A universal autoregulatory mammalian cell line platform for biologics manufacturing

Abstract

Disclosed are genetically controlled circuits, cells and methods that use repressor polypeptides to reduce the rate of transcription of an exogenous therapeutic polypeptide encoding gene in response to changing conditions. [Selection diagram] None

Description

[Technical Field]

[0001] The present invention relates to methods and compositions for regulating the expression of products, such as recombinant proteins, by cells and cell lines using genetic regulatory circuits that respond to cellular stress. [Background technology]

[0002] Recombinant therapeutic proteins are commonly expressed in cell expression systems, such as mammalian cell expression systems. In 2014, the total number of approved biologics on the market was 212, and 56% of therapeutic products approved for marketing by the FDA were manufactured in mammalian cell lines. However, the high costs associated with manufacturing have contributed to the growth of medical transdermal therapy worldwide.

[0003] Furthermore, next-generation protein biologics (NGBs), such as next-generation fusion proteins, multimeric glycoproteins, or next-generation antibodies, often have complex and / or non-native structures and have proven more difficult to express than molecules such as monoclonal antibodies. Current host cell lines have not evolved pathways for the efficient synthesis and secretion of NGBs, resulting in significantly reduced growth, low productivity, and often a product with poor product quality (PQ). Therefore, these NGBs are considered difficult to express, and the productivity and product quality do not meet clinical and commercial requirements. Therefore, there is a growing need to develop and manufacture recombinant biological therapeutics rapidly, efficiently, and cost-effectively while maintaining final product quality.

[0004] Current gene expression systems for the synthesis of recombinant proteins using mammalian cell lines are constitutively active and direct the transcription of recombinant protein product genes regardless of cell culture conditions or host cell metabolism. Such systems are unable to coordinate product gene transcription with the intracellular conditions of the host cell line, as occurs for endogenous host cell proteins, leading to cellular stress and poor product results, particularly for NGB. Because NGB is pushing our current cell lines and gene expression systems to their limits, there is a need to better coordinate transcription of recombinant protein product genes with the overall metabolism of the host cell. This would reduce the level of cellular stress and help better utilize the existing ability of our mammalian cell factories to produce high levels of product with the correct product quality attributes (e.g., glycosylation profile, correct folding structure, etc.).

[0005] When mammalian host cell lines are constrained to constitutively synthesize high levels of recombinant protein products, particularly NGB, or when proteins are difficult to express, a cellular stress pathway called the unfolded protein response (UPR) is activated by the accumulation of misfolded proteins. This leads to a generalized decline in protein translation to allow the cell sufficient time to properly process and fold the current protein load. Activation of such a stress response is inhibitory not only to the overall yield of recombinant protein products, but also to the desired PQ profile.

[0006] [Summary of the Invention] In one aspect, the disclosure features a genetic regulatory circuit that uses a repressor polypeptide (e.g., a version of the Cas9 protein (derived from a CRISPR-Cas9 gene editing system) lacking nuclease activity (dCas9)) to reduce the rate of transcription of an exogenous therapeutic polypeptide encoding a gene in response to a change in condition (e.g., increased cellular stress). In response to the change in condition, the condition-dependent gene promoter increases the rate of transcription of the repressor polypeptide gene. The resulting repressor polypeptide binds to the exogenous therapeutic polypeptide encoding the gene or to a control element operably linked to the exogenous therapeutic polypeptide encoding the gene. In some embodiments, when the repressor polypeptide comprises a version of Cas9, the repressor polypeptide binds to the exogenous therapeutic polypeptide encoding the gene or to a control element operably linked to the exogenous therapeutic polypeptide encoding the gene for co-expression of at least one guide RNA (gRNA) having homology to the exogenous therapeutic polypeptide encoding the gene or to a control element operably linked thereto. When the repressor polypeptide binds to an exogenous therapeutic polypeptide encoding gene or a regulatory element operably linked thereto, the transcription rate of the exogenous therapeutic polypeptide encoding gene is reduced, leading to a decrease in the intracellular mRNA copy number of the therapeutic polypeptide. In some embodiments, the change in state is a change in cellular stress, for example, an increase in cellular stress or a transition from a non-stressed state to a stressed state, and the change in cellular stress is activation of the mammalian UPR, although other cellular stress responses can also be used for this purpose. In one embodiment, the exogenous therapeutic polypeptide encoding gene is transcribed under the control of the hCMV promoter, although other promoters may also be used (e.g., mCMV and hybrid CMV promoters). By reducing the transcription rate of the exogenous therapeutic polypeptide encoding gene, the biosynthetic burden of the exogenous therapeutic polypeptide in the host cell is reduced, thereby alleviating the initial cellular stress response.In this way, the host cell line can autoregulate the transcription rate of the recombinant protein product gene and avoid prolonged activation of the early cellular stress response. Once the early stress response is alleviated, the transcription rate of the exogenous therapeutic polypeptide is derepressed over time. This can result in an overall increase in recombinant protein yield over time, as the cell optimally coordinates recombinant gene expression with the overall physiological context of the cell to better utilize the cell's biosynthetic capacity.

[0007] In one aspect, the disclosure features a genetic control circuit that includes a first control element, e.g., a first promoter element, operably linked to a sequence encoding an exogenous therapeutic polypeptide and a second control element, e.g., a second promoter element, operably linked to a sequence encoding a repressor polypeptide, wherein the second control element has a first level of activity under a first condition and a second level of activity under a second condition, and wherein, in the presence of the second condition, expression of the therapeutic polypeptide is modulated, e.g., decreased. In some embodiments, the genetic control circuit further optionally includes a third control element, e.g., a third promoter, operably linked to a sequence encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) gRNAs. In some embodiments, the third control element has a first level of activity under a first condition and a second level of activity under a second condition.

[0008] In one aspect, the disclosure features a cell, e.g., a CHO cell, comprising a first control element, e.g., a first promoter, operably linked to a sequence encoding an exogenous therapeutic polypeptide and a second control element, e.g., a second promoter, operably linked to a sequence encoding a repressor polypeptide, wherein the second control element has a first level of activity under a first condition and a second level of activity under a second condition, and wherein, in the presence of the second condition, expression of the therapeutic polypeptide is modulated, e.g., decreased. In some embodiments, the cell further optionally comprises a third control element, e.g., a third promoter, operably linked to a sequence encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) gRNAs. In some embodiments, the third control element has a first level of activity under a first condition and a second level of activity under a second condition. In some embodiments, the cell further optionally comprises a fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more control elements. In one embodiment, an entire signaling pathway is controlled by controlling a single node in the pathway using the methods disclosed herein. In some embodiments, an entire signaling pathway is controlled by controlling multiple metabolic branches of the signaling pathway, for example, by using different promoters that regulate different sequences in the pathway. Thus, the methods of the present invention provide several layers of control in autoregulatory cells. For example, a translation elongation initiation factor is an example of a global node that can control multiple pathways. Alternatively, an example of a local node is a gene encoding the enzyme galactosyltransferase, which is required to add a galactose residue to the glycan attached to Asn297 of a recombinant antibody heavy chain polypeptide, creating an N-glycan with both galactose and sialic acid residues.

[0009] In one aspect, the disclosure features a cell, e.g., a CHO cell, comprising a first control element, e.g., a first promoter, operably linked to an insertion site, e.g., a restriction site or SSI site, and a second control element, e.g., a second promoter, operably linked to a sequence encoding a repressor polypeptide, wherein the insertion site is suitable for insertion of a sequence encoding an exogenous therapeutic polypeptide, and the second control element has a first level of activity under a first condition and a second level of activity under a second condition, and in the presence of the second condition, expression of the therapeutic polypeptide is modulated, e.g., decreased. In some embodiments, the cell further optionally comprises a third control element, e.g., a third promoter, operably linked to a sequence encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) gRNAs. In some embodiments, the third control element has a first level of activity under a first condition and a second level of activity under a second condition.

[0010] In one aspect, the disclosure features a kit for expression of a therapeutic polypeptide, comprising cells, e.g., CHO cells, comprising a first control element, e.g., a first promoter, operably linked to a sequence encoding an exogenous therapeutic polypeptide and a second control element, e.g., a second promoter, operably linked to a sequence encoding a repressor polypeptide, wherein the second control element has a first level of activity under a first condition and a second level of activity under a second condition, and in the presence of the second condition, expression of the therapeutic polypeptide is modulated, e.g., decreased. In some embodiments, the cells further optionally comprise a third control element, e.g., a third promoter, operably linked to a sequence encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) gRNAs. In some embodiments, the third control element has a first level of activity under a first condition and a second level of activity under a second condition.

[0011] In one aspect, the disclosure features a kit for expression of a therapeutic polypeptide, comprising cells, e.g., CHO cells, comprising a first control element, e.g., a first promoter operably linked to an insertion site, e.g., a restriction site or SSI site, and a second control element, e.g., a second promoter, operably linked to a sequence encoding a repressor polypeptide, wherein the insertion site is suitable for insertion of a sequence encoding an exogenous therapeutic polypeptide, and the second control element has a first level of activity under a first condition and a second level of activity under a second condition, and in the presence of the second condition, expression of the therapeutic polypeptide is modulated, e.g., decreased. In some embodiments, the cells further optionally comprise a third control element, e.g., a third promoter, operably linked to a sequence encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) gRNAs. In some embodiments, the third control element has a first level of activity under a first condition and a second level of activity under a second condition.

[0012] In one aspect, the disclosure features a kit for expression of a therapeutic polypeptide, the kit including one or more nucleic acids comprising a first control element, e.g., a first promoter, operably linked to a sequence encoding an exogenous therapeutic polypeptide, and a second control element, e.g., a second promoter, operably linked to a sequence encoding a repressor polypeptide, the second control element having a first level of activity under a first condition and a second level of activity under a second condition, and in the presence of the second condition, expression of the therapeutic polypeptide is modulated, e.g., decreased. In some embodiments, the kit further optionally includes a third control element, e.g., a nucleic acid comprising a third promoter, operably linked to a sequence encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) gRNAs. In some embodiments, the third control element has a first level of activity under a first condition and a second level of activity under a second condition.

[0013] In one aspect, the disclosure features a kit for expressing a therapeutic polypeptide, comprising one or more nucleic acids comprising a first control element, e.g., a first promoter, operably linked to an insertion site, e.g., a restriction site, and a second control element, e.g., a second promoter, operably linked to a sequence encoding a repressor polypeptide, wherein the insertion site is suitable for insertion of a sequence encoding an exogenous therapeutic polypeptide, and the second control element has a first level of activity under a first condition and a second level of activity under a second condition, and in the presence of the second condition, expression of the therapeutic polypeptide is modulated, e.g., decreased. In some embodiments, the kit further optionally comprises a nucleic acid comprising a third control element, e.g., a third promoter, operably linked to a sequence encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) gRNAs. In some embodiments, the third control element has a first level of activity under a first condition and a second level of activity under a second condition.

[0014] In one aspect, the disclosure features a method of producing a therapeutic polypeptide, the method including: a) obtaining cells, e.g., CHO cells, comprising a first control element, e.g., a first promoter, operably linked to a sequence encoding an exogenous therapeutic polypeptide and a second control element, e.g., a second promoter, operably linked to a sequence encoding a repressor polypeptide, wherein the second control element has a first level of activity under first conditions and a second level of activity under second conditions, and in the presence of the second conditions, expression of the therapeutic polypeptide is modulated, e.g., decreased; and b) culturing the cells under conditions that allow production of the therapeutic polypeptide, thereby producing the therapeutic polypeptide. In some embodiments, the cells of a) further optionally comprise a third control element, e.g., a third promoter, operably linked to a sequence encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) gRNAs. In some embodiments, the third control element has a first level of activity under a first condition and a second level of activity under a second condition.

[0015] In one aspect, the disclosure features a method of making a therapeutic polypeptide, the method including: a) obtaining a cell, e.g., a CHO cell; b) forming or providing in the cell a first nucleic acid sequence encoding a first control element, e.g., a first promoter, operably linked to a sequence encoding an exogenous therapeutic polypeptide; c) forming or providing in the cell a second nucleic acid encoding a second control element, e.g., a second promoter, operably linked to a sequence encoding a repressor polypeptide, wherein the second control element has a first level of activity under first conditions and a second level of activity under second conditions, and wherein in the presence of the second conditions, expression of the therapeutic polypeptide is modulated, e.g., decreased; and d) culturing the cell under conditions that allow the therapeutic polypeptide to be made, thereby making the therapeutic polypeptide. In some embodiments, the method optionally further comprises a further step between c) and d) comprising forming or providing in the cell a third control element, e.g., a third nucleic acid encoding a third promoter, operably linked to a sequence encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) gRNAs. In some embodiments, the third control element has a first level of activity under first conditions and a second level of activity under second conditions.

[0016] In one aspect, the disclosure provides a method of making a therapeutic polypeptide, comprising the steps of: a) obtaining a cell, e.g., a CHO cell; b) forming or providing in the cell a first nucleic acid sequence encoding a first control element, e.g., a first promoter, operably linked to a sequence encoding an exogenous therapeutic polypeptide; c) forming or providing in the cell a second nucleic acid encoding a second control element, e.g., a second promoter, operably linked to a sequence encoding a repressor polypeptide; and, optionally, d) forming in the cell one or more (e.g., e) forming or providing a third nucleic acid encoding a third control element operably linked to a sequence encoding a gRNA (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more), wherein the second control element has a first level of activity under first conditions and a second level of activity under second conditions, and in the presence of the second conditions, expression of the therapeutic polypeptide is modulated, e.g., decreased; and f) culturing the cells under conditions that allow for production of the therapeutic polypeptide, thereby producing the therapeutic polypeptide. In embodiments, steps a-d can be performed in any order. In some embodiments, the third control element has a first level of activity under first conditions and a second level of activity under second conditions.

[0017] In one aspect, the disclosure features a nucleic acid including a first control element, e.g., a first promoter, operably linked to a sequence encoding an exogenous therapeutic polypeptide and a second control element, e.g., a second promoter, operably linked to a sequence encoding a repressor polypeptide, wherein the second control element has a first level of activity under a first condition and a second level of activity under a second condition, and wherein, in the presence of the second condition, expression of the therapeutic polypeptide is modulated, e.g., decreased. In some embodiments, the nucleic acid further optionally includes a third control element, e.g., a third promoter, operably linked to a sequence encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) gRNAs. In some embodiments, the third control element has a first level of activity under a first condition and a second level of activity under a second condition.

[0018] In one aspect, the disclosure features a nucleic acid including a first control element, e.g., a first promoter, operably linked to an insertion site, e.g., a restriction site, and a second control element, e.g., a second promoter, operably linked to a sequence encoding a repressor polypeptide, wherein the insertion site is suitable for insertion of a sequence encoding an exogenous therapeutic polypeptide, and the second control element has a first level of activity under a first condition and a second level of activity under a second condition, and in the presence of the second condition, expression of the therapeutic polypeptide is modulated, e.g., decreased. In some embodiments, the nucleic acid further includes a third control element, e.g., a third promoter, operably linked to a sequence encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) gRNAs. In some embodiments, the third control element has a first level of activity under a first condition and a second level of activity under a second condition.

[0019] In one aspect, the present disclosure features a method of making a cell of the disclosure, the method including: a) forming or providing in the cell a first nucleic acid sequence encoding a first control element, e.g., a first promoter, operably linked to a sequence encoding an exogenous therapeutic polypeptide; and b) forming or providing in the cell a second control element, e.g., a second nucleic acid encoding a second promoter, operably linked to a sequence encoding a repressor polypeptide, wherein the second control element has a first level of activity under first conditions and a second level of activity under second conditions, and wherein in the presence of the second conditions, expression of the therapeutic polypeptide is modulated, e.g., decreased or increased, thereby producing the cell. In some embodiments, the method further includes step c) comprising forming or providing in the cell a third control element, e.g., a third nucleic acid sequence encoding a third promoter, operably linked to a sequence encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) gRNAs. In embodiments, steps a-c can be performed in any order. In some embodiments, the third control element has a first level of activity under first conditions and a second level of activity under second conditions.

[0020] In one aspect, the present disclosure features a method of producing a cell capable of providing economically enhanced yields of a polypeptide having desired product quality attributes, e.g., an exogenous therapeutic polypeptide, the method including: a) forming or providing in the cell a first nucleic acid sequence encoding a first control element, e.g., a first promoter, operably linked to a sequence encoding the exogenous therapeutic polypeptide; and b) forming or providing in the cell a second nucleic acid encoding a second control element, e.g., a second promoter, operably linked to a sequence encoding a repressor polypeptide, wherein the second control element has a first level of activity under first conditions and a second level of activity under second conditions, and wherein in the presence of the second conditions, expression of the therapeutic polypeptide is modulated, e.g., decreased or increased, thereby producing a cell capable of providing economically enhanced yields of a polypeptide having desired product quality attributes, e.g., an exogenous therapeutic polypeptide. In some embodiments, the method further includes step c) comprising forming or providing in the cell a third control element, e.g., a third nucleic acid sequence encoding a third promoter, operably linked to a sequence encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) gRNAs. In embodiments, steps a-c can be performed in any order. In some embodiments, the third control element has a first level of activity under first conditions and a second level of activity under second conditions.

[0021] In one aspect, the present disclosure features a cell comprising a first control element selected from Table 5 operably linked to a sequence encoding an exogenous therapeutic polypeptide selected from Tables 1-4, a second control element selected from Table 6 operably linked to a sequence encoding a Cas9 polypeptide, and one or more constitutively expressed gRNA sequences, wherein the second control element has a first level of activity under a first condition and a second level of activity under a second condition, and wherein expression of the therapeutic polypeptide is modulated in the presence of the second condition.

[0022] In one aspect, the present disclosure features a plurality of cells described herein, wherein one or more cells comprise a first condition and one or more cells comprise a second condition. [Brief explanation of the drawings]

[0023] [Figure 1A] Figures 1A, 1B, 1C, and 1D show a schematic representation of the design principles of a genetic control circuit that regulates transcription of a recombinant or therapeutic polypeptide product gene in response to a condition, e.g., cellular stress. In Figure 1A, production of the recombinant protein induces stress / toxicity, which activates production of a repressor, thereby directly inhibiting recombinant or therapeutic polypeptide expression. In Figure 1B, the resulting inhibition of recombinant or therapeutic polypeptide expression in Figure 1A ultimately eliminates stress / toxicity, removing activation of the repressor and indirectly leading to a passive increase in recombinant or therapeutic polypeptide expression. In effect, this system can lead to a time-dependent oscillation of recombinant or therapeutic polypeptide expression around a specific level that induces stress / toxicity in the host cell (Figure 1C). An additional, optional layer of control can be applied to this basic circuit, including a positive activator of recombinant or therapeutic polypeptide expression that becomes active when cellular stress / toxicity is alleviated (Figure 1D). [Figure 1B]Figures 1A, 1B, 1C, and 1D show a schematic representation of the design principles of a genetic control circuit that regulates transcription of a recombinant or therapeutic polypeptide product gene in response to a condition, e.g., cellular stress. In Figure 1A, production of the recombinant protein induces stress / toxicity, which activates production of a repressor, thereby directly inhibiting recombinant or therapeutic polypeptide expression. In Figure 1B, the resulting inhibition of recombinant or therapeutic polypeptide expression in Figure 1A ultimately eliminates stress / toxicity, removing activation of the repressor and indirectly leading to a passive increase in recombinant or therapeutic polypeptide expression. In effect, this system can lead to a time-dependent oscillation of recombinant or therapeutic polypeptide expression around a specific level that induces stress / toxicity in the host cell (Figure 1C). An additional, optional layer of control can be applied to this basic circuit, including a positive activator of recombinant or therapeutic polypeptide expression that becomes active when cellular stress / toxicity is alleviated (Figure 1D). [Figure 1C] Figures 1A, 1B, 1C, and 1D show a schematic representation of the design principles of a genetic control circuit that regulates transcription of a recombinant or therapeutic polypeptide product gene in response to a condition, e.g., cellular stress. In Figure 1A, production of the recombinant protein induces stress / toxicity, which activates production of a repressor, thereby directly inhibiting recombinant or therapeutic polypeptide expression. In Figure 1B, the resulting inhibition of recombinant or therapeutic polypeptide expression in Figure 1A ultimately eliminates stress / toxicity, removing activation of the repressor and indirectly leading to a passive increase in recombinant or therapeutic polypeptide expression. In effect, this system can lead to a time-dependent oscillation of recombinant or therapeutic polypeptide expression around a specific level that induces stress / toxicity in the host cell (Figure 1C). An additional, optional layer of control can be applied to this basic circuit, including a positive activator of recombinant or therapeutic polypeptide expression that becomes active when cellular stress / toxicity is alleviated (Figure 1D). [Figure 1D]Figures 1A, 1B, 1C, and 1D show a schematic representation of the design principles of a genetic control circuit that regulates transcription of a recombinant or therapeutic polypeptide product gene in response to a condition, e.g., cellular stress. In Figure 1A, production of the recombinant protein induces stress / toxicity, which activates production of a repressor, thereby directly inhibiting recombinant or therapeutic polypeptide expression. In Figure 1B, the resulting inhibition of recombinant or therapeutic polypeptide expression in Figure 1A ultimately eliminates stress / toxicity, removing activation of the repressor and indirectly leading to a passive increase in recombinant or therapeutic polypeptide expression. In effect, this system can lead to a time-dependent oscillation of recombinant or therapeutic polypeptide expression around a specific level that induces stress / toxicity in the host cell (Figure 1C). An additional, optional layer of control can be applied to this basic circuit, including a positive activator of recombinant or therapeutic polypeptide expression that becomes active when cellular stress / toxicity is alleviated (Figure 1D). [Figure 2] 2 depicts an exemplary genetic control circuit showing recombinant or therapeutic polypeptide (rP) transcription driven by a first control element, e.g., a first promoter element, e.g., an hCMV promoter, is constitutive in the absence of a repressor polypeptide, e.g., dCas9, controlled from a second control element, e.g., a second promoter element, e.g., an unfolded protein response (UPR)-activated promoter. In this example, upon activation of the UPR, which can occur when rP is synthesized, a repressor polypeptide, e.g., dCas9, is produced. In combination with a gRNA, dCas9 binds to the hCMV promoter and inhibits rP production until the UPR stress response is alleviated. [Figure 3A]Figure 3A depicts a genetic regulatory circuit showing GFP transcription driven by a first control element, e.g., a first promoter element, e.g., an hCMV promoter, that is constitutive in the absence of a repressor polypeptide, e.g., dCas9, controlled from a second control element, e.g., a second promoter element, e.g., a constitutive mCMV promoter. In this example, constitutively expressed dCas9, in combination with gRNA1, 2, or 3 (specific for hCMV and constitutively generated from the U6 promoter), binds to the hCMV promoter and inhibits GFP expression. Figure 3B depicts flow cytometry histograms showing GFP fluorescence in CHO cells stably expressing hCMV-eGFP that were transfected with 1) an empty expression vector (UTC), 2) an expression vector encoding dCas9, or 3) an expression vector encoding dCas9 and three gRNAs specific for hCMV. [Figure 3B] Figure 3A depicts a genetic regulatory circuit showing GFP transcription driven by a first control element, e.g., a first promoter element, e.g., an hCMV promoter, that is constitutive in the absence of a repressor polypeptide, e.g., dCas9, controlled from a second control element, e.g., a second promoter element, e.g., a constitutive mCMV promoter. In this example, constitutively expressed dCas9, in combination with gRNA1, 2, or 3 (specific for hCMV and constitutively generated from the U6 promoter), binds to the hCMV promoter and inhibits GFP expression. Figure 3B depicts flow cytometry histograms showing GFP fluorescence in CHO cells stably expressing hCMV-eGFP that were transfected with 1) an empty expression vector (UTC), 2) an expression vector encoding dCas9, or 3) an expression vector encoding dCas9 and three gRNAs specific for hCMV. [Figure 4A]Figure 4A is a diagram depicting a genetic control circuit showing Mab HC and Mab LC transcription driven by two separate first control elements, e.g., a first promoter element, e.g., an hCMV promoter, that is constitutive in the absence of a repressor polypeptide, e.g., dCas9, controlled by a second control element, e.g., a second promoter element, e.g., a constitutive mCMV promoter. In this example, constitutively expressed dCas9, in combination with gRNA1, 2, or 3 (specific for hCMV and constitutively generated from the U6 promoter), binds to the hCMV promoter and inhibits Mab HC and Mab LC expression. Figure 4B is a graph depicting hCMV-Mab levels obtained from CHO cells transiently transfected with no DNA, an expression vector encoding dCas9, or an expression vector encoding dCas9 and one, two, or three gRNAs specific for hCMV, 3, 4, or 5 days after transfection. [Figure 4B] Figure 4A is a diagram depicting a genetic control circuit showing Mab HC and Mab LC transcription driven by two separate first control elements, e.g., a first promoter element, e.g., an hCMV promoter, that is constitutive in the absence of a repressor polypeptide, e.g., dCas9, controlled by a second control element, e.g., a second promoter element, e.g., a constitutive mCMV promoter. In this example, constitutively expressed dCas9, in combination with gRNA1, 2, or 3 (specific for hCMV and constitutively generated from the U6 promoter), binds to the hCMV promoter and inhibits Mab HC and Mab LC expression. Figure 4B is a graph depicting hCMV-Mab levels obtained from CHO cells transiently transfected with no DNA, an expression vector encoding dCas9, or an expression vector encoding dCas9 and one, two, or three gRNAs specific for hCMV, 3, 4, or 5 days after transfection. [Figure 5A]5A is a diagram depicting a genetic regulatory circuit showing GFP transcription driven by a first regulatory element, e.g., a first promoter element, e.g., an hCMV promoter, that is constitutive in the absence of a second regulatory element, e.g., a repressor polypeptide, e.g., dCas9, controlled from a second promoter element, e.g., an unfolded protein response (UPR) stress-inducible Grp78 promoter. In this example, UPR stress promotes expression of dCas9, which, in combination with gRNA1, 2, or 3 (specific for hCMV and constitutively generated from the U6 promoter), binds to the hCMV promoter and inhibits GFP expression. Figure 5B is a graph of flow cytometry data showing GFP fluorescence of CHO cells stably expressing hCMV-eGFP that were transiently transfected with either 1) an empty expression vector (WT), 2) an expression vector encoding dCas9 under the Grp78 promoter (Grp78 dCas9 control), or 3) an expression vector encoding dCas9 under the Grp78 promoter and an expression vector encoding a gRNA with specificity for the hCMV promoter, and treated with 400 ng / mL tunicamycin (TM) or not treated with TM (0TM). [Figure 5B]5A is a diagram depicting a genetic regulatory circuit showing GFP transcription driven by a first regulatory element, e.g., a first promoter element, e.g., an hCMV promoter, that is constitutive in the absence of a second regulatory element, e.g., a repressor polypeptide, e.g., dCas9, controlled from a second promoter element, e.g., an unfolded protein response (UPR) stress-inducible Grp78 promoter. In this example, UPR stress promotes expression of dCas9, which, in combination with gRNA1, 2, or 3 (specific for hCMV and constitutively generated from the U6 promoter), binds to the hCMV promoter and inhibits GFP expression. Figure 5B is a graph of flow cytometry data showing GFP fluorescence of CHO cells stably expressing hCMV-eGFP that were transiently transfected with either 1) an empty expression vector (WT), 2) an expression vector encoding dCas9 under the Grp78 promoter (Grp78 dCas9 control), or 3) an expression vector encoding dCas9 under the Grp78 promoter and an expression vector encoding a gRNA with specificity for the hCMV promoter, and treated with 400 ng / mL tunicamycin (TM) or not treated with TM (0TM). [Figure 6A]Figures 6A-6C show the effect of the genetic control circuit on transient recombinant protein expression in CHO host cell lines. Figure 6A shows the genetic control circuit contained in an expression vector. The dCas9 gene is under the control of the Grp78 promoter, and three gRNA sequences (gRNA1, 2, and 3) with specificity for the hCMV promoter are each under a separate constitutive U6 promoter (gRNA123 circuit). A variant of this vector contained the gRNA14 sequence instead of the gRNA1, 2, and 3 sequences (sgRNA14 circuit). Figure 6B shows the recombinant protein concentration produced from stable CHO pools containing the control circuit after transient transfection with expression vectors encoding several difficult-to-express recombinant proteins. Recombinant protein concentrations were determined 6 days after transient transfection. The parental CHO cell line lacks the genetic control circuit. Error bars represent the standard deviation of triplicate transfections for all data points, except for transfection of the parental cell line with the blinatumomab vector, which was performed in duplicate. Figure 6C shows the recombinant protein concentration on day 6 produced from stable CHO pools containing the regulatory circuit after transient transfection with an expression vector encoding the highly aggregating Mab H9K7. 24 hours after transfection, half of the transiently transfected flasks were treated with the UPR-inducer tunicamycin™ at a concentration of 0.1 μg / mL. Error bars represent the standard deviation of triplicate transfections. [Figure 6B]Figures 6A-6C show the effect of the genetic control circuit on transient recombinant protein expression in CHO host cell lines. Figure 6A shows the genetic control circuit contained in an expression vector. The dCas9 gene is under the control of the Grp78 promoter, and three gRNA sequences (gRNA1, 2, and 3) with specificity for the hCMV promoter are each under a separate constitutive U6 promoter (gRNA123 circuit). A variant of this vector contained the gRNA14 sequence instead of the gRNA1, 2, and 3 sequences (sgRNA14 circuit). Figure 6B shows the recombinant protein concentration produced from stable CHO pools containing the control circuit after transient transfection with expression vectors encoding several difficult-to-express recombinant proteins. Recombinant protein concentrations were determined 6 days after transient transfection. The parental CHO cell line lacks the genetic control circuit. Error bars represent the standard deviation of triplicate transfections for all data points, except for transfection of the parental cell line with the blinatumomab vector, which was performed in duplicate. Figure 6C shows the recombinant protein concentration on day 6 produced from stable CHO pools containing the regulatory circuit after transient transfection with an expression vector encoding the highly aggregating Mab H9K7. 24 hours after transfection, half of the transiently transfected flasks were treated with the UPR-inducer tunicamycin™ at a concentration of 0.1 μg / mL. Error bars represent the standard deviation of triplicate transfections. [Figure 6C]Figures 6A-6C show the effect of the genetic control circuit on transient recombinant protein expression in CHO host cell lines. Figure 6A shows the genetic control circuit contained in an expression vector. The dCas9 gene is under the control of the Grp78 promoter, and three gRNA sequences (gRNA1, 2, and 3) with specificity for the hCMV promoter are each under a separate constitutive U6 promoter (gRNA123 circuit). A variant of this vector contained the gRNA14 sequence instead of the gRNA1, 2, and 3 sequences (sgRNA14 circuit). Figure 6B shows the recombinant protein concentration produced from stable CHO pools containing the control circuit after transient transfection with expression vectors encoding several difficult-to-express recombinant proteins. Recombinant protein concentrations were determined 6 days after transient transfection. The parental CHO cell line lacks the genetic control circuit. Error bars represent the standard deviation of triplicate transfections for all data points, except for transfection of the parental cell line with the blinatumomab vector, which was performed in duplicate. Figure 6C shows the recombinant protein concentration on day 6 produced from stable CHO pools containing the regulatory circuit after transient transfection with an expression vector encoding the highly aggregating Mab H9K7. 24 hours after transfection, half of the transiently transfected flasks were treated with the UPR-inducer tunicamycin™ at a concentration of 0.1 μg / mL. Error bars represent the standard deviation of triplicate transfections. [Figure 7A]Figures 7A and 7B show the levels of recombinant protein aggregation for proteins synthesized by stable CHO pools containing the control circuit after transient transfection with expression vectors encoding several difficult-to-express recombinant proteins, such as those described in Figures 6A-6C. The levels of recombinant protein aggregation were determined from cell culture supernatant samples by oligomer detection assay (ODA) as described in Obrezanova et al. MAbs. 2015;7(2):352-63. Using this assay, a decrease in protein aggregation is represented by a decrease in absorbance at 450 nm. Figure 7A shows the aggregation data for the cell culture supernatant samples assayed for concentration in Figure 6B, and Figure 7B shows the aggregation data for the cell culture supernatant samples assayed for concentration in Figure 6C. [Figure 7B] Figures 7A and 7B show the levels of recombinant protein aggregation for proteins synthesized by stable CHO pools containing the control circuit after transient transfection with expression vectors encoding several difficult-to-express recombinant proteins, such as those described in Figures 6A-6C. The levels of recombinant protein aggregation were determined from cell culture supernatant samples by oligomer detection assay (ODA) as described in Obrezanova et al. MAbs. 2015;7(2):352-63. Using this assay, a decrease in protein aggregation is represented by a decrease in absorbance at 450 nm. Figure 7A shows the aggregation data for the cell culture supernatant samples assayed for concentration in Figure 6B, and Figure 7B shows the aggregation data for the cell culture supernatant samples assayed for concentration in Figure 6C. DETAILED DESCRIPTION OF THE INVENTION

