Sustained epigenetic gene silencing

Abstract

Products are provided for silencing a gene of interest or for editing the epigenetic state of a genetic element of interest. [Solution] A product comprising two or more ATRs selected from (a) an artificial transcriptional repressor (ATR) comprising a DNA-binding domain operably linked to a KRAB domain or its homolog; (b) an ATR comprising a DNA-binding domain operably linked to a DNMT3A, DNMT3B or DNMT1 domain or its homolog; (c) an ATR comprising a DNA-binding domain operably linked to a DNMT3L domain or its homolog; and (d) an ATR comprising a DNA-binding domain operably linked to a SETDB1 domain or its homolog, or polynucleotides encoding them, wherein at least two of the ATRs are selected from different groups (a), (b), (c) or (d).

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 for 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] The 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. Classic examples include Huntington's disease, most types of spinocerebellar ataxias, and some collagen disorders. Furthermore, gene silencing has emerged as a promising strategy to treat 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 (CC 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 obvious 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 been recently proposed that hemoglobinopathies, the most common inherited recessive disorders of the hematopoietic system and prime targets 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 detailed 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 affected by 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 it maintains 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 techniques are used to silence gene expression: RNA interference (RNAi; Davidson, BL et al. (2011) Nat. Rev. Genet. 12: 329-40), which uses single short hairpin RNAs (shRNAs), and gene targeting using artificial nucleases (AN; Carroll, D. (2014) Annu. Rev. Biochem. 83: 409-39). RNAi exploits 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 the AN target gene (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 the low efficiency with which they produce homozygous disruptions in diploid mammalian cells can compromise the efficacy of these therapies. These shortcomings are particularly important in applications where residual levels of gene activity are sufficient for biological function.

[0008] Furthermore, the safe use of these techniques requires overcoming the issues of a) off-target (non-specific) gene silencing, b) altering the transcriptional profile of cells by inhibiting endogenous miRNA pathways, and c) overactivating DNA damage response to trigger apoptosis or alter cell cycle progression (Ciccia, A. et al. (2010) Mol. Cell 40: 179-204). In addition, RNAi and AN 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 the function of the genome without modifying 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; HPTM). Alternatively, they may constitute long-term instructions (e.g., DNA methylation; Smith, ZD et al. (2013) Nat. Rev. Genet. 14: 204-20) that stably contribute to cellular identity and memory. Current research is elucidating the composition and function of molecular complexes that are recruited to chromatin to induce epigenetic repressive states and the mechanisms by which these states propagate indefinitely 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 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 have not demonstrated 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 repressive state at ATR target sites.

[0011] It has also been shown that silencing induced by artificial Kruppel-associated box (KRAB) system 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; [Non-licensed document 2] Kay, MA (2011) Nat. Rev. Genet. 12: 316-28; [Non-licensed document 3] Biffi, A.(2013) Science 341: 1233158;

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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 a robust and heritable state of transcriptional suppression of desired target genes.Importantly, this allows the persistent (permanent) inactivation of genes of therapeutic interest (e.g., disease-causing) or biotechnological interest.

[0015] Due to the above challenges regarding 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 fulfill all of the following criteria: 1. It functions through a combinatorial collection of two or more different effector modules; 2. Establish a robust and persistent 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, since the activity of each ATR at off-target sites would be transient, if not absent.

[0017] In one embodiment, the present invention relates to a compound of group (a), (b), (c) or (d): (a) An artificial transcriptional repressor (ATR) that contains a DNA-binding domain operably linked to a KRAB domain or its homologue; (b) an ATR that contains a DNA-binding domain operably linked to a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof; (c) an ATR that includes a DNA-binding domain operably linked to a DNMT3L domain or a homolog thereof; and (d) an ATR that contains a DNA-binding domain operably linked to a SETDB1 domain or a homolog thereof; The present invention provides a product (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), (c) or (d).

[0018] In another embodiment, the present invention provides a method for treating a pulmonary artery disease comprising administering to a patient in group (a), (b) or (c): (a) An artificial transcriptional repressor (ATR) that contains a DNA-binding domain operably linked to a KRAB domain or its homologue; (b) an ATR that includes 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 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, the product of the invention comprises ATR(a) and (b) or polynucleotides encoding them. In another embodiment, the product of the invention comprises ATR(a) and (c) or polynucleotides encoding them. In another embodiment, the product of the invention comprises ATR(b) and (c) or polynucleotides encoding them. In a preferred embodiment, the product of the invention comprises ATR(a), (b) and (c) or polynucleotides encoding them. In another embodiment, the product of the invention comprises ATR(a), (b) and (d) or polynucleotides encoding them. In another embodiment, the product of the invention comprises ATR(b) and (d) or polynucleotides encoding them. In another embodiment, the product of the invention comprises ATR(c) and (d) or polynucleotides encoding them. In another embodiment, the product of the invention comprises ATR(b), (c) and (d) or polynucleotides encoding them.

[0020] A KRAB 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: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 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: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 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: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., a TALE DNA binding domain or a CRISPR / Cas system, may 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 of a target gene (eg, a promoter or enhancer sequence).

[0029] The DNA binding domain can bind to a binding site within the splice site. Splice variants of a given gene can be regulated by DNA methylation / demethylation at the splice site. These modifications can then lead to exon exclusion / inclusion in the mature transcript. This exclusion / inclusion can be of therapeutic importance, for example in the case of Duchenne muscular dystrophy. In Duchenne muscular dystrophy, exclusion (by genetic ablation or exon skipping) of exons containing the most frequent disease-causing mutations from the mature mRNA 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 ATR of the present invention may also target genetic elements (e.g., sequences that control the topological arrangement, stability and replication of the genome, e.g., insulators, laminin-associated domains, telomeres and centromeric regions), repetitive elements or mobile elements that may or may not be actively transcribed. 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 product of the present invention and ATR for epigenetic editing of regulatory DNA elements (e.g., those described herein). Epigenetic editing of a target gene or of a genetic element may also be associated with its transcriptional activation or activity, respectively. The present invention may also include the use of the product of the present invention and ATR 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 included in a single vector; the polynucleotides encoding (a), (b) and (c) can be included in separate vectors; the polynucleotides encoding (a) and (b) can be included in a single vector and the polynucleotide encoding (c) can be included in a separate vector; the polynucleotides encoding (a) and (c) can be included in a single vector and the polynucleotide encoding (b) can be included in a separate vector; or the polynucleotides encoding (b) and (c) can be included in a single vector and the polynucleotide encoding (a) can be included in a separate vector.

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

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

[0039] The vector may 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 pharma- ceutically acceptable carrier, diluent or excipient.

[0042] In one embodiment, a 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 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.The use can be, for example, in vitro or ex vivo.For example, the target gene can be silenced in a population of cells (for example, cell line or primary cell) to enhance the production of a substance (for example, a biotherapeutic substance) by the cell or to give the cell a growth advantage.Alternatively, for example, the target gene can be silenced to create a knockout animal for the target gene.The epigenetic approach of the present invention provides an alternative to existing methods of knocking out genes (for example, those that utilize homologous recombination).Alternatively, for example, the target gene can be silenced in plant cells.

[0050] In the above uses, including therapeutic uses, the 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 by expression of two or more ATRs in a cell, for example, from a polynucleotide encoding an ATR. The 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 effect on splice sites. The delivery of two or more ATRs of the present invention to a cell can also allow silencing and / or editing of genetic elements as 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 expression.

[0052] In one embodiment, the delivery of two or more ATRs of the present invention to a cell (e.g., by expression in the cell) causes persistent (permanent) silencing of a target gene.In another embodiment, the delivery of two or more ATRs of the present invention to a cell (e.g., by expression in the cell) causes persistent (permanent) silencing of 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 the cell resulting from differentiation of the stem cell).

[0053] For example, the cells can be of animal (e.g., mammalian, e.g., human), fungal (e.g., yeast) or plant origin. 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 spacing (separation distance) of the binding sites selected for binding to the DNA binding domains of different ATRs is not particularly limited in size. For example, the DNA binding domains of different ATRs can be selected to bind to binding sites separated by about 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. In one embodiment, the DNA binding domains of different ATRs can be selected to bind to binding sites separated by 1 to 30 bp. Preferably, the DNA binding domains of different ATRs can be selected to bind to binding sites separated by about 15 to 25 bp. For example, the DNA binding domains of different ATRs can be selected to bind to binding sites separated by 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.

[0055] The DNA binding domains of different ATRs may also be selected to bind to the same binding site. For example, the DNA binding domains of different ATRs may be selected to bind to binding sites that are 0 bp apart. Thus, for example, the DNA binding domains of different ATRs may be selected to bind to binding sites that are about 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 may be selected to bind to binding sites that are about 0-15 or 15-25 bp apart.

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

[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 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 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 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 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 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 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 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 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 a polynucleotide encoding the same, for use in therapy.

[0063] In another aspect, the 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 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 a polynucleotide encoding the same, for use in therapy.

[0064] In another aspect, the 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 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 a polynucleotide encoding the same, for use in therapy.

[0065] In another aspect, the 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 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 a polynucleotide encoding the same, for use in therapy.

[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 a polynucleotide 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 a cell that is a descendant of a cell that comprises two or more artificial transcriptional repressors (ATRs) of the invention. In one embodiment, the descendant cell no longer comprises two or more ATRs of the invention. In another aspect, the invention provides a cell 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 method for the treatment of a pulmonary artery disease comprising administering to a patient in group (a), (b) or (c): (a) An artificial transcriptional repressor (ATR) that contains a DNA-binding domain operably linked to a KRAB domain or its homologue; (b) an ATR that includes 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 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 following, wherein at least two of the ATRs are selected from different groups (a), (b) or (c). The present invention also provides a method of gene therapy comprising administering to a subject two or more artificial transcriptional repressors (ATRs) or polynucleotides encoding the same selected from the following groups (a), (b), (c) or (d): (a) An artificial transcriptional repressor (ATR) that contains a DNA-binding domain operably linked to a KRAB domain or its homologue; (b) an ATR that contains a DNA-binding domain operably linked to a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof; (c) an ATR that includes a DNA-binding domain operably linked to a DNMT3L domain or a homolog thereof; and (d) an ATR that contains a DNA-binding domain operably linked to a SETDB1 domain or a homolog thereof; 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 following, 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 method for the treatment of a pulmonary artery disease comprising administering to a patient in group (a), (b) or (c): (a) An artificial transcriptional repressor (ATR) that contains a DNA-binding domain operably linked to a KRAB domain or its homologue; (b) an ATR that includes 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 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) An artificial transcriptional repressor (ATR) that contains a DNA-binding domain operably linked to a KRAB domain or its homologue; (b) an ATR that contains a DNA-binding domain operably linked to a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof; (c) an ATR that includes a DNA-binding domain operably linked to a DNMT3L domain or a homolog thereof; and (d) an ATR that contains a DNA-binding domain operably linked to a SETDB1 domain or a homolog thereof; The present invention 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), (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 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, uses of the product in therapy, cells and their progeny comprising the product, 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 an ATR comprising a DNA binding domain operably linked to 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 same, wherein the ATR is selected from the 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), where 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 of group (a), a domain of group (b), and a domain of group (c). The present invention also relates to a DNA binding domain operably linked to a domain of group (a), (b), (c), or (d): (a) KRAB domain or its homologues; (b) a DNMT3A, DNMT3B or DNMT1 domain or a homolog 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 consisting of: (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 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 the 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 Each ATR comprises a DNA binding domain operably linked to two or more domains selected from group (a), (b), (c), where 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 of group (a), a domain of group (b), and a domain of group (c). The present invention also provides a product comprising two or more different artificial transcriptional repressors (ATRs) or polynucleotides encoding same, where the two or more different ATRs are selected from groups (a), (b), (c), or (d): (a) KRAB domain or its homologues; (b) a DNMT3A, DNMT3B or DNMT1 domain or a homolog 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 embodiment, the present invention provides a product comprising only one ATR and another effector protein not operably linked to a 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 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 as 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. Preferably, the other effector protein is selected such that it does not contain a domain belonging to the same group (a), (b) or (c) as the effector domain constituting the ATR.

