JP-2025516294-A5 -

Dates

Publication Date

20260511

Application Date

20230501

Description

To better understand this disclosure, the following examples are provided. These examples are illustrative only and should not be construed as limiting the scope of this disclosure. The present invention includes, for example, the following embodiments: [Embodiment 1] A system for suppressing the transcription of the human PCSK9 gene in human cells, and possibly human hepatocytes, a) A domain of DNA methyltransferase (DNMT) and/or a domain that recruits DNMT, wherein the DNMT domain and/or recruiter domain may include a DNMT3A domain and/or a DNMT3L domain, and the recruited DNMT may be DNMT3A, and Transcriptional repressor domain It collectively includes, A fusion protein in which each domain is linked to a DNA-binding domain that binds to a target region in the human PCSK9 gene, or b) One or more nucleic acid molecules encoding one or more fusion proteins A system that includes this. [Embodiment 2] The system according to Embodiment 1, wherein the DNA-binding domain binds to the target sequence in SEQ ID NO: 1488 or 1489. [Embodiment 3] The system according to Embodiment 1 or 2, wherein the DNA-binding domain targets the fusion protein to one or more sequences in the PCSK9 gene selected from SEQ ID NOs. 700-747 and 1036-1261. [Embodiment 4] The system according to any one of Embodiments 1 to 3, wherein the DNA-binding domain includes a dead CRISPR Cas (dCas) domain, a ZFP domain, or a TALE domain. [Embodiment 5] The system according to Embodiment 4, wherein the DNA-binding domain comprises a dCas9 domain, and the system further comprises (i) one or more guide RNAs comprising any one of sequence numbers 1262 to 1487, or (ii) nucleic acid molecules encoding one or more guide RNAs. [Embodiment 6] The system according to Embodiment 4 or 5, wherein the dCas domain comprises a sequence having at least 90% identity with the dCas9 sequence, and optionally sequence number 12 or 13. [Embodiment 7] The system according to Embodiment 4, wherein the ZFP domain targets a nucleotide sequence selected from SEQ ID NOs. 700 to 747. [Embodiment 8] The system according to Embodiment 7, wherein the ZFP domain contains one of the F1 to F6 amino acid sequences from ZF001 to ZF048 shown in Table 1. [Embodiment 9] The system according to any one of Embodiments 1 to 8, wherein the DNMT3A domain includes a sequence having at least 90% identity with sequence number 574 or 575. [Embodiment 10] The system according to any one of Embodiments 1 to 9, wherein the DNMT3L domain includes a sequence having at least 90% identity with a sequence selected from sequence numbers 578 to 581. [Embodiment 11] The system according to any one of Embodiments 1 to 9, wherein the DNMT3L domain includes a sequence having at least 90% identity with a sequence selected from sequence numbers 582 to 603. [Embodiment 12] The system according to any one of Embodiments 1 to 8, wherein the DNMT domain includes a sequence having at least 90% identity with a sequence selected from sequence numbers 601 to 603. [Embodiment 13] The system according to any one of Embodiments 1 to 12, wherein the transcriptional repressor domain includes a sequence having at least 90% identity with a sequence selected from Sequence ID No. 33 to 570. [Embodiment 14] The system according to any one of Embodiments 1 to 12, wherein the transcriptional repressor domain includes a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627. [Embodiment 15] The system according to Embodiment 14, wherein the KRAB domain comprises a sequence having at least 90% identity with a sequence selected from sequence numbers 89, 116, 245, and 255. [Embodiment 16] The system according to any one of Embodiments 1 to 12, wherein the transcriptional repressor domain comprises a fusion of the N-terminal and C-terminal regions of ZIM3 and KOX1 KRAB, and optionally comprises the amino acid sequence of SEQ ID NO: 571 or 572. [Embodiment 17] The system according to any one of Embodiments 1 to 12, wherein the transcriptional repressor domain is derived from KAP1, MECP2, HP1a/CBX5, HP1b, CBX8, CDYL2, TOX, TOX3, TOX4, EED, EZH2, RBBP4, RCOR1, or SCML2. [Embodiment 18] The system a) comprising a DNMT3A domain, a DNMT3L domain, a transcriptional repressor domain, and a DNA-binding domain, In some cases, one or both of the DNMT3A and DNMT3L domains are human. In some cases, the DNA-binding domain may include a dead CRISPR-Cas domain or a ZFP domain in the fusion protein, or b) Nucleic acid molecules encoding a fusion protein A system according to any one of embodiments 1 to 17, including the system described above. [Embodiment 19] The system according to Embodiment 18, wherein the fusion protein comprises, from N-terminus to C-terminus, a DNMT3A domain, a first peptide linker, a DNMT3L domain, a second peptide linker, a DNA-binding domain, a third peptide linker, and a transcriptional repressor domain. [Embodiment 20] The system according to Embodiment 19, wherein the fusion protein comprises, from N-terminus to C-terminus,