Expression regulation using controllable introns

Abstract

The present invention relates to the use of regulatory nucleic acid sequences that can control gene expression in eukaryotic cells and are responsive to the unfolded protein response (UPR). Controllable introns and UPR-inducible promoters that can control gene expression are disclosed. Recombinant expression constructs containing such regulatory nucleic acid sequences, whereby expression of an encoded expression product can be induced by inducing the unfolded protein response (UPR) in eukaryotic cells containing the constructs, methods for using such constructs, and related vectors, cells, and the like are also disclosed.

Description

Technical Field

[0001] The present invention relates to the use of regulatory nucleic acid sequences that can control gene expression in eukaryotic cells and are responsive to the unfolded protein response (UPR). In some embodiments, the present invention relates to the use of controllable introns for regulating gene expression. In other embodiments, the present invention relates to UPR-inducible promoters. The present invention also relates to recombinant expression constructs containing such regulatory nucleic acid sequences, wherein the expression of the encoded expression product can be induced by inducing the unfolded protein response (UPR) in eukaryotic cells containing the construct, as well as methods of using such constructs, and related vectors, cells, etc.

Background Art

[0002] Controllable gene expression is desirable in a great many situations where it is beneficial or necessary to regulate the expression level of an expression product. For example, in the biotechnology industry, it can be highly advantageous to be able to induce the production of an expression product (e.g., a protein) at a desired time in a fermentation process. In another example, in gene therapy, it can be desirable to be able to induce the expression of a therapeutic product (e.g., a therapeutic protein) at a desired time and / or location of treatment.

[0003] Inducible promoters are known in the art, for example, the tetracycline (Tet)-on and -off inducible expression systems (Gossen M and Bujard H. PNAS. June 15, 1992; 89(12):5547-5551; Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, and Bujard H. Science. June 23, 1995; 268(5218)).

[0004] Inducible promoters work by regulating expression at the transcriptional level, thereby regulating the amount of mRNA produced from the associated expression system. This provides a useful level of gene expression, but requires additional, ideally improved, systems to regulate expression.

[0005] In particular, improved expression regulatory systems are needed for toxic proteins, i.e., proteins that are toxic to the cells on which they are produced. In the case of toxic proteins, even small amounts of expression can often cause cell death, or production may be very poor.

[0006] Furthermore, in the case of genes used in cell therapy, a system is needed that allows the expression of therapeutic proteins or RNA, or other expression products, to be induced at the desired time and / or location.

[0007] This invention relates to the regulation of expression that acts at the time of translation. In particular, it relates to the use of introns excised by splicing as a result of unfolding protein responses (UPRs) in eukaryotic cells to regulate expression.

[0008] The unfolding protein response (UPR) is a cellular mechanism for coping with endoplasmic reticulum (ER) stress. UPR is activated in response to the accumulation of unfolded or misfolded proteins in the lumen of the ER. Through various mechanisms, UPR aims to restore normal cellular function. If these objectives are not achieved within a specific time span, or if the damage is prolonged, UPR aims for apoptosis. UPR can be triggered by increased protein synthesis and folding, such as heterologous protein production, or by other cellular stresses, such as chemically induced stresses, including blockage of glycosylation pathways or disulfide bond formation. UPR is highly conserved across all eukaryotes.