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

[0087] In another embodiment, the present invention provides a product comprising only one ATR and another effector protein not operably linked to a DNA binding domain, or a polynucleotide encoding the same. The ATR may comprise a DNA binding domain operably linked to a SETDB1 effector domain or a homologue 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 homologue thereof. The other effector protein may be an effector domain / protein as 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 another effector protein can be in the form of a single vector or can be contained in separate vectors.

[0091] The vector may 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 the transient expression of ATR and / or another effector protein in the cell.

[0092] The ATR and / or another effector protein of the 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 pharma- ceutically 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 a polynucleotide 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 for simultaneous, sequential or separate administration to a subject.

[0096] In another embodiment, the present invention provides 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-mentioned use, including use in therapy, the 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 a transient delivery. The delivery can be by the expression of the ATR of the present invention, or the ATR of the present invention and another effector protein, in the cell, for example, by 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, the 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) will persistently silence the target gene.In another embodiment, the 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) will persistently silence the target gene in the descendants of the cell.For example, the cell can be a stem cell, and the target gene can be silenced in the descendants of the stem cell (e.g., the target gene can be silenced in the cell that is generated from the differentiation of the stem cell).

[0100] For example, the cells can be of animal (e.g., mammalian, e.g., human), fungal (e.g., yeast) or plant origin. 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 same, for use in therapy, in which the ATR is administered to a subject simultaneously, sequentially or separately in combination with a first separate 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 separate 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 same, for use in therapy, in which the ATR is administered to a subject simultaneously, sequentially or separately in combination with a first, separate effector protein that is not operably linked to a DNA binding domain comprising a KRAB domain or a homolog thereof, and / or a second, separate 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 same, for use in therapy, in which the ATR is administered to a subject simultaneously, sequentially or separately in combination with a first, separate effector protein that is not operably linked to a DNA binding domain comprising a KRAB domain or a homolog thereof, and / or a second, separate 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 that contains 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 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 invention provides a cell that is a progeny of a cell comprising the ATR of the invention or the ATR of the invention and another effector protein. In one embodiment, the progeny cell no longer comprises the ATR of the invention and / or another effector protein. In another aspect, the invention provides a cell of the 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 administering to a subject, simultaneously, sequentially or separately, only one ATR and another effector protein not functionally linked to a 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 functionally 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 as 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. Preferably, the other effector protein is selected such 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 embodiment, the present invention provides a kit comprising only one ATR and another effector protein not operably linked to a 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 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 as 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. Preferably, the other effector protein is selected such 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 the ATR of the present invention, or the 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) comprises a DNMT3A, DNMT3B or DNMT1 domain or a homolog thereof, the ATR or another effector protein of the invention may comprise a SETDB1 domain or a homolog thereof.

[0116] The method and use of the present invention, for example, gene therapy or target gene silencing method, can also include a step of inactivating endogenous genes 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 of this method step can be, for example, transient or permanent.The inactivation can be achieved, for example, by genetic deletion, for example, by using CRISPR / Cas9-based approaches, 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.The inactivation of DNMT3B will be particularly preferred when using three ATRs that individually contain KRAB, DNMT3A and DNMT3L domains. [Brief description 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. A vector expressing ATR (with a candidate repression (Rep) domain) is then introduced into single cell-derived clones containing homozygous insertion of the cassette, and 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 evaluated by measuring eGFP expression. [Diagram 2]Comparison of epigenetic silencing induced by tetR:K and tetR:D3A. A. Diagram of bidirectional lentiviral vector (Bid.LV) expressing tetR:K and marker gene mOrange (left) or tetR:DNMT3A and marker gene ΔLNGFR (right). B. TetO7.eGFP reporter clones were transduced with Bid.LV-tetR:K or Bid.LV-tetR:D3A in the presence or absence of doxycycline (right and left 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 transduced with Bid.LV-tetR:K or Bid.LV-tetR:D3A in the presence or absence of doxycycline at the indicated time points. 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 the absence of doxycycline in (B) were sorted to pure state and maintained in culture in the presence or absence of doxycycline to evaluate the reactivation of eGFP. Representative dot plots of the cells in the presence or absence of the drug are 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 (left histogram). Representative dot plots of vehicle and AZA treated cells are shown. [Diagram 3] TetR:D3A-induced transcriptional repression is localized to the target locus. A. Diagram of the AAVS1 site. Genes surrounding the reporter cassette (red arrows) 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. Relative expression levels of each gene were normalized to expression of B2M and expressed as fold change relative to untreated cells (control) (n=3). [Figure 4]Synergistic activity of tetR:K and tetR:D3A for their transient co-delivery. A. 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 after 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 sorted eGFP-silenced cells from the mixed conditions shown in (A) and untreated cells. The relative expression levels of each gene were normalized to B2M and expressed as fold change relative to untreated cells (standard) (n=3). c. eGFP-negative cells from the mixed treatment conditions in (A) were sorted and then treated with AZA or DMSO (histograms show the percentage of eGFP-positive cells 7 days after the indicated treatments; n=3). D. Experiments similar to (A) but 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 conditions shown in (D) (n=3). [Diagram 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 the study. The TetO7 sequence was cloned upstream of the hPGK promoter driving the expression of the eGFP reporter transgene. B. Graphs showing the kinetics of eGFP silencing (% of eGFP-negative cells by flow cytometry) in K562 LV / TET07 reporter cell lines transfected with in vitro transcribed mRNAs 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 U937LV / TETO7 cell lines (C) or B-lymphoblastoid LV / TETO7 reporter cell lines (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 (% of eGFP negative cells by flow cytometry) in K562 LV / TETO7 reporter cell lines upon transduction of lentiviral vectors expressing the indicated tetR-based ATR (n=3). B. Graph showing the percentage of cells positive for the indicated LV over time in culture (n=3). C. Graph showing the kinetics of eGFP silencing (% of eGFP negative cells by flow cytometry) in K562 LV / TETO7 reporter cell lines transfected with lentiviral vectors stably expressing the indicated tetR-based ATR 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 (% of 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 presented as mean ± SEM), or with a plasmid encoding tetR:D3A and one of the other tetR-based ATRs (B; n=3; data presented as mean ± SEM), or with a plasmid encoding tetR:K and one of the other tetR-based ATRs (C; n=3; data presented as mean ± SEM), or with a plasmid encoding tetR:D3A + tetR:K in combination with one of the other tetR-based ATRs (D; n=3; data presented as mean ± SEM). E. Histogram showing the potency 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 potency 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 of 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 mouse NIH / 3T3 LV / TETO7 reporter cell lines electroporated with mRNAs 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 (ATR) containing tailored DNA-binding domains (head-to-tail orientation). A. Schematic diagram of different artificial Tail (Tale) binding sites (head-to-tail).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 binding to the target. B. Graph showing silencing efficiency (% of eGFP-negative cells 34 days after electroporation) in relation to spacer length in K→D3A (left) and D3A→K (right) relative ATR order of binding to the target. 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 (ATR) containing tailored DNA-binding domains (head-to-head orientation). A. Schematic diagram of different artificial Tail (Tale) binding sites (head-to-head).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 are fused separately to each epigenetic effector resulting in two alternative co-delivery methods with different D3A-K relative order of binding to the target. B. Graph showing silencing efficiency (% of eGFP-negative cells 34 days after electroporation) in relation to spacer length in D3A→K (left) and K→D3A (right) relative ATR order of binding to the target. 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).hPGK.eGFP cassettes semi-randomly integrated into the genome of K562 cells by LV transduction, bound by 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 FIG. 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) by using different 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 TetO7 reporter LV (vector diagram in FIG. 5A). Then, on day 3 after thawing, cells were washed and electroporated with in vitro transcribed mRNA. The next 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 indicated in (B) (n=3; data presented as mean±SEM). [Figure 12]Sustained epigenetic silencing in human T lymphocytes by using different 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 introduced with reporter TetO.LV. On day 6, the cells were transfected with in vitro transcribed mRNAs encoding the indicated ATRs, and the expression of eGFP was analyzed by flow cytometry at the indicated time points. Three weeks after transfection, 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 system ATRs. B. Graph showing the kinetics of B2M silencing in HEK-293T cells electroporated with plasmids encoding the indicated TALE system 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 CRISPR / dCas9-based ATR plasmid (n=3; data are presented as mean ± SEM). G. B2M-silenced cells from Figure 2C (referred to as TALE B2M- in this panel), B2M-silenced cells sorted from the triple CRISPR / dCas9-based ATR combination in Figure 2F (referred to as TALE B2M- in this panel), and wild-type HeK-293T cells (referred to as WT B2M+ in this panel) were exposed or not to IFN-γ and then analyzed to measure the expression levels of B2M and OAS1. Histogram showing the fold change in expression levels of B2M and OAS1 genes between IFN-γ 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 plots) or after 4 days of IFN-γ treatment (right plots). 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 plasmids encoding the triple TALE:ATR combination, and the cell sorting method used to enrich for double positive and double negative cells. B. ChlP analysis performed in untreated cells (upper histogram) and silenced cells from (A) (lower histogram) in the presence of RNA PolII. The histograms show the fold enrichment in RNA PolII relative to input with respect 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 B2M CpG islands 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 Ct values ​​of 37 or higher were excluded from the analysis. The relative expression levels of each gene were normalized to HPRT and expressed as fold change relative to untreated cells (standard). [Figure 15]Silencing of beta2-microglobulin (B2M) is effective in another human cell line. A. Diagram of the CRISPR / Cas9-based gene targeting method used to insert a 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 (left panel and dot plot) after transfection with in vitro transcribed mRNA encoding the triple TALER:ATR combination (i) and 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 the 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 the binding of TALE-based ATR 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 the grey boxes. As already shown in Figure 13A, three different RDVs each equipped with another effector domain 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 lymphoblast LV-TetO7 reporter cell line 27 days after transfection with in vitro transcribed mRNAs encoding the indicated tetR-based ATRs delivered in the presence or absence of tetR:D3L or untargeted full-length DNMT3L-encoding mRNA (n=2; data 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, for each module, the relative order of the effector domains can be swapped. For example, for module 1, site A can be bound by KRAB-based ATR and site B can be bound by DNMT3A-based ATR, 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-targeted 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 (as shown in FIG. 13E) delivered alone or in combination with dCas9:D3L or non-targeted 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, whereas 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 upon doxycycline treatment (left) or to express the gRNA of interest (right). Representative flow cytometry analysis of bi) eGFP-positive K-562 cells transfected with the lentiviral vector described in FIG. 5A and sorted to near-pure for eGFP expression; ii) cell line from (i) transfected with LV encoding an 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); 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 by 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-lymphoblastoid 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. B-cell lymphoma / leukemia 11A (BCL11A) gene showing two transcript variants of this gene. The dashed boxes highlight gene regulatory elements. In particular, the gene promoter / enhancer region at the level of the transcription start site, with a cluster of four different CpG islands varying in size and number of CpG residues (yellow box), as well as an erythroid-specific enhancer (red box) that results in lineage-restricted expression of the gene within the gene in the second intron. To investigate the function of both the gene promoter and the erythroid-specific enhancer, a tdTomato transgene linked to the BCL11A transcript via the 2A autocatalytic peptide was targeted within the third exon of the gene. A representative dot plot of B-lymphoblast cells after sorting of tdTomato-positive cells is shown on the right. B. Histograms 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, 10 gRNAs for CpG115) (n=3; data are presented as mean ± SEM). Untreated cells or cells transfected with only the pool of gRNAs or only the dCas9-based ATR were used as controls. C. tdTomato reporter cell lines were co-transfected with plasmids encoding the dCas9-based ATR alone or in double or triple combinations and a plasmid with a pool of 9 gRNAs targeting CpG38 or a plasmid with a pool of 8 gRNAs against CpG31. Silencing efficiency was measured 2 weeks after transfection and is represented as a histogram (n=3; data are 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 orientation 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 shown on the x-axis of the histograms. Silencing efficiency is shown as the percentage of dTomato-negative cells. Analysis was performed 18 days after transfection of the plasmids (n=3; data are presented as mean ± SEM). E. Top: Diagram of the interferon (alpha, beta and omega) receptor 1 (IFNAR1) gene. The green box highlights the CpG islands at the level of the gene promoter / enhancer region (numbers of CpG residues are shown). Bottom: Histogram showing the fold change in expression levels of the IFNAR1 gene 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 levels of the IFNAR1 gene were normalized to the expression of DNMT1 and expressed as fold change relative to untreated cells (standard) (n=1). F. Top: Diagram of the vascular endothelial growth factor A (VEGFA) gene. The green box highlights the CpG island (numbers of CpG residues are indicated) at the level of the gene promoter / enhancer region. Bottom: Histogram showing the fold change in expression levels of the VEGFA gene 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. The relative expression levels of the VEGFA gene were normalized to the expression of DNMT1 and expressed as fold change relative to untreated cells (standard) (n=1).

