Synthetic transcription factors

Abstract

The present invention is generally in the fields of synthetic biology, gene therapy, and cell therapy.The present invention particularly relates to a synthetic transcription factor comprising a DNA binding domain derived from a mitochondrial DNA binding protein (for example, MTERF1) and a transcriptional regulatory domain derived from one or more other proteins; a nucleic acid or a combination of nucleic acids encoding the synthetic transcription factor of the present invention; a DNA construct comprising an MTERF1 binding site and a minimal promoter; a system comprising the synthetic transcription factor of the present invention and the DNA construct of the present invention; and its use, for example, medical use.

Description

[Technical Field]

[0001] The present invention generally relates to the fields of synthetic biology, gene therapy and cell therapy.The present invention particularly relates to a synthetic transcription factor comprising a DNA binding domain derived from mitochondrial DNA binding protein (for example, MTERF1) and a transcriptional regulatory domain derived from one or more other proteins; a nucleic acid or a combination of nucleic acids encoding the synthetic transcription factor of the present invention; a DNA construct comprising an MTERF1 binding site and a minimal promoter; a system comprising the synthetic transcription factor of the present invention and the DNA construct of the present invention; and its use, for example, medical use.Furthermore, the present invention also relates to a library of DNA constructs and their use in the method of optimizing promoter binding to transcription factor. [Background technology]

[0002] In gene and cell therapy (GCT), human-made gene sequences are at the heart of therapeutic formulations. In gene therapy, a delivery vector carrying the therapeutic gene sequence (the "payload") is administered directly to the patient, delivering the therapeutic payload to multiple cells in the patient's body. In cell therapy, the payload is introduced into cells, e.g., patient-derived cells, ex vivo, which are then (re)infused back into the patient [1].

[0003] Therapeutic transgenes encoded on the gene payload of GCTs are typically driven by constitutive or tissue-specific promoters. However, the repertoire of naturally occurring promoters for specific transgene expression is limited [2]. Alternatively, tissue or cell-type specificity can be achieved with the aid of engineered genetic networks (i.e., "biocomputing circuits") that process multiple cellular inputs (also entirely encoded on the gene payload) in a programmable, logical manner. For example, a cancer cell classifier circuit was engineered to restrict the expression of proapoptotic genes to cancer cells while sparing healthy ones [3]. In the context of chimeric antigen receptor (CAR) T-cell therapy, an "AND" gate was assembled, resulting in increased specificity for cancer cells that co-express two defined antigens on their surface [4]. Such circuits may lead to safer and more effective GCTs. However, circuits typically encode protein components ("auxiliary proteins") in addition to the therapeutic transgene. These protein components help implement a logical regulatory process for precise activation of therapeutic transgenes when multiple conditions are met in the target cell.

[0004] Auxiliary proteins, such as transcription factors, can increase toxicity and reduce the efficacy of biomedical treatments in human patients, particularly when these proteins are of non-human origin. The use of auxiliary proteins from non-human sources is driven by the so-called "orthogonality requirement": these proteins should not interfere with endogenous gene regulation in human cells, and they, or the processes they control, should not be modifiable by or interfere with endogenous human factors. A priori, there is concern that fully human proteins employed as auxiliary proteins in gene therapy payloads will engage with endogenous components in human cells, resulting in side effects and toxicity. To achieve orthogonality of auxiliary proteins, particularly when the proteins are transcription factors, prior art relies on non-human DNA-binding domains (DBDs) that do not have binding sites on the regulatory sequences of human genes. Non-human DBDs are often derived from prokaryotes and are further fused to transactivation domains of viral or human origin [5]. While meeting the orthogonality requirement, it has been found in animal models and human clinical trials that non-human proteins can elicit immune responses, leading to the elimination of cells expressing such proteins.[6][7] This results in reduced therapeutic efficacy of GCT products. Therefore, while the orthogonality requirement points to the use of proteins of non-human origin, the low immunogenicity requirement suggests the opposite, i.e., the use of human or nearly human proteins, which in turn may lead to reduced orthogonality. Thus, reconciling these two opposing requirements is difficult.

[0005] There are two current approaches to addressing the immunogenicity of transgenes in general, and proteins of non-human origin in particular.

[0006] The first approach aims to suppress the immune response while accepting potentially immunogenic protein sequences. In many cases, this results in systemic pharmacological suppression of the immune system. However, this strategy can increase the patient's risk of infection. Other strategies are inspired by or copy viral strategies against host defense, including prevention of proteasomal degradation and epitope production [9], downregulation of MHC class I

[10] , surface enzymes that degrade antibodies

[11] , and combinations thereof. Among the drawbacks of these strategies are the difficulty of fine-tuning and reliability, as well as the large DNA cargo, typically in cases where immune-evasive viral proteins are encoded on the delivery vector. A more targeted approach involves introducing antigen-presenting cell (APC)-specific miRNA targeting sites into the 5' or 3' UTR of the transgene

[12] . This prevents transgene expression in APCs, a central step in the induction of an immune response. Although shown to be effective in some conditions, this approach is ineffective in others, preventing its application to a wide range of diseases.

[13]

[0007] For certain delivery vectors, such as AAV, it has been shown that inhibiting AAV interaction with TLR9 on dendritic cells reduces the development of adaptive immunity against the viral capsid and transgene product

[14] .

[0008] Another approach is to attempt to reduce the immunogenicity of the protein itself by modifying its sequence or by intelligent selection. One strategy is to "humanize" the transgene, i.e., replace non-human protein domains with human-derived domains that are not recognized as foreign / non-self by the immune system. For synthetic transcription factors, this strategy is applicable to transactivation domains because these domains are generally not expected to crosstalk with endogenous processes by themselves and do not lead to such crosstalk. However, it is very difficult to apply the strategy to DNA-binding domains (DBDs), because a DBD of human origin poses the risk of interfering with endogenous gene expression. In one previously proposed solution for "humanizing" DBDs, zinc finger (ZNF) protein domains derived from human proteins, each with a DNA-binding specificity of 2–3 DNA base pairs, were fused to each other to recognize longer DNA sequences not naturally found in the human genome, thereby fulfilling the orthogonality criterion

[15]

[16] . However, the fusion of some ZNF domains results in multiple domain junction regions of non-human sequences that may themselves be immunogenic. Therefore, such artificial DBDs are largely non-human. Especially given the large patient pool with highly diverse MHC, TCR, and antibody repertoires, immune responses to junction-derived peptides are likely to still occur. Another approach to humanizing the DBD of a synthetic transcription factor (TF) is to utilize a naturally occurring human DBD. This reduces the number of novel (non-human) junctions to one—the junction between the human-derived DBD and the human-derived TAD—thus minimizing the potential for immunogenicity. Typically, this involves employing the DBD of a human TF that is not expressed in the cell type in which the synthetic TF is expressed

[17] . However, there is a risk that chimeric proteins assembled according to this strategy will bind to their corresponding response elements in the human genome, thereby leading to unwanted expression of endogenous human genes and violating the requirement of orthogonality.

[0009] Thus, there remains a need for improved means and methods for regulating gene expression, particularly in humans. Summary of the Invention

[0010] The present invention relates to the embodiments characterized in the claims and described herein below.

[0011] Thus, the present invention relates to synthetic transcription factors comprising (i) a DNA-binding domain (DBD) derived from a mitochondrial DNA-binding protein and (ii) a transcriptional regulatory domain derived from one or more other proteins.

[0012] The present invention is based, at least in part, on the surprising discovery that synthetic transcription factors that contain as their DNA-binding domain (DBD) a DBD from a mitochondrial DNA-binding protein, such as MTERF1, are capable of regulating transcription in cells, specifically in the nucleus of the cell.

[0013] As illustrated in the accompanying examples, synthetic transcription factors, specifically (i) a C-terminal fragment of the human MTERF1 protein containing the DNA binding domain but lacking the mitochondrial transit peptide (MTP), and (ii) the human transactivation domain RelA, 430-551 It has been found that a fusion protein comprising the synthetic transcription factor (referred to as "MTF") can promote the transcription of a gene of interest (Gol) in a cell, specifically in the nucleus of the cell. Furthermore, in this context, a DNA construct, i.e., a gene expression construct, has been developed that comprises a promoter containing a response element (RE) for binding of this synthetic transcription factor and a minimal promoter. Here, the promoter can be operably linked to a gene of interest.

[0014] As further illustrated in the accompanying examples, it has surprisingly been found that the synthetic transcription factors of the invention, e.g., MTF proteins, and transcription systems of the invention, e.g., including synthetic transcription and DNA constructs, have high orthogonality in cells of interest, e.g., human cells; see e.g., Example 3.

[0015] Specifically, we were able to demonstrate that the synthetic transcription factor, i.e., MTF protein, diffuses within the cell, including the nucleus (see, e.g., Figure 7A). This functionality is in stark contrast to the WT MTERF1 protein, which is localized exclusively to mitochondria. Furthermore, the ability of the synthetic transcription factor to promote transcription of a gene of interest from a synthetic DNA construct was not affected in cells when the WT MTERF1 protein was overexpressed (see, e.g., Figure 7C).

[0016] Using RNA-Seq, we further found that MTF does not retain the gene regulatory functionality of WT MTERF1. Surprisingly, only a very small number of genes were differentially expressed upon transfection of the MTF construct. This demonstrates high orthogonality to endogenous gene regulatory processes in human cells (see, for example, Figure 8).

[0017] Thus, the present inventors have unexpectedly developed a synthetic transcription factor containing a human DNA-binding domain that has extremely high orthogonality in human cells. Specifically, (i) endogenous human gene expression is not substantially modulated by the synthetic transcription factor of the present invention, and (ii) endogenously expressed wild-type MTERF1 does not regulate, i.e., disrupt, the expression of a gene of interest driven by a promoter containing a corresponding response element (i.e., the DNA construct of the present invention) in human cells. In other words, the inventive means of the present invention illustrated in the accompanying examples do not substantially interfere with endogenous gene regulation in human cells, and transcription of the gene of interest is not interfered with by the endogenous human factor, i.e., WT MTERF1.

[0018] Thus, the present invention provides, inter alia, improved components for improved transcription systems, specifically improved synthetic transcription factors and corresponding gene expression constructs, which function in a substantially orthogonal manner in cells of certain biological species, e.g., humans.

[0019] High orthogonality in cells, e.g., human cells, is advantageous for ensuring reliable control of the expression of a gene of interest, which may encode, for example, a pro-apoptotic protein. Specifically, high orthogonality ensures that a gene of interest is expressed only in the cells in which it should be expressed and only when it should be expressed. In addition, the high orthogonality of synthetic transcription systems ensures that endogenous gene expression is not perturbed or disrupted in an undesired manner, thereby reducing the risk of side effects or toxicity.

[0020] Specifically, high orthogonality is highly advantageous in the context of engineered genetic networks (i.e., "biocomputing circuits") that process multiple cellular inputs in a programmable, logical manner, e.g., in cancer cell classifier circuits.

[0021] Gene therapy products that require engineered transcription factors as part of their mechanism of action, such as gene therapy products that operate as multi-component networks otherwise known as "biocomputing gene circuits," are expected to have a favorable safety profile when using synthetic transcription factors according to the present invention compared to alternatives.

[0022] Thus, the present invention further enables more effective and / or safer means as described herein for gene and / or cell therapy, for example, for therapies employing biocomputing circuitry.

[0023] As used herein, gene refers to the sequence of nucleotides on DNA that is transcribed to produce functional RNA.Gene can be a protein-coding gene or a non-coding gene.In addition, gene is usually associated with at least one regulatory sequence, such as promoter and optionally enhancer, which can be involved in the transcription of gene.The promoter that can be involved in the transcription of gene can also be considered to be operably linked to gene.As further described herein, regulatory sequence (for example, promoter) usually comprises at least one response element, and response element comprises at least one binding site for transcription factor.

[0024] As used herein, transcription factor refers to a protein that regulates the transcription of one or more genes. Specifically, transcription factor controls the transcription rate of a gene, i.e., the transcription of genetic information from DNA to messenger RNA, for example, by binding to a specific DNA sequence on a promoter, i.e., a response element (RE). A "response element" can also be referred to as a "transcription factor binding site" or can contain at least one transcription factor binding site.

[0025] A defining feature of transcription factors is that they contain at least one DNA-binding domain (DBD), which binds to or attaches to specific sequences of DNA (i.e., regulatory sequences) adjacent to or at some distance from the genes they regulate. Specifically, the DBD binds to response elements, such as promoters or enhancers, contained in the regulatory sequences.

[0026] Furthermore, transcription factors contain transcriptional regulatory domains, e.g., activation or repression domains, which typically contain interaction sites for other proteins, such as transcriptional coregulators. Activation domains may also be referred to as "transactivation domains (TADs)" or "transcriptional activation domains." Similarly, repression domains may be referred to as "transcriptional repression domains."

[0027] Optionally, transcription factors may further contain a signal sensing domain (SSD) (e.g., a ligand binding domain) that senses and responds to external signals, transmitting these signals to the rest of the transcription complex, resulting in up- or down-regulation of gene expression.

[0028] Although transcription factors can act alone, they often work in complexes with other proteins by promoting (as activators) or inhibiting (as repressors) the recruitment of RNA polymerase, the enzyme that transcribes genetic information from DNA to RNA, to specific genes, such as the target genes described herein. Thus, the DNA-binding domain of a transcription factor guides the transcription factor to the response elements contained in the gene's regulatory sequences, specifically the promoter or enhancer associated with the gene, and the transcriptional regulatory domain typically promotes or inhibits gene transcription in cooperation with endogenous transcriptional regulators that bind to or interact with the transcriptional regulatory domain, i.e., coregulators described further herein below. Specifically, a transcription factor can stimulate transcription initiation, particularly when it has an activation domain, or can inhibit transcription initiation, particularly when it has a repression domain. For example, a transcription factor can facilitate RNA polymerase binding to DNA, which can promote transcription, particularly transcription initiation. Alternatively, a transcription factor can inhibit RNA polymerase binding to DNA, which can inhibit transcription, particularly transcription initiation.

[0029] In this specification and in the context of the present invention, the term "synthetic" specifically means that a compound, e.g., a transcription factor, is composed of at least two moieties that do not naturally occur together in such a manner. Specifically, a synthetic protein, e.g., a synthetic transcription factor, contains one moiety derived from one type of protein and at least one other moiety derived from at least one other protein.

[0030] Specifically, as used herein, the synthetic transcription factors of the present invention comprise (i) a DNA-binding domain (DBD) derived from a certain protein, namely, a mitochondrial DNA-binding protein, and (ii) a transcriptional regulatory domain derived from one or more other proteins.

[0031] In a preferred embodiment of the present invention, the synthetic transcription factor is a fusion protein.

[0032] Fusion protein as used herein and in the context of the present invention refers to a synthetic protein, such as a synthetic transcription factor, in which at least two or all parts of the protein, particularly parts that do not naturally occur together on a single polypeptide, are contained on one amino acid chain, i.e., one polypeptide.

[0033] Thus, in a preferred embodiment, the synthetic transcription factor of the present invention is a fusion protein comprising a DNA-binding domain according to the present invention and a transcriptional regulatory domain according to the present invention. Specifically, the two domains are connected in the fusion protein via a peptide bond, either directly or via a peptide linker. In other words, in a preferred embodiment, the DNA-binding domain according to the present invention and the transcriptional regulatory domain according to the present invention are contained on a single amino acid chain, i.e., a single polypeptide. More preferably, in the context of these embodiments, essentially all portions of the synthetic transcription factor of the present invention are contained on a single polypeptide.

[0034] In a further embodiment of the present invention, a synthetic transcription factor comprises or consists of a first and a second polypeptide, wherein said first polypeptide comprises a DNA-binding domain in accordance with the present invention and said second polypeptide comprises a transcriptional regulatory domain in accordance with the present invention. Specifically, each of the first and second polypeptides comprises a multimerization domain as described herein, wherein the multimerization domains of the first and second polypeptides are capable of binding to and / or interacting with each other.

[0035] In the present specification and the context of the present invention, mitochondrial DNA binding protein refers to the DNA binding protein that normally resides in mitochondria.Specifically, mitochondrial DNA binding protein binds to mitochondrial DNA in a sequence-specific manner.For example, mitochondrial DNA binding protein can be mitochondrial transcription factor or mitochondrial transcription termination factor.

[0036] Preferably, herein and in the context of the present invention, the mitochondrial DNA binding protein is derived from a mammalian species. Preferably, herein, the mammalian species is human.

[0037] In the context of the present invention, it has further been found that MTERF1 has large recognition sites that are rare or absent on gene regulatory sequences on the nuclear genome of human cells, and that MTERF1 does not have any perfect binding sites on the human nuclear genome, which is particularly beneficial for high orthogonality, e.g., in human cells, as described herein.

[0038] Therefore, the mitochondrial DNA binding protein herein and in the context of the present invention is preferably MTERF1, preferably human MTERF1, i.e., Uniprot Q99551. Specifically, human MTERF1, i.e., wild-type (WT) MTERF1, has the amino acid sequence shown in SEQ ID NO: 108.

[0039] However, MTERF1 can also be from other species, such as mouse or dog. Specifically, mouse MTERF1 has the amino acid sequence set forth in SEQ ID NO: 111 or 113. Furthermore, canine MTERF1 has the amino acid sequence set forth in SEQ ID NO: 115. Furthermore, MTERF1 orthologous sequences from other species, such as mammalian species, are readily available to those skilled in the art and can also be used herein and in the context of the present invention.

[0040] In the present specification and the context of the present invention, the term "derived from" should be interpreted in a technically meaningful manner.For example, a domain derived from a certain protein refers to a part or fragment of said protein, which may further comprise at least one modification, particularly at least one amino acid substitution, deletion, and / or insertion.The degree of modification can be determined by the sequence identity with reference sequence.

[0041] Furthermore, a domain derived from a certain protein specifically has qualitatively similar functionality to a domain on said protein (although functionality may be enhanced or reduced to some extent). Specifically, the DNA binding domain derived from certain DNA binding proteins, for example MTERF1, has the ability to bind to DNA in a sequence-specific manner, specifically to the response element of said DNA binding protein, for example MTERF1.

[0042] Similarly, a transcriptional regulatory domain, e.g., an activation domain, derived from a transcription factor such as certain transactivator proteins, e.g., RELA, specifically has the ability to regulate, e.g., promote, the transcription of a gene when it binds to or is located in close proximity to a regulatory sequence, e.g., a response element, of the gene.

[0043] Generally, herein and in the context of the present invention, an amino acid sequence (e.g., of a certain protein domain or motif) or a DNA sequence (e.g., of a binding site or minimal protein) can be defined by a certain % sequence identity to a reference sequence. Thus, the term "sequence identity" is used herein specifically to describe the sequence relationship between two or more amino acid sequences, proteins (or fragments thereof), or polypeptides (or fragments thereof). Specifically, a sequence can have at least n% sequence identity to a reference sequence, where n is an integer between 60 and 100, e.g., 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99.

[0044] For example, an amino acid sequence may have at least 60%, 70%, 80%, or 90%, preferably at least 80%, 85%, 90%, or 95%, more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in a certain SEQ ID NO. The same applies mutatis mutandis to nucleic acid sequences, e.g., DNA sequences.

[0045] Generally, the higher the % sequence identity, the more preferred the sequence. However, additional preferred % identities are directly described in the context of certain embodiments herein. Furthermore, it should be noted that the present invention is in no way limited to high or preferred sequence identities, and that, for example, any of the % sequence identities described above are contemplated.

[0046] The term "sequence identity" as used herein and in the context of the present invention has essentially the same meaning as commonly used and understood by those skilled in the art. The degree of sequence identity can be determined according to methods well known in the art, preferably using a suitable computer algorithm such as CLUSTAL.

[0047] When using the Clustal analysis method to determine whether a particular sequence is, for example, at least 60% identical to a reference sequence, default settings can be used.

[0048] In a preferred embodiment, Clustal Omega (Madeira F, Park YM, Lee J, et al. The EMBL-EBI search and sequence analysis tools APls in 2019. Nucleic Acids Research. 2019 Jul;47(W1):W636-W641. DOI:10.1093 / nar / gkz268. PMID:30976793; PMCID:PMC6602479) is used for comparing amino acid sequences. In the case of pairwise comparison / alignment, the following default settings are preferably chosen:Program:clustalo;Version:1.2.4;Input Parameters:Output guide tree:true;Output distance matrix:false;Dealign input sequences:false;mBed-like clustering guide tree:true;mBed-like clustering iteration:true;Number of iterations:O;Maximum guide tree iterations:-1;Maximum HMM iterations:-1;Output alignment format:clustal_num;Output order:aligned;Sequence Type:protein. Preferably, the degree of identity is calculated over the entire length of the sequences.

[0049] Furthermore, the amino acid residues located at a position corresponding to a position on the reference sequence can be identified by those skilled in the art by methods known in the art. Alignment can be performed by means and methods known to those skilled in the art, for example, by using a known computer algorithm such as the Lipman-Pearson method (Science 227 (1985), 1435) or the CLUSTAL algorithm. In such alignment, it is preferred that maximum homology be assigned to conserved amino acid residues present in the amino acid sequence.

[0050] In a preferred embodiment, Clustal Omega is used for comparing amino acid sequences. In the case of pairwise comparison / alignment, the following default settings are preferably selected: Program: clustalo; Version: 1.2.4; Input Parameters: Output guide tree: true; Output distance matrix: false; Dealign input sequences: false; mBed-like clustering guide tree: true; mBed-like clustering iteration: true; Number of iterations: 0; Maximum guide tree iterations: -1; Maximum HMM iterations: -1; Output alignment format: clustal_num; Output order: aligned; Sequence Type: protein.

[0051] In the context of the present invention, "amino acid substitution" means that the respective amino acid residue at the indicated position can be substituted by any other possible amino acid residue, e.g., a naturally occurring or non-naturally occurring amino acid (Brustad and Arnold, Curr. Opin. Chem. Biol. 15 (2011), 201-210).

[0052] Generally, in the context of this specification and the present invention, a feature that "has" a certain sequence may "comprise" said sequence, may "be defined by" said sequence, or may "consist of" said sequence.

[0053] In the context of this specification and the present invention, a DNA-binding domain according to the invention may have at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to SEQ ID NO: 1. In certain preferred embodiments, the DNA-binding domain has the sequence shown in SEQ ID NO: 1.

[0054] As illustrated in the accompanying examples, in the context of the present invention, it has further surprisingly been found that the DNA-binding domain of MTERF1 can be, for example, N-terminally truncated and still retain sufficient functionality, i.e., bind to its response element and provide functionality to a synthetic transcription factor (see, e.g., Figure 3). Shorter DNA-binding domains can be advantageous because they have a reduced DNA footprint / gene payload, for example, for viral transduction. Furthermore, adjusting the length of the MTERF1-binding domain allows for altering the expression level of a gene of interest.

[0055] Thus, a DNA binding domain according to the present invention may comprise MTERF1 subdomain B having the sequence shown in SEQ ID NO:7 or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO:7.

[0056] Surprisingly, it has further been found that one MTERF1 motif on the MTERF1 DNA binding domain can be omitted while the functionality of the DNA binding domain is substantially retained.

[0057] Thus, a DNA binding domain according to the present invention may comprise MTERF1 subdomain A (i.e., a shorter subdomain) having the sequence shown in SEQ ID NO: 9 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO: 9.

[0058] Thus, a DNA-binding domain according to the present invention may comprise (i) a first MTERF1 motif having the sequence set forth in SEQ ID NO: 104 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO: 104, and / or (ii) a second MTERF1 motif having the sequence set forth in SEQ ID NO: 106 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO: 106. Preferably, the first MTERF1 motif is N-terminal to the second MTERF1 motif. Furthermore, the first and second MTERF1 motifs may be directly adjacent to each other (preferably due to a peptide bond) or may be connected via a linker (e.g., by a peptide linker).

[0059] Preferably, the DNA binding domain comprising the first and / or second MTERF1 motif further comprises (iii) an MTERF1 C-terminal domain having the sequence shown in SEQ ID NO: 11, or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 11.

[0060] Specifically, the first MTERF1 motif and / or the second MTERF1 motif described herein is contained in subdomain A described herein. Furthermore, MTERF1 subdomain A described herein is specifically contained in MTERF1 subdomain B described herein.

[0061] Furthermore, it is possible that a small number of amino acids, for example, about 1 to 30 or about 1 to 10 amino acids, can be deleted from the C-terminus of an MTERF1-derived DNA-binding domain or MTERF1-derived DNA-binding subdomain in accordance with the invention. It may also be possible to delete the entire C-terminal most subdomain of an MTERF1-derived DNA-binding domain or MTERF1-derived DNA-binding subdomain in accordance with the invention.