[0009] In mammalian cells, there are three mechanisms by which ER stress is sensed and UPR is activated: 1) IRE-1 splicing of XBP-1 mRNA ER stress, resulting from increased protein folding demands or chemical inhibition of the ER process, is detected by the ER transmembrane protein IRE1 via the dissociation of the chaperone BiP. This dissociation of BiP activates IRE1 by enabling protein oligomerization and phosphorylation, thereby exposing the RNase domain facing the ER lumen. Subsequently, the RNase domain catalyzes the removal of non-standard introns from XBP-1 mRNA in a spliceosome-independent manner. Under non-stress conditions, XBP-1 mRNA is not spliced ​​(XBP1u, which, when translated, forms a 261-amino acid ORF that is a non-functional protein). However, when ER stress is detected, XBP1u is spliced ​​by the IRE1 RNase to form XBP1s, which encodes a functional 376-amino acid protein. This functional protein, XBP1, is a transcription factor that regulates the expression of several genes involved in protein homeostasis, such as chaperones, disulfide isomerases, and enzymes involved in phospholipid biosynthesis. It controls these processes by binding to specific sequences, ER stress response elements (ERSEs), or unfolding protein response elements (UPREs), thereby enhancing gene expression. Therefore, the regulation of gene expression is the removal of mRNA introns from XBP1u by IRE1. A very similar system functions in non-mammalian eukaryotic cells. 2) ATF6 Activated transcription factor (ATF6) is a type II transmembrane protein that possesses a transcription factor domain in its cytoplasmic region. It is synthesized as an inactive precursor and retained in the ER through association with BiP / GRP78. In response to stress conditions, ATF6 dissociates and is transported to the Golgi apparatus, where processing occurs and the transcription factor domain is released. This domain is then transported to the nucleus, where it can bind to ERSE and UPRE to enhance the expression of genes involved in protein homeostasis. 3) Double-stranded RNA-activated protein kinase (PERK) PERK is a type 1 ER transmembrane protein containing an ER lumen stress sensor and a cytoplasmic protein kinase domain. PERK is activated in response to ER stress and inhibits normal protein translation in the ER of mammalian cells by inactivating eukaryotic initiation factor (elF2a) through phosphorylation.

[0010] Mechanisms 1) and 2) are most relevant to the present invention. [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] U.S. Patent No. 4,683,195 [Non-patent literature]

[0012] [Non-Patent Document 1] Gossen M and Bujard H. PNAS. June 15, 1992; 89(12):5547-51 [Non-Patent Document 2] Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, and Bujard H. Science, June 23, 1995; 268(5218) [Non-Patent Document 3] Yoshida ら, Cell, volume 107, pages 881~891, December 28, 2001 [Non-licensed Document 4] Lu, Mol Cell. September 4, 2014; 55(5): 758~770 [Non-licensed Document 5] Samaliら, International Journal of Cell Biology, 2010 volume, Article ID 830307, ​​11 pages doi:10.1155 / 2010 / 830307 [Non-licensed Document 6] Chakrabortyら、Appl Biol Chem DOI、10.1007 / s13765-016-0167-6、オンラインISSN 2468-0842、プリントISSN 2468-0834 [Non-licensed Document 7] Nagashima, Scientific Reports 1, Article number: 29 (2011), DOI: 10.1038 / srep00029 [Non-licensed Document 8] "Therapeutic potentials of short interfering RNAs", Appl Microbiol Biotechnol, DOI 10.1007 / s00253-017-8433-z [Non-licensed Document 9] "MicroRNA therapeutics: towards a new era for the management of cancer and other diseases", Nature Reviews Drug Discovery; 16, pp. 203~222 (2017) [Non-licensed Document 10] Iwao and Shidoj, PLOS ONE DOI:10.1371 / journal.pone.0132761 July 17, 2015 [Non-licensed Document 11] Robblee et al., "Saturated Fatty Acids Engage an IRE1a-Dependent Pathway to Activate the NLRP3 Inflammasome in Myeloid Cells", Cell Reports 14, pp. 2611 - 2623, March 22, 2016 [Non-Patent Document 12] Ariyama et al., "Decrease in Membrane Phospholipid Unsaturation Induces Unfolded Protein Response", THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 285, No. 29, pp. 22027 - 22035, July 16, 2010 [Non-Patent Document 13] Basseri and Austin, "Endoplasmic Reticulum Stress and Lipid Metabolism: Mechanisms and Therapeutic Potential", Biochemistry Research International 2012, Article ID 841362, 13 pages, doi:10.1155 / 2012 / 841362 [Non-Patent Document 14] Kitai et al., "Membrane lipid saturation activates IRE1a without inducing clustering", Genes to Cells (2013) 18, pp. 798 - 809 [Non-Patent Document 15] Current Protocols in Molecular Biology (Ausubel, 2000, Wiley and son Inc, Library of Congress, USA) [Non-Patent Document 16] Molecular Cloning: A Laboratory Manual, 3rd Edition, (Sambrook et al., 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press)

