WO2024112773A1 - Constructions d'édition d'adn et procédés les utilisant - Google Patents

Constructions d'édition d'adn et procédés les utilisant Download PDF

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WO2024112773A1
WO2024112773A1 PCT/US2023/080718 US2023080718W WO2024112773A1 WO 2024112773 A1 WO2024112773 A1 WO 2024112773A1 US 2023080718 W US2023080718 W US 2023080718W WO 2024112773 A1 WO2024112773 A1 WO 2024112773A1
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ssdna
dna
construct
nls
cell
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PCT/US2023/080718
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Hao Wu
Richard Shan
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Fc Ip Holdings, Llc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the DNA editing construct comprises a DNA editing motif having a site-specific DNA endonuclease activity; a nuclear localization signal (NLS); and a single stranded DNA (ssDNA) binding motif.
  • the DNA editing motif introduces a double strand break, a single strand break, or no double strand break in double stranded DNA molecules.
  • the DNA editing motif introduces the double-strand break in double stranded DNA molecules, and the double-strand break is a blunt-ended break or a sticky-ended break.
  • the DNA editing motif comprises a Cas nuclease, a zinc-finger nuclease, a meganuclease, or a transcription activator-like effector nuclease (TALEN).
  • the DNA editing motif comprises a class I Cas protein or a class II Cas protein.
  • the DNA editing motif comprises Cas9, Cas3, Cas10, Cas11, CasX, Cas12a, or MAD7.
  • the DNA editing motif comprises a catalytic dead form thereof.
  • the DNA editing motif comprises nickase forms thereof.
  • the nuclear localization signal comprises a 53BP1 NLS, a CXCR4 NLS, an ERK5 NLS, an EWS NLS, an IER5 NLS, an ING4 NLS, an Hrp1 NLS, an MSX1 NLS, an NLS-RAR ⁇ NLS, a nucleoplasmin NLS, a Pho4 NLS, a PTHrP NLS, an rpL23a NLS, a simian virus 40 (SV40) large T antigen NLS, a UL79 NLS, a VACM-1/CUL5 NLS, a VP1 NLS, a PABPN1 NLS, a STAT1 NLS, a FGF2 NLS, a RARa NLS, a tandem phenylalanine-glycine repeat (FG repeat) from a nuclear pore complex, a histone H2a NLS
  • the construct comprises a plurality of the nuclear localization signals.
  • the ssDNA binding motif is or comprises a K homology (KH) domain ssDNA binding motif, an oligonucleotide/oligosaccharide binding (OB) fold ssDNA binding motif, a RecA/Rad51/DMC1 loop L2 homologous ssDNA binding motif, an RNA recognition motif (RRM) that binds ssDNA, or a whirly domain ssDNA binding motif.
  • KH K homology
  • OB oligonucleotide/oligosaccharide binding
  • RRM RNA recognition motif
  • the ssDNA binding motif is or comprises a bacterial single- stranded DNA-binding (SSB) protein or ssDNA binding portions thereof, a RecA/Rad51/DMC family protein or ssDNA binding portions thereof, a replication protein A (RPA) or ssDNA binding portions thereof, or a single-stranded annealing protein (SSAP) or ssDNA binding portions thereof.
  • SSB bacterial single- stranded DNA-binding
  • RPA replication protein A
  • SSAP single-stranded annealing protein
  • the ssDNA binding motif is or comprises an ssDNA binding motif derived from a RecA/Rad51/DMC family protein, and wherein the ssDNA binding motif does not have recombinase activity.
  • the ssDNA binding motif is or comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:11. 2 51354128.1 Attorney Docket No.385722-1001WO1 (00005) [0018]
  • the construct comprises a plurality of the ssDNA binding motifs. [0019] In some embodiments, the construct further comprises one or more linkers connecting the DNA editing motif, the nuclear localization signal, and the ssDNA binding motif to each other. [0020] In some embodiments, the one or more linkers connects the DNA editing motif, the nuclear localization signal, and the ssDNA binding motif to each other covalently.
  • the one or more linkers independently are polypeptide linkers. [0022] In some embodiments, each of the polypeptide linkers independently has a length ranging from about 2 amino acid residues to about 100 amino acid residues. [0023] In some embodiments, the construct is a protein. Nucleic acid [0024] In some aspects, the present invention is directed to a nucleic acid. [0025] In some embodiments, the nucleic acid encodes the construct here, such as the protein construct herein. [0026] In some embodiments, the nucleic acid further encodes a guide RNA (gRNA) that provides the site-specificity to the DNA editing motif. [0027] In some embodiments, the nucleic acid is comprised in an expression vector.
  • gRNA guide RNA
  • the expression vector is a plasmid vector, an RNA vector, or a viral vector.
  • the nucleic acid is packaged in a viral particle, a lipid nanoparticle, or a non-lipid nanoparticle.
  • Composition [0030] In some aspects, the present invention is directed to a composition. [0031] In some embodiments, the composition is a composition for editing DNA. [0032] In some embodiments, the composition comprises a DNA editing construct; and a single stranded DNA (ssDNA) having a DNA insert, a 5’-homology arm, and a 3’-homology arm.
  • ssDNA single stranded DNA
  • the DNA editing construct is the same as or similar to those described elsewhere herein. 3 51354128.1 Attorney Docket No.385722-1001WO1 (00005) [0034]
  • the 5’ homology arm and the 3’ homology arm are complementary to the nuclear DNA in a target region.
  • the ssDNA is a circular ssDNA (cssDNA), a linear ssDNA (lssDNA), a double stranded DNA/circular single stranded DNA (dsDNA/cssDNA) hybrid having an ssDNA section, or an RNA/DNA hybrid having an ssDNA section.
  • the ssDNA is chemically modified.
  • the ssDNA is produced from a cell free system or a cell culture system.
  • each of the 5’-homology arm and the 3’-homology arm independently has a length ranging from about 25 nucleotides to about 5000 nucleotides.
  • the DNA insert has a length ranging from 1 nucleotide to about 50,000 nucleotides.
  • Kit [0040] In some aspects, the present invention is directed to a kit. [0041] In some embodiments, the kit is a kit for editing DNA.
  • the kit comprises a construct and/or a nucleic acid of; and a single stranded DNA (ssDNA), a nucleic acid that encodes the ssDNA, or a nucleic acid for constructing the ssDNA.
  • the construct is a DNA editing construct here.
  • the nucleic acid is a nucleic acid encoding a DNA editing construct herein.
  • the ssDNA comprises a DNA insert, a 5’-homology arm, and a 3’-homology arm, wherein the 5’ homology arm and the 3’ homology arm are complementary to the nuclear DNA in a target region.
  • the ssDNA is a circular ssDNA (cssDNA), a linear ssDNA (lssDNA), a double stranded DNA/circular single stranded DNA (dsDNA/cssDNA) hybrid having an ssDNA section, or an RNA/DNA hybrid having an ssDNA section.
  • the ssDNA is chemically modified;
  • the ssDNA is produced from a cell free system or a cell culture system.
  • each of the 5’-homology arm and the 3’-homology arm independently has a length ranging from about 25 nucleotides to about 5000 nucleotides.
  • the DNA insert has a length ranging from 1 nucleotide to about 50,000 nucleotides.
  • the nucleic acid for constructing the ssDNA donor template encodes every elements of the ssDNA donor template, except for the DNA insert.
  • the nucleic acid for constructing ssDNA donor template encodes every elements of the ssDNA donor template, except for the 5’ homology arm and the 3’ homology arm. [0052] In some embodiments, the nucleic acid for constructing ssDNA donor template does not encode the 5’ homology arm, the 3’ homology arm, and the DNA insert. [0053] In some embodiments, the nucleic acid includes a multiple cloning site for inserting the 5’ homology arm, the 3’ homology arm, and/or the DNA insert. [0054] In some embodiments, the nucleic acid for constructing ssDNA donor template is a plasmid or a viral vector.
  • the kit further comprises a component for delivering the construct, the nucleic acid for encoding the construct, the ssDNA, and/or the nuclei acid for encoding or constructing the ssDNA into a cell.
  • Method of editing a nuclear DNA is directed to a method of editing a nuclear DNA, such as a nuclear DNA in a cell.
  • the method comprises delivering into the cell the construct herein and/or the nucleic acid herein; and a single stranded DNA (ssDNA) having a DNA insert, a 5’-homology arm, and a 3’-homology arm, wherein the 5’ homology arm and the 3’ homology arm are complementary to the nuclear DNA in a target region.
  • the ssDNA is a circular ssDNA (cssDNA), a linear ssDNA (lssDNA), a double stranded DNA/circular single stranded DNA (dsDNA/cssDNA) hybrid having an ssDNA section, or an RNA/DNA hybrid having an ssDNA section.
  • the ssDNA is chemically modified. 5 51354128.1 Attorney Docket No.385722-1001WO1 (00005) [0060] In some embodiments, the ssDNA is produced from a cell free system or a cell culture system. [0061] In some embodiments, each of the 5’-homology arm and the 3’-homology arm independently has a length ranging from about 25 nucleotides to about 5000 nucleotides. [0062] In some embodiments, the DNA insert has a length ranging from 1 nucleotide to about 50,000 nucleotides.
  • the construct and/or nucleic acid and the ssDNA are delivered into the cell by a viral vector, a lipid or non-lipid nanoparticle delivery, an electroporation delivery, a gene gun delivery, or an injection.
  • the nuclear DNA is a genomic DNA.
  • the cell is a prokaryotic cell, a cultured plant cell, a eukaryotic cell, a cultured mammalian cell, a primary cell, an embryonic stem cell, an adult stem cell, a hematopoietic stem cell, an induced pluripotent stem cell, or a CAR-T cell.
  • the cell is in the body of a subject.
  • the subject is a mammal, such as a human.
  • Non-natural polypeptide [0068]
  • the present invention is directed to a non-natural polypeptide comprising an amino acid sequence having 90% or higher identity to the amino acid sequence set forth in SEQ ID NO:11.
  • the non-natural polypeptide is a ssDNA-binding polypeptide.
