RNAi targeting KIF1A missense mutations for treatment of KIF1A-associated neurological disorders

Abstract

Provided are RNAi involving siRNA, shRNA or antisense oligonucleotide (ASO) that reduce the expression of toxic KIF1A alleles, thereby reducing the production of mutant KIF1A, for use in the treatment of KAND. Also provided are RNA targeting oligonucleotides that reduce the expression of toxic KIF1A alleles herein incorporate sequences that target common benign single nucleotide polymorphisms (SNPs) that are cis present with one or more pathogenic KIF1A mutations.

Description

Cross Reference to Related Applications

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 506,460, filed June 6, 2023, and U.S. Provisional Application No. 63 / 603,847, filed November 29, 2023, both of which are incorporated by reference in their entireties. TECHNICAL FIELD

[0002] The present disclosure relates to reducing toxicity in treating KIF1A-associated neurological disorders KIF1A RNAi compositions that reduce expression of the toxic allele. Reference to a Sequence Listing

[0003] The instant application contains a Sequence Listing which has been submitted electronically in.XML format and which is hereby incorporated by reference in its entirety. The.XML copy, created on June 4, 2024, is named “2262-101 PCT.xml” and is 695,054 bytes in size. The Sequence Listing contained in the.XML file is part of the specification and is hereby incorporated by reference in its entirety. BACKGROUND

[0004] KIF1A-associated neurological disorders (KAND) encompass a group of rare, progressive neurodegenerative conditions caused by KIF1A mutations in the KIF1A gene. KIF1A is a member of the kinesin-3 family of microtubule motor proteins. KIF1A functions primarily as a protein dimer and is involved in anterograde axonal transport of synaptic vesicle precursors and dense-core secretory vesicles along axonal microtubules. The majority of known pathogenic KIF1A mutations are located within the motor domain, which means a defect in the transport function. KIF1A Mutations in the gene can disrupt KIF1A’s ability to transport synaptic vesicles, leading to a variety of neuropathologies. In KIF1A particular, the spectrum of mutations leading to the toxic allele is broad.

[0005] KAND is associated with a variety of symptoms. See, e.g., https: / / rarediseases.org / rare-diseases / kifl la-related-disorder. Symptoms can include intellectual disability, delays in reaching developmental milestones (developmental delay), reduced muscle tone (hypotonia), and delayed language development. Additional symptoms include exaggerated reflexes (hyperreflexia), and increased muscle tone (hypertonia) as the infant or child ages. Ultimately, affected children can develop spastic paraplegia, a condition in which a person has difficulty walking due to weakness and muscle tightness (spasticity) in the legs. Spastic paraplegia can worsen over time, severely impacting the ability to walk and move around. Some affected individuals have optic atrophy and experience narrowing of the visual field. Some affected individuals experience progressive worsening of cerebellar nerve cells (cerebellar atrophy), which can lead to problems with balance and coordination, and experience peripheral neuropathy. Other symptoms can include poor coordination (ataxia), rapid, involuntary eye movements (nystagmus), crossed eyes (strabismus), drooping of the upper eyelid (ptosis), weakness or paralysis of half the facial muscles (bilateral facial paralysis), clumsiness in attempting to use the hands to manipulate or hold objects, and tremors that occur when trying to make a voluntary movement (intention tremor) and small head deformity. Various different seizure types can also occur.

[0006] There are currently no effective treatments or cures for KAND. Because there are excess mutations leading to toxicity KIF1A Effective treatment options become complicated due to the excess mutations of the allele leading to toxicity SUMMARY

[0007] Provided herein are novel RNA-targeting oligonucleotides, i.e., RNAi involving small interfering RNA (siRNA), short hairpin RNA (shRNA), and antisense oligonucleotides (ASO), that reduce expression of the toxic KIF1A allele, thereby reducing production of mutant KIF1A, for the treatment of KAND. In embodiments, the RNA-targeting oligonucleotides herein that reduce expression of the toxic KIF1A allele incorporate sequences that target common benign single nucleotide polymorphisms (SNPs) that are in cis with one or more pathogenic KIF1A mutations.

[0008] Accordingly, provided herein are five separate KIF1ARNAi sequences for one or more of the missense mutations (T99M, E253K, P305L, R316W, and R203S). The RNAi sequences can be delivered as short interfering RNA (siRNA) duplexes or transcribed from plasmid DNA as short hairpin RNA (shRNA). Expression vectors encoding the RNAi sequences are provided. In embodiments, the expression vectors are adeno-associated virus (AAV) vectors or lentivirus vectors. In embodiments, the siRNAs are delivered by nanoparticle vehicles or by polymeric vehicles. Pharmaceutical compositions comprising the foregoing are provided.

[0009] In embodiments, provided herein are siRNAs targeting individual KIF1A siRNAs for missense mutations comprising a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or 100% complementary to any one of SEQ ID NOs: 2-229.

[0010] In embodiments, provided herein are siRNAs targeting SNP rs1063353 (L331L) for which one or more pathogenic KIF1A mutations are present in cis, thereby inducing knockdown of the targeted protein. In embodiments, the pathogenic KIF1A missense mutations are one or more of T99M, E253K, P305L, R316W, and R203S that are pathogenic for KAND. In embodiments, provided herein are siRNAs targeting SNP rs1063353 (L331L) comprising a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or 100% complementary to any one of SEQ ID NOs: 230-253.

[0011] In embodiments, provided herein are antisense oligonucleotides (ASOs) targeting SNP rs1063353 (L331L) for which one or more pathogenic KIF1A mutations are present in cis, thereby inducing knockdown of the targeted protein. In embodiments, the pathogenic KIF1A missense mutations are one or more of T99M, E253K, P305L, R316W, and R203S that are pathogenic for KAND. In embodiments, provided herein are antisense oligonucleotides targeting SNP rs1063353 (L331L) comprising a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or 100% identical to any one of SEQ ID NOs: 254-283, 536, 537, and 538.

[0012] In embodiments, provided herein are expression vectors encoding siRNAs targeting individualKIF1A Polynucleotides of siRNA of missense mutations, the siRNA comprising a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or 100% complementary to any one of SEQ ID NOs: 2-229. In embodiments, the polynucleotides encoding SEQ ID NOs: 2-229 are SEQ ID NOs: 284-511, respectively.

[0013] In embodiments, provided herein are polynucleotides encoding siRNA targeting SNP rs1063353 (L331L), which SNP rs1063353 (L331L) is present in cis with one or more pathogenic KIF1A mutations to induce knockdown of the targeted protein, the siRNA comprising a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or 100% complementary to any one of SEQ ID NOs: 230-253. In embodiments, the polynucleotides encoding SEQ ID NOs: 230-253 are SEQ ID NOs: 512-535.

[0014] In embodiments, shRNAs and polynucleotides encoding shRNAs are provided that relate to SEQ ID NOs: 2-253, SEQ ID NOs: 284-511, and SEQ ID NOs: 512-535.

[0015] Expression vectors comprising the polynucleotides, siRNAs, shRNAs, or ASOs are provided. In embodiments, the expression vectors are adeno-associated virus (AAV) vectors or lentiviral vectors. Pharmaceutical compositions comprising the foregoing are provided. DETAILED DESCRIPTION

[0016] The compositions described herein are intended to target toxic KIF1A alleles, thereby reducing the production of mutant KIF1A for the treatment of KAND. Effective inhibition of mutant KIF1A by the RNAi oligonucleotides described herein results in a reduction in the expression level of mutant KIF1A without interfering with non-mutant wild-type KIF1A. The genome KIF1ARelevant information is available at the following public disclosures: Gene ID. 547 (https: / / www.ncbi.nlm.nih.gov / gene / 547); kinin family member 1A (KIF1A), GenBank accession number NC_000002.12; NCBI reference sequence: NG_029724.1. The RNAi described in this article is based on Homo sapiens kinin family member 1A (KIF1A), transcript variant 1, mRNA, GenBank accession number NM_001244008.2, designated as SEQ ID NO: 1 in this article. KIF1A Proteins possessing the UniProtKB identifier Q12756. As used herein, "mutant" KIF1A "Including five separate" KIF1A Any one or more of the missense mutations (T99M, E253K, P305L, R316W, and R203S) KIF1A Variants. Some KIF1A The variants are described as 52 transcripts mentioned in KIF1AENSG00000130294 (https: / / useast.ensembl.org / Homo_sapiens / Gene / Summary?g=ENSG00000130294;r=2:240713761-240824293).

[0017] The RNAi oligonucleotides described herein are siRNA, shRNA, or ASO. RNAi as used herein may also be referred to as short interfering nucleic acid (siNA). In the implementation, a specific RNAi sequence (which may be delivered as a siRNA duplex or ASO or transcribed from plasmid DNA as shRNA) targets five separate pathogenicity markers of KAND. KIF1A Missense mutations (T99M, E253K, P305L, R316W, and R203S) (“ KIF1A One or more of the "missense mutations". In embodiments, this document provides a double-stranded RNA molecule incorporating an antisense strand and a sense strand, wherein the nucleotide sequence of the antisense strand is identical to that of a human mutant. KIF1A The nucleotide sequences are regionally complementary. In the implementation, this document provides a double-stranded RNA molecule incorporating an antisense strand and a sense strand, wherein the nucleotide sequence of the antisense strand is regionally complementary to that of a human mutant. KIF1A The nucleotide sequence of transcript variant 1, mRNA, SEQ ID NO: 1 is regionally complementary. In the implementation scheme, human... KIF1A The region of the nucleotide sequence contains five separate KIF1A One or more missense mutations.

[0018] To avoid being bound by specific theories, the RNAi described in this paper can suppress mutants through the following methods. KIF1A (1) Cutting is made by having one or more KIF1A Mutants of missense mutations KIF1A (2) Reduced by having one or more KIF1A Mutants of missense mutations KIF1A The steady-state level of the encoded RNA transcript (i.e., the baseline level at steady state); and / or (3) termination with one or more KIF1A Mutants of missense mutations KIF1A Transcription.

[0019] siRNA molecules can be composed of a characteristic 19+2mer structure (i.e., a double helix of two 21-nucleotide RNA molecules with 19 complementary bases and a terminal 2-nucleotide 3' overhang, such as dTdT at the 3' end). One strand of the siRNA (guide strand or antisense strand) is complementary to the target transcript, while the other strand is designated as the transit strand or sense strand. As part of the RNA-induced silencing complex (RISC), the siRNA acts as a guide for the Argonaute 2 protein (AGO2) to its complementary target transcript. The complementarity between the siRNA and the target transcript leads to the cleavage of the target relative position of the guide strand, catalyzed by AGO2, resulting in gene silencing.

