Regulatory sequences containing microRNA target sites

Abstract

The present invention relates to an isolated nucleic acid sequence comprising at least two copies of a miRNA target site of the miR183 family, having a sequence shown in SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24, or a sequence having at least 90% identity with any one of SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24. The present invention also relates to a vector comprising a regulatory element comprising at least one copy of the miRNA target site of the miR183 family. The present isolated nucleic acid sequence and the present vector may be particularly useful for controlling the expression of a gene of interest, for example, when designing and developing gene therapy.

Description

Technical Field

[0001] The present invention relates to regulatory sequences containing microRNA target sites, particularly miRNA target sites of the miR183 family, and their use for regulating the expression of target nucleic acid sequences such as target genes.

Background Art

[0002] MicroRNAs (miRNAs or miRs) are a rich class of short, naturally occurring non-coding RNAs approximately 20 nucleotides in length that play important roles in gene expression. Most miRNAs are transcribed from DNA sequences into large RNA precursors called primary miRNAs (or pri-miRNAs) by RNA polymerase II or III. Pri-miRNAs are processed into hairpin precursor miRNAs (or pre-miRNAs) approximately 70 to approximately 120 nucleotides in length, which themselves are further processed into mature miRNAs approximately 20 nucleotides in length. In most cases, after recognizing their target sites, which are generally located in the 3' untranslated transcript regions (UTRs), mature miRNAs achieve complete or incomplete base complementary pairs, thereby resulting in mRNA cleavage and inhibition of mRNA translation into protein. Thus, miRNAs play important roles in the regulation of post-transcriptional gene expression in a sequence-specific manner.

[0003] Accordingly, strategies for selective gene silencing based on the use of vectors containing one or more copies of miRNA target sites operably linked to or inserted into the nucleic acid sequence of a target gene have been developed and described in the art. In these strategies, the miRNA target sites are in short nucleotide sequences complementary to specific mature miRNAs. Introduction of the vector into cells that express miRNAs that specifically recognize the miRNA target sites results in the suppression of the expression of the target gene in said cells through the interaction of the miRNA with its miRNA target site.

[0004] Thousands of miRNAs, including over 2,000 mature miRNAs that have been identified in humans, have been discovered in many organisms. miRNAs can be grouped into clusters defined as several miR genes that are located adjacent to each other on a chromosome, and these are transcribed as one long primary miRNA and then processed into individual hairpin precursor miRNAs. Furthermore, due to the high sequence homology among the miRNAs in a cluster, they are classified as a family, enabling both common and unique mRNA targets for the miRNAs of that family. Among the identified miRNA families, the miR183 family consists of three homologous miRNAs, namely miR183, miR96, and miR182. The miRNAs of the miR183 family are highly expressed in the sensory cells of the eye, nose, and inner ear and are essential for their development.

[0005] Many sensory disorders, particularly blindness and deafness, are of genetic origin, i.e., caused by the presence of mutations in genes. Such sensory disorders are usually referred to as hereditary or congenital sensory disorders. Currently, many gene therapies have been developed for the purpose of treating sensory disorders caused by single gene mutations. Treatments based on gene therapy rely on providing intact genes to complement the defective genes that cause congenital sensory disorders. It should be noted that it is most important that the gene introduced by gene therapy is specifically expressed only in the cells and tissues where the gene's expression occurs naturally. In particular, it is important to prevent the gene introduced by gene therapy from being expressed in cells and tissues where the expression of that gene may cause any adverse effects. Therefore, regulatory sequences and vectors that enable the control of the expression of the gene of interest are required. In particular, regulatory sequences and vectors that enable the prevention or suppression of the expression of the gene of interest in cells and tissues where the expression may cause any adverse effects are required.

[0006] The inventors have surprisingly demonstrated that introducing a vector containing at least one copy of a gene and a "precursor miR183 target site" into cells that express miR183 significantly suppresses the expression of the gene in said cells. As used herein, the so-called "precursor miR183 target site" refers to a miR183 target site having a sequence complementary to the human sequence of precursor miR183. Similar regulatory effects were observed when using other "precursor miRNA target sites" of the miR183 family, namely the so-called "precursor miR182 target site" that refers to a miR182 target site having a sequence complementary to the human sequence of precursor miR182, and the so-called "precursor miR96 target site" that refers to a miR96 target site having a sequence complementary to the human sequence of precursor miR96. Surprisingly, the so-called precursor miR183 target site was significantly more efficient than a shorter miR183 target site having a sequence complementary to the human sequence of mature miR183 in suppressing gene expression in cells that express miR183. The inventors have also demonstrated that injection of a vector containing at least one copy of a gene and the so-called precursor miR183 target site into the inner ear of mice significantly suppresses the expression of the gene in inner and outer hair cells of the cochlea.

[0007] Therefore, from these results, it is established that a vector containing at least one copy of a so-called precursor miRNA target site of the miR183 family can be a useful tool for selectively and effectively suppressing unwanted gene expression in sensory cells. Such vectors can be particularly interesting in gene therapy aimed at treating congenital sensory disorders.

Summary of the Invention

[0008] Accordingly, the present invention relates to an isolated nucleic acid sequence comprising at least two copies of an miRNA target site of the miR183 family, wherein the miRNA target site has a sequence shown in SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24, or a sequence having at least 90% identity with any one of SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24. In some embodiments, the present invention relates to an isolated nucleic acid sequence comprising at least two copies of an miR183 target site having a sequence shown in SEQ ID NO: 1 or a sequence having at least 90% identity with SEQ ID NO: 1. In some embodiments, the isolated nucleic acid sequence comprises 2 to 6 copies of an miRNA target site of the miR183 family, for example, 2 to 6 copies of an miR183 target site having a sequence shown in SEQ ID NO: 1 or a sequence having at least 90% identity with SEQ ID NO: 1. In some embodiments, the isolated nucleic acid sequence comprises 3 copies of an miRNA target site of the miR183 family, for example, 3 copies of an miR183 target site having a sequence shown in SEQ ID NO: 1 or a sequence having at least 90% identity with SEQ ID NO: 1. In some embodiments, the isolated nucleic acid sequence comprises a sequence shown in SEQ ID NO: 7, SEQ ID NO: 23 or SEQ ID NO: 26, or a sequence having at least 90% identity with any one of SEQ ID NO: 7, SEQ ID NO: 23 or SEQ ID NO: 26. In some embodiments, the isolated nucleic acid sequence comprises a sequence shown in SEQ ID NO: 7 or a sequence having at least 90% identity with SEQ ID NO: 7. In some embodiments, the copies of the miRNA target site of the miR183 family, for example, the copies of the miR183 target site are separated by a spacer.

[0009] The present invention relates to an expression cassette comprising a promoter, a gene of interest, and a regulatory element comprising at least one copy of an miRNA target site of the miR183 family, wherein the miRNA target site has a sequence shown in SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24 or a sequence having at least 90% identity with any one of SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24. The present invention also relates to a vector comprising a regulatory element comprising at least one copy of an miRNA target site of the miR183 family, wherein the miRNA target site has a sequence shown in SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24 or a sequence having at least 90% identity with any one of SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 2. In some embodiments, the regulatory element comprises at least one copy of an miR183 target site having a sequence shown in SEQ ID NO: 1 or a sequence having at least 90% identity with SEQ ID NO: 1. In some embodiments, the regulatory element comprises 2 to 6 copies, preferably 3 copies, of an miRNA target site of the miR183 family (e.g., an miR183 target site having a sequence shown in SEQ ID NO: 1 or a sequence having at least 90% identity with SEQ ID NO: 1), and these copies are optionally separated by a spacer. In some embodiments, the regulatory element comprises a sequence shown in SEQ ID NO: 7, SEQ ID NO: 23 or SEQ ID NO: 26 or a sequence having at least 90% identity with any one of SEQ ID NO: 7, SEQ ID NO: 23 or SEQ ID NO: 26. In some embodiments, the regulatory element comprises a sequence shown in SEQ ID NO: 7 or a sequence having at least 90% identity with SEQ ID NO: 7. In some embodiments, the regulatory element further comprises at least one copy of another miRNA target site, preferably at least one copy of another miRNA target site of the miR183 family.In some embodiments, the regulatory element comprises at least one copy of a miR183 target site having the sequence shown in SEQ ID NO:1 or a sequence having at least 90% identity with SEQ ID NO:1 and at least one copy of another miRNA target site, preferably at least one copy of another miRNA target site of the miR183 family, more preferably a miR182 target site.

[0010] In some embodiments, the regulatory element contained within the expression cassette described herein or the vector described herein that further comprises a gene of interest is operably linked to or inserted into the gene of interest, preferably inserted into the 3'UTR of the gene of interest.

[0011] The present invention also relates to a pharmaceutical composition comprising the isolated nucleic acid sequence described herein, the expression cassette described herein, or the vector described herein, and at least one pharmaceutically acceptable excipient.

[0012] The present invention also relates to the isolated nucleic acid sequence described herein, the expression cassette described herein, the vector described herein, or the pharmaceutical composition described herein for use as a medicament.

[0013] The present invention also relates to the use of the isolated nucleic acid sequence described herein, the expression cassette described herein, or the vector described herein for specifically expressing a gene of interest in cells that do not express an miRNA of the miR183 family, such as cells that do not express miR183.

[0014] Definitions In the present invention, the following terms have the following meanings.

[0015] The terms "a" and "an" refer to one or more than one (i.e., at least one) element of the grammatical object of the article. By way of example, "an element" means one or more than one element.

[0016] "About" before a number encompasses up to ±10% of the value of the number. It should be understood that the value itself to which the term "about" refers is also specifically and preferably disclosed.

[0017] "Code" as in a coding sequence refers to the unique properties of a specific sequence of nucleotides in a nucleic acid such as a gene, complementary DNA (cDNA) or messenger RNA (mRNA) and the biological properties resulting therefrom, which function as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., ribosomal RNA (rRNA), transfer RNA (tRNA) and mRNA) or a defined sequence of amino acids (e.g., polypeptide or protein).

[0018] "Expression" refers to the transcription and / or translation of a specific nucleotide sequence such as a gene.

[0019] As used herein, the term "gene" broadly refers to a coding nucleic acid sequence that can be transcribed into either an RNA molecule, i.e., a coding RNA molecule such as mRNA that can subsequently be translated into a polypeptide or protein, or a non-coding RNA molecule such as rRNA or tRNA. "Transgene" specifically refers to a gene derived from one species that is introduced into an organism belonging to a different species. Thus, it should be noted that a gene may or may not include a coding sequence (or CDS), i.e., a nucleic acid sequence that actually codes for a protein. Genes, especially those that include a CDS, preferably may also include untranslated transcribed regions (UTRs) such as 3' UTR and / or 5' UTR, as well as other sequences such as regulatory elements and / or introns that are transcribed but not translated. Thus, the term "gene" as used herein may refer to a coding nucleic acid sequence that includes a coding sequence (or CDS), as well as at least one regulatory element that is transcribed but not translated, such as 3' UTR, 5' UTR, and / or intron.

[0020] As used herein, the expression "having the sequence shown in SEQ ID NO: X" when referring to a given sequence (such as a miRNA target site) means that the given sequence (such as a miRNA target site) contains or consists of the sequence shown in SEQ ID NO: X.

