Method and expression system for producing biologically active molecules

Abstract

The invention relates to a method, expression system and related kit for producing at least one biologically active molecule, in particular a biotherapeutic, by optogenetic control of gene expression in at least one eukaryotic or prokaryotic cell, wherein the biologically active molecule comprises at least one protein of interest comprising at least two different subunits. In particular, one embodiment of the expression system according to the invention comprises three expression cassettes (2, 3, 4) for optogenetic control of the production of a protein of interest comprising two subunits (5, 6). A first expression cassette (2) comprises a first nucleic acid sequence (7) that is operably linked to a first promoter (8) and encodes a photosensitive transcription factor (9) comprising a gene expression controlling domain (10). The expression system (1) further comprises a second expression cassette (3) comprising a second nucleic acid sequence (11) encoding the first subunit (5) of the protein of interest. The second nucleic acid sequence (11) is operably linked to a second promoter (12) being operably linked to a transcription factor binding site (13) designed to bind the photosensitive transcription factor (9). The expression system (1) also comprises a third expression cassette (4) comprising a third nucleic acid sequence (15) encoding the second subunit (6) of the protein of interest and being operably linked to a third promoter (16).

Description

[0001] Ningaloo Biosystems GmbH, UNIV Bonn 24539WO

[0002] Method and expression system for producing biologically active molecules

[0003] Field of the invention:

[0004] The invention relates to a method, expression system and related kit for producing at least one biologically active molecule, in particular a biotherapeutic, by optogenetic control of gene expression in at least one eukaryotic or prokaryotic cell, wherein the biologically active molecule comprises at least one protein of interest comprising at least two different subunits. In particular, the invention concerns biological production systems, primarily based on living cells cultivated in bioreactors or other culture vessels, and more particularly a novel genetic expression system tailored for optimizing biotherapeutics production through optogenetic regulation.

[0005] Background of the Invention

[0006] Biopharmaceuticals are a class of medical drugs produced using biotechnology. They are typically made from proteins and / or nucleic acids that are derived from living organisms, including humans, animals, plants, and microorganisms. These drugs are used for therapeutic or diagnostic purposes and include a wide range of products such as:

[0007] • Antibodies and antibody derivatives: Targeted therapies for conditions like cancer and autoimmune diseases.

[0008] • Vaccines: Preventative treatments for infectious diseases.

[0009] • Recombinant proteins: Engineered proteins used to treat diseases like diabetes (e.g., insulin) and growth hormone deficiencies.

[0010] • Gene therapies: Treatments that involve modifying a person's genes to treat or cure disease.

[0011] • Cell therapies: Using living cells to treat or repair tissues and organs.

[0012] In recent years, biopharmaceuticals have demonstrated their potential to provide breakthrough treatments for many life-threatening and chronic conditions, offering more precise and effective therapeutic options compared to traditional small molecule drugs.

[0013] Many of these biopharmaceuticals are made in cell culture, either using microbial systems or mammalian cell lines as production host. These cell systems are typically genetically modified to express recombinant genes encoding the genes for the protein or RNA subcomponents of the drug substance. Until now, genetic expression systems for producing proteins, especially biotherapeutics, have been either constitutively expressed or controlled solely through conventional chemical signals, predominantly by the addition of specific chemicals to the culture. For instance, the addition of cumate (4-isopropylbenzoic acid) can induce the production of certain proteins toxic or challenging for cells to express, thereby initiating the biosynthesis of these proteins only once the cell has attained specific desirable metabolic and / or physiological states. However, such control mechanisms are severely limited: the introduction of chemical compounds is irreversible or may require laborious removal to be reversed, the related regulation is inflexible, typically single-channeled (addressing only a single gene per time), and often characterized by limited dynamic range. Moreover, modern biotherapeutics frequently comprise multiple components, such as in the case of bispecific antibodies, consisting of three or more protein chains. Unbalanced quantities or concentrations of subcomponents of the biotherapeutic can lead to increased levels of unintended by-products (e.g., greater amounts of unassembled subcomponents, homomultimers, assemblies with out-of-balance numbers of subcomponents) and reduced yields of the intended product. To ameliorate the unbalanced expression of different genes within a cell, countermeasures have been employed, e.g., through supertransfection (Carver et al., 2020). Nevertheless, such a readjustment of relative or absolute amounts of gene products is entirely static.

[0014] Optogenetics represents a relatively novel method for controlling the activity of living cells using light. An example are the light-sensitive systems which provide rapid, noninvasive and reversible control of gene expression upon light exposure (Shimizu-Sato et al., 2002; Pastrana, 2011 ). As a consequence, light-activated enzymes and transcription factors allow precise control of the biochemical pathways at the level of individual cells. While the temporal response to biochemicals at the genetic level is rather slow, as the biochemical processes take hours or days to be effective, optogenetic methods operate on a millisecond timescale, thus allowing to control gene expression not only in real-time.

[0015] Prior art

[0016] US 2021 / 0147855 A1 discloses optogenetic circuits for controlling protein production with light in Escherichia coli. Using these circuits, gene expression can be induced in darkness and repressed under blue light. The disclosed system thus uses light as a suitable alternative to chemical induction for microbial production of chemicals and proteins. In particular, a system is provided, containing (1 ) a first sequence encoding a first repressor, under a first promoter, the first promoter being a light-controllable promoter, (2) a second sequence encoding a second repressor, under a second promoter, the second promoter being controllable by the first repressor, and (3) a third sequence encoding a gene of interest under a third promoter, the third promoter being controllable by the second repressor.

[0017] WO 2013 / 074911 A1 discloses a system to control gene expression with blue light, in particular methods for light-dependent gene regulation using a light- responsive DNA-binding protein. The system includes the photosensitive protein EL222 (E. Htoralis 222) containing a LOV-domain as well as a DNA binding domain (DBD) and being fused both with one nuclear localization signal (NLS) and one activation domain. The system further includes a recombinant nucleic acid molecule containing five EL222 DNA binding sites fused to a minimal promoter and allowing moderate to strong blue light induced transcription. The EL222 protein is modified for use in eukaryotic cells by adding a nuclear localization sequence (NLS) and a transcriptional activation domain (VP16-AD) from the herpes simplex virus VP 16 protein. The DNA binding sites are assembled in tandem repeats upstream of a minimal promoter. In the dark, the VP16-EL222 chimera cannot activate a luciferase reporter construct under the control of five-tandem copies of EL222 DNA binding sites (EL222-DBS), while exposure to blue light activates the VP16-EL222 protein allowing it to turn on luciferase transcription.

[0018] Accordingly, optogenetics represents a method for controlling numerous processes within living cells through the genetic integration of photoreceptors (e.g., Rost et al., 2017; Hansen et al, 2020). By coupling photoreceptors with effector proteins such as enzymes, protein dimerization domains, ion channels, transcription factors, or signal transduction factors different processes within cells can be governed using light.

[0019] Summary of the invention

[0020] It is the object of the invention to provide a method and expression system for producing complex biologically active molecules, in particular complex biopharmaceuticals / biotherapeutics, enabling full flexibility in process control as well as real-time regulation of the entire bioproduction process.

[0021] The object is met by providing a method for producing at least one biologically active molecule, in particular a biotherapeutic, by optogenetic control of gene expression in at least one eukaryotic or prokaryotic cell, wherein the biologically active molecule comprises at least one protein of interest comprising at least two different subunits, and wherein the method comprises: introducing into the cell with at least one first expression cassette comprising at least one first nucleic acid sequence linked to a first promoter, wherein the first nucleic acid sequence encodes at least one photosensitive transcription factor, introducing into the cell with a second expression cassette comprising at least one second nucleic acid sequence encoding a first of the at least two subunits of the protein of interest and being operably linked to a second promoter, the second promoter being operably linked to at least one transcription factor binding site, and introducing into the cell a third expression cassette comprising at least one third nucleic acid sequence encoding at least a second of the at least two subunits of the protein of interest and being operably linked to a third promoter, wherein expression of the second nucleic acid sequence is controlled through activation or repression of the second promoter by illuminating the cell with light comprising at least one wavelength that triggers at least one structural change of the photosensitive transcription factor.

[0022] Since illumination of the cells can be controlled through a software controlling a hardware for generating or regulating light, the method according to the invention thus may use, for example, an expression system comprising control interfaces operating bio-digitally instead of purely chemically-analog, particularly a polynucleotide encoding a transcription factor under the control of a promoter, alongside an optimized light-activatable protein-encoding polynucleotide that modulates promoter activity in response to light stimuli. This innovative approach enables dynamic modulation of gene expression, addressing key challenges in biotherapeutics manufacturing. The genetic expression system and associated methodologies used in the method according to the invention represent a significant advancement in biotherapeutics production, offering unparalleled control and optimization opportunities for the development of complex nextgeneration biopharmaceuticals. That is, the method according to the invention enables full flexibility in process control as well as real-time process regulation of the entire production of complex biopharmaceuticals, so that it is possible to balance or individually temporally dose / pulse multiple components (subunits) within complex protein products, enhancing product quality and therapeutic efficacy. Basically, the application of optogenetics to biopharmaceutical production not only allows to manipulate bioprocesses with unparalleled temporal precision and favorable side effect profiles but also offers light sensors introduced into cells via genetical manipulation as novel control elements, providing the opportunity to establish unique bio-physical interfaces for more automated bioproduction of the future. Additionally, feeding analytical data recorded during the cell culture process as data inputs into the control system of an illumination apparatus executing illumination programs used to temporally control intracellular processes permit the establishment of closed real-time bioprocess control loops as well as seamless integration of advanced data processing methods like machine learning / systems for artificial intelligence into future, fully controlled bioproduction environments, e.g. to automatically avoid process deviations and achieve optimal output.

[0023] It is a particular benefit of the method according to the invention that expression / secretion of a protein of interest comprising at least two different subunits can be regulated by the presence and relative concentrations and / or temporally controlled expression of different amounts of at least one light- dependently expressed subunit. For example, limiting expression of a lightdependent subunit restrains the production of the assembled protein of interest, while enhancing expression of a light-dependent subunit promotes the production of the assembled protein of interest. Thus, expression / secretion of complex biologically active molecules can be reliably and precisely regulated by different combination of light-dependent subunits of a protein or protein-nucleic acid complex of interest and the expression of the protein or protein-nucleic acid complex of interest and by-products can be influenced by the relative amounts and / or timing of the subunits expressed. Exact balancing and / or timing of expression of the subunits of a product (protein or protein-nucleic acid complex of interest) has a striking effect on the formation of intended product vs byproducts, their relative concentrations, and thus on unpurified product quality, which has an impact on necessary purification efforts, yields of purified product and thus in sum production costs per unit of the product.

[0024] “Structural change” as used herein in the sense of the invention refers to any change in the photosensitive transcription factor’s conformation and / or secondary or tertiary structure that causes, for example, an exposition of a formerly hidden domain, a dimerization, a multimerization, or the like. For example, the light induction may cause di- / multimerization of a photosensitive transcription factor, whereas said di- / multimerization is a prerequisite to either bind to a transcription factor binding site or to another factor that activates gene expression.

[0025] In an advantageous embodiment of the invention, the structural change causes binding or release of the photosensitive transcription factor to / from the transcription factor binding site. For example, if the photosensitive transcription factor binds to the transcription factor binding site when the cell is illuminated using a light source emitting at least one wavelength that triggers this binding, the gene expression controlling domain can control expression of the second nucleic acid sequence encoding a first of the at least two subunits of the protein of interest. That is, expression of the second nucleic acid sequence can be directly (i.e. in real-time) controlled through light dose-dependent activation (upregulation) or repression (downregulation) of the second promoter by switching on a suitable light source. Vice versa, if the light triggers release of the photosensitive transcription factor from the transcription factor binding site, the activating or repressing effect of the gene expression controlling domain can be promptly terminated by switching the light source on. The third nucleic acid sequence encoding the at least second of the at least two subunits of the protein of interest is expressed under the control of the third promoter which may be a constitutive promoter. Since light dose or pulsing can be used to modulate the stimulus, regulation of the production of a complex protein of interest comprising at least two subunits can thus be controlled in real-time, e.g. by balancing or temporally controlling at least the expression of the second nucleic acid sequence. However, it is also possible to control expression of the third (and / or further) nucleic acid sequence(s) through illumination by using a third (and / or further) promoter being operably linked to at least one photosensitive or chemically controlled transcription factor binding site. In such an embodiment of the invention, additional fine-tuning of the production process can be achieved.

[0026] In another advantageous embodiment of the invention, the structural change causes nuclear trafficking, i.e. nuclear import and / or export, of the photosensitive transcription factor. Accordingly, the photosensitive transcription factor can bind to a promoter or to another factor binding to a promotor to activate or repress gene expression (e.g., expression of the second nucleic acid) when imported into the nucleus, or reduce transcriptional activation or repression when exported from the nucleus.

[0027] The biologically active molecules may be, for example, blood factors, thrombolytic agents, hormones such as insulin or growth hormones, cytokines, peptides, receptors, chaperons, structural proteins, motor proteins, signaling peptides or proteins, adhesion proteins, defense or transport proteins, protease inhibitors, toxins or venoms, vaccines, viral vectors, virus-like particles, therapeutic enzymes, antibodies, nanobodies or protein-nucleic acid complexes, hematopoietic growth factors such as erythropoietin, interferons, interleukins, chimeric antigen receptors, protein nanoparticles, or protein scaffolds. Basically, the biologically active moleculecan be any biopharmaceutical or biotherapeutic, i.e. any pharmaceutical drug product manufactured in, extracted from, and / or semisynthesized by biological sources such as living cells, cell extracts, cell components, and / or artificial biochemical compounds or systems. In an advantageous approach, the method according to the invention can further be employed for the development of a stably transfected cell line optimized for consistent biopharmaceutical production, ensuring reliability and scalability in manufacturing processes.

[0028] In a further advantageous embodiment of the invention, the photosensitive transcription factor comprises at least one gene expression controlling domain and / or forms a complex with at least one gene expression controlling domain, wherein the gene expression controlling domain comprises a transcriptional activation domain or a transcriptional repression domain. For example, if the photosensitive transcription factor binds to the transcription factor binding site when the cell is illuminated using a light source emitting at least one wavelength that triggers this binding, the gene expression controlling domain can control expression of the second nucleic acid sequence encoding a first of the at least two subunits of the protein of interest. To this end, the gene expression controlling domain may comprise a transcriptional activation domain, such as VP16, for activating the second promoter, or a transcriptional repression domain, such as KRAB, for repressing the second promoter. In an advantageous embodiment of the invention, the transcriptional activation domain is selected from the group consisting of VP16, VP64, p65, RTA, and any combination thereof. In a preferred embodiment, the transcriptional activation domain comprises VP64 as four repetitions of one region of the herpes simplex virus VP 16 transcription activation domain (TAD), each separated by one Glycine-Serine (GS) linker, p65 as a fragment of the human RelA TAD, and RTA as the transcriptional activation domain from the human Epstein-Barr virus. Additionally, VP64 and p65 sequences are separated by an alanine-containing linker, while p65 and RTA are separated by a triple GS-linker. The combination of these activation domains results in significantly enhanced activation of the second promoter.

