Compositions and methods for site-directed mutagenesis

Abstract

The present disclosure provides improved genome editing compositions and methods for editing double-stranded DNA target sites. The present disclosure further provides genome edited cells produced by the compositions and methods described.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 128,391, filed December 21, 2020, which is incorporated by reference in its entirety.

[0002] Description of sequence listing The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference. The name of the text file containing the sequence listing is BLUE-132PC_ST25.txt. The text file is 541 KB, was created on December 15, 2021, and has been submitted electronically via EFS-Web. [Background technology]

[0003] The present disclosure relates to improved genome editing compositions. More specifically, the present disclosure relates to fusion polypeptides comprising a DNA binding domain and a homing endonuclease variant linked to an exonuclease for site-specific mutagenesis of dsDNA in cells, compositions, and methods of using the same.

[0004] Description of related fields The relatively recent proliferation of genome editing technologies has opened the possibility of directly targeting and modifying genomic sequences in almost all eukaryotic cells and mammals. Such technologies include, but are not limited to, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), clustered regularly interspaced short repeating sequences (CRISPR)-Cas-associated nucleases, and homing endonucleases (HEs). What all of these editing technologies have in common is that they create breakpoints in the target nucleotide sequence, while the natural cellular repair mechanism is left to religate the nucleotide sequence by either non-homologous end joining (NHEJ) or homology-directed repair (HDR). However, the repair is not always perfect. Thus, the end product is a nucleotide sequence that contains any one of a variety of genetic lesions.

[0005] The most frequently observed genetic lesions resulting from the application of gene editing nucleases are insertions and deletions (commonly referred to as "indels"). Indels arise when double-stranded DNA breaks (DSBs) are processed and resealed by the NHEJ DNA repair mechanism. NHEJ indels typically predominate gene editing events in the absence of excess delivered homologous DNA sequences, which can convert DSBs into a variety of homologous recombination outcomes.

[0006] The specific characteristics of each genetic lesion can lead to different phenotypic outcomes.For example, any given genetic lesion can lead to a wide range of phenotypic outcomes, ranging from total gene knockout, to gain or loss of function, to no phenotypic effect at all.Therefore, there is an increasing need to further understand the mechanisms that lead to different mutations during gene editing, and to develop compositions and methods for carrying out therapeutically meaningful gene editing. Summary of the Invention [Means for solving the problem]

[0007] The present disclosure relates generally, in part, to a fusion polypeptide comprising a DNA binding domain, a homing endonuclease variant that cleaves a target site in a human gene, a linker domain, and an exonuclease, and methods of using the same.

[0008] In one aspect, a fusion peptide is provided that comprises a DNA binding domain and a homing endonuclease (HE) variant that binds to and cleaves a selected double-stranded DNA (dsDNA) target site in a cell, a linker domain, and an exonuclease or a biologically active fragment thereof.

[0009] In certain embodiments, the exonuclease is Trex2, ExoI, or ExoX, or a biologically active fragment thereof. In some embodiments, the exonuclease is ExoX or a biologically active fragment thereof. In some embodiments, the exonuclease is ExoI or a biologically active fragment thereof. In some embodiments, the exonuclease is Trex2 or a biologically active fragment thereof.

[0010] In various embodiments, the homing endonuclease is an engineered homing endonuclease.

[0011] In various embodiments, the selected dsDNA target site is a non-natural homing endonuclease target site.

[0012] In various embodiments, the DNA binding domain binds to a dsDNA target site upstream of the endonuclease dsDNA target site. In some embodiments, the DNA binding domain comprises a TALE DNA binding domain. In some embodiments, the TALE DNA domain comprises about 9.5 TALE repeat units to about 15.5 TALE repeat units. In some embodiments, the TALE DNA domain comprises 11.5 TALE repeat units or 12.5 TALE repeat units. In some embodiments, the DNA binding domain comprises a zinc finger DNA binding domain. In some embodiments, the zinc finger DNA binding domain comprises 2, 3, 4, 5, 6, 7, or 8 zinc finger motifs.

[0013] In various embodiments, the linker domain is a peptide linker. In some embodiments, the peptide linker is a self-cleaving peptide linker. In some embodiments, the peptide linker comprises about 4 to about 30 amino acids. In some embodiments, the peptide linker comprises about 10 to about 16 amino acids. In some embodiments, the peptide linker comprises about 12 amino acids. In some embodiments, the peptide linker comprises the amino acid sequence (GGGGS) 1-4 linker (SEQ ID NO: 117, 150-152). In some embodiments, the peptide linker comprises a (GGGGS)2 linker (SEQ ID NO: 150).

[0014] In various embodiments, the HE variant is a LAGLIDADG homing endonuclease (LHE) variant. In some embodiments, the HE variant lacks 1, 2, 3, 4, 5, 6, 7, or 8 N-terminal amino acids compared to the corresponding wild-type HE. In some embodiments, the HE variant lacks 4 N-terminal amino acids compared to the corresponding wild-type HE. In some embodiments, the HE variant lacks 8 N-terminal amino acids compared to the corresponding wild-type HE. In some embodiments, the HE variant lacks 1, 2, 3, 4, or 5 C-terminal amino acids compared to the corresponding wild-type HE. In some embodiments, the HE variant lacks a C-terminal amino acid compared to the corresponding wild-type HE. In some embodiments, the HE variant lacks 2 C-terminal amino acids compared to the corresponding wild-type HE.

[0015] In certain embodiments, the HE variants are I-AabMI, I-AaeMI, I-AniI, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-GpiI, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-LtrII, I-LtrII, I-LtrIII ... The HE variant is a variant of LHE selected from the group consisting of trI, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI, I-OheMI, I-OnuI, I-OsoMI, I-OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-SceI, I-ScuMI, I-SmaMI, I-SscMI, and I-Vdi141I. In some embodiments, the HE variant is a variant of LHE selected from the group consisting of I-CpaMI, I-HjeMI, I-OnuI, I-PanMI, and I-SmaMI. In a preferred embodiment, the HE variant is an I-OnuI LHE variant.

[0016] In various embodiments, the HE target site is in an immune system checkpoint gene, a globin gene, a gene encoding a polypeptide that contributes to the suppression of gamma-globin gene expression and HbF, or an immunosuppressive signaling gene. In some embodiments, the HE target site is in ..., such as programmed cell death protein 1 (PD-1, PDCD1), lymphocyte activation gene 3 protein (LAG-3), T cell immunoglobulin domain and mucin domain protein 3 (TIM-3), cytotoxic T lymphocyte antigen-4 (CTLA-4), band T lymphocyte attenuator (BTLA), T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), V domain Ig inhibitor of T cell activation (VISTA), and and killer cell immunoglobulin-like receptors (KIR), CCR5, TRAC (TCRα), TCRβ, IL10Rα, IL10Rβ, TGFBR1, TGFBR2, CBL-B, PCSK9, AHR, BTK, α-globin, β-globin, γ-globin, BCL11A, KLF1, SOX6, GATA1, LSD1, alpha folate receptor (FRα), αvβ6 integrin, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase IX (CAIX), CD16, and CD19. , CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, carcinoembryonic antigen (CEA), C-type lectin-like molecule-1 (CLL-1), CD2 subset 1 (CS-1), chondroitin sulfate proteoglycan 4 (CSPG4), cutaneous T-cell lymphoma-associated antigen 1 (CTAGE1), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGF) EGFR family including GFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), epithelial cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EPHA2), fibroblast activation protein (FAP), Fc receptor-like 5 (FCRL5), fetal acetylcholinesterase receptor (AchR), ganglioside G2 (GD2), ganglioside G3 (GD3), glypican-3 (GPC3), ErbB2 (HER2), IL-11Rα, IL-13Rα2, kappa,Cancer / testis antigen 2 (LAGE-1A), lambda, Lewis-Y (LeY), L1 cell adhesion molecule (L1-CAM), melanoma antigen gene (MAGE)-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, melanoma antigen recognized by T cells 1 (MelanA or MART1), mesothelin (MSLN), MUC1, MUC16, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), neural cell adhesion molecule (NCAM), cancer / testis antigen 1 (NY-ESO-1), polysialic acid; placenta specific 1 (PLAC1), predominantly expressed antigen in melanoma (PRAME), prostate stem cell and in a gene selected from the group consisting of prostate cancer cell antigen (PSCA), prostate specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), synovial sarcoma, X breakpoint 2 (SSX2), survivin, tumor associated glycoprotein 72 (TAG72), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7 related (TEM7R), TEM5, TEM8, trophoblast glycoprotein (TPBG), UL16 binding protein (ULBP) 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms' tumor 1 (WT-1) genes. In some embodiments, the HE target site is in a gene selected from the group consisting of programmed cell death protein 1 (PD-1, PDCD1), lymphocyte activation gene 3 protein (LAG-3), T cell immunoglobulin domain and mucin domain protein 3 (TIM-3), cytotoxic T lymphocyte antigen-4 (CTLA-4), band T lymphocyte attenuator (BTLA), T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), V domain Ig inhibitor of T cell activation (VISTA), and killer cell immunoglobulin-like receptor (KIR), CCR5, TRAC (TCRα), IL10Rα, TGFBR2, CBL-B, PCSK9, AHR, BTK, α-globin, β-globin, γ-globin, and BCL11A genes. In some embodiments, the HE target site is in the TRAC (TCRα) gene, the CBL-B gene, or the PDCD1 (PD-1) gene. In certain embodiments,The TCR alpha gene target site comprises the amino acid sequence set forth in SEQ ID NO: 1. In a specific embodiment, the CBL-B gene target site comprises the amino acid sequence set forth in SEQ ID NO: 2. In a specific embodiment, the PD-1 gene target site comprises the amino acid sequence set forth in SEQ ID NO: 3.

[0017] In various embodiments, the DNA binding domain comprises a TALE DNA binding domain having a target site as set forth in SEQ ID NO:4.

[0018] In various embodiments, ExoX or a biologically active fragment thereof comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 109. In certain embodiments, ExoX or a biologically active fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 109.

[0019] In various embodiments, the fusion polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 46, 64, 73, and 82. In certain embodiments, the fusion polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 46, 64, 73, and 82.

[0020] In various embodiments, ExoI or a biologically active fragment thereof comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 112. In certain embodiments, ExoI or a biologically active fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:112.

[0021] In various embodiments, the fusion polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 43. In certain embodiments, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 43.

[0022] In various embodiments, a polynucleotide is provided that encodes a fusion polypeptide contemplated herein. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the polynucleotide sequence set forth in any one of SEQ ID NOs: 44, 62, 71, and 80. In certain embodiments, the polynucleotide comprises the polynucleotide sequence set forth in any one of SEQ ID NOs: 44, 62, 71, and 80.

[0023] In various embodiments, an mRNA is provided that encodes a fusion polypeptide as contemplated herein. In some embodiments, the mRNA comprises an RNA sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the RNA sequence shown in any one of SEQ ID NOs: 45, 63, 72, and 81. In certain embodiments, the mRNA comprises an RNA sequence shown in any one of SEQ ID NOs: 45, 63, 72, and 81. In some embodiments, the mRNA comprises an RNA sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the RNA sequence shown in SEQ ID NO: 42. In certain embodiments, the mRNA comprises an RNA sequence shown in any one of SEQ ID NOs: 42.

[0024] In various embodiments, a vector is provided that encodes a fusion polypeptide as contemplated herein. In some embodiments, the vector comprises a polynucleotide that encodes a fusion polypeptide as contemplated herein.

[0025] In various embodiments, a cell is provided that comprises a fusion polypeptide encoding a fusion polypeptide as contemplated herein. In some embodiments, the cell comprises a polynucleotide encoding a fusion polypeptide as contemplated herein. In some embodiments, the cell comprises an mRNA encoding a fusion polypeptide as contemplated herein. In some embodiments, the cell comprises a vector as contemplated herein. In some embodiments, the cell comprises one or more modifications.

[0026] In various embodiments, the cell is a hematopoietic cell. In some embodiments, the cell is a hematopoietic stem or progenitor cell. In some embodiments, the cell is a CD34+ cell. In some embodiments, the cell is a CD133+ cell.

[0027] In various embodiments, the cell is an immune effector cell. In some embodiments, the immune effector cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell. In some embodiments, the immune effector cell is a T cell. In some embodiments, the immune effector cell is an αβ T cell, a γδ T cell, a natural killer (NK) cell, or a natural killer T (NKT) cell.

[0028] In various embodiments, a cell population contemplated herein is provided.

[0029] In various embodiments, a composition is provided that includes a fusion polypeptide as contemplated herein. In some embodiments, a composition is provided that includes a polynucleotide as contemplated herein. In some embodiments, a composition is provided that includes an mRNA as contemplated herein. In some embodiments, a composition is provided that includes a vector as contemplated herein. In some embodiments, a composition is provided that includes a cell as contemplated herein. In some embodiments, a composition is provided that includes a cell population as contemplated herein. In certain embodiments, the composition includes a pharmaceutically acceptable carrier.

[0030] In another aspect, a method of site-directed mutagenesis is provided, the method comprising: (a) selecting a double-stranded DNA (dsDNA) target site; and (b) introducing into a cell a fusion polypeptide, polynucleotide, mRNA, or vector as contemplated herein, wherein the fusion peptide generates a directionally biased deletion with a deletion center position near the selected dsDNA target cleavage site in the cell.

[0031] In various embodiments, more than 50%, more than 51%, more than 52%, more than 53%, more than 54%, more than 55%, more than 56%, more than 57%, more than 58%, more than 59%, more than 60%, more than 65%, more than 70%, more than 75%, or more than 80% of the directionally biased deletions have a deletion center position to one side of the center position of the HE target site.

[0032] In various embodiments, the central location of the deletion is on the same side as the DNA binding domain target site relative to the central location of the HE target site. In certain embodiments, the central location of the deletion is 5' relative to the central location of the HE target site.

[0033] In various embodiments, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the deletions have a deletion center more than 4 nucleotides away from the central position of the HE target site.

[0034] In various embodiments, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, or at least 35% of the deletions have a deletion center more than 8 nucleotides away from the central position of the HE target site.

[0035] In various embodiments, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 50%, at least 59%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the deletions are 6 bp or more in length.

[0036] In various embodiments, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% of the deletions are 12 bp or more in length.

[0037] In various embodiments, the directionally biased deletion comprises about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length.

[0038] In various embodiments, the deletion extends within the DNA-binding domain target site. In some embodiments, the central position of the deletion is within the DNA-binding domain target site.

[0039] In various embodiments, the method further comprises introducing a terminal processing enzyme, or a biologically active fragment thereof, into the cell. In some embodiments, the terminal processing enzyme, or a biologically active fragment thereof, is selected from the group consisting of Trex2, Trex1, Trex1 without a transmembrane domain, Apollo, Artemis, DNA2, ExoI, ExoT, ExoIII, ExoX, Fen1, Fan1, MreII, Rad2, Rad9, TdT (terminal deoxynucleotidyl transferase), PNKP, RecE, RecJ, RecQ, lambda exonuclease, Sox, vaccinia DNA polymerase, exonuclease I, exonuclease II, exonuclease III, exonuclease II, exonuclease III, exonuclease I, exonuclease II ... The terminal processing enzyme is selected from the group consisting of exonuclease III, exonuclease VII, NDK1, NDK5, NDK7, NDK8, WRN, T7-exonuclease gene 6, avian myeloblastosis virus integration protein (IN), Bloom, Antarctic phosphatase, alkaline phosphatase, polynucleotide kinase (PNK), ApeI, mung bean nuclease, Hex1, TTRAP (TDP2), Sgs1, Sae2, CUP, Pol mu, Pol lambda, MUS81, EME1, EME2, SLX1, SLX4, and UL-12. In some embodiments, the terminal processing enzyme is an exonuclease, or a biologically active fragment thereof. In certain embodiments, the exonuclease is Trex2, or a biologically active fragment thereof.

[0040] In various embodiments, the method is an in vitro method. In various embodiments, the method is an ex vivo method. In various embodiments, the method is an in vivo method.

[0041] In another aspect, a method of treating, preventing, or ameliorating at least one symptom of a disease or a condition related thereto is provided, the method comprising harvesting a cell population from a subject, editing the cell population according to the methods contemplated herein, and administering the edited cell population to the subject.

[0042] In another aspect, there is provided a use of a cell as contemplated herein for treating, preventing, or ameliorating at least one symptom of a disease or condition associated therewith.

[0043] In another aspect, there is provided a use of the populations contemplated herein for treating, preventing, or ameliorating at least one symptom of a disease or a condition related thereto.

[0044] In another aspect, there is provided a use of a composition contemplated herein for treating, preventing, or ameliorating at least one symptom of a disease or a condition related thereto.

[0045] In various embodiments, the disease or condition is an immune disorder or cancer. [Brief description of the drawings]

[0046] [Figure 1A] A diagram of a TALE DNA binding domain linked to a homing endonuclease reprogrammed to target a TCRα nucleotide sequence (TCRα megaTAL). [Figure 1B] Next generation sequencing (NGS) indel activity of the TCRα megaTAL. NGS data reads containing indels characteristic of gene editing events are tabulated according to their length. [Diagram 2] Figure 1 shows the indel position distribution (fingerprint) assessed by NGS for both low and high activity TCRα megaTALs in two different donors. NGS data are tabulated according to both their length and their longitudinal position relative to the breakpoint center resulting from the megaTAL. Insertions are excluded from this analysis. [Figure 3A] Figure 3A shows indel activity by NGS of TCRα megaTAL (Figure 3A), TCRα megaTAL-Trex2 fusion (Figure 3B), and TCRα megaTAL+Trex2 co-expression (Figure 3C). NGS data reads containing indels characteristic of gene editing events are tabulated according to their length. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 4] The indel position distribution ("fingerprint") assessed by NGS is shown for TCRα megaTAL, TCRα megaTAL-Trex2 fusion, and TCRα megaTAL+Trex2 co-expression. NGS data are tabulated according to both their length and their longitudinal position relative to the breakpoint center resulting from the megaTAL. Insertions are excluded from this analysis. [Diagram 5] Shown is the editing efficiency of TCRα megaTALs fused to various Trex2 homologs assessed by staining of CD3 components associated with the TCR alpha and beta chains on the cell surface followed by flow cytometry analysis. [Figure 6] Figure 2 shows indel activity by NGS of TCRα megaTAL fused to Trex2 homolog. NGS data reads containing indels characteristic of gene editing events are tabulated according to their length. [Figure 7] Figure 1 shows the indel position distribution ("fingerprint") assessed by NGS for TCRα megaTALs fused to Trex2 platypus, opossum, human, and mouse homologs. NGS data are tabulated according to both their length and their longitudinal position relative to the breakpoint center resulting from the megaTAL. Insertions are excluded from this analysis. [Figure 8] Figure 2 shows the editing efficiency of TCRα megaTAL fused to exonucleases RAD1, RAD9A, ExoI, ExoX, T5FEN, lambdaExo, and RecJ with or without co-expression of mouse Trex2, the Trex2 homologue assessed by staining for CD3 expression followed by flow cytometry analysis. [Figure 9]Figure 1 shows the indel position distribution ("fingerprint") assessed by NGS for the TCRα megaTAL alone and fused to the exonucleases ExoI or ExoX, with or without co-expression of Trex2. The NGS data are tabulated according to both their length and their longitudinal position relative to the breakpoint center resulting from the megaTAL. Insertions are excluded from this analysis. [Figure 10] Figure 1 shows the indel position distribution ("fingerprint") assessed by NGS for the highly active CBL-B megaTAL alone or fused to Trex2 or ExoX. The NGS data are tabulated according to both their length and their longitudinal position relative to the breakpoint center resulting from the megaTAL. Insertions are excluded from this analysis. [Figure 11] Figure 1 shows the indel position distribution ("fingerprint") assessed by NGS for low activity CBL-B megaTALs alone or fused to Trex2 or ExoX. NGS data are tabulated according to both their length and their longitudinal position relative to the breakpoint center caused by the megaTAL. Insertions are excluded from this analysis. [Figure 12] Figure 1 shows the indel position distribution ("fingerprint") assessed by NGS for PD-1 megaTALs alone or fused to Trex2 or ExoX. NGS data are tabulated according to both their length and their longitudinal position relative to the breakpoint center caused by the megaTAL. Insertions are excluded from this analysis. [Figure 13] The PDCD1 gene, the 22 base pair PD-1 megaTAL target site, and the 13 base TALE array binding site (sequence numbers 154 and 159), as well as four representative deletion species (sequence numbers 155-158 and 160-162), from the experiment depicted in Figure 12 are shown. [Figure 14]The indel position distribution ("fingerprint") assessed by NGS is shown for the PD-1 megaTAL alone or fused to Trex2 or ExoX. Deletion species are coded to indicate the resulting reading frame category. NGS data are tabulated according to both their length and their longitudinal position relative to the breakpoint center resulting from the megaTAL. Insertions are excluded from this analysis. [Figure 15] Stacked histograms quantifying the normalized proportions of each of the four deletion species categories shown in FIG. 14 are shown. [Figure 16] Shown is a cyan fluorescent protein (CFP) assay to track transfection efficiency, and flow cytometry analysis of activated primary human T cells electroporated with polyadenylated mRNA encoding either wild-type or mock-edited PD-1 alleles, or both, in each of the three possible reading frames. [Figure 17] The indel position distribution ("fingerprint") assessed by NGS is shown for the low-edited TCRα megaTAL, the high-edited TCRα megaTAL (TCRα 2.2), and each direct fusion to Trex2 or ExoX. The NGS data are tabulated according to both their length and their longitudinal position relative to the breakpoint center resulting from the megaTAL. Insertions are excluded from this analysis. [Figure 18] Figure 1 shows the indel position distribution ("fingerprint") at known KAT2B off-target sites of low-edited TCRα megaTALs with or without direct fusion to Trex2 or ExoX. NGS data are tabulated according to both their length and their longitudinal position relative to the breakpoint center resulting from the megaTAL. Insertions are excluded from this analysis. [Figure 19]Figure 1 shows the indel position distribution ("fingerprint") at known AC016700.3 off-target sites (SEQ ID NOs: 163 and 164) of the hyper-edited TCRα megaTAL (TCRα 2.2) with or without direct fusion to ExoX. NGS data are tabulated according to both their length and their longitudinal position relative to the breakpoint center resulting from the megaTAL. Insertions are excluded from this analysis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] A brief explanation of sequence numbers SEQ ID NOs:1-3 are exemplary homing endonuclease target sites.

[0048] SEQ ID NO:4 is the PD-1 TALE array target site.

[0049] SEQ ID NOs:5-7 are the low activity TCRα megaTAL DNA, RNA, and protein sequences.

[0050] SEQ ID NOs:8-10 are the highly active TCRα megaTAL DNA, RNA, and protein sequences.

[0051] SEQ ID NOs: 11-13 are the low activity TCRα megaTAL-Trex2 DNA, RNA, and protein sequences.

[0052] SEQ ID NOs:14-16 are the Trex2 exonuclease DNA, RNA, and protein sequences.

[0053] SEQ ID NOs: 17-55 are the DNA, RNA, and protein sequences of low activity TCRα megaTAL fused to a Trex2 homologue.

[0054] SEQ ID NOs: 56-64 are the DNA, RNA, and protein sequences of highly active CBL-B megaTALs with or without fusions to Trex2 or ExoX.

[0055] SEQ ID NOs: 65-73 are the DNA, RNA, and protein sequences of low activity CBL-B megaTALs with or without fusion to Trex2 or ExoX.

[0056] SEQ ID NOs: 74-82 are the DNA, RNA, and protein sequences of PD-1 megaTAL with or without fusion to Trex2 or ExoX.

[0057] SEQ ID NO:83 is the mRNA sequence encoding PD-1.

[0058] SEQ ID NO:84 is an mRNA sequence encoding a mock-edited PD-1 open reading frame (ORF) containing a 1 bp deletion at codon 3.

[0059] SEQ ID NO:85 is an mRNA sequence encoding a mock-edited PD-1 open reading frame (ORF) containing a 2 bp deletion at codon 3.

[0060] SEQ ID NO:86 is an mRNA sequence encoding a mock-edited PD-1 open reading frame (ORF) containing a 3 bp deletion at codon 3.

[0061] SEQ ID NOs:87-101 are the DNA, RNA, and protein sequences of TCRα, CBL-B, and PD-1 homing endonucleases.

[0062] SEQ ID NOs: 102 to 106 are wild-type I-OnuI endonuclease and portions thereof.

[0063] SEQ ID NOs:107-109 are the ExoX exonuclease DNA, RNA, and protein sequences.

[0064] SEQ ID NOs:110-112 are the ExoI exonuclease DNA, RNA, and protein sequences.

[0065] SEQ ID NOs: 113 to 123 show the amino acid sequences of various linkers.

[0066] SEQ ID NOs: 124 to 148 show the amino acid sequences of the protease cleavage site and the self-cleaving polypeptide cleavage site.

[0067] In the above sequences, X, if present, refers to any amino acid or to the absence of an amino acid. [Mode for carrying out the invention]

[0068] A. Overview The present disclosure generally relates, in part, to improved genome editing compositions and methods of use thereof. Without wishing to be bound by any particular theory, the genome editing compositions contemplated herein are used to 1) increase the size of the deletion induced by gene editing to a specific target size, and 2) bias the deletion center position. In certain embodiments, the deletion center position is predominantly on the same side as the DNA binding domain target site relative to the original breakpoint center or endonuclease target site center. In certain embodiments, the deletion center position is biased toward the 5' side of the original breakpoint center or endonuclease target site center. It is further contemplated that by controlling the size and location of the genetic lesion at or proximal to the breakpoint, the phenotypic outcome of genome editing, such as disruption of regulatory DNA sequences and / or target gene expression, can be more precisely controlled.

[0069] The most frequently observed genetic lesion resulting from the application of gene editing nucleases is the NHEJ indel. Little is known about how the NHEJ machinery arrives at a given indel that is resealed and resistant to further DNA cleavage, thus becoming enshrined as a permanent genotype in the pool of gene edited alleles. However, it is known that indel events are observed almost exclusively at and directly proximal to the DNA sequence where the nuclease-generated breakpoint occurs. Previous studies using various nuclease platforms (ZFN, TALEN, and CRISPR) suggest that gene edited alleles show some consistency in terms of the qualitative characteristics of the observed indels. This suggests that there are critical biophysical and / or biochemical processes that determine the outcome of gene editing. However, while there are some nuances in the observed spectrum of edited alleles resulting from different gene editing nucleases across different DNA target sites, the ability to influence indel characteristics remains elusive.

[0070] The qualitative characteristics that define a given indel are (i) its length in number of inserted or deleted bases, (ii) its longitudinal location along the chromosome, usually indicated relative to the nuclease target site or breakpoint, and (iii) with respect to the insertion, the length and composition of the inserted sequence. Deletions are the most prominent outcome, typically comprising 90-95% of the observed events. Their most commonly reported size characteristics tend to be small (i.e., 1-20 base pairs in length, with frequencies biased towards the low end of that range), and their location distribution is found to be evenly distributed, encompassing the DNA breakpoint and diverging outward in either direction without significant bias. Exceptions to these characteristics are frequently assumed to be driven by microhomologies (small overlapping tracts roughly 3-6 base pairs in length) located on either side of the DNA breakpoint. Little has been reported regarding the characteristics of insertions, which occur at much lower frequencies during the application of genome editing tools. Furthermore, the genotypic features linking each indel species to a phenotype (e.g., how it affects an open reading frame or whether it disrupts a transcription factor binding motif) are potentially vast and specific to each given application.

[0071] Contemplated herein are unique fusion polypeptides with distinct editing patterns (e.g., deletions) that are useful for precisely controlling the phenotypic outcome of genome editing. In various embodiments, the fusion polypeptide comprises a DNA-binding domain, a homing endonuclease variant, and an end-processing enzyme (e.g., an exonuclease). In certain embodiments, the exonuclease is ExoX, ExoI, or a biologically active fragment thereof. In certain embodiments, the fusion polypeptide induces an extended gene deletion with a deletion center that is directionally biased on the same side, i.e., 5', of the DNA-binding domain target site relative to the original edit / breakpoint. In certain embodiments, the fusion polypeptide generates a deletion center position that is encompassed by the DNA-binding domain target site. In certain embodiments, the fusion polypeptide generates a deletion that extends into (or is encompassed by) the DNA-binding domain target site.

[0072] In certain embodiments, the fusion polypeptide comprises a DNA-binding domain and a homing endonuclease (HE) variant that binds and cleaves a selected double-stranded DNA (dsDNA) target site in a cell, a polypeptide linker, and an exonuclease (e.g., ExoX or ExoI), or a biologically active fragment thereof.

[0073] In various embodiments, a vector, polynucleotide, mRNA, or cDNA encoding the fusion polypeptide is contemplated. In various embodiments, a genome-edited cell is contemplated. In various embodiments, a composition comprising the fusion polypeptide, vector, polynucleotide, mRNA, cDNA, or cell is contemplated.

[0074] In various embodiments, methods of genome editing, site-directed mutagenesis, site-directed mutagenesis increasing the length of deletions, positional bias of deletions, and treatment of subjects in need thereof are contemplated.

[0075] Thus, the compositions and methods contemplated herein represent a significant improvement over existing gene editing strategies because they allow for strategic control and selection of desired mutagenic outcomes.

