Oligonucleotide modulators activate utrophin expression

Abstract

The present disclosure relates to an oligonucleotide modulator for preventing or treating dystrophin deficiency-associated disorders (DDD), including Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). The oligonucleotide modulator comprises a sense nucleic acid strand and an antisense nucleic acid strand, the sense nucleic acid strand and the antisense nucleic acid strand being independently oligonucleotide strands of 16 to 35 nucleotides in length, and one nucleotide strand has at least 75% base homology or complementarity to a target selected from the promoter region of the target gene UTRN.

Description

[Technical field]

[0001] The present disclosure relates to the field of nucleic acids, particularly with respect to oligonucleotide modulators related to gene activation and their pharmaceutical uses. [Background technology]

[0002] Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disorder that occurs in approximately 1 in every 3,500-5,000 male newborns. Approximately 20,000 children worldwide are diagnosed with DMD each year. DMD patients die early in their 20s-40s. The disease is caused by mutations in the DMD gene, which codes for the dystrophin protein, which is critical in muscle fibers; when it is missing, muscle cells are destroyed and gradually lost, causing muscle weakness to worsen over time. Approximately one-third of children develop DMD as a result of a spontaneous mutation in the dystrophin gene and have no family history of the disease.

[0003] Dystrophin is part of the dystrophin glycoprotein complex (DGC), which bridges the intracellular cytoskeleton (F-actin) with the extracellular matrix, thus providing stability to contracting muscle fibers. Becker muscular dystrophy (BMD) is a milder, less progressive form of the disease, also caused by alterations in the same DMD gene.

[0004] Generally, DMD patients carry mutations that result in a non-functional, incomplete dystrophin protein (nonsense or frameshift mutations), whereas in BMD, an internally deleted protein with reduced molecular weight (derived from an in-frame deletion) is expressed and is partially functional.

[0005] The DMD gene is highly complex, containing at least seven independent tissue-specific promoters and two polyadenylation sites. Furthermore, the dystrophin transcript is alternatively spliced ​​to generate a variety of different transcripts encoding multiple protein isoforms. Dystrophin is also expressed in the brain, where its function remains unknown. However, lack of dystrophin in the brain may underlie the cognitive impairment experienced by many DMD patients.

[0006] The utrophin gene (UTRN) encodes the utrophin protein, which is structurally and functionally similar to dystrophin. Utrophin contains an actin-binding N-terminus, a triple coiled-coil repeat central region, and a C-terminus consisting of protein-protein interaction motifs that interact with dystroglycan protein components. Utrophin is located at neuromuscular synapses and muscle-tendon junctions, where it is involved in postsynaptic membrane maintenance and acetylcholine receptor clustering.

[0007] In early developing human muscle, utrophin is present in the sarcolemma and is gradually replaced by dystrophin towards birth. In adult tissues, utrophin is expressed in a wide range of tissues, including lung, kidney, liver, and spleen, with limited expression in the sarcolemma of regenerating muscle fibers and blood vessels, as well as in the neuromuscular and myotendinous junctions of muscles. Two differentially regulated utrophin promoters, A and B, have been reported to drive two distinct mRNA isoforms that are translated into full-length utrophin protein with unique N-termini and distinct expression patterns.

[0008] Unlike the dystrophin tissue-specific promoter, the utrophin A promoter, which drives genes encoding full-length proteins, is associated with a CpG island at the 5' end of the gene. Utrophin A, expressed in many tissues, is an isoform expressed in muscle at the neuromuscular and myotendinous junctions, choroid plexus, pia mater and renal glomerulus, and is found in the sarcolemma in regenerating muscle fibers. Utrophin B differs from A by a slightly different N-terminal site of action, but is restricted to endothelial cells and blood vessels. Both dystrophin and utrophin have smaller transcripts driven by similar internal promoters (Dp71, Dp140, Up71, Up140).

[0009] Utrophin is an autosomal and functional paralog of dystrophin and can correct the primary dystrophin defect in DMD and BMD. Mouse studies suggest that the utrophin gene may functionally replace the dystrophin gene and thus be a potential therapeutic target for muscular dystrophies caused by dystrophin deficiency. Thus, utrophin-based strategies may provide treatment for all patients with DMD / BMD, regardless of genetic defects.

[0010] Inducing utrophin expression at both transcriptional and post-transcriptional levels using oligonucleotides is an attractive approach to develop novel treatments for DMD and BMD, regardless of the DMD gene mutation site, but such approaches are limited by gene upregulation tools [US 20120122953 A1 and WO2019183005A1]. Summary of the Invention [Problem to be solved by the invention]

[0011] To address the aforementioned problems, the present disclosure provides oligonucleotide regulatory agents, such as small activating RNA (saRNA) molecules, to treat diseases or conditions caused by insufficient or insufficient levels of dystrophin, such as DMD and BMD, by targeting the UTRN gene promoter, then activating UTRN gene transcription, and then increasing expression levels of utrophin protein to correct the deficiency of dystrophin via an RNA activation (RNAa) mechanism. [Means for solving the problem]

[0012] In particular, the inventors have found that such SaRNAs capable of activating / upregulating the expression of UTRN mRNA are not randomly distributed on the promoter but are clustered in specific hotspot regions. Only some regions on the promoter of the UTRN gene support gene activation by saRNA, such as, for example, the regions at -636 to -496, -351 to -294, -236 to -187, and -101 to -65 upstream of the transcription start site of the UTRN gene. The inventors have also found that the optimal target sequence / sense strand of saRNA in the UTRN promoter region includes sequences with the following criteria: (1) 35% to 70% GC content, (2) less than five consecutive identical nucleotides, (3) no more than three dinucleotide repeats, and (4) no more than three trinucleotide repeats. As a beneficial result of the criteria, the target sequence (e.g., an isolated nucleic acid sequence containing the target sequence) upon interaction with saRNA can activate / upregulate the expression of UTRN mRNA by at least 10% compared to the baseline level of UTRN mRNA. Based at least in part on these discoveries, the present disclosure features saRNAs, compositions, and pharmaceutical compositions for activating / upregulating the expression of UTRN mRNA by at least 10% compared to the baseline level of the UTRN gene.Also provided herein are methods for preventing or treating diseases or conditions induced by insufficient expression of dystrophin, dystrophin gene mutations, and / or low-functional dystrophin levels in an individual, comprising administering any of the saRNAs, compositions, and / or pharmaceutical compositions described herein.

[0013] In one aspect of the disclosure, an oligonucleotide regulatory agent (such as the saRNA molecule) capable of activating / upregulating expression of the UTRN gene in a cell is provided, the oligonucleotide regulatory agent (e.g., the saRNA) consisting of an oligonucleotide sequence of 16-35 contiguous nucleotides, the contiguous oligonucleotide sequence having at least 75%, or at least 80%, or at least 85%, or at least 90% sequence homology or complementarity to an equal-length region of SEQ ID NO: 1200, thereby activating or upregulating expression of the gene by at least 10% compared to baseline expression of the UTRN gene. In some embodiments, the equal-length region of SEQ ID NO: 1200 is located upstream of the region -636 to -496 (SEQ ID NO: 1207), the region -351 to -294 (SEQ ID NO: 1208), the region -236 to -187 (SEQ ID NO: 1209), or the region -101 to -65 (SEQ ID NO: 1210) of the transcription start site of the UTRN gene.

[0014] In certain embodiments, the saRNA disclosed in this disclosure comprises a sense strand and an antisense strand, the sense strand and the antisense strand each comprising a complementary region, and the complementary regions of the sense strand and the antisense strand form a double-stranded nucleic acid structure. In certain embodiments, the sense strand and the antisense strand disclosed in this disclosure have at least 90% complementarity. In certain embodiments, the sense strand and the antisense strand disclosed in this disclosure are arranged on two different nucleic acid strands. In certain embodiments, the sense strand and the antisense strand disclosed in this disclosure are arranged on a contiguous nucleic acid strand, optionally a hairpin single-stranded nucleic acid molecule, and the complementary regions of the sense strand and the antisense strand form a double-stranded nucleic acid structure. In certain embodiments, the sense strand and the antisense strand disclosed in this disclosure comprise a 3' overhang ranging from 0 to 6 nucleotides in length, or 2 to 3 nucleotides in length. In certain embodiments, at least one of the nucleotides of the overhang is a thymine deoxyribonucleotide. In certain embodiments, the overhang is a natural overhang that is selected from or is a complementary nucleotide at a corresponding position on the DNA target. In certain embodiments, the sense strand and the antisense strand disclosed in the present disclosure independently comprise a length of about 16 to about 35, about 17 to about 30, about 18 to about 25, or about 19 to about 22 contiguous nucleotides.

[0015] In certain embodiments, the sense strand disclosed in this disclosure has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NOs: 400-797, and the antisense strand disclosed in this disclosure has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NOs: 800-1197. In certain embodiments, the sense strand disclosed in this disclosure comprises a nucleotide sequence selected from SEQ ID NOs: 400-797, and the antisense strand disclosed in this disclosure comprises a nucleotide sequence selected from SEQ ID NOs: 800-1197.

[0016] In certain embodiments, the oligonucleotide sequences disclosed in this disclosure have at least 75% sequence homology or complementarity to a nucleotide sequence selected from SEQ ID NOs: 1-398. In certain embodiments, the sense strand of the oligonucleotide sequences disclosed in this disclosure has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NOs: 1-398. In certain embodiments, the antisense strand of the oligonucleotide sequences disclosed in this disclosure has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NOs: 1-398.

[0017] In certain embodiments, at least one nucleotide of the saRNA disclosed in this disclosure is a chemically modified nucleotide.In certain embodiments, at least one nucleotide of the antisense strand and / or the sense strand disclosed in this disclosure is chemically modified.In certain embodiments, the chemically modified nucleotide disclosed in this disclosure is a nucleotide that has at least one of the following modifications: a) modification of the phosphodiester bonds linking the nucleotides in the nucleotide sequence of the saRNA; b) modification of the 2'-OH of ribose in the nucleotide sequence of the saRNA; and c) Base modifications in the nucleotide sequence of the saRNA.

[0018] In certain embodiments, at least one nucleotide of the saRNA disclosed in the present disclosure is a locked nucleic acid, a basic nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, or a non-natural base containing nucleotide.

[0019] In certain embodiments, the chemical modification of the at least one chemically modified nucleotide disclosed in the present disclosure is the addition of an (E)-vinylphosphonate moiety to the 5'-end of the sense or antisense strand.

[0020] In certain embodiments, the present disclosure provides oligonucleotide modulators, wherein the sense and / or antisense strands of the saRNA disclosed in the present disclosure are conjugated to one or more conjugation moieties selected from lipids, fatty acids, fluorophores, ligands, sugars, peptides, and antibodies.

[0021] In certain embodiments, the sense or antisense strand of the saRNA disclosed in the present disclosure is conjugated to one or more conjugation moieties selected from cell membrane penetrating peptides, polyethylene glycol, alkaloids, tryptamines, benzimidazoles, quinolones, amino acids, cholesterol, glucose, and N-acetylgalactosamine.

[0022] In certain embodiments of the oligonucleotide modulator, the sense or antisense strand of the saRNA disclosed in the present disclosure is conjugated to one or more conjugation moieties selected from cell membrane penetrating peptides, polyethylene glycol, alkaloids, tryptamines, benzimidazoles, quinolones, amino acids, cholesterol, glucose, and N-acetylgalactosamine.

[0023] In another aspect of the present disclosure, an isolated polynucleotide of saRNA is provided, the isolated polynucleotide being a contiguous nucleotide sequence having a length of 16-35 nucleotides in SEQ ID NO: 1200. Specifically, the isolated polynucleotide is a nucleic acid sequence selected from SEQ ID NOs: 1-398. In another aspect of the present disclosure, a method of using the isolated polynucleotide of saRNA is provided.

[0024] In another aspect of the present disclosure, an isolated oligonucleotide complex is provided, the isolated oligonucleotide complex comprises the antisense strand of the saRNA disclosed herein and the sense strand of the isolated polynucleotide disclosed herein.In some embodiments, the isolated oligonucleotide complex activates the expression of UTRN gene by at least 10% compared to the baseline level of the gene.

[0025] Another aspect of the present disclosure provides an isolated polynucleotide encoding the saRNA disclosed herein.In one embodiment, the saRNA disclosed herein is a small activating RNA (saRNA) molecule.In one embodiment, the polynucleotide is a DNA molecule.Another aspect of the present disclosure provides a vector comprising the isolated polynucleotide disclosed herein.

[0026] In another aspect of the present disclosure, an isolated nucleic acid complex is provided, the isolated nucleic acid complex comprises the antisense strand of the saRNA disclosed herein and the sense strand of the isolated polynucleotide disclosed herein.In some embodiments, the isolated nucleic acid complex activates the expression of UTRN gene by at least 10% compared to the baseline expression of UTRN gene in cells.

[0027] The present disclosure also relates to isolated polynucleotides encoding the saRNAs disclosed herein in this disclosure. Also disclosed are vectors comprising the isolated polynucleotides disclosed herein.

[0028] Another aspect of the present disclosure provides a cell comprising the saRNA disclosed herein, an isolated polynucleotide encoding the saRNA disclosed herein, or a vector disclosed herein. In one embodiment, the cell is a mammalian cell, optionally a human cell. In some embodiments, the cell is a host cell. The aforementioned cell may be in vitro, such as a cell line or cell line, or may be present in a mammalian body, such as a human body. In some embodiments, the isolated polynucleotide is DNA. In some embodiments, the vector is AAV.

[0029] Another aspect of the disclosure provides compositions, such as pharmaceutical compositions, comprising an isolated polynucleotide encoding the saRNA disclosed herein, or optionally a pharma- ceutically acceptable carrier. In some embodiments, the pharma- ceutically acceptable carrier comprises an aqueous carrier, a liposome, a polymeric polymer, or a polypeptide. In some embodiments, the pharma- ceutically acceptable carrier is selected from an aqueous carrier, a liposome, a polymeric polymer, and a polypeptide. In some embodiments, the aqueous carrier may be, for example, RNase-free water or an RNase-free buffer. In some embodiments, the composition may comprise 0.001-200 nM (e.g., 0.001-200 nM, 0.001-100 nM, 0.001-50 nM, 0.001-20 nM, 10-100 nM, 10-50 nM, 20-50 nM, 20-100 nM, or 50-150 nM), or optionally, 1-150 nM of the saRNA described herein, or an isolated polynucleotide encoding the saRNA.

[0030] Another aspect of the present disclosure relates to the use of the aforementioned saRNA, an isolated polynucleotide encoding the saRNA disclosed herein, or a composition comprising the aforementioned saRNA or isolated polynucleotide disclosed herein in preparation for activating / upregulating expression of the UTRN gene in a cell.

[0031] The present disclosure also relates to a method for activating / upregulating the expression of UTRN gene in a cell, comprising administering to the cell the above-mentioned saRNA, the isolated polynucleotide disclosed herein, or the composition comprising the above-mentioned saRNA, or the isolated polynucleotide disclosed herein.Meanwhile, a method for increasing utrophin levels in a cell or functional utrophin levels in muscle is also provided, comprising introducing into the cell an effective amount of saRNA, nucleic acid, or the composition disclosed herein.

[0032] The isolated polynucleotide disclosed herein, or the composition comprising the above-mentioned saRNA disclosed herein, or the isolated polynucleotide may be directly introduced into a cell, or may be produced in the cell after the nucleotide sequence encoding the saRNA is introduced into the cell. The cell is, for example, a mammalian cell, such as a human cell. The aforementioned cell may be in vitro, such as a cell line or cell line, or may be present in a mammalian body, such as a human body. The human body may be a subject suffering from a disease or condition caused by insufficient expression of dystrophin in an individual, a mutation in the dystrophin gene, and / or a low functional dystrophin level, and the isolated polynucleotide disclosed herein, or the composition comprising the above-mentioned saRNA or isolated polynucleotide disclosed herein is administered in an amount sufficient to treat the disease or condition. Specifically, the condition caused by the lack of dystrophin due to dystrophin gene mutation and / or the insufficient expression of functional dystrophin includes, for example, DMD and BMD. In one embodiment, the disease caused by insufficient expression of dystrophin, dystrophin gene mutation, and / or a low functional dystrophin level is DMD. In another embodiment, the disease caused by under-expression of dystrophin, dystrophin gene mutations, and / or low functional dystrophin levels is BMD.