[0029] In another advantageous embodiment of the invention, the first promoter is a constitutive promoter, or the first promoter is operably linked to at least one regulatory sequence. If the first promoter is a constitutive promoter such as the cytomegalovirus (CMV) immediate-early promoter, RNA Pol III promoter H1 or phosphoglycerate kinase 1 (PGK) promoter, the photosensitive transcription factor is continually expressed without the need to induce the expression. The first expression cassette thus represents a non-regulated photoswitch providing a constant level of photosensitive molecules within the cell sufficient to activate or repress at least one of the second or the third expression cassette. In this case, the amount of the photosensitive transcription factor within the cell is always high, so that the transcription factor level in its active state induced by illumination with a light source is not a limiting factor for the production of the protein of interest.

[0030] Alternatively, if the first promoter is operably linked to at least one regulatory sequence, expression of the photosensitive transcription factor can be controlled, for example, to avoid unnecessary expression thereof. In the latter embodiment, the regulatory sequence may comprise a transcription factor binding site, wherein the first promoter and thus expression of the first nucleic acid sequence encoding the photosensitive transcription factor is induced or stimulated (upregulated) by illuminating the cell with light comprising at least one wavelength that triggers binding of the photosensitive transcription factor to a transcription factor binding site of the regulatory sequence. In this advantageous embodiment of the invention, the first expression cassette represents a self-stimulating photoswitch providing a high number of photosensitive transcription factor molecules within the cell only upon illumination. In this embodiment a lower number of photosensitive transcription factor molecules is expressed in the dark through basal activity of the first promoter that is sufficient to initiate higher expression of the gene encoding itself upon commencing illumination. By limiting the expression of the photosensitive transcription factor, possible cytotoxic effects or metabolic burden of high-level expression can be eliminated, or at least reduced and higher dynamic ranges can be achieved though light-dependent signal amplification via self-regulation of the photosensitive transcription factor.

[0031] In another advantageous embodiment of the invention, the third promoter is a constitutive promoter. If the third promoter is a constitutive promoter, the third nucleic acid sequence is continually expressed without the need to induce the expression. In this embodiment, expression of the third nucleic acid is not controlled and thus fine-tuning of the production (assembly) of the protein of interest is essentially accomplished by controlling the expression of the second nucleic acid. Alternatively, the third promoter can be operably linked to at least one transcription factor binding site, wherein expression of the third nucleic acid sequence is controlled through activation or repression of the third promoter by illuminating the cell with light comprising at least one wavelength that triggers at least one structural change of said photosensitive transcription factor or another photosensitive transcription factor. Preferably, the photosensitive transcription factor is the one encoded by the first nucleic acid sequence. But optionally, it might also be advantageous in certain applications that a different photosensitive transcription factor is provided in order to control the third promoter. That is, the further promoter sequence might be controlled by the same or another independent photosensitive transcription factor, e.g., sensitive to another wavelength of light.

[0032] The structural change may cause binding or release of the photosensitive transcription factor to / from the transcription factor binding site, or binding or release between at least one DNA binding subunit and at least one transcription activating subunit of the transcription factor. Accordingly, if light triggers the binding of a photosensitive transcription factor to the transcription factor binding site, expression of the third nucleic acid sequence can be controlled in real-time through activation (upregulation) or repression (downregulation) of the third promoter by switching-on a suitable light source. If light triggers the release of a photosensitive transcription factor from the transcription factor binding site, switching-on the light source promptly terminates the activating or repressing effect of the gene expression controlling domain. Since light dose or pulsing schemes can be used to modulate the stimulus, regulation of the production of a complex protein of interest comprising at least two subunits can thus be controlled in real-time by balancing or temporally dose / pulse the expression of both the second and the third nucleic acid sequence. In this advantageous embodiment of the invention, additional fine-tuning of the production process can be achieved.

[0033] The structural change may also cause nuclear trafficking, i.e. nuclear import and / or export, of the photosensitive transcription factor. Accordingly, the photosensitive transcription factor can bind to a promoter or to another factor binding to a promotor to activate or repress expression of the third nucleic acid sequence when imported into the nucleus, or reduce transcriptional activation or repression when exported from the nucleus.

[0034] For example, if the protein of interest is an antibody, one of the at least two subunits of the protein of interest may be a light chain of the antibody and another one of the at least two subunits of the protein of interest may be a heavy chain or a fusion of a light and a heavy chain of the antibody. That is, for example, the first of the at least two subunits of the protein of interest may be a light chain of the antibody and the second of the at least two subunits of the protein of interest may be a heavy chain of the antibody. Vice versa, the first of the at least two subunits of the protein of interest may be a heavy chain of the antibody and the second of the at least two subunits of the protein of interest may be a light chain of the antibody. Consequently, the method according to the invention allows for optogenetic control of monoclonal antibody synthesis, offering tailored production strategies and enhanced antibody engineering capabilities.

[0035] In another advantageous embodiment of the invention, at least one additional expression cassette is introduced into the cell, the additional expression cassette comprising at least one further nucleic acid sequence encoding at least one further subunit of the at least two subunits of the protein of interest and being operably linked to a further promoter sequence. That is, it is also possible to control expression of any further nucleic acid sequence and thus further subunits of the protein of interest through illumination by using at least one further promoter being operably linked to at least one transcription factor binding site. In such an embodiment of the invention, additional fine-tuning of the entire production process can be achieved. Moreover, full flexibility in process control as well as real-time process regulation of the production of complex biopharmaceuticals is enabled, so that it is possible to balance multiple subunits within complex protein products, enhancing product quality and therapeutic efficacy.

[0036] In this embodiment, the further promoter sequence may comprise a constitutive promoter. If the further promoter is constitutive, the third nucleic acid sequence is continually expressed without the need to induce the expression. In this embodiment, the expression of each further nucleic acid is not controlled and thus fine-tuning of the entire production (assembly) of the protein of interest which may comprise several subunits is essentially accomplished by controlling the expression of the second, and optionally the third, nucleic acid. Alternatively, at least one of the further promoter sequences can be operably linked to at least one transcription factor binding site, wherein expression of the further nucleic acid sequence is controlled through activation or repression of the further promoter sequence by illuminating the cell with light comprising at least one wavelength that triggers at least one structural change of said photosensitive transcription factor or another photosensitive transcription factor. Preferably, the photosensitive transcription factor is the one encoded by the first nucleic acid sequence. But optionally, it might also be advantageous in certain applications that a different photosensitive transcription factor is provided in order to control the further promoter sequence. That is, the further promoter sequence might be controlled by the same or another independent photosensitive transcription factor, e.g., sensitive to another wavelength of light.

[0037] The structural change may cause binding or release of the photosensitive transcription factor to / from the transcription factor binding site, or binding or release between at least one DNA binding subunit and at least one transcription activating subunit of the transcription factor. Accordingly, if light triggers the binding of a photosensitive transcription factor to the transcription factor binding site, expression of at least one of the further nucleic acid sequences can be controlled in real-time through activation (upregulation) or repression (downregulation) of the further promoter by switching on a suitable light source. If the light triggers release of the photosensitive transcription factor from the transcription factor binding site, switching-on the light source promptly terminates the activating or repressing effect of the gene expression controlling domain. Regulation of the production of complex proteins of interest comprising several subunits can thus be controlled in real-time by balancing the expression of the second, optionally the third, and at least one of the further nucleic acid sequences. In this advantageous embodiment of the invention, additional fine- tuning of the entire production process can be achieved.

[0038] The structural change may also cause nuclear trafficking, i.e. nuclear import and / or export, of the photosensitive transcription factor. Accordingly, the photosensitive transcription factor can bind to a promoter or to another factor binding to a promotor to activate or repress expression of the further nucleic acid sequence(s) when imported into the nucleus, or reduce transcriptional activation or repression when exported from the nucleus.

[0039] For example, the protein of interest may be a bispecific antibody, wherein one of the at least two subunits of the protein of interest may be a light chain of the antibody, another one of the at least two subunits of the protein of interest may be a heavy chain of the antibody, and at least one further subunit of the at least two subunits of the protein of interest may be a single chain variable fragment (scFv), an antigen binding fragment (Fab), or a fusion of a light and a heavy chain of the antibody. For example, the first of at least two subunits might be a first light chain, the second of the at least two subunits a first heavy chain, the third of the at least two subunits a second light chain and the fourth of the at least two subunits a second heavy chain. This option includes the production of two different heavy chains and two different light chains (w / o hybrid). Moreover, the method according to the invention might also be used to produce, for example, at least two different single chain variable fragments (= hybrid chains) together (w / o light chain or heavy chain). The method according to the invention can thus modulate bispecific antibody production, enabling the improved production of advanced therapeutic molecules with multiple binding specificities. According to the invention, all expression cassettes encoding a subunit of the protein of interest can be controlled by the same photosensitive transcription factor. Alternatively, different expression cassettes can be controlled by different photosensitive transcription factors, or at least one of the expression cassettes can be controlled by a different photosensitive transcription factor while the other expression cassettes are controlled by the same photosensitive transcription factor, and so on.

[0040] For example, the wavelength of the light may be selected from a wavelength between 400 nm and 500 nm (blue light) or between 640 and 780 nm (red and far-red light).

[0041] Basically, the optogenetic method according to the invention can be combined with other methods, for example, with chemical control methods, especially for the production of different subcomponents of the biologically active molecule.

[0042] The object is further met by providing an expression system for producing at least one biologically active molecule, in particular a biotherapeutic, by optogenetic control of gene expression in at least one eukaryotic or prokaryotic cell, wherein the biologically active molecule comprises at least one protein of interest comprising at least two subunits, said expression system comprising: at least one first expression cassette comprising at least one first nucleic acid sequence linked to a first promoter, wherein the first nucleic acid sequence encodes at least one photosensitive transcription factor comprising a gene expression controlling domain and / or having a structure that enables forming a complex with at least one gene expression controlling domain, a second expression cassette comprising at least one second nucleic acid sequence encoding a first of the at least two subunits of the protein of interest and being operably linked to a second promoter, wherein the second promoter is operably linked to at least one transcription factor binding site and activatable or repressible by illumination with light comprising at least one wavelength that triggers at least one structural change of the photosensitive transcription factor, and a third expression cassette comprising at least one third nucleic acid sequence encoding at least a second of the at least two subunits of the protein of interest and being operably linked to a third promoter.

[0043] For example, since illumination of the cells can be controlled through a software controlling a hardware for generating or regulating light, the expression system according to the invention may comprise control interfaces operating bio-digitally instead of purely chemically-analog, in particular at least one polynucleotide encoding at least one transcription factor under the control of at least one promoter, alongside at least one light-activatable protein-encoding polynucleotide that modulates promoter activity in response to light stimuli. This innovative approach enables dynamic modulation of gene expression, addressing key challenges in biotherapeutics manufacturing. The expression system according to the invention thus represents a significant advancement in biotherapeutics production, offering unparalleled control and optimization opportunities for the development of complex next-generation biopharmaceuticals. That is, the expression system according to the invention enables full flexibility in process control as well as real-time process regulation of the entire production of complex biopharmaceuticals, so that it is possible to balance and / or individually temporally dose / pulse multiple components (subunits) within complex protein products, enhancing product quality and thus purity of the product before purification and / or production efficiency by reducing by-products that are either inactive or show unintended activities. Reducing by-products also leads to increased therapeutic efficacy, since more effective molecules are provided with the same amount of protein. In turn, if smaller relative amounts of by-products need to be separated from the intended protein or protein-nucleic acid complex of interest, subsequent purification steps might be simplified or omitted, resulting in time and / or material savings as well as reduced loss of the intended protein or protein-nucleic acid complex.

[0044] It is a particular benefit of the expression system according to the invention that expression / secretion of a protein of interest comprising at least two different subunits can be regulated by the presence and relative concentrations and / or temporally controlled expression of different amounts of at least one light- dependently expressed subunit. For example, limiting expression of a lightdependent subunit restrains the production of the assembled protein of interest, while enhancing expression of a light-dependent subunit promotes the production of the assembled protein of interest. Thus, expression / secretion of complex biologically active molecules can be reliably and precisely regulated by different combination of light-dependent subunits of a protein of interest and the expression of the protein of interest and by-products can be influenced by the relative amounts and / or timing of the subunits expressed. Exact balancing and / or timing of expression of the subunits of a product (protein of interest) has a striking effect on the formation of intended product vs by-products, their relative concentrations, and thus on unpurified product quality, which has an impact on necessary purification efforts, yields of purified product and thus in sum production costs per unit of the product.

[0045] “Structural change” as used herein in the sense of the invention refers to any change in the photosensitive transcription factor’s conformation and / or tertiary structure that causes, for example, an exposition of a formerly hidden domain, a dimerization, a multimerization, or the like. For example, the light induction may cause di- / multimerization of a photosensitive transcription factor, whereas said di- / multimerization is a prerequisite to either bind to a transcription factor binding site or binding to another factor that activates gene expression.

[0046] The structural change may cause, for example, binding or release of the photosensitive transcription factor to / from the transcription factor binding site. For example, if the photosensitive transcription factor binds to the transcription factor binding site when the cell is illuminated using a light source emitting at least one wavelength that triggers this binding, the gene expression controlling domain can control expression of the second nucleic acid sequence encoding a first of the at least two subunits of the protein of interest. That is, expression of the second nucleic acid sequence can be directly (i.e. in real-time) controlled through light dose-dependent activation (upregulation) or repression (downregulation) of the second promoter by switching on a suitable light source. Vice versa, if the light triggers release of the photosensitive transcription factor from the transcription factor binding site, the activating or repressing effect of the gene expression controlling domain can be promptly terminated by switching the light source on. The third nucleic acid sequence encoding the at least second of the at least two subunits of the protein of interest is expressed under the control of the third promoter which may be a constitutive promoter. Since light dose or pulsing can be used to modulate the stimulus, regulation of the production of a complex protein of interest comprising at least two subunits can thus be controlled in realtime by balancing at least the expression of the second nucleic acid sequence. However, it is also possible to control expression of the third (and / or further) nucleic acid sequence(s) through illumination by using a third (and / or further) promoter being operably linked to at least one photosensitive or chemically controlled transcription factor binding site. In such an embodiment of the invention, additional fine-tuning of the production process can be achieved.

[0047] The structural change may further cause, alternatively or additionally, nuclear trafficking, i.e. nuclear import and / or export, of the photosensitive transcription factor. Accordingly, the photosensitive transcription factor can bind to a promoter or to another factor binding to a promoter to activate or repress gene expression only when located in the nucleus, which in turn can be controlled by exposure to light.

[0048] The expression system according to the invention may further comprise, for example, at least one artificial polynucleotide designed for fine-tuning of the expression of complex biotherapeutic products, allowing for precise control over product characteristics and performance. The expression system according to the invention may also comprise, for example, at least one artificial polynucleotide encoding a light-activatable (photo-sensitive) transcription factor efficiently activating a specific promoter in living cells such as, for example, mammalian, insect, bacterial, fungal or plant cells. Moreover, the expression system according to the invention may comprise at least one artificial polynucleotide encoding a red or blue light activated transcription factor efficiently activating a specific promoter in mammalian cells.

[0049] The biologically active molecules may be, for example, blood factors, thrombolytic agents, hormones such as insulin or growth hormones, cytokines, peptides, receptors, chaperons, structural proteins, motor proteins, signaling peptides or proteins, adhesion proteins, defense or transport proteins, protease inhibitors, toxins or venoms, nanobodies or protein-nucleic acid complexes, hematopoietic growth factors such as erythropoietin, interferons, interleukins as well as vaccines, therapeutic enzymes, chimeric antigen receptors, protein nanoparticles, protein scaffolds, virus-like particles, viral vectors, or antibodies. Basically, the biologically active molecule can be any biopharmaceutical and / or biotherapeutic, i.e. any pharmaceutical drug product manufactured in, extracted from, and / or semi synthesized by biological sources such as living cells, cell extracts, cell components, and / or artificial biochemical compounds or systems. In an advantageous approach, the method according to the invention can further be employed for the development of a stably transfected cell line optimized for consistent biopharmaceutical production, ensuring reliability and scalability in manufacturing processes.

