functional nucleic acids

Abstract

The present invention relates to a functional nucleic acid that targets FAN1 mRNA or miRNAs that bind to FAN1 mRNA, thereby releasing the microRNA-mediated repression of FAN1. The present invention also includes methods for enhancing FAN1 protein expression using the functional nucleic acid of the present invention, and methods for treating or improving FAN1-related diseases or disorders, such as triplet elongation disorders, including those of the polyglutamine family.

Description

Technical Field

[0001] (Field of the Invention) The present invention relates to a functional nucleic acid that targets FAN1 mRNA or a miRNA that binds to FAN1 mRNA, thereby relieving microRNA-mediated inhibition of FAN1. The present invention also encompasses a method of enhancing FAN1 protein expression and a method of treating or ameliorating a disease or disorder associated with FAN1, such as a triplet expansion disorder, using the functional nucleic acid of the present invention.

Background Art

[0002] (Background of the Invention) Triplet or trinucleotide repeat disorders are a major group of human diseases caused by the expansion of repetitive trinucleotide sequences within the genome, which results in disease-specific tandem repeat regions. These diseases are usually neurological and vary in terms of the position of the repeat region on the genome, symptoms, and the stage of development at which they occur.

[0003] The expansion of these triplet repeats ultimately results in abnormal proteins, which can lead to either a gain-of-function mutation or a loss-of-function mutation. The number of triplet repeats is usually associated with the age of onset (AAO) of the disease, with larger expansions of the repeat sequence corresponding to earlier AAO.

[0004] One group of triplet repeat disorders is the polyglutamine (polyQ) disease group, which consists of a group of hereditary neurological diseases associated with the expansion of the triplet "CAG" repeat. Further disorders are associated with the expansion of "CGG" repeats, such as fragile X-related disorders (FXD); the expansion of "CTG" repeats, such as myotonic dystrophy type 1; the expansion of "GAA" repeats, such as Friedreich's ataxia; the expansion of "GCC" repeats, such as FRAXE mental retardation; and the expansion of "GCG" repeats, such as oculopharyngeal muscular dystrophy.

[0005] The polyglutamine group includes disorders that are among the most common genetic neurological disorders, including many spinocerebellar ataxias (e.g., type 1 (SCA1), type 2 (SCA2), type 3 (SCA3), type 6 (SCA6), type 7 (SCA7), and type 17 (SCA17), dentatorubral-pallidoluysian atrophy (DRPLA), X-linked spinal muscular atrophy 1 (SMAX1 / SBMA), and Huntington's disease (HD).

[0006] Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an elongation of the CAG trinucleotide repeat in the huntingtin gene. HD causes widespread cognitive, motor, and mental impairments with a variety of symptoms.

[0007] The onset of HD symptoms often varies considerably among individuals. Both CAG repeat length and genetic variations at other locations within the genome are known to influence the age of disease onset. In fact, genome-wide association studies (GWAS) have identified many single nucleotide polymorphisms (SNPs) associated with regulating the age of HD symptom onset.

[0008] Some of the identified SNPs are located on chromosome 15 and are linked to the Fan1 gene. Fan1 encodes the protein FAN1 (FANCD2 / FANCI-associated nuclease 1), an enzyme that possesses both endonuclease and exonuclease activity, which is important in DNA interstrand crosslink repair.

[0009] Increased FAN1 expression has been shown to limit somatic CAG repeat elongation in model systems and is associated with delayed onset and progression of HD, while Fan1 knockdown increases CAG repeat elongation (Goold R et al., FAN1 modifies Huntington's disease progression by stabilizing the expanded HTT CAG repeat. Hum Mol Genet. 2019 Feb 15;28(4):650-661. doi: 10.1093 / hmg / ddy375. PMID: 30358836; PMCID: PMC6360275). Therefore, FAN1 is thought to be involved in regulating the onset and progression of HD; McAllister B et al., Exome sequencing of individuals with Huntington's disease implicates FAN1 nuclease activity in slowing CAG expansion and disease onset (Nat Neurosci. 2022 Apr;25(4):446-457. doi: 10.1038 / s41593-022-01033-5. Epub 2022 Apr 4. PMID: 35379994; PMCID: PMC8986535).

[0010] The SNP identified as being associated with delayed onset of HD motor dysfunction is rs3512. rs3512 is located in the 3'-UTR (exon 15) of the Fan1 gene, and is also found within the intron of the MTMR10 gene, which is encoded on the antisense strand. Transcriptome analysis of eQTLs has revealed that rs3512 is associated with increased Fan1 gene expression (Genetic Modifiers of Huntington's Disease (GeM-HD) Consortium. Identification of Genetic Factors that Modify Clinical Onset of Huntington's Disease). Cell. 2015 Jul 30;162(3):516-26. doi: 10.1016 / j.cell.2015.07.003. PMID: 26232222; PMCID: PMC4524551), suggesting that rs3512 delays HD onset by influencing regulatory mechanisms that inhibit Fan1 expression.

[0011] Gene regulatory mechanisms are diverse and complex. One group of regulatory mechanisms is mediated by microRNAs (miRNAs). miRNAs are short, non-coding RNAs expressed within cells that negatively regulate target gene expression at the mRNA level through their ability to bind to the 3'-UTR of specific target mRNAs, thereby repressing translation initiation / elongation or inducing mRNA degradation.

[0012] Hemoglobin (HD) is associated with debilitating symptoms that worsen throughout the individual's life, often leading to premature death. Despite being a well-known disease, therapeutic interventions for controlling its onset and progression are lacking. Furthermore, disease-modifying interventions are generally absent for both HD and the broader triplet repeat diseases.

[0013] The object of the present invention is to provide target-specific functional nucleic acids that can function as disease modifiers in the treatment and prevention of triplet repeat diseases, particularly hemoglobin (HD). [Overview of the project]

[0014] (Summary of the invention) The inventors sought to develop functional nucleic acid molecules that could function as disease modifiers for triplet repeat diseases, particularly HD.

[0015] Fan1 is thought to be involved in HD, as well as in a broader range of polyQ family disorders, and even in triplet repeat disorders associated with the extension of other motifs. Therefore, the inventors conducted a literature and database search to identify the regulatory pathways involved in the control of Fan1.

[0016] The inventors identified miR-124-3p, a microRNA that targets Fan1. Interestingly, the binding site of miR-124-3p located at the Fan1 3'-UTR is altered in HD patients with rs3512, which exhibits delayed onset of HD motor dysfunction.

[0017] In summary, these observations suggest that increased Fan1 expression in HD patients with rs3512 is associated with the loss of miR-124-3p-mediated Fan1 suppression. Therefore, we attempted to reproduce the naturally observed suppressive effect of the rs3512 SNP by designing a functional nucleic acid molecule that can reverse the suppressive effect of miR-124-3p on Fan1, thereby allowing Fan1 to act as a disease modifier in the triplet elongation disorder in which it is associated.

[0018] In a first embodiment, the functional nucleic acid molecule provided herein is having a length of 10 to 40 nucleotides and comprising one or more consecutive nucleotide sequences, each independently consisting of nucleotides of length 5 or more, wherein each consecutive nucleotide sequence is at least 80% complementary to a consecutive nucleotide sequence in the 3'-UTR of Fan1.

[0019] In one embodiment, the present invention provides a functional nucleic acid molecule and a conjugate comprising one or more portions covalently bonded to the functional nucleic acid molecule.

[0020] In one embodiment, a pharmaceutically acceptable salt of a functional nucleic acid molecule or conjugate of the present invention is provided.

[0021] In one embodiment, compositions comprising a functional nucleic acid molecule, conjugate, or pharmaceutically acceptable salt of the present invention, as well as diluents, solvents, carriers, salts, and / or adjuvants are provided.

[0022] In one embodiment, a pharmaceutical composition is provided comprising a functional nucleic acid molecule, conjugate, or pharmaceutically acceptable salt of the present invention, as well as a pharmaceutically acceptable diluent, solvent, carrier, salt, and / or adjuvant.

[0023] In another embodiment, a method is provided for regulating Fan1 protein expression in target cells containing Fan1 and microRNA miR-124-3p, comprising the step of exposing the cells to a functional nucleic acid molecule, conjugate, pharmaceutically acceptable salt, composition, or pharmaceutical composition of the present invention.

[0024] In a further embodiment, a method is provided for treating, preventing or delaying the onset of a Fan1 protein-related disease in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of a functional nucleic acid molecule, conjugate, pharmaceutically acceptable salt, or pharmaceutical composition of the present invention.

[0025] In one aspect, there is provided a functional nucleic acid molecule, conjugate, pharmaceutically acceptable salt, or pharmaceutical composition of the invention for use in medicine.

[0026] In one aspect, there is provided a functional nucleic acid molecule, conjugate, pharmaceutically acceptable salt, or pharmaceutical composition of the invention for use in the treatment, prevention, or delay of the onset of triplet repeat expansion diseases.

[0027] In one aspect, there is provided the use of a functional nucleic acid molecule, conjugate, pharmaceutically acceptable salt, or pharmaceutical composition of the invention for the preparation of a medicament for the treatment, prevention, or delay of triplet repeat expansion diseases.

[0028] In some embodiments of the invention according to the foregoing methods and uses, the disease is a polyglutamine (polyQ) disease, where the disease is selected from the group consisting of Huntington's disease (HD), spinocerebellar ataxia type 1 (SCA1), spinocerebellar ataxia type 2 (SCA2), spinocerebellar ataxia type 3 (SCA3), spinocerebellar ataxia type 6 (SCA6), spinocerebellar ataxia type 7 (SCA7), spinocerebellar ataxia type 17 (SCA17), dentatorubral-pallidoluysian atrophy (DRPLA), and X-linked spinal and bulbar muscular atrophy 1 (SMAX1 / SBMA).

[0029] In one embodiment, the disease is Huntington's disease (HD).

Brief Description of the Drawings

[0030] (Brief Description of the Drawings) [Figure 1]Figure 1 - miR-124-3p mimic downregulation of Fan1 mRNA. Transfection with the miR-124-3p oligonucleotide mimic results in downregulation of Fan1 mRNA (Figure 1A). Figure 1A shows the downregulation of Fan1 mRNA mediated by the miR-124-3p mimic with both 50 nM and 100 nM mimics. Figure 1B shows the downregulation of Fan1 mediated by siRNA, which serves as a positive control for RNA transfection and Fan1 detection. Data represent the mean of all experiments performed (n=3). Statistical analysis consisted of two-way ANOVA with Tukey's multiple comparison test for mimic treatment and an unpaired t-test for siRNA treatment. [Figure 2] Figure 2 - miR-124-3p mimic downregulation of Fan1 protein. Transfection with the oligonucleotide mimic of miR-124-3p results in downregulation of Fan1 protein (Figures 2A and 2B). Figure 2A shows the bands of the imaged Western blot, where three independent replicate experiments are shown for the untargeted mimic control (+) group and the miR-124-3p mimic (+) group, respectively. Figure 2B shows the intensity of the Fan1 band observed at 24 and 48 hours post-treatment. Data represent the mean and SD of all experiments performed (n=3 at 24 hours, n=2 at 48 hours). Statistical analysis of the 24-hour data consisted of an unpaired t-test. [Figure 3]Figure 3 - SNP rs3512 reduces miR-124-3p-mediated repression. Introduction of the rs3512 SNP into the 3'UTR sequence of Fan1 reduces miR-124-3p-mediated repression. Figure 3A shows the dual reporter system used to investigate the effect of rs3512 on the repressive activity of miRNA miR-124-3p on mRNA with a 3'UTR sequence derived from Fan1. Figure 3B shows the multiplicative change in FLuc reporter gene fluorescence normalized to RLuc in the presence of a non-targeted miRNA mimic control and a miR-124-3p mimic. Data represent the mean and standard deviation of all experiments performed (n=5). Statistical analysis consisted of two-way ANOVA with Tukey's multiple comparison test. [Figure 4] Figure 4 - Schematic representation of a functional nucleic acid molecule targeting the Fan1 3'-UTR at the miR-124-3p binding site. The functional nucleic acid molecule was designed for the 3'-UTR region of human Fan1 at the predicted miR-124-3p seed sequence (i.e., the location where miR-124-3p is predicted to bind). The functional nucleic acid molecule was designed using a tiling approach. In this approach, each functional nucleic acid molecule is complementary to a target sequence located one nucleotide downstream (i.e., 3') of its preceding target sequence. The Fan1 mRNA is shown in a magnified view of the approximate region encompassing the functional nucleic acid molecule binding site. [Figure 5] Figure 5 - ASO-mediated steric hindrance of miR-124-3p on the FAN1 3'-UTR results in upregulation of FAN1 in cortical neurons. (A) Schematic outline of the cortical neuron differentiation and gymnosis protocol. Neural stem cells were differentiated and matured for 20 days. Mature cortical neurons were treated for 10 days with gymnosis using either a 5 μM untargeted control or a steric hindrance-type ASO of miR-124-3p. (B) Measured FAN1 band density after imaging Western blot and normalization by TUBB3. Multiple changes were calculated compared to the untargeted ASO control. Data represent the mean and SD of two replicates in a single experiment. [Figure 6]Figure 6 - ASO-mediated steric hindrance of the miRNA target site on the FAN1 3'-UTR leads to upregulation of FAN1. Multiple changes in HiBiT luminescence signal normalized by cell viability compared to mock-transfected samples after treatment with PS-Me (A), PS-Me-LNA (B), PS-MOE (C), or PS-MOE-LNA miRNA steric-hindrance ASO. Data represent the mean and SD of the mean values ​​in each experiment. N=2 for PS-Me, PS-MOE, and PS-MOE-LNA libraries; N=4 for PS-Me-LNA library. Figures 6A-D include compounds 26-57. [Figure 7] Figure 7 - ASO-mediated steric hindrance of miR-197-3p on the FAN1 3'-UTR leads to upregulation of FAN1. (A) Functional nucleic acid molecules were designed for the 3'-UTR region of human Fan1 at the miR-197-3p predicted binding site. Functional nucleic acid molecules were designed using a tiling approach, in which each functional nucleic acid molecule is complementary to a target sequence located 3 nucleotides downstream (i.e., 3') of its preceding target sequence. The Fan1 3'-UTR is illustrated with the approximate region encompassing the functional nucleic acid molecule binding site. (B) Multiple changes in HiBiT luminescence signal normalized by cell viability (right) and unnormalized (left) compared to mock-transfected samples after treatment with PS-Me-LNA miR-197-3p steric-hindrance ASO. Data represent mean and SD of the mean values ​​in each experiment, N=2. Optimization of ASO chemical modification results in ASOs with retained or improved functionality (C). Since mRNA does not change intrinsically upon ASO treatment (D), ASO-mediated upregulation is mediated at the translational level. [Figure 8] Figure 8 - Functional nucleic acid molecules targeting different miRNA seed regions in the 3'-UTR of Fan1 increase FAN1 protein expression. ASOs targeting the miR-197-3p, miR-181-5p, and miR-145-5p seed regions cause upregulation of FAN1 (A), which is mediated at the translational level because the mRNA does not change intrinsically upon ASO treatment (B). [Figure 9] Figure 9 - Functional nucleic acid molecules targeting different miRNA seed regions in the 3'-UTR of Fan1 increase FAN1 protein expression in medium-sized spiny neurons derived from HD patients. The Fan1 upregulatory activity of sterically insufficient miRNA ASOs is reproduced in HD-associated cell models, in which case ASOs can upregulate FAN1 in disease-associated cell types (A and B). [Figure 10] Figure 10 - FAN1 eQTL data extracted from the GTEx Portal show Fan1 upregulation only in brain-derived tissue from individuals carrying the rs3512 SNP. Increased levels of Fan1 transcript are detected only in brain-derived tissue from individuals carrying the rs3512 SNP in one allele (G / C) or both alleles (C / C), compared to individuals homozygous for the reference allele (G / G). Similar levels of Fan1 transcript are detected in whole blood and adipose tissue where miR-124-3p is not expressed, regardless of genotype. [Modes for carrying out the invention]

[0031] (Detailed description of the invention) The object of the present invention is to provide a functional nucleic acid molecule that acts to release the post-transcriptional repression of Fan1 mRNA.

[0032] For this purpose, the inventors have devised functional nucleic acid molecules each independently comprising one or more consecutive nucleotide sequences, each consisting of nucleotides of length 5 or more, wherein each consecutive nucleotide sequence is at least 80% complementary to a consecutive nucleotide sequence in the 3'UTR of Fan1.

[0033] The complementarity of the functional nucleic acid molecule of the present invention to Fan1's 3'UTR mRNA and / or other RNAs that bind to Fan1's 3'UTR, such as miRNA, e.g., miR-124-3p, allows the functional nucleic acid molecule to bind to or anneal to the RNA, thereby preventing post-transcriptional repression of Fan1.

[0034] By either directly targeting Fan1 mRNA or targeting other RNAs that themselves target Fan1 mRNA, miRNA binding to Fan1 mRNA can be blocked, thereby preventing the repressive effect of Fan1 expression.

[0035] Therefore, the functional nucleic acid molecule of the present invention can be used, for example, for targeted modulation of Fan1 expression in diseases or disorders in which Fan1 modulates the disease. Fan1 is known to be involved in the modulation of triplet extension disease of the polyglutamine family. Accordingly, the functional nucleic acid molecule of the present invention can be used as a disease modifier in polyglutamine disorders such as Huntington's disease (HD), spinocerebellar ataxia type 1 (SCA1), spinocerebellar ataxia type 2 (SCA2), spinocerebellar ataxia type 3 (SCA3), spinocerebellar ataxia type 6 (SCA6), spinocerebellar ataxia type 7 (SCA7), spinocerebellar ataxia type 17 (SCA17), dentatorubral-pallidoluysian atrophy (DRPLA), and X-linked spinal muscular atrophy 1 (SMAX1 / SBMA).

[0036] (Functional nucleic acid molecule) Provided herein are functional nucleic acid molecules having a length of 10 to 40 nucleotides, each independently comprising a sequence of nucleotides of 5 or more nucleotides in length, wherein each sequence of nucleotides is at least 80% complementary to a sequence of nucleotides in the 3'UTR of Fan1.

[0037] The functional nucleic acid molecules according to the present invention may also be defined by other terms in the art, depending on the structural and functional characteristics of the specific functional nucleic acid. For example, functional nucleic acid molecules can be considered as antisense oligonucleotides (ASOs), long non-coding RNAs (lncRNAs), sterically hindered oligonucleotides (SBOs), or anti-miRNA oligonucleotides (AMOs).

[0038] In one embodiment, the functional nucleic acid molecule is an antisense oligonucleotide (ASO).

[0039] In one embodiment, the functional nucleic acid molecule is an anti-miRNA oligonucleotide (AMO).

[0040] In one embodiment, the functional nucleic acid molecule is a sterically hindered oligonucleotide (SBO).

[0041] Further examples include functional nucleic acid molecules such as small activated RNA (saRNA), small interfering RNA (siRNA), or heterodouble-stranded oligonucleotides (HDOs).

[0042] In one embodiment, the functional nucleic acid molecule is a small activated RNA (saRNA).

[0043] In one embodiment, the functional nucleic acid molecule is a small interfering RNA (siRNA).

[0044] In one embodiment, the functional nucleic acid molecule is a heterodouble-stranded oligonucleotide (HDO).

[0045] As used herein, “functional nucleic acid molecules” refer to the synthetic molecules of the present invention. In particular, the term “functional nucleic acid molecules” refers to nucleic acid molecules (e.g., DNA, RNA, or mixtures thereof, or synthetic analogs thereof) that can release the post-translational repression of Fan1.

[0046] It will be understood that terms such as "releasing the post-translational repression of Fan1" may also be used to describe other functional effects related to this effect. For example, it could also be said that the functional nucleic acid molecule of the present invention increases the expression of Fan1 (i.e., by releasing its repression).

[0047] The term "functional RNA molecule" also refers to functional nucleic acid molecules formed from any combination of RNA and its modified versions; any combination of DNA and its modified versions; any combination of a mixture of RNA and DNA and its modified versions; and any combination of chemical analogues of nucleotides.

[0048] The functional nuclear molecule of the present invention may be a DNA molecule that is transcribed into a functional RNA molecule according to the present invention.

[0049] The functional nuclear molecule of the present invention may be a DNA molecule that is transcribed into a functional RNA molecule according to the present invention and subsequently further modified by chemical or enzymatic modification.

[0050] In one embodiment, the functional nuclear molecule is a single-chain molecule.

[0051] In one embodiment, the functional nuclear molecule is a double-stranded molecule.

[0052] In one embodiment, the functional nucleus includes regions of single-stranded and double-stranded oligonucleotides and / or analogs thereof.

[0053] In relation to the above, it will be understood that a double helix refers to the complementary base pairing between two or more different molecules. Therefore, a single-stranded molecule (i.e., a single molecule) is considered single-stranded according to the above definition, even if the molecule forms a double-stranded secondary structure (e.g., a stem-loop).

[0054] In one embodiment, the functional nuclear molecule consists of DNA.

[0055] In one embodiment, the functional nuclear molecule includes DNA.

[0056] DNA molecules may not be functional in the same way that RNA molecules are. For example, a functional DNA molecule may be functional in that it encodes a functional RNA molecule, where the RNA molecule is a functional nucleic acid molecule according to the present invention.

[0057] In one embodiment, the functional nuclear molecule consists of RNA.

[0058] In one embodiment, the functional nuclear molecule includes RNA.

[0059] In one embodiment, the functional nuclear molecule consists of modified RNA nucleotides.

[0060] In one embodiment, the functional nuclear molecule includes modified RNA nucleotides.

[0061] In one embodiment, the functional nuclear molecule consists of a synthetic nucleotide analog.

[0062] In one embodiment, the functional nuclear molecule includes a synthetic nucleotide analog.

[0063] In one embodiment, the functional nuclear molecule contains or consists of morpholinonucleotides.

[0064] In one embodiment, a functional nucleic acid molecule having a length of 10 to 40 nucleotides is provided, which independently comprises one or more consecutive nucleotide sequences consisting of nucleotides of length 5 or more, wherein each consecutive nucleotide sequence is at least 80% complementary to a consecutive nucleotide sequence in the 3'UTR of Fan1.

[0065] One or more consecutive nucleotide sequences can be selected independently of each other if multiple consecutive nucleotide sequences exist. The characteristics of any one consecutive nucleotide sequence (e.g., length, composition, and % complementarity) can be selected completely independently of any one other (i.e., all other) consecutive nucleotide sequences within the same functional nucleic acid molecule. As a result, there are no restrictions imposed on any one consecutive nucleotide sequence by any other consecutive nucleotide sequences within the functional nucleic acid molecule.

[0066] Each of one or more consecutive nucleotide sequences may target a discontinuous sequence within the 3'-UTR of Fan1. That is, a functional nucleic acid molecule can bind to a target oligonucleotide at separate target sites, where these target sites are not contiguous. Therefore, a functional nucleic acid molecule can bind to a target oligonucleotide at multiple positions, causing the intervening region of the functional nucleic acid molecule to "loop out."

