EP4010004A2 - Zusammensetzungen und verfahren zur modulation von genexpression - Google Patents

Zusammensetzungen und verfahren zur modulation von genexpression

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Publication number
EP4010004A2
EP4010004A2 EP20851042.0A EP20851042A EP4010004A2 EP 4010004 A2 EP4010004 A2 EP 4010004A2 EP 20851042 A EP20851042 A EP 20851042A EP 4010004 A2 EP4010004 A2 EP 4010004A2
Authority
EP
European Patent Office
Prior art keywords
dbd
gene
nucleic acid
terminus
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20851042.0A
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English (en)
French (fr)
Other versions
EP4010004A4 (de
Inventor
Fyodor Urnov
John A. Stamatoyannopoulos
Joycelynn PEARL
Matthew WILKEN
Christie CIARLO
Shon GREEN
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Altius Institute for Biomedical Sciences
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I Altius Institute For Biomedical Sciences
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Application filed by I Altius Institute For Biomedical Sciences filed Critical I Altius Institute For Biomedical Sciences
Publication of EP4010004A2 publication Critical patent/EP4010004A2/de
Publication of EP4010004A4 publication Critical patent/EP4010004A4/de
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/71—Fusion polypeptide containing domain for protein-protein interaction containing domain for transcriptional activaation, e.g. VP16
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/005—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination repressible enhancer/promoter combination, e.g. KRAB
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

Definitions

  • Modulating gene expression has been a strategy for enhancing the success of cancer and infectious disease therapies.
  • cell therapies such as CAR T cell therapies
  • immune checkpoint inhibitors there exists a need for agents that can modulate the expression of target genes, such as, immune checkpoint inhibitors.
  • the present disclosure provides engineered polypeptides comprising DNA binding domains and repressor domains for repressing a target gene.
  • the present disclosure provides polypeptides, compositions thereof, and methods for suppressing expression of a target gene such as PDCD1, CTLA4, LAG3, or TIM-3.
  • the polypeptides disclosed herein include a DNA binding domain (DBD) that binds to a sequence of the target gene and a transcriptional repressor domain that suppresses expression of the target gene.
  • DBD DNA binding domain
  • the transcriptional repressor domain may be a known transcriptional repressor or may be a novel transcriptional repressor disclosed herein.
  • sequences of novel transcriptional repressor domains that are conjugated to a heterologous DNA binding domain. As shown herein, these novel transcriptional repressor domains mediate suppression of expression of a target gene bound by the heterologous DNA binding domain.
  • FIGS. 1A-1C illustrate the locations in the PDCD1 gene to which the DBDs of the indicated recombinant polypeptides were designed to bind. Recombinant polypeptides that repressed expression of PDCD1 in at least 50% of cells treated with the recombinant polypeptides are indicated by clear arrows .
  • Recombinant polypeptides that repressed expression of PDCD1 in less than 50% of the cells treated with the recombinant polypeptides are indicated by solid arrows .
  • the orientation of the arrows indicates the DNA strand to which the recombinant polypeptide is designed to bind. Arrows having the orientation are designed to bind to the anti-sense strand.
  • Arrows having the orientation are designed to bind to the sense strand.
  • FIG. 2 shows the fold change in number of PD-1 expressing cells 2 days after transfection of mRNA encoding the indicated recombinant polypeptides into CD3+ T cells.
  • FIG. 3 shows effect of dose of mRNA encoding the recombinant polypeptide, pAL040 and pAL043, on the percent of CD3+ T cells expressing PD-1 3 days after transfection.
  • FIG. 4 shows the fold change in number of PD-1 -positive cells at the indicated number of days post-transfection of mRNA encoding the indicated recombinant polypeptide relative to control.
  • FIGS. 5 A and 5B show that PD-1 repression with pAL043 in anti-CD 19 CAR-T cells is sustained after in vivo expansion and clearance of CD 19-positive NALM-6 B-ALL tumor model in NOD SCID Gamma (NSG) mice.
  • FIG. 6 illustrates the locations in the TIM3 gene at which the DBDs of the indicated recombinant polypeptides bind.
  • Recombinant polypeptides that repressed expression of TIM3 in at least 50% of the cells are indicated by unfilled arrows .
  • Recombinant polypeptides that repressed expression of TIM3 in less than 50% of the cells are indicated by filled arrows
  • FIG. 7 shows the fold change in number of cells expressing TIM3 at 2 days, 5 days, 8 days, or 14 days after transfection of mRNA encoding the indicated recombinant polypeptides into CD3+ T cells.
  • FIG. 8 shows the fold change in number of cells expressing TIM3 at 3 days or 6 days after transfection of mRNA encoding the indicated recombinant polypeptides into CD3+ T cells.
  • FIG. 9 illustrates the locations in the CTLA4 gene at which the DBDs of the indicated recombinant polypeptides bind. Recombinant polypeptides that repressed expression of CTLA4 in at least
  • FIG. 10 shows the fold change in number of cells expressing CTLA4 at 3 days after transfection of mRNA encoding the indicated recombinant polypeptides into CD3+ T cells.
  • FIG. 11 illustrates the locations in the LAG3 gene at which the DBDs of the indicated recombinant polypeptides bind. Recombinant polypeptides that repressed expression of LAG3 in at least 50% of the cells are indicated by unfdled arrows . Recombinant polypeptides that repressed expression of LAG3 in less than 50% of the cells are indicated by fdled arrows
  • FIG. 12 shows the fold change in number of cells expressing LAG3 at 2 days, 7 days, or 12 days after transfection of mRNA encoding the indicated recombinant polypeptides into CD3+ T cells.
  • FIG. 13 shows the fold change in number of cells expressing LAG3 at 2 days after transfection of mRNA encoding the indicated recombinant polypeptides into CD3+ T cells.
  • FIGS. 14A and 14B show multiplexing of recombinant polypeptides to simultaneously suppress expression of PD-1, LAG3, and TIM3 is a single cell.
  • FIGS. 15A-15C illustrates specificity of the recombinant polypeptides as indicated by lack of significant off-target effect as measured by RNA-seq.
  • FIG. 16 shows characterization of repression of TIM3 expression by the listed candidate transcriptional repressors.
  • FIG. 17 shows characterization of repression of LAG3, TIM3, or PD-1 expression by the listed candidate transcriptional repressors.
  • FIG. 18 shows characterization of repression of TIM3 expression by the listed candidate transcriptional repressors.
  • FIG. 19 shows a schematic of an anti-CD 19 CAR-T cell in which expression of PD1, TIM3, and LAG3 has been repressed using the engineered polypeptides (pAL043+TL8188+TL8222) described herein.
  • FIG. 20 shows flow cytometry data confirming repression of PD1, TIM3, and LAG3 expression in the multiplex-treated CAR-T cells.
  • FIG. 21 provides an overview of in vivo leukemia xenograft model and treatment using indicated CAR-T cells.
  • FIG. 22 demonstrates that multiplexed repression of immune checkpoint genes is sustained in vivo.
  • FIG. 23 demonstrates that multiplexed repression of immune checkpoint genes enhances CAR-Ts ability to resist tumor re-challenge.
  • FIG. 24 shows expansion of CAR-Ts in the mouse blood.
  • FIG. 25 TALE-KRAB split system.
  • FIG. 26 Large-scale analysis of functional domains enabled by split encoding of DNA targeting and functional activities.
  • FIG. 27 Repression of TIM3 expression using TALE-KRAB split system.
  • FIGS. 28 and 29 Control of gene expression using CIPHR logic gates.
  • the present disclosure provides recombinant polypeptides, compositions and methods for suppressing target gene expression for therapeutic purposes.
  • engineered polypeptides comprising a DNA-binding domain (DBD) and a transcription repressor.
  • the DBD mediates binding of the disclosed polypeptides to a sequence in the target gene.
  • the target gene may be PDCD1, LAG3, TIM3, or CTLA4.
  • regions in these target genes have been identified that can be targeted for repression of expression of these gene when these regions are bound by the polypeptides disclosed herein. These regions may be located in the target gene within an expression control region, such as, a coding region, a non-coding region, such as, a regulatory region (e.g., promoter region) or an intron.
  • an expression control region such as, a coding region, a non-coding region, such as, a regulatory region (e.g., promoter region) or an intron.
  • novel transcriptional repressors that are conjugated to a heterologous DNA binding domain and mediate suppression of expression of a target gene bound by the DNA binding domain.
  • split systems for modulating gene expression where the DBD and the functional domain are provided as separated polypeptides and are assembled using dimerization of a heterodimer pair, where the DBD and the functional domain are each fused to a member of the heterodimer pair.
  • polypeptide derived in the context of a polypeptide refers to a polypeptide that has a sequence that is based on that of a protein from a particular source (e.g., Xanthomonas or Legionella).
  • a polypeptide derived from a protein from a particular source may be a variant of the protein from the particular source.
  • a polypeptide derived from a protein from a particular source may have a sequence that is modified with respect to the protein's sequence from which it is derived.
  • a polypeptide derived from a protein from a particular source shares at least 30% sequence identity with, at least 40% sequence identity with, at least 50% sequence identity with, at least 60% sequence identity with, at least 70% sequence identity with, at least 80% sequence identity with, or at least 90% sequence identity with the protein from which it is derived.
  • the term “modular” as used herein in the context of a DNA binding domain e.g., a modular animal pathogen derived nucleic acid binding domain (MAP-NBD) indicates that the plurality of repeat units present in the DBD can be rearranged and/or replaced with other repeat units and can be arranged in an order such that the DBD binds to the target nucleic acid.
  • MAP-NBD modular animal pathogen derived nucleic acid binding domain
  • any repeat unit in a modular nucleic acid binding domain can be switched with a different repeat unit.
  • modularity of the DNA binding domains disclosed herein allows for switching the target nucleic acid base for a particular repeat unit by simply switching it out for another repeat unit.
  • modularity of the DNA binding domains disclosed herein allows for swapping out a particular repeat unit for another repeat unit to increase the affinity of the repeat unit for a particular target nucleic acid.
  • the modular nature of the DNA binding domains disclosed herein enables the development of genome editing complexes that can precisely target any nucleic acid sequence of interest.
  • polypeptide refers to a polymeric form of amino acids of any length, which can include genetically coded and non-genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified polypeptide backbones.
  • the terms include fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusion proteins with heterologous and homologous leader sequences, with or without N-terminus methionine residues; immunologically tagged proteins; and the like.
  • the terms refer to a polymeric form of amino acids of any length which include genetically coded amino acids.
  • the terms refer to a polymeric form of amino acids of any length which include genetically coded amino acids fused to a heterologous amino acid sequence.
  • heterologous refers to two components that are defined by structures derived from different sources.
  • a “heterologous” polypeptide may include operably linked amino acid sequences that are derived from different polypeptides (e.g., a DBD and a functional domain, e.g., a transcriptional repressor, derived from different sources).
  • a “heterologous” polynucleotide may include operably linked nucleic acid sequences that can be derived from different genes.
  • heterologous nucleic acids include expression constructs in which a nucleic acid comprising a coding sequence is operably linked to a regulatory element (e.g., a promoter) that is from a genetic origin different from that of the coding sequence (e.g., to provide for expression in a host cell of interest, which may be of different genetic origin than the promoter, the coding sequence or both).
  • a regulatory element e.g., a promoter
  • heterologous can refer to the presence of a nucleic acid (or gene product, such as a polypeptide) that is of a different genetic origin than the host cell in which it is present.
  • operably linked refers to linkage between molecules to provide a desired function.
  • operably linked in the context of nucleic acids refers to a functional linkage between nucleic acid sequences.
  • a nucleic acid expression control sequence such as a promoter, signal sequence, or array of transcription factor binding sites
  • the expression control sequence affects transcription and/or translation of the second polynucleotide.
  • operably linked refers to a functional linkage between amino acid sequences (e.g., different domains) to provide for a described activity of the polypeptide.
  • a “target nucleic acid,” “target sequence,” or “target site” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule, such as, the DBD disclosed herein will bind.
  • the target nucleic acid may be present in an isolated form or inside a cell.
  • a target nucleic acid may be present in a region of interest.
  • a “region of interest” may be any region of cellular chromatin, such as, for example, a gene or a non-coding sequence within or adjacent to a gene, in which it is desirable to bind an exogenous molecule.
  • a region of interest can be present in a chromosome, an episome, an organellar genome (e.g., mitochondrial, chloroplast), or an infecting viral genome, for example.
  • a region of interest can be within the coding region of a gene, within transcribed non-coding regions such as, for example, promoter sequences, leader sequences, trailer sequences or introns, or within non-transcribed regions, either upstream or downstream of the coding region.
  • a region of interest can be as small as a five nucleotide pair or up to 200 nucleotide pairs in length, or any integral value of nucleotide pairs.
  • exogenous molecule is a molecule that is not normally present in a cell but can be introduced into a cell by one or more genetic, biochemical or other methods.
  • An exogenous nucleic acid can be present in an infecting viral genome, a plasmid or episome introduced into a cell.
  • Methods for the introduction of exogenous molecules into cells include, but are not limited to, lipid-mediated transfer (i.e., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, 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.
  • an endogenous nucleic acid can comprise a chromosome, the genome of a mitochondrion, chloroplast or other organelle, or a naturally-occurring episomal nucleic acid.
  • Additional endogenous molecules can include proteins, for example, transcription factors and enzymes.
  • a “gene,” for the purposes of the present disclosure, includes 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. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control region.
  • 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, shRNA, RNAi, miRNA or any other type of RNA) or a protein produced by translation of a mRNA.
  • 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, myristylation, and glycosylation.
  • conjugating refers to an association of two entities, for example, of two molecules such as two proteins, two domains (e.g., a binding domain and a transcription repressor domain), or a protein and an agent, e.g., a protein binding domain and a small molecule.
  • the association can be, for example, via a direct or indirect (e.g., via a linker) covalent linkage or via non-covalent interactions. In some embodiments, the association is covalent. In some embodiments, two molecules are conjugated via a linker connecting both molecules.
  • the two proteins may be conjugated via a polypeptide linker, e.g., an amino acid sequence connecting the C-terminus of one protein to the N- terminus of the other protein.
  • a polypeptide linker e.g., an amino acid sequence connecting the C-terminus of one protein to the N- terminus of the other protein.
  • conjugated proteins may be expressed as a fusion protein.
  • an effective amount refers to an amount of a biologically active agent that is sufficient to elicit a desired biological response.
  • an effective amount of a polypeptide comprising a transcriptional repressor may refer to the amount of the polypeptide that is sufficient to induce repression of expression from a gene specifically bound by the polypeptide.
  • an agent e.g., a recombinant polynucleotide
  • the effective amount of an agent e.g., a recombinant polynucleotide, may vary depending on various factors as, for example, on the desired biological response, the specific allele, genome, target site, cell, or tissue being targeted, and the agent being used.
  • strand refers to a nucleic acid made up of nucleotides covalently linked together by covalent bonds, e.g., phosphodiester bonds.
  • DNA usually exists in a double-stranded form, and as such, has two complementary strands of nucleic acid referred to herein as the “top” and “bottom” strands or the “Watson” and “Crick” strands.
  • Watson strand refers to 5' to 3' top strand (5'
  • Crick strand refers to 3' to 5' bottom strand (3' 5').
  • the assignment of a strand as being a top or bottom strand is arbitrary and does not imply any particular orientation, function or structure.
  • complementary strands of a chromosomal DNA may be interchangeably referred to as “top” and “bottom” strands, “plus” and “minus” strands, the “first” and “second” strands, the “coding” and “noncoding” strands, the “Watson” and “Crick” strands, or the “sense” and “antisense” strands.
  • on-target repression refers repression of expression of a gene containing the genomic sequence that is the target of the recombinant polypeptide comprising the DBD and the transcription repressor.
  • the DBD determines the specificity of the polypeptide for the binding the target site.
  • An on-target repression site refers to a nucleic acid sequence that includes the DNA sequence specifically bound by the DBD of the recombinant polypeptide.
  • off-target repression refers to repression of expression of a gene containing the genomic sequence that is not the target of the recombinant polypeptide comprising the DBD and the transcription repressor but is repressed due to non-specific binding of the DBD of the recombinant polypeptide.
  • domain refers to a part of a protein sequence that may exist and function independently of the rest of the protein chain.
  • these recombinant polypeptides function as transcriptional repressors by virtue of the DBD that mediates binding to a target gene and a repressor domain that suppresses target gene expression upon binding of the polypeptide to the target gene.
  • the recombinant polypeptides disclosed herein may also be referred to as transcriptional repressors.
  • sequences provided herein may be specified to be at least 30%, 40%, 50%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or a 100% identical to another sequence provided herein.
  • Percent identity between a pair of sequences may be calculated by multiplying the number of matches in the pair by 100 and dividing by the length of the aligned region, including gaps. Identity scoring only counts perfect matches and does not consider the degree of similarity of amino acids to one another. Only internal gaps are included in the length, not gaps at the sequence ends.
  • Conservative amino acid substitutions refers to substitution of amino acid residues within the following groups: 1) L, I, M, V, F; 2) R, K; 3) F, Y, H, W, R; 4) G, A, T, S; 5) Q, N; and 6) D, E.
  • Conservative amino acid substitutions may preserve the activity of the protein by replacing an amino acid(s) in the protein with an amino acid with a side chain of similar acidity, basicity, charge, polarity, or size of the side chain.
  • Guidance for substitutions, insertions, or deletions may be based on alignments of amino acid sequences of proteins from different species or from a consensus sequence based on a plurality of proteins having the same or similar function.
  • the terms “patient” or “subject” are used interchangeably to refer to a human or a non-human animal (e.g., a mammal).
  • the terms “treat”, “treating”, treatment” and the like refer to a course of action (such as administering a polypeptide comprising a DBD fused to a heterologous transcription repressor domain or a nucleic acid encoding the polypeptide) initiated after a disease, disorder or condition, or a symptom thereof, has been diagnosed, observed, and the like so as to eliminate, reduce, suppress, mitigate, or ameliorate, either temporarily or permanently, at least one of the underlying causes of a disease, disorder, or condition afflicting a subject, or at least one of the symptoms associated with a disease, disorder, condition afflicting a subject.
  • prevent refers to a course of action (such as administering a polypeptide comprising a DBD fused to a heterologous functional domain or a nucleic acid encoding the polypeptide) initiated in a manner (e.g., prior to the onset of a disease, disorder, condition or symptom thereof) so as to prevent, suppress, inhibit or reduce, either temporarily or permanently, a subject's risk of developing a disease, disorder, condition or the like (as determined by, for example, the absence of clinical symptoms) or delaying the onset thereof, generally in the context of a subject predisposed to having a particular disease, disorder or condition. In certain instances, the terms also refer to slowing the progression of the disease, disorder or condition or inhibiting progression thereof to a harmful or otherwise undesired state.