[0020] In the implementation scheme, the sense strand of the siRNA is any one of SEQ ID NO: 2-115. In the implementation scheme, the antisense strand of the siRNA is any one of SEQ ID NO: 116-229.

[0021] antisense siRNA strand and KIF1A Non-complementary nucleobases between nucleotide sequences can be tolerated, provided that the antisense siRNA remains able to interact with... KIF1A Nucleotide sequence-specific hybridization.

[0022] In some embodiments, the siRNA may comprise a nucleotide sequence of at least 85% complement and of equal length to any one of SEQ ID NO: 2-115. In some embodiments, the siRNA may comprise a nucleotide sequence of at least 90% complement and of equal length to any one of SEQ ID NO: 2-115. In some embodiments, the siRNA may comprise a nucleotide sequence of at least 95% complement and of equal length to any one of SEQ ID NO: 2-115. In some embodiments, the siRNA may cover a nucleotide sequence of 100% complement and of equal length to any one of SEQ ID NO: 2-115 (in this case, SEQ ID NO: 116-229). The percentage of complementarity is used herein in a conventional sense to refer to the base pairing between adenine and thymine, adenine and uracil (RNA), and guanine and cytosine.

[0023] In the implementation scheme, the siRNA or a designated portion thereof provided herein is similar to... KIF1A RNA nucleotide sequences that are 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary or compatible with each other. KIF1A The RNA nucleotide sequence must be at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary. shRNA and KIF1A The percentage of complementarity in nucleotide sequences can be determined using conventional methods.

[0024] For example, 18 of the 20 nucleobases are... KIF1A The antisense strand of the siRNA, with complementary regions and thus specific hybridization, will represent 90% complementarity. In this example, the remaining non-complementary nucleotides can be clustered or scattered with complementary nucleotides and do not need to be adjacent to each other or adjacent to complementary nucleotides. Therefore, an siRNA of 18 nucleotides in length with four non-complementary nucleotides flanked by two regions perfectly complementary to the target nucleotide sequence will have 77.8% overall complementarity to the target nucleotide sequence and will therefore fall within the scope of the subject matter disclosed herein. siRNA and KIF1AThe percentage of complementarity of regions of a nucleotide sequence can be routinely determined using BLAST (Basic Local Alignment Search Tool) and PowerBLAST (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), both known in the art. The percentage of homology, sequence identity, or complementarity can be determined using, for example, the Gap program (Wisconsin sequence analysis package for Unix, version 8, Genetics Computer Group, University Research Park, Madison Wis.) with default settings, employing the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

[0025] In one embodiment, the siRNA may comprise a nucleotide sequence of at least 85% complement and of equal length to an RNA sequence encoded by any of SEQ ID NO: 284-397. In another embodiment, the siRNA may comprise a nucleotide sequence of at least 90% complement and of equal length to an RNA sequence encoded by any of SEQ ID NO: 284-397. In yet another embodiment, the siRNA may comprise a nucleotide sequence of at least 95% complement and of equal length to an RNA sequence encoded by any of SEQ ID NO: 284-397. In yet another embodiment, the siRNA may encompass a nucleotide sequence of 100% complement and of equal length to an RNA sequence encoded by any of SEQ ID NO: 284-397 (in this case, SEQ ID NO: 398-511).

[0026] In the implementation, the antisense strand is a shortened or truncated modified oligonucleotide. The shortened or truncated modified oligonucleotide may have a single nucleoside deleted from the 5' end (5' truncated), the central portion, or optionally from the 3' end (3' truncated). The shortened or truncated oligonucleotide may have one or more nucleosides deleted from the 5' end, one or more nucleosides deleted from the central portion, or optionally one or more nucleosides deleted from the 3' end. Alternatively, the deleted nucleosides may be dispersed throughout the modified oligonucleotide, for example, dispersed in the antisense strand, having one or more nucleosides deleted from the 5' end, one or more nucleosides deleted from the central portion, and / or one or more nucleosides deleted from the 3' end.

[0027] In the implementation scheme, siRNA may include, but is not limited to, modified siRNA, including siRNA with enhanced in vivo stability. Modified siRNA includes molecules containing nucleotide analogs, including those with additions, deletions, and / or substitutions in the nucleotides, sugars, or backbone; and cross-linked or otherwise chemically modified molecules. One or more modified nucleotides may be within a portion of the siRNA molecule or throughout the entire siRNA molecule. For example, the siRNA molecule may be modified, or contain modified nucleic acids in a region at its 5' end, its 3' end, or both, and / or within the guide strand, the transit strand, or both, and / or within nucleotides protruding beyond the 5' end, 3' end, or both. In the implementation scheme, the nucleic acid may be chemically modified at the backbone, nucleotides, ribose, and 2'-ribose substitutions, for example, by cEt (restrained ethyl-bridged nucleic acid); ENA (ethylene-bridged nucleic acid); 2'-F (2'-fluorine); LNA (locked nucleic acid); 2'-MOE (2'-... O -Methoxyethyl;2'-OMe,2'- O -Methyl; PMO, phosphodiamide morpholino oligonucleotide; PNA, peptide nucleic acid; the phosphodiester bond between nucleotides can be replaced by a thiophosphate bond, PS, thiophosphate; tcDNA, tricyclic DNA.

[0028] shRNA is also involved in RISC. After the vector carrying the shRNA genomic material integrates into the host genome, the shRNA genomic material is transcribed into pri-microRNA in the host. pri-microRNA is processed by ribonucleases such as Drosha into pre-shRNA and exported from the nucleus. Pre-shRNA is processed by endonucleases such as Dicer to form siRNA. siRNA is loaded into RISC, where the sense strand is degraded and the antisense strand acts as a guide to direct the RISC to the complementary sequence in the mRNA. When the sequence is perfectly complementary, RISC cleaves the mRNA, and when the sequence is imperfectly complementary, RISC represses the translation of the mRNA. Therefore, shRNA reduces or eliminates the translation of mRNA by having one or more of these sequences. KIF1A Mutants of missense mutations KIF1A Expression of encoded RNA transcripts.

[0029] As used herein, “short hairpin RNA (shRNA)” includes conventional stem-loop shRNA, which forms precursor microRNA (premiRNA). “shRNA” also includes shRNA that embeds microRNA (miRNA-based shRNA), in which the guide strand and guest strand of the miRNA duplex are integrated into an existing (or natural) miRNA or into a modified or synthetic (designed) miRNA. During transcription, conventional shRNA forms a primary miRNA (pri-miRNA) or a structure very similar to the natural pri-miRNA. The pri-miRNA is then processed into preshRNA by Drosha and its cofactors. Therefore, the term “shRNA” includes both pri-miRNA (shRNA-mir) molecules and preshRNA molecules.

[0030] A “stem-loop structure” refers to a nucleic acid having a secondary structure comprising a region of double-stranded or duplexed nucleotides (stem portion) connected on one side by a region consisting primarily of single-stranded nucleotides (loop portion). It is known in the art that the loop portion is at least 4 nucleotides long, 6 nucleotides long, 8 nucleotides long, or longer. The terms “hairpin” and “fold-back” are also used herein to refer to stem-loop structures. Such structures are well known in the art, and the use of the term is consistent with its known meaning in the art. For example, DNA loop portions CTCGAG (SEQ ID NO: 539), TCAAGAG (SEQ ID NO: 540), TTCG (SEQ ID NO: 541), and GAAGCTTG (SEQ ID NO: 542) or RNA loop portions CUCGAG (SEQ ID NO: 543), UCAAGAG (SEQ ID NO: 544), UUCG (SEQ ID NO: 545), and GAAGCUUG (SEQ ID NO: 546) are suitable stem-loop structures. As is known in the art, secondary structures do not require precise base pairing. Therefore, stems may include one or more base mismatches or protrusions. Alternatively, base pairing may be precise, i.e., without any mismatches.

[0031] In the implementation, an shRNA sequence is provided comprising a first part, a second part, and a third part, wherein the first part comprises any one of SEQ ID NO: 2-115, the second part comprises any one of SEQ ID NO: 539-546, and the third part comprises corresponding nucleotide sequences complementary to those in SEQ ID NO: 2-115 (i.e., SEQ ID NO: 116-229).

[0032] In the implementation, an shRNA sequence is provided comprising a first part, a second part, and a third part, wherein the first part comprises any one of SEQ ID NO: 230-241, the second part comprises any one of SEQ ID NO: 539-546, and the third part comprises corresponding nucleotide sequences complementary to those in SEQ ID NO: 230-241 (i.e., SEQ ID NO: 242-253).

[0033] In the implementation scheme, shRNA may include, but is not limited to, modified shRNA, including shRNA with enhanced in vivo stability. Modified shRNA includes molecules containing nucleotide analogs, including those with additions, deletions, and / or substitutions in the nucleobases, sugars, or backbone; and cross-linked or otherwise chemically modified molecules as discussed above. One or more modified nucleotides may be within a portion of the shRNA molecule or throughout the entire shRNA molecule. For example, the shRNA molecule may be modified, or contain modified nucleic acids in a region at its 5' end, its 3' end, or both, and / or within the guide strand, the transit strand, or both, and / or within nucleotides protruding from the 5' end, 3' end, or both.

[0034] The implementation plan provides separate encoding targets. KIF1A Missense mutations in shRNA oligonucleotide sequences result in mutants. KIF1A Expression is reduced. The polynucleotide can be a DNA polynucleotide suitable for cloning into a suitable vector (e.g., a plasmid) for culturing and subsequently producing a virus or viral particle. The viral particle can then contain a DNA polynucleotide having a nucleotide-coding sequence in a form suitable for infection. Therefore, the polynucleotide can be a DNA sequence cloned into a plasmid for producing a virus or viral particle, or encapsulated within a virus or viral particle. Since retroviruses carry nucleotide-coding sequences in the form of RNA polynucleotides, retroviral particles (e.g., lentiviruses) can contain shRNA, which includes the nucleotide portion, second portion, and third portion as described above.

