Compositions and methods for treating duchenne muscular dystrophy

Abstract

The present application provides an engineered circular RNA capable of recruiting adenosine deaminase acting on RNA (ADAR; arRNA) and its application in treating Duchenne muscular dystrophy (DMD).

Description

Cross-references to related applications

[0001] This application claims priority to international application PCT / CN2023 / 118647, filed on September 13, 2023, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] This application belongs to the medical field. In particular, this application relates to an engineered circular RNA capable of recruiting adenosine deaminase (ADAR; arRNA) that acts on RNA, and its use in the treatment of Duchenne muscular dystrophy (DMD).

[0003] Information on sequence listings submitted electronically This application contains a sequence listing submitted electronically. The information contained in this electronic sequence listing (FH01123PCT-SEQListing.xml; size: 47KB; creation date: September 13, 2024) is incorporated herein by reference in its entirety. Background Technology

[0004] RNA splicing is a crucial step in gene expression in eukaryotes, during which introns are excised from primary transcripts and exons are joined sequentially. Different splicing patterns express functional proteins of varying lengths, greatly increasing protein diversity. RNA splicing is influenced by various factors, such as cis-acting element sequences on RNA, splicing regulatory proteins or factors, and post-transcriptional modifications. Abnormal splicing can lead to physiological dysfunction and is a direct cause of many diseases. It is estimated that 35-50% of human diseases are caused by gene splicing abnormalities, including some cancers and genetic diseases. Targeted regulation or modulation of the RNA splicing process offers new insights into the treatment of these diseases.

[0005] One such inherited disease, Duchenne muscular dystrophy (DMD), is caused by a defect in the expression of the protein dystrophin. The gene encoding dystrophin contains 79 exons spread across more than 2 million nucleotides of DNA. Any exon mutation, such as altering the reading frame, introducing a stop codon, or causing the deletion or duplication of one or more intact exons, can disrupt the production of functional dystrophin, leading to DMD. There are also less severe muscular dystrophys, such as Becker's muscular dystrophy (BMD). In this disease variant, the deletion of one or more exons does not alter the correct reading frame, so the translation of mRNA into the protein does not terminate prematurely. During the processing of the mutated dystrophin precursor mRNA, if splicing around a key exon preserves the correct reading frame of the gene, a protein-encoding mRNA with a short internal deletion is produced, which can retain some activity, resulting in the Becker phenotype (see, for example, Monaco et al., 1988, Genomics, 2: 90-95).

[0006] Targeting exon skipping may be particularly suitable for long genes. Long genes contain numerous exons and introns, with redundant exon genetic components, or one or more specific exons that are not essential for the expression of functional proteins. Recently, researchers have developed new tools for RNA editing using deaminase proteins, such as adenosine deaminase (ADAR), which acts on RNA. In mammalian cells, there are three types of ADAR proteins: Adar1 (two isoforms, p110 and p150), Adar2, and Adar3 (non-catalytically active). The catalytic substrate for ADAR proteins is double-stranded RNA. ADAR proteins can remove the NH2 group from the nucleotide base of adenosine (A), converting A to inosine (I); in subsequent cellular transcription and translation, inosine is recognized as guanosine (G) and pairs with cytidine (C). Researchers have constructed the λN-ADARDD system by fusing a λN peptide with the domains of human Adar1 or Adar2 deaminases. This system can be guided to specific RNA targets for binding via fusion RNA containing a BoxB stem-loop and antisense RNA. (See, for example, Montiel-Gonzalez et al., 2013, Proc Natl Acad Sci US A, 110: 18285-18290; Montiel-Gonzalez et al., 2016, Nucleic Acids Res, 44:E157; Sinnamon et al., 2017, Proc Natl Acad Sci USA, 114: E9395-E9402). This method can edit target A to I by introducing an AC mismatch at the target A base, thus achieving A-to-G base editing at the RNA level. Other methods of RNA editing include: fusing antisense RNA with an R / G motif (ADAR recruiting RNA scaffold); editing target RNA by overexpressing Adar1 or Adar2 proteins in mammalian cells; and using dCas13-ADAR for precise targeting and editing of RNA.Currently, the main tools reported for regulating DMD precursor mRNA splicing in vitro and in vivo are antisense oligonucleotides (see, for example, Matsuo et al., 1991, J Clin Invest, 87: 2127-2131; Takeshima et al., 1995, J Clin Invest, 95: 515-520; Pramono et al. 1996, Biochem Biophys ResCommun, 226: 445-449; Dunckley et al., 1996, Biochem Soc Trans, 24: 276S; Dunckley et al., 1998, Hum Mol Genet, 7: 1083-1090; Errington, et al., 2003, J Gene Med, 5, 518-527). The first example of specific and reproducible exon skipping in the MDX mouse model was achieved by Wilton et al. using antisense oligonucleotides (Wilton et al., 1999, Neuromuscle Disord, 9:330-338). However, the results showed that antisense oligonucleotides were less effective in immortalized cell cultures expressing higher levels of dystrophin.

[0007] More effective tools targeting DMD exons are still needed for efficient and long-term treatment of DMD. Summary of the Invention

[0008] In one general aspect, this document provides a circular RNA molecule for the treatment of DMD, having at least 50 nucleotides and comprising a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with at least one nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In some embodiments, the circular RNA molecule comprises at least one nucleotide sequence from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0009] In some embodiments, the RNA molecule has at least 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides.

[0010] In some embodiments, the RNA molecule comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence of SEQ ID NO:1. In some embodiments, the circular RNA molecule comprises the nucleotide sequence of SEQ ID NO:1. Preferably, the RNA molecule comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence of SEQ ID NO:4. In some embodiments, the circular RNA molecule comprises the nucleotide sequence of SEQ ID NO:4.

[0011] In some embodiments, the RNA molecule comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence of SEQ ID NO:2. Preferably, the RNA molecule comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence of SEQ ID NO:5. In some embodiments, the circular RNA molecule comprises the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:5.

[0012] In some embodiments, the RNA molecule comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence of SEQ ID NO:1, and the second nucleotide sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence of SEQ ID NO:2. In some embodiments, the circular RNA molecule comprises the first nuclear nucleotide sequence of SEQ ID NO:1 and the second nucleotide sequence of SEQ ID NO:2. Preferably, the RNA molecule comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence of SEQ ID NO:4, and the second nucleotide sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence of SEQ ID NO:5. In some embodiments, the circular RNA molecule comprises the first nucleotide sequence shown in SEQ ID NO:4 and the second nucleotide sequence shown in SEQ ID NO:5.

[0013] In some embodiments, the RNA molecule comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence of SEQ ID NO:3. Preferably, the RNA molecule comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence of SEQ ID NO:6. In some embodiments, the circular RNA molecule comprises the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:6.

[0014] In some embodiments, the RNA molecule comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a nucleotide sequence selected from SEQ ID NO:7 to SEQ ID NO:48. Preferably, the RNA molecule comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a nucleotide sequence selected from the group consisting of SEQ ID NO:13-15, 23, 24, 31-39, and 46. In some embodiments, the circular RNA molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:7 to SEQ ID NO:48, preferably, a nucleotide sequence selected from the group consisting of SEQ ID NO:13-15, 23, 24, 31-39, and 46.

[0015] In some embodiments, the RNA molecule comprises one or more modified nucleotides, such as deoxyribonucleotides, nucleotide analogs, abase nucleotides (referred to herein as Ab), 2'-modified nucleotides, 3' to 3' linked (reverse) nucleotides (referred to herein as invdN, invN, invn, invAb), nucleotides containing non-natural bases, bridging nucleotides, peptide nucleic acids (PNAs), 2',3'-open-ring nucleotide analogs (unlocked nucleobase analogs, referred to herein as NUNA), locked nucleotides (referred to herein as NLNA), 3'-O-methoxy (2' nucleoside-linked) nucleotides (referred to herein as 3'-OMen), 2'-F-arabinonucleotides (referred to herein as NfANA), 5'-Me-2'-fluoronucleotides (referred to herein as 5Me-Nf), morpholinonucleotides, vinylphosphonic acid deoxyribonucleotides (referred to herein as vpdN), nucleotides containing vinylphosphonic acid, and nucleotides containing cyclopropylphosphonic acid (cPrpN). 2'-Modified nucleotides (i.e., nucleotides with a group other than a hydroxyl group at the 2' position of the five-membered sugar ring) include, but are not limited to: 2'-O-methyl nucleotides (represented by the lowercase letter 'n' in the nucleotide sequence herein), 2'-deoxy-2'-fluoronucleotides (represented as Nf, or 2'-fluoronucleotides herein), 2'-deoxynucleotides (represented as dN herein), 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotides (represented as NM or 2'-MOE herein), 2'-amino nucleotides, or 2'-alkyl nucleotides.

[0016] In some embodiments, the RNA molecule contains one or more cytidine mismatches or guanosine mismatches that directly correspond to adenosine in the target sequence. In some embodiments, the RNA molecule contains a cytidine mismatch that directly corresponds to adenosine in the target sequence. In another embodiment, the RNA molecule contains a cytidine mismatch located at least 5 nucleotides away from the 3' end of the RNA molecule. In another embodiment, the RNA molecule contains a cytidine mismatch located at least 20 nucleotides away from the 5' end of the RNA molecule. In yet another embodiment, the RNA molecule contains a cytidine mismatch located at least 5 nucleotides away from the 3' end of the RNA molecule and at least 20 nucleotides away from the 5' end of the RNA molecule. In one embodiment, the RNA molecule contains a guanosine mismatch that directly corresponds to adenosine in the target sequence. In some embodiments, the RNA molecule contains two or more consecutive mismatched nucleotides corresponding to one or more adenosine residues in the target sequence.

[0017] Another general aspect of this application relates to a vector containing a nucleotide sequence encoding the RNA molecule of this application.

[0018] In some embodiments, the vector further comprises a 3' twisted nuclease sequence linked to the 3' end of the nucleotide sequence encoding the RNA molecule; and a 5' twisted nuclease sequence linked to the 5' end of the nucleotide sequence encoding the RNA molecule.

[0019] In some embodiments, the vector further comprises a promoter operatively linked to a nucleotide sequence encoding the RNA molecule. Preferably, the promoter is a polymerase II promoter (“Pol II promoter”), such as a CMV promoter or a U7 promoter; or a Pol III promoter, such as a U6 promoter.

[0020] In some embodiments, the vector is a viral vector or a non-viral vector.

