity. If this assumption is correct, it is possible to create even smaller MSECs by deleting a certain length of base pairs, and such miniaturized MSECs may be efficiently packaged in AAV vectors in combination with large cDNAs. Furthermore, even if the size of the cDNA components is not an issue when packaging into vectors, it is possible to create MSECs that contain miniaturized CKM enhancers and CKM promoters, allowing the addition of additional control elements within the miniaturized regulatory region, or the addition of multimerized CKM enhancers or enhancers from other muscle genes (see the following section and representative embodiments). This concept of compacting regulatory regions was tested by deleting portions of non-conserved sequence regions between adjacent regulatory elements in the CKM 5'-enhancer region (-1256 to -1050) (Figure 8) and the CKM promoter region (-358 to +7) (Figure 9) by sequentially deleting the size of the deletion sites in stepwise order. In Figures 8 and 9, the test regions that could be deleted without significantly reducing transcriptional activity are shown as sequence regions marked with "x", but only a few test regions could be deleted without losing transcriptional activity. The largest region that could be deleted in the 5'-enhancer region was a 63 bp region between the right E-box and the 3'-MEF2 site; the largest region that could be deleted in the proximal promoter region was a 104 bp region from the 5' end to residue -254.By repeating this deletion procedure starting from the original 605 bp MSEC derived from CKM, we first obtained the design of MSEC-571 (similar to the 580 bp cassette described in PMID:17235310), and then obtained a number of MSECs (e.g., MSEC-400) in the size range of 400-500 bp that showed very high muscle-specific expression both in cell culture and in vivo.

[0067] Identification of regulatory regions and control elements in the regulatory region of intron 1 of the CKM gene In early CKM gene expression studies, the regulatory region of intron 1 of CKM (MR1) was serendipitously discovered when a 1 kb restriction fragment in intron 1 was inserted 5' of the proximal promoter of the CKM gene, and this MR1 ​​was found to have enhancer-like properties, increasing expression in skeletal muscle cultures and transgenic mice (PMID:3336366; PMID:2796990). Although many of the MSECs with high overall expression were significantly less active in slow-twitch fibers than in fast-twitch fibers (PMID:17235310, Figure 6), a 6.5 kb restriction fragment encompassing the -3349 to +3236 region of the native mouse CKM gene was able to express full-length dystrophin in both muscle fiber types in transgenic mice (PMID:8355788). Based on this result, we initiated a detailed analysis of MR1. We hypothesized that an enhancer within the MR1 region is required for expression of full-length CKM in slow-twitch fibers (PMID:21797989). Sequence alignment of the MR1 region of mouse CKM with intron 1 sequences from five other mammalian species revealed several subregions containing conserved base pair clusters (Fig. S1 in PMID:21797989). Of these, a 95 bp cluster in the region +901 to +995 was particularly interesting because it contained four known muscle gene regulatory elements (two E-boxes, one MEF2, and one sequence overlapping MAF and AP1) (Fig. 1 in PMID:21797989). We then performed cell culture studies of this 95 bp region in various MSECs (e.g., MSEC-732; MSEC-734; MSEC-804) and demonstrated that this 95 bp region behaves like an enhancer (hence the name "small intronic enhancer (SIE)") and showed high transcriptional activity when combined with the proximal promoter region of the mouse CKM gene from -358 to +7 (Figure 2 in PMID:21797989).Next, we performed mutational studies of the regulatory elements, which showed that two E-box regulatory elements and one MEF2 regulatory element were important for the transcriptional activity of the full-length SIE in skeletal muscle cultures, but base pair deletions within the half-site sequence of the MAF and AP1 overlap region did not result in loss of transcriptional activity (Fig. 3 in PMID:21797989). The results of this deletion experiment did not indicate whether the highly conserved half-site has a functional role in vivo, but suggested that SIE could be further miniaturized by deleting part of this half-site sequence. This approach resulted in the design of a 74 bp sequence (MSEC-74) that exhibits high transcriptional activity in skeletal muscle cultures and can exhibit additional activity when ligated to another MSEC, such as MSEC-571 (e.g., MSEC-732, MSEC-734, MSEC-804, MSEC-809, MSEC-892a / b, MSEC-966, MSEC-970 and MSEC-1204) (see the following section and representative embodiments).

[0068] MSEC containing regulatory elements of slow muscle fiber genes As mentioned above, many of the CKM-based MSECs with high overall expression were found to be significantly less active in slow-twitch fibers than in fast-twitch fibers (Figure 6 in PMID:17235310). This fiber-type transcriptional bias was not optimal for the use of MSECs, which require gene products to be expressed to the same extent in all types of skeletal muscle fibers. To overcome this issue, we considered ligating enhancer regions from slow-twitch specific genes, such as slow-twitch troponin I (TNNI1), to CKM-based MSECs (e.g., MSEC-550, MSEC-563, or MSEC-757) or TNNT2-based MSECs (e.g., MSEC-587).

[0069] Identification of regulatory regions and control elements in the human cardiac troponin T (TNNT2) gene The CKM gene is expressed at high levels in both skeletal and cardiac muscles, but the property of CKM being highly expressed in skeletal muscles means that MSECs based on CKM may not be optimal for treating health problems restricted to the cardiac muscle. For this reason, it would be useful to design MSECs that show cardiac-specific expression. Since transcription from the cardiac troponin T gene (TNNT2) is known to be cardiac-specific (PMID:26774798), and furthermore, other researchers have identified regulatory regions of the TNNT2 gene in birds and rodents (PMID:2993302; PMID:7982978; PMID:18951515; PMID:9689598), we cloned the analogous region of the human TNNT2 gene. A 495 bp construct (MSEC-495) (Figure 10) was tested in fetal rat cardiac myocyte cultures and showed high transcriptional activity. Next, MSEC-495 was downsized to generate MSEC-320 by repeated partial deletion of the less conserved sequence region of the CKM 5'-enhancer-promoter genomic fragment from -1256 to +7, as described above, and the 130 bp enhancer region obtained by the downsizing was then multimerized to obtain the highly active MSEC-455a. As expected, MSEC-455a was highly expressed in cardiomyocytes and only background levels of expression were observed in non-muscle cells, but it was also expressed in skeletal muscle cultures during differentiation. This behavior is consistent with the fact that activation of many cardiac genes occurs during the early stages of skeletal muscle differentiation, followed by repression of cardiac genes as myofibers mature (PMID:1728592). Also consistent with this fact, MSEC-455a only shows background levels of transcriptional activity when delivered systemically to adult mice by AAV (Figure 16).Furthermore, the compact TNNT2 enhancer has been shown to synergize with MSECs containing CKM enhancer and promoter components (e.g., MSEC-571a) by increasing expression in cardiac muscle, and also with some, but not all, types of skeletal muscle (e.g., MSEC-725a and MSEC-875) (FIG. 16). These TNNT2-based MSECs are highly significant (see representative embodiments).

[0070] Enhancer multimerization in MSECs The transcriptional activity of MSEC can be enhanced by ligating additional enhancers to the 5' side of the promoter or the 3' side of the cDNA, and the orientation of the enhancer sequence can be in either direction relative to the transcription start point. The multiple enhancers added can be the same or different from each other, and do not have to be from the same natural gene from which the promoter is derived. For example, MSEC-455a, MSEC-590 and MSEC-725a have two, three and four miniaturized TNNT2 enhancers ligated to the miniaturized TNNT2 promoter, respectively. MSEC-1204 has eight 74bp CKM SIEs ligated to the 5'-enhancer of the CKM gene. The proximal CKM region that constitutes MSEC-571 and MSEC-770 has a MYH6 enhancer ligated to a miniaturized CKM enhancer ligated to the CKM promoter. In addition, MSEC-518 has the TNNI1 enhancer ligated to a synthetic enhancer ligated to a truncated ACTA1 promoter.

[0071] MSEC containing the ACTA1 promoter region Since the enhancer and promoter of the CKM gene show higher expression in fast muscle fibers, a strategy to circumvent this problem is to replace the promoter of the CKM gene with a miniaturized promoter of the ACTA1 gene (since α-skeletal actin is produced at approximately equal expression in all types of fibers), and then combine this miniaturized promoter of the ACTA1 gene with a synthetic enhancer, such as MSEC-285, MSEC-319b, or MSEC-429a (see representative embodiments). Transcriptional activity can be further enhanced by ligating to the "distal regulatory element (DRE)" of ACTA1 present in the enhancer-like region at -1282 to -1177 of the human ACTA1 gene (PMID:1633435).

[0072] Changing the type of control element in MSEC Previous analyses have shown that enhancers and promoters from various muscle genes contain different types and numbers of control elements that confer positive transcriptional activity to the regulatory fragments, and these control elements are located at different spatial positions relative to adjacent control elements. This suggests that transcriptional activity can be modified in beneficial ways by adding or substituting virtually any type of muscle gene control element and / or universally active control element at various positions in the native or miniaturized enhancer and promoter regions. For example, the activity of the original MSEC was increased by inserting additional MEF2 and E-box control elements in the proximal promoter of the CKM gene (see Figure 9). Modifications with these control elements can increase or decrease the activity of MSEC in any type of striated muscle, increase or decrease the relative activity of MSEC in selected muscle types, or increase the "leakage" of expression in cell types other than muscle cells, resulting in low expression of the gene product in these cells or even higher expression in striated muscle cells.

[0073] Regulatory elements responsive to hormones or vitamins Modification of enhancer and promoter regions of MSECs with other types of control elements may include the insertion of DNA response elements that are responsive to glucocorticoid and steroid hormones and function via DNA glucocorticoid / steroid response elements (GRE / SRE) (PMID:32822588); thyroid hormones and function via DNA thyroid response elements (TRE) (PMID:1318069); or vitamin D and function via DNA vitamin D response elements (VDRE) (PMID:31203824). The therapeutic benefit of inserting these types of control elements into MSECs is that they can be made responsive to external / pharmacological signals, thereby activating the transcriptional activity of MSECs when this is beneficial.

[0074] Artificial control elements for muscle-specific and externally controllable gene product expression Based on the possibility of designing proteins containing DNA-binding domains that bind to DNA sequences and domains that can promote dimerization with partner proteins upon drug induction (PMID:25989233, PMID:19933107, PMID:22753599), MSECs targeted by such proteins can be designed to contain one or more special DNA-binding sequences (i.e., sequences that are not present in the genome and to which the designed protein specifically binds) in addition to other essential control elements. Such MSECs are then fused to cDNAs that encode the desired gene product. Transcription from such MSECs can then be performed in a muscle-specific manner by co-expressing the special DNA-binding proteins with a second protein that contains a dimerization domain and a transcription activation domain that allows functional interaction with the DNA-binding subunit only in the presence of the drug. Since the artificial transcription factor components are produced only in muscle cells and dimerization of the two transcription factor subunits occurs only in response to the drug, transcription of the gene product can be externally regulated via the concentration of the drug. An externally controllable system of this general type has been designed and extensively tested by Ariad Pharmaceuticals (PMID:22753599), but has never been formulated in a tightly regulated muscle-specific manner. We adapted parts of the Ariad Pharmaceuticals system to target muscle-specific expression and secretion of parathyroid hormone in cell culture and mice (see figure in Salva, MZ 2007, PhD thesis, University of Washington).