[0035] In the implementation plan, encoding targets are separate. KIF1A The missense mutated shRNA oligonucleotide sequence encodes a first part, a second part, and a third part, wherein the first part contains any one of SEQ ID NO: 2-115, the second part contains any one of SEQ ID NO: 536-542, and the third part contains the corresponding nucleotide sequences complementary to those in SEQ ID NO: 2-115 (i.e., SEQ ID NO: 116-229).

[0036] In the implementation scheme, the polynucleotide encoding the shRNA oligonucleotide sequence targeting SNP rs1063353 (L331L) encodes a first part, a second part, and a third part, wherein the first part comprises any one of SEQ ID NO: 512-523, the second part comprises any one of SEQ ID NO: 539-546, and the third part comprises the corresponding nucleotide sequences complementary to those in SEQ ID NO: 512-523 (i.e., SEQ ID NO: 524-535).

[0037] "Reduce expression," "decrease expression," or "inhibit expression" refers to a decrease or blockage in the expression or activity of mutant KIF1A, and does not necessarily indicate a complete elimination of expression or activity. Mechanisms for reducing target expression include the availability of RNA polynucleotides and the extraction of mutant KIF1A from larger genomes. KIF1A Hybridization of one or more target sequences transcribed from one or more sequences within a sequence, wherein the result or effect of the hybridization is target degradation or target occupancy, accompanied by stagnation of cellular mechanisms involving, for example, transcription or splicing.

[0038] As discussed below, targeted and multiple KIF1A Common SNPs associated with missense mutations reduce mutant types. KIF1A Expression of ASOs. Classical single-stranded ASOs primarily function in the nucleus by selectively cleaving pre-mRNA with complementary sites via an RNase H-dependent mechanism. Although ASOs can also function via translational arrest through steric hindrance, in practice they are used as 'gapmers' with a central region supporting RNase H activity, flanked by chemically modified ends that increase affinity and reduce sensitivity to nucleases. The endogenous RNase H enzyme RNASEH1 recognizes RNA-DNA heteroduplex substrates formed when DNA-based oligonucleotides bind to their homologous mRNA transcripts and catalyzes RNA degradation. Cleavage at the ASO binding site leads to the destruction of the target RNA, thereby silencing target gene expression.

[0039] The gapmer ASO consists of a DNA-based internal 'gap' and an RNA-like flanking region (optionally composed of 2'- O The RNA-DNA duplex, composed of bases modified with methyl (2'-OMe) or locked nucleic acid (LNA), binds to the target transcript with high affinity. The resulting RNA-DNA duplex acts as a substrate for RNASEH1, leading to the degradation of the target transcript.

[0040] Those skilled in the art will understand that a typical nucleotide containing ribose, phosphate, and one of the bases adenine, guanine, cytosine, and uracil can be used to establish a relationship with...KIF1A The complementarity of mRNA, wherein the bases are linked to phosphodiester bonds representing naturally occurring nucleic acids. In embodiments, the nucleic acid can be chemically modified at the RNA backbone, nucleobases, ribose, and 2'-ribose substitutions, for example, by cEt (constrained ethyl-bridged nucleic acid); ENA (ethylene-bridged nucleic acid); 2'-F (2'-fluorine); LNA (locked nucleic acid); 2'-MOE (2'-... O -Methoxyethyl;2'-OMe,2'- O -Methyl; PMO, phosphodiamide morpholino oligonucleotide; PNA, peptide nucleic acid; the phosphodiester bond between nucleotides can be replaced by a thiophosphate bond, PS, thiophosphate; tcDNA, tricyclic DNA.

[0041] As used herein, the term "nucleic acid" refers to a molecule comprising monomeric nucleotides. Examples of nucleic acids include ribonucleic acid (RNA), deoxyribonucleic acid (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering RNA (siRNA) and short hairpin RNA (shRNA), microRNA, pri-microRNA, pre-shRNA, and ASO. "Nucleic acid" includes oligonucleotides and polynucleotides. "Nucleotide" refers to a nucleoside having a phosphate group covalently linked to the sugar moiety of the nucleoside. "Oligonucleotide" or "polynucleotide" refers to a polymer of linked nucleotides, each of which may be modified independently of the others.

[0042] Table 1 lists novel siRNA oligonucleotide sequences that target individual KIF1A missense mutations.

[0043]

[0044] Table 2 lists novel polynucleotides encoding siRNA oligonucleotide sequences that target individual KIF1A missense mutations.

[0045]

[0046] In the implementation scheme, as described above, common SNPs can be used to generate interfering nucleic acids that selectively reduce or silence mutant KIF1A expression. This alternative approach to allele-specific silencing of mutant KIF1A is particularly convenient when a large number of different patient-specific pathogenic mutations are present. In the implementation scheme, RNAi sequences targeting specific benign SNPs (rs1063353; L331L) are described, which are used to target at least five individual SNPs. KIF1A The mutation's "handle." This involves identifying which nucleotide (A or G) at the L331L SNP is present in a given patient. Hum Gene Ther On alleles with the same pathogenic mutation, one of two potential oligonucleotide drug candidates could be used to specifically reduce RNA expression of the mutant allele.

[0047] Table 3 lists the novel siRNA oligonucleotide sequences targeting SNP rs1063353 (L331L).

[0048]

[0049] Table 4 lists novel polynucleotides encoding siRNA oligonucleotide sequences targeting SNP rs1063353 (L331L).

[0050]

[0051] Table 5 shows the 20nt MOE Gapmer ASO targeting either of the two alleles at rs1063353. In the implementation, the ASO can be truncated, i.e., 19nt, 18nt, 17nt, 16nt, or 15nt. They can be in the form of 5-10-5 or in the forms of 5-10-4, 4-10-4, 4-10-3, 3-10-3, 3-10-2, 5-9-6, 5-9-5, 4-9-5, 4-9-4, 3-9-4, 3-9-3, 6-8-6, 6-8-5, 5-8-5, 5-8-4, 4-8-4, or 4-8-3.

[0052]

[0053] Table 6 shows examples of ASOs targeting SNP rs1063353, with stabilization chemistry illustrated. It should be understood that the stabilization chemistry shown for the ASOs in Table 5 is merely representative, and those skilled in the art will understand that the same or similar modifications can be applied to other oligonucleotides described herein.

[0054]

[0055] The oligonucleotides described herein can be conveniently and routinely prepared using known techniques such as solid-phase synthesis. Equipment for such synthesis is available from several vendors, including, for example, Applied Biosystems (Foster City, Calif.). Alternatively or alternatively, any other means known in the art for such synthesis can be employed. It is well known that similar techniques are used to prepare oligonucleotides, such as substituted sugars, phosphate thioesters, and alkylated derivatives.

[0056] In this embodiment, lipid nanoparticles can be used to deliver oligonucleotides. In this embodiment, anionic oligonucleotides are complexed with cationic lipids to form lipid nanoparticles (LNPs). LNPs for in vivo use can be 100 nm–200 nm in size and include a surface coating of a neutral polymer such as PEG to minimize protein binding and uptake by RES cells. The role of LNPs involves initial uptake via endocytosis. Subsequently in vivo, the cationic lipids of the LNP interact with anionic membrane lipids to disrupt the membrane structure. This results in both increased membrane permeability and dissolution of the LNP, and is the basis for the delivery of oligonucleotides into the cytoplasm.

[0057] In the implementation scheme, stable nucleic acid lipid particles (SNALP) can be used for siRNA delivery. Optimized cationic lipids can be obtained by: (i) altering the pKa so that the lipids are almost uncharged in circulation but become charged in low-pH endosomes and (ii) using readily biodegradable bonds. This results in a significant improvement in efficacy, allowing for significant target silencing at doses as low as 0.005 mg / kg of siRNA or ASO, accompanied by low toxicity.

[0058] In the implementation scheme, polymer nanocarriers can be used for oligonucleotide delivery. Biomedically compatible polymers such as polylactide, polyglycolic acid, or poly(lactic-co-glycolic acid) (PLGA) can be used to form solid nanoparticles via oil-in-water emulsion technology. Because PLGA is anionic, positively charged side chains in the polymer can be incorporated, or the anionic oligonucleotide can be complexed with a positively charged moiety such as polyethyleneimine (PEI).

[0059] In embodiments, micelle polymer nanocarriers can be used for oligonucleotide delivery. These can be formed through the self-assembly of amphiphilic polymers in an aqueous environment. For example, polymer micelles can be formed from triblock polymers comprising a hydrophobic portion driving self-assembly, a cationic portion binding oligonucleotides, and a PEG or other neutral polymer providing a protective coating. In embodiments, another type of polymer nanocarrier is a nanohydrogel. These nanoparticles have an open, water-filled polymer lattice that readily incorporates biomacromolecules such as peptides and oligonucleotides, and their release kinetics are controlled by the degree of cross-linking of the lattice.

[0060] In the implementation scheme, the oligonucleotides described herein may be incorporated into plasmids, viral vectors, or viral particles.

[0061] A "vector" is a replicon, such as a plasmid, bacteriophage, or granule, into which a DNA or RNA segment can be inserted to induce replication of the inserted segment. Typically, a vector is capable of replication when associated with appropriate control elements. Suitable vector backbones include, for example, those conventionally used in the art, such as plasmids, plasmids containing a viral genome, viruses, or artificial chromosomes. The term "vector" includes cloning and expression vectors, as well as viral vectors and integration vectors.

[0062] It will be apparent to those skilled in the art that the term "viral vector" is widely used to refer to nucleic acid molecules (e.g., transfer plasmids) that include viral nucleic acid elements that typically facilitate the transfer of nucleic acid molecules to cells or viral particles, mediating the transfer of nucleic acid sequences and / or the integration of nucleic acid sequences into the cellular genome.

[0063] Viral vectors contain structural and / or functional genetic elements primarily derived from viruses. Desiredly, viral vectors are non-toxic, non-immunogenic, readily produced, and effectively protect and deliver DNA or RNA to target cells. According to the compositions and methods described herein, viral vectors may contain DNA encoding one or more of the siRNA, shRNA, or dsRNA described herein. In embodiments, the viral vector is a lentiviral vector or an adeno-associated virus (AAV) vector.

[0064] As used herein, the term "lentivirus" refers to a complex group (or genus) of retroviruses. Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); Vesnamedi virus (VMV); Caprine arthritis-encephalitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Simian immunodeficiency virus (SIV). As used herein, the term "lentivirus" includes lentiviral particles. Lentivirals transduce dividing and postmitotic cells.

