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EP-4739781-A1 - SCAN-BASED FUSION PROTEINS FOR MODULATING GENE EXPRESSION AND METHODS OF USE

EP4739781A1EP 4739781 A1EP4739781 A1EP 4739781A1EP-4739781-A1

Abstract

The invention provides fusion protein comprising a SCAN domain bearing member of a zinc finger-containing protein, a fragment or variant thereof and a catalytically inactive RNA- guided endonuclease or a DNA adenine methyltransferase. Preferably, the fusion protein is for use in the treatment and/or prevention of cancer, neurodegenerative disease, neurodevelopmental disease, aging and aging-related disease.

Inventors

  • TRONO, DIDIER
  • BEGNIS, Martina

Assignees

  • ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE (EPFL)

Dates

Publication Date
20260513
Application Date
20240703

Claims (18)

  1. 1. A fusion protein comprising i) a SCAN domain bearing member of a zinc finger-containing protein (ZFP), a fragment or a biologically active variant having at least 80% nucleotide sequence identity thereto , and ii) a catalytically inactive RNA-guided endonuclease or iii) a DNA adenine methyltransferase.
  2. 2. The fusion protein according to claim 1 , wherein the catalytically inactive endonuclease is a Cas9 protein or a biologically active variant having at least 80% nucleotide sequence identity thereto.
  3. 3. The fusion protein according to claim 1 or 2, wherein the SCAN domain is able to drive nucleolar localization and/or protein dimerization of a transcription factor.
  4. 4. The fusion protein according to any one of claims 1 to 3, wherein the SCAN domain is selected from ZNF274 or the group comprising any other SCAN-containing proteins capable of interacting with the SCAN domain of ZNF274 or whose perinucleolar tethering depends on ZNF274.
  5. 5. The fusion protein according to claim 4, wherein the SCAN domain of ZNF274 comprisesan amino acid sequence as set forth in SEQ ID NO: 1, a fragment or a a biologically active variant having at least 80% nucleotide sequence identity thereto.
  6. 6. The fusion protein according to any one of claims 1 to 5, wherein the DNA adenine methyltransferase is selected from the group comprising the E.coli adenine methyltransferase (Dam), a fragment or a biologically active variant having at least 80% nucleotide sequence identity thereto.
  7. 7. The fusion protein according to claim 6, wherein the Dam domain comprises an amino acid sequence as set forth in SEQ ID NO: 2, a fragment or a biologically active variant having at least 80% nucleotide sequence identity thereto.
  8. 8. The fusion protein according to any one of claims 1 to 7, further comprising a repressor domain selected from the group comprising a Krtippel-associated box (KRAB) domain, a transcription repression domain of methyl-CpG binding protein 2, or regulatory domains of other proteins (e.g. a transcriptional repression domain) or a combination of one or more thereof.
  9. 9. The fusion protein according to any one of claims 1 to 8, wherein the Cas9 protein comprises at least one domain selected from the group comprising a Reel domain, a bridge helix domain, and/or a protospacer adjacent motif (PAM) interacting domain.
  10. 10. The fusion protein according to any one of claims 1 to 9, wherein the Cas9 is a dCas9 protein comprising a D10A mutation in a RuvCl domain and/or a H840A mutation in a HNH domain.
  11. 11. The fusion protein according to claim 9 or 10, wherein the dCas9 protein comprises an amino acid sequence as set forth in SEQ ID NO: 3, a fragment or a biologically active variant having at least 80% nucleotide sequence identity thereto.
  12. 12. The fusion protein according to any one of claims 1 to 11, further comprising one or more linkers.
  13. 13. The fusion protein according to any one of claims 1 to 12, wherein the fusion protein is selected from the group comprising i) a dCas9-KRAB-SCAN fusion protein as set forth in SEQ ID NO. 4 or an amino acid fragment sequence having at least 80%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto; ii) a dCas9 -SCAN fusion protein as set forth in SEQ ID NO. 5 or an amino acid fragment sequence having at least 80%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto; or iii) a E. coli DNA adenine methyltransferase (Dam)-SCAN fusion protein as set forth in SEQ ID NO. 6 or an amino acid fragment sequence having at least 80%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.
  14. 14. An isolated polynucleotide encoding a fusion protein according to any one of claims 1 to 13.
  15. 15. A plasmid or a vector comprising a nucleic acid sequence of claim 14.
  16. 16. A prokaryotic or eukaryotic host cell, or population of cells, comprising the plasmid or vector of claim 15.
  17. 17. A pharmaceutical composition comprising a therapeutically effective amount of at least one fusion protein according to any one of claims 1 to 13 and at least one or more gRNAs that bind the catalytically inactive endonuclease, a polynucleotide of claim 14, a plasmid or vector of claim 15, or a prokaryotic or eukaryotic host cell, or population of cells, of claim 16, and a pharmaceutically acceptable carrier, diluent and/or excipient.
  18. 18. The pharmaceutical composition according to claim 17 for use in the treatment and / or prevention of cancer, neurodegenerative disease, neurodevelopmental disease, aging and aging-related disease.