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[0013] The present invention provides a synthetic nucleic acid expression construct for producing an expression product in a cell, wherein the nucleic acid expression construct comprises a promoter sequence operably linked to a nucleic acid sequence encoding an expression product, the nucleic acid sequence encoding the expression product comprises a sequence encoding a controllable intron, and the controllable intron is an intron containing a cleavable sequence that has the ability to be cleaved by splicing from a transcript produced from the synthetic expression construct by an unfolding protein response (UPR) system in the cell, thereby resulting in a transcript encoding a functional expression product. [Means for solving the problem]

[0014] Thus, the present invention is based on the use of a controllable intron that has the ability to be spliced ​​out from the RNA transcript of the nucleic acid sequence encoding the expression product by a UPR mechanism that controls expression. The UPR mechanism is ubiquitous across eukaryotes, and its mechanism and associated sequences are remarkably conserved. Therefore, the present invention can be implemented across all eukaryotic cells. The controllable intron is preferably capable of being spliced ​​out by the IRE1 protein or its homolog or ortholog (homogenos or orthologs of IRE1 are present in all eukaryotes, including fungi, plants, and mammals).

[0015] Promoters are typically heterogeneous with respect to the nucleic acid sequence encoding the expression product. That is, promoters are not found in nature operably ligated to the sequence encoding the expression product. For example, promoters and the nucleic acid sequence encoding the expression product are not typically found together in naturally occurring genes. In some embodiments of the present invention, the promoter is a synthetic promoter, i.e., a promoter that does not exist in nature. There are various constitutive and non-constitutive promoters known in the art that are suitable for use in eukaryotic cells, and non-limiting examples include the CAG promoter, the CMV promoter, and SV40.

[0016] In a preferred embodiment, the expression product is a protein. In such embodiments, the unspliced ​​transcript produced from the nucleic acid sequence encoding the expression product encodes a truncated or separately deleted version of the protein as a result of the presence of a controllable intron. However, when the transcript is processed by the UPR mechanism in the cell, the excisable sequence of the intron is cleaved by splicing, and a functional protein can be produced from the transcript. Thus, the excision of the controllable intron by splicing results in a functional mRNA encoding a functional protein expression product.

[0017] However, in some cases, the expression product may be a product other than a protein. Appropriately, the expression product may be an RNA molecule, such as a ribozyme, RNA aptamer, siRNA, antisense RNA, or miRNA. In such embodiments, a non-functional form of the RNA molecule is produced as an unspliced ​​transcript, and the excision of the cleavable portion of its introns by splicing converts the RNA molecule into an active form.

[0018] In a preferred embodiment of the present invention, the excision of controllable introns in the splicing of excisable sequences enables the correct translation of the transcript from the nucleic acid sequence encoding the expression product, thereby enabling the production of the desired expression product (e.g., a protein). In such embodiments, the presence of introns in the transcript from the nucleic acid sequence encoding the expression product results in the translation of a protein from a non-functional transcript. Typically, this results from the insertion of an amino acid encoded by the intron in the translated protein, or more preferably, from the introduction of a stop codon on the 3' side of the intron in the transcript or as a result of a frameshift (relative to a normal transcript encoding a functional protein). Proteins encoded by unspliced ​​transcripts may be non-functional for a number of reasons, e.g., the following: - Introns can cause frameshifts downstream of the intron (i.e., in the 3' direction). This typically occurs when the cleavable sequence of the intron is not a multiple of 3 nucleotides in length. Such frameshifts often result in the introduction of a stop codon, leading to a truncated protein. In other cases, it can simply result in a complete alteration of the encoded amino acid sequence downstream of the intron. - The introduction of a coding sequence in which amino acids that are destructive to the function of the protein will be present in the translated protein. In this case, the amino acid sequence downstream of the intron is not altered, but the amino acid sequence coded by the intron, which would be present in the translated protein in the unspliced ​​form, will disrupt the function of the protein. - Introduction of a stop codon in an intron sequence. In this case, the intron itself may contain a stop codon, which would result in premature termination of translation and the production of a truncated protein.