  • Figs.1A-1F demonstrate that fusion of Cas9 and homologous recombination proteins enhance the ssDNA mediated knock-in in K562 cells, in accordance with some embodiments.
  • Fig.1A Schematic diagram of various Cas9-homologous recombination protein fusion 6 51354128.1 Attorney Docket No.385722-1001WO1 (00005) constructs (enGagers) in all-in-one (AIO) plasmid format modified from Addgene plasmid #42230.
  • RecA is the bacteria homologous DNA repair protein and Rad51(AE)/Rad51(SEAD) are two mutant variants from eukaryotes.
  • Brex is a 36 amino acid peptide reported to recruit Rad51 in mammalian cells.
  • Fig.1B Schematic diagram of knock-in strategy of a 2Kb (left) and 4Kb (right) cssDNA donor template for RAB11A locus.
  • Fig.1C representative FACS profiles with gating strategy showing % of GFP transgene cassette knock-in on RAB11A locus at day 3 post electroporation for various enGagers listed in A.
  • Fig.1D Quantification of 2Kb GFP transgene cassette knock-in fold change of various enGagers as compared to wild type (WT) Cas9 at day 3 (left), 8 (middle) and 14 (right) post electroporation.
  • Fig.1E representative FACS profiles with gating strategy showing % of 4Kb GFP transgene cassette knock-in on RAB11A locus at day 3 post electroporation for various enGagers listed in A.
  • Fig.1F Quantification of 4Kb GFP transgene cassette knock-in fold change of various enGagers as compared to WT Cas9 at day 3 (left), 7 (right) post electroporation.
  • Figs.2A-2D Identification of small enGagers with Cas9 fused ssDNA binding motifs in K562 cells, in accordance with some embodiments.
  • Fig.2A Schematic diagram of various Cas9-ssDNA binding motifs fusion constructs (enGagers) in all-in-one (AIO) plasmid format modified from Addgene plasmid #42230.
  • FECO, WECO and YECO are 20 amino acid sequences previously identified as ssDNA binding motifs in various bacteria species of RecA, FECO3X, WECO3X and YECO3X are 3 tandem copies of the 20 AA peptides separated by multi-GS peptide linkers.
  • Fig.2B Schematic diagram of knock-in strategy of a 2Kb cssDNA donor template for RAB11A locus.
  • Fig.2C representative FACS profiles with gating strategy showing % of GFP transgene cassette knock-in on RAB11A locus at day 7 post electroporation for various small enGagers listed in A.
  • Fig.2D Quantification of 2Kb GFP transgene cassette knock-in fold change of various enGagers as compared to WT Cas9 at day 7 post electroporation. Note that Cas9-FECO fusion performs similarly with Cas9-RecA fusion in cssDNA mediated transgene integration (1.59- vs 1.57-fold).
  • Figs.3A-3E Identification of additional novel enGagers from Cas9-ssDNA binding module chimeras in K562 cells, in accordance with some embodiments.
  • Fig.3A Schematic diagram of various Cas9-ssDNA binding protein and peptide fusion constructs (enGagers) in all- in-one (AIO) plasmid format modified from Addgene plasmid #42230.
  • Fig.3B Schematic diagram of knock-in strategy of a 2Kb cssDNA donor template for RAB11A locus.
  • Fig.3C Quantification of 2Kb GFP transgene cassette knock-in fold change of various enGagers as compared to WT Cas9 at day 7 post electroporation.
  • Cas9-DrRecA and Cas9-DrRecA20AA fusion has the highest performance in knock-in with 2.17- and 2.43-fold as compared to WT Cas9, respectively.
  • Fig. 3D Quantification of cell viability day 7 post electroporation. Bars represents mean ⁇ SD from 3 biological replicates.
  • Figs.3E Amino acid sequence alignment of 20AA of multiple E. coli RecA mutant variants and RecA from archaea and mammalian organism.
  • the amino acid sequences of Figs.3E are listed below in Table 1: Table 1 S. solfataricus RadA ssDNA binding motif: 8 51354128.1 Attorney Docket No.385722-1001WO1 (00005) H. sapiens DMC1 ssDNA binding motif: NQMTADPGATMTFQADPKKPIGG (SEQ ID NO:6) [00 ] gs.
  • Fig.4A Schematic diagram of various Cas9-ssDNA binding protein and peptide fusion constructs (enGagers) in mRNA form. Two nuclear localization signals were added to the N’ and C’-termini of the Cas9 protein.
  • GS is a shortened multi-GS peptide linker “EFGGGGS” (SEQ ID NO:12).
  • Fig.4B Schematic diagram of knock-in strategy of a 2Kb cssDNA donor template for RAB11A locus.
  • Fig.4C Dose titration of cssDNA at 0.3, 1, 1.5, 2 and 3ug for 2Kb GFP transgene knock-in at day 3 post electroporation. Cas9-RecA mRNA enhance around 25-30% knock-in efficiency than WT Cas9 mRNA at all the cssDNA dose tested in K562 cells.
  • Fig.4D representative FACS profiles with gating strategy showing % of 2Kb GFP transgene cassette knock-in on RAB11A locus by cssDNA donor at day 5 post electroporation for various enGagers listed in Fig.4A in K562 cells.
  • Fig.4E Quantification of 2Kb GFP transgene cassette knock-in fold change (left) and cell viability (right) of various enGagers as compared to WT Cas9 at day 5 post electroporation from Fig.4D.
  • Fig.4F representative FACS profiles with gating strategy showing % of 2Kb GFP transgene cassette knock-in on RAB11A locus by dsDNA donor at day 5 post electroporation for various enGagers listed in Fig.4A in K562 cells.
  • Fig.4G Quantification of 2Kb GFP transgene cassette knock-in fold change (left) and cell viability (right) of various enGagers as compared to WT Cas9 at day 5 post electroporation from Fig.4F.
  • Fig.4H Quantification of 2Kb GFP 9 51354128.1 Attorney Docket No.385722-1001WO1 (00005) transgene cassette knock-in fold change (left) and cell viability (right) of various enGagers mRNA as compared to WT Cas9 mRNA with cssDNA donor at 5 days post-delivery in HEK293 cells by lipofectamine 3000 transfection.
  • Fig.4I Quantification of 2Kb GFP transgene cassette knock-in fold change (left) and cell viability (right) of various enGagers mRNA as compared to WT Cas9 mRNA with dsDNA donor at 5 days post-delivery in HEK293 cells by lipofectamine 3000 transfection. Bars represents mean ⁇ SD from 3 biological replicates. [0075] Figs.5A-5F demonstrate that enGagers in mRNA form enhance genome integration on various locus and large payload, in accordance with some embodiments.
  • Fig.5A Schematic diagram of knock-in strategy of 2Kb (css76), 4Kb (css116) and 8Kb (css167) cssDNA donor templates for RAB11A locus.
  • Fig.5B Quantification of % of 2Kb (left), 4Kb (middle) and 8Kb (right) GFP transgene cassette knock-in for various mRNA enGagers RAB11A day 13 post electroporation in K562 cells.
  • Fig.5C Quantification of cell viability for 2Kb (left), 4Kb (middle) and 8Kb (right) GFP transgene cassette knock-in for various mRNA enGagers on RAB11A locus day 13 post electroporation in K562 cells.
  • Fig.5D Schematic diagram of knock- in strategy of 2Kb (css27) and 4Kb (css88) cssDNA donor templates for B2M locus.
  • Fig.5E Quantification of % of 2Kb (left) and 4Kb (right) GFP transgene cassette knock-in for various mRNA enGagers on B2M locus day 13 post electroporation in K562 cells.
  • Fig.5F Quantification of cell viability for 2Kb (left) and 4Kb (right) GFP transgene cassette knock-in for various mRNA enGagers on B2M locus day 13 post electroporation in K562 cells. Bars represents mean ⁇ SD from 3 biological replicates.
  • Figs.6A-6F demonstrate that CAR-T engineered by enGager has superior efficiency than those engineered by WT Cas9, in accordance with some embodiments.
  • Fig.6A Schematic diagram of knock-in strategy of 3Kb CD19/CD22 dual CAR (css62) cssDNA donor templates for TRAC locus in primary T cells.
  • Fig.6B Quantification of % of CD19/CD22 CAR knock-in using cssDNA donor analyzed by Protein-L for various doses of WT Cas9 and Cas9-GS-FECO enGager mRNA at 1 ug, 2ug and 4ug at day 7 post electroporation of primary T cells.
  • Cas9-GS- FECO enGager achieves ⁇ 4- to 6-fold higher CAR-T engineering efficiency than WT Cas9.
  • Fig. 6C Characterization of the engineered CAR-T cells or mock treated T cells for total cell count, cell proliferation fold change and cell viability over time (at day 1, 4, 7 and 11 days post electroporation).
  • Fig.6D NALM6 leukemia lymphocyte killing curve of unengineered T cells, CD19-CD22 dual CAR-T cells engineered with 2 ug of WT Cas9 mRNA and 2 ug of GS-FECO 10 51354128.1 Attorney Docket No.385722-1001WO1 (00005) enGager mRNA over the course of 96 hrs. Effect (T cells): Target (NALM6 cells) are at 2.25:1 for left panel, 4.5:1 for middle panel and 9:1 for right panel. Bars represents mean ⁇ SD from 3 biological replicates.
  • Fig.6E NALM6 cell killing function of CAR-T cells at 24 hrs for E:T ratio at 2.25:1, 4.5:1 and 9:1.
  • Fig.6F Schematic diagram of engineered enGagers with single stranded DNA binding protein (SSBP) can recruit cssDNA donor template and form a tripartite editing machinery for efficient translocation of the entire editing complex from cytoplasm to nucleus and tethering to the targeted genomic locus.
  • SSBP single stranded DNA binding protein
  • the donor DNA has higher effective local concentration in the nucleus for more efficient homologous directed genome integration. This process works more prominently with cssDNA.
  • Figs.7A-7B illustration of a non-limiting DNA editing construct, as well as method of editing nuclear DNA using the construct, in accordance with some embodiments.
  • Fig.7A the DNA editing construct 100 includes a DNA editing motif 110, a nuclear localization signal 130, and a single stranded DNA binding motif 150 with linkers 170.
