Artificial transcription factors

Abstract

The present invention is directed to engineered systems and genetic circuits for controlling expression of genes in a cell, based on competitive dimerization between a transcription factor and an inhibitory polypeptide configured to dimerize with said transcription factor. The invention is also directed to cells comprising said systems and genetic circuits, to vectors for expressing said systems and genetic circuits, and to uses thereof.

Description

[0001] ARTIFICIAL TRANSCRIPTION FACTORS

[0002] FIELD OF THE INVENTION

[0003] The present invention is directed to engineered systems and genetic circuits for controlling expression of genes in a cell, based on competitive dimerization between a transcription factor and an inhibitory polypeptide configured to dimerize with said transcription factor. The invention is also directed to cells comprising said systems and genetic circuits, to vectors for expressing said systems and genetic circuits, and to uses thereof.

[0004] BACKGROUND

[0005] Engineering human cells for biotechnology or therapeutic purposes typically involves introducing in the cells a single molecular component, optimized for a specific function. For example, in biotechnology, the production of biopharmaceuticals in cell lines often depends on the constitutive expression of recombinant proteins. Similarly, in therapeutic applications, T cell immunotherapy for cancer relies on the constitutive expression of synthetic receptors. Moreover, gene therapy of haploinsufficiency disorders aims to achieve constitutive expression of the therapeutic gene, whether through direct gene replacement or by means of synthetic gene regulatory programs, employing artificial transcription factor such as Transcription Activator-Like Effectors (TALEs), synthetic Zinc Fingers, or nuclease-dead CRISPR-Cas9 systems. However, constitutive expression is static and does not respond to changes in the cellular environment, leading to significant limitations; moreover, competition for resources between native proteins and recombinant proteins limits protein production in cell lines. In fact, constitutive expression of CARs in immunotherapy leads to exhaustion. Moreover, state of the art approaches to gene replacement therapy are limited to those diseases where uncontrolled maximal expression of the therapeutic gene suffices, thus excluding a large group of rare genetic disorders requiring the therapeutic gene to be expressed in a tight therapeutic window because of overexpression-induced toxicity, or those resulting from gain-of-function (GOF) mutations leading to proteins with enhanced or new function. For example, in the context of gene replacement therapy, when the therapeutic gene is driven by a constitutive tissue-specific promoter and delivered in individual cells by a viral vector, heterogeneity in virus uptake causes differences in gene expression levels across cells, which in the case of dosage sensitive genes can lead to toxic effects caused by overexpression; this occurs for instance in the context of gene replacement therapy of Friedreich's ataxia (FRDA; OMIM 22930), when the therapeutic gene encoding for mitochondrial protein Frataxin (FXN) is driven by a constitutive tissuespecific promoter and delivered in individual cells by a viral vector. In contrast, dynamic regulation allows gene expression or receptor activity to adjust, based on the cellular state, environmental variables or external inputs, being crucial for achieving more precise and effective outcomes, especially in the above-mentioned applications.

[0006] To unleash the full power of synthetic biology, the focus must therefore shift from single molecular components to system of components and synthetic gene circuits, interconnecting multiple components to enable engineering of “smart” cells with programmed “sense and respond” functions.

[0007] However, multiple challenges have so far prevented this transition. The foremost obstacle is the absence of a versatile engineering platform for providing systems and gene circuits, that can support diverse functions, modular components, and scalability, while minimizing circuit’s size in terms of composing elements and total DNA base pairs; this is required for compatibility with delivery systems, such as viral vectors, and to reduce cellular burden. Furthermore, in the context of therapeutic applications, minimization of non-human derived parts is also necessary to prevent immunogenicity.

[0008] There is therefore the need to provide improved engineered systems and genetic circuits for gene expression that overcome the above limitations.

[0009] BRIEF DESCRIPTION OF THE INVENTION

[0010] The limitations of the prior art are overcome by the present invention, providing novel transcription factors (TFs) and transcription factors’ systems for dynamically controlling gene expression in a cell, preferably in an eukaryotic cell, most preferably in a mammalian cell. In particular, the present invention provides an engineered system for controlling gene expression in a cell, which is based on competitive dimerization between a transcription factor polypeptide and an inhibitory polypeptide thereof. Dimerization is a powerful tool to achieve dynamic control of protein function and to quickly integrate information from different signalling pathways. Indeed, dimerizing proteins are extensively used in many cellular processes such as cell growth and differentiation, and response to stress. Competitive dimerization networks where proteins competitively bind to form dimers, can be used to implement complex regulatory functions with a limited number of different proteins, thus offering great scalability.

[0011] The system of the invention comprises:

[0012] a) monomers of an engineered transcription factor (Modular Artificial Dimerizing Transcription Factor, MADTFs), preferably a dimeric transcription factor, said monomers (MADTF monomers) being polypeptides configured to dimerize with each other and each comprising, or consisting of: a nucleic acid binding domain, a first dimerization domain, a second dimerization domain different from the first dimerization domain, and an effector domain configured to activate or repress transcription of a gene, upon binding of the MADTF dimer to said gene, or to induce epigenetic and / or genomic changes, upon binding of the MADTF dimer to a gene regulatory element; and

[0013] b) an engineered transcription factor’s inhibitory polypeptide (ATF), configured to inhibit the MADTF, preferably by inhibiting dimerization of MADTF monomers with each other, said inhibitory polypeptide comprising, or consisting of, a first dimerization and a second dimerization domain, different from the first dimerization domain, wherein at least one dimerization domain of ATF is configured to dimerize with a dimerization domain of the MADTF monomer;

[0014] wherein the affinity of dimerization between the ATF and a MADTF monomer is higher than the affinity of dimerization of MADTF monomers with each other.

[0015] The MADTF monomer of the invention is configured to dimerize with another MADTF monomer for activating or repressing transcription of a gene, but it is also configured to preferentially dimerize with the inhibitory polypeptide ATF, thanks to dimerization domains of ATF that dimerize with MADTF domains with higher affinity compared to the affinity of dimerization of MADTF monomers’ domains with each other. Dimerization of MADTF monomers with ATF then blocks the activity of MADTF, which is dependent on dimerization of MADTF monomers with each other. In other words, by competing for dimerization with MADTF monomers, ATF acts as a dominant negative inhibitor.

[0016] Preferably, the ATF of the invention does not comprise any nucleic acid binding domain and / or any effector domain, being thus not capable of modulating per se transcription of a gene, e.g. by homodimerization with another ATF.

[0017] Inclusion of transcription factor dimers in competitive dimerization networks significantly expands the potential functions of said networks. Promiscuously dimerizing TFs can give rise to multi stability. Therefore, the competitive dimerization among MADTFs and ATFs unlocks synthetic circuits with a vast functional diversity, enabling the design of highly sophisticated cellular behaviors that far exceed current capabilities.

[0018] Moreover, the modular nature of the MADTF of the invention, facilitates the interchangeability of domains, allowing for customization of DNA binding specificity, dimerization strength, and effector domains. For instance, substitution of the nucleic acid binding domain enables targeted binding to endogenous genomic loci, while different effector domains can be used to activate or repress gene transcription, or to induce epigenetic and genomic changes.

[0019] The system of the present invention then provides a versatile, modular and scalable technological platform that can be used for the construction of synthetic genetic circuits with complex regulatory functions.

[0020] The present invention is also directed to novel engineered genetic circuits, comprising the system of the invention, for controlling gene expression in a cell, as defined in the enclosed claims. Preferably, said circuits are Gene Dosage Compensation (GDC) circuits particularly suitable for gene replacement therapies: GDC circuits can be engineered to tune the expression of a target gene in a tight expression window and make it independent of virus uptake.

[0021] The present invention is also directed to expression vectors for expressing the system and / or circuits of the invention and to cells that incorporate said system, genetic circuit or expression vectors.

[0022] Advantageously, the transcription factors and inhibitory polypeptide of the system of the invention are compact in size, in terms of DNA sequence. This makes the system and circuits of the invention suitable for being delivered to a cell through viral or non-viral particles. Moreover, the system of the invention is orthogonal to mammalian cells, and does not rely on proteins with potential off-target effects, such as members of the Cas family.

[0023] Therefore, the invention is also directed to methods for controlling gene expression in a cell by introducing in the cell the system, genetic circuit or expression vectors of the invention, preferably by means of viral or non-viral delivery, and to the use of said systems, genetic circuits, expression vectors and cells in the claimed applications.

[0024] The features and advantages of the present invention will become apparent from the following detailed description, from the embodiments provided by way of illustrative and non-limiting examples, and from the attached figures.

[0025] BRIEF DESCRIPTION OF FIGURES

[0026] Figure 1 shows schemes of MADTF / ATF systems according to two preferred embodiments of the invention (a-d and e-h, respectively): (a) shows the structure of a modular MADTF comprising: a DNA Binding Domain (DBD), a first dimerization domain (DD) being a homodimerization domain, a second dimerization domain being a synthetic orthogonal domain (SOD), and effector domain (ED); MADTF is active when in dimeric form; (b) shows the structure of a ATF inhibitor of the MADTF in (a), comprising the same DD and a cognate synthetic orthogonal heterodimerization domain SOD*, which binds the complementary SOD of the MADTF; (c) MADTF monomer preferentially binds to ATF thanks to the higher affinity complementarity of SOD / SOD* cognate domains, forming an inactive heterodimer, (d) the system according to the invention (with SOD) leverages the higher affinity between MADTF and ATF monomers provided by the SOD / SOD* over the homodimerization of MADTF monomers by DD, thus selectively promoting non-functional heterodimers, that effectively inhibit the transcription factor activity; conversely, with state-of-the art TFs, lacking a second domain (without SOD), the dominant negative approach would result in a mixed population of inactive MADTF / ATF heterodimers, inactive ATF / ATF homodimers, and active MADTF homodimers, because of the single shared dimerization domain, thus leading to poor transcription factor inhibition, (e) shows an artificial zinc finger DBD consisting of six fingers (6F-ZF), each one recognizing three DNA bases; the artificial zinc finger DBD can be used as DBD in two modular MADTF monomers, as shown in (f): each monomer comprises a first half of the six fingers DBD (3F-ZF-MADTFs), then, from bottom to the top: a first synthetic orthogonal dimerization domain called SOD Z, or its complementary SOD Z*, acting as a heterodimerization domains to form the full 6F-ZF-MADTF, a second dimerization domain being a SOD in both monomers, and the TD or other effector domains, (g) shows the structure of the ATF inhibitor of the MADTF shown in (e), consisting of either one of SOD Z or SOD Z* of MADTF monomers as first dimerization domains (here a ATF with SOD Z is shown), and of a second dimerization domain being SOD*, which binds the complementary SOD on both the ZF-MAD-TF monomers; (h) from left to right panel: a single monomer of 3F-ZF-MADTF, having only a 3F-ZF DBD, unable to bind DNA and activate gene expression because of weak binding affinity; when both 3F-ZF-MAD-TF monomers are present, they ca heterodimerize thanks to the binding of the complementary first dimerization domains SOD Z / SOD Z* and the MADTF heterodimer can efficiently activate transcription, as it will effectively bind DNA as a 6F-ZF; the activity of the 6F-ZF-MADTF heterodimer can still be fully inhibited by the ATF, as both 3F-ZF-MAD-TFs bind to ATF due to the stronger affinity between the interaction of the second dimerization domains SOD and SOD*, relative to affinity of SOD Z and the SOD Z* domains of the MADTF monomers; 3F-ZF-MAD-TFs bound to ATF are thus unable to form a MADTF dimer.

[0027] Figure 2 shows the feasibility of a system according to a preferred embodiment of the invention: (a) shows the structure of the state-of-the-art artificial GAIN TF, comprising, as depicted from bottom to top: GAL4 DNA binding domain (GAIN DBD) of 35 aminoacids (aa), a linker of 14 aa, the first dimerization domain being a Gal4 dimerization domain (DD) of 98 aa, made by 3 alpha helices, a linker of 7 aa, and a VP 16 effector domain from herpes simplex virus acting of 78 aa as transactivation domain (TD). (b) shows the GAIN TF monomers of (a) in the homodimeric form, alongside a cognate promoter containing four upstream activating sequences (UAS) driving a firefly luciferase reporter, (c) shows the MADTF MAD-GAL4 monomer, according to preferred embodiments of the invention, including as depicted from bottom to top: the GAL4 DBD of 35 aa, a linker of 14 aa, a truncated Gal4 dimerization domain (Custom Gal4 DD) of 23 aa, as first dimerization domain, a linker of 10 aa, a SOD composed of the synthetic coiled-coil domain N7 of 33 aa, as second dimerization domain, a Nuclear Localization Signal (NLS) of 8 aa, the VP 16 Transactivation Domain of 78 aa and a Aul tag of 6 aa. (d) The ATF monomer, according to the invention, formed by the truncated GAL4 DD (Custom Gal4 DD) of 23 aa, a linker of 10 aa long, and either one, two, or four repetitions of the synthetic coiled-coils domain N8 of 33 aa, separated by linkers of 10 aa, serving as the second dimerization domain of the ATF, SOD*. The ATF ends with the NLS and the Aul tag. (e) shows the experimental results of the Luciferase assays (GLOMAX®) performed in Hek293T cells transfected with a CMV-MAD-GAL4 plasmid, the UAS-fLuc plasmid and either one of the three CMV-ATF plasmids, or negative control without N8 (no SOD*). The amount of CMV-MAD-GAL4 and UAS-ff_luciferase plasmids were fixed at 50 ng. The ATF plasmid’s concentration ranged from 0 ng to 100 ng. The repression of luciferase increases with the number of synthetic N8 coiled coils. When 50 ng of the ATF plasmid containing four N8s is used, the repression of luciferase is approximately 50-fold compared to the condition without the SOD*.

[0028] Figure 3 shows the feasibility of a further system according to the invention, shown in a): the MADTF monomer comprises a ERa DBD (MAD-ERa), consisting of the ERa DBD of 83 aa, a linker of 14 aa, a truncated GAL4 DD of 23 aa, a GS linker of 10 aa, a coiled coil N7 SOD of 33 aa, a NLS of 8 aa and the VP16 ED of 78 aa, with an Aul tag of 6 aa; b) shows the reduction in luciferase activity, by the addition of ATF, with and without a SOD*, to MAD-ERa and reporter ERE-fluc. The amount of MAD-ERa is fixed at 50 ng. The ATF goes from 0, to 50 ng, to 100 ng. Only the inhibitor with SOD* can successfully reduce luciferase activity, showing that the system is functional also with different DBDs.

[0029] Figure 4 shows a scheme of a synthetic gene circuits based on the MADTF / ATF system: a) shows the structure of a CIL-ON circuit and its control: CIL-ON circuit is a doxycycline-inducible gene expression circuit based on a Coherent Inhibitory Loop (CIL) topology (see De Carluccio, G., Fusco, V. & di Bernardo, D. Engineering a synthetic gene circuit for high-performance inducible expression in mammalian systems. Nat Commun 15, 3311, 2024). In both CIL ON and control circuits, the transcription factor rtTA, under the control of the constitutive pCMV promoter and inducible by doxycycline, drives the expression of the gene downstream of the TRE3G promoter, which is MAD-GAL4 thus driving the expression of firefly luciferase (ff luciferase) upon binding to ff luciferase inducible pUAS promoter. ATF with 4N8 SOD* domain is under the control of the constitutive pCMV / TO promoter, inhibiting the leaky expression of MAD-GAL4. In the control, instead of ATF, UbGFP is present. In the CIL ON system, the leakiness of MAD-GAL4 and consequently of the reporter is reduced due to ATF activity, while in the control, this reduction does not occur, b) shows luciferase expression of the CIL ON system compared to the control, in log and linear scale: when the system is off, the leaky expression of luciferase is more than one-fold lower compared to the control, but when the system is active at 1000 ng / ml of doxycycline, the leaky expression of the reporter gene is almost the same as the control, c) shows fold change in linear scale of the luciferase expression of the CIL ON circuit, compared to the control, between the 0 doxycycline condition and 1000 ng / ml doxycycline condition. At 1000 ng / ml doxycycline, the fold change of the CIL ON is one-fold higher compared to the control, d) shows the structure of the CIL OFF system and its control. In both CIL OFF and control, the transcription factor rtTA, inducible by doxycycline and under the control of the constitutive pCMV promoter, drives the expression of the gene downstream of the TRE3G promoter, which is ATF with 4N8 SOD* for CIL OFF, UbGFP for the CIL OFF control. MAD-GAL4, under the control of the constitutive pCMV / TO promoter, drives the expression of firefly luciferase (ff luciferase) under the control of the inducible pUAS promoter. In the CIL OFF system, MAD-GAL4 is inhibited by ATF, while in the control, this inhibition does not occur, e) shows the luciferase expression of the CIL OFF system compared to the control in log and linear scale. When the system is off, the maximum expression of luciferase is the same as the control, but when the system is active at 1000 ng / ml of doxycycline, the leaky expression of the reporter gene is almost one-fold lower compared to the control, f) shows fold change in linear scale of the luciferase expression of the CIL OFF compared to the control between the 0 doxycycline condition and 1000 doxycycline condition: at 1000 doxycycline, the fold change of the CIL OFF is one-fold higher compared to the control.

[0030] Figure 5 shows a scheme of a potential application of GDC circuit of the invention to gene therapy for Friedrich’s Ataxia (FRDA). LV: lentiviral vector; AAV: Adeno Associated Vector; hIPSC: human induced pluripotent stem cells; DRGO: Dorsal Root Ganglion Organoid.

[0031] Figure 6 shows different configurations of MADTF / ATF pairs, obtainable by CLASSIC high-throughput hierarchical combinatorial cloning. The MADTF design space (from N to C): 4 TD, 1 SOD, 10 linkers either rigid(r) or flexible (f); 1 DD, 10 linkers, 1 DBD. The ATF design space (from N to C): 4 SOD* number variants; 10 linkers, 1 DD. The SOD / SOD*, DD and DBD can be exchanged for alternative ones (indicated in gray).

[0032] Figure 7 is a scheme of synthetic gene circuits with MADTFs and ATFs as components: (a) MADTF dimers can bind DNA and control transcription of target genes, whereas MADTF / ATF heterodimers are inactive, (b) shows the Boolean logic representation of MADTF / ATF which is a NIMPLY logic function, (c-d) show examples of two synthetic circuits with input-output functions relating the concentration of the MADTF monomer (input) to that of the homodimer (output): a negative feedback loop (c), and a positive feedback loop (d); (e-f) show examples of synthetic networks with multiple MADTF / ATF pairs, which can give rise to more complex functions: a bistable genetic toggle switch (e) and a putative synthetic network (f) with three MADTFs and two ATFs.

[0033] Figure 8 shows implementations of a logic circuit of the invention: a) Logic formalism describing the MAD-TF / ATF experimental system; 0 or 1 represent presence or absence of the indicated protein, b) The logic circuit of the Set-Reset Flip-Flop circuit implementing a binary memory unit: when both SET and REST are 0 the system maintains its previous state (i.e. either 0 or 1). When only SET or RESET are equal to 1, the system will switch to the indicated state. SET and RESET can never be both equal to 1 otherwise there will be a NOT DEFINED state (ND), c) An alternative logic circuit of the Set-Reset Flip-Flop using two different MADTF / ATF pairs, d) possible experimental implementation of the circuit in (c) wherein the pERE promoter is recognised by the MAD-ER (MADTF with ERalpha as DBD) equipped with P3 coiled-coil domain as SOD Z. At the same time, MAD-GAL4 (MADTF with GAL4 DBD) equipped with N7 coiled coil domain as SOD recognize pUAS promoter. Both MAD-ER and MAD-GAL4 are equipped also with two different destabilization domains, one stabilized by molecule shieldl, one by molecule dTAG-13. Shield 1 act as SET molecule, dTAG-13 as RESET molecule. When one of the SET or RESET molecules is present, the corresponding stabilized MAD-TF activates the ATF inhibitor of the opposite MAD-TF, equipped with the corresponding SOD-Z* (coiled-coil P4, that binds to SOD Z P3) or SOD* (coiled-coil N8, that binds to SOD N7). This enables the functioning of the Set-Reset Filp-Flop circuit, as described in (c).

[0034] Figure 9 shows the feasibility of systems according to preferred embodiments of the invention, wherein the previously tested MADTFs are modified by replacing their DNA-binding domain (DBD) with artificial zinc finger proteins, ZFA and ZFC, (resulting in MAD-ZFA and MAD-ZFC), while keeping the other components of the MADTF unchanged. These MAD-TFs regulate the expression of a flue reporter gene placed under the control of promoters that are specifically recognized by the corresponding zinc fingers. Further to addition of ATF (ΔGAL4) inhibitor, the luciferase activity of any one of the MAD-ZFs is reduced. The amount of each MAD-ZFs (A or C) was fixed at 50 ng, and the inhibitor was co-transfected at an equimolar ratio.

[0035] Figure 10 shows the feasibility of systems according to preferred embodiments of the invention, wherein the MADTFs is a modified MAD-GAL4, wherein the VP 16 activator domain is substituted with p65 ( MAD-GAL4 (p65)): further to addition of ATF (ΔGAL4) inhibitor expression of the reporter UAS-fluc, measured as luciferase activity, under the control of MAD-GAL4(p65) is substantially absent. MAD-GAL4 (p65) was used at 50 ng, and the inhibitor was co-transfected at an equimolar ratio.