[0021] "Identity" or "identical," as used herein in the context of the relationship between the sequences of two or more nucleic acids, refers to the degree of sequence relatedness between said nucleic acids, determined by the number of matches between the sequences consisting of two or more nucleotides. "Identity" is a measure of the percentage of exact matches between the shorter of two or more sequences with gap alignments (if any) handled by a particular mathematical model or computer program (i.e., "algorithm"). The identity of related nucleic acid sequences can be readily calculated by known methods. Such methods include, but are not limited to, those described in "Computational Molecular Biology," Lesk, A.M. ed., Oxford University Press, New York, 1988; "Biocomputing: Informatics and Genome Projects," Smith, D.W. ed., Academic Press, New York, 1993; "Computer Analysis of Sequence Data, Part 1," Griffin, A.M. and Griffin, H.G. eds., Humana Press, New Jersey, 1994; "Sequence Analysis in Molecular Biology," von Heinje, G., Academic Press, 1987; "Sequence Analysis Primer," Gribskov, M. and Devereux, J. eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988). Preferred methods for determining identity are designed to give the maximum match between the sequences being tested. Methods for determining identity are described in publicly available computer programs.Preferred computer program methods for determining identity between two arrays include the GCG program package, including GAP (Devereux et al., Nucleic Acids Res. January 11, 1984; 12(1 Pt 1):387-95, Genetics Computer Group, University of Wisconsin, Madison, Wisconsin), BLASTP, BLASTN and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al., NCB / NLM / NIH, Bethesda, Maryland 20894; Altschul et al., J. Mol. Biol. 215, 403-410 (1990)).

[0022] "Isolated" with respect to a nucleic acid refers to a nucleic acid that has been changed or removed from its natural state. For example, a nucleic acid that naturally exists in a living organism is "not isolated", but a nucleic acid that has been partially or completely separated from the coexisting substances in its natural state is "isolated". Thus, an "isolated nucleic acid" is a nucleic acid that is substantially separated from other nucleic acid sequences such as genomic DNA or RNA, as well as proteins or complexes such as ribosomes and polymerases that are naturally associated with the native sequence. An isolated nucleic acid can exist in a substantially purified form or, for example, in a non-natural environment such as a host cell. Typically, a preparation of isolated nucleic acid may contain nucleic acid that is at least about 80% pure, at least about 85% pure, at least about 90% pure, at least about 95% pure, greater than about 95% pure, greater than about 96% pure, greater than about 97% pure, greater than about 98% pure or greater than about 99% pure. Thus, isolated nucleic acids include nucleic acids purified by standard purification methods and nucleic acid sequences removed from their naturally occurring environment. Isolated nucleic acids also include chemically synthesized nucleic acids and nucleic acids biologically synthesized by heterologous systems.

[0023] "MicroRNA" or "miRNA" refers to endogenous small non-coding RNA molecules of about 18 to about 24 nucleotides that play an important role in the post-transcriptional regulation of gene expression in eukaryotic cells. A single miRNA can regulate up to hundreds of different mRNAs, and most mRNAs are expected to be targeted by multiple miRNAs. miRNA genes are transcribed by RNA polymerase II or III and then processed to yield single-stranded mature miRNAs, which are incorporated into the RNA-induced silencing complex (RISC). The miRNA, as the central part of the RISC complex, guides the RISC to its mRNA target, where the miRNA usually binds to the 3' untranslated region (3'UTR) of the mRNA transcript by partial complementary base pairing. Note that complete base pairing must occur over a short length of 7 or 8 nucleotides complementary to the so-called miRNA "seed" region located at positions 2 to 8 from the 5' end of the mature miRNA. Gene silencing can be achieved by Argonaute-2 (AGO2)-mediated mRNA cleavage or translational repression promoted by AGO1-4, and in either case, a decrease in the level of the corresponding protein ultimately occurs. As used herein, "miRNA", such as "miR183", refers to the mature miRNA, such as mature miR183. In contrast, "precursor miRNA" or "pre-miRNA", such as "precursor miR183", refers to the hairpin precursor sequence from which the mature miRNA is processed.

[0024] As used herein, the term "microRNA target site" or "miRNA target site" or "miR target site" refers to a nucleic acid sequence that can bind to an miRNA (i.e., a mature miRNA). The term "microRNA target site" or "miRNA target site" or "miR target site" as used herein encompasses both endogenous target sites that may be present in native transcripts and artificial or engineered target sites (i.e., target sites that do not occur naturally) that can be inserted as regulatory elements (or regulatory sequences) into a vector, particularly an expression vector, to control the expression of a nucleic acid sequence of interest such as a gene of interest. Particularly in a vector, the miRNA target site can be operably linked to or inserted into the sequence of a gene, particularly into the transcribed sequence of a gene. By definition, a miRNA target site must contain a nucleic acid sequence that is at least partially complementary to the corresponding miRNA, for example, a nucleic acid sequence that is complementary to the corresponding miRNA over a length of at least 5 nucleotides, usually 6 - 7 nucleotides. Thus, a miR183 target site must contain a nucleic acid sequence that is at least partially complementary to miR183, for example, a nucleic acid sequence that is complementary to miR183 over a length of at least 7 - 8 nucleotides. Similarly, a miR182 target site must contain a nucleic acid sequence that is at least partially complementary to miR182, for example, a nucleic acid sequence that is complementary to miR182 over a length of at least 7 - 8 nucleotides, and a miR96 target site must contain a nucleic acid sequence that is at least partially complementary to miR96, for example, a nucleic acid sequence that is complementary to miR96 over a length of at least 7 - 8 nucleotides. Also, a miRNA target site, particularly an artificial or engineered miRNA target site (i.e., a target site that does not occur naturally), may contain or consist of a nucleic acid sequence that is complementary to the miRNA over the entire length of the miRNA (i.e., over 18 - 24 nucleotides of the miRNA). When inserted into a vector, the miR target site can enable binding of the corresponding miRNA, so that when this vector is introduced into a host cell that expresses the miRNA, it can mediate miRNA-induced silencing of the expression of a nucleic acid of interest such as a gene of interest.For example, when inserted into a vector, the miR183 target site enables binding of miR183. Thus, when this vector is introduced into a host cell that expresses miR183, it can mediate miR183-induced silencing of the expression of a target nucleic acid such as a target gene. Similarly, when inserted into a vector, the miR182 target site enables binding of miR182. Thus, when this vector is introduced into a host cell that expresses miR182, it can mediate miR182-induced silencing of the expression of a target nucleic acid such as a target gene. When inserted into a vector, the miR96 target site enables binding of miR96. Thus, when this vector is introduced into a host cell that expresses miR96, it can mediate miR96-induced silencing of the expression of a target nucleic acid such as a target gene. Methods for evaluating whether a nucleic acid sequence can be a suitable miRNA target site (e.g., miR183 target site, miR182 target site, or miR96 target site) are well known in the art. Examples of such methods are described in the following experimental sections.

[0025] As used herein, the term "microRNA target site of the miR183 family" or "miRNA target site of the miR183 family" or "miR target site of the miR183 family" refers to a nucleic acid sequence that can bind a miRNA (i.e., mature miRNA) belonging to the miR183 family (sometimes referred to as the miR183 cluster). Thus, the terms "microRNA target site of the miR183 family" or "miRNA target site of the miR183 family" or "miR target site of the miR183 family" refer to a nucleic acid sequence that can bind miR183, miR182, and / or miR96.

[0026] "Nucleic acid" refers to a polymer of nucleotides (i.e., polynucleotide) covalently linked by phosphodiester bonds, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in either single-stranded or double-stranded form. Thus, the nucleic acids used herein may be single-stranded, partially double-stranded or fully double-stranded. The nucleotides constituting the nucleic acids of the present disclosure may be unmodified (natural) nucleotides or unnatural, i.e., modified nucleotides. Examples of unmodified (i.e., natural or occurring in nature) nucleotides include adenosine monophosphate (AMP), deoxyadenosine monophosphate (dAMP), cytidine monophosphate (CMP), deoxycytidine monophosphate (dCMP), guanosine monophosphate (GMP), deoxyguanosine monophosphate (dGMP), thymidine monophosphate (TMP), deoxythymidine monophosphate (dTMP), and uridine monophosphate (UMP). The term "nucleic acid" also encompasses nucleic acids that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to natural nucleotides, including known analogs of natural nucleotides.

[0027] "Nucleic acid sequence" or "nucleotide sequence" refers to the continuous sequence of nucleotides in a single nucleic acid. Unless otherwise specified, a particular nucleic acid sequence implicitly includes its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs (single nucleotide polymorphisms), and complementary sequences, as well as the explicitly recited sequence. In particular, the specific nucleic acid sequences described herein implicitly include their corresponding complementary sequences. It should be noted that the specific nucleic acid sequences described herein implicitly include DNA sequences and the corresponding RNA sequences.

[0028] "Operably linked" refers to the functional linkage between a regulatory array and a heterologous nucleic acid sequence, such as a gene, such that the former regulates the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. In particular, when a promoter affects the transcription or expression of a gene, the promoter is operably linked to the gene. Similarly, when a regulatory sequence affects the expression of a gene (i.e., induces or inhibits (or represses) it), the regulatory sequence is operably linked to the gene. The operably linked sequences may be adjacent to each other.

[0029] "Pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to an excipient or carrier that does not produce side effects, allergic reactions or other adverse reactions when administered to an animal, preferably a human. It includes all kinds of solvents such as dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents. Pharmaceutically acceptable excipients or carriers refer to all kinds of non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating agents or formulation aids. For administration to humans, the formulation must meet the sterility, pyrogenicity, general safety and purity standards required by regulatory authorities such as the FDA (US Food and Drug Administration) or the EMA (European Medicines Agency).

[0030] "Vector" refers to a medium that can transform, transfect or transduce a host cell and promote the expression (e.g., transcription and / or translation) of the introduced nucleic acid sequence, thereby introducing a nucleic acid sequence (e.g., a DNA or RNA molecule), such as an RNA or a nucleic acid encoding a polypeptide or protein of interest, into the host cell.

[0031] "Expression vector" refers to a vector containing regulatory elements (or regulatory sequences) that are operably linked or can be operably linked to a nucleic acid sequence of interest to be expressed, such as a gene of interest. Thus, an expression vector contains sufficient cis-acting regulatory elements to control the expression of the nucleic acid sequence of interest (present or inserted therein), and other elements that may be required to control the expression of the nucleic acid sequence of interest may be provided by the host cell or in vitro expression system (such as miRNA that binds to the miR target site). Examples of cis-acting regulatory elements include promoters and miR target sites such as the miR183 target site, miR182 target site, and miR96 target site described herein.

Mode for Carrying Out the Invention

[0032] Human microRNA 183 (miR183 or miR-183) belongs to the miR183 family (sometimes referred to as the miR183 cluster), which consists of three homologous miRNAs, namely miR183 (or miR-183), miR96 (or miR-96), and miR182 (or miR-182). The miRNAs of the miR183 family are particularly required for the proper development of the sensory organs. In particular, the miRNAs of the miR183 family are expressed in vertebrate sensory neurons and hair cells, as well as in sensory cells of all species of animals. Among vertebrates, the miRNAs of the miR183 family are expressed in the olfactory epithelium, eyes, sensory thalamus, and ears.

