WO2025126049A2 - Édition génétique multiplex - Google Patents

Édition génétique multiplex Download PDF

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Publication number
WO2025126049A2
WO2025126049A2 PCT/IB2024/062469 IB2024062469W WO2025126049A2 WO 2025126049 A2 WO2025126049 A2 WO 2025126049A2 IB 2024062469 W IB2024062469 W IB 2024062469W WO 2025126049 A2 WO2025126049 A2 WO 2025126049A2
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gene
cells
target
sequence
seq
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WO2025126049A3 (fr
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John KULMAN
Daniel FERULLO
Brian FOCHTMAN
Aishwarya VISHWANATHAN
Vasudevan ACHUTHAN
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CRISPR Therapeutics AG
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CRISPR Therapeutics AG
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
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    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
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    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4214—Receptors for cytokines
    • A61K40/4215—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
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    • 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
    • C12N15/1137—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 against enzymes
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    • 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
    • C12N15/1138—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 against receptors or cell surface proteins
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    • 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)
    • C12Y305/04001—Cytosine deaminase (3.5.4.1)
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    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
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    • 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
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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)
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Definitions

  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • CRISPR associated CRISPR associated systems
  • gRNA customizable guide RNA
  • Genome editing technologies involving non-homologous end-joining (NHEJ) and homology-directed repair (HDR) can lead to gene disruption through the introduction of insertions, deletions, translocations or other DNA rearrangements at the site of a double-stranded DNA break (DSB).
  • NHEJ non-homologous end-joining
  • HDR homology-directed repair
  • DSB double-stranded DNA break
  • the method comprises contacting a plurality of DNA molecules or a plurality of cells comprising the plurality of DNA molecules with (a) a base editor protein or a nucleic acid encoding the base editor protein, and (b) two or more guide RNAs (gRNAs) targeting one or more first target nucleotide sequence of the plurality of DNA molecules, thereby editing the plurality of DNA molecules by deaminating a nucleotide base within the one or more first target nucleotide sequence of the plurality of DNA molecules.
  • the two or more gRNAs can comprise two, three, four, five or more gRNAs.
  • the two or more gRNAs target two or more different target nucleotide sequences in a same target gene. In some embodiments, the two or more gRNAs target two or more different target nucleotide sequences in different target genes. In some embodiments, the two or more gRNAs target a same target nucleotide sequence.
  • the two or more gRNAs can, e.g., target a target gene selected from Regnase-1 (Reg1) gene, Transforming Growth Factor Beta Receptor II (TGFBRII) gene, beta-2-microglobulin ( ⁇ 2M) gene, CD70 gene, T cell receptor alpha chain constant region (TRAC) gene, Cbl Proto-Oncogene B (Cblb) gene, poliovirus receptor (PVR) gene, MHC class I chain-related A/B (MCIA/B) gene, transporter associated with antigen processing 1 (TAP-1) gene, Fas cell surface death receptor (FAS) gene, or a combination thereof.
  • a target gene selected from Regnase-1 (Reg1) gene, Transforming Growth Factor Beta Receptor II (TGFBRII) gene, beta-2-microglobulin ( ⁇ 2M) gene, CD70 gene, T cell receptor alpha chain constant region (TRAC) gene, Cbl Proto-Oncogene B (Cblb) gene, polio
  • the contacting is in vivo in a subject suspected to have, having or diagnosed with a disease or disorder.
  • the contacting can be, for example, ex vivo or in vitro.
  • the method further comprises providing the plurality of cells comprising the plurality of DNA molecules.
  • the method further comprises delivering to the plurality of cells a nucleic acid encoding a chimeric antigen receptor (CAR).
  • the nucleic acid encoding the base editor protein is provided at a concentration of at least about 1 pmol/million cells.
  • each of the two or more gRNAs is provided at a concentration of at least 90 pmol/million cells.
  • the plurality of cells are T cells or precursor cells thereof.
  • the method further comprises generating a population of genetically engineered cells.
  • the population of genetically engineered cells comprise at least two gene edits.
  • at least 50% of the cells in the population of genetically engineered cells comprise at least two genome edits and wherein fewer than 1%, 0.5%, 0.2% or 0.1% of the cells in the population of genetically engineered cells have an insertion, deletion, translocation or other DNA rearrangement.
  • the one or more first target nucleotide sequence encodes a protein and wherein the deamination results in a reduction or inhibition of the expression level of the encoded protein.
  • the one or more first target nucleotide sequence is associated with a disease or disorder.
  • the deamination corrects a point mutation in the at least one target nucleotide sequence associated with the disease or disorder.
  • the one or more first target nucleotide sequence comprises a T to C point mutation associated with a disease or disorder and wherein the deamination of the mutant C base results in a sequence that is not associated with the disease or disorder.
  • the method can, for example, comprises: administering to the subject a therapeutically effective amount of a composition or a pharmaceutical composition comprising: (a) a base editor protein or a nucleic acid encoding the base editor protein, and (b) two or more guide RNAs (gRNAs) targeting one or more target gene, wherein the one or more target gene is associated with the disease or disorder.
  • gRNAs guide RNAs
  • the two or more gRNAs target a same target gene.
  • the two or more gRNAs target two or more different target genes.
  • the disease or disorder can be, for example, a cancer, an autoimmune disease, an allergy, Graves’ diseases, multiple sclerosis, or an infection.
  • Non-limiting examples of cancer include: pancreatic cancer, gastric cancer, ovarian cancer, uterine cancer, breast cancer, prostate cancer, testicular cancer, thyroid cancer, nasopharyngeal cancer, non-small cell lung (NSCLC), glioblastoma, neuronal, soft tissue sarcomas, leukemia, lymphoma, melanoma, colon cancer, colon adenocarcinoma, brain glioblastoma, hepatocellular carcinoma, liver hepatocholangiocarcinoma, osteosarcoma, gastric cancer, esophagus squamous cell carcinoma, advanced stage pancreas cancer, lung adenocarcinoma, lung squamous cell carcinoma, lung small cell cancer, renal carcinoma, intrahepatic biliary cancer, and a combination thereof.
  • NSCLC non-small cell lung
  • the autoimmune disease can be, for example, type 1 diabetes or an autoimmune thyroid disease.
  • the one or more target gene comprises a Cbl Proto-Oncogene B (Cblb) gene, poliovirus receptor (PVR) gene, MHC class I chain-related A/B (MCIA/B) gene, transporter associated with antigen processing 1 (TAP-1) gene, or a combination thereof.
  • the one or more target gene comprise a Reg1 gene, a TGFBRII gene, a ⁇ 2M gene, a TRAC gene, or a combination thereof.
  • the base editor protein is a fusion protein comprising: a Cas9 domain, wherein the Cas9 domain when associated with a guide RNA (gRNA) specifically binds to a target nucleic acid sequence; and a cytidine deaminase domain capable of deaminating a cytosine base in a single-stranded portion of the target nucleic acid sequence.
  • the cytidine deaminase domain comprises a deaminase from an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase, optionally the APOBEC family deaminase is an APOBEC1 deaminase.
  • APOBEC apolipoprotein B mRNA-editing complex
  • the cytidine deaminase domain comprises a rat deaminase, an armadillo deaminase, a bat deaminase, an orangutan deaminase, or a variant thereof.
  • the cytidine deaminase domain comprises (1) an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity to any one of SEQ ID NOs: 1-18, or (2) an amino acid sequence having one, two, three, four, five, six, seven, eight, nine, or ten mismatches relative to any one of SEQ ID NOs: 1-18.
  • the cytidine deaminase domain comprises amino acid mutation(s) at one or more positions functionally equivalent to R30, E31, L32, R33, K34, E35, T36, R52, Q56, N57, N59, K60, H61, V62, L88, S89, W90, R118, Y120, H121, H122, R126, R128, R169, I195, R197, R198, K199, Q200, P201, Q202, and L203 in the deaminase of SEQ ID NO: 1.
  • the cytidine deaminase is encoded by (1) a nucleic acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity to any one of SEQ ID NOs: 19-36; or (2) a nucleic acid sequence having one, two, three, four, five, six, seven, eight, nine, or ten mismatches relative to any one of SEQ ID NOs: 19-36.
  • the Cas9 domain of is a nuclease-inactive Cas9, a dead Cas9, a Cas9 nickase, or a fragment or a variant thereof.
  • the Cas9 domain can be, or can comprise, Streptococcus pyogenes (SpyCas9), Staphylococcus lugdunensis (SluCas9), or derived from Staphylococcus aureus (SaCas9), Neisseria meningitides Cas9, Streptococcus thermophilus Cas9, Treponema denticola Cas9, Campylobacter jejuni Cas9, or a variant thereof.
  • the fusion protein further comprises an uracil glycosylase inhibitor (UGI) domain, wherein the UGI domain inhibits a uracil-DNA glycosylase.
  • UGI uracil glycosylase inhibitor
  • the fusion protein further comprises a nuclear localization sequence.
  • the nucleic acid encoding the base editor protein can, for example, comprise a sequence encoding the cytidine deaminase domain having at least 85%, 90%, 95%, 98%, or 99% identity to any one of SEQ ID NOs: 19-36.
  • one of the two or more gRNAs targets Cblb gene and comprises a spacer sequence (a) having a RNA sequence corresponding to the target sequence of SEQ ID NO: 87; (b) having 1, 2, or 3 mismatches relative to the RNA sequence corresponding to the target sequence of SEQ ID NO: 87; or (c) having about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to the RNA sequence corresponding to the target sequence of SEQ ID NO: 87.
  • one of the two or more gRNAs targets PVR gene and comprises a spacer (a) having any one of the sequences set forth in SEQ ID NOs: 101, 103, 105, and 113-116; (b) having 1, 2, or 3 mismatches relative to any one of the sequences set forth in SEQ ID NOs: 101, 103, 105, and 113-116; or (c) having about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of the sequences set forth in SEQ ID NOs: 101, 103, 105, and 113-116.
  • one of the two or more gRNAs targets MICA gene and comprises a spacer (a) having any one of the sequences set forth in SEQ ID NOs: 127, 128 and 131, (b) having 1, 2, or 3 mismatches relative to any one of the sequences set forth in SEQ ID NOs: 127, 128 and 131, or (c) having about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any one of the sequences set forth in SEQ ID NOs: 127, 128 and 131.
  • one of the two or more gRNAs targets TAP-1 gene and comprises a spacer (a) having a sequence set forth in SEQ ID NO: 174, (b) having 1, 2, or 3 mismatches relative to the sequence set forth in SEQ ID NO: 174, or (c) having about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to the sequence set forth in SEQ ID NO: 174.
  • the method further comprises contacting the plurality of DNA molecules or the plurality of cells comprising the plurality of DNA molecules with (c) a RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease; and (d) one or more guide RNAs targeting one or more second target nucleotide sequence of the plurality of DNA molecules, thereby genetically editing the plurality of DNA molecules within the one or more second target nucleotide sequence of the plurality of DNA molecules.
  • the two or more guide RNAs (gRNAs) targeting the one or more first target nucleotide sequence of the plurality of DNA molecules target a target gene selected from the group consisting of Regnase-1 (Reg1) gene, Transforming Growth Factor Beta Receptor II (TGFBRII) gene, beta-2- microglobulin ( ⁇ 2M) gene, CD70 gene, Cbl Proto-Oncogene B (Cblb) gene, poliovirus receptor (PVR) gene, MHC class I chain-related A/B (MCIA/B) gene, transporter associated with antigen processing 1 (TAP-1) gene, FAS gene, or a combination thereof; and the one or more gRNAs targeting the one or more second target nucleotide sequence of the plurality of DNA molecules target TRAC gene.
  • a target gene selected from the group consisting of Regnase-1 (Reg1) gene, Transforming Growth Factor Beta Receptor II (TGFBRII) gene, beta-2- microglobulin ( ⁇ 2M) gene, CD
  • contacting (a), (b), (c) and (d) with the plurality of DNA molecules or the plurality of cells comprising the plurality of DNA molecules is performed sequentially or simultaneously.
  • (a), (b), (c), and (d) are delivered to the plurality of cells comprising the plurality of DNA molecules in a single electroporation event.
  • (a) and (b) are delivered to the plurality of cells comprising the plurality of DNA molecules in a first electroporation event, followed by the delivery of (c) and (d) in a second electroporation event.
  • the RNA-guided endonuclease is Streptococcus pyogenes (SpyCas9), Staphylococcus lugdunensis (SluCas9), Staphylococcus aureus (SaCas9), or a variant thereof.
  • SpyCas9 Streptococcus pyogenes
  • SluCas9 Staphylococcus lugdunensis
  • SaCas9 Staphylococcus aureus
  • FIG.1 shows the degree of B2M and FAS knockdown by sgRNA using seven BE4 cytosine base editor (CBE) mRNAs in Jurkat cells (determined by flow cytometry).
  • FIGS.2A-2B show on-target activity based on Amp-Seq.
  • FIG.2A is a diagram showing the percentage of knockdown from flow cytometry by deaminase enzymes from various species.
  • FIG.2B is a diagram showing the percentage of cytidine (C) to thymine (T) conversion rate by deaminases from various species. The C to T conversion rates correlate well with the percentage of protein loss.
  • FIGS.3A-3B depict diagrams showing the off-target activity based on Amp- Seq results from the R-loop assay.
  • FIGS.3A-3B show C to T conversion rate by deaminases from various species. Higher on-target C->T rates with Rat & Orangutan APOBEC1 ( ⁇ 70%) were observed.
  • FIG. 4 depicts a diagram showing the sum of C to T conversion rates across the on-target spacer regions and sum of C to T off-target activity across the R-loop amplicons by deaminases from various species.
  • Deaminase from armadillo e.g., nine-banded armadillo
  • orangutan rat, bat (e.g., little brown rat)
  • Mongolian gerbil exhibit high on-target editing efficiency and low off-target editing efficiency.
  • FIG.5 depicts a plot showing that RNP & BE CTX131 CAR-T Cells achieved comparable editing efficiencies. About 70% CAR insertion, greater than 98% TCR & CD70 KO, about 70% B2M1 RNP editing and showing superior B2M1 editing by base editor (BE).
  • FIG.6 depicts a plot showing absence of indels in base edited CTX 131 CAR T cells.
  • FIGS.7A-7B and FIGS.8A-8B show base edited CTX131 CAR T cell health. Equivalent proliferative capacity of BE & RNP CTX131 were observed. CAR T Cell viability was improved with Modified CBE mRNA.
  • FIG. 9 depicts a graph showing RNP and BE Anti-CD70 CAR-Ts are comparably efficacious in vitro in cytotoxicity assay.
  • FIGS.10A-10B depict plots showing that base edited CAR-T cells are highly efficacious in in vivo xenograft model ( Figure 10A) without any associated toxicity ( Figure 10B).
  • Group 2 CTX131-RNP
  • Group 4 CTX131-BE.
  • FIG. 11A shows % base editing in human T cells using CBEs comprising deaminases from rat orangutan, and armadillo, respectively.
  • FIG. 11B shows on-target vs. off- target spurious deamination by the same three CBEs.
  • FIG.12 shows a sequence alignment of rat APOBEC1 (rAPOBEC1), wildtype deaminase from Nine-banded armadillo (SEQ ID NO: 1), wildtype deaminase from Mongolian gerbil (SEQ ID NO: 7), and wildtype deaminase from Little brown bat (SEQ ID NO: 13). Row 5 of the sequence alignment shows a sequence of an exemplary homology model of rAPOBEC1.
  • Amino acids at various positions were selected based on proximity to modeled ssDNA strand for making mutant deaminases (including the residues colored in purple, e.g., one or more amino acids of RELRKET (positions 30-36), R at position 52, QN (positions 56 and 57), NKHV (positions 59-62), LSW (positions 88-90), R at position 118, YHH at positions 120-122, R at position 126, R at position 128, R at position 169, I at position 195, R at position 197, R at position 198, and KQPQL at positions 199-203).
  • FIG. 13 illustrates a modified R-loop assay used for evaluating spurious off- target deamination.
  • the modified R-loop assay eliminates the need for using nSaCas9-2XUGI mRNA.
  • Top panel illustrates the process used for generating the cell lines stably expressing the nSaCas9-2XUGI protein using lentiviruses encoding nSaCas9-2XUGI-P2A-Hygromycin resistance.
  • FIG. 14A is a table showing C to T base editing efficiencies for Group A knockout (FAS, TRAC, B2M, CD70, and optionally REG1) and Group B knockout (FAS, TRAC, B2M, CD70, and optionally RFX5).
  • FIG. 14B-C are graphically representation of the five-plex editing data of Group A (FIG.14B) and Group B (FIG.14C).
  • FIG. 15 illustrates an exemplary experiment layout used in multiplex editing optimization disclosed herein.
  • FIG.16A depicts a schematic representation of a deaminase homology model constructed from homology modeling.
  • FIG. 16B shows an alignment of potential sites for mutations identified by different molecular modeling programs.
  • FIG.17 shows a table providing a list of exemplary gRNA sequences and their target regions in Cblb gene.
  • FIG. 18 is an exemplary graph showing the percentage of cells expressing B2M and TCR.
  • FIGS. 19A-B provide graphs showing results from Western blot analysis of Cblb BE gRNA screen in Donor 1 and Donor 2, respectively.
  • FIG. 19C is a graph showing a summary of Western blot analysis of Cblb BE gRNA screen in both donors.
  • FIG.20A depicts total cell counts of Cblb edited cells.
  • FIG.20B depicts the CD4+/CD8+ ratio of Cblb edited cells.
  • FIG.21 depicts editing efficiency of three exemplary Cblb gRNAs.
  • FIGs.22A-C provide editing analysis for each of the Cblb gRNAs.
  • FIG.23 shows a knockout of Cblb gene by the Cblb guides.
  • FIG.24 is a graph showing CD4+/CD8+ ratios in CD155 edited cells.
  • FIG.20A depicts total cell counts of Cblb edited cells.
  • FIG.20B depicts the CD4+/CD8+ ratio of Cblb edited cells.
  • FIG.21 depicts editing efficiency of three exemplary Cblb gRNAs.
  • FIGs.22A-C provide editing analysis for each of the
  • FIG. 25 is a graph showing the percentage of CD155+ cells following gene editing.
  • FIGS.26A-B provide editing analysis for exemplary lead CD155 gRNAs.
  • FIG. 27 includes graphs showing CD4+/CD8+ ratios in CD155 edited cells after 24-hr activation and 48-hr activation in comparison to the data shown in FIG.24
  • FIG.28 is a graph showing the expression percentage of CD155 protein after 24-hr activation and 48-hr activation in compasiron to the data shown in FIG.25
  • FIGs.29A-B provide editing analysis for each lead CD155 gRNA.
  • FIGs.30A-B are graphs showing CD3 and CD133 levels in CD155 edited cells after 24-hr activation (FIG.30A) and 48-hr activation (FIG.30B).
  • FIG.30C is a table presenting the data plotted in FIGs.30A-B.
  • FIG.31 illustrates a schematic gene structure of MICA gene.
  • FIG. 32 provides graphs showing the expression of MICA at day 0 after thawing of cells and day 2 post thaw.
  • FIG.33 is a graph showing ⁇ 2m expression relative to the RNP control sample.
  • FIG.34 is a graph showing cell counts of MICA edited cells at day 7.
  • FIGs. 36A-F are graphs providing detailed analysis of base editing by exemplary MICA gRNAs.
  • FIG. 37 is a graph showing total cell counts at day 6 following TAP-1 gene editing.
  • FIG.38 is a plot summarizing target and bystander editing for exemplary TAP- 1 gRNAs.
  • FIG.39 is a plot showing the HLA ABC knock down percentage by exemplary TAP-1 gRNAs at day 8 in comparison to ⁇ 2M gRNA control.
  • FIG.40 is a plot showing HLA-ABC mean fluorescence intensity (MFI) values following TAP-1 gene editing at day 8.
  • MFI mean fluorescence intensity
  • FIG.41 shows a non-limiting exemplary experiment outline for BCMA CAR T generation.
  • FIGs. 42A-C are plots showing the fold increase of multi-edited cells via a single-step electroporation process (FIG.42A) or a two-step electroporation process (FIG.42B) in comparison to cells containing a single gene edit (FIG.42C).
  • FIG. 43 is a plot demonstrating the binding between SluCas9 sgRNA and SluCas9 or SpCas9.
  • FIGs. 44A-B show results from binding assays (FIG.
  • FIG. 44A is a plot showing the indel frequency by different Cas9 and gRNA combinations.
  • FIG. 45 is a plot showing the indel frequency by different Cas9 and gRNA combinations.
  • a base editing fusion protein comprises a Cas9 domain, wherein the Cas9 domain when associated with a guide RNA (gRNA) specifically binds to a target nucleic acid sequence, and a deaminase domain (e.g., a cytidine deaminase domain) capable of deaminating a nucleotide base (e.g., from C to U) in a target nucleic acid sequence.
  • gRNA guide RNA
  • a deaminase domain e.g., a cytidine deaminase domain
  • genomic editing is a type of genetic engineering in which nucleotide(s)/nucleic acid(s) is/are inserted, deleted, and/or substituted in a DNA sequence, such as in the genome of a targeted cell.
  • Targeted gene editing enables insertion, deletion, and/or substitution at pre-selected sites in the genome of a targeted cell (e.g., in a targeted gene or targeted DNA sequence).
  • an sequence of an endogenous gene is edited, for example by deletion, insertion or substitution of nucleotide(s)/nucleic acid(s)
  • the endogenous gene comprising the affected sequence can be knocked-out or knocked-down due to the sequence alteration.
  • a “CRISPR-Cas9” system is a naturally-occurring defense mechanism in prokaryotes that has been repurposed as a RNA-guided DNA-targeting platform used for gene editing. It relies on the DNA nuclease Cas9, and two noncoding RNAs-crisprRNA (crRNA) and trans-activating RNA (tracrRNA) to target the cleavage of DNA. crRNA drives sequence recognition and specificity of the CRISPR-Cas9 complex through Watson-Crick base pairing typically with a 20 nucleotide (nt) sequence in the target DNA.