[0062] Preferably, in the present specification and in the context of the present invention, the MTERF1-derived DNA binding domain of the present invention exhibits an arginine (R) at a position corresponding to position 387 on the sequence of SEQ ID NO: 108, for example at position 330 on the sequence of SEQ ID NO: 1.

[0063] Preferably, herein and in the context of the present invention, a synthetic transcription factor does not comprise a mitochondrial transit peptide having the sequence shown in SEQ ID NO: 37 or a sequence having at least 90% sequence identity to SEQ ID NO: 37. Preferably, the synthetic transcription factor does not have any functional mitochondrial transit peptide. Specifically, the synthetic transcription factors of the present invention do not comprise a mitochondrial transit peptide at the N-terminus.

[0064] As used herein, a "mitochondrial transit peptide" may also refer to a "mitochondrial targeting signal." Specifically, a mitochondrial transit peptide directs a protein to mitochondria, enabling the protein to enter and / or localize in mitochondria.

[0065] However, the synthetic transcription factors of the present invention are preferably unable to enter or localize to mitochondria. Highly preferably, in the context of this specification and the present invention, the synthetic transcription factors of the present invention are capable of entering and / or localizing in the cell nucleus, which is particularly advantageous for achieving the high orthogonality described herein.

[0066] Therefore, the synthetic transcription factor of the present invention can have the ability to localize more efficiently in the nucleus of a cell than in the mitochondria of said cell. This ability can be determined by separately measuring the amount of the synthetic transcription factor in the nucleus and mitochondria of the same cell(s). This can be done by conventional methods in the art, such as immunostaining, separation of nuclei and mitochondria, followed by Western blot or ELISA.

[0067] A synthetic transcription factor of the invention can enter and / or localize to the cell nucleus in the absence of a functional mitochondrial transit peptide described herein, for example, as set forth in SEQ ID NO: 37. Furthermore, a synthetic transcription factor of the invention can enter and / or localize to the cell nucleus when it comprises a nuclear localization signal.

[0068] Thus, the synthetic transcription factor of the present invention may contain a nuclear localization signal (NLS), which is also referred to as a "nuclear localization peptide." NLS sequences are well known in the art, and in principle, any of them can be employed.

[0069] As used herein, the term "gene expression" always encompasses the term "gene transcription" or "transcription of a gene," but it may also encompass post-transcriptional mechanisms in certain circumstances. As used herein, the term "gene transcription" or "transcription of a gene" refers to gene expression in a more specific manner. However, since the present invention specifically relates to means and methods for regulating gene transcription, the term "gene expression" may be replaced herein by the term "gene transcription" or "transcription of a gene," unless explicitly indicated differently.

[0070] Specifically, herein and in the context of the present invention, a synthetic transcription factor is capable of regulating the transcription of at least one gene of interest in a cell, preferably said synthetic transcription factor is capable of regulating the transcription of at least one gene of interest in the nucleus of a cell.

[0071] The synthetic transcription factors of the invention can regulate transcription of a gene of interest in the same manner as described herein in the context of transcription factors generally. Specifically, the synthetic transcription factors of the invention can control the rate of transcription of a gene of interest. Furthermore, the synthetic transcription factors of the invention can induce or initiate transcription of a gene of interest.

[0072] The gene of interest used in the present specification and the present invention is not limited to any gene.Preferably, in the present specification and the present invention, the gene of interest encodes, for example, cell death-promoting protein such as hBAX or HSV-TK, immune-stimulating cytokine such as IL-2 or IL-12, or antigen receptor such as CAR or TCR.

[0073] hBAX refers to a proapoptotic protein, which can be used in cell sorters to kill cells, for example, in cancer cell sorters to kill cancer cells. HSV-TK refers to a protein that metabolizes ganciclovir into toxic metabolites. It can be used in cell sorters to kill cells, for example, in cancer cell sorters to kill cancer cells. IL-2 or IL-12 are immune-stimulating cytokines, which can be used in cell sorters, for example, in cancer cell sorters, to attract and induce proliferation of T cells. CAR and TCR refer to antigen receptors that recognize cells with corresponding surface antigens. They can be used, for example, in conjunction with the SynNotch system; Morsut (2016), Cell 164(4):780-91.

[0074] Preferably, in the context of the present specification and the present invention, the synthetic transcription factor regulates the transcription of a gene of interest in a cell by (i) promoting transcription of the gene of interest or (ii) suppressing transcription of the gene of interest. The synthetic transcription factors of the present invention described herein preferably promote or suppress transcription of the gene of interest in the nucleus of the cell. This is particularly advantageous for achieving the high orthogonality described herein.

[0075] Specifically, the synthetic transcription factors of the present invention can promote the transcription of genes by increasing the rate of transcription. Furthermore, the synthetic transcription factors of the present invention can repress the transcription of genes by decreasing the rate of transcription.

[0076] As used herein, promoting gene transcription can include, for example, inducing, initiating, and / or enhancing gene transcription. Furthermore, suppressing gene transcription can include, for example, blocking or repressing gene transcription, for example, blocking or repressing the induction or initiation of gene transcription.

[0077] Preferably, herein and in the context of the present invention, the synthetic transcription factor is capable of binding to a response element in a cell, preferably in the nucleus of a cell. Specifically, the DNA binding domain comprised in the synthetic transcription factor of the present invention is capable of binding to a response element in a cell, preferably in the nucleus of a cell.

[0078] In the present specification and in the context of the present invention, a response element may comprise an MTERF1 binding site having the sequence shown in SEQ ID NO: 42 or a sequence having at least 60%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 42. Preferably, an MTERF1 binding site according to the present invention consists of the sequence shown in SEQ ID NO: 42 or a sequence having at least 60%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 42.

[0079] Specifically, a synthetic transcription factor and / or DNA binding domain according to the invention can bind to at least one MTERF1 binding site on a response element described herein.

[0080] It has further been found that the expression level of a gene of interest can be regulated by modifying the design of the response element-containing promoter and the amount of synthetic transcription factor expressed in cells. Furthermore, it has been observed that the copy number of REs and the spacing between them also affect GoI expression at a certain MTF level (see, for example, Figure 1). It should be noted that in Figure 1, the term "response element" (RE) corresponds to the term "binding site" used herein, specifically the MTERF1 binding site defined by SEQ ID NO: 42.

[0081] A response element according to the invention, in the context of a DNA construct of the invention, can also comprise multiple copies of the MTERF1 binding site described herein, for example, 2 to 50, 2 to 25, or 2 to 15 copies, preferably 2 to 5 copies. Moreover, the individual copies of the binding site need not be identical to each other and can include variation.

[0082] Moreover, the copies of the binding site on the response element, for example two or more or all copies, may be immediately adjacent to each other or may be separated by one or more, for example 1 to 100, 1 to 50, 1 to 20, 1 to 10, for example 6 or 10 nucleotides.

[0083] The synthetic transcription factor of the present invention can specifically bind to a promoter comprising a response element as described herein in a cell, preferably in the nucleus of the cell.The promoter may further comprise a minimal promoter, for example, a minimal TATA box, preferably having the sequence shown in SEQ ID NO: 103, or a minimal CMV promoter, preferably having the sequence shown in SEQ ID NO: 136.Preferably, the minimal promoter is located 3' of the response element.For example, the promoter has the sequence shown in SEQ ID NO: 110.

[0084] Additional promoters, response elements, and minimal promoters are described herein in the context of the DNA constructs of the present invention and may also be considered and employed in that particular context herein.

[0085] Preferably, herein and in the context of the present invention, the promoter is operably linked to a gene of interest as described herein, preferably in the cell nucleus as described herein.

[0086] Specifically, in the context of this specification and the present invention, binding of a synthetic transcript of the present invention (particularly of a DNA binding domain) to a promoter of the present invention (particularly to a response element of the present invention on said promoter) regulates transcription of a gene of interest that is operably linked to said promoter as described herein, preferably in the nucleus of a cell.

[0087] The orthogonality of the synthetic transcription factors of the invention or transcription systems of the invention described herein is specifically assessed in cells that are derived from the same species, e.g., the same mammalian species, from which the mitochondrial DNA-binding protein according to the invention (and thus the DNA-binding domain contained in the synthetic transcription factors of the invention) is derived. In this specification, such cells are also referred to simply as "allogenic cells."

[0088] The synthetic transcription factors of the invention and transcription systems of the invention described herein can function in a manner that is highly orthogonal to endogenous processes, particularly gene regulatory processes, in cells (i.e., cells of the same species).

[0089] Thus, in the context of this specification and the present invention, a cell, particularly a cell whose transcription is regulated by a synthetic transcription factor of the present invention, is derived from the same species, e.g., the same mammalian species, from which the mitochondrial DNA-binding protein employed in the context of the present invention (and thus the DNA-binding domain on the synthetic transcription factor of the present invention) is derived. That is, as described herein, it is an allogeneic cell. Preferably, the DNA-binding domain is derived from a human mitochondrial DNA-binding protein (e.g., human MTERF1), and thus the cell (i.e., the allogeneic cell) is preferably a human cell.

[0090] Preferably, the synthetic transcription factors of the invention herein essentially do not modulate transcription, ie the transcriptome, in the same species of cell other than transcription of the gene(s) of interest.

[0091] Furthermore, it is possible that the synthetic transcription factors of the invention do not specifically bind to essentially any endogenous DNA sequences in the same species of cell. Preferably, the synthetic transcription factors of the invention do not specifically bind to essentially any DNA sequences in the cell other than the promoter, particularly the promoter containing the response element according to the invention.

[0092] Furthermore, a DNA-binding domain in accordance with the present invention (i.e., a DNA-binding domain contained in a synthetic transcription factor of the present invention) preferably does not specifically bind to essentially any endogenous DNA sequences in the nucleus of a cell of the same species.

[0093] Furthermore, it is possible that the synthetic transcription factors of the invention do not essentially compete with the mitochondrial DNA binding protein from which the DBD according to the invention is derived for sequence-specific DNA binding in homologous cells.

[0094] Furthermore, it is possible that the synthetic transcription factors of the present invention do not essentially interfere with the function of the mitochondrial DNA binding protein from which the DBD according to the present invention is derived.

[0095] It is also possible that the synthetic transcription factors of the invention do not essentially interfere with the function of the proteins from which the transcriptional regulatory domains according to the invention are derived.

[0096] The transcriptional regulatory domains described herein are typically involved in promoting or repressing transcription. Typically, transcriptional regulatory domains contain interaction sites for other proteins, such as transcriptional coregulators. A transcriptional coregulator is a protein that interacts with a transcription factor to either promote or repress the transcription of a specific gene. A transcriptional coregulator that activates gene transcription is called a coactivator, while one that represses is known as a corepressor. An activation domain as used herein may bind to a coactivator rather than a corepressor, while a repression domain as used herein may bind to a corepressor rather.

[0097] The primary mechanism of action of transcriptional coregulators is to modify chromatin structure, thereby making the associated DNA more or less accessible to transcription. In humans, anywhere from several dozen to several hundred coregulators are known, depending on the level of confidence with which proteins can be characterized as coregulators. For example, one class of transcriptional coregulators alters chromatin structure through covalent modifications of histones, while a second, ATP-dependent class alters chromatin conformation.

[0098] Exemplary coactivators include, inter alia, preinitiation complexes containing, for example, transcription factor IID (TFIID), the Mediator complex, histone acetyltransferases, and chromatin remodeling complexes. Exemplary corepressors include, inter alia, Polycomb repressive complexes, e.g., PRC1 or PRC2, histone deacetylases, and histone methyltransferases.

[0099] In the context of this specification and the present invention, a transcriptional regulatory domain is specifically capable of regulating the transcription of a gene, and specifically wherein said transcriptional regulatory domain is part of, binds to or interacts with a DNA binding protein that is capable of binding to or interacting with a regulatory sequence, such as a promoter or enhancer, of said gene.

[0100] In the context of the present invention, since a transcriptional regulatory domain is contained in the synthetic transcription factor of the present invention together with a DNA-binding domain according to the present invention, the transcriptional regulatory domain can specifically interact with said DNA-binding domain and thus be directed to a gene regulatory sequence, e.g., a promoter as described herein, and regulate the transcription of the corresponding gene.

[0101] Specifically, a transcriptional regulatory domain in accordance with the present invention may be capable of binding to and / or interacting with an RNA polymerase, preferably RNA polymerase II; at least one other transcription factor, such as a general transcription factor (e.g., TFIID); and / or at least one transcriptional coregulator, such as a transcriptional coactivator (e.g., the Mediator complex and / or a histone acetyltransferase) and / or a transcriptional corepressor (e.g., a Polycomb repressive complex or a histone deacetylase), as described herein.

[0102] In the present specification and in the context of the present invention, the transcriptional regulatory domain may be (i) an activation domain or (ii) a repression domain as described herein. Preferably, the transcriptional regulatory domain according to the present invention is an activation domain.

[0103] Specifically, a transcriptional regulatory domain can either (i) promote transcription of a gene (specifically when defined as an activation domain) or (ii) repress transcription of a gene (specifically when defined as a repression domain). Preferably, a transcriptional regulatory domain according to the present invention can (i) promote transcription of a gene.

[0104] Thus, a transcriptional regulatory domain according to the present invention may be (i) an activation domain that binds and / or interacts with at least one co-activator to promote gene transcription, or (ii) a repression domain that binds and / or interacts with at least one co-repressor to repress gene transcription; preferably, an activation domain as described in (i) above.

[0105] The present invention is not particularly limited to the transcriptional regulatory domain; many suitable activation and repression domains are readily available and can be employed in the context of the present invention. The functionality of the transcriptional regulatory domain in the context of the synthetic transcription factors of the invention provided herein can be easily tested and verified by routine means, for example, as described in the accompanying Examples and as shown, for example, in Figure 4. For example, to assess the functionality of an activation domain in the context of the present invention, the following simple assay can be performed: A reporter DNA construct (e.g., a plasmid) containing the promoter set forth in SEQ ID NO: 110 operably linked to a gene encoding a detectable protein (e.g., a fluorescent protein) is introduced (e.g., transfected) into suitable cells. In addition, a DNA construct encoding the transcriptional regulatory domain to be tested fused to the human MTERF1-binding domain set forth in SEQ ID NO: 1 under the control of a constitutive promoter, such as EF1a (SEQ ID NO: 137), CMV (SEQ ID NO: 138), or UbC (SEQ ID NO: 139), is introduced (e.g., transfected) into the same cells. Furthermore, a construct encoding an additional constitutively expressed protein that is detectable independently of the detectable protein encoded on the reporter construct is introduced into the same cells. The amounts of both detectable proteins are then quantified (e.g., by flow cytometry, microscopy, ELISA, or Western blot). The "signal" is determined as the ratio between the reporter and constitutively expressed detectable proteins. As a negative control, the same assay is performed, but the construct encoding the transcriptional regulatory domain-MTERF1 fusion protein is not introduced into the cells.

[0106] The signal of the experimental condition is then compared to that of the negative control, and if the signal is higher relative to the negative control, it is determined that the activation domain is functional in the context of the synthetic transcription factor of the invention (i.e., it is capable of promoting transcription of a gene of interest).

[0107] In cases where the transcriptional regulatory domain is a repression domain, the following assay can be performed to assess its functionality: A promoter to be repressed, e.g., EF1a (SEQ ID NO: 137), CMV (SEQ ID NO: 138), or UbC (SEQ ID NO: 139), which also contains a binding site for a transcription factor of the invention either within the promoter sequence or within 0-2000 bases adjacent to the 5' or 3' end of the promoter, is operably linked to a gene encoding a detectable protein on a reporter DNA construct (e.g., a plasmid), and this reporter DNA construct is introduced (e.g., transfected) into a suitable cell.

[0108] In addition, a DNA construct encoding the transcriptional regulatory domain to be tested fused to the human MTERF1 binding domain set forth in SEQ ID NO: 1 under the control of a constitutive promoter, e.g., EF1a (SEQ ID NO: 137), CMV (SEQ ID NO: 138), or UbC (SEQ ID NO: 139), is introduced (e.g., transfected) into the same cells.

[0109] Furthermore, a construct encoding a constitutively expressed additional protein that can be detected independently of the detectable protein encoded on the reporter construct is introduced into the same cells.Then, the amount of both detectable proteins is quantified (for example, by flow cytometry, microscopy, ELISA, or Western blot).The "signal" is determined as the ratio between the reporter and the constitutively expressed detectable protein.As a negative control, the same assay is performed, but the construct encoding the transcriptional regulatory domain-MTERF1 fusion protein is not introduced into cells.

[0110] The signal of the experimental condition is then compared to that of the negative control, and if the signal is lower relative to the negative control, it is determined that the repression domain is functional in the context of the synthetic transcription factor of the invention (i.e., it is capable of preventing transcription of the gene of interest).

[0111] Furthermore, in cases where a certain transcriptional regulatory domain, such as an activation domain, is found to have little functionality by itself, it may provide good functionality when included multiple times on a synthetic transcription factor and / or in combination with additional transcriptional regulatory domains. For example, as described herein and illustrated in the accompanying Examples (see, e.g., Figures 4 and 9), the use of two FOXO domains strongly enhanced transactivation activity compared to a single FOXO domain.

[0112] The transcriptional regulatory domain of the present invention can be an activation domain comprising at least one transactivation domain independently selected from the group consisting of: a RELA domain (e.g., RelA 430-551 , RelA 342-551 , RelA 361-551 , or RelA 521-551 ) (i.e. "TA1"), WW domain (WWC1 2-81 ), KRAB domain (ZNF473 5-48 ), NucRecCoAct domain (NCOA3 1045-1092 ), LMSTEN domain (MYB 251-330 ), and FoxoTAD (FOXO3 604-644 ),

[0113] In the context of this specification and the present invention, the transcriptional regulatory domain, in particular the activation domain, may have at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:33, SEQ ID NO:52, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:13, SEQ ID NO:64, SEQ ID NO:66, and SEQ ID NO:68; or; or the transcriptional regulatory domain, in particular the activation domain, may comprise at least one sequence having at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:33, SEQ ID NO:52, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:13, SEQ ID NO:64, SEQ ID NO:66, and SEQ ID NO:68.

[0114] In some preferred embodiments of the present invention, the transcriptional regulatory domain, particularly the activation domain, has at least 60%, preferably at least 70%, and more preferably at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:33, SEQ ID NO:52, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:54, SEQ ID NO:56, and SEQ ID NO:58.

[0115] In a more preferred embodiment of the present invention, the transcriptional regulatory domain, particularly the activation domain, has at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 33, and SEQ ID NO: 52.

[0116] In some preferred embodiments, the transcriptional regulatory domain of the present invention comprises a first, second, and / or third RELA transactivation domain; wherein the first RELA transactivation domain (TA1) has the sequence set forth in SEQ ID NO: 31 or a sequence having at least 80% sequence identity to SEQ ID NO: 31; wherein the second RELA transactivation domain has the sequence set forth in SEQ ID NO: 132 or a sequence having at least 80% sequence identity to SEQ ID NO: 132; and wherein the third RELA transactivation domain has the sequence set forth in SEQ ID NO: 134 or a sequence having at least 80% sequence identity to SEQ ID NO: 134. Preferably, the transcriptional regulatory domain comprises at least the first RELA transactivation domain described herein.

[0117] Furthermore, the transcriptional regulatory domain may comprise multiple copies, for example, two or three copies, of the first, second, and / or third RELA domain, preferably the first RELA domain (TA1). As illustrated in the accompanying examples, this may increase the expression level of a gene(s) of interest (see, for example, Figures 4 and 9). Furthermore, for example, in the context of these embodiments, the transcriptional regulatory domain according to the present invention may comprise a RELA subdomain A having the sequence shown in SEQ ID NO: 3 or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 3.

[0118] In the context of the present invention, it has further been found that employing larger subunits of the RELA transactivation domain can increase the expression level of a gene of interest (see, eg, FIGS. 4 and 10C).

[0119] Thus, for example, in the context of these "RELA" embodiments, a transcriptional regulatory domain according to the present invention may comprise a RELA subdomain B having the sequence shown in SEQ ID NO: 27 or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 27.

[0120] Furthermore, for example, in the context of these embodiments, a transcriptional regulatory domain according to the present invention may comprise a RELA subdomain C having the sequence shown in SEQ ID NO:29 or a sequence having at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to SEQ ID NO:29.

[0121] Specifically, the first, second, and / or third RELA motif described herein, the RELA subdomain A, and / or the RELA subdomain B are contained in the RELA subdomain C. Furthermore, the RELA subdomain A is specifically contained in the RELA subdomain B.

[0122] Furthermore, the transcriptional regulatory domain of the present invention may comprise a FOXO3 transactivation domain (FOXO TAD) having the sequence shown in SEQ ID NO:17 or a sequence having at least 80% sequence identity to SEQ ID NO:17.

[0123] Even more surprisingly, in the context of the present invention, it was found that the use of two copies of the FOXO TAD provides high expression levels of the gene of interest while maintaining a small size (and therefore low gene payload) (see, e.g., Figures 4 and 9).

[0124] Thus, in a further preferred embodiment, the transcriptional regulatory domain of the present invention may comprise multiple copies, such as 2 or 3 copies, of said FOXO3 transactivation domain (FOXO TAD). Preferably, said transcriptional regulatory domain has the sequence set forth in SEQ ID NO: 33 or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 33.

[0125] Furthermore, the transcriptional regulatory domain of the present invention may comprise a MYB transactivation domain (LMSTEN) having the sequence shown in SEQ ID NO:17 or a sequence having at least 80% sequence identity to SEQ ID NO:17.

[0126] In some embodiments, the transcriptional regulatory domain comprises multiple copies, e.g., 2 or 3 copies, of the MYB transactivation domain (LMSTEN). Preferably, the transcriptional regulatory domain has the sequence set forth in SEQ ID NO: 196, or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 196.

[0127] In some embodiments, the transcriptional regulatory domain comprises two copies of: (i) a first RELA transactivation domain (TA1) having the sequence set forth in SEQ ID NO: 31 or a sequence having at least 80% sequence identity to SEQ ID NO: 31; (ii) a FOXO3 transactivation domain (FOXO TAD) having the sequence set forth in SEQ ID NO: 33 or a sequence having at least 80% sequence identity to SEQ ID NO: 33; or (iii) a MYB transactivation domain (LMSTEN) having the sequence set forth in SEQ ID NO: 196 or a sequence having at least 80% sequence identity to SEQ ID NO: 196.

[0128] Furthermore, the transcriptional regulatory domain according to the present invention may comprise three copies of the first RELA transactivation domain (TA1) having the sequence shown in SEQ ID NO: 31 or a sequence having at least 80% sequence identity to SEQ ID NO: 31.

[0129] Thus, in some embodiments, the transcriptional regulatory domain has a sequence set forth in SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 194, or SEQ ID NO: 196, or a sequence having at least 70%, preferably at least 80%, and more preferably at least 90% sequence identity to any of these sequences.

[0130] Even more surprisingly, in the context of the present invention, it was found that two copies of a FOXO TAD enhance the transcriptional activity of a synthetic TF more than two copies of TA1 or two copies of LMSTEN (see Figure 9).

[0131] Thus, in the context of the present invention, a transcriptional regulatory domain comprising multiple copies of the same transactivation domain preferably comprises at least two copies of the FOXO3 transactivation domain (FOXO TAD) set forth in SEQ ID NO: 33, or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 33.

[0132] In some preferred embodiments, the transcriptional regulatory domain of the present invention comprises at least two domains independently selected from the group consisting of a TA1 described herein, a FOXO TAD described herein, and a LMSTEN described herein. Preferably, the transcriptional regulatory domain comprises at least a FOXO TAD. More preferably, the transcriptional regulatory domain comprises at least a FOXO TAD, a TA1, and a LMSTEN described herein.

[0133] In some preferred embodiments, the transcriptional regulatory domain comprises, as described herein, (i) two FOXO TADs, (ii) a FOXO TAD and LMSTEN, (iii) LMSTEN and TA1, (iv) a FOXO TAD and TA1, or (v) a FOXO TAD, LMSTEN, and TA1. More preferably, the transcriptional regulatory domain comprises a FOXO TAD, specifically option (i), (ii), (iv), or (v) above.

[0134] In a further preferred embodiment of the invention, the transcriptional regulatory domain has the sequence set forth in SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, or SEQ ID NO: 58, or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to any of these sequences.