[0065] The term "lentiviral vector" refers to a viral vector (e.g., a viral plasmid) that contains structural and functional genetic elements or portions thereof primarily derived from lentiviruses, including long terminal repeat (LTR) sequences. A lentiviral vector is a hybrid vector (e.g., in the form of a transfer plasmid) having a retroviral (e.g., lentivirus) sequence for reverse transcription, replication, integration, and / or packaging of a nucleic acid sequence (e.g., a coding sequence). The term "retroviral vector" refers to a viral vector (e.g., a transfer plasmid) that contains structural and functional genetic elements or portions thereof primarily derived from retroviruses.

[0066] Adenoviral vectors are designed for direct administration to live subjects. Unlike retroviral vectors, most adenoviral vector genomes do not integrate into the host cell's chromosome. Instead, the gene introduced into the cell using the adenoviral vector is maintained in the nucleus as an extrachromosomal element (epistome) for an extended period. Adenoviral vectors can transduce dividing and non-dividing cells in many different tissues in vivo, including airway epithelial cells, endothelial cells, hepatocytes, and various tumors.

[0067] The term “adeno-associated virus” (AAV) refers to a small ssDNA virus that infects humans and some other primate species, is known not to cause disease, and elicits only a very mild immune response. As used herein, the term “AAV” is intended to include AAV particles. AAVs can infect both dividing and non-dividing cells and can integrate their genome into the host cell’s genome. These characteristics make AAVs an attractive candidate for generating viral vectors for gene therapy, although the cloning capacity of the vectors is relatively limited. In the implementation scheme, the vectors used are derived from adeno-associated viruses (i.e., AAV vectors). More than 30 naturally occurring AAV serotypes are available. Numerous natural variants exist in the AAV capsid, allowing the identification and use of AAVs with properties particularly suited to specific target cell types. AAV viruses can be engineered using conventional molecular biology techniques, enabling the optimization of these particles for cell-specific delivery of shRNA DNA sequences, for minimizing immunogenicity, for modulating stability and particle lifespan, for efficient degradation, and for precise delivery to the nucleus, among other things.

[0068] An "expression vector" is a vector that contains a regulatory region. Many vectors and expression systems are commercially available from companies such as Novagen (Madison, Wis.), Clontech (Palo Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen / Life Technologies (Carlsbad, Calif.). Expression vectors can be viral expression vectors derived from a specific virus.

[0069] The vectors provided herein may also include, for example, origins of replication, scaffold attachment regions (SARs), and / or markers. Marker genes can confer selectable phenotypes to host cells. For example, markers can confer resistance to biocides, such as resistance to antibiotics (e.g., kanamycin, G418, bleomycin, or hygromycin). Expression vectors may include tag sequences designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences include, for example, green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FIag. TM Tag (Kodak, New Haven, Conn.) sequences are typically expressed as fusions with the encoded polypeptide. Such tags can be inserted at any position within the polypeptide, including the carboxyl or amino terminus.

[0070] Other expression vectors may include, for example, segments of chromosomal, non-chromosomal, and synthetic DNA sequences. Suitable vectors include pLK0.1 puro, derivatives of SV40, and plasmids such as RP4; phage DNA, such as many derivatives of phage 1, such as NM989, and other phage DNAs, such as M13 and filamentous single-stranded phage DNA; vectors that can be used in eukaryotic cells, such as vectors that can be used in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNA, such as plasmids that have been modified to use phage DNA or other expression control sequences.

[0071] Vectors may also include regulatory regions. The term "regulatory region" refers to a nucleotide sequence that affects the initiation and rate of transcription or translation, as well as the stability and / or mobility of the transcribed or translated product. Regulatory regions include, but are not limited to, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein-binding sequences, 5' and 3' untranslated regions (UTRs), transcription start sites, termination sequences, polyadenylation sequences, nuclear localization signals, and introns.

[0072] As used herein, the term "operably linked" refers to the localization of regulatory regions and sequences of nucleic acids to be transcribed in order to influence the transcription or translation of such sequences. For example, to place a coding sequence under the control of a promoter, the translation initiation site of a polypeptide's translation reading frame is typically located between one and about 50 nucleotides downstream of the promoter. However, the promoter can be located up to about 5,000 nucleotides upstream of the translation initiation site or up to about 2,000 nucleotides upstream of the transcription initiation site. Promoters typically include at least a core (basic) promoter. Promoters may also include at least one control element, such as an enhancer sequence, an upstream element, or an upstream activation region (UAR). The selection of promoters to be included depends on several factors, including but not limited to efficiency, selectivity, inducibility, desired expression levels, and cellular or tissue-preferred expression. Regulation of coding sequence expression can be achieved by appropriately selecting promoters and other regulatory regions and appropriately localizing them relative to the coding sequence.

[0073] The vector may also contain additional components or functions that further regulate gene delivery and / or gene expression or otherwise provide beneficial properties to the targeted cells. Such additional components include, for example, components that influence binding to or targeting cells (including components mediating cell type or tissue-specific binding); components that influence cellular uptake of the vector nucleic acid; components that influence the localization of intracellular polynucleotides after uptake (such as agents mediating nuclear localization); and components that influence polynucleotide expression. Such components may also include biomarkers, such as detectable and / or selectable biomarkers that can be used to detect or select cells that have taken up the vector and expressed nucleic acids delivered by the vector. Such components may be provided as a natural feature of the vector (e.g., using certain viral vectors with components or functions that mediate binding and uptake), or the vector may be modified to provide such functions. Numerous such vectors are known in the art and are generally available.

[0074] "Recombinant viral vector" refers to a viral vector containing one or more heterologous gene products or sequences. Because many viral vectors exhibit packaging-related size limitations, heterologous gene products or sequences are typically introduced by replacing one or more parts of the viral genome. Such viruses may become replication-deficient, requiring one or more missing functions in a trans-form during viral replication and capsidation (e.g., by using helper viruses or packaging cell lines carrying gene products essential for replication and / or capsidation).

[0075] In the implementation scheme, the viral vector used herein can, for example, be at least 10 5 Use at a concentration of one viral genome per cell.

[0076] The selection of an appropriate promoter can be easily accomplished. Examples of suitable promoters include RNA polymerase II or III promoters. For example, candidate shRNA sequences can be selected from RNA polymerase III promoters U6 or H1 or neuron-specific RNA polymerase II promoters (including neuron-specific enolase (NSE), synaptic protein I (Syn), or Ca2+ promoters). 2+ / CaM-activated protein kinase II α (CaMKII α) is expressed under the control of the protein kinase II α (CaMKII α).

[0077] Other suitable promoters that can be used for gene expression include, but are not limited to, the 763-base-pair cytomegalovirus (CMV) promoter and the Rous sarcoma virus (RSV) promoter (Davis et al.). Escherichia coli 4:151 (1993)), SV40 early promoter region, herpes simplex kinase promoter, regulatory sequence of metallothionein (MMT) gene, PGK (glycerol phosphate kinase) promoter, alkaline phosphatase promoter; and animal transcription control region, which exhibits tissue specificity and has been used in transgenic animals; myelin basic protein gene control region, which is active in oligodendrocytes in the brain, and gonadotropin-releasing hormone gene control region, which is active in the hypothalamus. Some proteins can be expressed using their native promoters. Other elements that enhance expression may also be included, such as enhancers or systems leading to high levels of expression, such as the tat gene and tar element. The assembly or cassette can then be inserted into a vector, such as a plasmid vector, such as pLK0.1, pUC19, pUC118, pBR322, or other known plasmid vectors. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory press, (1989). Plasmid vectors may also include selective markers, such as β-lactamase genes for ampicillin resistance, provided that the marker peptide does not adversely affect the metabolism of the treated organism.

[0078] The coding sequence of the RNAi described herein can be cloned into a viral vector using any suitable genetic engineering technique well known in the art, including but not limited to standard techniques such as PCR, polynucleotide synthesis, restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, as described in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY (1989)), Coffin et al. (Retroviruses. Cold Spring Harbor Laboratory Press, NY (1997)), and "RNAViruses: A Practical Approach" (Alan J. Cann, Ed., Oxford University Press, (2000)). In embodiments, the RNAi, for example, shRNA DNA sequence contains flanking sequences at the 5' and 3' ends that are complementary to the sequences on the plasmid and / or vector that are cleaved by restriction endonucleases. As is well known in the art, the flanking sequences depend on the restriction endonuclease used during the restriction digestion of the plasmid and / or vector. Therefore, those skilled in the art can select the flanking sequences at the 5' and 3' ends of the RNAi DNA sequence accordingly. In the implementation scheme, the target site can be cloned into the vector using nucleic acid fusion and exchange technologies known in the art, including Gateway, PCR infusion, Cre-lox P, and Creator.

[0079] In an embodiment, the expression vector comprises a promoter and a polynucleotide comprising a first nucleotide sequence encoding the shRNA described herein. In an embodiment, the promoter and the polynucleotide comprising the first nucleotide sequence are operatively linked. In an embodiment, the promoter is a U6 promoter. In an embodiment, the first nucleotide sequence contained in the expression vector may be any polynucleotide encoding SEQ ID NO: 2 to 229. In an embodiment, the first nucleotide sequence contained in the expression vector may comprise any polynucleotide encoding SEQ ID NO: 230-253. In an embodiment, the first nucleotide sequence contained in the expression vector may comprise any of SEQ ID NO: 254-283. In an embodiment, the first nucleotide sequence contained in the expression vector may comprise any of SEQ ID NO: 535, 537, and 537. In an embodiment, the polynucleotide containing the first nucleotide sequence in the expression vector is a DNA polynucleotide. In an embodiment, the first nucleotide sequence of the expression vector is a DNA nucleotide sequence. The siRNA or shRNA encoded by the first nucleotide sequence contained in the expression vector may be described as any of the variations disclosed herein.

[0080] As discussed below, the recombinant viral vector, along with the components required for packaging the recombinant viral particles, is transfected into packaging cells or cell lines. The recombinant viral particles, collected from the supernatant of the transfected cells, are used to infect target cells or organisms to express shRNA. The transduced cells or organisms are used for transient expression or selected for stable expression.

[0081] In this implementation, viral particles are used to deliver the encoding nucleotide sequence of siRNA or shRNA. The terms virus and viral particle are used interchangeably herein. Viral particles typically comprise a variety of viral components and sometimes include host cell components in addition to one or more nucleic acids. Nucleic acid sequences can be packaged into viral particles that are capable of delivering the siRNA or shRNA nucleic acid sequence to the target cells of a patient in need.