  • nt nucleotide
  • the CRISPR-Cas9 complex only binds DNA sequences that contain a sequence match to the first 20 nt of the crRNA, single-guide RNA (sgRNA), if the target sequence is followed by a specific short DNA motif (with the sequence NGG) referred to as a protospacer adjacent motif (PAM).
  • sgRNA single-guide RNA
  • PAM protospacer adjacent motif
  • TracrRNA hybridizes with the 3’ end of crRNA to form an RNA-duplex structure that is bound by the Cas9 endonuclease to form the catalytically active CRISPR-Cas9 complex, which can then cleave the target DNA.
  • CRISPR-Cas9 gene editing system comprises an RNA-guided nuclease and one or more guide RNAs targeting one or more target genes.
  • RNA-guided endonuclease refers to a polypeptide capable of binding a RNA (e.g., a gRNA) to form a complex targeted to a specific DNA sequence (e.g., in a target DNA).
  • RNA-guided endonuclease is a Cas polypeptide (e.g., a Cas endonuclease, such as a Cas9 endonuclease).
  • the RNA-guided endonuclease as described herein is targeted to a specific DNA sequence in a target DNA by an RNA molecule to which it is bound.
  • the RNA molecule can include a sequence that is complementary to and capable of hybridizing with a target sequence within the target DNA, thus allowing for targeting of the bound polypeptide to a specific location within the target DNA.
  • guide RNA refers to a site-specific targeting RNA that can bind an RNA-guided endonuclease to form a complex, and direct the activities of the bound RNA-guided endonuclease (such as a Cas endonuclease) to a specific target sequence within a target nucleic acid.
  • the guide RNA can include one or more RNA molecules.
  • nuclease and “endonuclease” are used interchangeably herein to refer to an enzyme which possesses endonucleolytic catalytic activity for polynucleotide cleavage.
  • the term “Cas endonuclease” or “Cas nuclease” refers to an RNA-guided DNA endonuclease associated with the CRISPR adaptive immunity system.
  • the term “deaminase” refers to an enzyme that catalyzes a deamination reaction.
  • the deaminase is a cytidine deaminase, catalyzing the hydrolytic deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively.
  • the term “invariable region” of a gRNA refers to the nucleotide sequence of the gRNA that associates with the RNA-guided endonuclease.
  • the gRNA comprises a crRNA and a transactivating crRNA (tracrRNA), wherein the crRNA and tracrRNA hybridize to each other to form a duplex.
  • the crRNA comprises 5’ to 3’: a spacer sequence and minimum CRISPR repeat sequence (also referred to as a “crRNA repeat sequence” herein); and the tracrRNA comprises a minimum tracrRNA sequence complementary to the minimum CRISPR repeat sequence (also referred to as a “tracrRNA anti- repeat sequence” herein) and a 3’ tracrRNA sequence.
  • the invariable region of the gRNA refers to the portion of the crRNA that is the minimum CRISPR repeat sequence and the tracrRNA.
  • target DNA refers to a DNA that includes a “target site” or “target sequence.”
  • target sequence is used herein to refer to a nucleic acid sequence present in a target DNA to which a DNA-targeting sequence or segment (also referred to herein as a “spacer”) of a gRNA can hybridize, provided sufficient conditions for hybridization exist.
  • the target sequence 5'-GAGCATATC-3' within a target DNA is targeted by (or is capable of hybridizing with, or is complementary to) the RNA sequence 5'-GAUAUGCUC- 3'.
  • Hybridization between the DNA-targeting sequence or segment of a gRNA and the target sequence can, for example, be based on Watson-Crick base pairing rules, which enables programmability in the DNA-targeting sequence or segment.
  • the DNA-targeting sequence or segment of a gRNA can be designed, for instance, to hybridize with any target sequence.
  • polynucleotide and nucleic acid are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
  • a polynucleotide can be single-, double-, or multi- stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids/triple helices, or a polymer including purine and pyrimidine bases (e.g., the five biologically occurring bases adenine, guanine, thymine, cytosine and uracil) or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • purine and pyrimidine bases e.g., the five biologically occurring bases adenine, guanine, thymine, cytosine and uracil
  • other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases e.g., the five biologically occurring bases adenine, guanine, thymine, cytosine and uracil
  • a nucleic acid or polynucleotide can refer to any nucleic acid, whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sultone linkages, and combinations of such linkages.
  • phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphorami
  • a “secondary structure” of a nucleic acid molecule refers to the base pairing interactions within the nucleic acid molecule.
  • the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds).
  • the term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product.
  • a polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis. [0073] As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to the nucleotide bases or amino acid residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • a functionally equivalent residue of an amino acid used herein typically can refer to other amino acid residues having physiochemical and stereochemical characteristics substantially similar to the original amino acid.
  • the physiochemical properties include water solubility (hydrophobicity or hydrophilicity), dielectric and electrochemical properties, physiological pH, partial charge of side chains (positive, negative or neutral) and other properties identifiable to a person skilled in the art.
  • the stereochemical characteristics include spatial and conformational arrangement of the amino acids and their chirality.
  • fusion protein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins. One protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) protein thus forming an “amino-terminal fusion protein” or a “carboxy-terminal fusion protein,” respectively.
  • a protein may comprise different domains, for example, a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9 that directs the binding of the protein to a target site) and a nucleic acid cleavage domain or a catalytic domain of a recombinase.
  • a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain, and an organic compound, e.g., a compound that can act as a nucleic acid cleavage agent.
  • a protein is in a complex with, or is in association with, a nucleic acid, e.g., RNA.
  • any of the proteins provided herein can be produced by any method known in the art, for example via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker.
  • Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.
  • binding refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid).
  • Binding interactions can be characterized by a dissociation constant (Kd), for example a Kd of, or a Kd less than, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10- 11 M, 10 -12 M, 10 -13 M, 10 -14 M,10 -15 M, or a number or a range between any two of these values.
  • Kd dissociation constant
  • Kd can be dependent on environmental conditions, e.g., pH and temperature. “Affinity” refers to the strength of binding, and increased binding affinity is correlated with a lower Kd.
  • hybridizing or “hybridize” refers to the pairing of substantially complementary or complementary nucleic acid sequences within two different molecules. Pairing can be achieved by any process in which a nucleic acid sequence joins with a substantially or fully complementary sequence through base pairing to form a hybridization complex. “Hybridizing” or “hybridize” can comprise denaturing the molecules to disrupt the intramolecular structure(s) (e.g., secondary structure(s)) in the molecule.
  • denaturing the molecules comprises heating a solution comprising the molecules to a temperature sufficient to disrupt the intramolecular structures of the molecules. In some instances, denaturing the molecules comprises adjusting the pH of a solution comprising the molecules to a pH sufficient to disrupt the intramolecular structures of the molecules.
  • two nucleic acid sequences or segments of sequences are “substantially complementary” if at least 80% of their individual bases are complementary to one another.
  • a splint oligonucleotide sequence is not more than about 50% identical to one of the two polynucleotides (e.g., RNA fragments) to which it is designed to be complementary.
  • each sequence can be referred to herein as a ‘segment’, and the segments are substantially complementary if they have 80% or greater identity.
  • the terms “complementarity” and “complementary” mean that a nucleic acid can form hydrogen bond(s) with another nucleic acid based on traditional Watson-Crick base paring rule, that is, adenine (A) pairs with thymine (U) and guanine (G) pairs with cytosine (C).
  • Complementarity can be perfect (e.g. complete complementarity) or imperfect (e.g. partial complementarity).
  • Perfect or complete complementarity indicates that each and every nucleic acid base of one strand is capable of forming hydrogen bonds according to Watson-Crick canonical base pairing with a corresponding base in another, antiparallel nucleic acid sequence. Partial complementarity indicates that only a percentage of the contiguous residues of a nucleic acid sequence can form Watson-Crick base pairing with the same number of contiguous residues in another, antiparallel nucleic acid sequence. In some embodiments, the complementarity can be at least 70%, 80%, 90%, 100% or a number or a range between any two of these values. In some embodiments, the complementarity is perfect, i.e. 100%.
  • the complementary candidate sequence segment is perfectly complementary to the candidate sequence segment, whose sequence can be deducted from the candidate sequence segment using the Watson-Crick base pairing rules.
  • the term "vector” refers to a polynucleotide construct, typically a plasmid or a virus, used to transmit genetic material to a host cell.
  • Vectors can be, for example, viruses, plasmids, cosmids, or phage.
  • a vector as used herein can be composed of either DNA or RNA.
  • a vector is composed of DNA.
  • An "expression vector” is a vector that is capable of directing the expression of a protein encoded by one or more genes carried by the vector when it is present in the appropriate environment.
  • Vectors are preferably capable of autonomous replication.
  • an expression vector comprises a transcription promoter, a gene, and a transcription terminator. Gene expression is usually placed under the control of a promoter, and a gene is said to be “operably linked to” the promoter.
  • transfection or “infection” refer to the introduction of a nucleic acid into a host cell, such as by contacting the cell with a recombinant MVA virus or a gutless picornaviral particle as described herein.
  • the term “transgene” refers to any nucleotide or DNA sequence that is integrated into one or more chromosomes of a target cell by human intervention.
  • the transgene comprises a polynucleotide that encodes a protein of interest.
  • the protein-encoding polynucleotide is generally operatively linked to other sequences that are useful for obtaining the desired expression of the gene of interest, such as transcriptional regulatory sequences.
  • the transgene can additionally comprise a nucleic acid or other molecule(s) that is used to mark the chromosome where it has integrated.
  • the term “prophylaxis,” “prevent,” “preventing,” “prevention,” and grammatical variations thereof as used herein refers the preventive treatment of a subclinical disease-state in a subject, e.g., a mammal (including a human), for reducing the probability of the occurrence of a clinical disease-state.
  • the method can partially or completely delay or preclude the onset or recurrence of a disorder or condition and/or one or more of its attendant symptoms or barring a subject from acquiring or reacquiring a disorder or condition or reducing a subject’s risk of acquiring or requiring a disorder or condition or one or more of its attendant symptoms.
  • the subject is selected for preventative therapy based on factors that are known to increase risk of suffering a clinical disease state compared to the general population.
  • “Prophylaxis” therapies can be divided into (a) primary prevention and (b) secondary prevention.
  • Primary prevention is defined as treatment in a subject that has not yet presented with a clinical disease state, whereas secondary prevention is defined as preventing a second occurrence of the same or similar clinical disease state.
  • treatment refers to a clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible.
  • the aim of treatment includes, but is not limited to, the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and/or the remission of the disease, disorder or condition.
  • Treatment refer to one or both of therapeutic treatment and prophylactic or preventative measures.
  • Subjects in need of treatment include those already affected by a disease or disorder or undesired physiological condition as well as those in which the disease or disorder or undesired physiological condition is to be prevented.
  • a treatment is considered "effective treatment,” if any one or all of the signs or symptoms of, as but one example, levels of functional target are altered in a beneficial manner (e.g., increased by at least 10%), or other clinically accepted symptoms or markers of disease (e.g., cancer) are improved or ameliorated.
  • Efficacy can also be measured by failure of a subject to worsen as assessed by hospitalization or need for medical interventions (e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and/or described herein.
  • Treatment includes any treatment of a disease in subject and includes: (1) inhibiting the disease, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.
  • the terms "effective amount” or “pharmaceutically effective amount” or “therapeutically effective amount” refer to an amount sufficient to effect beneficial or desirable biological and/or clinical results.
  • An effective amount also includes an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease.
  • pharmaceutically acceptable excipient refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive or diluent for administration of a compound(s) of interest to a subject.
  • Pharmaceutically acceptable excipient can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers.
  • a "subject" refers to an animal for whom a diagnosis, treatment, or therapy is desired. I some embodiments, the subject is a mammal.
  • “Mammal,” as used herein, refers to an individual belonging to the class Mammalia and includes, but not limited to, humans, domestic and farm animals, zoo animals, sports and pet animals.
  • Non-limiting examples of mammals include mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cows; horses; primates, such as monkeys, chimpanzees and apes, and, in particular, humans.
  • the mammal is a primate.
  • the mammal is a human.
  • the mammal is not a human.
  • RNA-guided nucleases from CRISPR systems generate precise breaks in DNA or RNA at specified positions. In cells, this activity can lead to changes in DNA sequence or RNA transcript abundance.
  • Base editing is a genome editing approach that uses components from CRISPR systems together with DNA editing enzymes, such as deaminase enzymes, to directly install point mutations into cellular DNA or RNA by converting one base or base pair into another, while minimizing the formation of double-stranded DNA breaks (DSBs).
  • DNA editing enzymes such as deaminase enzymes
  • DSBs double-stranded DNA breaks
  • the fusion protein described herein can comprise a Cas9 domain capable of binding to a guide RNA (gRNA) which in turn binds a target nucleic acid sequence of a nucleic acid via hybridization; and a deaminase domain that can deaminate a nucleobase, such as, cytidine.
  • gRNA guide RNA
  • a deaminase domain that can deaminate a nucleobase, such as, cytidine.
  • the deamination of a nucleobase by a deaminase can lead to a point mutation at the respective residue (e.g., from cytosine to uracil), which is referred to herein as base editing or nucleic acid editing.
  • Fusion proteins comprising a Cas9 variant or domain and a deaminase domain can thus be used for the targeted editing of nucleic acid sequences.
  • Such fusion proteins are useful for targeted editing of DNA in vitro, e.g., for the generation of mutant cells or animals; for the introduction of targeted mutations, e.g., for the correction of genetic defects in cells ex vivo, e.g., in cells obtained from a subject that are subsequently re-introduced into the same or another subject; and for the introduction of targeted mutations in vivo, e.g., the correction of genetic defects or the introduction of deactivating mutations in disease-associated genes in a subject.
  • the nucleobase editors or base editors (BEs) described herein comprise fusions between a catalytically impaired Cas nuclease and a base-modification enzyme that operates on single-stranded DNA (ssDNA).
  • the fusion protein comprises a nuclease-inactive Cas9 (dCas9) fused to a deaminase. In some embodiments, the fusion protein comprises a Cas9 nickase fused to a deaminase.
  • the fusion protein comprises a Cas9 nickase fused to a deaminase and further fused to a uracil glycosylase inhibitor (UGI) domain.
  • the base editing fusion proteins described herein exhibit enhanced on-target editing efficiency and reduced off-target editing efficiency.
  • the on-target editing efficiency is at least 50%, 60%, 70%, 80%, 90%, 95% or greater.
  • the off-target editing is reduced by about, at least or at least about 80%, 85%, 90%, 95%, 98%, 99% or 100%.
  • the base editing fusion protein described herein can comprise a nucleic acid editing domain such as a deaminase or deaminase domain.
  • the term “deaminase” refers to an enzyme that catalyzes a deamination reaction.
  • the deaminase or deaminase domain belongs to the deaminase superfamily.
  • the deaminase superfamily encompasses zinc-dependent enzymes catalyzing the deamination of bases in free nucleotides and nucleic acids.
  • the deaminase superfamily displays a conserved ⁇ -sheet with five ⁇ -strands arranged in 2-1-3-4-5 order interleaved with three ⁇ -helices forming an ⁇ / ⁇ -fold.
  • the active site comprises two zinc- chelating motifs, respectively represented by a motif of HxE/CxE/DxE at the end of helix 2 and Cx n C (where x is any amino acid and n is ⁇ 2) located in loop 5 and the beginning of helix 3.
  • the zinc ion is coordinated by the side chains of residues His and Cys, such as His257, Cys291 and Cys288 in APOBEC3G.
  • the deaminase can be a cytidine deaminase, catalyzing the hydrolytic deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively.
  • the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase.
  • APOBEC apolipoprotein B mRNA-editing complex
  • an APOBEC family deaminase contains a conserved ⁇ / ⁇ -fold core domain at the N-terminal and a C-terminal domain.
  • the APOBEC deaminase core domain comprises three active loops (e.g., loops 1, 3 and 7 in APOBEC1 and loops 1, 5 and 7 in APOBEC3) containing amino acid residues known to form interactions with a nucleic acid upon its binding to the deaminase.
  • the C-terminal domain of an APOBEC family deaminase comprises a ⁇ -hairpin and three small helical domains.
  • the deaminase is a dimer formed by two APOBEC deaminase monomers mediated through interactions between the C-terminal domains.
  • amino acid residues in the active loops of the deaminase core domain and/or residues in the C-terminal domain can be mutated to generate deaminase variants with desired properties such as low spurious off-target editing activity and improved on-target editing precision (e.g., by narrowing editing window).
  • the APOBEC family of cytosine deaminase enzymes encompasses eleven proteins that serve to initiate mutagenesis in a controlled and beneficial manner as will be understood by one skilled in the art.
  • the deaminase is an APOBEC1 family deaminase.
  • the deaminase is an APOBEC3 family deaminase.
  • the deaminase is an activation-induced cytidine deaminase (AID), which is responsible for the maturation of antibodies by converting cytosines in ssDNA to uracils in a transcription-dependent, strand-biased fashion.
  • the deaminase is an ACF1/ASE deaminase. Additional suitable nucleic acid-editing enzymes or domains will be apparent to the skilled artisan based on this disclosure.
  • the deaminase or deaminase domain can comprise, or can be, a naturally- occurring deaminase from an organism, mammals, fungus, reptiles, amphibians and birds.
  • the deaminase or deaminase domain is a variant of a naturally-occurring deaminase from an organism, that does not occur in nature.
  • the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring deaminase from an organism.
  • the deaminase or deaminase domain is a variant of a naturally- occurring deaminase comprising one or more mutations or a deletion or insertion of one or more residues within a sequence.
  • the one or more mutations/deletions/insertions result in altered catalytic deaminase activity.
  • the one or more mutations/deletions/insertions result in reduced catalytic deaminase activity such that the deaminase or deaminase domain is less likely to catalyze the deamination of a residue adjacent to a target residue, thereby narrowing the deamination window.
  • the deaminase or deaminase domain can be from a mammal, for example, rat, bat, armadillo, or orangutan; or be a variant thereof.
  • the deaminase or deaminase domain is a rat deaminase.
  • the deaminase or deaminase domain is a bat deaminase.
  • the deaminase or deaminase domain is an armadillo deaminase. In some embodiments, the deaminase or deaminase domain is an orangutan deaminase. In some embodiments, the deaminase or deaminase domain is from Dasypus novemcinctus (nine-banded armadillo), Meriones unguiculatus (Mongolian gerbil) or Myotis lucifugus (little brown bat). In some embodiments, the deaminase or deaminase domain is from Dasypus novemcinctus.
  • the deaminase can be, for example, a deaminase (SEQ ID NO: 1) from Nine- banded Armadillo (Dasypus novemcinctus) encoded by a nucleic acid sequence of SEQ ID NO: 19.
  • SEQ ID NO: 1 The residues in bold and underlined in SEQ ID NO: 1 shown below are non-limiting exemplary residues that can be mutated (e.g., by substitution, deletion or insertion) to generate variants with one or more desirable properties.
  • residues in bold and underlined in SEQ ID NO: 7 shown below are non-limiting exemplary residues that can be mutated (e.g., by substitution, deletion or insertion) to generate variants with one or more desirable properties.
  • MSSETGPAADPTLRRRIEPQEFGAFFDPQLLRKETCLLYEINWGGRHSVWRHTGQNTDRHAEINFIEKFT SERYFCPFTRCSITWFLSWSPCGECCRAIVEFLSRYPNVTLFIYVARLYHHTDERNRQGLRDLCRRGVTI RIMTEQECYYCWRNFVNYSPSNEAHWPRYPHLWVRMYVLELYCILLGLPPCLKILRRNQNQLTIFNLAFQ HCHFQRLPYYIF (SEQ ID NO: 7)
  • residues in bold and underlined in SEQ ID NO: 13 shown below are non-limiting exemplary residues that can be mutated (e.g., by substitution, deletion or insertion) to generate variants with one or more desirable properties.
  • the deaminase or deaminase domain comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more or more mutations compared to any one of the amino acid sequences set forth in SEQ ID NOs: 1-18.
  • the deaminase or deaminase domain comprises an amino acid sequence having one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty mismatches relative to any one of SEQ ID NOs: 1-18. In some embodiments, the deaminase or deaminase domain comprises an amino acid sequence of any one of SEQ ID NOs: 1-18.
  • the deaminase or deaminase domain described herein comprises one or more mutations as compared to a parent deaminase or deaminase domain (e.g., a wild type deaminase).
  • the deaminase or deaminase domain can comprise one or more mutations with respect to any sequence of SEQ ID NOs: 1, 7, and 13.
  • the deaminase or deaminase domain can comprise one or more mutations of R33A, K34A, W90Y, H126E and H122A with respect to wildtype deaminase (SEQ ID NO: 1).
  • SEQ ID NO: 2 is the R33A variant of SEQ ID NO: 1
  • SEQ ID NO: 3 is the K34A variant of SEQ ID NO: 1
  • SEQ ID NO: 4 is the W90Y variant of SEQ ID NO: 1
  • SEQ ID NO: 5 is the W90Y/H126E variant of SEQ ID NO: 1
  • SEQ ID NO: 6 is the H122A variant of SEQ ID NO: 1.
  • the deaminase or deaminase domain can comprise one or more mutations of R32A, K33A, W89Y, R125E and H121A with respect to wildtype deaminase (SEQ ID NO: 7).
  • SEQ ID NO: 8 is the R32A variant of SEQ ID NO: 7
  • SEQ ID NO: 9 is the K33A variant of SEQ ID NO: 7
  • SEQ ID NO: 10 is the W89Y variant of SEQ ID NO: 7
  • SEQ ID NO: 11 is the W89Y/R125E variant of SEQ ID NO: 7
  • SEQ ID NO: 12 is the H121A variant of SEQ ID NO: 7.
  • the deaminase or deaminase domain can comprise one or more mutations of R33A, K34A, W90Y, Q126E and H122A with respect to wildtype deaminase (SEQ ID NO: 13).