[0033] The human miR-183 gene consists of one exon on chromosome 7q32.2. Mature miR183 is generated by the processing of a hairpin precursor called precursor miR183 or pre-miR183. Human precursor miR183 is 110 nucleotides long and has the sequence shown in SEQ ID NO: 2, which is referred to as NR_029615.1 in the NCBI database.

[0034] Processing of the hairpin precursor miR183 folded into a stem-loop structure results in mature miR183. Mature human miR183, called miR183-5p (or hsa-miR183-5p or hsa-miR-183-5p), is 22 nucleotides in length and has the sequence shown in SEQ ID NO: 3, which is referenced as MIMAT0000261 in miRBase (https: / / www.mirbase.org). The sequence of miR183-5p corresponds to nucleotides 27-48 of the human precursor miR183 of SEQ ID NO: 2. Mature human miR183, called miR183-3p (or hsa-miR183-3p or hsa-miR-183-3p), is also 22 nucleotides in length and has the sequence shown in SEQ ID NO: 4, which is referenced as MIMAT0004560 in miRBase. The sequence of miR183-3p corresponds to nucleotides 66-87 of the human precursor miR183 of SEQ ID NO: 2. As used herein, the term "mature miR183" (or "mature miR-183") encompasses both miR183-5p and miR183-3p.

[0035] In vivo, mature miR183 can bind to a target mRNA containing a short sequence complementary to the seed region of the mature miR183. For example, the seed region of hsa-miR183-5p is AUGGCAC, which corresponds to nucleotides 2-8 of hsa-miR183-5p (SEQ ID NO: 3).

[0036] The human miR-182 gene consists of one exon on chromosome 7q32.2. Mature miR182 results from the processing of a hairpin precursor called precursor miR182 or pre-miR182. The human precursor miR182 is 110 nucleotides in length and has the sequence shown in SEQ ID NO: 22, which is referenced as NR_029614.1 in the NCBI database.

[0037] Processing of the hairpin precursor miR182 folded into a stem-loop structure results in mature miR182. Mature human miR182, called miR182-5p (or hsa-miR182-5p or hsa-miR-182-5p), is 24 nucleotides in length and has the sequence shown in SEQ ID NO: 11, which is referenced as MIMAT0000259 in miRBase (https: / / www.mirbase.org). The sequence of miR182-5p corresponds to nucleotides 23 to 46 of the human precursor miR182 of SEQ ID NO: 22. Mature human miR182, called miR182-3p (or hsa-miR182-3p or hsa-miR-182-3p), is 21 nucleotides in length and has the sequence shown in SEQ ID NO: 13, which is referenced as MIMAT0000260 in miRBase. The sequence of miR182-3p corresponds to nucleotides 67 to 87 of the human precursor miR182 of SEQ ID NO: 22. As used herein, the term "mature miR182" (or "mature miR-182") encompasses both miR182-5p and miR182-3p.

[0038] In vivo, mature miR182 can bind to a target mRNA containing a short sequence complementary to the seed region of the mature miR182. For example, the seed region of hsa-miR182-5p is UUGGCAA corresponding to nucleotides 2 to 8 of hsa-miR182-5p (SEQ ID NO: 11).

[0039] The human miR-96 gene consists of one exon on chromosome 7q32.2. Mature miR96 results from the processing of a hairpin precursor called precursor miR96 or pre-miR96. The human precursor miR96 is 78 nucleotides in length and has the sequence shown in SEQ ID NO: 25, which is referenced as NR_029512.1 in the NCBI database.

[0040] Processing of the hairpin precursor miR96 folded into a stem-loop structure results in mature miR96. Mature human miR96, called miR96-5p (or hsa-miR96-5p or hsa-miR-96-5p), is 23 nucleotides in length and has the sequence shown in SEQ ID NO: 28, which is referenced as MIMAT0000095 in miRBase (https: / / www.mirbase.org). The sequence of miR96-5p corresponds to nucleotides 9-31 of the human precursor miR96 of SEQ ID NO: 25. Mature human miR96, called miR96-3p (or hsa-miR96-3p or hsa-miR-96-3p), is 22 nucleotides in length and has the sequence shown in SEQ ID NO: 30, which is referenced as MIMAT0004510 in miRBase. The sequence of miR96-3p corresponds to nucleotides 52-73 of the human precursor miR96 of SEQ ID NO: 25. As used herein, the term "mature miR96" (or "mature miR-96") encompasses both miR96-5p and miR96-3p.

[0041] In vivo, mature miR96 can bind to a target mRNA region that contains a short sequence complementary to the seed of said mature miR96. For example, the seed region of hsa-miR96-5p is UUGGCAC, which corresponds to nucleotides 2-8 of hsa-miR96-5p (SEQ ID NO: 28).

[0042] As detailed in the following experimental section, the inventors have surprisingly demonstrated that when inserted into a vector, an miR183 target site 110 nucleotides in length having the sequence shown in SEQ ID NO: 1, which is complementary to the human sequence of precursor miR183 (i.e., SEQ ID NO: 2), can be successfully used as a regulatory element for regulating the expression of a nucleic acid of interest in cells expressing miR183. As shown above, an miR183 target site having a sequence complementary to the human sequence of precursor miR183 (i.e., SEQ ID NO: 2) may be referred to herein as a "precursor miR183 target site". When introduced into cells, the miR183 target site contained within the expression vector is transcribed along with the nucleic acid of interest and thus is present in the resulting mRNA. The 110-nucleotide-long miR183 target site having the sequence shown in SEQ ID NO: 1 is expected to fold into a stem-loop structure. The inventors have surprisingly demonstrated that, contrary to expectations, the stem-loop structure does not prevent the binding of miR183 to the miR183 target site of SEQ ID NO: 1.

[0043] Similarly, the present inventors have demonstrated that when inserted into a vector, an miR182 target site 110 nucleotides in length having the sequence shown in SEQ ID NO: 21, which is complementary to the human sequence of precursor miR182 (i.e., SEQ ID NO: 22), can be successfully used as a regulatory element for controlling the expression of a nucleic acid of interest in cells expressing miR182. As shown above, an miR182 target site having a sequence complementary to the human sequence of precursor miR182 (i.e., SEQ ID NO: 22) may be referred to herein as a "precursor miR182 target site". When introduced into cells, the miR182 target site contained within the expression vector is transcribed along with the nucleic acid of interest and thus is present in the resulting mRNA. The present inventors have also demonstrated that when inserted into a vector, an miR96 target site 78 nucleotides in length having the sequence shown in SEQ ID NO: 24, which is complementary to the human sequence of precursor miR96 (i.e., SEQ ID NO: 25), can be successfully used as a regulatory element for controlling the expression of a nucleic acid of interest in cells expressing miR96. As shown above, an miR96 target site having a sequence complementary to the human sequence of precursor miR96 (i.e., SEQ ID NO: 25) may be referred to herein as a "precursor miR96 target site". When introduced into cells, the miR96 target site contained within the expression vector is transcribed along with the nucleic acid of interest and thus is present in the resulting mRNA. The 110-nucleotide-long mir182 target site having the sequence shown in SEQ ID NO: 21 and the 78-nucleotide-long mir96 target site having the sequence shown in SEQ ID NO: 24 are also expected to fold into a stem-loop structure. The present inventors have surprisingly demonstrated that, contrary to expectations, the stem-loop structure does not interfere with either the binding of miR182 to the mir182 target site of SEQ ID NO: 21 or the binding of miR96 to the mir96 target site of SEQ ID NO: 24.

[0044] The inventors have also demonstrated that more potent suppression of nucleic acid expression occurs by inserting several copies of the miR183 target site of SEQ ID NO: 1. Unexpectedly, the suppression of nucleic acid expression caused by the use of the miR183 target site of SEQ ID NO: 1 is significantly more potent than the suppression caused by the use of shorter target sites such as the miR183 target site having the sequence shown in SEQ ID NO: 5, which is complementary to the sequence of hsa-miR183-5p (i.e., SEQ ID NO: 3).

[0045] As exemplified in the following experimental section, as a method for evaluating whether a nucleic acid sequence can be a suitable miR target site, for example, a suitable miR183 target site, under the control of a promoter such as a ubiquitin promoter or a constitutive promoter, inserting the nucleic acid sequence to be evaluated into a vector containing a reporter gene such as a gene encoding a GFP protein (green fluorescent protein). The nucleic acid sequence to be evaluated may be operably linked to the gene or inserted into the gene, for example, in the 3'UTR of the gene. Then, a vector (e.g., a plasmid) containing the nucleic acid sequence to be evaluated and the reporter gene is introduced into a host cell that expresses miR183, such as HEK293 cells. Inhibition of the expression of the reporter gene in the host cell indicates that the nucleic acid sequence to be evaluated is a suitable miR183 target site, particularly as compared to a control condition where the vector contains only the reporter gene under the control of a promoter. A similar method may be used to evaluate whether a nucleic acid sequence can be a suitable miR182 target site or a suitable miR96 target site.

[0046] Accordingly, a first aspect of the present invention is an isolated nucleic acid sequence comprising or consisting of at least two copies of an miR target site of the miR183 family, wherein the miR target site has the sequence shown in SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24.

[0047] The isolated nucleic acid sequences described herein may comprise or consist of at least two copies of a miR183 target site having the sequence shown in SEQ ID NO: 1 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1. In some embodiments, the miR183 target site having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1 described herein is a functional miR183 target site, i.e., it enables binding of miR183.

[0048] In some embodiments, the miR183 target site having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1 has a length of at least 100 nucleotides. In some embodiments, the miR183 target site having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1 has a length of at least 60, 65, 70, 75, 80, 85, 90, 95, 100 or 105 nucleotides. In some embodiments, the miR183 target site having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1 has a length of 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 or 110 nucleotides.

[0049] In some embodiments, the mirR183 target site has the sequence shown in SEQ ID NO: 1, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more nucleotides are replaced by different nucleotides with reference to the corresponding nucleotides of SEQ ID NO: 1. In some embodiments, the mirR183 target site has the sequence shown in SEQ ID NO: 1, wherein up to 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotide is replaced by a different nucleotide with reference to the corresponding nucleotide of SEQ ID NO: 1. In some embodiments, such a mirR183 target site having the sequence shown in SEQ ID NO: 1 with nucleotide substitutions is a functional mirR183 target site, i.e., it enables binding of miR183.

[0050] The isolated nucleic acid sequence described herein may comprise or consist of at least two copies of a miR182 target site having the sequence shown in SEQ ID NO: 21 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 21. In some embodiments, a mirR182 target site having a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 21 as described herein is a functional mirR182 target site, i.e., it enables binding of miR182.

[0051] In some embodiments, the mirR182 target site having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 21 has a length of at least 100 nucleotides. In some embodiments, the mirR182 target site having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 21 has a length of at least 60, 65, 70, 75, 80, 85, 90, 95, 100 or 105 nucleotides. In some embodiments, the mirR182 target site having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 21 has a length of 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 or 110 nucleotides.

[0052] In some embodiments, the mirR182 target site has the sequence shown in SEQ ID NO: 21, where 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more nucleotides are replaced by different nucleotides with reference to the corresponding nucleotides of SEQ ID NO: 21. In some embodiments, the mirR182 target site has the sequence shown in SEQ ID NO: 21, where up to 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotide is replaced by different nucleotides with reference to the corresponding nucleotides of SEQ ID NO: 21. In some embodiments, such a mirR182 target site having the sequence shown in SEQ ID NO: 21 with nucleotide substitutions is a functional mirR182 target site, i.e., it enables binding of miR182.