[0033] Another aspect of the present disclosure relates to a method for preventing or treating a disorder caused by insufficient expression of dystrophin, a dystrophin gene mutation, and / or an insufficient level of functional dystrophin in an individual, comprising administering a therapeutically effective dose of a saRNA disclosed herein. The method comprises an isolated polynucleotide encoding said saRNA disclosed herein and a vector disclosed herein, or a composition comprising the saRNA disclosed herein to an individual. In a particular embodiment, the disease or condition is DMD. In a particular embodiment, the disease or condition is BMD. The individual may be a mammal, such as a human. In one embodiment, the individual may include symptoms caused by insufficient expression of dystrophin, a mutation in the dystrophin gene, and / or low functional dystrophin levels in muscle, e.g., BMD. In one embodiment, the disease caused by insufficient muscle levels of functional dystrophin due to a dystrophin gene mutation is DMD or BMD. In one embodiment, the disease described herein includes DMD and BMD. In certain embodiments, the saRNA disclosed herein, the isolated polynucleotide disclosed herein, the vector disclosed herein, or the composition disclosed herein is administered to an individual by a route of administration selected from one or more of parenteral injection, oral administration, intranasal administration, inhalation administration, vaginal administration, and rectal administration.In certain embodiments, the route of administration is selected from one or more of intrathecal administration, intramuscular administration, intravenous administration, intraarterial administration, intraperitoneal administration, intravesical administration, intraventricular administration, intravitreal administration, and subcutaneous administration.In certain embodiments, the method disclosed herein activates / upregulates the expression of the UTRN gene or UTRN mRNA in an individual by at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%) compared to the baseline level of the gene.In certain embodiments, the method disclosed herein increases the utrophin level in an individual by at least 10% compared to the baseline level of the gene.

[0034] Another aspect of the present disclosure relates to the use of the saRNA disclosed herein, the isolated polynucleotide disclosed herein, or the composition comprising the saRNA disclosed herein for preparing a medicament for preventing or treating a disorder or condition that causes the expression of dystrophin, dystrophin gene mutation, and / or low-functional dystrophin level in an individual. The individual may be a mammal, such as a human. In one embodiment, the disease or condition may include, for example, DMD or BMD. In one embodiment, the disease caused by insufficient expression of dystrophin, dystrophin gene mutation, and / or low-functional dystrophin level is DMD. In one embodiment, the disease described herein includes DMD and BMD.

[0035] In addition, the present disclosure further provides a kit for carrying out the prevention or treatment method disclosed herein, the kit includes a) saRNA, b) instructions, and c) optionally, a means for administering the saRNA to an individual.Specifically, the kit can be packaged in a labeled package, and the label on the package indicates that the molecule or composition can be used for the prevention or treatment of disorders or conditions caused by insufficient expression of dystrophin, or for DMD or BMD.

[0036] Kits are provided by the present disclosure for carrying out the methods disclosed herein, the kits including a) the saRNA disclosed herein, and b) instructions for use. In certain embodiments, the instructions include a means or method for administering the saRNA disclosed herein to an individual.

[0037] Aspects of the present disclosure include kits comprising the saRNA disclosed herein, the isolated polynucleotide disclosed herein, the vector disclosed herein, or the composition disclosed herein, where the label on the package indicates that the saRNA, the isolated polynucleotide, the vector, or the composition can be used to prevent or treat a disease or condition induced by insufficient expression of dystrophin, or for DMD or BMD.

[0038] The disclosure further provides kits for detecting dystrophin, utrophin, or utrophin-related proteins (e.g., dystroglycan) in muscle or plasma, or in cells disclosed herein, transfected with the above-described SaRNA, or the above-described nucleic acid, or the above-described composition.

[0039] The saRNA that activates / upregulates the expression of the UTRN gene (such as the saRNA molecule) provided herein can efficiently and specifically upregulate the expression of the UTRN gene and increase the expression level of UTRN mRNA with less toxicity and adverse effects, and can be used in the preparation of a medicament for preventing or treating disorders associated with insufficient expression of dystrophin, and diseases or conditions caused by dystrophin gene mutations. [Brief description of the drawings]

[0040] [Figure 1]Figure 1 shows the changes in expression levels of human UTRN mRNA mediated by saRNA. 398 human UTRN promoter-targeting saRNAs were individually transfected into human malignant embryonic rhabdomyomatosis cell line (RD) at a concentration of 25 nM for 3 days. The sequences of saRNA strands and duplex composition are shown in Table 1. Mock transfected in the absence of oligonucleotides (not shown). dsCon2 duplex was used as a non-specific duplex control (not shown). DS18-si8 is a duplex siRNA targeting the UTRN gene and was transfected as a silencing dsRNA control (not shown). The mRNA levels of UTRN on day 3 were quantified by one-step RT-qPCR using each gene-specific primer set (shown in Table 3) in the PCR reaction. The geometric mean values ​​of the mRNA amounts of TBP and B2M were used as internal references. Values ​​(y-axis, log2 fold change) indicate the relative fold change in UTRN mRNA expression levels by each 398 saRNA relative to mock treatment after normalization to TBP and B2M (mean ± SEM of duplicate transfection wells). saRNAs are sorted in descending order on the x-axis by their activity (log2) in inducing UTRN mRNA expression. [Diagram 2]Figure 2 sorts saRNAs by their target location and hotspot region on the human UTRN promoter. 398 human UTRN promoter-targeting saRNAs were individually transfected into RD cells at 25 nM for 3 days. Mock transfected in the absence of oligonucleotides (not shown). dsCon2 duplex was used as a non-specific duplex control (not shown). DS18-si8 is a double-stranded siRNA targeting the UTRN gene and was transfected as a silencing dsRNA control (not shown). UTRN mRNA levels on day 3 were quantified by one-step RT-qPCR using gene-specific primer sets (shown in Table 3) in individual PCR reactions. The geometric mean of TBP and B2M mRNA amounts were used as internal references. Values ​​(y-axis, log2 fold change) indicate the relative fold change in UTRN mRNA expression levels by each saRNA relative to mock treatment (mean ± SEM of two replicate transfection wells) after normalization to B2M and TBP. The saRNAs are sorted on the x-axis by their position on the promoter from -666 bp upstream and 334 bp downstream of the UTRN transcription start site (TSS). The positions of the four saRNA hotspot regions were marked H1–H4 in rectangular dotted boxes. Numbers on the boxes indicate the boundaries of the hotspot regions relative to the UTRN TSS (+1 position), which span the 5' end of the first saRNA target and the 3' end of the last saRNA target within each hotspot region. [Diagram 3]Figure 3 shows the activating effect of lead saRNA on the expression of human UTRN gene RD cells. Cells were treated with 25 nM saRNA concentration for 3 days. Mock transfected in the absence of oligonucleotide. dsCon2 was transfected as a non-specific double-stranded control. DS18-si8 is a double-stranded siRNA targeting the UTRN gene and was transfected as a silencing dsRNA control. Figure 3 shows that the mRNA levels of UTRN in RD cells at day 3 were quantified by two-step RT-qPCR using gene-specific primer sets (shown in Table 3). The geometric mean values ​​of the mRNA amounts of TBP and B2M were used as internal references. Values ​​(y-axis) are expressed as UTRN mRNA expression levels relative to mock treatment after normalization to TBP and B2M (mean ± SEM of two replicate transfection wells). [Figure 4] Figure 4 shows the activating effect of lead saRNAs on protein expression of UTRN in RD cells. Cells were treated with the indicated saRNAs (see Table 1) at 25 nM for 5 days. Mock transfected in the absence of oligonucleotides. dsCon2 was transfected as a non-specific double-stranded control. DS18-si8 is a double-stranded siRNA targeting the UTRN gene and was transfected as a silencing dsRNA control. Utrophin protein levels in RD cells at day 5 were determined by Western blotting using a primary antibody against human utrophin protein. Antibodies against α / β tubulin protein were also blotted and used as a control for protein loading. Figure 4 shows the relative fold change in utrophin protein levels derived from quantifying band intensity. Values ​​(y-axis) are the relative band intensity of utrophin normalized to α / β tubulin. All SaRNAs are sorted on the x-axis in descending order by their activity (fold change) in inducing utrophin protein expression. [Diagram 5]Figure 5 shows the activation effect of saRNA on the expression of human UTRN mRNA in RD cells. The indicated saRNA (see Table 10) was transfected into RD cells at 25 nM for 3 days. Mock was transfected in the absence of oligonucleotide. dsCon2 was transfected as a non-specific double-stranded control. DS18-si8 is a double-stranded siRNA targeting the UTRN gene and was transfected as a silencing dsRNA control. UTRN mRNA levels were quantified by two-step RT-qPCR using gene-specific primer sets (shown in Table 3). The geometric mean values ​​of TBP and HPRT1 mRNA amounts were used as internal references. Values ​​(y-axis) are expressed as UTRN mRNA expression levels relative to mock treatment after normalization to TBP and HPRT1 (mean ± SEM of two replicate transfection wells). [Figure 6] Figures 6A-6B show the activation effect of saRNA on the expression of human utrophin protein in RD cells. The indicated saRNA (see Table 10) was transfected into RD cells at 25 nM for 3 days. Mock was transfected in the absence of oligonucleotide. dsCon2 was transfected as a non-specific double-stranded control. Utrophin protein levels were determined by JESS using a primary antibody against human utrophin protein. An antibody against α / β tubulin protein also served as a control for protein loading. Figure 6A shows the protein bands of utrophin and α / β tubulin protein. Figure 6B shows the relative fold change in utrophin levels derived from quantifying the band intensities in Figure 6A. Values ​​(y-axis) are the relative band intensities of utrophin after normalization to α / β tubulin (mean ± SEM of two replicate transfection wells). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Double-stranded RNA (dsRNA) targeting gene regulatory sequences, including promoters, has been shown to upregulate target genes in a sequence-specific manner at the transcriptional level through a mechanism known as RNA activation (RNAa) (Li, LC et al. Small dsRNAs induce transcriptional activation in human cells. PNAS (2006)). Such dsRNAs are called small activating RNAs (saRNAs).

[0042] Embodiments of the present disclosure are based in part on the surprising discovery that oligonucleotide modulators (e.g., also referred to herein as "UTRN gene saRNAs" or "UTRN saRNAs") can activate or upregulate expression of the UTRN gene in cells. Increased production of functional UTRN gene mRNA following administration with the saRNAs of the present disclosure can achieve significant increases or upregulation of levels of UTRN mRNA and utrophin protein.

[0043] In particular, the present inventors have discovered that functional saRNAs capable of activating / upregulating the expression of UTRN mRNA are not randomly distributed on the promoter, but are clustered in specific hotspot regions. Only some regions on the promoter of the UTRN gene are favorable for gene activation by saRNA, such as, for example, the regions at -636 to -496, -351 to -294, -236 to -187, and -101 to -65 upstream of the transcription start site of the UTRN gene. These specific promoter regions (herein referred to as "hotspot regions") identified by the present disclosure are optionally at least 37 nt in length, or alternatively have lengths ranging from about 37 to about 200 nt.

[0044] The inventors have also discovered that optimal target sequences / sense strands of saRNA within the UTRN promoter region include sequences having the following criteria: (1) 35%-70% GC content, (2) less than five consecutive identical nucleotides, (3) no more than three dinucleotide repeats, and (4) no more than three trinucleotide repeats. As a beneficial consequence of the criteria, the target sequence (e.g., an isolated nucleic acid sequence comprising the target sequence), upon interaction with the saRNA, can activate / upregulate expression of UTRN mRNA by at least 10% or 1.1-fold compared to baseline levels of UTRN mRNA.

[0045] A "hotspot region" herein is defined by a nucleic acid region on the target gene of a SaRNA in which at least 25% of the SaRNAs designed according to criterion (1) span the 5' end of the first SaRNA target and the 3' end of the last SaRNA target in each hotspot. (2), (3), and (4) are as described above. If found to be functional, i.e., capable of inducing a 1.1-fold or greater change in the mRNA expression of the target gene compared to the baseline level of mRNA expression. In some embodiments, at least 28%, at least 30%, about 35%, about 40%, or more than 50% of the SaRNAs designed to be functional, i.e., capable of inducing a 1.1-fold or greater change in the mRNA transcription level or protein expression level of the target gene compared to the baseline level of the gene.

[0046] Based at least in part on these findings, the present disclosure features saRNAs, compositions, and pharmaceutical compositions for activating / upregulating the expression of UTRN mRNA by at least 10% compared to the baseline level of UTRN mRNA.Also provided herein is a method for preventing or treating a disease or condition caused by insufficient expression of dystrophin, dystrophin gene mutation, and / or low-functional dystrophin level in an individual, comprising administering to the individual any saRNA, composition, and / or pharmaceutical composition described herein.

[0047] The embodiments of the present disclosure are also based in part on the surprising discovery that saRNAs capable of activating or upregulating expression of the UTRN gene in cells are clustered in specific UTRN gene promoter regions, as shown in FIG. 2. The inventors have identified these clusters of UTRN gene promoter regions that are considered to be "hotspot" promoter regions that enrich target sites for evolved functional saRNAs (see, e.g., Table 9). For example, four hotspot regions of the human UTRN promoter located in the regions -636 to -496 (H1), -351 to -294 (H2), -236 to -187 (H3), and -101 to -65 (H4) from the TSS of the promoter were detected and found to be optimal target sites for saRNAs that activate UTRN gene expression by an RNA activation mechanism.

[0048] This saRNA-UTRN mRNA-utrophin pathway can provide an alternative therapeutic approach to current treatments for dystrophin deficiency disorders (DDD), for example, for patients with Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD).

[0049] In this disclosure, relevant terms are defined as follows:

[0050] The term "complementarity" as used herein refers to the ability to form base pairs between two oligonucleotide strands. Base pairs are generally formed through hydrogen bonds between nucleotides in antiparallel oligonucleotide strands. The bases of complementary oligonucleotide strands can be paired in the Watson-Crick manner (e.g., A-T, A-U, and C-G) or in any other manner that allows the formation of a double strand (e.g., Hoogsteen or reverse Hoogsteen base pairs).

[0051] Complementarity includes perfect complementarity and incomplete complementarity. Perfect complementarity or 100% complementarity means that each nucleotide of a first oligonucleotide strand can form a hydrogen bond with the corresponding position of a second oligonucleotide strand in the double-stranded region of a double-stranded oligonucleotide molecule, and no base pairs are "mispaired". "Incomplete complementarity" means that not all nucleotide units of the two strands are bonded to each other by hydrogen bonds. For example, for two oligonucleotide strands with a length of 20 nucleotides in the double-stranded region, if only two base pairs in this double-stranded region can be formed through hydrogen bonds, the oligonucleotide strands have 10% complementarity. In the same example, if 18 base pairs in this double-stranded region can be formed through hydrogen bonds, the oligonucleotide strands have 90% complementarity. Substantial complementarity refers to at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95%, or 99% complementarity.

[0052] The term "oligonucleotide" or "polynucleotide" can be used interchangeably and refers to a polymer of nucleotides, including, but not limited to, single-stranded or double-stranded nucleic acid molecules of DNA, RNA, or DNA / RNA hybrids, oligonucleotide chains that contain alternating deoxyribosyl and ribosyl moieties in regular and irregular manners, and modified and naturally or unnaturally occurring frameworks of such oligonucleotides. The oligonucleotide for activating target gene transcription described herein is a small activating nucleic acid molecule (saRNA).

[0053] As used herein, the terms "oligonucleotide chain," "chain," and "oligonucleotide sequence" can be used interchangeably to refer collectively to short nucleotide sequences having less than 35 bases (including nucleotides in deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)). In a non-limiting example, the length of the chain can be any length between 16 and 35 nucleotides.

[0054] The term "target gene" as used herein can refer to a nucleic acid sequence, a transgene, a viral or bacterial sequence, a chromosomal or extrachromosomal gene naturally occurring in an organism, and / or can be transiently or stably transfected or integrated into its cells and / or chromatin. The target gene can be a protein-coding gene or a non-protein-coding gene (such as microRNA genes and long non-coding RNA genes). The target gene generally contains a promoter sequence, and positive regulation of the target gene can be achieved by designing a saRNA with sequence identity (also called homology) to the promoter sequence characterized as upregulation of the expression of the target gene. A "target sequence" or "target site," as used interchangeably herein, refers to a sequence segment of a target gene sequence, such as a target gene promoter, that is homologous or complementary to the sense or antisense strand of a saRNA according to the present disclosure. The target gene can also include one or more regulatory elements, and one or more saRNAs are designed to have sequence identity with the regulatory elements. Non-limiting examples of the one or more regulatory elements include promoters, enhancers, silencers, insulators, TATA boxes, GC boxes, CAAT boxes, transcription initiation sites, DNA binding motifs for transcription factors or other proteins that regulate transcription, and 5' untranslated regions.