[0082] Viral particles can be generated by: (a) introducing a viral expression vector into a suitable cell line; (b) culturing the cell line under suitable conditions to allow the generation of viral particles; (c) recovering the generated viral particles; and (d) optionally purifying the recovered infectious viral particles.

[0083] Expression vectors containing nucleotide sequences encoding one or more siRNAs or shRNAs described herein can be introduced into suitable cell lines for proliferation or expression using well-known techniques readily available to those skilled in the art. These include, but are not limited to, microinjection of microparticles of DNA into the cell nucleus, CaPO4-mediated transfection, DEAE-dextran-mediated transfection, electroporation, liposome transfection / liposome fusion, particle bombardment, gene gun, transduction, infection (e.g., with infectious viral particles), and other techniques, such as those described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

[0084] In embodiments where the expression vector is defective, infectious particles can be generated in complementary cell lines or by using helper viruses that provide nonfunctional viral genes in a trans configuration. Suitable cell lines for complementation with adenovirus vectors include 293 cells and PER-C6 cells, commonly used for complementation with E1. Infectious viral particles can be recovered from culture supernatant or from cells after lysis and optionally further purified using standard techniques such as chromatography, ultracentrifugation in a cesium chloride gradient, etc.

[0085] In the implementation scheme, this document provides host cells that include the nucleic acid molecules, vectors, or infectious viral particles described herein. The term "host cell" should be interpreted broadly without any limitation regarding the tissues, organs, or isolated cells of a specific organism. Such cells can be a unique type of cell or a group of different types of cells, including cultured cell lines, primary cells, and proliferating cells.

[0086] Therefore, host cells include prokaryotic cells, lower eukaryotic cells such as yeast and other eukaryotic cells such as insect cells, plant cells and higher eukaryotic cells such as vertebrate cells, and mammalian (e.g., human or non-human) cells are particularly preferred. Suitable mammalian cells include, but are not limited to, hematopoietic cells (totipotent stem cells, leukocytes, lymphocytes, monocytes, macrophages, APCs, dendritic cells, non-human cells, etc.), lung cells, tracheal cells, hepatocytes, epithelial cells, endothelial cells, muscle (e.g., skeletal muscle, cardiac muscle, or smooth muscle) cells, or fibroblasts. For example, host cells may include *Escherichia coli* (…). Bacillus ), Bacillus ( Listeria Listeria () Saccharomyces ),yeast(KIF1A The host cells include BHK (juvenile hamster kidney) cells, MDCK (Madin-Darby canine kidney cell line), CRFK (Crandell cat kidney cell line), CV-1 (African monkey kidney cell line), COS (e.g., COS-7) cells, Chinese hamster ovary (CHO) cells, mouse NIH / 3T3 cells, HeLa cells, and Vero cells. The host cells also include complementary cells capable of complementing at least one defective function of a replication-defective vector (e.g., a defective adenovirus vector) available herein, such as those described above.

[0087] In this implementation, the host cell can be encapsulated. For example, transfected or infected eukaryotic host cells can be encapsulated with a compound that forms a microporous membrane, and the encapsulated cells can be further implanted into the body. The capsule containing the cells of interest can be prepared using a hollow microporous membrane having a molecular weight cutoff value suitable for allowing proteins and nutrients to pass freely between the inside and outside of the capsule, while preventing the implanted cells from contacting the host cells.

[0088] The viral particles used in this study include AAV particles and lentiviral particles. AAV particles carry the coding sequence of the siRNA or shRNA of this study in the form of genomic DNA. On the other hand, lentiviral particles belong to the retrovirus class and carry the coding sequence of the siRNA or shRNA of this study in the form of RNA.

[0089] Contains coding target mutant ​ Recombinant engineered viral particles of RNA (siRNA, shRNA, or ASO DNA, or RNA in the case of lentiviral particles), such as AAV particles, artificial AAV particles, self-complementary AAV particles, and lentiviral particles, can be delivered to target cells to reduce KIF1A expression. The use of AAV is a common DNA delivery method because it is relatively non-toxic, provides efficient gene transfer, and can be easily optimized for specific purposes. In the implementation, the selected AAV serotype has natural neurotropism. In the implementation, the AAV serotype can be AAV9 or AAV10.

[0090] Suitable recombinant AAV can be produced by culturing host cells containing: a nucleotide sequence encoding the AAV serotype capsid protein or a fragment thereof, as defined herein; a functional rep gene; a small gene containing at least an AAV inverted terminal repeat (ITR) and encoding a nucleotide sequence; and sufficient helper functions to allow the small gene to be packaged into the AAV capsid protein. The components required for culturing in host cells to package the AAV small gene into the AAV capsid can be provided to the host cells in a trans configuration. Alternatively, any one or more of the desired components (e.g., the small gene, rep sequence, cap sequence, and / or helper functions) can be provided by stable host cells that have been engineered to contain one or more of the desired components using methods known to those skilled in the art.

[0091] Unless otherwise stated, the AAV inverted terminal repeat (ITR) sequences and other selected AAV components described herein can be readily selected from any AAV serotype, including but not limited to AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVRec3, or other known and unknown AAV serotypes. These ITRs or other AAV components can be readily isolated from AAV serotypes using techniques available to those skilled in the art. Such AAVs can be isolated or obtained from academic, commercial, or public sources (e.g., the U.S. Center for Type Culture Collection, Manassas, Va.). Alternatively, AAV sequences can be obtained by synthesis or other suitable means by referencing published sequences, such as those available in literature or databases such as GenBank, PubMed, etc.

[0092] The small gene, rep sequence, cap sequence, and accessory function required to generate the rAAV described herein can be delivered to the packaging host cell in the form of any genetic element carrying the sequence thereon. The selected genetic element can be delivered by any suitable method. Methods used to construct the embodiments described herein are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for generating rAAV virions are well known, and the choice of suitable method is not limiting. See, for example, K. Fisher et al., 1993 J. Viral., 70:520-532 and U.S. Patent No. 5,478,745, etc. All citations herein are incorporated herein by reference.

[0093] The selection of these and other common vectors and regulatory elements is routine, and many such sequences are available. See, for example, Sambrook et al., and the references cited therein, e.g., pp. 3.18–3.26 and 16.17–16.27, and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989). Of course, not all vectors and expression control sequences will function equally well to express all the transgenes described herein. However, those skilled in the art can make selections among these and other expression control sequences.

[0094] Viruses containing the desired siRNA or shRNA coding sequence can be formulated for administration to patients or humans in need via any suitable method of administration. Such formulation includes the use of pharmaceutically and / or physiologically acceptable media or vectors, particularly those suitable for administration to the brain (e.g., via subcranial or spinal injection). Furthermore, more than one of the siRNAs, shRNAs, or ASOs described herein can be administered in combination therapy. In combination therapy, different siRNAs, shRNAs, or ASOs can be administered simultaneously, separately, sequentially, and in any order.

[0095] The pharmaceutical compositions described herein comprise a carrier and / or diluent suitable for delivery to human or animal organisms by injection. Such carriers and / or diluents should generally be non-toxic at the dosage and concentration used. They may be selected from those commonly used to formulate compositions for parenteral administration in single or multiple doses or for direct infusion by continuous or periodic administration. In embodiments, it is isotonic, hypotonic, or weakly hypertonic and has a relatively low ionic strength, such as that provided by sugars, polyols, and isotonic saline solutions. Representative examples include sterile water, physiological saline (e.g., sodium chloride), antibacterial water, Ringer's solution, glucose or sucrose solution, Hank's solution, and other physiologically balanced saline solutions (see, for example, the latest edition of Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams & Wilkins). The pH of the composition is appropriately adjusted and buffered to suit human or animal use, for example, at a physiological pH or a slightly alkaline pH (between about pH 8 and about pH 9, with pH 8.5 being particularly preferred). Suitable buffers include phosphate-buffered saline (e.g., PBS), bicarbonate buffer, and / or Tris buffer. In some embodiments, for example, the composition is formulated in 1 M sucrose, 150 mM NaCl, 1 mM MgCl2, 54 mg / L Tween 80, 10 mM Tris at pH 8.5. In other embodiments, for example, the composition is formulated in 10 mg / ml mannitol, 1 mg / ml HSA, 20 mM Tris (pH 7.2), and 150 mM NaCl. These compositions are stable at -70°C for at least 6 months.

[0096] The pharmaceutical compositions described herein can be in various forms, such as solids (e.g., powders, lyophilized forms) or liquids (e.g., aqueous). In the case of solid compositions, preparation methods include, for example, vacuum drying and lyophilization, which produce an active agent plus a powder of any additional desired components from its previously sterile filtered solution. If desired, such solutions can be stored in sterile ampoules, ready for reconstitution by adding sterile water for ready injection.

[0097] Nebulized or aerosolized formulations are also suitable. Methods of intranasal administration are well known in the art, including the application of the composition in drop, spray, or dry powder form from a pressurized container or dispenser (containing a suitable propellant, such as a gas like carbon dioxide) or nebulizer to the nasopharynx of the individual to be treated. Enteric-coated formulations, such as gastric-tolerant capsules and granules for oral administration, and suppositories for rectal or vaginal administration, may also be suitable. For non-parenteral administration, the composition may also include absorption enhancers that increase mucosal pore size. Such absorption enhancers include sodium deoxycholate, sodium glycocholate, dimethyl-β-cyclodextrin, lauroyl-1-lysophosphatidylcholine, and other substances with structural similarity to the phospholipid domains of the mucosa.