[0135] In the context of the present invention, it has further been found that certain transcriptional regulatory domains, for example comprising a FOXO-TAD and additional domains, specifically an additional FOXO-TAD, a TA1, and / or a LMSTEN domain, confer high transcriptional activity to synthetic transcription factors; see, e.g., Figure 9C.

[0136] Furthermore, such combinatorial transcriptional regulatory domains (e.g., comprising a FOXO-TAD and additional domains, particularly additional FOXO-TADs, TA1, and / or LMSTEN domains) can be synthesized using N-terminally truncated MTERF1 DNA-binding domains, e.g., MTERF1 104-399 (SEQ ID NO: 9) were found to compensate or overcompensate for the slight loss of transcriptional activity; see, eg, Figure 9F.

[0137] Therefore, in a further preferred embodiment of the present invention, the transcriptional regulatory domain has a sequence as set forth in SEQ ID NO: 52, SEQ ID NO: 33, SEQ ID NO: 54, or SEQ ID NO: 58, or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to any of these sequences.

[0138] Furthermore, a DNA binding domain according to the present invention may comprise MTERF1 subdomain A having the sequence shown in SEQ ID NO:9 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO:9.

[0139] In some of the most preferred embodiments of the invention, the transcriptional regulatory domain has the sequence set forth in SEQ ID NO: 52, or a sequence having at least 70%, preferably at least 80%, and more preferably at least 90% sequence identity to SEQ ID NO: 52. Furthermore, a DNA-binding domain in accordance with the invention may comprise MTERF1 subdomain A having the sequence set forth in SEQ ID NO: 9, or a sequence having at least 80%, preferably at least 90%, and more preferably at least 95% sequence identity to SEQ ID NO: 9.

[0140] Furthermore, preferably, in addition to the first, second, and / or third RELA motif described herein, such as the TA1, RELA subdomain A, B, or C, FOXO TAD, and / or LMSTEN, the transcriptional regulatory domain, particularly the activation domain, of the present invention may comprise a sequence having at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 13, SEQ ID NO: 64, SEQ ID NO: 66, and SEQ ID NO: 68.

[0141] Alternatively, the transcriptional regulatory domain, particularly the repression domain, of the present invention may have at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, and SEQ ID NO:98; or wherein the transcriptional regulatory domain, particularly the repression domain, may comprise at least one sequence having at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, and SEQ ID NO:98.

[0142] Preferably, the transcriptional regulatory domain, particularly the repression domain, preferably has at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, or SEQ ID NO:76.

[0143] More preferably, the transcriptional regulatory domain, particularly the repression domain, has at least 60%, preferably at least 70%, more preferably sequence identity to SEQ ID NO:70.

[0144] Furthermore, the synthetic transcription factors of the present invention may further comprise a regulatable domain, preferably a regulatable destabilization domain or a regulatable localization domain.

[0145] Specifically, the controllable domain used herein and in the context of the present invention is controllable by a compound or by light (e.g., infrared, visible, and / or UV light). Preferably, the compound is a small molecule. Preferably, the light is light of a specific wavelength or a specific range of wavelengths. Contacting a synthetic transcription factor containing a compound- or light-controllable domain can modulate, e.g., promote or repress, the transcriptional activity of the transcription factor.

[0146] As described herein, a synthetic transcription factor comprising a controllable domain as described herein may also be referred to as an "inducible transcription factor" because its activity (or inactivity) can be induced by an external stimulus, specifically a chemical compound or light. Many suitable controllable domains are known in the art, and any of these may be used in the context of the present invention. Furthermore, the induction of the activity (or inactivity) of a transcription factor is not limited to a specific mechanism. For example, certain controllable domains (e.g., the NS3 domain from hepatitis C virus) function as destabilization domains that destabilize proteins to which they are fused, where the fusion protein can be stabilized by a small molecule that binds to the destabilization domain. Other controllable domains function in the opposite manner, where the fusion protein can be destabilized by a small molecule that binds to the controllable domain.

[0147] Again, other regulatable domains (e.g., ERT2 from the human estrogen receptor) control the configuration of proteins to which they are fused, where configuration can be modulated by small molecules that bind to the regulatable domain. Synthetic transcription factors in the context of the present invention specifically function in the nucleus of a cell. Therefore, the activity of a transcription factor can be induced when it is localized into the nucleus from outside the nucleus (e.g., the cytoplasm).

[0148] Some regulatable domains (e.g., FRB and FKBP domains) bind to each other in the presence of chemical compounds or light, thereby forcing proteins to which they are fused together. In cases where a functional protein contains both moieties, its activity is induced by dimerization in the presence of chemical compounds or light.

[0149] Other controllable domains and methods of inducing the synthetic transcription factors of the present invention that are known in the art or that will be developed can be used in the context of the present invention.

[0150] In some embodiments, the synthetic transcription factors of the invention comprise a regulatable destabilization domain and are stabilized or destabilized (preferably stabilized) by a compound or light. Preferably, the regulatable destabilization domain comprises an NS3 domain having the sequence set forth in SEQ ID NO: 158 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO: 158. Specifically, the synthetic transcription factors comprising the NS3 domain according to the invention are stabilized by the small molecule grazoprevir.

[0151] In some embodiments, the synthetic transcription factor comprises a controllable localization domain and is localized to either the nucleus or cytoplasm of a cell, preferably the nucleus, by the compound or light. Preferably, the controllable localization domain comprises an ERT2 domain having a sequence set forth in SEQ ID NO: 152 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO: 152. Specifically, the synthetic transcription factor comprising the ERT2 domain is localized to the nucleus of a cell by 4-hydroxytamoxifen.

[0152] In a further embodiment, particularly when the DNA-binding domain and the transcriptional regulatory domain according to the invention are comprised in separate polypeptides, the regulatable domain comprises an FRB domain and an FKBP domain, wherein said FRB domain has the sequence set forth in SEQ ID NO: 140 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO: 140, and / or wherein said FKBP domain has the sequence set forth in SEQ ID NO: 142 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO: 142. Specifically, said FRB domain and said FKBP domain bind to each other in the presence of C16-(S)-7-methylindolerapamycin.

[0153] In some embodiments, the synthetic transcription factor of the present invention comprises a synNotch core having the sequence set forth in SEQ ID NO:160 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO:160.

[0154] The synNotch core is a surface receptor fused at its N-terminus to a single-chain variable fragment (scFv). Once the scFc::synNotch core::transcription factor fusion protein binds to its target (via the scFv), the transcription factor is released, localized to the nucleus, and regulates (e.g., activates) gene expression; see, e.g., Morsut (2016), Cell, 164(4).

[0155] It is further desirable herein and in the context of the present invention that the synthetic transcription factor be of low immunogenicity in the organism in which it is desirably employed, for example in humans.

[0156] Even more surprisingly, it has been found that in the context of the present invention, synthetic transcription factors of human origin can be assembled in human cells that retain the high orthogonality described herein. Specifically, in the synthetic transcription factors of human origin according to the present invention, both the DNA-binding domain according to the present invention and the transcriptional regulatory domain according to the present invention are derived from human proteins, preferably human MTERF1 and at least one other human protein.

[0157] In the context of synthetic transcription factors that include an activation domain, these synthetic transcription factors are also referred to herein as "human-derived transcriptional activator proteins" or simply "HumTAPs."

[0158] Preferably, herein and in the context of the present invention, synthetic transcription factors of human origin, such as HumTAP, consist exclusively of domains derived from human proteins, more preferably they consist exclusively of human protein domains.

[0159] Also, what is described herein in the context of orthogonality generally, and in the context of humans (or human cells) specifically, applies to the human-derived synthetic transcription factors described herein, particularly since orthogonality was tested in the accompanying examples with HumTAP (see, e.g., Example 3).

[0160] As illustrated in the accompanying Examples, to assess the immunogenicity of proteins, the present inventors employed an assay to examine immune responses to peptides derived from proteins of interest using primary PBMCs from normal human donors. It was found that PBMCs reacted to HumTAP (i.e., MTF)-derived peptides (specifically, corresponding to domain junctions of HumTAP) as they did to self-peptides (i.e., non-immunogenic control peptides) rather than to immunogenic positive control peptides (see, e.g., Example 2). This suggests low immunogenicity of the human-derived synthetic transcription factors of the present invention, e.g., HumTAP, in humans.

[0161] Thus, the present inventors have further developed a class of synthetic transcription factors made entirely from human protein subunits. Data presented in the accompanying examples demonstrate favorable profiles of GoI transactivation, immunogenicity, and orthogonality. As illustrated in the accompanying examples, the immunogenic potential of synthetic transcription factors created using two fully human protein domains (i.e., a human DNA-binding domain and a human transcriptional regulatory domain) is minimized. Thus, it is expected that fewer or no local or systemic immunosuppressive effects and additional genetic elements will be required to counteract a patient's immune response to a gene and cell therapy (GCT) payload containing a human-derived synthetic transcription factor, such as a gene encoding the HumTAP protein. This may further result in improved clinical performance of GCTs compared to prior art alternatives.

[0162] Thus, the human-derived synthetic transcription factors according to the present invention successfully reconcile the described requirements of high orthogonality (eg in human cells) and low immunogenicity (eg in humans). Thus, the development of a human-derived synthetic transcription factor, such as HumTAP, that has high orthogonality in human cells is a particularly remarkable (and unexpected) achievement by the inventors. Moreover, the present invention is of particular value for synthetic gene circuits that can be used in the therapies described herein, such as gene therapy or cell therapy.

[0163] Thus, the protein or proteins from which the transcriptional regulatory domain is derived are preferably from the same species, e.g., the same mammalian species, as the mitochondrial DNA-binding protein according to the invention is derived from, as defined herein and in the context of the present invention. Furthermore, the synthetic transcription factors of the present invention may be composed essentially or entirely of parts of proteins from the same species, e.g., the same mammalian species.

[0164] Preferably, herein and in the context of the present invention, the mitochondrial DNA-binding protein according to the invention, e.g., MTERF1, and one or more other proteins according to the invention from which the transcriptional regulatory domain is derived, e.g., RELA, FOXO3, and / or MYB, are of human origin. Moreover, the synthetic transcription factors of the present invention can be composed essentially or entirely of parts of human proteins.

[0165] In certain embodiments, the transcriptional regulatory domain of the present invention is derived from a single human protein. Preferably, the transcriptional regulatory domain, particularly the activation domain, has at least 90%, preferably at least 95%, more preferably at least 99% sequence identity to SEQ ID NO:3, SEQ ID NO:27, or SEQ ID NO:29, preferably SEQ ID NO:3.

[0166] Alternatively, the transcriptional regulatory domain, particularly the repression domain, of the present invention may have at least 90%, preferably at least 95%, more preferably at least 99% sequence identity to SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, or SEQ ID NO:98, preferably to SEQ ID NO:70.

[0167] It is possible that the synthetic transcription factors of the present invention are essentially non-immunogenic in the mammalian species from which the mitochondrial DNA binding protein is derived. Preferably, in the context of this specification and the present invention, the synthetic transcription factors are essentially non-immunogenic in humans.

[0168] As described hereinabove, in some preferred embodiments, the synthetic transcription factors of the invention are fusion proteins comprising a DNA-binding domain in accordance with the invention and a transcriptional regulatory domain in accordance with the invention. Also, as described hereinabove, the DNA-binding domain and the transcriptional regulatory domain are connected to each other on the fusion protein either directly by means of a direct peptide bond or by means of a peptide linker (and two corresponding peptide bonds at either end of the linker).

[0169] Thus, the synthetic transcription factor fusion proteins of the present invention may further comprise a peptide linker between the DNA-binding domain and the transcriptional regulatory domain of the synthetic transcription factor. Many suitable peptide linkers are known in the art, and any may be employed in the context of the present invention, for example, in the context of the fusion proteins of the present invention. Suitable peptide linkers include, among others, the two-amino acid linker "GS," G4S as set forth in SEQ ID NO: 118, AP6 as set forth in SEQ ID NO: 120, cMycNLS as set forth in SEQ ID NO: 122, "EAAAK" as set forth in SEQ ID NO: 124, and the SV40 linker as set forth in SEQ ID NO: 126. Furthermore, the cMycNLS and the SV40 linker also function as nuclear localization sequences (NLS) and therefore may also be employed for that purpose herein and in the context of the present invention.

[0170] Furthermore, the present invention relates to nucleic acids encoding a fusion protein of the invention comprising a DNA binding domain of the invention and a transcriptional regulatory domain of the invention (ie, a fusion protein comprising a synthetic transcription factor of the invention).

[0171] The nucleic acid of the present invention can be DNA or RNA.Furthermore, the nucleic acid can be single-stranded or double-stranded, for example, dsDNA, ssRNA, ssDNA, or dsRNA.Preferably, the nucleic acid of the present invention comprises a coding strand (i.e., a sense strand).However, the nucleic acid of the present invention can also refer to (or even consist of) an antisense strand, and therefore can be characterized by a reverse complementary sequence.The same applies to the DNA construct of the present invention.

[0172] Furthermore, the nucleic acid of the invention can be mRNA, for example, mRNA contained in a lipid nanoparticle.

[0173] In the context of the present invention, it has further been found that DNA sequences that are codon-optimized for humans provide higher expression of the gene of interest (see, eg, FIG. 2).

[0174] Thus, in a preferred embodiment, the nucleic acid of the invention comprises the DNA sequence set forth in SEQ ID NO: 5, or a DNA sequence having at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to SEQ ID NO: 5; wherein said DNA sequence encodes a DNA-binding domain having at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to SEQ ID NO: 1. Preferably, said DNA-binding domain has the sequence set forth in SEQ ID NO: 1.

[0175] Furthermore, the nucleic acid of the present invention may comprise the DNA sequence set forth in SEQ ID NO: 6, or a DNA sequence having at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to SEQ ID NO: 6, wherein said DNA sequence encodes a transcriptional regulatory domain having at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to SEQ ID NO: 3. Preferably, said transcriptional regulatory domain has the sequence set forth in SEQ ID NO: 3.

[0176] Furthermore, the present invention relates to a DNA plasmid comprising a nucleic acid of the invention, preferably said plasmid being suitable for expressing a synthetic transcription factor in a cell.

[0177] Furthermore, the present invention relates to a viral vector comprising a nucleic acid of the present invention or a plasmid of the present invention. The nucleic acids described herein may also be defined by the corresponding reverse complement sequences.

[0178] Suitable viral vectors that can be employed in the context of the present invention include, inter alia, adeno-associated viral (AAV) vectors, lentiviral vectors, adenoviral vectors, herpes simplex viral vectors, and VSV vectors. Preferably, the viral vector is an adeno-associated viral vector or a lentiviral vector.

[0179] Furthermore, the present invention relates to cells comprising a nucleic acid of the invention, a plasmid of the invention, and / or a viral vector of the invention.

[0180] Generally, any cell can be used in the context of this specification and the present invention.Preferably, cell is mammalian cell, preferably human cell.For example, cell can be immune cell such as T cell, B cell or NK cell.In the context of in vivo, cell can also be preferably cancer or tumor cell.

[0181] As described hereinabove, in some embodiments, a synthetic transcription factor of the present invention comprises or consists of a first and a second polypeptide, wherein the first polypeptide comprises a DNA-binding domain in accordance with the present invention and the second polypeptide comprises a transcriptional regulatory domain in accordance with the present invention. In other words, a synthetic transcription factor can be a multimeric protein comprising a first polypeptide (i.e., a second amino acid chain) comprising a DNA-binding domain in accordance with the present invention and a second polypeptide (i.e., a second amino acid chain) comprising a transcriptional regulatory domain in accordance with the present invention. Specifically, the first and second polypeptides can bind and / or interact with each other. The binding or interaction can be reversible and / or inducible, for example, by a compound or light, as described herein.

[0182] Preferably, when the synthetic transcript is a multimeric protein as described above, it comprises a multimerization domain, wherein the multimerization domains of the first and second polypeptides can bind and / or interact with each other. Preferably, the multimerization domain is a dimerization domain.

[0183] In some embodiments, the multimerization domain is a homodimerization domain, in which case the multimerization domains of the first and second polypeptides are essentially identical to one another.

[0184] In a preferred embodiment, the multimerization domain is a heterodimerization domain, in which case the multimerization domains of the first and second polypeptides are different from each other.

[0185] In some embodiments, (i) the multimerization domain of the first polypeptide comprises or consists of a SYNZIP1 domain, and the multimerization domain of the second polypeptide comprises or consists of a SYNZIP2 domain; or (ii) the multimerization domain of the first polypeptide comprises or consists of a SYNZIP2 domain, and the multimerization domain of the second polypeptide comprises or consists of a SYNZIP1 domain.

[0186] Specifically, the SYNZIP1 domain has the sequence set forth in SEQ ID NO: 154, or a sequence having at least 80%, preferably at least 90%, and more preferably at least 95% sequence identity to SEQ ID NO: 154; and the SYNZIP2 domain has the sequence set forth in SEQ ID NO: 156, or a sequence having at least 80%, preferably at least 90%, and more preferably at least 95% sequence identity to SEQ ID NO: 156.

[0187] In some embodiments, the multimerization domain is a controllable domain as described herein. Specifically, the multimerization domain can be a dimerization domain, where a compound or light as described herein controls the dimerization of the multimerization domains of the first and second polypeptides, i.e., the controllable dimerization domains. Preferably, the first and second polypeptides bind and / or interact with each other in the presence of the small molecule or light.

[0188] In a preferred embodiment, the multimerization domain of the first polypeptide comprises or consists of an FKBP domain, and the multimerization domain of the second polypeptide comprises or consists of an FRB domain; alternatively, the multimerization domain of the first polypeptide comprises or consists of an FRB domain, and the multimerization domain of the second polypeptide comprises or consists of an FKBP domain. Preferably, the multimerization domain of the first polypeptide comprises or consists of an FKBP domain, and the multimerization domain of the second polypeptide comprises or consists of an FRB domain. The first and second polypeptides can bind and / or interact with each other in the presence of C16-(S)-7-methylindolerapamycin, specifically, wherein C16-(S)-7-methylindolerapamycin induces heterodimerization of the FKBP domain and the FRB domain.

[0189] Specifically, as used herein, an FKBP domain has the sequence shown in SEQ ID NO:142, or a sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO:142.

[0190] Moreover, the FRB domain specifically has the sequence set forth in SEQ ID NO: 140, or a sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO: 140. Preferably, the DNA-binding domain is N-terminal to the FKBP domain on the first polypeptide, and / or the transcriptional regulatory domain is C-terminal to the FRB domain on the second polypeptide.

[0191] Furthermore, the DNA binding domain and the FKBP domain can be linked to each other via a first peptide linker, and / or the transcriptional regulatory domain and the FRB domain can be linked to each other via a second peptide linker. The first and second peptide linkers can be independently selected from the group consisting of, for example, the cMyc NLS linker shown in SEQ ID NO: 122, the 6AP (AP6) linker shown in SEQ ID NO: 120, the AP8 linker shown in SEQ ID NO: 144, the G4S linker shown in SEQ ID NO: 118, the EAAAK3 linker shown in SEQ ID NO: 146, the EAAAK2 linker shown in SEQ ID NO: 148, and the G4S4 linker shown in SEQ ID NO: 150. It should be noted that the terms "6AP" and "AP6" are used interchangeably herein.

[0192] In a preferred embodiment, the first peptide linker is a cMyc NLS linker shown in SEQ ID NO: 122 or a 6AP (AP6) linker shown in SEQ ID NO: 120, and / or the second peptide linker is a 6AP (AP6) linker shown in SEQ ID NO: 120.

[0193] In a further preferred embodiment, for example in the context of the FKBP and FRB domains, the transcriptional regulatory domain has the sequence set forth in SEQ ID NO:52, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:54, or SEQ ID NO:58, or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to any of these sequences. More preferably, the transcriptional regulatory domain has the sequence set forth in SEQ ID NO:52 or SEQ ID NO:29, or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO:52 or SEQ ID NO:29.

[0194] In some preferred embodiments, the first polypeptide of the synthetic transcription factor comprises the sequence set forth in SEQ ID NO: 174, 176, 178, 180, 182, 184, 186, 188, or 192, or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 174, 176, 178, 180, 182, 184, 186, 188, or 192; and / or the second polypeptide of the synthetic transcription factor comprises the sequence set forth in SEQ ID NO: 162, 164, 166, 168, 170, 172, or 190, or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 162, 164, 166, 168, 170, 172, or 190.

[0195] Furthermore, the present invention relates to a combination of nucleic acids encoding the synthetic transcription factors of the present invention, wherein one nucleic acid encodes a DNA-binding domain in accordance with the present invention and another nucleic acid encodes a transcriptional regulatory domain in accordance with the present invention. Specifically, the nucleic acid encoding the DNA-binding domain in accordance with the present invention corresponds to a first nucleic acid encoding a first polypeptide in accordance with the present invention described herein. Furthermore, the nucleic acid encoding the transcriptional regulatory domain in accordance with the present invention corresponds to a second nucleic acid encoding a second polypeptide in accordance with the present invention described herein.

[0196] Therefore, the present invention further relates to a combination of nucleic acids encoding a synthetic transcription factor of the present invention comprising a first and a second polypeptide as described herein, wherein said combination of nucleic acids comprises a first and a second nucleic acid, wherein said first nucleic acid encodes said first polypeptide and said second nucleic acid encodes said second polypeptide.

[0197] Furthermore, the nucleic acids of the invention can be contained in multiple plasmids, viral vectors, cells, or kits, as described herein. In certain embodiments, a combination of nucleic acids according to the invention refers to a kit comprising said combination of nucleic acids.

[0198] In certain embodiments of the combination of nucleic acids, one nucleic acid has the DNA sequence set forth in SEQ ID NO:5, or a DNA sequence having at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to SEQ ID NO:5, wherein said DNA sequence encodes a DNA-binding domain having at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to SEQ ID NO:1.

[0199] Preferably, the DNA binding domain has the sequence shown in SEQ ID NO:1.

[0200] Furthermore, another nucleic acid in the combination may have the DNA sequence shown in SEQ ID NO:6, or a DNA sequence having at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to SEQ ID NO:6, wherein the DNA sequence encodes a transcriptional regulatory domain having at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to SEQ ID NO:3.

[0201] Preferably, the transcriptional regulatory domain has the sequence shown in SEQ ID NO:3.

[0202] Furthermore, the present invention relates to a DNA construct comprising a promoter (P) comprising a response element and a minimal promoter characterized by: said response element comprises an MTERF1 binding site, which has (specifically comprises or consists of) the sequence shown in SEQ ID NO: 42 or SEQ ID NO: 200, or a sequence having at least 60%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 42 or SEQ ID NO: 200.

[0203] SEQ ID NO:200 corresponds to the reverse complement (ie, antisense) sequence of SEQ ID NO:42.

[0204] In the present specification and the context of the present invention, a sequence is read from the 5' to the 3' end, i.e., in sense. Therefore, a DNA construct comprising an MTERF1 binding site in sense relative to a minimal promoter comprises, for example, the sequence shown in SEQ ID NO: 42, or a sequence having at least 60%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 42. Following the same logic, a DNA construct comprising an MTERF1 binding site in antisense relative to a minimal promoter comprises, in particular, the sequence shown in SEQ ID NO: 200, or a sequence having at least 60%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 200.

[0205] Nevertheless, specifically in the context of a dsDNA construct in accordance with the present invention, SEQ ID NO: 42, or a sequence having at least 60%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 42, may be included in the 5' to 3' direction on one strand of the dsDNA construct, and the sequence of a minimal promoter described herein may be included in the 5' to 3' direction on the other strand of the dsDNA construct.

[0206] Preferably, herein, particularly in the context of the DNA constructs of the present invention provided herein, the MTERF1 binding site comprises or consists of the sequence shown in SEQ ID NO: 42 or a sequence having at least 60%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 42, particularly MTERF1 binding in sense relative to the minimal promoter.

[0207] Preferably, as used herein, for example, in the context of the DNA construct of the present invention, the response element of the present invention consists of: (i) one or more copies, for example, 2 to 50 copies (preferably 2 to 5 copies), of the MTERF1 binding site, wherein the multiple copies are immediately adjacent to each other, or (ii) multiple copies, for example, 2 to 50 copies (preferably 2 to 5 copies), of the MTERF1 binding site, and a BS-BS spacer between at least two, preferably all, consecutive copies of the binding site. Specifically, the spacer has a length of 1 to 1,000 nucleotides, preferably 1 to 100 nucleotides, more preferably 1 to 10, for example, 1 to 6 nucleotides. Preferably, the BS-BS spacer consists of 1 to 10 nucleotides at the 5' end of the sequence set forth in SEQ ID NO:198, i.e., the first 1, 2, 3, 4, 5, 6, 7, 8, or 9, or all, of the 5' end of the sequence set forth in SEQ ID NO:198. Moreover, as described herein, in cases where the MTERF1 binding site is in a sense orientation relative to the minimal promoter, the BS-BS spacer can consist of 1, 4, 5, or 8 nucleotides, preferably the first 1, 4, 5, or 8 nucleotides at the 5' end of the sequence set forth in SEQ ID NO: 198.

