Regulatory sequence comprising microRNA target site

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, the miRNA target site of the miR183 family having a sequence as shown in SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24, or having 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 invention also relates to a vector comprising a regulatory element, said regulatory element comprising at least one copy of said miRNA target site of the miR183 family. The isolated nucleic acid sequences and vectors can be particularly used to control the expression of genes of interest, such as in the design and development of gene therapies.

Description

Field of the Invention

[0001] The present invention relates to a regulatory sequence comprising a microRNA target site, in particular a miRNA target site of the miR183 family, and use thereof for controlling the expression of a target nucleic acid sequence (eg a target gene). Background of the Invention

[0003] MicroRNA (miRNA or miR) is a class of abundant short, naturally occurring non-coding RNAs, about 20 nucleotides in length, that play an important role in regulating gene expression. Most miRNAs are transcribed from DNA sequences by RNA polymerase II or III into large RNA precursors, called primary miRNAs or pri-miRNAs. Pri-miRNAs are processed into hairpin precursor miRNAs (precursor miRNAs or pre-miRNAs) of about 70 to 120 nucleotides in length, which themselves are further processed into mature miRNAs of about 20 nucleotides in length. In most cases, after recognizing their target sites (usually located in the 3'-untranslated transcription region (UTR)), mature miRNAs achieve complete or incomplete base complementary pairing, leading to mRNA cleavage and inhibiting the translation of mRNA into proteins. Therefore, miRNAs play an important role in regulating post-transcriptional gene expression in a sequence-specific manner.

[0004] Therefore, the art has developed and described selective gene silencing strategies based on the use of vectors containing one or more copies of miRNA target sites, which are operably linked to or inserted into the nucleic acid sequence of the target gene. In these strategies, the miRNA target site is composed of a short nucleotide sequence complementary to a specific mature miRNA. After the vector is introduced into a cell expressing the miRNA, the miRNA specifically recognizes the miRNA target site, and the miRNA inhibits the expression of the target gene in the cell by interacting with the miRNA target site.

[0005] Thousands of miRNAs have been found in many organisms, and more than 2,000 mature miRNAs have been identified in Homo sapiens. MiRNAs can be grouped into clusters, which are defined as several miR genes located adjacent to each other on the chromosome, which are transcribed into a long primary miRNA and then processed into individual hairpin precursor miRNAs. In addition, the high sequence homology between miRNAs in a cluster classifies them into a family and allows the miRNAs of this family to have common and unique mRNA targets. Among the identified miRNA families, the miR183 family consists of three homologous miRNAs: miR183, miR96, and miR182. The miR183 family of miRNAs is highly expressed in sensory cells of the eye, nose, and inner ear and is essential for their development.

[0006] Many sensory impairments, especially blindness and deafness, are of hereditary origin, that is, caused by the presence of gene mutations. Such sensory impairments are often referred to as hereditary or congenital sensory impairments. Currently, many gene therapy approaches are being developed to treat sensory impairments caused by single gene mutations. Treatments based on gene therapy rely on providing complete genes to compensate for defective genes that cause congenital sensory impairments. It is noteworthy that it is crucial that the genes introduced by gene therapy are specifically expressed only in cells and tissues that naturally express the gene. In particular, it is crucial to ensure that the genes introduced by gene therapy are not expressed in cells and tissues where gene expression may cause any harmful effects. Therefore, regulatory sequences and vectors that can control the expression of target genes are needed. Specifically, regulatory sequences and vectors are needed to prevent or inhibit the expression of target genes in cells and tissues where their expression may cause any harmful effects.

[0007] The inventors surprisingly confirmed that the introduction of a vector comprising a gene and at least one copy of a "precursor miR183 target site" into a cell expressing miR183 significantly inhibited the expression of the gene in the cell. The so-called "precursor miR183 target site" used herein refers to a miR183 target site having a sequence complementary to the human sequence of precursor miR183. Similar regulatory effects were observed in other "precursor miRNA target sites" of the miR183 family, i.e., the so-called "precursor miR182 target site" 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" 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 is more effective than the shorter miR183 target site having a sequence complementary to the human sequence of mature miR183 in inhibiting gene expression in cells expressing miR183. The inventors also demonstrated that injection of a vector containing the gene and at least one copy of the so-called pro-miR183 target site into the inner ear of mice significantly suppressed gene expression in both inner and outer hair cells of the cochlea.

[0008] These results therefore demonstrate that vectors containing at least one copy of a so-called pre-miRNA target site of the miR183 family can be a useful tool for selectively and effectively inhibiting unwanted gene expression in sensory cells. Such vectors may be particularly useful in gene therapy approaches seeking to treat congenital sensory disorders. Summary of the invention

[0009] Therefore, the present invention relates to an isolated nucleic acid sequence comprising at least two copies of a miRNA target site of the miR183 family, wherein the miRNA target site has a sequence as shown in SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24, or a sequence having at least 90% identity to 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 a miR183 target site, wherein the miR183 target site has a sequence as shown in SEQ ID NO: 1 or a sequence having at least 90% identity to SEQ ID NO: 1. In some embodiments, the isolated nucleic acid sequence comprises 2 to 6 copies of a miRNA target site of the miR183 family, for example, 2 to 6 copies of a miR183 target site having a sequence as shown in SEQ ID NO: 1 or a sequence having at least 90% identity to SEQ ID NO: 1. In some embodiments, the isolated nucleic acid sequence comprises three copies of a miRNA target site of the miR183 family, such as three copies of a miR183 target site having a sequence as set forth in SEQ ID NO: 1 or a sequence having at least 90% identity to SEQ ID NO: 1. In some embodiments, the isolated nucleic acid sequence comprises a sequence as set forth in SEQ ID NO: 7, SEQ ID NO: 23, or SEQ ID NO: 26, or a sequence having at least 90% identity to 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 as set forth in SEQ ID NO: 7, or a sequence having at least 90% identity to SEQ ID NO: 7. In some embodiments, the copies of the miRNA target site of the miR183 family, such as the copies of the miR183 target site, are separated by spacers.

[0010] The present invention also relates to an expression cassette comprising a promoter, a gene of interest, and a regulatory element, wherein the regulatory element comprises at least one copy of a miRNA target site of the miR183 family, wherein the miRNA target site has a sequence as shown in SEQ ID NO: 1, SEQ ID NO: 21, or SEQ ID NO: 24, or a sequence having at least 90% identity to 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, wherein the regulatory element comprises at least one copy of a miRNA target site of the miR183 family, wherein the miRNA target site has a sequence as shown in SEQ ID NO: 1, SEQ ID NO: 21, or SEQ ID NO: 24, or a sequence having at least 90% identity to 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 a miR183 target site having a sequence as shown in SEQ ID NO: 1, or a sequence having at least 90% identity to SEQ ID NO: 1. In some embodiments, the regulatory element comprises 2 to 6 copies, preferably 3 copies, of a miRNA target site of the miR183 family (e.g., a miR183 target site having a sequence as shown in SEQ ID NO: 1 or having a sequence at least 90% identical to SEQ ID NO: 1), and the copies are optionally separated by a spacer. In some embodiments, the regulatory element comprises a sequence as shown in SEQ ID NO: 7, SEQ ID NO: 23, or SEQ ID NO: 26, or a sequence at least 90% identical to 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 as shown in SEQ ID NO: 7, or a sequence at least 90% identical to 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 a miR183 target site having a sequence shown in SEQ ID NO:1 or a sequence having at least 90% identity to 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.

[0011] In some embodiments, the regulatory element contained in the expression cassette described herein or in the vector described herein (the expression cassette or vector also contains the target gene) is operably linked to the target gene or inserted into the target gene, preferably into the 3'-UTR of the target gene.

[0012] The present invention also relates to a pharmaceutical composition comprising the isolated nucleic acid sequence described herein, or the expression cassette described herein, or the vector described herein, and at least one pharmaceutically acceptable excipient.

[0013] The present invention also relates to an isolated nucleic acid sequence as described herein, or an expression cassette as described herein, or a vector as described herein, or a pharmaceutical composition as described herein, for use as a medicament.

[0014] The present invention also relates to the use of the isolated nucleic acid sequence described herein, or the expression cassette described herein, or the vector described herein, for specifically expressing a target gene in cells that do not express miRNA of the miR183 family (eg, in cells that do not express miR183).

[0015] definition

[0016] In the present invention, the following terms have the following meanings:

[0017] The terms "a" and "an" refer to one or to more than one (ie, to at least one) of the grammatical object of the article. For example, "an element" refers to one element or to more than one element.

[0018] "About" preceding a number encompasses plus or minus 10% or less of the numerical value. It is to be understood that the value referred to by the term "about" itself is also specifically and preferably disclosed.

[0019] "Encoding", as in coding sequence, refers to the inherent property of a specific nucleotide sequence in a nucleic acid (e.g., a gene, complementary DNA (cDNA), or messenger RNA (mRNA)) to serve as a template for the synthesis of other polymers and macromolecules having a defined nucleotide sequence (e.g., ribosomal RNA (rRNA), transfer RNA (tRNA), and mRNA) or a defined amino acid sequence (e.g., polypeptides or proteins) in biological processes, and the resulting biological properties.

[0020] "Expression" refers to the transcription and / or translation of a specific nucleotide sequence (eg, a gene).

[0021] The term "gene" as used herein refers broadly to a coding nucleic acid sequence that can be transcribed into an RNA molecule, which is a coding RNA molecule (e.g., mRNA) that can be subsequently translated into a polypeptide or protein, or a non-coding RNA molecule (e.g., rRNA or tRNA). "Transgenic" refers in particular to a gene derived from a species that will be introduced into an organism belonging to a different species. Therefore, 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 encodes a protein. A gene, particularly a gene comprising a CDS, may also preferably include an untranslated transcription region (UTR), such as a 3'-UTR and / or a 5'-UTR, and other sequences that are transcribed but not translated, such as regulatory elements and / or introns. Therefore, as used herein, the term "gene" may refer to a coding nucleic acid sequence comprising a coding sequence (or CDS) and at least one regulatory element (e.g., 3'-UTR, 5'-UTR, and / or introns) that is transcribed but not translated.

[0022] As used herein, when referring to a given sequence (e.g., a miRNA target site), the expression "having a sequence shown in SEQ ID NO: X" means that the given sequence (e.g., a miRNA target site) comprises or consists of the sequence shown in SEQ ID NO: X.

[0023] "Identity" or "sameness" when used herein to describe the relationship between the sequences of two or more nucleic acids refers to the degree of sequence relatedness between the nucleic acids, which is determined by the number of matches between fragments of two or more nucleotides. "Identity" measures the percentage of identical matches between the smaller sequences in two or more sequences, where gap comparisons (if any) are solved by a specific mathematical model or computer program (i.e., "algorithm"). The identity of related nucleic acid sequences can be easily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM, and Griffin, HG, 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 maximize the match between the sequences tested. Methods for determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucleic Acids Res. 1984 Jan 11; 12(1Pt1): 387-95; Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN and FASTA (Altschul et al., J. MoI. Biol. 215, 403-410 (1990)).The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLASTX Annual, Altschul et al. NCB / NLM / NIH Bethesda, Md. 20894; Altschul et al., J. MoI. Biol. 215, 403-410 (1990)).

