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  • C12N2510/00—Genetically modified cells

Definitions

• the present disclosure relates to improved genome editing compositions. More particularly, the disclosure relates to fusion polypeptides comprising a DNA-binding domain and a homing endonuclease variant linked to an exonuclease, compositions, and methods of using the same for site-directed mutagenesis of dsDNA within a cell.
• TALENs transcription activator-like effector nucleases
• ZFNs zinc-finger nucleases
• CRISPR clustered regularly interspaced short palindromic repeat
• HEs homing endonucleases
• NHEJ non-homologous end-joining
• HDR homology-directed repair
• Indels The most frequently observed genetic lesions resulting from the application of gene editing nucleases are insertions and deletions (customarily referred to as ‘indels’). Indels arise when double-stranded DNA breaks (DSB) are processed and re-sealed by NHEJ DNA repair machinery. NHEJ indels typically predominate gene editing events in the absence of a delivered excess of homologous DNA sequence that can divert DSBs toward various homologous recombination outcomes.
• DSB double-stranded DNA breaks
• each genetic lesion can lead to different phenotypic outcomes.
• any given genetic lesion may result in a wide array of phenotypic outcomes spanning from a total knockout of a gene, to a gain or loss of function, to no phenotype affect at all. Accordingly, there is a growing need to further understand the mechanisms that lead to different mutations upon genetic editing, and to develop compositions and methods to effectuate therapeutically meaningful genetic edits.
• the present disclosure generally relates, in part, to fusion polypeptides comprising a DNA-binding domain, a homing endonuclease variant that cleaves a target site in the human gene, a linker domain, and an exonuclease, and methods of using the same.
• a fusion polypeptide comprising, a DNA-binding domain and a homing endonuclease (HE) variant that binds and cleaves a selected double strand DNA (dsDNA) target site in a cell; a linker domain; and an exonuclease or biologically active fragment thereof.
• HE homing endonuclease
• the exonuclease is Trex2, Exol, or ExoX, or biologically active fragment thereof. In some embodiments, the exonuclease is ExoX, or biologically active fragment thereof. In some embodiments, the exonuclease is Exol, or biologically active fragment thereof. In some embodiments, the exonuclease is Trex2, or biologically active fragment thereof.
• the homing endonuclease is an engineered homing endonuclease.
• the selected dsDNA target site is a non-native homing endonuclease target site.
• the DNA-binding domain binds a dsDNA target site upstream of the endonuclease dsDNA target site.
• the DNA- binding domain comprises a TALE DNA-binding domain.
• the TALE DNA domain comprising about 9.5 TALE repeat units to about 15.5 TALE repeat units.
• the TALE DNA domain comprising 11.5 TALE repeat units or 12.5 TALE repeat units.
• the DNA-binding domain comprises a zinc finger DNA-binding domain.
• the zinc finger DNA-binding domain comprises 2, 3, 4, 5, 6, 7, or 8 zinc finger motifs.
• the linker domain is a peptide linker.
• the peptide linker is a self-cleaving peptide linker.
• the peptide linker comprises about 4 to about 30 amino acids.
• the peptide linker comprises about 10 to about 16 amino acids.
• the peptide linker comprises about 12 amino acids.
• the peptide linker is a (GGGGS)i-i linker (SEQ ID NOs: 117, 150-152).
• the peptide linker comprises a (GGGGS)2 linker (SEQ ID NO: 150).
• the HE variant is an LAGLID ADG homing endonuclease (LHE) variant.
• LHE LAGLID ADG homing endonuclease
• the HE variant lacks the 1, 2, 3, 4, 5, 6, 7, or 8 N-terminal amino acids compared to a corresponding wild type HE.
• the HE variant lacks the 4 N-terminal amino acids compared to a corresponding wild type HE.
• the HE variant lacks the 8 N- terminal amino acids compared to a corresponding wild type HE.
• the HE variant lacks the 1, 2, 3, 4, or 5 C-terminal amino acids compared to a corresponding wild type HE.
• the HE variant lacks the C- terminal amino acid compared to a corresponding wild type HE.
• the HE variant lacks the 2 C-terminal amino acids compared to a corresponding wild type HE.
• the HE variant is a variant of an LHE selected from the group consisting of: I-AabMI, I-AaeMI, I-Anil, 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-Gpil, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-Ltrll, I-Ltrl, I- LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI, I-OheMI, I-Onul, I-OsoMI, I- OsoMII, I-OsoMIII, I-
• the HE variant is a variant of an LHE selected from the group consisting of: I-CpaMI, I-HjeMI, I-Onul, I- PanMI, and I-SmaMI. In a preferred embodiments, the HE variant is an I-Onul LHE variant.
• the HE target site is within a immune system checkpoint gene, globin gene, gene that encodes a polypeptide that contributes to repression of y-globin gene expression and HbF, or immunosuppressive signaling gene.
• the HE 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 suppressor of T cell activation (VISTA), and killer cell immunoglobulin-like receptor (KIR), CCR5, TRAC (TCRa), TCRP, ILlORa, IL10RP, TGFBR1, TGFBR2, CBL-B, PCSK9
• PD-1 programmed
• the HE 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 suppressor of T cell activation (VISTA), and killer cell immunoglobulin-like receptor (KIR), CCR5, TRAC (TCRa), ILlORa, TGFBR2, CBL-B, PCSK9, AHR, BTK, a- globin, P-globin, y-globin, and BCL11 A gene.
• PD-1 programmed cell death protein 1
• LAG-3 lymphocyte activation gene 3 protein
• TIM-3 T cell immunoglobulin domain and mucin domain protein 3
• CTLA-4
• the HE target site is within a TRAC (TCRa) gene, a CBL-B gene, or a PDCD1 (PD-1) gene.
• TCRa 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.
• the PD-1 gene target site comprises the amino acid sequence set forth in SEQ ID NO: 3.
• the DNA-binding domain comprises a TALE DNA- binding domain having a target site as set forth in SEQ ID NO: 4.
• the ExoX, or biologically active fragment thereof comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence as set forth in SEQ ID NO: 109.
• the ExoX, or biologically active fragment thereof comprises an amino acid an amino acid sequence as set forth in SEQ ID NO: 109.
• the fusion polypeptide comprises an ammo acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 46, 64, 73, and 82.
• the fusion polypeptide comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 46, 64, 73, and 82.
• the Exol, or biologically active fragment thereof comprises an amino acid an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence as set forth in SEQ ID NO: 112.
• the Exol, or biologically active fragment thereof comprises an amino acid an amino acid sequence as set forth in SEQ ID NO: 112.
• the fusion polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence as set forth SEQ ID NOs: 43.
• the fusion polypeptide comprises an amino acid sequence as set forth SEQ ID NOs: 43.
• a polynucleotide encoding the fusion polypeptide contemplated herein is provided.
• the polynucleotide comprises a nucleotide sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a polynucleotide sequence as set forth in any one of SEQ ID NOs: 44, 62, 71, and 80.
• the polynucleotide comprises a polynucleotide sequence as set forth in any one of SEQ ID NOs: 44, 62, 71, and 80.
• an mRNA encoding the fusion polypeptide contemplated herein is provided.
• the mRNA comprises an RNA sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to an RNA sequence as set forth in any one of SEQ ID NOs: 45, 63, 72, and 81.
• the mRNA comprises an RNA sequence as set forth in any one of SEQ ID NOs: 45, 63, 72, and 81.
• the mRNA comprises an RNA sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to an RNA sequence as set forth in SEQ ID NOs: 42.
• the mRNA comprises an RNA sequence as set forth in any one of SEQ ID NOs: 42.
• a vector encoding the fusion polypeptide contemplated herein is provided.
• the vector comprises a polynucleotide encoding the fusion polypeptide contemplated herein.
• a cell comprising the fusion polypeptide encoding the fusion polypeptide contemplated herein.
• the cell comprises a polynucleotide encoding the fusion polypeptide contemplated herein.
• the cell comprises a vector contemplated herein.
• the cell comprises one or more genome modifications.
• 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.
• the cell is an immune effector cell.
• the immune effector cell is a cytotoxic T lymphocytes (CTLs), a tumor infiltrating lymphocytes (TILs), or a helper T cell.
• CTLs cytotoxic T lymphocytes
• TILs tumor infiltrating lymphocytes
• helper T cell a helper T cell.
• the immune effector cell is a T cell.
• the immune effector cell is an aP T cell, a y6 T cell, a natural killer (NK) cell, or a natural killer T (NKT) cell.
• a population of cells contemplated herein is provided.
• a composition comprising the fusion polypeptide contemplated herein is provided.
• a composition comprising the polynucleotides contemplated herein is provided.
• a composition comprising the mRNA contemplated herein is provided.
• a composition comprising the vector contemplated herein is provided.
• a composition comprising the cell contemplated herein is provided.
• a composition comprising the population of cells contemplated herein is provided.
• the composition comprises a pharmaceutically acceptable carrier.
• a method of site-directed mutagenesis comprising: (a) selecting a double-stranded DNA (dsDNA) target site, and (b) introducing into the cell a fusion polypeptide, polynucleotide, mRNA, or vector contemplated herein; wherein the fusion peptide generates directionally biased deletions having a deletion center location near the selected dsDNA target cut site in the cell.
• dsDNA double-stranded DNA
• greater than 50%, greater than 51%, greater than 52%, greater than 53%, greater than 54%, greater than 55%, greater than 56%, greater than 57%, greater than 58%, greater than 59%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, or greater than 80% of the directionally biased deletions have a deletion center location on one side of the HE target site center location.
• the deletion center location is on the same side as the DNA-binding domain target site relative to the HE target site center location. In a particular embodiments, the deletion center location is 5’ to the HE target site center location.
• 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 deletions have a deletion center greater than 4 nucleotides away from the HE target site center location.
• 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 deletions have a deletion center greater than 8 nucleotides away from the HE target site center location.
• 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 deletions are 6 bps in length or greater.
• 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 deletions are 12 bps in length or greater.
• the directionally biased deletions comprise a length of about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides.
• the deletion extends into the DNA-binding domain target site.
• the deletion center location is within the DNA- binding domain target site.
• the method further comprises introducing into the cell an end-processing enzyme, or or biologically active fragment thereof.
• the end-processing enzyme, or biologically active fragment thereof is selected from the group consisting of: Trex2, Trexl, Trexl without transmembrane domain, Apollo, Artemis, DNA2, Exol, ExoT, ExoIII, ExoX, Fenl, Fanl, Mrell, Rad2, Rad9, TdT (terminal deoxynucleotidyl transferase), PNKP, RecE, RecJ, RecQ, Lambda exonuclease, Sox, Vaccinia DNA polymerase, exonuclease I, exonuclease III, exonuclease VII, NDK1, NDK5, NDK7, NDK8, WRN, T7-exonuclease Gene 6, avian myeloblastosis virus integration protein (IN), Bloom, Antartic Phophata
• 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.
• a method of treating, preventing, or ameliorating at least one symptom of a disease, or condition associated therewith comprising harvesting a population of cells from a subject; editing the population of cells according to a method contemplated herein, and administering the edited population of cells to the subject.
• use of the population contemplated herein for treating, preventing, or ameliorating at least one symptom of a disease or condition associated therewith is provided.
• use of the compositions contemplated herein for treating, preventing, or ameliorating at least one symptom of a disease or condition associated therewith is provided.
• the disease or condition is an immune disorder or cancer.
• Figure 1A shows a cartoon of a TALE DNA-binding domain linked to a homing endonuclease reprogramed to target a TCRa nucleotide sequence (TCRa megaTAL).
• Figure IB shows the indel activity by next-gen sequencing (NGS) for a TCRa megaTAL.
• NGS next-gen sequencing
• Figure 2 shows indel position distributions (“fingerprints”) as assessed by NGS for both a low activity and high activity TCRa megaTALs, in two different donors.
• the NGS data are tabulated according to both their length and their longitudinal location relative to the megaTAL caused breakpoint center. Insertions are excluded from this analysis.
• Figure 3A - 3C show indel activity by NGS for a TCRa megaTAL (Figure 3A), TCRa megaTAL-Trex2 fusion ( Figure 3B), and a TCRa megaTAL + Trex2 co-expression ( Figure 3C).
• the NGS data reads containing indels characteristic of gene editing events are tabulated according to their length.
• Figure 4 shows indel position distributions (“fingerprints”) as assessed by NGS for a TCRa megaTAL, TCRa megaTAL-Trex2 fusion, and TCRa megaTAL + Trex2 coexpression.
• the NGS data are tabulated according to both their length and their longitudinal location relative to the megaTAL caused breakpoint center. Insertions are excluded from this analysis.
• Figure 5 shows editing efficiency of TCRa megaTALs fused to various Trex2 homologs as assessed by staining for the CD3 components that combine with the TCR alpha and beta chains on the cell surface, followed by flowcytometry analysis.
• Figure 6 shows indel activity by NGS for TCRa megaTALs fused to Trex2 homologs.
• the NGS data reads containing indels characteristic of gene editing events are tabulated according to their length.
• Figure 7 shows indel position distributions (“fingerprints”) as assessed by NGS for a TCRa megaTALs fused to Trex2 platypus, opossum, human, and mouse homologues.
• the NGS data are tabulated according to both their length and their longitudinal location relative to the megaTAL caused breakpoint center. Insertions are excluded from this analysis.
• Figure 8 shows editing efficiency of TCRa megaTALs fused to exonucleases RADI, RAD9A, Exol, ExoX, T5FEN, lanbdaExo, and RecJ, with or without co-expression of mouse Trex2.
• Trex2 homologs as assessed by staining for the CD3 expression followed by flowcytometry analysis.
• Figure 9 shows indel position distributions (“fingerprints”) as assessed by NGS for a TCRa megaTALs alone, and TCRa megaTALs fused to exonucleases Exol or ExoX, with or without co-expression of Trex2.
• the NGS data are tabulated according to both their length and their longitudinal location relative to the megaTAL caused breakpoint center. Insertions are excluded from this analysis.
• Figure 10 shows indel position distributions (“fingerprints”) as assessed by NGS for high-activity CBL-B megaTALs alone or fused to Trex2 or ExoX.
• the NGS data are tabulated according to both their length and their longitudinal location relative to the megaTAL caused breakpoint center. Insertions are excluded from this analysis.
• Figure 11 shows indel position distributions (“fingerprints”) as assessed by NGS for low-activity CBL-B megaTALs alone or fused to Trex2 or ExoX.
• the NGS data are tabulated according to both their length and their longitudinal location relative to the megaTAL caused breakpoint center. Insertions are excluded from this analysis.
• Figure 12 shows indel position distributions (“fingerprints”) as assessed by NGS for PD-1 megaTALs alone or fused to Trex2 or ExoX.
• the NGS data are tabulated according to both their length and their longitudinal location relative to the megaTAL caused breakpoint center. Insertions are excluded from this analysis.
• Figure 13 shows the PDCD1 gene, the 22 base pair PD-1 megaTAL target site, and the 13 base TALE array binding site (SEQ ID NOs 154 and 159), as well as 4 representative deletion species (SEQ ID NOs: 155-158 and 160-162) from the experiment shown in Figure 12.
• Figure 14 shows indel position distributions (“fingerprints”) as assessed by NGS for PD-1 megaTALs alone or fused to Trex2 or ExoX.
• the deletion species are coded to indicate their resulting reading frame category.
• the NGS data are tabulated according to both their length and their longitudinal location relative to the megaTAL caused breakpoint center. Insertions are excluded from this analysis.
• Figure 15 shows stacked histograms quantifying the normalized fraction of each of the four deletion species categories shown in Figure 14.
• Figure 16 shows flow cytometry analysis of activated primary human T cells electroporated with poly-adenylated mRNA encoding either or both a cyan fluorescent protein (CFP) to track transfection efficiency, and wild-type or mock-edited PD-1 alleles in each of the three possible reading frames.
• CFP cyan fluorescent protein
• Figure 17 shows indel position distributions (“fingerprints”) as assessed by NGS for a low editing TCRa megaTAL, high editing TCRa megaTAL (TCRa 2.2), as well as direct fusions of each to Trex2 or ExoX.
• the NGS data are tabulated according to both their length and their longitudinal location relative to the megaTAL caused breakpoint center. Insertions are excluded from this analysis.
• Figure 18 shows indel position distributions (“fingerprints”) at the known KAT2B off-target site for the low-editing TCRa megaTAL, with and without direct fusion to Trex2 or ExoX.
• the NGS data are tabulated according to both their length and their longitudinal location relative to the megaTAL caused breakpoint center. Insertions are excluded from this analysis.
• Figure 19 shows indel position distributions (“fingerprints”) at the known AC016700.3 off-target site (SEQ ID NOs: 163 and 164) for the high-editing TCRa megaTAL (TCRa 2.2), with and without direct fusion to ExoX.
• the NGS data are tabulated according to both their length and their longitudinal location relative to the megaTAL caused breakpoint center. Insertions are excluded from this analysis.
• SEQ ID NOs: 1-3 are illustrative homing endonuclease target sites.
• SEQ ID NO: 4 is PD-1 TALE array target site.
• SEQ ID NO: 5-7 are low activity TCRa megaTAL DNA, RNA, and protein sequences.
• SEQ ID NO: 8-10 are high activity TCRa megaTAL DNA, RNA, and protein sequences.
• SEQ ID NO: 11-13 are low activity TCRa megaTAL-Trex2 DNA, RNA, and protein sequences.
• SEQ ID NOs: 14-16 are Trex2 exonuclease DNA, RNA, and protein sequences.
• SEQ ID NOs: 17-55 are DNA, RNA and protein sequences for low activity TCRa megaTALs fused to Trex2 homologs.
• SEQ ID NOs: 56-64 are DNA, RNA, and protein sequences for high activity CBL- B megaTALs with and without fusion to Trex2 or ExoX.
• SEQ ID NO: 65-73 are DNA, RNA, and protein sequences for low activity CBL-B megaTALs with and without fusion to Trex2 or ExoX.
• SEQ ID NO: 74-82 are DNA, RNA, and protein sequences for PD-1 megaTALs with and without fusion to Trex2 or ExoX.
• SEQ ID NO: 83 is an mRNA sequence encoding PD-1.
• SEQ ID NO: 84 is an mRNA sequence encoding a mock-edited PD-1 open reading frame (ORF) comprising a 1 bp deletion at codon 3.
• SEQ ID NO: 85 is an mRNA sequence encoding a mock-edited PD-1 open reading frame (ORF) comprising a 2 bp deletion at codon 3.
• SEQ ID NO: 86 is an mRNA sequence encoding a mock-edited PD-1 open reading frame (ORF) comprising a 3 bp deletion at codon 3.
• SEQ ID NOs: 87-101 are DNA, RNA, and protein sequences for TCRa, CBL-B, and PD-1 homing endonucleases.
• SEQ ID NOs: 102-106 are wild type I-Onul endonucleases and portions thereof.
• SEQ ID NOs: 107-109 are ExoX exonuclease DNA, RNA, and protein sequences.
• SEQ ID NOs: 110-112 are Exol exonuclease DNA, RNA, and protein sequences.
• SEQ ID NO: 113-123 sets forth the amino acid sequence of various linkers.
• SEQ ID NOs: 124-148 sets forth the amino acid sequence of protease cleavage sites and self-cleaving polypeptide cleavage sites.
• X refers to any amino acid or the absence of an amino acid.
• the present disclosure generally relates to, in part, improved genome editing compositions and methods of use thereof.
• the genome editing compositions contemplated herein are used to 1) increase the size of deletions induced by a genetic edit to a specific target size and 2) bias the deletion center location.
• the deletion center locations are predominantly on the same side as the DNA-binding domain target site relative to the original breakpoint center or endonuclease target site center.
• the deletion center locations are biased to 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 genetic lesions at or proximal to a breakpoint, one can more precisely control the phenotypic outcome of the genomic edit, for example, disruption of regulatory DNA sequences and/or target gene expression.
• NHEJ indels The most frequently observed genetic lesions resulting from the application of gene editing nucleases are NHEJ indels. Little is known regarding how the NHEJ machinery arrives at a given indel that is re-sealed and resistant to further DNA cleavage, thus becoming enshrined as a permanent genotype within a pool of gene edited alleles. However, it is known that indel events are almost exclusively observed at, and directly proximal to, the DNA sequence where nuclease generated breakpoint(s) occur. Previous studies with various nuclease platforms (ZFN, TALEN, and CRISPR) have suggested that the gene edited alleles show some measure of consistency with respect to the qualitative properties of the observed indels.
• the qualitative properties that define a given indel are: (i) its length, in number of bases inserted or deleted; (ii) its longitudinal position along the chromosome, usually stated relative to the nuclease target site or breakpoint; and (iii) for insertions, the inserted sequence length and composition.