[0053] The isolated nucleic acid sequences described herein may comprise or consist of at least two copies of a miR96 target site having the sequence shown in SEQ ID NO: 24 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 24. In some embodiments, a mirR96 target site having a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 24 as described herein is a functional mirR183 target site, i.e., it enables binding of miR96.

[0054] In some embodiments, a mirR96 target site having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 24 has a length of at least 70 nucleotides. In some embodiments, a mir96 target site having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 24 has a length of at least 40, 45, 50, 55, 60 or 65 nucleotides. In some embodiments, a mir96 target site having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 24 has a length of 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 or 78 nucleotides.

[0055] In some embodiments, a mirR96 target site has a sequence shown in SEQ ID NO: 24 in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 or more nucleotides are substituted by different nucleotides with reference to the corresponding nucleotides of SEQ ID NO: 24. In some embodiments, a mirR96 target site has a sequence shown in SEQ ID NO: 24 in which up to 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotide is substituted by a different nucleotide with reference to the corresponding nucleotide of SEQ ID NO: 24. In some embodiments, such a mirR96 target site having a sequence shown in SEQ ID NO: 24 with nucleotide substitutions is a functional mirR96 target site, i.e., it enables binding of miR96.

[0056] As used herein, the expression "at least two copies" includes 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of the miR target sites of the miR183 family described herein (e.g., the sequence shown in SEQ ID NO:1 described herein or an miR183 target site having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:1).

[0057] In some embodiments, the isolated nucleic acid sequence comprises or consists of 2 to 6 copies of the miR target sites of the miR183 family described herein (e.g., the sequence shown in SEQ ID NO:1 described herein or an miR183 target site having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:1). Thus, the isolated nucleic acid sequence may comprise or consist of 2, 3, 4, 5 or 6 copies of the miR target sites of the miR183 family described herein (e.g., the sequence shown in SEQ ID NO:1 described herein or an miR183 target site having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:1).

[0058] In some embodiments, the isolated nucleic acid sequence comprises or consists of 3 copies of the miR target sites of the miR183 family described herein. Thus, the isolated nucleic acid sequence may comprise or consist of a sequence shown in SEQ ID NO:7, SEQ ID NO:23 or SEQ ID NO:26, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NO:7, SEQ ID NO:23 or SEQ ID NO:26.

[0059] In particular, the isolated nucleic acid sequence may comprise or consist of three copies of a miR183 target site having the sequence shown in SEQ ID NO: 1 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1. Accordingly, the isolated nucleic acid sequence may comprise or consist of the sequence shown in SEQ ID NO: 7 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 7. In some embodiments, the isolated nucleic acid sequence having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 7 described herein corresponds to a functional mirR183 target site, i.e., it allows binding of miR183.

[0060] In some embodiments, the isolated nucleic acid sequence having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 7 has a length of at least 300 nucleotides. In some embodiments, the isolated nucleic acid sequence having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 7 has a length of at least 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 305, 310, 315, 320, 325 or 330 nucleotides. In some embodiments, the isolated nucleic acid sequence having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 7 has a length of 300, 305, 310, 315, 320, 325 or 330 nucleotides.

[0061] In some embodiments, the isolated nucleic acid sequence has the sequence shown in SEQ ID NO: 7, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides are replaced by different nucleotides with reference to the corresponding nucleotides of SEQ ID NO: 7. In some embodiments, the isolated nucleic acid sequence has the sequence shown in SEQ ID NO: 7, wherein up to 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotide is replaced by a different nucleotide with reference to the corresponding nucleotide of SEQ ID NO: 7. In some embodiments, such an isolated nucleic acid sequence having the sequence shown in SEQ ID NO: 7 with nucleotide substitutions corresponds to a functional mirR183 target site, i.e., it enables binding of miR183.

[0062] The isolated nucleic acid sequence may comprise or consist of three copies of the miR182 target site having the sequence shown in SEQ ID NO: 21 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 21. Thus, the isolated nucleic acid sequence may comprise or consist of the sequence shown in SEQ ID NO: 23 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 23. In some embodiments, the isolated nucleic acid sequence having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 23 described herein corresponds to a functional mirR182 target site, i.e., it enables binding of miR182.

[0063] In some embodiments, the isolated nucleic acid sequence having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 23 has a length of at least 300 nucleotides. In some embodiments, the isolated nucleic acid sequence having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 23 has a length of at least 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 305, 310, 315, 320, 325 or 330 nucleotides. In some embodiments, the isolated nucleic acid sequence having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 23 has a length of 300, 305, 310, 315, 320, 325 or 330 nucleotides.

[0064] In some embodiments, the isolated nucleic acid sequence has a sequence shown in SEQ ID NO: 23 in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides are substituted by different nucleotides with reference to the corresponding nucleotides of SEQ ID NO: 23. In some embodiments, the isolated nucleic acid sequence has a sequence shown in SEQ ID NO: 23 in which up to 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotide is substituted by a different nucleotide with reference to the corresponding nucleotide of SEQ ID NO: 23. In some embodiments, such an isolated nucleic acid sequence having a sequence shown in SEQ ID NO: 23 with nucleotide substitutions corresponds to a functional mirR182 target site, i.e., it enables binding of miR182.

[0065] The isolated nucleic acid sequence may comprise or consist of three copies of the miR96 target site having the sequence set forth in SEQ ID NO: 24 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 24. Accordingly, the isolated nucleic acid sequence may comprise or consist of the sequence set forth in SEQ ID NO: 26 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 26. In some embodiments, the isolated nucleic acid sequence having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 26 described herein corresponds to a functional mirR96 target site, i.e., it enables binding of miR96.

[0066] In some embodiments, the isolated nucleic acid sequence having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 26 has a length of at least 210 nucleotides. In some embodiments, the isolated nucleic acid sequence having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 26 has a length of at least 160, 170, 180, 190, 200, 205, 210, 215, 220, 225, 230 or 234 nucleotides. In some embodiments, the isolated nucleic acid sequence having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 26 has a length of 200, 205, 210, 215, 220, 225, 230 or 234 nucleotides.

[0067] In some embodiments, the isolated nucleic acid sequence has the sequence set forth in SEQ ID NO: 26, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides are substituted by different nucleotides with reference to the corresponding nucleotides of SEQ ID NO: 26. In some embodiments, the isolated nucleic acid sequence has the sequence set forth in SEQ ID NO: 26, wherein a maximum of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotide is substituted by a different nucleotide with reference to the corresponding nucleotide of SEQ ID NO: 26. In some embodiments, such an isolated nucleic acid sequence having the sequence set forth in SEQ ID NO: 26 with nucleotide substitutions corresponds to a functional mirR96 target site, i.e., it enables binding of miR96.

[0068] In some embodiments, in the isolated nucleic acid sequences described herein, copies of the miR target sites of the miR183 family described herein (e.g., miR183 target sites, etc.) are separated by spacers. As used herein, "spacer" refers to a non-coding sequence. In some embodiments, the spacer is in the range of about 5 nucleotides to about 25 nucleotides, preferably in the range of about 10 nucleotides to about 20 nucleotides, more preferably about 20 nucleotides in length. Spacers are well used in the art and are well known to those skilled in the art. For example, spacers are described in Hammarsten et al., "Herpes simplex virus: selection of origins of DNA replication. Nucleic Acids Res. May 1, 1997; 25(9):1753-60). Examples of spacers include spacers having the sequences shown in SEQ ID NO: 14 (ATAACTAAAAGATTCGGA), SEQ ID NO: 15 (AATATATATATATTATTA), SEQ ID NO: 16 (AAAAACATATAAAATAAT) or SEQ ID NO: 17 (CTTTCTTTTCCCAATTTT). In some embodiments, the spacer has the sequence shown in SEQ ID NO: 14.

[0069] In some embodiments, the isolated nucleic acid sequence is a single-stranded sequence. In some embodiments, the isolated nucleic acid sequence is a double-stranded sequence.

[0070] In some embodiments, the isolated nucleic acid sequences described herein further comprise at least one copy of another miR target site. In some embodiments, the isolated nucleic acid sequence · At least two copies of the miR183 target site having the sequence shown in SEQ ID NO: 1 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1, or at least two copies of the miR182 target site having the sequence shown in SEQ ID NO: 21 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 21, or at least two copies of the miR96 target site having the sequence shown in SEQ ID NO: 24 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 24, and · At least one copy of another miR target site comprising or consisting of.

[0071] For example, the other miR target site may be a miR target site recognized by a miRNA expressed in hair cells such as sensory neurons and / or hair cells of the inner ear. Other miR targets may be the miR182 target site, the miR96 target site, the miR194 target site, the miR140 target site, the miR18a target site, the miR99a target site, the miR30b target site, the miR15a target site or the miR210 target site. In some embodiments, the other miR target site is the miR182 target site.

[0072] The miR182 target site may be a miR182 target site having a sequence complementary to mature miR182. For example, the miR182 target site may have the sequence shown in SEQ ID NO: 10 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 10. The sequence of SEQ ID NO: 10 is complementary to the sequence of hsa-miR182-5p. Hsa-miR182-5p is 24 nucleotides in length and has the sequence shown in SEQ ID NO: 11, which is referred to as MIMAT0000259 in miRBase as described above. The mature miR182 target site may have the sequence shown in SEQ ID NO: 12 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 12. The sequence of SEQ ID NO: 12 is complementary to the sequence of hsa-miR182-3p. Hsa-miR182-3p is 21 nucleotides in length and has the sequence shown in SEQ ID NO: 13, which is referred to as MIMAT0000260 in miRBase as shown above.

[0073] In some embodiments, the isolated nucleic acid sequences described herein further comprise at least one copy of another miR target site of the miR183 family described herein. For example, the isolated nucleic acid sequence · at least two copies of a miR183 target site having the sequence shown in SEQ ID NO: 1 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1, and · At least one copy of the miR182 target site having the sequence shown in SEQ ID NO: 21 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 21, and / or at least one copy of the miR96 target site having the sequence shown in SEQ ID NO: 24 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 24 may comprise or consist of.

[0074] This isolated nucleic acid sequence · At least two copies of the miR182 target site having the sequence shown in SEQ ID NO: 21 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 21, and · At least one copy of the miR183 target site having the sequence shown in SEQ ID NO: 1 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1, and / or at least one copy of the miR96 target site having the sequence shown in SEQ ID NO: 24 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 24 may comprise or consist of.

[0075] This isolated nucleic acid sequence · At least two copies of the miR96 target site having the sequence shown in SEQ ID NO: 24 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 24, and · At least one copy of the miR183 target site having the sequence shown in SEQ ID NO: 1 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1, and / or at least one copy of the miR182 target site having the sequence shown in SEQ ID NO: 21 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 21 It may comprise or consist of.

[0076] Another aspect of the present invention is an isolated nucleic acid coding sequence (also referred to as an isolated coding sequence) comprising at least one copy of a miR target site of the miR183 family, wherein the miR target site has a sequence shown in SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24.