[0118] Detailed Description of the Invention Preferred features and embodiments of the present invention will now be illustrated 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 are general texts incorporated herein by reference.

[0120] In one embodiment, the present invention relates to a method for the treatment of a cancer of the present invention comprising administering to a patient a cancer-related product selected from the group (a), (b) or (c): (a) an artificial transcriptional repressor (ATR) that contains 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; The present invention provides a product containing two or more ATRs, or polynucleotides encoding them, selected from the following: wherein at least two of the ATRs are selected from non-identical groups (distinct groups) of (a), (b) or (c).

[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 article of manufacture 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 transcriptional repression of the target gene. Alternatively or additionally, the effector domain can recruit other active agents in 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 such a way that they can perform their functions (e.g., binding to DNA, catalyzing a reaction, or recruiting additional active substances from within the cell) substantially unhindered. For example, the DNA-binding domain can be conjugated to the effector domain, for example, to form a fusion protein. Methods for conjugating polypeptides are known in the art, such as providing a linker protein that connects the polypeptides. Another method for conjugating polypeptides known in the art is chemical photoinduced conjugation (e.g., using chemical crosslinkers). The DNA-binding domain and the effector domain (e.g., KRAB domain, DNMT3A, DNMT3B or DNMT1 domain, or DNMT3L domain, or homologs thereof) of ATR preferably 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 the ATR that retains a certain function. A domain can be a separate domain (e.g., a catalytic domain) isolated from a naturally occurring protein, but can also be the complete full-length naturally occurring protein. In other words, a 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 the ATR that includes 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 the practice of 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) play an important role in silencing endogenous retroviruses. These transcription factors bind to specific ERV sequences via the DNA-binding domain of their ZFPs while recruiting KRAB Associated Protein 1 (KAP1) through its conserved KRAB domain. KAP1 further binds 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). At the same time, 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, as well as 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. There, DNA methylation pretargeted to ERVs by postimplantation 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 therapeutics based on the transient expression of ATRs in somatic cells, the KRAB-ZFP / KAP1 machinery is expected to be non-functional if used alone. On the other hand, we consider a preferred method to simultaneously introduce two different ATRs: one based, for example, on the KRAB domain, the initiator of the epigenetic cascade that occurs in ERVs in embryonic stem cells, and the other based, for example, on DNMT3A, the final lock of this process. This approach allows to recapitulate, on preselected target genes, the repressive chromatin state established in ERVs of preimplantation embryos, and then to be permanently inherited throughout mammalian development and adulthood.

[0130] The ATR of the present invention may, for example, contain 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 have 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 be similarly used in the ATR of the present invention. For example, the ATR 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 (Human DNMT1 catalytic domain; 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, for example, contain 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 ATR of the present invention contains, 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 may be encoded by a polynucleotide comprising a nucleic acid sequence encoding, for example, a 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] The ATR of the present invention contains, 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 may be encoded by a polynucleotide comprising a nucleic acid sequence encoding, for example, a 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 ATR of the present invention contains a DNA-binding domain that binds to a specific nucleic acid sequence, enabling 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] 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 the temporal control of tetR binding to the target nucleotide sequence, the tetracycline operon (TetO), by administration of doxycycline (doxy). This allows for the investigation of whether the chromatin state induced by ATR can be maintained after release of ATR from the target site.

[0143] Moreover, methods are known in the art for engineering DNA binding domains to bind 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 targeted binding site: 5′-TACCTAGAGGAGAAAGGTT-3′ (SEQ ID NO: 35).

[0145] Example sequences of TALE domains designed to target the promoter region of the β2-microglobulin gene include: (SEQ ID NO: 16), its target binding site: 5′-TCTCTCCTACCCTCCCGCT-3′ (SEQ ID NO: 17) MGKPIPNPLLGLDSTGGMAPKKKRKVDGGVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSGGG (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 targeted 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 GVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNNGKQALETVQRLLPVLCQDHGLTPDQVVA IASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIA SNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASH DGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDG GKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGK QALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSGGG (SEQ ID NO: 39), its targeted binding site: 5′-TCTCCCGGGAATCGTTTT-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′- TCCCGCTGCCTTTTGTGCC -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′- TCCCCCGGCCCTAGCTCCT -3′ (SEQ ID NO: 50) TALE BCL11A #8 MGKPIPNPLLGLLDSTGGMAPKKKRKVDGGVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHERAIV GVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVA IASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIA SHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASN GGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDG GKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNHGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGK QALETVQRLLPVLCQDHGLTPDQVVAIASNHGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSGGG (SEQ ID NO: 51), its targeted binding site: 5′- TCCTGGTCCGCCCCCAGCA -3′ (SEQ ID NO: 52) TALE BCL11A #9 MGKPIPNPLLGLLDSTGGMAPKKKRKVDGGVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHERAIV GVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVA IASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIA SNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASN GGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDG GKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGK QALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSGGG (SEQ ID NO: 53), its targeted 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 targeted 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 targeted 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-based DNA binding system (van der Oost et al. (2014) Nat. Rev. Microbiol. 12: 479-92), and the guide RNA (gRNA) can be selected to allow the ATR containing the Cas9 domain to target a specific sequence. Thus, to use the CRISPR / Cas system as the DNA binding domain of the present invention, it is understood that the ATR effector domain can be operably linked to the 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, an enhancer, or a splice site). Methods for designing gRNAs are known in the art. Moreover, the entirely heterologous Cas9 protein, as well as Cas9 / gRNA ribonucleoprotein complexes and modifications of gRNA structure / composition that bind different proteins, have been developed in recent years to simultaneously and directly target different 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, the ATR of the present invention may 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, which 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 by a polynucleotide containing a nucleic acid sequence, for example, encoding the protein of SEQ ID NO:22 or encoding a protein having 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% amino acid identity to SEQ ID NO:22, 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 having at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% identity to SEQ ID NO:22, which 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 encoding the protein of SEQ ID NO:22 or encoding a protein having at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% amino acid identity to SEQ ID NO:22, whose amino acid sequence substantially retains the native function of the protein represented by SEQ ID NO:22.

[0154] An example sequence of a genomic target site 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] Example sequences of gRNAs targeting the BCL11A gene include: 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] Example sequences of gRNAs targeting the IFNAR1 gene include: 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] Example sequences of gRNAs targeting the VEGFA gene include: 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] Targeted 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 expression of the target gene or that the remaining target gene expression level is sufficiently low to ameliorate or prevent the disease condition.

[0162] The reduced expression may be sufficient to allow studies of the function of the gene 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% compared to 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 silencing a target gene. Thus, two or more ATRs of the present invention may exhibit synergistic effects, such as therapeutic synergy, 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 containing 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 following transient introduction or expression of the ATR of the present invention into a cell.

[0167] By "transient expression", it is understood that the expression of ATR is not stable over a long 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 introducing the nucleotide sequence encoding ATR into a cell. Preferably, expression is substantially lost within 12, 6, 4, or 2 weeks after introducing 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 (i.e., the cell substantially loses it) for a substantial extended period of time. 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 the ATR is introduced into the cell. Preferably, the ATR is lost substantially within 12, 6, 4, or 2 weeks after the ATR is introduced into the cell.

[0169] Methods for measuring the transcription of genes, such as the target of ATR, are 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 genes, methods for measuring the expression of genes are known in the art.Other suitable methods include Western blot-based methods or flow cytometry methods.

[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 a cell, in the presence and absence of an 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 such that when the vector transfects a cell, the reporter gene is expressed. Suitable reporter genes include, for example, genes encoding fluorescent proteins, such as green, yellow, cherry, cyan, or orange fluorescent proteins. Additionally, 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 a suitable 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 silenced permanently. By "permanent silencing", it is understood that the transcription or expression of the target gene is reduced (e.g., reduced by 100%) for at least 2 months, 6 months, 1 year, 2 years, or the life 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 (i.e., the silencing of the target gene is inherited by the progeny of the cells to which two or more ATRs of the present invention have been administered). For example, two or more ATRs of the present invention can be administered to a stem cell (e.g., a hematopoietic stem cell) 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] Target gene can be silenced by using ATR that binds to the target gene itself or to the target gene's regulatory sequence (e.g., promoter or enhancer sequence).Furthermore, ATR that binds to the splice site of the target gene itself can be used to change the alternative splicing of the target gene.The ability to silence target gene or change its splicing variant by using ATR that binds to 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] The therapeutic use may be, for example, for the treatment of beta thalassemia or sickle cell anemia.

[0178] Therapeutic uses can be, for example, for the preparation of "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 contemporaneously, 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 within the same time frame, even if not simultaneously. Thus, if the half-life in the blood of the first agent administered is such that both agents are present simultaneously in therapeutically effective amounts, "sequential" administration may permit administration of one agent after the other, within 5 minutes, 10 minutes, or even hours. The time delay between administration of the components may 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 administration of one agent and 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, when silenced, produces a therapeutic effect.

[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 cancer genes.

[0184] Silencing of 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] By way of example, the products, artificial transcriptional repressors (ATRs), polynucleotides and cells of the invention can be used to treat, for example, Huntington's disease, spinocerebellar ataxias, collagenopathies, hemoglobinopathies, and diseases caused by trinucleotide repeat expansions. Moreover, the products of the 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 diseases as 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 reactions, cachexia, anorexia, acute infections, HIV infection, shock states, graft-versus-host reactions, 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 as 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, as well as for preventing transplant rejection or inducing tumor immunity); control of hematopoiesis, e.g., for 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, burns, ulcers and the treatment of periodontal disease and neurodegeneration; inhibition or activation of follicle-stimulating hormone (regulation of fertility); migration / chemokinesis (e.g., to recruit specific cell types to the site 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 behavior 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 invention may be useful in the treatment of diseases as 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 thus anti-inflammatory activity; anti-immune activity, i.e. an inhibitory effect on cellular and / or humoral immune responses, including responses unrelated to inflammation; inhibition of the adhesion ability of macrophages and T cells to extracellular matrix components and fibronectin, and of upregulated fas receptor expression in T cells; related to 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, liver 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), habitual abortion, 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 due to infection, proliferative vitreoretinopathy, acute ischemic optic neuropathy, e.g., excessive scarring after 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 the 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', post-polio syndrome, immune and inflammatory components of psychiatric disorders, myelitis, encephalitis, subacute sclerosing panencephalitis, cerebrospinal fluid inflammatory disease ... 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, the inflammatory component of muscle atrophy and 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 transplantation, e.g. 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 for the treatment of cancer, 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. It is understood by those skilled in the art that many different polynucleotides can code for the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is understood by those skilled in the art that nucleotide substitutions that do not affect the polypeptide sequence encoded by the nucleotides of the present invention can be made by routine methods to reflect the codon usage of any particular 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 life span 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 an animal or human cell, carrying out 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 using an agarose gel), and recovering the amplified DNA. The 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 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 which are linked to each other by peptide bonds or disulfide bonds.

[0195] Variants, derivatives, analogues, homologues 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 in question 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, ie, a compound that has at least one of the inherent functions of the polypeptide or polynucleotide that it mimics.

