Persistent epigenetic gene silencing

Abstract

To provide more powerful and safer gene silencing technologies.SOLUTION: A product comprising two or more artificial transcription repressors (ATRs), or polynucleotides encoding them, selected from the groups (a), (b), (c) or (d): (a) an ATR comprising a DNA-binding domain operably linked to a KRAB domain or homolog thereof; (b) an ATR comprising a DNA-binding domain operably linked to a DNMT3A, DNMT3B or DNMT1 domain or homolog thereof; (c) an ATR comprising a DNA-binding domain operably linked to a DNMT3L domain or homolog thereof; and (d) an ATR comprising a DNA-binding domain operably linked to a SETDB1 domain or homolog thereof, wherein at least two of the ATRs are selected from different groups (a), (b), (c) or (d).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to gene silencing and / or epigenetic editing. More particularly, the present invention relates to improved methods for silencing a gene of interest or editing the epigenetic state of a genetic element of interest, including during gene therapy applications. [Background technology]

[0002] Gene therapy involves the introduction of genetic material into cells to treat or prevent disease. The genetic material may replace defective genes with functional copies of those genes, inactivate improperly functioning genes, or introduce new therapeutic genes into cells.

[0003] A classic example of gene therapy is gene replacement, in which a DNA sequence encoding a functional therapeutic gene is used to replace a dysfunctional gene (Naldini, L. (2011) Nat. Rev. Genet. 12: 301-15; Kay, MA (2011) Nat. Rev. Genet. 12: 316-28; Biffi, A. et al. (2013) Science 341: 1233158; Aiuti, A. et al. (2013) Science 341: 1233151; Aiuti, A. et al. (2009) N. Engl. J. Med. 360: 447-58). However, there are several genetic diseases in which the goal of gene therapy is to silence gene function rather than replace it. Typical examples include Huntington's disease, most types of spinocerebellar ataxia, and some collagen disorders. Furthermore, gene silencing has emerged as a promising strategy for treating certain infectious diseases (Younan, P. et al. (2014) Mol. Ther. 22: 257-64), by inactivating pathogen-associated gene products or host genes required for the pathogen life cycle.

[0004] For example, silencing of the chemokine (C-C motif) receptor type 5 (CCR) gene, one of two cellular coreceptors required for HIV entry into T cells, has attracted considerable attention because spontaneous deletions in CCR5 confer resistance to infection by CCR5-tropic HIV strains without causing any apparent pathological effects (Liu, R. et al. (1996) Cell 86: 367-77; Hutter, G. et al. (2009) N. Engl. J. Med. 360: 692-8).

[0005] It has also recently been suggested that hemoglobinopathies, the most common inherited recessive disorders of the hematopoietic system and a prime target for therapeutic gene replacement (Weatherall, DJ (2013) Annu. Rev. Genomics Hum. Genet. 14: 1-24), may also be amenable to therapeutic gene silencing. This intriguing concept is based on our ever-growing understanding of the mechanisms regulating the switch from fetal to adult hemoglobin during development (Stamatoyannopoulos, G. (2005) Exp. Hematol. 33: 259-71; Bauer, DE et al. (2011) Curr. Opin. Pediatr. 23: 1-8), as well as extensive clinical evidence showing that sustained expression of fetal hemoglobin (HbF) significantly improves morbidity and mortality in patients with sickle cell disease (SCD; Platt, OS et al. (1994) N. Engl. J. Med. 330: 1639-44) and β-thalassemia (β-Thal; Andreani, M. et al. (2011) Haematologica 96: 128-33). In particular, genome-wide association studies performed in patients with hereditary hyperfetal hemoglobinemia (HbF) have shown that the transcription factor B-cell lymphoma / leukemia 11A (BCL11A) is a master regulator of the hemoglobin switch (Sankaran, VG et al. (2008) Science 322: 1839-42; Uda, M. et al. (2008) Proc. Natl. Acad. Sci. USA 105: 1620-5; Galarneau, G. et al. (2010) Nat. Genet. 42: 1049-51), and that inactivating mutations in this gene result in enhanced HbF expression (Wilber, A. et al. (2011) Blood 117: 2817-26; Xu, J. et al. (2011) Science 334: 993-6). Furthermore, an erythroid-specific enhancer within the second intron of BCL11A has recently been identified (Bauer, DE et al. (2013) Science 342: 253-7).Genetic inactivation of this regulatory element impairs BCL11A expression specifically in erythroid precursors, leading to HbF reactivation, while maintaining the activity of this protein required for proper B cell ontogeny (Canver, MC et al. (2015) Nature Sep 16 doi: 10.1038 / nature15521 [Epub ahead of print]; Vierstra, J. et al. (2015) Nat. Methods 12: 927-30).

[0006] Currently, two major targeting technologies are used to silence gene expression: RNA interference (RNAi; Davidson, BL et al. (2011) Nat. Rev. Genet. 12: 329-40), which uses a single short hairpin RNA (shRNA), and gene targeting using artificial nucleases (AN; Carroll, D. (2014) Annu. Rev. Biochem. 83: 409-39). RNAi utilizes the endogenous microRNA (miRNA) pathway to downregulate the expression of target transcripts complementary to the shRNA (Davidson, BL et al. (2011) Nat. Rev. Genet. 12: 329-40). The AN approach exploits the error-prone nature of the non-homologous end joining DNA repair process to persistently disrupt the coding frame of AN target genes (Ciccia, A. et al. (2010) Mol. Cell 40: 179-204).

[0007] Although promising preclinical and clinical data have been obtained using these technologies (DiGiusto, DL et al. (2013) Viruses 5: 2898-919; DiGiusto, DL et al. (2010) Sci. Transl. Med. 2: 36ra43; Ramachandran, PS et al. (2013) Neurotherapeutics 10: 473-85; McBride, JL et al. (2011) Mol. Ther. 19: 2152-62), the partial reduction of gene expression by shRNAs and their low efficiency in producing homozygous disruption in diploid mammalian cells can compromise the efficacy of these therapies. These drawbacks are particularly important in applications where residual levels of gene activity are sufficient for biological function.

[0008] Furthermore, the safe use of these technologies requires solving the issues of a) off-target (non-specific) gene silencing, b) altering the transcriptional profile of cells by inhibiting endogenous miRNA pathways, and c) inducing apoptosis or altering cell cycle progression by overactivating the DNA damage response (Ciccia, A. et al. (2010) Mol. Cell 40: 179-204). In addition, RNAi and RNAi are not suitable for inactivating a wide range of non-transcriptional regulatory elements, such as promoters or enhancers.

[0009] Epigenetic mechanisms are also utilized to silence gene expression. Epigenetics refers to mechanisms that transmit heritable changes in genome function without altering the primary DNA sequence. These changes may mediate short-term instructions that can be rapidly reversed in response to external stimuli (e.g., histone post-transcriptional modifications; HPTMs). Alternatively, they may constitute long-term instructions that stably contribute to cellular identity and memory (e.g., DNA methylation; Smith, ZD et al. (2013) Nat. Rev. Genet. 14: 204-20). Current research is elucidating the composition and function of molecular complexes recruited to chromatin to induce epigenetic repression states and the mechanisms by which these states propagate unchecked throughout cell division (Cedar, H. et al. (2009) Nat. Rev. Genet. 10: 295-304; Chen, T. et al. (2014) Nat. Rev. Genet. 15: 93-106; Probst, AV et al. (2009) Nat. Rev. Mol. Cell Biol. 10: 192-206).

[0010] Several studies have established gene silencing using stably expressed artificial transcriptional repressors (ATRs) formed from DNA-binding domains fused to the effector domains of chromatin-remodeling enzymes (de Groote, ML et al. (2012) Nucleic Acids Res. 40: 10596-613; Mendenhall, EM et al. (2013) Nat. Biotechnol. 31: 1133-6; Zhang, F. et al. (2011) Nat. Biotechnol. 29: 149-53; Konermann, S. et al. (2013) Nature 500: 472-6; Sera, T. (2009) Adv. Drug Deliv. Rev. 61: 513-26; Qi, LS et al. (2013) Cell 152: 1173-83). However, these studies did not demonstrate sustained epigenetic silencing in the absence of continuous expression of ATR, presumably because the selected effector domains are essentially unable to recreate a self-replicating chromatin repression state at ATR target sites.

[0011] It has also been shown that silencing induced by artificial Kruppel-associated box (KRAB) repressors is abolished in somatic cells when the repressor proteins are no longer expressed or no longer bind to their target sites (Szulc, J. et al. (2006) Nat. Methods 3: 109-16).

[0012] Therefore, there remains a significant need for the development of more powerful and safer gene silencing technologies. [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] Naldini, L. (2011) Nat. Rev. Genet. 12: 301-15;

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[0014] The present inventors have developed a novel approach for gene silencing that utilizes endogenous epigenetic mechanisms.Unexpectedly, the present inventors' approach transmits robust and inheritable transcriptional suppression of desired target genes.Importantly, this allows the sustained (permanent) inactivation of genes that are of therapeutic interest (e.g., disease-causing) or biotechnology interest.

[0015] Because of the aforementioned challenges with sustaining robust gene silencing, and because long-term persistent expression of artificial transcriptional repressors (ATRs) from integrating vectors can pose a significant safety threat to cells, we chose to use only ATRs that satisfy all of the following criteria: 1. Functions through a combinatorial set of two or more different effector modules; 2. Establish a robust and sustained state of epigenetic repression; and 3. It exerts its biological function when transiently expressed in cells.

[0016] This approach allowed us to improve both the efficiency and safety of gene silencing, because the activity of each ATR at off-target sites would be transient, if not absent.

[0017] In one embodiment, the present invention provides a compound of group (a), (b), (c) or (d): (a) Artificial transcriptional repressors (ATRs) containing a DNA-binding domain operably linked to a KRAB domain or its homologue; (b) ATR containing a DNA-binding domain operably linked to a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof; (c) an ATR comprising a DNA-binding domain operably linked to a DNMT3L domain or a homolog thereof; and (d) ATR containing a DNA-binding domain operably linked to a SETDB1 domain or its homologue. The present invention provides a product (product) comprising two or more artificial transcriptional repressors (ATRs) or polynucleotides encoding them selected from the following, wherein at least two of the ATRs are selected from different groups (a), (b), (c) or (d).

[0018] In another embodiment, the present invention provides a compound according to group (a), (b) or (c): (a) Artificial transcriptional repressors (ATRs) containing a DNA-binding domain operably linked to a KRAB domain or its homologue; (b) an ATR comprising a DNA-binding domain operably linked to a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof; and (c) ATR containing a DNA-binding domain operably linked to a DNMT3L domain or its homologue. The present invention provides a product comprising two or more artificial transcriptional repressors (ATRs) selected from, or polynucleotides encoding them, wherein at least two of the ATRs are selected from different groups (a), (b), or (c).

[0019] In one embodiment, a product of the invention comprises ATR(a) and (b) or polynucleotides encoding them. In another embodiment, a product of the invention comprises ATR(a) and (c) or polynucleotides encoding them. In another embodiment, a product of the invention comprises ATR(b) and (c) or polynucleotides encoding them. In a preferred embodiment, a product of the invention comprises ATR(a), (b), and (c) or polynucleotides encoding them. In another embodiment, a product of the invention comprises ATR(a), (b), and (d) or polynucleotides encoding them. In another embodiment, a product of the invention comprises ATR(b) and (d) or polynucleotides encoding them. In another embodiment, a product of the invention comprises ATR(c) and (d) or polynucleotides encoding them. In another embodiment, a product of the invention comprises ATR(b), (c), and (d) or polynucleotides encoding them.

[0020] A KRAB domain or homolog thereof can comprise an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% identity to SEQ ID NO: 1, 2, 3, 4, 5, 6 or 7, wherein the amino acid sequence substantially retains the native function of the protein represented by SEQ ID NO: 1, 2, 3, 4, 5, 6 or 7.

[0021] The DNMT3A domain or homolog thereof can comprise an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% identity to SEQ ID NO:8, wherein the amino acid sequence substantially retains the native function of the protein represented by SEQ ID NO:8.

[0022] The DNMT3B domain or a homolog thereof can comprise an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% identity to SEQ ID NO: 9 or 36, wherein the amino acid sequence substantially retains the native function of the protein represented by SEQ ID NO: 9 or 36.

[0023] The DNMT1 domain or homolog thereof can comprise an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% identity to SEQ ID NO: 10, wherein the amino acid sequence substantially retains the native function of the protein represented by SEQ ID NO: 10.

[0024] The DNMT3L domain or a homolog thereof can comprise an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% identity to SEQ ID NO:11, wherein the amino acid sequence substantially retains the native function of the protein represented by SEQ ID NO:11.

[0025] The SETDB1 domain or a homolog thereof can comprise an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% identity to SEQ ID NO: 12 or 13, wherein the amino acid sequence substantially retains the native function of the protein represented by SEQ ID NO: 12 or 13.

[0026] In one embodiment, the DNA binding domain of (a), (b), (c) or (d) comprises a domain independently selected from a TALE DNA binding domain, a zinc finger domain, a tetR DNA binding domain, a meganuclease or a CRISPR / Cas system. In a preferred embodiment, the DNA binding domain of (a), (b), (c) or (d) comprises a TALE DNA binding domain or a CRISPR / Cas system.

[0027] The DNA binding domains of (a), (b), (c) or (d), e.g., TALE DNA binding domains or CRISPR / Cas systems, can be selected or engineered to bind to different binding sites.

[0028] The DNA binding domain may bind to a binding site within a target gene or within a regulatory sequence (e.g., a promoter or enhancer sequence) of a target gene.

[0029] DNA-binding domains can bind to binding sites within splice sites. Splice variants of a given gene can be regulated by DNA methylation / demethylation at splice sites. These modifications can then lead to exon exclusion / inclusion in the mature transcript. This exclusion / inclusion can be therapeutically important, for example, in the case of Duchenne muscular dystrophy. In Duchenne muscular dystrophy, elimination of exons containing the most frequent disease-causing mutations from mature mRNA (by genetic ablation or exon skipping) has been proposed for treatment (Ousterout, DG et al. (2015) Mol. Ther. 23: 523-32; Ousterout, DG et al. (2015) Nat. Commun. 6: 6244; Kole, R. et al. (2015) Adv. Drug Deliv. Rev. 87: 104-7; Touznik, A. et al. (2014) Expert Opin. Biol. Ther. 14: 809-19).

[0030] The ATRs of the present invention may also target genetic elements, which may or may not be actively transcribed (e.g., sequences that control the topological arrangement, stability, and replication of the genome, such as insulators, laminin-associated domains, telomeres, and centromeric regions), repetitive elements, or mobile elements. Thus, the present invention may relate to epigenetic editing, e.g., silencing / editing of genetic elements. Thus, the present invention may include the use of the products of the present invention and ATRs for epigenetic editing of regulatory DNA elements (e.g., those described herein). Epigenetic editing of target genes or genetic elements may also be associated with their transcriptional activation or activity, respectively. The present invention may also include the use of the products of the present invention and ATRs for simultaneous epigenetic silencing of multiple target genes or regulatory DNA elements (e.g., those described herein).

[0031] In one embodiment, the polynucleotides encoding two or more ATRs are in the form of a single vector or are contained within separate vectors.

[0032] In one embodiment using two ATRs, the polynucleotides encoding (a) and (b) can be contained in a single vector; the polynucleotides encoding (a) and (c) can be contained in a single vector; or the polynucleotides encoding (b) and (c) can be contained in a single vector.

[0033] In another embodiment using two ATRs, the polynucleotides encoding (a) and (d) can be contained in a single vector; the polynucleotides encoding (b) and (d) can be contained in a single vector; or the polynucleotides encoding (c) and (d) can be contained in a single vector.

[0034] In another embodiment using two ATRs, the polynucleotides encoding (a) and (b) can be contained in separate vectors; the polynucleotides encoding (a) and (c) can be contained in separate vectors; or the polynucleotides encoding (b) and (c) can be contained in separate vectors.

[0035] In another embodiment using two ATRs, the polynucleotides encoding (a) and (d) can be contained in separate vectors; the polynucleotides encoding (b) and (d) can be contained in separate vectors; or the polynucleotides encoding (c) and (d) can be contained in separate vectors.

[0036] In one embodiment using three ATRs, the polynucleotides encoding (a), (b), and (c) can be contained in a single vector; the polynucleotides encoding (a), (b), and (c) can be contained in separate vectors; the polynucleotides encoding (a) and (b) can be contained in a single vector and the polynucleotide encoding (c) can be contained in a separate vector; the polynucleotides encoding (a) and (c) can be contained in a single vector and the polynucleotide encoding (b) can be contained in a separate vector; or the polynucleotides encoding (b) and (c) can be contained in a single vector and the polynucleotide encoding (a) can be contained in a separate vector.

[0037] In one embodiment using three ATRs, the polynucleotides encoding (a), (b), and (d) can be contained in a single vector; the polynucleotides encoding (a), (b), and (d) can be contained in separate vectors; the polynucleotides encoding (a) and (b) can be contained in a single vector and the polynucleotide encoding (d) can be contained in a separate vector; the polynucleotides encoding (a) and (d) can be contained in a single vector and the polynucleotide encoding (b) can be contained in a separate vector; or the polynucleotides encoding (b) and (d) can be contained in a single vector and the polynucleotide encoding (a) can be contained in a separate vector.

[0038] In one embodiment using three ATRs, the polynucleotides encoding (b), (c), and (d) can be contained in a single vector; the polynucleotides encoding (b), (c), and (d) can be contained in separate vectors; the polynucleotides encoding (b) and (c) can be contained in a single vector and the polynucleotide encoding (d) can be contained in a separate vector; the polynucleotides encoding (b) and (d) can be contained in a single vector and the polynucleotide encoding (c) can be contained in a separate vector; or the polynucleotides encoding (c) and (d) can be contained in a single vector and the polynucleotide encoding (b) can be contained in a separate vector.

[0039] The vector can be, for example, a plasmid vector, an mRNA vector (e.g., an in vitro transcribed mRNA vector), or a viral vector. Preferably, the vector allows for transient expression of ATR in cells.

[0040] As an alternative to delivering a polynucleotide encoding ATR to a cell, the ATR of the present invention can be delivered to a cell by protein transduction, which can be, for example, by vector delivery or direct protein delivery.

[0041] In one embodiment, the product of the invention is in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, diluent or excipient.

[0042] In one embodiment, the product of the invention further comprises a KRAB domain or a homolog thereof or a polynucleotide encoding same, wherein the KRAB domain or homolog thereof is not operably linked to a DNA binding domain.

[0043] In one embodiment, the product of the invention further comprises a DNMT3A, DNMT3B or DNMT1 domain or a homolog thereof or a polynucleotide encoding same, wherein the DNMT3A, DNMT3B or DNMT1 domain or a homolog thereof is not operably linked to a DNA binding domain.

[0044] In one embodiment, the product of the invention further comprises a DNMT3L domain or a homolog thereof or a polynucleotide encoding same, wherein the DNMT3L domain or homolog thereof is not operably linked to a DNA-binding domain.

[0045] In one embodiment, the product of the invention further comprises a SETDB1 domain or a homolog thereof or a polynucleotide encoding same, wherein the SETDB1 domain or homolog thereof is not operably linked to a DNA-binding domain.

[0046] In another aspect, the present invention provides a product of the invention for use in therapy.

[0047] In another aspect, the present invention provides a product of the invention for use in therapy, wherein two or more artificial transcriptional repressors (ATRs) or polynucleotides encoding them are in a combined preparation (combined preparation) for simultaneous, sequential or separate administration to a subject.

[0048] In this case, administration to a subject includes administration to a subject, for example, during ex vivo therapy.

[0049] In another aspect, the present invention provides the use of the product of the present invention for silencing target gene.This use can be, for example, in vitro or ex vivo use.For example, target gene can be silenced in a group of cells (for example, cell line or primary cell) to enhance the production of substance (for example, biotherapeutic substance) by the cell or to give the cell a growth advantage.Alternatively, for example, target gene can be silenced to create knockout animals for the target gene.The epigenetic approach of the present invention provides an alternative to existing methods of gene knockout (for example, those that utilize homologous recombination).Alternatively, for example, target gene can be silenced in plant cells.

[0050] In the above uses, including therapeutic uses, delivery of two or more ATRs of the present invention to a cell can silence a target gene. The delivery can be transient delivery. The delivery can be due to expression of two or more ATRs in the cell, for example, expression from a polynucleotide encoding the ATRs. Delivery of two or more ATRs of the present invention to a cell can also cause exon exclusion / inclusion in the mature transcript, for example, by effects on splicing sites. Delivery of two or more ATRs of the present invention to a cell can also enable silencing and / or editing of genetic elements described herein.

[0051] In one embodiment, expression of two or more ATRs of the present invention in a cell silences a target gene. The expression may be transient.

[0052] In one embodiment, delivery of two or more ATRs of the present invention to a cell (e.g., by expression in the cell) persistently silences a target gene. In another embodiment, delivery of two or more ATRs of the present invention to a cell (e.g., by expression in the cell) persistently silences a target gene in the progeny of the cell. For example, the cell can be a stem cell, and the target gene can be silenced in the progeny of the stem cell (e.g., the target gene can be silenced in cells resulting from the differentiation of the stem cell).

[0053] For example, the cells can be derived from animals (e.g., mammals, e.g., humans), fungi (e.g., yeast), or plants. For example, the cells can be hematopoietic stem and progenitor cells, T lymphocytes, mesenchymal stem cells, fibroblasts, monocytes, epidermal, or neural stem cells.

[0054] The size of the separation distance between the binding sites selected for binding by the DNA-binding domains of different ATRs is not particularly limited. For example, the DNA-binding domains of different ATRs can be selected to bind to binding sites that are approximately 1 to 100 bp, 1 to 50 bp, 1 to 30 bp, 5 to 30 bp, 10 to 30 bp, or 15 to 30 bp apart. In one embodiment, the DNA-binding domains of different ATRs can be selected to bind to binding sites that are 1 to 30 bp apart. Preferably, the DNA-binding domains of different ATRs can be selected to bind to binding sites that are approximately 15 to 25 bp apart. For example, the DNA-binding domains of different ATRs can be selected to bind to binding sites that are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 bp apart.

[0055] The DNA-binding domains of different ATRs can also be selected to bind to the same binding site. For example, the DNA-binding domains of different ATRs can be selected to bind to binding sites that are 0 bp apart. Thus, for example, the DNA-binding domains of different ATRs can be selected to bind to binding sites that are approximately 0-100 bp, 0-50 bp, 0-30 bp, 5-30 bp, 10-30 bp, or 15-30 bp apart. The DNA-binding domains of different ATRs can be selected to bind to binding sites that are approximately 0-15 or 15-25 bp apart.

[0056] The order in which different ATRs bind to a target gene is not particularly important. In one embodiment, the two or more ATRs include an ATR containing a KRAB domain or a homolog thereof and an ATR containing a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof, and the DNA binding domain (e.g., a TALE DNA binding domain) of each ATR is selected so that the ATR containing a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof binds to the DNA upstream of the ATR containing a KRAB domain or a homolog thereof.

[0057] In one embodiment, the DNA binding domain is a TALE DNA binding domain or a CRISPR / Cas system.

[0058] Selection of a DNA binding domain can involve engineering the DNA binding domain to bind to a specific desired DNA sequence.

[0059] In another aspect, the present invention provides an artificial transcriptional repressor (ATR) comprising a DNA-binding domain operably linked to a KRAB domain or a homolog thereof, or a polynucleotide encoding the same, for use in therapy, wherein the ATR is administered to a subject simultaneously, sequentially or separately in combination with a second ATR comprising a DNA-binding domain operably linked to a DNMT3A, DNMT3B or DNMT1 domain or a homolog thereof, and / or a third ATR comprising a DNA-binding domain operably linked to a DNMT3L domain or a homolog thereof, or a polynucleotide encoding the same.

[0060] In another aspect, the present invention provides an artificial transcriptional repressor (ATR) comprising a DNA-binding domain operably linked to a DNMT3A, DNMT3B or DNMT1 domain or a homolog thereof, or a polynucleotide encoding the same, for use in therapy, wherein the ATR is administered to a subject simultaneously, sequentially or separately in combination with a second ATR comprising a DNA-binding domain operably linked to a KRAB domain or a homolog thereof, and / or a third ATR comprising a DNA-binding domain operably linked to a DNMT3L domain or a homolog thereof, or a polynucleotide encoding the same.

[0061] In another aspect, the present invention provides an artificial transcriptional repressor (ATR) comprising a DNA-binding domain operably linked to a DNMT3L domain or a homolog thereof, or a polynucleotide encoding the same, for use in therapy, wherein the ATR is administered to a subject simultaneously, sequentially or separately in combination with a second ATR comprising a DNA-binding domain operably linked to a KRAB domain or a homolog thereof, and / or a third ATR comprising a DNA-binding domain operably linked to a DNMT3A, DNMT3B or DNMT1 domain or a homolog thereof, or a polynucleotide encoding the same.

[0062] In another aspect, the present invention provides an artificial transcriptional repressor (ATR) comprising a DNA-binding domain operably linked to a KRAB domain or a homolog thereof, or a polynucleotide encoding the same, for use in therapy, wherein the ATR is administered to a subject simultaneously, sequentially, or separately in combination with a second ATR comprising a DNA-binding domain operably linked to a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof, and / or a third ATR comprising a DNA-binding domain operably linked to a DNMT3L domain or a homolog thereof, and / or a fourth ATR comprising a DNA-binding domain operably linked to a SETDB1 domain or a homolog thereof, or polynucleotides encoding the same.

[0063] In another aspect, the present invention provides an artificial transcriptional repressor (ATR) comprising a DNA-binding domain operably linked to a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof, or a polynucleotide encoding the same, for use in therapy, wherein the ATR is administered to a subject simultaneously, sequentially, or separately in combination with a second ATR comprising a DNA-binding domain operably linked to a KRAB domain or a homolog thereof, and / or a third ATR comprising a DNA-binding domain operably linked to a DNMT3L domain or a homolog thereof, and / or a fourth ATR comprising a DNA-binding domain operably linked to a SETDB1 domain or a homolog thereof, or polynucleotides encoding the same.

[0064] In another aspect, the present invention provides an artificial transcriptional repressor (ATR) comprising a DNA-binding domain operably linked to a DNMT3L domain or a homolog thereof, or a polynucleotide encoding the same, for use in therapy, wherein the ATR is administered to a subject simultaneously, sequentially or separately in combination with a second ATR comprising a DNA-binding domain operably linked to a KRAB domain or a homolog thereof, and / or a third ATR comprising a DNA-binding domain operably linked to a DNMT3A, DNMT3B or DNMT1 domain or a homolog thereof, and / or a fourth ATR comprising a DNA-binding domain operably linked to a SETDB1 domain or a homolog thereof, or polynucleotides encoding the same.

[0065] In another aspect, the present invention provides an artificial transcriptional repressor (ATR) comprising a DNA-binding domain operably linked to a SETDB1 domain or a homolog thereof, or a polynucleotide encoding the same, for use in therapy, wherein the ATR is administered to a subject simultaneously, sequentially or separately in combination with a second ATR comprising a DNA-binding domain operably linked to a DNMT3A, DNMT3B or DNMT1 domain or a homolog thereof, and / or a third ATR comprising a DNA-binding domain operably linked to a DNMT3L domain or a homolog thereof, and / or a fourth ATR comprising a DNA-binding domain operably linked to a KRAB domain or a homolog thereof, or polynucleotides encoding the same.

[0066] In another aspect, the present invention provides a cell comprising two or more artificial transcriptional repressors (ATRs) of the present invention. The cell can be transfected with polynucleotides encoding two or more ATRs of the present invention. The polynucleotides can be in the form of a single vector or can be contained in separate vectors.

[0067] In another aspect, the invention provides cells that are progeny of cells that contain two or more artificial transcriptional repressors (ATRs) of the invention. In one embodiment, the progeny cells no longer contain two or more ATRs of the invention. In another aspect, the invention provides cells of the invention for use in therapy.

[0068] In another aspect, the present invention provides a method of gene therapy comprising transfecting a cell with polynucleotides encoding two or more artificial transcriptional repressors (ATRs) of the present invention, wherein the polynucleotides are in the form of a single vector or are contained within separate vectors.

[0069] In one embodiment, the transfection is performed ex vivo.

[0070] In another aspect, the present invention provides a compound selected from group (a), (b) or (c): (a) Artificial transcriptional repressors (ATRs) containing a DNA-binding domain operably linked to a KRAB domain or its homologue; (b) an ATR comprising a DNA-binding domain operably linked to a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof; and (c) ATR containing a DNA-binding domain operably linked to a DNMT3L domain or its homologue. The present invention provides a method of gene therapy comprising simultaneously, sequentially or separately administering to a subject two or more artificial transcriptional repressors (ATRs) or polynucleotides encoding them selected from the group (a), (b), (c), or (d), wherein at least two of the ATRs are selected from different groups (a), (b), or (c). (a) Artificial transcriptional repressors (ATRs) containing a DNA-binding domain operably linked to a KRAB domain or its homologue; (b) ATR containing a DNA-binding domain operably linked to a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof; (c) an ATR comprising a DNA-binding domain operably linked to a DNMT3L domain or a homolog thereof; and (d) ATR containing a DNA-binding domain operably linked to a SETDB1 domain or its homologue. The present invention provides a method of gene therapy comprising administering to a subject simultaneously, sequentially or separately two or more artificial transcriptional repressors (ATRs) or polynucleotides encoding them selected from the group (a), (b), (c) or (d), wherein at least two of the ATRs are selected from different groups (a), (b), (c) or (d).

[0071] In another aspect, the present invention provides a compound selected from group (a), (b) or (c): (a) Artificial transcriptional repressors (ATRs) containing a DNA-binding domain operably linked to a KRAB domain or its homologue; (b) an ATR comprising a DNA-binding domain operably linked to a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof; and (c) ATR containing a DNA-binding domain operably linked to a DNMT3L domain or its homologue. The present invention also provides a kit comprising two or more artificial transcriptional repressors (ATRs) or polynucleotides encoding them, wherein at least two of the ATRs are selected from different groups (a), (b), or (c). The present invention also provides a kit comprising two or more artificial transcriptional repressors (ATRs) or polynucleotides encoding the same, wherein at least two of the ATRs are selected from different groups (a), (b), (c), or (d): (a) Artificial transcriptional repressors (ATRs) containing a DNA-binding domain operably linked to a KRAB domain or its homologue; (b) ATR containing a DNA-binding domain operably linked to a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof; (c) an ATR comprising a DNA-binding domain operably linked to a DNMT3L domain or a homolog thereof; and (d) ATR containing a DNA-binding domain operably linked to a SETDB1 domain or its homologue. The present invention provides a kit comprising two or more artificial transcriptional repressors (ATRs) or polynucleotides encoding them selected from the following groups, wherein at least two of the ATRs are selected from different groups (a), (b), (c), or (d).

[0072] In another aspect, the present invention provides a method for silencing a target gene, comprising administering to a cell two or more ATRs of the present invention or polynucleotides encoding them. The method may be an in vitro method.

[0073] In another aspect, the present invention provides a product comprising an artificial transcriptional repressor (ATR) comprising a DNA-binding domain operably linked to a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof, preferably a DNMT3A domain or a homolog thereof, and an ATR comprising a DNA-binding domain operably linked to a SETDB1 domain or a homolog thereof, or a polynucleotide encoding the same. The present invention also provides uses of the product, use of the product in therapy, cells comprising the product and their progeny, methods of using the product, and kits comprising the product (as described herein). The product may also further comprise an ATR comprising a DNA-binding domain operably linked to a DNMT3L or KRAB domain or a homolog thereof, or a polynucleotide encoding the same.

[0074] In one embodiment, the product comprises an artificial transcriptional repressor (ATR) comprising a DNA-binding domain operably linked to a DNMT3A, DNMT3B or DNMT1 domain or a homolog thereof, preferably a DNMT3A domain or a homolog thereof, an ATR comprising a DNA-binding domain operably linked to a SETDB1 domain or a homolog thereof, and an ATR comprising a DNA-binding domain operably linked to a DNMT3L domain or a homolog thereof, or polynucleotides encoding them.

[0075] In one embodiment, the product comprises an artificial transcriptional repressor (ATR) comprising a DNA-binding domain operably linked to a DNMT3A, DNMT3B or DNMT1 domain or a homolog thereof, preferably a DNMT3A domain or a homolog thereof, an ATR comprising a DNA-binding domain operably linked to a SETDB1 domain or a homolog thereof, and an ATR comprising a DNA-binding domain operably linked to a KRAB domain or a homolog thereof, or polynucleotides encoding them.