[0075] N-box regulatory elements for expression of synapse-specific transcripts N-box control elements are present in the promoters and enhancers of muscle genes that are highly transcribed in myofiber nuclei in the vicinity of neuromuscular synapses. Thus, N-boxes are a special kind of MSEC insertion control element that can selectively enhance transcription in myonuclei of skeletal muscle fibers in synaptic regions and suppress expression of MSECs in non-synaptic regions. These short control elements (e.g., -TTCCGG- multimers found in enhancer and promoter regions of acetylcholinesterase (ACHE), acetylcholine receptor subunits (e.g., CHRM1, CHRND, CHRNG) and acetylcholine receptor genes) respond to neurally mediated signals such as agrin and neuregulin that stimulate productive binding of transcription factors, such as GABP binding to N-box control elements (PMID:11498047; PMID:7479853; and references listed in the figures). Thus, N-boxes are useful for expressing therapeutic gene products locally in myonuclei at the subsynaptic region for the treatment of various myogenic neuromuscular junction diseases (PMID:27112691), and for secreting neurotrophic factors that enhance motor neuron survival through the neuromuscular junction, such as in amyotrophic lateral sclerosis (ALS) (PMID:8860837). A specific advantage of the MSEC design with respect to N-box regulatory elements is that N-boxes are known to function both 5' and 3' to the transcription start site and over long distances (Figure 21), and therefore can be inserted at various positions that can enhance or prevent steric interactions of N-box transcription factors with other transcription factors associated with MSEC. This suggests that the N-box can exert its function even if it is inserted at various positions in MSEC. The N-box may be a compact N-box-containing enhancer isolated from a muscle gene such as the acetylcholinesterase (ACHE) gene or the acetylcholine receptor subunit (CHRM1, CHRND, CHRNG) gene, or a multimerized N-box cluster such as the 43-bp sequence in which three N-boxes are clustered together, as shown in Figure 21.In vitro assays have demonstrated that such N-box-containing MSECs exert their functions in differentiated skeletal muscle cells in response to the addition of specific ligands, such as agrin and neuregulin, to the culture medium (PMID:11498047; and references listed in the figure). Based on this, we determined the optimal location of the N-box within MSECs by quantitatively comparing the expression levels of N-box-containing MSECs in response to the external addition of neurotrophic factors (PMID:11498047).

[0076] TATA box modification to promote gradual increase in MSEC expression A particularly useful feature of MSECs is the enhancer / promoter design, which allows them to have nearly identical signaling responses but express gene products at various levels in response to the same environmental input. Since the DNA sequence of the TATA box is known to affect the transcription rate (see Figures 19 and 20) (PMID: 10617571), the transcription rate can be varied by inserting various TATA box sequences into the same MSEC. Since the MSECs with common features contain the same enhancer / promoter region, the insertion of the TATA box sequence not only allows MSECs to produce gene products in a certain concentration range, but also makes the signaling response of such MSECs uniform, facilitating the evaluation of safety parameters of MSECs for gene therapy. The consensus core sequence for the vertebrate TATA box is gTATAAAA, and while the TATA box of ACTA1, the most highly transcribed human skeletal muscle gene (Figure 6), is a perfect match to the vertebrate consensus TATA sequence (see the sequence of MSEC-239), the TATA box sequence of the mouse CKM gene differs from the vertebrate "consensus" TATA sequence (Figure 19). These comparisons are consistent with the hypothesis that the TATA box sequence is a key determinant of the transcriptional activity of MSECs, and that by varying the TATA box in one MSEC, MSECs with different transcriptional activities can be produced.

[0077] Effect of exon 1 variation of MSEC on MSEC-mediated gene product expression Many of the MSECs described herein based on the CKM gene have their 3' terminus at +7 relative to the transcription start site (TSS) at +1. However, the short exon 1 sequence of the CKM gene is highly conserved, and since transcriptional and translational regulatory mechanisms are known to occur 3' from the transcription start site, we investigated the effect of extending the MSEC sequence to the +50 position of the mouse CKM gene. By extending the MSEC sequence to the +50 position of the mouse CKM gene, the expression level of the gene product from the MSEC increased almost two-fold in skeletal muscle cultures and five-fold in cardiomyocyte cultures (PMID:17235310). In the next study, we investigated how long the region from +1 to +50 is required to obtain maximum activity of MSEC-438a. By sequentially truncating the sequence from the 3' end, it was found that the region from +1 to +12 of the exon 1 sequence of the CKM gene was optimal, and transcriptional activity in skeletal muscle culture was increased by approximately 2-fold compared to MSEC-400. Furthermore, transcriptional activity in vivo upon systemic delivery was also increased by approximately 2-fold, while no change in expression in the myocardium was observed (see Figure 26).

[0078] Human MSEC: At least two-thirds of the sequence entries in the MSEC library contain partial and recombinant sequences from the mouse CKM gene, but for comparison purposes we designed several MSEC sequences based on the human CKM gene and found that the general regularity of the transcriptional activity of these MSECs was nearly the same when tested in rodent skeletal and cardiac muscle cultures. This was expected, since the sequences of the control elements of mouse and human muscle genes are very similar and human transcription factors can easily bind to mouse control elements. Furthermore, we observed that MSECs constructed from mouse CKM sequences performed well in human clinical trials, suggesting that the species-specific differences between mouse-based MSEC sequences and the ability of human transcription factors to recognize DNA are quite small. Many other recombinant sequences in the MSEC library are derived from human genes. The cardiac-specific MSEC sequence is derived from human TNNT2, the sequence enhancing expression in slow-twitch muscle fibers is derived from human TNNI1, and most of the minimal promoter sequences used in combination with the synthetic MSEC enhancer are derived from human ACTA1. However, it is important to emphasize that, regardless of the species origin of each MSEC component, all MSECs are artificial compared to the entire DNA regulatory elements of the original gene.

[0079] Synthetic MSEC The many variations in the type, number and spacing of regulatory elements in muscle gene enhancers and promoters allow the creation of rationally designed synthetic MSECs (see Figures 2 and 24). A particular advantage of designing synthetic MSECs over stepwise modification of natural MSECs is that regulatory elements with known interacting transcription factors (e.g., E-boxes and MEF2 transcription factors, TEF1 and MEF2 transcription factors, NKX2.5 and MEF2c transcription factors) or transcription factors whose dimerization is driven by adjacent DNA binding sites (e.g., TEF1-TEF1 interaction spaced 3 bp apart) (PMID:10975468) can be placed adjacent to each other and spaced appropriately to maximize protein-protein interactions. Synthetic MSECs can consist of synthetic enhancers (MSEC-503, as well as MSEC-285, MSEC-322, and MSEC-361) ligated to natural or recombinant / miniaturized gene promoters (e.g., CKM and ACTA1 promoters), natural or recombinant / miniaturized enhancers ligated to synthetic promoters, or combinations of synthetic enhancer and synthetic promoter regions. The enhancer portions of synthetic MSECs can also be multimerized and positioned 5' and 3' to the transcription start site.

[0080] Combination of cDNA containing miRNA target sequences and MSEC (combinatorial use) Gene therapy methods for treating neuromuscular and cardiac diseases mostly require systemic delivery of gene delivery vectors, so that both skeletal and cardiac muscle cells are transduced. When a particular disease affects both skeletal and cardiac muscle tissues, treatment can be performed using a single MSEC with optimal expression characteristics in both skeletal and cardiac muscles. However, in situations where only one type of muscle is affected by the disease and treatment is required, the muscles not affected by the disease may be adversely affected by the production of the therapeutic gene product. This problem can be avoided if there are MSECs that exhibit sufficiently skeletal muscle-specific or sufficiently cardiac-specific expression characteristics; for example, MSEC-455a is highly cardiac muscle-specific (see FIG. 16). However, when the therapeutic gene product is toxic to skeletal or cardiac muscles even at very low levels of expression, this situation can be avoided by using MSECs with beneficial expression characteristics in the diseased tissue. This problem can be partially solved by improving the targeting of the tissue-specific vector, but even such improvements may not be sufficient to solve the problem of toxicity due to very low levels of gene product expression in the wrong type of muscle.

[0081] Problems with the toxicity of such therapeutic gene products can be ameliorated by the combinatorial use of MSECs in combination with cDNAs that have muscle type-specific miRNA target sequences inserted into them. The miRNA target sequences are usually, but not necessarily, inserted into the 3' region of the cDNA. The mRNA products that contain the miR target sequences are selectively degraded in muscle cells that express the miR complementary to the target site, but not in muscles that do not express the miR complementary to the target site. This process occurs naturally in skeletal muscle through the expression of miR-206 (PMID:25678853; PMID:32620696) and in cardiac muscle through the expression of miR-208a (PMID:25678853; PMID:32620696; PMID:19726871). This hypothesis was tested and verified in skeletal and cardiac muscle cultures by inserting multimerized miR-206 or miR-208a target sites into the 3' end of the reporter gene cDNA (see page 1 of Figure 27 for miR target site sequences). The expression of the mRNA product could be reduced by 90-95% in a muscle type-specific manner (see Figures 17 and 18). Thus, the combinatorial use of muscle type-specific miR target sites and MSECs can be functional in vivo and can be applied to muscle type-specific treatments. Regarding the use of miR-208a target sites to reduce the expression of potentially toxic mRNA products in myocardium, it is interesting to note that various types of cardiac stress, such as ischemia and diabetes, increase myocardial mir-208a expression (PMID:30696455), suggesting that the function of the miR-208a target site is enhanced in response to cardiac toxicity problems.

[0082] Linker sequences as components contributing to the transcriptional function of MSECs DNA linkers of various lengths and sequences were included in various MSECs as insertion sequences between enhancer and promoter regions, between enhancers, and between multimerized miRNA target sites. Figure 15 shows examples of short and long linker sequences of various lengths and sequences included in various MSECs. The insertion of linkers into MSEC sequences has three basic aspects: (1.) the linker introduces an artificial sequence between the ligated components; (2.) the linker introduces various spatial distances between the ligated components; and (3.) both of these two factors can affect the transcriptional activity of each MSEC, regardless of the intrinsic activity of the enhancer and promoter sequences of the MSEC. Thus, the linker sequence can be considered as a functional component in each MSEC that is related to the degree of translational activity. These linker sequences are not related to the selectivity of expression in muscle cells and non-muscle cell types.

[0083] Overall value of the entire MSEC library The current MSEC library contains about 350 MSEC sequences that show muscle-specific expression over a thousand-fold range in muscle culture. Although the MSECs that show very low expression in muscle culture have not been tested in vivo, it is expected that these MSECs will also show a three-order of magnitude range in gene product expression in vivo if all MSECs were tested with the same vector dosing protocol. Thus, if very low expression levels are required in vivo, MSECs for that purpose are already available. Furthermore, if dose-response assessment of therapeutic gene products in vivo is required, the use of MSECs that show a transcriptional activity range of about 30-fold in skeletal and cardiac muscles will provide less ambiguous results than the traditional method of simply varying the vector dose over a dose range of more than 30-fold. For this reason, vector dosing protocols include two variables: the percentage of cells transduced and the expression level of the gene product in each transduced cell, whereas MSEC dosing protocols include only one variable: the expression level of the gene product in each transduced cell. The availability of MSECs that can obtain optimal expression of gene products at vector doses that transduce sufficient cells, both because they eliminate the safety hazard of excessive vector doses and because low vector doses do not transduce many cells, is important in the development of effective gene therapies.

[0084] How to Select an MSEC for a Specific Application Selecting the best MSECs for the treatment of various neuromuscular diseases from among the currently available MSECs in the libraries disclosed herein requires a multi-step process that is different for each disease, and even different for each disease's various alleles. This is because different proteins are affected by different neuromuscular diseases, and the normal concentrations of such proteins can vary over three orders of magnitude (e.g., some proteins, such as skeletal and cardiac actins, are more abundant than others, such as myosin and titin, which are even more abundant than regulatory proteins, such as kinases and phosphatases). Furthermore, each mRNA and the protein it encodes has an inherently different half-life, and therefore the amount of mRNA required to produce normal amounts of each therapeutic protein is different for each neuromuscular disease.

[0085] A further complication is that different patients suffering from the same neuromuscular disease may have different diseased gene alleles, which may result in the production of non-functional or less functional mutant proteins that compete with the therapeutic protein for binding to the partner protein, and thus require higher expression levels of the therapeutic protein than in normal muscle cells to "outcome" the deleterious protein. Another complication is that it is difficult to predict how much of the therapeutic gene product is needed to obtain optimal benefit before efficacy is demonstrated in a biomarker or functional improvement assay in a pilot study where graded vector doses or MSEC activity levels are tested. In this regard, it is important to recognize that although graded vector doses and increased MSEC activity can prevent suboptimal expression of the therapeutic protein, the biomarker and functional assays used to evaluate it do not necessarily detect toxic amounts. To be precise, toxicity can only be detected by using assays that target the problems that are logically related to excessive expression of the therapeutic gene product. Moreover, such toxic phenotypes may require long periods of time to be observed. Thus, there are safety risks to patients when discounting high levels of a therapeutic protein simply because of an observed functional benefit.