  • the phrase “therapeutically effective amount” refers to the administration of an agent to a subject, either alone or as a part of a pharmaceutical composition or as a companion therapy and either in a single dose or as part of a series of doses, in an amount that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease, disorder or condition when administered to a patient.
  • the therapeutically effective amount can be ascertained by measuring relevant physiological effects.
  • the recombinant polypeptides include a DBD that mediates binding to a sequence in a target gene and a heterologous transcriptional repressor.
  • the DBD includes a plurality of RUs ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the target gene, where binding of the recombinant polypeptide to the nucleic acid sequence results in decreased expression of the target gene.
  • a recombinant polypeptide disclosed herein may include from N- to C- terminus: a N-cap region, a DBD comprising a plurality of RUs ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the target gene, a C-cap region, an optional linker, and a transcription repressor domain.
  • the transcriptional repressor domain may be at the N- terminus of the recombinant polypeptide instead of the C-terminus.
  • the RUs may have the sequence (X 1-11 X 12 X 13 X 14-33, 34, or 35 ) z (SEQ ID NO: 453), where X 1-11 is a chain of 11 contiguous amino acids, X 14-33 or 34 or 35 is a chain of 20, 21 or 22 contiguous amino acids, X 12 X 13 is selected from: (a) NH, HH, KH, NK, NQ, RH, RN, SS, NN, SN, or KN for recognition of guanine (G); (b) NI, KI, RI, HI, or SI for recognition of adenine (A); (c) NG, HG, KG, or RG for recognition of thymine (T); (d) HD, RD, SD, ND, KD, or YG for recognition of cytosine (C); and (e) NV or HN for recognition of A or G; and (f) H*, HA, KA, N*
  • Any suitable RU such as those based upon the RUs from Xanthomonas transcription activator- like effector (TALE) systems, Ralstonia solanacearum (modular Ralstonia nucleic acid binding domain; RNBD), or an animal pathogen (e.g., Legionella quateirensis, Legionella maceachernii, Burkholderia, Paraburkholderia, or Francisella) (modular animal pathogen nucleic acid binding domain; MAP-NBD) may be used for binding to the regions of the target genes provided herein.
  • TALE Xanthomonas transcription activator- like effector
  • RNBD modular Ralstonia solanacearum
  • an animal pathogen e.g., Legionella quateirensis, Legionella maceachernii, Burkholderia, Paraburkholderia, or Francisella
  • MAP-NBD modular animal pathogen nucleic acid binding domain
  • the arrangement of the RUs in the DBD may be based upon the sequence identified in the target gene to which binding of the recombinant polypeptide results in decreased expression of the target gene. These sequences identified in PDCD-1 gene, TIM3 gene, CTLA4 gene, and LAG3 gene, and the corresponding DBDs are described in detail below.
  • PDCD-1 programmed cell death 1 gene
  • PD-1 gene is also known as PD-1 gene and encodes a cell surface membrane protein of the immunoglobulin superfamily, which is also referred to as PDCD-1 or PD-1.
  • PD- 1 binds to the ligands PD-L1 and PD-L2.
  • the PD-l/PD-1 ligands pathway plays a role in immunosuppression.
  • Recent studies have shown that PD-L1 and PD-L2 are widely expressed on various cancer cells (Keir ME, et al. Annu Rev Immunol. 2008; 26 (677-704)).
  • Expression of PD-ligands prevents cancer cells from being killed by T cells and promotes cancer progression. Targeting the PD-1 pathway has been recognized as an effective immunotherapy for different cancers (Ostrand-Rosenberg S, et al. J Immunol. 2014; 193(8): 3835- 41).
  • TIM3 T-Cell Immunoglobulin Mucin Receptor 3 gene is also referred to as Hepatitis A Virus Cellular Receptor 2 (HAVCR2) and encodes a cell surface membrane protein of the immunoglobulin superfamily of the same name.
  • HAVCR2 Hepatitis A Virus Cellular Receptor 2
  • CTLA4 gene (Cytotoxic T-Lymphocyte Associated Protein 4) encodes an immunoglobulin superfamily protein of the same name which transmits an inhibitory signal to T cells.
  • LAG3 (Lymphocyte-Activation Gene 3) gene encodes the Lymphocyte Activating 3 protein which is also known as LAG3 protein.
  • CTLA-4, PD-1, LAG-3, and TIM3 are known immune checkpoint proteins. The pathways involving LAG3 and TIM3 are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al. 2011. Nature 480:480-489).
  • a DBD may include a plurality of RUs ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the PDCD1 gene.
  • the plurality of RUs may be a number of repeat units sufficient to bind to a target sequence, which number may range from 7 to 40. In certain aspects, the number of RUs may range from 9 to 35. In certain aspects, the number of RUs may range from 12 to 30, 14 to 25, or 16 to 25.
  • the recombinant polypeptides disclosed herein all reduce the expression of the target gene in at least 50% of the cells transfected with a nucleic acid encoding the recombinant polypeptides while cells not transfected with a nucleic acid encoding the recombinant polypeptides do not show a significant decrease in the expression of the target gene.
  • the recombinant polypeptides disclosed herein all reduce the expression of the target gene in at least 80% of the cells transfected with a nucleic acid encoding the recombinant polypeptides while cells not transfected with a nucleic acid encoding the recombinant polypeptides do not show a significant decrease in the expression of the target gene.
  • recombinant polypeptides that bind to sequences in the PDCD-1 gene that have been identified to be present in regions of the gene that when bound by the recombinant polypeptides comprising a transcriptional repressor domain lead to suppression of PD-1 expression from the PDCD-1 gene.
  • FIG. 1A The sequences in the PDCD-1 gene that were tested to determine repression by a transcriptional repressor domain bound to the sequence are pictorially depicted in Figure 1A.
  • the analysis of repression by the disclosed recombinant polypeptides that are designed to bind to these sequences identified certain regions that provide repression of PDCD-1 expression in at least 50% of the cells expressing these recombinant polypeptides. These regions are depicted in Figures 1B-1C and include regions 1-4. In regions 1, 2, 3, the anti-sense strand of the PDCD-1 gene was successfully targeted to significantly repress expression of PD-1. In region 4, the sense strand was identified as the region of the PDCD-1 gene that can be successfully target for repression. In addition, certain sequences in the sense strand in region 1 were also identified a region that can be successfully targeted for repression. [0076] Region 1:
  • Table 1 illustrates the identification of region 1 which includes sequences that can be targeted for repression.
  • region 1 which includes sequences that can be targeted for repression.
  • the indicated recombinant polypeptides that included RUs arranged from N-terminus to C-terminus to bind to the listed target sequence, repressed expression of PD- 1 by at least 80% as compared to a negative control.
  • the location of these target sequences when aligned reveals a region (Region 1) in minus strand of the PDCD-1 gene that may be targeted for repressing PDCD-1 expression.
  • the alignment of the target sequences also reveals the minimal sequences within Region 1 that can be targeted for binding by the DBD for repressing PDCD-1 expression.
  • a recombinant polypeptide that suppresses expression of PD 1 receptor encoded by the PDCD 1 gene may include a DNA binding domain (DBD) and a transcriptional repressor domain.
  • the DBD may include a plurality of RUs ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the PDCD 1 gene, wherein the nucleic acid sequence is present within the sequence: TGGTGGGGCTGCTCCAGGCATGCAGATCCCACAGGCGCCCTGG (SEQ ID NO: 1). As explained in the Examples section of the application, this sequence corresponds to Region 1 in the PDCD1 gene.
  • the RUs may include the sequence (X 1-11 X 12 X 13 X 14-33, 34, or 35 ) z (SEQ ID NO: 453), where X 1-11 is a chain of 11 contiguous amino acids, X 14-33 or 34 or 35 is a chain of 20, 21 or 22 contiguous amino acids,X 12 X 13 is selected from: (a) NH, HH, KH, NK, NQ, RH, RN, SS, NN, SN, or KN for recognition of guanine (G); (b) NI, KI, RI, HI, or SI for recognition of adenine (A); (c) NG, HG, KG, or RG for recognition of thymine (T); (d) HD, RD, SD, ND, KD, or YG for recognition of cytosine (C); and (e) NV or HN for recognition of A or G; and (f) H*, HA, KA, N*,
  • the RUs are ordered from N-terminus to the C-terminus to bind to the sequence: GGGGCTGCTCC (SEQ ID NO:2), wherein the first RU at the N-terminus binds to the G at the 5’ end of the sequence and the last RU at the C-terminus binds to the C at the 3’ end of the sequence.
  • the X 12 X 13 in the RUs from N-terminus to C-terminus may be NH, NH, NH, HD, NG, NH, HD, NG, HD, and HD.
  • the DBD may include at least an additional RU at the N-terminus such that the DBD binds to the nucleic acid sequence TGGGGCTGCTCC (SEQ ID NO:3), wherein X 12 X 13 in the additional RU is NG, HG, KG, or RG for recognition of the T.
  • the RUs are ordered from N-terminus to the C-terminus to bind to the sequence: GGTGGGGCTGCTCC (SEQ ID NO:4), wherein the first RU at the N-terminus binds to the G at the 5’ end of the sequence and the last RU at the C-terminus binds to the C at the 3’ end of the sequence.
  • the DBD comprises at least fourteen RUs, wherein X 12 X 13 in the RUs from N-terminus to C-terminus are NH, NH, NG, NH, NH, NH, HD, NG, NH, HD, NG, HD, and HD.
  • the DBD comprises three additional RU at the N-terminus such that the DBD binds to the nucleic acid sequence TGGTGGGGCTGCTCC (SEQ ID NO:5). In certain aspects, the DBD comprises three additional RUs at the C-terminus such that the DBD binds to the sequence GGTGGGGCTGCTCCAGG (SEQ ID NO:6).
  • the RUs are arranged from N-terminus to C-terminus to bind to the sequence: GC AGAT CCC AC AGGCGC (SEQ ID NO:7).
  • the RUs are arranged from N-terminus to C-terminus to bind to the sequence: CCCACAGGCGCCCTGG (SEQ ID NO: 8).
  • the RUs are arranged from N-terminus to C-terminus to bind to the sequence: GGGGCTGCTCCAGGCATGC (SEQ ID NO: 9).
  • the RUs may be arranged from N-terminus to C-terminus to bind to a sequence that is a complement of a sequence in region 1.
  • the complementary sequence may be the sequence: GGAGCAGCCCC (SEQ ID NO: 105).
  • the DBD that binds to the complementary sequence may include RUs ordered from N-terminus to C-terminus to bind to the sequence: GGAGCAGCCCCACCAGAGT (SEQ ID NO: 106).
  • Table 2 illustrates the identification of region 2 which includes sequences that can be targeted for repression.
  • region 2 which includes sequences that can be targeted for repression.
  • the indicated recombinant polypeptides that included RUs arranged from N-terminus to C-terminus to bind to the listed target sequence, repressed expression of PD- 1 by at least 80% as compared to a negative control.
  • the location of these target sequences when aligned reveals a region (Region 2) in the minus strand of the PDCD-1 gene that may be targeted for repressing PDCD-1 expression.
  • the alignment of the target sequences also reveals the minimal sequence that can be targeted for binding by the DBD for repressing PDCD-1 expression.
  • the DBD includes a plurality of RUs ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the PDCD 1 gene, wherein the nucleic acid sequence is present within the sequence:
  • the DBD includes a plurality of RUs ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the PDCD 1 gene, wherein the nucleic acid sequence is present within the sequence: CCTCCCCCAGCACTGCCTCTGTCACTCTCGCCCACGT (SEQ ID NO: 454).
  • the nucleic acid sequence is present within the sequence: CCTCCCCCAGCACTGCCTCTGTCACTCTCGCCCACGT (SEQ ID NO: 454).
  • all of the eight DBD-repressor domains that bound to a nucleic acid sequence within this sequence repressed expression of PD-1 in at least 50% of the cells treated with the DBD-repressor domain as compared to mock treated cells.
  • the DBD includes a plurality of RUs ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the PDCD 1 gene, wherein the nucleic acid sequence is present within the sequence: GCCTCTGTCACTCTCGCCCAC (SEQ ID NO: 444).
  • GCCTCTGTCACTCTCGCCCAC SEQ ID NO: 444
  • the RUs are ordered from N-terminus to C-terminus of the DBD to bind to the nucleic acid sequence TCTGTCACTCTCG (SEQ ID NO: 11).
  • the DBD comprises at least thirteen RUs, wherein X 12 X 13 in the RUs from N-terminus to C-terminus are NG, HD, NG, NH, NG, HD, NI, HD, NG, HD, NG, HD, and NH.
  • the DBD further comprises three additional RUs at the N-terminus such that the DBD binds to the nucleic acid sequence GCCTCTGTCACTCTCG (SEQ ID NO: 12).
  • the DBD further comprises three additional RUs at the C-terminus such that the DBD binds to the nucleic acid sequence GCCTCTGTCACTCTCGCCC (SEQ ID NO: 13).
  • the DBD comprises at least nineteen RUs, wherein X 12 X 13 in the RUs from N-terminus to C-terminus are NH, HD, HD, NG, HD, NG, NH, NG, HD, NI, HD, NG, HD, NG, HD, NH, HD, HD, and HD.
  • the DBD further comprises five additional RUs at the C-terminus such that the DBD binds to the nucleic acid sequence TCTGTCACTCTCGCCCAC (SEQ ID NO: 14).
  • the DBD comprises at least eighteen RUs, wherein X 12 X 13 in the RUs from N-terminus to C- terminus are NG, HD, NG, NH, NG, HD, NI, HD, NG, HD, NG, HD, NG, NH, HD, HD, HD, NI, and HD.
  • the DBD comprises thirteen RUs ordered from N-terminus to C-terminus of the DBD to bind to the nucleic acid sequence: CCCCCAGCACTGC (SEQ ID NO: 15). In certain aspects, the DBD further comprises three additional RUs at the N-terminus such that the DBD binds to the nucleic acid sequence: CCTCCCCCAGCACTGC (SEQ ID NO: 16). In certain aspects, the DBD further comprises an additional RU at the C-terminus such that the DBD binds to the nucleic acid sequence: CCTCCCCCAGCACTGCC (SEQ ID NO: 17).
  • Table 3 illustrates the identification of region 3 which includes sequences that can be targeted for repression.
  • the indicated recombinant polypeptides that included RUs arranged from N-terminus to C-terminus to bind to the listed target sequence, repressed expression of PD- 1 by at least 80% as compared to a negative control.
  • the location of these target sequences when aligned reveals a region (Region 3) in the minus strand in the PDCD-1 gene that may be targeted for repressing PDCD-1 expression.
  • the alignment of the target sequences also reveals the minimal sequence that can be targeted for binding by the DBD for repressing PDCD-1 expression.
  • the DBD includes at least nine RUs ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the PDCD 1 gene, wherein the nucleic acid sequence is present within the sequence:
  • the DBD comprises ten RUs ordered from N-terminus to C-terminus to bind to the nucleic acid sequence: TCCGCTCACC (SEQ ID NO:20). In certain aspects, the DBD comprises nine additional RUs at the C-terminus such that the DBD binds to the nucleic acid sequence: TCCGCTCACCTCCGCCTGA (SEQ ID NO:21). In certain aspects, the DBD comprises four additional RUs at the N-terminus such that the DBD binds to the nucleic acid sequence: CCCTTCCGCTCACC (SEQ ID NO:22).
  • the DBD comprises five additional RUs at the C-terminus such that the DBD binds to the nucleic acid sequence: CCCTTCCGCTCACCTCCGC (SEQ ID NO:23). In certain aspects, the DBD comprises two additional RUs at the N-terminus such that the DBD binds to the nucleic acid sequence: TTCCCTTCCGCTCACC (SEQ ID NO:24). [0101] In certain aspects, the DBD comprises twelve RUs ordered from N-terminus to C-terminus to bind to the nucleic acid sequence: GGGACAGTTTCC (SEQ ID NO:25).
  • the DBD further comprises four additional RUs at the C-terminus such that the DBD binds to the nucleic acid sequence: GGGACAGTTTCCCTTC (SEQ ID NO:26). In certain aspects, the DBD further comprises five additional RUs at the N-terminus such that the DBD binds to the nucleic acid sequence: GACCTGGGACAGTTTCC (SEQ ID NO: 27).
  • the DBD comprises eleven RUs ordered from N-terminus to C-terminus to bind to the nucleic acid sequence: CAACCTGACCT (SEQ ID NO:28). In certain aspects, the DBD comprises nine additional RUs at the C-terminus such that the DBD binds to the nucleic acid sequence: CAACCTGACCTGGGACAGTT (SEQ ID NO:29) In certain aspects, the DBD comprises five additional RUs at the N-terminus such that the DBD binds to the nucleic acid sequence: CCCTTCAACCTGACCT (SEQ ID NO:30).
  • Table 4 illustrates the identification of region 4 which includes sequences that can be targeted for repression.
  • region 4 includes sequences that can be targeted for repression.
  • the indicated recombinant polypeptides that included RUs arranged from N-terminus to C-terminus to bind to the listed target sequence, repressed expression of PD- 1 by at least 80% as compared to a negative control.
  • the location of these target sequences when aligned reveals a region (Region 4) in the plus strand of the PDCD-1 gene that may be targeted for repressing PDCD-1 expression.
  • the alignment of the target sequences also reveals the minimal sequence that can be targeted for binding by the DBD for repressing PDCD-1 expression.
  • the DBD includes at least nine RUs ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the PDCD 1 gene, wherein the nucleic acid sequence is present within the sequence: GCCGCCTTCTCCACTGCTCAGGCGGAGGT (SEQ ID NO:31). [0106] As explained in the Examples section of the application, this sequence corresponds to Region 4. [0107] In certain aspects, the DBD comprises RUs arranged from N-terminus to C-terminus such that the DBD binds to the nucleic acid sequence: GCCGCCTTCTCCACT (SEQ ID NO:32).
  • the DBD comprises RUs arranged from N-terminus to C-terminus such that the DBD binds to the nucleic acid sequence: CCACTGCTCAGGCG (SEQ ID NO:33). In certain aspects, the DBD further comprises three additional RUs at the N-terminus such that the DBD binds to the nucleic acid sequence: TCTCCACTGCTCAGGCG (SEQ ID NO:34). In certain aspects, the DBD further comprises five additional RUs at the C-terminus such that the DBD binds to the nucleic acid sequence: CCACTGCTCAGGCGGAGGT (SEQ ID NO:35).
  • the present disclosure provides additional recombinant polypeptides for repressing PDCD1 expression.
  • the DBD of the recombinant polypeptide includes at least nine RUs ordered from N- terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the PDCD1 gene, wherein the nucleic acid sequence is present within the sequence: CCCAGGTCAGGTTGAAG (SEQ ID NO:63).