[0076] The SETDB1 domain or a homolog thereof can comprise an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% identity to SEQ ID NO: 12 or 13, wherein the amino acid sequence substantially retains the native function of the protein represented by SEQ ID NO: 12 or 13.

[0077] In another aspect, the present invention provides an artificial transcriptional repressor (ATR) or a polynucleotide encoding the same, wherein the ATR is selected from group (a), (b), or (c): (a) KRAB domain or its homologues; (b) a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof; and (c) DNMT3L domain or its homologue The ATR may comprise a DNA-binding domain operably linked to two or more domains selected from group (a), (b), (c), and (d), wherein at least two of the domains operably linked to the DNA-binding domain are selected from different groups (a), (b), or (c). The ATR may, for example, comprise a DNA-binding domain operably linked to a domain from group (a), a domain from group (b), and a domain from group (c). The present invention also provides a DNA-binding domain operably linked to a domain from group (a), (b), (c), or (d): (a) KRAB domain or its homologues; (b) DNMT3A, DNMT3B, or DNMT1 domains or homologs thereof; (c) a DNMT3L domain or a homolog thereof; and (d) SETDB1 domain or its homologue The present invention provides an artificial transcriptional repressor (ATR) comprising a DNA-binding domain operably linked to two or more domains selected from the group (a), (b), (c), or (d), wherein at least two of the domains operably linked to the DNA-binding domain are selected from different groups (a), (b), (c), or (d).

[0078] In one embodiment, the DNA binding domain comprises a TALE DNA binding domain, a zinc finger domain, a tetR DNA binding domain, a meganuclease, or a CRISPR / Cas system.

[0079] The present invention also provides uses of this ATR, uses of this ATR in therapy, cells and their progeny comprising this ATR, methods of using this ATR, and kits comprising this ATR (as described herein).

[0080] In another aspect, the present invention provides a product comprising two or more different artificial transcriptional repressors (ATRs) or polynucleotides encoding same, wherein the two or more different ATRs are selected from group (a), (b), or (c): (a) KRAB domain or its homologues; (b) a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof; and (c) DNMT3L domain or its homologue wherein at least two of the domains operably linked to each individual DNA-binding domain are selected from different groups (a), (b), or (c). Each ATR may, for example, comprise a DNA-binding domain operably linked to a domain from group (a), a domain from group (b), and a domain from group (c). The present invention also provides products comprising two or more different artificial transcriptional repressors (ATRs) or polynucleotides encoding same, wherein the two or more different ATRs are selected from groups (a), (b), (c), or (d): (a) KRAB domain or its homologues; (b) DNMT3A, DNMT3B, or DNMT1 domains or homologs thereof; (c) a DNMT3L domain or a homolog thereof; and (d) SETDB1 domain or its homologue wherein at least two of the domains operably linked to each individual DNA-binding domain are selected from different groups (a), (b), (c) or (d).

[0081] In one embodiment, the DNA binding domains of the two or more different ATRs are individually selected from the group consisting of a TALE DNA binding domain, a zinc finger domain, a tetR DNA binding domain, a meganuclease, or a CRISPR / Cas system.

[0082] The DNA binding domains of two or more different ATRs, such as TALE DNA binding domains or CRISPR / Cas systems, can be selected or engineered to bind to different sites.

[0083] The DNA binding domain may bind to a binding site within a target gene or within a regulatory sequence of a target gene (e.g., a promoter or enhancer sequence). The DNA binding domain may bind to a binding site within a splice site.

[0084] The invention also provides uses of this product, uses of this product in therapy, cells and their progeny comprising this product, methods of using this product, and kits comprising this product (as described herein).

[0085] In another aspect, the present invention provides a product comprising only one ATR and another effector protein, or a polynucleotide encoding the same, that is not operably linked to a DNA-binding domain. The ATR may comprise a DNA-binding domain operably linked to an effector domain selected from (a) a KRAB domain or a homolog thereof; (b) a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof; or (c) a DNMT3L domain or a homolog thereof (i.e., the ATR may be as described herein). The other effector protein not operably linked to a DNA-binding domain may comprise a KRAB, DNMT3A, DNMT3B, DNMT1, or DNMT3L domain or a homolog thereof. The other effector protein may be an effector domain / protein described herein. The other effector protein may be a full-length protein or a functional fragment thereof. Preferably, the other effector protein is different from the effector domain of the ATR. Preferably, the other effector protein is of a different class from the effector domain of the ATR, and preferably, the other effector protein is selected so that it does not contain a domain belonging to the same group (a), (b), or (c) as the effector domain constituting the ATR.

[0086] The additional effector protein not operably linked to the DNA-binding domain may also include SETDB1 or a homolog thereof.

[0087] In another aspect, the present invention provides a product comprising only one ATR and another effector protein, or a polynucleotide encoding the same, that is not operably linked to a DNA-binding domain. The ATR may comprise a DNA-binding domain operably linked to a SETDB1 effector domain or a homolog thereof (i.e., the ATR may be as described herein). The other effector protein not operably linked to a DNA-binding domain may comprise a KRAB, DNMT3A, DNMT3B, DNMT1, or DNMT3L domain or a homolog thereof. The other effector protein may be an effector domain / protein described herein. The other effector protein may be a full-length protein or a functional fragment thereof.

[0088] In one embodiment, the DNA binding domain of ATR is selected from the group consisting of a TALE DNA binding domain, a zinc finger domain, a tetR DNA binding domain, a meganuclease, or a CRISPR / Cas system.

[0089] The invention also provides uses of this product, uses of this product in therapy, cells and their progeny comprising this product, methods of using this product, and kits comprising this product (as described herein).

[0090] When a product of the invention comprises only one ATR and another effector protein that is not operably linked to a DNA-binding domain, the polynucleotides encoding the ATR and the other effector protein can be in the form of a single vector or can be contained in separate vectors.

[0091] The vector can be, for example, a plasmid vector, an mRNA vector (e.g., an in vitro transcribed mRNA vector), or a viral vector. Preferably, the vector allows for transient expression of ATR and / or another effector protein in the cell.

[0092] ATR and / or another effector protein of the present invention can also be delivered to cells by protein transduction, as described herein.

[0093] The ATR and / or another effector protein of the present invention, or the polynucleotides encoding them, may be in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, diluent or excipient.

[0094] In another aspect, the present invention provides an ATR of the present invention, or an ATR of the present invention and another effector protein, or polynucleotides encoding them, for use in therapy.

[0095] In another aspect, the present invention provides the ATR of the present invention and another effector protein or polynucleotide encoding the same for use in therapy, wherein the ATR and another effector protein or polynucleotide encoding the same are a combination agent for simultaneous, sequential or separate administration to a subject.

[0096] In another aspect, there is provided an ATR of the present invention, or an ATR of the present invention and another effector protein, or a polynucleotide encoding the same, for silencing a target gene. The use may be, for example, in vitro or ex vivo.

[0097] In the above uses, including therapeutic uses, delivery of the ATR of the present invention, or the ATR of the present invention and another effector protein, to a cell can silence a target gene. The delivery can be transient. The delivery can be due to expression of the ATR of the present invention, or the ATR of the present invention and another effector protein, in the cell, for example, expression from a polynucleotide encoding the ATR of the present invention or the ATR of the present invention and another effector protein.

[0098] In one embodiment, expression of the ATR of the present invention, or the ATR of the present invention and another effector protein, in a cell silences a target gene. The expression may be transient.

[0099] In one embodiment, delivery of the ATR of the present invention, or the ATR of the present invention and another effector protein, to a cell (e.g., by expression in the cell) persistently silences the target gene. In another embodiment, delivery of the ATR of the present invention, or the ATR of the present invention and another effector protein, to a cell (e.g., by expression in the cell) persistently silences the target gene in the progeny of the cell. For example, the cell can be a stem cell, and the target gene can be silenced in the progeny of the stem cell (e.g., the target gene can be silenced in a cell resulting from differentiation of the stem cell).

[0100] For example, the cells can be derived from animals (e.g., mammals, e.g., humans), fungi (e.g., yeast), or plants. For example, the cells can be hematopoietic stem and progenitor cells, T lymphocytes, mesenchymal stem cells, fibroblasts, monocytes, epidermal, or neural stem cells.

[0101] In another aspect, the present invention provides an artificial transcriptional repressor (ATR) comprising a DNA-binding domain operably linked to a KRAB domain or a homolog thereof, or a polynucleotide encoding the same, for use in therapy, in which the ATR is administered to a subject simultaneously, sequentially or separately in combination with a first, distinct effector protein that is not operably linked to a DNA-binding domain comprising a DNMT3A, DNMT3B or DNMT1 domain or a homolog thereof, and / or a second, distinct effector protein that is not operably linked to a DNA-binding domain comprising a DNMT3L domain or a homolog thereof, or a polynucleotide encoding the same.

[0102] In another aspect, the present invention provides an artificial transcriptional repressor (ATR) comprising a DNA-binding domain operably linked to a DNMT3A, DNMT3B or DNMT1 domain or a homolog thereof, or a polynucleotide encoding the same, for use in therapy, in which the ATR is administered to a subject simultaneously, sequentially or separately in combination with a first, distinct effector protein that is not operably linked to a DNA-binding domain comprising a KRAB domain or a homolog thereof, and / or a second, distinct effector protein that is not operably linked to a DNA-binding domain comprising a DNMT3L domain or a homolog thereof, or a polynucleotide encoding the same.

[0103] In another aspect, the present invention provides an artificial transcriptional repressor (ATR) comprising a DNA-binding domain operably linked to a DNMT3L domain or a homolog thereof, or a polynucleotide encoding the same, for use in therapy, in which the ATR is administered to a subject simultaneously, sequentially or separately in combination with a first, distinct effector protein that is not operably linked to a DNA-binding domain comprising a KRAB domain or a homolog thereof, and / or a second, distinct effector protein that is not operably linked to a DNA-binding domain comprising a DNMT3A, DNMT3B or DNMT1 domain or a homolog thereof, or a polynucleotide encoding the same.

[0104] A third, separate effector protein that is not operably linked to the DNA-binding domain containing the SETDB1 domain or a homolog thereof may also be used in these combinations.

[0105] In another aspect, the present invention provides an artificial transcriptional repressor (ATR) comprising a DNA-binding domain operably linked to a SETDB1 domain or a homolog thereof, or a polynucleotide encoding the same, for use in therapy, wherein the ATR is administered to a subject simultaneously, sequentially or separately in combination with a first additional effector protein that is not operably linked to a DNA-binding domain comprising a KRAB domain or a homolog thereof, a second additional effector protein that is not operably linked to a DNA-binding domain comprising a DNMT3A, DNMT3B or DNMT1 domain or a homolog thereof, and / or a third additional effector protein that is not operably linked to a DNA-binding domain comprising a DNMT3L domain or a homolog thereof, or a polynucleotide encoding the same.

[0106] In another aspect, the present invention provides a cell comprising the ATR of the present invention or the ATR of the present invention and another effector protein. The cell can be transfected with a polynucleotide encoding the ATR of the present invention or the ATR of the present invention and another effector protein. The polynucleotides encoding the ATR of the present invention and another effector protein can be in the form of a single vector or can be contained in separate vectors.

[0107] In another aspect, the present invention provides cells that are progeny of cells comprising the ATR of the present invention or the ATR of the present invention and another effector protein. In one embodiment, the progeny cells no longer comprise the ATR of the present invention and / or another effector protein. In another aspect, the present invention provides cells of the present invention for use in therapy.

[0108] In another aspect, the present invention provides a method of gene therapy comprising transfecting a cell with a polynucleotide encoding the ATR of the present invention or the ATR of the present invention and another effector protein. The polynucleotides encoding the ATR of the present invention and another effector protein can be in the form of a single vector or can be contained in separate vectors.

[0109] In one embodiment, the transfection is performed ex vivo.

[0110] In another aspect, the present invention provides a method of gene therapy comprising simultaneously, sequentially, or separately administering to a subject a single ATR and another effector protein not operably linked to the DNA-binding domain, or a polynucleotide encoding the same. The ATR may comprise a DNA-binding domain operably linked to an effector domain selected from (a) a KRAB domain or a homolog thereof; (b) a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof; or (c) a DNMT3L domain or a homolog thereof (i.e., the ATR may be as described herein). The other effector protein not operably linked to the DNA-binding domain may comprise a KRAB, DNMT3A, DNMT3B, DNMT1, or DNMT3L domain or a homolog thereof. The other effector protein may be an effector domain / protein described herein. The other effector protein may be a full-length protein or a functional fragment thereof. Preferably, the other effector protein is different from the effector domain of the ATR. Preferably, the other effector protein is of a different class from the effector domain of the ATR, and preferably, the other effector protein is selected so that it does not contain a domain belonging to the same group (a), (b), or (c) as the effector domain constituting the ATR.

[0111] The ATR may also comprise a DNA-binding domain operably linked to a SETDB1 effector domain or a homolog thereof. The other effector protein not operably linked to a DNA-binding domain may also comprise a SETDB1 domain or a homolog thereof.

[0112] In another aspect, the present invention provides a kit comprising a single ATR and another effector protein, or a polynucleotide encoding the same, that is not operably linked to a DNA-binding domain. The ATR may comprise a DNA-binding domain operably linked to an effector domain selected from (a) a KRAB domain or a homolog thereof; (b) a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof; or (c) a DNMT3L domain or a homolog thereof (i.e., the ATR may be as described herein). The other effector protein not operably linked to a DNA-binding domain may comprise a KRAB, DNMT3A, DNMT3B, DNMT1, or DNMT3L domain or a homolog thereof. The other effector protein may be an effector domain / protein described herein. The other effector protein may be a full-length protein or a functional fragment thereof. Preferably, the other effector protein is different from the effector domain of the ATR. Preferably, the other effector protein is of a different class from the effector domain of the ATR, and preferably, the other effector protein is selected so that it does not contain a domain belonging to the same group (a), (b), or (c) as the effector domain constituting the ATR.

[0113] The ATR may also comprise a DNA-binding domain operably linked to a SETDB1 effector domain or a homolog thereof. The other effector protein not operably linked to a DNA-binding domain may also comprise a SETDB1 domain or a homolog thereof.

[0114] In another aspect, the present invention provides a method for silencing a target gene, comprising administering to a cell an ATR of the present invention, or an ATR of the present invention and another effector protein, or a polynucleotide encoding the same. The method may be an in vitro method.

[0115] It is also contemplated that when another component of a product of the invention (i.e., ATR or another effector protein) contains a DNMT3A, DNMT3B or DNMT1 domain or a homolog thereof, the ATR or another effector protein of the invention may contain a SETDB1 domain or a homolog thereof.

[0116] The methods and uses of the present invention, such as gene therapy or target gene silencing methods, can also include a step of inactivating an endogenous gene that can oppose the activity of the ATR or another effector protein of the present invention. For example, the DNMT3B gene can be inactivated. The inactivation step of this method can be, for example, transient or permanent. The inactivation can be achieved, for example, by genetic deletion, for example, by using a CRISPR / Cas9-based approach, or by post-transcriptional downregulation, for example, by using sh / siRNA, or by transcriptional downregulation, for example, by using individual KRAB-based ATRs targeted to the regulatory sequence of the gene of interest. Inactivation of DNMT3B is particularly preferred when using three ATRs that individually contain the KRAB, DNMT3A, and DNMT3L domains. [Brief explanation of the drawings]

[0117] [Figure 1] Diagram showing details of the experimental cell model. An eGFP expression cassette (based on the hPGK promoter) followed by a TetO7 sequence is integrated into the first intron of the PPP1R12C gene (also known as the AAVS1 site) of the K562 cell line. Single-cell-derived clones containing homozygous insertions of the cassette are then transfected with a vector expressing ATR (with a candidate repression (Rep) domain). After deposition of a repressive epigenetic mark (red lollipop), the cells are treated with or without doxycycline. Maintenance of silencing or reactivation of eGFP expression is then assessed by measuring eGFP expression. [Figure 2]Comparison of epigenetic silencing induced by tetR:K and tetR:D3A. A. Diagram of a bidirectional lentiviral vector (Bid.LV) expressing tetR:K and the marker gene mOrange (left) or tetR:DNMT3A and the marker gene ΔLNGFR (right). B. TetO7.eGFP reporter clones were transfected with Bid.LV-tetR:K or Bid.LV-tetR:D3A in the presence or absence of doxycycline (left and right graphs, respectively) and analyzed by flow cytometry over time to measure the percentage of eGFP-negative cells. C. Representative dot plot analysis of TetO7.eGFP reporter cell lines transfected with Bid.LV-tetR:K or Bid.LV-tetR:D3A in the presence or absence of doxycycline at the indicated time points. The mean fluorescence intensity (MFI) of eGFP-silenced cells is compared to the MFI of untreated wild-type cells. D. Silenced cells from the condition in (B) without doxycycline were sorted to a pure state and maintained in culture in the presence or absence of doxycycline to evaluate the reactivation of eGFP. A representative dot plot of the cells in the presence or absence of the drug is shown below. E. Silenced and sorted cells from the condition in (B) were treated with AZA (1 μM) or vehicle (DMSO) for 7 days and analyzed for eGFP expression (histogram on the left). A representative dot plot of vehicle- and AZA-treated cells is shown. [Figure 3] TetR:D3A-induced transcriptional repression is localized to the target gene locus. A. Diagram of the AAVS1 site. Genes surrounding the reporter cassette (red arrow) are indicated. B. Histogram showing the fold change in expression levels of the indicated genes between eGFP-negative cells silenced with Bid.LV-tetR:K or Bid.LV-tetR:D3A and untreated cells. The relative expression level of each gene was normalized to the expression of B2M and expressed as the fold change relative to untreated cells (control) (n = 3). [Figure 4]Synergistic activity of tetR:K and tetR:D3A upon their transient co-delivery. A. The TetO7.eGFP reporter cell line was transiently transfected with plasmids encoding tetR:K and tetR:D3A, either alone or in combination. The cells were analyzed over time by flow cytometry, and silencing efficiency was measured as the percentage of eGFP-negative cells post-transfection. Representative dot plots for each transfection condition are shown below the histograms (n=3). B. Histogram showing the fold change in expression levels of the indicated genes between selected eGFP-silenced cells from the mixed condition shown in (A) and untreated cells. The relative expression level of each gene was normalized to B2M and expressed as the fold change relative to untreated cells (control) (n=3). c. eGFP-negative cells from the mixed treatment condition in (A) were sorted and then treated with AZA or DMSO (histograms show the percentage of eGFP-positive cells 7 days after the indicated treatment; n=3). D. Experiments similar to (A) were performed using in vitro transcribed mRNAs encoding tetR:K and tetD3A delivered alone or in combination. E. Histograms showing the fold change in expression levels of the indicated genes between sorted eGFP-silenced cells and untreated cells from the mixed condition shown in (D) (n=3). [Figure 5]Gene silencing with the combination of tetR:K and tetD3A is site (locus) and cell type independent. A. Diagram of the TetO7 reporter LV used in this study. The TetO7 sequence was cloned upstream of the hPGK promoter, which drives expression of the eGFP reporter transgene. B. Graphs showing the kinetics of eGFP silencing (% of eGFP-negative cells by flow cytometry) in the K562 LV / TET07 reporter cell line transfected with in vitro transcribed mRNA encoding tetR:K and tetD3A delivered alone or in combination (n=3; data are presented as mean ± SEM). C-D. Graphs showing the kinetics of eGFP silencing (% of eGFP-negative cells by flow cytometry) in the U937LV / TET07 cell line (C) or the B lymphoblastoid LV / TET07 reporter cell line (D). Cells were transfected as indicated in (B) (n=1 for U937 and n=3 for B lymphoblast cells). [Figure 6] Screening for additional epigenetic effector domains for ATR. A. Graph showing the kinetics of eGFP silencing (% eGFP-negative cells by flow cytometry) in K562 LV / TETO7 reporter cell lines upon transduction of lentiviral vectors expressing the indicated tetR-based ATRs (n=3). B. Graph showing the percentage of cells positive for the indicated LVs in culture over time (n=3). C. Graph showing the kinetics of eGFP silencing (% eGFP-negative cells by flow cytometry) in K562 LV / TETO7 reporter cell lines transfected with lentiviral vectors stably expressing the indicated tetR-based ATRs before and after doxycycline treatment (n=3). [Figure 7]Screening of additional combinations of artificial transcriptional repressors (ATRs) in various mammalian cells. A–D. Graphs showing the kinetics of eGFP silencing (% eGFP-negative cells by flow cytometry) in the K562 LV / TETO7 reporter cell line upon electroporation with individual plasmids encoding the indicated tetR-based ATRs (A; n=3; data are presented as mean ± SEM), or with a plasmid encoding tetR:D3A and one of the other tetR-based ATRs (B; n=3; data are presented as mean ± SEM), or with a plasmid encoding tetR:K and one of the other tetR-based ATRs (C; n=3; data are presented as mean ± SEM), or with a plasmid encoding tetR:D3A + tetR:K in combination with a plasmid encoding one of the other tetR-based ATRs (D; n=3; data are presented as mean ± SEM). E. Histogram showing the efficacy of eGFP silencing 21 days after electroporation of the K562 LV / TETO7 reporter cell line with plasmids encoding the indicated tetR-based ATRs (time points after A–D; n=3; data are presented as mean ± SEM). F. Histogram showing the efficacy of eGFP silencing 30 days after electroporation of the K562 LV / TETO7 reporter cell line with plasmids encoding the indicated tetR-based ATRs, including those based on SETDB1 (n=3; data are presented as mean ± SEM). G. Graph showing the kinetics of eGFP silencing in the B lymphoblast LV / TETO7 reporter cell line electroporated with mRNA encoding the indicated tetR-based ATRs (n=3; data are presented as mean ± SEM).H. Graph showing the kinetics of eGFP silencing in the mouse NIH / 3T3 LV / TETO7 reporter cell line electroporated with mRNA encoding the indicated tetR-based ATRs ( n = 2; data are presented as mean ± range). [Figure 8] Gene silencing by transient co-delivery of artificial transcriptional repressors (ATRs) containing tailored DNA-binding domains (head-to-tail orientation). A. Schematic diagram of various artificial tail (Tale)-binding sites (head-to-tail) integrated semi-randomly into the genome of K562 cells by LV transduction. The two ATR-binding sites are separated by a TALE domain, resulting in two alternative co-delivery strategies with different D3A-K relative target binding orders. B. Graph showing silencing efficiency (% of eGFP-negative cells 34 days after electroporation) as a function of spacer length in the K→D3A (left) and D3A→K (right) relative ATR orders of target binding orders. C. Graph showing the kinetics of eGFP silencing in cell lines with a 25-bp spacer (the best-performing spacer tested in the experiment shown in B) in the K → D3A (left) and D3A → K (right) relative ATR order of binding to the target (n = 3; data are presented as mean ± SEM). [Figure 9]Gene silencing by transient co-delivery of artificial transcriptional repressors (ATRs) containing tailored DNA-binding domains (head-to-head orientation). A. Schematic diagram of various artificial tail (Tale)-binding sites (head-to-head) of hPGK-eGFP cassettes semi-randomly integrated into the genome of K562 cells by LV transduction, with different spacer lengths between the two ATR-binding sites. Two different TALE domains were fused separately to each epigenetic effector, resulting in two alternative co-delivery methods with different D3A-K relative order of target binding. B. Graph showing silencing efficiency (% of eGFP-negative cells 34 days after electroporation) as a function of spacer length in the D3A→K (left) and K→D3A (right) relative ATR orders of target binding. C. Graph showing the kinetics of eGFP silencing in cell lines with a 15-bp spacer (the best-performing spacer tested in the experiment shown in B) in the D3A→K (left) and K→D3A (right) relative ATR order of binding to the target (n=3; data are presented as mean ± SEM). [Figure 10] Gene silencing with artificial transcriptional repressors (ATRs) containing two or more effector domains. A. Schematic diagram of various artificial tail-binding sites (head-to-head) integrated into the genome of K562 cells by LV transduction, linked to a chimeric K:tetR:D3A ATR (Bi-Partite; BiP). In this case, KRAB and DNMT3A domains were fused to the N- and C-termini (respectively) of the same DNA-binding domain. B. Graph showing the kinetics of eGFP silencing in cell lines (same cell lines as used in Figure 8B) transfected alone or in combination with plasmids encoding Bi-Partite (BiP) fusion proteins with a 25-bp spacer (n = 3; data are presented as mean ± SEM). [Figure 11]Sustained epigenetic silencing in human hematopoietic stem and progenitor cells (HSPCs) using various combinations of artificial transcriptional repressors (ATRs). A. Timeline diagram of the method used to evaluate the efficiency of silencing in HSPCs. Briefly, on day 0, human CD34+ cells were thawed in stimulation medium containing early-acting cytokines, and on day 1, they were transfected with the TetO7 reporter LV (vector diagram in Figure 5A). Then, on day 3 after thawing, cells were washed and electroporated with in vitro transcribed mRNA. The following day, 800 cells were plated for CFC-U assay, while the remaining cells were expanded in liquid culture and analyzed by flow cytometry at the indicated time points. CFC-U analysis was performed 14 days after thawing. B. Graph showing the kinetics of eGFP silencing in liquid-cultured human CD34+ cells transfected with in vitro-transcribed mRNAs encoding the indicated ATRs delivered alone or in double or triple combinations (data normalized to non-electroporated but LV-transduced controls; n=3; data presented as mean ± SEM). C. Histogram showing the percentage of eGFP-silencing in erythroid and myeloid colonies derived from human CD34+ cells transfected with in vitro-transcribed mRNAs as shown in (B) (n=3; data presented as mean ± SEM). [Figure 12]Sustained epigenetic silencing in human T lymphocytes using various combinations of artificial transcriptional repressors (ATRs). A. Timeline diagram of the method used in this study to evaluate the efficiency of silencing in human primary T cells. Briefly, on day 0, T cells were isolated with anti-CD3 / CD28-coated beads and left in culture for 3 days before being transfected with the reporter TetO.LV. On day 6, the cells were transfected with in vitro transcribed mRNA encoding the indicated ATRs, and eGFP expression was analyzed by flow cytometry at the indicated time points. Three weeks after transfection, the cells were restimulated, and the stability of eGFP silencing was measured by flow cytometry. B. Graph showing the kinetics of eGFP silencing in human primary T cells transfected with in vitro transcribed mRNAs encoding the indicated ATRs, delivered alone or in double or triple combinations, normalized to untreated cells (data normalized to non-electroporated but LV-transduced controls; n = 2; data presented as mean ± range). [Figure 13]Sustained epigenetic silencing of the human beta2-microglobulin (B2M) gene using a combination of artificial transcriptional repressors (ATRs). A. Schematic diagram of the B2M locus showing the binding sites of the TALE-based ATRs. B. Graph showing the kinetics of B2M silencing in HEK-293T cells electroporated with plasmids encoding the indicated TALE-based ATRs delivered alone or in combination (n=3; data are presented as mean ± SEM). C. Representative flow cytometry dot plots of HEK-293T cells transfected with plasmids encoding triple TALE:ATR combinations, and the cell sorting method used to enrich for double-negative (lower plot on the left) and double-positive (lower plot on the right) cells. D. Histogram showing the fold change in expression levels of the B2M gene in sorted cells from (C) and untreated HEK293T cells (n=3; data are presented as mean ± SEM). E. Diagram of gRNA and dCas9-based ATR selected to target a CpG island located within the B2M promoter region. F. Histogram showing silencing efficiency 33 days after electroporation of the CRISPR / dCas9-based ATR plasmid (n=3; data are presented as mean ± SEM). G. B2M-silenced cells from Figure 2C (referred to in this panel as TALE B2M-), B2M-silenced cells sorted from the triple CRISPR / dCas9-based ATR combination in Figure 2F (referred to in this panel as TALE B2M-), and wild-type HeK-293T cells (referred to in this panel as WT B2M+) were exposed to or not IFN-γ and then analyzed to measure the expression levels of B2M and OAS1. Histogram showing the fold change in expression levels of the B2M and OAS1 genes between IFN-γ-treated and untreated cells. Expression of the hypoxanthine phosphoribosyltransferase 1 (HPRT1) gene was used as a standard (n=3; data are presented as mean±SEM).H. Representative flow cytometry dot plots of the indicated HEK-293T populations either untreated (left plot) or after 4 days of IFN-γ treatment (right plot). Numbers indicate MFI B2M. [Figure 14] Silencing of beta2-microglobulin (B2M) is associated with significant epigenetic editing of the gene. A. Representative flow cytometry dot plots of HEK-293T cells transfected with a plasmid encoding the triple TALE:ATR combination, along with the cell sorting method used to enrich for double-positive and double-negative cells. B. ChlP analysis performed in the presence of RNA PolII in untreated cells (top histogram) and silenced cells from (A) (bottom histogram). The histograms show the fold enrichment in RNA PolII relative to input, relative to the distance of the qPCR assay from the transcription start site (TSS; set to +1) of the gene (n=3; data are presented as mean ± SEM). The ubiquitously transcribed AAVS1 locus was used as a positive control (PC) for RNA PolII enrichment, and the silent CCR5 gene was used as a negative control (NC). C. Bisulfite analysis of the B2M CpG island in untreated (UT) and silenced cells. The relative positions of the gene's TSS and the binding sites of the three TALE:ATR (D;L;K) are indicated. D. Histogram showing the percentage of B2M-positive cells at day 7 after AZA treatment (n=3; data are presented as mean±SEM). E. Top: Schematic of the B2M locus. CpG islands within this locus are shown in green. Bottom: Histogram showing the fold change in gene expression levels of the indicated genes between silenced and untreated cells. Genes with a Ct value of 37 or higher were excluded from the analysis. The relative expression level of each gene was normalized to HPRT and expressed as a fold change relative to untreated cells (standard). [Figure 15]Silencing of beta2-microglobulin (B2M) is effective in other human cell lines. A. Diagram of the CRISPR / Cas9-based gene targeting method used to insert the td / Tomato transgene under the control of the B2M promoter (left). Representative flow cytometry dot plots of K-562 cells before and after gene targeting (top right and bottom right, respectively). B. Histogram showing B2M silencing efficiency (i.e., dtTomato-negative cells) 30 days after electroporation with plasmids encoding the indicated TALE-based ATRs containing wild-type (WT) or codon-optimized effector domains (n=1). C. Graph showing the kinetics of B2M silencing (measured as % of dtTomato-negative cells) in K-562 cells electroporated with plasmids encoding the indicated CRISPR / dCas9-based ATRs (n=1). D. Representative flow cytometry analysis (n=1) of sorted tdTomato-negative cells after transfection with in vitro transcribed mRNA encoding the triple TALER:ATR combination (left panel and dot plot), and (ii) cells from (i) after transfection with a plasmid encoding dCas9:Tet1 in combination with a plasmid for B2M gRNA (left panel and dot plot). [Figure 16] Silencing of beta2-microglobulin (B2M) is effective in primary T lymphocytes. A. Schematic of the experimental workflow. B. Graph showing the kinetics of B2M silencing in human T lymphocytes electroporated with mRNA encoding the triple-TALE system ATR (n=1). C. Representative flow cytometry dot plots of the indicated T lymphocyte populations after 14 days of treatment. The percentage of cells within the indicated gates and B2M MFI are shown. [Figure 17]A single ATR binding site is sufficient for effective silencing of endogenous genes with both Cas9 and TALE-based ATR. A. Top: Diagram of the B2M gene showing the relative position of the gRNA (red arrow) selected to target the CpG island of the B2M gene. Bottom: Histogram (n=1) showing the efficiency of B2M silencing (calculated as % of tdTomato-negative cells) 18 days after electroporation of the CRISPR / dCas9-based ATR plasmid in the K562 B2MtdTomato reporter cell line. B. Left: Diagram of TALE-based ATR binding to DNA. Three different DNA-binding domains (forms #1-#3; referred to in this figure as RDVs (Repeat Variable Diresidues)) each equipped with both of the three different effector domains are shown in gray boxes. As previously shown in Figure 13A, three different RDVs equipped with separate effector domains are shown below this figure. Right: Histogram showing the percentage of tdTomato-negative cells after transfection with plasmids encoding the indicated TALE-ATR combinations (n=3; data are presented as mean±SEM). [Figure 18]Transient expression of untargeted DNMT3L improves and rescues the silencing efficiency of DNMT3A + KRAB-based ATR in refractory cell types. A. Histogram showing the percentage of eGFP silencing in the B lymphoblastoid LV-TetO7 reporter cell line 27 days after transfection with in vitro transcribed mRNA encoding the indicated tetR-based ATR delivered in the presence or absence of tetR:D3L or untargeted full-length DNMT3L-encoding mRNA (n = 2; data are presented as mean ± range). B. Diagram of the B2M locus showing the relative arrangement and binding sites of the indicated TALE-based ATRs. Note that each module can bind a pair of TALE-based ATRs. Furthermore, the relative order of the effector domains within each module can be swapped. For example, for module 1, a KRAB-based ATR can bind to site A and a DNMT3A-based ATR can bind to site B, or vice versa. C. Representative flow cytometry dot plots of B2M silencing in HEK-293T cells 21 days after transfection with plasmids encoding the indicated pairs of TALE-based ATRs (module 1 or module 2; shown in the two possible relative orders of ATR binding) delivered alone (top row plots) or in combination with non-targeting DNMT3L (bottom row plots). D. Histograms showing the percentage of B2M silencing in HEK-293T cells 45 days after transfection with plasmids encoding the indicated dCas9-based ATRs and cognate gRNAs (shown in Figure 13E) delivered alone or in combination with dCas9:D3L or non-targeting full-length DNMT3L-encoding plasmids (n=3; data are presented as mean ± SEM). [Figure 19]Genetic inactivation of DNMT3B increases the silencing efficiency of the triple ATR combination in permissive cell lines, while transient expression of non-targeted DNMT3B rescues the silencing efficiency of the DNMT3A + KRAB combination in refractory cell types. A. Diagram of the lentiviral vectors used to conditionally express Cas9 (left) or the gRNA of interest (right) upon doxycycline administration. Representative flow cytometry analysis of bi) eGFP-positive K-562 cells transfected with the lentiviral vector described in Figure 5A and sorted to near-pure status for eGFP expression; ii) cell line from (i) transfected with LV encoding inducible Cas9 and LV encoding DNMT3B-gRNA (ΔLNGFR was used as a marker for transfection of the latter LV; middle plot) (note that this second cell line was then exposed to doxycycline for 7 days to activate Cas9 expression and disrupt the coding region of the endogenous DNMT3B gene); and iii) cells from (ii) electroporated with plasmids encoding the double tetR:K+tetR:D3A (top right plot) or triple tetR:K+tetR:D3A+tetR:D3L (bottom right plot) ATR combinations. C. Histogram showing the percentage of eGFP-silenced cells 19 days after genetic disruption of the DNMT3B gene using the CRISPR / inducible Cas9 system (n=1). These figures were obtained by calculating the silencing efficiency in ΔLNGFR-positive cells (cells with disruption of DNMT3B; red bars) and ΔLNGFR-negative cells (wild-type K-562 cells; blue bars). D. Histogram showing the silencing efficiency (% of eGFP-negative cells) in the B lymphoblast TetO7 reporter cell line 27 days after transfection with mRNA encoding the indicated tetR-based ATR delivered in the presence or absence of mRNA encoding the non-targeted wild-type DNMT3B sequence (data shown as the average of two experiments). [Figure 20]Sustained epigenetic silencing of additional human endogenous genes using a combination of artificial transcriptional repressors (ATRs). A. The B-cell lymphoma / leukemia 11A (BCL11A) gene showing two transcript variants of this gene. Dashed boxes highlight gene regulatory elements. In particular, the gene promoter / enhancer region at the level of the transcription start site (yellow box), which contains a cluster of four distinct CpG islands varying in size and number of CpG residues, and an erythroid-specific enhancer (red box) that confers lineage-restricted expression of the gene within the second intron of the gene. To investigate the function of both the gene promoter and the erythroid-specific enhancer, we targeted a tdTomato transgene linked to the BCL11A transcript via the 2A autocatalytic peptide within the third exon of the gene. A representative dot plot of B lymphoblastoid cells after sorting for tdTomato-positive cells is shown on the right. B. Histogram showing the percentage of tdTomato-negative cells 32 days after transfection with the indicated dCas9-based ATR and the corresponding pools of gRNAs targeting the indicated CpG islands of BCL11A (i.e., 11 gRNAs for CpG105, 8 gRNAs for CpG31, 9 gRNAs for CpG38, and 10 gRNAs for CpG115) (n=3; data are presented as mean ± SEM). Untreated cells or cells transfected with only the gRNA pool or dCas9-based ATR served as controls. C. tdTomato reporter cell lines were cotransfected with a single or double or triple combination of plasmids encoding dCas9-based ATR and a plasmid with a pool of nine gRNAs targeting CpG38 or a plasmid with a pool of eight gRNAs against CpG31. Silencing efficiency was measured 2 weeks after transfection and presented as a histogram (n=3; data presented as mean±SEM).D. Top: Diagram of the binding sites of TALE-based ATRs targeting CpG31 (top left) or CpG31 (top right) in the BCL11A promoter region, and their relative orientations of binding to DNA (+ indicates Watson strand, - indicates Crick strand). Bottom: dtTomato reporter cell lines were transfected with plasmids encoding TALE:KRAB alone or with the indicated combinations of triple TALE-based ATRs indicated on the x-axis of the histogram. Silencing efficiency is shown as the percentage of dTomato-negative cells. Analysis was performed 18 days after plasmid transfection (n=3; data are presented as mean ± SEM). E. Top: Diagram of the interferon (alpha, beta, and omega) receptor 1 (IFNAR1) gene. Green boxes highlight CpG islands at the level of the gene promoter / enhancer region (CpG residue numbers are indicated). Bottom: Histogram showing the fold change in IFNAR1 gene expression levels between cells electroporated with a pool of 13 gRNAs against the IFNAR1 CpG island and a plasmid encoding dCas9:K+dCas9:D3A+dCas9:D3L ATR (18 days after treatment) and untreated cells. The relative expression level of the IFNAR1 gene was normalized to the expression of DNMT1 and expressed as the fold change compared to untreated cells (n=1). F. Top: Diagram of the vascular endothelial growth factor A (VEGFA) gene. The green box highlights the CpG island (CpG residue numbers are indicated) at the level of the gene promoter / enhancer region. Bottom: Histogram showing the fold change in VEGFA gene expression levels between cells electroporated with a pool of three gRNAs against the VEGFA CpG island and plasmids encoding dCas9:K + dCas9:D3A + dCas9:D3L ATR (14 days after treatment) and untreated cells. Relative VEGFA gene expression levels were normalized to DNMT1 expression and expressed as fold change compared to untreated cells (control) (n=1).