[0021] In some embodiments, the vector is a viral vector, such as an adeno-associated virus (AAV) vector, a lentiviral vector, an adenovirus vector, an RNA replicon, a poxvirus vector, an enterovirus vector, a Venezuelan equine encephalitis virus vector, a Semliki forest virus vector, or a tobacco mosaic virus vector.

[0022] This application also provides a host cell containing the RNA molecule or vector described in this application.

[0023] This application also provides a pharmaceutical composition comprising the RNA molecule or carrier described in this application, and a pharmaceutically acceptable carrier.

[0024] In some embodiments, the pharmaceutical composition of this application comprises one or more lipids. Preferably, the pharmaceutical composition comprises lipid nanoparticles having one or more cationic lipids, non-cationic lipids, and polyethylene glycol (PEG) or PEG-modified lipids.

[0025] In some embodiments, the method of modulating RNA splicing includes administering an effective amount of an RNA molecule, vector, or pharmaceutical composition according to any embodiment of the present application to a subject in need, thereby modulating RNA splicing in precursor mRNA, for example, inducing exon skipping.

[0026] In some implementations, the subject is required to be treated for Duchenne muscular dystrophy, and the precursor mRNA is a precursor mRNA of the dystrophin gene or a fragment thereof.

[0027] It should be understood that one, some, or all of the features described in the various embodiments herein can be combined to form other embodiments of this application. The following detailed description will further describe these and other embodiments of this application. Attached Figure Description

[0028] The above and other objects, aspects, features and advantages of exemplary embodiments will become more apparent and better understood by taking into account the accompanying drawings and the following description.

[0029] Figure 1A and Figure 1B The exon skipping efficiency of arRNA constructs of different lengths is shown in HEK293T cells expressing the WT(A) or δ49 / 50(B) reporter genes.

[0030] Figure 2A and Figure 2B The exon skipping efficiency of arRNA constructs, each 100 nucleotides in length and targeting different locations, is shown in HEK293T cells expressing the WT(A) or δ49 / 50(B) reporter genes.

[0031] Figure 3A and Figure 3B The exon skipping efficiency of arRNA constructs is shown in HEK293T cells expressing the WT(A) or δ49 / 50(B) reporter genes when the AC mismatch is located at different positions.

[0032] Figure 4 The exon skipping efficiency of various arRNA constructs at different concentrations was shown in HEK293T cells expressing the WT reporter gene.

[0033] Figure 5 The structures of the δ49 / 50 and WT reporter genes are shown.

[0034] Figures 6A-6D The sequencing chromatograms of the intron regions between exons 48 and 51, and between exons 51 and 52, in the δ49 / 50 reporter gene plasmid are shown. Figure 6A and 6B ), and sequencing chromatograms of the intron regions between exons 50 and 51 and between exons 51 and 52 in the WT reporter gene plasmid ( Figure 6C and 6D ).

[0035] Figure 7 This demonstrates exon skipping mediated by arRNA at the protein level.

[0036] Figures 8A-8B It showed 4 weeks after the injection ( Figure 8A ) and 26 weeks ( Figure 8B The exon jumping efficiency detected in non-human primate tissues at the time of ( ).

[0037] Figure 9 The number of arRNAs detected in different non-human primate tissues is shown at 4 and 26 weeks post-injection. Detailed Implementation

[0038] Background Art and This specification references or describes in its entirety numerous publications, articles, patents, and patent applications; each of these references is incorporated herein by reference in its entirety. Discussions of documents, actions, materials, devices, articles, etc., included in this specification are intended to provide background information for the invention. Such discussions do not constitute an admission that any part or all of these matters constitutes prior art to any invention disclosed or claimed herein.

[0039] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art. All patents, patent applications, published applications, and other publications cited herein are incorporated herein by reference in their entirety. In the event of any discrepancy or inconsistency between the definitions set forth in this section and those in the patents, applications, or other publications incorporated herein by reference, the definitions set forth in this section shall prevail.

[0040] It should be understood that certain features of this application described in different implementation contexts for clarity may also be provided in combination in a single embodiment. Conversely, various features of this application, described in a single implementation context for brevity, may also be provided individually or in any suitable sub-combination. This application specifically covers and discloses all combinations of embodiments involving particular method steps, reagents, or conditions, as if each combination had been separately and explicitly disclosed.

[0041] As in this document and the appended claims, unless the context clearly specifies otherwise, the singular forms “a” or “an” and “the” include plural references. It should also be noted that claims can be drafted to exclude any optional elements. Therefore, this statement is intended to provide a basis for the use of exclusive terms such as “only” or “unique”, or for the use of negative qualifiers, in the formulation of claim elements.

[0042] Unless otherwise stated, any numerical values ​​described herein, such as concentrations or concentration ranges, should be understood to be modified by the term "about" in all cases. Therefore, numerical values ​​typically include ±10% of the stated value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges explicitly includes all possible subranges, all individual values ​​within that range, including integers and fractions of values ​​within that range, unless the context clearly specifies otherwise.

[0043] Unless otherwise stated, when the term "at least" is used before a series of elements, it should be understood to refer to each element in that series. Those skilled in the art will recognize, or can determine by conventional experimentation alone, numerous equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be included within the scope of the invention.

[0044] As used herein, the terms “comprise / comprising,” “include / including,” “have / having,” “contain / containing,” or any other variations thereof, shall be understood to include the stated integers or groups of integers, but not exclude any other integers or groups of integers intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that comprises a list of elements may not be limited to those elements, but may include other elements not expressly listed, or other elements inherent in the composition, mixture, process, method, article, or apparatus itself. Furthermore, unless expressly stated otherwise, “or” shall be understood as an inclusive “or,” not an exclusive “or.” For example, conditions “A or B” may be satisfied in any of the following ways: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); and both A and B are true (or exist).

[0045] It should also be understood that when terms such as “about,” “approximately,” “usually,” “substantially,” and similar terms are used herein to describe the size or characteristics of a component in a preferred embodiment of this application, it means that the described size / characteristic is not a strict limit or parameter, and does not exclude minor deviations that are functionally identical or similar as understood by one of ordinary skill in the art. At a minimum, deviations covered by such expressions, including numerical parameters, should be deviations that do not change the least significant figure of the value when applying mathematically and industrially recognized principles (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.).

[0046] For sequence alignment, a reference sequence is typically used, and the sequence to be tested is compared to it. When using a sequence alignment algorithm, the sequence to be tested and the reference sequence must be input into the computer. If necessary, the coordinates of subsequences must be specified, and the parameters of the algorithm program must be set. Subsequently, the sequence alignment algorithm will calculate the percentage of sequence identity between the sequence to be tested and the reference sequence based on the set program parameters.

[0047] Optimal alignment of sequences for comparison can be achieved through various methods, such as Smith & Waterman's local homology algorithm (Adv. Appl. Math. 1981; 2:482), Needleman and Wunsch's homology alignment algorithm (J. Mol. Biol. 1970; 48:443), Pearson and Lipman's similarity search method (Proc. Nat'l. Acad. Sci. USA 1988; 85:2444), computer program implementations of the above algorithms (such as the GAP, BESTFIT, FASTA, and TFASTA programs in the Wisconsin genetics software package, Genetics Computer Group, 575 Science Dr., Madison, WI), or visual inspection (generally see: Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., 1995Supplement (Ausubel)).

[0048] Examples of algorithms suitable for determining sequence identity percentages and sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., J. Mol. Biol. 1990; 215: 403-410 and Altschul et al., Nucleic Acids Res. 1997; 25: 3389-3402, respectively. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (NCBI). The algorithm first identifies high-scoring sequence pairs (HSPs) by recognizing short words of length W in the query sequence; when these short words are compared to words of the same length in the database sequence, they either match exactly or satisfy a positive threshold score T. T is called the neighbor word score threshold (Altschul et al., ibid.). These initial neighbor word hits serve as seeds to initiate a search for longer HSPs containing them. Subsequently, these word hits extend in both directions along each sequence until the accumulated alignment score can no longer increase.

[0049] For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for matching residue pairs; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Word matching extension in each direction is terminated when any of the following conditions are met: the cumulative alignment score decreases by X from its maximum value; the cumulative score drops to zero or below due to the accumulation of one or more negatively scored residue alignments; or the ends of either sequence are aligned. The parameters W, T, and X of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) defaults to a word length (W) of 11, an expected value (E) of 10, M=5, N=-4, and compares both strands. For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 score matrix by default (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 1989; 89:10915).

[0050] In addition to calculating the percentage of sequence identity, the BLAST algorithm also performs statistical analysis on the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 1993; 90: 5873-5787). One similarity metric provided by the BLAST algorithm is the minimum sum probability (P(N)), which reflects the probability that a match between two nucleotide or amino acid sequences is accidental. For example, when a test nucleic acid is compared with a reference nucleic acid sequence, if its minimum sum probability is less than about 0.1, the nucleic acid is considered similar to the reference sequence; if it is less than about 0.01, the similarity is higher; and if it is less than about 0.001, the similarity is the highest.

[0051] Another piece of evidence for the high degree of identity between two nucleic acid sequences or peptides is that, as described below, the peptide encoded by the first nucleic acid and the peptide encoded by the second nucleic acid exhibit immune cross-reactivity. Therefore, when one peptide differs from another peptide (for example) only in conserved amino acid substitutions, they are generally considered to be highly identical. Another piece of evidence for the high degree of identity between two nucleic acid sequences is that the two molecules can hybridize under stringent conditions.

[0052] As used herein, the terms “deaminase recruiting RNA,” “arRNA,” “ADAR recruiting RNA,” and “arRNA” are used interchangeably and all refer to engineered RNAs capable of recruiting ADAR to target exons and / or adjacent 5' introns.

[0053] As used herein, the terms “peptide,” “polypeptide,” or “protein” refer to a molecule composed of amino acids that can be recognized as a protein by those skilled in the art. Amino acid residues are represented herein by conventional single-letter or three-letter codes. The terms “peptide,” “polypeptide,” and “protein” are used interchangeably in this document to refer to an amino acid polymer of any length. This polymer may be linear or branched, and may contain modified amino acids or be interrupted by non-amino acid sequences. These terms also cover amino acid polymers modified by natural or artificial means, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (e.g., coupling with a labeled component). This definition also includes polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids), and other modifications known in the art.

[0054] The peptide sequences described herein follow conventional writing rules, with the N-terminal region on the left and the C-terminal region on the right. Although isoforms of amino acids are known, all references are to the L-form unless otherwise explicitly stated.