[0055] As used herein, the term "sense strand" of a saRNA refers to the strand having sequence homology or sequence identity to a segment of the coding strand of the target gene promoter sequence in the saRNA duplex.

[0056] As used herein, the term "antisense strand" of a saRNA refers to the strand complementary to the sense strand of the saRNA duplex or target gene sequence region.

[0057] The term "coding strand" as used herein refers to the DNA strand of a target gene unavailable for transcription, the nucleotide sequence of this strand being the same as that of the RNA produced from transcription (in the RNA, the Ts of the DNA are replaced by Us). The coding strand of the double-stranded DNA sequence of a target gene promoter described herein refers to the promoter sequence on the same DNA strand as the DNA coding strand of the target gene.

[0058] The term "template strand" as used herein refers to the other strand complementary to the coding strand in the double-stranded DNA of a target gene, i.e., the strand that can be transcribed into RNA as a template, which is complementary to the transcribed RNA (A-U and G-C). In the transcription process, RNA polymerase binds to the template strand, moves along the template strand in the 3'→5' direction, and catalyzes the synthesis of RNA along the 5'→3' direction. The template strand of the double-stranded DNA sequence of the target gene promoter described herein refers to the promoter sequence on the same DNA strand as the DNA template strand of the target gene.

[0059] As used herein, the term LNA refers to a locked nucleic acid in which the 2'-oxygen atom and the 4'-carbon atom are linked by an extra bridge.As used herein, the term BNA refers to a 2'-O and 4'-aminoethylene bridged nucleic acid that can contain a 5-membered or 6-membered bridge structure with NO linkage.As used herein, the term PNA refers to a nucleic acid mimicked with a pseudopeptide backbone consisting of N-(2-aminoethyl)glycine units with the nucleobase linked to the glycine nitrogen via a carbonyl methylene linker.

[0060] The term "promoter" as used herein refers to a sequence that is spatially associated with a protein-coding or RNA-coding nucleic acid sequence and plays a regulatory role in the transcription of the protein-coding or RNA-coding nucleic acid sequence. Generally, eukaryotic gene promoters contain 100-5000 base pairs, although this length range is not intended to be limiting for the term "promoter" as used herein. Promoter sequences are generally located at the 5' end of the protein-coding or RNA-coding sequence, but may also be present in exon and intron sequences.

[0061] The term "transcription start site" as used herein refers to the nucleotide that marks the start of transcription on the template strand of a gene. A transcription start site can appear on the template strand in the promoter region. A gene can have multiple transcription start sites.

[0062] The term "identity" or "homology" as used herein means that one oligonucleotide strand (sense or antisense) of the saRNA has sequence similarity with the coding or template strand in a region of a target gene. As used herein, "identity" or "homology" may be at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95% or 99%.

[0063] The term "isometric portion" refers to a portion of a sequence that is compared to a subject sequence (e.g., a contiguous oligonucleotide sequence from a saRNA) and has the same length (the same number of bases) as the subject sequence.

[0064] The term "sequence-specific mode" as used herein refers to the method of binding or hybridization of two nucleic acid fragments according to their nucleotide sequences, such as the Watson-Crick method (e.g., A-T, A-U, and C-G), or any other method that allows the formation of a duplex (e.g., Hoogsteen or reverse Hoogsteen base pairing).

[0065] As used herein, the term "overhang" refers to non-base-paired nucleotides at the end (5' or 3') of an oligonucleotide strand, which is formed in a double-stranded oligonucleotide by one strand extending from the other strand. The single-stranded region extending from the 3' and / or 5' ends of the double strand is called an overhang. As used herein, the term "natural overhang" refers to an overhang consisting of one or more nucleotides that are identical or complementary to a corresponding position on a DNA target. A natural overhang on the sense strand consists of one or more nucleotides that are identical to a corresponding position on a DNA target. A natural overhang on the antisense strand consists of one or more nucleotides that are complementary to a corresponding position on a DNA target.

[0066] As used herein, the terms "gene activation" or "activated gene expression" and "gene upregulation" or "upregulation gene expression" may be used interchangeably and refer to an increase in the transcription, translation, expression, or activity of a nucleic acid compared to a baseline level of the nucleic acid, as determined by measuring the gene's transcription level, mRNA level, protein level, enzyme activity, methylation state, chromatin state or configuration, translation level, or activity or state in a cell or biological system. These activities or states can be determined directly or indirectly. Also, "gene activation", "activated gene expression", "gene upregulation", and "upregulation gene expression" refer to an increase in activity associated with a nucleic acid sequence, regardless of the mechanism of its activation. For example, gene activation occurs at the transcription level, increasing transcription into RNA and translating the RNA into protein, thereby increasing protein expression.

[0067] The term "baseline expression" or "baseline level" of a nucleic acid or gene refers to the expression level of a nucleic acid or gene without any artificial regulation, e.g., before or without administration of a saRNA according to the present disclosure.

[0068] As used herein, the terms "oligonucleotide modulator", "small activating RNA", "saRNA" and "small activating nucleic acid molecule" can be used interchangeably and refer to a nucleic acid molecule capable of upregulating the expression of a target gene, and can be composed of a first nucleic acid fragment (sense strand) containing a nucleotide sequence having sequence identity with a non-coding nucleic acid sequence (e.g., promoter or enhancer) of a target gene, and a second nucleic acid fragment (antisense strand) containing a nucleotide sequence complementary to the first nucleic acid fragment, and a second nucleic acid fragment (antisense strand) containing a nucleotide sequence complementary to the promoter or enhancer of the target gene, and the first and second nucleic acid fragments form a duplex. saRNA can also be composed of a synthetic or vector-expressed single-stranded RNA molecule capable of forming a hairpin structure by two complementary regions (first and second regions) within the molecule, the first region containing a nucleotide sequence having sequence identity with the target sequence of the promoter of the gene, and the second region containing a nucleotide sequence complementary to the first region. The length of the double-stranded region of the saRNA is typically about 10 to about 50, about 12 to about 48, about 14 to about 46, about 16 to about 44, about 18 to about 42, about 20 to about 40, about 22 to about 38, about 24 to about 36, about 26 to about 34, and about 28 to about 32 base pairs, and typically about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 base pairs. Additionally, the terms "oligonucleotide modulator," "saRNA," and "small activating RNA" also encompass nucleic acids other than ribonucleotides, including, but not limited to, modified nucleotides or analogs.

[0069] As used herein, the term "functional saRNA" refers to a saRNA that activates the expression of its intended target gene by at least 10% (or at least 1.1-fold) compared to the baseline level of the gene. A "non-functional saRNA" refers to a saRNA that regulates the expression of a UTRN gene by less than 10% (or less than 1.1-fold) compared to the baseline level of the gene.

[0070] As used herein, the term "hotspot region" refers to a gene promoter region that contains a hotspot and a continuous target sequence that spans the very 5' end of the first SaRNA and the 3' end of the last SaRNA within the hotspot.

[0071] As used herein, the terms "isolated target site" and "isolated polynucleotide" can be used interchangeably, and refer to a target site to which a SaRNA has complementarity or hybridization. For example, an isolated nucleic acid sequence of a target site can include a nucleic acid sequence to which a region of a saRNA has complementarity or hybridization. As used herein, the term "isolated polynucleotide" refers to a polynucleotide that encodes a saRNA.

[0072] As used herein, the term "synthesis" refers to methods for the synthesis of oligonucleotides and includes any method that allows for RNA synthesis, such as chemical synthesis, in vitro transcription, and / or vector-based expression.

[0073] As used herein, when referring to a dystrophin deficiency associated disorder, the terms "disease," "disorder," and "condition" can be used interchangeably.

[0074] As used herein, capitalized "UTRN" or "UTRN gene" refers to the human gene.

[0075] As used herein, the term UTRN mRNA refers to expression of the UTRN gene or the message RNA (mRNA) produced from transcription of the UTRN gene.

[0076] As used herein, the terms "UTRN protein" and "utrophin" may be used interchangeably and refer to the protein produced from expression of the UTRN gene or translation of UTRN mRNA.

[0077] saRNA In the present disclosure, the expression of the UTRN gene is upregulated by RNA activation, and the associated disease (such as DMD) is treated by increasing the expression level of utrophin. Because the UTRN gene encodes utrophin, increasing UTRN mRNA expression leads to increasing the expression of utrophin, thereby treating the disease (such as DMD). Therefore, the UTRN gene is, in some cases, the target gene in the present disclosure.

[0078] Embodiments of the present disclosure include oligonucleotide modulators (e.g., saRNA) comprising an oligonucleotide sequence having a length ranging from 16 to 35 contiguous nucleotides, where the contiguous oligonucleotide sequence has at least 75%, or at least 80%, or at least 85%, or at least 90% sequence homology or complementarity to an equal length portion of SEQ ID NO: 1200, and where the saRNA activates / upregulates expression of the UTRN gene by at least 10% compared to its baseline expression.

[0079] In some embodiments, the equal length portion of SEQ ID NO: 1200 disclosed herein is located in the region -636 to -496 (SEQ ID NO: 1207), in the region -351 to -294 (SEQ ID NO: 1208), in the region -236 to -187 (SEQ ID NO: 1209), or in the region -101 to -65 (SEQ ID NO: 1210) upstream of the transcription start site of the UTRN gene.

[0080] In some embodiments, the continuous oligonucleotide sequence of the saRNA has 5 or less nucleotide differences or mismatches with respect to the equal length portion of SEQ ID NO: 1200, i.e., 5, 4, 3, 2, 1, or 0 nucleotide differences or mismatches. In some embodiments, the differences or mismatches are located in the middle or at the 3' end of the oligonucleotide sequence of the saRNA. Methods and principles of saRNA molecule design are well known to those skilled in the art and are described in detail in, for example, Place et al., Molecular Therapy-Nucleic Acids (2012) 1, e15; and Li et al., PNAS, 2006, vol. 103, no. 46, 17337-17342, which are incorporated herein by reference in their entirety.

[0081] In some embodiments, the saRNA disclosed herein comprises a sense strand and an antisense strand. The sense strand and the antisense strand comprise complementary regions capable of forming a double-stranded nucleic acid structure that activates expression of the UTRN gene in a cell via the RNAa mechanism. As used herein, the RNAa mechanism (also known as RNA activation) refers to a mechanism by which a double-stranded nucleic acid structure can upregulate a target gene in a sequence-specific manner at the transcriptional level. The sense and antisense strands of the saRNA can be on either two different nucleic acid strands or on one nucleic acid strand (e.g., contiguous nucleic acid sequences). When the sense and antisense strands are disposed on two different strands, at least one strand of the saRNA has an overhang of 0, 1, 2, 3, 4, 5 or 6 nucleotides in length, and optionally, a 3' overhang of 0-6 nucleotides in length, such that both strands have a 3' overhang of 2 or 3 nucleotides in length. The nucleotides of the overhang are optionally thymine deoxyribonucleotides (dT) and optionally natural overhangs that are selected from or complementary nucleotides at the corresponding positions on the DNA target. When the sense strand and the antisense strand are arranged on one nucleic acid strand, in some cases, the saRNA is a hairpin single-stranded nucleic acid molecule, and the complementary regions of the sense strand and the antisense strand form a double-stranded nucleic acid structure with each other. In the above-mentioned saRNA, in some embodiments, the sense strand and the antisense strand each have a length in the range of 16 to 35 nucleotides. For example, in some embodiments, the sense strand and the antisense strand independently comprise a length of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides.

[0082] In certain embodiments, one strand of the saRNA has at least 75% (e.g., at least about 79%, about 80%, about 85%, about 90%, about 95% or about 99%) sequence homology or complementarity to a nucleotide sequence selected from SEQ ID NOs: 1 to 398. Specifically, the sense strand of the saRNA disclosed herein has at least 75% (e.g., at least about 79%, about 80%, about 85%, about 90%, about 95% or about 99%) sequence homology to any nucleotide sequence selected from SEQ ID NOs: 400 to 797, and the antisense strand of the saRNA disclosed herein has at least 75% (e.g., at least about 79%, about 80%, about 85%, about 90%, about 95% or about 99%) sequence homology to any nucleotide sequence selected from SEQ ID NOs: 800 to 1197. More specifically, the sense strand of the saRNA disclosed herein consists of, or is any nucleotide sequence selected from SEQ ID NOs: 400-797, and the antisense strand of the saRNA disclosed herein consists of, or is any nucleotide sequence selected from SEQ ID NOs: 800-1197.

[0083] In certain embodiments, one strand of the saRNA may have 5 or less nucleotide differences or mismatches, i.e., 5, 4, 3, 2, 1, or 0 nucleotides, with respect to a nucleotide sequence selected from SEQ ID NOs: 1-398. Specifically, the sense strand of the saRNA disclosed herein may have 5 or less nucleotide differences or mismatches, i.e., 5, 4, 3, 2, 1, or 0 nucleotides, with respect to a nucleotide sequence selected from SEQ ID NOs: 1-398, and the antisense strand of the saRNA disclosed herein may have 5 or less nucleotide differences or mismatches, i.e., 5, 4, 3, 2, 1, or 0 nucleotides, with respect to a nucleotide sequence selected from SEQ ID NOs: 800-1197. In some embodiments, the differences or mismatches are located at the middle or 3' end of the sense or antisense strand of the saRNA.

[0084] In certain embodiments, the antisense strand disclosed herein can interact with a target nucleic acid sequence of a promoter of a gene in a sequence-specific manner, meaning that the antisense strand can hybridize to the target nucleic acid via hydrogen bonds. In certain embodiments, the antisense strand has a nucleotide sequence that, when written in the 5' to 3' direction, comprises the reverse complement of the target portion of the target nucleic acid to which it targets. In one such embodiment, the antisense strand has a nucleotide sequence that, when written in the 5' to 3' direction, comprises the reverse complement of the target portion of SEQ ID NO: 1200, specifically, the target portion is a nucleic acid sequence selected from SEQ ID NOs: 1 to 398.

[0085] In the saRNA disclosed herein, all nucleotides may be natural nucleotides or non-chemically modified nucleotides, or at least one nucleotide is a chemically modified nucleotide.Non-limiting examples of chemical modifications include one or more of the following combinations: (1) Modification of the phosphodiester bond of the nucleotide in the nucleotide sequence of the saRNA, (2) modification of the 2'-OH of ribose in the nucleotide sequence of saRNA; (3) modification of the bases in the nucleotides of the saRNA; and (4) The method of claim 1, wherein at least one nucleotide of the nucleotide sequence of the small activator nucleic acid molecule is a locked nucleic acid.

[0086] The chemical modifications described herein are well known to those skilled in the art, and the modification of phosphodiester bond refers to the modification of oxygen in phosphodiester bond, including phosphorothioate modification and boranophosphate modification.The modifications disclosed herein stabilize saRNA structure while maintaining high specificity and high affinity for base pairing.

[0087] In some embodiments, the saRNA of the present disclosure comprises at least one chemically modified nucleotide that is modified at the 2'-OH in the pentose of the nucleotide, i.e., introduction of a specific substituent at the hydroxyl position of the ribose, such as a 2'-fluoro modification, a 2'-oxymethyl modification, a 2'-oxyethylidenemethoxy modification, a 2,4'-dinitrophenol modification, a Locked Nucleic Acid (LNA) 2'-amino modification or a 2'-deoxy modification, e.g., a 2'-deoxy-2'-fluoro modified nucleotide and a 2'-deoxy modified nucleotide.

[0088] In some embodiments, the saRNA of the present disclosure comprises at least one chemically modified nucleotide that is modified at the base of the nucleotide, such as a 5'-bromouracil modification, a 5'-iodouracil modification, an N-methyluracil modification, or a 2,6-diaminopurine modification.

[0089] In some embodiments, the chemical modification of the saRNA is the addition of an (E)-vinylphosphonate moiety at the 5' end of the sense or antisense sequence. In some embodiments, the chemical modification of at least one chemically modified nucleotide is the addition of a 5'-methylcytosine moiety at the 5' end of the sense or antisense sequence.

[0090] In some embodiments, the saRNA disclosed herein comprises at least one nucleotide in the nucleotide sequence of the small activated nucleic acid molecule that is a chemically modified nucleic acid, such as a locked nucleotide, a basic nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base containing nucleotide. In some embodiments, the saRNA disclosed herein comprises an "endolite" modification of a 2'-O-methyl modified nucleotide and a nucleotide that contains a 5'-phosphorothioate group.