  • the DBD of the recombinant polypeptide includes at least nine RUs ordered from N- terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the PDCD1 gene, wherein the nucleic acid sequence is present within the sequence: GGCCAGGGCGCCTGT (SEQ ID NO:36). In certain aspects, the DBD of the recombinant polypeptide includes at least nine RUs ordered from N- terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the PDCD1 gene, wherein the nucleic acid sequence is present within the sequence: CTGCATGCCTGGAGCAG (SEQ ID NO:37).
  • the DBD of the recombinant polypeptide includes at least nine RUs ordered from N- terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the PDCD1 gene, wherein the nucleic acid sequence is present within the sequence: GCTCCCGCCCCCTCTTCCT (SEQ ID NO:38). In certain aspects, the DBD of the recombinant polypeptide includes at least nine RUs ordered from N- terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the PDCD1 gene, wherein the nucleic acid sequence is present within the sequence: CTTCCTCCACATCCACG (SEQ ID NO:39).
  • the DBD of the recombinant polypeptide includes at least nine RUs ordered from N- terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the PDCD1 gene, wherein the nucleic acid sequence is present within the sequence: CCTCCACATCCACGTGGGC (SEQ ID NO:40).
  • the RUs of the recombinant polypeptide may be arranged from N-terminus to C-terminus to bind to a sequence present in a target sequence listed in Table 9 and shown to have a PD-1 suppression of at least 50%. As noted herein the RUs may range from 7 to 40 in number.
  • the RUs of the recombinant polypeptide may be arranged from N-terminus to C-terminus to bind to the target sequence listed in Table 9 and shown to have a PD-1 suppression of at least 50%.
  • the recombinant polypeptides disclosed herein all reduce the expression of PDCD1 gene in at least 50% of the cells transfected with a nucleic acid encoding the recombinant polypeptides while cells not transfected with a nucleic acid encoding the recombinant polypeptides do not show a significant decrease in the expression of the target gene.
  • the recombinant polypeptides disclosed herein all reduce the expression of the PDCD1 gene in at least 80% of the cells transfected with a nucleic acid encoding the recombinant polypeptides while cells not transfected with a nucleic acid encoding the recombinant polypeptides do not show a significant decrease in the expression of the PDCD1 gene.
  • a recombinant polypeptide of the present disclosure may include a DBD and a transcriptional repressor domain, the DBD comprising a plurality of RUs ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the PDCD1 gene, wherein the nucleic acid sequence is present within one of the following sequences:
  • TTCCCTTCCGCTCACC (SEQ ID NO:24);
  • GCCTCTGTCACTCTCGCCC (SEQ ID NO: 13);
  • GGTGGGGCTGCTCCAGG SEQ ID NO:6
  • TCTCCACTGCTCAGGCG SEQ ID NO:34
  • TCCGCTCACCTCCGCCTGA SEQ ID NO:21
  • GCCTCTGTCACTCTCG (SEQ ID NO: 12);
  • each of the RU comprises the sequence X 1-11 X 12 X 13 X 14-33, 34, or 35 (SEQ ID NO: 455), wherein X 1-11 is a chain of 11 contiguous amino acids, X 14-33 or 34 or 35 is a chain of 20, 21 or 22 contiguous amino acids, X 12 X 13 is selected from (a) NH, HH, KH, NK, NQ, RH, RN, SS, NN, SN, or KN for recognition of guanine (G); (b) NI, KI, RI, HI, or SI for recognition of adenine (A); (c) NG, HG, KG, or RG for recognition of thymine (T); (d) HD, RD, SD, ND, KD, or YG for recognition of cytosine (C); and (e) NV or HN for recognition of A or G; and (f) H*, HA, KA, N*, NA
  • the DBD comprises at least fourteen RUs, at least sixteen, or at least seventeen RUs and optionally, up to 25 RUs.
  • DBD binds to the nucleic acid sequence selected from SEQ ID NOs.: 5, 27, 24, 32, 13, 63, 6, 34, 21, 23, 26, 12, 7, 16, 17, 14, and 29.
  • recombinant polypeptides that bind to sequences in the TIM3 gene that have been identified to be present in regions of the gene that when bound by the recombinant polypeptides comprising a transcriptional repressor domain lead to suppression of TIM3 expression from the TIM3 gene.
  • a recombinant polypeptide that suppresses expression of TIM3 encoded by the TIM3 gene may include a DNA binding domain (DBD) and a transcriptional repressor.
  • the DBD may include a plurality of RUs ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the TIM3 gene, wherein the nucleic acid sequence is present within the sequence: GGCAGTGTTACTATAAGAATCACTGGCAATCAGACACCCGGGTG (SEQ ID NO: 41) or a complement thereof.
  • the DBD comprises RUs that bind to the nucleic acid sequence TGTTACTATA (SEQ ID NO:42). In certain aspects, the DBD comprises an additional RU at the C- terminus such that the DBD binds to the nucleic acid sequence TGTTACTATA (SEQ ID NO:43). In certain aspects, the DBD comprises three additional RUs at the N-terminus such that the DBD binds to the nucleic acid sequence CAGTGTTACTATAA (SEQ ID NO:44). the DBD comprises two additional RUs at the N-terminus such that the DBD binds to the nucleic acid sequence GGCAGTGTTACTATAA (SEQ ID NO:45).
  • the DBD comprises RUs that bind to the nucleic acid sequence TCAGACACCCGGGTG (SEQ ID NO:46). In certain aspects, the DBD comprises three additional RUs at the N-terminus such that the DBD binds to the nucleic acid sequence CAATCAGACACCCGGGTG (SEQ ID NO:47). In certain aspects, the DBD comprises three additional RUs at the N-terminus such that the DBD binds to the nucleic acid sequence TGGCAATCAGACACCCGGGTG (SEQ ID NO:48).
  • a recombinant polypeptide that represses TIM3 expression may bind to a sequence that is a complement of
  • the DBD comprises RUs that are ordered to bind to the sequence TGCCAGTGATT (SEQ ID NO:50). In certain aspects, the DBD comprises eight additional RUs at the C-terminus such that the DBD binds to the sequence TGCCAGTGATTCTTATAGT (SEQ ID NO:51). In certain aspects, the DBD comprises RUs that are ordered to binds to the sequence TGATTGCCAGTGATT (SEQ ID NO:52). In certain aspects, the DBD comprises four additional RUs at the N-terminus such that the DBD binds to the sequence TGTCTGATTGCCAGTGATT (SEQ ID NO:53).
  • the present disclosure provides additional recombinant polypeptides for repressing TIM3 expression.
  • the DBD of such a recombinant polypeptide may include a plurality of RUs ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of TIM3 gene, wherein the nucleic acid sequence is: TACACACAT (SEQ ID NO:54).
  • the DBD comprises four additional RUs at the N-terminus such that the DBD binds to the sequence ACACTACACACAT (SEQ ID NO:55).
  • the DBD comprises four additional RUs at the N-terminus such that the DBD binds to the sequence TGCCACACTACACACAT (SEQ ID NO:56).
  • the RUs of the recombinant polypeptide may be arranged from N-terminus to C-terminus to bind to a sequence present in a target sequence listed in Table 10 and shown to have a TIM3 suppression of at least 50%. As noted herein the RUs may range from 7 to 40 in number. In certain aspects, the RUs of the recombinant polypeptide may be arranged from N-terminus to C-terminus to bind to the target sequence listed in Table 10 and shown to have a TIM3 suppression of at least 50%.
  • the recombinant polypeptides disclosed herein all reduce the expression of TIM3 gene in at least 50% of the cells transfected with a nucleic acid encoding the recombinant polypeptides while cells not transfected with a nucleic acid encoding the recombinant polypeptides do not show a significant decrease in the expression of the TIM3.
  • the recombinant polypeptides disclosed herein all reduce the expression of the TIM3 gene in at least 80% of the cells transfected with a nucleic acid encoding the recombinant polypeptides while cells not transfected with a nucleic acid encoding the recombinant polypeptides do not show a significant decrease in the expression of the TIM3 gene.
  • a recombinant polypeptide of the present disclosure may include a DBD and a transcriptional repressor domain, the DBD comprising a plurality of RUs ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the TIM3 gene, wherein the nucleic acid sequence is present within one of the following sequences:
  • GGCAGTGTTACTATAA SEQ ID NO:45
  • TGCCAGTGATTCTTATAGT SEQ ID NO:51
  • TGCCACACTACACACAT SEQ ID NO:56
  • TGTCTGATTGCCAGTGATT SEQ ID NO:53
  • each of the RU comprises the sequence X 1-11 X 12 X 13 X 14-33, 34, or 35 (SEQ ID NO: 455), wherein X 1-11 is a chain of 11 contiguous amino acids, X 14-33 or 34 or 35 is a chain of 20, 21 or 22 contiguous amino acids, X 12 X 13 is selected from (a) NH, HH, KH, NK, NQ, RH, RN, SS, NN, SN, or KN for recognition of guanine (G); (b) NI, KI, RI, HI, or SI for recognition of adenine (A); (c) NG, HG, KG, or RG for recognition of thymine (T); (d) HD, RD, SD, ND, KD, or YG for recognition of cytosine (C); and (e) NV or HN for recognition of A or G; and (f) H*, HA, KA, N*, NA
  • the DBD comprises at least fourteen RUs, at least sixteen, or at least seventeen RUs and optionally, up to 25 RUs.
  • DBD binds to the nucleic acid sequence selected from SEQ ID NOs:45, 51, 48, 56, and 53.
  • recombinant polypeptides that bind to sequences in the LAG3 gene that have been identified to be present in regions of the gene that when bound by the recombinant polypeptides comprising a transcriptional repressor domain lead to suppression of LAG3 expression from the LAG3 gene.
  • the recombinant polypeptide that binds to this region may include a DBD in which the RUs are ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the LAG3 gene, wherein the nucleic acid sequence is present within the sequence: GCCGTTCTGCTGGTCTCTGGGCCTTCACCCCTGTGCCCGGCCTTCC (SEQ ID NO: 57).
  • the DBD comprises RUs that bind to the sequence TCTGCTGGTCT (SEQ ID NO:58). In certain aspects, the DBD comprises five additional RUs at the N-terminus such that the DBD binds to the sequence GCCGTTCTGCTGGTCT (SEQ ID NO:59). In certain aspects, the DBD comprises two additional RUs at the C-terminus such that the DBD binds to the sequence GCCGTTCTGCTGGTCTCT (SEQ ID NO:60). In certain aspects, the DBD comprises four additional RUs at the C-terminus such that the DBD binds to the sequence TCTGCTGGTCTGGGC (SEQ ID NO:61).
  • the DBD comprises an additional RUs at the C-terminus such that the DBD binds to the sequence TCTGCTGGTCTGGGCC (SEQ ID NO:62). In certain aspects, the DBD comprises three additional RUs at the C-terminus such that the DBD binds to the sequence TCTGCTGGTCTGGGCCTTC (SEQ ID NO:63).
  • the DBD comprises RUs that bind to the sequence TCTCTGGGCCTTCA (SEQ ID NO:64). In certain aspects, the DBD comprises two additional RUs at the N-terminus such that the DBD binds the sequence GGTCTCTGGGCCTTCA (SEQ ID NO:65). In certain aspects, the DBD comprises three additional RUs at the C-terminus such that the DBD binds the sequence GGTCTCTGGGCCTTCACCC (SEQ ID NO:66). In certain aspects, the DBD comprises an additional RUs at the N-terminus such that the DBD binds the sequence TGGTCTCTGGGCCTTCACC (SEQ ID NO:67).
  • the DBD comprises RUs that bind to the sequence TTCACCCCTGTG (SEQ ID NO:68). In certain aspects, the DBD comprises four additional RUs at the C-terminus such that the DBD binds to the sequence TTCACCCCTGTGCCCG (SEQ ID NO:69). In certain aspects, the DBD comprises four additional RUs at the C-terminus such that the DBD binds to the sequence TTCACCCCTGTGCCCGGCCT (SEQ ID NO:70). In certain aspects, the DBD comprises three additional RUs at the C-terminus such that the DBD binds to the sequence TTCACCCCTGTGCCCGGCCTTCC (SEQ ID NO:71).
  • the DBD of such a recombinant polypeptide may include a plurality of RUs ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of LAG3 gene, wherein the nucleic acid sequence is: TGCTCTGTCTGC (SEQ ID NO:72).
  • the DBD comprises two additional RUs at the C-terminus such that the DBD binds to the sequence TGCTCTGTCTGCTC (SEQ ID NO:73).
  • the DBD comprises two additional RUs at the N-terminus such that the DBD binds to the sequence TTTGCTCTGTCTGCTC (SEQ ID NO:74).
  • the RUs of the recombinant polypeptide may be arranged from N-terminus to C-terminus to bind to a sequence present in a target sequence listed in Table 12 and shown to have a LAG3 suppression of at least 50%. As noted herein the RUs may range from 7 to 40 in number. In certain aspects, the RUs of the recombinant polypeptide may be arranged from N-terminus to C-terminus to bind to the target sequence listed in Table 12 and shown to have a LAG3 suppression of at least 50%.
  • the recombinant polypeptides disclosed herein all reduce the expression of LAG3 gene in at least 50% of the cells transfected with a nucleic acid encoding the recombinant polypeptides while cells not transfected with a nucleic acid encoding the recombinant polypeptides do not show a significant decrease in the expression of the LAG3.
  • the recombinant polypeptides disclosed herein reduce the expression of the LAG3 gene in at least 80% of the cells transfected with a nucleic acid encoding the recombinant polypeptides while cells not transfected with a nucleic acid encoding the recombinant polypeptides do not show a significant decrease in the expression of the TIM3 gene.
  • a recombinant polypeptide of the present disclosure may include a DBD and a transcriptional repressor domain, the DBD comprising a plurality of RUs ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the TIM3 gene, wherein the nucleic acid sequence is present within one of the following sequences:
  • TTCACCCCTGTGCCCGGCCTTCC SEQ ID NO:71
  • TCTGCTGGTCTCTGGGCCTTC SEQ ID NO:450
  • TTTGCTCTGTCTGCTC SEQ ID NO:74
  • each of the RU comprises the sequence X 1-11 X 12 X 13 X 14-33, 34, or 35 (SEQ ID NO: 455), wherein X 1-11 is a chain of 11 contiguous amino acids, X 14-33 or 34 or 35 is a chain of 20, 21 or 22 contiguous amino acids, X 12 X 13 is selected from (a) NH, HH, KH, NK, NQ, RH, RN, SS, NN, SN, or KN for recognition of guanine (G); (b) NI, KI, RI, HI, or SI for recognition of adenine (A); (c) NG, HG, KG, or RG for recognition of thymine (T); (d) HD, RD, SD, ND, KD, or YG for recognition of cytosine (C); and (e) NV or HN for recognition of A or G; and (f) H*, HA, KA, N*, NA
  • the DBD comprises at least fourteen RUs, at least sixteen, or at least seventeen RUs and optionally, up to 25 RUs.
  • DBD binds to the nucleic acid sequence selected from SEQ ID NOs: 65, 448, 60, 59, 71, 449, 450, and 74.
  • recombinant polypeptides that bind to sequences in the CTLA4 gene that have been identified to be present in regions of the gene that when bound by the recombinant polypeptides comprising a transcriptional repressor domain lead to suppression of CTLA4 expression from the CTLA4 gene.
  • the DBD of the recombinant polypeptide may include at least nine RUs ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the CTLA4 gene, wherein the nucleic acid sequence is present in the sequence: ACATATCTGGGATCAAAGCT (SEQ ID NO:75); ATATAAAGTCCTTGAT (SEQ ID NO:76); or TTCTATTCAAGTGCC (SEQ ID NO:77).
  • the RUs of the recombinant polypeptide may be arranged from N-terminus to C-terminus to bind to a sequence present in a target sequence listed in Table 11 and shown to have a CTLA4 suppression of at least 50%. As noted herein the RUs may range from 7 to 40 in number. In certain aspects, the RUs of the recombinant polypeptide may be arranged from N-terminus to C-terminus to bind to the target sequence listed in Table 11 and shown to have a CTLA4 suppression of at least 50%.
  • the DBD may be extended at the N-terminus, the C-terminus, or both to increase the number of RUs that contact the nucleic acid sequence is present in the sequence of SEQ ID NOs: 75-77.
  • the DBD may include at least 10, at least 12, at least 13, at least 14, at least 16, or more and up to 20, 25, 35, or 40 RUs. Repeat Units
  • the repeat unit may have the sequence X 1-11 X 12 X 13 X 14-33, 34, or 35 (SEQ ID NO: 455), where X 1-11 is a chain of 11 contiguous amino acids, X 14-33 or 34 or 35 is a chain of 20, 21 or 22 contiguous amino acids, X 12 X 13 is selected from: (a) NH, HH, KH, NK, NQ, RH, RN, SS, NN, SN, or KN for recognition of guanine (G); (b) NI, KI, RI, HI, or SI for recognition of adenine (A); (c) NG, HG, KG, or RG for recognition of thymine (T); (d) HD, RD, SD, ND, KD, or YG for recognition of cytosine (C); and (e) NV or HN for recognition of A or G; and (f) H*, HA, KA, N*,
  • Any suitable RU such as those based upon the RUs from Xanthomonas transcription activator- like effector (TALE) systems, Ralstonia solanacearum (modular Ralstonia nucleic acid binding domain; RNBD), or an animal pathogen (e.g., Legionella quateirensis, Legionella maceachernii, Burkholderia, Paraburkholderia, or Francisella) (modular animal pathogen nucleic acid binding domain; MAP-NBD) may be arranged to bind to the nucleotide sequences in the target genes as disclosed herein.
  • TALE Xanthomonas transcription activator- like effector
  • RNBD modular Ralstonia solanacearum
  • an animal pathogen e.g., Legionella quateirensis, Legionella maceachernii, Burkholderia, Paraburkholderia, or Francisella
  • MAP-NBD modular animal pathogen nucleic acid binding domain
  • the DNA binding domains of the disclosed recombinant polypeptides may be engineered to include 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more, e.g., up to 30, 40 or 50 repeat units arranged in a N-terminal to C-terminal direction to bind to a predetermined 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotide length nucleic acid sequence, such as, a sequence disclosed herein.
  • DNA binding domains may be engineered to include 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more or more, e.g., up to 30, 40 or 50 repeat units that are specifically ordered or arranged to bind to target nucleic acid sequences of length 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more or more, e.g., up to 30, 40 or 50, respectively.
  • the RUs are contiguous.
  • half-RUs may be used in the place of one or more RUs.
  • the last RU in a DBD may be a half RU.
  • the RUs and the half-RU, if present, are derived from Xanthomonas TALE.