[0024] definition The disclosures of any and all patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. While the present invention has been disclosed with reference to particular embodiments, it will be apparent that other embodiments and variations of the present invention may be devised by others skilled in the art without departing from the true spirit and scope of the present invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Furthermore, the materials, methods, and examples are merely illustrative and not intended to be limiting. Headings, subheadings, or numbered or lettered elements, e.g., (a), (b), (i), etc., are presented merely for ease of reading. The use of headings or numbered or lettered elements in this document does not require that the steps or elements be performed in alphabetical order or that the steps or elements are necessarily separate from one another. Other features, objects, and advantages of the present invention will be apparent from the description of the figures and from the claims. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0026] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "a cell" can mean one cell or more than one cell.

[0027] As used herein, the term "genetic regulatory circuit" refers to a sequence of gene expression elements, e.g., a protein-coding sequence, a control element, or a promoter element, wherein the genetic regulatory circuit includes at least one protein-coding sequence that encodes a recombinant or therapeutic polypeptide product, and the genetic regulatory circuit includes other gene expression elements that conditionally regulate expression of the recombinant or therapeutic polypeptide product. In one embodiment, the genetic regulatory circuit may include suitable insertion sites, e.g., restriction sites, recombination target sites, or landing pads, for insertion of one or more protein-coding sequences in place of the at least one protein-coding sequence that encodes a recombinant or therapeutic polypeptide product. In some embodiments, the genetic regulatory circuit may comprise a contiguous portion of a single nucleic acid molecule, multiple distinct portions of a single nucleic acid molecule, or may be distributed across more than one nucleic acid molecule.

[0028] As used herein, the term "control element" refers to a nucleic acid suitable for regulating (e.g., increasing or decreasing) expression of a coding sequence, e.g., a gene. A control element can comprise a promoter sequence, an enhancer sequence, or both a promoter and an enhancer sequence. A control element can comprise a contiguous nucleic acid sequence, a discontinuous nucleic acid sequence (a sequence interrupted by other coding or non-coding nucleic acid sequences), or both. A single control element can be contained in a single nucleic acid or in more than one nucleic acid. In one embodiment, a control element can comprise a sequence 5' or 3' of a coding sequence, e.g., a coding sequence for a recombinant, therapeutic, or repressor polypeptide. In one embodiment, a control element can comprise a sequence within one or more introns of a gene, e.g., a gene encoding a recombinant, therapeutic, or repressor polypeptide. In one embodiment, a control element can be contained, in part or in its entirety, within a coding sequence, e.g., a coding sequence for a recombinant, therapeutic, or repressor polypeptide. In one embodiment, a control element may be contained, partially or entirely, within a coding sequence, e.g., the coding sequence for a recombinant, therapeutic, or repressor polypeptide. In one embodiment, a control element may be contained, partially or entirely, within one or more introns of a gene, e.g., a gene encoding a recombinant, therapeutic, or repressor polypeptide. In one embodiment, a single control element may comprise a nucleic acid sequence i) proximal to (e.g., adjacent to or contained within) a gene, e.g., a gene encoding a recombinant, therapeutic, or repressor polypeptide, or ii) distal to (e.g., separated by 10 or more, 100 or more, 1000 or more, or 10,000 or more bases, located in separate and distinct nucleic acids) a gene, e.g., a gene encoding a recombinant, therapeutic, or repressor polypeptide.

[0029] As used herein, the term "promoter element" refers to a sequence having sufficient sequence derived from a naturally occurring or engineered promoter such that operably linking a coding sequence to the promoter element results in expression of the coding sequence. For example, a cytomegalovirus (CMV) promoter element comprises all or an active fragment of a CMV promoter, e.g., all or an active fragment of a CMV promoter, optionally including intron A and / or UTR sequences. In one embodiment, the CMV promoter element differs by no more than 5, 10, 20, 30, 50, or 100 nucleotides from a naturally occurring or engineered variant CMV promoter. In one embodiment, the CMV promoter element differs by no more than 1, 5, 10, or 50% of its nucleotides from a naturally occurring or engineered variant CMV promoter. An engineered promoter is a promoter that comprises synthetic (non-naturally occurring) sequence. In one embodiment, an engineered promoter comprises a non-naturally occurring rearrangement of naturally occurring transcriptional regulatory elements (e.g., as described in Brown et al., Biotechnology and Bioengineering, Vol. 111, No. 8, August 2014). In one embodiment, a promoter element for use in the cells, nucleic acids, and methods of the disclosure comprises sufficient sequence from an engineered promoter, e.g., a promoter comprising synthetic (non-naturally occurring) sequence, such that operably linking a coding sequence to the promoter element results in expression of the coding sequence. As used herein, a promoter element can be constitutive, regulated, repressible, strong, weak, or other characteristic of the promoter sequence it comprises. In one embodiment, a promoter element can comprise sequence 5' or 3' of a coding sequence, e.g., a coding sequence for a recombinant, therapeutic, or repressor polypeptide. In one embodiment, a promoter element can comprise sequence within one or more introns of a gene, e.g., a gene encoding a recombinant, therapeutic, or repressor polypeptide.In one embodiment, a promoter element may be contained, in part or in whole, within a sequence 5' or 3' of a coding sequence, e.g., a coding sequence for a recombinant, therapeutic, or repressor polypeptide. In one embodiment, a promoter element may be contained, in part or in whole, within a coding sequence, e.g., a coding sequence for a recombinant, therapeutic, or repressor polypeptide. In one embodiment, a promoter element may be contained, in part or in whole, within one or more introns of a gene, e.g., a gene encoding a recombinant, therapeutic, or repressor polypeptide.

[0030] As used herein, the term "operably linked" refers to the relationship between a polypeptide-encoding nucleic acid sequence and a control element; a polypeptide-encoding sequence and a control element are operably linked when they are positioned in a manner suitable for the control element to regulate expression of the polypeptide-encoding sequence. Thus, for different control elements, operably linked refers to different placements of the polypeptide-encoding sequence relative to the control element. For example, a polypeptide-encoding sequence can be operably linked to a control element containing a promoter element when the promoter element and the polypeptide-encoding sequence are positioned proximal to each other on the same nucleic acid. In another example, a polypeptide-encoding sequence can be operably linked to a control element containing a distally acting enhancer sequence when the enhancer sequence and the polypeptide-encoding sequence are positioned an appropriate number of bases apart on the same nucleic acid or even on separate and distinct nucleic acids. An insertion site, e.g., a restriction site, landing pad, or SSI site, can also be operably linked to a control element when the polypeptide-encoding sequence inserted at the insertion site is operably linked to the control element.

[0031] As used herein, the term "endogenous" refers to any material that originates from or is naturally produced within an organism, cell, tissue, or system.

[0032] As used herein, the term "recombination target site" is a stretch of nucleotides that is essential for targeted recombination, that together with a recombinase allows it, and that defines the location of such recombination.

[0033] As used herein, the term "recombination target sites" used in conjunction with a gene "flanked by" or "flanked by" a gene, e.g., a gene encoding a recombinant, e.g., therapeutic, repressor, or selectable marker, polypeptide, means that the recombination target sites are located 5' and 3' of the gene, meaning that one target site is located 5' and the other target site is located 3' of the gene coding sequence of interest. Recombination target sites may be located immediately adjacent to the gene coding sequence of interest or at a specified distance. The flanking sequences, particularly those flanking the recombination target site, are located in either the forward or reverse orientation, preferably both forward, or preferably both reverse.

[0034] As used herein, the term "exogenous" refers to any material that is introduced into or produced outside of an organism, cell, tissue, or system. Accordingly, an "exogenous nucleic acid" refers to a nucleic acid that is introduced into or produced outside of an organism, cell, tissue, or system. In one embodiment, the sequence of an exogenous nucleic acid is not naturally produced or cannot be found in nature inside the organism, cell, tissue, or system into which the exogenous nucleic acid is introduced. Similarly, an "exogenous polypeptide" refers to a polypeptide that is not naturally produced or cannot be found in nature inside the organism, cell, tissue, or system into which the exogenous polypeptide is introduced, e.g., by expression from an exogenous nucleic acid sequence.

[0035] As used herein, the term "xenogeneic" refers to any material from one species that may be introduced into an organism, cell, tissue or system from a different species.

[0036] As used herein, the terms "nucleic acid," "polynucleotide," or "nucleic acid molecule" are used interchangeably and refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or combinations of DNA or RNA, and polymers thereof, in either single- or double-stranded form. The term "nucleic acid" includes, but is not limited to, genes, cDNA, or mRNA. In one embodiment, a nucleic acid molecule is synthetic (e.g., chemically synthesized or artificial) or recombinant. Unless specifically limited, the term encompasses molecules containing analogs or derivatives of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring or non-naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0037] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds or by means other than peptide bonds. A protein or peptide must contain at least two amino acids, and no limit is placed on the maximum number of amino acids that a protein or peptide sequence may comprise. In one embodiment, a protein can comprise more than one polypeptide, e.g., two, three, four, five, or more polypeptides, each associated with another by covalent or non-covalent bonds / interactions. A polypeptide includes any peptide or protein comprising two or more amino acids joined to one another by peptide bonds or by means other than peptide bonds. As used herein, the term "polypeptide" refers to both short chains, which are commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers, and to longer chains, which are commonly referred to in the art as proteins, of which there are numerous types, including, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins, among others.

[0038] As used herein, "recombinant polypeptide" or "recombinant protein" refers to a polypeptide that can be produced by a cell described herein. A recombinant polypeptide is one in which at least one nucleotide of a sequence encoding the polypeptide or at least one nucleotide of a sequence controlling expression of the polypeptide has been created by genetic engineering (of a cell, or of a precursor cell). For example, it is the product of a rearrangement in which at least one nucleotide has been altered, e.g., introduced into or genetically engineered into a cell. For example, a recombinant polypeptide can also be a therapeutic polypeptide.

[0039] As used herein, "therapeutic polypeptide" refers to a polypeptide having utility for human or animal health or medicine that is produced, e.g., expressed, by a cell that has been modified or genetically engineered to produce the therapeutic polypeptide. In one embodiment, the therapeutic polypeptide is a naturally occurring polypeptide or a non-naturally occurring polypeptide, e.g., a synthetic polypeptide. In one embodiment, a portion of the therapeutic polypeptide is naturally occurring and another portion of the therapeutic polypeptide is non-naturally occurring. In one embodiment, the therapeutic polypeptide is a recombinant polypeptide. In one embodiment, the therapeutic polypeptide is suitable for diagnostic or preclinical use. In another embodiment, the therapeutic polypeptide is suitable for therapeutic use, e.g., for the treatment of a disease. In one embodiment, the therapeutic polypeptide is selected from Tables 1-4. In some embodiments, the modified or genetically engineered cell comprises an exogenous nucleic acid that controls the expression of or encodes the therapeutic polypeptide. In other embodiments, the modified or genetically engineered cell comprises other molecules other than nucleic acids that, for example, control the expression or assembly of the therapeutic polypeptide in the cell.

[0040] As used herein, a "repressor polypeptide" refers to a polypeptide that controls the expression of another polypeptide (e.g., a therapeutic polypeptide) produced, e.g., expressed, by a cell that has been modified or genetically engineered to produce the repressor polypeptide. In one embodiment, the repressor polypeptide is a naturally occurring polypeptide or a non-naturally occurring polypeptide, e.g., a synthetic polypeptide. In one embodiment, a portion of the repressor polypeptide is naturally occurring and another portion of the repressor polypeptide is non-naturally occurring. In one embodiment, the repressor polypeptide is a recombinant polypeptide. In some embodiments, the repressor polypeptide reduces expression of a therapeutic polypeptide. In some embodiments, the repressor polypeptide completely eliminates expression of a therapeutic polypeptide. In some embodiments, expression of the repressor polypeptide is regulated. For example, the repressor polypeptide is highly expressed under one set of conditions, and under another set of conditions, expression of the repressor polypeptide is inhibited, e.g., reduced or completely eliminated.

[0041] As used herein, "level of activity" refers to a measure of the strength of expression induced by a control or promoter element. For example, a control element may have a high level of activity such that a coding sequence operably linked to the control element is strongly expressed.

[0042] As used herein, "condition" refers to the value of a cellular and / or environmental parameter that can affect the level of activity of a control element or promoter element. A condition can include one value of a cellular and environmental parameter, or a condition can include more than one value of a cellular and environmental parameter (e.g., two, three, four, five, six, or more). For example, a control element can have a first level of activity under a first condition and a second level of activity under a second condition. Cellular and environmental parameters include, but are not limited to, the level of one or more polypeptides, the compartment-localized level of one or more polypeptides (e.g., localized in the nucleus, cytoplasm, or endoplasmic reticulum), the level of activation of a cellular signaling pathway, such as a stress response, unfolded protein response, heat shock response, etc., the level of activation of a signaling molecule (e.g., Ca +2 These include the levels of ATP, cAMP, glucose, ATP, etc., temperature, pH, cell cycle / growth phase, cell density and nutrient availability of the culture.

[0043] As used herein, a Cas9 molecule or Cas9 polypeptide refers to a molecule or polypeptide that can interact with a guide RNA (gRNA) molecule and, in cooperation with the gRNA molecule, directs or localizes to a site containing a target domain and a PAM sequence. Cas9 molecules and Cas9 polypeptides, as used herein, include naturally occurring Cas9 molecules and engineered, altered, or modified Cas9 molecules, or Cas9 polypeptides that differ from a reference sequence by, for example, at least one amino acid residue, e.g., the most similar naturally occurring Cas9 molecule or sequence. Exemplary Cas9 molecule or Cas9 polypeptide sequences can be found in WO2015 / 157070, the entire contents of which are incorporated herein by reference. Cas9 molecules or Cas9 polypeptides include Cas9 molecules with DNA cleavage and nicking activity and others, such as dCas9 molecules or dCas9 polypeptides that do not specifically cleave or nick DNA.

[0044] overview In one aspect, the present disclosure provides genetic control circuits, nucleic acids, cells, methods, and methods for generating cells or cell lines for fine-tuning the transcription rate of a recombinant or therapeutic protein product gene(s) in response to changes in cellular or environmental conditions, e.g., changes in a cellular stress response, e.g., the unfolded protein response (UPR). An example of the general design principles of the present disclosure for a genetic control circuit is depicted in Figures 1A and 1B. In this non-limiting schematic example, production of a recombinant protein product induces stress / toxicity, which activates production of a repressor polypeptide, thereby inhibiting recombinant polypeptide product expression. Removal of the stress signal inactivates expression of the repressor polypeptide. Relief of inhibition is accompanied by reactivation of recombinant polypeptide product production.

[0045] product Provided herein are genetic control circuits, cells, and methods for identifying, selecting, or producing cells or cell lines capable of producing high yields of a product, e.g., an exogenous therapeutic polypeptide. Products encompassed by the present disclosure include, but are not limited to, molecules, nucleic acids, polypeptides (e.g., recombinant and / or therapeutic polypeptides), or hybrids thereof, that can be produced, e.g., expressed, in a cell. In some embodiments, cells are genetically engineered or modified to produce a product. Such modifications include introducing a molecule that controls or results in the production of the product. For example, cells are modified by introducing an exogenous nucleic acid encoding a polypeptide, e.g., a recombinant polypeptide, and the cells are cultured under conditions suitable for the production, e.g., expression and secretion, of the polypeptide, e.g., the recombinant polypeptide. In another example, cells are modified by introducing an exogenous nucleic acid that controls, e.g., increases, the expression of a polypeptide endogenously expressed by the cell, such that the cell produces a level or amount of the polypeptide that is higher than the level or amount endogenously produced in an unmodified cell. In embodiments, cells or cell lines identified, selected, or generated by the methods described herein produce a product, e.g., a recombinant polypeptide, useful in the treatment of a medical condition, disorder, or disease, including, but not limited to, metabolic diseases or disorders (e.g., metabolic enzyme deficiencies), endocrine disorders (e.g., hormone deficiencies), hemostasis, thrombosis, hematopoietic disorders, pulmonary disorders, gastrointestinal disorders, immunoregulation (e.g., immunodeficiencies), infertility, transplantation, cancer, and infectious diseases.

[0046] A recombinant polypeptide is an exogenous protein, e.g., a protein that is not naturally expressed by a cell. A recombinant polypeptide can be, for example, a therapeutic or diagnostic protein useful for drug screening. The therapeutic or diagnostic protein can be an antibody molecule, e.g., an antibody or antibody fragment, a fusion protein, a hormone, a cytokine, a growth factor, an enzyme, a glycoprotein, a lipoprotein, a reporter protein, a therapeutic peptide or structural and / or functional fragment thereof, or a hybrid of any of these. In embodiments, the product, e.g., an exogenous therapeutic polypeptide, comprises an antibody or antibody fragment comprising multiple polypeptide chains, e.g., a heavy chain and a light chain.

[0047] In one embodiment, the product, e.g., a recombinant polypeptide, is an antibody molecule. Products encompassed herein include diagnostic antibody molecules useful for imaging techniques, e.g., monoclonal antibodies or antibody fragments thereof, and therapeutic antibody molecules suitable for administration to a subject, e.g., useful for treating a disease or disorder. An antibody molecule is a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. In one embodiment, the antibody molecule is a full-length antibody or antibody fragment. Antibodies and multiformat proteins can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural or recombinant sources. An antibody can be a tetramer of immunoglobulin molecules. In one embodiment, the antibody is a monoclonal antibody. The antibody can be a human or humanized antibody. In one embodiment, the antibody is an IgA, IgG, IgD, or IgE antibody. In one embodiment, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.