[0024] When referring to nucleic acids, "isolated" refers to nucleic acids that are altered or removed from their natural state. For example, nucleic acids naturally present in living organisms are not "isolated", but nucleic acids that are partially or completely separated from coexisting substances in their natural state are "isolated". Therefore, "isolated nucleic acids" are nucleic acids that are substantially separated from other nucleic acid sequences (e.g., genomic DNA or RNA) and proteins or complexes (e.g., ribosomes and polymerases) that naturally accompany natural sequences. Isolated nucleic acids can exist in a substantially purified form or in a non-natural environment such as a host cell. Typically, preparations of isolated nucleic acids can contain nucleic acids with a purity of at least about 80%, a purity of at least about 85%, a purity of at least about 90%, a purity of at least about 95%, a purity of greater than about 95%, a purity of greater than about 96%, a purity of greater than about 97%, a purity of greater than about 98%, or a purity of greater than about 99%. Therefore, isolated nucleic acids include nucleic acids purified by standard purification methods and include nucleic acid sequences that have been removed from their naturally occurring environment. Isolated nucleic acids also include chemically synthesized nucleic acids and nucleic acids biologically synthesized by heterologous systems.

[0025] "microRNA" or "miRNA" refers to an endogenous small non-coding RNA molecule of about 18 to about 24 nucleotides in length, which plays a key 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. The miRNA gene is transcribed by RNA polymerase II or III and then processed to produce single-stranded mature miRNAs, which are integrated into the RNA-induced silencing complex (RISC). As the central part of the RISC complex, miRNA guides RISC to its mRNA target, where miRNA usually binds to the 3' untranslated region (3'UTR) of the mRNA transcript through partially complementary base pairing. It is worth noting that complete base pairing must occur on a short length of 7 or 8 nucleotides complementary to the so-called miRNA "seed" region located at positions 2-8 of the 5' end of the mature miRNA. Gene silencing can occur through mRNA cleavage mediated by argonaute-2 (AG02) or translation inhibition promoted by AG01 to 4, both of which ultimately lead to reduced levels of the corresponding protein. As used herein, "miRNA", such as "miR183", refers to 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.

[0026] As used herein, "microRNA target site" or "miRNA target site" or "miR target site" refers to a nucleic acid sequence that can bind to miRNA (i.e., mature miRNA). The term "microRNA target site" or "miRNA target site" or "miR target site" as used herein includes both endogenous target sites that can be found in natural transcripts and artificial or engineered target sites (i.e., non-natural target sites) that can be inserted into a vector (especially an expression vector) as a regulatory element (or regulatory sequence) to control the expression of a target nucleic acid sequence (e.g., a target gene). Specifically, in a vector, a miRNA target site can be operably linked to a gene sequence or inserted into a gene sequence, especially into a transcribed sequence of an inserted gene. By definition, a miRNA target site must include 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 at least 5 nucleotides in length, typically 6-7 nucleotides in length. Therefore, the miR183 target site must include a nucleic acid sequence that is at least partially complementary to miR183, for example, a nucleic acid sequence that is complementary to miR183 at least 7-8 nucleotides in length. Similarly, the miR182 target site must contain a nucleic acid sequence that is at least partially complementary to miR182, e.g., a nucleic acid sequence that is complementary to miR182 over a length of at least 7-8 nucleotides; and the miR96 target site must contain a nucleic acid sequence that is at least partially complementary to miR96, e.g., a nucleic acid sequence that is complementary to miR96 over a length of at least 7-8 nucleotides. miRNA target sites, particularly artificial or engineered miRNA target sites (i.e., non-naturally occurring target sites), may also 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 to 24 nucleotides of the miRNA). When inserted into a vector, the miR target site allows binding of the corresponding miRNA and is therefore able to mediate miRNA-induced silencing of expression of a target nucleic acid (e.g., a target gene) when the vector is introduced into a host cell expressing the miRNA. For example, when inserted into a vector, the miR183 target site allows the binding of miR183, and thus can mediate miR183-induced silencing of target nucleic acid (e.g., target gene) expression when the vector is introduced into a host cell expressing miR183. Similarly, when inserted into a vector, the miR182 target site allows the binding of miR182, and thus can mediate miR182-induced silencing of target nucleic acid (e.g., target gene) expression when the vector is introduced into a host cell expressing miR182; when inserted into a vector, the miR96 target site allows the binding of miR96, and thus can mediate miR96-induced silencing of target nucleic acid (e.g., target gene) expression when the vector is introduced into a host cell expressing miR96.Methods for assessing whether a nucleic acid sequence may 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 experimental section below.

[0027] As used herein, "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 to miRNA (i.e., mature miRNA) belonging to the miR183 family (sometimes also referred to as the miR183 cluster). Therefore, 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 to miR183, miR182 and / or miR96.

[0028] "Nucleic acid" refers to a nucleotide polymer (i.e., polynucleotide) covalently linked by a phosphodiester bond, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), in the form of a single strand or double strand. As used herein, nucleic acids can therefore be single-stranded, partially double-stranded, or completely double-stranded. The nucleotides constituting the nucleic acids of the present disclosure can be unmodified (natural) nucleotides or non-natural or modified nucleotides. Unmodified (or natural or naturally occurring) 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 includes nucleic acids containing known analogs of natural nucleotides, which have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.

[0029] "Nucleic acid sequence" or "nucleotide sequence" refers to a continuous sequence of nucleotides in a single nucleic acid. Unless otherwise indicated, a specific nucleic acid sequence also implicitly includes its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs (single nucleotide polymorphisms) and complementary sequences and sequences explicitly indicated. It should be noted that specific nucleic acid sequences as described herein implicitly include its corresponding complementary sequences. It should be noted that specific nucleic acid sequences as described herein implicitly include DNA sequences and corresponding RNA sequences.

[0030] "Operably linked (operatively linked)" refers to the functional connection between a regulatory sequence and a heterologous nucleic acid sequence (e.g., a gene), resulting in the former regulating the expression of the latter. For example, when a first nucleic acid sequence is in a functional relationship with a second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. In particular, if a promoter affects the transcription or expression of a gene, the promoter is operably linked to the gene. Similarly, if a regulatory sequence affects (i.e., induces or inhibits (or represses)) the expression of a gene, the regulatory sequence is operably linked to the gene. Operably linked sequences can be adjacent to each other.

[0031] "Pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to an excipient or carrier that does not produce adverse, allergic or other undesirable reactions when administered to a mammal, preferably a human. It includes any and all solvents, such as dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents and absorption delaying agents. A pharmaceutically acceptable excipient or carrier refers to any type of non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation adjuvant. For human administration, the formulation should meet the sterility, pyrogenicity, general safety and purity standards required by regulatory agencies such as the FDA (U.S. Food and Drug Administration) or EMA (European Medicines Agency).

[0032] "Vector" refers to a vector that can introduce a nucleic acid sequence (e.g., a DNA or RNA molecule) (e.g., a nucleic acid encoding a target RNA or polypeptide or protein) into a host cell, thereby transforming, transfecting or transducing the host cell and promoting the expression (e.g., transcription and / or translation) of the introduced nucleic acid sequence.

[0033] "Expression vector" refers to a vector comprising a regulatory element (or regulatory sequence) that is operably connected to or will be operably connected to a target nucleic acid sequence to be expressed (e.g., a target gene). Thus, the expression vector comprises sufficient cis-acting regulatory elements to control the expression of the target nucleic acid sequence (present in the expression vector or to be inserted into the expression vector); other elements that may be required to control the expression of the target nucleic acid sequence may be provided by a host cell or an in vitro expression system (e.g., a miRNA that will bind to a miR target site). Cis-acting regulatory elements include, for example, promoters and miR target sites, such as the miR183 target site, miR182 target site, and miR96 target site described herein. DETAILED DESCRIPTION OF THE INVENTION

[0035] Human microRNA183 (miR183 or miR-183) belongs to the miR183 family (sometimes also referred to as the miR183 cluster), which consists of three homologous miRNAs: miR183 (or miR-183), miR96 (or miR-96), and miR182 (or miR-182). The miR183 family of miRNAs is particularly important for the normal development of sensory organs. Specifically, miR183 family of miRNAs are expressed in sensory neurons and hair cells of vertebrates and sensory cells of all animal species. In vertebrates, miR183 family of miRNAs are expressed in the olfactory epithelium, eyes, neuromasts, and ears.

[0036] The human miR-183 gene consists of one exon on chromosome 7q32.2. Mature miR183 is processed from a hairpin precursor called precursor miR183 or pre-miR183. Human precursor miR183 is 110 nucleotides long, has a sequence shown in SEQ ID NO: 2, and is cited as NR_029615.1 in the NCBI database.

[0037] The hairpin precursor miR183 folded into a stem-loop structure is processed to obtain mature miR183. The human mature miR183 is called miR183-5p (or hsa-miR183-5p or hsa-miR-183-5p), is 22 nucleotides long, has the sequence shown in SEQ ID NO:3, and is referenced in miRBase (https: / / www.mirbase.org) as MIMAT0000261. The sequence of miR183-5p corresponds to nucleotides 27 to 48 of the human precursor miR183 of SEQ ID NO:2. The human mature miR183, called miR183-3p (or hsa-miR183-3p or hsa-miR-183-3p), is also 22 nucleotides long and has the sequence shown in SEQ ID NO:4, and is referenced in miRBase as MIMAT0004560. The sequence of miR183-3p corresponds to nucleotides 66 to 87 of human precursor miR183 of SEQ ID NO: 2. As used herein, the term "mature miR183" (or "mature miR-183") encompasses miR183-5p and miR183-3p.

[0038] In vivo, mature miR183 can bind to a target mRNA comprising a short sequence complementary to the seed region of mature miR183. For example, the seed region of hsa-miR183-5p is AUGGCAC, corresponding to nucleotides 2 to 8 of hsa-miR183-5p (SEQ ID NO: 3).

[0039] The human miR-182 gene consists of an exon on chromosome 7q32.2. Mature miR182 is processed from a hairpin precursor called precursor miR182 or pre-miR182. Human precursor miR182 is 110 nucleotides long, has a sequence shown in SEQ ID NO: 22, and is cited as NR_029614.1 in the NCBI database.

[0040] The hairpin precursor miR182 folded into a stem-loop structure is processed to obtain mature miR182. The human mature miR182 is called miR182-5p (or hsa-miR182-5p or hsa-miR-182-5p), is 24 nucleotides long, has the sequence shown in SEQ ID NO: 11, and is referenced in miRBase (https: / / www.mirbase.org) as MIMAT0000259. The sequence of miR182-5p corresponds to nucleotides 23 to 46 of the human precursor miR182 of SEQ ID NO: 22. The human mature miR182, called miR182-3p (or hsa-miR182-3p or hsa-miR-182-3p), is 21 nucleotides long, has the sequence shown in SEQ ID NO: 13, and is referenced in miRBase as MIMAT0000260. The sequence of miR182-3p corresponds to nucleotides 67 to 87 of human precursor miR182 of SEQ ID NO: 22. As used herein, the term "mature miR182" (or "mature miR-182") encompasses miR182-5p and miR182-3p.

[0041] 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).

[0042] The human miR-96 gene consists of one exon on chromosome 7q32.2. Mature miR96 is processed from a hairpin precursor called precursor miR96 or pre-miR96. Human precursor miR96 is 78 nucleotides long, has a sequence shown in SEQ ID NO: 25, and is cited as NR_029512.1 in the NCBI database.