[0208] Furthermore, instead of the exemplary 2-50 copies, other ranges may be envisaged, such as 2-25 copies, 2-15 copies, 2-10 copies, or preferably 2-5 copies.

[0209] In some preferred embodiments, the response element consists of multiple copies, eg, 2-15 copies, of the MTERF1 binding site, which are immediately adjacent to each other.

[0210] Furthermore, the DNA constructs of the present invention may be, for example, at most about 10 6 , preferably at most 10 5 , and more preferably may have a length of at most about 10,000 nucleotides.

[0211] Preferably, as used herein, e.g., in the context of the DNA construct of the present invention, the response element and minimal promoter of the present invention are separated from each other by at most about 2000 nucleotides (i.e., by an RE-minP spacer having a length of at most about 2000 nucleotides), preferably at most about 200 nucleotides, more preferably at most about 20 nucleotides, e.g., about 6 or 8 nucleotides. Preferably, the RE-minP spacer consists of 1 to 10 nucleotides at the 5' end of the sequence set forth in SEQ ID NO: 199; preferably, the MTERF1 binding site comprises or consists of the sequence set forth in SEQ ID NO: 42, or a sequence having at least 60%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 42, particularly in the sense relative to the minimal promoter.

[0212] Furthermore, as used herein, for example in the context of the DNA constructs of the present invention, the minimal promoter may be 3' or 5' to said response element. Preferably, the minimal promoter is 3' to said response element.

[0213] In the context of this specification and the present invention, a minimal promoter may be a minimal TATA box having at least 60%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO:103.

[0214] Furthermore, the minimal promoter may be a minimal CMV promoter having at least 60%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO:136.

[0215] Specifically, in the present specification and in the context of the present invention, a promoter (P) according to the present invention is capable of binding to a synthetic transcription factor according to the present invention. Specifically, a response element according to the present invention is capable of binding to a DNA binding domain according to the present invention.

[0216] Specifically, herein and in the context of the present invention, a DNA binding domain according to the present invention binds to a response element of the present invention in a sequence-specific manner.

[0217] Preferably, herein and in the context of the present invention, the DNA construct of the present invention further comprises at least one gene of interest, preferably located 3' of the minimal promoter.

[0218] In preferred embodiments, the gene(s) of interest encode a cell death-promoting protein such as hBAX or HSV-TK described herein, an immune-stimulating cytokine such as IL-2 or IL-12, and / or an antigen receptor such as a CAR or TCR.

[0219] Preferably, herein and in the context of the present invention, the promoter (P) according to the invention is operably linked to at least one gene of interest as described herein.

[0220] Specifically, when said promoter (P), specifically the response element of the invention, is bound by a synthetic transcription factor of the invention in a cell, e.g., in the nucleus of a human cell, at least one of said gene(s) of interest is transcribed.

[0221] In certain embodiments, a DNA construct of the invention does not comprise the sequence shown in SEQ ID NO:117 or a sequence having at least 90% sequence identity to SEQ ID NO:117.

[0222] As illustrated in the accompanying Examples, the strength of the promoter (P) contained in the DNA construct of the present invention can be adjusted as desired, i.e., stronger or weaker promoter variants can be employed; see Figures 14 and 15 and SEQ ID NOs: 201-1190.

[0223] Therefore, the DNA construct of the present invention may comprise a sequence selected from the group consisting of SEQ ID NOs: 201 to 1190. Furthermore, the spacer sequence therein, i.e., consecutive nucleotides (specifically, 1 to 10 nucleotides in length) that do not belong to either the MTERF1 binding site (SEQ ID NO: 42) or the minimal promoter sequence (SEQ ID NO: 103), may be replaced with another corresponding spacer sequence of the same length.

[0224] As shown in Figure 15 and reflected in SEQ ID NO: 300, a particular promoter variant of particular interest has two MTERF1 binding sites directly adjacent to each other in sense orientation relative to the minimal promoter, and an 8 nucleotide spacer between the response element (or 3'-most MTERF1 binding site) and the minimal promoter sequence. This promoter variant is relatively strong, but has a relatively small size.

[0225] Therefore, in some preferred embodiments, the response element comprises or consists of two copies of the MTERF1 binding site, each having the sequence shown in SEQ ID NO: 42 or a sequence having at least 60%, preferably at least 80%, and more preferably at least 90% sequence identity to SEQ ID NO: 42; wherein the two copies of the MTERF1 binding site are directly adjacent to each other; and the response element and the minimal promoter are separated from each other by 8 nucleotides.

[0226] In some preferred embodiments, a DNA construct of the present invention comprises the sequence shown in SEQ ID NO:300.

[0227] As shown in Figure 15 and reflected in SEQ ID NO: 693, a further particular promoter variant of particular interest has five MTERF1 binding sites in a sense orientation relative to the minimal promoter, separated from each other by spacers 8 nucleotides in length, and a 10 nucleotide spacer between the response element (or the 3'-most MTERF1 binding site) and the minimal promoter sequence. This promoter variant is particularly strong.

[0228] Therefore, in another preferred embodiment, the response element consists of five copies of the MTERF1 binding site, said copies being separated from each other by 8 nucleotides, wherein each MTERF1 binding site has the sequence shown in SEQ ID NO: 42 or a sequence having at least 60%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 42; and wherein the response element and the minimal promoter are separated from each other by 10 nucleotides.

[0229] In some preferred embodiments, a DNA construct of the present invention comprises the sequence shown in SEQ ID NO:693.

[0230] Furthermore, the DNA construct of the present invention can be single-stranded or double-stranded, i.e., dsDNA or ssDNA or dsRNA.Furthermore, the present invention relates to the RNA corresponding to the DNA construct of the present invention (i.e., having the same sequence except for uracil versus thymine).Corresponding RNA can also be single-stranded or double-stranded, i.e., ssRNA or dsRNA.Furthermore, RNA can be, for example, the mRNA contained in lipid nanoparticles described herein.

[0231] Preferably, the DNA constructs (or corresponding RNA) of the present invention comprise the coding strand (i.e., the sense strand). However, the DNA constructs (or corresponding RNA) of the present invention may also refer to (or even consist of) the antisense strand and thus may be characterized by a reverse complementary sequence.

[0232] Thus, the present invention further relates to single- or double-stranded nucleic acids comprising the sense strand of the DNA construct of the present invention and / or the antisense strand of the DNA construct of the present invention.

[0233] Furthermore, the present invention relates to single- or double-stranded nucleic acids, such as DNA or RNA, comprising a sequence corresponding to the sense strand of a DNA construct of the present invention and / or a sequence corresponding to the antisense strand of a DNA construct of the present invention.

[0234] Furthermore, the present invention relates to a plasmid comprising the DNA construct of the present invention.

[0235] The present invention also relates to viral vectors comprising the DNA constructs of the invention, the corresponding RNA, or the corresponding plasmids.

[0236] Furthermore, the present invention relates to a cell comprising the DNA construct of the present invention, for which the same applies as disclosed herein in the context of the nucleic acid of the present invention encoding the fusion protein (i.e., synthetic transcription factor) of the present invention.

[0237] Furthermore, the present invention relates to a system (in particular a transcription system) comprising (i) a synthetic transcription factor of the invention, a corresponding nucleic acid of the invention, a corresponding DNA plasmid of the invention, and / or a corresponding viral vector of the invention, and (ii) a DNA construct of the invention, a DNA plasmid of the invention, and / or a viral vector of the invention. Preferably, said system comprises a synthetic transcription factor of the invention and a DNA construct of the invention.

[0238] Furthermore, the system can be an engineered genetic network, for example a biocomputing circuit.

[0239] Specifically, the system of the present invention is suitable for regulating the transcription of at least one gene of interest and can be used for this purpose. Preferably, the gene of interest is contained in the DNA construct of the present invention described herein. Preferably, the gene of interest (singular or plural) encodes a cell death-promoting protein such as hBAX or HSV-TK described herein, an immunostimulatory cytokine such as IL-2 or IL-12, and / or an antigen receptor such as CAR or TCR.

[0240] Furthermore, the present invention relates to a cell comprising the synthetic transcription factor of the present invention and the DNA construct of the present invention.Preferably, the cell is a mammalian cell, preferably a human cell.For example, when the target gene encodes an antigen receptor such as CAR or TCR as described herein, the cell according to the present invention can be an immune cell such as a T cell, a B cell, or a NK cell.In an in vivo context, the cell can also be preferably a cancer or tumor cell.

[0241] Furthermore, the present invention relates to kits comprising a synthetic transcription factor of the invention, a corresponding nucleic acid of the invention, a corresponding DNA plasmid of the invention, a corresponding viral vector of the invention, a combination of nucleic acids of the invention, a DNA construct of the invention, a single- or double-stranded nucleic acid of the invention, a DNA plasmid of the invention, a viral vector of the invention, and / or a system of the invention.

[0242] In certain embodiments, the kit comprises (i) a nucleic acid of the invention (i.e., encoding a fusion protein / synthetic transcription factor of the invention), a corresponding DNA plasmid of the invention, or a corresponding viral vector of the invention, and (ii) a DNA construct of the invention, a corresponding DNA plasmid of the invention, or a corresponding viral vector of the invention.

[0243] Furthermore, the present invention relates to a pharmaceutical composition comprising a synthetic transcription factor of the invention, a corresponding nucleic acid of the invention, a corresponding DNA plasmid of the invention, a corresponding viral vector of the invention, a combination of nucleic acids of the invention, a DNA construct of the invention, a single- or double-stranded nucleic acid of the invention, a corresponding DNA plasmid of the invention (i.e., corresponding to a DNA construct of the invention), a corresponding viral vector of the invention, a system of the invention, or any cell of the invention.

[0244] In certain embodiments, the pharmaceutical composition comprises (i) a nucleic acid of the invention (corresponding to a fusion protein / synthetic transcription factor of the invention), a corresponding DNA plasmid of the invention, or a corresponding viral vector of the invention, and (ii) a DNA construct of the invention, a corresponding DNA plasmid of the invention, or a corresponding viral vector of the invention.

[0245] In certain embodiments, the pharmaceutical composition comprises the cells of the invention comprising the systems of the invention.

[0246] Furthermore, the pharmaceutical compositions of the present invention may further comprise a pharmaceutically acceptable excipient.

[0247] Furthermore, the pharmaceutical composition of the present invention can be used to treat disease, wherein target cells are killed and / or manipulated.Specifically, the treatment can involve the cancer cell classifier circuit described and / or referred to herein.Preferably, in this context, at least one target gene can code for cell death-promoting protein such as hBAX or HSV-TK, immune-stimulating cytokine such as IL-2 or IL-12, and / or antigen receptor such as CAR or TCR.

[0248] Furthermore, the pharmaceutical composition of the present invention can be used in a method for treating tumors or cancer. Specifically, the treatment can involve the cancer cell classifier circuit described herein. Preferably, in this context, at least one gene of interest encodes a cell death-promoting protein such as hBAX or HSV-TK, an immune-stimulating cytokine such as IL-2 or IL-12, and / or an antigen receptor such as CAR or TCR.

[0249] Furthermore, the nucleic acids of the invention (corresponding to the fusion proteins / synthetic transcription factors of the invention), the corresponding DNA plasmids of the invention, the corresponding viral vectors of the invention, the combinations of nucleic acids of the invention, the DNA constructs of the invention, the corresponding single- or double-stranded nucleic acids of the invention, the corresponding DNA plasmids of the invention, the corresponding viral vectors of the invention, or the systems of the invention may be used in gene therapy.

[0250] Furthermore, the cells of the present invention may be used in cell therapy. Preferably, the cells are T cells, e.g., CAR T cells, and the cell therapy is T cell therapy, e.g., CAR T cell therapy.

[0251] Furthermore, the nucleic acids of the invention (corresponding to the fusion proteins / synthetic transcription factors of the invention), the corresponding DNA plasmids of the invention, the corresponding viral vectors of the invention, combinations of nucleic acids of the invention, DNA constructs of the invention, the corresponding single- or double-stranded nucleic acids of the invention, the corresponding DNA plasmids of the invention, the corresponding viral vectors of the invention, or the systems of the invention may be used as part of or in combination with engineered genetic networks, in particular biocomputing circuits.

[0252] Furthermore, the nucleic acids of the invention (corresponding to the fusion proteins / synthetic transcription factors of the invention), the corresponding DNA plasmids of the invention, the corresponding viral vectors of the invention, the combinations of nucleic acids of the invention, the DNA constructs of the invention, the corresponding single- or double-stranded nucleic acids of the invention, the corresponding DNA plasmids of the invention, the corresponding viral vectors of the invention, or the systems of the invention can be used to transcribe a gene of interest in vitro or in vivo, for example in a cell in vitro or in vivo.

[0253] As illustrated in the accompanying examples, the present inventors further generated a library of promoter variants and developed a method for screening promoters optimized for transcription factor binding; see Example 6 and Figures 11-15. The present inventors have found, inter alia, promoter variants that are relatively small in size and provide relatively high transcriptional activity, i.e., relatively strong (e.g., SEQ ID NO: 300). Furthermore, the present inventors have found, inter alia, particularly strong promoter variants (e.g., SEQ ID NO: 693). Furthermore, the present inventors have surprisingly found that in addition to the number of transcription factor (TF) binding sites and the orientation of the transcription factor binding sites relative to the minimal promoter, the presence or length of spacers between TF binding sites and between the 3'-most TF binding site and the minimal promoter (e.g., the spacer between the 3'-most TF binding site and the minimal promoter) influences promoter strength.

[0254] Accordingly, the present invention further relates to a library of DNA constructs comprising at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, 100000, 500000, or 1000000, preferably at least about 100, 500, 900, 950, 990, or 1000 different DNA constructs, wherein each DNA construct in said library comprises: (i) a promoter (P) consisting of a response element (RE), a minimal promoter (minP) 3' of the response element, and an optional RE-minP spacer between the response element and the minimal promoter, wherein each response element consists of one or more copies of a transcription factor binding site (BS) and an optional BS-BS spacer between at least two, and preferably all, consecutive copies of the binding site; (ii) an output sequence (which is preferably 3' of the minimal promoter); Wherein, all DNA constructs in the library differ from each other in the sequence of their promoter (P). Optionally, each DNA construct in the library contains a unique barcode sequence that distinguishes all DNA constructs in the library from each other. Barcoding means and methods are well known in the art.

[0255] The DNA constructs in the library, specifically the promoters, response elements, transcription factor binding sites, minimal promoters, and spacers, can be designed as described herein in the context of the DNA constructs of the present invention.

[0256] Therefore, the transcription factor binding site is preferably an MTERF1 binding site comprising or consisting of the sequence set forth in SEQ ID NO: 42 or SEQ ID NO: 200 (preferably SEQ ID NO: 42), or a sequence having at least 60%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 42 or SEQ ID NO: 200 (preferably SEQ ID NO: 42). Preferably, said MTERF1 binding site consists of the sequence set forth in SEQ ID NO: 42.

[0257] The DNA constructs in the library can be identical to each other, except for promoter sequence and any barcode sequence.What's more, the minimal promoters on different DNA constructs, particularly on different promoters, can be identical to each other.What's more, the transcription factor binding sites on different DNA constructs, particularly on different promoters, can have the same sequence, preferably the sequence shown in SEQ ID NO: 42 or SEQ ID NO: 200, respectively, in either sense or antisense orientation relative to the minimal promoter.

[0258] In some embodiments, 20%, 30%, 40%, or 50% of the promoters of the DNA constructs differ from each other in (i) the binding site copy number and / or (ii) the presence or length of the RE-minP spacer; and / or at least 80%, at least 90%, or all of the promoters of the DNA constructs in the library differ from each other in at least one parameter selected from the group consisting of (i) the binding site copy number, (ii) the presence or length of the RE-minP spacer, (iii) the presence or length of the BS-BS spacer, and (iv) the orientation of the sense or antisense binding site sequence relative to the minimal promoter.

[0259] In a preferred embodiment of the library, the minimal promoter is a minimal TATA box having at least 60%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO:103.

[0260] In some embodiments of the library, the minimal promoter is a minimal CMV promoter having at least 60%, preferably at least 80%, and more preferably at least 90% sequence identity to SEQ ID NO:136.

[0261] In particular, the promoters (P) on the DNA constructs in the library, and in particular the response elements, are (or are suspected to be) capable of binding to the synthetic transcription factors of the present invention.

[0262] In a further aspect, the present invention relates to a method of optimizing a promoter for binding to a transcription factor comprising the steps of: a) preparing a library of DNA constructs according to the invention; b) combining the library of DNA constructs with said transcription factors in a cell or in an in vitro transcription system, preferably in a cell; c) determining the transcriptional activity of the promoter of each DNA construct in the library, preferably by determining the amount of mRNA produced from each DNA construct in the library, specifically wherein said mRNA comprises a sequence corresponding to the output sequence of the DNA construct; and d) Selecting promoters based on their transcriptional activity, thereby obtaining promoters that are optimized for binding to said transcription factors.

[0263] Preferably, the transcriptional activity of the promoter of each DNA construct in the library is determined by RNA sequencing, preferably by next generation RNA sequencing, e.g., as illustrated in Example 6. More preferably, the method comprises a massively parallel reporter assay, e.g., as described in Example 6. In a preferred embodiment of the method, the transcription factor is a synthetic transcription factor according to the present invention.

[0264] Sequence Listing The following sequences and SEQ ID NOs refer to SEQ ID NOs described herein and in the context of the present invention:

[0265] [Table 1]

[0266] [Table 2]

[0267] [Table 3]

[0268] [Table 4]

[0269] [Table 5]

[0270] Table 6

[0271] Table 7

[0272] Table 8

[0273] Table 9

[0274] Table 10

[0275] Table 11

[0276] Table 12

[0277] Table 13

[0278] Table 14

[0279] Table 15

[0280] Table 16

[0281] Table 17

[0282] Table 18

[0283] Table 19

[0284] Table 20

[0285] Table 21

[0286] Table 22

[0287] Table 23

[0288] Table 24

[0289] Table 25

[0290] Table 26

[0291] Table 27

[0292] Table 28

[0293] Table 29

[0294] Table 30

[0295] Table 31

[0296] Table 32

[0297] Table 33

[0298] Table 34

[0299] Table 35

[0300] Table 36

[0301] Table 37

[0302] Table 38

[0303] Table 39

[0304] Table 40

[0305] Table 41

[0306] Table 42

[0307] Table 43

[0308] Table 44

[0309] Table 45

[0310] Table 46

[0311] [Table 47]

[0312] [Table 48]

[0313] [Table 49]

[0314] [Table 50]

[0315] [Table 51]

[0316] [Table 52]

[0317] [Table 53]

[0318] [Table 54]

[0319] [Table 55]

[0320] The sequences of SEQ ID NOs: 1 to 200 are also set out in the attached sequence listing in accordance with WIPO St. 26.

[0321] The following promoter (sensor) sequences, SEQ ID NOs: 201 to 1190, are shown only in the attached sequence listing in accordance with WIPO St. 26. These promoter sequences have the following key: (a) orientation of binding site (BS) relative to the minimal promoter (TATA) - (b) number of BS - (c) distance BS-BS - (d) distance BS-TATA).

[0322] [Table 56]

[0323] [Table 57]

[0324] [Table 58]

[0325] [Table 59]

[0326] [Table 60]

[0327] [Table 61]

[0328] [Table 62]

[0329] [Table 63]

[0330] [Table 64]

[0331] [Table 65]

[0332] The present invention is also characterized by the following figures, figure descriptions, and the following non-limiting examples. [Brief explanation of the drawings]

