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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
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/102—Mutagenizing nucleic acids
• • 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
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
• • 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
  • C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding
• • 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
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
  • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
  • C12Y305/04005—Cytidine deaminase (3.5.4.5)

Definitions

• the base editor system further comprises a nickase that creates a single-stranded DNA break on the unedited or edited strand, wherein the DNA break is no more than about 500 bps, optionally no more than 200 bps, optionally about 10-50 bps, from the cytosine to be edited.
• the nickase may be, e.g., a ZFP-based nickase, a TALE- based nickase, or a CRISPR-based nickase.
• the nickase is a ZFP- based nickase formed by dimerization of a first nickase domain and a second nickase domain fused respectively to two ZFP domains that bind to the target genomic region, wherein the first and second nickase domains are inactive, or lack significant or specific nickase activity, on their own.
• one of the nickase domains is fused to the first or second ZFP-cytidine deaminase fusion protein, and the other nickase domain is fused to a third ZFP domain that binds to a third sequence in the target genomic region.
• the base editor system further comprises an inhibitory component of the cytidine deaminase, e.g., a toxin-derived deaminase inhibitor (TDDI) where the cytidine deaminase is a TDD.
• TDDI toxin-derived deaminase inhibitor
• the inhibitor may be a DddI component where the cytidine deaminase is DddA.
• the protein components of the present base editor systems are provided to the cells by means of expression cassettes or constructs.
• Such cassettes or constructs may be provided to the cells on the same or separate expression vectors such as viral vectors.
• the viral vectors may be, e.g., adeno-associated viral (AAV) vectors, adenoviral vectors, or lentiviral vectors.
• AAV adeno-associated viral
• the cytidine deaminase is DddA that has a mutation at one or more residues selected from Y1307, T1311, S1331 , V1346, H1366, N1367, N1368, P1369, E1370, G1371, T1372, F1375, V1392, P1394, P1395, I1399, P1400, V1401, K1402, A1405, and T1406 in SEQ ID NO: 72.
• the present disclosure provides a pair of fusion proteins comprising a) a first fusion protein that comprises i) a zinc finger protein (ZFP) domain that binds to a gene (which may be a eukaryotic, e.g., human, gene), and ii) a first dimerization domain, and b) a second fusion protein that comprises i) a cytidine deaminase inhibitory domain, e.g., wherein the cytidine deaminase is a TDD comprising an amino acid sequence at least 90% identical to SEQ ID NO: 49, 81, 92, 95, 98, 101, 104, 107, 134, 143, 152, 157, 162, 167, 172, 177, 184, 189, 194, 199, 204, 209, 214, or 219, and ii) a second dimerization domain, wherein the first and second dimerization domains can dim
• a cell which may be a eukaryotic cell, e.g., a mammalian cell or a plant cell
• a cell comprising a base editor system as described herein, fusion protein(s) as described herein, isolated nucleic acid molecule(s) as described herein, expression construct(s) as described herein, or viral vector(s) as described herein.
• the mammalian cell is a human cell, such as a human embryonic stem or a human induced pluripotent stem cell.
• genetically engineered cells which may be eukaryotic cells, e.g., mammalian cells such as human iPSCs or plant cells
• eukaryotic cells e.g., mammalian cells such as human iPSCs or plant cells
• FIG. 1 is a schematic illustrating a pair of ZFP-TDD fusion proteins for C to T base editing.
• the rectangles represent DNA-binding zinc fingers in the ZFP domains of the fusion proteins.
• the arrow shapes above the underlined C nucleotide represent dimerized TDD domains of the fusion proteins.
• the black lines between the zinc finger domains and the TDD domains represent peptide linkers.
• FIG. 2A is a schematic showing ZFP designs for CCR5- targeting ZFP-TDD fusion protein pairs.
• C9, C10, C18, and C24 are target nucleotides for base editing. Top strand (left to right): SEQ ID NO: 227.
• FIG. 2B is a schematic showing an example of a construct design for a dimerized ZFP-DddA pair.
• FLAG FLAG tag.
• NLS nuclear localization sequence.
• UGI uracil DNA glycosylase inhibitor.
• FIG. 3 is a table showing the heatmap results of C to T base editing at a human CCR5 locus by a series of ZFP-DddA fusion protein pairs.
• the degree of editing activity corresponds to the darkness of shading within a cell.
• L0, L7A, and L26 represent peptide linkers used to fuse the DddA domain to the C-terminus of the ZFP domain in the fusion protein.
• FIG. 5 is a schematic showing ZFP designs for CCR5 -targeting ZFP-TDD fusion proteins.
• C9, C10, C18, and C24 are target nucleotides for base editing. From top to bottom: SEQ ID NO: 229 (left to right), SEQ ID NO: 230 (right to left), SEQ ID NO: 231 (left to right), SEQ ID NO: 232 (right to left), SEQ ID NO: 233 (left to right), and SEQ ID NO: 234 (right to left).
