KR20250009923A - Composition for regulating activity or expression of gap43 - Google Patents

Abstract

The present invention relates to compounds and compositions comprising the same for modulating the expression level and/or activity of the gap 43 gene, and to their use for preventing, treating, or alleviating diseases, disorders, and conditions associated with gap43.

Description

COMPOSITION FOR REGULATING ACTIVITY OR EXPRESSION OF GAP43

The present invention relates to compounds and compositions comprising the same for modulating the expression level and/or activity of the gap 43 gene, wherein the compounds and compositions are useful for preventing, treating, or alleviating diseases, disorders, and conditions associated with gap43.

Brain tumors are one of the tumors of the central nervous system (CNS), and are tumors that occur in the brain. Brain tumors can be classified into primary brain tumors and metastatic brain tumors depending on the origin of the tumor, and can also be classified as benign or malignant tumors. Brain tumors can occur in various cells that occur in the brain, such as brain cells, glial cells, and meningeal cells. Tumors that occur in glial cells include glioma.

According to a study in the United States, diffuse gliomas account for approximately 72% of malignant brain tumors in adults and 21% of all primary brain tumors and other central nervous system (CNS) tumors in adults. In addition, glioblastoma, the most malignant type in adults, is reported to account for 73% of diffuse gliomas, 52% of malignant nonmetastatic brain tumors, and 15% of all primary brain and other CNS tumors.

Diffuse gliomas are a group of neoplasms in which tumor cells exhibit infiltrative growth, and these diseases are very difficult to treat. Surgical resection is an important treatment method, but it is not enough to eradicate the disease. Patients often require adjuvant chemoradiation after surgery. However, in most cases, local recurrence and distant tumor progression occur.

In the case of brain tumors, the presence of the brain blood barrier makes it difficult for therapeutic drug delivery to reach the target area, and the development of therapeutic agents is not active due to the relative lack of understanding of brain neurobiology. Furthermore, compared to other brain tumors, glioblastomas exhibit aggressive variants, and if not treated promptly, they can result in fatal outcomes within a few weeks.

Therefore, in addition to surgical treatment, radiation therapy and chemotherapy are performed in the treatment of glioblastoma, but there is no perfect cure due to the occurrence of resistant mutations and recurrence by tumor stem cells. Therefore, there is a need to develop a new treatment for glioblastoma.

One example of the present invention relates to a compound for modulating the activity and/or expression level of GAP43, a pharmaceutical composition or method for preventing, ameliorating, and/or treating a disease, disorder, or condition associated with GAP43, comprising the compound as an active ingredient.

A further example of the present invention relates to a composition or method for modulating the activity and/or expression level of GAP43 using a compound for modulating the activity and/or expression level of GAP43.

The compound for regulating the activity and/or expression level of the above GAP43 may be an oligomeric compound and/or an RNAi agent, wherein the oligomeric compound may be an antisense oligonucleotide, and the RNAi agent may include, but is not limited to, ssRNAi, siRNA, shRNA, and miRNA.

One example of the present invention relates to a compound for modulating the activity and/or expression level (amount) of GAP43, a pharmaceutical composition or method for preventing, ameliorating, and/or treating a disease, disorder, or condition associated with GAP43, comprising the compound as an active ingredient.

A specific embodiment of the present invention relates to a method for preventing, ameliorating, and/or treating a disease, disorder, or condition associated with GAP43, comprising administering to a subject or patient in need thereof a compound for modulating the activity and/or expression level of GAP43.

As used herein, “subject,” “patient,” or “subject” means a human or non-human animal selected for treatment or therapy, wherein non-human animals may include, but are not limited to, mammals such as mice, rats, and monkeys.

In a specific embodiment of the present invention, the compound for modulating the activity and/or expression level (amount) of GAP43 relates to an oligomeric compound and/or an RNAi agent. One embodiment provides methods, compounds, and compositions for inhibiting GAP43 mRNA and protein expression, inhibiting the activity of GAP43 protein, or reducing mRNA and protein levels. In a specific embodiment, the compound for modulating the activity and/or expression level (amount) of GAP43 can be an inhibitor that reduces or inhibits the activity and/or expression level (amount) of GAP43.

GAP43, growth associated factor (neuromodulin) protein 43 (hereinafter referred to as "GAP43"), which is abundantly present in growth cones, plays an important role in peduncle extension and neurite branching. GAP43 is phosphorylated by protein kinase C and dephosphorylated by calcineurin. GAP43 is known as a neural tissue-specific cytoplasmic protein that can be attached to the membrane through a double palmitoylation sequence of cysteines 3 and 4. Two transcripts of GAP43, transcript 1 and transcript 2, have been reported.

GAP43 is included in the Genome Reference Consortium Human Build 38 patch release 14 Homo sapiens (assembly GRCh38.p14). The human GAP43 gene is available under NCBI Nucleotide ID NC_000003.12 ((https://www.ncbi.nlm.nih.gov/gene/2596)) and is located at genomic location 115,623,510 to 115,721,483. In addition, the DNA genetic information of GAP43 is available at the UCSC Genome Browser (https://genome.ucsc.edu) under Human (GRCh38/hg38). Information about the sequence and target genes (collectively, gene records) can be found in genetic sequence databases. Genetic sequence databases include the NCBI Reference Sequence database, GenBank, the European Nucleotide Archive, and the DNA Data Bank of Japan (the latter three forming the International Nucleotide Sequence Database Collaboration or INSDC).

In this specification, the GAP43 gene can be a human GPA43 gene sequence, a known reference transcript or a variant thereof, and can be found in various genetic databases. "RNA" in this specification means an RNA transcript, and unless otherwise specified, includes both pre-mRNA and mature mRNA.

As used herein, "target nucleic acid" and "target RNA" refer to a nucleic acid that an antisense compound is designed to affect. "Target RNA" refers to an RNA transcript, including pre-mRNA and mRNA, unless otherwise specified. As used herein, "target region" refers to a portion of a target nucleic acid to which an oligomeric compound is designed to hybridize.

The above compounds include, but are not limited to, antisense oligonucleotides and RNA interference agents (RNAi agents) for GAP43 nucleic acids.

The above RNAi agent refers to an antisense agent that acts at least in part through RISC or Ago2 to regulate a target nucleic acid and/or a protein encoded by the target nucleic acid. RNAi agents include, but are not limited to, double-stranded siRNA, single-stranded RNAi (ssRNAi), and microRNA including microRNA mimics. It is intended that the RNAi agent does not include antisense oligonucleotides.

The GAP43 inhibitor according to the present invention can comprise a conjugate group and/or a terminal group. In certain embodiments, the GAP43 inhibitor modulates the amount and/or activity of a target nucleic acid. The conjugate moiety modifies one or more properties of the attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, uptake, tissue distribution, cellular distribution, cellular uptake, charge, and clearance. In certain embodiments, the conjugate moiety imparts new properties to the attached oligonucleotide. As used herein, "conjugate group" means a group of atoms that is directly attached to an oligonucleotide. The conjugate group comprises a conjugate moiety and a conjugate linker that attaches the conjugate moiety to the oligonucleotide.

As used herein, the term "inhibiting expression or activity" refers to a decrease or inhibition of expression or activity compared to expression or activity in an untreated or control sample, and does not necessarily refer to a complete elimination of expression or activity. Specifically, the antisense oligomer or RNAi agent according to the present invention may be capable of reducing the level of GAP43 expression to a level of 70% or less, 65% or less, 60% or less, or 50% or less.

In certain embodiments, the oligomeric compounds and oligomeric duplexes are capable of hybridizing to a target nucleic acid to produce at least one antisense activity; and such oligomeric compounds and oligomeric duplexes are antisense compounds. In certain embodiments, the antisense compounds have antisense activity when they reduce or inhibit the amount or activity of a target nucleic acid by at least 25% in a standard cell assay. In certain embodiments, the antisense compounds selectively affect one or more target nucleic acids.

In certain embodiments, the GAP43 inhibitor can cause a decrease in the activity or expression level of GAP43 RNA or GAP43 protein of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% relative to 100% prior to treatment. Or, the activity or expression level of the GAP43 protein may be about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, or about 5% or less, based on 100% before treatment.

The GAP43 inhibitor according to the present invention may be an antisense oligonucleotide, wherein the antisense oligonucleotide is an oligonucleotide having a nucleic acid sequence complementary to a target nucleic acid or a region or segment thereof. In certain embodiments, the antisense oligonucleotide is capable of specifically hybridizing to a target nucleic acid or a region or segment thereof. As used herein, "antisense oligonucleotide" means an oligonucleotide comprising an oligonucleotide portion of an antisense compound capable of hybridizing to a target nucleic acid and having at least one antisense activity. The terms "antisense oligonucleotide" or "antisense compound" or "antisense oligonucleotide" or "antisense RNase H oligonucleotide" are used interchangeably and each have a base-pairing moiety linked by a nucleobase bond that allows the base-pairing moieties to hybridize. In such embodiments, the portion of the oligonucleotide has a nucleobase sequence complementary to a target nucleobase sequence. In specific embodiments, a region or the entire length of the nucleobase sequence of the oligonucleotide is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100% complementary to a target gene or target region. The term "antisense RNase H oligonucleotide" means an oligonucleotide comprising a region that is complementary to a target sequence and includes at least one chemical modification suitable for RNase H-mediated nucleic acid reduction.

In the present specification, compounds, for example, oligonucleotides, for modulating the activity and/or expression level (amount) of GAP43 also include pharmaceutically acceptable salts thereof, and the “pharmaceutically acceptable salt” means a physiologically and pharmaceutically acceptable salt of a compound, for example, an oligomeric compound or an oligonucleotide, i.e., a salt that retains the desired biological activity of the parent compound and does not cause undesirable toxicological effects.

One embodiment relates to an oligomeric compound comprising a modified oligonucleotide consisting of 8 to 80 linked nucleosides, wherein the nucleic acid sequence of the modified oligonucleotide is at least 80% complementary to an identical length portion of a GAP43 nucleic acid sequence, wherein the modified oligonucleotide comprises one or more modifications selected from the group consisting of a modified sugar moiety, a modified internucleoside linkage, and a modified base.

Another embodiment relates to an oligomeric compound comprising a modified oligonucleotide consisting of 8 to 80 linked nucleosides, wherein the nucleic acid sequence of the modified oligonucleotide comprises 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases from the nucleic acid sequence of SEQ ID NO : 1 to SEQ ID NO: 206, wherein the modified oligonucleotide comprises one or more modifications selected from the group consisting of a modified sugar moiety, a modified internucleoside linkage and a modified base.

"Oligomer compound" means a compound comprising a single oligonucleotide and optionally one or more additional features, e.g., a conjugating group or a terminal group. "Oligonucleotide" means a polymer of linked nucleosides, each of which may be independently modified or unmodified. Unless otherwise specified, the oligonucleotide comprises 8 to 80 subunits, 8 to 70 subunits, 8 to 60 subunits, 8 to 50 subunits, 8 to 40 subunits, 8 to 30 subunits, 8 to 25 subunits, 8 to 22 subunits, 8 to 20 subunits, 10 to 80 subunits, 10 to 70 subunits, 10 to 60 subunits, 10 to 50 subunits, 10 to 40 subunits, 10 to 30 subunits, 10 to 25 subunits, 10 to 22 subunits, 10 to 20 subunits, 12 to 80 subunits, 12 to 70 subunits, 12 to 60 subunits, 12 to 50 subunits, 12 to 40 subunits, 12 to It may comprise at least one subunit selected from the group consisting of 30, 12 to 25, 12 to 22, 12 to 20, 14 to 80, 14 to 70, 14 to 60, 14 to 50, 14 to 40, 14 to 30, 14 to 25, 14 to 22, 14 to 20, 16 to 80, 16 to 70, 16 to 60, 16 to 50, 16 to 40, 16 to 30, 16 to 25, 16 to 22, or 16 to 20.

The length of the oligonucleotide can be increased or decreased without abolishing the activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of oligonucleotides 13 to 25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. Oligonucleotides 25 nucleobases in length with 8 or 11 mismatched bases near the ends of the oligonucleotide were able to direct specific cleavage of the target RNA, although to a lesser extent than oligonucleotides that did not contain mismatches.

As used herein, "adjacent" in the context of an oligonucleotide refers to nucleosides, nucleobases, sugar moieties, or internucleoside linkages (internucleoside linkages) that are immediately adjacent to each other. For example, "adjacent nucleobases" means nucleobases that are immediately adjacent to each other in the sequence.

As exemplary oligomeric compounds of the present invention, nucleobase sequences of SEQ ID NO: 1 to SEQ ID NO: 206 are shown in Tables 1 and 2 below, and the start point and end point of Tables 1 and 2 below indicate the base positions on the genome of human GAP43 provided in Human (GRCh38/hg38) in the UCSC Genome browser (https://genome.ucsc.edu).