[0043] The hairpin precursor miR96 folded into a stem-loop structure is processed to obtain mature miR96. The human mature miR96 is called miR96-5p (or hsa-miR96-5p or hsa-miR-96-5p), is 23 nucleotides long, has the sequence shown in SEQ ID NO:28, and is referenced as MIMAT0000095 in miRBase (https: / / www.mirbase.org). The sequence of miR96-5p corresponds to nucleotides 9 to 31 of the human precursor miR96 of SEQ ID NO:25. The human mature miR96, called miR96-3p (or hsa-miR96-3p or hsa-miR-96-3p), is 22 nucleotides long, has the sequence shown in SEQ ID NO:30, and is referenced as MIMAT0004510 in miRBase. The sequence of miR96-3p corresponds to nucleotides 52 to 73 of the human precursor miR96 of SEQ ID NO:25. As used herein, the term "mature miR96" (or "mature miR-96") encompasses miR96-5p and miR96-3p.

[0044] In vivo, mature miR96 can bind to a target mRNA containing a short sequence complementary to the seed region of the mature miR96. For example, the seed region of hsa-miR96-5p is UUGGCAC, corresponding to nucleotides 2 to 8 of hsa-miR96-5p (SEQ ID NO: 28).

[0045] As described in the experimental section below, the inventors have surprisingly demonstrated that when inserted into a vector, a 110-nucleotide-long miR183 target site having a sequence shown in SEQ ID NO:1 complementary to the human sequence of precursor miR183 (i.e., SEQ ID NO:2) can be successfully used as a regulatory element to control the expression of the target nucleic acid in cells expressing miR183. As described above, the miR183 target site having a sequence complementary to the human sequence of precursor miR183 (i.e., SEQ ID NO:2) is sometimes referred to herein as a "precursor miR183 target site". After being introduced into the cell, the mirR183 target site contained in the expression vector is transcribed together with the target nucleic acid and is therefore present in the resulting mRNA. The mir183 target site having a 110-nucleotide-long sequence shown in SEQ ID NO:1 is expected to fold into a stem-loop structure. The inventors surprisingly showed that the stem-loop structure does not prevent miR183 from binding to the mir183 target site of SEQ ID NO:1.

[0046] Similarly, the inventors have demonstrated that when inserted into a vector, a 110 nucleotide long miR182 target site having a sequence shown in SEQ ID NO:21 complementary to the human sequence of precursor miR182 (i.e., SEQ ID NO:22) can be successfully used as a regulatory element to control the expression of a target nucleic acid in a cell expressing miR182. As described above, a miR182 target site having a sequence complementary to the human sequence of precursor miR182 (i.e., SEQ ID NO:22) is sometimes referred to herein as a "precursor miR182 target site". After introduction into a cell, the mirR182 target site contained in the expression vector is transcribed together with the target nucleic acid and is therefore present in the resulting mRNA. The inventors have also demonstrated that when inserted into a vector, a 78 nucleotide long miR96 target site having a sequence shown in SEQ ID NO:24 complementary to the human sequence of precursor miR96 (i.e., SEQ ID NO:25) can be successfully used as a regulatory element to control the expression of a target nucleic acid in a cell expressing miR96. As described above, the miR96 target site having a sequence complementary to the human sequence of precursor miR96 (i.e., SEQ ID NO: 25) is sometimes referred to herein as a "precursor miR96 target site". After introduction into the cell, the mirR96 target site contained in the expression vector is transcribed together with the target nucleic acid and is therefore present in the resulting mRNA. The mir182 target site (110 nucleotides long, with the sequence shown in SEQ ID NO: 21) and the mir96 target site (78 nucleotides long, with the sequence shown in SEQ ID NO: 24) are also expected to fold into a stem-loop structure. The inventors surprisingly showed that, contrary to expectations, the stem-loop structure neither prevented miR182 from binding to the mir182 target site of SEQ ID NO: 21, nor prevented miR96 from binding to the mir96 target site of SEQ ID NO: 24.

[0047] The inventors also demonstrated that inserting multiple copies of the miR183 target site of SEQ ID NO: 1 resulted in stronger inhibition of nucleic acid expression. Unexpectedly, the inhibition of nucleic acid expression induced by using the miR183 target site of SEQ ID NO: 1 was significantly stronger than the inhibition induced by using a shorter target site, such as the miR183 target site having a sequence shown in SEQ ID NO: 5 complementary to the hsa-miR183-5p sequence (i.e., SEQ ID NO: 3).

[0048] As shown in the experimental section below, the method for evaluating whether a nucleic acid sequence may be a suitable miR target site (e.g., a suitable miR183 target site) includes inserting the nucleic acid sequence of the evaluation into a vector comprising a reporter gene (e.g., a gene encoding GFP protein (green fluorescent protein)), which is controlled by a promoter (e.g., an ubiquitous promoter or a constitutive promoter). The nucleic acid sequence of the evaluation can be operably connected to a gene or inserted into a gene, such as in the 3'-UTR of a gene. The vector (e.g., plasmid) comprising the nucleic acid sequence of the evaluation and the reporter gene is then introduced into a host cell expressing miR183, such as a HEK293 cell. The inhibition of reporter gene expression in the host cell (particularly compared to the control condition in which the vector only comprises a reporter gene controlled by a promoter) shows that the nucleic acid sequence of the evaluation is a suitable miR183 target site. A similar method can be used to evaluate whether a nucleic acid sequence may be a suitable miR182 target site or a suitable miR96 target site.

[0049] Therefore, a first aspect of the present invention is an isolated nucleic acid sequence comprising or consisting of at least two copies of a miR target site of the miR183 family, wherein the miR target site has a sequence as 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% or 99% or higher identity to any one of SEQ ID NO:1, SEQ ID NO:21 or SEQ ID NO:24.

[0050] The isolated nucleic acid sequence described herein may comprise or consist of at least two copies of a miR183 target site having a sequence as set forth in SEQ ID NO: 1, or having a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to SEQ ID NO: 1. In some embodiments, the mirR183 target site described herein having a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to SEQ ID NO: 1 is a functional mirR183 target site, that is, it allows miR183 to bind.

[0051] In some embodiments, the mirR183 target site having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to SEQ ID NO: 1 has a length of at least 100 nucleotides. In some embodiments, the mirR183 target site having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical 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 mirR183 target site having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical 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.

[0052] In some embodiments, the mirR183 target site has a sequence as shown in SEQ ID NO:1, and 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 or 60 or more nucleotides are substituted by different nucleotides compared to the corresponding nucleotides of SEQ ID NO:1. In some embodiments, the mirR183 target site has a sequence as shown in SEQ ID NO: 1, and at most 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 substituted by a different nucleotide compared to the corresponding nucleotide of SEQ ID NO: 1. In some embodiments, this mirR183 target site having the sequence shown in SEQ ID NO: 1 and having nucleotide substitutions is a functional mirR183 target site, that is, it allows miR183 binding.

[0053] The isolated nucleic acid sequence described herein may comprise or consist of at least two copies of a miR182 target site having a sequence as set forth in SEQ ID NO: 21, or having a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to SEQ ID NO: 21. In some embodiments, the mirR182 target site described herein having a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to SEQ ID NO: 21 is a functional mirR182 target site, that is, it allows miR182 to bind.

[0054] In some embodiments, the mirR182 target site having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to SEQ ID NO: 21 has a length of at least 100 nucleotides. In some embodiments, the mirR182 target site having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical 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 at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical 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.

[0055] In some embodiments, the mirR182 target site has a sequence as shown in SEQ ID NO:21, and 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 or 60 or more nucleotides are substituted by different nucleotides compared to the corresponding nucleotides of SEQ ID NO:21. In some embodiments, the mirR182 target site has a sequence as shown in SEQ ID NO:21, and at most 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 nucleotides are substituted by different nucleotides compared to the corresponding nucleotides of SEQ ID NO:21. In some embodiments, this mirR182 target site having the sequence shown in SEQ ID NO:21 and having nucleotide substitutions is a functional mirR182 target site, that is, it allows miR182 binding.

[0056] The isolated nucleic acid sequence described herein may comprise or consist of at least two copies of a mi96 target site having a sequence as set forth in SEQ ID NO: 24, or having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 24. In some embodiments, the mirR96 target site described herein having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 24 is a functional mirR183 target site, that is, it allows miR96 to bind.

[0057] In some embodiments, the mirR96 target site having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 24 has a length of at least 70 nucleotides. In some embodiments, the mir96 target site having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 24 has a length of at least 40, 45, 50, 55, 60, or 65 nucleotides. In some embodiments, the mir96 target site having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical 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.

[0058] In some embodiments, the mirR96 target site has a sequence as shown in SEQ ID NO:24, and 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 compared to the corresponding nucleotides of SEQ ID NO:24. In some embodiments, the mirR96 target site has a sequence as set forth in SEQ ID NO: 24, and wherein at most 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 nucleotides are substituted with different nucleotides compared to the corresponding nucleotides of SEQ ID NO: 24. In some embodiments, the mirR96 target site having a sequence as set forth in SEQ ID NO: 24 and having nucleotide substitutions is a functional mirR96 target site, that is, it allows miR96 to bind.

[0059] As used herein, the expression "at least two copies" includes two, three, four, five, six, seven, eight, nine, ten or more copies of a miR target site of the miR183 family described herein (e.g., a miR183 target site having a sequence shown in SEQ ID NO: 1 or having a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to SEQ ID NO: 1, as described herein).

[0060] In some embodiments, the isolated nucleic acid sequence comprises or consists of two to six copies of a miR target site of the miR183 family described herein (e.g., a miR183 target site having a sequence as set forth in SEQ ID NO: 1 or having a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher identity to SEQ ID NO: 1, as described herein). The isolated nucleic acid sequence may therefore comprise or consist of two, three, four, five or six copies of a miR target site of the miR183 family described herein (e.g., a miR183 target site having a sequence as set forth in SEQ ID NO: 1 or having a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher identity to SEQ ID NO: 1, as described herein).

[0061] In some embodiments, the isolated nucleic acid sequence comprises three copies of the miR target site of the miR183 family described herein or consists of it. Therefore, the isolated nucleic acid sequence can include the sequence shown in SEQ ID NO:7, SEQ ID NO:23 or SEQ ID NO:26 or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher identity to any one of SEQ ID NO:7, SEQ ID NO:23 or SEQ ID NO:26, or consists of it.

[0062] Specifically, the isolated nucleic acid sequence may comprise or consist of three copies of a miR183 target site having a sequence as set forth in SEQ ID NO: 1 or having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher identity to SEQ ID NO: 1, as described herein. Thus, the isolated nucleic acid sequence may comprise or consist of a sequence as set forth in SEQ ID NO: 7 or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher identity to SEQ ID NO: 7. In some embodiments, the isolated nucleic acid sequence described herein having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to SEQ ID NO:7 corresponds to a functional mirR183 target site, that is, it allows miR183 to bind.

[0063] In some embodiments, the isolated nucleic acid sequence having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 7 has a length of at least 300 nucleotides. In some embodiments, the isolated nucleic acid sequence having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical 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 at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO:7 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 as set forth in SEQ ID NO: 7, and 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 or 30 or more nucleotides are substituted with different nucleotides compared to the corresponding nucleotides of SEQ ID NO: 7. In some embodiments, the isolated nucleic acid sequence has a sequence as set forth in SEQ ID NO: 7, and wherein at most 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 nucleotides are substituted with different nucleotides compared to the corresponding nucleotides of SEQ ID NO: 7. In some embodiments, this isolated nucleic acid sequence having the sequence shown in SEQ ID NO:7 and having nucleotide substitutions corresponds to a functional mirR183 target site, that is, it allows miR183 binding.