• Deletions are the most prominent outcomes, typically comprising 90-95% of the observed events. Their most commonly reported size characteristics tend to small (i.e. 1-20 base pairs in length, with the frequency biased toward the low end of that range) and their positional distributions have been found to be evenly distributed, covering the DNA breakpoint and emanating outward in either direction without significant bias.
• the fusion polypeptides comprise a DNA- binding domain, a homing endonuclease variant, and an end-processing enzyme (e.g, exonuclease).
• the exonuclease is ExoX, Exol, or a biologically active fragment thereof.
• the fusion polypeptides induce elongated genetic deletions having directionally biased deletion centers on the same side as the DNA-binding domain target site, or 5’, to that of the original edit/breakpoint.
• the fusion polypeptides generate deletion center locations encompassed by the DNA-binding domain target site.
• the fusion polypeptides generate deletions that extend into (or are encompassed by) the DNA- binding domain target site.
• the fusion polypeptide comprises a DNA-binding domain and a homing endonuclease (HE) variant that binds and cleaves a selected double strand DNA (dsDNA) target site in a cell, a polypeptide linker and an exonuclease (e.g., ExoX or Exol), or biologically active fragment thereof.
• HE homing endonuclease
• vectors, polynucleotides, mRNA, or cDNA encoding the fusion polypeptides are contemplated.
• genome edited cells are contemplated.
• compositions comprising the fusion polypeptides, vectors, polynucleotides, mRNA, cDNA or cells are contemplated.
• methods of genome editing, site-directed mutagenesis, increasing the length of site-directed mutagenic deletions, biasing the location of deletions, and treating subjects in need thereof are contemplated.
• compositions and methods contemplated herein represent a significant improvement compared to existing gene editing strategies because it allows for strategic control and selection of desired mutagenic outcomes.
• Techniques for 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 as cited and discussed throughout the present specification.
• the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
• the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ⁇ 15%, ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, or ⁇ 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
• a range e.g., 1 to 5, about 1 to 5, or about 1 to about 5, refers to each numerical value encompassed by the range.
• the range “1 to 5” is equivalent to the expression 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.
• the term “substantially” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
• “substantially the same” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that produces an effect, e.g., a physiological effect, that is approximately the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
• ex vzvo refers generally to activities that take place outside an organism, such as experimentation or measurements done in or on living tissue in an artificial environment outside the organism, preferably with minimum alteration of the natural conditions.
• ex vivo procedures involve living cells or tissues taken from an organism and cultured or modulated in a laboratory apparatus, usually under sterile conditions, and typically for a few hours or up to about 24 hours, but including up to 48 or 72 hours, depending on the circumstances.
• tissues or cells can be collected and frozen, and later thawed for ex vivo treatment. Tissue culture experiments or procedures lasting longer than a few days using living cells or tissue are typically considered to be “z « vitro.” though in certain embodiments, this term can be used interchangeably with ex vivo.
• zzz vivo refers generally to activities that take place inside an organism.
• cellular genomes are engineered, edited, or modified in vivo.
• promote or “promote” or “increase” or “expand” or “potentiate” refers generally to the ability of a fusion polypeptide, nuclease variant, genome editing composition, or genome edited cell contemplated herein to produce, elicit, or cause a greater response (i.e., physiological response) compared to the response caused by either vehicle or control.
• a measurable response may include an increase in editing events (e.g., indels), deletions, insertions, deletion length, and/or target gene expression, among others apparent from the understanding in the art and the description herein.
• An “increased” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g, 1.5, 1.6, 1.7. 1.8, etc.) the response produced by vehicle or control.
• a measurable response may include a decrease in editing events (e.g, indels), deletions, insertions, deletion length, target gene expression, and/or one or more symptoms associated with a disease.
• a “decrease” or “reduced” amount is typically a “statistically significant” amount, and may include a decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g, 1.5, 1.6, 1.7. 1.8, etc.) the response (reference response) produced by vehicle, or control.
• maintain or “preserve,” or “maintenance,” or “no change,” or “no substantial change,” or “no substantial decrease” refers generally to the ability of a fusion polypeptide, nuclease variant, genome editing composition, or genome edited cell contemplated herein to produce, elicit, or cause a substantially similar or comparable physiological response (i.e., downstream effects) in as compared to the response caused by either vehicle or control.
• a comparable response is one that is not significantly different or measurable different from the reference response.
• binding affinity or “specifically binds” or “specifically bound” or “specific binding” or “specifically targets” as used herein, describe binding of one molecule to another, e.g, DNA-binding domain of a polypeptide binding to DNA, at greater binding affinity than background binding.
• a binding domain “specifically binds” to a target site if it binds to or associates with a target site with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) of, for example, greater than or equal to about 10 5 M 4 .
• a binding domain binds to a target site with a Ka greater than or equal to about 10 6 M 4 , 10 7 M 4 , 10 8 M 4 , 10 9 M 4 , IO 10 M 4 , 10 11 M 4 , 10 12 M 4 , or 10 13 M 4 .
• “High affinity” binding domains refers to those binding domains with a Ka of at least 10 7 M 4 , at least 10 8 M 4 , at least 10 9 M’ J , at least IO 10 M 4 , at least 10 11 M 4 , at least 10 12 M 4 , at least 10 13 M 4 , or greater.
• affinity may be defined as an equilibrium dissociation constant (Ka) of a particular binding interaction with units of M (e.g, 10' 5 M to 10 43 M, or less).
• Ka equilibrium dissociation constant
• Affinities of nuclease variants comprising one or more DNA-binding domains for DNA target sites contemplated in particular embodiments can be readily determined using conventional techniques, e.g, yeast cell surface display, or by binding association, or displacement assays using labeled ligands.
• the affinity of specific binding is about 2 times greater than background binding, about 5 times greater than background binding, about 10 times greater than background binding, about 20 times greater than background binding, about 50 times greater than background binding, about 100 times greater than background binding, or about 1000 times greater than background binding or more.
• an HE or megaTAL selectively binds an on-target DNA-binding site about 5, 10, 15, 20, 25, 50, 100, or 1000 times more frequently than the HE or megaTAL binds an off-target DNA target binding site.
• On-target refers to a target site sequence.
• Off-target refers to a sequence similar to but not identical to a target site sequence.
• 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 will bind and/or cleave, provided sufficient conditions for binding and/or cleavage exist.
• a polynucleotide sequence or SEQ ID NO. that references only one strand of a target site or target sequence, it would be understood that the target site or target sequence bound and/or cleaved by a nuclease variant is double-stranded and comprises the reference sequence and its complement.
• the target site is in an immune system checkpoint gene, globin gene, gene that encodes a polypeptide that contributes to repression of y-globin gene expression and/or HbF, or immunosuppressive signaling gene.
• the target site is a sequence in the human TRAC gene, CBL-B gene, or the PDCD1 gene.
• Recombination refers to a process of exchange of genetic information between two polynucleotides, including but not limited to, donor capture by non-homologous end joining (NHEJ) and homologous recombination.
• NHEJ non-homologous end joining
• HR homologous recombination
• HDR homology- directed repair
• This process requires nucleotide sequence homology, uses a “donor” molecule as a template to repair a “target” molecule (i.e., the one that experienced the double-strand break), and is variously known as “non-crossover gene conversion” or “short tract gene conversion,” because it leads to the transfer of genetic information from the donor to the target.
• a “donor” molecule i.e., the one that experienced the double-strand break
• non-crossover gene conversion or “short tract gene conversion”
• such transfer can involve mismatch correction of heteroduplex DNA that forms between the broken target and the donor, and/or “synthesis-dependent strand annealing,” in which the donor is used to resynthesize genetic information that will become part of the target, and/or related processes.
• Such specialized HR often results in an alteration of the sequence of the target molecule such that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.
• “Cleavage” refers to the breakage 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 a phosphodiester bond. Both single-stranded cleavage and double-stranded cleavage are possible. Double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends.
• polypeptides and nuclease variants are used for targeted double-stranded DNA cleavage.
• Endonuclease cleavage recognition sites may be on either DNA strand.
• “Directionally biased” or “directionally biased deletion” refers to the location of deletions created by a cell’s endogenous repair machinery in response to a double-strand DNA break induced by an endonuclease. If the deletions are directionally biased, they will predominately occur either 5’ or 3’ relative to the target cut site or double-strand DNA break (the breakpoint or breakpoint center).
• the fusion polypeptides induce genetic deletions having directionally bias deletions occurring on the same side as DNA-binding domain target site, relative to the breakpoint center or HE target site center location.
• the fusion polypeptides induce genetic deletions having directionally biased deletion centers 5’ to that of the original breakpoint center or HE target site center location.
• the directionally biased deletions are also elongated.
• the amount of directionally biased deletions or elongation induced by the fusion proteins contemplated herein can be compared to the distribution of deletions or length of deletions produced by the co-expression of the same exonuclease and a fusion polypeptide comprising the same DNA-binding domain and same homing endonuclease.
• exogenous molecule is a molecule that is not normally present in a cell, but that is introduced into a cell by one or more genetic, biochemical or other methods.
• exogenous molecules include, but are not limited to small organic molecules, protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules.
• lipid-mediated transfer i.e., liposomes, including neutral and cationic lipids
• electroporation direct injection, cell fusion, particle bombardment, biopolymer nanoparticle, calcium phosphate coprecipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer.
• 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.
• a “gene,” refers to a DNA region encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences.
• a gene includes, but is not limited to, promoter sequences, enhancers, silencers, insulators, boundary elements, terminators, polyadenylation sequences, post-transcription response elements, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, replication origins, matrix attachment sites, and locus control regions.
• Gene expression refers to the conversion of the information, contained in a gene, into a gene product.
• a gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA).
• Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.
• 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. Genetic modifications may be targeted or non-targeted to a particular site in a cell's genome. In one embodiment, genetic modification is site-specific. In one embodiment, genetic modification is not site-specific.
• Genome editing refers to the substitution, deletion, and/or introduction of genetic material at a target site in the cell's genome, which restores, corrects, disrupts, and/or modifies expression of a gene or gene product.
• Genome editing contemplated in particular embodiments comprises introducing one or more nuclease variants into a cell to generate DNA lesions at or proximal to a target site in the cell's genome, optionally in the presence of a donor repair template.
• gene therapy refers to the introduction of extra genetic material into the total genetic material in a cell that restores, corrects, or modifies expression of a gene or gene product, or for the purpose of expressing a therapeutic polypeptide.
• introduction of genetic material into the cell's genome by genome editing that restores, corrects, disrupts, or modifies expression of a gene or gene product, or for the purpose of expressing a therapeutic polypeptide is considered gene therapy.
• Fusion polypeptides contemplated in particular embodiments herein that are suitable for editing a target site comprise a DNA-binding domain, a homing endonuclease, and an end-processing domain (e.g., an exonuclease).
• the fusion polypeptides comprise a DNA-binding domain and a homing endonuclease variant, a polypeptide linker, and an exonuclease (e.g, ExoX), or biologically active fragment thereof.
• the fusion polypeptides comprise, in N-terminal to C- terminal order, 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.
• an exonuclease e.g, ExoX
• the DNA-binding domain is a TALE DNA-binding domain or a zinc finger DNA binding domain.
• the homing endonuclease variant is an engineered nuclease.
• the terms “reprogrammed nuclease,” “engineered nuclease,” or “nuclease variant” are used interchangeably and refer to a nuclease comprising one or more DNA- binding domains and one or more DNA cleavage domains (e.g. , a nuclease or homing endonuclease), wherein the nuclease has been designed and/or modified from a parental 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-native target sequence or site.
• fusion polypeptides that bind and cleave a target sequence include, but are not limited to, megaTALs linked by a linker domain (e.g., a polypeptide linker) to an exonuclease (e.g, an ExoX exonuclease), or biologically active fragment thereof.
• linker domain e.g., a polypeptide linker
• exonuclease e.g, an ExoX exonuclease
• the fusion polypeptides are useful for creating positionally biased and/or elongated deletions compared to deletions induced by similar non-fused/linked polypeptides, e.g, a separate megaTAL and exonuclease when introduced into a cell together.
• the deletions are more directionally biased compared to deletions induced by similar non-fused/linked polypeptides.
• the fusion polypeptides contemplated herein create substantially more deletions on one side of a breakpoint over the other.
• the described fusion polypeptides induce substantially more mutagenic deletions having a deletion center 5’ to the breakpoint or nuclease target site center location.
• a homing endonuclease or meganuclease is reprogrammed to introduce double-strand breaks (DSBs) at a target site within a target gene.
• DLBs double-strand breaks
• “Homing endonuclease” and “meganuclease” are used interchangeably and refer to naturally- occurring nucleases that recognize 12-45 base-pair cleavage sites (e.g., a target site) and are commonly grouped into five families based on sequence and structure motifs: LAGLID ADG, GIY-YIG, HNH, His-Cys box, and PD-(D/E)XK.
• 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 basal activity.
• an “engineered homing endonuclease,” “reprogrammed homing endonuclease,” “homing endonuclease variant,” “engineered meganuclease,” “reprogrammed meganuclease,” or “meganuclease variant” refers to a homing endonuclease comprising one or more DNA-binding domains and one or more DNA cleavage domains, wherein the homing endonuclease has been designed and/or modified from a parental or naturally occurring homing endonuclease, to bind and cleave a DNA target sequence or site.
• the homing endonuclease variant may be designed and/or modified from a naturally occurring homing endonuclease or from another homing endonuclease variant.
• HE variants do not exist 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 alterations, e.g., mutating, substituting, adding, or deleting one or more amino acids, in a naturally occurring HE or HE variant.
• a HE variant comprises one or more amino acid alterations to the DNA recognition interface.
• HE variants contemplated in particular embodiments may further comprise one or more linkers and/or additional functional domains, e.g, an end-processing enzymatic domain of an end-processing enzyme that exhibits 5 '-3' exonuclease, 5 '-3' alkaline exonuclease, 3'-5' exonuclease (e.g., Trex2, Exol, or ExoX), 5' flap endonuclease, helicase, template-dependent DNA polymerase or template-independent DNA polymerases activity.
• polypeptides comprising an HE variant linked by a linker domain (e.g., a polypeptide linker) to an end-processing enzyme are provided.
• the end-possessing enzyme exhibits 3 '-5' exonuclease activity.
• the end-processing enzyme is a Trex2, Exol, or ExoX.
• the HE variant and end-processing enzyme may be introduced separately, e.g, in different vectors or separate mRNAs, or together, e.g, as a fusion protein, or in a polycistronic construct separated by a viral self-cleaving peptide or an IRES element.
• a “DNA recognition interface” refers to the HE amino acid residues that interact with nucleic acid target bases as well as those residues that are adjacent.
• the DNA recognition interface comprises an extensive network of side chain-to-side chain and side chain-to-DNA contacts, most of which is necessarily unique to recognize a particular nucleic acid target sequence.
• the amino acid sequence of the DNA recognition interface corresponding to a particular nucleic acid sequence varies significantly and is a feature of any natural or HE variant.
• a HE variant contemplated in particular embodiments may be derived by constructing libraries of HE variants in which one or more amino acid residues localized in the DNA recognition interface of the natural HE (or a previously generated HE variant) are varied.
• the libraries may be screened for target cleavage activity against each predicted BTK target site using cleavage assays (see e.g., Jaijour et al., 2009. Nuc. Acids Res. 37(20): 6871-6880).
• LAGLID ADG homing endonucleases are the most well studied family of homing endonucleases, are primarily encoded in archaea and in organellar DNA in green algae and fungi, and display the highest overall DNA recognition specificity. LHEs comprise one or two LAGLID ADG catalytic motifs per protein chain and function as homodimers or single chain monomers, respectively. Structural studies of LAGLID ADG proteins identified a highly conserved core structure (Stoddard 2005), characterized by an aPPaPPa fold, with the LAGLID ADG motif belonging to the first helix of this fold.
• LHEs The highly efficient and specific cleavage of LHEs represents a protein scaffold to derive novel, highly specific endonucleases.
• engineering LHEs to bind and cleave a nonnatural or non-canonical target site requires selection of the appropriate LHE scaffold, examination of the target locus, selection of putative target sites, and extensive alteration of the LHE to alter its DNA contact points and cleavage specificity, at up to two-thirds of the base-pair positions in a target site.
• LHEs from which reprogrammed LHEs or LHE variants may be designed include, but are not limited to I-Crel and I-Scel.
• LHEs from which reprogrammed LHEs or LHE variants may be designed include, but are not limited to I-AabMI, I-AaeMI, I-Anil, 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-Gpil, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-Ltrll, I- Ltrl, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI, I-OheMI, I-Onul, I-OsoMI, I- OsoMII, I
• the reprogrammed LHE or LHE variant is selected from the group consisting of: an I-CpaMI variant, an I-HjeMI variant, an I-Onul variant, an I-PanMI variant, and an I-SmaMI variant.
• reprogrammed I-Onul LHEs or I-Onul variants targeting a target gene can be generated from a natural I-Onul or biologically active fragment thereof (SEQ ID NOs: 102-106).
• target gene is an immune system checkpoint gene, a globin gene, a gene that encodes a polypeptide that contributes to repression of y-globin gene expression and/or HbF, or an immunosuppressive signaling gene.
• 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 suppressor of T cell activation (VISTA), and killer cell immunoglobulin-like receptor (KIR), CCR5, TRAC (TCRa), TCR , ILlORa, IL10RP, TGFBR1, TGFBR2, CBL-B, PCSK9, AHR, BTK, a-globin, 0-globin, y-globin, BCL11A, KLF1, SOX6, GATA1, LSD, alpha folate receptor (FRa), avP6 integrin, B cell maturation antigen (BCMA), B
• 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 suppressor of T cell activation (VISTA), and killer cell immunoglobulin-like receptor (KIR), CCR5, TRAC (TCRa), ILlORa, TGFBR2, CBL- B, PCSK9, AHR, BTK, a-globin, P-globin, y-globin, and BCL11A gene.
• PD-1 programmed cell death protein 1
• LAG-3 lymphocyte activation gene 3 protein
• TIM-3 T cell immunoglobulin domain and mucin domain protein 3
• CTLA-4 cytotoxic T lymphocyte antigen
• the target gene is TRAC (TCRa), CBL-B, or PDCD1 (PD-1) gene.
• the target gene is a TRAC gene, CBL-B gene, or PD-1 gene.
• the target gene/ site comprises the nucleotide sequences set forth in SED ID NO: 1, 2, or 3.
• reprogrammed I-Onul LHEs or I-Onul variants targeting the human TRAC (TCRa) gene were generated from an existing I-Onul variant.
• reprogrammed I-Onul LHEs were generated against a human TRAC gene target site set forth in SEQ ID NO: 1.
• reprogrammed I-Onul LHEs or I-Onul variants targeting the human PDCD1 (PD-1) gene were generated from an existing I-Onul variant.
• reprogrammed I-Onul LHEs were generated against a human PD-1 gene target site set forth in SEQ ID NO: 2.
• reprogrammed I-Onul LHEs or I-Onul variants targeting the human CBL-B gene were generated from an existing I-Onul variant.
• reprogrammed I-Onul LHEs were generated against a human CBL-B gene target site set forth in SEQ ID NO: 3.
• an I-Onul LHE variant that binds and cleaves the a target gene comprises one or more amino acid substitutions or modifications in the DNA recognition interface of an I-Onul as set forth in any one of SEQ ID NOs: 89, 92, 95, 98, 101 or 102-106, biologically active fragments thereof, and/or further variants thereof.
• the reprogrammed I-Onul LHE or I-Onul variant that binds and cleaves a target gene comprises one or more amino acid substitutions in the DNA recognition interface.
• the I-Onul LHE that binds and cleaves a target gene comprises 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 with the DNA recognition interface of I-Onul (Taekuchi etal.
• the I-Onul LHE that binds and cleaves a 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 with the DNA recognition interface of I-Onul (Taekuchi et al. 2011. Proc Natl Acad Sci U S. A. 2011 Aug 9; 108(32): 13077-13082) or an I-Onul LHE variant as set forth in SEQ ID NOs: 89, 92, 95, 98, 101 or 102-106, or further variants thereof.
• an I-Onul LHE variant that binds and cleaves the target gene comprises one or more amino acid substitutions or modifications in the DNA recognition interface, particularly in the subdomains situated from positions 24-50, 68-82, 180-203 and 223-240 of I-Onul (SEQ ID NOs: 102-106) an I-Onul variant as set forth in SEQ ID NOs: 89, 92, 95, 98, and 101, biologically active fragments thereof, and/or further variants thereof.