[0118] Detailed Description of the Invention Preferred features and embodiments of the present invention will now be described by way of non-limiting examples.

[0119] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology, and immunology, which are within the capabilities of those skilled in the art. Such techniques are explained in the literature. For example, Sambrook, J., Fritsch, EF, and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, FM et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J., and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, JM, and McGee, J.O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, MJ (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, DM, and Dahlberg, JE (1992) Methods in Enzymology: DNA Structures Part See A: Synthesis and Physical Analysis of DNA, Academic Press, both of which general texts are incorporated herein by reference.

[0120] In one embodiment, the present invention provides a method for treating a cancer cell comprising administering to a subject a cancer cell of group (a), (b), or (c): (a) an artificial transcriptional repressor (ATR) containing a DNA-binding domain operably linked to a KRAB domain or its homologue; (b) an ATR containing a DNA-binding domain operably linked to a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof; and (c) ATR containing a DNA-binding domain operably linked to a DNMT3L domain or a homolog thereof. The present invention provides a product containing two or more ATRs, or polynucleotides encoding them, selected from the group consisting of (a), (b), or (c), wherein at least two of the ATRs are selected from non-identical groups (distinct groups).

[0121] The product of the present invention can be, for example, a composition containing a mixture of two or more ATRs selected from group (a), (b), or (c): (a) an ATR containing a DNA-binding domain operably linked to a KRAB domain or a homolog thereof; (b) an ATR containing a DNA-binding domain operably linked to a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof; and (c) an ATR containing a DNA-binding domain operably linked to a DNMT3L domain or a homolog thereof, or polynucleotides encoding them, wherein at least two of the ATRs are selected from non-identical groups (a), (b), or (c). Alternatively, the product may be a kit comprising a formulation of two or more ATRs, or polynucleotides encoding them, selected from group (a), (b), or (c): (a) an ATR containing a DNA-binding domain operably linked to a KRAB domain or a homolog thereof; (b) an ATR containing a DNA-binding domain operably linked to a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof; and (c) an ATR containing a DNA-binding domain operably linked to a DNMT3L domain or a homolog thereof, wherein at least two of the ATRs are selected from non-identical groups (a), (b), or (c), and the kit further optionally includes instructions for simultaneous, sequential, or separate administration of the formulations to a subject in need thereof.

[0122] Artificial transcriptional repressors (ATRs) are active agents that act to reduce the transcription of target genes. ATRs can be chimeric proteins consisting of a DNA-binding domain operably linked to an effector domain (e.g., a KRAB domain, a DNMT3A, DNMT3B, or DNMT1 domain, or a DNMT3L domain, or a homolog thereof). The DNA-binding domain allows ATR to bind to a specific nucleic acid sequence and can be engineered to bind to a selected nucleic acid sequence. The effector domain can possess catalytic activity that allows for transcriptional repression of the target gene. Alternatively, or in addition, the effector domain can recruit other active agents within the cell to the target gene, resulting in transcriptional repression of the target gene.

[0123] By "operably linked," it is understood that the individual components are joined together in a manner that allows them to perform their functions (e.g., binding to DNA, catalyzing a reaction, or recruiting additional active substances from within a cell) substantially unimpeded. For example, a DNA-binding domain can be conjugated to an effector domain to form a fusion protein. Methods for conjugating polypeptides are known in the art, such as providing a linker protein connecting the polypeptides. Another method for conjugating polypeptides known in the art is chemical photo-induced conjugation (e.g., using a chemical cross-linking agent). Preferably, the DNA-binding domain and effector domain of ATR (e.g., the KRAB domain, the DNMT3A, DNMT3B, or DNMT1 domain, or the DNMT3L domain, or homologs thereof) form a fusion protein.

[0124] Effector domain The term "effector domain" should be understood to refer to a portion of the ATR that exerts a silencing effect on a target gene, for example, by catalyzing a reaction on DNA or chromatin (e.g., DNA methylation) or by recruiting additional active substances from within the cell, resulting in the repression of gene transcription.

[0125] A "domain," in this context, should be understood as a portion of ATR that retains a certain function. A domain can be a discrete domain (e.g., a catalytic domain) isolated from a naturally occurring protein, but can also be the entire full-length naturally occurring protein. In other words, the full-length protein or a functional fragment thereof can be used as an effector domain. Thus, for example, a "KRAB domain" refers to a portion of ATR that contains an amino acid sequence having the function of a KRAB domain.

[0126] Chromatin-remodeling enzymes known to be involved in permanent epigenetic silencing of endogenous retroviruses (ERVs; Feschotte, C. et al. (2012) Nat. Rev. Genet. 13: 283-96; Leung, DC et al. (2012) Trends Biochem. Sci. 37: 127-33) can provide suitable effector domains for practicing the present invention.

[0127] The Kruppel-associated box family of zinc finger proteins (KRAB-ZFPs; Huntley, S. et al. (2006) Genome Res. 16: 669-77) plays an important role in silencing endogenous retroviruses. These transcription factors bind to specific ERV sequences via the DNA-binding domain of their ZFPs and recruit KRAB Associated Protein 1 (KAP1) via its conserved KRAB domain. KAP1 further binds to multiple effectors that promote the local formation of repressive chromatin (Iyengar, S. et al. (2011) J. Biol. Chem. 286: 26267-76).

[0128] During early embryonic development, KAP1 is known to recruit the histone methyltransferase SET domain bifurcated 1 (SETDB1), which accumulates two histone marks associated with transcriptional repression: histone H3 lysine-9 di- and trimethylation (H3K9me2 and H3K9me3, respectively). Concurrently, KAP1 binds heterochromatin protein 1α (HP1α), which reads H3K9me2 and H3K9me3 and stabilizes KAP1-containing complexes. KAP1 can also interact with other well-known epigenetic silencers, such as lysine-specific histone demethylase 1 (LSD1), which inhibits transcription by removing histone H3 lysine-4 methylation, and the nucleosome remodeling and deacetylase complex (NURD), which removes acetyl groups from histones. Finally, KAP1-containing complexes contribute to the recruitment of de novo DNA methyltransferase 3A (DNMT3A), which methylates cytosines at CpG sites (Jones, PA (2012) Nat. Rev. Genet. 13: 484-92). Together, these data suggest a model in which the KAP1 complex ensures ERV silencing in preimplantation embryos through the concerted action of histone-modifying enzymes and DNA-binding domain methylation. Thus, postimplantation, DNA methylation pretargeted to ERVs by KRAB-ZFPs becomes stable (Reik, W. (2007) Nature 447: 425-32) and is inherited through mitosis and somatic differentiation without the need for sustained expression of ERV-specific KRAB-ZFPs. Contrary to embryonic stem cells, the KAP1 complex cannot efficiently induce DNA methylation in somatic cells and can only accumulate H3K9 methylation. However, this histone mark is not maintained unless it is persistently deposited at target sites by KRAB-ZFPs (Hathaway, NA et al. (2012) Cell 149: 1447-60).

[0129] Therefore, when considering epigenetic therapies based on transient expression of ATRs in somatic cells, the KRAB-ZFP / KAP1 machinery is expected to be ineffective if used alone. Instead, we consider a preferred method of simultaneously introducing two distinct ATRs: one based on, for example, the KRAB domain, which is the initiator of the epigenetic cascade that occurs in ERVs in embryonic stem cells, and the other based on, for example, DNMT3A, the final lock of this process. This approach could recapitulate, at preselected target genes, the repressive chromatin state established in ERVs in preimplantation embryos, which can then be permanently inherited throughout mammalian development and adulthood.

[0130] The ATR of the present invention may contain, for example, a KRAB domain. Various KRAB domains are known as the KRAB-ZFP protein family. For example, the ATR of the present invention may contain the KRAB domain of human zinc finger protein 10 (ZNF10; Szulc, J. et al. (2006) Nat. Methods 3: 109-16): ALSPQHSAVTQGSIIKNKEGMDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIKSSV (SEQ ID NO: 1)

[0131] Other examples of KRAB domains suitable for use in the present invention include: ITLEDVAVDFTWEEWQLLGAAQKDLYRDVMLENYSNLVAVGYQASKPDALFKLEQGEQLWTIEDGIHSGACS (KRAB domain of ZNF350 protein; SEQ ID NO: 2) VMFEEVSVCFTSEEWACLGPIQRALYWDVMLENYGNVTSLEWETMTENEEVTSKPSSSQRADSHKGTSKRLQG (KRAB domain of ZNF197 protein; SEQ ID NO: 3) VSFKDVAVDFTQEEWQQLDPDEKITYRDVMLENYSHLVSVGYDTTKPNVIIKLEQGEEPWIMGGEFPCQHSP (KRAB domain of RBAK protein; SEQ ID NO: 4) VKIEDMAVSLILEEWGCQNLARRNLSRDNRQENYGSAFPQGGENRNENEESTSKAETSEDSASRGETTGRSQKE (KRAB domain of ZKSCAN1 protein; SEQ ID NO: 5) LTFKDVFVDFTLEEWQQLDSAQKNLYRDVMLENYSHLVSVGYLVAKPDVIFRLGPGEESWMADGGTPVRTCA (KRAB domain of KRBOX4 protein; SEQ ID NO: 6) VTFEDVTLGFTPEEWGLLDLKQKSLYREVMLENYRNLVSVEHQLSKPDVVSQLEEAEDFWPVERGIPQDTIP (KRAB domain of ZNF274 protein; SEQ ID NO: 7)

[0132] The ATR of the present invention can contain, for example, a domain, preferably a catalytic domain, of human DNA methyltransferase 3A (DNMT3A; Law, JA et al. (2010) Nat. Rev. Genet. 11: 204-20). For example, the ATR of the present invention can contain the sequence: TYGLLRRREDWPSRLQMFFANNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDDRPF FWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACV (SEQ ID NO: 8) It may contain:

[0133] DNA methyltransferases 3B and 1 (DNMT3B and DNMT1), like DNMT3A, contribute to the accumulation and maintenance of DNA methylation and can similarly be used in the ATRs of the present invention. For example, the ATRs of the present invention can contain any of the following sequences: CHGVLRRRKDWNVRLQAFFTSDTGLEYEAPKLYPAIPAARRRPIRVLSLFDGIATGYLVLKELGIKVGKYVASEVCEESIAVGTVKHEGNIKYVNDVRNITKKNIEEWGPFDLVIGGSPCNDLSNVNPARKGLYEGTGRLFFEFYHLLNYSRPKEGDDDRPF FWMFENVVAMKVGDKRDISRFLECNPVMIDAIKVSAAHRARYFWGNLPGMNRPVIASKNDKLELQDCLEYNRIAKLKKVQTITTKSNSIKQGKNQLFPVVMNGKEDVLWCTELERIFGFPVHYTDVSNMGRGARQKLLGRSWSVPVIRHLFAPLKDYFACE (catalytic domain of human DNMT3B; SEQ ID NO: 9) MVAELISEEDLEFMKGDTRHLNGEEDAGGREDSILVNGACSDQSSDSPPILEAIRTPEIRGRRSSSRLSKREVSSLLSYTQDLTGDGDGEDGDGSDTPVMPKLFRETRTRSESPAVRTRNNNSVSSRERHRPSPRSTRGRQGRNHVDESPVEFPATRSLRRRATASAGTPWPSPPSSYLTIDLTDDTEDTHGTPQSSSTPYARLAQDSQQG GMESPQVEADSGDGDSSEYQDGKEFGIGDLVWGKIKGFSWWPAMVVSWKATSKRQAMSGMRWVQWFGDGKFSEVSADKLVALGLFSQHFNLATFNKLVSYRKAMYH ALEKARVRAGKTFPSSPGDSLEDQLKPMLEWAHGGFKPTGIEGLKPNNTQPENKTRRRTADDSATSDYCPAPKRLKTNCYNNGKDRGDEDQSREQMASDVANNKSS LEDGCLSCGRKNPVSFHPLFEGLCQTCRDRFLELFYMYDDDGYQSYCTVCCEGRELLLCSNTSCCRCFCVECLEVLVGTGTAEAAKLQEPWSCYMCLPQRCHGV LRRRKDWNVRLQAFFTSDTGLEYEAPKLYPAIPAARRRPIRVLSLFDGIATGYLVLKELGIKVGKYVASEVCEESIAVGTVKHEGNIKYVNDVRNITKKNIEEWGP FDLVIGGSPCNDLSNVNPARKGLYEGTGRLFFEFYHLLNYSRPKEGDDRPFFWMFENVVAMKVGDKRDISRFLECNPVMIDAIKVSAAHRARYFWGNLPGMNRPVI ASKNDKLELQDCLEYNRIAKLKKVQTITTKSNSIKQGKNQLFPVVMNGKEDVLWCTELERIFGFPVHYTDVSNMGRGARQKLLGRSWSVPVIRHLFAPLKDYFACE (DNMT3B; SEQ ID NO: 36) LRTLDVFSGCGGLSEGFHQAGISDTLWAIEMWDPAAQAFRLNNPGSTVFTEDCNILLKLVMAGETTNSRGQRLPQKGDVEMLCGGPPCQGFSGMNRFNSRTYSKFKNSLVVSFLSYCDY YRPRFFLLENVRNFVSFKRSMVLKLTLRCLVRMGYQCTFGVLQAGQYGVAQTRRRAIILAAAPGEKLPLFPEPLHVFAPRACQLSVVVDDKKFVSNITRLSSGPFRTITVRDTMSDLPEV RNGASALEISYNGEPQSWFQRQLRGAQYQPILRDHICKDMSALVAARMRHIPLAPGSDWRDLPNIEVRLSDGTMARKLRYTHHDRKNGRSSSGALRGVCSCVEAGKACDPAARQFNTLI PWCLPHTGNRHNHWAGLYGRLEWDGFFSTTVTNPEPMGKQGRVLHPEQHRVVSVRECARSQGFPDTYRLFGNILDKHRQVGNAVPPPLAKAIGLEIKLCMLAKARESASAKIKEEEAAKD (catalytic domain of human DNMT1; SEQ ID NO: 10)

[0134] The ATR of the present invention can contain, for example, DNA (cytosine-5)-methyltransferase 3-like (DNMT3L), which is a catalytically inactive DNA methyltransferase that activates DNMT3A by binding to the catalytic domain of DNMT3A. For example, the ATR of the present invention can contain the following sequence: MAAIPALDPEAEPSMDVILVGSSELSSSVSPGTGRDLIAYEVKANQRNIEDICICCGSLQVHTQHPLFEGGICAPCKDKFLDALFLYDDDGYQSYCSICCSGETLLICGNPDCTRCYCFECVDSLVGPGTSGKVHAMSNWVCYLCLPSSRSGLLQRRRKWRSQLKAFYDRESENPLEMFETVPVWRRQPVRVL SLFEDIKKELTSLGFLESGSDPGQLKHVVDVTDTVRKDVEEWGPFDLVYGATPPLGHTCDRPPSWYLFQFHRLLQYARPKPGSPRPFFWMFVDNLVLNKEDLDVASRFLEMEPVTIPDVHGGSLQNAVRVWSNIPAIRSRHWALVSEEELSLLAQNKQSSKLAAKWPTKLVKNCFLPLREYFKYFSTELTSSL (SEQ ID NO: 11)

[0135] The ATR of the present invention can contain, for example, a SETDB1 domain. For example, the ATR of the present invention can contain any of the following sequences: (SEQ ID NO: 12) VGCDCKDGCRDKSKCACHQLTIQATACTPGGQINPNSGYQYKRLEECLPTGVYECNKRCKCDPNMCTNRLVQHGLQVRLQLFKTQNKGWGIRCLDDIAKGSFVCIYAGKILTDDFADKEGLEMGDEYFANLDHIESVENFK EGYESDAPCSSDSSGVDLKDQEDGNSGTEDPEESNDDSSDNFCKDEDFSTSSVWRSYATRRQTRGQKENGLSETTSKDSHPPDLGPPHIPVPPSIPVGGCNPPSSEETPKNKVASWLSCNSVSEGGFADSDSHSSFKTNE GGEGRAGGSRMEAEKASTSGLGIKDEGDIKQAKKEDTDDRNKMSVVTESSRNYGYNPSPVKPEGLRRPPSKTSMHQSRRLMASAQSNPDDVLTLSSSTESEGESGTSRKPTAGQTSATAVDSDDIQTISSGSEGDDFEDKK NMTGPMKRQVAVKSTRGFALKSTHGIAIKSTNMASVDKGESAPVRKNTRQFYDGEESCYIIDAKLEGNLGRYLNHSCSPNLFVQNVFVDTHDLRFPWVAFFASKRIRAGTELTWDYNYEVGSVEGKELLCCCGAIECRGRLL (Catalytic domain of human SETDB1: SEQ ID NO: 13)

[0136] The ATRs of the present invention contain, for example, an amino acid sequence having 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, which amino acid sequence substantially retains the native function of the protein represented by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13.

[0137] The ATR of the present invention can be encoded by a polynucleotide comprising a nucleic acid sequence encoding, for example, the protein of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, or a protein having 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% amino acid identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, whose amino acid sequence substantially retains the native function of the protein represented by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13.

[0138] ATRs of the present invention contain, for example, an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13, which amino acid sequence substantially retains the native function of the protein represented by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13.

[0139] The ATR of the present invention can be encoded by a polynucleotide comprising a nucleic acid sequence encoding, for example, the protein of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, or a protein having at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% amino acid identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, whose amino acid sequence substantially retains the native function of the protein represented by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13.

[0140] DNA-binding domain The ATRs of the present invention contain a DNA-binding domain that binds to a specific nucleic acid sequence, allowing the ATR to target a specific site within a polynucleotide, such as the genome of a cell. The DNA-binding domain can be, for example, protein-, DNA-, RNA-based, or chemical.

[0141] Several suitable DNA-binding domains are known in the art, such as transcription activator-like effector (TALE) domains and zinc finger proteins (ZEPs) (Gaj, T. et al. (2013) Trends Biotechnol. 31: 397-405).

[0142] The tetracycline-regulated repressor (tetR) DNA-binding domain, such as the E. coli tetR DNA-binding domain (Gossen, M. et al. (1992) Proc. Natl. Acad. Sci. USA 89: 5547-51), may be used as a suitable DNA-binding domain in the ATR of the present invention. The tetR system is particularly advantageous for use as a model system because it allows for temporal control of tetR binding to its target nucleotide sequence, the tetracycline operon (TetO), by administration of doxycycline (doxy). This allows for the investigation of whether the ATR-induced chromatin state can be maintained even after ATR release from the target site.

[0143] Furthermore, methods are known in the art for engineering DNA binding domains to bind to desired nucleic acid sequences.

[0144] Examples of suitable TALE domain sequences include: MGKPIPNPLLGLDSTGGMAPKKKRKVDGGVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVA (SEQ ID NO: 14), its target binding site: 5'-TACCCAGATTGGCCCCACT-3' (SEQ ID NO: 34) and: (SEQ ID NO: 15), its target binding site: 5′-TACCTAGAGGAGAAAGGTT-3′ (SEQ ID NO: 35).

[0145] An example sequence of a TALE domain designed to target the promoter region of the β2-microglobulin gene is: (SEQ ID NO: 16), its target binding site: 5'-TCTCTCCTACCCTCCCGCT-3' (SEQ ID NO: 17) MGKPIPNPLLGLDSTGGMAPKKKRKVDGGVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSGGG (SEQ ID NO: 18), its target binding site: 5'-TGGTCCTTCCTCTCCCGCT-3' (SEQ ID NO: 19) MGKPIPNPLLGLLDSTGGMAPKKKRKVDGGVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHERAIV GVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVA IASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIA SHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGKQALETVQRLLPVLCQDHGLTPDQVVAIASN GGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDG GKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGK QALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSGGG (SEQ ID NO: 20), its target binding site: 5′-TCGCTCCGTGACTTCCCTT-3′ (SEQ ID NO: 21).

[0146] Example sequences of TALE domains designed to target the BCL11A gene include: TALE BCL11A #1 (SEQ ID NO: 37), its target binding site: 5′- TCCAAAAGCCAGTCTCACC -3′ (SEQ ID NO: 38) TALE BCL11A #2 MGKPIPNPLLGLLDSTGGMAPKKKRKVDGGVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHERAIV GVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVA IASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIA SNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASH DGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDG GKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGK QALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSGGG (SEQ ID NO: 39), its target binding site: 5′-TCTCCCCGGGAATCGTTTT-3′ (SEQ ID NO: 40) TALE BCL11A #3 (SEQ ID NO: 41), its target binding site: 5'-TCCTCCCGCTGCACACTTG-3' (SEQ ID NO: 42) TALE BCL11A #4 (SEQ ID NO: 43), its target binding site: 5′- TAGTCATCCCCACAATAGT -3′ (SEQ ID NO: 44) TALE BCL11A #5 (SEQ ID NO: 45), its target binding site: 5'-TCCCCGCTGCCTTTTGTGCC -3' (SEQ ID NO: 46) TALE BCL11A #6 (SEQ ID NO: 47), its target binding site: 5'-TCCTCGCGCTTGCCCTCCC -3' (SEQ ID NO: 48) TALE BCL11A #7 (SEQ ID NO: 49), its target binding site: 5′-TCCCCCCGGCCCTAGCTCCT-3′ (SEQ ID NO: 50) TALE BCL11A #8 MGKPIPNPLLGLLDSTGGMAPKKKRKVDGGVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHERAIV GVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVA IASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIA SHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASN GGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDG GKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNHGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGK QALETVQRLLPVLCQDHGLTPDQVVAIASNHGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSGGG (SEQ ID NO: 51), its target binding site: 5′-TCCTGGTCCGCCCCCAGCA -3′ (SEQ ID NO: 52) TALE BCL11A #9 MGKPIPNPLLGLLDSTGGMAPKKKRKVDGGVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHERAIV GVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVA IASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIA SNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASN GGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDG GKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGK QALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSGGG (SEQ ID NO: 53), its target binding site: 5′- TGCCGAGACCTCTTCTCGA -3′ (SEQ ID NO: 54) TALE BCL11A #10 MGKPIPNPLLGLLDSTGGMAPKKKRKVDGGVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHERAIV GVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNNGKQALETVKRLLPVLCQDHGLTPDQVVA IASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIA SHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASH DGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNG GKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGK QALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSGGG (SEQ ID NO: 55), its target binding site: 5′-TCGGCTTTGCAAAGCATTT-3′ (SEQ ID NO: 56) TALE BCL11A #11 MGKPIPNPLLGLLDSTGGMAPKKKRKVDGGVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHERAIV GVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVA IASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIA SHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGKQALETVQRLLPVLCQDHGLTPDQVVAIASN NGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIG GKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGK QALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSGGG (SEQ ID NO: 57), its target binding site: 5′- TGCAAAGCCGAGTTTCACC -3′ (SEQ ID NO: 58) TALE BCL11A #12 MGKPIPNPLLGLLDSTGGMAPKKKRKVDGGVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHERAIV GVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVA IASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIA SNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNHGGKQALETVQRLLPVLCQDHGLTPDQVVAIASH DGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNG GKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGK QALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSGGG (SEQ ID NO: 59), its target binding site: 5′- TACAGTTGCCCTGCAAAAT -3′ (SEQ ID NO: 60) TALE BCL11A #13 MGKPIPNPLLGLLDSTGGMAPKKKRKVDGGVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHERAIV GVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVA IASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIA SHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGKQALETVQRLLPVLCQDHGLTPDQVVAIASH DGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDG GKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGK QALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSGGG (SEQ ID NO: 61), its target binding site: 5'-TCCGCCCTGGGTACTTTCT -3' (SEQ ID NO: 62) TALE BCL11A #14 MGKPIPNPLLGLLDSTGGMAPKKKRKVDGGVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHERAIV GVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVA IASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIA SNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASH DGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIG GKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGK QALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSGGG (SEQ ID NO: 63), its target binding site: 5′-TCTCTTGTCCACAGCTCGG-3′ (SEQ ID NO: 64) TALE BCL11A #15 MGKPIPNPLLGLLDSTGGMAPKKKRKVDGGVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHERAIV GVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVA IASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIA SHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGKQALETVQRLLPVLCQDHGLTPDQVVAIASH DGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGG GKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGK QALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSGGG (SEQ ID NO: 65), its target binding site: 5'- TCTCCCGCTGACTGCGCCT -3' (SEQ ID NO: 66) TALE BCL11A #16 MGKPIPNPLLGLLDSTGGMAPKKKRKVDGGVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHERAIV GVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVA IASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIA SNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGKQALETVQRLLPVLCQDHGLTPDQVVAIASH DGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNG GKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGK QALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSGGG (SEQ ID NO: 67), its target binding site: 5′- TCCCTTGCTGCCAAACTTT -3′ (SEQ ID NO: 68) TALE BCL11A #17 MGKPIPNPLLGLDSTGGMAPKKKRKVDGGVDLRTLGYSQQQQEKIKKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVA IASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASH DGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHD

[0147] Meganucleases (Silve, G. et al. (2011) Cur. Gene Ther. 11: 11-27) and CRISPR / Cas systems (Sander, JD et al. (2014) Nat. Biotechnol. 32: 347-55) can also be used as suitable DNA-binding domains of the ATR of the present invention.

[0148] The CRISPR / Cas system is a guide RNA-mediated DNA binding system (van der Oost et al. (2014) Nat. Rev. Microbiol. 12: 479-92), and the guide RNA (gRNA) can be selected to enable the ATR containing the Cas9 domain to target a specific sequence. Therefore, to use the CRISPR / Cas system as the DNA binding domain of the present invention, it is understood that an ATR effector domain can be operably linked to a Cas9 endonuclease. Preferably, the ATR effector domain is operably linked to an inactivated Cas9 endonuclease that has substantially no nuclease activity. The ATR containing the Cas9 endonuclease can be introduced into a target cell in combination with one or more guide RNAs (gRNAs). The guide RNA is designed to direct the ATR to a desired target gene or a regulatory element of the target gene (e.g., a promoter, enhancer, or splice site). Methods for designing gRNAs are known in the art. Moreover, completely heterologous Cas9 proteins, as well as Cas9 / gRNA ribonucleoprotein complexes and modified gRNA structures / compositions that bind to various proteins, have been developed in recent years to simultaneously and directly target various effector domains to target genomic sites in cells (Esvelt et al. (2013) Nat. Methods 10: 1116-21; Zetsche, B. et al. (2015) Cell pii: S0092-8674(15)01200-3; Dahlman, JE et al. (2015) Nat. Biotechnol. 2015 Oct 5. doi: 10.1038 / nbt.3390. [Epub ahead of print]; Zalatan, JG et al. (2015) Cell 160: 339-50; Paix, A. et al. (2015) Genetics 201: 47-54), which are suitable for use in the present invention.