[0333] [Figure 1-1]MTF activity for various reporter constructs in human cell lines. (A) DNA sequence of the response element (RE) repeat and spacer, which are repeated n times. In Figure 1, the term "response element" (RE) corresponds to the term "binding site" as used herein, specifically the MTERF1 binding site defined by SEQ ID NO: 42. (B) Dependence of gene of interest (GoI) expression on the number of REs on the reporter construct in the Hela human cell line. Microscopic images of Hela cells show expression of the mCerulean GoI (top) and the transfection reporter control mCherry (bottom). Fluorescence channel designations are indicated on the left. The number of RE repeats on each promoter construct is indicated above each pair of images. (C) Quantitative analysis of mCerulean levels normalized to mCherry levels in Hela cells transfected with reporter constructs with different numbers of RE-spacer repeats as illustrated in panel A. Analysis is based on flow cytometry data. The number of RE / MTERF1 binding sites (SEQ ID NO: 42) on each reporter construct is indicated on the x-axis. (D) Dependence of GoI expression on the number of REs on the reporter construct in the HEK293 human cell line. Microscopic images of HEK293 cells show expression of the mCerulean GoI (top) and the transfection reporter control mCherry (bottom). Fluorescence channel designations are indicated on the left. The number of RE repeats on each promoter construct is indicated above each pair of images. (E) Quantitative analysis of mCerulean levels normalized to mCherry levels in HEK293 cells shown in panel D. Analysis is based on flow cytometry data. The number of RE / MTERF1 binding sites on each reporter construct is indicated on the x-axis. [Figure 1-2]MTF activity for various reporter constructs in human cell lines. (F) DNA sequences of 5x RE repeats spaced 10, 6, or 0 base pairs (bp) apart. (G) Effect of spacing length between REs on reporter constructs on GoI expression in HeLa cells. Schematics of the promoter region and variable spacer arrangements are shown at the top. Microscopic images of HeLa cells show expression of the mCerulean GoI (top) and the transfection reporter control mCherry (bottom). Fluorescence channel names are shown on the left. The spacer length between REs on each promoter construct is indicated above each pair of images. (H) Quantitative analysis of mCerulean levels normalized to mCherry levels in HeLa cells transfected with reporter constructs with the spacers illustrated in panel F. Analysis is based on flow cytometry data. The spacer length between REs on each reporter construct is shown on the x-axis. (i) Effect of spacing length between REs on reporter constructs on GoI expression in HEK293 cells. A schematic of the promoter region and variable spacer arrangement is shown at the top. Microscopic images of HEK293 cells show expression of the mCerulean GoI (top) and the transfection reporter control mCherry (bottom). Fluorescence channel names are shown on the left. The spacer length between REs on each promoter construct is indicated above each pair of images. (J) Quantitative analysis of mCerulean levels normalized to mCherry levels in HeLa cells transfected with the reporter construct with the spacer illustrated in panel F. The analysis is based on flow cytometry data. The spacer length between REs on each reporter construct is shown on the x-axis. (K) DNA sequence of a scrambled (Scr) RE repeat repeated five times in the negative control promoter (SEQ ID NO: 51). (L) Microscopic image showing HeLa cells transfected with the negative control (Neg. ctrl) reporter design containing five scrambled (Scr) REs spaced 10 bp apart. (M) Analysis of flow cytometry measurements of the cells illustrated in panel L.(N) Microscopic image showing HEK293 cells transfected with a reporter design containing five scrambled REs spaced 10 bp apart. (O) Flow cytometry analysis of the cells illustrated in panel N. Scale bars in the microscopic images indicate 600 μm. Panels B, G, L: mCerulean, 100 ms exposure, LUT range 0-26,000; mCherry, 75 ms exposure, LUT range 0-35,000. Panels D, I, N: mCerulean, 75 ms exposure, LUT range 0-65k; mCherry, 50 ms exposure, LUT range 0-40,000. All micrographs are at 100x magnification. RE, response element; bp, base pair; Rel, relative; Scr, scrambled; Neg. ctrl, negative control. [Figure 2]Codon optimization of the MTF coding sequence (CDS). (A) An exemplary 6-amino acid subsequence of MTERF1 and its genetic code in wild-type (WT) and codon-optimized (CO) forms. Different DNA bases are shown on a light gray background. Each triplet is underlined, and the encoded amino acid is indicated below. (B) Micrographs of HeLa cells transfected with a 5xRE 6-bp spacer reporter (SEQ ID NO: 44) construct, MTF encoded by either the wild-type or codon-optimized DNA sequence, and a transfection control construct encoding constitutively expressed mCitrine. The upper image shows mCerulean, and mCitrine expression is indicated below. Fluorescence channels are indicated on the left. (C) Quantitative analysis of mCerulean levels normalized to mCitrine levels in the cells shown in panel B. Analysis is based on flow cytometry data. (D) Microscopic images illustrating upper mCerulean and lower mCitrine fluorescence levels in HEK293 cells. Fluorescence channel names are indicated on the left. (E) Quantitative analysis of mCerulean levels normalized to mCitrine levels in the cells shown in panel D. Analysis is based on flow cytometry data. Scale bars in B and D indicate 600 μm. Panel B: mCerulean, 2 s exposure, LUT range 5000-30'000; mCitrine, 500 ms exposure, LUT range 0-65'000. Panel D: mCerulean, 500 ms exposure, LUT range; mCitrine, 300 ms exposure, 0-65'000 LUT range. All micrographs are at 10x magnification. WT, wild type; CO, codon optimized; CDS, coding sequence. [Figure 3]Expression levels of reporter protein as a function of varying amounts of transfected MTF construct. The y-axis shows mCerulean expression relative to the transfection control mCherry. Error bars indicate standard deviation from triplicates of HEK293 cells transfected with the amount of MTF construct shown on the x-axis in nanograms. The line represents a dose-response curve fitted to the Hill equation, n=1. Analysis is based on flow cytometry data. [Figure 4-1] Comparing HumTAP variants with different transactivation domains. (A) Relative reporter expression in HEK293 cells after cotransfection of the indicated HumTAP constructs (names are shown on the x-axis using HUGO gene nomenclature and amino acid numbering according to UniProt) with the 5xRE 0 bp interval (SEQ ID NO: 48) promoter driving mCerulean GoI. Normalized to expression of the transfection control mCherry. [Figure 4-2] (B) Comparison of HumTAP variants with different transactivation domains. (C) DNA length in base pairs (bp) required to encode each TAD. The dotted lines in both panels indicate the mCerulean GoI expression level or the DNA size of the RelA430-551 TAD. Rel., relative; bp, base pairs. [Figure 5-1] Construction and testing of HumTAP with a reduced gene footprint. (A) Schematic diagram depicting the different domains of MTERF1. Numbers above the horizontal bars indicate amino acid numbers, starting with 1 at the N-terminus. Bar widths are proportional to their amino acid lengths, with the exception of dotted bars, which indicate stretches of amino acids that are not represented. [Figure 5-2]Construction and testing of HumTAP with a reduced gene footprint. (B) Quantitative analysis of mCerulean levels normalized to mCherry levels in cells transfected with HumTAP constructs assembled from the DNA-binding domains illustrated in panel A. Bars colored by different shades of gray represent different amounts of HumTAP construct transfected. The MTERF1 domains used are indicated on the x-axis. Analysis is based on flow cytometry data. (C) The length in base pairs (bp) of DNA required to encode the different DNA-binding domains. Peptide ranges from MTERF1 are indicated on the x-axis. The dotted line indicates the length of the benchmark MTERF1 58-399 domain. MTP, mitochondrial transit peptide; WT, wild-type; Rel., relative; bp, base pairs. [Figure 6-1] Experimental evaluation of immunogenicity. (A) Scheme of the experimental process. Peripheral blood mononuclear cells (PBMCs) are depicted in various shapes inside the drawing of the culture well, and peptides are depicted as rods. Dimethyl sulfoxide (DMSO) is the solvent for the peptides. [Figure 6-2] Experimental evaluation of immunogenicity. (B) P values ​​of a t-test comparing the number of spot-forming units (SFU) from cells primed and recalled with the same peptide pool and from the same PBMCs that were primed but not recalled. The x-axis indicates the protein from which the peptide pools used for priming and recall were derived. Light shades of gray depict p-values ​​above 0.05, and donors are classified as non-responders. Darker shades of gray indicate the area with p-values ​​below 0.05. Samples from donors that fall within this area are considered responders. The dotted line indicates a p-value of 0.05. Different dot signs indicate different PBMC donors. DMSO, dimethyl sulfoxide; ELISPot, enzyme-linked immunospot; H0: null hypothesis; SFU, spot-forming unit. [Figure 7]Orthogonal assessment of MTF. A) Confocal microscopy of HeLa cells transfected with either Flag-tagged wild-type MTERF1 or MTF. Scale bar indicates 10 μm. MitoRed is a dye that stains mitochondria, anti-Flag Ab is used to stain Flag-tagged transfected proteins, and Hoechst 33342 stains DNA. B) Diagram showing the purported interactions of the plasmids and their encoded proteins. C) Reporter fluorescence levels of HEK293 cells transfected with an MTF construct and a reporter construct containing a spacerless 5xRE (SEQ ID NO: 48) on its promoter, with or without WT MTERF1 driven by EF1a. [Figure 8] Volcano plot of differential gene expression analysis of transfected HEK293 cells. (A) Volcano plot showing differentially regulated genes between cells transfected with WT MTERF1 and junk plasmids. The y-axis shows the log10 of the false discovery rate (FDR). The log2 of the fold change (FC) is shown on the x-axis. Light gray dots indicate genes that are not significantly differentially expressed, medium shades of gray dots indicate down-regulated genes, and dark shades represent up-regulated genes. Labels indicate the ENSEMBL symbol of the gene corresponding to the nearest spot or "-" for transcripts not related to the named gene. (B) Volcano plot comparing gene expression between cells transfected with MTF constructs and cells that received junk DNA plasmids. The y-axis shows the log10 of the false discovery rate (FDR). The log2 of the fold change (FC) is shown on the x-axis. Light gray dots indicate genes that are not significantly differentially expressed, medium shades of gray dots indicate downregulated genes, and dark shades represent upregulated genes. Labels show the ENSEMBL symbol of the gene corresponding to the nearest spot, or "-" for transcripts not related to the named gene. WT, wild type; FDR, false discovery rate; FC, fold change. [Figure 9-1]Optimization of transcriptional activation strength and protein size. A) Overview of TAD domains. Subscripted numbers indicate the amino acid range encompassing the TAD domains used in this study. B, C, E, F) Transcriptional activation mediated by individual transactivation domains. The number of base pairs required to encode the HumTAP variants is shown in the upper panel. The lower panel shows flow cytometry analysis of HEK293 cells cotransfected with a plasmid encoding pEF1a-driven mCherry, the Design 6 reporter, and equimolar amounts of a plasmid encoding pEF1a-driven MTERF158-399 fused to the TAD domain indicated below each bar. Symbols below the bars correspond to those in panels A and D. Bars correspond to the average of three replicates, and each dot represents one replicate. The horizontal dashed line indicates rel. mCerulean levels transfected with the benchmark MTERF158-399::RelA430-551 HumTAP variant, i.e., "MTF" (SEQ ID NO: 100). [Figure 9-2]Optimization of transcriptional activation strength and protein size. B, C, E, F) Transcriptional activation mediated by individual transactivation domains. The number of base pairs required to encode the HumTAP variants is shown in the upper panels. The lower panels show flow cytometry analysis of HEK293 cells co-transfected with a plasmid encoding mCherry driven by pEF1a, the Design 6 reporter, and equimolar amounts of a plasmid encoding MTERF158-399 driven by pEF1a fused to the TAD domain indicated below each bar. Symbols below the bars correspond to those in panels A and D. Bars correspond to the average of three replicates, and each dot represents one replicate. The horizontal dashed line indicates rel. mCerulean levels transfected with the benchmark MTERF158-399::RelA430-551 HumTAP variant, i.e., "MTF" (SEQ ID NO: 100). D) Illustration of the MTERF1 structure and amino acid ranges retained for the different variants. The amino acid sequence omitted from the illustration is indicated by a dotted outline, and the vertical dashed line indicates the starting amino acid position for each variant. TAD, transactivation domain; DBD, DNA-binding domain; MTP, mitochondrial transit peptide; bp, base pair. [Figure 10-1] Construction of a rapalog-inducible HumTAP-based gene expression system. A) Illustration of A / C heterodimerizer-mediated dimerization of MTERF158-399::FKBP (SEQ ID NO: 192) and FRB::TAD. B) Flow cytometry analysis of HEK293 cells co-transfected with the Design 6 reporter plasmid, a plasmid encoding mCherry driven by pEF1a, and varying amounts of each plasmid encoding pEF1a-driven expression of the respective dimerization proteins. Axis units refer to molar equivalents of plasmid amounts, and relative mCerulean was calculated by normalizing the mCerulean signal to the mCherry signal. [Figure 10-2]Construction of a rapalog-inducible HumTAP-based gene expression system. C) Relative mCerulean expression levels in HEK293 cells co-transfected with the Design 6 reporter plasmid, a plasmid with pEF1a-driven mCherry expression, and a plasmid encoding pEF1a-driven expression of each of two dimerization proteins. The dots represent mCerulean levels normalized to mCherry levels, and the dashed line represents a logistic model fit. The color indicates which TAD was used to construct the FRB::TAD fusion protein transfected into the cells from which the data was obtained. D) Relative mCerulean expression levels in HEK293 cells co-transfected with the Design 6 reporter plasmid, a plasmid with pEF1a-driven mCherry expression, and a plasmid encoding pEF1a-driven expression of each of two dimerization proteins. The dots represent mCerulean levels normalized to mCherry levels, and the line represents data fitted using a logistic model. Colors indicate which linkers were used to construct the MTERF158-399::FKBP fusion protein (linker 1) and FRB::TAD fusion protein (linker 2) transfected into the cells from which the data were obtained. TAD, transactivation domain; Rv1, RelA430-551 (SEQ ID NO: 3). [Figure 11] Schematic diagram of the massively parallel reporter assay to determine the design principles of corresponding promoters. A) Illustration of the components and various properties in the plasmid library. B) Overview of the experimental procedure. BS, binding site; bp, base pair; BC, barcode; RT, reverse transcription; PCR, polymerase chain reaction. [Figure 12]Quality control of promoter design libraries. A) Chimera ratio indicates the ratio of reads containing unexpected promoter designs coupled to a given barcode to the total number of reads containing the barcode. Barcode read count indicates the total number of reads in which the barcode was present. The distribution of points along each axis is shown above and in the left margin. R2 refers to the squared Pearson correlation coefficient. B) The distribution of design parameters is shown above each stack. The percentage indicates the frequency of each given parameter identified from all reads, and the box is the corresponding height. dir., direction. [Figure 13-1] Quality control of sequencing results. A) Histogram showing how often each barcode was read in the plasmid DNA samples. B) The height of the stacked bars indicates the distribution of barcodes originating from either library members or the UbC control for each sample. C) Dot plot showing the activity scores for each barcode in two replicates of cells transfected with the MTF plasmid amount corresponding to EC90. The marginal density plot shows the distribution of barcode activity scores for replicate 2. D) Histogram of promoter-level activity scores at the MTF level corresponding to EC90. [Figure 13-2]Quality control of sequencing results. E) Dot plot showing on the y-axis Citrine fluorescence levels normalized to mCherry expression in HEK293 cells co-transfected with individually picked plasmids, a pEF1a-driven MTF-encoding plasmid at an amount corresponding to the EC90, and a pEF1a-driven mCherry-encoding plasmid. The x-axis shows the activity score for the design corresponding to the individually picked plasmids. R2 is the squared Pearson correlation, ρ is the Spearman correlation coefficient, and the linear regression fit is shown as a dashed line. F) Trajectories of activity scores obtained from RNA samples of cells co-transfected with the plasmid library, a UbC-driven Citrine-encoding plasmid, and various amounts of pEF1a-driven MTF-encoding plasmid. EC, effective concentration. [Figure 14-1] Promoter activity at the MTF level corresponding to its EC90. A) Scheme of the gene regulatory cascade underlying the measured promoter design activity scores. B) Rank assigned to promoters according to their activity score in descending order. Marginal density plot represents the distribution of promoter activity scores. C) Heat map of all activity scores for all designs. For designs with one BS, the "BS-BS distance" was not applied and was set to 0. [Figure 14-2] Promoter activity at MTF levels corresponding to its EC90. D) Distribution of activity scores. Violin contains all designs that share the indicated design parameters, and black dots represent the median activity score. P-values ​​were calculated using analysis of variance (ANOVA). E, F) Distribution of activity scores. Violin contains all designs that have binding sites in either sense (E) or antisense (F) orientation relative to the minimal promoter and share the indicated design parameters. Black dots represent the median activity score. P-values ​​were calculated using analysis of variance (ANOVA). BS, binding site; bp, base pair [Figure 15]Characterization of individual promoter variants. A) Variants were either picked randomly or specifically selected for their high activity scores and small size. Dark dots indicate promoter design variants that were picked and transfected individually; dots with different fill patterns indicate designs represented by the same pattern in panels B, C, and D. B, D) Average relative mCitrine levels of three or six replicates measured by flow cytometry in cells cotransfected with a plasmid encoding MTF at a level corresponding to the EC90. C) Relative mCitrine levels from cells transfected with a plasmid encoding MTF in the amount indicated in the axis labels. D) White dots indicate individual replicates. [Example]

[0334] Methods and materials for use in the present disclosure are described herein; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. Example 1. Engineering a synthetic transactivation system based on MTERF1

[0335] The present inventors engineered an exemplary "human-derived transcription activator protein" (HumTAP) transactivation system by engineering a HumTAP transactivator and a promoter containing a response element / MTERF1 binding site operably linked to a gene of interest. A peptide consisting of amino acids 58-399 of the human MTERF1 gene (MTERF1) was synthesized. 58-399 (SEQ ID NO: 1, Uniprot Q99551) was synthesized by PCR using a peptide consisting of amino acids 430 to 551 of the RELA protein (RelA 430-551 (SEQ ID NO: 3, Uniprot Q04206) to generate a prototype HumTAP protein. This fusion is hereafter referred to as MTERF1. 58-399 ::RelA 430-551(or referred to as "MTF"; SEQ ID NO: 100). A DNA construct was made with the strong EF1A promoter that drives constitutive expression of MTF (SEQ ID NO: 101) in human cells (the "MTF construct").

[0336] To construct a promoter with a response element / MTERF1-binding site driving a gene of interest, we placed an MTERF1-binding site (SEQ ID NO: 42) upstream of a minimal TATA box (SEQ ID NO: 103). An mCerulean fluorescent reporter representing the gene of interest (GoI) was placed downstream of the TATA box. Each of these is referred to as a reporter construct. Five different reporter constructs were created, each containing 3, 5, 7, 9, and 11 MTERF1-binding sites spaced 6 base pairs apart upstream of the TATA box (Figure 1A; SEQ ID NOs: 41, 44, 45, 46, and 47, respectively). These were cotransfected with the MTF construct into HeLa cells. Microscopic analysis showed the highest mCerulean signal for 11 repeats of the MTERF1-binding site (Figure 1B). Relative mCerulean levels were measured using flow cytometry and calculated by normalization to the transfection control in HeLa cells (Figure 1C). The same transfection conditions in HEK293 cells revealed an increase in reporter fluorescence levels with the number of RE / MTERF1 binding sites in the promoter design. Microscopy (Figure 1D) and flow cytometry (Figure 1E) were used. Next, reporter plasmids with various spacer lengths (0, 6, and 10 bp; SEQ ID NOs: 48, 44, and 49, respectively) between a fixed number of REs (i.e., five RE / MTERF1 binding sites) (Figure 1F) were co-transfected with the MTF construct. In HeLa cells, microscopy revealed a large increase in reporter expression when there was no spacing at all, and similar amounts of mCerulean with 6- and 10-bp spacing (Figure 1G). This was confirmed using flow cytometry (Figure 1H). A different trend was observed in HEK293 cells, with the highest reporter levels observed with 6-bp spacing and very little mCerulean signal observed with 10-bp spacing. Microscopy (Figure 1I) and flow cytometry (Figure 1J) were used.Confirming sequence specificity, a reporter construct with a promoter containing five scrambled DNA binding sites (SEQ ID NO: 51) spaced 10 bp apart (Fig. 1K) did not result in mCerulean expression upon cotransfection with the MTF construct in HeLa cells, as assessed by microscopy (Fig. 1L) and flow cytometry (Fig. 1M). HEK293 cells exhibited the same behavior under microscopy (Fig. 1N) and flow cytometry (Fig. 1O).

[0337] Next, we optimized the DNA sequence encoding MTF by changing the codon for each amino acid from the naturally occurring one to the most common one in humans (SEQ ID NO: 102; Figure 2A). This optimization increased reporter gene expression approximately three-fold in HeLa cells using a reporter construct (SEQ ID NO: 44) with 5xRE / MTERF1 binding sites separated by a 6-bp spacer on its promoter. This was assessed by microscopy (Figure 2B) and flow cytometry (Figure 2C). In HEK cells, this modification had no significant effect on reporter fluorescence levels, as illustrated by micrographs (Figure 2D) and flow cytometry analysis (Figure 2E).

[0338] By transfecting HEK293 cells with different amounts of the codon-optimized MTF construct, we determined the dose-dependence of mCerulean reporter (GoI) expression on MTF expression levels (Figure 3). HEK293 cells on 24-well plates were transfected with 0.75, 3, 6, 12, 24, 48, 96, 192, and 384 ng of the MTF construct, 100 ng of the reporter construct, and 50 ng of a constitutive mCherry transfection control construct. Two days after transfection, cells were analyzed using flow cytometry. The dose-response curve was modeled as the Hill equation, assuming a Hill coefficient of 1. The EC10 and EC50 were achieved at 1.25 ng and 11.24 ng of the transfected MTF construct, respectively. The theoretical EC90 was achieved at 101 ng, but a slight drop in relative reporter fluorescence levels is observed with higher amounts of transfected MTF construct.

[0339] With the goal of engineering a stronger and possibly shorter HumTAP, we investigated the MTERF1 58-399 The peptide (SEQ ID NO: 1) was fused to various transactivation domains

[18] , and HEK293 cells were transfected with these constructs and a reporter construct (SEQ ID NO: 48) containing the 5xRE / MTERF1 binding site without a spacer between them.

[0340] First, MTERF1 58-399 (SEQ ID NO: 1) was fused to different subsequences of RelA. 361-551 (SEQ ID NO: 27) corresponds to the sequence used to construct the bacterially derived synthetic TF PIT2

[19] , and RelA 342-551 (SEQ ID NO: 29) contains all annotated transactivation domains according to the domain annotation of Uniprot Q04206 (https: / / www.uniprot.org / uniprotkb / Q04206 / entry). 430-551Both lead to stronger reporter gene expression (Fig. 4A) compared to TA1 of RelA (SEQ ID NO: 3; Fig. 4B), but also require a larger gene footprint. 521-551 , SEQ ID NO: 31) domain

[20] did not significantly activate reporter gene expression. 58-399 (SEQ ID NO: 1) was fused to a set of transactivation domains previously found to be potent activators with a small genetic footprint

[18] (Figure 4B).

[0341] The WW domain of WWC1 (SEQ ID NO: 21; WWC1 2-81 , Uniprot Q8IX03), the KRAB domain of ZNF473 (ZNF473 5-48 , Uniprot M0R032, SEQ ID NO: 23), and the Nuc_rec_co-act domain of NCOA3 (SEQ ID NO: 25; NCOA3 1045-1092 , Uniprot Q9Y6Q9) did not lead to high reporter expression. Conversely, the LMSTEN domain of MYB (SEQ ID NO: 19; MYB 251-330 , Uniprot P10242) and the FOXO-TAD domain of FOXO3 (SEQ ID NO: 17; FOXO3 604-644 , Uniprot O43524) mediated significant mCerulean expression. A commonly used strong viral transactivation domain, the transactivation domain of HHV11 (SEQ ID NO: 15) linked by a GS linker (SEQ ID NO: 13; HHV11 437-447 VP64, consisting of three repeats of the nucleotide sequence (Uniprot P06492;), was used as the benchmark RelA 430-551 Interestingly, FOXO3 mediated lower reporter expression than the FOXO3 domain. 604-644 (SEQ ID NO: 33) or RelA 521-551Fusion of two copies of (SEQ ID NO: 35) resulted in 12- and 201-fold greater fluorescence than that conferred by only one copy of each TAD (FIG. 4A). These results indicate that the DBD of MTERF1 can be used as a versatile building block for regulating gene expression when fused to TADs that may or may not be derived from RelA.

[0342] To further reduce the DNA footprint of HumTAP, we investigated to what extent the DNA-binding domain could be reduced. 430-551 Three versions of the MTERF1 partial sequence fused to (SEQ ID NO: 3) were created. 73-399 (SEQ ID NO: 7), MTERF1 104-399 (SEQ ID NO: 9), and MTERF1 135-399 (SEQ ID NO: 11) is a mitochondrial transit peptide (MTERF1 1-57 ; SEQ ID NO: 37). These are subunits of MTERF1 without the first 14 N-terminal amino acids (MTERF 58-72 ; SEQ ID NO: 39) or in addition to the first (MTERF1 73-98 ;SEQ ID NO: 104) or the first and second (MTERF1 104-134 ; SEQ ID NO: 106) (Figure 5A)

[21] . Removal of the N-terminal domain reduces the gene footprint of HumTAP (Figure 5B). These constructs were co-transfected into HEK293 cells with a reporter construct (SEQ ID NO: 48) containing a spacerless 5xRE on its promoter. By truncating MTERF1 from the N-terminus up to amino acid 104, i.e., MTERF1 73-399 (SEQ ID NO: 7) and MTERF1 104-399 (SEQ ID NO: 9), output expression was found to be reduced, but not eliminated. However, further truncation to amino acid 135 (MTERF1 135-399(SEQ ID NO: 11)) led to a complete loss of transcriptional activation (Figure 5B), demonstrating that the gene footprint of synthetic TFs can be reduced while activity is retained.

[0343] Example 2. Immunogenicity assessment

[0344] T cells that tightly bind self-peptide-MHC complexes are deleted in the thymus during maturation, allowing tolerance to self-peptides.

[22] Therefore, because HumTAP is composed entirely of human protein subunits, it is expected to have a favorable immunogenicity profile compared to proteins of non-human origin.

[0345] We evaluated whether immune responses could be primed by protein subsequences. Using NetMHCpan 4.0

[23] , we predicted which peptides derived from the MTERF-RelA junction were most likely to bind strongly to the most common MHC I allele, HLA-A02:01, in the human population. A pool of the five strongest predicted binding peptides was synthesized. Furthermore, for each of these "core" binders, we synthesized 15-mers containing flanking sequences, resulting in 10 peptides. As a positive control, we used a peptide pool spanning the immunogenic cancer-associated protein NY-ESO1. Peptides derived from the self portion (i.e., RelA and MTERF, but not the junction) served as a negative control. Peripheral blood mononuclear cells (PBMCs) were isolated from normal blood donors and transferred to 24-well plates, and each peptide pool was added to each of three replicate wells. The cells were then incubated for 14 days in the presence of IL-2 and IL-7. During this stage, PBMCs, specifically T cells bearing T cell receptors (TCRs) that recognize peptides bound to MHC class I or class II on the surface of antigen-presenting cells, are stimulated and begin to proliferate, a process called "priming." Next, PBMCs in each well were restimulated overnight with the same peptide pool and subjected to IFNγ-ELISPOT in quadruplicate. Spots were then generated and counted (Figure 6A). Spots were induced by T cells previously primed with one of the peptides in the priming peptide mixture; therefore, more spots correspond to the more immunogenic peptide mix in the priming step. As recommended in Moodie (2010), Cancer Immunology, Immunotherapy volume 59, pp. 1489-1501, donors are classified as "responders" if significantly more spot-forming units (SFU) (p<0.05) are observed for the restimulated cells than their respective primed but non-restimulated background controls.Several donors did not show a significant response to the NY-ESO1 peptide pool and were ignored in the analysis. Seven PBMC samples showed a response to NY-ESO1 (positive control) and were included in the analysis. Of these samples, one mounted a significant response to the junction-derived peptide pool, and three PBMC samples were considered responders to the negative control peptide (Figure 6B). This suggests that the MTF junction-derived peptide pool behaves much more similarly to the negative control consisting of self-peptides than to the positive control of known immunogenicity, indicating low immunogenicity in humans. Example 3. Orthogonality evaluation

[0346] We checked whether the absence of the mitochondrial transit peptide (MTP) in HumTAP leads to orthogonality of HumTAP to human host cells, and vice versa: (i) whether endogenous human gene expression is not substantially modulated by HumTAP, and (ii) whether endogenously expressed wild-type MTERF1 (i.e., including the MTP set forth in SEQ ID NO: 37) lacks the ability to regulate expression of a gene of interest driven by a response element-containing promoter.