• an I-Onul LHE variant that binds and cleaves the target gene comprises one or more amino acid substitutions or modifications in the DNA recognition interface at amino acid positions selected from the group consisting of: 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 68, 70, 72, 75, 76, 78, 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 of I-Onul (SEQ ID NOs: 103-107) or an I-Onul variant as set forth in SEQ ID NOs: 89, 92, 95, 98, and 101, biologically active fragments thereof, and/or further variants thereof.
• an I-Onul LHE variant that binds and cleaves the target gene comprises one or more amino acid substitutions or modifications at additional positions situated anywhere within the entire I-Onul sequence.
• the residues which may be substituted and/or modified include but are not limited to amino acids that contact the nucleic acid target or that interact with the nucleic acid backbone or with the nucleotide bases, directly or via a water molecule.
• a I-Onul LHE variant contemplated herein that binds and cleaves the target gene comprises one or more substitutions and/or modifications, 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 in at least one position selected from the position group consisting of 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, 195, 197, 199, 201, 203, 210, 223, 225, 227, 229, 231, 232, 234, 236, 238, 240, and 246, of
• an I-Onul LHE variant that binds and cleaves the target gene comprises 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: 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, 195, 197, 199, 201, 203, 210, 223, 225, 227, 229, 231, 232, 234, 236, 238, 240, and 246 of I-Onul SEQ ID NOs: 102-106 or an I-Onul variant as set forth in SEQ ID NOs: 89, 92
• an I-Onul LHE variant that binds and cleaves the 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.
• an I-Onul 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.
• an I-Onul LHE variant comprises an amino acid sequence set forth in SEQ ID NO: 89, or a biologically active fragment thereof.
• an I-Onul LHE variant comprises an amino acid sequence set forth in SEQ ID NO: 92, or a biologically active fragment thereof.
• an I-Onul LHE variant comprises an amino acid sequence set forth in SEQ ID NO: 95, or a biologically active fragment thereof.
• an I-Onul LHE variant comprises an amino acid sequence set forth in SEQ ID NO: 98, or a biologically active fragment thereof.
• an I-Onul LHE vanant compnses an amino acid sequence set forth in SEQ ID NO: 101, or a biologically active fragment thereof.
• a fusion polypeptide contemplated herein comprises a DNA-binding domain.
• the DNA-binding domain is located N- terminal to that of the homing endonuclease.
• the fusion polypeptide comprises a DNA-binding domain positioned N-terminal to that of a homing endonuclease, which is N-terminal to that of an end-processing domain (e.g. , an exonuclease).
• an end-processing domain e.g. , an exonuclease
• an illustrative fusion polypeptide contemplated herein comprises a DNA-binding domain, a first polypeptide linker, a homing endonuclease, a second polypeptide linker, and an exonuclease.
• the DNA-binding domain comprises a TALE DNA-binding domain.
• a fusion polypeptide comprises a megaTAL.
• a “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 further comprises one or more linkers and/or additional functional domains, e.g, an endprocessing enzymatic domain of an end-processing enzyme that exhibits 5 '-3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonuclease (e.g, Trex2, Exol or ExoX), 5' flap endonuclease, helicase or template-independent DNA polymerases activity.
• a megaTAL comprising a homing endonuclease variant is reprogrammed to introduce double-strand breaks (DSBs) in a target gene.
• a megaTAL comprising a homing endonuclease variant is reprogrammed to introduce a double-strand break in a target sequence in an immune system checkpoint gene, a globin gene, a gene that encodes a polypeptide that contributes to repression of y-globin gene expression and/or HbF, or an immunosuppressive signaling gene.
• a megaTAL comprising a homing endonuclease variant is reprogrammed to introduce a double-strand break in a target sequence in a human TRAC gene, PD-1 gene, or CBL-B gene (e.g, SEQ ID NOs: 1-3, respectively).
• a “TALE DNA-binding domain” is the DNA-binding portion of transcription activator-like effectors (TALE or TAL-effectors), which mimics plant transcriptional activators to manipulate the plant transcriptome (see e.g, Kay etal., 2007. Science 318: 648-651).
• TALE DNA-binding domains contemplated in particular embodiments are engineered de novo or from naturally occurring TALEs, e.g., AvrBs3 from Xanthomonas campestris pv. vesicatoria, Xanthomonas gardneri, Xanthomonas translucens, Xanthomonas axonopodis, Xanthomonas perforans, Xanthomonas alfalfa, Xanthomonas citri, Xanthomonas euvesicatoria, and Xanthomonas oryzae and brgl l and hpxl7 from Ralstonia solanacearum.
• TALE proteins for deriving and designing DNA-binding domains are disclosed in U.S. Patent No. 9,017,967, and references cited therein, all of which are incorporated herein by reference in their entireties.
• the TALE DNA-binding domain comprising one or more repeat units that are involved in 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 in length.
• Each TALE DNA-binding domain repeat unit includes 1 or 2 DNA-binding residues making up the Repeat Variable Di -Residue (RVD), typically at positions 12 and/or 13 of the repeat.
• RVD Repeat Variable Di -Residue
• the natural (canonical) code for DNA recognition of these TALE DNA-binding domains has been determined such that an HD sequence at positions 12 and 13 leads to a binding to cytosine (C), NG binds to T, NI to A, NN binds to G or A, and NG binds to T.
• C cytosine
• NG binds to T
• NI to A NI to A
• NN binds to G or A
• NG binds to T.
• non-canonical (atypical) RVDs are contemplated.
• Non-canonical RVDs suitable for use in particular megaTALs include, but are not limited to HH, KH, NH, NK, NQ, RH, RN, SS, NN, SN, KN for recognition of guanine (G); NI, KI, RI, HI, SI for recognition of adenine (A); NG, HG, KG, RG for recognition of thymine (T); RD, SD, HD, ND, KD, YG for recognition of cytosine (C); NV, HN for recognition of A or G; and H*, HA, KA, N*, NA, NC, NS, RA, S* for recognition of A or T or G or C, wherein (*) means that the amino acid at position 13 is absent.
• fusion polypeptide or megaTAL contemplated herein comprises a TALE DNA-binding domain comprising 3 to 30 repeat units.
• 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.
• the TALE DNA-binding domain comprising 5-15 repeat units, more preferably 7-15 repeat units, more preferably 9-15 repeat units, and more preferably 9, 10, 11, 12, 13, 14, or 15 repeat units.
• fusion polypeptide or a megaTAL contemplated herein comprises a TALE DNA-binding domain comprising 3 to 30 repeat units and an additional single truncated TALE repeat unit comprising 20 amino acids located at the C-terminus of a 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 elsewhere herein, infra).
• a fusion polypeptide or megaTAL contemplated herein comprises a TALE DNA-binding domain comprising 3.5 to 30.5 repeat units.
• a fusion polypeptide or megaTAL comprises 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5,
• a fusion polypeptide or megaTAL contemplated herein comprises a TALE DNA-binding domain comprising 5.5-15.5 repeat units, more preferably 7.5-15.5 repeat units, more preferably 9.5-15.5 repeat units, and more preferably 9.5, 10.5, 11.5, 12.5, 13.5,
• a fusion polypeptide or megaTAL contemplated herein comprises a TAL effector architecture 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.
• NTD N-terminal domain
• CTD C-terminal domain
• the NTD, TALE repeats, and/or CTD domains are from the same species.
• one or more of the NTD, TALE repeats, and/or CTD domains are from different species.
• NTD N-terminal domain
• the NTD sequence if present, may be of any length as long as the TALE DNA-binding domain repeat units retain the ability to bind DNA.
• the NTD polypeptide comprises at least 120 to at least 140 or more amino acids N-terminal to the TALE DNA-binding domain (0 is amino acid 1 of the most N- terminal repeat unit).
• 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.
• a fusion polypeptide or megaTAL contemplated herein comprises an NTD polypeptide of at least about amino acids +1 to +122 to at least about +1 to +137 of a Xanthomonas TALE protein (0 is amino acid 1 of the most N-terminal repeat unit).
• 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.
• a fusion polypeptide or megaTAL contemplated herein comprises an NTD polypeptide of at least amino acids +1 to +121 of aRalstonia TALE protein (0 is amino acid 1 of the mostN- terminal repeat unit).
• 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 aRalstonia TALE protein.
• CTD C -terminal domain
• the CTD sequence if present, may be of any length as long as the TALE DNA-binding domain repeat units retain the ability to bind DNA.
• the CTD polypeptide comprises at least 20 to at least 85 or more amino acids C-terminal to the last full repeat of the TALE DNA-binding domain (the first 20 amino acids are the half-repeat unit C-terminal to the last C-terminal full repeat unit).
• 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, 443, 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 full repeat of the TALE DNA-binding domain.
• a fusion polypeptide or megaTAL contemplated herein comprises a CTD polypeptide of at least about amino acids -20 to -1 of Xanthomonas TALE protein (-20 is amino acid 1 of a half-repeat unit C- terminal to the last C-terminal full repeat unit).
• 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 full repeat of the TALE DNA-binding domain of aXanthomonas TALE protein.
• a fusion polypeptide or megaTAL contemplated herein comprises a CTD polypeptide of at least about amino acids -20 to -1 of aRalstonia TALE protein (-20 is amino acid 1 of a half-repeat unit C-terminal to the last C-terminal full repeat unit).
• 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 full repeat of the TALE DNA-binding domain of a Ralstonia TALE protein.
• a fusion polypeptide or megaTAL contemplated herein comprises a TALE DNA-binding domain engineered to bind a target sequence, a homing endonuclease reprogrammed to bind and cleave a target sequence/site, and optionally an NTD and/or CTD polypeptide, optionally joined to each other with one or more linker polypeptides contemplated elsewhere herein. It is further contemplated that a fusion polypeptide or megaTAL comprising TALE DNA-binding domain, and optionally an NTD and/or CTD polypeptide, may be fused to a linker polypeptide which is further fused to a homing endonuclease variant.
• the TALE DNA-binding domain binds a DNA target sequence that is within 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.
• the fusion polypeptides or megaTALs contemplated herein increase the specificity and efficiency of genome editing.
• a fusion polypeptide or megaTAL comprises a homing endonuclease variant and a TALE DNA-binding domain that binds a nucleotide/target sequence/site that is within about 4, 5, or 6 nucleotides, preferably, 6 nucleotides upstream of the binding/target site of the reprogrammed homing endonuclease.
• a fusion polypeptide or megaTAL contemplated herein comprises one or more TALE DNA-binding repeat units and an LHE variant designed or reprogrammed from an LHE selected from the group consisting of: I-AabMI, I-AaeMI, I- Anil, 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-Gpil, I-GzeMI, I-GzeMII, I-GzeMIII, I- HjeMI, I-Ltrll, I-Ltrl, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI, I-OheMI
• a fusion polypeptide or megaTAL contemplated herein comprises an NTD, one or more TALE DNA-binding repeat units, a CTD, and an LHE variant selected from the group consisting of: I-AabMI, I-AaeMI, I-Anil, 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-Gpil, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-Ltrll, I- Ltrl, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI, I-OheMI
• a fusion polypeptide or megaTAL contemplated herein comprises an NTD, about 9.5 to about 15.5 TALE DNA-binding repeat units, and an LHE variant selected from the group consisting of: I-AabMI, I-AaeMI, I-Anil, 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-Gpil, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-Ltrll, I- Ltrl, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI, I-OheMI,
• 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-Onul LHE variant.
• NTD NTD of about 122 amino acids to 137 amino acids
• CTD CTD of about 20 amino acids to about 85 amino acids
• I-Onul LHE variant any one of, two of, or all of the NTD, DNA-binding domain, and CTD can be designed from the same species or different species, in any suitable combination.
• 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 an end-processing domain (e.g., exonuclease), or biologically active fragment thereof, contemplated elsewhere herein.
• a fusion polypeptide contemplated herein comprises, in N-terminal to C-terminal order, 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-Onul LHE variant, a second linker domain, and an end-processing enzyme (e.g, 3’ to 5’ exonuclease) or biologically active fragment thereof.
• any one of, two of, or all of the TALE binding repeats can be designed from the same species or different species, in any suitable combination.
• the exonuclease is an ExoX exonuclease, or biologically active fragment thereof.
• a fusion polypeptide or megaTAL contemplated herein comprises the amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 7, 10, 58, 67, or 76.
• a fusion polypeptide or megaTAL contemplated herein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 7, 10, 58, 67, or 76.
• 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 an amino acid sequence as set forth in any one of SEQ ID NO: 13, 61, 70, or 79.
• a fusion polypeptide or megaTAL-Trex2 fusion protein contemplated herein comprises the amino acid sequence set forth in any one of SEQ ID NO: 13, 61, 70, or 79.
• 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 sequences as set forth in any one of SEQ ID NO: 46, 64, 73, or 82.
• a fusion polypeptide or megaTAL-ExoX fusion protein contemplated herein comprises the amino acid sequence set forth in any one of SEQ ID NO: 46, 64, 73, or 82.
• a fusion polypeptide or megaTAL-Exol 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 as set forth in SEQ ID NO: 43.
• a fusion polypeptide or megaTAL-Exol fusion protein contemplated herein comprises the amino acid sequence set forth in SEQ ID NO: 43.
• 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 a mRNA sequence as set forth in any one of SEQ ID NO: 6, 9, 57, 66, or 75.1n certain embodiments, a fusion polypeptide or megaTAL contemplated herein, is encoded by an mRNA sequence set forth in any one of SEQ ID NO: 6, 9, 57, 66, or 75.
• 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 a mRNA sequence as set forth in any one of SEQ ID NO: 12, 60, 69, or 78.
• a fusion polypeptide or megaTAL-Trex2 contemplated herein is encoded by an mRNA sequence set forth in any one of SEQ ID NO: 12, 60, 69, or 78.
• 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 a mRNA sequence as set forth in any one of SEQ ID NO: 45, 63, 72, or 81.
• a fusion polypeptide or megaTAL-ExoX contemplated herein is encoded by an mRNA sequence set forth in any one of SEQ ID NO: 45, 63, 72, or 81.
• a fusion polypeptide or megaTAL-Exol contemplated herein is encoded by an mRNA sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a mRNA sequence as set forth in SEQ ID NO: 42.
• a fusion polypeptide or megaTAL-Exol contemplated herein is encoded by an mRNA sequence set forth in SEQ ID NO: 42.
• 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 a DNA sequence as set forth in any one of SEQ ID NO: 5, 8, 56, 65, or 74.
• a fusion polypeptide or megaTAL contemplated herein is encoded by a DNA sequence set forth in any one of SEQ ID NO: 5, 8, 56, 65, or 74.
• 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 a DNA sequence as set forth in any one of SEQ ID NO: 11, 59, 68, or 77.
• a fusion polypeptide or megaTAL-Trex2 contemplated herein is encoded by a DNA sequence set forth in any one of SEQ ID NO: 11, 59, 68, or 77.
• 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 as set forth in any one of SEQ ID NO: 44, 62, 71, or 80.
• a fusion polypeptide or megaTAL-ExoX contemplated herein is encoded by a DNA sequence set forth in any one of SEQ ID NO: 44, 62, 71, or 80.
• a fusion polypeptide or megaTAL-Exol contemplated herein is encoded by a DNA sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a DNA sequence as set forth in SEQ ID NO: 41.
• a fusion polypeptide or megaTAL-Exol contemplated herein is encoded by a DNA sequence set forth in SEQ ID NO: 41.
• a megaTAL comprises a TALE DNA-binding domain and an I-Onul LHE variant the binds and cleaves the nucleotide sequence set forth in any one of SEQ ID NOs: 1-3.
• the DNA-binding domain comprises a zinc finger DNA-binding domain.
• the fusion polypeptides contemplated herein comprise a zinc finger DNA-binding domain, a homing endonuclease domain contemplated elsewhere herein, and an end-processing domain (e.g., exonuclease) contemplated elsewhere herein.
• the zinc finger DNA-binding domain that has one, two, three, four, five, six, seven, or eight or more zinc finger motifs.
• a single zinc finger motif is about 30 amino acids in length.
• Zinc fingers motifs include both canonical C2H2 zinc fingers, and non-canonical zinc fingers such as, for example, CsH zinc fingers and C4 zinc fingers.
• Zinc finger DNA-binding domains can be engineered to bind any DNA sequence.
• Candidate zinc finger DNA-binding domains for a given 3 bp DNA target sequence have been identified and modular assembly strategies have been devised for linking a plurality of the domains into a multi-finger peptide targeted to the corresponding composite DNA target sequence.
• Other suitable methods known in the art can also be used to design and construct nucleic acids encoding zinc finger DNA-binding domains, e.g., phage display, random mutagenesis, combinatorial libraries, computer/rational design, affinity selection, PCR, cloning from cDNA or genomic libraries, synthetic construction and the like. (See, e.g, U.S. Pat. No.
• Individual zinc finger motifs bind to a three or four nucleotide sequence.
• the length of a sequence to which a zinc finger binding domain is engineered to bind (e.g. , a target sequence) will determine the number of zinc finger motifs in an engineered zinc finger DNA-binding domain. For example, when the zinc finger motifs do not bind to overlapping subsites, a six-nucleotide target sequence is bound by a two-finger DNA- binding domain; a nine-nucleotide target sequence is bound by a three-finger DNA-binding domain, etc.
• DNA-binding sites for individual zinc fingers motifs in a target site need not be contiguous, but can be separated by one or several nucleotides, depending on the length and nature of the linker sequences between the zinc finger motifs in a multi-finger binding domain.
• 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 an end-processing domain (e.g, exonuclease).
• the homing endonuclease is a LHE variant LHE variant designed or reprogrammed from an LHE selected from the group consisting of: I-AabMI, I-AaeMI, I-Anil, 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-Gpil, I-GzeMI, I-GzeMII, I-GzeMIII, I- HjeMI, I-Ltrll, I-Ltrl, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI, I-OheMI, I- Onul, I-OsoMI
• fusion polypeptides contemplated herein comprise, a zinc finger DNA-binding domain comprising 2, 3, 4, 5, 6, 7, or 8 or more zinc finger motifs, a homing endonuclease variant contemplated elsewhere herein, and an end-processing domain (e.g, exonuclease), or biologically active fragment thereof, contemplated elsewhere herein.
• a fusion polypeptide contemplated herein comprises, in N-terminal to C-terminal order, 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-Onul LHE vanant, a second linker domain, and an end-processing domain (e.g., exonuclease) or biologically active fragment thereof.
• exonuclease is an ExoX exonuclease, or biologically active fragment thereof.
• Genome editing compositions e.g., fusion polypeptides
• methods contemplated in particular embodiments comprise editing cellular genomes using a DNA- binding domain, a homing endonuclease variant, and an end-processing enzyme.
• a 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).
• end-processing enzyme refers to an enzyme that modifies the 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 (for example, containing bases other than A, C, G, and T).
• An end-processing enzyme 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.
• An end-processing enzyme may modify ends at endonuclease cut sites or at ends generated by other chemical or mechanical means, such as shearing (for example by passing through fine-gauge needle, heating, sonicating, mini bead tumbling, and nebulizing), ionizing radiation, ultraviolet radiation, oxygen radicals, chemical hydrolysis and chemotherapy agents.
• genome editing compositions and methods contemplated in particular embodiments comprise editing cellular genomes using a fusion polypeptide comprising a DNA-binding domain, a homing endonuclease variant, and a DNA end-processing enzyme.
• DNA end-processing enzyme refers to an enzyme that modifies the exposed ends of DNA.
• a DNA end-processing enzyme may modify blunt ends or staggered ends (ends with 5' or 3' overhangs).
• a DNA end-processing enzyme may modify single stranded or double stranded DNA.
• a DNA end-processing enzyme may modify ends at endonuclease cut sites or at ends generated by other chemical or mechanical means, such as shearing (for example by passing through fine-gauge needle, heating, sonicating, mini bead tumbling, and nebulizing), ionizing radiation, ultraviolet radiation, oxygen radicals, chemical hydrolysis and chemotherapy agents.
• DNA end-processing enzyme 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.
• DNA end-processing enzymes suitable for use in particular embodiments contemplated herein include, but are not limited to: 5'-3' exonucleases, 5'-3' alkaline exonucleases, 3 '-5' exonucleases, 5' flap endonucleases, helicases, phosphatases, hydrolases and template-independent DNA polymerases.