[0036] Figure 11 shows the feasibility of a Gene Dosage Compensation (GDC) circuit, according to a preferred embodiment of the invention, wherein the MADTF (MAD-GAL4) drives expression of both the gene of interest and of ATF through a cassette comprising the gene of interest and the polynucleotide encoding the ATF with a P2A self-cleaving sequence in between; ATF inhibits MAD-GAL4 activity, establishing a self-regulating loop, (a) schematic of the GDC; (b) (b) Comparison of different GDC configurations with the control (pCMV-fluc).

[0037] DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention is directed to an engineered system to control gene expression in a cell, based on competitive dimerization between an artificial transcription factor (TF) and a transcription factor’s inhibitory polypeptide (TF inhibitor). In particular, the system of the invention comprises a modular artificial transcription factor, MADTF (sometimes indicated also as MAD-TF) and a transcription factor inhibitor configured to dimerize with MADTF monomers and lacking any effector domains and / or nucleic acid binding domains, ATF (sometimes indicated also as DTF).

[0039] As typically used in the art, the term “expression of a gene” can be also used in place of “expression of a transcript” or “expression of a protein” to mean that the gene is transcribed and / or translated. Equally, unless otherwise indicated, “expression of a protein”, such as expression of a protein, is a short term for indicating transcription and translation of a gene encoding the protein.

[0040] The gene whose expression is meant to be modulated by the system of the invention can be a gene encoding a natural protein, endogenous to a cell comprising the system of the invention, or a recombinant protein artificially expressed in the cell comprising the system of the invention.

[0041] MADTF and ATF

[0042] A transcription factor is a protein comprising nucleic acid binding domain, such as DNA binding domain (DBD), effector domain, typically being an activation domains (ADs) capable of promoting transcription of a gene or set of genes upon binding of the TF to a binding site in the promoter of the gene or set of genes. Many eukaryotic transcription factors regulate gene expression by forming dimeric (2:1) protein-DNA complexes.

[0043] The modular artificial transcription factor of the invention (MADTF) is characterized by the presence of at least two different dimerization domains, further to a nucleic acid binding domain and an effector domain, which can be an activator domain, activating transcription of a gene of interest, or a repressor domain, inhibiting transcription of a gene of interest.

[0044] The TF inhibitor of the invention (ATF) is configured to dimerize with the MADTF, and comprises as well two dimerization domains. MADTF and ATF are part of an engineered system for controlling gene expression in a cell.

[0045] The engineered system of the invention for controlling gene expression in a cell then comprises:

[0046] a) at least two monomers of an engineered dimeric transcription factor (Modular Artificial Dimerizing Transcription Factor, MADTF), preferably a dimeric transcription factor, each monomer (MADTF monomer) being a polypeptide comprising, or consisting of, a nucleic acid binding domain, a first dimerization domain, a second dimerization domain different from the first dimerization domain, and an effector domain configured to activate or repress gene transcription upon dimerization of the MADTF monomer with another MADTF monomer; and

[0047] b) an engineered transcription factor’s inhibitory polypeptide (ATF), configured to inhibit the MADTF, comprising, or consisting of, a first dimerization domain, and a second dimerization domain different from the first dimerization domain, wherein at least one dimerization domain of ATF is configured to dimerize with a complementary dimerization domain in a MADTF monomer;

[0048] wherein the affinity of dimerization between at least one dimerization domain of the ATF and at least one dimerization domain of the MADTF monomer, preferably between the second dimerization domain of ATF and the second dimerization domain of MADTF monomer, is higher than the affinity of dimerization between each dimerization domain of a MADTF monomer with each dimerization domain of another MADTF monomer, i.e. higher than the affinity of dimerization between the first dimerization domain of MADTF monomer with the first dimerization domain of another MADTF monomer and between the second dimerization domain of MADTF monomer with the second dimerization domain of another MADTF monomer. Preferably, the inhibitory polypeptide ATF inhibits the MADTF transcription factor by sequestrating the MADTF monomers, thus preventing dimerization of MADTF and consequently binding of MADTF to a nucleic acid of interest.

[0049] The term “Dimerization affinity” or “affinity of dimerization” means the propensity of two proteins to bind and form a stable complex. For instance, MADTF has a higher propensity to form dimers with ATF than with another MADTF, since the affinity of dimerization between at least one dimerization domain of the ATF and at least one dimerization domain of the MADTF monomer is higher than the affinity of dimerization between any dimerization domain of the MADTF monomers with each other.

[0050] Dimerization affinity can be measured by determining the dissociation constant (KD) of two molecules. KD can be measured by isothermal titration calorimetry (see e.g. Dutta AK, et al. Using isothermal titration calorimetry to determine thermodynamic parameters of protein-glycosaminoglycan interactions. Methods Mol Biol. 2015;1229:315-24).

[0051] According to the present invention a couple of molecules or domains that dimerize with a higher affinity when compared to a different couple molecules or domains means that the former couple have a lower KD than the latter couple. Therefore, according to the invention the KD of MADTF with ATF is higher than the KD of MADTF dimers.

[0052] In MADTF monomers, the “first” dimerization domain preferably indicates the dimerization domain linked to the nucleic acid binding domain. Therefore, the ’’second” dimerization domain preferably indicates the dimerization domain that is linked to the first dimerization domain and to the effector domain.

[0053] In accordance with the above, the “first” dimerization domain of ATF indicates the dimerization domain configured to dimerize with the first dimerization domain of MADTF, while the “second” dimerization domain of ATF indicates the dimerization domain configured to dimerize with the second dimerization domain of MADTF.

[0054] Preferably, MADTF monomers dimerize solely through their first dimerization domains. More preferably said first dimerization domains are homodimerization domains.

[0055] In preferred embodiments, ATF’s first dimerization domain is identical to the first dimerization domain of at least one MADTF monomer, while ATF’s second dimerization domain is different from the second dimerization domains of the MADTF, but complementary to it.

[0056] The terms “homodimer / homodimerization” and “heterodimer / heterodimerization” can be used to refer to dimerization of the macromolecules of the system of the invention, i.e. MADTF and ATF: identical molecules “homodimerize” (as in the MADTF dimer of Figure 1 a), and different molecules “heterodimerize” (as in the MADTF / ATF pairs of Figure 1 c).

[0057] Furthermore, the terms “homodimerization / homodimerizing” and “heterodimerization / heterodimerizing” can be used with reference to the domains of the macromolecules: a domain that can dimerize only with another identical domain is an homodimerizing domain, while two different domains that can dimerize only with each other are heterodimerizing domains. A pair of heterodimerization domains that are able to heterodimerize between themselves are also named “cognate” or “complementary” dimerization domains.

[0058] In some embodiments, MADTF / ATF dimers comprise one pair of homodimerizing domains and one pair of heterodimerizing domains. In other embodiments MADTF / ATF dimers do not comprise any homodimerization domains.

[0059] Examples of preferred embodiments of the system of the invention are depicted in Figure 1 a-d. Figure la shows MADTF monomers consisting of a DNA Binding Domain (DBD), a first dimerization domain being a low-affinity Homo-dimerization Domain (DD), a second Heterodimerization domain being a high-affinity Synthetic Orthogonal Domain (SOD) and an Effector Domain (ED). The MADTF exclusively operates in its dimeric state, as two intact DBDs will be necessary to bind the cognate DNA sequence. This property enables the engineering of a dominant-negative inhibitor (ATF), as shown in Figure lb, consisting of a first homodimerization domain identical to the first homodimerization domain of MADTF (the low-affinity homodimerization domain DD in Figure lb) and a second heterodimerization domain being a Synthetic Orthogonal Domain (SOD*) with a high affinity for the cognate SOD of the MADTF. Hence, when only the MADTF monomers are expressed, they will be able to homodimerize and bind the DNA (solely through their homodimerization domains DD, as in Fig. la); however upon expression of the ATF, the latter will form a non-functional heterodimer with monomeric MADTF, as the SOD affinity for SOD* is much higher than that DD affinity for itself (Figure 1c), thereby strongly reducing the pool of active homodimers and effectively blocking MADTF activity.

[0060] The system of the invention is more advantageous than the systems of the TF / inhibitory pairs of the prior art, comprising a single homodimerization domain, as shown in Figure Id (right panel): in fact in the latter case, a dominant negative inhibitor would be insufficient to prevent DNA binding, because expression of both the inhibitory polypeptide and the TF would result in a mixed scenario in which non-functional heterodimers, functional TF homodimers and nonfunctional inhibitor homodimers would co-exist, yielding to a poor inhibition of TF activity. On the contrary, in the system of the invention, wherein MADTF / ATF dimers comprises low affinity homodimerization domains (such as DD in Figure 1) together with high affinity strong heterodimerization domains (such as SOD / SOD* in Figure 1) guarantees that MADTF / ATF heterodimerization will be favored over MADTF homodimerization, leading to a complete inhibition of MADTF activity, as schematized in Figure Id (left panel).

[0061] Furthermore, the modular nature of the MADTF facilitates the interchangeability of domains, allowing for customization of DNA binding specificity, dimerization strength, and effector domains.

[0062] Figure If shows further preferred embodiments of the system of the invention, wherein MADTF is a heterodimer whose monomers have different nucleic acid binding domains and first dimerization domains, while having the same second dimerization domain: the first homodimerization domain of the MADTF of Figure la is here replaced by a weak synthetic heterodimerization domain, with one monomer having one domain (SOD Z), and the other having the complementary domain (SOD Z*); the rest of the MADTF structure remains the same, including the original second dimerization domain (SOD). Here the ATF, as shown in Figure 1g, consists of a first dimerization domain being a Synthetic Orthogonal Domain identical to the first dimerization domain of one of the MADTF monomers (SOD Z) and a second dimerization domain being a different and stronger Synthetic Orthogonal Domain (SOD*) with a high affinity for the cognate SODs of the MADTF. When only the MADTF monomers are expressed, they will be able to heterodimerize through the cognate first heterodimerization domains SOD Z / SOD Z* and to thus bind the DNA (Fig. Ih, middle panel); however upon expression of the ATF, the latter will form a non-functional heterodimer with each monomeric MADTF, at least through the high affinity SOD* / SOD second domains, as the SOD affinity for SOD* is higher than that SOD Z / SOD Z* affinity and heterodimerization domains such as SODs cannot homodimerize (Figure Ih, right panel). The terms “DD”, “SOD”, “SOD Z”, are only used for illustrative purposes to identify different dimerization domains; “SOD*” and “SOD Z*”, with asterisk, are used to identify heterodimerization domains cognate to SOD and SOD Z, respectively.

[0063] The term “consisting of’ when referred to MADTF or ATF molecules of the system of the invention is meant to indicate that no functional domains (or “primary” domains) are included in the molecule other than those listed. However, MADTF and ATF molecules “consisting of’ the listed domains preferably include spacers and linker peptides between said domains.

[0064] For instance, a ATF “consists of’ a first dimerization domain and a second dimerization domain in the sense that said ATF does not comprise further functional domains, in particular said ATF does not comprise a nucleic acid binding domain and / or an effector domain. Accordingly, a MADTF “consists of’ a nucleic acid binding domain, a first dimerization domain, a second dimerization domain and an effector domain, in the sense that said ATF does not comprise further functional domains. Still, said MADTF or ATF preferably comprise linker peptides, e.g. linking the first and second dimerization domains, and / or linking the first dimerization domain and the nucleic acid biding domain (in case of MADTF).

[0065] Therefore, MADTFs and ATFs of the system of the invention preferably comprise spacers or linker peptides linking the first and second dimerization domains; preferably, MADTFs further comprises a spacer or linker peptide between the nucleic acid binding domain and one of the dimerization domains, preferably the first dimerization domain.

[0066] Linkers with different length and flexibility can be included in the MADTFs and ATFs of the system of the invention.

[0067] Preferably, the nucleic acid binding domain and the first dimerization domain of MADTF monomers are linked by a peptidic linker of from 5 to 50 aminoacids, more preferably of from 10 to 30 aminoacids, most preferably of from 10 to 30 aminoacids and / or wherein the first and second dimerization domain of the MADTF monomers of ATF are linked by a peptidic linker of from 5 to 50 aminoacids, more preferably of from 10 to 30 aminoacids, most preferably of from 10 to 30 aminoacids.

[0068] The skilled person would know how to design linkers and spacers suitable for being employed in the MADTFs and ATFs of the systems of the invention (Huang, Z., Zhang, C. & Xing, X -H. Design and construction of chimeric linker library with controllable flexibilities for precision protein engineering, in 23-49, 2021).

[0069] In some embodiments MADTF and / or ATF comprise dimerization domains formed by multiple copies of a dimerizing peptide, said multiple copies being linked by peptide linkers.

[0070] Advantageously, MADTFs and ATFs according to the invention are compact in size, consisting of polypeptides of no more than 400 aminoacids, preferably no more than 300 aminoacids more preferably of no more than 200 aminoacids. For instance, preferred MADTFs comprise a nucleic acid binding domain of from 20 to 50 aminoacids (aa), preferably of from 30 to 40 aa; a first dimerization domain of from 10 to 100 aa, preferably of from 20 to 50 aa, more preferably of from 30 to 40 aa; a second dimerization domain comprising oner or more peptides of from 20 to 50 aa, preferably of from 30 to 40 aa; an effector domain of from 50 to 100 aa, more preferably of 70-90 and one or more linkers each of from 5 to 50 aa. Accordingly preferred ATF comprise a first dimerization domain of from 10 to 100 aa, preferably of from 20 to 50 aa, more preferably of from 30 to 40 aa; and a second dimerization domain comprising one or more peptides of from 20 to 50 aa, preferably of from 30 to 40 aa.

[0071] First dimerization domains

[0072] In preferred embodiments, MADTF monomers comprise a first dimerization domain being a low-affinity Dimerization Domain (DD), preferably a homodimerization domain and a second heterodimerization domain being a high-affinity Synthetic Orthogonal Domain (SOD).

[0073] More preferably said monomers consist of a DNA binding domain (DBD), a first dimerization domain being a low-affinity homodimerization domain, a second heterodimerization domain being a high-affinity synthetic orthogonal domain (SOD) and an effector domain (ED).

[0074] The first dimerization domain is a low-affinity domain being configured to form MADTF dimers that are stable enough to permit transcriptional activity, but unstable enough to be displaced when a ATF harboring dimerizes with at least one MADTF monomer through the high-affinity heterodimerization SODs.

[0075] Dimerization domains suitable for being employed in the system of the invention can be of natural or synthetic origin. For instance in some embodiments a dimerization domain is a dimerization domain of a transcriptional activator of yeast.

[0076] Preferably, at least one dimerization domain of a MADTF monomer and / or of ATF, more preferably the first dimerization domain of a MADTF monomer and / or of ATF, most preferably the first homodimerization domain of MADTF and the first dimerization domain of ATF, is a natural dimerization domain.

[0077] Preferably said natural dimerization domain is a Gal4 dimerization domain (Gal4 DD) encoded by a polynucleotide having sequence comprising, or consisting of, SEQ ID NO: 1, or a variant thereof. The complete dimerization domain of Gal4 (residues 50-106 of Gal4 protein, NCBI Reference Sequence NP 015076.1) contains three a helices. The first helix (residues 51-71 of Gal4 protein) forms a typical coiled-coil and the second and third helices (residues 74-82 and 86-95 of Gal4 protein, respectively) fold back in an antiparallel fashion to form a helical bundle interaction near the C-terminal end of the first helix of the opposing monomer (Hong M, Structural basis for dimerization in DNA recognition by Gal4. Structure, 2008 Jul; 16(7): 1019-26).

[0078] More preferably, said first homodimerization domain of MADTF and the first dimerization domain of ATF is a modified Gal4 dimerization domain, most preferably a truncated GAL4 dimerization domain comprising 16-22 aa of the Gal4 dimerization domain, more preferably comprising only the first alpha-helix of the full dimerization domain of Gal4 protein.

[0079] Preferably said first homodimerization domain of MADTF and the first dimerization domain of ATF is a truncated GAL4 dimerization domain comprising or consisting of aminoacids 50 to 72 of Gal4 dimerization protein (NP 015076.1), more preferably, said truncated GAL4 dimerization domain being encoded by a polynucleotide having sequence comprising or consisting of SEQ ID NO: 2, or variants thereof.

[0080] The term "variant" refers to biologically active derivatives of the reference molecule that retain desired activity. In general, the term "variant" refers to molecules having a native sequence and structure with one or more additions, substitutions (generally conservative in nature) and / or deletions, relative to the native molecule, so long as the modifications do not destroy biological activity, and which are "substantially homologous" to the reference molecule. In general, the sequences of such variants will have a high degree of sequence homology to the reference sequence, e.g., sequence homology of more than 50%, generally more than 60%-70%, even more particularly 80%-85% or more, such as at least 90%-95% or more, when the two sequences are aligned.

[0081] In accordance with the present invention, a variant of any biomolecule is a biomolecule that has a nucleic acid or aminoacidic sequence having a % of identity of 50%, 60%, 70%, 80%, 90%, 95%, or 99% to the wild-type nucleic acid or aminoacidic sequence and that retains the biological activity of the wild-type biomolecule. In preferred aspects, the term “variant” of a polynucleotide sequence is used herein to indicate a sequence having a % of identity of at least 90%, 95% or 99% to said polynucleotide sequence. In preferred aspects, the term “variant” of a polynucleotide sequence is used herein to indicate a sequence that is a codon-optimized sequence for expressing the biomolecule encoded by said sequence. The terms “% sequence identity”, “% identity” or “% sequence homology” refer to the percentage of nucleotides or amino acids of a candidate sequence that are identical to the nucleotides or amino acids in the sequence of reference, after aligning the sequences to achieve the maximum % sequence identity. In a preferred embodiment, sequence identity is calculated based on the full length of two given sequences or on part thereof. The % sequence identity can be determined by any methods or algorithms established in the art, such as the ALIGN, BLAST and BLAST 2.0 algorithms. Herein, the “% sequence identity”, “% identity” “or “% sequence homology” is calculated dividing the number of nucleotides or amino acids that are identical after aligning the sequence of reference and the candidate sequence, by the total number of nucleotides or amino acids in the sequence of reference and multiplying the result by 100. In accordance with degeneration of genetic code, variants include sequences where at least one base of the base sequence of a gene is replaced with a different type of base, without changing the amino acid sequence of the polypeptide expressed from the gene. Variants also include codon-optimized sequences and sequences comprising mutated or added nucleotides, e.g., for cloning needs. In accordance with the present invention, variants also include sequences encoding fragments of any biomolecule, i.e., a shorter form of the biomolecule, such as a truncated form, that retains the biological activity of the wild-type biomolecule.

[0082] In some preferred embodiments the system of the invention comprises: a) a MADTF homodimer comprising a first dimerization domain being a GAL4 dimerization domain encoded by a polynucleotide having sequence comprising, or consisting of, SEQ ID NO: 1 or a truncated GAL4 dimerization domain encoded by a polynucleotide having sequence comprising or consisting of SEQ ID NO: 2, or a variant thereof; preferably, said system comprises b) a ATF comprising the same first dimerization domain of MADTF.

[0083] Further preferred domains that can be employed in the system of the invention as first dimerization domain of a MADTF monomer, and / or as first dimerization domain of ATF, more preferably as first homodimerization domain of both, including: leucine zipper domain responsible for the dimerization of the GCN4 transcription factor (O’Shea, E. K., et al. X-Ray Structure of the GCN4 Leucine Zipper, a Two-Stranded, Parallel Coiled Coil. Science (1979) 254, 539-544, 1991), encoded by a polynucleotide having sequence comprising, or consisting of, SEQ ID NO: 3 (GCN4 DD), or the dimerization domain of GCN3 transcription factor, encoded by a polynucleotide having sequence comprising, or consisting of, SEQ ID NO: 4 (GCN3 DD); or a variant thereof.

[0084] Second dimerization domain

[0085] The second dimerization domain of MADTF and of ATF in the system of the invention can be of natural or synthetic origin.

[0086] The second dimerization domain of MADTF and of ATF is preferably a synthetic orthogonal heterodimerization domain (SODs), designed to have a high affinity for its complementary partner, forming stable heterodimers while remaining orthogonal to the native functions of the host cell.

[0087] Preferably, said synthetic orthogonal heterodimerization domain is a coiled-coiled dimerization domain specifically designed to form heterodimers. Therefore, preferably the ATFs of the system of the invention comprise second dimerization domains being cognate heterodimerization domains of the second dimerization domain of a MADTF monomer.

[0088] Preferred cognate heterodimerization domain that can be employed as second dimerization domains in MADTF and ATF pairs are dimerization domains comprising, or consisting of, N7 and N8 dimerization peptides (Plaper, T. et al. Coiled-coil heterodimers with increased stability for cellular regulation and sensing SARS-CoV-2 spike protein-mediated cell fusion. Sci Rep 11, 1-16, 2021) or variants thereof. Preferably, said N7 and N8 dimerization peptides are respectively encoded by a polynucleotide having sequence comprising, or consisting of, SEQ ID NO: 5 (N7) and SEQ ID NO: 6 (N8), or variants thereof.