[0149] For example, an ATR of the present invention can contain the following sequence: (Cas9 without catalytic activity: SEQ ID NO: 22)

[0150] The ATR of the present invention can contain, for example, an amino acid sequence having 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% identity to SEQ ID NO:22, while the amino acid sequence substantially retains the native function of the protein represented by SEQ ID NO:22.

[0151] The ATR of the present invention may be encoded, for example, by a polynucleotide containing a nucleic acid sequence that encodes the protein of SEQ ID NO:22 or that encodes a protein having 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% amino acid identity to SEQ ID NO:22, and whose amino acid sequence substantially retains the native function of the protein represented by SEQ ID NO:22.

[0152] The ATR of the present invention can, for example, contain an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% identity to SEQ ID NO:22, while the amino acid sequence substantially retains the native function of the protein represented by SEQ ID NO:22.

[0153] The ATR of the present invention may be encoded, for example, by a polynucleotide containing a nucleic acid sequence that encodes the protein of SEQ ID NO:22 or that encodes a protein having at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% amino acid identity to SEQ ID NO:22, and whose amino acid sequence substantially retains the native function of the protein represented by SEQ ID NO:22.

[0154] An example of a genomic target site sequence recognized by a guide RNA (gRNA) used to target the β2-microglobulin gene is: gRNA #1: TATAAGTGGAGGCGTCGCGC (SEQ ID NO: 23) gRNA #2: GCCCGAATGCTGTCAGCTTC (SEQ ID NO: 24) gRNA #3: TGCGTCGCTGGCTTGGAGAC (SEQ ID NO: 25) gRNA #4: CCAATCAGGACAAGGCCCGC (SEQ ID NO: 26) gRNA #5: AGGGTAGGAGAGACTCACGC (SEQ ID NO: 27) gRNA #6: GCGGGCCACCAAGGAGAACT (SEQ ID NO: 28) gRNA #7: GCTACTCTCTCTTTCTGGCC (SEQ ID NO: 29) gRNA #8: CTCCCGCTCTGCACCCTCTG (SEQ ID NO: 30) gRNA #9: TTTGGCCTACGGCGACGGGA (SEQ ID NO: 31) gRNA #10: GGGGCAAGTAGCGCGCGTCC (SEQ ID NO: 32) gRNA #11: TAGTCCAGGGCTGGATCTCG (SEQ ID NO: 33)

[0155] Examples of guide RNAs (gRNAs) used to target the β2-microglobulin gene include: gRNA #1: UAUAAGUGGAGGCGUCGCGC gRNA #2: GCCCGAAUGCUGUCAGCUUC gRNA #3: UGCGUCGCUGGCUUGGAGAC gRNA #4: CCAAUCAGGACAAGGCCCGC gRNA #5: AGGGUAGGAGAGACUCACGC gRNA #6: GCGGGCCACCAAGGAGAACU gRNA #7: GCUACUCUCUCUUUCUGGCC gRNA #8: CUCCCGCUCUGCACCCUCUG gRNA #9: UUUGGCCUACGGCGACGGGA gRNA #10: GGGGCAAGUAGGCGCGCGUCC gRNA #11: UAGUCCAGGGCUGGAUCUCG

[0156] All of the above gRNAs can be fused to a gRNA scaffold with the following sequence: GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUUUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU.

[0157] An example of a gRNA sequence targeting the BCL11A gene is: gRNA #1 against CpG 105: GCCUUUCUGCAGACGUUCCC (SEQ ID NO: 71) gRNA #2 against CpG 105: UGGGUGUGCGCCUUGGCCGG (SEQ ID NO: 72) gRNA #3 against CpG 105: CGGUGGUGAGAUGACCGCCU (SEQ ID NO: 73) gRNA #4 against CpG 105: GGAAUGUGCUCACGGCGCCG (SEQ ID NO: 74) gRNA #5 against CpG 105: GACUGCCCGCGCUUUGUCCU (SEQ ID NO: 75) gRNA #6 against CpG 105: CCAGAGUCUGGCCCCCGGAG (SEQ ID NO: 76) gRNA #7 against CpG 105: UCUGCGACCCUUAGGAGCCG (SEQ ID NO: 77) gRNA #8 against CpG 105: GAGCGCCCCGCCAAGCGACU (SEQ ID NO: 78) gRNA #9 against CpG 105: CAAGUCUCCAGGAGCCCGCG (SEQ ID NO: 79) gRNA #10 against CpG 105: CGCGGAAUCCAGCCUAAGUU (SEQ ID NO: 80) gRNA #11 against CpG 105: CCCGCUGCGGAGCUGUAACU (SEQ ID NO: 81) gRNA #1 against CpG 31: CGCUCCUGAGUCCGCGGAGU (SEQ ID NO: 82) gRNA #2 against CpG 31: CACGGCUCUCCCCGUCGCCG (SEQ ID NO: 83) gRNA #3 against CpG 31: CCGCCUUUUGUUCCGGCCAG (SEQ ID NO: 84) gRNA #4 against CpG 31: GCGCGAGGAGCCGGCACAAA (SEQ ID NO: 85) gRNA #5 against CpG 31: GCCACUUUCUCACUAUUGUG (SEQ ID NO: 86) gRNA #6 against CpG 31: GCUGCCUCUGAGGUUCGGUC (SEQ ID NO: 87) gRNA #7 against CpG 31: AAGGGCAGGAGCUAGGGCCG (SEQ ID NO: 88) gRNA #8 against CpG 31: GAGCCCGGACUGCUGCCUCC (SEQ ID NO: 89) gRNA #1 against CpG 38: GUUUACAAGCACCGCGUGUG (SEQ ID NO: 90) gRNA #2 against CpG 38: AACAGACAGAGGACCGAGCG (SEQ ID NO: 91) gRNA #3 against CpG 38: GGCGCCGGGUGGGCGAUCCG (SEQ ID NO: 92) gRNA #4 against CpG 38: GGUCGGGCAAGGCCCGGGCG (SEQ ID NO: 93) gRNA #5 against CpG 38: AAGAGGUCUCGGCAUUGUGC (SEQ ID NO: 94) gRNA #6 against CpG 38: GUUCCACAGCUUCGGGACCGCG (SEQ ID NO: 95) gRNA #7 against CpG 38: GAAAUCGGCUGGGUGAAACU (SEQ ID NO: 96) gRNA #8 against CpG 38: GCAGUGUCUCCGCGCCAGCC (SEQ ID NO: 97) gRNA #9 against CpG 38: CCUCCCCUCCCCUCCGCCCUGGG (SEQ ID NO: 98) gRNA #1 against CpG 115: UCCUCCUGUCCCGGGGUUAAAGG (SEQ ID NO: 99) gRNA #2 against CpG 115: CAUCUUUUGGGACACUCUAGGCUGG (SEQ ID NO: 100) gRNA #3 against CpG 115: AAGUCAGGCCCUUCUUCGGAAGG (SEQ ID NO: 101) gRNA #4 against CpG 115: GCAGCCUGGACUGCGCGCCCCGG (SEQ ID NO: 102) gRNA #5 against CpG 115: UGCCCGGCGAUUCUCGUCCG (SEQ ID NO: 103) gRNA #6 against CpG 115: UGAGCCAUUCGGUCGCUAGG (SEQ ID NO: 104) gRNA #7 against CpG 115: GGUGGUACUGAGGACCGGGA (SEQ ID NO: 105) gRNA #8 against CpG 115: AUUUUCUGGGUGCUCAGAGG (SEQ ID NO: 107) gRNA #9 against CpG 115: UGGUCUCAGCUCGCGCACGG (SEQ ID NO: 108) gRNA #10 against CpG 115: ACAAAGACAUACGGGGUGAU (SEQ ID NO: 109)

[0158] An example of a gRNA sequence targeting the IFNAR1 gene is: gRNA #1:AGGAACGGCGCGUGCGCGGA gRNA #2:AAGAGGCGGCGCGUGCGTAG gRNA #3:GGGCGGUGUGACUUAGGACG gRNA #4:CCAGAUGAUGGUCGUCCUCC gRNA #5: GACCCUAGUGCUCGUCGCCG gRNA #6:UGGGUGUUGUCCGCAGCCGC gRNA #7:ACGGGGGCGGCGAUGCUGUU gRNA #8: GACCGAAGGUUUCCCAGACU gRNA #9: GUCGGGUUUAAUCUUUGGCG gRNA #10:CGCUCCCGAGGACCCGUACA gRNA #11:CGGGUCCCACCCCCGUGAAA gRNA #12:UCAAACUCGACACAAAGCUC gRNA #13:GCGGAGCCGCGGUACUUUCC

[0159] An example of a gRNA sequence targeting the VEGFA gene is: gRNA #1:GGCGCGCGCGCUAGGUGGGA gRNA #2:AGAGAGGCUCACCGCCCACG gRNA #3:GUACGUGCGGUGACUCCGGU

[0160] All of the above gRNAs can be fused to a gRNA scaffold with the following sequence: GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUUUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU.

[0161] Target gene suppression By "silencing a target gene," it is understood that expression of the target gene is reduced to an extent sufficient to achieve a desired effect. The reduced expression may be sufficient to achieve a therapeutically meaningful effect, such as prevention or treatment of a disease. For example, a dysfunctional target gene that causes a disease is preferably suppressed to the extent that there is no target gene expression, or to the extent that the remaining target gene expression level is low enough to ameliorate or prevent the disease state.

[0162] The reduced expression may be sufficient to allow studies of gene function by examining cells with reduced or absent gene function.

[0163] After administration of two or more ATRs of the present invention, the level of transcription or expression of the target gene may be reduced, for example, by at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% below the transcription or expression level in the absence of the two or more ATRs.

[0164] Preferably, two or more ATRs of the present invention have a synergistic effect on target gene silencing. Thus, two or more ATRs of the present invention may exhibit synergistic effects, e.g., therapeutic synergism, when used as described herein.

[0165] For example, two or more ATRs of the present invention may result in a synergistic increase in the proportion of a cell population containing two or more ATRs that exhibits target gene silencing, compared to a cell population lacking the two or more ATRs (e.g., containing only one ATR or a different combination of ATRs). Alternatively, or in addition, two or more ATRs of the present invention may result in a synergistic increase in the duration that a target gene is silenced in a cell population containing two or more ATRs, compared to a cell population lacking the two or more ATRs.

[0166] Preferably, silencing of the target gene occurs after transient introduction or transient expression of the ATR of the present invention into a cell.

[0167] By "transient expression," it is understood that expression of ATR is not stable over an extended period of time. Preferably, the polynucleotide encoding ATR is not integrated into the host genome. More specifically, transient expression can be expression that is substantially lost within 20 weeks after introduction of the nucleotide sequence encoding ATR into a cell. Preferably, expression is substantially lost within 12, 6, 4, or 2 weeks after introduction of the nucleotide sequence encoding ATR into a cell.

[0168] Similarly, by "transient introduction," it is understood that the ATR does not remain in the cell for a substantially extended period of time (i.e., the cell substantially loses it). More specifically, transient introduction can be said to result in a state in which the ATR is lost from the cell substantially within 20 weeks after introduction of the ATR into the cell. Preferably, the ATR is lost substantially within 12, 6, 4, or 2 weeks after introduction of the ATR into the cell.

[0169] For example, the method for measuring the transcription of gene, which is the target of ATR, is known in the art.Suitable methods include reverse transcription PCR and Northern blot-based methods.In addition to the method for measuring the transcription of gene, the method for measuring gene expression is known in the art.Other suitable methods include Western blot-based methods or flow cytometry.

[0170] The effect of one ATR, or a combination of multiple ATRs, can be examined by comparing the transcription or expression of a target gene, e.g., a gene endogenous to the cell, in the presence and absence of the ATR or combination of ATRs.

[0171] The effect of one ATR, or a combination of ATRs, can also be studied using model systems that monitor the expression of a reporter gene, e.g., a gene encoding a fluorescent protein. Suitable methods for monitoring the expression of such reporter genes include flow cytometry, fluorescence-activated cell sorting (FACS), and fluorescence microscopy.

[0172] For example, a cell population can be transfected with a vector carrying a reporter gene. The vector can be constructed so that the reporter gene is expressed when the vector transfects cells. Suitable reporter genes include, for example, genes encoding fluorescent proteins, such as green, yellow, cherry, cyan, or orange fluorescent proteins. Furthermore, the cell population can be transfected with a vector encoding the ATR of interest. The number of cells expressing and not expressing the reporter gene, as well as the expression level of the reporter gene, can then be quantified using an appropriate method, such as FACS. The reporter gene expression level can then be compared in the presence and absence of ATR.

[0173] Preferably, the target gene is permanently silenced. By "permanent silencing", it is understood that the transcription or expression of the target gene is reduced (for example, reduced by 100%) for at least 2 months, 6 months, 1 year, 2 years, or the lifetime of the cell / organism, compared to the transcription or expression level in the absence of two or more ATRs.Preferably, the permanently silenced target gene remains silenced for the rest of the cell's life.

[0174] Preferably, the target gene remains silenced in the progeny of cells to which two or more ATRs of the present invention have been administered (i.e., the silencing of the target gene is inherited by subsequent cell generations). For example, two or more ATRs of the present invention can be administered to stem cells (e.g., hematopoietic stem cells) to silence the target gene in the stem cell and also in the progeny of the stem cell, which may include cells differentiated from the stem cell.

[0175] A target gene can be silenced by using ATR to bind to the target gene itself or to the regulatory sequence (for example, promoter or enhancer sequence) of the target gene. Furthermore, by using ATR to bind to the splice site of the target gene itself, the alternative splicing of the target gene can be changed. The ability to silence a target gene or change its splicing variant by using ATR to bind to a regulatory sequence is something that cannot be achieved by other gene silencing techniques, and is a significant advantage of the present invention.

[0176] Therapeutic Use In another aspect, the present invention provides the products, artificial transcriptional repressors (ATRs), polynucleotides, and cells of the invention for use in therapy.

[0177] Therapeutic use may be, for example, use for the treatment of beta thalassemia or sickle cell anemia.

[0178] Therapeutic uses can be, for example, for preparing "universally" allogeneic transplantable cells (e.g., by silencing β2-microglobulin, B2M), which can be applied, for example, to the preparation of hematopoietic stem and / or progenitor cells (HSPCs), whole organ transplantation, and cancer immunotherapy.

[0179] Two or more ATRs or polynucleotides encoding them can be administered simultaneously (as part of a dosing regimen), in combination, sequentially, or separately.

[0180] By "concurrently," it is understood that two agents are administered concurrently, whereas the term "in combination" is used to mean that they are administered "sequentially" within a time frame in which they are both effective, so that they act therapeutically, even if not simultaneously, within the same time frame. Thus, "sequential" administration may allow for the administration of one agent after the other within 5 minutes, 10 minutes, or even a few hours, provided that the serum half-life of the agent administered first is such that both agents are simultaneously present in therapeutically effective amounts. The time delay between administration of the components can vary depending on the exact nature of the components, the interaction between the components, and their respective half-lives.

[0181] "Separately," as opposed to "in combination" or "sequentially," is understood to mean that the time interval between the administration of one agent and the administration of the other is significant, i.e., the agent administered first may no longer be present in the bloodstream in therapeutically effective amounts when the second agent is administered.

[0182] Target gene Preferably, the target gene produces a therapeutic effect when silenced.

[0183] By way of example, the products, artificial transcriptional repressors (ATRs) and polynucleotides of the present invention can be used to silence beta2-microglobulin (B2M), BCL11A, KLF1, globin genes, CCR5, CXCR4, TCR genes, miR126, PDL1, CTLA4, COL1A1, viral sequences, and oncogenes.

[0184] Silencing the TCR genes, PDL1 and CTLA4, can increase the efficacy of cancer immunotherapy. Silencing of B2M can generate allogeneic HSPCs, T cells, or mesenchymal cells for transplantation.

[0185] Silencing miR126 can increase the more primitive hematopoietic stem cell pool before or after hematopoietic stem cell infusion.

[0186] For example, the products, artificial transcriptional repressors (ATRs), polynucleotides, and cells of the present invention can be used to treat, for example, Huntington's disease, spinocerebellar ataxias, collagenopathy-related diseases, hemoglobinopathies, and diseases caused by trinucleotide repeat expansions. Furthermore, the products of the present invention can be used to treat or prevent certain infectious diseases (e.g., CCR5-tropic HIV infections) by inactivating pathogen-associated gene products or host genes required for the pathogen life cycle.

[0187] Additionally or alternatively, the products, artificial transcriptional repressors (ATRs), polynucleotides and cells of the invention may be useful in the treatment of the diseases described in WO 1998 / 005635, a partial list of which is provided here for ease of reference: cancer, inflammation or inflammatory diseases, skin diseases, fever, cardiovascular events, bleeding, coagulation and acute phase response, cachexia, anorexia, acute infections, HIV infection, shock states, graft-versus-host reaction, autoimmune diseases, reperfusion injury, meningitis, migraine and aspirin-dependent antithrombosis; tumor growth, invasion and spread, angiogenesis, metastasis, malignant ascites and malignant pleural effusion; cerebral ischemia, ischemic heart disease. Diseases, osteoarthritis, rheumatoid arthritis, osteoporosis, asthma, multiple sclerosis, neurodegeneration, Alzheimer's disease, atherosclerosis, stroke, vasculitis, Crohn's disease and ulcerative colitis; periodontitis, gingivitis; psoriasis, atopic dermatitis, chronic ulcers, epidermolysis bullosa; corneal ulcers, retinopathy and surgical wound healing; rhinitis, allergic conjunctivitis, eczema, anaphylaxis; restenosis, congestive heart failure, endometriosis, atherosclerosis or internal sclerosis.

[0188] Additionally or alternatively, the products, artificial transcriptional repressors (ATRs), polynucleotides and cells of the invention may be useful in the treatment of diseases described in WO 1998 / 007859. For ease of reference, a partial list is provided here: cytokine and cell proliferation / differentiation activity; immunosuppressive or immunostimulatory activity (e.g., for the treatment of immune deficiencies such as human immunodeficiency virus infection; control of lymphocyte proliferation; for the treatment of cancer and many autoimmune diseases, and for preventing transplant rejection or inducing tumor immunity); control of hematopoiesis, e.g., in the treatment of bone marrow or lymphatic system diseases; promotion of growth of bone, cartilage, tendon, ligament and nerve tissue, e.g., for wound healing, treatment of burns, ulcers and periodontal disease and neurodegeneration; inhibition or activation of follicle-stimulating hormone (regulation of fertility); migration / chemokinesis (e.g., to recruit specific cell types to sites of injury or infection); hemostatic and thrombolytic activity (e.g., to treat hemophilia and stroke); anti-inflammatory activity (e.g., to treat septic shock or Crohn's disease); as antibacterial agents; e.g., metabolism or behavioral regulators; as analgesics; treatment of certain deficiencies; in human or veterinary medicine, e.g., in the treatment of psoriasis.

[0189] Additionally or alternatively, the products, artificial transcriptional repressors (ATRs), polynucleotides and cells of the present invention may be useful in the treatment of diseases described in WO 1998 / 009985, a partial list of which is provided here for ease of reference: macrophage inhibitory activity and / or T cell inhibitory activity, and thereby anti-inflammatory activity; anti-immune activity, i.e., inhibitory effects on cellular and / or humoral immune responses, including responses unrelated to inflammation; inhibition of the ability of macrophages and T cells to adhere to extracellular matrix components and fibronectin, and upregulated Fas receptor expression in T cells; arthritis, including rheumatoid arthritis, hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other autoimmune diseases. inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, inflammatory vascular disease, respiratory distress syndrome or other cardiopulmonary disease; inflammation associated with peptic ulcer, ulcerative colitis and other gastrointestinal diseases, liver fibrosis, cirrhosis or other liver diseases, thyroiditis or other glandular diseases, glomerulonephritis or other renal and urinary diseases, otitis or other ear, nose and throat diseases, dermatitis or other skin diseases, periodontal disease or other dental diseases, orchitis or epididymitis, infertility, testicular trauma or other immune-related testicular diseases, placental dysfunction,insufficiency), recurrent miscarriage, eclampsia, preeclampsia and other immune and / or inflammatory related gynecological diseases, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, retino-uveitis, optic neuritis, endophthalmitis, e.g., retinitis or cystoid macular edema, sympathetic ophthalmia, scleritis, retinitis pigmentosa, immune and inflammatory components of degenerative fundus diseases, inflammatory components of ocular trauma, ocular inflammation caused by infection, proliferative vitreoretinopathy, acute ischemic optic neuropathy, excessive scarring after e.g., glaucoma filtration surgery, immune and / or inflammatory responses to intraocular implants, and and other immune and inflammation-related eye diseases, inflammation associated with autoimmune diseases or conditions or disorders in the central nervous system (CNS) or any other organ where suppression of immunity and / or inflammation may be beneficial, Parkinson's disease, complications and / or side effects from treatment of Parkinson's disease, AIDS-related dementia complex, HIV-associated encephalopathy, Devic's disease, Sydenham's chorea, Alzheimer's disease and other degenerative diseases, conditions or disorders of the CNS, the inflammatory component of Stokes' syndrome, post-polio syndrome, immune and inflammatory components of psychiatric disorders, myelitis, encephalitis, subacute sclerosing panencephalitis, cerebrospinal fluid dysplasia ... inflammation, acute neuropathy, subacute neuropathy, chronic neuropathy, Guillain-Barré syndrome, Sydenham chorea, myasthenia gravis, pseudotumor cerebri, Down's syndrome, Huntington's disease, amyotrophic lateral sclerosis, the inflammatory component of CNS compression or CNS trauma or CNS infection, muscle atrophy and the inflammatory component of muscular dystrophies, and immune- and inflammatory-related diseases, conditions, or disorders of the central and peripheral nervous system, post-traumatic inflammation, septic shock, infectious diseases, inflammatory complications or side effects of surgery, complications and / or side effects of bone marrow transplantation or other transplants, for example due to infection with a viral carrier inhibiting undesirable immune responses and inflammation, including inflammatory and / or immune complications and side effects of gene therapy or inflammation associated with AIDS; suppressing or inhibiting humoral and / or cellular immune responses by reducing the amount of monocytes or lymphocytes, for the purpose of preventing and / or treating graft rejection in the case of transplantation of natural or artificial cells, tissues and organs, such as corneas, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue, and treating or ameliorating monocyte or leukocyte proliferative disorders, such as leukemia.

[0190] Polynucleotides The polynucleotides of the present invention can be DNA or RNA. They can be single-stranded or double-stranded. Those skilled in the art will understand that many different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, those skilled in the art will understand that nucleotide substitutions that do not affect the polypeptide sequence encoded by the nucleotides of the present invention can be made by conventional methods to reflect the codon usage frequency of any specific host organism in which the polypeptide of the present invention is to be expressed.

[0191] The polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or duration of polynucleotides of the invention.

[0192] Polynucleotides, such as DNA polynucleotides, can be produced recombinantly, synthetically, or by any method available to those of skill in the art.

[0193] Relatively long polynucleotides are typically produced recombinantly, for example, using polymerase chain reaction (PCR) cloning techniques. This involves creating a pair of primers (e.g., about 15-30 nucleotides) that flank the target sequence to be cloned, contacting the primers with mRNA or cDNA obtained from animal or human cells, performing the polymerase chain reaction under conditions that result in amplification of the desired region, isolating the amplified fragment (e.g., by purifying the reaction mixture on an agarose gel), and recovering the amplified DNA. Primers can be designed to contain appropriate restriction enzyme recognition sites so that the amplified DNA can be cloned into an appropriate vector.

[0194] protein As used herein, the term "protein" includes single polypeptide chain molecules as well as multi-polypeptide complexes in which the individual constituent polypeptides are linked by covalent or non-covalent bonds. As used herein, the terms "polypeptide" and "peptide" refer to polymers in which the monomers are amino acids and are linked to each other by peptide or disulfide bonds.

[0195] Variants, derivatives, analogs, homologs and fragments In addition to the specific proteins and nucleotides mentioned herein, the present invention also encompasses the use of variants, derivatives, analogs, homologs, and fragments thereof.

[0196] In the context of the present invention, a variant of any given sequence is one in which a particular sequence of residues (whether amino acid or nucleic acid residues) has been altered such that the polypeptide or polynucleotide substantially retains at least one of its inherent functions. Variant sequences can be obtained by addition, deletion, substitution, modification, replacement and / or mutation of at least one residue present in the naturally occurring protein.

[0197] The term "derivative" as used herein in reference to a protein or polypeptide of the invention includes any substitution, mutation, modification, replacement, deletion, and / or addition of one or more amino acid residues to or from the sequence, provided that the resulting protein or polypeptide retains at least one inherent function thereof.

[0198] The term "analog" as used herein with respect to a polypeptide or polynucleotide includes any mimetic, i.e., a compound that retains at least one of the inherent functions of the polypeptide or polynucleotide that it mimics.

[0199] Typically, from one, two, or three to ten or twenty amino acid substitutions can be made, provided that the altered sequence substantially retains the required activity or ability. Amino acid substitutions can include the use of non-naturally occurring analogues.

[0200] The proteins used in the present invention may have deletions, insertions, or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins. Deliberate amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues, so long as the underlying function is preserved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0201] For example, conservative substitutions can be made according to the following table: Amino acids in the same block in the second column and preferably in the same line in the third column can be substituted: JPEG2025063201000002.jpg47120

[0202] As used herein, the term "homolog" means something that has a certain degree of homology with the wild-type amino acid sequence and the wild-type nucleotide sequence. The term "homology" can be considered equivalent to "identity."

[0203] A homologous sequence can include an amino acid sequence that is at least 50%, 55%, 65%, 75%, 85%, or 90% identical to the reference sequence, but is preferably at least 95%, 97%, or 99% identical. Typically, a homolog will have the same active site, etc., as the reference amino acid sequence. Although homology can be considered in terms of similarity (i.e., amino acid residues with similar chemical properties / functions), for the purposes of the present invention, it is preferred to express homology in terms of sequence identity.

[0204] Homologous sequences can include nucleic acid sequences that are at least 50%, 55%, 65%, 75%, 85%, or 90% identical to the subject sequence, but are preferably at least 95%, 97%, or 99% identical. Although homology can also be considered in terms of similarity (i.e., amino acid residues having similar chemical properties / functions), for the present invention, it is preferred to express homology in terms of sequence identity.

[0205] Reference to a sequence having a percent identity to any one of the SEQ ID NOs detailed herein refers to a sequence having the stated percent identity over the full length of the sequence of the referenced SEQ ID NO.

[0206] Homology comparisons can be performed visually, or more usually, with the aid of readily available sequence comparison programs. These commercial computer programs can calculate the percent homology or identity between two or more sequences.

[0207] Percent homology can be calculated over contiguous sequences, i.e., by aligning one sequence with the other and directly comparing each amino acid in one sequence with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed over only a relatively small number of residues.

[0208] While this is a very simple and consistent method, it fails to take into account that, for example, in a pair of otherwise identical sequences, a single insertion or deletion in a nucleotide sequence can cause subsequent codons to fall out of alignment, resulting in a significant decrease in percent homology when a global alignment is performed. For this reason, most sequence comparison methods are designed to produce optimal alignments that take into account possible insertions and deletions without unduly penalizing the overall homology score. This is done by inserting "gaps" in the sequence alignment to maximize local homology.

[0209] However, these more complex methods assign a "gap penalty" to each gap that occurs in the alignment, so that a sequence alignment with as few gaps as possible for the same number of identical amino acids will score higher than one with many gaps, reflecting a higher relatedness between the two compared sequences. "Affine gap costs" are typically used, which impose a relatively high cost for the existence of a gap and a relatively small penalty for each subsequent residue in the gap. This is the most widely used gap scoring system. High gap penalties naturally result in optimized alignments with fewer gaps. Most alignment programs allow the gap penalty to be modified. However, when using such software for sequence comparisons, it is preferred to use the default values. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0210] Therefore, calculation of maximum percent homology first requires the creation of an optimal alignment, taking into account gap penalties. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res. 12: 387). Examples of other software capable of performing sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid - Ch. 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410), and the GENEWORKS comparison tool suite. Both BLAST and FASTA are available for offline and online searches (see Ausubel et al. (1999) ibid, pp. 7-58 to 7-60). However, depending on the application, it may be preferable to use the GCG Bestfit program. Another tool called BLAST 2 Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174: 247-50; FEMS Microbiol. Lett. (1999) 177: 187-8).

[0211] Although the final percent homology can be measured in terms of identity, the alignment process itself is not typically based on an all-or-nothing pairwise comparison. Instead, a scaled similarity score matrix is ​​used, which assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is BLOSUM62, the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values ​​or a custom symbol comparison table if specified (see user manual for further details). For some applications, it is preferable to use the public default values ​​for the GCG package, while for other software it is preferable to use a default matrix such as BLOSUM62.

[0212] Once the software has produced an optimal alignment, it is possible to calculate percent homology, preferably percent sequence identity, which the software will typically do as part of the sequence comparison and generate a result.

[0213] A "fragment" is also a variant, and the term typically refers to a selected region of a polypeptide or polynucleotide that is of functional or, for example, assay interest. Thus, a "fragment" refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

[0214] Such variants can be prepared using standard recombinant DNA techniques, such as site-directed mutagenesis. When inserting, synthetic DNA can be generated that encodes the insert along with 5' and 3' flanking sequences corresponding to the naturally occurring sequence on either side of the insertion site. The flanking regions contain convenient restriction enzyme sites corresponding to sites within the naturally occurring sequence, such that the naturally occurring sequence can be cleaved with the appropriate restriction enzyme(s) and the synthetic DNA ligated to the cleavage sites. The DNA can then be expressed in accordance with the invention to produce the encoded protein. This method is but one example of many standard techniques known in the art for manipulating DNA sequences; other known techniques can also be used.

[0215] Codon optimization The polynucleotides used in the present invention can be codon-optimized. Codon optimization has already been described in WO 1999 / 41397 and WO 2001 / 79518. Different cells have different frequencies of use of certain codons. This codon bias corresponds to the bias in the relative abundance of certain tRNAs in that cell type. Expression can be increased by changing the codons in the sequence to balance with the relative abundance of the corresponding tRNA. Similarly, expression can be decreased by deliberately selecting codons whose corresponding tRNAs are known to be rare in a particular cell type. This allows additional translational control to be utilized.

[0216] vector A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. According to the present invention, by way of example, vectors used in recombinant nucleic acid technology include those that allow the transfer of an entity such as a nucleic acid segment (e.g., a heterologous DNA segment, such as a heterologous cDNA segment) into a target cell. A vector can serve the purpose of maintaining a heterologous nucleic acid (DNA or RNA) within a cell, promoting replication of a vector containing a nucleic acid segment, or promoting expression of a protein encoded by the nucleic acid segment. A vector can be non-viral or viral. Examples of vectors used in recombinant nucleic acid technology include, but are not limited to, plasmids, mRNA molecules (e.g., in vitro transcribed mRNA), chromosomes, artificial chromosomes, and viruses. A vector can be, for example, a naked nucleic acid (e.g., DNA). In its simplest form, a vector can be the nucleic acid of interest itself.

[0217] The vector used in the present invention may be, for example, a plasmid, mRNA or viral vector, which may contain a promoter for expression of the polynucleotide and optionally a regulator of the promoter.

[0218] The vectors containing the polynucleotides used in the present invention can be introduced into cells using various techniques known in the art, such as transfection, transformation, and transduction. Several such techniques are known, such as infection with recombinant viral vectors, such as retroviruses, lentiviruses (e.g., integration-defective lentiviruses), adenoviruses, adeno-associated viruses, baculoviruses, and herpes simplex virus vectors; direct injection of nucleic acids; and biolistic (biolistics) transformation.

[0219] Non-viral delivery systems include, but are not limited to, DNA transfection, which involves the use of non-viral vectors to introduce genes into target cells. Typical transfection methods include electroporation, DNA particle bombardment (biolistic methods), lipid-mediated transfection, compacted DNA transfection, liposomes, immunoliposomes, lipofection, cationic transfection, cationic facial amphiphiles (CFAs) (Nat. Biotechnol. (1996) 14: 556), and combinations thereof.