• DNA end-processing enzymes suitable for use in particular embodiments contemplated herein include, but are not limited to, Trex2, Trexl, Trexl without transmembrane domain, Apollo, Artemis, DNA2, Exol, ExoT, ExoIII, ExoX, Fenl, Fanl, Mrell, Rad2, Rad9, TdT (terminal deoxynucleotidyl transferase), PNKP, RecE, RecJ, RecQ, Lambda exonuclease, Sox, Vaccinia DNA polymerase, exonuclease I, exonuclease III, exonuclease VII, NDK1, NDK5, NDK7, NDK8, WRN, T7-exonuclease Gene 6, avian myeloblastosis virus integration protein (IN), Bloom, Antartic Phophatase, Alkaline Phosphatase, Poly nucleotide Kina
• genome editing compositions and methods for editing cellular genomes contemplated herein comprise fusion polypeptides comprising a DNA- binding domain, a homing endonuclease variant, and an exonuclease.
• exonuclease refers to enzymes that cleave phosphodi ester bonds at the end of a polynucleotide chain via a hydrolyzing reaction that breaks phosphodiester bonds at either the 3' or 5' end.
• the exonuclease is a 3 '-5' exonuclease.
• the exonuclease is an ExoX exonuclease, or biologically active fragment thereof.
• the exonuclease is an Exol exonuclease, or biologically active fragment thereof. In some embodiments, the exonuclease is a Trex2 exonuclease, or biologically active fragment thereof.
• ExoX is an 3 -5 distributive exonuclease from Escherichia coh (E. coli) and a member of the DnaQ superfamily. ExoX is also referred to as Exodeoxyribonuclease 10, Exodeoxyribonuclease X, Exonuclease X, and Exo X.
• Exemplary ExoX reference sequence numbers used in particular embodiments include, but are not limited to:NP_416358.1; NC_000913.3; WP_000944256.1; NZ_STEB01000009.1AAC74914.
• a fusion polypeptide comprises a DNA-binding domain, a homing endonuclease variant, and an ExoX exonuclease, or a biologically active fragment thereof.
• the fusion polypeptide comprises, a DNA-binding domain and a homing endonuclease variant, linked by a linker domain (e.g., a polypeptide linker) to an ExoX exonuclease, or a biologically active fragment thereof.
• the ExoX comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence as set forth in SEQ ID NO: 109. In some embodiments, the ExoX comprises an amino acid sequence having at least 85% identity to an amino acid sequence as set forth in SEQ ID NO: 109. In some embodiments, the ExoX comprises an amino acid sequence having at least 90% identity to an amino acid sequence as set forth in SEQ ID NO: 109. In some embodiments, the ExoX comprises an amino acid sequence having at least 95% identity to an amino acid sequence as set forth in SEQ ID NO: 109.
• the ExoX comprises an amino acid sequence having at least 96% identity to an amino acid sequence as set forth in SEQ ID NO: 109. In some embodiments, the ExoX comprises an amino acid sequence having at least 97% identity to an amino acid sequence as set forth in SEQ ID NO: 109. In some embodiments, the ExoX comprises an amino acid sequence having at least 98% identity to an amino acid sequence as set forth in SEQ ID NO: 109. In some embodiments, the ExoX comprises an amino acid sequence having at least 99% identity to an amino acid sequence as set forth in SEQ ID NO: 109.
• the ExoX or biologically active fragment thereof, comprises an amino acid an amino acid sequence as set forth in SEQ ID NO: 109.
• Exol is an 3 '-5' processive exonuclease from Escherichia coli (E. coli) and a member of the DnaQ superfamily. Exol is also referred to as Exodeoxyribonuclease I, Exonuclease I, DNA deoxyribophosphodiesterase, and dRPase. Exemplary Exol reference sequence numbers used in particular embodiments include, but are not limited to: NP_416515.1, NC_000913.3, WP_000980589.1, NZ_LN832404.1.
• a fusion polypeptide comprises a DNA-binding domain, a homing endonuclease variant, and an Exol exonuclease, or a biologically active fragment thereof.
• the fusion polypeptide comprises, a DNA-binding domain and a homing endonuclease variant, linked by a linker domain (e.g., a polypeptide linker) to an Exol exonuclease, or a biologically active fragment thereof.
• the Exol comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence as set forth in SEQ ID NO: 112. In some embodiments, the Exol comprises an amino acid sequence having at least 85% identity to an amino acid sequence as set forth in SEQ ID NO: 112. In some embodiments, the Exol comprises an amino acid sequence having at least 90% identity to an amino acid sequence as set forth in SEQ ID NO: 112. In some embodiments, the Exol comprises an amino acid sequence having at least 95% identity to an amino acid sequence as set forth in SEQ ID NO: 112.
• the Exol comprises an amino acid sequence having at least 96% identity to an amino acid sequence as set forth in SEQ ID NO: 112. In some embodiments, the Exol comprises an amino acid sequence having at least 97% identity to an amino acid sequence as set forth in SEQ ID NO: 112. In some embodiments, the Exol comprises an amino acid sequence having at least 98% identity to an amino acid sequence as set forth in SEQ ID NO: 112. In some embodiments, the Exol comprises an amino acid sequence having at least 99% identity to an amino acid sequence as set forth in SEQ ID NO: 112.
• the Exol, or biologically active fragment thereof comprises an amino acid an amino acid sequence as set forth in SEQ ID NO: 112.
• Homing endonuclease variants contemplated in particular embodiments can be designed to bind to any suitable target sequence (e.g., a sequence within the human genome) and can have a novel binding specificity, compared to a naturally-occurring nuclease.
• the target site is a regulatory region of a gene including, but not limited to promoters, enhancers, repressor elements, and the like.
• the target site is a coding region of a gene or a splice site.
• a nuclease variant and donor repair template can be designed to insert a therapeutic polynucleotide.
• nuclease variant and donor repair template can be designed to insert a therapeutic polynucleotide under control of endogenous gene regulatory elements or expression control sequences.
• nuclease variants bind to and cleave a target sequence in a immune system checkpoint gene, globin gene, gene that encodes a polypeptide that contributes to repression of y-globin gene expression and/or HbF, or immunosuppressive signaling gene.
• immune system checkpoint genes include, but are not limited to: PD-1, LAG-3, TIM-3, CTLA-4, BTLA, TIGIT, VISTA, and KIRs.
• genes encoding immunosuppressive signaling components include, but are not limited to: IL-lORa, TGF R1, TGF R2, AHR, SGK1, TSC2, VHL, A2AR, and CBL-B.
• polypeptides that repress y-globin gene expression and HbF include, but are not limited to: BCL 11 A, KLF 1 , SOX6, GATA1 , and LSD 1.
• nuclease variants bind to and cleave a target sequence 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 suppressor of T cell activation (VISTA), and killer cell immunoglobulin-like receptor (KIR), CCR5, TRAC (TCRa), TCR , ILlORa, IL10RP, TGFBR1, TGFBR2, CBL-B, PCSK9, AHR, BTK, a-globin, 0-globin, y-globin, BCL11A, KLF1, SOX6, GATA1, LSD, alpha folate receptor (FRa),
• 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 suppressor of T cell activation (VISTA), and killer cell immunoglobulin- like receptor (KIR), CCR5, TRAC (TCRa), ILlORa, TGFBR2, CBL-B, PCSK9, AHR, BTK, a-globin, P-globin, y-globin, and BCL11A gene.
• PD-1 programmed cell death protein 1
• LAG- 3 lymphocyte activation gene 3 protein
• TIM-3 T cell immunoglobulin domain and mucin domain protein 3
• CTLA-4 cytotoxic T lymphocyte antigen-4
• the target gene is TRAC (TCRa), CBL-B, or PDCD1 (PD-1) gene.
• T-cell receptor alpha (TRAC) locus is a protein that in humans is encoded by the TRA gene.
• TRAC is also referred to as TRA, IMD7, TCRA, TRA@, TRAC, T-cell receptor alpha locus, TCRD, T cell receptor alpha locus, TCRa. It contributes the alpha chain to the larger TCR protein (T-cell receptor).
• Alpha-beta T cell receptors are antigen specific receptors which are essential to the immune response and are present on the cell surface of T lymphocytes. They recognize peptide-major histocompatibility (MH) (pMH) complexes that are displayed by antigen presenting cells (APC), a prerequisite for efficient T cell adaptive immunity against pathogens.
• MH peptide-major histocompatibility
• a nuclease variant introduces a DSB in exon 1 of the constant region of the human TCRa gene, preferably at SEQ ID NO: 1 in exon 1 of the constant region of the human TCRa gene, and more preferably at the sequence “ATTC” in SEQ ID NO: 1 in exon 1 of the constant region of the human TCRa gene.
• the TCRa gene is a human TCRa gene.
• the homing endonuclease variants bind to and cleave a target sequence in a program 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 which has potential phosphorylation sites located with immune tyrosine-based inhibitory motif (ITIM) and immune receptor inhibitory tyrosine-based switch motif (ITSM).
• ITIM immune tyrosine-based inhibitory motif
• ITDM immune receptor inhibitory tyrosine-based switch motif
• 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 through 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-y (IFN-y), tumor necrosis factor-a, and IL-2 production, and reduces T-cell survival.
• IFN-y interferon-y
• IL-2 tumor necrosis factor-a
• IL-2 interferon-y
• 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 infections and cancer, is characterized by T-cell dysfunction, resulting in suboptimal control of infections and tumors.
• a homing endonuclease variant introduces a doublestrand break (DSB) in exon 1 of a PD-1 gene, preferably at SEQ ID NO: 2 in exon 1 of a PD-1 gene, and more preferably at the sequence “ATCC” in SEQ ID NO: 2 in exon 1 of a PD-1 gene.
• a homing endonuclease variant or megaTAL cleaves double-stranded DNA and introduces a DSB into the polynucleotide sequence set forth in SEQ ID NO: 2.
• the PD-1 gene is a human PD-1 gene.
• homing endonuclease variants bind to and cleave a target sequence in a human casitas B-lineage (Cbl) lymphoma proto-oncogene B (CBLB) gene.
• Cbl casitas B-lineage
• CBLB proto-oncogene B
• 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, folic acid type, rare, fra (11 )(q23.3) (FRA1 IB); E3 ubiquitin-protein ligase CBL; Cas-Br-M (murine) 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.
• This gene is a proto-oncogene that encodes a RING finger E3 ubiquitin ligase.
• the encoded protein is one of the enzymes required for targeting substrates for degradation by the proteasome. 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 that allows it to interact with numerous tyrosine- phosphorylated substrates and target them for proteasome degradation.
• CBLB functions as a negative regulator of many signal transduction pathways, including T cell activation and persistence.
• a homing endonuclease variant introduces a doublestrand break (DSB) in a target site in a CBLB gene.
• a homing endonuclease variant or megaTAL introduces a DSB in exon 6 of a CBLB gene, preferably at SEQ ID NO: 3 in exon 6 of a CBLB gene, and more preferably at the sequence “ATTC” in SEQ ID NO: 3 in exon 6 of a CBLB gene.
• a homing endonuclease variant cleaves double-stranded DNA and introduces a DSB into the polynucleotide sequence set forth in SEQ ID NO: 3.
• the CBLB gene is a human CBLB gene.
• Fusion polypeptides may be used to introduce a DSB in a target sequence; the DSB may be repaired through homology directed repair (HDR) mechanisms in the presence of one or more donor repair templates.
• the donor repair template is used to insert a sequence into the genome.
• the donor repair template is used to insert a polynucleotide sequence encoding a therapeutic polypeptide.
• the donor repair template is used to insert a polynucleotide sequence encoding a therapeutic polypeptide, such that the expression of the polypeptide is under control of the endogenous promoter and/or enhancers.
• a donor repair template is introduced into a hematopoietic cell, e.g, a hematopoietic stem or progenitor cell, or CD34 + cell, by transducing the cell with an adeno-associated virus (AAV), retrovirus, e.g., lentivirus, IDLV, etc., herpes simplex virus, adenovirus, or vaccinia virus vector comprising the donor repair template.
• AAV adeno-associated virus
• retrovirus e.g., lentivirus, IDLV, etc.
• herpes simplex virus e.g., lentivirus, IDLV, etc.
• vaccinia virus vector comprising the donor repair template.
• the donor repair template comprises one or more homology arms that flank the DSB site.
• the term “homology arms” refers to a nucleic acid sequence in a donor repair template that is identical, or nearly identical, to DNA sequence flanking the DNA break introduced by the nuclease at a target site.
• the donor repair template comprises a 5' homology arm that comprises a nucleic acid sequence that is identical or nearly identical to the DNA sequence 5' of the DNA break site.
• the donor repair template comprises a 3 ' homology arm that comprises a nucleic acid sequence that is identical or nearly identical to the DNA sequence 3' of the DNA break site.
• the donor repair template comprises a 5 ' homology arm and a 3' homology arm.
• the donor repair template may comprise homology to the genome sequence immediately adjacent to the DSB site, or homology to the genomic sequence within any number of base pairs from the DSB site.
• the donor repair template comprises a nucleic acid sequence that is homologous to a genomic sequence 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 length of homologous sequence.
• suitable lengths of homology arms may be independently selected, and 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, 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 longer homology arms, including all intervening lengths of homology arms.
• 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 homology arms.
• the lengths of the 5' and 3' homology arms are independently selected from about 500 bp to about 1500 bp.
• the 5 'homology arm is about 1500 bp and the 3' homology arm is about 1000 bp.
• the 5 'homology arm is from about 200 bp to about 600 bp and the 3' homology arm is from about 200 bp to about 600 bp.
• the 5 'homology arm is about 200 bp and the 3 ' homology arm is about 200 bp.
• the 5 'homology arm is about 300 bp and the 3' homology arm is about 300 bp.
• 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.
• polypeptides are contemplated herein, including, but not limited to, fusion polypeptides, including homing endonuclease variants and/or megaTALs.
• a polypeptide comprises the 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, unless specified to the contrary, and according to conventional meaning, i.e., as a sequence of amino acids.
• a “polypeptide” includes fusion polypeptides and other variants.
• Polypeptides can be prepared using any of a variety of well-known recombinant and/or synthetic techniques. Polypeptides are not limited to a specific length, e.g., they may comprise a full-length protein sequence, a fragment of a full length protein, or a fusion protein, and may include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
• isolated protein refers to in vitro synthesis, isolation, and/or purification of a peptide or polypeptide molecule from a cellular environment, and from association with other components of the cell, i.e., it is not significantly associated with in vivo substances.
• polypeptides contemplated in particular embodiments include, but are not limited to homing endonuclease variants, megaTALs, end-processing nucleases, exonucleases, fusion polypeptides and variants thereof.
• Polypeptides include “polypeptide variants.” Polypeptide variants may differ from a naturally occurring polypeptide in one or more amino acid substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more amino acids of the above polypeptide sequences. For example, in particular embodiments, it may be desirable to improve the biological properties of a fusion polypeptide, homing endonuclease, megaTAL or the like that binds and cleaves a target site by introducing one or more substitutions, deletions, additions and/or insertions into the polypeptide.
• polypeptides include polypeptides 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, typically where the variant maintains at least one biological activity of the reference sequence.
• Polypeptides variants include biologically active “polypeptide fragments.”
• biologically active polypeptide fragments include DNA-binding domains, nuclease domains, endo-processing domains (e.g, exonucleases) and the like.
• 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.
• the biological activity is binding affinity and/or cleavage activity for a target sequence.
• a polypeptide fragment can comprise an amino acid chain at least 5 to about 1700 amino acids long. It will be appreciated that in certain embodiments, fragments are 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 long.
• a polypeptide comprises a biologically active fragment of a homing endonuclease variant.
• the polypeptides set forth herein may comprise one or more amino acids denoted as “X.” “X” if present in an amino acid SEQ ID NO, refers to any amino acid. One or more “X” residues may be present at the N- and C-terminus of an amino acid sequence set forth in particular SEQ ID NOs contemplated herein. If the “X” amino acids are not present the remaining amino acid sequence set forth in a SEQ ID NO may be considered a biologically active fragment.
• a polypeptide comprises a biologically active fragment of ahoming endonuclease variant, e.g, SEQ ID NOs: 89, 92, 95, 98, or 101, or a megaTAL (e.g., SEQ ID NOs: 7, 10, 58, 67, and 76).
• the biologically active fragment may comprise an N-terminal truncation and/or C-terminal truncation.
• a biologically active fragment lacks or comprises a deletion of the 1, 2, 3, 4, 5, 6, 7, or 8 N- terminal amino acids of a homing endonuclease variant compared to a corresponding wild type homing endonuclease sequence, more preferably a deletion of the 4 N-terminal amino acids of a homing endonuclease variant compared to a corresponding wild type homing endonuclease sequence.
• a biologically active fragment lacks or comprises a deletion of the 1, 2, 3, 4, or 5 C-terminal amino acids of ahoming endonuclease variant compared to a corresponding wild type homing endonuclease sequence, more preferably a deletion of the 2 C-terminal amino acids of a homing endonuclease variant compared to a corresponding wild type homing endonuclease sequence.
• a biologically active fragment lacks or comprises a deletion of the 4 N-terminal amino acids and 2 C-terminal amino acids of a homing endonuclease variant compared to a corresponding wild type homing endonuclease sequence.
• an I-Onul vanant compnses a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 the following N-terminal amino acids: M, A, Y, M, S, R, R, E; and/or a deletion of the following 1, 2, 3, 4, or 5 C-terminal amino acids: R, G, S, F, V.
• an I-Onul variant comprises a deletion or substitution of 1, 2, 3, 4, 5, 6, 7, or 8 the following N-terminal amino acids: M, A, Y, M, S, R, R, E; and/or a deletion or substitution of the following 1, 2, 3, 4, or 5 C-terminal amino acids: R, G, S, F, V.
• an I-Onul variant comprises a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 the following N-terminal amino acids: M, A, Y, M, S, R, R, E; and/or a deletion of the following 1 or 2 C-terminal amino acids: F, V.
• an I-Onul variant comprises a deletion or substitution of 1, 2, 3, 4, 5, 6, 7, or 8 the following N-terminal amino acids: M, A, Y, M, S, R, R, E; and/or a deletion or substitution of the following 1 or 2 C-terminal amino acids: F, V.
• polypeptides may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art.
• amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel etal., (1987 , Methods inEnzymol, 154: 367-382), U.S. Pat. No. 4,873,192, Watson, J. D.
• a variant will contain one or more conservative substitutions.
• a “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Modifications may be made in the structure of the polynucleotides and polypeptides contemplated in particular embodiments, polypeptides include polypeptides having at least about and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics.
• amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids.
• a conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.
• Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule.
• polynucleotide sequences encoding them can be separated by and IRES sequence as disclosed elsewhere herein.
• Polypeptides contemplated in particular embodiments include fusion polypeptides, e.g, 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.
• the fusion polypeptide comprises an amino acid sequence as set forth in any one of SEQ ID NOs: and 46, 64, 73, and 82.
• Fusion polypeptides and polynucleotides encoding fusion polypeptides are provided. Fusion polypeptides and fusion proteins refer to a polypeptide having at least two, three, four, five, six, seven, eight, nine, or ten polypeptide segments.
• two or more polypeptides can be expressed as a fusion protein that comprises one or more self-cleaving polypeptide sequences as disclosed elsewhere herein.
• a fusion protein contemplated herein comprises one or more DNA-binding domains and one or more nucleases, and one or more linker and/or selfcleaving polypeptides.
• a fusion protein contemplated herein comprises a nuclease variant; a linker or self-cleaving peptide; and an end-processing enzyme including but not limited to a 5'-3' exonuclease, a 5'-3' alkaline exonuclease, and a 3'-5' exonuclease e.g, Trex2, Exol, or ExoX).
• an end-processing enzyme including but not limited to a 5'-3' exonuclease, a 5'-3' alkaline exonuclease, and a 3'-5' exonuclease e.g, Trex2, Exol, or ExoX).
• Fusion polypeptides can comprise one or more polypeptide domains or segments including, but are not limited to signal peptides, cell permeable peptide domains (CPP), DNA-binding domains, nuclease domains, etc., 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 linked C-terminus to N-terminus, although they can also be linked C-terminus to C- terminus, N-terminus to N-terminus, or N-terminus to C-terminus.
• the polypeptides of the fusion protein can be in any order. Fusion polypeptides or fusion proteins can also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, so long as the desired activity of the fusion polypeptide is preserved. Fusion polypeptides may be produced by chemical synthetic methods or by chemical linkage between the two moieties or may generally be prepared using other standard techniques. Ligated DNA sequences comprising the fusion polypeptide are operably linked to suitable transcriptional or translational control elements as disclosed elsewhere herein.
• Fusion polypeptides may optionally comprise a linker that can be used to link the one or more polypeptides or domains within a polypeptide.
• a peptide linker sequence may be employed to separate any two or more polypeptide components by a distance sufficient to ensure that each polypeptide folds into its appropriate secondary and tertiary structures so as to allow the polypeptide domains to exert their desired functions.