  • Fig.7B the DNA editing construct 100, when introduced into a cell 500 together with a ssDNA 200 (shown as a circular single stranded DNA or cssDNA) that serves as a donor template for a homology-directed repair, are able to bind to the ssDNA 200 and forms the DNA editing complex 300.
  • a ssDNA 200 shown as a circular single stranded DNA or cssDNA
  • the DNA editing complex 300 moves into the nucleus 510 via active transportation in addition to passive diffusion, and edits the nuclear DNA 530 (at a locus 531) inside the nucleus 510 through the combined effects of the DNA editing motif 110 and the ssDNA 200.
  • DETAILED DESCRIPTION [0078] The following disclosure provides many different embodiments, or examples, for implementing different features of the provided subject matter. Specific examples of components and arrangements are described below to simplify the present disclosure. These are, of course, merely examples and are not intended to be limiting.
  • first and second features are formed in direct contact
  • additional features may be formed between the first and second features, such that the first and second features may not be in direct contact
  • present disclosure may repeat reference numerals and/or letters in the various examples. This repetition is for the purpose of 11 51354128.1 Attorney Docket No.385722-1001WO1 (00005) simplicity and clarity and does not in itself dictate a relationship between the various embodiments and/or configurations discussed. Definitions [0079] As used herein, each of the following terms has the meaning associated with it in this section.
  • cssDNA Circular single stranded DNA.
  • DSB Double stranded DNA break.
  • gRNA guide RNA.
  • HDR Homology directed repair.
  • HR Homologous recombination.
  • KI knock-in.
  • sgRNA Single guide RNA.
  • ssDNA Single stranded DNA.
  • Constructs for DNA Editing [0085] The study described herein (“the present study”) developed non-limiting novel DNA editing constructs that edit DNA using single stranded DNA (ssDNA) as a donor template.
  • the present invention is directed to DNA editing constructs.
  • the present invention is directed to DNA editing constructs that edit nuclear DNA using a ssDNA as a template.
  • the DNA editing construct 100 includes a DNA editing motif 110, a nuclear localization signal 130, and a single stranded DNA binding motif.
  • the DNA editing construct 100 when introduced into a cell 500 together with a ssDNA 200 (shown as a circular single stranded DNA or cssDNA) that serves as a donor template for a homology-directed repair, are able to bind to the ssDNA 200 and forms the DNA editing complex 300.
  • a ssDNA 200 shown as a circular single stranded DNA or cssDNA
  • the DNA editing complex 300 moves into the nucleus 510 via active transportation in addition to passive diffusion, and edits the nuclear DNA 530 inside the nucleus 510 through the combined effects of the DNA editing motif 110 and the ssDNA 200.
  • 13 51354128.1 Attorney Docket No.385722-1001WO1 (00005)
  • DNA Editing Motifs [0089]
  • the DNA editing motif 110 edits the DNA molecule via homologous recombination (HR) or homology-directed repair (HDR) using the ssDNA as the template.
  • the DNA editing motif 110 has a nuclease activity, such as a DNA nuclease activity, such as a DNA endonuclease activity, such as a site-specific DNA endonuclease activity.
  • the DNA editing motif 110 is, or includes, a nuclease, such as a DNA nuclease, such as a DNA endonuclease, such as a site-specific DNA endonuclease.
  • DNA endonuclease activity refers to the enzymatic activity of introducing a break, such as a double strand break (either a blunt-ended break or a sticky- ended break) or a single strand break (also referred to as a “nick”) into a DNA molecule, such as a double stranded DNA molecule, at locations other than the two ends thereof.
  • site-specific DNA endonuclease activity refers to the enzymatic activity of introducing a break into a DNA molecule at or near a specific site, such as a site as defined by a specific nucleotide sequence.
  • DNA endonuclease Any proteins that possess DNA endonuclease activity is referred to herein as a DNA endonuclease, regardless of their origins (natural, recombinant, engineered, truncated, etc.) [0092] It is worth noting that, the sequence specificities of some site-specific DNA endonucleases are, at least partially, derived from external components that are not considered as part of the nucleases per se by the art. For example, the site-specificities of some Cas proteins are largely derived from guide RNAs, which are not normally considered part of the Cas proteins.
  • nuclease is considered a “site-specific” regardless of whether the external components are actually present.
  • DNA editing construct 100 is able to bring ssDNA donor templates to the proximity of the target site on the DNA molecule.
  • DNA editing motif 110 includes a catalytic dead form of the site-specific nuclease.
  • the DNA editing motif is not a catalytic dead forms of a site- specific DNA endonuclease, as such form of endonucleases generally result in lower editing efficiencies than their enzymatically active counterparts.
  • the DNA editing motif 110 is a site-specific DNA endonuclease, or portions thereof that possess the site-specific DNA endonuclease activity, such as one of more protein domains (such as the enzyme domains) of the site-specific DNA endonuclease.
  • Non-limiting examples of site-specific DNA endonucleases include the Cas nucleases, the zinc-finger nucleases, the meganucleases, the transcription activator-like effector nucleases (TALENs), and the like.
  • a large number of site-specific DNA endonucleases are genetically engineered proteins, such as those described in Bogdanove et al. (Nucleic Acids Research, Volume 46, Issue 10, 1 June 2018, Pages 4845–4871, the entirety of this reference is hereby incorporated herein by reference).
  • Non-limiting examples of Cas nucleases include class I and Class II Cas proteins such as Cas9, CasX, Cas12a, MAD7, or derivatives thereof, such as catalytic dead forms or nickase forms thereof.
  • Class I and class II proteins are described in, for example, Makarova et al. (Nature Reviews Microbiology volume 18, pages67–83 (2020)). The entirety of this reference is hereby incorporated herein by reference.
  • Nuclear Localization Signals [0098]
  • the nuclear localization signal 130 includes or is a nuclear localization signal commonly known in the art, which is a peptide motif that mediates the transportation, such as the importin-mediated transportation, of proteins from the cytoplasm into the nucleus.
  • Non-limiting examples of nuclear localization signals include a 53BP1 NLS, a CXCR4 NLS, an ERK5 NLS, an EWS NLS, an IER5 NLS, an ING4 NLS, an Hrp1 NLS, an MSX1 NLS, an NLS-RAR ⁇ NLS, a nucleoplasmin NLS, a Pho4 NLS, a PTHrP NLS, an rpL23a NLS, a simian virus 40 (SV40) large T antigen NLS, a UL79 NLS, a VACM-1/CUL5 NLS, a VP1 NLS, 15 51354128.1 Attorney Docket No.385722-1001WO1 (00005) PABPN1 NLS, STAT1 NLS, FGF2 NLS, RARa NLS or the like, and their homologous sequences from different species.
  • SV40 simian virus 40
  • Nuclear localization signals also include one or more tandem phenylalanine-glycine repeats (FG repeats) from nuclear pore complex such as importin- ⁇ / ⁇ and their homologous sequence from different species.
  • Nuclear localization signals further include those found in histone H2a and H2b, which are described in, for example Mosammaparast et al. (J Cell Biol.2001 Apr 16; 153(2): 251–262). The entirety of this reference is hereby incorporated herein by reference. [00100] Nuclear localization signals are further described in, for example, Lu et al. (Cell Communication and Signaling volume 19, Article number: 60 (2021)).
  • the construct 100 includes a plurality of the nuclear localization signals 130, such as 2 or more, 3 or more, 4 or more, or 5 or more of the nuclear localization signals 130.
  • ssDNA Binding Motifs the ssDNA binding motif 150 is a motif that directly binds to ssDNA molecules. In some embodiments, the ssDNA binding motif 150 is not an adaptor for recruiting intrinsic ssDNA binding proteins in the cell.
  • the present study has discovered that, when an ssDNA binding motif 150 is included in the construct 100, the DNA editing efficiency can increase significantly, sometimes by about 50% or more, about 100% or more, or about 150% or more, as compared to similar DNA editing constructs without the ssDNA binding motif (see e.g., Figs.1D, 1E, 3C, 4E, 4H, 5B, and 5D).
  • the present study further discovered that, as long as a ssDNA binding motif 150 is present, the DNA editing efficiencies can be increased.
  • the structure or evolutionary paths of the ssDNA binding motif, or manners by which the ssDNA binding motif interacts with ssDNA molecules do not appear to matter.
  • RecA protein interact with ssDNA molecules via a 20 amino acid residue loop (“loop L2”)
  • bacteria SSB protein interact with ssDNA molecules via an oligonucleotide/oligosaccharide binding (OB) fold structural feature, which is unrelated to the loop L2 of RecA either structurally or evolutionarily.
  • OB oligonucleotide/oligosaccharide binding
  • the ssDNA Binding Motif 150 is a K homology (KH) domain ssDNA binding motif, an oligonucleotide/oligosaccharide binding (OB) fold ssDNA binding motif, a RecA/Rad51/DMC1 loop L2 homologous ssDNA binding motif, an RNA recognition motif (RRM) that binds ssDNA, a whirly domain ssDNA binding motif, or the like.
  • KH domain, OB folds, RRM and whirly domain are described in, for example, Dickey et al.
  • RecA/Rad51/DMC1 loop L2 homologous ssDNA binding motif is described herein (see e.g., Fig.3E).
  • the ssDNA binding motif 150 is a bacterial single-stranded DNA-binding (SSB) protein or ssDNA binding portions thereof (such as the OB fold), a DNA meiotic recombinase 1 (DMC1) protein or ssDNA binding portions thereof (such as the L2 homologous motif), a RecA/Rad51 family protein or ssDNA binding portions thereof (such as the L2 homologous motif), a replication protein A (RPA) or ssDNA binding portions thereof (such as the RPA1, RPA2, or RPA3 subunit of RPA, or the OB folds thereof), a single-stranded annealing protein (SSAP), SAAV2152, or a ring-shaped MCM helicase or ssDNA binding portions, and their interspecies homologs thereof.