  • X 1-11 is at least 80%, at least 90%, or 100% identical to LTPEQVVAIAS (SEQ ID NO: 458), LTPAQVVAIAS (SEQ ID NO: 459), LTPDQVVAIAN (SEQ ID NO: 460), LTPDQVVAIAS (SEQ ID NO: 461), LTPYQVVAIAS (SEQ ID NO: 462), LTREQVVAIAS (SEQ ID NO: 463), or LSTAQVVAIAS (SEQ ID NO: 464).
  • X 14-20 or 21 or 22 is at least 80%, at least 90%, at least 95%, or 100% identical to GGKQALETV QRLLPVLCQDHG (SEQ ID NO:79),
  • the RU is at least 80%, at least 90%, at least 95%, or 100% identical to:
  • X 12 X 13 is repeat variable diresdue (RVD) and is selected from: (a) NH, HH, KH, NK, NQ, RH, RN, SS, NN, SN, or KN for recognition of guanine (G); (b) NI, KI, RI, HI, or SI for recognition of adenine (A); (c) NG, HG, KG, or RG for recognition of thymine (T); (d) HD, RD, SD, ND, KD, or YG for recognition of cytosine (C); and (e) NV or HN for recognition of A or G; and (f) H*, HA, KA, N*, NA, NC, NS, RA, or S*for recognition of A or T or G or C, wherein (*) means that the amino acid at X
  • the DBD may include a N-cap region at N-terminus of the recombinant polypeptide which N-cap region is derived from the N-cap region of a Xanthomonas TALE protein.
  • the DBD may include a N-cap region at the N-terminus which may be present immediately adjacent the first RU.
  • the N-cap region at the N-terminus which may be linked to the first RU via a linker.
  • An N-cap region may be any length, e.g., may comprise from about 0 to about 136 amino acid residues in length.
  • An N-terminal cap may be about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, or about 130 amino acid residues in length.
  • the DBD comprises a N-cap region comprising an amino acid sequence at least 80% (e.g., at least 90%, at least 95%, or 100%) identical to the amino acid sequence:
  • the N-cap region is from TALE proteins like those expressed in Burkholderia, Paraburkholderia, or Xanthomonas.
  • the N-cap regions may be derived from N-cap domain used in conjunction with DNA binding domains disclosed in US20180010152.
  • the N-cap regions may be derived from the N-terminal regions disclosed in US20150225465, e.g., SEQ ID NOs.:7, 8, or 9 disclosed therein.
  • the N-cap region may include the amino acid residues from position 1 (N) through position 137 (M) of the naturally occurring Xanthomonas TALE protein (numbered backwards with N(l) being the residue immediately adjacent the first RU:
  • This amino acid sequence includes a M added to the N-terminus which is not present in the wild type N-cap region of a Xanthomonas TALE protein.
  • This amino acid sequence is generated by deleting amino acids N+288 through N+137 of the N-terminus region of a TALE protein, adding a M, such that amino acids N+136 through N+l of the N-terminus region of the TALE protein are present.
  • the N-terminus can be truncated such that the fragment of the N-terminus includes amino acids from position 1 (N) through position 120 (K) of the naturally occurring Xanthomonas spp. -derived protein as follows:
  • the N-cap region can be truncated such that the fragment of the N-terminus includes amino acids from position 1 (N) through position 115 (S) of the naturally occurring Xanthomonas spp. -derived protein as follows:
  • the N-cap region can be truncated may include amino acids from position 1 (N) through position 110 (H) of the naturally occurri ng Xanthomonas spp. -derived protein as follows: HHEALVGHGFTHAHIVALSQHPAALGTVAVKY QDMIAALPEATHEAIVGVGKQW SGARALEAL LTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLN (SEQ ID NO: 447).
  • the DBD may include a C-cap region at C-terminus of the recombinant polypeptide which C-cap region is derived from the C-cap region of a Xanthomonas TALE protein.
  • the C-cap region at the C-terminus which may be present immediately adjacent the last RU or the last half-RU, if present.
  • the C-cap region at the C-terminus which may be linked to the last RU or the last half-RU, if present, via a linker.
  • a C-cap may be any length and may comprise from about 0 to about 278 amino acid residues in length.
  • a C-terminal cap may be about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 80, about 100, about 150, about 200, or about 250 amino acid residues in length.
  • the DBD comprises a C-cap region comprising an amino acid sequence at least 80% (e.g., at least 90%, at least 95%, or 100%) identical to the amino acid sequence: SIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVA (SEQ ID NO:452).
  • the C-cap region is from TALE proteins like those expressed in Burkholderia, Parahurkholderia, or Xanthomonas. [0194] In some aspects, the C-Cap region can be positions 1 (S) through position 278 (Q) of the naturally occurring Xanthomonas spp. -derived protein as follows:
  • the predetermined N-terminus to C-terminus order of the plurality of RUs of the DNA binding domain determines the corresponding predetermined target nucleic acid sequence to which the recombinant polypeptides may bind.
  • the RUs and at least one or more half RU are specifically ordered to target the genomic locus or gene of interest.
  • the natural TALE-binding sites always begin with a thymine (T), which may be specified by a cryptic signal within the non-repetitive N-cap region of the TALE polypeptide; in some cases this region may be referred to as repeat 0.
  • T thymine
  • TALE binding sites do not necessarily have to begin with a thymine (T) and recombinant polypeptides disclosed herein may target DNA sequences that begin with T, A, G or C.
  • the recombinant polypeptides disclosed herein may target DNA sequences that begin with T and hence include a RU that contains a RVD that mediated binding to T.
  • the tandem repeat of TALE RUs ends with a half-length repeat or a stretch of sequence that may share identity with only the first 20 amino acids of a repetitive full length TALE RU and this half repeat may be referred to as a half-monomer, a half RU, or a half repeat.
  • DBD may be engineered to include X number (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) full length RUs that are specifically ordered or arranged to target nucleic acid sequences of X+2 length (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
  • the last RU in the DBD may be a half repeat.
  • the half repeat may comprise the amino acid sequence X 1-11 X 12 X 13 X 14-19 ,20 or 21 (SEQ ID NO: 471), wherein X 1-11 is a chain of 11 contiguous amino acids, X 14-19 ,20 or 21 is a chain of 7, 8 or 9 contiguous amino acids, and X 12 X 13 is selected from: (a) NH, HH, KH, NK, NQ, RH, RN, SS, NN, SN, or KN for recognition of guanine (G); (b) NI, KI, RI, HI, or SI for recognition of adenine (A); (c) NG, HG, KG, or RG for recognition of thymine (T); (d) HD, RD, SD, ND, KD, or YG for recognition of cytosine (C); (e) NV or HN for
  • X 1-11 is at least 80% identical, at least 90% identical, or 100% identical to LTPEQVVAIAS (SEQ ID NO:458).
  • X 14-20 or 21 or 22 is at least 80% identical to GGRPALE (SEQ ID NO: 472).
  • a recombinant polypeptide disclosed herein may include from N- to C-terminus, a N-cap region, a DBD comprising a plurality of RUs, a C-cap region, an optional linker, and a transcription repressor domain.
  • the RUs are derived from a TALE protein
  • the recombinant polypeptide may be referred to as TALE-TF.
  • the recombinant polypeptides, such as, TALE- TFs, of the present disclosure can further include a linker connecting the DBD or the C-cap region, if present, to the repressor domain.
  • the linker can serve to provide flexibility between the TALE protein and the repressor domain, allowing for the repressor domain (e.g., KRAB to efficiently inhibit transcriptional machinery).
  • a linker used herein can be a short flexible linker comprising an amino acid sequence comprising 0 residues, 1-3 residues, 4-7 residues, 8-10 residues, 10-12 residues, 5-20 residues, 12-15 residues, or 1-15 residues.
  • Linkers can include, but are not limited to, residues such as glycine, methionine, aspartic acid, alanine, lysine, serine, leucine, threonine, tryptophan, or any combination thereof.
  • the linker can have the amino acid sequence of GGGGGMDAKSLTAWS (SEQ ID NO: 109).
  • a Xanthomonas spp. -derived repeat units can have a sequence of LTPDOVVAIASNHGGKOALETVORLLPVLCODHG (SEQ ID NO: 438) comprising an RVD of NH, which recognizes guanine.
  • a Xanthomonas spp. -derived repeat units can have a sequence of LTPDOVVAIASNGGGKOALETVORLLPVLCODHG (SEQ ID NO: 439) comprising an RVD of NG, which recognizes thymidine.
  • -derived repeat units can have a sequence of LTPDQVVAIASNIGGKQALETV QRLLPVLCQDHG (SEQ ID NO: 440) comprising an RVD of NI, which recognizes adenosine.
  • a Xanthomonas spp. -derived repeat units can have a sequence of LTPDOVVAIASHDGGKOALETVORLLPVLCODHG (SEQ ID NO: 441) comprising an RVD of HD, which recognizes cytosine.
  • the RUs and one or both N-Cap and C-Cap regions may be derived from a transcription activator like effector-like protein (TALE-like protein) of Ralstonia solanacearum.
  • TALE-like protein transcription activator like effector-like protein
  • Repeat units derived from Ralstonia solanacearum can be 33-35 amino acid residues in length.
  • the repeat can be derived from the naturally occurring Ralstonia solanacearum TALE-like protein.
  • the RUs may have the sequence X 1-11 X 12 X 13 X 14-33, 34, or 35 (SEQ ID NO: 455), where X 1-11 is a chain of 11 contiguous amino acids, X 14-33 or 34 or 35 is a chain of 20, 21 or 22 contiguous amino acids, X 12 X 13 is RVD and is selected from: (a) NH, HH, KH, NK, NQ, RH, RN, SS, NN, SN, or KN for recognition of guanine (G); (b) NI, KI, RI, HI, or SI for recognition of adenine (A); (c) NG, HG, KG, or RG for recognition of thymine (T); (d) HD, RD, SD, ND, KD, or YG for recognition of cytosine (C); and (e) NV or HN for recognition of A or G; and (f) H*, HA,
  • X 1-11 may include a stretch of amino acids at least 80%, at least 90%, or a 100% identical to the X 1-11 residues of the following RUs from Ralstonia.
  • X 14-33 or 34 or 35 may include a stretch of 20, 21, or 22 amino acids at least 80%, at least 90%, or a 100% identical to the X 14-33 or 34 or 35 residues of the following RUs from Ralstonia:
  • a Ralstonia solanacearum- repeat unit can have at least 80% sequence identity with any one of the Ralstonia RUs provided herein.
  • the DBD may include a N-cap region at the N-terminus which may be present immediately adjacent the first RU or may be linked to the first RU via a linker.
  • an DBD of the present disclosure can have the full length naturally occurring N-terminus of a naturally occurring Ralstonia solanacearum-derived protein.
  • any truncation of the full length naturally occurring N-terminus of a naturally occurring Ralstonia solanacearum-derived protein can be used at the N-terminus of a DBD of the present disclosure.
  • amino acid residues at positions 1 (H) to position 137 (F) of the naturally occurring Ralstonia solanacearum-derived protein N-terminus can be used as the N-cap region.
  • the truncated N-terminus from position 1 (H) to position 137 (F) can have a sequence as follows:
  • the naturally occurring N-terminus of Ralstonia solanacearum can be truncated to any length and used as the N-cap of the engineered DNA binding domain.
  • the naturally occurring N-terminus of Ralstonia solanacearum can be truncated to include amino acid residues at position 1 (H) to position 120 (K) as follows:
  • the naturally occurring N-terminus of Ralstonia solanacearum can be truncated to amino acid residues at positions 1 to 50, 1 to 70, 1 to 100, 1 to 120, 1 to 130, 10 to 40, 60 to 100, or 100 to 120 and used as the N-cap of the engineered DNA binding domain.
  • N-cap region derived from Xanthomonas TALE the amino acid residues are numbered backward from the first repeat unit such that the amino acid (H in this case) of the N-cap adjacent the first RU is numbered 1 while the N-terminal amino acid of the N-cap is numbered 137 (and is F in this case) or 120 (and is K in this case).
  • the N-cap referred to as the amino terminus or the “NH2” domain, can recognize a guanine.
  • the N-cap can be engineered to bind a cytosine, adenosine, thymidine, guanine, or uracil.
  • an DBD of the present disclosure can include a plurality of RUs followed by a final single half-repeat also derived from Ralstonia solanacearum.
  • the half repeat can have 15 to 23 amino acid residues, for example, the half repeat can have 19 amino acid residues.
  • the half-repeat can have a sequence as follows: LSTAQVVAIACISGQQALE (SEQ ID NO:229).
  • an DBD of the present disclosure can have the full length naturally occurring C-terminus of a naturally occurring Ralstonia solanacearum-derived protein as a C-cap region that is conjugated to the last RU.
  • any truncation of the full length naturally occurring C-terminus of a naturally occurring Ralstonia solanacearum-derived protein can be used as the C-cap.
  • the DBD can comprise amino acid residues at position 1 (A) to position 63 (S) as follows:
  • AIEAHMPTLRQASHSLSPERVAAIACIGGRSAVEAVRQGLPVKAIRRIRREKAPVAGPPPAS (SEQ ID NO:230) of the naturally occurring Ralstonia solanacearum- derived protein C-terminus.
  • the naturally occurring C-terminus of Ralstonia solanacearum can be truncated to any length and used as the C-cap of the DBD.
  • the naturally occurring C-terminus of Ralstonia solanacearum can be truncated to amino acid residues at positions 1 to 63 and used as the C-terminus of the DBD.
  • the naturally occurring C-terminus of Ralstonia solanacearum can be truncated amino acid residues at positions 1 to 50 and used as the C-cap of the DBD.
  • the naturally occurring C-terminus of Ralstonia solanacearum can be truncated to amino acid residues at positions 1 to 63, 1 to 50, 1 to 70, 1 to 100, 1 to 120, 1 to 130, 10 to 40, 60 to 100, or 100 to 120 and used as the C-cap of the DBD.
  • TABLE 7 shows N-Cap, C-Cap, and half-repeats derived from Ralstonia.
  • the present disclosure provides DNA binding domains in which the repeat units can be derived from a Legionellales bacterium, a species of the genus of Legionella, such as L. quateirensis or L. maceachernii, the genus of Burkholderia, the genus of Paraburkholderia, or the genus of Francisella.
  • the RUs may have the sequence X 1-11 X 12 X 13 X 14-33, 34, or 35 (SEQ ID NO: 455), where X 1-11 is a chain of 11 contiguous amino acids, X 14-33 or 34 or 35 is a chain of 20, 21 or 22 contiguous amino acids, X 12 X 13 is selected from: (a) NH, HH, KH, NK, NQ, RH, RN, SS, NN, SN, HN, or KN for recognition of guanine (G); (b) NI, KI, RI, HI, HA, or SI for recognition of adenine (A); (c) NG, HG, KG, or RG for recognition of thymine (T); (d) HD, RD, SD, ND, KD, or YG for recognition of cytosine (C); and (e) NV or HN for recognition of A or G; and (f) H*,
  • X 1-11 may include a stretch of amino acids at least 80%, at least 90%, or a 100% identical to the X 1-11 residues of the following RUs from animal pathogens, Legionella, Burkholderia, Paraburkholderia, or Francisella.
  • X 14-33 or 34 or 35 may include a stretch of 20, 21, or 22 amino acids at least 80%, at least 90%, or a 100% identical to the X 14-33 or 34 or 35 residues of the RUs from animal pathogens, Legionella (e.g ., L. quateirensis or L. maceachernii), Burkholderia, Paraburkholderia, or Francisella listed in Table 8.
  • Table 8 Repeat Unit Sequences
  • Residues X 12 X 13 of the RU may include base contacting residues (BCR) as listed in the table 8 and may be chosen based upon the target nucleic acid sequence.
  • the last RU in the DBD may be a half RU.
  • the half RU may include a sequence that is at least 80%, at least 90%, at least 95% or a 100% identical to the half RU from L. quateirensis (FNAEQIVRMVSX 12 X 13 GGSKNU) (SEQ ID NO:333).
  • the half RU may include a sequence that is at least 80%, at least 90%, at least 95% or a 100% identical to the half RU from Francisella (YNKKQIVLIASX 12 X 13 SGG) (SEQ ID NO:334).
  • the polypeptide comprises an N-cap region, where the C-terminus (i.e., the last amino acid) of the N-cap region is covalently linked to the N-terminus (i.e., the first amino acid) of the first RU of the DBD either directly or via a linker.
  • the N-cap region is the N-terminus of L. quateirensis protein and may have an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, 95%, or 99%, or a 100%) identical to the amino acid sequence:
  • the N-cap region comprises a fragment of SEQ ID NO:335.
  • the N-cap region is a N-terminal domain or a fragment thereof from TALE proteins like those expressed in Burkholderia, Paraburkholderia, or Xanthomonas.
  • the polypeptide comprises a C-cap region, where the N-terminus (i.e., the first amino acid) of the C-terminal domain is covalently linked to the C-terminus (i.e., the last amino acid) of the last RU or the half-repeat unit, if present, in the DBD either directly or via a linker.
  • the C-cap region is the C-terminal domain of L. quateirensis protein and may have an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, 95%, or 99%, or a 100%) identical to the amino acid sequence:
  • the C-cap region comprises a fragment of SEQ ID NO:336, such as a fragment having the amino acid sequence
  • the C-cap region domain is a C-terminal domain or a fragment thereof from TALE proteins like those expressed in Burkholderia, Paraburkholderia, or Xanthomonas.
  • the present disclosure provides DNA binding domains in which the repeat units, the N-cap, and the C-ap can be derived from any one of Ralstonia solanacearum, Xanthomonas spp., Legionella quateirensis, Burkholderia, Paraburkholderia, or Francisella.
  • the present disclosure provides a DNA binding domain wherein the plurality of repeat units are selected from any one of the RUs as provided herein and can further comprise an N-cap and/or C-cap as provided herein.
  • repressor repressor domain
  • transcriptional repressor domain refers to a portion of the recombinant polypeptide as disclosed herein which portion decreases expression of a gene when the recombinant polypeptide is bound to the target gene.
  • the repressor domain comprises Kriippel-associated box (KRAB) protein.
  • the repressor domain comprises KRAB, Sin3a, LSD1, SUV39H1, G9A (EHMT2), DNMT1, DNMT3A-DNMT3L, DNMT3B, KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, Rb, or MeCP2.
  • the repressor domain comprises an amino acid sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or a 100% to the amino acid sequence set forth in one of SEQ ID NOs:84-101.
  • the repressor domain includes a KRAB domain comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or a 100% to the amino acid sequence set forth in SEQ ID NO:338:
  • the N-cap region or the C-cap region included in the disclosed DBD may include a nuclear localization sequence (NLS) to facilitate entry into the nucleus of a cell, e.g., an animal cell, such as, a human cell.
