Products and methods for treating diseases or conditions associated with variant or pathogenic KCNQ3 expression

Abstract

Products, methods, and uses for treating, ameliorating, or delaying the progression of, and / or preventing seizures, epileptic disorders or diseases, intellectual or developmental disorders, autism, or autism spectrum disorders associated with the expression of mutant or pathogenic potassium channels, voltage-dependent KQT-like subfamily Q, member 3 (KCNQ3) are disclosed herein. More specifically, RNA interference-based products, methods, and uses for reducing or inhibiting the expression of the KCNQ3 gene and the resulting mRNA and / or protein are disclosed herein. Even more specifically, the present disclosure provides microRNAs (miRNAs) for reducing or inhibiting the expression of KCNQ3, and methods of using such miRNAs for reducing or inhibiting mutant or pathogenic KCNQ3 expression in cells having a genetic mutation in the KCNQ3 gene and / or in cells of a subject that result in disease symptoms including, but not limited to, seizures, epilepsy, intellectual and / or developmental disorders, autism, or autism spectrum disorders. Such disease symptoms result, in some embodiments, from developmental epileptic encephalopathy (DEE) caused by various mutations in the KCNQ3 gene that result in the expression of various mutant or pathogenic forms of the KCNQ3 protein.

Description

【Technical Field】 【0001】 Incorporation by reference of a sequence listing This application includes, as a separate part of the disclosure, a sequence listing in computer-readable form (filename: 57884_Seqlisting.XML, size: 92,046 bytes, created on June 1, 2023), which is hereby incorporated by reference in its entirety into this specification. 【0002】 The present disclosure relates to the field of treating diseases associated with mutations or pathogenic expression of potassium channels, voltage-dependent KQT-like subfamily Q, member 3 (KCNQ3) gene and the resulting proteins. More specifically, the present disclosure provides RNA interference-based products, methods, and uses for treating, ameliorating, delaying the progression of, and / or preventing diseases or conditions associated with mutant or pathogenic expression of KCNQ3 protein resulting from one or more mutations of the KCNQ3 gene. Specifically, the present disclosure provides products and methods for reducing or inhibiting the expression of one or more mutant or pathogenic forms of KCNQ3 by interfering with KCNQ3 gene expression mainly by binding to messenger RNA (mRNA) in the cytoplasm. More specifically, the present disclosure provides microRNA (miRNA) for reducing or inhibiting mutant or pathogenic expression of KCNQ3, and methods of using such miRNA to reduce or inhibit mutant or pathogenic KCNQ3 expression in cells and / or subjects having neuronal excitability or disease symptoms resulting from such mutant or pathogenic KCNQ3 expression. Such disease symptoms include, but are not limited to, seizures, epilepsy, intellectual and / or developmental disorders, autism, or autism spectrum disorders. Such disease symptoms are, in some aspects, due to developmental epileptic encephalopathy (DEE) resulting from various mutations of the KCNQ3 gene, which result in various mutant or pathogenic forms of the KCNQ3 protein. 【Background Art】 【0003】 Standard drug therapies are generally ineffective in children with developmental and epileptic encephalopathy (DEE), despite the fact that over 30% of cases are now accurately genetically diagnosed as novel single gene variants (Non-Patent Document 1). Approximately 40% of DEE genes with known pathogenic variants appear to require the expression of defective gene products (i.e., gain of function or dominant negative), as opposed to encoding partial, e.g., haploinsufficiency, or complete loss of expression, and these tend to encode more severe diseases (Non-Patent Document 2). Facing this challenge and considering the recent rapid progress of gene therapy technology, due to the precise specificity that gene therapy has for genetic lesions, RNAi technology offers great promise for patients with gain-of-function variants and no other many options for effective treatment. 【0004】 This disclosure provides products, compositions, and methods for an RNAi approach to reducing the expression of a pathogenic variant (KCNQ3-R230H) involved in a form of DEE. This approach is scaled down to be implemented using a mouse model that expresses the orthologous genotype (i.e., Kcnq3R231 H / + ). Since mice that completely lack Kcnq3 from conception have very mild impairments with respect to overt clinical phenotypes or seizures (Non-Patent Document 3), an RNAi construct was developed to target both mutant and wild-type copies of Kcnq3 mRNA. Using this approach, reducing wild-type Kcnq3 mRNA will likely have little or no harmful effects in unaffected subjects, but reducing mutant Kcnq3 mRNA will significantly reduce phenotypic characteristics in subjects that model or suffer from human disease. 【0005】 RNA interference (RNAi) is a mechanism of gene regulation in eukaryotic cells and has been considered for the treatment of various diseases. RNAi refers to the post-transcriptional regulation of gene expression mediated by microRNA (miRNA). miRNA is a small (21-25 nucleotides), non-coding RNA that shares sequence homology and base pairs with the 3'untranslated region of cognate messenger RNA (mRNA). The interaction between miRNA and mRNA supports a cellular gene silencing mechanism that blocks mRNA translation. The RNAi pathway is summarized in Non-Patent Document 4. 【0006】 As the understanding of the natural RNAi pathway has advanced, researchers have designed artificial miRNAs for use in regulating the expression of target genes for the treatment of diseases. As described in Section 7.4 of Duan above, artificial miRNAs can be transcribed from DNA expression cassettes. The miRNA sequence specific to the target gene is transcribed together with the sequences necessary to direct miRNA processing in the cell. Viral vectors such as adeno-associated virus (AAV) have been used to deliver miRNAs to muscle and the brain and nervous system [Non-Patent Document 5]. 【0007】 AAV has unique features that make it attractive as a vector for delivering foreign DNA into cells, for example, in gene therapy. AAV infection of cells in culture is non-cytopathic, and natural infection in humans and other animals is asymptomatic and latent. Furthermore, AAV can infect many mammalian cells, giving the potential to target many different tissues in vivo. Additionally, AAV can transduce both slowly dividing and non-dividing cells and persist essentially throughout the lifespan of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is infectious as cloned DNA in a plasmid, enabling the construction of recombinant genomes. Moreover, since the signals that direct AAV replication, genome capsid formation, and integration are contained within the ITRs of the AAV genome, part or all of the internal ~4.3 kb genome (encoding the replication and structural capsid proteins rep-cap) can be replaced with foreign DNA. The rep and cap proteins can be provided in trans. Another important feature of AAV is that it is a very stable and long-lasting virus. This allows it to easily withstand the conditions (several hours at 56 - 65 °C) used to inactivate adenovirus, reducing the importance of cryopreservation for AAV. AAV can also be lyophilized. Finally, AAV-infected cells are resistant to superinfection. 【0008】 Care of patients suffering from DEE is limited to treatments that attempt to address the symptoms of the disorder and usually have limited results. Patients are examined by physical and occupational therapists, speech and language therapists, and developmental specialists for neurodevelopmental delays and autistic symptoms. In addition, patients can be treated with medications to address behavioral problems, sleep disorders, and / or seizures. Despite these attempts, patients are left substantially disabled, non-verbal, and ultimately unable to care for themselves as they reach adulthood. Therefore, the development of products and methods for effective disease-modifying therapies for the form of DEE associated with pathogenic variants of the KCNQ3 gene represents an important unmet need. 【Prior Art Documents】 【Non-Patent Documents】 【0009】 [Non-Patent Document 1] Stefanski et al., Epilepsia, 2021. 62(1): p. 143-151 [Non-Patent Document 2] Wang et al. (2021) Neurobiol Dis 148:105220 [Non-Patent Document 3] Soh et al. (2014) J Neurosci. 34:5311-21 [Non-Patent Document 4] Duan (Ed.), Muscle Gene Therapy, Chapter 7, Section 7.3, Springer Science+Business Media, LLC (2010) [Non-Patent Document 5] Fechner et al., J. Mol. Med., 86:987-997 (2008) [Summary of the Invention] 【0010】 The present disclosure provides products, methods, and uses for reducing or inhibiting KCNQ3 gene expression and ultimately preventing the translation of mutant or pathogenic KCNQ3 in order to treat, improve, delay the progression of, and / or prevent seizures, epilepsy, intellectual and / or developmental disorders, autism, or autism spectrum disorders associated with mutant or pathogenic KCNQ3 expression, including but not limited to developmental epileptic encephalopathy (DEE). The present disclosure provides products, methods, and uses for reducing or inhibiting the KCNQ3 gene expression of mutant or pathogenic variants of KCNQ3. Such mutations include various missense mutations, gain-of-function mutations, and any mutations that alter the expression of the KCNQ3 protein. In some embodiments, such mutations in the KCNQ3 gene are known variants or pathogenic variants including, but not limited to, KCNQ3-R230C, KCNQ3-R230H, KCNQ3-R230S, and / or KCNQ3-R227Q. 【0011】 The present disclosure provides nucleic acids designed to reduce or inhibit the expression of KCNQ3, or mutant or pathogenic expression of KCNQ3, viral vectors comprising the nucleic acids, compositions comprising the nucleic acids and vectors, methods for using these products to reduce, inhibit, and / or prevent the expression of mutant or pathogenic KCNQ3 genes in cells, and methods for treating or improving a disease in a subject suffering from a disease resulting from the expression of a mutant or pathogenic variant of KCNQ3 including, but not limited to, KCNQ3-R230C, KCNQ3-R230H, KCNQ3-R230S, and / or KCNQ3-R227Q. 【0012】 The present disclosure provides a nucleic acid encoding a potassium channel, voltage-dependent KQT-like subfamily Q, member 3 (KCNQ3)-targeting microRNA (miRNA), comprising a nucleotide sequence that (a) comprises at least 90% identity with a sequence shown in any one of SEQ ID NOs: 3-9, (b) is a nucleotide sequence shown in any one of SEQ ID NOs: 3-9, (c) is a nucleotide sequence encoding an RNA sequence shown in any one of SEQ ID NOs: 17-23, or (d) is a nucleotide sequence that specifically hybridizes to a KCNQ3 sequence shown in any one of SEQ ID NOs: 24-30. Such nucleic acids further comprise a promoter and / or enhancer in some embodiments. In some embodiments, such a promoter and / or enhancer is any one of a U6 promoter and / or enhancer, a U7 promoter and / or enhancer, a tRNA promoter and / or enhancer, an H1 promoter and / or enhancer, a CMV promoter and / or enhancer, a minimal CMV promoter and / or enhancer, a T7 promoter and / or enhancer, an EF1-alpha promoter and / or enhancer, a minimal EF1-alpha promoter and / or enhancer, an unc45b promoter and / or enhancer, a CK1 promoter and / or enhancer, a CK6 promoter and / or enhancer, a CK7 promoter and / or enhancer, a CK8 promoter and / or enhancer, a ubiquitous promoter and / or enhancer, a neuron-specific promoter and / or enhancer, or a brain-specific promoter and / or enhancer. In some embodiments, the promoter and / or enhancer is U6. In some embodiments, the nucleic acid comprises a nucleotide sequence that (a) comprises at least 90% identity with a sequence shown in any one of SEQ ID NOs: 10-16, or (b) is a nucleotide sequence shown in any one of SEQ ID NOs: 10-16.In some embodiments, the brain-specific promoter and / or enhancer is human synapsin 1 (hSyn1), neuron-specific enolase (Nse), MeCP2, mDLX, mDLX5 / 6, or calmodulin-dependent kinase II (CaMKII or Camk2a). 【0013】 The present disclosure provides an adeno-associated virus comprising a nucleic acid encoding a potassium channel, voltage-dependent KQT-like subfamily Q, member 3 (KCNQ3)-targeting microRNA (miRNA), the nucleic acid comprising (a) a nucleotide sequence having at least 90% identity with a sequence shown in any one of SEQ ID NOs: 3-9, (b) a nucleotide sequence shown in any one of SEQ ID NOs: 3-9, (c) a nucleotide sequence encoding an RNA sequence shown in any one of SEQ ID NOs: 17-23, or (d) a nucleotide sequence that specifically hybridizes to a KCNQ3 sequence shown in any one of SEQ ID NOs: 24-30. Such nucleic acids further comprise a promoter and / or enhancer in some embodiments. In some embodiments, such promoter and / or enhancer is any one of a U6 promoter and / or enhancer, a U7 promoter and / or enhancer, a tRNA promoter and / or enhancer, an H1 promoter and / or enhancer, a CMV promoter and / or enhancer, a minimal CMV promoter and / or enhancer, a T7 promoter and / or enhancer, an EF1-alpha promoter and / or enhancer, a minimal EF1-alpha promoter and / or enhancer, an unc45b promoter and / or enhancer, a CK1 promoter and / or enhancer, a CK6 promoter and / or enhancer, a CK7 promoter and / or enhancer, a CK8 promoter and / or enhancer, a ubiquitous promoter and / or enhancer, a neuron-specific promoter and / or enhancer, or a brain-specific promoter and / or enhancer. In some embodiments, the promoter and / or enhancer is U6. In some embodiments, the nucleic acid comprises (a) a nucleotide sequence having at least 90% identity with a sequence shown in any one of SEQ ID NOs: 10-16, or (b) a nucleotide sequence shown in any one of SEQ ID NOs: 10-16.In some embodiments, the brain-specific promoter and / or enhancer is human synapsin 1 (hSyn1), neuron-specific enolase (Nse), MeCP2, mDLX, mDLX5 / 6, or calmodulin-dependent kinase II (CaMKII or Camk2a). In some embodiments, the adeno-associated virus lacks the rep and cap genes. In some embodiments, the virus is a recombinant AAV (rAAV) or self-complementary recombinant AAV (scAAV). In some embodiments, the virus is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rh10, AAV11, AAV12, AAV13, AAV-anc80, AAV-B1, AAV.PHP.EB, or AAVv66. In some embodiments, the virus is AAV9. 【0014】 The present disclosure provides a nanoparticle, extracellular vesicle, or exosome comprising a nucleic acid encoding a potassium channel, voltage-dependent KQT-like subfamily Q, member 3 (KCNQ3)-targeting microRNA (miRNA), the nanoparticle, extracellular vesicle, or exosome comprising: (a) a nucleotide sequence having at least 90% identity with a sequence shown in any one of SEQ ID NOs: 3-9; (b) a nucleotide sequence shown in any one of SEQ ID NOs: 3-9; (c) a nucleotide sequence encoding an RNA sequence shown in any one of SEQ ID NOs: 17-23; or (d) a nucleotide sequence that specifically hybridizes to a KCNQ3 sequence shown in any one of SEQ ID NOs: 24-30. Such nucleic acids further comprise a promoter and / or enhancer in some embodiments. In some embodiments, such promoter and / or enhancer is any one of a U6 promoter and / or enhancer, a U7 promoter and / or enhancer, a tRNA promoter and / or enhancer, an H1 promoter and / or enhancer, a CMV promoter and / or enhancer, a minimal CMV promoter and / or enhancer, a T7 promoter and / or enhancer, an EF1-alpha promoter and / or enhancer, a minimal EF1-alpha promoter and / or enhancer, an unc45b promoter and / or enhancer, a CK1 promoter and / or enhancer, a CK6 promoter and / or enhancer, a CK7 promoter and / or enhancer, a CK8 promoter and / or enhancer, a ubiquitous promoter and / or enhancer, a neuron-specific promoter and / or enhancer, or a brain-specific promoter and / or enhancer. In some embodiments, the promoter and / or enhancer is U6. In some embodiments, the nucleic acid comprises: (a) a nucleotide sequence having at least 90% identity with a sequence shown in any one of SEQ ID NOs: 10-16; or (b) a nucleotide sequence shown in any one of SEQ ID NOs: 10-16.In some embodiments, the brain-specific promoter and / or enhancer is human synapsin 1 (hSyn1), neuron-specific enolase (Nse), MeCP2, mDLX, mDLX5 / 6, or calmodulin-dependent kinase II (CaMKII or Camk2a). 【0015】 The present disclosure provides a composition comprising (a) any one or more of the nucleic acids described hereinabove or throughout the present disclosure, (b) any one or more of the adeno-associated viruses described hereinabove or throughout the present disclosure, or (c) any one or more of the nanoparticles, extracellular vesicles, or exosomes described hereinabove or throughout the present disclosure, and a pharmaceutically acceptable carrier. 