SED ID
NO Name Start point End point Nucleotide sequence 1 SEQ_ID_12104510 115,623,692 115,623,711 TTCTTCTCATACAGCACAGC 2 SEQ_ID_12104511 115,676,022 115,676,041 ATCTTTTGGTCGTCATCATT 3 SEQ_ID_12104512 115,676,023 115,676,042 AATCTTTTGGTCGTCATCAT 4 SEQ_ID_12104513 115,676,027 115,676,046 GTTCAATCTTTTGGTCGTCA 5 SEQ_ID_12104514 115,676,028 115,676,047 TGTTCAATCTTTTGGTCGTC 6 SEQ_ID_12104515 115,676,029 115,676,048 TTGTTCAATCTTTTGGTCGT 7 SEQ_ID_12104516 115,676,065 115,676,084 GGCCTTATGAGCTTTATCTT 8 SEQ_ID_12104517 115,676,066 115,676,085 CGGCCTTATGAGCTTTATCT 9 SEQ_ID_12104518 115,676,067 115,676,086 GCGGCCTTATGAGCTTTATC 10 SEQ_ID_12104519 115,676,068 115,676,087 TGCGGCCTTATGAGCTTTAT 11 SEQ_ID_12104520 115,676,069 115,676,088 TTGCGGCCTTATGAGCTTTA 12 SEQ_ID_12104521 115,676,070 115,676,089 GTTGCGGCCTTATGAGCTTT 13 SEQ_ID_12104522 115,676,071 115,676,090 GGTTGCGGCCTTATGAGCTT 14 SEQ_ID_12104523 115,676,072 115,676,091 TGGTTGCGGCCTTATGAGCT 15 SEQ_ID_12104524 115,676,091 115,676,110 CGGAAGCTAGCCTGAATTTT 16 SEQ_ID_12104525 115,676,092 115,676,111 ACGGAAGCTAGCCTGAATTT 17 SEQ_ID_12104526 115,676,104 115,676,123 TGTTATGTGTCCACGGAAGC 18 SEQ_ID_12104527 115,676,105 115,676,124 TTGTTATGTGTCCACGGAAG 19 SEQ_ID_12104528 115,676,106 115,676,125 CTTGTTATGTGTCCACGGAA 20 SEQ_ID_12104529 115,676,107 115,676,126 CCTTGTTATGTGTCCACGGA 21 SEQ_ID_12104530 115,676,108 115,676,127 TCCTTGTTATGTGTCCACGG 22 SEQ_ID_12104531 115,676,166 115,676,185 TTATTAGCTTCAGCCTCAGC 23 SEQ_ID_12104532 115,676,172 115,676,191 TCCTTCTTATTAGCTTCAGC 24 SEQ_ID_12104533 115,676,180 115,676,199 GGGCTTCATCCTTCTTATTA 25 SEQ_ID_12104534 115,676,229 115,676,248 TCGGCAGTAGTGGTGCCTTC 26 SEQ_ID_12104535 115,676,230 115,676,249 TTCGGCAGTAGTGGTGCCTT 27 SEQ_ID_12104536 115,676,231 115,676,250 CTTCGGCAGTAGTGGTGCCT 28 SEQ_ID_12104537 115,676,403 115,676,422 GAAGCTTTAGTGGCACTTTC 29 SEQ_ID_12104538 115,676,404 115,676,423 GGAAGCTTTAGTGGCACTTT 30 SEQ_ID_12104539 115,676,405 115,676,424 TGGAAGCTTTAGTGGCACTT 31 SEQ_ID_12104540 115,676,407 115,676,426 AGTGGAAGCTTTAGTGGCAC 32 SEQ_ID_12104541 115,676,411 115,676,430 TATCAGTGGAAGCTTTAGTG 33 SEQ_ID_12104542 115,676,416 115,676,435 CGAGTTATCAGTGGAAGCTT 34 SEQ_ID_12104543 115,676,417 115,676,436 GCGAGTTATCAGTGGAAGCT 35 SEQ_ID_12104544 115,676,419 115,676,438 CGGCGAGTTATCAGTGGAAG 36 SEQ_ID_12104545 115,676,420 115,676,439 ACGGCGAGTTATCAGTGGAA 37 SEQ_ID_12104546 115,676,421 115,676,440 GACGGCGAGTTATCAGTGGA 38 SEQ_ID_12104547 115,676,422 115,676,441 GGACGGCGAGTTATCAGTGG 39 SEQ_ID_12104548 115,720,869 115,720,888 TAGAGTTCAGGCATGTTCTT 40 SEQ_ID_12104549 115,720,870 115,720,889 TTAGAGTTCAGGCATGTTCT 41 SEQ_ID_12104550 115,720,875 115,720,894 ATTTCTTAGAGTTCAGGCAT 42 SEQ_ID_12104551 115,721,081 115,721,100 TTTAAGCCACACTGTTTGAC 43 SEQ_ID_12104552 115,721,404 115,721,423 TTACGCTAATTGGCACATTT 44 SEQ_ID_12104553 115,721,405 115,721,424 TTTACGCTAATGGCACATT 45 SEQ_ID_12104554 115,721,406 115,721,425 GTTTACGCTAATTGGCACAT 46 SEQ_ID_12104555 115,721,407 115,721,426 AGTTTACGCTAATTGGCACA 47 SEQ_ID_12104556 115,721,408 115,721,427 AAGTTTACGCTAATTGGCAC 48 SEQ_ID_12104557 115,721,410 115,721,429 GCAAGTTTACGCTAATTGGC 49 SEQ_ID_12104558 115,721,411 115,721,430 CGCAAGTTTACGCTAATTGG 50 SEQ_ID_12104559 115,721,412 115,721,431 CCGCAAGTTTACGCTAATTG 51 SEQ_ID_12104560 115,721,413 115,721,432 GCCGCAAGTTTACGCTAATT 52 SEQ_ID_12104561 115,721,414 115,721,433 AGCCGCAAGTTTACGCTAAT 53 SEQ_ID_12104562 115,721,418 115,721,437 TTAGAGCCGCAAGTTTACGC 54 SEQ_ID_12104563 115,721,419 115,721,438 CTTAGAGCCGCAAGTTTACG 55 SEQ_ID_12104564 115,721,420 115,721,439 CCTTAGAGCCGCAAGTTTAC 56 SEQ_ID_12104565 115,721,421 115,721,440 GCCTTAGAGCCGCAAGTTTA 57 SEQ_ID_12104566 115,721,422 115,721,441 AGCCTTAGAGCCGCAAGTTT 58 SEQ_ID_12104567 115,721,424 115,721,443 GGAGCCTTAGAGCCGCAAGT 59 SEQ_ID_12104568 115,721,446 115,721,465 TATTAATATTCGGGTTGAAA 60 SEQ_ID_12104569 115,623,698 115,623,717 GTTTGGTTCTTCTCATACAG 61 SEQ_ID_12104570 115,623,700 115,623,719 CTGTTTGGTTCTTCTCATAC 62 SEQ_ID_12104571 115,676,030 115,676,049 CTTGTTCAATCTTTTGGTCG 63 SEQ_ID_12104572 115,676,043 115,676,062 GGTTTGATACCATCTTGTTC 64 SEQ_ID_12104573 115,676,073 115,676,092 TTGGTTGCGGCCTTATGAGC 65 SEQ_ID_12104574 115,676,074 115,676,093 TTTGGTTGCGGCCTTATGAG 66 SEQ_ID_12104575 115,676,075 115,676,094 TTTTGGTTGCGGCCTTATGA 67 SEQ_ID_12104576 115,676,076 115,676,095 ATTTTGGTTGCGGCCTTATG 68 SEQ_ID_12104577 115,676,077 115,676,096 AATTTTGGTTGCGGCCTTAT 69 SEQ_ID_12104578 115,676,078 115,676,097 GAATTTTGGTTGCGGCCTTA 70 SEQ_ID_12104579 115,676,079 115,676,098 TGAATTTTGGTTGCGGCCTT 71 SEQ_ID_12104580 115,676,082 115,676,101 GCCTGAATTTTGGTTGCGGC 72 SEQ_ID_12104581 115,676,084 115,676,103 TAGCCTGAATTTTGGTTGCG 73 SEQ_ID_12104582 115,676,093 115,676,112 CACGGAAGCTAGCCTGAATT 74 SEQ_ID_12104583 115,676,094 115,676,113 CCACGGAAGCTAGCCTGAAT 75 SEQ_ID_12104584 115,676,095 115,676,114 TCCACGGAAGCTAGCCTGAA 76 SEQ_ID_12104585 115,676,100 115,676,119 ATGTGTCCACGGAAGCTAGC 77 SEQ_ID_12104586 115,676,101 115,676,120 TATGTGTCCACGGAAGCTAG 78 SEQ_ID_12104587 115,676,103 115,676,122 GTTATGTGTCCACGGAAGCT 79 SEQ_ID_12104588 115,676,109 115,676,128 TTCCTTGTTATGTGTCCACG 80 SEQ_ID_12104589 115,676,115 115,676,134 AGCTTTTTCCTTGTTATGTG 81 SEQ_ID_12104590 115,676,205 115,676,224 TTCTTCTCCACCCCATCGGC 82 SEQ_ID_12104591 115,676,206 115,676,225 CTTCTTCTCCACCCCATCGG 83 SEQ_ID_12104592 115,676,233 115,676,252 TGCTTCGGCAGTAGTGGTGC 84 SEQ_ID_12104593 115,676,234 115,676,253 CTGCTTCGGCAGTAGTGGTG 85 SEQ_ID_12104594 115,676,235 115,676,254 GCTGCTTCGGCAGTAGTGGT 86 SEQ_ID_12104595 115,676,236 115,676,255 GGCTGCTTCGGCAGTAGTGG 87 SEQ_ID_12104596 115,676,238 115,676,257 GGGGCTGCTTCGGCAGTAGT 88 SEQ_ID_12104597 115,676,266 115,676,285 GGGCTCATCAGGCTTGGAGC 89 SEQ_ID_12104598 115,676,268 115,676,287 CCGGGCTCATCAGGCTTGGA 90 SEQ_ID_12104599 115,676,270 115,676,289 TGCCGGGCTCATCAGGCTTG 91 SEQ_ID_12104600 115,676,301 115,676,320 TTCTTCTCCTCGGAAGGAGT 92 SEQ_ID_12104601 115,676,475 115,676,494 GGCACATCGGCTTGTTTAGG 93 SEQ_ID_12104602 115,676,476 115,676,495 AGGCACATCGGCTTGTTTAG 94 SEQ_ID_12104603 115,676,477 115,676,496 CAGGCACATCGGCTTGTTTA 95 SEQ_ID_12104604 115,676,478 115,676,497 GCAGGCACATCGGCTTGTTT 96 SEQ_ID_12104605 115,676,479 115,676,498 AGCAGGCACATCGGCTTGTT 97 SEQ_ID_12104606 115,676,480 115,676,499 CAGCAGGCACATCGGCTTGT 98 SEQ_ID_12104607 115,676,552 115,676,571 TTGGAGGCTGGGCTGTTGCC 99 SEQ_ID_12104608 115,676,555 115,676,574 CCGTTGGAGGCTGGGCTGTT 100 SEQ_ID_12104609 115,676,568 115,676,587 CTCTCCCCAGTCTCCGTTGG 101 SEQ_ID_12104610 115,676,587 115,676,606 GTTCTCTTCAGCTTGGCTGC 102 SEQ_ID_12104611 115,720,810 115,720,829 GGGCACTTTCCTTAGGTTTG 103 SEQ_ID_12104612 115,720,811 115,720,830 CGGGCACTTTCCTTAGGTTT