[0048] "Antibody fragment" refers to at least a portion of an intact antibody or a recombinant variant thereof, and refers to the antigen-binding domain of the intact antibody, e.g., the antigen-determining variable region, sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies such as sdAb (either VL or VH), multispecific antibodies formed from antibody fragments such as camelid VHH domains and bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, and isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, bispecific antibodies, triabodies, tetrabodies, v-NAR, and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted onto polypeptide-based scaffolds, such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0049] In embodiments, the recombinant or therapeutic polypeptide is, for example, BOTOX, Myobloc, Neurobloc, Dysport (or other serotypes of botulinum neurotoxin), alglucosidase alfa, daptomycin, YH-16, choriogonadotropin alfa, filgrastim, cetrorelix, interleukin-2, aldesleukin, teselorin, denileukin diftitox, interferon alfa-n3 (injection), ... Lon Alpha-nl, DL-8234, Interferon, Suntory (Gamma-1a), Interferon Gamma, Thymosin Alpha 1, Tasonermin, DigiFab, ViperaTAb, EchiTAb, CroFab, Nesiritide, Abatacept, Alefacept, Rebif, Eptothermin Alpha, Teriparatide, Calcitonin, Etanercept, Hemoglobin Glutamer 250 (Bovine), Drotrecogin Alpha, Collagenase, Carperitide, Recombinant Human epidermal growth factor, DWP401, darbepoetin alfa, epoetin omega, epoetin beta, epoetin alfa, desirudin, lepirudin, bivalirudin, nonacog alfa, mononine, eptacog alfa (activated), recombinant factor VIII + VWF, Recombinate, recombinant factor VIII, factor VIII (recombinant), alphnmate, octocog alfa, factor VIII, palifermin, indikinase, tenecteplase , alteplase, pamiteplase, reteplase, nateplase, monteplase, follitropin alfa, rFSH, hpFSH, micafungin, pegfilgrastim, lenograstim, nartograstim, sermorelin, glucagon, exenatide, pramlintide, iniglucerase, galsulfase, leucotropin, molgramostim, triptorelin acetate, histrelin (Hydron), deslorelin, histrelin, nafarelin,Leuprolide (ATRIGEL), leuprolide (DUROS), goserelin, eutropin, somatropin, mecasermin, enlfavirtide, Org-33408, insulin glargine, insulin glulisine, insulin (inhaled), insulin lispro, insulin deternir, insulin (RapidMist), mecasermin linfabate, anakinra, cermoleukin, 99 mTc-apsitide, myelopid, Betaseron, glatiramer acetate, Gepon, sargramostim, oprelvekin, human leukocyte-derived alpha interferon, Bilive, insulin (recombinant), recombinant human insulin, insulin aspart, mecasenin, Roferon-A, interferon-alpha 2, alfaferone, interferon alfacon-1, interferon alpha, Avonex recombinant human luteinizing hormone, dornase alfa, trafermin, ziconotide, taltirelin, divotermin alfa, atosiban, becaplermin, eptifibatide, Zemaira, CTC-111, Shanvac-B, octreotide, ran Reotide, ancestim, agalsidase beta, agalsidase alfa, laronidase, prezatide copper acetate, rasburicase, ranibizumab, Actimmune, PEG-Intron, Tricomin, recombinant human parathyroid hormone (PTH) 1-84, epoetin delta, transgenic antithrombin III, granditropin, Vitrase, recombinant insulin, interferon-alpha, GEM-21S, vapreotide, idursulfase, omapatrilat, recombinant serum albumin, certolizumab pegol, glucarpidase, human recombinant C1 esterase inhibitor, lanoteplase, recombinant human growth hormone, enfuvirtide, VGV-1, interferon (alpha),Lucinactant, aviptadil, icatibant, ecallantide, omiganan, Aurograb, pexigananacetate, ADI-PEG-20, LDI-200, degarelix, cintredelin besudotox, Favld, MDX-1379, ISAtx-247, liraglutide, teriparatide, tifacogin facogin, AA4500, T4N5 liposome lotion, catumaxomab, DWP413, ART-123, Chrysalin, desmoteplase, amediplase, corifollitropin alfa, TH-9507, teduglutide, Diamyd, DWP-412, growth hormone, recombinant G-CSF, insulin, insulin (Technosphere), insulin (AERx), RGN -303, DiaPep277, interferon beta, interferon alpha-n3, belatacept, transdermal insulin patch, AMG-531, MBP-8298, Xerecept, opebacan, AIDSVAX, GV-1001, LymphoScan, ranpirnase, Lipoxysan, lusupultide, MP52, sipuleucel -T, CTP-37, Insegia, vitespen, human thrombin, thrombin, TransMID, alfimeprase, Puricase, terlipressin, EUR-1008M, recombinant FGF-I, BDM-E, rotigaptide, ETC-216, P-113, MBI-594AN, duramycin, SCV-07, OPI-45, endostatin, angiostatin, ABT-510, Bowman Birk inhibitor, XMP-629, 99 mTc-Hynic-Annexin V, kahalalide F, CTCE-9908, teverelix, ozarelix, romidepsin, BAY-504798, interleukin 4,PRX-321, Pepscan, ivoctadekin, rh-lactoferrin, TRU-015, IL-21, ATN-161, cilengitide, albuferon, Biphasix, IRX-2, omega interferon, PCK-3145, CAP-232, pasireotide, huN901-DMI, SB-249553, Oncovax-CL, Oncovax-P, BLP-25, CerVax-16, MART-1, gp100, tyrosinase, nemifitide, rAAT, CGRP, pegsnercept pegsunercept, thymosin beta 4, plitidepsin, GTP-200, ramoplanin, GRASPA, OBI-1, AC-100, salmon calcitonin (Eligen), examorelin, capromorelin, Cardeva, velafermin, 131I-TM-601, KK-220, T-10, ularitide, depelestat, hematide, chrysalin, rNAPc2, recombinant factor VIII (PEGylated liposomal), bFGF, PEGylated recombinant staphylokinase variant, V-10153, sonolysis protease Prolyse, NeuroVax, CZEN-002, rGLP-1, BIM-51077, LY-548806, exenatide (extended-release, Medisorb), AVE-0010, GA-GCB, avorelin, ACM-9604, linaclotide acetate acetate), CETi-1, Hemospan, VAL, rapid-acting insulin (injectable, Viadel), insulin (Eligen), recombinant methionyl human leptin, pitrakinra, Multikine, RG-1068, MM-093, NBI-6024, AT-001, PI-0824, Org-39141, Cpn10, talactoferrin, rEV-131, recombinant human insulin, RPI-78M, oprelvekin, CYT-99007 CTLA4-Ig, DTY-001,Valategrast, interferon alfa-n3, IRX-3, RDP-58, tauferon, bile salt-stimulated lipase, merispase, alkaline phosphatase, EP-2104R, melanotan-II, bremelanotide, ATL-104, recombinant human microplasmin, AX-200, SEMAX, ACV-1, Xen-2174, CJC-1008, dynorphin A, SI-6603, LAB GHRH, AER-002, BGC-728, ALTU-135, recombinant neuraminidase, Vacc-5q, Vacc-4x, Tat toxoid, YSPSL, CHS-13340, PTH(1-34) (Novasome), Ostabolin-C, PTH analogs, MBRI-93.02, MTB72F, MVA-Ag85A, FARA04, BA-210, recombinant plague FIV, AG-702, OxSODrol, rBetV1, Der-p1 / Der-p2 / Der-p7, PR1 peptide antigen, mutant ras vaccine, HPV-16 E7 lipopeptide vaccine, labyrinthin, WT1-peptide, IDD-5, CDX-110, Pentrys, Norelin, CytoFab, P-9808, VT-111, icrocaptide, telbermin, rupintrivir, reticulose, rGRF, HA, alpha-galactosidase A, ACE-011, ALTU-140, CGX-1160, angiotensin, D-4F, ETC-6 42, APP-018, rhMBL, SCV-07, DRF-7295, ABT-828, ErbB2-specific immunotoxin, DT3SSIL-3, TST-10088, PRO-1762, Combotox, cholecystokinin-B / gastrin-receptor binding peptide, 111In-hEGF, AE-37, trasnizumab-DM1, antagonist G, IL-12, PM-02734, IMP-321, rhIGF-BP3, BLX-883, CUV-1647, L-19-based ra, Re-188-P-2045,AMG-386, DC / 1540 / KLH, VX-001, AVE-9633, AC-9301, NY-ESO-1 (peptide), NA17.A2 peptide, CBP-501, recombinant human lactoferrin, FX-06, AP-214, WAP-8294A, ACP-HIP, SUN-11031, peptide YY[3-3, 6], FGLL, atacicept, BR3-Fc, BN-003, BA-058, human parathyroid hormone 1-34, F-18-CCR1, AT-1100, JPD-003, PTH(7-34) (Novasome), duramycin, CAB-2, CTCE-0214, glycopegylated erythropoietin, EPO-Fc, CNTO-528, AMG-114, JR-013, factor XIII, aminocandin ( aminocandin), PN-951, 716155, SUN-E7001, TH-0318, BAY-73-7977, teverelix, EP-51216, hGH, OGP-I, sifuvirtide, TV4710, ALG-889, Org-41259, rhCC10, F-991, thymopentin, r(m)CRP, hepatoselective insulin, subalin, L 19-IL-2 fusion protein, elafin, NMK-150, ALTU-139, EN-122004, rhTPO, thrombopoietin receptor agonist, AL-108, AL-208, nerve growth factor antagonist, SLV-317, CGX-1007, INNO-105, teriparatide (Eligen), GEM-OS1, AC-162352, PRX-302, LFn-p24 fusion, EP-1043, gpE1, gpE2, These include MF-59, hPTH(1-34), 768974, SYN-101, PGN-0052, aviscumnine, BIM-23190, multi-epitope tyrosinase peptide, enkastim, APC-8024, GI-5005, ACC-001, TTS-CD3, vascular-targeting TNF, desmopressin, onercept, and TP-9201.

[0050] In some embodiments, the polypeptide is adalimumab (HUMIRA), infliximab (REMICADE™), rituximab (RITUXAN™ / MAB THERA™), etanercept (ENBREL™), bevacizumab (AVASTIN™), trastuzumab (HERCEPTIN™), pegfilgrastim (NEULASTA™), or any other suitable polypeptide, including biosimilars and biobetters.

[0051] Other suitable polypeptides are those listed below and in Table 1 of U.S. Patent Application Publication No. 2016 / 0097074:

[0052] [Table 1] TIFF0007780858000002.tif214149 TIFF0007780858000003.tif209149 TIFF0007780858000004.tif209147 TIFF0007780858000005.tif104149

[0053] In embodiments, the polypeptide is a hormone, blood clotting / clotting factor, cytokine / growth factor, antibody molecule, fusion protein, protein vaccine, or peptide, as shown in Table 2.

[0054] [Table 2] TIFF0007780858000007.tif201149 TIFF0007780858000008.tif202149 TIFF0007780858000009.tif193149

[0055] In embodiments, the protein is a multispecific protein, such as a bispecific antibody, as shown in Table 3.

[0056] [Table 3] TIFF0007780858000011.tif151149 TIFF0007780858000012.tif154149 TIFF0007780858000013.tif157149 TIFF0007780858000014.tif148149 TIFF0007780858000015.tif133149 TIFF0007780858000016.tif153149 TIFF0007780858000017.tif154149

[0057] [Table 4] TIFF0007780858000019.tif206149 TIFF0007780858000020.tif206149 TIFF0007780858000021.tif211149 TIFF0007780858000022.tif206149 TIFF0007780858000023.tif211149 TIFF0007780858000024.tif91149

[0058] In some embodiments, the recombinant or therapeutic polypeptide is an antigen expressed by cancer cells. In some embodiments, the recombinant or therapeutic polypeptide is a tumor-associated antigen or a tumor-specific antigen. In some embodiments, the recombinant or therapeutic polypeptide is an antigen such as HER2, CD20, 9-O-acetyl-GD3, βhCG, A33 antigen, CA19-9 marker, CA-125 marker, calreticulin, carboanhydrase IX (MN / CA) IX), CCR5, CCR8, CD19, CD22, CD25, CD27, CD30, CD33, CD38, CD44v6, CD63, CD70, CC123, CD138, carcinoembryonic antigen (CEA; CD66e), desmoglein 4, E-cadherin neoepitope, endosialin, ephrin A2 (EphA2), epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), ErbB2, fetal acetylcholine receptor, fibroblast activation antigen (FAP), fucosyl GM1, GD2, GD3, GM2, ganglioside GD3, Globo H, glycoprotein 100, HER2 / neu, HER3, HER4, insulin-like growth factor receptor 1, Lewis-Y, LG, Ly-6, melanoma-specific chondroitin sulfate proteoglycan (MCSCP), mesothelin, MUC1, MUC1 variants (e.g., MUC1 A, B, C, D, X, Y, Z, REP, or SEC), MUC2, MUC3, MUC4, MUC5 AC , MUC5 B , MUC7, MUC16, Mullerian inhibitory factor (MIS) receptor type II, plasma cell antigen, polySA, PSCA, PSMA, sonic hedgehog (SHH), SAS, STEAP, sTn antigen, TNF-alpha precursor, and combinations thereof.

[0059] In some embodiments, the recombinant or therapeutic polypeptide is an activating receptor, such as 2B4 (CD244), α4β1 integrin, β2 integrin, CD2, CD16, CD27, CD38, CD96, CD100, CD160, CD137, CEACAM1 (CD66), CRTAM, CS1 (CD319), DNAM-1 (CD226), GITR (TNFRSF18), KIR, NKG2C, NKG2D, NKG2 E, one or more natural cytotoxicity receptors, NTB-A, PEN-5, and combinations thereof; optionally, the β2 integrin comprises CD11a-CD18, CD11b-CD18, or CD11c-CD18; optionally, the activating form of KIR comprises KIR2DS1, KIR2DS4, or KIR-S; and optionally, the natural cytotoxicity receptor comprises NKp30, NKp44, NKp46, or NKp80.

[0060] In some embodiments, the recombinant or therapeutic polypeptide is an inhibitory receptor and is selected from KIR, ILT2 / LIR-1 / CD85j, and inhibitory forms of KIR, KLRG1, LAIR-1, NKG2A, NKR-P1A, Siglec-3, Siglec-7, Siglec-9, and combinations thereof, and optionally, the inhibitory forms of KIR include KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, or KIR-L.

[0061] In some embodiments, the recombinant or therapeutic polypeptide is an activating receptor, such as CD3, CD2 (LFA2, OX34), CD5, CD27 (TNFRSF7), CD28, CD30 (TNFRSF8), CD40L, CD84 (SLAMF5), CD137 (4-1BB), CD226, CD229 (Ly9, SLAMF3), CD244 (2B4, SLAMF4), CD319 (CRACC, BLAME), Selected from CD352 (Lyl08, NTBA, SLAMF6), CRTAM (CD355), DR3 (TNFRSF25), GITR (CD357), HVEM (CD270), ICOS, LIGHT, LTβR (TNFRSF3), OX40 (CD134), NKG2D, SLAM (CD150, SLAMF1), TCRα, TCRβ, TCRδγ, TIM1 (HAVCR, KIM1), and combinations thereof.

[0062] In some embodiments, the recombinant or therapeutic polypeptide is an inhibitory receptor and is selected from PD-1 (CD279), 2B4 (CD244, SLAMF4), B71 (CD80), B7H1 (CD274, PD-L1), BTLA (CD272), CD160 (BY55, NK28), CD352 (Ly108, NTBA, SLAMF6), CD358 (DR6), CTLA-4 (CD152), LAG3, LAIR1, PD-1H (VISTA), TIGIT (VSIG9, VSTM3), TIM2 (TIMD2), TIM3 (HAVCR2, KIM3), and combinations thereof.

[0063] Other exemplary therapeutic or diagnostic proteins include, but are not limited to, any of the proteins listed in Tables 1-10 of Leader et al., "Protein therapeutics: a summary and pharmacological classification," Nature Reviews Drug Discovery, 2008, No. 7:21-39 (incorporated herein by reference), or any conjugate, variant, analog, or functional fragment of the recombinant polypeptides described herein.

[0064] Other recombinant products include, but are not limited to, non-antibody or alternative protein scaffolds such as DARPins, affibodies, and adnectins, which can be engineered to recognize or bind one or two or more, e.g., 1, 2, 3, 4, or 5 or more, different targets or antigens.

[0065] nucleic acid Also provided herein are nucleic acids, e.g., exogenous nucleic acids, encoding products, e.g., recombinant polypeptides described herein. Nucleic acid sequences encoding desired recombinant polypeptides can be obtained using recombinant methods known in the art, for example, by screening libraries obtained from cells expressing the gene using standard techniques, by deriving the nucleic acid sequence from a vector known to contain it, or by isolating it directly from cells and tissues containing it. Alternatively, nucleic acids encoding recombinant polypeptides may be produced synthetically rather than by cloning. Recombinant DNA techniques and technologies are highly advanced and well established in the art. Thus, one skilled in the art, armed with knowledge of the amino acid sequences of the recombinant polypeptides described herein, can readily envision or create nucleic acid sequences that will encode the recombinant polypeptides.

[0066] Expression of a recombinant polypeptide is usually achieved by operably linking a nucleic acid encoding the recombinant polypeptide or a portion thereof to a promoter and incorporating the construct into an expression vector. The vector can be suitable for replication and integration in eukaryotes or prokaryotes. Typical cloning vectors contain other regulatory elements, such as transcription and translation terminators, initiation sequences, and promoters, which are useful for regulating the expression of the desired nucleic acid sequence.

[0067] In embodiments, the product, e.g., an exogenous therapeutic polypeptide, comprises an antibody or antibody fragment comprising multiple polypeptide chains, e.g., a heavy chain and a light chain. Nucleic acid sequences encoding exogenous therapeutic polypeptides comprising multiple polypeptide chains can be located together (e.g., sequences encoding each polypeptide chain located on the same nucleic acid) or separately (e.g., sequences encoding each polypeptide chain located on different nucleic acids). Sequences encoding exogenous therapeutic polypeptides comprising multiple polypeptide chains can be operably linked to a single control element, e.g., a first control element, or to separate, distinct control elements (e.g., sequences encoding each polypeptide chain operably linked to its own first control element). In one embodiment in which a sequence encoding an exogenous therapeutic polypeptide comprising multiple polypeptide chains is operably linked to individual, distinct control elements, one or more (e.g., 1, 2, 3, 4, 5, 6, or all) of the control elements may have a first level of activity under a first condition and a second level of activity under a second condition, and one or more (e.g., 1, 2, 3, 4, 5, 6, or more) of the control elements may be constitutive.

[0068] Nucleic acid sequences encoding recombinant polypeptides can be cloned into several types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. In embodiments, expression vectors can be provided to cells in the form of viral vectors. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, Vols. 1-4, Cold Spring Harbor Press, NY, and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, a suitable vector contains an origin of replication functional in at least one organism, control elements including a promoter element and optionally an enhancer element, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584, WO 01 / 29058, and U.S. Pat. No. 6,326,193). Virus-derived vectors are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of the transgene and its propagation in daughter cells.

[0069] Vectors may also include, for example, a signal sequence to facilitate secretion, a polyadenylation signal and a transcription terminator (e.g., from the bovine growth hormone (BGH) gene), elements allowing episomal replication and replication in prokaryotes (e.g., SV40 origin and ColE1 or others known in the art), and / or elements allowing selection, such as a selectable marker or reporter gene.

[0070] Contemplated vectors may contain an insertion site suitable for inserting a sequence encoding a polypeptide, for example, an exogenous therapeutic polypeptide or a repressor polypeptide.

[0071] The insertion site may include a restriction endonuclease site.

[0072] The insertion site may include recombination target sites, which flank the sequence encoding a polypeptide, e.g., an exogenous therapeutic polypeptide or a repressor polypeptide. In one embodiment, the recombination target sites are lox sites. When the recombination target sites are lox sites, the host cell requires the presence and expression of Cre recombinase to achieve a crossover or recombination event.

[0073] In one embodiment, the recombination target site is an FRT site. When the recombination target site is an FRT site (cite), the host cell requires the presence and expression of FLP (FLP recombinase) to achieve a crossover or recombination event.

[0074] The insertion site can include a short, approximately 25 bp unique sequence and / or a landing pad, e.g., a portion of DNA, e.g., a selectable marker, flanked by restriction sites. Materials and methods contemplated include the landing pad site-specific integration techniques known in the art, e.g., in U.S. Provisional Patent Application No. 62 / 460,420, the entire contents of which are incorporated herein by reference.

[0075] In some embodiments, the vector comprises at least one (e.g., one, two, or more) of the isolated nucleotide sequences of SEQ ID NOs: 17, 18, 19, or homologs thereof. In one embodiment, the vector comprises at least one sequence encoding a selectable marker that is itself flanked at its 5' and 3' ends by one recombination target site, and at least one of the nucleotide sequences of SEQ ID NOs: 17 or 18, or a homologous sequence thereof, is located at the 3' end of the sequence encoding the selectable marker. In one embodiment, the vector comprises at least one sequence encoding a selectable marker that is itself flanked at its 5' and 3' ends by one recombination target site, and at least one nucleotide sequence as provided in SEQ ID NO: 19, or a homologous sequence thereof, is located at the 5' end of the sequence encoding the selectable marker.

[0076] First Control Element In one embodiment, a vector comprising a nucleic acid sequence encoding a product, e.g., a polypeptide, e.g., a recombinant or therapeutic polypeptide, further comprises a first control element, e.g., a first promoter element, responsible for recruitment of a polymerase that allows transcription initiation of expression of the polypeptide, e.g., the recombinant or therapeutic polypeptide. The first control element can include distal elements, e.g., elements that regulate polypeptide expression at some distance, e.g., several bases away, from the polypeptide-encoding sequence, and proximal elements, e.g., elements that regulate polypeptide expression in part due to their location adjacent to or within the polypeptide-encoding sequence. In some embodiments, a first control element, e.g., a promoter element, operably linked to a sequence encoding a polypeptide, e.g., a recombinant or therapeutic polypeptide, is a constitutive control element. In some embodiments, a first control element, e.g., a promoter element, operably linked to a sequence encoding a polypeptide, e.g., a recombinant or therapeutic polypeptide, is a regulated control element, e.g., a control element regulated by an endogenous or exogenous polypeptide. In some embodiments, a first control element, e.g., a first promoter element, operably linked to a sequence encoding a polypeptide, e.g., a recombinant or therapeutic polypeptide, has a first level of activity under a first condition, e.g., a first stage of cell growth, e.g., logarithmic growth, and a second level of activity under a second condition, e.g., a second stage of cell growth, e.g., a phase with sub-logarithmic growth, e.g., a stationary growth phase. In one embodiment, a control element suitable for the methods described herein typically associates with an enhancer to drive high levels of transcription and thus deliver high levels of copies of the target exogenous mRNA. In one embodiment, the first control element, e.g., a first promoter element, comprises the cytomegalovirus (CMV) major immediate-early promoter (Xia, Bringmann et al. 2006) and the SV40 promoter (Chernajovsky, Mory et al. 1984), both of which are derived from the eponymous viruses or promoters from which they are derived.Several other less common viral promoters have been successfully used to drive transcription when included in expression vectors, including the Rous sarcoma virus long terminal repeat (RSV-LTR) and Moloney murine leukemia virus (MoMLV) LTR (Papadakis, Nicklin, et al. 2004). In another embodiment, certain endogenous mammalian promoters can be utilized to drive constitutive transcription of genes of interest (Pontiller, Gross, et al. 2008). The CHO-specific Chinese hamster elongation factor 1-alpha (CHEF1α) promoter has provided a high-yield alternative to viral-based sequences (Deer, Allison 2004). In some embodiments, the first control element, e.g., first promoter element, used to drive transcription of a recombinant, e.g., therapeutic, polypeptide can include composite promoters, basal promoters, and tripartite composite promoters, such as the thymidine kinase (TK) promoter, actin promoter (e.g., β-actin promoter), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, cyclin T1 promoter, CAG promoter, RNA polymerase III U3 ​​promoter, cyclophilin promoter, Autographa californica nuclear polyhedrosis virus (AcNPV) P10 promoter, β3-galactosyltransferase 5 (β3GAL-T5) promoter, Fer1 promoter, and CMV-EF1α promoter. The promoter elements are summarized in Table 5 and are known in the art. It is contemplated that the present invention is not limited to a particular promoter or promoters. The promoters and transcriptional control mechanisms described in WO 2004 / 009823, WO 2006 / 111387, and WO 2014044845 (incorporated herein by reference in their entireties) are also contemplated in connection with the first and / or second control elements. In some embodiments, the first control element, e.g., promoter element, is an engineered promoter that includes a synthetic (non-naturally occurring) sequence.For example, the first control element, e.g., a promoter element, can include a promoter described in Brown et al., Biotechnology and Bioengineering, Vol. 111, No. 8, August 2014.

[0077] [Table 5]

[0078] Second Control Element In one embodiment, a vector comprising a nucleic acid sequence encoding a polypeptide, e.g., a recombinant or repressor polypeptide, further comprises a second control element, e.g., a second promoter element, operably linked to the polypeptide-encoding sequence, where the second control element is responsible for recruiting a polymerase that allows for transcription initiation of expression of the polypeptide, e.g., the recombinant or repressor polypeptide. The second control element can include distal elements, e.g., elements that regulate expression of the polypeptide at some distance from the polypeptide-encoding sequence, e.g., several bases apart, or on a separate and distinct nucleic acid, as well as proximal elements, e.g., elements that regulate expression of the polypeptide due in part to their location adjacent to or within the polypeptide-encoding sequence. In one embodiment, the second control element, e.g., the second promoter element, operably linked to the polypeptide-encoding sequence, e.g., the recombinant or repressor polypeptide, is a constitutive control element. In some embodiments, the second control element, e.g., the second promoter element, operably linked to the polypeptide-encoding sequence, e.g., the recombinant or repressor polypeptide, is a regulated control element, e.g., a promoter regulated by an endogenous or exogenous polypeptide. In some embodiments, a second control element, e.g., a second promoter element, operably linked to a sequence encoding a polypeptide, e.g., a recombinant or repressor polypeptide, has a first level of activity under first conditions and a second level of activity under second conditions.

[0079] In some embodiments, a second control element, e.g., a second promoter element, operably linked to a sequence encoding a polypeptide, e.g., a recombinant or repressor polypeptide, has a first level of activity under first conditions and a second level of activity under second conditions, where the second level of activity is regulated, e.g., up or down, relative to the first level of activity.

[0080] In some embodiments, the pair of first and second conditions is a first, e.g., low, level of stress and a second, e.g., high, level of stress; a first, e.g., low, level of unfolded or misfolded polypeptides and a second, e.g., high, level of unfolded or misfolded polypeptides; a first, e.g., low, level of unfolded or misfolded polypeptides in the cytosol and a second, e.g., high, level of unfolded or misfolded polypeptides in the cytosol; a first, e.g., low, level of unfolded or misfolded polypeptides in the endoplasmic reticulum (ER). a first, e.g., low level of an ER chaperone, e.g., BiP, and a second, e.g., high level of an ER chaperone, e.g., BiP; a first, e.g., low temperature and a second, e.g., high temperature; a first, e.g., low level of oxidative stress and a second, e.g., high level of oxidative stress; an ER Ca 2+ First, for example, high levels of ER Ca 2+a first, e.g., high ATP level and a second, e.g., low ATP level; a first, e.g., high glucose level and a second, e.g., low glucose level; a first, e.g., low level of activated Hsfl polypeptide and a second, e.g., high level of activated Hsfl polypeptide; a first, e.g., low level of phosphorylated trimeric Hsfl polypeptide and a second, e.g., high level of phosphorylated trimeric Hsfl polypeptide. a first, e.g., low level of an active, e.g., spliced ​​Xbp1 polypeptide and a second, e.g., high level of an active, e.g., spliced ​​Xbp1 polypeptide; a first, e.g., low level of an ATF4 polypeptide and a second, e.g., high level of an ATF4 polypeptide; a first, e.g., low level of an NRF2 polypeptide and a second, e.g., high level of an NRF2 polypeptide; and a first, e.g., low level of an ATF6 (e.g., ATF6α or ATF6β) polypeptide and a second, e.g., high level of an ATF6 (e.g., ATF6α or ATF6β) polypeptide.