[0067] Consecutive nucleotide sequences may bind to Fan1 simultaneously or separately (i.e., not simultaneously).

[0068] A single functional nucleic acid molecule may bind to a single Fan1 mRNA. Alternatively, in embodiments in which the functional nucleic acid contains multiple consecutive nucleotide sequences, a single functional nucleic acid molecule may bind to multiple Fan1 mRNAs, for example, where one consecutive nucleotide sequence may bind to one Fan1 mRNA molecule, while a second consecutive nucleotide sequence may simultaneously bind to a second Fan1 mRNA molecule at either the same target sequence or a different target sequence.

[0069] In one embodiment, a functional nucleic acid molecule having a length of 10 to 40 nucleotides is provided, comprising a sequence of nucleotides of 5 or more in length, wherein the sequence of nucleotides is at least 80% complementary to the sequence of nucleotides in the 3'UTR of Fan1.

[0070] In one embodiment, a functional nucleic acid molecule having a length of 10 to 35 nucleotides is provided, which independently comprises one or more consecutive nucleotide sequences consisting of nucleotides of length 5 or more, wherein each consecutive nucleotide sequence is at least 80% complementary to a consecutive nucleotide sequence in the 3'UTR of Fan1.

[0071] In one embodiment, a functional nucleic acid molecule having a length of 10 to 35 nucleotides is provided, comprising a sequence of nucleotides of 5 or more in length, wherein the sequence of nucleotides is at least 80% complementary to the sequence of nucleotides in the 3'UTR of Fan1.

[0072] In one embodiment, the functional nucleic acid molecule consists of one or more consecutive nucleotide sequences.

[0073] In one embodiment, the functional nucleic acid molecule has a length of 10 to 40 nucleotides, for example, 10 to 35, 10 to 30, 10 to 25, 10 to 20, or 10 to 15 nucleotides.

[0074] In one embodiment, the functional nucleic acid molecule is longer than 10 nucleotides, for example, longer than 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, or longer than 39 nucleotides.

[0075] In one embodiment, the functional nucleic acid molecule has a length of 10 nucleotides. In another embodiment, the functional nucleic acid molecule has a length of 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, or 40 nucleotides.

[0076] In one embodiment, one or more consecutive nucleotide sequences are independently 5 to 40 nucleotides in length, for example, 10 to 35, 10 to 30, 10 to 25, 10 to 20, or 10 to 15 nucleotides in length.

[0077] In one embodiment, one or more consecutive nucleotide sequences are independently longer than 5 nucleotides, for example, longer than 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, or longer than 39 nucleotides.

[0078] In one embodiment, one or more consecutive nucleotide sequences are independently 5 nucleotides long. In another embodiment, one or more consecutive nucleotide sequences are independently 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, or 40 nucleotides long.

[0079] In one embodiment, one or more consecutive nucleotide sequences are independently at least 80% complementary to consecutive nucleotide sequences within the 3'UTR of Fan1.

[0080] In one embodiment, one or more consecutive nucleotide sequences are independently at least 80% complementary to consecutive nucleotide sequences in the 3'UTR of Fan1, for example, at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary.

[0081] In one embodiment, one or more consecutive nucleotide sequences are independently at least 85% complementary to consecutive nucleotide sequences within the 3'UTR of Fan1.

[0082] In one embodiment, one or more consecutive nucleotide sequences are independently at least 90% complementary to consecutive nucleotide sequences within the 3'UTR of Fan1.

[0083] In one embodiment, one or more consecutive nucleotide sequences are independently at least 95% complementary to consecutive nucleotide sequences within the 3'UTR of Fan1.

[0084] In one embodiment, one or more consecutive nucleotide sequences are independently 100% complementary to consecutive nucleotide sequences within the 3'UTR of Fan1.

[0085] In one embodiment, the functional nucleic acid molecule contains one or more consecutive nucleotide sequences.

[0086] In another embodiment, the functional nucleic acid molecule contains or consists of a single sequence of nucleotides.

[0087] In another embodiment, the functional nucleic acid molecule contains or consists of two, three, four, five, or more consecutive nucleotide sequences.

[0088] It will be understood that the 3'UTR of Fan1 is intended to be a complete 3'UTR sequence that exists on both the coding and non-coding strands of the DNA. Therefore, each consecutive nucleotide sequence may or may not be complementary to the mRNA, and may or may not be complementary to the miRNA (which is complementary to the 3'UTR mRNA of Fan1).

[0089] It will be understood that the functional nucleic acid molecules of the present invention may be either DNA molecules or RNA molecules. Therefore, sequences implicitly represented as DNA in this specification (i.e., by containing T rather than U) should be understood to also represent the corresponding RNA molecules in which each T nucleotide in the sequence is replaced by a U nucleotide. Thus, the sequences according to the present invention may be either DNA sequences or RNA sequences. In this specification, representing a sequence as either one or both is merely for simplification and does not mean that the sequence is limited to DNA or RNA sequences unless explicitly stated otherwise.

[0090] References to nucleotides or nucleosides herein may be equivalent to ribo or deoxyribonucleotides / nucleosides and their modified versions.

[0091] By referring to the target sequence, those skilled in the art will readily understand what the substituent portion of such a molecule is. For example, if the target molecule is mRNA, then the nucleotide / nucleoside is a ribonucleotide / nucleoside, and so on.

[0092] In this specification, a polypeptide or polynucleotide sequence is said to be the same as or "identical" to another polypeptide or polynucleotide sequence if it shares 100% sequence identity. Residues in a sequence are numbered from left to right, i.e., from the N-terminus to the C-terminus in the case of polypeptides; and from the 5' end to the 3' end in the case of polynucleotides. If closely related sequences are not identical, they may be similar, i.e., they may have a certain degree of quantifiable sequence identity; for example, a sequence may have 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence identity with another sequence. Unless a specific reference range is given, for example, with respect to nucleotide positions, the sequence identity cited is understood to be calculated over a residue range over which the two sequences are aligned. The aligned residue range can represent the entirety of one or more input sequences, or a contiguous sequence region of one or more input sequences, and is usually determined by a standard tool known in the art, e.g., NCBI BLAST.

[0093] To compare two closely related polynucleotide sequences, the "% sequence identity" between the first and second nucleotide sequences can be calculated using NCBI BLAST with a standard setting for nucleotide sequences (BLASTN). To compare two closely related polypeptide sequences, the "% sequence identity" between the first and second polypeptide sequences can be calculated using NCBI BLAST with a standard setting for polypeptide sequences (BLASTP). A "difference" between sequences refers to a single nucleotide insertion, deletion, or substitution at the position of the second sequence compared to the first sequence. An insertion, deletion, or substitution in the second sequence that is otherwise identical to the first sequence (100% sequence identity) results in a decrease in % sequence identity.

[0094] "Complementarity" relates to the Watson-Crick base-pairing rule, in which an "A" nucleotide forms a hydrogen bond with a "T" (or "U") nucleotide, and a "G" nucleotide forms a hydrogen bond with a "C" nucleotide, forming a double-stranded structure through the "complementary" nucleotides. In this specification, a "complementary" sequence is a sequence that is closely related to another sequence, such that such base-pairing can occur. A complementary sequence may be 100% complementary, so that base-pairing can occur along its entire length, or it may be 99%, 90%, 80%, 70%, or 60% complementary, for example, so that base-pairing can occur along a portion of its sequence. Here, as is common in the art, a complementary sequence may also be called a "reverse complementary" sequence.

[0095] Functional nucleic acids contain a sequence that is long enough to bind to a target (e.g., mRNA or miRNA).

[0096] In one embodiment, the functional nucleic acid molecule includes a sequence selected from the group consisting of SEQ ID NOs: 949-973; 974-998; 999-1064; 1065-1130; 1137-1138; and / or 1139-1148, or includes a continuous nucleotide sequence consisting of said sequence.

[0097] In one embodiment, the functional nucleic acid molecule includes a sequence selected from the group consisting of SEQ ID NOs: 949 to 973, or a continuous nucleotide sequence consisting of said sequence.

[0098] In one embodiment, the functional nucleic acid molecule includes a sequence selected from the group consisting of SEQ ID NOs: 974 to 998, or a continuous nucleotide sequence consisting of said sequence.

[0099] In one embodiment, the functional nucleic acid molecule includes a sequence selected from the group consisting of SEQ ID NOs: 999 to 1064, or a continuous nucleotide sequence consisting of said sequence.

[0100] In one embodiment, the functional nucleic acid molecule includes a sequence selected from the group consisting of SEQ ID NOs: 1065 to 1130, or a continuous nucleotide sequence consisting of said sequence.

[0101] In one embodiment, the functional nucleic acid molecule includes a sequence selected from the group consisting of SEQ ID NOs: 1137 to 1138, or a continuous nucleotide sequence consisting of said sequence.

[0102] In one embodiment, the functional nucleic acid molecule includes a sequence selected from the group consisting of SEQ ID NOs: 1139 to 1148, or a continuous nucleotide sequence consisting of said sequence.

[0103] In one embodiment, the functional nucleic acid molecule includes a sequence having 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 949-973; 974-998; 999-1064; 1065-1130; 1137-1138; and / or 1139-1148, or includes a continuous nucleotide sequence consisting of said sequence.

[0104] In one embodiment, the functional nucleic acid molecule contains a sequence having 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 949-973; 974-998; 999-1064; 1065-1130; 1137-1138; and / or 1139-1148, or contains a continuous nucleotide sequence consisting of said sequence.

[0105] In one embodiment, the functional nucleic acid molecule contains a sequence having 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 949-973; 974-998; 999-1064; 1065-1130; 1137-1138; and / or 1139-1148, or contains a continuous nucleotide sequence consisting of said sequence.

[0106] In one embodiment, the functional nucleic acid molecule is selected from the group consisting of ASO 849, ASO 850, ASO 851, ASO 852, ASO 853, ASO 917, ASO 918, ASO 919, ASO 920, ASO 921, ASO 922, ASO 864, ASO 968, ASO 970, and ASO 969.

[0107] In one embodiment, the functional nucleic acid molecule includes a 5'-cap. The "5'-cap" refers to a modified nucleotide at the 5'-terminus of the transcript that provides the molecule with stability from degradation by exonucleases and can facilitate translation. Most commonly, the 5'-cap may be a 7-methylguanylate cap (m7G), i.e., a guanine nucleotide that is attached to RNA via a 5'-to-5' tripphosphate bond and methylated at position 7.

[0108] (FAN1) Fan1 encodes the protein FAN1 (FANCD2 / FANCI-related nuclease 1), an enzyme that possesses both endonuclease and exonuclease activity, which are important in DNA interstrand crosslink repair.

[0109] In this specification, Fan1 is used to refer to genes (i.e., DNA) and any other sequences that encode such sequences (e.g., mRNA). Proteins are referred to as FAN1. However, the context of any reference to Fan1 will be readily apparent to those skilled in the art in determining the type of molecule being referred to. Therefore, the aforementioned nomenclature is not intended to be limiting in any way.

[0110] The 3'-UTR sequences of four human Fan1 variants are listed below. [ka] [ka] [ka] [ka]

[0111] In one embodiment, the sequence derived from the 3'UTR of Fan1 is the sequence derived from the code strand of Fan1.

[0112] In one embodiment, the 3'UTR of Fan1 is as described in Sequence IDs 1-4.

[0113] In one embodiment, the 3'UTR of Fan1 is as described in Sequence IDs 1-4.

[0114] In one embodiment, one or more consecutive nucleotide sequences are independently at least 80% complementary to one or more of sequence numbers 1 to 4, for example, at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary.

[0115] In one embodiment, one or more consecutive nucleotide sequences are independently at least 85% complementary to any one or more consecutive nucleotide sequences in sequence numbers 1 to 4.

[0116] In one embodiment, one or more consecutive nucleotide sequences are independently at least 90% complementary to one or more consecutive nucleotide sequences in any one of sequence numbers 1 to 4.

[0117] In one embodiment, one or more consecutive nucleotide sequences are independently at least 95% complementary to any one or more consecutive nucleotide sequences in sequence numbers 1 to 4.

[0118] In one embodiment, one or more consecutive nucleotide sequences are 100% complementary to any one or more consecutive nucleotide sequences in sequence numbers 1 to 4.

[0119] Four more sequences of human Fan1 3'-UTR are listed below. Each of these sequences represents the reverse complement of sequence numbers 1-4 above. Therefore, sequence numbers 1-4 and 5-8 are 100% complementary to each other. [ka] [ka] [ka] [ka]

[0120] In one embodiment, the sequence derived from the 3'UTR of Fan1 is a sequence derived from the non-coding strand of Fan1.

[0121] In one embodiment, the 3'UTR of Fan1 is as described in Sequence IDs 5-8.

[0122] In one embodiment, one or more consecutive nucleotide sequences are independently at least 80% complementary to one or more of sequence numbers 5-8, for example, at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary.

[0123] In one embodiment, one or more consecutive nucleotide sequences are independently at least 85% complementary to any one or more consecutive nucleotide sequences in sequence numbers 5 to 8.

[0124] In one embodiment, one or more consecutive nucleotide sequences are independently at least 90% complementary to any one or more consecutive nucleotide sequences in sequence numbers 5 to 8.

[0125] In one embodiment, one or more consecutive nucleotide sequences are independently at least 95% complementary to any one or more consecutive nucleotide sequences in sequence numbers 5 to 8.

[0126] In one embodiment, one or more consecutive nucleotide sequences are independently 100% complementary to any one or more consecutive nucleotide sequences in sequence numbers 5 to 8.

[0127] The sequence derived from the 3'UTR of Fan1 may originate from either the coding strand or the non-coding strand of Fan1.

[0128] (microRNA) MicroRNAs (miRNAs) are short, non-coding RNAs that, through their ability to bind to the 3'-UTR of target mRNA, act to post-transcriptionally regulate target gene expression, thereby repressing translation initiation / elongation or inducing mRNA degradation.

[0129] miRNAs typically contain a seed region (nucleotides 1-8) embedded within it, consisting of the first 2-7 nucleotides (i.e., counting from the 5' end of the miRNA). This seed region is involved in Watson-Crick base pairing with its target mRNA (standard site). Not all nucleobases within the seed region are required for Watson-Crick base pairing with the target nucleobase; some nucleotides within the seed may involve G:U wobble pairing or contain incomplete seed matches (non-standard sites). Therefore, the seed region may not be 100% complementary to its target.

[0130] The miRNA designated "miR-124-3p" is the mature active form of a miRNA that targets Fan1. miR-124-3p is produced from three genes encoded on different chromosomes. A further mature miRNA, miR-506-3p, has the same seed sequence as miR-124-3p, but its 3'-sequence outside this seed region is different.

[0131] Furthermore, there is also an "isomiR" for miRNAs, which differs from the reference sequence by only a few nucleotides and is generated by various processes. It will be understood that the isomiR of any particular miRNA mentioned herein, including miR-124-3p, will be encompassed only by references to the reference miRNA. Therefore, a reference to miR-124-3p will encompass the isomiR of miR-124-3p.

[0132] In one embodiment, the functional nucleic acid molecule of the present invention competes with microRNA for binding to Fan1 mRNA.

[0133] In one embodiment, the consecutive nucleotide sequence of the functional nucleic acid of the present invention is complementary to the consecutive nucleotide sequence within the 3'UTR of Fan1 that overlaps with the nucleotide sequence bound by a microRNA targeting the 3'UTR of Fan1.

[0134] In one embodiment, one or more consecutive nucleotide sequences are independently at least 80% complementary to consecutive nucleotide sequences in a microRNA targeting the 3'-UTR of Fan1, for example, at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary.

[0135] In one embodiment, microRNAs are associated with regulating the development of triplet repeat disorders.

[0136] In one embodiment, microRNAs are associated with reducing the age at which triplet repeat disorders develop (AAO).

[0137] In one embodiment, microRNA is associated with increasing the age at which triplet repeat disorders develop (AAO).

[0138] In one embodiment, the microRNAs are hsa-miR-299-3p, hsa-miR-3940-5p, hsa-miR-4265, hsa-miR-4507, hsa-miR-4657, hsa-miR-6748-5p, hsa-miR-6759-5p, hsa-miR-6793-5p, hsa-miR-6796-5p, hsa-miR-6839-3p, hsa-miR-629-5p, hsa-miR-1275, hsa-miR-193b -5p, hsa-miR-3675-5p, hsa-miR-4665-5p, hsa-miR-6751-5p, hsa-miR-6803-5p, hsa-miR-6835-5p, hsa-miR-6842-5p, hsa- miR-6890-5p, hsa-miR-7109-5p, hsa-miR-7110-5p, hsa-miR-198, hsa-miR-1911-3p, hsa-miR-6753-5p, hsa-miR-1256, hsa- miR-3910, hsa-miR-1910-3p, hsa-miR-2682-5p, hsa-miR-34a-5p, hsa-miR-34b-5p, hsa-miR-34c-5p, hsa-miR-449a, hsa-m iR-449b-5p, hsa-miR-449c-5p, hsa-miR-548au-3p, hsa-miR-584-3p, hsa-miR-6511a-5p, hsa-miR-6808-5p, hsa-miR-6893- The following are selected from the group consisting of 5p, hsa-miR-940, hsa-miR-3714, hsa-miR-125b-1-3p, hsa-miR-155-5p, hsa-miR-3621, hsa-miR-3610, hsa-miR-4665-3p, hsa-miR-4755-3p, hsa-miR-3622a-5p, hsa-miR-5582-5p, hsa-miR-124-3p, and hsa-miR-506-3p, or these isomiR:.