[0050] In an advantageous embodiment of the invention, the expression system further comprises additional expression cassettes, each additional expression cassette comprising at least one further nucleic acid sequence encoding at least one further subunit of the at least two subunits of the protein of interest and being operably linked to a further promoter sequence. That is, by providing additional expression cassettes, controlling the expression of further nucleic acid sequences and thus further subunits of the protein of interest through illumination can be ensured, preferably by using at least one further promoter being operably linked to at least one transcription factor binding site. This allows for precise and effective production of complex biopharmaceuticals, wherein it is possible to balance or temporally dose / pulse multiple subunits within complex protein products, enhancing product quality and thus purity of the product before purification and / or production efficiency by reducing by-products that are either inactive or show unintended activities. Reducing by-products also leads to increased therapeutic efficacy, since more effective molecules are provided with the same amount of protein. In turn, if smaller relative amounts of by-products need to be separated from the intended protein or protein-nucleic acid complex of interest, subsequent purification steps might be simplified or omitted, resulting in time and / or material savings as well as reduced loss of the intended protein or protein-nucleic acid complex.

[0051] Each promotor I promoter sequence of the expression system according to the invention may comprise a constitutive promoter or may be operably linked to at least one transcription factor binding site, wherein expression of the nucleic acid sequence (gene) regulated by the promoter is controlled through activation or repression of the promoter sequence by illuminating the cell with light comprising at least one wavelength that triggers at least one structural change of a photosensitive transcription factor.

[0052] In another advantageous embodiment of the invention, the photosensitive transcription factor may comprise at least one light-oxygen-voltage-sensing domain (LOV domain), preferably EL222, or at least one cryptochrome (CRY), or at least one phytochrome, preferably phytochrome B. Alternatively or additionally, the transcription factor binding site comprises at least binding site selected from the group consisting of at least one (C120), GAL4, TetR, lexA, and lacR binding site.

[0053] In a further advantageous embodiment of the invention, the gene expression controlling domain of the photosensitive transcription factor may comprise at least one transcriptional activation domain, preferably selected from the group consisting of VP 16, VP64, p65, RTA, and any combination thereof, or a transcriptional repression domain, preferably KRAB. The photosensitive transcription factor may further comprise at least one nuclear localization signal (NLS) and / or at least one DNA-binding domain (DBD). An advantageous combination of transcriptional activation domains is VP64-p65-RTA, preferably operably linked to EL222. This combination surprisingly provides a significantly enhanced, very effective activation of the second promoter, in particular of an inducible minimum promoter.

[0054] The object is also met by providing a kit or composition for producing at least one biologically active molecule, in particular a biotherapeutic, wherein the biologically active molecule comprises at least one protein of interest comprising at least two subunits, by optogenetic control of gene expression of at least one of the at least two subunits in at least one eukaryotic or prokaryotic cell, comprising the expression system as described above.

[0055] The object is further met by providing a photosensitive transcription factor (photoswitch) comprising the blue light-activated light-oxygen-voltage receptor EL222 from Erythrobacter I itoral is and the three transcriptional activator domains VP64, p65, and Rta in tandem. The resultant photoswitch, dubbed “DEL-VPR”, allows an up to 570-fold induction of target gene expression by blue light, thereby achieving expression levels of strong constitutive promoters. DEL-VPR can be used, for example, to enable light-induced expression of complex monoclonal and bispecific antibodies with reduced byproduct expression and increased yield of functional protein complexes. This approach thus offers temporally controlled yet strong gene expression and applies to academic and industrial settings.

[0056] For example, the photosensitive transcription factor according to the invention may comprise the light-oxygen-voltage-sensing domain EL222, a transcriptional activation domain comprising VP64, p65, and RTA, and two nuclear localization signals. Preferably, the photosensitive transcription factor comprises the amino acid sequence according to SEQ ID NO: 1 set forth hereunder.

[0057] PKKKRKVDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDMLAAAEFQYLPDTDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPR RIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAP PQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGT LSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHT TEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMD FSALLSQISSGSGSGSRDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFH PPGSPWANRPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHL LEDPDEETSQAVKALREMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTL ESMTEDLNLDSPLTPELNEILDTFLNDECLLHAMHISTGLSIFDTSLFEFGADDT RVE VQ P P AQ WVL D L I E AS P I AS VVS DPRLADNPLIAINQAFTD LTG YS E E E C VG RNCRFLAGSGTEPWLTDKIRQGVREHKPVLVEILNYKKDGTPFRNAVLVAPIY DDDDELLYFLGSQVEVDDDQPNMGMARRERAAEMLRTLSPRQLEVTTLVAS GLRNKEVAARLGLSEKTVKMHRGLVMEKLNLKTSADLVRIAVEAGIEFSASKR

[0058] PAATKKAGQAKKKK (SEQ ID NO: 1 )

[0059] SEQ ID NO: 1 consists of the following sub-sequences:

[0060] PKKKRKV (SV40 NLS, SEQ ID NO: 2)

[0061] DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDML (VP64, SEQ ID NO: 3),

[0062] AAA (Linker, SEQ ID NO: 4),

[0063] EFQYLPDTDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPRRIAVPSRS SASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQA PAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQ LQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLME YPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQI SS (RelA (p65) AD, SEQ ID NO: 5),

[0064] GSGSGS (Linker, SEQ ID NO: 6),

[0065] RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWANRPLPASL APTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKALR EMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPE LNEILDTFLNDECLLHAMHISTGLSIFDTSLF (Rta AD, SEQ ID NO: 7),

[0066] PKKKRKVDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDMLAAAEFQYLPDTDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPR RIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAP PQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGT LSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHT TEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMD FSALLSQISSGSGSGSRDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFH PPGSPWANRPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHL LEDPDEETSQAVKALREMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTL ESMTEDLNLDSPLTPELNEILDTFLNDECLLHAMHISTGLSIFDTSLFEFGADDT RVE VQ P P AQ WVL D L I E AS P I AS VVS DPRLADNPLIAINQAFTD LTG YS E E E C VG RNCRFLAGSGTEPWLTDKIRQGVREHKPVLVEILNYKKDGTPFRNAVLVAPIY DDDDELLYFLGSQVEVDDDQPNMGMARRERAAEMLRTLSPRQLEVTTLVAS GLRNKEVAARLGLSEKTVKMHRGLVMEKLNLKTSADLVRIAVEAGI (EL222, SEQ ID NO: 8),

[0067] EFSAS (Linker, SEQ ID NO: 9), and

[0068] KRPAATKKAGQAKKKK (Nucleoplasmin NLS, SEQ ID NO: 10) The object is further met by providing a nucleic acid molecule comprising a nucleotide sequence encoding the photosensitive transcription factor as described above.

[0069] Another aspect of the invention is the provision of a method for producing at least one biologically active molecule, in particular a biotherapeutic, by optogenetic control of gene expression in at least one eukaryotic or prokaryotic cell, wherein the biologically active molecule comprises at least one protein of interest comprising at least two different subunits, wherein the method comprises: introducing into the cell at least one first expression cassette comprising at least one first nucleic acid sequence linked to a first promoter, wherein the first nucleic acid sequence encodes at least one photosensitive transcription factor, and introducing into the cell a second expression cassette comprising at least one second nucleic acid sequence encoding a first of the at least two subunits of the protein of interest and being operably linked to a second promoter, the second promoter being operably linked to at least one transcription factor binding site, wherein expression of the second nucleic acid sequence is controlled through activation or repression of the second promoter by illuminating the cell with light comprising at least one wavelength that triggers at least one structural change of the photosensitive transcription factor, wherein the first promoter is operably linked to at least one regulatory sequence, the regulatory sequence comprising at least one transcription factor binding site, wherein the first promoter and thus expression of the first nucleic acid sequence encoding the photosensitive transcription factor is induced or stimulated (upregulated) by illuminating the cell with light comprising at least one wavelength that triggers binding of the photosensitive transcription factor to a transcription factor binding site of the regulatory sequence.

[0070] As the first promoter is operably linked to at least one regulatory sequence, expression of the photosensitive transcription factor can be controlled, for example, to avoid unnecessary expression thereof. Expression of the first nucleic acid sequence is directly (i.e. in real-time) controlled through activation (upregulation) or repression (downregulation) of the first promoter by switching on a suitable light source. Vice versa, if the light triggers the release of the photosensitive transcription factor from the transcription factor binding site, the activating or repressing effect of the gene expression controlling domain can be promptly terminated by switching the light source on. That is, the first expression cassette represents a self-stimulating photoswitch providing a high number of photosensitive transcription factor molecules within the cell only upon illumination. In the dark, a lower number of photosensitive transcription factor molecules is expressed, which is, however, still sufficient to initiate higher expression of the gene encoding itself upon commencing illumination. By controlling (limiting) the expression of the photosensitive transcription factor, possible cytotoxic effects can be eliminated, or at least reduced and higher dynamic ranges can be ensured though light-dependent signal amplification via self-regulation of the photosensitive transcription factor..

[0071] A further aspect of the invention is the provision of an artificial polynucleotide, in particular for use in a method for producing at least one biologically active molecule, in particular a biotherapeutic, by optogenetic control of gene expression in at least one eukaryotic or prokaryotic cell, wherein the biologically active molecule comprises at least one protein of interest comprising at least two different subunits, wherein the artificial polynucleotide comprises at least one nucleic acid sequence encoding at least one photosensitive transcription factor linked to at least one promoter sequence, the photosensitive transcription factor comprising a gene expression controlling domain, wherein the gene expression controlling domain optionally comprises a transcriptional activation domain or a transcriptional repression domain, wherein the promoter sequence is operably linked to at least one regulatory sequence comprising a transcription factor binding site, wherein the promoter sequence and thus expression of the nucleic acid sequence encoding the photosensitive transcription factor is induced and / or stimulated (upregulated) by illuminating the cell with light comprising at least one wavelength that triggers binding of the photosensitive transcription factor to the transcription factor binding site of the regulatory sequence. As the promoter sequence is operably linked to at least one regulatory sequence, expression of the photosensitive transcription factor can be controlled, for example, to avoid unnecessary expression thereof. Expression of the nucleic acid sequence can be directly (i.e. in real-time) controlled through activation (upregulation) or repression (downregulation) of the promoter sequence by switching on a suitable light source. Vice versa, if the light triggers the release of the photosensitive transcription factor from the transcription factor binding site, the activating or repressing effect of the gene expression controlling domain can be promptly terminated by switching the light source on. That is, the first expression cassette represents a self-stimulating photoswitch providing a high number of photosensitive transcription factor molecules within the cell only upon illumination. In the dark, a lower number of photosensitive transcription factor molecules is expressed, which is, however, still sufficient to initiate higher expression of the gene encoding itself upon commencing illumination. By controlling (limiting) the expression of the photosensitive transcription factor, possible cytotoxic effects can be eliminated, or at least reduced and higher dynamic ranges can be ensured though light-dependent signal amplification via self-regulation of the photosensitive transcription factor.

[0072] “Introducing into the cell” as used herein in the sense of the invention refers to the transfer of nucleic acid molecules such as DNA and RNA (genetic material) into at least one cell, especially for either transcription into RNA and / or translation into proteins, including but not limited to transformation (transfer of non-viral genetic material into bacteria), transfection (transfer of non-viral genetic material into eukaryotic cells), and transduction (transfer of genetic material into eukaryotic cells using viral vectors).

[0073] The invention is further described in detail with reference to the following examples and figures.

[0074] Brief description of the figures

[0075] Figure 1 shows a schematic representation of an exemplary embodiment of an expression system according to the invention comprising expression cassettes for optogenetic control of the production of a monoclonal antibody.

[0076] Figure 2 shows a schematic representation of an exemplary embodiment of an expression system according to the invention comprising expression cassettes for optogenetic control of the production of a bispecific antibody.

[0077] Figure 3 shows a schematic representation of an exemplary embodiment of a self-stimulating photoswitch according to the invention.

[0078] Figure 4 shows a schematic representation of an exemplary embodiment of an expression system according to the invention comprising expression cassettes for balancing several components of a complex protein product.

[0079] Figure 5 shows a schematic representation of an exemplary embodiment of a method according to the invention using genetically engineered constructs according to Figure 4 for generating a stably transfected cell line for the production of biopharmaceuticals.

[0080] Figure 6 shows the result of a Western blot (WB) analysis under reducing conditions demonstrating light intensity-dependent increase in bispecific antibody (bsAb) expression / secretion upon blue light stimulation.

[0081] Figure 7 shows bar diagrams demonstrating the performance of a first expression cassette according to the invention (blue-sensitive photoswitch bluePS “DV”).

[0082] Figure 8 shows bar diagrams demonstrating the results of a comparison of the protein expression levels mediated by an exemplary embodiment of a first expression cassette according to the invention (blue-sensitive photoswitch bluePS “DV”) and different constitutive promoters.

[0083] Figure 9 shows the results of Western blot (WB) analyses demonstrating light dose- or pulse-dependent differential expression / secretion of subunits of a bispecific antibody (bsAb).

[0084] Figure 10 shows the result of a Western blot (WB) analysis under nonreducing conditions demonstrating light intensity-dependent increase in bispecific antibody (bsAb) expression / secretion upon blue light stimulation.

[0085] Figure 11 shows the result of a Western blot (WB) under non-reducing conditions demonstrating that expression / secretion of bispecific antibody (bsAb) complexes is regulated by different amounts of light-dependent chains.

[0086] Detailed of exemplary and preferred embodiments of the invention

[0087] Figure 1 shows an exemplary embodiment of an expression system 1 comprising three expression cassettes 2, 3, 4 for optogenetic control of the production of a protein of interest such as, in this embodiment, a monoclonal antibody comprising two subunits 5, 6. A first expression cassette 2 comprises a first nucleic acid sequence 7 (“ bluePS “DV” ”) that is operably linked to a first promoter 8 being disposed upstream of the first nucleic acid sequence 7. The first promoter 8 may be a constitutive promoter such as the cytomegalovirus promoter (“PCMV”), allowing for continual transcription of the first nucleic acid sequence 7. The first nucleic acid sequence 7 encodes a photosensitive transcription factor 9 comprising a gene expression controlling domain 10. The photosensitive transcription factor 9 may comprise at least one light-oxygen-voltage-sensing domain (LOV domain), preferably EL222. The gene expression controlling domain 10 of the photosensitive transcription factor 9 may comprise a transcriptional activation domain such as VP 16, VP64, p65, RTA or any combination thereof. The first expression cassette 2 thus represents a constitutively expressed photoswitch providing a constantly sufficient level of photosensitive transcription factors 9 within a cell. For example, the first nucleic acid sequence 7 “ bluePS “DV” ” may encode a photosensitive transcription factor comprising the light-oxygen-voltage-sensing domain EL222, a transcriptional activation domain comprising VP64, p65, and RTA, and two nuclear localization signals, preferably the amino acid sequence according to SEQ ID NO: 1.