[0077] The isolated coding sequence may comprise at least one copy of the miR183 target site having the sequence shown in SEQ ID NO: 1 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1. The isolated coding sequence may comprise at least one copy of the miR182 target site having the sequence shown in SEQ ID NO: 21 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 21. The isolated coding sequence may comprise at least one copy of the miR96 target site having the sequence shown in SEQ ID NO: 24 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 24.

[0078] In some embodiments, the isolated nucleic acid coding sequence encodes a polynucleotide or a protein. In some embodiments, the isolated coding sequence is a cDNA sequence. In some embodiments, the isolated coding sequence is an RNA coding sequence. For example, in some embodiments, the isolated coding sequence is an mRNA sequence. In some embodiments, at least one copy of the miR target site of the miR183 family described herein is inserted into the untranslated region of the isolated coding sequence described herein, such as the 5'UTR or 3'UTR of the isolated coding sequence.

[0079] In some embodiments, the isolated nucleic acid coding sequence comprises at least two copies of the miR target site of the miR183 family described herein (e.g., the sequence shown in SEQ ID NO: 1 described herein or a miR183 target site having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1).

[0080] In some embodiments, the isolated nucleic acid coding sequence comprises 2 to 6 copies of the miR target site of the miR183 family described herein (e.g., the sequence shown in SEQ ID NO: 1 described herein or a miR183 target site having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1).

[0081] In some embodiments, the isolated nucleic acid coding sequence comprises three copies of the miR target site of the miR183 family described herein. Thus, the isolated nucleic acid sequence may comprise or consist of the sequence shown in SEQ ID NO: 7, SEQ ID NO: 23 or SEQ ID NO: 26, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NO: 7, SEQ ID NO: 23 or SEQ ID NO: 26.

[0082] In particular, the isolated nucleic acid coding sequence may comprise three copies of the miR183 target site having the sequence shown in SEQ ID NO: 1 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1. Thus, the isolated nucleic acid coding sequence may comprise the sequence shown in SEQ ID NO: 7 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 7.

[0083] The isolated nucleic acid coding sequence may comprise three copies of the miR182 target site having the sequence shown in SEQ ID NO: 21 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 21. Thus, the isolated nucleic acid coding sequence may comprise the sequence shown in SEQ ID NO: 23 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 23.

[0084] The isolated nucleic acid coding sequence may comprise three copies of the miR96 target site having the sequence shown in SEQ ID NO: 24 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 24. Accordingly, the isolated nucleic acid coding sequence may comprise the sequence shown in SEQ ID NO: 26 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 26.

[0085] In some embodiments, copies of the miR target sites of the miR183 family described herein (such as miR183 target sites) are separated by the spacers described herein. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 14.

[0086] In some embodiments, the isolated nucleic acid coding sequence further comprises at least one copy of another miR target site. In some embodiments, the isolated nucleic acid coding sequence is · at least one copy of the miR183 target site having the sequence shown in SEQ ID NO: 1 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1, or at least one copy of the miR182 target site having the sequence shown in SEQ ID NO: 21 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 21, or at least one copy of the miR96 target site having the sequence shown in SEQ ID NO: 24 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 24, and · at least one copy of another miR target site including.

[0087] For example, other miR target sites may be miR target sites recognized by miRNAs expressed in hair cells such as sensory neurons and / or hair cells of the inner ear. Other miR targets may be miR182 target sites, miR96 target sites, miR194 target sites, miR140 target sites, miR18a target sites, miR99a target sites, miR30b target sites, miR15a target sites or miR210 target sites. In some embodiments, the other miR target site is a miR182 target site.

[0088] The miR182 target site may have a sequence complementary to mature miR182, such as the sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 10 or SEQ ID NO: 12.

[0089] In some embodiments, the isolated nucleic acid coding sequence further comprises at least one copy of another miR target site of the miR183 family described herein. For example, the isolated nucleic acid coding sequence is · at least one copy of a miR183 target site having the sequence shown in SEQ ID NO: 1 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 1, and · At least one copy of the miR182 target site having the sequence shown in SEQ ID NO: 21 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 21, and / or at least one copy of the miR96 target site having the sequence shown in SEQ ID NO: 24 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 24 may be included.

[0090] Alternatively, the isolated nucleic acid coding sequence may include at least one copy of the miR182 target site having the sequence shown in SEQ ID NO: 21 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 21, and at least one copy of the miR96 target site having the sequence shown in SEQ ID NO: 24 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 24.

[0091] Another aspect of the invention is an expression cassette comprising a promoter, a nucleic acid sequence of interest, and a regulatory element (or regulatory sequence) comprising or consisting of at least one copy of a miR target site of the miR183 family, wherein the miR target site has the sequence shown in SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24.

[0092] The regulatory element (or regulatory sequence) may comprise or consist of at least one copy of a miR183 target site having the sequence shown in SEQ ID NO: 1 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1. The regulatory element (or regulatory sequence) may comprise or consist of at least one copy of a miR182 target site having the sequence shown in SEQ ID NO: 21 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 21. The regulatory element (or regulatory sequence) may comprise or consist of at least one copy of a miR96 target site having the sequence shown in SEQ ID NO: 24 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 24.

[0093] As used herein, the expression "at least one copy" includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of a miR target site of the miR183 family described herein (e.g., a miR183 target site having the sequence shown in SEQ ID NO: 1 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1).

[0094] In some embodiments, the regulatory element (or regulatory sequence) comprises or consists of 1 to 6 copies of the miR target site of the miR183 family described herein (e.g., the sequence shown in SEQ ID NO: 1 described herein or an miR183 target site having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1). Thus, the regulatory element (or regulatory sequence) may comprise or consist of 1, 2, 3, 4, 5 or 6 copies of the miR target site of the miR183 family described herein (e.g., the sequence shown in SEQ ID NO: 1 described herein or an miR183 target site having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1).

[0095] In some embodiments, the regulatory element (or regulatory sequence) comprises or consists of at least 2 copies of the miR target site of the miR183 family described herein (e.g., the sequence shown in SEQ ID NO: 1 described herein or an miR183 target site having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1). In some embodiments, the regulatory element (or regulatory sequence) comprises or consists of 2 to 6 copies of the miR target site of the miR183 family described herein (e.g., the sequence shown in SEQ ID NO: 1 described herein or an miR183 target site having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1).

[0096] In some embodiments, the regulatory element (or regulatory sequence) comprises or consists of three copies of the miR target site of the miR183 family described herein. Thus, the regulatory element (or regulatory sequence) may comprise or consist of a sequence shown in SEQ ID NO: 7, SEQ ID NO: 23 or SEQ ID NO: 26 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with any one of SEQ ID NO: 7, SEQ ID NO: 23 or SEQ ID NO: 26.

[0097] In particular, the regulatory element (or regulatory sequence) may comprise or consist of three copies of the miR183 target site having a sequence shown in SEQ ID NO: 1 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 1. Thus, the regulatory element (or regulatory sequence) may comprise or consist of a sequence shown in SEQ ID NO: 7 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 7.

[0098] In some embodiments, the regulatory element (or regulatory sequence) having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 7 has a length of at least 300 nucleotides. In some embodiments, the regulatory element (or regulatory sequence) having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 7 has a length of 300, 305, 310, 315, 320, 325 or 330 nucleotides.

[0099] In some embodiments, the regulatory element (or regulatory sequence) has the sequence shown in SEQ ID NO: 7, where 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides are replaced by different nucleotides with reference to the corresponding nucleotides of SEQ ID NO: 7 described herein. In some embodiments, the regulatory element (or regulatory sequence) has the sequence shown in SEQ ID NO: 7, where a maximum of 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotide is replaced by a different nucleotide with reference to the corresponding nucleotide of SEQ ID NO: 7 described herein.

[0100] The regulatory element (or regulatory sequence) may comprise or consist of three copies of the miR182 target site having the sequence shown in SEQ ID NO: 21 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 21. Thus, the regulatory element (or regulatory sequence) may comprise or consist of the sequence shown in SEQ ID NO: 23 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 23.

[0101] In some embodiments, a regulatory element (or regulatory sequence) having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 23 has a length of at least 300 nucleotides. In some embodiments, a regulatory element (or regulatory sequence) having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 23 has a length of 300, 305, 310, 315, 320, 325 or 330 nucleotides.

[0102] In some embodiments, a regulatory element (or regulatory sequence) has a sequence shown in SEQ ID NO: 23 in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides are replaced by different nucleotides with reference to the corresponding nucleotides of SEQ ID NO: 23 described herein. In some embodiments, a regulatory element (or regulatory sequence) has a sequence shown in SEQ ID NO: 23 in which a maximum of 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotide is replaced by a different nucleotide with reference to the corresponding nucleotide of SEQ ID NO: 23 described herein.

[0103] The regulatory element (or regulatory sequence) may comprise or consist of three copies of the miR96 target site having the sequence shown in SEQ ID NO: 24 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 24. Accordingly, the regulatory element (or regulatory sequence) may comprise or consist of the sequence shown in SEQ ID NO: 26 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 26.

[0104] In some embodiments, the regulatory element (or regulatory sequence) having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 26 has a length of at least 210 nucleotides. In some embodiments, the regulatory element (or regulatory sequence) having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 26 has a length of 200, 205, 210, 215, 220, 225, 230 or 234 nucleotides.

[0105] In some embodiments, the regulatory element (or regulatory sequence) has the sequence shown in SEQ ID NO: 26 where 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides are substituted with different nucleotides with reference to the corresponding nucleotides of SEQ ID NO: 26 described herein. In some embodiments, the regulatory element (or regulatory sequence) has the sequence shown in SEQ ID NO: 26 where a maximum of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotide is substituted with a different nucleotide with reference to the corresponding nucleotide of SEQ ID NO: 26 described herein.

[0106] In some embodiments, in the regulatory element (or regulatory sequence) described herein that contains several copies of the miR target site of the miR183 family (such as the miR183 target site) described herein, said copies are separated by the spacers described herein. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 14.

[0107] In some embodiments, the regulatory element (or regulatory sequence) described herein further comprises at least one copy of another miR target site. Thus, in some embodiments, the regulatory element (or regulatory sequence) is · at least one copy of the miR183 target site having the sequence shown in SEQ ID NO: 1 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 1, or at least one copy of the miR182 target site having the sequence shown in SEQ ID NO: 21 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 21, or at least one copy of the miR96 target site having the sequence shown in SEQ ID NO: 24 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 24, and · at least one copy of another miR target site comprises or consists of.

[0108] For example, other miR target sites may be miR target sites recognized by miRNAs expressed in hair cells such as sensory neurons and / or hair cells of the inner ear. Other miR targets may be miR182 target sites, miR96 target sites, miR194 target sites, miR140 target sites, miR18a target sites, miR99a target sites, miR30b target sites, miR15a target sites or miR210 target sites. In some embodiments, other miR target sites are miR182 target sites.

[0109] The miR182 target site may have a sequence complementary to mature miR182, such as the sequence shown in SEQ ID NO: 10 or SEQ ID NO: 12, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 10 or SEQ ID NO: 12.

[0110] In some embodiments, the regulatory element (or regulatory sequence) described herein further comprises at least one copy of another miR target site of the miR183 family described herein. For example, the regulatory element (or regulatory sequence) described herein is · at least one copy of a miR183 target site having the sequence shown in SEQ ID NO: 1 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 1, and · at least one copy of a miR182 target site having the sequence shown in SEQ ID NO: 21 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 21, and / or at least one copy of a miR96 target site having the sequence shown in SEQ ID NO: 24 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 24 It may comprise or consist of.