[0139] In another embodiment, the microRNA is the miRNA defined in Table 1, namely: hsa-miR-299-3p, hsa-miR-3940-5p, hsa-miR-4265, hsa-miR-4507, hsa-miR-4657, hsa-miR-6748-5p, hsa-miR-6759-5p, hsa-miR-6793-5p, hsa-miR-6796-5p, hsa-miR-6839-3p, hsa-miR-629-5p, hsa-miR-1275, hsa-miR-193b-5p, hsa-miR-3675-5p, hsa-miR-4665-5p, hsa-miR-6751-5p, hsa-miR-6803-5p, hsa-miR-6835-5p, hsa-miR-6842-5p, hsa-miR-6890-5p, hsa-miR-7109-5p, hsa-miR-7110-5p, hsa-miR-198, hsa-miR-1911-3p, hsa-miR-6753-5p, hsa-miR-1256, hsa-miR-3910, hsa-miR-1910-3p, hsa-miR-2682-5p, hsa-miR-34a-5p, hsa-miR-34b-5p, hsa-miR-34c-5p, hsa-miR-449a, hsa-miR-449b-5p, hsa-miR-449c-5p, hsa-miR-548au-3p, hsa-miR-584-3p, hsa-miR-6511a-5p, hsa-miR-6808-5p, hsa-miR-6893-5p, hsa-miR-940, hsa-miR-3714, hsa-miR-125b-1-3p, hsa-miR-155-5p, hsa-miR-3621, hsa-miR-3610, hsa-miR-4665-3p, hsa-miR-4755-3p, hsa-miR-3622a-5p, hsa-miR-5582-5p, hsa-miR-124-3p, hsa-miR-5(...)hsa-miR-186-5p, hsa-miR-1914-3p, hsa-miR-200a-3p, hsa-miR-204-5p, hsa-miR-211-5p, hsa-miR-23a-5p, hsa-miR-23b-5p, hsa-miR-3120-3p, hsa-miR-3122, hsa-miR-3182, hsa-miR-3199, hsa-miR-335-3p, hsa-miR-3681-5p, hsa-miR-3913-5p, hsa-miR-3925-3p, hsa-miR-4284, hsa-miR- 4469, hsa-miR-450b-5p, hsa-miR-4640-5p, hsa-miR-4649-3p, hsa-miR-4650-3p, hsa-miR-4667-3p, hsa-miR-4677-5p, hsa-miR-4693-3p, hsa-mi R-4701-3p, hsa-miR-4726-5p, hsa-miR-4763-3p, hsa-miR-4768-5p, hsa-miR-4771, hsa-miR-4775, hsa-miR-4776-3p, hsa-miR-4797-3p, hsa-miR- 485-5p, hsa-miR-5002-3p, hsa-miR-5008-5p, hsa-miR-5089-5p, hsa-miR-513b-5p, hsa-miR-5190, hsa-miR-5191, hsa-miR-5193, hsa-miR-5194, hsa-miR-543, hsa-miR-545-3p, hsa-miR-548a-3p, hsa-miR-548aa, hsa-miR-548an, hsa-miR-548ap-3p, hsa-miR-548ar-3p, hsa-miR-548az-3p, hs a-miR-548bc, hsa-miR-548e-3p, hsa-miR-548n, hsa-miR-548t-3p, hsa-miR-589-3p, hsa-miR-6730-5p, hsa-miR-6736-5p, hsa-miR-6738-5p, hsa -miR-6740-5p, hsa-miR-676-3p, hsa-miR-6770-5p, hsa-miR-6826-3p, hsa-miR-6831-5p, hsa-miR-6833-3p, hsa-miR-6845-3p, hsa-miR-6870-3phsa-miR-6875-3p, hsa-miR-6878-3p, hsa-miR-6884-3p, hsa-miR-7108-3p, hsa-miR-7974, hsa-miR-802, hsa-miR-939-5p, hsa-let-7f-2-3p, hsa- miR-11181-3p, hsa-miR-1185-1-3p, hsa-miR-1185-2-3p, hsa-miR-1197, hsa-miR-12129, hsa-miR-12136, hsa-miR-1228-3p, hsa-miR-124-3p, hsa- miR-1264, hsa-miR-1265, hsa-miR-1276, hsa-miR-1288-5p, hsa-miR-1290, hsa-miR-1299, hsa-miR-130a-5p, hsa-miR-135a-2-3p, hsa-miR-135b-3 p, hsa-miR-138-1-3p, hsa-miR-145-5p, hsa-miR-15b-3p, hsa-miR-16-1-3p, hsa-miR-181a-5p, hsa-miR-181b-5p, hsa-miR-181c-5p, hsa-miR-181d -5p, hsa-miR-187-3p, hsa-miR-194-5p, hsa-miR-196a-3p, hsa-miR-196b-3p, hsa-miR-197-3p, hsa-miR-19a-5p, hsa-miR-19b-1-5p, hsa-miR-216 b-5p, hsa-miR-2276-3p, hsa-miR-2276-5p, hsa-miR-2278, hsa-miR-2355-3p, hsa-miR-27a-5p, hsa-miR-29a-5p, hsa-miR-3064-5p, hsa-miR-3085- 3p, hsa-miR-3125, hsa-miR-3127-3p, hsa-miR-3135a, hsa-miR-3143, hsa-miR-3150a-3p, hsa-miR-3154, hsa-miR-3158-3p, hsa-miR-3159, hsa-miR -3160-5p, hsa-miR-3163, hsa-miR-3184-3p, hsa-miR-3189-5p, hsa-miR-3191-5p, hsa-miR-3202, hsa-miR-331-3p, hsa-miR-342-3p, hsa-miR-346hsa-miR-3605-3p, hsa-miR-3663-5p, hsa-miR-3677-5p, hsa-miR-372-5p, hsa-miR-376b-5p, hsa-miR-378a-5p, hsa-miR-3916, hsa-miR-3929, hsa-miR-3938, hsa-miR-424-3p, hsa-miR-4254, hsa-miR-4282, hsa-miR-4323, hsa-miR-4428, hsa-miR-4443, hsa-miR-4451, hsa-miR-4476, hsa-miR -4478, hsa-miR-4502, hsa-miR-4503, hsa-miR-4515, hsa-miR-4679, hsa-miR-4694-5p, hsa-miR-4699-3p, hsa-miR-4729, hsa-miR-4738-3p, hsa- miR-4739, hsa-miR-4745-5p, hsa-miR-4755-5p, hsa-miR-4756-5p, hsa-miR-4766-5p, hsa-miR-4773, hsa-miR-4781-3p, hsa-miR-4803, hsa-miR-4 88-3p, hsa-miR-498-5p, hsa-miR-5001-3p, hsa-miR-5006-3p, hsa-miR-5008-3p, hsa-miR-505-3p, hsa-miR-506-3p, hsa-miR-506-5p, hsa-miR-5 088-3p, hsa-miR-5089-3p, hsa-miR-5094, hsa-miR-5189-3p, hsa-miR-5195-3p, hsa-miR-539-5p, hsa-miR-548a-5p, hsa-miR-548ab, hsa-miR-548 ad-5p, hsa-miR-548ae-5p, hsa-miR-548ag, hsa-miR-548ai, hsa-miR-548ak, hsa-miR-548am-5p, hsa-miR-548ap-5p, hsa-miR-548aq-5p, hsa-miR-548ar-5p, hsa-miR-548as-5p, hsa-miR-548au-5p, hsa-miR-548ay-5p, hsa-miR-548az-5p, hsa-miR-548b-5p, hsa-miR-548ba, hsa-miR-548bb-5phsa-miR-548c-5p、hsa-miR-548d-5p、hsa-miR-548g-3p、hsa-miR-548h- 5p、hsa-miR-548i、hsa-miR-548j-5p、hsa-miR-548m、hsa-miR-548o-5p、h sa-miR-548p, hsa-miR-548t-5p, hsa-miR-548v, hsa-miR-548w, hsa-miR-548y, hsa-miR-5586-5p, hsa-miR-559, hsa-miR-5591-5p, hsa-miR-5693, hsa-miR-570-5p, hsa-miR-578, hsa-miR-596, hsa-miR-601, hsa-miR-6081, hsa-miR-643, hsa-miR-6501-3p, hsa-miR-6504-5p, hsa-miR-6511a-3p hsa-miR-6511b-3p hsa-miR-6513-5p hsa-miR-660-3p hsa-miR-661 hsa-miR-663b hsa-miR-664a-3p hsa-miR-6726-5p hsa-miR-6734-5p hs a-miR-6736-3p, hsa-miR-6737-3p, hsa-miR-6738-3p, hsa-miR-6739-3p, hsa-miR-6742-3p, hsa-miR-6746-3p, hsa-miR-6756-3p, hsa-miR-676-5p, hsa-miR-6761-5p, hsa-miR-6763-3p, hsa-miR-6763-5p, hsa-miR-6791-3p, hsa-miR-6796-3p, hsa-miR-6818-3p, hsa-miR-6825-5p, hsa-miR-68 29-3p, hsa-miR-6833-5p, hsa-miR-6843-3p, hsa-miR-6848-3p, hsa-miR-6854-5p, hsa-miR-6859-5p, hsa-miR-6876-5p, hsa-miR-6894-5p, hsa-mi R-6895-3p, hsa-miR-7114-5p, hsa-miR-7157-3p, hsa-miR-765, hsa-miR-766-5p, hsa-miR-7852-3p, hsa-miR-7977, hsa-miR-7978, hsa-miR-8485hsa-miR-888-5p, hsa-miR-920, hsa-miR-943, hsa-miR-10398-5p, hsa-miR-105-5p, hsa-miR-10a-3p, hsa-miR-12115, hsa-miR-1236-3p, hsa-miR-1253, hsa-miR-125b-2-3p, hsa-miR-1286, hsa-miR-129-5p, hsa-miR-1293, hsa-miR-1302, hsa-miR-1324, hsa-miR-1343-3p, hsa-miR-1470, hsa -miR-147b-5p, hsa-miR-186-3p, hsa-miR-1912-5p, hsa-miR-196a-5p, hsa-miR-196b-5p, hsa-miR-1976, hsa-miR-205-5p, hsa-miR-2116-5p, hsa-miR-22-5p, hsa-miR-221-5p, hsa-miR-24-3p, hsa-miR-2467-5p, hsa-miR-2682-3p, hsa-miR-26b-3p, hsa-miR-30a-3p, hsa-miR-30d-3p, hsa-miR -30e-3p、hsa-miR-31-3p、hsa-miR-3133、hsa-miR-3145-3p、hsa-miR-3152-5p、hsa-miR-3155a、hsa-miR-3155b、hsa-miR-3166、hsa-miR-3171、hs a-miR-3175, hsa-miR-3177-3p, hsa-miR-3184-5p, hsa-miR-3188, hsa-miR-3190-3p, hsa-miR-324-5p, hsa-miR-326, hsa-miR-330-5p, hsa-miR-35 29-5p, hsa-miR-3612, hsa-miR-362-5p, hsa-miR-3621, hsa-miR-363-5p, hsa-miR-3655, hsa-miR-365a-5p, hsa-miR-365b-5p, hsa-miR-3675-5p hsa-miR-3680-3p, hsa-miR-3682-3p, hsa-miR-3682-5p, hsa-miR-3686, hsa-miR-3688-5p, hsa-miR-376a-3p, hsa-miR-376b-3p, hsa-miR-379-5phsa-miR-3925-5p, hsa-miR-3940-3p, hsa-miR-3942-3p, hsa-miR-423-5p, hsa-miR-4273, hsa-miR-4287, hsa-miR-4298, hsa-miR-4457, hsa-miR-44 83, hsa-miR-4484, hsa-miR-450a-1-3p, hsa-miR-4632-3p, hsa-miR-4659a-3p, hsa-miR-4659a-5p, hsa-miR-4659b-3p, hsa-miR-4660, hsa-miR-466 8-3p, hsa-miR-4685-3p, hsa-miR-4685-5p, hsa-miR-4687-3p, hsa-miR-4695-5p, hsa-miR-4707-5p, hsa-miR-4722-5p, hsa-miR-4724-5p, hsa-miR- 4727-3p, hsa-miR-4733-5p, hsa-miR-4736, hsa-miR-4740-3p, hsa-miR-4742-3p, hsa-miR-4757-5p, hsa-miR-4762-3p, hsa-miR-4764-3p, hsa-miR- 4769-3p, hsa-miR-4774-3p, hsa-miR-4779, hsa-miR-4799-5p, hsa-miR-484, hsa-miR-486-5p, hsa-miR-495-3p, hsa-miR-500b-5p, hsa-miR-501-5p 、hsa-miR-507、hsa-miR-518a-5p、hsa-miR-526b-5p、hsa-miR-527、hsa-m iR-542-5p、hsa-miR-548as-3p、hsa-miR-548at-3p、hsa-miR-548aw、hsa-m iR-548ay-3p, hsa-miR-551b-5p, hsa-miR-557, hsa-miR-5582-3p, hsa-miR-5584-5p, hsa-miR-5587-3p, hsa-miR-5590-5p, hsa-miR-5687, hsa-miR- 5688, hsa-miR-572, hsa-miR-576-5p, hsa-miR-590-3p, hsa-miR-6074, hsa-miR-6089, hsa-miR-6128, hsa-miR-623, hsa-miR-629-3p, hsa-miR-650hsa-miR-6508-3p、hsa-miR-6511b-5p、hsa-miR-6515-3p、hsa-miR-6516-5p、hsa-miR-6721-5p、hsa-miR-6728-5p、hsa-miR-6729-3p、hsa-miR-6741-5p、hsa-miR-6745、hsa-miR-6750-5p、hsa-miR-6756-5p、hsa-miR-6764-3p、hsa-miR-6765-5p、hsa-miR-6766-5p、hsa-miR-6768-3p、hsa-miR-6774-5p、hsa-miR-6777-5p、hsa-miR-6781-3p、hsa-miR-6783-3p、hsa-miR-6792-5p、hsa-miR-6795-3p、hsa-miR-6807-5p、hsa-miR-6809-3p、hsa-miR-6811-5p、hsa-miR-6817-5p、hsa-miR-6820-5p、hsa-miR-6823-3p、hsa-miR-6824-3p、hsa-miR-6837-5p、hsa-miR-6844、hsa-miR-6873-3p、hsa-miR-6889-5p、hsa-miR-6890-5p、hsa-miR-6891-3p、hsa-miR-7-1-3p、hsa-miR-7-2-3p、hsa-miR-7113-3p、hsa-miR-7113-5p、hsa-miR-7151-5p、hsa-miR-7156-3p、hsa-miR-7156-5p、hsa-miR-7162-3p、hsa-miR-766-3p、hsa-miR-769-3p、hsa-miR-7853-5p、hsa-miR-8057、hsa-miR-8073、hsa-miR-874-5p、hsa-miR-885-5p、hsa-miR-887-5p、hsa-miR-95-3p、hsa-miR-9500、hsa-miR-9903、hsa-miR-9983-3p、hsa-let-7a-3p、hsa-let-7a-5p、hsa-let-7b-3p、hsa-let-7b-5p、hsa-let-7c-5p、hsa-let-7d-5p、hsa-let-7e-5p、hsa-let-7f-1-3p、hsa-let-7f-5p、hsa-let-7g-5p、hsa-let-7i-5p、hsa-miR-1-3p, hsa-miR-10392-3p, hsa-miR-10526-3p, hsa-miR-106a-5p, hsa-miR-106b-5p, hsa-miR-10b-3p, hsa-miR-1183, hsa-miR-1185-5p, hsa-miR-1199-3p, hsa-miR-1199-5p, hsa-miR-1200, hsa-miR-1208, hsa-miR-12124, hsa-miR-12132, hsa-miR-12135, hsa-miR-122-3p, hsa-miR-1 226-5p、hsa-miR-122b-3p、hsa-miR-122b-5p、hsa-miR-1237-3p、hsa-mi R-1245a、hsa-miR-1245b-5p、hsa-miR-1247-5p、hsa-miR-1248、hsa-miR- 1251-3p, hsa-miR-1252-3p, hsa-miR-125b-1-3p, hsa-miR-1261, hsa-miR-1285-3p, hsa-miR-1288-3p, hsa-miR-1291, hsa-miR-1296-3p, hsa-miR- 1298-5p, hsa-miR-1306-5p, hsa-miR-130b-5p, hsa-miR-132-3p, hsa-miR-1323, hsa-miR-134-3p, hsa-miR-135a-5p, hsa-miR-135b-5p, hsa-miR- 136-3p, hsa-miR-136-5p, hsa-miR-139-5p, hsa-miR-140-3p, hsa-miR-1468-3p, hsa-miR-149-3p, hsa-miR-149-5p, hsa-miR-153-5p, hsa-miR-153 7-3p, hsa-miR-155-5p, hsa-miR-17-5p, hsa-miR-181a-2-3p, hsa-miR-181d-3p, hsa-miR-187-5p, hsa-miR-1909-3p, hsa-miR-191-5p, hsa-miR-1 94-3p, hsa-miR-200c-5p, hsa-miR-202-3p, hsa-miR-203a-3p, hsa-miR-2052, hsa-miR-206, hsa-miR-208b-5p, hsa-miR-20a-5p, hsa-miR-20b-3phsa-miR-20b-5p, hsa-miR-21-3p, hsa-miR-211-3p, hsa-miR-2110, hsa-miR-2115-3p, hsa-miR-212-3p, hsa-miR-216a-5p, hsa-miR-216b-3p, hsa-miR-217-3p, hsa-miR-218-2-3p, hsa-miR-218- 5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-222-5p, hsa-miR-2277-3p, hsa-miR-2392, hsa-miR-25-3p, hsa-miR-27a-3p, hsa-miR-27b-3p, hsa-miR-29b-2-5p, hsa-miR-302a-5p, hsa-miR-30 64-3p, hsa-miR-3074-5p, hsa-miR-3115, hsa-miR-3116, hsa-miR-3130-3p, hsa-miR-3136-3p, hsa-miR-3136-5p, hsa-miR-3137, hsa-miR-3142, hsa-miR-3144-5p, hsa-miR-3148, hsa-miR-3149 hsa-miR-3150b-3p, hsa-miR-3152-3p, hsa-miR-3157-5p, hsa-miR-3160-3p, hsa-miR-3162-5p, hsa-miR-3167, hsa-miR-3173-3p, hsa-miR-3179, hsa-miR-3180-5p, hsa-miR-3187-5p, hsa-miR-3 191-3p、hsa-miR-3194-5p、hsa-miR-32-5p、hsa-miR-3200-3p、hsa-miR- 320a-3p、hsa-miR-320a-5p、hsa-miR-320b、hsa-miR-320c、hsa-miR-320d hsa-miR-324-3p hsa-miR-328-3p hsa-miR-329-3p hsa-miR-330-3p hsa-miR-331-5p hsa-miR-335-5p hsa-miR-33a-3p hsa-miR-340-3p hsa -miR-345-5p, hsa-miR-34a-3p, hsa-miR-34b-3p, hsa-miR-3605-5p, hsa-miR-3609, hsa-miR-361-3p, hsa-miR-3610, hsa-miR-3613-3p, hsa-miR-3 614-3p, hsa-miR-3616-5p, hsa-miR-3617-3p, hsa-miR-362-3p, hsa-miR-3620-3p, hsa-miR-3622a-5p, hsa-miR-363-3p, hsa-miR-365a-3p, hsa-mi R-365b-3p, hsa-miR-3662, hsa-miR-3663-3p, hsa-miR-3671, hsa-miR-3675-3p, hsa-miR-3679-3p, hsa-miR-3679-5p, hsa-miR-3688-3p, hsa-miR -3689a-5p, hsa-miR-3689b-5p, hsa-miR-3689e, hsa-miR-3689f, hsa-miR-369-3p, hsa-miR-3690, hsa-miR-3691-5p, hsa-miR-370-3p, hsa-miR-37 14, hsa-miR-371a-5p, hsa-miR-371b-5p, hsa-miR-373-5p, hsa-miR-375-3p, hsa-miR-377-3p, hsa-miR-378g, hsa-miR-383-5p, hsa-miR-3910, hsa -miR-3919, hsa-miR-3920, hsa-miR-3928-3p, hsa-miR-3934-3p, hsa-miR-3940-5p, hsa-miR-3943, hsa-miR-409-3p, hsa-miR-421, hsa-miR-4270hsa-miR-4288, hsa-miR-4290, hsa-miR-4292, hsa-miR-4429, hsa-miR-4446-3p, hsa-miR-4456, hsa-miR-4458, hsa-miR-4474-3p, hsa-miR-4475, hsa-miR-4477a, hsa-miR-4496, hsa-miR-4500, hsa-miR-4507, hsa-miR-4516, hsa-miR-4519, hsa-miR-4633-3p, hsa-miR-4639-5p, hsa-miR-4646-3 p, hsa-miR-4652-3p, hsa-miR-4652-5p, hsa-miR-4653-3p, hsa-miR-4661-3p, hsa-miR-4665-3p, hsa-miR-4676-5p, hsa-miR-4677-3p, hsa-miR-46 90-5p, hsa-miR-4691-3p, hsa-miR-4709-5p, hsa-miR-4711-3p, hsa-miR-4712-3p, hsa-miR-4713-5p, hsa-miR-4714-3p, hsa-miR-4715-3p, hsa-mi R-4719, hsa-miR-4720-3p, hsa-miR-4728-3p, hsa-miR-4728-5p, hsa-miR-4731-3p, hsa-miR-4731-5p, hsa-miR-4732-5p, hsa-miR-4734, hsa-miR- 4738-5p, hsa-miR-4740-5p, hsa-miR-4743-3p, hsa-miR-4744, hsa-miR-4747-3p, hsa-miR-4752, hsa-miR-4753-3p, hsa-miR-4755-3p, hsa-miR-47 58-3p, hsa-miR-4763-5p, hsa-miR-4764-5p, hsa-miR-4772-3p, hsa-miR-4772-5p, hsa-miR-4782-5p, hsa-miR-4784, hsa-miR-4788, hsa-miR-4793 -3p、hsa-miR-4793-5p、hsa-miR-4794、hsa-miR-4796-3p、hsa-miR-4797-5p、hsa-miR-4801、hsa-miR-4804-3p、hsa-miR-488-5p、hsa-miR-490-3p、hsa-miR-499b-5p, hsa-miR-5002-5p, hsa-miR-5088-5p, hsa-miR-513a-3p, hsa-miR-513c-3p, hsa-miR-515-5p, hsa-miR-516b-5p, hsa-miR-5189-5p, hsa-miR-5196-3p, hsa-miR-519d-3p, hsa-miR-520g-3p, hsa-miR-520h, hsa-miR-526b-3p, hsa-miR-541-5p, hsa-miR-544b, hsa-miR-548ac hsa-miR-548ad-3p, hsa-miR-548ae-3p, hsa-miR-548ah-3p, hsa-miR-548ah-5p, hsa-miR-548aj-3p, hsa-miR-548aj-5p, hsa-miR-548am-3p, hsa-miR-548aq-3p, hsa-miR-548av-3p, hsa-miR-548bb-3p, hsa-miR-548d-3p, hsa-miR-548e-5p, hsa-miR-548f-5p, hsa-miR-548g-5p, hsa-miR-548h- 3p、hsa-miR-548j-3p、hsa-miR-548k、hsa-miR-548l、hsa-miR-548o-3p、 hsa-miR-548q、hsa-miR-548x-5p、hsa-miR-548z、hsa-miR-550a-3p、hsa- miR-558, hsa-miR-5582-5p, hsa-miR-5680, hsa-miR-5690, hsa-miR-5692a, hsa-miR-5692c, hsa-miR-5696, hsa-miR-5699-5p, hsa-miR-5702, hsa-miR-5703, hsa-miR-5706, hsa-miR-573, hsa-miR-579-3p, hsa-miR-580-3p, hsa-miR-583, hsa-miR-603, hsa-miR-6090, hsa-miR-612, hsa-miR-61 5-5p, hsa-miR-616-3p, hsa-miR-616-5p, hsa-miR-624-5p, hsa-miR-627-3p, hsa-miR-629-5p, hsa-miR-632, hsa-miR-642a-3p, hsa-miR-642a-5phsa-miR-642b-3p, hsa-miR-642b-5p, hsa-miR-647, hsa-miR-649, hsa-miR-6499-3p, hsa-miR-6505-5p, hsa-miR-6507-3p, hsa-miR-6507-5p, hsa-m iR-651-3p, hsa-miR-6510-3p, hsa-miR-6510-5p, hsa-miR-6512-3p, hsa-miR-6515-5p, hsa-miR-6529-3p, hsa-miR-656-3p, hsa-miR-664a-5p, hsa- miR-664b-3p, hsa-miR-6715a-3p, hsa-miR-6720-5p, hsa-miR-6722-3p, hsa-miR-6724-5p, hsa-miR-6727-5p, hsa-miR-6735-3p, hsa-miR-6749-3p hsa-miR-6751-3p, hsa-miR-6754-5p, hsa-miR-6757-3p, hsa-miR-6758-3p, hsa-miR-6758-5p, hsa-miR-6760-3p, hsa-miR-6760-5p, hsa-miR-6767- 3p, hsa-miR-6769a-3p, hsa-miR-6771-3p, hsa-miR-6773-5p, hsa-miR-6774-3p, hsa-miR-6775-3p, hsa-miR-6778-3p, hsa-miR-6782-5p, hsa-miR-6 783-5p, hsa-miR-6784-3p, hsa-miR-6785-5p, hsa-miR-6787-3p, hsa-miR-6791-5p, hsa-miR-6792-3p, hsa-miR-6798-5p, hsa-miR-6799-5p, hsa-mi R-6800-3p, hsa-miR-6801-3p, hsa-miR-6810-3p, hsa-miR-6810-5p, hsa-miR-6813-3p, hsa-miR-6827-3p, hsa-miR-6827-5p, hsa-miR-6830-3p, hsa -miR-6830-5p、hsa-miR-6832-5p、hsa-miR-6834-5p、hsa-miR-6836-3p、hsa-miR-6836-5p、hsa-miR-6839-3p、hsa-miR-6842-3p、hsa-miR-6851-5p、hsa-miR-6852-5p, hsa-miR-6856-3p, hsa-miR-6856-5p, hsa-miR-6857-5p, hsa-miR-6858-3p, hs a-miR-6860, hsa-miR-6861-5p, hsa-miR-6862-3p, hsa-miR-6865-3p, hsa-miR-6868-3p, hsa-miR -6885-3p, hsa-miR-6889-3p, hsa-miR-6890-3p, hsa-miR-6891-5p, hsa-miR-6892-3p, hsa-miR-6 893-3p, hsa-miR-7108-5p, hsa-miR-7109-3p, hsa-miR-7152-3p, hsa-miR-7152-5p, hsa-miR-7154 -3p, hsa-miR-7155-3p, hsa-miR-718, hsa-miR-758-5p, hsa-miR-7704, hsa-miR-8062, hsa-miR-8 73-5p, hsa-miR-876-5p, hsa-miR-877-3p, hsa-miR-889-5p, hsa-miR-892a, hsa-miR-892c-5p, hsa -miR-9-3p, hsa-miR-92a-3p, hsa-miR-92b-3p, hsa-miR-93-5p, hsa-miR-939-3p, hsa-miR-98-3p, hsa-miR-98-5p, hsa-miR-9985, hsa-miR-99a-3p, hsa-miR-99b-3p, or selected from the group consisting of these isomiRs.

[0140] In one embodiment, the microRNA is miR-124-3p or its isomiR.

[0141] In one embodiment, the microRNA contains a sequence selected from the group consisting of SEQ ID NOs: 9-60 or SEQ ID NOs: 61-944.

[0142] In one embodiment, the microRNA consists of a sequence selected from the group consisting of SEQ ID NOs: 9-60 or SEQ ID NOs: 61-944.