[0088] The expression system 1 further comprises a second expression cassette 3 comprising a second nucleic acid sequence 11 encoding the first subunit 5 (“light chain (LC)”) of the monoclonal antibody (first gene of interest). The second nucleic acid sequence 11 is operably linked to a second promoter 12 such as an inducible minimal promoter (“Pminimai”) which is disposed upstream of the second nucleic acid sequence 11. The second promoter 12 is operably linked to a transcription factor binding site 13 (“(C120)s”) arranged upstream of the second promoter 12. Illumination of the cell with blue light (A = 465 nm) triggers dimerization of the photosensitive transcription factors 9 provided by the photoswitch, allowing the resulting dimers 14 to bind to the transcription factor binding site 13. These structural changes (described by Motta-Mena et al., 2014) ensure that the gene expression controlling domain 10 is placed in the vicinity of the second promoter 12 which is, in this embodiment, activated by the transcriptional activation domain. Accordingly, transcription of the second nucleic acid sequence 11 encoding the first subunit 5 (“light chain (LC)”) of the monoclonal antibody is rapidly induced by the second promoter 12 upon switching on a suitable light source emitting blue light. If this light source is switched off, the structural changes described above spontaneously reverse in the dark so that binding of the dimer of the transcription factor 14 to the second transcription factor binding site 13 and expression of the second nucleic acid sequence 11 is stopped immediately. Fast and precise optogenetic control of the production of a monoclonal antibody can thus be ensured using the method and expression system according to the invention.

[0089] The expression system 1 also comprises a third expression cassette 4 comprising a third nucleic acid sequence 15 encoding the second subunit 6 (“heavy chain (HC)”) of the monoclonal antibody (second gene of interest). The third nucleic acid sequence 15 is operably linked to a third promoter 16 being disposed upstream of the third nucleic acid sequence 15. In this embodiment, the third promoter 16 is a constitutive promoter ( P constitutive ), allowing for continual transcription of the third nucleic acid sequence 15. The monoclonal antibody (protein of interest) is then assembled by association of the two subunits 5, 6, wherein production of this complex protein can be precisely balanced and / or its secretion effectively controlled by the optogenetic gene expression system according to the invention, in this embodiment especially by controlling expression of the second nucleic acid sequence 11 (first subunit 5).

[0090] Figure 2 shows an exemplary embodiment of an expression system 20 comprising four expression cassettes 2, 3, 21 , 22 for optogenetic control of the production of a protein of interest such as, in this embodiment, a bispecific antibody comprising three subunits 23, 24, 25. The first and the second expression cassettes 2, 3 are the same as the ones depicted in Figure 1 , respectively. Accordingly, the first expression cassette 2 represents a constitutive photoswitch providing a constantly sufficient level of photosensitive transcription factors 9 within a cell and the second expression cassette 3 comprises a second nucleic acid sequence 11 encoding the first subunit 23 (“light chain (LC)”) of the bispecific antibody. The first and the second expression cassettes 2, 3 and their respective effects within the cell are described in detail above with reference to Figure 1 .

[0091] The expression system 20 also comprises a third expression cassette 21 comprising a third nucleic acid sequence 26 encoding the second subunit 24 (“heavy chain (HC)”) of the bispecific antibody (second gene of interest). The third nucleic acid sequence 26 is operably linked to a third promoter 27 such as an inducible minimal promoter (“Pminimai”) which is disposed upstream of the third nucleic acid sequence 26. The third promoter 27 is operably linked to a transcription factor binding site 28 (“(C120)s”) arranged upstream of the third promoter 27. Illumination of the cell with blue light (A = 465 nm) triggers dimerization of the photosensitive transcription factors 9 provided by the photoswitch, allowing the resulting dimers 14 to bind to the transcription factor binding site 28. These structural changes (described by Motta-Mena et al., 2014) ensure that the gene expression controlling domain 10 is placed in the vicinity of the third promoter 27 which is, in this embodiment, activated by the transcriptional activation domain. Accordingly, transcription of the third nucleic acid sequence 26 encoding the second subunit 24 (“heavy chain (HC)”) of the bispecific antibody is rapidly induced by the third promoter 27 upon switching on a suitable light source emitting blue light. If this light source is switched off, the structural changes described above spontaneously reverse in the dark so that expression of the third nucleic acid sequence 26 is stopped immediately. Fast and precise optogenetic control of the production of a bispecific antibody can thus be ensured using the method and expression system according to the invention.

[0092] The expression system 20 further comprises an additional (fourth) expression cassette 22 comprising a further (fourth) nucleic acid sequence 29 encoding an additional (third) subunit 25 (“hybrid chain (hybC)”) of the bispecific antibody (further (third) gene of interest). The fourth nucleic acid sequence 29 is operably linked to a further (fourth) promoter sequence 30 being disposed upstream of the fourth nucleic acid sequence 29. In this embodiment, the fourth promoter 30 is a constitutive promoter ( P constitutive ), allowing for continual transcription of the fourth nucleic acid sequence 29. The bispecific antibody (protein of interest) is then assembled by the association of the three subunits 23, 24, 25, wherein the production of this complex protein can be precisely balanced by the optogenetic gene expression system according to the invention, in this embodiment, especially by controlling expression of the second and the third nucleic acid sequences 11 , 26 (first and second subunit 23, 24).

[0093] Figure 3 shows an exemplary embodiment of a self-stimulating photoswitch according to the invention that can be used, for example, in the method according to the invention for optogenetically controlling the production of a protein of interest. The self-stimulating photoswitch comprises an expression cassette 31 comprising a first nucleic acid sequence 32 that is operably linked to a first promoter 33 such as an inducible minimal promoter ( P minimal ) being disposed upstream of the first nucleic acid sequence 32. The first nucleic acid sequence 32 encodes a photosensitive transcription factor 9 (“ bluePS “DV” ”) comprising a gene expression controlling domain 10. The photosensitive transcription factor 9 may comprise at least one light-oxygen-voltage-sensing domain (LOV domain), preferably EL222. The gene expression controlling domain 10 of the photosensitive transcription factor 9 may comprise a transcriptional activation domain such as VP16, VP64, p65, RTA or any combination thereof. In this embodiment, the first promoter 33 is operably linked to at least one regulatory sequence 34 so as to control expression of the photosensitive transcription factor 9, for example, in order to avoid unnecessary expression thereof and increase the dynamic range of either activation or repression of promoters harboring transcription factor binding sites (not shown in this figure) for the photosensitive transcription factor 9. The regulatory sequence 34 comprises a transcription factor binding site 35 (“(C120)5”), wherein the first promoter 33 and thus the expression of the first nucleic acid sequence 32 encoding the photosensitive transcription factor 9 is induced or stimulated (upregulated) by illuminating the cell with light (A = 465 nm) that triggers the binding of the photosensitive transcription factor 9 to the transcription factor binding site 35 of the regulatory sequence 34.

[0094] Illumination of the cell with blue light (e.g., A = 465 nm) triggers dimerization of the photosensitive transcription factor 9, allowing the resulting dimers 14 to bind to the transcription factor binding site 35. These structural changes (described by Motta-Mena et al., 2014) ensure that the gene expression controlling domain 10 is placed in the vicinity of the first promoter 33 which is activated by the transcriptional activation domain. Accordingly, transcription of the first nucleic acid sequence 32 encoding the photosensitive transcription factor 9 is rapidly induced and self-stimulated by the first promoter 33 upon switching on a suitable light source emitting blue light. If this light source is switched off, the structural changes described above spontaneously reverse in the dark so that binding of the dimer of the transcription factor 14 to the first transcription factor binding site 35 and expression of the first nucleic acid sequence 32 is stopped immediately. Fast and precise optogenetic control of the production of the photosensitive transcription factor 9 can thus be ensured using this advantageous optogenetic switch according to the invention, wherein the expression cassette 31 represents a self-stimulating photoswitch providing a sufficient number of photosensitive molecules within the cell only upon illumination but can be rapidly stopped by switching-off the light. Accordingly, as the expression of the photosensitive transcription factor 9 can be limited, possible cytotoxic effects can be eliminated, or at least reduced and higher dynamic ranges can be ensured though lightdependent signal amplification via self-regulation of the photosensitive transcription factor.

[0095] The expression cassette 31 can be used, for example, with the expression systems 1 and 20 according to Figures 1 and 2, respectively, replacing the first expression cassette 2. Thus, both, the photoswitch (comprising expression cassette 31 ) and the genes of interest (second, third, and optionally further first nucleic acid sequences), are expressed to a very limited extent without exposure to light of the respective wavelength, leading to less cytotoxicity and higher dynamic range.

[0096] Figures 4 and 5 show an exemplary embodiment of an expression system 40 according to the invention comprising two genetically engineered constructs 41 , 42 including four expression cassettes 43, 44, 45, 46 for balancing or independently temporally controlling several components of a complex protein product (protein of interest). The genetically engineered constructs 41 , 42 can be used in a method for generating a stable cell line, for example, for the production of biopharmaceuticals. The expression system 40 comprises genetically engineered constructs 41 , 42 (e.g., plasmid vectors), each comprising at least one multiple cloning site, into which at least one gene of interest (“GOI 1” and “GOI 2”, respectively) is cloned. Each gene of interest encodes at least one subunit of the complex protein of interest. The first construct 41 comprises two expression cassettes 43, 44 that are similar to the expression cassettes 2 and 3 according to Figures 1 and 2. Accordingly, the first expression cassette 43 comprises a first nucleic acid sequence 47 (“bluePS “DV””) encoding a photosensitive transcription factor 9 and thus also represents a constitutively expressed photoswitch providing a constantly sufficient level of photosensitive transcription factors 9 within a cell. The second expression cassette 44 comprises a second nucleic acid sequence 48 (“GOI 1”) encoding at least one first subunit of the protein of interest. The first and the second expression cassette 43, 44 and their respective effects within the cell are described in detail above with reference to the expression cassettes 2 and 3 according to Figure 1 , respectively.

[0097] The second construct 42 comprises a different first expression cassette 45 comprising a first nucleic acid sequence 49 (“Two-component red photocontroller”) encoding two components 50, 51 of a photosensitive transcription factor 52 and thus also represents a constitutive photoswitch providing a constantly sufficient level of photosensitive transcription factors 52 within the cell. To this end, the first nucleic acid sequence 49 is operably linked to a first promoter 53 being disposed upstream of the first nucleic acid sequence 49. The first promoter 53 may be a constitutive promoter such as the cytomegalovirus promoter (“PCMV”), allowing for continual transcription of the first nucleic acid sequence 49. The first nucleic acid sequence 49 encodes two components 50, 51 of the photosensitive transcription factor 52, wherein the first component 50 may comprise a phytochrome B and a DNA binding domain, and the second component 51 may comprise a phytochrome-interacting domain and a gene expression controlling domain 54 comprising a transcriptional activation domain such as VP16, VP64, p65, RTA or any combination thereof.

[0098] The second construct 42 further comprises a third expression cassette 46 comprising a third nucleic acid sequence 55 (“GOI 2”) encoding at least one second subunit of the protein of interest. The third nucleic acid sequence 55 is operably linked to a third promoter 56 such as an inducible minimal promoter (“Pminimai”) which is disposed upstream of the third nucleic acid sequence 55. The third promoter 56 is operably linked to a transcription factor binding site 57 (“(GAL4)s”) arranged upstream of the third promoter 56. For example, an optogenetic switch as described by Redchuck et al. (2018) may be used in this embodiment of the invention. That is, upon far-red light (A > 730 nm) stimulation, phytochrome B (PhyB), which remains in its inactive form in the dark, undergoes a conformational change, turning into the active form. The active PhyB heterodimerizes with the phytochrome-interacting domain and thus a complex of the two components 50, 51 is formed, said complex representing the (active) photosensitive transcription factor 52, wherein the (GAL4) DNA binding domain and the transcriptional activation domain are brought together at the transcription factor binding site 57, triggering transcription of the gene of interest (“GOI 2”). Upon red (A = 630 nm) illumination, the complex dissociates, halting transcription immediately (Redchuck et al., 2018). Accordingly, transcription of the third nucleic acid sequence 55 encoding the at least one second subunit of the protein of interest can be rapidly induced by the third promoter 56 upon switching on a suitable light source emitting far-red light. If this light source is switched off, the structural changes described above spontaneously reverse in the dark so that expression of the third nucleic acid sequence 55 is stopped immediately. Fast and precise optogenetic control of the production of a complex protein product such as a biopharmaceutical can thus be ensured using the method and expression system according to the invention.

[0099] Figure 6 shows the control of product composition and output with light, in particular a light intensity-dependent increase in bispecific antibody (bsAb) expression / secretion upon blue light stimulation. The Western blot (WB) analysis demonstrates the expression / secretion of bispecific antibodies (bsAb) regulated by different light intensities. The heavy chain (HC, 52 kD) and the single chain variable fragment (hybC; 54 kD) were expressed in a light dependentendentmanner using the expression system 20 according ti Figure 2 including the blue photoswitch (“bluePS”). The light chain (LC; 25 kD) was expressed constitutively under the control of the H1 promoter. The WB shows the bsAb under reducing conditions which results in the disassembly of the bsAb complex into the individual light (LC), heavy (HC) and hybrid (hybC) chains. Accordingly, it can be demonstrated that bispecific antibody sidechains can be induced with light and different light intensities can be used to titrate sidechains individually.

[0100] Material and Methods:

[0101] 800,000 HEK293T cells / well were seeded in a 6-well plate in 1265 pl OptiMEM (Gibco). After 4 h cells were transfected with 2.5 pg total DNA / well using polyethyleneimine (PEI) in a ratio 1 :2 (DNA:PEI). The following genes were transfected: the blue photoswitch bluePS “DV” (encoding the light-oxygen- voltage-sensing domain EL222, a transcriptional activation domain comprising VP64, p65, and RTA, and two nuclear localization signals, preferably the amino acid sequence according to SEQ ID NO: 1 ), the constitutively expressed light chain pH1 -LC, and the light-dependent heavy (C120-HC) and hybrid (C120- hybC) chains in a ratio 3:1 :1 :1 . Cells were excited 24 h after transfection for 24 h at indicated intensities with blue light (A = 465 nm). Cell culture supernatant was collected, and cellular debris was removed. Proteins were precipitated by adding three times the volume of acetone overnight at -20 °C, followed by centrifugation for 10 min at 4,000 g and 4 °C. Pellets were airdried for 30 min and resuspended in Tris / 1 % SDS (pH 6.8). Protein concentrations were determined via BCA assay. Loading dye (200 mM Tris / HCI pH 6.8, 8% (w / v) SDS, 50% (v / v) glycerin, 4% (v / v) mercaptoethanol, 0.04% bromophenol blue) was added to the samples. Samples were denatured for 5 min at 95 °C and loaded on a 4-20% SDS-PAGE. Proteins were blotted on PVDF membranes (Merck Millipore) using a semi-dry blotter (BioRad). Membranes were blocked in 5% BSA (Sigma-Aldrich). Produced antibodies were detected with a goat-anti-human IgG antibody coupled to HRP (1 :5000) and ECL (Perkin Elmer).

[0102] Figure 7 shows the stimulating effect of a first expression cassette according to the invention (blue-sensitive photoswitch bluePS “DV”), resulting in up to almost 1000-fold induction. The data represent the means of three samples luminescence values light-exposed or kept in the dark samples, plotted against each other. Each value is the result of the firefly luminescence normalized by its corresponding renilla luminescence. It is demonstrated that the photoswitch bluePS “DV” according to the invention shows unprecedented dynamic range.