[0019] In a particular preferred embodiment of the present invention, the synthetic nucleic acid sequence construct is formed such that the excision of the intronic sequence during splicing removes an intermediate stop codon in the transcript. In this context, “intermediate” means a stop codon that is upstream (i.e., in the 5' direction) of a stop codon in a normal transcript, i.e., a transcript encoding a functional protein (e.g., wild-type mRNA).

[0020] Preferably, the controllable intron is formed such that the excision of the excisable sequence of the intron during splicing results in a shift in the reading frame for the sequence in the transcript located downstream (i.e., 3') of the controllable intron.

[0021] In a particular preferred embodiment of the present invention, controllable introns are formed such that, when splicing occurs, sequences having a length of nucleotides that are not multiples of 3 are excised from the transcript. In other words, the excisable sequences have a length of n nucleotides, where n is not divisible by 3.

[0022] In a preferred embodiment of the present invention, the controllable intron comprises the sequence CNG / CNG-Xn-CNG / CNG, where Xn represents a sequence of n bases in length, and / represents a cleavage site, and the sequence CNG-Xn-CNG is excised from the transcript.

[0023] Therefore, in other words, a controllable intron appropriately includes a central sequence (Xn) adjacent to two splice site target sequences, each having the sequence CNG / CNG, where / represents the cleavage site.

[0024] CNG / CNG is a consensus splice site sequence targeted in a highly conserved manner by the UPR system in eukaryotic cells. As is known in the art, this splice site consensus sequence is targeted by the IRE1 protein (its homologs or orthologues are present in all eukaryotes, including fungi, plants, and mammals) when the UPR response is induced. In nature, introns containing this consensus splice site target sequence are found in mRNA encoding transcription factors activated in the UPR, such as the XBP1 protein (in metazoans), the Hac1 protein (in yeast), and the bZIP60 protein (in plants). The endoribonuclease activity of IRE1 or its homolog or ortholog acts to cleave the RNA transcript at the position indicated by / , removing the cleavable intron sequence, and the cleaved RNA is then ligated together by RNA ligase proteins (RNA ligase Rlg1p in S. cerevisiae, RNA ligase RtcB in mammalian cells). The UPR system in mammalian cells, yeast cells, and plant cells has been extensively studied, and its mechanisms are relatively well characterized (e.g., Yoshida et al., Cell, vol. 107, pp. 881-891, December 28, 2001; Lu et al., Mol Cell. September 4, 2014; 55(5): pp. 758-770; Samali et al., International Journal of Cell Biology, vol. 2010, Article ID 830307, ​​p. 11 doi:10.1155 / 2010 / 830307; Chakraborty et al., Appl Biol Chem DOI, 10.1007 / s13765-016-0167-6, online ISSN 2468-0842, print ISSN 2468-0834; and Nagashima et al., Scientific Reports 1, Article number: (See 29 (2011), DOI: 10.1038 / srep00029). Therefore, the mechanism of UPR will not be discussed in detail herein.However, given the high level of conservation of IRE1-mediated splicing, it is important to note that introns originating from one species can be successfully spliced ​​by another species that are evolutionarily highly diversified; for example, Yoshida et al. (ibid.) explain how mammalian cells can successfully splice introns from yeast Hac1 mRNA (Hac1 corresponds to XBP1 in mammalian cells).

[0025] It should be noted that the controllable length of introns can vary considerably. For example, the XBP1 intron in mammals and plants is typically 26 nucleotides long, but variants of 20 and 23 nucleotides have been observed. The Hac1 intron in yeast is considerably longer, typically around 252 nucleotides long (although, as mentioned above, this much longer Hac1 intron can still be excised by splicing in mammalian cells).