[0143] MicroRNAs are listed in Table 1 below. Table 1 - Presumed Fan1-targeted microRNAs [Table 1] TIFF2026528923000010.tif236170TIFF2026528923000011.tif235170TIFF2026528923000012.t if235170TIFF2026528923000013.tif236170TIFF2026528923000014.tif235170TIFF20265289230 00015.tif236170TIFF2026528923000016.tif236170TIFF2026528923000017.tif235170TIFF202 6528923000018.tif236170TIFF2026528923000019.tif236170TIFF2026528923000020.tif235170 TIFF2026528923000021.tif235170TIFF2026528923000022.tif235170TIFF2026528923000023.t if235170TIFF2026528923000024.tif236170TIFF2026528923000025.tif236170TIFF20265289230 00026.tif235170TIFF2026528923000027.tif235170TIFF2026528923000028.tif235170TIFF202 6528923000029.tif236170TIFF2026528923000030.tif236170TIFF2026528923000031.tif251170

[0144] The seed sequences shown in Table 1 represent the 2nd to 7th nucleotides of the miRNA (read from 5' to 3'). The "target sequence" corresponds to the sequence to which the miRNA binds, e.g., the Fan1 sequence, and will be understood to represent the reverse complement of the sequence identified as the "seed sequence". Thus, both the seed sequence and the target sequence are encompassed by this invention.

[0145] In one embodiment, the functional nucleic acid molecule of the present invention competes for binding to Fan1 mRNA with one or more of the microRNAs specified in Table 1, or their isomiR.

[0146] In one embodiment, the consecutive nucleotide sequence of the functional nucleic acid of the present invention is complementary to the consecutive nucleotide sequence in the 3'UTR of Fan1 that overlaps with one or more of the microRNAs specified in Table 1, or the nucleotide sequence bound by its isomiR (i.e., the target sequence).

[0147] In one embodiment, one or more consecutive nucleotide sequences are independently at least 80% complementary to one or more microRNAs as defined in Table 1, or consecutive nucleotide sequences within their isomiR, for example, at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary.

[0148] In one embodiment, one or more consecutive nucleotide sequences are independently at least 80% complementary to one or more microRNAs specified in Table 1, or consecutive nucleotide sequences within their isomiR.

[0149] In one embodiment, one or more consecutive nucleotide sequences are independently at least 85% complementary to one or more microRNAs specified in Table 1, or consecutive nucleotide sequences within their isomiR.

[0150] In one embodiment, one or more consecutive nucleotide sequences are independently at least 90% complementary to one or more microRNAs specified in Table 1, or consecutive nucleotide sequences within their isomiR.

[0151] In one embodiment, one or more consecutive nucleotide sequences are independently at least 95% complementary to one or more microRNAs specified in Table 1, or consecutive nucleotide sequences within their isomiR.

[0152] In one embodiment, one or more consecutive nucleotide sequences are independently 100% complementary to one or more microRNAs specified in Table 1, or consecutive nucleotide sequences within their isomiR.

[0153] In one embodiment, one or more consecutive nucleotide sequences are independently at least 80% complementary to consecutive nucleotide sequences in SEQ ID NOs. 9-60 and / or SEQ ID NOs. 61-944, for example, at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary.

[0154] In one embodiment, one or more consecutive nucleotide sequences are independently at least 80% complementary to consecutive nucleotide sequences in SEQ ID NOs. 9-60 and / or SEQ ID NOs. 61-944.

[0155] In one embodiment, one or more consecutive nucleotide sequences are independently at least 85% complementary to consecutive nucleotide sequences in SEQ ID NOs. 9-60 and / or SEQ ID NOs. 61-944.

[0156] In one embodiment, one or more consecutive nucleotide sequences are independently at least 90% complementary to consecutive nucleotide sequences in SEQ ID NOs: 9-60 and / or SEQ ID NOs: 61-944.

[0157] In one embodiment, one or more consecutive nucleotide sequences are independently at least 95% complementary to consecutive nucleotide sequences in SEQ ID NOs: 9-60 and / or SEQ ID NOs: 61-944.

[0158] In one embodiment, one or more consecutive nucleotide sequences are independently 100% complementary to consecutive nucleotide sequences in SEQ ID NOs: 9-60 and / or SEQ ID NOs: 61-944.

[0159] In one embodiment, the functional nucleic acid molecule binds to one or more microRNAs specified in Table 1, or their isomiR, thereby preventing binding to Fan1 mRNA.

[0160] In one embodiment, the functional nucleic acid molecule comprises one or more consecutive nucleotide sequences, wherein the one or more consecutive nucleotide sequences are complementary to a consecutive nucleotide sequence in the 3'UTR of Fan1 that overlaps with one or more microRNAs specified in Table 1, or the nucleotide sequence bound by its isomiR.

[0161] In one embodiment, one or more consecutive nucleotide sequences are independently complementary to one or more microRNAs specified in Table 1, or to a consecutive nucleotide sequence in the 3'UTR of Fan1 that overlaps with one nucleotide in the nucleotide sequence bound by its isomiR.

[0162] In one embodiment, one or more consecutive nucleotide sequences are independently complementary to one or more microRNAs specified in Table 1, or to two nucleotides in the nucleotide sequence bound by its isomiR, and to consecutive nucleotide sequences in the 3'UTR of Fan1.

[0163] In one embodiment, one or more consecutive nucleotide sequences are independently complementary to one or more microRNAs specified in Table 1, or to a consecutive nucleotide sequence in the 3'UTR of Fan1 that overlaps with three nucleotides in the nucleotide sequence bound by its isomiR.

[0164] In one embodiment, one or more consecutive nucleotide sequences are independently complementary to one or more microRNAs specified in Table 1, or to a consecutive nucleotide sequence in the 3'UTR of Fan1 that overlaps with four nucleotides in the nucleotide sequence bound by its isomiR.

[0165] In one embodiment, one or more consecutive nucleotide sequences are independently complementary to one or more microRNAs specified in Table 1, or to a consecutive nucleotide sequence in the 3'UTR of Fan1 that overlaps with five nucleotides in the nucleotide sequence bound by its isomiR.

[0166] In one embodiment, one or more consecutive nucleotide sequences are independently complementary to a consecutive nucleotide sequence in the 3'UTR of Fan1 that overlaps with one or more microRNAs specified in Table 1, or the nucleotide sequence bound by its isomiR.

[0167] In one embodiment, one or more consecutive nucleotide sequences are independently complementary to a consecutive nucleotide sequence in the 3'UTR of Fan1 that overlaps with seven nucleotides in a nucleotide sequence bound by one or more microRNAs specified in Table 1, or its isomiR.

[0168] In one embodiment, one or more consecutive nucleotide sequences are independently complementary to a consecutive nucleotide sequence in the 3'UTR of Fan1 that overlaps with all the nucleotides in one or more microRNAs specified in Table 1, or the nucleotide sequence bound by its isomiR.

[0169] The endogenous microRNA miR-124-3p targets Fan1. The sequence of miR-124-3p is shown in Sequence ID No. 59.

[0170] In one embodiment, one or more consecutive nucleotide sequences are independently at least 80% complementary to consecutive nucleotide sequences within the miR-124-3p microRNA, for example, at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary.

[0171] In one embodiment, one or more consecutive nucleotide sequences are independently at least 80% complementary to consecutive nucleotide sequences within the miR-124-3p microRNA.

[0172] In one embodiment, one or more consecutive nucleotide sequences are independently at least 85% complementary to consecutive nucleotide sequences within the miR-124-3p microRNA.

[0173] In one embodiment, one or more consecutive nucleotide sequences are independently at least 90% complementary to consecutive nucleotide sequences within the miR-124-3p microRNA.

[0174] In one embodiment, one or more consecutive nucleotide sequences are independently at least 95% complementary to consecutive nucleotide sequences within the miR-124-3p microRNA.

[0175] In one embodiment, one or more consecutive nucleotide sequences are 100% complementary to consecutive nucleotide sequences within the miR-124-3p microRNA.

[0176] In one embodiment, one or more consecutive nucleotide sequences are independently at least 80% complementary to the consecutive nucleotide sequences in Sequence ID No. 59, for example, at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary.

[0177] In one embodiment, one or more consecutive nucleotide sequences are independently at least 80% complementary to the consecutive nucleotide sequences in SEQ ID NO: 59.

[0178] In one embodiment, one or more consecutive nucleotide sequences are independently at least 85% complementary to the consecutive nucleotide sequences in SEQ ID NO: 59.

[0179] In one embodiment, one or more consecutive nucleotide sequences are independently at least 90% complementary to the consecutive nucleotide sequences in SEQ ID NO: 59.

[0180] In one embodiment, one or more consecutive nucleotide sequences are independently at least 95% complementary to the consecutive nucleotide sequences in SEQ ID NO: 59.

[0181] In one embodiment, one or more consecutive nucleotide sequences are 100% complementary to the consecutive nucleotide sequences in Sequence ID No. 59.

[0182] In one embodiment, the functional nucleic acid molecule competes with the microRNA miR-124-3p for binding to Fan1 mRNA.

[0183] In one embodiment, the functional nucleic acid molecule binds to the microRNA miR-124-3p and prevents it from binding to Fan1 mRNA.

[0184] In one embodiment, the functional nucleic acid molecule comprises one or more consecutive nucleotide sequences, wherein the one or more consecutive nucleotide sequences are complementary to consecutive nucleotide sequences in the 3'UTR of Fan1 that overlap with the nucleotide sequence bound by the microRNA miR-124-3p.

[0185] In one embodiment, one or more consecutive nucleotide sequences are independently complementary to consecutive nucleotide sequences in the 3'UTR of Fan1 that overlap with one nucleotide in the nucleotide sequence bound by the microRNA miR-124-3p.

[0186] In one embodiment, one or more consecutive nucleotide sequences are independently complementary to consecutive nucleotide sequences in the 3'UTR of Fan1 that overlap with two nucleotides in the nucleotide sequence bound by microRNA miR-124-3p.

[0187] In one embodiment, one or more consecutive nucleotide sequences are independently complementary to consecutive nucleotide sequences in the 3'UTR of Fan1 that overlap with three nucleotides in the nucleotide sequence bound by microRNA miR-124-3p.

[0188] In one embodiment, one or more consecutive nucleotide sequences are independently complementary to consecutive nucleotide sequences in the 3'UTR of Fan1 that overlap with four nucleotides in the nucleotide sequence bound by microRNA miR-124-3p.

[0189] In one embodiment, one or more consecutive nucleotide sequences are independently complementary to consecutive nucleotide sequences in the 3'UTR of Fan1 that overlap with five nucleotides in the nucleotide sequence bound by the microRNA miR-124-3p.

[0190] In one embodiment, one or more consecutive nucleotide sequences are independently complementary to consecutive nucleotide sequences in the 3'UTR of Fan1 that overlap with six nucleotides in the nucleotide sequence bound by microRNA miR-124-3p.

[0191] In one embodiment, one or more consecutive nucleotide sequences are independently complementary to consecutive nucleotide sequences in the 3'UTR of Fan1 that overlap with seven nucleotides in the nucleotide sequence bound by microRNA miR-124-3p.

[0192] In one embodiment, one or more consecutive nucleotide sequences are independently complementary to consecutive nucleotide sequences in the 3'UTR of Fan1 that overlap with all the nucleotides in the nucleotide sequence bound by the microRNA miR-124-3p.

[0193] The intention behind the duplication is that any given nucleotide bound by a specific miRNA will also be bound by a functional nucleic acid.

[0194] (chemical modification) The functional nucleic acid molecules provided herein include or may consist of chemically modified substituents, that is, substituents that differ from those found in typical naturally occurring DNA and RNA nucleotides.

[0195] Chemically modified functional nucleic acid molecules contain one or more modifications compared to typical naturally occurring (i.e., "unmodified") functional nucleic acid molecules consisting of DNA and RNA nucleotides. These modified functional nucleic acid molecules contain one or more modified nucleosides and / or independently, one or more modified nucleoside bonds.

[0196] The terms “modification” or “chemical modification” refer to structural changes in or on the most common natural ribonucleotides: adenosine, guanosine, cytidine, thymidine, or uridine ribonucleotides. In particular, the chemical modifications described herein may be changes in or on nucleobases (i.e., chemical base modifications) or changes in or on sugars (i.e., chemical sugar modifications). Chemical modifications may be introduced transcriptionally (e.g., by substitution of one or more nucleotides with the modified nucleotide during synthesis), post-transcriptionally (e.g., by enzymatic action), or through chemical (i.e., non-transcriptional) oligonucleotide synthesis.

[0197] Chemical modifications typically involve the chemical modification of nucleobases containing nucleosides, the chemical modification of sugar moieties containing oligonucleotides, and / or the formation of modified nucleoside bonds that connect nucleosides (which themselves may be modified or unmodified).

[0198] In one embodiment, the functional nucleic acid molecule comprises DNA nucleotides, RNA nucleotides, modified DNA nucleotides, and / or modified RNA nucleotides. In another embodiment, the functional nucleic acid comprises DNA and / or RNA analogs.

[0199] In one embodiment, the functional nucleic acid molecule includes a DNA nucleoside, an RNA nucleoside, a modified DNA nucleoside, and / or a modified RNA nucleoside.

[0200] It will be understood that the functional RNA molecules or functional DNA molecules of the present invention may refer to functional RNA / DNA containing only unmodified RNA / DNA nucleotides; functional RNA / DNA containing both modified and unmodified RNA / DNA nucleotides; and functional RNA / DNA containing fully modified RNA / DNA nucleotides. Therefore, references to RNA or DNA shall include references to the modified RNA / DNAs according to the present invention.

[0201] In one embodiment, the functional nucleic acid molecule includes one or more chemical modifications of a nucleobase containing a nucleoside.

[0202] In one embodiment, the functional nucleic acid molecule includes one or more chemical modifications of a sugar moiety containing a nucleoside.

[0203] In one embodiment, the functional nucleic acid molecule includes one or more chemical modifications of the internucleoside bonds that connect nucleosides.

[0204] In one embodiment, the functional nucleic acid molecule includes one or more modified nucleobases, a modified sugar moiety, and / or modified nucleoside bonds.

[0205] In one embodiment, the functional nucleic acid molecule contains 1 to 40 modified nucleosides.

[0206] In one embodiment, the functional nucleic acid molecule contains one or more modified nucleosides.

[0207] In one embodiment, the functional nucleic acid molecule does not contain modified nucleosides.

[0208] In one embodiment, the functional nucleic acid molecule contains one or more modified nucleosides, for example, 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, or 40 nucleosides.

[0209] In one embodiment, a functional nucleic acid molecule comprises a modified nucleoside, where at least 5%, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% of the nucleoside is a modified nucleoside.

[0210] Chemical modifications are publicly known in the art and are listed, for example, in The RNA Modification Database provided by The RNA Institute (https: / / mods.rna.albany.edu / mods / ).

[0211] Exemplary chemical modifications are described in detail elsewhere, including, but are not limited to, WO 2023 / 092057, WO 2023 / 064707, and WO 2023 / 023550, which are incorporated herein by reference.

[0212] (Modified nucleobases) In one embodiment, the chemical modification is a chemical base modification, i.e., a modification of the nucleobase moiety. The chemical base modification may be selected from modifications of adenine, cytosine, and / or uracil nucleobases.

[0213] In one embodiment, the modified nucleobase is modified by alkylation, for example, methylation and / or isomerization.

[0214] In some embodiments, a modified nucleobase refers to a nucleoside that has been modified to lack a nucleobase. Therefore, in some embodiments, a modified nucleoside is a debased nucleoside.

[0215] The modified nucleobase can be selected from the group consisting of 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl-substituted pyrimidines, alkyl-substituted purines, and N-2, N-6, and O-6-substituted purines.

[0216] Modified nucleobases include 2-aminopropyladenine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azocymine, 5-ribosyluracil (pseudracil), 4-thiouracil, 8-halo, 8-amino, and 8-thiol The following can be selected from the group consisting of 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, in particular 5-bromo, 5-trifluoromethyl, 5-halouracil and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-extended bases and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxadin-2-one, 1,3-diazaphenothiazine-2-one, and 9-(2-aminoethoxy)-1,3-diazaphenoxadin-2-one (G-clamp). Modified nucleobases may also include those in which a purine or pyrimidine base is replaced by another heterocycle, such as 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.

[0217] Further nucleobases that may be known to those skilled in the art.

[0218] In a further embodiment, the chemical base modification is selected from the group consisting of pseudouridine (Ψ), N1-methylpseudridine (N1mΨ), 5-methylcytidine (m5C), and N6-methyladenosine (m6A). In a further embodiment, the chemical base modification is selected from the group consisting of pseudouridine, N1-methylpseudridine, and N6-methyladenosine.

[0219] (modified sugar moiety) In one embodiment, the chemical modification is a chemical sugar modification, that is, a modification of the sugar portion.

[0220] The modified sugar moiety contains a modified ribose ring structure compared to unmodified or naturally occurring ribose sugars (i.e., deoxy and non-deoxy) found in DNA or RNA.

[0221] Sugars can be modified, for example, by substitution with a hexose ring (HNA) or a bicyclic ring (where the bicyclic ring has a bridge between the C2 and C4 carbon atoms on the ribose ring and can form locked nucleic acid (LNA)).

[0222] Sugar modifications also include modifications performed by changing substituents on the ribose ring to groups other than naturally occurring hydrogen or 2'-OH groups in DNA and RNA nucleosides. Substituents may be introduced, for example, at the 2', 3', 4', or 5' positions.

[0223] In some embodiments, the modified sugar moiety is a non-bicyclic modified sugar moiety.

[0224] In some embodiments, the modified sugar moiety is a bicyclic or tricyclic sugar moiety.

[0225] In further embodiments, the modified sugar moiety is a sugar substitute (i.e., non-sugar) or a non-ribose sugar, which may optionally include one or more substitutions as described for other types of modified sugar moieties.

[0226] In some embodiments, the modified sugar moiety is a non-bicyclic modified sugar moiety comprising a furanosyl ring having one or more substituents, none of which bridge two atoms of the furanosyl ring to form a bicyclic structure. Such non-bridged substituents may be at any position of the furanosyl ring, including but not limited to substituents at the 2', 3', 4', and / or 5' positions. Examples of suitable 2'- substituents for the non-bicyclic modified sugar moiety include, but are not limited to, 2'-F, 2'-OCH3 ("OMe" or "O-methyl"), and 2'-O(CH2)2OCH3 ("MOE" or "O-methoxyethyl"), and 2'-ON-alkylacetamides, such as 2'-ON-methylacetamide ("NMA"), 2'-ON-dimethylacetamide, 2'-ON-ethylacetamide, or 2'-ON-propylacetamide.

[0227] It will be understood that the modified sugar moiety may be further defined by its isomer configuration in relation to specific modifications. Therefore, isomer variants of the modified sugar moiety may also be used within the functional nucleic acid molecules described herein.

[0228] In one embodiment, the modification is a 2' modification, which is a chemical sugar modification.

[0229] 2' sugar modifications produce nucleosides in which substituents other than H or -OH are located at the 2' position (i.e., 2'-substituted). These 2'-modified sugars include 2'-linked biradicals that can form a bridge between the 2' carbon and the second carbon in the ribose ring, thereby forming LNAs (2'-4' biradical bridges).

[0230] In some embodiments, the 2' sugar modification is independently selected from the group consisting of 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA, and 2'-F-ANA nucleoside.

[0231] In a preferred embodiment, the chemical sugar modification is a 2'-O-methyl (2-OMe) modification.

[0232] In a further embodiment, the chemical sugar modification is 2'-O-methyladenosine (Am).

[0233] In a further embodiment, the chemical sugar modification is a 2'-deoxy sugar.

[0234] In a further embodiment, the chemical sugar modification is 2'-methoxyethyl sugar (2'-MOE).

[0235] In a further embodiment, the chemical sugar modification is 2'-fluoride modification (2'-F).

[0236] In a further embodiment, the modified nucleoside is a bicyclic sugar moiety.

[0237] In one embodiment, the modified nucleoside is a locked nucleic acid (LNA).

[0238] In one embodiment, the modified nucleoside is a 2'-O-ethyl (cET) nucleotide.

[0239] (Inter-modified nucleoside bonding) The functional nucleic acid molecule of the present invention may contain one or more modified nucleoside bonds.

[0240] It will be understood that the modified nucleoside bond is a bond other than a phosphodiester bond, and that it functions similarly to a phosphodiester bond in order to covalently link two nucleosides.

[0241] Modified nucleoside bonds can confer advantageous properties to functional nucleic acid molecules, such as increased resistance to nucleases. These properties can be used, for example, to improve the in vitro or in vivo half-life of functional nucleic acid molecules.

[0242] In one embodiment, the modified nucleoside bond is an analogue of a phosphodiester bond.

[0243] In one embodiment, the bond between modified nucleosides is a phosphonoacetate (PACE) bond.

[0244] In one embodiment, the bond between modified nucleosides is a phosphoramidite bond.

[0245] In one embodiment, the modified nucleoside bond is a phosphorothioate bond or a variant or derivative thereof.

[0246] In one embodiment, the bond between modified nucleosides is a diphosphorothioate bond.

[0247] In one embodiment, the modified nucleoside bond is an alkyl, aryl, or heteroarylphosphorothioate bond.

[0248] In one embodiment, the bond between modified nucleosides is a methyl phosphorothioate bond.

[0249] In one embodiment, the bond between modified nucleosides is a boranophosphate bond.

[0250] In one embodiment, the modified nucleoside bond is a phosphonate bond or a variant or derivative thereof.

[0251] In one embodiment, the modified nucleoside bond is an alkyl, aryl, or heteroarylphosphonate bond.

[0252] In one embodiment, the bond between modified nucleosides is a methylphosphonate bond.

[0253] In one embodiment, the bond between modified nucleosides is a phosphorylguanidine bond.

[0254] In one embodiment, each nucleoside bond in the functional nucleic acid is independently selected from the group consisting of: phosphodiester bonds; phosphonoacetate (PACE) bonds; phosphoramidite bonds; phosphorothioate bonds; diphosphorothioate bonds; alkylphosphorothioate bonds; arylphosphorothioate bonds; heteroarylphosphorothioate bonds; boranophosphate bonds; phosphonate bonds; alkylphosphonate bonds; arylphosphonate bonds; heteroarylphosphonate bonds; methylphosphonate bonds; and phosphorylguanidine bonds, and / or their variants or derivatives.

[0255] In one embodiment, the modified nucleoside bond is independently selected from the group consisting of: phosphodiester bond; phosphonoacetate (PACE) bond; phosphoramidite bond; phosphorothioate bond; diphosphorothioate bond; alkylphosphorothioate bond; arylphosphorothioate bond; heteroarylphosphorothioate bond; boranophosphate bond; phosphonate bond; alkylphosphonate bond; arylphosphonate bond; heteroarylphosphonate bond; methylphosphonate bond; and phosphorylguanidine bond, and / or their variants or derivatives.

[0256] In one embodiment, the functional nucleic acid molecule contains one or more modified nucleoside bonds.

[0257] In one embodiment, the functional nucleic acid molecule contains 1 to 39 modified nucleoside bonds.

[0258] In one embodiment, the functional nucleic acid molecule does not contain modified nucleoside bonds.

[0259] In another embodiment, the functional nucleic acid molecule contains one or more modified nucleoside bonds, for example, 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, or 39 modified nucleoside bonds.