[0065] The isolated nucleic acid sequence may comprise or consist of three copies of a miR182 target site having a sequence as set forth in SEQ ID NO: 21, or having a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to SEQ ID NO: 21, as described herein. Thus, the isolated nucleic acid sequence may comprise or consist of a sequence as set forth in SEQ ID NO: 23, or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to SEQ ID NO: 23. In some embodiments, the isolated nucleic acid sequence described herein having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to SEQ ID NO:23 corresponds to a functional mirR182 target site, that is, it allows miR182 to bind.

[0066] In some embodiments, the isolated nucleic acid sequence having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 23 has a length of at least 300 nucleotides. In some embodiments, the isolated nucleic acid sequence having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical 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 at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to SEQ ID NO:23 has a length of 300, 305, 310, 315, 320, 325 or 330 nucleotides.

[0067] In some embodiments, the isolated nucleic acid sequence has a sequence as set forth in SEQ ID NO: 23, and 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 or 30 or more nucleotides are substituted with different nucleotides compared to the corresponding nucleotides of SEQ ID NO: 23. In some embodiments, the isolated nucleic acid sequence has a sequence as set forth in SEQ ID NO: 23, and wherein at most 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 nucleotides are substituted with different nucleotides compared to the corresponding nucleotides of SEQ ID NO: 23. In some embodiments, this isolated nucleic acid sequence having the sequence shown in SEQ ID NO: 23 and having nucleotide substitutions corresponds to a functional mirR182 target site, that is, it allows miR182 binding.

[0068] The isolated nucleic acid sequence may comprise or consist of three copies of a miR96 target site having a sequence as set forth in SEQ ID NO:24 or having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO:24, as described herein. Thus, the isolated nucleic acid sequence may comprise or consist of a sequence as set forth in SEQ ID NO:26 or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO:26. In some embodiments, the isolated nucleic acid sequence described herein having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to SEQ ID NO:26 corresponds to a functional mirR96 target site, that is, it allows miR96 binding.

[0069] In some embodiments, the isolated nucleic acid sequence having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 26 has a length of at least 210 nucleotides. In some embodiments, the isolated nucleic acid sequence having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical 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 that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO:26 has a length of 200, 205, 210, 215, 220, 225, 230, or 234 nucleotides.

[0070] In some embodiments, the isolated nucleic acid sequence has a sequence as set forth in SEQ ID NO: 26, and wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more nucleotides are substituted with different nucleotides compared to the corresponding nucleotides of SEQ ID NO: 26. In some embodiments, the isolated nucleic acid sequence has a sequence as set forth in SEQ ID NO: 26, and wherein at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides are substituted with different nucleotides compared to the corresponding nucleotides of SEQ ID NO: 26. In some embodiments, this isolated nucleic acid sequence having a sequence as set forth in SEQ ID NO: 26 and having nucleotide substitutions corresponds to a functional mirR96 target site, that is, it allows miR96 to bind.

[0071] In some embodiments, in the isolated nucleic acid sequence described herein, a copy of the miR target site of the miR183 family described herein (e.g., a copy of the miR183 target site) is separated by a spacer. As used herein, "spacer" refers to a non-coding sequence. In some embodiments, the spacer is characterized in that its length ranges from about 5 nucleotides to about 25 nucleotides, preferably from about 10 nucleotides to about 20 nucleotides, more preferably from about 20 nucleotides. Spacers are commonly 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. 1997 May 1; 25 (9): 1753-60). Examples of spacers include spacers having a sequence as 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 a sequence as shown in SEQ ID NO: 14.

[0072] 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.

[0073] In some embodiments, the isolated nucleic acid sequence described herein further comprises at least one copy of another miR target site. In some embodiments, the isolated nucleic acid sequence comprises or consists of:

[0074] - at least two copies of a miR183 target site, the miR183 target site having a sequence as shown in SEQ ID NO: 1, or having a sequence that is at least 70%, 75%, 80%,

[0075] %, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to SEQ ID NO:21, as described herein; or at least two copies of the miR182 target site having the sequence set forth in SEQ ID NO:21, or having a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to SEQ ID NO:21, as described herein; or at least two copies of the miR96 target site having the sequence set forth in SEQ ID NO:24, or having a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to SEQ ID NO:24.

[0076] 98% or 99% or more identical sequences, as described herein; and

[0077] - at least one copy of another miR target site.

[0078] For example, another miR target site can be a miR target site identified by a miRNA expressed in sensory neurons and / or hair cells (e.g., hair cells of the inner ear). Another miR target site can be a miR182 target site, a miR96 target site, a miR194 target site, a miR140 target site, a miR18a target site, a miR99a target site, a miR30b target site, a miR15a target site, or a miR210 target site. In some embodiments, another miR target site is a miR182 target site.

[0079] The miR182 target site can be a miR182 target site having a sequence complementary to mature miR182. For example, the miR182 target site can have a sequence as shown in SEQ ID NO: 10, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher 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, has a sequence as shown in SEQ ID NO: 11, and is referenced in miRBase as MIMAT0000259, as described above. The mature miR182 target site can have the sequence set forth in SEQ ID NO: 12, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical 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, has the sequence set forth in SEQ ID NO: 13, and is referenced in miRBase as MIMAT0000260, as described above.

[0080] In some embodiments, the isolated nucleic acid sequence described herein also comprises at least one copy of another miR target site of the miR183 family described herein. For example, the isolated nucleic acid sequence may comprise or consist of the following:

[0081] - at least two copies of a miR183 target site, the miR183 target site having a sequence as shown in SEQ ID NO: 1, or having a sequence that is at least 70%, 75%, 80%,

[0082] 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical sequences as described herein; and

[0083] - at least one copy of a miR182 target site having a sequence as shown in SEQ ID NO: 21, or having a sequence that is at least 70%, 75%, or 100% identical to SEQ ID NO: 21.

[0084] 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical sequence, and / or at least one copy of the miR96 target site, the miR96 target site having the sequence set forth in SEQ ID NO:24, or having a sequence that is at least 70%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to SEQ ID NO:24.

[0085] Sequences that are 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical.

[0086] The isolated nucleic acid sequence may comprise or consist of:

[0087] - at least two copies of a miR182 target site having a sequence as shown in SEQ ID NO: 21, or having a sequence that is at least 70%, 75%, or 100% identical to SEQ ID NO: 21.

[0088] 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical sequences as described herein; and

[0089] - at least one copy of a miR183 target site having a sequence as shown in SEQ ID NO: 1, or having a sequence that is at least 70%, 75%, 80%,

[0090] 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical sequence, and / or at least one copy of the miR96 target site, the miR96 target site having the sequence set forth in SEQ ID NO:24, or having a sequence that is at least 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO:24.

[0091] Sequences that are 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical.

[0092] The isolated nucleic acid sequence may comprise or consist of:

[0093] - at least two copies of a miR96 target site having SEQ ID NO:

[0094] 24, or having at least 70%, 75%, 80%,

[0095] 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical sequences as described herein; and

[0096] - at least one copy of a miR183 target site having a sequence as shown in SEQ ID NO: 1, or having a sequence that is at least 70%, 75%, 80%,

[0097] 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher sequence identity, and / or at least one copy of the miR182 target site, the miR182 target site having the sequence set forth in SEQ ID NO:21, or having a sequence that is at least 70%, 75%, 76%, 77%, 78%, 79% or higher identical to SEQ ID NO:21.

[0098] Sequences that are 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical.

[0099] Another aspect of the invention is an isolated nucleic acid coding sequence (also referred to as an isolated coding sequence), which comprises at least one copy of a miR target site of the miR183 family, wherein the miR target site has a sequence as 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% or 99% or higher identity to any one of SEQ ID NO:1, SEQ ID NO:21 or SEQ ID NO:24.

[0100] The isolated coding sequence may comprise at least one copy of a miR183 target site having a sequence as set forth in SEQ ID NO: 1, or having a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to SEQ ID NO: 1, as described herein. The isolated coding sequence may comprise at least one copy of a miR182 target site having a sequence as set forth in SEQ ID NO: 21, or having a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to SEQ ID NO: 21, as described herein. The isolated coding sequence can comprise at least one copy of a miR96 target site having a sequence as set forth in SEQ ID NO:24, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to SEQ ID NO:24, as described herein.

[0101] In some embodiments, the nucleic acid coding sequence of separation encodes polynucleotides or proteins. In some embodiments, the coding sequence of separation is a cDNA sequence. In some embodiments, the coding sequence of separation is an RNA coding sequence. For example, in some embodiments, the coding sequence of separation is an mRNA sequence. In some embodiments, at least one copy of the miR target site of the miR183 family described herein is inserted in the untranslated region of the coding sequence of separation described herein, for example, in the 5'-UTR or 3'-UTR of the coding sequence of separation.

[0102] In some embodiments, the isolated nucleic acid coding sequence comprises at least two copies of a miR target site of the miR183 family described herein (e.g., a miR183 target site having a sequence set forth in SEQ ID NO: 1 or having a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to SEQ ID NO: 1, as described herein).

[0103] In some embodiments, the isolated nucleic acid coding sequence comprises two to six copies of a miR target site of the miR183 family described herein (e.g., a miR183 target site having a sequence set forth in SEQ ID NO: 1 or having a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 1, as described herein).

[0104] In some embodiments, the isolated nucleic acid coding sequence comprises three copies of the miR target site of the miR183 family as described herein. Thus, the isolated nucleic acid sequence may comprise or consist of the following: 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% or 99% or higher identity with any one of SEQ ID NO:7, SEQ ID NO:23 or SEQ ID NO:26.

[0105] In particular, the isolated nucleic acid coding sequence may comprise three copies of a miR183 target site having a sequence as set forth in SEQ ID NO: 1, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to SEQ ID NO: 1, as described herein. Thus, the isolated nucleic acid coding sequence may comprise a sequence as set forth in SEQ ID NO: 7, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to SEQ ID NO: 7, as described herein.

[0106] The isolated nucleic acid coding sequence may comprise three copies of a miR182 target site having a sequence as set forth in SEQ ID NO: 21, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to SEQ ID NO: 21, as described herein. Thus, the isolated nucleic acid coding sequence may comprise a sequence as set forth in SEQ ID NO: 23, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to SEQ ID NO: 23, as described herein.

[0107] The isolated nucleic acid coding sequence may comprise three copies of a miR96 target site having a sequence as set forth in SEQ ID NO: 24, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO: 24, as described herein. Thus, the isolated nucleic acid coding sequence may comprise a sequence as set forth in SEQ ID NO: 26, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO: 26, as described herein.

[0108] In some embodiments, the copies of the miR target sites of the miR183 family described herein (eg, miR183 target sites) are separated by spacers described herein. In some embodiments, the spacer has a sequence as shown in SEQ ID NO:14.

[0109] 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 comprises:

[0110] - at least one copy of a miR183 target site having a sequence as shown in SEQ ID NO: 1, or having a sequence that is at least 70%, 75%, 80%,

[0111] %, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to SEQ ID NO:21, as described herein; or at least one copy of the miR182 target site having the sequence set forth in SEQ ID NO:21, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to SEQ ID NO:21, as described herein; or at least one copy of the miR96 target site having the sequence set forth in SEQ ID NO:24, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to SEQ ID NO:24.

[0112] 98% or 99% or more identical sequences, as described herein; and

[0113] - at least one copy of another miR target site.