  • SSB bacterial single-stranded DNA-binding
  • DMC1 DNA meiotic recombinase 1
  • DMC1 DNA meiotic recombina
  • the ssDNA binding motif 150 is the RecA DNA recombinase, a functional derivative, or a protein from the RecA phylogeny (including paralogous and orthologous proteins from the prokaryotes or the eukaryotes, such as Rad51 or DMC1), or the ssDNA binding portions thereof.
  • the ssDNA binding loop L2 of some non-limiting RecA protein e.g., WECO peptide
  • increases DNA editing efficiency at levels comparable to the full length RecA protein see e.g., Fig.2D
  • the recombinase activity of RecA is not important.
  • the RecA/Rad51/DMC1 proteins or ssDNA binding portions thereof ssDNA binding motif 150 does not have recombinase activity.
  • the ssDNA binding motif 150 is the loop L2 of the RecA/Rad51/DMC1 proteins.
  • the binding motif 150 includes, or is a polypeptide set forth in SEQ ID NOs:1-11. In some embodiments, the binding motif 150 includes, or is a polypeptide set forth in SEQ ID:1.
  • the ssDNA binding motif 150 is the bacterial SSB protein, a functional derivative, a protein from the SSB phylogeny, or the ssDNA binding portions thereof. [00111] In some embodiments, the ssDNA binding motif 150 is the RecT protein, a functional derivative, a protein from the RecT phylogeny, or the ssDNA binding portions thereof. [00112] In some embodiments, a length of the ssDNA binding motif 150 ranges from 5-25 amino acid residues. In some embodiments, the length of the ssDNA binding motif 150 ranges from 25-100 amino acid residues.
  • the length of the ssDNA binding motif 150 has more than 100 amino acid residues.
  • the present study tested the inclusion of multiple ssDNA binding motifs 150 in one DNA editing construct 100, and found that the multiple ssDNA binding motifs 150 at least did not affect the DNA editing efficiency when comparing to constructs 100 that include only one ssDNA binding motif 150 (see e.g., Fig.2D). Accordingly, in some embodiments, the construct 100 includes a plurality of the ssDNA binding motifs 150.
  • the construct 100 includes 2 or more of the ssDNA binding motifs 150, such as 3 or more of the ssDNA binding motifs 150, 4 or more of the ssDNA binding motifs 150, or 5 or more of the ssDNA binding motifs 150.
  • the DNA editing construct 100 includes two different types or more of the ssDNA binding motifs 150.
  • Linkers [00114] Referring to Fig.7A, in some embodiments, the construct 100 further include one or more linkers 170 connecting the DNA editing motif 110, the nuclear localization signal 130, and the ssDNA binding motif 150 to each other.
  • linkers 170 are not limited, as long as they are able to survive the intracellular environment (and the extracellular environment if the storage, delivery, etc. of the construct 100 requires substantial exposure to such environments) without breaking substantially, and strong enough that the DNA editing motif 110, the NLS 130 and the ssDNA binding motif 150 do not substantially dissociated with each other before or during DNA editing.
  • the one or more linkers 170 connect the DNA editing motif 110, the nuclear localization signal 130, and the ssDNA binding motif 150 to each other covalently. 18 51354128.1 Attorney Docket No.385722-1001WO1 (00005) [00117]
  • the one or more linkers 170 are polypeptide linkers.
  • each of the polypeptide linkers has the length ranging from about 2 amino acid residues to about 100 amino acid residues.
  • the DNA binding motif 110, the NLS 130, the ssDNA binding motif 150, and the linker 170 are each of them independently selected polypeptides.
  • the DNA binding motif 110, the NLS 130, the ssDNA binding motif 150, and the linker 170 are all part of the same polypeptide chain, and the construct 100 is one protein.
  • the DNA binding motif 110, the NLS 130, the ssDNA binding motif 150, and the linker 170 are not part of the same polypeptide chain and the construct 100 includes two or more separate polypeptide chains.
  • the construct 110 includes two or more proteins.
  • one or more of the linkers 170 are multi-piece design such that components of the construct 100 can be delivered separately and form an integral construct 100 when inside cells or before entering the cytoplasm.
  • one or more of the linkers 170 are formed by two or more components that are able to associate with each other to form the linkers 170. Examples of multi-piece configurations of linkers 170 include linkers composed of protein A/G and antibody Fc domain, linkers composed of a single-chain variable fragment (scFv) and the target thereof, etc.
  • scFv single-chain variable fragment
  • nucleic Acid Encoding Constructs for DNA Editing [00123] The study described herein (“the present study”) developed a novel DNA editing construct that edits DNA using a circular single stranded DNA (cssDNA) as a donor template, which has significantly higher DNA editing efficiency than similar conventional DNA editing constructs that also use cssDNA as donor templates.
  • the novel DNA editing construct can be one or more proteins, which can be encoded by a nucleic acid such as a DNA molecule or an RNA molecule.
  • the present invention is directed to a nucleic acid encoding a construct for DNA editing.
  • the construct for DNA editing comprises one or more proteins that are the same as or similar to those described elsewhere, such as in the “Construct for DNA Editing” section. 19 51354128.1 Attorney Docket No.385722-1001WO1 (00005) [00125]
  • the DNA editing motif 110 sometimes do not have site- specificity alone and depends on external components to provide the specificity.
  • the external components are sometimes guide RNAs.
  • the nucleic acid further encodes a guide RNA (gRNA) that provides the site-specificity to the DNA editing motif.
  • gRNA guide RNA
  • the function of the construct 100 sometimes includes more than one protein, and/or requires guide RNA.
  • the nucleic acid encodes more than one protein, or encodes one or more proteins and at least one guide RNA. In some embodiments, the nucleic acid is polycistronic. [00127] In some embodiments, the nucleic acid is comprised in an expression vector. Non- limiting examples of expression vector include plasmid vectors, RNA vectors, viral vectors, or the like. [00128] In some embodiments, the nucleic acid is packaged in a viral particle, a lipid nanoparticle, a non-lipid nanoparticle, or the like.
  • composition for Editing DNA [00129] The study described herein (“the present study”) developed novel compositions for editing DNA molecules, which include the novel DNA editing constructs and a single stranded DNA (ssDNA) as a donor template. When introduced into cells, the DNA editing composition herein achieved significantly higher DNA editing efficiency than those achieved with conventional DNA editing compositions including the same ssDNA and conventional DNA editing constructs. [00130] Accordingly, in some embodiments, the present invention is directed to a composition for editing DNA. In some embodiments, the composition edits nuclear DNA, such as genomic DNA. In some embodiments, the composition edits DNA via homologous recombination or homology directed repair, using the ssDNA as a donor template.
  • the composition for editing DNA includes a DNA editing construct and a ssDNA donor template.
  • the composition including both the DNA editing construct and the ssDNA donor template is prepared prior to the delivery into a cell to be edited. 20 51354128.1 Attorney Docket No.385722-1001WO1 (00005)
  • the DNA editing construct (or a nucleic acid encoding the same) and the ssDNA donor template are prepared separately and the composition is formed when, for example, both of the components are delivered into the cell.
  • the DNA editing construct when the composition is delivered into a cell and/or formed inside the cell, the DNA editing construct (sometimes in concert with cellular machineries endogenous to the cell) inserts the DNA insert into the target location in the nuclear DNA via homologous recombination or homology directed repair.
  • the DNA editing construct is the same as or similar to those as described elsewhere herein, such as in the “Constructs for DNA Editing” section.
  • the nucleic acid encoding the DNA editing construct is the same as or similar to those described elsewhere herein, such as in the “Nucleic Acid Encoding Constructs for DNA Editing” section.
  • the ssDNA includes a DNA insert, a 5’-homology arm, and a 3’- homology arm. In some embodiments, the 5’ homology arm and the 3’ homology arm are complementary to the nuclear DNA in a target region.
  • the ssDNA is a circular ssDNA (cssDNA), a linear ssDNA (lssDNA), a double stranded DNA/circular single stranded DNA (dsDNA/cssDNA) hybrid including a single stranded DNA portion, an RNA/DNA hybrid including a single stranded DNA portion, or the like.
  • the ssDNA is chemically modified.
  • the ssDNA includes a modified base, a modified sugar group, a modified backbone, or the like.
  • the ssDNA is produced from a cell free system or a cell culture system.
  • each of the 5’-homology arm and the 3’-homology arm has a length ranging from about 25 nucleotides to about 5000 nucleotides, such as from about 50 nucleotides to about 5000 nucleotides, from about 75 nucleotides to about 2500 nucleotides, from about 100 nucleotides to about 2000 nucleotides, from about 150 nucleotides to about 1500 nucleotides, from about 200 nucleotides to about 1000 nucleotides, or from about 300 nucleotides to about 500 nucleotides.
  • the DNA editing composition including the novel DNA editing construct herein can increase editing efficiencies for all tested insert sizes ( ⁇ 2kb, 21 51354128.1 Attorney Docket No.385722-1001WO1 (00005) ⁇ 4kb, ⁇ 8kb). Accordingly, it is expected that the composition herein can be used to insert DNA inserts of all size insertable by conventional DNA editing compositions.
  • the DNA insert in the ssDNA donor template has a length ranging from 1 nucleotide to about 50,000 nucleotides, such as from 1 nucleotide to about 40,000 nucleotides, from 1 nucleotide to about 30,000 nucleotides, from 1 nucleotide to about 20,000 nucleotides, from 1 nucleotide to about 10,000 nucleotides, or from 1 nucleotide to about 8,000 nucleotides.
  • the ssDNA, such as cssDNA suitable for being included in the DNA editing composition herein are also described in, for example, WO2020142730A1. The entirety of this reference is hereby incorporated herein by reference.
  • Kit for Editing DNA The study described herein (“the present study”) developed novel kit for editing DNA molecules, which include the novel DNA editing constructs (or nucleic acids encoding the constructs) and a single stranded DNA (ssDNA) as a donor template. When both components were introduced into cells, the combination of the DNA editing construct herein and the ssDNA donor template achieved significantly higher DNA editing efficiency than those achieved with conventional DNA editing combinations including the same ssDNA and conventional DNA editing constructs (or nucleic acids encoding such conventional editing constructs). [00144] Accordingly, in some embodiments, the present invention is directed to a kit for editing DNA. In some embodiments, the components of the kit, when combined, edit nuclear DNA, such as genomic DNA.