[0260] In one embodiment, a functional nucleic acid molecule comprises modified nucleoside bonds, where at least 5%, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% of the nucleoside bonds are modified nucleoside bonds.

[0261] It will be understood that any suitable nucleoside bond known to those skilled in the art may be selected for use in the functional nucleic acid molecules of the present invention. The nucleoside bonds described herein are merely examples and are not intended to be limiting.

[0262] It will also be understood that the choice of any given nucleoside bond is independent of any other choice of nucleoside bond, i.e., any other choice of bond between any other consecutive nucleoside pairs.

[0263] (Nucleotides and nucleic acid analogs) Nucleotides and nucleic acid analogs are compounds that are structurally and / or functionally similar to naturally occurring (or "unmodified") RNA and DNA.

[0264] In one embodiment, the functional nucleic acid molecule contains or consists of nucleotide analogs.

[0265] Nucleotide analogs may include nucleoside analogs in which the sugar portion is replaced by a non-sugar portion, such as peptide nucleic acid (PNA) or morpholino nucleic acid, thereby providing structural similarity to an unmodified nucleoside, but without containing the modified sugar itself.

[0266] In one embodiment, the nucleotide analog is independently selected from one or more of peptide nucleic acid (PNA) nucleotides, morpholino nucleotides, locked nucleic acid (LNA) nucleotides, glycol nucleic acid (GNA) nucleotides, threose nucleic acid (TNA) nucleotides, and / or hexitol nucleic acid (HNA) nucleotides.

[0267] In one embodiment, the nucleotide analog is a morpholino nucleotide.

[0268] In one embodiment, the functional nucleic acid molecule comprises one or more nucleotide analogs.

[0269] In one embodiment, the functional nucleic acid molecule contains 1 to 40 nucleotide analogs.

[0270] In one embodiment, the functional nucleic acid molecule either contains no nucleotide analogs or contains 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, or 40 nucleotide analogs.

[0271] In one embodiment, the functional nucleic acid molecule comprises nucleotide analog binding, wherein at least 5%, such as at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% of the nucleotides are nucleotide analogs.

[0272] (Conjugates and Linkers) In one aspect, there is provided a conjugate comprising a functional nucleic acid molecule of the invention and one or more moieties covalently attached to the functional nucleic acid molecule.

[0273] In one embodiment, the conjugate is a non-nucleotide-based moiety.

[0274] In one embodiment, the conjugate is a polypeptide or polypeptide analog.

[0275] In one embodiment, the conjugate is a polypeptide or protein or a region thereof (e.g., one or more domains), a domain, or a fragment.

[0276] Suitable polypeptides and proteins can be selected from any known source and can include, for example, viral proteins such as viral envelope / capsid proteins; toxins; and cell receptors.

[0277] In one embodiment, the conjugate is an antibody, scFv, nanobody, antibody-based moiety, or a fragment of any of the foregoing.

[0278] In one embodiment, the conjugate is a ligand or receptor, or a fragment thereof, that can bind to the corresponding receptor or ligand. In a preferred embodiment, the ligand and / or receptor is a cell surface ligand / receptor.

[0279] In one embodiment, the conjugate is a carbohydrate portion, for example, an N-acetylglucosamine portion.

[0280] In one embodiment, the conjugate is a lipid portion.

[0281] In one embodiment, the conjugate is a lipophilic portion.

[0282] In one embodiment, the conjugate is a small molecule, such as a small heterocyclic molecule.

[0283] In one embodiment, the conjugate is a small molecule, such as a small heterocyclic molecule.

[0284] Any combination or one or more of the aforementioned conjugates can be independently selected. Furthermore, the conjugates can be covalently bonded to functional nucleic acids at any preferred position within the molecule.

[0285] The conjugate portion can impart desirable properties to functional nucleic acids. For example, the conjugate may improve bioavailability, increase stability, improve cell targeting, and / or improve cell uptake.

[0286] In a particularly preferred embodiment, the conjugate facilitates or improves delivery across the blood-brain barrier.

[0287] In one embodiment, the conjugate is an oligonucleotide or an analogue thereof.

[0288] In a preferred embodiment, the conjugate is a functional nucleic acid molecule according to the invention. Thus, the functional nucleic acid molecules according to the invention can be conjugated together, with or without the use of an intervening linker. In such cases, the aforementioned conjugate moiety can also be considered a linker when used to space apart two or more functional nucleic acid molecules of the invention.

[0289] In one embodiment, the functional nucleic acid molecule comprises a linker. It will be understood that the linker can be used independently of the conjugate moiety discussed above.

[0290] A linker can be any moiety that serves to link, e.g., covalently link, two or more distinct moieties (such as a functional nucleic acid molecule and a conjugate moiety).

[0291] Advantageously, the linker can be cleavable, e.g., by an enzyme, etc., to facilitate temporal and / or spatial control of the moiety bound to the functional nucleic acid molecule. For example, the conjugate moiety can be bound to the functional nucleic acid molecule via a cleavable linker that is cleaved upon contact with a cellular protease to release the functional nucleic acid molecule from the conjugate.

[0292] In one embodiment, the linker is an oligonucleotide or an analogue thereof.

[0293] It will be understood that the functional nucleic acids of the invention can be represented by a basic sequence utilizing the standard IUB / IUPAC nucleic acid codes (e.g., A, T / U, G, C, etc.) to represent nucleotide sequences, or simply a "sequence", and / or both codes where chemical modifications of the components are detailed. When the functional nucleic acids of the invention are represented herein by a basic sequence, this is to allow for any chemical modification of the components as detailed herein.

[0294] (DNA Molecules and Vectors) A further aspect of the present invention provides a DNA molecule encoding a functional nucleic acid molecule of the present invention.

[0295] A further aspect of the present invention specifies an expression vector comprising the DNA molecule.

[0296] Exemplary expression vectors are known in the art and may include, for example, plasmid vectors, viral vectors (e.g., adenoviruses, adeno-associated viruses, retroviruses, or lentiviral vectors), phage vectors, cosmid vectors, and the like. The choice of expression vector may depend on the type of host cell in which it will be used and its intended use. In particular, but not limited to, the following plasmids have been used for the expression of functional nucleic acid molecules: Mammalian expression plasmids: -pCDNA3.1(-) -pDUAL-eGFPΔ (modified from peGFP-C2) Virus vectors: - pAAV (adeno-associated virus vector) -rcLV-TetOne-Puro (Third-generation lentiviral vector) -pLPCX-link (third-generation retrovirus vector)

[0297] In one embodiment, the mammalian expression plasmid is pCDNA3.1(-).

[0298] In another embodiment, the mammalian expression plasmid is pDUAL-eGFPΔ.

[0299] The plasmid of the present invention may include one or more features selected from the list including: a CMV promoter, an H1 promoter, and / or a BGH poly(A) terminator.

[0300] In one embodiment, the viral vector is pAAV.

[0301] In one embodiment, the viral vector is rcLV-TetOne-Puro.

[0302] In one embodiment, the viral vector is a pLPCX-link.

[0303] The vector of the present invention may include one or more features selected from the list including: a CAG promoter, a CMV enhancer, an SV40 late poly(A) terminator, an LTR-TREt (Tre-Tight) promoter, and / or a BGH poly(A) terminator.

[0304] It should be noted that any promoter may be used in the vector. Since the activity of the functional nucleic acids of the present invention is independent of the promoter, they are assumed to function in exactly the same way as those exemplified above.

[0305] (Compositions and Methods) The present invention also relates to compositions comprising functional nucleic acid molecules, DNA molecules, or expression vectors according to the present invention.

[0306] The composition may contain components that enable the delivery of the functional nucleic acid molecule by viral vectors (such as AAV and lentiviruses) and non-viral vectors (such as nanoparticles and lipid particles). Alternatively, the functional nucleic acid molecule of the present invention may be administered as naked or unpackaged DNA and / or RNA.

[0307] The composition contains components known in the art to help stabilize nucleic acid molecules, such as salts (e.g., Mg 2+ It may include (those that provide ions).

[0308] Functional nucleic acid molecules may be administered as part of a composition, for example, a composition comprising a suitable carrier. In one embodiment, the carrier is selected based on its ability to facilitate the transfection of target cells with one or more functional nucleic acid molecules.

[0309] Therefore, according to further aspects of the present invention, a composition is provided comprising a functional nucleic acid molecule, a DNA molecule, or an expression vector as described herein.

[0310] In one embodiment, a pharmaceutical composition is provided comprising at least one functional nucleic acid molecule, at least one DNA molecule, or at least one expression vector according to the present invention.

[0311] Preferably, the pharmaceutical composition may contain at least one functional nucleic acid molecule, at least one DNA molecule, or at least one expression vector according to the present invention, together with a suitable pharmaceutical excipient, diluent, carrier, and / or salt.

[0312] Suitable excipients, diluents, carriers, and / or salts may depend on the intended route of administration and standard pharmacopoeia.

[0313] Suitable carriers include any standard pharmaceutical carriers, vehicles, diluents, or excipients that are known in the art and are generally intended for use in facilitating the delivery of nucleic acids, such as RNA. Liposomes, exosomes, lipid particles, or nanoparticles are examples of suitable carriers that can be used for RNA delivery. In a preferred embodiment, the carrier or vehicle delivers its contents to a target cell, thereby delivering the functional nucleic acid molecule to a suitable intracellular compartment, such as the cytoplasm.

[0314] (Methods, treatment methods, and medical uses) The functional nucleic acids of the present invention, which can increase Fan1 expression through post-transcriptional inhibition of miRNA-mediated repression of Fan1, can be used for therapeutic and non-therapeutic purposes.

[0315] In one embodiment, an in vitro or in vivo method is provided for regulating Fan1 protein expression in target cells containing Fan1 and Fan1-targeting microRNAs, the method comprising the step of exposing the cells to a functional nucleic acid molecule, conjugate, pharmaceutically acceptable salt, composition, or pharmaceutical composition of the present invention.

[0316] In one embodiment, an in vitro or in vivo method is provided for regulating Fan1 protein expression in target cells containing Fan1 and microRNA miR-124-3p, the method comprising the step of exposing the cells to a functional nucleic acid molecule, conjugate, pharmaceutically acceptable salt, composition, or pharmaceutical composition of the present invention.

[0317] In one embodiment, an in vivo method is provided for regulating Fan1 protein expression in target cells containing Fan1 and Fan1-targeting microRNAs, comprising administering to the cells a therapeutically effective amount of the functional nucleic acid molecule, conjugate, pharmaceutically acceptable salt, composition, or pharmaceutical composition of the present invention.

[0318] One embodiment provides an in vivo method for regulating Fan1 protein expression in target cells containing Fan1 and microRNA miR-124-3p, comprising administering to the cells a therapeutically effective amount of the functional nucleic acid molecule, conjugate, pharmaceutically acceptable salt, composition, or pharmaceutical composition of the present invention.

[0319] In a further embodiment, a method is provided for treating, preventing or delaying the onset of a Fan1 protein-related disease in a subject, comprising administering to the subject a therapeutic or prophylactic effective amount of a functional nucleic acid molecule, conjugate, pharmaceutically acceptable salt, composition, or pharmaceutical composition of the present invention.

[0320] In a further embodiment, functional nucleic acid molecules, conjugates, pharmaceutically acceptable salts, or pharmaceutical compositions of the present invention are provided for use in medicine or therapy.

[0321] The methods and uses described herein are applicable to the treatment, prevention, or delay of the onset of diseases associated with the elongation of disease-related polynucleotide regions.

[0322] In one embodiment, the disease is a triplet repeat disorder.

[0323] In one embodiment, the disease is a disease related to or caused by CAG triplet repeat elongation, CGG triplet repeat elongation, CTG triplet repeat elongation, GAA triplet repeat elongation, GCC triplet repeat elongation, or GCG triplet repeat elongation.

[0324] In one embodiment, the disease is a CAG, CGG, CTG, GAA, GCC, or GCG triplet repeat disorder.

[0325] In one embodiment, the disease is a CGG repeat disorder.

[0326] In one embodiment, the disease is a polyglutamine (polyQ) disease.

[0327] In a further embodiment, the disease is selected from the group consisting of Huntington's disease (HD), spinocerebellar ataxia type 1 (SCA1), spinocerebellar ataxia type 2 (SCA2), spinocerebellar ataxia type 3 (SCA3), spinocerebellar ataxia type 6 (SCA6), spinocerebellar ataxia type 7 (SCA7), spinocerebellar ataxia type 17 (SCA17), dentatorubral-pallidoluysian atrophy (DRPLA), and X-linked spinal muscular atrophy 1 (SMAX1 / SBMA).

[0328] In a preferred embodiment, the disease is Huntington's disease (HD).

[0329] In one embodiment, the disease is a CGG repeat disorder.

[0330] In one embodiment, the CGG repeat failure is a vulnerability X-related failure (FXD).

[0331] In one embodiment, the disease is a CTG repeat disorder.

[0332] In one embodiment, CTG repeat disorder is myoric rigidity dystrophy type 1.

[0333] In one embodiment, the disease is a GAA repeat disorder.

[0334] In one embodiment, the GAA repeat disorder is Friedreich's ataxia.

[0335] In one embodiment, the disease is a GCC repeat disorder.

[0336] In one embodiment, the GCC repeat disorder is FRAXE intellectual disability.

[0337] In one embodiment, the disease is a GCG repeat disorder.

[0338] In one embodiment, GCG repeat disorder is oculopharyngeal muscular dystrophy.

[0339] In one embodiment, the present invention provides functional nucleic acid molecules, conjugates, pharmaceutically acceptable salts, or pharmaceutical compositions for use in the treatment, prevention, or delay of the onset of triplet repeat disorders.

[0340] In one embodiment, the triplet repeat fault is a CAG, CGG, CTG, GAA, GCC, or GCG triplet repeat fault.

[0341] In one embodiment, the present invention provides a functional nucleic acid molecule, conjugate, pharmaceutically acceptable salt, or pharmaceutical composition for use in the treatment, prevention, or delay of the onset of CAG repeat disorders.

[0342] In one embodiment, the present invention provides functional nucleic acid molecules, conjugates, pharmaceutically acceptable salts, or pharmaceutical compositions for use in the treatment, prevention, or delay of the onset of polyglutamine (polyQ) disease.

[0343] In one embodiment, the present invention provides functional nucleic acid molecules, conjugates, pharmaceutically acceptable salts, or pharmaceutical compositions for use in the treatment, prevention, or delay of the onset of diseases selected from the group consisting of Huntington's disease (HD), spinocerebellar ataxia type 1 (SCA1), spinocerebellar ataxia type 2 (SCA2), spinocerebellar ataxia type 3 (SCA3), spinocerebellar ataxia type 6 (SCA6), spinocerebellar ataxia type 7 (SCA7), spinocerebellar ataxia type 17 (SCA17), dentatorubral-pallidoluysian atrophy (DRPLA), and X-linked spinal muscular atrophy 1 (SMAX1 / SBMA).

[0344] In a preferred embodiment, the disease is Huntington's disease (HD).

[0345] In one embodiment, the present invention provides a functional nucleic acid molecule, conjugate, pharmaceutically acceptable salt, or pharmaceutical composition for use in the treatment, prevention, or delay of the onset of CGG repeat disorders.

[0346] In one embodiment, the CGG repeat failure is a vulnerability X-related failure (FXD).

[0347] In one embodiment, the present invention provides a functional nucleic acid molecule, conjugate, pharmaceutically acceptable salt, or pharmaceutical composition for use in the treatment, prevention, or delay of the onset of CTG repeat disorder.

[0348] In one embodiment, CTG repeat disorder is myoric rigidity dystrophy type 1.

[0349] In one embodiment, the present invention provides a functional nucleic acid molecule, conjugate, pharmaceutically acceptable salt, or pharmaceutical composition for use in the treatment, prevention, or delay of the onset of GAA repeat disorder.

[0350] In one embodiment, the GAA repeat disorder is Friedreich's ataxia.

[0351] In one embodiment, the present invention provides a functional nucleic acid molecule, conjugate, pharmaceutically acceptable salt, or pharmaceutical composition for use in the treatment, prevention, or delay of the onset of GCC repeat disorder.

[0352] In one embodiment, the GCC repeat disorder is FRAXE intellectual disability.

[0353] In one embodiment, the present invention provides a functional nucleic acid molecule, conjugate, pharmaceutically acceptable salt, or pharmaceutical composition for use in the treatment, prevention, or delay of the onset of GCG repeat disorder.

[0354] In one embodiment, GCG repeat disorder is oculopharyngeal muscular dystrophy.

[0355] In one embodiment, the present invention provides functional nucleic acid molecules, conjugates, pharmaceutically acceptable salts, or pharmaceutical compositions for the preparation of pharmaceuticals for the treatment, prevention, or delay of triplet repeat disorders.

[0356] In one embodiment, the pharmaceutical product is for the treatment, prevention, or delay of CAG triplet repeat disorders.

[0357] In one embodiment, the pharmaceutical product is for the treatment, prevention, or delay of polyglutamine disease (PolyQ).

[0358] In one embodiment, the pharmaceutical is for the treatment, prevention, or delay of a disease selected from the group consisting of Huntington's disease (HD), spinocerebellar ataxia type 1 (SCA1), spinocerebellar ataxia type 2 (SCA2), spinocerebellar ataxia type 3 (SCA3), spinocerebellar ataxia type 6 (SCA6), spinocerebellar ataxia type 7 (SCA7), spinocerebellar ataxia type 17 (SCA17), dentateburubral-pallidoluysian atrophy (DRPLA), and X-linked spinal muscular atrophy 1 (SMAX1 / SBMA).

[0359] In one embodiment, the pharmaceutical product is for the treatment, prevention, or delay of Huntington's disease (HD).

[0360] In one embodiment, the pharmaceutical product is for the treatment, prevention, or delay of CGG repeat disorders.

[0361] In one embodiment, the CGG repeat failure is a vulnerability X-related failure (FXD).

[0362] In one embodiment, the pharmaceutical product is for the treatment, prevention, or delay of CTG repeat disorders.

[0363] In one embodiment, CTG repeat disorder is myoric rigidity dystrophy type 1.

[0364] In one embodiment, the pharmaceutical product is for the treatment, prevention, or delay of GAA repeat disorder.

[0365] In one embodiment, the GAA repeat disorder is Friedreich's ataxia.

[0366] In one embodiment, the pharmaceutical product is for the treatment, prevention, or delay of GCC repeat disorders.

[0367] In one embodiment, the GCC repeat disorder is FRAXE intellectual disability.

[0368] In one embodiment, the pharmaceutical product is for the treatment, prevention, or delay of GCG repeat disorders.

[0369] In one embodiment, GCG repeat disorder is oculopharyngeal muscular dystrophy.

[0370] In all of the methods and uses described above, the action of functional nucleic acids can be considered to be disease modification.

[0371] Furthermore, in each of the embodiments described above, the DNA or vector encoding the functional nucleic acid molecule of the present invention may be used in combination with or alone with any other component.

[0372] The method of the present invention can be carried out in vitro, ex vivo, or in vivo.

[0373] The methods described herein may include transfecting cells with a functional nucleic acid molecule, DNA molecule, expression vector, composition, or pharmaceutical composition as defined herein. The functional nucleic acid molecule, DNA molecule, expression vector, composition, or pharmaceutical composition may be delivered to target cells using methods known in the art, such as microinjection, lipofection, electroporation, the use of calcium phosphate, vector-based autoinfection, or viral transduction.

[0374] Functional nucleic acid molecules, DNA molecules, compositions, and / or pharmaceutical compositions can preferably be used as pharmaceuticals for triplet repeat elongation diseases / disorders, such as polyglutamine (PolyQ) disease.

[0375] Since these disorders are hereditary, prevention will be understood to refer to the prevention of triplet elongation and any associated pathological consequences, rather than complete prevention of the disease itself.

[0376] Further prevention also includes adjusting the age of onset (AAO).

[0377] The functional nucleic acid molecules of the present invention will ultimately be found to be useful in increasing the levels of Fan1 protein. This increase in Fan1 is preferably within cells, for example, within the cells of interest.

[0378] In a preferred embodiment, the subject is a human subject.

[0379] In a more preferred embodiment, the subjects are human subjects with hereditary triplet repeat disorders.

[0380] In one embodiment, the disease or disorder is a neurological disease or disorder.

[0381] In one embodiment, the disease or disorder is Huntington's disease (HD).

[0382] In one embodiment, the disease or disorder is spinocerebellar ataxia type 1 (SCA1).

[0383] In one embodiment, the disease or disorder is spinocerebellar ataxia type 2 (SCA2).

[0384] In one embodiment, the disease or disorder is spinocerebellar ataxia type 3 (SCA3). Spinocerebellar ataxia type 3 is also known as Machado-Joseph disease (MJD).

[0385] In one embodiment, the disease or disorder is spinocerebellar ataxia type 6 (SCA6).

[0386] In one embodiment, the disease or disorder is spinocerebellar ataxia type 7 (SCA7).

[0387] In one embodiment, the disease or disorder is spinocerebellar ataxia type 17 (SCA17).

[0388] In one embodiment, the disease or disorder is dentatorubral-pallidoluysian atrophy (DRPLA).

[0389] In one embodiment, the disease or disorder is X-linked spinal and bulbar muscular atrophy 1 (SMAX1 / SBMA).

[0390] In this specification, examples of the plural form of a word should be interpreted as also including the singular form of the word, and vice versa, unless the context clearly indicates otherwise.

[0391] The present invention will now be described with reference to the following non-limiting examples. [Examples]

[0392] (Examples) (Example 1 - miRNA mimics downregulate Fan1 mRNA and protein levels) To investigate whether the miRNA miR-124-3p targets Fan1 mRNA and thereby mediates FAN1 expression, HCT116 cells were transfected with an oligonucleotide mimic of miR-124-3p. Oligonucleotides lacking the targeting sequence were used as controls.

[0393] (qRT-PCR analysis) HCT116 cells were cultured in DMEM GlutaMAX medium (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were seeded at 100,000 cells per well in 12-well plates and incubated overnight at 37°C, 95% humidity, and 5% CO2.

[0394] Cells were transfected with hsa-miR-124-3p mirVana® miRNA mimic (Invitrogen, 4464066, assay ID MC10060) or mirVana® miRNA mimic negative control #1 (Invitrogen, 4464058) at a final concentration of 50 nM or 100 nM using Lipofectamine RNAiMax (Invitrogen). As a control, cells were similarly transfected with Fan1-specific siRNA or untargeted control siRNA (Dharmacon, L-020327-00-0005 and D-001810-10-05) at a final concentration of 10 nM.

[0395] The day after transfection, the culture medium was removed from the wells, and the wells were washed three times with phosphate-buffered saline (PBS). The cells were lysed in RLT buffer, and total RNA was extracted according to the manufacturer's instructions, including the use of a gDNA Eliminator column (RNeasy Plus Mini Kit, Qiagen).