[0026] Accordingly, in various embodiments of the present invention, the controllable intron or Xn may be 10 to 500 nucleotides long, more preferably 15 to 350 nucleotides long, even more preferably 15 to 100 nucleotides long, even more preferably 15 to 35 nucleotides long, and even more preferably 20 to 25 nucleotides long. Accordingly, the excisable sequence of the controllable intron may appropriately have a length of 16 to 506 nucleotides, more preferably 21 to 356 nucleotides long, even more preferably 21 to 106 nucleotides long, even more preferably 21 to 41 nucleotides long, and even more preferably 26 to 31 nucleotides long. As mentioned above, in some embodiments of the present invention, it is preferable that the length Xn is selected so that the length of the excisable sequence is not divisible by 3.

[0027] There is considerable flexibility regarding the specific sequence of Xn. Preferred sequences are shown below, but of course, many other variants can be used, provided that the controllable intron remains functional, i.e., it is cut out from the transcript at an appropriate level by splicing via the UPR system. Of course, some possible sequences may be suboptimal or interfere with the splicing process, for example, as a result of the formation of undesirable secondary structures, but those skilled in the art can easily evaluate the effect of any given sequence and determine whether it produces any adverse effects on splicing.

[0028] The functionality of controllable introns, i.e., their ability to be successfully spliced ​​and cleaved from the transcript upon UPR induction, can be readily assessed by those skilled in the art using various approaches, which can be configured to suit the specific expression system in which the construct is intended to be used. One preferred example is the methodology described in the following examples, for instance, Example 1. For example, the functionality of any candidate controllable intron to be evaluated can be replaced in the construct described in Example 1 (called SYNP-XBP-01) in place of the exemplary intron used in Example 1, and the ability of the intron to be successfully spliced ​​and cleaved upon UPR induction can be measured by evaluating the level of EGFP expression before and after UPR induction by 2 mM DTT, as is done in Example 1. A functional controllable intron can be successfully spliced ​​and cleaved after UPR induction, resulting in the expression of functional EGFP. Preferably, the functional intron gives at least a 5-fold increase in EGFP expression 24 hours after UPR induction at 2 mM DTT, more preferably at least a 10-fold increase, more preferably at least a 100-fold increase, and even more preferably at least a 1000-fold increase. Before UPR induction, the EGFP expression level is preferably minimal, preferably negligible. Minimal expression can be defined as less than 50%, preferably less than 20%, more preferably less than 10%, even more preferably less than 5%, and even more preferably less than 1% of the expression level of the control construct (i.e., a construct without a controllable intron, in which the expression of the EGFP-coding sequence is driven by CMV-mp) as used in Example 1. A negligible expression level is a level that is essentially undetectable using the methodology of Example 1.

[0029] However, it will be understood that those skilled in the art can easily modify the approach employed in Example 1. For example, this may include using different cell types, different expression products, different indicators of splicing success (e.g., measuring the level of spliced ​​mRNA encoding the functional expression product, or using a different reporter protein), and different inducers of UPR. In such modified approaches, it is maintained that functionally controllable introns are successfully cleaved by splicing after UPR induction, resulting in the expression of the functional expression product after UPR induction. Preferably, this results in at least a 5-fold increase in the expression of the transcript encoding the functional expression product 24 hours after UPR induction, preferably at least a 10-fold increase, more preferably at least a 100-fold increase, and even more preferably at least a 1000-fold increase. It is preferable that the expression level of the transcript encoding the functional expression product is minimal or negligible before UPR induction.

[0030] CNG / CNG[CG] is a preferred splice site consensus target sequence for mammalian cells, and the presence of C or G at the indicated position in the sequence is preferred (but not required). The presence of C at this position is typically preferred over G. Therefore, in some preferred embodiments of the present invention, in particular when the synthetic nucleic acid expression construct is intended for mammalian cells, the intron contains the sequence CNG / CNG[CG] at one, the other, or both (preferably both) ends of the intron.