  • the components of the kit edit DNA via homologous recombination or homology directed repair, using the ssDNA as a donor template.
  • the kit for editing DNA includes a DNA editing construct (or a nucleic acid encoding the same) and a ssDNA donor template.
  • the DNA editing construct is the same as or similar to those as described elsewhere herein, such as in the “Constructs for DNA Editing” section.
  • the nucleic acid encoding the DNA editing construct is the same as or similar to those described elsewhere herein, such as in the “Nucleic Acid Encoding Constructs for DNA Editing” section.
  • the DNA editing construct (or a nucleic acid encoding the same) and the ssDNA donor templates are mixed together prior to delivery, or are delivered together or 22 51354128.1 Attorney Docket No.385722-1001WO1 (00005) separately into the cell.
  • a composition for editing DNA (which, in some embodiments, is the same as or similar to those described elsewhere herein, such as in the “Composition for Editing DNA” section) is formed before, during, or after the components are delivered into the cell.
  • the DNA editing construct when the components of the kits are delivered into a cell and/or formed inside the cell, the DNA editing construct (sometimes in concert with cellular machineries endogenous to the cell) inserts the DNA insert into the target location in the nuclear DNA via homologous recombination or homology directed repair.
  • the DNA editing construct is the same as or similar to those as described elsewhere herein, such as in the “Constructs for DNA Editing” section.
  • the nucleic acid encoding the DNA editing construct is the same as or similar to those described elsewhere herein, such as in the “Nucleic Acid Encoding Constructs for DNA Editing” section.
  • the kit for editing DNA does not include the ssDNA donor template. Rather, the kit includes a nucleic acid for producing the ssDNA donor template, such as in a cell free system or a cell culture system. [00151] In some embodiments, the nucleic acid for producing the ssDNA donor template needs to be engineered to construct a nucleic acid that encodes the ssDNA donor template. [00152] In some embodiments, the nucleic acid for constructing the ssDNA donor template encodes every elements of the ssDNA donor template, except for the DNA insert.
  • nucleic acid for constructing ssDNA donor template encodes every elements of the ssDNA donor template, except for the 5’ homology arm and the 3’ homology arm.
  • nucleic acid for constructing ssDNA donor template encodes every elements of the ssDNA donor template, except for the 5’ homology arm, the 3’ homology arm, and the DNA insert. This way, the nucleic acid for constructing ssDNA donor template can be fully customized for the site of the editing as well as the sequence to be inserted.
  • the nucleic acid for constructing ssDNA donor template includes a multiple cloning site for inserting the 5’ homology arm, the 3’ homology arm, and/or the DNA insert.
  • the nucleic acid for constructing ssDNA donor template is a plasmid or a viral vector.
  • the kit further includes a component for delivering the DNA editing construct (or the nucleic acid encoding the same) and the ssDNA.
  • Non-limiting examples of components for delivering including a microinjector (for injecting directly into cells), a lipid for forming lipid nanoparticles, a gene gun and metal nanoparticles (such as gold or tungsten nanoparticles), a cuvette for electroporation, and the like.
  • Methods of Editing DNA [00158] The study described herein (“the present study”) developed novel DNA editing constructs that edit DNA using a single stranded DNA (ssDNA) as a donor template.
  • the DNA editing constructs (or nucleic acids encoding the same) can be introduced into cells together with the ssDNA, and the DNA editing efficiency of this method is significantly higher than the similar conventional DNA editing methods that also use ssDNA as donor templates.
  • the present invention is directed to methods of editing DNA, such as nuclear DNA in a cell.
  • the method includes introducing a DNA editing construct or a nucleic acid encoding the DNA editing construct, as well as a ssDNA donor template into the cell.
  • the DNA editing construct and/or the nucleic acid is the same as or similar to those described elsewhere herein, such as in the “Construct for DNA Editing” and “Nucleic Acid Encoding Construct for DNA Editing” sections.
  • the ssDNA includes a DNA insert, a 5’-homology arm, and a 3’- homology arm.
  • the 5’ homology arm and the 3’ homology arm are complementary to the nuclear DNA in a target region.
  • the method inserts 24 51354128.1 Attorney Docket No.385722-1001WO1 (00005) the DNA insert into the target location in the nuclear DNA via homologous recombination/homology directed repair.
  • the ssDNA is a circular ssDNA (cssDNA), a linear ssDNA (lssDNA), a double stranded DNA/circular single stranded DNA (dsDNA/cssDNA) hybrid including a single stranded DNA portion, an RNA/DNA hybrid including a single stranded DNA portion, or the like.
  • the ssDNA is chemically modified.
  • the ssDNA includes a modified base, a modified sugar group, a modified backbone, or the like.
  • the ssDNA is produced from a cell free system or a cell culture system.
  • each of the 5’-homology arm and the 3’-homology arm has a length ranging from about 25 nucleotides to about 5000 nucleotides, such as from about 50 nucleotides to about 3000 nucleotides, from about 75 nucleotides to about 2500 nucleotides, from about 100 nucleotides to about 2000 nucleotides, from about 150 nucleotides to about 1500 nucleotides, from about 200 nucleotides to about 1000 nucleotides, or from about 300 nucleotides to about 500 nucleotides.
  • the DNA insert has a length ranging from 1 nucleotide to about 50,000 nucleotides, such as from 1 nucleotide to about 40,000 nucleotides, from 1 nucleotide to about 30,000 nucleotides, from 1 nucleotide to about 20,000 nucleotides, from 1 nucleotide to about 10,000 nucleotides, or from 1 nucleotide to about 8,000 nucleotides.
  • the ssDNA, such as cssDNA suitable for used in the DNA editing method herein are also described in, for example, WO2020142730A1. The entirety of this reference is hereby incorporated herein by reference.
  • the constructs or nucleic acids and the ssDNAs are delivered into the cell by a viral vector, a lipid or 25 51354128.1 Attorney Docket No.385722-1001WO1 (00005) non-lipid nanoparticle delivery, a gold particle mediated delivery, an electroporation delivery, and/or an injection.
  • the DNA editing construct is delivered into the cell as the construct (such as one or more proteins), or as a nucleic acid such as an RNA molecule or a DNA molecule.
  • the nuclear DNA is a genomic DNA.
  • the cell is a prokaryotic cell, a cultured plant cell, a eukaryotic cell, a cultured mammalian cell, a primary cell, an embryonic stem cell, an adult stem cell, an induced pluripotent stem cell, a hematopoietic stem cell or a CAR-T cell.
  • the cell is in the body of a subject.
  • the subject is a mammal, such as a human.
  • the method is a method for in vivo DNA engineering.
  • the method is an ex vivo DNA editing method.
  • the method edits DNA in a cell free system.
  • Non-Natural Polypeptides [00174] The present study discovered that L2 region of the RecA protein from the bacterium D. radiodurans is able to significantly increase the DNA editing efficiency when incorporated into the DNA editing construct herein (see e.g., Fig.3C). Without wishing to be bound by theory, it is hypothesized that the L2 region of D. radiodurans RecA (having the amino acid sequence NQVREKIGVMYGNPETTTGG, SEQ ID NO:11) can form stable complex with ssDNA molecules. [00175] Accordingly, in some aspects, the present invention is directed to a non-natural polypeptide including the D.
  • the non-natural polypeptide includes an amino acid sequence having 90% or higher identity, such as 95% or higher identity or identical to the amino acid sequence set forth in SEQ ID NO:11.
  • the non-natural polypeptide is a ssDNA-binding polypeptide. Examples 26 51354128.1 Attorney Docket No.385722-1001WO1 (00005) [00178] The instant specification further describes in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless so specified.
  • Non-viral DNA donor template has been widely used for targeted genomic integration by homologous recombination (HR). This process has become more efficient with RNA guided endonuclease editor system such as CRISPR/Cas9. Circular single stranded DNA (cssDNA) has been harnessed previously as a superior genome engineering catalyst (GATALYST) for efficient and safe targeted gene knock-in.
  • HR homologous recombination
  • cssDNA Circular single stranded DNA
  • GATALYST superior genome engineering catalyst
  • the present study developed enGager, a set of enhanced GATALYST associated genome editors by fusion of nucleus localization single (NLS) peptide tagged Cas9 with various single stranded DNA binding protein modules. These enGagers form tripartite complex with sgRNA and cssDNA donors as integrative genome integration machinery, thereby facilitate the nucleus shuttling of DNA donors and increase their active local concentration. When applied for targeted genome integration with cssDNA donor templates to diverse genomic loci in various cell types, these enGagers outperform unfused editors.
  • NLS nucleus localization single
  • the enhancement of integration efficiency ranges from 1.5- to more than 6-fold, with the effect being more prominent for larger than 4Kb transgene knock-in.
  • the present study further demonstrated that enGager mediated enhancement for genome integration is ssDNA, but not dsDNA dependent. Using one of the small enGagers, the present study demonstrated large chimeric antigen receptor (CAR) transgene integration in primary T cells with exceptional efficiency and anti-tumor cell function.
  • CAR chimeric antigen receptor
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas9 protein combined with 20 nucleotides (nt) guide RNA finds and induces precise double-stranded breaks (DSB) to the complementary region of the guide RNA for gene deletion, repair or insertion in prokaryotes and eukaryotes.
  • CRISPR/Cas9 gene knock-out using Non-Homogenous End Joining (NHEJ)
  • NHEJ Non-Homogenous End Joining
  • dCas9 catalytically dead Cas9 fused epigenetic regulators
  • KI targeted gene knock-in
  • HR homologous recombination
  • HDR Homology directed repair
  • DSB double stranded DNA break
  • HDR is one of the most efficient ways to insert a few bases to 2Kb length of genes but is extremely inefficient for > 4Kb large payload.
  • Adeno-Associated Virus Type 6 AAV6 has been demonstrated as a prevalent donor carrier for HDR mediated genome engineering. For instance, AAV6 have been demonstrated for efficient beta-globin gene integration in human and rodent hematopoietic cells as well as human stem cells.