[0055] As used herein, the term "domain" refers to a structure in a biomolecule that participates in its specific function. A domain may comprise a single, continuous region (e.g., a continuous sequence) or multiple separate, discontinuous regions (e.g., discontinuous sequences) in a biomolecule. Examples of protein domains include, but are not limited to, endonuclease domains, DNA-binding domains, and reverse transcription domains; examples of nucleic acid domains are regulatory domains, such as transcription factor-binding domains. In some embodiments, a domain (e.g., a Cas domain) may comprise two or more smaller domains (e.g., a DNA-binding domain and an endonuclease domain).

[0056] The term "heterogeneous," when used to describe a first element relative to a second element, means that the first element does not exist in nature in the manner described for the second element. For example, heterologous peptides, nucleic acid molecules, constructs, or sequences refer to: (a) peptides, nucleic acid molecules, or portions thereof that are not naturally present in the cells in which they are expressed; (b) peptides or nucleic acid molecules, or portions thereof, that have been altered or mutated relative to their native state; or (c) peptides or nucleic acid molecules whose expression has been altered compared to their native expression levels under similar conditions. For example, heterologous regulatory sequences (such as promoters or enhancers) can be used to regulate the expression of a gene or nucleic acid molecule in a manner different from its normal expression in nature. In another example, heterologous domains of a peptide or nucleic acid sequence (e.g., the DNA-binding domain of the peptide or the nucleic acid encoding the peptide's DNA-binding domain) may be arranged differently relative to other domains, or may have different sequences or origins relative to other domains or portions thereof of the peptide or its encoded nucleic acid. In some embodiments, heterologous nucleic acid molecules may be present in the genome of the native host cell, but their expression levels may be altered, or their sequences may be different, or both. In other embodiments, the heterologous nucleic acid molecule may not be inherent to the host cell or the host genome, but may be introduced into the host cell through transformation (e.g., transfection, electroporation), wherein the introduced molecule may be integrated into the host genome, or may exist temporarily as extrachromosomal genetic material (e.g., mRNA), or exist in a semi-stable state for more than one generation (e.g., augmented viral vectors, plasmids or other self-replicating vectors).

[0057] The terms “polynucleotide,” “nucleotide sequence,” and “nucleic acid” are used interchangeably. They refer to polymeric forms of nucleotides of any length, whether they are deoxyribonucleotides, ribonucleotides, or their analogues.

[0058] As used herein, the term "nucleic acid molecule" refers to RNA and DNA molecules, including but not limited to cDNA, genomic DNA, and mRNA, as well as synthetic nucleic acid molecules, such as those produced by chemical synthesis or recombination as described herein, such as RNA templates. Nucleic acid molecules can be double-stranded or single-stranded, circular or linear. If single-stranded, the nucleic acid molecule can be a sense strand or an antisense strand.

[0059] As used herein, the terms “adenine,” “guanine,” “cytosine,” “thymidine,” “uracil,” and “hypoxanthine” all refer to the corresponding bases themselves. The terms “adenosine,” “guanosine,” “cytidine,” “thymidine,” “uridine,” and “inosine” refer to the base linked to the ribose or deoxyribose portion. The term “nucleoside” refers to the base linked to the ribose or deoxyribose portion. The term “nucleotide” refers to the corresponding base-ribose-phosphate or base-deoxyribose-phosphate. Sometimes, the terms adenosine and adenine (abbreviated as “A”), guanosine and guanine (abbreviated as “G”), cytosine and cytidine (abbreviated as “C”), uracil and uridine (abbreviated as “U”), thymidine and thymidine (abbreviated as “T”), and inosine and hypoxanthine (abbreviated as “I”) are used interchangeably to refer to the corresponding bases, nucleosides, or nucleotides. Unless the context explicitly requires a distinction, the terms base, nucleoside, and nucleotide are sometimes used interchangeably.

[0060] The term "precursor mRNA" as used in this application refers to the primary transcript of a gene, which may contain introns and exons and requires further splicing to produce an mRNA molecule containing only exons.

[0061] In the context of this application, "target sequence" refers to a sequence in the precursor mRNA to which an arRNA sequence designed is fully or highly complementary, and the hybridization between the target sequence and the arRNA forms a double-stranded RNA (dsRNA) region containing the target adenosine. This region recruits ADAR, which selectively deaminates the target adenosine. In some embodiments, the adenosine deaminase is naturally present in host cells, such as eukaryotic cells (preferably mammalian cells, more preferably human cells). In some embodiments, the ADAR is introduced into the host cell. In some embodiments, the target sequence comprises a sequence in exon 51 of the dystrophin precursor mRNA, to which the binding of the RNA molecule in this application causes exon 51 to be skipped during splicing.

[0062] As used herein, “complementarity” refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid through conventional Watson-Crick base pairing. The percentage of complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairing) with a second nucleic acid (e.g., approximately 5, 6, 7, 8, 9, and 10 out of 10 represent approximately 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively). “Complete complementarity” means that all consecutive residues in one nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence. As used herein, “substantially complementary” means that the complementarity is at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% in regions of approximately 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides, or refers to two nucleic acids that can hybridize under stringent conditions.

[0063] As used herein, “carrier” includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed thereto at the doses and concentrations used. Physiologically acceptable carriers are typically pH buffer solutions. Non-limiting examples of physiologically acceptable carriers include: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. (Continuing here...) As used herein, an "effective amount" or "therapeutic effective amount" means the minimum concentration required to produce a measurable improvement or prevention of a particular disease. Effective amounts as used herein can vary depending on factors such as the patient's disease state, age, sex, weight, and the ability of a substance to elicit a desired response in an individual. An effective amount also refers to a dose in which the beneficial effects of treatment outweigh any toxic or adverse effects of treatment. In the context of cancer, an effective amount comprises a dose sufficient to shrink a tumor and / or reduce its growth rate (e.g., inhibit tumor growth), or to prevent or delay the proliferation of other unwanted cells in cancer. In some embodiments, an effective amount is a dose sufficient to delay cancer development. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. In some embodiments, an effective amount is an amount sufficient to reduce an individual's recurrence rate. An effective amount can be administered in one or more doses. An effective amount of a drug or composition may: (i) reduce the number of cancer cells; (ii) shrink the size of a tumor; (iii) inhibit, hinder, slow down, and preferably prevent cancer cell infiltration into surrounding organs to a certain extent; (iv) inhibit (i.e., slow down and preferably prevent) tumor metastasis to a certain extent; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of a tumor; (vii) reduce the tumor recurrence rate; and / or (viii) alleviate one or more cancer-related symptoms to a certain extent. An effective amount may be administered in one or more doses. For the purposes of this disclosure, an effective amount of a drug, compound, or pharmaceutical composition means a dose sufficient to directly or indirectly achieve preventative or therapeutic treatment. As is known in clinical practice, an effective amount of a drug, compound, or pharmaceutical composition may be achieved in combination with another drug, compound, or pharmaceutical composition, or may not be achieved. Therefore, an "effective amount" may be considered in the context of administration of one or more therapeutic agents, where the dose of the single drug may be considered an effective dose if the desired result can or has been achieved when used in combination with one or more other agents.

[0064] As described herein, "host cell" means any cell type that can be used as a host cell, provided that it can be modified as described herein. For example, a host cell can be a host cell that endogenously expresses ADAR, or a host cell that has ADAR introduced by methods known in the art. For example, a host cell can be a prokaryotic cell, a eukaryotic cell, or a plant cell. In some embodiments, the host cell is derived from an established cell line, such as a mammalian cell line, including human or non-human cell lines. In some embodiments, the host cell is derived from an individual, such as a human individual.

[0065] A “recombinant AAV vector (rAAV vector)” refers to a polynucleotide vector containing one or more heterologous sequences (i.e., non-AAV-derived nucleic acid sequences), with at least one flanking, in some embodiments, two AAV inverted terminal repeats (ITRs). When such an rAAV vector is present in a host cell infected with a suitable helper virus (or expressing a suitable helper function) and expressing the AAV rep and cap gene products (i.e., AAV Rep and Cap proteins), it can be replicated and packaged into infectious viral particles. When an rAAV vector is integrated into a larger polynucleotide (e.g., into a chromosome or other vectors used for cloning or transfection, such as plasmids), it can be referred to as a “precursor vector” and, in the presence of AAV packaging function and a suitable helper function, can be “rescued” through replication and capsidation processes. rAAV vectors can be in any of a variety of forms, including but not limited to plasmids, linear artificial chromosomes, lipid complexes, encapsulated in liposomes, and encapsulated in viral particles (especially AAV particles). The rAAV vector can be packaged into the capsid of an AAV virus to generate "recombinant adeno-associated virus particles (rAAV particles)".

[0066] "AAV inverted terminal repeat (ITR) sequence" is a well-known term in the art; it is a sequence of approximately 145 nucleotides located at both ends of a natural single-stranded AAV genome. The outermost 125 nucleotides of the ITR can be oriented in two different directions, resulting in heterogeneity between different AAV genomes and between the ends of a single AAV genome. The outermost 125 nucleotides also contain several shorter self-complementary regions (designated as A, A', B, B', C, C', and D regions), allowing intrastrand base pairing to occur in this part of the ITR.

[0067] "Instructions for use of a drug" refers to the instructions for use that are usually included in the commercial packaging of a drug. It contains information on indications, dosage, administration method, contraindications, other drugs that can be used in combination with the packaged product, and / or warnings about the use of such drugs.

[0068] The terms "subject," "patient," or "individual" include mammals, such as humans or other animals, and generally refer to humans. In some embodiments, the subject (e.g., patient) receiving the therapeutic agent and composition is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or ape. The subject can be male or female and can be of any suitable age, including infants, children, adolescents, adults, and elderly subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent, dog, cat, or farm animal, such as a cow or horse.

[0069] Engineered RNA for exon skipping For most eukaryotic genes, their DNA is transcribed to form precursor mRNA containing introns and exons. This precursor mRNA must be processed to remove introns to become mature messenger RNA (mRNA), which then guides protein synthesis. "RNA splicing" (also known as "exon splicing") refers to the process of removing or "splicing away" certain spacer sequences, thereby linking exons together. In some implementations, RNA splicing removes the intron between two adjacent exons. In other implementations, RNA splicing removes both the intron and the exon between two exons. "Exon skipping" refers to a type of RNA splicing in which one or more exons or segments thereof are removed. The final mRNA contains the remaining exons linked together.