  • NLS nuclear localization sequence
  • the polypeptide may be produced in a host cell and expressed with a translocation signal at the N-terminus which translocation signal may be cleaved during translocation.
  • the RUs may be linked C-terminus to N-terminus with no additional amino acids separating immediately adjacent RUs.
  • immediately adjacent RUs may be separated by a spacer sequence of at least one amino acid.
  • the spacer sequence includes at least 2, 3, 4, 5, 6, or 7 amino acids, or up to 5, or up to 10 amino acids.
  • the spacer sequence may include amino acids that have small side chains.
  • the spacer sequence is a flexible linker.
  • a DBD of the present disclosure can comprise between 2 to 50 RUs, e.g., between 5 and 36, between 9 and 36, between 9 and 40, between 12 and 30, between 5 to 10, between 10 to 15, between 15 to 20, between 20 to 25, between 25 to 30, between 30 to 35 animal pathogen-derived repeat domains, or between 35 to 40 animal pathogen-derived repeat domains.
  • a MAP- NBD described herein can comprise up to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
  • a recombinant polypeptide as disclosed herein can be linked to a fluorophore, such as Hydroxycoumarin, methoxycoumarin, Alexa fluor, aminocoumarin, Cy2, FAM, Alexa fluor 488, Fluorescein FITC, Alexa fluor 430, Alexa fluor 532, HEX, Cy3, TRITC, Alexa fluor 546, Alexa fluor 555, R-phycoerythrin (PE), Rhodamine Red-X, Tamara, Cy3.5, Rox, Alexa fluor 568, Red 613, Texas Red, Alexa fluor 594, Alexa fluor 633, Allophycocyanin, Alexa fluor 633, Cy5, Alexa fluor 660, Cy5.5, TruRed, Alexa fluor 680, Cy7, GFP, or mCHERRY.
  • a fluorophore such as Hydroxycoumarin, methoxycoumarin, Alexa fluor, aminocoumarin, Cy2, FAM, Alexa fluor 488, Flu
  • a recombinant polypeptide as disclosed herein can be linked to a biotinylation reagent.
  • a recombinant polypeptide labeled with an imaging moiety as disclosed herein may be used to image binding and/locabzation of the recombinant polypeptide to a site in the genome of a cell.
  • the polypeptides and the nucleic acids described herein may be present in a pharmaceutical composition comprising a pharmaceutically acceptable excipient.
  • the polypeptides and the nucleic acids are present in a therapeutically effective amount in the pharmaceutical composition.
  • a therapeutically effective amount can be determined based on an observed effectiveness of the composition.
  • a therapeutically effective amount can be determined using assays that measure the desired effect in a cell, e.g., in a reporter cell line in which expression of a reporter is modulated in response to the polypeptides of the present disclosure.
  • the pharmaceutical compositions can be administered ex vivo or in vivo to a subject in order to practice the therapeutic and prophylactic methods and uses described herein.
  • compositions of the present disclosure can be formulated to be compatible with the intended method or route of administration; exemplary routes of administration are set forth herein.
  • Suitable pharmaceutically acceptable or physiologically acceptable diluents, carriers or excipients include, but are not limited to, nuclease inhibitors, protease inhibitors, a suitable vehicle such as physiological saline solution or citrate buffered saline.
  • the pharmaceutical composition may include a plurality of the polypeptides provided herein.
  • the composition may include two, three, four, or more of the polypeptides provided herein, wherein the polypeptides all bind to sequences in regulatory region of the same gene or sequences in regulatory regions of different genes.
  • the composition may include a plurality of polypeptides that bind to a sequence of a target gene as disclosed herein (e.g., PD1, TIM3, or LAG3 gene).
  • the composition may include a first polypeptide that binds to regulatory region of a first gene and a second polypeptide that binds to regulatory region of a second gene, where the first and second genes are independently selected from PD1, TIM3, and LAG3.
  • the composition may include a first polypeptide that binds to regulatory region of PD1 gene, a second polypeptide that binds to regulatory region of TIM3 gene, and a third polypeptide that binds to regulatory region of LAG3 gene.
  • the composition may include a plurality of polypeptides that bind to regulatory region of PD1 gene, a plurality of polypeptides that bind to regulatory region of TIM3 gene, and a plurality of polypeptides that bind to regulatory region of LAG3 gene.
  • compositions comprising the disclosed polypeptides, and nucleic acids encoding the disclosed polypeptides can be delivered into a target cell by any suitable means, including, for example, by injection, infection, transfection, and vesicle or liposome mediated delivery.
  • a mRNA or a vector encoding the polypeptides disclosed herein may be injected, transfected, or introduced via viral infection into a target cell, where the cell is ex vivo or in vivo.
  • Any vector systems may be used including, but not limited to, plasmid vectors, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirus vectors and adeno-associated virus vectors, etc.
  • the nucleic acids encoding the polypeptides may be carried on the same vector or on different vectors.
  • Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer.
  • Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
  • Vectors suitable for introduction of polynucleotides as described herein include described herein include non-integrating lentivirus vectors (IDLV).
  • IDLV non-integrating lentivirus vectors
  • Non-viral vector delivery systems include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA.
  • Primary cells may be isolated and used ex vivo for reintroduction into the subject to be treated.
  • Suitable primary cells include peripheral blood mononuclear cells (PBMC), and other blood cell subsets such as, but not limited to, CD4+ T cells or CD8+ T cells.
  • the cell may be a CART cell.
  • Suitable cells also include stem cells such as, by way of example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells, mesenchymal stem cells, muscle stem cells and skin stem cells.
  • the stem cells may be isolated from a subject to be treated or may be derived from a somatic cell of a subject to be treated using the polypeptides disclosed herein.
  • the cells into which the polypeptides of the present disclosure or a nucleic acid encoding a polypeptide of the present disclosure may be an animal cell, e.g., from a human needing treatment.
  • the polypeptide of the present disclosure is only transiently present in a target cell.
  • the polypeptide is expressed from a nucleic acid that expressed the polypeptide for a short period of time, e.g., for up to 1 day, 3 days, 1 week, 3 weeks, or 1 month.
  • adenoviral based systems may be used.
  • Adeno-associated virus (“AAV”) vectors can also be used to transduce cells with nucleic acids encoding the polypeptide of the present disclosure, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures.
  • rAAV recombinant adeno- associated virus vectors
  • rAAV recombinant adeno- associated virus vectors
  • nucleic acids encoding the polypeptides disclosed herein can be delivered using a gene therapy vector with a high degree of specificity to a particular tissue type or cell type.
  • a viral vector is typically modified to have specificity for a given cell type by including a sequence encoding a ligand expressed as a fusion protein with a viral coat protein on the viruses' outer surface.
  • the ligand is chosen to have affinity for a receptor known to be present on the cell type of interest.
  • gene therapy vectors can be delivered in vivo by administration to an individual patient.
  • administration involves systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion), direct injection (e.g., intrathecal), or topical application, as described below.
  • vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector or which have been modified by expression of the polypeptide of the present disclosure encoded by the vector.
  • the nucleic acid encoding the polypeptides provided herein may be codon optimized to enhance expression of the polypeptide in the target cell.
  • the sequence of the nucleic acid can be varied to provide codons that are known to be highly used in animal cells, such as, human cells to enhance production of the polypeptide in a human cell.
  • silent mutations may be made in the nucleotide sequence encoding a polypeptide disclosed herein for codon optimization in mammalian cells.
  • a method of suppressing expression of PDCD-1 gene in a cell comprising introducing into the cell the recombinant polypeptide that comprises the DBD and the transcriptional repressor domain as provided herein, where the DBD binds to a target nucleic acid sequence present in the PDCD-1 gene and the transcriptional repressor suppresses expression of the PDCD-1 gene.
  • a method of suppressing expression of TIM3 gene in a cell comprising introducing into the cell the recombinant polypeptide that comprises the DBD and the transcriptional repressor domain as provided herein, where the DBD binds to a target nucleic acid sequence present in the TIM3 gene and the transcriptional repressor suppresses expression of the TIM3 gene.
  • a method of suppressing expression of LAG3 gene in a cell comprising introducing into the cell the recombinant polypeptide that comprises the DBD and the transcriptional repressor domain as provided herein, where the DBD binds to a target nucleic acid sequence present in the LAG3 gene and the transcriptional repressor suppresses expression of the LAG3 gene.
  • the polypeptide is introduced as a nucleic acid encoding the polypeptide.
  • the nucleic acid is a deoxyribonucleic acid (DNA).
  • the nucleic acid is a ribonucleic acid (RNA).
  • the sequence of the nucleic acid is codon optimized for expression in a human cell.
  • the cell is an animal cell. In certain aspects, the cell is a human cell. In certain aspects, the cell is a cancer cell. In certain aspects, the cell is an ex vivo cell.
  • the introducing comprises administering the polypeptide or a nucleic acid encoding the polypeptide to a subject.
  • the administering comprises parenteral administration.
  • the administering comprises intravenous, intramuscular, intrathecal, or subcutaneous administration.
  • the administering comprises direct injection into a site in a subject.
  • the administering comprises direct injection into a tumor.
  • the introducing may induce a repression of expression of the target gene for a period of at least 2 days, at least 3 days, at least 9 days, at least at least 15 days, at least 1 month, at least 6 months, at least 1 year to up to 5 years.
  • the introducing may suppress expression of gene expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or more.
  • the introducing may be repeated to maintain suppression of target gene expression.
  • the introducing may be performed as a combination therapy with, for example, a cancer therapy.
  • the combination therapy may involve introducing the recombinant polypeptide into the cell prior to, concurrently with, or after administration of cancer therapy.
  • An animal cell can include a cell from a marine invertebrate, fish, insects, amphibian, reptile, or mammal.
  • a mammalian cell can be obtained from a primate, ape, equine, bovine, porcine, canine, feline, or rodent.
  • a mammal can be a primate, ape, dog, cat, rabbit, ferret, or the like.
  • a rodent can be a mouse, rat, hamster, gerbil, hamster, chinchilla, or guinea pig.
  • a bird cell can be from a canary, parakeet or parrots.
  • a reptile cell can be from a turtle, lizard or snake.
  • a fish cell can be from a tropical fish.
  • the fish cell can be from a zebrafish (e.g., Danio rerio).
  • a worm cell can be from a nematode (e.g., C. elegans).
  • An amphibian cell can be from a frog.
  • An arthropod cell can be from a tarantula or hermit crab.
  • a mammalian cell can also include cells obtained from a primate (e.g., a human or a non-human primate).
  • a mammalian cell can include an epithelial cell, connective tissue cell, hormone secreting cell, a nerve cell, a skeletal muscle cell, a blood cell, an immune system cell, or a stem cell.
  • Exemplary mammalian cells can include, but are not limited to, 293A cell line, 293FT cell line, 293F cells , 293 H cells, HEK 293 cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, Expi293FTM cells, Flp-InTM T-RExTM 293 cell line, Flp-InTM-293 cell line, Flp-InTM-3T3 cell line, Flp-InTM-BHK cell line, Flp-InTM-CHO cell line, Flp-InTM-CV-l cell line, Flp-InTM-Jurkat cell line, FreeStyleTM 293-F cells, FreeStyleTM CHO-S cells, GripTiteTM 293 MSR cell line, GS-CHO cell line, HepaRGTM cells, T-RExTM Jurkat cell line, Per.C6 cells, T-RExTM-293 cell line, T-RExTM-CHO cell line, T-RExTM-HeLa cell line, NC-HI
  • a target cell is a cancerous cell, e.g., in a human.
  • Cancer can be a solid tumor or a hematologic malignancy.
  • the solid tumor can include a sarcoma or a carcinoma.
  • Exemplary sarcoma target cell can include, but are not limited to, cell obtained from alveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastoma, angiosarcoma, chondrosarcoma, chordoma, clear cell sarcoma of soft tissue, dedifferentiated liposarcoma, desmoid, desmoplastic small round cell tumor, embryonal rhabdomyosarcoma, epithelioid fibrosarcoma, epithelioid hemangioendothelioma, epithelioid sarcoma, esthesioneuroblastoma, Ewing sarcoma, extrarenal rhabdoid tumor, extraskeletal myxoid chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, giant cell tumor, hemangiopericytoma, infantile fibrosarcoma, inflammatory myofibroblastic
  • Exemplary carcinoma target cell can include, but are not limited to, cell obtained from anal cancer, appendix cancer, bile duct cancer (i.e., cholangiocarcinoma), bladder cancer, brain tumor, breast cancer, cervical cancer, colon cancer, cancer of Unknown Primary (CUP), esophageal cancer, eye cancer, fallopian tube cancer, gastroenterological cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, melanoma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid disease, penile cancer, pituitary tumor, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer.
  • CUP Unknown Primary
  • the cancerous cell can comprise cells obtained from a hematologic malignancy.
  • Hematologic malignancy can comprise a leukemia, a lymphoma, a myeloma, a non-Hodgkin's lymphoma, or a Hodgkin's lymphoma.
  • the hematologic malignancy can be a T-cell based hematologic malignancy.
  • the hematologic malignancy can be a B-cell based hematologic malignancy.
  • Exemplary B-cell based hematologic malignancy can include, but are not limited to, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risk CLL, a non-CLL/SLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B -lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, s
  • Exemplary T-cell based hematologic malignancy can include, but are not limited to, peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), anaplastic large cell lymphoma, angioimmunoblastic lymphoma, cutaneous T-cell lymphoma, adult T-cell leukemia/lymphoma (ATLL), blastic NK-cell lymphoma, enteropathy-type T-cell lymphoma, hematosplenic gamma-delta T-cell lymphoma, lymphoblastic lymphoma, nasal NK/T-cell lymphomas, or treatment-related T-cell lymphomas.
  • PTCL-NOS peripheral T-cell lymphoma not otherwise specified
  • anaplastic large cell lymphoma angioimmunoblastic lymphoma
  • ATLL adult T-cell leukemia/lymphoma
  • blastic NK-cell lymphoma enteropathy-type T-cell lymphoma
  • a cell can be a tumor cell line.
  • Exemplary tumor cell line can include, but are not limited to, 600MPE, AU565, BT-20, BT-474, BT-483, BT-549, Evsa-T, Hs578T, MCF-7, MDA-MB- 231, SkBr3, T-47D, HeLa, DU145, PC3, LNCaP, A549, H1299, NCI-H460, A2780, SKOV-3/Luc, Neuro2a, RKO, RKO-AS45-1, HT-29, SW1417, SW948, DLD-1, SW480, Capan-1, MC/9, B72.3, B25.2, B6.2, B38.1, DMS 153, SU.86.86, SNU-182, SNU-423, SNU-449, SNU-475, SNU-387, Hs 817.T, LMH, LMH/2A, SNU-398, PLHC-1, HepG2/SF
  • polypeptides disclosed herein are produced using a suitable method including recombinant and non-recombinant methods (e.g., chemical synthesis).
  • a polypeptide is chemically synthesized
  • the synthesis may proceed via liquid-phase or solid-phase.
  • Solid-phase peptide synthesis allows the incorporation of unnatural amino acids and/or peptide/protein backbone modification.
  • Various forms of SPPS such as Fmoc and Boc, are available for synthesizing polypeptides of the present disclosure. Details of the chemical synthesis are known in the art (e.g., Ganesan A. 2006 Mini Rev. Med. Chem. 6:3-10; and Camarero J.A. et al. 2005 Protein Pept Lett. 12:723-8).
  • the polypeptide may be produced as an intracellular protein or as a secreted protein, using any suitable construct and any suitable host cell, which can be a prokaryotic or eukaryotic cell, such as a bacterial (e.g., E. coli) or a yeast host cell, respectively.
  • eukaryotic cells that are used as host cells for production of the polypeptides include insect cells, mammalian cells, and/or plant cells.
  • mammalian host cells may include human cells (e.g., HeLa, 293, H9 and Jurkat cells); mouse cells (e.g., NIH3T3, L cells, and C127 cells); primate cells (e.g., Cos 1, Cos 7 and CV1) and hamster cells (e.g., Chinese hamster ovary (CHO) cells).
  • human cells e.g., HeLa, 293, H9 and Jurkat cells
  • mouse cells e.g., NIH3T3, L cells, and C127 cells
  • primate cells e.g., Cos 1, Cos 7 and CV1
  • hamster cells e.g., Chinese hamster ovary (CHO) cells.
  • the polypeptide disclosed herein are produced in CHO cells.
  • a variety of host-vector systems suitable for the expression of a polypeptide may be employed according to standard procedures known in the art. See, e.g., Sambrook et al. 1989 Current Protocols in Molecular Biology Cold Spring Harbor Press, New York; and Ausubel et al. 1995 Current Protocols in Molecular Biology, Eds. Wiley and Sons. Methods for introduction of genetic material into host cells include, for example, transformation, electroporation, conjugation, calcium phosphate methods and the like. The method for transfer can be selected so as to provide for stable expression of the introduced polypeptide -encoding nucleic acid.
  • the polypeptide -encoding nucleic acid can be provided as an inheritable episomal element (e.g., a plasmid) or can be genomically integrated.
  • an inheritable episomal element e.g., a plasmid
  • genomically integrated e.g., a variety of appropriate vectors for use in production of a polypeptide of interest are commercially available.
  • Vectors can provide for extrachromosomal maintenance in a host cell or can provide for integration into the host cell genome.
  • the expression vector provides transcriptional and translational regulatory sequences and may provide for inducible or constitutive expression where the coding region is operably-linked under the transcriptional control of the transcriptional initiation region, and a transcriptional and translational termination region.
  • the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. Promoters can be either constitutive or inducible, and can be a strong constitutive promoter (e.g., T7).
  • nucleic acids encoding the polypeptides disclosed herein.
  • a nucleic acid encoding the polypeptides disclosed herein is operably linked to a promoter sequence that confers expression of the polypeptide.
  • the sequence of the nucleic acid is codon optimized for expression of the polypeptide in a human cell.
  • the nucleic acid is a deoxyribonucleic acid (DNA).
  • the nucleic acid is a ribonucleic acid (RNA).
  • a vector comprising the nucleic acid encoding the polypeptides for binding a target nucleic acid as described herein. In certain aspects, the vector is a viral vector.
  • a host cell comprising the nucleic acid or the vector encoding the polypeptides disclosed herein is provided. In certain aspects, a host cell comprising the polypeptides disclosed herein is provided. In certain aspects, a host cell that expresses the polypeptide is also disclosed.
  • the present disclosure also provides recombinant polypeptide comprising a DNA binding domain and a transcriptional repressor domain, wherein the DNA binding domain and the transcriptional repressor domain are heterologous, wherein the transcriptional repressor domain comprises an amino acid sequence at least 80% identical to any one of the sequences set out in SEQ ID NOs: 84-101.