[0089] In preferred embodiments, the second dimerization domain of MADTF monomer is N7 encoded by a polynucleotide having sequence SEQ ID NO: 5 and the second dimerization domain of ATF monomers is N8 encoded by a polynucleotide having sequence SEQ ID NO: 6, or vice versa.

[0090] In some embodiments, a dimerization domain of the MADTFs and / or ATFs of the system of the invention is formed by multiple copies of a dimerization peptide. Preferably, the second dimerization domains of MADTF and / or of ATF is a dimerization domain consisting 2, 3, or 4 copies of a dimerization peptide, such as 2, 3, or 4 copies of a N8 peptide, each copy being separated from the other(s) by a linker, preferably said linker being 5 to 20 aminoacids, more preferably a linker of 8 to 15 aminoacids, most preferably of 10 to 12 aminoacids. Preferably the linker is a peptide having a number of aminoacids that is one half, one third or one fourth of the number of aminoacids of the dimerization peptide.

[0091] Preferably, the second dimerization domain of MADTF and / or of ATF is a dimerization domain comprising a single N8 peptide, encoded by a polynucleotide having sequence comprising, or consisting of, SEQ ID NO: 6, or it is a dimerization domain comprising two N8 peptides joined by a linker, encoded by a polynucleotide having sequence comprising, or consisting of, SEQ ID NO: 7 (2N8), or it is a dimerization domain comprising four N8 peptides joined by a linker, encoded by a polynucleotide having sequence comprising, or consisting of, SEQ ID NO: 8 (4N8), or variants thereof. More preferably, the second dimerization domain of MADTF or of ATF is a dimerization domain comprising four N8 peptides encoded by a polynucleotide having sequence comprising, or consisting of, SEQ ID NO: 8.

[0092] Preferred pairs of second heterodimerization domains that can be used in MADTF / ATF pairs according to the system of the invention are listed in Table 1, wherein the column “SOD” lists preferred second heterodimerization domains of MADTF monomers and the column “SOD*” lists, row by row, preferred cognate second heterodimerization domain of the ATF (or vice versa). Sequences are indicated in the right columns (SEQ ID NO: 5-8 are nucleotide sequences encoding for the dimerization domains, including linker where applicable; SEQ ID NO: 9-32 are aminoacidic sequences of the dimerization domains).

[0093]

[0094] Table 1

[0095] Effector domain

[0096] In preferred embodiments, the MADTF monomer of the system of the invention comprises an effector domain being an activation domain, capable of activating transcription of a bound gene upon dimerization with another MADTF.

[0097] In other preferred embodiments, the MADTF monomer of the system of the invention comprises an effector domain being repression domain, capable of repressing transcription of a bound gene upon dimerization with another MADTF.

[0098] MADTF effector domains can be derived from both viral and human proteins.

[0099] Preferably, the MADTF of the system of the invention comprises an effector domain being selected from:

[0100] VP16, encoded by a polypeptide having sequence comprising or consisting of SEQ ID NO: 33; VP64, encoded by a polypeptide having sequence comprising or consisting of SEQ ID NO: 34; VPR, encoded by a polypeptide having sequence comprising or consisting of SEQ ID NO: 35; p65, encoded by a polypeptide having sequence comprising or consisting of SEQ ID NO: 36; or variants thereof.

[0101] Nucleic acid binding domain

[0102] In accordance with the present invention, the MADTF can bind through its nucleic acid binding domains to any gene of interest, encoding any transcript or protein, either natural or artificial, e.g., recombinant. As an example, the gene of interest can be a gene encoding a reporter molecule (such as a luminescent or fluorescent reporter molecule), whose expression is regulated by the MADTF or a recombinant therapeutic gene.

[0103] For instance, selection of the nucleic acid binding domain (e.g. TALE, ZF) enables targeted binding to endogenous genomic loci; moreover, different EDs can be used to activate or repress gene transcription, or to induce epigenetic and genomic changes.

[0104] Preferably, the nucleic acid binding domain of MADTF is orthogonal to the ones of the cells comprising the system of the invention.

[0105] Preferably, the nucleic acid binding domain of MADTF is a DNA binding domain (DBD) of Gal4 (GAL4 DBD), encoded by a polynucleotide having sequence comprising, or consisting of, SEQ ID NO: 37, or a variant thereof.

[0106] Preferably, the nucleic acid binding domain of MADTF is a DNA binding domain (DBD) of ERalpha (ERalpha DBD), encoded by a polynucleotide having sequence comprising, or consisting of, SEQ ID NO: 56, or a variant thereof.

[0107] Preferably, the nucleic acid binding domain of MADTF is DBD with Zinc Finger (ZF) or TALE.

[0108] Synthetic transcription factors with ZF DBD are versatile tools that can be customized to bind any DNA sequence of interest, as each finger can be engineered to bind a specific DNA triplet. Preferably, the nucleic acid binding domain of MADTF or of a MADTF monomer is a DBD consisting of at least three zinc fingers (3F-ZF), more preferably of at least four zinc fingers (4F-ZF), in place of 3F-ZF-DBDs, to increase specificity. Alternative preferred orthogonal DBDs are DBD with transcription activator-like effector domains (TALEs).

[0109] As uniquely addressing a specific genomic locus, as necessary in gene therapy applications, typically requires recognizing at least 18 base pairs (bp) of DNA, hence at least six ZFs (6F-ZF), in preferred embodiments the MADTF of the system of the invention comprises monomers each comprising a set of 3 ZFs (3F-ZF), which provide the required 6 ZFs upon dimerization of the MADTF, as shown in Figure le-h. Preferred MADTF / ATF pairs

[0110] In some preferred embodiments, the system of the invention comprises:

[0111] a) MADTF monomers of a MADTF homodimer, said monomers comprising, or consisting of, a DNA binding domain, a first homodimerization domain, a second homodimerization domain being a synthetic orthogonal dimerization domain and an effector domain, and

[0112] b) a ATF comprising, or consisting of, a first dimerization domain being a homodimerization domain to the first dimerization domain of MADTF, and a second homodimerization domain being a synthetic orthogonal dimerization domain cognate to the second dimerization domain of the MADTF.

[0113] Preferably, the first homodimerization domain of MADTF and ATF is a dimerization domain encoded by a polynucleotide having sequence comprising, or consisting of, SEQ ID NO: 1-4, or variants thereof, more preferably a Gal4 dimerization domain encoded by a polynucleotide having sequence comprising, or consisting of, SEQ ID NO: 1 or 2, or variants thereof; and / or the second dimerization domain of MADTF is a dimerization domain encoded by a polynucleotide having sequence comprising or consisting of anyone of: SEQ ID NO: 5, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or variants thereof, and the second dimerization domain of ATF is a dimerization domain encoded by a polynucleotide having sequence comprising or consisting of anyone of: SEQ ID NO: 6, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or variants thereof, or vice versa.

[0114] Preferably, the system of the invention comprises:

[0115] a) MADTF monomers comprising, or consisting of, a Gal4 DNA binding domain encoded by a polynucleotide having sequence comprising or consisting of SEQ ID NO: 37 or a ERalpha DNA binding domain encoded by a polynucleotide having sequence comprising or consisting of SEQ ID NO: 56, a first homodimerization domain being a Gal4 dimerization domain encoded by a polynucleotide having sequence comprising, or consisting of, SEQ ID NO: 1 or 2, or variants thereof, a second dimerization domain being a synthetic orthogonal dimerization domain encoded by a polynucleotide having sequence selected from anyone of sequences comprising or consisting of SEQ ID NO: 5-8, and an effector domain encoded by a polynucleotide having sequence selected from anyone of sequences comprising or consisting of SEQ ID NO: 33-36, or variants thereof, and

[0116] b) a ATF comprising, or consisting of, a first dimerization domain being a Gal4 dimerization domain encoded by a polynucleotide having sequence comprising, or consisting of, SEQ ID NO: 1 or 2, or variants thereof, a second dimerization domain being a synthetic orthogonal dimerization domain encoded by a polynucleotide having sequence selected from anyone of sequences comprising or consisting of SEQ ID NO: 5-8,

[0117] provided that the second dimerization domains of MADTF and ATF are cognate heterodimerization domains. For example when the second dimerization domains of MADTF is N7, the second dimerization domains of ATF is N8 and vice versa.

[0118] As previously noted, the modular nature of the MADTF of the invention, facilitates the interchangeability of domains, allowing for customization of DNA binding specificity, dimerization strength, and effector domains.

[0119] Further combinations of MADTF / ATF pairs can thus be provided, by exchanging different pairs of first and second domains and different nucleic acid binding domains.

[0120] For instance, a library of MADTF / ATF pairs modulating the expression of reporter genes can be prepared and their efficacy can be measure by expressing them in cells: e.g. MADTF / ATF pairs can be expressed in a cell transiently by transfection or stably by single-copy genomic integration to modulate expression of a reporter, then said cells can be subjected to luminescence and fluorescence assays to evaluate the performance of said pairs. As an example, Hek293 cells can be engineered to constitutively express the doxycycline inducible rtTA transcription factor together with genomic integration of a synthetic promoter upstream of an eGFP fluorescent protein, said promoter harboring binding elements recognized by the DBD of choice in the MADTF. Upon doxycycline administration, rtTA will drive expression of the ATF from a pTRE3G promoter, which will thus allow to phenotypically screen the MADTF / ATF pairs maximizing fold-change between induced and uninduced conditions. This procedure would enable the skilled person to select of the best performing MADTF / ATF pairs, i.e. those maximizing expression of eGFP in the absence of the ATF, while minimizing eGFP expression in its presence.

[0121] Further MADTFs and ATFs pairs can thus be designed by the person skilled in the art. Libraries of MADTFs and ATFs can be also prepared using the gate cloning approach named CLASSIC as described by O’Connell, R. W. et al. (Ultra-high throughput mapping of genetic design space. bioRxiv, 2023).

[0122] As shown in Figure 7, the MAD-TF approach can be scaled to include multiple interacting MAD-TFs and ATFs due to its modularity and the availability of additional pairs of Synthetic Orthogonal Domains. This scalability allows for the implementation of more complex regulatory functions, such as genetic toggle switches and complex boolean logic functions. Control Engineering principles can be used to develop dynamical system models to encapsulate their key features such as competitive dimerization of varying strengths, DBDs with different affinities, and effector domains with either activating or repressing activity, and then computationally explore the range of unique functions that can be performed across all physiologically reasonable parameter values. Control Engineering abstracts synthetic circuits as dynamical input-output systems. In this setting, the inputs are a subset of monomers, while the output is a gene of interest whose expression is regulated by a MADTF dimer.

[0123] Specifically, in a Boolean logic formalism, the combination of MAD-TF and ATF can be represented by a two-input AND logic gate, with one of the inputs passing through a NOT gate, as shown in Figure 8a. Thanks to this formalism, it is possible to design complex regulatory functions; for example, the NAND-based Set-Reset Flip-Flop circuit in Figure 8b, is a classic electronic circuit acting as a memory device, also known as a toggle switch in synthetic biology. Using the logic formalism of the MAD-TF system, the flip-flop can be designed as shown in Figure 8c, and experimentally implemented as shown in Figure 8d, thus obtaining a biological toggle switch in mammalian cells.

[0124] The compact size of MADTF monomers and ATFs of the invention, the former being encoded by polynucleotides of few hundreds of base pairs, enhances their utility in industrial applications where genomic stability and minimal burden on the host organism are critical. Moreover, their modularity allows the selection of more suitable domains, based on the application. For instance, in biomedical settings, MAD-TF systems can be fully humanized by substituting domains with those derived from human proteins, such as replacing ta VP 16 activation domain with a p65 domain or by using DNA binding domains from zinc-finger proteins or TALENs instead of GAL4. This humanization reduces immunogenicity and improves compatibility with human cells, making MAD-TFs ideal candidates for gene therapy. Moreover, their compact size makes them compatible with viral delivery by Adeno Associated Vectors (AAV) and lentiviral vectors.

[0125] Genetic circuits

[0126] The present invention is also directed to novel engineered genetic circuits comprising the system of the invention for fine controlling of gene expression in a cell, as defined in the enclosed claims.

[0127] The skilled person would understand beyond any doubt that the sentence “a genetic circuit comprising the system of the invention” is meant to concisely indicate a genetic circuit comprising the elements of the system of the invention, or the polynucleotides encoding the same.

[0128] The systems and genetic circuits of the invention are engineered as at least one component of the system and / or genetic circuit is recombinant (i.e., non-natural), such as a recombinant gene or protein. Preferably all the component of the systems and / or genetic circuits of the invention are recombinant.

[0129] The genetic circuit of the invention is preferably a genetic circuit having a closed loop configuration, wherein the transcription factor’s inhibitory polypeptide, with its regulatory activity on the transcription factor, closes the loop.

[0130] Preferably, the systems of the invention are configured to implement a Gene Dosage Compensation (GDC) function, providing circuits being capable to regulate the level of expression of a gene within a specified range, and to implement Inducible Genetic Switches, providing circuits that guarantee a low basal expression when uninduced and a high maximal expression in response to a specific input signal or a specific combination of multiple inputs. Preferred circuits that can be implemented by the system of the invention are those described in WO2024023776, the content of which is incorporated herein by reference, in particular the circuits having a Coherent Feed Forward Loop (CFFL) configuration or a MI-FFL configuration (circuits based on a Coherent Inhibitory Loop (CIL) topology as disclosed by De Carluccio, G., et al. Nat Commun 15, 3311, 2024).

[0131] The invention is also directed to a genetic circuit comprising the system of the invention and at least one inducible transcriptional activator, preferably wherein expression of MADTF and ATF is under the control of promoters that comprise binding sites for said transcriptional activator. In some preferred embodiments, the expression of MADFT is activated by binding of the transcriptional activator to its promoter, said MADTF activating the expression of a gene of interest, optionally an exogenous gene, while expression of ATF is inhibited by binding of the transcriptional activator to its promoter (CIL-ON genetic circuit); in other preferred embodiments, the expression of MADTF and ATF is under the control of promoters comprising binding sites for the transcriptional activator, wherein binding of the transcriptional activator is wherein MADTF activates the expression of a gene of interest, and wherein expression of MADFT is inhibited by binding of the transcriptional activator to its promoter, while expression of ATF is activated by binding of the transcriptional activator to its promoter (CIL-OFF genetic circuit). Preferably, the inducible transcriptional activator is rtTA inducible transcriptional activator, encoded by a polynucleotide having sequence SEQ ID NO: 43, the promoter that is activated by the inducible transcriptional activator is TRE3G promoter having sequence SEQ ID NO: 45, and the promoter that is inhibited by the inducible transcriptional activator is pCMV / TO promoter having sequence SEQ ID NO: 46, or variants thereof.

[0132] Advantageously, genetic circuits as described above, can be used to control expression of toxic genes in a cell.

[0133] In particular, the genetic circuits of the invention offer significant advantages in both bioproduction and biomedical settings: in bioproduction, genetic circuits such as CIL-ON and CIL-OFF circuits can be used to engineer producer cells to improve production of biologicals such as antibodies and viral vectors that are toxic for cells, by effectively minimizing leakiness and allowing for inducible expression. These circuits reduce basal expression while maintaining high levels of inducible expression, which is crucial for the safe and efficient production of toxic proteins. This circuit design relies on the coherent inhibitory loop (CIL) topology to achieve tight regulation, thereby reducing unintended protein expression that could be harmful or inefficient. Another application is in the construction of whole cell biosensors, as the CIL-ON and CIL-OFF configurations yield very large fold changes. Hence, simply swapping the promoter regulating the expression of a transcriptional activator from a constitutive promoter to an analyte-sensitive promoter will yield highly sensitive biosensors.

[0134] The systems and genetic circuits of the invention can be advantageously used to regulate expression of a therapeutic gene for gene therapy.

[0135] Furthermore, the systems and circuits of the invention can be used in immune cell engineering for cancer therapies to enable cell activation only when specific conditions are met, such as the presence (or absence) of specific antigens.

[0136] In biotechnology, the systems and circuits of the invention can be used to engineer cell factories for biotechnology applications endowed with high-performance gene expression systems capable of sensing cellular and environmental cues and adapt production of recombinant proteins accordingly.

[0137] The components of the system or genetic circuit of the invention are preferably introduced in a cell by means of one or more expression vectors bearing one or more transcription units for expressing said components. For example, one or more plasmids bearing a transcription unit for expressing MADTF, a transcription unit for expressing a ATF, a transcription unit for expressing a gene of interest, a transcription unit for expressing a transcriptional activator controlling the expression of the MADTF or of the ATF, can be introduced in a cell, thus providing the cell with a genetic circuit according to the invention.

[0138] Therefore, the present invention is also directed to expression vectors comprising the transcription units for expressing the components of the system of the invention, or of the genetic circuits of the invention, and to a cell that incorporates said system, genetic circuit or expression vectors.

[0139] Preferably, the invention is directed to an expression vector comprising a transcription unit for expressing a MADTF monomer comprising a GAL4 DBD, a truncated Gal4 first dimerization domain, a second dimerization domain comprising a N7 peptide and a VP16 effector domain (MAD-GAL4), more preferably said vector having sequence comprising or consisting of SEQ ID NO: 38, or a variant thereof. Preferably, the invention is directed to an expression vector comprising a transcription unit for expressing a MADTF monomer comprising a ERalpha DBD, a truncated Gal4 first dimerization domain, a second dimerization domain comprising a N7 peptide and a VP 16 effector domain (MAD-ERa), more preferably said vector having sequence comprising or consisting of SEQ ID NO: 39, or a variant thereof.

[0140] Preferably, the invention is directed to an expression vector comprising a transcription unit for expressing a ATF comprising one or more N8 dimerization domain, said vector having sequence comprising or consisting of SEQ ID NO: 40 (ATF comprising one N8, ATF-N8), SEQ ID NO: 41 (ATF comprising two N8, ATF-2N8), SEQ ID NO: 42 (ATF comprising four N8, ATF-4N8), or a variant thereof.

[0141] In some embodiments, the invention is directed to an expression vector for expressing the MADTF and the ATF, said expression vector having sequence selected from SEQ ID NO: 57, 58, 59, and variants thereof.

[0142] Preferably, the invention is directed to an expression vector, or to an expression vector system, for expressing the components of a genetic circuits of the invention.

[0143] Preferably the genetic circuit of the invention has a CIL-ON configuration and comprises: an expression vector bearing a polynucleotide for expressing a rtTA having sequence SEQ ID NO: 43, under the control of CMV promoter having sequence SEQ ID NO: 44, more preferably said expression vector having sequence SEQ ID NO: 47;

[0144] an expression vector bearing a polynucleotide for expressing a ATF comprising from 1 to 4 N8 peptides in the second dimerization domain having sequence SEQ ID NO: 40, 41, or 42, more preferably bearing a polynucleotide for expressing a ATF comprising 4 N8 peptides and having sequence SEQ ID NO: 42, under the control of a pCMV / TO promoter having sequence SEQ ID NO: 46, more preferably said expression vector having sequence SEQ ID NO: 48;

[0145] an expression vector bearing a polynucleotide for expressing a MADTF, said MADTF comprising a first dimerization domain being a truncated Gal4, a second dimerization domain comprising a N7 peptide, more preferably said MADTF comprising a GAL4 DBD, a first dimerization domain being a truncated Gal4, a second dimerization domain comprising a N7 peptide and a VP 16 effector domain (MAD-GAL4), under the control of TRE3G promoter, more preferably said expression vector having sequence SEQ ID NO: 49.

[0146] Preferably the genetic circuit of the invention has a CIL-OFF configuration and comprises: an expression vector having sequence SEQ ID NO: 47 for expressing a rtTA under the control of CMV promoter;

[0147] an expression vector having sequence SEQ ID NO: 50 for expressing a ATF having 4 N8 peptides in the second dimerization domain, under the control of a TRE3G promoter; an expression vector having sequence SEQ ID NO: 51 for expressing a MADTF having a Gal4 effector domain, under the control of pCMV / TO promoter.

[0148] The present invention is also directed to a cell comprising the system and / or the genetic circuit of the invention.

[0149] The cell may be a prokaryotic cell (particularly bacterial) or a eukaryotic cell (particularly fungus, plant or animal, more particularly mammalian) cell. Any suitable expression system known in the art may be used for expressing the system or genetic circuit of the invention in a cell of interest. For example, the expression system may comprise one or several DNA vectors, such as plasmids, viruses or artificial chromosomes, known in the art of molecular biology. Preferably the cell is a eukaryotic cell, particularly a mammalian cell, more preferably a human cell.

[0150] The invention is also directed to in vitro, ex vivo or in vivo methods for controlling gene expression in a cell by introducing in the cell the system, genetic circuit of the invention, or the expression vectors thereof, and to the use of said systems, genetic circuits, expression vectors and cells in the claimed applications.

[0151] Such a method can be carried out by any means known in the art.