[0347] First, we assessed subcellular localization by transfecting HeLa cells with a plasmid constitutively expressing Flag-tagged MTF (SEQ ID NO: 128) or Flag-tagged wild-type MTERF1 (SEQ ID NO: 130). Cells were stained with anti-Flag tag antibody and imaged by confocal microscopy. Indeed, WT MTERF1 was found to be restricted to mitochondria, whereas MTF was distributed throughout the cell, including the nucleus, without apparent localization to mitochondria (Figure 7A).

[0348] To assess host orthogonality to a response element-driven gene of interest (Gol), we transfected MTF (SEQ ID NO: 100), representing HumTAP, together with a response element-driven mCerulean fluorescent reporter (SEQ ID NO: 48), with or without a construct encoding wild-type MTERF1, to assess whether it could interfere with induction of the GoI by MTF (Figure 7B). We found no difference in GoI fluorescence levels with or without concomitant WT MTERF1 expression (Figure 7C).

[0349] Alignment of MTERF1 binding sites against the human genome using BLAST did not reveal a perfect match on the nuclear genome, but the existence of non-perfect binding sites to which MTF may bind could not be excluded. Therefore, we used RNA-Seq to examine whether MTF (SEQ ID NO: 100) activates the expression of endogenous human genes. We also transfected WT MTERF1 (SEQ ID NO: 108) to compare the gene regulatory effects of MTERF1 with those of MTF. Differential gene expression analysis was performed on cells transfected with the "junk" plasmid using the edgeR software package for R

[24] . Genes were considered differentially expressed (DE) if they were found to be significantly differentially expressed by at least a fold change of 2 (FDR < 0.5). With WT MTERF1, seven genes were found to be downregulated and three were upregulated (not including MTERF1 expressed from the transfected plasmid) (Figure 8A). MTF downregulated 24 genes and upregulated 4 genes (Figure 8B). Notably, none of the genes found to be downregulated by WT MTERF1 were found to be affected by MTF. Only one gene, HSPA7, was upregulated in both conditions (Table 1). Although there were a few genes significantly downregulated by MTF, none of them contained sequences similar to MTERF1 binding sites in their genomic vicinity. Similarly, none of the upregulated genes in MTF-transfected cells possessed a classical MTERF1 binding motif. We conclude from this experiment that MTF does not retain the gene regulatory function of wild-type MTERF1 protein and does not exert widespread off-target gene activation. The lack of MTERF1 binding sites in the genomic vicinity of downregulated genes may suggest a mechanism independent of classical MTF binding. Future studies will assess the dose-dependence of differentially expressed (DE) genes on MTF dosage, which may help clarify the origin of DE genes.

[0350] Table 1. List of all DE genes for MTF (top) and WT MTERF1 (bottom). MTF [Table 66] WT MTERF1 [Table 67] Example 4. Optimizing humTAP

[0351] This example is an extension of Example 1 herein above, which describes some of the same data and results.

[0352] In many applications, viral delivery methods impose the need for components to be as small as possible, while at the same time robust transactivation of target genes by synthetic transcription factors (synTFs) is required.

[25] Therefore, we investigated a wide variety of human and engineered chimeric transactivation domains (TADs) for their size and transactivation strength and compared them with RelA. 430-551 It was benchmarked against the peptide (SEQ ID NO: 3).

[0353] RelA 430-551 In addition to v1), we tested three alternative subsequences: v2) RelA 342-551 (SEQ ID NO: 29), which contains all the transactivation domains annotated by UniProt Q04206; v3) RelA 361-551 (SEQ ID NO: 27), which is identical to the peptide used to construct PIT2 [2]; and v4) RelA 521-551(SEQ ID NO: 31), which corresponds to only the transactivation domain 1 (TA1)

[26] . Protein domains WW (SEQ ID NO: 21), KRAB (SEQ ID NO: 23) of ZNF473, Nuc-rec-co-Act (SEQ ID NO: 25), LMSTEN (SEQ ID NO: 19), and FoxoTAD (SEQ ID NO: 17) were recently identified as potent transactivators

[27] . To assess their functionality in the context of HumTAP, we compared each of them with MTERF1. 58-399 (SEQ ID NO: 1) was used to create a fusion protein (FIG. 9A).

[0354] Cotransfection of HEK293 cells with plasmids encoding pEF1a-driven expression of fused HumTAP, a response element-driven mCerulean reporter protein, and an mCherry transfection control demonstrated that all peptides derived from RelA, with the exception of TA1, mediated high levels of reporter gene expression. Inclusion of larger fragments of the RelA protein led to higher reporter expression. Conversely, the core transactivation domains TA1, WW, ZNF473 KRAB, and NucRecCoAct mediated little or no reporter expression. The LMSTEN and FoxoTAD domains produced some mCerulean expression, but at levels 11.3- and 8.4-fold lower than the benchmark RelA430-551 TAD, respectively (Figure 9B).

[0355] Interestingly, fusion of two TA1 domains (SEQ ID NO: 35) led to reporter expression, and addition of a third domain (SEQ ID NO: 194) further increased output expression (Figure 9C). Similarly, two copies of FoxoTAD (SEQ ID NO: 33) were 20-fold more efficient than just one FoxoTAD copy, compared with the benchmark RelA 430-551The FoxoTAD mediated approximately two-fold greater mCerulean expression than the TAD. This effect was less pronounced for the LMSTEN domain, for which two copies (SEQ ID NO: 196) mediated approximately 2.7-fold greater reporter expression than one copy. Fusing FoxoTAD to LMSTEN (SEQ ID NO: 54), LMSTEN to TA1 (SEQ ID NO: 56), or FoxoTAD to TA1 (SEQ ID NO: 58) simultaneously enhanced the expression of the benchmark RelA 430-551 The chimeric TAD (SEQ ID NO: 52) consisting of FoxoTAD, LMSTEN, and TA1 produces 3.7-fold higher reporter gene expression than RelA430-551 (Figure 9C). Thus, without incorporating protein domains of non-human origin, the commonly used RelA 430-551 It is possible to engineer both transactivation domains that are both stronger and smaller than the TADs described above. Given our observation of synergy between identical and heterotypic transactivation domains, it is likely that further studies exploring this effect will yield human-based chimeric TADs that mediate even higher levels of transgene expression without increasing the size of the protein domains.

[0356] To further reduce the size of the HumTAP protein, we deleted the N-terminus (SEQ ID NO: 39), optional Mterf motif 1 (SEQ ID NO: 104), and optional Mterf motif 2 (SEQ ID NO: 106)

[28] , in addition to the MTP (SEQ ID NO: 37) of MTERF1 (Figure 9D). Given the activity-killing effect of the R387A mutation, located just 12 amino acids from the MTERF1 C-terminus

[28] , we did not delete any of the C-terminal domains. 73-399 ; SEQ ID NO: 7) and both the N-terminus and Mterf motif 1 (MTERF1 104-399 ; SEQ ID NO: 9) resulted in approximately 1.5-fold less reporter expression, but reduced the DNA size by 45 and 135 bases, respectively.

[0357] Omitting also Mterf motif 2 (as in SEQ ID NO: 11) resulted in a non-functional synTF (Figure 9E).

[0358] To check whether the reduced transactivation activity of HumTAP with a smaller DBD could be compensated for, we fused a novel chimeric TAD to MTERF1 104-399 In this context, the FoxoTAD::TA1 chimeric TAD (SEQ ID NO: 58) produced a synTF as strong as MTF (SEQ ID NO: 100), but required 288 fewer DNA bases to encode on the vector. FoxoTAD::FoxoTAD (SEQ ID NO: 33) and FoxoTAD::LMSTEN (SEQ ID NO: 54) were fused to the MTERF1 peptide. 104-399 This combination results in a synTF that is approximately 1.5-fold more potent than MTF (SEQ ID NO: 100), but is 96 and 86 amino acids smaller, respectively. Using FoxoTAD::LMSTEN::TA1 (SEQ ID NO: 52) in this context led to a synTF that was similar in size to MTF, but mediated 3.5-fold more reporter gene expression (Figure 9F).

[0359] Overall, this data set supports the idea that MTERF1 retains DNA-binding functionality even when the N-terminal amino acids are missing. The reduced reporter levels could be explained by a reduced affinity of the protein for DNA or a less stable protein. Either way, high reporter gene expression levels could be restored through the use of optimized TADs. Example 5. Engineering an MTF-based inducible gene expression system

[0360] The tunability of output expression strength with small molecules is recognized as an important feature of synthetic transcription activation systems

[29] . Therefore, we sought to engineer an inducible HumTAP system based on dimerization.

[0361] One well-characterized small molecule-based dimerization system is based on the rapamycin analog A / C (C16-(S)-7-methylindolerapamycin), which induces heterodimerization between the human protein FK506-binding protein 12 (FKBP; SEQ ID NO: 142) and the FKBP12-rapamycin binding domain (FRB) mutant FRBT2098L (SEQ ID NO: 140)

[30] ,

[31] . To achieve A / C heterodimerizer-inducible gene expression, we coupled FKBP (SEQ ID NO: 142) to MTERF1. 58-399 FRB (SEQ ID NO: 140) was fused to the N-terminus of MTERF1 (SEQ ID NO: 1) and to the C-terminus of different transactivation domains (Figure 10A). At a constant A / C heterodimerization agent (i.e., C16-(S)-7-methylindolerapamycin) concentration of 100 nM, we observed that MTERF1 58-399 4-fold molar excess of FRB::RelA compared to ::FKBP (SEQ ID NO: 192) plasmid 430-551 We found that the (SEQ ID NO: 170) plasmid led to the highest mCerulean concentrations in HEK293 cells co-transfected with an inducible TF component and a mCerulean reporter protein driven by a response element (FIG. 10B). 58-399 In HEK cells co-transfected with ::FKBP and a fusion protein between FRB and the most potent of the newly developed TADs (see Example 4), mCerulean levels increased dose-dependently with increasing inducer concentration (Figure 10C). In accordance with the results described above, the chimeric transactivation domains enhanced the RelA 430-551 mediated higher maximal output expression levels than

[0362] Although the physiological blood concentration of rapamycin derived from the A / C dimer reaches approximately 22 nM upon ingestion

[32] , this system exhibits an EC50 between 100 and 120 nM. Previously reported inducible gene expression systems based on A / C-induced FRB and FKBP dimerization exhibited an EC50 value of approximately 1 nM

[31] ,

[33] ,

[34] . Thus, this system is inducible at pharmacologically achievable inducer concentrations. We reasoned that dimerization in this system could be prevented by either the FRB or FKBP fusion partner and examined whether the addition of a linker between the protein domains could impact the EC50 value (Figure 10D). Combining the cMyc nuclear localization signal (NLS) (SEQ ID NO: 122) linking MTERF158-399 and FKBP with the rigid linker AP6 (SEQ ID NO: 120) linking FRB and RelA430-551 led to a reduction in EC50 to approximately 60 nM, but increased the maximum output by approximately 3-fold compared to the variant with RelA430-551 as the TAD and no linker (Figure 10D). Example 6. Engineering a collection of promoters (sensors) for HumTAP

[0363] Several factors interact in a complex manner to impact the strength of synthetic promoters. We performed massively parallel reporter assays (MPRA)

[35] -

[37] to quantify the impact of four promoter design parameters on transactivation strength: the number of MTERF1 binding sites (BS) (SEQ ID NO: 42), their orientation relative to the minimal promoter, the number of bp spacing between BSs, and the number of bp spacing between the minimal promoter and the proximal BS (Figure 11A).

[0364] We considered a wide range of 1 to 5 BS (SEQ ID NO: 42) copies, each in both sense (SEQ ID NO: 42) or antisense (SEQ ID NO: 200) orientation relative to the yb_TATA minimal promoter (SEQ ID NO: 103), with distances of 0 to 10 bp to the minimal promoter (see SEQ ID NO: 199 for a 10 bp spacer; shorter spacers truncate the 3' end of SEQ ID NO: 199), and with spacings of 0 to 10 bp between BSs (see SEQ ID NO: 198 for a 10 bp spacer; shorter spacers truncate the 3' end of SEQ ID NO: 198). For each of the 990 possible parameter combinations, we assigned 10 unique 11-bp barcodes. The resulting library of 9,910 distinct DNA sequences was cloned such that each encoded promoter variant drove expression of mCitrine, which has a barcode attached to its 3' UTR. The relative frequency of each barcode in the plasmid library was measured using next-generation sequencing. To control for differences in transfection efficiency and expression levels between different conditions, we cloned 10 plasmids encoding the constitutive UbC promoter (SEQ ID NO: 139) driving mCitrine, each with a unique barcode on the 3'UTR. Then, amounts of MTF (SEQ ID NO: 100) plasmid corresponding to EC10, EC50, and EC90 (see Example 1 and Figure 3) were cotransfected into HEK293 cells in triplicate with the promoter library and 10 UbC control plasmids. To quantify the expression level of each barcode, RNA was extracted from the cells, reverse transcribed, amplified, and sequenced. For each barcode, a score for each condition and replicate was calculated as the barcode count from the RNA sample. This was normalized to the barcode frequency in the plasmid library and the median of the 10 barcodes associated with the UbC control. The "barcode-level activity score" was the average of the barcode scores from the triplicate replicates. For each promoter design, a final activity score was calculated as the median of the 10 concatenated barcode-level activity scores (Figure 11B).

[0365] We performed quality control of plasmid libraries using nanopore long-read sequencing, which was analyzed by an algorithm that extracted promoter design parameters and barcodes for each read. A common problem in MPRAM library construction is the decoupling of barcodes from their assigned variants due to the formation of chimeric DNA sequences during PCR amplification of DNA oligo pools

[38] ,

[39] . In our libraries, this effect occurs at an average rate of 17%. This means that, on average, 17% of plasmids encoding a given barcode are coupled to a promoter design different from the one assigned. The lack of correlation between barcode read counts and chimeric rates suggests that our analysis does not underestimate the true chimeric rate (Figure 12A). While chimeras likely cause noise in screening, we did not expect it to be high enough to hinder our ability to draw conclusions. Furthermore, analysis of design parameter distributions did not reveal any strong bias for any certain parameters, other than a skew toward promoters containing BSs in the antisense orientation (Figure 12B). Therefore, we chose to proceed with screening using a plasmid library.

[0366] We then performed the entire workflow outlined in Figure 11B. Analysis of barcode frequencies in the plasmid library indicated complete coverage of the library, with no barcodes read less than 10 times, and an average of approximately 27,000 reads per barcode, tailing toward a maximum of 224,959 reads (Figure 13A). As the MTF amount increases, so should the proportion of reads from the library compared to those from the UbC control. Indeed, we found that between 75% and 97.6% of reads were attributable to the plasmid library for 0 MTF and EC90 levels, respectively (Figure 13B). Calculation of barcode-level activity scores in each sample revealed a bimodal distribution of scores: barcodes either fell into the "low" group, having scores below approximately 0.001, or they fell into the "high" group, achieving scores above this threshold. Comparison of the two replicates showed that some barcodes fell into the "low" group in one replicate and the "high" group in the other, and vice versa (Figure 13C). After calculation of the mean and promoter-level activity scores across replicates, no further values ​​below 0.001 were observed in the EC90 condition (Figure 13D). We then isolated 39 individual plasmids from the library, cotransfected each plasmid into HEK293 cells with the amount of MTF corresponding to the EC90 and an mCherry transfection control plasmid, and measured fluorescence levels using flow cytometry. mCitrine fluorescence levels and promoter-level activity scores from the screening correlated well, with an R of 0.77. 2 and a Spearman correlation efficiency (which does not assume a linear relationship) of 0.9 (Figure 13E). Confirming promoter responsiveness to MTF, activity scores for most designs increased with MTF levels, although no consistent increase was observed for the negative control design based on scrambled BS (Figure 13F).

[0367] We concluded that promoter design-level activity scores at the EC90 condition are informative of the reporter expression level mediated by the promoter variant. Therefore, we analyzed promoter-level activity scores to discern the effect of design parameters on promoter activity scores at the MTF level corresponding to EC90 (Figure 14A). At the MTF level corresponding to EC90, the highest promoter-level score was approximately 32-fold higher than the lowest score (Figure 14B). While most promoters scored in the lower half of the total activity range, there was a tail of high-scoring promoters (Figure 14B), and differences and trends were visually apparent when presented as a heatmap (Figure 14C). Promoters assembled from binding sites in the sense orientation relative to the minimal promoter yielded significantly higher scores than designs with the opposite orientation (i.e., binding sites in the antisense orientation relative to the minimal promoter) (Figure 14D). As previously observed in other contexts

[35] , each additional binding site copy led to a higher score, independent of their orientation (Figure 14E). However, this effect appears to saturate in sense-oriented BS designs, meaning that more than five binding sites provide only a relatively modest increase in activity. For example, for sense-oriented BSs, the median activity score for all designs based on two BSs is 1.69-fold higher than that for designs based on one BS, but going from four to five BSs increases the median activity score by only 1.09-fold. Analysis of variance (ANOVA) suggests that the distance between BSs does not have a significant effect on the activity scores of both sense- and antisense-based designs. Nevertheless, the highest scores were achieved by promoters with sense-oriented BSs spaced 1, 4, 5, and 8 bp apart (Figure 14E). On the other hand, the impact of base pair spacing between the minimal promoter and the proximal BS on activity scores is highly significant. For sense-oriented designs, spacings greater than 5 bp have a positive effect, peaking at 8 bp (Figure 14E).Notably, in designs where the BS is in the antisense orientation relative to the minimal promoter, a greater distance between the proximal BS and the minimal promoter is associated with a lower activity score (Figure 14F).

[0368] Next, we sought to facilitate potential future applications by creating a collection of promoters with different strengths to match the requirements of potential future applications. Randomly selecting 20 variants yielded mostly weak promoters. To maximize diversity in size and strength, we selected 19 additional promoters based on their high activity scores or small gene size. Collectively, we created a promoter collection spanning a wide range of sizes and strengths (Figures 15A and 15B). After cotransfecting HEK293 cells with each variant and an mCherry transfection control plasmid, either with or without the MTF plasmid amount corresponding to the EC90, we measured relative mCitrine levels using flow cytometry. Recapitulating the screening findings, the number of base pairs between promoter components had a strong effect on reporter expression levels, allowing us to identify small-sized promoters that mediated high expression levels. For example, one promoter with the configuration sense-2-0-8 (orientation-base position number-base position distance-minimal promoter distance (SEQ ID NO: 300)) mediated as much mCitrine expression as the sense-5-9-8 design (SEQ ID NO: 672), but required 102 bp less coding region. Conversely, two promoter designs, sense-5-9-8 (SEQ ID NO: 672) and sense-5-8-10 (SEQ ID NO: 693), differ in length by only 2 bp, yet the latter mediated twice the amount of mCitrine expression as the former (Figures 15B, D). Confirming responsiveness to MTF, reporter expression was observed only in cells cotransfected with a plasmid encoding MTF, but not in cells lacking this plasmid (Figure 15C).

[0369] Although most attention has been focused on proteins in mammalian synTF engineering, the present inventors have now investigated and highlighted the significant impact of corresponding promoter design parameters. In addition to the importance of the number of binding sites, the spacing between promoter components has a significant impact on the output expression strength. These surprising findings allowed the present inventors to build a collection of promoters with a wide range of output strengths and find a variant (i.e., sense-2-0-8; SEQ ID NO: 300) that mediates high expression levels but requires only 52 bases to code, without considering the minimum promoter. reference

[0370] The following references refer to the reference numbers in parentheses disclosed herein, including the accompanying examples, namely [1]-

[24] . [1] L.Naldini, "Gene therapy returns to center stage," Nature, vol.526, no.7573, pp.351-360, Oct.2015, doi:10.1038 / nature15818. [2] S. Merlin and A Follenzi, "Transcriptional Targeting and MicroRNA Regulation of Lentiviral Vectors," Mol.Ther.- Methods Clin.Dev., vol.12, pp.223--232, Mar.2019, doi:10.1016 / j.omtm.2018.12.013. [3] Z.Xie, L.Wroblewska, L.Prochazka, R.Weiss, and Y.Benenson, "Multi-Input RNAi-Based Logic Circuit for Identification of Specific Cancer Cells," Science, vol.333, no.6047, pp.1307-1311, Sep.2011, doi:10.1126 / science.1205527. [4] L.Morsut et al., "Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors," Cell, vol.164, no.4, pp.780-791, Feb.2016, doi:10.1016 / j.cell.2016.01.012. [5] M.Gossen and H.Bujard, "Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.," Proc.Natl.Acad.Sci., vol.89, no.12, pp.5547-5551, Jun.1992, doi:10.1073 / pnas.89.12.5547. [6] M.Latta-Mahieu et al., "Gene Transfer of a Chimeric Trans-Activator Is lmmunogenic and Results in Short Lived Transgene Expression," Hum.Gene Ther., vol.13, no.13, pp.1611-1620, Sep.2002, doi:10.1089 / 10430340260201707. L.E.Mays and J.M.Wilson, "The Complex and Evolving Story of T cell Activation to AAV Vector-encoded Transgene Products," Mol.Ther., vol.19, no.1, pp.16-27, Jan.2011, doi:10.1038 / mt.2010.250. [9] S.A Hoyng et al., "Developing a potentially immunologically inert tetracycline-regulatable viral vector for gene therapy in the peripheral nerve," Gene Ther., vol.21, no.6, pp.549-557, Jun.2014, doi:10.1038 / gt.2014.22.

[10] X.Wang, F.G.Cabrera, K.L.Sharp, D.M.Spencer, A E.Foster, and J.H.Bayle, "Engineering Tolerance toward Allogeneic CAR-T Cells by Regulation of MHC Surface Expression with Human Herpes Virus-8 Proteins," Mol.Ther., Oct.2020, doi:10.1016 / j.ymthe.2020.10.019.

[11] L.Peraro et al., "Incorporation of bacterial immunoevasins to protect cell therapies from host antibody- mediated immune rejection," Mol.Ther., Jul.2021, doi:10.1016 / j.ymthe.2021.06.022.

[12] A Annoni, B.D.Brown, A Cantore, L.S.Sergi, L.Naldini, and M.-G.Roncarolo, "In vivo delivery of a microRNA-regulated transgene induces antigen-specific regulatory T cells and promotes immunologic tolerance," Blood, vol.114, no.25, pp.5152-5161, Dec.2009, doi:10.1182 / blood-2009-04-214569.

[13] F.Boisgerault et al., "Prolonged Gene Expression in Muscle Is Achieved Without Active Immune Tolerance Using MicrorRNA 142.3p-Regulated rAAV Gene Transfer," Hum.Gene Ther., vol.24, no.4, pp.393-405, Feb.2013, doi:10.1089 / hum.2012.208.

[14] Y.K.Chan et al., "Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses," Sci.Transl.Med., vol.13, no.580, p.eabd3438, Feb.2021, doi:10.1126 / scitranslmed.abd3438.

[15] V.M.Rivera et al., "A humanized system for pharmacologic control of gene expression," Nat.Med., vol.2, no.9, pp.1028--1032, Sep.1996, doi:10.1038 / nm0996-1028.

[16] I.Zhu et al., "Modular design of synthetic receptors for programmed gene regulation in cell therapies," Cell, vol.185, no.8, pp.1431-1443.e16, Apr.2022, doi:10.1016 / j.cell.2022.03.023.

[17] I.Zhu et al., "Design and modular assembly of synthetic intramembrane proteolysis receptors for custom gene regulation in therapeutic cells," bioRxiv, p.2021.05.21.445218, May 2021, doi:10.1101 / 2021.05.21.445218.

[18] J.Tycko et al., "High-Throughput Discovery and Characterization of Human Transcriptional Effectors," Cell, vol.0, no.0, Dec.2020, doi:10.1016 / j.cell.2020.11.024.

[19] M.Fussenegger et al., "Streptogramin-based gene regulation systems for mammalian cells," Nat.Biotechnol., vol.18, no.11, Art.no.11, Nov.2000, doi:10.1038 / 81208.

[20] J.M.O'shea and N.D.Perkins, "Regulation of the RelA (p65) transactivation domain," Biochem.Soc.Trans., vol.36, no.4, pp.603-608, Jul.2008, doi:10.1042 / BST0360603.

[21] E.Yakubovskaya, E.Mejia, J.Byrnes, E.Hambardjieva, and M.Garcia-Diaz, "Helix Unwinding and Base Flipping Enable Human MTERF1 to Terminate Mitochondrial Transcription," Cell, vol.141, no.6, pp.982-993, Jun.2010, doi:10.1016 / j.cell.2010.05.018.