  • SEQ ID NO: 14 is the R33A variant of SEQ ID NO: 13
  • SEQ ID NO: 15 is the K34A variant of SEQ ID NO: 13
  • SEQ ID NO: 16 is the W90Y variant of SEQ ID NO: 13
  • SEQ ID NO: 17 is the W90Y/Q126E variant of SEQ ID NO: 13
  • SEQ ID NO: 18 is the H122A variant of SEQ ID NO: 13.
  • One or more amino acid residues of any one of the deaminases disclosed herein can be mutated (e.g., substituted, deleted, or with insertion) to generate variants of the deaminases.
  • one or more of the residues highlighted in the sequence alignment of FIG.12 can be mutated to generate variants.
  • non-limiting exemplary residues include any one of the amino acid residues of RELRKET (positions 30-36), R at position 52, QN (positions 56 and 57), NKHV (positions 59-62), LSW (positions 88-90), R at position 118, YHH at positions 120-122, R at position 126, R at position 128, R at position 169, I at position 195, R at position 197, R at position 198, and KQPQL at positions 199-203) of rAPOBEC1 or of armadillo deaminase (SEQ ID NO: 1); any combination thereof, or functional equivalent(s) thereof.
  • deaminases or deaminase domains disclosed herein can comprise, for example, amino acid mutation(s) (e.g., substitution(s), deletion(s), or insertion(s)) at one or more positions functionally equivalent to F23, P29, R30, E31, L32, R33, K34, E35, T36, C37, L38, L39, R52, S55, Q56, N57, N59, K60, H61, V62, N65, F66, I67, E68, K69, Y75, N79, L88, S89, W90, S91, R106, Y107, P108, H109, T111, I114, R118, Y120, H121, H122, R126, R128, S137, G138, V139, T140, R169, I195, R197, R198, K199, Q200, P201, Q202, and L203 in the deaminase of SEQ ID NO: 1.
  • amino acid mutation(s)
  • the deaminases or deaminase domains disclosed herein can one or more amino acid substitutions of R33A, K34A, W90Y, H126E and H122A.
  • the amino acid substitution can be, for example, a mutation to nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, and/or a hydrophilic amino acid.
  • identification of potential amino acid residues for mutation in a deaminase can be accomplished using molecular modeling such as homology modeling or ab initio modeling to construct a three-dimensional structure of the deaminase.
  • Amino acid residues in a deaminase involved in binding with ssDNA or in close proximity to the ssDNA binding site can be identified as potential sites for mutations.
  • the term “homology model” refers to an structural model derived from known three-dimensional structure(s).
  • Known three-dimensional structures can be any deaminase structure (e.g., an APOBEC3 or APOBEC1 family deaminase) derived from experimental data such as crystallographic and NMR structure determination.
  • Generation of the homology model termed “homology modeling”, can include sequence alignment, structural alignment, residue replacement, residue conformation adjustment through energy minimization, or a combination thereof.
  • FIG. 16A depicts a schematic representation of a deaminase homology model constructed from homology modeling. Three-dimensional structures can also be obtained from ab initio modeling using empirical or semi-empirical techniques.
  • the deaminase or deaminase domain is encoded by a nucleic acid sequence that is about, at least, or at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to any one of amino acid sequences set forth in SEQ ID NOs: 19-36.
  • the deaminase or deaminase domain is encoded by a nucleic acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more or more mutations compared to any one of the nucleic acid sequences set forth in SEQ ID NOs: 19-36.
  • the deaminase or deaminase domain is encoded by a nucleic acid sequence having one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty mismatches relative to any one of SEQ ID NOs: 19-36.
  • the deaminase or deaminase domain is encoded by a nucleic acid sequence of any one of SEQ ID NOs: 19-36.
  • Cas9 domain [0104]
  • the fusion proteins described herein also comprise a Cas9 domain, when in conjunction with a bound guide RNA (gRNA), capable of specifically binding to a target nucleic acid sequence.
  • gRNA bound guide RNA
  • Cas9 or “Cas9 nuclease” refers to an RNA-guided nuclease comprising a Cas9 protein, or a fragment thereof (e.g., a protein comprising an active or inactive DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9).
  • a Cas9 nuclease is also referred to sometimes as a casn1 nuclease or a CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease.
  • a Cas9 refers to Cas9 from Corynebacterium ulcerans, Corynebacterium diphtheria, Spiroplasma syrphidicola, Prevotella intermedia, Spiroplasma taiwanense, Streptococcus iniae, Belliella baltica, Psychroflexus torquisI, Streptococcus thermophilus, Listeria innocua, Campylobacter jejuni, Neisseria. Meningitidis, Streptococcus pyogenes, or Staphylococcus aureus.
  • the Cas9 domain of a fusion protein can comprise a full-length amino acid of a Cas9 protein or a fragment thereof.
  • the Cas9 domain can comprise a truncated version of a nuclease domain or no nuclease domain at all.
  • the Cas9 domain can be a nuclease active Cas9 domain, a nuclease inactive Cas9 domain, a dead Cas9, a Cas9 fragment, or a Cas9 nickase.
  • the Cas9 domain is a nuclease active domain.
  • the Cas9 domain may be a Cas9 domain that cuts both strands of a duplexed nucleic acid (e.g., both strands of a duplexed DNA molecule).
  • the Cas9 domain is a nuclease-inactive Cas9 domain or dead Cas9 (dCas9).
  • the dCas9 domain can bind to a duplexed nucleic acid molecule (e.g., via a gRNA molecule) without cleaving either strand of the duplexed nucleic acid molecule.
  • Cas9 domains will be apparent to those of skill in the art based on this disclosure and knowledge in the field, and are within the scope of this disclosure.
  • Methods for generating a Cas9 protein (or a fragment thereof) having an inactive DNA cleavage domain are known in the art (e.g., Jinek et al., Science.337:816- 821(2012); Qi et al., “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression” (2013) Cell.28; 152(5):1173-83, the entire contents of each of which are incorporated herein by reference).
  • the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain.
  • the HNH subdomain cleaves the strand complementary to the gRNA
  • the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9.
  • the mutations D10A and H841A completely inactivate the nuclease activity of Streptococcus pyogenes Cas9 (Jinek et al., Science.337:816-821(2012); Qi et al., Cell.28; 152(5):1173-83 (2013).
  • nuclease-inactive Cas9 domains include, but are not limited to, D10A, D10A/D839A/H840A, and D10A/D839A/H840A/N863A mutant domains with respect to wild type Cas9 (e.g., SpCas9) (e.g., Mali et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology.2013; 31(9): 833-838, the entire contents of which are incorporated herein by reference).
  • wild type Cas9 e.g., SpCas9
  • SpCas9 e.g., Mali et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology.2013; 31(9): 833-838, the entire contents of which are incorporated herein by reference.
  • Mutations can be introduced to any suitable Cas9 known in the art, including but not limited to Cas9 from Corynebacterium ulcerans, Corynebacterium diphtheria, Spiroplasma syrphidicola, Prevotella intermedia, Spiroplasma taiwanense, Streptococcus iniae, Belliella baltica, Psychroflexus torquisI, Streptococcus thermophilus, Listeria innocua, Campylobacter jejuni, Neisseria. Meningitidis, Streptococcus pyogenes or Staphylococcus aureus.
  • the Cas9 domain is a Cas9 nickase.
  • the Cas9 nickase can be a Cas9 protein that is capable of cleaving only one strand of a duplexed nucleic acid molecule (e.g., a duplexed DNA molecule).
  • a Cas9 nickase can have an active HNH nuclease domain and is able to cleave the non-targeted strand of DNA, i.e., the strand bound by the gRNA.
  • the Cas9 nickase can have an inactive RuvC nuclease domain and is not able to cleave the targeted strand of the RNA, i.e., the strand where base editing is desired.
  • the Cas9 domain is a variant sharing homology to Cas9 or a fragment thereof.
  • a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% to wild type Cas9.
  • the Cas9 domain comprises one or more point mutations with respect to the corresponding wild type Cas9. Mutations may comprise substituting one or more charged and/or polar residues (e.g., aspartic acid, histidine, asparagine) to alanine.
  • the Cas9 domains described herein can have different PAM specificities.
  • Cas9 proteins such as Cas9 from S. pyogenes (spCas9), require a canonical NGG PAM sequence to bind a particular nucleic acid region. This may limit the ability to edit desired bases within a genome.
  • the base editing fusion proteins provided herein may need to be placed at a precise location, for example where a target base is placed within a 4 base region (e.g., a “deamination window”), which is approximately 15 bases upstream of the PAM. See Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double- stranded DNA cleavage” Nature 533, 420-424 (2016), the entire contents of which are hereby incorporated by reference. Accordingly, in some embodiments, any of the fusion proteins provided herein can contain a Cas9 domain that is capable of binding a nucleotide sequence that does not contain a canonical (e.g., NGG) PAM sequence.
  • a canonical e.g., NGG
  • Cas9 domains that bind to non-canonical PAM sequences have been described in the art and would be apparent to the skilled artisans.
  • Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver, B. P., et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015); and Kleinstiver, B. P., et al., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015); the entire contents of each are hereby incorporated by reference.
  • the fusion protein comprises one or more uracil glycosylase inhibitor (UGI) domain.
  • uracil glycosylase inhibitor or “UGI,” as used herein, refers to a protein that is capable of inhibiting a uracil-DNA glycosylase base-excision repair enzyme.
  • cellular DNA-repair response to the presence of U:G heteroduplex DNA may be responsible for the decrease in nucleobase editing efficiency in cells.
  • UGI uracil DNA glycosylase
  • fusion proteins comprising a UGI domain can inhibit human UDG activity, enabling increased deamination efficiency.
  • a UGI domain can comprise a wild-type UGI or a UGI variant including a fragment of UGI or a protein homologous to a UGI or UGI fragment.
  • the present disclosure includes a fusion protein comprising a deaminase and Cas9 nickase domain further fused to at least one UGI domain.
  • the UGI domain can be fused to the Cas9 domain and/or the deaminase domain either directly or via an optional linker.
  • the fusion protein comprises two UGI domains.
  • the one or more UGI domains, the deaminase domain, and the Cas9 domain can be connected to one another in any suitable configuration.
  • the fusion protein comprises the structure of [deaminase domain]-[Cas9 domain]-[UGI]-[UGI] connected either directly or via an optional linker.
  • a UGI domain can be any protein or fragment thereof capable of inhibiting (e.g., sterically blocking) a uracil-DNA glycosylase base-excision repair enzyme.
  • a UGI is a protein that binds uracil. In some embodiments, a UGI is a protein that binds uracil in DNA. In some embodiments, a UGI is a catalytically inactive uracil DNA-glycosylase protein. In some embodiments, a UGI is a catalytically inactive uracil DNA- glycosylase protein that does not excise uracil from the DNA.
  • UGI protein and nucleotide sequences are known to those in the art, and include, for example, those published in Wang et al., Uracil-DNA glycosylase inhibitor gene of bacteriophage PBS2 encodes a binding protein specific for uracil-DNA glycosylase. J. Biol. Chem.264:1163-1171(1989); Lundquist et al., Site-directed mutagenesis and characterization of uracil-DNA glycosylase inhibitor protein. Role of specific carboxylic amino acids in complex formation with Escherichia coli uracil-DNA glycosylase. J. Biol.
  • the fusion proteins comprising a UGI further comprise a nuclear targeting sequence, for example a nuclear localization sequence.
  • fusion proteins provided herein further comprise a nuclear localization sequence (NLS).
  • the NLS can be fused to the N-terminus or C-terminus of the fusion protein.
  • the NLS can be fused to the N-terminus or C-terminus of the UGI, the N-terminus or C-terminus of the Cas9 domain, or the N-terminus or C-terminus of the deaminase domain.
  • the fusion can be either direct or via a linker.
  • the Cas9 domain, the deaminase domain, and optionally the UGI and NLS can be fused to one another via a linker.
  • a linker joins a Cas9 domain (e.g., a Cas9 nickase) and a deaminase domain.
  • a linker joins a Cas9 domain with a UGI domain.
  • a linker join the deaminase domain with a UGI domain.
  • a linker joins one UGI domain with another UGI domain.
  • a linker can be an amino acid, a peptide or protein, an organic molecule, a polymer or chemical moiety.
  • the sequence, length and flexibility of the linker can vary in different embodiments. In some embodiments, the linker can have about 3-100 amino acids in length.
  • Suitable linker motifs and configurations are described in published literatures (see, e.g., Chen et al., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013; 65(10):1357-69 and Guilinger J P, Thompson D B, Liu D R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat.
  • the linker comprises a (GGS) n , (G)n, ((GGGGS)n motif, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 19, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
  • the optional linker comprises a (GGS)n motif, where n is 1, 3, or 7.
  • the deaminase domain can be fused to the N-terminus of the Cas9 domain, the C-terminus of the Cas9 domain, or both.
  • a base editing fusion protein described herein comprises the structure from the N-terminus to the C-terminus: [deaminase domain]-[Cas9 domain], in which the deaminase domain and the Cas9 domain are connected directly or via a linker.
  • a base editing fusion protein described herein comprises a structure from the N- terminus to the C-terminus selected from the following: [deaminase domain]-[Cas9 domain]- [UGI], [UGI]-[deaminase domain]-[Cas9 domain] or [deaminase domain]-[UGI]-[Cas9 domain].
  • more than one UGI domain can be fused to the deaminase and/or Cas9 domain.
  • a base editing fusion protein described herein can have a structure of [deaminase domain]-[Cas9 domain]-[UGI]-[UGI].
  • the deaminase domain is a mammalian deaminase (e.g., any one of SEQ ID NOs: 1-18 or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identify to any one of SEQ ID NOs: 1-18) or a fragment thereof and the Cas9 domain is a Cas9 nickase or a fragment thereof.
  • a mammalian deaminase e.g., any one of SEQ ID NOs: 1-18 or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identify to any one of SEQ ID NOs: 1-18
  • the Cas9 domain is a Cas9 nickase or a fragment thereof.
  • the base editing fusion protein used herein can be a portion or variant of BE1 base editor, BE2 base editor, BE3 base editor, HF-BE3, BE4, BE4-GAM, YE1-BE3, EE-BE3, YE2-BE3, YEE-BE3, VQR-BE3, VRER-BE3, VRER-BE3, Sa-BE3, Sa-BE4, SaBE4-Gam, SaKKH-BE3, Cas12a-BE, xBE3 described in Rees and Liu, Nat Rev Genet.2018 December; 19 (12): 770-788, the contents of which are incorporated herein by reference.
  • the base editing fusion protein described herein can efficiently generate an intended mutation, such as a point mutation from C to T, in a nucleic acid without generating an unintended mutation such as an unintended point mutation.
  • the base editing fusion protein described herein can modify a specific nucleotide base without generating undesired byproducts including indels, translocations and other DNA rearrangements.
  • the term “indel”, as used herein, refers to the insertion or deletion of one or more nucleotide base within a nucleic acid.
  • the fusion protein described herein can generate fewer than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.01%, 0.001%, or less indels. In some embodiments, the fusion protein described herein can generate a ratio of intended point mutations to indels that is at least 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 100:1, 200:1, 400:1, 600:1, 800:1, 1000:1 or higher.
  • the fusion protein described herein can generate fewer than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.01%, 0.001%, or less translocations or other DNA rearrangement. In some embodiments, the fusion protein described herein does not generate any indel, DNA translocation or other DNA rearrangement.
  • CRISPR-Cas9 Gene Editing System [0120] In some embodiments, targeted gene editing can be achieved through CRISPR- Cas gene editing system.
  • the CRISPR-Cas9 system is a naturally-occurring defense mechanism in prokaryotes that has been repurposed as a RNA-guided DNA-targeting platform used for gene editing. It relies on the DNA nuclease Cas9, and two noncoding RNAs-crisprRNA (crRNA) and trans-activating RNA (tracrRNA) to target the cleavage of DNA. crRNA drives sequence recognition and specificity of the CRISPR-Cas9 complex through Watson-Crick base pairing typically with a 20 nucleotide (nt) sequence in the target DNA.
  • nt nucleotide
  • the CRISPR-Cas9 complex only binds DNA sequences that contain a sequence match to the first 20 nt of the crRNA, single-guide RNA (sgRNA), if the target sequence is followed by a specific short DNA motif (with the sequence NGG) referred to as a protospacer adjacent motif (PAM).
  • sgRNA single-guide RNA
  • PAM protospacer adjacent motif
  • TracrRNA hybridizes with the 3’ end of crRNA to form an RNA-duplex structure that is bound by the Cas9 endonuclease to form the catalytically active CRISPR-Cas9 complex, which can then cleave the target DNA.
  • CRISPR-Cas9 gene editing system comprises an RNA-guided nuclease and one or more guide RNAs targeting one or more target genes.
  • the RNA-guided endonuclease can be naturally-occurring or non-naturally occurring.
  • RNA-guided endonuclease examples include a Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cpf1 endonuclease, and functional derivatives thereof.
  • Cas1, Cas1B Cas2, Cas3, Cas4, Cas5, Cas6, Cas
  • the RNA-guided endonuclease is a Cas9 endonuclease.
  • the Cas9 endonuclease can be from, e.g., Streptococcus pyogenes (SpyCas9), Staphylococcus lugdunensis (SluCas9), Staphylococcus aureus (SaCas9), or Staphylococcus schleiferi.
  • the RNA-guided endonuclease is a variant of Cas9, including but not limited to, a small Cas9, a dead Cas9 (dCas9), and a Cas9 nickase.
  • the RNA-guided endonuclease can be a small RNA-guided endonuclease.
  • the small RNA-guided endonucleases can be engineered from portions of RNA-guided endonucleases derived from any of the RNA-guided endonucleases described herein and known in the art.
  • the small RNA-guided endonucleases can be, e.g., small Cas endonucleases.
  • a small RNA-guided nuclease is shorter than about 1,100 amino acids in length.
  • the RNA-guided endonuclease can be a mutant RNA-guided endonuclease.
  • the RNA-guided endonuclease can be a mutant of a naturally occurring RNA-guided endonuclease.
  • the mutant RNA-guided endonuclease can also be a mutant RNA-guided endonuclease with altered activity compared to a naturally occurring RNA-guided endonuclease, such as altered endonuclease activity (e.g., altered or abrogated DNA endonuclease activity without substantially diminished binding affinity to DNA).
  • Such modification can allow for the sequence-specific DNA targeting of the mutant RNA-guided endonuclease for the purpose of transcriptional modulation (e.g., activation or repression); epigenetic modification or chromatin modification by methylation, demethylation, acetylation or deacetylation, or any other modifications of DNA binding and/or DNA-modifying proteins known in the art.
  • the mutant RNA-guided endonuclease has no DNA endonuclease activity.
  • the RNA-guided endonuclease can be a nickase that cleaves the complementary strand of the target DNA but has reduced ability to cleave the non-complementary strand of the target DNA, or that cleaves the non-complementary strand of the target DNA but has reduced ability to cleave the complementary strand of the target DNA. In some embodiments, the RNA-guided endonuclease has a reduced ability to cleave both the complementary and the non- complementary strands of the target DNA. [0125] The RNA-guided endonuclease can be derived from different types of CRISPR/Cas systems.
  • the CRISPR/Cas system comprises components derived from a Type-I, a Type-II, or a Type-III system.
  • Updated classification schemes for CRISPR/Cas loci define Class 1 and Class 2 CRISPR/Cas systems, having Types I to V or VI (Makarova et al., (2015) Nat Rev Microbiol, 13(11):722-36; Shmakov et al., (2015) Mol Cell, 60:385-397).
  • Class 2 CRISPR/Cas systems have single protein effectors.
  • Cas proteins of Types II, V, and VI are single-protein, RNA-guided endonucleases, herein called “Class 2 Cas nucleases.”
  • Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins.
  • the Cpf1 nuclease (Zetsche et al., (2015) Cell 163:1-13) is homologous to Cas9, and contains a RuvC-like nuclease domain.
  • the Cas nuclease is from a Type-I CRISPR/Cas system.
  • the Cas nuclease is a component of the Cascade complex of a Type-I CRISPR/Cas system.
  • the Cas nuclease is a Cas3 nuclease.
  • the Cas nuclease is derived from a Type-III CRISPR/Cas system.
  • the Cas nuclease is derived from Type-IV CRISPR/Cas system.
  • the Cas nuclease is derived from a Type-V CRISPR/Cas system.
  • the Cas nuclease is derived from a Type-VI CRISPR/Cas system.
  • gRNAs Guide RNAs
  • the gene editing systems described herein e.g., the base editing fusion protein or CRISPR-Cas9 can, for example, be in complex with or associated with a guide RNA (gRNA).
  • gRNA guide RNA
  • disclosed herein also includes a fusion protein and a guide RNA bound to the Cas9 domain of the fusion protein.
  • Disclosed herein can also comprises a composition comprising a fusion protein described herein or a nucleic acid sequence encoding the fusion protein and one or more guide RNAs targeting one or more target genes.
  • the gRNA comprise 5’ to 3’: a crRNA and a tracrRNA, wherein the crRNA and tracrRNA hybridize to form a duplex.
  • the crRNA comprises a spacer sequence capable of targeting a target sequence in a target nucleic acid (e.g., genomic DNA molecule) and a crRNA repeat sequence.
  • the tracrRNA comprises a tracrRNA anti-repeat sequence and a 3’ tracrRNA sequence.
  • the 3’ end of the crRNA repeat sequence is linked to the 5’ end of the tracrRNA anti-repeat sequence, e.g., by a tetraloop, where the crRNA repeat sequence and the tracrRNA anti-repeat sequence hybridize to form the sgRNA.
  • the sgRNA comprises 5’ to 3’: a spacer sequence, a crRNA repeat sequence, a tetraloop, a tracrRNA anti-repeat sequence, and a 3’ tracrRNA sequence.
  • the sgRNA comprise a 5’ spacer extension sequence.
  • the sgRNA comprise a 3’ tracrRNA extension sequence.
  • the 3’ tracrRNA can comprise, or consist of, one or more stem loops, for example one, two, three, or more stem loops.
  • the guide RNA can be from 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence.
  • the guide RNA is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long.
  • the guide RNA is a single-guide RNA (sgRNA).
  • the guide RNA disclosed herein can target any sequence of interest via the spacer sequence in the crRNA.