[0081] In some embodiments, the second control element has an Nth level of activity under an Nth condition, where N is 3, 4, 5, 6, 7, 8, 9, 10, or more, and in the presence of the Nth condition, expression of the therapeutic polypeptide is modulated, e.g., decreased or increased, relative to expression of the therapeutic polypeptide under the previous conditions (e.g., conditions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.). For each pair of first and second conditions listed herein, an additional Nth (e.g., a third, fourth, fifth, etc.) condition is considered, and the additional Nth conditions are further related conditions. For example, a first, e.g., low, level of stress and a second, e.g., high, level of stress are listed above, and an additional Nth (e.g., third, fourth, fifth, etc.) (e.g., low or high) level of stress are also considered, along with the corresponding Nth level of activity.

[0082] In some embodiments, the first conditions inhibit expression of a polypeptide, e.g., a recombinant or repressor polypeptide. In some embodiments, the second conditions induce expression of a polypeptide, e.g., a recombinant or repressor polypeptide. In some embodiments, the second conditions induce expression of a polypeptide, e.g., a repressor polypeptide, and the repressor polypeptide inhibits expression of another polypeptide, e.g., an exogenous therapeutic polypeptide. In some embodiments, under the second conditions, expression, e.g., transcription levels, of the exogenous therapeutic polypeptide is reduced by at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to expression under the first conditions.

[0083] In some embodiments, the second control element does not induce expression of a polypeptide, e.g., a recombinant or repressor polypeptide, under a first set of conditions (e.g., the recombinant or repressor polypeptide is not visibly expressed), and induces expression of a polypeptide, e.g., a recombinant or repressor polypeptide, under a second set of conditions (e.g., the recombinant or repressor polypeptide is visibly expressed). Obvious expression can be detectable (e.g., by methods known in the art) accumulation of the polypeptide, e.g., the recombinant or repressor polypeptide, or detectable accumulation of mRNA encoding the polypeptide, e.g., the recombinant or repressor polypeptide.

[0084] In some embodiments, the second control element has a first level of activity under a first condition, a second level of activity under a second condition, and a third level of activity under a third condition. The first level of activity can result in a lack of apparent expression of the polypeptide, e.g., a recombinant or repressor polypeptide. The second level of activity can result in apparent expression of the polypeptide, e.g., a recombinant or repressor polypeptide. The third level of activity can result in modulation (e.g., an increase or decrease) of expression of the polypeptide, e.g., a recombinant or repressor polypeptide, relative to the second level of activity.

[0085] In some embodiments, the second control element has an Nth level of activity under Nth condition, where N is 3, 4, 5, 6, 7, 8, 9, 10, or more, and in the presence of the Nth condition, expression of the therapeutic polypeptide is modulated, e.g., decreased or increased, relative to expression of the therapeutic polypeptide under previous conditions (e.g., conditions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.). In one embodiment, the Nth level of activity of the second control element cycles based on the cycling of the Nth condition. For example, given a first condition that is a first level of cellular stress and a second condition that is a second level of cellular stress, the second control element can have a first level of activity and a second, higher level of activity (e.g., this exemplary second control element has activity proportional to cellular stress). The second, higher level of activity can increase expression of a polypeptide, e.g., a repressor polypeptide, which, upon accumulation, alters, e.g., decreases, expression of the recombinant or therapeutic polypeptide. A change, e.g., a decrease, in expression of the recombinant or therapeutic polypeptide creates a third condition (e.g., a third level of cellular stress that is lower than the second level of cellular stress). The third condition has a corresponding third level of activity of the second control element, which in this example, may have activity at the third level that is decreased relative to the activity at the second level, resulting in decreased expression of the repressor polypeptide. Decreased expression of the repressor polypeptide under the third condition may lead to increased expression of the recombinant or therapeutic polypeptide, creating a fourth condition (e.g., a fourth level of cellular stress that is higher than the third level of cellular stress), etc. In some embodiments, the cycling of the activity of the second control element associated with the cycling of conditions may, over time, approach equilibrium, e.g., a situation where the difference between the activity of the second control element at the Nth condition and the N+1th condition is negligible.In one embodiment, the second control element, e.g., the second promoter element, includes one or more (e.g., two, three, four, or more): a heat shock element (HSE) comprising one or more sequences corresponding to SEQ ID NOs: 8-11, a cAMP response element (CRE) comprising a sequence corresponding to SEQ ID NO: 12, an antioxidant response element (ARE) comprising a sequence corresponding to SEQ ID NO: 13, or an unfolded protein response element (ERSE) comprising a sequence corresponding to SEQ ID NO: 14. In some embodiments, the second control element, e.g., the second promoter element, can include one or more (e.g., two, three, four, or more) HSEs, CREs, AREs, or ERSEs that include sequences that include zero, one, two, three, four, or five substitutions relative to a related consensus sequence known in the art. In some embodiments, the second control element, e.g., the second promoter element, can include one or more (e.g., two, three, four, or more) HSEs, CREs, AREs, or ERSEs that comprise the consensus sequences listed in Table 6. In some embodiments, the second control element, e.g., the second promoter element, can include one or more (e.g., two, three, four, or more) HSEs, CREs, AREs, or ERSEs that comprise sequences that include zero, one, two, three, four, or five substitutions relative to the corresponding consensus sequences listed in Table 6 or known in the art. It is contemplated that the present invention is not limited to a particular promoter or promoters.

[0086] [Table 6]

[0087] In some embodiments, the second control element, e.g., the second promoter element, comprises one or more (e.g., two, three, four, or more) elements that are regulated, e.g., activated, by elements of the heat shock response or the unfolded protein response (UPR). In some embodiments, the second control element, e.g., the second promoter element, comprises one or more (e.g., two, three, four, or more) elements that are regulated, e.g., activated, by the accumulation of misfolded proteins. In some embodiments, the second control element, e.g., the second promoter element, comprises one or more (e.g., two, three, four, or more) Xbp1-responsive promoter elements. In some embodiments, the second control element, e.g., the second promoter element, comprises one or more (e.g., two, three, four, or more) ATF6-responsive promoter elements. In some embodiments, the second control element, e.g., the second promoter element, comprises one or more (e.g., two, three, four, or more) ATF4-responsive promoter elements. In some embodiments, the second control element, e.g., the second promoter element, comprises one or more (e.g., two, three, four, or more) NRF2-responsive promoter elements. In some embodiments, the second control element, e.g., the second promoter element, comprises one or more (e.g., two, three, four, or more) Hsf1-responsive promoter elements.

[0088] Third Control Element In some embodiments, the cells, vectors, nucleic acids, and kits and methods comprising the same of the invention further comprise or employ a nucleic acid sequence encoding one or more gRNAs (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more gRNAs) operably linked to a third control element, e.g., a third promoter element. The third control element is responsible for recruitment of a polymerase that allows transcription initiation of expression of the one or more gRNAs. In some embodiments, a third control element, e.g., a third promoter element, is operably linked to a sequence encoding multiple gRNAs, wherein the multiple gRNAs and / or the sequence encoding the multiple gRNAs are each incorporated by reference herein. Gao, Y. and Y. Zhao (2014). J Integr Plant Biol 56(4):343-349; Martick, M. et al. (2008). Nature 454(7206):899-902; Xie, K. et al. (2015). Proc Natl Acad Sci USA 112(11):3570-3575; Nissim, L. et al. (2014). Mol Cell 54(4):698-710; and Port, F. and S.L. Bullock (2016). "Augmenting CRISPR applications in Drosophila with tRNA-flanked The third control element can be provided, manufactured, positioned, and processed as described in "sgRNAs." Nat Meth [Prior to online publication]. The third control element can include distal elements, e.g., elements that regulate expression of a polypeptide at some distance, e.g., several bases in length, or on a separate and distinct nucleic acid, from the sequence encoding one or more gRNAs, as well as proximal elements, e.g., elements that regulate expression of one or more gRNAs due in part to their location adjacent to or within the sequence encoding one or more gRNAs. In one embodiment, a third control element, e.g., a third promoter element, operably linked to a sequence encoding one or more gRNAs is a constitutive control element.In some embodiments, the third control element, e.g., a third promoter element, operably linked to a sequence encoding one or more gRNAs is a regulated control element, e.g., a promoter regulated by an endogenous or exogenous polypeptide. In some embodiments, the third control element, e.g., a third promoter element operably linked to a sequence encoding one or more gRNAs, has a first level of activity under a first condition and a second level of activity under a second condition.

[0089] In some embodiments, a third control element, e.g., a third promoter element, operably linked to a sequence encoding one or more gRNAs is a copy of the first control element described herein. In some embodiments, a third control element, e.g., a third promoter element, operably linked to a sequence encoding one or more gRNAs is a copy of the second control element described herein.

[0090] In some embodiments, the third control element, for example, the third promoter element, is an engineered promoter that includes a synthetic (non-naturally occurring) sequence. For example, the third control element, for example, the third promoter element, can include a promoter as described in Brown et al., Biotechnology and Bioengineering, Vol. 111, No. 8, August 2014.

[0091] In addition to the promoter, the vectors described herein also contain an enhancer region, as described above, adjacent to the core promoter; a specific nucleotide motif region, which can recruit transcription factors and upregulate the rate of transcription (Riethoven 2010). Like the promoter sequence, these regions are often derived from viruses, and are contained within promoter sequences such as hCMV and SV40 enhancer sequences, or even sequences derived from adenovirus (Gaillet, Gilbert et al. 2007).

[0092] Other nucleic acid features In one embodiment, a vector comprising a nucleic acid sequence encoding a product, e.g., a polypeptide, e.g., a recombinant polypeptide described herein, further comprises a nucleic acid sequence encoding a selectable marker. In one embodiment, the selectable marker comprises glutamine synthetase (GS), dihydrofolate reductase (DHFR), an enzyme conferring resistance to, e.g., methotrexate (MTX), or an antibiotic marker, e.g., an enzyme conferring resistance to an antibiotic such as hygromycin, neomycin (G418), zeocin, puromycin, or blasticidin. In another embodiment, the selectable marker comprises or is compatible with the Selexis selection system (e.g., SUREtechnology Platform™ and Selexis Genetic Elements™, commercially available from Selexis SA) or the Catalant selection system.

[0093] In one embodiment, a vector comprising a nucleic acid sequence encoding a recombinant product described herein comprises a selectable marker that is useful for identifying a cell or cells that comprise a nucleic acid sequence encoding a recombinant product described herein. In another embodiment, the selectable marker is useful for identifying a cell or cells that comprise a nucleic acid sequence encoding a recombinant product as described herein integrated into their genome. Identification of a cell or cells that have integrated a nucleic acid sequence encoding a recombinant protein can be useful for selection and genetic engineering of cells or cell lines that stably express the product.

[0094] Suitable vectors for use are commercially available and include those associated with the GS Expression System™, GS Xceed™ Gene Expression System, or Potelligent® CHOK1SV technology, available from Lonza Biologics, PLC, such as those described in Fan et al., Pharm. Bioprocess. (2013); vol. 1(5):487-502, the entire contents of which are incorporated herein by reference. The GS expression vector contains a GS gene or a functional fragment thereof (e.g., a GS minigene) and one or more, e.g., one, two, or three or more, high-efficiency transcription cassettes for expression of a gene of interest, e.g., a nucleic acid encoding a recombinant polypeptide described herein. The minigene contains a single intron of the GS gene and approximately 1 kb of DNA located at the 3' end, and is transcribed from the SV40 late promoter. In one embodiment, the GS vector contains a GS gene operably linked to the SV40 L promoter and one or two poly(A) signals. In another embodiment, the GS vector comprises a GS gene operably linked to an SV40E promoter and an SV40 intron splicing and polyadenylation signal. In such an embodiment, for example, a transcription cassette for expression of a gene of interest or a recombinant polypeptide described herein comprises the hCMV-MIE promoter and 5' untranslated sequence from the hCMV-MIE gene, including the first intron. Other vectors can be constructed based on the GS expression vector, for example, in which other selectable markers are substituted for the GS gene in the expression vectors described herein.

[0095] Suitable vectors for use in the methods described herein include, but are not limited to, pcDNA3.1 / Zeo, pcDNA3.1 / CAT, pcDNA3.3TOPO (Thermo Fisher, formerly Invitrogen); pTarget, HaloTag (Promega); pUC57 (GenScript); pFLAG-CMV (Sigma-Aldrich); pCMV6 (Origene); pEE12 or pEE14 (Lonza Biologics) or other commercially available vectors such as pBK-CMV / pCMV-3Tag-7 / pCMV-Tag2B (Stratagene).

[0096] cell Recombinant proteins or polypeptides, e.g., therapeutic polypeptides, can be produced by recombinant DNA technology, expressed by host cells, and purified from the host cells (e.g., CHO cells), or secreted into a fluid, e.g., the cell medium in which the host cells are cultured, and purified from the fluid. Cells capable of producing recombinant proteins or polypeptides in high yields and of suitable quality are highly desirable in the art. Recombinant, e.g., therapeutic polypeptide-producing, cells, methods for producing cells, methods, and related kits are useful for producing cells with improved viability, highly productive cells to obtain high yields of recombinant, e.g., therapeutic polypeptide product, or to provide high-quality preparations of recombinant polypeptide products, e.g., preparations of recombinant polypeptide products containing high amounts of correctly folded protein, low amounts of aggregated protein, a desired glycosylation pattern, or a desired level of glycosylation. The recombinant, e.g., therapeutic polypeptide-producing cells, methods for producing cells, methods and related kits are particularly useful for the production of recombinant, e.g., therapeutic polypeptides, where there is a demand for efficient cell line development, large quantities of recombinant therapeutic polypeptide product, and high-grade quality for therapeutic use in patients.

[0097] Cells and cell culture In one aspect, the disclosure relates to methods for evaluating, classifying, identifying, selecting, or generating cells or cell lines that produce a product, e.g., a recombinant or therapeutic polypeptide as described herein, hi another aspect, the disclosure relates to methods and compositions for evaluating, classifying, identifying, selecting, or generating cells or cell lines with improved, e.g., enhanced, productivity and product quality.

[0098] In embodiments, the cell is a mammalian cell. In other embodiments, the cell is a non-mammalian cell. In one embodiment, the cell is derived from a mouse, rat, Chinese hamster, Syrian hamster, monkey, ape, dog, horse, ferret, or cat. In embodiments, the cell is a mammalian cell, e.g., a human cell or a rodent cell, e.g., a hamster cell, a mouse cell, or a rat cell. In another embodiment, the cell is derived from a duck, a parrot, a fish, an insect, a plant, a fungus, or a yeast. In one embodiment, the cell is an archaebacterium. In one embodiment, the cell is a species of actinomycete, e.g., Mycobacterium tuberculosis.

[0099] In one embodiment, the cell is a Chinese hamster ovary (CHO) cell. In one embodiment, the cell is a CHO-K1 cell, a CHOK1SV cell, a DG44 CHO cell, a DUXB11 CHO cell, a CHO-S, a CHO GS knockout cell, a CHOK1SV FUT8 knockout cell, a CHOZN, or a CHO-derived cell. An example of a CHO GS knockout cell (e.g., a GSKO cell) is a CHO-K1SV GS knockout cell (Lonza Biologics, Inc.). An example of a CHO FUT8 knockout cell is Potelligent® CHOK1SV FUT8 knockout (Lonza Biologics, PLC.).

[0100] In one embodiment, the cell is a site-specific integration (SSI) host cell. In one embodiment, the SSI host cell comprises an endogenous Fer1L4 gene and an exogenous nucleotide sequence is integrated into the Fer1L4 gene. In some embodiments, the exogenous nucleotide sequence comprises at least one gene coding sequence of interest, e.g., a gene encoding a therapeutic, repressor, or selectable marker polypeptide. In some embodiments, the exogenous nucleotide sequence comprises at least two recombination target sites. In some embodiments, the recombination target sites are located on both sides of the at least one gene coding sequence of interest. In other embodiments, the recombination target sites are located adjacent to, but not on both sides of, the at least one gene coding sequence of interest. In some embodiments, the gene coding sequence of interest comprises at least one selectable marker gene.

[0101] In one embodiment, the SSI host cell is characterized by the presence of an exogenous nucleotide sequence, i.e., a sequence encoding at least one recombinant, e.g., therapeutic or repressor polypeptide, flanked at its 5' and 3' ends by one recombination target site each, and at least one of the nucleotide sequences of SEQ ID NO: 17 or 18 or a homologous sequence thereof is located at the 3' end of the exogenous nucleotide sequence integrated into the genome of the host cell. In one embodiment, the SSI host cell is characterized by the presence of an exogenous nucleotide sequence, i.e., a sequence encoding a recombinant, e.g., therapeutic or repressor polypeptide, flanked at its 5' and 3' ends by one recombination target site each, and at least one nucleotide sequence as given in SEQ ID NO: 19 or a homologous sequence thereof is located at the 5' end of the exogenous nucleotide sequence integrated into the genome of the host cell.

[0102] In one embodiment, the cell is a site-specific integration (SSI) host cell. In one embodiment, the SSI host cell is characterized by the presence of an exogenous nucleotide sequence, i.e., at least one sequence encoding a selectable marker, flanked at its 5' and 3' ends by one recombination target site each, and at least one nucleotide sequence of SEQ ID NO: 17 or 18 or a homologous sequence thereof is located at the 3' end of the exogenous nucleotide sequence integrated into the genome of the host cell. In one embodiment, the SSI host cell is characterized by the presence of an exogenous nucleotide sequence, i.e., at least one sequence encoding a selectable marker, flanked at its 5' and 3' ends by one recombination target site each, and at least one nucleotide sequence as given in SEQ ID NO: 19 or a homologous sequence thereof is located at the 5' end of the exogenous nucleotide sequence integrated into the genome of the host cell.

[0103] In another embodiment, the cell is HeLa, HEK293, HT1080, H9, HepG2, MCF7, Jurkat, NIH3T3, PC12, PER.C6, BHK (baby hamster kidney cells), VERO, SP2 / 0, NS0, YB2 / 0, YO, EB66, C127, L cells, COS, e.g., COS1 and COS7, QC1-3, CHOK1, CHOK1SV, Potelligent™ (CHOK1SV FUT8-KO), CHO GS knockout, Xceed™ (CHOK1SV GS-KO), CHOS, CHO DG44, CHO DXB11, and CHOZN, or any cell derived therefrom.

[0104] In one embodiment, the eukaryotic cell is a stem cell. The stem cell can be, for example, a pluripotent stem cell, including embryonic stem cells (ESCs), adult stem cells, induced pluripotent stem cells (iPSCs), tissue-specific stem cells (e.g., hematopoietic stem cells), and mesenchymal stem cells (MSCs).

[0105] In one embodiment, the cell is a differentiated form of any of the cells described herein. In one embodiment, the cell is a cell derived from any primary cell in culture.

[0106] In embodiments, the cells are hepatocytes or non-parenchymal cells, such as human hepatocytes, animal hepatocytes, etc. For example, the cells can be adherent metabolically competent human hepatocytes, adherent induction-competent human hepatocytes, adherent Qualyst Transporter Certified™ human hepatocytes, suspension-competent human hepatocytes (including pooled hepatocytes from 10 donors and 20 donors), human hepatic Kupffer cells, human hepatic stellate cells, dog hepatocytes (including single and pooled beagle hepatocytes), mouse hepatocytes (including CD-1 and C57BI / 6 hepatocytes), rat hepatocytes (including Sprague-Dawley, Wistar Han, and Wistar hepatocytes), monkey hepatocytes (including cynomolgus or rhesus hepatocytes), cat hepatocytes (including Domestic Shorthair hepatocytes), and rabbit hepatocytes (including New Zealand White hepatocytes). Exemplary hepatocytes are commercially available from Triangle Research Labs, LLC, 6 Davis Drive, Research Triangle Park, North Carolina 27709, USA.

[0107] In one embodiment, the eukaryotic cell is, for example, a yeast cell (e.g., a cell of the genus Pichia (e.g., Pichia pastoris, Pichia methanolica, Pichia kluyveri, and Pichia angusta), a cell of the genus Komagataella (e.g., Komagataella pastoris, Komagataella pseudopastoris, or Komagataella phaffii), a cell of the genus Saccharomyces (e.g., Saccharomyces cerevisae, Saccharomyces cerevisiae, Saccharomyces kluyveri, Saccharomyces kluyveri, Saccharomyces uvarum), Kluyveromyces genus (e.g., Kluyveromyces lactis, Kluyveromyces marxianus), Candida genus (e.g., Candida utilis, Candida cacaoi, Candida boidinii), Geotrichum genus (e.g., Geotrichum fermentans), Hansenula polymorpha, Yarrowia lipolytica The preferred species is Pichia pastoris. Examples of Pichia pastoris strains include X33, GS115, KM71, KM71H, and CBS7435.

[0108] In one embodiment, the eukaryotic cell is a fungal cell (e.g., a fungal cell of the genus Aspergillus (such as A. niger, A. fumigatus, A. orzyae, A. nidula), Acremonium (such as A. thermophilum), Chaetomium (such as C. thermophilum), Chrysosporium (such as C. hrysosporium species (e.g., C. thermophile), Cordyceps species (e.g., C. militaris), Corynascus species, Ctenomyces species, Fusarium species (e.g., F. oxysporum), Glomerella species (e.g., G. graminicola), Hypocrea species pocrea species (e.g., H. jecorina), Magnaporthe species (e.g., M. orzyae), Myceliophthora species (e.g., M. thermophile), Nectria species (e.g., N. heamatococca), Neurospora species (e.g., N. crassa), Penicillium species (e.g., nicillium species, Sporotrichum species (such as S. thermophile), Thielavia species (such as T. terrestris, T. heterothallica), Trichoderma species (such as T. reesei) or Verticillium species (such as V. dahlia).

[0109] In one embodiment, the eukaryotic cell is an insect cell (e.g., Sf9, Mimic™ Sf9, Sf21, High Five™ (BT1-TN-5B1-4), or BT1-Ea88). cells), algal cells (e.g., from Amphora species, Bacillariophyceae species, Dunaliella species, Chlorella species, Chlamydomonas species, Cyanophyta species (cyanobacteria), Nannochloropsis species, Spirulina species or Ochromonas species) or plant cells (e.g., from monocotyledonous plants (e.g., maize, rice, wheat or Setaria species) or from dicotyledonous plants (e.g., cassava, potato, soybean, tomato, tobacco, alfalfa, Physcomitrella The cells are derived from species of the genus Arabidopsis (Arabidopsis patens).

[0110] In one embodiment, the cell is a bacterial cell or a prokaryotic cell.

[0111] In embodiments, the prokaryotic cell is a Gram-positive cell, such as a Bacillus species, Streptomyces species, Streptococcus species, Staphylococcus species, or Lactobacillus species. Bacillus species that can be used include, for example, B. subtilis, B. amyloliquefaciens, B. licheniformis, B. natto, or B. megaterium. In embodiments, the cell is B. subtilis, such as B. subtilis 3NA and B. subtilis 168. Bacillus species can be obtained, for example, from the Bacillus Genetic Stock Center, Biological Sciences 556, 484 West 12th Avenue, Columbus, Ohio 43210-1214.

[0112] In one embodiment, the prokaryotic cell is a Gram-negative cell, for example, a Salmonella species such as TG1, TG2, W3110, DH1, DHB4, DH5a, HMS 174, HMS174(DE3), NM533, C600, HB101, JM109, MC4100, XL1-Blue, and Origami, or Escherichia coli and those derived from E. coli B strains, such as BL-21 or BL21(DE3) or BL21(DE3)pLysS, all of which are commercially available.

[0113] Suitable host cells are commercially available from culture collections such as, for example, DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany) or the American Type Culture Collection (ATCC).

[0114] In one embodiment, the cell comprises an exogenous nucleic acid encoding a recombinant polypeptide, e.g., any one of the cells described herein that expresses a recombinant polypeptide, e.g., a recombinant polypeptide selected from Tables 1-4.

[0115] In one embodiment, the cell culture is performed as a batch culture, a fed-batch culture, a draw and fill culture, or a continuous culture. In one embodiment, the cell culture is a suspension culture. In one embodiment, the cells or cell cultures are placed in vivo, e.g., into a model organism or a human subject, for expression of the recombinant polypeptide.

[0116] In one embodiment, the culture medium is serum-free. Serum-free, protein-free and chemically defined, animal component-free (CDACF) medium is commercially available, for example, from Lonza Biologics.

[0117] Suitable culture media and culture methods for mammalian cell lines are well known in the art, for example, as described in US Pat. No. 5,633,162. Examples of standard cell culture media for laboratory flasks or low-density cell culture and adapted to the needs of specific cell types include, for example, Roswell Park Memorial Institute (RPMI) 1640 medium (Morre, G., The Journal of the American Medical Association, Vol. 199, pp. 519-520, 1967), L-15 medium (Leibovitz, A. et al., Amer. J. of Hygiene, Vol. 78, No. 1, pp. 173-, 1963), "Dulbecco's Modified Eagle's Medium" (DMEM), Eagle's Minimum Essential Medium (MEM), Ham's F12 medium (Ham, R. et al., Proc. Natl. Acad. Sc. Vol. 53, pp. 288-, 1965), or Iscove's Modified DMEM lacking albumin, transferrin, and lecithin (Iscoves et al., J. Exp. med. Vol. 1, pp. 923-, 1978). For example, Ham's F10 or F12 medium was specially designed for CHO cell culture. Other media specially adapted for CHO cell culture are described in European Patent No. 481791. Such culture media may be supplemented with fetal bovine serum (FBS, also called fetal calf serum FCS), the latter being known to provide a natural source of excess hormones and growth factors. Cell culture of mammalian cells is nowadays a common operation well described in scientific textbooks and manuals, and is described in detail, for example, in R. Ian Fresney, Culture of Animal Cells, a manual, 4th edition, Wiley-Liss / NY, 2000.