[0103] Left diagram (a): Comparison of the protein expression levels mediated by different versions (“D”, “M”, “O”, and “DV”) of the first expression cassette according to the invention (blue-sensitive photoswitch “bluePS “DV””) tested during the development of an improved photo-controller (bluePS “DV”). Cells were excited using constant blue light (A = 465 nm) for 9 h at the intensity of 400 pW / cm2

[0104] Right diagram (b): Light intensity dependency of the protein expression levels mediated by the improved blue-sensitive photoswitch bluePS “DV”. Cells were excited using constant blue light (A = 465 nm) for 9 h at the intensity of 350 pW / cm2(A), 870 pW / cm2(B), and 1570 pW / cm2(C). The expression level mediated by bluePS “DV” increases with increasing light intensity, demonstrating that the induction of reporter gene expression is a light-dependent process. Material and Methods:

[0105] 50,000 HEK293T cells / well were seeded in 50 l DMEM+10% FCS / well in two 96-well plates, one exposed to the blue light and one kept in the dark. 1 h after seeding, cells were transfected with 0,1 pg total DNA / well using Lipofectamine 3000 in a ratio of 1 :3 (DNA: Lipofectamine). The following genes were transfected in the ratio 5: 1 :0,1 : as blue-sensitive photoswitch, the bluePS “DV”; as lightsensitive luminescent reporter, (C120)s-Firefly Luciferase; as constitutive luminescent reporters, SV40p-Renilla Luciferase for the normalization of the firefly measures, and HSV TPk / SV40p / CMVp / H1 p-Firefly Luciferase as references for constitutive protein expression. Cells were excited with constant blue light (A = 465 nm) for 9 h, starting 8 h after transfection, and the intensity of 400 pW / cm2Terminating the light excitation protocol, the dual luciferase assay was performed on the cells following the instruction of the Dual-Glo® kit (Promega; Cat. Nr. E2940). As the first step, the culture medium was removed and substituted by 40 pL / well of Opti-MEM. To each plate well was then added an equal volume of Dual-Glo® Reagent and let it incubate for 30 minutes, to allow the cell lysis to occur. At the end of the incubation, the firefly luminescence was measured with the FLUOstar Omega microplate reader. Afterward, the Renilla luminescence was activated and the firefly luminescence was quenched by adding a volume equal to the original culture medium volume of Dual-Glo® Stop & Gio® Reagent. Again, the incubation time was of 30 minutes, at the end of which the Renilla luminescence was measured. The ratio between the experimental reporter Firefly and the control reporter Renilla luminescence allowed to normalize the measured values.

[0106] Figure 8 shows a comparison of the protein expression levels mediated by an exemplary embodiment of a first expression cassette according to the invention (blue-sensitive photoswitch bluePS “DV”) and different constitutive promoters (“HSV”, “TPk”, “SV40p”, “RSVp”, “CMVp”, and “H1 p”), demonstrating that this “DV” photo-controller in combination with the (C120)s transcription factor binding element has superior strength. Cells were excited using constant blue light (A = 465 nm) for 9 h at the intensities of 350 pW / cm2(A), 870 pW / cm2(B) and 1570 pW / cm2(C). The data represent the means of three samples of firefly luminescence values light-exposed (for bluePS “DV” samples) or kept in the dark (for the constitutive promoters' samples), normalized by their corresponding Renilla luminescence. It is demonstrated that the photoswitch bluePS “DV” according to the invention shows a fast and tunable response to illumination, allows for dynamic gene control, and at least matches or even outperforms state- of-the-art industry expression systems.

[0107] Material and Methods:

[0108] 50,000 HEK293T cells / well were seeded in 50 pl DMEM+10% FCS / well in two 96-well plates, one exposed to the blue light and one kept in the dark. 1 h after seeding, cells were transfected with 0,1 pg total DNA / well using Lipofectamine 3000 in a ratio of 1 :3 (DNA: Lipofectamine). The following genes were transfected in the ratio 5: 1 :0,1 : as blue-sensitive photoswitch, the bluePS “DV”; as lightsensitive luminescent reporter, (C120)s-Firefly Luciferase; as constitutive luminescent reporters, SV40p-Renilla Luciferase for the normalization of the firefly measures and HSV TPk / SV40p / CMVp / H1 p-Firefly Luciferase as references for constitutive protein expression. Cells were excited with constant blue light (A = 465 nm) for 9 h, starting 8 h after transfection, and at the intensity of 350 pW / cm2(A), 870 pW / cm2(B) and 1570 pW / cm2(C). . Terminating the light excitation protocol, the dual luciferase assay was performed on the cells following the instruction of the Dual-Glo® kit (Promega; Cat. Nr. E2940). As first step, the culture medium was removed and substituted by 40 pL / well of Opti-MEM. To each plate well was then added an equal volume of Dual-Glo® Reagent and let it incubate for 30 minutes, to allow the cell lysis to occur. At the end of the incubation, the firefly luminescence was measured with the FLUOstar Omega microplate reader. Afterward, the Renilla luminescence was activated and the firefly luminescence was quenched by adding a volume equal to the original culture medium volume of Dual-Glo® Stop & Gio® Reagent. Again, the incubation time was of 30 minutes, at the end of which the Renilla luminescence was measured. The ratio between the experimental reporter firefly and the control reporter Renilla luminescence allowed to normalize the measured values.

[0109] Figure 9 shows the results of Western blot analyses demonstrating light dosedependent expression / secretion of bispecific antibodies (bsAb), in particular titration of an antibody composition for better output. The light (LC) and the heavy (HC) chains are expressed in a light-dependent manner, the hybrid chain (hybC) is constitutively expressed (Light chain (LC): ca. 25 kDa, heavy chain (HC): ca. 52 kDa, hybrid chain (hybC): ca. 54 kDa).

[0110] Left picture (a): Western blot (WB) demonstrating a light intensity-dependent increase in LC and HC expression / secretion upon blue light stimulation, while the hybC expression is not influenced by light excitation.

[0111] Right picture (b): Western blot (WB) demonstrating a light pulse-dependent increase in LC and HC expression / secretion upon blue light stimulation, while the hybC expression is not influenced by light excitation. WB shows bsAb under reducing conditions which results in the disassembly of the bsAb complex into the individual light (LC), heavy (HC) and hybrid (hybC) chains.

[0112] Material and Methods:

[0113] 140,000 HEK293T cells / well were seeded in a 24-well plate in 300 pl OptiMEM. After 4 h cells were transfected with 0.5 pg total DNA / well using PEI in a ratio 1 :2 (DNA:PEI). The following genes were transfected: the blue photoswitch bluePS “DV” (encoding the light-oxygen-voltage-sensing domain EL222, a transcriptional activation domain comprising VP64, p65, and RTA, and two nuclear localization signals, preferably the amino acid sequence according to SEQ ID NO: 1 ), the consitutively expressed hybrid chain pH1 -hybC and the light-dependent light (C120-LC) and heavy (C120-HC) chains in a ratio 3:1 :1 :1 . Cells were excited 24 h after transfection for 24 h continuously at indicated intensities (left) or at indicated pulses at intensity of 400 pW / cm2(right) with blue light (A = 465 nm). Cell culture supernatant was collected, and cellular debris was removed. Loading dye (200 mM Tris / HCI pH 6.8, 8% (w / v) SDS, 50% (v / v) glycerin, 4% (v / v) mercaptoethanol, 0.04% bromophenol blue) was added to the samples. Samples were denatured for 5 min at 95 °C and loaded on a 4-20% SDS-PAGE. Proteins were blotted on PVDF membranes (Merck Millipore) using a semi-dry blotter (BioRad). Membranes were blocked in 5% BSA (Sigma-Aldrich). Produced antibodies were detected with a goat-anti-human IgG antibody coupled to HRP (1 :5000) and ECL (Perkin Elmer). Figure 10 shows the result of a Western blot (WB) analysis under non-reducing conditions demonstrating light intensity-dependent increase in bispecific antibody (bsAb) expression / secretion upon blue light stimulation.

[0114] Figure 10.1 shows light-dependent influence of expression of all side chains on bsAb complex formation HEK293T and CHO-K1 . A) The pDEL-VPR is activated by blue light excitation (A = 465 nm) and in turn dimerizes and binds to the C120 sequence. Subsequently, the light (LC) heavy (HC) and single variable fragment (hybC) chains are expressed in a light dose-dependent manner, followed by their assembly into bsAb in the ER. The bsAb was either expressed in HEK293T (B) or CHO-K1 (C) either light-dependently under the control of the C120 promoter or constitutively under the control of the CMV promoter. The light-dependent conditions were excited at different intensities (pW / cm2) for 24 h (always ON). The samples were analyzed on a Western blot (WB) under non-reducing conditions using an anti-human lgG1 detection antibody coupled to HRP. The bsAb bands of the WB for the HEK293T and CHO-K1 were quantified in D) and E), respectively. F) and G) show the fold change of bsAb titer of the light-induced (C120 light; 1500 pW / cm2), non-induced (C120 dark; 0 pW / cm2) or the constitutive (CMV) conditions after 24 h expression in HEK293T or CHO-K1 , respectively, quantified by ELISA. The values were normalized to the CMV samples. H) and I) show the fold change of bsAb productivity of the experiment presented in F-G) in HEK293T and CHO-K1 , respectively. The values were normalized to the CMV samples. D-l) Means of biological independent samples ± STD are presented as bars, dots indicate individual values of each sample. Statistical analysis was performed using one-way ANOVA with Holm-Sidak post- hoc test. Plots contain five (n=5) in C-E) and six (n=6) independent samples in F-

[0115] I)

[0116] Figure 10.2 shows light-dependent influence of expression of the light (LC) and heavy (HC) chains on bsAb complex formation HEK293T and CHO-K1 .

[0117] A) In the dark, the pDEL-VPR stays inactive, therefore, the light-dependent expression of the light (LC) and heavy (HC) chain is not induced, while the single chain variable fragment (hybC) in constitutively expressed. Consequently, in the absence of the LC and HC, the hybC is assembled in dimers. B) The pDEL-VPR is activated by blue light excitation (A = 465 nm) and in turn dimerizes and binds to the C120 sequence. Subsequently, the LC and HC are expressed in light intensity-dependent manner. Additionally, the hybC is expressed constitutively. Consequently, in the light the trimeric bsAb is assembled. The bsAb was expressed in HEK293T (C) or CHO-K1 (D) either partially light-dependently (lightdependent LC / HC + constitutive hybC) under the control of the C120 promoter or all chains constitutively under the control of the CMV promoter. The lightdependent conditions were excited at different intensities (pW / cm2) for 24 h (always ON). The different bsAb complexes were analyzed on a Western blot (WB) under non-reducing conditions using an anti-human lgG1 detection antibody coupled to HRP. The bsAb bands of the WB for the HEK293T and CHO- K1 were quantified in E) and F), respectively. The bands of hybC dimers for the HEK293T and CHO-K1 were quantified in G) and H), respectively. The ratio of bsAb:2hybC dimers for the HEK293T and CHO-K1 were quantified in I) and J), respectively. E-J) Means of biological independent samples ± STD are presented as bars, dots indicate individual values of each sample. Statistical analysis was performed using ANOVA with Holm-Sidak post-hoc test. Plots contain five (n=5) independent samples.

[0118] Material and Methods:

[0119] 800,000 HEK293T cells / well were seeded in a 6-well plate in 1265 pl OptiMEM. After 4 h cells were transfected with 2.5 pg total DNA / well using PEI in a ratio 1 :2 (DNA:PEI). Following genes were transfected: pDEL-VPR, pH1 -LC, C120-HC and C120-hybC in a ratio 3:1 :1 :1 . Cells were excited 24 h after transfection for 24h at indicated intensities with blue light (A = 465 nm). Cell culture supernatant was collected, and cellular debris was removed. Proteins were precipitated by adding three times the volume of acetone overnight at -20 °C, followed by centrifugation for 10 min at 4,000 g and 4 °C. Pellets were airdried for 30 min and resuspenden in Tris / 1 % SDS (pH 6.8). Protein concentrations were determined via BCA assay. Loading dye (200 mM Tris / HCI pH 6.8, 8% (w / v) SDS, 50% (v / v) glycerin, 4% (v / v) mercaptoethanol, 0.04% bromophenol blue) was added to the samples. Samples were denatured for 5 min at 95 °C and loaded on a 4-20% SDS-PAGE. Proteins were blotted on PVDF membranes (Merck Millipore) using a semi-dry blotter (BioRad). Membranes were blocked in 5% BSA (Sigma- Aldrich). Produced antibodies were detected with a goat-anti-human IgG antibody coupled to HRP (1 :5000) and ECL (Perkin Elmer).

[0120] Light chain (LC) ca. 25 kD, heavy chain (HC) ca. 52 kD, single chain fragment (hybC) ca. 54 kD

[0121] Figure 11 shows the result of a western blot (WB) under non-reducing conditions demonstrating that expression / secretion of bispecific antibodies (bsAb) complexes is regulated by the presence / relative concentrations and / or temporally controlled expression of different amounts of light-dependent chains.

[0122] Figure 11 demonstrates that expression / secretion of bispecific antibodies (bsAb) complexes is regulated by different combination of light-dependent chains and the expression of the main product bsAB and by-products are influenced by the relative amounts and / or timing of sidechains expressed. bsAb complexes resulted from light-dependent expression of 1 ) all three bsAb chains, 2) the LC, 3) the HC and 4) the hybC in the light (24 h always ON, 1500 pW / cm2) or 5) constitutive expression.

[0123] With light-dependent expression of the light chain (LC, lanes 2) the formation of by-products, in particular the HCcsFv dimer is largely favored, while with expression of the light chain (LC) initiated 24 h after transfection with the constitutively expressed heavy chain (HC) and the hybC chain, the products are shifted in favor of the intended product bsAb. Similar effects can be seen in case of the light-induced heavy chain (lanes 3), while with full light-induction of the hybC chain still a significant amount of hybC dimer by-product can be detected (lanes 4). The latter might be due to overexpression of hybCrelative to the LC and HC chains. Thus, exact balancing and / or timing of expression the three bsAb components has a striking effect on the formation of intended product vs byproducts, their relative concentrations, and thus on unpurified product quality, which as an impact on necessary purification efforts, yields of purified product and thus in sum production costs per unit of bsAb product.

[0124] Material and Methods:

[0125] 140,000 HEK293T cells / well were seeded in a 24-well plate in 400 pl OptiMEM. After 4 h cells were transfected with 0.5 pg total DNA / well using PEI in a ratio 1 :2 (DNA:PEI). Following genes were transfected: 1 ) pDEL-VPR + C120-LC + C120- HC + C120- hybC, 2) pDEL-VPR + C120-LC + H1 -HC + H1 - hybC, 3) pDEL-VPR + H1 -LC + C120-HC + H1 - hybC, 4) pDEL-VPR + H1 -LC + H1 -HC + C120- hybC in a ratio 3: 1 : 1 : 1 or 5) CMV-LC + CMV-HC + CMV- hybC. Cells were excited 24 h after transfection for 24 h at indicated intensities with blue light (A = 465 nm). Cell culture supernatant was collected, and cellular debris was removed. Non-reducing loading dye (200 mM Tris / HCI pH 6.8, 8% (w / v) SDS, 50% (v / v) glycerin, 0.04% bromophenol blue) was added to the samples. Samples were denatured for 5 min at 95 °C and loaded on a 4-20% SDS-PAGE. Proteins were blotted on PVDF membranes (Merck Millipore) using a semi-dry blotter (BioRad). Membranes were blocked in 5% BSA (Sigma-Aldrich). Produced antibodies were detected with a goat-anti-human IgG antibody coupled to HRP (1 :5000) and ECL (Perkin Elmer).