[0076] Recombinant (i.e., engineered) DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture, transformation (e.g., electroporation, lipofection), enzymatic reactions, purification, and related techniques and procedures may be generally performed as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology, and immunology, which are cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008), Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience, Glover, DNA. Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford Univ. Press USA, 1985), Current Protocols in Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M.Shevach,Warren Strober 2001 John Wiley & Sons,NY,NY)、Real-Time PCR:Current Technology and Applications,Edited by Julie Logan,Kirstin Edwards and Nick Saunders,2009,Caister Academic Press,Norfolk,UK、Anand,Techniques for the Analysis of Complex Genomes,(Academic Press,New York,1992)、Guthrie and Fink,Guide to Yeast Genetics and Molecular Biology(Academic Press,New York,1991)、Oligonucleotide Synthesis(N.Gait,Ed.,1984)、Nucleic Acid The Hybridization(B.Hames & S.Higgins,Eds.,1985)、Transcription and Translation(B.Hames & S.Higgins,Eds.,1984)、Animal Cell Culture(R.Freshney,Ed.,1986)、Perbal,A Practical Guide to Molecular Cloning(1984)、Next-Generation Genome Sequencing(Janitz,2008 Wiley-VCH)、PCR Protocols(Methods in Molecular Biology)(Park,Ed.,3rd Edition,2010 Humana Press)、Immobilized Cells And Enzymes(IRL Press,1986)、the treatise,Methods In Enzymology(Academic Press,Inc.,N.Y.)、Gene Transfer Vectors For Mammalian Cells(J.H.Miller and M.P.Calos eds.Please refer to research papers in specialist journals such as Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987), Handbook Of Experimental Immunology, Volumes I-IV (DM Weir and CC Blackwell, eds., 1986), Roitt, Essential Immunology, 6th Edition, (Blackwell Scientific Publications, Oxford, 1988), Current Protocols in Immunology (QE Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1991), Annual Review of Immunology, and Advances in Immunology.

[0077] B. Definition Before describing this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein.

[0078] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although similar or equivalent methods and materials to those described herein can be used to practice or test specific embodiments, preferred compositions, methods and materials embodiments are disclosed herein. For purposes of this disclosure, the following terms are defined below. Additional definitions are set forth throughout this disclosure.

[0079] The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one or to more than one) of the grammatical object of the article. By way of example, "an element" means one element or one or more than one element.

[0080] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination of the alternatives.

[0081] The term "and / or" should be understood to mean either one or both of the alternatives.

[0082] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0083] In one embodiment, a range, for example, 1 to 5, about 1 to 5, or about 1 to about 5, refers to every number subsumed within the range. For example, in one non-limiting and merely exemplary embodiment, the range "1 to 5" is equivalent to the expressions 1, 2, 3, 4, 5, or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0, or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0084] As used herein, the term "substantially" refers to a quantity, level, value, number, frequency, percentage, size, size, amount, weight, or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more compared to a reference quantity, level, value, number, frequency, percentage, size, size, amount, weight, or length. In one embodiment, "substantially the same" refers to a quantity, level, value, number, frequency, percentage, size, size, amount, weight, or length that produces an effect, e.g., a physiological effect, that is approximately the same as the reference quantity, level, value, number, frequency, percentage, size, size, amount, weight, or length.

[0085] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises", and "comprising" will be understood to imply the inclusion of the specified step or component or group of steps or components, but not the exclusion of any other step or component or group of steps or components. "Consisting of" means including and limited to all that precedes the word "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are necessary or mandatory, and that no other elements may be present. "Consisting essentially of" means including any elements that are limited to any elements listed after the phrase, and other elements that do not interfere with or contribute to the activity or action specified in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are necessary or mandatory, but that there are no other elements that materially affect the activity or action of the recited elements.

[0086] References throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a particular embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. As such, the appearances of such phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It should also be understood that the affirmative recitation of a feature in an embodiment serves as a basis for the exclusion of that feature in certain embodiments.

[0087] The term "in vitro" generally refers to activities that occur at a location outside of an organism, such as experiments or measurements performed in an artificial environment outside of the organism, preferably with minimal changes to natural conditions, or experiments or measurements on living tissue. In certain embodiments, "in vitro" procedures include living cells or tissues taken from an organism and cultured or conditioned in a laboratory setup, usually under sterile conditions, typically for a few hours or up to about 24 hours, but up to 48 or 72 hours depending on the circumstances. In certain embodiments, such tissues or cells can be collected, frozen, and then thawed for in vitro processing. Tissue culture experiments or procedures that use living cells or tissues and last for several days or more are typically considered to be "in vitro", although in certain embodiments, the term can be used interchangeably with in vitro.

[0088] The term "in vivo" generally refers to activities that occur inside an organism. In one embodiment, the genome of a cell is manipulated, edited, or modified in vivo.

[0089] "Enhancement" or "promotion" or "increase" or "expand" or "enhancement" generally refers to the ability of a fusion polypeptide, nuclease variant, genome editing composition, or genome edited cell contemplated herein to produce, induce, or cause a greater response (i.e., a physiological response) compared to the response caused by either a vehicle or a control. Measurable responses may include an increase in editing events (e.g., indels), deletions, insertions, deletion length, and / or target gene expression, particularly as will be apparent from an understanding of the art and the description herein. An "increased" or "enhanced" amount is typically a "statistically significant" amount and can include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30-fold or more (e.g., 500, 1000-fold) (including all integers and decimal points above 1 in between, e.g., 1.5, 1.6, 1.7, 1.8, etc.) over the response produced by vehicle or control.

[0090] "Reduce" or "lower" or "reducing" or "reducing" or "attenuating" or "ablate" or "inhibiting" or "attenuating" generally refers to the ability of a fusion polypeptide, nuclease variant, genome editing composition, or genome edited cell contemplated herein to produce, induce, or cause a lesser response (i.e., a physiological response) compared to the response caused by either a vehicle or a control. A measurable response may include a reduction in one or more symptoms associated with an editing event (e.g., indels), deletions, insertions, deletion length, target gene expression, and / or disease. The amount of "reduction" or "decreased" is typically a "statistically significant" amount and can include a reduction that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30-fold or more (e.g., 500, 1000-fold) (including all integers and decimal points above 1 in between, e.g., 1.5, 1.6, 1.7, 1.8, etc.) than the response produced by the vehicle or control (reference response).

[0091] "Maintain" or "preserve" or "maintain", or "no change" or "no substantial change" or "no substantial decrease" generally refers to the ability of a fusion polypeptide, nuclease variant, genome editing composition, or genome edited cell contemplated herein to produce, induce, or cause a substantially similar or equivalent physiological response (i.e., a downstream effect) compared to the response caused by either a vehicle or a control. An equivalent response is one that is not substantially different or not measurably different from a reference response.

[0092] As used herein, the terms "specific binding affinity" or "specifically binds" or "specifically bound" or "specific binding" or "specifically targeted" describe the binding of one molecule to another molecule, e.g., a DNA-binding domain of a polypeptide that binds to DNA, with a binding affinity higher than background binding. A binding domain may have a binding affinity of, for example, about 10 5 M -1 Affinity or K a (i.e., the equilibrium association constant for a particular binding interaction in units of 1 / M). In certain embodiments, a binding domain "specifically binds" to a target site if it binds to or associates with the target site with a 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , 10 12 M -1 , or 10 13 M -1 More than K a A "high affinity" binding domain binds to a target site with at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M-1 , at least 10 11 M -1 , at least 10 12 M -1 , at least 10 13 M -1 , or more than K a It refers to a binding domain having the following structure:

[0093] Alternatively, the affinity may be expressed in M ​​units (e.g., 10 -5 M~10 -13 The equilibrium dissociation constant (K d The affinity of a nuclease variant comprising one or more DNA binding domains for a DNA target site as contemplated in certain embodiments can be readily determined using conventional techniques, such as yeast cell surface display, or by binding association or displacement assays using labeled ligands.

[0094] In one embodiment, the affinity of specific binding is about 2-fold higher than background binding, about 5-fold higher than background binding, about 10-fold higher than background binding, about 20-fold higher than background binding, about 50-fold higher than background binding, about 100-fold higher than background binding, or about 1000-fold higher than background binding, or more.

[0095] The terms "selectively bind" or "selectively bound" or "selective binding" or "selectively target" describe preferential binding of one molecule to a target molecule in the presence of multiple off-target molecules (on-target binding). In certain embodiments, the HE or megaTAL selectively binds to a DNA binding site on the target about 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, 100-fold, or 1000-fold more frequently than the HE or megaTAL binds to an off-target DNA target binding site.

[0096] "On target" refers to the target site sequence.

[0097] "Off-target" refers to a sequence that is similar, but not identical, to the target site sequence.

[0098] A "target site" or "target sequence" is a chromosomal or extrachromosomal nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule binds and / or cleaves, provided that sufficient conditions for binding and / or cleavage exist. When referring to a polynucleotide sequence or SEQ ID NO: that references only one strand of a target site or target sequence, it is understood that the target site or target sequence that is bound and / or cleaved by a nuclease variant is double-stranded and includes the reference sequence and its complementary strand. In various embodiments, the target site is in an immune system checkpoint gene, a globin gene, a gene that encodes a polypeptide that contributes to the suppression of gamma-globin gene expression and / or HbF, or an immunosuppressive signaling gene. In certain embodiments, the target site is a sequence in the human TRAC gene, the CBL-B gene, or the PDCD1 gene.

[0099] "Recombination" refers to the process of exchange of genetic information between two polynucleotides, including, but not limited to, non-homologous end joining (NHEJ) and donor capture by homologous recombination. For the purposes of this disclosure, "homologous recombination (HR)" refers to a specific form of such exchange that occurs, for example, during the repair of double-strand breaks in cells via the homology-directed repair (HDR) mechanism. This process is known variously as "non-crossover gene conversion" or "short tract gene conversion" because it requires nucleotide sequence homology and uses the "donor" molecule as a template to repair the "target" molecule (i.e., the one that experienced the double-strand break), leading to the transfer of genetic information from the donor to the target. Without wishing to be bound by any particular theory, such transfer may involve mismatch correction of heteroduplex DNA formed between the destroyed target and the donor, and / or "synthesis-dependent strand annealing," and / or related processes, in which the donor is used to resynthesize the genetic information that will become part of the target. Such specified HR often results in an alteration of the sequence of the target molecule such that some or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.

[0100] "Cleavage" refers to the disruption of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods, including, but not limited to, enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-strand and double-strand cleavage are possible. Double-strand cleavage can result from two separate single-strand cleavage events. DNA cleavage can result in the generation of either blunt or staggered ends. In certain embodiments, the polypeptides and nuclease variants contemplated herein, such as homing endonuclease variants, megaTALs, and related fusion polypeptides, are used for targeted double-stranded DNA cleavage. The endonuclease cleavage recognition site may be on either DNA strand.

[0101] "Directionally biased" or "directionally biased deletion" refers to the location of deletions made by the cell's endogenous repair mechanisms in response to endonuclease-induced double-stranded DNA breaks. When deletions are directionally biased, they will occur predominantly either 5' or 3' to the target cleavage site or double-stranded DNA break (breakpoint or breakpoint center). That is, compared to the breakpoint center or center position of the target site, substantially more deletions occur on one side than the other. In certain embodiments, the fusion polypeptide induces gene deletions with directionally biased deletions that occur on the same side as the DNA-binding domain target site, relative to the breakpoint center or center position of the HE target site. In further embodiments, the fusion polypeptide induces gene deletions with directionally biased deletion centers 5' to that of the original breakpoint center or center position of the HE target site. In preferred embodiments, the directionally biased deletions are also extended. Furthermore, the amount of directionally biased deletions or extensions induced by the fusion proteins contemplated herein can be compared to the distribution of deletions or deletion lengths produced by co-expression of the same exonuclease, as well as fusion polypeptides containing the same DNA binding domain and the same homing endonuclease.

[0102] A "foreign" molecule is a molecule that is not normally present in a cell, but is introduced into a cell by one or more genetic, biochemical, or other methods. Exemplary foreign molecules include, but are not limited to, small organic molecules, proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, polysaccharides, any modified derivatives of the above molecules, or any complexes containing one or more of the above molecules. Methods for introducing foreign molecules into cells are known to those skilled in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes containing neutral and cationic lipids), electroporation, direct injection, cell fusion, bioprojectile bombardment, biopolymer nanoparticles, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer, and viral vector-mediated transfer.

[0103] An "endogenous" molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions. Additional endogenous molecules can include proteins.

[0104] "Gene" refers to a DNA region that encodes a gene product, as well as all DNA regions that regulate the production of the gene product, whether or not such regulatory sequences are adjacent to the coding and / or transcribed sequence. Genes include, but are not limited to, promoter sequences, enhancers, silencers, insulators, border regions, terminators, polyadenylation sequences, post-transcriptional response elements, translation control sequences such as ribosome binding sites and internal ribosome entry sites, origins of replication, substrate binding sites, and locus control regions.

[0105] "Gene expression" refers to the conversion of the information contained in a gene into a gene product. A gene product can be the direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA). Gene products also include RNA that is modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins that are modified, for example, by methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

[0106] As used herein, the term "genetically engineered" or "genetically modified" refers to the chromosomal or extrachromosomal addition of extra genetic material in the form of DNA or RNA to the total genetic material in a cell. The genetic modification can be targeted to a specific site in the genome of the cell or can be non-targeted. In one embodiment, the genetic modification is site-specific. In one embodiment, the genetic modification is not site-specific.

[0107] As used herein, the term "genome editing" refers to the replacement, deletion, and / or introduction of genetic material at a target site in the genome of a cell, which restores, corrects, disrupts, and / or modifies the expression of a gene or gene product. In certain embodiments, genome editing contemplated includes introducing one or more nuclease variants into a cell to generate a DNA lesion at or near a target site in the genome of the cell, optionally in the presence of a donor repair template.

[0108] As used herein, the term "gene therapy" refers to the introduction of excess genetic material into the total genetic material in a cell to restore, correct, or modify the expression of a gene or gene product, or for the purpose of expressing a therapeutic polypeptide. In certain embodiments, the introduction of genetic material into the genome of a cell by genome editing to restore, correct, disrupt, or modify the expression of a gene or gene product, or for the purpose of expressing a therapeutic polypeptide, is considered gene therapy.

[0109] C. Fusion Polypeptides Fusion polypeptides contemplated in certain embodiments herein that are suitable for editing target sites include a DNA binding domain, a homing endonuclease, and a terminal processing domain (e.g., an exonuclease). In various embodiments, the fusion polypeptide includes a DNA binding domain and a homing endonuclease variant, a polypeptide linker, and an exonuclease (e.g., ExoX), or a biologically active fragment thereof. In certain embodiments, the fusion polypeptide includes, in order from N-terminus to C-terminus, a DNA binding domain, a first linker domain, a homing endonuclease variant, a second linker domain, and an exonuclease (e.g., ExoX), or a biologically active fragment thereof.

[0110] In various embodiments, the DNA binding domain is a TALE DNA binding domain or a zinc finger DNA binding domain.

[0111] In various embodiments, the homing endonuclease is an engineered nuclease. The terms "reprogrammed nuclease", "engineered nuclease", or "nuclease variant" are used interchangeably and refer to a nuclease (e.g., a nuclease or homing endonuclease) that includes one or more DNA binding domains and one or more DNA cleavage domains, and the nuclease is designed and / or modified from a parent or naturally occurring nuclease to bind and cleave a double-stranded DNA target sequence or site. The nuclease variant may be designed and / or modified from a naturally occurring nuclease or from a previous nuclease variant. In some embodiments, the nuclease variant is designed to bind and cleave a non-natural target sequence or site.

[0112] An example of a fusion polypeptide that binds to and cleaves a target sequence includes, but is not limited to, a megaTAL, or a biologically active fragment thereof, linked to an exonuclease (e.g., ExoX exonuclease) by a linker domain (e.g., a polypeptide linker).

[0113] In a preferred embodiment, the fusion polypeptides, when introduced together into a cell, are useful for generating positionally biased and / or extended deletions compared to deletions induced by similar non-fused / linked polypeptides, such as separate megaTALs and exonucleases. In certain embodiments, the deletions are more directionally biased compared to deletions induced by similar non-fused / linked polypeptides. In more specific embodiments, the fusion polypeptides contemplated herein generate substantially more deletions on one side of the breakpoint than the other. For example, the fusion polypeptides described induce substantially more mutagenic deletions with the deletion center 5' to the center position of the breakpoint or nuclease target site.

[0114] 1. Homing endonuclease (meganuclease) variants In various embodiments, the homing endonucleases or meganucleases are reprogrammed to introduce a double-stranded break (DSB) at a target site within a target gene. "Homing endonucleases" and "meganucleases" are used interchangeably and refer to naturally occurring nucleases that recognize 12-45 base pair cut sites (e.g., target sites) and are generally grouped into five families based on sequence and structural motifs: LAGLIDADG, GIY-YIG, HNH, His-Cys box, and PD-(D / E)XK.

[0115] A "reference homing endonuclease" or "reference meganuclease" refers to a wild-type homing endonuclease or a homing endonuclease found in nature. In one embodiment, a "reference homing endonuclease" refers to a wild-type homing endonuclease that has been modified to increase its basal activity.

[0116] "Engineered homing endonuclease," "reprogrammed homing endonuclease," "homing endonuclease variant," "engineered meganuclease," "reprogrammed meganuclease," or "meganuclease variant" refers to a homing endonuclease that comprises one or more DNA binding domains and one or more DNA cleavage domains, where the homing endonuclease has been designed and / or modified from a parent or naturally occurring homing endonuclease to bind and cleave a DNA target sequence or site. A homing endonuclease variant may be designed and / or modified from a naturally occurring homing endonuclease or from another homing endonuclease variant.

[0117] Homing endonuclease (HE) variants do not occur in nature and can be obtained by recombinant DNA technology or by random mutagenesis. HE variants may be obtained by making one or more amino acid changes in a naturally occurring HE or HE variant, for example, by mutating, substituting, adding, or deleting one or more amino acids. In certain embodiments, the HE variant comprises one or more amino acid changes to the DNA recognition interface.

[0118] In certain embodiments, the HE variants contemplated may further comprise one or more linkers and / or additional functional domains, e.g., a terminal processing enzyme domain of a terminal processing enzyme that exhibits 5' to 3' exonuclease, 5' to 3' alkaline exonuclease, 3' to 5' exonuclease (e.g., Trex2, ExoI, or ExoX), 5' flap endonuclease, helicase, template-dependent DNA polymerase, or template-independent DNA polymerase activity. In various embodiments, a polypeptide is provided that comprises an HE variant linked to a terminal processing enzyme by a linker domain (e.g., a polypeptide linker). In various embodiments, the terminal carrying enzyme exhibits 3' to 5' exonuclease activity. In certain embodiments, the terminal processing enzyme is Trex2, ExoI, or ExoX. The HE variant and the terminal processing enzyme may be introduced separately, for example on different vectors or separate mRNAs, or together, for example as a fusion protein, or in a polycistronic construct separated by a viral self-cleaving peptide or an IRES element.

[0119] "DNA recognition interface" refers to the HE amino acid residues that interact with the nucleic acid target base as well as adjacent residues. For each HE, the DNA recognition interface contains an extensive network of side chain-side chain and side chain-DNA contacts, most of which are necessarily unique for recognizing a particular nucleic acid target sequence. Thus, the amino acid sequence of the DNA recognition interface corresponding to a particular nucleic acid sequence varies widely and is characteristic of any natural or HE variant. As a non-limiting example, the HE variants contemplated in certain embodiments can be derived by constructing a library of HE variants in which one or more amino acid residues localized in the DNA recognition interface of a natural HE (or a previously generated HE variant) are altered. The library can be screened for target cleavage activity against each predicted BTK target site using a cleavage assay (see, e.g., Jarjour et al., 2009. Nuc. Acids Res. 37(20):6871-6880).

[0120] LAGLIDADG homing endonucleases (LHEs) are the best studied family of homing endonucleases, mainly encoded in archaea and in organelle DNA in green algae and fungi, and show the highest overall DNA recognition specificity. LHEs contain one or two LAGLIDADG catalytic motifs per protein chain and function as homodimers or single-chain monomers, respectively. Structural studies of LAGLIDADG proteins have identified a highly conserved core structure (Stoddard 2005), which is characterized by an αββαββα fold with the LAGLIDADG motif belonging to the first helix of this fold. The highly efficient and specific cleavage of LHEs represents a protein scaffold for deriving novel, highly specific endonucleases. However, engineering an LHE to bind and cleave a non-native or non-canonical target site requires selection of an appropriate LHE scaffold, exploration of the target locus, selection of a putative target site, and extensive modification of the LHE to alter its DNA contact points and cleavage specificity, up to two-thirds of the base pair positions in the target site.

[0121] In one embodiment, LHEs from which reprogrammed LHE or LHE variants can be designed include, but are not limited to, I-CreI and I-SceI.

[0122] Illustrative examples of LHEs from which reprogrammed LHEs or LHE variants can be engineered include I-AabMI, I-AaeMI, I-AniI, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-GpiI, I-GzeMI, I-GzeMII, I-GzeMIII, I These include, but are not limited to, I-HjeMI, I-LtrII, I-LtrI, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI, I-OheMI, I-OnuI, I-OsoMI, I-OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I-SscMI, and I-Vdi141I.

[0123] In one embodiment, the reprogrammed LHE or LHE variant is selected from the group consisting of an I-CpaMI variant, an I-HjeMI variant, an I-OnuI variant, an I-PanMI variant, and an I-SmaMI variant.

[0124] In one embodiment, a reprogrammed I-OnuI LHE or I-OnuI variant targeting a target gene can be generated from native I-OnuI or a biologically active fragment thereof (SEQ ID NOs: 102-106).

[0125] In various embodiments, the target gene is an immune system checkpoint gene, a globin gene, a gene encoding a polypeptide that contributes to the suppression of γ-globin gene expression and / or HbF, or an immunosuppressive signaling gene.

[0126] In one embodiment, the target genes are programmed cell death protein 1 (PD-1, PDCD1), lymphocyte activation gene 3 protein (LAG-3), T cell immunoglobulin domain and mucin domain protein 3 (TIM-3), cytotoxic T lymphocyte antigen-4 (CTLA-4), band T lymphocyte attenuator (BTLA), T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), V domain Ig inhibitor of T cell activation (VISTA), and killer cell immunoglobulin-like receptor (KIR), CCR5, TRAC (TCRα), TCRβ, IL10Rα, IL10Rβ, TGFBR1, TGFBR2, CBL-B, PCSK9, AHR, BTK, α-globin, β-globin, γ-globin, BCL11A, KLF1, SOX6, GATA1, LSD, alpha folate receptor (FR α), αvβ6 integrin, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase IX (CAIX), CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, C D70, CD79a, CD79b, CD123, CD133, CD138, CD171, carcinoembryonic antigen (CEA), C-type lectin-like molecule-1 (CLL-1), CD2 subset 1 (CS-1), chondroitin sulfate proteoglycan 4 (CSPG4), cutaneous T-cell lymphoma-associated antigen 1 (CTAGE1), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), epithelial cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EPHA2) , fibroblast activation protein (FAP), Fc receptor-like 5 (FCRL5), fetal acetylcholinesterase receptor (AchR), ganglioside G2 (GD2), ganglioside G3 (GD3), glypican-3 (GPC3), EGFR family including ErbB2 (HER2), IL-11Rα, IL-13Rα2, kappa, cancer / testis antigen 2 (LAGE-1A), lambda, Lewis-Y (LeY), L1 cell adhesion molecule (L1-CAM), melanoma antigen gene (MAGE)-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10,Melanoma antigen recognized by T cells 1 (MelanA or MART1), mesothelin (MSLN), MUC1, MUC16, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), neural cell adhesion molecule (NCAM), cancer / testis antigen 1 (NY-ESO-1), polysialic acid; placenta-specific 1 (PLAC1), predominantly expressed antigen in melanoma (PRAME), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor The gene is selected from the group consisting of resorcinolone 1 (ROR1), synovial sarcoma, X breakpoint 2 (SSX2), survivin, tumor associated glycoprotein 72 (TAG72), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7 related (TEM7R), TEM5, TEM8, trophoblast glycoprotein (TPBG), UL16 binding protein (ULBP) 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms' tumor 1 (WT-1) genes.

[0127] In certain embodiments, the target gene is selected from the group consisting of programmed cell death protein 1 (PD-1, PDCD1), lymphocyte activation gene 3 protein (LAG-3), T cell immunoglobulin domain and mucin domain protein 3 (TIM-3), cytotoxic T lymphocyte antigen-4 (CTLA-4), band T lymphocyte attenuator (BTLA), T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), V domain Ig inhibitor of T cell activation (VISTA), and killer cell immunoglobulin-like receptor (KIR), CCR5, TRAC (TCRα), IL10Rα, TGFBR2, CBL-B, PCSK9, AHR, BTK, α-globin, β-globin, γ-globin, and BCL11A genes.

[0128] In certain embodiments, the target gene is the TRAC (TCRα), CBL-B, or PDCD1 (PD-1) gene.

[0129] In one embodiment, the target gene is the TRAC gene, the CBL-B gene, or the PD-1 gene. In a particular embodiment, the target gene / site comprises the nucleotide sequence set forth in SEQ ID NO: 1, 2, or 3.

[0130] In one embodiment, a reprogrammed I-OnuI LHE or I-OnuI variant targeting the human TRAC (TCR alpha) gene was generated from an existing I-OnuI variant. In some embodiments, a reprogrammed I-OnuI LHE was generated against the human TRAC gene target site shown in SEQ ID NO:1.

[0131] In another embodiment, a reprogrammed I-OnuI LHE or I-OnuI variant targeting the human PDCD1 (PD-1) gene was generated from an existing I-OnuI variant. In some embodiments, the reprogrammed I-OnuI LHE was generated against the human PD-1 gene target site shown in SEQ ID NO:2.

[0132] In another embodiment, a reprogrammed I-OnuI LHE or I-OnuI variant targeting the human CBL-B gene was generated from an existing I-OnuI variant, hi some embodiments, a reprogrammed I-OnuI LHE was generated against the human CBL-B gene target site shown in SEQ ID NO:3.

[0133] In certain embodiments, the I-OnuI LHE variant that binds to and cleaves a target gene comprises one or more amino acid substitutions or modifications in the DNA recognition interface of I-OnuI set forth in any one of SEQ ID NOs: 89, 92, 95, 98, 101, or 102-106, a biologically active fragment thereof, and / or a further variant thereof.

[0134] In certain embodiments, the reprogrammed I-OnuI LHE or I-OnuI variant that binds and cleaves the target gene comprises one or more amino acid substitutions in the DNA recognition interface. In certain embodiments, the I-OnuI LHE that binds and cleaves the target gene comprises I-OnuI (Taekuchi et al. 2011. Proc Natl Acad Sci USA 2011 Aug 9; 108(32): 13077-13082) or I-OnuI (Taekuchi et al. 2011. Proc Natl Acad Sci USA 2011 Aug 9; 108(32): 13077-13082) set forth in SEQ ID NO: 89, 92, 95, 98, 101, or 102-106. The DNA recognition interface of the LHE variant or further variant thereof has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0135] In one embodiment, the I-OnuI LHE that binds to and cleaves the target gene comprises at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97%, more preferably at least 99% sequence identity to the DNA recognition interface of I-OnuI (Taekuchi et al. 2011. Proc Natl Acad Sci USA 2011 Aug 9; 108(32): 13077-13082) or an I-OnuI LHE variant set forth in SEQ ID NO: 89, 92, 95, 98, 101, or 102-106, or a further variant thereof.

[0136] In certain embodiments, I-OnuI LHE variants that bind to and cleave target genes comprise one or more amino acid substitutions or modifications in the DNA recognition interface, in particular in the subdomains located at positions 24-50, 68-82, 180-203, and 223-240 of I-OnuI (SEQ ID NOs: 102-106), the I-OnuI variants set forth in SEQ ID NOs: 89, 92, 95, 98, and 101, biologically active fragments thereof, and / or further variants thereof.

[0137] In certain embodiments, the I-OnuI LHE variant that binds to and cleaves the target gene is selected from the group consisting of I-OnuI (SEQ ID NOs: 103-107) or I-OnuI variants set forth in SEQ ID NOs: 89, 92, 95, 98, and 101, biologically active fragments thereof, and / or further variants thereof, such as 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 68, 70, 72, 75, 76, 78, 80, 82, 84, 86, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 120, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 140, 142, 144, 146, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164 , 80, 82, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238, and 240.

[0138] In one embodiment, an I-OnuI LHE variant that binds and cleaves a target gene comprises one or more amino acid substitutions or modifications at additional positions located anywhere within the entire I-OnuI sequence. Residues that can be substituted and / or modified include amino acids that contact the nucleic acid target, directly or via water molecules, or interact with the nucleic acid backbone or nucleotide bases. In one non-limiting example, the I-OnuI LHE variant contemplated herein that binds and cleaves a target gene comprises one or more amino acid substitutions or modifications at additional positions located anywhere within the entire I-OnuI sequence. LHE variants include I-OnuI variants as set forth in SEQ ID NOs: 102 to 106 or SEQ ID NOs: 89, 92, 95, 98, and 101, biologically active fragments thereof, and / or further variants thereof at positions: 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 61, 68, 70, 72, 75, 76, 78, 80, 82, 85, 116, 135, 138, 143, 147, 159, 164, 168, 178, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 300, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 343 and preferably at least 5, preferably at least 10, preferably at least 15, preferably at least 20, more preferably at least 25, more preferably at least 30, even more preferably at least 35, or even more preferably at least 40, at least one position selected from the group consisting of: 3, 195, 197, 199, 201, 203, 210, 223, 225, 227, 229, 231, 232, 234, 236, 238, 240, and 246.