[0118] Other suitable culture methods are known to those skilled in the art and may vary depending on the recombinant polypeptide product and the host cell used. It is within the skill of one of ordinary skill in the art to determine or optimize conditions suitable for expression and production of a recombinant or therapeutic polypeptide to be expressed by a cell.

[0119] In one aspect, the cell or cell line comprises an exogenous nucleic acid encoding a product, e.g., a recombinant or therapeutic polypeptide. In one embodiment, the cell or cell line expresses a product, e.g., a therapeutic or diagnostic agent. Methods for genetically modifying or engineering cells to express a desired polypeptide or protein are well known in the art and include, for example, transfection, transduction (e.g., viral transduction), or electroporation.

[0120] Physical methods for introducing nucleic acids, e.g., exogenous nucleic acids or vectors described herein, into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, Vols. 1-4, Cold Spring Harbor Press, NY).

[0121] Chemical means for introducing nucleic acids, e.g., exogenous nucleic acids or vectors described herein, into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo include liposomes (e.g., artificial membrane vesicles). Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of polynucleotides using targeted nanoparticles or other suitable submicron-sized delivery systems.

[0122] In embodiments, integration of an exogenous nucleic acid into a host cell's nucleic acid, e.g., the host cell's genome or chromosomal nucleic acid, is desired. A method for determining whether integration of an exogenous nucleic acid into a host cell's genome has occurred can include the GS / MSX selection method. The GS / MSX selection method uses complementation of glutamine auxotrophy with a recombinant GS gene to select for high-level protein expression from cells. Briefly, the GS / MSX selection method involves including a nucleic acid encoding glutamine synthetase in a vector containing an exogenous nucleic acid encoding a recombinant polypeptide product. Administration of methionine sulfoximine (MSX) selects for cells that have stably integrated into their genome exogenous nucleic acids encoding both a recombinant, therapeutic, or repressor polypeptide and GS. Because GS can be endogenously expressed by some host cells, e.g., CHO cells, the concentration and duration of selection using MSX can be optimized to identify high-producing cells that have stably integrated into the host genome exogenous nucleic acids encoding a recombinant, therapeutic, or repressor polypeptide product. GS selection and systems are further described in Fan et al., Pharm. Bioprocess. (2013); 1(5):487-502, which is incorporated herein by reference in its entirety.

[0123] Other methods for identifying and selecting cells that have stably integrated the exogenous nucleic acid into the host cell genome can include, but are not limited to, including a reporter gene in the exogenous nucleic acid and assessing the presence of the reporter gene in the cells and PCR analysis and detection of the exogenous nucleic acid.

[0124] In one embodiment, cells selected, identified, or generated using the methods described herein (e.g., cells comprising a first control element, e.g., a first promoter element, operably linked to a sequence encoding an exogenous therapeutic polypeptide and a second control element, e.g., a second promoter element, operably linked to a sequence encoding a repressor polypeptide, wherein the second control element has a first level of activity under a first condition and a second level of activity under a second condition, and wherein in the presence of the second condition, expression of the therapeutic polypeptide is modulated, e.g., decreased) are capable of producing higher or more consistent yields of protein product than cells selected using only one selection method for recombinant or stable expression, e.g., integration, of an exogenous nucleic acid encoding a therapeutic polypeptide. In one embodiment, cells selected, identified, or generated using the methods described herein produce 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more of a product, e.g., a recombinant or therapeutic polypeptide, compared to cells that were only selected, identified, or generated for stable expression, e.g., integration, of an exogenous nucleic acid encoding a recombinant or therapeutic polypeptide. In one embodiment, cells selected, identified, or generated using the methods described herein produce a product, e.g., a recombinant or therapeutic polypeptide, for a period or number of cell passages that is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more increased compared to cells that were only selected, identified, or generated for stable expression, e.g., integration, of an exogenous nucleic acid encoding a recombinant or therapeutic polypeptide. In one embodiment, cells selected, identified, or generated using the methods described herein produce 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300% more correctly folded products, e.g., recombinant or therapeutic polypeptides, compared to cells that have only been selected, identified, or generated for stable expression, e.g., integration, of an exogenous nucleic acid encoding a recombinant or therapeutic polypeptide.In one embodiment, cells selected, identified, or generated using the methods described herein produce 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% less aggregated protein or product, e.g., a recombinant or therapeutic polypeptide, compared to cells that were only selected, identified, or generated for stable expression, e.g., integration, of an exogenous nucleic acid encoding a recombinant or therapeutic polypeptide. In one embodiment, cells selected, identified, or generated using the methods described herein produce 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300% more glycosylated product, e.g., a recombinant or therapeutic polypeptide, compared to cells that were only selected, identified, or generated for stable expression, e.g., integration, of an exogenous nucleic acid encoding a recombinant or therapeutic polypeptide. In one embodiment, the population of cells selected, identified or generated using the methods described herein and used to produce a product are 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 or 300% more viable compared to cells that were only selected, identified or generated for stable expression, e.g., integration, of an exogenous nucleic acid encoding a recombinant or therapeutic polypeptide and used to produce the product.

[0125] Cell evaluation, classification, selection or identification In one aspect, the disclosure features a method of evaluating a cell, e.g., a candidate cell, for its ability to produce a product, e.g., a recombinant or therapeutic polypeptide. The results of such an evaluation can provide information useful for the selection or identification of cells for generating cells or cell lines that are high-producing cells or cell lines. In another embodiment, depending on the evaluation described herein, a cell or cell line can be classified, e.g., as having high production potential.

[0126] A high-producing cell or cell line is capable of producing a high yield of recombinant or therapeutic polypeptide product compared to a reference cell or a cell that has not been selected or generated by the methods described herein. In one embodiment, a high producing cell line is capable of producing 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, 500 mg / L, 600 mg / L, 700 mg / L, 800 mg / L, 900 mg / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, 50 g / L, 55 g / L, 60 g / L, 65 g / L, 70 g / L, 75 g / L, 80 g / L, 85 g / L, 90 g / L, 95 g / L, or 100 g / L or more of a product, e.g., a recombinant polypeptide product. In one embodiment, a high producing cell line produces 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, 500 mg / L, 600 mg / L, 700 mg / L, 800 mg / L, 900 mg / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, 50 g / L, 55 g / L, 60 g / L, 65 g / L, 70 g / L, 75 g / L, 80 g / L, 85 g / L, 90 g / L, 95 g / L, or 100 g / L or more of a product, e.g., a recombinant or therapeutic polypeptide product. The amount of product produced can vary depending on the cell type, e.g., species, and recombinant or therapeutic polypeptide to be expressed. By way of example, a high-producing cell can produce at least 1 g / L, 2 g / L, 5 g / L, 10 g / L, 15 g / L, 20 g / L, or 25 g / L or more of a recombinant or therapeutic polypeptide, e.g., as described herein.

[0127] In embodiments where the product is difficult to express, the high-producing cells may produce lower concentrations of product, e.g., less than 0.1 g / L, 0.5 g / L, or 1 g / L, but productivity will be higher or increased than that observed for cells that do not contain a nucleic acid comprising a regulatory element operably linked to a sequence encoding a repressor polypeptide. For example, the level, amount, or quantity of product produced by the identified or selected cells may be increased, e.g., 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, or 100-fold or more, compared to the level, amount, or quantity produced by cells that do not contain a cell comprising a regulatory element operably linked to a sequence encoding a repressor polypeptide.

[0128] The methods described herein for evaluating cells include assessing the effect of a repressor polypeptide on one or more parameters associated with cell function. Parameters associated with cell function include, but are not limited to, cell survival, culture viability, ability to grow, ability to produce a product, and protein degradation. In embodiments, to determine the effect of a repressor polypeptide on a parameter associated with cell function, for example, to determine whether a cell containing a nucleic acid comprising a regulatory element operably linked to a sequence encoding a repressor polypeptide results in an increase or decrease in one of the parameters associated with cell function, the value of the effect of expression of the repressor polypeptide on one or more parameters associated with cell function is compared to a reference value. In one embodiment, depending on the determination of an increase or decrease in one or more parameters associated with cell function, cells can be selected or identified for development as a cell production line. In one embodiment, depending on the determination of an increase or decrease in one or more parameters associated with cell function, cells can be identified as high-producing cells, e.g., cells capable of producing a high yield of a product.

[0129] In any of the embodiments described herein, the reference value can be the value of the effect of the repressor polypeptide on a parameter associated with cellular function of a reference cell, e.g., a cell having a predetermined productivity. Alternatively, or additionally, in any of the embodiments described herein, the reference value can be the value of the parameter associated with cellular function of the same cell being tested, where the cell does not contain a nucleic acid comprising a regulatory element operably linked to a sequence encoding a repressor polypeptide, e.g., the value of the parameter is measured before the cell is contacted with a nucleic acid comprising a regulatory element operably linked to a sequence encoding a repressor polypeptide, or a separate aliquot of cells that has not been contacted with a nucleic acid comprising a regulatory element operably linked to a sequence encoding a repressor polypeptide.

[0130] In one embodiment, cell survival can be measured by determining or quantifying cell viability, e.g., the number or amount of cells that survive expression of a recombinant or therapeutic polypeptide in cells that also comprise a regulatory element operably linked to a sequence encoding a repressor polypeptide. Increased cell survival includes a 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold or more increase in the number of cells, e.g., intact or viable cells, remaining after expression of a recombinant or therapeutic polypeptide in cells that also comprise a regulatory element operably linked to a sequence encoding a repressor polypeptide compared to after expression of the recombinant or therapeutic polypeptide in cells that do not comprise a regulatory element operably linked to a sequence encoding a repressor polypeptide. Alternatively, increased cell survival includes a 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more decrease in the number of apoptotic cells following expression of a recombinant or therapeutic polypeptide in cells that also contain a regulatory element operably linked to a sequence encoding a repressor polypeptide, compared to following expression of the recombinant or therapeutic polypeptide in cells that do not contain a regulatory element operably linked to a sequence encoding a repressor polypeptide. Methods for detecting cell survival or apoptosis, such as Annexin V assay, time integral of viable cell concentration (IVC), maximum viable cell concentration, and cell-specific productivity rates, are known in the art.

[0131] In one embodiment, culture viability can be measured by determining or quantifying the number or amount of living cells, e.g., living cells or cells that do not display a characteristic associated with viability, e.g., proliferation markers, cells with intact DNA, or cells that do not display apoptotic markers, in a culture or population of cells. Increased culture viability includes a 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold or greater increase in the number of cells, e.g., intact or living cells, remaining after expression of a recombinant or therapeutic polypeptide in cells that also contain a regulatory element operably linked to a sequence encoding a repressor polypeptide, compared to after expression of the recombinant or therapeutic polypeptide in cells that do not contain a regulatory element operably linked to a sequence encoding a repressor polypeptide. Methods for assessing culture viability are known in the art. Other methods for assessing culture viability include, but are not limited to, trypan blue exclusion followed by counting using a hemocytometer or Vi-CELL (Beckman-Coulter). Other methods for assessing culture viability may involve examining viable biomass, including using high frequency impedance or capacitance (e.g., Carvell and Dowd, 2006, Cytotechnology 50:35-48) or Raman spectroscopy (e.g., Moretto et al., 2011, American Pharmaceutical Review 14).

[0132] In one embodiment, the ability of cells to proliferate can be measured by quantifying or counting cell number, cell doublings, or cell growth rate. Alternatively, proliferating cells can be identified by analyzing the genomic content of the cells (e.g., replicating DNA), for example, by flow cytometry analysis, or by analyzing the presence of proliferation markers involved in the cell cycle, e.g., Ki67, phosphorylated cyclin-CDK complexes. An increase in the ability to proliferate includes a 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold or more increase in the number of cells or the number of cells expressing a proliferation marker after expression of a recombinant or therapeutic polypeptide in cells that also contain a regulatory element operably linked to a sequence encoding a repressor polypeptide, compared to after expression of the recombinant or therapeutic polypeptide in cells that do not contain a regulatory element operably linked to a sequence encoding a repressor polypeptide. Alternatively, an increased ability to proliferate includes a 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold or more increase in cell doubling or growth rate following expression of a recombinant or therapeutic polypeptide in a cell that also comprises a regulatory element operably linked to a sequence encoding a repressor polypeptide, compared to following expression of the recombinant or therapeutic polypeptide in a cell that does not comprise a regulatory element operably linked to a sequence encoding a repressor polypeptide. Methods for assessing culture viability are known in the art.

[0133] The methods provided herein are useful for identifying, selecting, or generating cells or cell lines with improved ability to produce recombinant or therapeutic polypeptides, e.g., products. In one embodiment, the methods provided herein are also useful for identifying, selecting, or generating cells or cell lines that result in improved quality of recombinant or therapeutic polypeptides.

[0134] In one embodiment, the ability of a cell to produce a product can be measured by examining or quantifying the amount or concentration of product produced. An increase in ability to produce a product includes a 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold or more increase in protein production following expression of a recombinant or therapeutic polypeptide in a cell that also includes a regulatory element operably linked to a sequence encoding a repressor polypeptide compared to following expression of the recombinant or therapeutic polypeptide in a cell that does not include a regulatory element operably linked to a sequence encoding a repressor polypeptide.

[0135] In one embodiment, the quality of a product, e.g., an expressed recombinant or therapeutic polypeptide, can be measured by examining or quantifying the amount or concentration of properly folded, functional, or non-aggregated product. An increase in the quality of a product produced by a cell includes a 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold or more increase in the amount or concentration of properly folded, functional, or non-aggregated product, e.g., an expressed recombinant or therapeutic polypeptide, following expression of a recombinant or therapeutic polypeptide in a cell that also includes a regulatory element operably linked to a sequence encoding a repressor polypeptide, compared to following expression of the recombinant or therapeutic polypeptide in a cell that does not include a regulatory element operably linked to a sequence encoding a repressor polypeptide.

[0136] In one embodiment, the quality of a product, e.g., an expressed recombinant or therapeutic polypeptide, can be measured by determining or quantifying the amount or concentration of product with the correct glycosylation profile, macroheterogeneity (i.e., site occupancy), and glycosylation consistency. An increase in the quality of a product produced by a cell includes a 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold or more increase in the amount or concentration of a product with a glycosylation profile, increased site occupancy, or increased glycosylation consistency after expression of a recombinant or therapeutic polypeptide in a cell that also includes a regulatory element operably linked to a sequence encoding a repressor polypeptide, compared to after expression of the recombinant or therapeutic polypeptide in a cell that does not include a regulatory element operably linked to a sequence encoding a repressor polypeptide.

[0137] Methods for measuring increased protein production are well known to those skilled in the art. For example, increased recombinant or therapeutic protein production can be assessed on a small scale by measuring titer in tissue culture medium by ELISA (Smales et al., 2004, Biotechnology Bioengineering 88:474-488). It can also be quantitatively assessed on a large scale by, for example, a ForteBio Octet for high-throughput measurement of recombinant monoclonal antibody (mAb) concentration in medium (Mason et al., 2012, Biotechnology Progress 28:846-855) or by Protein A HPLC (Stansfield et al., 2007, Biotechnology Bioengineering 97:410-424). Other methods for assessing the production of a product, e.g., a recombinant or therapeutic polypeptide described herein, can refer to the specific production rate (qP) and / or the time integral of the viable cell concentration (IVC) of a product, particularly a recombinant or therapeutic polypeptide, in cells. Recombinant or therapeutic polypeptide production or productivity, defined as the concentration of polypeptide in the culture medium, is a function of these two parameters (qP and IVC), calculated according to Porter et al. (Porter et al. 2010 Biotechnology Progress 26:1446-1455).

[0138] Methods for measuring the improved quality of products produced by cell lines engineered as described herein are known in the art. In one embodiment, methods for examining the fidelity of the primary sequence of an expressed recombinant or therapeutic polypeptide product, such as mass spectrometry, HPLC, SDS-PAGE, peptide mapping, and IEF, are known in the art. The increased amount or concentration of properly folded product, e.g., expressed recombinant or therapeutic polypeptide, can be determined by assessing circular dichroism or the intrinsic fluorescence of the expressed recombinant or therapeutic polypeptide. Depending on the identity of the recombinant or therapeutic polypeptide, various functional assays can be used to test for the increased amount or concentration of functional product. For example, antibodies can be tested by ELISA or other immunoaffinity assays.

[0139] Cell lines and methods for recombinant polypeptide production The current state of the art in both mammalian and microbial selection systems is to apply selection pressure at the level of transcription of DNA into RNA. A gene of interest is linked to a selection marker to create a high level of expression of the selection marker that is likely to result in high expression of the gene of interest. Cells that express the selection marker at a high enough level to survive and grow are those that do not, and are unlikely to survive and grow. In this way, a population of cells can be enriched for cells that express the selection marker, and by implication, high levels of the gene of interest. This method has proven extremely successful for expressing proteins that are not difficult to express.

[0140] In some embodiments, further steps may be performed to improve product expression, e.g., transcription, translation, and / or secretion of the product, or product quality, e.g., proper folding and / or primary sequence fidelity. Such further steps include introducing a substance that improves product expression or product quality. In one embodiment, the substance that improves product expression or product quality can be a small molecule, a polypeptide, or a polypeptide that improves protein folding, e.g., a nucleic acid encoding a chaperone protein. In one embodiment, the substance that assists protein folding comprises a nucleic acid encoding a chaperone protein, e.g., BiP, PD1, or ERO1 (Chakravarthi & Bulleid 2004; Borth et al. 2005; Davis et al. 2000). Other additional steps to improve product yield and quality include overexpression of transcription factors such as SBP1 and ATF6 (Tigges & Fussenegger 2006; Cain et al. 2013; Ku et al. 2008), and lectin-binding chaperone proteins such as calnexin and calreticulin (Chung et al. 2004). Overexpression of substances that aid or improve protein folding and product quality and yield proteins described herein can be achieved by introduction of exogenous nucleic acids encoding the proteins. In another embodiment, substances that improve product expression or product quality are small molecules that can be added to cell culture to increase product expression or product quality. In one embodiment, cells are cultured at a lower temperature, for example, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, or 10°C lower than the temperature at which the cells are normally grown.

[0141] Any of the methods described herein can further include a selection step to identify cells with high productivity and high-quality product. For example, FAC selection can be used to select specific cells with a desired characteristic, such as higher expression of a protein folding protein, e.g., a chaperone.

[0142] In one aspect, the disclosure provides a method comprising recovering or removing a recombinant or therapeutic polypeptide product. In embodiments in which the recombinant or therapeutic polypeptide is secreted from the cell, the method may comprise removing, collecting, or separating the recombinant or therapeutic polypeptide from the cell, a cell population, or the culture medium in which the cells are cultured. In embodiments in which the recombinant or therapeutic polypeptide is intracellular, purifying the recombinant or therapeutic polypeptide product comprises separating the recombinant or therapeutic polypeptide produced by the cell from one or more of any of the following: host cell proteins, host cell nucleic acids, host cell lipids, and / or other debris derived from the host cell.

[0143] In embodiments, the processes described herein provide a substantially pure protein product. As used herein, "substantially pure" means substantially free of pyrogenic materials, substantially free of nucleic acids, and / or substantially free of endogenous cellular protein enzymes and components derived from host cells, such as polymerases, ribosomal proteins, and chaperone proteins. A substantially pure protein product contains, for example, less than 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of contaminating endogenous proteins (also known as host cell proteins), nucleic acids, or other macromolecules derived from host cells.

[0144] Methods for recovering and purifying products, such as recombinant or therapeutic polypeptides, are well established in the art. Physical, chemical, or physico-chemical methods are used to recover recombinant or therapeutic polypeptide products. The physical, chemical, or physico-chemical method can be a filtration method, a centrifugation method, an ultracentrifugation method, an extraction method, a lyophilization method, a precipitation method, a chromatography method, or a combination of two or more thereof. In one embodiment, the chromatography method comprises one or more of size exclusion chromatography (or gel filtration), ion exchange chromatography, e.g., anion or cation exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, and / or multimodal chromatography.

[0145] Repressor Polypeptides Provided herein are repressor polypeptides and sequences encoding repressor polypeptides useful in genetic control circuits, cells, and methods for identifying, selecting, or generating cells or cell lines capable of producing high yields of a product, e.g., a recombinant or therapeutic polypeptide. Generally, a repressor polypeptide inhibits expression of a product, e.g., a recombinant or therapeutic polypeptide, in a regulated manner. In some embodiments, the sequence encoding the repressor polypeptide is under the transcriptional control of a control element that activates transcription of the sequence encoding the repressor polypeptide in response to one or more conditions. In some embodiments, the repressor polypeptide binds to a control element, e.g., a promoter element, operably linked to a sequence encoding a recombinant or therapeutic polypeptide. In some embodiments, binding of the repressor polypeptide to the control element inhibits transcription of the sequence encoding the operably linked recombinant or therapeutic polypeptide. In some embodiments, the repressor polypeptide binds to a sequence encoding an untranslated region of a recombinant or therapeutic polypeptide transcript. In some embodiments, binding of the repressor polypeptide to an untranslated region of a recombinant or therapeutic polypeptide transcript inhibits translation of the sequence encoding the recombinant or therapeutic polypeptide. In some embodiments, the repressor polypeptide binds to the coding sequence of a sequence encoding a recombinant or therapeutic polypeptide, hi some embodiments, binding of the repressor polypeptide to the coding sequence of the recombinant or therapeutic polypeptide inhibits the transcription, translation, or transcription and translation of the sequence encoding the recombinant or therapeutic polypeptide.

[0146] It is contemplated that the present disclosure is not specific to individual repressor polypeptides. Exemplary repressor polypeptides include, but are not limited to, Cas9 molecules, TALE molecules, and zinc finger molecules. In some embodiments, the repressor polypeptide is a Cas-associated protein known in the art. In some embodiments, the repressor polypeptide is a protein from a type I, II, or III CRISPR / Cas system (e.g., as described in K.S. Makarova et al., Nat. Rev. Microbiol. 9:467 (2011); K.S. Makarova, N.V. Grishin, S.A. Shabalina, Y.I. Wolf, E.V. Koonin, Biol. Direct 1:7 (2006); or K.S. Makarova, L. Aravind, Y.I. Wolf, E.V. Koonin, Biol. Direct 6:38 (2011)).

[0147] In some embodiments, the repressor polypeptide is a Cas9 molecule. A repressor polypeptide that is a Cas9 molecule requires one or more (e.g., 1, 2, 3, 4, or more) suitable gRNAs to inhibit expression of a recombinant or therapeutic polypeptide.

[0148] In some embodiments, the repressor polypeptide is a TALE molecule.

[0149] In some embodiments, the repressor polypeptide is a zinc finger molecule.

[0150] In some embodiments, the repressor polypeptide is an endogenous repressor of the first control element, e.g., the first promoter element. In one embodiment, the endogenous gene encoding the repressor polypeptide is inactive, e.g., knocked out or mutated to result in a loss of function.

[0151] Cas9 molecule Cas9 molecules to be used in the genetic control circuits, cells, and methods of the present disclosure can comprise polypeptides originating from a variety of species, and furthermore, one or more domains from a Cas9 molecule in one species can be combined with one or more domains from a Cas9 molecule in another species, for example, in a fusion protein. Additional species containing Cas9 polypeptides include Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces species, cycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, and the like. thuringiensis, Bacteroides spp., Blastopirellula marina, Bradyrhizobium spp., Brevibacillus laterosporus, Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Candidatus Puniceispirillum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium acores accolens), Corynebacterium diphtheriaediphtheria, Corynebacterium matruchotii, Dinoroseobacter shibae, Eubacterium dolichum, Gammaproteobacteria, Gluconacetobacter diazotrophicus, Haemophilus parainfluenzae, Haemophilus sputorum, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, Ilyobacter polytropus, Kingella kingae kingae, Lactobacillus crispatus, Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium, Methylocystis species, Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica, Neisseria meningitidis meningitidis, Neisseria species, Neisseria wadsworthii, Nitrosomonas species, Parvibaculum labamentivoranslavamentivorans, Pasteurella multocida, Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodovulum species, Simonsiella muelleri, Sphingomonas species, Sporolactobacillus vineae, Staphylococcus lugdunensis lugdunensis, Streptococcus species, Subdoligranulum species, Tistrella mobilis, Treponema species or Verminephrobacter eiseniae.

[0152] Cas9 structure and activity Crystal structures are available for the naturally occurring Cas9 polypeptide (Jinek et al., Science 343(6176):1247997, 2014) and for S. pyogenes Cas9 with guide RNAs (e.g., synthetic fusions of crRNA and tracrRNA) (Nishimasu et al., Cell 156:935-949, 2014; and Anders et al., Nature 2014, doi:10.1038 / nature13579).

[0153] In one embodiment, the Cas9 molecule or polypeptide comprises one or more of the following domains: a RuvC-like domain and an HNH-like domain. In one embodiment, the Cas9 molecule or polypeptide is a dCas9 molecule or polypeptide, which comprises a RuvC-like domain, e.g., a RuvC-like domain that lacks nuclease activity, and / or an HNH-like domain, e.g., an HNH-like domain that lacks nuclease activity.

[0154] In one embodiment, a Cas9 molecule or polypeptide can comprise more than one RuvC-like domain (e.g., 1, 2, 3, or more RuvC-like domains). In one embodiment, the RuvC-like domain comprises one or more mutations that alter its activity, such that the RuvC domain does not cleave DNA or has reduced DNA cleavage activity. In one embodiment, the RuvC-like domain is at least 5, 6, 7, or 8 amino acids in length, but not more than 20, 19, 18, 17, 16, or 15 amino acids in length. In one embodiment, a Cas9 molecule or polypeptide comprises an N-terminal RuvC-like domain of about 10-20 amino acids in length, e.g., about 15 amino acids in length.