[0031] Accordingly, in some preferred embodiments of the present invention, the controllable intron comprises the sequence CNG / CNG-Xn-CNG / CNG[CG], where Xn represents a sequence of n nucleotides in length, and / represents a cleavage site such that the excisable sequence CNG-Xn-CNG is excised from the transcript by splicing, and the nucleotide at the 5' end of sequence Xn is C or G. Appropriate lengths for Xn are shown above.

[0032] In some preferred embodiments of the present invention, Xn includes the sequence CACUCAGACUACGUGCACCU (Sequence ID 1), or a sequence that is at least 60% identical thereto, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, and even more preferably at least 95%, 96%, 97%, 98%, or 99% identical thereto.

[0033] In some preferred embodiments of the present invention, Xn consists of the sequence CACUCAGACUACGUGCACCU (Sequence ID 1), or a sequence that is at least 60% identical thereto, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, and even more preferably at least 95%, 96%, 97%, 98%, or 99% identical thereto.

[0034] The sequence CACUCAGACUACGUGCACCU (SEQ ID NO: 1) corresponds to a region of the mammalian XBP1 intron located inside the IRE1 cleavage site as shown above. Therefore, this represents a preferred embodiment of the present invention, particularly when the synthetic nucleic acid expression construct is intended for mammalian cells. However, sequences very similar to this are also found across various non-mammalian XBP1 introns.

[0035] In some embodiments of the present invention, Xn includes or consists of one of the following sequences: - CACUCAGACUACGUGCACCU(Sequence ID 1); - CACUCAGACUACGUGCUCCU(Sequence ID 2); - CACUCAGACUACGUGCCCCU(Sequence ID 3); - CACUCAGACUACGUGCGCCU(Sequence ID 4); and - CACUCAGACUAUGUGCACCU (Sequence ID 5).

[0036] In some embodiments of the present invention, Xn includes or comprises the sequence ACGGGCAACUUUACACGACG (SEQ ID NO: 49), or a sequence that is at least 60% identical thereto, more preferably at least 70% identical thereto, even more preferably at least 80% identical thereto, even more preferably at least 90% identical thereto, and even more preferably at least 95%, 96%, 97%, 98%, or 99% identical thereto.

[0037] In a particularly preferred embodiment of the present invention, the controllable intron includes or comprises the sequence CNG / CNGCACUCAGACUACGUGCACCUCNG / CNGC (SEQ ID NO: 6), or a sequence that is at least 60% identical thereto, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, and even more preferably at least 95%, 96%, 97%, 98%, or 99% identical thereto, where / represents a cleavage site. In the variant sequences according to the above levels of sequence identity, the splice site target sequence preferably remains as CNG / CNGC, with sequence variations present in other regions.

[0038] Appropriately, the controllable intron includes or consists of the sequence CAG / CAGCACUCAGACUACGUGCACCUCUG / CUGC (SEQ ID NO: 7), or a sequence that is at least 60% identical thereto, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, and even more preferably at least 95%, 96%, 97%, 98%, or 99% identical thereto, where / represents a cleavage site. In the variant sequences with the above levels of sequence identity, the splice site target sequence preferably remains as CAG / CUGC, with sequence variations present in other regions.

[0039] In some preferred embodiments of the present invention, a controllable intron comprises or consists of one of the following sequences: - CNG / CAGCACUCAGACUACGUGCACCUCUG / CNG(Sequence ID 8); - CNG / CAGCACUCAGACUACGUGCUCCUCUG / CNG(Sequence ID 9); - CNG / CAGCACUCAGACUACGUGCCCCUCUG / CNG(Sequence ID 10); - CNG / CAGCACUCAGACUACGUGCGCCUCUG / CNG(Sequence ID 11); and - CNG / CAGCACUCAGACUAUGUGCACCUCUG / CNG (Sequence ID 12).