[22] L.Klein, B.Kyewski, P.M.Allen, and K.A Hogquist, "Positive and negative selection of the T cell repertoire:what thymocytes see (and don't see)," Nat.Rev.lmmunol., vol.14, no.6, pp.377-391, Jun.2014, doi:10.1038 / nri3667.

[23] V.Jurtz, S.Paul, M.Andreatta, P.Marcatili, B.Peters, and M.Nielsen, "NetMHCpan-4.0:Improved Peptide-MHC Class I Interaction Predictions Integrating Eluted Ligand and Peptide Binding Affinity Data," J.lmmunol., vol.199, no.9, pp.3360-3368, Nov.2017, doi:10.4049 / jimmunol.1700893.

[24] M.D.Robinson, D.J.McCarthy, and G.K.Smyth, "edgeR:a Bioconductor package for differential expression analysis of digital gene expression data," Bioinformatics, vol.26, no.1, pp.139-140, Jan.2010, doi:10.1093 / bioinformatics / btp616.

[25] J.T.Bulcha, Y.Wang, H.Ma, P.W.L.Tai, and G.Gao, "Viral vector platforms within the gene therapy landscape," Sig Transduct Target Ther, vol.6, no.1, Art.no.1, Feb.2021, doi:10.1038 / s41392-021-00487-6.

[26] J.M.O'shea and N.D.Perkins, "Regulation of the RelA (p65) transactivation domain," Biochemical Society Transactions, vol.36, no.4, pp.603--608, Jul.2008, doi:10.1042 / BST0360603.

[27] J.Tycko et al., "High-Throughput Discovery and Characterization of Human Transcriptional Effectors," Cell, vol.0, no.0, Dec.2020, doi:10.1016 / j.cell.2020.11.024.

[28] E.Yakubovskaya, E.Mejia, J.Byrnes, E.Hambardjieva, and M.Garcia-Diaz, "Helix Unwinding and Base Flipping Enable Human MTERF1 to Terminate Mitochondrial Transcription," Cell, vol.141, no.6, pp.982-993, Jun.2010, doi:10.1016 / j.cell.2010.05.018.

[29] H.-S.Li et al., "Multidimensional control of therapeutic human cell function with synthetic gene circuits," Science, Dec.2022, doi:10.1126 / science.ade0156.

[30] J.H.Bayle, J.S.Grimley, K.Stankunas, J.E.Gestwicki, T.J.Wandless, and G.R.Crabtree, "Rapamycin Analogs with Differential Binding Specificity Permit Orthogonal Control of Protein Activity," Chemistry & Biology, vol.13, no.1, pp.99-107, Jan.2006, doi:10.1016 / j.chembiol.2005.10.017.

[31] V.M.Rivera et al., "A humanized system for pharmacologic control of gene expression," Nat Med, vol.2, no.9, pp.1028-1032, Sep.1996, doi:10.1038 / nm0996-1028.

[32] M.J.Drummond et al., "Rapamycin administration in humans blocks the contraction-induced increase in skeletal muscle protein synthesis," The Journal of Physiology, vol.587, no.7, pp.1535-1546, 2009, doi:10.1113 / jphysiol.2008.163816.

[33] A Maze and Y.Benenson, "Artificial signaling in mammalian cells enabled by prokaryotic two-component system," Nat Chem Biol, vol.16, no.2, pp.179-187, Feb.2020, doi:10.1038 / s41589-019-0429-9.

[34] P.S.Donahue, J.W.Draut, J.J.Muldoon, H.I.Edelstein, N.Bagheri, and J.N.Leonard, "The COMET toolkit for composing customizable genetic programs in mammalian cells," Nat Commun, vol.11, no.1, Art.no.1, Feb.2020, doi:10.1038 / s41467-019-14147-5.

[35] R.P.Smith et al., "Massively parallel decoding of mammalian regulatory sequences supports a flexible organizational model," Nat Genet, vol.45, no.9, Art.no.9, Sep.2013, doi:10.1038 / ng.2713.

[36] J.E.Davis, K.D.lnsigne, E.M.Jones, Q.B.Hastings, and S.Kosuri, "Multiplexed dissection of a model human transcription factor binding site architecture." bioRxiv, p.625434, May 13, 2019.doi:10.1101 / 625434.

[37] E.Sharon et al., "Inferring gene regulatory logic from high-throughput measurements of thousands of systematically designed promoters," Nat Biotechnol, vol.30, no.6, Art.no.6, Jun.2012, doi:10.1038 / nbt.2205.

[38] M.Hegde, C.Strand, R.E.Hanna, and J.G.Doench, "Uncoupling of sgRNAs from their associated barcodes during PCR amplification of combinatorial CRISPR screens," PLOS ONE, vol.13, no.5, p.e0197547, May 2018, doi:10.1371 / journal.pone.0197547.

[39] J.Liu et al., "Extensive Recombination Due to Heteroduplexes Generates Large Amounts of Artificial Gene Fragments during PCR," PLOS ONE, vol.9, no.9, p.e106658, Sep.2014, doi:10.1371 / journal.pone.0106658.

Claims

1. A synthetic transcription factor comprising (i) a DNA-binding domain (DBD) derived from a mitochondrial DNA-binding protein and (ii) a transcriptional regulatory domain derived from one or more other proteins.

2. 2. The synthetic transcription factor of claim 1, wherein the mitochondrial DNA binding protein is derived from a mammalian species.

3. 3. The synthetic transcription factor of claim 2, wherein the mammalian species is human.

4. The synthetic transcription factor of any one of claims 1 to 3, wherein the mitochondrial DNA binding protein is MTERF1.

5. The DNA binding domain (i) a first MTERF1 motif having the sequence set forth in SEQ ID NO: 104 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO: 104, and / or (ii) a second MTERF1 motif having the sequence set forth in SEQ ID NO: 106 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO: 106; Including; Preferably, the first MTERF1 motif is N-terminal to the second MTERF1 motif. A synthetic transcription factor according to any one of claims 1 to 4.

6. The synthetic transcription factor of claim 5, wherein the DNA binding domain further comprises (iii) an MTERF1 C-terminal domain having the sequence shown in SEQ ID NO: 11 or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO:

11.

7. the DNA-binding domain comprises MTERF1 subdomain A having the sequence set forth in SEQ ID NO:9 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO:9; Specifically, wherein the first MTERF1 motif and / or the second MTERF1 motif is contained in said subdomain A; A synthetic transcription factor according to any one of claims 1 to 6.

8. A synthetic transcription factor described in any one of claims 1 to 7, wherein the DNA binding domain comprises MTERF1 subdomain B having the sequence shown in SEQ ID NO: 7 or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 7, and specifically wherein MTERF1 subdomain A is contained in said MTERF1 subdomain B.

9. 8. A synthetic transcription factor according to any one of claims 1 to 7, wherein the DNA-binding domain has at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to SEQ ID NO: 1; preferably wherein said DNA-binding domain has the sequence shown in SEQ ID NO:

1.

10. 10. The synthetic transcription factor of any one of claims 1 to 9, which does not comprise a mitochondrial transit peptide having the sequence shown in SEQ ID NO: 37 or a sequence with at least 90% sequence identity to SEQ ID NO:

37.

11. 11. The synthetic transcription factor of any one of claims 1 to 10, which does not have a functional mitochondrial transit peptide.

12. 12. The synthetic transcription factor of any one of claims 1 to 11, comprising a nuclear localization signal.

13. 13. The synthetic transcription factor according to any one of claims 1 to 12, capable of modulating the transcription of at least one gene of interest in a cell, preferably in the nucleus of a cell; preferably wherein the gene(s) of interest encode a cell death-promoting protein, such as hBAX or HSV-TK, an immunostimulatory cytokine, such as IL-2 or IL-12, and / or an antigen receptor, such as a CAR or TCR.

14. 14. The synthetic transcription factor of claim 13, wherein the synthetic transcription factor regulates transcription of a gene of interest in a cell, preferably in the cell nucleus, by (i) promoting transcription of the gene of interest or (ii) suppressing transcription of the gene of interest.

15. The synthetic transcription factor of any one of claims 1 to 14, wherein the synthetic transcription factor and / or DNA binding domain is capable of binding to a response element in a cell, preferably in the nucleus of a cell.

16. 16. The synthetic transcription factor of claim 15, wherein the response element comprises an MTERF1 binding site having the sequence shown in SEQ ID NO: 42 or a sequence having at least 60%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO:

42.

17. 17. The synthetic transcription factor of claim 16, wherein the response element comprises multiple copies of the binding site, for example 2 to 15 copies; preferably 2 to 5 copies.

18. 18. A synthetic transcription factor according to claim 17, wherein two or more or all copies of the binding site on the response element are either immediately adjacent to each other or separated by one or more, e.g. 6 or 10, nucleotides.

19. A synthetic transcription factor according to any one of claims 15 to 18, capable of binding to a promoter comprising a response element in a cell, preferably in the nucleus of a cell.

20. 20. The synthetic transcription factor of claim 19, wherein the promoter further comprises a minimal promoter, such as a minimal TATA box preferably having the sequence set forth in SEQ ID NO: 103 or a minimal CMV promoter preferably having the sequence set forth in SEQ ID NO: 136; preferably, the minimal promoter is 3' to the response element.

21. 21. The synthetic transcription factor of any one of claims 19 to 20, wherein the promoter has the sequence shown in SEQ ID NO:

110.

22. 22. The synthetic transcription factor according to any one of claims 19 to 21, wherein the promoter is operably linked to a gene of interest, preferably in the cell nucleus.

23. 23. The synthetic transcription factor of claim 22, wherein binding of the synthetic transcription factor to a promoter, specifically binding of the DNA binding domain to a response element within the promoter, regulates transcription of a gene of interest operably linked to said promoter, for example in the nucleus of a cell; specifically, wherein said binding regulates transcription of a gene of interest (singular or plural) in the nucleus of a cell as defined in claim 13 or 14.

24. 24. The synthetic transcription factor of any one of claims 1 to 23, which is capable of entering the nucleus of a cell.

25. 25. The synthetic transcription factor of any one of claims 1 to 24, having the ability to localize to the nucleus in a cell more efficiently than to the mitochondria in said cell; specifically, wherein said ability is determined by measuring the amount of synthetic transcription factor separately in the nucleus and mitochondria of the same cell(s).

26. 26. The synthetic transcription factor of any one of claims 1 to 25, which is unable to enter mitochondria.

27. the cell is from the same species as the mitochondrial DNA binding protein is from, 27. The synthetic transcription factor of any one of claims 13 to 26, for example from the same mammalian species.

28. 28. The synthetic transcription factor of claim 27, which does not essentially modulate transcription, i.e., the transcriptome, in a cell other than the transcription of the gene(s) of interest.

29. 29. The synthetic transcription factor of claim 27 or 28, which does not specifically bind to essentially any endogenous DNA sequence in a cell.

30. 30. The synthetic transcription factor of any one of claims 27 to 29, wherein the DNA-binding domain does not specifically bind to essentially any endogenous DNA sequence in the nucleus of a cell.

31. 31. The synthetic transcription factor of any one of claims 27 to 30, which does not essentially compete for sequence-specific DNA binding in a cell with the mitochondrial DNA-binding protein from which the DBD is derived.

32. 32. A synthetic transcription factor according to any one of claims 27 to 31, which does not essentially interfere with the function of the mitochondrial DNA binding protein from which the DBD is derived.

33. 33. The synthetic transcription factor of any one of claims 1 to 32, wherein the transcriptional regulatory domain is capable of regulating the transcription of a gene, in particular wherein said transcriptional regulatory domain is part of, binds to or interacts with a DNA binding protein that is capable of binding to or interacting with a regulatory sequence, such as a promoter or enhancer, of said gene.

34. 34. The synthetic transcription factor of any one of claims 1 to 33, wherein the transcriptional regulatory domain is capable of binding to and / or interacting with an RNA polymerase, preferably RNA polymerase II; at least one other transcription factor, such as a general transcription factor; and / or at least one transcriptional co-regulator, such as a transcriptional co-activator and / or a transcriptional co-repressor.

35. 35. The synthetic transcription factor of any one of claims 1 to 34, wherein the transcriptional regulatory domain is (i) an activation domain or (ii) a repression domain.

36. 35. The synthetic transcription factor of any one of claims 1 to 34, wherein the transcriptional regulatory domain is capable of (i) promoting transcription of a gene when specifically defined as an activation domain, or (ii) repressing transcription of a gene when specifically defined as a repression domain.

37. 37. The synthetic transcription factor of any one of claims 1 to 36, wherein the transcriptional regulatory domain, in particular the activation domain, has at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:33, SEQ ID NO:52, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:13, SEQ ID NO:64, SEQ ID NO:66, and SEQ ID NO:68; or wherein said transcriptional regulatory domain, in particular said activation domain, comprises at least one sequence having at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:33, SEQ ID NO:52, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:13, SEQ ID NO:64, SEQ ID NO:66, and SEQ ID NO:

68.

38. A synthetic transcription factor described in any one of claims 1 to 37, wherein the transcriptional regulatory domain comprises a first, second, and / or third RELA transactivation domain; wherein the first RELA transactivation domain (TA1) has the sequence shown in SEQ ID NO: 31 or a sequence having at least 80% sequence identity to SEQ ID NO: 31; the second RELA transactivation domain has the sequence shown in SEQ ID NO: 132 or a sequence having at least 80% sequence identity to SEQ ID NO: 132; and the third RELA transactivation domain has the sequence shown in SEQ ID NO: 134 or a sequence having at least 80% sequence identity to SEQ ID NO: 134; preferably, the transcriptional regulatory domain comprises at least the first RELA transactivation domain.

39. 39. A synthetic transcription factor as described in claim 38, wherein the transcriptional regulatory domain comprises multiple copies, for example two or three copies, of the first, second, and / or third RELA domain, preferably the first RELA domain (TA1).

40. 40. A synthetic transcription factor according to any one of claims 1 to 39, wherein the transcriptional regulatory domain comprises a RELA subdomain A having the sequence shown in SEQ ID NO: 3 or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO:

3.

41. A synthetic transcription factor described in any one of claims 1 to 40, wherein the transcriptional regulatory domain comprises a RELA subdomain B having the sequence shown in SEQ ID NO: 27 or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 27; specifically, wherein a RELA subdomain A is contained in the RELA subdomain B.

42. A synthetic transcription factor described in any one of claims 1 to 41, wherein the transcriptional regulatory domain comprises a RELA subdomain C having the sequence shown in SEQ ID NO: 29 or a sequence having at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to SEQ ID NO: 29; specifically, wherein the first, second, and / or third RELA motif, RELA subdomain A, and / or RELA subdomain B are contained in the RELA subdomain C.

43. 43. The synthetic transcription factor of any one of claims 1 to 42, wherein the transcriptional regulatory domain comprises a FOXO3 transactivation domain (FOXO TAD) having the sequence shown in SEQ ID NO: 17 or a sequence with at least 80% sequence identity to SEQ ID NO:

17.

44. 44. The synthetic transcription factor of claim 43, wherein the transcriptional regulatory domain comprises multiple copies, for example two or three copies, of the FOXO3 transactivation domain (FOXO TAD); preferably wherein the transcriptional regulatory domain has the sequence shown in SEQ ID NO: 33 or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO:

33.

45. 45. The synthetic transcription factor of any one of claims 1 to 44, wherein the transcriptional regulatory domain comprises a MYB transactivation domain (LMSTEN) having the sequence shown in SEQ ID NO: 17 or a sequence with at least 80% sequence identity to SEQ ID NO:

17.

46. 46. ​​The synthetic transcription factor of claim 45, wherein the transcriptional regulatory domain comprises multiple copies, for example two or three copies, of the MYB transactivation domain (LMSTEN); preferably wherein the transcriptional regulatory domain has the sequence set forth in SEQ ID NO: 196 or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO:

196.

47. The transcriptional regulatory domain is (i) a first RELA transactivation domain (TA1) having the sequence set forth in SEQ ID NO: 31 or a sequence having at least 80% sequence identity to SEQ ID NO: 31; (ii) a FOXO3 transactivation domain (FOXO TAD) having the sequence set forth in SEQ ID NO: 33 or a sequence having at least 80% sequence identity to SEQ ID NO: 33; or (iii) a MYB transactivation domain (LMSTEN) having the sequence set forth in SEQ ID NO: 196 or a sequence having at least 80% sequence identity to SEQ ID NO: 196; 2 copies of Preferably, two copies of the FOXO3 transactivation domain (FOXO TAD) set forth in SEQ ID NO: 33 or a sequence having at least 80% sequence identity to SEQ ID NO: 33; 47. The synthetic transcription factor of any one of claims 1 to 46, comprising:

48. 48. A synthetic transcription factor according to any one of claims 1 to 47, wherein the transcriptional regulatory domain comprises three copies of a first RELA transactivation domain (TA1) having the sequence shown in SEQ ID NO: 31 or a sequence having at least 80% sequence identity to SEQ ID NO:

31.

49. 49. The synthetic transcription factor of any one of claims 1 to 48, wherein the transcriptional regulatory domain has the sequence set forth in SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 194, or SEQ ID NO: 196, or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to any of these sequences.

50. 49. The synthetic transcription factor of any one of claims 1 to 48, wherein the transcriptional regulatory domain has the sequence shown in SEQ ID NO: 33 or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO:

33.

51. 51. The synthetic transcription factor of any one of claims 38 to 50, wherein the transcriptional regulatory domain comprises at least two domains independently selected from the group consisting of TA1, a FOXO TAD, and LMSTEN, preferably wherein the transcriptional regulatory domain comprises at least a FOXO TAD; more preferably wherein the transcriptional regulatory domain comprises at least a FOXO TAD, TA1, and LMSTEN.

52. The transcriptional regulatory domains are: (i) two FOXO TADs, (ii) a FOXO TAD and a LMSTEN, (iii) a LMSTEN and a TA1, (iv) a FOXO TAD and a TA1, or (v) a FOXO TAD, a LMSTEN, and a TA1; preferably a FOXO TAD, specifically options (i), (ii), (iv), or (v).

52. The synthetic transcription factor of claim 51, comprising:

53. 52. The synthetic transcription factor of any one of claims 1 to 51, wherein the transcriptional regulatory domain has the sequence set forth in SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, or SEQ ID NO: 58, or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to any of these sequences.

54. 53. The synthetic transcription factor of any one of claims 1 to 52, wherein the transcriptional regulatory domain has the sequence set forth in SEQ ID NO: 52, SEQ ID NO: 33, SEQ ID NO: 54, or SEQ ID NO: 58, or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to any of these sequences.

55. 55. The synthetic transcription factor of any one of claims 51 to 54, wherein the transcriptional regulatory domain has the sequence shown in SEQ ID NO: 52 or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO:

52.

56. 56. A synthetic transcription factor according to any one of claims 51 to 55, wherein the DNA binding domain comprises or consists of MTERF1 subdomain A having the sequence shown in SEQ ID NO: 9 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO: 9; and preferably the transcriptional regulatory domain comprises or consists of a sequence as defined in claim 54 or 55.

57. 54. The synthetic transcription factor of any one of claims 37 to 45, 51 and 53, wherein the transcriptional regulatory domain has at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:33, SEQ ID NO:52, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:54, SEQ ID NO:56 and SEQ ID NO:

58.

58. 58. The synthetic transcription factor of any one of claims 37 to 45, 51, 53 and 57, wherein the transcriptional regulatory domain has at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:33 and SEQ ID NO:

52.

59. 59. The synthetic transcription factor of any one of claims 1 to 58, wherein the transcriptional regulatory domain comprises a sequence having at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 13, SEQ ID NO: 64, SEQ ID NO: 66, and SEQ ID NO: 68, preferably in addition to the first, second, and / or third RELA motif, such as TA1, RELA subdomain A, B, or C, FOXO TAD, and / or LMSTEN.

60. 37. The synthetic transcription factor of any one of claims 1 to 36, wherein the transcriptional regulatory domain, in particular the repression domain, has at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96 and SEQ ID NO:98; or wherein said transcriptional regulatory domain, in particular said repression domain, comprises at least one sequence having at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96 and SEQ ID NO:

98.

61. 61. The synthetic transcription factor of claim 60, wherein the transcriptional regulatory domain has at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, or SEQ ID NO:

76.

62. 62. The synthetic transcription factor of claim 60 or 61, wherein the transcriptional regulatory domain has at least 60%, preferably at least 70%, more preferably sequence identity to SEQ ID NO:

70.

63. 63. The synthetic transcription factor of any one of claims 1 to 62, wherein the one or more proteins from which the transcriptional regulatory domain is derived are from the same species, such as the same mammalian species, from which the mitochondrial DNA binding protein is derived.

64. 64. A synthetic transcription factor according to any one of claims 1 to 63, which is essentially composed of parts of proteins from the same biological species, such as the same mammalian species.

65. 65. The synthetic transcription factor of any one of claims 1 to 58 and 60 to 64, wherein the mitochondrial DNA binding protein, such as MTERF1, and one or more other proteins from which the transcriptional regulatory domain is derived, such as RELA, FOXO3, and / or MYB, are of human origin.

66. 66. A synthetic transcription factor according to any one of claims 1 to 58 and 60 to 65, which consists essentially of portions of human proteins.

67. 67. The synthetic transcription factor of claim 65 or 66, wherein the transcriptional regulatory domain is derived from a single human protein.

68. 68. The synthetic transcription factor according to any one of claims 65 to 67, wherein the transcriptional regulatory domain, in particular the activation domain, has at least 90%, preferably at least 95%, more preferably at least 99% sequence identity to SEQ ID NO: 3, SEQ ID NO: 27 or SEQ ID NO: 29, preferably SEQ ID NO:

3.

69. 68. The synthetic transcription factor according to any one of claims 65 to 67, wherein the transcriptional regulatory domain, in particular the repression domain, has at least 90%, preferably at least 95%, more preferably at least 99% sequence identity to SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96 or SEQ ID NO:98, preferably SEQ ID NO:

70.

70. 70. The synthetic transcription factor of any one of claims 1 to 69, which is essentially non-immunogenic in humans.

71. 70. The synthetic transcription factor of any one of claims 1 to 69, wherein the mitochondrial DNA binding protein is essentially non-immunogenic in the mammalian species from which it is derived.

72. 70. The synthetic transcription factor of any one of claims 65 to 69, which is essentially non-immunogenic in humans.

73. 72. The synthetic transcription factor of any one of claims 1 to 71, further comprising a regulatable domain, preferably a regulatable destabilization domain or a regulatable localization domain.

74. 74. The synthetic transcription factor of claim 73, wherein the controllable domain is controllable (i) by a chemical compound, preferably by a small molecule, or (ii) by light, preferably by a specific wavelength or range of wavelengths.

75. 75. A synthetic transcription factor according to claim 73 or 74, wherein the synthetic transcription factor comprising a controllable destabilization domain is stabilized or destabilized by a compound or light; and / or the synthetic transcription factor comprising a controllable localization domain is localized to either the nucleus or cytoplasm of a cell, preferably to the nucleus, by said compound or light.

76. 76. The synthetic transcription factor of any one of claims 73 to 75, wherein the regulatable destabilization domain comprises an NS3 domain having the sequence shown in SEQ ID NO: 158 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO:

158.

77. 77. The synthetic transcription factor of claim 76, wherein the synthetic transcription factor comprising an NS3 domain is stabilized by grazoprevir.

78. The regulatable localization domain is selected from the group consisting of the sequence shown in SEQ ID NO: 152 and the sequence shown in SEQ ID NO:

153.

78. The synthetic transcription factor of any one of claims 73 to 77, comprising an ERT2 domain having a sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO:

152.

79. 79. The synthetic transcription factor of claim 78, wherein the synthetic transcription factor comprising an ERT2 domain is localized to the nucleus of a cell by 4-hydroxytamoxifen.

80. 81. The synthetic transcription factor of any one of claims 73 to 80, wherein the regulatable domain comprises an FRB domain and an FKBP domain, wherein the FRB domain has the sequence shown in SEQ ID NO: 140 or a sequence with at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO: 140, and / or the FKBP domain has the sequence shown in SEQ ID NO: 142 or a sequence with at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO:

142.

81. 81. The synthetic transcription factor of claim 80, wherein the FRB domain and the FKBP domain bind to each other in the presence of C16-(S)-7-methylindolerapamycin.

82. 82. The synthetic transcription factor of any one of claims 1 to 81, further comprising a synNotch core having the sequence shown in SEQ ID NO: 160 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO:

160.

83. 83. The synthetic transcription factor of any one of claims 1 to 82, which is a fusion protein comprising a DNA-binding domain and a transcriptional regulatory domain.