[0155] In one embodiment, a Cas9 molecule or polypeptide can comprise more than one HNH-like domain (e.g., 1, 2, 3, or more HNH-like domains). In one embodiment, the HNH-like domain comprises one or more mutations that alter its activity, such that the HNH-like domain does not cleave DNA or has reduced DNA cleavage activity. In one embodiment, the HNH-like domain is at least 15, 20, or 25 amino acids in length, but is at most 40, 35, or 30 amino acids in length, e.g., 20-35 amino acids in length, e.g., 25-30 amino acids in length.

[0156] In embodiments, a Cas9 molecule or Cas9 polypeptide has the ability to interact with a gRNA molecule and is co-localized with the gRNA molecule in the core targeting domain, but is unable to cleave the target nucleic acid or is unable to cleave it at an efficient rate. A Cas9 molecule that has no or substantially no cleavage activity is referred to herein as a dCas9 molecule or dCas9 polypeptide. For example, a dCas9 molecule or dCas9 polypeptide may lack or have substantially reduced cleavage activity, e.g., less than 20, 10, 5, 1, or 0.1% of the cleavage activity of a reference Cas9 molecule or Cas9 polypeptide, as measured by assays known in the art or described herein.

[0157] Targeting and PAM A Cas9 molecule or Cas9 polypeptide is a polypeptide that can interact with a guide RNA (gRNA) molecule and localize, together with the gRNA molecule, to a site that contains a target domain and a PAM sequence.

[0158] In one embodiment, the ability of a Cas9 molecule or Cas9 polypeptide to interact with a target nucleic acid is dependent on the PAM sequence. The PAM sequence is a sequence in the target nucleic acid. Cas9 molecules from different bacterial species may recognize different sequence motifs (e.g., PAM sequences). Cas9 molecules can be genetically engineered to alter the PAM specificity of the Cas9 molecule. Exemplary naturally occurring Cas9 molecules are described in Chylinski et al., RNA Biology 2013, vol. 10:5, pp. 727-737.

[0159] Altering the Cas9 structure In some embodiments, for example, one or more mutations can be present in one or more RuvC-like domains of a Cas9 molecule or polypeptide, e.g., the N-terminal RuvC-like domain, the HNH-like domain, or a region outside the RuvC-like and HNH-like domains. In some embodiments, the mutation(s) are present in a RuvC-like domain, e.g., the N-terminal RuvC-like domain. In some embodiments, the mutation(s) are present in the HNH-like domain. In some embodiments, the mutation(s) are present in a RuvC-like domain, e.g., both the N-terminal RuvC-like domain and the HNH-like domain.

[0160] In one embodiment, the Cas9 molecule or Cas9 polypeptide, e.g., a dCas9 molecule or dCas9 polypeptide, can be any Cas9 molecule sequence described herein or a naturally occurring Cas9 molecule sequence from a species listed herein or described in Chylinski et al., RNA Biology 2013, 10:5, 727-737; Hou et al., PNAS Early Edition 2013, 1-6, e.g., a Cas9 molecule. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology When compared, they differ in no more than 2, 5, 10, 15, 20, 30, or 40% of the amino acid residues. differ by at least 1, 2, 5, 10, or 20 amino acids, but not more than 100, 80, 70, 60, 50, 40, or 30 amino acids; or The Cas9 molecule or polypeptide comprises an identical amino acid sequence, and in one embodiment, the Cas9 molecule or polypeptide comprises one or more of the following activities: helicase activity or the ability to localize to a target nucleic acid together with a gRNA molecule. In one embodiment, the Cas9 molecule or polypeptide does not comprise nickase activity or double-strand cleavage activity (e.g., endonuclease and / or exonuclease activity).

[0161] Exemplary mutations that may be made in the RuvC or HNH domain relative to the S. pyogenes sequence include D10A, E762A, H840A, N854A, N863A and / or D986A.

[0162] Exemplary Cas9 polypeptide and Cas9 domain sequences can be found in Tables 50-54 of WO 2015 / 157070.

[0163] dCas9 repressor polypeptide In one embodiment, the Cas9 molecule or Cas9 polypeptide is a dCas9 molecule or dCas9 polypeptide that includes one or more differences in the RuvC domain and / or the HNH domain compared to a reference Cas9 molecule, and the dCas9 molecule or dCas9 polypeptide does not cleave nucleic acids or cleaves them significantly less efficiently than a wild-type molecule, e.g., cleaves less than 50, 25, 10, or 1% of the nucleic acid of the reference Cas9 molecule as measured by an assay described herein, when compared to a wild-type molecule in a cleavage assay as described herein.

[0164] Mutating key residues in both DNA cleavage domains of the Cas9 protein (e.g., D10A and H840A mutations) results in the generation of catalytically inactive Cas9 (dCas9, also known as dead Cas9) molecules. Enzymatically inactive Cas9, e.g., dCas9, forms a complex with a gRNA and localizes to the DNA sequence specified by the gRNA's targeting domain, but does not cleave the target DNA. Enzymatically inactive (e.g., dCas9) Cas9 molecules can block transcription when recruited to early regions in coding sequences. Further repression can be achieved by fusing a transcriptional repression domain (e.g., KRAB, SID, or ERD) to an enzymatically inactive Cas9, e.g., dCas9, and recruiting it to a target sequence, e.g., a sequence within 1000 bp 3' of the start codon of a gene, or a regulatory element, e.g., a promoter element, e.g., within 500 bp 5' of the start codon. Targeting promoter DNase I hypersensitive sites (DHSs) (e.g., by generating a gRNA complementary to a DHS) may be an additional strategy for gene suppression, e.g., inhibition of sequences encoding recombinant or therapeutic polypeptides, because these regions are more likely to be accessible to enzymatically inactive Cas9, e.g., dCas9, and are also more likely to harbor sites for endogenous transcription factors. Without wishing to be bound by theory, it is contemplated herein that blocking binding sites for endogenous transcription factors or RNA polymerases will be useful in downregulating gene expression, e.g., expression of sequences encoding recombinant or therapeutic polypeptides. In one embodiment, one or more enzymatically inactive Cas9, e.g., dCas9 molecules, may be used to block the binding of one or more endogenous transcription factors. In another embodiment, an enzymatically inactive Cas9, e.g., dCas9 molecule, can be fused to an effector domain, e.g., a repression domain, an activation domain, a methylation enzyme, or the like. Fusion of an enzymatically inactive Cas9, such as dCas9, with an effector domain allows the effector to be recruited to any DNA site specified by the gRNA. Altering the chromatin state can result in reduced expression of the target gene.One or more enzymatically inactive Cas9, e.g., dCas9 molecules fused to one or more chromatin-modifying proteins may be used to alter the chromatin state.

[0165] In one embodiment, the gRNA molecule is capable of targeting a control element (e.g., a promoter element), e.g., a control element operably linked to a sequence encoding a recombinant or therapeutic polypeptide. In one embodiment, the gRNA molecule is capable of targeting a sequence encoding a recombinant or therapeutic polypeptide.

[0166] gRNA molecule A gRNA molecule, as that term is used herein, refers to a nucleic acid that facilitates specific targeting or homing of a gRNA molecule / Cas9 molecule complex to a target sequence. A gRNA molecule can be unimolecular (comprising a single RNA molecule), sometimes referred to herein as a "chimeric" gRNA, or modular (comprising more than one, usually two separate RNA molecules). A gRNA molecule comprises several domains.

[0167] In one embodiment, the unimolecular or chimeric gRNA comprises, typically from 5' to 3': Targeting domain, a first complementarity domain, Concatenated domains, a second complementarity domain (complementary to the first complementarity domain), the proximal domain, and Optionally, a tail domain Includes.

[0168] In one embodiment, the modular gRNA comprises: Usually from 5' to 3' Targeting domain, First complementarity domain and a first strand comprising: Usually from 5' to 3' optionally a 5' extension domain; a second complementarity domain, Proximal domain Optionally, a tail domain and a second strand comprising Includes.

[0169] In one embodiment, the gRNA comprises a first strand comprising a tracrRNA and a second strand comprising a crRNA. Exemplary tracrRNAs and crRNAs and methods for designing them can be found in the art, for example, in Jinek et al., Science 2012, Aug. 17:337, No. 6096, pp. 816-821.

[0170] Exemplary gRNAs and methods for designing gRNAs can be found in WO 2015 / 157070, Xu, H. et al., Genome Res. 2015 Aug;25(8):1147-57, and methods known in the art.

[0171] gRNA domain The targeting domain comprises a nucleotide sequence that is complementary to a target sequence on a target nucleic acid, e.g., at least 80, 85, 90, or 95% complementary, e.g., fully complementary. The targeting domain is part of an RNA molecule and therefore contains the base uracil (U), whereas any DNA encoding a gRNA molecule will contain the base thymine (T). In one embodiment, the complementarity of the target domain to the target sequence is believed to contribute to the specificity of the interaction of the gRNA molecule / Cas9 molecule complex with the target nucleic acid. In one embodiment, the targeting domain is 5 to 50 nucleotides in length. In some embodiments, the targeting domain has complementarity to a first control element, e.g., a first promoter element, a sequence encoding a recombinant or therapeutic polypeptide, or to an untranslated region or intron contained within a first control element, e.g., a first promoter element, or a sequence encoding a recombinant or therapeutic polypeptide. The strand of a target nucleic acid to which the targeting domain is complementary is referred to herein as the complementary strand.

[0172] The first complementarity domain is complementary to the second complementarity domain, and in one embodiment has sufficient complementarity to the second complementarity domain to form a double-stranded region under at least some physiological conditions.

[0173] The first complementarity domain may share homology with or be derived from a naturally occurring first complementarity domain. In one embodiment, it has at least 50% homology with a first complementarity domain derived from S. pyogenes, S. aureus, or S. thermophilus. The linking domain serves to link the first complementarity domain of the unimolecular gRNA to the second complementarity domain. The linking domain can link the first and second complementarity domains covalently or non-covalently. In one embodiment, the linkage is covalent. Typically, the linking domain comprises one or more nucleotides, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0174] In modular gRNA molecules, the two molecules associate through hybridization of complementary domains.

[0175] In one embodiment, the modular gRNA may comprise an additional sequence 5' of the second complementarity domain, referred to herein as a 5' extension domain. In one embodiment, the 5' extension domain is 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length.

[0176] The second complementarity domain is complementary to the first complementarity domain and, in one embodiment, has sufficient complementarity to form a double-stranded region under at least some physiological conditions. In one embodiment, the second complementarity domain may contain a sequence that lacks complementarity with the first complementarity domain, e.g., a sequence that loops out from the double-stranded region. In one embodiment, the second complementarity domain is 5-27 nucleotides in length. In one embodiment, it is longer than the first complementarity domain. The second complementarity domain may share homology with or be derived from a naturally occurring second complementarity domain. In one embodiment, it has at least 50% homology to a second complementarity domain from S. pyogenes, S. aureus, or S. thermophilus.

[0177] In one embodiment, the proximal domain is 5-20 nucleotides in length. In one embodiment, the proximal domain may share homology with or be derived from a naturally occurring proximal domain. In one embodiment, it has at least 50% homology with a proximal domain from S. pyogenes, S. aureus, or S. thermophilus.

[0178] In one embodiment, the tail domain is 0 (none), 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In one embodiment, the tail domain nucleotides can share homology with or be derived from a sequence derived from the 5' end of a naturally occurring tail domain. In one embodiment, the tail domain has at least 50% homology to a tail domain disclosed herein, e.g., an S. pyogenes, S. aureus, or S. thermophilus tail domain. In one embodiment, the tail domains comprise sequences that are complementary to each other and form a double-stranded region under at least some physiological conditions.

[0179] In one embodiment, the tail domain comprises nucleotides at the 3' end that are associated with methods of in vitro or in vivo transcription. When a U6 promoter is used for in vivo transcription, these nucleotides can be the sequence UUUUUU.

[0180] How to design gRNAs Methods for selecting and validating gRNA target sequences and off-target analysis are described, for example, in Mali et al., 2013, Science 339(6121):823-826; Hsu et al., Nat Biotechnol 31(9):827-32; Fu et al., 2014, Nat Biotechnol, doi:10.1038 / nbt.2808 PubMed PMID:24463574; Heigwer et al., 2014, Nat METHODS 11(2):122-3 doi:10.1038 / nmeth.2812 PubMed PMID:24481216; Bae et al., 2014, BIOINFORMATICS PubMed PMID:24463181; Xiao A et al., 2014, BIOINFORMATICS PubMed PMID:24463182. It is described in PMID:24389662.

[0181] For example, a software tool can be used to optimize the selection of gRNAs within a user's target sequence to, for example, minimize total off-target activity in the genome. Off-target activity can be DNA binding, DNA cleavage, DNA nicking, or another activity. For each possible gRNA selection using S. pyogenes Cas9, the tool can identify all off-target sequences in the genome (preceded by either a NAG or NGG PAM) that contain up to a certain number of mismatched base pairs (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Each possible gRNA is then ranked according to its total predicted off-target cleavage, with the highest-ranked gRNA representing the most likely to have the greatest on-target and least off-target cleavage. Other features can be included in the tool, such as automated reagent design for CRISPR assembly, primer design for on-target Surveyor assays, and primer design for high-throughput detection and quantification of off-target cleavage by next-generation sequencing. Candidate gRNA molecules can be evaluated by methods known in the art.

[0182] TALE molecule Transcription Activator-Like Effector (TALE) molecule or TALE polypeptide, as the term is used herein, refers to a molecule or polypeptide comprising multiple TALE DNA-binding repeat domains (TALE DBDs) that can home to or localize to a nucleic acid location specified by the TALE DBD. TALE molecule and TALE polypeptide, as the terms are used herein, refer to, for example, naturally occurring TALE molecules and engineered, altered, or modified TALE molecules or TALE polypeptides that differ by at least one amino acid residue from a reference sequence, e.g., the most similar naturally occurring TALE molecule known in the art.

[0183] A TALE DBD, as that term is used herein, refers to a 33-35 amino acid motif that contains two hypervariable residues (i.e., repeat variable dinucleotides, RVDs) at positions 12 and 13 of the motif. The RVDs of a TALE DNA binding domain (DBD) specify the DNA base pair or base pairs for which the TALE DBD has binding affinity. When TALE DBDs are combined in an array within a TALE molecule or TALE polypeptide, the order of the TALE DBDs (and their RVDs) determines the DNA sequences for which the TALE molecule or TALE polypeptide has binding affinity. Naturally occurring TALE polypeptides and TALE DBDs are produced by bacteria of the genus Xanthomonas.

[0184] Repeat variable dinucleotides (RVDs), as that term is used herein, refer to the two hypervariable amino acid residues at positions 12 and 13 of the TALE DBD. RVDs determine the DNA base-pairing affinity of the TALE DBD. All possible combinations of RVDs and their respective base-pairing affinities are known in the art. See, for example, Cong L. et al. Nat Commun. 2012 July 24; 3():968; Juillerat A. et al. Sci Rep. 2015 January 30; 5():8150; Miller JC et al. Nat Methods 12, 465-471 (2015); Streubel J. et al. Nat Biotechnol 30, 593-595 (2012); and Yang J. et al. Cell Res 24, 628-631 (2014), the entire contents of which are incorporated herein by reference. All possible RVDs are considered for use with repressor polypeptides, such as the TALE molecules described herein.

[0185] A TALE DBD array, as that term is used herein, refers to the identity and order of the TALE DBDs, e.g., the RVDs of each TALE DBD, within a TALE molecule or TALE polypeptide. The TALE DBD array determines the sequence-specific binding affinity of the TALE molecule or TALE polypeptide.

[0186] In some embodiments, the repressor polypeptide is a TALE molecule or TALE polypeptide. TALE DBDs and TALE polypeptides from any species of Xanthomonas can be used in genetic control circuits, cells, and methods for identifying, selecting, or generating cells or cell lines capable of producing high yields of products, such as the recombinant or therapeutic polypeptides described herein. In some embodiments, the repressor polypeptide is a naturally occurring TALE molecule or TALE polypeptide. In some embodiments, the repressor polypeptide is an engineered TALE molecule or TALE polypeptide, i.e., a TALE molecule or TALE polypeptide that differs by one or more amino acids from a naturally occurring TALE molecule or TALE polypeptide, or from another engineered TALE molecule or TALE polypeptide known in the art.

[0187] In some embodiments, the engineered TALE molecule or TALE polypeptide is: any TALE molecule sequence described herein or a naturally occurring TALE molecule sequence, e.g., a TALE molecule derived from a species listed herein or described in a publication referenced herein; 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology When compared, they differ in no more than 2, 5, 10, 15, 20, 30, or 40% of the amino acid residues. differ by at least 1, 2, 5, 10, or 20 amino acids, but not more than 100, 80, 70, 60, 50, 40, or 30 amino acids; or They contain identical amino acid sequences.

[0188] In some embodiments, a TALE molecule localizes to a target DNA sequence specified by the TALE DBD array of that TALE molecule. In some embodiments, a TALE molecule can block transcription when recruited to a coding sequence, e.g., an early region in the coding sequence of a recombinant or therapeutic polypeptide. In some embodiments, a TALE molecule can block transcription when recruited to a control element, e.g., a promoter element, operably linked to a sequence encoding a recombinant or therapeutic polypeptide. In some embodiments, further repression can be achieved by fusing a transcriptional repression domain (e.g., KRAB, SID, or ERD) to the TALE molecule, allowing for the recruitment of an effector to any DNA site specified by the TALE DBD array.

[0189] In some embodiments, the TALE molecule comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more) TALE DBDs.

[0190] In some embodiments, a repressor polypeptide, e.g., a TALE DBD array of a TALE molecule, specifies a target DNA sequence. In some embodiments, the target sequence specified by the TALE DBD array is contained within a regulatory element, e.g., a promoter element, operably linked to a sequence encoding a recombinant or therapeutic polypeptide. In some embodiments, the target sequence specified by the TALE DBD array is contained within a sequence encoding a recombinant or therapeutic polypeptide.

[0191] Exemplary naturally occurring and engineered TALE polypeptide sequences and methods for designing and testing TALE polypeptides for use with genetic regulatory circuits, cells, and methods for identifying, selecting, or generating cells or cell lines capable of producing high yields of product, e.g., recombinant or therapeutic polypeptides described herein, are described in the art, e.g., Zhang F et al. Nat Biotechnol. 2011;29:149-153; Geissler R et al. PLoS One. 2011;6:e19509; Garg A et al. Nucleic Acids Res. 2012; Bultmann S et al. Nucleic Acids Res. 2012;40:5368-5377; Cermak T et al. Efficient design and assembly of custom TALEN and other TAL effector based constructs for DNA targeting. Nucleic Acids Res. 2011;39:e82; Cong, which are incorporated herein by reference in their entireties. L et al. Nat Commun. 2012;3:968; and Miller JC et al. Nat Biotechnol. 2011;29:143-148.

[0192] zinc finger molecules A zinc finger molecule, as that term is used herein, refers to a molecule or polypeptide that contains multiple zinc finger domains (ZFDs). A zinc finger molecule has affinity for a particular DNA sequence, determined by the identity and order of the ZFDs it contains.

[0193] As used herein, zinc finger domain (ZFD) refers to any of a family of polypeptides that specifically bind to DNA in a sequence and require a zinc ion ligand for DNA binding. Numerous families of ZFDs have been studied and characterized (see, e.g., Krishna, SS. et al., Nucl. Acids Res. (2003) 31(2):532-550). The present disclosure contemplates zinc finger molecules that may include ZFDs of any type or origin known to those skilled in the art. While not intending to be limited to any particular type of ZFD, the present disclosure contemplates zinc finger molecules that include the Cys2His2ZFD, the most prevalent and well-studied ZFD in the art. The Cys2His2ZFD contains two beta strands that form an antiparallel beta sheet and an alpha helix. Positions -1, 1, 2, 3, 5, and 6 of the alpha helix are known to specify DNA sequence-specific binding by interacting with DNA base pairs. In one embodiment, the Cys2His2ZFD can have specific binding affinity for a three base pair target sequence. In one embodiment, the Cys2His2ZFD can interact specifically with additional base pairs adjacent to the target sequence in a context-specific manner, i.e., depending on the presence or identity of adjacent ZFDs within the zinc finger molecule.

[0194] A zinc finger domain array or ZFD array, as that term is used herein, refers to the identity and order of ZFDs within a zinc finger molecule or zinc finger polypeptide. The ZFD array determines the sequence-specific binding affinity of the zinc finger molecule or zinc finger polypeptide.

[0195] In some embodiments, the repressor polypeptide is a zinc finger molecule or zinc finger polypeptide. ZFDs and zinc finger polypeptides from any species (e.g., mammalian species, e.g., human) can be used in the genetic control circuits, cells, and methods for identifying, selecting, or generating cells or cell lines capable of producing high yields of product, e.g., recombinant or therapeutic polypeptides described herein. In some embodiments, the repressor polypeptide is a naturally occurring zinc finger molecule or zinc finger polypeptide. In some embodiments, the repressor polypeptide is an engineered zinc finger molecule or zinc finger polypeptide, i.e., a zinc finger molecule or zinc finger polypeptide that differs by one or more amino acids from a naturally occurring zinc finger molecule or zinc finger polypeptide or from another engineered zinc finger molecule or zinc finger polypeptide known in the art.

[0196] In some embodiments, the engineered zinc finger molecule or zinc finger polypeptide is a combination of any zinc finger molecule sequence described herein or a naturally occurring zinc finger molecule sequence, e.g., a zinc finger molecule derived from a species listed herein or described in the publications referenced herein, and 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology When compared, they differ in no more than 2, 5, 10, 15, 20, 30, or 40% of the amino acid residues. differ by at least 1, 2, 5, 10, or 20 amino acids, but not more than 100, 80, 70, 60, 50, 40, or 30 amino acids; or They contain identical amino acid sequences.

[0197] In some embodiments, a zinc finger molecule localizes to a target DNA sequence specified by the ZFD array of that zinc finger molecule. In some embodiments, a zinc finger molecule can block transcription when recruited to an early region in a coding sequence, e.g., a coding sequence for a recombinant or therapeutic polypeptide. In some embodiments, a zinc finger molecule can block transcription when recruited to a regulatory element, e.g., a promoter element, operably linked to a sequence encoding a recombinant or therapeutic polypeptide. In some embodiments, further repression can be achieved by fusing a transcriptional repression domain (e.g., KRAB, SID, or ERD) to the zinc finger molecule, allowing for the recruitment of an effector to any DNA site specified by the ZFD array.

[0198] In some embodiments, a zinc finger molecule comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more) ZFDs. In some embodiments, ZFD arrays can be constructed from ZFDs with known target sequence affinities to generate zinc finger molecules or zinc finger polypeptides with desired specific target sequences.

[0199] In some embodiments, a ZFD array of repressor polypeptides, e.g., zinc finger molecules, specifies a target DNA sequence. In some embodiments, the target sequence specified by the ZFD array is contained within a control element, e.g., a promoter element, operably linked to a sequence encoding a recombinant or therapeutic polypeptide. In some embodiments, the target sequence specified by the ZFD array is contained within a sequence encoding a recombinant or therapeutic polypeptide.

[0200] Exemplary naturally occurring and engineered zinc finger polypeptide sequences and methods for designing and testing zinc finger polypeptides for use with genetic regulatory circuits, cells, and methods for identifying, selecting, or generating cells or cell lines capable of producing high yields of product, e.g., recombinant or therapeutic polypeptides described herein, are well known in the art, e.g., Wolfe SA et al. Annu Rev Biophys Biomol Struct. 2000;29:183-212; Pabo CO et al. Annu Rev Biochem. 2001;70:313-340; Greisman HA, Pabo CO. Science. 1997;275:657-661; Isalan M et al. Proc Natl Acad Sci U S A. 1997;94:5617-5621; Wolfe SA et al. J Mol Biol. 1999;285:1917-1934.

[0201] Methods for designing ZFDs and ZFD arrays that bind to specific target DNA sequences can be found in the art, for example, in Maeder ML et al. Mol Cell. 2008;31:294-301; Sander JD et al. Nat METHODs. 2011;8:67-69; and Meng X et al. Nat Biotechnol. 2008;26:695-701, the entire contents of which are incorporated herein by reference.

[0202] Manufacturing Applications The cells, methods, kits, reaction mixtures, and nucleic acids disclosed herein can be used in bioreactors or process vessels or tanks, or more generally, with any source. The devices, equipment, and methods described herein are suitable for culturing any desired cell line, including prokaryotic and / or eukaryotic cell lines. Also included are industrial equipment containing components suitable for culturing suspension cells or anchorage-dependent (adherent) cells, and suitable for production operations set up for the manufacture of pharmaceutical and pharmaceutical products—such as polypeptide products, nucleic acid products (e.g., DNA or RNA), or cells and / or viruses, such as those used in cell therapy and / or viral therapy.

[0203] In embodiments, the cells express or produce a product, such as a recombinant therapeutic or diagnostic product. As described in more detail below, examples of products produced by the cells include, but are not limited to, antibody molecules (e.g., monoclonal antibodies, bispecific antibodies), antibody mimetics (polypeptide molecules that specifically bind to an antigen but are not structurally related to antibodies, such as DARPins, affibodies, adnectins, or IgNARs), fusion proteins (e.g., Fc fusion proteins, chimeric cytokines), other recombinant proteins (e.g., glycosylated proteins, enzymes, hormones), viral therapeutics (e.g., anti-oncolytic viruses, viral vectors for gene therapy and viral immunotherapy), cell therapy (e.g., pluripotent stem cells, mesenchymal stem cells, and adult stem cells), vaccines or lipid-encapsulated particles (e.g., exosomes, virus-like particles), RNA (e.g., siRNA, etc.) or DNA (e.g., plasmid DNA, etc.), antibiotics, or amino acids. In embodiments, the devices, equipment, and methods can be used to manufacture biosimilars.

[0204] Also included are industrial facilities comprising components that enable the production of eukaryotic cells, e.g., mammalian cells, or lower eukaryotic cells, e.g., yeast cells or filamentous fungal cells, or prokaryotic cells, e.g., gram-positive or gram-negative cells, and / or products of eukaryotic cells or prokaryotic cells, e.g., proteins, peptides, antibiotics, amino acids, nucleic acids (e.g., DNA or RNA), synthesized by eukaryotic cells on a large scale. Unless otherwise indicated herein, devices, facilities, and methods can include any desired capacity or manufacturing capability, including, but not limited to, bench scale, pilot scale, and full manufacturing scale capabilities.