[0139] In certain embodiments, the I-OnuI LHE variant that binds to and cleaves the target gene is selected from the group consisting of I-OnuI variants set forth in SEQ ID NOs: 102-106 or SEQ ID NOs: 89, 92, 95, 98, and 101, biologically active fragments thereof, and / or further variants thereof, such as 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 61, 68, 70, 72, 75, 76, 78, 80, 82, 85, 116, 135, 138, 143, 147, 159, 164, 168, 178, 180, 182, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 198, 199, 198, 199, 198, 199, 199, 198, 199, 199, 198, 199, 199, 199, 200, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220 At least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more amino acid substitutions at amino acid positions selected from the group consisting of 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 210, 223, 225, 227, 229, 231, 232, 234, 236, 238, 240, and 246.

[0140] In certain embodiments, an I-OnuI LHE variant that binds to and cleaves a target gene comprises an amino acid sequence that is at least 80%, preferably at least 85%, more preferably at least 90%, or even more preferably at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 89, 92, 95, 98, and 101, or a biologically active fragment thereof.

[0141] In certain embodiments, the I-OnuI LHE variant comprises an amino acid sequence set forth in any one of SEQ ID NOs: 89, 92, 95, 98, and 101, or a biologically active fragment thereof.

[0142] In certain embodiments, the I-OnuI LHE variant comprises the amino acid sequence set forth in SEQ ID NO:89, or a biologically active fragment thereof.

[0143] In certain embodiments, the I-OnuI LHE variant comprises the amino acid sequence set forth in SEQ ID NO: 92, or a biologically active fragment thereof.

[0144] In certain embodiments, the I-OnuI LHE variant comprises the amino acid sequence set forth in SEQ ID NO: 95, or a biologically active fragment thereof.

[0145] In certain embodiments, the I-OnuI LHE variant comprises the amino acid sequence set forth in SEQ ID NO: 98, or a biologically active fragment thereof.

[0146] In certain embodiments, the I-OnuI LHE variant comprises the amino acid sequence set forth in SEQ ID NO: 101, or a biologically active fragment thereof.

[0147] 2.DNA-binding domain In various embodiments, the fusion polypeptide contemplated herein comprises a DNA binding domain. In certain embodiments, the DNA binding domain is located N-terminal to that of the homing endonuclease. In certain embodiments, the fusion polypeptide comprises a DNA binding domain located N-terminal to that of the homing endonuclease, which is N-terminal to that of the terminal processing domain (e.g., exonuclease). In other words, the homing endonuclease is sandwiched between the DNA binding domain and the exonuclease. Thus, in order from N-terminus to C-terminus, an exemplary fusion polypeptide contemplated herein comprises a DNA binding domain, a first polypeptide linker, a homing endonuclease, a second polypeptide linker, and an exonuclease.

[0148] In one aspect, the DNA binding domain comprises a TALE DNA binding domain. In certain embodiments, the fusion polypeptide comprises a megaTAL. "megaTAL" refers to a polypeptide comprising a TALE DNA binding domain and a homing endonuclease variant that binds and cleaves a DNA target sequence in a target gene and comprises one or more linkers and / or additional functional domains, such as a 5' to 3' exonuclease, a 5' to 3' alkaline exonuclease, a 3' to 5' exonuclease (e.g., Trex2, ExoI, or ExoX), a 5' flap endonuclease, a helicase, or an end-processing enzyme domain of an end-processing enzyme exhibiting template-independent DNA polymerase activity.

[0149] In various embodiments, a megaTAL comprising a homing endonuclease variant is reprogrammed to introduce a double-stranded break (DSB) in a target gene. In some embodiments, a megaTAL comprising a homing endonuclease variant is reprogrammed to introduce a double-stranded break in a target sequence in an immune system checkpoint gene, a globin gene, a gene encoding a polypeptide that contributes to the suppression of γ-globin gene expression and / or HbF, or an immunosuppressive signaling gene. In some embodiments, a megaTAL comprising a homing endonuclease variant is reprogrammed to introduce a double-stranded break in a target sequence in the human TRAC gene, the PD-1 gene, or the CBL-B gene (e.g., SEQ ID NOs: 1-3, respectively).

[0150] A "TALE DNA binding domain" is the DNA binding portion of a transcription activator-like effector (TALE or TAL effector), which mimics plant transcription activators to manipulate the plant transcriptome (see, e.g., Kay et al., 2007. Science 318:648-651). In certain embodiments, contemplated TALE DNA binding domains are, for example, engineered de novo or derived from naturally occurring TALEs, such as AvrBs3 from Xanthomonas campestris pv.vesicatoria, Xanthomonas gardneri, Xanthomonas translucens, Xanthomonas axonopodis, Xanthomonas perforans, Xanthomonas alfalfa, Xanthomonas citri, Xanthomonas euvesicatoria, and Xanthomonas oryzae, and brg11 and hpx17 from Ralstonia solanacearum. Illustrative examples of TALE protein deriving and engineering DNA binding domains are disclosed in U.S. Pat. No. 9,017,967 and the references cited therein, all of which are incorporated herein by reference in their entirety.

[0151] In certain embodiments, a TALE DNA binding domain comprises one or more repeat units that are involved in the binding of the TALE DNA binding domain to its corresponding target DNA sequence. A single "repeat unit" (also referred to as a "repeat") is typically 33-35 amino acids long. Each TALE DNA binding domain repeat unit typically comprises one or two DNA binding residues that constitute a repeat variable dipeptide (RVD) at positions 12 and / or 13 of the repeat. The natural (canonical) code for DNA recognition of these TALE DNA binding domains has been determined such that the HD sequence at positions 12 and 13 directs binding to cytosine (C), NG binds to T, NI binds to A, NN binds to G or A, and NG binds to T. In certain embodiments, non-canonical (atypical) RVDs are contemplated.

[0152] Illustrative examples of non-canonical RVDs suitable for use in certain megaTALs contemplated in certain embodiments include, but are not limited to, HH, KH, NH, NK, NQ, RH, RN, SS, NN, SN, KN for recognizing guanine (G); NI, KI, RI, HI, SI for recognizing adenine (A); NG, HG, KG, RG for recognizing thymine (T); RD, SD, HD, ND, KD, YG for recognizing cytosine (C); NV, HN for recognizing A or G; and H*, HA, KA, N*, NA, NC, NS, RA, S* for recognizing A or T or G or C, where (*) denotes the absence of the amino acid at position 13. Further illustrative examples of RVDs suitable for use in certain megaTALs contemplated in certain embodiments further include those disclosed in U.S. Pat. No. 8,614,092, which is incorporated herein by reference in its entirety.

[0153] In certain embodiments, a fusion polypeptide or megaTAL contemplated herein comprises a TALE DNA binding domain comprising 3-30 repeat units. In certain embodiments, the TALE DNA binding domain comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 TALE DNA binding domain repeat units. In preferred embodiments, the TALE DNA binding domain comprises 5-15 repeat units, more preferably 7-15 repeat units, more preferably 9-15 repeat units, more preferably 9, 10, 11, 12, 13, 14, or 15 repeat units.

[0154] In certain embodiments, a fusion polypeptide or megaTAL contemplated herein comprises a TALE DNA binding domain comprising 3-30 repeat units and an additional single truncated TALE repeat unit comprising 20 amino acids located at the C-terminus of the set of TALE repeat units, i.e., an additional C-terminal half TALE DNA binding domain repeat unit (amino acids -20 to -1 of the C-cap disclosed above and elsewhere herein). Thus, in certain embodiments, a fusion polypeptide or megaTAL contemplated herein comprises a TALE DNA binding domain comprising 3.5-30.5 repeat units. In certain embodiments, the fusion polypeptide or megaTAL comprises 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 10.5, 11.5, 12.5, 13.5, 14.5, 15.5, 16.5, 17.5, 18.5, 19.5, 20.5, 21.5, 22.5, 23.5, 24.5, 25.5, 26.5, 27.5, 28.5, 29.5, or 30.5 TALE DNA binding domain repeat units. In a preferred embodiment, the fusion polypeptide or megaTAL contemplated herein comprises a TALE DNA binding domain comprising 5.5 to 15.5 repeat units, more preferably 7.5 to 15.5 repeat units, more preferably 9.5 to 15.5 repeat units, more preferably 9.5, 10.5, 11.5, 12.5, 13.5, 14.5 or 15.5 repeat units.

[0155] In certain embodiments, a fusion polypeptide or megaTAL contemplated herein comprises a TAL effector construct comprising an "N-terminal domain (NTD)" polypeptide, one or more TALE repeat domains / units, a "C-terminal domain (CTD)" polypeptide, and a homing endonuclease variant. In some embodiments, the NTD, TALE repeats, and / or CTD domains are from the same species. In other embodiments, one or more of the NTD, TALE repeats, and / or CTD domains are from different species.

[0156] As used herein, the term "N-terminal domain (NTD)" polypeptide refers to a sequence adjacent to the N-terminal portion or fragment of a naturally occurring TALE DNA binding domain. The NTD sequence, if present, may be of any length so long as the TALE DNA binding domain repeat unit retains the ability to bind DNA. In certain embodiments, the NTD polypeptide comprises at least 120 to at least 140 or more amino acids N-terminal to the TALE DNA binding domain (where 0 is amino acid 1 of the most N-terminal repeat unit). In certain embodiments, the NTD polypeptide comprises at least about 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, or at least 140 amino acids N-terminal to the TALE DNA binding domain. In one embodiment, a fusion polypeptide or megaTAL contemplated herein comprises an NTD polypeptide from at least about amino acids +1 to +122 to at least about +1 to +137 of a Xanthomonas TALE protein (where 0 is amino acid 1 of the most N-terminal repeat unit). In certain embodiments, the NTD polypeptide comprises at least about 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, or 137 amino acids N-terminal to the TALE DNA binding domain of a Xanthomonas TALE protein. In one embodiment, a fusion polypeptide or megaTAL contemplated herein comprises an NTD polypeptide from at least amino acids +1 to +121 of a Ralstonia TALE protein (where 0 is amino acid 1 of the most N-terminal repeat unit). In certain embodiments, the NTD polypeptide comprises at least about 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, or 137 amino acids N-terminal to the TALE DNA binding domain of a Ralstonia TALE protein.

[0157] As used herein, the term "C-terminal domain (CTD)" polypeptide refers to a sequence adjacent to the C-terminal portion or fragment of a naturally occurring TALE DNA binding domain. The CTD sequence, if present, may be of any length so long as the TALE DNA binding domain repeat unit retains the ability to bind DNA. In certain embodiments, the CTD polypeptide comprises at least 20 to at least 85 or more amino acids C-terminal to the last complete repeat of the TALE DNA binding domain (the first 20 amino acids are the C-terminal half repeat unit to the last C-terminal complete repeat unit). In certain embodiments, the CTD polypeptide comprises at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or at least 85 amino acids C-terminal to the last complete repeat of the TALE DNA binding domain. In one embodiment, a fusion polypeptide or megaTAL contemplated herein comprises a CTD polypeptide of at least about amino acids -20 to -1 of a Xanthomonas TALE protein (-20 being amino acid 1 of the half-repeat unit at the C-terminus of the C-terminal perfect repeat unit). In a particular embodiment, the CTD polypeptide comprises at least about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid at the C-terminus of the last perfect repeat of the TALE DNA binding domain of a Xanthomonas TALE protein. In one embodiment, a fusion polypeptide or megaTAL contemplated herein comprises a CTD polypeptide of at least about amino acids -20 to -1 of a Ralstonia TALE protein (-20 being amino acid 1 of the half-repeat unit at the C-terminus of the C-terminal perfect repeat unit).In certain embodiments, the CTD polypeptide comprises at least about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids C-terminal to the last complete repeat of the TALE DNA binding domain of a Ralstonia TALE protein.

[0158] In certain embodiments, the fusion polypeptide or megaTAL contemplated herein comprises a TALE DNA binding domain engineered to bind to a target sequence, a homing endonuclease reprogrammed to bind and cleave the target sequence / site, and an NTD and / or CTD polypeptide, optionally linked together with one or more linker polypeptides contemplated elsewhere herein. It is further contemplated that a fusion polypeptide or megaTAL comprising a TALE DNA binding domain and optionally an NTD and / or CTD polypeptide can be fused to a linker polypeptide, which is further fused to a homing endonuclease variant. Thus, the TALE DNA binding domain binds to a DNA target sequence about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides away from the target sequence bound by the DNA binding domain of the homing endonuclease variant. Thus, the fusion polypeptide or megaTAL contemplated herein increases the specificity and efficiency of genome editing.

[0159] In one embodiment, the fusion polypeptide or megaTAL comprises a homing endonuclease variant and a TALE DNA binding domain that binds to a nucleotide / target sequence / site approximately 4, 5, or 6 nucleotides upstream, preferably within 6 nucleotides, of the binding / target site of the reprogrammed homing endonuclease.

[0160] In certain embodiments, a fusion polypeptide or megaTAL contemplated herein comprises one or more TALE DNA-binding repeat units, as well as I-AabMI, I-AaeMI, I-AniI, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-GpiI, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-LtrII, I-LtrI, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI, I-O ... The LHE variants include LHE variants engineered or reprogrammed from an LHE selected from the group consisting of heMI, I-OnuI, I-OsoMI, I-OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I-SscMI, I-Vdi141I, and variants thereof, or preferably, I-CpaMI, I-HjeMI, I-OnuI, I-PanMI, SmaMI, and variants thereof, or preferably, I-OnuI and variants thereof.

[0161] In certain embodiments, the fusion polypeptide or megaTAL contemplated herein comprises an NTD, one or more TALE DNA-binding repeat units, a CTD, and one or more of the following: I-AabMI, I-AaeMI, I-AniI, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-GpiI, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-LtrII, I-LtrI, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl ... I-NcrMI, I-OheMI, I-OnuI, I-OsoMI, I-OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I-SscMI, I-Vdi141I, and variants thereof, or preferably, I-CpaMI, I-HjeMI, I-OnuI, I-PanMI, SmaMI, and variants thereof, or preferably, I-OnuI and variants thereof.

[0162] In certain embodiments, a fusion polypeptide or megaTAL contemplated herein comprises an NTD, about 9.5 to about 15.5 TALE DNA-binding repeat units, and one or more of I-AabMI, I-AaeMI, I-AniI, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-GpiI, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-LtrII, I-LtrI, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I- The LHE variants include those selected from the group consisting of NcrMI, I-OheMI, I-OnuI, I-OsoMI, I-OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I-SscMI, I-Vdi141I, and variants thereof, or preferably I-CpaMI, I-HjeMI, I-OnuI, I-PanMI, SmaMI, and variants thereof, or preferably I-OnuI and variants thereof.

[0163] In certain embodiments, a fusion polypeptide or megaTAL contemplated herein comprises an NTD of about 122 amino acids to 137 amino acids, about 9.5, about 10.5, about 11.5, about 12.5, about 13.5, about 14.5, or about 15.5 binding repeat units, a CTD of about 20 amino acids to about 85 amino acids, and an I-OnuI LHE variant. In certain embodiments, any one, two, or all of the NTD, DNA binding domain, and CTD can be designed from the same or different species, in any suitable combination.

[0164] In certain embodiments, a fusion polypeptide or megaTAL contemplated herein comprises a TALE DNA binding domain comprising about 9.5, about 10.5, about 11.5, about 12.5, about 13.5, about 14.5, or about 15.5 binding repeat units, a homing endonuclease variant contemplated elsewhere herein, and a terminal processing domain (e.g., an exonuclease) contemplated elsewhere herein, or a biologically active fragment thereof.

[0165] In certain embodiments, the fusion polypeptide contemplated herein comprises, in order from N-terminus to C-terminus, a TALE DNA binding domain comprising about 9.5, about 10.5, about 11.5, about 12.5, about 13.5, about 14.5, or about 15.5 binding repeat units, a first linker domain, an I-OnuI LHE variant, a second linker domain, and a terminal processing enzyme (e.g., a 3' to 5' exonuclease), or a biologically active fragment thereof. In certain embodiments, any one, two, or all of the TALE binding repeats can be designed from the same or different species, in any suitable combination. In a preferred embodiment, the exonuclease is an ExoX exonuclease, or a biologically active fragment thereof.

[0166] In certain embodiments, a fusion polypeptide or megaTAL contemplated herein comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 7, 10, 58, 67, or 76. In certain embodiments, a fusion polypeptide or megaTAL contemplated herein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 7, 10, 58, 67, or 76. In certain embodiments, a fusion polypeptide or megaTAL-Trex2 fusion protein contemplated herein comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 13, 61, 70, or 79. In certain embodiments, a fusion polypeptide or megaTAL-Trex2 fusion protein contemplated herein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 13, 61, 70, or 79. In certain embodiments, a fusion polypeptide or megaTAL-ExoX fusion protein contemplated herein comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 46, 64, 73, or 82. In certain embodiments, a fusion polypeptide or megaTAL-ExoX fusion protein contemplated herein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 46, 64, 73, or 82. In certain embodiments, a fusion polypeptide or megaTAL-ExoI fusion protein contemplated herein comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence set forth in SEQ ID NO: 43. In certain embodiments, a fusion polypeptide or megaTAL-ExoI fusion protein contemplated herein comprises the amino acid sequence shown in SEQ ID NO:43.

[0167] In certain embodiments, a fusion polypeptide or megaTAL contemplated herein is encoded by an mRNA sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the mRNA sequence set forth in any one of SEQ ID NOs: 6, 9, 57, 66, or 75. In certain embodiments, a fusion polypeptide or megaTAL contemplated herein is encoded by an mRNA sequence set forth in any one of SEQ ID NOs: 6, 9, 57, 66, or 75. In certain embodiments, a fusion polypeptide or megaTAL-Trex2 contemplated herein is encoded by an mRNA sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the mRNA sequence set forth in any one of SEQ ID NOs: 12, 60, 69, or 78. In certain embodiments, a fusion polypeptide or megaTAL-Trex2 contemplated herein is encoded by an mRNA sequence set forth in any one of SEQ ID NOs: 12, 60, 69, or 78. In certain embodiments, a fusion polypeptide or megaTAL-ExoX contemplated herein is encoded by an mRNA sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to an mRNA sequence set forth in any one of SEQ ID NOs: 45, 63, 72, or 81. In certain embodiments, a fusion polypeptide or megaTAL-ExoX contemplated herein is encoded by an mRNA sequence set forth in any one of SEQ ID NOs: 45, 63, 72, or 81. In certain embodiments, a fusion polypeptide or megaTAL-ExoI contemplated herein is encoded by an mRNA sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the mRNA sequence set forth in SEQ ID NO: 42. In certain embodiments, a fusion polypeptide or megaTAL-ExoI contemplated herein is encoded by the mRNA sequence set forth in SEQ ID NO: 42.

[0168] In certain embodiments, a fusion polypeptide or megaTAL contemplated herein is encoded by a DNA sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the DNA sequence set forth in any one of SEQ ID NOs: 5, 8, 56, 65, or 74. In certain embodiments, a fusion polypeptide or megaTAL contemplated herein is encoded by a DNA sequence set forth in any one of SEQ ID NOs: 5, 8, 56, 65, or 74. In certain embodiments, a fusion polypeptide or megaTAL-Trex2 contemplated herein is encoded by a DNA sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the DNA sequence set forth in any one of SEQ ID NOs: 11, 59, 68, or 77. In certain embodiments, a fusion polypeptide or megaTAL-Trex2 contemplated herein is encoded by a DNA sequence set forth in any one of SEQ ID NOs: 11, 59, 68, or 77. In certain embodiments, a fusion polypeptide or megaTAL-ExoX contemplated herein is encoded by a DNA sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a DNA sequence set forth in any one of SEQ ID NOs: 44, 62, 71, or 80. In certain embodiments, a fusion polypeptide or megaTAL-ExoX contemplated herein is encoded by a DNA sequence set forth in any one of SEQ ID NOs: 44, 62, 71, or 80. In certain embodiments, a fusion polypeptide or megaTAL-ExoI contemplated herein is encoded by a DNA sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a DNA sequence set forth in SEQ ID NO: 41. In certain embodiments, the fusion polypeptide or megaTAL-ExoI contemplated herein is encoded by the DNA sequence shown in SEQ ID NO:41.

[0169] In certain embodiments, the megaTAL comprises a TALE DNA binding domain and an I-OnuI LHE variant that binds to and cleaves a nucleotide sequence set forth in any one of SEQ ID NOs: 1-3.

[0170] In another aspect, the DNA binding domain comprises a zinc finger DNA binding domain. In certain embodiments, a fusion polypeptide contemplated herein comprises a zinc finger DNA binding domain, a homing endonuclease domain as contemplated elsewhere herein, and a terminal processing domain (e.g., an exonuclease) as contemplated elsewhere herein.

[0171] In certain embodiments, a zinc finger DNA binding domain has one, two, three, four, five, six, seven, eight or more zinc finger motifs. Typically, a single zinc finger motif is about 30 amino acids in length. Zinc finger motifs include both C2H2 zinc fingers and non-canonical zinc fingers, e.g., C3H zinc fingers and C4 zinc fingers.

[0172] Zinc finger DNA binding domains can be engineered to bind any DNA sequence. Candidate zinc finger DNA binding domains for a given 3bp DNA target sequence have been identified, and a modular assembly strategy has been devised to link multiple domains into a multi-finger peptide targeted to the corresponding complex DNA target sequence. Other suitable methods known in the art can be used to design and construct nucleic acids encoding zinc finger DNA binding domains, such as phage display, random mutagenesis, combinatorial libraries, computer / rational design, affinity selection, PCR, cloning from cDNA or genomic libraries, synthetic construction, etc. (See, e.g., U.S. Pat. No. 5,786,538; Wu et al., PNAS 92:344-348 (1995); Jamieson et al., Biochemistry 33:5689-5695 (1994); Rebar & Pabo, Science 263:671-673 (1994); Choo & Klug, PNAS 91:11163-11167 (1994); Choo & Klug, PNAS 91:11168-11172 (1994); Desjarlais & Berg, PNAS 90:2256-2260 (1993); Desjarlais & Berg, PNAS 89:7345-7349 (1992); Pomerantz et al., Science 267:93-96 (1995); Pomerantz et al, PNAS 92:9752-9756 (1995); Liu et al, PNAS 94:5525-5530 (1997); Griesman & Pabo, Science 275:657-661 (1997); Desjarlais & Berg, PNAS 91:1 1-99-1 1103 (1994).

[0173] Each zinc finger motif binds to three or four nucleotide sequences. The length of the sequence (e.g., target sequence) that the zinc finger binding domain is engineered to bind determines the number of zinc finger motifs in the engineered zinc finger DNA binding domain. For example, if the zinc finger motifs do not bind to overlapping subsites, a 6 nucleotide target sequence is bound by a 2 finger DNA binding domain, a 9 nucleotide target sequence is bound by a 3 finger DNA binding domain, and so on. In certain embodiments, the DNA binding sites of the individual zinc finger motifs in the target site do not have to be contiguous, but can be separated by one or several nucleotides, depending on the length and nature of the linker sequence between the zinc finger motifs in the multi-finger binding domain.

[0174] In certain embodiments, a fusion polypeptide contemplated herein comprises a zinc finger DNA binding domain comprising one or more zinc finger motifs, a linker, a homing endonuclease variant, a linker, and a terminal processing domain (e.g., an exonuclease). In certain embodiments, the homing endonuclease comprises an LHE variant, the LHE variant being I-AabMI, I-AaeMI, I-AniI, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-GpiI, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-LtrII, I-LtrI, I-LtrWI, I-MpeMI, I-MveMI, I-N The LHE is designed or reprogrammed from a LHE selected from the group consisting of I-crII, I-Ncrl, I-NcrMI, I-OheMI, I-OnuI, I-OsoMI, I-OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I-SscMI, I-Vdi141I, and variants thereof, or preferably, I-CpaMI, I-HjeMI, I-OnuI, I-PanMI, SmaMI, and variants thereof, or preferably, I-OnuI and variants thereof.

[0175] In certain embodiments, a fusion polypeptide contemplated herein comprises a zinc finger DNA binding domain comprising two, three, four, five, six, seven, eight or more zinc finger motifs, a homing endonuclease variant contemplated elsewhere herein, and a terminal processing domain (e.g., an exonuclease) contemplated elsewhere herein, or a biologically active fragment thereof.

[0176] In certain embodiments, the fusion polypeptides contemplated herein comprise, in order from N-terminus to C-terminus, a zinc finger DNA binding domain comprising about 2, 3, 4, 5, 6, 7, or 8 or more zinc finger motifs, a first linker domain, an I-OnuI LHE variant, a second linker domain, and a terminal processing domain (e.g., an exonuclease), or a biologically active fragment thereof. In certain embodiments, any one, two, or all of the zinc finger motifs can be designed from the same or different species, in any suitable combination. In a preferred embodiment, the exonuclease is an ExoX exonuclease, or a biologically active fragment thereof.

[0177] 3. Terminal processing enzymes Genome editing compositions (e.g., fusion polypeptides) and methods contemplated in certain embodiments include editing a cell's genome using a DNA binding domain, a homing endonuclease variant, and an end-processing enzyme. In certain embodiments, the fusion polypeptide encodes a DNA binding domain, a homing endonuclease variant, and one or more end-processing enzymes (e.g., exonucleases), each separated by a linker domain (e.g., a peptide linker).

[0178] The term "end-processing enzyme" refers to an enzyme that modifies exposed ends of a polynucleotide chain. The polynucleotide may be double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), RNA, double-stranded hybrids of DNA and RNA, and synthetic DNA (e.g., containing bases other than A, C, G, and T). End-processing enzymes may modify exposed polynucleotide chain ends by adding one or more nucleotides, removing one or more nucleotides, removing or modifying a phosphate group, and / or removing or modifying a hydroxyl group. End-processing enzymes may modify ends at endonuclease cleavage sites or at ends generated by shearing (e.g., by passing through a fine gauge needle, heating, sonication, mini-bead tumbling, and spraying), ionizing radiation, ultraviolet radiation, oxygen radicals, chemical hydrolysis, and other chemical or mechanical means such as chemotherapeutic agents.

[0179] In certain embodiments, genome editing compositions and methods contemplated in certain embodiments include editing a cell's genome using a fusion polypeptide comprising a DNA binding domain, a homing endonuclease variant, and a DNA end-processing enzyme.

[0180] The term "DNA end-processing enzyme" refers to an enzyme that modifies exposed ends of DNA. DNA end-processing enzymes may modify blunt or staggered ends (ends with 5' or 3' overhangs). DNA end-processing enzymes may modify single-stranded or double-stranded DNA. DNA end-processing enzymes may modify ends at endonuclease cleavage sites or at ends generated by shearing (e.g., by passing through a fine gauge needle, heating, sonication, mini-bead tumbling, and spraying), ionizing radiation, ultraviolet radiation, oxygen radicals, chemical hydrolysis, and other chemical or mechanical means such as chemotherapeutic agents. DNA end-processing enzymes may modify exposed DNA ends by adding one or more nucleotides, removing one or more nucleotides, removing or modifying a phosphate group, and / or removing or modifying a hydroxyl group.

[0181] Illustrative examples of DNA end-processing enzymes suitable for use in certain embodiments contemplated herein include, but are not limited to, 5' to 3' exonucleases, 5' to 3' alkaline exonucleases, 3' to 5' exonucleases, 5' flap endonucleases, helicases, phosphatases, hydrolases and template-dependent DNA polymerases.

[0182] Additional examples of DNA end-processing enzymes suitable for use in certain embodiments contemplated herein include Trex2, Trex1, transmembrane domain-free Trex1, Apollo, Artemis, DNA2, ExoI, ExoT, ExoIII, ExoX, Fen1, Fan1, MreII, Rad2, Rad9, TdT (terminal deoxynucleotidyl transferase), PNKP, RecE, RecJ, RecQ, lambda exonuclease, Sox, vaccinia DNA polymerase, exonuclease I, exonuclease II, exonuclease III, exonuclease II, exonuclease III, exonuclease I, exonuclease II ... Examples of exonuclease III, exonuclease VII, NDK1, NDK5, NDK7, NDK8, WRN, T7-exonuclease gene 6, avian myeloblastosis virus integration protein (IN), Bloom, Antarctic phosphatase, alkaline phosphatase, polynucleotide kinase (PNK), ApeI, mung bean nuclease, Hex1, TTRAP (TDP2), Sgs1, Sae2, CUP, Pol mu, Pol lambda, MUS81, EME1, EME2, SLX1, SLX4, and UL-12.

[0183] In certain embodiments, genome editing compositions and methods for editing a cell genome contemplated herein include a fusion polypeptide comprising a DNA binding domain, a homing endonuclease variant, and an exonuclease. The term "exonuclease" refers to an enzyme that cleaves a phosphodiester bond at the end of a polynucleotide chain via a hydrolysis reaction that cleaves the phosphodiester bond at either the 3' or 5' end. In certain embodiments, the exonuclease is a 3' to 5' exonuclease. In some embodiments, the exonuclease is an ExoX exonuclease, or a biologically active fragment thereof. In some embodiments, the exonuclease is an ExoI exonuclease, or a biologically active fragment thereof. In some embodiments, the exonuclease is a Trex2 exonuclease, or a biologically active fragment thereof.

[0184] ExoX is a 3'-5' partitioning exonuclease from Escherichia coli (E. coli) and a member of the DnaQ superfamily. ExoX is also referred to as exodeoxyribonuclease 10, exodeoxyribonuclease X, exonuclease X, and ExoX. Exemplary ExoX reference sequences for use in certain embodiments include, but are not limited to, NP_416358.1, NC_000913.3, WP_000944256.1, NZ_STEB01000009.1AAC74914.