[0086] Step 1. Selection of MSECs based on cDNA size A stepwise approach to identifying optimal MSECs can begin by determining the size of the cDNA for the therapeutic gene product, including the size of the 5' and / or 3' untranslated regions, as well as the size of any introns that may be required to obtain high levels of functional protein. Information about the size of these cDNAs, combined with information about the limits of what can be packaged into a vector, determines the size range of MSECs that can be efficiently packaged with a particular cDNA.

[0087] For AAV vectors, which have a size limit of 4.8 kb for efficient packaging and require two inverted terminal repeat (ITR) sequences of about 145 bases for genome packaging, the combined size limit for MSEC and cDNA is about 4.5 kb. Therefore, the size of MSECs that are compatible with cDNAs of less than 0.5 kb, less than 1 kb, less than 2 kb, less than 3 kb, or less than 4 kb must be less than 4 kb, less than 3.5 kb, less than 2.5 kb, less than 1.5 kb, or less than 0.5 kb. Many MSECs have been miniaturized to accommodate packaging of large cDNAs, so multiple MSECs are available for the expression of almost all therapeutic proteins for neuromuscular diseases. However, this does not preclude the use of MSEC to express larger proteins, since AAV vectors can package constructs as large as 5.2 kb with very low efficiency, and cDNAs encoding large proteins can be split into two or more fragments, and the proteins encoded by these fragments can be joined in the correct order from N- to C-terminus using appropriate intein technology (PMID:32251274).

[0088] Step 2. Selection of MSECs based on muscle type-specific expression The second step in identifying the most suitable MSECs for the treatment of a particular neuromuscular disease is based on the knowledge of which muscle types require treatment. For example, whether the muscle types require treatment are skeletal and cardiac muscles, only skeletal muscles, only cardiac muscles, or whether the expression of a therapeutic gene product is beneficial in one muscle type but toxic in other muscle types. The selection of MSECs can be further narrowed based on the knowledge of which human skeletal muscles or human cardiac muscles are most severely affected by the disease (e.g., Duchenne muscular dystrophy affects virtually all striated muscles, whereas limb-girdle muscular dystrophy affects only one subset of anatomically classified muscles, and facioscapulohumeral muscular dystrophy mainly affects a different muscle group than the muscles affected by the disease). Similarly, some neuromuscular diseases have a relatively large effect on certain types of human skeletal muscle fibers, such as type I fibers, type IIa fibers, and type IIx fibers.

[0089] Although there are no MSECs that show the same transcriptional activity in all striated muscles, there are many MSECs that show several-fold higher expression in skeletal muscles than in cardiac muscles, and there are many MSECs that show several-fold higher expression in cardiac muscles than in skeletal muscles, some of which show 10-fold to several-hundred-fold activity in one or another type of muscle. In addition to the differences in expression in skeletal and cardiac muscles, there are no existing MSECs that show more than several-fold differences in expression between various anatomically different skeletal muscles of mice, a finding that probably also applies to anatomically different muscles in humans. On the other hand, some MSECs have different expression levels among various types of skeletal muscle fibers (e.g., the relative expression levels of reporter proteins by MSEC-438a in mouse muscle fibers are in the ratio of type IIb:type IIx:type IIa:type I = 8:4:2:1). Thus, when transduced with an AAV vector containing a construct consisting of MSEC-438a and a therapeutic cDNA, expression of the therapeutic gene product is expected to be higher in human skeletal muscles that contain primarily fast-twitch fibers (types IIx and IIa) than in muscles that contain primarily slow-twitch fibers (type I).Currently, MSEC-438a is the only MSEC for which myofiber-specific transcriptional activity has been measured, but research on this type of MSEC is ongoing, as are newly designed MSECs.

[0090] Step 3. Selection of MSECs based on concentration levels of functional gene products After narrowing the selection of MSECs based on the two aforementioned criteria, it is necessary to estimate the concentration of the therapeutic protein required to provide a functional benefit for a particular neuromuscular disease. In addition, if data are available, it is necessary to investigate whether this therapeutic protein exerts toxicity in excessive amounts. Unfortunately, conclusive data on the concentration of various neuromuscular disease proteins in various human striated muscles and muscle fibers, the translation efficiency of the mRNAs encoding them, and the steady-state half-lives of these proteins and mRNAs have yet to be established (Steed et al., 2020. PMID:32403418), and information on the toxicity of therapeutic proteins is limited. However, relative mRNA expression from the gtexportal.org website and other RNA-seq studies may be useful as a starting point, and these websites and studies have shown that there is at least a 5,000-fold difference in abundance between the most abundant and least abundant muscle gene mRNAs. That is, the relative expression of therapeutic mRNAs, whether thousands, hundreds or tens of transcripts per million (TPM), appears to be at least somewhat proportional to the normal steady-state levels of each neuromuscular disease protein. Similarly, the relative protein abundances of "high, medium or low" based on biochemical and immunomicroscopic data and knowledge of the function of each neuromuscular disease protein are also worth considering, and from such information one can infer whether one should focus one's efforts on screening MSECs with "high, medium or low" relative transcriptional activity. Information on the transport of each neuromuscular disease mRNA and protein, as well as diffusion parameters from transduced myonuclei, are also important in selecting MSECs with the transcriptional activity required for optimal treatment of each neuromuscular disease.

[0091] Unless it is clear that a neuromuscular disease requires the highest possible expression of therapeutic protein, simply testing the most active MSECs at the highest tolerated vector dose is not a sensible starting point. Although initial therapeutic effects obtained in this way may appear beneficial, such positive outcomes will likely mask better responses obtained with lower protein doses and will likely cause toxicities that were not evident during the initial short-term screening period.

[0092] A further informative strategy to identify MSECs with optimal transcriptional activity for each neuromuscular disease would be to use a safe intermediate vector dose (e.g., 7 × 10 as previously described). 13 This is a round-robin approach to determine the "best guess" for the amount of mRNA expression required by testing MSECs that are likely to express 5-, 20-, 100-, or 500-fold higher mRNA expression levels than the native transcript in neuromuscular diseases when delivered at 1000 mg / kg (vg / kg). Selection of these MSECs can be based on a table showing the transcriptional activity of MSECs, taking into account the muscle type expression level criteria for MSECs with the native M-type creatine kinase enhancer and promoter (whose transcriptional activity in skeletal and cardiac muscle cell culture studies is set at 1.0) and the package size of the MSEC (see Figures 5, 7, and 23).

[0093] In the actual screening, since the mRNA transcript from the native CKM gene in adult skeletal muscle is 25,000 TPM, in the primary screening for neuromuscular diseases where the mRNA ranges from 50 TPM, 500 TPM, or 5,000 TPM, if the transcriptional activity of the MSEC is 5-fold, 20-fold, 100-fold, or 500-fold compared to the enhancer and promoter activity of the native CKM, the MSEC selected will be (0.01, 0.04, 0.2, 1), (0.1, 0.4, 2.0, 10), or (1, 4, 20, highest value), respectively. The data from these preliminary tests are then carefully analyzed to identify the approximate range of expression levels of the MSEC that appears to be most beneficial and likely not toxic. In the second screening, the transcriptional activity of several MSECs found to be "best" in the first screening is tested.

[0094] Representative Embodiments - Set 1

[0095] 1. In some embodiments, the MSECs of the invention are based on the sequence of the region from -3,356 to +7 of the native mouse muscle creatine kinase (CKM) gene (MSEC-3363), as well as on small genomic segments within this region. Examples of this include MSEC-1263a, which consists of a region from -1,256 to +7 including the 5'-enhancer region of the mouse CKM gene within the region from -1256 to -1051; and various mouse CKM proximal promoters that do not contain enhancers, namely MSEC-1057, which consists of a region from -1050 to +7, MSEC-1027, which consists of a region from -1020 to +7, MSEC-783, which consists of a region from -776 to +7, MSEC-730, which consists of a region from -723 to +7, and MSEC-365, which consists of a region from -358 to +7; and MSEC-87, which consists of a region from -80 to +7, which is the mouse CKM basal promoter (PMID:3990682; PMID:3785216; PMID:3336366). These enhancerless and other similar enhancerless mouse CKM proximal promoters show varying degrees of low-level muscle-specific expression when used as stand-alone MSECs, whereas the basal promoters show only background levels of expression. However, the basal promoters such as MSEC-87 and other promoters of somewhat larger size work well in concert with the CKM promoter and other muscle gene promoters and enhancers to confer varying levels of muscle-specific transcriptional activity, and their small size makes them useful as components of MSECs.

[0096] 2. In some embodiments, the enhancer-free mouse CKM promoter described in embodiment 1 and other promoters generated based on sequences within the region -1050 to +7 can both be ligated to the native mouse CKM 5'-enhancer (e.g., MSEC-584 and MSEC-297a) or variants thereof (e.g., MSEC-297b derived from MSEC-297k) (PMID:3336366; PMID:8474439).

[0097] It is important to note that most of the MSECs described in this disclosure contain short non-genomic linker DNA segments of various lengths between the enhancer and promoter, utilizing molecular biology techniques to ligate the two components (see Figure 15 and Figures 12-14). For this reason, the bp length of the MSECs shown in the sequences in Figure 27 does not always match perfectly with the predicted bp length of the combined ligated components. For example, MSEC-297a of embodiment 2 is 297 bp long, but its "predicted length" from ligation of the 206 bp 5'-enhancer segment and the 87 bp basic promoter segment is only 293 bp. This apparent discrepancy is due to the 2 bp linker sequence "GA" that is inserted between the enhancer and promoter when ligating the two regulatory regions (PMID:8474439). Importantly, since the linker sequence can be considered an integral part of the overall MSEC sequence, the linker sequence within each MSEC should not be considered to have a "neutral effect" on transcription, but rather to have a beneficial, detrimental, or no effect on the transcriptional activity of the MSEC. These effects may be due to specific linker base pairs having intrinsic transcriptional activity by themselves, or in cooperation with contiguous sequences 5' and / or 3' to the linker sequence, or due to more or less efficient binding between various transcription factors bound to sequences 5' and 3' to the linker. Using a simple method to synthesize new multi-component MSECs based on the full length of the linker-free sequence, it was possible to remove the linker sequence to generate slightly smaller MSECs. However, these MSECs did not necessarily exhibit the same degree of transcriptional activity as similar MSECs containing linkers. In some, but not all, of the MSECs shown in Figure 27, the linker sequences are underlined.

[0098] A further aspect of the MSEC description regarding bp length is that many of the MSECs are assembled from genomic fragments obtained from various restriction enzyme digests. Thus, depending on the enzymes used, slightly different fragment lengths are obtained, such as the proximal promoter fragments [MSEC-1030 consisting of the region from -1020 to +7; MSEC-783 consisting of the region from -776 to +7; and MSEC-730 consisting of the region from -723 to +7]; and the basal promoter [MSEC-118 consisting of the region from -117 to +1; and MSEC-87 consisting of the region from -80 to +7]. The various proximal promoters were then miniaturized to generate 318 bp long (-268 to +50) or 280 bp long (-268 to +12) promoter regions derived from the mouse CKM region, which are used in the various MSECs described below.

[0099] 3. In some embodiments, the enhancer component described in embodiment 2 may have one or more mutated control elements or one or more deleted control elements. The individual enhancer and promoter control elements of the CKM gene are shown in Figure 3, Figure 8 and Figure 9. Depending on the mutated control elements and the resulting mutated sequences, MSECs are obtained that have reduced transcriptional activity, usually ranging from about 1% to about 95% of the activity of the native MSEC (see, for example, Figure 7 and tables and figures in PMID:8474439 and PMID:876664). However, some mutations, such as in the AP2 control element of the 5'-enhancer, may result in high activity (PMID:8474439). MSECs with such ranges of low or high activity may be useful for low or high expression of gene products, as required for a particular application (Figure 7).