[0152] Preferably, said system or genetic circuits of the invention, or the expression vectors thereof, are delivered into a cell by means of viral or non-viral vectors, most preferably by AAV vectors, or by lentiviral vectors, being delivered by non-viral vectors, more preferably by lipid- or nonlipid- nanoparticles. Therefore, the invention is also directed to vectors for delivering the system or genetic circuits of the invention, or the expression vectors thereof, in a cell, more preferably said vectors being non-viral vectors, such as lipid or non-lipid nanoparticles, or viral vectors, such as AAV or lentiviral vectors, most preferably AAV vectors.

[0153] Further preferred uses of the systems and genetic circuits of the present invention include their use for controlling expression of genes for gene therapy in a cell, such a gene therapy being preferably delivered to a cell by viral vectors, most preferably by AAV vectors, or by lentiviral vectors, being delivered by non-viral vectors, more preferably by lipid- or non-lipid-nanoparticles.

[0154] Preferably, the system or genetic circuits of the invention are used for the treatment of an inherited disease by gene therapy or gene editing.

[0155] The invention also provides Gene Dosage Compensation (GDC) circuits for gene replacement therapies, that can be engineered to tune the expression of a target gene in a tight expression window and make it independent of virus uptake.

[0156] Further preferred uses of the genetic circuits of the present invention include their use for controlling expression of genes in a method of gene editing.

[0157] Further preferred uses of the genetic circuits of the present invention include their use for controlling expression of genes for cancer therapy.

[0158] In fact, therapeutic applications for the system and genetic circuits of the invention include treating Mendelian disorders where precise gene dosage is essential: MADTFs can provide regulated expression to avoid the detrimental effects of over-expression or under-expression. In autoimmune diseases, MAD-TFs can be used for ex-vivo gene therapy to engineer immune cells to transiently suppress immune responses, reducing the risk of chronic immunosuppression that can lead to infections or malignancies. In cancer gene therapy, MADTF-based logic circuits can be designed to express toxic genes selectively in cancer cells enhancing the specificity and efficacy of treatments while minimizing off-target effects (on target / off tumor toxicity). In CAR-T cell therapy, MADTF based biomolecular circuits can be used to augment CAR-T cells to activate only if specific conditions are met. For example, the Tumor Associated Antigen (TAA) may be expressed also in healthy cells thus increasing toxicity; using the MADTF system it is possible to engineer a CAR-T using synthetic receptors, so that its activation happens only when the TAA is recognized in the absence of a protective antigen expressed only by healthy cells. Similarly, it is possible to engineer Natural Killer (NK) cells with MADTF based-circuits using synthetic receptors, so that its activation happens only when the TAA is recognized in the absence of a protective antigen expressed only by healthy cells.

[0159] In preferred embodiments, a genetic circuit according to the invention is adapted to express a therapeutic gene for the gene therapy. Preferably, the circuit comprises a MADTF preferably engineered with a genome orthogonal ZF-DBD to drive expression of the a therapeutic gene from an artificial cognate promoter.

[0160] In some embodiments, the components of the circuit are cloned into a LTR “transgene” cassette of a lentiviral plasmid to yield lentiviruses encoding the genetic circuit (LV. GDC). In other embodiments, the components of the circuit are cloned into a AAV ITR “transgene” cassette to yield AAV encoding the genetic circuit (AAV. GDC).

[0161] Overall, the modularity, tunability and scalability of MAD-TF systems, combined with their compact size and the ability to humanize their components, make them highly versatile tools in both industrial bioproduction and therapeutic gene regulation. These systems not only provide precise control over gene expression but also align with the safety and efficiency requirements of modern biotechnology and medicine.

[0162] It should be understood that all the possible combinations of the preferred aspects of the present invention are also described, and therefore similarly preferred.

[0163] Examples of preferred embodiments of the present invention and analyses of their efficacy are provided below for illustrative and non-limiting purposes.

[0164] EXAMPLES MA TERI ATS AND METHODS

[0165] Plasmid Construction

[0166] Plasmids of sequences SEQ ID NO: 38-42, 47-55, as detailed hereafter, have been used in the for expressing the systems and circuits employed in the examples that follow.

[0167] Plasmids were constructed using the Gibson assembly method (Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6, 343-345, 2009). The Tet-On®3G system, comprising the TRE3G promoter and the rtTA protein, has been acquired from TakaraBio.

[0168] Cell culture and transfection

[0169] The HEK293T cell line (ATCC) was cultured in DMEM Glutamax (Gibco) supplemented with 10% Tet-Free Fetal Bovine Serum (Euroclone) and 1% Penicillin-Streptomycin (Euroclone). The cell line has been kept at 37°C in a 5% CO2 environment. For luciferase experiment 2xl04(HEK293T) cells per well were seeded in CoStar White 96-well plates (Coming) to perform standard transfection, while 4,5xl04(HEK293T) were seeded when performing reverse transfection. After 18 hours of seedling, for standard transfection, or immediately after seedling, for reverse transfection, cells have been transfected using a home-made solution of PEI (MW 25000, Polysciences, stock concentration 0,324 mg / ml, pH 7.5) using from 200 to 250ng of DNA per well. For HEK293T cells, 5 pl of PEI solution for each pg of DNA were used.

[0170] Luciferase assay

[0171] To normalize reporter values to transfection efficiency, lOng of pRL-TK (encoding for Renilla Luciferase) have been used for each experiment. The cells were collected 48 hours after transfection and lysed with 5X Passive Lysis Buffer (Biotin) diluted in water. Firefly Luciferase and Renilla Luciferase expression were measured using the Dual Luciferase AssayPromega) on a Glomax Explorer plate reader (Promega). Firefly Luciferase Arbitrary Units (Luciferase [A. U.]) were calculated by normalizing each sample’s Firefly Luciferase activity to the constitutive Renilla activity detected in the same sample.

[0172] The Fold Activation has been obtained by dividing the average of each datapoint of Luciferase [A. U.] by the average of Luciferase [A. U.] in the absence of copper.

[0173] EXAMPLE 1

[0174] The feasibility of the MADTF / DTF technology has been demonstrated as shown in Figure 2. A MADTF artificial transcription factor, based on GAL4-VP16 transcriptional activator (Sadowski, I., Ma, J., Triezenberg, S. & Ptashne, M. GAL4-VP16 is an unusually potent transcriptional activator. Nature 335, 563-564, 1988), was provided, as shown in Figure 2a, b. The yeast GAL4 is a homodimeric TF and the presence of the VP 16 transactivation domain makes it a potent transcriptional activator of gene expression in mammalian cells from the canonical pUAS promoter. To engineer the MAD-GAL4, as in Figure 2c, the affinity of its homodimerization domain (DD) was first decreased by removing two out of the three alphahelices. Then a synthetic orthogonal heterodimerization domain (SOD) was added, consisting of the synthetic N7 coiled-coil peptide, and a Nuclear Localisation Signal upstream of the VP 16. Then an inhibitory protein was engineered in accordance with the present invention Different versions of the ATF were designed as shown in Figure 2d, consisting of the same DD domain as in the MAD-GAL4 but with different numbers of the N8 synthetic coiled-coil peptide (SOD*) which specifically heterodimerizes with N7. The MAD-GAL4 is only 207 aa long while the ATFs ranges from 80 aa for one N8 to less than 200 aa for four N8s, thus making them compact in term of size and compatible with viral delivery such as Adeno Associated Vectors (AAVs). Results in Figure 2e show the luminescence levels of the firefly Luciferase expressed from the pUAS promoter when co-transfecting HEK293T cells with a fixed amount of MAD-GAL4 plasmid (50ng) and increasing amounts of ATF with a different number of N8 repetitions. A dominant negative ATF without SOD* but consisting of the DD domain only is used as a negative control. As depicted in Figure 2e, these results demonstrate a strong inhibition of MAD-GAL4 proportional to the number of the coiled-coils N8 domains present in the inhibitor protein, with ATF-4N8 exhibiting the highest inhibitory potency, and achieving nearly a fiftyfold reduction in luciferase expression when transfected in equimolar concentration with the plasmid encoding for the MAD-GAL4.

[0175] EXAMPLE 2

[0176] To prove the modular nature of the MAD-TF, the DNA binding domain (DBD) of GAL4 was replaced with that of the human transcription factor ERa. Notably, ERa functions exclusively as a homodimer. We evaluated the functionality of the newly engineered MAD-TF by switching the GAL4 DBD with the ERa DBD to obtain the MAD-ERa, as shown in Figure 3 a. By means of a firefly luciferase under the control of an ERa -responsive promoter (3x ERE flue), we assessed the functionality of the MAD-ERa and the effectiveness of the inhibitor ATF 4N8 SOD*, which acts as a universal inhibitor regardless of the DBD present. As illustrated in Figure 3b, ATF 4N8 SOD* effectively suppressed the activity of MAD- ERa.

[0177] EXAMPLE 3

[0178] MADTFs and DTFs according to the invention have been used to engineer two biomolecular circuits based on the Coherent Inhibitory Loop (CIL) topology: the CIL-ON biomolecular circuit is shown on Figure 4a. Its function is to augment the state-of-the-art doxycycline-inducible gene expression system based on the rtTA transactivator by lowering the basal “leaky” expression in the absence of doxycycline without compromising the maximal achievable expression in the presence of doxycycline: the doxycycline-sensitive reverse tetracycline transactivator (rtTA) is driven by the constitutive promoter pCMV, the MAD-GAL4 is expressed by the pTRE3G promoter upon binding of rtTA in the presence of doxycycline, and the ATF-4N8 is placed downstream of the pCMV / TO promoter, which in the presence of doxycycline can be repressed by rtTA thanks to Tet-responsive-elements flanking the TATA box. A reporter plasmid encoding the pUAS promoter upstream of the firefly luciferase (fLuc) is used to monitor the circuit behavior at different concentrations of Doxycycline. To compare the performance of the CIL-ON, a circuit was also built, wherein the ATF-4N8 was replaced with a GFP, which acts as a negative control, as shown in Figure 4a.

[0179] As shown in Figure 4b, in the CIL-ON system, in the absence of doxycycline, MAD-GAL4 is expressed at a basal level because of the pTRE3G leaky expression, however ATF-4N8 is fully expressed thus blocking MAD-GAL4 and resulting in a strong repression of fLuc expression compared to that of the state-of-the-art system (Control CIL). At a saturating concentration of doxycycline (1000ug / mL), the CIL-ON system maximally expresses the fLuc reporter, as in this condition the rtTA drives expression of MAD-GAL4 from the pTRE3G promoter while reducing expression of the ATF-4N8 from the CMV / TO promoter; moreover, the high-expression of MAD-GAL4 effectively sponges out residual expression of ATF-4N8, thus maintaining the maximal expression of the fLuc reporter at a comparable level to that of the state-of-the-art system. Thanks to the combination of low leakiness and high maximal expression, the CIL-ON circuit confers very high fold-change in terms of response to doxycycline administration, as shown in Figure 4c, with more than one-log increase when compared to the state-of-the-art system (control CIL).

[0180] The second circuit implemented is the CIL OFF system, as shown in Figure 4d. The CIL OFF was obtained by swapping the positions of the MAD-GAL4 and the ATF-4N8, so that the pCMV / TO promoter now drives expression of MAD-GAL4, while the pTRE3G promoter drives expression of ATF-4N8. To compare the performance of the CIL-OFF, a circuit was also built wherein the ATF-4N8 is replaced with a GFP, which acts as a negative control, as shown in Figure 4d.

[0181] The CIL OFF exhibits dual properties to the of the CIL ON, and effectively acts as an “inverter”. Specifically, in the absence of doxycycline, MAD-GAL4 is maximally expressed, whereas ATF-4N8 is expressed only at a basal “leaky” level, so that luciferase is fully expressed at a comparable level to that of the negative control circuit lacking ATF-4N8, as shown in Figure 4e. Upon administration of doxycycline, rtTA inhibits MAD-GAL4 expression by binding to the PCMV / TO promoter, while inducing ATF-4N8 expression by binding to the TRE3G promoter. This results in a double inhibition of MAD-Gal4, both transcriptional and by protein sequestration, leading to an almost complete inhibition of luciferase expression unlike the negative control lacking the ATF-4N8 (CIL OFF control). This property results in more than one-fold change difference in repression of the CIL OFF compared to the control, as reported in Figure 4f.

[0182] EXAMPLE 4 To further demonstrate the compatibility of the system of the invention with different DNA-binding domains (DBDs), two engineered Zinc Finger (ZF) proteins were employed as MAD-TF in respective experiments; specifically each of homodimeric ZF transcription factors BCRZFR39A and ErbB2ZFR2AR39A (herein MAD-ZFA and MAD-ZFC, respectively), developed by Zhu et al. (Science. 2022 Jan 21;375(6578):eabg9765), and available from Addgene, USA, were used to drive the expression of a luciferase reporter gene by ZF-mediated promoter binding.

[0183] As shown in Figure 9, the MAD-TFs drove expression of the luciferase reporter gene while the corresponding ATF effectively inhibited the reporter’s expression. Inhibition occurred via binding of ATF to a single MAD-TF (MAD-ZF in this case) monomer, disrupting homodimer formation and preventing MAD-TF activity.

[0184] EXAMPLE 5

[0185] To further demonstrate the modularity of the system of the invention, the VP 16 activation domain in MAD-GAL4 of Example 1 was replaced with the p65 activation domain derived from the human NF-KB transcription factor. Substitution of VP16 with the human-derived p65 domain was designed to preserve transcriptional activity while reducing immunogenic risk in therapeutic applications in humans.

[0186] As shown in Figure 10, the MAD-TF containing the p65 activation domain effectively activated reporter gene expression and exhibited inhibition by ATF comparable to that of original MAD-GAL4 construct. These results confirm that the system of the invention maintains its functionality independently from the specific activation domain and that p65 effector domain provides a viable option for applications requiring reduced immunogenicity.

[0187] EXAMPLE 6

[0188] The circuits of the present invention can be used to treat Mendelian disorders where precise gene dosage is essential, providing regulated expression that prevents harmful effects from either overexpression or underexpression.

[0189] As a proof of concept, a Gene Dosage Compensation (GDC) circuit was designed in which MAD-TF drives the expression of both a therapeutic gene and of its inhibitory ATF. This configuration creates a self-regulating feedback loop, where MAD-TF activity is fine-tuned through competitive dimerization, keeping expression levels within a safe and effective range. To evaluate GDC performance, firefly luciferase was used as a reporter for the therapeutic gene. Various ATFs with different inhibitory strengths were tested to modulate the loop (SEQ ID NO: 57-59). For comparison, a constitutive pCMV-fluc construct served as a standard gene therapy model, and a GDC “open” configuration expressing the gene of interest under MAD-TF without the inhibitory component was also used.

[0190] As illustrated in Figure 11, adjusting the inhibitory strength of ATFs enables tight control of gene expression.

[0191] Luciferase expression (proxy for the therapeutic gene) is maintained within a narrow therapeutic range. Different ATF variants modulate loop strength, enabling precise control of gene expression.

[0192] The GDC circuit supports enhanced yet regulated expression, maintaining the gene product below the toxicity threshold and within a narrow, stable range. Since the GDC circuit amplifies the expression of the therapeutic gene, thanks to the presence of the transcription factor (MADTF), this system allows for lower viral dosages as compared with standard constructs used in gene replacement therapy, where the therapeutic gene is driven by a strong constitutive promoter. Simultaneously, the inhibitory ATF prevents excessive expression in those cells transduced by higher viral copy numbers (i.e. those close to the site of injection), ensuring safety. Thus, the GDC design achieves two key goals: it boosts transgene expression efficiency while preventing toxic overexpression.

[0193] In conclusion, the modularity of the system of the invention enables tuning of gene expression by selecting transcription factors with varying inhibitory strengths, providing a flexible strategy for adapting the circuit to different therapeutic contexts through distinct Gene Dosage Compensation architectures. Moreover, the system is sufficiently compact to be packaged within a single AAV vector, supporting efficient in vivo delivery.

[0194] Sequences disclosed in conjunction with the present invention are enclosed and displayed hereafter.

[0195] SEQUENCES SEQ ID NO: 1 Gal4 DD actagggcacatctgacagaagtggaatcaaggctagaaagactggaacagctatttctactgatttttcctcga gaagaccttgacatgattttgaaaatggattctttacaggatataaaagcattgttaacaggattatttgtacaa gataatgtgaataaagat

[0196] SEQ ID NO: 2 Gal4 truncated DD actagggcacatctgacagaagtggaatcaaggctagaaagactggaacagctatttctactgattttt SEQ ID NO: 3 GCN4 DD cagcgcatgaagcagctggaggacaaggtggaggagctgctgagcaagaactaccacctggagaacgaggtggcc cgcctgaagaagctggtgggcgacgccgcccgc SEQ ID NO: 4 GCN3 DD gagatcgccgccctgaagcaggagatcgccgccctgaagaaggagaacgccgccctgaagttcgagatcgccgcc ctgaagcag

[0197] SEQ ID NO: 5 N7 SOD atgggggaaattgctgctctggaagccaaaaatgcggcgttgaaagccgagattgcggccttggaagctaagatc gctgctttaaaggccggatactaa

[0198] SEQ ID NO: 6 N8 SOD atgttaaaggcggagaacgcagctctggaagccaagattgcagccttaaaagcggagattgctgcgttagaggca ggctactaa

[0199] SEQ ID NO: 7 2N8 SOD (with linker) ggcggctctggcggcggctccggaggctcttacgggaaaatcgcggcattaaaggcggagaacgcagctctggaa gccaagattgcagccttaaaagcggagattgctgcgttagaggcaggctacggcggctctggcggcggctccgga ggctcttacgggaaaatcgcggcattaaaggcggagaacgcagctctggaagccaagattgcagccttaaaagcg gagattgctgcgttagaggcaggctac

[0200] SEQ ID NO: 8 4N8 SOD (with linkers) ggcggctctggcggcggctccggaggctcttacgggaaaatcgcggcattaaaggcggagaacgcagctctggaa gccaagattgcagccttaaaagcggagattgctgcgttagaggcaggctacggcggctctggcggcggctccgga ggctcttacgggaaaatcgcggcattaaaggcggagaacgcagctctggaagccaagattgcagccttaaaagcg gagattgctgcgttagaggcaggctacggcggctctggcggcggctccggaggctcttacgggaaaatcgcggca ttaaaggcggagaacgcagctctggaagccaagattgcagccttaaaagcggagattgctgcgttagaggcaggc tacggcggctctggcggcggctccggaggctcttacgggaaaatcgcggcattaaaggcggagaacgcagctctg gaagccaagattgcagccttaaaagcggagattgctgcgttagaggcaggctac

[0201] SEQ ID NO: 9 Pl SOD

[0202] SPEDEIQALEEENAQLEQENAALEEEIAQLEYG SEQ ID NO: 10 P2 SOD

[0203] SPEDKIAQLKEKNAALKEKNQQLKEKIQALKYG SEQ ID NO: 11 P3 SOD

[0204] SPEDEIQQLEEEIAQLEQKNAALKEKNQALKYG SEQ ID NO: 12 P4 SOD

[0205] SPEDKIAQLKQKIQALKQENQQLEEENAALEYG SEQ ID NO: 13 P5 SOD

[0206] SPEDENAALEEKIAQLKQKNAALKEEIQALEYG SEQ ID NO: 14 P6 SOD

[0207] SPEDKNAALKEEIQALEEENQALEEKIAQLKYG SEQ ID NO: 15 P7 SOD

[0208] SPEDEIQALEEKNAQLKQEIAALEEKNQALKYG SEQ ID NO: 16 P8 SOD

[0209] SPEDKIAQLKEENQQLEQKIQALKEENAALEYG SEQ ID NO: 17 P9 SOD

[0210] SPEDENQALEQKNAQLKQEIAALEQEIAQLEYG SEQ ID NO: 18 PIO SOD

[0211] SPEDKNAQLKEENAALEEKIQQLKEKIQALKYG SEQ ID NO: 19 P11 SOD

[0212] SPEDENQALEQEIAQLEQEIAALEQKNAQLKYG SEQ ID NO: 20 P12 SOD

[0213] SPEDKNAQLKEKIAALKEKIQQLKEENQALEYG SEQ ID NO: 21 N5 SOD

[0214] YEIAALEAKIAALKAKNAALKAEIAALEAGC SEQ ID NO: 22 N6 SOD

[0215] YKIAALKAEIAALEAENAALEAKIAALKAGC SEQ ID NO: 23 P5A SOD

[0216] YGENAALEAKIAALKAKNAALKAEIAALEAGC SEQ ID NO: 24 P6A SOD

[0217] YGKNAALKAEIAALEAENAALEAKIAALKAGGC SEQ ID NO: 25 P7A SOD

[0218] YGEIAALEAKNAALKAEIAALEAKNAALKAGC SEQ ID NO: 26 P8A SOD

[0219] YGKIAALKAENAALEAKIAALKAENAALEAGGC SEQ ID NO: 27 SYNZIP1 SOD NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEE SEQ ID NO: 28 SYNZIP2 SOD ARNAYLRKKIARLKKDNLQLERDEQNLEKIIANLRDEIARLENEVASHEQ SEQ ID NO: 29 SYNZIP3 SOD