【0016】 The present disclosure provides a method of reducing, inhibiting, and / or preventing the expression of the potassium channel, voltage-gated KQT-like subfamily Q, member 3 (KCNQ3) gene or a variant thereof in a cell, the method comprising contacting the cell with (a) any one or more of the nucleic acids described hereinabove or throughout the present disclosure, (b) any one or more of the adeno-associated viruses described hereinabove or throughout the present disclosure, or (c) any one or more of the nanoparticles, extracellular vesicles, or exosomes described hereinabove or throughout the present disclosure, or (d) a composition described hereinabove or throughout the present disclosure. 【0017】 The present disclosure provides a method of treating a subject having a KCNQ3 mutation that results in the expression of a mutant or pathogenic KCNQ3, the method comprising administering to the subject an effective amount of (a) any one or more of the nucleic acids described hereinabove or throughout the present disclosure, (b) any one or more of the adeno-associated viruses described hereinabove or throughout the present disclosure, or (c) any one or more of the nanoparticles, extracellular vesicles, or exosomes described hereinabove or throughout the present disclosure, or (d) any one or more of the compositions described hereinabove or throughout the present disclosure. In some embodiments, the mutation is a missense mutation or a gain-of-function mutation. In some embodiments, the mutation is a point mutation, a frameshift mutation, a base substitution, a deletion, or an insertion, or any combination of these mutations. In some embodiments, the mutation is a base substitution, a deletion, or an insertion, or any combination thereof. In some embodiments, the mutation is any one or more mutations in the KCNQ3 gene that result in a substitution of R230C, R230H, R230S, and / or R227Q in the KCNQ3 polypeptide. In some embodiments, the mutation results in a subject suffering from any of the various symptoms associated with mutant or pathogenic expression of KCNQ3. In some embodiments, the subject suffers from seizures, epileptic disease or disorder, intellectual or developmental disorder, autism, or autism spectrum disorder associated with mutant or pathogenic KCNQ3 expression. 【0018】 The present disclosure provides a method of treating or ameliorating a subject suffering from seizures, epileptic disorders or diseases, intellectual or developmental disorders, autism, or autism spectrum disorder associated with mutant or pathogenic KCNQ3 expression, the method comprising administering to the subject an effective amount of (a) any one or more of the nucleic acids described hereinabove or throughout the present disclosure, (b) any one or more of the adeno-associated viruses described hereinabove or throughout the present disclosure, or (c) any one or more of the nanoparticles, extracellular vesicles, or exosomes described hereinabove or throughout the present disclosure, or (d) any one or more of the compositions described hereinabove or throughout the present disclosure. In some embodiments, the subject has or is at risk of having developmental epileptic encephalopathy (DEE). In some embodiments, the subject has any mutation in the KCNQ3 gene. Such mutations include, but are not limited to, missense mutations and gain-of-function mutations. In some embodiments, the subject has a mutation in the KCNQ3 gene, the mutation being any one or more mutations in the KCNQ3 gene that result in substitution of R230C, R230H, R230S, and / or R227Q of the KCNQ3 polypeptide. 【0019】 The present disclosure provides the use of (a) any one or more of the nucleic acids described hereinabove or throughout the present disclosure, (b) any one or more of the adeno-associated viruses described hereinabove or throughout the present disclosure, or (c) any one or more of the nanoparticles, extracellular vesicles, or exosomes described hereinabove or throughout the present disclosure, or (d) any one or more of the compositions described hereinabove or throughout the present disclosure, for the preparation of a medicament for reducing or inhibiting the expression of the potassium channel, voltage-dependent KQT-like subfamily Q, member 3 (KCNQ3) gene or a variant thereof in a cell. 【0020】 The present disclosure provides the use of (a) any one or more of the nucleic acids described hereinabove or throughout the present disclosure, (b) any one or more of the adeno-associated viruses described hereinabove or throughout the present disclosure, or (c) any one or more of the nanoparticles, extracellular vesicles, or exosomes described hereinabove or throughout the present disclosure, or (d) any one or more of the compositions described hereinabove or throughout the present disclosure, for treating or ameliorating seizures, epileptic diseases or disorders, intellectual or developmental disorders, autism, or autism spectrum disorders associated with mutant or pathogenic KCNQ3 expression. In some embodiments, the seizures, epileptic diseases or disorders, intellectual or developmental disorders, autism, or autism spectrum disorders associated with mutant or pathogenic KCNQ3 expression are developmental epileptic encephalopathy (DEE). In some embodiments, the seizures, epileptic diseases or disorders, intellectual or developmental disorders, autism, or autism spectrum disorders associated with mutant or pathogenic KCNQ3 expression are due to any mutation in the KCNQ3 gene. In some embodiments, the mutation is any one or more mutations in the KCNQ3 gene that result in substitution of R230C, R230H, R230S, and / or R227Q of the KCNQ3 polypeptide. 【0021】 The present disclosure provides for the use of (a) any one or more of the nucleic acids described hereinabove or throughout the present disclosure, (b) any one or more of the adeno-associated viruses described hereinabove or throughout the present disclosure, or (c) any one or more of the nanoparticles, extracellular vesicles, or exosomes described hereinabove or throughout the present disclosure, or (d) any one or more of the compositions described hereinabove or throughout the present disclosure, for the preparation of a medicament for treating or ameliorating seizures, epileptic diseases or disorders, intellectual or developmental disorders, autism, or autism spectrum disorders associated with mutant or pathogenic KCNQ3 expression. In some embodiments, the seizures, epileptic diseases or disorders, intellectual or developmental disorders, autism, or autism spectrum disorders associated with mutant or pathogenic KCNQ3 expression are developmental epileptic encephalopathy (DEE). In some embodiments, the seizures, epileptic diseases or disorders, intellectual or developmental disorders, autism, or autism spectrum disorders associated with mutant or pathogenic KCNQ3 expression are caused by any mutation in the KCNQ3 gene. In some embodiments, the mutation is any one or more mutations in the KCNQ3 gene that result in substitution of R230C, R230H, R230S, and / or R227Q of the KCNQ3 polypeptide. 【0022】 The present disclosure provides for the use of (a) any one or more of the nucleic acids described hereinabove or throughout the present disclosure, (b) any one or more of the adeno-associated viruses described hereinabove or throughout the present disclosure, or (c) any one or more of the nanoparticles, extracellular vesicles, or exosomes described hereinabove or throughout the present disclosure, or (d) any one or more of the compositions described hereinabove or throughout the present disclosure, to reduce, inhibit, and / or prevent the expression of potassium channels, voltage-dependent KQT-like subfamily Q, member 3 (KCNQ3) gene or its variants in cells. In some embodiments, the cells are in a subject. In some embodiments, the variant of KCNQ3 results from any mutation in the KCNQ3 gene. In some embodiments, the variant of KCNQ3 results in substitutions of R230C, R230H, R230S, and / or R227Q of the KCNQ3 polypeptide. 【0023】 In some embodiments, the nucleic acids, AAVs, nanoparticles, extracellular vesicles, exosomes, or compositions, or pharmaceuticals of the present disclosure are formulated for intracerebroventricular injection, intrathecal injection, injection into the bloodstream, aerosol administration, or oral administration. 【0024】 Further aspects and advantages of the present disclosure will be apparent to those skilled in the art from a review of the following detailed description, taken in conjunction with the drawings. However, the detailed description, including the drawings and specific examples, illustrates embodiments of the disclosed subject matter and is provided by way of example only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS 【0025】 【Figure 1】Disclosed are the microRNA design parameters used to design the KCNQ3 miRNA of the present disclosure. Artificial microRNAs were designed using the aforementioned algorithms (Wallace et al. (2017) Mol Ther Methods Clin. Dev. Dec 24, 8:121-130, Boudreau et al. (2011) RNA Interference Methods. Ed. S.Q. Harper. Humana Springer Press, 2011, pages 19-37). Briefly, all microRNAs contain processing sites for the RNAse III enzymes Drosha and Dicer, resulting in a mature 22 nucleotide (nt) duplex RNA containing 2 nt 3' overhangs on both strands. The antisense guide strand of the microRNA is incorporated into the RNA-induced silencing complex (RISC), instructing the cellular gene silencing machinery to cleave the target mRNA, in this case human KCNQ3 or murine Kcnq3. 【Figure 2】Shows the sequence and structure of the miKCNQ3 construct. The upper left shows the native human mir30a sequence and structure. The artificial miRNA incorporates some sequence and structural features from mir30a, but with modifications. In particular, the underlined mature mir30a and corresponding sense strand located between the arrows in this figure are replaced with a 22-nucleotide sequence that targets conserved regions on the human, mouse, and rat KCNQ3 open reading frames. All microRNAs were designed as DNA constructs and cloned into the U6T6 plasmid using the indicated restriction enzymes (the SpeI site is ligated to the XbaI site located in the U6T6 polylinker). The upper right shows the RNA sequence of miKCNQ3-A, and the mature duplex sequence is located between the arrows. The gray arrow indicates the cleavage site for Drosha, while the black arrow indicates the Dicer cleavage site. The mismatches at positions 13 and 75 help facilitate proper Drosha processing, and the indicated mismatches were incorporated into each miRNA as shown. Positions 35 and 53 are shown for orientation. The antisense guide strands of all microRNAs (miKCNQ3 A-G) contain complete 22-nt basepair homology with the human, mouse, and rat KCNQ3 / Kcnq3 sequences. 【Figure 3】 Shows the constructed luciferase reporter plasmid containing the human KCNQ3 or rodent Kcnq3 sequence as the 3’UTR of Renilla luciferase used to measure the silencing of human or rodent KCNQ3. The reporter plasmid contained the second gene, firefly luciferase, used as a normalization control. 【Figure 4】Shows the results of luciferase assay screening. Various KCNQ3 miRNAs were cloned as the 3’UTR of Renilla luciferase. The second reporter, firefly luciferase, was present on the same plasmid and served as a non-targeted transfection control. HEK293 cells were transfected with the indicated reporter plasmid together with the U6.miKCNQ3 expression cassette or the miGFP control. The luciferase assay showed that each of the miKCNQ3 microRNAs of the present disclosure was able to knockdown human and rodent KCNQ3 / Kcnq3 transcripts. The luciferase assay identified miKCNQ3-A as a lead microRNA for initial experiments to knockdown human and rodent KCNQ3 / Kcnq3 transcripts. The data shown represent three independent experiments for each of the indicated reporter constructs. Error bars indicate S.E.M. 【Figure 5】 Shows representative clusters of three spike-wave discharges (arrows) in the EEG of Kcnq3R231H / + adult mice (lower two traces) compared to wild-type Kcnq3+ / + mice (upper two traces). “FR” and “FL” refer to the signals of bilateral electrodes located over the right (R) or left (L) hemisphere in front of the Bregma line relative to a reference electrode located over the cerebellum. By characterizing the clinically relevant phenotypic features of Kcnq3R231H / + mice, it was determined that the heterozygotes have a form of generalized epilepsy in the form of frequent, spontaneous spike-wave discharges (SWD) in the electroencephalogram (EEG), rather than wild-type littermates. Also, it was determined that the heterozygotes have a significantly lower threshold (increased sensitivity) to the maximum seizures induced electrically compared to wild-type littermates, indicating a general tendency to seizures. Each of these electroclinical features is quantitative and reproducible and represents a powerful and clinically relevant endpoint for measuring the effectiveness of new therapies. 【Figure 6】The Kcnq3R231H / + genotype does not cause growth retardation in mouse offspring (Figure 6A), or change the expression of Kcnq3 mRNA (Figure 6B) or total protein (Figure 6C), but is associated with an increase in KCNQ3 protein in the neuronal membrane (Figure 6D). 【Figure 7】 Shown is a significant decrease in the SWD incidence (Figure 7A) and average SWD duration (Figure 7B) in adult Kcnq3R231H / + mice transduced with scAAV9-miKCNQ3 as neonates. 【Figure 8】 Shown is a decrease in SWD incidence (upper panel) and a decrease in SWD duration (lower panel) in Kcnq3R231H / + adult mice transduced with scAAV9-miKcnq3-A as neonates. The dotted line indicates the same mice tested at both ages. The p-values shown are based on a one-sided Fisher's exact test. 【Figure 9】It is shown that there was a significant decrease in Kcnq3 mRNA in adult Kcnq3R231H / + mice transduced with scAAV9-miKCNQ3 as neonates. qPCR data were generated from mice previously treated with scAAV9-U6-miKcnq3a-eGFP or scAAV9-eGFP control virus and evaluated for EEG activity to measure endogenous Kcnq3 mRNA and endogenous Actb as an internal control. Exogenous eGFP mRNA was also measured as a control for AAV9 transduction. The number of different amplification cycles between Kcnq3 and Actb (ΔCt, upper panel), eGFP and Actb (ΔCt, middle panel), or Kcnq3-Actb-eGFP (ΔCt, lower panel) was determined and plotted. Figure 9 shows separate marker shapes or shadings reflecting different biological replicates (technical replicates share the marker shape). Since eGFP is an internal control for transduced cells, the statistical significance shown is between miKcnq3a and eGFP-treated mice (lower panel), using least squares regression and post hoc Dunnett's test with non-parametric transformed data. Excluding heterozygotes treated with one miKcnq3 still having some SWD, the fold difference in mRNA expression between heterozygotes treated with miKcnq3 and control virus was 6.78-fold. 【Figure 10】 Western blot (Figure 10A) with densitometric quantification (Figure 10B) measuring KCNQ3 levels and loading control, β-actin, in adult mouse brain after treatment as neonates with scAAV9-miKCNQ3 is shown. Figure 10B provides the results after densitometric quantification of KCNQ3 / β-actin. There was a significant decrease in KCNQ3 protein in adult mice transduced with scAAV9-miKCNQ3 as neonates. 【Figure 11】 The sequence of miKCNQ3A is shown, including the mature duplex sequence and the guide strand (antisense) sequence. 【Figure 12】 The sequence of miKCNQ3B is shown, including the mature duplex sequence and the guide strand (antisense) sequence. 【Figure 13】The sequence of miKCNQ3C is shown, including the mature duplex array and the guide strand (antisense) array. 【Figure 14】 The sequence of miKCNQ3D is shown, including the mature duplex array and the guide strand (antisense) array. 【Figure 15】 The sequence of miKCNQ3E is shown, including the mature duplex array and the guide strand (antisense) array. 【Figure 16】 The sequence of miKCNQ3F is shown, including the mature duplex array and the guide strand (antisense) array. 【Figure 17】 The sequence of miKCNQ3G is shown, including the mature duplex array and the guide strand (antisense) array. **DETAILED DESCRIPTION OF THE INVENTION** 【0026】 The present disclosure provides a novel strategy for achieving control of potassium channel, voltage-dependent KQT-like subfamily Q, member 3 (KCNQ3) gene expression by binding to KCNQ3 messenger RNA (mRNA) after transcription and suppressing or reducing or inhibiting KCNQ3 protein production because the expression of variants or pathogenic forms of KCNQ3 is due to gain-of-function mutations of KCNQ3. For example, such KCNQ3 mutations, known as KCNQ3 R230C / H / S and KCNQ3 R227Q mutations, are known to cause seizures and epilepsy, including but not limited to developmental epileptic encephalopathy (DEE), resulting in the production of pathogenic forms of KCNQ3 protein in the brain. Thus, in some aspects, the products and methods described herein are used to treat, improve, delay the progression of, and / or prevent seizures, epileptic disorders or disabilities, intellectual or developmental disabilities, neurodevelopmental disorders (NDDs), autism, or autism spectrum disorders, including but not limited to DEE. 