[0091] In some embodiments, the saRNA of the present disclosure is chemically modified to enhance stability or other beneficial properties. The nucleic acids featured in the present disclosure can be synthesized and / or modified by conventional methods, such as those described in "Current protocols in nucleic acid chemistry", Beaucage, SL et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, incorporated herein by reference. Modifications include, for example, (a) terminal modifications, e.g., 5'-terminal modifications (phosphorylation, conjugation, reverse linkage, etc.) 3'-terminal modifications (linkage, DNA nucleotide, reverse linkage, etc.); (b) base modifications, e.g., destabilizing bases replacing stabilizing bases, or base pairing with an expanded repertoire of partners, removing bases (basic nucleotides) or replacing linked bases; (c) sugar modifications (e.g., at the 2' or 4' position) or sugar replacement; (d) backbone modifications, including modification or replacement of phosphodiester bonds. Specific examples of saRNA molecules that can be used in the present disclosure include, but are not limited to, RNA that contains modified backbones or does not have natural internuclear bonds.In some embodiments, RNA with modified backbones includes, among others, those that do not have phosphorus atoms in their backbones.In some embodiments, modified RNA that does not have phosphorus atoms in its internuclear backbones can also be considered as oligonucleosides.In some embodiments, modified oligonucleotides have phosphorus atoms in their internucleoside backbones.

[0092] The method of claim 1, wherein the modified oligonucleotide backbone has an inverted polarity of, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates and other alkylphosphonates including 3'-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs thereof, and the adjacent nucleotide unit pairs are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0093] Non-limiting examples of the preparation of phosphorus-containing linkages include: Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5, 550, 111; 5, 563, 253; 5, 571, 799; 5, 587, 361; 5, 625, 050; 6, 028, 188; 6, 124, 445; 6, 160, 109; 6, 169, 170; 6, 172, 209; 6, 239, 265; 6, 277, 603; 6, 326, 199; 6, 346, 614; 6, 444, 423; 6, 531, 590; 6, 534, 639; 6, 608, 035; 6, 683, 167; 6, 858, 715; 6, 867, 294; 6, 878, 805; See, 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Patent RE39464.

[0094] In certain embodiments, the small activating nucleic acid molecule is RNA, DNA, BNA, LNA or peptide nucleic acid (PNA).

[0095] Furthermore, to facilitate the entry of saRNA into cells, chemical conjugate moieties can be introduced into the ends of the sense or antisense strands of saRNA based on the modifications described above, to facilitate their action through the lipid bilayer that constitutes the cell membrane and gene promoter regions in the nucleus.

[0096] In certain embodiments, the saRNA disclosed in the present disclosure is covalently linked to one or more conjugate groups.In certain embodiments, the conjugate group modifies one or more properties of the oligonucleotide that is linked to it, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance.In certain embodiments, the conjugate group imparts new properties to the oligonucleotide that is linked to it, such as a fluorophore or reporter group that allows the oligonucleotide to be detected. Specific conjugation groups and conjugation sites have been described previously, for example: cholesterol sites (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), thioethers, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids, 19 ...esters, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 4, 1053-1060), thioesters, e.g Res., 1992, 20, 533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO 1, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett, 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid with a palmityl moiety (Mishra et al., Biochim, Biophys. Acta, 1995, 1264, 229-237), octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937), tocopherol group (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et al., Molecular Therapy, 2008, 16, 734-740), or GalNAc clusters (e.g., WO2014 / 179620).

[0097] In some embodiments, the saRNA of the disclosure relates to intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folic acid, lipids, phospholipids, biotin, phenazines, phenanthridines, anthraquinones, adamantanes, acridines, fluoresceins, rhodamines, coumarins, fluorophores, and dyes.

[0098] In some embodiments, the conjugation moiety comprises an active drug substance, e.g., aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, benzothiadiazide, chlorothiazide, diazepines, indomethicine, barbiturates, cephalosporins, sulfa drugs, antidiabetics, antibacterials, antibiotics.

[0099] In some embodiments, the saRNA of the present disclosure is conjugated to one or more conjugation moieties selected from lipids, fatty acids, fluorophores, ligands, sugars, peptides, and antibodies.

[0100] In some embodiments, the saRNA of the present disclosure relates to a sense or antisense strand of the saRNA conjugated to one or more conjugation moieties selected from a cell membrane-permeable peptide, polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, cholesterol, glucose, and N-acetylgalactosamine.

[0101] In some embodiments, the saRNA conjugated to one or more conjugate moieties disclosed in the embodiments is directly contacted, transferred, delivered, or administered to a cell or subject. As used interchangeably herein, a "patient," "individual," or "subject" can refer to a non-human (e.g., mammalian) subject or a human subject.

[0102] In some embodiments, the sense and antisense strands of the saRNA independently have at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of nucleotides that are chemically modified nucleotides.

[0103] In some embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of the nucleotides of the saRNA are chemically modified nucleotides.

[0104] These modifications can increase the bioavailability of the saRNA, improve affinity for target sequences, and enhance resistance to nuclease hydrolysis in cells.

[0105] In some embodiments, the saRNA of the present disclosure that is effective upon contact with a cell to activate or upregulate expression of one or more genes in the cell is, for example, at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 500%, at least 800%, at least 1000%, at least 2000%, or at least 5000%).

[0106] In a non-limiting example, the saRNA is designed at least in part based on the following criteria: (1) having a GC content of 35%-70%; (2) having less than five consecutive identical nucleotides; (3) having no more than three dinucleotide repeats; and (4) having no more than three trinucleotide repeats. In some embodiments, the saRNA is designed / selected, at least in part, based on criteria that allow for the production of functional saRNA. For example, in some cases, a sequence located upstream of the TSS may contain a sequence that does not favor the synthesis of saRNA, even though it is located in a hotspot region.

[0107] In some embodiments, the saRNA is designed / selected, at least in part, based on criteria including sequences having a particular GC content (e.g., 25%-75% GC content) and lacking consecutive identical nucleotides, consecutive dinucleotides, or consecutive trinucleotides. In some embodiments, the saRNA sequence contains sequences having 35%-70% GC content, (1) sequences having less than five consecutive identical nucleotides, (2) sequences having three or fewer dinucleotide repeats, and (3) sequences having three or fewer trinucleotide repeats.

[0108] In some embodiments, the saRNA sequence comprises a sequence with a GC content of 25%-75%, 30%-70%, 35%-70%, 40%-60%, or 45%-55%. In some embodiments, the saRNA comprises a sequence with a GC content of 35%-70%.

[0109] In some embodiments, the saRNA sequence comprises a sequence having less than 7 consecutive identical nucleotides, less than 6 consecutive identical nucleotides, less than 5 consecutive identical nucleotides, less than 4 consecutive identical nucleotides, or less than 3 consecutive identical nucleotides. In some embodiments, the saRNA comprises a sequence having less than 5 consecutive identical nucleotides.

[0110] In some embodiments, the saRNA sequence contains a sequence with 5 or fewer dinucleotide repeats, 4 or fewer dinucleotide repeats, 3 or fewer dinucleotide repeats, or 2 or fewer dinucleotide repeats. In some embodiments, the saRNA contains a sequence with 3 or fewer dinucleotide repeats.

[0111] In some embodiments, the saRNA sequence contains a sequence with 5 or fewer trinucleotide repeats, 4 or fewer trinucleotide repeats, 3 or fewer trinucleotide repeats, or 2 or fewer trinucleotide repeats. In some embodiments, the saRNA contains a sequence with 3 or fewer trinucleotide repeats.

[0112] Target sequence In certain embodiments, the present disclosure relates to an isolated target site of the saRNA of the present disclosure, specifically, the isolated target site is a nucleotide sequence having a length ranging from 16 to 35 nucleotides in the nucleotide sequence of SEQ ID NO: 1200. In certain embodiments, the isolated target site is a nucleic acid sequence selected from SEQ ID NOs: 1 to 398. The isolated target site can interact with the antisense strand of the saRNA disclosed in the present disclosure, and thus can activate expression of the UTRN gene (e.g., mRNA expression, protein expression, UTRN expression). In some embodiments, the target site is selected at least in part based on a gene sequence. In some embodiments, the target site is selected at least in part based on a sequence close to the transcription start site (TSS) of the gene. In some embodiments, the target site is selected at least in part based on a promoter sequence upstream of the TSS. In some embodiments, the target site is selected at least in part based on a sequence from -5000bp, -4000bp, -3000bp, -2000bp, -1000bp, or -500bp upstream of the TSS. In some embodiments, the target site is selected at least in part by shifting 1 bp each time toward the TSS, resulting in a target sequence, and then repeating this process, increasing toward the TSS by an additional base pair (e.g., n+1). In some embodiments, the target site has a length of about 8 to about 35 nucleotides. In some embodiments, the target site has a length of about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides.

[0113] In certain embodiments, the present disclosure relates to an isolated oligonucleotide complex comprising the saRNA disclosed herein and an isolated target site disclosed herein.In certain embodiments, the isolated oligonucleotide complex activates the expression of the UTRN gene by at least 10% (e.g., activates / upregulates the expression of the UTRN gene compared to baseline UTRN gene expression level).

[0114] Hotspots In certain embodiments, the present disclosure relates to an isolated nucleic acid sequence located upstream of the transcription start site of the UTRN gene, i.e., a "hotspot region". In certain embodiments, the isolated nucleic acid sequence disclosed herein is an oligonucleotide sequence having a length of at least 37 consecutive nucleotides and having at least 75%, or at least 80%, or at least 85%, or at least 90% sequence homology to an equivalent length region within the nucleotide sequence of SEQ ID NO: 1200. In some embodiments, at least 25% (e.g., 28%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of the SaRNA designed to target a sequence within the hotspot is functional, i.e., can induce at least a 1.1-fold change in mRNA expression of the target gene. In a non-limiting example, at least 50% of the saRNAs designed to target hotspots are functional, i.e., capable of inducing at least a 1.1-fold change in mRNA expression of the target gene. In a non-limiting example, the saRNAs are designed at least in part based on the following criteria: (1) have a GC content of 35%-70%; (2) have less than five consecutive identical nucleotides; (3) have no more than three dinucleotide repeats; and (4) have no more than three trinucleotide repeats. In some embodiments, the same or similar criteria are used to select the isolated nucleic acid sequences and / or target sequences. In a non-limiting example, the isolated nucleic acid sequences upstream of the TSS of the UTRN gene are selected based on at least the following criteria: (1) have a GC content of 35%-70%; (2) have less than five consecutive identical nucleotides; (3) have no more than three dinucleotide repeats; and (4) have no more than three trinucleotide repeats. In some embodiments, the isolated nucleic acid has a length of about 19 to about 250 (e.g., about 27 to about 200, about 30 to about 200, about 33 to about 200, about 36 to about 150, about 39 to about 100, about 42 to about 75, about 45 to about 70, or about 48 to about 55) nucleotides. In some embodiments, the hotspot region is a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1207-1210.In some embodiments, the hotspot region is a nucleic acid sequence selected from the group consisting of region -636 to -496 (SEQ ID NO: 1207), region -351 to -294 (SEQ ID NO: 1208), region -236 to -187 (SEQ ID NO: 1209), or region -101 to -65 (SEQ ID NO: 1210) of the transcription start site of the UTRN gene. The present disclosure also provides a method for designing a saRNA, the method providing a saRNA that targets the isolated nucleic acid sequence of the present disclosure.

[0115] In some embodiments, the target sequence is designed / selected at least in part based on the criteria that allow the production of functional saRNA.For example, in some cases, the sequence located upstream of the TSS may contain a sequence that does not favor the synthesis of the target sequence, even though it is located in a hotspot region.

[0116] In some embodiments, target sequences within hotspot regions are selected, at least in part, based on criteria including sequences having a particular GC content (e.g., 25%-75% GC content) and lacking consecutive identical nucleotides, consecutive dinucleotides, or consecutive trinucleotides. In some embodiments, target sequences within hotspot regions include sequences having 35%-70% GC content, (1) sequences having less than five consecutive identical nucleotides, (2) sequences having three or fewer dinucleotide repeats, and (3) sequences having three or fewer trinucleotide repeats.

[0117] In some embodiments, the target sequence comprises a sequence with a GC content of 25%-75%, 30%-70%, 35%-70%, 40%-60%, or 45%-55%. In some embodiments, the saRNA comprises a sequence with a GC content of 35%-70%.

[0118] In some embodiments, the target sequence comprises a sequence having less than 7 consecutive identical nucleotides, less than 6 consecutive identical nucleotides, less than 5 consecutive identical nucleotides, less than 4 consecutive identical nucleotides, or less than 3 consecutive identical nucleotides. In some embodiments, the saRNA comprises a sequence having less than 5 consecutive identical nucleotides.

[0119] In some embodiments, the target sequence contains a sequence with 5 or fewer dinucleotide repeats, 4 or fewer dinucleotide repeats, 3 or fewer dinucleotide repeats, or 2 or fewer dinucleotide repeats. In some embodiments, the target sequence contains a sequence with 3 or fewer dinucleotide repeats.

[0120] In some embodiments, the target sequence contains a sequence with 5 or fewer trinucleotide repeats, 4 or fewer trinucleotide repeats, 3 or fewer trinucleotide repeats, or 2 or fewer trinucleotide repeats. In some embodiments, the target sequence contains a sequence with 3 or fewer trinucleotide repeats.

[0121] The RNAa activity of each designed saRNA depends on a number of complex factors, including the chromatin environment, sequence features of the target itself and neighboring regions, and transcription factor binding. The underlying determinant may be the accessibility of the DNA target. Regions with high availability may show high activity of the RNA. dsRNAs designed to target other regions of motility promotion may be non-functional, or may show transcriptional silencing effects. This may explain the existence of hotspot regions where functional SaRNAs are clustered together. For example, target sequences designed at least in part based on the following criteria: (1) have GC content between 35% and 70%, (2) have less than five consecutive identical nucleotides, (3) have three or fewer dinucleotide repeats; and (4) have three or fewer trinucleotide repeats may not activate / upregulate the expression of the UTRN gene by at least 10% compared to the baseline expression of the UTRN gene. This is because the target sequence to which the saRNA binds is not within a hotspot region (e.g., any of the hotspot regions described herein).

[0122] In certain embodiments, the present disclosure relates to an isolated nucleic acid complex comprising the saRNA disclosed in the present disclosure and the isolated nucleic acid sequence disclosed herein. In certain embodiments, the isolated nucleic acid complex activates expression of the UTRN gene by at least 10% compared to baseline expression of the UTRN gene.

[0123] In some aspects, methods are also provided that use isolated nucleic acids upstream of transcription target sites in the UTRN gene.

[0124] DNA encoding saRNA In certain embodiments, the present disclosure relates to a nucleic acid or polynucleotide encoding a saRNA that can activate or upregulate the expression of UTRN gene in a cell by at least 10% (e.g., compared to the baseline expression of UTRN gene).In certain embodiments, the nucleic acid is a DNA encoding the saRNA.In certain embodiments, the nucleic acid is a recombinant vector, particularly a recombinant AAV vector.The vector disclosed herein comprises a fragment of the DNA encoding the saRNA of the present disclosure.

[0125] Cells containing saRNA After contacting a cell, the saRNA disclosed herein can effectively activate or upregulate expression of the UTRN gene in the cell, e.g., upregulate expression by at least 10% (e.g., compared to baseline expression of the UTRN gene).

[0126] In certain embodiments, the present disclosure relates to a cell comprising the saRNA disclosed herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell, such as a human malignant embryonic rhabdomyomatosis cell (e.g., RD ​​cell). The cell disclosed herein may be in vitro or ex vivo, such as a cell line or cell line, or may be present in a mammalian body, such as a human body. The human body disclosed herein is a subject suffering from a disease or condition caused by UTRN gene mutation, low utrophin levels, and / or insufficient levels of functional utrophin in muscle. In some embodiments, the cell is from a subject suffering from DMD.

[0127] Compositions containing saRNA In certain embodiments, the present disclosure relates to a composition or pharmaceutical composition comprising the saRNA or nucleic acid of the present disclosure. In some embodiments, the composition comprises at least one pharma- ceutically acceptable carrier. In some embodiments, the composition comprises at least one pharma- ceutically acceptable carrier selected from an aqueous carrier, a liposome or LNP, a polymer, a micelle, a colloid, a metal nanoparticle, a non-metal nanoparticle, a bioconjugate (e.g., GalNAc), a polypeptide, an antibody, and any combination thereof. In one embodiment, the aqueous carrier may be, for example, RNase-free water or an RNase-free buffer solution. In some embodiments, the composition may contain 0.001-200 nM (e.g., 0.01-100 nM, 0.1-50 nM, 1-150 nM, 1-200 nM, 1-20 nM, 0.001-1 nM, 1-10 nM, 10-100 nM, 10-50 nM, 20-50 nM, 20-100 nM) of saRNA or isolated polynucleotide as described herein. In some embodiments, the composition contains 25 nM of saRNA or isolated polynucleotide as described herein.