  • the transcriptional repressor domain comprises an amino acid sequence at least 85% identical, at least 90% identical, at least 95% identical, or a 100% identical to any one of the sequences set out in SEQ ID NOs: 84-101.
  • the DNA binding domain may be a zinc finger protein (ZFP), a transcription activator-like effector (TALE), or a guide RNA.
  • ZFP zinc finger protein
  • TALE transcription activator-like effector
  • the DNA binding domain may be a DBD as disclosed herein that binds to a target sequence provided herein.
  • the DNA binding domain may bind to a target nucleic acid sequence in a gene.
  • the target nucleic acid sequence may be present in a PDCD 1 gene, a CTLA4 gene, a LAG3 gene, a TET2 gene, a ETLA gene, a HA VCR2 gene, a CCR5 gene, a CXCR4 gene, a TRA gene, a TRE gene, a E2M gene, an albumin gene, a HEE gene, a HEA1 gene, a TTR gene, a NR3C1 gene, a CD52 gene, an erythroid specific enhancer of the BCL11A gene, a CELE gene, a TGFER1 gene, a SERPINAl gene, a HEV genomic DNA in infected cells, a CEP290 gene, a DMD gene, a CFTR gene, or an IL2RG gene.
  • the present disclosure also provides a nucleic acid encoding the recombinant polypeptide.
  • the nucleic acid may be operably linked to a promoter sequence that confers expression of the polypeptide.
  • the sequence of the nucleic acid is codon optimized for expression of the polypeptide in a human cell.
  • the nucleic acid is a deoxyribonucleic acid (DNA).
  • the nucleic acid is a ribonucleic acid (RNA).
  • the present disclosure also provides a vector comprising the nucleic acid disclosed herein.
  • the vector may be a viral vector.
  • the present disclosure also provides a host cell comprising the nucleic acid or the vector disclosed herein.
  • the host cell may include the polypeptide.
  • the host cell may express the polypeptide.
  • composition comprising the polypeptide and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition may include the nucleic acid or the vector and a pharmaceutically acceptable excipient.
  • a method of suppressing expression of an endogenous gene in a cell may include introducing into the cell the recombinant polypeptide, wherein the DBD of the polypeptide binds to a target nucleic acid sequence present in the endogenous gene and the heterologous transcriptional repressor domain suppresses expression of the endogenous gene.
  • the recombinant polypeptide is introduced as a nucleic acid encoding the polypeptide.
  • the nucleic acid may be a deoxyribonucleic acid (DNA) or RNA.
  • the nucleic acid may be codon optimized for expression in a human cell.
  • the target gene may be a PDCD 1 gene, a CTLA4 gene, a LAG3 gene, a TET2 gene, a ETLA gene, a HA VCR2 gene, a CCR5 gene, a CXCR4 gene, a TRA gene, a TRE gene, a E2M gene, an albumin gene, a HEE gene, a HEA1 gene, a TTR gene, a NR3C1 gene, a CD52 gene, an erythroid specific enhancer of the ECL11A gene, a CELE gene, a TGFER1 gene, a SERPINA1 gene, a HEV genomic DNA in infected cells, a CEP290 gene, a DMD gene, a CFTR gene, or an IL2RG gene.
  • the cell may be an animal cell.
  • the cell may be a human cell.
  • the cell may be a cancer cell.
  • the cell may be an ex vivo cell or an in vivo cell.
  • the introducing may include administering the polypeptide or a nucleic acid encoding the polypeptide to a subject.
  • the administering may include parenteral administration.
  • the administering may include intravenous, intramuscular, intrathecal, or subcutaneous administration.
  • the administering may include direct injection into a site in a subject.
  • the administering may include direct injection into a tumor.
  • a DBD and a functional domain are provided as separate polypeptides instead of a single polypeptide and are assembled into a functional complex using dimerization of a heterodimer pair, where the DBD and the functional domain are each fused to a member of the heterodimer pair.
  • indirect dimerization may also be utilized by using a fused polypeptide comprising two individual members of a heterodimer pair that act as a bridge to bring a DBD and a functional domain together, as explained in detail below.
  • split systems find use in screens for a DBD or a functional domain by, e.g., using a DBD fused to a first member of a heterodimer pair and screening a plurality of candidate functional domains each fused to a second member of the heterodimer pairs and vice versa.
  • control of modulation of gene expression may be achieved by having the DBD and functional domain expression on board (e.g., constitutive expression) a cell as separate polypeptides and assembling a functional DBD and functional domain complex by introducing a bridging construct into the cell, when modulation of gene expression is desired.
  • the bridging construct may be expressed transiently thereby modulating gene expression transiently.
  • control of modulation of gene expression may be achieved by disrupting the DBD and functional domain complex by introducing a disruptor comprising a heterodimer pair or an individual member of a heterodimer pair as explained below.
  • the individual components of a split system may be introduced into a cell as nucleic acids encoding the individual components or as polypeptides or a combination thereof.
  • the split systems may be used for modulating gene expression in any cell such as a mammalian cell having a target site at which the DBD binds. Examples of such cells are provided herein, e.g., in the preceding sections of the application.
  • the heterodimer pairs of the split system include: 37A, 37B; 13A, 13B; DHD37-BBB-A, DHD37-BBB-B; DHD150-A, DHD150-B; DHD154-A, DHD-154B; 37A, 9B; 13A, 37B; 13A, DHD150- B; 37A, DHD37-BBB-B; and DHD37-BBB-A, 37B, where each of 37A, 37B, 13A, 13B, DHD37-BBB- A, DHD37-BBB-B, DHD150-A, DHD150-B, DHD154-A, and DHD-154B, are the individual members of the listed heterodimer pairs.
  • the term first member and second member refers to either of the individual members of a listed heterodimer pair.
  • 37A and the numeral “1” are used herein interchangeably and in the context of a member of a heterodimer pair refer to a polypeptide comprising an amino acid sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical) to the amino acid sequence:
  • DSDEHLKKLKTFLENLRRHLDRLDKHIKQLRDILSENPEDERVKDVIDLSERSVRIVKTVIKIFEDS VRKKE (SEQ ID NO: 473), and is capable of binding to 37B, 9B, and DHD37-BBB-B.
  • 37B and “1” are used herein interchangeably and in the context of a member of a heterodimer pair refers to a polypeptide comprising an amino acid sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical) to the amino acid sequence:
  • GSDDKELDKLLDTLEKILQTATKIIDDANKLLEKLRRSERKDPKVVETYVELLKRHEKAV KELLEIAKTHAKKVE (SEQ ID NO: 474), and is capable of binding to 37A, 13A, and DHD37-BBB-A.
  • the term “13 A” and the numeral “9” are used herein interchangeably and in the context of a member of a heterodimer pair refer to a polypeptide comprising an amino acid sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical) to the amino acid sequence:
  • GTKEDILERQRKIIERAQEIHRRQQEILEELERIIRKPGSSEEAMKRMLKLLEESLRLLKELL ELSEESAQLLYEQR (SEQ ID NO: 475), and is capable of binding to 13B, 37B, and DHD150-B.
  • 13B and 9' are used herein interchangeably and in the context of a member of a heterodimer pair refers to a polypeptide comprising an amino acid sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical) to the amino acid sequence:
  • DHD37-BBB-A in the context of a member of a heterodimer pair refers to a polypeptide comprising an amino acid sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical) to the amino acid sequence:
  • DHD37-BBB-B in the context of a member of a heterodimer pair refers to a polypeptide comprising an amino acid sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical) to the amino acid sequence:
  • GDVKELTKILDTLTKILETATKVIKDATKLLEEHRKSDKPDPRLIETHKKLVEEHETLVRQHKELA EEHLKRTR (SEQ ID NO: 478), and is capable of binding to DHD37-BBB-A and 37A.
  • DHD150-A in the context of a member of a heterodimer pair refers to a polypeptide comprising an amino acid sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical) to the amino acid sequence: GDVKELTKILDTLTKILETATKVIKDATKLLEEHRKSDKPDPRLIETHKKLVEEHETLVRQHKELA EEHLKRTR (SEQ ID NO: 478), and is capable of binding to DHD150-B.
  • GDVKELTKILDTLTKILETATKVIKDATKLLEEHRKSDKPDPRLIETHKKLVEEHETLVRQHKELA EEHLKRTR SEQ ID NO: 478
  • DHD150-B in the context of a member of a heterodimer pair refers to a polypeptide comprising an amino acid sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical) to the amino acid sequence: DNEEIIKEARRVVEEYKKAVDRLEELVRRAENAKHASEKELKDIVREILRISKELNKVSERLIELW ERSQERAR (SEQ ID NO: 479), and is capable of binding to DHD150-A and 13A.
  • DHD154-A and “DHD-154-A” in the context of a member of a heterodimer pair refers to a polypeptide comprising an amino acid sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical) to the amino acid sequence:
  • TAEELLEVHKKSDRVTKEHLRVSEEILKVVEVLTRGEVSSEVLKRVLRKLEELTDKLRRVTEEQR RVVEKLN (SEQ ID NO: 480), and is capable of binding to DHD-154-B.
  • DHD154-B and “DHD-154-B” in the context of a member of a heterodimer pair refers to a polypeptide comprising an amino acid sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical) to the amino acid sequence:
  • DLEDLLRRLRRLVDEQRRLVEELERVSRRLEKAVRDNEDERELARLSREHSDIQDKHDKLAREIL EVLKRLLERTE SEQ ID NO: 481
  • the present disclosure provides two or more nucleic acids encoding one or more of the members of the heterodimer pairs.
  • the nucleic acid encoding a fusion protein comprising a DBD and a member of a heterodimer pair and another nucleic acid encoding a fusion protein comprising a functional domain and a member of the heterodimer pair are provided.
  • a plurality of nucleic acids encode (i) polypeptides that dimerize via direct dimerization, comprising: (A) a DBD fused to a first member of a heterodimer pair and a functional domain fused to a second member of the heterodimer pair, or (B) a DBD fused to a second member of a heterodimer pair and a functional domain fused to a first member of the heterodimer pair, wherein the first and second members of the heterodimer pair bind to each other thereby directly dimerizing the DBD and the functional domain, and wherein the heterodimer pair is selected from one of the following heterodimer pairs: 37A, 37B; 13A, 13B; DHD37-BBB-A, DHD37-BBB-B; DHD150-A, DHD150-B; DHD154-A, DHD-154B; 37A, 9B; 13A, 37B
  • the DBD in (i) (A) or (i) (B) may be fused to a first member of a first heterodimer pair and the functional domain is a first functional domain fused a second member of the first heterodimer pair and to a first member of a second heterodimer pair, and may be used with a second functional domain fused to a second member of the second heterodimer pair, wherein the members of the first heterodimer pair mediate dimerization of the DBD and the first functional domain and members of the second heterodimer pair mediate dimerization of the first functional domain and the second functional domain.
  • the DBD is fused to a first member of a first heterodimer pair and to a first member of a second heterodimer pair
  • the functional domain is fused a second member of the first heterodimer pair the system further comprising a second functional domain fused to a second member of the second heterodimer pair, wherein the members of the first heterodimer pair mediate assembly of the DBD and the first functional domain and members of the second heterodimer pair mediate assembly of the DBD and the second functional domain.
  • a plurality of nucleic acids are provided, where the plurality of nucleic acids encode (ii) polypeptides that dimerize indirectly via a bridging construct, comprising: (A) a DBD fused to a first member of a first heterodimer pair; a bridging construct comprising a second member of the first heterodimer pair fused to a first member of a second heterodimer pair; and a functional domain fused to a second member of the second heterodimer pair; or (B) a DBD fused to a second member of a first heterodimer pair; a bridging construct comprising a first member of the first heterodimer pair fused to a first member of a second heterodimer pair; and a functional domain fused to a second member of the second heterodimer pair; or (C) a DBD fused to a second member of a first heterodimer pair; a bridging construct comprising a first
  • the DBD may bind to a target nucleic acid sequence present in an endogenous gene in a cell.
  • the functional domain may be an enzyme, a transcriptional activator, a transcriptional repressor, or a DNA nucleotide modifier.
  • the enzyme may be a nuclease, a DNA modifying protein, or a chromatin modifying protein.
  • the nuclease may be a cleavage domain or a half cleavage domain.
  • the cleavage domain or half-cleavage domain may be a type IIS restriction enzyme.
  • the type IIS restriction enzyme may be Fokl or Bfil.
  • the chromatin modifying protein may be lysine- specific histone demethylase 1 (LSD1).
  • the transcriptional activator may be VP16, VP64, p65, p300 catalytic domain, TET1 catalytic domain, TDG, Ldbl self-associated domain, SAM activator (VP64, p65, HSF1), or VPR (VP64, p65, Rta).
  • the transcriptional repressor may be KRAB, Sin3a, LSD1, SUV39H1, G9A (EHMT2), DNMT1, DNMT3A-DNMT3L, DNMT3B, KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, Rb, MeCP2, or a novel transcriptional repressor as disclosed herein.
  • the DNA nucleotide modifier may be an adenosine deaminase.
  • the target nucleic acid sequence may be within a PDCD 1 gene, a CTLA4 gene, a LAG3 gene, a TET2 gene, a ETLA gene, a HA VCR2 gene, a CCR5 gene, a CXCR4 gene, a TRA gene, a TRE gene, a E2M gene, an albumin gene, a HEE gene, a HEA1 gene, a TTR gene, a NR3C1 gene, a CD52 gene, an erythroid specific enhancer of the ECL11A gene, a CELE gene, a TGFER1 gene, a SERPINAl gene, a HEV genomic DNA in infected cells, a CEP290 gene, a DMD gene, a CFTR gene, or an IL2RG gene.
  • the DBD may be a transcription activator-like effector (TALE).
  • a DBD fused to a member of a heterodimer pair fused to a member of a heterodimer pair, a functional domain fused to a member of a heterodimer pair, a bridging construct comprising a member of a heterodimer pair fused to another member, such as those described in the preceding paragraphs and further described below and those encoded by the plurality of nucleic acids described above.
  • a DBA and a functional domain is as set forth in (i)(A) or (i)(B).
  • a DBD, a bridging construct, and a functional domain is as set forth in (ii)(A), (ii)(B), or (ii)(C).
  • host cells that include (a) nucleic acids encoding the polypeptides as set forth in (i)(A) or (i)(B); or (b) nucleic acids encoding the polypeptides as set forth in (ii)(A), (ii)(B), or
  • host cells that include host cells that include (a) the polypeptides as set forth in (i)(A) or (i)(B); or (b) the polypeptides as set forth (ii)(A), (ii)(B), or (ii)(C).
  • kits comprising: (a) nucleic acids encoding the polypeptides as set forth in (i)(A) or (i)(B); or (b) nucleic acids encoding the polypeptides as set forth in (ii)(A), (ii)(B), or (ii)(C).
  • kit comprising: (a) a first vector comprising a nucleic acid encoding the DBD set forth in (i)(A); and (b) a second vector comprising a nucleic acid encoding the functional domain set forth in (i)(A); or (a) a first vector comprising a nucleic acid encoding the DBD set forth in
  • kits comprising: a first vector comprising a nucleic acid encoding the DBD set forth in (ii)(A); a second vector comprising a nucleic acid encoding the bridging construct set forth in (ii)(A); and a third vector comprising a nucleic acid encoding the functional domain set forth in
  • compositions comprising the nucleic acids disclosed herein or the polypeptides disclosed herein.
  • the pharmaceutical composition may also include a pharmaceutically acceptable excipient.
  • the pharmaceutical composition may include (a) nucleic acids encoding the polypeptides as set forth in (i)(A) or (i)(B); or (b) nucleic acids encoding the polypeptides as set forth in (ii)(A), (ii)(B), or (ii)(C).
  • the pharmaceutical composition may include (a) a first vector comprising a nucleic acid encoding the DBD set forth in (i)(A); and (b) a second vector comprising a nucleic acid encoding the functional domain set forth in (i)(A); or (a) a first vector comprising a nucleic acid encoding the DBD set forth in (i)(B); and (b) a second vector comprising a nucleic acid encoding the functional domain set forth in (i)(B).
  • the pharmaceutical composition may include: (a) a first vector comprising a nucleic acid encoding the DBD set forth in (ii)(A); (b) a second vector comprising a nucleic acid encoding the bridging construct set forth in (ii)(A); and (c) a third vector comprising a nucleic acid encoding the functional domain set forth in (ii)(A); or (a) a first vector comprising a nucleic acid encoding the DBD set forth in (ii)(B); (b) a second vector comprising a nucleic acid encoding the bridging construct set forth in (ii)(B); and (c) a third vector comprising a nucleic acid encoding the functional domain set forth in (ii)(B); or (a) a first vector comprising a nucleic acid encoding the DBD set forth in (ii)(C); (b) a second vector comprising a nucleic acid
  • the pharmaceutical composition may include the DBD and a functional domain or a DNA binding domain, a functional domain and a bridging construct as provided herein and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition may include the host cell as provided herein and a pharmaceutically acceptable excipient.
  • the split systems of DBD and functional domains and heterodimer pairs may be used in a method for modulating expression from a target gene in a cell.
  • the method may include (i) introducing into the cell a first nucleic acid encoding a DNA binding domain fused to a first member of a heterodimer pair and a second nucleic acid encoding a functional domain fused to a second member of the heterodimer pair; or (ii) introducing into the cell a first nucleic acid encoding a DNA binding domain fused to a second member of a heterodimer pair and a second nucleic acid encoding a functional domain fused to a first member of the heterodimer pair; or (iii) introducing into the cell a DNA binding domain fused to a first member of a heterodimer pair and a functional domain fused to a second member of the heterodimer pair; or (iv) introducing into the cell a DNA binding domain fused to a second
  • the heterodimer pair may be selected from one of the following heterodimer pairs: 37A, 37B; 13A, 13B; DHD37-BBB-A, DHD37- BBB-B; DHD150-A, DHD150-B; DHD154-A, DHD-154B; 37A, 9B; 13A, 37B; 13A, DHD150-B; 37A, DHD37-BBB-B; and DHD37-BBB-A, 37B, wherein the DBD dimerizes with the functional domain via dimerization of the members of the heterodimer pair and wherein binding of the DBD to a target nucleic acid sequence in the target gene results in modulation of expression of the target gene via the functional domain dimerized to the DBD.
  • the method may be used for screening a candidate DBD or a candidate functional domain or for ranking DBDs or functional domains based on specificity, activity, and the like.
  • the modulation of expression of the target gene may be assessed to determine whether a DBD is specific for the target gene and/or whether the functional domain is active in repressing or activating expression of the target gene.