[0098] The composition may also contain other pharmaceutically acceptable excipients to provide desired pharmaceutical or pharmacokinetic properties, including, for example, altering or maintaining the pH, osmotic pressure, viscosity, transparency, color, sterility, stability, and dissolution rate of the formulation, and altering or maintaining release or absorption into human or animal organisms. For example, polymers such as polyethylene glycol can be used to obtain desired solubility, stability, half-life, and other pharmaceutically advantageous properties. Representative examples of stabilizing components include polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose, and combinations thereof. Other stabilizing components particularly suitable for plasmid-based compositions include hyaluronidase, chloroquine, protonated compounds such as propylene glycol, polyethylene glycol, glycerol, ethanol, 1-methyl-L-2-pyrrolidone or their derivatives, aprotonated compounds such as dimethyl sulfoxide (DMSO), diethyl sulfoxide, di-n-propyl sulfoxide, dimethyl sulfone, sulfolane, dimethylformamide, dimethylacetamide, tetramethylurea, acetonitrile, nuclease inhibitors such as actin G, and cationic salts such as magnesium (Mg²⁺). 2+ ) and lithium (Li + ( ), and any derivatives thereof. The amount of cationic salt in the compositions herein preferably ranges from about 0.1 mM to about 100 mM, and still more preferably from about 0.1 mM to about 10 mM. Viscosity enhancers include sodium carboxymethyl cellulose, sorbitol, and dextran. The compositions may also contain substances known in the art that promote permeation or transport across the blood barrier or membrane of a particular organ, such as antibodies against transferrin receptors. A gel complex of polylysine and lactose or poloxamer 407 can be used to facilitate administration to arterial cells.

[0099] Viral particles and pharmaceutical compositions can be administered to patients in therapeutically effective amounts. As used herein, the term "therapeutically effective amount" refers to an amount sufficient to achieve the desired biological effect. For example, a therapeutically effective amount for treating KAND is an amount sufficient to improve one or more symptoms of KAND as described herein (e.g., intellectual disability, hypotonia, language skills, hypertonia, spasticity, peripheral neuropathy, tremor, or seizures).

[0100] Appropriate dosages can vary based on known factors such as the pharmacokinetic characteristics of the specific active agent, the age, health, and weight of the host organism; one or more conditions to be treated, the nature and severity of symptoms, the types of concurrent treatments, the frequency of treatment, the need for prevention or treatment, and / or the desired effect. Dosage is also calculated based on the specific route of administration chosen. Practitioners can further refine the calculations required to determine the appropriate dosage for treatment based on relevant circumstances. As a general guideline, virus-particle-based compositions can be, for example, at least 10 5 The composition can be formulated in doses of one viral genome / cell. The titer can be determined using conventional techniques. Vector plasmid-based compositions can be formulated in doses between 1 μg and 100 mg, for example, between 10 μg and 10 mg, or between 100 μg and 1 mg. Administration can be administered as a single dose or as one or more doses repeated at time intervals.

[0101] The pharmaceutical compositions described herein may be encapsulated in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. In all cases, the composition shall be sterile and flowable to the extent that it is readily injectable. It shall be stable under the conditions of manufacture and storage and protected against contamination by microorganisms such as bacteria and fungi. Sterile injectable solutions may be prepared by incorporating an active agent (e.g., infectious granules) in the required amount into one or a combination of the ingredients listed above, followed by filtration and sterilization.

[0102] The viral particles and pharmaceutical compositions described herein can be administered via parenteral routes, including intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, intrathecal, or intracranial (e.g., intracerebral or intraventricular) administration. In some embodiments, the viral particles or pharmaceutical compositions are administered intracerebrally or intraventricularly. In some embodiments, the viral particles or pharmaceutical compositions described herein are administered intrathecally.

[0103] In the implementation scheme, the viral particles and pharmaceutical composition described above are administered to a subject via subcranial injection into the brain or spinal cord of a patient or human in need. In this implementation scheme, subcranial administration to the brain results in the direct delivery of the encoded nucleotide sequences described herein to brain cells, including glial cells and neurons. As used herein, the term "neuron" means any cell in the brain or any cell associated with brain function. The term can refer to any type of neuron, including unipolar, bipolar, multipolar, and pseudounipolar.

[0104] The nucleotide sequence encoding kinin family member 1A (KIF1A), mRNA transcript variant 1 GenBank accession number NM_001244008.2 SEQ ID NO: 1 acagcgcaga gcagccggcg agcggcagca gctccgggct cgagagcccg cgcccatgcc 60 agtgcccatg cggggccgcc gccagtgacg ccggagaggt gttcccccca cactggggct 120 cccactactg cgaggagtga cccacgaagg ccacagagat ggccggggct tcggtgaagg 180 tggcggtgcg ggtccgcccc ttcaattccc gggaaatgag ccgtgactcc aagtgcatca 240 ttcagatgtc tggaagcacc accaccattg ttaaccccaa acagcccaag gagacgccca 300 aaagcttcag ctttgactac tcctactggt cgcacacctc acctgaggac atcaactacg 360[[ID=