[0070] The splicing process is guided by a complex, multi-component mechanism. Splice sites are mainly divided into three types: the 5' splice site (5'ss) located at the 5' end of the intron, the 3' splice site (3ss) located at the 3' end of the intron, and the branch point (BPS) located 18-40 nt upstream of the 3'ss. The first step in splicing is that the U1 small nuclear ribonucleoprotein (snRNP) recognizes the 5'ss and binds to it through complementary base pairing. Simultaneously, the U2 small nuclear RNA cofactor 2 (U2AF2) binds to the polypyrimidine region and interacts with splicing factor 1 (SF1) to assist in its binding to the BPS. Furthermore, U2 small nuclear RNA cofactor 1 (U2AF1) binds to the 3'ss, forming the E complex. Then, with the assistance of U2AF, the U2 snRNP replaces SF1 and binds to the branch point, forming the A complex. Numerous splicing factors play important roles in this process. After complex A is formed and stabilized, the addition of trimeric snRNP particles (U4, U6, U5) causes a rearrangement of complex A, bringing the three splicing sites closer together. U4 and U6 snRNPs bind together through complementary pairing of their RNA components, while U5 snRNP binds loosely through protein-protein interactions, forming complex B. Then, through a series of conformational changes, U1 snRNP dissociates, and U6 snRNP binds to the 5'ss. Simultaneously, U4 snRNP dissociates, allowing U6 and U2 snRNPs to pair via snRNA. This rearrangement process forms the catalytically active complex B, which then undergoes two transesterification reactions. The first transesterification reaction connects the 5'ss and BPS, and cleaves the RNA strand to form a lasso structure, forming complex C. In the second transesterification reaction, the exon cleaved in the first step attacks the 3'ss; the exons connect to form the mRNA to be released, while the introns are released in a lasso structure, subsequently undergoing rapid degradation. The snRNPs released during the splicing process will again participate in the assembly, rearrangement, and catalysis of the spliceosome.

[0071] One general aspect of this application relates to an RNA molecule, specifically a circular RNA molecule, that can be used to regulate RNA splicing, such as exon skipping, in the precursor mRNA of the dystrophin gene. The dystrophin gene is also known as the Duchenne muscular dystrophy (DMD) gene. Mutations in the DMD gene (2.24 million base pairs) can lead to the biochemical loss or abnormality of dystrophin. The DMD gene has multiple gene promoters that drive the expression of different mRNA (and their encoded protein) isoforms. The human DMD gene produces a “full-length” 14 kb mRNA containing 79 exons using three gene promoters closest to the 5' end (Dp427B, Dp427M, Dp427P), encoding a 427 kDa membrane cytoskeletal protein (dystrophin). This protein is expressed in all skeletal muscle, smooth muscle (blood vessels and viscera), the heart, peripheral nerves, and some neurons. See, for example, Takeda et al., Neuromuscul Dis. 2021; 8(Suppl 2): ​​S343–S358, which is incorporated herein by reference in its entirety.

[0072] In some embodiments, the RNA molecule contains a target RNA sequence that can hybridize with a target RNA associated with a disease or condition; wherein the RNA is capable of recruiting ADAR to deaminate a target adenosine residue in the target RNA, and wherein the arRNA is a circular RNA or capable of forming a circular RNA.

[0073] In some embodiments, mutations in the DMD pre-mRNA may cause frameshifts during translation, and alternative RNA splicing can produce a mature mRNA that corrects the frameshift. In other embodiments, nonsense mutations exist in the DMD pre-mRNA, and alternative RNA splicing can produce a mature mRNA that does not contain an exon with a premature stop codon. Therefore, exon skipping can increase the content of the functional dystrophin peptide, thereby treating or preventing DMD. This functional dystrophin peptide can be a truncated peptide compared to dystrophin produced in healthy subjects whose DMD mRNA does not contain a frameshift or premature stop codon. In some embodiments, the RNA molecule of this application can be used to induce or promote skipping of one or more exons in the dystrophin pre-mRNA. For example, the RNA molecule in this application can be used to induce a single skipping of exon 51 in dystrophin pre-mRNA, or simultaneously with skipping of exons 53, 45, 44, 46, 50, 52, 55, 43, 54, 8, 7, or 6, exons 45-55, or exons 3-9. In some embodiments, an increase in the frequency of skipping of one or more exons in dystrophin mRNA can lead to an increase in the expression of functional dystrophin in the subject or in the subject's cells. In some embodiments, DMD in the subject can be treated or prevented by administering engineered polynucleotides to the subject to edit DMD precursor mRNA to achieve exon skipping.

[0074] In one general aspect, this application provides a circular RNA molecule having at least 50 nucleotides and comprising a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with at least one nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0075] In some embodiments, the RNA molecule has at least 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides.

[0076] In one embodiment, the circular RNA molecule comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:1. For example, the RNA molecule may comprise the nucleotide sequence of SEQ ID NO:1, such as nucleotide sequences selected from the group consisting of SEQ ID NO:7-39. The RNA molecule may also comprise the nucleotide sequence of SEQ ID NO:4, such as nucleotide sequences selected from the group consisting of SEQ ID NO:13-17, 23, 24, and 31-39.

[0077] In another embodiment, the circular RNA molecule comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:2. For example, the RNA molecule may comprise a nucleotide sequence of SEQ ID NO:2, such as nucleotide sequences selected from the group consisting of SEQ ID NO:9-14, 17-23, and 25-44. The RNA molecule may also comprise a nucleotide sequence of SEQ ID NO:5, such as nucleotide sequences selected from the group consisting of SEQ ID NO:13, 14, and 30-44.

[0078] In another embodiment, the circular RNA molecule comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:3. For example, the RNA molecule may comprise a nucleotide sequence of SEQ ID NO:3, such as a nucleotide sequence selected from SEQ ID NO:45-48. The RNA molecule may also comprise a nucleotide sequence of SEQ ID NO:6, such as a nucleotide sequence selected from SEQ ID NO:45 and 46.

[0079] In some embodiments, the circular RNA molecule comprises a first nucleotide sequence and a second nucleotide sequence; wherein the first nucleotide sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of the sequences in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and the second nucleotide sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with another sequence in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0080] In one embodiment, the circular RNA molecule comprises a first nucleotide sequence and a second nucleotide sequence; wherein the first nucleotide sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:1, and the second nucleotide sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:2. For example, the RNA molecule may comprise the nucleotide sequences of SEQ ID NO:1 and SEQ ID NO:2, such as nucleotide sequences selected from the group consisting of SEQ ID NO:9-14, 17-23, and 25-39. The RNA molecule may also comprise the nucleotide sequences of SEQ ID NO:4 and SEQ ID NO:5, such as nucleotide sequences selected from the group consisting of SEQ ID NO:13, 14, and 31-38.

[0081] In some embodiments, the RNA molecule contains a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence selected from SEQ ID NOs:7-48.

[0082] Preferably, the RNA molecule contains a nucleotide sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with nucleotide sequences selected from the group consisting of SEQ ID NO: 13-15, 23, 24, 31-39 and 46.

[0083] In some embodiments, the RNA molecule comprises one or more modified nucleotides, such as deoxyribonucleotides, nucleotide analogs, abase nucleotides (referred to herein as Ab), 2'-modified nucleotides, 3' to 3' linked (reverse) nucleotides (referred to herein as invdN, invN, invn, invAb), nucleotides containing non-natural bases, bridging nucleotides, peptide nucleic acids (PNAs), 2',3'-open-ring nucleotide analogs (unlocked nucleobase analogs, referred to herein as NUNA), locked nucleotides (referred to herein as NLNA), 3'-O-methoxy (2' nucleoside-linked) nucleotides (referred to herein as 3'-OMen), 2'-F-arabinonucleotides (referred to herein as NfANA), and 5'-Me-2'-fluoronucleotides. Acids (referred to as 5Me-Nf in this article), morpholinonucleotides, vinylphosphonic acid deoxyribonucleotides (referred to as vpdN in this article), nucleotides containing vinylphosphonic acid, and nucleotides containing cyclopropylphosphonic acid (cPrpN), 2'-modified nucleotides (i.e., nucleotides with a group other than a hydroxyl group at the 2' position of the five-membered sugar ring), including but not limited to: 2'-O-methyl nucleotides (referred to as lowercase 'n' in the nucleotide sequence), 2'-deoxy-2'-fluoronucleotides (referred to as Nf in this article, also referred to as 2'-fluoronucleotides), 2'-deoxynucleotides (referred to as dN in this article), 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotides (referred to as NM or 2'-MOE in this article), 2'-amino nucleotides, or 2'-alkyl nucleotides.

[0084] According to this disclosure, the RNA molecules in this application can be prepared by methods known in the art. For example, they can be prepared by chemical synthesis, in vitro transcription, in vivo transcription, or a combination thereof.

[0085] Vector and host cell A vector containing a nucleotide sequence encoding the RNA molecule of this application is also provided.

[0086] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked to it. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends, or without free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and other types of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA fragments can be inserted, for example, using standard molecular cloning techniques. Some vectors are capable of autonomous replication in the host cell they are introduced into (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors). Other vectors (e.g., non-free mammalian vectors) integrate into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome. Furthermore, some vectors are capable of directing the transcription or expression of coding nucleotide sequences operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0087] Recombinant expression vectors may contain nucleic acids as described in this application, in a form suitable for transcription or expression in host cells. In some embodiments, the recombinant expression vector includes one or more regulatory elements that may be selected according to the host cell used for transcription or expression and are operatively linked to the nucleic acid sequence to be transcribed or expressed. In recombinant expression vectors, "operatively linked" means that the target nucleotide sequence is linked to the regulatory element in a manner that allows the expression of that nucleotide sequence (e.g., in an in vitro transcription / translation system, or within the host cell after the vector has been introduced into the host cell).

[0088] In some embodiments, the vector further comprises a 3' twisted ribozyme sequence linked to the 3' end of the nucleotide sequence encoding the arRNA. In some embodiments, the vector further comprises a 5' twisted ribozyme sequence linked to the 5' end of the nucleotide sequence encoding the arRNA. In some embodiments, the vector further comprises a 3' twisted ribozyme sequence linked to the 3' end of the nucleotide sequence encoding the arRNA; and a 5' twisted ribozyme sequence linked to the 5' end of the nucleotide sequence encoding the arRNA. In some embodiments, the 3' twisted sequence is twisted P3 U2A, and the 5' twisted sequence is twisted P1. In some embodiments, the 5' twisted sequence is twisted P3 U2A, and the 3' twisted sequence is twisted P1. A twisted ribozyme is a self-cleaving catalytic RNA structure. In some embodiments, the arRNA can undergo autocatalytic cleavage. In some embodiments, the catalytic arRNA product contains a 5'-hydroxyl group and a 2',3'-cyclic phosphate group at the 3' end.