SED ID
NO Name Start point End point Nucleotide sequence 104 SEQ_ID_12104613 115,720,813 115,720,832 GCCGGGCACTTTCCTTAGGT 105 SEQ_ID_12104614 115,720,858 115,720,877 CATGTTCTTGGTCAGCCTCA 106 SEQ_ID_12104615 115,720,860 115,720,879 GGCATGTTCTTGGTCAGCCT 107 SEQ_ID_12104616 115,720,861 115,720,880 AGGCATGTTCTTGGTCAGCC 108 SEQ_ID_12104617 115,720,863 115,720,882 TCAGGCATGTTCTTGGTCAG 109 SEQ_ID_12104618 115,721,079 115,721,098 TAAGCCACACTGTTTGACTT 110 SEQ_ID_12104619 115,721,127 115,721,146 ATCATTACCAAAAACTTGCC 111 SEQ_ID_12104620 115,721,184 115,721,203 ACGCACCAGATCAAATAACT 112 SEQ_ID_12104621 115,721,255 115,721,274 GAACGGAACATTGCACACAC 113 SEQ_ID_12104622 115,721,256 115,721,275 TGAACGGAACATTGCACACA 114 SEQ_ID_12104623 115,721,400 115,721,419 GCTAATTGGCACATTTGCTT 115 SEQ_ID_12104624 115,721,401 115,721,420 CGCTAATTGGCACATTTGCT 116 SEQ_ID_12104625 115,721,402 115,721,421 ACGCTAATTGGCACATTTGC 117 SEQ_ID_12104626 115,721,403 115,721,422 TACGCTAATTGGCACATTTG 118 SEQ_ID_12104627 115,721,415 115,721,434 GAGCCGCAAGTTTACGCTAA 119 SEQ_ID_12104628 115,721,416 115,721,435 AGAGCCGCAAGTTTACGCTA 120 SEQ_ID_12104629 115,721,417 115,721,436 TAGAGCCGCAAGTTTACGCT 121 SEQ_ID_12104630 115,721,427 115,721,446 AAAGGAGCCTTAGAGCCGCA 122 SEQ_ID_12104631 115,721,433 115,721,452 GTTGAAAAAGGAGCCTTAGA 123 SEQ_ID_12104632 115,721,434 115,721,453 GGTTGAAAAAGGAGCCTTAG 124 SEQ_ID_12104633 115,721,437 115,721,456 TCGGGTTGAAAAAAGGAGCCT 125 SEQ_ID_12104634 115,721,438 115,721,457 TTCGGGTTGAAAAAGGAGCC 126 SEQ_ID_12104635 115,721,439 115,721,458 ATTCGGGTTGAAAAAAGGAGC 127 SEQ_ID_12104636 115,721,440 115,721,459 TATTCGGGTTGAAAAAGGAG 128 SEQ_ID_12104637 115,676,096 115,676,115 GTCCACGGAAGCTAGCCTGA 129 SEQ_ID_12104638 115,676,097 115,676,116 TGTCCACGGAAGCTAGCCTG 130 SEQ_ID_12104639 115,676,098 115,676,117 GTGTCCACGGAAGCTAGCCT 131 SEQ_ID_12104640 115,676,099 115,676,118 TGTGTCCACGGAAGCTAGCC 132 SEQ_ID_12104641 115,676,271 115,676,290 TTGCCGGGCTCATCAGGCTT 133 SEQ_ID_12104642 115,676,272 115,676,291 TTTGCCGGGCTCATCAGGCT 134 SEQ_ID_12104643 115,676,273 115,676,292 CTTTGCCGGGCTCATCAGGC 135 SEQ_ID_12104644 115,676,274 115,676,293 GCTTTGCCGGGCTCATCAGG 136 SEQ_ID_12104645 115,676,275 115,676,294 TGCTTTGCCGGGCTCATCAG 137 SEQ_ID_12104646 115,676,291 115,676,310 CGGAAGGAGTTTCTCCTGCT 138 SEQ_ID_12104647 115,676,292 115,676,311 TCGGAAGGAGTTTCTCCTGC 139 SEQ_ID_12104648 115,676,293 115,676,312 CTCGGAAGGAGTTTCTCCTG 140 SEQ_ID_12104649 115,676,294 115,676,313 CCTCGGAAGGAGTTTCTCCT 141 SEQ_ID_12104650 115,676,295 115,676,314 TCCTCGGAAGGAGTTTCTCC 142 SEQ_ID_12104651 115,676,297 115,676,316 TCTCCTCGGAAGGAGTTTCT 143 SEQ_ID_12104652 115,676,298 115,676,317 TTCTCCTCGGAAGGAGTTTC 144 SEQ_ID_12104653 115,676,299 115,676,318 CTTCTCCTCGGAAGGAGTTT 145 SEQ_ID_12104654 115,676,300 115,676,319 TCTTCTCCTCGGAAGGAGTT 146 SEQ_ID_12104655 115,676,423 115,676,442 AGGACGGCGAGTTATCAGTG 147 SEQ_ID_12104656 115,676,424 115,676,443 GAGGACGGCGAGTTATCAGT 148 SEQ_ID_12104657 115,676,425 115,676,444 GGAGGACGGCGAGTTATCAG 149 SEQ_ID_12104658 115,676,426 115,676,445 TGGAGGACGGCGAGTTATCA 150 SEQ_ID_12104659 115,676,427 115,676,446 TTGGAGGACGGCGAGTTATC 151 SEQ_ID_12104660 115,676,428 115,676,447 CTTGGAGGACGGCGAGTTAT 152 SEQ_ID_12104661 115,676,429 115,676,448 CCTTGGAGGACGGCGAGTTA 153 SEQ_ID_12104662 115,676,556 115,676,575 TCCGTTGGAGGCTGGGCTGT 154 SEQ_ID_12104663 115,676,558 115,676,577 TCTCCGTTGGAGGCTGGGCT 155 SEQ_ID_12104664 115,676,559 115,676,578 GTCTCCGTTGGAGGCTGGGC 156 SEQ_ID_12104665 115,676,565 115,676,584 TCCCCAGTCTCCGTTGGAGG 157 SEQ_ID_12104666 115,720,809 115,720,828 GGCACTTTCCTTAGGTTTGG 158 SEQ_ID_12104667 115,721,010 115,721,029 GAGAAAGAGGGCTAGTGGGT 159 SEQ_ID_12104668 115,721,077 115,721,096 AGCCACACTGTTTGACTTGG 160 SEQ_ID_12104669 115,721,183 115,721,202 CGCACCAGATCAAATAACTT 161 SEQ_ID_12104670 115,721,185 115,721,204 CACGCACCAGATCAAATAAC 162 SEQ_ID_12104671 115,721,251 115,721,270 GGAACATTGCACACACACTT 163 SEQ_ID_12104672 115,721,252 115,721,271 CGGAACATTGCACACACACT 164 SEQ_ID_12104673 115,721,253 115,721,272 ACGGAACATTGCACACACAC 165 SEQ_ID_12104674 115,721,258 115,721,277 GATGAACGGAACATTGCACA 166 SEQ_ID_12104675 115,721,259 115,721,278 AGATGAACGGAACATTGCAC 167 SEQ_ID_12104676 115,721,260 115,721,279 CAGATGAACGGAACATTGCA 168 SEQ_ID_12104677 115,721,261 115,721,280 TCAGATGAACGGAACATTGC 169 SEQ_ID_12104678 115,721,262 115,721,281 CTCAGATGAACGGAACATTG 170 SEQ_ID_12104679 115,721,423 115,721,442 GAGCCTTAGAGCCGCAAGTT 171 SEQ_ID_12104680 115,623,699 115,623,718 TGTTTGGTTCTTCTCATACA 172 SEQ_ID_12104681 115,676,040 115,676,059 TTGATACCATCTTGTTCAAT 173 SEQ_ID_12104682 115,676,032 115,676,051 ATCTTGTTCAATCTTTTGGT 174 SEQ_ID_12104683 115,623,667 115,623,686 TGTCTCGTCCCTTGCCTTCT 175 SEQ_ID_12104684 115,623,693 115,623,712 GTTCTTCTCATACAGCACAG 176 SEQ_ID_12104685 115,623,689 115,623,708 TTCTCATACAGCACAGCATG 177 SEQ_ID_12104686 115,676,039 115,676,058 TGATACCATCTTGTTCAATC 178 SEQ_ID_12104687 115,623,695 115,623,714 TGGTTCTTCTCATACAGCAC 179 SEQ_ID_12104688 115,676,034 115,676,053 CCATCTTGTTCAATCTTTTG 180 SEQ_ID_12104689 115,676,048 115,676,067 CTTCTGGTTTGATACCATCT 181 SEQ_ID_12104690 115,721,332 115,721,351 TTGTTCATTCCATCACATTG 182 SEQ_ID_12104691 115,721,177 115,721,196 AGATCAAATAACTTGGATAC 183 SEQ_ID_12104692 115,721,136 115,721,155 GATTGAATCATCATTACCAA 184 SEQ_ID_12104693 115,721,138 115,721,157 ATGATTGAATCATCATTACC 185 SEQ_ID_12104694 115,721,399 115,721,418 CTAATTGGCACATTTGCTTT 186 SEQ_ID_12104695 115,721,139 115,721,158 AATGATTGAATCATCATTAC 187 SEQ_ID_12104696 115,623,694 115,623,713 GGTTCTTCTCATACAGCACA 188 SEQ_ID_12104697 115,623,691 115,623,710 TCTTCTCATACAGCACAGCA 189 SEQ_ID_12104698 115,721,254 115,721,273 AACGGAACATTGCACACACA 190 SEQ_ID_12104699 115,623,615 115,623,634 CTTATTCCCCCCCTCTCCAC 191 SEQ_ID_12104700 115,721,186 115,721,205 ACACGCACCAGATCAAATAA 192 SEQ_ID_12104701 115,721,171 115,721,190 AATAACTTGGATACAGTGCA 193 SEQ_ID_12104702 115,720,838 115,720,857 GGTTCCTCTTCTTTACCCTC 194 SEQ_ID_12104703 115,676,201 115,676,220 TCTCCACCCCATCGGCAACA 195 SEQ_ID_12104704 115,721,161 115,721,180 ATACAGTGCAAGAATTTCCC 196 SEQ_ID_12104705 115,721,188 115,721,207 CCACACGCACCAGATCAAAT 197 SEQ_ID_12104706 115,721,189 115,721,208 GCCACACGCACCAGATCAAA 198 SEQ_ID_12104707 115,721,190 115,721,209 GGCCACACGCACCAGATCAA 199 SEQ_ID_12104708 115,721,196 115,721,215 CCACAGGGCCACACGCACCA 200 SEQ_ID_12119278 115,721,078 115,721,095 GCCACACTGTTTGACTTG 201 SEQ_ID_12119279 115,721,077 115,721,094 CCACACTGTTTGACTTGG 202 SEQ_ID_12119280 115,721,079 115,721,096 AGCCACACTGTTTGACTT 203 SEQ_ID_12119288 115,721,252 115,721,269 GAACATTGCACACACACT 204 SEQ_ID_12119289 115,721,251 115,721,268 AACATTGCACACACACTT 205 SEQ_ID_12119290 115,721,253 115,721,270 GGAACATTGCACACACAC 206 SEQ_ID_12119300 115,721,254 115,721,271 CGGAACATTGCACACACA

In a specific embodiment, the oligonucleotide is an oligomeric compound comprising a modified oligonucleotide consisting of 8 to 80 linked nucleosides, wherein the nucleic acid sequence of the modified oligonucleotide can be an oligomeric compound comprising 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases selected from a region of a nucleic acid sequence of SEQ ID NO: 207 to SEQ ID NO: 212, more specifically, a region selected from the group consisting of SEQ ID NOs: 213, 214, 209, 215, 211, and 212. The oligomeric compound can be a modified oligonucleotide comprising one or more modifications selected from the group consisting of a modified sugar moiety, a modified internucleoside linkage and a modified base. The above specific oligonucleotide may be more specifically a TR7 region for the TR1 region, more specifically a TR8 region for the TR2 region, and more specifically a TR 9 region for the TR4 region.

The above region may be shown to be a region with very effective inhibitors of GAP43 expression in an antisense oligonucleotide screen, and this region on GAP43 mRNA may be a good target region for targeting with ASOs.

SED ID
NO Name Start point End point Nucleotide sequence 207 TR1 115,676,065 115,676,117 AAGATAAAGCTCATAAGGCCGCAACCAAAATTCAGGCTAGCTTCCGTGGACAC 208 TR2 115,676,104 115,676,134
AAGATAAAGCTCATAAGGCCGCAACCAAAATTCAGGCTAGCTTCCGTGGACACATAACAAGGAAAAAAGCT 209 TR3 115,676,291 115,676,312
AGCAGGAGAAACTCCTTCCGAG 210 TR4 115,676,403 115,676,430
GAAAGTGCCACTAAAGCTTCCACTGATAACTCGCCGTCC 211 TR5 115,721,077 115,721,096 AGCCACACTGTTTGACTTGG 212 TR6 115,721,189 115,721,275
TTTGATCTGGTGCGTGTGGCCCTGTGGGAGTCCACTTTCctctctctctctctctctGTTCCAAGTGTTGTGTGCAATGTTCCGTTCA 213 TR7 115,676,072
115,676,097
AGCTCATAAGGCCGCAACCAAAATTC 214 TR8 115,676,104 115,676,128
AGCTTCCGTGGACACATAACAAGGAA 215 TR9 115,676,404 115,676,430
AAAGTGCCACTAAAGCTTCCACTGATA

As used herein, the term "modified oligonucleotide" means an oligonucleotide having a modification of at least one sugar, nucleobase or internucleoside linkage, wherein the modified oligonucleotide may comprise one or more modifications selected from the group consisting of a modified sugar moiety, a modified internucleoside linkage and a modified base. The term "unmodified oligonucleotide" means an oligonucleotide that does not comprise a modification of any sugar, nucleobase or internucleoside linkage.

"Nucleic acid" refers to a molecule composed of monomeric nucleotides. Nucleic acids include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), single-stranded nucleic acids, and double-stranded nucleic acids.

As used herein, “modified nucleoside” means a nucleoside comprising a modified nucleobase and/or a modified sugar moiety.

As used herein, "nucleobase" means an unmodified nucleobase or a modified nucleobase. A nucleobase is a heterocyclic moiety. As used herein, an "unmodified nucleobase" is adenine (A), thymine (T), cytosine (C), uracil (U), or guanine (G). As used herein, a "modified nucleobase" is a group of atoms other than unmodified A, T, C, U, or G that can pair with at least one other nucleobase. "5-methyl cytosine" is a modified nucleobase.

The oligonucleotide may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 modified nucleobases of any sequence.

The above modified oligonucleotide has a sugar motif, wherein the sugar motif is

It may comprise a 5'-region consisting of 1 to 6 linked 5'-region nucleosides; a central region consisting of 6 to 10 linked central region nucleosides; and a 3'-region consisting of 1 to 6 linked 3'-region nucleosides.

In an embodiment, the modified sugar moiety can comprise one or more selected from the group consisting of a non-bicyclic modified sugar moiety and a bicyclic or tricyclic sugar moiety. In a specific embodiment, the modified sugar moiety is a sugar surrogate. Such sugar surrogates can comprise one or more substitutions corresponding to those of other types of modified sugar moieties.

In certain embodiments, the modified sugar moiety is a non-bicyclic modified sugar moiety comprising a furanosyl ring having one or more substituents, none of which substituents bridge two atoms of the furanosyl ring to form a bicyclic structure. Such non-bridging substituents can be at any position of the furanosyl, including but not limited to substituents at the 2', 3', 4' and/or 5' positions. Examples of 2'-substituents suitable for such non-bicyclic modified sugar moieties include, but are not limited to: 2'-F, 2'-OCH3 ("OMe" or "O-methyl"), and 2'-O(CH2)2OCH3 ("MOE"). In certain embodiments, the 2'-substituent is halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O-C1-C10 alkoxy, O-C1-C10 substituted alkoxy, O-C1-C10 alkyl, O-C1-C10 substituted alkyl, S-alkyl, N(Rm)-alkyl, O-alkenyl, S-alkenyl, N(Rm)-alkenyl, O-alkynyl, S-alkynyl, N(Rm)-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2ON(Rm)(Rn), or OCH2C(=O)-N(Rm)(Rn), wherein each of Rm and Rn is independently H, an amino protecting group. or substituted or unsubstituted C1-C10 alkyl), -O(CH2)2ON(CH3)2 (“DMAOE”), 2'-OCH2OCH2N(CH2)2 (“DMAEOE”), etc.

In certain embodiments, the non-bicyclic modified sugar moiety comprises a substituent at the 3'-position. Examples of suitable substituents at the 3'-position of the modified sugar moiety include, but are not limited to, alkoxy (e.g., methoxy), alkyl (e.g., methyl, ethyl). In certain embodiments, the non-bicyclic modified sugar moiety comprises a substituent at the 4'-position. Examples of suitable 4'-substituents for non-bicyclic modified sugar moieties include, but are not limited to, alkoxy (e.g., methoxy), alkyl, and those described in WO 2015/106128 (Manoharan et al.). Examples of suitable 5'-substituents for non-bicyclic modified sugar moieties include, but are not limited to: 5'-methyl (R or S), 5'-vinyl, ethyl, and 5'-methoxy. In certain embodiments, the non-bicyclic modified sugar moiety comprises more than one non-bridging sugar substituent, for example, but not limited to, a 2'-F-5'-methyl sugar moiety.

In certain embodiments, the 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2'-substituent selected from F, OCH3, and OCH2CH2OCH3.

Certain modified sugar moieties include substituents that bridge two atoms of the furanosyl ring to form a second ring, thereby forming a bicyclic sugar moiety. Nucleosides comprising such bicyclic sugar moieties have been referred to as bicyclic nucleosides (BNAs), locked (locked) nucleosides, or conformationally constrained nucleotides (CRNs). Certain such compounds are described in U.S. Patent Publication No. 2013/0190383; and PCT Publication No. WO 2013/036868. In certain such embodiments, the bicyclic sugar moiety includes a bridge between the 4' and 2' furanose ring atoms. In certain such embodiments, the furanose is a ribose ring. Examples of such 4' to 2' bridging sugar substituents include, but are not limited to: 4'-CH ₂ -2', 4'-(CH ₂ ) ₂ -2', 4'-(CH ₂ ) ₃ -2', 4'-CH ₂ -O-2'("LNA"),4'-CH ₂ -S-2', 4'-(CH ₂ ) ₂ -O-2'("ENA"),4'-CH(CH ₃ )-O-2' (referred to as "constrained ethyl" or "cEt" when in the S configuration), 4'-CH ₂ -O-CH ₂ -2', 4'-CH ₂ -N(R)-2', 4'-CH(CH ₂ OCH ₃ )-O-2'("constrainedMOE" or "cMOE") and analogs thereof, 4'-C(CH ₃ )(CH ₃ )-O-2' and analogs thereof, 4'-CH ₂ -N(OCH ₃ )-2' and analogs thereof, 4'-CH ₂ -ON(CH ₃ )-2', 4'-CH ₂ -C-(H)(CH ₃ )-2', 4'-CH ₂ -C-(=CH ₂ )-2' and analogs thereof, 4'-C(R _a R _b )-N(R)-O-2', 4'-C(R _a R _b )-ON(R)-2', 4'-CH ₂ -ON(R)-2' and 4'-CH ₂ -N(R)-O-2' (wherein R, R _a and R _b are each independently H, a protecting group or C ₁ -C ₁₂ alkyl).