【0027】 The KCNQ3 gene belongs to a large family of genes that provide instructions for making potassium channels. These channels transport positively charged potassium atoms (ions) across cell membranes and play an important role in the ability of cells to generate and transmit electrical signals. The specific function of a potassium channel depends on its protein components and its location within the body. Channels made from the KCNQ3 protein are active in nerve cells (neurons) in the brain, where they transport potassium ions out of the cell. These channels carry a specific type of electrical signal called the M-current, which prevents neurons from continuously sending signals to other neurons. The M-current ensures that neurons are not constantly active or excitable. Potassium channels are composed of several protein components (subunits). Each channel contains four alpha subunits that form a pore through which potassium ions move. Four alpha subunits from the KCNQ3 gene can form a channel. 【0028】 Several mutations in the KCNQ3 gene that result in mutant or pathogenic expression of the KCNQ3 protein have been identified in people suffering from DEE and / or seizures, epileptic disorders or diseases, intellectual or developmental disorders, neurodevelopmental disorders (NDDs), autism, or autism spectrum disorder. For example, various mutations in the KCNQ3 gene such as R230C, R230H, R230S, and R227Q have each been reported to be gain-of-function mutations in human patients (Sands et al., Ann. Neurol. 2019;86:181-92). Patients identified with such heterozygous KCNQ3 novel variants (DNVs) exhibited developmental delay, features of autism, autism spectrum disorder, and epileptiform discharges or epileptic spikes (Sands et al., Ann. Neurol. 2019;86:181-92). Gain-of-function mutations are a type of mutation in which the modified gene product has a new molecular function or a new pattern of gene expression. Gain-of-function (GoF) mutations are almost always dominant or semi-dominant. The present disclosure includes such various KCNQ3 gene mutations and products and methods for treating patients having such gene mutations. Although only a limited number of mutations are known, the present disclosure includes products and methods for treating any KCNQ3 gene mutation and patients having such gene mutations that result in mutant or pathogenic forms of the KCNQ3 protein. In some embodiments, such heritable mutations include gain-of-function mutations (R230C, R230H, R230S, and R227Q of KCNQ3) that cause DEE. Sands et al. predicted that the R227Q or R230C / S / H substitutions selectively destabilize the resting (closed) structure of the KCNQ3 voltage-sensing domain (VSD) and presumably account for the observed GoF effect. The miRNAs of the present disclosure are not allele-specific, but the products and methods of the present disclosure are designed to reduce or inhibit the expression of mutant forms of the KCNQ3 gene that result in mutant or pathogenic expression of the KCNQ3 protein. This is because patients with normal KCNQ3 gene expression do not require such therapeutic inventions. 【0029】 The present disclosure provides the use of a mouse model as a mechanistic basis for studying the effect of miRNAs on GoF by KCNQ3 R227 and R230 variants. The R231H variant in mice is equivalent to the R230 variant in humans. C57BL / 6J and FVB / NJ mice were purchased from The Jackson Laboratory and maintained by sibling mating in the animal facility of Columbia University. Kcnq3 R231H mice were developed at the Transgenic Core at Columbia Herbert Irving Comprehensive Cancer Center by using CRISPR / Cas9 mutagenesis with a donor oligonucleotide in C57BL / 6J conjugates having sgRNA 5'-GCAGGAUCUGCAGGAAGCGA-3' (SEQ ID NO: 38) to change the Arg231 CGC codon to CAC His and also remove the PstI restriction enzyme site for convenient genotyping. Founder mice were mated to wild-type C57BL / 6J and then backcrossed to wild-type C57BL / 6J to maintain the strain. For RNAi studies, male Kcnq3R231H / + heterozygotes were mated to wild-type FVB / NJ to generate an F1 hybrid population segregating for the Kcnq3 R231H mutation and used for viral injection, EEG testing, and assessment of mRNA and protein abundance (Sands et al., www.aesnet.org / abstractslisting / kcnq3-gain-of-function-mouse-model--electroclinical-and-behavioral-phenotype). 【0030】 The present disclosure provides products and methods designed to treat seizures, epileptic disorders or seizures, intellectual or developmental disorders, neurodevelopmental disorders (NDDs), autism, or autism spectrum disorder resulting from a variant or pathogenic expression of KCNQ3. The present disclosure provides products and methods for preventing, treating, or ameliorating conditions resulting from any mutation in the KCNQ3 gene that results in a variant or pathogenic expression of KCNQ3. More specifically, the present disclosure provides products and methods for preventing, treating, or ameliorating conditions resulting from genetic and / or novel missense mutations in the KCNQ3 gene. In some embodiments, the condition or disease resulting from a variant or pathogenic expression of KCNQ3 is DEE. In some embodiments, such genetic and / or novel mutations include gain-of-function mutations (R230C, R230H, R230S, and R227Q of KCNQ3) that cause DEE. Accordingly, the products and methods of the present disclosure are designed to treat diseases or disorders resulting from any mutation in the KCNQ3 gene, including but not limited to the R230C, R230H, R230S, and / or R227Q mutations that result in a variant or pathogenic expression of KCNQ3. 【0031】 Hereditary missense variants that result in loss of function cause autosomal dominant syndromes with neonatal seizures that respond to treatment and do not grow over time, and thus are not expected to benefit from the products and methods of the present disclosure. Another form of DEE is caused by homozygous mutations that result in loss of function, and similarly, this form is not expected to benefit from treatment. 【0032】 The KCNQ3 gene (Gene ID: 3786; ncbi.nlm.nih.gov / gene / 3786) encodes a protein that functions in the regulation of neuronal excitability. The encoded protein forms M channels by associating with the products of the related KCNQ2 or KCNQ5 genes, both of which also encode integral membrane proteins. M-channel currents are inhibited by the M1 muscarinic acetylcholine receptor and activated by retigabine, a novel antiepileptic drug. Defects in this KCNQ3 gene are the cause of epilepsy, benign familial neonatal type 2 (BFNC2), also known as benign neonatal convulsions type 2 (EBN2). Alternative splicing of this gene results in multiple transcript variants. 【0033】 In some embodiments, a nucleic acid encoding human KCNQ3 is represented by the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of human KCNQ3 is represented by the amino acid sequence set forth in SEQ ID NO: 2. In various embodiments, the methods of the present disclosure also target isoforms and variants of the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, a variant comprises 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, and 70% identity to the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the methods of the present disclosure target isoforms and variants of a nucleic acid comprising a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, a variant comprises 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, and 70% identity to the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2. 【0034】 【Table 1】 【0035】 In some embodiments, the products and methods are designed to treat KCNQ3-related disorders resulting from mutations in the KCNQ3 gene. Such KCNQ3-related disorders include, but are not limited to, DEE. The products and methods are designed to treat or reduce or inhibit mutant or pathogenic expression of KCNQ3 resulting from various mutations in the KCNQ3 gene. In some embodiments, such mutations in the KCNQ3 gene include, but are not limited to, R230C, R230H, R230S, and R227Q. Each of these specific mutations has been reported to be a gain-of-function mutation. The present disclosure includes products and methods for treating KCNQ3-related disorders resulting from such various KCNQ3 gene mutations. 【0036】 The present disclosure provides nucleic acids encoding microRNAs (miRNAs) that target KCNQ3 and variants of KCNQ3 and reduce or inhibit the expression of KCNQ3 and variants of KCNQ3. The nucleic acids include nucleotide sequences encoding microRNAs (miRNAs) that target KCNQ3 and variants of KCNQ3. The miRNA nucleotide sequences were specifically designed and selected using algorithms developed to predict effective artificial microRNAs (Figure 1 shows the criteria for selection). Using human KCNQ3 cDNA as a query sequence, the algorithm identified 152 promising microRNAs that meet the criteria listed in Figure 1. A second layer of selection was added by incorporating species conservation. Specifically, mouse, rat, and human KCNQ3 cDNAs were aligned. Of the 152 promising microRNAs, only 15 miRNAs contained a perfect 22-nucleotide base pairing between the antisense guide strand of the three species (i.e., mouse, rat, and human) and the KCNQ3 target site. As described herein, 7 of the 15 miRNAs were selected for construction and proof-of-concept testing. 【0037】 The present disclosure provides nucleic acids encoding miRNAs targeting KCNQ3 and variants of KCNQ3, and the nucleic acids also include promoter nucleotide sequences. The present disclosure provides nucleic acids comprising RNA sequences targeted by miRNAs. 【0038】 The present disclosure provides KCNQ3 sequences designed to be targeted by miRNA sequences. The present disclosure includes various nucleic acids comprising, consisting essentially of, or consisting of the various nucleotide sequences described herein. In some embodiments, the nucleic acids comprise nucleotide sequences. In some embodiments, the nucleic acids consist essentially of nucleotide sequences. In some embodiments, the nucleic acids consist of nucleotide sequences. 【0039】 Exemplary nucleotide sequences used for miRNA targeting of KCNQ3 described herein include, but are not limited to, those specified in Table 2 below and Figures 2 and 11 - 17. 【0040】 【Table 2 - 1】 【0041】 【Table 2 - 2】 【0042】 【Table 2 - 3】 【0043】 【Table 2 - 4】 【0044】 【Table 2 - 5】 【0045】 【Table 2-6】 【0046】 【Table 2-7】 【0047】 【Table 2-8】 【0048】 【Table 2-9】 【0049】 【Table 2-10】 【0050】 Exemplary nucleotide sequences are shown in Table 2 above and in FIGS. 2 and 11-17. In some examples, the miRNA has one binding site to KCNQ3. In some embodiments, the nucleic acids of the disclosure comprise a nucleotide sequence having at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequences shown in any one of SEQ ID NOs: 3-37. See Table 2. 【0051】 In some embodiments, the nucleic acids of the present disclosure include a nucleotide sequence having at least 90% identity with the sequence shown in any one of SEQ ID NOs: 3-9, the nucleotide sequence shown in any one of SEQ ID NOs: 3-9, a nucleotide sequence having at least 90% identity with the sequence shown in any one of SEQ ID NOs: 10-16, the nucleotide sequence shown in any one of SEQ ID NOs: 10-16, a nucleotide sequence encoding an RNA sequence shown in any one of SEQ ID NOs: 17-23, or a nucleotide sequence that specifically hybridizes to a KCNQ3 sequence shown in any one of SEQ ID NOs: 24-30. 【0052】 In some embodiments, the nucleic acids of the present disclosure include a nucleotide sequence having at least 90% identity with the sequence shown in any one of SEQ ID NOs: 31-37, or the nucleotide sequence shown in any one of SEQ ID NOs: 31-37. As shown in Figure 2, SEQ ID NOs: 31-37 are DNA sequences encoding miRNAs containing 5' XhoI (CTCGAG) and 3' hybrid Xba / SpeI (ACTAGA) restriction sites. These DNA sequences include an underlined 22-base nucleotide sequence (SEQ ID NOs: 3-9) encoding a 22-nucleotide miRNA sequence (SEQ ID NOs: 17-23). 【0053】 In some embodiments, the nucleic acids of the present disclosure include, or consist of, a nucleotide sequence shown in any one of SEQ ID NOs: 17-23, or a nucleotide sequence shown in any one of SEQ ID NOs: 24-30. 【0054】 In some aspects, the present disclosure includes the use of RNA interference to reduce or inhibit KCNQ3 expression. RNA interference (RNAi) is a mechanism of gene regulation in eukaryotic cells and has been considered for the treatment of various diseases. RNAi refers to the post-transcriptional regulation of gene expression mediated by miRNAs. miRNAs are small (21-25 nucleotides), non-coding RNAs that share sequence homology with and base pair with the sequence target sites of cognate messenger RNAs (mRNAs). The interaction between miRNAs and mRNAs leads to a cellular gene silencing mechanism that induces mRNA decay and / or inhibits mRNA translation into protein. 【0055】 Understanding of the natural RNAi pathway has advanced, and researchers are designing artificial shRNAs and snRNAs for use in regulating the expression of target genes for the treatment of diseases. Several classes of small RNAs that trigger the RNAi process in mammalian cells are known, including small (or short) interfering RNAs (siRNAs), as well as small (or short) hairpin RNAs (shRNAs) and microRNAs (miRNAs), and these constitute similar classes of vector expression triggers [Davidson et al., Nat. Rev. Genet. 12:329-40, 2011, Harper, Arch. Neurol. 66:933-8, 2009]. Since shRNAs and miRNAs are expressed in vivo from plasmid or virus-based vectors, long-term gene expression silencing can be achieved with a single administration as long as the vector is present in the target cell nucleus and the driving promoter is active (Davidson et al., Methods Enzymol. 392:145-73, 2005). Importantly, this vector expression approach builds on decades of progress already made in the field of gene therapy, but instead of expressing protein-coding genes, the vector cargo in RNAi therapy strategies is an artificial shRNA or miRNA cassette that targets the disease gene of interest. 【0056】 In some embodiments, the products and methods of the present disclosure include microRNA (miRNA). MicroRNA (miRNA) is a class of non-coding RNAs that play an important role in regulating RNA silencing and gene expression. The majority of miRNAs are transcribed from DNA sequences into primary miRNAs, processed into precursor miRNAs, and ultimately into mature miRNAs. In most cases, miRNAs interact with the 3'untranslated region (3'UTR) of target mRNAs to induce mRNA degradation and translational repression. However, interactions of miRNAs with other regions, including the 5'UTR, coding sequences, and gene promoters, have also been reported. Under certain conditions, miRNAs can also activate translation or regulate transcription. The interactions between miRNAs and their target genes are dynamic and depend on many factors, such as the intracellular location of miRNAs, the abundance of miRNAs and target mRNAs, and the affinity of miRNA-mRNA interactions. 【0057】 Most tests to date have shown that miRNAs bind to specific sequences in the 3'UTR of their target mRNAs, inducing translational repression and mRNA deadenylation and decapping. miRNA binding sites have also been detected in other mRNA regions, including the 5'UTR and coding sequences, as well as within promoter regions. Binding of miRNAs to the 5'UTR and coding regions has a silencing effect on gene expression, while miRNA interactions with promoter regions have been reported to induce transcription. 【0058】 In various embodiments, polymerase II promoters and polymerase III promoters such as U6 and H1 are used. In some embodiments, U6 miRNA is used. In some embodiments, H1 miRNA is used. Thus, in some embodiments, U6 miRNA or H1 miRNA is used to further reduce, inhibit, knockdown, or interfere with KCNQ3 gene expression. Conventional small / short hairpin RNA (shRNA) sequences are typically transcribed in the cell nucleus from vectors containing a Pol III promoter such as U6. The endogenous U6 promoter normally controls the expression of U6 RNA, a small nuclear RNA (snRNA) involved in splicing, which is well characterized [Kunkel et al., Nature. 322(6074):73-7(1986), Kunkel et al., Genes Dev. 2(2):196-204(1988), Paule et al., Nucleic Acids Res. 28(6):1283-98(2000)].In some embodiments, the U6 or H1 promoter is used to control vector - based expression of shRNA molecules in mammalian cells [Paddison et al., Proc. Natl. Acad. Sci. USA 99(3):1443 - 8(2002), Paul et al., Nat. Biotechnol. 