[0205] Furthermore, unless otherwise indicated herein, equipment may include any suitable reactor(s), including, but not limited to, stirred tank, airlift, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and / or spouted bed bioreactors. As used herein, a "reactor" may include a fermentor or fermentation unit or any other reaction vessel, and the term "reactor" is used synonymously with "fermentor." For example, in some embodiments, an exemplary bioreactor unit may perform one or more or all of the following: supplying nutrients and / or carbon sources, injecting a suitable gas (e.g., oxygen), inlet and outlet flow of fermentation or cell culture medium, separating gas and liquid phases, maintaining temperature, maintaining oxygen and CO2 levels, maintaining pH levels, agitation (e.g., stirring), and / or cleaning / sterilization. An exemplary reactor unit, such as a fermentation unit, may contain multiple reactors within the unit; for example, a unit can have 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more bioreactors in each unit, and / or a facility may contain multiple units with single or multiple reactors within a facility. In various embodiments, the bioreactors can be suitable for batch, semi-fed-batch, fed-batch, perfusion, and / or continuous fermentation processes. Any suitable reactor diameter can be used. In embodiments, the bioreactors can have a volume between about 100 mL and about 50,000 L.Non-limiting examples include 100 mL, 250 mL, 500 mL, 750 mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters, and 550 liters. 10,000 liters, 15,000 liters, 20,000 liters, and / or 50,000 liters. Further, suitable reactors may be multi-use, single-use, disposable, or non-disposable, and may be formed from any suitable material, including stainless steel (e.g., 316L or any other suitable stainless steel) and alloys such as Inconel, plastic, and / or glass.

[0206] In embodiments, unless otherwise noted herein, a facility may also include any suitable unit operations and / or equipment not otherwise mentioned, such as operations and / or equipment for separation, purification, and isolation of such products. Any suitable facility and environment can be used, such as traditional field-assembled facilities, modular, mobile, and temporary facilities, or any other suitable structure, facility, and / or layout. For example, in some embodiments, a modular cleanroom can be used. Furthermore, unless otherwise noted, the devices, systems, and methods described herein can be housed and / or performed in a single location or facility, or alternatively, can be housed and / or performed in separate or multiple locations and / or facilities.

[0207] By way of non-limiting example, exemplary equipment, devices and / or systems that may be suitable are described in U.S. Patent Application Publication Nos. 2013 / 0280797, 2012 / 0077429, 2011 / 0280797, 2009 / 0305626, and U.S. Patent Nos. 8,298,054, 7,629,167, and 5,656,491, which are incorporated by reference in their entireties.

[0208] Exemplary Sequences Exemplary guide RNA target sequences in HCMV promoters and introns (PAM sequence underlined) All 5' to 3'

[0209] gRNA 1 TGTCAACATGGCGGTAATGT TGG (SEQ ID NO: 1)

[0210] gRNA 2 TACCGCCCATTTGCGTCAAT GGG (SEQ ID NO: 2)

[0211] gRNA 3 CTACCGCCCATTTGCGTCAA TGG (SEQ ID NO: 3)

[0212] gRNA14 ACCGTTAACAGCACCGCAAC GGG (SEQ ID NO: 4)

[0213] Sequence 5 - hCMV-MIE region targeted by gRNA >pEE12.4 (5421bp~7528bp, direct) 2108bp

[0214] Sequence 6-U6 promoter TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAG ATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACC (SEQ ID NO: 6)

[0215] Sequence 7-Grp78 promoter >GA_Grp78_dCas9_Ub_Puro_U6_RGR_2+1+3(15bp~1508bp, direct)1494bp

[0216] HRE consensus sequence 1: nGAAnnTTCnnGAA (SEQ ID NO: 8)

[0217] HRE consensus sequence 2: nGAAnnGAAnnTTCn (SEQ ID NO: 9)

[0218] HRE consensus sequence 3: nGAAnnGAAnnGAAn (SEQ ID NO: 10)

[0219] HRE consensus sequence 4: nTTCnnGAAnnGAAn (SEQ ID NO: 11)

[0220] CRE consensus sequence: TGACGTCA (SEQ ID NO: 12)

[0221] ARE consensus sequence: TGAG / CnnnGC (SEQ ID NO: 13)

[0222] ERSE consensus sequence: CCAAT(N9)CCACG (SEQ ID NO: 14)

[0223] Wild-type S. pyogenes Cas9: MDKKYSIGLD IGTNSVGWAV ITDEYKVPSK KFKVLGNTDRHSIKKNLIGA LLFDSGETAE ATRLKRTARR RYTRRKNRIC YLQEIFSNEM AKVDDSFFHRLEESFLVEED KKHERHPIFG NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAHMIKFRGHFLI EGDLNPDNSD VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSRRLENLIAQLP GEKKNGLFGN LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLAQIGDQYADLF LAAKNLSDAI LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVRQQLPEKYKEI FFDQSKNGYA GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLRKQRTFDNGSI PHQIHLGELH AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNSRFAWMTRKSE ETITPWNFEE VVDKGASAQS FIERMTNFDK NLPNEKVLPK HSLLYEYFTVYNELTKVKYV TEGMRKPAFL SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEISGVEDRFNAS LGTYHDLLKI IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYAHLFDDKVMKQ LKRRRYTGWG RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDDSLTFKEDIQK AQVSGQGDSL HEHIANLAGS PAIKKGILQT VKVVDELVKV MGRHKPENIVIEMARENQTT QKGQKNSRER MKRIEEGIKE LGSQILKEHP VENTQLQNEK LYLYYLQNGRDMYVDQELDI NRLSDYDVDH IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMKNYWRQLLNAK LITQRKFDNL TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMNTKYDENDKLI REVKVITLKS KLVSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKKYPKLESEFVY GDYKVYDVRK MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEIRKRPLIETNGETG EIVWDKGRDF ATVRKVLSMP QVNIVKKTEV QTGGFSKESI LPKRNSDKLIARKKDWDPKK YGGFDSPTVA YSVLVVAKVE KGKSKKLKSV KELLGITIME RSSFEKNPIDFLEAKGYKEV KKDLIIKLPK YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLASHYEKLKGSPE DNEQKQLFVE QHKHYLDEII EQISEFSKRV ILADANLDKV LSAYNKHRDKPIREQAENII HLFTLTNLGA PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRIDLSQLGGD(SEQ ID NO: 15)

[0224] dCas9: MDKKYSIGL A IGTNSVGWAV ITDEYKVPSKKFKVLGNTDR HSIKKNLIGA LLFDSGETAE ATRLKRTARR RYTRRKNRIC YLQEIFSNEM AKVDDSFFHRLEESFLVEED KKHERHPIFG NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAHMIKFRGHFLI EGDLNPDNSD VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSRRLENLIAQLP GEKKNGLFGN LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLAQIGDQYADLF LAAKNLSDAI LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVRQQLPEKYKEI FFDQSKNGYA GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLRKQRTFDNGSI PHQIHLGELH AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNSRFAWMTRKSE ETITPWNFEE VVDKGASAQS FIERMTNFDK NLPNEKVLPK HSLLYEYFTVYNELTKVKYV TEGMRKPAFL SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEISGVEDRFNAS LGTYHDLLKI IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYAHLFDDKVMKQ LKRRRYTGWG RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDDSLTFKEDIQK AQVSGQGDSL HEHIANLAGS PAIKKGILQT VKVVDELVKV MGRHKPENIVIEMARENQTT QKGQKNSRER MKRIEEGIKE LGSQILKEHP VENTQLQNEK LYLYYLQNGRDMYVDQELDI NRLSDYDVD A IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMKNYWRQLLNAK LITQRKFDNL TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMNTKYDENDKLI REVKVITLKS KLVSDRFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKKYPKLESEFVY GDYKVYDVRK MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEIRKRPLIETNGETG EIVWDKGRDF ATVRKVLSMP QVNIVKKTEV QTGGFSKESI LPKRNSDKLIARKKDWDPKK YGGFDSPTVA YSVLVVAKVE KGKSKKLKSV KELLGITIME RSSFEKNPIDFLEAKGYKEV KKDLIIKLPK YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLASHYEKLKGSPE DNEQKQLFVE QHKHYLDEII EQISEFSKRV ILADANLDKV LSAYNKHRDKPIREQAENII HLFTLTNLGA PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRIDLSQLGGD (SEQ ID NO: 16)

[0225] GAAGTTACTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC (SEQ ID NO: 17)

[0226] GAAGTTACTATTCCGAAGTTCCTATTCTCTAGATAGTATAGGAACTTC (SEQ ID NO: 18)

[0227] GAAGTTACTATTCCGAAGTTCCTATTCTCTACTTAGTATAGGAACTTC (SEQ ID NO: 19)

[0228] Numbered Embodiments 1. A first control element operably linked to a sequence encoding an exogenous therapeutic polypeptide; a second control element operably linked to the sequence encoding the repressor polypeptide; Optionally, a third control element operably linked to the sequence encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) gRNAs; and A cell comprising: i) the second control element has a first level of activity under a first condition and a second level of activity under a second condition; or ii) the third control element has a first level of activity under a first condition and a second level of activity under a second condition; The expression of the therapeutic polypeptide is regulated in the presence of a second condition, the cell.

[0229] 2. a first control element operably linked to the insertion site; a second control element operably linked to the sequence encoding the repressor polypeptide; Optionally, a third control element operably linked to the sequence encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) gRNAs; and A cell comprising: i) the second control element has a first level of activity under a first condition and a second level of activity under a second condition; or ii) the third control element has a first level of activity under a first condition and a second level of activity under a second condition; the insertion site is suitable for insertion of a sequence encoding an exogenous therapeutic polypeptide; The expression of the therapeutic polypeptide is regulated in the presence of a second condition, the cell.

[0230] 3. The cell of paragraph 1 or 2, wherein the modulation is reversible.

[0231] 4. The cell of paragraph 1 or 2, wherein the modulation is irreversible.

[0232] 5. The cell of any preceding paragraph, wherein the second control element has an Nth level of activity under an Nth condition, where N is 3, 4, 5, 6, 7, 8, 9, or 10, and wherein in the presence of the Nth condition, expression of the therapeutic polypeptide is modulated relative to expression of the therapeutic polypeptide under the preceding conditions.

[0233] 6. The cell of any preceding paragraph, wherein the third control element has an Nth level of activity under an Nth condition, where N is 3, 4, 5, 6, 7, 8, 9, or 10, and wherein in the presence of the Nth condition, expression of the therapeutic polypeptide is modulated relative to expression of the therapeutic polypeptide under the preceding conditions.

[0234] 7. The cell of any preceding paragraph, wherein the first control element and the sequence encoding the exogenous therapeutic polypeptide are located in a first nucleic acid, and the second control element and the sequence encoding the repressor polypeptide are located in a second nucleic acid.

[0235] 8. The cell of paragraph 7, wherein the sequence encoding the third regulatory element and one or more gRNAs is located in the first nucleic acid.

[0236] 9. The cell of paragraph 7, wherein the sequence encoding the third regulatory element and the one or more gRNAs is located on the second nucleic acid.

[0237] 10. The cell of paragraph 7, wherein the sequence encoding the third regulatory element and the one or more gRNAs is located on a third nucleic acid.

[0238] 11. The cell of any of paragraphs 1 to 6, wherein the first control element, the sequence encoding the exogenous therapeutic polypeptide, the second control element, and the sequence encoding the repressor polypeptide are located on the same nucleic acid.

[0239] 12. The cell of paragraph 11, wherein the sequence encoding the third control element and one or more gRNAs is located on the same nucleic acid as the first control element, the sequence encoding the exogenous therapeutic polypeptide, the second control element, and the sequence encoding the repressor polypeptide.

[0240] 13. The cell of paragraph 11, wherein the sequence encoding the third control element and one or more gRNAs is located on a nucleic acid that is separate from the first control element, the sequence encoding the exogenous therapeutic polypeptide, the second control element, and the sequence encoding the repressor polypeptide.

[0241] 14. The cell of any of paragraphs 7 to 13, wherein the nucleic acid(s) are contained within a vector, e.g., a plasmid, suitable for stable expression.

[0242] 15. The cell of any of paragraphs 7 to 13, wherein the one or more nucleic acids are contained within a vector suitable for transient expression.

[0243] 16. The cell of paragraph 14 or 15, wherein the one or more nucleic acids are contained within the same vector.

[0244] 17. The cell of paragraph 14 or 15, wherein each nucleic acid is contained in a different vector.

[0245] 18. The cell of paragraphs 7 to 13, wherein the one or more nucleic acids are contained within a single chromosome.

[0246] 19. The cell of any of paragraphs 7 to 13, wherein each nucleic acid is contained within a different chromosome.

[0247] 20. The cell of any of paragraphs 7-10, wherein the first nucleic acid is contained in a vector and the second nucleic acid is contained in a chromosome.

[0248] 21. The cell of any of paragraphs 7-10, wherein the first nucleic acid is contained within a chromosome and the second nucleic acid is contained within a vector.

[0249] 22. The cell of paragraph 10, wherein the first nucleic acid is contained in a vector, the second nucleic acid is contained in a chromosome, and the third nucleic acid is contained in a vector.

[0250] 23. The cell of paragraph 10, wherein the first nucleic acid is comprised in a chromosome, the second nucleic acid is comprised in a vector, and the third nucleic acid is comprised in a vector.

[0251] 24. The cell of paragraph 10, wherein the first nucleic acid is comprised in a vector, the second nucleic acid is comprised in a chromosome, and the third nucleic acid is comprised in a chromosome.

[0252] 25. The cell of paragraph 10, wherein the first nucleic acid is comprised in a chromosome, the second nucleic acid is comprised in a vector, and the third nucleic acid is comprised in a chromosome.

[0253] 26. The cell of any preceding paragraph, wherein the stress response induces expression of a repressor polypeptide from the second control element or the third control element.

[0254] 27. The cell of any preceding paragraph, wherein the repressor polypeptide inhibits expression of the therapeutic polypeptide.

[0255] 28. The cell of any preceding paragraph, wherein the first control element is responsive to a repressor polypeptide.

[0256] 29. The first control element comprises a first promoter element, wherein the first promoter element, in the absence of a repressor polypeptide, has the following properties: a) It is constitutive, b) regulated, or c) having a first level of expression during a first stage of cell growth and a second level of expression during a second stage of growth; The cell of any preceding paragraph having one of:

[0257] 30. The cell of any preceding paragraph, wherein the first promoter element is constitutive in the absence of the repressor polypeptide.

[0258] 31. The cell of any preceding paragraph, wherein the first promoter element is selected from Table 5.

[0259] 32. The therapeutic polypeptide is fusion proteins, multi-domain polypeptides, bispecific antibody molecules, multispecific antibody molecules, Multispecific molecules and Molecules including ligand and antibody molecules The cells of any preceding paragraph, including:

[0260] 33. The cell of any preceding paragraph, wherein the therapeutic polypeptide is selected from Tables 1-4.

[0261] 34. The cell of any preceding paragraph, wherein the second control element or the third control element comprises a promoter comprising a sequence selected from Table 5 or Table 6, or having 0, 1, 2, or 3 base substitutions compared to a sequence selected from Table 5 or Table 6.

[0262] 35. The cell of paragraph 34, wherein the third control element is selected from Table 6 and the second control element is selected from Table 5.

[0263] 36. The cell of paragraph 34, wherein the third control element is selected from Table 5 and the second control element is selected from Table 6.

[0264] 37. The cell of any of paragraphs 1-34, wherein the second control element comprises a second promoter element, the second promoter element being constitutive, and the third control element comprises a third promoter element having a first level of activity under first conditions and a second level of activity under second conditions.

[0265] 38. The cell of any of paragraphs 1-34, wherein the second control element comprises a second promoter element having a first level of activity under first conditions and a second level of activity under second conditions, and the third control element comprises a third promoter element, wherein the third promoter element is constitutive.

[0266] 39. The cell of any of paragraphs 1 to 38, wherein the second control element or the third control element comprises one or more of a heat shock element (HSE), a cAMP response element (CRE), an antioxidant response element (ARE), or an endoplasmic reticulum response element (ERSE).

[0267] 40. The cell of any of paragraphs 1-38, wherein the second control element or the third control element is regulated by an element of the heat shock response or the unfolded protein response (UPR).

[0268] 41. The cell of any of paragraphs 1-38, wherein the second control element or the third control element is regulated by the accumulation of misfolded proteins.

[0269] 42. The cell of any of paragraphs 1 to 38, wherein the second control element or the third control element comprises an Xbp1-responsive promoter element.

[0270] 43. The cell of any of paragraphs 1 to 38, wherein the second control element or the third control element comprises a Grp78 promoter element.

[0271] 44. The cell of any of paragraphs 1 to 38, wherein the second control element or the third control element comprises an ATF6-responsive promoter element, an ATF4-responsive promoter element, an NRF2-responsive promoter element, or an Hsf1-responsive promoter element.

[0272] 45. The cell of any preceding paragraph, wherein the repressor polypeptide results in a reduction in the activity, level, or expression of the exogenous therapeutic polypeptide.

[0273] 46. ​​The cell of any preceding paragraph, wherein the repressor polypeptide specifically binds to a target nucleic acid sequence.

[0274] 47. The cell of any of paragraphs 1-46, wherein the repressor polypeptide specifically binds to the regulatory element.

[0275] 48. The cell of any of paragraphs 1-46, wherein the repressor polypeptide specifically binds to the promoter.

[0276] 49. The cell of any preceding paragraph, wherein the repressor polypeptide results in reduced transcription of the exogenous therapeutic polypeptide.

[0277] 50. The cell of any preceding paragraph, wherein the repressor polypeptide binds to a nucleic acid encoding an exogenous therapeutic polypeptide or to a first promoter operably linked to a nucleic acid encoding an exogenous therapeutic polypeptide.

[0278] 51. The cell of any preceding paragraph, wherein the repressor polypeptide reduces translation of the exogenous therapeutic polypeptide.

[0279] 52. The cell of any preceding paragraph, wherein the repressor polypeptide comprises a Cas9 molecule.

[0280] 53. The cell of any preceding paragraph, wherein the repressor polypeptide comprises a Cas9 molecule that has modified cleavage activity compared to naturally occurring Cas9.

[0281] 54. The cell of any preceding paragraph, wherein the repressor polypeptide comprises a Cas9 molecule that lacks cleavage activity in one or both of the HNH and RuvC domains.

[0282] 55. The cell of any preceding paragraph, wherein the repressor polypeptide comprises a dCas9 molecule.

[0283] 56. The cell of any preceding paragraph, wherein the repressor polypeptide comprises a Cas9 molecule further comprising a heterologous repressor domain that enhances repression of the exogenous therapeutic polypeptide.

[0284] 57. The cell of paragraph 56, wherein the heterologous repressor domain is selected from the group consisting of the KRAB (Krüppel-associated box) domain of Kox1, the CS (chromoshadow) domain of HP1α, the WPRW domain of Hes1, and four concatenated copies of the mSin3 interacting domain (SID4X).

[0285] 58. The cell of any of paragraphs 52 to 57, wherein the Cas9 molecule, when complexed with the gRNA, binds to the target nucleic acid in a sequence-specific manner.

[0286] 59. The cell of any of paragraphs 52 to 58, wherein the Cas9 molecule, when complexed with the gRNA, binds to the untranslated sequence.

[0287] 60. The cell of any of paragraphs 52-59, wherein the Cas9 molecule:gRNA complex binds to a first control element.

[0288] 61. The cell of any of paragraphs 52 to 60, wherein the Cas9 molecule:gRNA complex binds to a sequence encoding an exogenous therapeutic polypeptide.

[0289] 62. The cell of any preceding paragraph, wherein the cell further comprises an Nth sequence encoding an Nth gRNA operably linked to a third control element, where N is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0290] 63. The cell of any preceding paragraph, wherein the third control element is a further copy of the second control element.

[0291] 64. The cell of any of paragraphs 1-62, wherein the third control element is a further copy of the first control element.

[0292] 65. The cell of any of paragraphs 1 to 63, wherein the third control element has a first level of activity under a first condition and a second level of activity under a second condition, and in the presence of the second condition, expression of the gRNA is regulated.

[0293] 66. The third control element has the following characteristics: a) It is constitutive, b) regulated, or c) having a first level of expression during a first stage of cell growth and a second level of expression during a second stage of growth; The cell of any preceding paragraph having one of:

[0294] 67. The cell of any preceding paragraph, wherein the second level of activity is greater than the first level of activity.

[0295] 68. The cell of any preceding paragraph, wherein the first condition is a first level of stress and the second condition is a second level of stress.

[0296] 69. The cell of any preceding paragraph, wherein the first condition is a first level of unfolded or misfolded polypeptide and the second condition is a second level of unfolded or misfolded polypeptide.

[0297] 70. The cell of any preceding paragraph, wherein the first condition is a first level of folded exogenous therapeutic polypeptide and the second condition is a second level of folded exogenous therapeutic polypeptide.

[0298] 71. The cell of any preceding paragraph, wherein the first condition is a first level of unfolded or misfolded polypeptide in the cytosol, and the second condition is a second level of unfolded or misfolded polypeptide in the cytosol.

[0299] 72. The cell of any preceding paragraph, wherein the first condition is a first level of unfolded or misfolded polypeptides in the endoplasmic reticulum (ER), and the second condition is a second level of unfolded or misfolded polypeptides in the ER.

[0300] 73. The first condition / second condition pair is a first level of protein aggregation and a second level of protein aggregation; a first glycosylation pattern at a first level on an exogenous therapeutic polypeptide and a first glycosylation pattern at a second level on an exogenous therapeutic polypeptide; a first glycosylation pattern at a level on the exogenous therapeutic polypeptide and a second glycosylation pattern at a level on the exogenous therapeutic polypeptide; a first level of cell viability and a second level of cell viability; Activation of the heat shock response (HSR) at a first level and activation of the HSR at a second level, Activation of the first level of the unfolded protein response (UPR) and activation of the second level of the UPR; a first level free ER chaperone and a second level free ER chaperone; a first temperature and a second temperature; a first level of oxidative stress and a second level of oxidative stress; First level ER Ca +2 and second-level ER Ca +2 , a first ER oxidation state and a second ER oxidation state; a first cellular energy level and a second cellular energy level; a first ATP level and a second ATP level; a first glucose level and a second glucose level; a first level of activated Hsf1 polypeptide and a second level of activated Hsf1 polypeptide; a first level of phosphorylated trimeric Hsf1 polypeptide and a second level of phosphorylated trimeric Hsf1 polypeptide; a first level of active Xbp1 polypeptide and a second level of activated Xbp1 polypeptide; a first level ATF4 polypeptide and a second level ATF4 polypeptide; a first level NRF2 polypeptide and a second level NRF2 polypeptide, and First level ATF6 polypeptide and second level ATF6 polypeptide The cell of any preceding paragraph selected from the group consisting of:

[0301] 74. The cell of any preceding paragraph, wherein the stress response induces expression of a repressor polypeptide, and the repressor polypeptide inhibits expression of the therapeutic polypeptide.

[0302] 75. The cell of any preceding paragraph, wherein, in the second condition, expression of the exogenous therapeutic polypeptide is reduced by at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to expression in the first condition.

[0303] 76. A kit for expression of a therapeutic polypeptide comprising the cells of any preceding paragraph.

[0304] 77. A first control element operably linked to a sequence encoding an exogenous therapeutic polypeptide; a second control element operably linked to the sequence encoding the repressor polypeptide; Optionally, a third control element operably linked to the sequence encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) gRNAs; and A nucleic acid comprising: i) the second control element has a first level of activity under a first condition and a second level of activity under a second condition; or ii) the third control element has a first level of activity under a first condition and a second level of activity under a second condition; The nucleic acid, wherein expression of the therapeutic polypeptide is regulated in the presence of a second condition.

[0305] 78. A first control element operably linked to the insertion site; a second control element operably linked to the sequence encoding the repressor polypeptide; Optionally, a third control element operably linked to the sequence encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) gRNAs; and A nucleic acid comprising: i) the second control element has a first level of activity under a first condition and a second level of activity under a second condition; or ii) the third control element has a first level of activity under a first condition and a second level of activity under a second condition; the insertion site is suitable for insertion of a sequence encoding an exogenous therapeutic polypeptide; The nucleic acid, wherein expression of the therapeutic polypeptide is regulated in the presence of a second condition.

[0306] 79. The nucleic acid of paragraph 77 or 78, wherein the first control element and the sequence encoding the exogenous therapeutic polypeptide are contained in a first nucleic acid and a second control element, and the sequence encoding the repressor polypeptide is contained in a second nucleic acid.

[0307] 80. The nucleic acid of paragraph 79, wherein the sequence encoding the third regulatory element and one or more gRNAs is located in the first nucleic acid.

[0308] 81. The nucleic acid of paragraph 79, wherein the sequence encoding the third regulatory element and one or more gRNAs is located in the second nucleic acid.

[0309] 82. The nucleic acid of paragraph 79, wherein the sequence encoding the third regulatory element and one or more gRNAs is located in a third nucleic acid.

[0310] 83. The nucleic acid of paragraph 77 or 78, wherein the first control element, the sequence encoding the exogenous therapeutic polypeptide, the second control element, and the sequence encoding the repressor polypeptide are contained in the same nucleic acid.

[0311] 84. The nucleic acid of paragraph 83, wherein the sequence encoding the third control element and one or more gRNAs is located on the same nucleic acid as the first control element, the sequence encoding the exogenous therapeutic polypeptide, the second control element, and the sequence encoding the repressor polypeptide.

[0312] 85. The nucleic acid of paragraph 83, wherein the sequence encoding the third control element and one or more gRNAs is located on a nucleic acid separate from the first control element, the sequence encoding the exogenous therapeutic polypeptide, the second control element, and the sequence encoding the repressor polypeptide.

[0313] 86. The nucleic acid of any of paragraphs 79 to 85, wherein the one or more nucleic acids are contained within a vector suitable for stable expression.