[0185] In preferred embodiments contemplated herein, the fusion polypeptide comprises a DNA binding domain, a homing endonuclease variant, and an ExoX exonuclease, or a biologically active fragment thereof. In various embodiments, the fusion polypeptide comprises a DNA binding domain and a homing endonuclease variant, or a biologically active fragment thereof, linked to an ExoX exonuclease by a linker domain (e.g., a polypeptide linker).

[0186] In various embodiments, ExoX comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 109. In some embodiments, ExoX comprises an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 109. In some embodiments, ExoX comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 109. In some embodiments, ExoX comprises an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 109. In some embodiments, ExoX comprises an amino acid sequence having at least 96% identity to the amino acid sequence set forth in SEQ ID NO: 109. In some embodiments, ExoX comprises an amino acid sequence having at least 97% identity to the amino acid sequence set forth in SEQ ID NO: 109. In some embodiments, ExoX comprises an amino acid sequence having at least 98% identity to the amino acid sequence set forth in SEQ ID NO: 109. In some embodiments, ExoX comprises an amino acid sequence having at least 99% identity to the amino acid sequence set forth in SEQ ID NO:109.

[0187] In certain embodiments, ExoX, or a biologically active fragment thereof, comprises the amino acid sequence set forth in SEQ ID NO:109.

[0188] ExoI is a 3' to 5' processive exonuclease from Escherichia coli (E. coli) and a member of the DnaQ superfamily. ExoI is also referred to as exodeoxyribonuclease I, exonuclease I, DNA deoxyribophosphodiesterase, and dRPase. Exemplary ExoI reference sequences for use in certain embodiments include, but are not limited to, NP_416515.1, NC_000913.3, WP_000980589.1, NZ_LN832404.1.

[0189] In preferred embodiments contemplated herein, the fusion polypeptide comprises a DNA binding domain, a homing endonuclease variant, and an ExoI exonuclease, or a biologically active fragment thereof. In various embodiments, the fusion polypeptide comprises a DNA binding domain and a homing endonuclease variant, or a biologically active fragment thereof, linked to the ExoI exonuclease by a linker domain (e.g., a polypeptide linker).

[0190] In various embodiments, ExoI comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:112. In some embodiments, ExoI comprises an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:112. In some embodiments, ExoI comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:112. In some embodiments, ExoI comprises an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO:112. In some embodiments, ExoI comprises an amino acid sequence having at least 96% identity to the amino acid sequence set forth in SEQ ID NO:112. In some embodiments, ExoI comprises an amino acid sequence having at least 97% identity to the amino acid sequence set forth in SEQ ID NO:112. In some embodiments, ExoI comprises an amino acid sequence having at least 98% identity to the amino acid sequence set forth in SEQ ID NO:112. In some embodiments, ExoI comprises an amino acid sequence having at least 99% identity to the amino acid sequence set forth in SEQ ID NO:112.

[0191] In a particular embodiment, ExoI, or a biologically active fragment thereof, comprises the amino acid sequence set forth in SEQ ID NO:112.

[0192] D. Target site Homing endonuclease variants contemplated in certain embodiments may be designed to bind to any suitable target sequence (e.g., sequences in the human genome) and may have novel binding specificity compared to naturally occurring nucleases. In certain embodiments, the target site is a regulatory region of a gene, including but not limited to a promoter, enhancer, repressor element, etc. In certain embodiments, the target site is a coding region of a gene or a splice site. In certain embodiments, the nuclease variant and donor repair template may be designed to insert a therapeutic polynucleotide. In certain embodiments, the nuclease variant and donor repair template may be designed to insert a therapeutic polynucleotide under the control of an endogenous gene regulatory element or expression control sequence. In various embodiments, the nuclease variant binds and cleaves a target sequence in an immune system checkpoint gene, a globin gene, a gene encoding a polypeptide that contributes to the suppression of gamma-globin gene expression and / or HbF, or an immunosuppressive signaling gene.

[0193] Illustrative examples of immune system checkpoint genes include, but are not limited to, PD-1, LAG-3, TIM-3, CTLA-4, BTLA, TIGIT, VISTA, and KIR.

[0194] Illustrative examples of genes encoding immunoinhibitory signaling components include, but are not limited to, IL-10Rα, TGFβR1, TGFβR2, AHR, SGK1, TSC2, VHL, A2AR, and CBL-B.

[0195] Illustrative examples of polypeptides that inhibit γ-globin gene expression and HbF include, but are not limited to, BCL11A, KLF1, SOX6, GATA1, and LSD1.

[0196] In various embodiments, the nuclease variants are selected from the group consisting of programmed cell death protein 1 (PD-1, PDCD1), lymphocyte activation gene 3 protein (LAG-3), T cell immunoglobulin domain and mucin domain protein 3 (TIM-3), cytotoxic T lymphocyte antigen-4 (CTLA-4), band T lymphocyte attenuator (BTLA), T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), V domain Ig inhibitor of T cell activation (VISTA), and killer cell immunoglobulin-like receptor (KIR). , CCR5, TRAC (TCRα), TCRβ, IL10Rα, IL10Rβ, TGFBR1, TGFBR2, CBL-B, PCSK9, AHR, BTK, α-globin, β-globin, γ-globin, BCL11A, KLF1, SOX6, GATA1, LSD, alpha folate receptor FRα, αvβ6 integrin, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase IX (CAIX), CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, carcinoembryonic antigen (CEA), C-type lectin-like molecule-1 (CLL-1), CD2 subset 1 (CS-1), chondroitin sulfate proteoglycan 4 (CSPG4), cutaneous T-cell lymphoma-associated antigen 1 (CTAGE1), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), epithelial cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EPHA), 2), fibroblast activation protein (FAP), Fc receptor-like 5 (FCRL5), fetal acetylcholinesterase receptor (AchR), ganglioside G2 (GD2), ganglioside G3 (GD3), glypican-3 (GPC3), EGFR family including ErbB2 (HER2), IL-11Rα, IL-13Rα2, kappa, cancer / testis antigen 2 (LAGE-1A), lambda, Lewis-Y (LeY), L1 cell adhesion molecule (L1-CAM), melanoma antigen gene (MAGE)-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10,Melanoma antigen recognized by T cells 1 (MelanA or MART1), mesothelin (MSLN), MUC1, MUC16, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), neural cell adhesion molecule (NCAM), cancer / testis antigen 1 (NY-ESO-1), polysialic acid; placenta-specific 1 (PLAC1), predominantly expressed antigen in melanoma (PRAME), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), glioma The antibody binds to and cleaves a target sequence in a gene selected from the group consisting of sarcoma sarcoma, X breakpoint 2 (SSX2), survivin, tumor-associated glycoprotein 72 (TAG72), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), TEM5, TEM8, trophoblast glycoprotein (TPBG), UL16-binding protein (ULBP) 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms' tumor 1 (WT-1) genes.

[0197] In certain embodiments, the target gene is selected from the group consisting of programmed cell death protein 1 (PD-1), lymphocyte activation gene 3 protein (LAG-3), T cell immunoglobulin domain and mucin domain protein 3 (TIM-3), cytotoxic T lymphocyte antigen-4 (CTLA-4), band T lymphocyte attenuator (BTLA), T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), V domain Ig inhibitor of T cell activation (VISTA), and killer cell immunoglobulin-like receptor (KIR), CCR5, TRAC (TCRα), IL10Rα, TGFBR2, CBL-B, PCSK9, AHR, BTK, α-globin, β-globin, γ-globin, and BCL11A genes.

[0198] In certain embodiments, the target gene is the TRAC (TCRα), CBL-B, or PDCD1 (PD-1) gene.

[0199] In various embodiments, the nuclease variant binds and cleaves a target sequence in the TRAC gene. The T-cell receptor alpha (TRAC) locus is a protein encoded by the TRA gene in humans. TRAC is also referred to as TRA, IMD7, TCRA, TRA@, TRAC, T-cell receptor alpha locus, TCRD, T-cell receptor alpha locus, TCRα. It contributes an alpha chain to the larger TCR protein (T-cell receptor). Alpha-beta T-cell receptors are antigen-specific receptors that are essential for immune responses and are present on the cell surface of T lymphocytes. They recognize peptide major histocompatibility (MH) (pMH) complexes presented by antigen-presenting cells (APCs) and are a prerequisite for efficient T-cell adaptive immunity against pathogens.

[0200] In a particular embodiment, the nuclease variant introduces a DSB in exon 1 of the constant region of the human TCR alpha gene, preferably at SEQ ID NO: 1 in exon 1 of the constant region of the human TCR alpha gene, and more preferably at the sequence "ATTC" in SEQ ID NO: 1 in exon 1 of the constant region of the human TCR alpha gene. In a preferred embodiment, the TCR alpha gene is the human TCR alpha gene.

[0201] In various embodiments, the homing endonuclease variant binds and cleaves a target sequence in the programmed death receptor 1 (PD-1) gene. PD-1 is also referred to as programmed cell death 1 (PDCD1), systemic lupus erythematosus susceptibility 2 (SLEB2), CD279, HPD1, PD1, HPD-L, and HSLE1. PD-1 is a member of the B7 / CD28 family of costimulatory receptors. The PD-1 molecule consists of an extracellular ligand-binding IgV domain, a transmembrane domain, and an intracellular domain with potential phosphorylation sites located at immunotyrosine-based inhibitory motifs (ITIMs) and immunoreceptor inhibitory tyrosine-based switch motifs (ITSMs). PD-1 is an inhibitory co-receptor expressed on T cells, Tregs, exhausted T cells, B cells, activated monocytes, dendritic cells (DCs), natural killer (NK) cells, and natural killer T (NKT) cells. PD-1 negatively regulates T cell activation by binding to its ligands, programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2). PD-1 binding inhibits T cell proliferation and interferon-γ (IFN-γ), tumor necrosis factor-α, and IL-2 production, reducing T cell survival. PD-1 expression is a hallmark of "exhausted" T cells that have experienced high levels of stimulation. This state of exhaustion, which occurs during chronic infection and cancer, is characterized by T cell dysfunction, resulting in suboptimal control of infection and tumors.

[0202] In a particular embodiment, the homing endonuclease variant introduces a double stranded break (DSB) in exon 1 of the PD-1 gene, preferably at SEQ ID NO: 2 in exon 1 of the PD-1 gene, and more preferably at the sequence "ATCC" at SEQ ID NO: 2 in exon 1 of the PD-1 gene. In a preferred embodiment, the homing endonuclease variant or megaTAL breaks double stranded DNA and introduces a DSB in the polynucleotide sequence shown in SEQ ID NO: 2. In a preferred embodiment, the PD-1 gene is the human PD-1 gene.

[0203] In various embodiments, the homing endonuclease variant binds and cleaves a target sequence in the human Casitas B lineage (Cbl) lymphoma proto-oncogene B (CBLB) gene. CBL is also referred to as CBL2; Noonan syndrome-like disorder with or without juvenile myelomonocytic leukemia (NSLL); C-CBL; RING finger protein 55 (RNF55); fragile site, folate type, rare, fra(11)(q23.3)(FRA11B); E3 ubiquitin-protein ligase CBL; Cas-Br-M (mouse) ecotropic retroviral transforming sequence; Cbl proto-oncogene, E3 ubiquitin protein ligase; RING-type E3 ubiquitin transferase CBL; Casitas B lineage lymphoma proto-oncogene; oncogene CBL2; proto-oncogene c-Cbl; and signal transduction protein CBL.

[0204] This gene is a proto-oncogene that encodes a RING finger E3 ubiquitin ligase. The encoded protein is one of the enzymes required to target substrates for degradation by the proteasome. This protein mediates the transfer of ubiquitin from ubiquitin conjugating enzymes (E2) to specific substrates. This protein also contains an N-terminal phosphotyrosine binding domain, which allows it to interact with and target a number of tyrosine-phosphorylated substrates for proteasomal degradation. CBLB functions as a negative regulator of many signaling pathways, including T cell activation and persistence.

[0205] In certain embodiments, the homing endonuclease variant introduces a double stranded break (DSB) at the target site of the CBLB gene. In certain embodiments, the homing endonuclease variant or megaTAL introduces a DSB in exon 6 of the CBLB gene, preferably at SEQ ID NO: 3 in exon 6 of the CBLB gene, and more preferably at the sequence "ATCC" in SEQ ID NO: 3 in exon 6 of the CBLB gene. In a preferred embodiment, the homing endonuclease variant breaks double stranded DNA and introduces a DSB at the polynucleotide sequence shown in SEQ ID NO: 3. In a preferred embodiment, the CBLB gene is a human CBLB gene.

[0206] E. Donor repair template A fusion polypeptide comprising a homing endonuclease variant may be used to introduce DSBs into a target sequence, which may be repaired via a homology-directed repair (HDR) mechanism in the presence of one or more donor repair templates. In certain embodiments, a donor repair template is used to insert a sequence into a genome. In particularly preferred embodiments, a donor repair template is used to insert a polynucleotide sequence encoding a therapeutic polypeptide. In particularly preferred embodiments, a donor repair template is used to insert a polynucleotide sequence encoding a therapeutic polypeptide such that expression of the polypeptide is under the control of an endogenous promoter and / or enhancer.

[0207] In various embodiments, the donor repair template is administered to hematopoietic cells, e.g., hematopoietic stem or progenitor cells, or CD34 cells by transducing the cells with an adeno-associated virus (AAV), retrovirus, e.g., lentivirus, IDLV, herpes simplex virus, adenovirus, or vaccinia virus vector that contains the donor repair template. + is introduced into cells.

[0208] In certain embodiments, the donor repair template comprises one or more homology arms flanking the DSB site.

[0209] As used herein, the term "homologous arm" refers to a nucleic acid sequence in the donor repair template that is identical or nearly identical to the DNA sequence adjacent to the DNA break introduced by the nuclease at the target site. In one embodiment, the donor repair template comprises a 5' homologous arm that comprises a nucleic acid sequence that is identical or nearly identical to the DNA sequence 5' of the DNA break site. In one embodiment, the donor repair template comprises a 3' homologous arm that comprises a nucleic acid sequence that is identical or nearly identical to the DNA sequence 3' of the DNA break site. In a preferred embodiment, the donor repair template comprises a 5' homologous arm and a 3' homologous arm. The donor repair template may comprise homology to a genomic sequence immediately adjacent to the DSB site or within any number of base pairs from the DSB site. In one embodiment, the donor repair template comprises a nucleic acid sequence that is homologous to a genomic sequence of about 5 bp, about 10 bp, about 25 bp, about 50 bp, about 100 bp, about 250 bp, about 500 bp, about 1000 bp, about 2500 bp, about 5000 bp, about 10000 bp or more, including any intervening lengths of homologous sequence.

[0210] Illustrative examples of suitable lengths of the homology arms contemplated in certain embodiments include, but are not limited to, about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp, about 1200 bp, about 1300 bp, about 1400 bp, and about 1600 bp, which may be independently selected, including all intervening lengths of the homology arms. Homologous arms include, but are not limited to, 0 bp, about 1500 bp, about 1600 bp, about 1700 bp, about 1800 bp, about 1900 bp, about 2000 bp, about 2100 bp, about 2200 bp, about 2300 bp, about 2400 bp, about 2500 bp, about 2600 bp, about 2700 bp, about 2800 bp, about 2900 bp, or about 3000 bp or more.

[0211] Additional illustrative examples of suitable homology arm lengths include, but are not limited to, about 100 bp to about 3000 bp, about 200 bp to about 3000 bp, about 300 bp to about 3000 bp, about 400 bp to about 3000 bp, about 500 bp to about 3000 bp, about 500 bp to about 2500 bp, about 500 bp to about 2000 bp, about 750 bp to about 2000 bp, about 750 bp to about 1500 bp, or about 1000 bp to about 1500 bp, including all intervening lengths of the homology arms.

[0212] In certain embodiments, the length of the 5' and 3' homology arms are independently selected from about 500 bp to about 1500 bp. In one embodiment, the 5' homology arm is about 1500 bp and the 3' homology arm is about 1000 bp. In one embodiment, the 5' homology arm is about 200 bp to about 600 bp and the 3' homology arm is about 200 bp to about 600 bp. In one embodiment, the 5' homology arm is about 200 bp and the 3' homology arm is about 200 bp. In one embodiment, the 5' homology arm is about 300 bp and the 3' homology arm is about 300 bp. In one embodiment, the 5' homology arm is about 400 bp and the 3' homology arm is about 400 bp. In one embodiment, the 5' homology arm is about 500 bp and the 3' homology arm is about 500 bp. In one embodiment, the 5' homology arm is about 600 bp and the 3' homology arm is about 600 bp.

[0213] F. Polypeptides A variety of polypeptides are contemplated herein, including but not limited to fusion polypeptides comprising homing endonuclease variants and / or megaTALs. In a preferred embodiment, the polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 7, 10, 13, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 89, 92, 95, 98, and 101. "Polypeptide," "polypeptide fragment," "peptide," and "protein" are used interchangeably in their conventional sense, i.e., as amino acid sequences, unless otherwise specified to the contrary. In one embodiment, "polypeptide" includes fusion polypeptides and other variants. Polypeptides may be prepared using any of a variety of well-known recombinant and / or synthetic techniques. Polypeptides are not limited to a particular length, for example, they can include full-length protein sequences, fragments of full-length proteins, or fusion proteins, and can include post-translational modifications of the polypeptides, such as glycosylation, acetylation, phosphorylation, and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.

[0214] As used herein, "isolated protein," "isolated peptide," or "isolated polypeptide," etc., refer to the in vitro synthesis, isolation, and / or purification of a peptide or polypeptide molecule from its cellular environment and from association with other components of a cell, i.e., not significantly associated with substances in vivo.

[0215] Illustrative examples of polypeptides contemplated in certain embodiments include, but are not limited to, homing endonuclease variants, megaTALs, terminal processing nucleases, exonucleases, fusion polypeptides, and variants thereof.

[0216] Polypeptides include "polypeptide variants". Polypeptide variants may differ from naturally occurring polypeptides in one or more amino acid substitutions, deletions, additions and / or insertions. Such variants may be naturally occurring or synthetically generated, for example, by modifying one or more amino acids of the above polypeptide sequences. For example, in certain embodiments, it may be desirable to improve the biological properties of fusion polypeptides, homing endonucleases, megaTALs, etc., that bind and cleave target sites by introducing one or more substitutions, deletions, additions and / or insertions into the polypeptide. In certain embodiments, polypeptides include those having at least about 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to any of the reference sequences contemplated herein, and typically the variants retain at least one biological activity of the reference sequence.

[0217] Polypeptide variants include biologically active "polypeptide fragments." Illustrative examples of biologically active polypeptide fragments include DNA binding domains, nuclease domains, endoprocessing domains (e.g., exonucleases), and the like. As used herein, the term "biologically active fragment" or "minimal biologically active fragment" refers to a polypeptide fragment that retains at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% of the naturally occurring polypeptide activity. In preferred embodiments, the biological activity is binding affinity and / or cleavage activity for a target sequence. In certain embodiments, the polypeptide fragment may comprise an amino acid chain of at least 5 to about 1700 amino acids in length. In certain embodiments, the fragment comprises at least 5, 6, 7, 8, 9, 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, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more amino acids in length. In certain embodiments, the polypeptide comprises a biologically active fragment of a homing endonuclease variant. In certain embodiments, the polypeptides described herein may comprise one or more amino acids designated as "X". "X" refers to any amino acid when present in an amino acid sequence. One or more "X" residues may be present at the N-terminus and C-terminus of the amino acid sequence set forth in a particular SEQ ID NO contemplated herein. When the "X" amino acid is not present, the remaining amino acid sequence set forth in the SEQ ID NO: may be considered a biologically active fragment.

[0218] In certain embodiments, the polypeptide comprises a biologically active fragment of a homing endonuclease variant, such as SEQ ID NO: 89, 92, 95, 98, or 101, or a megaTAL (e.g., SEQ ID NO: 7, 10, 58, 67, and 76). The biologically active fragment may comprise an N-terminal truncation and / or a C-terminal truncation. In certain embodiments, the biologically active fragment lacks or comprises a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 N-terminal amino acids of the homing endonuclease variant compared to the corresponding wild-type homing endonuclease sequence, and more preferably comprises a deletion of 4 N-terminal amino acids of the homing endonuclease variant compared to the corresponding wild-type homing endonuclease sequence. In certain embodiments, the biologically active fragment lacks or comprises a deletion of 1, 2, 3, 4, or 5 C-terminal amino acids of a homing endonuclease variant compared to the corresponding wild-type homing endonuclease sequence, and more preferably comprises a deletion of 2 C-terminal amino acids of a homing endonuclease variant compared to the corresponding wild-type homing endonuclease sequence. In certain preferred embodiments, the biologically active fragment lacks or comprises a deletion of 4 N-terminal amino acids and 2 C-terminal amino acids of a homing endonuclease variant compared to the corresponding wild-type homing endonuclease sequence.

[0219] In certain embodiments, the I-OnuI variant comprises a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 of the following N-terminal amino acids: M, A, Y, M, S, R, R, E, and / or a deletion of 1, 2, 3, 4, or 5 of the following C-terminal amino acids: R, G, S, F, V.

[0220] In certain embodiments, an I-OnuI variant comprises deletions or substitutions of 1, 2, 3, 4, 5, 6, 7, or 8 of the following N-terminal amino acids: M, A, Y, M, S, R, R, E, and / or deletions or substitutions of 1, 2, 3, 4, or 5 of the following C-terminal amino acids: R, G, S, F, V.

[0221] In certain embodiments, the I-OnuI variant comprises a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 of the following N-terminal amino acids: M, A, Y, M, S, R, R, E, and / or a deletion of 1 or 2 of the following C-terminal amino acids: F, V.

[0222] In certain embodiments, an I-OnuI variant comprises deletions or substitutions of 1, 2, 3, 4, 5, 6, 7, or 8 of the following N-terminal amino acids: M, A, Y, M, S, R, R, E, and / or deletions or substitutions of 1 or 2 of the following C-terminal amino acids: F, V.

[0223] As mentioned above, a polypeptide may be modified in various ways, including amino acid substitution, deletion, truncation, and insertion. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be generated by mutations in DNA. Methods for mutagenesis and nucleotide sequence modification are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82:488-492), Kunkel et al., (1987, Methods in Enzymol, 154:367-382), U.S. Patent No. 4,873,192, Watson, JD et al., (Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987), and references cited therein. Guidance regarding appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0224] In certain embodiments, the variant comprises one or more conservative substitutions. A "conservative substitution" is one in which an amino acid is replaced with another amino acid having similar properties, so that a person skilled in the art of peptide chemistry would expect that the secondary structure and hydropathic properties of the polypeptide will not be substantially altered. Modifications may be made in the polynucleotide and polypeptide structures contemplated in certain embodiments, including polypeptides that have at least approximately the functional molecule encoding a variant or derivative polypeptide with desired characteristics, yet still obtain. When it is desired to change the amino acid sequence of a polypeptide to generate an equivalent or improved variant polypeptide, a person skilled in the art can change one or more of the codons of the encoding DNA sequence, for example, according to Table 1. [Table 1]

[0225] Guidance for determining which amino acid residues may be substituted, inserted or deleted without abolishing biological activity can be found using computer programs well known in the art, such as DNASTAR, DNA Strider, Geneious, Mac Vector, or Vector NTI software. The amino acid changes in the protein variants disclosed herein are preferably conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes include substitutions of one of a family of amino acids that are related at their side chains. Naturally occurring amino acids are generally divided into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan and tyrosine are sometimes classified jointly as aromatic amino acids. Suitable conservative substitutions of amino acids in peptides or proteins are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide generally do not significantly alter biological activity (see, for example, Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0226] In one embodiment where expression of more than one polypeptide is desired, the polynucleotide sequences encoding them may be separated by an IRES sequence, as disclosed elsewhere herein.

[0227] In certain embodiments, contemplated polypeptides include fusion polypeptides, such as SEQ ID NOs: 7, 10, 13, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, and 82. In preferred embodiments, the fusion polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 46, 64, 73, and 82. In certain embodiments, fusion polypeptides and polynucleotides encoding the fusion polypeptides are provided. Fusion polypeptides and fusion proteins refer to polypeptides having at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more polypeptide segments.

[0228] In another embodiment, two or more polypeptides may be expressed as a fusion protein that contains one or more self-cleaving polypeptide sequences, as disclosed elsewhere herein.

[0229] In one embodiment, a fusion protein contemplated herein comprises one or more DNA binding domains and one or more nucleases, and one or more linker and / or self-cleaving polypeptides.

[0230] In one embodiment, a fusion protein contemplated herein comprises a nuclease variant, a linker or self-cleaving peptide, and a terminal processing enzyme, including, but not limited to, a 5' to 3' exonuclease, a 5' to 3' alkaline exonuclease, and a 3' to 5' exonuclease (e.g., Trex2, ExoI, or ExoX).

[0231] Fusion polypeptides may contain one or more polypeptide domains or segments, including, but not limited to, signal peptides, cell penetrating peptide domains (CPPs), DNA binding domains, nuclease domains, and the like, epitope tags (e.g., maltose binding protein ("MBP"), glutathione S-transferase (GST), HIS6, MYC, FLAG, V5, VSV-G, and HA), polypeptide linkers, and polypeptide cleavage signals. Fusion polypeptides are typically joined C-terminus to N-terminus, but they may also be joined C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. In certain embodiments, the polypeptides of the fusion protein may be in any order. Fusion polypeptides or fusion proteins may include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, provided that the desired activity of the fusion polypeptide is preserved. Fusion polypeptides may be made by chemical synthesis or by chemical conjugation between two moieties, or may be generally prepared using other standard methods. The ligated DNA sequence comprising the fusion polypeptide is operably linked to suitable transcriptional or translational control elements as disclosed elsewhere herein.

[0232] A fusion polypeptide may optionally include a linker that can be used to link one or more polypeptides or domains within the polypeptide. A peptide linker sequence may be utilized to separate any two or more polypeptide components by a distance sufficient to ensure that each polypeptide folds into its appropriate secondary and tertiary structure so that the polypeptide domains can perform their desired functions. Such peptide linker sequences are incorporated into the fusion polypeptide using standard techniques in the art. Suitable peptide linker sequences may be selected based on the following factors: (1) their ability to adopt a flexible extended conformation, (2) their inability to adopt a secondary structure that may interact with functional epitopes on the first and second polypeptides, and (3) their lack of hydrophobic or charged residues that may react with the polypeptide functional epitopes. Preferred peptide linker sequences include Gly, Asn, and Ser residues. Other near-neutral amino acids, such as Thr and Ala, may also be used in the linker sequence. Amino acid sequences that may be usefully employed as linkers include those described in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. No. 4,935,233; and U.S. Pat. No. 4,751,180. Linker sequences are not required if a particular fusion polypeptide segment contains a non-essential N-terminal amino acid region that can be used to separate functional domains and prevent steric interference. Preferred linkers are flexible amino acid subsequences that are typically synthesized as part of a recombinant fusion protein. Linker polypeptides may be 1-200 amino acids long, 1-100 amino acids long, or 1-50 amino acids long, including all integer values ​​between those values.

[0233] Exemplary linkers include, but are not limited to, the following amino acid sequences: glycine polymer (G) n , glycine-serine polymer (G 1-5 S 1-5 ) n(wherein n is an integer of at least 1, 2, 3, 4, or 5), glycine-alanine polymers, alanine-serine polymers, GGG (SEQ ID NO:113), DGGGS (SEQ ID NO:114), TGEKP (SEQ ID NO:115) (see, e.g., Liu et al., PNAS 5525-5530 (1997)), GGRR (SEQ ID NO:116) (Pomerantz et al. 1995, supra), (GGGGS) n (wherein n = 1, 2, 3, 4, or 5 (SEQ ID NO: 117 and 150-153)) (Kim et al., PNAS 93, 1156-1160 (1996.)), EGKSSGSGSESKVD (SEQ ID NO: 118) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87: 1066-1070), KESGSVSSEQLAQFRSLD (SEQ ID NO: 119) (Bird et al., 1988, Science 242: 423-426), GGRRGGGS (SEQ ID NO: 120), LRCRDGERP (SEQ ID NO: 121), LRQKDGGGSERP (SEQ ID NO: 122), LRQKD(GGGS)2ERP (SEQ ID NO: 123). Alternatively, flexible linkers can be rationally designed using computer programs that can model both the DNA binding site and the peptide itself (Desjarlais & Berg, PNAS 90:2256-2260 (1993), PNAS 91:11099-11103 (1994)), or by phage display methods.

[0234] The fusion polypeptide may further comprise a polypeptide cleavage signal between each of the polypeptide domains described herein, or between the endogenous open reading frame and the polypeptide encoded by the donor repair template. In addition, a polypeptide cleavage site can be inserted into any linker peptide sequence. Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites such as protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004.Traffic, 5(8);616-26).

[0235] Suitable protease cleavage sites and autocleaving peptides are known to those skilled in the art (see, for example, Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Examples of protease cleavage sites include, but are not limited to, cleavage sites for potyvirus NIa protease (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus P1 (P35) protease, byovirus NIa protease, biovirus RNA-2-encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (Waika virus) 3C-like protease, PYVF (Parsnip yellow mottle virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. Due to its high cleavage stringency, in one embodiment, the TEV (Tobacco Etch Virus) protease cleavage site is preferred, e.g., EXXYXQ(G / S) (SEQ ID NO: 121), e.g., ENLYFQG (SEQ ID NO: 122) and ENLYFQS (SEQ ID NO: 123) (X represents any amino acid) (cleavage by TEV occurs between Q and G or Q and S).

[0236] In certain embodiments, the self-cleaving polypeptide site comprises a 2A or 2A-like site, sequence or domain (Donnelly et al., 2001. J. Gen. Virol. 82:1027-1041). In certain embodiments, the viral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide.