[0100] 4. In some of the various types of MSECs described in embodiment 2 and embodiment 3, the native 5'-enhancer or recombinant 5'-enhancer of the CKM gene can be ligated in reverse to the transcription start site of the MSECs (see PMID:3336366 and PMID:8474439 for many examples (e.g. MSEC-310)). Depending on the enhancer and its modification, this may increase or decrease the transcription activity of the MSECs in different relative amounts in skeletal and cardiac muscle cultures, without necessarily affecting the overall size of the MSECs (PMID:8474439). Such MSECs are expected to show similar activity changes in vivo.

[0101] 5. In some of the various types of MSECs described in embodiment 2, embodiment 3 and embodiment 4, a single copy of the natural 5'-enhancer or recombinant 5'-enhancer of the CKM gene can be ligated to the 3' end of the cDNA in forward or reverse orientation relative to the transcription start point of the MSEC (PMID:3336366 and PMID:8474439, Figure 2). Depending on the type of enhancer or its modification, this operation can increase or decrease the transcription activity of the MSEC (PMID:8474439). Based on the data described in PMID:8474439 and embodiment 6, it is expected that activity can also be increased by placing a single enhancer on both the 5' and 3' sides.

[0102] 6. In some of the embodiments of various kinds of MSECs described in embodiment 2, embodiment 3 and embodiment 4, the native 5'-enhancer or recombinant 5'-enhancer of CKM gene can be multimerized to incorporate two, three or more enhancers arranged in tandem at the 5' side of the promoter region of MSECs (e.g., MSEC-507; MSEC 746; MSEC-749). Furthermore, in each multimerized enhancer group, the orientation of each enhancer group relative to the transcription start site can be forward or reverse (see the sequence shown in FIG. 27). Depending on the type of enhancer or its modification, this operation usually increases the transcription activity of MSECs several fold. Based on the data described in PMID:8474439 and embodiment 5, it is expected that the activity can also be increased by placing multimerized enhancers at the 3' side or both the 5' and 3' sides of the cDNA.

[0103] 7. Based on the data described in PMID:8474439 and embodiments 5 and 6, it is expected that activity can also be increased by placing multimerized enhancers on both the 5' and 3' sides of a contiguous enhancer group in forward or reverse orientation.

[0104] 8. In some embodiments, the proximal and basal promoter regions of the CKM gene can be designed to be similar in humans and other vertebrates (embodiment 1), and these can be used in the same manner as embodiments 2, 3, 4, 5, and 6 as a means to provide control elements with DNA binding sites that may have improved affinity for transcription factors from the same species (e.g., MSEC-463; MSEC-464a; MSEC-463b).

[0105] 9. In some of the embodiments of the various types of MSECs described in embodiments 2-8, any native or recombinant 5'-enhancer of the CKM gene can be ligated to a heterologous promoter (e.g., thymidine kinase promoter or SV40 promoter) in the forward or reverse direction relative to the transcription start point of the MSECs (see several examples in PMID:33363666 and FIG. 7 in PMID:8474439). Depending on the type of enhancer or heterologous promoter or its modification, this may increase or decrease the transcriptional activity of the MSECs (PMID:8474439). However, combining a heterologous promoter with the CKM enhancer may also reduce the muscle specificity of the MSECs and may have high relative activity in cells other than muscle cells.

[0106] 10. In some embodiments, intron 1 (3'-enhancer, also called regulatory region 1 (MR1)) of the mouse CKM gene located in the genomic region +740 to +1,721 of the mouse CKM gene (see FIG. 3 of this application and FIG. 2 of PMID:21797989) is used in combination with a proximal promoter segment of the CKM gene, such as the region -358 to +7 (e.g., MSEC-365). This large MSEC [(+740 to +1,721)+(-358 to +7)] shows in vitro activity in differentiated skeletal muscle cultures and also shows high transcriptional activity in transgenic mice in vivo (FIG. 5 of PMID:21797989).

[0107] In some embodiments, the intron 1 MR1 enhancer region of the mouse CKM gene is miniaturized to obtain a 95 bp mini intronic enhancer (SIE) sequence located at +904 to +998 of the native mouse CKM gene (see FIG. 3 of this application and FIG. 2 of PMID:21797989). When this SIE (MSEC-95) is used with a proximal promoter segment of the CKM gene, such as the region from -358 to +7 (e.g., MSEC-365), the resulting MSEC-476b shows high in vitro activity in skeletal muscle cultures comparable to the 5'-enhancer of the CKM gene (FIG. 2 of PMID:21797989).

[0108] 11b. The miniaturized SIE can be multimerized and combined with a variety of additional MSEC promoters to further increase activity stepwise (see FIG. 5, "data in blue", FIG. 11, and the sequences of MSEC-681, MSEC-804, and MSEC-805).

[0109] 12. In some embodiments, enhancers and / or promoters from vertebrate muscle genes other than CKM can be designed and used similarly to embodiments 2-8. For example, a number of MSECs have been constructed from the enhancer and promoter regions of the human cardiac troponin T gene (TNNT2) (see FIG. 10 and MSEC-495). These TNNT2-based MSECs are particularly useful for any purpose in which cardiac muscle-specific gene expression is desired, but skeletal muscle expression is not. In particular, MSEC-320, MSEC-455a and MSEC-590, which contain one, two and three copies, respectively, of the truncated TNNT2 enhancer and the truncated TNNT2 promoter, can be used to increase the expression level of cardiac muscle-specific expression.

[0110] 13. In some embodiments, enhancers and / or promoters from different types of vertebrate muscle genes can be combined in one MSEC. This was first done in an MSEC named MH-CK7 (MSEC-770), which combined a human α-myosin heavy chain enhancer with a recombinant 5'-enhancer and recombinant proximal promoter of mouse CKM gene (PMID: 17235310). This method resulted in MSECs that showed high transcriptional activity in both skeletal and cardiac muscles. The MH-CK7 / MSEC-770 construct, consisting of α-myosin heavy chain and CK7 parts, was further miniaturized to generate smaller constructs with similar activity, such as MSEC-745, MSEC-720, MSEC-714, MSEC-697, MSEC-689, and MSEC-672 (see FIG. 22). Other combinatorial MSECs combined enhancers (PMID:8900050) from the slow and fast troponin I genes (TNNI1 and TNNI2) with enhancers and promoters from the mouse CKM gene to yield, for example, MSEC-481, MSEC-518, MSEC-541a, MSEC-550, MSEC-555, MSEC-563, MSEC-569, MSEC-587, MSEC-726, MSEC-757, MSEC-758, MSEC-768, MSEC-773, MSEC-778, MSEC-855, and MSEC-960.

[0111] 14. In some embodiments, one or more conserved E-boxes in the region −1,256 to +7 of the native mouse CKM gene were mutated. Depending on the type of mutation and the test system, this resulted in a significant, 1000-fold differential reduction in relative expression of MSEC in the cardiac muscle of transgenic mice, with less reduction in expression in skeletal muscles containing relatively more type I muscle fibers and little effect in muscles with predominantly fast-twitch fibers (PMID:8756664;12968024). Other MSECs, such as MSEC-1263f, have been obtained, as well as similar MSEC constructs that can be made by similarly mutating or deleting E-boxes within the 5′-enhancer regions of other MSECs that contain regulatory elements. These types of MSECs may be useful for treating diseases such as limb-girdle muscular dystrophy type 2A, X-linked myotubularin, nemaline myopathy and other neuromuscular diseases in which expression of a therapeutic gene product in skeletal muscle is desirable and expression in cardiac muscle may cause toxicity.

[0112] 15. In some embodiments, the CArG / SRF regulatory element in the 5'-enhancer region of MSEC-1263a was mutated, resulting in MSECs with differentially reduced relative expression in the cardiac muscle of transgenic mice, approximately 1000-fold lower than in skeletal muscle (PMID:8657140). Similar MSEC constructs can be generated by similarly mutating or deleting CArG / SRF in the 5'-enhancer region of other MSECs that contain such regulatory elements, e.g., mutating CArG / SRF in the 206 bp enhancer of the CKM gene results in almost complete loss of in vitro activity in cardiomyocytes, with little loss of in vitro activity in skeletal muscle (PMID:8474439). These types of MSECs may be useful in developing treatments for diseases such as limb-girdle muscular dystrophy type 2A, X-linked myotubularin, nemaline myopathy and other neuromuscular diseases where expression of the therapeutic gene product in skeletal muscle is desirable and expression in cardiac muscle may cause toxicity.

[0113] 16. In some embodiments, the AT-rich regulatory element in the 5'-enhancer region of MSEC-1263a was mutated, resulting in MSECs that are differentially expressed in cardiac muscle at approximately 10-fold lower relative expression levels than in skeletal muscle (PMID:8657140). As noted above in embodiment "A" (several representative MSECs are listed in the sequence listing), similar mutations in the CArG / SRF regulatory element in the 5'-enhancer region of other MSECs that contain such regulatory elements can be used to generate similar MSEC constructs that may be useful in the development of treatments for diseases such as limb-girdle muscular dystrophy type 2A, X-linked myotubularin, nemaline myopathy and other neuromuscular diseases where myocardial toxicity is an issue.

[0114] 17. In some embodiments, the Six4 regulatory element in the 5'-enhancer region of MSEC-1263a was mutated, resulting in MSECs that are differentially expressed in cardiac muscle at approximately 20-fold lower relative expression levels than in skeletal muscle (PMID:12779122). As noted above in embodiment "A" (several representative MSECs are listed in the sequence listing), similar mutations in the Six4 regulatory element in the 5'-enhancer region of other MSECs that contain such regulatory elements can be used to generate similar MSEC constructs that may be useful in the development of treatments for diseases such as limb-girdle muscular dystrophy type 2A, X-linked myotubularin, nemaline myopathy and other neuromuscular diseases where myocardial toxicity is an issue.

[0115] 18. In some embodiments, MSECs are miniaturized by reducing the number of intervening base pairs between functional control elements. These types of modifications are shown in Figures 8 and 9. In some cases, such changes in the design of MSECs have the additional benefit of facilitating protein-protein interactions between transcription factors bound to adjacent control elements, resulting in increased transcriptional activity of the MSECs. An example of such a design property of MSECs is a CK7 regulatory cassette (MSEC-580) created by deleting most of the intervening base pairs between the E-box to the right of the 5'-enhancer of the CKM gene and the 5'-MEF2 control element. Such miniaturization also has the advantage that additional control elements can be inserted within the native enhancer and native promoter that increase the activity of the MSECs, such as a miniaturized CK9 promoter with the addition of adjacent MEF2 and E-box elements (Figure 9). Another form of miniaturization of MSECs can be achieved by deleting control elements whose activity is relatively more important for the production of therapeutic gene products in cardiac muscle than in skeletal muscle, as shown in embodiments 15-18. This approach has the advantage of generating smaller MSECs that can be used in combination with natural cDNAs that are too large for efficient packaging into AAV, or therapeutic cDNAs whose size makes them difficult to package efficiently into AAV, such as cDNAs encoding additional functional domains and Cas9, as long as expression levels in skeletal muscle remain high enough to produce a beneficial outcome.

[0116] 19. In some embodiments, MSEC is engineered by adding the first 50 bp (+1 to +50) of the exon 1 region of the mouse CKM gene (see Figures 3 and 9 of this application and Figure 1 of PMID: 17235310) to increase transcriptional activity over MSEC containing only sequences from the +1 to +7 region of exon 1 of the CKM gene. However, subsequent studies investigating the exon 1 region consisting of +1 to +50 found that incorporation of only the exon 1 region consisting of +1 to +12 was optimal.

[0117] 20. In some embodiments, MSEC is engineered by removing one or more low activity control elements of the enhancer or promoter of the CKM gene to further reduce the size of the MSEC without sacrificing the required transcriptional activity. Such an example is the deletion of the AP2 control element of the 5'-enhancer of the CKM gene. Interestingly, this method can further increase the overall activity of the enhancer of the CKM gene (see Figure 5 of PMID:8474439). Furthermore, by deleting the AP2 control element and the CArG / SRF control element as well as their adjacent and intervening sequences, the exon 1 region (embodiment 19) can be reduced in size to +1 to +12, reducing the overall size of the already reduced CK8e MSEC-438 to about 340 bp.

