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CN-122012459-A - Epigenetic editing system

CN122012459ACN 122012459 ACN122012459 ACN 122012459ACN-122012459-A

Abstract

The invention belongs to the field of nucleic acid editing, in particular to the technical field of regularly clustered interval short palindromic repeat (CRISPR). In particular, the present invention utilizes Cas9 or smaller Cas-SF01 (a Cas12i3 variant) to rationally design and engineer construction of compact, mRNA delivered epigenetic inhibitors, combined with optimized mRNA structure and Lipid Nanoparticle (LNP) delivery, single intravenous injection of optimized OFF-EE-V2 mRNA and selected guide RNAs (gRNA) targeting mouse PCSK9, resulting in a reduction of circulating PCSK9 protein of about 90%, a corresponding reduction of LDL-C levels of about 55%, with an effect lasting at least 180 days. The Cas-SF01 based editor shows higher specificity than the Cas9 based counterpart with fewer off-target methylation events. The optimized LNP formulation also showed good safety. These findings establish a powerful and versatile platform for advancing in vivo therapies that implement precise and durable epigenetic silencing based on an engineered mRNA editor for transient delivery.

Inventors

  • ZHU JIANKANG
  • LIU PENGPENG
  • XU CHAO
  • ZENG CHEN
  • LIU XIAOYI

Assignees

  • 南方科技大学

Dates

Publication Date
20260512
Application Date
20260119
Priority Date
20251028

Claims (13)

  1. 1. A fusion protein is characterized by comprising a DNA methyltransferase domain, a Cas protein domain and a transcription repression domain from the N-terminal to the C-terminal in sequence; The DNA methyltransferase domain includes DNMT3A and DNMT3L; The Cas protein domain is selected from a nuclease-activity-inactivated Cas9 protein or a nuclease-activity-inactivated Cas12 protein; the transcription repression domain is KRAB.
  2. 2. The fusion protein of claim 1, further comprising a Nuclear Localization Sequence (NLS); Optionally, the fusion protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs 1-6, or an amino acid sequence having at least 95% homology with the amino acid sequences set forth in SEQ ID NOs 1-6.
  3. 3. The fusion protein of claim 1, wherein the nuclease-activity-inactivated Cas9 protein is dCas9 and the nuclease-activity-inactivated Cas12 protein is Cas12i protein.
  4. 4. A fusion protein according to claim 3, characterized in that the Cas12i protein is a Cas-SF01 protein, preferably the Cas-SF01 fusion protein comprises the amino acid sequence shown in SEQ ID No. 6.
  5. 5. An isolated nucleic acid molecule encoding the fusion protein of any one of claims 1-4.
  6. 6. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule is mRNA; Preferably, the nucleic acid molecule comprises the nucleotide sequence shown as SEQ ID NO. 136 or SEQ ID NO. 137.
  7. 7. A vector comprising the nucleic acid molecule of claim 5 or 6.
  8. 8. An epigenetic editing system, comprising: (i) The fusion protein of any one of claims 1 to 4, or the nucleic acid molecule of any one of claims 5 to 6, or the vector of claim 7, and (Ii) One or more guide RNAs (grnas) or nucleic acids encoding the same, the grnas being capable of targeting a promoter region or a region upstream of a transcription initiation site of a target gene.
  9. 9. The epigenetic editing system of claim 8, wherein the target gene is a proprotein convertase subtilisin/Kexin type 9 (PCSK 9) gene; Preferably, the gRNA comprises a targeting sequence complementary to a target sequence selected from any one of SEQ ID NOs 76-135; More preferably, the targeting sequence of the gRNA is selected from one or more of SEQ ID NOs 81, 82, 84, 88, 91, 100, 134.
  10. 10. A pharmaceutical composition, in the form of a capsule, characterized by comprising: (a) The epigenetic editing system as set forth in claim 8 or 9, and (B) A pharmaceutically acceptable carrier, preferably a Lipid Nanoparticle (LNP).
  11. 11. The pharmaceutical composition of claim 10, wherein the epigenetic editing system comprises: an mRNA encoding the fusion protein of claim 4, and PCSK 9-targeting gRNA having a targeting sequence selected from one or more of SEQ ID NOs 81, 82, 84, 88, 91, 100, 134.
  12. 12. The pharmaceutical composition of claim 10, wherein the Lipid Nanoparticle (LNP) consists of cationic lipids, helper phospholipids, cholesterol, and polyethylene glycol lipids (PEG-lipid); Preferably, the mole percentage ranges of the cationic lipid, the auxiliary phospholipid, the cholesterol and the polyethylene glycol lipid are respectively: 40-60% of cationic lipid; 5-15% of auxiliary phospholipids; 30-45% of cholesterol; polyethylene glycol lipid 1-3%.
  13. 13. Use of the fusion protein of any one of claims 1-4, the nucleic acid molecule of any one of claims 5-6, or the epigenetic editing system as set forth in any one of claims 8-9, in the manufacture of a medicament for modulating gene expression; Preferably, the medicament is for inhibiting PCSK9 gene expression in a subject for the treatment of hypercholesterolemia or cardiovascular disease.