[0126] Molecular weights: Light chain (LC) ca. 25 kD, heavy chain (HC) ca. 52 kD, single chain fragment (hybC) ca. 54 kD

[0127] Detailed description of a further exemplary and preferred embodiment of the invention

[0128] In the following the inventors describe an advantageous embodiment of the invention concerning the design of a modular blue-light sensitive transcription factor (photoswitch “DEL-VPR”). “DEL-VPR” comprises the amino acid sequence according to SEQ ID NO: 1.

[0129] Description of the figures

[0130] Figure 12: Comparison of various LOV-based photoswitches in HEK293T and CHO-K1 cells.

[0131] A) EL222-based photoswitches consist of a blue light-dependent LOV domain and an HTH DNA binding domain and mediated gene expression by binding to the C120 sequence, which is located upstream of a minimal promoter (minP). The photoswitch VP-EL222 consists of an N-terminal SV40 nuclear location sequence (NLS) followed by VP16 and EL222. VEL consists of an N-terminal SV40 NLS, VP16, nucleoplasmin (NP) NLS and EL222. TAEL consists of the transactivation domain TA4 and EL222. DEL-VPR compromises an SV40 NLS, the three transactivation domains VP64, p65 and Rta as well as EL222. The VP- EL222 of DEL-VPR was synthesized (Integrated DNA Technology) based on the sequence from Motta Mena et al. (2014). B) WD-based photoswitches consist of the DNA binding domain Gal4 and the blue light-dependent WD domain. They mediate gene expression by binding to the UAS sequence, which is upstream of a minP. GAVPO consists of p65 fused to Gal4 and VVD. GAVTA contains TA4, Gal4 and VVD. Induction of light-mediated luciferase activity in C) HEK293T and D) CHO-K1 cells. The values indicate the induction of luciferase activity compared to the dark activity of various photoswitches and constitutive promoters. The cells were excited with continuous blue light (470 nm) for 9 h at 1500 pW / cm2. The data represents the ratio of light versus dark from firefly luciferase (fLuc) normalized to SV40-driven renilla luciferase (rFluc) expression. Relative luciferase activity of firefly vs. renilla luciferase of various experiments shown in (C-D) in HEK293T (E) and CHO-K1 cells (F). C-F) Bars represent the mean of six independent experiments (n=6) ± SD. Dots represent means of triplicates from individual experiments. Statistical analysis was performed using one-way ANOVA and the Holm-Sidak post-hoc test. Indicated p-values represent comparison to DEL-VPR. (C-D) Red written numbers show means of induction.

[0132] Figure 13: Single-cell analysis of DEL-VPR-mediated induction.

[0133] A) DEL-VPR is activated by blue light excitation (A= 470 nm) and in turn, dimerizes and binds to the C120 sequence to induce mScarlet expression. B) hiPSC-derived smNPCs transduced with AAV2 / 1 -mScarlet, either controlled via the light inducible DEL-VPR, or the constitutive CMV promoter, were exposed to blue light for 24 h before fixation, staining, and imaging. Light exposed smNPCs transduced with the C120 construct display robust mScarlet expression (red), as opposed to the dark control. CMV driven constructs display expression independent of light exposure. Nuclei were stained with DAPI (blue). Scale bar: 10 pm. C) HEK293T cells transfected with either DEL-VPR and C120-mScarlet (encoded on the same plasmide) or CMV-mScarlet exposed to 24 h blue light at 1500 pW / cm2 (always ON) or kept in the dark. Scale bar: 100 pm. mScarlet intensity of of total D) HEK293T or E) CHO-K1 cells expressing DEL-VPR- or CMV- mediated mScarlet plus respective dark controls. Cells were continuously excited with blue light 24 h after transfection for 24 hours at 1500 pW / cm2. Mean mScarlet intensity of mScarlet+ F) HEK293T or G) CH0-K1 cells from experiment presented in (D-E). Viability of light-excited H) HEK293T or I) CHO-K1 from experiment presented in (D-E) using Zombie Violet as viability stain. D-l) Bars represent the mean of five independent experiments (n=5) ± SD. Dots represent means of triplicates from individual experiments. Statistical analysis was performed using two-way ANOVA and the Holm-Sidak post-hoc test.

[0134] Figure 14: DEL-VPR mediates light-dose dependent expression of functional recombinant proteins.

[0135] A) DEL-VPR is activated by blue light excitation (A= 470 nm) and in turn, dimerizes and binds to the C120 sequence to induce Interferon (IFN) expression. Functional IFN induces via an artificial IFN receptor (IFNR)ZSTAT signaling pathway the expression of secreted embryonal alkaline phosphatase (SEAP) which can be detected in a colorimetric assay in high throughput. Dose-response curves of SEAP activity induced at indicated light intensities in B) HEK293T and C) CHO-K1 cells. Cells were excited 24 h after transfection for 24 h at indicated blue light intensities (always ON). The supernatant of these cells was added at a dilution of 1 :100 on HEKblue IFN cells and incubated for 24 h. Values were normalized to CMV-driven SEAP activity of respective experiments. Dots represent the mean of six (n=6) independent experiments ± SD. Statistical analysis was performed using one-way ANOVA and the Holm-Sidak post-hoc test. Light-dependent SEAP activity induced at indicated light intensities and pulses in D) HEK293T and E) CHO-K1 cells. Cells were excited 24 h after transfection for 24 h at indicated blue light intensities and pulses. The supernatant of these cells was added at a dilution of 1 :100 on HEKblue IFN cells and incubated for 24 h. Values were normalized to CMV-driven SEAP activity of respective experiments. Bars represent the mean of six (n=6) independent experiments ± SD, dots show the mean of technical triplicates of individual experiments.

[0136] Figure 15: Light-dependent titration of mAb expression in HEK293T and CHO-K1.

[0137] A) DEL-VPR is activated by blue light excitation (A= 470 nm) and in turn, dimerizes and binds to the C120 sequence. Subsequently, the light (LC) and heavy (HC) chains are expressed in a light dose-dependent manner, followed by their assembly into mAb in the ER. B) Assessment of DEL-VPR and CMV-driven mAb functionality in HEK293T and CHO-K1 cells. The mAb antigen HER2 was coated on ELISA plates and bound mAb was detected. HER1 serves as a negative control. The absorbance was subtracted from the one of non-transfected cell supernatants. The mAb, consisting of the light and heavy chains, was expressed in HEK293T (C) or CHO-K1 (D) either light-dependently under the control of the C120 promoter or constitutively under the control of the CMV promoter. The light-dependent conditions were excited at different intensities (pW / cm2) for 24 h (always ON). The samples were analyzed on a Western blot (WB) under non-reducing conditions using an anti-human lgG1 detection antibody coupled to HRP. The mAb bands of the WB for the HEK293T and CHO- K1 were quantified in E) and F), respectively. G) and H) show the fold change of mAb titers of the light-induced (DEL-VPR light; 1500 pW / cm2), non-induced (DEL-VPR dark), or the constitutive (CMV) conditions after 24 h expression in HEK293T or CHO-K1 , respectively, quantified by ELISA. The values were normalized to the CMV values. I) and J) show the fold change of mAb productivity of the experiment presented in G-H) in HEK293T and CHO-K1 , respectively. The values were normalized to the CMV values. B, E-J) Means of biological independent samples ± SD are presented as bars, and dots indicate individual values of each sample. Statistical analysis was performed using one-way ANOVA with the Holm-Sidak post-hoc test. Plots contain four (n=4, B), five (n=5, E-F) or six (n=6; G-J) independent samples.

[0138] Figure 16: Light-dependent titration of bsAb expression in HEK293T and CHO-K1.

[0139] A) DEL-VPR is activated by blue light excitation (A= 470 nm) and in turn, dimerizes and binds to the C120 sequence. Subsequently, the light (LC), heavy (HC) and single variable fragment (HCscFv) chains are expressed in a light dosedependent manner, followed by their assembly into bsAb in the ER. B) Assessment of DEL-VPR and CMV-driven bsAb functionality in HEK293T and CHO-K1 cells. The two distinct antigens were separately coated on independent ELISA plates and bound bsAb was detected. The absorbance was subtracted from the one of non-transfected cell supernatant. The bsAb was either expressed in HEK293T (C) or CHO-K1 (D) either light-dependently under the control of DEL- VPR or constitutively under the control of the CMV promoter. Cells were excited at different intensities (pW / cm2) for 24 h (always ON). The samples were analyzed on a Western blot (WB) under non-reducing conditions using an antihuman IgG 1 detection antibody coupled to HRP. The bsAb bands of the WB for the HEK293T and CHO-K1 were quantified in E) and F), respectively. G) and H) show the fold change of bsAb titer of the light-induced (DEL-VPR; 1500 pW / cm2), non-induced (DEL-VPR dark) or constitutive (CMV) conditions after 24 h expression in HEK293T or CHO-K1 , respectively, quantified by ELISA. The values were normalized to the CMV samples. I) and J) show the fold change of bsAb productivity of the experiment presented in G-H) in HEK293T and CHO-K1 , respectively. The values were normalized to the CMV samples. E-J) Means of biological independent samples ± SD are presented as bars, dots indicate individual values of each sample. Statistical analysis was performed using oneway ANOVA with the Holm-Sidak post-hoc test. Plots contain four (n=4, B) five (n=5, D-F) or six (n=6, G-J) independent samples.

[0140] Figure 17: Light-dependent titration modulation of bsAb complexes in HEK293T and CHO-K1.

[0141] A) In the dark, the DEL-VPR stays inactive. Therefore, the light-dependent expression of the light (LC) and heavy (HC) chain is not induced, while the single chain variable fragment (HCscFv) is constitutively expressed. Consequently, without the LC and HC, the HCscFv is assembled in dimers. B) DEL-VPR is activated by blue light excitation (A= 470 nm) and dimerizes and binds to the C120 sequence. Subsequently, the LC and HC are expressed in a light intensitydependent manner. Additionally, the HCscFv is expressed constitutively. Consequently, in the light, the trimeric bsAb is assembled. The bsAb was expressed in HEK293T (C) or CHO-K1 (D) either partially light-dependently (lightdependent LC / HC + constitutive HCscFv) under the control of DEL-VPR or all chains constitutively under the control of the CMV promoter. The light-dependent conditions were excited at different intensities (pW / cm2) for 24 h (always ON). The different bsAb complexes were analyzed on a Western blot (WB) under nonreducing conditions using an anti-human lgG1 detection antibody coupled to HRP. The bsAb bands of the WB for the HEK293T and CHO-K1 were quantified in E) and F), respectively. The bands of HCscFv dimers for the HEK293T and CH0-K1 were quantified in G) and H), respectively. The ratio of bsAb: HCscFv dimers for the HEK293T and CH0-K1 were quantified in I) and J), respectively. E-J) Means of biological independent samples ± SD are presented as bars; dots indicate the individual values of each sample. Statistical analysis was performed using ANOVA with the Holm-Sidak post-hoc test. Plots contain five (n=5) independent samples.

[0142] Figure 18: Comparison of dark activity of various LOV-based photoswitches.

[0143] Comparison of dark fLuc activity normalized to rLuc from various photoswitches in A) HEK293T and B) CHO-K1 cells. Bars represent the mean of six independent experiments (n=6) ± SD. Dots represent means of triplicates from individual experiments. Statistical analysis was performed using one-way ANOVA and the Holm-Sidak post-hoc test. Indicated p-Values represent comparison to DEL-VPR.

[0144] Figure 19: mScarlet induction in HEK293T and CHO-K1.

[0145] Brightfield and fluorescent (red, mScarlet) images of A) HEK293T and B) CHO- K1 expressing mScarlet either in a DEL-VPR mediated manner or CMV-driven in after 24 h of blue light exposure (1500 pW / cm2, always ON) or kept in the dark. Scale bar: 100 pm.

[0146] Figure 20; Transient mScarlet expression in HEK293T and CHO-K1.

[0147] DEL-VPR is activated by blue light excitation (A= 470 nm) and in turn, dimerizes and binds to the C120 sequence to induce mScarlet expression. Percentage of m Scarlet-positive (mScarlet+) A) HEK293T or B) CHO-K1 expressing DEL-VPR- or CMV- mediated mScarlet plus respective dark and non-transfected (NT) controls. Cells were continuously excited with blue light 24 h after transfection for 24 hours at 1500 pW / cm2. Bars represent the mean of five independent experiments (n=5) ± SD. Dots represent means of triplicates from individual experiments. Statistical analysis was performed using two-way ANOVA and the Holm-Sidak post-hoc test. Figure 21 : Light-dependent fluorescent reporter expression in HEK293T and CHO-K1.

[0148] Fluorescence levels were measured in HEK293T (A, C, E) and CHO-K1 (B, D, F) cells, which transiently expressed mCherry either in a light-dependent fashion or under the control of different constitutive promoters. We compared different versions of the blue-sensitive photoswitch EL222-VP-EL222, VEL, DEL-VPR and EL222 without additional activation domain and the strong and medium constitutive promoters CMV and H1 , respectively. For the light condition, samples were excited for 32 h using blue light (A= 470 nm) with a constant intensity (1500 pW / cm2), while for the dark condition, samples were kept in the dark for the entire time. Live cell image collection started 8 h after the beginning of the light excitation (+) and continued for a total of 48 h. For the last 24 h, all the samples were kept in the dark (-). In A) and B) are shown examples of the images collected and analyzed. Scale bar 100 pm. C) and D) top panels represent the time course of the mean number of mCherry fluorescence positive cells per mm2 detected in samples exposed to the light, while the bottom panels show the mean values relative to the last time point (48 h) of the light and the dark conditions compared. The graphs E) and F) represent the fold difference of the mean number of mCherry fluorescent cells transfected with the different versions of the photoswitch EL222 versus the one detected in the samples transfected with the strong constitutive promoter CMV. Data are presented as lines with markers or as bar graphs, showing mean ± SD, and with dots indicating individual values of each sample. Statistical analysis was performed in C), D), E) and F) using a oneway ANOVA with Holm-Sidak post-hoc test. Statistically significant differences among the different photoswitches with p<0,05 were indicated as follows: a=VP- EL222 vs. VEL; b=VP-EL222 vs. DEL-VPR; c=VP-EL222 vs. EL222 0 AD; d=VP- EL222 vs. CMV; e=VEL vs. DEL-VPR; f=VEL vs. EL222 0 AD; g=VEL vs. CMV; h=DEL-VPR vs. EL222 0 AD; i=DEL-VPR vs CMV; j=EL222 0 AD vs CMV. In the bottom panels of C) and D), the statistical analysis was performed using the Mann-Whitney test after having verified the distribution with a Shapiro-Wilk test. Plots contain four (n=4) independent experiments, each representing the means of three samples (total n=12). Figure 22: Light-dose-dependent SEAP activity.