  • a spacer sequence in a gRNA is a sequence (e.g., a 20 nucleotide sequence) that defines the target sequence (e.g., a DNA target sequences, such as a genomic target sequence) of a target gene of interest.
  • the spacer sequence range from 15 to 30 nucleotides.
  • the spacer sequence can be, can be about, can be at least, or can be at most 10, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, or a number or a range between any of these values, of nucleotides in length.
  • a spacer sequence contains 20 nucleotides.
  • the “target sequence” is in a target gene that is adjacent to a PAM sequence and is the sequence to be modified by an RNA-guided nuclease (e.g., Cas9 nickase).
  • the “target sequence” is on the so-called PAM-strand in a “target nucleic acid,” which is a double-stranded molecule containing the PAM-strand and a complementary non-PAM strand.
  • the gRNA spacer sequence can hybridize to the complementary sequence located in the non-PAM strand of the target nucleic acid of interest.
  • the gRNA spacer sequence is the RNA equivalent of the target sequence.
  • the spacer of a gRNA interacts with a target nucleic acid of interest in a sequence-specific manner via hybridization (i.e., base pairing).
  • the nucleotide sequence of the spacer thus varies depending on the target sequence of the target nucleic acid of interest.
  • the spacer sequence is designed to hybridize to a region of the target nucleic acid that is located 5' of a PAM recognizable by a Cas9 domain used in the system.
  • the spacer can perfectly match the target sequence or can have mismatches.
  • Each Cas9 domain has a particular PAM sequence that it recognizes in a target DNA. For example, S.
  • the target sequence is a DNA sequence.
  • the target sequence is a sequence in the genome of a mammal.
  • the target sequence is a sequence in the genome of a human.
  • the target nucleic acid sequence has 20 nucleotides in length. In some embodiments, the target nucleic acid has less than 20 nucleotides in length.
  • the target nucleic acid has more than 20 nucleotides in length. In some embodiments, the target nucleic acid has at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides in length. In some embodiments, the target nucleic acid has at most: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides in length. In some embodiments, the 3′ end of the target sequence is immediately adjacent to a canonical PAM sequence (NGG).
  • NVG canonical PAM sequence
  • the target nucleic acid in a sequence comprising 5'-NNNNNNNNNNNNNNNNNNNNNNNNNRG-3' (SEQ ID NO: 55), can be the sequence that corresponds to the Ns, wherein N can be any nucleotide, and the underlined NRG sequence (R is G or A) is the S. pyogenes PAM.
  • the 3’ end of the target sequence is not immediately adjacent to a canonical PAM sequence.
  • the PAM sequence used in the compositions and methods of the present disclosure as a sequence recognized by SpCas9 is NGG.
  • the percent complementarity between the spacer sequence and the target nucleic acid is about, at least, at least about, at most or at most about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%.
  • the spacer sequence of the guide RNA and the target nucleic acid in the target gene is 100% complementary.
  • the percent complementarity between the spacer sequence and the target nucleic acid is 100% over the six contiguous 5'-most nucleotides of the target sequence of the complementary strand of the target nucleic acid.
  • the percent complementarity between the spacer sequence and the target nucleic acid is at least 60% over about 20 contiguous nucleotides.
  • the spacer sequence of the guide RNA and the target sequence in the target gene can contain up to 10 mismatches, e.g., up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 mismatch.
  • the guide RNA can be complementary to a sequence associated with a disease or disorder.
  • the guide RNA is complementary to a sequence associated with a disease or disorder having a mutation in a gene.
  • the gRNA is a chemically modified gRNA.
  • RNA modifications can be introduced to the gRNAs to enhance stability, reduce the likelihood or degree of innate immune response, and/or enhance other attributes as described in the art.
  • the gRNAs described herein can comprise one or more modifications including internucleoside linkages, purine or pyrimidine bases, or sugar.
  • a modification is introduced at the terminal of a gRNA with chemical synthesis or with a polymerase enzyme. Examples of modified nucleic acids and their synthesis are disclosed in WO2013/052523. Synthesis of modified polynucleotides is also described in Verma and Eckstein, Annual Review of Biochemistry, vol.76, 99-134 (1998).
  • the chemically-modified gRNA can comprise phosphorothioated 2'-O-methyl nucleotides at the 3' end and the 5' end of the gRNA. In some embodiments, the chemically- modified gRNA comprises phosphorothioated 2'-O-methyl nucleotides at the 3' end of the gRNA. In some embodiments, the chemically-modified gRNA comprises phosphorothioated 2'-O-methyl nucleotides at the 5 'end of the gRNA.
  • the chemically-modified gRNA comprises three or four phosphorothioated 2'-O-methyl nucleotides at the 3' end and/or three or four at the 5' end of the gRNA.
  • more than one guide RNA can be used with a fusion protein described herein.
  • two, three, four, five, six or more guide RNA can be used with a fusion protein described herein.
  • Each guide RNA can contain a different targeting sequence, such that the deaminase cleaves more than one target nucleic acid, thus generating more than one gene edits to the target nucleic acid sequences.
  • RNAs described herein can be used in a gene editing system (e.g., a base editor and/or CRISPR/Cas9 system) to disrupt one or more targeted genes.
  • a disrupted gene refers to a gene comprising an insertion, deletion or substitution relative to an endogenous gene such that expression of a functional protein from the endogenous gene is reduced or inhibited.
  • Reg1 gene editing [0141]
  • a guide RNA used herein can disrupt a Reg1 (Regnase 1) gene. Reg1 contains a zinc finger motif, binds RNA and exhibits ribonuclease activity.
  • Reg1 plays roles in both immune and non-immune cells and its expression can be rapidly induced under diverse conditions including microbial infections, treatment with inflammatory cytokines and chemical or mechanical stimulation.
  • Human Reg1 gene is located on chromosome 1p34.3. Additional information can be found in GenBank under Gene ID: 80149.
  • gRNAs used for disrupting Reg1 gene can target exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, or a combination thereof.
  • one or more genetic editing may occur in exon 2 or exon 4.
  • Such genetic editing can be induced by base editing or CRISPR/Cas technology using a suitable guide RNA.
  • a gRNA spacer sequence is the RNA equivalent of a target sequence.
  • a gRNA targeting Reg1 gene that can be used in base editing can comprise a spacer having a RNA sequence corresponding to the target sequence of SEQ ID NO: 62, in which a “T” is substituted with a “U”.
  • a gRNA targeting Reg1 gene that can be used in a CRISPR/Cas9 system can comprise a spacer having a RNA sequence corresponding to the target sequence of SEQ ID NO: 67.
  • the spacer sequence comprised in a gRNA sequence can be a variant of SEQ ID NO: 62 or 67.
  • the spacer sequence of the gRNA can have 1, 2, or 3 mismatches relative to the RNA sequence corresponding to the target sequence set forth in SEQ ID NO: 62 or SEQ ID NO: 67.
  • the spacer sequence of the guide RNA has about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to the RNA sequence corresponding to the target sequence set forth in SEQ ID NO: 62 or SEQ ID NO: 67.
  • TGFBRII gene editing [0143]
  • a guide RNA used herein can disrupt a TGFBRII gene, which encodes Transforming Growth Factor Receptor Type II (TGFBRII).
  • TGFBRII receptors are a family of serine/threonine kinase receptors involved in the TGF ⁇ signaling pathway. These receptors bind growth factor and cytokine signaling proteins in the TGF ⁇ family, for example, TGF ⁇ s (TGF ⁇ 1, TGF ⁇ 2, and TGF ⁇ 3), bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), activin and inhibin, myostatin, anti-Müllerian hormone (AMH), and NODAL.
  • gRNAs used for disrupting TGFBRII gene can target exon 1, exon 2, exon 3, exon 4, exon 5, or a combination thereof.
  • Such genetic editing may be induced by base editing or CRISPR/Cas technology using a suitable guide RNA.
  • a gRNA targeting TGFBRII gene that can be used in base editing can comprise a spacer having a RNA sequence corresponding to the target sequence of SEQ ID NO: 61 or a variant thereof.
  • a gRNA targeting TGFBRII gene that can be used in a CRISPR/Cas9 system can comprise a spacer having a RNA sequence corresponding to the target sequence of SEQ ID NO: 66 or a variant thereof.
  • the spacer sequence of the guide RNA contains 1, 2, or 3 mismatches relative to the RNA sequence corresponding to the target sequence set forth in SEQ ID NO: 61 or SEQ ID NO: 66.
  • the spacer sequence of the guide RNA has about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to the RNA sequence corresponding to the target sequence set forth in SEQ ID NO: 61 or SEQ ID NO: 66.
  • CD70 gene editing [0144]
  • a guide RNA used herein can disrupt a gene involved in T cell exhaustion, e.g., disruption of a gene that positively regulates T cell exhaustion.
  • T cell exhaustion is a process of stepwise and progressive loss of T cell functions, which may be induced by prolonged antigen stimulation or other factors. Genes involved in T cell exhaustion refer to those that either positively regulate or negatively regulate this biological process.
  • a guide RNA described herein can disrupt a Cluster of Differentiation 70 (CD70) gene.
  • CD70 is a member of the tumor necrosis factor superfamily and its expression is restricted to activated T and B lymphocytes and mature dendritic cells.
  • CD70 is implicated in tumor cell and regulatory T cell survival through interaction with its ligand, CD27.
  • CD70 and its receptor CD27 have multiple roles in immune function in multiple cell types including T cells (activated and T reg cells), and B cells.
  • CD70 gene results in enhanced cytotoxicity of immune cells engineered to express an antigen targeting moiety at lower ratios of engineered immune cells to target cells, indicating the potential efficacy of low doses of engineered immune cells. See, e.g., WO2019/215500, the relevant disclosures of which are incorporated by reference for the purpose and subject matter referenced herein.
  • Structures of CD70 genes are known in the art. For example, human CD70 gene is located on chromosome 19p13.3. The gene contains four protein encoding exons. Additional information can be found in GenBank under Gene ID: 970.
  • gRNAs used for disrupting CD70 gene can target exon 1, exon 2, exon 3, exon 4, or a combination thereof. Such genetic editing may be induced by base editing or CRISPR/Cas technology using a suitable guide RNA.
  • a gRNA targeting CD70 gene that can be used in base editing can comprise a spacer having a RNA sequence corresponding to the target sequence of SEQ ID NO: 63 or a variant thereof.
  • a gRNA targeting CD70 gene that can be used in a CRISPR/Cas9 system can comprise a spacer having a RNA sequence corresponding to the target sequence of SEQ ID NO: 68 or a variant thereof.
  • the spacer sequence of the guide RNA contains 1, 2, or 3 mismatches relative to the RNA sequence corresponding to the target sequence set forth in SEQ ID NO: 63 or SEQ ID NO: 68. In some embodiments, the spacer sequence of the guide RNA has about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to the RNA sequence corresponding to the target sequence set forth in SEQ ID NO: 63 or SEQ ID NO: 68.
  • a guide RNA used herein can disrupt a ⁇ 2M (beta-2- microglobulin) gene.
  • ⁇ 2M is a common (invariant) component of MHC I complexes. Disrupting its expression by gene editing will prevent host versus therapeutic allogeneic T cells responses leading to increased allogeneic T cell persistence. In some embodiments, expression of the endogenous ⁇ 2M gene is eliminated to prevent a host-versus-graft response.
  • a gRNA targeting ⁇ 2M gene that can be used in base editing can comprise a spacer having a RNA sequence corresponding to the target sequence of SEQ ID NO: 64 or a variant thereof.
  • a gRNA targeting ⁇ 2M gene that can be used in a CRISPR/Cas9 system can comprise a spacer having a RNA sequence corresponding to the target sequence of SEQ ID NO: 69 or a variant thereof.
  • the spacer sequence of the guide RNA contains 1, 2, or 3 mismatches relative to the RNA sequence corresponding to the target sequence set forth in SEQ ID NO: 64 or SEQ ID NO: 69.
  • the spacer sequence of the guide RNA has about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to the RNA sequence corresponding to the target sequence set forth in SEQ ID NO: 64 or SEQ ID NO: 69.
  • TRAC gene editing [0148]
  • a guide RNA used herein can disrupt a TRAC gene. This disruption leads to loss of function of the TCR and renders T cell non-alloreactive and suitable for allogeneic transplantation, minimizing the risk of graft versus host disease.
  • expression of the endogenous TRAC gene is eliminated to prevent a graft-versus-host response.
  • a gRNA targeting TRAC gene that can be used in a Crispr/Cas9 system can comprise a spacer having a RNA sequence corresponding to the target sequence of SEQ ID NO: 65 or a variant thereof.
  • an AAV insertion into the TRAC locus can take place in an electroporation event by double stranded breaks and HDR using conventional CRISPR-Cas9 methods.
  • the spacer sequence of the guide RNA contains 1, 2, or 3 mismatches relative to the RNA sequence corresponding to the target sequence set forth in SEQ ID NO: 65.
  • the spacer sequence of the guide RNA has about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to the RNA sequence corresponding to the target sequence set forth in SEQ ID NO: 65.
  • Cblb gene editing [0149]
  • a guide RNA used herein can disrupt a Cblb (Cbl Proto- Oncogene B) gene.
  • Cblb gene is a proto-oncogene that encodes a RING finger E3 ubiquitin ligase.
  • the encoded protein is one of the enzymes required for targeting substrates for degradation by the proteasome.
  • the Cblb protein mediates the transfer of ubiquitin from ubiquitin conjugating enzymes (E2) to specific substrates.
  • Cblb protein also contains an N-terminal phosphotyrosine binding domain that allows it to interact with numerous tyrosine-phosphorylated substrates and target them for proteasome degradation.
  • Cblb protein functions as a negative regulator of many signal transduction pathways.
  • Cblb gene has been found to be mutated or translocated in many cancers including acute myeloid leukaemia, and expansion of CGG repeats in the 5' UTR has been associated with Jacobsen syndrome. Mutations in this gene are also the cause of Noonan syndrome-like disorder.
  • Cblb proteins has 982 amino acid residues and contains a highly conserved tyrosine kinase-binding domain, a linker domain, a RING finger, a proline-rich domains, and an ubiquitin-associated domain. It has been demonstrated that Cblb ablation can enhance cytotoxicity of human placental stem cell-derived natural killer cell for cancer immunotherapy. Cblb deficiency can also overcome endogenous CD8+ T cell exhaustion and deletion of Cblb in CAR T cells can render them resistant to exhaustion. Thus, targeting Cblb can offer therapeutic advantages through the development of efficient CAR T cell therapy for solid tumors.
  • gRNAs used for disrupting Cblb gene can target any one of the Cblb exons.
  • Exemplary gRNAs targeting Cblb gene are provided in FIG.17.
  • gRNAs targeting Cblb gene can have a spacer having a RNA sequence corresponding to any one of the target sequences set forth in SEQ ID NOs: 77-100 or a variant thereof.
  • a gRNA targeting the Cblb gene can comprises a spacer having a RNA sequence corresponding to any one of the target sequences set forth in SEQ ID NOs: 79, 81, 87, 91, 92, 96, and 97 or a variant thereof.
  • the spacer sequence of the guide RNA contains 1, 2, or 3 mismatches relative to the RNA sequence corresponding to any one of the target sequences set forth in SEQ ID NOs: 79, 81, 87, 91, 92, 96, and 97. In some embodiments, the spacer sequence of the guide RNA has about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or higher identity to the RNA sequence corresponding to any one of the target sequences set forth in SEQ ID NOs: 79, 81, 87, 91, 92, 96, and 97.
  • gRNAs used for disrupting Cblb gene can target exon 7 of the Cblb gene.
  • the gRNA targeting the Cblb gene can comprise a spacer (a) having a RNA sequence corresponding to the target sequence of SEQ ID NO: 87; (b) having 1, 2, or 3 mismatches relative to the RNA sequence corresponding to the target sequence of SEQ ID NO: 87; or (c) having about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or higher identity to the RNA sequence corresponding to the target sequence of SEQ ID NO: 87.
  • a guide RNA used herein can disrupt a PVR (poliovirus receptor) gene encoding CD155 protein.
  • Human CD155 ⁇ is a transmembrane protein comprising an N-terminal ectodomain divided into the Ig-like D1, D2, and D3 domains, followed by a transmembrane domain and a C-terminal cytoplasmic domain.
  • the PVR gene contains 8 exons, encoding a CD155 protein of 417 amino acid in length.
  • gRNAs used for disrupting PVR gene can target any one of the PVR exons. Exemplary gRNAs targeting PVR gene are provided in Table 23.
  • gRNAs targeting PVR gene can have a spacer having any one of the sequences set forth in SEQ ID NOs: 101-121 or a variant thereof.
  • gRNAs used for disrupting PVR gene can target one of the Ig-like D1, D2, or D3 domains or a region adjacent to D1, D2 or D3 domains of the CD155 protein.
  • gRNAs used for disrupting PVR gene can target exon 1, 2, 3, 4, 5 or 6 of the PVR gene.
  • a gRNA targeting the PVR gene can comprise a spacer having any one of the sequences set forth in SEQ ID NOs: 101, 103, 105, and 113-116 or a variant thereof.
  • the spacer sequence of the guide RNA contains 1, 2, or 3 mismatches relative to any one of the sequences set forth in SEQ ID NOs: 101, 103, 105, and 113-116. In some embodiments, the spacer sequence of the guide RNA has about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or higher identity to any one of the sequences set forth in SEQ ID NOs: 101, 103, 105, and 113-116. In some embodiments, gRNAs used for disrupting PVR gene can target exon 6 of the PVR gene.
  • the gRNA targeting the PVR gene can comprise a spacer (a) having a sequence of SEQ ID NO: 116; (b) having 1, 2, or 3 mismatches relative to the sequence co of SEQ ID NO: 116; or (c) having about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or higher identity to the sequence of SEQ ID NO: 116.
  • MHC class I chain-related (MIC) gene editing [0153]
  • a guide RNA used herein can disrupt a MIC gene. MIC genes are located within the MHC class I region of chromosome 6 p21.3.
  • MICA to MICG There are a total of seven genes designated as MICA to MICG, of which MICA and MICB are the only functional genes, while MICC to MICG are essentially pseudogenes. Similar to MHC class I molecules, MICA/B has 3 extracellular Ig-like domains, ⁇ 1, ⁇ 2 and ⁇ 3, encoded by exon 2, exon 3 and exon 4, respectively. MICA/B further contains a leader sequence (encoded by exon 1) at the 5’ end of the extracellular Ig-like domains and a transmembrane region (encoded by exon 5) and a cytoplasmic tail (encoded by exon 6) at the 3’ end of the extracellular Ig-like domains.
  • FIG. 31 illustrates a schematic gene structure of MICA.
  • MICA gene encodes a MICA protein having 383 amino acids.
  • MICA/B functions as a stress-induced antigen that binds NK cell activating receptor NKG2D (tumor surveillance or viral infection) resulting in target cell lysis and secretion of inflammatory cytokines.
  • Ablation of MICA/B expression from allogeneic CAR T cells ⁇ 2m KO or other MHC Class I modulating edit
  • gRNAs used for disrupting MICA or MICB gene can target any one of the exons. Exemplary gRNAs targeting MICA and MICB gene are provided in Table 26 and Table 27, respectively.
  • the spacer sequence of the guide RNA contains 1, 2, or 3 mismatches relative to any one of the sequences set forth in SEQ ID NOs: 127, 128 and 131. In some embodiments, the spacer sequence of the guide RNA has about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or higher identity to any one of the sequences set forth in SEQ ID NOs: 127, 128 and 131.
  • TAP gene editing [0154] In some embodiments, a guide RNA used herein can disrupt a TAP (transporter associated with antigen processing) gene.
  • TAP plays a crucial role in the processing and presentation of the major histocompatibility complex (MHC) class I restricted antigens.
  • MHC major histocompatibility complex
  • TAP transports peptides from the cytosol into the endoplasmic reticulum, selecting peptides matching in length and sequence to respective MHC class I molecules.
  • Abnormalities in MHC class I surface expression have been found in a number of different malignancies, including tumors of distinct histology, viral infections, and autoimmune diseases, and therefore represent an important mechanism of malignant or virus-infected cells to escape proper immune response. In many cases, this downregulation has been attributed to impaired TAP expression.
  • TAP possesses two hydrophobic N-terminal domains crossing the membrane multiple times, forming a translocation pore and two highly conserved C-terminal cytosolic adenosine triphosphate (ATP)-binding cassettes.
  • TAP consists of two submits, TAP-1 and TAP-2.
  • gRNAs used for disrupting TAP-1 or TAP-2 gene can target any one of TAP exons (e.g., exons 1, 2, 3, 4, 5, 6, 8, 9, 10 or 11).
  • Exemplary gRNAs targeting TAP-1 gene are provided in Table 29.
  • gRNAs targeting TAP-1 gene can comprise a spacer having any one of the sequences set forth in SEQ ID NOs: 150-198 or a variant thereof.
  • gRNAs targeting TAP-1 gene can have a spacer having any one of the sequences set forth in SEQ ID NOs: 150-174 or a variant thereof.
  • gRNAs used for disrupting TAP-1 gene can target exon 5 of the TAP-1 gene.
  • a gRNA targeting TAP-1 gene can comprise a spacer having a sequence set forth in SEQ ID NO: 174 or a variant thereof.
  • the spacer sequence of the guide RNA contains 1, 2, or 3 mismatches relative to the sequence set forth in SEQ ID NO: 174.
  • the spacer sequence of the guide RNA has about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or higher identity to the sequence set forth in SEQ ID NO: 174.
  • Non-limiting gRNAs used herein are provided in Table 34.
  • the gRNAs can comprise a spacer having any one of the sequences set forth in SEQ ID Nos: 247-254 or a variant thereof.
  • the spacer sequence of the guide RNA contains 1, 2, or 3 mismatches relative to any one of the sequences set forth in SEQ ID NO: 247-254.
  • the spacer sequence of the guide RNA has about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or higher identity to any one of the sequences set forth in SEQ ID NO: 247-254.
  • a guide RNA used herein can disrupt a Fas cell surface death receptor (FAS) gene.
  • FAS Fas cell surface death receptor
  • the FAS is a member of the TNF- receptor superfamily and contributes to the regulation of programmed cell death.