20(5):505 - 8(2002), Medina et al., Curr. Opin. Mol. Ther. 1:580 - 94(1999)], which is because (1) the promoter is recognized by RNA polymerase III (poly III) and controls high - level constitutive expression of shRNA, (2) the Pol III promoter has a greater ability than RNA polymerase II to synthesize high yields of shRNA [Boden et al., Nucleic Acids Res. 32:1154 - 8(2004), Xia et al., Neurodegenerative Dis. 2:220 - 31(2005)], (3) the Pol III promoter is consistent with small, easy - to - handle sequences and simple terminators [Medina et al. (1999) supra], and (2) the promoter is active in most mammalian cell types. In some embodiments, the promoter is a type III Pol III promoter in which all elements necessary to control shRNA expression are located upstream of the transcription start site [Paule et al., Nucleic Acids Res. 28(6):1283 - 98(2000)]. The present disclosure includes both murine and human U6 promoters. shRNA containing sense and antisense sequences from a target gene, connected by a loop, is transported from the nucleus to the cytoplasm, where Dicer processes it into small / short interfering RNA (siRNA). 【0059】 In various aspects, the miRNA is expressed under various promoters and / or enhancers including, but not limited to, the U6 promoter, U7 promoter, H1 promoter, T7 promoter, tRNA promoter, EF1-alpha promoter, minimal EF1-alpha promoter, unc45b promoter, CK1 promoter, CK6 promoter, CK7 promoter, CK8 promoter, miniCMV promoter, CMV enhancer and / or promoter, ubiquitous promoter, neuron-specific promoter or brain-specific promoter. In some aspects, such neuron-specific or brain-specific promoters are human synapsin 1 (hSyn1), neuron-specific enolase (Nse), MeCP2, mDLX, mDLX5 / 6, calmodulin-dependent kinase II (CaMKII or Camk2a). In some aspects, the promoter and / or enhancer includes, but is not limited to, the promoters and enhancers disclosed in Tables 3 and 4 of Haery et al., any of the promoters and / or enhancers disclosed by Haery et al. (Front Neuroanat. 2019;13:93; PMID:31849618, which is hereby incorporated by reference in its entirety). 【0060】 In some embodiments, the disclosure includes a vector comprising any of the nucleic acids described herein. Accordingly, embodiments of the disclosure utilize viral vectors (e.g., adeno-associated virus (AAV), adenovirus, retrovirus, lentivirus, equine-related virus, alphavirus, poxvirus, herpesvirus, herpes simplex virus, poliovirus, sindbis virus, vaccinia virus, or synthetic virus, such as chimeric virus, mosaic virus, or pseudotyped virus, and / or virus containing foreign proteins, synthetic polymers, nanoparticles, or small molecules) to deliver the nucleic acids disclosed herein. 【0061】 In some embodiments, the vector is an AAV vector. In some aspects, the vector is a single-stranded AAV vector. In some aspects, the AAV is recombinant AAV (rAAV). In some aspects, the rAAV lacks the rep and cap genes. In some aspects, the rAAV is self-complementary (sc) AAV. 【0062】 In some aspects, the viral vector is an adeno-associated virus (AAV), such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rh10, AAV11, AAV12, AAV13, AAV-anc80, AAV-B1, AAV.PHP.EB, or AAVv66. 【0063】 In some embodiments, the viral vector is an adeno-associated virus (AAV), e.g., AAV1 (i.e., AAV containing AAV1 inverted terminal repeats (ITRs) and / or AAV1 capsid protein), AAV2 (i.e., AAV containing AAV2 ITRs and / or AAV2 capsid protein), AAV3 (i.e., AAV containing AAV3 ITRs and / or AAV3 capsid protein), AAV4 (i.e., AAV containing AAV4 ITRs and / or AAV4 capsid protein), AAV5 (i.e., AAV containing AAV5 ITRs and / or AAV5 capsid protein), AAV6 (i.e., AAV containing AAV6 ITRs and / or AAV6 capsid protein), AAV7 (i.e., AAV containing AAV7 ITRs and / or AAV7 capsid protein), AAV8 (i.e., AAV containing AAV8 ITRs and / or AAV8 capsid protein), AAV9 (i.e., AAV containing AAV9 ITRs and / or AAV9 capsid protein), AAV.rh74 (i.e., AAV containing AAV.rh74 ITRs and / or AAV.rh74 capsid protein), AAV.rh8 (i.e., AAV containing AAV.rh8 ITRs and / or AAV.rh8 capsid protein), AAV.rh10 (i.e., AAV containing AAV.rh10 ITRs and / or AAV.rh10 capsid protein), AAV11 (i.e., AAV containing AAV11 ITRs and / or AAV11 capsid protein), AAV12 (i.e., AAV containing AAV12 ITRs and / or AAV12 capsid protein), AAV13 (i.e., AAV containing AAV13 ITRs and / or AAV13 capsid protein), AAV-anc80 (i.e., AAV containing AAV-anc80 ITRs and / or AAV-anc80 capsid protein), AAV-B1 (i.e., AAV containing AAV-B1 ITRs and / or AAV-B1 capsid protein), AAV.PHP.EB (i.e., AAV-PHP.EB ITRs and / or AAV-PHP.AAV containing an EB capsid protein, or AAVv66 (i.e., AAV containing an AAVv66 ITR and / or an AAVv66 capsid protein). 【0064】 In some embodiments, the present disclosure utilizes adeno-associated virus (AAV) to deliver nucleic acids encoding miRNAs. AAV is a replication-defective parvovirus, and its single-stranded DNA genome is approximately 4.7 kb in length and contains 145-nucleotide inverted terminal repeats (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the complete genome of AAV1 is provided in GenBank accession number NC_002077. The complete genome of AAV2 is provided in GenBank accession numbers NC_001401 and Srivastava et al., J. Virol., 45:555-564 (1983), the complete genome of AAV3 is provided in GenBank accession number NC_1829, the complete genome of AAV4 is provided in GenBank accession number NC_001829, the AAV5 genome is provided in GenBank accession number AF085716, the complete genome of AAV6 is provided in GenBank accession number NC_001862, at least a portion of the AAV7 and AAV8 genomes are provided in GenBank accession numbers AX753246 and AX753249, respectively (see also U.S. Pat. Nos. 7,282,199 and 7,790,449 related to AAV8), the AAV9 genome is provided in Gao et al., J. Virol., 78:6381-6388 (2004). The genome of AAV10 is provided in Mol. Ther., 13(1):67-76 (2006). The genome of AAV11 is provided in Virology, 330(2):375-383 (2004). The genome of AAV12 is provided in J. Virol. 2008 Feb;82(3):1399-406. The genome of AAV13 is provided in J. Virol. 2008;82:8911. Cis-acting sequences that induce viral DNA replication (rep), capsid formation / packaging, and integration into the host cell chromosome are contained within the AAV ITRs. Three AAV promoters (named p5, p19, and p40 at their relative map positions) drive the expression of two AAV internal open reading frames encoding the rep and cap genes.Two rep promoters (p5 and p19), in conjunction with differential splicing of a single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins have multiple enzymatic properties that ultimately participate in replicating the viral genome. The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are involved in the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka (Current Topics in Microbiology and Immunology, 158:97-129 (1992)). 【0065】 AAV has unique features that make it attractive as a vector for delivering foreign DNA into cells, for example, in gene therapy. AAV infection of cells in culture is non-cytopathic, and natural infection in humans and other animals is asymptomatic and latent. AAV can infect many mammalian cells, giving the potential to target many different tissues in vivo. AAV transduces both slowly dividing and non-dividing cells and can persist essentially throughout the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is infectious as cloned DNA in a plasmid, enabling the construction of recombinant genomes. Further, since the signals that direct AAV replication, genome capsid formation, and integration are contained within the ITRs of the AAV genome, part or all of the internal approximately 4.3 kb genome (encoding the replication and structural capsid proteins rep-cap) can be replaced with foreign DNA. In some embodiments, the rep and cap proteins are provided in trans. Another important feature of AAV is that it is a very stable and robust virus. This easily withstands the conditions (several hours at 56 °C to 65 °C) used to inactivate adenovirus, reducing the importance of cryopreservation of AAV. AAV can be lyophilized, and AAV-infected cells are not resistant to superinfection. 【0066】 In some embodiments, provided is a DNA plasmid of the present disclosure that includes an rAAV genome of the present disclosure. The DNA plasmid is introduced into cells permissive for infection by an AAV helper virus (e.g., an adenovirus, an E1-deleted adenovirus, or a herpesvirus) to assemble the rAAV genome into infectious virus particles. Techniques for producing rAAV particles that provide a cell with an AAV genome to be packaged, the rep and cap genes, and helper virus functions are standard in the art. Production of rAAV requires the following components present within a single cell (referred to herein as a packaging cell): an rAAV genome, AAV rep and cap genes that are separate from (i.e., not present within) the rAAV genome, and helper virus functions. The AAV rep gene can be from any AAV serotype from which the recombinant virus can be derived, including, but not limited to, AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rh10, AAV11, AAV12, AAV13, AAV-anc80, AAV-B1, AAV.PHP.EB, or AAVv66, and can be from an AAV serotype different from the rAAV genome ITR. In some aspects, the ITRs in AAV are from different AAV serotypes. In some aspects, AAV includes ITRs or capsid proteins from different serotypes, i.e., different from the rest of the vector. For example, in some aspects, AAV2 or AAV2-based ITRs are used not only in AAV2 or AAV2-based serotypes, but also in various AAV vectors. In some aspects, various ITRs are interchangeable between different AAV serotypes. For example, in some aspects, AAV2 ITRs are interchangeable between different AAV serotypes. Thus, in some aspects, AAV2 ITRs are used in different AAV serotypes of AAV vectors, including, but not limited to, AAV9. In some aspects, the AAV2 Rep helper gene is used. 【0067】 In some embodiments, the AAV DNA in the rAAV genome is from any AAV serotype including, but not limited to, AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rh10, AAV11, AAV12, AAV13, AAV-anc80, AAV-B1, AAV.PHP.EB, or AAVv66 from which the recombinant virus can be derived. Other types of rAAV variants, e.g., rAAV having capsid mutations, are also included in the present disclosure. See, e.g., Marsic et al., Molecular Therapy 22(11):1900-1909 (2014). As noted above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. The use of homologous components is specifically contemplated. The production of pseudotyped rAAV is disclosed, e.g., in WO01 / 83692, which is incorporated herein by reference in its entirety. 【0068】 The recombinant AAV genome of the present disclosure includes at least one KCNQ3-targeting polynucleotide or one or more AAV ITRs adjacent to the nucleotide sequence. In some embodiments, the polynucleotide is an miRNA or a polynucleotide encoding an miRNA. In some embodiments, the miRNA is administered together with other polynucleotide constructs that target KCNQ3. The AAV DNA in the rAAV genome can be from any AAV serotype including, but not limited to, AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rh10, AAV11, AAV12, AAV13, AAV-anc80, AAV-B1, AAV.PHP.EB, or AAVv66 from which the recombinant virus can be derived. As shown above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. 【0069】 The DNA plasmids of the present disclosure contain the rAAV genomes of the present disclosure. The DNA plasmids are introduced into cells that permit infection by an AAV helper virus (e.g., an adenovirus, an E1-deleted adenovirus, or a herpes virus) in order to assemble the rAAV genome into infectious virus particles. Techniques for producing rAAV particles that provide the cell with the AAV genome to be packaged, the rep and cap genes, and the helper virus functions are standard in the art. The production of rAAV requires the following components present within a single cell (referred to herein as a packaging cell): an rAAV genome, AAV rep and cap genes that are separate from (i.e., not present within) the rAAV genome, and helper virus functions. The AAV rep gene can be from any AAV serotype from which the recombinant virus can be derived, including, but not limited to, AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rh10, AAV11, AAV12, AAV13, AAV-anc80, AAV-B1, AAV.PHP.EB, or AAVv66, different from the AAV serotype of the rAAV genome ITR. In some embodiments, the AAV DNA in the rAAV genome is from any AAV serotype, including, but not limited to, AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rh10, AAV11, AAV12, AAV13, AAV-anc80, AAV-B1, AAV.PHP.EB, or AAVv66, from which the recombinant virus can be derived. Other types of rAAV variants, e.g., rAAV having capsid mutations, are also included in the present disclosure. See, e.g., Marsic et al., Molecular Therapy 22(11):1900-1909 (2014). As noted above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. The use of cognate components is specifically contemplated. The production of pseudotyped rAAV is disclosed, e.g., in WO01 / 83692, which is hereby incorporated by reference in its entirety. 【0070】 In some embodiments, packaging cells are provided. The packaging cells are generated to have a cell line that stably expresses all the components necessary for AAV particle production. The retroviral vector is generated by removing the retroviral gag, pol, and env genes. These are replaced by the therapeutic gene. Packaging cells are essential for producing vector particles. The packaging cell line provides all the viral proteins necessary for capsid production and virion maturation of the vector. Therefore, the packaging cell line is generated such that they contain the gag, pol, and env genes. After inserting the desired gene into the retroviral DNA vector and maintaining the appropriate packaging cell line, it is now straightforward to prepare the retroviral vector. 【0071】 For example, a plasmid (or plasmids) containing an rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes separated from the rAAV genome, and a selectable marker such as the neomycin resistance gene is integrated into the genome of the cell. The AAV genome has been introduced into the bacterial plasmid by procedures such as GC tailing [Samulski et al., 1982, Proc. Natl. Acad. Sci. USA, 79:2077-2081], addition of synthetic linkers containing restriction endonuclease cleavage sites [Laughlin et al., 1983, Gene, 23:65-73], or direct blunt-end ligation [Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666]. The packaging cell line is then infected with a helper virus such as adenovirus. The advantage of this method is that the cells are selectable and suitable for large-scale production of rAAV. Another example of a suitable method is to use an adenovirus or baculovirus instead of a plasmid to introduce the rAAV genome and / or the rep and cap genes into the packaging cells. 【0072】 Accordingly, in some embodiments, a method of generating a packaging cell that results in a cell line that stably expresses all the components necessary for AAV particle production is provided. For example, a plasmid (or plasmids) containing an rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes separated from the rAAV genome, and a selectable marker such as the neomycin resistance gene is integrated into the genome of the cell. The AAV genome has been introduced into the bacterial plasmid by procedures such as GC tailing [Samulski et al., 1982, Proc. Natl. Acad. Sci. USA, 79:2077-2081], addition of a synthetic linker containing a restriction endonuclease cleavage site (Laughlin et al., 1983, Gene, 23:65-73), or direct blunt-end ligation (Senapathy et al., 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus such as adenovirus. The advantage of this method is that the cells are selectable and suitable for large-scale production of rAAV. Another example of a suitable method is to use an adenovirus or baculovirus rather than a plasmid to introduce the rAAV genome and / or the rep and cap genes into the packaging cell. 【0073】 The general principles of rAAV production are described, for example, in Carter, 1992, Current Opinions in Biotechnology, 1533-539, and Muzyczka, 1992, Curr. Topics in Microbiol. and Immunol. 158:97-129). Various approaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072 (1984), Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984), Tratschin et al., Mo1. Cell. Biol. 5:3251 (1985), McLaughlin et al., J. Virol., 62:1963 (1988), and Lebkowski et al., 1988 Mol. Cell. Biol., 7:349 (1988), Samulski et al., J. Virol., 63:3822-3828 (1989), U.S. Patent No. 5,173,414, WO95 / 13365 and corresponding U.S. Patent No. 5,658,776, WO95 / 13392, WO96 / 17947, PCT / US98 / 18600, WO97 / 09441 (PCT / US96 / 14423), WO97 / 08298 (PCT / US96 / 13872), WO97 / 21825 (PCT / US96 / 20777), WO97 / 06243 (PCT / FR96 / 01064), WO99 / 11764, Perrin et al., Vaccine, 13:1244-1250 (1995), Paul et al., Human Gene Therapy, 4:609-615 (1993), Clark et al., Gene Therapy, 3:1124-1132 (1996), U.S. Patent No. 5,786,211, No. 5,871,982, No. 6,258,595, and McCarty, Mol. Ther., 16(10):1648-1656 (2008). The foregoing documents are hereby incorporated by reference in their entirety and these portions of the documents regarding rAAV production are specifically emphasized. The production and use of various types of rAAV are specifically contemplated and exemplified. Accordingly, recombinant AAV (i.e., infectious, capsidated rAAV particles) are provided herein.In some embodiments, the genome of the rAAV lacks the AAV rep and cap genes, i.e., there is no AAV rep or cap DNA between the ITRs of the rAAV genome. In some embodiments, the AAV is recombinant linear AAV (rAAV), single-stranded AAV (ssAAV), or recombinant self-complementary AAV (scAAV). 