The modified sugar moiety comprises a substituent that bridges two atoms of the furanosyl ring to form a second ring, thereby producing a bicyclic sugar moiety. A nucleoside comprising such a bicyclic sugar moiety has been referred to as a bicyclic nucleoside (BNA), or a constrained nucleoside. The bicyclic sugar moiety comprises a bridge between the 4' and 2' furanose ring atoms. In certain such embodiments, the furanose is a ribose ring. Examples of such 4' to 2' bridging sugar substituents include, but are not limited to: 4'-CH ₂ -2', 4'-(CH ₂ ) ₂ -2', 4'-(CH ₂ ) ₃ -2', 4'-CH ₂ -O-2'("LNA"),4'-CH ₂ -S-2', 4'-(CH ₂ ) ₂ -O-2'("ENA"),4'-CH(CH ₃ )-O-2' (referred to as "constrained ethyl" or "cEt" when in the S configuration), 4'-CH ₂ -O-CH ₂ -2', 4'-CH ₂ -N(R)-2', 4'-CH(CH ₂ OCH ₃ )-O-2'("constrainedMOE" or "cMOE") and analogs thereof, 4'-C(CH ₃ )(CH ₃ )-O-2' and analogues thereof, 4'-CH ₂ -N(OCH ₃ )-2' and analogues thereof, 4'-CH ₂ -ON(CH ₃ )-2', 4'-CH ₂ -C-(H)(CH ₃ )-2', 4'-CH ₂ -C-(=CH ₂ )-2' and analogues thereof, 4'-C(R _a R _b )-N(R)-O-2', 4'-C(R _a R _b )-ON(R)-2', 4'-CH ₂ -ON(R)-2' and 4'-CH ₂ -N(R)-O-2' (wherein R, R _a and R _b are each independently H, a protecting group or C ₁ -C ₁₂ alkyl) (e.g., US 7,427,672 (Imanishi Includes, but is not limited to, etc.

In this specification, the general description of bicyclic nucleosides includes both isomeric configurations. When the positions of particular bicyclic nucleosides (e.g., LNA or cEt) are identified in the embodiments exemplified herein, they are present in the β-D configuration, unless otherwise specified.

In certain embodiments, the modified sugar moiety is a sugar surrogate. In such certain embodiments, an oxygen atom of the sugar moiety is replaced by, for example, a sulfur, carbon or nitrogen atom.

Many other bicyclic and tricyclic sugars and sugar substitutes that can be used in modified nucleosides are known in the art.

In one embodiment, the oligonucleotide compound can comprise an unmodified internucleoside linkage (a phosphodiester internucleoside linkage) and/or one or more modified internucleoside linkages. In certain embodiments, each internucleoside linkage can be a phosphodiester internucleoside linkage (P=O), or a phosphorothioate internucleoside linkage (P=S). In certain embodiments, each internucleoside linkage of the modified oligonucleotide can be independently selected from a phosphorothioate internucleoside linkage and a phosphodiester internucleoside linkage. In one embodiment, each phosphorothioate internucleoside linkage is independently selected from stereorandom phosphorothioate, phosphorothioate, and phosphorothioate.

In certain embodiments, the modified oligonucleotide comprises or consists of a region having a fully modified sugar motif, wherein each nucleoside within the fully modified region comprises an identical modified sugar moiety, referred to herein as a uniformly modified sugar motif. In certain embodiments, the fully modified oligonucleotide is a uniformly modified oligonucleotide. In certain embodiments, each nucleoside of the uniformly modified nucleotide comprises an identical 2'-modification.

In certain embodiments, the modified oligonucleotide comprises and consists of a region having a gapmer motif defined by two external regions or "wings" and a central or internal region or "gap". The three regions of the gapmer motif (the 5'-wing, the gap, and the 3'-wing) form a contiguous sequence of nucleosides, wherein at least a portion of the sugar moiety of the nucleosides of each wing can be different from at least a portion of the sugar moiety of the nucleosides of the gap. "GAPMER" means an oligonucleotide comprising an internal region having a plurality of nucleosides that support RNase H cleavage located between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as the "gap" and the external regions may be referred to as the "wings". Here, the lengths (number of nucleosides) of the three regions of the gapmer can be provided using the notation [number of nucleosides in 5' wing] - [number of nucleosides in gap] - [number of nucleosides in 3' wing]. For example, a 5-10-5 gapmer consists of 5 linked nucleosides in each wing and 10 linked nucleosides in the gap. When chemical modifications are included in the nomenclature, for example, a 5-10-5 MOE gapmer consists of 5 linked MOE modified nucleosides in the 5' wing, 10 linked deoxynucleosides in the gap and 5 linked MOE nucleosides in the 3' wing. In certain embodiments, the modified oligonucleotide can include a 5-10-5 MOE gapmer, a 3-10-3 BNA gapmer, a 3-10-3 cEt gapmer or a 3-10-3 LNA gapmer.

One example of the present invention relates to a pharmaceutical composition for preventing, alleviating or treating glioblastoma, comprising a compound for modulating the activity and/or expression level of GAP43, an oligomeric compound and/or an RNAi agent, or a method for preventing, alleviating or treating glioblastoma, comprising a step of administering a compound for modulating the activity and/or expression level of GAP43, an oligomeric compound and/or an RNAi agent to a subject or individual in need thereof.

Another example of the present invention provides a composition for reducing human GAP43 protein synthesis or human GAP43 mRNA levels in a human subject having an increased expression level of a human GAP43 gene associated with glioblastoma. Alternatively, the present invention provides a method for reducing human GAP43 protein synthesis or human GAP43 mRNA levels in a human subject having an increased expression level of a human GAP43 gene associated with glioblastoma.

In certain embodiments, the method according to the present invention comprises administering to a subject a compound for modulating the activity and/or expression level of GAP43, an oligomeric compound and/or an RNAi agent, and detecting or quantifying the amount of GAP43 RNA or GAP43 protein in a cell or biological fluid of the subject. In certain embodiments, the method comprises detecting/quantifying a first amount of GAP43 RNA or GAP43 protein in a first biological sample obtained prior to the administration, detecting/quantifying a second amount of GAP43 RNA or GAP43 protein in a second biological sample obtained after the administration, and comparing the first amount to the second amount to detect or quantify a decrease in GAP43 RNA or GAP43 protein.

Inhibition of GAP43 nucleic acid levels or expression can be assayed in a variety of ways known in the art. For example, target nucleic acid levels can be quantified, for example, by Northern blot analysis, competitive polymerase chain reaction (PCR), or quantitative real-time PCR. Antisense inhibition of GAP43 nucleic acid can be assessed by measuring GAP43 protein levels. GAP43 protein levels can be assessed or quantified in a variety of ways well known in the art, such as by immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (e.g., caspase activity assays), immunohistochemistry, immunocytochemistry, or fluorescence-activated cell sorting (FACS).

The above GAP43 associated disease, disorder or condition may be, for example, diffuse gliomas. The present invention is a GAP43 inhibitor which acts as an epigenetic priming factor for diffuse gliomas, such as glioblastomas. The diffuse gliomas include diffuse gliomas, particularly glioblastoma, IDH-wild type, according to the fifth edition of the WHO Classification of Tumors of the Central Nervous System 2021.

The above glioblastoma may have one or more characteristics selected from the group consisting of the following (1) to (5): (1) microvascular proliferation, (2) necrosis, (3) TERT promoter mutation, (4) EGFR hyperactivation mutation, and (5) chromosome 7 gain and chromosome 10 deletion.

In addition, the glioblastoma may include one or more genetic mutations selected from the group consisting of EGFR, PDGF, INK4α, MDM2, RB, CDK4/6, PTEN, TP53, and NF1 genes, and specifically, may include one or more mutations selected from the group consisting of EGFR, PTEN, and TP53. The glioblastoma may include one or more alterations selected from the group consisting of increased expression of EGFR, mutation of PTEN, and mutation of p53, and more specifically, may include activation alteration of EGFR. The glioblastoma includes an activating mutation of epidermal growth factor receptor (EGFR) gene or increased expression level of EGFR, and further may include one or more genetic mutations selected from the group consisting of PTEN and p53 mutations. More specifically, the glioblastoma may comprise a TERT promoter mutation together with a gene alteration for epidermal growth factor receptor (EGFR), and may additionally comprise one or more gene alterations selected from the group consisting of PTEN and p53 mutations.

In the present specification, EGFR tyrosine kinase activity in glioblastoma cells can be dysregulated by EGFR gene mutations (e.g., amplification, indel (insertion and deletion), single nucleotide variation (SNV)), overexpression of EGFR protein, increased gene copy number, chromosomal rearrangement, and activation by autocrine function. Most GBMs characterized by EGFR amplification are positive for mutant EGFRvIII, EGFRvV, and EGFR SNV. For example, EGFR, TP53, or PTEN mutations are disclosed in, but are not limited to, Cimino et al. Exp Mol Pathol. 2015 Jun; 98(3): 568-573. The "copy number variation (CNV)" in the present invention refers to a form of structural variation in the genome, which is an amplification or deletion of a DNA fragment of 1 Kb or more.

p53 is a tumor suppressor known to play a role in suppressing abnormal cell proliferation and inducing cancer cell death. It is also expressed as TP53, and can be expressed as Trp53 in mouse genes.

In one example, the glioblastoma may originate from the differentiation process of oligodendrocyte progenitor cells into oligodendrocytes through differentiation of neuronal stem cells into oligodendrocytes.

In one example, the glioblastoma can be a primary glioblastoma and/or a recurrent glioblastoma, for example, the recurrent glioblastoma can be other than a pseudo-progression for a tumor determined to be progressive disease by the RANO criteria. In some embodiments, the recurrent glioblastoma is characterized by a new lesion outside the irradiated field following radiation therapy. In some embodiments, the recurrent glioblastoma is characterized by a recurrence following radiation therapy. In some embodiments, the recurrent glioblastoma is temporally separated from a prior occurrence of the glioblastoma. In some embodiments, the recurrent glioblastoma can be a recurrent glioblastoma having a new lesion outside the irradiated field, a recurrence following radiation therapy, or a recurrent glioblastoma that occurs within a time interval of a prior occurrence of the glioblastoma.

In some embodiments, the subject or patient may have received one or more treatments selected from the group consisting of drug therapy, radiation therapy, and surgical therapy prior to treatment. The subject may have received an antiangiogenic agent, or an alkylating agent (e.g., temozolomide) prior to treatment, or may have been exposed to both radiation therapy and an alkylating agent (e.g., temozolomide) prior to treatment.

In certain embodiments, the present disclosure provides a pharmaceutical composition comprising one or more oligomeric compounds. In certain embodiments, the one or more oligomeric compounds each comprise a modified oligonucleotide. In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable diluent or carrier. In certain embodiments, the pharmaceutical composition comprises the modified oligonucleotide and artificial cerebrospinal fluid. In certain embodiments, the pharmaceutical composition comprises the modified oligonucleotide and artificial cerebrospinal fluid. In certain embodiments, the pharmaceutical composition comprises one or more oligomeric compounds and one or more excipients. In certain embodiments, the excipients are selected from water, a salt solution, an alcohol, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone.

In certain embodiments, the pharmaceutical composition comprising an oligomeric compound comprises any pharmaceutically acceptable salt of the oligomeric compound, an ester of the oligomeric compound, or a salt of such an ester.

In certain embodiments, the pharmaceutical composition comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions.

In certain embodiments, the pharmaceutical composition is prepared for oral administration. In certain embodiments, the pharmaceutical composition is prepared for buccal administration. In certain embodiments, the pharmaceutical composition is prepared for administration by injection (e.g., intravenously, subcutaneously, intramuscularly, intrathecally (IT), intracerebroventricularly (ICV), etc.). In certain such embodiments, the pharmaceutical composition includes a carrier and is formulated in an aqueous solution, such as water, or a physiologically compatible buffer, such as Hank's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients (e.g., ingredients that aid solubility or act as preservatives) are included. In certain embodiments, an injectable suspension is prepared using a suitable liquid carrier, suspending agent, etc. Certain injectable pharmaceutical compositions are presented in unit dosage form, such as ampoules or multi-dose containers. Certain injectable pharmaceutical compositions are suspensions, solutions or emulsions in oily or aqueous vehicles and may contain formulatory agents such as suspending agents, stabilizers and/or dispersing agents. Specific solvents suitable for use in the injectable pharmaceutical compositions include, but are not limited to, lipophilic solvents and fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, and liposomes.

The formulation composition and method of a pharmaceutical composition depend on several criteria including, but not limited to, the route of administration, the extent of the disease, or the dosage to be administered.

In certain embodiments, the oligomeric compound may be mixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations. The compositions and methods for formulating pharmaceutical compositions depend on a number of criteria, including but not limited to the route of administration, the extent of the disease, or the dosage administered.

Maintenance doses or maintenance doses are administered following the doses or loading doses.

The compounds for regulating the activity and/or expression level of GAP43 according to the present invention, oligomeric compounds and/or RNAi agents, can be administered orally or parenterally, and parenteral administration means administration via injection (e.g., bolus injection) or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, for example, intrathecal or intracerebroventricular administration.

The present invention relates to compounds for modulating the expression level and/or activity of the gap 43 gene and compositions comprising the same, which are useful for preventing, treating, or alleviating diseases, disorders, and conditions associated with gap43.

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not intended to be limited to the following examples.

Example 1: Effect of 5-10-5 MOE gapmer modified oligonucleotides on human GAP43 RNA in vitro, transfection technique, single dose

Modified oligonucleotides complementary to human GAP43 nucleic acid were tested for their effect on GAP43 RNA levels in the U87-MG cell line (glioblastoma cell line).