[0128] How saRNA is used Another aspect of the present disclosure relates to an SARNA for activating / upregulating UTRN gene expression in a cell. The saRNA comprises an oligonucleotide sequence having a length of 16-35 contiguous nucleotides. In some embodiments, the oligonucleotide sequence has at least 75%, or at least 80%, or at least 85%, or at least 90% homology or complementarity with the isometric region of SEQ ID NO: 1200, and specifically, the saRNA activates / upregulates the expression of the UTRN gene by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 500%, at least 800%, at least 1000%, at least 2000%, or at least 5000%) compared to baseline expression of the UTRN gene. In certain embodiments, when the saRNA disclosed in the embodiments is administered to, for example, a cell or a subject, the expression of the UTRN gene is activated / upregulated by at least 1.1-fold (e.g., at least 1.2-fold, at least 1.5-fold, at least 1.8-fold, at least 2.0-fold, or at least 2.2-fold compared to the baseline expression of the UTRN gene). In certain embodiments, the saRNA activates or upregulates the expression of the UTRN gene by about 2.2-fold. In certain embodiments, expression of the UTRN gene is activated / upregulated by administering the disclosed saRNA to cells at a concentration of at least 0.01 nM, e.g., 0.02 nM, 0.05 nM, 0.08 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.8 nM, 1 nM, 5 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100 nM, 150 nM, 200 nM.In certain embodiments, induction of UTRN gene-encoded protein (utrophin) is activated / upregulated by administering the saRNA disclosed in the embodiments, for example, to a cell or subject, which increases the expression of UTRN gene-encoded protein (utrophin) by at least 1.1-fold (e.g., at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 6-fold, at least 7-fold, or at least 8-fold compared to baseline). In certain embodiments, the saRNA activates or upregulates the expression of utrophin protein by about 8.0-fold. In certain embodiments, induction of the protein encoding the UTRN gene (utrophin) is activated / upregulated by administering to cells a saRNA disclosed in the embodiments at a concentration of at least 0.01 nM, e.g., 0.02 nM, 0.05 nM, 0.08 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.8 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100 nM, 150 nM or 200 nM.

[0129] Another aspect of the present disclosure relates to a method for preventing or treating a disorder or condition induced by insufficient expression of dystrophin, dystrophin gene mutation, and / or low functional dystrophin level in an individual, comprising administering to the individual an effective amount of saRNA, a nucleic acid or isolated polynucleotide encoding the saRNA, or a composition comprising the saRNA disclosed herein. In certain embodiments, the effective amount of the saRNA disclosed herein is a concentration ranging from 0.01 nM to 50 nM, such as 0.01 nM, 0.02 nM, 0.05 nM, 0.08 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.8 nM, 1 nM, 5 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100 nM, 150 nM, or 200 nM. In some embodiments, the disorder or condition is DMD. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human.

[0130] In any of the embodiments provided herein, such saRNA, nucleic acid encoding the saRNA of the present disclosure, or composition comprising such saRNA of the present disclosure may be directly introduced into a cell, or may be produced in a cell when a nucleotide sequence encoding the saRNA is introduced into a cell, such as a mammalian cell, including but not limited to a RD cell, or a human cell. Such cells may be ex vivo, such as a cell line, or may be present in a mammalian body, such as a human. In some embodiments, the human is a subject or individual suffering from a dystrophin deficiency-associated condition or DMD or BMD. In certain embodiments, a nucleic acid or isolated polynucleotide encoding the saRNA described herein or a composition comprising the aforementioned saRNA, in an amount sufficient to treat DMD or BMD, respectively.

[0131] Another aspect of the present disclosure relates to administering an effective implementation of the saRNA or composition to an individual using the administration route described herein. In some embodiments, the administration route is selected from one or more of parenteral injection, oral administration, intranasal administration, inhalation administration, vaginal administration, and rectal administration. In some embodiments, the administration route is selected from one or more of intrathecal, intramuscular, intravenous, intraarterial, intraperitoneal, intravesical, intraventricular, intravitreal, and subcutaneous administration.

[0132] Dosage regimen and route of administration An aspect of the present disclosure relates to a pharmaceutical composition comprising the saRNA of the present disclosure.In some embodiments, the pharmaceutical composition comprises the saRNA of the present disclosure and pharmaceutically acceptable carrier, therapeutically inert carrier, diluent or pharmaceutically acceptable excipient.The pharmaceutical composition disclosed herein should be developed for the agent of preventing or treating dystrophin deficiency or DMD or BMD.

[0133] Aspects of the present disclosure also relate to methods of using the saRNAs of the present disclosure to prepare such compositions.

[0134] Another aspect of the present disclosure relates to the use of the saRNA of the present disclosure in the manufacture of a pharmaceutical composition disclosed herein.

[0135] Another aspect of the present disclosure relates to the use of saRNA or isolated polynucleotide according to any one of the embodiments described herein or according to the composition according to any one of the embodiments described herein in the manufacture of a medicament for the prevention or treatment of a gene or protein-related condition caused by insufficient expression of dystrophin, dystrophin gene mutation, and / or low functional dystrophin level in an individual.For use according to certain embodiments, the condition can include dystrophin mutation-related disorders or conditions, including DMD.For use according to certain embodiments, the condition caused by insufficient expression of utrophin is BMD or DMD.Also relevant is the use according to certain embodiments, where the individual is a mammal, for example a human.

[0136] The dosage that the saRNA or composition of the present disclosure can be administered can vary within wide limits and is adapted to individual requirements in each case.In certain embodiments, the first dosage of the pharmaceutical composition of the present disclosure is administered when the subject is less than 1 week old, less than 1 month old, less than 3 months old, less than 6 months old, less than 1 year old, less than 2 years old, less than 15 years old, or more than 15 years old.

[0137] A single dose of saRNA can be in the range of 0.01 mg / kg to 1000 mg / kg, for example, about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25, 30, 40, 50, 75, 100, 120, 150, 200, 250, 300, 400, 500, 750, or 1000 mg / kg. The doses described herein may contain two or more of the saRNA sequences described herein.

[0138] In some embodiments, the proposed dosing frequency is approximate.For example, in certain embodiments, if the proposed dosing frequency is a dose on day 1 and a second dose on day 29, the DMD patient can be administered the second dose 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 days after the administration of the first dose.In certain embodiments, if the proposed dosing frequency is a dose on day 1 and a second dose on day 15, the DMD patient can be administered the second dose 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after the administration of the first dose.In certain embodiments, if the proposed dosing frequency is a dose on day 1 and a second dose on day 85, the DMD patient can be administered the second dose 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 days after the administration of the first dose.

[0139] In certain embodiments, the dose and / or volume of the injection is adjusted based on the age of the subject, the weight of the subject, and / or other factors that may require adjustment of the parameters of the injection.

[0140] In certain embodiments, the pharmaceutical composition includes a co-solvent system. Some such co-solvent systems include, for example, benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of 3% w / v benzyl alcohol, 8% w / v of the non-polar surfactant Polysorbate 80™, and 65% w / v polyethylene glycol 300 in absolute ethanol. The proportions of such a co-solvent system may vary widely without significantly changing the solubility and toxicity characteristics. Additionally, the identity of the co-solvent components may vary, for example, with the addition of Polysorbate 80™. TM Other surfactants may be used in place of the polyethylene glycol. The fraction size of the polyethylene glycol may be varied. Other biocompatible polymers may be substituted for the polyethylene glycol, e.g., polyvinylpyrrolidone, and other sugars or polysaccharides may be substituted for the dextrose.

[0141] Examples of other compositions or components relevant to the saRNA, compositions, pharmaceutical compositions, and methods described herein include, but are not limited to, diluents, salts, buffers, chelating agents, preservatives, desiccants, antimicrobial agents, needles, syringes, packaging materials, tubes, bottles, flasks, beakers, etc., for example, using, modifying, assembling, storing, packaging, preparing, mixing, diluting, and / or preserving the components for a particular use. In embodiments in which a liquid form of any component is used, the liquid form may be concentrated or ready to use.

[0142] In some embodiments, lipid moieties used in nucleic acid therapy can be applied to the present disclosure for delivery of saRNA molecules disclosed herein. In such methods, nucleic acids (e.g., one or more saRNAs described herein) are introduced into preformed liposomes or lipoplexes made from a mixture of cationic lipids and neutral lipids. In certain methods, saRNA complexes with monocationic or polycationic lipids are formed without the presence of neutral lipids. In certain embodiments, lipid moieties are selected to increase distribution of pharmaceuticals to specific cells or tissues. In certain embodiments, lipid moieties are selected to increase drug distribution to adipose tissue. In certain embodiments, lipid moieties are selected to increase drug distribution to muscle tissue.

[0143] In certain embodiments, the pharmaceutical composition comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions, including those that comprise hydrophobic compounds. In certain embodiments, certain organic solvents, such as dimethylsulfoxide, are used.

[0144] In certain embodiments, a pharmaceutical composition comprises one or more tissue-specific delivery molecules designed to deliver one or more agents of the present disclosure to a particular tissue or cell type, for example, in certain embodiments, a pharmaceutical composition comprises a liposome coated with a tissue-specific antibody.

[0145] In some embodiments, saRNA can be delivered or administered via vector.Can use any vector that can be used for gene delivery.Can use viral vector in some embodiments.Non-limiting examples of viral vector that can be used in the present disclosure include but are not limited to human immunodeficiency virus;HSV, herpes simplex virus;MMSV, Moloney murine sarcoma virus;MSCV, mouse stem cell virus;SFV, Semliki Forest virus;SIN, Sindbis virus;VEE, Venezuelan equine encephalitis virus;VSV, vesicular stomatitis virus;VV, vaccinia virus;AAV, adeno-associated virus;Adenovirus;Lentivirus;and retrovirus.

[0146] In some embodiments, the vector is a recombinant AAV vector (rAAV). AAV vectors are DNA viruses of relatively small size that can stably and site-specifically integrate into the genome of infected cells. They can infect a wide range of cells without affecting cell growth, morphology, or differentiation, and are not thought to be involved in human pathology. The AAV genome has been cloned, sequenced, and characterized. It encompasses approximately 4700 bases and contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as the origin of viral replication. The remaining genome is divided into two essential regions: the left part of the genome contains the rep gene, which is involved in viral replication and expression, and the right part of the genome contains the cap gene, which encodes the viral capsid protein.

[0147] AAV vectors may be prepared using methods standard in the art. Adeno-associated viruses of any serotype are suitable (see, for example, Blacklow, "Parvoviruses and Human Diseases" JR Pattison, ed. (1988) p. 165-174; Rose, Comprehensive Virology 3:1, 1974; P. Tattersall "The Evolution of Parvovirus Taxonomy" In Parvoviruses (JR Kerr, SF Cotmore. ME Bloom, RM Linden, CR Parrish, Eds.) p 5-14, Hudder Arnold, London, UK (2006); and DE Bowles, JE Rabinowitz, RJ Samulski "The Genus Dependovirus" (JR Kerr, SF Cotmore. ME Bloom, RM Linden, CR Parrish, Eds.) p 15-23, Hudder Arnold, London, UK). (2006), the disclosures of which are incorporated herein by reference in their entirety. Vector purification methods are described, for example, in U.S. Patent Nos. 6,566,118, 6,989,264, and 6,995,006, and in International Publication No. WO 1999 / 011764, entitled "High-titer helper-free preparation of recombinant AAV vectors," the disclosures of which are incorporated herein by reference in their entirety. Hybrid vector preparations are described, for example, in PCT Application No. PCT / US2005 / 027091, the disclosures of which are incorporated herein by reference in their entirety. The use of AAV-derived vectors to transfer genes in vitro and in vivo has been described (see, e.g., International Patent Application Publication Nos. WO 91 / 18088 and WO 93 / 09239, U.S. Pat. Nos. 4,797,368, 6,596,535, and 5,139,941, and European Patent No. 0488528, the disclosures of which are incorporated by reference in their entireties herein).These publications describe various AAV-derived constructs in which the rep and / or cap genes are deleted and replaced by a gene of interest, and the use of these constructs to transfer the gene of interest in vitro (into cultured cells) or in vivo (directly into an organism). Replication-defective recombinant AAV (rAAV) according to the present disclosure can be prepared by co-transfecting a plasmid containing a nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions and a plasmid carrying the AAV packaging genes (rep and cap genes) into a cell line infected with a human helper virus (e.g., adenovirus). The resulting AAV recombinant is then purified by standard techniques.

[0148] In some embodiments, the vector(s) for use in the methods of the present disclosure are packaged in a viral particle (e.g., AAV viral particle, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16). Thus, the present disclosure may include a recombinant viral particle (containing a recombinant polynucleotide, and therefore a recombinant viral particle) comprising any of the vectors described herein. Methods for producing such particles are known in the art and are described in U.S. Pat. No. 6,596,535.

[0149] The preparations, pharmaceutical compositions, or medicaments of the present disclosure are formulated, administered, and administered in a manner consistent with proper medical practice.Factors to be considered in this context include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual subject, the cause of the disorder, the site of delivery of the drug, the method of administration, the schedule of administration, and other factors known to medical practitioners.

[0150] For formulations, pharmaceutical compositions, or medicaments of the disclosure, delivery can optionally be via parenteral injection, including intrathecal, intramuscular, intravenous, intraarterial, intraperitoneal, intravesical, intraventricular, intravitreal, or subcutaneous administration, or via oral, intranasal, inhalation, vaginal, or rectal administration.

[0151] The typical formulation of the oligonucleotide modulator of the present disclosure is prepared by mixing the saRNA of the present disclosure with carrier or excipient.Suitable carrier and excipient are well known to those skilled in the art and are described in detail in, for example, Ansel HC et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems (2004) Lipincott, Williams & Wilkins, Philadelphia; Gennaro AR et al., Remington: The Science and Practice of Pharmacy (2000) Lippincott, Williams & Wilkins, Philadelphia; and Wowe R. C, Handbook of Pharmaceutical Excipients (2005) Pharmaceutical Press, Chicago. The formulation may also contain one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifying agents, glidants, processing aids, colorants, sweeteners, germinants, flavorings, diluents and other known additives to provide an elegant display of a medicament (i.e., a saRNA of the present disclosure or a pharmaceutical composition thereof), or a pharmaceutical agent (i.e., a drug).

[0152] Diagnostic methods Another aspect of the present disclosure relates to a method for detecting dystrophin, utrophin or dystrophin-associated proteins (e.g., dystroglycan) in cells. In certain embodiments, the method comprises detecting dystrophin, utrophin or dystrophin-associated proteins (e.g., dystroglycan) in cells transfected with the saRNA, isolated polynucleotide or composition comprising the saRNA disclosed herein. In certain embodiments, the method disclosed herein can be applied to detect a specific subgroup of subjects suffering from disorders or conditions caused by insufficient expression of dystrophin, dystrophin gene mutations, and / or low functional dystrophin levels in individuals. As an alternative embodiment of the method disclosed herein, the method can be used for efficacy or safety monitoring of the aforementioned subjects treated with the saRNA, nucleic acid, or isolated polynucleotide encoding the saRNA disclosed herein.

[0153] In certain embodiments, baseline measurements are taken from biological samples as defined herein before carrying out treatment as described herein from an individual.In certain embodiments, the baseline expression of dystrophin or DMD gene is the expression of dystrophin or DMD gene obtained from biological samples before administration of SaRNA as described herein.In certain embodiments, the baseline expression of utrophin or UTRN gene is the expression of utrophin or UTRN gene obtained from biological samples before administration of Saran as described herein.In certain embodiments, the biological sample is peripheral blood cells, plasma, muscle cells, serum, skin tissue, cerebrospinal fluid (CSF).

[0154] In some embodiments, the saRNA provided herein activates the amount of functional utrophin in a cell by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 500%, at least 800%, at least 800%, at least 1000%, at least 2000%, or at least 5000%) compared to the baseline measurements described above.

[0155] In some embodiments, saRNA shows more than additive or synergistic effects in treating, preventing, delaying progression and / or improving diseases caused by dystrophin gene mutation.In some embodiments, saRNA shows greater additive or synergistic effects in protecting cells involved in the pathophysiology of disease, particularly for treating, preventing, delaying progression and / or improving DDD (e.g., DMD or BMD).

[0156] Another aspect of the present disclosure relates to a method for activating / upregulating the expression of UTRN gene in a cell, which comprises administering saRNA, or isolated polynucleotide, or composition of the embodiment disclosed herein.In some embodiments, saRNA, isolated polynucleotide, or composition is directly introduced into a cell.In some embodiments, saRNA of the embodiment disclosed herein is produced in the cell after nucleotide sequence encoding saRNA is introduced into the cell.In some embodiments, the cell disclosed herein is a mammalian cell, for example a human cell.