[0118] 21. In some embodiments, the size of the MSEC is further reduced without sacrificing the necessary transcriptional activity by removing one or more less active control elements in the enhancer or promoter of the CKM gene, and then the MSEC is modified by inserting a sequence of a more active control element, for example, the MEF2 control element (Figure 9), to obtain MSEC-336a, MSEC-336b, or MSEC-336c.

[0119] 22. [Spare column]

[0120] 23. In some embodiments, the enhancer and / or promoter regions of MSECs contain entirely novel arrangements of regulatory elements. Such "synthetic" MSECs have the important properties of high transcriptional activity and small size and can be used in combination with miniaturized enhancers and promoters from multiple muscle genes (see Figures 12, 13, 14; MSEC-259, MSEC-285, MSEC-319b, MSEC-322, MSEC-341, MSEC-361, MSEC-367, MSEC-383, MSEC-386, MSEC-388, MSEC-3 39a, MSEC-339b, MSEC-405a, MSEC-425, MSEC-474, MSEC-529a, MSEC-531a, MSEC-531b, MSEC-586, MSEC-562, MSEC-812a, MSEC-812b), and can also be used in combination with promoters derived from genes other than muscle genes, for example, MSEC using the thymidine kinase promoter can be obtained (Figure 7 in PMID:8474439).

[0121] 24. In some embodiments, MSECs may contain inserted N-box regulatory elements that enhance transcriptional activity in myonuclei at the neuromuscular synapse and reduce transcriptional activity in non-synaptic regions (Figure 21). Such MSECs contain 1-5 43 bp enhancer-like clusters of multiple N-Box consensus sequences ligated 5' and / or 3' to a transcriptionally permissive muscle gene promoter (e.g., MSEC-118, MSEC-239, etc.), and may be useful in treating a subset of neuromuscular disorders in which the disorder is caused by genes selectively expressed in myonuclei localized at the neuromuscular junction. Such genes include acetylcholine receptor subunits (multiple CHRN genes), acetylcholinesterase (ACHE), agrin (AGRN), muscle-associated receptor tyrosine kinase (MUSK), collagen-like tail subunit (COLQ), and utrophin (UTRN). Local expression of essential neurotrophic factors, such as BDNF, may also be beneficial in the treatment of spinal and bulbar muscular atrophy (SBMA) (PMID:308775922).

[0122] 25. In some embodiments, MSECs may contain a TATA box with a sequence different from that of the native gene in order to alter the transcriptional activity of the MSEC without affecting reception by the MSEC of transcriptional signals acting on all other enhancer and promoter regulatory elements (see Figures 19 and 20). TATA box sequences that confer 20-fold or more transcriptional activity (e.g., MSEC-438a or MSEC-455a with a TATA box consisting of cTATAAAAg or cTATAAAGg) have the therapeutic advantage of being able to produce quantitatively different levels of expression of a therapeutic gene product in the qualitatively same muscle type.

[0123] 26. In some embodiments, MSEC may contain a TATA box with an altered position relative to the transcription start site, thus altering the efficiency of transcription initiation, thereby altering the overall transcription activity of MSEC and the amount of gene product produced, without affecting the reception by MSEC of transcription signals acting on all other enhancer and promoter regulatory elements. For example, MSEC-438a and MSEC-455a variants with a distance of 27-34 bp between the 5' T of the TATA box sequence and the transcription start site can confer more than 50-fold transcription activity (PMID: 16916456). As described in embodiment 25, these MSECs have the therapeutic advantage of being able to produce quantitatively different expression levels of therapeutic gene products in the qualitatively same type of muscle. Moreover, MSECs containing TATA box and distance changes (embodiment 25 and embodiment 26) show even greater quantitative differences.

[0124] 27. In some embodiments, MSEC can be used in combination with miRNA target sites ligated to the 3' end of any cDNA component that is expressed as an RNA or protein product. When this type of construct is expressed in a cell in vitro or in vivo in the presence of the appropriate miRNA, the miRNA binds to a target site in the transcribed RNA and degrades the target site, thereby reducing the expression of the RNA or protein product in a particular type of cell by as much as 20-fold. In contrast, RNA transcripts produced in cells that do not express a miRNA that can bind to the miRNA target site are not degraded. Using this method, it is easy to achieve high expression of in vivo products in skeletal muscle while greatly reducing the expression of in vivo products in cardiac muscle (because cardiac muscle contains miR208a) (PMID:34957257). Conversely, it is easy to achieve high expression of in vivo products in cardiac muscle while greatly reducing the expression of in vivo products in skeletal muscle since skeletal muscle contains miR206 (PMID:23439498). By implementing this method with three tandem miRNA208a target sites in combination with MSEC-438a, it is possible to obtain high expression of the in vivo product in skeletal muscle and low expression of the in vivo product in cardiac muscle (as shown in FIG. 17). By implementing this method with three tandem miRNA206 target sites in combination with MSEC-438a or MSEC-455 (with minor modifications to enhance specificity), it is possible to obtain high expression of the in vivo product in cardiac muscle and low expression of the in vivo product in skeletal muscle (as shown in FIG. 18). Importantly, these two differential targeting methods can be easily used with any type of MSEC, specifically, appropriate tandem mir208a or miR206 target sites can be ligated to any cDNA encoding a beneficial therapeutic gene product to be expressed in one type of striated muscle and low expression in the other type of striated muscle.

[0125] 28. In relation to all of the above embodiments, it is important to highlight that the overall characteristics of the MSEC library as a whole show that the range of expression between the least active and the most active MSECs varies by more than three orders of magnitude (Figure 7). Given the package size limitations of which MSECs can be used in combination with a particular vector, this means that any one or more MSECs in the MSEC library are likely suitable for almost any use where a specific production level is required in skeletal and / or cardiac muscle tissue. This characteristic of the MSEC library as a whole is important because the mRNA expression levels of natural genes whose mutations cause neuromuscular diseases vary by more than three orders of magnitude. Thus, the MSEC library contains MSECs with activities that appear to match the activities required to obtain near-normal expression levels of each therapeutic gene product for each neuromuscular disease.

[0126] 29. In relation to all of the above embodiments, it is important to emphasize that the overall properties of the entire MSEC library can be tuned to have some degree of responsiveness to signaling pathways acting on transcription factors that bind to one or more control elements within each MSEC. As an example, it is expected that by increasing the number of MEF2 control elements within the MSEC, the responsiveness of the MSEC to external signals acting through the MEF2 signaling pathway can be increased. Thus, various MSECs can be engineered to have some degree of responsiveness to external signals as further information on how various skeletal and cardiac muscle signaling pathways act on specific transcription factors, and such synthetic MSECs can be constructed.

[0127] 30. A general embodiment of all the aforementioned MSECs is that they can be used to produce proteins, RNA products, metabolites or synthetic products derived from the expression of enzymes or synthetic proteins. These products can remain within the differentiated myofibers or can be recombinantly secreted with secretion signals to reach extracellular matrix regions and / or the circulatory system. This is evidenced by the fact that to date, more than 30 cDNAs have been successfully ligated into various MSECs. To date, cDNAs have been successfully encoded and produced, including full-length mouse dystrophin, Dp260 dystrophin, mini-dystrophin, more than 10 micro-dystrophins, micro-utrophins; hormones and cytokines such as human parathyroid hormone, human growth hormone, human placental alkaline phosphatase, mouse interleukin-10; clotting factors such as factor IX; human TDP43; and other proteins that reside and function within myofibers, such as mouse ribonucleotide reductase subunit 1, mouse ribonucleotide reductase subunit 2, mouse α-glucosidase, mouse glycogen synthase, and human liver arginase. In particular, the use of secretion signals is of interest because they allow skeletal muscle to perform biochemical functions that are normally found in the liver. Furthermore, MSECs have been used to express a variety of bacterial and invertebrate proteins, including Cas9, chloramphenicol acetyltransferase, β-galactosidase, luciferase, Renilla luciferase, green fluorescent protein, mCherry fluorescent protein, and mTmG-2a puromycin resistance fusion protein. Importantly, no cDNA ligated to MSECs to date has failed to result in muscle-specific expression of the appropriate product.

[0128] Representative Embodiments - Set 2

[0129] In some embodiments, the MSEC is based on a small sequence segment within the region from -3,300 to +7 relative to the transcription start site (TSS) of the native mouse muscle creatine kinase (CKM) gene sequence and a small sequence segment within the intron 1 region from +740 to +1721 of the native mouse CKM gene sequence.

[0130] In some embodiments, the MSEC is based on a 1,260 bp 5'-end portion of the -1,256 to +7 region of the native mouse CKM gene, which includes the 5'-enhancer and "proximal" promoter regions.

[0131] In some embodiments, the 5'-enhancer is brought into close proximity to the "proximal" promoter region by removing the MSEC-internal portion of the region from -1256 to +7 of the native mouse CKM gene.

[0132] In some embodiments, the 206 bp 5'-enhancer region (-1256 to -1050) is directly ligated to proximal promoter regions of various lengths, such as the 776 to +7 region, the 356 to +7 region, the 220 bp portion after removal of multiple internal sequences from within the 356 to +7 promoter region, the -117 to +7 region, or the -80 to +7 region.

[0133] In some embodiments, the 5'-enhancer region of the MSEC is positioned in a reverse orientation relative to the promoter fragment.

[0134] In some embodiments, the 5'-enhancer region of the MSEC is positioned at various linear distances from the promoter.

[0135] In some embodiments, the native or engineered intron 1 enhancer of the creatine kinase gene is added to the MSEC.

[0136] In some embodiments, MSECs can be used in combination with multiple recombinant miRNA target sites to silence expression in selected muscle or other tissue types.

[0137] In some embodiments, the MSEC is engineered to change the number of nucleotides between the functional regulatory elements.

[0138] In some embodiments, the MSEC is engineered to insert additional regulatory elements.

[0139] In some embodiments, the MSEC is engineered to remove one or more regulatory elements.

[0140] In some embodiments, MSEC is engineered by deleting a particular control element sequence and then inserting a different control element sequence.

[0141] In some embodiments, the MSEC is recombined by rearrangement of the linear order of regulatory elements within the enhancer and promoter regions of the MSEC.

[0142] In some embodiments, the 3'-promoter region of the MSEC is ligated to separate portions of CKM exon 1 to provide MSECs that are differentially expressed in skeletal and cardiac muscle.

[0143] In some embodiments, MSECs that contain a recombinant enhancer or promoter from one or more muscle genes further contain one or more regulatory elements identified in an enhancer or promoter of another muscle gene.

[0144] For example, in some embodiments, the MSEC contains an inserted N-box regulatory element that enhances transcriptional activity in myonuclei at neuromuscular synaptic regions and decreases transcriptional activity in non-synaptic regions.

[0145] In some embodiments, the MSEC contains a TATA box having a sequence different from that of the native gene for the purpose of altering the transcriptional activity of the MSEC without affecting reception by the MSEC of transcriptional signals that act on all other regulatory elements.

[0146] In some embodiments, the MSEC contains a TATA box that is altered in its relative position to the transcription start site, thereby altering the efficiency of transcription initiation, thereby altering the overall transcriptional activity of the MSEC and the amount of gene product produced.

[0147] In some embodiments, the MSEC contains a recombinant portion of the enhancer and / or promoter sequence of another mouse or human skeletal or cardiac muscle gene, such as the human alpha myosin heavy chain gene (hMYH6) or the human cardiac troponin T gene (hTNNT2).

[0148] In some embodiments, MSEC contains regulatory elements that respond to hormones and vitamins.

[0149] In the various types of MSECs mentioned above, any embodiment of recombination of MSECs engineered from CKM components can also be applied.

[0150] In some embodiments, the MSEC comprises a truncated human TNNT2 enhancer region (MSEC-130).