The modified oligonucleotides in the table below are 5-10-5 MOE gapmers having mixed internucleoside linkages. The gapmer is 20 nucleosides in length, wherein the central cap segment is composed of 10 2'-deoxynucleotides and the 3' and 5' wings are each composed of 5 2'-MOE nucleotides. The motif for the gapmer is (from 5' to 3'): eeeeeddddddddddeeeee; where 'd' represents a 2'-deoxyribosyl sugar moiety and 'e' represents a 2'-MOE sugar moiety. The internucleoside linkage for the gapmer is a phosphothioester bond.

U87-MG cells cultured at a density of 20,000 cells/well were treated with 400 nM of modified oligonucleotides using Lipofectamine ^TM 3000. Total RNA was extracted from the cells after 24 h of treatment. The RNA expression level of the GAP43 gene relative to GAPDH, one of the cell maintenance genes, was measured by quantitative PCR. The RNA level was measured using human GAPDH primer probe set (forward sequence GACTTCAACAGCGACAC (SEQ ID N: 216) ; reverse sequence CGTTGTCATACCAGGAAAT (SEQ ID N: 217)) and human GAP43 primer probe set (forward sequence AGCTGAAGAGAACATAGAAGCTGTA (SEQ ID N: 218); reverse sequence TGGGGATGTGGAAAGCCATTT (SEQ ID NO: 219)).

The expression levels of GAP43 RNA of the modified oligonucleotides were calculated as the ratio (relative expression) of the GAP43 RNA levels to the untreated control (UC) cells and are presented in Tables 4 to 8 below. The experiments were repeated three times only for the experimental batches in which the negative control (NC, ACTACACCTTACTTATCACA (SEQ ID NO: 220)) showed the expected level of GAP43 RNA expression.

Compound Relative expression UC 1 NC 0.91 SEQ_ID_12104510 0.65 SEQ_ID_12104511 0.62 SEQ_ID_12104512 0.48 SEQ_ID_12104513 0.31 SEQ_ID_12104514 0.35 SEQ_ID_12104515 0.44 SEQ_ID_12104516 0.29 SEQ_ID_12104517 0.35 SEQ_ID_12104518 0.37 SEQ_ID_12104519 0.32 SEQ_ID_12104520 0.25 SEQ_ID_12104521 0.28 SEQ_ID_12104522 0.34 SEQ_ID_12104523 0.35 SEQ_ID_12104524 0.38 SEQ_ID_12104525 0.28 SEQ_ID_12104526 0.34 SEQ_ID_12104527 0.33 SEQ_ID_12104528 0.25 SEQ_ID_12104529 0.25 SEQ_ID_12104530 0.32 SEQ_ID_12104531 0.64 SEQ_ID_12104532 0.47 SEQ_ID_12104533 0.48 SEQ_ID_12104534 0.32 SEQ_ID_12104535 0.39 SEQ_ID_12104536 0.4 SEQ_ID_12104537 0.26 SEQ_ID_12104538 0.27 SEQ_ID_12104539 0.32 SEQ_ID_12104540 0.36 SEQ_ID_12104541 0.39 SEQ_ID_12104542 0.31 SEQ_ID_12104543 0.33 SEQ_ID_12104544 0.29 SEQ_ID_12104545 0.33 SEQ_ID_12104546 0.46 SEQ_ID_12104547 0.29

Compound Relative expression UC 1.01 NC 0.94 SEQ_ID_12104548 0.43 SEQ_ID_12104549 0.54 SEQ_ID_12104550 0.46 SEQ_ID_12104551 0.4 SEQ_ID_12104552 0.54 SEQ_ID_12104553 0.46 SEQ_ID_12104554 0.45 SEQ_ID_12104555 0.39 SEQ_ID_12104556 0.37 SEQ_ID_12104557 0.37 SEQ_ID_12104558 0.46 SEQ_ID_12104559 0.44 SEQ_ID_12104560 0.44 SEQ_ID_12104561 0.44 SEQ_ID_12104562 0.54 SEQ_ID_12104563 0.4 SEQ_ID_12104564 0.43 SEQ_ID_12104565 0.42 SEQ_ID_12104566 0.42 SEQ_ID_12104567 0.44 SEQ_ID_12104568 0.67 SEQ_ID_12104569 0.38 SEQ_ID_12104570 0.43 SEQ_ID_12104571 0.33 SEQ_ID_12104572 0.39 SEQ_ID_12104573 0.37 SEQ_ID_12104574 0.33 SEQ_ID_12104575 0.28 SEQ_ID_12104576 0.28 SEQ_ID_12104577 0.37 SEQ_ID_12104578 0.3 SEQ_ID_12104579 0.33 SEQ_ID_12104580 0.34 SEQ_ID_12104581 0.3 SEQ_ID_12104582 0.3 SEQ_ID_12104583 0.24 SEQ_ID_12104584 0.26 SEQ_ID_12104585 0.4 SEQ_ID_12104586 0.42 SEQ_ID_12104587 0.38 SEQ_ID_12104588 0.38 SEQ_ID_12104589 0.36 SEQ_ID_12104590 0.46 SEQ_ID_12104591 0.52

Compound Relative expression NA 1 NC 0.95 SEQ_ID_12104592 0.42 SEQ_ID_12104593 0.43 SEQ_ID_12104594 0.27 SEQ_ID_12104595 0.43 SEQ_ID_12104596 0.56 SEQ_ID_12104597 0.33 SEQ_ID_12104598 0.38 SEQ_ID_12104599 0.46 SEQ_ID_12104600 0.61 SEQ_ID_12104601 0.4 SEQ_ID_12104602 0.51 SEQ_ID_12104603 0.42 SEQ_ID_12104604 0.38 SEQ_ID_12104605 0.43 SEQ_ID_12104606 0.36 SEQ_ID_12104607 0.53 SEQ_ID_12104608 0.55 SEQ_ID_12104609 0.6 SEQ_ID_12104610 0.42 SEQ_ID_12104611 0.51 SEQ_ID_12104612 0.55 SEQ_ID_12104613 0.32 SEQ_ID_12104614 0.38 SEQ_ID_12104615 0.39 SEQ_ID_12104616 0.37 SEQ_ID_12104617 0.49 SEQ_ID_12104618 0.39 SEQ_ID_12104619 0.34 SEQ_ID_12104620 0.38 SEQ_ID_12104621 0.21 SEQ_ID_12104622 0.3 SEQ_ID_12104623 0.36 SEQ_ID_12104624 0.41 SEQ_ID_12104625 0.38 SEQ_ID_12104626 0.44 SEQ_ID_12104627 0.42 SEQ_ID_12104628 0.51 SEQ_ID_12104629 0.54 SEQ_ID_12104630 0.52 SEQ_ID_12104631 0.58 SEQ_ID_12104632 0.54 SEQ_ID_12104633 0.54 SEQ_ID_12104634 0.61 SEQ_ID_12104635 0.74

Compound Relative expression UC 1.01 NC 0.92 SEQ_ID_12104636 0.66 SEQ_ID_12104637 0.32 SEQ_ID_12104638 0.3 SEQ_ID_12104639 0.29 SEQ_ID_12104640 0.4 SEQ_ID_12104641 0.51 SEQ_ID_12104642 0.48 SEQ_ID_12104643 0.43 SEQ_ID_12104644 0.41 SEQ_ID_12104645 0.42 SEQ_ID_12104646 0.34 SEQ_ID_12104647 0.34 SEQ_ID_12104648 0.31 SEQ_ID_12104649 0.35 SEQ_ID_12104650 0.45 SEQ_ID_12104651 0.55 SEQ_ID_12104652 0.54 SEQ_ID_12104653 0.5 SEQ_ID_12104654 0.58 SEQ_ID_12104655 0.38 SEQ_ID_12104656 0.43 SEQ_ID_12104657 0.41 SEQ_ID_12104658 0.44 SEQ_ID_12104659 0.53 SEQ_ID_12104660 0.35 SEQ_ID_12104661 0.38 SEQ_ID_12104662 0.54 SEQ_ID_12104663 0.57 SEQ_ID_12104664 0.46 SEQ_ID_12104665 0.46 SEQ_ID_12104666 0.34 SEQ_ID_12104667 0.66 SEQ_ID_12104668 0.26 SEQ_ID_12104669 0.46 SEQ_ID_12104670 0.39 SEQ_ID_12104671 0.23 SEQ_ID_12104672 0.26 SEQ_ID_12104673 0.23 SEQ_ID_12104674 0.41 SEQ_ID_12104675 0.36 SEQ_ID_12104676 0.32 SEQ_ID_12104677 0.37 SEQ_ID_12104678 0.27 SEQ_ID_12104679 0.44

Compound Relative expression UC 1 NC 0.93 SEQ_ID_12104680 0.69 SEQ_ID_12104681 0.54 SEQ_ID_12104682 0.49 SEQ_ID_12104683 0.6 SEQ_ID_12104684 0.55 SEQ_ID_12104685 0.55 SEQ_ID_12104686 0.64 SEQ_ID_12104687 0.57 SEQ_ID_12104688 0.77 SEQ_ID_12104689 0.77 SEQ_ID_12104690 0.45 SEQ_ID_12104691 0.56 SEQ_ID_12104692 0.63 SEQ_ID_12104693 0.66 SEQ_ID_12104694 0.57 SEQ_ID_12104695 0.79 SEQ_ID_12104696 0.54 SEQ_ID_12104697 0.64 SEQ_ID_12104698 0.51 SEQ_ID_12104699 1 SEQ_ID_12104700 0.55 SEQ_ID_12104701 0.51 SEQ_ID_12104702 0.64 SEQ_ID_12104703 0.67 SEQ_ID_12104704 0.51 SEQ_ID_12104705 0.46 SEQ_ID_12104706 0.26 SEQ_ID_12104707 0.3 SEQ_ID_12104708 0.34

Example 2: Experimental study of the effect of 5-10-5 MOE gapmer modified oligonucleotides on human GAP43 RNA in vitro (mixed culture, single dose)

GAP43 RNA expression levels were tested in SK-N-AS (neuroblastoma) cell line by treating modified oligonucleotides complementary to human GAP43 nucleic acid.

The modified oligonucleotide in the table below is a 5-10-5 MOE gapmer having mixed internucleoside linkages. The gapmer is 20 nucleosides in length, wherein the central cap segment is composed of 10 2’-deoxynucleotides and the 3’ and 5’ wings are composed of 5 2’-MOE nucleotides each. The motif for the gapmer is (from 5’ to 3’ direction): eeeeeddddddddddddeeeee; where ‘d’ represents a 2’-deoxyribosyl sugar moiety and ‘e’ represents a 2’-MOE sugar moiety. The internucleoside linkage for the gapmer is a phosphothioester bond.

SK-N-AS cell culture medium cultured at a density of 20,000 cells/well was treated with 5 μM of the modified oligonucleotide. Total RNA was extracted from the cells after 24 hours of treatment. The RNA expression level of the GAP43 gene relative to GAPDH, one of the cell maintenance genes, was measured by quantitative PCR. The RNA level was measured using a human GAPDH primer probe set (forward sequence GACTTCAACAGCGACAC (SEQ ID NO: 216); reverse sequence CGTTGTCATACCAGGAAAT (SEQ ID NO: 217)) and a human GAP43 primer probe set (forward sequence AGCTGAAGAGAACATAGAAGCTGTA (SEQ ID NO: 218); reverse sequence TGGGGATGTGGAAAGCCATTT (SEQ ID NO: 219)).

The reduction of GAP43 RNA was calculated as the ratio of GAP43 RNA level to untreated control (UC) cells (Aver). The level of reduction in GAP43 RNA expression due to modified oligonucleotide treatment is presented in the table and figure below. The average Ct value of GAPDH (GAPHD) of each well when treated with modified oligonucleotides was plotted to predict cytotoxicity. The experiment was performed in triplicate only for experimental batches in which the negative control (ACTACACCTTACTTATCACA) presented the expected level of GAP43 RNA expression level.

Compound Relative expression UC 1.00 NC 1.10 SEQ_ID_12104513 0.21 SEQ_ID_12104514 0.31 SEQ_ID_12104516 0.67 SEQ_ID_12104517 0.63 SEQ_ID_12104518 0.53 SEQ_ID_12104519 0.39 SEQ_ID_12104520 0.35 SEQ_ID_12104521 0.42 SEQ_ID_12104522 0.25 SEQ_ID_12104523 0.29 SEQ_ID_12104524 0.35 SEQ_ID_12104525 0.41 SEQ_ID_12104526 0.22 SEQ_ID_12104527 0.26 SEQ_ID_12104528 0.40 SEQ_ID_12104529 0.31 SEQ_ID_12104530 0.36 SEQ_ID_12104534 0.94 SEQ_ID_12104535 0.88 SEQ_ID_12104536 0.82 SEQ_ID_12104537 0.30 SEQ_ID_12104538 0.19 SEQ_ID_12104539 0.20 SEQ_ID_12104540 0.37 SEQ_ID_12104541 0.44 SEQ_ID_12104542 0.52 SEQ_ID_12104545 0.32 SEQ_ID_12104547 0.46 SEQ_ID_12104555 0.51 SEQ_ID_12104556 0.63 SEQ_ID_12104557 0.69 SEQ_ID_12104569 0.49 SEQ_ID_12104571 0.45 SEQ_ID_12104572 0.27 SEQ_ID_12104573 0.32 SEQ_ID_12104574 0.32 SEQ_ID_12104575 0.31 SEQ_ID_12104576 0.38 SEQ_ID_12104577 0.45 SEQ_ID_12104578 0.47 SEQ_ID_12104579 0.59 SEQ_ID_12104580 0.69 SEQ_ID_12104581 0.48 SEQ_ID_12104582 0.35 SEQ_ID_12104583 0.45 SEQ_ID_12104584 0.61 SEQ_ID_12104587 0.35 SEQ_ID_12104588 0.35 SEQ_ID_12104589 0.54 SEQ_ID_12104594 0.88 SEQ_ID_12104597 0.83 SEQ_ID_12104598 0.93 SEQ_ID_12104601 0.51 SEQ_ID_12104604 0.80 SEQ_ID_12104606 0.65 SEQ_ID_12104613 0.69 SEQ_ID_12104614 0.91 SEQ_ID_12104615 0.61 SEQ_ID_12104616 0.74 SEQ_ID_12104618 0.74 SEQ_ID_12104619 0.64 SEQ_ID_12104620 0.45 SEQ_ID_12104621 0.18 SEQ_ID_12104622 0.40 SEQ_ID_12104623 0.47 SEQ_ID_12104625 0.54 SEQ_ID_12104637 0.43 SEQ_ID_12104638 0.44 SEQ_ID_12104639 0.50 SEQ_ID_12104640 0.70 SEQ_ID_12104646 0.64 SEQ_ID_12104647 0.64 SEQ_ID_12104648 0.90 SEQ_ID_12104649 0.90 SEQ_ID_12104655 0.78 SEQ_ID_12104660 0.85 SEQ_ID_12104661 0.71 SEQ_ID_12104666 0.75 SEQ_ID_12104668 0.10 SEQ_ID_12104670 0.61 SEQ_ID_12104671 0.15 SEQ_ID_12104672 0.14 SEQ_ID_12104673 0.15 SEQ_ID_12104675 0.41 SEQ_ID_12104676 0.54 SEQ_ID_12104677 0.39 SEQ_ID_12104678 0.31 SEQ_ID_12104706 0.32 SEQ_ID_12104707 0.49 SEQ_ID_12104708 0.55

Example 3: Effect of 5-10-5 MOE gapmer modified oligonucleotides on human GAP43 RNA in vitro, mixed culture, multiple doses

Modified oligonucleotides selected from the above examples were tested at various doses in SK-N-AS (neuroblastoma cell line).