[0220] The term "transfection" should be understood to include polynucleotide delivery into cells by both viral and non-viral delivery.

[0221] Protein delivery method As an alternative to introducing a polynucleotide into a cell, the products and artificial transcriptional repressors (ATRs) of the present invention can be introduced into a cell by protein introduction.

[0222] Protein introduction can be achieved by vector delivery (Cai, Y. et al. (2014) Elife 3: e01911; Maetzig, T. et al. (2012) Curr. Gene Ther. 12: 389-409). Vector delivery requires engineering a viral particle (e.g., a lentiviral particle) to contain the protein to be introduced into a cell. Thus, when the engineered viral particle enters a cell as part of its natural life cycle, the protein contained in the particle is delivered into the cell.

[0223] Protein introduction can be achieved by protein delivery (Gaj, T. et al. (2012) Nat. Methods 9: 805-7). Protein delivery can be achieved, for example, by using a vehicle (e.g., liposomes) or by administering the protein itself directly to the cells.

[0224] Pharmaceutical Composition The products, artificial transcriptional repressors (ATRs), polynucleotides, and cells of the present invention can be formulated with a pharmaceutically acceptable carrier, excipient, or additive for administration to a subject. Suitable carriers and excipients include isotonic saline, such as phosphate-buffered saline, which may contain human serum albumin.

[0225] Handling of cell therapy products is preferably conducted in accordance with the FACT-JACIE International Standards for Cell Therapy.

[0226] kit In one embodiment, the present invention provides a kit comprising two or more artificial transcriptional repressors (ATRs) or polynucleotides encoding same selected from group (a), (b), or (c), wherein at least two of the ATRs are selected from different groups (a), (b), or (c): (a) an ATR containing a DNA-binding domain or a homolog thereof operably linked to a KRAB domain; (b) an ATR containing a DNA-binding domain or a homolog thereof operably linked to a DNMT3A, DNMT3B, or DNMT1 domain; and (c) an ATR containing a DNA-binding domain or a homolog thereof operably linked to a DNMT3L domain.

[0227] The two or more ATRs or polynucleotides encoding them can be provided in a suitable container.

[0228] The kit can include instructions for use, eg, instructions for administering two or more ATRs or polynucleotides encoding same to a subject in need thereof simultaneously, sequentially, or separately.

[0229] Treatment method It will be understood that all references herein to treatment include curative, symptomatic, and preventative treatment; however, in the context of the present invention, reference to prevention relates more broadly to prophylactic treatment. Treatment of mammals, particularly humans, is preferred. Both human and animal treatments are within the scope of the present invention. (Example) Example 1

[0230] To recapitulate the endogenous epigenetic mechanisms that permanently silence endogenous retroviruses (ERVs) during development, we used the Kruppel-associated box (KRAB) domain of human zinc finger protein 10 (ZNF10; Szulc, J. et al. (2006) Nat. Methods 3: 109-16) and the catalytic domain of human DNA methyltransferase 3A (DNMT3A; Law, JA et al. (2010) Nat. Rev. Genet. 11: 204-20). The amino acid sequences of these domains are shown in Table 1.

[0231] To examine the activity and stability of gene silencing induced by the two effector domains, we used the tetracycline (tet) response system. The two effector domains were separately fused to the DNA-binding domain of the Escherichia coli (E. coli) tetracycline-regulated repressor (tetR) (Gossen, M. et al. (1992) Proc. Natl. Acad. Sci. USA 89: 5547-51) to generate the tetR:KRAB and tetR:DNMT3A artificial transcriptional repressors (ATRs, hereafter referred to as tetR:K and tetR:D3A, respectively). The advantage of the tetR system is that binding of tetR to its target nucleotide sequence, the tetracycline operon (TetO), can be temporally controlled by doxycycline (doxy) administration. This will allow us to examine whether the chromatin state induced by ATR is maintained after ATR release from target loci.

[0232] To rapidly assess ATR activity, we devised a cellular experimental model that allows easy time-dependent monitoring of ATR activity by flow cytometry (Figure 1). Specifically, we generated single-cell-derived clones of K562 cells engineered to contain a homozygous insertion of an eGFP expression cassette followed by seven tandem repeats of TetO (TetO7; Figure 1, top diagram) within the first intron of the PPP1R12C gene (Lombardo, A. et al. (2011) Nat. Methods 8: 861-9; also known as the AAVS1 locus). Expression of the eGFP marker in this reporter construct is driven by the ubiquitously expressed human phosphoglycerate kinase (hPGK) gene promoter. This reporter cell line is hereafter referred to as the AAVS1 / TetO7 cell line.

[0233] Upon ATR expression, these chimeric proteins bind to the TetO7 element via its tetR DNA-binding domain, ultimately leading to the accumulation of repressive epigenetic marks on nearby chromatin (represented as red lollipops on the hPGK promoter; Fig. 1, middle schematic). This causes transcriptional silencing of the cassette. Upon conditional release of ATR from the TetO7 element by doxycycline administration, the repressive marks are either erased or heritably transmitted to progeny cells by endogenous cellular mechanisms, thus resulting in transcriptional reactivation of eGFP expression or permanent silencing, respectively (Fig. 1, bottom diagram). The major advantages of using such an experimental model are: i) ATR activity can be rapidly and easily monitored by observing eGFP expression by flow cytometry analysis; and ii) because these clones were engineered to contain homozygous insertions of the cassette, the epigenetic and transcriptional effects of silencing on genes at and near the integration site can be examined without the indistinguishable effects of the unmodified wild-type locus.

[0234] To assess whether the new ATRs were biologically active, we introduced tetR:K and tetR:D3A into the AAVS1 / TetO7 cell line using standard integration of bidirectional lentiviral vectors (Amendola, M. et al. (2005) Nat. Biotechnol. 23: 108-16; Bid.LV; Figure 2A). The advantage of this vector is that ATR and a marker gene (truncated low-affinity nerve growth factor receptor - ΔLNGFR- or monomeric orange - mOrange-) are constitutively coexpressed from the same promoter, thus limiting silencing analysis exclusively to cells expressing ATR.

[0235] In summary, the described experimental setup allows us to investigate whether constitutive binding of a candidate ATR to a TetO7 cassette can accumulate repressive epigenetic marks on nearby chromatin and induce transcriptional silencing of a reporter cassette. Subsequent conditional release of ATR binding by doxycycline treatment allows us to determine whether the artificially induced repressive marks are subsequently erased by endogenous mechanisms (thus leading to transcriptional reactivation) or are heritably transmitted to progeny cells (thus establishing permanent, heritable epigenetic silencing).

[0236] Based on molecular characterization, we transduced the AAVS1 / TetO7 cell line with either Bid.LV-tetR:K or Bid.LV-tetR:D3A in the presence or absence of doxycycline and then maintained it in these culture conditions for up to 200 days. During this time, cells were periodically analyzed by flow cytometry to determine the percentage of eGFP-negative (eGFP-) cells within the Bid.LV-transduced cell population. As shown in Figure 2B, constitutive binding of ATR to the TetO7 sequence (doxy- conditions) ultimately resulted in eGFP silencing in 100% of transduced cells, although the kinetics differed between the two ATRs. Specifically, tetR:K-transduced cells rapidly became eGFP- (Figure 2B, left histogram), and this effect was independent of the level of transduction (Figure 2C, left flow cytometry dot plot). In contrast, tetR:D3A-mediated silencing was significantly slower (Figure 2B, right histogram). In this case, cells with high levels of marker gene expression (presumably cells with high vector copy number VCN) were silenced first (Figure 2C, right flow cytometry dot plot), indicating that a certain level of tetR:D3A expression was required to ensure earlier suppression. Importantly, at later time points (approximately 200 days), flow cytometry analysis revealed that the mean fluorescence intensity (MFI) of eGFP was superimposable between silenced and wild-type (WT) K562 cells (compare MFI in Figure 2C), indicating complete silencing of eGFP expression. When doxycycline was present in the culture (doxy+ condition), the transformed cells did not silence eGFP at all, indicating that ATR binding to the target sequence is required for silencing. Collectively, these data indicate that the two ATRs mediate silencing with different kinetics, but that both are functional.

[0237] Next, we assessed whether release of ATR from the locus resulted in eGFP reactivation. To this end, eGFP cells were sorted 21 days after Bid.LV transfection and then cultured in the presence or absence of doxycycline for an additional 170 days. Interestingly, doxycycline administration produced two opposing results, depending on the ATR used: tetR:K-induced silencing rapidly (within 15 days after doxycycline administration) and completely disappeared in the entire cell population (Figure 2D, left histogram and representative flow cytometry analysis below); on the other hand, tetR:D3A-induced silencing remained unchanged throughout the experimental period (Figure 2D, right histogram and representative flow cytometry analysis below). This clearly demonstrates that, contrary to tetR:K, which must be continuously active at the locus to repress it, tetR:D3A can establish a repressive epigenetic modification that can be permanently propagated by endogenous intracellular mechanisms even in the absence of the initial stimulus. This discrepancy can be explained by the fact that in somatic cells, KRAB-based mechanisms are unable to induce efficient (stably heritable) DNA methylation, but only accumulate reversible epigenetic marks such as H3K9 methylation (Hathaway, NA et al. (2012) Cell 149: 1447-60).

[0238] Overall, these experiments clearly demonstrate that reporter cassette silencing can remain unchanged over several cell generations in the absence of tetR:D3A binding to the TetO7 element, whereas conditional release of tetR:K from the TetO7 element results in rapid and complete reactivation of eGFP expression in tetR:K-transduced cells.

[0239] DNA methylation is involved in maintaining permanent tetR:D3-induced silencing To understand whether DNA methylation is required for maintaining the repressed state induced by tetR:D3, eGFP- cells obtained from the doxycycline-condition (Figure 2D) were treated with either 5-aza-2'-deoxycytidine (5-Aza) or vehicle (i.e., dimethyl sulfoxide, DMSO) and then analyzed by flow cytometry to measure eGFP expression. 5-Aza is a cytosine analog that, after incorporation into DNA, is recognized as a substrate by DNA methyltransferases. Unlike cytosine, it establishes a covalent bond that cannot be broken down, thus blocking DNMT activity (Issa, JP et al. (2005) Nat. Rev. Drug Discov. Suppl. S6-7). As shown in Figure 2E, treatment with 5-Aza resulted in complete reactivation of eGFP expression. As expected, DMSO treatment did not alter eGFP silencing, and the eGFP+ cells in the cultures were contaminating cells from the cell sorting procedure.

[0240] Unlike tetR:K, repression caused by tetR:D3A is restricted to the target locus One of the requirements for a safe epigenetic therapy is that silencing should not spread to genes surrounding the desired target gene. It should be noted that site-specific integration of the reporter cassette into the AAVS1 locus facilitates analysis of the effect of the silencing platform of the present invention on the expression of genes integrated near the reporter cassette integration site. Therefore, the expression levels of genes at and near the AAVS1 integration site (Figure 3A) are compared between the eGFP- cells from Figure 2 and the untreated AAVS1 / TetO7 cell line.

[0241] eGFP- cells transduced with tetR:K significantly downregulated all analyzed genes (Figure 3B, left histogram; data are presented as mean ± SEM, n = 3), indicating that this ATR accumulates a repressive mark that can extend at least 340 kb (approximately 170 kb on either side of the ATR binding site). This finding is consistent with previous studies performed in somatic cell lines, demonstrating that tetR:K can silence promoters located tens of kilobases away from the ATR binding site by long-range spreading of H3K9me3 (Groner, AC et al. (2010) PLoS Genet. 6: e1000869). Importantly, analysis of eGFP- cells transduced with tetR:D3A and grown with doxycycline revealed significant downregulation of not only the eGFP gene, but also, to a lesser extent, the PPP1R12C gene (which hosts a reporter cassette in its first intron) (Fig. 3B, right histogram; data are presented as mean ± SEM, n = 3; ***p < 0.0001 and **p < 0.001, one-way ANOVA and Bonferroni post-hoc test), indicating highly localized epigenetic repression.

[0242] In summary, these experiments demonstrate that tetR:K induces rapid and robust transcriptional repression that can spread over long distances and is reversible upon ATR release from the locus, whereas tetR:D3A induces silencing at a slower rate, but transcriptional repression is clearly restricted to the target locus and is maintained durably in the absence of an initial stimulus.

[0243] Synergistic activity of multiple ATRs by transient co-infusion We next investigated whether transient co-transfection of these two ATRs was sufficient to induce rapid (as in tetR:K) or permanent (as in tetR:D3A) epigenetic silencing. To answer this question, we transfected AAVS1 / TetO7 cells with plasmids encoding these ATRs alone or in combination and then monitored eGFP expression in the cells by flow cytometry over time. A representative example of such an experiment is shown in Figure 4A, where we report the kinetics of silencing of the eGFP expression cassette in cells transfected with the indicated ATR-encoding plasmids (% of eGFP- cells; data are expressed as mean ± SEM, n = 3), with corresponding flow cytometry dot plot analysis performed at the end of the experiment. These analyses revealed that: i) none of the cells transfected with either tetR:K or tetR:D3A-encoding plasmids became eGFP-negative, although transient introduction of tetR:K was associated with a brief increase in repression that rapidly returned to control levels by 10 days posttransfection (the latter data suggesting a transient accumulation of H3K9me3, which subsequently disappeared as the tetR:K-encoding plasmid was diluted by cell division); and ii) surprisingly, up to 20% of the cells cotransfected with tetR:K and tetR:D3A-encoding plasmids became stably silenced. These data revealed significant synergy between the DNMT3A- and KRAB-based repressors, providing the first demonstration of permanent epigenetic silencing by transient cotransfection of ATR.

[0244] Next, we investigated whether the silencing induced by the tetR:K / tetR:D3A combination was limited to the reporter cassette or spread along the AAVS1 locus, thereby affecting genes near the reporter cassette insertion site. To answer this question, we compared the expression profiles of genes at and near the AAVS1 insertion site (a schematic of the locus is shown in Figure 3A) between eGFP-negative and eGFP-positive populations selected from the tetR:K / tetR:D3A treatment condition. This analysis revealed that this treatment resulted in significant silencing of only the reporter transgene (Figure 4B; data are presented as mean ± SEM, n = 3; **p < 0.001, one-way ANOVA and Bonferroni post-hoc test). These important data demonstrate that the tetR:K / tetR:D3A combination accumulates punctuated epigenetic silencing only at the intended target gene, highlighting the safety of this method. Finally, treatment of eGFP-negative sorted cells from the tetR:K / tetR:D3A condition with 5-Aza completely reactivated eGFP expression in these cells (Figure 4C; data are presented as mean ± SEM, n = 3; ***p < 0.0001, two-tailed unpaired t-test), indicating that DNA methylation plays an important role in maintaining this repressed epigenetic state. Similar results were obtained by transfecting the AAVS1 / TetO7 reporter cell line with in vitro-transcribed mRNA encoding ATR (Figure 4D). Surprisingly, the degree of silencing measured in these experiments was approximately twofold higher than that measured by plasmid transfection, likely reflecting the better tolerance and higher expression levels achieved by mRNA transfection. Gene expression analysis showed that only eGFP and the eGFP reporter cassette harboring its gene (i.e., PPP1R12C) were downregulated by treatment (Figure 4E; data are presented as mean ± SEM, n = 3; ***p < 0.0001 and *p < 0.01, one-way ANOVA and Bonferroni post-hoc test).

[0245] Combinatorial silencing by ATR is locus and cell type independent Having found that the two ATRs induce permanent silencing even when transiently introduced into cells, we investigated whether this effect is locus-independent. Indeed, the efficacy of epigenetic therapeutics may depend on the chromatin environment in which the target locus is embedded, and theoretically, some loci may be more refractory to specific repression mechanisms than others. For example, published evidence suggests that loci enriched for H3K4 methylation may be protected from DNA methylation (Ooi, SK et al. (2007) Nature 448: 714-7). Consistent with this, endogenous epigenetic factors naturally present at or adjacent to the target locus may interfere with ATR activity but may also restore the original physiological epigenetic profile of the target gene.

[0246] To address this issue, we inserted the TetO7 sequence upstream of the hPGK promoter of an eGFP expression cassette, and then semirandomly introduced this construct into the genome of K562 cells by standard lentiviral vector transduction (a schematic diagram of the provirus used is shown in Figure 5A). Subsequently, eGFP-expressing cells were selected for purity (hereafter referred to as the TetO7.LV reporter cell line) and transfected with in vitro-transcribed mRNA encoding tetR:K or tetR:D3A, alone or in combination. Time-course flow cytometry analysis of these cells (Fig. 5B; data are presented as mean ± SEM, n = 3; ***p < 0.0001, two-way ANOVA and Bonferroni post-hoc test) revealed that: i) up to 32% of cells transfected with mRNA encoding tetR:D3A gradually became eGFP-negative, reaching a plateau of suppression 2 weeks after transfection; ii) up to 80% of cells transfected with a plasmid encoding tetR:K rapidly became eGFP-negative, but shortly thereafter, the majority of these cells reactivated eGFP expression (unlike experiments performed with the AAVS1 / TetO7 cell line, in which up to 19% of cells remained eGFP-negative); and iii) strikingly, up to 80% of cells cotransfected with mRNA encoding tetR:K / tetR:D3A became permanently silenced. Interestingly, even though comparable silencing efficiencies were recorded between the tetR:K and tetR:K / tetR:D3A conditions shortly after transfection, only the combination of the two factors resulted in high levels of permanent epigenetic silencing. Similar results were observed in U937 cells with random insertion of the TetO7 / eGFP cassette (Figure 5C). However, in this case, silencing efficiencies for all treatment conditions were lower than those obtained in K562 cells, although overall transfection efficiencies between these two cell types were comparable.Unexpectedly, when similar experiments were performed in B lymphoblastoid cells containing randomly inserted TetO7 / eGFP cassettes, long-term stable silencing was observed only under tetR:D3A treatment (Fig. 5D; data are presented as mean ± SEM, n = 3; ***p < 0.0001, two-way ANOVA and Bonferroni post-hoc test). Contrary to the results of all the above experiments, silencing induced by multiple ATR combinations was transient and showed kinetics superimposable to those measured under tetR:K treatment.

[0247] Collectively, these results clearly demonstrate that the two ATRs act cooperatively to establish a steady state of epigenetic repression, even when their target sites are randomly distributed throughout the genomes of various cell types accessible to lentiviral vectors, thus demonstrating that the silencing mechanism is locus-independent. Nevertheless, these studies suggest that several cell-intrinsic factors can modulate the in vivo activity of these proteins.

[0248] Identification of novel ATRs that can increase the silencing efficiency of the platform of the present invention While the above data provide initial evidence for our findings of permanent epigenetic silencing by transient expression of ATR, they also indicate that several cell-specific factors can modulate the in vivo activity of these proteins. For example, the low level of silencing observed in the U937 cell line and the unexpected lack of silencing activity of the ATR combination in B lymphoblastoid cells may be explained by the absence of cofactors involved in the silencing process or the presence of cell-type-specific repressors. Therefore, including additional ATRs in the mixtures of the present invention may be useful for enhancing the silencing efficiency of the KRAB / DNMT3A combination, even when those ATRs are present at low concentrations, by avoiding the absence of cofactors or by enabling proper function of the repression complex. Therefore, we investigated whether other effector domains (or combinations thereof) from chromatin-remodeling enzymes involved in establishing a permanent epigenetic repression state could be used to enhance the silencing efficiency of the ATR of the present invention. To this end, by utilizing literature on known interactions of DNMT3A or KRAB-ZFP proteins (Chen, T. et al. (2014) Nat. Rev. Genet. 15: 93-106), as well as molecules more broadly involved in the transcriptional regulation of cell fate specification and development (Schwartz, YB et al. (2013) Nat. Rev. Genet. 14: 853-64), we identified the following candidates: · Euchromatin histone lysine N-methyltransferase 2 (EHMT2, also known as G9a): a histone methyltransferase that catalyzes the dimethylation of histone H3 lysine-9 and recruits several histone deacetylases; · SET domain bifurcated 1 (SETDB1): a histone methyltransferase that accumulates di- and trimethylated histone H3 lysine-9 (two histone marks involved in transcriptional repression); · Chromobox protein homolog 5 (CBX5, also known as HP1α): a component of heterochromatin that recognizes and binds to histone H3K9me, resulting in epigenetic repression; · DNA (cytosine-5)-methyltransferase 3-like (DNMT3L): a catalytically inactive DNA methyltransferase that activates DNMT3A by binding to its catalytic domain; · Enhancer of Zeste homolog 2 (EZH2): catalytic subunit of polycomb repressive complex 2, which methylates lysine-9 and lysine-27 of histone H3 to create binding sites for classical polycomb repressive complex 1; Suppressor of variegation 4-20 homolog 2 (SUV420H2): a histone methyltransferase that specifically trimethylates lysine-20 of histone H4 (a specific histone mark involved in transcriptional repression in pericentromeric heterochromatin). · Transducin-like enhancer protein 1 (TLE1): A chromatin-bound transcriptional corepressor that binds to and inhibits the activity of many transcription factors.

[0249] We constructed novel ATRs containing the effector domains of the above proteins and the DNA-binding domain of tetR. These novel ATRs are hereafter referred to as tetR:SET (SETDB1); tetR:H (HP1-α); tetR:T (TLE1); tetR:GS or tetR:GL (depending on the length of the effector domain cloned from G9α); tetR:ES or tetR:EL (depending on the length of the effector domain cloned from EZH2); tetR:D3L (DNMT3L); and tetR:SUV (SUV420H2). The amino acid sequences of the effector domains are listed in Table 1.

[0250] We first examined the activity of these novel ATRs in the LV / TetO7 K562 reporter cell line using standard Bid.LV integration. Among the novel ATRs, we found that tetR:SET, tetR:GS, and tetR:H effectively silencing proteins when stably expressed individually (Figure 6A; data are presented as mean ± SEM, n = 3). However, none of these ATRs achieved the silencing efficiency of tetR:K and tetR:D3A. Unlike tetR:GS, tetR:GL was ineffective in this experimental setting, suggesting that the presence of ankyrin repeats in this relatively long form of G9a negatively impacts silencing efficiency. The ineffectiveness of tetR:T, tetR:SUV, tetR:ES, and tetR:EL in this experimental setting may be due to the intrinsic biological inactivity of the selected domains or the absence of endogenous interactors required for the activity of these proteins in this cell line. Furthermore, we found that for some of the ATRs used, the percentage of transduced cells decreased over time (Figure 6B; data are expressed as mean ± SEM, n = 3). This data indicates a growth disadvantage for cells stably expressing ATR and therefore supports the rationale for using transient transfection methods to safely express ATR.

[0251] We next assessed whether silencing induced by the novel ATR persisted in the absence of ATR at the target locus. To this end, 18 days after Bid.LV-ATR transduction, samples were treated with doxycycline, and eGFP expression was monitored by flow cytometry (Figure 6C; data are presented as mean ± SEM, n = 3). As expected, silencing induced by tetR:D3A was maintained after doxycycline treatment. Considering the other samples, silencing was maintained only in a portion of initially silenced cells, which varied depending on the ATR. Specifically, silencing induced by tetR:K yielded more stable results, remaining in up to 45.8% of initially silenced cells. This contrasts with results observed using the AAVS1 / TetO7 K562 reporter cell line, in which eGFP was fully reactivated in all transduced cells 7 days after doxycycline treatment. This data suggests a role in which the positioning of TetO7 relative to the hPGK promoter and / or the epigenetic environment in which the cassette is integrated plays an important role in maintaining the repressive state caused by tetR:K.

[0252] Next, we examined the efficacy of ATR during transient transfection in the same LV / TetO7 K562 reporter cell line. Specifically, we tested ATR individually (Figure 7A) or in combination with tetR:D3A (Figure 7B), tetR:K (Figure 7C), or the combination of tetR:K and tetR:D3A (Figure 7D). To better understand the ultimate increase in silencing efficiency beyond that measured in the positive control, these experiments were performed using non-saturating doses of the ATR expression plasmid. tetR:T was not examined in this experiment because the same plasmid backbone as the other ATRs was not available for tetR:T at the time of the experiment.

[0253] By tracking eGFP expression in treated cells over time by flow cytometry, we found that none of the ATRs effectively silencing tetR:K, tetR:D3A, and tetR:SET suppressed silencing in less than 1% of cells when expressed individually (Figure 7A; data are presented as mean ± SEM, n = 3). When combined with tetR:D3A (Figure 7B; data are presented as mean ± SEM, n = 3), all of the novel ATRs conferred increased silencing efficiency. However, efficiencies similar to those measured in the tetR:K + tetR:D3A condition were only achieved when tetR:D3A was combined with either tetR:SET or tetR:D3L. Furthermore, when co-transfected with tetR:K (Figure 7C; data are presented as mean ± SEM, n = 3), only tetR:D3L among the novel ATRs conferred greater synergistic activity than tetR:D3A. Finally, adding one of the novel ATRs to the tetR:K + tetR:D3A combination (Figure 7D; data are presented as mean ± SEM, n = 3) enhanced silencing efficiency for most ATRs, demonstrating biological activity even for ATRs that were stable but not functional when introduced individually (Figure 6A). Importantly, this experiment identified the tetR:K + tetR:D3A + tetR:D3L combination as the best combination, demonstrating remarkable efficiency considering the low plasmid dose used in this experiment. Specifically, the tetR:K + tetR:D3A + tetR:D3L combination resulted in a 4.1-fold increase in silencing efficiency compared to the tetR:K + tetR:D3A combination (Figure 7E; data are presented as mean ± SEM, n = 3; ***p < 0.0001, one-way ANOVA and Bonferroni post-hoc test). Considering the increased silencing efficiency compared with the tetR:D3A + tetR:D3L, tetR:K + tetR:D3A, and tetR:D3L + tetR:K combinations, all three ATRs play a relevant role in the tetR:K + tetR:D3A + tetR:D3L mixture.Interestingly, starting from the confirmation that tetR:SET can synergize with both tetR:D3A and tetR:D3A + tetR:K (see Figure 7E), we reloaded the same experiment with a lower ATR dose and found that tetR:SET also significantly synergized with the combination of tetR:D3A + tetR:D3L (Figure 7F; tetR:D3L is labeled as tetR:L). This data indicates that the combination of tetR:D3A + tetR:D3L + tetR:SET can be an effective alternative to the combination of tetR:D3A + tetR:D3L + tetR:K, even though the silencing efficiency is lower.

[0254] Inclusion of tetR:D3L in the tetR:K+tetR:D3A combination can restore silencing efficiency in refractory cell types We therefore investigated whether the combination of tetR:K, tetR:D3A, and tetR:D3L could overcome the block seen in B lymphoblastoid cells (see Figure 5D). To address this question, we transfected the TetO7.LV reporter B lymphoblastoid cell line with in vitro transcribed mRNAs encoding the three ATRs, either alone or in different combinations (Figure 7G; tetR:D3A is labeled tetR:D; tetR:D3L is labeled tetR:L; data are presented as mean ± SEM, n = 3). As expected from previous experiments, cotransfection of tetR:K and tetR:D3A resulted in a transient increase in silencing that completely disappeared after dilution of the transfected mRNA, resulting in the absence of eGFP-negative cells. However, both tetR:D3A + tetR:D3L and tetR:D3L + tetR:K were able to induce high levels of silencing (50% and 60%, respectively). These levels were significantly higher than those observed when ATR was introduced alone (14% for tetR:D3A, and levels comparable to untreated samples for tetR:K- and tetR:D3L-transfected cells). Surprisingly, co-introduction of the three ATRs resulted in a majority of cells becoming eGFP-negative (up to 80%), clearly demonstrating that the addition of a single factor to the tetR:D3A / tetR:K mix was sufficient to restore the induction and maintenance of silencing in previously refractory cell lines. Based on these promising results, we also investigated whether our silencing platform would be effective in experimentally relevant cell lines derived from other organisms, such as mice. To answer this question, we first transduced mouse NIH / 3T3 cells with TetO7.LV, sorted the cells to obtain a pure eGFP-positive population (containing an average of one copy of vector per cell), and finally transfected the cells with mRNA encoding the tetR-based ATR, either individually or in combination.Surprisingly, flow cytometry analysis of treated cells demonstrated effective and long-lasting silencing in this cell model: a single administration of tetR:D3A + tetR:D3L or the triple ATR combination resulted in 45% or 80% gene silencing efficiency, respectively (Figure 7H), whereas the tetR:D3A + tetR:K combination was ineffective, as previously observed in B lymphoblastoid cells.

[0255] Efficient silencing by transient co-transfection of ATR with a custom-made DNA-binding domain The primary goal of this project is to develop an epigenetic therapeutic platform that can be used to silence the expression of any gene of interest. Although we have already identified an effector domain that, when fused to tetR, acts synergistically to silence promoters near the TetO7 element, the artificial nature of the prokaryotic TetO7 / tetR system hinders the therapeutic application of this technology. Furthermore, the TetO7 element can accommodate seven tetR dimers with high affinity, which may lead to stochastic homo- or heterodimerization of ATR on this element. This may promote positive interactions between repressors. For these reasons, several issues remain to be addressed in order to translate the knowledge gained from the TetO7 / tetR system to a situation where each ATR has a single, independent binding site on the target gene. In particular, it remains unclear whether a single element (defined as a specific genomic sequence containing the binding sites of each repressor, hereafter referred to as a "silencing element") is sufficient to silence a gene of interest. Furthermore, the relative order and orientation of two repressors on a silencing element, as well as the distance between their binding sites, may be important determinants of the activity of the repression complex. Notably, these determinants cannot be clarified based on the literature or by experimentally examining them on endogenous genes, as this would require designing several different ATRs with unique binding sites and affinities.

[0256] To address these issues, we developed an engineered cell model for our current purpose that readily reports the silencing activity of ATR containing a transcription activator-like effector (TALE; Gaj, T. et al. (2013) Trends Biotechnol. 31: 397-405) DNA-binding domain. In this set of experiments, we first examined ATR corresponding to the tetR:K + tetR:D3A combination.

[0257] Briefly, KRAB and DNMT3A domains were fused to the DNA-binding domains of two TALEs that recognize two distinct genomic target sites with high efficiency (the amino acid sequences of the two TALEs are shown in Table 2). This method yielded two TALE:KRAB fusion proteins (hereafter referred to as TALE:K) and two TALE:DNMT3A fusion proteins (hereafter referred to as TALE:D3A), corresponding to each of the two genomic target sites. In parallel, two TALE target sites were inserted upstream of the hPGK promoter of an eGFP expression cassette, spaced by nucleotide sequences of increasing length (5, 10, 15, 20, 25, and 30 bp), and these constructs were then semi-randomly introduced into the genome of the K562 cell line using standard lentiviral vector transduction methods. Specifically, the target sites of the two TALEs were positioned so that TALE-repressor binding occurred in a head-to-tail (HT) configuration. A schematic diagram of these vectors is shown in Figure 8A (the left side shows a vector containing a binding site in the TALE:K → TALE:D3A configuration; the right side shows a vector containing a binding site in the TALE:D3A → TALE:K configuration). eGFP-expressing cells were then selected for purity, and this cell line was transfected with in vitro transcribed mRNA encoding TALE:K or TALE:D3A, alone or in combination. The cells were then analyzed by time-lapse flow cytometry to assess the extent and duration of silencing. A representative example of such an analysis can be seen in Figure 8, where we report the silencing efficiency (% of eGFP-negative cells) of the indicated ATRs versus spacer length (Figure 8B; data are expressed as mean ± SEM, n = 3), as well as the kinetics of silencing of an eGFP expression cassette measured in cell lines with a 25 bp spacer (Figure 8C; data are expressed as mean ± SEM, n = 3; ***p < 0.0001 and **p < 0.001, two-way ANOVA and Bonferroni post-hoc test).

[0258] These experiments revealed the following: i) co-transfection of TALE:D3A and TALE:K resulted in complete silencing of the eGFP expression cassette in up to 25% of treated cells; ii) the relative order of binding of the two ATRs at the target locus affected the overall silencing efficiency, with the TALE:D3A → TALE:K configuration performing 2.2 to 5.4 times better than the opposite configuration; iii) among the spacer lengths examined, 25 and 30 bp outperformed the others; and iv) individual introduction of TALE:K or TALE:D3A resulted in low (3%) or nonexistent silencing of the eGFP expression cassette, respectively.