[0089] In some embodiments, the arRNA transcript is further flanked by 5' and / or 3' linker sequences, with 5'-twitch nuclease and / or 3'-twitch nuclease respectively linked to the outer side of these linker sequences. In some embodiments, the arRNA contains a 3' linker sequence. In some embodiments, the arRNA contains a 5' linker sequence.

[0090] In some embodiments, the vector further comprises a promoter operatively linked to a nucleotide sequence encoding the RNA molecule. Preferably, the promoter is a polymerase II promoter (“Pol II promoter”), such as the CMV promoter or the U7 promoter; or a Pol III promoter, such as the U6 promoter.

[0091] In some embodiments, the vector is a viral vector or a non-viral vector. In some embodiments, the vector is a viral vector, such as an adeno-associated virus (AAV) vector, a lentiviral vector, an adenovirus vector, an RNA replicon, a poxvirus vector, an enterovirus vector, a Venezuelan equine encephalitis virus vector, a Semliki forest virus vector, or a tobacco mosaic virus vector.

[0092] In some embodiments, the vector is an rAAV vector. In some embodiments, the rAAV vector is a vector derived from an AAV serotype, including but not limited to: its ITR derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAVDJ, goat AAV, bovine AAV, or mouse AAV capsid serotypes, etc. In some embodiments, the nucleic acid in the AAV comprises the ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAVDJ, goat AAV, bovine AAV, or mouse AAV capsid serotypes. In some embodiments, the nucleic acid in the AAV also encodes the arRNA described herein. The use of any AAV serotype is within the scope of this disclosure. In some embodiments, the vector is encapsulated in rAAV particles. In some embodiments, the AAV virus particles comprise AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAVDJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV2V708K, AAV2-HBKO, AAVDJ8, AAVPHP.B, AAVPHP.eB, AAVBR1, AAVHSC15, AAVHSC17, goat AAV, AAV1 / AAV2 chimera, bovine AAV, mouse AAV, or rAAV2 / HBoV1 serotype capsid.

[0093] A host cell is also provided, comprising the RNA molecule or vector of this application. In some embodiments, the host cell is a cell line, such as HEK293T, HT29, A549, HepG2, RD, SF268, SW13, and HeLa cells. In some embodiments, the host cell is a primary cell, such as a fibroblast, epithelial cell, or immune cell. In some embodiments, the host cell is a T cell. In some embodiments, the host cell is a post-mitotic cell. In some embodiments, the host cell is a cell of the central nervous system (CNS), such as brain cells, for example, cerebellar cells.

[0094] Compositions, kits and products This document also provides compositions (e.g., pharmaceutical compositions) containing any of the RNA, vectors, or host cells of this application.

[0095] In some embodiments, a pharmaceutical composition is provided comprising the RNA molecule or carrier of this application, and a pharmaceutically acceptable carrier.

[0096] Acceptable carriers, excipients, or stabilizers are non-toxic to the receptor at the doses and concentrations used and include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride, hexamethyl ammonium chloride, benzalkonium chloride, benzyl chloride, phenol, butanol, or benzyl alcohol; alkyl parabens, such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); and low molecular weight (less than approximately 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium ions; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as Tween. TM Prunnick TM Alternatively, polyethylene glycol (PEG) may be used. In some embodiments, lyophilized formulations are provided. Pharmaceutical compositions intended for in vivo administration must be sterile. This can be easily achieved, for example, by filtration through a sterile filter membrane.

[0097] In some embodiments, the pharmaceutical composition comprises one or more lipids; preferably, the pharmaceutical composition comprises lipid nanoparticles having one or more cationic lipids, non-cationic lipids, and polyethylene glycol (PEG) or PEG-modified lipids.

[0098] The RNA molecule or the nucleic acid encoding the RNA molecule in this application may be conjugated to lipids, nanoparticles, polymers, liposomes, micelles, dodecyl phosphate choline (DPC), or other delivery systems available in the art. The RNA molecule or its encoded nucleic acid may also be chemically conjugated to targeting groups, lipids (including but not limited to cholesterol and cholesterol derivatives), nanoparticles, polymers, liposomes, micelles, DPC (see, for example, WO 2000 / 053722, WO 2008 / 0022309, WO 2011 / 104169, WO 2012 / 083185, WO 2013 / 032829, WO 2013 / 158141, WO 2017 / 067640, WO 2009 / 092612, the entire contents of which are incorporated herein by reference) or other delivery systems available in the art.

[0099] In addition, kits are provided for use with any of the RNA editing methods or treatments described herein, comprising any of the arRNAs, vectors, compositions or edited host cells described herein.

[0100] In some embodiments, a kit for regulating exon jumping in a host cell is provided, comprising the arRNA molecule of this application or a construct comprising a nucleic acid encoding said arRNA.

[0101] In some embodiments, a kit for regulating exon jumping in a host cell is provided, comprising arRNA or a construct comprising a nucleic acid encoding arRNA; wherein the arRNA comprises a target RNA sequence hybridized to a target RNA, wherein the arRNA is capable of recruiting ADAR to deaminate a target adenosine residue in the target RNA, and wherein the construct comprises a polymerase II promoter (“Pol II promoter”) operatively linked to the nucleic acid encoding the arRNA.

[0102] In some embodiments, the kit further comprises ADAR or a construct comprising a nucleic acid encoding ADAR. In some embodiments, the kit further comprises an ADAR3 inhibitor or a construct thereof. In some embodiments, the kit further comprises an interferon stimulator or a construct thereof. In some embodiments, the kit further comprises instructions for performing any of the RNA editing methods or therapeutic methods described herein.

[0103] The kits in this application are packaged appropriately. Appropriate packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags), etc. The kits may optionally include additional components such as transfection or transduction reagents, cell culture media, buffers, and explanatory information.

[0104] Therefore, this application also provides an article of manufacture. This article of manufacture may comprise a container and a label or packaging instructions located on or associated with the container. Suitable containers include vials (e.g., sealed vials), bottles, jars, flexible packaging, etc. In some embodiments, the container contains a pharmaceutical composition and may have a sterile access port (e.g., the container may be an intravenous infusion bag or a vial with a stopper that can be punctured by a hypodermic needle). The container containing the pharmaceutical composition may be a reusable vial, allowing for multiple administrations of the reconstituted formulation (e.g., 2-6 doses). A pharmaceutical package insert refers to instructions for use typically included in the commercial packaging of a pharmaceutical product, containing information about the product's indications, usage, dosage, route of administration, contraindications, and / or warnings. Furthermore, the article of manufacture may also comprise a second container containing pharmaceutically acceptable buffer solutions, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. It may also include other materials required from a commercial and user perspective, including additional buffer solutions, diluents, filters, needles, and syringes.

[0105] The kit or product may include multiple unit doses of the pharmaceutical composition and instructions for use, and the packaging quantity is sufficient to meet the storage and use needs of pharmacies (such as hospital pharmacies and dispensing pharmacies).

[0106] Treatment In a general aspect, this application also provides a method for regulating RNA splicing (e.g., exon skipping). More specifically, a method for regulating RNA splicing (e.g., exon skipping) is provided, comprising administering an effective amount of the RNA molecule, vector, or pharmaceutical composition of this application to a subject in need, thereby inducing RNA splicing (e.g., exon skipping) in pre-mRNA.

[0107] In some embodiments, the subject requires treatment for a disease or condition, including but not limited to hereditary diseases. In some embodiments, the subject requires treatment for Duchenne muscular dystrophy, and the pre-mRNA is the pre-mRNA of the dystrophin gene or a fragment thereof. The subject may have a mutation, such as a deletion, in the dystrophin (DMD) gene. In some embodiments, the subject has one or more frameshift deletions that disrupt the reading frame in the dystrophin (DMD) gene. In some embodiments, the subject has one or more in-frame deletions in the DMD gene. Preferably, the subject has one or more frameshift and / or in-frame deletions in exons 1 to 22 and / or exons 43 to 55. See, for example, Echigoya et al., 2018, J.Pers. Med., 8: 41.

[0108] In some embodiments, this application provides a method for preventive treatment of a subject to prevent or at least alleviate Duchenne muscular dystrophy, comprising administering to the subject an effective amount of the RNA molecule, carrier, or pharmaceutical composition of this application.

[0109] An effective amount of the RNA molecule, carrier, or pharmaceutical composition described in this application can be administered to the subject in need by any suitable method in the art. For example, the RNA molecule, carrier, or pharmaceutical composition can be delivered via intravenous, intra-arterial, intraperitoneal, intramuscular, or subcutaneous routes of administration.

[0110] Implementation methods listed item by item The exemplary implementation of this application is as follows: 1. A circular ribonucleic acid (RNA) molecule having at least 50 nucleotides and comprising a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with at least one nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0111] 1a. A circular ribonucleic acid (RNA) molecule having at least 50 nucleotides and comprising at least one nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

[0112] 2. An RNA molecule according to embodiment 1 or 1a, having at least 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 nucleotides.

[0113] 3. An RNA molecule according to any one of embodiments 1 to 2, comprising a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:1.

[0114] 3a. The RNA molecule according to Embodiment 3, comprising the nucleotide sequence of SEQ ID NO:1.

[0115] 4. The RNA molecule according to Embodiment 3, comprising a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a nucleotide sequence selected from SEQ ID NO:7-39.

[0116] 4a. The RNA molecule according to embodiment 3a, comprising nucleotide sequences selected from the group consisting of: SEQ ID NO:7-39.

[0117] 5. The RNA molecule according to Embodiment 4, comprising a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:4.

[0118] 5a. The RNA molecule according to embodiment 4a, comprising the nucleotide sequence of SEQ ID NO:4.

[0119] 6. The RNA molecule according to embodiment 5, comprising a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a nucleotide sequence selected from the group consisting of: SEQ ID NO: 13-17, 23, 24, and 31-39.

[0120] 6a. The RNA molecule according to embodiment 5a, comprising nucleotide sequences selected from the group consisting of: SEQ ID NO: 13-17, 23, 24 and 31-39 7. An RNA molecule according to any one of embodiments 1 to 2, comprising a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:2.

[0121] 7a. An RNA molecule according to any one of embodiments 1 to 2, comprising the nucleotide sequence of SEQ ID NO:2.

[0122] 8. The RNA molecule according to embodiment 7, comprising a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a nucleotide sequence selected from the group consisting of: SEQ ID NO: 9-14, 17-23, and 25-44.

[0123] 8a. The RNA molecule according to embodiment 7a, comprising nucleotide sequences selected from the group consisting of: SEQ ID NO: 9-14, 17-23 and 25-44.

[0124] 9. The RNA molecule according to Embodiment 8, comprising a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:5. 9a. An RNA molecule according to embodiment 8a, comprising the nucleotide sequence of SEQ ID NO:5.