[0151] The present disclosure describes MSECs that exhibit similar, high, medium or low transcript expression in all types of muscle fibers; MSECs that exhibit fast, medium, slow or "off" transcription rates depending on muscle fiber type (e.g., fast transcription rates in type I, medium transcription rates in type IIa, and slow or "off" transcription in type IIx); and MSECs that exhibit any combination of such activity levels. Similar transcription factor changes result in differential gene expression levels in atrial, ventricular and conduction system cardiomyocytes of the heart, and graded increases in gene product expression in various types of skeletal and cardiac muscle. Also, by varying the enhancers and promoters of muscle genes, and the types of control elements contained in these enhancers and promoters; their sequences; and the linear order of the control elements, the spacing between the control elements, and the adjacent control elements, synthetic MSECs that respond differently to a wide variety of physiological signals associated with different muscle types and changes in workload could be generated to obtain the various MSEC transcriptional activities seen in the MSEC libraries disclosed herein. Furthermore, MSECs could be miniaturized by deleting DNA sequences between the enhancers and promoters, or deleting DNA sequences between the control elements, with the aim of increasing the length of cDNA that can be efficiently packaged into adeno-associated viruses and other types of vectors. As described below, each therapeutic gene product has unique properties, and pathological differences between various diseases result in various optimal MSECs for various therapeutic goals.

[0152] The relevant experimental methods are described in various publications by Stephen D. Hauschka. If not specified in detail, these experimental methods are always based on standard cell molecular biology, biochemical and immunological protocols used at a particular time.

[0153] MSEC Design and Construction: The design of MSEC sequences was based on partial genomic sequences from various skeletal and cardiac muscle genes, where basic regulatory regions had already been identified. The designed native sequences were used to test for muscle-specific expression in skeletal and cardiac muscle cultures as well as in non-muscle cultures. Sequences that showed muscle-specific transcriptional activity were then used to perform a miniaturization protocol with iterative deletions to reduce the size of the MSEC sequences. The miniaturized enhancer and promoter regions, combined to yield a large number of different gene identities, were then ligated to generate a variety of MSECs containing components from different muscle genes. In all iterative steps, the MSECs were sequenced by standard protocols and the determined sequences were entered into the MSEC sequence library (Figure 27).

[0154] In vitro MSEC assay: The transcriptional activity of MSECs was tested by performing transient transfection assays using standard transfection techniques. Skeletal muscle cultures were performed using the MM14 mouse myoblast cell line as a representative skeletal muscle cell, and cardiac muscle cultures were performed using neonatal rat cardiomyocytes as a representative cardiac muscle cell (PMID: 8474439). The protocol with data correction for different transfection efficiencies and different differentiation stages is shown in Figure 4. In another in vitro skeletal muscle MSEC assay, isolated mouse flexor digitorum brevis (FDB) muscle fibers were transfected by electroporation. MSEC expression was also tested in various cell types other than muscle cells (Figure 25), and reporter gene expression was measured using the same method as used for skeletal muscle and cardiac muscle cultures, except that the cultured cells were transiently transduced using standard methods and activity was not corrected for muscle differentiation stage.

[0155] Quantification of in vitro data: All comparisons of MSECs were performed by transfecting one type of MSEC into four or more culture dishes and calculating the average expression level of the reporter gene. MSEC-1263a (containing the genomic region from -1263 to +7 of the native mouse CKM gene) was included in all experiments, its average activity level was considered as 1.0, and used as a normalization standard for the expression levels of all MSECs tested in the same experiment. Based on preliminary data, the analysis of MSECs was repeated one or more times, and the normalized mean data values ​​obtained from each replicate were averaged. Although it was not possible to compare hundreds of types of MSECs in a single experiment, such a method provided a standard for comparing the transcriptional activities of all MSECs.

[0156] In vivo MSEC assay: The in vivo transcriptional activity of MSECs was tested in normal and dystrophic mice using transgenic mice, injection of vectors into muscle, and systemic vector delivery protocols. Standard protocols were used in all studies and have been described in various publications by Stephen D. Hauschka. Prior to systemic injection, recipient mice were weighed and vectors were delivered at the same number of vector genomes per kg body weight to all mice in each experimental group. Typically, AAV6 was used as the vector, but other AAV serotypes were also tested and found to show similar levels of transcriptional activity in MSECs with different AAV serotypes, although the relative transduction efficiency of individual AAV serotypes varied depending on the tissue type.

[0157] Vector production and quantification: All vectors were produced and titered using standard procedures at the Vector Core of the Wellstone Center at the University of Washington.

[0158] promoter Preferred promoters for use in MSECs are provided in the MSEC library disclosed herein (e.g., promoters contained within each MSEC in FIG. 27). However, the disclosure is not limited to these preferred promoters. MSECs or MSEC-derived components, whether combined with virally derived promoter components, artificial promoter components, or other gene promoter components from virtually any vertebrate, can express gene products at desired levels in not only striated muscle, but all other tissues or specific other tissues, e.g., striated and smooth muscle, liver, connective tissue, etc., and in subsets of muscle tissue cells such as satellite cells where standard MSECs are inactive.

[0159] As described herein, certain embodiments utilize promoter sequences contained within MSECs depicted in the library depicted in FIG. 27. Certain examples also utilize basal or proximal promoters from CKM, other muscle genes such as ACTA1 and TNNI1, or non-muscle genes such as thymidine kinase. Promoters selected for use within MSECs may contain TATA box mutations (FIGS. 19 and 20) and / or have multiple N boxes (FIG. 21) ligated. Examples of TATA box mutations that affect transcriptional activity are described in PMID:2342467 and PMID:10617571 and FIG. 20.

[0160] A transcription product of a coding sequence is selectively expressed in a target cell when a gene is selectively expressed in a target muscle cell but is not substantially expressed in a non-target cell. In certain embodiments, selective expression is more than 50% expression compared to a reference specific type of cell; more than 60% expression compared to a reference specific type of cell; more than 70% expression compared to a reference specific type of cell; more than 80% expression compared to a reference specific type of cell; or more than 90% expression compared to a reference specific type of cell. In certain embodiments, the reference specific type of cell refers to a non-muscle cell or a non-target muscle cell. In certain embodiments, the reference specific type of cell is present in an anatomical structure adjacent to the anatomical structure containing the target muscle cell.

[0161] In certain embodiments, the transcription product of the coding sequence may be under-expressed in unselected cells and / or in a reference cell type, for example, less than 1% or 1%, 2%, 3%, 5%, 10%, 15% or 20% of the expression of the transcript in the target cell. In certain embodiments, the targeted muscle cell is the only cell type capable of expressing the correct combination of transcription factors that can bind to the enhancers disclosed herein and induce gene expression. Thus, in certain embodiments, expression occurs exclusively in the targeted cell type.

[0162] In certain instances, CK7 enhancers (e.g., MSEC-770) and CK8 enhancers (e.g., MSEC-438a) induce preferential expression in skeletal and cardiac muscle compared to non-muscle cells. In another instance, a cTnT enhancer (e.g., MSEC-455a) induces preferential expression in cardiac muscle compared to skeletal and other non-muscle cells.

[0163] Further exemplary enhancers may be derived from slow-twitch gene enhancers (TNNC1, TNNT1, TNNI1 and the slow-twitch intronic enhancer (SIE) of CKM), fast-twitch gene enhancers (TNNI2 and TNNC2), or from genes thought to be expressed at similar levels in all skeletal muscle fibers (e.g., ACTA1).

[0164] In the present disclosure, the enhancer can be multimerized. In certain embodiments, the active segment of the enhancer can be multimerized. The multimerized enhancer can include 2 copies, 3 copies, 4 copies, 5 copies, 6 copies, 7 copies, 8 copies, 9 copies or 10 copies of the enhancer or its active segment.

[0165] Genes and gene products Nucleic acid sequences (e.g., cDNA) encoding the protein or RNA of interest can be prepared and assembled into complete coding sequences by standard molecular cloning techniques (genomic library screening, polymerase chain reaction (PCR), primer-assisted ligation, yeast or bacterial libraries, site-directed mutagenesis, etc.). The resulting coding region can be inserted into an expression vector as described herein.

[0166] "Gene" refers to a nucleic acid sequence that codes for a protein or RNA of interest (the term is used interchangeably with "polynucleotide" or "nucleotide sequence"). The definition of the term includes various sequence polymorphisms, mutations and / or sequence variants, where such changes do not substantially affect the function of the encoded gene product. The term "gene" may also include regulatory regions, such as promoters, enhancers, and termination regions, in addition to the coding sequence. Furthermore, the term may include any introns and other DNA sequences spliced ​​from the mRNA transcript, as well as variants resulting from alternative splice sites. These sequences may further include sequences that may be introduced to confer codon preference in a particular type of cell, or degenerate codons of the native sequence. Codon-optimized gene sequences may also be used.

[0167] "Encode" refers to the property that a particular nucleotide sequence within a gene, such as complementary DNA (cDNA) or messenger RNA (mRNA), serves as a template for the synthesis of another macromolecule, such as a defined amino acid sequence. Thus, a particular gene encodes a protein when its corresponding mRNA is transcribed and translated to produce the protein in a cell or other biological system. A "gene sequence encoding a protein" includes any degenerate nucleotide sequence that encodes the same amino acid sequence or an amino acid sequence having substantially similar form and function.

[0168] Certain embodiments of MSECs are used to selectively express proteins or RNAs. Representative proteins include dystrophin; actin (skeletal or cardiac); kinases; phosphatases (e.g., P13P phosphatase (myotubularin)); proteases (e.g., calpain 3); reporter proteins such as luciferase, alkaline phosphatase, chloramphenicol acetyltransferase, GFP, mCherry; nucleases for gene editing (e.g., Cas9 and Cpf1); hormones; cytokines; extracellular matrix proteins; enzymes; antibodies; viral antigens for vaccines; and clotting factors; as well as small forms of such proteins. Representative RNAs include guide RNAs for gene editing (e.g., CRISPR RNA), microRNAs, and long non-coding RNAs.

[0169] Further exemplary expression products include dystrophin (e.g., full length mouse dystrophin, Dp260 dystrophin, mini-dystrophin and micro-dystrophin, as well as dystrophin fragments containing sequences for assembling inteins), human liver arginase, human parathyroid hormone, human growth hormone, human placental alkaline phosphatase, coagulation factor IX, human TDP43, mouse ribonucleotide reductase subunit 1, mouse ribonucleotide reductase subunit 2, mouse interleukin 10, mouse α-glucosidase, mouse glycogen synthase, bacterial Cas / 9, bacterial chloramphenicol acetyltransferase, bacterial β-galactosidase, bacterial luciferase, bacterial Renilla, green fluorescent protein, mCherry fluorescent protein, and mTmG-2a puromycin resistance fusion protein.

[0170] MicroRNA target sites Alternatively, the expression level of gene products is controlled by various microRNAs (miRNAs) expressed in proliferative skeletal myoblasts, differentiated fibers, and differentiated cardiomyocytes. In general, miRNAs affect the amount of a particular mRNA translated into protein by increasing the rate of degradation of the mRNA to which the miRNA binds. The selectivity of mRNA degradation is determined by the qualitative and concentration differences of miRNAs produced by each type of cell, and also by the binding affinity of the miRNA to various miR target site (miRTS) sequences that are present in the mRNA of various muscle genes, which are slightly different. In light of this, by inserting a variable number of miRNA target sites (miRTs) with low to high binding affinity into the non-coding region of the cDNA of the desired gene product, the expression level of the gene product in a specific muscle type can be regulated with even greater precision than transcriptional regulation alone. Thus, even finer adjustment of the expression level of gene products among various muscle types can be achieved by selecting the appropriate MSECs and miRTSs for the treatment of each disease. As an example, microRNA208a can reduce the expression level of a gene product in cardiomyocytes. Further examples of relevant microRNA target sites are listed on page 1 of FIG. 27 and in FIGS. 17 and 18.

[0171] Barcode In certain instances, MSECs may include or encode barcodes linked to cDNA. Barcodes linked to MSECs are typically used to track the location of specific MSECs after transfection of the MSECs in research studies. Barcodes linked to cDNAs are also typically used to compare the transcriptional activity of two or more MSECs ligated to cDNAs labeled with different barcodes. The transcriptional activity of each MSEC is then determined by administering a mixture of multiple AAVs or other vectors containing various cDNAs indirectly labeled with barcodes to a cell culture or tissue, followed by next-generation sequencing of the various mRNAs produced by the various types of MSECs.