[0314] 87. The nucleic acid of any of paragraphs 79 to 85, wherein the nucleic acid(s) is / are contained within a vector suitable for transient expression.

[0315] 88. The nucleic acid of paragraph 86 or 87, wherein one or more nucleic acids are contained within the same vector.

[0316] 89. The nucleic acid of paragraph 86 or 87, wherein each nucleic acid is contained in a different vector.

[0317] 90. The nucleic acid of any of paragraphs 79 to 85, wherein the one or more nucleic acids are contained within a single chromosome.

[0318] 91. The nucleic acid of any of paragraphs 79-85, wherein each nucleic acid is contained within a different chromosome.

[0319] 92. The nucleic acid of any of paragraphs 79-82, wherein the first nucleic acid is contained in a vector and the second nucleic acid is contained in a chromosome.

[0320] 93. The nucleic acid of any of paragraphs 79-82, wherein the first nucleic acid is contained within a chromosome and the second nucleic acid is contained within a vector.

[0321] 94. The nucleic acid of paragraph 82, wherein the first nucleic acid is comprised in a vector, the second nucleic acid is comprised in a chromosome, and the third nucleic acid is comprised in a vector.

[0322] 95. The nucleic acid of paragraph 82, wherein the first nucleic acid is comprised in a chromosome, the second nucleic acid is comprised in a vector, and the third nucleic acid is comprised in a vector.

[0323] 96. The nucleic acid of paragraph 82, wherein the first nucleic acid is comprised in a vector, the second nucleic acid is comprised in a chromosome, and the third nucleic acid is comprised in a chromosome.

[0324] 97. The nucleic acid of paragraph 82, wherein the first nucleic acid is comprised in a chromosome, the second nucleic acid is comprised in a vector, and the third nucleic acid is comprised in a chromosome.

[0325] 98. A kit for expressing a therapeutic polypeptide comprising a nucleic acid of any of paragraphs 77 to 97.

[0326] 99. A method for producing a cell according to any one of paragraphs 1 to 75, comprising: a) forming or providing in a cell a first nucleic acid sequence encoding a first control element operably linked to a sequence encoding an exogenous therapeutic polypeptide; b) forming or providing in the cell a second nucleic acid encoding a second control element operably linked to a sequence encoding a repressor polypeptide; c) optionally forming or providing in the cell a third nucleic acid encoding a third control element operably linked to a sequence encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) gRNAs; Including, i) the second control element has a first level of activity under a first condition and a second level of activity under a second condition; or ii) the third control element has a first level of activity under a first condition and a second level of activity under a second condition; expression of the therapeutic polypeptide is regulated in the presence of a second condition; This method produces cells.

[0327] 100. The method of paragraph 99, wherein the step of forming or providing the first nucleic acid sequence in the cell comprises introducing the first nucleic acid sequence into the cell.

[0328] 101. The method of paragraph 100, wherein introducing the first nucleic acid sequence into the cell comprises a technique selected from transient transfecting, stable transfecting, transduction, and transformation.

[0329] 102. The method of any of paragraphs 99-101, wherein the step of forming or providing a second nucleic acid sequence in the cell comprises introducing the second nucleic acid sequence into the cell.

[0330] 103. The method of paragraph 102, wherein introducing the second nucleic acid sequence into the cell comprises a technique selected from transient transfecting, stable transfecting, transduction, and transformation.

[0331] 104. The method of any of paragraphs 99-102, wherein the step of forming or providing a third nucleic acid sequence in the cell comprises introducing the third nucleic acid sequence into the cell.

[0332] 105. The method of paragraph 104, wherein introducing the third nucleic acid sequence into the cell comprises a technique selected from transient transfecting, stable transfecting, transduction, and transformation.

[0333] 106. The method of paragraph 99, wherein (a), (b) and optionally (c) comprise simultaneously introducing the first, second and third nucleic acids into the cell.

[0334] 107. The method of paragraph 99 in which (a), (b) and optionally (c) occur sequentially.

[0335] 108. The method of paragraph 99, wherein the step of forming or providing a first nucleic acid sequence in the cell comprises inserting a sequence encoding an exogenous therapeutic polypeptide into a suitable insertion site in the cell, the insertion site being operably linked to a first control element.

[0336] 109. The method of paragraph 99, wherein the step of forming or providing a second nucleic acid sequence in the cell comprises inserting a sequence encoding a repressor polypeptide into a suitable insertion site in the cell, the sequence being operably linked to a second control element.

[0337] 110. The method of paragraph 99, wherein the step of forming or providing a third nucleic acid sequence in the cell comprises inserting, into a suitable insertion site in the cell, a sequence encoding one or more gRNAs operably linked to a third control element.

[0338] 111. A method for producing a therapeutic polypeptide, comprising: a) obtaining a cell according to any of paragraphs 1 to 75; b) culturing the cells under conditions that allow for the production of a therapeutic polypeptide; Including, thereby producing a therapeutic polypeptide.

[0339] 112. A method for producing a therapeutic polypeptide, comprising: a) obtaining cells; b) forming or providing in the cell a first nucleic acid sequence encoding a first control element operably linked to a sequence encoding an exogenous therapeutic polypeptide; c) forming or providing in the cell a second nucleic acid encoding a second control element operably linked to a sequence encoding a repressor polypeptide; d) optionally forming or providing in the cell a third nucleic acid encoding a third control element operably linked to a sequence encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) gRNAs, i) the second control element has a first level of activity under a first condition and a second level of activity under a second condition; or ii) the third control element has a first level of activity under a first condition and a second level of activity under a second condition; expression of the therapeutic polypeptide is regulated in the presence of a second condition; e) culturing the cells under conditions that allow for the production of a therapeutic polypeptide; thereby producing a therapeutic polypeptide.

[0340] 113. Any cell in paragraphs 1 to 75 Culture medium and A reaction mixture comprising: A reaction mixture, wherein the culture medium is suitable for expressing a therapeutic polypeptide.

[0341] 114. A first control element operably linked to a sequence encoding an exogenous therapeutic polypeptide; a second control element operably linked to the sequence encoding the repressor polypeptide; Optionally, a third control element operably linked to the sequence encoding one or more gRNAs. A genetic control circuit comprising: i) the second control element has a first level of activity under a first condition and a second level of activity under a second condition; or ii) the third control element has a first level of activity under a first condition and a second level of activity under a second condition; A genetic regulatory circuit, wherein expression of the therapeutic polypeptide is regulated in the presence of a second condition.

[0342] 115. The genetic control circuit of paragraph 114, wherein the regulation is reversible.

[0343] 116. The genetic regulatory circuit of paragraph 114 or 115, wherein the stress response induces expression of a repressor polypeptide from the second regulatory element.

[0344] 117. The genetic control circuit of any of paragraphs 114-116, wherein the repressor polypeptide inhibits expression of the therapeutic polypeptide.

[0345] 118. The genetic control circuit of any of paragraphs 114-117, wherein the first control element is responsive to a repressor polypeptide.

[0346] 119. The therapeutic polypeptide is fusion proteins, multi-domain polypeptides, bispecific antibody molecules, multispecific antibody molecules, multispecific molecules, and Molecules including ligand and antibody molecules The genetic control circuit of any of paragraphs 114 to 118, comprising:

[0347] 120. The genetic control circuit of any of paragraphs 114-119, wherein the therapeutic polypeptide is selected from Tables 1-4.

[0348] 121. The genetic control circuit of any of paragraphs 114-120, wherein the second control element or the third control element is selected from Table 5 or Table 6.

[0349] 122. The genetic control circuit of any of paragraphs 114-120, wherein the third control element is selected from Table 6 and the second control element is selected from Table 5.

[0350] 123. The genetic control circuit of any of paragraphs 114-120, wherein the third control element is selected from Table 5 and the second control element is selected from Table 6.

[0351] 124. The genetic control circuit of any of paragraphs 114-120, wherein the second control element comprises a second promoter element, wherein the second promoter element is constitutive, and the third control element comprises a third promoter element having a first level of activity under first conditions and a second level of activity under second conditions.

[0352] 125. The genetic control circuit of any of paragraphs 114-120, wherein the second control element comprises a second promoter element having a first level of activity under first conditions and a second level of activity under second conditions, and the third control element comprises a third promoter element, wherein the third promoter element is constitutive.

[0353] 126. The genetic control circuit of any of paragraphs 114-121, wherein the third control element is a copy of the first control element.

[0354] 127. The genetic control circuit of any of paragraphs 114-121, wherein the third control element is a copy of the second control element.

[0355] 128. The genetic regulatory circuit of any of paragraphs 114-127, wherein the repressor polypeptide results in a reduction in the activity, level, or expression of the exogenous therapeutic polypeptide.

[0356] 129. A first control element selected from Table 5 operably linked to a sequence encoding an exogenous therapeutic polypeptide selected from Tables 1-4; a second regulatory element selected from Table 6 operably linked to a sequence encoding a Cas9 polypeptide; and one or more constitutively expressed gRNA sequences; A cell comprising: The second control element has a first level of activity under a first condition and a second level of activity under a second condition, and expression of the therapeutic polypeptide is regulated in the presence of the second condition.

[0357] 130. A first control element selected from Table 5 operably linked to a sequence encoding an exogenous therapeutic polypeptide selected from Tables 1-4; a second regulatory element selected from Table 5 operably linked to a sequence encoding a Cas9 polypeptide; and a third regulatory element operably linked to one or more gRNA sequences selected from Table 6; A cell comprising: The third control element has a first level of activity under a first condition and a second level of activity under a second condition, and expression of the therapeutic polypeptide is regulated in the presence of the second condition.

[0358] 131. The plurality of cells of any one of paragraphs 1-75, 129 or 130, wherein one or more cells comprise a first condition and one or more cells comprise a second condition.

[0359] 132. The cell, method, nucleic acid, or genetic control circuit of any of paragraphs 1-30, 32-75, 77-97, 99-112, and 114-128, wherein the first control element is an engineered promoter.

[0360] 133. The cell, method, nucleic acid, or genetic control circuit of any of paragraphs 1-62, 65-75, 77-97, 99-112, 114-121, 124-126, and 128, wherein the third control element is an engineered promoter. [Example]

[0361] Example Example 1 The following example utilizes the design principles of the genetic control circuit depicted in Figures 1A and 1B. In this example, the principle of using a repressor (i.e., dCas9) to repress expression of a recombinant protein gene (i.e., GFP) was tested using the circuit depicted in Figure 3A. Here, a CHOK1SV-derived GS-KO (Xceed™) cell line stably expressing a recombinant polypeptide gene, GFP, operably linked to a first control element, e.g., a first promoter element, hCMV, was transiently transfected with either 1) an expression vector encoding the repressor polypeptide, dCas9, operably linked to a constitutive mCMV promoter alone, or 2) an expression vector encoding the repressor polypeptide (dCas9) and vectors expressing gRNAs 1, 2, and 3, each controlled from a separate U6 promoter (Figure 3A). Four days after transfection, GFP fluorescence was determined by flow cytometry, and it was observed that transfection with dCas9 plus gRNAs 1-3 resulted in a decrease in population GFP fluorescence compared to cells transfected with dCas9 alone or untransfected control cells (UTC) (Figure 3B). This demonstrates the suppression of recombinant polypeptide (GFP) expression using the repressor polypeptide, dCas9, and gRNAs.

[0362] Example 2 In this example, the principle of using a repressor (i.e., dCas9) to suppress expression of recombinant monoclonal antibody heavy chain (HC) and light chain (LC) genes was tested using the circuit depicted in Figure 4A. In this example, we demonstrate the inhibition of expression of a therapeutic polypeptide, IgG4 Mab cB72.3, operably linked to a first control element, e.g., promoter element, hCMV, by a repressor polypeptide operably linked to the constitutive mCMV promoter. Using a CHO cell line pool stably expressing the IgG4 Mab cB72.3 HC and LC genes, each driven by a separate hCMV promoter, we tested the ability to downregulate Mab expression using dCas9 and gRNAs 1-3 targeting the hCMV promoter. Pools were transiently transfected with either dCas9 plasmid alone (dCas9) or dCas9 plasmid + / - gRNA-encoding plasmid (dCas9+g1-g3, see Figure 4B), and Mab concentrations were examined 3, 4, and 5 days after transfection (Figure 4B). Error bars represent the standard deviation across triplicate transfections. Cells were also transfected with buffer alone as a negative control (no DNA). This demonstrates the suppression of Mab expression using dCas9 and gRNA.

[0363] Example 3 In this example, we demonstrated inhibition of expression of a recombinant polypeptide, GFP, using the genetic control circuit depicted in Figure 5A. CHOK1SV-derived GS-KO (Xceed™) cells stably expressing GFP operably linked to a first control element, e.g., a promoter element, hCMV, were transiently transfected with a set of plasmids: a second control element, e.g., a promoter element, a sequence encoding a repressor polypeptide operably linked to the Grp78 promoter, a plasmid containing dCas9, and a plasmid encoding expression of gRNAs 1-3, each under the control of a separate U6 promoter. The Grp78 promoter is activated by the unfolded protein response, which in this case was artificially activated by the addition of tunicamycin™ 24 hours after transfection. Four days after transient transfection, GFP output was measured by flow cytometry. It can be seen that in cells transfected with both dCas9 and gRNA plasmids 1-3, GFP output was suppressed after 400 ng / mL tunicamycin (TM) treatment (Grp78 dCas9 + gRNA) compared to cells similarly transfected but not treated with tunicamycin (0 TM) or cells transfected only with dCas9 (Grp78 dCas9 control) (Figure 5).

[0364] Example 4 This example demonstrates the ability of the genetic control circuit depicted in Figure 2 to enhance the production of several recombinant proteins, including proteins that are difficult to express. Using the vector depicted in Figure 6A, CHOK1SV-derived GS-KO (Xceed™) cells stably expressing the genetic control circuit were constructed. This vector contained the dCas9 gene under the Grp78 promoter and three gRNAs (gRNA1, 2, and 3), each with specificity for the hCMV promoter under a separate constitutive U6 promoter. A variant of this vector contained a single gRNA14 sequence instead of the gRNA1, 2, and 3 sequences, and this variant vector was used to construct CHOK1SV-derived GS-KO (Xceed™) cells stably expressing the genetic control circuit as well. The genetic control circuit vector also contained a puromycin resistance gene (puromycin N-acetyltransferase ("puromycin")) under the SV40 promoter, allowing for positive selection of cells that had stably integrated the genetic control circuit after transfection by treatment with the antibiotic puromycin. Stable CHOK1SV-derived GS-KO (Xceed™) pools expressing genetic control circuits with either a single gRNA14 sequence or gRNAs 1, 2, and 3 were then transiently transfected with expression vectors encoding several difficult-to-express recombinant proteins: H1K1 and H9K7 (both highly aggregating MAbs), etanercept (a composite Fc-fusion protein), and blinatumomab (a composite bispecific T-cell engager (BiTE)), as well as the IgG4 MAb cB72.3. The recombinant protein concentrations produced 6 days after transfection, as determined using an Octet Bioanalyzer, are shown in Figure 6B. The results show that for all recombinant proteins, including H9K7, the presence of at least one genetic control circuit was associated with an increased average recombinant protein concentration compared to the parental CHOK1SV-derived GS-KO (Xceed™) cell line lacking the control circuit. This suggests that genetic control circuits can increase productivity for some proteins, including complex, difficult-to-express molecules.To further investigate the ability of genetic regulatory circuits to increase recombinant protein expression, CHO cells stably expressing the circuits and transiently transfected with a vector encoding H9K7 were subjected to an increase in the UPR by adding tunicamycin (TM) (0.1 μg / mL) 24 hours after transfection (Figure 6C). Cells containing the genetic regulatory circuit resulted in an increase in the average concentration of H9K7 6 days after transfection when TM was added, whereas the parental CHO host cell line lacking the regulatory circuit showed no effect. This indicates that genetic regulatory circuits can increase the expression and yield of proteins that are difficult to express exogenously in the presence of an activated UPR. It also indicates that the effects of genetic regulatory circuits are linked to the UPR.

[0365] For the above transfection sets, the level of protein aggregation in the cell culture supernatants was also examined by ODA assay (Obrezanova et al. MAbs. 2015;7(2):352-63) (Figures 7A and 7B). In this assay, cB72.3 is known to exhibit low levels of aggregation, whereas H1K1, H9K7, and etanercept are known to be highly aggregated in the parental cell line CHOK1SV-derived GS-KO (Xceed™), thus exhibiting high absorbance values ​​at 450 nm (Figure 7A). The control circuit was not predicted to show any substantial benefit in reducing the aggregation of H9K7 and etanercept, as these are known to exhibit high-to-severe aggregation and thus may exceed the dynamic range of the circuit's control of this parameter. However, H1K1 is known to exhibit slightly lower, though still high, levels of aggregation by comparison, and may be amenable to improvement (i.e., reduced aggregation). Indeed, in this assay, both variants of the control circuit were associated with reduced mean H1K1 aggregation, despite increased overall product concentrations compared to the parental host cell line (compare Figures 6B and 7A). In the presence of tunicamycin, no improvement (i.e., reduction) in H9K7 aggregation was observed with the gRNA14 and gRNA123 variants using the control circuit, despite increased overall product concentrations (compare Figures 6C with 7B), again suggesting that the aggregation behavior of the H9K7 antibody exceeds the dynamic range of the control circuit's influence.

[0366] In the above transfection set, it may be possible to measure other important product quality (PQ) attributes such as N-glycan micro / macroheterogeneity (e.g., by UPLC or LC-MS), and improvements in PQ are expected in cells containing regulatory circuits.

[0367] [Related Applications] This application claims priority to U.S. Provisional Patent Application No. 62 / 521,005, filed June 16, 2017, the entire contents of which are incorporated herein by reference.

Claims

1. (a) a first control element operably linked to a sequence encoding an exogenous therapeutic polypeptide; (b) a second control element operably linked to the sequence encoding a repressor polypeptide, wherein the repressor polypeptide is a Cas9 molecule, a TALE molecule, or a zinc finger molecule; and optionally (c) a third control element operably linked to a sequence encoding an exogenous therapeutic polypeptide or a sequence encoding one or more gRNAs having homology to the first control element operably linked thereto; A cell comprising: (i) a repressor polypeptide, alone or in combination with the one or more gRNAs, inhibits expression of the exogenous therapeutic polypeptide; (ii) the second control element or the third control element has a first level of activity under a first condition and a second level of activity under a second condition; and (iii) the second control element or the third control element comprises a second promoter element or a third promoter element comprising the sequence of SEQ ID NO: 14, and is an unfolded protein response element (ERSE), is regulated by the unfolded protein response (UPR), or is regulated by the accumulation of misfolded proteins; The first and second conditions are: (i) low levels of unfolded or misfolded polypeptides in the endoplasmic reticulum (ER) and high levels of unfolded or misfolded polypeptides in the ER, respectively; (ii) a low level of activation of the unfolded protein response (UPR) and a high level of activation of the UPR, respectively; or (iii) a low level of protein aggregation and a high level of protein aggregation, respectively; and wherein in the presence of said second condition, a repressor polypeptide, alone or in combination with said one or more gRNAs, inhibits expression of said therapeutic polypeptide.

2. (a) the first control element and a sequence encoding an exogenous therapeutic polypeptide are located on a first nucleic acid, and the second control element and a sequence encoding a repressor polypeptide are located on a second nucleic acid; (i) the third control element and one or more gRNA-encoding sequences, if present, are located in the first nucleic acid; or (ii) the third regulatory element and one or more gRNA-encoding sequences, if present, are located on the second nucleic acid; or (iii) the third regulatory element and one or more gRNA-encoding sequences, if present, are located on a third nucleic acid; or (b) the first control element, the sequence encoding the exogenous therapeutic polypeptide, the second control element, and the sequence encoding the repressor polypeptide are located on the same nucleic acid; (i) if present, the third control element and the sequence encoding one or more gRNAs are located on the same nucleic acid as the first control element, the sequence encoding the exogenous therapeutic polypeptide, the second control element, and the sequence encoding the repressor polypeptide; or (ii) the third control element and the sequence encoding one or more gRNAs, if present, are located on a nucleic acid that is separate from the first control element, the sequence encoding the exogenous therapeutic polypeptide, the second control element, and the sequence encoding the repressor polypeptide.

3. (a) the first nucleic acid is contained in a vector and the second nucleic acid is contained in a chromosome; or (b) the first nucleic acid is contained within a chromosome and the second nucleic acid is contained within a vector; or (c) the first nucleic acid is contained in a vector, the second nucleic acid is contained in a chromosome, and the third nucleic acid is contained in a vector; or (d) the first nucleic acid is contained within a chromosome, the second nucleic acid is contained within a vector, and the third nucleic acid is contained within a vector; or (e) the first nucleic acid is contained in a vector, the second nucleic acid is contained in a chromosome, and the third nucleic acid is contained in a chromosome; or (f) the first nucleic acid is contained within a chromosome, the second nucleic acid is contained within a vector, and the third nucleic acid is contained within a chromosome; The cell of claim 2.

4. The cell of any one of claims 1 to 3, wherein the first control element is responsive to the repressor polypeptide.

5. The cell of any one of claims 1 to 4, wherein the first control element is selected from Table 5.

6. the exogenous therapeutic polypeptide is fusion proteins, multi-domain polypeptides, bispecific antibody molecules, multispecific antibody molecules, multispecific molecules, and / or Molecules including ligand and antibody molecules The cell according to any one of claims 1 to 5, comprising:

7. The cell of any one of claims 1 to 6, comprising the third control element operably linked to a sequence encoding one or more gRNAs.

8. The cell of any one of claims 1 to 7, wherein the second control element or the third control element comprises a Grp78 promoter element.

9. 9. The cell of any one of claims 1 to 8, wherein the repressor polypeptide, alone or in combination with the one or more gRNAs, is capable of binding to the first control element or a sequence encoding the exogenous therapeutic polypeptide.

10. the repressor polypeptide (a) Cas9 molecule, (b) a Cas9 molecule having modified cleavage activity compared to naturally occurring Cas9; (c) a Cas9 molecule lacking cleavage activity in one or both of the HNH and RuvC domains; (d) a dCas9 molecule, or 10. The cell of any one of claims 1 to 9, comprising: (e) a Cas9 molecule further comprising a heterologous repressor domain that enhances repression of the exogenous therapeutic polypeptide, wherein the heterologous repressor domain is selected from the group consisting of the KRAB (Krüppel-associated box) domain of Kox1, the CS (chromoshadow) domain of HP1α, the WPRW domain of Hes1, and four concatenated copies of the mSin3-interacting domain (SID4X).

11. The cell according to any one of claims 1 to 10, which is a mammalian cell line.

12. 1. A kit for generating cells for expressing a therapeutic polypeptide, comprising: (a) a first nucleic acid comprising a first control element operably linked to an insertion site suitable for insertion of an exogenous sequence such that the exogenous sequence encoding a therapeutic polypeptide is operably linked to the first control element; (b) a second nucleic acid comprising a second control element operably linked to a sequence encoding a repressor polypeptide, wherein the repressor polypeptide is a Cas9 molecule, a TALE molecule, or a zinc finger molecule; and optionally (c) a third nucleic acid comprising a third control element operably linked to a sequence encoding a therapeutic polypeptide or a sequence encoding one or more gRNAs having homology to the first control element operably linked thereto; Including, (i) a repressor polypeptide, alone or in combination with the one or more gRNAs, inhibits expression of the therapeutic polypeptide; (ii) the second control element or the third control element has a first level of activity under a first condition and a second level of activity under a second condition; and (iii) the second control element or the third control element comprises a second promoter element or a third promoter element comprising the sequence of SEQ ID NO: 14, is an unfolded protein response element (ERSE), and is regulated by the unfolded protein response (UPR) or by the accumulation of misfolded proteins; The first and second conditions are: (i) low levels of unfolded or misfolded polypeptides in the endoplasmic reticulum (ER) and high levels of unfolded or misfolded polypeptides in the ER, respectively; (ii) a low level of activation of the unfolded protein response (UPR) and a high level of activation of the UPR, respectively; or (iii) a low level of protein aggregation and a high level of protein aggregation, respectively; and In the presence of the second condition, a repressor polypeptide, alone or in combination with the one or more gRNAs, inhibits expression of the therapeutic polypeptide.

13. The kit of claim 12, comprising the third nucleic acid.

14. 14. Use of the kit according to claim 12 or 13 for generating a cell line expressing a therapeutic polypeptide.

15. 1. A method of making a therapeutic polypeptide, comprising: A method comprising culturing the cell of any one of claims 1 to 11 under conditions that allow the production of said therapeutic polypeptide, thereby producing said therapeutic polypeptide.

16. A nucleic acid comprising: (i) a sequence encoding a first control element and an exogenous therapeutic polypeptide; (ii) a sequence encoding a second control element and a repressor polypeptide, wherein the repressor polypeptide is a Cas9 molecule, a TALE molecule, or a zinc finger molecule; and, optionally, (iii) a sequence encoding a third control element and one or more gRNAs. An expression vector comprising: wherein the sequence encoding the one or more gRNAs has homology to the sequence encoding the exogenous therapeutic polypeptide or the first control element operably linked thereto; a repressor polypeptide, alone or in combination with the one or more gRNAs, inhibits expression of the therapeutic polypeptide; the second control element or the third control element has a first level of activity under a first condition and a second level of activity under a second condition; and the second control element or the third control element comprises a second promoter element or a third promoter element comprising the sequence of SEQ ID NO: 14, and is an unfolded protein response element (ERSE), or is regulated by the unfolded protein response (UPR), or is regulated by the accumulation of misfolded proteins; The first and second conditions are: (i) low levels of unfolded or misfolded polypeptides in the endoplasmic reticulum (ER) and high levels of unfolded or misfolded polypeptides in the ER, respectively; (ii) a low level of activation of the unfolded protein response (UPR) and a high level of activation of the UPR, respectively; or (iii) a low level of protein aggregation and a high level of protein aggregation, respectively; and an expression vector wherein, in the presence of said second condition, a repressor polypeptide, alone or in combination with said one or more gRNAs, inhibits expression of said therapeutic polypeptide.

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