[0199] Typically, amino acid substitutions can be made from one, two or three to ten or twenty substitutions, 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] 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: TIFF0007623272000001.tif47120

[0202] The term "homolog" as used herein means something that has a certain 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% or 97% or 99% identical. Typically, a homolog has 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 that have similar chemical properties / functions), in the context 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 preferably are at least 95% or 97% or 99% identical. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties / functions), in the context of 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 sequence of 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] Although this is a very simple and consistent method, it fails to take into account that, for example, in a pair of sequences that would otherwise be identical, a single insertion or deletion in a nucleotide sequence may cause subsequent codons to be misaligned, resulting in a large 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, such 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 degree of relatedness between the two compared sequences. An "affine gap cost" is usually used, which imposes a relatively high cost on the existence of a gap and a relatively small penalty on each subsequent residue in the gap. This is the most widely used gap scoring system. High gap penalties will naturally result in an optimized alignment with fewer gaps. Most alignment programs allow the gap penalty to be modified. However, it is preferred to use the default values ​​when using such software for sequence comparisons. 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] Thus, the calculation of maximum percent homology first requires the creation of an optimal alignment, taking into account gap penalties. A suitable computer program for carrying out 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 suite of comparison tools. Both BLAST and FASTA are available for offline and online searches (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60). However, for some applications, 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 usually based on an all-or-nothing pairwise comparison. Instead, a scaled similarity scoring matrix is ​​used that 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 preferred to use the public default values ​​for the GCG package, while for other software it is preferred to use a default matrix such as BLOSUM62.

[0212] Once the software has produced an optimal alignment, it can calculate percent homology, preferably percent sequence identity. The software will typically do this as part of the sequence comparison and generate a calculation.

[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 an insertion is made, synthetic DNA can be made that encodes the insert along with 5' and 3' flanking sequences that correspond to the naturally occurring sequence on either side of the insertion site. The flanking regions contain convenient restriction enzyme sites that correspond to sites within the naturally occurring sequence so that the naturally occurring sequence can be cleaved with the appropriate restriction enzyme or enzymes and the synthetic DNA ligated to the cleavage sites. The DNA is then 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 the manipulation of DNA sequences, and 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. These codon biases correspond to biases in the relative abundance of certain tRNAs in that cell type. Expression can be increased by changing 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. In this way, additional translational control is available.

[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) in a cell, facilitating the replication of a vector containing a nucleic acid segment, or facilitating the expression of a protein encoded by a 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 vector containing the polynucleotide 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 virus vectors, such as retrovirus, lentivirus (e.g., integration-defective lentivirus), adenovirus, adeno-associated virus, baculovirus, and herpes simplex virus vectors; direct injection of nucleic acid, and biolistic (biolistic) transformation.

[0219] Non-viral delivery systems include, but are not limited to, DNA transfection methods. Transfection includes the process of using 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 delivery of a polynucleotide into a cell by both viral and non-viral delivery.

[0221] Protein delivery method As an alternative to introducing a polynucleotide into a cell, the products of the invention and the artificial transcriptional repressor (ATR) 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 the cell. Thus, when the engineered viral particle enters the 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 utilizing a vehicle (e.g., liposomes) or by administering the protein itself directly to the cells.

[0224] Pharmaceutical Compositions The products, artificial transcriptional repressors (ATRs), polynucleotides, and cells of the present invention can be formulated with pharma- ceutically acceptable carriers, excipients, or additives 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 performed 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 same 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 preventative 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 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, the tetracycline (tet) response system was used. 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 the binding of tetR to its target nucleotide sequence, the tetracycline operon (TetO), can be temporally controlled by administration of doxycycline (doxy). This will allow us to examine whether the chromatin state induced by ATR is maintained following release of ATR from its target loci.

[0232] To rapidly assess the activity of ATR, we devised a cellular experimental model in which ATR activity can be easily observed over time 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 schematic) 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 expression of ATR, these chimeric proteins bind to the TetO7 element via its tetR DNA-binding domain, ultimately leading to the accumulation of repressive epigenetic marks on the neighboring chromatin (shown as red lollipops on the hPGK promoter; Fig. 1, middle schematic), which causes transcriptional silencing of the cassette. Upon conditional release of ATR from the TetO7 element by doxycycline treatment, the repressive marks are either erased or genetically transmitted to progeny cells by endogenous intracellular machinery, thus leading to transcriptional reactivation of eGFP expression or permanent silencing, respectively (Fig. 1, bottom schematic). 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 cytometric 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 studied without the indistinguishable effects of the unmodified wild-type locus.

[0234] To assess whether the new ATRs are biologically active, tetR:K and tetR:D3A were introduced 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; Fig. 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 co-expressed from the same promoter, thus limiting the analysis of silencing exclusively to cells expressing ATR.

[0235] In summary, the described experimental setup allows us to test whether constitutive binding of a candidate ATR to a TetO7 cassette can accumulate repressive epigenetic marks on nearby chromatin as well as induce transcriptional silencing of a reporter cassette, where subsequent conditional release of ATR binding by doxycycline treatment allows us to discern whether the artificially induced repressive marks are subsequently erased by endogenous mechanisms (thus resulting in transcriptional reactivation) or are heritably transmitted to progeny cells (thus indicating the establishment of a permanent heritable epigenetic silencing).

[0236] Based on molecular characterization, AAVS1 / TetO7 cell lines were transduced with either Bid.LV-tetR:K or Bid.LV-tetR:D3A in the presence or absence of doxycycline and then maintained in these culture conditions for up to 200 days. During this time, cells were periodically analyzed by flow cytometry to measure the percentage of eGFP-negative (eGFP-) cells in the Bid.LV-transduced cell population. As shown in Figure 2B, constitutive binding of ATR to the TetO7 sequence (doxy- conditions) ultimately led to 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). On the other hand, silencing by tetR:D3A was significantly slower (Fig. 2B, right histogram). In this case, cells with higher expression levels of the marker gene (presumably cells with higher vector copy number VCN) were silenced first (Fig. 2C, right flow cytometry dot plot), indicating that a certain level of expression of tetR:D3A was required to ensure earlier suppression. Importantly, at a later time point (~200 days), flow cytometry analysis revealed that the mean fluorescence intensity (MFI) of eGFP was superimposed between silenced and wild-type (WT) K562 cells (compare MFI in Fig. 2C), indicating complete silencing of eGFP expression. When doxycycline was present in the culture (doxy+ condition), transformed cells did not silence eGFP at all, indicating that binding of ATR to the target sequence is required to cause silencing. Collectively, these data indicate that the two ATRs mediate silencing with different kinetics, but that both are functional.

[0237] We next assessed whether release of ATR from the locus would result in eGFP reactivation. To this end, eGFP cells were sorted 21 days after Bid.LV transduction and then cultured in the presence or absence of doxycycline for an additional 170 days. Interestingly, doxycycline treatment led to two opposing outcomes, depending on the ATR used: silencing caused by tetR:K rapidly (within 15 days after doxycycline treatment) and completely disappeared in the entire cell population (Figure 2D, left histogram and representative flow cytometry analysis below); on the other hand, silencing caused by tetR:D3A remained unchanged throughout the experimental period (Figure 2D, right histogram and representative flow cytometry analysis below). This clearly indicates that, contrary to tetR:K, which must be continuously active at the locus to repress it, tetR:D3A is able to 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 machinery cannot efficiently induce (stably heritable) DNA methylation, but only accumulates reversible epigenetic marks such as H3K9 methylation (Hathaway, NA et al. (2012) Cell 149: 1447-60).

[0238] Overall, these experiments clearly show 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 leads to rapid and complete reactivation of eGFP expression in tetR:K-transduced cells.

[0239] DNA methylation is involved in the maintenance of permanent silencing caused by tetR:D3 To understand whether DNA methylation is necessary for the maintenance of the repressed state caused by tetR:D3, eGFP- cells obtained from doxycycline-conditions in 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 analogue that, after incorporation into DNA, is recognized as a substrate by DNA methyltransferases, which establishes a covalent bond that, unlike cytosine, 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 a 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 the surrounding genes of the desired target gene. It should be noted that the site-specific integration of the reporter cassette into the AAVS1 locus allows the silencing platform of the present invention to easily analyze the effect of the integrated gene near the reporter cassette integration site. Therefore, the expression levels of the 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 of the 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, showing that tetR:K can silence promoters located tens of kilobases away from the ATR binding site by long-distance 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 showed that only the eGFP gene, and to a lesser extent the PPP1R12C gene (which hosts a reporter cassette in its first intron), was significantly downregulated (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 release of ATR from the locus, whereas tetR:D3A induces silencing at a slower rate but that transcriptional repression is well 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 asked whether transient co-introduction of the two ATRs above was sufficient to induce rapid (like tetR:K) and permanent (like tetR:D3A) epigenetic silencing. To answer this question, we followed eGFP expression in AAVS1 / TetO7 cells by flow cytometry analysis over time after transfection with plasmids encoding these ATRs alone or in combination. A representative example of such an experiment is shown in Figure 4A, where we report the kinetics of silencing of an eGFP expression cassette in cells transfected with plasmids encoding the indicated ATRs (% of eGFP- cells; data presented 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 plasmids encoding either tetR:K or tetR:D3A 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 indicating a transient accumulation of H3K9me3 that was subsequently lost as the tetR:K-encoding plasmid was diluted by cell division); and ii) surprisingly, up to 20% of the cells cotransfected with plasmids encoding tetR:K and tetR:D3A became stably silenced. These data revealed significant synergy between DNMT3A- and KRAB-based repressors, providing the first demonstration of permanent epigenetic silencing by transient cotransfection of ATR.

[0244] We next investigated whether the silencing caused by the tetR:K / tetR:D3A combination was restricted to the reporter cassette or otherwise spread along the AAVS1 locus, thus affecting genes in the vicinity of the insertion site of the reporter cassette. To answer this question, the expression profiles of genes at and in the vicinity of the AAVS1 insertion site (a schematic of the locus is shown in Figure 3A) were compared between eGFP-negative and eGFP-positive populations sorted from the tetR:K / tetR:D3A treatment condition. In these analyses, we found that the treatment resulted in significant silencing of only the reporter transgene (Figure 4B; data presented as mean ± SEM, n = 3; **p < 0.001, one-way ANOVA and Bonferroni post-hoc test). These important data indicate that the tetR:K / tetR:D3A combination accumulates punctuated epigenetic silencing only in the target genes of interest, highlighting the safety of the method. Finally, treatment of eGFP-negative sorted cells from tetR:K / tetR:D3A conditions with 5-Aza completely reactivated eGFP expression in these cells (Figure 4C; data presented as mean ± SEM, n = 3; ***p < 0.0001, two-tailed unpaired t test), indicating that DNA methylation plays a key role in maintaining this repressed epigenetic state. Similar results were found 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 about two-fold 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 it (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 Since the two ATRs were found to cause permanent silencing even when transiently introduced into cells, we investigated whether this effect was locus independent. Indeed, the efficacy of epigenetic therapeutics may depend on the chromatin environment in which the target locus is embedded, and in theory, some loci may be more refractory to certain repression mechanisms than others. For example, some 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 the activity of ATR 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 introduced this construct semi-randomly into the genome of K562 cells by standard lentiviral vector transduction (a schematic diagram of the provirus used is shown in Figure 5A). eGFP-expressing cells were then selected for purity (hereafter referred to as the TetO7.LV reporter cell line) and transfected with in vitro transcribed mRNAs encoding tetR:K or tetR:D3A alone or in combination. Time-course flow cytometric analysis of the cells (Figure 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 inhibition 2 weeks after transfection; ii) up to 80% of cells transfected with a plasmid encoding tetR:K rapidly became eGFP-negative, but the majority of these cells reactivated eGFP expression shortly thereafter (unlike experiments performed with the AAVS1 / TetO7 cell line, where up to 19% of cells remained eGFP-negative); and iii) surprisingly, up to 80% of cells co-transfected with mRNA encoding tetR:K / tetR:D3A became permanently silenced. Interestingly, even though comparable silencing efficiencies were recorded between 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 carrying a random insertion of the TetO7 / eGFP cassette (Figure 5C). In this case, however, the efficiency of silencing for all treatment conditions was lower than that 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 random insertions of the TetO7 / eGFP cassette, long-term stable silencing was observed only under tetR:D3A treatment (Figure 5D; data are presented as mean ± SEM, n = 3; ***p < 0.0001, two-way ANOVA and Bonferroni post-hoc test). Contrary to all the results of the above experiments, silencing caused by multiple ATR combinations was transient and showed kinetics superimposable to those measured under tetR:K treatment conditions.