[0396] RNA concentration and purity were measured using a Nanodrop spectrophotometer (Thermo Scientific), and 250 ng of RNA was reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer's instructions. To eliminate the possibility of gDNA contamination, a reverse transcription reaction product (-RT control) without reverse transcriptase was prepared.

[0397] After synthesis, the cDNA was diluted 1:5. qPCR was performed using the SYBR Green PCR Master Mix (Applied Biosystems) on a QuantStudio 1 Real-Time PCR System (Applied Biosystems) with the following primers and conditions: Table 2 - Primer sequences (IDT) [Table 2] Table 3 - qPCR reaction composition [Table 3] Table 4-qPCR conditions [Table 4]

[0398] The analysis was performed using the comparative Ct method with B2M as the normalization factor. The multiplicative changes were calculated between samples transfected with the untargeted control mimic and samples transfected with the miR-124-3p mimic, or between samples transfected with the untargeted control siRNA and samples transfected with FAN1 siRNA. The experiment was independently reproduced three times.

[0399] (Western blot analysis) Cell transfection was performed at a final concentration of 100 nM as described in the qRT-PCT section above. 24 or 48 hours after transfection, the cells were washed with ice-cold PBS and collected in 500 μl of PBS. The cell suspension was centrifuged at 5,000 rpm at 4°C for 5 minutes, and the supernatant was removed. The cell pellet was resuspended in 100 μl of RIPA buffer (Thermo Scientific) containing protease and phosphatase inhibitors (PhosStop tablets and cOmplete Mini, EDTA-free protease inhibitor cocktail, Roche).

[0400] The sample was incubated on ice for 15 minutes with periodic mixing. Then, the sample was centrifuged at 13,000 rpm at 4°C for 15 minutes, and the supernatant was collected. The supernatant corresponds to the recovered soluble protein fraction. The total protein content in the sample was determined by performing a Pierce BCA protein assay (Thermo Scientific) according to the manufacturer's instructions.

[0401] The samples were mixed with 4×LDS sample buffer and DTT (50 mM final concentration), boiled at 95°C for 5 minutes, and then subjected to SDS-PAGE analysis using NuPAGE 4-12% Bis Tris Midi Gels (ThermoFisher). 10 μg of protein was loaded per well.

[0402] The isolated proteins were transferred to a nitrocellulose membrane using BOLT transfer buffer (ThermoFisher). The membrane was blocked in 5% BSA (Sigma Aldrich), then probed overnight with the primary antibody, and subsequently incubated with the secondary antibody for 1 hour (see Table 5 below). The proteins were visualized using Clarity Western ECL Substrate (Bio-Rad) and imaged using Odyssey Imager (Li-Cor). Protein bands were quantified using Empiria Studio 2.2. The analysis was performed using tubulin as the normalization factor. The multiplicative changes were calculated between samples transfected with the untargeted control mimic and samples transfected with the miR-124-3p mimic, or between samples transfected with the untargeted control siRNA and samples transfected with FAN1 siRNA. The experiment was independently reproduced twice (48 hours) or three times (24 hours). Table 5: Antibodies used in Western blotting [Table 5]

[0403] When normalized against their respective control values, the data revealed that both Fan1 mRNA (Figure 1) and protein (Figure 2) levels were lower in cells receiving the miR-124-3p mimic compared to cells receiving only the control oligonucleotide.

[0404] These data suggest that the naturally occurring microRNA miR-124-3p has the ability to downregulate Fan1 mRNA and protein levels. Therefore, considering the decrease in both mRNA and protein levels, it is quite possible that Fan1 repression can be achieved via mRNA-targeted post-transcriptional repression achieved by miRNA-induced mRNA degradation.

[0405] (Example 2 - The rs3512 SNP affects miRNA-mediated Fan1 downregulation.) To investigate the effect of rs3512 SNPs on miR-124-3p-mediated Fan1 regulation, a dual luciferase assay was performed.

[0406] In short, the putative miR-124-3p target region of Fan1 3'-UTR was cloned downstream of a firefly luciferase (FLuc) expression cassette within a construct that also contains an independent sea urchin (Renilla) luciferase (RLuc) expression cassette for use as a normalization factor.

[0407] The following constructs were produced and tested in combination with a non-targeted miRNA mimic control or the miR-124-3p mimic: wt FAN1 3'-UTR: wild-type sequence of the estimated miR-124-3p target region; • rs3512 FAN1 3'-UTR: Estimated miR-124-3p target region containing the rs3512(G>C) SNP; • Pairing of mutant seeds: Negative controls of the putative miR-124-3p target region in which nucleotide mutations were induced to eliminate pairing with positions 2, 3, 5, and 6 of the miR-124-3p seed. • miR-124-3p sensor: A putative miR-124-3p target region with nucleotide mutations that make it completely complementary to miR-124-3p (i.e., siRNA-like). Positive control.

[0408] A diagram of the construct can be found in Figure 3A.

[0409] (pmirGLO dual luciferase-FAN1 3'-UTR reporter cloning) Forward and reverse oligonucleotides were designed and synthesized to contain the target sequence and the restriction enzyme extrusion (IDT) necessary for cloning.

[0410] Oligonucleotides were hybridized, and the pmirGLO vector (Promega) was linearized with PmeI and XbaI (NEB). After ligation, the constructs were transformed into Dh5-alpha-competent Escherichia coli (NEB) and selected in LB-ampicillin (50 μg / ml). Positive clones were amplified in culture, and the final constructs were purified using the PureLink® Rapid Low-Endotoxin Maxi Plasmid Purification Kit (Thermo Scientific). Plasmid sequences were confirmed by Sanger sequencing (Genewiz).

[0411] (Transfection and dual luciferase reporter assay) HCT116 cells were cultured in DMEM GlutaMAX medium (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. 10,000 cells per well were seeded into 96-well white-walled plates and incubated overnight at 37°C, 95% humidity, and 5% CO2.

[0412] The morning after seeding, cells were transfected with 25 ng of each reporter using Trans-IT 2020 (Mirus Bio). In the afternoon, cells were transfected with either the hsa-miR-124-3p miRCURY LNA miRNA mimic (Qiagen, 339173, assay YM00471256-ADA) or the negative control miRCURY LNA miRNA mimic (Qiagen, 339173, assay YM00479902-ADA) at a final concentration of 100 nM using Lipofectamine RNAiMax (Invitrogen).

[0413] Twenty-four hours after the final transfection, a dual-luciferase assay was performed using the Dual-Luciferase® reporter assay system (Promega) according to the manufacturer's instructions.

[0414] For analysis, after background removal, the FLuc signal from each well was normalized by the RLuc signal, and the multiplicative change between samples transfected with the miR-124-3p mimic and samples transfected with the untargeted control mimic was calculated. The experiment was reproduced five times.

[0415] The SNP rs3512 can mitigate the miR-124-3p mimic-induced suppression of Fluc reporter gene expression. The introduction of a Fan1 3'-UTR-derived sequence into the Fluc reporter gene's 3'-UTR makes the reporter gene more susceptible to downregulation by the miR-124-3p mimic. The downregulation observed in the Fluc reporter gene is affected by the insertion of the rs3512 SNP into the Fan1 3'-UTR sequence (Figure 3B).

[0416] In summary, these data and the data from Example 1 indicate that miR-124-3p induces degradation of mRNA containing the Fan1 3'-UTR sequence (Figure 1), resulting in a decrease in Fan1 protein (Figure 2) or the protein of the chimeric construct encoded by the mRNA containing the Fan1 3'-UTR sequence (e.g., FLuc) (Figure 3). Furthermore, it is clear that the SNP rs3512 can reverse the repressive effect of miR-124-3p, thereby increasing protein expression in the presence of miR-124-3p compared to proteins encoded by sequences containing the wild-type Fan1 3'-UTR.

[0417] (Example 3 - Functional nucleic acids targeting Fan1 3'-UTR inhibit miRNA-mediated downregulation of Fan1) To investigate whether synthetic functional nucleic acids can inhibit the miR-124-3p-mediated inhibition of Fan1, we designed and synthesized a panel of functional nucleic acids.

[0418] Functional nucleic acid molecules were designed using a tiling approach to target the 3'-UTR region of human Fan1, where the miR-124-3p seed region is predicted to bind. In this approach, each antisense oligonucleotide is complementary to a target sequence located one nucleotide downstream (i.e., 3') of its preceding target sequence. Figure 4 shows a magnified view of the Fan1 mRNA, illustrating the approximate region encompassing the predicted miR-124-3p targeting site.

[0419] The sequences are shown in Table 10. In this table, "m" represents a 2'-O-methyl modified nucleoside; " / 52MOEr_ / , / i2MOEr_ / , / 32MOEr" represent 5'_2'-O-methoxyethyl nucleoside, internal_2'-O-methoxyethyl nucleoside, and 3'_2'-O-methoxyethyl nucleoside, respectively; * The symbol "+" represents a phosphorothioate bond between adjacent nucleotides; and "+" represents an LNA nucleotide.

[0420] (Example 4 - ASO-mediated steric hindrance of miR-124-3p to FAN1 3'-UTR results in FAN1 upregulation) miR-124 is highly conserved and tissue-specifically expressed, accounting for 25-50% of all miRNAs in the mouse brain (Lagos-Quintana et al., 2002). As a proof-of-concept for the usefulness of miR-124-3p steric-hindrance ASO for FAN1 upregulation, human cortical neurons were used as a cell model.

[0421] (Cortical neuronal differentiation and gymnosis) Wild-type neural stem cells (axoCells, Axol Bioscience Ltd) were thawed and seeded at a rate of 32,000 cells per well in 96-well plates according to the manufacturer's instructions. Unless otherwise specified, cells were maintained overnight at 37°C, 95% humidity, and 5% CO2. Cells were differentiated according to the manufacturer's instructions (days 1-6) and matured into cerebral cortical neurons (days 7-14).

[0422] On day 20 post-differentiation, neurons were initially treated by gymnosis using either a non-targeted control or miR-124-3p sterically insufficient ASO. The ASO was manufactured by Integrated DNA Technologies (IDT) and resuspended in TE buffer. Briefly, 50% of the medium was changed every other day, excluding weekends, using pre-warmed medium (neuronal maturation basal medium, 20 ng / mL BDNF, 0.5 mM cAMP, and 0.2 mM ascorbic acid) supplemented with ASO to achieve a final concentration of 5 μM. After five administrations to neurons, samples were collected on day 31 post-plating.

[0423] The miR-124-3p sterically hindered ASO used herein corresponds to compound 58 (SEQ ID NOs: 1031 and 1097).

[0424] (Western blotting) The culture medium was removed, and each well was dissolved in 10 μl of Laemmli SDS buffer (Thermo Scientific). Two wells were pooled per sample. The samples were boiled at 95°C for 5 minutes, and then subjected to SDS-PAGE analysis using NuPAGE 4-12% Bis Tris Midi Gels (ThermoFisher) at 150 V for 80 minutes. 10 μl of the dissolved solution was loaded per well.

[0425] The isolated proteins were transferred to a nitrocellulose membrane at 350 mA for 1 hour using BOLT transfer buffer (ThermoFisher). The membrane was blocked in 5% BSA (Sigma Aldrich), probed with primary antibody for 3 hours, and then incubated with fluorescent secondary antibody at room temperature for 1 hour (see Table 6 below). The proteins were imaged using Odyssey Imager (Li-Cor). Protein bands were quantified using Empiria Studio 2.2 software. The analysis was performed using tubulin-β3 as the normalization factor. The multiplicative change between the untargeted ASO control sample and the miR-124-3p sterically hindered ASO sample was calculated.

[0426] Table 6: Antibodies used in Western blotting [Table 6]

[0427] When normalized against each control value, the data revealed that FAN1 levels were increased in neurons treated with miR-124-3p steric-inhibited ASO compared to those treated with control ASO (Figure 5B).

[0428] These data suggest the ability of sterically hindered ASOs to bind to Fan1 3'-UTR, compete with miR-124-3p, deactivate miRNA-mediated repression, and thereby upregulate FAN1 protein levels.

[0429] These data further support the proof of concept that blocking or competing with miRNAs can be used as a strategy to upregulate FAN1.

[0430] (Example 5 - ASO-mediated steric hindrance of the miRNA target site on the FAN1 3'-UTR leads to FAN1 upregulation) To further demonstrate the validity of the concept of miRNA steric hindrance as a strategy for FAN1 upregulation, other candidate miRNAs were searched for. A set of brain-enriched miRNAs was identified and prioritized according to the intensity and expression of their target sites in HCT116: miR-186-5p, miR-335-3p, miR-145-5p, miR-181-5p, miR-197-3p, and miR-194-5p.

[0431] To facilitate screening, we created a CRISPR-modified HCT116 cell line expressing a C-terminal HiBiT-tagged version of FAN1 that can be quantified by a luminescence assay.

[0432] ASOs were designed as 18-mer or 22-mer sequences containing antisense sequences for the expected binding region of each miRNA on the Fan1 3'-UTR. Each sequence was produced with four different sets of chemical modifications: • Phosphothioate skeleton (PS), 2'-O-methyl (Me): Referred to as "PS-Me". • The phosphorothioate skeleton (PS), 2'-O-methyl (Me), and the three nucleotides at the 3' end of the locked nucleic acid (LNA) are referred to as "PS-Me-LNA". • Phosphothioate skeleton (PS), 2'-O-methoxyethyl (MOE): Referred to as "PS-MOE". • A phosphorothioate skeleton (PS), 2'-O-methoxyethyl (MOE), and the three nucleotides at the 3' end of the locked nucleic acid (LNA): referred to as "PS-MOE-LNA".

[0433] (Cell culture and transfection) ASO was manufactured by Integrated DNA Technologies (IDT), resuspended in TE buffer, and dispensed into 96-well white-walled plates using an Assist Plus Pipetting Robot (INTEGRA Biosciences Ltd). The plates were stored at -20°C until use.

[0434] HCT116 FAN1-HiBiT cells were artificially generated by Synthego. The cells were cultured in DMEM GlutaMAX medium (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

[0435] The transfection mixture was prepared using HiPerFect (Qiagen) and dispensed into wells containing ASO. The plates were shaken and incubated at room temperature for 10 minutes. After incubation, 10,000 cells per well were seeded into each transfection plate and incubated at 37°C, 95% humidity, and 5% CO2 for 48 hours. The volume was adjusted to achieve a final ASO concentration of 50 nM.

[0436] (Fluorescence and luminescence assays) Forty-eight hours after transfection, an insoluble CellTiter-Fluor® Cell Viability Assay was performed according to the manufacturer's instructions (Promega). HiBiT expression, as a substitute for FAN1 expression, was measured using the Nano-Glo® HiBiT Lytic Detection System (Promega) according to the manufacturer's instructions.

[0437] For analysis, after removing the background, the HiBiT emission signals from each well were normalized by their respective CellTiter-Fluor fluorescence signals, and the multiplicative changes were calculated between samples transfected with miRNA sterically hindrance-type ASOs and mock-transfected samples.

[0438] When normalized against their respective control values, the data reveals that steric hindrance at multiple miRNA binding sites, most notably miR-145-5p, miR-181-5p, and miR-197-3p, can individually lead to FAN1 upregulation (Figures 6A-D).

[0439] To attempt to improve the observed upregulation, a small tiling library was designed for the miR-197-3p site. Sequences were designed using a tiling approach for the 3'-UTR region of human Fan1 where the miR-197-3p seed is expected to bind. In this approach, each antisense oligonucleotide is complementary to a target sequence located 3 nucleotides downstream (i.e., 3') of its preceding target sequence (Figure 7A). Cell culture, transfection, and analysis were performed as previously described.

[0440] When normalized against their respective control values, the data revealed that all ASOs tested resulted in FAN1 upregulation, and up to 30% more protein was detected by the treatment compared to mock-transfected cells (Figure 7B).

[0441] The ASO used in this specification is ASO 849 = Compound 77 (SEQ ID NOs. 1050 and 1116) ASO 850 = Compound 78 (SEQ ID NOs: 1051 and 1117) ASO 851 = Compound 79 (SEQ ID NOs: 1052 and 1118) ASO 852 = Compound 80 (SEQ ID NOs: 1053 and 1119) ASO 853 = Compound 81 (SEQ ID NOs: 1054 and 1120) Corresponds to (see also Tables 10 and 11).

[0442] (Example 6 - Chemical modification of functional nucleic acids can increase FAN1 upregulation) Functional nucleic acids, also known as antisense oligonucleotides (ASOs), were designed using a tiling approach across the miR-197-3p target site (Figure 7A), and their ability to upregulate FAN1 was evaluated as described above, both with and without normalization (Figure 7B; right).

[0443] In this specification, one particularly effective functional nucleic acid, referred to as ASO_849 (see, for example, Figure 7B), was selected for further iteration of chemical modifications in an attempt to further improve its effects.

[0444] We designed ASO 849 as an ASO that targets the site also targeted by miRNA miR-197-3p, and therefore acts as a sterically hindrance-dependent ASO for this miRNA.

[0445] (Functional nucleic acid molecule design) We designed a set of ASOs, each having the same underlying nucleotide sequence (i.e., SEQ ID NO: 1050), but modified to include the following different chemical modifications: • Phosphothioate skeleton (PS), 2'-O-methoxyethyl (MOE): "PS-MOE" • Phosphothioate skeleton (PS), 2'-O-methoxyethyl (MOE), and the three nucleotides at the 3' end as locked nucleic acid (LNA) • Phosphothioate skeleton (PS), 2'-O-methoxyethyl (MOE), locked nucleic acid (LNA) consisting of the two nucleotides at the 3' end and one nucleotide at the 5' end • Phosphothioate skeleton (PS), 2'-O-methoxyethyl (MOE), locked nucleic acid (LNA) consisting of the two nucleotides at the 3' end and the two nucleotides at the 5' end • Phosphothioate backbone (PS), 2'-O-methyl (Me), locked nucleic acid (LNA) consisting of the two nucleotides at the 3' end and one nucleotide at the 5' end • Phosphothioate skeleton (PS), 2'-O-methyl (Me), locked nucleic acid (LNA) consisting of the two nucleotides at the 3' end and the two nucleotides at the 5' end.

[0446] To facilitate the screening of the above compounds, we created a CRISPR-modified HCT116 cell line expressing a C-terminal HiBiT-tagged version of FAN1 that facilitates quantification by luminescence assays.

[0447] The ability of these ASOs to upregulate FAN1 is shown in Figure 7C, where all compounds can upregulate FAN1.

[0448] (Cell culture and transfection) ASO was manufactured by Integrated DNA Technologies (IDT), resuspended in TE buffer, and dispensed into 96-well white-walled plates using an Assist Plus Pipetting Robot (INTEGRA Biosciences Ltd). The plates were stored at -20°C until use.

[0449] HCT116 FAN1-HiBiT cells were artificially generated by Synthego. The cells were cultured in DMEM GlutaMAX medium (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

[0450] The transfection mixture was prepared using HiPerFect (Qiagen) and dispensed into wells containing ASO. The plates were shaken and incubated at room temperature for 10 minutes. After incubation, 10,000 cells per well were seeded into each transfection plate and incubated at 37°C, 95% humidity, and 5% CO2 for 48 hours. The volume was adjusted to achieve a final ASO concentration of 50 nM.

[0451] (Fluorescence and luminescence assays) Forty-eight hours after transfection, an insoluble CellTiter-Fluor® Cell Viability Assay was performed according to the manufacturer's instructions (Promega). HiBiT expression, as a substitute for FAN1 expression, was measured using the Nano-Glo® HiBiT Lytic Detection System (Promega) according to the manufacturer's instructions.

[0452] For analysis, after removing the background, the HiBiT emission signals from each well were normalized by their respective CellTiter-Fluor fluorescence signals, and the multiplicative changes were calculated between samples transfected with miRNA sterically hindrance-type ASOs and mock-transfected samples.

[0453] When normalized against their respective control values, the data reveals that changes in ASO chemical modification, most notably the two LNAs on the PS-Me+ 3' side and the one LNA on the 5' side, can lead to more pronounced FAN1 upregulation (Table 7 and Figure 7C). Table 7. The respective effects of miR-197-3p on steric-impaired ASO iterations and FAN1 levels. [Table 7]

[0454] The multiplicative change in FAN1 levels compared to mocked cells is expressed as follows: 1.0~1.09: -; 1.1~1.19: +; 1.2~1.29: ++; >1.3: +++

[0455] To determine whether the observed effects occurred at the RNA level or the protein level, cells were treated with ASO 920, 921, 922 (miR-197 steric hindrance) and 864 (untargeted), followed by qRT-PCR analysis (Figure 7D). Cell culture and transfection were carried out as previously described.

[0456] (qRT-PCR) qRT-PCR was performed in three independent wells per condition using the Cells-to-CT® 1-Step TaqMan® Kit (Ambion) according to the manufacturer's instructions. Primers and probes were synthesized by IDT.

[0457] The analysis was performed using the comparative Ct method with B2M as the normalization factor. The multiplier change between mock-treated samples and samples treated with miR-197-3p steric hindrance or untargeted ASO was calculated. The experiment was independently reproduced twice. Table 8: Primer and probe arrangements [Table 8]

[0458] qRT-PCR analysis showed that RNA levels remained unchanged between treatments, suggesting that the FAN1 upregulation after ASO treatment occurs at the translational level (Figure 7D). This is consistent not only with the standard mechanism of action in which many animal miRNAs act to repress translation, but also with effects previously observed using miRNA-sterically hindrance ASOs (Iwakawa et al., 2015; Aggarwal et al., 2023).

[0459] As used in this specification, ASO refers to ASO 849 = Sequence ID 1116 ASO 850 = Sequence ID 1117 ASO 851 = Sequence ID 1118 ASO 852 = Sequence ID 1119 ASO 853 = Sequence ID 1120 ASO 917 = Sequence ID 1139 ASO 918 = Sequence ID 1140 ASO 919 = Sequence ID 1141 ASO 920 = Sequence ID 1142 ASO 921 = Sequence ID 1143 ASO 922 = Sequence ID 1144 ASO 864 = Sequence ID 1145 Corresponds to (see also Tables 10 and 11).

[0460] (Example 7 - Optimized miRNA sterically incompatible functional nucleic acid chemical properties and delivery result in improved FAN1 upregulation) To demonstrate that a similar strategy can be applied to design sterically inaccurate miRNA ASOs for other miRNA sites on the Fan1 3'-UTR, novel sterically inaccurate miRNA ASOs were designed for the miR-181-5p and miR-145-5p sites. Furthermore, ASO delivery was further improved by increasing or decreasing the ASO dose and the amount of transfection reagent.

[0461] ASO was manufactured by Integrated DNA Technologies (IDT), resuspended in TE buffer, and dispensed into 96-well white-walled plates using an Assist Plus Pipetting Robot (INTEGRA Biosciences Ltd). The plates were stored at -20°C until use.