[0149] Single values of dose-response curves of SEAP activity induced at indicated light intensities in A) HEK293T and B) CHO-K1 cells. Cells were excited 24 h after transfection for 24 h at indicated blue light intensities (always ON). The supernatant of these cells was added at a dilution of 1 : 100 on HEKblue IFN cells and incubated for 24 h. Values were normalized to CMV-driven SEAP activity of respective experiments. Bars represent means of six (n=6) independent experiments ± SD. Dots show means of independent triplicates from independent experiments. Statistical analysis was performed using one-way ANOVA and the Holm-Sidak post-hoc test.

[0150] Figure 23: Light-dependent titration of mAb expression in HEK293T and CHO-K1.

[0151] A) and B) show the titer of mAb of the light-induced (DEL-VPR light; 1500 pW / cm2), non-induced (DEL-VPR dark), or the constitutive (CMV) conditions after 24 h expression in HEK293T or CHO-K1 , respectively, quantified by ELISA. H) and I) show the productivity of mAb production of the experiment presented in C-D) in HEK293T and CHO-K1 , respectively. A-D) Means of biological independent samples ± STD are presented as bars, dots indicate individual values of each sample. Statistical analysis was performed using one-way ANOVA with the Holm-Sidak post-hoc test. Plots contain six (n=6) independent samples.

[0152] Figure 24: Stable light-depedent titration of bsAb expression in HEK293T. A) The construct for piggyBac-mediated integration contains flanking inverted terminal repeats (ITR) at both ends. The 5’ITR is followed by C120-LC, C120-HC and C120-HCscFv. It also contains a CMV-driven floxed-m Cherry with a stop codon followed by DEL-VPR. Under basal conditions, during the selection and expansion phase, only mCherry is expressed. Upon TAT-Cre treatment, mCherry gets cut out, including its stop codon, therefore, DEL-VPR is getting expressed and can induce the expression of the three bsAb chains. B) Western Blot of DEL- VPR-mediated expression in the non-activated (- Cre) and the activated (+ Cre) state with various blue light intensities to induce bsAb expression in stably integrated HEK293T cells. Quantification of the blots shown in (B) of the bsAb (C) and the HCscDv dimer (2HCscFv) (D). Data is normalized to bsAb intensity of the CMV control. (C-D) Means of independent samples ± SD are presented as bars, dots indicate individual values of each sample. Statistical analysis was performed using three-way ANOVA with the Holm-Sidak post-hoc test. Plots contain five (n=3).

[0153] Figure 25: Light-dependent titration of bsAb expression in HEK293T and CHO-K1.

[0154] A) and B) show the titer of bsAb of the light-induced (DEL-VPR; 1500 pW / cm2), non-induced (DEL-VPR) or the constitutive (CMV) conditions after 24 h expression in HEK293T or CHO-K1 , respectively, quantified by ELISA. C) and D) show the productivity of bsAb production of the experiment presented in A-B) in HEK293T and CHO-K1 , respectively. A-D) Means of biological independent samples ± STD are presented as bars, dots indicate individual values of each sample. Statistical analysis was performed using one-way ANOVA with the Holm- Sidak post-hoc test. Plots contain six (n=6) independent samples.

[0155] Design of the modular blue-light sensitive photoswitch DEL-VPR

[0156] To optimize inducible gene expression for protein production, we developed an optogenetic tool for precise temporal control that achieves high expression levels while promoting protein functionality. We identified the bacterial LOV-based expression systems as promising candidates due to their compact size, natural occurrence of the chromophore, and proven performance. We investigated LOV- based photoswitches derived from EL222, fused to the transactivation domain VP16 with either a single SV40 NLS (VP-EL222) or a modified version, incorporating an additional nucleoplasmin NLS (VEL; Figure 12A). Additionally, we examined TAEL, a fusion of EL222 with a triplicated VP16 core (TA4). These tools were primarily optimized for the application in zebrafish. To enhance activation potential, we replaced VP 16 with VPR (VP64-p65-Rta), known for its superior transcriptional activity compared to its constituent components. We then compared the EL222-based photoswitches with GAVPO (p65-Gal4-WD; Figure 12B) and GAVTA (TA4-Gal4-WD), which are based on the WD LOV receptor and have also been used in zebrafish. Performance was assessed in a luciferase assay and benchmarked against common constitutive promoters. Firefly luciferase (fLuc) activity, induced by photoswitches or constitutive promoters, was measured and normalized to SV40-driven, constitutively expressed Renilla luciferase (rLuc) to account for transfection efficiency and cell number. DEL-VPR achieved a 570-fold induction in HEK293T and 160-fold in CHO-K1 under light versus dark conditions (Figure 12C-D). For reference, in HEK293T cells, the other photoswitches induced luciferase activity by 30-100- fold upon light stimulation. In CHO-K1 , GAVTA induced a 150-fold increase, while the other optogenetic tools led to inductions of merely 5-30-fold. As to be expected, the constitutive promoters and non-transfected controls showed no light dependency.

[0157] When fully induced by blue light, DEL-VPR reached levels comparable to or even exceeding common constitutive promoters (Figure 12E-F). DEL-VPR significantly outperformed all other photoswitches in HEK293T and CHO-K1 cells, with no significant differences in dark activity among photoswitches in HEK293T were detected (Figure 18A). In CHO-K1 , DEL-VPR dark activity was comparable to GAVPO, TAEL, and VEL, but higher compared to GAVTA and VP-EL222 (Figure 18B).

[0158] These results demonstrate that DEL-VPR is a highly effective optogenetic tool for light-induced gene expression, achieving expression levels comparable to strong constitutive promoters like CMV and H1 while maintaining low dark activity, with variations depending on the cell type.

[0159] DEL-VPR drives light-inducible protein expression in iPSC-derived cells via AAV transduction

[0160] Building on our initial success using DEL-VPR to induce protein expression in transfectable cell lines, we next sought to explore whether this optogenetic system could be delivered via adeno-associated virus (AAV), a critical step for applications in cell types that require viral vectors for genetic manipulation. To investigate this, we engineered AAV encoding either DEL-VPR alongside the light-responsive reporter 0120-mScarlet, or a constitutively active CMV-m Scarlet construct as a control (Figure 13A). Upon transduction of iPSC-derived neuronal progenitor cells (NPC), robust red fluorescence was observed in cells expressing DEL-VPR and 0120-mScarlet following blue light stimulation, while no signal was detected in the absence of light (Figure 13B). In contrast, CMV-mScarlet expression was consistently visible regardless of light exposure. These results demonstrate that DEL-VPR can be efficiently packaged into AAVs and remain functional in cell types dependent on viral transduction, paving the way for its application in more physiologically relevant systems such as primary cells.

[0161] Single-Cell analysis of DEL-VPR-mediated fluorescent reporter expression The luciferase activity in Figure 12 reflects cumulative expression from all cells in the well but lacks insights into cell-to-cell variability in expression levels and distribution. Thus, we conducted single-cell analysis using flow cytometry by measuring the fluorescence intensity of mScarlet, driven by DEL-VPR (Figure 13A). Of note, DEL-VPR and C120-mScarlet were encoded on the same plasmid to minimize variability from co-transfection.

[0162] In HEK293T and CHO-K1 , a strong induction of mScarlet upon light stimulation was observed (Figure 13C, Figure 19). The fluorescence intensity after light stimulation in DEL-VPR cells was comparable to CMV-driven mScarlet expression. This observation could also be confirmed in live-cell imaging data of mCherry expression (Figure 21 ).

[0163] The transfection efficiency did not vary between experiments and reached 81 % in HEK293T cells and 62% in CHO-K1 cells for CMV-m Scarlet, and 76% in HEK293T and 63% in CHO-K1 for DEL-VPR (Figure 20). Without light stimulation, m Scarlet-positive cells (mScarlet+) were barely observed for DEL- VPR, whereas after illumination they reached almost the level of CMV-driven mScarlet+ cells. This indicates the low basal activity of DEL-VPR in the fluorescent reporter assay.

[0164] Analysis of mScarlet intensity in the total cell population revealed no significant effect of light exposure in CMV-mScarlet-expressing HEK293T or CHO-K1 cells (Figure 13D-E). Induced by blue light, DEL-VPR-transfected cells displayed mScarlet intensities comparable to CMV-driven expression in both the total and mScarlet+ populations (Figure 13D-G). By contrast, cells kept in the dark exhibited low mScarlet intensities. Notably, in DEL-VPR-transfected cells, even the mScarlet+ population showed significantly lower fluorescence in the dark compared to light-exposed cells (Figure 13F-G). These findings confirm that DEL- VPR has low basal activity, both in terms of the proportion of affected cells and expression intensity. Single-cell characterization of cyto- and phototoxicity

[0165] Photoactivation, transactivation domains, and blue light exposure can all contribute to cytotoxicity. To assess this, we performed a viability assay using flow cytometry on HEK293T and CHO-K1 cells transfected with either DEL-VPR and C120-mScarlet (on a single plasmid; Figure 13A) or CMV-m Scarlet after 24 h of constant blue light exposure (1500 pW / cm2) or in the dark.

[0166] Strikingly, the significantly higher viability of DEL-VPR cells kept in the dark compared to both CMV-transfected cells and light-exposed DEL-VPR cells in both cell types indicates that DEL-VPR itself does not exhibit cytotoxicity under the applied conditions (Figure 13H-I). Furthermore, in HEK293T blue-light exposure mildly but significantly reduces viability in HEK293T independently of the presence of DEL-VPR, whereas in CHO-K1 only in the DEL-VPR condition viability is reduced in the light compared to the dark condition. Taken together, these data show a limited cyto- and phototoxicity of DEL-VPR and the blue light exposure under the used conditions.

[0167] Optimization of light dose for maximal expression with minimal light exposure

[0168] Although we did not observe substantial phototoxicity under the tested conditions, further minimizing cellular stress is an important goal for the potential use of DEL- VPR in long-term large-scale bioproduction. To reduce the light exposure while maintaining the high inducibility of DEL-VPR, we investigated the lowest effective light dose. First, we conducted a dose-response experiment by varying light intensity under continuous exposure. To assess expression efficiency, we used light-induced expression of interferon beta (IFN|3), a high-throughput approach that requires functional IFN|3, a protein relevant to bioproduction. In this assay, secreted IFN|3 activates SEAP expression in reporter cells, with SEAP activity directly correlating to functionally active IFN|3 levels (Figure 14A).

[0169] SEAP expression was significantly induced at light intensities above 333 pW / cm2in HEK293T and above 100 pW / cm2in CHO-K1 (Figure 14B-C). Single values of individual experiments are presented in Figure 22. SEAP activity increased with higher light intensities, reaching saturation at 1000 pW / cm2in HEK293T and 833 pW / cm2in CHO-K1 . At these intensities, SEAP levels matched those of the CMV condition, to which samples were normalized. Thus, blue light applied at 1000 pW / cm2is sufficient for maximal DEL-VPR induction.

[0170] After identifying these minimum light intensities for maximal DEL-VPR induction, we tested different light-pulse regimens to further reduce light exposure and minimize potential phototoxicity. These intensities were benchmarked against the previously used 1500 pW / cm2and normalized to CMV-driven expression levels in each experiment.

[0171] As observed in continuous light conditions, no significant differences were detected between 1000 and 1500 pW / cm2in HEK293T or between 833 and 1500 pW / cm2in CHO-K1 under any pulsed-light regimen (Figure 14D-E). In HEK293T, light pulses of 10 s / 50 s, 1 min / 1 min, 2 min / 1 min, and 1 min / 3 min (ON / OFF) at 1000 pW / cm2maintained IFN[3 / SEAP expression comparable to continuous illumination (Figure 14D). At 1500 pW / cm2, pulses could be reduced to 10 s / 50 s, 0.5 min / 0.5 min, 1 min / 1 min, 2 min / 2 min, or 1 min / 3 min without significant loss of expression.

[0172] In CHO-K1 , all pulse conditions except 10 s / 50 s at 833 pW / cm2 preserved SEAP activity without significant reduction (Figure 14E).

[0173] In summary, overall light exposure can be reduced by resorting to intermittent illumination with a 1 min / 3 min on-off duty cycle at 1000 pW / cm2for HEK293T and 833 pW / cm2for CHO-K1 , thus decreasing total light dose by 4-fold. This offers a segue to also reducing overall energy and thus heat input into the cell culture volume by optogenetic irradiation, which becomes particularly important in large-scale formats with low surface to volume ratios and thus unfavourable efficiencies of heat dissipation caused by optogenetic control regimes.

[0174] Optogenetic production of a monoclonal antibody

[0175] As our data pinpointed DEL-VPR as a powerful tool for modulating gene expression in mammalian cells, we investigated whether more complex proteins, such as monoclonal antibodies (mAb), widely used in biotherapeutics, could also be efficiently produced using this optogenetic strategy. We chose the human epidermal growth factor receptor 2 (HER2)-targeted mAb Trastuzumab (IgG), also known as Herceptin, which is used to treat HER2-positive breast and gastric cancer by inhibiting the HER2 signalling pathway and thereby limiting proliferation. The mAb comprises two light (LC) and two heavy (HC) chains that assemble into a heterotetramer. We cloned the LC and HC downstream of the light-regulated promoter C120 and co-expressed the two chains under the control of DEL-VPR (Figure 15A).

[0176] To confirm the functionality of light-induced and CMV-driven mAb, we performed an ELISA using HER2-coated plates as the target antigen. Both DEL-VPR (light) and CMV-driven mAb in HEK293T and CHO-K1 showed clear absorbance signals for HER2, whereas DEL-VPR samples incubated in the dark showed no signal (Figure 15B). Specificity was verified using HER1 , a non-target antigen, which displayed no signal above the non-transfected control (used as normalization).

[0177] After confirming the mAb functionality, the dose-dependent inducibility was examined. In both HEK293T and CHO-K1 cells, mAb expression was not detectable in the dark (Figure 15C-F). However, blue-light exposure increased mAb expression, assembly, and secretion into the supernatant in a light dosedependent manner. In HEK293T cells, mAb expression was maximally induced at 1500 pW / cm2 blue light, while in CHO-K1 cells, it was already reached at 1000 pW / cm2 blue light and slightly dropped again at 1500 pW / cm2. At the maximal blue-light intensity, the expression levels reached or exceeded those afforded by the constitutive CMV promoter.

[0178] ELISA against human IgG demonstrated a significant increase of antibody titer upon light induction by DEL-VPR in HEK293T and CHO-K1 , while in the dark the titer barely exceeded the value of the negative control used for normalization (Figure 15G-H, Figure 23A-B). The same holds for the productivity measured in pg / cell / day (Figure 151-J, I, Figure 23C-D). The cells were incubated in the dark for the first 24 h after transfection, before switching to light conditions to initiate protein production for 24 h. By contrast, for the constitutive promoters (CMV-DEL- VPR, CMV-mAb) the protein production went on for a total of 48 h. Even despite the shorter production time, the mAb titer and productivity were significantly increased by DEL-VPR compared to the constitutive CMV-driven expression in HEK293T (Figure 15G, I, Figure 23A, C). However, in CHO-K1 cells, the titer and productivity were not significantly different between the DEL-VPR (light) and CMV (Figure 15H, J, Figure 23B, D). Thus, our data demonstrate that DEL-VPR allows a tightly controlled, light-dependent induction of mAb expression, reaching or even surpassing the level of strong constitutive expression mediated by a CMV promoter.