[0237] In one embodiment, the viral 2A peptide is selected from the group consisting of a foot and mouth disease virus (FMDV) 2A peptide, an equine rhinitis A virus (ERAV) 2A peptide, a zosea signa virus (TaV) 2A peptide, a porcine teschovirus-1 (PTV-1) 2A peptide, a tylovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.

[0238] Illustrative examples of 2A sites are provided in Table 2. [Table 2]

[0239] G. Polynucleotides In certain embodiments, polynucleotides are provided that encode one or more of the fusion polypeptides, homing endonuclease variants, megaTALs, terminal processing enzymes, and exonucleases contemplated herein. As used herein, the term "polynucleotide" or "nucleic acid" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and DNA / RNA hybrids.

[0240] In various embodiments, the polynucleotide encodes one or more fusion polypeptides, such as SEQ ID NOs: 5, 8, 11, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, and 80. In some embodiments, the polynucleotide encodes one or more fusion polypeptides is an RNA polynucleotide. In some embodiments, the RNA polynucleotide encodes one or more fusion polypeptides comprises any one of the sequences set forth in SEQ ID NOs: 6, 9, 12, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, and 81. In some embodiments, the polynucleotide encodes an amino acid sequence set forth in any one of SEQ ID NOs: 7, 10, 13, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, and 82.

[0241] Polynucleotides may be single-stranded or double-stranded and may be either recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to, pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), synthetic RNA, synthetic mRNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, and recombinant DNA. Polynucleotide refers to a polymeric form of nucleotides at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, or at least 15000 or more nucleotides in length, either ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide, as well as all intermediate lengths. In the present context, it will be readily understood that "intermediate length" means any length between the recited values, e.g., 6, 7, 8, 9, etc., 101, 102, 103, etc., 151, 152, 153, etc., 201, 202, 203, etc. In certain embodiments, the polynucleotide or variant has at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference sequence.

[0242] In certain embodiments, a polynucleotide may be codon optimized. As used herein, the term "codon optimization" refers to the substitution of codons in a polynucleotide encoding a polypeptide to increase expression, stability, and / or activity of the polypeptide. Factors that influence codon optimization include one or more of the following, but are not limited to: (i) variation in codon bias between two or more organisms or genes or synthetically constructed bias tables, (ii) variation in the degree of codon bias within an organism, gene or set of genes, (iii) systematic variation of a codon with its context, (iv) variation of a codon with its decoding tRNA, (v) variation of a codon with the GC % either in the entire triplet or in one position of the triplet, (vi) variation in similarity to a reference sequence, e.g., a natural sequence, (vii) variation in the codon frequency cutoff, (viii) structural properties of the mRNA transcribed from the DNA sequence, (ix) prior knowledge of the function of the DNA sequence on which the design of the codon substitution set is based, and / or (x) synthetic variation of the codon set for each amino acid, and / or (xi) isolated elimination of incorrect translation start positions.

[0243] As used herein, the term "nucleotide" refers to a heterocyclic nitrogenous base in N-glycosidic linkage with a phosphorylated sugar. Nucleotides are understood to include natural bases and a wide variety of modified bases recognized in the art. Such bases are typically located at the 1' position of the nucleotide sugar moiety. Nucleotides generally include a base, a sugar, and a phosphate group. In ribonucleic acid (RNA), the sugar is ribose, and in deoxyribonucleic acid (DNA), the sugar is deoxyribose, i.e., deoxyribose is a sugar lacking the hydroxyl group present in ribose. Examples of natural nitrogenous bases include the purines adenosine (A) and guanidine (G), and the pyrimidines cytidine (C) and thymidine (T) (or uracil (U) in the case of RNA). The C-1 atom of deoxyribose is linked to the N-1 of a pyrimidine or the N-9 of a purine. Nucleotides are usually mono-, di-, or triphosphates. Nucleotides may be unmodified or modified at the sugar, phosphate, and / or base moieties (interchangeably referred to as nucleotide analogs, nucleotide derivatives, modified nucleotides, non-natural nucleotides, and non-standard nucleotides, see, for example, WO92 / 07065 and WO93 / 15187). Examples of modified nucleobases are summarized by Limbach et al., (1994, Nucleic Acids Res. 22, 2183-2196).

[0244] Nucleotides may also be considered as phosphate esters of nucleosides, with esterification occurring on the hydroxyl group attached to C-5 of the sugar. As used herein, the term "nucleoside" refers to a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar. Nucleosides are recognized in the art to include natural bases, as well as modified bases, which are well known. Such bases are typically located at the 1' position of the nucleoside sugar moiety. Nucleosides generally include a base and a sugar. Nucleosides may be unmodified or modified at the sugar and / or base moieties (interchangeably referred to as nucleoside analogs, nucleoside derivatives, modified nucleosides, non-natural nucleosides, or non-standard nucleosides). As mentioned above, examples of modified nucleobases are summarized by Limbach et al., (1994, Nucleic Acids Res. 22, 2183-2196).

[0245] Illustrative examples of polynucleotides include SEQ ID NOs: 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 89, 92, 95, 98, and 101, as well as SEQ ID NOs: 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 89, 92, 95, 98, and 101. Polynucleotides encoding the polynucleotide sequences set forth in any of the preceding claims, but are not limited to, those encoding the polynucleotide sequences set forth in any of the following claims: 3, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80, 81, 87, 88, 90, 91, 93, 94, 96, 97, 99, and 100.

[0246] In various exemplary embodiments, polynucleotides contemplated herein include, but are not limited to, polynucleotides encoding fusion polypeptides, homing endonuclease variants, megaTALs, terminal processing enzymes, exonucleases, and expression vectors, viral vectors, and transfer plasmids that comprise the polynucleotides contemplated herein.

[0247] As used herein, the terms "polynucleotide variant" and "variant" and the like refer to a polynucleotide that exhibits a comparable sequence identity with a reference polynucleotide sequence or hybridizes with a reference sequence under stringent conditions as defined herein. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion, substitution, or modification of at least one nucleotide. Thus, the terms "polynucleotide variant" and "variant" include polynucleotides in which one or more nucleotides are added or deleted, or modified or replaced with another nucleotide. In this regard, it is well understood in the art that certain modifications, including mutations, additions, deletions, and substitutions, can be made to a reference polynucleotide, and the modified polynucleotide can retain the biological function or biological activity of the reference polynucleotide.

[0248] In one embodiment, the polynucleotide comprises a nucleotide sequence that hybridizes to a target nucleic acid sequence under stringent conditions. To hybridize under "stringent conditions", a hybridization protocol is described in which nucleotide sequences that are at least 60% identical to each other remain hybridized. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) of a particular sequence at a defined ionic strength and pH. Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes that are complementary to the target sequence hybridize to the target sequence at equilibrium. The target sequence is generally present in excess, so that at Tm, 50% of the probes are occupied at equilibrium.

[0249] "Sequence identity", or phrases such as "a sequence 50% identical to", as used herein, refers to the degree to which sequences are identical nucleotide-by-nucleotide or amino acid-by-amino acid over a comparison window. Thus, "percentage of sequence identity" may be calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions where identical nucleobases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein, and typically, a polypeptide variant retains at least one biological activity of the reference polypeptide.

[0250] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity." A "reference sequence" comprises nucleotides and amino acid residues that are at least 12 monomeric units in length, and often 15-18 monomeric units in length, and often at least 25 monomeric units in length. Two polynucleotides may each contain (1) sequences that are similar between the two polynucleotides (i.e., only a portion of the complete polynucleotide sequence), and (2) sequences that diverge between the two polynucleotides, and sequence comparison between two (or more) polynucleotides is typically performed by comparing the sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 consecutive positions, usually about 50 to about 100, more usually about 100 to about 150, in which a sequence is compared to a reference sequence at the same number of consecutive positions after the two sequences are optimally aligned. The comparison window may contain no more than about 20% additions or deletions (i.e., gaps) compared to the reference sequence (not including additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences to align the comparison window may be performed by computerized implementation of algorithms (GAP, BESTFIT, FASTA, and TFASTA) in Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, or by inspection and best alignment (i.e., resulting in the highest homology over the comparison window) generated by any of a variety of selected methods. Reference may also be made to the BLAST family of programs disclosed, for example, by Altschul et al., 1997, Nucl. Acids Res. 25:3389.A detailed discussion of sequence analysis can be found in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994-1998, Chapter 15, Unit 19.3.

[0251] As used herein, an "isolated polynucleotide" refers to a polynucleotide that has been purified from sequences that flank it in its naturally occurring state, e.g., a DNA fragment that has been removed from sequences that normally flank the fragment. In certain embodiments, an "isolated polynucleotide" refers to a complementary DNA (cDNA), recombinant polynucleotide, synthetic polynucleotide, or other polynucleotide that is not found in nature and has been created by the hand of man.

[0252] In various embodiments, the polynucleotide comprises an mRNA encoding a polypeptide contemplated herein, including but not limited to a fusion polypeptide, a homing endonuclease variant, a megaTAL, a terminal processing enzyme, and an exonuclease. In certain embodiments, the mRNA comprises a cap, one or more nucleotides and / or modified nucleotides, and a poly(A) tail.

[0253] In certain embodiments, the mRNAs contemplated herein comprise a poly(A) tail to help protect the mRNA from exonuclease degradation, stabilize the mRNA, and facilitate translation. In certain embodiments, the mRNA comprises a 3' poly(A) tail structure.

[0254] In certain embodiments, the length of the poly(A) tail is at least about 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or at least about 500 or more adenine nucleotides, or any intervening number of adenine nucleotides. 8, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 300, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 35 9, 200, 201, 202, 202, 203, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 30 40, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, or 275 or more adenine nucleotides.

[0255] In certain embodiments, the length of the poly(A) tail is about 10 to about 500 adenine nucleotides, about 50 to about 500 adenine nucleotides, about 100 to about 500 adenine nucleotides, about 150 to about 500 adenine nucleotides, about 200 to about 500 adenine nucleotides, about 250 to about 500 adenine nucleotides, about 300 to about 500 adenine nucleotides, about 50 to about 450 adenine nucleotides, about 50 to about 500 adenine nucleotides, about 60 to about 650 adenine nucleotides, about 70 to about 750 adenine nucleotides, about 80 to about 850 adenine nucleotides, about 90 to about 950 adenine nucleotides, about 100 to about 500 adenine nucleotides, about 150 to about 500 adenine nucleotides, about 200 to about 500 adenine nucleotides, about 250 to about 500 adenine nucleotides, about 300 to about 500 adenine nucleotides, about 50 to about 450 adenine nucleotides, about 50 to about 500 adenine nucleotides, about 60 to about 750 adenine nucleotides, about 70 to about 850 adenine nucleotides, about 80 to about 950 adenine nucleotides, about 90 to about 1000 adenine nucleotides, about 100 to about 1000 adenine nucleotides, about 150 to about 500 adenine nucleotides, about 200 to about 500 adenine nucleotides, about 250 to about 500 adenine nucleotides, about 300 nucleotides, about 50 to about 400 adenine nucleotides, about 50 to about 350 adenine nucleotides, about 100 to about 500 adenine nucleotides, about 100 to about 450 adenine nucleotides, about 100 to about 400 adenine nucleotides, about 100 to about 350 adenine nucleotides, about 100 to about 300 adenine nucleotides, about 150 to about 500 adenine nucleotides, about 150 to about 450 adenine nucleotides, about 150 to about 400 adenine nucleotides, about 150 to about 350 adenine nucleotides, about 150 to about 300 adenine nucleotides, about 150 to about 250 adenine nucleotides, about 150 to about 200 adenine nucleotides, about 200 to about 500 adenine nucleotides, about 200 to about 450 adenine nucleotides, about 200 to about 400 adenine nucleotides nucleotides, about 200 to about 350 adenine nucleotides, about 200 to about 300 adenine nucleotides, about 250 to about 500 adenine nucleotides, about 250 to about 450 adenine nucleotides, about 250 to about 400 adenine nucleotides, about 250 to about 350 adenine nucleotides, or about 250 to about 300 adenine nucleotides, or any intervening range of adenine nucleotides.

[0256] Terms describing the orientation of a polynucleotide include 5' (usually the end of a polynucleotide having a free phosphate group) and 3' (usually the end of a polynucleotide having a free hydroxyl (OH) group). A polynucleotide sequence may be annotated in a 5' to 3' orientation or a 3' to 5' orientation. For DNA and mRNA, the 5' to 3' strand is designated the "sense", "plus", or "coding" strand because its sequence is identical to that of the pre-messenger (pre-mRNA) [except for uracil (U) in RNA instead of thymine (T) in DNA]. For DNA and mRNA, the complementary 3' to 5' strand, which is the strand transcribed by RNA polymerase, is designated as the "template", "antisense", "minus", or "non-coding" strand. As used herein, the term "reverse orientation" refers to a 5' to 3' sequence written in a 3' to 5' orientation or a 3' to 5' sequence written in a 5' to 3' orientation.

[0257] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the complementary strand of the DNA sequence 5'AGTCATG 3' is 3'TCAGTAC 5'. The latter sequence is often written as the reverse complement, 5'CATGACT 3', with the 5' end on the left and the 3' end on the right. A sequence equivalent to its reverse complement is said to be a palindromic sequence. Complementarity can be "partial," where only a portion of the nucleic acid bases match according to the base-pairing rules. Alternatively, there can be "complete" or "total" complementarity between nucleic acids.

[0258] As used herein, the term "nucleic acid cassette" or "expression cassette" refers to a genetic sequence in a vector capable of expressing an RNA and subsequently expressing a polypeptide. In one embodiment, the nucleic acid cassette comprises a gene of interest, e.g., a polynucleotide of interest. In another embodiment, the nucleic acid cassette comprises one or more expression control sequences, e.g., a promoter, an enhancer, a poly(A) sequence, and a gene of interest, e.g., a polynucleotide of interest. A vector may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleic acid cassettes. The nucleic acid cassettes are positionally and sequentially oriented within the vector, such that the nucleic acid in the cassette can be transcribed into RNA, translated into a protein or polypeptide if necessary, subjected to appropriate post-translational modifications required for activity in a transformed cell, targeted to an appropriate intracellular compartment for transfer to a compartment suitable for biological activity, or secreted into an extracellular compartment. Preferably, the cassette has its 3' and 5' ends adapted for preparation for insertion into a vector, e.g., it has a restriction endonuclease site at each end. In a preferred embodiment, the nucleic acid cassette contains the sequence of a therapeutic gene used to treat, prevent, or ameliorate a genetic disorder. The cassette can be extracted and inserted as a single unit into a plasmid or viral vector.

[0259] Polynucleotides include polynucleotides of interest. As used herein, the term "polynucleotide of interest" refers to a polynucleotide that encodes a polypeptide or a fusion polypeptide, or serves as a template for transcription of an inhibitory polynucleotide, as contemplated herein.

[0260] Furthermore, those skilled in the art will understand that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that can code for fragments of the polypeptides or variants thereof contemplated herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage, such as polynucleotides optimized for human and / or primate codon preferences, are specifically contemplated in certain embodiments. In one embodiment, a polynucleotide is provided that comprises a specific allele sequence. An allele is an endogenous polynucleotide sequence that is altered as a result of one or more mutations, such as deletion, addition, and / or substitution of nucleotides.

[0261] In certain embodiments, the polynucleotide of interest comprises a donor repair template.

[0262] Polynucleotides contemplated in certain embodiments may be combined with other DNA sequences, regardless of the length of the coding sequence itself, and include promoters and / or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), stop codons, transcription termination signals, post-transcriptional response elements, such as woodchuck hepatitis virus post-transcriptional response element (WPRE), hepatitis B virus post-transcriptional response element (HPRE), and polynucleotides encoding self-cleaving polypeptides, epitope tags, as disclosed elsewhere herein or known in the art, whereby the total length may vary widely. Thus, in certain embodiments, polynucleotide fragments of almost any length may be used, with the total length preferably being limited by the ease of preparation and use in the recombinant DNA protocol contemplated.

[0263] Polynucleotides may be prepared, manipulated, expressed, and / or delivered using any of a variety of established techniques known and available in the art. To express a desired polypeptide, a nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector. A desired polypeptide can also be expressed by delivering mRNA encoding the polypeptide into a cell.

[0264] Illustrative examples of vectors include, but are not limited to, plasmids, autonomously replicating sequences, and transposable elements, such as Sleeping Beauty, PiggyBac, and the like.

[0265] Additional illustrative examples of vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses.

[0266] Illustrative examples of viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), pox viruses, baculoviruses, papilloma viruses, and papova viruses (e.g., SV40).

[0267] Illustrative examples of expression vectors include, but are not limited to, pClneo vector (Promega) for expression in mammalian cells, pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In certain embodiments, coding sequences for the polypeptides disclosed herein may be ligated into such expression vectors for expression of the polypeptides in mammalian cells.

[0268] In certain embodiments, the vector is an episomal vector, or a vector that is maintained extrachromosomally. As used herein, the term "episomal" refers to a vector that can replicate without integrating into the host chromosomal DNA and without being gradually lost with the division of the host cell, which also means that the vector replicates extrachromosomally or episomally.

[0269] "Expression control sequences," "control elements," or "regulatory sequences" present in an expression vector are the vector's origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno or Kozak sequences), introns, post-transcriptional regulatory elements, polyadenylation sequences, untranslated regions of the 5' and 3' untranslated regions that interact with host cell proteins to effect transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous and inducible promoters, may be used.

[0270] In certain embodiments, polynucleotides include vectors, including, but not limited to, expression vectors and viral vectors. Vectors may contain one or more exogenous, endogenous, or heterologous control sequences, such as promoters and / or enhancers. An "endogenous control sequence" is a sequence that is naturally linked to a given gene in the genome. An "exogenous control sequence" is a sequence that is placed in juxtaposition to a gene by genetic engineering means (i.e., molecular biology techniques), whose transcription is induced by its linked enhancer / promoter. A "heterologous control sequence" is an exogenous sequence that originates from a different species than the cell being genetically engineered. A "synthetic" control sequence may contain one or more endogenous and / or exogenous sequence elements and / or in vitro or in silico determined sequence elements that provide optimal promoter and / or enhancer activity for a particular therapy.

[0271] As used herein, the term "promoter" refers to a recognition site in a polynucleotide (DNA or RNA) to which an RNA polymerase binds. The RNA polymerase initiates and transcribes a polynucleotide operably linked to the promoter. In certain embodiments, a promoter operated in a mammalian cell contains an AT-rich region located approximately 25-30 bases upstream from the site where transcription is initiated, and / or another sequence found 70-80 bases upstream from the start of transcription is a CNCAAT region, where N can be any nucleotide.

[0272] The term "enhancer" refers to a DNA segment that contains a sequence that can provide enhanced transcription, and in some cases can function regardless of orientation relative to another control sequence. Enhancers can function in concert with or additively with a promoter and / or other enhancer elements. The term "promoter / enhancer" refers to a DNA segment that contains a sequence that can provide both promoter and enhancer functions.

[0273] The term "operably linked" refers to a juxtaposition where the components described are in a relationship permitting them to function in their intended manner. In one embodiment, the term refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter and / or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide of interest, where the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0274] As used herein, the term "structural expression control sequence" refers to a promoter, enhancer, or promoter / enhancer that allows for continuous or sequential transcription of an operably linked sequence. A constitutive expression control sequence may be a "ubiquitous" promoter, enhancer, or promoter / enhancer that allows for expression in a wide variety of cell and tissue types, or it may be a "cell-specific," "cell type-specific," "cell lineage," or "tissue-specific" promoter, enhancer, or promoter / enhancer that allows for expression in a restricted variety of cell and tissue types, respectively.

[0275] Exemplary ubiquitous expression control sequences suitable for use in certain embodiments include the cytomegalovirus (CMV) immediate early promoter, the viral Simian Virus 40 (SV40) (e.g., early or late), Moloney Murine Leukemia Virus (MoMLV) LTR promoter, Rous Sarcoma Virus (RSV) LTR, Herpes Simplex Virus (HSV) (thymidine kinase) promoter, the H5, P7.5 and P11 promoters from vaccinia virus, the short elongation factor 1 alpha (EF1a-short) promoter, the long elongation factor 1 alpha (EF1a-long) promoter, early growth response protein 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde triphosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa beta, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), β-kinesin (β-KIN), human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, β-actin promoter and myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site replacement (MND) promoter (Challita et al., J Virol. 69 (2): 748-55 (1995)), but are not limited to these.

[0276] In certain embodiments, it may be desirable to use cell-, cell type-, cell lineage-, or tissue-specific expression control sequences to achieve cell type-, cell lineage-, or tissue-specific expression of a desired polynucleotide sequence (e.g., expressing a nucleic acid encoding a particular polypeptide only in a subset of cell types, cell lineages, or tissues, or only at a particular stage of development).

[0277] As used herein, "conditional expression" may refer to any type of conditional expression, including, but not limited to, inducible expression, repressible expression, expression in cells or tissues having a particular physiological, biological or disease state, etc. This definition is not intended to exclude cell type-specific or tissue-specific expression. Certain embodiments provide for conditional expression of a polynucleotide of interest, expression of which is controlled, for example, by subjecting a cell, tissue, organism, etc. to a treatment or condition that causes expression of the polynucleotide, or that causes increased or decreased expression of a polynucleotide encoded by the polynucleotide of interest.

[0278] Illustrative examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters, such as promoters of genes encoding glucocorticoid receptors or estrogen receptors (inducible by treatment with the corresponding hormones), metallothionein promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible by interferon), the "GeneSwitch" mifepristone-regulated system (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switches (WO2002 / 088346), tetracycline-dependent regulatory systems, and the like.

[0279] Conditional expression can also be achieved by using site-specific DNA recombinases. According to certain embodiments, the polynucleotide comprises at least one (typically two) site for recombination mediated by a site-specific recombinase. As used herein, the term "recombinase" or "site-specific recombinase" includes excision or integration proteins, enzymes, cofactors or associated proteins involved in a recombination reaction involving one or more recombination sites (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more), which may be wild-type proteins (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or mutants, derivatives (e.g., fusion proteins comprising the recombination protein sequence or fragments thereof), fragments and variants thereof. Illustrative examples of recombinases suitable for use in certain embodiments include, but are not limited to, Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, ΦC31, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCE1, and ParA.

[0280] The polynucleotide may contain one or more recombination sites for any of a wide range of site-specific recombinases. It should be understood that the target site for the site-specific recombinase is in addition to any site required for integration of the vector, for example, a retroviral or lentiviral vector. As used herein, the term "recombination sequence", "recombination site" or "site-specific recombination site" refers to a specific nucleic acid sequence that a recombinase recognizes and binds to.

[0281] In certain embodiments, the polynucleotides contemplated herein include one or more polynucleotides of interest that encode one or more polypeptides. In certain embodiments, to achieve efficient translation of each of the multiple polypeptides, the polynucleotide sequences may be separated by one or more IRES sequences or polynucleotide sequences that encode self-cleaving polypeptides.

[0282] As used herein, "internal ribosome entry site" or "IRES" refers to an element that promotes direct entry of an internal ribosome into an initiation codon, such as ATG, of a cistron (protein coding region), resulting in cap-independent gene translation. See, e.g., Jackson et al., 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA 1(10):985-1000. Examples of IRES commonly used by those of skill in the art include those described in U.S. Patent No. 6,692,736. Further examples of "IRES" known in the art include, but are not limited to, IRES obtainable from picornaviruses (Jackson et al., 1990) and IRES obtainable from viral or cellular mRNA sources, such as, for example, immunoglobulin heavy chain binding protein (BiP), vascular endothelial growth factor (VEGF) (Huez et al. 1998. Mol. Cell. Biol. 18(11):6178-6190), fibroblast growth factor 2 (FGF-2), and insulin-like growth factor (IGFII), the translation initiation factor eIF4G, and the yeast transcription factors TFIID and HAP4, the encephalomyocarditis virus (EMCV) available commercially from Novagen (Duke et al., 1992. J. Virol 66(3):1602-9) and the VEGF IRES (Huez et al., 1998. Mol Cell Biol 18(11):6178-90). IRESes have also been reported in the viral genomes of species from the Picornaviridae, Dicistroviridae, and Flaviviridae families, as well as in HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV).

[0283] In certain embodiments, the polynucleotide comprises a polynucleotide having a consensus Kozak sequence and encoding a desired polypeptide. As used herein, the term "Kozak sequence" refers to a short nucleotide sequence that greatly promotes initial binding of mRNA to the small subunit of the ribosome and increases translation. The consensus Kozak sequence is (GCC)RCCATGG (SEQ ID NO: 149), where R is a purine (A or G) (Kozak, 1986. Cell. 44(2):283-92, and Kozak, 1987. Nucleic Acids Res. 15(20):8125-48).

[0284] Elements that direct efficient termination and polyadenylation of heterologous nucleic acid transcripts increase expression of heterologous genes. Transcription termination signals are generally found downstream of polyadenylation signals. In certain embodiments, the vector includes a polyadenylation sequence 3' of the polynucleotide encoding the expressed polypeptide. As used herein, the term "polyA site" or "polyA sequence" refers to a DNA sequence that directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by adding a polyA tail to the 3' end of the coding sequence, thus contributing to improved translation efficiency. Cleavage and polyadenylation are directed by poly(A) sequences in the RNA. The core poly(A) sequence of mammalian pre-mRNA has two recognition elements flanking the cleavage polyadenylation site. Typically, a nearly invariant AAUAAA hexamer is present 20-50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the initial transcript occurs between these two elements, adding up to 250 adenosines to the 5' cleavage product. In certain embodiments, the core poly(A) sequence is a polyA sequence of choice (e.g., AATAAA, ATTAAA, AGTAAA). In certain embodiments, the poly(A) sequence is the SV40 polyA sequence, bovine growth hormone polyA sequence (BGHpA), rabbit β-globin polyA sequence (rβgpA), variants thereof, or another suitable heterologous or endogenous polyA sequence known in the art.

[0285] In certain embodiments, a polynucleotide encoding one or more fusion polypeptides, homing endonuclease variants, megaTALs, terminal processing enzymes, or exonucleases is delivered to hematopoietic cells, e.g., CD34 cells, by both non-viral and viral methods. + In certain embodiments, delivery of one or more polynucleotides encoding a nuclease and / or a donor repair template can be provided by the same method or by different methods and / or by the same vector or by different vectors.

[0286] The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally inserted, for example, into a vector nucleic acid molecule. The vector may contain sequences that direct autonomous replication in a cell, or may contain sequences sufficient to allow integration into host cell DNA. In certain embodiments, non-viral vectors are used to deliver one or more of the polynucleotides contemplated herein to CD34. + Deliver to cells.

[0287] Illustrative examples of non-viral vectors include, but are not limited to, plasmids (eg, DNA or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.

[0288] Exemplary methods of non-viral delivery of polynucleotides contemplated in certain embodiments include, but are not limited to, electroporation, sonoporation, lipofection, microinjection, biolistec, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipid:nucleic acid complexes, naked DNA, artificial virions, DEAE-dextran mediated transfer, gene guns, and heat shock.

[0289] Illustrative examples of polynucleotide delivery systems suitable for use in certain contemplated embodiments include, but are not limited to, systems provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. Lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides are described in the literature. See, for example, Liu et al. (2003) Gene Therapy. 10:180-187, and Balazs et al. (2011) Journal of Drug Delivery. 2011:1-12. Antibody-targeted delivery, bacteria-guided delivery, and non-biological nanocell-based delivery are also contemplated in certain embodiments.

[0290] In certain embodiments, polynucleotides (e.g., DNA or RNA) encoding the fusion polypeptides contemplated herein can be directly introduced into cells, for example, by electroporation. In the electroporation method, polynucleotides, or a complex of site-specific polypeptides and polynucleotides, are mixed with target cells in an electroporation buffer to form a suspension. This suspension is then subjected to an electric pulse at an optimized voltage, which generates temporary pores in the phospholipid bilayer of the cell membrane, allowing charged molecules such as DNA and proteins to be driven through the pores into the cells. Reagents and devices for performing electroporation are commercially available. The electroporation medium can be any suitable medium known in the art. Suitable methods of electroporation are available and known to those skilled in the art.

[0291] Viral vectors comprising polynucleotides contemplated in certain embodiments may be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous or intracranial infusion) or local application as described below. Alternatively, vectors may be delivered ex vivo to cells, such as, for example, cells explanted from an individual patient (e.g., mobilized peripheral blood, lymphocytes, bone marrow aspirates, tissue biopsies, etc.) or hematopoietic stem cells of a universal donor, followed by reimplantation of the cells into the patient.

[0292] In one embodiment, viral vectors containing nuclease variants and / or donor repair templates are directly administered to organisms for transduction of cells in vivo. Alternatively, naked DNA or mRNA may be administered. Administration is by any route that is normally used to introduce molecules into blood or tissue cells for final contact, including, but not limited to, injection, infusion, topical application, and electroporation. Suitable methods for administering such nucleic acids are available and well known to those skilled in the art, and although a particular composition can be administered using more than one route, a particular route may often provide a more immediate and more effective response than another route.

[0293] Illustrative examples of viral vector systems suitable for use in certain embodiments contemplated herein include, but are not limited to, adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors.

[0294] H. Genome-edited cells Genome-edited cells produced and / or edited by the contemplated methods are provided in certain embodiments. Genome-edited cells contemplated in certain embodiments may be autogeneic (self) or non-autologous ("non-self", e.g., allogeneic, syngeneic, or xenogeneic). As used herein, "autogeneic" refers to cells derived from the same subject. As used herein, "allogeneic" refers to cells of the same species that are genetically different from the compared cell. As used herein, "syngeneic" refers to cells of a different subject that are genetically identical to the compared cell. As used herein, "xenogeneic" refers to cells of a different species from the compared cell. In a preferred embodiment, the cells are obtained from a mammalian subject. In a more preferred embodiment, the cells are obtained from a primate subject, optionally a non-human primate. In a most preferred embodiment, the cells are obtained from a human subject.