[0172] Barcodes are well known to those skilled in the art. In certain embodiments, barcodes refer to DNA sequences that are used to identify MSECs. In certain embodiments, such barcodes can be designed to be unique. In certain embodiments, DNA barcodes can include standardized short DNA sequences. See, for example, Kress and Erickson, Proc. Natl. Acad. Sci. USA, 105(8): 2761-2762; Savolainen et al., Trans R Soc London Ser B. 2005; 360:1805-1811.

[0173] In a particular example, different types of MSECs include or code for different types of barcodes. In another example, MSECs are grouped based on common characteristics, and MSECs in each group have a common barcode. One representative common characteristic is that enhancers are the same. In this example, MSECs with the same enhancer have a common barcode, while MSECs with different enhancers have different barcodes.

[0174] Barcodes of various lengths can be used. Typically, the longer the sequence, the more barcodes and the different types of barcodes can be incorporated. In certain examples, the barcoded MSECs may all have the same barcode length (even if the MSECs have different sequences), but different barcode lengths can be used in different types of MSECs. The barcode sequence may be at least 2 nucleotides long, at least 4 nucleotides long, at least 6 nucleotides long, at least 8 nucleotides long, at least 10 nucleotides long, at least 12 nucleotides long, at least 15 nucleotides long, at least 20 nucleotides long or longer (e.g., 3-6 nucleotides long). In certain embodiments, the length of the barcode sequence may be up to 20 nucleotides long, up to 15 nucleotides long, up to 12 nucleotides long, up to 10 nucleotides long, up to 8 nucleotides long, up to 6 nucleotides long, up to 4 nucleotides long or less. In certain embodiments, the length of the barcode sequence may be 4-36 nucleotides long, 6-30 nucleotides long, or 8-20 nucleotides long. Barcode sequences are described, for example, in U.S. Patent Publication No. 5,635,400; Brenner et al., Proc. Natl. Acad. Sci., 97:1665-1670, 2000; Shoemaker et al., Nature Genetics 14: 450-456, 1996; European Patent Publication No. 0799897; U.S. Patent Publication No. 5,981,179; U.S. Patent Publication No. 20140342921; and U.S. Patent Publication No. 8,460,865.

[0175] Delivery of MSECs In certain embodiments, the MSEC ligated to a cDNA encoding a protein or RNA of interest can be incorporated into a vector and introduced into a cell. A "vector" is a nucleic acid molecule capable of transporting another nucleic acid.

[0176] Various types of viral vectors are available and have been developed for gene therapy delivery (PMID: 29883422). Any of the MSECs described herein are suitable for incorporation into and delivery using one or more of such viruses, with the only limitations being the size of the genome and the size of the cDNA of interest that each vector can package. Such MSEC properties are not only available for currently available viral vectors, but also for any newly developed viral vectors. A specific advantage of the MSEC technology of the present invention is that small muscle-specific MSECs, several hundred bp long, can be used for viruses with small package sizes that have limited packaging capacity, while larger MSECs are compatible with vectors with large packaging capacity. Furthermore, the latter type of vector has the advantage of being able to accommodate even larger cDNAs. This means that full-length cDNAs of the largest known natural proteins, cDNAs encoding multiple proteins and / or RNAs, and even very large artificial proteins can be packaged and expressed at appropriate expression levels using various types of MSECs and delivery vectors.

[0177] In another embodiment, the MSEC-cDNA construct can be delivered by various formulations consisting of "naked DNA" (e.g., Froehner, March 2021 MDA Clinical & Scientific Conference: Non-viral Delivery in Neuromuscular Disease) or by artificial lipid and / or protein nanoparticles encapsulating the MSEC-cDNA that contain various modifications, such as tissue-specific ligands or antibodies, to enhance binding to and uptake by muscle cells and / or promote transport of the internalized MSEC-cDNA complex to the cell nucleus (PMID:30186185).

[0178] Additionally, MSECs can be used to express protein or RNA components required for activation or repression of gene expression from targeted loci or genome modification.

[0179] composition The vectors described herein can be formulated into compositions for administration to a subject, the compositions comprising a therapeutically effective amount of MSEC (e.g., in the form of a vector) and a pharma- ceutically acceptable carrier.

[0180] Representative pharma- ceutically acceptable carriers in common use include any absorption retarders, antioxidants, binders, buffers, bulking or fillers, chelating agents, coatings, disintegrants, dispersion media, gels, isotonicity agents, and lubricants.

[0181] "Prophylactic treatment" includes treatment administered to a subject who does not exhibit signs or symptoms of a muscle-related disease, or who exhibits only early signs or symptoms of a muscle-related disease, with the intent of reducing or decreasing the risk of the muscle-related disease developing further. Thus, prophylactic treatment serves as treatment to prevent a muscle-related disease. In certain embodiments, prophylactic treatment inhibits, slows, or prevents the progression of a muscle-related disease.

[0182] "Therapeutic treatment" includes treatment administered to a subject exhibiting symptoms or signs of a muscle-related disease, and is administered to a subject with the intent of reducing or eliminating the signs or symptoms of the muscle-related disease. Therapeutic treatment can prevent, control, or eliminate the presence or activity of the muscle-related disease, and / or can prevent, control, or eliminate the side effects of the muscle-related disease.

[0183] Functioning as a prophylactic or therapeutic treatment is not mutually exclusive, and in certain embodiments, an administered dose may serve more than one purpose.

[0184] Skeletal and cardiac muscles are affected by hundreds of genetic diseases and can undergo various types of physical damage, leading to a gradual decline in their function due to disuse and aging. Furthermore, skeletal muscles undergo exhaustive catabolic degradation associated with cancer. Since all skeletal muscle fibers are innervated and the function of the myocyte synaptic area, where muscle contraction is stimulated by neuronal axons, depends on nerve interactions, muscle cells also exhibit various neuromuscular junction (NMJ) disorders. Furthermore, muscle plays a key role in the normal function of neurons innervating skeletal muscle fibers, since their maintenance depends in part on muscle-mediated signals. MSECs can play an important role in therapeutic strategies to combat all of these medical challenges and similar challenges in the veterinary field.

[0185] Age-related sarcopenia, cancer cachexia, and spinal cord injury tend to affect type II fibers over type I fibers. Duchenne muscular dystrophy (DMD) and facioscapulohumeral muscular dystrophy (FSHD) also frequently affect type II fibers, but FKRP-mediated dystroglycanopathies (MDDGA5, MDDGB5, and MDDGC5) result in a relative increase in type I fibers, possibly due to a gradual conversion of type II fibers to type I fibers. In contrast, myotonic dystrophies and some types of limb-girdle muscular dystrophies (e.g., calpain 3 deficiency-induced limb-girdle muscular dystrophy type 2A) are associated with a decrease in type I fibers. Neuromuscular junction (NMJ) diseases also result in a conversion of skeletal muscle fiber types. For example, infants with the most severe forms of spinal muscular atrophy (SMA) have a very low number of type II fibers, with a concomitant increase in type I fibers. In patients with advanced amyotrophic lateral sclerosis (ALS), type II fibers are converted to type I fibers. Some striated muscle diseases, such as Duchenne muscular dystrophy (DMD), affect both skeletal and cardiac muscles, whereas other striated muscle diseases affect primarily skeletal or cardiac muscles, while some cardiac diseases affect most prominently the ventricles, atria, or conduction system. These disease-specific changes in muscle of various types highlight the importance of MSECs, which can be optimized for various diseases in order to target gene therapy to the most affected muscle and fiber types.

[0186] Specific muscle-related diseases that can be treated by MSECs include myocardial diseases (e.g., hypertrophic cardiomyopathy), and striated muscle diseases, including dystrophies and dystroglycanopathies. Dystrophies include muscular dystrophies and myotonic dystrophies. Examples of muscular dystrophies include limb-girdle muscular dystrophy (LGMD), calpain 3 deficiency limb-girdle muscular dystrophy type 2A (LGMD2A), myotubular myopathy (MTM1), ACTA1 LGMD, Duchenne muscular dystrophy (DMD) and facioscapulohumeral muscular dystrophy (FSHD). Examples of dystroglycanopathies include MDC1A, MDDGA5, MDDGB5, MDDGC5 and MDDGC14 (GMPPB diseases). Neuromuscular diseases (NMD) and neuromuscular junction diseases (NMJ) can also be treated. These diseases include spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), myasthenic neuromuscular diseases (e.g., congenital myasthenia (congenital myasthenia affecting acetylcholine receptor subunits (CHRNA1; CHRNB1; CHRND; CHRNE), COLQ, or DOK7), limb-girdle muscular dystrophy type 2A (LGMD2A), and myotubular myopathy (MTM1). Additional examples of diseases that can be treated with MSEC include amyopathies, nemaline myopathies (e.g., nemaline myopathy type 2), myofibrillar myopathy type 5, Miyoshi myopathy, scapuloperoneal myopathy, X-linked myotubular myopathy, central core disease, paramyotonia, Pompe disease, cancer cachexia, and diseases of aging (age-related sarcopenia), as well as other diseases or disorders as described elsewhere herein.

[0187] "Muscle" refers to structures composed of myoblasts, myotubes, myofibers, stem cells that can give rise to myoblasts, and the proteins that support these structures. Muscle includes skeletal muscle, cardiac muscle, and smooth muscle.

[0188] "Muscle injury" refers to a condition in which a muscle does not function normally. Muscle injury can occur when a muscle is subjected to excessive force, causing the muscle fibers to become compressed, inflamed, and eventually rupture. Muscle injury can also occur when a muscle is stretched beyond its capacity, or when a muscle is asked to contract forcefully and quickly.

[0189] "Muscle atrophy" and "sarcopenia" refer to conditions caused by disuse of muscles, e.g., lack of physical activity. For example, subjects with conditions that limit mobility can lose muscle tone and develop atrophy.

[0190] "Muscular strength" refers to the amount of force a muscle can exert through maximal effort.

[0191] "Cancer-associated cachexia" and "infection-induced cachexia" refer to persistent loss of skeletal muscle mass that cannot be restored by normal nutritional support and leads to gradual functional impairment. Cachexia caused by cancer is termed "cancer-associated cachexia," and cachexia induced by infection is defined as "infection-induced cachexia."

[0192] "Aging" refers to a physiological process involving a gradual decline in function or the gradual decline of physiological function with age.

[0193] "Cardiovascular disease" refers to diseases of the circulatory system, including the heart and blood vessels. The four main types of cardiovascular disease are coronary heart disease, stroke, peripheral artery disease, and aortic disease.

[0194] "Regeneration" refers to the repair of cells, tissues, or organs. In this disclosure, "regeneration" refers to the repair of myoblasts, muscle fibers, and the muscle environment, which refers to an environment that can provide an optimal environment for the development of muscle fibers.

[0195] As previously mentioned, the most immediate use of MSECs is in the development of treatments for muscle-related disorders. However, MSECs can be used in a number of other applications, including, but not limited to, in the development of treatments for a variety of disorders in which skeletal muscle tissue can be utilized for the production of beneficial secretory products, such as hormones, clotting factors, antibodies, and other beneficial proteins or metabolites.