[0220] NEVTTLENDAAFIENENAYLEKEIARLRKEKAALRNRLAHKK SEQ ID NO: 30 SYNZIP4 SOD

[0221] Q KVAE L KN RVAVKLN RN EQ L KN KVE E L KN RNAYL KN E LAT L EN E VARL EN D VAE SEQ ID NO: 31 SYNZIP5 SOD

[0222] NTVKELKNYIQELEERNAELKNLKEHLKFAKAELEFELAAHKFE SEQ ID NO: 32 SYNZIP6 SOD QKVAQLKNRVAYKLKENAKLENIVARLENDNANLEKDIANLEKDIANLERDVAR SEQ ID NO: 33 VP16ED gcacccccgaccgatgtcagcctgggggacgagctccacttagacggcgaggacgtggcgatggcgcatgccgac gcgctagacgatttcgatctggacatgttgggggacggggattccccgggtccgggatttaccccccacgactcc gccccctacggcgctctggatatggccgacttcgagtttgagcagatgtttaccgatgcccttggaattgacgag tacggtggg

[0223] SEQ ID NO: 34 VP64 ED gacgcattggacgattttgatctggatatgctgggaagtgacgccctcgatgattttgaccttgacatgcttggt tcggatgcccttgatgactttgacctcgacatgctcggcagtgacgcccttgatgatttcgacctggacatgctg SEQ ID NO: 35 VPRED gacgcattggacgattttgatctggatatgctgggaagtgacgccctcgatgattttgaccttgacatgcttggt tcggatgcccttgatgactttgacctcgacatgctcggcagtgacgcccttgatgatttcgacctggacatgctg attaactctagaagttccggatctccgaaaaagaaacgcaaagttggtagccagtacctgcccgacaccgacgac cggcaccggatcgaggaaaagcggaagcggacctacgagacattcaagagcatcatgaagaagtcccccttcagc ggccccaccgaccctaggcctccacctagaagaatcgccgtgcccagcagatccagcgccagcgtgccaaaacct gccccccagccttaccccttcaccagcagcctgagcaccatcaactacgacgagttccctaccatggtgttcccc agcggccagatctctcaggcctctgctctggccccagcccctcctcaggtgctgcctcaggctcctgctcctgca ccagctccagccatggtgtctgcactggctcaggcaccagcacccgtgcctgtgctggctcctggacctccacag gctgtggctccaccagcccctaaacctacacaggccggcgagggcacactgtctgaagctctgctgcagctgcag ttcgacgacgaggatctgggagccctgctgggaaacagcaccgatcctgccgtgttcaccgacctggccagcgtg gacaacagcgagttccagcagctgctgaaccagggcatccctgtggcccctcacaccaccgagcccatgctgatg gaataccccgaggccatcacccggctcgtgacaggcgctcagaggcctcctgatccagctcctgcccctctggga gcaccaggcctgcctaatggactgctgtctggcgacgaggacttcagctctatcgccgatatggatttctcagcc ttgctgggctctggcagcggcagccgggattccagggaagggatgtttttgccgaagcctgaggccggctccgct attagtgacgtgtttgagggccgcgaggtgtgccagccaaaacgaatccggccatttcatcctccaggaagtcca tgggccaaccgcccactccccgccagcctcgcaccaacaccaaccggtccagtacatgagccagtcgggtcactg accccggcaccagtccctcagccactggatccagcgcccgcagtgactcccgaggccagtcacctgttggaggat cccgatgaagaaacgagccaggctgtcaaagccctt egggaga tggccgat a ctgtgattccccagaaggaagag gctgcaatctgtggccaaatggacctttcccatccgcccccaaggggccatctggatgagctgacaaccacactt gagtccatgaccgaggatctgaacctggactcacccctgaccccggaattgaacgagattctggataccttcctg aacgacgagtgcctcttgcatgccatgcatatcagcacaggactgtccatcttcgacacatctctgttt SEQ ID NO: 36 p65 ED

[0224] cccacccaggctggggaaggaa eget gtcagaggccct get gcagctgcagtttgatgatgaagacctgggggcc ttgcttggcaacagcacagacccagctgtgttcacagacctggcatccgtcgacaactccgagtttcagcagctg ctgaaccagggcatacctgtggccccccacacaactgagcccatgctgatggagtaccctgaggctataactcgc ctagtgacaggggcccagaggccccccgacccagctcctgctccactgggggccccggggctccccaatggcctc ctttcaggagatgaagacttctcctccattgcggacatggacttctcagccctgctgagtcagatcagctcc SEQ ID NO: 37 GAL4 DBD aagctactgtcttctatcgaacaagcatgcgatatttgccgacttaaaaagctcaagtgctccaaagaaaaaccg aagtgcgccaagtgtctgaagaacaactgggagtgtcgctactctcccaaaaccaaaaggtctccgctg SEQ ID NO: 38 MAD-GAL4 tgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcgacggatcg ggagatctcccgatcccctatggtcgactctcagtacaatctgctctgatgccgcatagttaagccagtatctgc tccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccg acaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgtt gacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttcc gcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatga cgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggactatttacggtaaactgccc acttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcct ggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctatta ccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctcc accccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccg ccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagag aacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagcttggtaccgagctcgga tccactagtaacggccgccagtgtgctggaattcatgaagctactgtcttctatcgaacaagcatgcgatatttg ccgacttaaaaagctcaagtgctccaaagaaaaaccgaagtgcgccaagtgtctgaagaacaactgggagtgtcg ctactctcccaaaaccaaaaggtctccgctgactagggcacatctgacagaagtggaatcaaggctagaaagact ggaacagctatttctactgatttttggcggctctggcggcggctccggaggctctggggaaattgctgctctgga agccaaaaatgcggcgttgaaagccgagattgcggccttggaagctaagatcgctgctttaaaggccggatacga tccaaaaaagaagagaaaggtagcacccccgaccgatgtcagcctgggggacgagctccacttagacggcgagga cgtggcgatggcgcatgccgacgcgctagacgatttcgatctggacatgttgggggacggggattccccgggtcc gggatttaccccccacgactccgccccctacggcgctctggatatggccgacttcgagtttgagcagatgtttac cgatgcccttggaattgacgagtacggtggggacacctaccgctacatctaatctagagggccctattctatagt gtcacctaaat get agaget eget gatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccct cccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgc attgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagaca atagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggt atccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttg ccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaag ctctaaatcggggcatccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagg gtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttcttta atagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattt tggggatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgt gtgtcagttagggtgtggaaagtccccaggctccccaggcaggcagaagtatgcaaagcatgcatctcaattagt cagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcag caaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatg gctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggag gcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcaagaga caggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggc tattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggc gcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgt ggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctat tgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatg caatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgag cacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccg aactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgc cgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatc aggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgcttt acggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactct ggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatga aaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagtt cttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaa taaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtatacc gtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaat tccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaat tgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgc ggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggct gcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaa catgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccg cccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagatacca ggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctt tctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctc caagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtc caacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtagg cggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctct gctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtgg tttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggg gtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcaccta gatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttacca atgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgt gtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcacc ggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgc ctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgt tgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatc aaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaag taagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaag atgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttg cccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttc ggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatc ttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaat aagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattg tctcatgagcggatacatatttgaa

[0225] SEQ ID NO: 39 MAD-ERalpha ctcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatt tagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcgacggatcgggagat ctcccgatcccctatggtcgactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctg cttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaatt gcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacatt gattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgtta cataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatg ttcccatagtaacgccaatagggactttccattgacgtcaatgggtggactatttacggtaaactgcccacttgg cagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcatt atgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatgg tgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccacccca ttgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccat tgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaaccca ctgcttactggcttatcgaaattaatacgactcactatagggagacccaagcttggtaccgagctcggatccact agtaacggccgccagtgtgctggaattcatgaaggagactcgctactgtgcagtgtgcaatgactatgcttcagg ctaccattatggagtctggtcctgtgagggctgcaaggccttcttcaagagaagtattcaaggacataacgacta tatgtgtccagccaccaaccagtgcaccattgataaaaacaggaggaagagctgccaggcctgccggctccgcaa atgctacgaagtgggaatgatgaaaggtgggatacgaaaagaccgaagaggatgggagtgtcgctactctcccaa aaccaaaaggtctccgctgactagggcacatctgacagaagtggaatcaaggctagaaagactggaacagctatt tctactgatttttggcggctctggcggcggctccggaggctctggggaaattgctgctctggaagccaaaaatgc ggcgttgaaagccgagattgcggccttggaagctaagatcgctgctttaaaggccggatacgatccaaaaaagaa gagaaaggtagcacccccgaccgatgtcagcctgggggacgagctccacttagacggcgaggacgtggcgatggc gcatgccgacgcgctagacgatttcgatctggacatgttgggggacggggattccccgggtccgggatttacccc ccacgactccgccccctacggcgctctggatatggccgacttcgagtttgagcagatgtttaccgatgcccttgg aattgacgagtacggtggggacacctaccgctacatctaatctagagggccctattctatagtgtcacctaaatg ctagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgcctt ccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagta ggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatg ctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgc cctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctag cgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcggg gcatccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcac gtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactct tgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttggggatttcgg cctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagg gtgtggaaagtccccaggctccccaggcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggt gtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcc cgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattt tttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggag gcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcaagagacaggatgaggat cgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatg actgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttcttt ttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacga cgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgc cggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggc tgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgga tggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgcca ggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatgg tggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgt tggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccg ctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaat gaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggctt cggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccc caacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcattttt ttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctag ctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacat acgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctc actgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcgg tttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggt atcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaa aggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacga gcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccc tggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcggg aagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctg tgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaag acacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacaga gttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagt taccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttg caagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctca gtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaa ttaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcag tgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactac gatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagattt atcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtc tattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctac aggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttac atgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgc agtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgt gactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaat acgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaact ctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttt tactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacg gaaatgttgaatactcata

[0226] SEQ ID NO: 40 DTF N8 SOD ccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatc gacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcg tgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgcttt ctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaacccc ccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgc cactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggt ggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaa gagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagatta cgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaact cacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagtt ttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatct cagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggct taccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaacc agccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgcc gggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgt cacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgt tgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactca tggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtact caaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccg cgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttac cgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcg tttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatac tcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaat gtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcgacggatcgg gagatctcccgatcccctatggtcgactctcagtacaatctgctctgatgccgcatagttaagccagtatctgct ccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccga caattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttg acattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccg cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgac gtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggactatttacggtaaactgccca cttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctg gcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattac catggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctcca ccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgc cccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagaga acccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagcttggtaccgagctcggat ccactagtaacggccgccagtgtgctggaattCGCCACcatgactagggcacatctgacagaagtggaatcaagg ctagaaagactggaacagctatttctactgatttttggcggctctggcggcggctccggaggctcttacgggaaa atcgcggcattaaaggcggagaacgcagctctggaagccaagattgcagccttaaaagcggagattgctgcgtta gaggcaggctacgatccaaaaaagaagagaaaggtagacacctaccgctacatctaatctagagggccctattct atagtgtcacctaaatgctagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttg cccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgc atcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattggga agacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctag ggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctac acttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccg tcaagctctaaatcggggcatccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttga ttagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgtt ctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagg gattttggggatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtgg aatgtgtgtcagttagggtgtggaaagtccccaggctccccaggcaggcagaagtatgcaaagcatgcatctcaa ttagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaatta gtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgcc ccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtg aggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatca agagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtgga gaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgca ggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggct atcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggct gctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggc tgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcga gcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgcc agccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctg cttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccg ctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgt gctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcggg actctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttc tatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctg gagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttc acaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgt ataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctc acaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcaca ttaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaa cgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgtt cggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcagga aagaacatgtgagcaaaaggccagcaaaagg

[0227] SEQ ID NO: 41 DTF 2N8 SOD gcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatca caaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaag ctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgt ggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgca cgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacga cttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttctt gaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttacctt cggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagca gcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaa cgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaa atgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggc acctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacg ggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagc aataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaa ttgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcat cgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatc ccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgtt atcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactgg tgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacggga taataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaag gatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttacttt caccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatg ttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacat atttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcga cggatcgggagatctcccgatcccctatggtcgactctcagtacaatctgctctgatgccgcatagttaagccag tatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggc ttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatat acgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatg gagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtca ataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggactatttacggtaa actgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatgg cccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatc gctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttcca agtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaac aactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggcta actagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagcttggtaccga gctcggatccactagtaacagccgccagtgtgctggaattCGCCACcatgactagggcacatctgacagaagtgg aatcaaggctagaaagactggaacagctatttctactgatttttggcggctctggcggcggctccggaggctctt acgggaaaatcgcggcattaaaggcggagaacgcagctctggaagccaagattgcagccttaaaagcggagattg ctgcgttagaggcaggctacggcggctctggcggcggctccggaggctcttacgggaaaatcgcggcattaaagg cggagaacgcagctctggaagccaagattgcagccttaaaagcggagattgctgcgttagaggcaggctacgatc caaaaaagaagagaaaggtagacacctaccgctacatctaatctagagggccctattctatagtgtcacctaaat gctagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgcct tccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagt aggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcat gctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcg ccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgcccta gcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgg ggcatccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttca cgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactc ttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttggggatttcg gcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttag ggtgtggaaagtccccaggctccccaggcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccagg tgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtc ccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaatt ttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttgga ggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcaagagacaggatgagga tcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctat gactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctt tttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacg acgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtg ccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcgg ctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcgg atggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgcc aggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatg gtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcg ttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgcc gctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaa tgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggct tcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccacc ccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttt tttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctcta gctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaaca tacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgct cactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcg gtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcgg tatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaa aaggcca

[0228] SEQ ID NO: 42 DTF 4N8 SOD ttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggt cattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgccca acgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtc aatgggtggactatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgcccccta ttgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggc agtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagc ggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaac gggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtct atataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactata gggagacccaagcttggtaccgagctcggatccactagtaacagccgccagtgtgctggaattCGCCACcatgac tagggcacatctgacagaagtggaatcaaggctagaaagactggaacagctatttctactgatttttggcggctc tggcggcggctccggaggctcttacgggaaaatcgcggcattaaaggcggagaacgcagctctggaagccaagat tgcagccttaaaagcggagattgctgcgttagaggcaggctacggcggctctggcggcggctccggaggctctta cgggaaaatcgcggcattaaaggcggagaacgcagctctggaagccaagattgcagccttaaaagcggagattgc tgcgttagaggcaggctacggcggctctggcggcggctccggaggctcttacgggaaaatcgcggcattaaaggc ggagaacgcagctctggaagccaagattgcagccttaaaagcggagattgctgcgttagaggcaggctacggcgg ctctggcggcggctccggaggctcttacgggaaaatcgcggcattaaaggcggagaacgcagctctggaagccaa gattgcagccttaaaagcggagattgctgcgttagaggcaggctacgatccaaaaaagaagagaaaggtagacac ctaccgctacatctaatctagagggccctattctatagtgtcacctaaatgctagagctcgctgatcagcctcga ctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactc ccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtg gggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgg cttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcgg cgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcc cttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcggggcatccctttagggttccgattta gtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgataga cggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactca accctatctcggtctattcttttgatttataagggattttggggatttcggcctattggttaaaaaatgagctga tttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctcccc aggcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccag caggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgc ccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgagg ccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcc cgggagcttgtatatccattttcggatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatg gattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggct gctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtg ccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgc tcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctc accttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacct gcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcagg atgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacg gcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggat tcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaag agcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgcct tctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaatgaccgaccaagcgacgcccaacct gccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccgg ctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataa tggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggttt gtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtc atagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaa agcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaa cctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgc ttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaat acggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccg taaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaag tcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcc tgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctc acgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcc cgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagc agccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaacta cggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtag ctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaa aaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagg gattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaat ctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctg tctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctgg ccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccgg aagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctag agtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtc gtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaa agcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggc agcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtc attctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatag cagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgag atccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtg agcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactctt cctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaa aaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcgacggatcgggagatctccc gatcccctatggtcgactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgt gtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatg aagaatctgcttagggttaggcgttttgcgctgc

[0229] SEQ ID NO: 43 rtTA3g atgggcagcatgtctagactggacaagagcaaagtcataaactctgctctggaattactcaatggagtcggtatc gaaggcctgacgacaaggaaactcgctcaaaagctgggagttgagcagcctaccctgtactggcacgtgaagaac aagcgggccctgctcgatgccctgccaatcgagatgctggacaggcatcatacccactcctgccccctggaaggc gagtcatggcaagactttctgcggaacaacgccaagtcataccgctgtgctctcctctcacatcgcgacggggct aaagtgcatctcggcacccgcccaacagagaaacagtacgaaaccctggaaaatcagctcgcgttcctgtgtcag caaggcttctccctggagaacgcactgtacgctctgtccgccgtgggccactttacactgggctgcgtattggag gaacaggagcatcaagtagcaaaagaggaaagagagacacctaccaccgattctatgcccccacttctgaaacaa gcaattgagctgttcgaccggcagggagccgaacctgccttccttttcggcctggaactaatcatatgtggcctg gagaaacagctaaagtgcgaaagcggcgggccgaccgacgcccttgacgattttgacttagacatgctcccagcc gatgcccttgacgactttgaccttgatatgctgcctgctgacgctcttgacgattttgaccttgacatgctcccc gggtcaggctaa

[0230] SEQ ID NO: 44 pCMV Cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgac gtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgccca cttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctg gcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattac catggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctcca ccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgc cccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagct

[0231] SEQ ID NO: 45 pTRE3g gagtttactccctatcagtgatagagaacgtatgaagagtttactccctatcagtgatagagaacgtatgcagac tttactccctatcagtgatagagaacgtataaggagtttactccctatcagtgatagagaacgtatgaccagttt actccctatcagtgatagagaacgtatctacagtttactccctatcagtgatagagaacgtatatccagtttact ccctatcagtgatagagaacgtataagctttaggcgtgtacggtgggcgcctataaaagcagagctcgtttagtg aaccgtcagatcgcctggagcaattccacaacacttttgtcttataccaactttccgtaccacttcctaccctcg taaa

[0232] SEQ ID NO: 46 pCMV / TO cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgac gtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgccca cttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctg gcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattac catggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctcca ccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgc cccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctccctatcagtgatag agatctccctatcagtgatagaga