  • the gRNAs used herein for disrupting FAS gene are provided in Table 35.
  • the gRNs can comprise a spacer having any one of the sequences set forth in SEQ ID Nos: 255-265 or a variant thereof.
  • the spacer sequence of the guide RNA contains 1, 2, or 3 mismatches relative to any one of the sequences set forth in SEQ ID NO: 255-265.
  • the spacer sequence of the guide RNA has about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or higher identity to any one of the sequences set forth in SEQ ID NO: 255-265.
  • the gRNAs described herein disrupts one or more of the genes described herein including Reg1, TGFBRII, FAS, CD70, ⁇ 2M, TRAC, Cblb, PVR, MCIA/B, and TAP (e.g., TAP-1) by introducing one or more genetic edits at one or more suitable target sites (e.g., in coding regions or in non-coding regulatory regions such as promoter regions), thus substantially reducing or completely eliminating the expression of Reg1, TGFBRII, FAS, CD70, ⁇ 2M, TRAC, Cblb, PVR, MCIA/B, and/or TAP (e.g., TAP-1).
  • TAP e.g., TAP-1
  • the gRNAs described herein are used in base editing together with a base editor described herein.
  • a base editor described herein For example, one or more of the gRNAs described herein and a base editor protein or a nucleic acid encoding the base editor protein can be introduced into cells (e.g., T cells) via any suitable means (e.g., via electroporation or chemical transfection) in one or more events.
  • base editors are utilized in combination with CRISPR-Cas9 editing to create cells having multiple edits in two or more target genes such as Reg1, TGFBRII, FAS, CD70, ⁇ 2M, TRAC, Cblb, PVR, MCIA/B, and/or TAP (e.g., TAP-1).
  • a base editor or a nucleic acid encoding the base editor and a RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease together with respective gRNAs can be introduced into the cells in one or more events (e.g., via electroporation or chemical transfection).
  • cells can be transfected in a single electroporation step in which a RNA-guided endonuclease or a nucleic acid encoding the RNA- guided endonuclease and one or more gRNAs are combined with base editing reagents comprising a base editor or a nucleic acid encoding the base editor and one or more gRNAs.
  • cells can be transfected in two or more electroporation events.
  • cells can be first transfected with base editing reagents comprising gRNAs and a base editor or a nucleic acid encoding the base editor in one or more events, followed by another transfection with a RNA- guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease and one or more guide RNAs.
  • Multiplex gene editing [0160] Provided herein also includes methods of making multiplex gene edits by introducing to DNA molecules or a cell comprising the DNA molecules two or more guide RNAs targeting at least one target nucleotide sequence (e.g., two or more target sequences) of the DNA molecules.
  • a method comprises contacting a plurality of DNA molecules with two or more guide RNAs described herein targeting two or more target nucleotide sequences of the plurality of DNA molecules.
  • the method further comprises contacting the plurality of DNA molecules with a gene editing system.
  • the gene editing system is a base editing system comprising a base editor protein described herein or a nucleic acid encoding the base editor protein.
  • the gene editing system is a CRISPR-Cas9 nuclease system comprising a RNA-guided endonuclease (e.g., SluCas9, SaCas9, SpCas9) or a nucleic acid encoding the RNA-guided endonuclease.
  • the gene editing system comprises both the CRISPR-Cas9 system and the base editing system.
  • any of the base editor and/or nucleases disclosed herein may be delivered using a vector system, including, but not limited to, plasmid vectors, DNA minicircles, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirus vectors and adeno-associated virus vectors, and combinations thereof.
  • plasmid vectors DNA minicircles, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirus vectors and adeno-associated virus vectors, and combinations thereof.
  • Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding nucleases and/or base editors and donor templates in cells (e.g., T cells).
  • Non-viral vector delivery systems include DNA plasmids, DNA minicircles, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer.
  • Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
  • Methods of non-viral delivery of nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, naked RNA, capped RNA, artificial virions, and agent-enhanced uptake of DNA.
  • a method of multiplex DNA editing uses the base editing fusion proteins, complexes and compositions described herein for editing nucleic acids.
  • the method can comprise contacting a plurality of DNA molecules with (a) a base editor fusion protein or a nucleic acid encoding the base editor fusion protein; and (b) two or more guide RNAs (gRNAs) targeting at least one target nucleotide sequence of the plurality of DNA molecules, thereby editing the plurality of DNA molecules by deaminating a nucleotide base within the at least one target nucleotide sequence of the plurality of DNA molecules.
  • the number of guide RNAs used in multiplex gene editing can be two, three, four, five or more.
  • the plurality of DNA molecules can be in contact with the base editor protein and gRNA in an effective amount and under conditions suitable for the deamination of a nucleotide base.
  • a method comprises contacting a composition comprising a base editor protein described herein or a nucleic acid sequence encoding the base editor protein and one or more guide RNAs targeting one or more target genes (e.g., two or more guide RNAs targeting one, two, or more target genes) in an effective amount and under conditions suitable for the deamination of a nucleotide base.
  • the two or more gRNAs can target two or more different target nucleotide sequences in a same target gene (e.g., targeting different exons in a target gene).
  • the two or more gRNAs can also target two or more different target nucleotide sequences in different target genes.
  • the two or more gRNAs can target a same target nucleotide sequence.
  • the method can further comprise providing a plurality of cells comprising the plurality of DNA molecules.
  • the cells can be T cells or precursor cells thereof.
  • the method can comprise delivering to the plurality of cells a nucleic acid encoding a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the method can comprise generating a population of genetically engineered cells comprising at least two or more edits in the DNA molecules.
  • the contacting can be performed in vivo, ex vivo, or in vitro.
  • the contacting is performed in vivo in a subject susceptible to having, having, or diagnosed with the disease or disorder.
  • the two or more gRNAs can target a same target gene or two or more different target genes associated with different diseases or disorders.
  • the disease or disorder is a disease associated with a point mutation, or a single- base mutation, in the genome.
  • the disease is a genetic disease, a cancer, a metabolic disease, or a lysosomal storage disease.
  • the disease or disorder is cancer.
  • the target DNA sequence and/or the target gene comprises a sequence associated with a disease or disorder and wherein the deamination of a nucleotide base results in a sequence that is not associated with a disease or disorder.
  • the target DNA sequence comprises a T to C point mutation associated with a disease or disorder and wherein the deamination of the mutant C base results in a sequence that is not associated with a disease or disorder.
  • the deamination of the target nucleobase results in the correction of a genetic defect, e.g., in the correction of a point mutation that leads to a loss of function in a gene product.
  • the genetic defect can be associated with a disease or disorder.
  • the methods provided herein are used to introduce a deactivating point mutation into a gene or allele that encodes a gene product that is associated with a disease or disorder (e.g., an oncogene).
  • a disease or disorder e.g., an oncogene
  • the sequence associated with a disease or disorder is a gene encoding a protein.
  • the deamination results in a disrupted or deactivated gene such that expression of a functional protein from the gene is reduced or inhibited.
  • the deamination can generate a premature stop codon in a coding sequence, which results in the expression of a truncated gene product, e.g., a truncated protein lacking the function of the full- length protein.
  • the methods described herein can restore the function of dysfunctional gene via base editing.
  • any single nucleotide polymorphisms (SNPs) involving a T to C point mutation while having a nearby Cas recognizable PAM sequence can be corrected using the method herein described. Deamination of the mutant C back to U corrects the mutation.
  • genes comprising a T to C point mutation associated with a disease or disorder include, but are not limited to, CBS, DPYS, AGA, ALDOB, RFX6, TMEM67, ERCC6, GJC2, PC, ADSL, TPP1, BEST1, ACAT1, SMPD1, LDLR, TLL1, SLC26A2, GBA, EIF2B3, GAN, ANKH, FBLN5, ROBO2, KLF6, DDX11, FOXF1, NOTCH3, MMP13, ITGB2, ABCA1, MT-TL2, MT-TI, MT- ND1, PRPS1, F8, F9, TAZ, BTK, FOXP3, CASK, MECP2, MVK, TEAD1, SOS1, FGFR2, GP9, GPI, PTH, KIT, MAPT, LMNA, KRT5, HBG2, GH1, GRN, GPD2, NR3C1, SMC1A, SGSH, MRPS22, CLN6, MFN2, RHBDF2, UVSSA, P
  • the target gene sequence does not comprise a T to C point mutation.
  • the deaminase can introduce a deactivating mutation or a premature stop codon in a coding sequence in the target gene sequence, resulting in the expression of a non-functional and/or truncated gene product.
  • the target sequence comprises a Cbl proto- oncogene B (Cblb) gene or a variant thereof.
  • CBL-B is an E3 ubiquitin-protein ligase that in humans is encoded by the Cblb gene.
  • the target sequence comprises an ANGPTL3 gene or a variant thereof.
  • the two or more target genes are selected from the group consisting of: Cblb, PVR, MICA/B, TAP (TAP-1 or TAP-2), TGFBRII, Reg1, CD70, ⁇ 2M, FAS or a combination thereof.
  • the two or more target genes comprise a Cblb gene.
  • the gRNA targeting the Cblb gene can comprise a spacer (a) having a RNA sequence corresponding to the target sequence of SEQ ID NO: 87; (b) having 1, 2, or 3 mismatches relative to the RNA sequence corresponding to the target sequence of SEQ ID NO: 87; or (c) having about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or higher identity to the RNA sequence corresponding to the target sequence of SEQ ID NO: 87.
  • the two or more target genes comprise a PVR gene.
  • a gRNA targeting the PVR gene can comprise a spacer (a) having a sequence set forth in any one of SEQ ID NOs: 101, 103, 105, and 113-116, (b) having 1, 2, or 3 mismatches relative to any one of the sequences set forth in SEQ ID NOs: 101, 103, 105, and 113-116, or (c) having about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or higher identity to any one of the sequences set forth in SEQ ID NOs: 101, 103, 105, and 113-116.
  • the gRNA targeting the PVR gene can comprise a spacer (a) having a sequence of SEQ ID NO: 116; (b) having 1, 2, or 3 mismatches relative to the sequence co of SEQ ID NO: 116; or (c) having about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or higher identity to the sequence of SEQ ID NO: 116.
  • the two or more target genes comprise a MICA gene.
  • a gRNA targeting MICA gene can comprise a spacer having any one of sequences set forth in SEQ ID NOs: 127, 128 and 131 or a variant thereof.
  • the spacer sequence of the guide RNA contains 1, 2, or 3 mismatches relative to any one of the sequences set forth in SEQ ID NOs: 127, 128 and 131. In some embodiments, the spacer sequence of the guide RNA has about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or higher identity to any one of the sequences set forth in SEQ ID NOs: 127, 128 and 131. [0175] In some embodiments, the two or more target genes comprise a TAP-1 gene.
  • a gRNA targeting TAP-1 gene can comprise a spacer (a) having a sequence set forth in SEQ ID NO: 174, (b) having 1, 2, or 3 mismatches relative to the sequence set forth in SEQ ID NO: 174, or (c) having about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or higher identity to the sequence set forth in SEQ ID NO: 174.
  • the two or more target genes are selected from the group consisting of Regnase-1 (Reg1) gene, Transforming Growth Factor Beta Receptor II (TGFBRII) gene, beta-2-microglobulin ( ⁇ 2M) gene, CD70 gene, FAS gene, T cell receptor alpha chain constant region (TRAC) gene, or a combination thereof.
  • the gRNA targeting TGFBRII, Reg1, CD70, or ⁇ 2M can comprise a spacer (a) having any one of the sequences set forth in SEQ ID NO: 61, 62, 63, or 64, respectively, (b) having 1, 2, or 3 mismatches relative to any one of the sequences set forth in SEQ ID NO: 61, 62, 63, or 64, respectively, or (c) having about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or higher identity to any one of the sequences set forth in SEQ ID NO: 61, 62, 63, or 64, respectively.
  • the method can further comprise contacting the plurality of DNA molecules with a RNA-guided DNA endonuclease (e.g., Cas9) or a nucleic acid encoding the RNA-guided DNA endonuclease.
  • a RNA-guided DNA endonuclease e.g., Cas9
  • the gRNA provided together with the RNA-guided DNA endonuclease is capable of guiding the RNA-guided DNA endonuclease to matching sequences of DNA.
  • the gRNA targeting TRAC, TGFBRII, Reg1, CD70, or ⁇ 2M can comprise a spacer (a) having any one of the sequences set forth in SEQ ID NO: 65, 66, 67, 68, or 69, respectively, (b) having 1, 2, or 3 mismatches relative to any one of the sequences set forth in SEQ ID NO: 65, 66, 67, 68, or 69, respectively, or (c) having about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or higher identity to any one of the sequences set forth in SEQ ID NO: 65, 66, 67, 68, or 69, respectively.
  • the method comprises contacting a plurality of DNA molecules or cells (e.g., T cells) comprising the plurality of DNA molecules with (a) a base editor fusion protein or a nucleic acid encoding the base editor fusion protein; and (b) one or more guide RNAs (gRNAs) targeting one or more first target gene of the plurality of DNA molecules, thereby editing the plurality of DNA molecules by deaminating a nucleotide base within the one or more first target gene of the plurality of DNA molecules.
  • gRNAs guide RNAs
  • the one or more first target gene is selected from the group consisting of: Cblb, PVR, MICA/B, TAP (TAP-1 or TAP-2), TGFBRII, Reg1, CD70, ⁇ 2M, FAS, and a combination thereof.
  • the method can further comprise contacting the plurality of DNA molecules or cells comprising the plurality of DNA molecules with (c) a RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease; and (d) one or more guide RNAs targeting one or more second target gene of the plurality of DNA molecules, thereby editing the one or more second target gene in the plurality of DNA molecules.
  • the one or more second target gene comprises TRAC gene.
  • the gene editing leads to a disrupted gene (e.g., a disrupted Cblb, PVR, MICA/B, TAP (TAP-1 or TAP-2), TGFBRII, Reg1, CD70, ⁇ 2M, FAS and/or TRAC) having an insertion, deletion or substitution relative to an endogenous gene such that expression of a functional protein from the endogenous gene is reduced or inhibited.
  • a disrupted gene e.g., a disrupted Cblb, PVR, MICA/B, TAP (TAP-1 or TAP-2), TGFBRII, Reg1, CD70, ⁇ 2M, FAS and/or TRAC
  • one or more of Cblb, PVR, MICA/B, TAP (TAP-1 or TAP-2), TGFBRII, Reg1, CD70, ⁇ 2M, and FAS genes may be disrupted first using base editing, followed by the editing of TRAC gene with CRISPR-Cas9 nuclease editing.
  • the disruption of the Cblb, PVR, MICA/B, TAP (TAP-1 or TAP-2), TGFBRII, Reg1, CD70, ⁇ 2M, and/or FAS genes can be performed in a single event or in multiple sequential events.
  • the genetically engineered cells can be produced by multiple, sequential electroporation events with multiple gRNAs targeting the genes of interest, e.g., Cblb, PVR, MICA/B, TAP (TAP-1 or TAP-2), TGFBRII, Reg1, CD70, ⁇ 2M, FAS, TRAC etc.
  • multiple gRNAs targeting the genes of interest e.g., Cblb, PVR, MICA/B, TAP (TAP-1 or TAP-2), TGFBRII, Reg1, CD70, ⁇ 2M, FAS, TRAC etc.
  • the multiple gRNAs, base editors and RNA-guided endonuclease are introduced into cells in two sequential electroporation events to produce resulting cell populations, in which (a) a base editor fusion protein or a nucleic acid encoding the base editor fusion protein and (b) one or more guide RNAs (gRNAs) targeting one or more a first target gene (e.g., Cblb, PVR, MICA/B, TAP (TAP-1 or TAP-2), TGFBRII, Reg1, CD70, ⁇ 2M, and FAS) are delivered to a target, followed by the delivery of (c) a RNA-guided endonuclease or a nucleic acid encoding the RNA- guided endonuclease, and (d) a gRNA targeting one or more second target gene (e.g., TRAC).
  • a first target gene e.g., Cblb, PVR, MICA/B, TAP (TAP-1 or
  • the genetic editing described herein can be carried out in a single electroporation event to produced modified cell populations, in which (a) a base editor fusion protein or a nucleic acid encoding the base editor fusion protein, (b) one or more guide RNAs (gRNAs) targeting one or more first target gene (e.g., Cblb, PVR, MICA/B, TAP (TAP-1 or TAP-2), TGFBRII, Reg1, CD70, ⁇ 2M, and FAS), (c) a RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease, and (d) a gRNA targeting one or more second target gene (e.g., TRAC) are delivered to a target simultaneously.
  • gRNAs guide RNAs
  • first target gene e.g., Cblb, PVR, MICA/B, TAP (TAP-1 or TAP-2), TGFBRII, Reg1, CD
  • the method can further comprise contacting the plurality of DNA molecules or cells comprising the plurality of DNA molecules with a nucleic acid encoding a chimeric antigen receptor (CAR).
  • the nucleic acid encoding the CAR can be inserted in the disrupted gene, such as in the disrupted Reg1 gene, the disrupted TGFBRII gene, the disrupted TRAC gene, the disrupted ⁇ 2M, or the disrupted CD70 gene.
  • the nucleic acid encoding the CAR is inserted in the disrupted TRAC gene.
  • the nucleic acid encoding the CAR may replace the deleted fragment in the TRAC gene.
  • any of the CAR constructs disclosed herein may comprise an extracellular antigen binding domain specific to a tumor antigen, a co-stimulatory signaling domain of 4-1BB or CD28, and a cytoplasmic signaling domain of CD3 ⁇ .
  • the tumor antigen can be CD19, CD70, CD33, PTK7, or BCMA (B-cell maturation antigen).
  • the CAR can bind BCMA (anti- BCMA CAR).
  • the extracellular antigen binding domain in the anti-BCMA CAR can be a single chain variable fragment (scFv) that binds BCMA (anti-BCMA CAR).
  • the guide RNA used herein can comprise two or more guide RNAs targeting two or more target genes and the methods provided here can edit two more target genes. In some embodiments, editing of the target genes results a significant reduction in the gene products of the two or more target genes (e.g., mRNA or protein level).
  • the gene products of the two or mor target genes are reduced by at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a number or a range between any two of these values.
  • the gRNAs, base editors, and/or nucleases can be introduced into cells (e.g., T cells) using any suitable mean identifiable to a skilled person and described herein.
  • delivery of gRNA, a base editor, an RNA-guided nuclease, an RNP may be through direct injection or cell transfection using known methods such as electroporation or chemical transfection. Other conventional cell transfection methods may also be used.
  • an RNA-guided nuclease and/or a base editor can be delivered to a cell in a DNA vector that expresses the RNA-guided nuclease and/or the base editor in the cell.
  • an RNA-guided nuclease and/or a base editor can be delivered to a cell in an RNA that encodes and expresses the RNA-guided nuclease and/or the base editor in the cell.
  • a gRNA targeting a gene can be delivered to a cell as a RNA, or a DNA vector that expresses the gRNA in the cell.
  • the concentration and/or amount of base editor proteins or mRNA encoding the base editor proteins can vary in different embodiments.
  • the mRNA encoding the base editor protein can be provided at an amount of at least about 0.5 pmol/million cells, 1 pmol/million cells (e.g., T cells), 1.2 pmol/million cells, 1.4 pmol/million cells, 1.6 pmol/million cells, 1.8 pmol/million cells, 2.0 pmol/million cells, 2.5 pmol/million cells, 3.0 pmol/million cells, 4.0 pmol/million cells, 5.0 pmol/million cells, 6.0 pmol/million cells, 7.0 pmol/million cells, 8.0 pmol/million cells, 9.0 pmol/million cells, 10.0 pmol/million cells, or a number or a range between any two of these values.
  • the mRNA encoding the base editor protein is provided at an amount of at least 1.15 pmol/million cells.
  • the cells can be immune cells, for example T cells, B cells, dendritic cells, or any combination thereof.
  • the cells are stem cells.
  • the cells are somatic cells.
  • the concentration and/or amount of the gRNAs can vary in different embodiments.
  • each of the gRNAs can be provided at an amount of at least about 70 pmol/million cells (e.g., T cells), 75 pmol/million cells, 80 pmol/million cells, 85 pmol/million cells, 90 pmol/million cells, 95 pmol/million cells, 100 pmol/million cells, 120 pmol/million cells, 140 pmol/million cells, 160 pmol/million cells, 180 pmol/million cells, 200 pmol/million cells, 220 pmol/million cells, 240 pmol/million cells, 260 pmol/million cells, 280 pmol/million cells, 300 pmol/million cells, or a number or a range between any two of these values.
  • T cells e.g., T cells
  • 75 pmol/million cells e.g., 80 pmol/million cells, 85 pmol/million cells, 90 pmol/million cells, 95 pmol/million cells, 100 pmol/million cells, 120 pmol/million cells
  • each gRNA is provided at a concentration of at least 90 pmol/million cells.
  • all the base editing guides e.g., gRNAs targeting Reg1 gene, CD70 gene, ⁇ 2M gene, TGFBRII gene, Cblb, PVR, MCIA/B, and/or TAP
  • multiplex gene editing with the base editor proteins described herein can result in an editing efficiency at least about 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% higher than with conventional CRISPR-Cas9 methods using ribonucleoproteins.
  • multiplex gene editing with the base editor proteins described herein can result in an editing efficiency for each target gene at an equivalent level compared to the editing efficiency in individual gene editing.
  • Methods for Analyzing Off-Target Deamination also includes methods of using the base editing fusion proteins and guide RNAs described herein to evaluate the ability of base editing proteins to deaminate cytosines in DNA regions unrelated to their on-target loci (i.e., spurious off-target deamination activity).
  • a method can comprise transfecting a cell with a first Cas9 domain or a nucleic acid encoding the first Cas9 domain to produce a cell expressing the first Cas9 domain and transfecting the cell expressing the first Cas9 domain with a first guide RNA and a fusion protein comprising: 1) a second Cas9 domain, wherein the second Cas9 domain when associated with a second guide RNA (gRNA) specifically binds to a target nucleic acid sequence and 2) a cytidine deaminase domain capable of deaminating a cytosine base in a single-stranded portion of the target nucleic acid sequence, or a nucleic acid sequence encoding the fusion protein.