[0259] Considering the significant impact of structural variables (e.g., spacer length and the relative binding order of the two ATRs on the target sequence) on silencing efficiency, we next investigated whether transitioning to a head-to-head (HH) configuration, in which the C-termini of the two ATRs face each other, would be advantageous for our strategy. To transition from a head-to-tail to a head-to-head configuration, we started with the reporter cassette shown in Figure 8 and changed the orientation of the 3' TALE binding site while keeping the 5' TALE binding site unchanged. This simple change allowed us to use the same four ATRs used in previous experiments. We also generated six eGFP reporter cassettes with different spacer lengths between the two TALE target sites (5, 10, 15, 20, 25, and 30 bp), and these constructs were introduced into K562 cells by lentiviral vector transduction (a schematic diagram of this vector is shown in Table 9A). Transduced cells were then sorted to obtain a pure eGFP+ population and transformed by electroporation with plasmids encoding TALE:K or TALE:D3A, either alone or in combination. Treated cells were then analyzed by time-lapse flow cytometry to measure the extent and duration of silencing. To strictly compare head-to-head and head-to-tail configurations, cell lines containing the 25 bp spacer and HT, TALE:D3A→TALE:K configuration described in Figure 8C, were included in this experiment. The results of these experiments showed that: i) co-transfection of TALE:K and TALE:D3A, even in the HH configuration, resulted in a clear synergistic effect, enabling long-term silencing of the reporter cassette in up to 34.7% of treated cells (Figure 9B; data are presented as mean ± SEM, n = 3); ii) individual transfection of TALE:K or TALE:D3A resulted in low (up to 7.1%) or non-existent permanent silencing, respectively; and iii) the relative order of binding of the two ATRs at the target locus affected the overall silencing efficiency, with the TALE:D3A → TALE:K configuration performing 1.3- to 1.7-fold better than the opposite configuration (Figure 9C; data are presented as mean ± SEM, n = 3).However, the relative order of attachment appears to have a greater impact on silencing in the head-to-tail configuration than in the head-to-head configuration (compare Figures 8 and 9). A bell-shaped trend indicates the effect of the spacer length tested on the silencing efficiency of the HH configuration, with a 15-bp spacer being the best for both the TALE:D3A→TALE:K and TALE:K→TALE:D3A configurations (even outperforming the 25-bp spacer in the head-to-tail experiments). However, the difference between the 15-bp head-to-head and 25-bp head-to-tail configurations was minimal (34.7% vs. 26.8% of long-term eGFP cells, respectively, i.e., a 1.3-fold increase).

[0260] Overall, these data demonstrate for the first time that transient introduction of a combination of multiple ATRs with custom DNA-binding domains may be able to achieve permanent epigenetic silencing of a desired target gene. Furthermore, these studies clarify rules for selecting TALE binding sites that can be used to identify silencing elements on a desired target gene. Targeting multiple silencing elements on the regulatory sequence of this gene should increase silencing efficiency.

[0261] In parallel with these studies, we developed a bipartite ATR by combining two effector domains on the same TALE: a KRAB domain at the N-terminus and a DNMT3A domain at the C-terminus (Figure 10A). Although transient transfection of individual proteins was not sufficient to induce appreciable levels of gene silencing, their combination was sufficient to silence eGFP in up to 7% of treated cells (Figure 10B; data are presented as mean ± SEM, n = 3). The advantage offered by such an approach is that multiple effector domains can be introduced into the same target site while reducing the number of different mRNAs that need to be generated and transfected.

[0262] Permanent epigenetic silencing in human HSPCs by using different combinations of ATR Although primary hematopoietic stem cells (HSPCs) are the clinically relevant human cell type for most ex vivo gene therapy applications (Biffi, A. et al. (2013) Science 341: 1233158; Aiuti, A. et al. (2013) Science 341: 1233151; Aiuti, A. et al. (2009) N. Engl. J. Med. 360, 447-458; Cartier, N. et al. (2009) Science 326: 818-23; Hacein-Bey-Abina, S. et al. (2010) N. Engl. J. Med. 363: 355-64; Cavazzana-Calvo, M. et al. (2010) Nature 467: 318-22), because these cells possess lifelong self-renewal and pluripotency. HSPC differentiation is accompanied by global chromatin remodeling, resulting in a gradual transition from an open chromatin morphology to a more compact, repressive morphology. Thus, this cell type is the most appropriate and rigorous model for examining the efficiency and demonstrating the stability of our epigenetic platform. To assess whether the introduction of various ATR combinations is sufficient to induce significant levels of silencing in human HSPCs, we transduced human umbilical cord blood-derived CD34+ cells obtained from healthy individuals with the TetO7 / eGFP reporter LV described in Figure 5A. The cells were then transfected with in vitro transcribed mRNA encoding tetR:D3A, tetR:K, or tetR:D3L, alone or in combination. Transfected and untransfected cells were then grown in liquid culture for 2 weeks under myeloid differentiation conditions and plated onto semi-solid medium for colony-forming unit-cell (CFU-C) assays (see Figure 11A for the layout of this experiment).

[0263] Flow cytometry analysis of cells grown in liquid culture showed that treatment with tetR:K resulted in a transient increase in eGFP suppression, which was then maintained in up to 20% of treated cells until the end of the experiment (Figure 11B; data are expressed as mean ± SEM, n = 3). A similar phenotype was observed in CD34+ cells transfected with mRNA encoding tetR:D3A and tetR:D3L. Treatment with the combination of tetR:K / tetR:D3A or tetR:D3A / tetR:D3L produced a synergistic effect, completely silencing eGFP expression in up to 40% of treated cells. Remarkably, by combining tetR:D3L / tetR:K or tetR:D3L / tetR:K / tetR:D3A, we achieved up to 90% silencing of the reporter gene. Importantly, similar levels of silencing were observed in erythroid and myeloid cells derived from CFU-C assays (Figure 11C; data are presented as mean ± SEM, n = 3), demonstrating that silencing is maintained upon HSPC differentiation.

[0264] Permanent epigenetic silencing in human T lymphocytes using various combinations of ATR To assess whether the introduction of various combinations of ATRs was sufficient to induce significant levels of silencing in human T lymphocytes, we transduced human T cells obtained from healthy individuals with the TetO7 / eGFP reporter LV described in Figure 5A. The cells were then transfected with in vitro transcribed mRNA encoding tetR:D3A, tetR:K, or tetR:D3L, alone or in combination. Transfected and untransfected cells were then maintained in liquid culture in medium supplemented with IL-15 and IL-7 for 3 weeks before reactivation (see Figure 12A for the layout of these experiments).

[0265] Flow cytometry analysis of cells showed that treatment with individual ATRs and tetR:D3A / tetR:K resulted in either no or transient eGFP expression, whereas treatment with all other possible ATR combinations resulted in permanent silencing of the reporter gene. Importantly, silencing levels measured during the early stages of cell growth and in the stationary phase were superimposable, indicating that silencing was maintained even after changes in the transcriptional and metabolic states of the cells (Figure 12B; data are presented as mean ± SEM, n = 3).

[0266] Permanent epigenetic silencing of human endogenous genes using custom-made ATRs To assess whether the results obtained with the eGFP reporter system could be transferred to endogenous genes embedded in their natural epigenetic context, we created custom TALEs targeting the promoter region of the β2-microglobulin (B2M) gene (the amino acid sequences of these TALEs and the nucleotide sequences of the corresponding binding sites are listed in Table 3). These TALEs were fused with the KRAB, DNMT3A, and DNMT3L effector domains (see Figure 13A for a schematic diagram of the system). The spacer length between the first and second TALEs, or between the second and third TALEs, was 1 or 20 bp, respectively. We then co-transfected HEK-293T cells with the plasmids encoding the novel ATRs described above and analyzed the cells for B2M expression by flow cytometry.

[0267] Fifty days after transfection, when the percentage of B2M-negative cells stabilized, significant proportions of B2M-negative cells were measured only in the conditions treated with the combinations TALE:D3A + TALE:D3L and TALE:D3A + TALE:D3L + TALE:K (Figure 13B; data are expressed as mean ± SEM, n = 3; ***p < 0.0001, one-way ANOVA and Bonferroni post-hoc test). Surprisingly, up to 80% of all ATR-treated cells permanently lost surface expression of B2M. In parallel experiments, B2M-negative and B2M-positive cells were sorted and analyzed for surface expression of MHC-I molecules, which requires B2M on the cell membrane. Unlike B2M-positive cells, almost all B2M-negative cells were also negative for MHC-I expression (Figure 13C). Gene expression analysis was performed on the sorted B2M-negative and B2M-positive cells, revealing approximately 100-fold lower expression in the negative cells than in the positive cells (Figure 13D; data are presented as mean ± SEM, n = 3). Next, we evaluated whether the three effector domains could also induce permanent epigenetic silencing when targeted to B2M by the guide RNA-mediated CRISPR / Cas9 system. To this end, we designed catalytically inactive Cas9 (D10A+H840A; dCas9; amino acid sequence listed in Table 4) in-frame with the KRAB, DNMT3A, or DNMT3L effector domains (Figure 13E; top diagram). Eleven guide RNAs (gRNAs; nucleotide sequences listed in Table 4) were designed to target the promoter region of the B2M gene (Figure 13E; bottom schematic; arrows indicate the locations of the CRISPR / dCas9 target sites). HEK-293T cells were then co-transfected with plasmids expressing the 11 B2M gRNAs along with all possible combinations of plasmids encoding dCas9 fusion proteins.Thirty-three days after transfection, flow cytometry analysis of the treated HEK-293T cells showed that only the combinations of dCas9:K + dCas9:D3L, dCas9:D3A + dCas9:D3L, and dCas9:K + dCas9:D3a + dCas9:D3L were able to silence the B2M gene (Figure 13F; data are expressed as mean ± SEM, n = 3). Next, we evaluated whether B2M silencing was resistant to IFN-γ treatment, a potent inducer of B2M expression (Vraetz, T. et al. (1999) Nephrol. Dial. Transplant. 14: 2137-43; Gobin, SJ et al. (2003) Blood 101: 3058-64). For this experiment, we used wild-type and B2M-negative cells, the latter selected from the triple ATR treatment conditions described in Figures 13B and 13F. As expected, IFN-γ treatment caused a significant (>100-fold) upregulation of 2'-5'-oligoadenylate synthetase 1 (OAS1) gene expression in all cell types tested (Figure 13G). Meanwhile, IFN-γ treatment significantly upregulated B2M expression at both the transcriptional and protein levels in wild-type cells, whereas no increase in expression of this gene was measured in B2M-negative cells (Figures 13G and 13H, respectively).

[0268] To assess whether the silencing induced by our ATR involves the accumulation of repressive epigenetic marks on the target gene, we analyzed the epigenetic status of the B2M gene in wild-type and silenced cells. To this end, cells treated with a plasmid encoding the triple TALE:ATR combination were sorted until purity was achieved to obtain a pure population of silenced cells (Figure 14A, representative FACS dot plots are shown). Chromatin immunoprecipitation (ChIP) followed by quantitative PCR analysis of RNA polymerase II (RNA Pol II) on the promoter region and gene body of B2M revealed a complete absence of this protein in silenced cells, whereas it was highly enriched in the promoter region of untreated cells (PPP1R12C and CCR5 genes were used as positive and negative controls, respectively; Figure 14B). We also performed bisulfite analysis of the B2M CpG island and found that the promoter region was largely depleted of 5mC at the CpG level (<1%) in untreated cells, whereas the same region in silenced cells was highly modified by de novo DNA methylation (>80% on average) (Figure 14C). DNA methylation also plays a role in maintaining silencing, as AZA treatment was associated with re-expression of the B2M gene in previously silenced cells (Figure 14D). Finally, to address whether silencing was limited to the B2M gene, we performed transcriptional analysis of the B2M locus by RT-qPCR (Figure 14E, upper panel). We found that B2M was the only gene downregulated by transient transfection of the triple ATR combination, while the expression of its neighboring genes was unaffected (Figure 14E).

[0269] In parallel with these experiments, we also investigated silencing the B2M gene in K-562 cells. Because this cell line does not express MHC-I, which is completely required for B2M surface expression, we targeted the coding sequence of the fluorescent marker tdTomato into the first intron of the B2M gene to accurately report on the B2M transcriptional status (Figure 15A). After gene targeting with CRISPR / Cas9, tdTomato-positive cells were selected and then electroporated with plasmids encoding either a TALE-based ATR or a CRISPR / dCas9-based ATR against the B2M promoter / enhancer (the target sequences of these ATRs are the same as in Figure 13). Regarding TALE-based ATRs, both the TALE:D3L+TALE:K combination and the TALE:D3A+TALE:D3L+TALE:K combination were able to stably silence B2M expression, but the triple ATR combination proved to be the most effective (Figure 15B). Codon optimization of the effector domain of TALE-based ATRs improved the silencing efficiency of the above combinations (Figure 15B; compare the red and green bars). Regarding the silencing activity of CRISPR / dCas9-based ATRs, we found that all but the dCas9:D3A+dCas9:K combination were able to achieve high silencing efficiency of the B2M gene (up to 55% of stably silenced cells; Figure 15C). Finally, targeting the B2M promoter / enhancer in selected silenced cells with dCas9 fused to the catalytic domain of the TET1 enzyme (known to demethylate DNA; Maeder, ML et al. (2013) Nat. Biotechnol. 31: 1137-42) resulted in reactivation of expression of this gene (Figure 15D), supporting the idea that silencing induced by the triple ATR combination is dependent on DNA methylation.

[0270] To assess whether B2M silencing could be effective in primary human T lymphocytes, in vitro transcribed mRNA encoding the TALE:K+TALE:D3A+TALE:D3L ATR was electroporated into human T cells obtained from a healthy donor. Transfected and untransfected cells were then maintained in liquid culture for 2 weeks in medium supplemented with IL-15 and IL-7 (experimental scheme shown in Figure 16A). Flow cytometry analysis of the cells revealed that treatment with TALE:K+TALE:D3A+TALE:D3L ATR resulted in silencing of the B2M gene (silencing kinetics shown in Figure 16B, FACS plot shown in Figure 16C).

[0271] Interestingly, functional deconvolution of seven gRNAs, from quadruplets to individual ones, revealed that even a single gRNA, a triple combination, and a combination of dCas9:D3A and dCas9:D3L, were sufficient to effectively silence B2M (Figure 17). Unexpectedly, some single gRNAs were able to produce silencing efficiencies comparable to those measured with the seven-gRNA pool. Furthermore, in some instances, triple ATR combinations were observed to perform better than dCas9:D3A and dCas9:D3L combinations. Overall, these data demonstrate that a gRNA bound to three ATRs, even one of which is appropriately positioned on a target gene, can induce efficient silencing.

[0272] We also investigated whether a single TALE protein is sufficient to induce efficient and permanent epigenetic silencing. To this end, we engineered four TALE proteins, each fused to three different effector domains: KRAB, DNMT3A, and DNMT3L (Figure 17B; schematic diagram on the left). As models, we used the previously described TALE proteins targeting the B2M gene and the K562 B2M tdTomato reporter cell line (Figures 13A and 15A, respectively). Unexpectedly, efficient and permanent gene silencing of the B2M gene was achieved under conditions in which the repressive domains competed for the same binding site on the B2M gene (Figure 17B; gray bars in the histogram). The differences in efficiency most likely reflect differences in the binding affinities of the TALEs, some of which functioned as efficiently as control conditions in which each effector domain was fused to a different DNA-binding domain (Figure 17B; dark blue bars in the histogram).

[0273] In summary, these data demonstrate, for the first time, the permanent silencing of an endogenous gene in human cells using a custom-designed ATR. Importantly, the silencing was completely resistant to external stimuli acting on the B2M promoter / enhancer, thus providing further evidence for the stability of the epigenetic modifications accumulated by the triple ATR combination. Furthermore, we provide evidence that our strategy can be broadly applied by tethering repressor domains to endogenous genes using two different DNA-binding technologies: TALE and CRISPR / Cas9.

[0274] Transient expression of untargeted DNMT3L improves and restores silencing efficiency of DNMT3A + KRAB-based ATR in refractory cell types To reduce the number of different ATRs to design and construct, we investigated whether at least one effector domain could be introduced into cells without a DNA-binding domain and still effectively act in conjunction with two other ATRs targeted to a desired gene of interest. To assess whether the introduction of untargeted DNMT3L (hereafter referred to as D3L) would be as effective at cooperating with the other two effector domains (specifically, DNMT3A and KRAB) as its targeted counterpart, we first utilized the TetO7 / tetR system. Therefore, we transfected TetO7.LV reporter B lymphoblastoid cells with in vitro transcribed mRNAs encoding the tetR-based ATR and untargeted D3L, and measured the percentage of eGFP-negative cells under various transfection conditions by time-lapse flow cytometry analysis (Figure 18A; data are presented as mean ± range, n = 2). At 27 days posttransfection, we found very little, if any, silencing in cells treated with individual ATRs or the tetR:K + tetR:D3A combination. Instead, up to 70% of cells treated with the triple ATR combination became eGFP-negative. Targeted tetR:D3L also synergized with tetR:K or tetR:D3A, although the silencing levels measured in these two experimental conditions were 3.5-fold lower than those measured with the triple ATR combination (approximately 20% eGFP-negative cells) (Figure 18A; compare with the plus tetR:D3L condition). These data are consistent with previous findings in the TetO7.LV reporter B-lymphoblastoid cell line, where the unexpected decrease in silencing efficiency of the tetR:D3A + tetR:K combination was fully restored by including tetR:D3L in the mixture (see Figure 7G for comparison). When non-targeting D3L was introduced alone or in combination with tetR:K, no eGFP-negative cells were observed, whereas D3L was able to effectively synergize with both the tetR:D3A and tetR:D3A + tetR:K combinations (Figure 18A; see plus D3L condition).Importantly, the silencing levels measured in these two experimental conditions were comparable to those observed by simultaneously tethering DNMT3L and DNMT3A to the TetO7 sequence, or DNMT3L, DNMT3A, and KRAB. These data demonstrate that untargeted DNMT3L can effectively synergize with the combination of KRAB and DNMT3A.

[0275] We evaluated whether these findings also hold true for ATRs based on custom-made DNA-binding domains. To this end, we selected four different TALE binding sites in the B2M promoter region and constructed corresponding TALE DNA-binding domains (a schematic diagram of the B2M locus showing the different TALE binding sites is shown in Figure 18B; the amino acid sequence of TALE A and the nucleotide sequence of its corresponding binding site are listed in Table 5; TALEs B, C, and D have already been described and correspond to TALEs #1, #2, and #3 in Table 3). These TALEs each carried KRAB or DNMT3A. The four different TALE binding sites constituted two independent silencing modules (Module 1: Site A plus Site B; Module 2: Site C plus Site D), to which TALE:D3A and TALE:K could bind in two different orders (Site A:K-Site B:D3A or Site A:D3A-Site B:K). Next, we transfected HEK-293T cells with plasmids encoding a TALE-based ATR and a non-targeted D3L, and measured the percentage of double-negative B2M / MHCI-negative cells in different transfection conditions by time-course flow cytometry analysis (Figure 18C). At 12 days posttransfection, low percentages of B2M / MHCI-negative cells were measured in all conditions treated with the TALE:D3A + TALE:K combination (top plot, Figure 18C). Meanwhile, co-treatment of cells with the combination of two ATRs plus a non-targeted D3L resulted in an average 5-fold increase in silencing efficiency above the level measured without D3L (bottom plot, Figure 18C). This increase was observed for both silencing modules, regardless of the relative order of binding of the TALE proteins on the B2M promoter.

[0276] Finally, we performed a similar experiment using the ATR-based CRISPR / Cas9 system (Figure 18D; data are presented as mean ± SEM, n = 3). Here, we found that transient expression of D3L in HEK-293T cells transfected with dCas9:K + dCas9:D3A ATR plus B2M gRNA (as used in Figure 13E) resulted in gene silencing levels comparable to those obtained with the triple combination of dCas9-based ATR plus B2M gRNA (Figure 18D). Similar results were obtained by introducing D3L via DNMT3A ATR.

[0277] Taken together, these data clearly demonstrate that untargeted DNMT3L is efficiently replaced by its targeted counterpart in the ATR mixture of the present invention.

[0278] Transient expression of untargeted DNMT3B restores the silencing efficiency of DNMT3A + KRAB-based ATR in refractory cell types Considering the role of DNMT3B in de novo DNA methylation, we investigated whether endogenous DNMT3B might cooperate with the ATR of the present invention. To answer this question, we performed gene knockout of DNMT3B using CRISPR / Cas9 in the TetO7.LV K562 reporter cell line. To do this, we transduced cells with two lentiviral vectors: one encoding the doxycycline-inducible Cas9 nuclease (Wang, T. et al. (2014) Science 343: 80-4), and the other encoding a gRNA targeting exon 2 of the DNMT3B gene and the ΔLNGFR marker (Figure 19A shows a schematic diagram of the vectors; the middle FACS plot shows the double-transduced cells). Cas9 was activated by doxycycline administration, and the cells were electroporated with various combinations of ATR-encoding plasmids. The silencing efficiency was then measured in ΔLNGFR-positive and -negative cells by flow cytometry. By comparing these figures, we can assess whether inactivation of the DNMT3B gene improves the silencing efficiency of various ATR combinations. Here, we observed that the subpopulation expressing gRNAs against DNMT3B (i.e., ΔLNGFR-positive cells) was less permissive than wild-type cells to silencing by the tetRK + tetR:D3A combination (Figures 19B and 19C; upper right FACS plots), thus demonstrating that endogenous DNMT3B is a suitable partner for these two ATRs. Surprisingly, gene knockout of DNMT3B increased the silencing efficiency of the tetR:K + tetR:D3A + tetR:D3L combination, suggesting that DNMT3B acts as a decoy for these ATRs in this case (Figures 19B and 19C; lower right FACS plots). For all other ATR combinations and individual ATRs, inactivation of DNMT3B did not result in significant differences in silencing efficiency compared to wild-type cells.Furthermore, given that the B lymphoblastoid cell line described above lacks DNMT3B expression (as measured by RT-qPCR analysis), in contrast to K562 cells, we investigated whether overexpression of DNMT3B could increase ATR silencing efficiency in this cell line, which is refractory to the DNMT3A+KRAB combination. Specifically, we transiently transfected the TetO7.LV B lymphoblastoid reporter cell line with mRNA encoding full-length DNMT3B (without fusing it to the tetR DNA-binding domain; the amino acid sequence of DNMT3B is listed in Table 1) with or without two ATRs. Surprisingly, overexpression of DNMT3B significantly restored the activity of the tetR:K+tetR:D3A combination in 52% of treated cells, enabling stable eGFP silencing, a 65-fold increase compared to tetR:K+tetR:D3A alone (Figure 19C). Of note, DNMT3B overexpression also resulted in a 2.9-fold increase in silencing efficiency in the tetR:D3A condition (Figure 19D).

[0279] Taken together, these data clearly demonstrate that untargeted DNMT3B can effectively restore the activity of the DNMT3A+KRAB combination in refractory cell lines.

[0280] Silencing the BCL11A gene using both CRISPR / dCas9-based and TALE-based ATR We utilized the combination of ATR to silence BCL11A, a gene whose inhibition has been proposed as a potential therapeutic intervention for β-thalassemia and sickle cell anemia. To readily assess the activity of ATR on the BCL11A gene, we targeted the tdTomato transgene located within the third exon of the gene in human B lymphoblastoid cells by CRISPR / Cas gene targeting (Figure 20A). This targeting strategy inhibits the expression of the tdTomato transgene from the regulatory sequences of the BCL11A gene. This allows for a steady report of the gene's expression level. Next, we enriched tdTomato-positive cells until they were nearly pure by cell sorting, and then targeted four CpG islands within the promoter / enhancer region of this gene using CRISPR / dCas9-based ATRs containing DNMT3A or DNMT3L. Each of the four CpG islands was investigated individually using a separate pool of gRNAs (also known as CRISPRs; the nucleotide sequences of the gRNAs are listed in Table 6). By comparing tdTomato expression between treated and untreated controls, we were able to assess the relative contribution of each island to BCL11A expression (Figure 20B). Compared to control-treated cells, silencing of each CpG island was associated with long-term, stable suppression of BCL11A expression (shown here as % of tdTomato-negative cells), although the extent of silencing of this gene varied depending on the CpG island targeted by the ATR. Next, we selected CpG islands 31 and 38 for further study to evaluate the activity of triple ATR combinations. These studies included all possible double ATR combinations and single KRAB-based ATRs. Surprisingly, all conditions tested were able to induce significant levels of gene silencing through epigenetic editing of CpG 38 (the island that responded best in previous experiments), with triple ATR combinations resulting in up to 55% gene silencing (Figure 20C). Finally, we designed 17 different TALE-based ATRs targeting CpG islands 31 and 38 (7 and 10 TALE proteins, respectively; the amino acid sequences of these TALEs and their associated target sequences are listed in Table 7; schematic diagram at the top of Figure 20D) and tested their silencing activity as triple ATR combinations or as KRAB-based ATRs.Silencing of both CpG islands with all triple ATR combinations resulted in effective and long-lasting silencing of BCL11A (reaching up to 55% of tdTomato-negative cells), whereas silencing with TALE:KRAB resulted in varying degrees of gene silencing, some as efficient as the triple ATR combination and others completely inactive. Together, these data demonstrate the feasibility of permanently silencing the human BCL11A gene.

[0281] Silencing additional human endogenous genes using CRISPR / dCas9-based ATR Finally, we further tested the epigenetic silencing technology of the present invention on two endogenous human genes: the interferon (α, β, and ω) receptor 1 (IFNAR1) gene and the vascular endothelial growth factor A (VEGFA) gene. Both genes exhibit CpG islands in the gene promoter / enhancer regions. Therefore, we designed 13 gRNAs against the IFNAR1 CpG island (Figure 20E, top) and three gRNAs against the VEGFA CpG island (Figure 20F, top) (the nucleotide sequences of the gRNAs are listed in Table 6). Interestingly, electroporation of K562 cells with a plasmid encoding the 13 gRNA pool against the IFNAR1 gene plus a triple dCas9-based ATR combination achieved long-term downregulation of IFNAR1 transcription levels (0.22-fold change) in treated cells compared to untreated samples (Figure 20E, bottom). Furthermore, electroporation of K562 cells with a plasmid encoding a combination of three gRNA pools against the VEGFA gene plus a triple dCas9-based ATR achieved long-term downregulation of VEGFA transcript levels (0.57-fold change) in treated cells compared to untreated samples (Figure 20F, bottom). Overall, these data demonstrate the feasibility of silencing various human endogenous genes with CRISPR / dCas9-based ATR.

[0282] material and method Lentiviral vector and ATR constructs ATR reporter lentiviral vectors (LVs) containing TetO7 sequences or TALE binding sites, and DNMT3B gRNA-expressing LVs were generated from the self-inactivating transfection construct pCCLsin.cPPT.hPGK.eGFP.Wpre (Follenzi, A. et al. (2000) Nat. Genet. 25: 217-22), while ATR-expressing Bid.LVs were generated from the transfection construct pCCLsin.cPPT.dLNGFR.mhCMV.hPGK.GFP.Wpre (Gentner, B. et al. (2010) Sci. Transl. Med. 2: 58ra84). The doxycycline-inducible Cas9 expression vector was purchased from Addgene (pCW-Cas9; #50661; Wang, T. et al. (2014) Science 343: 80-4). LV stocks were prepared as previously described (Follenzi, A. et al. (2002) Methods Mol. Med. 69:259-74). Briefly, HEK293T cells were cotransfected with 35 / 12.5 / 9 / 6.25 μg of the transfer construct plasmids pMD.Lg / pRRE packaging plasmid, pMD2.VSV-G envelope-encoding plasmid, and pRSV-Rev per 15 cm dish by calcium phosphate DNA precipitation. Vector particles were concentrated 300-fold by ultracentrifugation as previously described, and titers were measured on HEK293T cells by serial dilution (Cantore, A. et al. (2015) Sci. Transl. Med. 7:277ra28). All other tetR-based ATRs were generated by replacing the KRAB domain in tetR:KRAB (as discussed in Szulc, J. et al. (2006) Nat. Methods 3: 109-16) with other suitable effector domains.TALE-based ATRs were generated using a modified version of the Golden Gate TALEN Kit 2.0a (Addgene, Kit #1000000024; Cermak, T. et al. (2011) Nucleic Acids Res. 39: e82), which contained the following structural changes: the Golden Gate TALE C- and N-terminal subregions were replaced with +163 and +63 deletions, respectively. These constructs were configured to provide an in-frame effector domain. Cas9-based ATRs were generated by replacing the VP160 transactivator with the effector domain or the catalytic domain of TET1 from the plasmid pAC154-dual-dCas9VP160-sgExpression (Addgene #48240; Cheng, AW et al. (2013) Cell Res. 23: 1163-71).

[0283] Cell culture conditions and manipulations Human Epstein-Barr virus-immortalized B lymphocytes (B lymphoblastoid cells) and U-937 cells were maintained in RPMI-1640 (Sigma); HEK293T and K-562 cells were maintained in IMDM (Sigma); and NIH / 3T3 cells were maintained in DMEM (Sigma). All media were supplemented with 10% FBS (fetal bovine serum; EuroClone), L-glutamine (EuroClone), and 1% penicillin / streptomycin (100 U / mL final concentration; EuroClone). Cells were cultured at 37°C in a 5% CO2 humidified incubator. Reporter cell lines were generated by transducing cells with the indicated ATR reporter LV at a multiplicity of infection (MOI) of 0.1 and then enriched for eGFP expression using a MoFlo XDP Cell Sorter (Beckman Coulter). Reporter cell lines with targeted integration were generated as follows: i) To insert an eGFP cassette into the AAVS1 locus, a donor construct (containing a TetO7 sequence downstream or upstream of the cassette; 1.5 μg donor plasmid) and the AAVS1-ZFNs were cotransfected as mRNA (0.5 μg each ZFN; Lombardo, A. et al. (2011) Nat. Methods 8: 861-9).Single-cell-derived clones were obtained by limiting dilution plating and analyzed by Southern blot to confirm targeted integration of the cassette as previously described (Lombardo, A. et al. (2011) Nat. Methods 8: 861-9); ii) To insert tdTomato into the third exon of BCL11A, a donor construct (2 μg) containing the tdTomato transgene fused to the 2A autocatalytic peptide was cotransfected with a plasmid encoding Cas9 (1 μg) and another plasmid expressing a gRNA targeting exon 3 (125 ng; gRNA sequence: 5′-GGAGCTCTAATCCCCACGCCTGG-3′); iii) Using a targeting strategy similar to that used for BCL11A, a splice acceptor-IRES-tdTomato cassette was inserted into intron 1 of B2M (gRNA sequence: 5′-AGGCTACTAGCCCCATCAAGAGG-3′). Both tdTomato cell lines were generated by FACS sorting of positive cells.

[0284] To test ATR activity, reporter cell lines were transduced with ATR-expressing Bid.LV at an MOI of 10, or transfected with ATR-expressing plasmids or in vitro-transcribed mRNA (4D-Nucleofector™ System; Lonza) according to the manufacturer's instructions for K-562, U937, and NIH / 3T3 cells, and with pulse-programmed EW-113 and SF solutions for B lymphoblastoid cells. Except for experiments performed under nonsaturating conditions using 500 ng of the respective ATR-encoding plasmid, typically 2 μg of nucleic acid (both plasmid and in vitro-transcribed mRNA) was transfected for tetR-based or TALE-based ATRs, respectively. Meanwhile, 1–2 μg of the dCas9-based ATR-encoding plasmid and 125–250 ng of the gRNA expression plasmid were electroporated. In vitro transcribed mRNA was prepared as previously described (Genovese, P. et al. (2014) Nature 510: 235-40). Where necessary, cells were treated with 1 μg of 5-aza-2-deoxycytidine (AZA, Sigma) or 12 μg / mL of doxycycline (Sigma). The AZA-containing medium was changed daily, and cells were analyzed by flow cytometry 4 and 7 days after treatment. Where necessary, cells were treated with 500 U / mL of recombinant human IFN-γ (R&D Systems). The IFN-γ-containing medium was changed daily, and cells were analyzed by flow cytometry 2 and 4 days after treatment. Cord blood-derived CD34+ cells obtained from healthy donors were purchased from Lonza. The cells were cultured for 10 min in serum-free StemSpan medium (StemCell Technologies) supplemented with penicillin, streptomycin, and the following human early-acting cytokines: stem cell factor (SCF) 50 ng / mL, Flt3 ligand (Flt3-L) 50 ng / mL, thrombopoietin (TPO) 50 ng / mL, and interleukin-6 (IL-6) 50 ng / mL (all purchased from Peprotech). 6CD34+ cells / mL were stimulated overnight and then transduced with TetO7-reporter LV at an MOI of 30–50. Forty-eight hours later, cells were electroporated with 2 μg of ATR-encoding mRNA (P3 Primary Cell 4D-Nucleofector X Kit, program EO-100; Lonza). 1 μM SR1 (BioVision Inc.) was added with each medium change. After one week in stimulation medium, cells were cultured in liquid medium in IMDM with 10% FBS. For CFC assays, 800 cells / plate were seeded in methylcellulose-based medium (MethoCult H4434, StemCell Technologies) one day after electroporation. Two weeks after plating, colonies were counted, identified according to morphological criteria, and analyzed by flow cytometry.