[0295] An "isolated cell" refers to a cell obtained from an in vivo tissue or organ and substantially free of extracellular matrix, that is not naturally occurring, e.g., a non-naturally occurring cell, a modified cell, an engineered cell, etc.

[0296] In certain embodiments, cells are edited by a fusion polypeptide contemplated herein, e.g., a fusion polypeptide comprising a DNA-binding domain and a homing endonuclease (HE) variant that binds and cleaves a selected double-stranded DNA (dsDNA) target site in the cell, a polypeptide linker, and an exonuclease, or a biologically active fragment thereof.

[0297] In various embodiments, the cells contain directionally biased deletions induced by the fusion polypeptides contemplated herein. In some embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the directionally biased deletions have a deletion center position on one side of the HE target site center position. In some embodiments, the deletion center position is on the same side as the DNA binding domain target site relative to the center position of the HE target site. In some embodiments, the deletion center position is 5' to the center position of the HE target site.

[0298] In various embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the deletions have a deletion center that is more than 4 nucleotides away from the central position of the HE target site, hi various embodiments, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or at least 35% of the deletions have a deletion center that is more than 8 nucleotides away from the central position of the HE target site.

[0299] In various embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the deletions are 6 bp or more in length. In various embodiments, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 40%, at least 55%, or at least 60% of the deletions are 12 bp or more in length. In certain embodiments, the directionally biased deletions comprise about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length.

[0300] In various embodiments, the deletion extends within the DNA-binding domain target site. In some embodiments, the central position of the deletion is within the DNA-binding domain target site.

[0301] Illustrative examples of cell types whose genomes may be edited using the compositions and methods contemplated herein include, but are not limited to, cell lines, primary cells, stem cells, progenitor cells, and differentiated cells.

[0302] The term "stem cell" refers to a cell that is an undifferentiated cell capable of (1) long-term self-renewal, or the generation of at least one identical copy of the original cell, (2) differentiation at the single cell level into multiple, and in some cases, one specialized cell type, and (3) functional regeneration of tissue in vivo. Stem cells are subclassified as totipotent, pluripotent, multipotent, and oligo / unipotent depending on their developmental potential. "Self-renewal" refers to a cell that has the inherent ability to produce unmodified daughter cells and generate specialized cell types (capabilities). Self-renewal can be achieved in two ways: asymmetric cell division produces one daughter cell that is identical to the parent cell and one daughter cell that is different from the parent cell and is a progenitor or differentiated cell. Symmetric cell division produces two identical daughter cells. "Proliferation" or "expansion" of a cell refers to a cell that divides symmetrically.

[0303] As used herein, the term "progenitor" or "progenitor cell" refers to a cell that has the capacity for self-renewal and the ability to differentiate into a more mature cell. Many progenitor cells differentiate along a single lineage, but can have a fairly extensive proliferation capacity.

[0304] In certain embodiments, the cells are primary cells. As used herein, the term "primary cells" is known in the art to refer to cells that are isolated from tissues and established for in vitro or ex vivo growth. The corresponding cells undergo much less population overlap, if any, and are therefore more representative of the main functional components of the tissues from which they are derived, compared to continuous cell lines, and therefore represent a more representative model for in vivo conditions. Methods for obtaining samples from various tissues and for establishing primary cell lines are well known in the art (see, for example, Jones and Wise, Methods Mol Biol. 1997). Primary cells for use in the methods contemplated herein are derived from umbilical cord blood, placental blood, mobilized peripheral blood, and bone marrow. In one embodiment, the primary cells are hematopoietic stem or progenitor cells.

[0305] In one embodiment, the genome edited cell is an embryonic stem cell. In one embodiment, the genome edited cell is an adult stem or progenitor cell. In one embodiment, the genome edited cell is a primary cell.

[0306] In a preferred embodiment, the genome edited cell is a hematopoietic cell, e.g., a hematopoietic stem cell, a hematopoietic progenitor cell, e.g., a B cell progenitor cell, or a cell population comprising hematopoietic cells.

[0307] As used herein, the term "cell population" refers to a plurality of cells that may consist of any number and / or combination of homogenous or heterogeneous cell types, as described elsewhere herein. For example, for transduction of hematopoietic stem or progenitor cells, a cell population may be isolated or obtained from umbilical cord blood, placental blood, bone marrow, or mobilized peripheral blood. The cell population may comprise about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the target cell type to be edited. In certain embodiments, hematopoietic stem or progenitor cells may be isolated or purified from a heterogeneous cell population using methods known in the art.

[0308] Exemplary sources for obtaining hematopoietic cells include, but are not limited to, cord blood, bone marrow, or mobilized peripheral blood.

[0309] Hematopoietic stem cells (HSCs) give rise to committed hematopoietic progenitor cells (HPCs) that are capable of generating the entire repertoire of mature blood cells over the lifetime of an organism. The term "hematopoietic stem cell" or "HSC" refers to a pluripotent stem cell that gives rise to all blood cell types in an organism, including myeloid (e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid (e.g., T cells, B cells, NK cells) lineages, and others known in the art (Fei, R., et al., U.S. Pat. No. 5,635,387; McGlave, et al., U.S. Pat. No. 5,460,964; Simmons, P., et al., U.S. Pat. No. 5,677,136; Tsukamoto, et al., U.S. Pat. No. 5,750,397; Schwartz, et al., U.S. Pat. No. 5,759,793; DiGuisto, et al., U.S. Pat. No. 5,681,599; Tsukamoto, et al., U.S. Pat. No. 5,681,599). (See, e.g., U.S. Pat. No. 5,716,827 to al.) When transplanted into animals or humans exposed to lethal doses of radiation, hematopoietic stem and progenitor cells are capable of repopulating the erythroid, neutrophil-macrophage, megakaryocyte and lymphohematopoietic cell pools.

[0310] Additional illustrative examples of hematopoietic stem or progenitor cells suitable for use in the methods and compositions contemplated herein include CD34 + CD38 Lo CD90 + CD45 RA- Hematopoietic cells, CD34 + , CD59 + , Thy1 / CD90 + , CD38 Lo / - , C-kit / CD117 + , and Lin (-) Hematopoietic cells, as well as CD133 + Examples of hematopoietic cells include hematopoietic cells that are

[0311] In a preferred embodiment, CD133 + CD90 + In a preferred embodiment, the hematopoietic cell is CD133 + CD34 + In a preferred embodiment, the hematopoietic cell is CD133 + CD90 + CD34 + Hematopoietic cells.

[0312] As used herein, the term "CD34+ cells" refers to cells that express the CD34 protein on their cell surface. As used herein, "CD34" refers to a cell surface glycoprotein (e.g., sialomucin protein) that often acts as a cell-cell adhesion factor and is involved in T cell entry into lymph nodes. CD34+ cell populations include hematopoietic stem cells (HSCs), which when administered to a patient, differentiate and contribute to all hematopoietic lineages, including T cells, NK cells, NKT cells, neutrophils, and cells of the monocyte / macrophage lineage.

[0313] There are various ways to characterize the hematopoietic hierarchy. One way of characterization is the SLAM code. The SLAM (signaling lymphocyte activation molecule) family is a group of >10 molecules whose genes are located in tandem mainly at a single locus on chromosome 1 (mouse), all belonging to a subset of the immunoglobulin gene superfamily, and thought to be originally involved in T cell stimulation. The family includes CD48, CD150, CD244, etc., with CD150 being the founding member and therefore also referred to as slamF1, i.e., SLAM family member 1. The signature SLAM code for the hematopoietic hierarchy is: Hematopoietic stem cell (HSC)-CD150 + CD48 - CD244 - , Multipotent progenitor cells (MPP)-CD150 - CD48 - CD244 + , lineage restricted progenitor (LRP)-CD150 - CD48 + CD244 +;Common myeloid progenitor cell (CMP)-lin-SCA-1-c-kit + CD34 + CD16 / 32 mid ;Granulocyte-macrophage progenitor (GMP)-lin - SCA-1-c-kit + CD34 + CD16 / 32 hi ; and megakaryocyte-erythroid progenitor (MEP)-lin - SCA-1-c-kit + CD34 - CD16 / 32 low It is.

[0314] Preferred target cell types to be edited using the compositions and methods contemplated herein include hematopoietic cells, preferably human hematopoietic cells, more preferably human hematopoietic stem and progenitor cells, and even more preferably, CD34 + Human hematopoietic stem cells are included. As used herein, the term "CD34+ cells" refers to cells that express CD34 protein on their cell surface. As used herein, "CD34" refers to a cell surface glycoprotein (e.g., sialomucin protein) that often acts as a cell-cell adhesion factor. CD34+ is a cell surface marker for both hematopoietic stem cells and progenitor cells.

[0315] In one embodiment, the genome edited hematopoietic cells are + CD48 - CD244 - In one embodiment, the genome-edited hematopoietic cells are CD34 + CD133 + In one embodiment, the genome-edited hematopoietic cells are CD133 + In one embodiment, the genome-edited hematopoietic cells are CD34 + It is a cell.

[0316] In certain embodiments, the fusion polypeptides contemplated herein are introduced into and expressed in immune effector cells. An "immune effector cell" is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, cytokine secretion, induction of ADCC and / or CDC, etc.). Exemplary immune effector cells contemplated herein are T lymphocytes, including, but not limited to, cytotoxic T cells (CTL; ​​CD8+ T cells), TILs, and helper T cells (HTL; CD4+ T cells). In certain embodiments, the cells comprise αβ T cells. In certain embodiments, the cells comprise γδ T cells. In one embodiment, the immune effector cells comprise natural killer (NK) cells. In one embodiment, the immune effector cells comprise natural killer T (NKT) cells.

[0317] Immune effector cells can be autogeneic (self) or non-autologous ("non-self", e.g., allogeneic, syngeneic, or xenogeneic). As used herein, "autogeneic" refers to cells derived from the same subject. As used herein, "allogeneic" refers to cells of the same species that are genetically different from the compared cells. As used herein, "syngeneic" refers to cells of a different subject that are genetically identical to the compared cells. As used herein, "xenogeneic" refers to cells of a different species from the compared cells. In a preferred embodiment, the cells are autogeneic.

[0318] Exemplary immune effector cells for use with the fusion polypeptides contemplated in certain embodiments include T lymphocytes. The term "T cell" or "T lymphocyte" is art-recognized and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be helper T cells (HTL; CD4+ T cells), CD4+ T cells, cytotoxic T cells (CTL; ​​CD8+ T cells), CD4+ CD8+ T cells, CD4-CD8- T cells, or any other subset of T cells. Other exemplary populations of T cells suitable for use in certain embodiments include naive T cells (TN), T memory stem cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM), and effector T cells (TEFF).

[0319] As will be understood by those skilled in the art, other cells may be used herein as immune effector cells bearing fusion polypeptides. In particular, immune effector cells also include NK cells, NKT cells, neutrophils, and macrophages. Immune effector cells also include precursors of effector cells, and such precursor cells may be induced to differentiate into immune effector cells in vivo or in vitro. Thus, in certain embodiments, immune effector cells include precursors of immune effector cells, such as hematopoietic stem cells (HSCs), contained within the CD34+ population of cells derived from umbilical cord blood, bone marrow, or mobilized peripheral blood, which differentiate into mature immune effector cells when administered to a subject, or may be induced to differentiate into mature immune effector cells in vitro.

[0320] Methods for generating immune effector cells expressing a CAR contemplated herein are provided in certain embodiments. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells express one or more CARs contemplated herein. In certain embodiments, the immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. Such cells may then be readministered directly to the individual. In further embodiments, the immune effector cells are first activated and stimulated to proliferate in vitro before being genetically modified to express a CAR. In this regard, the immune effector cells may be cultured before and / or after being genetically modified (i.e., transduced or transfected to express a CAR contemplated herein).

[0321] In certain embodiments, a source of cells is obtained from a subject prior to in vitro manipulation or genetic modification of immune effector cells as contemplated herein, hi certain embodiments, the modified immune effector cells comprise T cells.

[0322] In certain embodiments, PBMCs may be directly genetically modified using the methods contemplated herein to express a CAR. In certain embodiments, following isolation of PBMCs, T lymphocytes are further isolated, and in certain embodiments, both cytotoxic and helper T lymphocytes can be preserved into naive, memory, and effector T cell subpopulations, either before or after genetic modification and / or expansion.

[0323] Immune effector cells, such as T cells, can be genetically modified after isolation using known methods, or the immune effector cells can be activated and expanded (or differentiated in the case of precursor cells) in vitro prior to being genetically modified. In certain embodiments, immune effector cells, such as T cells, are genetically modified with a chimeric antigen receptor contemplated herein (e.g., transduced with a nucleic acid encoding a CAR or a viral vector containing a polycistronic message encoding a CAR) and then activated and expanded in vitro. In various embodiments, T cells can be activated and expanded before or after genetic modification to express a CAR using methods such as those described in, for example, U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041, and U.S. Patent Application Publication No. 2006 / 0121005.

[0324] In one embodiment, CD34+ cells are transduced with a nucleic acid construct contemplated herein. In certain embodiments, the transduced CD34+ cells are differentiated in vivo into mature immune effector cells after being administered to a subject, typically the subject from whom the cells were originally isolated. In another embodiment, CD34+ cells can be stimulated in vitro with one or more of the following cytokines: Flt-3 Ligand (FLT3), Stem Cell Factor (SCF), Megakaryocyte Growth and Differentiation Factor (TPO), IL-3, and IL-6, according to previously described methods (Asheuer et al., 2004; Imren, et al., 2004), either before or after being genetically modified with a CAR contemplated herein.

[0325] In certain embodiments, the population of modified immune effector cells for the treatment of cancer comprises the CAR contemplated herein. For example, the population of modified immune effector cells is prepared from peripheral blood mononuclear cells (PBMCs) obtained from a patient (autologous donor) diagnosed with a B-cell malignancy as described herein. The PBMCs form a heterogeneous population of T lymphocytes that can be CD4+, CD8+, or CD4+ and CD8+.

[0326] PBMCs may also contain other cytotoxic lymphocytes such as NK cells or NKT cells. An expression vector carrying the coding sequence of a CAR contemplated in certain embodiments is introduced into a population of human donor T cells, NK cells, or NKT cells. In certain embodiments, successfully transduced T cells carrying the expression vector can be further expanded to increase the number of these CAR protein expressing T cells by isolating CD3 positive T cells using flow cytometry, followed by cell activation using anti-CD3 antibodies and / or anti-CD28 antibodies and IL-2 or any other method known in the art as described elsewhere herein. Standard procedures are used for cryopreservation of T cells expressing CAR protein T cells for storage and / or preparation for use in human subjects. In one embodiment, in vitro transduction, culture, and / or expansion of T cells is performed in the absence of non-human animal derived products such as fetal calf serum and fetal bovine serum. Because a heterogeneous population of PBMCs are genetically modified, the resulting transduced cells are a heterogeneous population of modified cells that contain a BCMA targeting CAR as contemplated herein.

[0327] In further embodiments, a mixture of, for example, one, two, three, four, five, or more different expression vectors can be used in genetically modifying a donor population of immune effector cells, each vector encoding a different chimeric antigen receptor protein as contemplated herein, and the resulting modified immune effector cells form a mixed population of modified cells.

[0328] Genetically engineered cells, including T cells, can be produced using a variety of methods known in the art, see, e.g., WO2016 / 094304, which is incorporated by reference in its entirety.

[0329] I. Compositions and Formulations Compositions contemplated in certain embodiments may include one or more of the polypeptides, polynucleotides, vectors comprising same, as well as genome editing compositions and genome edited cell compositions contemplated herein. Genome editing compositions and methods contemplated in certain embodiments are useful for editing a target site in a cell or cell population.

[0330] In various embodiments, compositions contemplated herein include a fusion polypeptide comprising a DNA binding domain, a homing endonuclease variant, and a terminal processing enzyme, e.g., a 3' to 5' exonuclease (ExoX). The nuclease variant may be in the form of an mRNA that is introduced into the cell via the polynucleotide delivery methods disclosed above, e.g., electroporation, lipid nanoparticles, etc. In one embodiment, a composition comprising a fusion polypeptide, a homing endonuclease variant, and a 3' to 5' exonuclease (e.g., ExoX) is introduced into the cell via the polynucleotide delivery methods disclosed above.

[0331] In certain embodiments, the compositions contemplated herein comprise a cell population, a nuclease variant, and optionally a donor repair template. In certain embodiments, the compositions contemplated herein comprise a cell population, a nuclease variant, a terminal processing enzyme, and optionally a donor repair template. The nuclease variant and / or the terminal processing enzyme may be in the form of mRNA that is introduced into the cell via the polynucleotide delivery method disclosed above. The donor repair template may also be introduced into the cell by a separate composition.

[0332] In certain embodiments, the compositions contemplated herein comprise a cell population, a fusion polypeptide comprising a DNA binding domain, a homing endonuclease variant, a 3'-5' exonuclease (e.g., ExoX), and, optionally, a donor repair template. The fusion polypeptide comprising a DNA binding domain, a homing endonuclease variant, and a 3'-5' exonuclease may be in the form of an mRNA that is introduced into the cell via the polynucleotide delivery methods disclosed above. The donor repair template may also be introduced into the cell by a separate composition.

[0333] In certain embodiments, the cell population comprises hematopoietic stem cells, hematopoietic progenitor cells, CD133 + cells, CD34 + The present invention includes genetically modified hematopoietic cells, including, but not limited to, immune effector cells.

[0334] Compositions include, but are not limited to, pharmaceutical compositions. A "pharmaceutical composition" refers to a composition that is formulated for administration to a cell or animal in a pharma- ceutical or physiologically acceptable solution, alone or in combination with one or more other therapeutic modalities. It should also be understood that, if desired, the composition may be administered in combination with other agents as well, such as, for example, cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or various other pharma- ceutical active agents. There is virtually no limit to the other components that may be included in the composition, provided that the added agents do not adversely affect the composition.

[0335] As used herein, the phrase "pharmacologically acceptable" is employed to refer to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0336] The term "pharmaceutical acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle in which the therapeutic cells are administered. Illustrative examples of pharmaceutical carriers may be sterile liquids, such as cell culture media, water, and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Saline solutions and dextrose and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients in certain embodiments include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated. Supplementary active ingredients may also be incorporated into the composition.

[0337] In one embodiment, the composition comprising a pharma- ceutically acceptable carrier is suitable for administration to a subject. In certain embodiments, the composition comprising a carrier is suitable for parenteral administration, such as, for example, intravascular (intravenous or intra-arterial), intraperitoneal or intramuscular administration. In certain embodiments, the composition comprising a pharma- ceutically acceptable carrier is suitable for intraventricular, intrathecal or intrathecal administration. Pharmaceutically acceptable carriers include sterile aqueous solutions, cell culture media, or dispersions. The use of such media and agents for pharma- ceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the transduced cells, its use in the pharmaceutical composition is contemplated.

[0338] In certain embodiments, compositions contemplated herein comprise genetically modified hematopoietic stem and / or progenitor cells or immune effector cells comprising an exogenous polynucleotide encoding a fusion polypeptide contemplated herein, and a pharma- ceutically acceptable carrier.

[0339] Compositions, including cell-based compositions, contemplated herein may be administered by parenteral methods of administration.

[0340] A pharma- ceutically acceptable carrier must be of sufficiently high purity and of sufficiently low toxicity to be suitable for administration to the human subject being treated, and must also maintain or enhance the stability of the composition. A pharma- ceutically acceptable carrier may be liquid or solid, and is selected to provide the desired volume, consistency, when mixed with the other components of the composition, having regard to the intended mode of administration. For example, the pharma- ceutically acceptable carrier may be, but is not limited to, a binder (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose, etc.), a filler (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, calcium hydrogen phosphate, etc.), a lubricant (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.), a disintegrant (e.g., starch, sodium starch glycolate, etc.), or a wetting agent (e.g., sodium lauryl sulfate, etc.). Other pharma- ceutically acceptable carriers suitable for the compositions contemplated herein include, but are not limited to, water, salt solutions, alcohol, polyethylene glycol, gelatin, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, etc.

[0341] Such carrier solutions may also include buffers, diluents, and other suitable additives. As used herein, the term "buffer" refers to a solution or liquid whose chemical composition neutralizes acids or bases without causing significant changes in pH. Examples of buffers contemplated herein include, but are not limited to, Dulbecco's phosphate buffered saline (PBS), Ringer's solution, 5% dextrose solution (D5W), and normal / physiological saline (0.9% NaCl).

[0342] The pharma- ceutically acceptable carrier may be present in an amount sufficient to maintain the pH of the composition at about 7. Alternatively, the composition has a pH in the range of about 6.8 to about 7.4, such as 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, and 7.4. In yet another embodiment, the composition has a pH of about 7.4.

[0343] The compositions contemplated herein may comprise a non-toxic pharma- ceutically acceptable medium.The compositions may be suspensions.As used herein, the term "suspension" refers to a non-adherent state in which cells do not bind to a solid support.For example, cells maintained as suspensions may be stirred or agitated, and do not adhere to a support, such as a culture dish.

[0344] In certain embodiments, compositions contemplated herein are formulated in suspension, where the genome-edited hematopoietic stem and / or progenitor cells are dispersed in an acceptable liquid medium or solution, such as saline or serum-free medium, such as in an intravenous (IV) bag. Acceptable diluents include, but are not limited to, water, PlasmaLyte, Ringer's solution, isotonic sodium chloride solution (saline), serum-free cell culture medium, and media suitable for cryogenic storage, such as Cryostor® medium.

[0345] In certain embodiments, the pharma- ceutically acceptable carrier is substantially free of naturally occurring proteins of human or animal origin and is suitable for storage of compositions comprising genome-edited cells, e.g., hematopoietic stem and progenitor cells. Therapeutic compositions are intended for administration to human patients and therefore are substantially free of cell culture components, such as, for example, bovine serum albumin, horse serum, and fetal bovine serum.

[0346] In some embodiments, the composition is formulated in a pharma- ceutically acceptable cell culture medium. Such a composition is suitable for administration to a human subject. In certain embodiments, the pharma-ceutically acceptable cell culture medium is a serum-free medium.

[0347] Serum-free media have several advantages over serum-containing media, including a simpler and more defined composition, lower amounts of contaminants, elimination of potential sources of infectious entities, and lower costs. In various embodiments, serum-free media may be animal-free and optionally protein-free. Optionally, the media may contain biopharmaceutical acceptable recombinant proteins. "Animal-free" media refers to media whose components are derived from non-animal sources. Recombinant proteins replace natural animal proteins in animal-free media, whose nutrients are derived from synthetic, plant, or microbial sources. "Protein-free" media, in contrast, are defined as being substantially protein-free.

[0348] Illustrative examples of serum-free media for use in certain compositions include, but are not limited to, QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.

[0349] In a preferred embodiment, the composition comprising genome-edited hematopoietic stem and / or progenitor cells is formulated in PlasmaLyte.

[0350] In various embodiments, the compositions comprising hematopoietic stem and / or progenitor cells are formulated in a cryopreservation medium. For example, a cryopreservation medium containing a cryopreservative may be used to maintain high cell activity after thawing. Illustrative examples of cryopreservation media used in certain compositions include, but are not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.

[0351] In one embodiment, the composition is formulated in a solution comprising 50:50 PlasmaLyte A and CryoStor CS10.

[0352] In certain embodiments, the compositions are substantially free of mycoplasma, endotoxin, and microbial contamination. "Substantially free" with respect to endotoxin means lower endotoxin per cell dose than permitted by the FDA for biologics, which is 5 EU / kg total endotoxin per day, and 350 EU per total cell dose for an average 70 kg human. In certain embodiments, compositions comprising retroviral vector-transduced hematopoietic stem or progenitor cells contemplated herein contain about 0.5 EU / mL to about 5.0 EU / mL, or about 0.5 EU / mL, 1.0 EU / mL, 1.5 EU / mL, 2.0 EU / mL, 2.5 EU / mL, 3.0 EU / mL, 3.5 EU / mL, 4.0 EU / mL, 4.5 EU / mL, or 5.0 EU / mL.

[0353] In certain embodiments, compositions and formulations suitable for delivery of polynucleotides are contemplated, including, but not limited to, one or more mRNAs encoding one or more reprogrammed nucleases, and optionally, a terminal processing enzyme.

[0354] Exemplary formulations for ex vivo delivery may also include the use of various transfection agents known in the art, such as calcium phosphate, electroporation, heat shock, and various liposome formulations (i.e., lipid-mediated transfection). Liposomes are lipid bilayers that surround a fraction of aqueous fluid, as described in more detail below. DNA spontaneously associates with the outer surface of cationic liposomes (due to their charge), and these liposomes interact with cell membranes.

[0355] In certain embodiments, the formulation of pharma- ceutically acceptable carrier solutions is well known in the art, as is the development of suitable dosing and treatment regimens for use of certain compositions described herein in a variety of treatment regimens, including, for example, enteral and parenteral, e.g., intravascular, intravenous, intraarterial, intraosseous, intraventricular, intracerebral, intracranial, intrathecal, intrathecal, and intramedullary administration and formulations. Those of skill in the art will appreciate that certain embodiments contemplated herein are well known in the pharmaceutical arts and can be found, for example, in Remington: The Science and Practice of Pharmacy, volume I and volume II.22, which are incorporated herein by reference in their entirety. nd It will be understood that the present invention may include other formulations such as those described in the 2012 Edition. Edited by Loyd V. Allen Jr. Philadelphia, PA: Pharmaceutical Press;

[0356] J. Site-directed mutagenesis methods The qualitative characteristics that define a given deletion or insertion (indel) are (i) its length in number of inserted or deleted bases, (ii) its longitudinal location along the chromosome, usually indicated relative to a nuclease target site or breakpoint, and (iii) with respect to the insertion, the length and composition of the inserted sequence. Deletions are the most prominent outcome, typically comprising 90-95% of the observed events. Their most commonly reported size characteristics tend to be small (i.e., 1-20 base pairs in length, with frequencies biased towards the low end of that range), and their location distribution is found to be evenly distributed, encompassing the DNA breakpoint and diverging outward in either direction without significant bias. Exceptions to these characteristics are frequently assumed to be driven by microhomologies (small overlapping tracts roughly 3-6 base pairs in length) located on either side of the DNA breakpoint. Little has been reported regarding the characteristics of insertions, which occur at much lower frequencies during the application of genome editing tools. Furthermore, the genotypic features linking each indel species to a phenotype (e.g., how it affects an open reading frame or whether it disrupts a transcription factor binding motif) are potentially vast and specific to each given application.

[0357] Engineered megaTAL nucleases, as well as other fusion polypeptides containing a DNA binding domain and a homing endonuclease, have distinct features from other gene editing platforms. For example, megaTALs are monomeric, hybrid molecules that contain a modularly assembled transcription activator-like effector (TALE) array fused to a reprogrammed homing endonuclease (HE). The TALE array, which anchors the HE at the target site, can be sized to recognize binding sites of approximately 6 to 18 base pairs. The homing endonuclease recognizes and cleaves a 22-base pair target site. The two target sites are separated by a spacer region that can be anywhere from 0 to approximately 12 base pairs in length. Two unique properties of megaTAL nucleases result from their distinct compositions: (i) the immobilization mechanism of the TALE array provides a highly biased distribution of overall binding affinity to one side of the target site, and (ii) the product of the DNA cleavage reaction catalyzed by the homing endonuclease is a DNA end with a 3' overhang of 4 base pairs in length. In contrast, ZFNs and TALENs, whose zinc finger or TALE arrays are engineered by FokI nuclease, respectively, have a relatively uniform distribution of binding affinity and generate 5' overhang ends of 4 base pairs in length. The mechanisms of CRISPR DNA recognition and cleavage are fundamentally different in terms of DNA sequence recognition and affinity, however, the product of DNA cleavage is either a blunt DNA end (Cas9) or a 5' overhang end (Cpf1).

[0358] To assess quantitative and qualitative aspects of gene editing events, the inventors of the fusion polypeptides contemplated herein tested the co-delivery of various terminal processing enzymes to characterize the effect on the edited allele. Furthermore, the inventors designed a number of multipart megaTAL fusion proteins to assess how the editing outcome can be manipulated by in-line enzyme function.

[0359] Thus, the inventors surprisingly discovered that fusion polypeptides comprising a DNA-binding domain and a homing endonuclease variant linked to an exonuclease, in particular Trex2, ExoI, or ExoX, by a linker domain (e.g., a polypeptide linker), when expressed in a cell, created extended, directionally biased deletions when compared to fusion polypeptides comprising a DNA-binding domain and a homing endonuclease variant that are not linked to an exonuclease, i.e., when the DNA-binding domain / homing endonuclease fusion and the exonuclease are expressed separately.

[0360] Thus, there is provided a method of site-directed mutagenesis in a cell, comprising selecting a double-stranded DNA (dsDNA) target site and introducing into the cell a fusion polypeptide, or a polynucleotide, mRNA, or vector encoding a fusion polypeptide as contemplated herein, wherein the fusion peptide generates a directionally biased deletion with the deletion center near the selected dsDNA target cleavage site in the cell.

[0361] In various embodiments, the method comprises introducing a fusion peptide comprising a DNA-binding domain and a homing endonuclease (HE) variant that binds and cleaves a selected double-stranded DNA (dsDNA) target site in a cell, a linker domain, and an exonuclease or a biologically active fragment thereof. In some embodiments, the exonuclease is Trex2, ExoI, or ExoX. In some embodiments, the DNA-binding domain comprises a TALE DNA-binding domain (e.g., megaTAL) or a zinc finger DNA-binding domain.