【0074】 Accordingly, the present disclosure provides, in some embodiments, packaging cells that produce infectious rAAV. In one embodiment, the packaging cells are stably transformed cancer cells such as HeLa cells, 293 cells, and PerC.6 cells (isogenic 293 strain). In another embodiment, the packaging cells are not transformed cancer cells such as low passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus fetal lung cells). 【0075】 In some embodiments, rAAV is purified by methods standard in the art, e.g., by column chromatography or cesium chloride gradient. Methods for purifying rAAV vectors from helper virus are known in the art and are disclosed, for example, in Clark et al., Hum. Gene Ther., 10(6):1031-1039 (1999), Schenpp and Clark, Methods Mol. Med., 69:427-443 (2002), U.S. Patent No. 6,566,118, and WO98 / 09657. 【0076】 In some embodiments, the disclosure includes a composition comprising any of the nucleic acids or any of the vectors described herein in combination with a diluent, excipient, or buffer. In some embodiments, the disclosure provides a composition comprising a vector, such as a viral vector, as described herein. Accordingly, a composition comprising a delivery vehicle (such as rAAV) described herein is provided. In various aspects, such compositions also include a pharmaceutically acceptable carrier. Generally, as used herein, "pharmaceutically acceptable carrier" means all aqueous and non-aqueous solutions, sterile solutions, solvents, buffers, such as phosphate buffered saline (PBS) solution, water, suspensions, emulsions such as oil / water emulsions, various types of wetting agents, liposomes, dispersion media, and coatings, which are compatible with pharmaceutical administration, particularly parenteral administration. The use of such media and agents in pharmaceutical compositions is well known in the art, and compositions containing such carriers can be formulated by well-known conventional methods. 【0077】 In various aspects, any composition of the disclosure also includes other components such as diluents, excipients, and / or adjuvants. Acceptable carriers, diluents, excipients, and adjuvants are non-toxic to the recipient and are preferably inert at the dosages and concentrations used, and include buffers such as phosphoric acid, citric acid, or other organic acids, antioxidants such as ascorbic acid, low molecular weight polypeptides, proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming counterions such as sodium, and / or nonionic surfactants such as Tweens, pluronics, or polyethylene glycol (PEG). 【0078】 In some embodiments, the nucleic acid is introduced into a vector for delivery. In some embodiments, the vector for delivery is AAV or rAAV. Accordingly, embodiments of the present disclosure include a nucleotide sequence comprising at least 90% identity with the sequence shown in any one of SEQ ID NOs: 3-9, the nucleotide sequence shown in any one of SEQ ID NOs: 3-9, a nucleotide sequence comprising at least 90% identity with the sequence shown in any one of SEQ ID NOs: 10-16, the nucleotide sequence shown in any one of SEQ ID NOs: 10-16, a nucleotide sequence encoding the RNA sequence shown in any one of SEQ ID NOs: 17-23, or a nucleotide sequence that specifically hybridizes to the KCNQ3 sequence shown in any one of SEQ ID NOs: 24-30, and include an rAAV genome. 【0079】 In some other embodiments, the nucleic acid is introduced into cells via non-vectorized delivery. Accordingly, in embodiments, the present disclosure includes non-vectorized delivery of a nucleic acid encoding a KCNQ3-targeted miRNA. In some embodiments, in this context, a synthetic carrier that forms a complex with the nucleic acid and can protect them from extracellular and intracellular nucleases is an alternative to viral vectors. In some embodiments, such non-vectorized delivery includes the use of nanoparticles, extracellular vesicles, or exosomes containing the nucleic acids of the present disclosure. The present disclosure also includes compositions comprising any of the constructs described herein, alone or in combination. 【0080】 A sterile injectable solution is prepared by incorporating the rAAV in the required amount into a suitable solvent, optionally together with various other ingredients enumerated above, and then filter sterilizing. Generally, a dispersion is prepared by incorporating the sterilized active ingredient into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredients from those solutions that have been previously sterile filtered. 【0081】 The titer of rAAV administered by the methods of the present disclosure can vary, for example, depending on the specific rAAV, the mode of administration, the treatment goal, the individual, and the cell type targeted, and can be determined by standard methods in the art. The titer of rAAV can be about 1×10 6 particles, about 1×10 7 particles, about 1×10 8 particles, about 1×10 9 particles, about 1×10 10 particles, about 1×10 11 particles, about 1×10 12 particles, about 1×10 13 to about 1×10 14 particles, or more, and can be in the range of DNase-resistant particles (DRP). The dosage can also be expressed in units of viral genome (vg) (e.g., 1×10 7 vg, 1×10 8 vg, 1×10 9 vg, 1×10 10 vg, 1×10 11 vg, 1×10 12 vg, 1×10 13 vg, and 1×10 14 vg). 【0082】 Accordingly, in some aspects, the present disclosure provides a method of delivering any one or more nucleic acids to a cell or subject, the nucleic acid comprising a nucleotide sequence having at least 90% identity to the sequence shown in any one of SEQ ID NOs: 3-9, the nucleotide sequence shown in any one of SEQ ID NOs: 3-9, a nucleotide sequence having at least 90% identity to the sequence shown in any one of SEQ ID NOs: 10-16, the nucleotide sequence shown in any one of SEQ ID NOs: 10-16, a nucleotide sequence encoding an RNA sequence shown in any one of SEQ ID NOs: 17-23, or a nucleotide sequence that specifically hybridizes to a KCNQ3 sequence shown in any one of SEQ ID NOs: 24-30. 【0083】 In some embodiments, the method comprises administering to a cell or a subject an AAV comprising any one or more nucleic acids comprising a nucleotide sequence having at least 90% identity to the sequence set forth in any one of SEQ ID NOs: 3-9, the nucleotide sequence set forth in any one of SEQ ID NOs: 3-9, a nucleotide sequence having at least 90% identity to the sequence set forth in any one of SEQ ID NOs: 10-16, the nucleotide sequence set forth in any one of SEQ ID NOs: 10-16, a nucleotide sequence encoding an RNA sequence set forth in any one of SEQ ID NOs: 17-23, or a nucleotide sequence that specifically hybridizes to a KCNQ3 sequence set forth in any one of SEQ ID NOs: 24-30. 【0084】 In yet another embodiment, the present disclosure provides a method of reducing the expression of the KCNQ3 gene or reducing the expression of functional KCNQ3 in a cell or a subject, the method comprising contacting the cell or the subject with any one or more nucleic acids comprising a nucleotide sequence having at least 90% identity to the sequence set forth in any one of SEQ ID NOs: 3-9, the nucleotide sequence set forth in any one of SEQ ID NOs: 3-9, a nucleotide sequence having at least 90% identity to the sequence set forth in any one of SEQ ID NOs: 10-16, the nucleotide sequence set forth in any one of SEQ ID NOs: 10-16, a nucleotide sequence encoding an RNA sequence set forth in any one of SEQ ID NOs: 17-23, or a nucleotide sequence that specifically hybridizes to a KCNQ3 sequence set forth in any one of SEQ ID NOs: 24-30. 【0085】 In some embodiments, the method comprises delivering the nucleic acid with one or more AAV vectors. In some embodiments, the method comprises delivering the nucleic acid to the cell by non-vectorized delivery. 【0086】 In some embodiments, the expression of KCNQ3 or the expression of a functional KCNQ3 is reduced in a cell or subject by at least or about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% by the methods provided herein. 【0087】 In some embodiments, the disclosure provides AAV transduced cells for delivery of a nucleic acid encoding a KCNQ3 miRNA described herein. Methods of transducing target cells with rAAV, either in vivo or in vitro, are included in the disclosure. The methods include administering to a subject in need thereof, such as a human, a composition comprising an effective dose or effective plural doses of the rAAV of the disclosure. When the dose is administered prior to the onset of a seizure or epileptic disorder, or other symptoms of a disease or disorder associated with a mutant or pathogenic expression of KCNQ3, the administration is prophylactic. When the dose is administered after the onset of a seizure or epileptic disorder, or other symptoms of a disease or disorder associated with a mutant or pathogenic expression of KCNQ3, the administration is therapeutic or ameliorative. In embodiments of the disclosure, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom of a disease or disorder associated with a mutant or pathogenic expression of KCNQ3 being treated, delays or prevents progression of symptoms of a disease or disorder associated with a mutant or pathogenic expression of KCNQ3, and / or results in remission (partial or complete) of symptoms of a disease or disorder associated with a mutant or pathogenic expression of KCNQ3. In some embodiments, the disease or disorder associated with a mutant or pathogenic expression of KCNQ3 is developmental epileptic encephalopathy (DEE). 【0088】 In some embodiments, the disclosure provides non-vectored delivery of a nucleic acid encoding a KCNQ3 miRNA described herein. In some embodiments, the nucleic acid or composition comprising the nucleic acid is delivered by nanoparticles, extracellular vesicles, or exosomes. 【0089】 Combination therapies are also contemplated by the present disclosure. Accordingly, the present disclosure includes one or more other compounds or compositions comprising other RNA inhibitory compounds or small molecule compounds for downregulating KCNQ3 in the treatment of DEE or a variant of KCNQ3 or other conditions associated with pathogenic expression, including possible combination therapies or multiple combination therapies. As used herein, combination includes concurrent treatment or sequential treatment. Combinations of the methods of the present disclosure with standard medical treatments and supportive therapies are specifically contemplated, including physical and occupational therapy, speech therapy, treatment by developmental specialists for neurodevelopmental delays and autism symptoms, medications for addressing behavioral problems (including, but not limited to, alpha-2 adrenergic agonists, antipsychotics, selective serotonin reuptake inhibitors (SSRI), etc.), medications for addressing sleep disorders (including, but not limited to, melatonin, trazodone, benzodiazepines, doxepin, eszopiclone, lemborexant, ramelteon, suvorexant, zaleplon, zolpidem, etc.), and medications for addressing seizures and / or EEG abnormalities (including, but not limited to, carbamazepine, eslicarbazepine, ethosuximide, everolimus, gabapentin, lacosamide, oxcarbazepine, lamotrigine, phenobarbital, phenytoin, pregabalin, tiagabine, vigabatrin, valproic acid, acetazolamide, brivaracetam, cannabidiol, cenobamate, clobazam, clonazepam, chlordiazepate, diazepam, divalproex, felbamate, fenfluramine, lamotrigine, levetiracetam, lorazepam, mesuximide, perampanel, primidone, rufinamide, stiripentol, topiramate valproic acid, or zonisamide, including, but not limited to, any of many anti-seizure medications known in the art), or combinations of therapies with other inhibitory RNA constructs, etc. are likewise specifically contemplated. 【0090】 Other combination therapies included in the present disclosure are combinations of KCNQ3 miRNA with other miRNAs as described herein, or U7-snRNA-based gene therapies, small molecule inhibitors of KCNQ3 expression, oligonucleotides that inhibit KCNQ3 via RNAi, or combinations with RNAse H or exon skipping mechanisms, U7-snRNA plus a theoretical CRISPR-based gene therapy approach. 【0091】 Administration of an effective dose of the compositions of the present disclosure, including AAV, nanoparticles, extracellular vesicles, and exosomes containing the compositions and nucleic acids of the present disclosure, can be by routes standard in the art and can include, but are not limited to, intramuscular, parenteral, intravascular, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraventricular, intrathecal, intraosseous, intraocular, rectal, or vaginal. The route of administration and serotype of the AAV component of the rAAV of the present disclosure (in particular, AAV ITR and capsid proteins) can be selected and / or adapted by one of ordinary skill in the art considering the disease state being treated and the target cells / tissues such as cells expressing a mutant or pathogenic variant of the KCNQ3 gene that results in a mutant or pathogenic expression of KCNQ3. In some embodiments, the composition or pharmaceutical is formulated for intraventricular injection, intrathecal injection, intramuscular injection, oral administration, subcutaneous, intradermal, or transdermal delivery, injection into the bloodstream, or for aerosol administration. In some embodiments, the route of administration is intraventricular. In some embodiments, the route of administration is intravenous. 【0092】 In some embodiments, the actual administration of the rAAV of the present disclosure can be achieved by using any physical method that transports the rAAV recombinant vector to the target tissue of an animal. Administration according to the present disclosure includes, but is not limited to, direct injection into the brain, bloodstream, central nervous system, and / or other organs. It has been demonstrated that simply resuspending rAAV in phosphate-buffered saline is sufficient to provide a vehicle useful for expression in the brain, and there are no known limitations on carriers or other components that can be co-administered with rAAV (although compositions that degrade DNA should be avoided by normal means using rAAV). The capsid protein of rAAV can be modified such that the rAAV is targeted to a specific target tissue of interest, such as the brain. See, for example, WO02 / 053703, which is incorporated herein by reference. The pharmaceutical composition can be prepared for oral administration, as an injectable formulation, or as a topical formulation delivered to muscle by subcutaneous, intradermal, and / or transdermal transport. Numerous formulations for both intramuscular injection and transdermal transport have already been developed and can be used to practice the present disclosure. rAAV can be used with any pharmaceutically acceptable carrier to facilitate administration and handling. 【0093】 For the purpose of injection, in some embodiments, solutions such as sterile aqueous solutions are used. Such aqueous solutions can be buffered as needed, and the liquid diluent can first be made isotonic with physiological saline or glucose. Solutions of rAAV as the free acid (where the DNA contains acidic phosphate groups) or as a pharmaceutically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropyl cellulose. Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oils. Under normal storage conditions and in use, these preparations contain preservatives to prevent the growth of microorganisms. In this regard, all of the sterile aqueous media used can be readily obtained by standard techniques well known to those skilled in the art. 【0094】 Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that it is readily injectable with a syringe. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. In some embodiments, appropriate fluidity is maintained, for example, by the use of coatings such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents (such as sugars or sodium chloride). Prolonged absorption of injectable compositions can be brought about by the use of agents that delay absorption, for example, aluminum monostearate and gelatin. 