[0157] Another aspect of the present disclosure relates to a method for increasing the level of utrophin in a cell or the level of functional utrophin in the muscle of a subject, comprising introducing into the cell or subject an effective amount of a saRNA, a nucleic acid or polynucleotide encoding the saRNA, or a composition of an embodiment disclosed herein.

[0158] kit Another aspect of the present disclosure relates to a kit for carrying out the method for increasing utrophin level in cells or functional utrophin level in muscle, comprising saRNA disclosed herein.In certain embodiments, the kit further comprises a means for administering said saRNA to an individual.In certain embodiments, the kit is in a labeled package, and the label on said package indicates that saRNA or composition can be used to prevent or treat the disease or condition caused by insufficient expression of dystrophin, dystrophin gene mutation, and / or low-functional dystrophin level in an individual or for DDD, such as DMD or BMD.

[0159] As used herein, a "kit" generally defines a package, assembly, or container (such as an insulated container) that contains one or more of the components or embodiments of the present disclosure and / or other components relevant to the present disclosure, e.g., as described above. Any agents or components of the kit may be provided in liquid form (e.g., in solution) or in solid form (e.g., dried powder, frozen, etc.).

[0160] In additional embodiments, the kits may include instructions or directions to a website or other source in any form provided for using the kit in connection with the components and / or methods described herein. For example, the instructions may include instructions for use, modification, mixing, dilution, preservation, assembly, storage, packaging, and / or preparation of the components and / or other components associated with the kit. In some cases, the instructions may also include instructions for delivery of the components, for example, for shipment or storage at room temperature, sub-zero temperature, cryogenic temperature, etc. The instructions may be provided in any form useful to a user of the kit, such as provided in any manner, written or oral (e.g., telephone), digital, optical, visual (e.g., videotape, DVD, etc.), and / or electronic communication (including internet or web-based communication).

[0161] Another aspect of the present disclosure relates to a kit for detecting dystrophin, utrophin or dystrophin-related protein (e.g., dystroglycan) in cells.In certain embodiments, the kit is for detecting dystrophin, utrophin or dystrophin-related protein (e.g., dystroglycan) in cells transfected with any one or more of the saRNAs, isolated polynucleotides, or compositions disclosed herein.Also provided herein is a kit for increasing utrophin levels in cells.

[0162] Specific Embodiments The present disclosure provides the following specific embodiments:

[0163] Embodiment 1 is a small activating RNA (saRNA) comprising an oligonucleotide sequence having a length ranging from 16 to 35 contiguous nucleotides, wherein the oligonucleotide sequence comprises a contiguous nucleotide sequence having at least 75%, at least 80%, at least 85%, or at least 90% homology or complementarity to an equal length portion of SEQ ID NO: 1200, and the saRNA upregulates expression of the UTRN gene by at least 10% compared to baseline expression of the UTRN gene.

[0164] Embodiment 2 is the saRNA described in embodiment 1, in which an equal length portion of SEQ ID NO: 1200 is located in the region -636 to -496 (SEQ ID NO: 1207), the region -351 to -294 (SEQ ID NO: 1208), the region -236 to -187 (SEQ ID NO: 1209), or the region -101 to -65 (SEQ ID NO: 1210) of the transcription start site of the UTRN gene.

[0165] Embodiment 3 is an saRNA described in any one of embodiments 1 to 2, wherein the saRNA (1) has a GC content of 35% to 70%; (2) has less than five consecutive identical nucleotides; (3) has three or fewer dinucleotide repeats; and (4) has three or fewer trinucleotide repeats.

[0166] Embodiment 4 is the saRNA according to any one of embodiments 1 to 3, wherein the saRNA comprises a sense strand and an antisense strand.

[0167] Embodiment 5 is the saRNA according to any one of embodiments 1 to 4, wherein the oligonucleotide sequence is a sense strand or an antisense strand of the saRNA.

[0168] Embodiment 6 is a saRNA described in any one of embodiments 1 to 5, in which the sense strand and the antisense strand each contain a complementary region, and the complementary regions of the sense strand and the antisense strand form a double-stranded nucleic acid structure.

[0169] Embodiment 7 is the saRNA according to any one of embodiments 4 to 6, wherein the sense strand and the antisense strand have at least 90% complementarity.

[0170] Embodiment 8 is the saRNA of embodiment 4, wherein the sense strand and the antisense strand are located on two different nucleic acid strands.

[0171] Embodiment 9 is the saRNA of embodiment 4, wherein the sense strand and the antisense strand are arranged on a continuous nucleic acid strand, optionally a hairpin single-stranded nucleic acid molecule, and the complementary regions of the sense strand and the antisense strand form a double-stranded nucleic acid structure.

[0172] Embodiment 10 is the saRNA of embodiment 4, wherein at least one of the sense strand and the antisense strand comprises a 3' overhang ranging from 0 to 6 nucleotides in length.

[0173] Embodiment 11 is the saRNA of embodiment 10, wherein the sense and antisense strands comprise 3' overhangs ranging from 2 to 3 nucleotides in length.

[0174] Embodiment 12 is the saRNA of embodiment 10, wherein at least one of the nucleotides of the overhang is selected from or complementary to a corresponding nucleotide in the UTRN gene.

[0175] Embodiment 13 is a saRNA according to any of embodiments 4 to 12, wherein the sense strand and the antisense strand independently comprise a length of about 16 to about 35, about 17 to about 30, about 18 to about 25, or about 19 to about 22 consecutive nucleotides.

[0176] Embodiment 14 is a saRNA described in any one of embodiments 4 to 12, wherein the sense strand has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NOs: 400 to 797, and the antisense strand has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NOs: 800 to 1197.

[0177] Embodiment 15 is the saRNA of embodiment 14, wherein the sense strand comprises a nucleotide sequence selected from SEQ ID NOs: 400 to 797, and the antisense strand comprises a nucleotide sequence selected from SEQ ID NOs: 800 to 1197.

[0178] Embodiment 16 is the saRNA of embodiment 1, wherein the oligonucleotide sequence has at least 75% sequence homology or complementarity to a nucleotide sequence selected from SEQ ID NOs: 1-398.

[0179] Embodiment 17 is the saRNA of embodiment 4, wherein the sense strand has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NOs: 1-398.

[0180] Embodiment 18 is the saRNA of embodiment 4, wherein the antisense strand has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NOs: 1-398.

[0181] Embodiment 19 is the saRNA according to any one of embodiments 1 to 18, wherein at least one nucleotide of the saRNA is a chemically modified nucleotide.

[0182] Embodiment 20 is the saRNA of embodiment 19, wherein at least one nucleotide of the antisense strand and / or the sense strand is chemically modified.

[0183] Embodiment 21 is the saRNA of embodiment 19, wherein the chemically modified nucleotide is a nucleotide having at least one of the following modifications: a) modification of the phosphodiester bonds linking the nucleotides in the nucleotide sequence of the saRNA; b) modification of the 2'-OH of ribose in the nucleotide sequence of the saRNA; and c) Base modifications in the nucleotide sequence of the saRNA.

[0184] Embodiment 22 is the saRNA of embodiment 19, wherein at least one nucleotide of the saRNA is a locked nucleic acid, a basic nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, or a non-natural base containing nucleotide.

[0185] Embodiment 23 is the saRNA of embodiment 19, wherein the chemical modification of at least one chemically modified nucleotide is the addition of an (E)-vinylphosphonate moiety at the 5' end of the sense strand or the antisense strand.

[0186] Embodiment 24 is an saRNA described in any one of embodiments 1 to 23, wherein the sense strand or antisense strand of the saRNA is conjugated to one or more conjugation moieties selected from lipids, fatty acids, fluorophores, ligands, sugars, peptides, and antibodies.

[0187] Embodiment 25 is the saRNA described in embodiment 24, wherein the sense strand or antisense strand of the saRNA is conjugated to one or more conjugate moieties selected from a cell membrane penetrating peptide, polyethylene glycol, an alkaloid, a tryptamine, a benzimidazole, a quinolone, an amino acid, cholesterol, glucose, and N-acetylgalactosamine, and any combination thereof.

[0188] Embodiment 26 is an oligonucleotide regulatory element comprising one or more saRNAs according to any one of embodiments 1 to 25.

[0189] Embodiment 27 is an oligonucleotide modulator according to embodiment 26, further comprising one or more moieties or components conjugated, bound or associated with said saRNA(s).

[0190] Embodiment 28 is an oligonucleotide modulator according to embodiment 27, wherein the sense strand and / or the antisense strand of the saRNA is conjugated to one or more conjugation moieties selected from the group consisting of lipids, fatty acids, fluorophores, ligands, sugars, peptides, and antibodies.

[0191] Embodiment 29 is the oligonucleotide modulator of embodiment 27, wherein the conjugate moieties are independently selected from lipids, cell membrane penetrating peptides, polyethylene glycol, alkaloids, tryptamines, benzimidazoles, quinolones, amino acids, cholesterol, glucose, N-acetylgalactosamine, and any combination thereof.

[0192] Embodiment 30 further includes the SARNA conjugated or combined with one or more other active moieties for treating a UTRN-related disease or disorder, wherein one or more of the other active moieties is independently selected from SARNA, single-stranded oligonucleotides, chemical moieties, polypeptides, and antibodies, which is the oligonucleotide modulator described in embodiment 26.

[0193] Embodiment 31 is an isolated polynucleotide, wherein the isolated polynucleotide comprises the contiguous nucleotide sequence of embodiment 1.

[0194] Embodiment 32 is the isolated polynucleotide of embodiment 31, wherein the isolated polynucleotide is a nucleic acid sequence selected from SEQ ID NOs: 1-398.

[0195] Embodiment 33 is an isolated oligonucleotide complex comprising an antisense strand according to any one of embodiments 1 to 25 and an isolated polynucleotide according to any one of embodiments 31 to 32.

[0196] Embodiment 34 is the isolated oligonucleotide complex of embodiment 33, wherein the isolated oligonucleotide complex activates expression of the UTRN gene by at least 10% compared to baseline expression of the UTRN gene.

[0197] Embodiment 35 is an isolated nucleic acid sequence upstream of the transcription start site of the UTRN gene, wherein the isolated nucleic acid sequence is selected from SEQ ID NOs: 1207-1210.

[0198] Embodiment 36 is the isolated nucleic acid sequence according to embodiment 35, wherein the isolated nucleic acid sequence comprises the isolated polynucleotide according to any one of embodiments 31 to 32.

[0199] Embodiment 37 is an isolated nucleic acid sequence described in embodiment 35, wherein at least 25% of the designed saRNAs targeting the isolated nucleic acid sequence can activate expression of the UTRN gene by at least 10%, and the designed saRNA (1) has a GC content of 35% to 70%, (2) has less than five consecutive identical nucleotides, (3) has three or fewer dinucleotide repeats, and (4) has three or fewer trinucleotide repeats.

[0200] Embodiment 38 is an isolated nucleic acid complex comprising an antisense strand of the saRNA according to any one of embodiments 1 to 25, and a sense strand of the isolated nucleic acid sequence according to any one of embodiments 35 to 37.

[0201] Embodiment 39 is the isolated nucleic acid complex of embodiment 38, wherein the isolated nucleic acid complex activates expression of the UTRN gene by at least 10% compared to baseline expression of the UTRN gene.

[0202] Embodiment 40 is an isolated polynucleotide encoding the saRNA according to any one of embodiments 1 to 25.

[0203] Embodiment 41 is the isolated polynucleotide of embodiment 40, wherein the isolated polynucleotide is DNA.

[0204] Embodiment 42 is a vector comprising the isolated polynucleotide according to any one of embodiments 40 to 41.

[0205] Embodiment 43 is a host cell comprising the saRNA according to any one of embodiments 1 to 25, the isolated polynucleotide according to any one of embodiments 40 to 41, or the vector according to embodiment 42.

[0206] Embodiment 44 is a composition comprising the saRNA of any one of embodiments 1 to 25, or the isolated polynucleotide of embodiment 40 or embodiment 41, and optionally a pharma- ceutically acceptable carrier.

[0207] Embodiment 45 is the composition of embodiment 44, wherein the pharma- ceutically acceptable carrier is selected from the group consisting of aqueous carriers, liposomes, macromolecular polymers, polypeptides, and antibodies.

[0208] Embodiment 46 is the composition according to embodiment 44 or 45, wherein the composition comprises 0.001 to 200 nM of saRNA.

[0209] Embodiment 47 is the composition described in embodiment 46, wherein the composition comprises 1 to 200 nM of saRNA.

[0210] Embodiment 48 is an saRNA comprising an oligonucleotide sequence having a length ranging from 16 to 35 consecutive nucleotides for activating / upregulating expression of the UTRN gene in a cell, wherein the oligonucleotide sequence has at least 75%, or at least 80%, or at least 85%, or at least 90% sequence homology or complementarity to an equal length portion of SEQ ID NO: 1200, and wherein the saRNA activates expression of the UTRN gene by at least 10% compared to its baseline expression.

[0211] Embodiment 49 is an saRNA described in embodiment 48, in which a region of equal length to SEQ ID NO: 1200 is located in the region -636 to -496 (SEQ ID NO: 1207), in the region -351 to -294 (SEQ ID NO: 1208), in the region -236 to -187 (SEQ ID NO: 1209), or in the region -101 to -65 (SEQ ID NO: 1210) of the transcription start site of the UTRN gene.

[0212] Embodiment 50 is the saRNA described in embodiment 49, wherein the saRNA comprises a sense strand and an antisense strand, the sense strand comprises a nucleotide sequence selected from SEQ ID NOs: 400 to 797, and the antisense strand comprises a nucleotide sequence selected from SEQ ID NOs: 800 to 1197.

[0213] Embodiment 51 is a product for activating / upregulating UTRN gene expression in a cell, the product activates expression of the UTRN gene by at least 10% compared to baseline expression of the UTRN gene, and the product comprises an active substance selected from the saRNA described in any one of embodiments 1 to 25, the isolated polynucleotide described in any one of embodiments 40 to 41, the vector of embodiment 42, or the composition described in any one of embodiments 44 to 47.

[0214] Embodiment 52 is a product for activating / upregulating UTRN gene expression in a cell, wherein the active substance is directly introduced into said cell, and / or the cell is in vitro, ex vivo or in vivo; and / or The product wherein the cell is a mammalian cell.

[0215] Embodiment 53 is a method for treating a pulmonary arthritis, comprising administering to a patient a therapeutically effective amount of 1) comprising the step of formulating the active agent in a physiologically or pharma- ceutically acceptable carrier, such as one or more carriers selected from the group consisting of aqueous carriers, liposomes, high molecular weight polymers, polypeptides, and antibodies; and / or 2) conjugating the active agent to one or more conjugation moieties selected from lipids, cell membrane penetrating peptides, polyethylene glycol, alkaloids, tryptamines, benzimidazoles, quinolones, amino acids, cholesterol, glucose, and N-acetylgalactosamine, and any combination thereof (e.g., two conjugation moieties, one of which is a lipid and the other of which is N-acetylgalactosamine).

[0216] Embodiment 54 is the product of embodiment 53, wherein the conjugation moieties are independently derivatized to a fluorophore, a ligand, a sugar, a peptide, and an antibody.

[0217] Embodiment 55 is a product for activating / upregulating UTRN gene expression in a cell, wherein the cell is derived from a patient suffering from or having a disease or condition caused by insufficient expression of UTRN protein, a UTRN gene mutation, and / or low functional UTRN levels, and the active agent is administered in an amount sufficient to prevent or treat the disease or condition, such as Duchenne muscular dystrophy (DMD) or Beck muscular dystrophy (DMD).

[0218] Embodiment 56 is a method for preventing or treating a disease or condition caused by insufficient expression of dystrophin, a dystrophin gene mutation, and / or a low-functional dystrophin level in an individual, comprising administering to the individual an effective amount of the saRNA described in any one of embodiments 1 to 25, the isolated polynucleotide described in any one of embodiments 40 to 41, the vector described in embodiment 42, or the composition described in any one of embodiments 44 to 46.

[0219] Embodiment 57 is the method of embodiment 56, wherein the disease or condition is dystrophin deficiency disorder (DDD).

[0220] Embodiment 58 is the method of embodiment 56, wherein the disease or condition is Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD).

[0221] Embodiment 59 is the method of embodiment 56, wherein the individual is a mammal, and optionally the individual is a human.

[0222] Embodiment 60 is the method of embodiment 56, wherein the individual suffers from a condition caused by insufficient expression of dystrophin, a dystrophin gene mutation, and / or low functional dystrophin levels in the individual.