[0111] Alternatively, the regulatory element (or regulatory sequence) described herein may comprise or consist of at least one copy of a miR182 target site having a sequence shown in SEQ ID NO: 21 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 21, and at least one copy of a miR96 target site having a sequence shown in SEQ ID NO: 24 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 24.

[0112] As used herein, the expression "nucleic acid sequence of interest" is intended to refer to a nucleic acid sequence that is expressed. In particular, the expression "nucleic acid sequence of interest" is intended to refer to a nucleic acid sequence that is expressed in a regulated manner when introduced into, for example, a host cell or host organism. The nucleic acid sequence of interest may be a nucleic acid sequence encoding a gene of interest, i.e., a functional nucleic acid (such as RNA) or a polypeptide or protein.

[0113] For example, a nucleic acid of interest such as a gene of interest may include an open reading frame encoding a polypeptide or protein. Thus, the nucleic acid sequence of interest may include the coding sequence of interest. Alternatively, the nucleic acid sequence of interest may encode a functional nucleic acid such as non-coding RNA.

[0114] In some embodiments, the nucleic acid of interest is a gene of interest. As used herein, the term gene may particularly refer to a coding nucleic acid sequence including a coding sequence (or CDS) and at least one regulatory element that is transcribed but not translated, such as a 3'UTR, 5'UTR and / or intron.

[0115] In this expression cassette, the regulatory element may be operably linked to or inserted into the nucleic acid of interest. Preferably, the regulatory element is inserted into the untranslated region of the nucleic acid of interest. Examples of untranslated regions include 5'UTR, 3'UTR, and introns. In some embodiments, the regulatory element is inserted into the 3'UTR of the nucleic acid of interest, particularly the 3'UTR of the gene of interest.

[0116] As shown above, the expression cassette contains a promoter. For example, the promoter may be a ubiquitously expressed promoter and / or a constitutive promoter. Promoters are well known to those skilled in the art, and those skilled in the art will know how to select a suitable promoter according to the nucleic acid sequence of interest to be expressed and the cells in which expression is desired.

[0117] In this expression cassette, the promoter is usually inserted upstream of the nucleic acid sequence of interest to be expressed, that is, at the 5' of the nucleic acid sequence of interest to be expressed.

[0118] This expression cassette may further contain any sequence or element that may be required for the expression of the nucleic acid sequence of interest. Such sequences or elements include, for example, poly(A) signals, poly(A) sites, enhancer sequences, terminator sequences, degrons, silencers, insulators, and / or operators.

[0119] Another aspect of the present invention is a vector containing the regulatory element (or regulatory sequence) described herein, the isolated nucleic acid coding sequence described herein, or the expression cassette described herein.

[0120] In particular, the present invention relates to a vector comprising or consisting of a regulatory element comprising at least one copy of a miR target site of the miR183 family, wherein the miR target site has the sequence shown in SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with any one of SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24. For example, the vector may comprise or consist of a regulatory element comprising at least one copy of a miR183 target site having the sequence shown in SEQ ID NO: 1 described herein or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 1.

[0121] In some embodiments, a vector comprising the regulatory element described herein also comprises a nucleic acid sequence of interest described herein, such as a gene of interest. In such vectors, the regulatory element may be operably linked to or inserted into the nucleic acid of interest. Preferably, the regulatory element is inserted into an untranslated region of the nucleic acid of interest, such as the 5’UTR, 3’UTR or intron. In some embodiments, the regulatory element is inserted into the 3’UTR of the nucleic acid of interest, particularly the 3’UTR of the gene of interest.

[0122] Vectors are well known to those skilled in the art, and those skilled in the art will know how to select a suitable vector depending on the nucleic acid sequence of interest to be expressed (e.g., the length of the nucleic acid sequence of interest) and the cell in which expression is desired.

[0123] Examples of vectors include cosmids, plasmids, episomes, artificial chromosomes, phages and viruses (such as lentiviruses, retroviruses, adenoviruses and adeno-associated viruses (AAV)), liposomes, lipid nanoparticles, niosomes, polymeric nanoparticles.

[0124] In some embodiments, the vector is an expression vector.

[0125] In some embodiments, the vector is one of a liposome, a lipid nanoparticle, a niosome, or a polymeric nanoparticle, and the vector comprises an isolated nucleic acid coding sequence described herein, preferably an RNA coding sequence.

[0126] In some embodiments, the vector, particularly the expression vector, is a plasmid. In some embodiments, the vector, particularly the expression vector, is a virus, such as an adeno-associated virus (AAV). Methods for producing viral vectors are well known in the art and include, for example, transfecting packaging cells and / or using transient transfection with a helper plasmid or virus.

[0127] Another aspect of the invention is a host cell comprising an isolated sequence described herein, a regulatory element described herein, an isolated nucleic acid coding sequence described herein, an expression cassette described herein, or a vector described herein.

[0128] In some embodiments, the host cell is an isolated host cell.

[0129] In some embodiments, the host cell may be a prokaryotic cell or a eukaryotic cell such as a yeast cell or an animal cell, particularly a mammalian cell.

[0130] The term "host cell" with respect to animal and human cells generally refers to cells of a cultured cell line. Animals and humans into which an isolated sequence described herein, a regulatory element described herein, an isolated nucleic acid coding sequence described herein, an expression cassette described herein, or a vector described herein has been introduced are expressly excluded from the definition of "host cell".

[0131] Another aspect of the present invention is a composition comprising, consisting essentially of, or consisting of an isolated sequence described herein, an isolated nucleic acid coding sequence described herein, a regulatory element described herein, an expression cassette described herein, a vector described herein, or a host cell described herein.

[0132] Another aspect of the present invention is a pharmaceutical composition comprising, consisting essentially of, or consisting of an isolated sequence described herein, an isolated nucleic acid coding sequence described herein, a regulatory element described herein, an expression cassette described herein, a vector described herein, or a host cell described herein and a pharmaceutically acceptable excipient or carrier.

[0133] Examples of pharmaceutically acceptable excipients or carriers that can be used in the pharmaceutical compositions described herein include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, protamine sulfate, sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, salts or electrolytes such as zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances (e.g., sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin.

[0134] Another aspect of the present invention is a medicament comprising, consisting essentially of, or consisting of an isolated sequence described herein, an isolated nucleic acid coding sequence described herein, a regulatory element described herein, an expression cassette described herein, a vector described herein, or a host cell described herein.

[0135] As used herein, "consisting essentially of" with reference to a composition, pharmaceutical composition or medicament means that the isolated nucleic acid sequence described herein, the isolated nucleic acid coding sequence described herein, the regulatory element described herein, the expression cassette described herein, the vector described herein or the host cell described herein is the only therapeutic agent or agent having biological activity in said composition, pharmaceutical composition or medicament.

[0136] Another aspect of the present invention is a kit comprising or consisting of the isolated sequences described herein, the regulatory elements described herein, the isolated nucleic acid coding sequences described herein, the expression cassettes described herein, the vectors described herein or the host cells described herein and optionally instructions for use.

[0137] "Kit" is intended to mean any product (e.g., a package or container) containing the isolated sequences described herein, the regulatory elements described herein, the isolated nucleic acid coding sequences described herein, the expression cassettes described herein, the vectors described herein or the host cells described herein. The kit can be promoted, distributed or sold as a unit for performing the uses or methods described herein.

[0138] Another aspect of the present invention is the isolated sequences described herein, the isolated nucleic acid coding sequences described herein, the regulatory elements described herein, the expression cassettes described herein, the vectors described herein, the host cells described herein, the compositions described herein or the pharmaceutical compositions described herein for use as a medicament.

[0139] Another aspect of the present invention is the isolated sequences described herein for use in gene therapy, the regulatory elements described herein, the isolated nucleic acid coding sequences described herein, the expression cassettes described herein, the vectors described herein, the host cells described herein, the compositions described herein, pharmaceutical compositions or the drugs described herein.

[0140] Another aspect of the present invention is the isolated sequences described herein for use in the treatment of sensory disorders, the isolated nucleic acid coding sequences described herein, the regulatory elements described herein, the expression cassettes described herein, the vectors described herein, the host cells described herein, the compositions described herein, pharmaceutical compositions or the drugs described herein.

[0141] Another aspect of the present invention is a pharmaceutical composition described herein for the treatment of sensory disorders or for use in the treatment of sensory disorders.

[0142] Another aspect of the present invention is the use of an isolated sequence described herein, an isolated nucleic acid coding sequence described herein, a regulatory element described herein, an expression cassette described herein, a vector described herein or a host cell described herein for the manufacture of a drug for the treatment of sensory disorders.

[0143] Another aspect of the present invention is a method of gene therapy in a subject in need thereof, the method comprising administering to the subject an isolated sequence described herein, an isolated nucleic acid coding sequence described herein, a regulatory element described herein, an expression cassette described herein or a vector described herein.

[0144] Another aspect of the present invention is a method for treating sensory disorders in a subject in need thereof, the method comprising administering to the subject an isolated sequence described herein, an isolated nucleic acid coding sequence described herein, a regulatory element described herein, an expression cassette described herein, or a vector described herein.

[0145] In some embodiments, the sensory disorder is a hereditary or congenital sensory disorder, i.e., a sensory disorder of genetic origin.

[0146] Another aspect of the present invention is the use of an isolated sequence described herein, an isolated nucleic acid coding sequence described herein, or an expression cassette described herein for producing a vector, particularly for producing an expression vector, especially for in vitro or ex vivo use.

[0147] Another aspect of the present invention is the use of a vector described herein for producing a host cell described herein, particularly for in vitro or ex vivo use.

[0148] Another aspect of the present invention is the use of the isolated sequences described herein, the isolated nucleic acid coding sequences described herein, the regulatory elements described herein, the expression cassettes described herein, or the vectors described herein for specifically expressing a gene of interest in cells that do not express miRNAs of the miR183 family, i.e., cells that do not express miR183, miR182, and / or miR96, particularly for in vitro or ex vivo use. For example, the isolated sequences described herein, the isolated nucleic acid coding sequences described herein, the regulatory elements described herein, the expression cassettes described herein, or the vectors described herein that contain the miR183 target sites described herein may be particularly used for specifically expressing a gene of interest in cells that do not express miR183. Similarly, the isolated sequences described herein, the isolated nucleic acid coding sequences described herein, the regulatory elements described herein, the expression cassettes described herein, or the vectors described herein that contain the miR182 target sites described herein may be particularly used for specifically expressing a gene of interest in cells that do not express miR182, and the isolated sequences described herein, the isolated nucleic acid coding sequences described herein, the regulatory elements described herein, the expression cassettes described herein, or the vectors described herein that contain the miR96 target sites described herein may be particularly used for specifically expressing a gene of interest in cells that do not express miR96.

[0149] Another aspect of the present invention is the use of the isolated sequences described herein, the isolated nucleic acid coding sequences described herein, the regulatory elements described herein, the expression cassettes described herein, or the vectors described herein for regulating or controlling the expression of a nucleic acid of interest, such as a gene of interest, particularly for in vitro or ex vivo use.