  • gRNA second guide RNA
  • the first guide RNA when associated with the first Cas9 domain binds to a non-target nucleic acid sequence (e.g., a nucleic acid sequence different from the target nucleic acid sequence).
  • a non-target nucleic acid sequence e.g., a nucleic acid sequence different from the target nucleic acid sequence.
  • target locus e.g., the non-target nucleic acid sequence
  • base pairing between the first gRNA and target DNA strand leads to displacement of a small segment of single-stranded DNA in an “R-loop”.
  • DNA bases within the R-loop formed by the first Cas9 domain and the first gRNA may be modified by the deaminase domain of the fusion protein (see, for example, FIG.13, the bottom panel).
  • the first Cas9 domain, the fusion protein and the guide RNAs can be introduced to the cell via any suitable transfection approach described herein and known in the art.
  • the first Cas9 domain, the first guide RNA, and the fusion protein can be co-transfected (e.g., transfected simultaneously) or transfected sequentially (e.g., one after another).
  • the method comprises contacting the cell with a plasmid or viral vector encoding a first Cas9 domain to produce a cell expressing the first Cas9 domain.
  • the cell is in vitro.
  • the cell expressing the first Cas9 domain can be further transfected (e.g., via nucleofection) with a nucleic acid encoding a fusion protein disclosed herein and a first guide RNA capable of binding to the first Cas9.
  • the first Cas9 domain can comprise a catalytically impaired Cas9.
  • the first Cas9 domain can comprise a truncated version of a nuclease domain or no nuclease domain at all.
  • the first Cas9 domain can comprise a Cas9 nickase, such as a SaCas9 nickase.
  • the first Cas9 domain can further comprise at least one UGI domain.
  • the first Cas9 domain can be fused to two UGI domains.
  • the second Cas9 domain of the fusion protein can be any Cas9 domain described herein in the context of a base editing fusion protein.
  • the first Cas9 domain and the second Cas9 domain are different.
  • the first Cas9 domain and the second Cas9 domain can be derived from different organisms.
  • the first Cas9 domain can comprise a SaCas9 domain while the second Cas9 domain can comprise a SpCas9 domain.
  • the first Cas9 domain and the second Cas9 can have different PAM specificities.
  • amplicon sequencing of the R-loop regions induced by the first Cas9 domain can be performed to analyze the spurious off-target deamination activity.
  • the base editing fusion proteins described herein have a spurious off-target deamination rate less than 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or lower.
  • Engineered Cells [0195] Provided herein includes a method for producing an engineered cells and a population of genetically engineered cells prepared by the method.
  • the method can, for example, comprise providing a plurality of cells, delivering to the plurality of cells (a) a fusion protein described herein or a nucleic acid encoding the fusion protein and (b) at least one guide RNA targeting one or more first target gene (e.g., , genetically editing the one or more first target gene, and producing one or more genetically engineered cells having at least one gene edit in the one or more first target gene.
  • the one or more first target gene is selected from the group consisting of: Cblb, PVR, MICA/B, TAP (TAP-1 or TAP-2), TGFBRII, Reg1, CD70, ⁇ 2M, FAS, and a combination thereof.
  • the method comprises delivering to the plurality of cells two or more guide RNAs targeting two or more target genes, therefore producing engineered cells having two more gene edits in the two or more target genes.
  • the method can further comprise delivering to the plurality of cells (c) a RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease; and (d) at least one guide RNA targeting one or more second target gene or at least one nucleic acid encoding the at least one guide RNA, wherein the at least one guide RNA is capable of guiding the RNA-guided endonuclease to matching sequences of DNA, thereby producing one or more genetically engineered cells having at least one gene edit in the one or more second target gene.
  • the one or more second target gene comprises TRAC gene.
  • the genetically engineered cells e.g., T cells
  • T cells can be produced by a single electroporation event or multiple, sequential electroporation events (e.g., two or more electroporation events).
  • the plurality of cells are immune cells.
  • the plurality of cells are T cells.
  • the plurality of cells (e.g., T cells) can be derived from parent T cells (e.g., non-edited wild-type T cells) obtained from a suitable source, for example, one or more mammal donors.
  • the parent T cells are primary T cells (e.g., non-transformed and terminally differentiated T cells) obtained from one or more human donors.
  • the parent T cells can be differentiated from precursor T cells obtained from one or more suitable donor or stem cells such as hematopoietic stem cells or inducible pluripotent stem cells (iPSC), which may be cultured in vitro.
  • T cells can be obtained from a number of sources, including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • T cells are obtained from a unit of blood collected from a subject using any number of techniques known to the skilled person, such as sedimentation, e.g., FICOLLTM separation.
  • T cells can be isolated from a mixture of immune cells (e.g., those described herein) to produce an isolated T cell population. For example, after isolation of peripheral blood mononuclear cells (PBMC), both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after activation, expansion, and/or genetic modification.
  • PBMC peripheral blood mononuclear cells
  • a specific subpopulation of T cells expressing one or more of the following cell surface markers: TCRab, CD3, CD4, CD8, CD27 CD28, CD38 CD45RA, CD45RO, CD62L, CD127, CD122, CD95, CD197, CCR7, KLRG1, MCH-I proteins and/or MCH-II proteins, can be further isolated by positive or negative selection techniques.
  • a specific subpopulation of T cells, expressing one or more of the markers selected from the group consisting of TCRab, CD4 and/or CD8, is further isolated by positive or negative selection techniques.
  • the engineered T cell populations do not express or do not substantially express one or more of the following markers: CD70, CD57, CD244, CD160, PD-1, CTLA4, H ⁇ 3, and LAG3.
  • subpopulations of T cells can be isolated by positive or negative selection prior to genetic engineering and/or post genetic engineering.
  • an isolated population of T cells can express one or more of the T cell markers, including, but not limited to a CD3+, CD4+, CD8+, or a combination thereof.
  • the T cells are isolated from a donor, or subject, and first activated and stimulated to proliferate in vitro prior to undergoing gene editing.
  • the T cell population comprises primary T cells isolated from one or more human donors. Such T cells are terminally differentiated, not transformed, depend on cytokines and/or growth factors for growth, and/or have stable genomes. Alternatively, the T cells can be derived from stem cells (e.g., HSCs or iPSCs) via in vitro differentiation. [0203] T cells from a suitable source can be subjected to one or more rounds of stimulation, activation and/or expansion. T cells can be activated and expanded generally using methods as described, for example, in U.S.
  • T cells can be activated and expanded for about, at least, at least about, at most, or at most about 4 hours, 6 hours, 12 hours, 24 hours, 1 day to 4 days, 1 day to 3 days, 1 day to 2 days, 2 days to 3 days, 2 days to 4 days, 3 days to 4 days, or 2 days, 3 days, or 4 days prior to introduction of the genome editing compositions into the T cells.
  • T cells are activated and expanded for about 4 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours prior to introduction of the gene editing compositions into the T cells.
  • T cells are activated at the same time that genome editing compositions are introduced into the T cells.
  • the T cell population can be expanded and/or activated after the genetic editing as disclosed herein.
  • T cell populations or isolated T cells generated by any of the gene editing methods described herein are also within the scope of the present disclosure.
  • the method herein described can further comprise delivering to the plurality of cells (e.g., T cells or precursor cells thereof described above) a nucleic acid encoding a chimeric antigen receptor (CAR).
  • the CAR can comprise an extracellular antigen binding domain specific to a tumor antigen, a co-stimulatory signaling domain of 4-1BB or CD28, and a cytoplasmic signaling domain of CD3 ⁇ .
  • the tumor antigen can be CD19, BCMA, CD70, CD33, or PTK7.
  • the CAR can binds CD19 (anti-CD19 CAR) and the extracellular antigen binding domain in the anti-CD19 CAR is a single chain variable fragment (scFv) that binds CD19 (anti-CD19 scFv).
  • the nucleic acid encoding a CAR can comprise an ectodomain that binds specifically to LIV1.
  • the ectodomain that binds specifically to LIV1 comprises an anti-LIV1 antigen-binding fragment, and optionally the anti- LIV1 antigen-binding fragment comprises an anti-LIV1 antibody.
  • the nucleic acid encoding a CAR can be delivered to the cells via conventional viral and non-viral based gene transfer methods known to a skilled person.
  • a nucleic acid encoding a CAR construct can be delivered to a cell using an adeno-associated virus (AAV) such as AAV6.
  • AAV adeno-associated virus
  • a nucleic acid encoding a CAR can be designed to insert into a genomic site of interest in the host T cells via a donor template.
  • a nucleic acid encoding a CAR (e.g., via a donor template, which can be carried by a viral vector such as an AAV vector) can be designed such that it can insert into a location within a TRAC gene to disrupt the TRAC gene in the engineered T cells and express the CAR polypeptide.
  • the method can comprise genetically editing one or more target genes, including the target genes described herein and known in the art, using the nucleic acid editing methods described.
  • the gRNA comprises two or more guide RNAs targeting two or more target genes and wherein the produced one or more genetically engineered cells have two or more gene edits in the two or more target genes.
  • the engineered cells can be produced by sequential targeting of the genes of interest. For example, in some embodiments, a first target gene can be edited first, followed by editing of a second, third, fourth and more target genes. In other embodiments, two or more target genes can be edited simultaneously. Accordingly, in some embodiments, the genetically engineered cells disclosed herein can be produced by multiple, sequential electroporation events with guide RNAs and the base editing fusion protein or complexes thereof targeting the genes of interest. In other embodiments, the engineered CAR cells disclosed herein can be produced by a single electroporation event with a complex comprising a base editing fusion protein and multiple gRNAs targeting the genes of interest.
  • the at least one target gene is selected from the group consisting of the Regnase-1 (Reg1) gene, the Transforming Growth Factor Beta Receptor II (TGFBRII) gene, the TRAC gene, the beta-2-microglobulin ( ⁇ 2M) gene, the CD70 gene, T cell receptor alpha chain constant region (TRAC) gene, Cbl Proto-Oncogene B (Cblb) gene, poliovirus receptor (PVR) gene, MHC class I chain-related A/B (MCIA/B) gene, transporter associated with antigen processing 1 (TAP-1) gene, or a combination thereof.
  • the Regnase-1 (Reg1) gene
  • TGFBRII Transforming Growth Factor Beta Receptor II
  • ⁇ 2M beta-2-microglobulin
  • CD70 CD70 gene
  • T cell receptor alpha chain constant region (TRAC) gene Cbl Proto-Oncogene B (Cblb) gene
  • poliovirus receptor (PVR) gene MHC class I chain-related A
  • the population of engineered cells comprise one or more gene edits.
  • the engineered cells comprise two or more gene edits.
  • at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more of the cells in the population of genetically engineered cells comprise at least two genome edits (e.g., two, three, four, five, six or more genome edits).
  • at least 50% of a population of engineered cells may not express a detectable level of a target protein.
  • At least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% or more of the engineered cells may not express a detectable level of a target protein (e.g., ⁇ 2M, TRAC, CD70, Reg1, TGFBRII, Cblb, CD155, MICA/B, TAP-1 protein).
  • a target protein e.g., ⁇ 2M, TRAC, CD70, Reg1, TGFBRII, Cblb, CD155, MICA/B, TAP-1 protein.
  • At least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more of the cells in the population of genetically engineered cells may not express a detectable level of two or more target proteins (e.g., two, three, four, five, six or more target proteins).
  • engineered cells (e.g., T cells) of the present disclosure exhibit at least 20% greater cellular proliferative capacity, relative to control cells.
  • engineered cells can exhibit about, at least, or at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% greater cellular proliferative capacity, relative to control cells.
  • engineered cells of the present disclosure exhibit 20%-100%, 20%- 90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 30%-100%, 30%-90%, 30%-80%, 30%-70%, 30%-60%, 30%-50%, 40%-100%, 40%-90%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%- 100%, 50%-90%, 50%-80%, 50%-70%, or 50%-60% greater cellular proliferative capacity, relative to control cells.
  • engineered cells of the present disclosure exhibit an at least 20% increase in cellular viability, relative to control cells.
  • engineered cells of the present disclosure can exhibit about, at least, or at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90% or a number between any two of the values, increase in cellular viability, relative to control cells.
  • engineered cells of the present disclosure exhibit a 20%-100%, 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 30%- 100%, 30%-90%, 30%-80%, 30%-70%, 30%-60%, 30%-50%, 40%-100%, 40%-90%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%-100%, 50%-90%, 50%-80%, 50%-70%, or 50%-60% increase in cellular viability, relative to control cells.
  • the control cells can be engineered cells or unedited cells.
  • the control cells are cells edited using a gene editing strategy different from the ones described herein.
  • the control cells can comprise one or more gene edits.
  • the control cells comprise one gene edit.
  • compositions and Therapeutic Applications Provided herein also include a pharmaceutical composition for carrying out the methods disclosed herein and related methods of using the base editing fusion protein, pharmaceutical compositions and cells described herein to prevent or treat a disease or disorder.
  • the composition can, for example, comprise (a) a fusion protein described herein or a nucleic acid sequence encoding the fusion protein and (b) one or more guide RNAs targeting one or more target genes.
  • a pharmaceutical composition comprising the composition and a pharmaceutical acceptable carrier or excipient.
  • a composition described above can further have one or more additional reagents, where such additional reagents are selected from a buffer, a buffer for introducing a polypeptide or polynucleotide into a cell, a wash buffer, a control reagent, a control vector, a control RNA polynucleotide, a reagent for in vitro production of the polypeptide from DNA, adaptors for sequencing and the like.
  • a buffer can be a stabilization buffer, a reconstituting buffer, a diluting buffer, or the like.
  • a composition can also include one or more components that can be used to facilitate or enhance the on-target binding or the cleavage of DNA by the endonuclease, or improve the specificity of targeting.
  • any components of a composition are formulated with pharmaceutically acceptable excipients such as carriers, solvents, stabilizers, adjuvants, diluents, etc., depending upon the particular mode of administration and dosage form.
  • guide RNA compositions are generally formulated to achieve a physiologically compatible pH, and range from a pH of about 3 to a pH of about 11, about pH 3 to about pH 7, depending on the formulation and route of administration.
  • Suitable excipients can include, for example, carrier molecules that include large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles.
  • excipients include antioxidants (for example and without limitation, ascorbic acid), chelating agents (for example and without limitation, EDTA), carbohydrates (for example and without limitation, dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose), stearic acid, liquids (for example and without limitation, oils, water, saline, glycerol and ethanol), wetting or emulsifying agents, pH buffering substances, and the like.
  • antioxidants for example and without limitation, ascorbic acid
  • chelating agents for example and without limitation, EDTA
  • carbohydrates for example and without limitation, dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose
  • stearic acid for example and without limitation, liquids (for example and without limitation, oils, water, saline, glycerol and ethanol), wetting or emulsifying agents, pH buffering substances, and the like.
  • Physiologically tolerable carriers are well known in the art.
  • Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline.
  • Aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes.
  • Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.
  • the amount of an active compound used in the cell compositions that is effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition and can be determined by standard clinical techniques.
  • the compounds herein described e.g., a base editing fusion protein or a nucleic acid encoding the base editing fusion protein and/or gRNAs
  • the compounds herein described can be delivered via transfection such as calcium phosphate transfection, DEAE- dextran mediated transfection, cationic lipid-mediated transfection, electroporation, electrical nuclear transport, chemical transduction, electrotransduction, Lipofectamine-mediated transfection, Effectene-mediated transfection, lipid nanoparticle (LNP)-mediated transfection, or any combination thereof.
  • the lipids comprised in the lipid nanoparticles comprise cationic lipids and/or ionizable lipids.
  • Any suitable cationic lipids and/or ionizable lipids known in the art can be used to formulate LNPs for delivery of gRNA and Cas endonuclease to the cells.
  • Exemplary cationic lipids include one or more amine group(s) bearing positive charge.
  • the cationic lipids are ionizable such that they can exist in a positively charged or neutral from depending on pH.
  • the cationic lipid of the lipid nanoparticle comprises a protonatable tertiary amine head group that shows positive charge at low pH.
  • the lipid nanoparticles can further comprise one or more neutral lipids (e.g., Distearoylphosphatidylcholine (DSPC), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-Dipalmitoyl-sn-glycero-3- phosphorylethanolamine (DPPE) etc. as a helper lipid), charged lipids, steroids, and polymers conjugated lipids.
  • neutral lipids e.g., Distearoylphosphatidylcholine (DSPC), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-Dipalmitoyl-sn-glycero-3- phosphorylethanolamine (DPPE) etc.
  • the lipid nanoparticles can have a mean diameter of about, at least, at least about, at most or at most about 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, or a number or a range between any of these values.
  • the lipid nanoparticle particle size is about 50 to about 100 nm in diameter, or about 70 to about 90 nm in diameter, or about 55 to about 95 nm in diameter.
  • the compounds of the composition described herein are encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle.
  • the encapsulation can be full encapsulation, partial encapsulation, or both.
  • the nucleic acid and/or polypeptides are fully or substantially encapsulated (e.g., greater than 90% of the RNA) in the lipid nanoparticle.
  • one or more compounds herein described are associated with a liposome or lipid nanoparticle via a covalent bond or non-covalent bond.
  • any of the compounds in the composition can be separately or together contained in a liposome or lipid nanoparticle.
  • the composition and/or pharmaceutical composition herein described can be administered to a subject in need thereof to prevent or treat a disease or disorder. Accordingly, the present disclosure also provides a method of preventing or treating a disease or disorder in a subject in need thereof. The method can comprise administering to the subject a therapeutically effective amount of the composition or pharmaceutical composition described herein, wherein the one or more target gene is associated with the disease or disorder.
  • a subject can be any subject for whom diagnosis, treatment, or therapy is desired. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.
  • the subject is suspected to have, having or diagnosed to have a disease or disorder.
  • Any suitable administration route capable of delivering the composition can be used herein.
  • the pharmaceutical composition thereof can be administered by aerosol delivery, nasal delivery, vaginal delivery, rectal delivery, buccal delivery, ocular delivery, local delivery, topical delivery, intracisternal delivery, intraperitoneal delivery, oral delivery, intramuscular injection, intravenous injection, subcutaneous injection, intranodal injection, intratumoral injection, intracardiac injection, intraperitoneal injection, intrathecal injection, intraventricular injection, intracerebroventricular injection, intradermal injection, or any combination thereof.
  • the administration can be local or systemic.
  • Systemic administration refers to the administration of a population of cells other than directly into a target site, tissue, or organ, such that it enters the subject's circulatory system and, thus, is subject to metabolism and other like processes.
  • Systemic administration can include enteral and parenteral administration.
  • more than one administration can be employed to achieve the desired level of gene expression over a period of various intervals, e.g., daily, weekly, monthly, or yearly.
  • the route is intravenous.
  • the pharmaceutical composition thereof can be administered to a subject in need thereof at a pharmaceutically effective amount.
  • the amount of the pharmaceutical composition can result in a desired reduction or loss of function in one or more gene products.
  • the disease or disorder can be, for example, a disease associated with a gene having a point mutation (e.g., a T to C point mutation) and the methods and compositions described herein can correct the point mutation therefore preventing or treating the disease or disorder.
  • a point mutation e.g., a T to C point mutation
  • Examples of genes comprising a pathogenic T to C point mutation associated with a disease or disorder are disclosed herein. Additional suitable gene sequences that can be corrected with the methods and compositions herein described will be apparent to those of skill in the art based on this disclosure.
  • the disease or disorder can be cancer.
  • Non-limiting examples of cancers that can be treated as provided herein include: breast cancer, e.g., estrogen receptor-positive breast cancer, prostate cancer, squamous tumors, e.g., of the skin, bladder, lung, cervix, endometrium, head neck, and biliary tract, and neuronal tumors.
  • the methods comprise delivering the CAR T cells of the present disclosure to a subject having cancer, including, breast cancer, e.g., estrogen receptor-positive breast cancer, prostate cancer, squamous tumors, e.g., of the skin, bladder, lung, cervix, endometrium, head neck, and biliary tract, and/or neuronal tumors.
  • the base editing fusion proteins, compositions, engineered T cells, methods and kits disclosed herein can be used to treat various types of cancer, including but are not limited to, melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC) and small-cell lung cancer (SCLC)), esophageal cancer, squamous cell carcinoma of the head and neck, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other neoplastic malignancies.
  • melanoma e.g., metastatic malignant melanoma
  • renal cancer e.g., clear cell carcinoma
  • prostate cancer e.g
  • the disease or condition provided herein includes refractory or recurrent malignancies whose growth may be inhibited using the methods and compositions disclosed herein.
  • the cancer is carcinoma, squamous carcinoma, adenocarcinoma, sarcomata, endometrial cancer, breast cancer, ovarian cancer, cervical cancer, fallopian tube cancer, primary peritoneal cancer, colon cancer, colorectal cancer, squamous cell carcinoma of the anogenital region, melanoma, renal cell carcinoma, lung cancer, non-small cell lung cancer, squamous cell carcinoma of the lung, stomach cancer, bladder cancer, gall bladder cancer, liver cancer, thyroid cancer, laryngeal cancer, salivary gland cancer, esophageal cancer, head and neck cancer, glioblastoma, glioma, squamous cell carcinoma of the head and neck, prostate cancer, pancreatic cancer, mesothelioma, sarcoma, hematological cancer, leuk
  • the cancer is carcinoma, squamous carcinoma (e.g., cervical canal, eyelid, tunica conjunctiva, vagina, lung, oral cavity, skin, urinary bladder, tongue, larynx, and gullet), and adenocarcinoma (for example, prostate, small intestine, endometrium, cervical canal, large intestine, lung, pancreas, gullet, rectum, uterus, stomach, mammary gland, and ovary).
  • the cancer is sarcomata (e.g., myogenic sarcoma), leukosis, neuroma, melanoma, and lymphoma.