[0114] For example, another miR target site can be a miR target site identified by a miRNA expressed in sensory neurons and / or hair cells (e.g., hair cells of the inner ear). Another miR target site can be a miR182 target site, a miR96 target site, a miR194 target site, a miR140 target site, a miR18a target site, a miR99a target site, a miR30b target site, a miR15a target site, or a miR210 target site. In some embodiments, another miR target site is a miR182 target site.

[0115] The miR182 target site can have a sequence complementary to mature miR182, such as a sequence shown in SEQ ID NO:10, SEQ ID NO:12, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to SEQ ID NO:10 or SEQ ID NO:12.

[0116] 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 may comprise:

[0117] - at least one copy of a miR183 target site having a sequence as shown in SEQ ID NO: 1, or having a sequence that is at least 70%, 75%, 80%,

[0118] 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical sequences as described herein; and

[0119] - at least one copy of a miR182 target site having a sequence as shown in SEQ ID NO: 21, or having a sequence that is at least 70%, 75%, or 100% identical to SEQ ID NO: 21.

[0120] 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical sequence as described herein; and / or at least one copy of a miR96 target site having a sequence as set forth in SEQ ID NO:24, or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical sequence to SEQ ID NO:24.

[0121] or a sequence of higher identity, as described herein.

[0122] Alternatively, the isolated nucleic acid coding sequence may comprise at least one copy of a miR182 target site having a sequence as set forth in SEQ ID NO:21, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity thereto, as described herein; and at least one copy of a miR96 target site having a sequence as set forth in SEQ ID NO:24, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity thereto, as described herein.

[0123] Another aspect of the present invention is an expression cassette comprising a promoter, a target nucleic acid sequence and a regulatory element (or regulatory sequence), wherein the regulatory element (or regulatory sequence) comprises or consists of: at least one copy of a miR target site of the miR183 family, wherein the miR target site has a sequence as 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% or 99% or higher identity to any one of SEQ ID NO:1, SEQ ID NO:21 or SEQ ID NO:24.

[0124] The regulatory element (or regulatory sequence) may comprise or consist of at least one copy of a miR183 target site having a sequence as set forth in SEQ ID NO: 1, or having a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to SEQ ID NO: 1, as described herein. The regulatory element (or regulatory sequence) may comprise or consist of at least one copy of a miR182 target site having a sequence as set forth in SEQ ID NO: 21, or having a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to SEQ ID NO: 21, as described herein. The regulatory element (or regulatory sequence) may comprise or consist of: at least one copy of a miR96 target site having a sequence as shown in SEQ ID NO:24, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to SEQ ID NO:24, as described herein.

[0125] 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 a sequence shown in SEQ ID NO: 1 or having a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to SEQ ID NO: 1, as described herein).

[0126] In some embodiments, the regulatory element (or regulatory sequence) comprises or consists of: 1 to 6 copies of a miR target site of the miR183 family described herein (e.g., a miR183 target site having a sequence as set forth in SEQ ID NO: 1 or having a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher identity to SEQ ID NO: 1, as described herein). Thus, the regulatory element (or regulatory sequence) may comprise or consist of: 1, 2, 3, 4, 5 or 6 copies of a miR target site of the miR183 family described herein (e.g., a miR183 target site having a sequence as set forth in SEQ ID NO: 1 or having a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher identity to SEQ ID NO: 1, as described herein).

[0127] In some embodiments, the regulatory element (or regulatory sequence) comprises or consists of: at least 2 copies of a miR target site of the miR183 family described herein (e.g., a miR183 target site having a sequence as shown in SEQ ID NO: 1 or having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher identity to SEQ ID NO: 1, as described herein). In some embodiments, the regulatory element (or regulatory sequence) comprises or consists of: 2 to 6 copies of a miR target site of the miR183 family described herein (e.g., a miR183 target site having a sequence as shown in SEQ ID NO: 1 or having a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher identity to SEQ ID NO: 1, as described herein).

[0128] In some embodiments, the regulatory element (or regulatory sequence) comprises or consists of: 3 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% or 99% or higher identity with any one of SEQ ID NO:7, SEQ ID NO:23 or SEQ ID NO:26.

[0129] In particular, the regulatory element (or regulatory sequence) may comprise or consist of: 3 copies of a miR183 target site having a sequence as set forth in SEQ ID NO: 1, or having a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher identity to SEQ ID NO: 1, as described herein. Thus, the regulatory element (or regulatory sequence) may comprise or consist of: a sequence as set forth in SEQ ID NO: 7, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher identity to SEQ ID NO: 7, as described herein.

[0130] In some embodiments, the regulatory element (or regulatory sequence) having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 7 has a length of at least 300 nucleotides. In some embodiments, the regulatory element (or regulatory sequence) having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 7 has a length of 300, 305, 310, 315, 320, 325, or 330 nucleotides.

[0131] In some embodiments, the regulatory element (or regulatory sequence) has a sequence as set forth in SEQ ID NO:7, and 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 or 30 or more nucleotides are substituted by different nucleotides compared to the corresponding nucleotides of SEQ ID NO:7, as described herein. In some embodiments, the regulatory element (or regulatory sequence) has a sequence as set forth in SEQ ID NO:7, and 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 nucleotides are substituted by different nucleotides compared to the corresponding nucleotides of SEQ ID NO:7, as described herein.

[0132] The regulatory element (or regulatory sequence) may comprise or consist of three copies of a miR182 target site having a sequence as set forth in SEQ ID NO: 21, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to SEQ ID NO: 21, as described herein. Thus, the regulatory element (or regulatory sequence) may comprise or consist of a sequence as set forth in SEQ ID NO: 23, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to SEQ ID NO: 23, as described herein.

[0133] In some embodiments, the regulatory element (or regulatory sequence) having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 23 has a length of at least 300 nucleotides. In some embodiments, the regulatory element (or regulatory sequence) having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 23 has a length of 300, 305, 310, 315, 320, 325, or 330 nucleotides.

[0134] In some embodiments, the regulatory element (or regulatory sequence) has a sequence as shown in SEQ ID NO:23, and 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 or 30 or more nucleotides are replaced by different nucleotides compared to the corresponding nucleotides of SEQ ID NO:23. In some embodiments, the regulatory element (or regulatory sequence) has a sequence as shown in SEQ ID NO:23, and at most 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 nucleotides are replaced by different nucleotides compared to the corresponding nucleotides of SEQ ID NO:23.

[0135] The regulatory element (or regulatory sequence) may comprise or consist of: 3 copies of a miR96 target site having a sequence as set forth in SEQ ID NO: 24, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO: 24, as described herein. Thus, the regulatory element (or regulatory sequence) may comprise or consist of: a sequence as set forth in SEQ ID NO: 26, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO: 26, as described herein.

[0136] In some embodiments, the regulatory element (or regulatory sequence) having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical 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 that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 26 has a length of 200, 205, 210, 215, 220, 225, 230, or 234 nucleotides.

[0137] In some embodiments, the regulatory element (or regulatory sequence) has a sequence as set forth in SEQ ID NO: 26, and wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more nucleotides are substituted with different nucleotides compared to the corresponding nucleotides of SEQ ID NO: 26, as described herein. In some embodiments, the regulatory element (or regulatory sequence) has a sequence as set forth in SEQ ID NO: 26, and wherein at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides are substituted with different nucleotides compared to the corresponding nucleotides of SEQ ID NO: 26, as described herein.

[0138] In some embodiments, in the regulatory element (or regulatory sequence) described herein, multiple copies of the miR target site (e.g., miR183 target site) of the miR183 family described herein are included, and the copies are separated by the spacer described herein. In some embodiments, the spacer has the sequence shown in SEQ ID NO:14.

[0139] In some embodiments, the regulatory elements (or regulatory sequences) described herein further comprise at least one copy of another miR target site. Thus, in some embodiments, the regulatory elements (or regulatory sequences) comprise or consist of the following:

[0140] - at least one copy of a miR183 target site having a sequence as shown in SEQ ID NO: 1, or having a sequence that is at least 70%, 75%, 80%,

[0141] %, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to SEQ ID NO:21, as described herein; or at least one copy of the miR182 target site having the sequence set forth in SEQ ID NO:21, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to SEQ ID NO:21, as described herein; or at least one copy of the miR96 target site having the sequence set forth in SEQ ID NO:24, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to SEQ ID NO:24.

[0142] 98% or 99% or more identical sequences, as described herein; and

[0143] - at least one copy of another miR target site.

[0144] For example, another miR target site can be a miR target site identified by a miRNA expressed in sensory neurons and / or hair cells (e.g., hair cells of the inner ear). Another miR target site can be a miR182 target site, a miR96 target site, a miR194 target site, a miR140 target site, a miR18a target site, a miR99a target site, a miR30b target site, a miR15a target site, or a miR210 target site. In some embodiments, another miR target site is a miR182 target site.

[0145] The miR182 target site can have a sequence complementary to mature miR182, such as a sequence shown in SEQ ID NO:10, SEQ ID NO:12, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher identity to SEQ ID NO:10 or SEQ ID NO:12.

[0146] 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 may comprise or consist of the following:

[0147] - at least one copy of a miR183 target site having a sequence as shown in SEQ ID NO: 1, or having a sequence that is at least 70%, 75%, 80%,

[0148] 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical sequences as described herein; and

[0149] - at least one copy of a miR182 target site having a sequence as shown in SEQ ID NO: 21, or having a sequence that is at least 70%, 75%, or 100% identical to SEQ ID NO: 21.

[0150] 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical sequence, as described herein, and / or at least one copy of a miR96 target site having a sequence as set forth in SEQ ID NO:24, or having a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24.

[0151] or a sequence of higher identity, as described herein.

[0152] 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 as set forth in SEQ ID NO:21, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to SEQ ID NO:21, as described herein, and at least one copy of a miR96 target site having a sequence as set forth in SEQ ID NO:24, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to SEQ ID NO:24, as described herein.

[0153] As used herein, the expression "target nucleic acid sequence" means a nucleic acid sequence to be expressed. Specifically, the expression "target nucleic acid sequence" means a nucleic acid sequence to be expressed in a controlled manner, for example when introduced into a host cell or host organism. The target nucleic acid sequence can be a target gene, i.e. a nucleic acid sequence encoding a functional nucleic acid (e.g., RNA) or a polypeptide or protein.

[0154] For example, a target nucleic acid (such as a target gene) may include an open reading frame encoding a polypeptide or protein. Thus, a target nucleic acid sequence may include a target coding sequence. Alternatively, a target nucleic acid sequence may encode a functional nucleic acid, such as a non-coding RNA.

[0155] In some embodiments, the target nucleic acid is a target gene. The term "gene" as used herein may specifically refer to a coding nucleic acid sequence, which comprises a coding sequence (or CDS) and at least one transcribed but untranslated regulatory element, such as a 3'-UTR, a 5'-UTR and / or an intron.

[0156] In the expression cassette, the regulatory element can be operably connected to the target nucleic acid or inserted into the target nucleic acid. Preferably, the regulatory element is inserted into the untranslated region of the target nucleic acid. The untranslated region includes, for example, 5'-UTR, 3'-UTR and introns. In some embodiments, the regulatory element is inserted into the 3'-UTR of the target nucleic acid, particularly the 3'-UTR of the target gene.

[0157] As mentioned above, the expression cassette comprises a promoter. For example, the promoter can be a ubiquitin promoter and / or a constitutive promoter. Promoters are well known to those skilled in the art, and they will know how to select a suitable promoter according to the target nucleic acid sequence to be expressed and the cell in which it is expressed.