84. 84. The synthetic transcription factor of claim 83, wherein the DNA binding domain is N-terminal to the transcription regulatory domain.

85. 85. The synthetic transcription factor of claim 83 or 84, wherein the fusion protein further comprises a controllable destabilization domain as defined in any one of claims 73 to 77 or a controllable localization domain as defined in any one of claims 73 to 75, 78 and 79.

86. 85. A synthetic transcription factor as described in claim 83 or 84, wherein the fusion protein further comprises a single-chain variable fragment (scFv) and a synNotch core as defined in claim 82; preferably, the order of domains on the fusion protein from N-terminus to C-terminus is as follows: (i) scFv, (ii) synNotch, and (iii) a DNA-binding domain and a transcriptional regulatory domain, wherein the DNA-binding domain can be N-terminal or C-terminal to the transcriptional regulatory domain.

87. 87. A nucleic acid encoding the synthetic transcription factor of any one of claims 83 to 86.

88. 88. The nucleic acid of claim 87, which is DNA or RNA.

89. 89. The nucleic acid of claim 87 or 88, which is mRNA, for example mRNA contained in a lipid nanoparticle.

90. 88. The nucleic acid of claim 87, comprising the DNA sequence set forth in SEQ ID NO: 5 or a DNA sequence having at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to SEQ ID NO: 5, wherein the DNA sequence encodes a DNA-binding domain having at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to SEQ ID NO: 1; preferably wherein the DNA-binding domain has the sequence set forth in SEQ ID NO:

1.

91. Preferably, a DNA plasmid comprising a nucleic acid according to any one of claims 87 to 90, wherein the plasmid is suitable for expressing a synthetic transcription factor in a cell.

92. 92. A viral vector comprising a nucleic acid according to any one of claims 87 to 90, a plasmid according to claim 91, or a nucleic acid having a sequence complementary to a nucleic acid according to any one of claims 87 to 90.

93. A cell comprising a nucleic acid according to any one of claims 87 to 90, a plasmid according to claim 91, or a viral vector according to claim 92.

94. 83. The synthetic transcription factor of any one of Claims 1 to 82, comprising or consisting of a first and a second polypeptide, wherein the first polypeptide comprises a DNA-binding domain and the second polypeptide comprises a transcriptional regulatory domain.

95. 95. The synthetic transcription factor of claim 94, wherein each of the first and second polypeptides comprises a multimerization domain, and wherein the multimerization domains of the first and second polypeptides are capable of binding to and / or interacting with each other.

96. 96. The synthetic transcription factor of claim 95, wherein the multimerization domain is a dimerization domain.

97. 97. The synthetic transcription factor of claim 95 or 96, wherein the multimerization domain is a homodimerization domain, in particular wherein the multimerization domains of the first and second polypeptides are identical to each other.

98. 97. The synthetic transcription factor of claim 95 or 96, wherein the multimerization domain is a heterodimerization domain, in particular wherein the multimerization domains of the first and second polypeptides are different from each other.

99. 99. The synthetic transcription factor of claim 98, wherein: (i) the multimerization domain of the first polypeptide comprises or consists of a SYNZIP1 domain and the multimerization domain of the second polypeptide comprises or consists of a SYNZIP2 domain; or (ii) a synthetic transcription factor, wherein the multimerization domain of the first polypeptide comprises or consists of a SYNZIP2 domain and the multimerization domain of the second polypeptide comprises or consists of a SYNZIP1 domain; wherein the SYNZIP1 domain has the sequence set forth in SEQ ID NO: 154, or a sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO: 154; wherein the SYNZIP2 domain is a synthetic transcription factor having the sequence shown in SEQ ID NO: 156 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO:

156.

100. 100. The synthetic transcription factor of any one of claims 95 to 99, wherein the multimerization domain is a controllable domain, in particular wherein said multimerization domain is controllable (i) by a chemical compound, preferably by a small molecule, or (ii) by light, preferably by a specific wavelength or a specific range of wavelengths.

101. 101. The synthetic transcription factor of claim 100, wherein the multimerization domain is a dimerization domain and a compound or light controls the dimerization of the multimerization domains of the first and second polypeptides; preferably, the first and second polypeptides bind and / or interact with each other in the presence of the small molecule or light.

102. 102. The synthetic transcription factor of any one of claims 94 to 96, 98, 100 and 101, wherein: (i) the multimerization domain of the first polypeptide comprises or consists of an FKBP domain and the multimerization domain of the second polypeptide comprises or consists of an FRB domain; or (ii) a synthetic transcription factor, wherein the multimerization domain of the first polypeptide comprises or consists of an FRB domain and the multimerization domain of the second polypeptide comprises or consists of an FKBP domain; wherein the FKBP domain has the sequence set forth in SEQ ID NO: 142, or a sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO: 142; wherein the FRB domain has the sequence set forth in SEQ ID NO: 140, or a sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to SEQ ID NO: 140; Preferably, wherein the multimerization domain of said first polypeptide comprises or consists of said FKBP domain and the multimerization domain of said second polypeptide comprises or consists of said FRB domain.

103. 103. The synthetic transcription factor of claim 102, wherein the DNA-binding domain is N-terminal to the FKBP domain on the first polypeptide and / or the transcriptional regulatory domain is C-terminal to the FRB domain on the second polypeptide.

104. 104. The synthetic transcription factor of claim 102 or 103, wherein the first and second polypeptides bind and / or interact with each other in the presence of C16-(S)-7-methylindolerapamycin, and specifically, wherein C16-(S)-7-methylindolerapamycin induces heterodimerization of the FKBP domain and the FRB domain.

105. 105. The synthetic transcription factor of any one of claims 102 to 104, wherein (i) the DNA-binding domain and the FKBP domain are linked to each other via a first peptide linker, and / or (ii) the transcriptional regulatory domain and the FRB domain are linked to each other via a second peptide linker.

106. 106. The synthetic transcription factor of claim 105, wherein the first and second peptide linkers are independently selected from the group consisting of a cMyc NLS linker set forth in SEQ ID NO: 122, a 6AP (AP6) linker set forth in SEQ ID NO: 120, an AP8 linker set forth in SEQ ID NO: 144, a G4S linker set forth in SEQ ID NO: 118, an EAAAK3 linker set forth in SEQ ID NO: 146, an EAAAK2 linker set forth in SEQ ID NO: 148, and a G4S4 linker set forth in SEQ ID NO:

150.

107. 106. The synthetic transcription factor of claim 105, wherein the first peptide linker is a cMyc NLS linker as set forth in SEQ ID NO: 122 or a 6AP (AP6) linker as set forth in SEQ ID NO: 120, and / or the second peptide linker is a 6AP (AP6) linker as set forth in SEQ ID NO:

120.

108. 108. The synthetic transcription factor of any one of claims 102 to 107, wherein the transcriptional regulatory domain is as defined in any one of claims 51 to 58.

109. 109. The synthetic transcription factor of any one of claims 102 to 108, wherein the transcriptional regulatory domain has the sequence set forth in SEQ ID NO: 52, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 54, or SEQ ID NO: 58, or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to any of these sequences.

110. 109. The synthetic transcription factor of any one of claims 102 to 108, wherein the transcriptional regulatory domain has the sequence shown in SEQ ID NO: 52 or SEQ ID NO: 29, or a sequence with at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 52 or SEQ ID NO:

29.

111. 111. The synthetic transcription factor of any one of claims 102 to 110, wherein the first polypeptide comprises the sequence set forth in SEQ ID NO: 174, 176, 178, 180, 182, 184, 186, 188, or 192, or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 174, 176, 178, 180, 182, 184, 186, 188, or 192.

112. 112. The synthetic transcription factor of any one of claims 102 to 111, wherein the second polypeptide comprises the sequence set forth in SEQ ID NO: 162, 164, 166, 168, 170, 172, or 190, or a sequence having at least 70%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 162, 164, 166, 168, 170, 172, or 190.

113. 83. A combination of nucleic acids encoding a synthetic transcription factor according to any one of claims 1 to 82, wherein one nucleic acid encodes the DNA binding domain and another nucleic acid encodes the transcriptional regulatory domain.

114. 113. A combination of nucleic acids encoding a synthetic transcription factor according to any one of claims 94 to 112, comprising a first and a second nucleic acid, wherein said first nucleic acid encodes a first polypeptide and said second nucleic acid encodes a second polypeptide.

115. 115. The combination of claim 113 or 114, wherein one nucleic acid, particularly the first nucleic acid, has the DNA sequence set forth in SEQ ID NO: 5 or a DNA sequence having at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to SEQ ID NO: 5, and wherein said DNA sequence encodes a DNA binding domain having at least 60%, preferably at least 70%, more preferably at least 80% sequence identity to SEQ ID NO: 1, preferably wherein said DNA binding domain has the sequence set forth in SEQ ID NO:

1.

116. 115. The combination of claim 113 or 114, wherein the nucleic acid is a DNA or RNA molecule.

117. 117. The combination of claim 116, wherein the nucleic acid is an mRNA molecule, e.g., an mRNA contained in a lipid nanoparticle.

118. Preferably, a DNA plasmid combination comprising a nucleic acid combination according to any one of claims 113 to 115, wherein the plasmid is suitable for expressing a synthetic transcription factor in a cell.

119. A viral vector combination comprising a nucleic acid according to any one of claims 113 to 115, a plasmid according to claim 119, or a nucleic acid having a sequence complementary to a nucleic acid according to any one of claims 113 to 115.

120. 120. A cell comprising a combination according to any one of claims 113 to 119.

121. A DNA construct comprising a promoter (P) including a response element and a minimal promoter, characterized in that the response element comprises an MTERF1 binding site and comprises or consists of the sequence shown in SEQ ID NO: 42 or SEQ ID NO: 200, or a sequence having at least 60%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 42 or SEQ ID NO:

200.

122. A DNA construct as described in claim 121, wherein the MTERF1 binding site comprises or consists of the sequence shown in SEQ ID NO: 42 or a sequence having at least 60%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO:

42.

123. the response element is: (i) one or more copies, for example 2 to 50 copies, preferably 2 to 5 copies, of an MTERF1 binding site, wherein said multiple copies are immediately adjacent to each other, or (ii) multiple copies, for example 2 to 50 copies, preferably 2 to 5 copies, of said MTERF1 binding site and a BS-BS spacer between at least two, preferably all consecutive copies of said binding site, wherein the BS-BS spacer has a length of 1 to 1000 nucleotides, preferably 1 to 100 nucleotides, more preferably 1 to 10, for example 1 to 6 nucleotides, preferably wherein said BS-BS spacer consists of 1 to 10 nucleotides at the 5' end of the sequence set forth in SEQ ID NO: 198; 123. The DNA construct of claim 121 or 122, consisting of:

124. 124. A DNA construct according to any one of claims 121 to 123, wherein the response element consists of multiple copies, for example 2 to 15 copies, of the MTERF1 binding site immediately adjacent to each other.

125. The DNA construct is at most about 10 6 , preferably at most 10 5 125. The DNA construct of any one of claims 121 to 124, having a length of at most about 10,000 nucleotides.

126. 126. A DNA construct according to any one of claims 121 to 125, wherein the response element and the minimal promoter are separated from each other by a RE-minP spacer having a length of at most about 2000 nucleotides, preferably at most about 200 nucleotides, more preferably at most about 20 nucleotides, and most preferably about 6 to 10 nucleotides, for example about 6 or 8 nucleotides; wherein preferably the RE-minP spacer consists of 1 to 10 nucleotides at the 5' end of the sequence set forth in SEQ ID NO: 199; and preferably, the MTERF1 binding site comprises or consists of the sequence set forth in SEQ ID NO: 42, or a sequence having at least 60%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO:

42.

127. 127. A DNA construct according to any one of claims 121 to 126, wherein the minimal promoter is 3' or 5', preferably 3', to the response element.

128. 128. The DNA construct of any one of claims 121 to 127, wherein the minimal promoter is a minimal TATA box having at least 60%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO:

103.

129. 128. The DNA construct of any one of claims 121 to 127, wherein the minimal promoter is a minimal CMV promoter having at least 60%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO:

136.

130. 130. A DNA construct according to any one of claims 121 to 129, wherein the promoter (P), in particular the response element, is capable of binding to a synthetic transcription factor according to any one of claims 1 to 86 and 94 to 112.

131. 131. The DNA construct of any one of claims 121 to 130, further comprising at least one gene of interest, preferably encoding a cell death-promoting protein, such as hBAX or HSV-TK, an immunostimulatory cytokine, such as IL-2 or IL-12, and / or an antigen receptor, such as a CAR or TCR.

132. 132. The DNA construct of claim 131, wherein the promoter (P) is operably linked to at least one gene of interest; preferably, wherein the at least one gene of interest is 3' of the minimal promoter.

133. 133. A DNA construct according to any one of claims 130 to 132, wherein at least one of the gene(s) of interest is transcribed in a cell when the promoter (P), in particular the response element, is bound by a synthetic transcription factor according to any one of claims 1 to 86 and 94 to 112.

134. 134. A DNA construct according to any one of claims 130 to 133, wherein at least one gene(s) of interest is transcribed when the promoter (P), in particular the response element, is bound by a synthetic transcription factor according to any one of claims 1 to 86 and 94 to 112 in the nucleus of a human cell.

135. 135. The DNA construct of any one of claims 121 to 134, which does not comprise the sequence set forth in SEQ ID NO: 117 or a sequence having at least 90% sequence identity to SEQ ID NO:

117.

136. 136. The DNA construct of any one of claims 121 to 135, comprising a sequence selected from the group consisting of SEQ ID NOs: 201 to 1190.

137. A DNA construct described in any one of claims 121 to 135, wherein the response element comprises or consists of two copies of an MTERF1 binding site, each having the sequence shown in SEQ ID NO: 42 or a sequence having at least 60%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 42; wherein the two copies of the MTERF1 binding site are directly adjacent to each other; and wherein the response element and the minimal promoter are separated from each other by 8 nucleotides.

138. 138. A DNA construct according to any one of claims 121 to 137, comprising the sequence set forth in SEQ ID NO:

300.

139. A DNA construct described in any one of claims 121 to 135, wherein the response element consists of five copies of the MTERF1 binding site, the copies being separated from each other by 8 nucleotides, and wherein each MTERF1 binding site has the sequence shown in SEQ ID NO: 42 or a sequence having at least 60%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 42; and wherein the response element and the minimal promoter are separated from each other by 10 nucleotides.

140. 140. The DNA construct of any one of claims 121 to 135 or 139, comprising the sequence set forth in SEQ ID NO:

693.

141. A single- or double-stranded nucleic acid comprising the sense strand of a DNA construct according to any one of claims 121 to 140 and / or the antisense strand of a DNA construct according to any one of claims 121 to 140.

142. 142. The single- or double-stranded nucleic acid of claim 141, which is DNA or RNA.

143. A DNA plasmid comprising the DNA construct of any one of claims 121 to 140.

144. 144. A viral vector comprising a DNA construct according to any one of claims 121 to 140, a single- or double-stranded nucleic acid according to claim 141 or 142, or a DNA plasmid according to claim 143.

145. A cell comprising the DNA construct of any one of claims 121 to 140.

146. 143. A system comprising: (i) a synthetic transcription factor according to any one of claims 1 to 86 and 94 to 112, a nucleic acid according to any one of claims 87 to 93, a DNA plasmid according to claim 94, a viral vector according to claim 95, or a combination according to any one of claims 113 to 119, and (ii) a DNA construct according to any one of claims 121 to 140, a DNA plasmid according to claim 143, and / or a viral vector according to claim 144, preferably wherein the system comprises a synthetic transcription factor according to any one of claims 1 to 86 and 94 to 112 and a DNA construct according to any one of claims 121 to 140; preferably wherein the system is an engineered genetic network, preferably a biocomputing circuit.

147. 147. The system of claim 146 for regulating the transcription of at least one gene of interest, preferably included on the DNA construct, preferably 3' of a minimal promoter; preferably the gene(s) of interest encode a cell death-promoting protein such as hBAX or HSV TK, an immunostimulatory cytokine such as IL-2 or IL-12, and / or an antigen receptor such as a CAR or TCR.

148. A cell comprising a synthetic transcription factor according to any one of claims 1 to 86 and 94 to 112 and a DNA construct according to any one of claims 121 to 140.

149. 143. A kit comprising a synthetic transcription factor according to any one of claims 1 to 86 and 94 to 112, a nucleic acid according to any one of claims 87 to 93, a DNA plasmid according to claim 94, a viral vector according to claim 95, a combination according to any one of claims 113 to 119, a DNA construct according to any one of claims 121 to 140, a DNA plasmid according to claim 143, a viral vector according to claim 144, and / or a system according to claim 146 or 147.

150. 150. A kit according to claim 149, comprising (i) a nucleic acid according to any one of claims 87 to 93, a DNA plasmid according to claim 94, a viral vector according to claim 95, or a combination according to any one of claims 113 to 119, and (ii) a DNA construct according to any one of claims 121 to 140, a DNA plasmid according to claim 143, or a viral vector according to claim 144.

151. 143. A pharmaceutical composition comprising a synthetic transcription factor according to any one of claims 1 to 86 and 94 to 112, a nucleic acid according to any one of claims 87 to 93, a DNA plasmid according to claim 94, a viral vector according to claim 95, a combination according to any one of claims 113 to 119, a DNA construct according to any one of claims 121 to 140, a single or double stranded nucleic acid according to claim 141 or 142, a DNA plasmid according to claim 143, a viral vector according to claim 144, a system according to claim 146 or 147, or a cell according to any one of claims 93, 120, 145 and 148.

152. 152. The pharmaceutical composition of claim 151, comprising (i) a nucleic acid according to any one of claims 87 to 93, a DNA plasmid according to claim 94, a viral vector according to claim 95, or a combination according to any one of claims 113 to 119, and (ii) a DNA construct according to any one of claims 121 to 140, a DNA plasmid according to claim 143, or a viral vector according to claim 144.

153. 153. A pharmaceutical composition according to claim 151 or 152, comprising the cell of claim 148.

154. 154. The pharmaceutical composition of any one of claims 151 to 153, further comprising a pharmaceutically acceptable excipient.

155. 155. A pharmaceutical composition according to any one of claims 151 to 154 for use in treating diseases in which target cells are killed and / or manipulated, in particular wherein the treatment involves a cancer cell classifier circuit; preferably wherein at least one gene of interest encodes a cell death-promoting protein such as hBAX or HSV-TK, an immune stimulating cytokine such as IL-2 or IL-12, and / or an antigen receptor such as a CAR or TCR.

156. In particular, the pharmaceutical composition of any one of claims 151 to 154 for use in a method of treating tumors or cancer, wherein the treatment involves a cancer cell classifier circuit; preferably wherein at least one gene of interest encodes a cell death-promoting protein, such as hBAX or HSV-TK, an immune stimulating cytokine, such as IL-2 or IL-12, and / or an antigen receptor, such as a CAR or TCR.

157. A nucleic acid according to any one of claims 87 to 93, a DNA plasmid according to claim 94, a viral vector according to claim 95, a combination according to any one of claims 113 to 119, a DNA construct according to any one of claims 121 to 140, a single or double stranded nucleic acid according to claim 141 or 142, a DNA plasmid according to claim 143, a viral vector according to claim 144 or a system according to claim 146 or 147 for use in gene therapy.

158. 149. The cell of any one of claims 93, 120, 145, and 148 for use in cell therapy.

159. 158. The cell for use according to claim 157, wherein the cell is a T cell, such as a CAR T cell, and the cell therapy is T cell therapy, such as CAR T cell therapy.

160. An engineered genetic network, in particular a synthetic transcription factor according to any one of claims 1 to 86 and 94 to 112, a nucleic acid according to any one of claims 87 to 93, a DNA plasmid according to claim 94, a viral vector according to claim 95, a combination according to any one of claims 113 to 119, a DNA construct according to any one of claims 121 to 140, a single or double stranded nucleic acid according to claim 141 or 142, a DNA plasmid according to claim 143, a viral vector according to claim 144 as part of or in combination with a biocomputing circuit. Use of the system of claim 146 or 147 or the cell of any one of claims 93, 120, 145 and 148.

161. Use of a synthetic transcription factor according to any one of claims 1 to 86 and 94 to 112, a nucleic acid according to any one of claims 87 to 93, a DNA plasmid according to claim 94, a viral vector according to claim 95, a combination according to any one of claims 113 to 119, a DNA construct according to any one of claims 121 to 140, a single or double stranded nucleic acid according to claim 141 or 142, a DNA plasmid according to claim 143, a viral vector according to claim 144, a system according to claim 146 or 147, or a cell according to any one of claims 93, 120, 145 and 148 for transcribing a gene of interest in vitro or in vivo, for example in a cell in vitro or in vivo.

162. A library of DNA constructs comprising at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, 100000, 500000, or 1000000, preferably at least about 100, 500, 900, 950, 990, or 1000 different DNA constructs, wherein each DNA construct in the library comprises: (i) a promoter (P) consisting of a response element (RE), a minimal promoter (minP) 3' of the response element, and an optional RE-minP spacer between the response element and the minimal promoter; wherein each response element consists of one or more copies of a transcription factor binding site (BS) and an optional BS-BS spacer between at least two, and preferably all, consecutive copies of the binding site; (ii) a sequence preferably 3' of said minimal promoter; wherein all DNA constructs in said library differ from one another in the sequence of their promoters (P); and optionally, wherein each DNA construct in said library comprises a unique barcode sequence that distinguishes all DNA constructs in the library from one another.

163. The library described in claim 162, wherein the transcription factor binding site is an MTERF1 binding site comprising or consisting of the sequence set forth in SEQ ID NO: 42 or SEQ ID NO: 200, or a sequence having at least 60%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO: 42 or SEQ ID NO: 200; preferably, wherein the MTERF1 binding site consists of the sequence set forth in SEQ ID NO:

42.

164. 164. The library of Claim 162 or 163, wherein the DNA constructs are identical to each other with the exception of the promoter sequence and any barcode sequence.

165. 165. The library of any one of claims 162 to 164, wherein the minimal promoters on different DNA constructs, in particular on different promoters, are identical to each other.

166. 166. The library of any one of claims 162 to 165, wherein transcription factor binding sites on different DNA constructs, in particular on different promoters, have the same sequence, preferably SEQ ID NO: 42 or SEQ ID NO: 200, respectively, in either sense or antisense orientation relative to the minimal promoter.

167. 167. The library of any one of Claims 162-166, wherein the promoters in 20%, 30%, 40%, or 50% of the DNA constructs differ from each other in (i) binding site copy number and / or (ii) the presence or length of a RE-minP spacer.

168. At least 80%, at least 90%, or all promoters in the DNA construct are identical in the following respects: (i) binding site copy number, (ii) presence or length of a RE-minP spacer, (iii) presence or length of a BS-BS spacer, and (iv) sense or antisense binding site sequence orientation relative to the minimal promoter.

168. The library of any one of claims 162 to 167, wherein the libraries differ from each other in at least one parameter selected from the group consisting of:

169. 169. The library of any one of claims 162 to 168, wherein the minimal promoter is a minimal TATA box having at least 60%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO:

103.

170. 169. The library of any one of claims 162 to 168, wherein the minimal promoter is a minimal CMV promoter having at least 60%, preferably at least 80%, more preferably at least 90% sequence identity to SEQ ID NO:

136.

171. 171. A library according to any one of claims 162 to 170, wherein the promoter (P), in particular the response element, is capable of binding to a synthetic transcription factor according to any one of claims 1 to 86 and 94 to 112.

172. Next steps: a) preparing a library of DNA constructs according to any one of claims 161 to 170, b) combining the library of DNA constructs with said transcription factors in a cell or in an in vitro transcription system; combining, preferably in a cell; c) determining the transcriptional activity of the promoter of each DNA construct in the library, preferably by determining the amount of mRNA produced from each DNA construct in the library, specifically wherein said mRNA comprises a sequence corresponding to the output sequence; and d) selecting promoters based on their transcriptional activity, thereby obtaining promoters that are optimized for binding to said transcription factors; A method for optimizing a promoter for binding to a transcription factor, comprising:

173. 173. The method of claim 172, wherein the transcriptional activity of the promoter of each DNA construct in the library is determined by RNA sequencing, preferably by next-generation RNA sequencing.

174. 174. The method of claim 172 or 173, wherein the method is a massively parallel reporter assay.

175. 175. The method of any one of claims 172 to 174, wherein the transcription factor is as defined in any one of claims 1 to 86 or 94 to 112.

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