【0095】 In some embodiments, the formulation includes a stabilizer. The term "stabilizer" refers to a substance or excipient that protects the formulation from harmful conditions such as those that occur during heating or freezing and / or extends the stability or shelf life of the formulation in a stable state. Examples of stabilizers include, but are not limited to, sugars such as sucrose, lactose, and mannose, sugar alcohols such as mannitol, amino acids such as glycine or glutamic acid, and proteins such as human serum albumin or gelatin. 【0096】 In some embodiments, the formulation includes an antimicrobial preservative. The term "antimicrobial preservative" refers to any substance added to a composition that inhibits the growth of microorganisms that may be introduced upon repeated puncture of the vial or container in use. Examples of antimicrobial preservatives include, but are not limited to, substances such as thimerosal, 2-phenoxyethanol, benzethonium chloride, and phenol. 【0097】 The term "transduction" is used to refer to the administration / delivery of one or more of the KCNQ3 targeting constructs described herein, such as a KCNQ3 miRNA or a nucleic acid encoding a KCNQ3 miRNA, to recipient cells, either in vivo or in vitro, via the replication-deficient rAAV of the present disclosure that results in decreased expression of KCNQ3 by the recipient cells. 【0098】 In one embodiment, transduction by rAAV is performed in vitro. In one implementation, the desired target cells are removed from the subject, transduced with rAAV, and reintroduced into the subject. Alternatively, syngeneic or xenogeneic cells can be used if they do not produce an inappropriate immune response in the subject. 【0099】 Suitable methods for transduction of a subject and reintroduction of the transduced cells are known in the art. In one embodiment, the cells are transduced in vitro, for example in a suitable medium, by combining the rAAV with the cells and screening these cells for the DNA of interest using conventional techniques such as Southern blot and / or PCR, or using a selectable marker. The transduced cells are then formulated into a pharmaceutical composition and the composition can be introduced into the subject by various techniques such as by intracerebroventricular, intramuscular, intravenous, subcutaneous, and intraperitoneal injection, or by injection into the brain or smooth and cardiac muscle using, for example, a catheter. 【0100】 The present disclosure provides a method of administering a therapeutically effective dose (or a dose administered essentially simultaneously or at intervals) of rAAV comprising DNA encoding a microRNA designed to reduce or inhibit the expression of KCNQ3 to a cell or a subject in need thereof. In some embodiments, the effective dose is thus a therapeutically effective dose. 【0101】 In some embodiments, the dose or effective dose of rAAV administered is from about 1.0×10 10 vg / kg to about 1.0×10 16 vg / kg. In some aspects, 1.0×10 10 vg / kg is also denoted as 1.0 E10 vg / kg, which is an alternative way of presenting scientific notation. Similarly, 10 11 is equivalent to E11, etc. In some aspects, the dose of rAAV administered is from about 1.0×10 11 vg / kg to about 1.0×10 15 vg / kg. In some aspects, the dose of rAAV is about 1.0×10 10 vg / kg, about 2.0×10 10 vg / kg, about 3.0×10 10 vg / kg, about 4.0×10 10 vg / kg, about 5.0×10 10 vg / kg, about 6.0×10 10 vg / kg, about 7.0×10 10 vg / kg, about 8.0×10 10 vg / kg, about 9.0×10 10 about 1.0×10 11 vg / kg, about 2.0×10 11 vg / kg, about 3.0×10 11 vg / kg, about 4.0×10 11 vg / kg, about 5.0×10 11 vg / kg, about 6.0×10 11 vg / kg, about 7.0×10 11 vg / kg, about 8.0×10 11 vg / kg, about 9.0×10 11 vg / kg, about 1.0×10 12vg / kg, about 2.0×10 12 vg / kg, about 3.0×10 12 vg / kg, about 4.0×10 12 vg / kg, about 5.0×10 12 vg / kg, about 6.0×10 12 vg / kg, about 7.0×10 12 vg / kg, about 8.0×10 12 vg / kg, about 9.0×10 12 vg / kg, about 1.0×10 13 vg / kg, about 2.0×10 13 vg / kg, about 3.0×10 13 vg / kg, about 4.0×10 13 vg / kg, about 5.0×10 13 vg / kg, about 6.0×10 13 vg / kg, about 7.0×10 13 vg / kg, about 8.0×10 13 vg / kg, about 9.0×10 13 vg / kg, about 1.0×10 14 vg / kg, about 2.0×10 14 vg / kg, about 3.0×10 14 vg / kg, about 4.0×10 14 vg / kg, about 5.0×10 14 vg / kg, about 6.0×10 14 vg / kg, about 7.0×10 14 vg / kg, about 8.0×10 14 vg / kg, about 9.0×10 14 vg / kg, about 1.0×10 15 vg / kg, about 2.0×10 15 vg / kg, about 3.0×10 15 vg / kg, about 4.0×10 15 vg / kg, about 5.0×10 15 vg / kg, about 6.0×10 15 vg / kg, about 7.0×10 15 vg / kg, about 8.0×10 15 vg / kg, about 9.0×10 15 vg / kg, or about 1.0×10 16 vg / kg. 【0102】 In some embodiments, the dosage is about 1.0×10 11 vg / kg to about 1.0×10 15 vg / kg. In some embodiments, the dosage is about 1.0×10 13 vg / kg to about 5.0×10 13 vg / kg. In some embodiments, the dosage is about 2.0×10 13 vg / kg to about 4.0×10 13 vg / kg. In some embodiments, the dosage is about 3.0×10 13 vg / kg. 【0103】 In some embodiments, after the initial dosage, a second, more dosage follows. In some embodiments, after the initial dosage, a second, same dosage follows. In some embodiments, after the initial dosage, one or more, less dosages follow. In some embodiments, after the initial dosage, multiple dosages that are the same dosage or a more dosage follow. 【0104】 Methods of transducing a delivery vehicle (e.g., rAAV) into target cells, in vivo or in vitro, are contemplated. Transduction of cells with the rAAV of the present disclosure results in sustained expression of the KCNQ3 miRNA sequence. Accordingly, the present disclosure provides rAAV, and methods of administering / delivering rAAV that expresses the KCNQ3 miRNA sequence in cells in a subject, in vitro or in vivo. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. These methods include transducing cells and tissues (including, but not limited to, tissues such as the brain) with one or more of the rAAV described herein. Transduction can be carried out using a gene cassette that includes cell-specific control elements. The term "transduction" is used, for example, to refer to the administration / delivery of a nucleic acid, e.g., a KCNQ3 miRNA, that includes a nucleotide sequence encoding the KCNQ3 miRNA sequence, via a replication-deficient rAAV described herein, either in vivo or in vitro, that results in reduced expression or inhibition of expression of KCNQ3 in target cells. 【0105】 An in vivo method includes administering an effective dose or effective multiple doses of a composition, such as rAAV, to a subject in need thereof, including a human subject. Thus, provided is a method of administering an rAAV as described herein at an effective dose (or doses administered essentially simultaneously or at intervals) to a patient in need thereof. When a dose or multiple doses are administered prior to the onset of a disorder / disease, the administration is prophylactic. When a dose or multiple doses are administered after the onset of a disorder / disease, the administration is therapeutic. An effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease state being treated, delays or prevents progression to the disorder / disease state, delays or prevents progression of the disorder / disease state, reduces the severity of the disease, results in remission (partial or complete) of the disorder / disease state, and / or extends survival. 【0106】 In some embodiments, the compositions and methods of the present disclosure are used in treating, ameliorating, or preventing a disease or disorder associated with the expression of a mutant form of the KCNQ3 protein or a mutant or pathogenic KCNQ3 gene that results in pathogenic expression. In some aspects, symptoms of such diseases or disorders resulting from mutant or pathogenic expression of the KCNQ3 protein include, but are not limited to, seizures, epileptic diseases or disorders, intellectual or developmental disorders, autism, or autism spectrum disorder. In some aspects, any of such symptoms exhibit developmental epileptic encephalopathy (DEE). 【0107】 Molecular, biochemical, histological, and functional outcome measures demonstrate the therapeutic efficacy of the products and methods disclosed herein for reducing mutant or pathogenic expression of KCNQ3 mRNA and protein and treating seizures, epileptic disorders or syndromes, intellectual or developmental disorders, autism, autism spectrum disorders, or DEE resulting from mutant or pathogenic expression of KCNQ3 mRNA and protein. Outcome measures include, but are not limited to, reduction or elimination of KCNQ3 mRNA or protein, or mutant or pathogenic variants thereof, in affected tissue. Absence of KCNQ3 expression in cells and / or downregulation of expression of mutant or pathogenic KCNQ3 mRNA or protein is detected by measuring the level of KCNQ3 protein by methods known in the art including, but not limited to, RT-PCR, QRT-PCR, RNAscope, Western blot, immunofluorescence, or immunohistochemistry, in brain biopsies before and after administration of microRNA or rAAV containing microRNA to determine improvement. 【0108】 In some embodiments, the level of KCNQ3 gene expression or protein expression in a target cell is decreased following administration of a nucleic acid or vector encoding a KCNQ3 miRNA, e.g., an rAAV, as compared to the level of KCNQ3 gene expression or protein expression prior to administration of the KCNQ3 miRNA-encoding nucleic acid or vector encoding a KCNQ3 miRNA, e.g., an rAAV. In some aspects, the expression of KCNQ3 is decreased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 100%, or more than at least about 100%. The number, frequency, or intensity of seizures or epileptic events is improved by at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 100%, or more than at least about 100%. 【0109】 Another outcome measure includes assessing the levels of membrane-bound KCNQ3 protein before and after treatment, as it has been shown to increase as a result of the mutation in adult mice. Thus, a positive treatment outcome for treatment by the products and methods of the present disclosure is a decrease in the level of membrane-bound KCNQ3 after administration of KCNQ3 miRNA (or AAV containing KCNQ3 miRNA) compared to before administration of KCNQ3 miRNA (or AAV containing KCNQ3 miRNA). 【0110】 Another outcome measure includes examination of intracranial EEG after administration of KCNQ3 miRNA (or AAV containing KCNQ3 miRNA) compared to before administration of KCNQ3 miRNA (or AAV containing KCNQ3 miRNA). There is significant seizure-like activity in the offspring of mutant mice at the second week after birth that have similarities to the electroclinical features of children with the pathogenic R231 variant of KCNQ3. Accordingly, the products and methods of the present disclosure are expected to improve or reduce seizure-like activity. 【0111】 Another outcome measure includes examination of sedentary activity behavior after administration of KCNQ3 miRNA (or AAV containing KCNQ3 miRNA) compared to before administration of KCNQ3 miRNA (or AAV containing KCNQ3 miRNA). Mice with the pathogenic R231 variant have moderate hyperactivity. Accordingly, the products and methods of the present disclosure are expected to improve or reduce hyperactivity. 【0112】 Administration of an effective amount of a nucleic acid, viral vector, or composition of the present disclosure can be by routes standard in the art and can include, but is not limited to, intraventricular, intrathecal, intravenous, intracranial, oral, buccal, nasal, intraosseous, intramuscular, parenteral, intravascular, transpulmonary, intraocular, rectal, or vaginal. In some embodiments, the effective amount is delivered by a systemic administration route, i.e., by systemic administration. Systemic administration is a route of administration to the circulatory system such that the entire body is affected. Such systemic administration can be effected in various embodiments via enteral administration (absorption of a drug through the gastrointestinal tract) or parenteral administration (generally via injection, infusion, or transplantation). In various embodiments, the effective amount is delivered by a combination of routes. For example, in various embodiments, the effective amount is delivered intravenously and / or intramuscularly, or intravenously and intraventricularly, etc. In some embodiments, the effective amount is delivered sequentially or continuously. In some embodiments, the effective amount is delivered simultaneously. The route of administration and serotype of the AAV components of the rAAV of the present disclosure (in particular, AAV ITRs and capsid proteins) are selected and / or adapted by one of ordinary skill in the art in view of the circumstances or condition of the disease or disorder being treated, the circumstances, condition, or age of the subject, and the target cell / tissue in which the nucleic acid or protein is to be expressed. 【0113】 In particular, the actual administration of the delivery vehicle (such as rAAV) can be achieved by using any physical method of transporting the delivery vehicle (such as rAAV) to the target cells of the animal. Administration includes, but is not limited to, injection into the brain, nervous system, liver, or bloodstream. It has been demonstrated that simply resuspending rAAV in phosphate-buffered saline is sufficient to provide a vehicle useful for expression in the brain, and there are no known limitations on carriers or other components that can be co-administered with rAAV (although compositions that degrade DNA should be avoided by normal means using rAAV). The capsid protein of rAAV can be modified so that rAAV is targeted to specific target tissues of interest such as nerve cells. See, for example, WO02 / 053703, which is incorporated herein by reference. The pharmaceutical composition can be prepared as an injectable formulation or as a topical formulation delivered to muscle by transdermal transport. Numerous formulations for both intramuscular injection and transdermal transport have already been developed and can be used to practice the present disclosure. The delivery vehicle (such as rAAV) can be used with any pharmaceutically acceptable carrier to facilitate administration and handling. 【0114】 Dispersions of the delivery vehicle (such as rAAV) can also be prepared in glycerol, sorbitol, liquid polyethylene glycol, and mixtures thereof, and in oils. Under normal storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms. In this regard, all of the sterile aqueous media used can be readily obtained by standard techniques known to those of ordinary skill in the art. 【0115】 Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability is possible. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, sorbitol, etc.), suitable mixtures thereof, and vegetable oils. Suitable fluidity can be maintained, for example, by the use of coating agents such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents (such as sugars or sodium chloride). Prolonged absorption of injectable compositions can be brought about by the use of agents that delay absorption, for example, aluminum monostearate and gelatin. 【0116】 Sterile injectable solutions are prepared by incorporating the rAAV in the required amount into a suitable solvent, with the various other ingredients enumerated above as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle containing the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredients from those solutions that have been previously sterile filtered. 【0117】 "Treating" includes ameliorating, reducing, or inhibiting one or more symptoms of a seizure or epileptic episode, and includes, but is not limited to, reducing or eliminating seizures, reducing seizure intensity, and / or reducing the number of seizures. Treatment also includes ameliorating or eliminating various symptoms associated with the expression of a KCNQ3 variant (i.e., a KCNQ3 pathogenic protein) disclosed herein, including, but not limited to, developmental delay, cognitive dysfunction, autism, behavioral problems, epilepsy, hypotension, and / or strabismus. 【0118】 The present disclosure also provides a kit comprising a nucleic acid, vector, or composition of the present disclosure, or manufactured according to the process of the present disclosure. In the context of the present disclosure, the term "kit" means two or more components, one of which corresponds to a nucleic acid, vector, or composition of the present disclosure, and the other corresponds to a container, recipient, instructions, or other item. Thus, a kit is a set of products sufficient to achieve a particular goal in various embodiments and can be sold as a single unit. 【0119】 The kit can include one or more recipients (vials, ampoules, containers, syringes, bottles, bags, etc.) of any suitable shape, size, and material containing the nucleic acid, vector, or composition of the present disclosure at a suitable dosage for administration (see above). The kit can additionally include instructions or directions for use (e.g., in the form of a leaflet or instruction manual), means such as syringes, pumps, injectors, etc. for administering the nucleic acid, vector, or composition, means for reconstituting the nucleic acid, vector, or composition, and / or means for diluting the nucleic acid, vector, or composition. 