[0285] Resting T lymphocytes were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors by leukocyte separation and Ficoll-Hypaque gradient separation. Cells were activated and sorted using magnetic beads conjugated with antibodies against CD3 and CD28 (ClinExVivo CD3 / CD28; Invitrogen) according to the manufacturer's instructions, and then cultured at 1 × 10 cells in RPMI (Sigma) supplemented with penicillin, streptomycin, 10% FBS, and 5 ng / mL IL-7 and IL-15 (PeproTech) as previously described (Kaneko, S. et al. (2009) Blood 113: 1006-15). 6The cells were cultured at a concentration of 10 cells / mL. After 3 days of culture, the cells were transduced with TetO7-reporter LV at an MOI of 10. Three days after transduction, the cells were washed and electroporated with 2 μg of mRNA encoding ATR. To examine silencing resistance to polyclonal TCR stimulation, bulk-treated T lymphocytes were cocultured with 6000 rad-irradiated PMBCs obtained from an unrelated donor and a pool of 10000 rad-irradiated JY cells in the presence of 30 ng / mL of anti-CD3 antibody (OKT3) (Orthoclone, Milan, Italy) and 50 U / mL of human recombinant IL-2 (PrepoTech). To investigate B2M silencing in primary T lymphocytes, these cells were isolated from PBMCs of healthy donors by leukocyte separation, Ficoll-Hypaque gradient separation, and final selection with the Pan T Cell Isolation Kit (Miltenyi Biotec). T cells were then activated with CD3- and CD28-conjugated magnetic beads (ClinExVivo CD3 / CD28; Invitrogen) according to the manufacturer's instructions and cultured at 1 × 10 cells in RPMI (Sigma) supplemented with penicillin, streptomycin, 10% FBS, and 5 ng / mL IL-7 and IL-15 (PeproTech) as previously described (Kaneko, S. et al. (2009) Blood 113: 1006-15). 6 The beads were cultured at a concentration of 1000 / mL. After 3 days in culture, the cells were electroporated with in vitro transcribed mRNA encoding the TALE-based ATR and maintained in culture for an additional 2 weeks; however, the beads were removed 4 days after electroporation. The use of human CB-derived CD34+ cells and primary T lymphocytes was approved by the San Raffaele Hospital Bioethical Committee.

[0286] Flow cytometry and gene expression analysis For immunophenotypic analysis of Bid.LV-transduced cells, CD34 + cells and their progeny, and T lymphocytes (performed by FACSCanto II; BD Pharmingen), the following antibodies were used: JPEG2025063201000003.jpg73150 Aminoactinomycin D (7-AAD)-positive nonviable cells were excluded from the analysis. 1-5 × 10 cells were used per analysis. 5 Viable cells were scored. Single stained and FMO stained cells were used as controls.

[0287] For gene expression analysis, 2-6 x 10 6 Total RNA extracted from cells (RNeasy Mini kit; Qiagen) was reverse transcribed using random hexamers using the SuperScript III First-Strand Synthesis System (Invitrogen) according to the manufacturer's protocol. 15–100 ng of cDNA obtained from K-562 and HEK293T cells was analyzed in triplicate using TaqMan Gene Expression Assays (Applied Biosystems). The gene expression assay used to detect eGFP transcripts was previously described (Lombardo, A. et al. (2011) Nat. Methods 8: 861-9). Real-time PCR was performed using a ViiA 7 Real-Time PCR System (Applied Biosystems), and raw data (Ct and raw fluorescence) were extracted using dedicated software. Genes with Ct values ​​≥ 37 were excluded from the analysis. The relative expression level of each gene was calculated by the ΔΔCt method and expressed as a fold change compared to mock-treated samples (calibrators), corrected for HPRT or B2M expression (housekeeping gene controls).

[0288] molecular analysis For bisulfite sequencing, genomic DNA was extracted using the DNeasy Blood & Tissue Kit or QIAamp DNA Mini Kit (QIAGEN) and then treated with the EpiTect Bisulfite kit (Qiagen) according to the manufacturer's instructions. For bisulfite sequencing, genomic DNA was extracted using the DNeasy Blood & Tissue Kit or QIAamp DNA Mini Kit (QIAGEN) and then treated with the EpiTect Bisulfite kit (Qiagen) according to the manufacturer's instructions. The B2M promoter region was then PCR-amplified using the following primers: PCR fragments were purified and cloned into pCRII-TOPO TA (Invitrogen), and 5-10 clones per sample were verified by sequencing using M13 universal primers. JPEG2025063201000005.jpg54133

[0289] Chromatin immunoprecipitation (ChIP) analysis was performed as previously described using 5-10 μg of ChIP-grade antibodies (Abcam) raised against human H3 or the RNA polymerase II CTD repeat YSPTSPS (Lombardo, A. et al. (2011) Nat. Methods 8: 861-9). IgG isotypes were also used as controls. The primers used in these experiments are listed below. The percentage of enrichment of RNA Pol II for each examined site was calculated by the ΔCt method using Input as the reference. JPEG2025063201000006.jpg113121

[0290] statistical analysis One-way ANOVA with Bonferroni's multiple comparison post hoc test was used to assess the statistical significance of differences in gene expression between all samples (P<0.05). JPEG2025063201000007.jpg182129JPEG2025063201000008.jpg192129JPEG2025063201000009.jpg25126Table 1

[0291] JPEG2025063201000010.jpg113135 Nucleotide sequence of the corresponding TALE binding site TALE forward 5′-TACCCAGATTGGCCCCACT-3′ TALE reverse 5′-TACCTAGAGGAGAAAGGTT-3′ Table 2

[0292] Amino acid sequence of TALE targeting the B2M promoter region JPEG2025063201000011.jpg162128 Nucleotide sequence of the corresponding TALE binding site TALE #1 5′-TCTCTCCTACCCTCCCGCT-3′ TALE #2 5′-TGGTCCTTCCTCTCCCGCT-3′ TALE #3 5′-TCGCTCCGTGACTTCCCTT-3′ Table 3

[0293] JPEG2025063201000012.jpg95143B2M Nucleotide sequence of the gRNA target site JPEG2025063201000013.jpg10351JPEG2025063201000014.jpg5949B2M Nucleotide sequence of gRNA JPEG2025063201000015.jpg10180Table 4

[0294] Amino acid sequence of TALE A targeting the B2M promoter region JPEG2025063201000016.jpg61143 Nucleotide sequence of the corresponding TALE binding site TALE A 5′-TGCTCGCGCTACTCTCTCT-3′ Table 5

[0295] Nucleotide sequence of BCL11A-targeting gRNA gRNA #1 against CpG 105: GCCUUUCUGCAGACGUUCCC gRNA #2 against CpG 105: UGGGUGUGCGCCUUGGCCGG gRNA #3 against CpG 105: CGGUGGUGAGAUGACCGCCU gRNA #4 against CpG 105: GGAAUGUGCUCACGGCGCCG gRNA #5 against CpG 105: GACUGCCCGCGCUUUGUCCU gRNA #6 against CpG 105: CCAGAGUCUGGCCCCCGGAG gRNA #7 against CpG 105: UCUGCGACCCUUAGGAGCCG gRNA #8 against CpG 105: GAGCGCCCCGCCAAGCGACU gRNA #9 against CpG 105: CAAGUCUCCAGGAGCCCGCG gRNA #10 against CpG 105: CGCGGAAUCCAGCCUAAGUU gRNA #11 against CpG 105: CCCGCUGCGGAGCUGUAACU gRNA #1 against CpG 31: CGCUCCUGAGUCCGCGGAGU gRNA #2 against CpG 31: CACGGCUCUCCCCGUCGCCG gRNA #3 against CpG 31: CCGCCUUUUGUUCCGGCCAG gRNA #4 against CpG 31: GCGCGAGGAGCCGGCACAAA gRNA #5 against CpG 31: GCCACUUUCUCACUAUUGUG gRNA #6 against CpG 31: GCUGCCUCUGAGGUUCGGUC gRNA #7 against CpG 31: AAGGGCAGGAGCUAGGGCCG gRNA #8 against CpG 31: GAGCCCGGACUGCUGCCUCC gRNA #1 against CpG 38: GUUUACAAGCACCGCGUGUG gRNA #2 against CpG 38: AACAGACAGAGGACCGAGCG gRNA #3 against CpG 38: GGCGCCGGGUGGGCGAUCCG gRNA #4 against CpG 38: GGUCGGGCAAGGCCCGGGCG gRNA #5 against CpG 38: AAGAGGUCUCGGCAUUGUGC gRNA #6 against CpG 38: GUUCCACAGCUUCGGGACCG gRNA #7 against CpG 38: GAAAUCGGCUGGGUGAAACU gRNA #8 against CpG 38: GCAGUGUCUCCGCGCCAGCC gRNA #9 against CpG 38: CCUCCCCUCCCCUCCGCCCU gRNA #1 against CpG 115: UCCUCCUGUCCCGGGGUUAA gRNA #2 against CpG 115: CAUCUUUUGGGACACUCUAGG gRNA #3 against CpG 115: AAGUCAGGCCCUUCUUCGGAA gRNA #4 against CpG 115: GCAGCCUGGACUGCGCGCCC gRNA #5 against CpG 115: UGCCCGGCGAUUCUCGUCCG gRNA #6 against CpG 115: UGAGCCAUUCGGUCGCUAGG gRNA #7 against CpG 115: GGUGGUACUGAGGACCGGGA gRNA #8 against CpG 115: AUUUUCUGGGUGCUCAGAGG gRNA #9 against CpG 115: UGGUCUCAGCUCGCGCACGG gRNA #10 against CpG 115: ACAAAGACAUACGGGGUGAU Nucleotide sequence of gRNA targeting IFNAR1 gRNA #1: AGGAACGGCGCGUGCGCGGA gRNA #2: AAGAGGCGGCGCGUGCGUAG gRNA #3: GGGCGGUGUGACUUAGGACG gRNA #4: CCAGAUGAUGGUCGUCCUCC gRNA #5: GACCCUAGUGCUCGUCGCCG gRNA #6: UGGGUGUUGUCCGCAGCCGC gRNA #7: ACGGGGGCGGCGAUGCUGUU gRNA #8: GACCGAAGGUUUCCCAGACU gRNA #9: GUCGGGUUUAAUCUUUGGCG gRNA #10: CGCUCCCGAGGACCCGUACA gRNA #11: CGGGUCCCACCCCCGUGAAA gRNA #12: UCAAACUCGACACAAAGCUC gRNA #13: GCGGAGCCGCGGUACUUUCC Nucleotide sequence of VEGFA-targeting gRNA gRNA #1: GGCGCGCGCGCUAGGUGGGA gRNA #2: AGAGAGGCUCACCGCCCACG gRNA #3:GUACGUGCGGUGACUCCGGU Table 6

[0296] Amino acid sequence of TALE targeting the BCL11A gene JPEG2025063201000017.jpg61142 Nucleotide sequence of the corresponding TALE binding site JPEG2025063201000018.jpg75142 Nucleotide sequence of the corresponding TALE binding site JPEG2025063201000019.jpg55142JPEG2025063201000020.jpg22143Nucleotide sequence of the corresponding TALE binding site JPEG2025063201000021.jpg76142 Nucleotide sequence of the corresponding TALE binding site JPEG2025063201000022.jpg74143 Nucleotide sequence of the corresponding TALE binding site JPEG2025063201000023.jpg20129JPEG2025063201000024.jpg53142Nucleotide sequence of the corresponding TALE binding site JPEG2025063201000025.jpg75143 Nucleotide sequence of the corresponding TALE binding site JPEG2025063201000026.jpg67143JPEG2025063201000027.jpg11141Nucleotide sequence of the corresponding TALE binding site JPEG2025063201000028.jpg74142 Nucleotide sequence of the corresponding TALE binding site JPEG2025063201000029.jpg75142 Nucleotide sequence of the corresponding TALE binding site JPEG2025063201000030.jpg33142JPEG2025063201000031.jpg46143Nucleotide sequence of the corresponding TALE binding site JPEG2025063201000032.jpg75142 Nucleotide sequence of the corresponding TALE binding site JPEG2025063201000033.jpg76143 Nucleotide sequence of the corresponding TALE binding site JPEG2025063201000034.jpg75142 Nucleotide sequence of the corresponding TALE binding site JPEG2025063201000035.jpg75143 Nucleotide sequence of the corresponding TALE binding site JPEG2025063201000036.jpg45143JPEG2025063201000037.jpg34142Nucleotide sequence of the corresponding TALE binding site JPEG2025063201000038.jpg76144 Nucleotide sequence of the corresponding TALE binding site 5′- TGGGCCCTCACGCCTTTCT -3′ Table 7

[0297] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described products, uses, methods, and kits of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described methods for carrying out the invention will be apparent to those skilled in biochemistry and biotechnology or related fields and are intended to be within the scope of the following claims.