• Such a peptide linker sequence is incorporated into the fusion polypeptide using standard techniques in the art.
• Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes.
• Preferred peptide linker sequences contain 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 which may be usefully employed as linkers include those disclosed in Maratea e/ o/.. Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci.
• Linker sequences are not required when a particular fusion polypeptide segment contains non- essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
• Preferred linkers are typically flexible amino acid subsequences which are synthesized as part of a recombinant fusion protein.
• Linker polypeptides can be between 1 and 200 amino acids in length, between 1 and 100 amino acids in length, or between 1 and 50 amino acids in length, including all integer values in between.
• Exemplary linkers include, but are not limited to the following amino acid sequences: glycine polymers (G) n ; glycine-serine polymers (Gi -581-5)11, where n is an integer of at least one, two, three, four, or five; 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 etal.
• GGG SEQ ID NO: 113
• DGGGS SEQ ID NO: 114
• TGEKP SEQ ID NO: 115
• KESGSVSSEQLAQFRSLD (SEQ ID NO: 119) (Bird et al., 1988, Science 242:423-426), GGRRGGGS (SEQ ID NO: 120) LRQRDGERP (SEQ ID NO: 121); LRQKDGGGSERP (SEQ ID NO: 122); LRQKD(GGGS) 2 ERP (SEQ ID NO: 123).
• flexible linkers can be rationally designed using a computer program capable of modeling both DNA-binding sites and the peptides themselves (Desjarlais & Berg, PNAS 90:2256-2260 (1993), PNAS 91:11099-11103 (1994) or by phage display methods.
• Fusion polypeptides may further comprise a polypeptide cleavage signal between each of the polypeptide domains described herein or between an endogenous open reading frame and a polypeptide encoded by a donor repair template.
• a polypeptide cleavage site can be put 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).
• Suitable protease cleavages sites and self-cleaving peptides are known to the skilled person (see, e.g., in Ryan etal., 1991. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594).
• Exemplary protease cleavage sites include, but are not limited to the cleavage sites of potyvirus NIa proteases (e.g., tobacco etch virus protease), potyvirus HC proteases, potyvirus Pl (P35) proteases, byovirus NIa proteases, byovirus RNA-2- encoded proteases, aphthovirus L proteases, enterovirus 2A proteases, rhinovirus 2A proteases, picoma 3C proteases, comovirus 24K proteases, nepovirus 24K proteases, RTSV (rice tungro spherical virus) 3C-like protease, PYVF (parsnip yellow fleck virus) 3C-like protease, heparin, thrombin, factor Xa and enterokinase.
• potyvirus NIa proteases e.g., tobacco etch virus protease
• potyvirus Pl (P35) proteases byo
• TEV tobacco etch vims protease cleavage sites
• EXXYXQ(GZS) SEQ ID NO: 121
• ENLYFQG SEQ ID NO: 122
• ENLYFQS SEQ ID NO: 123
• X represents any amino acid (cleavage by TEV occurs between Q and G or Q and S).
• 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).
• the viral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide.
• the viral 2A peptide is selected from the group consisting of: a foot-and-mouth disease vims (FMDV) 2A peptide, an equine rhinitis A vims (ERAV) 2A peptide, a Thosea asigna vims (TaV) 2A peptide, a porcine teschovirus-1 (PTV-1) 2A peptide, a Theilovims 2A peptide, and an encephalomyocarditis vims 2A peptide.
• FMDV foot-and-mouth disease vims
• EAV equine rhinitis A vims
• TaV Thosea asigna vims
• PTV-1 porcine teschovirus-1
• Exemplary 2 A sites include the following sequences:
• polynucleotides encoding one or more fusion polypeptides, homing endonuclease variants, megaTALs, end-processing enzymes, and exonucleases contemplated herein are provided.
• polynucleotide or “nucleic acid” refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and DNA/RNA hybrids.
• a polynucleotide encoding one or more fusion polypeptides e.g., SEQ ID NOs: 5, 8, 11, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53,
• the polynucleotide encoding one or more fusion polypeptides is an RNA polynucleotide. In some embodiments, the
• RNA polynucleotide encoding one or more fusion polypeptides comprises a sequence set forth in any one of SEQ ID NOs: 6, 9, 12, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54,
• the polynucleotide encodes an amino acid sequence as 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.
• Polynucleotides may be single-stranded or double-stranded and 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.
• Polynucleotides refer to a polymeric form of nucleotides of 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 a modified form of either type of nucleotide, as well as all intermediate lengths.
• intermediate lengths in this context, means any length between the quoted values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc.,' 151, 152, 153, etc.,' 201, 202, 203, etc.
• polynucleotides or variants have 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 a reference sequence.
• polynucleotides may be codon-optimized.
• codon-optimized refers to substituting codons in a polynucleotide encoding a polypeptide in order to increase the expression, stability and/or activity of the polypeptide.
• Factors that influence codon optimization include, but are not limited to one or more of: (i) variation of codon biases 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 codons including context, (iv) variation of codons according to their decoding tRNAs, (v) variation of codons according to GC %, either overall or in one position of the triplet, (vi) variation in degree of similarity to a reference sequence for example a naturally occurring sequence, (vii) variation in the codon frequency cutoff, (viii) structural properties of mRNAs transcribed from the DNA sequence, (ix) prior knowledge about the function of the DNA sequences upon which design of the codon substitution set is to be based, and/or (x) systematic variation of codon sets for each amino acid, and/or (xi) isolated removal of spurious translation initiation sites.
• 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 art-recognized modified bases. Such bases are generally located at the 1 ' position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. In ribonucleic acid (RNA), the sugar is a ribose, and in deoxyribonucleic acid (DNA) the sugar is a deoxyribose, i.e., a sugar lacking a hydroxyl group that is present in ribose.
• RNA ribonucleic acid
• DNA deoxyribonucleic acid
• Exemplary natural nitrogenous bases include the purines, adenosine (A) and guanidine (G), and the pyrimidines, cytidine (C) and thymidine (T) (or in the context of RNA, uracil (U)).
• the C-l atom of deoxyribose is bonded to N-l of a pyrimidine or N-9 of a purine.
• Nucleotides are usually mono, di- or triphosphates.
• the nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, nucleotide derivatives, modified nucleotides, non-natural nucleotides, and non-standard nucleotides; see for example, WO 92/07065 and WO 93/15187).
• modified nucleic acid bases are summarized by Limbach et al. , (1994, Nucleic Acids Res . 22, 2183-2196).
• a nucleotide may also be regarded as a phosphate ester of a nucleoside, with esterification occurring on the hydroxyl group attached to C-5 of the sugar.
• nucleoside refers to a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar. Nucleosides are recognized in the art to include natural bases, and also to include well known modified bases. Such bases are generally located at the 1 ' position of a nucleoside sugar moiety. Nucleosides generally comprise a base and sugar group.
• the nucleosides can be unmodified or modified at the sugar, and/or base moiety, (also referred to interchangeably as nucleoside analogs, nucleoside derivatives, modified nucleosides, non-natural nucleosides, or non-standard nucleosides).
• modified nucleic acid bases are summarized by Limbach et al., (1994, Nucleic Acids Res. 22, 2183-2196).
• polynucleotides include, but are not limited to polynucleotides encoding 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 and polynucleotide sequences set forth in SEQ ID NOs: 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 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.
• polynucleotides contemplated herein include, but are not limited to polynucleotides encoding fusion polypeptides, homing endonuclease variants, megaTALs, end-processing enzymes, exonucleases, and expression vectors, viral vectors, and transfer plasmids comprising polynucleotides contemplated herein.
• polynucleotide vanant and “vanant” and the like refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions that are defined hereinafter.
• polynucleotide variant and “variant” include polynucleotides in which one or more nucleotides have been added or deleted, or modified, or replaced with different nucleotides.
• certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide.
• a polynucleotide comprises a nucleotide sequence that hybridizes to a target nucleic acid sequence under stringent conditions.
• stringent conditions describes hybridization protocols in which nucleotide sequences at least 60% identical to each other remain hybridized.
• stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific 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 complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
• sequence identity or, for example, comprising a “sequence 50% identical to,” as used herein, refer to the extent that sequences are identical on a nucleotide- by -nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
• a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g, A, T, C, G, I) or the identical amino acid residue (e.g, Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
• the identical nucleic acid base e.g, A, T, C, G, I
• the identical amino acid residue e.g, Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His,
• 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, typically where the polypeptide variant maintains at least one biological activity of the reference polypeptide.
• references 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” is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length.
• two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides
• sequence comparisons between two (or more) polynucleotides are typically performed by comparing 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 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
• the comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
• Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
• GAP Garnier et al.
• BESTFIT Pearson FASTA
• FASTA Pearson's Alignment of sequences
• TFASTA Pearson's Alignin
• an “isolated polynucleotide,” as used herein, refers to a polynucleotide that has been purified from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment that has been removed from the sequences that are normally adjacent to the fragment.
• an “isolated polynucleotide” refers to a complementary DNA (cDNA), a recombinant polynucleotide, a synthetic polynucleotide, or other polynucleotide that does not exist in nature and that has been made by the hand of man.
• a polynucleotide comprises an mRNA encoding a polypeptide contemplated herein including, but not limited to, a fusion polypeptide, homing endonuclease variant, a megaTAL, an end-processing enzyme, and an exonuclease.
• the mRNA comprises a cap, one or more nucleotides and/or modified nucleotides, and a poly(A) tail.
• an mRNA contemplated herein comprises a poly(A) tail to help protect the mRNA from exonuclease degradation, stabilize the mRNA, and facilitate translation.
• an mRNA comprises a 3' poly(A) tail structure.
• 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.
• the length of the poly(A) tail is at least about 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,
• 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 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 nucleot
• Polynucleotide sequences can be annotated in the 5' to 3' orientation or the 3' to 5' orientation.
• the 5' to 3' strand is designated the “sense,” “plus,” or “coding” strand because its sequence is identical to the sequence of the pre-messenger (pre-mRNA) [except for uracil (U) in RNA, instead of thymine (T) in DNA],
• pre-mRNA pre-messenger
• the complementary 3' to 5' strand which is the strand transcribed by the RNA polymerase is designated as “template,” “antisense,” “minus,” or “non-coding” strand.
• reverse orientation refers to a 5 ' to 3 ' sequence written in the 3 ' to 5 ' orientation or a 3 ' to 5 ' sequence written in the 5' to 3' orientation.
• complementarity refers to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules.
• the complementary strand of the DNA sequence 5' A G T C A T G 3' is 3' T C A G T A C 5'.
• the latter sequence is often written as the reverse complement with the 5' end on the left and the 3' end on the right, 5' C A T G A C T 3'.
• a sequence that is equal to its reverse complement is said to be a palindromic sequence.
• Complementarity can be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules.
• nucleic acid cassete or “expression cassete” as used herein refers to genetic sequences within the vector which can express an RNA, and subsequently a polypeptide.
• the nucleic acid cassete contains a gene(s)-of-interest, e.g, a polynucleotide(s)-of-interest.
• the nucleic acid cassete contains one or more expression control sequences, e.g, a promoter, enhancer, poly(A) sequence, and a gene(s)-of-interest, e.g, a polynucleotide(s)-of-interest.
• Vectors may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleic acid cassetes.
• the nucleic acid cassete is positionally and sequentially oriented within the vector such that the nucleic acid in the cassete can be transcribed into RNA, and when necessary, translated into a protein or a polypeptide, undergo appropriate post-translational modifications required for activity in the transformed cell, and be translocated to the appropriate compartment for biological activity by targeting to appropriate intracellular compartments or secretion into extracellular compartments.
• the cassete has its 3' and 5' ends adapted for ready insertion into a vector, e.g, it has restriction endonuclease sites at each end.
• the nucleic acid cassete contains the sequence of a therapeutic gene used to treat, prevent, or ameliorate a genetic disorder.
• the cassete can be removed and inserted into a plasmid or viral vector as a single unit.
• Polynucleotides include polynucleotide(s)-of-interest.
• polynucleotide-of-interesf refers to a polynucleotide encoding a polypeptide or fusion polypeptide or a polynucleotide that serves as a template for the transcription of an inhibitory polynucleotide, as contemplated herein.
• nucleotide sequences that may encode a polypeptide, or fragment of variant thereof, as contemplated herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated in particular embodiments, for example polynucleotides that are optimized for human and/or primate codon selection. In one embodiment, polynucleotides comprising particular allelic sequences are provided.
• Alleles are endogenous polynucleotide sequences that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides.
• a polynucleotide-of-interest comprises a donor repair template.
• polynucleotides contemplated in particular embodiments may be combined with other DNA sequences, such as promoters and/or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g, LoxP, FRT, and Att sites), termination codons, transcriptional termination signals, post-transcription response elements, e.g, 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 as known in the art, such that their overall length may vary considerably. It is therefore contemplated in particular embodiments that a polynucleotide fragment of almost any length may be employed
• Polynucleotides can be prepared, manipulated, expressed and/or delivered using any of a variety of well-established techniques known and available in the art.
• a nucleotide sequence encoding the polypeptide can be inserted into appropriate vector.
• a desired polypeptide can also be expressed by delivering an mRNA encoding the polypeptide into the cell.
• vectors include, but are not limited to plasmid, autonomously replicating sequences, and transposable elements, e.g, Sleeping Beauty, PiggyBac.
• vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (Y AC), bacterial artificial chromosome (BAC), or Pl -derived artificial chromosome (PAC), bacteriophages such as lambda phage or Ml 3 phage, and animal viruses.
• artificial chromosomes such as yeast artificial chromosome (Y AC), bacterial artificial chromosome (BAC), or Pl -derived artificial chromosome (PAC)
• bacteriophages such as lambda phage or Ml 3 phage
• animal viruses include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (Y AC), bacterial artificial chromosome (BAC), or Pl -derived artificial chromosome (PAC), bacteriophages such as lambda phage or Ml 3 phage, and animal viruses.
• viruses useful as vectors include, without limitation, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g, herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g, SV40).
• expression vectors include, but are not limited to pClneo vectors (Promega) for expression in mammalian cells; pLenti4/V 5-DESTTM, pLenti6/V 5- DESTTM, and pLenti6.2/V 5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells.
• coding sequences of polypeptides disclosed herein can be ligated into such expression vectors for the expression of the polypeptides in mammalian cells.
• the vector is an episomal vector or a vector that is maintained extrachromosomally.
• episomal vector refers to a vector that is able to replicate without integration into host's chromosomal DNA and without gradual loss from a dividing host cell also meaning that said vector replicates extrachromosomally or episomally.
• “Expression control sequences,” “control elements,” or “regulatory sequences” present in an expression vector are those non-translated regions of the vector — origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgamo sequence or Kozak sequence) introns, post-transcriptional regulatory elements, a poly adenylation sequence, 5' and 3' untranslated regions — which interact with host cellular proteins to carry out 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 promoters and inducible promoters may be used.
• a polynucleotide comprises a vector, including but not limited to expression vectors and viral vectors.
• a vector may comprise one or more exogenous, endogenous, or heterologous control sequences such as promoters and/or enhancers.
• An “endogenous control sequence” is one which is naturally linked with a given gene in the genome.
• An “exogenous control sequence” is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer/promoter.
• a “heterologous control sequence” is an exogenous sequence that is from a different species than the cell being genetically manipulated.
• a “synthetic” control sequence may comprise elements of one more endogenous and/or exogenous sequences, and/or sequences determined in vitro or in silico that provide optimal promoter and/or enhancer activity for the particular therapy.
• promoter refers to a recognition site of a polynucleotide (DNA or RNA) to which an RNA polymerase binds. An RNA polymerase initiates and transcribes polynucleotides operably linked to the promoter.
• promoters operative in mammalian cells comprise an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated and/or another sequence found 70 to 80 bases upstream from the start of transcription, a CNCAAT region where N may be any nucleotide.
• enhancer refers to a segment of DNA which contains sequences capable of providing enhanced transcription and in some instances can function independent of their orientation relative to another control sequence.
• An enhancer can function cooperatively or additively with promoters and/or other enhancer elements.
• promoter/enhancer refers to a segment of DNA which contains sequences capable of providing both promoter and enhancer functions.
• operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
• 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, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
• constitutive expression control sequence refers to a promoter, enhancer, or promoter/enhancer that continually or continuously allows for transcription of an operably linked sequence.
• a constitutive expression control sequence may be a “ubiquitous” promoter, enhancer, or promoter/enhancer that allows expression in a wide variety of cell and tissue types or a “cell specific,” “cell type specific,” “cell lineage specific,” or “tissue specific” promoter, enhancer, or promoter/enhancer that allows expression in a restricted variety of cell and tissue types, respectively.
• Illustrative ubiquitous expression control sequences suitable for use in particular embodiments include, but are not limited to, a cytomegalovirus (CMV) immediate early promoter, a viral simian virus 40 (SV40) (e.g, early or late), a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and Pl 1 promoters from vaccinia virus, a short elongation factor 1 -alpha (EF la-short) promoter, a long elongation factor 1 -alpha (EFla-long) promoter, early growth response 1 (EGR1), femtin H (FerH), femtin L (FerL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation
• UBC Ubiquitin C promoter
• PGK phosphoglycerate kinase- 1
• CAG cytomegalovirus enhancer/chi
• a cell, cell type, cell lineage or tissue specific expression control sequence may be desirable to use to achieve cell type specific, lineage specific, or tissue specific expression of a desired polynucleotide sequence (e.g, to express a particular nucleic acid encoding a polypeptide in only a subset of cell types, cell lineages, or tissues or during specific stages of development).
• 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 or tissue specific expression. Certain embodiments provide conditional expression of a polynucleotide-of-interesf e.g, expression is controlled by subjecting a cell, tissue, organism, etc., to a treatment or condition that causes the polynucleotide to be expressed or that causes an increase or decrease in expression of the polynucleotide encoded by the polynucleotide-of-interest.
• inducible promoters/systems include, but are not limited to, steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionine promoter (inducible by treatment with various heavy metals), MX-1 promoter (inducible by interferon), the “GeneSwitch” mifepristone-regulatable system (Sirin et al., 2003, Gene, 323:67), the cumate inducible gene switch (WO 2002/088346), tetracycline-dependent regulatory systems, etc.
• steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionine promoter (inducible by treatment with various heavy metals), MX-1 promoter (inducible by interferon), the “GeneSwitch” m
• Conditional expression can also be achieved by using a site-specific DNA recombinase.
• polynucleotides comprise at least one (typically two) site(s) for recombination mediated by a site-specific recombinase.
• recombinase or “site-specific recombinase” include excisive or integrative proteins, enzymes, co-factors or associated proteins that are involved in recombination reactions involving one or more recombination sites (e.g, two, three, four, five, six, seven, eight, nine, ten 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 containing the recombination protein sequences or fragments thereof), fragments, and variants thereof.
• Illustrative examples of recombinases suitable for use in particular embodiments include, but are not limited to: Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, ⁇ DC31, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCEl, and ParA.
• the polynucleotides may comprise one or more recombination sites for any of a wide variety of site-specific recombinases. It is to be understood that the target site for a site-specific recombinase is in addition to any site(s) required for integration of a vector, e.g, a retroviral vector or lentiviral vector.
• the terms “recombination sequence,” “recombination site,” or “site-specific recombination site” refer to a particular nucleic acid sequence to which a recombinase recognizes and binds.
• polynucleotides contemplated herein include one or more polynucleotides-of-interest that encode one or more polypeptides.
• the polynucleotide sequences can be separated by one or more IRES sequences or polynucleotide sequences encoding self-cleaving polypeptides.
• an “internal ribosome entry site” or “IRES” refers to an element that promotes direct internal ribosome entry to the initiation codon, such as ATG, of a cistron (a protein encoding region), thereby leading to the cap-independent translation of the gene. See, e.g, Jackson et al. , 1990. Trends Biochem Set 15(12):477-83) and Jackson and Kaminski. 1995. RNA l(10):985-1000. Examples of IRES generally employed by those of skill in the art include those described in U.S. Pat. No. 6,692,736.
• IRES immunoglobulin heavy -chain binding protein
• VEGF vascular endothelial growth factor
• FGF-2 fibroblast growth factor 2
• IGFII insulin-like growth factor
• eIF4G translational initiation factor eIF4G and yeast transcription factors TFIID and HAP4
• EMCV encephelomycarditis virus
• IRES have also been reported in viral genomes of Picomaviridae, Dicistroviridae and Flaviviridae species and in HCV, Friend murine leukemia virus (FrMLV) and Moloney murine leukemia virus (MoMLV).
• the polynucleotides comprise polynucleotides that have a consensus Kozak sequence and that encode a desired polypeptide.