[0158] In the expression cassette, the promoter is usually inserted upstream of the target nucleic acid sequence to be expressed, that is, 5' of the target nucleic acid sequence to be expressed.

[0159] The expression cassette may also include any sequence or element that may be required for expressing the target nucleic acid sequence. Such sequences or elements may include, for example, poly (A) signals, poly (A) sites, enhancer sequences, terminator sequences, degrons, silencers, insulators and / or operators.

[0160] Another aspect of the invention is a vector comprising a regulatory element (or regulatory sequence) as described herein, or an isolated nucleic acid coding sequence as described herein, or an expression cassette as described herein.

[0161] In particular, the present invention relates to a vector comprising a regulatory element, the regulatory element comprising or consisting of at least one copy of a miR target site of the miR183 family, the miR target site having a sequence as shown in SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24, or having a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to any one of SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24. For example, the vector may comprise a regulatory element, the regulatory element comprising or consisting of at least one copy of a miR183 target site having a sequence as shown in SEQ ID NO: 1, or having a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to SEQ ID NO: 1, as described herein.

[0162] In some embodiments, the vector comprising the regulatory element as described herein also comprises a target nucleic acid sequence as described herein, such as a target gene. In such a vector, the regulatory element can be operably connected to the target nucleic acid or can be inserted into the target nucleic acid. Preferably, the regulatory element is inserted into the untranslated region of the target nucleic acid, such as 5'-UTR, 3'-UTR or intron. In some embodiments, the regulatory element is inserted into the 3'-UTR of the target nucleic acid, particularly the 3'-UTR of the target gene.

[0163] Vectors are well known to those skilled in the art, who know how to select a suitable vector according to the target nucleic acid sequence to be expressed (eg, according to the length of the target nucleic acid sequence) and the cells in which the expression is desired.

[0164] Examples of vectors include cosmids, plasmids, episomes, artificial chromosomes, bacteriophages and viruses (e.g., lentiviruses, retroviruses, adenoviruses and adeno-associated viruses (AAV)), liposomes, lipid nanoparticles, niosomes, polymer-based nanoparticles.

[0165] In some embodiments, the vector is an expression vector.

[0166] In some embodiments, the vector is one of a liposome, a lipid nanoparticle, a vesicle, or a polymer-based nanoparticle, and the vector comprises an isolated nucleic acid coding sequence, preferably an RNA coding sequence, as described herein.

[0167] 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, including, for example, transfection of packaging cells and / or transient transfection using helper plasmids or viruses.

[0168] Another aspect of the invention is a host cell comprising an isolated sequence as described herein, a regulatory element as described herein, an isolated nucleic acid coding sequence as described herein, an expression cassette as described herein, or a vector as described herein.

[0169] In some embodiments, the host cell is an isolated host cell.

[0170] In some embodiments, the host cell can be a prokaryotic cell or a eukaryotic cell, such as a yeast cell or an animal cell, particularly a mammalian cell.

[0171] It should be noted that, for animal and human cells, the term "host cell" 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 have been introduced are specifically excluded from the definition of "host cell".

[0172] Another aspect of the 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.

[0173] Another aspect of the 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.

[0174] Pharmaceutically acceptable excipients or carriers that can be used in the pharmaceutical compositions described herein include, for example, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (e.g., sodium carboxymethyl cellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin.

[0175] Another aspect of the 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.

[0176] As used herein, "consisting essentially of," with respect 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 biologically active agent in the composition, pharmaceutical composition, or medicament.

[0177] Another aspect of the invention is a kit comprising or consisting of an isolated sequence as described herein, a regulatory element as described herein, an isolated nucleic acid coding sequence as described herein, an expression cassette as described herein, a vector as described herein or a host cell as described herein, and optionally instructions for use.

[0178] "Kit" refers to any article (e.g., package or container) comprising the following: an isolated sequence described herein, a regulatory element described herein, an isolated nucleic acid coding sequence described herein, an expression cassette described herein, a vector described herein, or a host cell described herein. The kit can be promoted, distributed, or sold as a unit for performing the uses or methods described herein.

[0179] Another aspect of the invention is an isolated sequence as described herein, an isolated nucleic acid coding sequence as described herein, a regulatory element as described herein, an expression cassette as described herein, a vector as described herein, a host cell as described herein, a composition as described herein or a pharmaceutical composition as described herein for use as a medicament.

[0180] Another aspect of the invention is an isolated sequence as described herein, a regulatory element as described herein, an isolated nucleic acid coding sequence as described herein, an expression cassette as described herein, a vector as described herein, a host cell as described herein, a composition as described herein, a pharmaceutical composition or a medicament as described herein for use in gene therapy.

[0181] Another aspect of the invention is an isolated sequence as described herein, an isolated nucleic acid coding sequence as described herein, a regulatory element as described herein, an expression cassette as described herein, a vector as described herein, a host cell as described herein, a composition as described herein, a pharmaceutical composition or a medicament as described herein for use in treating a sensory disorder.

[0182] Another aspect of the invention is a pharmaceutical composition as described herein for use in the treatment of a sensory disorder or for use in the treatment of a sensory disorder.

[0183] Another aspect of the 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 in the manufacture of a medicament for treating a sensory disorder.

[0184] Another aspect of the invention is a method of performing gene therapy in a subject in need thereof, the method comprising administering to the subject an isolated sequence described herein, an isolated nucleic acid encoding sequence described herein, a regulatory element described herein, an expression cassette described herein, or a vector described herein.

[0185] Another aspect of the invention is a method of treating a sensory disorder in a subject in need thereof, the method comprising administering to the subject an isolated sequence described herein, an isolated nucleic acid encoding sequence described herein, a regulatory element described herein, an expression cassette described herein, or a vector described herein.

[0186] In some embodiments, the sensory disorder is a hereditary or congenital sensory disorder, ie, a sensory disorder of genetic origin.

[0187] Another aspect of the invention is the use of an isolated sequence as described herein, an isolated nucleic acid coding sequence as described herein or an expression cassette as described herein for producing a vector, in particular for producing an expression vector, in particular for use in vitro or ex vivo.

[0188] Another aspect of the invention is the use of a vector as described herein for producing a host cell as described herein, in particular for use in vitro or ex vivo.

[0189] Another aspect of the present invention is the use of the isolated sequence described herein, the isolated nucleic acid coding sequence described herein, the regulatory element described herein, the expression cassette described herein, or the vector described herein for specifically expressing a target gene in a cell that does not express a miRNA of the miR183 family (i.e., in a cell that does not express miR183, miR182, and / or miR96), particularly in vitro or ex vivo. For example, the isolated sequence described herein, the isolated nucleic acid coding sequence described herein, the regulatory element described herein, the expression cassette described herein, or the vector described herein comprising the miR183 target site described herein can be particularly used for specifically expressing a target gene in a cell that does not express miR183. Similarly, the isolated sequence described herein, the isolated nucleic acid coding sequence described herein, the regulatory element described herein, the expression cassette described herein, or the vector described herein comprising the miR182 target site described herein can be particularly used to specifically express the target gene in cells that do not express miR182; and the isolated sequence described herein, the isolated nucleic acid coding sequence described herein, the regulatory element described herein, the expression cassette described herein, or the vector described herein comprising the miR96 target site described herein can be particularly used to specifically express the target gene in cells that do not express miR96.

[0190] Another aspect of the invention is the use of an isolated sequence as described herein, an isolated nucleic acid coding sequence as described herein, a regulatory element as described herein, an expression cassette as described herein or a vector as described herein for regulating or controlling the expression of a target nucleic acid (e.g., a target gene), particularly in vitro or ex vivo.

[0191] Another aspect of the invention is the use of the isolated sequence described herein, the isolated nucleic acid coding sequence described herein, the regulatory element described herein, the expression cassette described herein or the vector described herein for reducing off-target expression of a target nucleic acid (e.g., a target gene), wherein the expression of the target nucleic acid is prevented (or repressed or inhibited) in cells and / or tissues expressing at least one miRNA of the miR183 family (i.e., in cells and / or tissues expressing miR183, miR182 and / or miR96).

[0192] Another aspect of the invention is a method of producing a vector as described herein, the method comprising inserting into the vector a regulatory element as described herein.

[0193] Another aspect of the present invention is a method for regulating or controlling the expression of a target nucleic acid (e.g., a target gene) in a tissue comprising at least two different cell types, particularly an in vitro or ex vivo method, the method comprising introducing into the tissue an isolated nucleic acid coding sequence as described herein, a regulatory element as described herein, an expression cassette as described herein, or a vector as described herein.

[0194] Another aspect of the invention is a method for regulating or controlling the expression of a target nucleic acid (e.g., a target gene) in a subject in need of gene therapy, the method comprising introducing an isolated nucleic acid coding sequence described herein, a regulatory element described herein, an expression cassette described herein, or a vector described herein into a tissue of the subject, wherein the tissue comprises at least two different cell types.

[0195] The subject in need of gene therapy may be a subject suffering from sensory disorders, particularly a subject suffering from congenital sensory disorders.

[0196] The expression "introduced into a tissue of a subject" used herein may correspond to systemic administration to a subject or local administration to a subject.

[0197] In particular, the tissue comprising at least two different cell types may include at least one cell type in which expression of the target nucleic acid is desired and at least one cell type in which expression of the target nucleic acid is not desired.

[0198] In some embodiments, the tissue comprising at least two different cell types comprises at least one cell type expressing at least one miRNA of the miR183 family (i.e., miR183, miR182, and / or miR96) and at least one cell type not expressing any miRNA of the miR183 family. Specifically, the tissue comprising at least two different cell types may comprise at least one cell type expressing miR183 and at least one cell type not expressing miR183. The tissue comprising at least two different cell types may comprise at least one cell type expressing miR182 and at least one cell type not expressing miR182. The tissue comprising at least two different cell types may comprise at least one cell type expressing miR96 and at least one cell type not expressing miR96.

[0199] As used herein, “does not express any miRNA of the miR183 family” means that any miRNA of miR183 is not expressed in an amount sufficient for the miRNA to exert biological activity, particularly in an amount sufficient to mediate gene silencing.

[0200] Another aspect of the present invention is a method for reducing off-target expression of a target nucleic acid (e.g., a target gene), wherein the expression of the target nucleic acid is prevented (or repressed or inhibited) in cells and / or tissues expressing at least one miRNA of the miR183 family (i.e., in cells and / or tissues expressing miR183, miR182 and / or miR96), the method comprising introducing the isolated nucleic acid coding sequence described herein, the regulatory element described herein, the expression cassette described herein, or the vector described herein into the tissue (e.g., in the tissue of a subject in need of gene therapy), wherein the tissue comprises at least two different cell types described herein.

[0201] Sequence Listing

[0202]

[0203]

[0204]

[0205]

[0206] BRIEF DESCRIPTION OF THE DRAWINGS

[0207] Figure 1A-1B is a schematic diagram of an AAV cassette comprising at least the following: a promoter, a transgene (i.e., GFP), and a polyadenylation or poly(A) signal (pA). When present, the dashed box represents a so-called "pre-miR183 target site" or "pre-miRT183" (a miR183 target site having a sequence as shown in SEQ ID NO: 1). Figure 1A represents an AAV-GFP construct that does not contain any miR183 target site. Figure 1B represents an AAV-GFP-premiRT183 construct comprising three copies of the premiR183 target site inserted 3' (ie, downstream) of the GFP coding sequence.