[0233] SEQ ID NO: 47 pCMV-rtTA3G acgcagaaaggcccacccgaaggtgagccaggtgattacatttgggccctcattaccaatgcttaatcagtgagg cacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatgc gggagggcttaccatctggccccagtgctgcaatgataccgcgagaaccacgctcaccggctccagatttatcag caataaaccagccagccgggagggccgagcgcagaagtgatcctgcaactttatccgcctccatccagtctatta attgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggca tcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgat cccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgccagaagtaagttggccgcagtgt tatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtgagatgcttttctgtgactg gtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacggg ataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgtaaactctcaa ggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactt tcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaat gttgaatactcattttagcttccttagctcctgaaaatctcgataactcaaaaaatacgcccggtagtgatctta tttcattatggtgaaagttggaacctcttacgtgccgatcaagtcaaaagcctccggtcggaggcttttgacttt ctgctatggaggtcaggtatgatttaaatggtcagtattgagcgatatctagagaattcgtataggtcttgtatt cagtctcgcactttagacatggtcttctgctgcccgtggtcctctattcgtcggatgttctgtcgattcttcctg tctatctggactcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgac gtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacg gtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaa atggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagt catcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatt tccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcg taacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctggact tagcctttagtgaaccgtcagaattaattcagatcgatctaccagaaccgtcagatccgctagagattacgccaa ccgccaccatgggcagcatgtctagactggacaagagcaaagtcataaactctgctctggaattactcaatggag tcggtatcgaaggcctgacgacaaggaaactcgctcaaaagctgggagttgagcagcctaccctgtactggcacg tgaagaacaagcgggccctgctcgatgccctgccaatcgagatgctggacaggcatcatacccactcctgccccc tggaaggcgagtcatggcaagactttctgcggaacaacgccaagtcataccgctgtgctctcctctcacatcgcg acggggctaaagtgcatctcggcacccgcccaacagagaaacagtacgaaaccctggaaaatcagctcgcgttcc tgtgtcagcaaggcttctccctggagaacgcactgtacgctctgtccgccgtgggccactttacactgggctgcg tattggaggaacaggagcatcaagtagcaaaagaggaaagagagacacctaccaccgattctatgcccccacttc tgaaacaagcaattgagctgttcgaccggcagggagccgaacctgccttccttttcggcctggaactaatcatat gtggcctggagaaacagctaaagtgcgaaagcggcgggccgaccgacgcccttgacgattttgacttagacatgc tcccagccgatgcccttgacgactttgaccttgatatgctgcctgctgacgctcttgacgattttgaccttgaca tgctccccgggtcaggctaataacagcttccggactctagaacatccctacaggtgatatcctcgggtaacttgt ttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgc attctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtcgtctatgatgaggatgttggtgga gagcat gtggaggaagtggatagggaaggttgtagagtagatccggttgaagtgatgaggataggaggaggactg cacatattgatcctcgtcacaaccacaccagcacacctcaaatcccacaccactcccacaattaccattcactca acaaactcacacatcccacgataacgaattcaagcttgatatcattcaggacgagcctcagactccagcgtaact ggactgcaatcaactcactggctcaccttcacgggtgggcctttcttcggtagaaaatcaaaggatcttcttgag atcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccgg atcaagagctaccaactctttttccgaggtaactggcttcagcagagcgcagataccaaatactgttcttctagt gtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttacc agtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgca gcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacct acagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggt cggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgcca cctctgacttgagcatcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagca SEQ ID NO: 48 pCMV / TO-DTF-4N8 aggtcaggtatgatttaaatggtcagtattgagcgatatctagagaattcgtataggtcttgtattcagtctcgc actttagacatggtcttctgctgcccgtggtcctctattcgtcggatgttctgtcgattcttcctgtctatctgg actcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataat gacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgc ccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgc ctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctat taccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtct ccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactc cgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctccctatcagtga tagagatctccctatcagtgatagagatcgtcgacgagggacttagcctttagtgaaccgtcagaattaattcag atcgatctaccagaaccgtcagatccgctagagattacgccaaccgccaccatgactagggcacatctgacagaa gtggaatcaaggctagaaagactggaacagctatttctactgatttttggcggctctggcggcggctccggaggc tcttacgggaaaatcgcggcattaaaggcggagaacgcagctctggaagccaagattgcagccttaaaagcggag attgctgcgttagaggcaggctacggcggctctggcggcggctccggaggctcttacgggaaaatcgcggcatta aaggcggagaacgcagctctggaagccaagattgcagccttaaaagcggagattgctgcgttagaggcaggctac ggcggctctggcggcggctccggaggctcttacgggaaaatcgcggcattaaaggcggagaacgcagctctggaa gccaagattgcagccttaaaagcggagattgctgcgttagaggcaggctacggcggctctggcggcggctccgga ggctcttacgggaaaatcgcggcattaaaggcggagaacgcagctctggaagccaagattgcagccttaaaagcg gagattgctgcgttagaggcaggctacgatccaaaaaagaagagaaaggtagacacctaccgctacatctaatgc tgaggctaactgaaacacggaaggagacaatacaggaaggaacggtaacttgtttattgcagcttataatggtta caaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaa actcatcaatgtatcttatcatgtctgtcgtctatgatgaggatgttggtggagagcatgtggaggaagtggata gggaaggttgtagagtagatccggttgaagtgatgaggataggaggaggactgcacatattgatcctcgtcacaa ccacaccagcacacctcaaatcccacaccactcccacaattaccattcactcaacaaactcacacatcccacgat aacgaattcaagcttgatatcattcaggacgagcctcagactccagcgtaactggactgcaatcaactcactggc tcaccttcacgggtgggcctttcttcggtagaaaatcaaaggatcttcttgagatcctttttttctgcgcgtaat ctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttt tccgaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccac ttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgat aagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggt tcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagc gccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagg gagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcatcgattt ttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcagaaaggcccacccgaaggtgagcc aggtgattacatttgggccctcattaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgt tcatccatagttgcctgactccccgtcgtgtagataactacgatgcgggagggcttaccatctggccccagtgct gcaatgataccgcgagaaccacgctcaccggctccagatttatcagcaataaaccagccagccgggagggccgag cgcagaagtgatcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagt tcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatg gcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagc tccttcggtcctccgatcgttgccagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcat aattctcttactgtcatgccatccgtgagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaa tagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaacttta aaagtgctcatcattggaaaacgttcttcggggcgtaaactctcaaggatcttaccgctgttgagatccagttcg atgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaaca ggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcattttagcttccttagct cctgaaaatctcgataactcaaaaaatacgcccggtagtgatcttatttcattatggtgaaagttggaacctctt acgtgccgatcaagtcaaaagcctccggtcggaggcttttgactttctgctatgg

[0234] SEQ ID NO: 49 TRE3G-MAD-GAL4 gatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtgg tttgtttgccggatcaagagctaccaactctttttccgaggtaactggcttcagcagagcgcagataccaaatac tgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgct aatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttacc ggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccga actgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggt aagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgt cgggtttcgccacctctgacttgagcatcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgc cagcaacgcagaaaggcccacccgaaggtgagccaggtgattacatttgggccctcattaccaatgcttaatcag tgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactac gatgcgggagggcttaccatctggccccagtgctgcaatgataccgcgagaaccacgctcaccggctccagattt atcagcaataaaccagccagccgggagggccgagcgcagaagtgatcctgcaactttatccgcctccatccagtc tattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctac aggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttac atgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgccagaagtaagttggccgc agtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtgagatgcttttctgt gactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaat acgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgtaaact ctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttt tactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacg gaaatgttgaatactcattttagcttccttagctcctgaaaatctcgataactcaaaaaatacgcccggtagtga tcttatttcattatggtgaaagttggaacctcttacgtgccgatcaagtcaaaagcctccggtcggaggcttttg actttctgctatggaggtcaggtatgatttaaatggtcagtattgagcgatatctagagaattcgtataggtctt gtattcagtctcgcactttagacatggtcttctgctgcccgtggtcctctattcgtcggatgttctgtcgattct tcctgtctatctggactgtacacgccacctcgacatactcgagtttactccctatcagtgatagagaacgtatga agagtttactccctatcagtgatagagaacgtatgcagactttactccctatcagtgatagagaacgtataagga gtttactccctatcagtgatagagaacgtatgaccagtttactccctatcagtgatagagaacgtatctacagtt tactccctatcagtgatagagaacgtatatccagtttactccctatcagtgatagagaacgtataagctttaggc gtgtacggtgggcgcctataaaagcagagctcgtttagtgaaccgtcagatcgcctggagcaattccacaacact tttgtcttataccaactttccgtaccacttcctaccctcgtaaaggacttagcctttagtgaaccgtcagaatta attcagatcgatctaccagaaccgtcagatccgctagagattacgccaaccgccaccatgaagctactgtcttct atcgaacaagcatgcgatatttgccgacttaaaaagctcaagtgctccaaagaaaaaccgaagtgcgccaagtgt ctgaagaacaactgggagtgtcgctactctcccaaaaccaaaaggtctccgctgactagggcacatctgacagaa gtggaatcaaggctagaaagactggaacagctatttctactgatttttggcggctctggcggcggctccggaggc tctggggaaattgctgctctggaagccaaaaatgcggcgttgaaagccgagattgcggccttggaagctaagatc gctgctttaaaggccggatacgatccaaaaaagaagagaaaggtagcacccccgaccgatgtcagcctgggggac gagctccacttagacggcgaggacgtggcgatggcgcatgccgacgcgctagacgatttcgatctggacatgttg ggggacggggattccccgggtccgggatttaccccccacgactccgccccctacggcgctctggatatggccgac ttcgagtttgagcagatgtttaccgatgcccttggaattgacgagtacggtggggacacctaccgctacatctaa taacagcttccggactctagaacatccctacaggtgatatcctcgggtaacttgtttattgcagcttataatggt tacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtcc aaactcatcaatgtatcttatcatgtctgtcgtctatgatgaggatgttggtggagagcatgtggaggaagtgga tagggaaggttgtagagtagatccggttgaagtgatgaggataggaggaggactgcacatattgatcctcgtcac aaccacaccagcacacctcaaatcccacaccactcccacaattaccattcactcaacaaactcacacatcccacg ataacgaattcaagcttgatatcattcaggacgagcctcagactccagcgtaactggactgcaatcaactcactg gctcaccttcacgggtgggcctttcttcggtagaaaatcaaag

[0235] SEQ ID NO: 50 TRE3G-DTF-4N8 tgggccctcattaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttg cctgactccccgtcgtgtagataactacgatgcgggagggcttaccatctggccccagtgctgcaatgataccgc gagaaccacgctcaccggctccagatttatcagcaataaaccagccagccgggagggccgagcgcagaagtgatc ctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaata gtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagct ccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctc cgatcgttgccagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactg tcatgccatccgtgagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggc gaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatca ttggaaaacgttcttcggggcgtaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactc gtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatg ccgcaaaaaagggaataagggcgacacggaaatgttgaatactcattttagcttccttagctcctgaaaatctcg ataactcaaaaaatacgcccggtagtgatcttatttcattatggtgaaagttggaacctcttacgtgccgatcaa gtcaaaagcctccggtcggaggcttttgactttctgctatggaggtcaggtatgatttaaatggtcagtattgag cgatatctagagaattcgtataggtcttgtattcagtctcgcactttagacatggtcttctgctgcccgtggtcc tctattcgtcggatgttctgtcgattcttcctgtctatctggactgtacacgccacctcgacatactcgagttta ctccctatcagtgatagagaacgtatgaagagtttactccctatcagtgatagagaacgtatgcagactttactc cctatcagtgatagagaacgtataaggagtttactccctatcagtgatagagaacgtatgaccagtttactccct atcagtgatagagaacgtatctacagtttactccctatcagtgatagagaacgtatatccagtttactccctatc agtgatagagaacgtataagctttaggcgtgtacggtgggcgcctataaaagcagagctcgtttagtgaaccgtc agatcgcctggagcaattccacaacacttttgtcttataccaactttccgtaccacttcctaccctcgtaaagga cttagcctttagtgaaccgtcagaattaattcagatcgatctaccagaaccgtcagatccgctagagattacgcc aaccgccaccatgactagggcacatctgacagaagtggaatcaaggctagaaagactggaacagctatttctact gatttttggcggctctggcggcggctccggaggctcttacgggaaaatcgcggcattaaaggcggagaacgcagc tctggaagccaagattgcagccttaaaagcggagattgctgcgttagaggcaggctacggcggctctggcggcgg ctccggaggctcttacgggaaaatcgcggcattaaaggcggagaacgcagctctggaagccaagattgcagcctt aaaagcggagattgctgcgttagaggcaggctacggcggctctggcggcggctccggaggctcttacgggaaaat cgcggcattaaaggcggagaacgcagctctggaagccaagattgcagccttaaaagcggagattgctgcgttaga ggcaggctacggcggctctggcggcggctccggaggctcttacgggaaaatcgcggcattaaaggcggagaacgc agctctggaagccaagattgcagccttaaaagcggagattgctgcgttagaggcaggctacgatccaaaaaagaa gagaaaggtagacacctaccgctacatctaataacagcttccggactctagaacatccctacaggtgatatcctc gggtaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatt tttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtcgtctatgatgagg atgttggtggagagcatgtggaggaagtggatagggaaggttgtagagtagatccggttgaagtgatgaggatag gaggaggactgcacatattgatcctcgtcacaaccacaccagcacacctcaaatcccacaccactcccacaatta ccattcactcaacaaactcacacatcccacgataacgaattcaagcttgatatcattcaggacgagcctcagact ccagcgtaactggactgcaatcaactcactggctcaccttcacgggtgggcctttcttcggtagaaaatcaaagg atcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggt ttgtttgccggatcaagagctaccaactctttttccgaggtaactggcttcagcagagcgcagataccaaatact gttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgcta atcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccg gataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaa ctgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggta agcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtc gggtttcgccacctctgacttgagcatcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgcc agcaacgcagaaaggcccacccgaaggtgagccaggtgattacatt

[0236] SEQ ID NO: 51 pCMV / TO-MAD-GAL4 accgcgagaaccacgctcaccggctccagatttatcagcaataaaccagccagccgggagggccgagcgcagaag tgatcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagt taatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcatt cagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcgg tcctccgatcgttgccagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctct tactgtcatgccatccgtgagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtat gcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgct catcattggaaaacgttcttcggggcgtaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacc cactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggca aaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcattttagcttccttagctcctgaaaa tctcgataactcaaaaaatacgcccggtagtgatcttatttcattatggtgaaagttggaacctcttacgtgccg atcaagtcaaaagcctccggtcggaggcttttgactttctgctatggaggtcaggtatgatttaaatggtcagta ttgagcgatatctagagaattcgtataggtcttgtattcagtctcgcactttagacatggtcttctgctgcccgt ggtcctctattcgtcggatgttctgtcgattcttcctgtctatctggactcgttacataacttacggtaaatggc ccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaata gggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcat atgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgacctta tgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtac atcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttg ttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtagg cgtgtacggtgggaggtctatataagcagagctctccctatcagtgatagagatctccctatcagtgatagagat cgtcgacgagggacttagcctttagtgaaccgtcagaattaattcagatcgatctaccagaaccgtcagatccgc tagagattacgccaaccgccaccatgaagctactgtcttctatcgaacaagcatgcgatatttgccgacttaaaa agctcaagtgctccaaagaaaaaccgaagtgcgccaagtgtctgaagaacaactgggagtgtcgctactctccca aaaccaaaaggtctccgctgactagggcacatctgacagaagtggaatcaaggctagaaagactggaacagctat ttctactgatttttggcggctctggcggcggctccggaggctctggggaaattgctgctctggaagccaaaaatg cggcgttgaaagccgagattgcggccttggaagctaagatcgctgctttaaaggccggatacgatccaaaaaaga agagaaaggtagcacccccgaccgatgtcagcctgggggacgagctccacttagacggcgaggacgtggcgatgg cgcatgccgacgcgctagacgatttcgatctggacatgttgggggacggggattccccgggtccgggatttaccc cccacgactccgccccctacggcgctctggatatggccgacttcgagtttgagcagatgtttaccgatgcccttg gaattgacgagtacggtggggacacctaccgctacatctaatgctgaggctaactgaaacacggaaggagacaat acaggaaggaacggtaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcaca aataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtcgt ctatgatgaggatgttggtggagagcatgtggaggaagtggatagggaaggttgtagagtagatccggttgaagt gatgaggataggaggaggactgcacatattgatcctcgtcacaaccacaccagcacacctcaaatcccacaccac tcccacaattaccattcactcaacaaactcacacatcccacgataacgaattcaagcttgatatcattcaggacg agcctcagactccagcgtaactggactgcaatcaactcactggctcaccttcacgggtgggcctttcttcggtag aaaatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgcta ccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaggtaactggcttcagcagagcgcaga taccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacc tcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagac gatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacga cctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggaca ggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatcttt atagtcctgtcgggtttcgccacctctgacttgagcatcgatttttgtgatgctcgtcaggggggcggagcctat ggaaaaacgccagcaacgcagaaaggcccacccgaaggtgagccaggtgattacatttgggccctcattaccaat gcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgt agataactacgatgcgggagggcttaccatctggccccagtgctgcaatgat

[0237] SEQ ID NO: 52 pCMV / TO:: Ub-GFP-DR ggcccacccgaaggtgagccaggtgattacatttgggccctcattaccaatgcttaatcagtgaggcacctatct cagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatgcgggagggct taccatctggccccagtgctgcaatgataccgcgagaaccacgctcaccggctccagatttatcagcaataaacc agccagccgggagggccgagcgcagaagtgatcctgcaactttatccgcctccatccagtctattaattgttgcc gggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgt cacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgt tgtgcaaaaaagcggttagctccttcggtcctccgatcgttgccagaagtaagttggccgcagtgttatcactca tggttatggcagcactgcataattctcttactgtcatgccatccgtgagatgcttttctgtgactggtgagtact caaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccg cgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgtaaactctcaaggatcttac cgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcg tttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatac tcattttagcttccttagctcctgaaaatctcgataactcaaaaaatacgcccggtagtgatcttatttcattat ggtgaaagttggaacctcttacgtgccgatcaagtcaaaagcctccggtcggaggcttttgactttctgctatgg aggtcaggtatgatttaaatggtcagtattgagcgatatctagagaattcgtataggtcttgtattcagtctcgc actttagacatggtcttctgctgcccgtggtcctctattcgtcggatgttctgtcgattcttcctgtctatctgg actcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataat gacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgc ccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgc ctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctat taccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtct ccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactc cgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctccctatcagtga tagagatctccctatcagtgatagagatcgtcgacgagggacttagcctttagtgaaccgtcagaattaattcag atcgatctaccagaaccgtcagatccgctagagattacgccaaccgccaccatgggcagcatgcagatcttcgtg aagaccctgaccggcaagaccatcaccctagaggtggagcccagtgacaccatcgagaacgtgaaggccaagatc caggataaagagggcatcccccctgaccagcagaggctgatctttgccggcaagcagctggaagatggccgcacc ctctctgattacaacatccagaaggagtcaaccctgcacctggtccttcgcctgagaggtgtcgtccatggatcc ggagcttggctgttgcccgtgtcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccc cgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgagaattctgcagtcgacggtaccgcggg cccgggatccaccggccggtcgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctg gtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggc aagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacc tacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaa ggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgag ggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaag ctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttc aagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgac ggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgc gatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtcaggc taataacagcttccggactctagaacatccctacaggtgatatcctcgtgctgcaagtaaacccctaccaactgg tcggggtttgaaacggtacctacaggtgatatcctcgggtaacttgtttattgcagcttataatggttacaaata aagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcat caatgtatcttatcatgtctgtcgtctatgatgaggatgttggtggagagcatgtggaggaagtggatagggaag gttgtagagtagatccggttgaagtgatgaggataggaggaggactgcacatattgatcctcgtcacaaccacac cagcacacctcaaatcccacaccactcccacaattaccattcactcaacaaactcacacatcccacgataacgaa ttcaagcttgatatcattcaggacgagcctcagactccagcgtaactggactgcaatcaactcactggctcacct tcacgggtgggcctttcttcggtagaaaatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctg cttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgag gtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaag aactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcg tgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgc acacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacg cttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagctt ccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcatcgatttttgtga tgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcagaaa

[0238] SEQ ID NO: 53 pUASJhic ttctgctatggaggtcaggtatgatttaaatggtcagtattgagcgatatctagagaattcgtataggtcttgta ttcagtctcgcactttagacatggtcttctgctgcccgtggtcctctattcgtcggatgttctgtcgattcttcc tgtctatctggactaggcctccaaggcggagtactgtcctccgggctggcggagtactgtcctccggcaaggtcg gagtactgtcctccgacactagaggtcggagtactgtcctccgacgcaaggcggagtactgtcctccgggctgcg gagtactgtcctccggcaaggtcggagtactgtcctccgacactagaggtcggagtactgtcctccgacgcaagg tcggagtactgtcctccgacactagaggtcggagtactgtcctccgacgcaaggtcggagtactgtcctccgaca ctagaggtcggagtactgtcctccgacgcaaggcggagtactgtcctccgggctggcggagtactgtcctccggc aagggtcgactctagagggtatataatggatcccatcgcgtctcagcctcactttgagctcctccacacgaattc cccgataccgtcgattcaaggagcttgcttgttctttttgcagaagctcagaataaacgctcaactttggcagat ctaccGACttagcctttagtgaaccgtcagaattaattcagatcgatctaccagaaccgtcagatccgctagaga ttacgccaaccgccaccatgggcagcgaagatgccaaaaacattaagaagggcccagcgccattctacccactcg aagacgggaccgccggcgagcagctgcacaaagccatgaagcgctacgccctggtgcccggcaccatcgccttta ccgacgcacatatcgaggtggacattacctacgccgagtacttcgagatgagcgttcggctggcagaagctatga agcgctatgggctgaatacaaaccatcggatcgtggtgtgcagcgagaatagcttgcagttcttcatgcccgtgt tgggtgccctgttcatcggtgtggctgtggccccagctaacgacatctacaacgagcgcgagctgctgaacagca tgggcatcagccagcccaccgtcgtattcgtgagcaagaaagggctgcaaaagatcctcaacgtgcaaaagaagc taccgatcatacaaaagatcatcatcatggatagcaagaccgactaccagggcttccaaagcatgtacaccttcg tgacttcccatttgccacccggcttcaacgagtacgacttcgtgcccgagagcttcgaccgggacaaaaccatcg ccctgatcatgaacagtagtggcagtaccggattgcccaagggcgtagccctaccgcaccgcaccgcttgtgtcc gattcagtcatgcccgcgaccccatcttcggcaaccagatcatccccgacaccgctatcctcagcgtggtgccat ttcaccacggcttcggcatgttcaccacgctgggctacttgatctgcggctttcgggtcgtgctcatgtaccgct tcgaggaggagctattcttgcgcagcttgcaagactataagattcaatctgccctgctggtgcccacactattta gcttcttcgctaagagcactctcatcgacaagtacgacctaagcaacttgcacgagatcgccagcggcggggcgc cgctcagcaaggaggtaggtgaggccgtggccaaacgcttccacctaccaggcatccgccagggctacggcctga cagaaacaaccagcgccattctgatcacccccgaaggggacgacaagcctggcgcagtaggcaaggtggtgccct tcttcgaggctaaggtggtggacttggacaccggtaagacactgggtgtgaaccagcgcggcgagctgtgcgtcc gtggccccatgatcatgagcggctacgttaacaaccccgaggctacaaacgctctcatcgacaaggacggctggc tgcacagcggcgacatcgcctactgggacgaggacgagcacttcttcatcgtggaccggctgaagagcctgatca aatacaagggctaccaggtagccccagccgaactggagagcatcctgctgcaacaccccaacatcttcgacgccg gggtcgccggcctgcccgacgacgatgccggcgagctgcccgccgcagtcgtcgtgctggaacacggtaaaacca tgaccgagaaggagatcgtggactatgtggccagccaggttacaaccgccaagaagctgcgcggtggtgttgtgt tcgtggacgaggtgcctaaaggactgaccggcaagttggacgcccgcaagatccgcgagattctcattaaggcca agaagggcggcaagatcgccgtgaattctgcttgcaagaactggttcagtagcttaagccactttgtgatccacc ttaacagccacggcttccctcccgaggtggaggagcaggccgccggcaccctgcccatgagctgcgcccaggaga gcggcatggatagacaccctgctgcttgcgccagcgccaggatcaacgtcTCAGGCtaataacagcttccggact ctagaacatccctacaggtgatatcctcgggtaacttgtttattgcagcttataatggttacaaataaagcaata gcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtat cttatcatgtctgtcgtctatgatgaggatgttggtggagagcatgtggaggaagtggatagggaaggttgtaga gtagatccggttgaagtgatgaggataggaggaggactgcacatattgatcctcgtcacaaccacaccagcacac ctcaaatcccacaccactcccacaattaccattcactcaacaaactcacacatcccacgataacgaattcaagct tgatatcattcaggacgagcctcagactccagcgtaactggactgcaatcaactcactggctcaccttcacgggt gggcctttcttcggtagaaaatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaa caaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaggtaactgg cttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgt agcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttac cgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcc cagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccga agggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccaggggg aaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcatcgatttttgtgatgctcgtc aggggggcggagcctatggaaaaacgccagcaacgcagaaaggcccacccgaaggtgagccaggtgattacattt gggccctcattaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgc ctgactccccgtcgtgtagataactacgatgcgggagggcttaccatctggccccagtgctgcaatgataccgcg agaaccacgctcaccggctccagatttatcagcaataaaccagccagccgggagggccgagcgcagaagtgatcc tgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatag tttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctc cggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctcc gatcgttgccagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgt catgccatccgtgagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcg accgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcat tggaaaacgttcttcggggcgtaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcg tgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgc cgcaaaaaagggaataagggcgacacggaaatgttgaatactcattttagcttccttagctcctgaaaatctcga taactcaaaaaatacgcccggtagtgatcttatttcattatggtgaaagttggaacctcttacgtgccgatcaag tcaaaagcctccggtcggaggcttttgact