[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 indicating that the silencing mechanism is locus independent. Nevertheless, these studies suggest that some 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 Although the above data provide the first proof of our findings of permanent epigenetic silencing by transient expression of ATR, they also show that some cell-specific factors can modulate the in vivo activity of these proteins. For example, the low level of silencing observed in U937 cell line, as well as the unexpected lack of silencing activity of ATR combinations 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. Thus, the inclusion of additional ATRs in the mixture of the present invention may be useful to enhance the silencing efficiency of KRAB / DNMT3A combinations by avoiding the absence of cofactors or by allowing proper function of repression complexes, even when these ATRs are present at low concentrations. 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 ATR of the present invention. To this end, by utilizing the 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 histone H3K9me, resulting in epigenetic silencing; · 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] New ATRs were generated that contain the effector domains of the above proteins and the DNA binding domain of tetR. Hereinafter, the new ATRs are 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 G9a); 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] First, we investigated the activity of the novel ATRs in the LV / TetO7 K562 reporter cell line by using standard Bid.LV integration and found that among the novel ATRs, tetR:SET, tetR:GS and tetR:H effectively induced silencing when stably expressed individually (Fig. 6A; data presented as mean ± SEM, n = 3). However, none of these ATRs reached the silencing efficiency of tetR:K and tetR:D3A. Unlike tetR:GS, tetR:GL was not effective in this experimental setting, suggesting that the inclusion of ankyrin repeats in this relatively long form of G9a negatively impacts the 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 in this cell line of endogenous interactors required for the activity of the proteins. Furthermore, we found that for some of the ATRs used, the percentage of transduced cells decreased over time (Figure 6B; data are presented as mean ± SEM, n = 3). This data indicates a growth disadvantage for cells stably expressing ATR and therefore supports the rationale for using a transient transfection method to safely express ATR.

[0251] We next assessed whether silencing caused by the novel ATR was maintained 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 cytometric analysis (Figure 6C; data are presented as mean ± SEM, n = 3). As expected, silencing caused 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 caused by tetR:K gave more stable results and was maintained in up to 45.8% of initially silenced cells. This is in contrast to the results observed using the AAVS1 / TetO7 K562 reporter cell line, where eGFP was fully reactivated in all transduced cells 7 days after doxycycline treatment. The data suggest a role in that 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] We next examined the efficacy of ATR during transient transfection in the same LV / TetO7 K562 reporter cell line. Specifically, we examined ATR individually (Figure 7A) or in combination with tetR:D3A (Figure 7B), tetR:K (Figure 7C), or the combination tetR:K+tetR:D3A (Figure 7D). To better understand the ultimate increase in silencing efficiency over the levels 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 was not available for tetR:T as the other ATRs at the time of the experiment.

[0253] By following eGFP expression in treated cells over time by flow cytometry, we found that none of the ATRs effectively silencing when expressed individually, with tetR:K, tetR:D3A, and tetR:SET suppressing less than 1% of cells (Figure 7A; data presented as mean ± SEM, n = 3). When combined with tetR:D3A (Figure 7B; data 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-introduced with tetR:K (Figure 7C; data presented as mean ± SEM, n = 3), only tetR:D3L among the novel ATRs conferred greater synergy than tetR:D3A. Finally, the addition of one of the novel ATRs to the tetR:K+tetR:D3A combination (Fig. 7D; data presented as mean ± SEM, n = 3) enhanced the silencing efficiency of most ATRs, thus demonstrating biological activity even for ATRs that were stable but not functional when introduced individually (Fig. 6A). Importantly, this experiment identified the tetR:K+tetR:D3A+tetR:D3L combination as the best combination, showing impressive efficiency considering the low plasmid dose used in this experiment. Specifically, the tetR:K+tetR:D3A+tetR:D3L combination led to a 4.1-fold increase in silencing efficiency compared to the tetR:K+tetR:D3A combination (Fig. 7E; data 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 combinations of tetR:D3A+tetR:D3L, tetR:K+tetR:D3A, and tetR:D3L+tetR:K, all three ATRs play their relevant roles 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 Fig. 7E), we reloaded the same experiment with a lower ATR dose and found that tetR:SET can also significantly synergize with the combination of tetR:D3A+tetR:D3L (Fig. 7F; tetR:D3L is denoted 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 if 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 use of the combination tetR:K+tetR:D3A+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 denoted as tetR:D; tetR:D3L is denoted as tetR:L; data are presented as mean ± SEM, n = 3). As expected from previous experiments, tetR:K+tetR:D3A co-delivery led to a transient increase in silencing that disappeared completely 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, the introduction of the three ATRs together resulted in the majority of cells being 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 induction and maintenance of silencing in previously refractory cell lines. Based on these promising results, we also investigated whether our silencing platform was 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 mRNAs encoding the tetR-based ATRs, either individually or in combination.Surprisingly, flow cytometry analysis of treated cells demonstrated effective and long-term silencing even in this cell model: a single dose of tetR:D3A+tetR:D3L or the combination of the three ATRs resulted in gene silencing efficiencies of 45% or 80%, respectively (Figure 7H), whereas the combination tetR:D3A+tetR:K did not work, as previously observed in B lymphoblastoid cells.

[0255] Effective silencing by transient co-transfection of ATR with a custom-made DNA-binding domain The main 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 with tetR, acts synergistically to silence promoters in the vicinity of the TetO7 element, the artificial nature of the prokaryotic TetO7 / tetR system hampers the therapeutic application of this technology. Moreover, the TetO7 element can accommodate seven tetR dimers with high affinity, which may lead to stochastic homo- or heterodimerization of ATR on the element. This occurrence may promote positive interactions between repressors. For these reasons, several questions remain to be addressed in order to transfer the knowledge gained in the TetO7 / tetR system to a situation where each ATR has a single and independent binding site on the target gene. In particular, it remains to be seen whether one element (defined as a certain genomic sequence containing the binding sites of each repressor, hereafter referred to as "silencing element") is sufficient to silence a gene of interest. Furthermore, the relative order and orientation of two repressors when placed on a silencing element, as well as the distance between their binding sites, may be important determinants for the activity of the repression complex. Notably, it is not possible to clarify these determinants based on the literature or by experimentally examining them on endogenous genes, since it would be necessary to design several different ATRs with their own binding sites and affinities.

[0256] To address these questions, we developed an engineered cellular model for the immediate 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 combination tetR:K+tetR:D3A.

[0257] Briefly, KRAB and DNMT3A domains were fused to the DNA-binding domains of two TALEs that recognize two different 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 the two genomic target sites, respectively. In parallel, the two TALE target sites were inserted upstream of the hPGK promoter of an eGFP expression cassette, spaced apart by progressively longer nucleotide sequences (5, 10, 15, 20, 25 and 30 bp), and these constructs were then semi-randomly introduced into the genome of the K562 cell line by standard lentiviral vector transduction methods. In particular, the target sites of the two TALEs were positioned such that the binding of the TALE-repressor occurred in a head-to-tail (HT) configuration. A schematic of these vectors is shown in Figure 8A (on the left, vectors containing binding sites in the TALE:K→TALE:D3A configuration; on the right, vectors containing binding sites in the TALE:D3A→TALE:K configuration). eGFP-expressing cells were then selected for purity, and the cell lines were transfected with in vitro transcribed mRNAs 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 presented 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 presented as mean ± SEM, n = 3; ***p < 0.0001 and **p < 0.001, two-way ANOVA and Bonferroni post-hoc test).

[0258] These experiments revealed that: 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 influenced the overall silencing efficiency, with the TALE:D3A→TALE:K configuration performing 2.2- to 5.4-fold 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 absent silencing of the eGFP expression cassette, respectively.

[0259] Considering the significant effect of structural variables (e.g., the length of the spacer and the relative order of binding of the two ATRs on the target sequence) on silencing efficiency, we next investigated whether the transition 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 head-to-tail to head-to-head configuration, we started with the reporter cassette described 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 introduced these constructs into K562 cells by lentiviral vector transduction (a schematic diagram of this vector is shown in Table 9A). The transduced cells were then sorted for 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 clear synergy, allowing long-term silencing of the reporter cassette in up to 34.7% of treated cells (Figure 9B; data presented as mean ± SEM, n=3); ii) individual transfection of TALE:K or TALE:D3A resulted in low (up to 7.1%) or absent permanent silencing, respectively; and iii) the relative order of binding of the two ATRs at the target locus influenced 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 presented as mean ± SEM, n=3).However, the relative order of binding appears to have a greater effect on silencing in the head-to-tail configuration than in the head-to-head configuration (compare Figures 8 and 9). A bell-shaped trend represents the effect of the spacer length examined on the silencing efficiency of the HH configuration, with the 15 bp spacer being the best in both the TALE:D3A→TALE:K and TALE:K→TALE:D3A configurations (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% long-term eGFP cells, respectively, i.e., a 1.3-fold increase).

[0260] Overall, these data reveal for the first time that a combination of multiple ATRs with custom-made DNA-binding domains can be transiently introduced 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. By targeting multiple silencing elements on the regulatory sequence of this gene, silencing efficiency should be increased.

[0261] In parallel with these studies, we developed a bipartite ATR by combining two effector domains on the same TALE, namely, a KRAB domain at the N-terminus and a DNMT3A domain at the C-terminus (Figure 10A). Even though 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 produced 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 have self-renewal and pluripotency throughout their life. HSPC differentiation is accompanied by global chromatin remodeling, resulting in a gradual transition from an open chromatin morphology to a more compacted and repressive one. Thus, this cell type is the most appropriate and rigorous model to examine the efficiency and prove the stability of our epigenetic platform. To assess whether the introduction of different ATR combinations is sufficient to cause significant levels of silencing in human HSPCs, the TetO7 / eGFP reporter LVs described in Figure 5A were used to transduce human umbilical cord blood-derived CD34+ cells obtained from healthy individuals. The cells were then transfected with in vitro transcribed mRNAs encoding tetR:D3A, tetR:K, or tetR:D3L, either alone or in combination. Transfected and untransfected cells were then grown in liquid culture for 2 weeks in 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 cytometric analysis of cells grown in liquid culture showed that treatment with tetR:K resulted in a transient increase in eGFP suppression that was then maintained until the end of the experiment in up to 20% of treated cells (Figure 11B; data are presented as mean ± SEM, n = 3). A similar phenotype was observed in CD34+ cells transfected with mRNAs encoding tetR:D3A and tetR:D3L. Treatment with the combinations tetR:K / tetR:D3A or tetR:D3A / tetR:D3L resulted in a synergistic effect, showing that up to 40% of treated cells completely silenced eGFP expression. Surprisingly, by combining tetR:D3L / tetR:K or tetR:D3L / tetR:K / tetR:D3A, up to 90% silencing of the reporter gene was reached. Importantly, similar levels of silencing were observed in erythroid and myeloid cells derived from CFU-C assays (Figure 11C; data presented as mean ± SEM, n = 3), indicating that silencing was maintained upon HSPC differentiation.