• Kozak sequence refers to a short nucleotide sequence that greatly facilitates the 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).
• vectors comprise a poly adenylation sequence 3' of a polynucleotide encoding a polypeptide to be expressed.
• polyA site or “polyA sequence” as used herein denotes a DNA sequence which directs both the termination and poly adenylation of the nascent RNA transcript by RNA polymerase II.
• Poly adenylation sequences can promote mRNA stability by addition of a polyA tail to the 3' end of the coding sequence and thus, contribute to increased translational efficiency.
• Cleavage and polyadenylation is directed by a poly(A) sequence in the RNA.
• the core poly(A) sequence for mammalian pre-mRNAs has two recognition elements flanking a cleavage- polyadenylation site. Typically, an almost invariant AAUAAA hexamer lies 20-50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled to the addition of up to 250 adenosines to the 5' cleavage product.
• the core poly(A) sequence is an ideal polyA sequence (e.g. , AATAAA, ATT AAA, AGTAAA).
• the poly(A) sequence is an SV40 polyA sequence, a bovine growth hormone polyA sequence (BGHpA), a rabbit P-globin polyA sequence (rPgpA), vanants thereof, or another suitable heterologous or endogenous polyA sequence known in the art.
• BGHpA bovine growth hormone polyA sequence
• rPgpA rabbit P-globin polyA sequence
• vanants thereof or another suitable heterologous or endogenous polyA sequence known in the art.
• polynucleotides encoding one or more fusion polypeptides, homing endonuclease variants, megaTALs, end-processing enzymes, or exonucleases may be introduced into hematopoietic cells, e.g., CD34 + cells, by both non-viral and viral methods.
• delivery of one or more polynucleotides encoding nucleases and/or donor repair templates may be provided by the same method or by different methods, and/or by the same vector or by different vectors.
• vector is used herein to refer to a nucleic acid molecule capable transferring or transporting another nucleic acid molecule.
• the transferred nucleic acid is generally linked to, e.g, inserted into, the vector nucleic acid molecule.
• a vector may include sequences that direct autonomous replication in a cell, or may include sequences sufficient to allow integration into host cell DNA.
• non-viral vectors are used to deliver one or more polynucleotides contemplated herein to a CD34 + cell.
• non-viral vectors include, but are not limited to plasmids (e.g, DNA plasmids or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.
• Illustrative methods of non-viral delivery of polynucleotides contemplated in particular embodiments include, but are not limited to: electroporation, sonoporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, nanoparticles, poly cation or lipidmucleic acid conjugates, naked DNA, artificial virions, DEAE-dextran-mediated transfer, gene gun, and heat-shock.
• polynucleotide delivery systems suitable for use in particular embodiments contemplated in particular embodiments include, but are not limited to those provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc.
• Lipofection reagents are sold commercially (e.g, TransfectamTM and LipofectinTM). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides have been described in the literature. See e.g., Liu et al. (2003) Gene Therapy. 10: 180-187; and Balazs et al. (2011) Journal of Drug Delivery. 2011:1-12.
• Antibody -targeted, bactenally derived, non-living nanocell-based delivery is also contemplated in particular embodiments.
• polynucleotides encoding the fusion polypeptides contemplated herein can be introduced directly into the cells, for example by electroporation.
• electroporation methods the polynucleotides, or the complexes of site-directed polypeptides and polynucleotides are mixed in an electroporation buffer with the target cells to form a suspension. This suspension is then subjected to an electrical pulse at an optimized voltage, which creates temporary pores in the phospholipid bilayer of the cell membrane, permitting charged molecules like DNA and proteins to be driven through the pores and into the cell. Reagents and equipment to perform electroporation are sold commercially.
• the electroporation medium can be any suitable medium known in the art. Suitable methods of electroporation are available and known to those of skill in the art
• Viral vectors comprising polynucleotides contemplated in particular embodiments can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g. intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below.
• vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g. mobilized peripheral blood, lymphocytes, bone marrow aspirates, tissue biopsy, etc.) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient.
• viral vectors comprising nuclease variants and/or donor repair templates are administered directly to an organism for transduction of cells in vivo.
• naked DNA or mRNA can be administered.
• Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application and electroporation. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
• viral vector systems suitable for use in particular embodiments contemplated herein include, but are not limited to adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors.
• AAV adeno-associated virus
• retrovirus retrovirus
• herpes simplex virus adenovirus
• vaccinia virus vectors vaccinia virus vectors.
• Genome edited cells manufactured and/or edited by the methods contemplated in particular embodiments are provided.
• Genome edited cells contemplated in particular embodiments may be autologous/autogeneic (“self’) or non-autologous (“non-self,” e.g, allogeneic, syngeneic or xenogeneic).
• Autologous refers to cells from the same subject.
• Allogeneic refers to cells of the same species that differ genetically to the cell in comparison.
• Syngeneic refers to cells of a different subject that are genetically identical to the cell in comparison.
• Xenogeneic refers to cells of a different species to the cell in comparison.
• the cells are obtained from a mammalian subject.
• the cells are obtained from a primate subject, optionally a non-human primate.
• the cells are obtained from a human subject.
• isolated cell refers to a non-naturally occurring cell, e.g, a cell that does not exist in nature, a modified cell, an engineered cell, etc., that has been obtained from an in vivo tissue or organ and is substantially free of extracellular matrix.
• the cells are edited by the fusion polypeptides 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 strand DNA (dsDNA) target site in a cell, a polypeptide linker and an exonuclease, or biologically active fragment thereof.
• a fusion polypeptide comprising a DNA-binding domain and a homing endonuclease (HE) variant that binds and cleaves a selected double strand DNA (dsDNA) target site in a cell
• dsDNA double strand DNA
• the cell comprises directionally biased deletions induced by the fusion polypeptides contemplated herein.
• 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 location on one side of the HE target site center location.
• the deletion center location is on the same side as the DNA-binding domain target site relative to the HE target site center location.
• the deletion center location is 5’ to the HE target site center location.
• At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of deletions have a deletion center greater than 4 nucleotides away from the HE target site center location. In various embodiments, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or at least 35% of deletions have a deletion center greater than 8 nucleotides away from the HE target site center location.
• At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of deletions are 6 bps in length or greater.
• 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 deletions are 12 bps in length or greater.
• the directionally biased deletions comprise a length of about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides.
• the deletion extends into the DNA-binding domain target site. In some embodiments, the deletion center location is within the DNA-binding domain target site.
• Illustrative examples of cell types whose genome can 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.
• stem cell refers to a cell which is an undifferentiated cell capable of (1) long term self -renewal, or the ability to generate at least one identical copy of the original cell, (2) differentiation at the single cell level into multiple, and in some instance only one, specialized cell type and (3) of in vivo functional regeneration of tissues.
• Stem cells are subclassified according to their developmental potential as totipotent, pluripotent, multipotent and oligo/unipotent.
• Self-renewal refers a cell with a unique capacity to produce unaltered daughter cells and to generate specialized cell types (potency). Selfrenewal can be achieved in two ways.
• Asymmetric cell division produces one daughter cell that is identical to the parental cell and one daughter cell that is different from the parental cell and is a progenitor or differentiated cell.
• Symmetric cell division produces two identical daughter cells.
• “Proliferation” or “expansion” of cells refers to symmetrically dividing cells.
• progenitor or “progenitor cells” refers to cells have the capacity to self-renew and to differentiate into more mature cells. Many progenitor cells differentiate along a single lineage, but may have quite extensive proliferative capacity.
• the cell is a primary cell.
• the term “primary cell” as used herein is known in the art to refer to a cell that has been isolated from a tissue and has been established for growth in vitro or ex vivo. Corresponding cells have undergone very few, if any, population doublings and are therefore more representative of the main functional component of the tissue from which they are derived in comparison to continuous cell lines, thus representing a more representative model to the in vivo state. Methods to obtain samples from various tissues and methods to establish primary cell lines are well-known in the art (see, e.g., 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 cell is a hematopoietic stem or progenitor cell.
• 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 primary cell.
• the genome edited cell is a hematopoietic cell, e.g, hematopoietic stem cell, hematopoietic progenitor cell, such as a B cell progenitor cell, or cell population comprising hematopoietic cells.
• the term “population of cells” refers to a plurality of cells that may be made up of any number and/or combination of homogenous or heterogeneous cell types, as described elsewhere herein.
• a population of cells may be isolated or obtained from umbilical cord blood, placental blood, bone marrow, or mobilized peripheral blood.
• a population of cells 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.
• hematopoietic stem or progenitor cells may be isolated or purified from a population of heterogeneous cells using methods known in the art.
• HSCs Hematopoietic stem cells
• HPCs committed hematopoietic progenitor cells
• HSC hematopoietic stem cell
• myeloid e.g, monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells
• lymphoid lineages e.g, T- cells, B-cells, NK-cells
• myeloid e.g, monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells
• lymphoid lineages e.g, T- cells, B-cells, NK-cells
• hematopoietic stem and progenitor cells can repopulate the erythroid, neutrophil-macrophage, megakaryocyte and lymphoid hematopoietic cell pool.
• hematopoietic stem or progenitor cells suitable for use with the methods and compositions contemplated herein include hematopoietic cells that are CD34 + CD38 Lo CD90 + CD45 RA ", hematopoietic cells that are CD34 + , CD59 + , Thyl/CD90 + , CD38 Lo/ ", C-kit/CD117 + , and LirU 1 . and hematopoietic cells that are CD133 + .
• the hematopoietic cells that are CD133 + CD90 + In a preferred embodiment, the hematopoietic cells that are CD133 + CD34 + . In a preferred embodiment, the hematopoietic cells that are CD133 + CD90 + CD34 + .
• CD34+ cell refers to a cell expressing the CD34 protein on its cell surface.
• 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 entrance into lymph nodes.
• the CD34+ cell population contains hematopoietic stem cells (HSC), which upon administration 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.
• HSC hematopoietic stem cells
• the SLAM (Signaling lymphocyte activation molecule) family is a group of >10 molecules whose genes are located mostly tandemly in a single locus on chromosome 1 (mouse), all belonging to a subset of immunoglobulin gene superfamily, and originally thought to be involved in T-cell stimulation.
• This family includes CD48, CD150, CD244, etc., CD150 being the founding member, and, thus, also called slamFl, i.e., SLAM family member 1.
• the signature SLAM code for the hematopoietic hierarchy is hematopoietic stem cells (HSC) - CD150 + CD48'CD244‘; multipotent progenitor cells (MPPs) - CD150'CD48'CD244 + ; lineage-restricted progenitor cells (LRPs) - CD150'CD48 + CD244 + ; common myeloid progenitor (CMP) - lin-SCA-l-c- kit + CD34 + CD16/32 nud ; granulocyte-macrophage progenitor (GMP) - lin'SCA-l-c- kit + CD34 + CD16/32 w ; and megakaryocyte-erythroid progenitor (MEP) - lin’SCA-l-c- kit + CD34'CD16/32 low .
• HSC hematopoietic stem cells
• MPPs multipotent progenitor cells
• Preferred target cell types edited with 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.
• the term “CD34+ cell,” as used herein refers to a cell expressing the CD34 protein on its cell surface.
• 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 of both hematopoietic stem and progenitor cells.
• the genome edited hematopoietic cells are CD150 + CD48‘ CD244' cells. In one embodiment, the genome edited hematopoietic cells are CD34 + CD133 + cells. In one embodiment, the genome edited hematopoietic cells are CD133 + cells. In one embodiment, the genome edited hematopoietic cells are CD34 + cells.
• the fusion polypeptides contemplated herein are introduced 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, secretion of cytokines, induction of ADCC and/or CDC).
• Illustrative immune effector cells contemplated herein are T lymphocytes, including but not limited to cytotoxic T cells (CTLs; CD8+ T cells), TILs, and helper T cells (HTLs; CD4+ T cells).
• the cells comprise a T cells.
• the cells comprise y6 T cells.
• immune effector cells include natural killer (NK) cells.
• immune effector cells include natural killer T (NKT) cells.
• Immune effector cells can be autologous/autogeneic (“self’) or non-autologous (“non-self,” e.g, allogeneic, syngeneic or xenogeneic).
• Autologous refers to cells from the same subject.
• Allogeneic refers to cells of the same species that differ genetically to the cell in comparison.
• Syngeneic refers to cells of a different subject that are genetically identical to the cell in companson.
• Xenogeneic refers to cells of a different species to the cell in comparison. In preferred embodiments, the cells are autologous.
• T lymphocytes include T lymphocytes.
• T cell or “T lymphocyte” are art-recognized and are intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes.
• a T cell can be a T helper (Th) cell, for example a T helper 1 (Thl) or a T helper 2 (Th2) cell.
• the T cell can be a helper T cell (HTL; CD4+ T cell) CD4+ T cell, a cytotoxic T cell (CTL; CD8+ T cell), CD4+CD8+ T cell, CD4-CD8- T cell, or any other subset of T cells.
• HTL helper T cell
• CTL cytotoxic T cell
• CD4+CD8+ T cell CD4-CD8- T cell
• Other illustrative populations of T cells suitable for use in particular 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).
• immune effector cells may also include NK cells, NKT cells, neutrophils, and macrophages.
• Immune effector cells also include progenitors of effector cells wherein such progenitor cells can be induced to differentiate into an immune effector cells in vivo or in vitro.
• immune effector cell includes progenitors of immune effectors cells such as hematopoietic stem cells (HSCs) contained within the CD34+ population of cells derived from cord blood, bone marrow or mobilized peripheral blood which upon administration in a subject differentiate into mature immune effector cells, or which can be induced in vitro to differentiate into mature immune effector cells.
• HSCs hematopoietic stem cells
• 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.
• the immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. Such cells can then be directly readministered into the individual.
• the immune effector cells are first activated and stimulated to proliferate in vitro prior to being genetically modified to express a CAR.
• 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).
• the source of cells is obtained from a subject.
• modified immune effector cells comprise T cells.
• PBMCs may be directly genetically modified to express a CAR using methods contemplated herein.
• T lymphocytes after isolation of PBMC, T lymphocytes are further isolated and in certain embodiments, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after genetic modification and/or expansion.
• the immune effector cells can be genetically modified following isolation using known methods, or the immune effector cells can be activated and expanded (or differentiated in the case of progenitors) in vitro prior to being genetically modified.
• the immune effector cells such as T cells
• T cells can be activated and expanded before or after genetic modification to express a CAR, using methods as described, for example, in U.S.
• CD34+ cells are transduced with a nucleic acid construct contemplated herein.
• the transduced CD34+ cells differentiate into mature immune effector cells in vivo following administration into a subject, generally the subject from whom the cells were originally isolated.
• CD34+ cells may be stimulated in vitro prior to exposure to or after being genetically modified with a CAR as contemplated herein, 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 the methods described previously (Asheuer et al., 2004; Imren, et al., 2004).
• a population of modified immune effector cells for the treatment of cancer comprises a CAR contemplated herein.
• a population of modified immune effector cells are prepared from peripheral blood mononuclear cells (PBMCs) obtained from a patient diagnosed with B cell malignancy described herein (autologous donors).
• PBMCs peripheral blood mononuclear cells
• the PBMCs form a heterogeneous population of T lymphocytes that can be CD4+, CD8+, or CD4+ and CD8+.
• the PBMCs also can include other cytotoxic lymphocytes such as NK cells or NKT cells.
• An expression vector carrying the coding sequence of a CAR contemplated in particular embodiments is introduced into a population of human donor T cells, NK cells or NKT cells.
• successfully transduced T cells that carry the expression vector can be sorted using flow cytometry to isolate CD3 positive T cells and then further propagated to increase the number of these CAR protein expressing T cells in addition to cell activation using anti-CD3 antibodies and or anti-CD28 antibodies and IL-2 or any other methods known in the art as described elsewhere herein. Standard procedures are used for cry opreservation of T cells expressing the CAR protein T cells for storage and/or preparation for use in a human subject.
• the in vitro transduction, culture and/or expansion of T cells are performed in the absence of nonhuman animal derived products such as fetal calf serum and fetal bovine serum. Since a heterogeneous population of PBMCs is genetically modified, the resultant transduced cells are a heterogeneous population of modified cells comprising a BCMA targeting CAR as contemplated herein.
• a mixture of, e.g., one, two, three, four, five or more, different expression vectors can be used in genetically modifying a donor population of immune effector cells wherein each vector encodes a different chimeric antigen receptor protein as contemplated herein.
• the resulting modified immune effector cells forms a mixed population of modified cells.
• T cells can be manufactured using various methods known in the art, see, e.g., WO 2016/094304 which is incorporated herein by reference in its entirety. I. COMPOSITIONS AND FORMULATIONS
• compositions contemplated in particular embodiments may comprise one or more polypeptides, polynucleotides, vectors comprising same, and genome editing compositions and genome edited cell compositions, as contemplated herein.
• the genome editing compositions and methods contemplated in particular embodiments are useful for editing a target site in a cell or a population of cells.
• compositions contemplated herein comprise a fusion polypeptide comprising a DNA-binding domain, a homing endonuclease variant, and an end-processing enzyme, e.g, a 3 '-5' exonuclease (ExoX).
• the nuclease variant may be in the form of an mRNA that is introduced into a cell via polynucleotide delivery methods disclosed supra, e.g, electroporation, lipid nanoparticles, etc.
• composition comprising an mRNA encoding a fusion polypeptide, homing endonuclease variant, and a 3 '-5' exonuclease (e.g, ExoX), is introduced in a cell via polynucleotide delivery methods disclosed supra.
• compositions contemplated herein comprise a population of cells, a nuclease variant, and optionally, a donor repair template.
• compositions contemplated herein comprise a population of cells, a nuclease variant, an end-processing enzyme, and optionally, a donor repair template.
• the nuclease variant and/or end-processing enzyme may be in the form of an mRNA that is introduced into the cell via polynucleotide delivery methods disclosed supra.
• the donor repair template may also be introduced into the cell by means of a separate composition.
• compositions contemplated herein comprise a population of cells, a fusion polypeptide comprising a DNA-binding domain, a homing endonuclease variant,, an 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 3 '-5' exonuclease may be in the form of an mRNA that is introduced into the cell via polynucleotide delivery methods disclosed supra.
• the donor repair template may also be introduced into the cell by means of a separate composition.
• the population of cells comprise genetically modified hematopoietic cells including, but not limited to, hematopoietic stem cells, hematopoietic progenitor cells, CD133 + cells, CD34 + cells, and immune effector cells.
• Compositions include, but are not limited to pharmaceutical compositions.
• a “pharmaceutical composition” refers to a composition formulated in pharmaceutically - acceptable or physiologically -acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy.
• compositions may be administered in combination with other agents as well, such as, e.g, cytokines, growth factors, hormones, small molecules, chemotherapeutics, pro-drugs, drugs, antibodies, or other various pharmaceutically -active agents.
• agents such as, e.g, cytokines, growth factors, hormones, small molecules, chemotherapeutics, pro-drugs, drugs, antibodies, or other various pharmaceutically -active agents.
• phrases “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which 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.
• pharmaceutically acceptable carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic cells are administered.
• pharmaceutical carriers can 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 aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
• Suitable pharmaceutical excipients 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 compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
• a composition comprising a pharmaceutically acceptable carrier is suitable for administration to a subject.
• a composition comprising a carrier is suitable for parenteral administration, e.g, intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.
• a composition comprising a pharmaceutically acceptable carrier is suitable for intraventricular, intraspinal, or intrathecal administration.
• Pharmaceutically acceptable carriers include sterile aqueous solutions, cell culture media, or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the transduced cells, use thereof in the pharmaceutical compositions is contemplated.
• 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 pharmaceutically acceptable carrier.
• composition comprising a cell-based composition contemplated herein can be administered by parenteral administration methods.
• the pharmaceutically acceptable carrier must be of sufficiently high purity and of sufficiently low toxicity to render it suitable for administration to the human subject being treated. It further should maintain or increase the stability of the composition.
• the pharmaceutically acceptable carrier can be liquid or solid and is selected, with the planned manner of administration in mind, to provide for the desired bulk, consistency, etc., when combined with other components of the composition.
• the pharmaceutically acceptable carrier can be, without limitation, a binding agent (e.g, pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.), a filler (e.g, lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, poly acrylates, calcium hydrogen phosphate, etc.), a lubricant (e.g, magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, com starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.), a disintegrant (e.g, starch, sodium starch glycolate, etc.), or a wetting agent (e.g, sodium lauryl sulfate, etc.).
• a binding agent e.g, pregelatinized maize starch, polyvinylpyr
• compositions contemplated herein include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatins, amyloses, magnesium stearates, talcs, silicic acids, viscous paraffins, hydroxymethylcelluloses, polyvinylpyrrolidones and the like.