[0179] Optogenetic production of a bispecific antibody

[0180] Having successfully demonstrated the light-dependent expression of mAb, we next applied our approach to more complex biopharmaceuticals like a bispecific antibody (bsAb). Since one of the driving factors for the high production costs of bsAb is a contamination of unwanted by-products (e.g. HCscFv homodimers), we investigated whether we could modulate bsAb complex assembly by light- mediated induction. To establish this, we initially tested the light regulation of all bsAb chains simultaneously.

[0181] We chose a trimeric bsAb (IgG), comprising an LC and HC on one side and an HCscFv on the other side. We cloned the three constituent bsAb chains downstream of the light-responsive promoters C120 and co-expressed them with DEL-VPR (Figure 16A).

[0182] To confirm the functionality of light-induced and CMV-driven bsAb, we performed an ELISA using coated plates with each of the antigens separately as the prey. Both DEL-VPR (light) and CMV-driven bsAb in HEK293T and CHO-K1 showed clear absorbance signals for both antigens, whereas DEL-VPR samples incubated in the dark showed no signal (Figure 16B). This data demonstrates the bispecific functionality of the produced antibodies.

[0183] After verifying the functionality of the bsAb we analyzed the light-regulated expression on protein level using Western Blots. In both HEK293T and CHO-K1 cells, bsAb expression driven by DEL-VPR and secretion were barely detectable in the dark but significantly increased in a light dose-dependent manner, as observed by trimeric bsAb complex assembly in the supernatant (Figure 16C-F), for both HEK293T and CHO-K1 cells, maximal bsAb expression was observed at 1500 pW / cm2. In HEK293T cells, the bsAb level was significantly higher in the DEL-VPR samples (1500 pW / cm2) compared to the CMV-bsAb control (Figure 16C, E). The light-dependent bsAb induction by DEL-VPR also emerged in stably integrated HEK293T (Figure 24). In CHO-K1 cells, the DEL-VPR-induced samples showed a tendency towards higher expression than the CMV samples (Figure 16D, F). Strikingly, DEL-VPR-mediated expression resulted in the predominant assembly of the trimeric bsAb in the supernatant. DEL-VPR led to a significant induction of the bsAb titer under blue light compared to the dark by 30-fold and 34-fold while increasing the productivity by 32-fold and 33-fold in HEK293T and CH0-K1 , respectively (Figure 16G-J, Figure 25). The antibody titer was not significantly different between the DEL-VPR-induced and CMV-driven expression in HEK293T cells (Figure 16G), whereas in CHO-K1 cells, the bsAb levels were slightly higher in the CMV-driven expression compared to the DEL-VPR-driven expression (Figure 16H, Figure 25B). The productivity in pg / cell / day did not differ between the constitutive and the DEL- VPR-induced expression (Figure 161, Figure 25C-D). As in the above experiments, the constitutive expression occurred over 48 h, whereas the DEL- VPR-induced expression only upregulated by light during the last 24 h of the experiment.

[0184] In summary, these data demonstrate that DEL-VPR can be used as an optogenetic tool for the tuneable expression of large protein complexes, reaching levels of constitutive protein expression driven by, e.g., a CMV promoter. By allowing to switch on bsAb expression during a particular desired time window, DEL-VPR stands to reduce the metabolic burden that can occur with a static high expression using constitutive promoters such as CMV. bsAb complex composition can be modulated by light

[0185] When we induced all three bsAb chains at the same time (Figure 16B-E), we observed the trimeric bsAb as the predominant product. In addition to regulating the production of the bsAb as such, we examined whether the composition of the bsAb complexes could be modified by light since by-products represent a major factor impeding purification and yield in bsAb production. To this end, we constitutively expressed the HCscFv chain combined with the light-dependent expression of LC / HC driven by DEL-VPR (Figure 17A-B). This enables a lightdependent transition from the HCscFv dimer in the dark to the trimeric bsAb complex in the presence of light, thereby establishing the proof of concept for light-dependent modulation of protein complex assembly.

[0186] In the dark, only the constitutively expressed HCscFv was produced and subsequently assembled and secreted as HCscFv dimers into the supernatant in both HEK293T and CHO-K1 cells (Figure 17C-F). We observed HCscFv dimers in all DEL-VPR conditions in the dark (Figure 17C-D), but the expression of the LC and HC in a light dose-dependent manner resulted in a switch from the HCscFv dimers to the trimeric bsAb. This was also demonstrated by the ratio of bsAb to HCscFv dimer, which increased in a light dose-dependent manner (Figure 17C-D, G-H). At maximal expression (1500 pW / cm2), both the bsAb levels and the ratio of bsAb to HCscFv dimers reached levels like for CMV-driven expression (Figure 17C-F, l-J).

[0187] In conclusion, our data show that DEL-VPR allows not only dynamic regulation of the amount of biopharmaceuticals produced but also their correct assembly into protein complexes. The use of DEL-VPR, therefore, offers the possibility to dynamically modify the composition of bsAb. Moreover, DEL-VPR opens new opportunities in bioproduction, namely the dynamically titrable expression of biopharmaceuticals without sacrificing the high production levels that have so far only been achieved by constitutive promoters. At the same time, the DEL-VPR could be used to reduce protein toxicity and to minimize the metabolic burden of protein overexpression since recombinant protein expression could be kept inactive during the clonal selection and expansion phase. Alongside absolute expression levels, the folding and post-translational maturation of the antibodies are the main bottlenecks in limiting the yield. Based on the fast response times and reversibility of optogenetic regulation, the prospective enhancement of realtime feedback could facilitate the production of proteins in a manner that aligns with the cells’ capacity for their processing, thereby paving the way for the next generation of bioproduction. In particular, DEL-VPR could help limit the expression to suitable levels such as not to overwhelm the cellular capabilities for protein translation, folding, assembly, and, if applicable, secretion. DEL-VPR could thus help to reduce the aggregation and toxicity of non-processed antibodies and increase their titer.

[0188] In summary, DEL-VPR has the potential to be employed in a multitude of applications within the field of bioprocessing. These include the induction of the expression at a desired point in time, the modulation of protein complex composition and the regulation of the expression of toxic proteins. Therefore, DEL-VPR could facilitate more efficient production of various biopharmaceuticals, reducing their production costs and increasing their accessibility as standard therapies for targeting difficult-to-treat conditions such as cancer and autoimmune diseases. Beyond bioproduction, DEL-VPR supports multiple use cases in basic research and cell biology, e.g., the control of cellular metabolism; the modulation of cell-cycle progression; the induction or prevention of apoptosis; the modulation of signaling pathways; and the control of host- and stem-cell differentiation for cell-based therapeutics.

[0189] Non-patent literature:

[0190] Carver J, Ng D, Zhou M, et al. Maximizing antibody production in a targeted integration host by optimization of subunit gene dosage and position. Biotechnol Progress. 2020; 36:e2967. https: / / doi.org / 10.1002 / btpr.2967

[0191] Jan N Hansen, Fabian Kaiser, Christina Klausen, Birthe Stuven, Raymond Chong, Wolfgang Bdnigk, David U Mick, Andreas Mdglich, Nathalie Jurisch- Yaksi, Florian I Schmidt, Dagmar Wachten (2020) Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium eLife 9:e57907; https: / / doi.org / 10.7554 / eLife.57907

[0192] Motta-Mena L.B., Reade A., Mallory M.J., Glantz S., Weiner O.D, Lynch K.W., Gardner K.H. An optogenetic gene expression system with rapid activation and deactivation kinetics. Nat Chem Biol. 10(3): 196-202 (2014). https: / / doi: 10.1038 / nchembio.1430

[0193] Pastrana, E. Optogenetics: controlling cell function with light. Nat Methods 8, 24-25 (2011). https: / / doi.Org / 10.1038 / nmeth.f.323

[0194] Redchuk, T., Kaberniuk, A. & Verkhusha, V. Near-infrared light-controlled systems for gene transcription regulation, protein targeting and spectral multiplexing. Nat Protoc 13, 1121-1136 (2018). https: / / doi.Org / 10.1038 / nprot.2018.022

[0195] Rost B.R., Schneider-Warme F., Schmitz D., Hegemann, P. Optogenetic Tools for Subcellular Applications in Neuroscience. Neuron 96(3), 572-603 (2017). https: / / doi.Org / 10.1016 / j . neuron.2017.09.047

[0196] Shimizu-Sato, S., Huq, E., Tepperman, J. et al. A light-switchable gene promoter system. Nat Biotechnol 20, 1041-1044 (2002). https: / / doi.Org / 10.1038 / nbt734

Claims

Ningaloo Biosystems GmbH, UNIV Bonn 24539WOClaims1 . Method for producing at least one biologically active molecule, in particular a biotherapeutic, by optogenetic control of gene expression in at least one eukaryotic or prokaryotic cell, wherein the biologically active molecule comprises at least one protein of interest comprising at least two different subunits (5, 6, 23, 24, 25), said method comprising:- introducing into the cell at least one first expression cassette (2, 31 , 43, 45) comprising at least one first nucleic acid sequence (7, 32, 47, 49) linked to a first promoter (8, 33, 53), wherein the first nucleic acid sequence (7, 32, 47, 49) encodes at least one photosensitive transcription factor (9, 52),- introducing into the cell a second expression cassette (3, 44) comprising at least one second nucleic acid sequence (11 , 48) encoding a first (5, 23) of the at least two subunits (5, 6, 23, 24, 25) of the protein of interest and being operably linked to a second promoter (12), the second promoter (12) being operably linked to at least one transcription factor binding site (13), and- introducing into the cell a third expression cassette (4, 21 , 46) comprising at least one third nucleic acid sequence (15, 26, 55) encoding at least a second (6, 24) of the at least two subunits (5, 6, 23, 24, 25) of the protein of interest and being operably linked to a third promoter (16, 27, 56), wherein expression of the second nucleic acid sequence (11 , 48) is controlled through activation or repression of the second promoter (12) by illuminating the cell with light comprising at least one wavelength that triggers at least one structural change of the photosensitive transcription factor (9).

2. Method according to claim 1 , the structural change causes binding or release of the photosensitive transcription factor (9) to / from the transcription factor binding site (13), or wherein the structural change causes nuclear trafficking of the photosensitive transcription factor (9).

3. Method according to claim 1 or 2, wherein the photosensitive transcription factor (9, 52) comprises at least one gene expression controlling domain (10, 54) and / or forms a complex with at least one gene expression controlling domain (10, 54), wherein the gene expression controlling domain (10, 54) comprises a transcriptional activation domain or a transcriptional repression domain.

4. Method according to any one of claims 1 to 3, wherein the first promoter (8, 53) is a constitutive promoter or wherein the first promoter (33) is operably linked to at least one regulatory sequence (34), wherein the regulatory sequence (34) comprises at least one transcription factor binding site (35), wherein the first promoter (33) and thus expression of the first nucleic acid sequence (32) encoding the photosensitive transcription factor (9) is upregulated by illuminating the cell with light comprising at least one wavelength that triggers binding of the photosensitive transcription factor (9) to a transcription factor binding site (35) of the regulatory sequence (34).

5. Method according to any one of claims 1 to 4, wherein the third promoter (16) is a constitutive promoter or wherein the third promoter (27, 56) is operably linked to at least one transcription factor binding site (28, 57), wherein expression of the third nucleic acid sequence (26, 55) is controlled through activation or repression of the third promoter (27, 56) by illuminating the cell with light comprising at least one wavelength that triggers at least one structural change of said photosensitive transcription factor (9, 52) or another photosensitive transcription factor.

6. Method according to claim 5, wherein the structural change causes binding or release of the photosensitive transcription factor (9, 52) to / from the transcription factor binding site (28, 57), binding or release between at least one DNA binding subunit and at least one transcription activating subunit of the transcription factor, or wherein the structural change causes nuclear trafficking of the photosensitive transcription factor (9, 52).

7. Method according to any one of claims 1 to 6, wherein at least one additional expression cassette (22) is introduced into the cell, the additional expression cassette (22) comprising at least one further nucleic acid sequence (29) encoding at least one further subunit (25) of the at least two subunits (5, 6, 23, 24, 25) of the protein of interest and being operably linked to a further promoter sequence (30).

8. Method according to claim 7, wherein the further promoter sequence (30) comprises a constitutive promoter or wherein the further promoter sequence is operably linked to at least one transcription factor binding site, wherein expression of the further nucleic acid sequence is controlled through activation or repression of the further promoter sequence by illuminating the cell with light comprising at least one wavelength that triggers at least one structural change of said photosensitive transcription factor (9, 52) or another photosensitive transcription factor.

9. Method according to claim 8, wherein the structural change causes binding or release of the photosensitive transcription factor to / from the transcription factor binding site, binding or release between at least one DNA binding subunit and at least one transcription activating subunit of the transcription factor, or wherein the structural change causes nuclear trafficking of the photosensitive transcription factor.

10. Expression system (1 , 20, 40) for producing at least one biologically active molecule, in particular a biotherapeutic, by optogenetic control of gene expression in at least one eukaryotic or prokaryotic cell, wherein the biologically active molecule comprises at least one protein of interest comprising at least two different subunits (5, 6, 23, 24, 25), said expression system comprising:- At least one first expression cassette (2, 31 , 43, 45) comprising at least one first nucleic acid sequence (7, 32, 47, 49) linked to a first promoter (8, 33, 53), wherein the first nucleic acid sequence (7, 32, 47, 49) encodes at least one photosensitive transcription factor (9, 52) comprising a gene expression controlling domain (10, 54) and / orhaving a structure that enables forming a complex with at least one gene expression controlling domain (10, 54),- a second expression cassette (3, 44) comprising at least one second nucleic acid sequence (11 , 48) encoding a first (5, 23) of the at least two subunits (5, 6, 23, 24, 25) of the protein of interest and being operably linked to a second promoter (12), wherein the second promoter (12) is operably linked to at least one transcription factor binding site (13) and activatable or repressible by illumination with light comprising at least one wavelength that triggers at least one structural change of the photosensitive transcription factor (9), and- a third expression cassette (4, 21 , 46) comprising at least one third nucleic acid sequence (15, 26, 55) encoding at least a second (6, 24) of the at least two subunits (5, 6, 23, 24, 25) of the protein of interest and being operably linked to a third promoter (16, 27, 56).11 . Expression system according to claim 10, further comprising additional expression cassettes (22), each additional expression cassette (22) comprising at least one further nucleic acid sequence (29) encoding at least one further subunit (25) of the at least two subunits (5, 6, 23, 24, 25) of the protein of interest and being operably linked to a further promoter sequence (30).

12. Expression system according to claim 10 or 11 , wherein the photosensitive transcription factor (9, 52) comprises at least one light- oxygen-voltage-sensing domain (LOV domain), preferably EL222, or at least one cryptochrome (CRY), or at least one phytochrome, preferably phytochrome B, and / or wherein the transcription factor binding site (13, 57) comprises at least one binding site selected from the group consisting of at least one (C120), GAL4, TetR, lexA, and lacR binding site.

13. Expression system according to any one of claims 10 to 12, wherein the gene expression controlling domain (10, 54) of the photosensitive transcription factor (9, 52) comprises at least one transcriptional activation domain, preferably selected from the group consisting of VP16, VP64,p65, RTA, and any combination thereof, or a transcriptional repression domain, preferably KRAB.

14. A kit or composition for producing at least one biologically active molecule, in particular a biotherapeutic, wherein the biologically active molecule comprises at least one protein of interest comprising at least two subunits, by optogenetic control of gene expression of at least one of the at least two subunits in at least one eukaryotic or prokaryotic cell, comprising the expression system according to any one of claims 10 to 13.

Images