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

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

[0266] Permanent epigenetic silencing of endogenous human genes using custom-made ATRs To evaluate whether the results obtained with the eGFP reporter system could be transferred to endogenous genes embedded in their natural epigenetic context, we generated custom-made 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) and fused these TALEs with the KRAB, DNMT3A, and DNMT3L effector domains (see Figure 13A for a schematic diagram of the system). The length of the spacer between the first and second TALEs or between the second and third TALEs is 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] 50 days after transfection, when the percentage of B2M-negative cells had stabilized, a significant proportion of B2M-negative cells was measured only in conditions treated with the combinations TALE:D3A+TALE:D3L and TALE:D3A+TALE:D3L+TALE:K (Figure 13B; data presented as mean ± SEM, n=3; ***p<0.0001, one-way ANOVA and Bonferroni post-hoc test). Strikingly, up to 80% of all ATR-treated cells had permanently lost surface expression of B2M. In parallel experiments, we sorted B2M-negative and B2M-positive cells and analyzed them for surface expression of MHC-I molecules, which require B2M to be present on the cell membrane. Unlike B2M-positive cells, almost all B2M-negative cells were also found to be negative for MHC-I expression (Figure 13C). Gene expression analysis was performed on sorted B2M-negative and B2M-positive cells, and negative cells were found to have approximately 100-fold lower expression than positive cells (Figure 13D; data are presented as mean ± SEM, n=3). We next assessed whether the three effector domains could also cause permanent epigenetic silencing when targeted to B2M by the guide RNA-directed CRISPR / Cas9 system. To this end, catalytically inactive Cas9 (D10A+H840A; dCas9; amino acid sequence listed in Table 4) was fused in frame with KRAB, DNMT3A or DNMT3L effector domains (Figure 13E; top diagram), and 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 location of the CRISPR / dCas9 target sites). We then co-transfected HEK-293T cells with plasmids expressing the 11 B2M gRNAs together with all possible combinations of plasmids encoding dCas9 fusion proteins. Thirty-three days after transfection, the treated HEK-293T cells were analyzed by flow cytometry to identify the most common B2M gRNAs: dCas9:K+dCas9:D3L, dCas9:D3A+dCas9:D3L, and dCas9:K+dCas9:D3a+dCas9: It was shown that only the combination of D3L was able to cause silencing of the B2M gene (Figure 13F; data are presented as mean ± SEM, n = 3). We next assessed 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, wild-type and B2M-negative cells were used, the latter selected from the triple ATR treatment conditions described in Figures 13B and 13F. As expected, IFN-γ treatment caused a significant upregulation (>100-fold) in the expression of the 2′-5′-oligoadenylate synthetase 1 (OAS1) gene in all cell types tested (Figure 13G). On the other hand, IFN-γ treatment in wild-type cells significantly upregulated B2M expression at both the transcriptional and protein levels, whereas no increased expression of this gene was measured in B2M-negative cells (Figures 13G and 13H, respectively).

[0268] To assess whether the silencing caused by the ATR of the present invention is accompanied by the accumulation of repressive epigenetic marks on the target gene, the epigenetic state of the B2M gene was analyzed in wild-type and silenced cells. For this purpose, cells treated with a plasmid encoding the triple TALE:ATR combination were sorted until pure 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 PolII) on the promoter region and gene body of B2M revealed a total absence of this protein in silenced cells, whereas this protein was highly enriched in the promoter region of untreated cells (PPP1R12C and CCR5 genes were used as positive and negative controls for the above experiments, 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 S14C). DNA methylation was also involved in maintaining silencing, since AZA treatment was associated with re-expression of the B2M gene in pre-silenced cells (Figure S14D). Finally, to address whether silencing was limited to the B2M gene, we performed transcriptional analysis of the B2M locus by RT-qPCR (Figure S14E, top panel) and found that B2M was the only gene downregulated upon transient introduction of the triple ATR combination, while the expression of its neighboring genes was unaffected (Figure S14E).

[0269] In parallel to these experiments, we also investigated silencing of the B2M gene in K-562 cells. Since this cell line does not express MHC-I, which is completely required for B2M surface expression, to properly report on the B2M transcriptional status, we targeted the coding sequence of the fluorescent marker tdTomato into the first intron of the B2M gene (Figure 15A). After gene targeting with CRISPR / Cas9, tdTomato-positive cells were selected and then electroporated with plasmids encoding either TALE-based ATRs or CRISPR / dCas9-based ATRs against the B2M promoter / enhancer (the target sequences of these ATRs are the same as in Figure 13). For TALE-based ATRs, both the combinations TALE:D3L+TALE:K and TALE:D3A+TALE:D3L+TALE:K were able to stably silence B2M expression, but the triple ATR combination proved to be the most successful (Figure 15B). Codon optimization of the effector domain of TALE-based ATR improved the silencing efficiency of the above combinations (Figure 15B; compare red and green bars). As for the silencing activity of CRISPR / dCas9-based ATR, we found that all combinations except dCas9:D3A+dCas9:K were able to produce high silencing efficiency of the B2M gene (up to 55% stably silenced cells; Figure 15C). Finally, targeting dCas9 fused to the catalytic domain of TET1 enzyme (known to demethylate DNA; Maeder, ML et al. (2013) Nat. Biotechnol. 31: 1137-42) to the B2M promoter / enhancer of selected silenced cells led to reactivation of the expression of this gene (Figure 15D), supporting the idea that silencing produced by triple ATR combinations 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 above 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 in Figure 16A). Flow cytometric analysis of the cells revealed that treatment with TALE:K+TALE:D3A+TALE:D3L ATR led to silencing of the B2M gene (silencing kinetics in Figure 16B, FACS plot in Figure 16C).

[0271] Interestingly, functional deconvolution of seven gRNAs from quadruplets to individual ones revealed that even a single gRNA, triple combinations, and dCas9:D3A+dCas9:D3L combinations, were sufficient to perform effective silencing of 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 cases, triple ATR combinations were observed to outperform dCas9:D3A+dCas9:D3L combinations. In conclusion, the above data showed that a gRNA associated with three ATRs, even one of which was sufficiently well positioned on the target gene, could induce efficient silencing.

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

[0273] In summary, the above data reveal, for the first time to our knowledge, the permanent silencing of endogenous genes in human cells using custom-made ATR. Importantly, the silencing was completely resistant to external stimuli acting on the B2M promoter / enhancer, thus providing another evidence for the stability of 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 techniques, namely 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 with the other two ATRs targeted on the desired gene of interest. We first utilized the TetO7 / tetR system to evaluate whether the introduction of untargeted DNMT3L (hereafter referred to as D3L) is as effective as its targeted counterpart in cooperating with the other two effector domains (specifically DNMT3A and KRAB). Thus, we transfected TetO7.LV reporter B-lymphoblastoid cells with in vitro transcribed mRNAs encoding tetR-based ATR and untargeted D3L, and measured the percentage of eGFP-negative cells in different transfection conditions by time-lapse flow cytometry analysis (Figure 18A; data are expressed as mean ± range, n=2). At 27 days post-transfection, little, if any, silencing was found 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 in the triple ATR combination (approximately 20% eGFP-negative cells) (Figure 18A; compare with the plus tetR:D3L condition). These data are in line with what was previously found 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 tetR:D3A and the tetR:D3A+tetR:K combination (Figure 18A; see plus D3L condition).Importantly, the silencing levels measured in these two experimental conditions were comparable to those found by simultaneously tethering TetO7 sequences to DNMT3L and DNMT3A; or DNMT3L, DNMT3A, and KRAB. These data indicate that untargeted D3L can effectively synergize with the combination of KRAB + 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 the 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), where TALE:D3A and TALE:K could be bound 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 TALE-based ATR and non-targeted D3L, and measured the percentage of double-negative B2M / MHCI-negative cells in the different transfection conditions by time-course flow cytometry analysis (Figure 18C). At 12 days post-transfection, a low percentage of B2M / MHCI-negative cells was measured in all conditions treated with the TALE:D3A+TALE:K combination (top plot in Figure 18C). Meanwhile, co-treatment of cells with the combination of two ATR plus non-targeted D3L resulted in an average 5-fold increase in silencing efficiency over the level measured without D3L (bottom plot in Figure 18C). This increase was independent of the relative order of binding of the TALE proteins on the B2M promoter and was confirmed for both silencing modules.

[0276] Finally, a similar experiment was performed using the ATR based CRISPR / Cas9 system (Figure 18D; data are presented as mean ± SEM, n = 3). Here, transient expression of D3L in HEK-293T cells transfected with dCas9:K+dCas9:D3A ATR plus B2M gRNA (as used in Figure 13E) was found to result 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 with DNMT3A ATR.

[0277] Taken together, these data clearly demonstrate that non-targeted 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 forming de novo DNA methylation, we investigated whether endogenous DNMT3B could cooperate with the ATR of the present invention. To answer this question, we performed gene knockout of DNMT3B by CRISPR / Cas9 in TetO7.LV K562 reporter cell line. To do this, we transduced cells with two lentiviral vectors, one encoding doxycycline-inducible Cas9 nuclease (Wang, T. et al. (2014) Science 343: 80-4) and the other encoding gRNA against exon 2 of DNMT3B gene and ΔLNGFR marker (schematic diagram of vectors in Figure 19A; middle FACS plot for double-transduced cells). Cas9 was activated by doxycycline administration, and the cells were electroporated with plasmids encoding various combinations of ATR, and silencing efficiency was measured in ΔLNGFR positive and negative cells by flow cytometry. By comparing these figures, it is possible to evaluate whether inactivation of the DNMT3B gene improves the silencing efficiency of various ATR combinations. Here, it was observed that the subpopulation expressing gRNA against DNMT3B (i.e., ΔLNGFR-positive cells) was less permissive than wild-type cells to silencing by the tetRK+tetR:D3A combination (Figure 19B and Figure 19C; top right FACS plot), thus indicating 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 in this case DNMT3B acts as a decoy for these ATRs (Figure 19B and Figure 19C; bottom right FACS plot). 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, considering that the above B lymphoblastoid cell line 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, allowing stable eGFP silencing, which was 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 of 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 gene targeting with CRISPR / Cas technology (Figure 20A). Such a targeting strategy inhibits the expression of the tdTomato transgene from the regulatory sequences of the BCL11A gene. This allows for a steady reporting of the expression level of this gene. tdTomato-positive cells were then enriched by cell sorting until nearly pure, and four CpG islands within the promoter / enhancer region of this gene were targeted using CRISPR / dCas9-based ATRs containing DNMT3A or DNMT3L. Each of the four CpG islands was examined individually using separate pools 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, the relative contribution of each island to BCL11A expression could be assessed (Figure 20B). Compared to control-treated cells, silencing of each CpG island was associated with a 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 ATR. We then selected CpG islands 31 and 38 for further study with the aim of evaluating the activity of triple ATR combinations. These studies included all possible double ATR combinations, as well as single KRAB-based ATRs. Surprisingly, all conditions tested were able to induce significant levels of gene silencing by epigenetic editing of CpG 38 (the island that responded best in the previous experiments), with the triple ATR combination 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 in 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-term silencing of BCL11A (reaching up to 55% of tdTomato-negative cells), whereas silencing with TALE:KRAB was accompanied by varying degrees of gene silencing, some as efficient as the triple ATR combination, and others completely inactive. Collectively, these data demonstrate the feasibility of permanently silencing the human BCL11A gene.