[0208] Figure 2A-2E is a schematic diagram of a plasmid comprising at least the following: a promoter (i.e., smCBA promoter), a transgene (i.e., GFP), and a polyadenylation or poly(A) signal (pA). When present, the dashed box represents a precursor miR183 target site comprising a sequence complementary to has-miR183-5p ("5p binding site" or 5P) and a sequence complementary to hashsa-miR183-3p ("3p binding site" or 3P). When present, the black box represents a 5p binding site (5P) and the white box represents a 3p binding site (3P). The smaller shaded box indicates the presence of a spacer (between the 5p and / or 3p binding sites). Figure 2A represents a plasmid construct that does not contain any miR183 target site (0xpre-miRT183 or no miRT183). Figure 2B Represents a plasmid construct containing 1 copy of the pre-miR183 target site downstream of the smCBA-GFP cassette (1x pre-miRT183). Figure 2C Represents a plasmid construct containing 3 copies of the pre-miR183 target site downstream of the smCBA-GFP cassette (3xpre-miRT183). Figure 2D Represents a plasmid construct containing 3 copies of the miR183 target site consisting of a 5P binding site, located downstream of the smCBA-GFP cassette (3xmiRT183 5P). There is a spacer between the copies, which is indicated by a smaller shaded box. Figure 2E Represents a plasmid construct containing 3 copies of the miR183 target site consisting of tandem 5P and 3P binding sites, located downstream of the smCBA-GFP cassette (3x miRT183 5+3P). The 5P and 3P binding sites are separated by a spacer, which is represented by a smaller shaded box.

[0209] Figures 3A-3E The regulatory role of precursor miR183 target sites on transgene expression in the mature mouse inner ear is demonstrated. Figures 3A-3D is a confocal microscopy image showing the Figure 1A The AAV-GFP construct shown in ( Figure 3A-3B ) or use Figure 1B The organ of Corti of mice treated with the AAV-GFP-precursor miRT183 constructs shown in FIG. 3C 3D . Figure 3B and 3D Shown is staining of hair cells present in the organ of Corti with myosin VIIa (MyoVIIa). Figure 3A and 3C GFP expression in the organ of Corti is shown. Scale bar is indicated. Figure 3E Quantification of GFP-positive inner hair cells (IHC) and outer hair cells (OHC) in mice treated with AAV-GFP construct or AAV-GFP-pre-miRT183 construct is shown. Data are presented as mean ± SD. Statistical analysis was performed using ANOVA (**** = p < 0.0001).

[0210] Figures 4A-4D The regulatory role of precursor miR183 target sites on transgene expression in HEK293 cells is illustrated. Figures 4A-4C is a string containing 0 ( Figure 4A ), 1( Figure 4B ) or 3 ( Figure 4C Confocal images of HEK293 cells transfected with a GFP-expressing plasmid that expressed 5 copies of the precursor miR183 target site. White dots represent GFP-positive cells. Figure 4D Shown is the quantification of GFP fluorescence measured in cell lysates and normalized to the control (ie, 0 copies of the precursor miR183 target site).

[0211] Figure 5A-5B is a diagram comparing the regulatory effects of so-called precursor miR183 target sites and so-called mature miR183 target sites on transgene expression. HEK293 cells were transfected with different plasmid constructs containing different miR183 target sites, as shown in FIG2 . Figure 5A Shown are the plasmid contents in cell lysates measured by quantitative polymerase chain reaction (qPCR). Figure 5B GFP fluorescence measured in cell lysates is shown. Statistical analysis was performed using Fisher's LSD multiple comparisons: ns = not significant; * = p < 0.05.

[0212] Figure 6A-6B are graphs comparing the regulatory effects of 2 and 3 copies of the so-called pre-miR183 target site on transgene expression. HEK293 cells were transfected with plasmids containing the pre-miR183 target site (2x pre-miRT183 or 3x pre-miRT183) downstream of the GFP expression cassette as indicated. These graphs show that 24 hours after medium replacement ( Fig. 6A ) or 48 hours after medium replacement ( Figure 6B ) Quantification of GFP fluorescence measured in cell lysates and normalized to control (i.e., no miR target site). Error bars represent standard error of the mean (SEM). *: p<0.05, **: p<0.01; one-way ANOVA.

[0213] Figure 7A-7B Figures show the regulatory effect of the so-called precursor miR182 target site on transgene expression. HEK293 cells were transfected with a plasmid containing 3 copies of the precursor miR182 target site (3x precursor miRT182) downstream of a GFP expression cassette. The figures show that 24 hours after medium replacement ( Fig. 7A ) or 48 hours after medium replacement ( Figure 7B ) Quantification of GFP fluorescence measured in cell lysates and normalized to control (ie, no miR target site). Error bars represent standard error of the mean. ****: p<0.0001; unpaired two-tailed T-test.

[0214] Figures 8A-8BFigures show the regulatory effect of the so-called precursor miR96 target site on transgene expression. HEK293 cells were transfected with a plasmid containing 3 copies of the precursor miR96 target site (3x precursor miRT96) downstream of a GFP expression cassette. The figures show that 24 hours after medium replacement ( Fig. 8A ) or 48 hours after medium replacement ( Figure 8B ) Quantification of GFP fluorescence measured in cell lysates and normalized to control (ie, no miR target site). Error bars represent standard error of the mean. *: p<0.05, ***: p<0.001; unpaired two-tailed T test. Example

[0215] The present invention is further illustrated by the following examples.

[0216] Example 1

[0217] Materials and methods

[0218] Mice and cell lines

[0219] Wild-type C57Bl / 6 mice were housed under standard conditions with a 12 h / 12 ​​h light / dark cycle and an enriched environment. Studies were conducted in compliance with animal health regulations, in particular Council Directive 2010 / 63 / EU of 22 September 2010 and French Decree No. 2013-118 of 1 February 2013 on the protection of animals used for scientific purposes.

[0220] HEK293 cells (human embryonic kidney 293 cells) were cultured under standard conditions in DMEM containing 10% fetal bovine serum (FBS).

[0221] Carrier

[0222] AAV vectors were constructed for administration to mice. The AAV-GFP control construct contained a GFP expression cassette in which the GFP coding sequence was under the control of the ubiquitin promoter (see Figure 1A In the AAV-GFP-precursor miRT183 construct, three copies of the miR183 target site were inserted 3' (i.e., downstream) of the GFP coding sequence, wherein the miR183 target site has the sequence shown in SEQ ID NO: 1 (the so-called "precursor miR183 target site"), which is complementary to the human sequence of precursor miR183 (i.e., SEQ ID NO: 2) (see Figure 1B ).

[0223] The pAAV-smCBA-eGFP-bGH backbone (Genscript) was used to construct a plasmid vector for transfection of HEK293 cells. Figure 2AAs shown, the "no miRT183" control construct contains a GFP expression cassette in which the eGFP coding sequence is under the control of the ubiquitin promoter smCBA (truncated chimeric cytomegalovirus (CMV)-chicken β-actin). Plasmids containing miR183 target sites were obtained by cloning the micro RNA target site into the pAAV-smCBA-eGFP-bGH backbone (Genscript):

[0224] - a plasmid containing 1 copy of the so-called precursor miR183 target site having the sequence shown in SEQ ID NO: 1, located downstream of the smCBA-eGFP cassette (1x precursor miRT183- Figure 2B );

[0225] - a plasmid containing 3 copies of the so-called pre-miR183 target site having the sequence shown in SEQ ID NO: 1, located downstream of the smCBA-eGFP cassette (3x pre-miRT183- Figure 2C );

[0226] - A plasmid containing 3 copies of a miR183 target site consisting of the sequence shown in SEQ ID NO: 5 (the so-called "5p binding site"), complementary to hsa-miR183-5p (i.e., SEQ ID NO: 3), located downstream of the smCBA-GFP cassette (3x miRT183 5P- Figure 2D ); 3 copies of the so-called "5p binding site" are separated by a spacer having a sequence as shown in SEQ ID NO: 14; and

[0227] - A plasmid containing 3 copies of a miR183 target site consisting of a sequence represented by SEQ ID NO:5 (the so-called "5p binding site", which is complementary to hsa-miR183-5p (i.e., SEQ ID NO:3)) and a sequence represented by SEQ ID NO:6 (the so-called "3p binding site", which is complementary to hsa-miR183-3p (i.e., SEQ ID NO:4)) in tandem, located downstream of the smCBA-GFP cassette (3x miRT183 5+3P- Figure 2E ); the so-called "5p binding site" and "3p binding site" are separated by a spacer having a sequence shown in SEQ ID NO:14.

[0228] exist Figure 2DIn the plasmid "3x miRT183 5P", the regulatory element containing 3 copies of the miR183 target site and 2 spacers has the sequence shown in SEQ ID NO:8, and the miR183 target site consists of the sequence shown in SEQ ID NO:5 (the so-called "5p binding site"). Figure 2E In the plasmid "3x miRT183 5+3P", the regulatory element containing 3 copies of the miR183 target site and 5 spacers has the sequence shown in SEQ ID NO:9, and the miR183 target site is composed of the sequence shown in SEQ ID NO:5 (the so-called "5p binding site") and the sequence shown in SEQ ID NO:6 (the so-called "3p binding site") in series.

[0229] In vivo experiments

[0230] On postnatal day 14 (P14), wild-type C57Bl / 6 mice were administered an adeno-associated viral vector (AAV) containing a nucleotide sequence encoding GFP (green fluorescent protein). A total volume of 1 μl of the AAV vector was injected into the inner ear of the mice through the round window membrane. As shown in Figure 1, AAV-GFP constructs or AAV-GFP-precursor miRT183 constructs were injected at a concentration of 5x10 13 vg / mL (vector genomes per mL). Two weeks after injection, mice were euthanized and temporal bones were collected and fixed for histological analysis. After dissection of the cochlea, the organ of Corti was immunolabeled with myosin VIIa (MyoVIIa). In the cochlea, myosin VIIa is specifically expressed in hair cells. The organ of Corti was then analyzed by confocal microscopy to evaluate the expression of MyoVIIa and GFP.

[0231] In vitro experiments

[0232] In order to evaluate the effect of regulatory elements containing miR183 target sites on gene expression, 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, PEI-DNA-OptiMEM medium was replaced with DMEM 2% FBS. Three days after transfection, cells were collected, washed with PBS and lysed with PBS-Tween 20 (0.2%). After clarifying the lysate by centrifugation, GFP fluorescence was measured using a Synergy plate reader (Agilent), and the results were provided in arbitrary units. At the same time, protein content was determined using a BCA assay kit (ThermoFisher). Plasmid copy number in cell lysate was measured by TaqMan qPCR using GFP primers and probes as shown below:

[0233] -eGFP fwd: 5'-GAGCGCACCATCTTCTTCA (SEQ ID NO: 18);

[0234] -eGFP rev: 5'-TTCAGCTCGATGCGGTTC (SEQ ID NO: 19); and

[0235] -eGFP probe: 5'-AGGACGACGGCAACTACAAGACC (SEQ ID NO: 20).