The modified oligonucleotides in the table below are 5-105 MOE gapmers having mixed internucleoside linkages. The gapmer is 20 nucleosides in length, wherein the central cap segment is composed of 10 2’-deoxynucleotides and the 3’ and 5’ wings are each composed of 5 2’-MOE nucleotides. The motif for the gapmer is (from 5’ to 3’): eeeeeddddddddddeeeee; where ‘d’ represents a 2’-deoxyribosyl sugar moiety and ‘e’ represents a 2’-MOE sugar moiety. The internucleoside linkage for the gapmer is a phosphotioester bond.

Cells were cultured at a density of 20,000 cells/well in a 96-well plate, and oligonucleotides were mixed with the culture medium at six different concentrations ranging from 0 to 10 μM.

After 24 hours of treatment, total RNA was extracted from the cells. The RNA expression level of the GAP43 gene relative to GAPDH, one of the cell maintenance genes, was measured by quantitative PCR. The probes for each gene were the same as those used in the above example.

The reduction of GAP43 RNA was calculated as the ratio of GAP43 RNA level to that of untreated control (UC) cells (Relative Expression of GAP43 mRNA). The half maximal inhibitory concentration (IC50) of each modified oligonucleotide is also presented. The average Ct value of GAPDH (GAPDH) in each well when treated with various concentrations of each modified oligonucleotide in order to predict cytotoxicity is presented in Table 10. The experiments were performed in triplicate only for experimental batches in which the negative control (NC) presented the expected level of GAP43 RNA expression.

Sample IC ₅₀ (nM) SEQ_ID_12104513 632.6 SEQ_ID_12104514 638.2 SEQ_ID_12104522 562.2 SEQ_ID_12104526 880.2 SEQ_ID_12104527 991.8 SEQ_ID_12104537 632.6 SEQ_ID_12104538 654 SEQ_ID_12104539 1138 SEQ_ID_12104620 2199 SEQ_ID_12104621 1616 SEQ_ID_12104668 368.8 SEQ_ID_12104671 631.8 SEQ_ID_12104672 631 SEQ_ID_12104673 1092

Example 4: Tolerability of modified oligonucleotides complementary to human GAP43 in (FOB) wild-type mice

The modified oligonucleotides described above were tested in wild-type male C57BL/6 mice to evaluate the tolerability of the oligonucleotides. Each wild-type male C57BL/6 mouse was given a single ICV dose of 300 μg of the modified oligonucleotides listed in the table below.

The modified oligonucleotide in the table below is a 5-10-5 MOE gapmer having mixed internucleoside linkages. This gapmer is a 20-nucleoside gapmer, wherein the central cap segment is composed of 10 2’-deoxynucleotides, all cytosine residues are 5-methylcytosines (5-mC), and the 3’ and 5’ wings are each composed of 5 2’-MOE nucleotides. The motif for the gapmer is (in the 5’ to 3’ direction): eeeeeddddddddddeeeee; where ‘d’ represents a 2’-deoxyribosyl sugar moiety and ‘e’ represents a 2’-MOE sugar moiety. The internucleoside linkage for the gapmer is a phosphothioester bond.

Each treatment group consisted of three mice. Artificial cerebrospinal fluid was provided as a negative control for the drug treatment group. The recovery of the mice administered the drug was checked at 30 minutes, 180 minutes, and 360 minutes after administration, respectively. Mice that recovered within 30 minutes are indicated as *** in the table below, mice that recovered within 180 minutes are indicated as **, and mice that recovered after more than 180 minutes are indicated as *, respectively. The average scores for the scores are presented in the table below. Artificial cerebrospinal fluid was used as a control in the above experiments. The compounds described in the table below have sequences substantially identical to those described in Tables 4 to 8, but have different chemical modifications and thus have different Compound IDs.

Compound (sample) Sequence Average recovery time Control NA *** SEQ_ID_12119207 GGCCTTATGAGCTTTATCTT * SEQ_ID_12119213 CCACGGAAGCTAGCCTGAAT * SEQ_ID_12119214 TCCACGGAAGCTAGCCTGAA * SEQ_ID_12119215 ACGCACCAGATCAAATAACT *** SEQ_ID_12119216 GAACGGAACATTGCACACAC ** SEQ_ID_12119217 AGCCACACTGTTTGACTTGG * SEQ_ID_12119218 CACGCACCAGATCAAATAAC *** SEQ_ID_12119219 GGAACATTGCACACACACTT * SEQ_ID_12119220 CGGAACATTGCACACACACT * SEQ_ID_12119221 ACGGAACATTGCACACACAC * SEQ_ID_12119222 GCCACACGCACCAGATCAAA *

Example 5: Testing the effect of locked nucleic acid modified oligonucleotides on human GAP43 RNA in vitro (single dose)

Changes in GAP43 RNA levels were tested by treating SK-N-AS (neuroblastoma) cell line with locked nucleic acid oligonucleotides complementary to human GAP43 nucleic acid.

The locked nucleic acid oligonucleotides in the table below are 5-9-6 gapmers having mixed internucleoside linkages. The gapmers are 20 nucleosides in length, wherein the central gap segment is nine 2’-deoxynucleotides and the 3’ and 5’ wings are composed of 2’-deoxynucleotides or locked nucleic acid sugar moieties (LNA). The motifs for the gapmers are listed in the table below in the 5’ to 3’ direction, where ‘d’ represents a 2’-deoxyribosyl sugar moiety and ‘k’ represents a 2’-4’ locked nucleic acid sugar moiety. The internucleoside linkages for the gapmers are phosphothioester bonds. When all cytosine residues in the oligonucleotide are 5'-methylcytosine, it is indicated as 5mC (+), and when only the cytosine residues in the wing of the gapmer are 5'-methylcytosine, it is indicated as 5mC (-).

Compound (sample) Sugar moiety
(5'->3') 5mC Sequence SEQ_ID_12119223 kdkdkddddddddddddkkkddk - ACGCACCAGATCAAATAACT SEQ_ID_12119224 kdkdkddddddddddkkdddk - ACGCACCAGATCAAATAACT SEQ_ID_12119225 kdkdkddddddddddddkdkddk - ACGCACCAGATCAAATAACT SEQ_ID_12119226 kdkdkddddddddddddddk - ACGCACCAGATCAAATAACT SEQ_ID_12119227 k(d)kdkddddddddddddkdkddk - ACGCACCAGATCAAATAACT SEQ_ID_12119228 kdkdkddddddddddddkkddk - ACGCACCAGATCAAATAACT SEQ_ID_12119229 kdkdkddddddddddddkddk - ACGCACCAGATCAAATAACT SEQ_ID_12119230 kdkdkddddddddddddddkddk - ACGCACCAGATCAAATAACT SEQ_ID_12119231 kdkdkddddddddddddddk - ACGCACCAGATCAAATAACT SEQ_ID_12119232 k(d)kdkddddddddddddddk - ACGCACCAGATCAAATAACT SEQ_ID_12119233 kdddkddddddddddkkkddk - ACGCACCAGATCAAATAACT SEQ_ID_12119234 kdddkddddddddddkkdddk - ACGCACCAGATCAAATAACT SEQ_ID_12119235 kdddkddddddddddddkdkddk - ACGCACCAGATCAAATAACT SEQ_ID_12119236 kdddkddddddddddddddk - ACGCACCAGATCAAATAACT SEQ_ID_12119237 kdddkddddddddddkkdddk - ACGCACCAGATCAAATAACT SEQ_ID_12119238 kdddkddddddddddddkkddk - ACGCACCAGATCAAATAACT SEQ_ID_12119239 kdddkddddddddddddkddk - ACGCACCAGATCAAATAACT SEQ_ID_12119240 kdddkddddddddddddddkddk - ACGCACCAGATCAAATAACT SEQ_ID_12119241 kdddkddddddddddddddk - ACGCACCAGATCAAATAACT SEQ_ID_12119256 kdkdkddddddddddddkkddk + ACGCACCAGATCAAATAACT SEQ_ID_12119257 kdkdkddddddddddddkddk + ACGCACCAGATCAAATAACT SEQ_ID_12119258 kdkdkddddddddddddddkddk + ACGCACCAGATCAAATAACT SEQ_ID_12119259 kdkdkddddddddddddddk + ACGCACCAGATCAAATAACT SEQ_ID_12119260 k(d)kdkddddddddddddddk + ACGCACCAGATCAAATAACT SEQ_ID_12119261 kdddkddddddddddddkkddk + ACGCACCAGATCAAATAACT SEQ_ID_12119262 kdddkddddddddddddkddk + ACGCACCAGATCAAATAACT SEQ_ID_12119263 kdddkddddddddddddddkddk + ACGCACCAGATCAAATAACT SEQ_ID_12119264 kdddkddddddddddddddk + ACGCACCAGATCAAATAACT

In substantially the same manner as in Example 2, 5 μM of the modified oligonucleotide prepared above was treated, and after 24 hours of treatment, total RNA was extracted from the cells and the RNA expression level was measured.

The reduction in GAP43 RNA was calculated as the ratio of GAP43 RNA levels to untreated control (UC) cells. Oligonucleotide treatment experiments were performed in triplicate, independent experiments.

Compound SK-N-AS
GAP43 RNA expression ratio SEQ_ID_12119223 0.27 SEQ_ID_12119224 0.23 SEQ_ID_12119225 0.25 SEQ_ID_12119226 0.35 SEQ_ID_12119227 0.44 SEQ_ID_12119228 0.16 SEQ_ID_12119229 0.14 SEQ_ID_12119230 0.15 SEQ_ID_12119231 0.23 SEQ_ID_12119232 0.30 SEQ_ID_12119233 0.26 SEQ_ID_12119234 0.29 SEQ_ID_12119235 0.24 SEQ_ID_12119236 0.41 SEQ_ID_12119237 0.51 SEQ_ID_12119238 0.18 SEQ_ID_12119239 0.20 SEQ_ID_12119240 0.21 SEQ_ID_12119241 0.22

Compared with the results according to Table 9 of Example 2 above, most of the locked nucleic acid modified oligonucleotides showed an increased efficiency in reducing GAP43 RNA expression compared to the corresponding 2'-MOE gapmer and the corresponding compound described in Table 8 above.

Example 6: Testing the effect of oligonucleotides with modified internucleoside linkages on human GAP43 RNA in vitro (single dose)

Oligonucleotides complementary to human GAP43 nucleic acid were treated in SK-N-AS (neuroblastoma) cell line to examine changes in GAP43 RNA levels.

The oligonucleotides of the table below comprise 5-10-5, 6-8-4, 4-8-6, or 5-8-5 gapmers having mixed internucleoside linkages. These gapmers are 20 or 18 nucleosides in length, wherein the central cap segment is a 2'-deoxynucleotide, and the 3' and 5' wings are each composed of 2'-MOE nucleotides. The motifs for the gapmers are (from 5' to 3'): as shown in the table below. In the table below, ‘d’ represents a 2'-deoxyribosyl sugar moiety, ‘e’ represents a 2'-MOE sugar moiety, and ‘f’ represents a 2'-OME sugar moiety. The internucleoside linkages for the gapmers are a mixmer of phosphothioester bonds and phosphoester bonds. The linkage motifs are as shown in the table below; In the formula, ‘s’ represents a phosphothioester bond and ‘o’ represents a phosphoester bond. When all cytosine residues are 5’-methylcytosine, it is represented as 5mC (+), and when only the cytosine residues in the wing of the gapmer are 5’-methylcytosine, it is represented as 5mC (-).