[0298] SEQUENCE LISTING <110> Ospedale San Raffaele Srl Fondazione Telethon ETS <120> PERMANENT EPIGENETIC GENE SILENCING <130> PA25-019 <141> 2015-10-23 <150> GB 1418965.8 <151> 2014-10-24 <160> 175 <170> PatentIn version 3.5 <210> 1 <211> 117 <212> PRT <213> Homo sapiens <400> 1 Ala Leu Ser Pro Gln His Ser Ala Val Thr Gln Gly Ser Ile Ile Lys 1 5 10 15 Asn Lys Glu Gly Met Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr 20 25 30 Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp 35 40 45 Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu 50 55 60 Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro 65 70 75 80 Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu 85 90 95 Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu 100 105 110 Ile Lys Ser Ser Val 115 <210> 2 <211> 72 <212> PRT <213> Homo sapiens <400> 2 Ile Thr Leu Glu Asp Val Ala Val Asp Phe Thr Trp Glu Glu Trp Gln 1 5 10 15 Leu Leu Gly Ala Ala Gln Lys Asp Leu Tyr Arg Asp Val Met Leu Glu 20 25 30 Asn Tyr Ser Asn Leu Val Ala Val Gly Tyr Gln Ala Ser Lys Pro Asp 35 40 45 Ala Leu Phe Lys Leu Glu Gln Gly Glu Gln Leu Trp Thr Ile Glu Asp 50 55 60 Gly Ile His Ser Gly Ala Cys Ser 65 70 <210> 3 <211> 73 <212> PRT <213> Homo sapiens <400> 3 Val Met Phe Glu Glu Val Ser Val Cys Phe Thr Ser Glu Glu Trp Ala 1 5 10 15 Cys Leu Gly Pro Ile Gln Arg Ala Leu Tyr Trp Asp Val Met Leu Glu 20 25 30 Asn Tyr Gly Asn Val Thr Ser Leu Glu Trp Glu Thr Met Thr Glu Asn 35 40 45 Glu Glu Val Thr Ser Lys Pro Ser Ser Ser Gln Arg Ala Asp Ser His 50 55 60 Lys Gly Thr Ser Lys Arg Leu Gln Gly 65 70 <210> 4 <211> 72 <212> PRT <213> Homo sapiens <400> 4 Val Ser Phe Lys Asp Val Ala Val Asp Phe Thr Gln Glu Glu Trp Gln 1 5 10 15 Gln Leu Asp Pro Asp Glu Lys Ile Thr Tyr Arg Asp Val Met Leu Glu 20 25 30 Asn Tyr Ser His Leu Val Ser Val Gly Tyr Asp Thr Thr Lys Pro Asn 35 40 45 Val Ile Ile Lys Leu Glu Gln Gly Glu Glu Pro Trp Ile Met Gly Gly 50 55 60 Glu Phe Pro Cys Gln His Ser Pro 65 70 <210> 5 <211> 74 <212> PRT <213> Homo sapiens <400> 5 Val Lys Ile Glu Asp Met Ala Val Ser Leu Ile Leu Glu Glu Trp Gly 1 5 10 15 Cys Gln Asn Leu Ala Arg Arg Asn Leu Ser Arg Asp Asn Arg Gln Glu 20 25 30 Asn Tyr Gly Ser Ala Phe Pro Gln Gly Gly Glu Asn Arg Asn Glu Asn 35 40 45 Glu Glu Ser Thr Ser Lys Ala Glu Thr Ser Glu Asp Ser Ala Ser Arg 50 55 60 Gly Glu Thr Thr Gly Arg Ser Gln Lys Glu 65 70 <210> 6 <211> 72 <212> PRT <213> Homo sapiens <400> 6 Leu Thr Phe Lys Asp Val Phe Val Asp Phe Thr Leu Glu Glu Trp Gln 1 5 10 15 Gln Leu Asp Ser Ala Gln Lys Asn Leu Tyr Arg Asp Val Met Leu Glu 20 25 30 Asn Tyr Ser His Leu Val Ser Val Gly Tyr Leu Val Ala Lys Pro Asp 35 40 45 Val Ile Phe Arg Leu Gly Pro Gly Glu Glu Ser Trp Met Ala Asp Gly 50 55 60 Gly Thr Pro Val Arg Thr Cys Ala 65 70 <210> 7 <211> 72 <212> PRT <213> Homo sapiens <400> 7 Val Thr Phe Glu Asp Val Thr Leu Gly Phe Thr Pro Glu Glu Trp Gly 1 5 10 15 Leu Leu Asp Leu Lys Gln Lys Ser Leu Tyr Arg Glu Val Met Leu Glu 20 25 30 Asn Tyr Arg Asn Leu Val Ser Val Glu His Gln Leu Ser Lys Pro Asp 35 40 45 Val Val Ser Gln Leu Glu Glu Ala Glu Asp Phe Trp Pro Val Glu Arg 50 55 60 Gly Ile Pro Gln Asp Thr Ile Pro 65 70 <210> 8 <211> 322 <212> PRT <213> Homo sapiens <400> 8 Thr Tyr Gly Leu Leu Arg Arg Arg Glu Asp Trp Pro Ser Arg Leu Gln 1 5 10 15 Met Phe Phe Ala Asn Asn His Asp Gln Glu Phe Asp Pro Pro Lys Val 20 25 30 Tyr Pro Pro Val Pro Ala Glu Lys Arg Lys Pro Ile Arg Val Leu Ser 35 40 45 Leu Phe Asp Gly Ile Ala Thr Gly Leu Leu Val Leu Lys Asp Leu Gly 50 55 60 Ile Gln Val Asp Arg Tyr Ile Ala Ser Glu Val Cys Glu Asp Ser Ile 65 70 75 80 Thr Val Gly Met Val Arg His Gln Gly Lys Ile Met Tyr Val Gly Asp 85 90 95 Val Arg Ser Val Thr Gln Lys His Ile Gln Glu Trp Gly Pro Phe Asp 100 105 110 Leu Val Ile Gly Gly Ser Pro Cys Asn Asp Leu Ser Ile Val Asn Pro 115 120 125 Ala Arg Lys Gly Leu Tyr Glu Gly Thr Gly Arg Leu Phe Phe Glu Phe 130 135 140 Tyr Arg Leu Leu His Asp Ala Arg Pro Lys Glu Gly Asp Asp Arg Pro 145 150 155 160 Phe Phe Trp Leu Phe Glu Asn Val Val Ala Met Gly Val Ser Asp Lys 165 170 175 Arg Asp Ile Ser Arg Phe Leu Glu Ser Asn Pro Val Met Ile Asp Ala 180 185 190 Lys Glu Val Ser Ala Ala His Arg Ala Arg Tyr Phe Trp Gly Asn Leu 195 200 205 Pro Gly Met Asn Arg Pro Leu Ala Ser Thr Val Asn Asp Lys Leu Glu 210 215 220 Leu Gln Glu Cys Leu Glu His Gly Arg Ile Ala Lys Phe Ser Lys Val 225 230 235 240 Arg Thr Ile Thr Thr Arg Ser Asn Ser Ile Lys Gln Gly Lys Asp Gln 245 250 255 His Phe Pro Val Phe Met Asn Glu Lys Glu Asp Ile Leu Trp Cys Thr 260 265 270 Glu Met Glu Arg Val Phe Gly Phe Pro Val His Tyr Thr Asp Val Ser 275 280 285 Asn Met Ser Arg Leu Ala Arg Gln Arg Leu Leu Gly Arg Ser Trp Ser 290 295 300 Val Pro Val Ile Arg His Leu Phe Ala Pro Leu Lys Glu Tyr Phe Ala 305 310 315 320 Cys Val <210> 9 <211> 322 <212> PRT <213> Homo sapiens <400> 9 Cys His Gly Val Leu Arg Arg Arg Lys Asp Trp Asn Val Arg Leu Gln 1 5 10 15 Ala Phe Phe Thr Ser Asp Thr Gly Leu Glu Tyr Glu Ala Pro Lys Leu 20 25 30 Tyr Pro Ala Ile Pro Ala Ala Arg Arg Arg Pro Ile Arg Val Leu Ser 35 40 45 Leu Phe Asp Gly Ile Ala Thr Gly Tyr Leu Val Leu Lys Glu Leu Gly 50 55 60 Ile Lys Val Gly Lys Tyr Val Ala Ser Glu Val Cys Glu Glu Ser Ile 65 70 75 80 Ala Val Gly Thr Val Lys His Glu Gly Asn Ile Lys Tyr Val Asn Asp 85 90 95 Val Arg Asn Ile Thr Lys Lys Asn Ile Glu Glu Trp Gly Pro Phe Asp 100 105 110 Leu Val Ile Gly Gly Ser Pro Cys Asn Asp Leu Ser Asn Val Asn Pro 115 120 125 Ala Arg Lys Gly Leu Tyr Glu Gly Thr Gly Arg Leu Phe Phe Glu Phe 130 135 140 Tyr His Leu Leu Asn Tyr Ser Arg Pro Lys Glu Gly Asp Asp Arg Pro 145 150 155 160 Phe Phe Trp Met Phe Glu Asn Val Val Ala Met Lys Val Gly Asp Lys 165 170 175 Arg Asp Ile Ser Arg Phe Leu Glu Cys Asn Pro Val Met Ile Asp Ala 180 185 190 Ile Lys Val Ser Ala Ala His Arg Ala Arg Tyr Phe Trp Gly Asn Leu 195 200 205 Pro Gly Met Asn Arg Pro Val Ile Ala Ser Lys Asn Asp Lys Leu Glu 210 215 220 Leu Gln Asp Cys Leu Glu Tyr Asn Arg Ile Ala Lys Leu Lys Lys Val 225 230 235 240 Gln Thr Ile Thr Thr Lys Ser Asn Ser Ile Lys Gln Gly Lys Asn Gln 245 250 255 Leu Phe Pro Val Val Met Asn Gly Lys Glu Asp Val Leu Trp Cys Thr 260 265 270 Glu Leu Glu Arg Ile Phe Gly Phe Pro Val His Tyr Thr Asp Val Ser 275 280 285 Asn Met Gly Arg Gly Ala Arg Gln Lys Leu Leu Gly Arg Ser Trp Ser 290 295 300 Val Pro Val Ile Arg His Leu Phe Ala Pro Leu Lys Asp Tyr Phe Ala 305 310 315 320 Cys Glu <210> 10 <211> 478 <212> PRT <213> Homo sapiens <400> 10 Leu Arg Thr Leu Asp Val Phe Ser Gly Cys Gly Gly Leu Ser Glu Gly 1 5 10 15 Phe His Gln Ala Gly Ile Ser Asp Thr Leu Trp Ala Ile Glu Met Trp 20 25 30 Asp Pro Ala Ala Gln Ala Phe Arg Leu Asn Asn Pro Gly Ser Thr Val 35 40 45 Phe Thr Glu Asp Cys Asn Ile Leu Leu Lys Leu Val Met Ala Gly Glu 50 55 60 Thr Thr Asn Ser Arg Gly Gln Arg Leu Pro Gln Lys Gly Asp Val Glu 65 70 75 80 Met Leu Cys Gly Gly Pro Pro Cys Gln Gly Phe Ser Gly Met Asn Arg 85 90 95 Phe Asn Ser Arg Thr Tyr Ser Lys Phe Lys Ser Leu Val Val Ser 100 105 110 Tyr Cys Asp Tyr Tyr Arg Pro Arg Phe Phe Leu Leu Glu 115 120 125 Asn Val Arg Asn Phe Will Be Phe Lys Arg Will Be Met Val Leu Lys Leu 130 135 140 Thr Leu Arg Cys Leu Val Arg Met Gly Tyr Gln Cys Thr Phe Gly Val 145 150 155 160 Leu Gln Ala Gly Gln Tyr Gly Val Ala Gln Thr Arg Arg Ala Ile 165 170 175 Ile Leu Ala Ala Ala Pro Gly Glu Lys Leu Pro Leu Phe Pro Glu Pro 180 185 190 Leu His Val Phe Ala Pro Arg Ala Cys Gln Leu Ser Val Val Val Asp 195 200 205 Asp Lys Lys Phe Val Ser Asn Ile Thr Arg Leu Ser Ser Gly Pro Phe 210 215 220 Arg Thr Ile Thr Val Arg Asp Thr Met Ser Asp Leu Pro Glu Val Arg 225 230 235 240 Asn Gly Ala Ser Ala Leu Glu Ile Ser Tyr Asn Gly Glu Pro Gln Ser 245 250 255 Trp Phe Gln Arg Gln Leu Arg Gly Ala Gln Tyr Gln Pro Ile Leu Arg 260 265 270 Asp His Ile Cys Lys Asp Met Ser Ala Leu Val Ala Ala Arg Met Arg 275 280 285 His Ile Pro Leu Ala Pro Gly Ser Asp Trp Arg Asp Leu Pro Asn Ile 290 295 300 Glu Val Arg Leu Ser Asp Gly Thr Met Ala Arg Lys Leu Arg Tyr Thr 305 310 315 320 His His Asp Arg Lys Asn Gly Arg Ser Ser Ser Gly Ala Leu Arg Gly 325 330 335 Val Cys Ser Cys Val Glu Ala Gly Lys Ala Cys Asp Pro Ala Ala Arg 340 345 350 Gln Phe Asn Thr Leu Ile Pro Trp Cys Leu Pro His Thr Gly Asn Arg 355 360 365 His Asn His Trp Ala Gly Leu Tyr Gly Arg Leu Glu Trp Asp Gly Phe 370 375 380 Phe Ser Thr Thr Val Thr Asn Pro Glu Pro Met Gly Lys Gln Gly Arg 385 390 395 400 Val Leu His Pro Glu Gln His Arg Val Val Ser Val Arg Glu Cys Ala 405 410 415 Arg Ser Gln Gly Phe Pro Asp Thr Tyr Arg Leu Phe Gly Asn Ile Leu 420 425 430 Asp Lys His Arg Gln Val Gly Asn Ala Val Pro Pro Pro Leu Ala Lys 435 440 445 Ala Ile Gly Leu Glu Ile Lys Leu Cys Met Leu Ala Lys Ala Arg Glu 450 455 460 Ser Ala Ser Ala Lys Ile Lys Glu Glu Glu Ala Ala Lys Asp 465 470 475 <210> 11 <211> 386 <212> PRT <213> Homo sapiens <400> 11 Met Ala Ala Ile Pro Ala Leu Asp Pro Glu Ala Glu Pro Ser Met Asp 1 5 10 15 Val Ile Leu Val Gly Ser Ser Glu Leu Ser Ser Ser Val Ser Pro Gly 20 25 30 Thr Gly Arg Asp Leu Ile Ala Tyr Glu Val Lys Ala Asn Gln Arg Asn 35 40 45 Ile Glu Asp Ile Cys Ile Cys Cys Gly Ser Leu Gln Val His Thr Gln 50 55 60 His Pro Leu Phe Glu Gly Gly Ile Cys Ala Pro Cys Lys Asp Lys Phe 65 70 75 80 Leu Asp Ala Leu Phe Leu Tyr Asp Asp Asp Gly Tyr Gln Ser Tyr Cys 85 90 95 Ser Ile Cys Cys Ser Gly Glu Thr Leu Leu Ile Cys Gly Asn Pro Asp 100 105 110 Cys Thr Arg Cys Tyr Cys Phe Glu Cys Val Asp Ser Leu Val Gly Pro 115 120 125 Gly Thr Ser Gly Lys Val His Ala Met Ser Asn Trp Val Cys Tyr Leu 130 135 140 Cys Leu Pro Ser Ser Arg Ser Gly Leu Leu Gln Arg Arg Arg Lys Trp 145 150 155 160 Arg Ser Gln Leu Lys Ala Phe Tyr Asp Arg Glu Ser Glu Asn Pro Leu 165 170 175 Glu Met Phe Glu Thr Val Pro Val Trp Arg Arg Gln Pro Val Arg Val 180 185 190 Leu Ser Leu Phe Glu Asp Ile Lys Lys Glu Leu Thr Ser Leu Gly Phe 195 200 205 Leu Glu Ser Gly Ser Asp Pro Gly Gln Leu Lys His Val Val Asp Val 210 215 220 Thr Asp Thr Val Arg Lys Asp Val Glu Glu Trp Gly Pro Phe Asp Leu 225 230 235 240 Val Tyr Gly Ala Thr Pro Pro Leu Gly His Thr Cys Asp Arg Pro Pro 245 250 255 Ser Trp Tyr Leu Phe Gln Phe His Arg Leu Leu Gln Tyr Ala Arg Pro 260 265 270 Lys Pro Gly Ser Pro Arg Pro Phe Phe Trp Met Phe Val Asp Asn Leu 275 280 285 Val Leu Asn Lys Glu Asp Leu Asp Val Ala Ser Arg Phe Leu Glu Met 290 295 300 Glu Pro Val Thr Ile Pro Asp Val His Gly Gly Ser Leu Gln Asn Ala 305 310 315 320 Val Arg Val Trp Ser Asn Ile Pro Ala Ile Arg Ser Arg His Trp Ala 325 330 335 Leu Val Ser Glu Glu Glu Leu Ser Leu Leu Ala Gln Asn Lys Gln Ser 340 345 350 Ser Lys Leu Ala Ala Lys Trp Pro Thr Lys Leu Val Lys Asn Cys Phe 355 360 365 Leu Pro Leu Arg Glu Tyr Phe Lys Tyr Phe Ser Thr Glu Leu Thr Ser 370 375 380 Leo Serum 385 <210> 12 <211> 1291 <212> PRT <213> Homo sapiens <400> 12 Met Ser Ser Leu Pro Gly Cys Ile Gly Leu Asp Ala Ala Thr Ala Thr 1 5 10 15 Val Glu Ser Glu Glu Ile Ala Glu Leu Gln Gln Ala Val Val Glu Glu 20 25 30 Leu Gly Ile Ser Met Glu Glu Leu Arg His Phe Ile Asp Glu Glu Leu 35 40 45 Glu Lys Met Asp Cys Val Gln Gln Arg Lys Lys Gln Leu Ala Glu Leu 50 55 60 Glu Thr Trp Val Ile Gln Lys Glu Ser Glu Val Ala His Val Asp Gln 65 70 75 80 Leu Phe Asp Asp Ala Ser Arg Ala Val Thr Asn Cys Glu Ser Leu Val 85 90 95 Lys Asp Phe Tyr Ser Lys Leu Gly Leu Gln Tyr Arg Asp Ser Ser Ser 100 105 110 Glu Asp Glu Ser Ser Arg Pro Thr Glu Ile Ile Glu Ile Pro Asp Glu 115 120 125 Asp Asp Asp Val Leu Ser Ile Asp Ser Gly Asp Ala Gly Ser Arg Thr 130 135 140 Pro Lys Asp Gln Lys Leu Arg Glu Ala Met Ala Ala Leu Arg Lys Ser 145 150 155 160 Ala Gln Asp Val Gln Lys Phe Met Asp Ala Val Asn Lys Lys Ser Ser 165 170 175 Ser Gln Asp Leu His Lys Gly Thr Leu Ser Gln Met Ser Gly Glu Leu 180 185 190 Ser Lys Asp Gly Asp Leu Ile Val Ser Met Arg Ile Leu Gly Lys Lys 195 200 205 Arg Thr Lys Thr Trp His Lys Gly Thr Leu Ile Ala Ile Gln Thr Val 210 215 220 Gly Pro Gly Lys Lys Tyr Lys Val Lys Phe Asp Asn Lys Gly Lys Ser 225 230 235 240 Leu Leu Ser Gly Asn His Ile Ala Tyr Asp Tyr His Pro Pro Ala Asp 245 250 255 Lys Leu Tyr Val Gly Ser Arg Val Val Ala Lys Tyr Lys Asp Gly Asn 260 265 270 Gln Val Trp Leu Tyr Ala Gly Ile Val Ala Glu Thr Pro Asn Val Lys 275 280 285 Asn Lys Leu Arg Phe Leu Ile Phe Phe Asp Asp Gly Tyr Ala Ser Tyr 290 295 300 Val Thr Gln Ser Glu Leu Tyr Pro Ile Cys Arg Pro Leu Lys Lys Thr 305 310 315 320 Trp Glu Asp Ile Glu Asp Ile Ser Cys Arg Asp Phe Ile Glu Glu Tyr 325 330 335 Val Thr Ala Tyr Pro Asn Arg Pro Met Val Leu Leu Lys Ser Gly Gln 340 345 350 Leu Ile Lys Thr Glu Trp Glu Gly Thr Trp Trp Lys Ser Arg Val Glu 355 360 365 Glu Val Asp Gly Ser Leu Val Arg Ile Leu Phe Leu Asp Asp Lys Arg 370 375 380 Cys Glu Trp Ile Tyr Arg Gly Ser Thr Arg Leu Glu Pro Met Phe Ser 385 390 395 400 Met Lys Thr Ser Ser Ala Ser Ala Leu Glu Lys Lys Gln Gly Gln Leu 405 410 415 Arg Thr Arg Pro Asn Met Gly Ala Val Arg Ser Lys Gly Pro Val Val 420 425 430 Gln Tyr Thr Gln Asp Leu Thr Gly Thr Gly Thr Gln Phe Lys Pro Val 435 440 445 Glu Pro Pro Gln Pro Thr Ala Pro Pro Ala Pro Pro Phe Pro Pro Ala 450 455 460 Pro Pro Leu Ser Pro Gln Ala Gly Asp Ser Asp Leu Glu Ser Gln Leu 465 470 475 480 Ala Gln Ser Arg Lys Gln Val Ala Lys Lys Ser Thr Ser Phe Arg Pro 485 490 495 Gly Ser Val Gly Ser Gly His Ser Ser Pro Thr Ser Pro Ala Leu Ser 500 505 510 Glu Asn Val Ser Gly Gly Lys Pro Gly Ile Asn Gln Thr Tyr Arg Ser 515 520 525 Pro Leu Gly Ser Thr Ala Ser Ala Pro Ala Pro Ser Ala Leu Pro Ala 530 535 540 Pro Pro Ala Pro Pro Val Phe His Gly Met Leu Glu Arg Ala Pro Ala 545 550 555 560 Glu Pro Ser Tyr Arg Ala Pro Met Glu Lys Leu Phe Tyr Leu Pro His 565,570,575 Val Cys Ser Tyr Thr Cys Leu Ser Arg Val Arg Pro Met Arg Asn Glu 580,585,590 Gln Tyr Arg Gly Lys Asn Pro Leu Val Pro Leu Tyr Asp Phe 595,600,605 Arg Arg Met Thr Ala Arg Arg Arg Val Asn Arg Lys Met Gly Phe His 610 615 620 The Tyrl Lys Thr Pro Cys Gly Leu Cys Leu Arg Thr With Gln Glu 625 630 635 640 Ile Glu Arg Tyr Leu Phe Glu Thr Gly Cys Asp Phe Leu Phe Leu Glu 645,650,655 Met Phe Cys Leu Asp Pro Tyr Val Leu Val Asp Arg Lys Phe Gln Pro 660,665,670 Tyr Lys Pro Phe Tyr Tyr Ile Leu Asp and Thr Tyr Gly Lys Glu Asp 675,680,685 Val Pro Leu Ser Cys Val Asn Glu Ile Asp Thr Thr Pro Pro Pro Gln 690 695 700 Val Ala Tyr Ser Lys Glu Arg Ile Pro Gly Lys Gly Val Phe Ile Asn 705 710 715 720 Thr Gly Pro Glu Phe Leu Val Gly Cys Asp Cys Lys Asp Gly Cys Arg 725 730 735 Asp Lys Ser Lys Cys Ala Cys His Gln Leu Thr Ile Gln Ala Thr Ala 740 745 750 Cys Thr Pro Gly Gly Gln Ile Asn Pro Asn Ser Gly Tyr Gln Tyr Lys 755 760 765 Arg Leu Glu Glu Cys Leu Pro Thr Gly Val Tyr Glu Cys Asn Lys Arg 770 775 780 Cys Lys Cys Asp Pro Asn Met Cys Thr Asn Arg Leu Val Gln His Gly 785 790 795 800 Leu Gln Val Arg Leu Gln Leu Phe Lys Thr Gln Asn Lys Gly Trp Gly 805 810 815 Ile Arg Cys Leu Asp Asp Ile Ala Lys Gly Ser Phe Val Cys Ile Tyr 820 825 830 Ala Gly Lys Ile Leu Thr Asp Asp Phe Ala Asp Lys Glu Gly Leu Glu 835 840 845 Met Gly Asp Glu Tyr Phe Ala Asn Leu Asp His Ile Glu Ser Val Glu 850 855 860 Asn Phe Lys Glu Gly Tyr Glu Ser Asp Ala Pro Cys Ser Ser Asp Ser 865 870 875 880 Ser Gly Val Asp Leu Lys Asp Gln Glu Asp Gly Asn Ser Gly Thr Glu 885 890 895 Asp Pro Glu Glu Ser Asn Asp Asp Ser Ser Asp Asp Asn Phe Cys Lys 900 905 910 Asp Glu Asp Phe Ser Thr Ser Ser Val Trp Arg Ser Tyr Ala Thr Arg 915 920 925 Arg Gln Thr Arg Gly Gln Lys Glu Asn Gly Leu Ser Glu Thr Thr Ser 930 935 940 Lys Asp Ser His Pro Pro Asp Leu Gly Pro Pro His Ile Pro Val Pro 945 950 955 960 Pro Ser Ile Pro Val Gly Gly Cys Asn Pro Pro Ser Ser Glu Glu Thr 965 970 975 Pro Lys Asn Lys Val Ala Ser Trp Leu Ser Cys Asn Ser Val Ser Glu 980 985 990 Gly Gly Phe Ala Asp Ser Asp Ser His Ser Ser Phe Lys Thr Asn Glu 995 1000 1005 Gly Gly Glu Gly Arg Ala Gly Gly Ser Arg Met Glu Ala Glu Lys 1010 1015 1020 Ala Ser Thr Ser Gly Leu Gly Ile Lys Asp Glu Gly Asp Ile Lys 1025 1030 1035 Gln Ala Lys Lys Glu Asp Thr Asp Asp Arg Asn Lys Met Ser Val 1040 1045 1050 Val Thr Glu Ser Ser Arg Asn Tyr Gly Tyr Asn Pro Ser Pro Val 1055 1060 1065 Lys Pro Glu Gly Leu Arg Arg Pro Pro Ser Lys Thr Ser Met His 1070 1075 1080 Gln Ser Arg Arg Leu Met Ala Ser Ala Gln Ser Asn Pro Asp Asp 1085 1090 1095 Val Leu Thr Leu Ser Ser Ser Thr Glu Ser Glu Gly Glu Ser Gly 1100 1105 1110 Thr Ser Arg Lys Pro Thr Ala Gly Gln Thr Ser Ala Thr Ala Val 1115 1120 1125 Asp Ser Asp Asp Ile Gln Thr Ile Ser Ser Gly Ser Glu Gly Asp 1130 1135 1140 Asp Phe Glu Asp Lys Lys Asn Met Thr Gly Pro Met Lys Arg Gln 1145 1150 1155 Val Ala Val Lys Ser Thr Arg Gly Phe Ala Leu Lys Ser Thr His 1160 1165 1170 Gly Ile Ala Ile Lys Ser Thr Asn Met Ala Ser Val Asp Lys Gly 1175 1180 1185 Glu Ser Ala Pro Val Arg Lys Asn Thr Arg Gln Phe Tyr Asp Gly 1190 1195 1200 Glu Glu Ser Cys Tyr Ile Ile Asp Ala Lys Leu Glu Gly Asn Leu 1205 1210 1215 Gly Arg Tyr Leu Asn His Ser Cys Ser Pro Asn Leu Phe Val Gln 1220 1225 1230 Asn Val Phe Val Asp Thr His Asp Leu Arg Phe Pro Trp Val Ala 1235 1240 1245 Phe Phe Ala Ser Lys Arg Ile Arg Ala Gly Thr Glu Leu Thr Trp 1250 1255 1260 Asp Tyr Asn Tyr Glu Val Gly Ser Val Glu Gly Lys Glu Leu Leu 1265 1270 1275 Cys Cys Cys Gly Ala Ile Glu Cys Arg Gly Arg Leu Leu 1280 1285 1290 <210> 13 <211> 565 <212> PRT <213> Homo sapiens <400> 13 Val Gly Cys Asp Cys Lys Asp Gly Cys Arg Asp Lys Ser Lys Cys Ala 1 5 10 15 Cys His Gln Leu Thr Ile Gln Ala Thr Ala Cys Thr Pro Gly Gly Gln 20 25 30 Ile Asn Pro Asn Ser Gly Tyr Gln Tyr Lys Arg Leu Glu Glu Cys Leu 35 40 45 Pro Thr Gly Val Tyr Glu Cys Asn Lys Arg Cys Lys Cys Asp Pro Asn 50 55 60 Met Cys Thr Asn Arg Leu Val Gln His Gly Leu Gln Val Arg Leu Gln 65 70 75 80 Leu Phe Lys Thr Gln Asn Lys Gly Trp Gly Ile Arg Cys Leu Asp Asp 85 90 95 Ile Ala Lys Gly Ser Phe Val Cys Ile Tyr Ala Gly Lys Ile Leu Thr 100 105 110 Asp Asp Phe Ala Asp Lys Glu Gly Leu Glu Met Gly Asp Glu Tyr Phe 115 120 125 Ala Asn Leu Asp His Ile Glu Ser Val Glu Asn Phe Lys Glu Gly Tyr 130 135 140 Glu Ser Asp Ala Pro Cys Ser Ser Asp Ser Ser Gly Val Asp Leu Lys 145 150 155 160 Asp Gln Glu Asp Gly Asn Ser Gly Thr Glu Asp Pro Glu Glu Ser Asn 165 170 175 Asp Asp Ser Ser Asp Asp Asn Phe Cys Lys Asp Glu Asp Phe Ser Thr 180 185 190 Ser Ser Val Trp Arg Ser Tyr Ala Thr Arg Arg Gln Thr Arg Gly Gln 195 200 205 Lys Glu Asn Gly Leu Ser Glu Thr Thr Ser Lys Asp Ser His Pro Pro 210 215 220 Asp Leu Gly Pro Pro His Ile Pro Val Pro Pro Ser Ile Pro Val Gly 225 230 235 240 Gly Cys Asn Pro Pro Ser Ser Glu Glu Thr Pro Lys Asn Lys Val Ala 245 250 255 Ser Trp Leu Ser Cys Asn Ser Val Ser Glu Gly Gly Phe Ala Asp Ser 260 265 270 Asp Ser His Ser Ser Phe Lys Thr Asn Glu Gly Gly Glu Gly Arg Ala 275 280 285 Gly Gly Ser Arg Met Glu Ala Glu Lys Ala Ser Thr Ser Gly Leu Gly 290 295 300 Ile Lys Asp Glu Gly Asp Ile Lys Gln Ala Lys Lys Glu Asp Thr Asp 305 310 315 320 Asp Arg Asn Lys Met Ser Val Val Thr Glu Ser Ser Arg Asn Tyr Gly 325 330 335 Tyr Asn Pro Ser Pro Val Lys Pro Glu Gly Leu Arg Arg Pro Pro Ser 340 345 350 Lys Thr Ser Met His Gln Ser Arg Arg Leu Met Ala Ser Ala Gln Ser 355 360 365 Asn Pro Asp Asp Val Leu Thr Leu Ser Ser Ser Thr Glu Ser Glu Gly 370 375 380 Glu Ser Gly Thr Ser Arg Lys Pro Thr Ala Gly Gln Thr Ser Ala Thr 385 390 395 400 Ala Val Asp Ser Asp Asp Ile Gln Thr Ile Ser Ser Gly Ser Glu Gly 405 410 415 Asp Asp Phe Glu Asp Lys Lys Asn Met Thr Gly Pro Met Lys Arg Gln 420 425 430 Val Ala Val Lys Ser Thr Arg Gly Phe Ala Leu Lys Ser Thr His Gly 435 440 445 Ile Ala Ile Lys Ser Thr Asn Met Ala Ser Val Asp Lys Gly Glu Ser 450 455 460 Ala Pro Val Arg Lys Asn Thr Arg Gln Phe Tyr Asp Gly Glu Glu Ser 465 470 475 480 Cys Tyr Ile Ile Asp Ala Lys Leu Glu Gly Asn Leu Gly Arg Tyr Leu 485 490 495 Asn His Ser Cys Ser Pro Asn Leu Phe Val Gln Asn Val Phe Val Asp 500 505 510 Thr His Asp Leu Arg Phe Pro Trp Val Ala Phe Phe Ala Ser Lys Arg 515 520 525 Ile Arg Ala Gly Thr Glu Leu Thr Trp Asp Tyr Asn Tyr Glu Val Gly 530 535 540 Ser Val Glu Gly Lys Glu Leu Leu Cys Cys Cys Gly Ala Ile Glu Cys 545 550 555 560 Arg Gly Arg Leu Leu 565 <210> 14 <211> 826 <212> PRT <213> Artificial Sequence <220> <223> Transcription-activator like effector (TALE) domain <400> 14 Met Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Gly 1 5 10 15 Gly Met Ala Pro Lys Lys Lys Arg Lys Val Asp Gly Gly Val Asp Leu 20 25 30 Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys 35 40 45 Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly 50 55 60 Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu 65 70 75 80 Gly Thr Val Ala Val Lys Tyr Gln Asp Met Ile Ala Ala Leu Pro Glu 85 90 95 Ala Thr His Glu Ala Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala 100 105 110 Arg Ala Leu Glu Ala Leu Leu Thr Val Ala Gly Glu Leu Arg Gly Pro 115 120 125 Pro Leu Gln Leu Asp Thr Gly Gln Leu Leu Lys Ile Ala Lys Arg Gly 130 135 140 Gly Val Thr Ala Val Glu Ala Val His Ala Trp Arg Asn Ala Leu Thr 145 150 155 160 Gly Ala Pro Leu Asn Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser 165 170 175 Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro 180 185 190 Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile 195 200 205 Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu 210 215 220 Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val 225 230 235 240 Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln 245 250 255 Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln 260 265 270 Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr 275 280 285 Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro 290 295 300 Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu 305 310 315 320 Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu 325 330 335 Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln 340 345 350 Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His 355 360 365 Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly 370 375 380 Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln 385 390 395 400 Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly 405 410 415 Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu 420 425 430 Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser 435 440 445 Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro 450 455 460 Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile 465 470 475 480 Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu 485 490 495 Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val 500 505 510 Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln 515 520 525 Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln 530 535 540 Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr 545 550 555 560 Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro 565 570 575 Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu 580 585 590 Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu 595 600 605 Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln 610 615 620 Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His 625 630 635 640 Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly 645 650 655 Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln 660 665 670 Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile 675 680 685 Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu 690 695 700 Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser 705 710 715 720 His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro 725 730 735 Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile 740 745 750 Ala Ser Asn Gly Gly Gly Arg Pro Ala Leu Glu Ser Ile Val Ala Gln 755 760 765 Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His Leu 770 775 780 Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Leu Asp Ala Val Lys 785 790 795 800 Lys Gly Leu Pro His Ala Pro Ala Leu Ile Lys Arg Thr Asn Arg Arg 805 810 815 Ile Pro Glu Arg Thr Ser His Arg Val Ala 820 825 <210> 15 <211> 826 <212> PRT <213> Artificial Sequence <220> <223> TALE domain <400> 15 Met Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Gly 1 5 10 15 Gly Met Ala Pro Lys Lys Lys Arg Lys Val Asp Gly Gly Val Asp Leu 20 25 30 Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys 35 40 45 Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly 50 55 60 Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu 65 70 75 80 Gly Thr Val Ala Val Lys Tyr Gln Asp Met Ile Ala Ala Leu Pro Glu 85 90 95 Ala Thr His Glu Ala Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala 100 105 110 Arg Ala Leu Glu Ala Leu Leu Thr Val Ala Gly Glu Leu Arg Gly Pro 115 120 125 Pro Leu Gln Leu Asp Thr Gly Gln Leu Leu Lys Ile Ala Lys Arg Gly 130 135 140 Gly Val Thr Ala Val Glu Ala Val His Ala Trp Arg Asn Ala Leu Thr 145 150 155 160 Gly Ala Pro Leu Asn Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser 165 170 175 Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro 180 185 190 Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile 195 200 205 Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu 210 215 220 Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val 225 230 235 240 Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln 245 250 255 Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln 260 265 270 Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr 275 280 285 Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro 290 295 300 Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu 305 310 315 320 Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu 325 330 335 Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln 340 345 350 Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His 355 360 365 Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly 370 375 380 Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln 385 390 395 400 Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Asn 405 410 415 Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu 420 425 430 Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser 435 440 445 Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro 450 455 460 Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile 465 470 475 480 Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu 485 490 495 Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val 500 505 510 Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln 515 520 525 Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln 530 535 540 Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr 545 550 555 560 Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro 565 570 575 Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu 580 585 590 Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu 595 600 605 Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln 610 615 620 Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His 625 630 635 640 Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly 645 650 655 Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln 660 665 670 Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Asn 675 680 685 Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu 690 695 700 Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser 705 710 715 720 Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro 725 730 735 Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile 740 745 750 Ala Ser Asn Gly Gly Gly Arg Pro Ala Leu Glu Ser Ile Val Ala Gln 755 760 765 Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His Leu 770 775 780 Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Leu Asp Ala Val Lys 785 790 795 800 Lys Gly Leu Pro His Ala Pro Ala Leu Ile Lys Arg Thr Asn Arg Arg 805 810 815 Ile Pro Glu Arg Thr Ser His Arg Val Ala 820 825 <210> 16 <211> 831 <212> PRT <213> Artificial Sequence <220> <223> TALE domain <400> 16 Met Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Gly 1 5 10 15 Gly Met Ala Pro Lys Lys Lys Arg Lys Val Asp Gly Gly Val Asp Leu 20 25 30 Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys 35 40 45 Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly 50 55 60 Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu 65 70 75 80 Gly Thr Val Ala Val Lys Tyr Gln Asp Met Ile Ala Ala Leu Pro Glu 85 90 95 Ala Thr His Glu Ala Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala 100 105 110 Arg Ala Leu Glu Ala Leu Leu Thr Val Ala Gly Glu Leu Arg Gly Pro 115 120 125 Pro Leu Gln Leu Asp Thr Gly Gln Leu Leu Lys Ile Ala Lys Arg Gly 130 135 140 Gly Val Thr Ala Val Glu Ala Val His Ala Trp Arg Asn Ala Leu Thr 145 150 155 160 Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser 165 170 175 His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro 180 185 190 Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile 195 200 205 Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu 210 215 220 Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val 225 230 235 240 Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln 245 250 255 Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln 260 265 270 Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr 275 280 285 Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro 290 295 300 Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu 305 310 315 320 Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu 325 330 335 Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln 340 345 350 Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His 355 360 365 Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly 370 375 380 Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln 385 390 395 400 Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile 405 410 415 Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu 420 425 430 Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser 435 440 445 His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro 450 455 460 Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile 465 470 475 480 Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu 485 490 495 Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val 500 505 510 Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln 515 520 525 Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln 530 535 540 Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr 545 550 555 560 Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro 565 570 575 Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu 580 585 590 Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu 595 600 605 Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln 610 615 620 Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His 625 630 635 640 Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly 645 650 655 Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln 660 665 670 Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn His 675 680 685 Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu 690 695 700 Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser 705 710 715 720 His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro 725 730 735 Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile 740 745 750 Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Ser Ile Val Ala Gln 755 760 765 Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His Leu 770 775 780 Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Leu Asp Ala Val Lys 785 790 795 800 Lys Gly Leu Pro His Ala Pro Ala Leu Ile Lys Arg Thr Asn Arg Arg 805 810 815 Ile Pro Glu Arg Thr Ser His Arg Val Ala Gly Ser Gly Gly Gly 820 825 830 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Binding site <400> 17 tctctcctac cctcccgct 19 <210> 18 <211> 831 <212> PRT <213> Artificial Sequence <220> <223> TALE domain <400> 18 Met Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Gly 1 5 10 15 Gly Met Ala Pro Lys Lys Lys Arg Lys Val Asp Gly Gly Val Asp Leu 20 25 30 Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys 35 40 45 Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly 50 55 60 Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu 65 70 75 80 Gly Thr Val Ala Val Lys Tyr Gln Asp Met Ile Ala Ala Leu Pro Glu 85 90 95 Ala Thr His Glu Ala Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala 100 105 110 Arg Ala Leu Glu Ala Leu Leu Thr Val Ala Gly Glu Leu Arg Gly Pro 115 120 125 Pro Leu Gln Leu Asp Thr Gly Gln Leu Leu Lys Ile Ala Lys Arg Gly 130 135 140 Gly Val Thr Ala Val Glu Ala Val His Ala Trp Arg Asn Ala Leu Thr 145 150 155 160 Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser 165 170 175 Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro 180 185 190 Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile 195 200 205 Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu 210 215 220 Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val 225 230 235 240 Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln 245 250 255 Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln 260 265 270 Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr 275 280 285 Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro 290 295 300 Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu 305 310 315 320 Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu 325 330 335 Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln 340 345 350 Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His 355 360 365 Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly 370 375 380 Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln 385 390 395 400 Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp 405 410 415 Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu 420 425 430 Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser 435 440 445 His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro 450 455 460 Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile 465 470 475 480 Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu 485 490 495 Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val 500 505 510 Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln 515 520 525 Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln 530 535 540 Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr 545 550 555 560 Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro 565 570 575 Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu 580 585 590 Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu 595 600 605 Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln 610 615 620 Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His 625 630 635 640 Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly 645 650 655 Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln 660 665 670 Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly 675 680 685 Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu 690 695 700 Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser 705 710 715 720 His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro 725 730 735 Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile 740 745 750 Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Ser Ile Val Ala Gln 755 760 765 Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His Leu 770 775 780 Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Leu Asp Ala Val Lys 785 790 795 800 Lys Gly Leu Pro His Ala Pro Ala Leu Ile Lys Arg Thr Asn Arg Arg 805 810 815 Ile Pro Glu Arg Thr Ser His Arg Val Ala Gly Ser Gly Gly Gly 820 825 830 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Binding site <400> 19 tggtccttcc tctcccgct 19 <210> 20 <211> 831 <212> PRT <213> Artificial Sequence <220> <223> TALE domain <400> 20 Met Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Gly 1 5 10 15 Gly Met Ala Pro Lys Lys Lys Arg Lys Val Asp Gly Gly Val Asp Leu 20 25 30 Arg Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys 35 40 45 Val Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly 50 55 60 Phe Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu 65 70 75 80 Gly Thr Val Ala Val Lys Tyr Gln Asp Met Ile Ala Ala Leu Pro Glu 85 90 95 Ala Thr His Glu Ala Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala 100 105 110 Arg Ala Leu Glu Ala Leu Leu Thr Val Ala Gly Glu Leu Arg Gly Pro 115 120 125 Pro Leu Gln Leu Asp Thr Gly Gln Leu Leu Lys Ile Ala Lys Arg Gly 130 135 140 Gly Val Thr Ala Val Glu Ala Val His Ala Trp Arg Asn Ala Leu Thr 145 150 155 160 Gly Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser 165 170 175 His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro 180 185 190 Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile 195 200 205 Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu 210 215 220 Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val 225 230 235 240 Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln 245 250 255 Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln 260 265 270 Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr 275 280 285 Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro 290 295 300 Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu 305 310 315 320 Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu 325 330 335 Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln 340 345 350 Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His 355 360 365 Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly 370 375 380 Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln 385 390 395 400 Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Gly 405 410 415 Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu 420 425 430 Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser 435 440 445 Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro 450 455 460 Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile 465 470 475 480 Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu 485 490 495 Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val 500 505 510 Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln 515 520 525 Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln 530 535 540 Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr 545 550 555 560 Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro 565 570 575 Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu 580 585 590 Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu 595 600 605 Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln 610 615 620 Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His 625 630 635 640 Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly 645 650 655 Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln 660 665 670 Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp 675 680 685 Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu 690 695 700 Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser 705 710 715 720 Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro 725 730 735 Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile 740 745 750 Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Ser Ile Val Ala Gln 755 760 765 Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His Leu 770 775 780 Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Leu Asp Ala Val Lys 785 790 795 800 Lys Gly Leu Pro His Ala Pro Ala Leu Ile Lys Arg Thr Asn Arg Arg 805 810 815 Ile Pro Glu Arg Thr Ser His Arg Val Ala Gly Ser Gly Gly Gly 820 825 830 <210> 21 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Binding site <400> 21 tcgctccgtg acttccctt 19 <210> 22 <211> 1441 <212> PRT <213> Artificial Sequence <220> <223> Artificial transcription repressor (ATR) sequence, catalytically inactive Cas9 <400> 22 Met Gly Gly Arg Arg Val Arg Trp Glu Val Tyr Ile Ser Arg Ala Leu 1 5 10 15 Trp Leu Thr Arg Glu Pro Thr Ala Tyr Trp Leu Ile Glu Ile Asn Thr 20 25 30 Thr His Tyr Arg Glu Thr Gln Ala Thr Gly Ala Thr Met Tyr Pro Tyr 35 40 45 Asp Val Pro Asp Tyr Ala Ser Pro Lys Lys Lys Arg Lys Val Glu Ala 50 55 60 Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val 65 70 75 80 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 85 90 95 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 100 105 110 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 115 120 125 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 130 135 140 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 145 150 155 160 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 165 170 175 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 180 185 190 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 195 200 205 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 210 215 220 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 225 230 235 240 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 245 250 255 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 260 265 270 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 275 280 285 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 290 295 300 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 305 310 315 320 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 325 330 335 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 340 345 350 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 355 360 365 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 370 375 380 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 385 390 395 400 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 405 410 415 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 420 425 430 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 435 440 445 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 450 455 460 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 465 470 475 480 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 485 490 495 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 500 505 510 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 515 520 525 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 530 535 540 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 545 550 555 560 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 565 570 575 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 580 585 590 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 595 600 605 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 610 615 620 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 625 630 635 640 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 645 650 655 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 660 665 670 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 675 680 685 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 690 695 700 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 705 710 715 720 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 725 730 735 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 740 745 750 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 755 760 765 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 770 775 780 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 785 790 795 800 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 805 810 815 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 820 825 830 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 835 840 845 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 850 855 860 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 865 870 875 880 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 885 890 895 Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys 900 905 910 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 915 920 925 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 930 935 940 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 945 950 955 960 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 965 970 975 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 980 985 990 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 995 1000 1005 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys 1010 1015 1020 Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys 1025 1030 1035 Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu 1040 1045 1050 Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu 1055 1060 1065 Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg 1070 1075 1080 Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala 1085 1090 1095 Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu 1100 1105 1110 Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu 1115 1120 1125 Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp 1130 1135 1140 Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile 1145 1150 1155 Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser 1160 1165 1170 Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys 1175 1180 1185 Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val 1190 1195 1200 Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser 1205 1210 1215 Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met 1220 1225 1230 Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala 1235 1240 1245 Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro 1250 1255 1260 Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu 1265 1270 1275 Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro 1280 1285 1290 Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys 1295 1300 1305 Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val 1310 1315 1320 Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser 1325 1330 1335 Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys 1340 1345 1350 Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu 1355 1360 1365 Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly 1370 1375 1380 Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys 1385 1390 1395 Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His 1400 1405 1410 Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln 1415 1420 1425 Leu Gly Gly Asp Ser Pro Lys Lys Lys Arg Lys Val Gly 1430 1435 1440 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Genomic target site for guide RNA (gRNA) #1 for beta2-microglobulin gene <400> 23 tataagtgga ggcgtcgcgc 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Genomic target site for gRNA #2 for beta2-microglobulin gene <400> 24 gcccgaatgc tgtcagcttc 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Genomic target site for gRNA #3 for beta2-microglobulin gene <400> 25 tgcgtcgctg gcttggagac 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Genomic target site for gRNA #4 for beta2-microglobulin gene <400> 26 ccaatcagga caaggcccgc 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Genomic target site for gRNA #5 for beta2-microglobulin gene <400> 27 agggtaggag agactcacgc 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Genomic target site for gRNA #6 for beta2-microglobulin gene <400> 28 gcgggccacc aaggagaact 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Genomic target site for gRNA #7 for beta2-microglobulin gene <400> 29 gctactctct ctttctggcc 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Genomic target site for gRNA #8 for beta2-microglobulin gene <400> 30 ctcccgctct gcaccctctg 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Genomic target site for gRNA #9 for beta2-microglobulin gene <400> 31 tttggcctac ggcgacggga 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Genomic target site for gRNA #10 for beta2-microglobulin gene <400> 32 ggggcaagta gcgcgcgtcc 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Genomic target site for gRNA #11 for beta2-microglobulin gene <400> 33 tagtccaggg ctggatctcg 20 <210> 34 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Binding site <400> 34 tacccagatt ggccccact 19 <210> 35 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Binding site <400> 35 tacctagagg agaaaggtt 19 <210> 36 <211> 846 <212> PRT <213> Homo sapiens <400> 36 Met Val Ala Glu Leu Ile Ser Glu Glu Asp Leu Glu...

Claims

1. An artificial transcriptional repressor (ATR) comprising a DNA-binding domain operably linked to two or more domains selected from group (a), (b), or (c), the two or more domains include (a) and (b), or (a) and (c); (a) is a Kruppel-associated box (KRAB) domain or a homologous gene thereof; (b) is a DNA methyltransferase 3A (DNMT3A), DNA methyltransferase 3B (DNMT3B), or DNA methyltransferase 1 (DNMT1) domain or a homologous gene thereof; (c) is a DNA (cytosine-5)-methyltransferase 3-like (DNMT3L) domain or a homologous gene thereof; Optionally, the DNA binding domain comprises a zinc finger domain or a CRISPR / Cas system. ATR.

2. The ATR of claim 1 , wherein the DNA binding domain comprises a zinc finger domain.

3. The ATR of claim 1, wherein the DNA binding domain comprises a CRISPR / Cas system.

4. The ATR of claim 3, wherein the CRISPR / Cas system comprises Cas9.

5. The ATR of claim 4, wherein Cas9 is derived from S. pyogenes.

6. The ATR of claim 4, wherein Cas9 has catalytic activity.

7. The ATR of claim 4, wherein Cas9 is catalytically inactive.

8. A polynucleotide encoding the ATR described in claim 3.

9. A polynucleotide encoding the ATR described in claim 4.

10. A polynucleotide encoding the ATR described in claim 5.

11. A polynucleotide encoding the ATR described in claim 6.

12. A polynucleotide encoding the ATR described in claim 7.

13. A composition for use in a method for silencing a target gene, the composition comprising an ATR according to any one of claims 1 to 7, the method comprising administering the ATR to a cell.

14. The ATR according to any one of claims 1 to 7 for use in gene therapy.

15. 13. A composition for use in a method for silencing a target gene, said composition comprising a polynucleotide according to any one of claims 8 to 12, said method comprising administering said polynucleotide to a cell.

16. A polynucleotide according to any one of claims 8 to 12 for use in gene therapy.

17. A cell comprising the ATR according to any one of claims 1 to 7.

18. A cell that is a descendant of the cell of claim 17.

19. The ATR of any one of claims 1 to 7, comprising a DNA binding domain operably linked to a KRAB domain or a homolog thereof, a DNTM3A domain or a homolog thereof, and a DNMT3L domain or a homolog thereof.

20. The ATR of any one of claims 1 to 7, comprising a DNA binding domain operably linked to a KRAB domain or a homolog thereof, and a DNMT3L domain or a homolog thereof.

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