[0150] Another aspect of the present invention is the use of the isolated sequences described herein, the isolated nucleic acid coding sequences described herein, the regulatory elements described herein, the expression cassettes described herein or the vectors described herein for reducing the off-target expression of a nucleic acid of interest, such as a gene of interest, wherein the expression of the nucleic acid of interest can be prevented (or suppressed or inhibited) in cells and / or tissues that express at least one miRNA of the miR183 family, i.e., cells and / or tissues that express miR183, miR182 and / or miR96.

[0151] Another aspect of the present invention is a method for producing the vectors described herein, the method comprising inserting the regulatory elements described herein into a vector.

[0152] Another aspect of the present invention is a method, particularly an in vitro or ex vivo method, for regulating or controlling the expression of a nucleic acid of interest, such as a gene of interest, in a tissue comprising at least two different cell types, the method comprising introducing into the tissue the isolated nucleic acid coding sequences described herein, the regulatory elements described herein, the expression cassettes described herein or the vectors described herein.

[0153] Another aspect of the present invention is a method for regulating or controlling the expression of a nucleic acid of interest, such as a gene of interest, in a subject in need of gene therapy, the method comprising introducing into the tissue of the subject the isolated nucleic acid coding sequences described herein, the regulatory elements described herein, the expression cassettes described herein or the vectors described herein, wherein the tissue comprises at least two different cell types.

[0154] The subject in need of gene therapy may be a subject suffering from a sensory disorder, particularly a congenital sensory disorder.

[0155] As used herein, the expression "introduce ~ into the target tissue" may correspond to systemic administration to the subject or local administration to the subject.

[0156] In particular, a tissue containing at least two different cell types may contain at least one cell type in which expression of the nucleic acid of interest is desired and at least one cell type in which expression of the nucleic acid of interest is not desired.

[0157] In some embodiments, a tissue containing at least two different cell types contains at least one cell type that expresses at least one miRNA of the miR183 family (i.e., miR183, miR182, and / or miR96) and at least one cell type that does not express any miRNA of the miR183 family. In particular, a tissue containing at least two different cell types may contain at least one cell type that expresses miR183 and at least one cell type that does not express miR183. A tissue containing at least two different cell types may contain at least one cell type that expresses miR182 and at least one cell type that does not express miR182. A tissue containing at least two different cell types may contain at least one cell type that expresses miR96 and at least one cell type that does not express miR96.

[0158] As used herein, "does not express any miRNA of the miR183 family" is intended to mean that none of the miRNAs of miR183 are expressed in an amount sufficient for the miRNA to confer biological activity, particularly in an amount sufficient to mediate gene silencing.

[0159] Another aspect of the present invention is a method for reducing off-target expression of a nucleic acid of interest, such as a gene of interest, wherein the expression of the nucleic acid of interest can be prevented (or suppressed or inhibited) in cells and / or tissues expressing at least one miRNA of the miR183 family, i.e., cells and / or tissues expressing miR183, miR182, and / or miR96, and the method comprises introducing into a tissue (e.g., a tissue of a subject in need of gene therapy) an isolated nucleic acid coding sequence described herein, a regulatory element described herein, an expression cassette described herein, or a vector described herein, and the tissue comprises at least two different cell types described herein. [Table 1] JPEG2025522638000002.jpg161132JPEG2025522638000003.jpg165132JPEG2025522638000004.jpg175132JPEG2025522638000005.jpg63132 [Brief Description of the Drawings]

[0160]

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Example

[0161] The present invention is further illustrated by the following examples.

[0162] Example 1 Materials and methods Mice and cell lines Wild-type C57Bl / 6 mice were maintained under standard conditions, i.e., a 12-hour / 12-hour light / dark cycle and environmental enrichment. The study was conducted in accordance with Council Directive 2010 / 63 / EU of 22 September 2010 on the protection of animals used for scientific purposes and French Act No. 2013-118 of 1 February 2013 on animal health regulations, particularly with regard to the protection of animals used for scientific purposes.

[0163] HEK293 cells (human embryonic kidney 293 cells) were cultured under standard conditions in DMEM containing 10% fetal bovine serum (FBS).

[0164] Vectors An AAV vector was constructed for administration to mice. The AAV-GFP control construct contains a GFP expression cassette in which the GFP coding sequence is under the control of the ubiquitous promoter (see Figure 1A). In the AAV-GFP-precursor miR-183 construct, three copies of the miR-183 target site (so-called "precursor miR-183 target site") having the sequence shown in SEQ ID NO: 1, which is complementary to the human sequence of precursor miR-183 (i.e., SEQ ID NO: 2), were inserted at the 3' (i.e., downstream) of the GFP coding sequence (see Figure 1B).

[0165] Using the pAAV-smCBA-eGFP-bGH backbone (Genscript), a plasmid vector was constructed for transfection into HEK293 cells. As shown in Figure 2A, the "miR-183-free" control construct contains a GFP expression cassette in which the eGFP coding sequence is under the control of the ubiquitous promoter smCBA (truncated chimeric cytomegalovirus (CMV)-chicken β-actin). By cloning the microRNA target site into the pAAV-smCBA-eGFP-bGH backbone (Genscript), a plasmid containing the miR-183 target site, i.e., · A plasmid (1× precursor miRT183 - Figure 2B) containing one copy of the so - called precursor miR183 target site having the sequence shown in SEQ ID NO: 1 downstream of the smCBA - eGFP cassette, · A plasmid (3× precursor miRT183 - Figure 2C) containing three copies of the so - called precursor miR183 target site having the sequence shown in SEQ ID NO: 1 downstream of the smCBA - eGFP cassette, · A plasmid (3× miRT183 5P - Figure 2D) containing three copies of the miR183 target site (so - called "5p binding site") consisting of the sequence shown in SEQ ID NO: 5 complementary to hsa - miR183 - 5p (i.e., SEQ ID NO: 3) downstream of the smCBA - GFP cassette (the three copies of the so - called "5p binding site" are separated by a spacer having the sequence shown in SEQ ID NO: 14), and · A plasmid (3× miRT183 - 5 + 3P - Figure 2E) containing three copies of the miR183 target site consisting of the sequence shown in SEQ ID NO: 5 (so - called "5p binding site" complementary to hsa - miR183 - 5p, i.e., SEQ ID NO: 3) in series and the sequence shown in SEQ ID NO: 6 (so - called "3p binding site" complementary to hsa - miR183 - 3p, i.e., SEQ ID NO: 4) downstream of the smCBA - GFP cassette (the so - called "5p binding site" and "3p binding site" are separated by a spacer having the sequence shown in SEQ ID NO: 14) were obtained. In the plasmid "3× miRT183 - 5P" of Figure 2D, the regulatory element containing three copies of the miR183 target site consisting of the sequence shown in SEQ ID NO: 5 (so - called "5p binding site") and two spacers has the sequence shown in SEQ ID NO: 8. In the plasmid "3× miRT183 - 5 + 3P" of Figure 2E, the regulatory element containing three copies of the miR183 target site consisting of the sequence shown in SEQ ID NO: 5 in series (so - called "5p binding site") and the sequence shown in SEQ ID NO: 6 (so - called "3p binding site") and five spacers has the sequence shown in SEQ ID NO: 9.

[0166] In vivo experiments On the 14th day after birth (P14), an adeno-associated virus vector (AAV) containing a nucleotide sequence encoding GFP (green fluorescent protein) was administered to wild-type C57Bl / 6 mice. 1 μL of the total volume of the AAV vector was injected into the inner ear of the mice through the round window membrane. Either the AAV-GFP construct or the AAV-GFP-precursor miRT183 construct shown in Figure 1 was injected at a concentration of 5×10 13 vg / mL (vector genome per mL). Two weeks after the injection, the mice were euthanized, the temporal bones were collected, and fixed for histological analysis. After the cochlea was dissected, the organ of Corti was immunolabeled with myosin VIIa (MyoVIIa). MyoVIIa was specifically expressed in hair cells in the cochlea. The organ of Corti was then analyzed by confocal microscopy to evaluate the expression of both MyoVIIa and GFP.

[0167] In vitro experiments To evaluate the effect on gene expression of regulatory elements containing miR183 target sites, several plasmids were synthesized and compared (see Figure 2). Each plasmid (20 μg) was transfected into HEK293 cells in a T75 flask using polyethyleneimine (PEI) diluted in OptiMEM medium. After 7 hours, the PEI-DNA-OptiMEM medium was replaced with DMEM 2% FBS. Three days after transfection, the cells were harvested, washed with PBS, and lysed with PBS-Tween20 (0.2%). After clarifying the lysate by centrifugation, GFP fluorescence was measured using a Synergy plate reader (Agilent), and the results were obtained as arbitrary units. In parallel, the protein content was determined using a BCA assay kit (ThermoFisher). The following GFP primers and probe: · eGFP fwd: 5’-GAGCGCACCATCTTCTTCA (SEQ ID NO: 18), · eGFP rev: 5’-TTCAGCTCGATGCGGTTC (SEQ ID NO: 19), and · eGFP probe: 5’-AGGACGACGGCAACTACAAGACC (SEQ ID NO: 20) Using this, the plasmid copy number in the cell lysate was measured by TaqMan qPCR.

[0168] Results To evaluate the regulatory effect of the so-called precursor miR183 target site (i.e., the miR183 target site having the sequence shown in SEQ ID NO: 1) on the expression of the transgene, mice were injected with the AAV-GFP-precursor miRT183 construct containing a regulatory element consisting of three copies of the precursor miR183 target site (see Fig. 1A). As a control, mice were injected with the AAV-GFP construct that did not contain any regulatory elements (see Fig. 1B). Two weeks after injection, the mice were euthanized, their cochleae were harvested, stained for myosin VIIA and GFP, and analyzed by confocal microscopy. Analysis of cochleae from mice injected with the AAV-GFP control construct revealed co-localization of GFP and MyoVIIA in the inner hair cell layer (Figs. 3A - 3B), indicating efficient GFP expression in hair cells (inner hair cells, IHCs and outer hair cells, OHCs). In contrast, the presence of the regulatory element containing three copies of the precursor miR183 target site inhibited GFP expression in the inner hair cells of cochleae from mice injected with the AAV-GFP-precursor miR183 construct, as demonstrated by the absence of co-localization between GFP and MyoVIIa and the persistence of the remaining GFP signal in the underlying supporting cells (Figs. 3C - 3D). The regulatory effect of the precursor miR183 target site was also demonstrated after quantification of GFP-positive hair cells from cochleae of mice injected with the AAV-GFP construct or the AAV-GFP-precursor miRT183 construct. As shown in Fig. 3E, for both inner hair cells (IHCs) and outer hair cells (OHCs) of mice injected with the AAV-GFP construct, i.e., in the absence of the regulatory element containing the miR183 target site, the percentage of GFP-positive cells was significantly higher (Fig. 3E).

[0169] These data demonstrate that GFP expression was significantly inhibited in the hair cells of mice treated with AAV-GFP-precursor miRT183 due to the presence of the so-called precursor miR183 target site with the sequence shown in SEQ ID NO: 1 downstream of the GFP expression cassette.