  • the cancer can include pancreatic cancer, gastric cancer, ovarian cancer, uterine cancer, breast cancer, prostate cancer, testicular cancer, thyroid cancer, nasopharyngeal cancer, non-small cell lung (NSCLC), glioblastoma, neuronal, soft tissue sarcomas, leukemia, lymphoma, melanoma, colon cancer, colon adenocarcinoma, brain glioblastoma, hepatocellular carcinoma, liver hepatocholangiocarcinoma, osteosarcoma, gastric cancer, esophagus squamous cell carcinoma, advanced stage pancreas cancer, lung adenocarcinoma, lung squamous cell carcinoma, lung small cell cancer, renal carcinoma, intrahepatic biliary cancer, and a combination thereof.
  • NSCLC non-small cell lung
  • the cancer is breast cancer, prostate cancer, squamous tumor cancer, neuronal tumor cancer, or a combination thereof.
  • the cancer comprises cancer cells expressing LIV1.
  • the cancer can be a solid tumor, a liquid tumor, or a combination thereof.
  • the cancer is a solid tumor, including but are not limited to, melanoma, renal cell carcinoma, lung cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, gall bladder cancer, laryngeal cancer, liver cancer, thyroid cancer, stomach cancer, salivary gland cancer, prostate cancer, pancreatic cancer, Merkel cell carcinoma, brain and central nervous system cancers, and any combination thereof.
  • the cancer is a liquid tumor.
  • the cancer is a hematological cancer.
  • hematological cancer include diffuse large B cell lymphoma (“DLBCL”), Hodgkin's lymphoma (“HL”), Non-Hodgkin's lymphoma (“NHL”), Follicular lymphoma (“FL”), acute myeloid leukemia (“AML”), and multiple myeloma (“MM”).
  • DLBCL diffuse large B cell lymphoma
  • HL Hodgkin's lymphoma
  • NHL Non-Hodgkin's lymphoma
  • FL Follicular lymphoma
  • AML acute myeloid leukemia
  • MM multiple myeloma
  • the base editing fusion proteins, compositions, engineered T cells, methods and kits disclosed herein can be used to treat arteriosclerosis, atherosclerosis, cardiovascular diseases, coronary heart disease, diabetes, diabetes mellitus, non- insulin-dependent diabetes mellitus, fatty liver, hyperinsulinism, hyperlipidemia, hypertriglyceridemia, hypobetalipoproteinemias, inflammation, insulin resistance, metabolic diseases, obesity, malignant neoplasm of mouth, lipid metabolism disorders, lip and oral cavity carcinoma, dyslipidemias, metabolic syndrome x, hypotriglyceridemia, opitz trigonocephaly syndrome, ischemic stroke, hypertriglyceridemia result, hypobetalipoproteinemia familial 2, familial hypobetalipoproteinemia, and ischemic cerebrovascular accident.
  • the base editing fusion proteins, compositions, engineered T cells, methods and kits disclosed herein can be used to treat a metabolic disease, a lipid metabolism disease, an autoimmune condition (e.g., type 1 diabetes), allergies, infections, obesity, atherosclerosis, hyperfattyacidemia, metabolic syndrome, dyslipidemia, hypobetalipoproteinemia, familial hypercholesterolemia (including homozygous familial hypercholesterolemia (HoFH) and heterozygous familial hypercholesterolemia (HeFH)), hypertriglyceridemia, familial combined hyperlipidemia, familial chylomicronemia syndrome, multifactorial chylomicronemia syndrome, familial combined hyperlipidemia (FCHL), metabolic syndrome (MetS), nonalcoholic fatty liver disease (NAFLD), elevated lipoprotein (a), elevated lipids such as total cholesterol, triglycerides, LDLs, HDLs, and/or other non-HDLs in the blood, or a combination thereof.
  • an autoimmune condition e.g., type 1 diabetes
  • NAFLD can be hepatic steatosis or steatohepatitis.
  • the diabetes can be type 2 diabetes or type 2 diabetes with dyslipidemia.
  • Dyslipidemia can be hyperlipidemia, for example hypercholesterolemia, hypertriglyceridemia, or both.
  • the base editing fusion proteins, compositions, engineered T cells, methods and kits disclosed herein can be used to treat a cardiovascular disease or disorder.
  • cardiovascular disease refers to a disorder of the heart and blood vessels, and includes disorders of the arteries, veins, arterioles, venules, and capillaries.
  • the cardiovascular disease is stroke, myocardial infarction, atherosclerosis, familial hypercholesterolemia, atherosclerosis, thrombosis, calcific aortic valve disease, coronary artery disease, peripheral arterial disease, cerebrovascular disease, renal artery stenosis, aortic aneurysm, cardiomyopathy, hypertensive heart disease, heart failure, pulmonary heart disease, congenital heart disease, or rheumatic heart disease.
  • the base editing fusion proteins, compositions, engineered T cells, methods and kits disclosed herein can be used to treat calcific aortic valve disease, myocardial infarctions, coronary heart disease, atherosclerosis, thrombosis, stroke or a combination thereof.
  • the methods and compositions herein described can reduce the plasma low- density lipoprotein (LDL) levels such as the plasms Lp(a) levels, therefore reducing the risk of cardiovascular disease, such as the risk of heart attack, stroke, blood clots, fatty build-up in veins and other coronary artery disease, the likelihood of mortality related to cardiovascular events, or a combination thereof.
  • LDL plasma low- density lipoprotein
  • the methods and compositions herein described can reduce or relieve one or more symptoms of the cardiovascular disease.
  • the plasma Lp(a) level in the subject following carrying out the method can be reduced by about, at least, or at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or a number or a range between any of these values.
  • the method can comprise administering to a subject an engineered cell herein described or a population of the engineered cells.
  • the step of administering can include introducing (e.g., transplantation) the cells, e.g., an engineered T cell or a population thereof described herein, into a subject, by a method or route that results in at least partial localization of the introduced cells at a desired site, such as tumor, such that a desired effect(s) is produced.
  • Engineered T cells can be administered by any appropriate route that results in delivery to a desired location in the subject where at least a portion of the implanted cells or components of the cells remain viable.
  • the period of viability of the cells after administration to a subject can be as short as a few hours, e.g., twenty-four hours, to a few days, to as long as several years, or even the life time of the subject, i.e., long-term engraftment.
  • an effective amount of engineered T cells is administered via a systemic route of administration, such as an intraperitoneal or intravenous route.
  • An engineered T cell population being administered according to the methods described herein can comprise allogeneic T cells obtained from one or more donors.
  • Allogeneic refers to a cell, cell population, or biological samples comprising cells, obtained from one or more different donors of the same species, where the genes at one or more loci are not identical to the recipient.
  • an engineered T cell population, being administered to a subject can be derived from one or more unrelated donors, or from one or more non-identical siblings.
  • syngeneic cell populations can used, such as those obtained from genetically identical donors, (e.g., identical twins).
  • the cells are autologous cells; that is, the engineered T cells are obtained or isolated from a subject and administered to the same subject, i.e., the donor and recipient are the same.
  • a donor as used herein is an individual who is not the subject being treated.
  • a donor is an individual who is not the patient. In some embodiments, a donor is an individual who does not have or is not suspected of having the cancer being treated. In some embodiments, multiple donors, e.g., two or more donors, are used. [0241] In some embodiments, an engineered T cell population being administered according to the methods described herein does not induce toxicity in the subject, e.g., the engineered T cells do not induce toxicity in non-cancer cells. In some embodiments, an engineered T cell population being administered does not trigger complement mediated lysis, or does not stimulate antibody-dependent cell mediated cytotoxicity (ADCC).
  • ADCC antibody-dependent cell mediated cytotoxicity
  • An effective amount refers to the amount of a population of engineered T cells needed to prevent or alleviate at least one or more signs or symptoms of a medical condition, and relates to a sufficient amount of a composition to provide the desired effect, e.g., to treat a subject having a medical condition.
  • an effective amount of cells comprises about, at least or at least about 10 2 cells, 5 ⁇ 10 2 cells, 10 3 cells, 5 ⁇ 10 3 cells, 10 4 cells, 5 ⁇ 10 4 cells, 10 5 cells, 2 ⁇ 10 5 cells, 3 ⁇ 10 5 cells, 4 ⁇ 10 5 cells, 5 ⁇ 10 5 cells, 6 ⁇ 10 5 cells, 7 ⁇ 10 5 cells, 8 ⁇ 10 5 cells, 9 ⁇ 10 5 cells, 1 ⁇ 10 6 cells, 2 ⁇ 10 6 cells, 3 ⁇ 10 6 cells, 4 ⁇ 10 6 cells, 5 ⁇ 10 6 cells, 6 ⁇ 10 6 cells, 7 ⁇ 10 6 cells, 8 ⁇ 10 6 cells, 9 ⁇ 10 6 cells, 10 ⁇ 10 6 cells, 12 ⁇ 10 6 cells, 14 ⁇ 10 6 cells, 16 ⁇ 10 6 cells, 18 ⁇ 10 6 cells, 20 ⁇ 10 6 cells, 25 ⁇ 10 6 cells, 30 ⁇ 10 6 cells, or a number between any two of the
  • the cells are derived from one or more donors, or are obtained from an autologous source. In some embodiments described herein, the cells are expanded in culture prior to administration to a subject in need thereof.
  • Combinational Cancer Therapy [0244]
  • the fusion proteins, complexes, compositions, engineered cells, methods, and kits disclosed herein can be used with additional cancer therapeutics or therapy to treat cancer.
  • the treatment can comprise administration of at least one additional cancer therapeutics or cancer therapy.
  • the treatment can comprise administration a therapeutically effective amount of at least one additional cancer therapeutics or cancer therapy.
  • the compositions, complexes, and engineered cells herein described and the cancer therapeutics or cancer therapy can, for example, co-administered simultaneously or sequentially.
  • the one or more additional agents comprise anti- hormonal agents capable of regulating or inhibiting hormone action on tumors such as anti- estrogens including for example tamoxifen, (NolvadexTM), raloxifene, aromatase inhibiting 4(5)- imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vin
  • the one or more additional therapeutic agents that can be administered to the subject receiving, has received, or will receive, the administration of the engineered T cells disclosed herein comprise currently prescribed anti-cancer drugs such as Herceptin®, Avastin®, Erbitux®, Rituxan®, Taxol®, Arimidex®, Taxotere®, ABVD, AVICINE, Abagovomab, Acridine carboxamide, Adecatumumab, 17-N-Allylamino-17- demethoxygeldanamycin, Alpharadin, Alvocidib, 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone, Amonafide, Anthracenedione, Anti-CD22 immunotoxins, Antineoplastic, Antitumorigenic herbs, Apaziquone, Atiprimod, Azathioprine, Belotecan, Bendamustine, BIBW 2992, Biricodar, Brostallicin, Bryostat
  • anti-cancer drugs
  • the anti- angiogenesis agents can be MMP-2 (matrix-metalloproteinase 2) inhibitors, MMP-9 (matrix- metalloprotienase 9) inhibitors, and COX-11 (cyclooxygenase 11) inhibitors.
  • Anti-angiogenesis agents include, for example, rapamycin, temsirolimus (CCI-779), everolimus (RAD001), sorafenib, sunitinib, and bevacizumab.
  • Non-limiting examples of COX-II inhibitors include alecoxib, valdecoxib, and rofecoxib.
  • Non-limiting examples of anti-neoplastic agents include acemannan, aclarubicin, aldesleukin, alemtuzumab, alitretinoin, altretamine, amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, ANCER, ancestim, ARGLABIN, arsenic trioxide, BAM 002 (Novelos), bexarotene, bicalutamide, broxuridine, capecitabine, celmoleukin, cetrorelix, cladribine, clotrimazole, cytarabine ocfosfate, DA 3030 (Dong-A), daclizumab, denileukin diftitox, deslorelin, dexrazoxane, dilazep, docetaxel, docosanol, doxercalciferol, doxiflur
  • anti-angiogenic agent examples include, but are not limited to, ERBITUXTM (IMC-C225), KDR (kinase domain receptor) inhibitory agents (e.g., antibodies and antigen binding regions that specifically bind to the kinase domain receptor), anti-VEGF agents (e.g., antibodies or antigen binding regions that specifically bind VEGF, or soluble VEGF receptors or a ligand binding region thereof) such as AVASTINTM or VEGF-TRAPTM, and anti-VEGF receptor agents (e.g., antibodies or antigen binding regions that specifically bind thereto), EGFR inhibitory agents (e.g., antibodies or antigen binding regions that specifically bind thereto) such as Vectibix (panitumumab), IRESSATM (gefitinib), TARCEVATM (erlotinib), anti-Ang1 and anti-Ang2 agents (e.g., antibodies or antigen binding regions specifically binding thereto or to their receptors, e.
  • Autophagy inhibitors include, but are not limited to, chloroquine, 3-methyladenine, hydroxychloroquine (PlaquenilTM), bafilomycin A1, 5-amino-4-imidazole carboxamide riboside (AICAR), okadaic acid, autophagy-suppressive algal toxins which inhibit protein phosphatases of type 2A or type 1, analogues of cAMP, and drugs which elevate cAMP levels such as adenosine, LY204002, N6-mercaptopurine riboside, and vinblastine.
  • Non-limiting chemotherapeutic agents include, natural products such as vinca alkaloids (e.g., vinblastine, vincristine, and vinorelbine), paclitaxel, epidipodophyllotoxins (e.g., etoposide and teniposide), antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin, doxorubicin, and idarubicin), anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin), mitomycin, enzymes (e.g., L-asparaginase which systemically metabolizes L- asparagine and deprives cells which do not have the capacity to synthesize their own asparagine), antiplatelet agents, antiproliferative/antimitotic alkylating agents such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide and
  • chemotherapeutic agents may include mechlorethamine, camptothecin, ifosfamide, tamoxifen, raloxifene, gemcitabine, navelbine, sorafenib, or any analog or derivative variant of the foregoing.
  • Non-limiting examples of steroids include 21-acetoxypregnenolone, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, deflazacort, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difuprednate, enoxolone, fluazacort, flucloronide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluocortolone, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticas
  • the compounds of the present invention can also be used in combination with additional pharmaceutically active agents that treat nausea.
  • agents that can be used to treat nausea include: dronabinol; granisetron; metoclopramide; ondansetron; and prochlorperazine; or a pharmaceutically acceptable salt thereof.
  • Proteasome inhibitors include, but are not limited to, Kyprolis®(carfilzomib), Velcade®(bortezomib), and oprozomib.
  • Monoclonal antibodies include, but are not limited to, Darzalex® (daratumumab), Herceptin® (trastuzumab), Avastin® (bevacizumab), Rituxan® (rituximab), Lucentis® (ranibizumab), and Eylea® (aflibercept).
  • the cancer can be, e.g., breast cancer and the additional cancer therapies include surgery (e.g., breast conserving surgery or mastectomy), chemotherapy, radiation, hormonal therapy, HER2 targeted therapies and other target therapies (e.g., monoclonal antibodies, TKIs, cyclin-dependent kinase inhibitors, mTOR inhibitors, PARP inhibitors, PD-L1 inhibitor).
  • the additional therapeutic agents can include Trastuzumab, Pembrolizumab, Capecitabine, Atezolizumab, Ipatasertib, Bevacizumab, Cobimetinib, Gemcitabine, Carboplatin, Eribulin, or a combination thereof.
  • Kits [0255] Provided herein also includes kits for use in producing the base editing fusion proteins alone or in complex with a gRNA, the compositions, and the engineered cells and carrying out the methods described herein for therapeutic uses.
  • a kit comprises components for performing base editing of one or more target sequences.
  • a kit can comprise a base editing fusion protein herein described or a nucleic acid sequence encoding the base editing fusion protein, and one or more guide RNAs targeting the one or more target sequences.
  • a kit can also comprises a nucleic acid construct comprising (a) a nucleotide sequence encoding a base editing fusion protein as provided herein; and (b) a heterologous promoter that drives expression of the sequence of (a).
  • a kit can comprise a complex of the base editing fusion protein bound with a gRNA.
  • a kit can also comprise cells comprising a deaminase protein, a fusion protein, a nucleic acid molecule encoding the fusion protein, a complex comprising the fusion protein bound with a gRNA, and/or a vector as provided herein.
  • a kit can further comprise a RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease.
  • a kit can comprise a ribonucleoprotein formed by a guide RNA and a RNA-guided endonuclease protein.
  • kit can be in separate containers, or combined in a single container.
  • Any kit described above can further comprise one or more additional reagents selected from a buffer, a buffer for introducing a polypeptide or polynucleotide into a cell, a wash buffer, a control reagent, a control vector, a control RNA polynucleotide, a reagent for in vitro production of the polypeptide from DNA, adaptors for sequencing and the like.
  • a buffer can be a stabilization buffer, a reconstituting buffer, a diluting buffer, or the like.
  • kits can also comprise one or more components that can be used to facilitate or enhance the on-target binding or the cleavage of DNA by the endonuclease, or improve the specificity of targeting.
  • a kit can further include instructions for using the components of the kit to practice the methods described herein.
  • the instructions for practicing the methods are generally recorded on a suitable recording medium.
  • the instructions can be printed on a substrate, such as paper or plastic, etc.
  • the instructions can be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging), etc.
  • the instructions can be present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, flash drive, etc.
  • a suitable computer readable storage medium e.g., CD-ROM, diskette, flash drive, etc.
  • the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the Internet), can be provided.
  • An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.
  • Example 1 Analyses of cytosine base editors comprising deaminases from different species [0259]
  • Five additional previously described APOBEC1 genes were chosen to evaluate in a base editor model (Table 1).
  • Seven cytosine base editor mRNAs in total including Rat APOBEC1 (protein sequence: SEQ ID NO: 73; nucleic acid sequence: SEQ ID NO: 74) and Rat- E63A APOBEC1) were produced by in vitro transcription (Table 1). These base editor mRNAs were introduced into Jurkat cells in conjunction with sgRNAs designed to create functional knockouts at the FAS and ⁇ 2M loci, and the degree of ⁇ 2M and FAS knockdown was determined by flow cytometry (FIG.1).
  • the CBE harboring the wild type Rat APOBEC1 gene showed the highest degree of protein knockdown for each locus.
  • the protein knockdown capability of the CBE with wild type Orangutan APOBEC1 gene was comparable to what is observed in the wild type Rat APOBEC1 CBE.
  • a catalytically impaired SaCas9 bound to an SaCas9 guide creates an R-loop at targeted loci, exposing ssDNA for the potential of guide-independent CBE deamination which is then quantitated by amplicon-seq.
  • R-loop assays involved transfecting plasmids encoding catalytically impaired SaCas9
  • the assay used herein includes transfecting mRNAs encoding SaCas9 nickase fused to two UGI domains (e.g., SaCas9(D10A)-2XUGI).
  • Table 5 also confirms that the modified R-loop assay shows similar spurious deamination trends as the previously described methods with the base editors listed in Table 1.
  • This modified R-loop assay can avoid variability between the R-loop assay experiments as the entire bulk-cell population express nSaCas9 and the stable expression of nSaCas9 can amplify the off-target rates compared to transient transfection methods, leading to higher confidence in the deaminase selected using this method for low spurious deamination activity.
  • SpCas9 CBE mRNA and SaCas9 sgRNAs were nucleofected into Jurkat lenti-SaCas9(D10A)- 2XUGI cells and cell pellets harvested for DNA extraction 96 hours after nucleofection. Genomic DNA was extracted from cell pellets and sent for amplicon sequencing of the targeted R-loop regions. Percent C->T conversion for all cytosines in each amplicon was quantitated.
  • Example 2 Use of an exemplary cytosine base editor for multiplex editing of a CAR T cell
  • the cytosine base editors described in Example 1 were tested in an exemplary CAR T cell to demonstrate efficiency multiplex editing.
  • the CAR T cell has edits in the TRAC, ⁇ 2M, CD70, Regnase, and TGFBRII genes to knock out gene expression and additionally expresses an anti-CD70 CAR.
  • the CAR T cell was generated either by CRISPR-Cas9 editing or by editing the genes by an exemplary cytosine base editor with an orangutan deaminase followed by incorporation of the CAR by AAV insertion.
  • Table 6 describes the study design and Table 7 has details of the gRNAs used. Table 6.
  • Editing efficiency of multiple base editing is shown in Table 16 and 17.
  • Table 16 Editing efficiency of multiplex base editing via genomic (ICE) analysis Groups % BE in a % Base editi t ing eff) Locus ng in ( %Edi Guide single edit multiplexing FAS saFASExon4 76 64 M1 TRAC saTRACExon3 100 99 (59%) B2M sdB2MExon1 98 97 CD70 sdCD70Exon1 96 97 REG1 pmSTOPREG1Exon1 98 99 M2 TRAC saTRACExon3 100 99 (91%) B2M sdB2MExon1 98 98 CD70 sdCD70Exon1 96 95 TGFBRII pmSTOPQTGFBRIIExon5 95 93 M3 B2M sdB2MExon1 98 96 (85%) CD70 sdCD70Exon1 96 97 REG1 pmSTOPReg1Exon
  • Table 18 shows an exemplary mRNA and guide RNA formulation.
  • the amount in Table.18 are for triplicate sample sets.
  • the data demonstrates a minimal amount of BE mRNA of 1.15 pmol/million T cells and amount of sufficient guide (93.2 pmol of each sgRNA per 1 million T cells) required for multiplex editing.
  • Table 18 shows an exemplary mRNA and guide RNA formulation. The amount in Table.18 are for triplicate sample sets. The data demonstrates a minimal amount of BE mRNA of 1.15 pmol/million T cells and amount of sufficient guide (93.2 pmol of each sgRNA per 1 million T cells) required for multiplex editing. Table 18.
  • BE mRNA and guide RNA mRNA ( ⁇ L Total Guide per target Groups equivalent of 1.15 amount of locus ( ⁇ L equivalent pmol/ sample) guide ( ⁇ L) of 93.2 pmol/sample) saFASExon4+ sdCD70 Exon1+TRACEx3SA + B2MEx1 8.5 7.215483871 1.80 per guide SD saFASExon4+sdCD70 Exon1+TRACEx3SA + B2MEx1 8.5 9.019354839 1.80 per guide SD + pmSTOPReg1Exon1 saFASExon4+ sdCD70 Exon1+TRACEx3SA + 8.5 9.019354839 1.80 per guide B2MEx1SD+ pmSTOPRFX5Exon7 [0283] Formulation and dose of mRNA are also optimized to find minimal amount of mRNA to guide ratio.