[0223] Embodiment 61 is the method described in embodiment 56, wherein the saRNA described in any one of embodiments 1 to 25, the isolated polynucleotide described in any one of embodiments 40 to 41, the vector of embodiment 42, or the composition described in any one of embodiments 44 to 47 is administered to an individual by a route of administration selected from one or more of parenteral injection, oral administration, intranasal administration, inhalation administration, vaginal administration, and rectal administration.

[0224] Embodiment 62 is the method of embodiment 61, wherein the route of administration is selected from one or more of intrathecal administration, intramuscular administration, intravenous administration, intraarterial administration, intraperitoneal administration, intravesical administration, intraventricular administration, intravitreal administration, and subcutaneous administration.

[0225] Embodiment 63 is the method of embodiment 56, wherein expression of UTRN gene mRNA in the individual is activated / upregulated by at least 10% compared to baseline expression of the UTRN gene.

[0226] Embodiment 64 is the method of embodiment 56, wherein the method increases utrophin levels in the individual by at least 10% compared to baseline expression of the UTRN gene.

[0227] Embodiment 65 is a method for detecting dystrophin, utrophin or a dystrophin-associated protein (e.g., dystroglycan) in a host cell according to embodiment 43.

[0228] Embodiment 66 is a kit for carrying out the method according to embodiment 56, comprising a) saRNA of any one of embodiments 1 to 25.

[0229] Embodiment 67 is the kit of embodiment 66, wherein the kit further comprises b) instructions for use, and c) optionally, a means for administering the saRNA of embodiments 1-25 to an individual.

[0230] Embodiment 68 is a kit comprising the saRNA described in any one of embodiments 1 to 25, the isolated polynucleotide described in any one of embodiments 40 to 41, the vector described in embodiment 42, or the composition described in any one of embodiments 44 to 47 in a labeled package and a label on the package, wherein the isolated polynucleotide, the vector or the composition can be used for the prevention or treatment of a disease or condition caused by insufficient expression of dystrophin, and is a therapeutic agent for Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD).

[0231] Embodiment 69 is a kit for detecting dystrophin, utrophin or a dystrophin-associated protein (e.g., dystroglycan) in a host cell according to embodiment 43.

[0232] Embodiment 70 is the use of a saRNA according to any one of embodiments 1 to 25, an isolated polynucleotide according to any one of embodiments 40 to 41, a vector according to any one of embodiments 42 or a composition according to any one of embodiments 44 to 47 for the preparation of a medicament for preventing or treating a disease or condition induced by insufficient expression of dystrophin, a dystrophin gene mutation and / or a low-functional dystrophin level in an individual.

[0233] Embodiment 71 is the use according to embodiment 70, wherein the disease or condition is Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD).

[0234] Embodiment 72 is the use of embodiment 70, wherein the individual is a mammal, optionally wherein the mammal is a human.

[0235] Embodiment 73 is the use of a saRNA according to any one of embodiments 1 to 25, an isolated polynucleotide according to any one of embodiments 40 to 41, a vector according to embodiment 42, or a composition according to any one of embodiments 44 to 47 in the preparation for activating / upregulating expression of the UTRN gene in a cell.

[0236] Embodiment 74 is the use according to embodiment 73, in which the saRNA according to any one of embodiments 1 to 25, or the isolated polynucleotide according to any one of embodiments 40 to 41, the vector according to embodiment 42, or the composition according to any one of embodiments 44 to 47 is directly introduced into a cell.

[0237] Embodiment 75 is the use described in embodiment 74, wherein the saRNA is produced in the cell after a nucleotide sequence encoding the saRNA is introduced into the cell.

[0238] Embodiment 76 is the use of any of embodiments 73 to 75, wherein the cell is a mammalian cell, optionally the mammalian cell is a human cell.

[0239] Embodiment 77 is the use according to embodiment 76, wherein the cell is in a human body.

[0240] Embodiment 78 is the use described in embodiment 77, wherein the human body is a subject suffering from a condition caused by insufficient expression of dystrophin, a dystrophin gene mutation, and / or low functional dystrophin levels in an individual, and the saRNA, isolated polynucleotide, or composition is administered in an amount sufficient to treat the condition.

[0241] Embodiment 79 is the use of embodiment 78, wherein the condition caused by insufficient expression of dystrophin is Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD).

[0242] Embodiment 80 is a method for activating / upregulating expression of the UTRN gene in a cell, comprising administering to the cell an effective amount of the saRNA described in any one of embodiments 1 to 25, the isolated polynucleotide described in any one of embodiments 40 to 41, the vector of embodiment 42, or the composition described in any one of embodiments 44 to 47.

[0243] Embodiment 81 is the method described in embodiment 80, in which the saRNA described in any one of embodiments 1 to 25, the isolated polynucleotide described in any one of embodiments 40 to 41, the vector of embodiment 42, or the composition described in any one of embodiments 44 to 47 is directly introduced into a cell.

[0244] Embodiment 82 is a method for directly introducing into a cell, comprising: 1) The saRNA according to any one of embodiments 1 to 25, the isolated polynucleotide according to any one of embodiments 40 to 41, the vector according to embodiment 42, or the saRNA in the composition according to any one of embodiments 44 to 47 is comprised in a pharma- ceutically acceptable carrier selected from the group consisting of an aqueous carrier, a liposome, a high molecular weight polymer, a polypeptide, and an antibody; 2) The saRNA described in any one of embodiments 1 to 25, the isolated polynucleotide described in any one of embodiments 40 to 41, the vector of embodiment 42, or the saRNA in the composition described in any one of embodiments 44 to 47 is conjugated to one or more conjugation moieties selected from a cell membrane-permeable peptide, polyethylene glycol, an alkaloid, tryptamine, benzimidazole, quinolone, an amino acid, cholesterol, glucose, and N-acetylgalactosamine.

[0245] Embodiment 83 is the method of any of embodiments 80 to 82, wherein the cell is a mammalian cell, such as a cell taken from a human.

[0246] Embodiment 84 is the method of embodiment 83, wherein the human body is a subject suffering from a condition caused by insufficient expression of dystrophin, a dystrophin gene mutation, and / or a low-functional dystrophin level in an individual, and the saRNA, isolated polynucleotide, or composition is administered in an amount sufficient to treat the condition.

[0247] Embodiment 85 is the method of embodiment 84, wherein the condition caused by insufficient expression of dystrophin is Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD).

[0248] Embodiment 86 is a method for increasing the level of utrophin in a cell, comprising introducing an effective amount of a saRNA described in any one of embodiments 1-25, an isolated polynucleotide described in any one of embodiments 40-41, a vector described in embodiment 42, or a saRNA in a composition described in any one of embodiments 44-47, wherein the saRNA, isolated polynucleotide, or composition activates expression of the UTRN gene by at least 10% compared to baseline UTRN gene expression. EXAMPLES

[0249] The present disclosure will be further illustrated with reference to the following specific examples and figures. It should be understood that these examples are intended to merely illustrate the present disclosure, rather than limiting the scope of the present disclosure. In the following examples, the test methods without specific conditions generally follow conventional conditions, such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or those recommended by manufacturers.

[0250] material and method dsRNA synthesis The present disclosure provides methods for preparing oligonucleotide modulators (dsRNA), including sequence design and synthesis.

[0251] dsRNA can be chemically synthesized or obtained from biotechnology companies that specialize in nucleic acid synthesis.Generally, the chemical synthesis of nucleic acid includes four steps: a) synthesis of oligomeric ribonucleotides, b) deprotection, c) purification and isolation, d) desalting and annealing.For example, the specific steps for the chemical synthesis of dsRNA described are as follows:

[0252] a) Synthesis of oligomeric ribonucleotides Synthesis of 1 μM RNA was performed on an automated DNA / RNA synthesizer (e.g., Applied Biosystems EXPEDITE8909), with a coupling time of 10–15 min for each cycle. Using solid-phase-bound 5'-Op-dimethoxytriphenylmethyl-thymidine substrate as the reaction initiator, one base was bound to the solid-phase substrate in the first cycle, followed by n ( One base was coupled in n-1 cycles (n ≥ 2) and this process was repeated until the synthesis of the entire nucleic acid sequence was completed.

[0253] b) Deprotection The solid-phase substrate bound to dsRNA was placed in a test tube, and 1 mL of a solution of a mixture of ethanol and ammonium hydroxide (volume ratio: 1:3) was added to the test tube. Then, the test tube was sealed and placed in an incubator, and the mixture was incubated at 25-70 °C for 2-30 hours. The solution containing the solid-phase substrate bound to dsRNA was filtered, and the filtrate was collected. The solid-phase substrate was rinsed twice (1 mL each time) with double-distilled water, and the filtrate was collected. The collected eluates were mixed and dried under vacuum for 1-12 hours. Next, 1 mL of a tetrabutylammonium fluoride in tetrahydrofuran solution (1 M) was added, and the mixture was left at room temperature for 4-12 hours, followed by the addition of 2 mL of n-butanol. The precipitate was collected and centrifuged at high speed to obtain a single-stranded dsRNA crude product.

[0254] c) Purification and separation The resulting crude dsRNA product was dissolved in 2 mL of 1 mol / mL aqueous ammonium acetate solution, and this solution was separated on a reversed-phase C18 column of high-pressure liquid chromatography to obtain a purified single-stranded dsRNA product.

[0255] d) Desalting and Annealing Salts were removed by gel filtration (size exclusion chromatography). A single sense oligomeric ribonucleic acid strand and a single antisense oligomeric ribonucleic acid strand were mixed in a 1:1 molar ratio in 1-2 mL of buffer (10 mM Tris, pH = 7.5-8.0, 50 mM NaCl). The solution was heated to 95 °C and then slowly cooled to room temperature to obtain a solution containing dsRNA.

[0256] Cell culture and treatment Human malignant embryonic rhabdomyomas cells (RD) (TCHu 45, Center for Excellence in Molecular Cell Science, Chinese Academy of Science, China) were cultured at 37 °C with 5% CO2 in modified DMEM medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA) supplemented with 10% bovine calf serum (Sigma-Aldrich) and 1% penicillin / streptomycin (Gibco). RD cells were seeded in 96-well plates at 4000 cells / well. Following the reverse transfection protocol, RD cells in each well were transfected individually with 0.3 μL of RNAiMAX (Invitrogen, Carlsbad, CA) at 25 nM, respectively, or any other concentration, with the transfection period being 3 or 5 days. Mock (blank control) was transfected without oligonucleotide. dsCon2 (SEQ ID NOs: 799 and 1199) was transfected as a non-specific double-stranded control. DS18-si8 (SEQ ID NOs: 399, 798 and 1198) is a double-stranded siRNA targeting the UTRN gene and was transfected as a silencing dsRNA control.

[0257] RNA isolation and reverse transcription quantitative polymerase chain reaction (RT-qPCR) (1) RNA isolation and one-step RT-qPCR At the end of transfection, the medium was discarded and the cells were washed with 150 μL of PBS per well. After discarding the PBS, 100 μL of cell lysis buffer (Power SYBR® Green Cells-to-Ct® Kit, Life Technologies) was added to each well and incubated at room temperature for 5 min. 0.5 μL of cell lysis was removed from each well and analyzed by RT-qPCR using One Step TB Green® PrimeScrip® RT-PCR Kit II (Takara, RR086A, Shlga, Japan) on a Roche Lightcycler 480 real-time PCR machine. PCR reactions were prepared using a Bravo automated liquid handling platform (Agilent, USA). Each transfection sample was amplified in triplicate wells. The PCR reaction conditions are shown in Table 2. [Table 2]

[0258] The reaction conditions were as follows: reverse transcription reaction (stage 1): 42°C for 5 min, 95°C for 10 s, PCR reaction (stage 2): 95°C for 5 s, 59°C for 20 s, 72°C for 10 s, 40 cycles of amplification, melting curve (stage 3). Human UTRN gene was amplified as the target gene. The geometric mean values ​​of TBP and B2M mRNA levels were used as internal references for RNA loading. Primer sequences are listed in Table 3. [Table 3]

[0259] (2) RNA isolation and two-step RT-qPCR To quantify mRNA expression in cells, total cellular RNA was isolated from treated cells using the RNeasy Plus Mini kit (Qiagen, Hilden, Germany) according to its manual. The obtained RNA (approximately 1 μg) was purified using PrimeScript with gDNA Eraser (Takara, RR047A, Shulga, Japan). TM The cDNA was reverse transcribed using a RT reagent kit. The resulting cDNA was then purified using B Green® Premix Ex Taq TM II (Takara, RR820A, Shulga, Japan) reagents and primers were used to amplify the target genes of interest specifically in a Roche LightCycler 480 Multiwell Plate 384 (Roche, Reference: 4729749001). The reaction conditions were as follows: reverse transcription reaction (stage 1): 42°C for 5 min, 95°C for 10 s, PCR reaction (stage 2): 95°C for 5 s, 60°C for 30 s, 72°C for 10 s, 40 cycles of amplification, melting curve (stage 3). The primer sequences are listed in Table 3. The PCR reaction conditions are shown in Tables 4 and 5. [Table 4] [Table 5]

[0260] Relative expression levels of UTRN gene in saRNA-transfected samples relative to the control treatment (mock) (E rel To calculate the Ct value, the Ct values ​​of the target gene and two internal reference genes were substituted into Equation I.

number

[0261] Western blotting Proteins were harvested from transfected cells using 1× RIPA buffer containing protease inhibitors. Protein concentrations were determined by BCA protein assay kit (Beyotime, P0010, Shanghai, China). Protein electrophoresis (10 μg protein / well) was performed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred to polyvinylidene difluoride (0.45 μm PVDF) membranes. Membranes were blotted with primary anti-UTRN antibody (Santa Cruz, sc-33700, USA) or anti-α / β tubulin antibody (CST, 2148s, USA) overnight at 4 °C. After washing three times with TBST buffer, they were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (CST, 7076s, USA) at room temperature for 1 h. The membrane was then washed three times for 10 min each with TBST buffer and analyzed with Image Lab (BIO-RAD, Chemistry Doctm MP Imaging System). The band densities of UTRN protein and α / β tubulin were quantified using ImageJ software.

[0262] Digital Western Blot Proteins were harvested from transfected cells using a denaturing cell lysis buffer containing protease inhibitors (Invent, SD-001, USA). Protein concentrations were determined by BCA protein assay kits. Proteins were detected and analyzed using Simple Western Automated Western Blot Systems (ProteinSimple, 004-600, USA). Protein electrophoresis was performed (0.1 μg / μl per well) using a separation module (ProteinSimple, SW004&SW008, USA) on a JESS. Capillaries were blotted with primary anti-utrophin (full length) antibody (Leica biosystems, NCL-DRP2, Germany) or anti-α / β tubulin antibody (CST, 2148S, USA). Afterwards, capillaries were blotted with HRP-conjugated secondary antibody and signals were detected with a detection kit (Protein simple, DM001&DM002, USA). Quantitative relative expression levels were calculated based on peak areas.

[0263] In the present disclosure, it was found that after being introduced into cells, the aforementioned saRNA can effectively increase the expression levels of UTRN mRNA and utrophin protein.

[0264] Example 1 Design and synthesis of dsRNA targeting the human UTRN promoter The 1000 nt coding strand of the promoter sequence of the human UTRN gene (SEQ ID NO: 1200) was extracted from the ENSEMBL genome database (www.ensembl.org). This sequence is located immediately upstream of the first nucleotide of the first exon of UTRN annotated by ENSEMBL (ENST00000433557.1). However, the 3' part of this sequence also contains the first exon of the NCBI annotated UTRN RefSeq mRNA sequence (NM_007124.3). Therefore, the first nucleotide of NM_007124.3 was considered as the true TSS (+1 position), and the downstream sequence was considered as the 5' untranslated region (UTR) (Table 6). [Table 6]

[0265] To identify functional dsRNAs capable of activating expression of the UTRN gene, we selected a series of 21-nt dsRNA targets on a 1000-bp UTRN promoter sequence, starting from -666 bp upstream of the TSS and moving 1 bp each time toward the TSS, resulting in 985 target sequences. We then filtered the target sequences to retain those that met the following criteria: (1) those with 35%–70% GC content, (2) those with fewer than five consecutive identical nucleotides, (3) those with three or fewer dinucleotide repeats, and (4) those with three or fewer trinucleotide repeats. After filtering, 398 target sequences remained and were used to determine the sense strand sequences of candidate saRNAs, of which 212 targets were on the promoter and 186 were on the 5'UTR. The strand composition and sequence of each dsRNA duplex containing its cognate target site in the UTRN promoter are listed in Table 1.