• FIG. 7B is a table showing the heatmap results of DddA C to T base editing at a human CCR5 locus using the approach of FIG. 7A.
• the degree of editing activity corresponds to the darkness of shading within a cell.
• FIG. 8 is a schematic illustrating the combined use of the ZFP-TDD base editing system and a CRISPR/Cas-based nickase system.
• FIG. 9 is a schematic illustrating an example of a trimeric ZFP-TDD + FokI nickase base editing system.
• FIG. 13 is a table showing the heatmap results of the highest frequency of C to T base editing for any C in the CCR5 base editing window by ZFP fusion protein pairs with TDD1-TDD6.
• FIG. 16 is a table showing the heatmap results of the highest frequency of C to T base editing at a human CIITA locus (“site 2”) by a series of ZFP-TDD fusion protein pairs.
• the degree of editing activity corresponds to the darkness of shading within a cell. 01: TDD1; 014: TDD14; etc.
• FIG. 17 is a table showing the heatmap results of the highest frequency of C to T base editing for any C (underlined) in the CIITA base editing window and its sequence motif for DddA, TDD4, TDD6, TDD9, TDD10, TDD14, TDD15 and TDD18.
• Amplicon SEQ ID NO: 244. 04: TDD4; 06: TDD6; etc.
• FIG. 19 is a schematic illustrating a design for inhibition of a TDD with a targeted ZFP-TDDI.
• the present systems and methods are advantageous in part due to the compact size of the ZFP domains in the fusion proteins.
• the large physical size of a TALE and the long C-terminal TALE linker may limit how small the base editing window can be, as well as design density.
• the size and highly repetitive nature of engineered TALEs also make it challenging to deliver TALE-based base editors to human cells using common viral vectors.
• the present ZFP-derived base editing systems circumvent these problems. For instance, the compactness of these ZFP-derived systems may allow for packaging within a single AAV vector, in contrast to TALE base editor systems (e.g., TALE-TDDs) or CRISPR/Cas base editor systems.
• a nickase in the editing system so as to allow the generation of a DNA nick near the edited base and thereby facilitate the DNA repair machinery to change the base opposite the edited C from G to a corresponding A, forming the correct T: A base pair.
• the inclusion of a nickase may greatly increase the base editing efficiency.
• the ZFP-cytidine deaminase fusion proteins of the present disclosure comprise a cytidine deaminase domain in addition to a ZFP domain.
• a cytidine deaminase domain for example, may catalyze the deamination of cytosine to uracil, wherein the uracil is replaced by a thymine base during DNA replication or repair.
• the deaminase domain may be naturally-occurring or may be engineered.
• a cytidine deaminase of the present disclosure operates on double-stranded DNA.
• the ZFP binds to a target site that is 1 to 100 (or any number therebetween) nucleotides on either side of the targeted base. In other embodiments, the ZFP binds to a target site that is 1 to 50 (or any number therebetween) nucleotides on either side of the targeted base.
• the TDD and TDDI may be any described herein.
• the TDD may be DddA and the TDDI may be Dddl.
• other cytidine deaminases and inhibitors may be used in place of the TDD and TDDI.
• a multiplex system described herein may comprise a first ZFP-cytidine deaminase pair and a second ZFP-cytidine deaminase pair, wherein the first and second pairs utilize different cytidine deaminases (e.g., selected from those described herein).
• the edited cells exhibit little to no off-target base editing (e.g., less than 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% off-target base editing); however, as base editing of off-target sites may not be prone to translocations or other genomic arrangements, higher percentages may also be contemplated.
• the present disclosure also provides nucleic acid molecules encoding the ZFP fusion proteins described herein, which may be part of a viral or non-viral vector. Further, the present disclosure provides a cell or population of cells comprising a base editor system as described herein, as well as descendants of such cells, wherein the cells comprise one or more edited bases.
• the AAV may be AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV8.2, AAV9, AAV. PHP. B, AAV. PHP. eB, or AAVrh10, or of a novel serotype or a pseudotype such as AAV2/8, AAV2/5, AAV2/6, AAV2/9, or AAV2/6/9.
• the systems can be used to modify pluripotent stem cells prior to their differentiation into multiple cell types.
• a lymphoid cell precursor may be modified prior to differentiation into lymphoid cell types such as regulatory T cells, effector T cells, natural killer cells, etc.
• the multiplex base editor systems of the present disclosure (comprising more than one ZFP-cytidine deaminase (e.g., ZFP-TDD) pair), in particular, can be used to prepare cells with multiple base edits at once, including pluripotent cells.
• the multiplex systems may be used to prepare, e.g., allogeneic T cells.
• the systems comprise a ZFP-cytidine deaminase inhibitor (e.g., ZFP-TDDI) that can be induced to assemble in the presence or absence of a dimerization-regulating agent, as described herein, it is contemplated that the edited cells may be placed under the control of a “kill switch” activated upon administration of the agent.