[0239] SEQ ID NO: 54 pTRE3G:: EGFP-DR(Rfx) caatgataccgcgagaaccacgctcaccggctccagatttatcagcaataaaccagccagccgggagggccgagc gcagaagtgatcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagtt cgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatgg cttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagct ccttcggtcctccgatcgttgccagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcata attctcttactgtcatgccatccgtgagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaat agtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaa aagtgctcatcattggaaaacgttcttcggggcgtaaactctcaaggatcttaccgctgttgagatccagttcga tgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacag gaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcattttagcttccttagctc ctgaaaatctcgataactcaaaaaatacgcccggtagtgatcttatttcattatggtgaaagttggaacctctta cgtgccgatcaagtcaaaagcctccggtcggaggcttttgactttctgctatggaggtcaggtatgatttaaatg gtcagtattgagcgatatctagagaattcgtataggtcttgtattcagtctcgcactttagacatggtcttctgc tgcccgtggtcctctattcgtcggatgttctgtcgattcttcctgtctatctggactgtacacgccacctcgaca tactcgagtttactccctatcagtgatagagaacgtatgaagagtttactccctatcagtgatagagaacgtatg cagactttactccctatcagtgatagagaacgtataaggagtttactccctatcagtgatagagaacgtatgacc agtttactccctatcagtgatagagaacgtatctacagtttactccctatcagtgatagagaacgtatatccagt ttactccctatcagtgatagagaacgtataagctttaggcgtgtacggtgggcgcctataaaagcagagctcgtt tagtgaaccgtcagatcgcctggagcaattccacaacacttttgtcttataccaactttccgtaccacttcctac cctcgtaaaggacttagcctttagtgaaccgtcagaattaattcagatcgatctaccagaaccgtcagatccgct agagattacgccaaccgccaccatgggcagcgtgagcaagggcgaggagctgttcaccggggtggtgcccatcct ggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacgg caagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgac ctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccga aggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcga gggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaa gctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaactt caagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcga cggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcg cgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtcagg ctaataacagcttccggactctagaacatccctacaggtgatatcctcgtgctgcaagtaaacccctaccaactg gtcggggtttgaaacggtaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttc acaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgt cgtctatgatgaggatgttggtggagagcatgtggaggaagtggatagggaaggttgtagagtagatccggttga agtgatgaggataggaggaggactgcacatattgatcctcgtcacaaccacaccagcacacctcaaatcccacac cactcccacaattaccattcactcaacaaactcacacatcccacgataacgaattcaagcttgatatcattcagg acgagcctcagactccagcgtaactggactgcaatcaactcactggctcaccttcacgggtgggcctttcttcgg tagaaaatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccg ctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaggtaactggcttcagcagagcgc agataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacat acctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaa gacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaa cgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcgg acaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatc tttatagtcctgtcgggtttcgccacctctgacttgagcatcgatttttgtgatgctcgtcaggggggcggagcc tatggaaaaacgccagcaacgcagaaaggcccacccgaaggtgagccaggtgattacatttgggccctcattacc aatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcg tgtagataactacgatgcgggagggcttaccatctggccccagtgctg

[0240] SEQ ID NO: 55 DTF no SOD ctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgcca gctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcac tgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccac agaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgc gttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcg aaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccct gccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggta tctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgc cttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaa caggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactag aaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccgg caaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctca agaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcat gagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatata tgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttc atccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgc aatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcg cagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttc gccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggc ttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctc cttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataa ttctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaata gtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaa agtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgat gtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacagg aaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaata ttattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaat aggggttccgcgcacatttccccgaaaagtgccacctgacgtcgacggatcgggagatctcccgatcccctatgg tcgactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtc gctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgctt agggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttat taatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaat ggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgcca atagggactttccattgacgtcaatgggtggactatttacggtaaactgcccacttggcagtacatcaagtgtat catatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgacc ttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcag tacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagt ttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggt aggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatc gaaattaatacgactcactatagggagacccaagcttggtaccgagctcggatccactagtaacggccgccagtg tgctggaattcgccaccatgactagggcacatctgacagaagtggaatcaaggctagaaagactggaacagctat ttctactgatttttggcggctctggcggcggctccggaggctctgatccaaaaaagaagagaaaggtataattct agagggccctattctatagtgtcacctaaatgctagagctcgctgatcagcctcgactgtgccttctagttgcca gccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaata aaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaa gggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaac cagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcg cagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgtt cgccggctttccccgtcaagctctaaatcggggcatccctttagggttccgatttagtgctttacggcacctcga ccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgac gttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattc ttttgatttataagggattttggggatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgc gaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccaggcaggcagaagtatgca aagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaag catgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttc cgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagct attccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccat tttcggatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctcc ggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgtt ccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcagga cgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagc gggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaa agtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagc gaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagca tcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgac ccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggct gggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggc tgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacga gttcttctgagcgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgat tccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgc ggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaat agcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgta tcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtga aattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgc SEQ ID NO: 56 ERalpha DBD Aaggagactcgctactgtgcagtgtgcaatgactatgcttcaggctaccattatggagtctggtcctgtgagggc tgcaaggccttcttcaagagaagtattcaaggacataacgactatatgtgtccagccaccaaccagtgcaccatt gataaaaacaggaggaagagctgccaggcctgccggctccgcaaatgctacgaagtgggaatgatgaaaggtggg atacgaaaagaccgaagagga

[0241] SEQ ID NO: 57 pCMV_MAD-GAL4r-pUASfluc-P2A-DGal4_1N8 tttagcttccttagctcctgaaaatctcgataactcaaaaaatacgcccggtagtgatcttatttcattatggtg aaagttggaacctcttacgtgccgatcaagtcaaaagcctccggtcggaggcttttgacttaagatacattgatg agtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttat ttgtaaccattataagctgcaataaacaagattagatgtagcggtaggtgtccccaccgtactcgtcaattccaa gggcatcggtaaacatctgctcaaactcgaagtcggccatatccagagcgccgtagggggcggagtcgtgggggg taaatcccggacccggggaatccccgtcccccaacatgtccagatcgaaatcgtctagcgcgtcggcatgcgcca tcgccacgtcctcgccgtctaagtggagctcgtcccccaggctgacatcggtcgggggtgctacctttctcttct tttttggatcgtatccggcctttaaagcagcgatcttagcttccaaggccgcaatctcggctttcaacgccgcat ttttggcttccagagcagcaatttccccagagcctccggagccgccgccagagccgccaaaaatcagtagaaata gctgttccagtctttctagccttgattccacttctgtcagatgtgccctagtcagcggggaccttttggttttgg gagagtagcgacactcccagttgttcttcagacacttggcgcacttcggtttttctttggagcacttgagctttt taagtcggcaaatatcgcatgcttgttcgatagaggacagtagcttcatgaattccagcacactggcggccgtta ctagtggatccgagctcggtaccaagcttgggtctgcctatagtgagtcgtattaatttcgataagccagtaagc agtgggttctctagttagccagagagctctgcttatatagacctcccaccgtacacgcctaccgcccatttgcgt caatggggcggagttgttacgacattttggaaagtcccgttgattttggtgccaaaacaaactcccattgacgtc aatggggtggagacttggaaatccccgtgagtcaaaccgctatccacgcccattgatgtactgccaaaaccgcat caccatggtaatagcgatgactaatacgtagatgtactgccaagtaggaaagtcccataaggtcatgtactgggc ataatgccaggcgggccatttaccgtcattgacgtcaatagggggcgtacttggcatatgatacacttgatgtac tgccaagtgggcagtttaccgtaaatactccacccattgacgtcaatggaaagtccctattggcgttactatggg aacatacgtcattattgacgtcaatgggcgggggtcgttgggcggtcagccaggcgggccatttaccgtaagtta tgtaacgcggaactccatatatgggctatgaactaatgaccccgtaattgattactattaataactagtcaataa tcaatgtttctgctatggaggtcaggtatgatttaaatggtcagtattgagcgatatctagagaattcgtatagg tcttgtattcagtctcgcactttagacatggtcttctgctgcccgtggtcctctattcgtcggatgttctgtcga ttcttcctgtctatctggactaggcctccaaggcggagtactgtcctccgggctggcggagtactgtcctccggc aaggtcggagtactgtcctccgacactagaggtcggagtactgtcctccgacgcaaggcggagtactgtcctccg ggctgcggagtactgtcctccggcaaggtcggagtactgtcctccgacactagaggtcggagtactgtcctccga cgcaaggtcggagtactgtcctccgacactagaggtcggagtactgtcctccgacgcaaggtcggagtactgtcc tccgacactagaggtcggagtactgtcctccgacgcaaggcggagtactgtcctccgggctggcggagtactgtc ctccggcaagggtcgactctagagggtatataatggatcccatcgcgtctcagcctcactttgagctcctccaca cgaattccccgataccgtcgattcaaggagcttgcttgttctttttgcagaagctcagaataaacgctcaacttt ggcagatctaccgacttagcctttagtgaaccgtcagaattaattcagatcgatctaccagaaccgtcagatccg ctagagattacgccaaccgccaccatgggcagcgaagatgccaaaaacattaagaagggcccagcgccattctac ccactcgaagacgggaccgccggcgagcagctgcacaaagccatgaagcgctacgccctggtgcccggcaccatc gcctttaccgacgcacatatcgaggtggacattacctacgccgagtacttcgagatgagcgttcggctggcagaa gctatgaagcgctatgggctgaatacaaaccatcggatcgtggtgtgcagcgagaatagcttgcagttcttcatg cccgtgttgggtgccctgttcatcggtgtggctgtggccccagctaacgacatctacaacgagcgcgagctgctg aacagcatgggcatcagccagcccaccgtcgtattcgtgagcaagaaagggctgcaaaagatcctcaacgtgcaa aagaagctaccgatcatacaaaagatcatcatcatggatagcaagaccgactaccagggcttccaaagcatgtac accttcgtgacttcccatttgccacccggcttcaacgagtacgacttcgtgcccgagagcttcgaccgggacaaa accatcgccctgatcatgaacagtagtggcagtaccggattgcccaagggcgtagccctaccgcaccgcaccgct tgtgtccgattcagtcatgcccgcgaccccatcttcggcaaccagatcatccccgacaccgctatcctcagcgtg gtgccatttcaccacggcttcggcatgttcaccacgctgggctacttgatctgcggctttcgggtcgtgctcatg taccgcttcgaggaggagctattcttgcgcagcttgcaagactataagattcaatctgccctgctggtgcccaca ctatttagcttcttcgctaagagcactctcatcgacaagtacgacctaagcaacttgcacgagatcgccagcggc ggggcgccgctcagcaaggaggtaggtgaggccgtggccaaacgcttccacctaccaggcatccgccagggctac ggcctgacagaaacaaccagcgccattctgatcacccccgaaggggacgacaagcctggcgcagtaggcaaggtg gtgcccttcttcgaggctaaggtggtggacttggacaccggtaagacactgggtgtgaaccagcgcggcgagctg tgcgtccgtggccccatgatcatgagcggctacgttaacaaccccgaggctacaaacgctctcatcgacaaggac ggctggctgcacagcggcgacatcgcctactgggacgaggacgagcacttcttcatcgtggaccggctgaagagc ctgatcaaatacaagggctaccaggtagccccagccgaactggagagcatcctgctgcaacaccccaacatcttc gacgccggggtcgccggcctgcccgacgacgatgccggcgagctgcccgccgcagtcgtcgtgctggaacacggt aaaaccatgaccgagaaggagatcgtggactatgtggccagccaggttacaaccgccaagaagctgcgcggtggt gttgtgttcgtggacgaggtgcctaaaggactgaccggcaagttggacgcccgcaagatccgcgagattctcatt aaggccaagaagggcggcaagatcgccgtgaattctgcttgcaagaactggttcagtagcttaagccactttgtg atccaccttaacagccacggcttccctcccgaggtggaggagcaggccgccggcaccctgcccatgagctgcgcc caggagagcggcatggatagacaccctgctgcttgcgccagcgccaggatcaacgtgtcaggcggcagcggcgcc accaacttcagcctgctgaagcaggccggcgacgtggaggagaaccccggccccatgactagggcacatctgaca gaagtggaatcaaggctagaaagactggaacagctatttctactgatttttggcggctctggcggcggctccgga ggctcttacgggaaaatcgcggcattaaaggcggagaacgcagctctggaagccaagattgcagccttaaaagcg gagattgctgcgttagaggcaggctacgatccaaaaaagaagagaaaggtagacacctaccgctacatctaactt gtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcact gcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtcgtctatgatgaggatgttggtg gagagcatgtggaggaagtggatagggaaggttgtagagtagatccggttgaagtgatgaggataggaggaggac tgcacatattgatcctcgtcacaaccacaccagcacacctcaaatcccacaccactcccacaattaccattcact caacaaactcacacatcccacgataacgaattcaagcttgatatcattcaggacgagcctcagactccagcgtaa ctggactgcaatcaactcactggctcaccttcacgggtgggcctttcttcggtagaaaatcaaaggatcttcttg agatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgcc ggatcaagagctaccaactctttttccgaggtaactggcttcagcagagcgcagataccaaatactgttcttcta gtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgtta ccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcg cagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatac ctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagg gtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgc cacctctgacttgagcatcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgca gaaaggcccacccgaaggtgagccaggtgattacatttgggccctcattaccaatgcttaatcagtgaggcacct atctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatgcgggag ggcttaccatctggccccagtgctgcaatgataccgcgagaaccacgctcaccggctccagatttatcagcaata aaccagccagccgggagggccgagcgcagaagtgatcctgcaactttatccgcctccatccagtctattaattgt tgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtg gtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatccccc atgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgccagaagtaagttggccgcagtgttatca ctcatggttatggcagcactgcataattctcttactgtcatgccatccgtgagatgcttttctgtgactggtgag tactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataat accgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgtaaactctcaaggatc ttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcacc agcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttga atactcat

[0242] SEQ ID NO: 58 pCMV_MAD-GAL4r-pUASfluc-P2A-DGal4_2N8 tttagcttccttagctcctgaaaatctcgataactcaaaaaatacgcccggtagtgatcttatttcattatggtg aaagttggaacctcttacgtgccgatcaagtcaaaagcctccggtcggaggcttttgacttaagatacattgatg agtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttat ttgtaaccattataagctgcaataaacaagattagatgtagcggtaggtgtccccaccgtactcgtcaattccaa gggcatcggtaaacatctgctcaaactcgaagtcggccatatccagagcgccgtagggggcggagtcgtgggggg taaatcccggacccggggaatccccgtcccccaacatgtccagatcgaaatcgtctagcgcgtcggcatgcgcca tcgccacgtcctcgccgtctaagtggagctcgtcccccaggctgacatcggtcgggggtgctacctttctcttct tttttggatcgtatccggcctttaaagcagcgatcttagcttccaaggccgcaatctcggctttcaacgccgcat ttttggcttccagagcagcaatttccccagagcctccggagccgccgccagagccgccaaaaatcagtagaaata gctgttccagtctttctagccttgattccacttctgtcagatgtgccctagtcagcggggaccttttggttttgg gagagtagcgacactcccagttgttcttcagacacttggcgcacttcggtttttctttggagcacttgagctttt taagtcggcaaatatcgcatgcttgttcgatagaggacagtagcttcatgaattccagcacactggcggccgtta ctagtggatccgagctcggtaccaagcttgggtctgcctatagtgagtcgtattaatttcgataagccagtaagc agtgggttctctagttagccagagagctctgcttatatagacctcccaccgtacacgcctaccgcccatttgcgt caatggggcggagttgttacgacattttggaaagtcccgttgattttggtgccaaaacaaactcccattgacgtc aatggggtggagacttggaaatccccgtgagtcaaaccgctatccacgcccattgatgtactgccaaaaccgcat caccatggtaatagcgatgactaatacgtagatgtactgccaagtaggaaagtcccataaggtcatgtactgggc ataatgccaggcgggccatttaccgtcattgacgtcaatagggggcgtacttggcatatgatacacttgatgtac tgccaagtgggcagtttaccgtaaatactccacccattgacgtcaatggaaagtccctattggcgttactatggg aacatacgtcattattgacgtcaatgggcgggggtcgttgggcggtcagccaggcgggccatttaccgtaagtta tgtaacgcggaactccatatatgggctatgaactaatgaccccgtaattgattactattaataactagtcaataa tcaatgtttctgctatggaggtcaggtatgatttaaatggtcagtattgagcgatatctagagaattcgtatagg tcttgtattcagtctcgcactttagacatggtcttctgctgcccgtggtcctctattcgtcggatgttctgtcga ttcttcctgtctatctggactaggcctccaaggcggagtactgtcctccgggctggcggagtactgtcctccggc aaggtcggagtactgtcctccgacactagaggtcggagtactgtcctccgacgcaaggcggagtactgtcctccg ggctgcggagtactgtcctccggcaaggtcggagtactgtcctccgacactagaggtcggagtactgtcctccga cgcaaggtcggagtactgtcctccgacactagaggtcggagtactgtcctccgacgcaaggtcggagtactgtcc tccgacactagaggtcggagtactgtcctccgacgcaaggcggagtactgtcctccgggctggcggagtactgtc ctccggcaagggtcgactctagagggtatataatggatcccatcgcgtctcagcctcactttgagctcctccaca cgaattccccgataccgtcgattcaaggagcttgcttgttctttttgcagaagctcagaataaacgctcaacttt ggcagatctaccgacttagcctttagtgaaccgtcagaattaattcagatcgatctaccagaaccgtcagatccg ctagagattacgccaaccgccaccatgggcagcgaagatgccaaaaacattaagaagggcccagcgccattctac ccactcgaagacgggaccgccggcgagcagctgcacaaagccatgaagcgctacgccctggtgcccggcaccatc gcctttaccgacgcacatatcgaggtggacattacctacgccgagtacttcgagatgagcgttcggctggcagaa gctatgaagcgctatgggctgaatacaaaccatcggatcgtggtgtgcagcgagaatagcttgcagttcttcatg cccgtgttgggtgccctgttcatcggtgtggctgtggccccagctaacgacatctacaacgagcgcgagctgctg aacagcatgggcatcagccagcccaccgtcgtattcgtgagcaagaaagggctgcaaaagatcctcaacgtgcaa aagaagctaccgatcatacaaaagatcatcatcatggatagcaagaccgactaccagggcttccaaagcatgtac accttcgtgacttcccatttgccacccggcttcaacgagtacgacttcgtgcccgagagcttcgaccgggacaaa accatcgccctgatcatgaacagtagtggcagtaccggattgcccaagggcgtagccctaccgcaccgcaccgct tgtgtccgattcagtcatgcccgcgaccccatcttcggcaaccagatcatccccgacaccgctatcctcagcgtg gtgccatttcaccacggcttcggcatgttcaccacgctgggctacttgatctgcggctttcgggtcgtgctcatg taccgcttcgaggaggagctattcttgcgcagcttgcaagactataagattcaatctgccctgctggtgcccaca ctatttagcttcttcgctaagagcactctcatcgacaagtacgacctaagcaacttgcacgagatcgccagcggc ggggcgccgctcagcaaggaggtaggtgaggccgtggccaaacgcttccacctaccaggcatccgccagggctac ggcctgacagaaacaaccagcgccattctgatcacccccgaaggggacgacaagcctggcgcagtaggcaaggtg gtgcccttcttcgaggctaaggtggtggacttggacaccggtaagacactgggtgtgaaccagcgcggcgagctg tgcgtccgtggccccatgatcatgagcggctacgttaacaaccccgaggctacaaacgctctcatcgacaaggac ggctggctgcacagcggcgacatcgcctactgggacgaggacgagcacttcttcatcgtggaccggctgaagagc ctgatcaaatacaagggctaccaggtagccccagccgaactggagagcatcctgctgcaacaccccaacatcttc gacgccggggtcgccggcctgcccgacgacgatgccggcgagctgcccgccgcagtcgtcgtgctggaacacggt aaaaccatgaccgagaaggagatcgtggactatgtggccagccaggttacaaccgccaagaagctgcgcggtggt gttgtgttcgtggacgaggtgcctaaaggactgaccggcaagttggacgcccgcaagatccgcgagattctcatt aaggccaagaagggcggcaagatcgccgtgaattctgcttgcaagaactggttcagtagcttaagccactttgtg atccaccttaacagccacggcttccctcccgaggtggaggagcaggccgccggcaccctgcccatgagctgcgcc caggagagcggcatggatagacaccctgctgcttgcgccagcgccaggatcaacgtgtcaggcggcagcggcgcc accaacttcagcctgctgaagcaggccggcgacgtggaggagaaccccggccccatgactagggcacatctgaca gaagtggaatcaaggctagaaagactggaacagctatttctactgatttttggcggctctggcggcggctccgga ggctcttacgggaaaatcgcggcattaaaggcggagaacgcagctctggaagccaagattgcagccttaaaagcg gagattgctgcgttagaggcaggctacggcggctctggcggcggctccggaggctcttacgggaaaatcgcggca ttaaaggcggagaacgcagctctggaagccaagattgcagccttaaaagcggagattgctgcgttagaggcaggc tacgatccaaaaaagaagagaaaggtagacacctaccgctacatctaacttgtttattgcagcttataatggtta caaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaa actcatcaatgtatcttatcatgtctgtcgtctatgatgaggatgttggtggagagcatgtggaggaagtggata gggaaggttgtagagtagatccggttgaagtgatgaggataggaggaggactgcacatattgatcctcgtcacaa ccacaccagcacacctcaaatcccacaccactcccacaattaccattcactcaacaaactcacacatcccacgat aacgaattcaagcttgatatcattcaggacgagcctcagactccagcgtaactggactgcaatcaactcactggc tcaccttcacgggtgggcctttcttcggtagaaaatcaaaggatcttcttgagatcctttttttctgcgcgtaat ctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttt tccgaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccac ttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgat aagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggt tcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagc gccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagg gagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcatcgattt ttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcagaaaggcccacccgaaggtgagcc aggtgattacatttgggccctcattaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgt tcatccatagttgcctgactccccgtcgtgtagataactacgatgcgggagggcttaccatctggccccagtgct gcaatgataccgcgagaaccacgctcaccggctccagatttatcagcaataaaccagccagccgggagggccgag cgcagaagtgatcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagt tcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatg gcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagc tccttcggtcctccgatcgttgccagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcat aattctcttactgtcatgccatccgtgagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaa tagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaacttta aaagtgctcatcattggaaaacgttcttcggggcgtaaactctcaaggatcttaccgctgttgagatccagttcg atgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaaca ggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaa tact cat