[15] ]] cgtcgcagaa gcaggtgtac cgggacatcg gcgaggagat gctgcagcat gcctttgagg 420 gatacaacgt gtgcatcttc gcctatgggc agacgggtgc cggcaagtcc tacaccatga {480} tgggcaagca ggagaaggac cagcagggca tcatcccaca gctctgcgag gacctcttct 540 ctcggatcaa cgacacgacc aacgacaaca tgtcctactc cgtggaggtc agctacatgg 600 agatttactg tgagcgcgtc cgtgacctcc tgaaccccaa gaacaagggc aaccttcgcg 660 Note: There seems to be a mistake in the original text where the tag is not correctly formatted in the translation. It should be as shown above. Also, the number 480 in the line with ID=18 seems to be in a wrong position in the original text's format, and I've adjusted it in the translation for better readability. If this is not allowed according to your strict rules, please let me know.tgagggagca cccactgctg gggccctacg tggaggacct ctccaagctg gctgtcacct 720 cctacaatga catccaggac ctcatggact cagggaacaa ggccaggacc gtggcggcca 780 ccaacatgaa tgagaccagc agtcgctccc acgccgtctt caacatcatc ttcacccaga 840 agcgccatga cgcagagacc aatatcacca cggagaaggt gagcaaaatc agcctggtgg 900 acctggctgg gagcgagcgg gctgactcca cgggagccaa gggcacgcgc ctcaaggagg 960 gggccaacat caacaagtcg ctgaccaccc tgggcaaggt catctccgcc ctggctgaaa 1020 tggactccgg acccaacaag aacaagaaaa agaagaagac agatttcatt ccgtaccgag 1080 attccgtgtt gacctggctc ctccgggaaa acctgggcgg taactcaagg acagctatgg 1140 tggcagcctt gagtcctgca gacatcaact acgatgagac ccttagcacg ctgaggtatg 1200 ctgaccgggc caagcagatc cgctgcaatg ctgtcatcaa tgaggacccc aacaacaagc 1260 tgatccgcga gctgaaggat gaggtgaccc ggctgcggga ccttctgtac gcccagggtc 1320 ttggcgacat cactgacacc aacactgtgc ctggaggacc caaattgacc aatgccctgg 1380 tgggtatgag cccctcatcc tcgctctcag ccctgtccag ccgcgcggcc tccgtgtcca 1440 gcctccacga gcgcatcttg tttgccccgg gcagcgagga ggccattgaa agactgaagg 1500 aaacagagaa gatcatagct gagctcaatg agacctggga ggagaagctg cggcggacag 1560 aagccatccg gatggagagg gaagccctgc tggccgagat gggtgtggcc atgagggagg 1620 atggcggcac cttgggcgta ttctctccca aaaagacacc acacctcgtc aacctgaacg 1680 aggacccgct gatgtctgag tgcctgctct actacatcaa ggatgggatc accagagtgg 1740 gcagggagga tggcgagagg cggcaggaca ttgttctgag tgggcacttc atcaaggagg 1800 agcactgcgt cttccggagc gactccaggg gaggcagcga agctgtggtg accttggagc 1860 cctgtgaggg ggcagacacc tacgtcaatg gcaagaaagt cacagagccc agcatcctgc 1920 gttcaggaaa ccgcatcatc atgggtaaga gccatgtgtt ccggttcaac caccccgagc 1980 aggcccggca ggagcgtgag cgcacgcctt gtgcggagac gccagctgag cctgtggact 2040 gggccttcgc ccagcgtgag ctgctggaga agcagggcat cgacatgaag caggagatgg 2100 agcagaggct ccaggactg gaggaccagt accggccgcga gcgggaggg gccacctacc 2160 tgctggagca gcagcggctg gactgaga gcaagctgga ggctctgcag aagcagatgg 2220 actccaggta ctacccggag gtgaacgagg aggaggag gcccgaggat gaagtccagt 2280 ggacagagcg ggagtgtgag ctggcgctct gggccttccg gaagtggaag tggtaccagt 2340 tcacgtctct gcgggacctg ctgtggggca acgccattct cctcaggg gccaatgcca 2400 tcagcgtgga gctgaaaaag aaggtacaat tccagttgt cctcctgacgzgacactct 2460 actccccctct gccacccgac ctgctgcccc cagaggccgc caagaccga gagacgcggc 2520 ccttcccccg caccattgtg gccgtggagg tccaggacca gagaacggg gccaccact 2580 actggacgct ggagaagctc aggcagcgtc tggactgat gcgggagatg tacgaccgcg 2640 ctgcagaggt gccctccagt gtcatcgagg actgtgacaa cgtggtgacc ggcggagacc 2700 ccttctatga ccgcttcccc tggttccggc tggtggcag ttcagccatc tctggctgca 2760 acagctaccc tcttctcaac acatgcatga gcgagcgcat ggctgctc accccctccc 2820 ccaccttctc gagccccgac tccgacgcca ccgagcctgc cgaggagcag agcgtggggg 2880 aggaggagga ggaggaggag gaggaggagg atgaggagga ggaggacctg gaggacgacg 2940 tctttccgga gcacgcgctg tgcgacggcc gggacccgtt ttacgaccgg ccccccctgt 3000 tcagtttagt aggaagggcc ttcgtgtacc tgagcaacct gctgtacccc gttcccctgg 3060 tacaccgtgt ggcaatcgtc agcgagaagg gcgaggtgaa gggcttcctc cgcgtggccg 3120 tccaggccat ctcagccgat gaagaggccc ctgattatgg ctctggcgtc cgccagtcgg 3180 gaactgctaa aatctccttt gatgaccagc attttgaaaa gttccagtcc gagtcttgcc 3240 ccgtggtggg gatgtcccgc tcgggaacct cccaggaaga gcttcgcatc gtggagggcc 3300 agggccaggg tgcagacgtg gggccctcag ccgatgaagt caacaacaac acctgttcag 3360 cagtgccccc agaaggcctc ctcctagaca gctctgagaa agccgccctg gatgggcccc 3420 tggatgctgc cctggaccac ctccgcctgg gcaacacctt caccttccgt gtgacagtcc 3480 tgcaggcgtc cagcatctct gccgaatatg ccgacatctt ctgccagttc aacttcatcc 3540 accgccacga cgaggccttc tccacagagc ccctgaagaa cacaggcaga ggccccccac 3600 ttggcttcta ccacgtccag aacatcgcag tggaggtgac caagtccttc attgagtaca 3660 tcaagagcca gcccattgtt ttcgaggtct ttggccacta ccagcagcac ccgttcccgc 3720 ccctctgcaa ggacgtgctc agccccctga ggccctcgcg ccgccacttc cctcgggtca 3780 tgccactgtc caagccagtg cccgccacca agctcagcac actgacgcgg ccctgtccgg 3840 gaccctgcca ctgcaagtac gacctgctgg tctacttcga gatctgtgag ctggaggcca 3900 acggcgatta catcccggcc gtggtggacc accgtggggg catgccatgc atggggacct 3960 tcctcctcca ccagggcatc cagcgacgga ttacggtgac actactgcat gagacaggca 4020 gccatatccg ctggaaggaa gtgcgcgagc tggtcgtggg ccgcatccga aacactccag 4080 agaccgacga gtccctgatc gaccccaaca tcttgtctct caacatcctc tcttccggat 4140 acatccaccc agcccaagat gaccggacct tttaccaatt tgaggctgcg tgggacagct 4200 ccatgcacaa ctctctcctg ctgaaccggg tcacccctta tcgagagaaa atctacatga 4260 cactctccgc ttatatcgag atggagaact gcacccagcc ggctgttgtc accaaggact 4320 tctgcatggt cttctattcc cgtgatgcca agctgccagc ctcgcgctcc atccgcaacc 4380 tctttggcag tgggagcctt cgggcctcag agagtaaccg tgtgactggt gtgtacgagc 4440 tcagcctgtg ccacgtggct gacgcgggca gcccagggat gcagcgccgg cgccgacgag 4500 tcctggacac atctgtggcc tatgtccggg gcgaggagaa cctggcaggc tggaggcccc 4560 ggagtgacag tctcattctg gaccaccagt gggagctgga gaagctgagc ctcctgcagg 4620 aggtggagaa gactaggcac tacctgctcc tgcgggagaa gctggagacc gcccagcggc 4680 ctgtcccgga ggcactgtcc ccggccttca gcgaggactc tgagtcccat ggctcctcca 4740 gcgcctcctc cccgctctcg gctgagggcc gcccatcacc cctggaggct cccaacgaga 4800 ggcagcggga gctggccgtc aagtgcttgc gcctgctcac gcacacattc aacagagagt 4860 acacacacag ccacgtctgc gtcagtgcca gcgagagcaa gctctccgag atgtctgtca 4920 ccctgctccg ggacccgtcg atgtcccctc taggggtggc cactctcacc ccctcctcca 4980 cttgcccctc tctggttgaa gggcggtacg gtgccactga cctgaggacc ccgcagccct 5040 gctcccggcc agccagccca gagcccgagc tgctgccaga ggccgactcc aagaagctcc 5100 cttcccctgc ccgggcaaca gagacagaca aggagcccca gcgcctgctg gtccctgaca 5160 tccaggagat ccgagtcagc ccgatcgttt ccaagaaggg gtacctgcac ttcctggagc 5220 cgcacacgtc aggctgggcc aggcgcttcg tggtggtgcg gcgccctat gcctacatgt 5280 acaacagcga caaggacacc gtggagcggt tcgtgctcaa cctggccact gcccaggtgg 5340 agtacagtga ggaccagcag gctatgctca agacacccaa cacattcgcg gtgtgcacgg 5400 aacaccgcgg catcctgctg caggccgcca gcgacaagga catgcatgac tggctgtacg 5460 ccttcaaccc cctcctggcc gggaccatac ggtccaagct ctccagaagg aggtctgccc 5520 agatgcgggt ctgaacctga gccctccgt gacagccggc aggcccagcc catcccctcc 5580 ctcatcctcg tctgtcctgt cacctgccgc ccagcccctc tcctgccaga cagcccacga 5640 ccgggtcgac cccccagggg acgcccatgc caggcccggg gacctgtgcc acacgaccag 5700 ctgtgctccc agcagaggct gtgcgtgtca gttcttcttg cagaatgtgc tctggtggaa 5760 caagttggga gaggctgggg gggccaaggg cacaggttac gggggttctt gctgccgttc 5820 tatatttt ttaagcatag acagacttat attaatata cgttagttag tgacattgaa 5880 acagtcaact cggaaattaa ctataagact tgttctattt ataagtattt atttctaatg 5940 cctccacata gccctgtaat attcagatgg aacccccaac cacctccacc ctgtttgttc 6000 ccacatgtgt ctcccaagcc tgctagggac aggcagggca gggacagcca ccttggaagg 6060 ccgcagtgag gagctgtctg gaccagtggg gcaccttggg gctagcacac gggtgtatcg 6120 cctgggcccc aggcttctcc atggccacat gggtcctggg tgtatgtgtg ggagagtggg 6180 ggggtgtctt tggtgcctga agtctgcgcg gcatggaggg tggtgtgagt tcctctggtg 6240 ggagggagaa cgcacatctc ttctgggcgg ccacctgagg agtgactcca agaagagttc 6300 cggcagcttt ccccaggaaa gggtgagggg tgacactcgg ctctggctct gagatgaggc 6360 agacggcacc caggctgtga tctgtcctgg gcggggacca ggagggagcg gggtcgggat 6420 cacctgccag tgtgcagact ctgggactgc gtgctgtctc cggaccatca gggtagggtg 6480 gtgggttgag accaggaagt cagggaagat cggaattcag ggcgacggtc taggtgtcga 6540 gggctgtggc gcagcctctt cagctgcggc gagaaatgga gtgagtcaag gtagcttctg 6600 ggaagaaatg ctgccattag caggtttctt gcaaagactt tcctctcttt gttcccaggg 6660 cagagagttt ctgtgagtcc cactgagaaa atcccatggg gtgggggtat cctggtcggt 6720 cggcaatgga gggtggctgg cttggtggtt attgtcttca aggagctctt cgctgctgca 6780 tctgcggtgt ccctttgttc ttgtcccatt tcaccccctc tgcagacacc aatgtccgag 6840 ggccacccag gacaggacgg gggtcagccc caagctgaga gtctggtcat aggagtcatg 6900 tccagaggcc tagggaggtt ttagggccct ccccacccac acccacaggt cgatttggtc 6960 tctttttagc tcaaggaaag acagtagcca agcaacagag cccctctccc gccgtggccc 7020 gtgggagcag ttacatcggg tctggtgctc cagacctagg gcccagcact ttcatcagat 7080 cctgcctcct ggagtggggg aaacgcagca ccccactggt tctgaggccc ctaccctccc 7140 aggctgtccc acgtgatgct gacatgagcc tcagagaccc caatcccatg cctgggggag 7200 agacagcggc tcaggagtgg gggagcacgg gccttctaca ccacatggga aggctctggc 7260 atgaggtttt cctttgggaa ggttgtttgg gcccctgaag ttccatctcc gagagtggtg 7320 tgcagggcag gccagggccc atgctggctg cagtctctgt ggctgcctgc ctgggccagc 7380 ctgtttggga gctgggactg tgggctcgcc ttttcgtacc tgggctcagg tgcggtgtgg 7440 ccaccgccac cctcatcccc tgcctgggag gctctcccag gggctgatgg gggggttctg 7500 tgagggagaa tcagggctcg ggaagccacg cctgggaagg caggacacag gacacagcag 7560 ctttctttgg aaatctccca ggtgaggatt cacatcccaa aataaaattc agaagccagg 7620 tggcctgtgc tccccatggg tgacctctgg aggcagtgga ccaagatgca gcaaggagag 7680 gatgcagaac agcttcttgc agaagcacct gctccggcat ccagcgctgc ctggaggcag 7740 gaaggagagg cagggcagga cacgctggtc tgagatgagg gggagcccca cgggccccag 7800 gcaggctaga ggaggcacag gccctgccac ggccaactca ggtcagccag cctgaggctg 7860 tggcctccaa agggtctggg cgcacccccc aggtcgcagg tgtctgaggc cagccaacct 7920 gcagagcact cgcggcgtgg gtgggctgag tggaggtgcc tgggagctgc ctaaattcag 7980 aagcctccac tgccatggag actgcctggc tgtgtcctct cagccaggtg ccgtgttggc 8040 cttggctagg acccagagcc acactgcagc ctccgccgac tccctccttc cgggcctgct 8100 ctagtgagga gtgcctaagc caggacccag aaactcagag ttgatggtga gaggaggccg 8160 cctgagtcag gacacctggc ttcttgaaaa gttcgctctg ccagaaacca cctagggacc 8220 acgttagctg ccttccttga gctccccagg agtcggtttc cacatctgtg aggtggaggg 8280 ctggtgtgga ggtgctcatg gggtgcggtg cttgggagac agccaggccc agggtggctg 8340 ctgcttcctg ctaagtgggg gaggtgagac agatctggaa agccgtctcc ctcagatggt 8400 ttcatttaat gctttatact gccgagtctg ggggcttgtt ttggtttggg ggcagccatc 8460 ctccaccaga aagggggagc tccttccgct gcggctccag atagatgggg atgccgggct 8520 ccaggccgac cagcacttgg gatctgatgg gacacggcca gtgcctaggg gtgccaagtc 8580 caaggcctcc cactggggagt catcgctgag aagatgccaa tgtttcatcc accggctgca 8640 caggcacaaa ctcccccacc caggacggct gtgatgaggt ggccctccct gtcaaccctg 8700 gtccctggag tccccagcac ctggggccct ggtggggctg atgtcacagg tgtttactgt 8760 gctgctgcac tggtcctatg ccagcctcac ccatgtgggg accacggaag gcacactccc 8820 ttaccccccgg tgccgggccg tgcggtcccc cagacggaca gcagctgtgg cgacctgcgt 8880 ttctccctgg gcctgtgctt cctgtagtta gtccgtcccc tggttcccct gtggctcaga 8940 ggccgcgtcc ctggcttgta catatgtgat tgctgtgggc acaccccaga ccccatgtca 9000 tagctgccgt cccgacgtca cgacgcctgt cccgatgtct tacacccgag tgttagccct 9060 aggctcctgt actgtgcgtg cacttgaggc tctgtccaat taagaaataa atgtggctct 9120 tactcaacac a 9131 It should be understood that the foregoing detailed description and appended examples are illustrative only and should not be considered as limiting the scope of the subject matter described herein, which is defined only by the appended claims and their equivalents. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the invention, including, but not limited to, those relating to chemical structure, substituents, derivatives, intermediates, synthesis, compositions, formulations, or methods of use.