[0266] Example 2 High-throughput screening of dsRNA targeting the human UTRN promoter To identify dsRNAs that could activate UTRN transcription, RD cells were transfected with each of the 398 dsRNAs described above at a transfection concentration of 25 nM for 72 h, followed by UTRN gene expression analysis by one-step RT-qPCR.

[0267] Of the 398 dsRNAs screened, 108 (27.1%) induced UTRN expression, and 23 (5.8%), 56 (14.1%), and 29 (7.3%) dsRNAs showed high (≥1.5-fold), moderate (1.2- to 1.5-fold), and mild (1.1- to 1.2-fold) activation of UTRN mRNA expression, respectively (Table 7 ).

[0268] Of the 212 dsRNAs located in bona fide promoters, 99 (46.7%) induced UTRN expression, of which 20 (9.5%), 52 (24.5%), and 27 (12.7%) showed high (≥1.5-fold), moderate (1.2- to 1.5-fold), and mild (1.1- to 1.2-fold) activation of UTRN mRNA expression, respectively (Table 8 ).

[0269] Of the 186 dsRNAs targeting the 5′UTR region, the majority inhibited UTRN mRNA expression ( Fig. 2 ).

[0270] dsRNAs with activating activity (≧1.1-fold) are exemplified in this disclosure as "functional saRNAs." The relative changes in UTRN mRNA expression resulting from saRNA treatment are also summarized in Table 1, while the expression data organized by gene transfer are plotted in FIG. [Table 7] [Table 8]

[0271] When dsRNAs are sorted by their location on the human UTRN promoter and 5′UTR (Fig. 2 ), it is clearly seen that almost half of the dsRNAs targeting the authentic UTRN promoter (−666 to −1) induce UTRN mRNA expression.

[0272] Categorizing the expression data by the location of the target sites within the human UTRN promoter revealed four "hotspot regions" enriched for dsRNA activity, including regions -636 to -496 (H1), -351 to -294 (H2), -236 to -187 (H3), and -101 to -65 (H4), relative to the TSS (Figure 2). Approximately 55% of the target sequences of functional dsRNAs were located in the indicated "hotspot regions." Each "hotspot region" corresponding to the promoter sequence is listed in Table 9.

[0273] Design criteria: (i) GC content between 35-65%; (ii) less than five consecutive identical nucleotides; (iii) no more than three total dinucleotide repeats; and (iv) no more than three total trinucleotide repeats; in this example, it was considered that at least 25% of the designed SaRNAs targeting the provided hotspot region sequences could activate the expression of the UTRN gene by at least 10%.

[0274] In the "hotspot region" H1 (-636 to -496), the dsRNA duplexes capable of upregulating human UTRN expression by 1.1-fold or more were as follows: DS18-0384, DS18-0383, DS18-0382, DS18-0380, DS18-0379,DS18-0378, DS18-0377, DS18-0376, DS18-0375, DS18-0374, DS18-0373, DS18-0372, DS18-0371, DS18-0370, DS18-0368, DS18-0363, DS18-0362, DS18-0358, DS18-0357, DS18-0355, DS18-0354, DS18-0352, DS18-0350, DS18-0349, DS18-0348, DS18-0347, DS18-0345, DS18-0344, DS18-0343, DS18-0335, DS18-0334, DS18-0333, DS18-0332, DS18-0329, DS18-0328, DS18-0327, DS18-0325, DS18-0324, DS18-0323, DS18-0321, DS18-0320, DS18-0315, DS18-0314, DS18-0313, DS18-0312, DS18-0311, DS18-0310, DS18-0308, DS18-0305, DS18-0304, DS18-0301, DS18-0298.

[0275] In the "hot spot region" H2 (-351 to -294), the dsRNA duplexes capable of upregulating human UTRN expression by 1.1-fold or more were as follows: DS18-0277, DS18-0272, DS18-0271, DS18-0264, DS18-0255, DS18-0253, DS18-0252, DS18-0251, DS18-0248, DS18-0243.

[0276] In the “hotspot region” H3 (−236 to −187), the dsRNA duplexes capable of upregulating human UTRN expression by 1.1-fold or more were as follows: DS18-0237, DS18-0236, DS18-0234, DS18-0233, DS18-0232, DS18-0231, DS18-0230, DS18-0229, DS18-0228, DS18-0225, DS18-0223, DS18-0222, DS18-0221, DS18-0216, DS18-0211, DS18-0209, DS18-0208, DS18-0207, DS18-0206.

[0277] In the "hot spot region" H4 (-101 to -65), the dsRNA duplexes capable of upregulating human UTRN expression by 1.1-fold or more were as follows: DS18-0205, DS18-0204, DS18-0202, DS18-0200, DS18-0199, DS18-0198, DS18-0197, DS18-0194, DS18-0193, DS18-0192, DS18-0191, DS18-0189, DS18-0188, DS18-0187.

[0278] These results indicated that functional dsRNAs were not randomly distributed on the promoters, but were clustered in specific hotspot regions. The corresponding DNA sequences are shown in Table 9. [Table 9]

[0279] Example 3. saRNA treatment increases UTRN mRNA levels in RD cells Based on the screening results of UTRN induction, the top 38 performing SaRNAs (see Table 1) were transfected into RD cells with 25 nM of each SaRNA for 3 days. The transfected RD cells were then analyzed for UTRN mRNA expression by RT-qPCR. Both dsCon2 and DS18-si8 acted as non-specific double-stranded controls for gene activating and silencing dsRNA controls, respectively. UTRN mRNA expression of individual saRNAs is shown in Figure 3.

[0280] Example 4. saRNA treatment increases utrophin protein levels in RD cells A subset of the top performers (18 saRNAs) were transfected into RD cells at a concentration of 25 nM and utrophin protein was quantified 5 days later by Western blotting. Both dsCon2 and DS18-si8 acted as non-specific double-stranded controls for gene activating and silencing dsRNA controls, respectively. Figure 4 summarizes the relative fold change in utrophin levels derived from quantifying band intensity.

[0281] Example 5 Immunoreactivity of saRNA Structures and Sequences for saRNA Activity in Vitro To evaluate the effect of duplex structure and sequence specificity on saRNA activity, a series of saRNA variants were synthesized based on the three best performers (i.e., DS18-0198, DS18-0305, and DS18-0324) for activating human UTRN mRNA (Figure 5) and utrophin protein (Figure 6A-B). Table 10 lists the sequence composition and design of each saRNA variant. Each duplex was transfected into RD cells for 3 days at a concentration of 25 nM. UTRN mRNA levels were analyzed by two-step RT-qPCR, and utrophin protein levels were detected by JESS. Treatment with dsCon2 and DS18-si8 acted as nonspecific duplex controls for gene activation and silencing dsRNA controls, respectively. α / β tubulin protein acted as a control for protein loading. UTRN mRNA levels are shown in Figure 5. Utrophin protein bands are shown in Figure 6A, and utrophin protein levels are shown in Figure 6B. [Table 10-1] [Table 10-2]

[0282] In summary, high-throughput screening data revealed multiple "hotspot regions" for saRNA activity in the promoter of the human UTRN gene. Exemplary saRNAs increased the expression of both UTRN mRNA and utrophin protein levels. These results provide evidence that targeted activation of UTRN expression is a promising strategy for treating DDD, such as DMD and BMD. [Table 1-1] [Table 1-2] [Table 1-3]

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Claims

1. A small activating RNA (saRNA) comprising an oligonucleotide sequence having a length of 16 to 35 consecutive nucleotides, wherein the oligonucleotide sequence comprises a consecutive nucleotide sequence having at least 75%, at least 80%, at least 85%, or at least 90% homology or complementarity to an equal length portion of SEQ ID NO: 1200, and wherein the saRNA upregulates expression of the UTRN gene by at least 10% compared to baseline expression of the UTRN gene.

2. The saRNA of claim 1, wherein the equal length portion of SEQ ID NO: 1200 is located in the region -636 to -496 (SEQ ID NO: 1207), the region -351 to -294 (SEQ ID NO: 1208), the region -236 to -187 (SEQ ID NO: 1209), or the region -101 to -65 (SEQ ID NO: 1210) upstream of the transcription start site of the UTRN gene.

3. The saRNA of claim 1, wherein the saRNA has (1) a GC content of 35% to 70%, (2) less than five consecutive identical nucleotides, (3) no more than three dinucleotide repeats, and (4) no more than three trinucleotide repeats.

4. The saRNA of claim 1, wherein the saRNA comprises a sense strand and an antisense strand, the sense strand and the antisense strand each comprising a complementary region, the complementary regions of the sense strand and the antisense strand form a double-stranded nucleic acid structure, and the oligonucleotide sequence is the sense strand or the antisense strand of the saRNA.

5. The saRNA of claim 4, wherein the sense strand and the antisense strand have at least 90% complementarity.

6. The saRNA described in claim 4, wherein (1) the sense strand and the antisense strand are located on two different nucleic acid strands, or (2) the sense strand and the antisense strand are arranged on a continuous nucleic acid strand, optionally a hairpin single-stranded nucleic acid molecule, and the complementary regions of the sense strand and the antisense strand form a double-stranded nucleic acid structure.

7. The saRNA of claim 4, wherein at least one of the sense strand and the antisense strand comprises a 3' overhang ranging from 0 to 6 or 2 to 3 nucleotides in length.

8. 8. The saRNA of claim 7, wherein at least one of the nucleotides of the overhang is a nucleotide selected from the corresponding nucleotide on the UTRN gene, or is complementary thereto.

9. 5. The saRNA of claim 4, wherein the sense strand and the antisense strand independently comprise a length of about 16 to about 35, about 17 to about 30, about 18 to about 25, or about 19 to about 22 consecutive nucleotides.

10. The saRNA of claim 4, wherein the sense strand has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NOs: 400 to 797, and the antisense strand has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NOs: 800 to 1197, preferably wherein the sense strand comprises a nucleotide sequence selected from SEQ ID NOs: 400 to 797, and the antisense strand comprises a nucleotide sequence selected from SEQ ID NOs: 800 to 1197.

11. The saRNA of claim 1, wherein the oligonucleotide sequence has at least 75% sequence homology or complementarity to a nucleotide sequence selected from SEQ ID NOs: 1 to 398, or the sense strand has at least 75% sequence homology to a nucleotide sequence selected from SEQ ID NOs: 1 to 398, or the antisense strand has at least 75% sequence complementarity to a nucleotide sequence selected from SEQ ID NOs: 1 to 398.

12. The saRNA of claim 1, wherein at least one nucleotide of the antisense strand and / or sense strand of the saRNA is chemically modified.

13. The saRNA of claim 12, wherein the chemically modified nucleotide is a nucleotide having at least one of the following modifications: a) modification of the phosphodiester bonds linking the nucleotides in the nucleotide sequence of the saRNA; b) modification of the 2'-OH of ribose in the nucleotide sequence of the saRNA, and c) Base modifications in the nucleotide sequence of saRNA.

14. 13. The saRNA of claim 12, wherein at least one nucleotide of the antisense strand and / or sense strand of the saRNA is a locked nucleic acid, a basic nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, or a non-natural base containing nucleotide.

15. 13. The saRNA of claim 12, wherein the chemical modification of the at least one chemically modified nucleotide is the addition of an (E)-vinylphosphonate moiety at the 5' end of the sense strand or the antisense strand.

16. 5. The saRNA of claim 4, wherein the sense strand or antisense strand of the saRNA is conjugated to one or more conjugation moieties selected from a lipid, a fatty acid, a fluorophore, a ligand, a sugar, a peptide, and an antibody.

17. 17. The saRNA of claim 16, wherein the sense strand or antisense strand of the saRNA is conjugated to one or more conjugate moieties selected from a cell membrane-penetrating peptide, polyethylene glycol, an alkaloid, tryptamine, benzimidazole, quinolone, an amino acid, cholesterol, glucose, and N-acetylgalactosamine, and any combination thereof.

18. An oligonucleotide modulator comprising one or more saRNAs described in any one of claims 1 to 17, and further comprising one or more moieties or components conjugated, combined, or bound to the saRNA(s).

19. 19. The oligonucleotide modulator of claim 18, wherein the sense strand and / or antisense strand of the saRNA is conjugated to one or more conjugation moieties selected from the group consisting of lipids, fatty acids, fluorophores, ligands, sugars, peptides, and antibodies.

20. 20. The oligonucleotide modulator of claim 19, wherein said conjugation moieties are independently selected from lipids, cell membrane-penetrating peptides, polyethylene glycol, alkaloids, tryptamines, benzimidazoles, quinolones, amino acids, cholesterol, glucose, N-acetylgalactosamine, and any combination thereof.

21. The oligonucleotide modulator of claim 18, wherein the oligonucleotide modulator further comprises a saRNA conjugated or combined with one or more other active moieties for treating a UTRN-related disease or disorder, wherein the one or more other active moieties are independently selected from a saRNA, a single-stranded oligonucleotide, a chemical moiety, a polypeptide, and an antibody.

22. 10. An isolated polynucleotide, wherein said isolated polynucleotide comprises a contiguous nucleotide sequence as defined in claim 1.

23. 23. The isolated polynucleotide of claim 22, wherein the isolated polynucleotide is a nucleic acid sequence selected from SEQ ID NOs: 1-398.

24. An isolated oligonucleotide complex comprising the antisense strand of the saRNA of any one of claims 1 to 17 and the isolated polynucleotide of any one of claims 22 to 23.

25. An isolated nucleic acid sequence upstream of the transcription start site of the UTRN gene, said isolated nucleic acid sequence being selected from SEQ ID NOs: 1207-1210.

26. The isolated nucleic acid sequence of claim 25, wherein at least 25% of the designed SaRNAs targeting the isolated nucleic acid sequence are capable of activating UTRN gene expression by at least 10%, and the designed SaRNAs (1) have a GC content of 35% to 70%, (2) have fewer than five consecutive identical nucleotides, (3) have three or fewer dinucleotide repeats, and (4) have three or fewer trinucleotide repeats.

27. An isolated nucleic acid complex comprising the antisense strand of the saRNA of any one of claims 1 to 17 and the sense strand of the isolated nucleic acid sequence of any one of claims 25 to 26.

28. An isolated polynucleotide encoding the saRNA of any one of claims 1 to 17.

29. 29. The isolated polynucleotide of claim 28, wherein the isolated polynucleotide is DNA.

30. 29. A vector comprising the isolated polynucleotide of claim 28.

31. A composition comprising the saRNA of any one of claims 1 to 17, and optionally a pharmaceutically acceptable carrier.

32. 32. The composition of claim 31, wherein the pharmaceutically acceptable carrier is selected from the group consisting of an aqueous carrier, a liposome, a high molecular weight polymer, a polypeptide, and an antibody.

33. The composition of claim 31, wherein the composition comprises 0.001 to 200 nM or 1 to 200 nM of the saRNA.

34. A product for activating / upregulating UTRN gene expression in a cell, said product activating UTRN gene expression by at least 10% compared to baseline expression of said UTRN gene, said product comprising an active substance selected from one or more of the saRNAs described in any one of claims 1 to 17.

35. 35. The product according to claim 34 for activating / upregulating UTRN gene expression in cells, wherein the active substance is introduced directly into the cells and / or the cell is in vitro, ex vivo or in vivo; and / or The product, wherein the cells are mammalian cells.

36. The product described in claim 34, used to prevent or treat a disease or condition (e.g., dystrophin deficiency syndrome (DDD), Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD)) caused by insufficient expression of dystrophin, mutations in the dystrophin gene, and / or low levels of functional dystrophin in an individual (e.g., a mammal, particularly a human).

37. A kit for preventing or treating a disease or condition (e.g., dystrophin deficiency syndrome (DDD), Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD)) caused by insufficient expression of dystrophin, mutations in the dystrophin gene, and / or low levels of functional dystrophin in an individual (e.g., a mammal, particularly a human), comprising: a) a saRNA described in claims 1 to 17; optionally b) instructions for use; and / or c) means for administering the saRNA described in claims 1 to 17 to an individual.

38. Use of the saRNA described in any one of claims 1 to 17 in preparing a medicament for preventing or treating a disease or condition (e.g., dystrophin deficiency syndrome (DDD), Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD)) caused by insufficient expression of dystrophin, mutations in the dystrophin gene, and / or low levels of functional dystrophin in an individual (e.g., a mammal, particularly a human).

39. Use of the saRNA of any one of claims 1 to 17 in the preparation of a formulation for activating / upregulating the expression of the UTRN gene or for increasing the level of utrophin in a cell.

40. A product as described in claim 34 for use in activating / upregulating the expression of the UTRN gene or increasing the level of utrophin in cells (e.g., mammalian cells or cells from the human body).

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