Compound Sugar moiety
(5'→ 3') Inter-nucleoside linkage (5’→ 3’) 5mC Sequence SEQ_ID_12119242 eeeeeddddddddddeeeee sosossssssssssssosos - AGCCACACTGTTTGACTTGG SEQ_ID_12119243 eeeeedfdddddddddeeeee sosossssssssssssosos - AGCCACACTGTTTGACTTGG SEQ_ID_12119244 eeeeeddddddddddeeeee sosossssssssssssosos - GGAACATTGCACACACACTT SEQ_ID_12119245 eeeeedfdddddddddeeeee sosossssssssssssosos - GGAACATTGCACACACACTT SEQ_ID_12119246 eeeeeddddddddddeeeee sosossssssssssssosos - CGGAACATTGCACACACACT SEQ_ID_12119247 eeeeedfdddddddddeeeee sosossssssssssssosos - CGGAACATTGCACACACACT SEQ_ID_12119248 eeeeeddddddddddeeeee soooosssssssssssooss - AGCCACACTGTTTGACTTGG SEQ_ID_12119249 eeeeeddddddddddeeeee sooosssssssssssssooss - AGCCACACTGTTTGACTTGG SEQ_ID_12119250 eeeeeddddddddddeeeee soossssssssssssssooss - AGCCACACTGTTTGACTTGG SEQ_ID_12119251 eeeeeddddddddddeeeee sosssssssssssssssooss - AGCCACACTGTTTGACTTGG SEQ_ID_12119252 eeeeeddddddddddeeeee soooosssssssssssooss - GGAACATTGCACACACACTT SEQ_ID_12119253 eeeeeddddddddddeeeee sooosssssssssssssooss - GGAACATTGCACACACACTT SEQ_ID_12119254 eeeeeddddddddddeeeee soossssssssssssssooss - GGAACATTGCACACACACTT SEQ_ID_12119255 eeeeeddddddddddeeeee sosssssssssssssssooss - GGAACATTGCACACACACTT SEQ_ID_12119265 eeeeeddddddddddeeeee sosossssssssssssosos + AGCCACACTGTTTGACTTGG SEQ_ID_12119266 eeeeedfdddddddddeeeee sosossssssssssssosos + AGCCACACTGTTTGACTTGG SEQ_ID_12119267 eeeeeddddddddddeeeee sosossssssssssssosos + GGAACATTGCACACACACTT SEQ_ID_12119268 eeeeeddddddddddeeeee sosossssssssssssosos + CGGAACATTGCACACACACT SEQ_ID_12119269 eeeeeddddddddddeeeee soooosssssssssssooss + AGCCACACTGTTTGACTTGG SEQ_ID_12119270 eeeeeddddddddddeeeee sooosssssssssssssooss + AGCCACACTGTTTGACTTGG SEQ_ID_12119271 eeeeeddddddddddeeeee soossssssssssssssooss + AGCCACACTGTTTGACTTGG SEQ_ID_12119272 eeeeeddddddddddeeeee sosssssssssssssssooss + AGCCACACTGTTTGACTTGG SEQ_ID_12119273 eeeeeddddddddddeeeee soooosssssssssssooss + GGAACATTGCACACACACTT SEQ_ID_12119274 eeeeeddddddddddeeeee sooosssssssssssssooss + GGAACATTGCACACACACTT SEQ_ID_12119275 eeeeeddddddddddeeeee soossssssssssssssooss + GGAACATTGCACACACACTT SEQ_ID_12119276 eeeeeddddddddddeeeee sosssssssssssssssooss + GGAACATTGCACACACACTT SEQ_ID_12119277 eeeeeddddddddddeeeee sooosssssssssssssooos - AGCCACACTGTTTGACTTGG SEQ_ID_12119278 eeeeddddddddddeeee sooossssssssssooos - GCCACACTGTTTGACTTG SEQ_ID_12119279 eeeeddddddddddeeee sosssssssssssssoss - CCACACTGTTTGACTTGG SEQ_ID_12119280 eeeeeddddddddddeeeee sooosssssssssssssooos - AGCCACACTGTTTGACTT SEQ_ID_12119281 eeeeddddddddddeeee sooossssssssssooos - GCCACACTGTTTGACTTG SEQ_ID_12119282 eeeeddddddddddeeee sosssssssssssssoss - CCACACTGTTTGACTTGG SEQ_ID_12119283 eeeeeddddddddddeeeee sooosssssssssssssooos - AGCCACACTGTTTGACTT SEQ_ID_12119284 eeeeddddddddddeeee sooossssssssssooos - GCCACACTGTTTGACTTG SEQ_ID_12119285 eeeeddddddddddeeee sosssssssssssssoss - CCACACTGTTTGACTTGG SEQ_ID_12119286 eeeeeddddddddddeeeee soooosssssssssssooss - AGCCACACTGTTTGACTT SEQ_ID_12119287 eeeeeddddddddddeeeee sooosssssssssssssooss - GGAACATTGCACACACACTT SEQ_ID_12119288 eeeeeddddddddddeeeee soossssssssssssssooss - GAACATTGCACACACACT SEQ_ID_12119289 eeeeeddddddddddeeeee sosssssssssssssssooss - AACATTGCACACACACTT SEQ_ID_12119290 eeeeeeddddddddeeee sooossssssssssooos - GGAACATTGCACACACAC SEQ_ID_12119291 eeeeeddddddddeeeee sosssssssssssssoss - GAACATTGCACACACACT SEQ_ID_12119292 eeeeddddddddeeeeee sosssssssssssssoss - AACATTGCACACACACTT SEQ_ID_12119293 eeeeeeddddddddeeee sosssssssssssssoss - GGAACATTGCACACACAC SEQ_ID_12119294 eeeeeddddddddeeeee sooossssssssssooss - GAACATTGCACACACACT SEQ_ID_12119295 eeeeddddddddeeeeee sooossssssssssooss - AACATTGCACACACACTT SEQ_ID_12119296 eeeeeeddddddddeeee sooossssssssssooss - GGAACATTGCACACACAC SEQ_ID_12119297 eeeeeddddddddddeeeee sooosssssssssssssooos - CGGAACATTGCACACACACT SEQ_ID_12119298 eeeeeddddddddeeeee sooossssssssssooos - GGAACATTGCACACACAC SEQ_ID_12119299 eeeeddddddddeeeeee sooossssssssssooos - GAACATTGCACACACACT SEQ_ID_12119300 eeeeeeddddddddeeee sooossssssssssooos - CGGAACATTGCACACACA SEQ_ID_12119301 eeeeeddddddddeeeee sosssssssssssssoss - GGAACATTGCACACACAC SEQ_ID_12119302 eeeeddddddddeeeeee sosssssssssssssoss - GAACATTGCACACACACT SEQ_ID_12119303 eeeeeeddddddddeeee sosssssssssssssoss - CGGAACATTGCACACACA SEQ_ID_12119304 eeeeeddddddddeeeee sooossssssssssooss - GGAACATTGCACACACAC SEQ_ID_12119305 eeeeddddddddeeeeee sooossssssssssooss - GAACATTGCACACACACT SEQ_ID_12119306 eeeeeeddddddddeeee sooossssssssssooss - CGGAACATTGCACACACA SEQ_ID_12119307 eeeeeddddddddddeeeee soooosssssssssssooss - CGGAACATTGCACACACACT SEQ_ID_12119308 eeeeeddddddddddeeeee sooosssssssssssssooss - CGGAACATTGCACACACACT SEQ_ID_12119309 eeeeeddddddddddeeeee soossssssssssssssooss - CGGAACATTGCACACACACT SEQ_ID_12119310 eeeeeddddddddddeeeee sosssssssssssssssooss - CGGAACATTGCACACACACT SEQ_ID_12119311 eeeeeddddddddddeeeee sooosssssssssssssooos + AGCCACACTGTTTGACTTGG SEQ_ID_12119312 eeeeeddddddddeeeee sooossssssssssooos + GCCACACTGTTTGACTTG SEQ_ID_12119313 eeeeddddddddeeeeee sooossssssssssooos + CCACACTGTTTGACTTGG SEQ_ID_12119314 eeeeeeddddddddeeee sooossssssssssooos + AGCCACACTGTTTGACTT SEQ_ID_12119315 eeeeeddddddddeeeee sosssssssssssssoss + GCCACACTGTTTGACTTG SEQ_ID_12119316 eeeeddddddddeeeeee sosssssssssssssoss + CCACACTGTTTGACTTGG SEQ_ID_12119317 eeeeeeddddddddeeee sosssssssssssssoss + AGCCACACTGTTTGACTT SEQ_ID_12119318 eeeeeddddddddeeeee sooossssssssssooss + GCCACACTGTTTGACTTG SEQ_ID_12119319 eeeeddddddddeeeeee sooossssssssssooss + CCACACTGTTTGACTTGG SEQ_ID_12119320 eeeeeeddddddddeeee sooossssssssssooss + AGCCACACTGTTTGACTT SEQ_ID_12119321 eeeeeddddddddeeeee sooossssssssssooos + GGAACATTGCACACACACTT SEQ_ID_12119322 eeeeeddddddddeeeee sooossssssssssooos + GAACATTGCACACACACT SEQ_ID_12119323 eeeeddddddddeeeeee sooossssssssssooos + AACATTGCACACACACTT SEQ_ID_12119324 eeeeeeddddddddeeee sooossssssssssooos + GGAACATTGCACACACAC SEQ_ID_12119325 eeeeeddddddddeeeee sosssssssssssssoss + GAACATTGCACACACACT SEQ_ID_12119326 eeeeddddddddeeeeee sosssssssssssssoss + AACATTGCACACACACTT SEQ_ID_12119327 eeeeeeddddddddeeee sosssssssssssssoss + GGAACATTGCACACACAC SEQ_ID_12119328 eeeeeddddddddeeeee sooossssssssssooss + GAACATTGCACACACACT SEQ_ID_12119329 eeeeddddddddeeeeee sooossssssssssooss + AACATTGCACACACACTT SEQ_ID_12119330 eeeeeeddddddddeeee sooossssssssssooss + GGAACATTGCACACACAC SEQ_ID_12119331 eeeeeddddddddddeeeee sooosssssssssssssooos + CGGAACATTGCACACACACT SEQ_ID_12119332 eeeeeddddddddeeeee sooossssssssssooos + GGAACATTGCACACACAC SEQ_ID_12119333 eeeeddddddddeeeeee sooossssssssssooos + GAACATTGCACACACACT SEQ_ID_12119334 eeeeeeddddddddeeee sooossssssssssooos + CGGAACATTGCACACACA SEQ_ID_12119335 eeeeeddddddddeeeee sosssssssssssssoss + GGAACATTGCACACACAC SEQ_ID_12119336 eeeeddddddddeeeeee sosssssssssssssoss + GAACATTGCACACACACT SEQ_ID_12119337 eeeeeeddddddddeeee sosssssssssssssoss + CGGAACATTGCACACACA SEQ_ID_12119338 eeeeeddddddddeeeee sooossssssssssooss + GGAACATTGCACACACAC SEQ_ID_12119339 eeeeddddddddeeeeee sooossssssssssooss + GAACATTGCACACACACT SEQ_ID_12119340 eeeeeeddddddddeeee sooossssssssssooss + CGGAACATTGCACACACA SEQ_ID_12119341 eeeeeddddddddddeeeee soooosssssssssssooss + CGGAACATTGCACACACACT SEQ_ID_12119342 eeeeeddddddddddeeeee sooosssssssssssssooss + CGGAACATTGCACACACACT SEQ_ID_12119343 eeeeeddddddddddeeeee soossssssssssssssooss + CGGAACATTGCACACACACT SEQ_ID_12119344 eeeeeddddddddddeeeee sosssssssssssssssooss + CGGAACATTGCACACACACT

In substantially the same manner as in Example 2, 5 μM of the modified oligonucleotide prepared above was treated, and after 24 hours of treatment, total RNA was extracted from the cells and the RNA expression level was measured.

The decrease in GAP43 RNA levels was calculated as the ratio of GAP43 RNA levels to untreated control (UC) cells.

Compound SK-N-AS
GAP43 RNA expression ratio SEQ_ID_12119242 0.09 SEQ_ID_12119243 0.12 SEQ_ID_12119244 0.29 SEQ_ID_12119245 0.41 SEQ_ID_12119246 0.25 SEQ_ID_12119247 0.45 SEQ_ID_12119248 0.28 SEQ_ID_12119249 0.21 SEQ_ID_12119250 0.19 SEQ_ID_12119251 0.15 SEQ_ID_12119252 0.43 SEQ_ID_12119253 0.39 SEQ_ID_12119254 0.34 SEQ_ID_12119255 0.20 SEQ_ID_12119277 0.31 SEQ_ID_12119278 0.59 SEQ_ID_12119279 0.64 SEQ_ID_12119280 0.87 SEQ_ID_12119281 0.29 SEQ_ID_12119282 0.45 SEQ_ID_12119283 0.55 SEQ_ID_12119284 0.34 SEQ_ID_12119285 0.44 SEQ_ID_12119286 0.62 SEQ_ID_12119287 0.49 SEQ_ID_12119288 0.76 SEQ_ID_12119289 1.05 SEQ_ID_12119290 0.66 SEQ_ID_12119291 0.97 SEQ_ID_12119292 1.05 SEQ_ID_12119293 0.58 SEQ_ID_12119294 0.94 SEQ_ID_12119295 0.82 SEQ_ID_12119296 0.53 SEQ_ID_12119297 0.38 SEQ_ID_12119298 0.60 SEQ_ID_12119299 0.86 SEQ_ID_12119300 0.59 SEQ_ID_12119301 0.55 SEQ_ID_12119302 0.69 SEQ_ID_12119303 0.63 SEQ_ID_12119304 0.90 SEQ_ID_12119305 0.91 SEQ_ID_12119306 0.61 SEQ_ID_12119307 0.51 SEQ_ID_12119308 0.54 SEQ_ID_12119309 0.41 SEQ_ID_12119310 0.41

Oligonucleotides with altered internucleoside linkages were confirmed to have a lower GAP43 RNA reduction efficiency compared to the original.

Example 7: Effect of oligonucleotides with modified locked nucleic acids or internucleoside linkages on human GAP43 RNA in vitro, mixed culture method, multiple doses

Modified oligonucleotides selected from the above examples were tested at various doses in SK-N-AS (neuroblastoma cell line).

The locked nucleic acid oligonucleotides in the table below are 5-9-6 gapmers having mixed internucleoside linkages. These gapmers are 20 nucleosides in length, wherein the central gap segment is nine 2'-deoxynucleotides, and the 3' and 5' wings are 2'-deoxynucleotides or LNA. The motifs for the gapmers are listed in the table below in the 5' to 3' direction, where ‘d’ represents a 2'-deoxyribosyl sugar moiety and ‘k’ represents a 2'-4' locked nucleic acid sugar moiety. The internucleoside linkages for the gapmers are phosphothioester bonds. When all cytosine residues are 5'-methylcytosines, it is indicated as 5mC (+), and when only the cytosine residues in the wings of the gapmer are 5'-methylcytosines, it is indicated as 5mC (-).

Compound Sugar moiety
(5'->3') 5mC Sequence SEQ_ID_12119232 k(d)kdkddddddddddddddk - ACGCACCAGATCAAATAACT SEQ_ID_12119240 kdddkddddddddddddddkddk - ACGCACCAGATCAAATAACT SEQ_ID_12119241 kdddkddddddddddddddk - ACGCACCAGATCAAATAACT SEQ_ID_12119256 kdkdkddddddddddddkkddk + ACGCACCAGATCAAATAACT SEQ_ID_12119257 kdkdkddddddddddddkddk + ACGCACCAGATCAAATAACT

The oligonucleotides of the table below comprise 5-10-5, 6-8-4, 4-8-6, or 5-8-5 gapmers having mixed internucleoside linkages. The gapmers are 20 or 18 nucleosides in length, wherein the central cap segment is a 2'-deoxynucleotide, and the 3' and 5' wings are each 2'-MOE nucleotides. The motifs for the gapmers are (from 5' to 3'): as shown in the table below. In the table below, ‘d’ represents a 2'-deoxyribosyl sugar moiety, ‘e’ represents a 2'-MOE sugar moiety, and ‘f’ represents a 2'-OME sugar moiety. The internucleoside linkages for the gapmers are a mixmer of phosphothioester bonds and phosphoester bonds. The linkage motifs are as shown in the table below; In the formula, ‘s’ represents a phosphothioester bond and ‘o’ represents a phosphoester bond. When all cytosine residues are 5’-methylcytosine, it is represented as 5mC (+), and when only the cytosine residues in the wing of the gapmer are 5’-methylcytosine, it is represented as 5mC (-).