  • the split systems of DBD and functional domains and heterodimer pairs may be used in a method for modulating expression from a target gene in a cell, where the method includes introducing into a cell expressing a DNA binding domain (DBD) fused to a first member of a first heterodimer pair and a functional domain fused to a second member of a second heterodimer pair, a bridging construct comprising a second member of the first heterodimer pair fused to a first member of the second heterodimer pair or a nucleic acid encoding the bridging construct; or introducing into a cell expressing a DNA binding domain (DBD) fused to a second member of a first heterodimer pair and a functional domain fused to a second member of a second heterodimer pair, a bridging construct comprising a first member of the first heterodimer pair fused to a first member of the second heterodimer pair or a nucleic acid encoding the bridging construct;
  • Such a system may be used for fine tuning control of modulation of gene expression by controlling expression of the different components required for modulating gene expression.
  • non-cognate heterodimer pair refers to a heterodimer pair whose members bind to each other with an affinity that is lower than the affinity with which members of a “cognate heterodimer pair” bind.
  • 37A,37B is a cognate heterodimer pair while 37A, 9B form a non-cognate heterodimer pair, since the binding affinity between 37A and 37B is higher than that between 37A and 9B.
  • cognate heterodimer pairs examples include 37A, 37B; 13A, 13B; DHD37- BBB-A, DHD37-BBB-B; DHD150-A, DHD150-B; and DHD154-A, DHD-154B. While members of a “non-cognate heterodimer” bind to each other, members that are not part of a “non-cognate heterodimer” or a “cognate heterodimer” do not significantly bind to each other and are not considered as members of a heterodimer pair.
  • the fusion polypeptides such as, DBD fused to a member of a heterodimer pair may be such that the C-terminus of the DBD is fused to the N-terminus of a member of a heterodimer pair and the N-terminus of the functional domain is fused to the C-terminus of a member of a heterodimer pair.
  • one or more components of the system may be expressed transiently while other component(s) are expressed stably. Stable and transient expression in a cell may be achieved by methods known in the art, such as, transient transfection, gene integration, constitutive and inducible promoters and the like.
  • Anti CD19 CAR-T cell manufacturing Primary T cells were thawed and activated with CD3/CD28 Dynabeads and cultured for 48 hours prior to electroporation with either no mRNA (control) or mRNA encoding the TALE-TFs against PD1. At 24 hours post electroporation T cells were transduced with a lentivirus vector encoding a 3 rd generation anti CD 19 CAR construct on Retronectin at an MOI of 5 to 10. After 24 hours the virus and beads were removed and T cells expanded in RPMI + 10%FBS + IL- 2 for up to 5 days.
  • Co-culture (killing) assay CAR-T cells and control T cells were incubated with CD 19- expressing NALM-6 cells or NALM-6 cells engineered to express PDL-1 (the ligand for PD-1) or NALM-6 cells in which the target antigen CD 19 was knocked out using TALENs (CD 19 KO) at an effector-to-target (E:T) ratio of 1: 1 in a 96-well round bottom culture plate for 16 hours at 37degrees with 5% C02. After 16 hours of incubation, specific target cell killing was measured by release of lactate dehydrogenase (LDH) into the supernatant (Promega kit #) or by flow cytometry analysis.
  • LDH lactate dehydrogenase
  • RNA samples were electroporated with TALETFs (either single or multiplexed) in triplicate. Cells were harvested at 2 days post-transfection for RNA extraction and parallel analysis of expression using flow cytometry. Total RNA was extracted from these samples and from control T cells electroporated without mRNA using Qiagen miRNeasy extraction kit. Total RNA samples were constructed into libraries using Illumina's TruSeq Stranded Total RNA Plurality of nucleic acids Prep Gold kit. Libraries were then sequenced using Illumina's Hiseq 4000 platform with 2x76bp read length to a depth of 25-50 million reads per sample. Reads were aligned using STAR paired alignment (RNA-STAR 2.3.1), mapped to the GRCh38 human genome assembly, and differential gene expression analysis was performed using edgeR.
  • TALETFs either single or multiplexed
  • Functional domains were added by Infusion cloning (Takara Bio; catalog # 638909) onto the C-terminal end of the TAF.
  • Functional domain constructs contained a 15 amino acid linker domain (GGGGGMDAKSFTAWS) (SEQ ID NO: 109) and either an epigenetic-functional domain (e.g. - KRAB) or heterodimer protein (e.g. - 9' of the 9:9' pair).
  • Table 9 TL11107 PDCD1 PROMOTER - GAGCGGAAGGGAAACTGTC 357 ⁇ 50%
  • FIG. 1A illustrates the locations in the PDCD1 gene to which the DBDs of the indicated recombinant polypeptides were designed to bind.
  • Recombinant polypeptides that repressed expression of PDCD1 in at least 50% of cells treated with the recombinant polypeptides are indicated by clear arrows .
  • Recombinant polypeptides that repressed expression of PDCD1 in less than 50% of the cells treated with the recombinant polypeptides are indicated by solid arrows .
  • the orientation of the arrows indicates the DNA strand to which the recombinant polypeptide is designed to bind. Arrows having the orientation are designed to bind to the anti-sense strand. Arrows having the orientation are designed to bind to the sense strand.
  • the analysis of repression by the disclosed recombinant polypeptides that are designed bind to these sequences identified certain regions that provide repression of PDCD-1 expression in at least 50% of the cells expressing these recombinant polypeptides. These regions are depicted in FIGS. 1B-1C and include regions 1-4. In regions 1, 2, 3, the anti-sense strand of the PDCD-1 gene was successfully targeted to significantly repress expression of PD-1. In region 4, the sense strand was identified as the region of the PDCD-1 gene that can be successfully target for repression. In addition, certain sequences in the sense strand in region 1 were also identified a region that can be targeted for repression.
  • Tables 1-4 illustrate the sequences present in each of Regions 1-4 that can be targeted for repression.
  • FIG. 2 shows the fold change in number of PD-1 expressing cells 2 days after transfection of mRNA encoding the indicated recombinant polypeptides into CD3+ T cells.
  • FIG. 3 shows effect of dose of mRNA encoding the recombinant polypeptide, pAF040 and pAF043, on the percent of CD3+ T cells expressing PD-1, 3 days after transfection.
  • CD3+ T cells were activated with beads and electroporated 48 hours post activation according to standard process with varying concentration of TAFE-TF mRNA from 3ng to 2ug per transfection (250,000 T cells per condition). PD-1 expression by flow was measured on day 3 post transfection.
  • FIG. 4 shows the fold change in number of PD-1 -positive cells at the indicated number of days post-transfection of mRNA encoding the indicated recombinant polypeptide relative to control, which are cells electroporated without repressor mRNA.
  • PD-1 repression is durable for about 2 weeks in culture and after freeze-thaw.
  • FIGS. 5 A and 5B show that PD-1 repression with pAF043 in anti-CD 19 CAR-T cells is sustained after in vivo expansion and clearance of CD19-positive NAFM-6 B-AFF tumor model in NSG mice.
  • targeting the sequence GGCCAGGGCGCCTGT (SEQ ID NO: 36) by TAFE-TF TL11084 also significantly suppressed PD-1 expression.
  • FIG. 6 illustrates the locations in the TIM3 gene at which the DBDs of the indicated recombinant polypeptides bind.
  • Recombinant polypeptides that repressed expression of TIM3 in at least 50% of the cells are indicated by unfilled arrows .
  • Recombinant polypeptides that repressed expression of TIM3 in less than 50% of the cells are indicated by filled arrows .
  • the orientation of the arrows indicates the DNA strand to which the recombinant polypeptide is designed to bind. Arrows having the orientation are designed to bind to the anti-sense strand.
  • Arrows having the orientation are designed to bind to the sense strand.
  • FIG. 7 shows the fold change in number of cells expressing TIM3 at 2 days, 5 days, 8 days, or 14 days after transfection of mRNA encoding the indicated recombinant polypeptides into CD3+ T cells.
  • FIG. 8 shows the fold change in number of cells expressing TIM3 at 3 days or 6 days after transfection of mRNA encoding the indicated recombinant polypeptides into CD3+ T cells.
  • FIG. 9 illustrates the locations in the CTLA4 gene at which the DBDs of the indicated recombinant polypeptides bind.
  • Recombinant polypeptides that repressed expression of CTLA4 in at least 50% of the cells are indicated by unfdled arrows .
  • Recombinant polypeptides that repressed expression of CTLA4 in less than 50% of the cells are indicated by filled arrows .
  • the orientation of the arrows indicates the DNA strand to which the recombinant polypeptide is designed to bind. Arrows having the orientation are designed to bind to the anti-sense strand. Arrows having the orientation are designed to bind to the sense strand.
  • FIG. 10 shows the fold change in number of cells expressing CTLA4 at 3 days after transfection of mRNA encoding the indicated recombinant polypeptides into CD3+ T cells.
  • EXAMPLE 4 Identification of TALE-TFs for LAG3 repression
  • This example illustrates identification of TALE-TFs that significantly repress LAG3 expression.
  • Figure 11 provides a pictorial map of all of the regions in the LAG3 gene that were tested for identifying TAFE-TFs that significantly repress LAG3 expression. The results are provided in Table 12 below:
  • FIG. 11 illustrates the locations in the LAG3 gene at which the DBDs of the indicated recombinant polypeptides bind. Recombinant polypeptides that repressed expression of LAG3 in at least
  • FIG. 12 shows the fold change in number of cells expressing LAG3 at 2 days, 7 days, or 12 days after transfection of mRNA encoding the indicated recombinant polypeptides into CD3+ T cells.
  • FIG. 13 shows the fold change in number of cells expressing LAG3 at 2 days after transfection of mRNA encoding the indicated recombinant polypeptides into CD3+ T cells.
  • FIGS. 14A and 14B show multiplexing of recombinant polypeptides to simultaneously suppress expression of PD-1, LAG3, and TIM3 is a single cell.
  • FIGS. 15A-15C illustrates specificity of the recombinant polypeptides as indicated by lack of significant off-target effect as measured by RNA-seq.
  • Anti-CD19 CAR-T cells were treated with epiTFs against PD-1, LAG3, and TIM3 and then used against a B-Cell Acute Lymphoblastic Leukemia (B-ALL) xenograft model in Non-obese Diabetic, NOD Scid Gamma (NSG) mice.
  • B-ALL B-Cell Acute Lymphoblastic Leukemia
  • CAR-T cells were manufactured using lentivirus delivery of a 3rd generation anti-CD 19 CAR containing FMC63 scFv, CD28 and 4-1BB co-stimulatory domains, and a truncated EGFRtag (Lenti- EF1a-CD19-EGFRt-3rd-CAR Vector, Creative Biolabs).
  • Primary human T cells were activated with Dynabeads as previously described and transfected by electroporation with repressor mRNA at 48 hours post activation.
  • Transfected cells along with no-mRNA transfected controls were allowed to recover for 24 hours after electroporation and then transduced with lentivirus encoding the CAR on RetroNectin (Takara Bio) according to manufacturer's protocol at an MOI of 5 and in the absence of serum.
  • RetroNectin RetroNectin
  • CAR-T cells were allowed to expand in media with IL-2 until day 11 post activation when they were washed with PBS and administered to mice.
  • CAR-T cells Prior to using in animals, CAR-T cells were analyzed by flow cytometry for CAR expression (via EGFR staining) and expression of immune checkpoint genes (PD-1, LAG3, and TIM3).
  • mice Animal experiments were conducted at the Fred Hutchinson Cancer Center, Comparative Medicine department (Seattle, WA) according to an approved IACCUC protocol.
  • Female NSG mice aged 6-8 weeks were implanted intravenously with 5x10 5 NAFM-6-luc-GFP tumor cells (human B-AFF cancer cells expressing CD 19) and tumors were measured by total bioluminescent flux using a Xenogen Imaging System (Perkin Elmer).
  • Each experimental arm contained 5 mice.
  • At 4 days post tumor implantation mice were imaged and randomized into treatment arms based on baseline tumor burden.
  • mice On day 5 post implantation mice were dosed intravenously with 250,000 anti-CD19 CAR-T cells either treated or untreated with repressor mRNA.
  • Peripheral blood was collected via retroorbital bleeding at weekly intervals into EDTA-coated tubes at room temperature.
  • Red blood cell lysis was performed using (1X RBC Lysis Buffer, eBiosciences Cat. # 333-57) according to manufacturer's protocol.
  • Flow cytometry was performed as previously described. At 3 weeks post initial dosing mice were re-challenged with 5x10 5 NAFM-6-luc-GFP tumor cells to test for persistence and activity of circulating CAR-T cells in the blood.
  • FIG. 19 shows a schematic of an anti-CD 19 CAR-T cell in which expression of PD1, TIM3, and LAG3 has been repressed using the engineered polypeptides (pAL043+TL8188+TL8222) described herein.
  • FIG. 20 shows flow cytometry data confirming repression of PD1, TIM3, and LAG3 expression in the multiplex-treated CAR-T cells.
  • FIG. 21 provides an overview of in vivo leukemia xenograft model and treatment using indicated CAR-T cells.
  • FIG. 22 demonstrates that multiplexed repression of immune checkpoint genes is sustained in vivo. Flow cytometry showed persistent repression of immune checkpoint genes at 1 week post dosing CAR-Ts into mice.
  • FIG. 23 demonstrates that multiplexed repression of immune checkpoint genes enhances CAR-Ts ability to resist tumor re-challenge. Tumor burden as measured by total flux (bioluminescence) showed all mice were initially “cured” of leukemia in all treatment arms, but upon re-challenge with leukemia cells only the mice treated with CAR-Ts in which all 3 immune checkpoint genes were repressed were able to completely resist tumor formation. This indicates superior persistence and resistance to exhaustion.
  • FIG. 24 shows expansion of CAR-Ts in the mouse blood.
  • Flow cytometry data showed expansion of CAR-T cells in the mouse blood (measured as human CD3+ T cells). After the re-challenge the multiplex-treated T cells expanded the best, in line with their enhanced proliferative capacity and resistance to exhaustion.
  • FIG. 16 shows characterization of repression of TIM3 expression by the listed candidate transcriptional repressors.
  • FIG. 17 shows characterization of repression of LAG3, TIM3, or PD-1 expression by the listed candidate transcriptional repressors.
  • FIG. 18 shows characterization of repression of TIM3 expression by the listed candidate transcriptional repressors.
  • CTBP1 [0370] CTBP1:
  • ZNF45 [0381] MTKSKEAVTFKDVAVVFSEEEFQFFDFAQRKFYRDVMFENFRNVVSVGHQSTPDGFPQ LEREEKLWMMKMATQRDNSSGAKNLKEMETLQEVGLRYLPHEELFCSQIWQQITRELIKYQDS VVNIQRTGCQLEKRDDLHYKDEGFSNQSSHLQVHRVHTGEKP (SEQ ID NO: 89)
  • ARQKLLGRSW SVPVIRHLFAPLKDYFACE SEQ ID NO:98
  • SSTFs synthetic split TFs
  • Fig. 26 Large-scale analysis of functional domains enabled by split encoding of DNA targeting and functional activities. Top panel. The DNA binding domain of the TIM3 repressor TL8188 was selected to screen additional functional domains.
  • TL8188-DBD was fused to heterodimer 37A, and a plurality of nucleic acids of functional domains was fused to heterodimer 37B. Both constructs were transiently expressed in primary human T cells by RNA electroporation, and fraction of cells with TIM3 repressed (%TIM3 negative cells in TL8188-treated cells relative to no RNA control) was evaluated periodically for 26 days by cell surface antibody staining and flow cytometry. Cells with greater fluorescence intensity than unstained control were considered TIM3+. Middle panel. Domains containing KRAB showed more durable repression, or relatively slow kinetics of decay, for several different KRAB domains. Bottom panel. Domains from methyl-DNA binding proteins showed less durable repression, or relatively fast kinetics of decay.
  • TIM3 expression was assayed using flow cytometry and plotted as %TIM3+cells at Day 2 post transfection with an mRNA encoding TIM3 targeting DBD (from TL8188) fused to one member of a heterodimer pair and an mRNA encoding another member of the heterodimer pair fused to a KRAB domain.
  • Cognate pairs 13A,13B; 37A,37B; DHD37-BBB-A, DHD37-BBB-B; DHD150A, DHD150B; DHD154A, DHD154B mediate dimerization and repression.
  • Non-cognate pairs 13,37; 13,DHD150; 37, DHD37-BBB; and 37, DHD150 also mediated dimerization and repression. See Fig. 27.
  • a recombinant polypeptide comprising: a DNA binding domain (DBD) and a transcriptional repressor domain, the DBD comprising a plurality of repeat units (RUs) ordered from N-terminus to C- terminus of the DBD to bind to a nucleic acid sequence of the PDCD1 gene, wherein the nucleic acid sequence is present within the sequence:
  • DBD DNA binding domain
  • RUs repeat units
  • each of the RU comprises the sequence X 1-11 X 12 X 13 X 14-33, 34, or 35 (SEQ ID NO: 455), wherein: X 1-11 is a chain of 11 contiguous amino acids, X 14-33 or 34 or 35 is a chain of 20, 21 or 22 contiguous amino acids, X 12 X 13 is selected from: (a) NH, HH, KH, NK, NQ, RH, RN, SS, NN, SN, or KN for recognition of guanine (G); (b) NI, KI, RI, HI, or SI for recognition of adenine (A); (c) NG, HG, KG, or RG for recognition of thymine (T); (d) HD, RD, SD, ND, KD, or YG for recognition of cytosine (C); and (e) NV
  • HD high definition, NG, NH, HD, NG, HD, and HD.
  • a recombinant polypeptide comprising: a DNA binding domain (DBD) and a transcriptional repressor, the DBD comprising a plurality of repeat units (RUs) ordered from N-terminus to C- terminus of the DBD to bind to a nucleic acid sequence of the PDCD1 gene, wherein the nucleic acid sequence is present within the sequence:
  • DBD DNA binding domain
  • RUs repeat units
  • each of the RU comprises the sequence X 1-11 X 12 X 13 X 14-33, 34, or 35 (SEQ ID NO: 455), wherein: X 1- 11 is a chain of 11 contiguous amino acids, X 14-33 or 34 or 35 is a chain of 20, 21 or 22 contiguous amino acids, X 12 X 13 is selected from: (a) NH, HH, KH, NK, NQ, RH, RN, SS, NN, SN, or KN for recognition of guanine (G); (b) NI, KI, RI, HI, or SI for recognition of adenine (A); (c) NG, HG, KG, or RG for recognition of thymine (T); (d) HD, RD, SD, ND, KD, or YG for recognition of cytosine (C); and (e)
  • the DBD comprises at least thirteen RUs, wherein X 12 X 13 in the RUs from N-terminus to C-terminus are NG, HD, NG, NH, NG, HD, NI, HD, NG, HD, NG, HD, and NH.