• ZFP-cytidine deaminase inhibitor e.g., ZFP-TDDI
• the edited cells may be placed under the control of a “kill switch” activated upon administration of the agent.
• the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier such as water, saline (e.g., phosphate-buffered saline), dextrose, glycerol, sucrose, lactose, gelatin, dextran, albumin, or pectin.
• a pharmaceutically acceptable carrier such as water, saline (e.g., phosphate-buffered saline), dextrose, glycerol, sucrose, lactose, gelatin, dextran, albumin, or pectin.
• the composition may contain auxiliary substances, such as, wetting or emulsifying agents, pH-buffering agents, stabilizing agents, or other reagents that enhance the effectiveness of the pharmaceutical composition.
• the pharmaceutical composition may contain delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, and vesicles.
• DddA-G1333 residues at residues G1404
• DddA-G1407 residues at residues G1404
• DddA-G1407 residues at residues G1404
• DddA-G1407 residues at residues at residues G1404
• DddA-G1407 residues at residues at residues G1404
• DddA-G1407 residues at residues at residues G1407
• Eight left ZFPs and five right ZFPs were designed to target the DddA halves to a site at the human CCR5 locus, such that the halves could dimerize at the target site and restore the catalytic activity of DddA.
• the left and right ZFP pairs cover a broad variety of different base editing windows from 2-bp to 24-bp (FIG. 2A).
• each split DddA pair was fused to the C-terminus of a left ZFP and the C-terminal half was fused to the C-terminus of a right ZFP, and vice-versa.
• DddA-G1333 one of three different linkers (L0, L7A and L26) was used, whereas for DddA- G1404 and DddA-G1407, the L26 linker was used.
• the L26 linker was used for all other experiments. unless otherwise indicated.
• a UGI (uracil DNA glycosylase inhibitor) domain was also fused to the C-terminus of each N-terminal and C-terminal half.
• All ZFP- DddA fusion constructs further contained a 3xFLAG tag as well as an SV40 nuclear localization signal fused to the N-terminus of the ZFP.
• An example of a left and right ZFP pair is shown in FIG. 2B.
• PCR primers for the CCR5 locus were designed using Primer3 with the following optimal conditions: amplicon size of 200 nucleotides; a melting temperature of 60 °C; primer length of 20 nucleotides; and GC content of 50%. Sequences for the primers and amplicon are shown in Table 3 below.
• DddA variants with mutations at positions E1370, N1368, and Y1307 were tested in K562 cells according to the protocols described above, using the left and right ZFP pairs shown in FIG.
• the efficiency of base editors can be increased by nicking the unmodified DNA strand with a nickase.
• the unmodified DNA strand then is recognized as newly synthesized by the cell, and the natural DNA repair machinery repairs the nicked DNA strand using the modified strand as a template.
• the unmodified strand can be nicked using a Fokl-derived ZFN or TALEN or a CRISPR/Cas-derived nickase.
• FIGs. 7A and 7B demonstrate a ZFP- TDD base editing design and results, respectively, with a CRISPR/Cas9 nickase.
• the trimeric ZFP-DddA-nickase system was tested in K562 cells according to the protocols described above. As shown in FIG. 11, the trimeric ZFP-DddA-nickase system demonstrated a higher level of base editing activity than CRISPR-based nickases, with around 70% base edits in some cases, and a lower level of indels that approached background. In addition to outperforming the CRISPR-based nickase system, the trimeric ZFP-TDD-nickase system may be highly advantageous in its compact size, which may fit into a single viral vector such as AAV, unlike other platforms such as CRISPR/Cas and TALE-TDD base editor systems.
• FIG. 13 shows a comparison of the highest frequency of editing for each deaminase for any C in the base editing window (based on data shown in FIG. 12 as well as additional replicates). At least three of the TDDs (TDD3, TDD4, and TDD6) demonstrated detectable base editing activity (>0.25% base editing), with TDD4 showing higher maximum activity than DddA.
• TDD enzymes may be inactivated by TDDIs.
• the natural DddA enzyme can be inactivated by the DddI inhibitor.
• a ZFP or TALE linked TDDI can be targeted to a potential TDD-derived cytosine base editor site, preventing that site from being edited (FIG. 19).
• the TDDI inhibitor may be linked to the ZFP using a dimerization domain potentiated by a small molecule, thus putting the editing activity under the control of the small molecule.
• editing can selectively be targeted to certain alleles, e.g., to knock out a detrimental mutant by editing in a stop codon only if the mutation is present.
• JAK2 V617F can be knocked out by editing in a stop codon only if the V617F mutation is present.
• This TDDI approach may also be used to reduce editing at off-target sites, particularly where it cannot be eliminated by other means.

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• 2021
  • 2021-09-24 CN CN202180066106.XA patent/CN116261594A/zh active Pending
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