[0236] result

[0237] To evaluate the regulatory effect of the so-called pre-miR183 target site (i.e., the miR183 target site having the sequence shown in SEQ ID NO: 1) on transgene expression, mice were injected with an AAV-GFP-pre-miRT183 construct containing a regulatory element consisting of three copies of the pre-miR183 target site (see Figure 1A As a control, mice were injected with an AAV-GFP construct that did not contain any regulatory elements (see Figure 1B Two weeks after injection, mice were euthanized and their cochleae were collected, stained for MyoVIIA and GFP, and analyzed by confocal microscopy. Analysis of the cochleae of mice injected with the AAV-GFP control construct showed colocalization of GFP and MyoVIIA in the inner hair cell layer ( Figure 3A-3B ), indicating that GFP is efficiently expressed in hair cells (inner hair cells IHC and outer hair cells OHC). In contrast, the presence of a regulatory element containing 3 copies of the pre-miR183 target site inhibited GFP expression in the inner hair cells of the cochlea of ​​mice injected with the AAV-GFP-pre-miR183 construct, as evidenced by the lack of colocalization between GFP and MyoVIIa and the residual GFP signal that persisted in the underlying supporting cells ( Figure 3C-3D ) The regulation of the precursor miR183 target site was also demonstrated after quantification of GFP-positive hair cells in the cochlea of ​​mice injected with AAV-GFP construct or AAV-GFP-pre-miRT183 construct. Figure 3E As shown, mice injected with the AAV-GFP construct (i.e., in the absence of the regulatory elements containing the miR183 target site) had significantly higher proportions of GFP-positive cells in both inner hair cells (IHCs) and outer hair cells (OHCs) ( Figure 3E ).

[0238] These data demonstrate that the so-called pre-miR183 target site having the sequence shown in SEQ ID NO: 1, present downstream of the GFP expression cassette, significantly inhibited GFP expression in hair cells of mice treated with AAV-GFP-pre-miRT183.

[0239] To further evaluate the regulatory effect of the precursor miR183 target site on transgene expression, a GFP expression cassette containing no miR183 target site (e.g. Figure 2A ), containing 1 copy of the precursor miR183 target site (as shown in Figure 2B ) or containing three copies of the precursor target site (as shown Figure 2C HEK293 cells were transfected with plasmid constructs (as shown). After cell lysis, GFP expression in transfected cells was assessed using a fluorescence plate reader. Figures 4A-4C As shown, an increase in the copy number of the precursor miR183 target site in the plasmid construct resulted in a decrease in the number of GFP-positive cells. Figure 4D Quantification of GFP fluorescence in transfected HEK293 cell lysates is shown and demonstrates that (i) the presence of one pre-miR183 target site is sufficient to strongly repress GFP expression and that (ii) increasing the number of pre-miR183 target sites enhances the repression of GFP expression.

[0240] These data demonstrate that effective inhibition of transgene expression can be obtained using vectors containing a regulatory element consisting of a so-called pre-miR183 target site having the sequence shown in SEQ ID NO: 1. Furthermore, these data demonstrate that vectors containing a regulatory element containing multiple copies of the pre-miR183 target site exhibit even stronger inhibition of transgene expression.

[0241] To compare the regulatory effects of the precursor miR183 target site with the so-called "mature miR183 target site" (composed of a sequence complementary to the mature miR183 sequence), a GFP expression cassette containing no miR183 target site (e.g. Figure 2A ), containing 3 copies of the precursor miR183 target site (as shown in Figure 2C ), comprising 3 copies of the so-called "mature miR183 target site" (composed of the sequence shown in SEQ ID NO: 5 complementary to hsa-miR183-5p (the so-called "5p binding site")) (as shown in Figure 2D ) or comprising three copies of the so-called "mature miR183 target site" (composed of a sequence shown in SEQ ID NO: 5 complementary to hsa-miR183-5p (the so-called "5p binding site") and a sequence shown in SEQ ID NO: 6 complementary to hsa-miR183-3p (the so-called "3p binding site") in tandem) (such as Figure 2E HEK293 cells were transfected with plasmid constructs (shown). First, the transfection efficiency was evaluated by quantitative PCR, and the results showed that similar transfection efficiencies were obtained using the four plasmid constructs ( Figure 5A ). GFP fluorescence in the cell lysates was then measured using a fluorescence plate reader, and the results showed that GFP expression in cells transfected with a plasmid containing three copies of the precursor miR183 target site was significantly reduced compared to GFP expression in cells transfected with a control construct that did not contain any miR183 target site ( Figure 5B Surprisingly, none of the plasmids containing three copies of the so-called "mature miR183 target site" induced a significant decrease in GFP expression compared to GFP expression in cells transfected with a control construct that did not contain any miR183 target site ( Figure 5B ).

[0242] These data unexpectedly demonstrate that the precursor miR183 target site having a sequence shown in SEQ ID NO: 1 complementary to the precursor miR183 sequence has a superior effect on transgene silencing compared to the so-called "mature miR183 target site" having a sequence complementary to the mature miR183 sequence.

[0243] Example 2

[0244] Materials and methods

[0245] Carrier

[0246] Plasmid vectors were constructed using the pAAV-smCBA-eGFP-bGH backbone (Genscript) for transfection of HEK293 cells. As described in Example 1, the "no miRT" control construct contained a GFP expression cassette in which the eGFP coding sequence was controlled by the ubiquitin promoter smCBA (truncated chimeric cytomegalovirus (CMV)-chicken β-actin). Plasmids containing miRNA target sites were obtained by cloning the micro RNA target sites into the pAAV-smCBA-eGFP-bGH backbone (Genscript):

[0247] - a plasmid containing 2 copies of the so-called pre-miR183 target site having the sequence shown in SEQ ID NO: 1, located downstream of the smCBA-eGFP cassette (3x pre-miRT183);

[0248] - a plasmid containing 3 copies of the so-called pre-miR183 target site having the sequence shown in SEQ ID NO: 1, located downstream of the smCBA-eGFP cassette;

[0249] - a plasmid comprising 3 copies of the so-called precursor miR182 target site having SEQ ID NO: 22 complementary to the human sequence of precursor miR182:

[0250] The sequence shown in 21 is located downstream of the smCBA-eGFP cassette (3x precursor miRT182);

[0251] - A plasmid comprising 3 copies of the so-called pre-miR96 target site having the sequence shown in SEQ ID NO: 24 complementary to the human sequence of pre-miR182 (ie SEQ ID NO: 25), located downstream of the smCBA-eGFP cassette.

[0252] In vitro experiments

[0253] In order to confirm the influence of the regulatory elements of the miR target sites of the miR183 family described herein 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 culture medium. After 12 hours, PEI-DNA-OptiMEM culture medium was replaced with DMEM 2% FBS (fetal bovine serum). Two or three days after transfection (24 hours or 48 hours after culture medium replacement), cells were collected, washed with PBS and lysed with PBS-Tween20 (0.2%). After the lysate was clarified by centrifugation, GFP fluorescence was measured using Synergy plate reader (Agilent). At the same time, protein content was determined using BCA detection kit (ThermoFisher) and used to normalize the data.

[0254] result

[0255] HEK293 cells were transfected with plasmid constructs containing no miR target site, 2 copies of the pre-miR183 target site, 3 copies of the pre-miR183 target site (corresponding to SEQ ID NO: 7), 3 copies of the pre-miR182 target site (corresponding to SEQ ID NO: 23), or 3 copies of the pre-miR96 target site (corresponding to SEQ ID NO: 26) downstream of the GFP expression cassette. GFP expression in transfected cells was assessed using a fluorescence plate reader 24 hours after medium change (i.e., two days after transfection) or 48 hours after medium change (i.e., three days after transfection), after cell lysis.

[0256] Figure 6 shows the quantification of GFP fluorescence in transfected HEK293 cell lysates and confirms that the presence of the precursor miR183 target site significantly inhibits GFP expression. Notably, the inhibition of GFP expression was maintained over time, with the expression of GFP being reduced 24 hours after medium replacement ( Fig. 6A ) and 48 h after medium replacement ( Figure 6B ) showed similar levels of repression as those observed in Figure 6. Figure 6 also demonstrates that the presence of 2 copies of the precursor miR183 target site is sufficient to significantly repress GFP expression.

[0257] Figure 7 shows the quantification of GFP fluorescence in transfected HEK293 cell lysates, indicating that the presence of the precursor miR182 target site significantly inhibited GFP expression. Notably, the inhibition of GFP expression was maintained over time, with the expression of GFP being reduced 24 hours after medium replacement ( Fig. 7A ) and 48 h after medium replacement ( Figure 7B ) observed similar levels of inhibition.

[0258] Figure 8 shows the quantification of GFP fluorescence in transfected HEK293 cell lysates, indicating that the presence of the precursor miR96 target site significantly inhibited GFP expression. Notably, the inhibition of GFP expression was maintained over time, with the expression of GFP being reduced 24 hours after medium replacement ( Fig. 8A ) and 48 h after medium replacement ( Figure 8B ) observed similar levels of inhibition.

[0259] These data demonstrate 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) are able to significantly inhibit the expression of genes in cells expressing miR182 or miR96, respectively. Therefore, the so-called precursor miR target sites of the miR183 family described herein can be used, for example, for insertion into expression cassettes or vectors to regulate the expression of target nucleic acids, in particular to prevent the expression of target nucleic acids in cells expressing miRNAs of the miR183 family.

Claims

1. An isolated nucleic acid sequence comprising at least 2 copies of a miRNA target site of the miR183 family, wherein the miRNA target site has a sequence as shown in SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO: 24, or a sequence having at least 90% identity to any one of SEQ ID NO: 1, SEQ ID NO: 21 or SEQ ID NO:

24.

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 as shown in SEQ ID NO:1 or a sequence having at least 90% identity with SEQ ID NO:

1.

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.

4. The isolated nucleic acid sequence according to any one of claims 1 to 3, wherein the isolated nucleic acid sequence comprises 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 to any one of SEQ ID NO:7, SEQ ID NO:23 or SEQ ID NO:

26.

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.

6. An expression cassette comprising a promoter, a target gene and a regulatory element, wherein the regulatory element comprises at least one copy of a miRNA target site of the miR183 family, wherein the miRNA target site has a sequence as shown in SEQ ID NO:1, SEQ ID NO:21 or SEQ ID NO:24, or a sequence that is at least 90% identical to any one of SEQ ID NO:1, SEQ ID NO:21 or SEQ ID NO:

24.

7. A vector comprising a regulatory element, wherein the regulatory element comprises at least one copy of a miRNA target site of the miR183 family, wherein the miRNA target site has a sequence as shown in SEQ ID NO:1, SEQ ID NO:21 or SEQ ID NO:24, or a sequence that is at least 90% identical to any one of SEQ ID NO:1, SEQ ID NO:21 or SEQ ID NO:

24.

8. The expression cassette according to claim 6 or the vector according to claim 7, wherein the regulatory element comprises at least one copy of a miR183 target site having a sequence as shown in SEQ ID NO:1 or a sequence having at least 90% identity with SEQ ID NO:

1.

9. An expression cassette according to claim 6 or 8 or a vector according to claim 7 or 8, wherein the regulatory element comprises 2 to 6 copies, preferably 3 copies, of a miRNA target site of the miR183 family, the copies being optionally separated by spacers.

10. The expression cassette according to any one of claims 6 or 8 to 9 or the vector according to any one of claims 7 to 9, wherein the regulatory element comprises the sequence shown in SEQ ID NO: 7, SEQ ID NO: 23 or SEQ ID NO: 26, or a sequence that is at least 90% identical to any one of SEQ ID NO: 7, SEQ ID NO: 23 or SEQ ID NO:

26.

11. An expression cassette according to any one of claims 6 or 8 to 10 or a vector according to any one of claims 7 to 10, wherein 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.

12. An expression cassette according to any one of claims 6 or 8 to 11 or a vector according to any one of claims 7 to 11, wherein the vector comprises a target gene, and wherein the regulatory element is operably linked to or inserted into the target gene, preferably into the 3'-UTR of the target gene.

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

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

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

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