[0125] 10. The RNA molecule according to embodiment 9, comprising a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a nucleotide sequence selected from the group consisting of SEQ ID NO: 13, 14, and 30-44.

[0126] 10a. An RNA molecule according to embodiment 9a, comprising nucleotide sequences selected from the group consisting of: SEQ ID NO: 13, 14 and 30-44.

[0127] 11. An RNA molecule according to any one of embodiments 1 to 2, comprising a first nucleotide sequence and a second nucleotide sequence; the first nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:1; and the second nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:2.

[0128] 11a. An RNA molecule according to any one of embodiments 1 to 2, comprising the nucleotide sequence of SEQ ID NO:1 and the nucleotide sequence of SEQ ID NO:2.

[0129] 12. The RNA molecule according to embodiment 11, comprising a first nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any nucleotide sequence selected from the group consisting of SEQ ID NO: 9-14, 17-23, and 25-39.

[0130] 12a. An RNA molecule according to embodiment 11a, comprising nucleotide sequences selected from the group consisting of: SEQ ID NO: 9-14, 17-23 and 25-39.

[0131] 13. The RNA molecule according to Embodiment 11, comprising a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:4, and the second nucleotide sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:5.

[0132] 13a. The RNA molecule according to embodiment 11a, comprising the nucleotide sequence of SEQ ID NO:4 and the nucleotide sequence of SEQ ID NO:5.

[0133] 14. The RNA molecule according to embodiment 13, comprising a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a nucleotide sequence selected from the group consisting of SEQ ID NO: 13, 14, and 31-38.

[0134] 14a. The RNA molecule according to embodiment 13a, comprising nucleotide sequences selected from the group consisting of SEQ ID NO: 13, 14 and 31-38.

[0135] 15. An RNA molecule according to any one of embodiments 1 to 2, comprising a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:3.

[0136] 15a. An RNA molecule according to any one of embodiments 1 to 2, comprising the nucleotide sequence of SEQ ID NO:3.

[0137] 16. The RNA molecule according to embodiment 15, comprising a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a nucleotide sequence selected from SEQ ID NO:45-48.

[0138] 16a. An RNA molecule according to embodiment 15a, comprising nucleotide sequences selected from the group consisting of: SEQ ID NO: 45-48.

[0139] 17. The RNA molecule according to embodiment 15, comprising a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:6.

[0140] 17a. An RNA molecule according to embodiment 15a, comprising the nucleotide sequence of SEQ ID NO:6.

[0141] 18. The RNA molecule according to embodiment 17, comprising a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a nucleotide sequence selected from the group consisting of SEQ ID NO: 45 and 46.

[0142] 18a. An RNA molecule according to embodiment 17a, comprising nucleotide sequences selected from the group consisting of SEQ ID NO: 45 and 46.

[0143] 19. The RNA molecule according to Embodiment 1, comprising a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with a nucleotide sequence selected from the group consisting of: SEQ ID NO: 13-15, 23, 24, 31-39 and 46.

[0144] 19a. An RNA molecule according to Embodiment 1, comprising nucleotide sequences selected from the group consisting of: SEQ ID NO: 13-15, 23, 24, 31-39 and 46.

[0145] 19b. An RNA molecule according to embodiment 1, comprising a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with a nucleotide sequence selected from the group consisting of: SEQ ID NO: 23, 35, 36, 37, 38 and 39.

[0146] 19c. The RNA molecule according to Embodiment 1, comprising nucleotide sequences selected from the group consisting of: SEQ ID NO: 23, 36, 37 and 39.

[0147] 20. An RNA molecule according to any of the foregoing embodiments, comprising one or more modified nucleotides, such as deoxyribonucleotides, nucleotide analogs, abase nucleotides (referred to herein as Ab), 2'-modified nucleotides, 3' to 3' linked (reverse) nucleotides (referred to herein as invdN, invN, invn, invAb), nucleotides containing non-natural bases, bridging nucleotides, peptide nucleic acids (PNAs), 2',3'-open-ring nucleotide analogs (unlocked nucleobase analogs, referred to herein as NUNA), locked nucleotides (referred to herein as NLNA), 3'-O-methoxy (2' nucleoside-linked) nucleotides (referred to herein as 3'-OMen), 2'-F-arabinonucleotides (referred to herein as NfANA), 5'-Me-2'-fluoronucleotides. Nucleotides (referred to herein as 5Me-Nf), morpholinonucleotides, vinylphosphonic acid deoxyribonucleotides (referred to herein as vpdN), nucleotides containing vinylphosphonic acid, and nucleotides containing cyclopropylphosphonic acid (cPrpN), 2'-modified nucleotides (i.e., nucleotides with a group other than a hydroxyl group at the 2' position of the five-membered sugar ring), including but not limited to: 2'-O-methyl nucleotides (referred to in the nucleotide sequence as lowercase 'n'), 2'-deoxy-2'-fluoronucleotides (referred to herein as Nf, also referred to as 2'-fluoronucleotides), 2'-deoxynucleotides (referred to herein as dN), 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotides (referred to herein as NM or 2'-MOE), 2'-amino nucleotides, or 2'-alkyl nucleotides.

[0148] 21. The RNA molecule according to any of the foregoing embodiments, comprising one or more cytidine mismatches or guanosine mismatches that are directly opposite to adenosine in the target sequence.

[0149] 22. The RNA molecule according to embodiment 21, wherein the RNA molecule contains a cytidine mismatch that is directly opposite to adenosine in the target sequence.

[0150] 23. The RNA molecule according to embodiment 21, wherein the cytidine mismatch is located at a position at least 5 nucleotides away from the 3' end of the RNA molecule.

[0151] 23a. An RNA molecule according to any one of embodiments 21 to 23, wherein the cytidine mismatch is located at a position at least 20 nucleotides away from the 5' end of the RNA molecule.

[0152] 23b. The RNA molecule according to embodiment 23a, wherein the cytidine mismatch is located at a position at least 5 nucleotides from the 3' end of the RNA molecule and at least 20 nucleotides from the 5' end of the RNA molecule.

[0153] 24. The RNA molecule according to embodiment 21, wherein the RNA molecule contains a guanosine mismatch that is directly opposite to adenosine in the target sequence.

[0154] 25. The RNA molecule according to any of the foregoing embodiments, wherein the RNA molecule comprises two or more consecutive mismatched nucleotides opposite to one or more adenosine residues in a target sequence.

[0155] 26. A vector comprising a nucleotide sequence encoding an RNA molecule according to any one of embodiments 1-25.

[0156] 27. The vector according to embodiment 26 further comprises a 3' twisted nuclease sequence linked to the 3' end of the nucleotide sequence encoding the RNA molecule; and a 5' twisted nuclease sequence linked to the 5' end of the nucleotide sequence encoding the RNA molecule.

[0157] 28. The vector according to embodiment 26 or 27 further comprises a promoter operatively linked to a nucleotide sequence encoding the RNA molecule. 28a. The vector according to embodiment 28, wherein the promoter is a polymerase II promoter (“Pol II promoter”). 28b. The carrier according to embodiment 28a, wherein the Pol II promoter is a CMV promoter or a U7 promoter.

[0158] 28c. The vector according to embodiment 28, wherein the promoter is a polymerase III promoter (“PolIII promoter”). 28d. The carrier according to embodiment 28c, wherein the Pol III promoter is the U6 promoter.

[0159] 29. The vector according to any one of embodiments 26-28d, wherein the vector is a viral vector.

[0160] 29a. The vector according to any one of embodiments 26-28d, wherein the vector is a non-viral vector.

[0161] 30. The vector according to embodiment 29, wherein the vector is a viral vector, such as an adeno-associated virus vector (AAV), a lentiviral vector, an adenovirus vector, an RNA replicon, a poxvirus vector, an enterovirus vector, a Venezuelan equine encephalitis virus vector, a Semliki forest virus vector, or a tobacco mosaic virus vector.

[0162] 31. A host cell comprising an RNA molecule according to any one of embodiments 1-25 or a vector according to any one of embodiments 26-30.

[0163] 32. A pharmaceutical composition comprising an RNA molecule according to any one of embodiments 1-25 or a carrier according to any one of embodiments 26-30, and a pharmaceutically acceptable carrier.

[0164] 33. The pharmaceutical composition according to embodiment 32, wherein the pharmaceutical composition comprises one or more lipids; preferably, the pharmaceutical composition comprises lipid nanoparticles having one or more cationic lipids, non-cationic lipids and polyethylene glycol (PEG) or PEG-modified lipids.

[0165] 34. A method for inducing RNA splicing (e.g., exon skipping) of the pre-mRNA of the dystrophin gene in human subjects in need, comprising administering to the subject an effective amount of the pharmaceutical composition according to embodiment 32 or 33.

[0166] 34a. The method according to embodiment 34, wherein the administration causes exon 51 skipping of the pre-mRNA of the dystrophin gene.

[0167] 35. The method according to embodiment 34 or 34a, wherein the subject requires treatment for Duchenne muscular dystrophy.

[0168] 35a. The method according to embodiment 34 or 34a or 35, wherein the dystrophin gene comprises the deletion of one or more exons.

[0169] 35b. The method according to embodiment 35a, wherein the dystrophin gene comprises a deletion of exon 49.

[0170] 35c. The method according to embodiment 35a, wherein the dystrophin gene comprises a deletion of exon 50.

[0171] 35d. The method according to embodiment 35a, wherein the dystrophin gene comprises the deletion of exon 49 and exon 50.

[0172] 35e. The method according to embodiment 35a, wherein the dystrophin gene does not contain the deletion of exon 49 and exon 50.

[0173] 35f. The method according to embodiment 35a, wherein the dystrophin gene comprises one or more frameshift deletions.

[0174] 35g. The method according to embodiment 35a, wherein the dystrophin gene comprises one or more in-frame deletions.

[0175] 35h. The method according to embodiment 35f or 35g, wherein the one or more deletions are located in the region of exons 1-22, preferably in the region of exons 2-20.

[0176] 35i. The method according to embodiment 35f or 35g, wherein the one or more deletions are located in the region of exon 43-55, preferably in the region of exon 45-55.

[0177] Example The following embodiments are intended to further illustrate the essence of this application. It should be understood that the following embodiments do not constitute a limitation of this application, and the scope of this application is defined by the appended claims. Unless otherwise stated, the experimental methods used in the following embodiments are conventional methods; unless otherwise stated, the reagents used in the following embodiments are all purchased from conventional reagent suppliers. Exemplary RNA constructs numbered 7-48 each have the RNA sequences shown in SEQ ID NO: 7-48.