  • Non- viral targeted gene KI relies primarily on double-stranded DNA (dsDNA) and linear single- stranded DNA (lssDNA) donors.
  • dsDNA double-stranded DNA
  • lssDNA linear single- stranded DNA
  • These nucleic acid donor templates have been demonstrated relatively efficient gene targeting in various primary cells, including T cells and natural killer cells.
  • CD19 CAR-T cells engineered on an immune checkpoint PD-1 locus with linear dsDNA donor template have been reported to treat B cell Non-Hodgkin lymphoma (B-NHL) patients with desired safty and efficacy.
  • B-NHL B cell Non-Hodgkin lymphoma
  • dsDNA donors are easy to access, it succumbs from low editing 28 51354128.1 Attorney Docket No.385722-1001WO1 (00005) efficiency and cGAS dependent cytotoxicity.
  • lssDNA has better performance for targeted gene integration but has major manufacture challenges.
  • cssDNA circular single stranded DNA
  • non-viral DNA donor template mediated genome integration is largely bottlenecked by the nucleus delivery of the donor DNAs, especially for large transgene integration. Therefore, developing a more efficient gene integration method, especially for long sequences with non-viral donor payload is an essential milestone to unlock the full potential for gene editing.
  • cssDNA Circular single stranded DNA was harnessed as a superior genome engineering catalyst (GATALYST) for efficient and safe targeted gene KI across multiple cell types to many targeted loci in the mammalian genome.
  • These enGagers form tripartite complex with sgRNA and cssDNA donors as integrative genome integration machinery, thus facilitate the nucleus translocation and increase the active local concentration of donor DNA.
  • these enGagers outperform unfused canonical endonuclease editors, in plasmid, mRNA and protein forms.
  • the enhancement of integration efficiency ranges from 1.5- to more than 6-fold, with the effect being more prominent for larger than 4Kb transgene KI and in primary cells such as human T cells.
  • the present study sought to develop a set of enhanced GATALYST associated genome editors (enGager) that could form an endonuclease/sgRNA/ssDNA tripartite complex by physically recruiting the cssDNA donors, and facilitate the nucleus shuttling of the DNA donor by virtue of the nucleus localization signal (NLS) peptide tagged on the endonuclease editors.
  • enGager enhanced GATALYST associated genome editors
  • NLS nucleus localization signal
  • the present study initially utilized an all-in-one (AIO) plasmid construct to engineer dual NLS tagged Cas9 fusion constructs for a GFP reporter screening (Cong et al., Science 339, 819–823, 2013).
  • These enGager constructs contain bi-cistronic expression cassettes for Cas9-fusion endonuclease proteins and a single-guide RNA (sgRNA) targeting against the human RAB11A locus (Roth et al., Nature 559, 405–409, 2018).
  • sgRNA single-guide RNA
  • the present study initially tested the Cas9 fusion with full length E. coli RecA and its human homolog protein Rad51. For potential better performance of Rad51-Cas9 fusion, the present study used two mutant forms of Rad51 (Rees et al., Nat Commun 10, 2212, 2019).
  • RecA and Rad51 As the full length RecA and Rad51 has both recombinase activity and DNA binding functions, to uncouple these two functions, the present study also engineered an AIO 30 51354128.1 Attorney Docket No.385722-1001WO1 (00005) construct by fusion of Cas9 with a 36 amino acid (AA) peptide “Brex” which is known as a RecA binding motif to recruit endogenous RecA/Rad51 recombinase, but does not have DNA binding capability in the cells (Fig.1A).
  • the KI efficiency by Cas9-Brex fusion nuclease is even lower than Cas9 alone (4.9% vs 6.8%) indicating that only deploying the recombination function is not sufficient to enhance cssDNA mediated genome targeting.
  • the present study found that the enGager induced knock-in enhancement is durable over time at 8 days and 14 days post-electroporation.
  • the Cas9-RecA fusion enGager is able to induce >3-fold KI enhancement as compared to unfused Cas9 (Fig.1D).
  • the present study further tested the knock-in efficiency at RAB11A locus in K562 cells for a larger cssDNA donor with a 4Kb GFP payload.
  • Example 4 Engineering small enGagers with Cas9 fused ssDNA binding motifs [00186] Based on the Brex- and full-length recombinase-Cas9 fusion results, it was hypothesized that the rate-limiting step to enhance the ssDNA mediated HDR is to increase the effective nucleoplasm delivery of the donor template, which in turn increase the local concentration of the tripartite HDR machinery on the targeted genomic locus including the DNA donor template. RecA family DNA recombinases have an evolutionarily conserved ssDNA binding L2 loop peptide that encompasses 20-24 amino acid with an extremely conserved central 31 51354128.1 Attorney Docket No.385722-1001WO1 (00005) aromatic residue.
  • the present study engineered smaller enGager editors that only capture the DNA binding feature.
  • the present study selected the 20 amino acid peptide sequences FECO, WECO and YECO from Voloshin et al. which demonstrated several fold higher binding affinity to ssDNA than to dsDNA in sequence independent manner (Science 272, 868–872, 1996).
  • the present study engineered smaller AIO constructs with Cas9 fused to either one copy or three tandem copies of these DNA binding motifs linked with multiple GS linker sequences (Fig.2A).
  • Example 5 Identification of additional enGagers from Cas9-ssDNA binding module chimeras
  • the present study performed an extensive search of RecA family recombinase and other protein families that has ssDNA binding capability, either small motif peptide or structured protein domain.
  • the present study tested DrRecA full length and its 20 amino acid L2 peptide, E. coli ssDNA binding protein, Lambda Red subunit of bacteriophage recombineering protein, E. coli ssDNA annealing protein RecT. (Figs .3A-3B).
  • Example 6 EnGager mediated genome integration enhancement is specific to ssDNA
  • mRNA has been an attractive approach to reduce cytotoxicity and enhance delivery using lipid nanoparticle technology for vaccine development and gene therapy.
  • the present study used mRNA form of RecA and FECO enGagers for the remaining of this study (Fig.4A). Initially, the RecA enGager mediated enhancement of a 2Kb RAB11A GFP reporter integration in K562 cells by electroporation delivery was validated.
  • RecA mRNA enGager When co-electroporated with various dose of cssDNA donor templates at 0.3, 1, 1.5, 2 to 3 ug per reaction, RecA mRNA enGager (at 1 ug per reaction) significantly increases the GFP KI compared to WT Cas9 mRNA (Figs.4B-4C). As RecA L2 peptides also have low binding affinity to dsDNA, the present study sought to investigate if these enGagers can facilitate HDR mediated KI by dsDNA donor templates.
  • Example 7 Engagers in mRNA form enhance ultra-large transgene integration on various genomic loci 33 51354128.1 Attorney Docket No.385722-1001WO1 (00005) [00189] The present study then assessed the effect of enGagers more broadly on various genomic loci and for donor payload with various size, especially 4 and 8Kb payloads that are around AAV6 and lentivirus packaging limit, respectively. Utilizing highly purified mRNA form of FECO and RecA enGagers, the present study tested the efficiency of GFP transgene KI on two genomic loci with various length of payload.
  • the present study designed 2Kb, 4Kb and 8Kb cssDNA payloads, for another target site, the clinically relevant immune cell therapy B2M locus (Ren et al., Clinical Cancer Research 23, 2255–2266, 2017), the present study designed 2Kb and 4Kb cssDNA payloads (Figs.5A and 5D).
  • Fig. 5B when mRNA enGager/sgRNA with cssDNA donor templates were co-electroporated to K562 cells, FECO and RecA enGagers increase the GFP transgene KI to 44.6-48.5% from 30.10% by WT Cas9 for 2Kb RAB11A GFP payload.
  • FECO and RecA enGagers increase the GFP transgene KI to 11.07-13.77% from 6.17% by WT Cas9.
  • FECO and RecA enGagers increase the GFP transgene KI to 3.73-5.17% from 2.97% by WT Cas9.
  • FECO and RecA enGagers increase the KI efficiency to 39.6-43.97% from 30.13% by WT Cas9 for 2Kb GFP transgene, and increase the KI efficiency to 10.67-14.07% from 6.27% by WT Cas9 for 4Kb GFP transgene (Fig.5E).
  • the present study chose a ⁇ 3Kb CD19-CD22 dual CAR construct that has been demonstrated anti-tumor function for potential treatment of patient with Acute Lymphoblastic Leukemia (ALL) (Fig.6A) (Fry et al., Nat Med 24, 20–28, 2018).
  • ALL Acute Lymphoblastic Leukemia
  • Fig.6A Acute Lymphoblastic Leukemia
  • the present study used PE conjugated Protein-L binder to analyze the KI efficiency.
  • GS- FECO mRNA enGager When delivered by co-electroporation with CD19-CD22 dual CAR cssDNA doner template in CD4+/CD8+ primary pan-T cells, GS- FECO mRNA enGager achieved 30.2% to 33.4% targeted CAR KI at day 7 post-delivery, whereas WT Cas9 mRNA only achieved 5% to 14.1% of CAR KI with various mRNA dosages. 34 51354128.1 Attorney Docket No.385722-1001WO1 (00005) Especially when lower dose of mRNA editors was used, GS-FECO enGager enhance cssDNA mediated CAR-T engineered by >6 fold (Fig.6B).
  • GS-FECO enGager mediated enhancement of dual CAR-T engineering did not compromise T cell counts, proliferation and cell viability compared to WT Cas9 (Fig.6C).
  • the present study used the Incucyte live imaging approach to monitor the cell killing functions over the course of 94 hours. The present study titrated the E:T ratio at 2:25:1, 4.5:1 and 9:1.
  • GS-FECO enGager mRNA engineered CD19-CD22 dual CAR-T cells demonstrated more effective and durable cancer cell killing function compared to WT Cas9 mRNA engineered dual CAR-T cells at every E:T ratio tested (Figs.6D-6E).
  • Example 9 [00191] In the present study, a set of enGager endonucleases by fusing Cas9 with various single stranded DNA binding protein modules, including some small versions based on as few as 20 amino acid motifs, was presented.