Claims

1. A short interfering ribonucleic acid for inhibiting the expression of a mutant allele of the kinin family member 1A (KIF1A) gene, comprising an antisense strand complementary to a nucleotide sequence selected from SEQ ID NO: 2 to 115.

2. The short interfering RNA according to claim 1, wherein the mutant allele contains a missense mutation selected from the group consisting of T99M, E253K, P305L, R316W and R203S.

3. The short interfering ribonucleic acid according to claim 1, wherein the antisense strand is at least 85%, at least 90%, at least 95%, or 100% complementary to a nucleotide sequence selected from SEQ ID NO: 2 to 115.

4. The short interfering ribonucleic acid according to claim 1, wherein the antisense strand is selected from SEQ ID NO: 116-229.

5. The short interfering RNA according to claim 1, wherein the short interfering RNA further comprises a sense strand, wherein the sense strand is complementary to the antisense strand, and wherein the sense strand comprises a nucleotide sequence selected from SEQ ID NO: 2 to 115.

6. The short interfering ribonucleic acid according to claim 1, wherein one or more nucleotides contain modifications selected from the group consisting of: restricted ethyl bridging nucleic acid (cEt), ethylidene bridging nucleic acid (ENA), 2'-fluorine (2'-F), locked nucleic acid (LNA), 2'- O -Methoxyethyl(2'-MOE), 2'- O -Methyl (2'-OMe), phosphodiamide morpholino oligonucleotide (PMO), peptide nucleic acid (PNA), and thiophosphate substitution (PS) between one or more phosphodiester bonds of nucleotides.

7. The short interfering ribonucleic acid according to claim 1, wherein the 3' end, the 5' end, or both the 3' end and the 5' end are shortened by one, two, or three nucleotides.

8. The short interfering ribonucleic acid according to claim 1 further comprises a terminal nucleotide overhang.

9. The short interfering ribonucleic acid according to claim 8, wherein the terminal overhang is dTdT at the 3' end.

10. A polynucleotide encoding a short interfering RNA for inhibiting the expression of a mutant allele of a kinin family member 1A (KIF1A) gene, said short interfering RNA comprising an antisense strand complementary to a nucleotide sequence selected from SEQ ID NO: 2 to 115.

11. The polynucleotide of claim 10, wherein the mutant allele contains a missense mutation selected from the group consisting of T99M, E253K, P305L, R316W and R203S.

12. The polynucleotide of claim 10, wherein the antisense strand is at least 85%, at least 90%, at least 95%, or 100% complementary to a nucleotide sequence selected from SEQ ID NO: 2 to 115.

13. The polynucleotide of claim 10, wherein the polynucleotide is selected from SEQ ID NO: 398 to 511.

14. The polynucleotide of claim 10, wherein the short interfering ribonucleic acid further comprises a sense strand, wherein the sense strand is complementary to the antisense strand, and wherein the sense strand comprises a nucleotide sequence selected from SEQ ID NO: 2 to 115.

15. The polynucleotide of claim 14, wherein the polynucleotide encoding the sense strand is selected from SEQ ID NO: 284 to 397.

16. The polynucleotide of claim 10, wherein the short interfering ribonucleic acid is shortened by one, two, or three nucleotides at the 3' end, the 5' end, or both the 3' end and the 5' end.

17. The polynucleotide of claim 10, wherein the short interfering ribonucleic acid further comprises a terminal nucleotide overhang.

18. The polynucleotide of claim 10, wherein the terminal overhang is dTdT at the 3' end.

19. A plasmid, viral vector, or viral particle comprising the polynucleotide of claim 10.

20. A short hairpin ribonucleic acid comprising a first part, a second part, and a third part, wherein the first part comprises any one of SEQ ID NO: 2 to 115, the second part comprises any one of SEQ ID NO: 539 to 546, and the third part comprises any one of the corresponding nucleotide sequences complementary to those in SEQ ID NO: 2 to 115.

21. The short hairpin ribonucleic acid according to claim 20, wherein the corresponding nucleotide sequence comprises any one of SEQ ID NO: 116 to 229.

22. The short hairpin ribonucleic acid of claim 20, wherein one or more nucleotides contain modifications selected from the group consisting of: restricted ethyl bridging nucleic acid (cEt), ethylidene bridging nucleic acid (ENA), 2'-fluoro(2'-F), locked nucleic acid (LNA), 2'- O -Methoxyethyl(2'-MOE), 2'- O -Methyl (2'-OMe), phosphodiamide morpholino oligonucleotide (PMO), peptide nucleic acid (PNA), and thiophosphate substitution (PS) between one or more phosphodiester bonds of nucleotides.

23. The short hairpin ribonucleic acid of claim 20, wherein the first portion is shortened by one, two, or three nucleotides at the 3' end, the 5' end, or both the 3' end and the 5' end.

24. The short hairpin ribonucleic acid of claim 20, wherein the third portion is shortened by one, two, or three nucleotides at the 3' end, the 5' end, or both the 3' end and the 5' end.

25. A polynucleotide encoding a short hairpin ribonucleic acid, the short hairpin ribonucleic acid comprising a first part, a second part, and a third part, the first part comprising any one of SEQ ID NO: 2 to 115, the second part comprising any one of SEQ ID NO: 539 to 546, and the third part comprising any one of the corresponding nucleotide sequences complementary to those in SEQ ID NO: 2 to 115.

26. The polynucleotide of claim 25, wherein the polynucleotide encoding the first portion is selected from SEQ ID NO: 284 to 397.

27. The polynucleotide of claim 25, wherein the polynucleotide encoding the third portion is selected from SEQ ID NO: 398 to 511.

28. The polynucleotide of claim 25, wherein the polynucleotide encoding the second portion is selected from SEQ ID NO: 539 to 546.

29. A plasmid, viral vector, or viral particle comprising the short hairpin ribonucleic acid according to claim 25.

30. A short interfering ribonucleic acid for inhibiting the expression of a mutant allele of the kinin family member 1A (KIF1A) gene, comprising an antisense strand complementary to a nucleotide sequence selected from SEQ ID NO: 230 to 241.

31. The short interfering RNA according to claim 30, wherein the mutant allele contains a missense mutation selected from the group consisting of T99M, E253K, P305L, R316W and R203S.

32. The short interfering ribonucleic acid according to claim 30, wherein the antisense strand is at least 85%, at least 90%, at least 95%, or 100% complementary to a nucleotide sequence selected from SEQ ID NO: 230 to 241.

33. The short interfering ribonucleic acid according to claim 30, wherein the antisense strand is selected from SEQ ID NO: 242 to 253.

34. The short interfering RNA of claim 30, wherein the short interfering RNA further comprises a sense strand, wherein the sense strand is complementary to the antisense strand, and wherein the sense strand comprises a nucleotide sequence selected from SEQ ID NO: 230 to 241.

35. The short interfering ribonucleic acid of claim 30, wherein one or more nucleotides contain modifications selected from the group consisting of: restricted ethyl bridging nucleic acid (cEt), ethylidene bridging nucleic acid (ENA), 2'-fluorine (2'-F), locked nucleic acid (LNA), 2'- O -Methoxyethyl(2'-MOE), 2'- O -Methyl (2'-OMe), phosphodiamide morpholino oligonucleotide (PMO), peptide nucleic acid (PNA), and thiophosphate substitution (PS) between one or more phosphodiester bonds of nucleotides.

36. The short interfering ribonucleic acid of claim 30, wherein the 3' end, the 5' end, or both the 3' end and the 5' end are shortened by one, two, or three nucleotides.

37. The short interfering ribonucleic acid according to claim 30, further comprising a terminal nucleotide overhang.

38. The short interfering ribonucleic acid according to claim 30, wherein the terminal overhang is dTdT at the 3' end.

39. A polynucleotide short interfering RNA encoding a mutant allele for inhibiting the expression of a kinin family member 1A (KIF1A) gene, said short interfering RNA comprising an antisense strand complementary to a nucleotide sequence selected from SEQ ID NO: 230 to 241.

40. The polynucleotide of claim 39, wherein the mutant allele contains a missense mutation selected from the group consisting of T99M, E253K, P305L, R316W and R203S.

41. The polynucleotide of claim 39, wherein the antisense strand is at least 85%, at least 90%, at least 95%, or 100% complementary to a nucleotide sequence selected from SEQ ID NO: 230 to 241.

42. The polynucleotide of claim 39, wherein the polynucleotide is selected from SEQ ID NO: 524 to 535.

43. The polynucleotide of claim 39, wherein the short interfering ribonucleic acid further comprises a sense strand, wherein the sense strand is complementary to the antisense strand, and wherein the sense strand comprises a nucleotide sequence selected from SEQ ID NO: 230 to 241.

44. The polynucleotide of claim 43, wherein the polynucleotide encoding the sense strand is selected from SEQ ID NO: 512 to 523.

45. The polynucleotide of claim 39, wherein the short interfering ribonucleic acid is shortened by one, two, or three nucleotides at the 3' end, the 5' end, or both the 3' end and the 5' end.

46. ​​The polynucleotide of claim 39, wherein the short interfering ribonucleic acid further comprises a terminal nucleotide overhang.

47. The polynucleotide of claim 39, wherein the terminal overhang is dTdT at the 3' end.

48. A plasmid, viral vector, or viral particle comprising the polynucleotide of claim 39.

49. An antisense oligonucleotide targeting the RNA SNP rs1063353 of the kinin family member 1A (KIF1A) gene, comprising gapmer oligonucleotides selected from SEQ ID NO: 254 to 283, 536, 537 and 538.

50. The antisense oligonucleotide of claim 49, wherein the gapmer oligonucleotide is truncated in the form of 5-10-4, 4-10-4, 4-10-3, 3-10-3, 3-10-2, 5-9-6, 5-9-5, 4-9-5, 4-9-4, 3-9-4, 3-9-3, 6-8-6, 6-8-5, 5-8-5, 5-8-4, 4-8-4 or 4-8-3.

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