[0281] Silencing additional human endogenous genes using CRISPR / dCas9-based ATR Finally, the epigenetic silencing technique of the present invention was further examined on two human endogenous genes: the interferon (alpha, beta, and omega) receptor 1 (IFNAR1) gene and the vascular endothelial growth factor A (VEGFA) gene. Both genes exhibit CpG islands in the gene promoter / enhancer regions. Thus, 13 gRNAs were designed against the IFNAR1 CpG island (Figure 20E, top) and 3 gRNAs against the VEGFA CpG island (Figure 20F, top) (nucleotide sequences of gRNAs are listed in Table 6). Interestingly, electroporation of K562 cells with plasmids encoding a combination of the 13 gRNA pool against the IFNAR1 gene plus triple dCas9-based ATR 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 by 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 transfer construct pCCLsin.cPPT.hPGK.eGFP.Wpre (Follenzi, A. et al. (2000) Nat. Genet. 25: 217-22), whereas ATR-expressing Bid.LVs were generated from the transfer construct pCCLsin.cPPT.dLNGFR.mhCMV.hPGK.GFP.Wpre (Gentner, B. et al. (2010) Sci. Transl. Med. 2: 58ra84). Doxycycline-inducible Cas9 expression vectors were 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 co-transfected with 35 / 12.5 / 9 / 6.25 μg of transfer construct plasmids pMD.Lg / pRRE packaging plasmid, pMD2.VSV-G envelope-encoding plasmid, and pRSV-Rev per 15 cm dish, respectively, by DNA calcium phosphate precipitation. Vector particles were concentrated 300-fold by ultracentrifugation as previously described and titered 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 such is 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) that contains the following construct changes: the Golden Gate TALE C- and N-terminal subregions were replaced with +163 and +63 deletions, respectively. These constructs were constructed to provide an effector domain in frame. Cas9-based ATRs were generated by replacing the VP160 transactivator with the effector domain or the catalytic domain of TET1 from 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 in IMDM (Sigma); and NIH / 3T3 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 maintained in 5% CO 2The cells were cultured at 37°C in a humidified incubator. Reporter cell lines were generated by transducing cells with the indicated ATR reporter LVs at a multiplicity of infection (MOI) of 0.1, followed by enrichment 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 co-transfected 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 within the third exon of BCL11A, a donor construct (2 μg) containing the tdTomato transgene fused to the 2A autocatalytic peptide was co-transfected 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 similar targeting strategy as 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 the activity of ATR, reporter cell lines were transduced with ATR-expressing Bid.LV at an MOI of 10 or transfected with ATR-expressing plasmid or in vitro-transcribed mRNA (4D-NucleofectorTM System; Lonza) according to the manufacturer's instructions for K-562, U937 and NIH / 3T3, and with pulse-programmed EW-113 and SF solutions for B-lymphoblastoid cells. Except for experiments performed in nonsaturating conditions with 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 ATR, respectively. Meanwhile, 1-2 μg of plasmid encoding dCas9-based ATR and 125-250 ng of expression plasmid for gRNA were introduced by electroporation. In vitro transcribed mRNA was generated as previously described (Genovese, P. et al. (2014) Nature 510: 235-40). When necessary, cells were treated with 1 μg 5-aza-2-deoxycytidine (AZA, Sigma) or 12 μg / mL doxycycline (Sigma). AZA-containing medium was changed daily and cells were analyzed by flow cytometry 4 and 7 days after treatment. When necessary, cells were treated with 500 U / mL recombinant human IFN-γ (R&D Systems). 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. They were then transduced with TetO7-reporter LV at an MOI of 30-50. After 48 h, the cells were electroporated with 2 μg of ATR-encoding mRNAs (P3 Primary Cell 4D-Nucleofector X Kit, program EO-100; Lonza). 1 μM SR1 (BioVision Inc.) was added at every medium change. After 1 week in stimulation medium, cells were cultured in liquid in IMDM 10% FBS. For CFC assays, 800 cells / plate were seeded in methylcellulose-based medium (MethoCult H4434, StemCell Technologies) 1 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). 6Cells were cultured at a concentration of 1000 cells / mL. After 3 days in culture, cells were transduced with TetO7-reporter LV at an MOI of 10. Three days after transduction, cells were washed and electroporated with 2 μg of mRNA encoding ATR. To examine silencing resistance to polyclonal TCR stimulation, bulk processed T lymphocytes were cocultured with 6000 rad-irradiated PMBCs from an unrelated donor and a pool of 10000 rad-irradiated JY cells in the presence of anti-CD3 antibody (OKT3) 30 ng / mL (Orthoclone, Milan, Italy) and human recombinant IL-2 50 U / mL (PrepoTech). In relation to silencing B2M 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 100 / 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, but 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: TIFF0007623272000002.tif73150 Aminoactinomycin D (7-AAD) positive nonviable cells were excluded from the analysis, and 1-5 × 10 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 with 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). TIFF0007623272000003.tif113115The gene expression assays used to detect eGFP transcripts were 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 were extracted (Ct and raw fluorescence) 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 fold change relative to mock-treated samples (calibrators), corrected for HPRT or B2M expression (housekeeping gene controls).

[0288] molecular analysis For bisulfite sequencing, genomic DNA was extracted with DNeasy Blood & Tissue Kit or QIAamp DNA Mini Kit (QIAGEN) and then processed with EpiTect Bisulfite kit (Qiagen) according to the manufacturer's instructions. For bisulfite sequencing, genomic DNA was extracted with DNeasy Blood & Tissue Kit or QIAamp DNA Mini Kit (QIAGEN) and then processed with EpiTect Bisulfite kit (Qiagen) according to the manufacturer's instructions. The B2M promoter region was then PCR amplified using the conversion products with the following primers: PCR fragments were purified and cloned into pCRII-TOPO TA (Invitrogen), and 5-10 clones for each sample were verified by sequencing with M13 universal primers. TIFF0007623272000004.tif54133

[0289] Chromatin immunoprecipitation (ChIP) analysis was performed as previously described (Lombardo, A. et al. (2011) Nat. Methods 8: 861-9) using 5-10 μg of ChIP-grade antibody (Abcam) raised against human H3 or the RNA polymerase II CTD repeat YSPTSPS. IgG isotypes were also used as controls. Primers used for these experiments are listed below. The percentage of enrichment of RNA PolII for each examined site was calculated by the ΔCt method using Input as the reference. TIFF0007623272000005.tif113121

[0290] statistical analysis One-way ANOVA with Bonferroni's multiple comparison post-hoc test was used to assess the statistical significance of gene expression differences between all samples (P<0.05). TIFF0007623272000006.tif182129TIFF0007623272000007.tif192129TIFF0007623272000008.tif25126Table 1

[0291] TIFF0007623272000009.tif113135 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 TIFF0007623272000010.tif162128 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] TIFF0007623272000011.tif95143B2M Nucleotide sequence of gRNA target site TIFF0007623272000012.tif10351TIFF0007623272000013.tif5949B2M Nucleotide sequence of gRNA TIFF0007623272000014.tif10180Table 4

[0294] Amino acid sequence of TALE A targeting the B2M promoter region TIFF0007623272000015.tif61143 Nucleotide sequence of the corresponding TALE binding site TALE A 5′-TGCTCGCGCTACTCTCTCT-3′ Table 5

[0295] Nucleotide sequence of gRNA targeting BCL11A 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 TIFF0007623272000016.tif61142 Nucleotide sequence of the corresponding TALE binding site TIFF0007623272000017.tif75142 Nucleotide sequence of the corresponding TALE binding site TIFF0007623272000018.tif55142TIFF0007623272000019.tif22143Nucleotide sequences of corresponding TALE binding sites TIFF0007623272000020.tif76142 Nucleotide sequence of the corresponding TALE binding site TIFF0007623272000021.tif74143 Nucleotide sequence of the corresponding TALE binding site TIFF0007623272000022.tif20129TIFF0007623272000023.tif53142Nucleotide sequence of the corresponding TALE binding site TIFF0007623272000024.tif75143 Nucleotide sequence of the corresponding TALE binding site TIFF0007623272000025.tif67143TIFF0007623272000026.tif11141Nucleotide sequence of the corresponding TALE binding site TIFF0007623272000027.tif74142 Nucleotide sequence of the corresponding TALE binding site TIFF0007623272000028.tif75142 Nucleotide sequence of the corresponding TALE binding site TIFF0007623272000029.tif33142TIFF0007623272000030.tif46143Nucleotide sequences of corresponding TALE binding sites TIFF0007623272000031.tif75142 Nucleotide sequence of the corresponding TALE binding site TIFF0007623272000032.tif76143 Nucleotide sequence of the corresponding TALE binding site TIFF0007623272000033.tif75142 Nucleotide sequence of the corresponding TALE binding site TIFF0007623272000034.tif75143 Nucleotide sequence of the corresponding TALE binding site TIFF0007623272000035.tif45143TIFF0007623272000036.tif34142Nucleotide sequences of corresponding TALE binding sites TIFF0007623272000037.tif76144 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 alterations 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 will 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 the art of biochemistry and biotechnology or related fields and are intended to be included within the scope of the following claims. The present invention includes the following embodiments. [1] Group (a), (b), (c) or (d): (a) An artificial transcriptional repressor (ATR) that contains a DNA-binding domain operably linked to a KRAB domain or its homologue; (b) an ATR that contains a DNA-binding domain operably linked to a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof; (c) an ATR that includes a DNA-binding domain operably linked to a DNMT3L domain or a homolog thereof; and (d) an ATR that contains a DNA-binding domain operably linked to a SETDB1 domain or a homolog thereof; 1. 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), (c) or (d). [2] Group (a), (b) or (c): (a) An artificial transcriptional repressor (ATR) that contains a DNA-binding domain operably linked to a KRAB domain or its homologue; (b) an ATR that includes 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. 1. 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). [3] The product described in [1] or [2], wherein the KRAB domain or a homolog thereof comprises an amino acid sequence having at least 60% identity to any one of SEQ ID NOs: 1 to 7. [4] The product described in any one of [1] to [3], wherein the DNMT3A domain or a homolog thereof comprises an amino acid sequence having at least 60% identity to SEQ ID NO: 8; the DNMT3B domain or a homolog thereof comprises an amino acid sequence having at least 60% identity to SEQ ID NO: 9 or 36; and / or the DNMT1 domain or a homolog thereof comprises an amino acid sequence having at least 60% identity to SEQ ID NO: 10. [5] The product described in any one of [1] to [4], wherein the DNMT3L domain or a homolog thereof comprises an amino acid sequence having at least 60% identity to SEQ ID NO:11. [6] The product described in any one of [1] to [5], wherein the SETDB1 domain or its homologue comprises an amino acid sequence having at least 60% identity to SEQ ID NO: 12 or 13. [7] The product described in any one of [1] to [6], wherein 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. [8] The product according to any one of [1] to [7], wherein the polynucleotides encoding two or more ATRs are in the form of a single vector or are contained in separate vectors. [9] The product described in any one of [1] to [8], further comprising another effector protein or a polynucleotide encoding the same that is not operably linked to the DNA-binding domain.

[10] The separate effector protein not operably linked to the DNA-binding domain is selected from group (a), (b), (c), or (d): (a) KRAB domain or its homologues; (b) a DNMT3A, DNMT3B or DNMT1 domain or a homolog thereof; (c) a DNMT3L domain or a homolog thereof; and (d) SETDB1 domain or its homologue The product according to [9], comprising a domain selected from:

[11] The product according to any one of [1] to

[10] , which is in the form of a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier, diluent or excipient.

[12] The product according to any one of [1] to

[11] for use in therapy.

[13] The product of any one of [1] to [8], for use in therapy, wherein two or more ATRs or polynucleotides encoding them are a combination agent for simultaneous, sequential or separate administration to a subject.

[14] A product for use in therapy according to

[12] or

[13] , wherein transient expression of two or more ATRs in a cell silences a target gene.

[15] A product for use in therapy described in any one of

[12] to

[14] , wherein delivery of two or more ATRs to a cell persistently silences a target gene.

[16] A product for use in therapy described in any one of

[12] to

[15] , wherein delivery of two or more ATRs to a cell persistently silences a target gene in progeny of the cell.

[17] A product for use in therapy described in any one of

[12] to

[16] , wherein the DNA binding domains of different ATRs are selected to bind to binding sites separated by 0 to 30 bp.

[18] The product for use in therapy described in

[17] , wherein the DNA binding domain is a TALE DNA binding domain or a CRISPR / Cas system.

[19] An artificial transcriptional repressor (ATR) comprising a DNA binding domain operably linked to a KRAB domain or a homolog thereof, or a polynucleotide encoding 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.

[20] 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 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.

[21] 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, for use in therapy.

[22] An artificial transcriptional repressor (ATR) comprising a DNA binding domain operably linked to a SETDB1 domain or a homolog thereof, or a polynucleotide encoding 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, for use in therapy.

[23] A cell comprising two or more ATRs according to any one of [1] to [7].

[24] A cell which is a descendant of the cell described in

[23] .

[25] The cell of

[23] or

[24] for use in therapy.

[26] A method of gene therapy comprising transfecting a cell with a polynucleotide encoding two or more ATRs according to [8].

[27] The method described in

[26] , wherein transfection is performed ex vivo.

[28] Group (a), (b), (c) or (d): (a) An artificial transcriptional repressor (ATR) that contains a DNA-binding domain operably linked to a KRAB domain or its homologue; (b) an ATR that contains a DNA-binding domain operably linked to a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof; (c) an ATR that includes a DNA-binding domain operably linked to a DNMT3L domain or a homolog thereof; and (d) an ATR that contains a DNA-binding domain operably linked to a SETDB1 domain or a homolog thereof; 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 consisting of: (a), (b), (c) or (d), wherein at least two of the ATRs are selected from different groups (a), (b), (c) or (d).