Description

SCAN-BASED FUSION PROTEINS FOR MODULATING GENE EXPRESSION AND METHODS OF USE FIELD OF THE INVENTION The invention provides fusion protein comprising a SCAN domain bearing member of a zinc finger-containing protein (ZFP), a fragment or variant thereof and a catalytically inactive RNA-guided endonuclease or a DNA adenine methyltransferase. Preferably, the fusion protein is for use in the treatment and/or prevention of cancer, neurodegenerative disease, neurodevelopmental disease, aging and aging-related disease. BACKGROUND OF THE INVENTION The eukaryotic genome is spatially organized to guarantee the orderly execution of transcriptional programs. The genome is dynamically distributed between euchromatin and heterochromatin, with hundreds of genes switching between these respectively active and inactive compartments during development and differentiation. Nuclear bodies can serve as a genomic scaffold for this reorganization by sequestering chromatin-regulating proteins. In the vast majority of nuclei, heterochromatin marked by H3K27me3 or H3K9me3 gathers at the nuclear lamina (NL) or around the nucleolus via an actively regulated dynamic process. While lamina-associated domains (LADs) have been extensively mapped, nucleolus-associated domains (NADs) are less characterized. Previous studies revealed that, in addition to centromeric and pericentromeric regions, NADs are enriched in specific subsets of human genes, including those coding for zinc finger proteins, protocadherins, immunoglobulins and olfactory receptors (OR), which are organised in large clusters and expressed in a tissue-specific manner. In mice, the association of developmentally regulated genes with NADs varies between different cell types. These observations led to the hypothesis that the nucleolus acts as a specialized compartment to establish repressive chromatin and controls the dynamic execution of lineage-specific expression programs. However, the molecular forces behind NADs organization have so far remained elusive. There exists a need for alternative methods and compounds to edit the spatial organization of target genomic loci, particularly with regards to membraneless nuclear compartments such as nucleoli. SUMMARY OF THE INVENTION The present invention provides fusion protein comprising i) a SCAN domain bearing member of a zinc finger-containing protein, a fragment or variant thereof and ii) a catalytically inactive RNA-guided endonuclease or iii) a DNA adenine methyltransferase. Further provided is an isolated polynucleotide encoding a fusion protein of the invention, a plasmid or a vector comprising said nucleic acid sequence, and a prokaryotic or eukaryotic host cell, or population of cells, comprising the plasmid or vector. Further provided is a pharmaceutical composition comprising a therapeutically effective amount of at least one fusion protein and at least one or more gRNAs that bind the catalytically inactive endonuclease, a polynucleotide, a plasmid or vector, or a prokaryotic or eukaryotic host cell, or population of cells, and a pharmaceutically acceptable carrier, diluent and/or excipient. Further provided are methods of treatment and/or prevention of a disease comprising administering to a subject in need thereof, a i) pharmaceutical composition of the invention, ii) a fusion protein along with at least one or more gRNAs of the invention, iii) a polynucleotide of the invention, iv) a plasmid or vector of the invention, or v) a prokaryotic or eukaryotic host cell, or population of cells. DESCRIPTION OF THE FIGURES Figure 1 : Schematic representation of dCas9-KRAB-SCAN fusion protein. Figure 2: Schematic representation of dCas9-SCAN fusion protein. Figure 3 : Schematic representation of E. coli DNA adenine methyltransferase (Dam)-SCAN fusion protein. Figure 4: CRISPR-S silencing of POU5F1B gene in LS1034 cell line, PCR primers Pl Figure 5: CRISPR-S silencing of POU5F1B gene in LS1034 cell line, PCR primers P2. DESCRIPTION OF THE INVENTION Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. In the case of conflict, the present specification, including definitions, will control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter