WO2023055893A1

WO2023055893A1 - Gene regulation

Info

Publication number: WO2023055893A1
Application number: PCT/US2022/045175
Authority: WO
Inventors: Chenzhong Kuang; Yan Xiao; Dirk Herman Antonius HONDMANN
Original assignee: Peter Biotherapeutics, Inc.
Priority date: 2021-09-30
Filing date: 2022-09-29
Publication date: 2023-04-06

Abstract

The present disclosure provides technologies for genetic regulation using sequence specificity modified molecules.

Description

GENE REGULATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to United States Provisional patent application number 63/250,995, filed on September 30, 2021, the entire disclosure of which is incorporated herein by reference.

BACKGROUND

[0002] Gene regulation and genome engineering hold great promise for the study of gene function and for the creation of new therapies for human diseases. There is a need for a greater variety of versatile methods that can perform a wide variety of gene and/or genome regulation, which may be used to treat human disease.

SUMMARY

[0003] The present disclosure provides technologies (e.g., systems, compositions, methods, etc.) for gene regulation. As will be appreciated by those of skill in the art, there is an unmet need for technologies that can precisely and specifically regulate one or more genes, but without necessarily modifying a polynucleotide or polypeptide sequence of the gene or administering a small molecule or biologic to otherwise interfere with function of a gene expression product. The present disclosure provides the insight that direct regulation of DNA transcription may have advantages over those technologies that regulate at the RNA level (e.g., RNAi, siRNA). The present disclosure provides technologies that enable precise, targeted gene regulation through use of particular gene regulation agents, Zinc Finger KRAB molecules, that alters transcription of DNA without sequence modification. In some embodiments, the present disclosure provides Zinc Finger KRAB molecules (also referred to as KRAB-X-Zfn molecules) with engineered domains (e.g., engineered zinc finger arrays) that have unexpectedly improved characteristics, e.g., gene regulation capabilities. The present disclosure provides the insight that Zinc Finger KRAB molecules can be engineered to alter the DNA target sequence specificity by making modifications to amino acids within one or more of its zinc finger alpha helices. The present disclosure encompasses a recognition that provided gene regulation agents (e.g., modified Zinc Finger KRAB molecules) are capable of efficiently generating a reduced level of nucleic acid expression at a precise target site(s). The present disclosure further provides the insight that zinc finger array specificity of modified Zinc Finger KRAB molecules can be assessed by a gene editing assay. For example, in some embodiments, target specificity of a modified Zinc Finger KRAB molecule can be assessed by RITDM (Replication Interrupted Template driven DNA Modification or Recombination Induced Template Driven DNA Modification, as described in as described in PCT/US2021/37113, which is herein incorporated by reference in its entirety).

[0004] In some embodiments, a modified Zinc Finger KRAB molecule is a human Zinc Finger KRAB molecule and/or comprises human sequences. The present disclosure further provides the insight that modified Zinc Finger KRAB molecules derived from human sequences (e.g., polypeptide and/or nucleic acid sequence) have beneficial immunogenicity characteristics. In some embodiments, provided gene regulation agents do not induce or induce a limited immunogenic response (e.g., in a subject) and, accordingly, provide increased safety for development of therapies. In some embodiments, a gene regulation agent (e.g., modified Zinc Finger KRAB molecule) is human and/or comprises human sequences and is applicable for use in human subjects.

[0005] Thus, the present disclosure provides, among other things, technologies to regulate expression of one or more polynucleotides (e.g., reduced levels of nucleic acid expression at a target site) using engineered gene regulation agents, such as Zinc Finger KRAB molecules that enable gene regulation (e.g., reduced levels of nucleic acid expression at a target site), for example, through inhibition of transcription. In some embodiments, use of a modified human Zinc Finger KRAB molecule regulates expression while also limiting formation of immunogenic responses. In some embodiments, a gene regulation agent modifies expression of one or more polynucleotides or polypeptides. In some embodiments, a gene regulation agent modifies (e.g., reduces) expression of a target DNA sequence. . In some embodiments, a gene regulation agent modifies (e.g., reduces) expression of a target RNA sequence (e.g., mRNA). In some embodiments, the regulation of gene expression is achieved via a system comprising one or more gene regulation agents, e.g., an agent comprising a zinc finger array and a KRAB-domain, to modify (e.g., eliminate, reduce, or inhibit) expression of one or more polynucleotides at a target site. In some embodiments, the modification is achieved using a system comprising one or more agents that in some way modifies a process (e.g., transcription) at or related to a target.

[0006] In some embodiments, provided are gene regulation agents comprising a ZFn element and a KRAB element. In some embodiments, a gene regulation agent comprises a structure represented by:

[KRAB] - X - [ZFn], where (i) the KRAB element is or comprises a KRAB domain or portion thereof; (ii) the X element is optional and is or comprises a functional domain; and (iii) the ZFn element is or comprises at least five zinc finger arrays, wherein at least one of the zinc finger arrays comprises at least one modified alpha helix, wherein the at least one modified alpha helix is engineered that comprises one or more amino acid modifications relative to a corresponding wild-type alpha helix sequence.

[0007] In some embodiments, provided are gene regulation agents where at least one modified alpha helix is engineered to bind to a first landing site, and where the landing site is associated with a target.

[0008] In some embodiments, provided are gene regulation agents that include a second zinc finger array comprises a second modified alpha helix, wherein the second modified alpha helix is engineered to bind to a second landing site, wherein the second landing site is associated with a target. In some embodiments, a first landing site and a second landing site are both associated with a single target (e.g., genetic target and/or locus in a genome). In some embodiments, a first landing site and a second landing site are associated with different targets (e.g., different genetic targets, different loci in a genome).

[0009] In some embodiments, provided gene regulation agents comprise a ZFn element that comprises or consists of at least six, seven, eight, nine, ten, or eleven, or more, zinc finger arrays. In some embodiments, a Zfn element comprises at least one zinc finger array that comprises at least one alpha helix engineered to comprise a modified amino acid sequence that differs from that of its corresponding wild type sequence. In some embodiments, provided gene regulation agents comprise a ZFn element comprises at least two zinc finger arrays that comprise an engineered alpha helix, at least three zinc finger arrays that comprise an engineered alpha helix, at least four zinc finger arrays that comprise an engineered alpha helix, at least give zinc finger arrays that comprise an engineered alpha helix, at least six zinc finger arrays that comprise an engineered alpha helix, at least seven zinc finger arrays that comprise an engineered alpha helix, at least eight zinc finger arrays that comprise an engineered alpha helix, at least nine zinc finger arrays that comprise an engineered alpha helix, or at least ten zinc finger arrays that comprise an engineered alpha helix.

[0010] In some embodiments, one or more modified alpha helix amino acid sequences comprises: one amino acid substitution mutation at a position selected from -1, +1, +2, +3, +4, +5, or +6 in the alpha helix. In some embodiments, one or more modified alpha helix amino acid sequences comprises: two amino acid substitution mutations at positions selected from -1, +1, +2, +3, +4, +5, or +6 in the alpha helix. In some embodiments, one or more modified alpha helix amino acid sequences comprises: three amino acid substitution mutations at positions selected from -1, +1, +2, +3, +4, +5, or +6 in the alpha helix. In some embodiments, one or more modified alpha helix amino acid sequences comprises: four amino acid substitution mutations at positions selected from -1, +1, +2, +3, +4, +5, or +6 in the alpha helix. In some embodiments, one or more modified alpha helix amino acid sequences comprises: five amino acid substitution mutation at positions selected from -1, +1, +2, +3, +4, +5, or +6 in the alpha helix. In some embodiments, one or more modified alpha helix amino acid sequences comprises: six amino acid substitution mutations at positions selected from -1, +1, +2, +3, +4, +5, or +6 in the alpha helix. In some embodiments, one or more modified alpha helix amino acid sequences comprises: an amino acid substitution mutation at each position in the alpha helix.

[0011] In some embodiments, at least one modified alpha helix amino acid sequences comprises one or more amino acid substitution mutations at positions selected from -1, +2, +3 and +6, or any combinations thereof.

[0012] In some embodiments, gene regulation agents comprise a ZFn element that comprises at least five zinc finger arrays. In some embodiments, a ZFn element that comprises five zinc finger arrays is derived from ZNF 434, ZKSCAN1, ZKSCAN2, ZNF 552, ZNF 562, ZNF 763 or ZNF 793. [0013] In some embodiments, gene regulation agents comprise a ZFn element that comprises up to seven zinc finger arrays. In some embodiments, a ZFn element that comprises seven zinc finger arrays is derived from ZNF 177, ZNF 233, ZNF 248, ZNF 554, ZNF 566, ZNF 577, ZNF 701, ZNF 812, or ZNF 891.

[0014] In some embodiments, gene regulation agents comprise a ZFn element that comprises up to eight zinc finger arrays. In some embodiments, a ZFn element that comprises eight zinc finger arrays is derived from: AC003682.1, ZNF 3, ZNF 124, ZNF 506, ZNF 525, ZNF 561, ZNF 610, ZNF 625, ZNF 766, ZNF 222, or ZNF 223.

[0015] In some embodiments, gene regulation agents comprise a ZFn element that comprises up to nine zinc finger arrays. In some embodiments, a ZFn element that comprises nine zinc finger arrays is derived from: ZIK 1, ZNF 324, ZNF 510, ZNF 519, ZNF 643, ZNF 773, ZNF 486, ZNF 732, ZNF 776, or ZNF 582.

[0016] In some embodiments, gene regulation agents comprise a ZFn element that comprises up to ten zinc finger arrays. In some embodiments, a ZFn element that comprises ten zinc finger arrays is derived from: ZNF 154, ZNF 37A, ZNF 468, ZNF 563, ZNF 614, ZNF 649, ZNF 677, ZNF 682, ZNF 689, ZNF 727, ZNF 730, or ZNF 578.

[0017] In some embodiments, gene regulation agents comprise a ZFn element that comprises up to eleven zinc finger arrays. In some embodiments, a ZFn element that comprises eleven zinc finger arrays is derived from: ZNF 155, ZNF 181, ZNF 253, ZNF 415, ZNF 416, ZNF 479, ZNF 485, ZNF 570, ZNF 675, ZNF 79 or ZIM 3.

[0018] In some embodiments, gene regulation agents comprise a Zfn element that comprises zinc finger arrays derived from one zinc finger polypeptide, where at least one alpha helix sequence originates from a different zinc finger polypeptide. In some embodiments, a gene regulation agent comprises a Zfn element comprising at least five zinc finger arrays from ZIM 3, wherein at least one of the zinc finger arrays from ZIM3 comprises at least one alpha helix originating from ZNF27.

[0019] In some embodiments, a gene regulation agent comprises a Zfn element comprising at least seven zinc finger arrays, wherein each of the zinc finger arrays from ZIM 3 comprises an alpha helix originating from ZF27. In some embodiments, sequences of alpha helices from ZF27 comprise sequences of SEQ ID NOs: 22-32. In some embodiments, sequences of alpha helices from ZF27 comprise sequences that each differ by no more than one amino acid from the sequences of SEQ ID NOs: 22-32. In some embodiments, sequences of alpha helices from ZF27 comprise sequences that each differ by no more than two amino acids from the sequences of SEQ ID NOs: 22-32.

[0020] In some embodiments, an X element is or comprises a polypeptide. In some embodiments, an X element is or comprises a polypeptide between 2 and 100 amino acids in length or between 0.2 kD and 10 kD in size.

[0021] In some embodiments, an X element is or comprises a linker. In some embodiments, an X element comprises a linker that has a sequence of about 1 to about 20 amino acids.

[0022] In some embodiments, gene regulation agents comprise an X element that is derived from or identical to a sequence present in a wild-type or naturally occurring KRAB-Zfn protein. In some embodiments, an X element comprises a polypeptide sequence of about 4 to about 20 amino acids, wherein the polypeptide sequence is identical to or differs by up to two amino acids from a sequence that found in a wild-type or naturally occurring KRAB-Zfn protein.

[0023] In some embodiments, an X element comprises a sequence of any one of SEQ ID NOs: 7 to 21. In some embodiments, an X element comprises a sequence that differs by up to one amino acid from any one of SEQ ID NOs: 7 to 21. In some embodiments, an X element comprises a sequence that differs by up to two amino acids from any one of SEQ ID NOs: 7 to 21. In some embodiments, an X element comprises a sequence that differs by up to three amino acids from any one of SEQ ID NOs: 7 to 21.

[0024] In some embodiments, a gene regulation agent does not comprise an X element.

[0025] In some embodiments, gene regulation agents comprise a KRAB element that comprises an amino acid sequence that is at least 90% identical to that of any one of SEQ ID NOs: 1-6. In some embodiments, gene regulation agents comprise a KRAB element that comprises an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to that of any one of SEQ ID NOs: 1-6. [0026] In some embodiments, a KRAB element comprises a KRAB-A domain. In some embodiments, a KRAB element comprises a KRAB-A domain that comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 4. In some embodiments, a KRAB element comprises a KRAB-A domain that comprises an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical that of SEQ ID NO: 4.

[0027] In some embodiments, a KRAB element comprises a KRAB-B domain. In some embodiments, a KRAB element comprises a KRAB-B domain that comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 6. In some embodiments, a KRAB element comprises a KRAB-B domain that comprises an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical that of SEQ ID NO: 6.

[0028] In some embodiments, a KRAB element comprises a KRAB-A and a KRAB-B domain. In some embodiments, a KRAB element comprises a KRAB-A and a KRAB-B domain that is at least 90% identical to that of SEQ ID NO: 5. In some embodiments, a KRAB element comprises a KRAB-A and a KRAB-B domain that is at least 95%, 96%, 97%, 98% or 99% identical that of SEQ ID NO: 5.

[0029] In some embodiments, a provided gene regulation agent is or comprises an engineered human Zinc Finger KRAB molecule or a portion thereof.

[0030] In some embodiments, an engineered human Zinc Finger KRAB molecule comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 50. In some embodiments, an engineered human Zinc Finger KRAB molecule comprises an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical that of SEQ ID NO: 50.

[0031] In some embodiments, an engineered human Zinc Finger KRAB molecule comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 57. In some embodiments, an engineered human Zinc Finger KRAB molecule comprises an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical that of SEQ ID NO: 57.

[0032] In some embodiments, an engineered human Zinc Finger KRAB molecule comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 61. In some embodiments, an engineered human Zinc Finger KRAB molecule comprises an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical that of SEQ ID NO: 61. [0033] In some embodiments, a provided gene regulation agent decreases or blocks expression of a genetic target. In some embodiments, a provided gene regulation agent decreases or blocks expression of a genetic target through binding to a landing site.

[0034] In some embodiments, provided are compositions comprising gene regulation agents of the present disclosure. In some embodiments, provided compositions comprise a gene regulation agent (e.g., a nucleic acid encoding a gene regulation agent) and one or more excipients.

[0035] In some embodiments, provided are vectors comprising a nucleic acid sequence encoding any one of the gene regulation agents of the present disclosure. In some embodiments, provided vectors a viral vector selected (e.g., lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno-associated viral vector).

[0036] In some embodiments, provided are compositions comprising vectors of the present disclosure. In some embodiments, provided compositions comprise vector encoding a gene regulation agent of the present disclosure one or more excipients.

[0037] In some embodiments, provided are kits comprising the gene regulation agents or vectors of the present disclosure, and instructions for use of the gene regulation agents or vectors.

[0038] In some embodiments, provided are methods of gene regulation, comprising: contacting a cell comprising a polynucleotide (e.g., contacting within a cell) with a gene regulation agent provided herein (e.g., a modified Zinc Finger KRAB molecule), wherein: (i) the polynucleotide (e.g., DNA) comprises a target; and (ii) the Zfin element of the gene regulation agent binds to a landing site associated with the target. In some embodiments, provided methods reduce expression of the target relative to: (i) a cell not contacted with a gene regulation agent and/or (ii) a cell contacted with an agent that does not comprise a KRAB domain. In some embodiments, the gene regulation agent reduces expression of the target through binding to the landing site. In some embodiments, the landing site is or comprises a target site. In some embodiments, the target site is an error site. In some embodiments, the landing site is or comprises all or a portion of a regulatory element. In some embodiments, the landing site is or comprises a regulatory element selected from the group consisting of: a promoter, an enhancer, a silencer, an insulator, a TATA box, a GC box, a CAAT box, a transcriptional start site, a DNA binding motif of a transcription factor or other protein that regulates transcription, a 5’ untranslated region, and a ‘3 untranslated region.

[0039] In some embodiments, the contacting of the cell with the gene regulation agent occurs prior to or concomitant with a reduction in rate and/or level of transcriptional activity of the target. In some embodiments, the contacting of the cell with the gene regulation agent occurs prior to or concomitant with a change in chromatin structure, wherein the change in chromatin structure reduces the rate and/or level of transcriptional activity of the target. In some embodiments, the contacting of the cell with the gene regulation agent further comprises contacting the cell with one or more of a DNA or histone modification factors or any other factors associated with a DNA modification and a histone modification. In some embodiments, where the one or more DNA or histone modification factor(s), factors associated with DNA modifications, or factors associated with histone modifications are selected from: methylases, methyltransferase, histone (de-)acylating enzymes helicases, recombinases, repair scaffold proteins, single strand DNA binding proteins, mismatch repair proteins.

[0040] In some embodiments, a rate and/or level of transcriptional activity of the target is reduced by at least 20%, at least 30%, at least 40%, at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, or more. In certain embodiments, a rate and/or level of transcriptional activity of the target is reduced by 50% or more.

[0041] In some embodiments, provided are methods of gene regulation, comprising: contacting a cell comprising a polynucleotide with a gene regulation agent provided herein, where the cell is present in vivo, and the contacting is achieved by administration by intravenous, parenchymal, intracranial, intracerebroventricular, intrathecal, or parenteral administration.

[0042] In some embodiments, provided are methods of gene regulation, comprising: contacting a cell or population of cells with a gene regulation agent provided herein, where the contacting occurs ex vivo (e.g., in vitro). In some embodiments, provided are methods of gene regulation, comprising: contacting a population of cells with a gene regulation agent provided herein, where the contacting is performed ex vivo or in vitro, resulting in a population of cells with reduced expression of the target and/or one or more histone modifications relative to the population of cells prior to the contacting. In some embodiments, at least a portion of the population of cells is administered to a subject in need thereof.

[0043] In some embodiments, a subject is a mammal, e.g., a human or non-human primate. In some embodiments, a subject is a human. In some embodiments, a subject is an adult human. In some embodiments, a subject is a fetal, infant, child, or adolescent human.

[0044] In some embodiments, a population of cells is or comprises a tissue and/or organ. In some embodiments, a population of cells is or comprises a tumor (e.g., a lung tumor, pancreatic tumor, or colon tumor).

[0045] In some embodiments, a population of cells is or comprises a specific cell lineage, e.g., that comprises one or more KRAS mutation(s). In some embodiments, the one or more KRAS mutation occurs at one or more positions selected from G12, G13, A18, Q61, KI 17, and A146.

[0046] In some embodiments, provided are methods of gene regulation, comprising: contacting a cell or population of cells with a gene regulation agent provided herein, where the contacting occurs in vivo. In some embodiments, the contacting occurs in a subject in need thereof. In some embodiments, a subject is a mammal, e.g., a human or non-human primate. In some embodiments, a subject is a human. In some embodiments, a subject is an adult human. In some embodiments, a subject is a fetal, infant, child, or adolescent human.

[0047] In some embodiments, provided are methods of gene regulation, comprising: contacting a cell or population of cells with at least two different gene regulation agents provided herein. In some embodiments, provided are methods of gene regulation, comprising: contacting a cell or population of cells with at least two different modified Zinc Finger KRAB molecules. In some embodiments, provided are methods of gene regulation, comprising: contacting a cell or population of cells with at least two gene regulations selected from a Zinc Finger KRAB molecule, a KRAB-DLR, and a KRAB-DLRR.

[0048] In some embodiments, the contacting with the at least two gene regulation agents is sequential or simultaneous.

[0049] In some embodiments, provided are methods of gene regulation, comprising: contacting a cell or population of cells with at least two different gene regulation agents that reduce expression of at least two targets. In some embodiments, expression of the at least two targets are reduced relative to: (i) a cell not contacted with a gene regulation agent(s) and/or (ii) a cell contacted with an agent(s) that does not comprise a KRAB domain.

[0050] In some embodiments, the at least two targets are associated with different genes.

In some embodiments, the different genes are located on the same chromosome. In some embodiments, the different genes are located on different chromosomes.

[0051] In some embodiments, the at least two targets are associated with the same gene.

[0052] In some embodiments, expression is measured by a level of mRNA, protein, coprecipitation assays, or chromatin accessibility.

[0053] In some embodiments, provided method result in a reduction in a level of a target as compared to the level of the target in the absence of the contacting.

[0054] In some embodiments, prior to or concomitant with the contacting, the target is being actively transcribed. In some embodiments, the rate and/or extent of transcription is quantified, e.g., using RT-PCR, qPCR, in situ hybridization, or other method known in the art to quantify transcription. In some embodiments, methods using gene regulation agents provided herein reduce transcription by at least 20%, at least 30%, at least 40%, at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, or more. In certain embodiments, transcription of the target is reduced by 50% or more.

[0055] In some embodiments, at least one target or sequence associated therewith is epigenetically modified. In some embodiments, an epigenetic modification is concomitant with a dissociation of an RNA polymerase from a DNA strand associated with the at least one target. In some embodiments, an epigenetic modification prevents association of an RNA polymerase with a DNA strand associated with the at least one target. In some embodiments, at least two targets or sequences associated therewith are epigenetically modified. BRIEF DESCRIPTION OF THE DRAWING

[0056] The Drawing included herein, which is composed of the following Figures, is for illustration purposes only and not for limitation.

[0057] Figure 1 is provides a schematic of DNA transcription in a normal (non- pathological) environment.

[0058] Figure 2 is an illustration of a mechanism of interaction between an exemplary KRAB-X-ZFn molecule and an RNA polymerase complex in which transcription is interrupted.

[0059] Figure 3 shows a partial amino acid alignment of mouse ZFP568 and human ZIM3. Zinc fingers 1 through 10 of mouse ZFP568 (upper amino acid sequence in the alignment, SEQ ID NO.: 65) are aligned with zinc fingers 2 through 11 of human ZIM3 (lower amino acid sequence in the alignment, SEQ ID NO.: 66). Zinc finger numbering of ZIM3 is indicated. Histidine (H) and Cysteine (C) zinc-atom interacting amino acids are indicated in underlined and bold.

[0060] Figure 4 provides an illustration with exemplary schematics representing exemplary gene regulation agents. Figure 4A provides an exemplary schematic of a DLRR construct, that includes from N-terminus to C-terminus: a D element comprising Zinc Finger alpha helices, an L element, and two R elements. Figure 4B provides an exemplary schematic of a KRAB-DLRR construct that includes from N-terminus to C-terminus: a KRAB domain, a D element comprising Zinc Finger alpha helices, an L element, and two R elements. Figure 4C provides an exemplary schematic of a Zinc Finger KRAB construct that includes from N- terminus to C-terminus: a KRAB domain and Zinc Finger alpha helices.

[0061] Figure 5A shows an exemplary targeting strategy used to demonstrate that validated exemplary gene regulation agents can be used to preselect binding sites for use in gene regulation. Figure 5B shows ddPCR detection of an exemplary target site.

[0062] Figure 6A and Figure 6B show relative changes in gene expression of a model gene (KRAS) by exemplary gene regulation agents that include DLR, DLRR, and DLRR molecules, pb74, pb75, and pb76, respectively. Figure 7A shows a representative RT-PCR results from agarose gel electrophoresis. Figure 7B illustrates relative suppression of KRAS gene expressions in U937 cells measured by four independent experiments in U937 cells. [0063] Figure 7 illustrates suppression of KRAS mRNA expression in U937 cell line over a period of time as assessed by RT-PCR.

[0064] Figure 8 illustrated quantification of relative suppression of KRAS mRNA expression by exemplary DLRR, KRAB-DLRR, and Sequence Specificity -Modified Zfn-KRAB molecules compared to no transfection control.

[0065] Figures 9A to 9C show that exemplary DLRR, KRAB-DLRR, and Sequence Specificity-Modified Zfn-KRAB molecules can efficiently suppress KRAS gene expression, as illustrated by reduced mRNA levels in a colon cancer cell line HCT116. Each of

[0066] Figure 10 shows relative suppression of KRAS mRNA by exemplary DLRR, KRAB-DLRR, and Sequence Specificity-Modified Zfn-KRAB molecules compared to no transfection control in a cancer cell line HCT116.

[0067] Figure 11 shows that exemplary DLRR, KRAB-DLRR, and Sequence Specificity-Modified Zfn-KRAB molecules can efficiently suppress KRAS gene expression, which results in a subsequent reduction in KRAS protein levels in HCT116 cells.

[0068] Figure 12 shows an exemplary targeting strategy used for BCL11 A gene regulation and illustrate BCL11 A gene structure, containing 5 exons. Two plasmids encode intramolecular modified zim3 recognizing special sequences in front of exon 1 of BCL11 A gene.

Beginning of Exon 1 of BCL11 A gene is labeled as position 1, and sequence before Exonl labeled minus number(s). One plasmid, pb89, recognizes sequence from position -19 to 2; while pb90, from position -29 to -8.

[0069] Figures 13A to 13C show that two exemplary ZIM3 based Sequence Specificity- modified Zfn-KRAB molecules can efficiently suppress BCL11 A gene expression, as illustrated by reduced mRNA levels.

[0070] Figure 14 illustrates relative suppression of BCL11 A mRNA by two exemplary ZIM3 based Sequence Specificity-Modified Zfn-KRAB molecules compared to no transfection control. DEFINITIONS

[0071] The scope of the present disclosure is defined by the claims appended hereto and is not limited by certain embodiments described herein. Those skilled in the art, reading the present specification, will be aware of various modifications that may be equivalent to such described embodiments, or otherwise within the scope of the claims. In general, terms used herein are in accordance with their understood meaning in the art, unless clearly indicated otherwise. In some instances, explicit definitions of certain terms are provided herein; meanings of these and other terms in particular instances throughout this specification will be clear to those skilled in the art from context.

[0072] As used herein, the term “adjacent” within a polynucleotide context, e.g., within a sequence context (e.g., genomic sequence, mRNA sequence, etc.), refers to adjacency of two things (e.g., components, molecules, etc.) in a linear polynucleotide (e.g., DNA) sequence and/or within a 3D chromosomal architecture of a folded genome. In some embodiments, at least one molecule as described herein comes into sufficiently close molecular proximity to, e.g., a polynucleotide, such as to be adjacent. In some such embodiments, such adjacency influences recombination events at a target site. In some embodiments, such adjacency influences gene activity (e.g. transcription) at or near a target site.

[0073] As used herein, the term “amino acid” refers to any compound and/or substance that can be incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has a general structure, e.g., H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and/or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with general structure as shown above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and/or substitution (e.g., of an amino group, a carboxylic acid group, one or more protons, and/or a hydroxyl group) as compared with a general structure. In some embodiments, such modification may, for example, alter circulating half-life of a polypeptide containing a modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing a modified amino acid, as compared with one containing an otherwise identical unmodified amino acid.

[0074] As used herein, the term “binding site” refers to a nucleic acid sequence within a nucleic acid molecule that is intended to be bound and/or bound by an element a gene regulation agent of the present disclosure (e.g., a KRAB element, a Zfn element, etc.). In some embodiments, a binding site is a site at which an element of an agent, e.g., a gene regulation agent. In some embodiments, a KRAB element (or portion thereof) binds to a specific binding site. In some embodiments, Zfn element (or part thereof) binds to a specific binding site. In some embodiments, a binding site is intended to be sequence-specific, but does not have to have 100% complementarity with an agent that binds to a binding site. For example, overall binding at a binding site is sequence-specific, which means that there is substantial sequence specificity of a given element for a particular binding site. For instance, for a given element to bind at a binding site, in some embodiments, there may be at least 15 nucleotides that are sequencespecific although the 15 nucleotides do not necessarily need to be contiguous with one another to confer specificity.

[0075] As used herein the term “associated” refers to a relationship of two events or entities with one another as related to presence, level, degree, type and/or form. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and/or form correlates with incidence of, susceptibility to, severity of, stage of, etc. the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof. For example, in some embodiments, a target sequence is associated with a gene if modification, in some way, of that target sequence impacts a particular gene. In some embodiments, a protein such as an RNA polymerase is associated with a transcript when it is actively transcribing mRNA from a polynucleotide. In some such embodiments, a disruption in the association causes a dissociation of the RNA polymerase from the transcript and subsequent degradation of any partially transcribed mRNA. In some embodiments, a polymeric modification agent (e.g., a gene regulation agent, e.g., a specificity-modified Zfn-KRAB molecule) is associated with one or more of a binding site, landing site, target site, target cell, target sequence, and/or target. In some embodiments, two events or entities may become dissociated from one another when their associated is disrupted or terminated.

[0076] As used herein, the term “epigenetic modification” refers to a modification that alters DNA accessibility and chromatin structure, thereby regulating gene expression. In some embodiments, an epigenetic modification has a direct impact on gene expression. In some embodiments, an epigenetic modification has an indirect impact on gene expression.

[0077] As used herein the term “KRAB domain” refers to a polynucleotide or polypeptide that corresponds to a sequence from a Kriippel associated box (KRAB).

[0078] As used herein, a “D-element” or “D domain” refers to a sequence-specific polynucleotide (e.g., DNA) binding element, as described in as described in in PCT/US2021/37113, which is herein incorporated by reference in its entirety. In some embodiments, a “D element” can be or comprise a naturally occurring sequence (e.g., represented by a polynucleotide) or a characteristic portion thereof, or a complement of a naturally occurring sequence or a characteristic portion thereof. In some embodiments, a D element can be or comprise one or more engineered (i.e., synthetic) nucleotides or characteristic portion(s) thereof. In some such embodiments, an engineered sequence (e.g., a sequence substantially composed of synthetic or engineered nucleotides) is analogous or corresponds to a naturally occurring sequence; however, any given engineered sequence is “produced by the hand of man.” In some embodiments D elements can include one or more of Zinc Finger proteins or domains, TALE-proteins or domains, Helix-loop-helix proteins or domains, Helix-turn-helix proteins or domains, Cas-proteins or domains (e.g., Cas9, dCas9, etc.), Leucine Zipper proteins or domains, beta-scaffold proteins or domains, Homeo-domain proteins or domains, High- mobility group box proteins or domains or characteristic portions thereof or combinations and/or parts thereof. Without being bound by any particular theory the present disclosure considers that, in some embodiments, a dissociation constant of 10E-6 or lower may confer sufficient binding strength for a given D element to bind and/or stay bound to a particular sequence.

[0079] As used herein, the term “KRAB-DLR” or “KRAB-DLRR” refers to a molecule that is or comprises at least one KRAB element and a DLR or DLRR molecule, as described in PCT/US2021/37113, which is herein incorporated by reference in its entirety. In some embodiments, a KRAB-DLR molecule comprises from N-terminus to C-terminus: a KRAB element, a D element, and one or more R elements. In some embodiments, a KRAB-DLR optionally comprises an L element between a D element and an R element. In some embodiments, a KRAB-DLR is a KRAB-DLRR and comprises at least two R elements.

[0080] As used herein, the term “DLR molecule” is or comprises a polymeric molecule, which molecule comprises at least one D element, an optional L element, and at least one R element, capable of binding a nucleic acid molecule, as described in PCT/US2021/37113, which is herein incorporated by reference in its entirety. In some embodiments, a DLR molecule is arranged in the order D-L-R. In some embodiments, one or more of the D, L, and/or R elements are in an order different from D-L-R. In some embodiments, where more than one unit of any particular element is present, one of skill in the art will understand that a numeral may be used to indicate a number of a particular element, e.g., DL2R2 or DL2R2 or D(LR)2, indicates a D element with two L elements bound to the D and two R elements, wherein the R elements may each be bound to the same or different L element. In some embodiments, an arrangement may also be shown as R-L-D-L-R, which would indicate that a single D element has two separate L elements bound to it, each of which has an R element bound to the L element. In some embodiments, a single D element may have more than one L element and more than one R element bound at a given time. In some embodiments, a single L element may have two R elements bound at the same time. In some embodiments, an R element may have, at either end, a sequence that functions as a linker. For example, in some embodiments, a given R element may have a sequence at an N or C-terminus a sequence that functions as a linker such that a polymeric agent (e.g., DLR molecule) is represented as DLRn, where n may be, e.g., an L element. In some embodiments, a DLR molecule has an overall dissociation constant in the same order as the lowest dissociation constant of any given component of the molecule (e.g., of a D unit, e.g., of an R unit, etc.) For example, in some embodiments, a D element and an R element of a given DLR molecule may have dissociation constants of 10E-6 or less and 10E-3 or less, respectively and, in such embodiments, a dissociation constant of a DLR molecule would be consistent with the lowest dissociation constant of a component of the molecule.

[0081] As used herein, the term “gene regulation” refers to a process comprising a change in gene expression, including via changing transcription and/or translation of a target, target sequence and/or target site.

[0082] As used herein, the term “gene regulation agent” refers to an agent capable of regulating expression of target by repressing expression and/or activity of the target. In some embodiments, a gene regulation agent refers to a DLR, DLRR, KRAB-DLR, or modified Zinc Finger KRAB molecule, protein, or agent. In some embodiments, a gene regulation agent binds to a landing site associated with the target and represses expression of the target.

[0083] As used herein, the term “genomic engineering” refers to a process that involves deliberate modification of one or more characteristics of genetic material or one or more mechanisms for expressing genetic material.

[0084] As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. As will be understood to those of skill in the art, comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

[0085] As used herein, the term “landing site” or “target site” refers to a nucleic acid sequence to which a sequence-specific element (e.g., ZFn element, a modified ZFn element comprising at least one sequence-modified alpha helix.) is targeted (e.g., to bind to it). In some embodiments a landing site may overlap with a target site (e.g., have nucleotides that are part of both a landing site and a target site).

[0086] As used herein, the term “nucleic acid” refers to any element that is or may be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid may be incorporated into a polynucleotide chain via phosphodiester linkage. In some embodiments, nucleic acids are polymers of deoxyribonucleotides or ribonucleotides. In some such embodiments, deoxyribonucleotides or ribonucleotides may be synthetic oligonucleotides. As will be clear from context, in some embodiments, “nucleic acid” refers to an individual nucleic acid residue (e.g., a nucleotide and/or nucleoside); in some embodiments, “nucleic acid” refers to a polynucleotide comprising individual nucleic acid residues. In some embodiments, a polymer or deoxyribonucleotides and/or ribonucleotides can be single-stranded or double-stranded and in in linear or circular form. Polynucleotides comprised of nucleic acids can also contain synthetic or chemically modified analogues of ribonucleotides, in which a sugar, phosphate and/or base units are modified. In some embodiments, a “nucleic acid” is or comprises RNA; in some embodiments, the RNA is or comprises mRNA. In some embodiments, a “nucleic acid” is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. Alternatively or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and/or 5’-N- phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleoside analogs. In some embodiments, a nucleic acid comprises one or more modified sugars as compared with those in natural nucleic acids. In some embodiments, a polynucleotide is comprised of at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues. In some embodiments, a polynucleotide is or comprises a partly or wholly single stranded molecule; in some embodiments, polynucleotide is or comprises a partly or wholly double stranded.

[0087] As used herein, the term “polynucleotide” refers to any polymeric chain of nucleic acids. In some embodiments, a polynucleotide is or comprises RNA. In some such embodiments, the RNA is or comprises mRNA. In some embodiments, a polynucleotide is or comprises DNA. In some embodiments, a polynucleotide is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a polynucleotide is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a polynucleotide analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. Alternatively or additionally, in some embodiments, a polynucleotide has one or more phosphorothioate and/or 5’-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, a polynucleotide is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, a polynucleotide is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5 -methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2- aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 - propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a polynucleotide comprises one or more modified sugars (e.g., 2’ -fluororibose, ribose, 2’ -deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a polynucleotide has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, a polynucleotide is prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a polynucleotide is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long. In some embodiments, a polynucleotide is partly or wholly single stranded. In some embodiments, a polynucleotide is partly or wholly double stranded. In some embodiments, a polynucleotide has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In some embodiments, a polynucleotide has enzymatic activity.

[0088] As used herein, the term “polypeptide” refers to any polymeric chain of residues (e.g., amino acids) that are typically linked by peptide bonds. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at a polypeptide’s N-terminus, at a polypeptide’s C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications may be acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. In some embodiments, useful modifications may be or include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, a protein may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, a protein is antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.

[0089] As used herein the term “ZFn element” or “Zinc Finger element” refers to an element comprising at least five zinc finger arrays derived from a human Zinc Finger KRAB protein. In some embodiments, a ZFn element is a polynucleotide (e.g., DNA)-binding molecule (e.g., a macromolecule, e.g., an oligonucleotide, etc.)

[0090] As used herein, the term “R element” or “X2 element” refers to a polynucleotide (e.g., DNA)-binding molecule (e.g., a macromolecule, e.g., an oligonucleotide, etc.) (e.g., as described in PCT/US2021/37113, which is herein incorporated by reference in its entirety), that binds to a polynucleotide that is different, e.g., opposite, a strand to which a sequence-specific D element binds. In some embodiments, an R-element binds to an opposite DNA strand than to where a D element is bound (i.e., lagging strand). In some embodiments, an R element can bind in a sequence specific manner or it can bind in a non-sequence specific (e.g., positional, etc.) manner. In some such embodiments, an R element may bind to DNA, RNA, mRNA, etc. In some embodiments, an R element is present within the same molecule as a given D element, but the D element and R element may be bound to two separate molecules, e.g., two separate DNA molecules; for example, a D element may be bound to a leading strand at or near a replication fork and an R element may be bound to a lagging strand at or near a replication fork, but on a separate DNA molecule than where the D element of a given DLR molecule is bound. In some embodiments, an R element binds to a polynucleotide with sufficient affinity (e.g., a dissociation constant of at least 10E-3 or less) to slow or stall polynucleotide processing (e.g., DNA replication, e.g., transcription, e.g., translation). In some embodiments, an R element of a given DLR molecule binds less strongly than a D element of the same molecule. In some embodiments, an R and D element of a given DLR molecule bind with similar affinities. In some embodiments, an R element binds in a sequence-specific manner; in some such embodiments, an R element and a D element of a given DLR molecule may bind with similar affinities (e.g., dissociation constant of 10E-6 or less, etc.). In some embodiments sequence specific interaction can be achieved through similar means as described and provided for and by a D element, however, in any given DLR molecule binding of an R element is different from that of a D element in that can be different from a D element (e.g., D element: engineered zinc finger protein combined with an R-element that comprises a CAS-protein). In some embodiments nonsequence specific interaction of sufficient affinity can be achieved through structures that can interact through various interactions such as, e.g., phosphate backbone interactions and/or hydrophobic/Van der Waals interactions with a major and/or minor groove of a DNA molecule. In some embodiments an R element can combine elements that result in non-sequence specific and -sequence-specific interactions. In some such embodiments, non-sequence specific and sequence specific interactions occur sequentially. In some embodiments, non-sequence specific and sequence specific interactions occur substantially simultaneously. In some embodiments, an R element can be or comprise a naturally occurring sequence or characteristic portion thereof. In some embodiments, an R element can.be or comprise an engineered sequence or characteristic portion thereof. In some such embodiments, an engineered sequence is analogous or corresponds to a naturally occurring sequence; however, any given engineered sequence is “produced by the hand of man.” In some embodiments an R-element binds to one or more regions which may be or comprise a Zinc Finger protein or domain, TALE protein or domain, Helix-loop-helix protein or domain, Helix-turn-helix protein or domain, CAS protein or domains Leucine Zipper protein or domain, beta-scaffold protein or domain, Homeo-domain protein or domain, High-mobility group box protein or domain or a combination thereof. In some embodiments, R elements may be engineered or designed such that binding interactions between R elements and a polynucleotide are different from naturally occurring binding interactions (e.g., an R element may bind to an engineered lagging DNA strand, etc.). In some embodiments R elements have little to no sequence specificity; for example, in some embodiments, R elements can be engineered, designed or selected to have little or no sequence specificity (e.g., no nucleotide and/or amino acid specificity). For instance, in some embodiments R elements can be engineered or designed to have a three-dimensional structure that can bind a given polynucleotide molecule (e.g., a DNA molecule) in a non-sequence specific manner. In some such embodiments such a structure can be based on a structural feature (e.g., fold) that may be present in a naturally occurring protein (e.g., polymerases, DNases, etc.) that interacts with a given polynucleotide (e.g., DNA, mRNA, etc.). In some embodiments specific amino acids are changed (as compared to those in a naturally occurring protein), for example an amino acid that may be involved in an active site may be changed such that the catalytic function is reduced and/or abolished. In some embodiments R elements are designed that are hybrids of naturally occurring folds and/or designed folds. In some embodiments, non-sequence specific binding by R elements can occur via one or more types of interactions known to those of skill in the art; for example, interactions of an R-element with a sugar phosphate backbone of a molecule to which it binds, hydrophobic interactions involving a minor or major groove of a DNA molecule to which an R-element binds or interacts, etc. As will be appreciated by one of skill in the art, such interactions are generally not explicitly sequence-specific, per se.

[0091] As used herein, the term “sample” refers to a portion or aliquot of a material obtained or derived from a source of interest, as described herein. In some embodiments, a source of interest is a biological or environmental source. In some embodiments, a source of interest may be or comprise a cell or an organism, such as a microbe, a plant, or an animal (e.g., a human). In some embodiments, an organism is a pathogen (e.g., an infectious pathogen, e.g., a bacterial pathogen, a viral pathogen, a parasitic pathogen, etc.). In some embodiments, a source of interest is or comprises biological tissue or fluid. In some embodiments, a biological tissue or fluid may be or comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humour, vomit, and/or combinations or component(s) thereof. In some embodiments, a biological fluid may be or comprise an intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, and/or a transcellular fluid. In some embodiments, a biological fluid may be or comprise a plant exudate. In some embodiments, a biological tissue or sample may be obtained, for example, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g., brocheoalveolar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage). In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, a sample is a primary sample in that it is obtained directly from a source of interest by any appropriate means. In some embodiments, as will be clear from context, a sample refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, processing a sample for testing to extract genetic material for genetic analyses such as by, e.g., applying one or more solutions, separating components using a semi- permeable membrane, etc. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques such as amplification or reverse transcription of nucleic acid, isolation and/or purification of certain components, etc. In some embodiments, a sample is used to design one or more KRAB-X-ZFn molecules and/or one or more KRAB-Xi-Zfn-Y-X2 molecules as provided herein.

[0092] As used herein, the term “sequence-specific binding” refers to an event that occurs when a macromolecule (e.g., a protein, peptide, polypeptide, nucleotide comprising protein) interacts with a polynucleotide (e.g., DNA, RNA, mRNA, etc.), and at least a sub-set (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of contacts between a macromolecule and a polypeptide is sequence-specific in that expected portions of each molecule interact with one another (e.g., Arginine interacting with Guanidine; other exemplary interactions will be known to those of skill in the art and can be found, for instance, in various descriptions throughout the literature describing DNA recognition codes for zinc fingers). As is understood by those of skill in the art, not every interaction between every portion of each molecule needs to be sequence specific; however the overall interaction between two molecules interacts, generally, in a manner that is sequence-specific. In some embodiments, an overall dissociation constant for interaction will be 10E-6 or less. As will be appreciated by those of skill in the art, a smaller dissociation constant indicates stronger binding. In some embodiments, sequence-specific binding will entail interaction in which at least three base pairs or nucleotides are bound with sufficient affinity and selectivity, such that other sequences will be bound at levels less than 50% of a desired or targeted DNA sequence.

[0093] As used herein, the term “subject” refers to an organism. In some embodiments, a subject is an individual organism. A subject may be of any chromosomal gender and at any stage of development, including prenatal development. In some embodiments a subject is comprised of, either wholly or partially, eukaryotic cells (e.g., an insect, a fly, a nematode). In some embodiments, a subject is a vertebrate. In some embodiments, a subject is a mammal. In some embodiments, a mammal is a human, including prenatal human forms. In some embodiments, a subject is suffering from a relevant disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and/or therapy is and/or has been and/or will be administered.

[0094] As used herein, the term “target” refers to a particular gene, region (e.g., promoter, enhancer, UTR, etc.), genomic target, or other location or component in a cell that is impacted by a gene regulation agent of the present disclosure. For example, in some embodiments, a target is a gene or genomic region and a gene regulation agent modifies expression of the target. In some embodiments, a target is a cell complex such as a polymerase and polynucleotide; for example, an RNA polymerase and strand of DNA and/or mRNA. A target may or may not be or comprise a landing site or a binding site or a portion thereof. In some embodiments, a target is or comprises a target sequence and/or target site. A target may or may not comprise a non-methylated, partially-methylated, or wholly-methylated region.

[0095] As used herein, the term “target cell” or “targeted cell” refers to a cell that has been contacted with at least one gene regulation agent (e.g., a KRAB-X-ZFn molecule or, e.g., a KRAB-Xi-Zfn-Y-X2 molecule). In some embodiments, a target cell comprises at least one nucleic acid change at a target site as compared to the same cell prior to the application of the at least one gene regulation agent, or, in some embodiments, as compared to another targeted cell or an untargeted cell. In some embodiments, a target cell does not comprise a nucleic acid change at a target site as compared to an untargeted cell. In some embodiments, a targeted cell may have one or more nucleic acid differences as compared to an untargeted cell, but is still not an edited cell as the one or more differences may not be at or within a target site. A targeted cell may or may not be an edited cell. In some embodiments, a targeted cell is an edited cell in that its nucleic acid sequence has been successfully edited in a specific and intended way, e.g., reflecting a designed genetic change. In some embodiments, a targeted but unedited cell and/or an untargeted cell may have one or more genetic changes as compared to an earlier version of a cell or a control. For example, in some embodiments, one or more SNPs may be detected but such SNPs may not be in a vicinity of a target site. In some embodiments, a target cell comprises a reduced level of transcription and/or mRNA of a target as compared to a cell that has not been contacted by a gene regulation agent.

[0096] As used herein, the terms “treat” or “treatment” refer to any technology as provided herein that is used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition. In some embodiments of the present disclosure a treatment may be or comprise changing a genotype in a subject. In some embodiments, treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and/or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition. In some embodiments, treatment refers to administration of a therapy (e.g., composition, pharmaceutical composition, e.g., gene regulation agent, e.g., a KRAB-X-Zfn molecule or, e.g., a KRAB-Xi-Zfn-Y-X2 molecule) that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition. In some embodiments, such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition. Thus, in some embodiments, treatment may be prophylactic; in some embodiments, treatment may be therapeutic.

[0097] As used herein, “Zinc Finger KRAB proteins” or “ZF KRAB proteins” or “ZF KRAB molecules” refer to proteins or molecules that comprise at least one human KRAB- domain and at least one human zinc finger.

[0098] As used herein, the term “Zinc Fingers” refer to small protein structures or motifs in which a zinc atom is present that helps to provide coordination and stability to the peptide fold. In some embodiments Zinc Fingers can be naturally occurring molecules, in some other embodiments zinc fingers can be designed. In some embodiments, Zinc Fingers are derived from human zinc finger proteins (e.g., a human Zinc Finger KRAB protein).

[0099] As used herein, the term “Zinc Finger Linkers” refer to amino acid sequences that connect Zinc Finger domains within a molecule. In some embodiments, Zinc Finger Linkers are derived from human zinc finger proteins (e.g., a human Zinc Finger KRAB protein).

[0100] As used herein, “modified Zinc Finger KRAB proteins” or “engineered Zinc Finger KRAB proteins” or “modified Zinc Finger KRAB molecules” or “engineered Zinc Finger KRAB molecules” or “modified Zinc Finger KRAB agent” or “engineered Zinc Finger KRAB agent” or “sequence specificity-modified Zinc Finger KRAB molecules” all refer to human Zinc Finger KRAB proteins or molecules, in which amino acids in one or more zinc fingers have been changed (e.g., substituted, inserted, deleted, etc.) relative to a reference (e.g., wild type) sequence. Amino acid sequence modifications include those that change or modify the DNA sequence specificity of such a protein or molecule, and include modifications in one or more Zinc Finger alpha helices.

DETAILED DESCRIPTION

[0101] Genomic engineering and gene regulation hold great promise. For instance, many types of gene regulation could be useful in treating one or more diseases, disorders or conditions. Genomic engineering and gene regulation offer an advantage that, in some embodiments, they can be very precise. The present disclosure recognizes that an ideal approach to gene regulation would encompass features such as being (1) safe and with few to no off-target effects; (2) versatile ability to regulate any gene and all types of gene variants (e.g., differences relative to wild-type) to a desired level of expression; (3) not be immunogenic, such that repeat dosing is possible when appropriate, desired or required; (4) operate at the DNA expression level, instead of having to suppress and/or interfere with a multitude of mRNA copies or other type of RNA molecules; and (5) be sufficiently effective to be of practical use. None of the currently existing methods for gene regulation fulfills all five criteria.

[0102] The present disclosure, among other things, provides insights and technologies for regulating expression of a target (e.g., mRNA associated with a disease state) with a gene regulation agent comprising molecules that include KRAB elements and bind to landing sites associated with the target. The present disclosure, among other things provides an insight that a gene regulation agent is capable of efficiently generating a reduced level of nucleic acid expression, for example, at a target site (or through interaction therewith), while limiting formation of immunogenic responses and, accordingly, provide increased safety for development of therapies applicable for use in human subjects.

[0103] The present disclosure appreciates that one challenge with currently available gene regulation approaches is that a number of current methods act by interfering at the RNA level. Such Inhibition may also occur at the mRNA level, e.g. by providing molecules that have “antisense” chemistry and that thereby can bind to a specific mRNA and reducing the amount of translated protein that can be produced in a cell. A wide variety of antisense technologies have been developed. Inhibition or repression at the DNA level is preferred, as it is more efficient than repression at the mRNA level. Typically a cell will only contain a few copies of a specific gene, while a gene can be transcribed many times resulting in a higher number of RNA molecules to be repressed than the number of DNA molecules that need to be repressed

[0104] Repression technologies that target the DNA or gene level exist. They include e.g. the usage of specific Peptide Nucleic Acid (PNA) molecules or artificial fusion molecules that combine a DNA-recognition domain with a repression domain.

[0105] While PNA repression can be used to repress certain genes, this approach suffers from a number of drawbacks: (1) PNA molecules are synthetic and cannot be produced in vivo. This prevents the possibility to use PNAs in applications where in vivo generation is beneficial or desired. As illustration, for example such application an could e.g. involve generation of repression molecules in the Central Nervous System, for example when suppression of alpha- synuclein build-up is required. (2) PNAs are synthetic molecules that are foreign to the body. This may imply that upon repeat dosing an immune reaction may be build up. Repeat dosing may be a desired feature when considering suppression over a longer time period. (3) PNA design limitations require amongst others that a target DNA sequence contains a long homopurine or homopyrimidine sequence, which further limits its versatility.

[0106] While certain fusion proteins comprising a DNA recognition domain and a gene repression domain (e.g. a KRAB-domain) have been described in literature, they suffer from limitations that greatly limit their use in development of human therapies. For example, certain fusion proteins include CAS9 and/or TALE DNA binding domains, which are of bacterial origin and are large structures. These proteins have significant immunogenic potential that limits the use fusion proteins with these domains for human therapies. This is particularly important for therapeutic context where repression over long time periods is requires within a human body. In such cases, it is likely that exposure to such a large foreign protein will start to elicit an immune reaction. This limits usage of such fusion proteins to single dosing, and/or short-term use, thereby significantly limiting their application scope.

[0107] Another class of fusion proteins comprises an artificial or designed zinc finger array coupled to a KRAB repressor domain. While these fusion proteins may reduce the risk of an immunogenic response, they still suffer from a number of important limitations: (1) In general they require fusing two or more domains and create at the fusion site none-naturally occurring amino acid sequences (for humans) that can create new linear epitopes that can become immunogenic upon extended exposure or repeat dosing in human therapies; (2) In specific examples said fusion proteins use additional functionalities, such as FLAG-tags or NLS sequences, which can create additional antigenic epitopes; (3) These fusion proteins may not allow a naturally occurring 3D folding, which may be required for optimal bio-molecular functioning; (4) These fusion proteins may lack other parts of naturally occurring human proteins, and those missing parts may have important functionality, even if these aspects are not fully understood; (5) Zinc fingers have been reported to interact with each other’s binding. Using zinc fingers within a naturally occurring protein scaffold can help with their proper orientation. As illustration, one indication of this limitation is that many naturally occurring human Zinc Finger KRAB proteins often use canonical five-amino-acid-long “TGEKP-linkers” (or variations thereof), whereas artificial zinc finger arrays often have to use modified linkers. The use of longer linkers has been described as needed to compensate for difference in phasing of the zinc fingers relative to the target DNA. The apparent need for “linker engineering” further complicates development of useful zinc finger arrays. This hindrance can be especially cumbersome when longer zinc finger arrays need to be created, that can target longer DNA recognition sequences. In addition, a need for “engineered” linkers has an inherent risk of deviating from naturally occurring peptide sequences, thus increasing potential immunogenicity.

[0108] The present disclosure recognizes that, among other things, it would be advantageous to be able to use naturally existing human Zinc Finger KRAB proteins and only having to make minimal changes to the amino acids that are directly or mostly involved in determining DNA sequence specificity of such a protein.

[0109] The present disclosure recognizes that, among other things, it would be advantageous to be able to use a naturally existing human Zinc Finger KRAB protein and only having to change amino acids residues that are present in alpha helix structure within zinc fingers of said protein.

[0110] Whilst Zinc Finger KRAB proteins with altered DNA sequence specificity have been developed, an important limitation of the current state of technology is that, given the size and complexity of the human genome, a recognition sequence of at least 15-17 nucleotides or preferably a longer recognition sequence of 18 or more nucleotides is required, when specific loci in a genome need to be targeted. Current technologies do not enable one to design and/or change DNA recognition properties of five or more zinc fingers concurrently, without having to make further modifications, for example to change zinc finger linker sequences, or for example by having to use zinc finger sequences from pre-selected zinc fingers, that are different (at least in part) of the zinc finger sequences in said human Zinc Finger KRAB protein. The current disclosure recognizes, amongst other things, that such additional changes may increase the immunogenic properties of said molecules and limit the desired ability for repeat dosing for certain therapeutic approaches. [oni] There is an unmet need is for a new approach that allows converting existing human Zinc Finger KRAB proteins or molecules by only making modifications to zinc finger alpha helix amino acids, to alter its desired or intended DNA target sequence specificity.

[0112] It would be beneficial to have a versatile technology that can target any gene, which uses human Zinc Finger KRAB protein scaffolds that can be modified to target (a) specific gene(s) and that are not immunogenic.

[0113] The present disclosure provides innovative technologies that are designed, among other things, to overcome limitations of current technologies. For example, in some embodiments, methods of the present disclosure are designed to function using a human Zinc Finger KRAB protein scaffold that is not immunogenic, that contains a naturally occurring repressor function and that can be designed to bind to a desired target site in a genome. For example, in some embodiments changes made to naturally occurring human Zinc Finger KRAB proteins are limited to specific amino acid substitutions in zinc finger alpha helix structures. For example, in some embodiments, a human Zinc Finger KRAB protein selected to be modified will have a naturally occurring number of zinc fingers that corresponds to a desired target sequence specificity, in which each zinc finger is designed to target 3 nucleotides. As illustration, a Zinc Finger KRAB protein containing nine zinc fingers may be used to target a sequence of up to 27 nucleotides (9 zinc fingers, each targeting 3 nucleotides allows a 27 nucleotides long target sequence).

[0114] The present disclosure appreciates that one challenge with currently available Zinc Finger KRAB protein development approaches is that measuring effects of a newly designed or experimentally new Zinc Finger KRAB protein may be challenging, especially when during the first stages of experimental design effects on repression can be relatively small. For example, as illustration, a (very) low gene repression effect obtained with a newly developed Zinc Finger KRAB protein (e.g., a modified Zinc Finger KRAB protein) may be caused by many experimental conditions, including, but not limited to properties of the KRAB domain, properties of the gene involved, properties of a zinc finger array used, properties of a specific cell line used for evaluation and/or combinations thereof. The present disclosure recognizes that, amongst other things, it would be advantageous to be able to test or evaluate properties of zinc fingers used, to confirm their suitability in targeting a desired DNA sequence. In some embodiments, a gene editing system can confirm suitability of targeting a particular DNA sequence. In some embodiments, an RITDM (Replication Interrupted Template driven DNA Modification or Recombination Induced Template Driven DNA Modification, as described in as described in PCT/US2021/37113) can be used to confirm suitability and/or efficacy of targeting of a particular DNA sequence. As illustrated in Example 1, a RITDM gene editing system can be used to confirm suitability of zinc fingers used, to target a specific gene. In some embodiments, as illustrated in Example 1, RITDM gene editing of KRAS may be used to test zinc finger suitability for usage in a gene regulation agent for KRAS. As may be appreciated by those skilled in the art, a gene editing test can be set up to provide sensitive gene editing diagnosis, e.g. by next generation or deep sequencing. As illustrated in Example 1, even relatively low-level gene editing frequencies in the order of a few percent can be detected. As illustrated in Example 1, zinc fingers used in a RITDM gene editing system can be used in subsequent gene regulations agents, e.g., gene regulation agents used to reduce expression of KRAS.

[0115] Thus, as described herein, the present disclosure provides technologies (e.g., systems, agents, methods, etc.) related to gene/genome regulation and/or genomic engineering. As will be appreciated by those of skill in the art, such technologies have a wide array of applications.

Transcription regulation by gene regulation agents

[0116] The present disclosure recognizes that, among other things, it would be advantageous to be able to achieve gene and/or genome regulation using naturally occurring human repressor proteins that have a designed DNA target specificity. As provided herein, technologies of the present disclosure are based upon the discovery that gene or genome regulation can be performed using newly developed modified Zinc Finger KRAB proteins that can achieve gene regulation or genome regulation. For example, in some embodiments the present disclosure provides one or more agents to achieve such gene or genome regulation.

[0117] In some embodiments, the present disclosure, among other things, provides a gene regulation agent including a structure of KRAB - X - ZFn, wherein a KRAB element is or comprises a KRAB domain or portion thereof; the X element is optional and is or comprises a functional domain; and the ZFn element is or comprises at least five zinc finger arrays, wherein at least one of the zinc finger arrays comprises at least one modified alpha helix, wherein the at least one modified alpha helix is engineered to comprise a modified amino acid sequence in that it differs from that of its corresponding wild type sequence. In some embodiments, a gene regulation agent is engineered by combination of various elements providing a sequence-specific DNA binding activity at a target sequence in a genome.

[0118] Without being bound by any particular theory, the present disclosure contemplates that binding of a gene regulation agent (e.g., comprising a modified Zinc Finger KRAB protein) to a given genomic target will slow down or stall DNA transcription. For example, as will be appreciated by one of skill in the art, Figure 1 illustrates a schematic of DNA transcription. Generally, during DNA transcription information encoded within genomic DNA sequences will be converted into RNA molecules, e.g. into mRNA molecules. In such cases, contacting transcription is actively occurring, a gene regulation agent described herein can inhibit (e.g., block) transcription, thereby reducing expression of a target.

[0119] Without being bound by any particular theory, the present disclosure contemplates that binding of a gene regulation agent (e.g., comprising a modified Zinc Finger KRAB protein) to a given genomic target may result in cellular processes that lead to a change in its chromatin structure, thereby changing the ability of a given cell to transcribe gene(s) in such a chromosomal section. In such cases, a change in chromatin structure in a region of the genome associated with a target results in repression of the target through means typically associated with repressed chromatin (e.g., increased DNA methylation, particular histone methylation patterns, etc.).

[0120] For example, the present disclosure contemplates that binding of a gene regulation agent (e.g., comprising a modified Zinc Finger KRAB protein) may block binding of requisite transcriptional machinery (e.g., RNA polymerase or e.g., transcription factors). In such cases, a gene regulation agent can block a transcription factor (e.g., TFIID) from binding to the TATA box. As RNA polymerase most often requires transcription factor binding in order to bind to and initiate transcription a gene regulation that blocks binding of a transcription factor thereby blocks transcription initiation. [0121] Accordingly, the present disclosure provides the insight that developing technologies (e.g., systems, compositions, methods) to temporarily block, slow or stall a polynucleotide process, (e.g., transcription) and/or its invoking changes in chromatin structure impacts the expression of specific genes. Thus, for example, in some embodiments, stalling of DNA transcription results in a lower amount of mRNA being produced. Thus, for example, in some embodiments, changes in chromatin structure may result in reduced DNA transcription, which results in a lower amount of mRNA being produced.

[0122] As is provided herein, in some embodiments, the present disclosure describes the development of gene regulation agents (e.g., comprising modified Zinc Finger KRAB molecules).

[0123] Thus, by way of non-limiting example, in some embodiments, the present disclosure provides a gene regulation agent (e.g., comprising a modified Zinc Finger KRAB protein) that can bind strongly enough to a DNA target sequence such that a RNA polymerase complex is temporarily blocked, slowed or stalled.

[0124] Thus, by way of non-limiting example, in some embodiments, the present disclosure provides a gene regulation agent (e.g., comprising a modified Zinc Finger KRAB protein) that can bind strongly enough to a DNA target sequence such that chromatin structure is modified and DNA transcription is reduced, slowed, stalled or prevented from initiating.

[0125] The present disclosure also recognizes that one challenge limiting genomic engineering is difficulty in precisely targeting gene regulation approaches. For example, in some embodiments, the present disclosure provides technologies that specifically target a gene regulation agent to a precise location in order to down-regulate a particular activity such as gene transcription.

[0126] Consistent with technologies of the present disclosure as described herein, another key aspect is ability to achieve gene regulation without having to modify a polynucleotide (e.g., a gene) sequence. For example, in some embodiments the present disclosure provides one or more agents to achieve such gene regulation. In some embodiments, an agent is a gene regulation agent (e.g., a modified Zinc Finger KRAB molecule). [0127] In some embodiments a cell is contacted with a gene regulation agent (e.g., comprising a modified Zinc Finger KRAB molecule) to genomically engineer a target. For example, in some embodiments, a modified Zinc Finger KRAB protein is capable of binding to a polynucleotide that is being transcribed. In such embodiments, the binding or association of the modified Zinc Finger KRAB molecule with a polynucleotide disrupts activity of, for example, an RNA polymerase, resulting in dissociation of tRNA polymerase and subsequent breakdown of a partially transcribed mRNA. In some such embodiments, a modified Zinc Finger KRAB protein is capable of annealing or otherwise associating to a polynucleotide and disrupting or interfering with transcription at a target site, e.g., in a genome. In some embodiments a gene regulation agent, e.g., a modified Zinc Finger KRAB molecule will be administered to a cell.

[0128] As will be understood by those of skill in the art, gene transcription is a process by which genetic information encoded in a polynucleotide (e.g., a strand of DNA) is copied into messenger RNA (mRNA). Transcription is carried out by an enzyme called RNA polymerase (RNAP) along with one or more accessory proteins called transcription factors, collectively referred as transcriptional machinery (Hahn, S., Nat. Struct. Mol. Biol. 2004; 11 : 394-403, which is herein incorporated by reference in its entirety). As depicted in Figure 1, transcription is initiated and RNAP moves along a DNA strand and begins mRNA synthesis by matching complementary bases to those of the DNA. Once mRNA is completely synthesized, transcription terminates. Newly formed mRNA copies of a gene then serve as blueprints for protein synthesis during the process of translation.

[0129] As will also be understood by those of skill in the art, RNAP progression may pause, stall, or be otherwise disrupted upon encountering any number of situations or “roadblocks” during movement of the polymerase along the DNA strand. A potential consequence of a stalled, paused, or otherwise disrupted RNAP activity is that transcription can be terminated immaturely, resulting in ineffective or incomplete mRNA synthesis. Generally, incomplete mRNA will not result in protein synthesis and, if it does, will not produce full-length or functional protein. Rather, it is more likely that RNAP disruption and dissociation from the DNA strand will result in mRNA that gets degraded.

[0130] The present disclosure provides, among other things, technologies to perform gene regulation (e.g., suppress gene expression, e.g., by site specific disruption of transcription) using a gene regulation agent (e.g., comprising a modified Zinc Finger KRAB molecule). Without being bound by any particular theory, the present disclosure contemplates that such a molecule may be further modified to increase DNA binding capacity and, thus, used to impact one or more aspects of gene regulation. For example, in some embodiments, the present disclosure contemplates that combining site-specific targeting with strengthened binding of a modified Zinc Finger KRAB proteins by adding one or more additional zinc fingers to such a molecule, will facilitate gene regulation (e.g., via disruption of transcription, e.g., by interference with transcriptional processes). For example, in some embodiments, one, two or three zinc fingers can be tethered together to enhance DNA binding. Linked zinc fingers can be used for gene regulation application can be multiples of the same or different zinc fingers. Thus, by way of non-limiting example, in some embodiments, when a modified Zinc Finger KRAB protein binds to a specific polynucleotide (e.g., DNA) target, it can block gene transcriptional complexes, interfering with RNAP progression along a polynucleotide (e.g., a gene), thereby disrupting transcription and ultimately reducing mRNA transcript levels.

[0131] In some embodiments, a gene regulation agent (e.g., comprising a modified Zinc Finger KRAB protein) can bind to a target site of a polynucleotide (e.g., in a genome). During gene expression, contact of a cell by such a molecule such as a modified Zinc Finger KRAB molecule with increased or altered DNA binding capacity, can create a situation where RNAP encounters a modified Zinc Finger KRAB molecule bound to DNA at the target site. By way of non-limiting example, the modified Zinc Finger KRAB molecule can then block the RNAP from continuing to transcribe the DNA. Without being bound by any particular theory, the present disclosure contemplates that upon transcription interruption, incompletely transcribed mRNA can then be subject to degradation. As a consequence, transcribed full-length mRNA from a target is reduced. Figure 1 depict mRNA transcription in the absence of exemplary modified Zinc Finger KRAB molecules. Figure 2 illustrates mRNA transcription of a DNA strand by RNAP. Figure 2 illustrates an exemplary gene regulation agent binding to a target sequence, thereby obstructing RNAP from moving along the same DNA strand. Consequently, in the presence of a gene regulation agent, transcription is down-regulated as evidenced by reduced mRNA transcripts detected (see, e.g., Example 1). [0132] Accordingly, the present disclosure provides the insight that developing technologies (e.g., systems, compositions, methods) to slow, stall, or otherwise disrupt a polynucleotide process such as transcription can regulate a gene in a sequence-specific manner to specifically reduce mRNA transcription of one or more targets. Thus, for example, in some embodiments, disruption of RNAP activity from a DNA strand that is being transcribed results in reduced mRNA production, which may, in some embodiments, reduce protein levels and/or function of one or more genes.

[0133] The present disclosure recognizes that, among other things, it would be advantageous to be able to achieve precise control over genetic activities (e.g., genomic engineering, e.g., gene regulation, e.g., gene transcription). To implement such programmed gene regulation at a target, gene regulation agents (e.g., comprising one or more modified Zinc Finger KRAB molecules) are introduced into cells in formats of DNA plasmids, RNA molecules, and/or proteins with or without modifications.

[0134] As described and demonstrated herein, in some embodiments, gene regulation agents can be used to modify and/or regulate one or more targets. For instance, without being bound by any particular theory, the present disclosure contemplates that gene regulation agents can change (e.g., slow, disrupt, terminate) transcription. Surprisingly, when gene regulation agents are designed and engineered in certain ways, they can achieve targeted programmed gene regulation (e.g., suppressing transcription or e.g. changing chromatin structure) at desired target loci and/or desired genes. For example, in some embodiments, gene regulation agents can be used to suppress or silence transcription. That is, without wishing to be bound by any particular theory, the present disclosure contemplates that a gene regulation agent can interfere with transcription during gene expression and/or alter chromatin structure. For instance, in some embodiments, a gene regulation agent can interfere, in a sequence-specific manner, with RNA polymerase activity and cause an RNA polymerase to dissociate from a polynucleotide strand, thus causing mRNA production to stop and result in breakdown of incompletely transcribed mRNA. For instance, in a sequence-specific manner, the binding of a gene regulation agent may result in chromatin structure changes, thus causing changes in the accessibility of genomic DNA sequences.

Compositions [0135] The present disclosure, among other things, provides compositions comprising one or more gene regulation agents. In some embodiments, a composition comprises a gene regulation agent as described herein (e.g., a modified Zinc Finger KRAB molecule, a KRAB- DLR, a KRAB-DLRR, etc.). In some embodiments, a gene regulation agent (e.g., a modified Zinc Finger KRAB molecule (e.g., a KRAB-X-ZFn molecule and/or a KRAB-Xi-ZFn-X2-Y molecule), a KRAB-DLR, a KRAB-DLRR, etc.) reduces, eliminates, lowered, and/or blocks gene expression. In some embodiments, a gene regulation agent of the present disclosure is a blocking agent, In some embodiments, a composition comprises one or more gene regulation agents that block expression of a nucleotide sequence (e.g., transcriptional blocking) as described herein. In some embodiments, a gene regulation agent of the present disclosure is a transcription modification agent. In some embodiments, a composition comprises one or more gene regulation agents that reduce, impair, or eliminate transcription. In some embodiments, a gene regulation agent of the present disclosure is an inhibiting agent (e.g., an agent that inhibits transcription and/or expression). In some embodiments, a composition comprises one or more gene regulation agents that inhibit transcription.

[0136] In some embodiments, a composition comprises a plurality of gene regulation agents. In some embodiments, a composition comprises at least 2 gene regulation agents. In some embodiments, a composition comprises a plurality of gene regulation agents comprising at least one modified Zinc Finger KRAB molecule and at least one of a DLR molecule, a DLRR molecule, a KRAB-DLR molecule, and a KRAB-DLRR molecule.

[0137] The present disclosure, among other things, provides a gene regulation agent comprising molecules that include a KRAB element, an optional linker element (e.g., an X, Xi and/or Y element), and a ZFn element. In some embodiments, a gene regulation agent is or comprises a Zinc Finger KRAB protein that includes a ZFn element including at least one modified alpha helix (e.g., the at least one modified alpha helix is engineered to comprise a modified amino acid sequence in that it differs from that of its corresponding wild type sequence). In some embodiments, a gene regulation agent is or comprises a KRAB domain fused to a DLR molecule (e.g., a DLR molecule as described in PCT/US2021/37113, which is herein incorporated by reference in its entirety). Modified Zinc Finger KRAB molecules

[0138] In some embodiments, a gene regulation agent comprises a modified Zinc Finger KRAB molecule. This disclosure provides, among other things, modified Zinc Finger KRAB molecules, e.g., Zinc Finger KRAB proteins. In some embodiments, Zinc Finger KRAB proteins include a KRAB element (e.g., any of the exemplary KRAB elements described herein), an optional X element (e.g., a linker), and a ZFn element (e.g., a ZFn that comprises at least five zinc finger arrays, wherein at least one of the zinc finger arrays comprises at least one modified alpha helix, wherein the at least one modified alpha helix is engineered to comprise a modified amino acid sequence in that it differs from that of its corresponding wild type sequence).

[0139] In some embodiments, a Zinc Finger KRAB protein refers to a class of molecules or domain structures that can act to repress gene transcription. A Zinc Finger KRAB (Kruppel Associated Box). The present disclosure encompasses a recognition that a Zinc Finger KRAB protein structure typically exists in nature as part of a larger protein or molecule in which a part of the molecule has a DNA binding function (e.g., one or more zinc finger arrays) that can target the repressing part of the molecule (e.g., a KRAB domain) to a specific genomic locus.

[0140] In some embodiments, Zinc Finger KRAB molecule may be derived from one or more naturally existing human Zinc Finger KRAB proteins. In some embodiments, Zinc Finger KRAB proteins may be non-naturally existing or designed protein or molecules. In some embodiments, changes may have been made to naturally existing human Zinc Finger KRAB molecules, for example, in some embodiments specific amino acid substitutions in alpha helices of (a) zinc finger(s) may have been made, for example to change the desired DNA sequence binding specificity of such a molecule. In some embodiments, Zinc Finger KRAB proteins may comprise zinc finger arrays that are continuous, for example, individual zinc fingers may be connected through five amino acid long canonical “TGEKP” linkers (or variants thereof); in some other embodiments, Zinc Finger KRAB proteins may comprise zinc finger arrays that are discontinuous, for example the may comprise zinc fingers that are separated by amino acid sequences of six amino acids or longer.

[0141] In some embodiments, a modified Zinc Finger KRAB molecule has or comprises a structure set forth as KRAB-X-ZFn. The present disclosure also provides, among other things, methods of making and using disclosed agents and/or molecules. In some such embodiments, a gene regulation agent (e.g., comprising a modified Zinc Finger KRAB molecule) reversibly binds to double-stranded DNA, in a sequence specific manner (see, e.g., Figure 2). In some embodiments, a gene regulation agent (e.g., comprising a modified Zinc Finger KRAB molecule) comprises at least two elements: at least one “KRAB” and at least one “ZFn”, with an optional “X” element. In some embodiments, a gene regulation agent (e.g., comprising a modified Zinc Finger KRAB molecule) may be ordered with KRAB, X, and ZFn elements placed consecutively. Thus, as described herein, in some embodiments, a gene regulation agent (e.g., comprising a modified Zinc Finger KRAB molecule) can be schematically represented as KRAB-X-ZFn or ZFn-X-KRAB.

[0142] In some embodiments, a given gene regulation agent may have more than one each of a given KRAB, X, or ZFn element. For example, in some embodiments, a KRAB element may be fused or otherwise connected to one or more X elements, which may each be fused or otherwise connected to one or more ZFn elements. In some embodiments, a given KRAB-X-ZFn molecule may have two ZFn elements, three ZFn elements, four ZFn elements or more. In some embodiments, a given KRAB-X-ZFn molecule may have two X elements, three X elements, four X elements, or more. In some embodiments, a KRAB-X-ZFn molecule may be schematically represented as, e.g., KRAB-X-ZFn; KRAB-X-ZFn-X; KRAB-X-ZFn-X-ZFn, etc.

[0143] In some embodiments, a ZFn element is comprised of multiple components or DNA binding elements. For example, in some embodiments, a ZFn element is “hybrid” comprising zinc-finger components and additional sequences. As provided herein, with regard to a KRAB-X-ZFn molecule, “ZFn” is a domain comprising a sequence-specific DNA binding element that binds to DNA; “X” is an optional linker element between segments “KRAB” and “ZFn”; and “KRAB” is a second domain that comprises a domain that can result in reduced expression of a certain gene or transcription element. In some embodiments, a KRAB element is or comprises a polypeptide that interacts with different cellular components than a ZFn element. In some embodiments, a KRAB element is bound to a polypeptide or polynucleotide on a different molecule as a ZFn element of a single KRAB-X-ZFn molecule. In certain aspects the three elements are able to be reversibly bound (element ZFn) or associated to a polynucleotide (e.g., DNA, e.g., RNA) molecule. [0144] In some embodiments, a KRAB-X-ZFn molecule may be or comprise a polypeptide. In some such embodiments, where a KRAB-X-ZFn is a polypeptide, a KRAB element can be located at either an N-terminal or C-terminal portion of a polypeptide, with a ZFn-element located at an opposite location (e.g., C-terminal or N-terminal location). In some embodiments, where a KRAB-X-ZFn molecule (e.g., polypeptide) comprises one or more X elements, such X elements are located in between or adjacent to KRAB elements and ZFn elements.

[0145] As described herein, technologies provided by the present disclosure (e.g., systems, methods, compositions, etc.) achieve one or more modifications (e.g., in expression) at one or more target sites. Accordingly, for example, in some embodiments, a gene regulation agent (e.g., comprising a modified Zinc Finger KRAB molecule) binds at a target site in a target genome wherein a ZFn element binds to a DNA double helix in a sequence-specific. Then, when DNA is being transcribed, such a modified Zinc Finger KRAB molecule is designed that it can interfere with transcription progression at a target site (e.g., via stalling or slowing).

Accordingly, for example, in some embodiments, a modified Zinc Finger KRAB molecule binds at a target site in a target genome wherein a ZFn element binds to a DNA double helix in a sequence-specific, resulting in cellular processes involving its KRAB domain, can result in genomic modifications that result in reduced expression. Thus gene regulation can be accomplished (see, e.g., Example 1).

KRAB Elements

[0146] This disclosure provides, among other things, gene regulation agents (e.g., modified Zinc Finger KRAB molecules and/or KRAB-DLR molecules) that include a KRAB element. In some embodiments, gene regulation agent is a modified Zinc Finger KRAB molecule that comprises at least one KRAB element.

[0147] In some embodiments, a modified Zinc Finger KRAB protein mediates repression through a KRAB domain and mediates DNA binding through one or more zinc finger arrays. In some embodiments, a KRAB domain may be combined with other naturally or non-naturally occurring protein sequences or structures. [0148] In some embodiments, a KRAB element comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 1.

[0149] In some embodiments, the KRAB element comprises or consists of an amino acid sequence of:

MDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQL TKPDVILRLEKGEEP (SEQ ID NO: 1).

[0150] In some embodiments, a KRAB element comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 2.

[0151] In some embodiments, the KRAB element comprises or consists of an amino acid sequence of: MNNSQGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLVSVGQGETTKPDV ILRLEQGKEPWLEEEEVLGSGRAEKNGDIGGQIWKPKDVKESLAREVPSINKETLTTQKG VECDGSKKILPLGIDDVSSLQHYVQNNSHDDNGYRKLVGNNPSK (SEQ ID NO: 2).

[0152] In some embodiments, the KRAB element comprises an amino acid sequence that differs by no more than 10 amino acids, no more than 9 amino acids, no more than 8 amino acids, no more than 7 amino acids, no more than 6 amino acids, no more than 5 amino acids, no more than 4 amino acids, no more than 3 amino acids, no more than 2 amino acids, or no more than 1 amino acid from a sequence of SEQ ID NO: 2.

[0153] In some embodiments, a KRAB element comprises a KRAB-A domain that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 3.

[0154] In some embodiments, the KRAB-A domain comprises or consists of an amino acid sequence of:

GSSGSSGVTYDDVHMNFTEEEWDLLDSSQKRLYEEVMLETYQNLTDIGYNWQDHHIEE SGPSSG (SEQ ID NO.: 3).

[0155] In some embodiments, a KRAB element comprises a KRAB-A domain that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 4.

[0156] In some embodiments, the KRAB-A domain comprises or consists of an amino acid sequence of: MNNSOGRVTFEDVTVNFTOGEWORLNPEQRNLYRDVMLENYSNLVSVG (SEQ ID NO: 4); underlined amino acids are consensus amino acids. In some embodiments, a KRAB element comprises a KRAB-A domain that comprises a sequence that differs by no more than 5 amino acids, no more than 4 amino acids, no more than 3 amino acids, no more than 2 amino acids, or no more than 1 amino acid from a sequence of SEQ ID NO: 4. In some embodiments, such differing amino acids do not comprise a consensus amino acid. For example, in some embodiments, a KRAB element comprises a sequence that differs by 3 amino acids from a sequence of SEQ ID NO.: 4, wherein said 3 differing amino acids are not consensus amino acids.

[0157] In some embodiments, a KRAB element comprises a KRAB-A domain and a KRAB-B domain where the KRAB element comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 5.

[0158] In some embodiments, the KRAB element comprises or consists of an amino acid sequence of: MNNSOGRVTFEDVTVNFTOGEWORLNPEORNLYRDYMLENYSNLVSVGOGETTKPDV ILRLEQGKE (SEQ ID NO: 5), wherein underlined amino acids are consensus amino acids.

[0159] In some embodiments, a KRAB element comprises a KRAB-A domain a KRAB- B domain, wherein said KRAB element comprises a sequence that differs by no more than 5 amino acids, no more than 4 amino acids, no more than 3 amino acids, no more than 2 amino acids, or no more than 1 amino acid from a sequence of SEQ ID NO: 5. In some embodiments, such differing amino acids do not comprise a consensus amino acid. For example, in some embodiments, a KRAB element comprises a sequence that differs by 2 amino acids from a sequence of SEQ ID NO.: 5, wherein said 2 differing amino acids are not consensus amino acids.

[0160] In some embodiments, a KRAB-B domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 6.

[0161] In some embodiments the KRAB-B domain comprises or consists of an amino acid sequence of : KPD VILRLEQGKE (SEQ ID NO: 6), wherein underlined amino acids are consensus amino acids.

[0162] In some embodiments, a KRAB element comprises a KRAB-B domain, wherein said KRAB element comprises a sequence that differs by no more than 5 amino acids, no more than 4 amino acids, no more than 3 amino acids, no more than 2 amino acids, or no more than 1 amino acid from a sequence of SEQ ID NO: 6. In some embodiments, such differing amino acids do not comprise a consensus amino acid. For example, in some embodiments, a KRAB element comprises a sequence that differs by 2 amino acids from a sequence of SEQ ID NO.: 6, wherein said 2 differing amino acids are not consensus amino acids.

X elements

[0163] In some embodiments, a gene regulation agent comprises an X element, where the X element comprises a linker. In some embodiments, a linker is or comprises a zinc finger linker. In some embodiments, a linker comprises an amino acid sequence of about 1 amino acid to about 20 amino acids.

[0164] In some embodiments, a linker is or comprises a polynucleotide. For example, in some embodiments, a polynucleotide linker is between about 2 to about 500 nucleic acid in length.

[0165] In some embodiments, a linker includes a polypeptide. For example, in some embodiments, a polypeptide linker is between 2 and 100 amino acids in length.

[0166] In some embodiments, a gene regulation agent (e.g., a Zinc Finger KRAB molecule) may comprise a “canonical” five amino acid sequence TGEKP (SEQ ID NO: 7). In some embodiments, a linker (e.g., a zinc finger linker) may be a variation of a five amino acid canonical linker, e g. TGERP (SEQ ID NO: 8), or TGDKP (SEQ ID NO: 9) or TGQKP (SEQ ID NO: 10). In some embodiments, a linker (e.g., a zinc finger linker) may comprise six amino acids or more. In some embodiments, a linker (e.g., a zinc finger linker) contains the same amino acid sequence as present in a human zinc finger protein. In some embodiments, a linker (e.g., a zinc finger linker) may be designed to be different from a naturally present linker. In some embodiments, a linker (e.g., a zinc finger linker) may be a non-canonical linker.

[0167] Exemplary X elements (e.g., linkers) include, without limitation, those listed in

Table 1 Table 1. Exemplary X Elements.

[0168] In some embodiments, a linker comprises a sequence that differs by no more than two amino acids from a sequence of any one of SEQ ID NOs: 7 to 21. In some embodiments, a linker comprises a sequence that differs by no more than one amino acid from a sequence of any one of SEQ ID NOs: 7 to 21. In some embodiments, a linker comprises or consists of a sequence of any one of SEQ ID NOs: 7 to 21.

ZFn elements

[0169] The present disclosure, among other things, provides one or more ZFn elements, wherein at least one of the zinc finger arrays of the ZFn element comprises at least one modified alpha helix (e.g., the at least one modified alpha helix is engineered to comprise a modified amino acid sequence in that it differs from that of its corresponding wild type sequence). For example, in some embodiments, a gene regulation agent comprising a ZFn element as described herein includes at least one zinc finger array that includes at least one modified alpha helix. The present disclosure provides, among other things, that a gene regulation agent comprising a ZFn having at least one zinc finger array that includes at least one modified alpha helix is effective at regulating expression of a target. [0170] A large number of naturally occurring proteins containing zinc fingers exist in nature. In many of these proteins zinc fingers are involved in some type of interaction with nucleic acids and/or other proteins. Protein chemistry and crystal structure experiments have elucidated many aspects of zinc finger structures and mechanisms by which they can bind to other molecules. An archetypical zinc finger structure that is often involved in DNA binding and DNA sequence recognition, comprises an alpha-helix structure with two anti-parallel beta-sheets that are oriented into a three-dimensional confirmation by a coordinating zinc atom. In these structures said zinc-atom interacts with cysteine and/or histidine amino acid side chains.

Specific amino acid side chains protrude from an alpha helix structure and these amino acids side chains are involved in (preferential) sequence specific binding (Choo and Klug, 1994, Proc Natl AcadSci USA, 91 : 11163-11167; Elrod-Erickson, et al., 1996, Structure 4 1171-1180, each of which is herein incorporated by reference in its entirety).

[0171] In some embodiments, zinc finger proteins have an ability to be used as modular units of approximately 30 amino acids, with each unit potentially able to bind to a DNA-triplet sequence. In some embodiments, zinc finger proteins have been combined into arrays of two or more zinc fingers, thus allowing for larger DNA sequences (i.e., additional DNA triplets) to be recognized and bound by Zn fingers/Zn-containing proteins (Choo and Klug, 1994, Proc Natl Acad Sci USA 91 : 11168-11172, which is herein incorporated by reference in its entirety).

[0172] Many sequence specific interactions between zinc fingers and DNA are known in the art. A number of studies have described how specific amino acid side chains in specific positions of alpha helices of zinc fingers allow for either more-specific or less-specific interactions binding to specific nucleotides in a DNA molecule (Klug, 2010, Annu. Rev. Biochem. 79 213-231, which is herein incorporated by reference in its entirety). Accordingly, such features may be incorporated when designing zinc finger units or zinc finger containing domains. Thus, in some embodiments, the present disclosure provides agents that incorporate zinc fingers and/or one or more features of zinc fingers that can be used to design or develop agents or approaches that preferentially recognize specific DNA sequences (Choo and Klu,.

1997, Curr. Opin. Struct. Biol. 7: 117-125; Klug, 2005, Proc. Japan Acad. 81 : 87-102; Sera and Uranga, 2002, Biochemistry 41 : 7074-7081, Zhu, et al., 2013. Nucleic Acids Res. 41 : 2455-2465, each of which is herein incorporated by reference in its entirety). [0173] In some embodiments, zinc fingers can influence behavior of adjacent zinc fingers. Accordingly, a series of preselected and pretested zinc finger dimers have been described (Isalan, et al., 1997, Proc Natl Acad Set USA 94: 5617-5621; Moore, et al., 2001, Proc Natl Acad Sci U S A 98: 1437-1441, each of which is herein incorporated by reference in its entirety) and a number of methods for the evaluation of interactions can be found in literature (Isalan, et al., 1998, Biochemistry 37: 12026-12033, which is herein incorporated by reference in its entirety). Thus, in some embodiments, when designing or selecting zinc finger arrays for use in one or more technologies of the present disclosure, such interactions, dimers, and/or methods can be taken into consideration.

[0174] The present disclosure recognizes that zinc finger array design principles as are known in the art may not always be sufficient to accurately predict how well a given zinc finger array will work for a given purposes, e.g., when designing a modified Zinc Finger KRAB molecule. Accordingly, among other things, the present disclosure provides agents and assays that may be used to design, evaluate and optimize zinc finger arrays for use in accordance with the present disclosure.

[0175] In some embodiments Cysteine and/or Histidine amino acid side-chains interact with the zinc atom. Zinc finger structure can function, amongst others, in protein-DNA interaction. As illustration, in some embodiments specific zinc finger amino acid side-chains may interact with DNA or other polynucleotides. In some embodiments Zinc Finger - DNA interactions can be dependent on a DNA nucleotide sequence, in other embodiments interactions may be non-sequence specific, e.g. as illustration by interacting with a DNA backbone. In some embodiments Zinc Finger motifs comprise an alpha helix. In some such embodiments, specific amino acids comprised in an alpha helix may interact preferentially with specific DNA nucleotides. As illustration, in some embodiments amino acid positions in a Zinc Finger alpha helix may be numbered. In some embodiments specific amino acids at specific alpha helix positions may have a preferential binding to a specific nucleotide (A, C, G or T) in a DNA molecule. As illustration, an Arginine amino acid at position +6 in a zinc finger alpha helix may preferentially bind to a G-nucleotide in a DNA target sequence.

[0176] In some embodiments, a gene regulation described herein includes a ZFn element including at least five, six, seven, eight, nine, ten, or eleven zinc finger arrays. In some embodiments, a Zfn element comprises one, two, three, or four zinc finger arrays. In some embodiments, a Zfn element comprises 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 zinc finger arrays.

[0177] In some embodiments, a Zfn element includes at least one zinc finger arrays that includes at least one alpha helix engineered to include a modified amino acid sequence that differs from that of its corresponding wild type sequence. For example, in some embodiments, a zinc finger array comprises (i) one amino acid substitution mutation at a position selected from - 1, +1, +2, +3, +4, +5, or +6 in the alpha helix; (ii) two amino acid substitution mutations at positions selected from -1, +1, +2, +3, +4, +5, or +6 in the alpha helix; (iii) three amino acid substitution mutations at positions selected from -1, +1, +2, +3, +4, +5, or +6 in the alpha helix; (iv) four amino acid substitution mutations at positions selected from -1, +1, +2, +3, +4, +5, or +6 in the alpha helix (v) five amino acid substitution mutation at positions selected from -1, +1, +2, +3, +4, +5, or +6 in the alpha helix; (vi) six amino acid substitution mutations at positions selected from -1, +1, +2, +3, +4, +5, or +6 in the alpha helix; or (vii) an amino acid substitution mutation at each position in the alpha helix, wherein the one or more amino acid substitutions in the alpha helix differ from that of its corresponding wild type sequence.

[0178] In some embodiments, human Zinc Finger KRAB protein sequences that contain six zinc finger structures or sequences can be used, in which a number of zinc finger alpha helix amino acids are substituted and/or replaced by different amino acids. In some such embodiments target sequences for such modified Zinc Finger KRAB molecules will be altered compared to the original Zinc Finger KRAB molecules. For example, human Zinc Finger KRAB proteins ZNF 434, ZKSCAN1, ZKSCAN2, ZNF 552, ZNF 562, ZNF 763 or ZNF 793 may be used.

[0179] In some embodiments, human Zinc Finger KRAB protein sequences that contain seven zinc finger structures or sequences can be used, in which a number of zinc finger alpha helix amino acids are substituted and/or replaced by different amino acids. In some such embodiments target sequences for such modified Zinc Finger KRAB molecules will be altered compared to the original Zinc Finger KRAB molecules. For example, human Zinc Finger KRAB proteins ZNF 177, ZNF 233, ZNF 248, ZNF 554, ZNF 566, ZNF 577, ZNF 701, ZNF 812 or ZNF 891 may be used. [0180] In some embodiments, human Zinc Finger KRAB protein sequences that contain eight zinc finger structures or sequences can be used, in which a number of zinc finger alpha helix amino acids are substituted and/or replaced by different amino acids. In some such embodiments target sequences for such modified Zinc Finger KRAB molecules will be altered compared to the original Zinc Finger KRAB molecules. For example, human Zinc Finger KRAB proteins AC003682.1, ZNF 3, ZNF 124, ZNF 506, ZNF 525, ZNF 561, ZNF 610, ZNF 625, ZNF 766, ZNF 222 or ZNF 223 may be used.

[0181] In some embodiments, human Zinc Finger KRAB protein sequences that contain nine zinc finger structures or sequences can be used, in which a number of zinc finger alpha helix amino acids are substituted and/or replaced by different amino acids. In some such embodiments target sequences for such modified Zinc Finger KRAB molecules will be altered compared to the original Zinc Finger KRAB molecules. For example, human Zinc Finger KRAB proteins ZIK 1, ZNF 324, ZNF 510, ZNF 519, ZNF 643, ZNF 773, ZNF 486, ZNF 732, ZNF 776 or ZNF 582 may be used.

[0182] In some embodiments, human Zinc Finger KRAB protein sequences that contain ten zinc finger structures or sequences can be used, in which a number of zinc finger alpha helix amino acids are substituted and/or replaced by different amino acids. In some such embodiments target sequences for such modified Zinc Finger KRAB molecules will be altered compared to the original Zinc Finger KRAB molecules. For example, human Zinc Finger KRAB proteins ZNF 154, ZNF 37A, ZNF 468, ZNF 563, ZNF 614, ZNF 649, ZNF 677, ZNF 682, ZNF 689, ZNF 727, ZNF 730 or ZNF 578 may be used.

[0183] In some embodiments, human Zinc Finger KRAB protein sequences that contain eleven zinc finger structures or sequences can be used, in which a number of zinc finger alpha helix amino acids are substituted and/or replaced by different amino acids. In some such embodiments target sequences for such modified Zinc Finger KRAB molecules will be altered compared to the original Zinc Finger KRAB molecules. For example, human Zinc Finger KRAB proteins ZNF 155, ZNF 181, ZNF 253, ZNF 415, ZNF 416, ZNF 479, ZNF 485, ZNF 570, ZNF 675, ZNF 79 or ZIM 3 may be used. [0184] In some embodiments, a gene regulation agent includes exemplary human zinc finger arrays and corresponding alpha helices selected from in Table 2, wherein underlined amino acids indicated Cys and His residues that coordinate zinc finger residues, bolded amino acids indicate the alpha helix, and the space indicates one or more intervening amino acids.

Table 2. Exemplary Human Zinc Finger Arrays and Alpha Helices

[0185] In some embodiments, human Zinc Finger KRAB protein sequences that contain twelve or more zinc finger structures or sequences can be used, in which a number of zinc finger alpha helix amino acids are substituted and/or replaced by different amino acids. In some such embodiments target sequences for such modified Zinc Finger KRAB molecules will be altered compared to the original Zinc Finger KRAB molecules.

[0186] In some embodiments, human Zinc Finger KRAB protein sequences can be used, in which a number of zinc finger alpha helix amino acids are substituted and/or replaced by different amino acids. Furthermore, in some embodiments additional human zinc finger may be tethered to such a human Zinc Finger KRAB protein. In some such embodiments target sequences for such modified Zinc Finger KRAB molecules will be altered compared to the original Zinc Finger KRAB molecules.

KRAB-DLR Fusion Proteins

[0187] In some embodiments, a gene regulation agent is or comprises a fusion protein comprising a KRAB element and a DLR molecule or a DLRR molecule, where the DLR and DLRR molecules are as described in PCT/US2021/37113, which is herein incorporated by reference in its entirety. In some embodiments where a gene regulation comprises a fusion protein comprising a KRAB element and a DLR molecule or a DLRR molecule, the gene regulation agent may have or comprise a structure set forth as KRAB-Xi-ZFn-Y-X2. The present disclosure also provides, among other things, methods of making and using disclosed agents and/or molecules. In some such embodiments, a gene regulation agent (e.g., a gene regulation agent having a structure of KRAB-Xi-ZFn-X2-Y) reversibly binds to double-stranded DNA, in a sequence specific manner (see, e.g., Figure 2). In some embodiments, a gene regulation agent (e.g., a gene regulation agent having a structure of KRAB-Xi-ZFn-Y-X2) comprises at least three elements: at least one “KRAB”, at least one “ZFn”, at least one “X2” element with optional “Xi” and “Y” elements. In some embodiments, a gene regulation agent (e.g., a gene regulation agent having a structure of KRAB-Xi-ZFn-Y-X2) may be ordered with KRAB, XI, ZFn. Y, and X2 elements placed consecutively. Thus, as described herein, in some embodiments, a gene regulation agent (e.g., a gene regulation agent having a structure of KRAB-Xi-ZFn-Y-X2) can be schematically represented as KRAB-Xi-ZFn-Y-X2 or X2-Y-ZFn-Xi-KRAB.

[0188] In some embodiments, a gene regulation agent (e.g., a gene regulation agent having a structure of KRAB-Xi-ZFn-Y-X2) may have more than one each of a given KRAB, Xi, ZFn, Y, or X2 element. For example, in some embodiments, a KRAB element may be fused or otherwise connected to one or more Xi elements, which may each be fused or otherwise connected to one or more ZFn elements, which may be fused or otherwise connected to one or more Y elements, which may be fused to or otherwise connected to one or more X2 elements. In some embodiments, a given KRAB-Xi-ZFn-Y-X2 molecule may have two KRAB elements, three KRAB elements, four KRAB elements, or more. In some embodiments, a given KRAB- Xi-ZFn-Y-X2 molecule may have two ZFn elements, three ZFn elements, four ZFn elements or more. In some embodiments, a given KRAB-Xi-ZFn-Y-X2 molecule may have two Xi elements, three Xi elements, four Xi elements, or more. In some embodiments, a given KRAB- Xi-ZFn-Y-X2 molecule may have two X2 elements, three X2 elements, four X2 elements, or more. In some embodiments, a given KRAB-Xi-ZFn-Y-X2 molecule may have two Y elements, three Y elements, four Y elements, or more.

[0189] In some embodiments, a ZFn element is comprised of multiple components or DNA binding elements. For example, in some embodiments, a ZFn element is “hybrid” comprising zinc-finger components and additional sequences. As provided herein, “ZFn” is a domain comprising a sequence-specific DNA binding element that binds to DNA; “Xi” and “Y” is are optional linker elements between segments “KRAB” and “ZFn” and “ZFn” and “X2”; and “KRAB” is a second domain that comprises a domain that can result in reduced expression of a certain gene or transcription element.

[0190] In some embodiments a KRAB-Xi-ZFn-Y-X2 molecule may be or comprise a polypeptide. In some such embodiments, where a KRAB-Xi-ZFn-Y-X2 is a polypeptide, a KRAB element can be located at either an N-terminal or C-terminal portion of a polypeptide, with a ZFn-element located at an opposite location (e.g., C-terminal or N-terminal location). In some embodiments, where a KRAB-Xi-ZFn-Y-X2 molecule (e.g., polypeptide) comprises one or more X elements, such X elements are located in between or adjacent to KRAB elements and ZFn elements. Methods of Using

[0191] Among other things, the present disclosure provides methods and compositions for carrying out targeted gene regulation such as, e.g., suppression of transcription. The present disclosure provides technologies that, in contrast to previously disclosed methods for gene repression, are making use of modified human proteins that are not immunogenic and can be used for repeated usage, which may be important for continued or prolonged therapeutic effect. The present disclosure provides the insight that such technologies reduce risks of creation of unwanted immunogenic responses, while being able to achieve the pharmaceutically desired effect of gene repression of target genes. In some embodiments, any gene or expressed segment of nucleic acid in a genome of a cell or organism can be targeted in accordance with technologies (e.g., methods) of the present disclosure.

Gene Conversion and Modification

[0192] In some embodiments as described herein, gene conversion and/or modification may be used to confirm binding specificity of a gene regulation agent of the present disclosure, as described in PCT/US2021/37113, which is herein incorporated by reference in its entirety. In some embodiments, provided is a method of producing a gene regulation agent, wherein the method comprises a step of confirming target specificity of a gene regulation agent of the present disclosure using a genetic modification assay, e.g., RITDM gene editing.

Gene Regulation

[0193] In some embodiments, gene regulation may or may not comprise genetic modification. In some embodiments, gene regulation is or comprises downregulation (e.g., silencing, suppression, repression). For example, in some embodiments, gene regulation is accomplished by interfering with one or more components of gene transcription. That is, in some embodiments, gene regulation occurs when a gene regulation agent binds to a particular location on a polynucleotide that is being transcribed. In some such embodiments, the association between the polynucleotide being transcribed and the RNA polymerase is disrupted, thus disrupting and reducing a level of transcription of a target gene as supported by reduction in a level of mRNA of the target. Therefore, in some embodiments, gene regulation is or comprises gene downregulation. In some embodiments, gene regulation is indirect in that is actually is or comprises gene upregulation (e.g., enhancement, increased transcription, etc.). In some such embodiments, such regulation (i.e., upregulation) of a target gene may be achieved by, for example, using a gene regulation agent to downregulate another gene that silences or represses or otherwise inhibits expression, thus by downregulating the inhibitory component, upregulation occurs.

[0194] In some embodiments, a gene regulation agent system provides methods of a targeted genetic (e.g., DNA, RNA or chromatin) modification. As described herein, targeted genetic (e.g., DNA, RNA or chromatin) modifications are, but are not limited to, changes that result in reduced gene or genetic unit expression. In some embodiments, these methods may include transfection of a cell with any of the gene regulation agents described herein. In some embodiments, a gene regulation agent comprises a modified Zinc Finger KRAB molecule, a KRAB-DLR molecule, or a KRAB-DLRR in accordance with the present disclosure.

[0195] In some embodiments, a gene regulation agent is capable of efficiently generating a reduced level of nucleic acid expression at a target site, while limiting formation of immunogenic responses and, accordingly, provide increased safety for development of therapies applicable for use in human subjects.

[0196] In some embodiments, contacting a cell with, or administering to a subject, a gene regulation agent occurs prior to or concomitant with a change in chromatin structure, wherein the change in chromatin structure reduces the rate and/or level of transcriptional activity of the target. In some embodiments, contacting a cell with, or administering to a subject, a gene regulation agent further comprises contacting the cell with one or more of a DNA or histone modification factors or any other factors associated with a DNA modification and a histone modification. In some embodiments, one or more DNA or histone modification factor(s), factors associated with DNA modifications, or factors associated with histone modifications are selected from: methylases, methyltransferase, histone (de-)acylating enzymes helicases, recombinases, repair scaffold proteins, single strand DNA binding proteins, mismatch repair proteins.

[0197] In some embodiments, provided are methods of reducing gene expression in a cell or population of cells. In some embodiments, provided are methods of gene regulation that include contacting of a cell or population of cells with a gene regulation agent of the present disclosure (e.g., a modified Zinc Finger KRAB molecule, a KRAB-DLR molecule, or a KRAB- DLRR). In some embodiments, provided methods include (i) contacting a cell or population of cells with a gene regulation agent (e.g., a modified Zinc Finger KRAB molecule, a KRAB-DLR molecule, or a KRAB-DLRR); and (ii) quantifying a level of transcription of a target (e.g., using RT-PCR, qPCR, in situ hybridization, or other method known in the art to quantify transcription).

[0198] In some embodiments, a level of transcription of a target is reduced relative to the level of transcription prior to the contacting with the gene regulation agent (e.g., a sample of the same population of cells prior the contacting). In some embodiments, a level of transcription of a target is reduced relative to comparable cell or population of cells that has not be contacted with the gene regulation agent (e.g., genetically identical cells cultured under the same or similar conditions). In some embodiments, a level of transcription of a target is reduced relative to a reference value.

[0199] In some embodiments, transcription of the target is reduced by at least 20%, at least 30%, at least 40%, at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, or more. In certain embodiments, transcription of the target is reduced by 50% or more.

[0200] In some embodiments, contacting of a cell or population of cells with a gene regulation agent further comprises contacting the cell with one or more of a DNA or histone modification factors or any other factors associated with a DNA modification and a histone modification. In some embodiments, where the one or more DNA or histone modification factor(s), factors associated with DNA modifications, or factors associated with histone modifications are selected from: methylases, methyltransferase, histone (de-)acylating enzymes helicases, recombinases, repair scaffold proteins, single strand DNA binding proteins, mismatch repair proteins.

[0201] In some embodiments, the contacting of a cell or population of cells is performed in vivo. In some embodiments, the contacting of a cell or population of cells is performed ex vivo. [0202] In some embodiments, provided are methods of gene regulation include: (i) contacting of a cell or population of cells with a modified Zinc Finger KRAB molecule, and (ii) quantifying a level of transcription of a target. In some embodiments, said contacting results in a reduction of transcription of a target sequence by at least 50%. In some embodiments, said contacting results in a sustained reduction of transcription of a target (e.g., for at least 3 days, 5 days, 7 days, 10 days, or more).

Target Sites and/or Landing Sites

[0203] In some embodiments, a gene regulation agent regulates expression of a target through binding to a landing site. In some embodiments, a landing site is or comprises one or more nucleotides. In some embodiments, a landing site is or comprises a target site. In some embodiments, a target sequence is or comprises one or more nucleotides. In some embodiments, a target sequence is modified by a change in its association with one or more other entities or elements. For example, in some embodiments, a target sequence is modified by a change that impacts gene regulation. For example, in some such embodiments, a target sequence is modified by dissociation of a protein (e.g., an RNA polymerase) from a transcript associated with or comprising a target sequence. That is, in some embodiments, a RNA polymerase is dissociated from a transcript that is associated, in some way, with a target sequence. In some embodiments, a target sequence is wholly naturally-occurring. In some embodiments, a target sequence is or comprises one or more synthetic nucleotides or components. In some embodiments, a target sequence is or comprises both naturally occurring and synthetic components (e.g., nucleic acid residues, etc.).

[0204] In some embodiments, a landing site is or comprises a nucleotide that is targeted for transcriptional regulation (e.g., repression of the target). In some embodiments, a landing site is a sequence-specific landing site. In some embodiments, a landing site is a structure specific landing site. In some embodiments, a landing site is both sequence and target specific. In some embodiments, a landing site is non-sequence and/or non-structure specific. In some embodiments, a landing site compromises a sequence associated with a disease, disorder or condition. In some embodiments, a landing site is or comprises a polynucleotide sequence, e.g., a DNA sequence, that comprises a point mutation associated with a disease, disorder or condition. In some such embodiments, a landing site may be or comprise an error site (e.g., a site where presence of one or more nucleotides is associated with existence, development or risk of a disease, disorder, or condition).

[0205] In some embodiments, a landing site comprises a small deletion, insertion and /or single nucleotide polymorphism within a coding sequence of a gene. In some embodiments, a landing site comprises more than one mutation, for example, a deletion and a point mutation wherein these two mutations are located adjacent to one another. In some embodiments, a deletion is associated with early termination of translation of a gene product (e.g., a protein) because of, e.g., generation of a premature stop codon and/or reading frame shift.

[0206] In some embodiments the landing site is or comprises all or a portion of a regulatory element. Non-limiting examples of regulatory elements include: a promoter, an enhancer, a silencer, an insulator, a TATA box, a GC box, a CAAT box, a transcriptional start site, a DNA binding motif of a transcription factor or other protein that regulates transcription, a 5’ untranslated region, and a ‘3 untranslated region. In some embodiments, by designing a gene regulation agent to bind to a regulatory element expression of a target gene is reduced, where the gene regulatory element regulates, at least in part, the transcription (i.e., expression) of the target.

[0207] In some embodiments, a landing site is or comprises all or a portion of a gene regulatory element. For example, in such cases, binding of a gene regulation agent to a gene regulatory element inhibits expression of a target. In some embodiments, a gene regulatory element includes one or more regulatory elements selected from: a promoter, an enhancer, a silencer, an insulator, a TATA box, a GC box, a CAAT box, a transcriptional start site, a DNA binding motif of a transcription factor or other protein that regulates transcription, a 5’ untranslated region, and a ‘3 untranslated region. For example, in some embodiments, a gene regulation agent binds to an enhancer thereby blocking or preventing a transcription factor or other transcriptional machinery from binding to an activating transcription of a target. Pharmaceutical Compositions

[0208] Pharmaceutical compositions of the present disclosure may include one or more gene regulation agents described herein. For example, in some embodiments, pharmaceutical compositions may comprise a gene regulation agent (e.g., comprising a modified Zinc Finger KRAB molecule, a KRAB-DLR molecule, or a KRAB-DLRR molecule). For example, a pharmaceutical composition of the present disclosure comprising one or more gene regulation agents (e.g., comprising a modified Zinc Finger KRAB molecule, a KRAB-DLR molecule, or a KRAB-DLRR molecule) as described herein, may be provided in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose, or dextrans; mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; and preservatives. In some embodiments, compositions of the present disclosure are formulated for intravenous administration. Any compositions described herein can be, e.g., a pharmaceutical composition.

[0209] In some embodiments, a composition, or, includes a pharmaceutically acceptable carrier such as, for example, phosphate buffered saline, saline, and/or bacteriostatic water (a “pharmaceutical composition”). Upon formulation, solutions will be administered in a manner compatible with a dosage formulation and in such amount as is therapeutically effective. Formulations are easily administered in a variety of dosage forms such as injectable solutions, injectable gels, drug-release capsules, and the like.

[0210] Compositions provided herein can be, e.g., formulated to be compatible with their intended route of administration. A non-limiting example of an intended route of administration is intravenous administration. In some embodiments, administration may occur ex vivo and cells may be provided post-administration, to a subject in need thereof.

[0211] In some embodiments, a pharmaceutical composition described herein is formulated for parenteral (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular) administration. In some embodiments, a pharmaceutical composition described herein is formulated for intravenous infusion or injection. In some embodiments, a pharmaceutical composition described herein is formulated for intramuscular or subcutaneous injection. Pharmaceutical compositions described herein can be formulated for administered by using infusion techniques that are commonly known in immunotherapy (See, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988, which is hereby incorporated by reference in its entirety).

[0212] As used herein, the terms “parenteral administration” and “administered parenterally” refer to modes of administration other than enteral and topical administration, usually by injection or infusion, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intratumoral, and intrastemal injection and infusion.

[0213] Also provided are kits including any compositions described herein. In some embodiments, a kit can include a solid composition (e.g., a lyophilized composition including at least one agent as described herein) and/or a liquid for solubilizing a lyophilized composition.

[0214] In some embodiments, a kit can include a pre-loaded syringe including any compositions described herein.

[0215] In some embodiments, a kit includes a vial comprising any of the compositions described herein (e.g., formulated as an aqueous composition, e.g., an aqueous pharmaceutical composition).

[0216] In some embodiments, a kit can include instructions for performing any methods described herein.

Cells

[0217] In some embodiments, the present disclosure provides technologies that can be used to contact one or more cells. In some embodiments, a cell is in vitro, ex vivo, or in vivo. In some embodiments, a cell (e.g., a mammalian cell) is autologous, meaning the cell is obtained, e.g., from a subject (e.g., a mammal) and cultured ex vivo.

[0218] In some embodiments, a cell is provided from a cell line, e.g., a stable cell line (e.g., HEK293, e.g., U937, etc.) In some embodiments, a cell is provided from a primary cell culture. In some embodiments, a cell is extracted from a subject in need of treatment. In some embodiments, cells are engineered to stably express exogenous genetic products. In some embodiments, a cell may be an artificial cell. In some embodiments, a cell may be an engineered cell.

[0219] In some embodiments, a cell is a human cell, a mouse cell, a porcine cell, a rabbit cell, a dog cell, a rat cell, a sheep cell, a cat cell, a horse cell, a non-human primate cell, or an insect cell.

[0220] In some embodiments, a cell is a stem cell. In some embodiments, a cell is a progenitor or precursor cell. In some embodiments, a cell is a differentiated cell. In some embodiments, a cell is a specialized cell type (e.g., a neuron, a cardiac cell, a kidney cell, an islet cell, etc.). In some embodiments, a cell is a post-mitotic cell (e.g., neuron).

[0221] In some embodiments, a host cell is transiently or non-transiently transfected with one or more vectors comprising a sequence encoding a gene regulation agent. In some embodiments, a cell is transfected in a substantially similar state as it occurs or exists in a subject. In some such embodiments, such a transfection may occur in vitro, ex vivo, or in vivo. In some embodiments, a cell is derived from one or more cells taken from a subject, such as development or a stable cell line and/or a primary cell culture. A wide variety of cell lines for tissue culture are known in the art. Examples of cells lines include, but are not limited to, HEK293 and U937. Cell lines are available from a variety of sources known to those with skill in the art, for example, the American Type Culture Collection (ATCC) (Manassas, VA, USA). In some embodiments, a cell transfected with one or more components of gene regulation agents as described as herein may be used establish a new cell line comprising one or more genetic modifications (e.g., any conceivable genetic modification including but not limited to loss-of- function, gain-of-function including one or more changes to create cellular models of known diseases, e.g., Alzheimer’s disease or various genotypically-characterized cancers, using, e.g., known pathological modifications, targeted gene regulation to change a level of transcription/gene expression, etc.) Methods of Making

[0222] In some embodiments, compositions, agents or systems of the present disclosure are prepared by any methods known to one of skill in the art. In some such embodiments, such preparations are formulated for delivery into a subject.

[0223] In some embodiments, compositions are prepared using any standard synthesis and/or purification system that will be known to one of skill in the art. For example, in some embodiments as described herein, one or more methods may include techniques such as de novo gene synthesis, DNA fragment assembly, PCR, mutagenesis, Gibson assembly, molecular cloning, standard single-stranded DNA synthesis, PCR, molecular cloning, digestion by restriction enzymes, small RNA molecule synthesis, cloning into plasmids with U6 promoter for RNA transcription, etc.

Design of modified Zinc Finger KRAB molecules

[0224] Among other things, the present disclosure provides technologies (e.g., systems, methods, compositions, etc.) such that gene regulation agents (e.g., comprising modified Zinc Finger KRAB molecules) can be designed. For example, in some embodiments as provided herein, a gene regulation agent (e.g., comprising a modified Zinc Finger KRAB molecule) may be designed using a known crystal structure of a (partially) homologous Zinc Finger KRAB molecule. In some other embodiments, as provided herein, gene regulation agent may be designed using known human Zinc Finger KRAB molecule sequences. As illustration, a zinc finger binding domain of murine ZFN568 can be used as homology model for human Zinc Finger KRAB protein ZIM3, as illustrated in Figure 3. As can be inferred from the crystal structure and published information an array of seven C-terminal zinc fingers can interact with DNA to create a specific recognition sequence. As illustrated in Example 1, the present disclosure shows that conversion of zinc finger alpha helices can be used to change the DNA recognition sequence of such a molecule, thus creating a modified Zinc Finger KRAB molecule.

[0225] In some certain embodiments, a target sequence for targeting human KRAS can comprise a sequence of: 5’- TTG-GAG-CTG-GTG-GCG-TAG-GCA -3’ (SEQ ID NO: 44) and is targeted by a zinc finger array that comprises a following zinc finger protein sequence: MNNSQGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLVSVGQGETTKPDV ILRLEQGKEPWLEEEEVLGSGRAEKNGDIGGQIWKPKDVKESLAREVPS1 KETLTTQKG VECDGSKKILPLGIDDVSSLQHYVQNNSHDDNGYRKLVGNNPSKFVGQQLKCNACRKL FSSKSRLQSHLRRH ACQKPFECHSC XJRAFGEKWKLDKHQK THAEERPYKCENCGN AYK QKSNLFQHQKMHTKEKPYQCKTCGKAFSWKSSCINHEKIHNAKKSYQCNECEKSFRQS GDLTRHKKVHTGQKPFQCTDCGKAFIRSDNLTTHQRIHTGEKPYKCSICEKAFSRSSDLT RHEKIHTGKRAYECDLCGNTFIRSDALTRHHKKIHTGEKPYECNRCGKAFFRSDALSEH

QKTHSGERTYRCSECGKTFIRSSNLTRHKKTHTGQKPYGCSECGKAFARSDALTTHQKR IHSR (SEQ ID NO: 50).

[0226] For example, in some embodiments, human Zinc Finger KRAB protein sequences can be used, in which a number of zinc finger alpha helix amino acids are substituted and/or replaced by different amino acids. In some such embodiments target sequences for such gene regulation agents (e.g., comprising one or more modified Zinc Finger KRAB molecules) will be altered compared to the original Zinc Finger KRAB molecules.

[0227] In some embodiments of the present disclosure human Zinc Finger KRAB proteins with continuous zinc finger arrays are used to design a modified Zinc Finger KRAB molecule.

[0228] As shown below, an exemplary ZF KRAB structure has zinc fingers connected through five amino acid long linker sequences to form a continuous zinc finger array (ZNF124; SEQ ID NO: 33). Histidine and Cysteine zinc interacting amino acids are shown in bold and underlined. Five amino acid zinc finger linkers are underlined.

[0229] MSGHPGSWEMNSVAFEDVAVNFTQEEWALLDPSQKNLYRDVMQETFRN LASIGNKGEDQSIEDQYKNSSRNLRHIISHSGNNPYGCEECGKKPCTCKQCQKTSLSVTR VHRDTVMHTGNGHYGCTICEKVFNIPSSFOIHQRNHTGEKPYECMECGKALGF SRSLN RHKRIHTGEKRYECKQCGKAF SRS SHLRDHERTHTGEKPYECKHCGKAFRYSNCLHY

HERTHTGEKPYVCMECGKAFSCLSSLQGHIKAHAGEEPYPCKOCGKAFRYASSLOKHE KTHIAQKPYVCNNCGKGFRCSSSLRDHERTHTGEKPYECOKCGKAF SRASTLWKHKK THTGEKPYKCKKM (SEQ ID NO: 33)

[0230] As shown below, an exemplary ZFn KRAB structure with a discontinuous zinc finger array in which some zinc fingers separated by more than five amino acids (ZNF597; SEQ ID NO: 34). Histidine and Cysteine zinc interacting amino acids are shown in bold and underlined. Five amino acid zinc finger linkers are underlined. An amino acid sequence interrupting a zinc finger array is shown in bold and italics

[0231] MASMPPTPEAQGPILFEDLAVYFSQEECVTLHPAQRSLSKDGTKESLEDA ALMGEEGKPEINQQLSLESMELDELALEKYPIAAPLVPYPEKSSEDGVGNPEAKILSGTPT YKRRVISLLVTIENHTPLVELSEYLGTNTLSEILDSPWEGAKNVYKCPECDQNFSDHSYL VLHQKIHSGEKKHKCGDCGKIFNHRANLRTHRRIHTGEKPYKCAKCSASFROHSHLSR HMNSHVKEKPYTCSICGRGFMWLPGLAOHQKSH dEATFEATWCDianWEinWLAZP EETFVSGPOYQHTKCMKSFRQSLYPALSEKSHDEDSERCSDDGDNFFSFSKFKPYQCPD CDMTFPCFSELISHONIHTEERPHKCKTCEESFALDSELACHQKSHMLAEPFKCTVCGK TFKSNLHLITHKRTHIKNTT* (SEQ ID NO: 34)

[0232] In some embodiments, for example when no suitable homology model is available, modified Zinc Finger KRAB molecules may be designed using an approach in which a desired (DNA) target sequence is selected, a desired zinc finger array length is selected and one or more human Zinc Finger KRAB molecules are selected for modification, which have the same number, or more, zinc fingers in their structure present as a continuous zinc finger array.

[0233] Without being bound by any particular theory, the present disclosure contemplates that when designing a modified Zinc Finger KRAB protein for a given genomic target, it may be useful to use a Zinc Finger KRAB molecule that has a continuous zinc finger array. In some embodiments such an array can be evaluated using a genomic modification assay, for example by using it in RITDM gene editing. In some embodiments such an array can be evaluated in other experimental assays.

Methods of Characterization

[0234] In some such embodiments, technologies of the present disclosure including a gene regulation as described herein, as will be understood by one of skill in the art given context, may be tested and/or characterized by one or more assays. For instance, by way of non-limiting example, in some embodiments, an agent of the present disclosure is tested as described in Example 1. [0235] In some embodiments gene repression can be demonstrated using reporter constructs such as by using fluorescence detection methods. By way of non-limiting example, the present disclosures contemplate that in some embodiments other types of reporter constructs can be used, such as, but not limited to reporters based on fluorescent detection, bioluminescence detection, the usage of antibiotics markers, markers that make use of antibody detection and/or use of a phenotypical feature.

[0236] In some embodiments, genomic engineering, can be demonstrated using RITDM- based validation and then gene repression assays as illustrated in Example 1, which allows for confirmation of targeting and confirmation of reduction in gene transcription.

[0237] In some embodiments, the present disclosure provides a programmed genomic engineering method, which may achieve gene modification through, for example, suppression of polynucleotide processing (e.g., transcription). Thus, in some embodiments, a transcriptional system in accordance with the present disclosure provides a specific method for targeted programmed gene regulation in cells, e.g., mammalian cells.

[0238] In some embodiments, methods in accordance with the present disclosure (e.g., gene regulation agent(s), e.g., transcriptional modification such as transcriptional suppression, with components and targets validated by RITDM) can be utilized in cell types in which a distinguishable sequence modification polynucleotide (e.g., donor template) can be efficiently analyzed if it has integrated into a targeted genome. Accordingly, in some embodiments, the present disclosure provides methods for evaluation of gene editing effects, e.g., on-target correction and off-targets mutations. In some embodiments, the present disclosure provides method for evaluation of gene regulation, e.g., suppression of gene transcription.

[0239] In some embodiments, analysis and/or identification of cells containing a desired genetic modification (e.g., gene repression) may be performed in a single cell, or in a population of cells (e.g., a batch of cells, e.g., several batches or pooled populations of cells, etc.).

[0240] In some embodiments, analysis and/or identification of cells containing a desired genetic modification may be performed in (a) specific clone(s).

[0241] In some embodiments, analysis and/or identification of cells containing a desired genetic modification may be performed using a digital PCR method. [0242] In some embodiments, analysis and/or identification of cells containing a desired genetic modification may be performed using a PCR method. In some embodiments, analysis and/or identification of cells containing a desired genetic modification may be performed using a Sanger Sequencing method. In some embodiments, analysis and/or identification of cells containing a desired genetic modification (e.g., gene conversion, e.g., transcript suppression, etc.) may be performed using a Next Generation Sequencing method. In some embodiments, analysis and/or identification of cells containing a desired genetic modification may be performed using any appropriate method to determine if one or more changes in one or more nucleotides has occurred. In some such embodiments, the present disclosure provides various methods of characterization, as described herein.

[0243] In some embodiments, analysis and/or identification of cells containing a desired genetic modification may be performed using an assay based on functionality.

[0244] In some embodiments, analysis and/or identification of cells containing a desired genetic modification may be performed using an assay based on phenotype.

[0245] In some embodiments, analysis and/or identification of cells containing a desired genetic modification (e.g., gene conversion, e.g., transcript suppression, etc.) may be performed using features of sequence modification polynucleotides (e.g., conversion polynucleotides) or other components that allow identification and potentially selection for converted cells. This may be done for example by making use of sequence modification polynucleotides (e.g., conversion polynucleotides) that contain a dye or chromophore or a chemical modification (e.g., biotin) that allows for detection.

[0246] In some such embodiments, prior to implementation of programmed gene regulation, genomic targeting capacity of a gene regulation agents may be tested via a RITDM system. In each test, components may comprise a DLR molecule and sequence modification polynucleotide. Detection of genetic conversion at a target gene is used to validate targeting capacity and specificity of a specific DLR molecule design, which, if successful, will then be used to use in the alteration and/or design of zinc fingers in a gene regulation agent to perform targeted gene regulation. In some embodiments, an agent (e.g., gene regulation agent) of this present disclosure is tested as described in Example 1. In some embodiments, DLR and/or gene regulation agents can be introduced into cells in forms of, but not limit to, DNA fragments, DNA plasmids, RNA with or without modification, and/or proteins.

[0247] In some embodiments, methods in accordance with the present disclosure can be utilized in cell types in which a targeted gene is actively transcribed into mRNA. Accordingly, in some embodiments, the present disclosure provides methods for suppressing targeted gene transcription by introduction of a gene regulation agent into cells, which may be validated by total RNA extraction and quantitation. For example, in some embodiments, total RNA is reversed transcribed into DNA, which is then used for templates for PCR reactions. These two processes are used together to perform reverse transcription-polymerase chain reaction RT-PCR, which, as is known to those of skill in the art, is a sensitive technique for mRNA detection and quantitation.

Genotyping and design of modified Zinc Finger KRAB molecules

[0248] In some embodiments, the present disclosure provides methods of making a change in genetic material (e.g., of a subject) based on analysis of a sample. For instance, in some embodiments, a sample is obtained. In some such embodiments, a sample may be tested to determine a genotype or change in expression level at one or more target sites and/or to determine a sequence or expression level of one or more target sequences using any number of methods known to those of skill in the art. In some embodiments, sequence analysis information is used to design and/or aid in selection of an appropriate gene regulation agent that can be used to change expression of a target. After analysis, a gene regulation agent may be introduced or administered such that it is has access to or contact with genetic material to which regulation of expression occurs.

[0249] In some embodiments, a sample is obtained or derived from a subject. In some embodiments, a subject is a control subject. In some embodiments, a subject has one or more diseases, disorders or conditions. In some embodiments, such a disease, disorder, or condition has one or more genetic changes associated therewith. In some embodiments, a subject is determined to have one or more genetic changes (e.g., genotype, expression level) associated with a particular disease, disorder or condition. [0250] In some embodiments, a subject does not have one or more genetic changes associated with a disease, disorder, or condition, but may have an acquired phenotype that would benefit from a change in expression level.

[0251] In some embodiments, a gene regulation agent is administered or introduced to a subject or sample derived therefrom, in need thereof. In some embodiments, a sample is acquired. In some embodiments, after acquisition, a sample may be optionally further processed (e.g., to purify, expand, test, etc.) to determine genotype and/or expression level information. In some embodiments, after genotypic information is determined, one or more gene regulation agents may be designed to modify one or more target sites and/or target expression levels.

[0252] In some embodiments, a gene regulation agent is administered or applied such that it contacts a polynucleotide (e.g., a polynucleotide comprising a target gene). In some embodiments, administration or application is ex vivo or in vitro. In some embodiments, administration or application is in vivo. In some embodiments, after one or more gene regulation agents contact the polynucleotide (e.g., a polynucleotide that includes a target), a change in expression level of a target is detectable. In some embodiments, a change in expression level of a target leads to a change in phenotype. In some embodiments, a change in phenotype is a reduction in one or more symptoms or manifestations of a disease, disorder, or condition, or risk thereof.

[0253] In some embodiments, after a polynucleotide (e.g., a polynucleotide that includes a target) is contacted by one or more gene regulation agents, no change in genotype is detectable. In some such embodiments, one or more of the polynucleotides is a control sequence designed to demonstrate no negative impact of administration of any composition comprising one or gene regulation agents.

[0254] In certain embodiments of this disclosure a desired genetic modification may entail other forms of genomic modification (e.g., epigenetic modification). For example, in some embodiments, activity of a gene regulation agent results in a genetic conversion of a chromatin structure.

[0255] The present disclosure provides the insight that successful regulation of disease causing gene variants (such as mutations) in genes associated with one or more diseases, disorders and/or conditions provides new strategies for treating conditions where gene regulation has become disrupted compared to a wild type counterpart.

[0256] In some embodiments, such technologies may be used, for example, in laboratory or research settings to design new cell lines for use in, e.g., development of therapeutics or screening of disease states or, e.g., screening of compound, etc.

[0257] In some embodiments, the present disclosure provides new methods and reagents for gene regulation. For instance, as illustrated in Example 1 a gene regulation agent-based gene-regulation system can yield important advantages such as specific reduction of gene expression of specific oncogenes.

Method of Treating

[0258] In some embodiments, technologies of the present disclosure are used to treat subjects with or at risk of a pathogenic phenotype due to an underlying (e.g., inherited, e.g., acquired) genotype.

[0259] In some embodiments, technologies of the present disclosure may be used to treat cancer. For example, in some embodiments, a cancer may be hereditary (e.g., KRAS gene mutation) or inherited (e.g., spontaneous mutation causing, e.g., leukemia). In some such embodiments, technologies of the present disclosure may be used to change expression levels of one or more cells comprising a cancer-associated (e.g., cancer causing) genetic sequence.

[0260] In some embodiments, technologies of the present disclosure may be used to achieve genetic modifications that result in removal of a gene regulation function. For example, in some embodiments, BCL11 A may silence fetal hemoglobin (HbF). In some such embodiments, reduction or removal of such silencing may increase production of HbF such that symptoms of disorders involving adult beta-hemoglobin, such as P-thalassemia and sickle cell disease may be ameliorated. Without being bound by any particular theory, the present disclosure contemplates that, in some embodiments, decreasing levels of BCL11 A using technologies provided by the present disclosure may increase HbF levels. In some embodiments technologies of the current disclosure may be used in immune-related treatments (e.g., immunooncology or other immune diseases, disorders or conditions). For example, in some embodiments genetic modifications may be made to one or more genes involved in immune function and/or immune regulation. In some embodiments, technologies of the present disclosure may be used to change expression levels of one or more genes comprising an immuno-associated genetic sequence (e.g., T-cell receptor alpha, T-cell receptor beta, PD-1 (i.e., PDCD-1), PD-L1 CTLA-4, TREM2). For example, in some embodiments, the present disclosure contemplates that reducing PDCD-1 expression may decrease or eliminate PD-1 signaling such that, in some embodiments, cancer activities are reduced or eliminated. In some embodiments, a cancer cell, after genomic modification and/or reduction in expression of (a) certain gene(s), may become more responsive or may become sensitive to a treatment (as compared to, e.g., prior to editing where, in some embodiments, a cancer cell may not have been sensitive or responsive to a particular treatment).

[0261] By way of non-limiting example, for instance, in some embodiments technologies of the present disclosure may be used to support development of cellular technologies that aim to treat cancer-associated conditions or immune-dysbiosis related conditions.

[0262] In some embodiments, technologies of the present disclosure may be used to treat one or more infectious diseases, disorders or conditions. For example, in some embodiments, an infectious disease may be caused by bacteria, parasites, and/or viruses. For example, the present disclosure provides technologies that may be used, e.g., to interfere with replication and/or proliferation of a virus or bacteria (e.g., by reducing expression of a key survival gene).

[0263] In some embodiments, the present disclosure provides methods of determining a genotype of a subject or a sample as described herein. In some embodiments, determining a genotype is used in diagnosing and/or treating a subject as described herein.

[0264] It will be understood by those in the art that many different changes (e.g., substitutions, deletions, additions, methylation patterns, chromatin modifications etc.) in any genetic material can result in or risk causing one or more pathogenic phenotypes.

[0265] In some embodiments, programmed gene regulation, as provided in accordance with the present disclosure, may be used to treat subjects with, or at risk of one or more pathogenic phenotype due to an underlying (e.g., inherited, e.g., acquired) genotype. For example, in some embodiments, a subject has mutation in a KRAS gene. In some such embodiments, a mutation in a KRAS gene results in an allele that generates a KRAS isoform that is associated with a higher risk of developing cancer. In some such embodiments, a cancer may include, but not be limited to, pancreatic cancer, colon cancer, and/or non-small cell lung cancer (NSCLC). In some embodiments, a gene regulation agent is engineered to reduce expression of a KRAS gene. For example, in some embodiments, a gene regulation agent includes a ZFn element includes at least one zinc finger array having at least one modified alpha helix, where the modified alpha helix mediates binding to the landing site, thereby reducing expression of a KRAS gene associated with the landing site. In some embodiments, a ZFn element engineered to bind to a landing site associated with a KRAS gene binds to a particular nucleic acid sequence associated with a mutation in the nucleic acid sequence (e.g., a nucleic acid sequence encoding a KRAS G12A, G12D, G12V, G12C, G12R, G13D, Q61K, Q61L, Q61K, Q61A, A146T, and/or K117N ).

[0266] This disclosure also provides, among other things, two gene regulation agents, where a first gene regulation agent and a second gene regulation agent each reduce expression of a target but are bind to different landing sites associated with the same target.

[0267] In some embodiments, programmed gene regulation as provided by the present disclosure may be used to treat one or more autosomal dominant genetic diseases in which a single copy of a disease-associated mutation has, will or is able to cause a disease. As provided herein, in some embodiments, a gene regulation agent is able to distinguish a mutated gene sequence from wild-type (“normal” or non-disease associated) loci and preferentially suppress expression of a mutated gene or related sequence. In some embodiments, technologies provided herein can be used to treat diseases that result from genetic mutations that are not amenable to treatment with approaches such as gene editing, including, but not limited to, autism or polycystic kidney disease.

[0268] In some embodiments, a gene regulation agent of the present disclosure is or comprises a modified Zinc Finger KRAB molecule that can be used to reduce expression levels of a target. In some such embodiments, methods comprise delivering (e.g., administering or contacting) one or more gene regulation agents, such as via one or more vectors and/or one or more transcripts thereof, and/or one or more proteins transcribed therefrom in accordance with the present disclosure, to a host cell and/or to a subject in need thereof. [0269] In some such embodiments, delivery of a gene regulation agent is achieved by contacting a cell with one or more components of a gene regulation agent, e.g., one or more agents of the present disclosure. In some embodiments conventional non-viral- or viral-based gene transfer methods that are known to those of skill in the art can be used to introduce nucleic acids (e.g., one or more components of a gene regulation agent as described herein) into cells, e.g., mammalian cells, e.g., human cells. In some embodiments, such methods can be used to administer nucleic acid encoding components of a gene regulation agent to cells in culture (e.g., in vitro or ex vivo), or in a host organism (e.g., in vivo or ex vivo).

[0270] By way of non-limiting example, in some embodiments non-viral vector delivery systems include DNA plasmids, RNA (e.g., a transcript of a vector described herein), naked nucleic acid, and/or nucleic acid complexed with a delivery vehicle, such as liposome. In some embodiments, viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cells.

[0271] In some embodiments, introduction of a gene regulation agent can be performed by transfection. In some embodiments, introduction of a gene regulation agent can be performed by nucleofection. In some embodiments, introduction of a gene regulation agent can be performed by any known or appropriate route of introduction into a target cell (e.g., a cell comprising at least one target site).

[0272] In some embodiments, a landing site comprises a small deletion, insertion and /or single nucleotide polymorphism within a coding sequence of a gene. In some embodiments, a landing site comprises more than one mutation, for example, a deletion and a point mutation wherein these two mutations are located adjacent to one another. In some embodiments, a deletion is associated with early termination of translation of a gene product (e.g., a protein) because of, e.g., generation of a premature stop codon and/or reading frame shift.

Combination therapy

[0273] In some embodiments, administration can occur in combination with other molecules. For example, in some embodiments, administration can occur in combination with an enhancing agent. In some embodiments, administration can occur in combination with an inhibiting agent. [0274] In some embodiments, an enhancing or inhibiting agent, when administered in conjunction with (e.g., sequentially or simultaneously) a gene regulation agent, may increase or decrease frequency of regulation events in a polynucleotide (e.g., DNA) contacted with the combination of an enhancing and/or inhibiting agent and gene regulation agent, relative to level of expression in a polynucleotide contacted with the gene regulation agent without the enhancing and/or inhibiting agent.

[0275] In some embodiments, administration of combinations may include more than one combination and may, in some embodiments, occur in stages. For example, a gene regulation agent may be combined with two additional agents, one of which enhances a particular process and another which inhibits a process. In some embodiments, administration may include one or more Sequence Specificity Modified Zinc Finger KRAB molecules administered in one or more stages or combinations. For instance, by way of non-limiting example, a first combination is administered comprising a gene regulation agent combined with an enhancing agent and a second combination is administered following a first combination, wherein the second combination combines the same or a different gene regulation agent with an inhibiting agent.

[0276] In some embodiments, any forms of combination therapy that enhances survival of cells that contain (a) desired genetic change(s) may be used.

[0277] In some embodiments, other forms of combination therapy that facilitate or provide detection of cells that contain (a) desired genetic change(s) may be used.

[0278] In some embodiments, other forms of combination therapy that facilitate or provide identification of cells that contain (a) desired genetic change(s) may be used.

[0279] Gene regulation and genome engineering can be useful for a wide variety of purposes. As a consequence, many different targets can be selected for gene regulation and/or for genome engineering. For example, in some embodiments a target chosen may be for the purpose of gene regulation or genome engineering to treat human diseases. For instance, in some embodiments, monogenic diseases can be targeted by conversion of underlying mutations or modifications to corresponding sequences and genome modifications found in a non-affected population [0280] In addition to monogenic diseases, gene mutations that are associated with increased risk for certain diseases can be modified to alter expression of certain genes that normalize or reduce that risk. For example, the ApoE gene has several variant alleles and certain variants (i.e., E4) are associated with increased risk for developing Alzheimer’s disease, whereas other variants normalize (i.e., E3 allele) or even reduce (i.e.E2 allele) the risk for Alzheimer’s diseases. In some embodiments, multigenic diseases could be targeted when multiple gene targets are being addressed either simultaneously or sequentially and either with one or multiple gene regulation agents.

[0281] In some embodiments, a gene may silence expression and/or function of another gene and/or protein. For instance, BCL11 A is a potent regulator of fetal-to-adult hemoglobin switch after birth. Generally, a higher level of BCL11 A is associated with adult hemoglobin, and in patients with sickle cell anemia or P-thalassemia, adult hemoglobin is damaged. Thus, without being bound by any particular theory and by way of non-limiting example, in some embodiments, BCL11 A may “silence” fetal hemoglobin (HbF) and in some embodiments, reduction or removal of such “silencing” may increase production of HbF such that symptoms of disorders involving adult beta-hemoglobin, such as P-thalassemia and sickle cell disease may be ameliorated. Accordingly, the present disclosure contemplates that, in some embodiments, decreasing levels of BCL11 A using technologies provided by the present disclosure may increase HbF levels.

[0282] In some embodiments, expression of a gene may result in signaling pathways that promote or maintain a disease state. For example, in some embodiments, PD-1 signaling in immune cells (e.g., T cells) maintain and expand a cancer phenotype. PDCD1 is an immune- inhibitory receptor expressed in activated T cells and can, in some embodiments, prevent activated T cells from killing cancer cells. In some embodiments, PDCD1 is expressed in tumors, e.g., melanoma. In some such embodiments, PDCD1 expression in tumors contributes to or causes immunotherapy resistance. Without being bound by any particular theory, in some embodiments, technologies of the present disclosure contemplate that introduction of a stop codon in the PD-1 gene (i.e., PDCD-1) will reduce or eliminate PD-1 signaling. For instance, in some embodiments, a stop codon can be introduced into PDCD1 using technologies of the present disclosure; in some such embodiments, the present disclosure contemplates that such a disruption will decrease or eliminate the impact of PDCD1 signaling and may, in some embodiments, improve or enhance impact of previously ineffective or less effective immunotherapies on cancer cells. In some embodiments, a decrease in PDCD1 signaling or expression may increase T-cell mediated responses to cancer cells; in some embodiments, such cells may become sensitive to a particular treatment after gene editing as compared to cell insensitivity prior to gene modification. In some such embodiments, such genetic modifications may reduce or eliminate cancer phenotypes and/or cellular behaviors.

[0283] In some such embodiments, expression of a gene may result in or promote or maintain a disease state, but a target or mutation may be difficult to access or “drug.” For example, in some embodiments KRAS, which is a frequent oncogenic driver in solid tumors including, but not limited to, pancreatic cancer, color cancer, non-small cell lung cancer (NSCLC), etc., is often considered “undruggable,” but targeted gene regulation can result in reduction of mutated KRAS expression levels by targeting those KRAS transcripts. While, in principle, a mutated KRAS gene can be edited to a wild type KRAS gene using one or more gene regulation agents, once a mutation in a KRAS gene occurs (and, e.g., tumor suppression function is lost), editing that gene is not necessarily a practical way to treat a cancer. Instead, repressing the expression of a mutant KRAS gene driving a particular cancer may be effective in treating the cancer. Decrease of KRAS transcripts may be accomplished, in some embodiments, using technologies of the present disclosure to selectively target and disrupt transcription of a mutated KRAS gene. Accordingly, in some such embodiments, decrease in pathogenic KRAS transcripts with technologies provided by the present disclosure may treat or improve a disease condition.

[0284] In some embodiments a target chosen may be for the purpose of creating models useful for the study of gene regulation or genome engineering to correct and/or ameliorate human diseases. These models can be cell-based models and/or animal models.

[0285] In some embodiments a target chosen may be for the purpose of creating models useful for the study of gene regulation or genome engineering. These models may be cell-based models and/or animal models. [0286] In some embodiments a target chosen may be for the purpose of creating models useful for the study of biological processes. These models may be cell-based and/or animal models.

[0287] In some embodiments a target chosen may be for the purpose of creating models useful for the study of disease causing processes. These models may be cell-based and/or animal models.

[0288] In some embodiments a target chosen may be for the purpose of gene regulation or genome engineering in mammalian cell lines involved in production of useful substances or features.

[0289] In some embodiments a target chosen may be for the purpose of gene regulation or genome engineering in plant cell lines involved in production of useful substances or features.

[0290] In some embodiments a target chosen may be for the purpose of gene regulation or genome engineering in eukaryotic cell lines involved in production of useful substances or features.

[0291] In some embodiments a target chosen may be for the purpose of gene regulation or genome engineering in one or more infectious agents (e.g., bacteria, parasite, virus, etc.).

[0292] In some embodiments a target chosen may be for the purpose of gene regulation or genome engineering in bacterial cell lines involved in production of useful substances or features.

[0293] In some embodiments a target chosen may be for the purpose of gene regulation or genome engineering in prokaryotic cell lines involved in production of useful substances or features.

[0294] In some embodiments a target chosen may be for the purpose of gene regulation or genome engineering in virus sequences.

Exemplary Embodiments

[0295] Exemplary Embodiments provided below are also within the scope of the present disclosure. [0296] Embodiment Al. A gene regulation agent comprising a structure represented by: KRAB - X - ZFn, wherein the KRAB element is or comprises a KRAB domain or portion thereof; the X element is optional and is or comprises a functional domain; and the ZFn element is or comprises at least five zinc finger arrays, wherein at least one of the zinc finger arrays comprises at least one modified alpha helix, wherein the at least one modified alpha helix is engineered to comprise a modified amino acid sequence in that it differs from that of its corresponding wild type sequence.

[0297] Embodiment A2. The gene regulation agent of embodiment Al, wherein the at least one modified alpha helix is engineered to bind a first landing site, wherein the landing site is associated with a target.

[0298] Embodiment A3. The gene regulation agent of embodiments Al or A2, wherein a second zinc finger array comprises a second modified alpha helix, wherein the second modified alpha helix is engineered to bind to a second landing site, wherein the second landing site is associated with a target.

[0299] Embodiment A4. The gene regulation agent of embodiment A3, wherein the first landing site and the second landing site are associated with a single target.

[0300] Embodiment A5. The gene regulation agent of embodiment A3, wherein the first landing site and the second landing site are associated with different targets.

[0301] Embodiment A6. The gene regulation agent of embodiment Al, wherein the ZFn element is or comprises at least six, seven, eight, nine, ten, or eleven, or more, zinc finger arrays.

[0302] Embodiment A7. The gene regulation agent of embodiment A6, wherein the zinc finger arrays comprise at least one alpha helix engineered to comprise a modified amino acid sequence that differs from that of its corresponding wild type sequence.

[0303] Embodiment A8. The gene regulation agent of any one of the preceding embodiments, wherein any of the at least one modified alpha helix amino acid sequences comprises: [0304] (i) one amino acid substitution mutation at a position selected from -1, +1, +2,

+3, +4, +5, or +6 in the alpha helix;

[0305] (ii) two amino acid substitution mutations at positions selected from -1, +1, +2, +3, +4, +5, or +6 in the alpha helix;

[0306] (iii) three amino acid substitution mutations at positions selected from -1, +1, +2, +3, +4, +5, or +6 in the alpha helix;

[0307] (iv) four amino acid substitution mutations at positions selected from -1, +1, +2, +3, +4, +5, or +6 in the alpha helix

[0308] (v) five amino acid substitution mutation at positions selected from -1, +1, +2,

+3, +4, +5, or +6 in the alpha helix;

[0309] (vi) six amino acid substitution mutations at positions selected from -1, +1, +2, +3, +4, +5, or +6 in the alpha helix; or

[0310] (vii) an amino acid substitution mutation at each position in the alpha helix.

[0311] Embodiment A9. The gene regulation of any one of embodiments A1-A7, wherein any of the at least one modified alpha helix amino acid sequences comprises one or more amino acid substitution mutations at positions selected from -1, +2, +3 and +6, or any combinations thereof.

[0312] Embodiment A10. The gene regulation agent of any one of embodiments A1-A9, wherein the ZFn element comprises at least five zinc finger arrays selected from ZNF 434, ZKSCAN1, ZKSCAN2, ZNF 552, ZNF 562, ZNF 763 or ZNF 793.

[0313] Embodiment All. The gene regulation agent of any one of embodiments A1-A9, wherein the ZFn element comprises up to seven zinc finger arrays selected from ZNF 177, ZNF 233, ZNF 248, ZNF 554, ZNF 566, ZNF 577, ZNF 701, ZNF 812, or ZNF 891.

[0314] Embodiment All. The gene regulation agent of any one of embodiments A1-A9, wherein the Zfn element comprises up to eight zinc finger arrays selected from AC003682.1, ZNF 3, ZNF 124, ZNF 506, ZNF 525, ZNF 561, ZNF 610, ZNF 625, ZNF 766, ZNF 222, or ZNF 223. [0315] Embodiment A13. The gene regulation agent of any one of embodiments A1-A9, wherein the Zfn element comprises up to nine zinc finger arrays selected from ZIK 1, ZNF 324, ZNF 510, ZNF 519, ZNF 643, ZNF 773, ZNF 486, ZNF 732, ZNF 776, or ZNF 582.

[0316] Embodiment A14. The gene regulation agent of any one of embodiments A1-A9, wherein the Zfn element comprises up to ten zinc finger arrays selected from ZNF 154, ZNF 37A, ZNF 468, ZNF 563, ZNF 614, ZNF 649, ZNF 677, ZNF 682, ZNF 689, ZNF 727, ZNF 730, or ZNF 578.

[0317] Embodiment A15. The gene regulation agent of any one of embodiments A1-A9, wherein the Zfn element comprises up to eleven zinc finger arrays selected from ZNF 155, ZNF 181, ZNF 253, ZNF 415, ZNF 416, ZNF 479, ZNF 485, ZNF 570, ZNF 675, ZNF 79 or ZIM 3.

[0318] Embodiment A16. The gene regulation agent of any one of the preceding embodiments, wherein the Zfn element comprises at least one zinc finger array selected from any one of the zinc finger arrays of embodiment A10, wherein the at least one zinc finger array is engineered to include at least one alpha helix originating from a second zinc finger array selected from any of the arrays of embodiment A10.

[0319] Embodiment A17. The gene regulation agent of embodiment Al 6, wherein the Zfn element comprises at least five zinc finger arrays from ZIM 3, wherein at least one of the zinc finger arrays from ZIM3 comprises at least one alpha helix originating from ZNF27.

[0320] Embodiment A18. The gene regulation agent of embodiment Al 7, wherein the Zfn element comprises at least seven zinc finger arrays from ZIM 3, wherein each of the zinc finger arrays from ZIM 3 comprises an alpha helix originating from ZF27.

[0321] Embodiment A19. The gene regulation agent of embodiment Al, wherein the X element is or comprises a polynucleotide.

[0322] Embodiment A20. The gene regulation agent of embodiment Al 9, wherein the X element is or comprises a polynucleotide between about 2 and 500 nucleic acids in length.

[0323] Embodiment A21. The gene regulation agent of embodiment Al, wherein the X element is or comprises engineered nucleic acids analogous to those present in a wild KRAB element. [0324] Embodiment A22. The gene regulation agent of embodiment Al, wherein the X element is or comprises a polypeptide.

[0325] Embodiment A23. The gene regulation agent of embodiment A22, wherein X element is or comprises a polypeptide between 2 and 100 amino acids in length or between 0.2 kD and 10 kD in size.

[0326] Embodiment A24. The gene regulation agent of embodiment Al, wherein the X element is or comprises a linker.

[0327] Embodiment A25. The gene regulation agent of embodiment A24, wherein the linker comprises a sequence of about 1 to about 20 amino acids.

[0328] Embodiment A26. The gene regulation agent of embodiment Al, wherein the gene regulation agent does not comprise the X element.

[0329] Embodiment A27. The gene regulation agent of embodiment Al, wherein the KRAB element comprises an amino acid sequence that is at least 95% identical to that of any one of SEQ ID NOs: 1-6.

[0330] Embodiment A28. The gene regulation agent of embodiment Al, wherein the KRAB element comprises a KRAB-A domain.

[0331] Embodiment A29. The gene regulation agent of embodiment Al, wherein the KRAB element comprises a KRAB-A and a KRAB-B domain.

[0332] Embodiment A30. The gene regulation agent of embodiments A28 or A29, wherein the KRAB-A domain comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 4.

[0333] Embodiment A31. The gene regulation agent of embodiment A29 or A30, wherein the KRAB-B domain comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 6.

[0334] Embodiment A32. The gene regulation agent of embodiment Al, wherein the gene regulation agent is or comprises an engineered human Zinc Finger KRAB molecule or a portion thereof. [0335] Embodiment A33. The gene regulation agent of embodiment A32, wherein the engineered human Zinc Finger KRAB molecule comprises an amino acid sequence that is at least 95% identical to that of any one of SEQ ID NOs: 50, 57 and 61.

[0336] Embodiment A34. The gene regulation agent of any of the preceding embodiments, wherein the gene regulation agent is or comprises a polypeptide between 80 and 10,000 amino acids in length or 8 kD and 1,000 kD in size.

[0337] Embodiment A35. The gene regulation agent of embodiment Al, wherein the gene regulation agent regulates expression of a target through binding to a landing site.

[0338] Embodiment Bl. A gene regulation agent comprising a structure represented by:

[0339] KRAB -XI - Zfn - Y - X2,

[0340] wherein: the KRAB element is or comprises a KRAB domain or portion thereof; the Xi element is optional and is or comprises a functional domain; the Zfn element is or comprises a sequence specific binding element; the Y element is or comprises an optional linker domain; and the X2 element is or comprises at least one binding element that is optionally sequence-specific.

[0341] Embodiment B2. The gene regulation agent of embodiment Bl, wherein the KRAB element comprises a KRAB-A domain.

[0342] Embodiment B3. The gene regulation agent of embodiment Bl, wherein the KRAB element comprises a KRAB-A and a KRAB-B domain.

[0343] Embodiment B4. The gene regulation agent of embodiment Bl, wherein the KRAB element comprises an amino acid sequence that is at least 95% identical to that of any one of SEQ ID NOs: 1-6.

[0344] Embodiment B5. The gene regulation agent of embodiments B3 or B4, wherein the KRAB-A domain comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 4.

[0345] Embodiment B6. The gene regulation agent of embodiment B4 or B5, wherein the KRAB-B domain comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 6. [0346] Embodiment B7. The gene regulation agent of embodiment Bl, wherein the gene regulation agent is or comprises an engineered human Zinc Finger KRAB molecule or a portion thereof.

[0347] Embodiment B8. The gene regulation agent of embodiment B7, wherein the engineered human Zinc Finger KRAB molecule comprises an amino acid sequence that is at least 95% identical to that of any one of SEQ ID NOs: 50, 57 and 61.

[0348] Embodiment B9. The gene regulation agent of embodiment Bl, wherein the XI element is or comprises a polynucleotide.

[0349] Embodiment B10. The gene regulation agent of embodiment B9, wherein the XI element is or comprises a polynucleotide between about 2 and 500 nucleic acids in length.

[0350] Embodiment Bll. The gene regulation agent of embodiment Bl, wherein the XI element is or comprises engineered nucleic acids analogous to those present in a wild-type KRAB element.

[0351] Embodiment Bll. The gene regulation agent of embodiment Bl, wherein the XI element is or comprises a polypeptide.

[0352] Embodiment B13. The gene regulation agent of embodiment Bl 2, wherein XI element is or comprises a polypeptide between 2 and 100 amino acids in length or between 0.2 kD and 10 kD in size.

[0353] Embodiment B14. The gene regulation agent of embodiment Bl, wherein the XI element is or comprises a linker.

[0354] Embodiment B15. The gene regulation agent of embodiment 14, wherein the linker comprises a sequence of about 1 to about 20 amino acids.

[0355] Embodiment B16. The gene regulation agent of embodiment Bl, wherein the gene regulation agent does not comprise the X element.

[0356] Embodiment B17. The gene regulation agent of embodiment Bl, wherein the Zfin element binds to a first landing site, wherein the landing site is associated with a target. [0357] Embodiment B18. The gene regulation agent of embodiment Bl, wherein a Zfn element binds to a second landing site, wherein the second landing site is associated with a target.

[0358] Embodiment B19. The gene regulation agent of embodiment Bl, wherein the ZFn element is or comprises at least five, six, seven, eight, nine, ten, or eleven, or more, zinc finger arrays.

[0359] Embodiment B20. The gene regulation agent of embodiment Bl, wherein the Zfn element is or comprises a polypeptide.

[0360] Embodiment B21. The gene regulation agent of embodiment Bl, where wherein the Zfn element is or comprises a polypeptide between 80 and 10,000 amino acids in length or between 8 kD and 1,000 kD in size.

[0361] Embodiment B22. The polymeric modification agent of embodiment Bl, wherein the sequence of the Zfn element is at least 50% identical to a sequence selected from SEQ ID NOS: 22-34.

[0362] Embodiment B23. The polymeric modification agent of embodiment Bl, wherein the Zfn element is or comprises a polynucleotide.

[0363] Embodiment B24. The polymeric modification agent of embodiment Bl, wherein the Zfn element is or comprises a polynucleotide between 20 and 50,000 nucleotides in length.

[0364] Embodiment B25. The gene regulation agent of embodiment Bl, wherein the gene regulation agent regulates expression of a target through binding to a landing site.

[0365] Embodiment B26. The gene regulation agent of embodiment Bl, wherein the Y element is or comprises a polypeptide.

[0366] Embodiment B27. The gene regulation agent of embodiment Bl, wherein the Y element is or comprises a polypeptide between 2 and 100 amino acids in length or between 0.2 kD and 10 kD in size. [0367] Embodiment B28. The gene regulation agent of embodiment Bl, wherein the sequence of the Y element is at least 50% identical to a sequence selected from SEQ ID NOS: 7- 21.

[0368] Embodiment B29. The gene regulation agent of embodiment Bl, wherein the Y element is or comprises a polynucleotide.

[0369] Embodiment B30. The gene regulation agent of embodiment Bl, wherein the Y element is or comprises a polynucleotide between 2 and 500 nucleic acids in length.

[0370] Embodiment B31. The gene regulation agent of embodiment Bl, wherein the X2 element is or comprises a polypeptide.

[0371] Embodiment B32. The gene regulation agent of embodiment Bl, wherein the X2 element is or comprises a polypeptide between 10 and 50,000 amino acids in length or between 1 kD and 5,000 kD in size.

[0372] Embodiment B33. The gene regulation agent of embodiment Bl, wherein the sequence of the X2 element is a sequence-specific DNA binding element.

[0373] Embodiment B34. The gene regulation agent of embodiment Bl, wherein the sequence of the X2 element is a non-sequence specific DNA binding element.

[0374] Embodiment B35. The gene regulation agent of embodiment Bl, wherein the X2 element is or comprises a polynucleotide.

[0375] Embodiment B36. The gene regulation agent of embodiment Bl, wherein the X2 element is or comprises a polynucleotide between about 2 and 50,000 nucleic acids in length.

[0376] Embodiment B37. The gene regulation agent of embodiment Bl, wherein the X2 element is or comprises one or more amino acids that bind at or near a site adjacent to a landing site, wherein regulates expression of a target through binding to a landing site.

[0377] Embodiment B38. A combination comprising at least two gene regulation agents wherein a first gene regulation agent reduces expression of at least a first target and a second gene regulation agent reduces expression of at least a second target, wherein the first and second targets are or comprise the same or different genes or mRNA products thereof. [0378] Embodiment Cl. A method comprising contacting a cell comprising a polynucleotide with at least one gene regulation agent of embodiment Al or Bl, wherein: (i) the polynucleotide comprises a target; and (ii) the Zfn element of the gene regulation agent binds to a landing site associated with the target; and wherein concomitant with or subsequent to the contacting with the gene regulation agent expression of the target is reduced relative to: (i) a cell not contacted with a gene regulation agent and/or (ii) a cell contacted with an agent that does not comprise a KRAB domain.

[0379] Embodiment C2. The method of embodiment Cl, wherein the gene regulation agent reduces expression of the target through binding to the landing site.

[0380] Embodiment C3. The method of embodiment Cl, wherein the landing site is or comprises a target site.

[0381] Embodiment C4. The method of embodiment C3, wherein the target site is an error site.

[0382] Embodiment C5. The method of embodiment Cl, wherein the landing site is or comprises all or a portion of a regulatory element.

[0383] Embodiment C6. The method of embodiment Cl, wherein the regulatory element is selected from the group consisting of: a promoter, an enhancer, a silencer, an insulator, a TATA box, a GC box, a CAAT box, a transcriptional start site, a DNA binding motif of a transcription factor or other protein that regulates transcription, a 5’ untranslated region, and a ‘3 untranslated region.

[0384] Embodiment C7. The method of embodiment Cl, wherein the polynucleotide is DNA.

[0385] Embodiment C8. The method of embodiment Cl, wherein the polynucleotide is mRNA.

[0386] Embodiment C9. The method of embodiment Cl, wherein the contacting of the cell with the gene regulation agent occurs prior to or concomitant with a reduction in rate and/or level of transcriptional activity of the target. [0387] Embodiment CIO. The method of embodiment Cl, wherein the contacting of the cell with the gene regulation agent occurs prior to or concomitant with a change in chromatin structure, wherein the change in chromatin structure reduces the rate and/or level of transcriptional activity of the target.

[0388] Embodiment Cll. The method of embodiment CIO, wherein the contacting of the cell with the gene regulation agent further comprises contacting the cell with one or more of a DNA or histone modification factors or any other factors associated with a DNA modification and a histone modification.

[0389] Embodiment Cll. The method of embodiment Cl, where the one or more DNA or histone modification factor(s), factors associated with DNA modifications, or factors associated with histone modifications are selected from: methylases, methyltransferase, histone (de-)acylating enzymes helicases, recombinases, repair scaffold proteins, single strand DNA binding proteins, mismatch repair proteins.

[0390] Embodiment C13. The method of any one of embodiments Cl-12, wherein the step of contacting comprises contacting within a cell.

[0391] Embodiment C14. The method of any one of embodiments Cl -Cl 3, wherein the contacting is achieved by administration of the at least one gene regulation agents of embodiment Al -A35 or B1-B38 by at least one of intravenous, parenchymal, intracranial, intracerebroventricular, intrathecal, or parenteral administration.

[0392] Embodiment C15. The method of embodiment C14, wherein the contacting is performed ex vivo or in vitro, resulting in a population of cells with reduced expression of the target and/or one or more histone modifications relative to the population of cells prior to the contacting.

[0393] Embodiment C16. The method of embodiment Cl 5, wherein at least a portion of the population of cells is administered to a subject in need thereof.

[0394] Embodiment C17 The method of any one of embodiments Cl -Cl 6, wherein the contacting comprises contacting a population of cells. [0395] Embodiment C18. The method of embodiment Cl 7, wherein the population of cells is or comprises a tissue.

[0396] Embodiment C19. The method of embodiment Cl 7, wherein the population of cells is or comprises an organ.

[0397] Embodiment C20. The method of embodiment Cl 7, wherein the population of cells is or comprises a tumor.

[0398] Embodiment C21. The method of embodiment C20, wherein the tumor is or comprises a lung tumor, pancreatic tumor, or colon tumor.

[0399] Embodiment C22 The method of embodiment Cl 7, wherein the population of cells is or comprises a specific cell lineage.

[0400] Embodiment C23. The method of embodiment C22, wherein the specific cell lineage is or comprises lung cells, pancreas cells, or colon cells.

[0401] Embodiment C24. The method of embodiment C22, wherein the specific cell lineage is or comprises cells comprising one or more KRAS mutation(s).

[0402] Embodiment C25. The method of embodiment C24, wherein the one or more KRAS mutation occurs at one or more positions selected from G12, G13, A18, Q61, KI 17, and A146.

[0403] Embodiment C26. The method of any one of embodiments C1-C25, wherein the contacting occurs in vivo.

[0404] Embodiment C27. The method of embodiment C26, wherein the contacting occurs in a subject in need thereof.

[0405] Embodiment C28. The method of embodiment C27, wherein the subject is a mammal.

[0406] Embodiment C29. The method of embodiment C28, wherein the mammal is a non-human primate.

[0407] Embodiment C30. The method of embodiment C28, wherein the mammal is a human. [0408] Embodiment C3166. The method of embodiment C30, wherein the human is an adult human.

[0409] Embodiment C32. The method of embodiment C30, wherein the human is a fetal, infant, child, or adolescent human.

[0410] Embodiment C33. The method of any one of embodiments C1-C32, wherein the contacting comprises contacting with at least two different gene regulation agents in accordance with embodiments 1 or 31.

[0411] Embodiment C34. The method of embodiment C34, wherein the contacting with the at least two gene regulation agents is sequential or simultaneous.

[0412] Embodiment C35. The method of embodiments C33 or C34, wherein the expression of at least two targets are reduced relative to: (i) a cell not contacted with a gene regulation agent(s) and/or (ii) a cell contacted with an agent(s) that does not comprise a KRAB domain.

[0413] Embodiment C36. The method of any one of embodiments C33-C35, wherein the at least two targets are associated with different genes.

[0414] Embodiment C37. The method of any one of embodiments C33-C36, wherein the at least two targets are associated with the same gene.

[0415] Embodiment C38. The method of embodiment C36, wherein the different genes are located on the same chromosome.

[0416] Embodiment C39. The method of embodiment C36, wherein the different genes are located on different chromosomes.

[0417] Embodiment C40. The method of embodiment Cl, wherein the expression is measured by a level of mRNA, protein, co-precipitation assays, or chromatin accessibility assays.

[0418] Embodiment C41. The method of any one of embodiments C1-C40, wherein after the contacting, there is a reduction in a level of a target as compared to the level of the target in the absence of the contacting. [0419] Embodiment C42. The method of any one of embodiments C1-C41, wherein prior to or concomitant with the contacting, the target is being actively transcribed.

[0420] Embodiment C43. The method of any one of embodiments C1-C42, wherein at least one target or sequence associated therewith is epigenetically modified.

[0421] Embodiment C44. The method of embodiment C43, wherein the epigenetic modification is concomitant with a dissociation of an RNA polymerase from a DNA strand associated with the at least one target.

[0422] Embodiment C45. The method of embodiment C43, wherein the epigenetic modification prevents association of an RNA polymerase with a DNA strand associated with the at least one target.

[0423] Embodiment C46. The method of embodiment C45, wherein the at least two targets or sequences associated therewith are epigenetically modified.

[0424] Embodiment C47. A vector comprising a nucleic acid sequence encoding any one of the gene regulation agents of any embodiments Al -A35 or B1-B35.

[0425] Embodiment C48. The vector of embodiment C47, wherein the vector comprises a viral vector selected from the group consisting of: a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno-associated viral vector.

[0426] Embodiment C49. A composition comprising any of the gene regulation agents of embodiments 1-35 or the vectors of embodiments C47 or C48.

[0427] Embodiment C50. A kit comprising the gene regulation agents of embodiments A1-A23 or B1-B35 or the compositions of embodiment C49 and instructions for use of the gene regulation agents or compositions.

EXAMPLES

[0428] The following examples are given for the purpose of illustrating various embodiments of the disclosure and are not meant to limit the present disclosure in any fashion. Changes therein and other uses which are encompassed within the spirit of the disclosure as defined by the scope of the claims will occur to those skilled in the art. EXAMPLE 1: Transcription modification mediated suppression of oncogenic KRAS gene expression in mammalian cells

[0429] This example describes regulation of human KRAS gene expression using exemplary gene regulation agents of the present disclosure. Specifically, the present example describes gene regulation (e.g., decreased expression) of human KRAS with exemplary gene regulation agents that include: DLR molecules, KRAB-DLR molecules, and modified Zinc Finger KRAB molecules. Human KRAS, was selected as a model genetic target for regulation in the present example because KRAS is a frequent oncogenic driver in solid tumors, including pancreatic cancer, colon cancer, non-small cell lung cancer (NSCLC), and many others (Salgia R. et.al. Cell Rep. Med. 2021; Jan 19; 2(1): 100186, which is herein incorporated by reference in its entirety). Few treatments are available for targeting KRAS directly, and KRAS mutations are often considered as “undruggable” targets. As demonstrated herein, gene regulation agents as described herein can be used to suppress KRAS gene expression as evidenced by reduced mRNA levels. Moreover, the present example shows additional beneficial characteristics of modified Zinc Finger KRAB molecules relative to other exemplary gene regulation agents.

[0430] An exemplary transcription modification strategy used in this example is to engineer gene regulation agents to specifically target KRAS genes in HEK293 cells. In this example, target specificity of zinc finger arrays of gene regulation agents was first confirmed using RITDM gene editing experiments (as described in PCT/US21/37113, which is herein incorporated by reference in its entirety) of human KRAS. After successful gene editing of human KRAS, engineered zinc finger alpha helices were used in designing a Sequence Specificity Modified Zinc Finger KRAB protein that could target KRAS.

[0431] As demonstrated in PCT/US21/37113, site-specific targeting of KRAS can be achieved using DLR molecules. Here, gene regulation agent comprising a modified Zinc Finger KRAB proteins were engineered and used to target KRAS and effects compared to targeting using DLR molecules (i.e., DLR molecules with no KRAB domain). This example also describes gene regulation using KRAB-DLR molecules. Exemplary schematics of DLR molecules, KRAB-DLR molecules, and Zinc Finger KRAB molecules is provided in Figure 4. [0432] In this example, three different exemplary DLR molecules, encoded on plasmid pb74, pb75, and pb76 (represented by SEQ ID NOs: 35-43, for full-length DNA, cDNA, and amino acid sequences) were developed (as described in PCT/US21/37113, which is herein incorporated by reference in its entirety).

[0433] pb74 DNA - SEQ ID NO : 35

GACTCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTA TTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGT TACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACG TCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCA TATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTA TGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTC ATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGG TTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTT GGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACG CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCT AACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGA GACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTATGGCGGCGATGGCCGAGCGGCC CTTCCAGTGCAGGATCTGTATGCGCAACTTTTCTCAGTCCGGCGACCTGACCCGGCA CATCAGAACCCATACAGGCGAAAAGCCTTTCGCCTGCGACATTTGTGGGAGAAAAT TTGCTCGGTCCGACAACCTGACCACCCATACCAAGATCCACACCGGCTCTCAGAAAC CATTCCAGTGCCGCATTTGTATGCGGAATTTTTCCCGGTCCTCCGACCTGACCCGGC ATATCCGCACTCACACCGGAGAGAAGCCCTTTGCTTGCGACATTTGTGGCAGGAAAT TTGCTCGGTCCGACGCCCTGACCCGGCACACTAAGATCCATACTGGGTCACAGAAAC CTTTCCAGTGCCGGATTTGTATGAGAAACTTTAGCCGGTCCGACGCCCTGTCCGAGC ACATCAGAACCCATACAGGCGAAAAGCCTTTCGCCTGCGACATTTGTGGGAGAAAA TTTGCTCGGTCCTCCAACCTGACCCGGCATACCAAGATCCACACCGGCTCTCAGAAA CCATTCCAGTGCCGCATTTGTATGCGGAATTTTTCCCGGTCCGACGCCCTGACCACC CACATCAGAACACATACTGGGCTGAGAGGATCCAATTCTGGTGATCCTCGGAGACA CAGTCTGGGCGGTTCTCGTAAACCCGATCTGATTGCCTATAAAAACTTTGATCTGCT GGTCATTGTTCTTAAGCCTTGAGCGGCCGCTCGAGTCTAGAGGGCCCGTTTAAACCC

GCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCC

CGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGA

GGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGG

GCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCG

GTGGGCTCTATGGCTTCTACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGC

CAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCT

TTCTCGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGCTCTGATCAAGAGACAGG

ATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGC

TTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTG

ATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCG

ACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTG

GCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAG

GGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGC

TCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGA

TCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTA

CTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGG

CTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGGA

TCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCG

CTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACAT

AGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTT

CCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTT

CTTGACGAGTTCTTCTGAATTATTAACGCTTACAATTTCCTGATGCGGTATTTTCTCC

TTACGCATCTGTGCGGTATTTCACACCGCATACAGGTGGCACTTTTCGGGGAAATGT

GCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATG

AGACAATAACCCTGATAAATGCTTCAATAATAGCACGTGCTAAAACTTCATTTTTAA

TTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAA

CGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCT

TGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTA

CCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACT

GGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGC CACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTA

CCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGA

TAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCC

CAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAG

AAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAG

GGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTT

ATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTC

AGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGG

GCTTTTGCTGGCCTTTTGCTCACATGTTCTT

[0434] pb74 cDNA - SEQ ID NO: 36

ATGGCGGCGATGGCCGAGCGGCCCTTCCAGTGCAGGATCTGTATGCGCAACTTTTCT

CAGTCCGGCGACCTGACCCGGCACATCAGAACCCATACAGGCGAAAAGCCTTTCGC

CTGCGACATTTGTGGGAGAAAATTTGCTCGGTCCGACAACCTGACCACCCATACCAA

GATCCACACCGGCTCTCAGAAACCATTCCAGTGCCGCATTTGTATGCGGAATTTTTC

CCGGTCCTCCGACCTGACCCGGCATATCCGCACTCACACCGGAGAGAAGCCCTTTGC

TTGCGACATTTGTGGCAGGAAATTTGCTCGGTCCGACGCCCTGACCCGGCACACTAA

GATCCATACTGGGTCACAGAAACCTTTCCAGTGCCGGATTTGTATGAGAAACTTTAG

CCGGTCCGACGCCCTGTCCGAGCACATCAGAACCCATACAGGCGAAAAGCCTTTCG

CCTGCGACATTTGTGGGAGAAAATTTGCTCGGTCCTCCAACCTGACCCGGCATACCA

AGATCCACACCGGCTCTCAGAAACCATTCCAGTGCCGCATTTGTATGCGGAATTTTT

CCCGGTCCGACGCCCTGACCACCCACATCAGAACACATACTGGGCTGAGAGGATCC

AATTCTGGTGATCCTCGGAGACACAGTCTGGGCGGTTCTCGTAAACCCGATCTGATT

GCCTATAAAAACTTTGATCTGCTGGTCATTGTTCTTAAGCCTTGA

[0435] pb74 amino acid - SEQ ID NO: 37

MAAMAERPFQCRICMRNFSQSGDLTRHIRTHTGEKPFACDICGRKFARSDNLTTHTKIHT GSQKPFQCRICMRNF SRS SDLTRHIRTHTGEKPF ACDICGRKF ARSDALTRHTKIHTGSQ KPFQCRICMRNFSRSDALSEHIRTHTGEKPFACDICGRKFARSSNLTRHTKIHTGSQKPFQ

CRICMRNFSRSDALTTHIRTHTGLRGSNSGDPRRHSLGGSRKPDLIAYKNFDLLVIVLKP*

[0436] pb75 DNA - SEQ ID NO: 38 GACTCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTA

TTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGT

TACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT

GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACG

TCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCA

TATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTA

TGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTC

ATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGG

TTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTT

GGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACG

CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCT

AACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGA

GACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTATGGCGGCGATGGCCGAGCGGCC

CTTCCAGTGCAGGATCTGTATGCGCAACTTTTCTCAGTCCGGCGACCTGACCCGGCA

CATCAGAACCCATACAGGCGAAAAGCCTTTCGCCTGCGACATTTGTGGGAGAAAAT

TTGCTCGGTCCGACAACCTGACCACCCATACCAAGATCCACACCGGCTCTCAGAAAC

CATTCCAGTGCCGCATTTGTATGCGGAATTTTTCCCGGTCCTCCGACCTGACCCGGC

ATATCCGCACTCACACCGGAGAGAAGCCCTTTGCTTGCGACATTTGTGGCAGGAAAT

TTGCTCGGTCCGACGCCCTGACCCGGCACACTAAGATCCATACTGGGTCACAGAAAC

CTTTCCAGTGCCGGATTTGTATGAGAAACTTTAGCCGGTCCGACGCCCTGTCCGAGC

ACATCAGAACCCATACAGGCGAAAAGCCTTTCGCCTGCGACATTTGTGGGAGAAAA

TTTGCTCGGTCCTCCAACCTGACCCGGCATACCAAGATCCACACCGGCTCTCAGAAA

CCATTCCAGTGCCGCATTTGTATGCGGAATTTTTCCCGGTCCGACGCCCTGACCACC

CACATCAGAACACATACTGGGCTGAGAGGATCCAATTCTGGTGATCCTCGGAGACA

CAGTCTGGGCGGTTCTCGTAAACCCGATCTGATTGCCTATAAAAACTTTGATCTGCT

GGTCATTGTTCTTAAGCCTAAATACTCCCAGAATTCTGGTGATCCTCGGAGACACAG

TCTGGGCGGTTCTCGTAAACCCGATGGTGCTATTTATACTGTTGGTTCTCCTATTGAT

TATGGTGTTATTGTTGTTACTAAACCTTGAGCGGCCGCTCGAGTCTAGAGGGCCCGT

TTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGC

CCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAAT

AAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTG GGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGG

GGATGCGGTGGGCTCTATGGCTTCTACTGGGCGGTTTTATGGACAGCAAGCGAACCG

GAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTG

GATGGCTTTCTCGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGCTCTGATCAAG

AGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCT

CCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGG

CTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTC

AAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATC

GTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGC

GGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTC

ACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATA

CGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGA

GCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCA

TCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACG

GCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAA

ATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATC

AGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCT

GACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCT

ATCGCCTTCTTGACGAGTTCTTCTGAATTATTAACGCTTACAATTTCCTGATGCGGTA

TTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACAGGTGGCACTTTTCGGG

GAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCC

GCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAGCACGTGCTAAAACTTCA

TTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATC

CCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGA

TCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCAC

CGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGG

TAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGT

TAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCC

TGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAA

GACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACA

CAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCT ATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGC GGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGT ATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATG CTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGT TCCTGGGCTTTTGCTGGCCTTTTGCTCACATGTTCTT

[0437] pb75 cDNA - SEQ ID NO: 39

ATGGCGGCGATGGCCGAGCGGCCCTTCCAGTGCAGGATCTGTATGCGCAACTTTTCT

CAGTCCGGCGACCTGACCCGGCACATCAGAACCCATACAGGCGAAAAGCCTTTCGC CTGCGACATTTGTGGGAGAAAATTTGCTCGGTCCGACAACCTGACCACCCATACCAA GATCCACACCGGCTCTCAGAAACCATTCCAGTGCCGCATTTGTATGCGGAATTTTTC CCGGTCCTCCGACCTGACCCGGCATATCCGCACTCACACCGGAGAGAAGCCCTTTGC TTGCGACATTTGTGGCAGGAAATTTGCTCGGTCCGACGCCCTGACCCGGCACACTAA

GATCCATACTGGGTCACAGAAACCTTTCCAGTGCCGGATTTGTATGAGAAACTTTAG

CCGGTCCGACGCCCTGTCCGAGCACATCAGAACCCATACAGGCGAAAAGCCTTTCG

CCTGCGACATTTGTGGGAGAAAATTTGCTCGGTCCTCCAACCTGACCCGGCATACCA

AGATCCACACCGGCTCTCAGAAACCATTCCAGTGCCGCATTTGTATGCGGAATTTTT

CCCGGTCCGACGCCCTGACCACCCACATCAGAACACATACTGGGCTGAGAGGATCC

AATTCTGGTGATCCTCGGAGACACAGTCTGGGCGGTTCTCGTAAACCCGATCTGATT GCCTATAAAAACTTTGATCTGCTGGTCATTGTTCTTAAGCCTAAATACTCCCAGAATT CTGGTGATCCTCGGAGACACAGTCTGGGCGGTTCTCGTAAACCCGATGGTGCTATTT ATACTGTTGGTTCTCCTATTGATTATGGTGTTATTGTTGTTACTAAACCTTGA

[0438] pb75 amino acid - SEQ ID NO: 40

MAAMAERPFQCRICMRNFSQSGDLTRHIRTHTGEKPFACDICGRKFARSDNLTTHTKIHT GSQKPFQCRICMRNF SRS SDLTRHIRTHTGEKPF ACDICGRKF ARSDALTRHTKIHTGSQ KPFQCRICMRNFSRSDALSEHIRTHTGEKPFACDICGRKFARSSNLTRHTKIHTGSQKPFQ CRICMRNFSRSDALTTHIRTHTGLRGSNSGDPRRHSLGGSRKPDLIAYKNFDLLVIVLKP KYSQNSGDPRRHSLGGSRKPDGAIYTVGSPIDYGVIVVTKP*

[0439] pb76 DNA - SEQ ID NO: 41 GACTCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTA

TTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGT

TACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT

GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACG

TCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCA

TATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTA

TGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTC

ATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGG

TTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTT

GGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACG

CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCT

AACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGA

GACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTATGGCGGCGATGGCCGAGCGGCC

CTTCCAGTGCAGGATCTGTATGCGCAACTTTTCTCAGTCCGGCGACCTGACCCGGCA

CATCAGAACCCATACAGGCGAAAAGCCTTTCGCCTGCGACATTTGTGGGAGAAAAT

TTGCTCGGTCCGACAACCTGACCACCCATACCAAGATCCACACCGGCTCTCAGAAAC

CATTCCAGTGCCGCATTTGTATGCGGAATTTTTCCCGGTCCTCCGACCTGACCCGGC

ATATCCGCACTCACACCGGAGAGAAGCCCTTTGCTTGCGACATTTGTGGCAGGAAAT

TTGCTCGGTCCGACGCCCTGACCCGGCACACTAAGATCCATACTGGGTCACAGAAAC

CTTTCCAGTGCCGGATTTGTATGAGAAACTTTAGCCGGTCCGACGCCCTGTCCGAGC

ACATCAGAACCCATACAGGCGAAAAGCCTTTCGCCTGCGACATTTGTGGGAGAAAA

TTTGCTCGGTCCTCCAACCTGACCCGGCATACCAAGATCCACACCGGCTCTCAGAAA

CCATTCCAGTGCCGCATTTGTATGCGGAATTTTTCCCGGTCCGACGCCCTGACCACC

CACATCAGAACACATACTGGGCTGAGAGGATCCAATTCTGGTGATCCTCGGAGACA

CAGTCTGGGCGGTTCTCGTAAACCCGATCTGATTGCCTATAAAAACTTTGATCTGCT

GGTCATTGTTCTTAAGCCTAAATACTCCCAGAATTCTGGTGATCCTCGGAGACACAG

TCTGGGCGGTTCTCGTAAACCCGATGGTGCTATTTATACTGTTGGTTCTCCTATTGAT

TATGGTGTTATTGTTGTTACTAAACCTAAGTACTCCCAGAACTCTGGTGATCCTCGGA

GACACAGTCTGGGCGGTTCTCGTAAACCCGATATTATTCTTGTTAATGATAATATTTC

TCTTATTCTTATTCTTGTTGCTAAACCTTGAGCGGCCGCTCGAGTCTAGAGGGCCCGT

TTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGC CCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAAT

AAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTG

GGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGG

GGATGCGGTGGGCTCTATGGCTTCTACTGGGCGGTTTTATGGACAGCAAGCGAACCG

GAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTG

GATGGCTTTCTCGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGCTCTGATCAAG

AGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCT

CCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGG

CTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTC

AAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATC

GTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGC

GGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTC

ACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATA

CGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGA

GCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCA

TCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACG

GCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAA

ATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATC

AGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCT

GACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCT

ATCGCCTTCTTGACGAGTTCTTCTGAATTATTAACGCTTACAATTTCCTGATGCGGTA

TTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACAGGTGGCACTTTTCGGG

GAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCC

GCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAGCACGTGCTAAAACTTCA

TTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATC

CCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGA

TCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCAC

CGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGG

TAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGT

TAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCC

TGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAA GACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACA

CAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCT

ATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGC

GGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGT

ATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATG

CTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGT

TCCTGGGCTTTTGCTGGCCTTTTGCTCACATGTTCTT

[0440] pb76 cDNA - SEQ ID NO: 42

ATGGCGGCGATGGCCGAGCGGCCCTTCCAGTGCAGGATCTGTATGCGCAACTTTTCT

CAGTCCGGCGACCTGACCCGGCACATCAGAACCCATACAGGCGAAAAGCCTTTCGC

CTGCGACATTTGTGGGAGAAAATTTGCTCGGTCCGACAACCTGACCACCCATACCAA

GATCCACACCGGCTCTCAGAAACCATTCCAGTGCCGCATTTGTATGCGGAATTTTTC

CCGGTCCTCCGACCTGACCCGGCATATCCGCACTCACACCGGAGAGAAGCCCTTTGC

TTGCGACATTTGTGGCAGGAAATTTGCTCGGTCCGACGCCCTGACCCGGCACACTAA

GATCCATACTGGGTCACAGAAACCTTTCCAGTGCCGGATTTGTATGAGAAACTTTAG

CCGGTCCGACGCCCTGTCCGAGCACATCAGAACCCATACAGGCGAAAAGCCTTTCG

CCTGCGACATTTGTGGGAGAAAATTTGCTCGGTCCTCCAACCTGACCCGGCATACCA

AGATCCACACCGGCTCTCAGAAACCATTCCAGTGCCGCATTTGTATGCGGAATTTTT

CCCGGTCCGACGCCCTGACCACCCACATCAGAACACATACTGGGCTGAGAGGATCC

AATTCTGGTGATCCTCGGAGACACAGTCTGGGCGGTTCTCGTAAACCCGATCTGATT

GCCTATAAAAACTTTGATCTGCTGGTCATTGTTCTTAAGCCTAAATACTCCCAGAATT

CTGGTGATCCTCGGAGACACAGTCTGGGCGGTTCTCGTAAACCCGATGGTGCTATTT

ATACTGTTGGTTCTCCTATTGATTATGGTGTTATTGTTGTTACTAAACCTAAGTACTC

CCAGAACTCTGGTGATCCTCGGAGACACAGTCTGGGCGGTTCTCGTAAACCCGATAT

TATTCTTGTTAATGATAATATTTCTCTTATTCTTATTCTTGTTGCTAAACCTTGA

[0441] pb76 amino acid - SEQ ID NO: 43

MAAMAERPFQCRICMRNFSQSGDLTRHIRTHTGEKPFACDICGRKFARSDNLTTHTKIHT

GSQKPFQCRICMRNF SRS SDLTRHIRTHTGEKPF ACDICGRKF ARSDALTRHTKIHTGSQ

KPFQCRICMRNFSRSDALSEHIRTHTGEKPFACDICGRKFARSSNLTRHTKIHTGSQKPFQ CRICMRNFSRSDALTTHIRTHTGLRGSNSGDPRRHSLGGSRKPDLIAYKNFDLLVIVLKP

KYSQNSGDPRRHSLGGSRKPDGAIYTVGSPIDYGVIVVTKPKYSQNSGDPRRHSLGGSR

KPDIILVNDNISLILILVAKP*

[0442] In the DLR molecules, sequence-specific D domains comprised a 7-zinc-finger- array designed to recognize a 21- nucleotide sequence of 5’-TTG-GAG-CTG-GTG-GCG-TAG- GCA-3’ (SEQ ID NO: 44) located on leading strand adjacent to codon A18 “GCC” within Exon 1 (See Figure 5A).

[0443] As exemplary proof of targeting specificity, RITDM was used to confirm KRAS targeting. In this embodiment, a 137-nucleotide sequence modification polynucleotide was first used to confirm targeting and is set forth as follows: 5’- AAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGTAGGCAAGAGTTG AGAATCCGTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAA CAATAGAGGTAAATCTTGTTTTAA-3’ (SEQ ID NO: 45). This sequence modification polynucleotide has a substitution sequence of “TGAGAATCCG” that was intended to replace “GCC” at its targeting locus of KRAS. Each of plasmid of pb74, pb75, and pb76 along with sequence modification polynucleotides (as described in PCT/US21/37113, which is herein incorporated by reference in its entirety) were introduced into HEK 293 cells by electroporation and reseeded into tissue culture vessels. Five days post transfection, genomic DNA was extracted, followed by ddPCR detection for genome editing effects. As shown in Figure 5B, ddPCR analysis demonstrates successful KRAS targeting. The upper panel of Figure 5B represents positive droplets with “TGAGAATCCG” genetic conversion; the lower panel of Figure 5B represents wild type droplets comprising “GCC.” All three DLR molecules with single (DLR) (pb74), double (DLRR) (pb75), or triple R (DLRRR) (pb76) elements, were able to successfully convert “GTT” into “TGAGAATCCG” at target site of KRAS gene in human genome in HEK293 cells, demonstrating that these DLR molecules are able to accurately target a human KRAS gene sequence. This also confirms site-specific binding of each of these DLR molecules as designed.

[0444] Then, programmed KRAS gene suppression was performed and analyzed. Plasmids including pb74, pb75, and pb76 were introduced into U937 cells by electroporation. A “no DNA” transfection was set to be used as a control for expression of KRAS without gene regulation. A positive control was obtained from reverse transcription (RT) reactions using total RNA extracted from cells unaltered in their KRAS expression. Seventy-two hours post electroporation, cells transfected with each plasmid were detached and collected. Total RNAs from each condition were then extracted by using Trizol reagent. Five hundred nanograms of total RNA was then converted into DNA by reverse transcription (RT) using a reverse transcriptase, appropriate buffer, and dNTPs. After this RT reaction, a PCR test was conducted using a primer set of Pop 133 (5’-GACTGAATATAAACTTGTGGTAGTTGGAGCT-3’, SEQ ID. NO: 46) and Popl34 (5’-TCCTCTTGACCTGCTGTGTCG-3’, SEQ ID. NO: 47). Primer Pop 133 is a forward primer binding within exonl of the human KRAS gene; and Pop 134 is a reverse primer binding on exon 2 of the human KRAS gene. When KRAS mRNA was present, a 184 bp RT-PCR amplicon was detected. Figure 6A shows a representative example of successful suppression of KRAS gene expression by pb74 (KRAS-DLR) and pb75 (DLRR), and pb76 (DLRRR). In each condition, RT-PCR conducted using a primer set of Popl33 and Pop 134 showed RT-PCR amplicons of 184bp in length, which is the same size as a positive control. After transfection of pb74, pb75, and pb76, intensities of KRAS RT-PCR bands were weaker than in negative controls. A further reference (ref-BMG) was generated by performing RT-PCR reaction for a house-keeping gene: beta-microglobin (BMG), which can be used for quantitation and normalization. Figure 6B shows quantification of effects of KRAS suppression by pb74, pb75, and pb76 which were measured in four independent experiments. All three constructs are able to suppress KRAS mRNA expression by more than 50%.

[0445] Next, human Zinc Finger KRAB protein ZIM 3 was used to create a gene regulation agent comprising a modified Zinc Finger KRAB molecule, in which in 7 zinc fingers alpha helix sequences were replaced with equivalent alpha helix sequences as evaluated in the experiments above. Plasmid, pb84 (represented by SEQ ID NOs: 48-50 (encoding full-length DNA, cDNA, and amino acid sequences) was constructed to have a cDNA of modified Zim3, in which changes were made to 7 alpha helix sequences of original Zim3. Specifically, the amino acid sequences of the alpha helices of Zim3 were modified to change its target sequences, such that this modified version of Zim3 recognize KRAS target sequence (e.g., a sequence of SEQ ID NO: 44).

[0446] pb84 DNA - SEQ ID NO: 48 GACTCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTA

TTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGT

TACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT

GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACG

TCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCA

TATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTA

TGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTC

ATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGG

TTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTT

GGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACG

CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCT

AACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGA

GACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTATGAACAATTCCCAGGGAAGAGT

GACCTTCGAGGATGTCACTGTGAACTTCACCCAGGGGGAGTGGCAGCGGCTGAATC

CCGAACAGAGAAACTTGTACAGGGATGTGATGCTGGAGAATTACAGCAACCTTGTC

TCTGTGGGACAAGGGGAAACCACCAAACCCGATGTGATCTTGAGGTTGGAACAAGG

AAAGGAGCCATGGTTGGAGGAAGAGGAAGTGCTGGGAAGTGGCCGTGCAGAAAAA

AATGGGGACATTGGAGGGCAGATTTGGAAGCCAAAGGATGTGAAAGAGAGTCTCGC

AAGAGAAGTCCCATCAATCAATAAGGAAACGCTGACTACGCAGAAAGGTGTAGAAT

GTGACGGATCTAAGAAAATACTTCCACTGGGCATAGATGATGTATCTTCCTTGCAAC

ACTATGTACAAAATAATTCTCACGATGATAATGGATACAGAAAATTAGTTGGCAATA

ATCCATCCAAATTTGTAGGACAACAACTGAAATGTAATGCCTGTAGAAAGTTATTCA

GTTCAAAGTCACGCCTTCAAAGTCACCTGAGGAGGCATGCCTGTCAAAAACCCTTTG

AATGTCATAGCTGTGGAAGAGCATTCGGGGAGAAGTGGAAACTTGATAAACATCAG

AAAACTCACGCAGAGGAAAGGCCCTATAAATGTGAGAACTGTGGAAATGCCTACAA

GCAGAAGTCAAATCTCTTTCAACATCAGAAAATGCATACTAAAGAGAAACCCTATC

AGTGTAAGACATGTGGAAAAGCCTTTTCCTGGAAATCATCCTGCATTAATCATGAGA

AAATTCATAATGCCAAGAAATCCTATCAGTGTAATGAATGTGAGAAATCCTTCAGGC

AGTCCGGCGACCTGACCCGGCATAAAAAAGTTCACACTGGACAAAAACCCTTTCAA

TGTACGGACTGTGGAAAGGCTTTCATTCGGTCCGACAACCTGACCACCCACCAGAG

AATACACACGGGAGAGAAACCCTATAAATGTAGCATATGTGAGAAGGCCTTTTCCC GGTCCTCCGACCTGACCCGGCATGAGAAAATTCACACTGGGAAGAGAGCTTATGAG

TGTGATCTATGTGGAAATACCTTTATCCGGTCCGACGCCCTGACCCGGCACCATAAA

AAAATCCATACTGGGGAAAAGCCCTATGAATGTAACAGATGTGGAAAAGCCTTCTT

TCGGTCCGACGCCCTGTCCGAGCATCAGAAAACTCATAGCGGAGAGAGGACCTATA

GATGTAGTGAATGTGGAAAAACCTTCATCCGGTCCTCCAACCTGACCCGGCATAAAA

AAACCCATACTGGACAAAAACCTTATGGATGTTCTGAATGCGGTAAAGCCTTCGCTC

GGTCCGACGCCCTGACCACCCACCAGAAAAGGATTCACTCCAGATAGGCGGCCGCT

CGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTG

CCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACT

CCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGT

CATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGA

CAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTACTGGGCGGTTTTA

TGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAA

GCCCTGCAAAGTAAACTGGATGGCTTTCTCGCCGCCAAGGATCTGATGGCGCAGGG

GATCAAGCTCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGAT

GGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGG

GCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGG

GCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGA

CGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCT

CGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGC

AGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATG

CAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGA

AACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGAT

GATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAA

GGCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGC

CGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGG

GTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGC

TTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATT

CGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAATTATTAACGCTTA

CAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATA

CAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAAT ACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATA

GCACGTGCTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGAT

AATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCC

GTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCT

TGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTA

CCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTC

CTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACA

TACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGT

CTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTG

AACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGA

GATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCG

GACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTC

CAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTG

AGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGC

AACGCGGCCTTTTTACGGTTCCTGGGCTTTTGCTGGCCTTTTGCTCACATGTTCTT

[0447] pb84 cDNA - SEQ ID NO: 49

ATGAACAATTCCCAGGGAAGAGTGACCTTCGAGGATGTCACTGTGAACTTCACCCA

GGGGGAGTGGCAGCGGCTGAATCCCGAACAGAGAAACTTGTACAGGGATGTGATGC

TGGAGAATTACAGCAACCTTGTCTCTGTGGGACAAGGGGAAACCACCAAACCCGAT

GTGATCTTGAGGTTGGAACAAGGAAAGGAGCCATGGTTGGAGGAAGAGGAAGTGCT

GGGAAGTGGCCGTGCAGAAAAAAATGGGGACATTGGAGGGCAGATTTGGAAGCCA

AAGGATGTGAAAGAGAGTCTCGCAAGAGAAGTCCCATCAATCAATAAGGAAACGCT

GACTACGCAGAAAGGTGTAGAATGTGACGGATCTAAGAAAATACTTCCACTGGGCA

TAGATGATGTATCTTCCTTGCAACACTATGTACAAAATAATTCTCACGATGATAATG

GATACAGAAAATTAGTTGGCAATAATCCATCCAAATTTGTAGGACAACAACTGAAA

TGTAATGCCTGTAGAAAGTTATTCAGTTCAAAGTCACGCCTTCAAAGTCACCTGAGG

AGGCATGCCTGTCAAAAACCCTTTGAATGTCATAGCTGTGGAAGAGCATTCGGGGA

GAAGTGGAAACTTGATAAACATCAGAAAACTCACGCAGAGGAAAGGCCCTATAAAT

GTGAGAACTGTGGAAATGCCTACAAGCAGAAGTCAAATCTCTTTCAACATCAGAAA

ATGCATACTAAAGAGAAACCCTATCAGTGTAAGACATGTGGAAAAGCCTTTTCCTGG AAATCATCCTGCATTAATCATGAGAAAATTCATAATGCCAAGAAATCCTATCAGTGT AATGAATGTGAGAAATCCTTCAGGCAGTCCGGCGACCTGACCCGGCATAAAAAAGT TCACACTGGACAAAAACCCTTTCAATGTACGGACTGTGGAAAGGCTTTCATTCGGTC CGACAACCTGACCACCCACCAGAGAATACACACGGGAGAGAAACCCTATAAATGTA GCATATGTGAGAAGGCCTTTTCCCGGTCCTCCGACCTGACCCGGCATGAGAAAATTC ACACTGGGAAGAGAGCTTATGAGTGTGATCTATGTGGAAATACCTTTATCCGGTCCG ACGCCCTGACCCGGCACCATAAAAAAATCCATACTGGGGAAAAGCCCTATGAATGT AACAGATGTGGAAAAGCCTTCTTTCGGTCCGACGCCCTGTCCGAGCATCAGAAAACT CATAGCGGAGAGAGGACCTATAGATGTAGTGAATGTGGAAAAACCTTCATCCGGTC CTCCAACCTGACCCGGCATAAAAAAACCCATACTGGACAAAAACCTTATGGATGTT CTGAATGCGGTAAAGCCTTCGCTCGGTCCGACGCCCTGACCACCCACCAGAAAAGG ATTCACTCCAGATAG

[0448] pb84 amino acid - SEQ ID NO: 50

MNNSQGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLVSVGQGETTKPDV ILRLEQGKEPWLEEEEVLGSGRAEKNGDIGGQIWKPKDVKESLAREVPSINKETLTTQKG VECDGSKKILPLGIDDVSSLQHYVQNNSHDDNGYRKLVGNNPSKFVGQQLKCNACRKL FSSKSRLQSHLRRHACQKPFECHSCGRAFGEKWKLDKHQKTHAEERPYKCENCGNAYK QKSNLFQHQKMHTKEKPYQCKTCGKAFSWKSSCINHEKIHNAKKSYQCNECEKSFRQS GDLTRHKKVHTGQKPFQCTDCGKAFIRSDNLTTHQRIHTGEKPYKCSICEKAFSRSSDLT RHEKIHTGKRAYECDLCGNTFIRSDALTRHHKKIHTGEKPYECNRCGKAFFRSDALSEH QKTHSGERTYRCSECGKTFIRSSNLTRHKKTHTGQKPYGCSECGKAFARSDALTTHQKR IHSR

[0449] Additionally, in another construct a KRAB domain is fused at N-terminus of a

DLRR molecule, encoded in plasmid 86 (represented by SEQ ID NOs: 51-53, for full-length DNA, cDNA, and amino acid sequences), so that this molecule could bind to a KRAS target sequence (e.g., a sequence of SEQ ID NO: 44).

[0450] pb86 DNA - SEQ ID NO: 51

GACTCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTA

TTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGT TACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT

GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACG

TCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCA

TATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTA

TGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTC

ATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGG

TTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTT

GGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACG

CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCT

AACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGA

GACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTATGGATGCTAAGTCACTAACTGCC

TGGTCCCGGACACTGGTGACCTTCAAGGATGTATTTGTGGACTTCACCAGGGAGGAG

TGGAAGCTGCTGGACACTGCTCAGCAGATCGTGTACAGAAATGTGATGCTGGAGAA

CTATAAGAACCTGGTTTCCTTGGGTTATCAGCTTACTAAGCCAGATGTGATCCTCCG

GTTGGAGAAGGGAGAAGAGCCCGGCGGTTCCGGCGGAGGGTCGATGGCGGCGATG

GCCGAGCGGCCCTTCCAGTGCAGGATCTGTATGCGCAACTTTTCTCAGTCCGGCGAC

CTGACCCGGCACATCAGAACCCATACAGGCGAAAAGCCTTTCGCCTGCGACATTTGT

GGGAGAAAATTTGCTCGGTCCGACAACCTGACCACCCATACCAAGATCCACACCGG

CTCTCAGAAACCATTCCAGTGCCGCATTTGTATGCGGAATTTTTCCCGGTCCTCCGAC

CTGACCCGGCATATCCGCACTCACACCGGAGAGAAGCCCTTTGCTTGCGACATTTGT

GGCAGGAAATTTGCTCGGTCCGACGCCCTGACCCGGCACACTAAGATCCATACTGG

GTCACAGAAACCTTTCCAGTGCCGGATTTGTATGAGAAACTTTAGCCGGTCCGACGC

CCTGTCCGAGCACATCAGAACCCATACAGGCGAAAAGCCTTTCGCCTGCGACATTTG

TGGGAGAAAATTTGCTCGGTCCTCCAACCTGACCCGGCATACCAAGATCCACACCG

GCTCTCAGAAACCATTCCAGTGCCGCATTTGTATGCGGAATTTTTCCCGGTCCGACG

CCCTGACCACCCACATCAGAACACATACTGGGCTGAGAGGATCCAATTCTGGTGATC

CTCGGAGACACAGTCTGGGCGGTTCTCGTAAACCCGATCTGATTGCCTATAAAAACT

TTGATCTGCTGGTCATTGTTCTTAAGCCTAAATACTCCCAGAATTCTGGTGATCCTCG

GAGACACAGTCTGGGCGGTTCTCGTAAACCCGATGGTGCTATTTATACTGTTGGTTC

TCCTATTGATTATGGTGTTATTGTTGTTACTAAACCTTGAGCGGCCGCTCGAGTCTAG

AGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATC TGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTC

CTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTC

TGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAG

GCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTACTGGGCGGTTTTATGGACAGCA

AGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAA

AGTAAACTGGATGGCTTTCTCGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGCT

CTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCAC

GCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACA

GACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGT

TCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGC

GCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGT

CACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCC

TGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGC

GGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCA

TCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGAC

GAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCAT

GCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCAT

GGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGA

CCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGA

ATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCAT

CGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAATTATTAACGCTTACAATTTCCTG

ATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACAGGTGGCAC

TTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAAT

ATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAGCACGTGCTA

AAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGA

CCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGA

TCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAA

AAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTT

TCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTA

GCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCT

GCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTT GGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTT

CGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAG

CGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCC

GGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAAC

GCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTT

TGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTT

TTACGGTTCCTGGGCTTTTGCTGGCCTTTTGCTCACATGTTCTT

[0451] pb86 cDNA - SEQ ID NO: 52

ATGGATGCTAAGTCACTAACTGCCTGGTCCCGGACACTGGTGACCTTCAAGGATGTA

TTTGTGGACTTCACCAGGGAGGAGTGGAAGCTGCTGGACACTGCTCAGCAGATCGT

GTACAGAAATGTGATGCTGGAGAACTATAAGAACCTGGTTTCCTTGGGTTATCAGCT

TACTAAGCCAGATGTGATCCTCCGGTTGGAGAAGGGAGAAGAGCCCGGCGGTTCCG

GCGGAGGGTCGATGGCGGCGATGGCCGAGCGGCCCTTCCAGTGCAGGATCTGTATG

CGCAACTTTTCTCAGTCCGGCGACCTGACCCGGCACATCAGAACCCATACAGGCGA

AAAGCCTTTCGCCTGCGACATTTGTGGGAGAAAATTTGCTCGGTCCGACAACCTGAC

CACCCATACCAAGATCCACACCGGCTCTCAGAAACCATTCCAGTGCCGCATTTGTAT

GCGGAATTTTTCCCGGTCCTCCGACCTGACCCGGCATATCCGCACTCACACCGGAGA

GAAGCCCTTTGCTTGCGACATTTGTGGCAGGAAATTTGCTCGGTCCGACGCCCTGAC

CCGGCACACTAAGATCCATACTGGGTCACAGAAACCTTTCCAGTGCCGGATTTGTAT

GAGAAACTTTAGCCGGTCCGACGCCCTGTCCGAGCACATCAGAACCCATACAGGCG

AAAAGCCTTTCGCCTGCGACATTTGTGGGAGAAAATTTGCTCGGTCCTCCAACCTGA

CCCGGCATACCAAGATCCACACCGGCTCTCAGAAACCATTCCAGTGCCGCATTTGTA

TGCGGAATTTTTCCCGGTCCGACGCCCTGACCACCCACATCAGAACACATACTGGGC

TGAGAGGATCCAATTCTGGTGATCCTCGGAGACACAGTCTGGGCGGTTCTCGTAAAC

CCGATCTGATTGCCTATAAAAACTTTGATCTGCTGGTCATTGTTCTTAAGCCTAAATA

CTCCCAGAATTCTGGTGATCCTCGGAGACACAGTCTGGGCGGTTCTCGTAAACCCGA

TGGTGCTATTTATACTGTTGGTTCTCCTATTGATTATGGTGTTATTGTTGTTACTAAAC

CTTGA*

[0452] pb86 amino acid - SEQ ID NO: 53 MDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQL TKPDVILRLEKGEEPGGSGGGSMAAMAERPFQCRICMRNFSQSGDLTRHIRTHTGEKPF ACDICGRKF ARSDNLTTHTKIHTGSQKPFQCRICMRNF SRS SDLTRHIRTHTGEKPF ACDI CGRKFARSDALTRHTKIHTGSQKPFQCRICMRNFSRSDALSEHIRTHTGEKPFACDICGR KF ARS SNLTRHTKIHTGSQKPFQCRICMRNF SRSD ALTTHIRTHTGLRGSNSGDPRRHSL GGSRKPDLIAYKNFDLLVIVLKPKYSQNSGDPRRHSLGGSRKPDGAIYTVGSPIDYGVIV VTKP*

[0453] Then, programmed KRAS gene suppression was performed and analyzed in a human U937 cell line over time. As a control, plasmid pb80 was created: its cDNA encodes a zinc finger array, recognizing a sequence not present in the KRAS gene: 5’- GGGGAGGACGCGGTG-3’ (SEQ ID NO.54). Plasmids including pb75 (i.e., DLRR), pb80 (i.e., mock), pb84 (i.e., ZIM3 KRAS Sequence Modified) or pb86 (i.e., KRAB-DLRR) were introduced into U937 cells by electroporation. A “no DNA” transfection was used as a negative control. At various intervals, here, three, five, and seven days post electroporation, cells transfected with each plasmid were detached and collected. Total RNA from each condition was then extracted by using Trizol reagent. Five hundred nanograms of total RNA was then converted into DNA by reverse transcription (RT) using a reverse transcriptase, appropriate buffer, and dNTPs. After this RT reaction, a PCR test was conducted using a primer set of Popl33 (SEQ ID. NO: 46) and Popl34 (SEQ ID. NO: 47). Primer Popl33 is a forward primer binding within Exonl of the human KRAS gene; and Pop 134 is a reverse primer binding on Exon2 of human KRAS gene. When KRAS mRNA was present, a 184 bp RT-PCR amplicon was detected. Figure 7 shows successful suppression of KRAS gene expression by each of pb84 (sequence specificity modified KRAS-ZIM3), pb86 (KRAB-DLRR) and pb75 (DLRR). At day 3 post transfection, pb75, pb84 and pb86 all inhibit KRAS gene expression in comparison to no treatment control. Over time course, at day 5, inhibitory effects from pb75, pb84 and pb86 continues, but mock control, pb80, starts losing inhibition of KRAS gene expression. At day 7, only pb84 (modified KRAS-ZIM3) has sustained inhibitory effects on KRAS gene expression. In addition, for analysis KRAS mRNA levels were also normalized by using RT-PCR reaction from a house-keeping gene: beta-microglobin (BMG), for quantification of KRAS gene regulation effects. As illustrated in Figure 8, measuring relative expression of KRAS in U937 cells over time showed that p84 (modified KRAS-ZIM3) maintained suppression of KRAS expression longer compared to all other conditions and gene regulatory agents tested. At the day 7, only 37.14% of KRAS mRNA can be expressed, significantly lower than others formats. Collectively, these results show that an exemplary sequence specificity modified KRAS-Zinc Finger molecule, modified KRAS-Zim3, showed the most effective inhibitory effects on KRAS gene expression in this experimental condition.

[0454] As noted above, KRAS is one the most frequently mutated oncogenes in human cancer, yet so far it has remained an “undruggable” target for most of the mutations involved. Thus, it is desirably for exemplary gene regulatory agents to regulate expression of KRAS in the context of cancer cells, Next, KRAS expression was measured in a human colorectal carcinoma cell line (i.e., HCT116) following electroporation with Pb75 (DLRR), Pb80 (mock), Pb84 (e.g., ZIM3 KRAS Sequence Modified) or Pb86 (KRAB-DLRR). Figures 9A to 9C show reduction in KRAS mRNA levels as measured by RT-PCR in cells treated with either Pb84 or Pb86, as compared to other conditions tested, including controls (n=3). Average reduction in expression of KRAS, using pb75, pb84 or pb86 is more than 50% (Figure 10). Moreover, Figure 11 shows robust reduction in KRAS protein levels (n=4) for cancer cells treated with either pb84 or pb86, thereby confirming the results from the RT-PCR as shown in Figures9A-9C and Figure 10.

[0455] Overall, these results demonstrate that KRAS gene expression was suppressed by an exemplary Sequence Specificity Modified KRAS-ZIM3 based molecule (e.g., a gene regulation agent having KRAB-X-ZFn structure) as well as molecules having KRAB-DLR structure (e.g., a KRAB-Xi-ZFn-Y-X2) as compared to DLRR molecules or controls.

Collectively this illustrates that gene regulation agent comprising a modified Zinc Finger KRAB molecule can be used to successfully achieve targeted, sustained reduction in gene suppression.

EXAMPLE 2: Transcription modification mediated suppression of Bell 1A gene expression in mammalian cells

[0456] In this example, human BCL11 A gene expression is inhibited by programmed gene regulation via a gene regulation agent comprising a modified Zinc Finger KRAB molecule. Reducing expression of the fetal hemoglobin (HbF) repressor BCL11 A leads to a simultaneous increase in y-globin expression and reduction in P-globin expression (Sankaran VG, et al. Science. 2008; 19 Dec, 322(5909): 1839-1842, which is herein incorporated by reference in its entirety). Thus, suppression of expression of BCL11 A can be used for development of a treatment for P-hemoglobinopathies, including sickle cell disease (SCD) and P-thalassemia. As demonstrated herein, gene regulation agents comprising an intramolecularly modified Zinc Finger KRAB molecule can be used to suppress KRAS gene expression as evidenced by reduced mRNA levels.

[0457] In this example, two different ZIM3 based gene regulation agents comprising a modified Zinc Finger KRAB molecule are designed and encoded on plasmids. One plasmid, pb89, comprises changes to seven zinc finger helices (represented by SEQ ID NOs: 55-57, for full-length DNA, cDNA, and amino acid sequences, respectively) and is designed to recognize a 21-nucleotide sequence, 5’- GCCCGCCCCGCAGCCCACCAT-3’ (SEQ ID NO.58) located on a lagging strand in front of Exon 1 of human BCL11 A (See Figure 12). Another plasmid, pb90, (represented by SEQ ID NOs: 59-61, for full-length DNA, cDNA, and amino acid sequences, respectively) comprises changes to seven zinc finger helices and is designed to recognize a 21- nucleotide sequence of 5 -CGCCGCCGCCGCCCGCCCCGC-3 (SEQ ID NO.62), located on a lagging strand of in front of Exon 1, comprising a different sequence than the first plasmid of human BCL11 A (See Figure 12).

[0458] Next, programmed BCL11 A gene suppression was performed and analyzed. Plasmids including pb89 (i.e., DLRR), or pb90 were introduced into U937 cells by electroporation. A “no DNA” transfection was used as a control. Seventy -two hours post electroporation, cells transfected with each plasmid were detached and collected. Total RNAs from each condition were then extracted by using Trizol reagent. Five hundred nanograms of total RNA was then converted into DNA by reverse transcription (RT) using a reverse transcriptase, corresponding buffer, and dNTPs. After this RT reaction, a PCR test was conducted using a primer set of Pop275 (5’-ATGTCTCGCCGCAAGCAAGG-3’, SEQ ID. NO: 63) and Pop276 (5’-AGGGGAAGGTGGCTTATCCA-3’, SEQ ID. NO: 64). Primer Pop275 is a forward primer binding within Exonl of the human BCL11 A gene; and Pop276 is a reverse primer binding on Exon2 of human BCL11 A gene. When BCL11 A mRNA was present, a 261 bp RT-PCR amplicon was detected. Figures 13A to 13C show successful suppression of BCL11 A gene expression by pb89 and pb90, in three independent experiments. In each condition, RT-PCR conducted using a primer set of Pop275 and Pop276 showed RT-PCR amplicons of 261bp in length, which is the same size as a positive control. After transfection pb89, and pb90, intensity of BCL11 A-ZIM3 RT-PCR bands was weaker than in controls. A further reference (ref-BMG) was generated by performing RT-PCR reaction for a house-keeping gene: beta-microglobin (BMG), which can be used for quantitation and normalization.

[0459] Measuring relative expression of BCL11 A in U937 cells over time showed that pb89 and pb90 (ZIM3 BCL11 A Sequence Modified) suppress BCL11 A gene transcript and reduced BCL1 la mRNA to 38.6% and 46.46% after normalization using a house-keeping gene beta-microglobin (BMG) expression as reference (Figure 14).

[0460] Overall, these results demonstrate that BCL11 A gene expression was suppressed by an exemplary Sequence Specificity Modified BCL11 A-ZIM3 based molecule (e.g., a gene regulation agent having KRAB-X-ZFn structure) as compared to controls. Collectively, these examples illustrate that gene regulation agents comprising an sequence specificity modified Zinc Finger KRAB molecule can be used to successfully perform targeted, programmed gene suppression of multiple targets.

[0461] pb89 DNA - SEQ ID NO: 55

GACTCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTA TTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGT TACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACG TCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCA TATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTA TGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTC ATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGG TTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTT GGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACG CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCT AACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGA GACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTATGAACAATTCCCAGGGAAGAGT

GACCTTCGAGGATGTCACTGTGAACTTCACCCAGGGGGAGTGGCAGCGGCTGAATC

CCGAACAGAGAAACTTGTACAGGGATGTGATGCTGGAGAATTACAGCAACCTTGTC

TCTGTGGGACAAGGGGAAACCACCAAACCCGATGTGATCTTGAGGTTGGAACAAGG

AAAGGAGCCATGGTTGGAGGAAGAGGAAGTGCTGGGAAGTGGCCGTGCAGAAAAA

AATGGGGACATTGGAGGGCAGATTTGGAAGCCAAAGGATGTGAAAGAGAGTCTCGC

AAGAGAAGTCCCATCAATCAATAAGGAAACGCTGACTACGCAGAAAGGTGTAGAAT

GTGACGGATCTAAGAAAATACTTCCACTGGGCATAGATGATGTATCTTCCTTGCAAC

ACTATGTACAAAATAATTCTCACGATGATAATGGATACAGAAAATTAGTTGGCAATA

ATCCATCCAAATTTGTAGGACAACAACTGAAATGTAATGCCTGTAGAAAGTTATTCA

GTTCAAAGTCACGCCTTCAAAGTCACCTGAGGAGGCATGCCTGTCAAAAACCCTTTG

AATGTCATAGCTGTGGAAGAGCATTCGGGGAGAAGTGGAAACTTGATAAACATCAG

AAAACTCACGCAGAGGAAAGGCCCTATAAATGTGAGAACTGTGGAAATGCCTACAA

GCAGAAGTCAAATCTCTTTCAACATCAGAAAATGCATACTAAAGAGAAACCCTATC

AGTGTAAGACATGTGGAAAAGCCTTTTCCTGGAAATCATCCTGCATTAATCATGAGA

AAATTCATAATGCCAAGAAATCCTATCAGTGTAATGAATGTGAGAAATCCTTCAGGC

GGTCCTCCCACCTGACCCGGCATAAAAAAGTTCACACTGGACAAAAACCCTTTCAAT

GTACGGACTGTGGAAAGGCTTTCATTCGGTCCGACACCCTGACCCGGCACCAGAGA

ATACACACGGGAGAGAAACCCTATAAATGTAGCATATGTGAGAAGGCCTTTTCCCG

GTCCGACCACCTGACCCGGCATGAGAAAATTCACACTGGGAAGAGAGCTTATGAGT

GTGATCTATGTGGAAATACCTTTATCGACCGGTCCCACCTGACCACCCATAAAAAAA

TCCATACTGGGGAAAAGCCCTATGAATGTAACAGATGTGGAAAAGCCTTCTTTGACC

GGTCCCACCTGACCCGGCATCAGAAAACTCATAGCGGAGAGAGGACCTATAGATGT

AGTGAATGTGGAAAAACCTTCATCCGGTCCGACGCCCTGACCCGGCATAAAAAAAC

CCATACTGGACAAAAACCTTATGGATGTTCTGAATGCGGTAAAGCCTTCGCTCGGTC

CGACGCCCTGTCCCAGCACCAGAAAAGGATTCACTCCAGATAGGCGGCCGCTCGAG

TCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAG

CCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCA

CTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATT

CTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAA

TAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTACTGGGCGGTTTTATGG ACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCC

CTGCAAAGTAAACTGGATGGCTTTCTCGCCGCCAAGGATCTGATGGCGCAGGGGAT

CAAGCTCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGA

TTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCA

CAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCG

CCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGA

GGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGA

CGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGG

ATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAAT

GCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAAC

ATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGAT

CTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGC

GAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGA

ATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTG

TGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTG

GCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGC

AGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAATTATTAACGCTTACAA

TTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACAG

GTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACA

TTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAGCA

CGTGCTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAAT

CTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTA

GAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGC

AAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCA

ACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTT

CTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATAC

CTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTA

CCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG

GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATA

CCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACA

GGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGG GGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCG

TCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACG

CGGCCTTTTTACGGTTCCTGGGCTTTTGCTGGCCTTTTGCTCACATGTTCTT*

[0462] pb89 cDNA - SEQ ID NO: 56

ATGAACAATTCCCAGGGAAGAGTGACCTTCGAGGATGTCACTGTGAACTTCACCCA

GGGGGAGTGGCAGCGGCTGAATCCCGAACAGAGAAACTTGTACAGGGATGTGATGC

TGGAGAATTACAGCAACCTTGTCTCTGTGGGACAAGGGGAAACCACCAAACCCGAT

GTGATCTTGAGGTTGGAACAAGGAAAGGAGCCATGGTTGGAGGAAGAGGAAGTGCT

GGGAAGTGGCCGTGCAGAAAAAAATGGGGACATTGGAGGGCAGATTTGGAAGCCA

AAGGATGTGAAAGAGAGTCTCGCAAGAGAAGTCCCATCAATCAATAAGGAAACGCT

GACTACGCAGAAAGGTGTAGAATGTGACGGATCTAAGAAAATACTTCCACTGGGCA

TAGATGATGTATCTTCCTTGCAACACTATGTACAAAATAATTCTCACGATGATAATG

GATACAGAAAATTAGTTGGCAATAATCCATCCAAATTTGTAGGACAACAACTGAAA

TGTAATGCCTGTAGAAAGTTATTCAGTTCAAAGTCACGCCTTCAAAGTCACCTGAGG

AGGCATGCCTGTCAAAAACCCTTTGAATGTCATAGCTGTGGAAGAGCATTCGGGGA

GAAGTGGAAACTTGATAAACATCAGAAAACTCACGCAGAGGAAAGGCCCTATAAAT

GTGAGAACTGTGGAAATGCCTACAAGCAGAAGTCAAATCTCTTTCAACATCAGAAA

ATGCATACTAAAGAGAAACCCTATCAGTGTAAGACATGTGGAAAAGCCTTTTCCTGG

AAATCATCCTGCATTAATCATGAGAAAATTCATAATGCCAAGAAATCCTATCAGTGT

AATGAATGTGAGAAATCCTTCAGGCGGTCCTCCCACCTGACCCGGCATAAAAAAGTT

CACACTGGACAAAAACCCTTTCAATGTACGGACTGTGGAAAGGCTTTCATTCGGTCC

GACACCCTGACCCGGCACCAGAGAATACACACGGGAGAGAAACCCTATAAATGTAG

CATATGTGAGAAGGCCTTTTCCCGGTCCGACCACCTGACCCGGCATGAGAAAATTCA

CACTGGGAAGAGAGCTTATGAGTGTGATCTATGTGGAAATACCTTTATCGACCGGTC

CCACCTGACCACCCATAAAAAAATCCATACTGGGGAAAAGCCCTATGAATGTAACA

GATGTGGAAAAGCCTTCTTTGACCGGTCCCACCTGACCCGGCATCAGAAAACTCATA

GCGGAGAGAGGACCTATAGATGTAGTGAATGTGGAAAAACCTTCATCCGGTCCGAC

GCCCTGACCCGGCATAAAAAAACCCATACTGGACAAAAACCTTATGGATGTTCTGA

ATGCGGTAAAGCCTTCGCTCGGTCCGACGCCCTGTCCCAGCACCAGAAAAGGATTC

ACTCCAGATAG [0463] pb89 amino acid - SEQ ID NO: 57

MNNSQGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLVSVGQGETTKPDV ILRLEQGKEPWLEEEEVLGSGRAEKNGDIGGQIWKPKDVKESLAREVPSINKETLTTQKG VECDGSKKILPLGIDDVSSLQHYVQNNSHDDNGYRKLVGNNPSKFVGQQLKCNACRKL FSSKSRLQSHLRRHACQKPFECHSCGRAFGEKWKLDKHQKTHAEERPYKCENCGNAYK QKSNLFQHQKMHTKEKPYQCKTCGKAFSWKSSCINHEKIHNAKKSYQCNECEKSFRRS SHLTRHKKVHTGQKPFQCTDCGKAFIRSDTLTRHQRIHTGEKPYKC SICEKAF SRSDHLT

RHEKIHTGKRAYECDLCGNTFIDRSHLTTHKKIHTGEKPYECNRCGKAFFDRSHLTRHQ KTHSGERTYRCSECGKTFIRSDALTRHKKTHTGQKPYGCSECGKAFARSDALSQHQKRI HSR

[0464] pb90 DNA - SEQ ID NO: 59

GACTCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTA TTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGT TACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACG TCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCA TATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTA

TGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTC ATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGG TTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTT GGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACG CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCT AACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGA

GACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTATGAACAATTCCCAGGGAAGAGT GACCTTCGAGGATGTCACTGTGAACTTCACCCAGGGGGAGTGGCAGCGGCTGAATC CCGAACAGAGAAACTTGTACAGGGATGTGATGCTGGAGAATTACAGCAACCTTGTC TCTGTGGGACAAGGGGAAACCACCAAACCCGATGTGATCTTGAGGTTGGAACAAGG AAAGGAGCCATGGTTGGAGGAAGAGGAAGTGCTGGGAAGTGGCCGTGCAGAAAAA AATGGGGACATTGGAGGGCAGATTTGGAAGCCAAAGGATGTGAAAGAGAGTCTCGC

AAGAGAAGTCCCATCAATCAATAAGGAAACGCTGACTACGCAGAAAGGTGTAGAAT GTGACGGATCTAAGAAAATACTTCCACTGGGCATAGATGATGTATCTTCCTTGCAAC

ACTATGTACAAAATAATTCTCACGATGATAATGGATACAGAAAATTAGTTGGCAATA

ATCCATCCAAATTTGTAGGACAACAACTGAAATGTAATGCCTGTAGAAAGTTATTCA

GTTCAAAGTCACGCCTTCAAAGTCACCTGAGGAGGCATGCCTGTCAAAAACCCTTTG

AATGTCATAGCTGTGGAAGAGCATTCGGGGAGAAGTGGAAACTTGATAAACATCAG

AAAACTCACGCAGAGGAAAGGCCCTATAAATGTGAGAACTGTGGAAATGCCTACAA

GCAGAAGTCAAATCTCTTTCAACATCAGAAAATGCATACTAAAGAGAAACCCTATC

AGTGTAAGACATGTGGAAAAGCCTTTTCCTGGAAATCATCCTGCATTAATCATGAGA

AAATTCATAATGCCAAGAAATCCTATCAGTGTAATGAATGTGAGAAATCCTTCAGGC

GGTCCTCCGACCTGACCCGGCATAAAAAAGTTCACACTGGACAAAAACCCTTTCAAT

GTACGGACTGTGGAAAGGCTTTCATTCGGTCCGACACCCTGACCCGGCACCAGAGA

ATACACACGGGAGAGAAACCCTATAAATGTAGCATATGTGAGAAGGCCTTTTCCCG

GTCCGACACCCTGACCCGGCATGAGAAAATTCACACTGGGAAGAGAGCTTATGAGT

GTGATCTATGTGGAAATACCTTTATCCGGTCCGACACCCTGACCCGGCATAAAAAAA

TCCATACTGGGGAAAAGCCCTATGAATGTAACAGATGTGGAAAAGCCTTCTTTCGGT

CCGACCACCTGACCCGGCATCAGAAAACTCATAGCGGAGAGAGGACCTATAGATGT

AGTGAATGTGGAAAAACCTTCATCGACCGGTCCCACCTGACCCGGCATAAAAAAAC

CCATACTGGACAAAAACCTTATGGATGTTCTGAATGCGGTAAAGCCTTCGCTCGGTC

CGACCACCTGTCCGAGCACCAGAAAAGGATTCACTCCAGATAGGCGGCCGCTCGAG

TCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAG

CCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCA

CTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATT

CTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAA

TAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTACTGGGCGGTTTTATGG

ACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCC

CTGCAAAGTAAACTGGATGGCTTTCTCGCCGCCAAGGATCTGATGGCGCAGGGGAT

CAAGCTCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGA

TTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCA

CAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCG

CCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGA

GGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGA CGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGG

ATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAAT

GCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAAC

ATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGAT

CTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGC

GAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGA

ATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTG

TGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTG

GCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGC

AGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAATTATTAACGCTTACAA

TTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACAG

GTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACA

TTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAGCA

CGTGCTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAAT

CTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTA

GAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGC

AAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCA

ACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTT

CTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATAC

CTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTA

CCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG

GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATA

CCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACA

GGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGG

GGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCG

TCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACG

CGGCCTTTTTACGGTTCCTGGGCTTTTGCTGGCCTTTTGCTCACATGTTCTT*

[0465] pb91 DNA - SEQ ID NO: 60

ATGAACAATTCCCAGGGAAGAGTGACCTTCGAGGATGTCACTGTGAACTTCACCCA

GGGGGAGTGGCAGCGGCTGAATCCCGAACAGAGAAACTTGTACAGGGATGTGATGC TGGAGAATTACAGCAACCTTGTCTCTGTGGGACAAGGGGAAACCACCAAACCCGAT

GTGATCTTGAGGTTGGAACAAGGAAAGGAGCCATGGTTGGAGGAAGAGGAAGTGCT

GGGAAGTGGCCGTGCAGAAAAAAATGGGGACATTGGAGGGCAGATTTGGAAGCCA

AAGGATGTGAAAGAGAGTCTCGCAAGAGAAGTCCCATCAATCAATAAGGAAACGCT

GACTACGCAGAAAGGTGTAGAATGTGACGGATCTAAGAAAATACTTCCACTGGGCA

TAGATGATGTATCTTCCTTGCAACACTATGTACAAAATAATTCTCACGATGATAATG

GATACAGAAAATTAGTTGGCAATAATCCATCCAAATTTGTAGGACAACAACTGAAA TGTAATGCCTGTAGAAAGTTATTCAGTTCAAAGTCACGCCTTCAAAGTCACCTGAGG

AGGCATGCCTGTCAAAAACCCTTTGAATGTCATAGCTGTGGAAGAGCATTCGGGGA

GAAGTGGAAACTTGATAAACATCAGAAAACTCACGCAGAGGAAAGGCCCTATAAAT

GTGAGAACTGTGGAAATGCCTACAAGCAGAAGTCAAATCTCTTTCAACATCAGAAA

ATGCATACTAAAGAGAAACCCTATCAGTGTAAGACATGTGGAAAAGCCTTTTCCTGG

AAATCATCCTGCATTAATCATGAGAAAATTCATAATGCCAAGAAATCCTATCAGTGT

AATGAATGTGAGAAATCCTTCAGGCGGTCCTCCGACCTGACCCGGCATAAAAAAGT

TCACACTGGACAAAAACCCTTTCAATGTACGGACTGTGGAAAGGCTTTCATTCGGTC

CGACACCCTGACCCGGCACCAGAGAATACACACGGGAGAGAAACCCTATAAATGTA

GCATATGTGAGAAGGCCTTTTCCCGGTCCGACACCCTGACCCGGCATGAGAAAATTC

ACACTGGGAAGAGAGCTTATGAGTGTGATCTATGTGGAAATACCTTTATCCGGTCCG

ACACCCTGACCCGGCATAAAAAAATCCATACTGGGGAAAAGCCCTATGAATGTAAC

AGATGTGGAAAAGCCTTCTTTCGGTCCGACCACCTGACCCGGCATCAGAAAACTCAT

AGCGGAGAGAGGACCTATAGATGTAGTGAATGTGGAAAAACCTTCATCGACCGGTC

CCACCTGACCCGGCATAAAAAAACCCATACTGGACAAAAACCTTATGGATGTTCTG

AATGCGGTAAAGCCTTCGCTCGGTCCGACCACCTGTCCGAGCACCAGAAAAGGATT CACTCCAGATAG

[0466] pb92 DNA - SEQ ID NO: 61

MNNSQGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLVSVGQGETTKPDV ILRLEQGKEPWLEEEEVLGSGRAEKNGDIGGQIWKPKDVKESLAREVPSINKETLTTQKG VECDGSKKILPLGIDDVSSLQHYVQNNSHDDNGYRKLVGNNPSKFVGQQLKCNACRKL FSSKSRLQSHLRRHACQKPFECHSCGRAFGEKWKLDKHQKTHAEERPYKCENCGNAYK QKSNLFQHQKMHTKEKPYQCKTCGKAFSWKSSCINHEKIHNAKKSYQCNECEKSFRRS SDLTRHKKVHTGQKPFQCTDCGKAFIRSDTLTRHQRIHTGEKPYKCSICEKAFSRSDTLT

RHEKIHTGKRAYECDLCGNTFIRSDTLTRHKKIHTGEKPYECNRCGKAFFRSDHLTRHQ

KTHSGERTYRCSECGKTFIDRSHLTRHKKTHTGQKPYGCSECGKAFARSDHLSEHQKRI HSR

EQUIVALENTS

[0467] It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the present disclosure, which is further defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A gene regulation agent comprising a structure represented by:

[KRAB] - X - [ZFn], wherein:

(i) the KRAB element is or comprises a KRAB domain or portion thereof;

(ii) the X element is optional and is or comprises a functional domain; and

(iii) the ZFn element is or comprises at least five zinc finger arrays, wherein at least one of the zinc finger arrays comprises at least one modified alpha helix, wherein the at least one modified alpha helix is engineered that comprises one or more amino acid modifications relative to a corresponding wild-type alpha helix sequence.

2. The gene regulation agent of claim 1, wherein the at least one modified alpha helix is engineered to bind to a first landing site, wherein the landing site is associated with a target.

3. The gene regulation agent of claim 1 or 2, wherein a second zinc finger array comprises a second modified alpha helix, wherein the second modified alpha helix is engineered to bind to a second landing site, wherein the second landing site is associated with a target.

4. The gene regulation agent of claim 3, wherein the first landing site and the second landing site are associated with a single target.

5. The gene regulation agent of claim 3, wherein the first landing site and the second landing site are associated with different targets.

6. The gene regulation agent of claim 1, wherein the ZFn element is or comprises at least six, seven, eight, nine, ten, or eleven, or more, zinc finger arrays.

7. The gene regulation agent of claim 6, wherein the zinc finger arrays comprise at least one alpha helix engineered to comprise a modified amino acid sequence that differs from that of its corresponding wild type sequence.

8. The gene regulation agent of any one of the preceding claims, wherein any of the at least one modified alpha helix amino acid sequences comprises:

(i) one amino acid substitution mutation at a position selected from -1, +1, +2, +3, +4, +5, or +6 in the alpha helix;

(ii) two amino acid substitution mutations at positions selected from -1, +1, +2, +3, +4, +5, or +6 in the alpha helix;

(iii) three amino acid substitution mutations at positions selected from -1, +1, +2, +3, +4, +5, or +6 in the alpha helix;

(iv) four amino acid substitution mutations at positions selected from -1, +1, +2, +3, +4, +5, or +6 in the alpha helix

(v) five amino acid substitution mutation at positions selected from -1, +1, +2, +3, +4, +5, or +6 in the alpha helix;

(vi) six amino acid substitution mutations at positions selected from -1, +1, +2, +3, +4, +5, or +6 in the alpha helix; or

(vii) an amino acid substitution mutation at each position in the alpha helix.

9. The gene regulation of any one of claim 1-7, wherein any of the at least one modified alpha helix amino acid sequences comprises one or more amino acid substitution mutations at positions selected from -1, +2, +3 and +6, or any combinations thereof.

10. The gene regulation agent of any one of claims 1-9, wherein the ZFn element comprises at least five zinc finger arrays selected from ZNF 434, ZKSCAN1, ZKSCAN2, ZNF 552, ZNF 562, ZNF 763 or ZNF 793.

11. The gene regulation agent of any one of claims 1-9, wherein the ZFn element comprises up to seven zinc finger arrays selected from ZNF 177, ZNF 233, ZNF 248, ZNF 554, ZNF 566, ZNF 577, ZNF 701, ZNF 812, or ZNF 891.

12. The gene regulation agent of any one of claims 1-9, wherein the Zfn element comprises up to eight zinc finger arrays selected from AC003682.1, ZNF 3, ZNF 124, ZNF 506, ZNF 525, ZNF 561, ZNF 610, ZNF 625, ZNF 766, ZNF 222, or ZNF 223.

13. The gene regulation agent of any one of claims 1-9, wherein the Zfn element comprises up to nine zinc finger arrays selected from ZIK 1, ZNF 324, ZNF 510, ZNF 519, ZNF 643, ZNF 773, ZNF 486, ZNF 732, ZNF 776, or ZNF 582.

14. The gene regulation agent of any one of claims 1-9, wherein the Zfn element comprises up to ten zinc finger arrays selected from ZNF 154, ZNF 37A, ZNF 468, ZNF 563, ZNF 614, ZNF 649, ZNF 677, ZNF 682, ZNF 689, ZNF 727, ZNF 730, or ZNF 578.

15. The gene regulation agent of any one of claims 1-9, wherein the Zfn element comprises up to eleven zinc finger arrays selected from ZNF 155, ZNF 181, ZNF 253, ZNF 415, ZNF 416, ZNF 479, ZNF 485, ZNF 570, ZNF 675, ZNF 79 or ZIM 3.

16. The gene regulation agent of any one of the preceding claims, wherein the Zfn element comprises at least one zinc finger array selected from any one of the zinc finger arrays of claim 10, wherein the at least one zinc finger array is engineered to include at least one alpha helix originating from a second zinc finger array selected from any of the arrays of claim 10.

17. The gene regulation agent of claim 16, wherein the Zfn element comprises at least five zinc finger arrays from ZIM 3, wherein at least one of the zinc finger arrays from ZIM3 comprises at least one alpha helix originating from ZNF27.

18. The gene regulation agent of claim 17, wherein the Zfn element comprises at least seven zinc finger arrays, wherein each of the zinc finger arrays from ZIM 3 comprises an alpha helix originating from ZF27, optionally wherein the alpha helices from ZF27 are selected from the sequences of SEQ ID NOs: 22-32.

19. The gene regulation agent of claim 1, wherein the X element is or comprises a polynucleotide.

20. The gene regulation agent of claim 19, wherein the X element is or comprises a polynucleotide between about 2 and 500 nucleic acids in length.

21. The gene regulation agent of claim 1, wherein the X element is or comprises engineered nucleic acids analogous to those present in a wild KRAB element.

22. The gene regulation agent of claim 1, wherein the X element is or comprises a polypeptide.

23. The gene regulation agent of claim 22, wherein X element is or comprises a polypeptide between 2 and 100 amino acids in length or between 0.2 kD and 10 kD in size.

24. The gene regulation agent of claim 1, wherein the X element is or comprises a linker.

25. The gene regulation agent of claim 24, wherein the linker comprises a sequence of about 1 to about 20 amino acids.

26. The gene regulation agent of claim 1, wherein the gene regulation agent does not comprise the X element.

27. The gene regulation agent of claim 1, wherein the KRAB element comprises an amino acid sequence that is at least 95% identical to that of any one of SEQ ID NOs: 1-6.

28. The gene regulation agent of claim 1, wherein the KRAB element comprises a KRAB-A domain.

29. The gene regulation agent of claim 1, wherein the KRAB element comprises a KRAB-A and a KRAB-B domain.

30. The gene regulation agent of claims 28 or 29, wherein the KRAB-A domain comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 4.

31. The gene regulation agent of claim 29 or 30, wherein the KRAB-B domain comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 6.

32. The gene regulation agent of claim 1, wherein the gene regulation agent is or comprises an engineered human Zinc Finger KRAB molecule or a portion thereof.

33. The gene regulation agent of claim 32, wherein the engineered human Zinc Finger KRAB molecule comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 50.

34. The gene regulation agent of any of the preceding claims, wherein the gene regulation agent is or comprises a polypeptide between 80 and 10,000 amino acids in length or 8 kD and 1,000 kD in size.

35. The gene regulation agent of claim 1, wherein the gene regulation agent regulates expression of a target through binding to a landing site.

36. A method comprising contacting a cell comprising a polynucleotide with at least one gene regulation agent of claim 1, wherein:

(i) the polynucleotide comprises a target; and

(ii) the Zfn element of the gene regulation agent binds to a landing site associated with the target; and wherein concomitant with or subsequent to the contacting with the gene regulation agent expression of the target is reduced relative to: (i) a cell not contacted with a gene regulation agent and/or (ii) a cell contacted with an agent that does not comprise a KRAB domain.

37. The method of claim 36, wherein the gene regulation agent reduces expression of the target through binding to the landing site.

38. The method of claim 36, wherein the landing site is or comprises a target site.

39. The method of claim 70, wherein the target site is an error site.

40. The method of claim 36, wherein the landing site is or comprises all or a portion of a regulatory element.

41. The method of claim 36, wherein the regulatory element is selected from the group consisting of: a promoter, an enhancer, a silencer, an insulator, a TATA box, a GC box, a CAAT box, a transcriptional start site, a DNA binding motif of a transcription factor or other protein that regulates transcription, a 5’ untranslated region, and a ‘3 untranslated region.

42. The method of claim 36, wherein the polynucleotide is DNA.

43. The method of claim 36, wherein the polynucleotide is mRNA.

44. The method of claim 36, wherein the contacting of the cell with the gene regulation agent occurs prior to or concomitant with a reduction in rate and/or level of transcriptional activity of the target.

45. The method of claim 36, wherein the contacting of the cell with the gene regulation agent occurs prior to or concomitant with a change in chromatin structure, wherein the change in chromatin structure reduces the rate and/or level of transcriptional activity of the target.

46. The method of claim 45, wherein the contacting of the cell with the gene regulation agent further comprises contacting the cell with one or more of a DNA or histone modification factors or any other factors associated with a DNA modification and a histone modification.

47. The method of claim 7, where the one or more DNA or histone modification factor(s), factors associated with DNA modifications, or factors associated with histone modifications are selected from: methylases, methyltransferase, histone (de-)acylating enzymes helicases, recombinases, repair scaffold proteins, single strand DNA binding proteins, mismatch repair proteins.

48. The method of any one of claims 36-47, wherein the step of contacting comprises contacting within a cell.

49. The method of any one of claims 36-48, wherein the contacting is achieved by administration of the at least one gene regulation agents of claim 1 or 33 by at least one of intravenous, parenchymal, intracranial, intracerebroventricular, intrathecal, or parenteral administration.

50. The method of claim 81, wherein the contacting is performed ex vivo or in vitro, resulting in a population of cells with reduced expression of the target and/or one or more histone modifications relative to the population of cells prior to the contacting.

51. The method of claim 82, wherein at least a portion of the population of cells is administered to a subject in need thereof.

52. The method of any one of claims 36-51, wherein the contacting comprises contacting a population of cells.

53. The method of claim 52, wherein the population of cells is or comprises a tissue.

54. The method of claim 52, wherein the population of cells is or comprises an organ.

55. The method of claim 52, wherein the population of cells is or comprises a tumor.

56. The method of claim 55, wherein the tumor is or comprises a lung tumor, pancreatic tumor, or colon tumor.

57. The method of claim 52, wherein the population of cells is or comprises a specific cell lineage.

58. The method of claim 57, wherein the specific cell lineage is or comprises lung cells, pancreas cells, or colon cells.

59. The method of claim 57, wherein the specific cell lineage is or comprises cells comprising one or more KRAS mutation(s).

60. The method of claim 59, wherein the one or more KRAS mutation occurs at one or more positions selected from G12, G13, A18, Q61, KI 17, and A146.

61. The method of any one of claims 36-60, wherein the contacting occurs in vivo.

62. The method of claim 61, wherein the contacting occurs in a subject in need thereof.

63. The method of claim 62, wherein the subject is a mammal.

64. The method of claim 63, wherein the mammal is a non-human primate.

65. The method of claim 63, wherein the mammal is a human.

66. The method of claim 65, wherein the human is an adult human.

67. The method of claim 65, wherein the human is a fetal, infant, child, or adolescent human.

68. The method of any one of claims 36-67, wherein the contacting comprises contacting with at least two different gene regulation agents in accordance with claims 1 or 31.

69. The method of claim 68, wherein the contacting with the at least two gene regulation agents is sequential or simultaneous.

70. The method of claims 68 or 69, wherein the expression of at least two targets are reduced relative to: (i) a cell not contacted with a gene regulation agent(s) and/or (ii) a cell contacted with an agent(s) that does not comprise a KRAB domain.

71. The method of any one of claims 68-70, wherein the at least two targets are associated with different genes.

72. The method of any one of claims 68-71, wherein the at least two targets are associated with the same gene.

73. The method of claim 71, wherein the different genes are located on the same chromosome.

74. The method of claim 71, wherein the different genes are located on different chromosomes.

75. The method of claim 36, wherein the expression is measured by a level of mRNA, protein, coprecipitation assays, or chromatin accessibility.

76. The method of any one of claims 36-75, wherein after the contacting, there is a reduction in a level of a target as compared to the level of the target in the absence of the contacting.

77. The method of any one of claims 36-76, wherein prior to or concomitant with the contacting, the target is being actively transcribed, e.g., using RT-PCR, qPCR, in situ hybridization, or other method known in the art to quantify transcription.

78. The method of any one of claims 36-77, wherein at least one target or sequence associated therewith is epigenetically modified.

79. The method of claim 78, wherein the epigenetic modification is concomitant with a dissociation of an RNA polymerase from a DNA strand associated with the at least one target.

80. The method of claim 78, wherein the epigenetic modification prevents association of an RNA polymerase with a DNA strand associated with the at least one target.

81. The method of claim 80, wherein the at least two targets or sequences associated therewith are epigenetically modified.

82. A vector comprising a nucleic acid sequence encoding any one of the gene regulation agents of claims 1-68.

83. The vector of claim 82, wherein the vector comprises a viral vector selected from the group consisting of: a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno-associated viral vector.

84. A composition comprising any of the gene regulation agents of claims 1-35 or the vectors of claims 82 or 83.

85. A kit comprising the gene regulation agents of claims 1-35 or the compositions of claim 84 and instructions for use of the gene regulation agents or compositions.

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