Description

Epigenetic editing system The application claims priority from China patent application with the application number CN2025115522676 and the name of 'an epigenetic editing system', the application date is 2025, 10, 28. The present application incorporates the entirety of the above-mentioned chinese patent application. Technical Field The invention relates to the field of gene editing, in particular to the technical field of regular clustered interval short palindromic repeat (CRISPR). In particular, the present invention relates to an epigenetic editing system. Background The realization of accurate programmable gene expression regulation by targeted epigenetic editing represents a revolutionary development of novel therapeutic methods, providing the potential for achieving long-term physiological changes without altering the underlying genomic sequence. The epigenetic editor based on CRISPR can make site-specific modification to the apparent genome, providing a powerful way to permanently silence pathogenic genes or activate therapeutic genes. This innovative strategy works by inducing a heritable chemical modification of chromatin structure (such as DNA methylation or histone modification), thereby altering the gene expression pattern. However, one significant obstacle to the widespread clinical use of many current CRISPR-based epigenomic editors is their bulky size, mainly due to the multi-domain nature of these fusion proteins (Cas protein, guide RNA and one or more effector domains). Such large size loads present a significant challenge for efficient in vivo delivery using size-limited vectors (e.g., adeno-associated viral AAV). The present invention seeks to address these key limitations by delivering mRNA of an engineered epigenetic inhibitory system via Lipid Nanoparticles (LNPs). The present invention is based on two different Cas platform developments and systems optimizing the CRISPR OFF-epigenomic editor (CRISPR OFF-EE) widely used streptococcus pyogenes Cas9 (SpCas 9) and smaller Cas12i3 variants Cas-SF01. These systems involve fusing a catalytically inactive Cas protein (dSpCas or dSF 01) with potent DNA methyltransferase effector domains (DNMT 3A and DNMT 3L) and a KRAB transcriptional repression domain to promote efficient, site-specific DNA methylation, thereby inducing sustained gene silencing. By using an mRNA delivery editor, the object of the present invention is to achieve transient protein expression, thereby programming persistent epigenetic changes, while circumventing the risks posed by viral vector DNA integration or long term expression of plasmid-based systems. This approach provides a potentially safer, more flexible model for in vivo application of these powerful epigenetic tools. Disclosure of Invention Fusion proteins In one aspect, the invention provides a fusion protein comprising a DNA methyltransferase, a Cas protein, and a transcription repression domain. In one embodiment, the DNA methyltransferase comprises DNMT3A and DNMT3L, and the transcription repression domain is KRAB. In one embodiment, the Cas protein is selected from Cas9 or Cas12 proteins. In one embodiment, the Cas protein is selected from Cas12i proteins. In one embodiment, the Cas protein is a Cas protein with inactive nuclease activity. In one embodiment, the Cas9 is dCas9 (Cas 9 with inactive nuclease activity). In one embodiment, the Cas12i protein is Cas-SF01. Specifically, CN116004573B discloses a Cas protein BC26312 with mutation of amino acid, which is called as Cas-SF01 in the invention. In one embodiment, the Cas-SF01 is a nuclease-activity-inactivated Cas protein, e.g., cas protein may be obtained by mutating E at 844 th E of Cas-SF01 to a or mutating D at 619 th to a. In one embodiment, the fusion protein comprises, in order from N-terminus to C-terminus, a DNA methyltransferase, a Cas protein, and a transcription repressing domain. In one embodiment, the fusion protein comprises, in order from the N-terminus to the C-terminus, a DNMT3A, DNMT3L, cas protein and a transcription repressing domain. In one embodiment, the elements of the fusion protein can be directly linked or linked through a linker (e.g., XTEN linker). In one embodiment, the fusion protein further comprises a Nuclear Localization Sequence (NLS). In one embodiment, the fusion protein optionally comprises one or two of the amino acid sequences as set forth in SEQ ID NO. 1-SEQ ID NO. 6 and/or comprises one or two of the amino acid sequences that are at least 95% homologous to the amino acid sequences as set forth in SEQ ID NO. 1-SEQ ID NO. 6. In a preferred embodiment, the amino acid sequence of the fusion protein is shown in SEQ ID NO. 1. In another preferred embodiment, the fusion protein comprises one or both of the amino acid sequences shown in SEQ ID NO. 2 and SEQ ID NO. 3. In another preferred embodiment, the fusion protein comprises one or both of the amino acid sequences shown in SEQ ID NO. 4 and SEQ ID NO. 5. In another pref