  • FIG.15 illustrates an experiment layout in an exemplary embodiment.1Mn T cells are electroporated with the BE4 mRNA and guide RNA.
  • BE4 mRNA of 1.15 pmol per million cells was used together with different amounts of sgRNA.
  • B2M, TRAC and FAS knockout rates and CD70 base editing rate were evaluated for each sample set.
  • TRAC, B2M and CD70 guides are agnostic to titration while the FAS guide follows a dose-dependent decrease.
  • Reduced translocations in BE CTX131 [0286] On-target indel rates and total translocation rates obtained from Anchor-Seq NGS analysis for RNP CTX131 and BE CTX131 (with CAR and without CAR) are presented in Table 21.
  • FIGS. 11A-11B Comparison of the three deaminases in % base editing and on-target vs. off-target activity are shown in FIGS. 11A-11B.
  • FIG. 11A shows % base editing (C>T conversion), demonstrating slightly improved multi-plex base editing for Armadillo CBE across 3 loci in T cells.
  • FIG. 11B shows that Armadillo CBE performed the best among the three deaminases tested in on-target vs. off-target spurious deamination, and Orangutan deaminase performed better than rat deaminase.
  • Cbl Proto-Oncogene B (Cblb) gRNA screen [0288] The cytosine base editors described herein (e.g., orangutan CBE or variants thereof) were tested for targeted knockdown of Cblb gene. Cblb gRNAs were designed to target different exon regions in Cblb gene.
  • FIG. 17 shows a table providing a list of gRNA target sequences (from the top row to the bottom row: SEQ ID NOs: 77-100) and target regions in the Cblb gene.
  • the gRNAs used in this example comprise a spacer having a RNA sequence corresponding to one of the target sequences set forth in SEQ ID NOs: 77-100.
  • the 27 gRNAs were tested in cells from 2 donors (10 gRNAs in Donor 1; 17 new and 3 repeat gRNAs in Donor 2). Cells were electroporated with the BE mRNA and guide RNA. Proliferation and viability of the cells were monitored. Cells were then taken down for protein lysates for Western blot assay. GAPDH is used as a loading control for Western blot assay to normalize the levels of Cblb protein.
  • FIG.18 is a graph showing the percentage of cells expressing B2M and TCR (control).
  • B2M1 BE gRNA shows expected knockout rates both with mSpyCas9 and mOrangutan APOBEC1 BE.
  • FIGS.19A-B provide graphs showing results from Western blot analysis of Cblb BE gRNA screen in Donor 1 and Donor 2, respectively.
  • FIG.19C is a graph showing a summary of Western blot analysis of Cblb BE gRNA screen in both donors.
  • the data indicates that 7 out of 27 Cblb BE gRNAs show certain level of protein expression loss by Western blot analysis: less than 10% Cblb expression by pmSTOPQCBLBExon7 (SEQ ID NO: 87) and sdCBLBExon13a (SEQ ID NO: 96); about 19% Cblb expression by sdCBLBExon8 (SEQ ID NO: 79); about 30%- 40% Cblb expression by pmSTOPQCBLBExon5a (SEQ ID NO: 81), saCBLBExon9c (SEQ ID NO: 91), sdCBLBExon13b (SEQ ID NO: 97), and sdCBLBexon9 (SEQ ID NO: 92).
  • sdCBLBexon12 caused some truncation in the protein but no expression loss.
  • 5’ region guides did not reduce Cblb expression, indicating that the editing window at 5’ region of Cblb gene is less optimum.
  • pmSTOPQCBLBExon5a gRNA showed better KO rate with mSpyCas9 (RNP) than with mOrangutan (mRNA). Further experiments can be performed to confirm Cblb protein function loss before implementing these gRNAs.
  • FIG. 21 depicts editing efficiency of the three exemplary Cblb gRNAs.
  • CBLB_1 sdCBLBExon8
  • CBLB_2 pmSTOPQCBLBExon7
  • CBLB_3 sdCBLBExon13a
  • CBLB_2 was selected as the lead guide as it did not have any bystander edits and shows great editing rate.
  • CBLB_3 was not chosen as it disrupts the splicing post Exon 13.
  • Example 6 CD155 (PVR) gRNA screen [0293] The cytosine base editors described herein were used for targeted knockdown of PVR (poliovirus receptor) gene encoding CD155. A number of CD155 gRNAs were designed to target different exon regions in PVR gene. Table 23 below provides a list of exemplary CD155 gRNA sequences. Table 24 below lists the mutation types, target nucleotide and protein regions for each gRNA of Table 23. Table 23.
  • 26A-B provide editing analysis for each lead guide including CD155 gRNA #1, #3, #5, #13, #14, #15 and #16.
  • CD155 gRNA the plotted positions on y-axis correspond with positions of edit in the sequence box shown in the top right of a panel.
  • the editing efficiency is demonstrated for both target editing (red column) and bystander editing (black column).
  • target editing red column
  • bystander editing black column.
  • CD155 BE guide #1 the bystander edit is in an intronic sequence, thus does not affect the amino acid sequence.
  • the target edit at position 14, 15, or both generates a premature stop codon.
  • CD155 BE guide #5 the bystander edit at position 3 or 5 occurs in the intronic sequence and does not affect the amino acid sequence, while the bystander edit at position 8 induces a missense mutation, GGG (G) to AGG (R), if spliced.
  • the target edit at position 13, 14 or both generates a premature stop codon, while the bystander edit at position 16 induces a missense mutation (Ser (AGC) to Asn (AAC)).
  • AAC Asn
  • CD155 BE guide #13 the bystander editing occurs in the intronic sequence and does not affect the amino acid sequence.
  • the target edit at position 13 generates a premature stop codon while the bystander edit at position 14 induces a silent mutation (Val to Val).
  • CD155 BE guide #16 the target edit at position 4 (C to T) disrupts splicing. No bystander edit was detected for CD155 BE guide #16.
  • CD155 BE guides targeting exon 2 of PVR gene show relatively poor editing and lack significant decrease in CD155 expression with the exception of guide #8 which shows some decrease in CD155 expression (3.98% vs an average of 11.22% in controls).
  • CD155 BE guides #18, #19, #20 and #21 demonstrated relatively higher editing efficiency (e.g., about 90% editing for #18 and #19) but an increase in CD155 expression, possibly because these guides edit the cytoplasmic domain of the protein, therefore creating truncated proteins.
  • CD155 BE guide #2, #6, #12 and #17 demonstrated the lowest editing efficiency among all the gRNAs screened.
  • the above CD155 guide screening experiments were repeated using a selected subset of guides including the lead guides identified above (CD155 gRNA # 1, #3, #5, #13, #14, #15 and #16) as well as CD155 gRNA #8, #18, and #12. Cells are activated for flow cytometric readout at Day 5. 100 ⁇ L cells were aliquoted and added with fresh 100 ⁇ L containing 2 ⁇ L of TransAct in each well. Gene expression levels were measured using flow cytometry following a 24-hr activation or 48-hr activation process.
  • FIG.27 includes graphs showing CD4+/CD8+ ratios in cells treated with each of the CD155 gRNAs following 24-hr activation and 48-hr activation in comparison to the data shown in FIG.24.
  • FIG. 28 is a graph showing the expression percentage of CD155 after 24 hr activation and 48 hr activation in comparison to the data shown in FIG.25.
  • FIGs.29A-B provide editing analysis for each lead guide including CD155 gRNA #1, #3, #5, #13, #14, #15 and #16.
  • FIGs.30A-B are graphs showing CD3 and CD155 levels in cells treated with CD155 BE guide #1, #3, #5, #13, #14, #15, #16, #8, #12 or #18 after 24 hr activation (FIG.30A) and 48 hr activation (FIG.30B).
  • the data plotted in FIGs.30A-B are further provided in the table shown in FIG.30C.
  • CD3 staining goes down with activation by 24 hrs, likely due to binding CD3/CD28 of TransAct with subsequent reduction of TCR/CD3.
  • CD3 staining slightly improved by 48 hrs. [0301]
  • ⁇ 2M gRNA was used as a quality control. At Day 5, flow cytometry results of gene expression levels were obtained.
  • An exemplary editing reagent used in the experiment is listed in Table 28 below. Table 28.
  • gRNA/mRNA used in the screening. Editing Reagent Vol/1e6 cells Vol for 3.33xMM MICA/B sgRNA (155 ⁇ M) (5 ⁇ g/mL) 0.6 ⁇ L 2 ⁇ L Trilink Armadillo mRNA (1.28 mg/mL) 1.5 ⁇ L 4.95 ⁇ L [0306] MICA was not expressed at detectable levels on day 6 or 7 post electroporation.
  • FIG.32 shows the expression of MICA at day 0 after thawing of cells and day 2 post thaw.
  • FIG.33 shows ⁇ 2m expression reduced by 97% via base editing compared to RNP- control on day 7 post editing.
  • FIG.34 is a graph showing cell counts at day 7, indicating that there was no impact on cell growth for any of the MICA gRNA.
  • FIGs.35A-B provide graphs showing MICA expression following 2-day activation with TransAct.
  • the data revealed that MICA gRNA #6, #7 and #10 resulted in consistent and substantial decrease (80-85% reduction) in MICA expression.
  • FIGs. 36A-F provide detailed analysis of base editing by MICA gRNA #1, #6, #7, #12, #8 and #9.
  • MICA BE gRNA pmSTOPMICAExon3 (MICA-6), pmSTOPWMICAExon2 (MICA-7) and pmSTOPQMICAEx3c(MICA-12) resulted in generation of a premature stop codon.
  • MICA gRNA #12 (pmSTOPQMICA_Exon 3c) initially showed high knock down of MICA protein following 1 day of TransAct restimulation.
  • TAP-1 gRNA screen [0308]
  • the cytosine base editors described herein were used for targeted knockdown of transporter associated with antigen processing 1 (TAP-1) gene.
  • TAP-1 gRNAs were designed to target different exon regions in TAP-1 gene. Table 29 below provides a list of exemplary TAP-1 gRNA sequences. Table 29.
  • TAP-1 gRNAs Guide # gRNA Sequence SEQ ID 1 saTAP1_Ex2_1 CCCCUGGAGAAAGAGAAGAG 150 2 sdTAP1_Ex2_1 AGACCUGGCUAUGGUGAGAA 151 3 sdTAP1_Ex3_1 AGAAACCUGUCUGGUUCUGU 152 4 pmSTOPWTAP1_Ex1_1 CAGCCAUGCGAGAGAAGCUC 153 5 pmSTOPWTAP1_Ex1_2 ACCGCCCAGACCCGGAGCAG 154 6 pmSTOPWTAP1_Ex1_3 CCAGCCAGAGCACGGCCCAG 155 7 pmSTOPRTAP1_Ex1_4 UGUUCCGAGAGCUGAUCUCA 156 8 pmSTOPRTAP1_Ex1_5 GUUCCGAGAGCUGAUCUCAU 157 9 pmSTOPRTAP1_Ex1_6 UUCCGAGAGCUGAUCUCAU 158 10 pmSTOPWTAP1_Ex1_7
  • FIG. 37 shows T cell counts at day 6. No impact on T cell expansion was observed with any of the TAP-1 BE gRNA screened. ⁇ 2M gRNA was used as a positive control and showed 98% knock out rate.
  • FIG.38 is a plot summarizing target and bystander editing for exemplary TAP-1 gRNAs.
  • FIG.39 is a plot showing the HLA ABC knock down percentage by exemplary TAP-1 gRNAs at day 8 in comparison to ⁇ 2M gRNA control.
  • FIG.40 is a plot showing HLA-ABC mean fluorescence intensity (MFI) values following TAP-1 gene editing at day 8.
  • MFI mean fluorescence intensity
  • TAP-1 gRNA sdTAP1_Ex2_1, pmSTOPRTAP1_Ex1_4, pmSTOPRTAP1_Ex1_5, pmSTOPWTAP1_Ex1_7, pmSTOPWTAP1_Ex1_8, pmSTOPQTAP1_Ex3_4, pmSTOPWTAP1_Ex4_3, and pmSTOPWTAP1_Ex5_5 resulted in a 50%-89% knock down of MHC class I proteins and a reduction in MHC class I MFI to levels similar to the ⁇ 2M KO sample.
  • RFX5 gRNAs were designed to target different exon and/or regulatory regions in RFX5 gene. Table 30 below provides a list of exemplary RFX5 gRNA sequences. Table 30. Exemplary RFX5 gRNAs gRNA Sequence SEQ ID NO sdRFX5_Ex1_1 GUACUUACGAAAUGGUACCU 199 sdRFX5_Ex3_1 CUACCUUUUGUCUCCAGUGG 200 sdRFX5_Ex3_2 AACCUACCUUUUGUCUCCAG 201 saRFX5_Ex4_1 CUCUGAGCUACAGAAACAAA 202 sdRFX5_Ex5_1 AAGGAUACUUGGACUGGCCC 203 sdRFX5_Ex6_1 UUACACUCUCAGAACCCUUU 204 sdRFX5_Ex7_1 CAACACACCAGCGAGCCCCA 205 saRFX5_Ex8_1 CUUCUGCA
  • SOCS1 gRNA screen [0313] The cytosine base editors described herein can also be used for targeted knockdown of SOCS1 (suppressor of cytokine signaling 1) gene.
  • SOCS1 gene encodes a member of the STAT-induced STAT inhibitor (SSI), also known as suppressor of cytokine signaling (SOCS), family.
  • SSI family members are cytokine-inducible negative regulators of cytokine signaling.
  • This gene can be induced by a subset of cytokines, including IL2, IL3 erythropoietin (EPO), CSF2/GM-CSF, and interferon (IFN)-gamma.
  • cytokines including IL2, IL3 erythropoietin (EPO), CSF2/GM-CSF, and interferon (IFN)-gamma.
  • EPO IL2
  • CSF2/GM-CSF CSF2/GM-CSF
  • IFN interferon
  • SOCS1 gRNAs gRNA Sequence SEQ ID NO SOCS1noSTART GUGCUACCAUCCUACAGAAG 229 Socs1noStart2b UGUGCUACCAUCCUACAGAA 230 SOCS1noStart3 GUGUGCUACCAUCCUACAGA 231 SOCS1pmSTOP1 GGGCCCCCAGUAGAAUCCGC 232 pmSTOPQSOCS1x CGCUGCGCCAGCGCCGCGUG 233 SOCS1-5 AGGAAGGUUCUGGCCGCCGU 234 SOCS1-6 CGGCGUGCGAACGGAAUGUG 235 SOCS1-7 GCCGGUAAUCGGCGUGCGAA 236 SOCS1-8 UCCGUUCGCACGCCGAUUAC 237 SOCS1-9 GCGCGUGAUGCGCCGGUAAU 238 SOCS1-10 CGCUGGCGCGCGUGAUGCGC 239 SOCS1-11 AGUAGAAUCCGCAGGCGUCC 240 SOCS1-12 GGACGCCUGCGGAUUCUACU 241 SO
  • the edits were to create CAR T cells with improved immune evasion capabilities and enhanced T cell potency.
  • the CAR T cells has edits in the TRAC, ⁇ 2M, Regnase, and TGFBRII genes as well as additional edits in the FAS, MICA, CD155, and RFX5 genes to knock out gene expression.
  • CRISPR-Cas9 editing was used to edit TRAC while base editing was used to edit ⁇ 2M, Regnase, TGFBRII, FAS, MICA, CD155, and RFX5 genes.
  • the CAR T cell was then generated by incorporation of the CAR by AAV insertion.
  • the RNA-guided endonuclease used in CRISPR editing can be SpCas9 or SluCas9.
  • SpCas9 with the TRAC guide specific for SpCas9 was compared with SluCas9 with the TRAC guide that was specific for SluCas9.
  • the two different Cas9s and their specific guides were evaluated to minimize the chances of base editing reagents (sgRNAs) being swapped with the SpCas9 of CRISPR editing.
  • Table 32 describes the study design. Table 32.
  • Exemplary multiplex study design Editors Target Type Function CRISPR TRAC (Sp/Slu) DSB GvHD T GFbR2 Premature Stop (Q) R egnase-1 Premature Stop (W) Potency B2M Splice Donor disruption T cell evasion (Rejection) Base Editors Fas Splice Donor disruption MICA Premature Stop (W) NK Evasion CD155 Premature Stop (W) RFX5 Premature Stop (R) T cell evasion - MHC Class II KO [0315] Furthermore, a single-step electroporation (1EP) and two-step electroporation (2EP) protocols were carried out to evaluate the difference in editing rates and cell growth rates.
  • 1EP single-step electroporation
  • 2EP two-step electroporation
  • FIG. 41 shows an experiment outline for BCMA CAR T generation. Four groups of cells were generated on Day 2.
  • Group 1 cells were generated using combined CRISPR-Cas9 editing and base editing according to Table 32, in which SpCas9 and TRAC guide specific for SpCas9 were used for CRISPR editing. Cells were generated with or without CAR insertion.
  • Group 2 cells were generated using combined CRISPR-Cas9 editing and base editing according to Table 32, in which SluCas9 and TRAC guide specific for SluCas9 were used for CRISPR editing. Cells were generated with or without CAR insertion.
  • Group 3 on Day 2 cells were base-edited in ⁇ 2M, Regnase, TGFBRII, FAS, MICA, CD155 and RFX5 genes with no CRISPR editing.
  • a second electroporation was carried out on Day 4 to edit TRAC gene using SpCas9 or SluCas9 followed by transduction with the CAR AAV. Accordingly, Group 3 followed the two-electroporation protocol while Groups 1 and 2 followed the one-electroporation protocol.
  • Cells in Group 4 contain a single edit in B2M, RFX5, TGFbR2, Regnase-1, CD155, MICA or FAS and were used as controls to compare the editing rates/growth rates with cells in the multiplexed samples in Groups 1-3. Cells in the control group were not transduced with AAV/CAR.
  • FIG. 42A-C are plots showing the fold increase of multi-edited cells via a single-step electroporation process (FIG.42A) or a two-step electroporation process (FIG.42B) in comparison to cells containing a single gene edit (FIG. 42C).
  • the fold expansion data demonstrates a better growth kinetics from cells generated via a single electroporation process.
  • the key to symbols is as follows.
  • Sp 2 EP Multi CAR+ electroporated with SpCas9 and sgRNA for TRAC; multi-edited with 7 sgRNAs and mRNA for Cas9; 2 electroporation reactions; has CAR inserted.
  • Sp 2 EP mRNA CAR+ control cells were electroporated with mRNA for Cas9 but not with sgRNAs; Trac was edited and CAR was inserted; used Sp Cas9 for Trac editing.
  • Slu 2 EP Multi CAR+ electroporated with Slu Cas9 and sgRNA for TRAC; multi-edited with 7 sgRNAs and mRNA for Cas9; involved 2 electroporation reactions; has CAR inserted.
  • Slu 2 EP mRNA CAR+ control cells were electroporated with mRNA for Cas9 but not with sgRNAs; Trac was edited and CAR was inserted; used Slu Cas9 for Trac editing.
  • Sp 2 EP Multi CAR- electroporated with Sp Cas9; multi-edited with 7 sgRNAs and mRNA for Cas9 and involved 2 electroporation reactions; no CAR.
  • Sp 2 EP mRNA CAR- control cells were electroporated with mRNA for Cas9 but not with sgRNAs; involved 2 electroporation reactions; no CAR.
  • Slu 2 EP Multi CAR- electroporated with Slu Cas9 and sgRNA for TRAC; multi-edited with 7 sgRNAs and mRNA for Cas9 and involved 2 electroporation reactions; CAR was not inserted.
  • Slu 2 EP mRNA CAR- control cells were electroporated with mRNA for Cas9 but not with sgRNAs; involved 2 electroporation reactions; Trac was edited but CAR was not inserted.
  • Unedited 1EP no sgRNA or mRNA or AAV.
  • Table 33 below shows the editing efficiencies of multi-edited cells compared to single-edited controls. Genomic data are represented as % conversion from C to T (on target conversion). FACS data is represented as % negative cells. Table 33. Editing efficiencies.
  • FIG. 43 is a plot demonstrating the binding between SluCas9 sgRNA and SluCas9 or SpCas9. SpCas9 showed no binding to SluCas9 guide RNA.
  • FIGs. 44A-B show results from binding assays (FIG. 44A) and in vitro cleavage assays (FIG.44B) of exemplary Staphylococcus Cas9 (SluCas9 and S.
  • gRNA sequences for base editing gRNA Sequence SEQ ID NO B2M-1sdB2MEx1 ACUCACGCUGGAUAGCCUCC 247 pmSTOPTGFBR2Qexon5T1 ACUCCAGUUCCUGACGGCUG 248 pmSTOPREG1Exon1 AAUUCCCACAGACUCAUGGU 249 pmSTOPMICAExon3 CUGUCCAUUCCUCAGUCUCC 250 pmSTOPCD155Ex4a GUGCUCCAAUUAUAGCCUGU 251 pmSTOPWTAP1_Ex5_5 GGUCCAGGAGUUGACUGCAU 252 pmSTOPRRFX5_Ex7_2 ACCUCGAGAUGAACUGGUGG 253 pmStopW_FAS_Ex1-3 GAGGGUCCAGAUGCCCAGCA 254 [0324] Table 35 below provides a list of exemplary lead gRNA sequences with chemical modification for targeting FAS gene.

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Abstract

La présente divulgation concerne des procédés de fabrication d'éditions de gènes ciblés multiplex par introduction, dans des molécules d'ADN ou une cellule comprenant des molécules d'ADN, d'au moins deux ARN guides. Dans des modes de réalisation, la présente divulgation concerne des protéines de fusion d'édition de base, des complexes de protéines, des compositions, des cellules et des procédés d'utilisation associées pour l'édition ciblée d'acides nucléiques. La protéine de fusion d'édition de base peut comprendre un domaine Cas9 et un domaine désaminase pouvant désaminer une base nucléotidique dans une séquence d'acide nucléique cible.
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