[0178] Example 1: Design of reporter gene plasmids PCR primers were designed to bind to intron sequences approximately 300 bp flanking exons 48, 50, 51, and 52 of dystrophin, respectively. The genome of HEK293T cells was used as a template for PCR. The PCR products of exons 48, 51, and 52 were sequentially cloned into the pcDNA3.1 vector plasmid to construct the δ49 / 50 reporter gene. Figure 5 The PCR products of exons 50, 51, and 52 were sequentially cloned into the pcDNA3.1 vector to construct the wild-type (WT) reporter gene (see...). Figure 5 The successfully constructed plasmid was transfected into HEK293T cells. Forty-eight hours after transfection, total RNA was collected and subjected to reverse transcription-polymerase chain reaction (RT-PCR). Subsequently, the PCR products were electrophoresed on a gel, the target band was excised, and sequenced to confirm correct exon insertion. Figures 6A-6D ).

[0179] Example 2: Jumping efficiency of arRNAs of different lengths Based on the predicted location of exon splicing enhancers (ESEs), arRNAs of varying lengths centered on the ESEs were designed. These arRNA sequences were then inserted into a circular cleavage sequence following the U6 promoter, and the circular arRNA plasmid was expressed in transfected cells for collection.

[0180] Cell plating Before cell plating, the state and density of cultured HEK293T cells were observed. Cells were digested with trypsin at 37°C for 5 minutes in a biosafety cabinet. 10 ml of complete culture medium was added to terminate trypsinization. The dissociated cell suspension was collected and centrifuged at 500 × g for 5 minutes. After cell counting, the cells were analyzed using a 1.5 × 10⁻⁶ plate. 5 Spread cells at a density of 100 cells / well onto a plate, gently shake to mix, and incubate in an incubator.

[0181] Cell transfection Observe the status and number of HEK293T cells 24 hours after plating and before transfection. Add 0.5 μg / well of WT reporter gene or δ49 / 50 plasmid and 1 μg / well of arRNA plasmid to Opti-MEM medium dilution. Then, add XtremeGENE transfection reagent and plasmid at a ratio of 3:1 (ul XtremeGENE:ug plasmid). Let the transfection reaction mixture stand for 15 minutes to form a complex. Add this transfection complex to the corresponding cell wells, gently shake to mix, and incubate. Collect cellular RNA 48 hours after transfection.

[0182] RT-PCR After extracting RNA from cells, 1 μg of RNA was used for reverse transcription, following the manufacturer's instructions. Subsequently, 2 μL of the reverse transcription product was used for PCR, again following the manufacturer's instructions. The obtained PCR products were subjected to agarose gel electrophoresis to verify correct band size, and then submitted for next-generation sequencing.

[0183] Exon skipping efficiency of RNA constructs of different lengths, such as Figure 1A and Figure 1B As shown.

[0184] Example 3: Jumping efficiency of arRNAs of the same length but different target locations Based on the experimental data above, an arRNA targeting exon 51 with a length of 100 bp was designed with 28 bp intervals. The designed arRNA sequence was ligated into the circular cleavage sequence after the U6 promoter, enabling cells transfected with this plasmid to express the circular arRNA.

[0185] Cell plating Before cell plating, the state and density of cultured HEK293T cells were observed. Cells were digested with trypsin at 37°C for 5 minutes in a biosafety cabinet. 10 ml of complete culture medium was added to terminate trypsinization. The dissociated cell suspension was collected and centrifuged at 500 × g for 5 minutes. After cell counting, the cells were analyzed using a 1.5 × 10⁻⁶ plate. 5Spread cells at a density of 100 cells / well onto a plate, gently shake to mix, and incubate in an incubator.

[0186] Cell transfection Observe the state and number of HEK293T cells 24 hours after plating and before transfection. Add 0.5 μg / well of WT reporter gene plasmid or δ49 / 50 plasmid and 1 μg / well of arRNA plasmid to Opti-MEM medium dilution. Then, add XtremeGENE transfection reagent and plasmid at a ratio of 3:1 (ul XtremeGENE:ug plasmid). Let the transfection reaction mixture stand for 15 minutes to form a complex. Add this transfection complex to the corresponding cell wells, gently shake to mix, and incubate. Collect cellular RNA 48 hours after transfection.

[0187] RT-PCR After extracting RNA from cells, 1 μg of RNA was used for reverse transcription, following the manufacturer's instructions. Subsequently, 2 μL of the reverse transcription product was used for PCR, again following the manufacturer's instructions. The obtained PCR products were subjected to agarose gel electrophoresis to verify correct band size, and then submitted for next-generation sequencing.

[0188] Exon skipping efficiencies of different constructs, such as Figure 2A and Figure 2B As shown.

[0189] Example 4: Jumping efficiency of arRNA with AC mismatch at different positions (ESE, BP, and AS) Sequences with or without AC mismatches were designed for the A region at the 3' splice site AG of exon 51; similarly, sequences with or without AC mismatches were designed for the A region at the branch point. These designs were combined with arRNA designs of varying lengths, and the designed arRNAs were ligated into the cleavage circular sequence following the U6 promoter. Cells were then transfected with plasmids to express the circular arRNAs. More specifically, RNA constructs 25-29 contained A / C mismatches in the predicted ESE region; RNA constructs 43, 30, 31, and 44 contained A / C mismatches in the AS (receptor splice site) region of the 3' splice site; and RNA constructs 34-37 contained A / C mismatches in the BP (branch point) region.

[0190] Cell plating Before cell plating, the state and density of cultured HEK293T cells were observed. Cells were digested with trypsin at 37°C for 5 minutes in a biosafety cabinet. 10 ml of complete culture medium was added to terminate trypsinization. The dissociated cell suspension was collected and centrifuged at 500 × g for 5 minutes. After cell counting, the cells were analyzed using a 1.5 × 10⁻⁶ plate.5 Spread cells at a density of 100 cells / well onto a plate, gently shake to mix, and incubate in an incubator.

[0191] Cell transfection Observe the status and number of HEK293T cells 24 hours after plating and before transfection. Add 0.5 μg / well of WT reporter gene plasmid or δ49 / 50 plasmid and 1 μg / well of arRNA plasmid to Opti-MEM medium dilution. Then, add XtremeGENE transfection reagent and plasmid at a ratio of 3:1 (ul XtremeGENE:ug plasmid). Let the transfection reaction mixture stand for 15 minutes to form a complex. Add this transfection complex to the corresponding cell wells, gently shake to mix, and incubate. Collect cellular RNA 48 hours after transfection.

[0192] RT-PCR After extracting RNA from cells, 1 μg of RNA was used for reverse transcription, following the manufacturer's instructions. Subsequently, 2 μL of the reverse transcription product was used for PCR, again following the manufacturer's instructions. The obtained PCR products were subjected to agarose gel electrophoresis to verify correct band size, and then submitted for next-generation sequencing.

[0193] like Figure 3A and 3B As shown, some AC mismatches lead to an increase in exon skipping efficiency, while other cases do not have this effect.

[0194] Example 5: Jumping efficiency of different concentrations of arRNA Based on the above data, we conclude that the trends of arRNA in the two reporter gene systems are consistent. Therefore, we selected the following conditions for concentration gradient experiments: 0.5 μg / well of WT reporter gene plasmid plus 1 μg / well of arRNA plasmid, and 0.5 μg / well of δ49 / 50 reporter plasmid plus 1 μg / well of arRNA plasmid. Fifteen arRNAs with high exon skipping efficiency were selected from the data. During the experiment, the reporter gene plasmid concentration was kept constant, and the arRNA plasmid concentration started at 1 μg / well and was serially diluted 4-fold, resulting in four concentration gradients.

[0195] Cell plating Before cell plating, the state and density of cultured HEK293T cells were observed. Cells were digested with trypsin at 37°C for 5 minutes in a biosafety cabinet. 10 ml of complete culture medium was added to terminate trypsinization. The dissociated cell suspension was collected and centrifuged at 500 × g for 5 minutes. After cell counting, the cells were analyzed using a 1.5 × 10⁻⁶ plate. 5Spread cells at a density of 100 cells / well onto a plate, gently shake to mix, and incubate in an incubator.

[0196] Cell transfection Observe the status and number of HEK293T cells 24 hours after plating and before transfection. Add 0.5 μg / well of WT reporter gene plasmid or δ49 / 50 plasmid, along with 1 μg, 0.25 μg, 0.0625 μg, or 0.015625 μg / well of arRNA plasmid to Opti-MEM medium dilution. Then, add XtremeGENE transfection reagent and plasmid at a ratio of 3:1 (μl XtremeGENE: μg plasmid). Let the transfection reaction mixture stand for 15 minutes to form a complex. Add this transfection complex to the corresponding cell wells, gently shake to mix, and incubate. Collect cellular RNA 48 hours after transfection.

[0197] RT-PCR After extracting RNA from cells, 1 μg of RNA was used for reverse transcription, following the manufacturer's instructions. Subsequently, 2 μL of the reverse transcription product was used for PCR, again following the manufacturer's instructions. The obtained PCR products were subjected to agarose gel electrophoresis to verify correct band size, and then submitted for next-generation sequencing.

[0198] like Figure 4 As shown, exon skipping was detected when using 0.0625 μg of arRNA plasmid.

[0199] Example 6. Evaluation and verification of arRNA-mediated exon skipping using the Eporter-2A-luciferase system. The effect of protein levels In addition to cDNA-level detection, we also used a 2A peptide to construct a reporter gene into the psicheck2 luciferase system to observe the protein-level expression of different arRNAs after exon skipping. We selected 0.5 μg luc reporter gene plasmid + 0.5 μg arRNA for evaluation. Based on the wild-type DMD sequence, exons 50, 51, and 52, along with their corresponding ±500 bp introns, were spliced ​​together and inserted into the René luciferase (RLuc) coding frame using a P2A self-cutting sequence. Furthermore, because the coding frames of exons 50, 51, and 52 were misaligned with the RLuc coding frame, the design was optimized to express RLuc only after exon 51 skipping, otherwise RLuc was not expressed. This represents the protein-level exon skipping mediated by arRNA ( Figure 7 (WT reporter gene in the middle).

[0200] As a positive control, the coding frame was also adjusted so that RLuc could be expressed without exon skipping, whereas RLuc would not be expressed if exon skipping occurred. Figure 7 (WT reporter gene within the frame).