[0462] HCT116 FAN1-HiBiT cells were artificially generated by Synthego. The cells were cultured in DMEM GlutaMAX medium (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

[0463] The transfection mixture was prepared using HiPerFect (Qiagen) and dispensed into wells containing ASO. The plates were shaken and incubated at room temperature for 10 minutes. After incubation, 25,000 cells per well were seeded into each 96-well transfection plate and incubated at 37°C, 95% humidity, and 5% CO2 for 24 hours. The volume was adjusted to achieve a final ASO concentration of 25 nM.

[0464] The miRNA steric-insensitive ASOs used herein are ASO 921 = Sequence ID 1143 ASO 968 = Sequence ID 1146 ASO 970 = Sequence ID 1147 Corresponds to: (see also Table 11).

[0465] (Fluorescence and luminescence assays) Twenty-four hours after transfection, an insoluble CellTiter-Fluor® Cell Viability Assay was performed according to the manufacturer's instructions (Promega). HiBiT expression, as a substitute for FAN1 expression, was measured using the Nano-Glo® HiBiT Lytic Detection System (Promega) according to the manufacturer's instructions.

[0466] For analysis, after removing the background, the HiBiT emission signals from each well were normalized by their respective CellTiter-Fluor fluorescence signals, and the multiplicative changes were calculated between samples transfected with miRNA sterically hindrance-type ASOs and mock-transfected samples.

[0467] After optimizing the chemical properties and delivery of the ASOs, higher levels of FAN1 upregulation were observed with miRNA steric-inhibited ASOs designed for miR-197-3p, miR-181-5p, and miR-145-5p (Figure 8A). In some cases, upregulation exceeding 40% was observed, which reinforces the potential of miRNA steric-inhibited ASOs as a tool for FAN1 regulation and therefore as a therapeutic agent for Huntington's disease.

[0468] To determine whether this effect occurs at the RNA level or the protein (translation) level, qRT-PCR analysis was performed as previously described. As seen earlier (Example 5), the data showed that RNA levels were essentially invariant between treatments, suggesting that the FAN1 upregulation after ASO treatment occurs at the translation level (Figure 8B).

[0469] (Example 8 - Functional nucleic acid-mediated steric hindrance of miRNA in Fan1 3'-UTR leads to FAN1 upregulation in medium spiny neurons derived from HD patients) To investigate whether the activity of optimized sterically incompatible miRNA ASOs could be observed in HD-associated cell models, gymnosis was performed in patient-derived medium-sized spiny neurons carrying a mutant Htt allele with >10⁹ CAG repeats. In addition to previously tested ASOs, a sterically incompatible miRNA ASO targeting the miR-124-3p site in the Fan1 UTR was also included because this miRNA is thought to be specifically expressed in neurons.

[0470] (Culture and gymnosis of medium spiny neurons (MSNs)) MSN (BrainXell, BX-0700) was cultured on plates coated with PDL (Thermo, A3890401) and SureBond (Axol, ax0053). Cell thawing and maintenance were performed according to the manufacturer's protocol. 40,000 cells were plated per well in a 96-well plate.

[0471] On day 7, MSNs were first treated by gymnosis using a non-targeted control ASO or a miRNA steric-inhibited ASO. The ASO was manufactured by Integrated DNA Technologies (IDT) and resuspended in TE buffer. Briefly, a 50% medium change was performed weekly using pre-warmed medium supplemented with ASO to achieve a final concentration of 5 μM. After five administrations to neurons, samples were collected on day 42.

[0472] The miRNA steric-insensitive ASOs used herein are ASO 921 = Sequence ID 1143 ASO 968 = Sequence ID 1146 ASO 969 = Sequence ID 1148 ASO 970 = Sequence ID 1147 Corresponds to: (see also Table 11).

[0473] (Western blotting analysis) The culture medium was removed, each well was washed with PBS, and the samples were dissolved in 20 μl of Laemmli SDS buffer (Thermo Scientific) diluted 1:1 with RIPA (ThermoFisher). The samples were boiled at 95°C for 5 minutes, and 13 μl of the dissolved solution was loaded into each well. The samples were electrophoresed on NuPAGE 4-12% Bis Tris Midi Gels (ThermoFisher) at 150 V for 80 minutes.

[0474] The isolated proteins were transferred to a nitrocellulose membrane at 350 mA for 1 hour using BOLT transfer buffer (ThermoFisher). The membranes were blocked in 5% BSA (Sigma Aldrich), and probed with the primary antibody at room temperature for 1 hour (for tubulin-β3) or overnight at 4°C (for FAN1). The membranes were then incubated with the fluorescent secondary antibody at room temperature for 1 hour (see Table 9 below). The proteins were imaged using an Odyssey Imager (Li-Cor), and the bands were quantified using Empiria Studio 2.2 software. Analysis was performed using tubulin-β3 as the normalization factor. The multiplicative change between the untargeted ASO control sample and the miRNA steric-hindrance ASO sample was calculated. Table 9: Antibodies used in Western blotting [Table 9]

[0475] When normalized against their respective control values, the data revealed that FAN1 levels were increased in neurons treated with miR-181-5p, miR-124-3p, and miR-145-5p steric-inhibited ASOs compared to those treated with the control ASO (Figure 9). FAN1 upregulation was not consistently observed when treated with miR-197-3p steric-inhibited ASO. This can be explained by the fact that the activity of this ASO modality is thought to be strongly correlated with the expression levels of target miRNAs, which are highly dependent on cell type and cell state.

[0476] These data suggest the ability of sterically hindered ASOs to bind to the Fan1 3'-UTR and compete with different miRNAs (miR-181-5p, miR-124-3p, and miR-145-5p in this embodiment) to deactivate miRNA-mediated repression, thereby upregulating FAN1 protein levels.

[0477] These data further support the proof of concept that blocking or competing with miRNAs can be used as a strategy to upregulate FAN1.

[0478] (Example 9 - Upregulation of FAN1 via miRNA steric hindrance ASO in medium-sized spiny neurons derived from HD patients reduces the rate of CAG repeat elongation.) Upregulation of FAN1 has been shown to reduce the rate of Htt CAG repeat elongation (Goold et al., 2019). To investigate whether steric-inhibiting miRNA ASO treatment in medium spiny neurons affects CAG elongation rate as a result of FAN1 upregulation, we will conduct experiments to measure the mean somatic cell length increase of the CAG region in these cells.

[0479] After 10 weeks of gymnosis, gDNA is extracted from neurons treated with sterically incompatible or untargeted ASOs. Neurons treated with untargeted or FAN1 gapmers are also included as controls. The gDNA is used for PCR amplification of the Htt locus. The PCR products are sent to Nanopore long-read sequencing, and the resulting data are used to calculate the mean increase in somatic cell length of the CAG region.

[0480] Data reveal that increased FAN1 levels are associated with slower CAG region elongation as a result of miRNA steric-inhibiting ASO treatment. Conversely, control neurons treated with FAN1 gapmers show faster CAG region elongation as a result of FAN1 downregulation. This reinforces the validity of miRNA regulatory blockade as an effective strategy for FAN1 upregulation in Huntington's disease treatment.

[0481] (Example 10 - Disruption of miR-124-3p-mediated regulation of FAN1 by introduction of SNP rs3512 or seed target site mutation results in decreased FAN1 upregulation and CAG elongation in HD patient-derived neurons.) Huntington's disease patients who possess the rs3512 SNP at the predictive binding site of miR-124-3p on the Fan1 3'-UTR exhibit delayed disease onset due to slower CAG elongation.

[0482] Transcriptome data obtained from the Genotype-Tissue Expression (GTEx) Portal suggest that individuals with the same SNP have higher levels of Fan1 mRNA in brain tissue where miR-124-3p is specifically expressed. Conversely, Fan1 mRNA levels remained unchanged across all genotypes in adipose tissue and whole blood, suggesting a correlation between miR-124-3p expression and FAN1 upregulation in the presence of rs3512 (Figure 10, data extracted from the Genotype-Tissue Expression (GTEx) Portal).

[0483] To investigate whether there is a correlation between rs3512, FAN1 levels, and CAG repeat elongation, we introduce an rs3512 SNP or another mutation within the miRNA seed region into patient-derived iPSCs possessing >127 CAG repeats on the Htt locus and a C-terminal HiBiT tag on the Fan1 locus to facilitate quantification.

[0484] A CRISPR single guide RNA (sgRNA) was designed to introduce a double-strand break in the DNA outside the 3'-UTR of Fan1. Repair templates were designed to either i. introduce an rs3512(G>C) SNP on the 3'-UTR (converting the predicted miR-124-3p 7mer-m8 site to a 6mer), or ii. introduce a two-nucleotide mutation at the predicted binding site of the miR-124-3p seed (thus eliminating the predicted miR-124-3p regulation). Furthermore, a PAM motif mutation was included in the sgRNA target site outside the 3'-UTR in both repair templates to avoid Cas9 re-cleavage after an HDR event.

[0485] Patient-derived iPSCs containing >127 CAG repeats were electroporated with a mixture of ribonucleoproteins consisting of Cas9 and sgRNA and an HDR template. Cells were serially diluted and sparsely plated to promote the formation of single-cell-derived colonies. Clones were harvested, plated in separate wells, grown, and genotyped for the presence or absence of desired editing. Homozygous and heterozygous clones containing two-nucleotide mutations on the rs3512 or miR-124-3p seed binding site were identified, validated, and grown.

[0486] Parental clones (wild-type) and validated iPSC clones of different genotypes are differentiated into excitatory neurons and maintained in culture for longer than 10 weeks to enable CAG repeat extension. At the end of the experiment, three wells per genotype are used for the HiBiT luminescence assay and three wells are used for gDNA extraction.

[0487] HiBiT luminescence was measured as a surrogate for FAN1 levels, revealing that introduction of rs3512 induces FAN1 upregulation in an allele-dependent manner; heterozygous and homozygous rs3512 clones exhibit 50% and 100% higher FAN1 levels compared to parental controls, as observed in datasets from the GTEx portal. Furthermore, the introduction of a two-nucleotide mutation into the miR-124-3p seed-binding site results in even higher FAN1 upregulation in an allele-dependent manner, revealing the maximum capacity for FAN1 upregulation when the interaction between miR-124-3p and Fan1 3'-UTR is blocked. This is expected to be achieved by using miRNA sterically hindrance functional nucleic acid molecules, such as those described herein.

[0488] gDNA extracted from a replica of the same clone is used for PCR amplification of the Htt locus. The PCR product is sent for Nanopore long-read sequencing, and the resulting data is used to calculate the mean increase in somatic cell length of the CAG region. The data reveals that an increase in FAN1 levels is proportionally associated with slower CAG region elongation as a result of disrupting the interaction between miR-124-3p and Fan1 3'-UTR, which reinforces the validity of miRNA regulatory blockade as an effective strategy for FAN1 upregulation in Huntington's disease treatment. [Table 10] TIFF2026528923000041.tif248170TIFF2026528923000042.tif248170TIFF2026528923000043.tif248170TIFF2026528923000044.tif248170 TIFF2026528923000045.tif248170TIFF2026528923000046.tif249170TIFF2026528923000047.tif249170TIFF2026528923000048.tif248170 [Table 11] TIFF2026528923000050.tif243170 Here, "m" represents a 2'-O-methyl modified nucleoside; * " represents a phosphorothioate bond between adjacent nucleotides; "+" represents an LNA nucleotide; and " / 52MOEr_ / , / i2MOEr_ / , / 32MOEr" represent 5'_2'-O-methoxyethyl nucleoside, internal_2'-O-methoxyethyl nucleoside, and 3'_2'-O-methoxyethyl nucleoside, respectively.

Claims

1. A functional nucleic acid molecule having a length of 10 to 40 nucleotides, wherein it independently comprises one or more consecutive nucleotide sequences consisting of 5 or more nucleotides in length, and each consecutive nucleotide sequence is at least 80% complementary to a consecutive nucleotide sequence in the 3'-UTR of Fan1.

2. The functional nucleic acid molecule according to claim 1, wherein the functional nucleic acid molecule competes with microRNA for binding to Fan1 mRNA.

3. A functional nucleic acid molecule according to claim 1 or 2, wherein one or more of the consecutive nucleotide sequences are complementary to consecutive nucleotide sequences in the 3'UTR of Fan1 that overlap with the nucleotide sequence bound by the microRNA.

4. The functional nucleic acid molecule according to any one of claims 1 to 3, wherein the microRNA is associated with regulating the development of triplet repeat disorders.