  • the DBD comprises at least eighteen RUs, wherein X 12 X 13 in the RUs from N-terminus to C-terminus are NG, HD, NG, NH, NG, HD, NI, HD, NG, HD, NG, HD, NG, HD, NG, NH, HD, HD, HD, NI, and HD.
  • a recombinant polypeptide comprising: a DNA binding domain (DBD) and a transcriptional repressor, the DBD comprising at least nine repeat units (RUs) ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the PDCD1 gene, wherein the nucleic acid sequence is present within the sequence:
  • DBD DNA binding domain
  • RUs repeat units
  • each of the RU comprises the sequence X 1- 11 X 12 X 13 X 14-33, 34, or 35 (SEQ ID NO: 455), wherein: X 1-11 is a chain of 11 contiguous amino acids, X 14-33 or 34 or 35 is a chain of 20, 21 or 22 contiguous amino acids, X 12 X 13 is selected from: (a) NH, HH, KH, NK, NQ, RH, RN, SS, NN, SN, or KN for recognition of guanine (G); (b) NI, KI, RI, HI, or SI for recognition of adenine (A); (c) NG, HG, KG, or RG for recognition of thymine (T); (d) HD, RD, SD, ND, KD, or YG for recognition of cytosine (C); and (e) NV or HN for recognition of A or G; and (
  • a recombinant polypeptide comprising: a DNA binding domain (DBD) and a transcriptional repressor, the DBD comprising at least nine repeat units (RUs) ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the PDCD1 gene, wherein the nucleic acid sequence is present within the sequence:
  • DBD DNA binding domain
  • RUs repeat units
  • each of the RU comprises the sequence X 1-11 X 12 X 13 X 14-33, 34, or 35 (SEQ ID NO: 455), wherein: X 1- 11 is a chain of 11 contiguous amino acids, X 14-33 or 34 or 35 is a chain of 20, 21 or 22 contiguous amino acids, X 12 X 13 is selected from: (a) NH, HH, KH, NK, NQ, RH, RN, SS, NN, SN, or KN for recognition of guanine (G); (b) NI, KI, RI, HI, or SI for recognition of adenine (A); (c) NG, HG, KG, or RG for recognition of thymine (T); (d) HD, RD, SD, ND, KD, or YG for recognition of cytosine (C); and (e
  • a recombinant polypeptide comprising: a DNA binding domain (DBD) and a transcriptional repressor, the DBD comprising at least nine repeat units (RUs) ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the PDCD 1 gene, wherein the nucleic acid sequence is present within the sequence: GCCGCCTTCTCCACTGCTCAGGCGGAGGT (SEQ ID NO:31), wherein each of the RU comprises the sequence X 1-11 X 12 X 13 X 14-33 or 34 or 35 (SEQ ID NO: 455), wherein: X 1-11 is a chain of 11 contiguous amino acids, X 14-33 or 34 or 35 is a chain of 20, 21 or 22 contiguous amino acids, X 12 X 13 is selected from: (a) NH, HH, KH, NK, NQ, RH, RN, SS, NN, SN, or KN for recognition of guanine (G);
  • a recombinant polypeptide comprising : a DNA binding domain (DBD) and a transcriptional repressor, the DBD comprising at least nine repeat units (RUs) ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the PDCD 1 gene, wherein the nucleic acid sequence is present within the sequence: GGCCAGGGCGCCTGT (SEQ ID NO:36);
  • each of the RU comprises the sequence X 1-11 X 12 X 13 X 14-33, 34, or 35 (SEQ ID NO: 455), wherein: X 1- 11 is a chain of 11 contiguous amino acids, X 14-33 or 34 or 35 is a chain of 20, 21 or 22 contiguous amino acids, X 12 X 13 is selected from: (a) NH, HH, KH, NK, NQ, RH, RN, SS, NN, SN, or KN for recognition of guanine (G); (b) NI, KI, RI, HI, or SI for recognition of adenine (A); (c) NG, HG, KG, or RG for recognition of th
  • a recombinant polypeptide comprising: a DNA binding domain (DBD) and a transcriptional repressor, the DBD comprising a plurality of repeat units (RUs) ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the TIM3 gene, wherein the nucleic acid sequence is present within the sequence:
  • DBD DNA binding domain
  • RUs repeat units
  • each of the RU comprises the sequence X 1-11 X 12 X 13 X 14-33, 34, or 35 (SEQ ID NO: 455), wherein: X 1-11 is a chain of 11 contiguous amino acids, X 14-33 or 34 or 35 is a chain of 20, 21 or 22 contiguous amino acids, X 12 X 13 is selected from: (a) NH, HH, KH, NK, NQ, RH, RN, SS, NN, SN, or KN for recognition of guanine (G); (b) NI, KI, RI, HI, or SI for recognition of adenine (A); (c) NG, HG, KG, or RG for recognition of thymine (T); (d) HD, RD, SD, ND, KD, or YG for recognition of cytosine (C);
  • a recombinant polypeptide comprising: a DNA binding domain (DBD) and a transcriptional repressor, the DBD comprising a plurality of repeat units (RUs) ordered from N-terminus to C-terminus of the DBD to bind a nucleic acid sequence of the TIM3 gene, wherein the nucleic acid sequence is present within the sequence:
  • DBD DNA binding domain
  • RUs repeat units
  • each of the repeat unit comprises the sequence X 1-11 X 12 X 13 X 14-33, 34, or 35 (SEQ ID NO: 455), wherein: X 1-11 is a chain of 11 contiguous amino acids, X 14-33 or 34 or 35 is a chain of 20, 21 or 22 contiguous amino acids, X 12 X 13 is selected from: (a) NH, HH, KH, NK, NQ, RH, RN, SS, NN, SN, or KN for recognition of guanine (G); (b) NI, KI, RI, HI, or SI for recognition of adenine (A); (c) NG, HG, KG, or RG for recognition of thymine (T); (d) HD, RD, SD, ND, KD, or YG for recognition of cytosine (C); and (e) NV or HN for recognition of A
  • a recombinant polypeptide comprising: a DNA binding domain (DBD) and a transcriptional repressor, the DBD comprising a plurality of repeat units (RUs) ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of TIM3 gene, wherein the nucleic acid sequence is: TACACACAT (SEQ ID NO:54), wherein each of the repeat unit comprises the sequence X 1- 11 X 12 X 13 X 14-33, 34, o ( r S 35 EQ ID NO: 455), wherein: X 1-11 is a chain of 11 contiguous amino acids, X 14-33 or 34 or 35 is a chain of 20, 21 or 22 contiguous amino acids, X 12 X 13 is selected from:(a) NH, HH, KH, NK, NQ, RH, RN, SS, NN, SN, or KN for recognition of guanine (G); (b) NI, KI, KI
  • a recombinant polypeptide comprising: a DNA binding domain (DBD) and a transcriptional repressor, the DBD comprising at least nine repeat units (RUs) ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the LAG3 gene, wherein the nucleic acid sequence is present within the sequence:
  • DBD DNA binding domain
  • RUs repeat units
  • each of the RU comprises the sequence X 1-11 X 12 X 13 X 14-33, 34, or 35 (SEQ ID NO: 455), wherein: X 1-11 is a chain of 11 contiguous amino acids, X 14-33 or 34 or 35 is a chain of 20, 21 or 22 contiguous amino acids, X 12 X 13 is selected from:
  • a recombinant polypeptide comprising: a DNA binding domain (DBD) and a transcriptional repressor, the DBD comprising a plurality of repeat units (RUs) ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of LAG3 gene, wherein the nucleic acid sequence is: TGCTCTGTCTGC (SEQ ID NO:72), wherein each of the repeat unit comprises the sequence X 1-11 X 12 X 13 X 14-33, 34, or 35 (SEQ ID NO: 455), wherein: X 1-11 is a chain of 11 contiguous amino acids, X 14-33 or 34 or 35 is a chain of 20, 21 or 22 contiguous amino acids, X 12 X 13 is selected from: (a) NH, HH, KH, NK, NQ, RH, RN, SS, NN, SN, or KN for recognition of guanine (G); (b) NI, KI, RI
  • a recombinant polypeptide comprising: a DNA binding domain (DBD) and a transcriptional repressor, the DBD comprising at least nine repeat units (RUs) ordered from N-terminus to C-terminus of the DBD to bind to a nucleic acid sequence of the CTLA4 gene, wherein the nucleic acid sequence is:
  • each of the RU comprises the sequence X 1-11 X 12 X 13 X 14-33, 34, or 35 (SEQ ID NO: 455), wherein: X 1-11 is a chain of 11 contiguous amino acids, X 14-33 or 34 or 35 is a chain of 20, 21 or 22 contiguous amino acids,
  • X 12 X 13 is selected from:
  • the DBD comprises a C-cap region comprising an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 452, wherein the recombinant polypeptide comprises from N-terminus to C-terminus: the N-cap region, the plurality of RUs, and the C-cap region.
  • X 1-11 is a chain of 11 contiguous amino acids
  • X 14-20 or 21 or 22 is a chain of 7, 8 or 9 contiguous amino acids
  • X 12 X 13 is selected from:(a) NH, HH, KH, NK, NQ, RH, RN, SS, NN, SN, or KN for recognition of guanine (G); (b) NI, KI, RI, HI, or SI for recognition of adenine (A); (c) NG, HG, KG, or RG for recognition of thymine (T); (d) HD, RD, SD, ND, KD, or YG for recognition of cytosine (C); and (e)
  • nucleic acid of clause 99 wherein the nucleic acid is operably linked to a promoter sequence that confers expression of the polypeptide.
  • nucleic acid of clause 99 or 100 wherein the sequence of the nucleic acid is codon optimized for expression of the polypeptide in a human cell.
  • RNA ribonucleic acid
  • a vector comprising the nucleic acid of any of clauses 99-103.
  • a host cell comprising the nucleic acid of any of clauses 99-103 or the vector of clause 104 or 105.
  • a pharmaceutical polypeptide comprising the polypeptide of any of clauses 1-98 and a pharmaceutically acceptable excipient.
  • a pharmaceutical polypeptide comprising the nucleic acid of any of clauses 99-103 or the vector of clause 104 or 105 and a pharmaceutically acceptable excipient.
  • a method of suppressing expression of PDCD-1 gene in a cell comprising: introducing into the cell the recombinant polypeptide of any one of clauses 1-48, wherein the recombinant polypeptide binds to a target nucleic acid sequence present in the PDCD-1 gene and the transcriptional repressor domain suppresses expression of the PDCD-1 gene.
  • a method of suppressing expression of TIM3 gene in a cell comprising: introducing into the cell the recombinant polypeptide of any one of clauses 49-64, wherein the recombinant polypeptide binds to a target nucleic acid sequence present in the TIM3 gene and the transcriptional repressor domain suppresses expression of the TIM3 gene.
  • a method of suppressing expression of LAG3 gene in a cell comprising: introducing into the cell the recombinant polypeptide of any one of clauses 65-82, wherein the recombinant polypeptide binds to a target nucleic acid sequence present in the LAG3 gene and the transcriptional repressor domain suppresses expression of the LAG3 gene.
  • a method of suppressing expression of CTLA4 gene in a cell comprising: introducing into the cell the recombinant polypeptide of any one of clause 83, wherein the recombinant polypeptide binds to a target nucleic acid sequence present in the CTLA4 gene and the transcriptional repressor domain suppresses expression of the CTLA4 gene.
  • nucleic acid is a deoxyribonucleic acid (DNA).
  • nucleic acid is a ribonucleic acid (RNA).
  • the transcriptional repressor domain comprises KRAB, Sin3a, LSD1, SUV39H1, G9A (EHMT2), DNMT1, DNMT3A-DNMT3L, DNMT3B, KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, Rb, or MeCP2.
  • the transcriptional repressor domain comprises KRAB, Sin3a, LSD1, SUV39H1, G9A (EHMT2), DNMT1, DNMT3A-DNMT3L, DNMT3B, KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, Rb, or MeCP2.
  • a recombinant polypeptide comprising a DNA binding domain and a transcriptional repressor domain, wherein the DNA binding domain and the transcriptional repressor domain are heterologous, wherein the transcriptional repressor domain comprises an amino acid sequence at least 80% identical to any one of the sequences set out in SEQ ID NOs: 84-101.
  • the target nucleic acid sequence is in a PDCD 1 gene, a CTLA4 gene, a LAG3 gene, a TET2 gene, a ETLA gene, a HAVCR2 gene, a CCR5 gene, a CXCR4 gene, a TRA gene, a TRE gene, a E2M gene, an albumin gene, a HEE gene, a HEA1 gene, a TTR gene, a NR3C1 gene, a CD52 gene, an erythroid specific enhancer of the BCL11A gene, a CELE gene, a TGFER1 gene, a SERPINAl gene, a HEV genomic DNA in infected cells, a CEP290 gene, a DMD gene, a CFTR gene, or an IL2RG gene.
  • nucleic acid of clause 135 or 136 wherein the sequence of the nucleic acid is codon optimized for expression of the polypeptide in a human cell.
  • RNA ribonucleic acid
  • a vector comprising the nucleic acid of any of clauses 135-138.
  • a host cell comprising the nucleic acid of any of clauses 135-139 or the vector of clause 140 or 141.
  • a host cell comprising the polypeptide of any of clauses 128-134.
  • a pharmaceutical composition comprising the polypeptide of any of clauses 128-134 and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprising the nucleic acid of any of clauses 135-139 or the vector of clause 140 or 141 and a pharmaceutically acceptable excipient.
  • a method of suppressing expression of an endogenous gene in a cell comprising: introducing into the cell the recombinant polypeptide of any one of clauses 128-134, wherein the DBD of the polypeptide binds to a target nucleic acid sequence present in the endogenous gene and the heterologous transcriptional repressor domain suppresses expression of the endogenous gene.
  • nucleic acid is a deoxyribonucleic acid (DNA).
  • nucleic acid is a ribonucleic acid (RNA).
  • the gene is a PDCD 1 gene, a CTLA4 gene, a LAG3 gene, a TET2 gene, a ETLA gene, a HAVCR2 gene, a CCR5 gene, a CXCR4 gene, a TRA gene, a TRE gene, a E2M gene, an albumin gene, a HEE gene, a HEA1 gene, a TTR gene, a NR3C1 gene, a CD52 gene, an erythroid specific enhancer of the ECL11A gene, a CELE gene, a TGFER1 gene, a SERPINAl gene, a HEV genomic DNA in infected cells, a CEP290 gene, a DMD gene, a CFTR gene, or an IL2RG gene.
  • polypeptides that dimerize via direct dimerization comprising:
  • A a DNA binding domain (DBD) fused to a first member of a heterodimer pair and a functional domain fused to a second member of the heterodimer pair, or
  • B a DNA binding domain (DBD) fused to a second member of a heterodimer pair and a functional domain fused to a first member of the heterodimer pair, wherein the first and second members of the heterodimer pair bind to each other thereby directly dimerizing the DBD and the functional domain, wherein the heterodimer pair is selected from one of the following heterodimer pairs:
  • DBD DNA binding domain
  • DHD37-BBB-A DHD37-BBB-B;
  • DHD154-A DHD-154B
  • polypeptides that dimerize indirectly via a bridging construct comprising:
  • A a DNA binding domain (DBD) fused to a first member of a first heterodimer pair; a bridging construct comprising a second member of the first heterodimer pair fused to a first member of a second heterodimer pair; and a functional domain fused to a second member of the second heterodimer pair; or
  • B a DNA binding domain (DBD) fused to a second member of a first heterodimer pair; a bridging construct comprising a first member of the first heterodimer pair fused to a first member of a second heterodimer pair; and a functional domain fused to a second member of the second heterodimer pair; or
  • C a DNA binding domain (DBD) fused to a second member of a first heterodimer pair; a bridging construct comprising a first member of the first heterodimer pair fused to a second member of a second heterodimer pair; and a functional domain fused to a first member of the second heterodimer pair, wherein the DBD and the functional domain dimerize indirectly via the bridging construct, wherein the first and second heterodimer pairs are different and are selected from the following heterodimer pairs:
  • DHD37-BBB-A DHD37-BBB-B;
  • DHD154-A DHD-154B
  • nuclease is a cleavage domain or a half-cleavage domain.
  • the transcriptional repressor comprises KRAB, Sin3a, LSD1, SUV39H1, G9A (EHMT2), DNMT1, DNMT3A-DNMT3L, DNMT3B, KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, Rb, MeCP2, or a transcriptional repressor provided in clauses 128-134.
  • the target nucleic acid sequence is within a PDCD 1 gene, a CTLA4 gene, a LAG
  • a host cell comprising: (a) nucleic acids encoding the polypeptides as set forth in clause 162 (i)(A) or (i)(B); or (b) nucleic acids encoding the polypeptides as set forth in clause 162 (ii)(A), ( ⁇ )(B), or (ii)(C). 181.
  • a host cell comprising: (a) the polypeptides as set forth in clause 162 (i)(A) or (i)(B); or (b) the polypeptides as set forth in clause 162 (ii)(A), (ii)(B), or (ii)(C).
  • a kit comprising:
  • nucleic acids encoding the polypeptides as set forth in clause 162 (ii)(A), (ii)(B), or (ii)(C).
  • a kit comprising:
  • a kit comprising:
  • a pharmaceutical composition comprising:
  • nucleic acids encoding the polypeptides as set forth in clause 162 (ii)(A), (ii)(B), or (ii)(C).
  • a pharmaceutical composition comprising:
  • a pharmaceutical composition comprising:
  • a pharmaceutical composition comprising the DBD and a functional domain or a DNA binding domain, a functional domain and a bridging construct of clause 178 and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprising the host cell of clause 180 or 181 and a pharmaceutically acceptable excipient.
  • a method for modulating expression from a target gene in a cell comprising:
  • DHD37-BBB-A DHD37-BBB-B;
  • DHD154-A DHD-154B
  • DBD DNA binding domain
  • a method of modulating expression of a target gene in a cell comprising:
  • a bridging construct comprising a second member of the first heterodimer pair fused to a first member of the second heterodimer pair or a nucleic acid encoding the bridging construct
  • a bridging construct comprising a first member of the first heterodimer pair fused to a first member of the second heterodimer pair or a nucleic acid encoding the bridging construct
  • a bridging construct comprising a second member of the first heterodimer pair fused to a second member of the second heterodimer pair or a nucleic acid encoding the bridging construct, wherein the DBD and the functional domain dimerize indirectly via the bridging construct, wherein binding of the DBD to a target nucleic acid sequence in a target gene in the cell results in in modulation of expression of the target gene via the functional domain dimerized to the DBD via the bridging construct, wherein the first and second heterodimer pairs are different and are selected from the following heterodimer pairs:
  • DHD37-BBB-A DHD37-BBB-B;
  • DHD154-A DHD-154B

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