【0120】 In some embodiments, the kit includes a label and / or instructions that explain the use of the reagents provided in the kit. The kit can also optionally include a catheter, syringe, or other delivery device for delivering one or more of the compositions used in the methods described herein. 【0121】 The present invention also provides a kit for a single-dose administration unit or for multiple doses. In some embodiments, the present disclosure provides a kit comprising single-chamber and multi-chamber prefilled syringes. 【0122】 The entire present document is intended to be related as a unified disclosure, and it should be understood that all combinations of the features described herein are contemplated, even if the combinations of features are not found together in the same sentence, or paragraph, or section of this document. The present disclosure also includes all embodiments of the present disclosure that are somewhat narrower in scope than, for example, the variations specifically mentioned above. With respect to the aspects of the present disclosure described as a genus, all individual species are considered separate aspects of the present disclosure. With respect to the aspects of the present disclosure described or claimed with "a" or "an", it should be understood that these terms mean "one or more" unless the context clearly requires a more limited meaning. 【0123】 Unless specifically stated otherwise, the term "at least" preceding a series of elements should be understood to refer to all elements in the series. One of ordinary skill in the art will recognize or be able to ascertain many equivalents to the specific embodiments of the present disclosure described herein without undue experimentation. Such equivalents are intended to be encompassed by the present disclosure. 【0124】 As used herein, the term "and / or" shall always include the meanings of "and", "or", and "all or any other combination of the elements connected by said term". As used herein, the term "about" or "approximately" means within 20%, preferably within 10%, more preferably within 5% of a given value or range. However, it also includes the specific number, for example, about 10 includes 10. 【0125】 Throughout this specification and the following claims, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", are to be interpreted as including the stated integer or step or group of integers or steps but not as excluding any other integer or step or group of integers or steps. As used herein, the term "comprising" can be replaced by the term "containing" or "including", or, where used occasionally herein, the term "having". 【0126】 As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claims. As used herein, "consisting essentially of" excludes materials or steps that do not substantially affect the basic and novel characteristics of the claims. 【0127】 In each example herein, any of the terms "comprising", "consisting essentially of", and "consisting of" can be replaced by either of the other two terms. 【0128】 It should be understood that the present disclosure is not limited to the particular methodologies, protocols, materials, reagents, substances, etc. described herein and can therefore vary. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the subject matter of the present disclosure, which is defined only by the claims. 【0129】 All publications and patents (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.) cited throughout the text of this specification are hereby incorporated by reference in their entirety, regardless of whether above or below. To the extent that the material incorporated by reference is inconsistent with or conflicts with this specification, this specification shall prevail over any such material. 【0130】 A better understanding of the present disclosure and its advantages will be obtained from the following examples provided for illustrative purposes only. The examples are not intended to limit the scope of the present disclosure. The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes will be suggested to those skilled in the art from those perspectives, but it should be understood that they are included within the spirit and scope of the present application and the appended claims. 【Examples】 【0131】 Additional aspects and details of the present disclosure will be apparent from the following examples, which are intended to be illustrative rather than limiting. Example 1 Materials and Methods Test design. The purpose of the test was to explore new strategies for treating epilepsy and related conditions caused by mutant or pathogenic expression of KCNQ3. More specifically, the purpose of the test was to explore new strategies for treating epilepsy and related conditions caused by hereditary or novel missense mutations in the KCNQ3 gene. In some aspects, such hereditary mutations include gain-of-function mutations (R230C, R230H, R230S, and R227Q of KCNQ3) that cause DEE. Accordingly, the products and methods of the present disclosure are designed to prevent the expression of mutant or pathogenic KCNQ3 gene mutations that result in the expression of various mutant or pathogenic forms of the KCNQ3 protein. 【0132】 An RNAi approach was taken to reduce the expression of a pathogenic variant (KCNQ3-R230H) involved in a form of developmental epileptic encephalopathy (DEE), and this approach was reduced using a mouse model of KCNQ3 developmental epileptic encephalopathy, an orthologous genotype (i.e., Kcnq3 R231H / + ). Mutations in humans that reliably cause DEE in the heterozygous state include R230C, R230H, R230S, and R227Q, and all of these mutations are included for treatment by the products and methods disclosed herein. R231 is the mouse residue corresponding to R230 in humans, and thus, a mouse model expressing the orthologous genotype disclosed herein (i.e., Kcnq3 R231H / + ) was developed and tested as disclosed herein. 【0133】 Since mice that completely lack Kcnq3 from conception have only very mild impairments with respect to overt clinical phenotypes or seizures (Soh et al. (2014) J Neurosci. 34:5311-21), the approach taken was to generate an RNAi construct (microRNA (miRNA)) that targets both mutant and wild-type copies of Kcnq3 mRNA. This hypothesis was that reduction of wild-type Kcnq3 mRNA would have little or no detrimental effect on the subject, while reduction of mutant Kcnq3 mRNA would significantly reduce the phenotypic features that model the human disease caused by the mutation. 【0134】 Design and cloning of artificial microRNA targeting KCNQ3. All design rules for artificial miRNAs were followed as described (Wallace, et al PMID 29387734, Wallace et al., Mol Ther Methods Clin Dev, 2018.8: p.121-130), including a 22-nucleotide mature miRNA length, antisense complementarity to human and rodent target mRNAs (KCNQ3 / Kcnq3), a GC content of less than 60% in the mature duplex, and guide strand bias, with the last four nucleotides at the antisense 5' end being A:U rich and the last four nucleotides at the antisense 3' end being G:C rich. The miRNA was cloned into the U6T6 expression vector (Boudreau, R.L., et al., Rapid Cloning and Validation of MicroRNA Shuttle Vectors: A Practical Guide., RNA Interference Methods, S.Q. Harper, Editor. 2011, Humana Springer Press. p.19-37). After in vitro testing using a luciferase assay (as described below), the lead candidate U6.miRNA was cloned into a self-complementary proviral AAV plasmid (scAAV) containing a CMV-driven eGFP reporter. Self-complementary AAV serotype 9 (scAAV9) virus was generated and titrated by Andelyn Biosciences (Columbus, OH). Vector titers were calculated using a linear DNA standard. 【0135】 HEK293 cell culture. HEK293 cells were grown using DMEM (Gibco) medium supplemented with 20% FBS (Corning), 1% L-glutamine (Gibco), and 1% penicillin-streptomycin (Gibco). Transfected cells were grown in DMEM medium that was the same but lacking penicillin-streptomycin. 【0136】 Dual luciferase assay. A dual luciferase plasmid was generated in the Psicheck2 vector (Promega) using firefly luciferase that functions as a control, and the human KCNQ3 or mouse / rat Kcnq3 target region was cloned downstream of the Renilla luciferase stop codon (Figure 3). HEK293 cells were co-transfected (Lipofectamine 2000, Invitrogen) with the appropriate reporter and individual U6.miRNA expression plasmids at a 1:5 molar ratio. KCNQ3 / Kcnq3 silencing was determined 48 hours after transfection using the Dual Luciferase Reporter Assay System (Promega). Individual assays were performed in triplicate and the results of three independent experiments were averaged. The results of the assay are shown in Figure 4 and presented as the mean ratio ± SEM of Renilla luciferase to firefly luciferase. 【0137】 Kcnq3 gain-of-function (GoF) mutant mice with the R231H mutation. C57BL / 6J and FVB / NJ mice were purchased from The Jackson Laboratory and maintained by sibling mating in the animal facility at Columbia. Kcnq3 R231H mice were developed at the Transgenic Core at Columbia Herbert Irving Comprehensive Cancer Center by using CRISPR / Cas9 mutagenesis with a donor oligonucleotide in C57BL / 6J conjugates with the sgRNA 5'-GCAGGAUCUGCAGGAAGCGA-3' (SEQ ID NO: 38) to change the Arg231 CGC codon to CAC His and also remove the PstI restriction enzyme site for convenient genotyping. Founder mice were mated to wild-type C57BL / 6J and then backcrossed to wild-type C57BL / 6J to maintain the strain. For RNAi studies, Kcnq3R231H / + heterozygous male mice were mated to wild-type FVB / NJ to obtain Kcnq3 R231HAn F1 hybrid population was generated to isolate the mutation and used for virus injection, EEG testing, and assessment of mRNA and protein abundance (Sands et al., www.aesnet.org / abstractslisting / kcnq3-gain-of-function-mouse-model--electroclinical-and-behavioral-phenotype). All mouse procedures were approved by Columbia University’s Institutional Animal Care and Use Committee and were conducted in accordance with the National Institute of Health guide for the care and use of laboratory animals. 【0138】 Treatment of mouse pups with scAAV9. On the day after birth of F1 hybrid pups, up to 10 μl of either scAAV9-U6-miKcnq3-A-GFP virus (8.6×10 10 vg / mouse) or scAAV9-eGFP virus (7.3×10 10 vg / mouse) was delivered by intracerebroventricular (icv) injection under hypothermic anesthesia using a sterile Hamilton syringe. The pups were returned to the home cage with their mother and raised for phenotypic evaluation. 【0139】 EEG study. After 40 days of age, subdural electrodes were surgically implanted into mice as described above (PMID: 32577763) and allowed to recover for at least 48 hours before EEG recording. Recordings were acquired on a Quantum 128 amplifier and Natus Neuroworks software (Natus, Inc), and EDF format files were exported and analyzed using Assyst version 3 software (Kaoskey, Inc). Detection and processing of SWD were performed using only automated algorithms. The following settings were used for initial detection. Processing window - 1.0 second, 0.25 second step. Frequency band - 14 Hz - 23 Hz, 20 FC samples. SBI processing was set to smoothed SBI. 2 passes. Threshold definition - relative position 0.1. Event processing - events less than 0.45 seconds were removed and events over 2.0 seconds were combined. Next, the following parameters were used for SWD processing. Event time refinement window size - 0.5 second, running beat spectrum calculation parameters - spectrum length - 1.0 second, window size - 0.05 second, SWD identification parameters - dominant frequency range 6.5 Hz - 10 Hz, 4 beat spectrum peaks of SWD, first peak relative amplitude average threshold - 0.35, number of first peaks - 3. According to these algorithms, SWD self-classified into positive and negative lists and manual review was performed to exclude false negative and false positive events. No manual event combination or trimming was performed in this analysis. The event list was uploaded for compilation, genotype treatment decoding, and statistical analysis using Microsoft Excel and JMP 16 software. 【0140】 RNA extraction. Brain tissues were snap-frozen in 2-methylbutane and stored at -80 °C. Samples were homogenized using a dounce and RNA was isolated using TRIzol reagent (ThermoFisher, Waltham, MA, catalog number 15596018). RNA was converted to cDNA using the Invitrogen SuperScript III First-Strand Synthesis System (Carlsbad, CA, catalog number 18080051). 【0141】 Quantitative RT-PCR analysis. Quantitative RT-PCR analysis was performed on a QuantStudio 5 RealTime PCR system (ThermoFisher Scientific, Inc) using the following primers: Kcnq3 (5’-CACCGTCAGAAGCACTTTGAG-3’ (SEQ ID NO: 39), 5’-CCTTTAGTATTGCTACCACGAGG-3’ (SEQ ID NO: 40)), Actb (5’-GGCTGTATTCCCCTCCATCG-3’ (SEQ ID NO: 41), 5’-CCAGGTAACAATGCCATGT-3’ (SEQ ID NO: 42)), and eGFP (5’-ACGTAAACGGCCACAAGTTC-3’ (SEQ ID NO: 43), 5’-CTGGGTGCTCAGGTAGTGGT-3’ (SEQ ID NO: 44)). For data analysis, threshold cycle (Ct) values were determined for endogenous Kcnq3 and Actb mRNAs, and eGFP mRNA introduced exogenously by the virus. Then, ΔCt was calculated for Kcnq3 and eGFP by subtracting Actb from each as an endogenous standard, and the transduction-specific ΔCt for Kcnq3 was calculated by further subtracting eGFP to enhance the analysis for transduced cells. Statistical evaluation was performed by converting the ΔCt values using JMP16 software to non-parametric and least-squares regression. 【0142】 Western blot analysis. The dissected mouse brain tissues were snap-frozen in liquid nitrogen and stored at -80 °C until extraction. The tissues were thawed on ice and homogenized using an electric mortar in RIPA buffer containing both protease and phosphatase inhibitor cocktails (Roche). The samples were centrifuged, and the resulting supernatants were collected and quantified using the BCA method (Pierce) with BSA as a standard. Using the Xcell Surelock Mini Cell system, a total of 15 μg of protein lysate per sample was loaded onto a 4-12% SDS-PAGE gel and subsequently transferred to a PVDF membrane. The membrane was incubated overnight at 4 °C with primary antibodies - KCNQ3 - 1:1000 (Synaptic systems - Kv7.3 - 368003); ACTB - 1:15,000 (Santa Cruz Biotechnology: sc - 47778), and then incubated for 1 hour at room temperature with secondary HRP-conjugated goat anti-rabbit (1:10,000) (Proteintech - SA00001 - 2). The signals were developed using Amersham ECL Western blotting detection reagent (GE Healthcare, RPN2106) and visualized using a Western blot imaging system (Azure Biosystems, Azure C400). 【0143】 Example 2 Design and functional in vitro screening of artificial microRNAs targeting conserved regions on human and rodent KCNQ3 transcripts The artificial microRNAs of the present disclosure were designed using the algorithms described by Wallace et al. ((2017) Mol Ther Methods Clin Dev. Dec 24, 8:121-130, see also Boudreau et al. (2011) “Rapid Cloning and Validation of MicroRNA Shuttle Vectors: A Practical Guide.” RNA Interference Methods. Ed. S.Q. Harper. Humana Springer Press, 2011, pages 19-37). Briefly, all microRNAs contain processing sites for the RNAse III enzymes Drosha and Dicer, resulting in mature 22 nucleotide (nt) duplex RNAs containing 2 nt 3’ overhangs on both strands (Figs. 1-2). The antisense guide strand of the microRNA is incorporated into the RNA-induced silencing complex (RISC), directing the cellular gene silencing machinery to cleave the target mRNA, in this case human KCNQ3 or murine Kcnq3. 【0144】 To identify microRNAs having the features listed above, human KCNQ3 cDNA was used as the query sequence (SEQ ID NO: 1). The longest full-length KCNQ3 transcript listed in ENSEMBL is 11,583 nucleotides long and contains a 563 nt and an 8,401 nt 3’UTR (ENSEMBL transcript ID ENST00000388996.10; KCNQ3-201). The open reading frame (ORF) is 2,619 nt long (ENSEMBL CCDS34943; SEQ ID NO: 1). Since species conservation (human, mouse, and rat) was included in the design of the miRNA constructs, and the protein coding region typically contains the highest amount of conservation among species, only the ORF was used as the query sequence. Thus, using the 2,619 nt human KCNQ3 ORF as the query sequence (SEQ ID NO: 1), 152 candidate microRNAs that met the desired criteria were identified as described above. The microRNAs target both the mutant and wild-type KCNQ3, rather than alleles specific only to known mutations. Since only patients with KCNQ3 mutations require treatment to reduce the expression of the mutant or pathogenic form of the KCNQ3 protein, allele specificity was not required. 