Compound Sugar moiety
(5'->3') Linkage moiety
(5'->3') 5mC Sequence SEQ_ID_12119246 eeeeeddddddddddeeeee sosossssssssssssosos - CGGAACATTGCACACACACT SEQ_ID_12119269 eeeeeddddddddddeeeee soooosssssssssssooss + AGCCACACTGTTTGACTTGG SEQ_ID_12119273 eeeeeddddddddddeeeee soooosssssssssssooss + GGAACATTGCACACACACTT SEQ_ID_12119274 eeeeeddddddddddeeeee sooosssssssssssssooss + GGAACATTGCACACACACTT SEQ_ID_12119275 eeeeeddddddddddeeeee soossssssssssssssooss + GGAACATTGCACACACACTT SEQ_ID_12119277 eeeeeddddddddddeeeee sooosssssssssssssooos - AGCCACACTGTTTGACTTGG SEQ_ID_12119281 eeeeeddddddddeeeee sosssssssssssssoss - GCCACACTGTTTGACTTG SEQ_ID_12119282 eeeeddddddddeeeeee sosssssssssssssoss - CCACACTGTTTGACTTGG SEQ_ID_12119284 eeeeeddddddddeeeee sooossssssssssooss - GCCACACTGTTTGACTTG SEQ_ID_12119285 eeeeddddddddeeeeee sooossssssssssooss - CCACACTGTTTGACTTGG SEQ_ID_12119287 eeeeeddddddddeeeee sooossssssssssooos - GGAACATTGCACACACACTT SEQ_ID_12119297 eeeeeddddddddddeeeee sooosssssssssssssooos - CGGAACATTGCACACACACT SEQ_ID_12119309 eeeeeddddddddddeeeee soossssssssssssssooss - CGGAACATTGCACACACACT SEQ_ID_12119310 eeeeeddddddddddeeeee sosssssssssssssssooss - CGGAACATTGCACACACACT

In a manner substantially identical to Example 3, the half maximal inhibitory concentration (IC50) of each modified oligonucleotide is presented. The reduction in GAP43 RNA was calculated as the ratio of GAP43 RNA levels to untreated control (UC) cells (Relative Expression of GAP43 mRNA).

Compound IC ₅₀ SEQ_ID_12119232 2739 SEQ_ID_12119240 1241 SEQ_ID_12119241 2758 SEQ_ID_12119256 922 SEQ_ID_12119257 1343 SEQ_ID_12119246 2425 SEQ_ID_12119269 1429 SEQ_ID_12119273 3933 SEQ_ID_12119274 6339 SEQ_ID_12119275 4178 SEQ_ID_12119277 1662 SEQ_ID_12119281 2410 SEQ_ID_12119282 3217 SEQ_ID_12119284 2884 SEQ_ID_12119285 9683 SEQ_ID_12119287 9941 SEQ_ID_12119297 5673 SEQ_ID_12119309 7554 SEQ_ID_12119310 5377

Some of the oligonucleotides with modified locked nucleic acids had lower IC50 values, while most of the oligonucleotides with modified internucleoside linkages had higher IC50 values.

Example 8: Tolerance of oligonucleotides with modified locked nucleic acids or internucleoside linkages to human GAP43 RNA in wild-type mice

Each ICV dose of the locked nucleic acid or nucleoside linkage-modified oligonucleotide listed in the table below was administered to wild-type 8-week-old male C57BL/6J mice. The mice administered the drug were checked for recovery at 30, 180, and 360 minutes after administration, respectively. Mice that recovered within 30 minutes are indicated as *** in the table below, mice that recovered within 180 minutes are indicated as **, and mice that recovered over 180 minutes are indicated as *, respectively, in the table below.

Compound Average recovery time Capacity (μg) SEQ_ID_12119256 ** 300 SEQ_ID_12119257 ** 300 SEQ_ID_12119258 * 300 SEQ_ID_12119259 * 300 SEQ_ID_12119260 ** 300 SEQ_ID_12119261 * 300 SEQ_ID_12119262 * 300 SEQ_ID_12119263 * 300 SEQ_ID_12119264 * 300 SEQ_ID_12119265 * 300 SEQ_ID_12119266 * 300 SEQ_ID_12119267 * 300 SEQ_ID_12119268 ** 300 SEQ_ID_12119269 ** 300 SEQ_ID_12119270 * 300 SEQ_ID_12119271 * 300 SEQ_ID_12119272 * 300 SEQ_ID_12119273 *** 300 SEQ_ID_12119274 *** 300 SEQ_ID_12119275 ** 300 SEQ_ID_12119276 ** 300 SEQ_ID_12119331 *** 300 SEQ_ID_12119344 ** 300 SEQ_ID_12119268 * 500 SEQ_ID_12119268 * 700 SEQ_ID_12119269 *** 100 SEQ_ID_12119269 * 200 SEQ_ID_12119273 *** 500 SEQ_ID_12119273 * 700 SEQ_ID_12119274 * 500 SEQ_ID_12119274 * 700 SEQ_ID_12119275 * 500 SEQ_ID_12119275 * 700 SEQ_ID_12119331 ** 500 SEQ_ID_12119331 * 700 SEQ_ID_12119344 * 500 SEQ_ID_12119344 * 700

The maximum tolerated dose based on acute neurotoxicity of each oligonucleotide was derived by observing the time to recovery of consciousness after ICV administration at different doses. It was observed that the tolerated dose was determined differently depending on the modification of the nucleoside linkage of the oligonucleotide.

Example 9: Neuroinflammation evaluation of oligonucleotides with altered internucleoside linkages to human GAP43 RNA in wild-type mice

Each ICV dose of each oligonucleotide with modified internucleoside linkages listed in the table below was administered to wild-type 8-week-old male C57BL/6J mice. To evaluate neuroinflammation, RNA expression levels of Gfap and Iba1 were calculated compared to the aCSF group and are shown in the table below.

Compound Gfap expression rate Iba1 expression ratio Dosage (μg) Control (artificial cerebrospinal fluid) 1.01 1.00 - SEQ_ID_12119268 1.24 1.10 300 SEQ_ID_12119268 0.83 1.31 500 SEQ_ID_12119268 0.94 1.47 700 SEQ_ID_12119269 0.89 1.38 100 SEQ_ID_12119269 0.92 1.11 200 SEQ_ID_12119269 1.09 1.30 300 SEQ_ID_12119273 1.34 1.67 300 SEQ_ID_12119273 1.11 1.31 500 SEQ_ID_12119273 1.32 1.52 700 SEQ_ID_12119274 1.36 1.50 300 SEQ_ID_12119274 0.99 1.16 500 SEQ_ID_12119274 1.31 1.08 700 SEQ_ID_12119275 1.54 1.29 300 SEQ_ID_12119275 0.81 0.91 500 SEQ_ID_12119275 1.14 1.06 700

Eight weeks after the administration of the sample, the mouse brains were extracted, RNA was extracted, and the expression of Gfap and Iba1 was measured. Among the evaluated oligonucleotides, there was no sequence in which an increase in the expression of Gfap or Iba1 genes was clearly observed. This means that the possibility of inducing neuroinflammation is low.

Example 10: Efficacy Evaluation Using Patient-Derived Glioblastoma Organoids

Modified oligonucleotides complementary to human GAP43 nucleic acid were treated with glioblastoma organoids derived from patient glioblastoma tissue to test the GAP43 inhibitory effect and tumor growth inhibitory effect.

Patient-derived glioblastoma organoids were prepared and dissociated into cell units according to the method of Fadi Jacob et al., (Cell. 2020 Jan 9;180(1):188-204.e22.). The dissociated cells were seeded at 1,000 cells/well on Aggrewell 400 plates and cultured for 7 days to form glioblastoma organoid spheres with a diameter of approximately 100 μm. The formed glioblastoma organoid spheres were transferred to Aggrewell 800 at 24 cells/well for each experimental group.

Organoids were treated with 10 μM of the modified oligonucleotide SEQ_ID_12119213 for 4 weeks, and PBS was treated at the same amount for the same period as a negative control. All organoids were photographed under a microscope on days 0, 3, 7, 14, 21, and 28 of treatment with the modified oligonucleotide or PBS, and their diameters were quantified. The diameters of organoids on days 0, 3, 7, 14, 21, and 28 are shown in the table below.

Number of days elapsed (days) 0 3 7 14 21 28 PBS 98.61 97.21 103.75 103.00 116.39 125.49 SEQ_ID_12119213 98.56 101.66 101.45 98.33 91.06 90.54

On day 28, total RNA was extracted from organoids of each condition. The RNA expression level of the GAP43 gene relative to GAPDH, one of the cell maintenance genes, was measured by quantitative PCR. The GAP43 RNA expression level of the modified oligonucleotide was calculated as the ratio (relative expression) of the GAP43 RNA level to the untreated control organoid. The primers for quantitative PCR used the primers used in the above example. The expression level of GAP43 was 1.22 in the PBS treatment group and 0.14 in the modified oligonucleotide treatment group.

Attach electronic file of sequence list

Claims (25)

A compound for modulating the activity and/or expression level of GAP43, wherein the compound is a modified oligonucleotide or an RNAi agent. In claim 1, the RNAi agent is a compound that is ssRNAi, siRNA, shRNA, or microRNA. An oligomeric compound comprising a modified oligonucleotide consisting of 8 to 80 linked nucleosides, wherein the nucleic acid sequence of the modified oligonucleotide is at least 80% complementary to a portion of the same length of a GAP43 nucleic acid sequence, and wherein the modified oligonucleotide comprises at least one modification selected from the group consisting of a modified sugar moiety, a modified internucleoside linkage, and a modified base. In the first paragraph, an oligomeric compound comprising a modified oligonucleotide consisting of 8 to 80 linked nucleosides, wherein the nucleic acid sequence of the modified oligonucleotide is a nucleic acid sequence of SEQ ID NO: 1 to SEQ ID NO: 206. An oligomeric compound comprising 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases, wherein the modified oligonucleotide comprises at least one modification selected from the group consisting of a modified sugar moiety, a modified internucleoside linkage and a modified base. An oligomeric compound comprising a modified oligonucleotide consisting of 8 to 80 linked nucleosides, wherein the nucleic acid sequence of the modified oligonucleotide comprises 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases from the nucleic acid sequences of SEQ ID NO: 207 to SEQ ID NO: 212, and wherein the modified oligonucleotide comprises at least one modification selected from the group consisting of a modified sugar moiety, a modified internucleoside linkage and a modified base. An oligomeric compound, wherein the nucleic acid sequence of the modified oligonucleotide in claim 5 comprises 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases selected from the group consisting of SEQ ID NOs: 213, 214, 209, 215, 211, and 212. An oligomeric compound according to any one of claims 3 to 6, wherein the modified oligonucleotide comprises at least one modified nucleoside comprising a modified sugar moiety. In any one of claims 3 to 7, the modified sugar moiety is a non-bicyclic modified sugar moiety consisting of a 2'-MOE sugar moiety and a 2'-OMe sugar moiety; and
An oligomeric compound comprising at least one selected from the group consisting of bicyclic modified sugar moieties comprising a 2',4'-bridge selected from O-CH ₂ -; and -O-CH(CH ₃ )-. An oligomeric compound according to any one of claims 3 to 8, wherein the oligomeric compound comprises at least one modified nucleoside comprising a modified sugar moiety of the bicyclic structure. An oligomeric compound according to any one of claims 3 to 9, wherein the modified oligonucleotide comprises at least one modified nucleoside comprising a bicyclic modified sugar moiety having a 2'-4' bridge and at least one modified nucleoside comprising a non-bicyclic modified sugar moiety. An oligomeric compound according to any one of claims 3 to 10, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage. An oligomeric compound according to any one of claims 3 to 11, wherein the modified oligonucleotide comprises at least one internucleoside linkage selected from the group consisting of a phosphorothioate internucleoside linkage, a mesyl phosphoramidate internucleoside linkage, and a phosphodiester internucleoside linkage. An oligomeric compound according to any one of claims 3 to 12, wherein the modified oligonucleotide comprises a modified nucleobase. An oligomeric compound in claim 13, wherein the modified nucleobase is 5-methyl cytosine. In any one of claims 3 to 14, the modified oligonucleotide comprises a sugar motif, wherein the sugar motif is:
A 5'-region consisting of 1 to 6 linked 5'-region nucleosides;
a central region consisting of 6 to 10 linked central region nucleosides; and
3'-region consisting of 1 to 6 linked 3'-region nucleosides
An oligomeric compound comprising: An oligomeric compound in claim 15, wherein each of the central region nucleosides is a 2'-β-D-deoxynucleoside. An oligomeric compound further comprising a conjugate according to any one of claims 3 to 16. A composition for reducing or inhibiting the activity or expression level of GAP 43, comprising an oligomeric compound according to any one of claims 3 to 17. A pharmaceutical composition for preventing, improving or treating a disease or disorder associated with GAP 43, comprising an oligomeric compound according to any one of claims 3 to 17. A pharmaceutical composition in claim 19, wherein the composition further comprises a pharmaceutically acceptable carrier or diluent. A pharmaceutical composition in claim 20, wherein the pharmaceutically acceptable diluent is artificial spinal fluid (aCSF) or phosphate-buffered saline (PBS). A pharmaceutical composition according to any one of claims 18 to 21, wherein the disease or disorder associated with GAP 43 is glioma. A pharmaceutical composition in claim 22, wherein the neuroglioma is glioblastoma. In claim 23, the pharmaceutical composition wherein the glioblastoma is IDH wild type and has at least one characteristic selected from the group consisting of the following (1) to (5):
(1) Microvascular proliferation,
(2) necrosis,
(3) TERT promoter mutation
(4) EGFR hyperactivation mutations, and
(5) Gain of chromosome 7 and deletion of chromosome 10. A pharmaceutical composition according to claim 23, wherein the glioblastoma comprises at least one genetic mutation selected from the group consisting of EGFR, PDGF, INK4α, MDM2, PRb, CDK4/6, PTEN, TP53, and NF1 genes.