[0362] In various embodiments, ExoX or a biologically active fragment thereof comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:109.

[0363] In various embodiments, ExoI or a biologically active fragment thereof comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:112.

[0364] In various embodiments, the linker domain is a peptide linker. In some embodiments, the peptide linker is a self-cleaving peptide linker. In some embodiments, the peptide linker comprises about 4 to about 30 amino acids. In some embodiments, the peptide linker is (GGGGS) 1-4 Linker (SEQ ID NOs: 117 and 150 to 152).

[0365] In various embodiments, the HE variant is a LAGLIDADG homing endonuclease (LHE) variant. In some embodiments, the HE variant is I-AabMI, I-AaeMI, I-AniI, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-GpiI, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-LtrII, I- A variant of LHE selected from the group consisting of I-LtrI, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI, I-OheMI, I-OnuI, I-OsoMI, I-OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-SceI, I-ScuMI, I-SmaMI, I-SscMI, and I-Vdi141I.

[0366] In various embodiments, contemplated methods include introducing a fusion polypeptide that targets a site within an immune system checkpoint gene, a globin gene, a gene encoding a polypeptide that contributes to the suppression of gamma-globin gene expression and HbF, or an immunosuppressive signaling gene. In some embodiments, the target site is programmed cell death protein 1 (PD-1, PDCD1), lymphocyte activation gene 3 protein (LAG-3), T cell immunoglobulin domain and mucin domain protein 3 (TIM-3), cytotoxic T lymphocyte antigen-4 (CTLA-4), band T lymphocyte attenuator (BTLA), T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), V domain Ig inhibitor of T cell activation. (VISTA), as well as killer cell immunoglobulin-like receptors (KIR), CCR5, TRAC (TCRα), TCRβ, IL10Rα, IL10Rβ, TGFBR1, TGFBR2, CBL-B, PCSK9, AHR, BTK, α-globin, β-globin, γ-globin, BCL11A, KLF1, SOX6, GATA1, LSD1, alpha folate receptor (FRα), αvβ6 integrin, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, decarboxylation Hydrogenase IX (CAIX), CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, carcinoembryonic antigen (CEA), C-type lectin-like molecule-1 (CLL-1), CD2 subset 1 (CS-1), chondroitin sulfate proteoglycan 4 (CSPG4), cutaneous T-cell lymphoma-associated antigen 1 (CTAGE1), epithelial Growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), epithelial cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EPHA2), fibroblast activation protein (FAP), Fc receptor-like 5 (FCRL5), fetal acetylcholinesterase receptor (AchR), ganglioside G2 (GD2), ganglioside G3 (GD3), glypican-3 (GPC3),EGFR family including ErbB2 (HER2), IL-11Rα, IL-13Rα2, kappa, cancer / testis antigen 2 (LAGE-1A), lambda, Lewis-Y (LeY), L1 cell adhesion molecule (L1-CAM), melanoma antigen gene (MAGE)-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, melanoma antigen recognized by T cells 1 (MelanA or MART1), mesothelin (MSLN), MUC1, MUC16, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), neural cell adhesion molecule (NCAM), cancer / testis antigen 1 (NY-ESO-1), polysialic acid; placenta specific 1 (PLAC1), melanoma Predominantly expressed antigen in ovarian cancer (PRAME), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), synovial sarcoma, X breakpoint 2 (SSX2), survivin, tumor associated glycoprotein 72 (TAG72), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7 related (TEM7R), TEM5, TEM8, trophoblast glycoprotein (TPBG), UL16 binding protein (ULBP) 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms' tumor 1 (WT-1) genes.

[0367] In some embodiments, the target site is within a gene selected from the group consisting of programmed cell death protein 1 (PD-1, PDCD1), lymphocyte activation gene 3 protein (LAG-3), T-cell immunoglobulin domain and mucin domain protein 3 (TIM-3), cytotoxic T-lymphocyte antigen-4 (CTLA-4), band T-lymphocyte attenuator (BTLA), T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), V-domain Ig inhibitor of T-cell activation (VISTA), and killer cell immunoglobulin-like receptor (KIR), CCR5, TRAC (TCRα), IL10Rα, TGFBR2, CBL-B, PCSK9, AHR, BTK, α-globin, β-globin, γ-globin, and BCL11A genes.

[0368] In various embodiments, the target site is in the TRAC (TCR alpha) gene, the CBL-B gene, or the PDCD1 (PD-1) gene. In certain embodiments, the TCR alpha gene target site comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the CBL-B gene target site comprises the amino acid sequence set forth in SEQ ID NO: 2. In certain embodiments, the PD-1 gene target site comprises the amino acid sequence set forth in SEQ ID NO: 3.

[0369] As contemplated throughout this disclosure, the fusion polypeptides and related methods generate directionally biased deletions in cells at selected dsDNA target sites. In various embodiments, the deletion center position is on the same side as the DNA binding domain target site relative to the center position of the HE target site. In certain embodiments, the deletion center position is 5' relative to the center position of the HE target site.

[0370] In various embodiments, more than 50% of the directionally biased deletions have a deletion central location to one side of the central location of the HE target site. In some embodiments, more than 51% of the directionally biased deletions have a deletion central location to one side of the central location of the HE target site. In some embodiments, more than 52% of the directionally biased deletions have a deletion central location to one side of the central location of the HE target site. In some embodiments, more than 53% of the directionally biased deletions have a deletion central location to one side of the central location of the HE target site. In some embodiments, more than 54% of the directionally biased deletions have a deletion central location to one side of the central location of the HE target site. In some embodiments, more than 55% of the directionally biased deletions have a deletion central location to one side of the central location of the HE target site. In some embodiments, more than 56% of the directionally biased deletions have a deletion central location to one side of the central location of the HE target site. In some embodiments, more than 57% of the directionally biased deletions have a deletion central location to one side of the central location of the HE target site. In some embodiments, more than 58% of the directionally biased deletions have a deletion central location to one side of the central location of the HE target site. In some embodiments, more than 59% of the directionally biased deletions have a deletion central location to one side of the central location of the HE target site. In some embodiments, more than 60% of the directionally biased deletions have a deletion central location to one side of the central location of the HE target site. In some embodiments, more than 65% of the directionally biased deletions have a deletion central location to one side of the central location of the HE target site. In some embodiments, more than 70% of the directionally biased deletions have a deletion central location to one side of the central location of the HE target site. In some embodiments, more than 75% of the directionally biased deletions have a deletion central location to one side of the central location of the HE target site. In some embodiments, more than 80% of the directionally biased deletions have a deletion central location to one side of the central location of the HE target site.

[0371] In various embodiments, at least 50% of the deletions have a deletion center that is more than 4 nucleotides away from the central position of the HE target site. In some embodiments, at least 51% of the deletions have a deletion center that is more than 4 nucleotides away from the central position of the HE target site. In some embodiments, at least 52% of the deletions have a deletion center that is more than 4 nucleotides away from the central position of the HE target site. In some embodiments, at least 53% of the deletions have a deletion center that is more than 4 nucleotides away from the central position of the HE target site. In some embodiments, at least 54% of the deletions have a deletion center that is more than 4 nucleotides away from the central position of the HE target site. In some embodiments, at least 55% of the deletions have a deletion center that is more than 4 nucleotides away from the central position of the HE target site. In some embodiments, at least 56% of the deletions have a deletion center that is more than 4 nucleotides away from the central position of the HE target site. In some embodiments, at least 57% of the deletions have a deletion center that is more than 4 nucleotides away from the central position of the HE target site. In some embodiments, at least 58% of the deletions have a deletion center that is more than 4 nucleotides away from the central position of the HE target site. In some embodiments, at least 59% of the deletions have a deletion center that is more than 4 nucleotides away from the central position of the HE target site. In some embodiments, at least 60% of the deletions have a deletion center that is more than 4 nucleotides away from the central position of the HE target site. In some embodiments, at least 65% of the deletions have a deletion center that is more than 4 nucleotides away from the central position of the HE target site. In some embodiments, at least 70% of the deletions have a deletion center that is more than 4 nucleotides away from the central position of the HE target site. In some embodiments, at least 75% of the deletions have a deletion center that is more than 4 nucleotides away from the central position of the HE target site. In some embodiments, at least 80% of the deletions have a deletion center that is more than 4 nucleotides away from the central position of the HE target site.

[0372] In various embodiments, at least 10% of the deletions have a deletion center that is more than 8 nucleotides away from the central position of the HE target site. In some embodiments, at least 11% of the deletions have a deletion center that is more than 8 nucleotides away from the central position of the HE target site. In some embodiments, at least 12% of the deletions have a deletion center that is more than 8 nucleotides away from the central position of the HE target site. In some embodiments, at least 13% of the deletions have a deletion center that is more than 8 nucleotides away from the central position of the HE target site. In some embodiments, at least 14% of the deletions have a deletion center that is more than 8 nucleotides away from the central position of the HE target site. In some embodiments, at least 15% of the deletions have a deletion center that is more than 8 nucleotides away from the central position of the HE target site. In some embodiments, at least 16% of the deletions have a deletion center that is more than 8 nucleotides away from the central position of the HE target site. In some embodiments, at least 17% of the deletions have a deletion center that is more than 8 nucleotides away from the central position of the HE target site. In some embodiments, at least 18% of the deletions have a deletion center that is more than 8 nucleotides away from the central position of the HE target site. In some embodiments, at least 19% of the deletions have a deletion center that is more than 8 nucleotides away from the central position of the HE target site. In some embodiments, at least 20% of the deletions have a deletion center that is more than 8 nucleotides away from the central position of the HE target site. In some embodiments, at least 25% of the deletions have a deletion center that is more than 8 nucleotides away from the central position of the HE target site. In some embodiments, at least 30% of the deletions have a deletion center that is more than 8 nucleotides away from the central position of the HE target site. In some embodiments, at least 35% of the deletions have a deletion center that is more than 8 nucleotides away from the central position of the HE target site.

[0373] In various embodiments, at least 50% of the deletions are 6 bp or more in length. In some embodiments, at least 51% of the deletions are 6 bp or more in length. In some embodiments, at least 52% of the deletions are 6 bp or more in length. In some embodiments, at least 53% of the deletions are 6 bp or more in length. In some embodiments, at least 54% of the deletions are 6 bp or more in length. In some embodiments, at least 55% of the deletions are 6 bp or more in length. In some embodiments, at least 56% of the deletions are 6 bp or more in length. In some embodiments, at least 57% of the deletions are 6 bp or more in length. In some embodiments, at least 58% of the deletions are 6 bp or more in length. In some embodiments, at least 59% of the deletions are 6 bp or more in length. In some embodiments, at least 60% of the deletions are 6 bp or more in length. In some embodiments, at least 65% of the deletions are 6 bp or more in length. In some embodiments, at least 70% of the deletions are 6 bp or more in length. In some embodiments, at least 75% of the deletions are 6 bp or more in length. In some embodiments, at least 80% of the deletions are 6 bp or more in length.

[0374] In various embodiments, at least 30% of the deletions are 12 bp or more in length. In some embodiments, at least 31% of the deletions are 12 bp or more in length. In some embodiments, at least 32% of the deletions are 12 bp or more in length. In some embodiments, at least 33% of the deletions are 12 bp or more in length. In some embodiments, at least 34% of the deletions are 12 bp or more in length. In some embodiments, at least 35% of the deletions are 12 bp or more in length. In some embodiments, at least 36% of the deletions are 12 bp or more in length. In some embodiments, at least 37% of the deletions are 12 bp or more in length. In some embodiments, at least 38% of the deletions are 12 bp or more in length. In some embodiments, at least 39% of the deletions are 12 bp or more in length. In some embodiments, at least 40% of the deletions are 12 bp or more in length. In some embodiments, at least 45% of the deletions are 12 bp or more in length. In some embodiments, at least 50% of the deletions are 12 bp or more in length. In some embodiments, at least 55% of the deletions are 12 bp or more in length. In some embodiments, at least 60% of the deletions are 12 bp or more in length.

[0375] In various embodiments, the directionally biased deletion comprises about 10 nucleotides in length. In some embodiments, the directionally biased deletion comprises about 11 nucleotides in length. In some embodiments, the directionally biased deletion comprises about 12 nucleotides in length. In some embodiments, the directionally biased deletion comprises about 13 nucleotides in length. In some embodiments, the directionally biased deletion comprises about 14 nucleotides in length. In some embodiments, the directionally biased deletion comprises about 15 nucleotides in length. In some embodiments, the directionally biased deletion comprises about 16 nucleotides in length. In some embodiments, the directionally biased deletion comprises about 17 nucleotides in length. In some embodiments, the directionally biased deletion comprises about 18 nucleotides in length. In some embodiments, the directionally biased deletion comprises about 19 nucleotides in length. In some embodiments, the directionally biased deletion comprises about 20 nucleotides in length.

[0376] In various embodiments, the deletion extends within the DNA-binding domain target site. In various embodiments, the central position of the deletion is within the DNA-binding domain target site.

[0377] In various embodiments, the method further comprises introducing into the cell, in addition to the fusion polypeptide, or a terminal processing enzyme or biologically active fragment thereof, or a polynucleotide, RNA, or vector encoding a terminal processing enzyme (e.g., an exonuclease). In some embodiments, the terminal processing enzyme, or biologically active fragment thereof, is selected from the group consisting of Trex2, Trex1, Trex1 without a transmembrane domain, Apollo, Artemis, DNA2, ExoI, ExoT, ExoIII, ExoX, Fen1, Fan1, MreII, Rad2, Rad9, TdT (terminal deoxynucleotidyl transferase), PNKP, RecE, RecJ, RecQ, lambda exonuclease, Sox, vaccinia DNA polymerase, exonuclease I, exonuclease II, exonuclease III, exonuclease II, exonuclease III, exonuclease I ... I, exonuclease II, exonuclease I, exonuclease I, exonuclease II, exonuclease I, exonuclease I, exonu The terminal processing enzyme is selected from the group consisting of exonuclease III, exonuclease VII, NDK1, NDK5, NDK7, NDK8, WRN, T7-exonuclease gene 6, avian myeloblastosis virus integration protein (IN), Bloom, Antarctic phosphatase, alkaline phosphatase, polynucleotide kinase (PNK), ApeI, mung bean nuclease, Hex1, TTRAP (TDP2), Sgs1, Sae2, CUP, Pol mu, Pol lambda, MUS81, EME1, EME2, SLX1, SLX4, and UL-12. In some embodiments, the terminal processing enzyme is an exonuclease. In certain embodiments, the exonuclease is Trex2, or a biologically active fragment thereof.

[0378] In certain embodiments, a method of site-directed mutagenesis in a cell is provided, comprising selecting a double-stranded DNA (dsDNA) target site and introducing into the cell a fusion polypeptide, or a polynucleotide, mRNA, or vector encoding a fusion polypeptide as contemplated herein, wherein the fusion peptide generates a directionally biased deletion with the deletion center near the selected dsDNA target cleavage site in the cell.

[0379] In certain embodiments, the method includes selecting a double-stranded DNA (dsDNA) target site; introducing into the cell a fusion polypeptide comprising a DNA binding domain and a homing endonuclease (HE) variant, a linker domain, and ExoI, or a biologically active fragment thereof, that binds to and cleaves the selected double-stranded DNA (dsDNA) target site in the cell; and introducing an exonuclease (e.g., Trex2), wherein the method generates a directionally biased deletion with a deletion center near the selected dsDNA target cleavage site in the cell.

[0380] In various embodiments, the method is an in vitro method. In various embodiments, the method is an ex vivo method. In various embodiments, the method is an in vivo method.

[0381] K. Treatment method The fusion polypeptides, gene-edited cells, compositions, and / or associated gene editing methods contemplated herein can be used to prevent, treat, and ameliorate a disease or disorder, or to ameliorate a disease state or symptom associated therewith. In some embodiments, the fusion polypeptides, gene-edited cells, compositions, and / or associated gene editing methods contemplated herein can be used in a method of treating, preventing, or inhibiting a disease (e.g., cancer, ischemia, diabetic retinopathy, macular degeneration, rheumatoid arthritis, psoriasis, HIV infection, sickle cell disease, Alzheimer's disease, muscular dystrophy, neurodegenerative disease, vascular disease, cystic fibrosis, stroke, hyper-IGE syndrome, hemophilia, etc.) or a method of ameliorating a disease state or symptom associated with a disease, such as cancer, ischemia, diabetic retinopathy, macular degeneration, rheumatoid arthritis, psoriasis, HIV infection, sickle cell disease, Alzheimer's disease, muscular dystrophy, neurodegenerative disease, vascular disease, cystic fibrosis, stroke, hyper-IGE syndrome, hemophilia, etc.

[0382] In some embodiments, the fusion polypeptides, gene edited cells, compositions, and / or related gene editing methods contemplated herein are useful for treating, preventing, or inhibiting autosomal dominant diseases such as achondroplasia, pseudoachondroplasia, multiple epiphyseal dysplasia, chondrodysplasia, osteogenesis imperfecta, Marfan syndrome, polydactyly, hereditary motor and sensory neuropathy I and II (Charcot-Marie-Tooth disease), myotonic dystrophy, and neurofibromatosis, or ameliorating a disease state or symptoms associated with autosomal dominant diseases such as achondroplasia, pseudoachondroplasia, multiple epiphyseal dysplasia, chondrodysplasia, osteogenesis imperfecta, Marfan syndrome, polydactyly, hereditary motor and sensory neuropathy I and II (Charcot-Marie-Tooth disease), myotonic dystrophy, and neurofibromatosis. In some embodiments, the fusion polypeptides, gene-edited cells, compositions, and / or related gene editing methods contemplated herein are useful for treating, preventing, or inhibiting diseases caused by gene misregulation.

[0383] In a preferred embodiment, the fusion polypeptides, gene-edited cells, compositions, and / or related gene editing methods contemplated herein may be used to prevent, treat, and / or ameliorate at least one symptom associated with an immune disorder or cancer.

[0384] "Immune disorder" refers to a disease that provokes a response from the immune system. In certain embodiments, the term "immune disorder" refers to cancer, graft-versus-host disease, an autoimmune disease, or an immune deficiency. In one embodiment, an immune disorder encompasses an infectious disease.

[0385] As used herein, the term "cancer" generally relates to a type of disease or condition in which abnormal cells divide uncontrollably and can invade nearby tissues.

[0386] As used herein, the term "malignant" refers to a cancer in which tumor cells exhibit one or more of the following: uncontrolled proliferation (i.e., dividing beyond normal limits), invasion (i.e., invading and destroying adjacent tissues), and metastasis (i.e., spreading to other parts of the body via the lymphatics or blood).

[0387] As used herein, the term "metastasize" refers to the spread of cancer from one part of the body to another. Tumors formed by the spread cells are called "metastatic tumors" or "metastatic cancers." Metastatic tumors contain cells that are similar to cells of the original tumor (primary tumor).

[0388] As used herein, the terms "benign" or "non-malignant" refer to tumors that may grow large but do not spread to other parts of the body. Benign tumors are self-limited and often do not invade or metastasize.

[0389] "Cancer cell" or "tumor cell" refers to an individual cell of a cancerous growth or cancerous tissue. "Tumor" generally refers to an expansion or lesion formed by the abnormal proliferation of cells, which may be benign, precancerous, or malignant. Most cancers form tumors, but some do not necessarily form tumors. For cancers that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably. The amount of tumor in an individual is the "tumor burden," and can be measured as the number, volume, or weight of tumors.

[0390] "Graft-versus-host disease" or "GVHD" refers to a complication that can occur after cell, tissue, or solid organ transplantation. GVHD can occur after stem cell or bone marrow transplantation, where transplanted donor cells attack the transplant recipient's body. Acute GVHD in humans occurs within about 60 days after transplantation, resulting in damage to the skin, liver, and intestine by the action of cytolytic lymphocytes. Chronic GVHD occurs later and is a systemic autoimmune disease that primarily affects the skin, resulting in polyclonal activation of B cells and overproduction of Ig and autoantibodies. Solid organ transplant graft-versus-host disease (SOT-GVHD) occurs in two forms. The more common type is antibody-mediated, where antibodies from a donor with blood type O attack the recipient's red blood cells in recipients with blood types A, B, or AB, resulting in mild, transient hemolytic anemia. The second form of SOT-GVHD is a cellular type associated with high mortality, in which donor-derived T cells mount an immunological attack against immunologically foreign host tissues, most frequently the skin, liver, gastrointestinal tract, and bone marrow, leading to complications in these organs.

[0391] "Graft-versus-leukemia" or "GVL" refers to an immune response against a person's leukemia cells by immune cells present in the donor's transplanted tissue, such as bone marrow or peripheral blood.

[0392] "Autoimmune disease" refers to a disease in which the body mounts an immunogenic (i.e., immune system) response against some of the constituents of its own tissues. In other words, the immune system loses the ability to recognize some of the tissues or systems within the body as "self" and attacks them as if they were foreign. Illustrative examples of autoimmune diseases include, but are not limited to, arthritis, inflammatory bowel disease, Hashimoto's thyroiditis, Graves' disease, lupus, multiple sclerosis, rheumatoid arthritis, hemolytic anemia, autoimmune thyroiditis, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, diabetes, scleroderma, psoriasis, and the like.

[0393] "Immune deficiency" refers to a condition in a patient in which the immune system is impaired by disease or exposure to chemicals. This condition results in an incomplete immune system in the number and type of blood cells required for defense against foreign agents. Immunodeficiency conditions or diseases are known in the art and include, for example, AIDS (acquired immune deficiency syndrome), SCID (severe combined immunodeficiency), selective IgA deficiency, common variable immunodeficiency, X-linked agammaglobulinemia, chronic granulomatous disease, hyper-IgM syndrome, Wiskott-Aldrich syndrome (WAS), and diabetes mellitus.

[0394] "Infectious disease" refers to a disease that can be transmitted from person to person or from organism to organism and is caused by a microorganism or virus (e.g., the common cold). Infectious diseases are known in the art and include, for example, hepatitis, sexually transmitted diseases (e.g., chlamydia, gonorrhea), tuberculosis, HIV / AIDS, diphtheria, hepatitis B, hepatitis C, cholera, and influenza.

[0395] As used herein, the terms "individual" and "subject" are often used interchangeably and refer to any animal exhibiting symptoms of an immune disorder that can be treated with the fusion polypeptides, gene-edited cells, compositions, and / or related gene editing methods contemplated elsewhere herein. Suitable subjects (e.g., patients) include laboratory animals (such as mice, rats, rabbits, or guinea pigs), farm animals, and domestic animals, or pets (such as cats or dogs). Non-human primates, and preferably human subjects, are also included. Exemplary subjects include human patients who have, have been diagnosed with, or are at risk of having an immune disorder.

[0396] As used herein, the term "patient" refers to a subject diagnosed with an immune disorder that can be treated with the fusion polypeptides, gene-edited cells, compositions, and / or related gene editing methods contemplated elsewhere herein.

[0397] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may include even a minimal reduction in one or more measurable markers of the disease or condition being treated, such as cancer, GVHD, infectious disease, autoimmune disease, inflammatory disease, and immune deficiency. Treatment may optionally include a delay in the progression of the disease or condition. "Treatment" does not necessarily indicate a complete elimination or cure of the disease or condition, or its associated symptoms.

[0398] As used herein, "prevent" and similar terms such as "prevention," "prevented," "preventing" refer to an approach for preventing, inhibiting, or reducing the likelihood of occurrence or recurrence of a disease or condition, such as cancer, GVHD, infectious disease, autoimmune disease, inflammatory disease, and immune disorders. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar terms also include reducing the intensity, effects, symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0399] As used herein, the phrase "amelioration of at least one symptom of" refers to a decrease in one or more symptoms of the disease or condition for which the subject is being treated, such as cancer, GVHD, infectious disease, autoimmune disease, inflammatory disease, and immune deficiency. In certain embodiments, the disease or condition being treated is cancer, in which case the one or more symptoms that are ameliorated include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged lymph nodes, abdominal distension or pain (caused by distended abdominal organs), bone or joint pain, bone fractures, unexpected weight loss, loss of appetite, night sweats, persistent low-grade fever, and decreased urination (caused by impaired kidney function).

[0400] In one embodiment, the fusion polypeptides, gene-edited cells, compositions, and / or related gene editing methods contemplated herein are used in methods of treating cancer. In certain embodiments, the fusion polypeptides, gene-edited cells, compositions, and / or related gene editing methods contemplated herein are used in methods of treating solid tumors or cancers.

[0401] In certain embodiments, the fusion polypeptides, gene-edited cells, compositions, and / or associated gene editing methods contemplated herein are useful in treating adrenal cancer, adrenal cortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid tumor / atypical rhabdomyoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain / CNS cancer, breast cancer, bronchial tumor, cardiac tumor, cervical cancer, bile duct cancer, chondrosarcoma, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma in situ (DCIS), endometrial cancer, epithelioma, esophageal cancer, nasal neuroblastoma, uterine cancer, thyroid ... -ing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, fibrous tissue sarcoma, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, glioma, glioblastoma, head and neck cancer, hemangioblastoma, hepatocellular carcinoma, hypopharyngeal cancer, intraocular melanoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, tongue cancer, liposarcoma, liver cancer, lung cancer, non-small cell lung cancer, embryonal carcinoid tumor, malignant mesothelioma, medullary carcinoma, medulloblastoma, meningioma, melanoma, Merkel cell carcinoma, midline carcinoma, mouth cancer, myxosarcoma, myelodysplastic syndrome, myeloproliferative neoplasm, cancer of the nasal cavity and paranasal sinuses, nasopharyngeal carcinoma, neuroblastoma, oligodendroglioma, oral cancer, oral cavity and sarcoma, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, islet cell tumor, papillary cancer, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pinealoma, pituitary tumor, pleuropulmonary blastoma, primary pleurima, prostate cancer, rectal cancer, retinoblastoma, renal cell carcinoma, cancer of the renal pelvis and ureter, rhabdomyosarcoma, salivary gland cancer, sebaceous gland cancer, skin cancer, soft tissue sarcoma, squamous cell carcinoma, small cell lung cancer, small intestine cancer, gastric cancer, sweat gland carcinoma, synovial tumor, testicular cancer, pharyngeal cancer, thymic cancer, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular cancer, vulvar cancer, and Wilms' tumor.

[0402] In certain embodiments, the fusion polypeptides, gene-edited cells, compositions, and / or related gene editing methods contemplated herein are used to treat solid tumors or cancers, including, but not limited to, liver cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, brain cancer, bone cancer, thyroid cancer, kidney cancer, or skin cancer.

[0403] In certain embodiments, the fusion polypeptides, gene-edited cells, compositions, and / or related gene editing methods contemplated herein are used to treat various cancers, including, but not limited to, pancreatic, bladder, and lung.

[0404] In certain embodiments, the fusion polypeptides, gene-edited cells, compositions, and / or related gene-editing methods contemplated herein are used to treat liquid or blood cancers.

[0405] In certain embodiments, the fusion polypeptides, gene-edited cells, compositions, and / or related gene editing methods contemplated herein are used to treat B-cell malignancies, including, but not limited to, leukemia, lymphoma, and multiple myeloma.

[0406] In certain embodiments, the fusion polypeptides, gene-edited cells, compositions, and / or associated gene editing methods contemplated herein are directed to the treatment of leukemias, lymphomas, and multiple myelomas: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, hairy cell leukemia (HCL), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML) and polycythemia vera, Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, Burkitt's lymphoma, and / or multiple myelomas. It is used to treat liquid cancers, including but not limited to lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, mycosis fungoides, anaplastic large cell lymphoma, Sezary syndrome, precursor T-lymphoblastic lymphoma, multiple myeloma, overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma, isolated plasmacytoma of bone, and extramedullary plasmacytoma.

[0407] In certain embodiments, the methods include administering a therapeutically effective amount of the fusion polypeptide, composition, and / or gene-edited cell to a patient in need thereof.

[0408] In certain embodiments, the fusion polypeptides, gene-edited cells, compositions, and / or related gene editing methods contemplated herein are used to treat a patient at risk of developing cancer. Accordingly, certain embodiments include treating or preventing or ameliorating at least one symptom of cancer by administering to a patient in need thereof a therapeutically effective amount of a fusion polypeptide, gene-edited cell, and / or composition contemplated herein.

[0409] In certain embodiments, methods are provided for treating, preventing, or ameliorating at least one symptom of a disease or a condition associated therewith, the methods comprising harvesting a cell population from a subject, editing the cell population according to the gene editing / mutagenesis methods provided herein, and administering the edited cell population to a subject in need thereof (e.g., a subject having cancer).

[0410] The amount and frequency of administration will be determined by factors such as the condition of the patient, the type and severity of the patient's disease, and appropriate dosages may be determined through clinical trials.

[0411] In one embodiment, the amount of gene-edited cells in the composition administered to a subject is at least 0.1 x 10 5 Cells, at least 0.5 x 10 5 Cells, at least 1 x 10 5 Cells, at least 5 x 10 5 Cells, at least 1 x 10 6 Cells, at least 0.5 x 10 7 Cells, at least 1 x 10 7 Cells, at least 0.5 x 10 8 Cells, at least 1 x 10 8 Cells, at least 0.5 x 10 9 Cells, at least 1 x 10 9細胞、 At least 2 × 10 9 Cells, at least 3 x 10 9 Cells, at least 4 x 10 9 Cells, at least 5 x 10 9 cells, or at least 1 × 10 10 It is a cell.