【0145】 The human KCNQ3 ORF was aligned with the ORFs of the rat and mouse Kcnq3 ORFs to identify miRNA binding sites located in conserved regions of each transcript. Seven miRNAs, namely, miKCNQ3-A-G, were identified and constructed. All candidate microRNAs were cloned into the U6T6 plasmid containing the U6 promoter and the RNA polymerase III termination signal (TTTTTT; SEQ ID NO: 45), and the sequences were verified. See Figure 2 for the binding sites, sequence alignments, and folded primary miRNA transcripts. 【0146】 Example 3 Reduction or inhibition of KCNQ3 protein level in vitro To measure the silencing of human or murine KCNQ3, a luciferase reporter plasmid containing the human KCNQ3 or murine Kcnq3 sequence was constructed as the 3’UTR of Renilla luciferase. The reporter plasmid contained firefly luciferase, a second gene used as a normalization control (Figure 3). 【0147】 To develop effective miRNA reagents targeting Kcnq3 mRNA, seven potential KCNQ3 / Kcnq3-specific miRNAs were tested for their effectiveness against the target after heterologous expression in HEK293 cells in vitro using a luciferase assay. More specifically, to perform luciferase assay screening, HEK293 cells were transfected with the U6.miKCNQ3 plasmids (miKCNQ3A-G), a non-targeting control plasmid (miGFP), and the KCNQ3 luciferase reporter plasmid. Luciferase activity was measured 48 hours later (Figure 4). 【0148】 All seven miRNAs tested resulted in a significant decrease in human target mRNA. However, only sequence A (miKCNQ3-A) caused silencing of the murine Kcnq3 sequence bound to Renilla luciferase. This was unexpected and without precedent. The 5’ end of the murine Kcnq3 cDNA is high GC-rich with repetitive sequences. Binding these difficult sequences (i.e., sequences with a high GC content are difficult to clone and often result in deletions) as the 3’UTR of Renilla luciferase may affect mRNA folding, thereby making the target site inaccessible to microRNA and the endogenous silencing machinery. Since sequence A caused silencing of both human and murine KCNQ3 / Kcnq3 transcripts in this initial test, it was selected as an initial lead and cloned into an scAAV9 proviral plasmid (scAAV9-miKCNQ3) that also contains a separate CMV-eGFP reporter gene as U6-miKQNC3-A. 【0149】 The self-complementary AAV9 vector was generated, purified, and titered by Andelyn Biosciences for transfection into HEK cells for large-scale viral production, purification, and concentration of AAV9 vector particles. The scAAV9-eGFP virus was similarly produced and used as a control virus for in vivo testing. 【0150】 Example 4 Characterization of the epileptic mouse model (Kcnq3 R231H / + mouse) Kcnq3 R231H / + In the characterization of the clinically relevant phenotypic features of Kcnq3 mice, it was determined that heterozygotes have a form of generalized epilepsy in the form of frequent, spontaneous spike-wave discharges (SWD) in the electroencephalogram (EEG), rather than wild-type littermates (Figure 5). EEG is considered the "gold standard" for seizures and is one of the strongest, clearest, and most quantitative measures among preclinical behaviors of all neurobehavioral behaviors other than epilepsy. 【0151】 Prior to the in vivo testing of scAAV9-miKcnq3-A, the basic molecular characteristics of Kcnq3 R231H / + mouse mutant mice were characterized. Heterozygous Kcnq3R R231H / + mice show a significantly reduced threshold (increased sensitivity) to electrically induced maximal seizures compared to wild-type littermates, indicating a general tendency towards seizures. Each of these electroclinical features is quantitative and reproducible and represents a powerful and clinically relevant endpoint for measuring the efficacy of new therapies. 【0152】 First, it was determined that the total Kcnq3 mRNA and protein levels in the brains of heterozygous mice were not significantly different from those of wild-type littermates (Figures 6A - D). Similarly, no differences were seen in Kcnq2 mRNA and protein levels. Kcnq2 is the primary subunit in the heterotetrameric Kcnq3 ion channel. These results suggest that Kcnq3 R231His consistent with the fact that it encodes a gain-of-function mechanism, as determined in previous heterologous expression studies (Miceli et al., Front Physiol. 2020 Sep 4;11:1040). 【0153】 An increase in membrane-bound Kcnq3 (but not Kcnq2) was observed. Without being bound by theory, this increase may be the result of compensation for physiological changes, which may be due to an increase in some transport, as has been reported for the KCNQ1 channel (Huang et al., J Biol Chem, 2021. 296: p. 100423). 【0154】 Example 5 Reduction or inhibition of KCNQ3 protein level in vivo The experiment was designed to test the scAAV9-miKcnq3 vector in vivo for its efficacy against the spontaneous seizure phenotype. Matings were set up between FVB / NJ females (The Jackson Laboratory) and C57BL / 6J-Kcnq3R231 H / + males (Columbia Herbert Irving Comprehensive Cancer Center) to generate approximately equal numbers of F1 hybrid Kcnq3R231 H / + (heterozygous) and Kcnq3 + / + (wild-type) littermates, which were genetically identical except for the Kcnq3 genotype. Due to known hybrid vigor and litter size, F1 hybrids were used instead of the inbred C57BL / 6J strain background, greatly facilitating the logistics of the study while maintaining a genetically homogeneous background in the test population (F1 hybrids have one chromosomal copy from each parental strain and are genetically identical to each other). Mouse pups were genotyped on postnatal day 0, and each mouse was injected with 10 μl of control virus (scAAV9-CMV-eGFP; 7.3×10 10 g), experimental virus (8.6×10 10On the first day after birth, they were treated by unilateral intracerebral injection with vg), or physiological saline. Eight heterozygotes were treated with the control virus, and ten heterozygotes were treated with the experimental virus. In addition, four wild-type mice were treated with the same amount of the experimental virus, and three wild-type mice were treated with physiological saline. 【0155】 Adult mice were surgically implanted with recording electrodes 40 days after birth and recorded by video EEG continuously for 24 hours between 47 and 61 days after birth (see Table 3 and Figure 8). Three heterozygous mice treated with the experimental virus, five heterozygous mice treated with the control virus, and one wild-type mouse treated with the control virus were bred for an additional period, and video EEG was recorded at about 15 weeks after birth. The results of these experiments are summarized in Table 3 and Figures 7 and 8. 【0156】 【Table 3】 【0157】 The results showed that only 3 out of 7 mutant mice (Kcnq3 R231H / + ) had any SWDs when treated with scAAV9-miKcnq3, compared to 8 / 8 mice that had SWDs when treated with the control eGFP virus. Three mutant mice (Kcnq3 R231H / + ) had SWDs, but their incidence was on average twice as low as that of mice treated with the control virus. The average incidence of SWDs was 15 times higher in mice treated with the control virus compared to mice treated with miKcnq3. 【0158】 These results indicate that scAAV9-miKcnq3-A was very effective in reducing the incidence, duration, and severity of this form of seizure in mice having an ortholog of the pathological clinical variant of KCNQ3. 【0159】 Figures 7A - B show adult Kcnq3 transduced with scAAV9-miKCNQ3 as neonatesR231H / + There was a significant decrease in the SWD incidence rate (Figure 7A) and the average SWD duration (Figure 7B) in mice. Figure 8 shows Kcnq3 transduced with scAAV9-miKcnq3-A as a neonate R231H / + Decrease in SWD incidence rate (upper panel) and decrease in SWD duration (lower panel) in adult mice are shown. The dotted line indicates the same mice tested at both ages. The p-values shown are based on a one-sided Fisher's exact test. 【0160】 Following the evaluation of SWD, brain samples were taken from five heterozygotes treated with the experimental virus, three heterozygotes treated with the control virus, and one wild type treated with the control virus to examine Kcnq3 mRNA levels after miKcnq3 or eGFP treatment. The results clearly show a reduced expression of Kcnq3 mRNA compared to eGFP associated with miKcnq3 treatment (Figure 9). 【0161】 After the EEG test, proteins were extracted from the brain samples and Western blot analysis (Figure 10A) and concentration measurement quantification (Figure 10B) were performed to measure KCNQ3 protein using an antibody against KCNQ3 and an antibody against β-actin as a loading control (Figure 10A - B). As expected for RNAi targeting mRNA regardless of the mutant allele, the amount of KCNQ3 protein in mice treated with scAAV9-miKcnq3-A was significantly reduced compared to mice treated with the scAAV9-eGFP control virus, regardless of whether the treated mice were heterozygous mutant or wild type (+ / +) littermates. 【0162】 This study showed that the scAAV9-miKcnq3-A vector was effective in reducing Kcnq3 mRNA and protein expression and was very effective in reducing the incidence, duration, and severity of this form of seizure in mice with an ortholog of the pathological clinical variant of KCNQ3. 【0163】 The foregoing description is given for clarity of understanding only, and modifications within the scope of the invention may be apparent to those skilled in the art, so no unnecessary limitations should be understood therefrom. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", are to be interpreted to mean the inclusion of the stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. 【0164】 Throughout this specification, where a composition is described as comprising a component or material, it is contemplated that the composition may consist essentially of, or consist of, any combination of the recited components or materials, unless otherwise stated. Similarly, where a method is described as comprising a particular step, it is contemplated that the method may also consist essentially of, or consist of, any combination of the recited steps, unless otherwise stated. The invention disclosed herein by way of example may be suitably practiced without the presence of any element or step specifically disclosed herein. 【0165】 The practice of the methods disclosed herein, and their individual steps, can be performed manually and / or using automation provided by or with the aid of electronic devices. Although the processes are described with reference to specific embodiments, those skilled in the art will readily understand that other ways of performing the acts associated with the methods may be used. For example, various orders of the steps may be changed without departing from the scope or spirit of the method, unless otherwise stated. In addition, some of the individual steps may be combined, omitted, or further subdivided into additional steps. 【0166】 All patents, publications, and references cited in this specification are hereby incorporated by reference in their entirety. In the event of any conflict between this disclosure and the incorporated patents, publications, and references, this disclosure shall prevail.

Claims

[Claim 1] A nucleic acid encoding a potassium channel, voltage-gated KQT-like subfamily Q, member 3 (KCNQ3) targeted microRNA (miRNA), (a) A nucleotide sequence having at least 90% identity with the sequence shown in any one of sequence numbers 3 to 9, (b) A nucleotide sequence shown in any one of sequence numbers 3 to 9, (c) A nucleotide sequence encoding an RNA sequence shown in any one of SEQ ID NOs: 17-23, or (d) A nucleic acid comprising a nucleotide sequence that specifically hybridizes with the KCNQ3 sequence shown in any one of sequence numbers 24 to 30. [Claim 2] The nucleic acid according to claim 1, further comprising a promoter and / or enhancer. [Claim 3] The nucleic acid according to claim 2, wherein the promoter and / or enhancer is any one of the following: U6 promoter and / or enhancer, U7 promoter and / or enhancer, tRNA promoter and / or enhancer, H1 promoter and / or enhancer, CMV promoter and / or enhancer, minimal CMV promoter and / or enhancer, T7 promoter and / or enhancer, EF1-alpha promoter and / or enhancer, minimal EF1-alpha promoter and / or enhancer, unc45b promoter and / or enhancer, CK1 promoter and / or enhancer, CK6 promoter and / or enhancer, CK7 promoter and / or enhancer, CK8 promoter and / or enhancer, ubiquitous promoter and / or enhancer, neuron-specific promoter and / or enhancer, or brain-specific promoter and / or enhancer. [Claim 4] The nucleic acid according to claim 3, wherein the promoter and / or enhancer is U6. [Claim 5] (a) A nucleotide sequence having at least 90% identity with the sequence shown in any one of SEQ ID NOs: 10 to 16, or (b) The nucleic acid according to claim 2, comprising the nucleotide sequence shown in any one of sequence numbers 10 to 16. [Claim 6] The nucleic acid according to claim 3, wherein the brain-specific promoter and / or enhancer is human synapsin 1 (hSyn1), neuron-specific enolase (Nse), MeCP2, mDLX, mDLX5 / 6, or calmodulin-dependent kinase II (CaMKII or Camk2a). [Claim 7] An adeno-associated virus comprising the nucleic acid described in any one of claims 1 to 6. [Claim 8] The adeno-associated virus according to claim 7, wherein the virus lacks the rep and cap genes. [Claim 9] The adeno-associated virus according to claim 7, wherein the virus is recombinant AAV (rAAV) or self-complementary recombinant AAV (scAAV). [Claim 10] The adeno-associated virus according to claim 7, wherein the virus is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rh10, AAV11, AAV12, AAV13, AAV-anc80, AAV-B1, AAV.PHP.EB, or AAVv66. [Claim 11] The adeno-associated virus according to claim 7, wherein the virus is AAV9. [Claim 12] Nanoparticles, extracellular vesicles, or exosomes comprising the nucleic acid described in any one of claims 1 to 6. [Claim 13] A nucleic acid according to any one of claims 1 to 6, A composition comprising a pharmaceutically acceptable carrier. [Claim 14] The composition according to claim 13 for reducing, inhibiting, and / or interfering with the expression of potassium channels, voltage-gated KQT-like subfamily Q, member 3 (KCNQ3) gene, or variants thereof in cells. [Claim 15] The composition according to claim 13 for treating a subject having a KCNQ3 mutation that results in the expression of a variant or pathogenic form of KCNQ3. [Claim 16] The composition according to claim 15, wherein the mutation is a base substation, deletion, or insertion. [Claim 17] The composition according to claim 15, wherein the mutation is any one or more mutations in the KCNQ3 gene resulting in substitutions of R230C, R230H, R230S, and / or R227Q in the KCNQ3 polypeptide. [Claim 18] The composition according to claim 15, wherein the subject suffers from seizures, epileptic disorders or disabilities, intellectual or developmental disabilities, autism, or autism spectrum disorder, which are associated with mutant or pathogenic KCNQ3 expression. [Claim 19] The composition according to claim 13 for treating or improving a subject suffering from seizures, epileptic disorders or disabilities, intellectual or developmental disabilities, autism, or autism spectrum disorder associated with mutant or pathogenic KCNQ3 expression. [Claim 20] The composition according to claim 19, wherein the subject is suffering from developmental epileptic encephalopathy (DEE). [Claim 21] The composition according to claim 19, wherein the subject is affected by a mutation in the KCNQ3 gene, and the mutation is any one or more mutations in the KCNQ3 gene that result in the substitution of R230C, R230H, R230S, and / or R227Q in the KCNQ3 polypeptide. [Claim 22] Use of nucleic acids according to any one of claims 1 to 6 for the preparation of pharmaceuticals for reducing or inhibiting the expression of potassium channels, voltage-gated KQT-like subfamily Q, member 3 (KCNQ3) genes or variants thereof in cells. [Claim 23] Use of the nucleic acid according to any one of claims 1 to 6 for the preparation of a pharmaceutical product for treating or improving seizures, epileptic disorders or disabilities, intellectual or developmental disabilities, autism, or autism spectrum disorders associated with mutant or pathogenic KCNQ3 expression. [Claim 24] The use according to claim 23, wherein the seizure, epileptic disorder or disability, intellectual or developmental disability, autism, or autism spectrum disorder associated with mutant or pathogenic KCNQ3 expression is developmental epileptic encephalopathy (DEE). [Claim 25] The use according to claim 23, wherein the seizures, epileptic disorders or disabilities, intellectual or developmental disabilities, autism, or autism spectrum disorders associated with mutant or pathogenic KCNQ3 expression are caused by any one or more mutations in the KCNQ3 gene resulting in substitutions of R230C, R230H, R230S, and / or R227Q in the KCNQ3 polypeptide. [Claim 26] The composition according to claim 13, formulated for intraventricular injection, intrathecal injection, intravascular injection, aerosol administration, or oral administration.

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