5. The aforementioned microRNA: (a)hsa-miR-299-3p、hsa-miR-3940-5p、hsa-miR-4265、hsa-miR-4507、hsa-miR-4657、hsa-miR-6748-5p、hsa-miR-6759-5p、hsa-miR-6793-5p、hsa -miR-6796-5p, hsa-miR-6839-3p, hsa-miR-629-5p, hsa-miR-1275, hsa-miR-193b-5p, hsa-miR-3675-5p, hsa-miR-4665-5p, hsa-miR-6751-5p, hsa -miR-6803-5p, hsa-miR-6835-5p, hsa-miR-6842-5p, hsa-miR-6890-5p, hsa-miR-7109-5p, hsa-miR-7110-5p, hsa-miR-198, hsa-miR-1911-3p, hsa -miR-6753-5p, hsa-miR-1256, hsa-miR-3910, hsa-miR-1910-3p, hsa-miR-2682-5p, hsa-miR-34a-5p, hsa-miR-34b-5p, hsa-miR-34c-5p, hsa-miR-4 49a, hsa-miR-449b-5p, hsa-miR-449c-5p, hsa-miR-548au-3p, hsa-miR-584-3p, hsa-miR-6511a-5p, hsa-miR-6808-5p, hsa-miR-6893-5p, hsa-miR -940, hsa-miR-3714, hsa-miR-125b-1-3p, hsa-miR-155-5p, hsa-miR-3621, hsa-miR-3610, hsa-miR-4665-3p, hsa-miR-4755-3p, hsa-miR-3622a-5p hsa-miR-5582-5p hsa-miR-124-3p hsa-miR-506-3p hsa-miR-10527-5p hsa-miR-1207-5p hsa-miR-1262 hsa-miR-1273h-3p hsa-miR-1287-3 p, hsa-miR-1301-3p, hsa-miR-1303, hsa-miR-1304-5p, hsa-miR-1343-5p, hsa-miR-141-3p, hsa-miR-186-5p, hsa-miR-1914-3p, hsa-miR-200a-3phsa-miR-204-5p, hsa-miR-211-5p, hsa-miR-23a-5p, hsa-miR-23b-5p, hsa-miR-3120-3p, hsa-miR-3122, hsa-miR-3182, hsa-miR-3199, hsa-miR-335-3p, hsa-miR-3681-5p, hsa-miR-3913-5p, hsa-miR-3925-3p, hsa-miR-4284, hsa-miR-4469, hsa-miR-450b-5p, hsa-miR-4640-5p, hsa-miR-464 9-3p, hsa-miR-4650-3p, hsa-miR-4667-3p, hsa-miR-4677-5p, hsa-miR-4693-3p, hsa-miR-4701-3p, hsa-miR-4726-5p, hsa-miR-4763-3p, hsa-mi R-4768-5p, hsa-miR-4771, hsa-miR-4775, hsa-miR-4776-3p, hsa-miR-4797-3p, hsa-miR-485-5p, hsa-miR-5002-3p, hsa-miR-5008-5p, hsa-miR-5 089-5p, hsa-miR-513b-5p, hsa-miR-5190, hsa-miR-5191, hsa-miR-5193, hsa-miR-5194, hsa-miR-543, hsa-miR-545-3p, hsa-miR-548a-3p, hsa-miR-548aa, hsa-miR-548an, hsa-miR-548ap-3p, hsa-miR-548ar-3p, hsa-miR-548az-3p, hsa-miR-548bc, hsa-miR-548e-3p, hsa-miR-548n, hsa-miR -548t-3p, hsa-miR-589-3p, hsa-miR-6730-5p, hsa-miR-6736-5p, hsa-miR-6738-5p, hsa-miR-6740-5p, hsa-miR-676-3p, hsa-miR-6770-5p, hsa- miR-6826-3p, hsa-miR-6831-5p, hsa-miR-6833-3p, hsa-miR-6845-3p, hsa-miR-6870-3p, hsa-miR-6875-3p, hsa-miR-6878-3p, hsa-miR-6884-3phsa-miR-7108-3p, hsa-miR-7974, hsa-miR-802, hsa-miR-939-5p, hsa-let-7f-2-3p, hsa-miR-11181-3p, hsa-miR-1185-1-3p, hsa-miR-1185-2-3p hsa-miR-1197 hsa-miR-12129 hsa-miR-12136 hsa-miR-1228-3p hsa-miR-124-3p hsa-miR-1264 hsa-miR-1265 hsa-miR-1276 hsa-miR-1288- 5p, hsa-miR-1290, hsa-miR-1299, hsa-miR-130a-5p, hsa-miR-135a-2-3p, hsa-miR-135b-3p, hsa-miR-138-1-3p, hsa-miR-145-5p, hsa-miR-15b-3p hsa-miR-16-1-3p hsa-miR-181a-5p hsa-miR-181b-5p hsa-miR-181c-5p hsa-miR-181d-5p hsa-miR-187-3p hsa-miR-194-5p hsa-miR-196a-3 p, hsa-miR-196b-3p, hsa-miR-197-3p, hsa-miR-19a-5p, hsa-miR-19b-1-5p, hsa-miR-216b-5p, hsa-miR-2276-3p, hsa-miR-2276-5p, hsa-miR-2277 8, hsa-miR-2355-3p, hsa-miR-27a-5p, hsa-miR-29a-5p, hsa-miR-3064-5p, hsa-miR-3085-3p, hsa-miR-3125, hsa-miR-3127-3p, hsa-miR-3135a, hs a-miR-3143, hsa-miR-3150a-3p, hsa-miR-3154, hsa-miR-3158-3p, hsa-miR-3159, hsa-miR-3160-5p, hsa-miR-3163, hsa-miR-3184-3p, hsa-miR-31 89-5p, hsa-miR-3191-5p, hsa-miR-3202, hsa-miR-331-3p, hsa-miR-342-3p, hsa-miR-346, hsa-miR-3605-3p, hsa-miR-3663-5p, hsa-miR-3677-5phsa-miR-372-5p, hsa-miR-376b-5p, hsa-miR-378a-5p, hsa-miR-3916, hsa-miR-3929, hsa-miR-3938, hsa-miR-424-3p, hsa-miR-4254, hsa-miR-4 282, hsa-miR-4323, hsa-miR-4428, hsa-miR-4443, hsa-miR-4451, hsa-miR-4476, hsa-miR-4478, hsa-miR-4502, hsa-miR-4503, hsa-miR-4515, hsa -miR-4679, hsa-miR-4694-5p, hsa-miR-4699-3p, hsa-miR-4729, hsa-miR-4738-3p, hsa-miR-4739, hsa-miR-4745-5p, hsa-miR-4755-5p, hsa-miR-4756-5p, hsa-miR-4766-5p, hsa-miR-4773, hsa-miR-4781-3p, hsa-miR-4803, hsa-miR-488-3p, hsa-miR-498-5p, hsa-miR-5001-3p, hsa-miR-500 6-3p, hsa-miR-5008-3p, hsa-miR-505-3p, hsa-miR-506-3p, hsa-miR-506-5p, hsa-miR-5088-3p, hsa-miR-5089-3p, hsa-miR-5094, hsa-miR-5189-3p, hsa-miR-5195-3p, hsa-miR-539-5p, hsa-miR-548a-5p, hsa-miR-548ab, hsa-miR-548ad-5p, hsa-miR-548ae-5p, hsa-miR-548ag, hsa-miR-548 ai, hsa-miR-548ak, hsa-miR-548am-5p, hsa-miR-548ap-5p, hsa-miR-548aq-5p, hsa-miR-548ar-5p, hsa-miR-548as-5p, hsa-miR-548au-5p, hsa-miR-548ay-5p, hsa-miR-548az-5p, hsa-miR-548b-5p, hsa-miR-548ba, hsa-miR-548bb-5p, hsa-miR-548c-5p, hsa-miR-548d-5p, hsa-miR-548g-3phsa-miR-548h-5p, hsa-miR-548i, hsa-miR-548j-5p, hsa-miR-548m, hsa-miR-548o-5p, hsa-miR-548p, hsa-miR-548t-5p, hsa-miR-548v, hsa-miR- 548w, hsa-miR-548y, hsa-miR-5586-5p, hsa-miR-559, hsa-miR-5591-5p, hsa-miR-5693, hsa-miR-570-5p, hsa-miR-578, hsa-miR-596, hsa-miR-601 hsa-miR-6081 hsa-miR-643 hsa-miR-6501-3p hsa-miR-6504-5p hsa-miR-6511a-3p hsa-miR-6511b-3p hsa-miR-6513-5p hsa-miR-660-3p hs a-miR-661, hsa-miR-663b, hsa-miR-664a-3p, hsa-miR-6726-5p, hsa-miR-6734-5p, hsa-miR-6736-3p, hsa-miR-6737-3p, hsa-miR-6738-3p, hsa-mi R-6739-3p, hsa-miR-6742-3p, hsa-miR-6746-3p, hsa-miR-6756-3p, hsa-miR-676-5p, hsa-miR-6761-5p, hsa-miR-6763-3p, hsa-miR-6763-5p, hsa -miR-6791-3p、hsa-miR-6796-3p、hsa-miR-6818-3p、hsa-miR-6825-5p、hsa-miR-6829-3p、hsa-miR-6833-5p、hsa-miR-6843-3p、hsa-miR-6848-3p、 hsa-miR-6854-5p, hsa-miR-6859-5p, hsa-miR-6876-5p, hsa-miR-6894-5p, hsa-miR-6895-3p, hsa-miR-7114-5p, hsa-miR-7157-3p, hsa-miR-765, h sa-miR-766-5p, hsa-miR-7852-3p, hsa-miR-7977, hsa-miR-7978, hsa-miR-8485, hsa-miR-888-5p, hsa-miR-920, hsa-miR-943, hsa-miR-10398-5phsa-miR-105-5p, hsa-miR-10a-3p, hsa-miR-12115, hsa-miR-1236-3p, hsa-miR-1253, hsa-miR-125b-2-3p, hsa-miR-1286, hsa-miR-129-5p, hsa-m iR-1293, hsa-miR-1302, hsa-miR-1324, hsa-miR-1343-3p, hsa-miR-1470, hsa-miR-147b-5p, hsa-miR-186-3p, hsa-miR-1912-5p, hsa-miR-196a-5 p, hsa-miR-196b-5p, hsa-miR-1976, hsa-miR-205-5p, hsa-miR-2116-5p, hsa-miR-22-5p, hsa-miR-221-5p, hsa-miR-24-3p, hsa-miR-2467-5p, hsa -miR-2682-3p, hsa-miR-26b-3p, hsa-miR-30a-3p, hsa-miR-30d-3p, hsa-miR-30e-3p, hsa-miR-31-3p, hsa-miR-3133, hsa-miR-3145-3p, hsa-miR-3 152-5p, hsa-miR-3155a, hsa-miR-3155b, hsa-miR-3166, hsa-miR-3171, hsa-miR-3175, hsa-miR-3177-3p, hsa-miR-3184-5p, hsa-miR-3188, hsa-m iR-3190-3p, hsa-miR-324-5p, hsa-miR-326, hsa-miR-330-5p, hsa-miR-3529-5p, hsa-miR-3612, hsa-miR-362-5p, hsa-miR-3621, hsa-miR-363-5p hsa-miR-3655 hsa-miR-365a-5p hsa-miR-365b-5p hsa-miR-3675-5p hsa-miR-3680-3p hsa-miR-3682-3p hsa-miR-3682-5p hsa-miR-3686 h sa-miR-3688-5p, hsa-miR-376a-3p, hsa-miR-376b-3p, hsa-miR-379-5p, hsa-miR-3925-5p, hsa-miR-3940-3p, hsa-miR-3942-3p, hsa-miR-423-5phsa-miR-4273, hsa-miR-4287, hsa-miR-4298, hsa-miR-4457, hsa-miR-4483, hsa-miR-4484, hsa-miR-450a-1-3p, hsa-miR-4632-3p, hsa-miR-4659a-3p, hsa-miR-4659a-5p, hsa-miR-4659b-3p, hsa-miR-4660, hsa-miR-4668-3p, hsa-miR-4685-3p, hsa-miR-4685-5p, hsa-miR-4687-3p, hsa-mi R-4695-5p, hsa-miR-4707-5p, hsa-miR-4722-5p, hsa-miR-4724-5p, hsa-miR-4727-3p, hsa-miR-4733-5p, hsa-miR-4736, hsa-miR-4740-3p, hsa- miR-4742-3p, hsa-miR-4757-5p, hsa-miR-4762-3p, hsa-miR-4764-3p, hsa-miR-4769-3p, hsa-miR-4774-3p, hsa-miR-4779, hsa-miR-4799-5p, hsa -miR-484, hsa-miR-486-5p, hsa-miR-495-3p, hsa-miR-500b-5p, hsa-miR-501-5p, hsa-miR-507, hsa-miR-518a-5p, hsa-miR-526b-5p, hsa-miR-527, hsa-miR-542-5p, hsa-miR-548as-3p, hsa-miR-548at-3p, hsa-miR-548aw, hsa-miR-548ay-3p, hsa-miR-551b-5p, hsa-miR-557, hsa-miR-5582 -3p, hsa-miR-5584-5p, hsa-miR-5587-3p, hsa-miR-5590-5p, hsa-miR-5687, hsa-miR-5688, hsa-miR-572, hsa-miR-576-5p, hsa-miR-590-3p, hsa -miR-6074, hsa-miR-6089, hsa-miR-6128, hsa-miR-623, hsa-miR-629-3p, hsa-miR-650, hsa-miR-6508-3p, hsa-miR-6511b-5p, hsa-miR-6515-3phsa-miR-6516-5p、hsa-miR-6721-5p、hsa-miR-6728-5p、hsa-miR-6729-3p、hsa-miR-6741-5p、hsa-miR-6745、hsa-miR-6750-5p、hsa-miR-6756-5p、hsa-miR-6764-3p、hsa-miR-6765-5p、hsa-miR-6766-5p、hsa-miR-6768-3p、hsa-miR-6774-5p、hsa-miR-6777-5p、hsa-miR-6781-3p、hsa-miR-6783-3p、hsa-miR-6792-5p、hsa-miR-6795-3p、hsa-miR-6807-5p、hsa-miR-6809-3p、hsa-miR-6811-5p、hsa-miR-6817-5p、hsa-miR-6820-5p、hsa-miR-6823-3p、hsa-miR-6824-3p、hsa-miR-6837-5p、hsa-miR-6844、hsa-miR-6873-3p、hsa-miR-6889-5p、hsa-miR-6890-5p、hsa-miR-6891-3p、hsa-miR-7-1-3p、hsa-miR-7-2-3p、hsa-miR-7113-3p、hsa-miR-7113-5p、hsa-miR-7151-5p、hsa-miR-7156-3p、hsa-miR-7156-5p、hsa-miR-7162-3p、hsa-miR-766-3p、hsa-miR-769-3p、hsa-miR-7853-5p、hsa-miR-8057、hsa-miR-8073、hsa-miR-874-5p、hsa-miR-885-5p、hsa-miR-887-5p、hsa-miR-95-3p、hsa-miR-9500、hsa-miR-9903、hsa-miR-9983-3p、hsa-let-7a-3p、hsa-let-7a-5p、hsa-let-7b-3p、hsa-let-7b-5p、hsa-let-7c-5p、hsa-let-7d-5p、hsa-let-7e-5p、hsa-let-7f-1-3p、hsa-let-7f-5p、hsa-let-7g-5p、hsa-let-7i-5p、hsa-miR-1-3p、hsa-miR-10392-3p、hsa-miR-10526-3p、hsa-miR-106a-5p, hsa-miR-106b-5p, hsa-miR-10b-3p, hsa-miR-1183, hsa-miR-1185-5p, hsa-miR-1199-3p, hsa-miR-1199-5p, hsa-miR-1200, hsa -miR-1208、hsa-miR-12124、hsa-miR-12132、hsa-miR-12135、hsa-miR-122-3p、hsa-miR-1226-5p、hsa-miR-122b-3p、hsa-miR-122b-5p、hsa-miR-12 37-3p、hsa-miR-1245a、hsa-miR-1245b-5p、hsa-miR-1247-5p、hsa-miR-1 248、hsa-miR-1251-3p、hsa-miR-1252-3p、hsa-miR-125b-1-3p、hsa-miR-1 261, hsa-miR-1285-3p, hsa-miR-1288-3p, hsa-miR-1291, hsa-miR-1296-3p, hsa-miR-1298-5p, hsa-miR-1306-5p, hsa-miR-130b-5p, hsa-miR-132- 3p, hsa-miR-1323, hsa-miR-134-3p, hsa-miR-135a-5p, hsa-miR-135b-5p, hsa-miR-136-3p, hsa-miR-136-5p, hsa-miR-139-5p, hsa-miR-140-3p, h sa-miR-1468-3p, hsa-miR-149-3p, hsa-miR-149-5p, hsa-miR-153-5p, hsa-miR-1537-3p, hsa-miR-155-5p, hsa-miR-17-5p, hsa-miR-181a-2-3p, hs a-miR-181d-3p, hsa-miR-187-5p, hsa-miR-1909-3p, hsa-miR-191-5p, hsa-miR-194-3p, hsa-miR-200c-5p, hsa-miR-202-3p, hsa-miR-203a-3p, hsa -miR-2052、hsa-miR-206、hsa-miR-208b-5p、hsa-miR-20a-5p、hsa-miR-20b-3p、hsa-miR-20b-5p、hsa-miR-21-3p、hsa-miR-211-3p、hsa-miR-2110、hsa-miR-2115-3p, hsa-miR-212-3p, hsa-miR-216a-5p, hsa-miR-216b-3p, hsa-miR-217-3p, hsa-miR-218-2-3p, hsa-miR-218-5p, hsa-miR-221-3p, hsa-miR-222-3p, hsa-miR-222-5p, hsa-miR-2277 -3p, hsa-miR-2392, hsa-miR-25-3p, hsa-miR-27a-3p, hsa-miR-27b-3p, hsa-miR-29b-2-5p, hsa-miR-302a-5p, hsa-miR-3064-3p, hsa-miR-3074-5p, hsa-miR-3115, hsa-miR-3116, hsa-miR-3130-3p hsa-miR-3136-3p hsa-miR-3136-5p hsa-miR-3137 hsa-miR-3142 hsa-miR-3144-5p hsa-miR-3148 hsa-miR-3149 hsa-miR-3150b-3p hsa-miR-3152-3p hsa-miR-3157-5p hsa-miR-3160-3p h sa-miR-3162-5p, hsa-miR-3167, hsa-miR-3173-3p, hsa-miR-3179, hsa-miR-3180-5p, hsa-miR-3187-5p, hsa-miR-3191-3p, hsa-miR-3194-5p, hsa-miR-32-5p, hsa-miR-3200-3p, hsa-miR-320a-3p hsa-miR-320a-5p hsa-miR-320b hsa-miR-320c hsa-miR-320d hsa-miR-324-3p hsa-miR-328-3p hsa-miR-329-3p hsa-miR-330-3p hsa-miR -331-5p, hsa-miR-335-5p, hsa-miR-33a-3p, hsa-miR-340-3p, hsa-miR-345-5p, hsa-miR-34a-3p, hsa-miR-34b-3p, hsa-miR-3605-5p, hsa-miR-36 09, hsa-miR-361-3p, hsa-miR-3610, hsa-miR-3613-3p, hsa-miR-3614-3p, hsa-miR-3616-5p, hsa-miR-3617-3p, hsa-miR-362-3p, hsa-miR-3620-3 p、hsa-miR-3622a-5p、hsa-miR-363-3p、hsa-miR-365a-3p、hsa-miR-365b-3p、hsa-miR-3662、hsa-miR-3663-3p、hsa-miR-3671、hsa-miR-3675-3p、 hsa-miR-3679-3p, hsa-miR-3679-5p, hsa-miR-3688-3p, hsa-miR-3689a-5p, hsa-miR-3689b-5p, hsa-miR-3689e, hsa-miR-3689f, hsa-miR-369-3p, hsa-miR-3690, hsa-miR-3691-5p, hsa-miR-370-3p, hsa-miR-3714, hsa-miR-371a-5p, hsa-miR-371b-5p, hsa-miR-373-5p, hsa-miR-375-3p, hsa -miR-377-3p、hsa-miR-378g、hsa-miR-383-5p、hsa-miR-3910、hsa-miR-3919、hsa-miR-3920、hsa-miR-3928-3p、hsa-miR-3934-3p、hsa-miR-3940- 5p, hsa-miR-3943, hsa-miR-409-3p, hsa-miR-421, hsa-miR-4270, hsa-miR-4288, hsa-miR-4290, hsa-miR-4292, hsa-miR-4429, hsa-miR-4446-3phsa-miR-4456, hsa-miR-4458, hsa-miR-4474-3p, hsa-miR-4475, hsa-miR-4477a, hsa-miR-4496, hsa-miR-4500, hsa-miR-4507, hsa-miR-4516, hs a-miR-4519, hsa-miR-4633-3p, hsa-miR-4639-5p, hsa-miR-4646-3p, hsa-miR-4652-3p, hsa-miR-4652-5p, hsa-miR-4653-3p, hsa-miR-4661-3p, h sa-miR-4665-3p, hsa-miR-4676-5p, hsa-miR-4677-3p, hsa-miR-4690-5p, hsa-miR-4691-3p, hsa-miR-4709-5p, hsa-miR-4711-3p, hsa-miR-4712-3p, hsa-miR-4713-5p, hsa-miR-4714-3p, hsa-miR-4715-3p, hsa-miR-4719, hsa-miR-4720-3p, hsa-miR-4728-3p, hsa-miR-4728-5p, hsa-miR-473 1-3p, hsa-miR-4731-5p, hsa-miR-4732-5p, hsa-miR-4734, hsa-miR-4738-5p, hsa-miR-4740-5p, hsa-miR-4743-3p, hsa-miR-4744, hsa-miR-4747 -3p, hsa-miR-4752, hsa-miR-4753-3p, hsa-miR-4755-3p, hsa-miR-4758-3p, hsa-miR-4763-5p, hsa-miR-4764-5p, hsa-miR-4772-3p, hsa-miR-477 2-5p, hsa-miR-4782-5p, hsa-miR-4784, hsa-miR-4788, hsa-miR-4793-3p, hsa-miR-4793-5p, hsa-miR-4794, hsa-miR-4796-3p, hsa-miR-4797-5p hsa-miR-4801 hsa-miR-4804-3p hsa-miR-488-5p hsa-miR-490-3p hsa-miR-499b-5p hsa-miR-5002-5p hsa-miR-5088-5p hsa-miR-513a-3phsa-miR-513c-3p, hsa-miR-515-5p, hsa-miR-516b-5p, hsa-miR-5189-5p, hsa-miR-5196-3p, hsa-miR-519d-3p, hsa-miR-520g-3p, hsa-miR-520h, hsa-miR-526b-3p, hsa-miR-541-5p, hsa-miR-544b, hsa-miR-548ac, hsa-miR-548ad-3p, hsa-miR-548ae-3p, hsa-miR-548ah-3p, hsa-miR-548ah-5 p, hsa-miR-548aj-3p, hsa-miR-548aj-5p, hsa-miR-548am-3p, hsa-miR-548aq-3p, hsa-miR-548av-3p, hsa-miR-548bb-3p, hsa-miR-548d-3p, hsa-miR-548e-5p, hsa-miR-548f-5p, hsa-miR-548g-5p, hsa-miR-548h-3p, hsa-miR-548j-3p, hsa-miR-548k, hsa-miR-548l, hsa-miR-548o-3p, hsa-miR -548q、hsa-miR-548x-5p、hsa-miR-548z、hsa-miR-550a-3p、hsa-miR-558 、hsa-miR-5582-5p、hsa-miR-5680、hsa-miR-5690、hsa-miR-5692a、hsa-m iR-5692c, hsa-miR-5696, hsa-miR-5699-5p, hsa-miR-5702, hsa-miR-5703, hsa-miR-5706, hsa-miR-573, hsa-miR-579-3p, hsa-miR-580-3p, hsa-mi R-583, hsa-miR-603, hsa-miR-6090, hsa-miR-612, hsa-miR-615-5p, hsa-miR-616-3p, hsa-miR-616-5p, hsa-miR-624-5p, hsa-miR-627-3p, hsa-mi R-629-5p, hsa-miR-632, hsa-miR-642a-3p, hsa-miR-642a-5p, hsa-miR-642b-3p, hsa-miR-642b-5p, hsa-miR-647, hsa-miR-649, hsa-miR-6499-3phsa-miR-6505-5p, hsa-miR-6507-3p, hsa-miR-6507-5p, hsa-miR-651-3p, hsa-miR-6510-3p, hsa-miR-6510-5p, hsa-miR-6512-3p, hsa-miR-6515- 5p, hsa-miR-6529-3p, hsa-miR-656-3p, hsa-miR-664a-5p, hsa-miR-664b-3p, hsa-miR-6715a-3p, hsa-miR-6720-5p, hsa-miR-6722-3p, hsa-miR-6 724-5p, hsa-miR-6727-5p, hsa-miR-6735-3p, hsa-miR-6749-3p, hsa-miR-6751-3p, hsa-miR-6754-5p, hsa-miR-6757-3p, hsa-miR-6758-3p, hsa-m iR-6758-5p, hsa-miR-6760-3p, hsa-miR-6760-5p, hsa-miR-6767-3p, hsa-miR-6769a-3p, hsa-miR-6771-3p, hsa-miR-6773-5p, hsa-miR-6774-3p hsa-miR-6775-3p, hsa-miR-6778-3p, hsa-miR-6782-5p, hsa-miR-6783-5p, hsa-miR-6784-3p, hsa-miR-6785-5p, hsa-miR-6787-3p, hsa-miR-6791-5p, hsa-miR-6792-3p, hsa-miR-6798-5p, hsa-miR-6799-5p, hsa-miR-6800-3p, hsa-miR-6801-3p, hsa-miR-6810-3p, hsa-miR-6810-5p, hsa-miR- 6813-3p, hsa-miR-6827-3p, hsa-miR-6827-5p, hsa-miR-6830-3p, hsa-miR-6830-5p, hsa-miR-6832-5p, hsa-miR-6834-5p, hsa-miR-6836-3p, hsa- miR-6836-5p, hsa-miR-6839-3p, hsa-miR-6842-3p, hsa-miR-6851-5p, hsa-miR-6852-5p, hsa-miR-6856-3p, hsa-miR-6856-5p, hsa-miR-6857-5phsa-miR-6858-3p、hsa-miR-6860、hsa-miR-6861-5p、hsa-miR-6862-3p、hsa-miR-6865-3 p、hsa-miR-6868-3p、hsa-miR-6885-3p、hsa-miR-6889-3p、hsa-miR-6890-3p、hsa-miR-6 891-5p、hsa-miR-6892-3p、hsa-miR-6893-3p、hsa-miR-7108-5p、hsa-miR-7109-3p、hsa- miR-7152-3p、hsa-miR-7152-5p、hsa-miR-7154-3p、hsa-miR-7155-3p、hsa-miR-718、hsa- miR-758-5p、hsa-miR-7704、hsa-miR-8062、hsa-miR-873-5p、hsa-miR-876-5p、hsa-miR- 877-3p、hsa-miR-889-5p、hsa-miR-892a、hsa-miR-892c-5p、hsa-miR-9-3p、hsa-miR-92a- 3p、hsa-miR-92b-3p、hsa-miR-93-5p、hsa-miR-939-3p、hsa-miR-98-3p、hsa-miR-98-5p、 hsa-miR-9985、hsa-miR-99a-3p、hsa-miR-99 b-3p (b) A sequence selected from the group consisting of sequence numbers 9 to 944, or a sequence consisting of such sequence A functional nucleic acid molecule according to any one of claims 1 to 4.

6. A functional nucleic acid molecule according to any one of claims 1 to 5, wherein the 3'UTR of Fan1 is as shown in any one of the sequences selected from the group consisting of SEQ ID NOs: 1 to 8.

7. A functional nucleic acid molecule according to any one of claims 1 to 6, wherein one or more of the consecutive nucleotide sequences include or consist of a sequence independently selected from the group consisting of SEQ ID NOs: 949-973; 974-998; 999-1064; 1065-1130; 1137-1138; and / or 1139-1148; or includes or consists of a sequence having at least 90% sequence identity with a sequence independently selected from the group consisting of SEQ ID NOs: 949-973; 974-998; 999-1064; 1065-1130; 1137-1138; and / or 1139-1148.

8. The functional nucleic acid molecule according to any one of claims 1 to 7, wherein the functional nucleic acid molecule comprises one or more modified nucleosides.

9. The functional nucleic acid molecule according to claim 8, wherein the one or more modified nucleosides independently comprise a modified sugar moiety or a modified nucleobase.

10. The functional nucleic acid molecule according to claim 8 or 9, wherein the one or more modified nucleosides independently comprise a modified sugar moiety independently selected from one or more of 2'-deoxy, 2'-MOE, 2'-OMe, and 2'-F:.

11. The functional nucleic acid molecule according to any one of claims 8 to 10, wherein the one or more modified nucleosides include a bicyclic sugar moiety.

12. A functional nucleic acid molecule according to any one of claims 8 to 10, comprising one or more modified nucleosides selected from LNA and cET.

13. The functional nucleic acid molecule according to any one of claims 1 to 12, wherein the functional nucleic acid molecule comprises one or more modified nucleoside interbonds.

14. The functional nucleic acid molecule according to any one of claims 1 to 13, wherein the functional nucleic acid molecule comprises one or more nucleotide analogs.

15. A functional nucleic acid molecule according to any one of claims 1 to 14 and a conjugate comprising one or more portions covalently bonded to the functional nucleic acid molecule.

16. A pharmaceutically acceptable salt of a functional nucleic acid molecule according to any one of claims 1 to 14 or a conjugate according to claim 15.

17. A composition comprising a functional nucleic acid molecule according to any one of claims 1 to 14, a conjugate according to claim 15, or a pharmaceutically acceptable salt according to claim 16, and a diluent, solvent, carrier, salt, and / or adjuvant.

18. A pharmaceutical composition comprising a functional nucleic acid molecule according to any one of claims 1 to 14, a conjugate according to claim 15, or a pharmaceutically acceptable salt according to claim 16, and a pharmaceutically acceptable diluent, solvent, carrier, salt, and / or adjuvant.

19. An in vivo or in vitro method for regulating Fan1 protein expression in target cells containing Fan1 and Fan1-targeting microRNAs, the method comprising the step of exposing the cells to a functional nucleic acid molecule according to any one of claims 1 to 14, a conjugate according to claim 15, or a pharmaceutically acceptable salt according to claim 16, a composition according to claim 17, or a pharmaceutical composition according to claim 18.

20. A method for treating, preventing, or delaying the onset of a Fan1 protein-related disease in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of a functional nucleic acid molecule according to any one of claims 1 to 14, a conjugate according to claim 15, a pharmaceutically acceptable salt according to claim 16, or a pharmaceutical composition according to claim 18.

21. A functional nucleic acid molecule according to any one of claims 1 to 14, a conjugate according to claim 15, a pharmaceutically acceptable salt according to claim 16, or a pharmaceutical composition according to claim 18, for use in treating, preventing, or delaying the onset of a disease.

22. Use of a functional nucleic acid molecule according to any one of claims 1 to 14, a conjugate according to claim 15, a pharmaceutically acceptable salt according to claim 16, or a pharmaceutical composition according to claim 18 for the preparation of a medicament for the treatment, prevention, or delay of a disease.

23. The method according to claim 20, wherein the disease is a triplet repeat disorder; a functional nucleic acid molecule, conjugate, pharmaceutically acceptable salt, or pharmaceutical composition for use according to claim 21; or the use of a functional nucleic acid molecule, conjugate, pharmaceutically acceptable salt, or pharmaceutical composition for use according to claim 22.

24. The method according to claim 20 or 23, wherein the disease is a disease related to or caused by CAG triplet repeat elongation, CGG triplet repeat elongation, CTG triplet repeat elongation, GAA triplet repeat elongation, GCC triplet repeat elongation, or GCG triplet repeat elongation; a functional nucleic acid molecule, conjugate, pharmaceutically acceptable salt, or pharmaceutical composition for use according to claim 21 or 23; or the use of a functional nucleic acid molecule, conjugate, pharmaceutically acceptable salt, or pharmaceutical composition for use according to claim 22 or 23.

25. The method according to any one of claims 20, 23, or 24, wherein the disease is selected from the group consisting of Huntington's disease (HD), spinocerebellar ataxia type 1 (SCA1), spinocerebellar ataxia type 2 (SCA2), spinocerebellar ataxia type 3 (SCA3), spinocerebellar ataxia type 6 (SCA6), spinocerebellar ataxia type 7 (SCA7), spinocerebellar ataxia type 17 (SCA17), dentatorubral-pallidoluysian atrophy (DRPLA), and spinal and bulbar muscular atrophy, X-linked 1 (SMAX1 / SBMA); a functional nucleic acid molecule, conjugate, pharmaceutically acceptable salt, or pharmaceutical composition for use according to any one of claims 21, 23, or 24; or the use of a functional nucleic acid molecule, conjugate, pharmaceutically acceptable salt, or pharmaceutical composition for use according to any one of claims 22, 23, or 24.

26. The method according to any one of claims 20 and 23-25, wherein the disease is Huntington's disease (HD); a functional nucleic acid molecule, conjugate, pharmaceutically acceptable salt, or pharmaceutical composition for use according to any one of claims 21 and 23-25; or the use of a functional nucleic acid molecule, conjugate, pharmaceutically acceptable salt, or pharmaceutical composition for use according to any one of claims 22 and 23-25.

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