[0412] In certain embodiments, about 1×10 7 cells ~ approx. 1 x 10 9 cells, approximately 2 x 10 7 Cells ~ approx. 0.9×10 9 cells, approximately 3 x 10 7 cells ~ approx. 0.8×10 9 cells, approximately 4 x 10 7 Cells ~ approx. 0.7×10 9 cells, approximately 5 x 10 7cells ~ approx. 0.6×10 9 cells, or approximately 5 × 10 7 Cells ~ approx. 0.5×10 9 The cells are administered to a subject.

[0413] In one embodiment, the amount of gene-edited cells in the composition administered to a subject is at least 0.1 x 10 4 At least 0.5 × 10 cells / kg body weight 4 Cells / kg body weight, at least 1 x 10 4 Cells / kg body weight, at least 5 × 10 4 Cells / kg body weight, at least 1 x 10 5 At least 0.5 × 10 cells / kg body weight 6 Cells / kg body weight, at least 1 x 10 6 At least 0.5 × 10 cells / kg body weight 7 Cells / kg body weight, at least 1 x 10 7 At least 0.5 × 10 cells / kg body weight 8 Cells / kg body weight, at least 1 x 10 8 At least 2 x 10 cells / kg body weight 8 Cells / kg body weight, at least 3 × 10 8 Cells / kg body weight, at least 4 x 10 8 Cells / kg body weight, at least 5 × 10 8 cells / kg body weight or at least 1 x 10 9 cells / kg body weight.

[0414] In certain embodiments, about 1×10 6 cells / kg body weight ~ approx. 1×10 8 cells / kg body weight, approximately 2×10 6 cells / kg body weight ~ approx. 0.9×10 8 cells / kg body weight, approximately 3×10 6 cells / kg body weight ~ approx. 0.8×10 8 cells / kg body weight, approximately 4×10 6 cells / kg body weight ~ approx. 0.7×10 8 cells / kg body weight, approximately 5×10 6 cells / kg body weight ~ approx. 0.6×10 8 cells / kg body weight, or approximately 5 x 10 6cells / kg body weight ~ approx. 0.5×10 8 Cells / kg body weight are administered to the subject.

[0415] Those skilled in the art will recognize that multiple administrations of the compositions contemplated in certain embodiments may be required to achieve the desired therapeutic effect. For example, the compositions may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a period of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, 10 years, or more.

[0416] Administration of the compositions contemplated in certain embodiments may be performed in any convenient manner, including aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. In preferred embodiments, the compositions are administered nasally, orally, enterally, or parenterally. As used herein, the phrases "parenteral administration" and "parenterally administered" refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intraarticular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.

[0417] In one embodiment, an effective amount of the composition is administered to a subject in need thereof to increase the cellular immune response against the cancer of the subject. The immune response may include cellular immune response mediated by cytotoxic T cells, regulatory T cells, and helper T cell responses that can kill infected cells. Humoral immune response may also be induced, mediated mainly by helper T cells that can activate B cells, thus resulting in antibody production. Various techniques may be used to analyze the type of immune response induced by the composition, which are well described in the art, for example, in Current Protocols in Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, NY.

[0418] All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, or issued patent was specifically and individually indicated to be incorporated by reference.

[0419] The foregoing embodiments have been described in detail with reference to figures and examples for purposes of clarity and understanding, but in light of the teachings contemplated herein, it will be readily apparent to one skilled in the art that certain changes and modifications may be made without departing from the spirit or scope of the appended claims. The following examples are provided for illustrative purposes only and not for purposes of limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to produce essentially similar results. EXAMPLES

[0420] Example 1 Reproducibility of megaTAL-induced indel length and distribution Gene editing at the TRAC locus (SEQ ID NO:1) was used as a model system to characterize the indel events generated by the megaTAL nuclease. Low / moderate efficiency megaTALs (e.g., SEQ ID NOs:5-7) were used in these studies to provide a dynamic range for any downstream manipulations hypothesized to alter editing rates. Several independent experiments were performed using activated and expanded primary human T cells from separate PBMC donors. After thawing, activation, and culture of PBMCs for a period of 3-4 days, in vitro transcribed, capped, and poly-adenylated mRNA encoding a megaTAL targeting exon 1 of the TRAC locus (TCRα megaTAL) was electroporated. The resulting cells were cultured for an additional growth period of 7-10 days to allow for dilution and degradation of the delivered mRNA and completion of the editing process. Polymerase chain reaction (PCR) was used to amplify the TRAC locus, and subsequent deep sequencing of the PCR amplicons allowed characterization of bulk editing events caused by the megaTAL. A frequency histogram summarizing the sizes of editing events is shown in Figure 1B. The most frequently observed indel length was 2 base pairs long (cumulatively representing approximately 40-45% of the total population of events), while 1 base pair deletions (approximately 20%) and 3, 8, and 9 base pair deletions (5-10%, respectively) were also frequently observed. The relative frequencies of these indel size populations were highly reproducible across technical replicates and independent experiments, indicating the consistency of indel length distribution during megaTAL gene editing.

[0421] Each bar in the histogram shown in FIG. 1B represents a population of edited alleles with the same indel length but potentially different positions relative to the megaTAL target site. FIG. 2 shows each unique edited species plotted according to its position relative to the megaTAL target site breakpoint center. These "fingerprints" plot each edited species according to its length (y-axis), position (x-axis), and frequency (circle size), thereby capturing both quantitative and qualitative characteristics of each edited species. The location of the deletion was calculated as the location of its midpoint 5' (negative) or 3' (positive) to the center of the 22 base pair homing endonuclease target site. In this way, the distribution of deletion species relative to the breakpoint center could be monitored for changes in both frequency and position. Independent experiments using separate PBMC donors and using TCRα megaTAL constructs with either low (e.g., SEQ ID NOs:5-7) or high (SEQ ID NOs:8-10) editing efficiencies show that the individual deletion species, i.e., each recurrent deletion event of a particular composition, occurs at a reproducible frequency. Thus, the overall megaTAL deletion species population has relative qualitative and quantitative properties that are highly consistent and independent of the enzymatic rate of the endonuclease reaction.

[0422] Example 2 Trex2 qualitatively alters megaTAL-edited alleles Co-delivery of 3 prime repair exonuclease-2 (Trex2) and gene editing nucleases has been shown to enhance gene editing efficiency, especially in megaTALs with 3' overhanging endonuclease reaction products. However, the qualitative impact of co-delivery of Trex2 on editing outcomes has not been fully evaluated. Therefore, an evaluation of the indel characteristics of two different co-delivery modes was performed; direct fusion of Trex2 to the C-terminus of megaTALs; as well as co-expression of Trex2 with megaTALs as independent polypeptides.

[0423] Activated primary human T cell samples were electroporated with in vitro transcribed, capped, and poly-adenylated mRNA encoding the TCRα megaTAL (Figure 3A), with mRNA encoding the TCRα megaTAL-Trex2 fusion (Figure 3B), or with two separate mRNAs, one encoding the TCRα megaTAL and one encoding Trex2 (Figure 3C). The resulting cells were cultured for an additional growth period of 7-10 days to allow dilution and degradation of the delivered mRNA and completion of the editing process. Polymerase chain reaction (PCR) was used to amplify the TRAC locus, and subsequent deep sequencing of the PCR amplicons allowed characterization of bulk editing events caused by the megaTAL. Frequency histograms tabulating the size of editing events are shown in Figures 3A-3C. Direct fusion of Trex2 exonuclease to the TCRα megaTAL (e.g., SEQ ID NOs: 11-13) led to a shift in the deletion length distribution, resulting in deletions up to 12 base pairs in length, including a significant proportion of indel species. Independent co-expression of TCRα megaTAL and Trex2 exonuclease (e.g., SEQ ID NOs: 14-16) resulted in a narrowing of indel species to deletions 1-4 base pairs in length, consistent with a model of partitioning exonuclease activity with a preference for single-stranded 3' overhangs. Each Trex2 delivery scenario increased the overall editing rate and the ratio of deletions to insertions compared to delivery of the TCRα megaTAL alone.

[0424] Figure 4 shows plots of the deletion fingerprints for each sample from Figures 3A-3C, capturing both quantitative and qualitative characteristics of each edited species. As in Figure 2, the location of the deletion was calculated as its midpoint 5' (negative) or 3' (positive) to the center of the 22 base pair homing endonuclease target site. The distribution of deletion species relative to the breakpoint center showed striking differences between TCRα megaTAL, TCRα megaTAL-Trex2 fusion, and TCRα megaTAL+Trex2 co-delivery editing outcomes (see Tables 3-5). In the megaTAL-Trex2 fusion, the appearance of deletion species whose location was highly skewed toward the 5' direction was observed. This pattern suggests that there is preferential exonuclease activity at the DNA breakpoint end, either in cis with the TALE array or staying on the same side of the breakpoint as the TALE array.

[0425] Furthermore, we observed a significant reduction in deletion length and location corresponding to the 3' edge of the TALE array binding site. Nevertheless, surprisingly, we observed deletion lengths that extended into the TALE binding site. This further suggests a mutually exclusive relationship between exonuclease activity and the presence of TALEs at the binding site, further solidifying the role of the TALE array in positioning Trex2 to perform its exonuclease function. However, the deletion center remained outside the TALE binding site. Conversely, co-expression of Trex2 as a separate polypeptide did not cause any substantial directional distortion, even though it shifted the species composition towards a deletion population in the size range of 1-4 bp centered around the DNA breakpoint. [Table 3] [Table 4] [Table 5]

[0426] Example 3 Evaluation of Trex2 homologue editing alleles To confirm that Trex2 enzymatic activity was directly involved in the observed changes in editing outcomes, versus potential confounding interactions with DNA repair machinery, several Trex2 homolog ORFs were fused to the TCRα megaTAL (e.g., SEQ ID NOs: 17-34). Activated primary human T cell samples were electroporated with in vitro transcribed, capped, polyadenylated mRNA encoding these fusion proteins, and target site editing was then monitored by flow cytometric analysis of expression of the TCR complex and by characterizing indel properties.

[0427] Figure 5 shows that many of the Trex2 homolog fusion proteins enhanced global editing to a similar extent as the human Trex2 ORF. Flow cytometry staining for the CD3 component, which combines with the TCR alpha and beta chains on the cell surface, was used to assess editing efficiency. Thus, the frequency of cells that lost CD3 staining correlates with the editing rate at the TCRα locus. Analysis of the distribution of indel species, as shown in Figure 6, further confirms that platypus, opossum, armadillo, and mouse Trex2 proteins, when fused to the TCRα megaTAL, elicit editing outcomes with characteristics consistent with those elicited by the human Trex2 ORF. Remarkably, ovine Trex2 elicited a unique indel spectrum enriched for 1-4 bp deletions and, surprisingly, a high frequency of 1 bp insertions.

[0428] As shown in the fingerprint plots shown in Figure 7, the deletion characteristics of the TCRα megaTAL-Trex2 homolog fusion protein mirror those observed for the human Trex2 protein. Without wishing to be bound by any particular theory, this data suggests that Trex2 exonuclease activity is the major determinant of the observed deletion characteristics. Thus, the outcome of extended deletion lengths is likely independent of interactions with other DNA repair mechanisms. The increased occurrence of insertions observed for the ovine Trex2 homolog indicates that this variant may potentially have an intrinsic enzymatic mechanism, such as residual template-independent polymerase activity. Finally, the highly consistent and clear boundaries in deletion size and position further suggest that the directional removal of bases may be limited by the association and dissociation kinetics and / or steric properties of the TALE array interaction with its binding site.

[0429] Example 4 Identifying exonucleases that have unique effects on megaTAL editing To determine whether other exonucleases with distinct substrate specificities and / or processing properties can uniquely alter gene editing outcomes, selected exonucleases or exonuclease domain ORFs were fused to TCRα megaTAL (e.g., SEQ ID NOs: 35-55) and their impact on gene editing outcomes was examined. Editing rates were examined as TCRα megaTAL fusion proteins operating independently as well as during co-expression with Trex2. The rationale for testing in both scenarios was two-fold: (i) to allow Trex2 activity to generate distinct and potentially preferential substrates for the tested exonucleases, and (ii) to look for loss in Trex2-mediated editing rate enhancement as a proxy for ends that are significantly processed by the tested exonuclease. Figure 8 shows the results of the exonuclease screen, where each sample was run in duplicate and tested in the absence (left) or presence (right) of Trex2 co-expression.

[0430] Of the exonucleases tested, most did not appreciably alter the editing rate or editing rate enhancement by Trex2 (measured as the ratio of CD3-negative cells in Trex2 samples relative to delivery of megaTAL exonuclease fusion alone). Some significantly reduced overall editing, indicating that they may have significantly affected protein stability and / or expression. Fusion of TCRα megaTAL to ExoI resulted in a modest increase in the Trex2 enhancement ratio, suggesting that these two exonucleases were modifying the DNA cleavage processing event, possibly in a synergistic manner. Notably, fusion of TCRα megaTAL to ExoX resulted in a complete loss of Trex2 enhancement.

[0431] Example 5 Evaluation of megaTAL-ExoI and megaTAL-ExoX editing profiles To further understand the qualitative aspects of the gene editing events described in Figure 8, amplicon sequencing and deletion fingerprint analysis were performed as previously described. These exonucleases, which did not affect the pattern of TCRα megaTAL editing rates, also showed no change in the fingerprint (data not shown). However, Figure 9 shows that fusion of TCRα megaTAL to ExoI did not change the deletion profile compared to TCRα megaTAL alone, but when Trex2 was co-expressed, a series of long, directionally biased deletion events appeared.

[0432] megaTAL-ExoI fusions generate deletion events with biased orientation of the TALE array: In contrast to the well-defined megaTAL-Trex2 fusion deletion events, the megaTAL-ExoI deletion events were longer and therefore appeared to extend beyond the TALE array binding site.

[0433] Fusion of TCRα megaTAL to ExoX resulted in a deletion profile that differed from that observed with the megaTAL-ExoI fusion. In the absence of Trex2, the TCRα megaTAL-ExoX fusion generated long, directionally biased deletions with high efficiency. Unlike the megaTAL-Trex2 fusion protein, the deletion centers observed were larger such that they progressed beyond the TALE array binding site with significant frequency. It is possible that the megaTAL-ExoX fusion has an inherent exonuclease mechanism that results in longer deletions with deletion centers further away from the cleavage site compared to the megaTAL-Trex2 fusion protein.

[0434] In FIG. 9, co-expression of Trex2 with the TCRα megaTAL-ExoX fusion resulted in rare, nearly undetectable editing events and abolished the long, directionally biased deletions that were evident in the megaTAL-ExoX fusion.

[0435] Example 6 Further evaluation of megaTAL-ExoX fusion protein editing profiles Given the observed pattern of deletions that emerged when the TCRα megaTAL-ExoX fusion was evaluated, several additional megaTAL-ExoX fusion proteins targeting different gene loci were tested. Activated primary human T cell samples were electroporated with in vitro transcribed, capped, and polyadenylated mRNA encoding either a highly active CBL-B targeted megaTAL, a highly active CBL-B megaTAL-Trex2 fusion, or a highly active CBL-B megaTAL-ExoX fusion (e.g., SEQ ID NOs: 56-64). The resulting cells were cultured for an additional growth period of 7-10 days to allow for dilution and degradation of the delivered mRNA and completion of the editing process. Polymerase chain reaction (PCR) was used to amplify the CBL-B locus, and subsequent deep sequencing of the PCR amplicons allowed characterization of the editing rate and indel characteristics proximal to the CBL-B target site (SEQ ID NO: 3). Figure 10 shows the deletion fingerprints observed for each of these three highly active CBL-B megaTAL formats (see also Tables 6-8). The highly active CBL-B megaTAL generated a distribution of deletion species similar to the TCRα megaTAL, with the most frequent events being 1-5 bp deletions, and no significant directional bias. Similarly, the highly active CBL-B megaTAL-Trex2 fusion lengthened the deletion species and skewed them significantly toward the 5' direction, confirming that the orthogonal megaTAL targets preferential exonuclease activity that occurs in cis with the TALE array or at DNA breakpoint ends that remain on the same side of the breakpoint as the TALE array. Also recurrent in the highly active CBL-B megaTAL-Trex2 fusion was a significant reduction in deletion length and location corresponding to the 3' edge of the TALE array binding site. [Table 6] [Table 7] [Table 8]

[0436] FIG. 11 shows representative deletion fingerprints from T cells treated with a similar set of CBL-B megaTAL, megaTAL-Trex2, and megaTAL-ExoX fusion proteins, however, using several-fold lower activity megaTALs (e.g., SEQ ID NOs: 65-73). See also Tables 9-11. There is substantial similarity in the spectrum of deletion species observed when comparing these low activity CBL-B megaTAL and megaTAL fusion proteins with their high activity counterparts shown in FIG. 10. This indicates that the unique deletion outcomes observed when these two exonucleases are fused to megaTALs are independent of the enzymatic rate of the endonuclease reaction. These results show both highly efficient exonuclease processing rates per endonuclease-generated break, further indicating that Trex2 and ExoX clearly affect gene editing outcomes through qualitatively different mechanisms, and not simply the same mechanism occurring at different efficiencies. [Table 9] [Table 10] [Table 11]

[0437] A third series of enzymes was constructed targeting the PD-1 locus. Figure 12 shows representative deletion fingerprints from T cell electroporated mRNAs encoding either the PD-1 megaTAL, the PD-1 megaTAL-Trex2 fusion, or the PD-1 megaTAL-ExoX fusion (e.g., SEQ ID NOs: 74-82). See also Tables 12-14. As described for the TCRα and CBL-B megaTALs and the exonuclease fusion proteins, the deletion species observed for each of the three PD-1 megaTAL samples followed a consistent trend in the independently operating PD-1 megaTALs to have non-directional small deletion species, while the longer 5' oriented species that appeared when the target was exposed to the PD-1 megaTAL-Trex2 fusion protein were still species resulting from processing of the PD-1 megaTAL-ExoX. The persistence of these observed patterns across several megaTALs tested indicates the robustness of different editing outcomes occurring upon megaTAL fusion to different exonuclease domains. [Table 12] [Table 13] [Table 14]

[0438] Example 7 Assessment of PD-1 megaTAL-ExoX fusion protein editing profile Figure 13 shows the PD-1 megaTAL target site proximal to the ATG start codon of the PDCD1 gene. The 22 bp PD-1 HE target site (SEQ ID NO:2) is approximately centered on the third codon that encodes an isoleucine residue that is part of the signal sequence that targets PD-1 for expression at the plasma membrane. The TALE array binding site (SEQ ID NO:4) is 5' to the ATG start codon. This target site orientation increases the likelihood that different megaTAL or megaTAL-exonuclease fusion compositions will differentially affect PD-1 expression depending on the respective distribution of their deletion species.

[0439] The categories of deletion species were assigned according to their impact on the PD-1 ORF: ATG deletion; in-frame; frame 2; or frame 3. Examples of each deletion type are shown in FIG. 13. For the PD-1 megaTAL and megaTAL-exonuclease fusion protein samples described in FIG. 12, the frame characteristics of each deletion species were bioinformatically analyzed. FIG. 14 shows the results of this analysis by overlaying the frame categories of gene-edited species on the deletion fingerprint plot. Because PD-1 megaTAL creates a broad spectrum of deletion species, the frame characteristics are more or less evenly distributed across the deletion fingerprint analysis, with ATG deletion species occupying the upper level of the plot where long deletions are represented. Conversely, PD-1 megaTAL-Trex2 fusion protein causes a shift in the position of in-frame, frame 2, and frame 3 species. When comparing the normalized proportions of each category of species, PD-1 megaTAL and PD-1 megaTAL-Trex2 fusion are not significantly different, as shown in FIG. 15. This is because the occurrence of long directional deletions reaching through the ATG start codon increased and effectively replaced the few long (but non-directional) deletions generated in the PD-1 megaTAL.

[0440] Conversely, increasing the deletion length caused by the PD-1 megaTAL-ExoX fusion protein substantially increases the small proportion of deletion species that exclude the ATG start codon. This increase in ATG deletion alleles occurs at the expense of deletions that produce species that fall into the other three categories, further suggesting that PD-1 megaTAL-ExoX fusions may disproportionately erase undesired PD-1 gene edited alleles, such as in-frame single amino acid deletions to the N-terminus of the signal sequence that may not also affect PD-1 surface expression or function. To assess the possibility that in-frame deletions do not functionally disable the PDCD1 locus, activated primary human T cell samples were electroporated with in vitro transcribed, capped, and polyadenylated mRNAs encoding either or both cyan fluorescent proteins (CFPs) and transfection efficiency was followed, as well as wild-type or mock-edited PD-1 alleles in each of the three possible reading frames. Flow cytometry was then performed to assess transfection efficiency and PD-1 protein expression, as shown in Figure 16. Electroporation of wild-type PD-1 mRNA (SEQ ID NO: 83) resulted in high levels of protein expression, whereas artificial mRNAs encoding one or two base pair deletions (SEQ ID NOs: 84 and 85), mimicking frame 2 or frame 3 deletion species, respectively, did not promote PD-1 protein expression, as would be expected for an mRNA encoding an out-of-frame protein transcript. In contrast, artificial mRNAs encoding a three base pair deletion in the PD-1 megaTAL target site (SEQ ID NO: 86) produced high levels of PD-1 surface expression, confirming, in principle, that some PD-1 gene edited allelic species are phenotypically non-penetrant.

[0441] Example 8 Assessment of editing at off-target loci To further understand the qualitative aspects of gene editing events at off-target sites, amplicon sequencing and deletion fingerprint analysis were performed at off-target sites specific to the low- and high-edited TCRα megaTAL. Primary human T cells activated and expanded from PBMCs were electroporated with 1.5ug of mRNA encoding the low-edited TCRα megaTAL, the high-edited TCRα megaTAL (TCRα 2.2), direct fusion of each to Trex2, and direct fusion of each to ExoX. Each construct was evaluated at its on-target locus (Figure 17, in replicates, only one replicate shown) or its off-target locus (in replicates, only one replicate shown). After a 7-10 day growth period, cells were harvested for genomic DNA isolation. PCR was performed over a region of approximately 300 base pairs encompassing the KAT2B off-target site (susceptible to editing by low-edited TCRα megaTALs, FIG. 18) or the 2.2 off-target site AC016700.3 (susceptible to editing by high-edited TCRα megaTALs, FIG. 19). PCR amplicons were then subjected to next-generation sequencing (NGS) and analyzed by tabulating deletion events according to both their frequency (circle size) and their length (y-axis) and their longitudinal position relative to the megaTAL that caused the breakpoint (x-axis). Insertions were excluded from this analysis. Fusions of TCRα 2.2 to Trex2 could not be edited, so no fingerprints are shown. These exonucleases that influenced the pattern of each TCRα megaTAL editing resulted in very low editing rates compared to the on-target editing efficiency of each TCRα megaTAL, indicating changes in the fingerprints. Due to the absence of substrates for the TALE array, there does not appear to be any biased affinity distribution with any of the megaTAL fusions tested.

Claims

1. A fusion polypeptide comprising a DNA-binding domain and an I-OnuI homing endonuclease (HE) variant that binds to and cleaves a selected double-stranded DNA (dsDNA) target site in a cell, a linker domain, and an exonuclease or a biologically active fragment thereof.

2. The fusion polypeptide of claim 1 , wherein the exonuclease is Trex2, ExoI, or ExoX, or a biologically active fragment thereof.

3. The fusion polypeptide of claim 1 or 2, wherein the DNA binding domain binds to a dsDNA target site upstream of the I-OnuI HE variant dsDNA target site.

4. i) the DNA-binding domain comprises a TALE DNA-binding domain; and / or ii) the DNA-binding domain comprises a zinc finger DNA-binding domain; and / or iii) the linker domain is a peptide linker; A fusion polypeptide according to any one of claims 1 to 3.

5. The fusion polypeptide of claim 4, wherein the TALE DNA domain comprises about 9.5 TALE repeat units to about 15.5 TALE repeat units.

6. The I-OnuIHE variant dsDNA target sites include programmed cell death protein 1 (PD-1, PDCD1), lymphocyte activation gene 3 protein (LAG-3), T cell immunoglobulin domain and mucin domain protein 3 (TIM-3), cytotoxic T lymphocyte antigen-4 (CTLA-4), band T lymphocyte attenuator (BTLA), T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), V domain Ig inhibitor of T cell activation (VISTA), and killer cell immunoglobulin-like receptor ( KIR), CCR5, TRAC (TCRα), TCRβ, IL10Rα, IL10Rβ, TGFBR1, TGFBR2, CBL-B, PC SK9, AHR, BTK, α-globin, β-globin, γ-globin, BCL11A, KLF1, SOX6, GATA1, LSD1, A rufafolate receptor (FRα), αvβ6 integrin, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase IX (CAIX), CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, carcinoembryonic antigen (CEA), C-type lectin-like molecule-1 (CLL-1), CD2 subset 1 (CS-1), chondroitin sulfate proteoglycan 4 (CSPG4), cutaneous T-cell lymphoma-associated antigen 1 (CTAGE1), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), epithelial cell adhesion molecule (EPCAM), ephrin type A receptor EGFR family including body 2 (EPHA2), fibroblast activation protein (FAP), Fc receptor-like 5 (FCRL5), fetal acetylcholinesterase receptor (AchR), ganglioside G2 (GD2), ganglioside G3 (GD3), glypican-3 (GPC3), ErbB2 (HER2), IL-11Rα, IL-13Rα2, kappa, cancer / testis antigen 2 (LAGE-1A), lambda, Lewis-Y (LeY), L1 cell adhesion molecule (L1-CAM), melanoma antigen gene (MAGE)-A1, MAGE-A3, MAGE-A4, MAGE-A6,MAGEA10, Melanoma antigen recognized by T cells 1 (MelanA or MART1), Mesothelin (MSLN), MUC1, MUC16, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), Neural cell adhesion molecule (NCAM), Cancer / Testis antigen 1 (NY-ESO-1), Polysialic acid; placenta specific 1 (PLAC1), Predominantly expressed antigen in melanoma (PRAME), Prostate stem cell antigen (PSCA), Prostate specific membrane antigen (PSMA), Receptor tyrosine kinase-like orphan receptor 1 (ROR1), Synovial sarcoma 6. The fusion polypeptide of any one of claims 1 to 5, wherein the fusion polypeptide is located in a gene selected from the group consisting of: , X breakpoint 2 (SSX2), survivin, tumor associated glycoprotein 72 (TAG72), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7 related (TEM7R), TEM5, TEM8, trophoblast glycoprotein (TPBG), UL16 binding protein (ULBP) 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms' tumor 1 (WT-1) genes.

7. 7. The fusion polypeptide of claim 6, wherein the TCRα gene target site comprises the amino acid sequence set forth in SEQ ID NO:1, the CBL-B gene target site comprises the amino acid sequence set forth in SEQ ID NO:2, or the PD-1 gene target site comprises the amino acid sequence set forth in SEQ ID NO:

3.

8. The fusion polypeptide of any one of claims 2 to 7, wherein the ExoX or a biologically active fragment thereof comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:

109.

9. The fusion polypeptide of claim 8, comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 46, 64, 73, and 82.

10. The fusion polypeptide of any one of claims 2 to 7, wherein the ExoI or a biologically active fragment thereof comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:

112.

11. The fusion polypeptide of claim 10, comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:

43.

12. A polynucleotide, mRNA or vector encoding the fusion polypeptide according to any one of claims 1 to 11.

13. A cell comprising a fusion polypeptide according to any one of claims 1 to 11.

14. 1. An in vitro or ex vivo method for site-directed mutagenesis, comprising: a) selecting a double-stranded DNA (dsDNA) target site; b) introducing into said cell a fusion polypeptide according to any one of claims 1 to 11 or a polynucleotide, an mRNA or a vector according to claim 12, The method of claim 1, wherein the fusion peptide generates a directionally biased deletion with a deletion center near the selected dsDNA target cleavage site in the cell.

15. A composition comprising a fusion polypeptide according to any one of claims 1 to 11, or a polynucleotide, mRNA, or vector according to claim 12, for use in a method for site-directed mutagenesis, the method comprising: a) selecting a double-stranded DNA (dsDNA) target site; b) introducing said fusion polypeptide, or said polynucleotide, mRNA, or vector into said cell, A composition, wherein the fusion peptide generates a directionally biased deletion with the deletion center near the selected dsDNA target cleavage site in the cell.

16. The method of claim 1, wherein i) more than 50% of the directionally biased deletions have a deletion center position to one side of the center position of the I-OnuIHE variant dsDNA target site; and / or ii) the central position of the deletion is on the same side as the target site of the DNA binding domain, relative to the central position of the I-OnuI HE variant dsDNA target site; and / or iii) the central position of the deletion is upstream relative to the central position of the I-OnuI HE variant dsDNA target site; and / or iv) at least 50% of the deletions have a deletion center that is more than 4 nucleotides away from the central position of the I-OnuI HE variant dsDNA target site; and / or v) at least 10% of the deletions have a deletion center that is more than 8 nucleotides away from the central position of the I-OnuI HE variant dsDNA target site; and / or vi) at least 35% of the deletions are 12 bp or longer in length; 16. The method of claim 14 or the composition of claim 15.

17. 16. The method of claim 14 or the composition of claim 15, wherein the deletion extends within the target site of the DNA-binding domain and / or the central position of the deletion is within the target site of the DNA-binding domain.

18. 18. A composition comprising an edited cell population for use in a method of treating, preventing, or ameliorating at least one symptom of a disease or a condition associated therewith, the method comprising: harvesting a cell population from a subject; editing the cell population according to the method of any one of claims 14, 16, or 17; and administering the composition to the subject.

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