[0201] Cell plating Before cell plating, the state and density of cultured HEK293T cells were observed. Cells were digested with trypsin at 37°C for 5 minutes in a biosafety cabinet. 10 ml of complete culture medium was added to terminate trypsinization. The dissociated cell suspension was collected and centrifuged at 500 × g for 5 minutes. After cell counting, the cells were analyzed using a 1.5 × 10⁻⁶ plate. 5 Spread cells at a density of 100 cells / well onto a plate, gently shake to mix, and incubate in an incubator.

[0202] Cell transfection Observe the status and number of HEK293T cells 24 hours after plating and before transfection. Add 0.5 μg / well of luc reporter gene plasmid or δ49 / 50 plasmid and 1 μg / well of arRNA plasmid to Opti-MEM medium dilution. Then, add XtremeGENE transfection reagent and plasmid at a ratio of 3:1 (ul XtremeGENE:ug plasmid). Let the transfection reaction mixture stand for 15 minutes to form a complex. Add this transfection complex to the corresponding cell wells, gently shake to mix, and incubate. Collect cellular RNA 48 hours after transfection.

[0203] Measurement of luciferase expression Cells were collected and lysed 72 hours after transfection. Luciferase activity was detected using a dual-luciferase assay kit and measured using a chemiluminescent microplate reader. The Rluc reading represents the level of protein recovery mediated by exon skipping, while the Fluc reading serves as an internal control for transfection. The Rluc / Fluc ratio was used to assess the efficiency of exon skipping recovery at the protein level; under these assay conditions, a higher Rluc / Fluc ratio indicates greater efficiency of exon skipping at the protein level.

[0204] like Figure 7 As shown, under these detection conditions, all arRNA constructs tested, except for constructs No. 7 and 47, effectively achieved exon skipping at the protein level. arRNA construct No. 7 had the shortest sequence length (50 nt) among all tested constructs, and its exon skipping efficiency in reporter gene detection was also relatively low. Figure 1A and 1BarRNA construct No. 47 is 100 nt in length and contains the sequence of SEQ ID NO:3, but not the sequences of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:4. arRNA construct No. 47 also contains multiple AC mismatches and, as detected in reporter gene assays, exhibits lower exon skipping efficiency compared to many other constructs. Figure 2A and 2B Furthermore, arRNA construct No. 28 achieved exon skipping at the protein level, but its efficiency was lower than that of arRNA constructs No. 27 and 29, which had similar sequences but contained different numbers of AC mismatches.

[0205] Example 7: Regulation of pre-mRNA splicing in vivo This embodiment demonstrates that exon skipping can be achieved in wild-type healthy monkeys by regulating the splicing of DMD pre-mRNA in vivo.

[0206] The circ-arRNA was packaged by PackGene Biotech using MyoAAV4E (Tabebordbar et al., Cell, 2022). The sequence of this arRNA is shown in SEQ ID NO:15. The AAV titer was 5E+12 vg / mL. MyoAAV4E was administered intravenously to DMD monkeys at a dose of 5E+13 vg / kg. The monkeys were monitored throughout the experiment.

[0207] At weeks 4 and 26 post-injection, cardiac muscle, gastrocnemius muscle, quadriceps femoris muscle, triceps brachii muscle, latissimus dorsi muscle, and diaphragm were collected from monkeys. The collected monkey tissues were homogenized in 1 ml of TRIzol, and RNA was extracted using the chloroform extraction method and reverse transcribed. PCR amplification products were analyzed by agarose gel electrophoresis to detect exon jumping efficiency. RT-PCR was used to detect the expression level of arRNA in each tissue.

[0208] The results showed that four weeks after injection, the exon skipping efficiency in various tissues was approximately 7% to 21%. Figure 8A Twenty-six weeks after injection, the exon skipping efficiency in various tissues was approximately 4% to 12%. Figure 8B Furthermore, a large amount of arRNA expression was detected in all tissues. Figure 9 ).

[0209] sequence list

Claims

1. A circular ribonucleic acid (RNA) molecule having at least 50 nucleotides and comprising at least one nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:

3.

2. The RNA molecule according to claim 1, wherein it has at least 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 nucleotides.

3. The RNA molecule according to claim 1 or 2, comprising the nucleotide sequence of SEQ ID NO:

1.

4. The RNA molecule according to claim 3, comprising any nucleotide sequence selected from the group consisting of: SEQ ID NO:7-39.

5. The RNA molecule according to claim 4, comprising the nucleotide sequence of SEQ ID NO:

4.

6. The RNA molecule according to claim 5, comprising any nucleotide sequence selected from the group consisting of: SEQ ID NO: 13-17, 23, 24 and 31-39.

7. The RNA molecule according to claim 1 or 2, comprising the nucleotide sequence of SEQ ID NO:

2.

8. The RNA molecule according to claim 7, comprising any nucleotide sequence selected from the group consisting of: SEQ ID NO: 9-14, 17-23 and 25-44.

9. The RNA molecule according to claim 8, comprising the nucleotide sequence of SEQ ID NO:

5.

10. The RNA molecule of claim 9, comprising any nucleotide sequence selected from the group consisting of: SEQ ID NO: 13, 14 and 30-44.

11. The RNA molecule according to claim 1 or 2, comprising the nucleotide sequence of SEQ ID NO:1 and the nucleotide sequence of SEQ ID NO:

2.

12. The RNA molecule of claim 11, comprising any nucleotide sequence selected from the group consisting of: SEQ ID NO: 9-14, 17-23 and 25-39.

13. The RNA molecule according to claim 12, comprising the nucleotide sequence of SEQ ID NO:4 and the nucleotide sequence of SEQ ID NO:

5.

14. The RNA molecule of claim 13, comprising any nucleotide sequence selected from the group consisting of: SEQ ID NO: 13, 14 and 31-38.

15. The RNA molecule according to claim 1 or 2, comprising the nucleotide sequence of SEQ ID NO:

3.

16. The RNA molecule of claim 15, comprising nucleotide sequences selected from the group consisting of: SEQ ID NO:45-48.

17. The RNA molecule of claim 15, comprising the nucleotide sequence of SEQ ID NO:

6.

18. The RNA molecule of claim 17, comprising nucleotide sequences selected from the group consisting of SEQ ID NO: 45 and 46.

19. The RNA molecule of claim 1, comprising any nucleotide sequence selected from the group consisting of: SEQ ID NO: 13-15, 16, 17, 23, 24, 31-39 and 46; preferably SEQ ID NO: 13, 15-17, 23, 36, 37, 38 or 39.

20. The RNA molecule according to any one of the preceding claims, comprising one or more modified nucleotides, such as deoxyribonucleotides, nucleotide analogs, abase-free nucleotides (referred to herein as Ab), 2'-modified nucleotides, 3' to 3' linked (reverse) nucleotides (referred to herein as invdN, invN, invn, invAb), nucleotides containing non-natural bases, bridging nucleotides, peptide nucleic acids (PNAs), 2',3'-open-ring nucleotide analogs (unlocked nucleobase analogs, referred to herein as NUNA), locked nucleotides (referred to herein as NLNA), 3'-O-methoxy (2' nucleoside-linked) nucleotides (referred to herein as 3'-OMen), 2'-F-arabinonucleotides (referred to herein as NfANA), 5'-Me -2'-Fluoronucleotides (referred to as 5Me-Nf in this article), morpholinonucleotides, vinylphosphonic acid deoxyribonucleotides (referred to as vpdN in this article), nucleotides containing vinylphosphonic acid, and nucleotides containing cyclopropylphosphonic acid (cPrpN), 2'-modified nucleotides (i.e., nucleotides with a group other than a hydroxyl group at the 2' position of the five-membered sugar ring), including but not limited to: 2'-O-methyl nucleotides (referred to as lowercase 'n' in the nucleotide sequence), 2'-deoxy-2'-fluoronucleotides (referred to as Nf in this article, also referred to as 2'-fluoronucleotides), 2'-deoxynucleotides (referred to as dN in this article), 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotides (referred to as NM or 2'-MOE in this article), 2'-amino nucleotides, or 2'-alkyl nucleotides.

21. The RNA molecule according to any of the preceding claims, comprising one or more cytidine mismatches or guanosine mismatches that are directly opposite to adenosine in the target sequence.

22. The RNA molecule according to claim 21, wherein, The RNA molecule contains a cytidine mismatch that is directly opposite to adenosine in the target sequence.

23. The RNA molecule according to claim 21, wherein, The cytidine mismatch is located at least 5 nucleotides from the 3' end of the RNA molecule and / or at least 20 nucleotides from the 5' end of the RNA molecule.

24. The RNA molecule according to claim 21, wherein, The RNA molecule contains a guanosine mismatch that is directly opposite to adenosine in the target sequence.

25. The RNA molecule according to any one of the preceding claims, wherein, The RNA molecule contains two or more consecutive mismatched nucleotides that correspond to one or more adenosine residues in the target sequence.

26. A vector comprising a nucleotide sequence encoding an RNA molecule according to any one of claims 1-25.

27. The vector of claim 26, further comprising a 3' twisted nuclease sequence linked to the 3' end of the nucleotide sequence encoding the RNA molecule; and a 5' twisted nuclease sequence linked to the 5' end of the nucleotide sequence encoding the RNA molecule.

28. The vector according to claim 26 or 27, further comprising a promoter operatively linked to a nucleotide sequence encoding the RNA molecule; preferably, the promoter is a polymerase II promoter ("Pol II promoter"), such as a CMV promoter or a U7 promoter; or a Pol III promoter, such as a U6 promoter.

29. The vector according to any one of claims 26-28, wherein the vector is a viral vector or a non-viral vector.

30. The vector according to claim 29, wherein the vector is a viral vector, such as an adeno-associated virus vector (AAV), a lentiviral vector, an adenovirus vector, an RNA replicon, a poxvirus vector, an enterovirus vector, a Venezuelan equine encephalitis virus vector, a Semliki forest virus vector, or a tobacco mosaic virus vector.

31. A host cell comprising an RNA molecule according to any one of claims 1-25, or a vector according to any one of claims 26-30.

32. A pharmaceutical composition comprising an RNA molecule according to any one of claims 1-25 or a carrier according to any one of claims 26-30, and a pharmaceutically acceptable carrier.

33. The pharmaceutical composition according to claim 32, wherein, The pharmaceutical composition comprises one or more lipids; preferably, the pharmaceutical composition comprises lipid nanoparticles having one or more cationic lipids, non-cationic lipids, and polyethylene glycol (PEG) or PEG-modified lipids.

34. A method for inducing dystrophin gene exon 51 skipping in a human subject in need, comprising administering to the subject an effective amount of the pharmaceutical composition according to claim 32 or 33.

35. The method of claim 34, wherein the subject requires treatment for Duchenne muscular dystrophy.

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