[0170] To further evaluate the regulatory effect of the precursor miR183 target site on transgene expression, HEK293 cells were transfected with plasmid constructs that did not contain the miR183 target site downstream of the GFP expression cassette (described in Figure 2A), plasmid constructs that contained one copy of the precursor miR183 target site (described in Figure 2B), or plasmid constructs that contained three copies of the precursor target site (described in Figure 2C). After cell lysis, GFP expression in the transfected cells was evaluated using a fluorescence plate reader. As shown in Figures 4A - 4C, increasing the number of copies of the precursor miR183 target site in the plasmid construct resulted in a decrease in the number of GFP-positive cells. Figure 4D shows the quantification of GFP fluorescence in the transfected HEK293 cell lysates, confirming that (i) the presence of one precursor miR183 target site was sufficient to strongly inhibit GFP expression, and (ii) increasing the number of precursor miR183 target sites enhanced the inhibition of GFP expression.

[0171] These data demonstrate that an efficient suppression of transgene expression can be obtained with a vector containing a regulatory element consisting of one so-called precursor miR183 target site with the sequence shown in SEQ ID NO: 1. Furthermore, these data demonstrate that vectors containing regulatory elements with several copies of the precursor miR183 target site exhibit an even more potent inhibitory effect on transgene expression.

[0172] To compare the regulatory effect of the precursor miR183 target site with that of so-called "mature miR183 target sites" consisting of sequences complementary to the sequence of mature miR183, a plasmid construct without a miR183 target site downstream of the GFP expression cassette (described in Figure 2A), a plasmid construct containing three copies of the precursor miR183 target site (described in Figure 2C), a plasmid construct containing three copies of so-called "mature miR183 target sites" consisting of the sequence shown in SEQ ID NO: 5 complementary to hsa-miR183-5p (so-called "5p binding site") (described in Figure 2D), or a plasmid construct containing three copies of so-called "mature miR183 target sites" consisting of the sequence shown in SEQ ID NO: 5 complementary to hsa-miR183-5p (so-called "5p binding site") and the sequence shown in SEQ ID NO: 6 complementary to hsa-miR183-3p (so-called "3p binding site") in series (described in Figure 2E) were transfected into HEK293 cells. First, the transfection efficiency was evaluated by quantitative PCR, and it was found that similar transfection efficiencies were obtained using each of the four plasmid constructs (Figure 5A). Then, GFP fluorescence was measured in cell lysates using a fluorescence plate reader, and a significant decrease in GFP expression was revealed in cells transfected with a plasmid containing three copies of the precursor miR183 target site when compared with GFP expression in cells transfected with a control construct without any miR183 target site (Figure 5B). Surprisingly, both plasmid constructs containing three copies of so-called "mature miR183 target sites" failed to cause a significant decrease in GFP expression when compared with GFP expression in cells transfected with a control construct without any miR183 target site (Figure 5B).

[0173] These data unexpectedly demonstrate an excellent effect in transgene silencing of the precursor miR183 target site having the sequence shown in SEQ ID NO: 1, which is complementary to the sequence of the precursor miR183, when compared to the so-called "mature miR183 target site" having a sequence complementary to the sequence of the mature miR183.

[0174] Example 2 Materials and Methods Vector Using the pAAV-smCBA-eGFP-bGH backbone (Genscript), a plasmid vector was constructed for transfection into HEK293 cells. As shown in Example 1, the "miRT-free" control construct contains a GFP expression cassette in which the eGFP coding sequence is under the control of the ubiquitous promoter smCBA (truncated chimeric cytomegalovirus (CMV)-chicken β-actin). By cloning the microRNA target site into the pAAV-smCBA-eGFP-bGH backbone (Genscript), plasmids containing the miRNA target site, namely · a plasmid containing two copies of the so-called precursor miR183 target site having the sequence shown in SEQ ID NO: 1 downstream of the smCBA-eGFP cassette (3× precursor miRT183), · a plasmid containing three copies of the so-called precursor miR183 target site having the sequence shown in SEQ ID NO: 1 downstream of the smCBA-eGFP cassette, · a plasmid containing three copies of the so-called precursor miR182 target site having the sequence shown in SEQ ID NO: 21, which is complementary to the human sequence of precursor miR182 (i.e., SEQ ID NO: 22) downstream of the smCBA-eGFP cassette (3× precursor miRT182), · a plasmid containing three copies of the so-called precursor miR96 target site having the sequence shown in SEQ ID NO: 24, which is complementary to the human sequence of precursor miR182 (i.e., SEQ ID NO: 25) downstream of the smCBA-eGFP cassette were obtained.

[0175] In Vitro Experiments To confirm the effect of the regulatory elements containing miR target sites of the miR183 family described in this specification on gene expression, several plasmids were synthesized. Each plasmid (2 μg) was transfected into HEK293 cells in 6-well plates using polyethyleneimine (PEI) diluted in OptiMEM medium. After 12 hours, the PEI-DNA-OptiMEM medium was replaced with DMEM 2% FBS (fetal bovine serum). Cells were harvested 2 or 3 days after transfection (24 or 48 hours after medium change), washed with PBS, and lysed with PBS-Tween20 (0.2%). After clarifying the lysate by centrifugation, GFP fluorescence was measured using a Synergy plate reader (Agilent). In parallel, the protein content was determined using a BCA assay kit (ThermoFisher), and the data was normalized using it.

[0176] Results HEK293 cells were transfected with plasmid constructs that did not contain miR target sites downstream of the GFP expression cassette or two copies of the precursor miR183 target site, three copies of the precursor miR183 target site (corresponding to SEQ ID NO: 7), three copies of the precursor miR182 target site (corresponding to SEQ ID NO: 23), or three copies of the precursor miR96 target site (corresponding to SEQ ID NO: 26). GFP expression in the transfected cells was evaluated using a fluorescence plate reader after cell lysis at either 24 hours after medium change (i.e., 2 days after transfection) or 48 hours after medium change (i.e., 3 days after transfection).

[0177] Figure 6 shows the quantification of GFP fluorescence in the transfected HEK293 cell lysates, and it is confirmed that the presence of the precursor miR183 target site significantly inhibits GFP expression. It should be noted that the inhibition of GFP expression is maintained over time by similar levels of inhibition observed 24 hours after medium change (Figure 6A) and 48 hours after medium change (Figure 6B). It is also confirmed from Figure 6 that the presence of two copies of the precursor miR183 target site is sufficient to significantly inhibit GFP expression.

[0178] Figure 7 shows the quantification of GFP fluorescence in the transfected HEK293 cell lysates, and it is also demonstrated that the presence of the precursor miR182 target site significantly inhibits GFP expression. It should be noted that the inhibition of GFP expression is maintained over time by similar levels of inhibition observed 24 hours after medium change (Figure 7A) and 48 hours after medium change (Figure 7B).

[0179] Figure 8 shows the quantification of GFP fluorescence in the transfected HEK293 cell lysates, and it is demonstrated that the presence of the precursor miR96 target site significantly inhibits GFP expression. It should be noted that the inhibition of GFP expression is maintained over time by similar levels of inhibition observed 24 hours after medium change (Figure 8A) and 48 hours after medium change (Figure 8B).

[0180] These data provide evidence that, similar to the so-called precursor miR183 target site (SEQ ID NO: 1), the so-called precursor miR182 target site (SEQ ID NO: 21) and the so-called precursor miR96 target site (SEQ ID NO: 24) can significantly inhibit gene expression in each of the cells expressing miR182 or miR96. Therefore, using the so-called miR target sites of the precursor miR183 family described herein, for example, inserting them into an expression cassette or vector to regulate the expression of a nucleic acid of interest, particularly preventing the expression of the nucleic acid of interest in cells expressing miRNAs of the miR183 family, can be achieved.

Claims

**Claim 1** An isolated nucleic acid sequence comprising at least two copies of an miRNA target site of the miR183 family, wherein the miRNA target site has a sequence shown in SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24, or a sequence having at least 90% identity with any one of SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24, said isolated nucleic acid sequence. **Claim 2** The isolated nucleic acid sequence according to claim 1, wherein the miRNA target site of the miR183 family is a miR183 target site having a sequence shown in SEQ ID NO: 1 or a sequence having at least 90% identity with SEQ ID NO:

1. **Claim 3** The isolated nucleic acid sequence according to claim 1 or 2, wherein the isolated nucleic acid sequence comprises 2 to 6 copies, preferably 3 copies, of the miRNA target site of the miR183 family. **Claim 4** The isolated nucleic acid sequence according to any one of claims 1 to 3, wherein the isolated nucleic acid sequence comprises a sequence shown in SEQ ID NO: 7, SEQ ID NO: 23 or SEQ ID NO: 26, or a sequence having at least 90% identity with any one of SEQ ID NO: 7, SEQ ID NO: 23 or SEQ ID NO:

26. **Claim 5** The isolated nucleic acid sequence according to any one of claims 1 to 3, wherein the copies of the miRNA target site of the miR183 family are separated by a spacer. **Claim 6** An expression cassette comprising a promoter, a gene of interest, and a regulatory element comprising at least one copy of an miRNA target site of the miR183 family, wherein the miRNA target site has a sequence shown in SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24, or a sequence having at least 90% identity with any one of SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24, said expression cassette. **Claim 7** A vector comprising a regulatory element comprising at least one copy of an miRNA target site of the miR183 family, wherein the miRNA target site has a sequence shown in SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24, or a sequence having at least 90% identity with any one of SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24, said vector. **Claim 8** The regulatory element according to claim 6 or the vector according to claim 7, comprising at least one copy of the miR183 target site having the sequence shown in SEQ ID NO: 1 or a sequence having at least 90% identity with SEQ ID NO:

1.

9. The regulatory element according to claim 6 or 8 or the vector according to claim 7 or 8, comprising 2 to 6 copies, preferably 3 copies, of the miRNA target site of the miR183 family, the copies being optionally separated by a spacer.

10. The regulatory element according to any one of claims 6 or 8 to 9 or the vector according to any one of claims 7 to 9, comprising the sequence shown in SEQ ID NO: 7, SEQ ID NO: 23 or SEQ ID NO: 26 or a sequence having at least 90% identity with any one of SEQ ID NO: 7, SEQ ID NO: 23 or SEQ ID NO:

26.

11. The regulatory element according to any one of claims 6 or 8 to 10 or the vector according to any one of claims 7 to 10, further comprising at least one copy of another miRNA target site, preferably at least one copy of another miRNA target site of the miR183 family.

12. The vector according to any one of claims 6 or 8 to 11 or the vector according to any one of claims 7 to 11, comprising a gene of interest, wherein the regulatory element is operably linked to or inserted into the gene of interest, preferably inserted into the 3'UTR of the gene of interest.

13. A pharmaceutical composition comprising the isolated nucleic acid sequence according to any one of claims 1 to 5, the expression cassette according to any one of claims 6 or 8 to 12 or the vector according to any one of claims 7 to 12, and at least one pharmaceutically acceptable excipient.

14. The isolated nucleic acid sequence according to any one of claims 1 to 5, the expression cassette according to any one of claims 6 or 8 to 12, the vector according to any one of claims 7 to 12 or the pharmaceutical composition according to claim 13, for use as a medicament.

15. Use of the isolated nucleic acid sequence according to any one of claims 1 to 5, the expression cassette according to any one of claims 6 or 8 to 12, or the vector according to claim 12 for specifically expressing a gene of interest in cells that do not express miRNA of the miR183 family.

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