• buffers refers to a solution or liquid whose chemical makeup neutralizes acids or bases without a significant change in pH.
• buffers contemplated herein include, but are not limited to, Dulbecco's phosphate buffered saline (PBS), Ringer's solution, 5% dextrose in water (D5W), normal/physiologic saline (0.9% NaCl).
• the pharmaceutically acceptable carriers may be present in amounts sufficient to maintain a pH of the composition of about 7.
• the composition has a pH in a range from about 6.8 to about 7.4, e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, and 7.4.
• the composition has a pH of about 7.4.
• compositions contemplated herein may comprise a nontoxic pharmaceutically acceptable medium.
• the compositions may be a suspension.
• the term “suspension” as used herein refers to non-adherent conditions in which cells are not attached to a solid support. For example, cells maintained as a suspension may be stirred or agitated and are not adhered to a support, such as a culture dish.
• compositions contemplated herein are formulated in a suspension, where the genome edited hematopoietic stem and/or progenitor cells are dispersed within an acceptable liquid medium or solution, e.g, saline or serum-free medium, in an intravenous (IV) bag or the like.
• acceptable liquid medium or solution e.g, saline or serum-free medium
• IV intravenous
• Acceptable diluents include, but are not limited to water, PlasmaLyte, Ringer's solution, isotonic sodium chloride (saline) solution, serum-free cell culture medium, and medium suitable for cryogenic storage, e.g., Cryostor® medium.
• a pharmaceutically acceptable carrier is substantially free of natural proteins of human or animal origin, and suitable for storing a composition comprising a population of genome edited cells, e.g., hematopoietic stem and progenitor cells.
• the therapeutic composition is intended to be administered into a human patient, and thus is substantially free of cell culture components such as bovine serum albumin, horse serum, and fetal bovine serum.
• compositions are formulated in a pharmaceutically acceptable cell culture medium.
• the pharmaceutically acceptable cell culture medium is a serum free medium.
• Serum-free medium has several advantages over serum containing medium, including a simplified and better-defined composition, a reduced degree of contaminants, elimination of a potential source of infectious agents, and lower cost.
• the serum-free medium is animal-free, and may optionally be protein-free.
• the medium may contain biopharmaceutically acceptable recombinant proteins.
• “Animal-free” medium refers to medium wherein the components are derived from non-animal sources. Recombinant proteins replace native animal proteins in animal-free medium and the nutrients are obtained from synthetic, plant or microbial sources.
• Protein-free in contrast, is defined as substantially free of protein.
• serum-free media used in particular compositions include, but are not limited to QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.
• compositions comprising genome edited hematopoietic stem and/or progenitor cells are formulated in PlasmaLyte.
• compositions comprising hematopoietic stem and/or progenitor cells are formulated in a cry opreservation medium.
• cry opreservation media with cry opreservation agents may be used to maintain a high cell viability outcome post-thaw.
• cry opreservation media used in particular compositions include, but are not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.
• compositions are formulated in a solution comprising 50:50 PlasmaLyte A to CryoStor CS10.
• the composition is substantially free of mycoplasma, endotoxin, and microbial contamination.
• substantially free with respect to endotoxin is meant that there is less endotoxin per dose of cells than is allowed by the FDA for a biologic, which is a total endotoxin of 5 EU/kg body weight per day, which for an average 70 kg person is 350 EU per total dose of cells.
• compositions comprising hematopoietic stem or progenitor cells transduced with a retroviral vector contemplated herein contains about 0.5 EU/mL to about 5.0 EU/mL, or about 0.5 EU/mL, E0 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.
• compositions and formulations suitable for the delivery of polynucleotides are contemplated including, but not limited to, one or more mRNAs encoding one or more reprogrammed nucleases, and optionally endprocessing enzymes.
• 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).
• transfection agents such as calcium phosphate, electroporation, heat shock and various liposome formulations (i.e., lipid-mediated transfection).
• Liposomes as described in greater detail below, are lipid bilayers entrapping a fraction of aqueous fluid. DNA spontaneously associates to the external surface of cationic liposomes (by virtue of its charge) and these liposomes will interact with the cell membrane.
• formulation of pharmaceutically-acceptable carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., enteral and parenteral, e.g., intravascular, intravenous, intraarterial, intraosseous, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, and intramedullary administration and formulation.
• enteral and parenteral e.g., intravascular, intravenous, intraarterial, intraosseous, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, and intramedullary administration and formulation.
• enteral and parenteral e.g., intravascular, intravenous, intraarterial, intraosseous, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, and intramedullary administration and formulation.
• particular embodiments contemplated herein
• the qualitative properties that define a given deletion or insertion are: (i) its length, in number of bases inserted or deleted; (ii) its longitudinal position along the chromosome, usually stated relative to the nuclease target site or breakpoint; and (iii) for insertions, the inserted sequence length and composition.
• Deletions are the most prominent outcomes, typically comprising 90-95% of the observed events. Their most commonly reported size characteristics tend to small (i.e., 1-20 base pairs in length, with the frequency biased toward the low end of that range) and their positional distributions have been found to be evenly distributed, covering the DNA breakpoint and emanating outward in either direction without significant bias.
• Engineered megaTAL nucleases, and other fusion polypeptides comprising a DNA- binding domain and a homing endonuclease have characteristics that are distinct from other gene editing platforms.
• megaTALs are monomeric, hybrid molecules comprising a modularly assembled transcription activator-like effector (TALE) array fused to a reprogrammed homing endonuclease (HE).
• TALE transcription activator-like effector
• HE reprogrammed homing endonuclease
• the TALE array which anchors the HE at the target site, can be sized to recognize a binding site 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 may be anywhere from 0 to approximately 12 base pairs in length.
• the inventors of the fusion polypeptides contemplated herein tested the co-delivery of various end-processing enzymes to characterize their impact on edited alleles. Furthermore, the inventors designed numerous multipartite megaTAL fusion proteins to evaluate how editing outcomes could be manipulated by in-line enzymatic functions.
• fusion polypeptides comprising a DNA-binding domain and a homing endonuclease variant, linked by a linker domain (e.g., a polypeptide linker) to an exonuclease, particularly Trex2, Exol, or ExoX, when expressed in a cell, created elongated and directionally biased deletions, as compared to a fusion polypeptide comprising a DNA-binding domain and a homing endonuclease variant not linked to a exonuclease, i.e., when the DNA-binding domain / homing endonuclease fusion and the exonuclease were expressed separately.
• a linker domain e.g., a polypeptide linker
• dsDNA double-stranded DNA
• methods 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 directionally biased deletions having a deletion center near the selected dsDNA target cut site in the cell.
• dsDNA double-stranded DNA
• the methods comprising introducing a fusion polypeptide comprising, a DNA-binding domain and a homing endonuclease (HE) variant that binds and cleaves a selected double strand DNA (dsDNA) target site in a cell; a linker domain; and an exonuclease or biologically active fragment thereof.
• the exonuclease is Trex2, Exol, or ExoX.
• the DNA-binding domain comprises a TALE DNA-binding domain (e.g., a megaTAL) or a zinc finger DNA-binding domain.
• the ExoX, or biologically active fragment thereof comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence as set forth in SEQ ID NO: 109.
• the Exol, or biologically active fragment thereof comprises an amino acid an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence as set forth in SEQ ID NO: 112.
• the linker domain is a peptide linker.
• the peptide linker is a self-cleaving peptide linker.
• the peptide linker comprises about 4 to about 30 ammo acids.
• the peptide linker is a (GGGGS)i-i linker (SEQ ID NOs: 117 and 150-152).
• the HE variant is an LAGLID ADG homing endonuclease (LHE) variant.
• the HE variant is a variant of an LHE selected from the group consisting of: I-AabMI, I-AaeMI, I-Anil, 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-Gpil, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I- Ltrll, I-Ltrl, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI, I-Ohe
• the contemplated methods comprise introducing a fusion polypeptide that targets a site within a immune system checkpoint gene, globin gene, gene that encodes a polypeptide that contributes to repression of y-globin gene expression and HbF, or immunosuppressive signaling gene.
• 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 suppressor of T cell activation (VISTA), and killer cell immunoglobulin-like receptor (KIR), CCR5, TRAC (TCRa), TCR , ILlORa, IL10RP, TGFBR1, TGFBR2, CBL-B, PCSK9, AHR, BTK, a-globin, -globin, y-globin, BCL11A, KLF1, SOX6, GATA1, LSD1, alpha folate receptor (FRa), avP6 integrin, B cell maturation antigen
• 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 suppressor of T cell activation (VISTA), and killer cell immunoglobulin-like receptor (KIR), CCR5, TRAC (TCRa), ILlORa, TGFBR2, CBL- B, PCSK9, AHR, BTK, a-globin, P-globin, y-globin, and BCL11A gene.
• PD-1 programmed cell death protein 1
• LAG-3 lymphocyte activation gene 3 protein
• TIM-3 T cell immunoglobulin domain and mucin domain protein 3
• CTLA-4 cytotoxic T
• the target site is within a TRAC (TCRa) gene, a CBL- B gene, or a PDCD1 (PD-1) gene.
• TCRa 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.
• the PD-1 gene target site comprises the amino acid sequence set forth in SEQ ID NO: 3.
• the fusion polypeptides and related methods generate directionally biased deletions in a cell at a selected dsDNA target site.
• the deletion center location is on the same side as the DNA- binding domain target site relative to the HE target site center location. In particular embodiments, the deletion center location is 5’ to the HE target site center location.
• greater than 50% of the directionally biased deletions have a deletion center location on one side of the HE target site center location. In some embodiments, greater than 51% of the directionally biased deletions have a deletion center location on one side of the HE target site center location. In some embodiments, greater than 52% of the directionally biased deletions have a deletion center location on one side of the HE target site center location. In some embodiments, greater than 53% of the directionally biased deletions have a deletion center location on one side of the HE target site center location. In some embodiments, greater than 54% of the directionally biased deletions have a deletion center location on one side of the HE target site center location.
• greater than 55% of the directionally biased deletions have a deletion center location on one side of the HE target site center location. In some embodiments, greater than 56% of the directionally biased deletions have a deletion center location on one side of the HE target site center location. In some embodiments, greater than 57% of the directionally biased deletions have a deletion center location on one side of the HE target site center location. In some embodiments, greater than 58% of the directionally biased deletions have a deletion center location on one side of the HE target site center location. In some embodiments, greater than 59% of the directionally biased deletions have a deletion center location on one side of the HE target site center location.
• greater than 60% of the directionally biased deletions have a deletion center location on one side of the HE target site center location. In some embodiments, greater than 65% of the directionally biased deletions have a deletion center location on one side of the HE target site center location. In some embodiments, greater than 70% of the directionally biased deletions have a deletion center location on one side of the HE target site center location. In some embodiments, greater than 75% of the directionally biased deletions have a deletion center location on one side of the HE target site center location. In some embodiments, greater than 80% of the directionally biased deletions have a deletion center location on one side of the HE target site center location.
• At least 50% of deletions have a deletion center greater than 4 nucleotides away from the HE target site center location.
• at least 51% of deletions have a deletion center greater than 4 nucleotides away from the HE target site center location.
• at least 52% of deletions have a deletion center greater than 4 nucleotides away from the HE target site center location.
• at least 53% of deletions have a deletion center greater than 4 nucleotides away from the HE target site center location.
• at least 54% of deletions have a deletion center greater than 4 nucleotides away from the HE target site center location.
• deletions have a deletion center greater than 4 nucleotides away from the HE target site center location. In some embodiments, at least 56% of deletions have a deletion center greater than 4 nucleotides away from the HE target site center location. In some embodiments, at least 57% of deletions have a deletion center greater than 4 nucleotides away from the HE target site center location. In some embodiments, at least 58% of deletions have a deletion center greater than 4 nucleotides away from the HE target site center location. In some embodiments, at least 59% of deletions have a deletion center greater than 4 nucleotides away from the HE target site center location.
• At least 60% of deletions have a deletion center greater than 4 nucleotides away from the HE target site center location. In some embodiments, at least 65% of deletions have a deletion center greater than 4 nucleotides away from the HE target site center location. In some embodiments, at least 70% of deletions have a deletion center greater than 4 nucleotides away from the HE target site center location. In some embodiments, at least 75% of deletions have a deletion center greater than 4 nucleotides away from the HE target site center location. In some embodiments, at least 80% of deletions have a deletion center greater than 4 nucleotides away from the HE target site center location.
• At least 10% of deletions have a deletion center greater than 8 nucleotides away from the HE target site center location. In some embodiments, at least 11% of deletions have a deletion center greater than 8 nucleotides away from the HE target site center location. In some embodiments, at least 12% of deletions have a deletion center greater than 8 nucleotides away from the HE target site center location. In some embodiments, at least 13% of deletions have a deletion center greater than 8 nucleotides away from the HE target site center location. In some embodiments, at least 14% of deletions have a deletion center greater than 8 nucleotides away from the HE target site center location.
• At least 15% of deletions have a deletion center greater than 8 nucleotides away from the HE target site center location. In some embodiments, at least 16% of deletions have a deletion center greater than 8 nucleotides away from the HE target site center location. In some embodiments, at least 17% of deletions have a deletion center greater than 8 nucleotides away from the HE target site center location. In some embodiments, at least 18% of deletions have a deletion center greater than 8 nucleotides away from the HE target site center location. In some embodiments, at least 19% of deletions have a deletion center greater than 8 nucleotides away from the HE target site center location.
• At least 20% of deletions have a deletion center greater than 8 nucleotides away from the HE target site center location. In some embodiments, at least 25% of deletions have a deletion center greater than 8 nucleotides away from the HE target site center location. In some embodiments, at least 30% of deletions have a deletion center greater than 8 nucleotides away from the HE target site center location. In some embodiments, at least 35% of deletions have a deletion center greater than 8 nucleotides away from the HE target site center location.
• At least 50% of deletions are 6 bps in length or greater. In some embodiments, at least 51% of deletions are 6 bps in length or greater. In some embodiments, at least 52% of deletions are 6 bps in length or greater. In some embodiments, at least 53% of deletions are 6 bps in length or greater. In some embodiments, at least 54% of deletions are 6 bps in length or greater. In some embodiments, at least 55% of deletions are 6 bps in length or greater. In some embodiments, at least 56% of deletions are 6 bps in length or greater. In some embodiments, at least 57% of deletions are 6 bps in length or greater. In some embodiments, at least 58% of deletions are 6 bps in length or greater.
• At least 59% of deletions are 6 bps in length or greater.
• at least 60% of deletions are 6 bps in length or greater.
• at least 65% of deletions are 6 bps in length or greater.
• at least 70% of deletions are 6 bps in length or greater.
• at least 75% of deletions are 6 bps in length or greater.
• at least 80% of deletions are 6 bps in length or greater.
• At least 30% of deletions are 12 bps in length or greater. In some embodiments, at least 31% of deletions are 12 bps in length or greater. In some embodiments, at least 32% of deletions are 12 bps in length or greater. In some embodiments, at least 33% of deletions are 12 bps in length or greater. In some embodiments, at least 34% of deletions are 12 bps in length or greater. In some embodiments, at least 35% of deletions are 12 bps in length or greater. In some embodiments, at least 36% of deletions are 12 bps in length or greater. In some embodiments, at least 37% of deletions are 12 bps in length or greater. In some embodiments, at least 38% of deletions are 12 bps in length or greater.
• At least 39% of deletions are 12 bps in length or greater. In some embodiments, at least 40% of deletions are 12 bps in length or greater. In some embodiments, at least 45% of deletions are 12 bps in length or greater. In some embodiments, at least 50% of deletions are 12 bps in length or greater. In some embodiments, at least 55% of deletions are 12 bps in length or greater. In some embodiments, at least 60% of deletions are 12 bps in length or greater.
• the directionally biased deletions comprise a length of about 10 nucleotides. In some embodiments, the directionally biased deletions comprise a length of about 11 nucleotides. In some embodiments, the directionally biased deletions comprise a length of about 12 nucleotides. In some embodiments, the directionally biased deletions comprise a length of about 13 nucleotides. In some embodiments, the directionally biased deletions comprise a length of about 14 nucleotides. In some embodiments, the directionally biased deletions comprise a length of about 15 nucleotides. In some embodiments, the directionally biased deletions comprise a length of about 16 nucleotides. In some embodiments, the directionally biased deletions comprise a length of about 17 nucleotides.
• the directionally biased deletions comprise a length of about 18 nucleotides. In some embodiments, the directionally biased deletions comprise a length of about 19 nucleotides. In some embodiments, the directionally biased deletions comprise a length of about 20 nucleotides. In various embodiments, the deletion extends into the DNA-binding domain target site. In various embodiments, the deletion center location is within the DNA- binding domain target site.
• the method further comprises introducing into the cell an end processing enzyme, or biologically active fragment thereof, or a polynucleotide, RNA, or vector encoding an end processing enzyme (e.g, an exonuclease) in addition to the fusion polypeptide, or.
• an end processing enzyme e.g, an exonuclease
• the end-processing enzyme, or biologically active fragment thereof is selected from the group consisting of: Trex2, Trexl, Trexl without transmembrane domain, Apollo, Artemis, DNA2, Exol, ExoT, ExoIII, ExoX, Fenl, Fanl, Mrell, Rad2, Rad9, TdT (terminal deoxynucleotidyl transferase), PNKP, RecE, RecJ, RecQ, Lambda exonuclease, Sox, Vaccinia DNA polymerase, exonuclease I, exonuclease III, exonuclease VII, NDK1, NDK5, NDK7, NDK8, WRN, T7-exonuclease Gene 6, avian myeloblastosis virus integration protein (IN), Bloom, Antartic Phophatase, Alkaline Phosphatase, Poly nucleotide Kinase (PNK), A
• methods of site-directed mutagenesis in a cell comprises 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 directionally biased deletions having a deletion center near the selected dsDNA target cut site in the cell.
• dsDNA double-stranded DNA
• the methods comprise selecting a double-stranded DNA (dsDNA) target site; introducing into a cell a fusion polypeptide comprising, a DNA-binding domain and a homing endonuclease (HE) variant that binds and cleaves a selected double strand DNA (dsDNA) target site in a cell, a linker domain, and an Exol, or biologically active fragment thereof; and introducing a exonuclease (e.g, Trex2); wherein the method generates directionally biased deletions having a deletion center near the selected dsDNA target cut site in the cell.
• the method is an in vitro method.
• the method is an ex vivo method.
• the method is an in vivo method.
• the fusion polypeptides, genetically edited cells, compositions, and/or associated methods of gene editing contemplated herein can be used in the prevention, treatment, and amelioration of a disease or disorder, or ameliorating a disease condition or symptom associated therewith.
• the fusion polypeptides, genetically edited cells, compositions, and/or associated methods of gene editing contemplated herein can be used in methods of treating, preventing, or inhibiting a disease (e.g., cancer, ischemia, diabetic retinopathy, macular degeneration, rheumatoid arthritis, psoriasis, HIV infection, sickle cell anemia, Alzheimer's disease, muscular dystrophy, neurodegenerative diseases, vascular disease, cystic fibrosis, stroke, hyper IGE syndrome, hemophilia) or ameliorating a disease condition or symptom associated with a disease, such as, cancer, ischemia, diabetic retinopathy, macular degeneration, rheumatoid arthritis, psorias
• the fusion polypeptides, genetically edited cells, compositions, and/or associated methods of gene editing contemplated herein are useful to treat, prevent, or inhibit an autosomal dominant disease, such as achondroplasia, pseudoachondroplasia, the multiple epiphyseal dysplasias, chondrodysplasias, osteogenesis imperfecta, Marfan syndrome, Polydactyly, hereditary motor sensory neuropathies I and II (Charcot-Marie-Tooth disease), myotonic dystrophy, and neurofibromatosis or ameliorate a disease condition or symptom associated with an autosomal dominant disease, such as achondroplasia, pseudoachondroplasia, the multiple epiphyseal dysplasias, chondrodysplasias, osteogenesis imperfecta, Marfan syndrome, Polydactyly, hereditary motor sensory neuropathies I and II (Charcot-Marie- Tooth disease), myotonic dystrophy, and neurofibromatosis.
• the fusion polypeptides, genetically edited cells, compositions, and/or associated methods of gene editing contemplated herein can be used to prevent, treat, and/or ameliorate of at least one symptom associated with and immune disorder or cancer.
• immune disorder refers to a disease that evokes a response from the immune system.
• the term “immune disorder” refers to a cancer, graft- versus-host disease, an autoimmune disease, or an immunodeficiency.
• immune disorders encompass infectious disease.
• cancer relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues.