  • the engineered enGagers with single stranded DNA binding protein can recruit cssDNA donor template and form a tripartite editing machinery for efficient translocation of the entire tripartite editing complex from cytoplasm to nucleoplasm.
  • the donor DNA has higher effective local concentration in the nucleus and to the targeted genomic locus for more efficient homologous directed genome integration (bottom).
  • Cas9 alone is not able to engage and recruit cssDNA donor template, therefore the donor DNA templates are restricted in the cytoplasm with relatively low level of gene KI efficiency.
  • Frist plasmids containing T7 promoter, 5’ UTR, Cas9 fusion sequence ORF 3’ UTR, bovine growth hormone polyadenylation signal (bGH polyA), and 64 poly adenine sequence were cloned.
  • restriction sites were inserted at the end of the 64 poly adenines.
  • plasmids were produced, ⁇ 50 ⁇ g of plasmids were digested using a restriction enzyme to cut the plasmids at the added restriction site for linearization. After 24 hours, the resulting linear DNA was cleaned through a DNA clean-up kit from zymo (Cat# D4029).
  • RNA Clean & concentrator from Zymo (Cat # R1019). DNase was also used during the purification to remove residual DNA templates from the solutions. After in vitro transcriptions, each mRNA was analyzed using agarose gel analysis.500ng of mRNA were diluted to 20ul of nuclease-free water then 40ul of 2 x RNA loading dye from ThermoFisher Scientific (Cat# R0641) was added.
  • HEK293 lipofectamine transfection [00194] For lipofectamine transfection of mRNA editors/sgRNA (mRNA cocktail) and DNA donors, 24 well plates are coated with PLO for 2hrs. Plates were washed 2X and dried before they were plated with cells. For each well 2X10 5 cells were plated on Day 0 using 500 ul of complete DMEM media. On Day 1, 250uls of media was slowly replaced with equal volume of serum free and antibiotic free DMEM media. Both mRNA cocktail and DNA were prepared separately as per the conditions.
  • mRNA cocktail To prepare the mRNA cocktail, individual mRNA construct (1ug/well) and RAB11A sgRNA (2.54ul/well from a stock of 80uM) were diluted in 1:1 ratio with lipofectamine 3000 and incubated for 10-15 minutes; for DNA prep, both single stranded and double stranded DNAs were packaged separately. For both, concentration of 1ug/well was used. Respective DNA was mixed with P3000 reagent (1ug/well) and the whole mix was diluted with lipofectamine 3000 in 1:1 dilution. The DNA cocktail was also incubated at RT for 10-15 mins. After 15 mins of incubation, mRNA cocktail and DNA cocktail were added in 1:1 ratio as per the plate map.
  • Donor template sequences (transgene sequence flanked with 5’ and 3’ homology arms at 300-500 nt in length) are constructed as dsDNA and placed into phagemid vector by Golden Gate Assembly. An XL1-Blue E.
  • Coli Strain was co-transformed with the M13 helper plasmid and phagemid containing donor template sequences and selected in agar plate with kanamycin (50 ⁇ g/mL), carbenicillin (100 ⁇ g/mL). Single colony was selected and grown for ⁇ 24 hours (37 58 51354128.1 Attorney Docket No.385722-1001WO1 (00005) °C, 225 rpm) in 250 mL 2xYT media (1.6% tryptone, 1% yeast extract, 0.25% NaCl) to reach OD600 between 2.5-3.0.
  • the bacteria were pelleted by centrifugation and the phage particles in the supernatant were precipitated by PEG-8000 by centrifugation, washed and lysed in 20 mM MOPS, 1M Guanidine-HCl and 2% Triton X-100.
  • the cssDNA released from phage were then extracted with NucleoBond Xtra Midi EF kit (Macherey-Nagel) following the manufacturer's instructions.
  • the concentration of cssDNA is determined by Nanodrop. Ratios of Absorbance (A260 nm/280 nm and 260nm/230nm) will reflect consistent purity (1.8 and > 2, respectively) from serial preps.
  • cssDNA is verified by DNA sequencing using custom-designed staggered sequencing primers for complete coverage.
  • Cell culture [00196] K562 cells (ATCC, CCL-243) were maintained in RPMI-1640 media with 10% FBS and 1% penicillin and streptomycin.
  • HEK293T (ATCC) cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin (Gibco).
  • iPSC Thermo Fisher
  • StemFlex Thermo Fisher
  • iPSC colonies were checked regularly and passaged using ReLeSR (StemCell Technologies) every 3-4 days of culture. iPSCs were ready for electroporation after 2- 3 passages.
  • T cells were cultured and expanded in TexMACS Medium (Miltenyi) supplemented with 200 IU/mL Human IL-2 IS (Miltenyi). T cells were activated for 2 days with T Cell TransAct (Miltenyi) before electroporation. All cells were maintained in a humidified incubator at 37 °C and 5 % CO2, unless otherwise specified. Cell count viability were determined using Via2-Cassette in NUCLEOCOUNTER® NC-202 (ChemoMetec) on specified days after engineering.
  • Electroporation of Cas9 RNP, AIO plasmid and mRNA/sgRNA complex with DNA donor All K562, HEK293T, iPSC and T cell electroporation were performed using the AmaxaTM 96-well ShuttleTM with the4D Nucleofector (Lonza). Cas9 nucleases and sgRNAs were precomplexed in supplemented NUCLEOFECTOR® Solution for 20 min at room temperature and the RNP solution was made up to a final volume of 2.5 ⁇ L (10X). For electroporating K562 cells, SF Cell Line 4D-NucleofectorTM Kit and 250,000-500,000 cells per reaction were used with program FF-120.
  • SF Cell Line 4D- 59 51354128.1 Attorney Docket No.385722-1001WO1 (00005) NUCLEOFECTORTM Kit and 200,000-300,000 cells per reaction were used with program FS- 100.
  • iPSC cells 100,000 cells per reaction were used with P3 Primary Cell 4D-NucleofectorTM Kit and program CA-137.
  • 2x106 cells per reaction were used with P3 Primary Cell 4D-NUCLEOFECTORTM Kit and program EO-115.
  • Indicated amount of HDR donor template (cssDNA or dsDNA) were co-electroporation with RNP.
  • mRNA or AIO plasmid enGager electroporation 1 ug-1.5ug of mRNA or DNA were electroporated together with indicated amount of HDR donor templates, with the same electroporation parameters used for RNP electroporation. After electroporation, cells were incubated in humidified 32°C incubator with 5 % CO 2 for 12-24 hours followed up transferring to 37°C incubator for additional days.
  • the ratio of Nucleofector solution to supplement is 4.5: 1 3.
  • cssDNA stock css76 (RAB11-GFP 2Kb payload) and css116 (RAB11-GFP 4kb payload).
  • the stock concentration is >2 ug/ul.
  • a) For FLO Prepare cells for FLO analysis. KI efficiency is checked by calculating the increase in GFP+ve cells over the control condition. b) To propagate cells for Day 7, take 40 ⁇ ls of cells solution and add to 160 ⁇ ls of respective media in the fresh 96 well round bottom plate. Outer wells should have water jacket to prevent evaporation. 61 51354128.1 Attorney Docket No.385722-1001WO1 (00005) 18. On Day6, again change the media of the cells and follow the same protocol for FLO on Day7. 19. Data analysis is performed using FlowJo_v10.8.0_CL software.
  • T cells were activated for 2 days with T Cell TransAct (Miltenyi) before electroporation.48 h after initiating T-cell initiation and activation, T cells were electroporated using AmaxaTM 96-well ShuttleTM in 4D Nucleofector.2 ⁇ 106 cells were mixed with 25 pmol of Cas9 WT protein or 1 ug of enGager mRNA and 50 pmol of gRNA (RNP) into each well.
  • RNP gRNA
  • For cssDNA engineered cells 2 ⁇ g of cssDNA encoding bi-specific CD19 ⁇ CD22 CAR was electroporated with RNP or mRNA/sgRNA cocktail targeting TRAC locus.
  • Embodiment 1 A DNA editing construct, comprising: a DNA editing motif having a site-specific DNA endonuclease activity; a nuclear localization signal (NLS); and a single stranded DNA (ssDNA) binding motif.
  • Embodiment 2. The construct of Embodiment 1, wherein the DNA editing motif introduces a double strand break, a single strand break, or no double strand break in double stranded DNA molecules.
  • Embodiment 4 The construct of any one of Embodiments 1-3, wherein the DNA editing motif is at least one site-specific nuclease selected from the group consisting of a Cas nuclease, a zinc-finger nuclease, a meganuclease, and a transcription activator-like effector nuclease (TALEN).
  • TALEN transcription activator-like effector nuclease
  • Embodiment 6 The construct of any one of Embodiments 1-4, wherein the DNA editing motif is a class I Cas protein or a class II Cas protein.
  • Embodiment 6 The construct of any one of Embodiments 1-5, wherein the DNA editing motif is Cas9, Cas3, Cas10, Cas11, CasX, Cas12a, MAD7, or catalytic dead forms or nickase forms thereof.
  • the nuclear localization signal is at least one selected from the group consisting of a 53BP1 NLS, a CXCR4 NLS, an ERK5 NLS, an EWS NLS, an IER5 NLS, an ING4 NLS, an Hrp1 NLS, an MSX1 NLS, an NLS-RAR ⁇ NLS, a nucleoplasmin NLS, a Pho4 NLS, a PTHrP NLS, an rpL23a NLS, a simian virus 40 (SV40) large T antigen NLS, a UL79 NLS, a VACM-1/CUL5 NLS, a VP1 NLS, 63 51354128.1 Attorney Docket No.385722-1001WO1 (00005) a PABPN1 NLS, a STAT1 NLS, a FGF2 NLS, a RARa NLS, a tandem phenylalanine-glycine repeat
  • Embodiment 8 The construct of any one of Embodiments 1-7, wherein the construct comprises a plurality of the nuclear localization signals.