[0196] Representative embodiment - set 3 1. A muscle-specific artificial expression cassette (MSEC), wherein a first coding sequence is selectively expressed in muscle cells contained in a heterogeneous cell population when the MSEC is administered to the heterogeneous cell population. 2. The MSEC of embodiment 1, as disclosed herein. 3. The MSEC of embodiment 1, comprising a promoter. 4. The MSEC of embodiment 2, wherein the promoter is a CKM proximal promoter, a TNNT2 promoter, a TNNT1 promoter, a thymidine kinase promoter, an SV40 promoter, or a CMV promoter. 5. The MSEC of embodiment 3 or 4, wherein the promoter is a truncated promoter. 6. The MSEC of any one of embodiments 3 to 5, wherein the promoter is a multimerized promoter. 7. The MSEC of embodiment 5, wherein the multimerized promoter has 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the promoter and / or the compacted promoter. 8. The MSEC of any one of embodiments 3 to 7, wherein the promoter comprises a TATA box mutation. 9. The MSEC of any one of embodiments 3 to 8, wherein an N-box is ligated to the promoter. 10. The MSEC of any one of embodiments 1 to 9, further comprising an enhancer. 11. The MSEC of embodiment 10, wherein the enhancer is a CKM intron 1 MR1 enhancer, a TNNT2 enhancer, a TNNT1 enhancer, a mouse CKM 5' enhancer, or an alpha myosin heavy chain enhancer. 12. The MSEC of embodiment 10 or 11, wherein the enhancer is a truncated enhancer. 13. The MSEC of embodiment 12, wherein said truncated enhancer is a truncated CKM intron 1 MR1 enhancer. 14. The MSEC of embodiment 12, wherein the truncated enhancer comprises the region from +904 to +998 of the native mouse CKM gene. 15. The MSEC of any one of embodiments 10 to 14, wherein the enhancer is a multimerized enhancer. 16. The MSEC of embodiment 15, wherein the multimerized enhancer has 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the enhancer and / or the compact enhancer. 17. The MSEC of any one of embodiments 1-16, wherein the sequence encoding the first nucleotide comprises a cDNA. 18. The MSEC of any one of embodiments 1-16, wherein the first nucleotide coding sequence or cDNA codes for a protein or RNA. 19. The MSEC of any one of embodiments 1 to 18, further comprising a sequence encoding a second nucleotide. 20. The MSEC of embodiment 19, wherein the second nucleotide coding sequence codes for a target site of a microRNA. 21. The MSEC of embodiment 20, wherein the second nucleotide coding sequence encodes 1 copy, 2 copies, 3 copies, 4 copies, 5 copies, 6 copies, 7 copies, 8 copies, 9 copies or 10 copies of the microRNA target site. 22. The MSEC of embodiment 20 or 21, wherein the microRNA target site reduces expression of the sequence encoding the first nucleotide in cardiac or skeletal muscle. 23. The MSEC of any one of embodiments 20 to 22, wherein the target site of the microRNA is a target site of a microRNA disclosed herein. 24. The MSEC of any one of embodiments 16 to 23, wherein the second nucleotide coding sequence encodes at least two microRNA target sites. 25. A sequence set forth in any one of SEQ ID NOs: 1 to 372, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1 to 372. 26. The MSEC or sequence of any one of embodiments 1-25, formulated for administration to a subject. 27. The MSEC of any one of embodiments 1 to 26, incorporated into a vector for delivery to a subject. 28. The MSEC of embodiment 27, wherein the delivery vector is a viral vector. 29. The MSEC of embodiment 28, wherein the viral vector is an adeno-associated viral (AAV) vector. 30. Use of an MSEC or sequence according to any one of embodiments 1 to 29 for inducing selective gene expression in muscle cells. 31. The use according to embodiment 30, wherein the muscle cells are striated, skeletal or cardiac muscles. 32. The use according to embodiment 30, wherein the muscle is a skeletal muscle or a cardiac muscle. 33. The use according to embodiment 30 for the development of a treatment for muscle-related diseases.

[0197] The headings of this disclosure are provided for organizational purposes only and do not limit the scope or interpretation of the disclosure.

[0198] The nucleic acid and / or amino acid sequences described herein are shown using standard abbreviations as defined in 37 CFR 1.822. In certain instances, only one strand for each nucleic acid sequence is shown, although the complementary strand, where appropriate, is also included in the embodiment.

[0199] Unless otherwise stated, the present disclosure can be carried out using conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA. These methods are described in the following publications. See, for example, Sambrook, et al. Molecular Cloning: A Laboratory Manual, 2nd Edition (1989); FM Ausubel, et al. eds., Current Protocols in Molecular Biology, (1987); the series Methods IN Enzymology (Academic Press, Inc.); M. MacPherson, et al., PCR: A Practical Approach, IRL Press at Oxford University Press (1991); MacPherson et al., eds. PCR 2: Practical Approach, (1995); Harlow and Lane, eds. Antibodies, A Laboratory Manual, (1988); and RI Freshney, ed. Animal Cell Culture (1987).

[0200] As will be appreciated by those of skill in the art, each embodiment disclosed herein comprises, consists essentially of, or consists of the particular components, steps, materials, or ingredients described. Thus, the terms "comprise" or "comprising" should be interpreted to mean "comprise, consist essentially of, or consist of." The transitional phrase "comprises" means, but is not limited to, the inclusion of any unrecited components, steps, materials, or ingredients, even if the amount is greater. The transitional phrase "consists of" excludes any unrecited components, steps, materials, or ingredients. The transitional phrase "consists essentially of" limits the scope of the embodiment to the recited components, steps, materials, or ingredients and those components, steps, materials, or ingredients that do not have a significant effect on the embodiment. A significant effect refers to an effect that statistically significantly reduces the ability to obtain the claimed effect according to the relevant experimental methods described in this disclosure.

[0201] Unless otherwise indicated, all numerical values ​​expressing quantities or properties of materials, such as molecular weight and reaction conditions, in the specification and claims are to be construed in all instances as modified by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. Without intending to limit the scope of the doctrine of equivalents to the scope of the claims, each numerical parameter should, at the very least, be construed in light of the number of reported significant digits and by applying ordinary rounding procedures. For clarity, the term "about," when used in conjunction with a stated value or range, has a meaning that would be reasonably interpreted by one of ordinary skill in the art, i.e., within ±20% of the stated value; within ±19% of the stated value; within ±18% of the stated value; within ±17% of the stated value; within ±16% of the stated value; within ±15% of the stated value; within ±14% of the stated value; within ±13% of the stated value; within ±12% of the stated value; within ±11% of the stated value; within ±10% of the stated value; within ±9% of the stated value; within ±8% of the stated value; within ±7% of the stated value; within ±6% of the stated value; within ±5% of the stated value; within ±4% of the stated value; within ±3% of the stated value; within ±2% of the stated value; or within ±1% of the stated value.

[0202] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations and approximate ranges, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain certain errors necessarily resulting from the standard deviation associated with their respective testing measurements.

[0203] In the description of the present invention (particularly in the description of the claims below), the terms "a", "an", "the" and similar modifiers are intended to include both the singular and the plural unless otherwise indicated or the context clearly indicates otherwise. Numerical ranges described herein are intended to be a shorthand way of referring to each numerical value falling within the range individually. Unless otherwise indicated, each numerical value is described herein as if it were individually described herein. Any method described herein can be performed in any suitable order unless otherwise indicated or the context clearly indicates otherwise. The use of any examples or language of examples (e.g., "etc.") provided herein is intended to be for the purpose of illustrating the invention only and does not limit the scope of the invention as described in the claims. No term described herein should be construed as indicating any non-claimed element essential to the practice of the invention.

[0204] Groupings of other elements of the invention disclosed herein or of various embodiments of the invention should not be construed as limiting the invention. Members of each group may be described herein or in the claims individually or in combination with other members of the group or other elements described herein. It is anticipated that for reasons of convenience and / or patentability, one or more members of a group may be added to another group, or one or more members may be deleted from a group. When such additions or deletions are made, the specification includes groups that are constructed to satisfy the recitation of all Markush groups set forth in the appended claims.

[0205] Specific embodiments of the present invention are described herein, including those embodiments known to the inventors to be the best mode for carrying out the invention. Of course, those skilled in the art will readily appreciate that the embodiments described herein may be modified in various ways upon review of the above detailed description. The inventors anticipate that such modifications may be adopted by those skilled in the art, and intend that the present invention may be practiced in other ways than as specifically described herein. Accordingly, the present invention includes all modifications of the subject matter recited in the appended claims and all equivalents of the subject matter of the present invention to the extent permitted within the scope of applicable law. Moreover, the present invention includes all combinations of the above-described elements in any and all variations thereof, unless otherwise indicated or the context clearly dictates otherwise.

[0206] Additionally, throughout this specification, various patents, publications, journal articles and other documents are cited (references herein). Each reference cited herein is individually incorporated herein by reference for the teachings thereof as if it were a part of this specification.

[0207] Finally, the embodiments of the invention disclosed herein are to be considered as illustrative of the principles of the invention. Other modifications may be adopted within the scope of the invention. Thus, by way of example, but not by way of limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Thus, the invention is not to be limited to what has been precisely shown and described herein.

[0208] The details described herein are by way of example and are presented solely for the purpose of illustrating preferred embodiments of the present invention, to provide what is believed to be the most useful, and to facilitate an understanding of the principles and conceptual aspects of various embodiments of the present invention. In this regard, no structural details of the present invention are described in more detail than is necessary for a basic understanding of the present invention, and those skilled in the art will be able to easily understand how to actually embody some forms of the present invention by reading the description of the present invention in conjunction with the drawings and / or examples.

[0209] The definitions and explanations used in this disclosure are intended to control future interpretations, unless clear and unambiguous changes are made in the examples, or the meaning of the terms makes the interpretation meaningless or substantially meaningless. If the definition of a term does not make sense or substantially meaningless from the interpretation of the term, please refer to the definition of the term from a dictionary known to those skilled in the art, such as Webster's Dictionary (3rd Edition) or Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

Claims

1. A muscle-specific expression cassette (MSEC) having the sequence set forth in SEQ ID NO:138, SEQ ID NO:102, SEQ ID NO:197, SEQ ID NO:252, SEQ ID NO:286, SEQ ID NO:1, SEQ ID NO:258, SEQ ID NO:259, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:290, SEQ ID NO:291, SEQ ID NO:296, SEQ ID NO:297, SEQ ID NO:304, SEQ ID NO:210, SEQ ID NO:268 or SEQ ID NO:170; or An MSEC having a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:138, SEQ ID NO:102, SEQ ID NO:197, SEQ ID NO:252, SEQ ID NO:286, SEQ ID NO:1, SEQ ID NO:258, SEQ ID NO:259, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:290, SEQ ID NO:291, SEQ ID NO:296, SEQ ID NO:297, SEQ ID NO:304, SEQ ID NO:210, SEQ ID NO:268 or SEQ ID NO:

170.

2. A muscle-specific expression cassette (MSEC), comprising an enhancer and a promoter operably linked to a first coding sequence, the promoter being a truncated promoter, such that when the MSEC is administered to a heterogeneous cell population, the first coding sequence is selectively expressed in muscle cells contained in the heterogeneous cell population.

3. The MSEC according to claim 2, characterized in that it is at least one selected from the following (i) to (iv): (i) the promoter contains a TATA box mutation; (ii) an N box is ligated to the promoter; (iii) the enhancer is a CKM intron 1 MR1 enhancer, a TNNT2 enhancer, a TNNT1 enhancer, a mouse CKM 5' enhancer, an alpha myosin heavy chain enhancer, or an ACTA1 enhancer; (iv) The enhancer is a miniaturized enhancer.

4. The MSEC of claim 2 , wherein the miniaturized promoter is multimerized.

5. The MSEC of claim 4, wherein the multimerized deflated promoter has 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the deflated promoter.

6. The MSEC of claim 2 , wherein the enhancer is a truncated enhancer, the truncated enhancer being a truncated CKM intron 1 MR1 enhancer.

7. The MSEC of claim 2 , formulated for administration to a subject or incorporated into a vector for delivery to a subject.

8. The MSEC of claim 7 , wherein the delivery vector is a viral vector.

9. The MSEC of claim 8 , wherein the viral vector is an adeno-associated viral (AAV) vector.

10. The MSEC of claim 2 , wherein the first coding sequence comprises a cDNA.

11. The MSEC of claim 10 , wherein the first coding sequence encodes a protein or RNA.

12. The MSEC of claim 2 further comprising a second coding sequence.

13. The MSEC of claim 12 , wherein the second coding sequence encodes a target site for a microRNA.

14. The MSEC of claim 13, wherein the second coding sequence encodes 1 copy, 2 copies, 3 copies, 4 copies, 5 copies, 6 copies, 7 copies, 8 copies, 9 copies or 10 copies of the microRNA target site.

15. The MSEC of claim 14, wherein the microRNA target site reduces expression of a first coding sequence in cardiac or skeletal muscle, or the second coding sequence encodes at least two microRNA target sites.

16. The MSEC of claim 1 for use in inducing selective gene expression in muscle cells.

17. 17. The MSEC for use according to claim 16, wherein the muscle cells are striated muscle cells, skeletal muscle cells or cardiac muscle cells.

18. 17. MSEC for use as claimed in claim 16 for the development of a treatment for a muscle-related disease.

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