• malignant refers to a cancer in which a group of tumor cells display one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., intrusion on and destruction of adjacent tissues), and metastasis (i.e., spread to other locations in the body via lymph or blood).
• metastatic tumor refers to the spread of cancer from one part of the body to another.
• a tumor formed by cells that have spread is called a “metastatic tumor” or a “metastasis.”
• the metastatic tumor contains cells that are like those in the original (primary) tumor.
• Benign or “non-malignanf ’ refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.
• a “cancer cell” or “tumor cell” refers to an individual cell of a cancerous growth or tissue.
• a tumor refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre-malignant, or malignant. Most cancers form tumors, but some, e.g., leukemia, do not necessarily form tumors. For those cancers that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.
• the amount of a tumor in an individual is the “tumor burden” which can be measured as the number, volume, or weight of the tumor.
• “Graft-versus-host disease” or “GVHD” refers complications that can occur after cell, tissue, or solid organ transplant.
• GVHD can occur after a stem cell or bone marrow transplant in which the transplanted donor cells attack the transplant recipient's body.
• Acute GVHD in humans takes place within about 60 days post-transplantation and results in damage to the skin, liver, and gut by the action of cytolytic lymphocytes.
• Chronic GVHD occurs later and is a systemic autoimmune disease that affects primarily the skin, resulting in the polyclonal activation of B cells and the hyperproduction of Ig and autoantibodies.
• Solid-organ transplant graft-versus-host disease (SOT-GVHD) occurs in two forms.
• the more common type is antibody mediated, wherein antibodies from a donor with blood type O attack a recipient’s red blood cells in recipients with blood type A, B, or AB, leading to mild transient, hemolytic anemias.
• the second form of SOT-GVHD is a cellular type associated with high mortality, wherein donor-derived T cells produce an immunological attack against immunologically disparate host tissue, most often in the skin, liver, gastrointestinal tract, and bone marrow, leading to complications in these organs.
• Graft-versus-leukemia or “GVL” refer to an immune response to a person’s leukemia cells by immune cells present in a donor’s transplanted tissue, such as bone marrow or peripheral blood.
• autoimmune disease refers to a disease in which the body produces an immunogenic (i.e., immune system) response to some constituent of its own tissue.
• immune system i.e., immune system
• the immune system loses its ability to recognize some tissue or system within the body as “self’ and targets and attacks it as if it were foreign.
• autoimmune diseases include, but are not limited to: arthritis, inflammatory bowel disease, Hashimoto’s thyroiditis, Grave’s disease, lupus, multiple sclerosis, rheumatic arthritis, hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus, celiac disease, Crohn’s disease, colitis, diabetes, scleroderma, psoriasis, and the like.
• Immunodeficiency means the state of a patient whose immune system has been compromised by disease or by administration of chemicals. This condition makes the system deficient in the number and type of blood cells needed to defend against a foreign substance.
• Immunodeficiency conditions or diseases are known in the art and include, for example, AIDS (acquired immunodeficiency syndrome), SCID (severe combined immunodeficiency disease), selective IgA deficiency, common variable immunodeficiency, X-linked agammaglobulinemia, chronic granulomatous disease, hyper-IgM syndrome, Wiskot-Aldrich Syndrome (WAS), and diabetes.
• infectious disease refers to a disease that can be transmited from person to person or from organism to organism, and is caused by a microbial or viral agent (e.g, common cold). Infectious diseases are known in the art and include, for example, hepatitis, sexually transmited diseases (e.g, Chlamydia, gonorrhea), tuberculosis, HIV/AIDS, diphtheria, hepatitis B, hepatitis C, cholera, and influenza.
• a microbial or viral agent e.g, common cold.
• Infectious diseases include, for example, hepatitis, sexually transmited diseases (e.g, Chlamydia, gonorrhea), tuberculosis, HIV/AIDS, diphtheria, hepatitis B, hepatitis C, cholera, and influenza.
• the terms “individual” and “subject” are often used interchangeably and refer to any animal that exhibits a symptom of an immune disorder that can be treated with the fusion polypeptides, genetically edited cells, compositions, and/or associated methods of gene editing contemplated elsewhere herein.
• Suitable subjects e.g, patients
• laboratory animals such as mouse, rat, rabbit, or guinea pig
• farm animals such as a cat or dog
• domestic animals or pets such as a cat or dog
• Non-human primates and, preferably, human subjects are included.
• Typical subjects include human patients that have, have been diagnosed with, or are at risk of having an immune disorder.
• the term “patient” refers to a subject that has been diagnosed with an immune disorder that can be treated with the fusion polypeptides, genetically edited cells, compositions, and/or associated methods of gene editing contemplated elsewhere herein.
• treatment includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated, e.g, cancer, GVHD, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency. Treatment can optionally involve delaying of the progression of the disease or condition. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.
• prevention and similar words such as “prevention,” “prevented,” “preventing” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of, a disease or condition, e.g, cancer, GVHD, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, “prevention” and similar words also includes reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to onset or recurrence of the disease or condition.
• the phrase “ameliorating at least one symptom of’ refers to decreasing one or more symptoms of the disease or condition for which the subject is being treated, e.g, cancer, GVHD, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency.
• the disease or condition being treated is a cancer, wherein the one or more symptoms ameliorated include, but are not limited to, weakness, fatigue, shortness of breath, easy bruising and bleeding, frequent infections, enlarged lymph nodes, distended or painful abdomen (due to enlarged abdominal organs), bone or joint pain, fractures, unplanned weight loss, poor appetite, night sweats, persistent mild fever, and decreased urination (due to impaired kidney function).
• the fusion polypeptides, genetically edited cells, compositions, and/or associated methods of gene editing contemplated herein are used in methods of treating cancer.
• m the fusion polypeptides, genetically edited cells, compositions, and/or associated methods of gene editing contemplated herein are used in methods of treating solid tumors or cancers.
• the fusion polypeptides, genetically edited cells, compositions, and/or associated methods of gene editing contemplated herein are used in the treatment of solid tumors or cancers including, but not limited to: adrenal cancer, adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid/rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain/CNS cancer, breast cancer, bronchial tumors, cardiac tumors, cervical cancer, cholangiocarcinoma, chondrosarcoma, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma in situ (DCIS) endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing’s sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube
• the fusion polypeptides, genetically edited cells, compositions, and/or associated methods of gene editing contemplated herein are used in the treatment of solid tumors or cancers including, without limitation, liver cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, brain cancer, bone cancer, thyroid cancer, kidney cancer, or skin cancer.
• the fusion polypeptides, genetically edited cells, compositions, and/or associated methods of gene editing contemplated herein are used in the treatment of various cancers including but not limited to pancreatic, bladder, and lung.
• the fusion polypeptides, genetically edited cells, compositions, and/or associated methods of gene editing contemplated herein are used in the treatment of liquid cancers or hematological cancers.
• the fusion polypeptides, genetically edited cells, compositions, and/or associated methods of gene editing contemplated herein are used in the treatment of B-cell malignancies, including but not limited to: leukemias, lymphomas, and multiple myeloma.
• the fusion polypeptides, genetically edited cells, compositions, and/or associated methods of gene editing contemplated herein are used in the treatment of liquid cancers including, but not limited to 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 myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML) and polycythemia vera, Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, Burkitt lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicular lymphoma, immuno
• a method comprises administering a therapeutically effective amount of fusion polypeptides, composition, and/or genetically edited cells to a patient in need thereof.
• the fusion polypeptides, genetically edited cells, compositions, and/or associated methods of gene editing contemplated herein are used in the treatment of patients at risk for developing a cancer.
• particular embodiments comprise the treatment or prevention or amelioration of at least one symptom of a cancer comprising administering to a patient in need thereof, a therapeutically effective amount of the fusion polypeptides, genetically edited cells, and/or compositions contemplated herein.
• a method of treating, preventing, or ameliorating at least one symptom of a disease, or condition associated therewith comprising harvesting a population of cells from a subject; editing the population of cells according to the methods of genetic editing/mutagenesis provided herein, and administering the edited population of cells to the subject in need thereof (e.g, a subject having cancer).
• the quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.
• the amount of genetically 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 cells, at least 2 x 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 x IO 10 cells.
• about 1 x 10 7 cells to about 1 x 10 9 cells, about 2 x 10 7 cells to about 0.9 x 10 9 cells, about 3 x 10 7 cells to about 0.8 x 10 9 cells, about 4 x 10 7 cells to about 0.7 x 10 9 cells, about 5 x 10 7 cells to about 0.6 x 10 9 cells, or about 5 x 10 7 cells to about 0.5 x 10 9 cells are administered to a subject.
• the amount of genetically edited cells in the composition administered to a subject is at least 0.1 x 10 4 cells/kg of body weight, at least 0.5 x 10 4 cells/kg of body weight, at least 1 x 10 4 cells/kg of body weight, at least 5 x 10 4 cells/kg of body weight, at least 1 x 10 5 cells/kg of body weight, at least 0.5 x 10 6 cells/kg of body weight, at least 1 x 10 6 cells/kg of body weight, at least 0.5 x 10 7 cells/kg of body weight, at least 1 x 10 7 cells/kg of body weight, at least 0.5 x 10 8 cells/kg of body weight, at least 1 x 10 8 cells/kg of body weight, at least 2 x 10 8 cells/kg of body weight, at least 3 x 10 8 cells/kg of body weight, at least 4 x 10 8 cells/kg of body weight, at least 5 x 10 8 cells/kg of body weight, or at least 1 x 10 9 cells/kg of body weight.
• compositions contemplated in particular embodiments may be required to affect the desired therapy.
• a composition may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a span 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.
• compositions contemplated in particular embodiments may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation.
• compositions are administered nasally, orally, enterally, or parenterally.
• parenteral administration and “administered parenterally” as used herein refers to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.
• the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.
• a subject in need thereof is administered an effective amount of a composition to increase a cellular immune response to a cancer in the subject.
• the immune response may include cellular immune responses mediated by cytotoxic T cells capable of killing infected cells, regulatory T cells, and helper T cell responses.
• Humoral immune responses mediated primarily by helper T cells capable of activating B cells thus leading to antibody production, may also be induced.
• a variety of techniques may be used for analyzing the type of immune responses induced by the compositions, which are well described in the art; e.g, 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, N.Y.
• TRAC locus SEQ ID NO: 1
• a low/moderate efficiency megaTAL for example SEQ ID NOs: 5-7
• SEQ ID NOs: 5-7 was used for these studies to provide dynamic range for any downstream manipulations that were hypothesized to alter editing rates.
• Several independent experiments were performed using primary human T cells activated and expanded from distinct PBMC donors. Following PBMC thawing, activation and culture 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 (TCRa megaTAL) were electroporated.
• the resulting cells were cultured for an additional outgrowth 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 amplicon enabled the characterization of the bulk editing events caused by the megaTAL. Frequency histograms tallying the sizes of the editing events are shown in Figure IB. The most frequently observed indel lengths were 2 base pairs long (cumulatively representing -40-45% of the total population of events), while 1 base pair deletions (-20%) and 3, 8, and 9 base pair deletions (5-10% each) were also observed at a high frequency. The relative frequencies of these indel size populations were highly reproducible across technical replicates and independent experiments, illustrating the consistency of the indel length distributions during megaTAL gene editing.
• Each bar in the histograms shown in Figure IB represents a population of edited alleles with the same indel length but potentially different positions relative to the megaTAL target site.
• Figure 2 shows each unique edited species plotted according to its positional location 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), thus capturing both quantitative and qualitative properties of each edited species.
• the position of a 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 location.
• Trex2 Three Prime Repair Exonuclease-2
• Rex2 Three Prime Repair Exonuclease-2
• the qualitative impact of Trex2 co-delivery on editing outcomes have not been thoroughly assessed. Therefore, evaluation of the indel properties of two distinct modes of co-delivery was performed; direct fusion of Trex2 to the C-terminus of a megaTAL; and co-expression of Trex2 and a megaTAL as independent polypeptides.
• Activated primary human T cell samples were electroporated with in vitro transcribed, capped and poly -adenylated mRNA encoding the TCRa megaTAL ( Figure 3 A), mRNA encoding TCRa megaTAL-Trex2 fusion ( Figure 3B), or two distinct mRNAs, one encoding TCRa megaTAL and the other encoding Trex2 ( Figure 3C).
• the resulting cells were cultured for an additional outgrowth 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 amplicon enabled the characterization of the bulk editing events caused by the megaTAL.
• Frequency histograms tallying the sizes of the editing events are shown in Figures 3A-3C.
• Direct fusion of the Trex2 exonuclease to the TCRa megaTAL resulted in a change in the deletion length distribution, with deletions up to 12 base pairs in length comprising a significant percentage of the indel species.
• Independent co-expression of the TCRa megaTAL and the Trex2 exonuclease resulted in a narrowing of the indel species toward deletions 1 to 4 base pairs in length, consistent with a model of distributive exonuclease activity with a preference for single-stranded 3’ overhangs.
• the overall editing rate and the ratio of deletions to insertions increased relative to the delivery of the TCRa megaTAL alone.
• Figure 4 shows the deletion fingerprint plots for each sample in Figures 3A-3C, capturing both quantitative and qualitative properties of each edited species.
• the position of a 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.
• the distribution of the deletion species relative to the breakpoint center showed marked differences between the TCRa megaTAL, TCRa megaTAL-Trex2 fusion, and TCRa megaTAL plus Trex2 co-delivery editing outcomes (see Tables 3-5).
• the megaTAL- Trex2 fusion emergence of deletion species whose positions were skewed heavily in the 5’ direction was observed. This pattern implies that there is preferential exonuclease activity occurring at the DNA breakpoint end that remains in cis with, or on the same side of the breakpoint as, the TALE array.
• Trex2 homolog ORFs were fused to the TCRa megaTAL (for example SEQ ID NOs: 17-34).
• Activated primary human T cell samples were electroporated with in vitro transcribed, capped and polyadenylated mRNA encoding these fusion proteins, then monitored target site editing by flow cytometric analysis of expression of the TCR complex and by characterizing indel properties.
• Figure 5 shows that many of the Trex2 homolog fusion proteins enhanced the overall editing to a similar extent as the human Trex2 ORF.
• Flow cytometry staining for the CD3 components that combine with the TCR alpha and beta chains on the cell surface was used to assess editing efficiency. The frequency of cells that have lost CD3 staining therefore correlates with the rate of editing at the TCRa locus.
• Analysis of the distribution of indel species, as shown in Figure 6, further confirms that the platypus, opossum, armadillo, and mouse Trex2 proteins cause editing outcomes with properties consistent with those caused by human Trex2 ORF when fused to the TCRa megaTAL.
• sheep Trex2 caused a unique indel spectrum enriched for 1-4 bp deletions and, surprisingly, a high frequency of 1 bp insertions.
• the deletion properties of the TCRa megaTAL-Trex2 homolog fusion proteins as shown in the fingerprint plots displayed in Figure 7, mirror those observed for the human Trex2 protein.
• this data suggests that Trex2 exonuclease activity is the primary determinant of the observed deletion properties.
• the extended deletion length outcomes are thus likely to be independent of interaction with other DNA repair machinery.
• the increased occurrence of insertions observed for the sheep Trex2 homolog indicates that this variant may have a unique enzymatic mechanism, such as potentially a residual template independent polymerase activity.
• the highly consistent and sharp demarcations in deletion size and position further suggest that the directional excision of bases may be limited by the association and dissociation kinetics and/or steric properties of the TALE array’s interaction with its binding site.
• the megaTAL-Exol fusion generates deletion events that are biased in the direction of the TALE array. In contrast with the sharply demarcated megaTAL-Trex2 fusion deletion events, the megaTAL-Exol deletion events appeared to be of greater length and thus extend beyond the TALE array binding site.
• the TCRa megaTAL-ExoX fusion resultsed in a deletion profile distinct from that observed with the megaTAL-Exol fusion.
• the TCRa megaTAL-ExoX fusion generates long, directionally -biased deletions with high efficiency.
• the deletion centers observed were greater such that they did progress beyond the TALE array binding site at an appreciable frequency. It is possible that the megaTAL-ExoX fusion has a unique exonuclease mechanism that leads to longer deletions with deletion centers further away from the cut site relative to megaTAL-Trex2 fusion proteins.
• PCR Polymerase chain reaction
• the high- activity CBL-B megaTAL-Trex2 fusion lengthened the deletions species and skewed them heavily in the 5’ direction, confirming for an orthogonal megaTAL target the preferential exonuclease activity occurring at the DNA breakpoint end that remains in cis with, or on the same side of the breakpoint as, the TALE array. Also recurring in the high-activity CBL-B megaTAL-Trex2 fusion was the steep drop-off in deletion length and position that corresponds with the 3’ edge of the TALE array binding site.
• Figure 11 shows representative deletion fingerprints from T cells treated with a similar series of CBL-B megaTAL, megaTAL-Trex2, and megaTAL-ExoX fusion proteins, however these employ a several-fold lower activity megaTAL (for example SEQ ID NOs: 65-73). See also Tables 9-11. There is a substantial similarity in the spectrum of deletion species observed when comparing between these low-activity CBL-B megaTALs and megaTAL fusion proteins to their high-activity counterparts displayed in Figure 10. This illustrates that the unique deletion outcomes observed when these two exonucleases are fused to a megaTAL are independent of the enzymatic rate of the endonuclease reaction.
• FIG. 12 shows representative deletion fingerprints from T cells electroporated mRNA encoding either a PD-1 megaTAL, PD-1 megaTAL-Trex2 fusion, or a PD-1 megaTAL-ExoX fusion (for example SEQ ID NOs: 74-82). See also Tables 12-14.
• the deletion species observed for each of the three PD-1 megaTAL samples followed the consistent trend of having non- directional, small deletion species in the stand-alone PD-1 megaTAL, 5’ directional and longer species emerged when that target was exposed to the PD-1 megaTAL-Trex2 fusion protein, and longer still were the species arising from treatment with PD-1 megaTAL-ExoX fusion protein.
• the persistence of these observed patterns across several megaTALs tested illustrates the robustness of the distinct editing outcomes that occur upon megaTAL fusion to different exonuclease domains.
• Figure 13 depicts the PD-1 megaTAL target site, which lies 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 encoding an isoleucine residue that is part of the signal sequence that targets PD-1 for expression on the plasma membrane.
• the TALE array binding site (SEQ ID NO: 4) lies 5’ of the ATG start codon. This target site orientation raises the potential for distinct megaTAL or megaTAL-exonuclease fusion compositions to differentially impact expression of PD-1 depending on their respective distributions of deletion species.
• the PD-1 megaTAL-Trex2 fusion protein causes a shift in the locations of the in-frame, frame 2, and frame 3 species.
• the normalized fraction of each category of species are compared, as shown in Figure 15, the PD-1 megaTAL and the PD-1 megaTAL-Trex2 fusion do not significantly differ. This is because the occurrence of long, directional deletions that reach through the ATG start codon, while increased, have effectively replaced the smattering of long (but non-directi onal) deletions that occur with the PD-1 megaTAL.
• the increased deletion lengths caused by the PD-1 megaTAL-ExoX fusion protein substantially increases the fractional proportion of deletion species that eliminate the ATG start codon.
• This increase in ATG-deleted alleles comes at the expense of deletions that produce species that fall into the other three categories, and further suggests that the PD-1 megaTAL-ExoX fusion may disproportionately erase undesired PD- 1 gene edited alleles, such as in-frame single amino acid deletions to the N-terminal end of the signal sequence that may not impact PD-1 surface expression nor function.
• activated primary human T cell samples were electroporated with in vitro transcribed, capped and poly-adenylated mRNA encoding either or both a cyan fluorescent protein (CFP) to track transfection efficiency, and 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, depicted in Figure 16.
• amplicon sequencing and deletion fingerprint analysis was performed at off-target sites specific to low and high editing TCRa megaTALs.
• Each construct was evaluated at the on-target locus ( Figure 17; in duplicate; only one replicate shown), or its off-target locus (in duplicate, only one replicate shown). Following an outgrowth period of 7-10 days, cells were harvested for genomic DNA isolation.
• PCR was performed over an approximately 300 base pair region encompassing the KAT2B off-target site (susceptible to editing by the low editing TCRa megaTAL, Figure 18) or the 2.2 off-target site AC016700.3 (susceptible to editing by the high editing TCRa megaTAL, Figure 19).
• the PCR amplicons were then subjected to next-generation sequencing (NGS) and analyzed by tabulating deletion events according to their frequency (circle size) as well as both their length (y-axis) and their longitudinal location relative to the megaTAL caused breakpoint (x-axis). Insertions were excluded from this analysis.
• NGS next-generation sequencing

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