[0243] SEQ ID NO: 59 pCMV_MAD-GAL4r-pUASfluc-P2A-DGal4_4N8 tttagcttccttagctcctgaaaatctcgataactcaaaaaatacgcccggtagtgatcttatttcattatggtg aaagttggaacctcttacgtgccgatcaagtcaaaagcctccggtcggaggcttttgacttaagatacattgatg agtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttat ttgtaaccattataagctgcaataaacaagattagatgtagcggtaggtgtccccaccgtactcgtcaattccaa gggcatcggtaaacatctgctcaaactcgaagtcggccatatccagagcgccgtagggggcggagtcgtgggggg taaatcccggacccggggaatccccgtcccccaacatgtccagatcgaaatcgtctagcgcgtcggcatgcgcca tcgccacgtcctcgccgtctaagtggagctcgtcccccaggctgacatcggtcgggggtgctacctttctcttct tttttggatcgtatccggcctttaaagcagcgatcttagcttccaaggccgcaatctcggctttcaacgccgcat ttttggcttccagagcagcaatttccccagagcctccggagccgccgccagagccgccaaaaatcagtagaaata gctgttccagtctttctagccttgattccacttctgtcagatgtgccctagtcagcggggaccttttggttttgg gagagtagcgacactcccagttgttcttcagacacttggcgcacttcggtttttctttggagcacttgagctttt taagtcggcaaatatcgcatgcttgttcgatagaggacagtagcttcatgaattccagcacactggcggccgtta ctagtggatccgagctcggtaccaagcttgggtctgcctatagtgagtcgtattaatttcgataagccagtaagc agtgggttctctagttagccagagagctctgcttatatagacctcccaccgtacacgcctaccgcccatttgcgt caatggggcggagttgttacgacattttggaaagtcccgttgattttggtgccaaaacaaactcccattgacgtc aatggggtggagacttggaaatccccgtgagtcaaaccgctatccacgcccattgatgtactgccaaaaccgcat caccatggtaatagcgatgactaatacgtagatgtactgccaagtaggaaagtcccataaggtcatgtactgggc ataatgccaggcgggccatttaccgtcattgacgtcaatagggggcgtacttggcatatgatacacttgatgtac tgccaagtgggcagtttaccgtaaatactccacccattgacgtcaatggaaagtccctattggcgttactatggg aacatacgtcattattgacgtcaatgggcgggggtcgttgggcggtcagccaggcgggccatttaccgtaagtta tgtaacgcggaactccatatatgggctatgaactaatgaccccgtaattgattactattaataactagtcaataa tcaatgtttctgctatggaggtcaggtatgatttaaatggtcagtattgagcgatatctagagaattcgtatagg tcttgtattcagtctcgcactttagacatggtcttctgctgcccgtggtcctctattcgtcggatgttctgtcga ttcttcctgtctatctggactaggcctccaaggcggagtactgtcctccgggctggcggagtactgtcctccggc aaggtcggagtactgtcctccgacactagaggtcggagtactgtcctccgacgcaaggcggagtactgtcctccg ggctgcggagtactgtcctccggcaaggtcggagtactgtcctccgacactagaggtcggagtactgtcctccga cgcaaggtcggagtactgtcctccgacactagaggtcggagtactgtcctccgacgcaaggtcggagtactgtcc tccgacactagaggtcggagtactgtcctccgacgcaaggcggagtactgtcctccgggctggcggagtactgtc ctccggcaagggtcgactctagagggtatataatggatcccatcgcgtctcagcctcactttgagctcctccaca cgaattccccgataccgtcgattcaaggagcttgcttgttctttttgcagaagctcagaataaacgctcaacttt ggcagatctaccgacttagcctttagtgaaccgtcagaattaattcagatcgatctaccagaaccgtcagatccg ctagagattacgccaaccgccaccatgggcagcgaagatgccaaaaacattaagaagggcccagcgccattctac ccactcgaagacgggaccgccggcgagcagctgcacaaagccatgaagcgctacgccctggtgcccggcaccatc gcctttaccgacgcacatatcgaggtggacattacctacgccgagtacttcgagatgagcgttcggctggcagaa gctatgaagcgctatgggctgaatacaaaccatcggatcgtggtgtgcagcgagaatagcttgcagttcttcatg cccgtgttgggtgccctgttcatcggtgtggctgtggccccagctaacgacatctacaacgagcgcgagctgctg aacagcatgggcatcagccagcccaccgtcgtattcgtgagcaagaaagggctgcaaaagatcctcaacgtgcaa aagaagctaccgatcatacaaaagatcatcatcatggatagcaagaccgactaccagggcttccaaagcatgtac accttcgtgacttcccatttgccacccggcttcaacgagtacgacttcgtgcccgagagcttcgaccgggacaaa accatcgccctgatcatgaacagtagtggcagtaccggattgcccaagggcgtagccctaccgcaccgcaccgct tgtgtccgattcagtcatgcccgcgaccccatcttcggcaaccagatcatccccgacaccgctatcctcagcgtg gtgccatttcaccacggcttcggcatgttcaccacgctgggctacttgatctgcggctttcgggtcgtgctcatg taccgcttcgaggaggagctattcttgcgcagcttgcaagactataagattcaatctgccctgctggtgcccaca ctatttagcttcttcgctaagagcactctcatcgacaagtacgacctaagcaacttgcacgagatcgccagcggc ggggcgccgctcagcaaggaggtaggtgaggccgtggccaaacgcttccacctaccaggcatccgccagggctac ggcctgacagaaacaaccagcgccattctgatcacccccgaaggggacgacaagcctggcgcagtaggcaaggtg gtgcccttcttcgaggctaaggtggtggacttggacaccggtaagacactgggtgtgaaccagcgcggcgagctg tgcgtccgtggccccatgatcatgagcggctacgttaacaaccccgaggctacaaacgctctcatcgacaaggac ggctggctgcacagcggcgacatcgcctactgggacgaggacgagcacttcttcatcgtggaccggctgaagagc ctgatcaaatacaagggctaccaggtagccccagccgaactggagagcatcctgctgcaacaccccaacatcttc gacgccggggtcgccggcctgcccgacgacgatgccggcgagctgcccgccgcagtcgtcgtgctggaacacggt aaaaccatgaccgagaaggagatcgtggactatgtggccagccaggttacaaccgccaagaagctgcgcggtggt gttgtgttcgtggacgaggtgcctaaaggactgaccggcaagttggacgcccgcaagatccgcgagattctcatt aaggccaagaagggcggcaagatcgccgtgaattctgcttgcaagaactggttcagtagcttaagccactttgtg atccaccttaacagccacggcttccctcccgaggtggaggagcaggccgccggcaccctgcccatgagctgcgcc caggagagcggcatggatagacaccctgctgcttgcgccagcgccaggatcaacgtgtcaggcggcagcggcgcc accaacttcagcctgctgaagcaggccggcgacgtggaggagaaccccggccccatgactagggcacatctgaca gaagtggaatcaaggctagaaagactggaacagctatttctactgatttttggcggctctggcggcggctccgga ggctcttacgggaaaatcgcggcattaaaggcggagaacgcagctctggaagccaagattgcagccttaaaagcg gagattgctgcgttagaggcaggctacggcggctctggcggcggctccggaggctcttacgggaaaatcgcggca ttaaaggcggagaacgcagctctggaagccaagattgcagccttaaaagcggagattgctgcgttagaggcaggc tacggcggctctggcggcggctccggaggctcttacgggaaaatcgcggcattaaaggcggagaacgcagctctg gaagccaagattgcagccttaaaagcggagattgctgcgttagaggcaggctacggcggctctggcggcggctcc ggaggctcttacgggaaaatcgcggcattaaaggcggagaacgcagctctggaagccaagattgcagccttaaaa gcggagattgctgcgttagaggcaggctacgatccaaaaaagaagagaaaggtagacacctaccgctacatctaa cttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttc actgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtcgtctatgatgaggatgttg gtggagagcatgtggaggaagtggatagggaaggttgtagagtagatccggttgaagtgatgagga taggaggag gactgcacatattgatcctcgtcacaaccacaccagcacacctcaaatcccacaccactcccacaattaccattc actcaacaaactcacacatcccacgataacgaattcaagcttgatatcattcaggacgagcctcagactccagcg taactggactgcaatcaactcactggctcaccttcacgggtgggcctttcttcggtagaaaatcaaaggatcttc ttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgttt gccggatcaagagctaccaactctttttccgaggtaactggcttcagcagagcgcagataccaaatactgttctt ctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctg ttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataag gcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgaga tacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggc agggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggttt cgccacctctgacttgagcatcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaac gcagaaaggcccacccgaaggtgagccaggtgattacatttgggccctcattaccaatgcttaatcagtgaggca cctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatgcgg gagggcttaccatctggccccagtgctgcaatgataccgcgagaaccacgctcaccggctccagatttatcagca ataaaccagccagccgggagggccgagcgcagaagtgatcctgcaactttatccgcctccatccagtctattaat tgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatc gtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcc cccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgccagaagtaagttggccgcagtgtta tcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtgagatgcttttctgtgactggt gagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggat aataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgtaaactctcaagg atcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttc accagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgt tgaatactcat

Claims

CLAIMS1. An engineered system for controlled expression of genes in a cell, comprising:a) monomers of an engineered dimeric transcription factor (Modular Artificial Dimerizing Transcription Factor, MADTF), each monomer (MADTF monomer) being a polypeptide comprising, or consisting of:a.1) a nucleic acid binding domain,a.2) a first dimerization domain,a.3) a second dimerization domain, different from the first dimerization domain, anda.4) an effector domain, configured to activate or repress gene transcription, upon binding of the MADTF dimer to a gene or to a gene regulatory element of the cell;andb) an engineered transcription factor’s inhibitory polypeptide (ATF), configured to inhibit MADTF, comprising, or consisting of:b.l) a first dimerization domain, andb.2) a second dimerization domain, different from the first dimerization domain,wherein at least one of the dimerization domains of ATF is configured to dimerize with a complementary dimerization domain of a MADTF monomer, preferably the second dimerization domain of ATF with the second dimerization domain of MADTF monomer; and wherein the affinity of dimerization between the at least one dimerization domain of ATF and the complementary dimerization domain of the MADTF monomer, is higher than the affinity of dimerization between each dimerization domain of a MADTF monomer with each dimerization domain of another MADTF monomer.

2. The system of claim 1, wherein ATF is configured to inhibit MADTF by inhibiting dimerization of MADTF monomers with each other.

3. The system of claims 1 or 2, wherein at least one dimerization domain of a MADTF monomer and / or of ATF, is a coiled-coiled homo-dimerization domain or a leucine zipper homo-dimerization domain.

4. The system of claim 3, wherein the first dimerization domain of MADTF (a.2) and / or the first dimerization domain of ATF (b.l) is selected from: a Gal4 homodimerization domain (Gal4 DD) encoded by a polynucleotide having sequence comprising, or consisting of, SEQ ID NO: 1, or a variant thereof; a truncated GAL4 homodimerization domain comprising or consisting of aminoacids 50 to 72 of the Gal4 protein; a truncated GAL4 dimerization domain encoded by a polynucleotide having sequence comprising or consisting of SEQ ID NO: 2, or variants thereof; a GCN4 dimerization domain encoded by a polynucleotide having sequence comprising, or consisting of, SEQ ID NO: 3, or a variant thereof; or a GCN3 dimerization domain, encoded by a polynucleotide having sequence comprising, or consisting of, SEQ ID NO: 4 (GCN4 DD); or a variant thereof.

5. The system of anyone of claims 1 to 4, wherein the second dimerization domain a MADTF monomer (a.3) or the second dimerization domain of ATF (b.2) is an heterodimerization domain selected from:at least one N7 peptide encoded by a polynucleotide having sequence comprising, or consisting of, SEQ ID NO: 5, or a variant thereof; at least one N8 peptide encoded by a polynucleotide having sequence comprising, or consisting of, SEQ ID NO: 6, or a variant thereof; a dimerization domain comprising from 2 to 4 copies of the N7 peptide or of the N8 peptide; a dimerization domain comprising two copies of the N8 peptides joined by a linker encoded by a polynucleotide having sequence comprising, or consisting of, SEQ ID NO: 7 (2N8), or a variant thereof; a dimerization domain comprising four copies of the N8 peptides joined by a linker encoded by a polynucleotide having sequence comprising, or consisting of, SEQ ID NO: 8 (4N8), or a variant thereof; a dimerization domain having sequence comprising, or consisting of, anyone of SEQ ID NO: 9-32;provided that the second dimerization domain of the MADTF monomer (a.3) and the second dimerization domain of ATF (b.2) are cognate heterodimerization domains.

6. The system of anyone of claims 1 to 5, wherein (a.l) the nucleic acid binding domain of MADTF is a DNA binding domain (DBD) with zinc fingers (ZF) or TALEs, preferably a ZF consisting of at least three zinc fingers (3F-ZF), at least four zinc fingers (4F-ZF), or at least six zinc fingers (6F-ZF), more preferably wherein each MADTF monomer of a MADTF dimer comprises one half of the DBD.

7. An engineered genetic circuit for controlled expression of genes in a cell comprising the system of anyone of claims 1 to 6, said genetic circuit further comprising at least one inducible transcriptional activator, wherein expression of MADTF and ATF is under the control of promoters that comprise binding sites for said transcriptional activator, wherein said inducible transcriptional activator is configured to bind to the promoter that drives the expression of the MADTF monomers and / or to the promoter that drives the expression of the ATF, wherein the MADTF dimer is configured to activate or repress the expression of a gene of interest, preferably an exogenous gene.

8. The genetic circuit of claim 7 configured to activate expression of MADTF and to inhibit the expression of ATF by binding of the transcriptional activator to their promoters (CIL-ON genetic circuit), wherein the transcriptional activator is rtTA inducible transcriptional activator, encoded by a polynucleotide having sequence SEQ ID NO: 43, the promoter that is activated by the inducible transcriptional activator is TRE3G promoter having sequence SEQ ID NO: 45, and the promoter that is inhibited by the inducible transcriptional activator is pCMV / TO promoter having sequence SEQ ID NO: 46, or variants thereof.

9. The genetic circuit of claim 7 configured to inhibit the expression of MADTF and to activate the expression of ATF by binding of the transcriptional activator to their promoters (CIL-OFF genetic circuit), wherein the transcriptional activator is rtTA inducible transcriptional activator, encoded by a polynucleotide having sequence SEQ ID NO: 43, the promoter that is activated by the inducible transcriptional activator is TRE3G promoter having sequence SEQ ID NO: 45, and the promoter that is inhibited by the inducible transcriptional activator is pCMV / TO promoter having sequence SEQ ID NO: 46, or variants thereof.

10. An expression vector for expressing the ATF of the system of anyone of claims 1-6.

11. The expression vector of claim 10 having sequence selected from anyone of SEQ ID NO: 40-42, and variants thereof.

12. The expression vector of anyone of claims 10 or 11 further expressing the MADTF of the system of anyone of claims 1-6.

13. The expression vector of claim 12 having sequence selected from SEQ ID NO: 57, 58 59, and variants thereof.

14. An expression vector system for expressing the MADTF and the ATF of the system of anyone of claims 1-6, comprising an expression vector having sequence selected from SEQ ID NO: 38 and 39, and an expression vector having sequence selected from SEQ ID NO: 40, 41 and 42.

15. An expression vector system for expressing the genetic circuit of anyone of claims 7-8, comprising an expression vector having sequence SEQ ID NO: 47, an expression vector having sequence SEQ ID NO: 48 and an expression vector having sequence SEQ ID NO: 49.

16. An expression vector system for expressing the genetic circuit of anyone of claims 7 or 9, comprising an expression vector having sequence SEQ ID NO: 47, an expression vector having sequence SEQ ID NO: 50 and an expression vector having sequence SEQ ID NO: 51.

17. A cell comprising the system of any one of claims 1-6, or the genetic circuits of anyone of claims 7-9, or the expression vector(s) of any one of claims 10-16, preferably said cell being an eukaryotic cell, more preferably being a mammalian cell.

18. Use of the system of any one of claims 1-6, or the genetic circuits of anyone of claims 7-9, or the expression vector(s) of any one of claims 10-16, or the cell of claim 17, in an in vitro or ex vivo method of production of recombinant proteins and / or in in vitro or ex vivo methods of engineering CAR-T cells and / or NK cells, and / or in an in vitro or ex vivo method of biosensing analytes.

19. The system of any one of claims 1-6, or the genetic circuits of anyone of claims 7-9, or the expression vector(s) of any one of claims 10-16, or the cell of claim 17, for use in a method of gene therapy.

20. The system of any one of claims 1-6, or the genetic circuits of anyone of claims 7-9, or the expression vector(s) of any one of claims 10-16, or the cell of claim 17, for use in a method of gene editing.

21. The system of any one of claims 1-6, or the genetic circuits of anyone of claims 7-9, or the expression vector(s) of any one of claims 10-16, or the cell of claim 17, for use in a method of cancer therapy.

22. An in vitro or ex vivo method for controlling gene expression in a cell, comprising introducing in the cell the system of any one of claims 1-6, or the genetic circuits of anyone of claims 7-9, or the expression vector(s) of any one of claims 10-16, by means of viral or non-viral delivery.

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