  • Embodiment 9. The construct of any one of Embodiments 1-8, wherein at least one of the following applies: (a) the ssDNA binding motif is or comprises a K homology (KH) domain ssDNA binding motif, an oligonucleotide/oligosaccharide binding (OB) fold ssDNA binding motif, a RecA/Rad51/DMC1 loop L2 homologous ssDNA binding motif, an RNA recognition motif (RRM) that binds ssDNA, or a whirly domain ssDNA binding motif, (b) the ssDNA binding motif is or comprises a bacterial single-stranded DNA-binding (SSB) protein or ssDNA binding portions thereof, a RecA/Rad51/DMC family protein or ssDNA binding portions thereof, a replication protein
  • KH K homo
  • Embodiment 10 The construct of any one of Embodiments 1-9, wherein the ssDNA binding motif is or comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:11. [00211] Embodiment 11. The construct of any one of Embodiments 1-10, wherein the construct comprises a plurality of the ssDNA binding motifs. [00212] Embodiment 12. The construct of any one of Embodiments 1-11, further comprising one or more linkers connecting the DNA editing motif, the nuclear localization signal, and the ssDNA binding motif to each other. [00213] Embodiment 13.
  • Embodiment 12 wherein the one or more linkers connects the DNA editing motif, the nuclear localization signal, and the ssDNA binding motif to each other covalently.
  • Embodiment 14 The construct of any one of Embodiments 12-13, wherein the one or more linkers independently are polypeptide linkers. 64 51354128.1 Attorney Docket No.385722-1001WO1 (00005) [00215] Embodiment 15. The construct of Embodiment 14, wherein each of the polypeptide linkers independently has a length ranging from about 2 amino acid residues to about 100 amino acid residues. [00216] Embodiment 16. The construct of any one of Embodiments 1-15, wherein the construct is a protein.
  • Embodiment 17 A nucleic acid encoding the construct of Embodiment 16.
  • Embodiment 18 The nucleic acid of Embodiment 17, further encodes a guide RNA (gRNA) that provides the site-specificity to the DNA editing motif.
  • gRNA guide RNA
  • Embodiment 19 The nucleic acid of any one of Embodiments 17-18, wherein the nucleic acid is comprised in an expression vector.
  • Embodiment 20 The nucleic acid of Embodiment 19, wherein the expression vector is a plasmid vector, an RNA vector, or a viral vector.
  • Embodiment 21 Embodiment 21.
  • a composition for editing DNA comprising: the construct of any one of Embodiments 1-16; and a single stranded DNA (ssDNA) having a DNA insert, a 5’-homology arm, and a 3’-homology arm, wherein the 5’ homology arm and the 3’ homology arm are complementary to the nuclear DNA in a target region.
  • ssDNA single stranded DNA
  • Embodiment 25 The composition of any one of Embodiments 22-24, wherein the DNA insert has a length ranging from 1 nucleotide to about 50,000 nucleotides. 65 51354128.1 Attorney Docket No.385722-1001WO1 (00005) [00226] Embodiment 26.
  • a kit for editing DNA comprising: the construct of any one of Embodiments 1-16 and/or the nucleic acid of any one of Embodiments 17-21; and a single stranded DNA (ssDNA), a nucleic acid that encodes the ssDNA, or a nucleic acid for constructing the ssDNA, wherein the ssDNA comprises a DNA insert, a 5’-homology arm, and a 3’-homology arm, wherein the 5’ homology arm and the 3’ homology arm are complementary to the nuclear DNA in a target region.
  • ssDNA single stranded DNA
  • the ssDNA comprises a DNA insert, a 5’-homology arm, and a 3’-homology arm, wherein the 5’ homology arm and the 3’ homology arm are complementary to the nuclear DNA in a target region.
  • the kit of Embodiment 26 wherein least one of the following applies: (a) the ssDNA is a circular ssDNA (cssDNA), a linear ssDNA (lssDNA), a double stranded DNA/circular single stranded DNA (dsDNA/cssDNA) hybrid having an ssDNA section, or an RNA/DNA hybrid having an ssDNA section; (b) the ssDNA is chemically modified; (c) the ssDNA is produced from a cell free system or a cell culture system. [00228] Embodiment 28.
  • each of the 5’- homology arm and the 3’-homology arm independently has a length ranging from about 25 nucleotides to about 5000 nucleotides.
  • Embodiment 29 The kit of any one of Embodiments 26-28, wherein the DNA insert has a length ranging from 1 nucleotide to about 50,000 nucleotides.
  • Embodiment 30 Embodiment 30.
  • kits of any one of Embodiments 26-29 wherein at least one of the following applies: (a) the nucleic acid for constructing the ssDNA donor template encodes every elements of the ssDNA donor template, except for the DNA insert, (b) the nucleic acid for constructing ssDNA donor template encodes every elements of the ssDNA donor template, except for the 5’ homology arm and the 3’ homology arm, (c) the nucleic acid for constructing ssDNA donor template does not encode the 5’ homology arm, the 3’ homology arm, and the DNA insert. [00231] Embodiment 31.
  • Embodiment 30 wherein the nucleic acid includes a multiple cloning site for inserting the 5’ homology arm, the 3’ homology arm, and/or the DNA insert.
  • 66 51354128.1 Attorney Docket No.385722-1001WO1 (00005)
  • Embodiment 32 The kit of any one of Embodiments 30-31, wherein the nucleic acid for constructing ssDNA donor template is a plasmid or a viral vector.
  • Embodiment 33 Embodiment 33.
  • kits of any one of Embodiment 27-32 further comprising a component for delivering the construct, the nucleic acid for encoding the construct, the ssDNA, and/or the nuclei acid for encoding or constructing the ssDNA into a cell.
  • a component for delivering the construct, the nucleic acid for encoding the construct, the ssDNA, and/or the nuclei acid for encoding or constructing the ssDNA into a cell comprising a component for delivering the construct, the nucleic acid for encoding the construct, the ssDNA, and/or the nuclei acid for encoding or constructing the ssDNA into a cell.
  • a method of editing a nuclear DNA in a cell comprising delivering into the cell the construct of any one of Embodiments 1-16 and/or the nucleic acid of any one of Embodiments 17-21; and a single stranded DNA (ssDNA) having a DNA insert, a 5’-homology arm, and a 3’-homology arm, wherein the 5’ homology arm and the 3’ homology arm are complementary to the nuclear DNA in a target region.
  • ssDNA single stranded DNA
  • the ssDNA is a circular ssDNA (cssDNA), a linear ssDNA (lssDNA), a double stranded DNA/circular single stranded DNA (dsDNA/cssDNA) hybrid having an ssDNA section, or an RNA/DNA hybrid having an ssDNA section;
  • the ssDNA is chemically modified;
  • the ssDNA is produced from a cell free system or a cell culture system.
  • Embodiment 37 The method of any one of Embodiments 34-35, wherein each of the 5’-homology arm and the 3’-homology arm independently has a length ranging from about 25 nucleotides to about 5000 nucleotides.
  • Embodiment 37 The method of any one of Embodiments 34-36, wherein the DNA insert has a length ranging from 1 nucleotide to about 50,000 nucleotides.
  • Embodiment 38 Embodiment 38.
  • Embodiment 39 The method of any one of Embodiments 34-38, wherein the nuclear DNA is a genomic DNA.
  • Embodiment 40 The method of any one of Embodiments 34-37, wherein the construct and/or nucleic acid and the ssDNA are delivered into the cell by a viral vector, a lipid or non-lipid nanoparticle delivery, an electroporation delivery, a gene gun delivery, or an injection.
  • Embodiment 41 The method of any one of Embodiments 34-40, wherein the cell is in the body of a subject.
  • Embodiment 42 The method of any one of Embodiments 34-40, wherein the cell is in the body of a subject.
  • Embodiment 41 wherein the subject is a mammal, such as a human.
  • Embodiment 43 A non-natural polypeptide comprising an amino acid sequence having 90% or higher identity to the amino acid sequence set forth in SEQ ID NO:11.
  • Embodiment 44 The non-natural polypeptide of Embodiment 43, wherein the non- natural polypeptide is a ssDNA-binding polypeptide.

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Abstract

L'invention concerne des constructions d'édition d'ADN comprenant un motif d'édition d'ADN, un signal de localisation nucléaire et un motif de liaison à l'ADN monocaténaire. Les constructions d'édition d'ADN, lorsqu'elles sont utilisées conjointement avec un modèle donneur d'ADN monocaténaire, peuvent obtenir une efficacité d'édition améliorée. L'invention concerne également des acides nucléiques pour coder la construction d'édition d'ADN, des compositions d'édition d'ADN et des kits comprenant les constructions d'édition d'ADN, ainsi que des procédés d'édition d'ADN à l'aide des constructions d'édition d'ADN.
PCT/US2023/080718 2022-11-22 2023-11-21 Constructions d'édition d'adn et procédés les utilisant WO2024112773A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996040765A1 (fr) * 1995-06-07 1996-12-19 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services ACTIVATION D'APPARIEMENTS D'ADN HOMOLOGUES PAR DES PEPTIDES DERIVES DU RecA
WO2018035495A1 (fr) * 2016-08-19 2018-02-22 Whitehead Institute For Biomedical Research Méthodes d'édition de la méthylation de l'adn
US20180171359A1 (en) * 2013-04-11 2018-06-21 Caribou Biosciences, Inc. Methods of modifying a target nucleic acid with an argonaute
WO2021113769A1 (fr) * 2019-12-07 2021-06-10 Scribe Therapeutics Inc. Compositions et méthodes pour le ciblage de htt

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996040765A1 (fr) * 1995-06-07 1996-12-19 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services ACTIVATION D'APPARIEMENTS D'ADN HOMOLOGUES PAR DES PEPTIDES DERIVES DU RecA
US20180171359A1 (en) * 2013-04-11 2018-06-21 Caribou Biosciences, Inc. Methods of modifying a target nucleic acid with an argonaute
WO2018035495A1 (fr) * 2016-08-19 2018-02-22 Whitehead Institute For Biomedical Research Méthodes d'édition de la méthylation de l'adn
WO2021113769A1 (fr) * 2019-12-07 2021-06-10 Scribe Therapeutics Inc. Compositions et méthodes pour le ciblage de htt

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