[29] Group (a), (b) or (c): (a) An artificial transcriptional repressor (ATR) that contains a DNA-binding domain operably linked to a KRAB domain or its homologue; (b) an ATR that includes 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. 1. 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 following, wherein at least two of the ATRs are selected from different groups (a), (b) or (c).

[30] Group (a), (b), (c) or (d): (a) An artificial transcriptional repressor (ATR) that contains a DNA-binding domain operably linked to a KRAB domain or its homologue; (b) an ATR that contains a DNA-binding domain operably linked to a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof; (c) an ATR that includes a DNA-binding domain operably linked to a DNMT3L domain or a homolog thereof; and (d) an ATR that contains a DNA-binding domain operably linked to a SETDB1 domain or a homolog thereof; A kit comprising two or more artificial transcriptional repressors (ATRs) selected from the following, or polynucleotides encoding them, wherein at least two of the ATRs are selected from different groups (a), (b), (c) or (d).

[31] Use of a product according to any one of [1] to

[11] for silencing a target gene, the use being in vitro or ex vivo.

[32] A method for silencing a target gene, comprising the step of administering to a cell the product described in any one of [1] to

[11] .

[33] An artificial transcriptional repressor (ATR) comprising a DNA-binding domain operably linked to a DNMT3A, DNMT3B or DNMT1 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 product comprising a polynucleotide encoding the same.

[34] The product described in

[33] , further comprising an ATR or a polynucleotide encoding the same, comprising a DNA-binding domain operably linked to a DNMT3L or KRAB domain or a homolog thereof.

[35] Group (a), (b), (c) or (d): (a) KRAB domain or its homologues; (b) a DNMT3A, DNMT3B or DNMT1 domain or a homolog thereof; (c) a DNMT3L domain or a homolog thereof; and (d) SETDB1 domain or its homologue 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).

[36] 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 An artificial transcriptional repressor (ATR) comprising a DNA-binding domain operably linked to two or more domains selected from the group (a), (b) or (c), wherein at least two of the domains operably linked to the DNA-binding domain are selected from different groups (a), (b) or (c).

[37] The ATR described in

[35] or

[36] , wherein 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.

[38] A polynucleotide encoding the ATR according to any one of

[35] to

[37] .

[39] 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 the group (a), (b), (c) or (d): (a) KRAB domain or its homologues; (b) a DNMT3A, DNMT3B or DNMT1 domain or a homolog 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).

[40] 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 the 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).

[41] The product of

[39] or

[40] , wherein 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.

[42] Group (a), (b), (c) or (d): (a) An artificial transcriptional repressor (ATR) that contains a DNA-binding domain operably linked to a KRAB domain or its homologue; (b) an ATR that contains a DNA-binding domain operably linked to a DNMT3A, DNMT3B, or DNMT1 domain or a homolog thereof; (c) an ATR that includes a DNA-binding domain operably linked to a DNMT3L domain or a homolog thereof; and (d) an ATR that contains a DNA-binding domain operably linked to a SETDB1 domain or a homolog thereof; A product comprising only one artificial transcriptional repressor (ATR) selected from, or a polynucleotide encoding the same, and one or more additional effector proteins or polynucleotides encoding them that are not operably linked to a DNA binding domain.

[43] Group (a), (b) or (c): (a) An artificial transcriptional repressor (ATR) that contains a DNA-binding domain operably linked to a KRAB domain or its homologue; (b) an ATR that includes 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. A product comprising only one artificial transcriptional repressor (ATR) selected from, or a polynucleotide encoding the same, and one or more additional effector proteins or polynucleotides encoding them that are not operably linked to a DNA binding domain.

[44] The one or more additional effector proteins not operably linked to the DNA-binding domain are selected from group (a), (b), (c) or (d): (a) KRAB domain or its homologues; (b) a DNMT3A, DNMT3B or DNMT1 domain or a homolog thereof; (c) a DNMT3L domain or a homolog thereof; and (d) SETDB1 domain or its homologue The product of

[42] or

[43] , comprising a domain selected from the group consisting of:

Claims

1. A method of using the product to silence a target gene, genetic element or splice variant in a cell in vitro, comprising the steps of: The product is a combination of groups (a) and (b), groups (a) and (c), groups (b) and (c), groups (b) and (d), and groups (c) and (d), or polynucleotides encoding them: (a) An artificial transcriptional repressor (ATR) that contains a DNA-binding domain operably linked to a KRAB domain; (b) an ATR comprising a DNA-binding domain operably linked to a DNMT3A, DNMT3B, or DNMT1 domain; (c) an ATR comprising a DNA-binding domain operably linked to a DNMT3L domain; and (d) an ATR that contains a DNA-binding domain operably linked to a SETDB1 domain; A combination of artificial transcriptional repressors (ATRs) selected from the group consisting of: The cells are selected from the group consisting of hematopoietic stem and progenitor cells, T lymphocytes, mesenchymal stem cells, fibroblasts, monocytes, epidermal and neural stem cells. method.

2. (i) the KRAB domain comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 1-7; (ii) the DNMT3A domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:8; the DNMT3B domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:9 or 36; and / or the DNMT1 domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:10; (iii) the DNMT3L domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 11; (iv) the SETDB1 domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 12 or 13; and / or (v) 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; The method of claim 1.

3. 3. The method of claim 1 or 2, wherein the polynucleotides encoding the combination of ATRs are in the form of a single vector or are contained within separate vectors.

4. the product further comprises another effector protein or a polynucleotide encoding the same that is not operably linked to the DNA-binding domain; The separate effector protein not operably linked to the DNA-binding domain is selected from group (a), group (b), group (c) or group (d): (a) KRAB domain; (b) a DNMT3A, DNMT3B or DNMT1 domain; (c) a DNMT3L domain; and (d) SETDB1 domain The method according to any one of claims 1 to 3, comprising a domain selected from:

5. The method according to any one of claims 1 to 4, wherein the product is in the form of a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier, diluent or excipient.

6. Group (a) and group (b), group (a) and group (c), group (b) and group (c), group (b) and group (d), and group (c) and group (d), or polynucleotides encoding them: (a) An artificial transcriptional repressor (ATR) that contains a DNA-binding domain operably linked to a KRAB domain; (b) an ATR comprising a DNA-binding domain operably linked to a DNMT3A, DNMT3B, or DNMT1 domain; (c) an ATR comprising a DNA-binding domain operably linked to a DNMT3L domain; and (d) an ATR that contains a DNA-binding domain operably linked to a SETDB1 domain; 1. A cell comprising a combination of artificial transcriptional repressors (ATRs) selected from the group consisting of: hematopoietic stem and progenitor cells, T lymphocytes, mesenchymal stem cells, fibroblasts, monocytes, epidermal and neural stem cells.

7. 7. A cell which is a progeny of the cell of claim 6, wherein the target gene, genetic element or splice variant is still silenced.

8. 8. A cell according to claim 6 or 7 for use in therapy.

9. 1. A method for generating cells for use in gene therapy, comprising the steps of: Groups (a) and (b), groups (a) and (c), groups (b) and (c), groups (b) and (d), and groups (c) and (d): (a) An artificial transcriptional repressor (ATR) that contains a DNA-binding domain operably linked to a KRAB domain; (b) an ATR comprising a DNA-binding domain operably linked to a DNMT3A, DNMT3B, or DNMT1 domain; (c) an ATR comprising a DNA-binding domain operably linked to a DNMT3L domain; and (d) an ATR that contains a DNA-binding domain operably linked to a SETDB1 domain; transfecting a cell with a polynucleotide encoding a combination of artificial transcriptional repressors (ATRs) selected from the group consisting of: The transfection is carried out ex vivo, the cells are selected from the group consisting of hematopoietic stem and progenitor cells, T lymphocytes, mesenchymal stem cells, fibroblasts, monocytes, epidermal and neural stem cells; The method.

10. 1. A method of using an artificial transcriptional repressor (ATR) comprising a DNA binding domain operably linked to a DNMT3A, DNMT3B or DNMT1 domain, and an ATR comprising a DNA binding domain operably linked to a SETDB1 domain, or a product comprising a polynucleotide encoding the same, to silence a target gene, genetic element or splice variant in a cell in vitro, comprising: the cells are selected from the group consisting of hematopoietic stem and progenitor cells, T lymphocytes, mesenchymal stem cells, fibroblasts, monocytes, epidermal and neural stem cells; method.

11. 1. A method of using an artificial transcriptional repressor (ATR) to silence a target gene, genetic element or splice variant in a cell in vitro, comprising: The ATR comprises a DNA binding domain operably linked to two or more domains selected from group (a), group (b), group (c) or group (d): (a) is the KRAB domain; (b) is a DNMT3A, DNMT3B, or DNMT1 domain; (c) is a DNMT3L domain; and (d) is the SETDB1 domain; The two or more domains include domains (a) and (b), (a) and (c), (b) and (d), or (c) and (d); the cells are selected from the group consisting of hematopoietic stem and progenitor cells, T lymphocytes, mesenchymal stem cells, fibroblasts, monocytes, epidermal and neural stem cells; method.

12. 1. A method of using a polynucleotide encoding an artificial transcriptional repressor (ATR) to silence a target gene, genetic element or splice variant in a cell in vitro, comprising: The ATR comprises a DNA binding domain operably linked to two or more domains selected from group (a), group (b), group (c) or group (d): (a) is the KRAB domain; (b) is a DNMT3A, DNMT3B, or DNMT1 domain; (c) is a DNMT3L domain; and (d) is the SETDB1 domain; The two or more domains include domains (a) and (b), (a) and (c), (b) and (d), or (c) and (d); The cells are selected from the group consisting of hematopoietic stem and progenitor cells, T lymphocytes, mesenchymal stem cells, fibroblasts, monocytes, epidermal and neural stem cells. method.

13. A method of using the product to silence a target gene, genetic element or splice variant in a cell in vitro, comprising the steps of: The product comprises two or more different artificial transcriptional repressors (ATRs) or polynucleotides encoding same, the two or more different ATRs being selected from group (a), group (b), group (c) or group (d): (a) KRAB domain; (b) a DNMT3A, DNMT3B or DNMT1 domain; (c) a DNMT3L domain; and (d) SETDB1 domain 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); The cells are selected from the group consisting of hematopoietic stem and progenitor cells, T lymphocytes, mesenchymal stem cells, fibroblasts, monocytes, epidermal and neural stem cells. method.

14. The method of any one of claims 1 to 5, wherein the product comprises an artificial transcriptional repressor (ATR) comprising a DNA binding domain operably linked to a KRAB domain, an ATR comprising a DNA binding domain operably linked to a DNMT3L domain, and an ATR comprising a DNA binding domain operably linked to a DNMT3A domain, or a polynucleotide encoding the same.

15. The method of any one of claims 1 to 5, wherein the product comprises an artificial transcriptional repressor (ATR) comprising a DNA binding domain operably linked to a KRAB domain, and an ATR comprising a DNA binding domain operably linked to a DNMT3L domain, or a polynucleotide encoding the same.

16. The method of any one of claims 1 to 5, wherein the product comprises an artificial transcriptional repressor (ATR) comprising a DNA-binding domain operably linked to a DNMT3L domain, and an artificial transcriptional repressor (ATR) comprising a DNA-binding domain operably linked to a DNMT3A domain, or a polynucleotide encoding the same.

17. The method of any one of claims 1 to 5, wherein the product comprises an artificial transcriptional repressor (ATR) comprising a DNA binding domain operably linked to a KRAB domain, and an artificial transcriptional repressor (ATR) comprising a DNA binding domain operably linked to a DNMT3A domain, or a polynucleotide encoding the same.

18. The method of claim 11, wherein the ATR comprises a DNA binding domain operably linked to a KRAB domain, a DNMT3A domain and a DNMT3L domain.

19. The method of claim 11, wherein the ATR comprises a DNA binding domain operably linked to a KRAB domain and a DNMT3L domain.

20. The method of claim 11, wherein the ATR comprises a DNA binding domain operably linked to a KRAB domain and a DNMT3A domain.

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