[0040] In a further preferred embodiment of the present invention, a controllable intron comprises or consists of one of the following sequences: - CAG / CAGCACUCAGACUACGUGCACCUCUG / CUGC(Sequence ID 7); - CAG / CAGCACUCAGACUACGUGCUCCUCUG / CUGC(Sequence ID 13); - CAG / CAGCACUCAGACUACGUGCCCCUCUG / CUGC(Sequence ID 14); - CAG / CAGCACUCAGACUACGUGCGCCUCUG / CUGC(Sequence ID 15); and - CAG / CAGCACUCAGACUAUGUGCACCUCUG / CUGC (Sequence ID 16).

[0041] In another embodiment of the present invention, the controllable intron is an array [ka] (Sequence ID 17) or [ka] This sequence includes (SEQ ID NO: 27), where / represents a cleavage site. This sequence results from the addition of a trinucleotide CUG to the mammalian XBP1 intron sequence at the underlined position. This trinucleotide addition is thought to slightly deoptimize the splicing of the intron, thereby reducing any undesirable splicing in the cell (and therefore the background expression of the expression product).

[0042] Therefore, in some preferred embodiments of the present invention, Xn includes or consists of CAGCACUCAGACUACGUGCACCU (SEQ ID NO: 23).

[0043] In another embodiment of the present invention, the controllable intron includes the sequence:CNG / CAGACGGGCAACUUUACACGACGCUG / CNG (SEQ ID NO: 50), or a sequence that is at least 60% identical thereto, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, and even more preferably at least 95%, 96%, 97%, 98%, or 99% identical thereto, where / represents a cleavage site. In the variant sequences with the above levels of sequence identity, the splice site target sequence preferably remains as CNG / CNG, with sequence variations present in other regions.

[0044] To avoid misunderstanding, it should be noted that in the intron sequences of the present invention, the splice site target sequences at each end of the intron are preferably constrained as CNG / CNG, more preferably as CNG / CNG[CG]. Thus, while several variations of the splice site target sequences are provided by these sequences, further variations, if necessary, should be accommodated in the central sequence located between the two splice site target sequences.

[0045] In some preferred embodiments of the present invention, the controllable intron is the XBP1 intron, the Hac1 intron, the bZIP60 intron, or a homolog thereof. This means that the intron may be the wild type of the XBP1, Hac1, or bZIP60 intron, or a naturally occurring homolog thereof.

[0046] In some embodiments of the present invention, it may be desirable that the splice site target sequences in the transcript (i.e., including the sequence CNG / CNG) be adjacent to sequences that can interact to form a stem-loop structure. Therefore, the splice site target sequences are preferably adjacent to complementary sequences that hybridize with each other to form a stem-loop structure, in which the splice site target sequences are located at least partially, preferably entirely, within the loop region of the stem-loop structure formed in the transcript.

[0047] For wild-type XBP1, HAC1, and bZIP60 introns, it is hypothesized that a stem-loop structure is formed at the splice site in the mRNA transcript. This involves hybridization between the intron sequence and the exon sequence, adjacent to the splice site target sequence, which are complementary in the nucleotide sequence when read in the opposite direction. Experiments reported herein have shown that splicing is successful when the intron is inserted into the coding sequence under circumstances where such a stem-loop structure is not expected to form. Therefore, the formation of a stem-loop structure does not appear to be necessary for the successful controllable intron splicing of the present invention.

[0048] That said, in some cases, the formation of a stem-loop structure may be desirable, for example, because it can result in optimal splicing activity. Conversely, in some cases, it may be desirable to avoid providing a sequence that can be modified to form a stem-loop structure, because the formation of a stem-loop structure may lead to splicing of undesirable intron activity, and consequently, to expression leakage.

[0049] In a particular embodiment of the present invention in which a stem-loop structure is to be formed, the stem-loop structure formed by the transcript preferably includes a loop containing 6 to 9 nucleotides and a stem having a length of 3 to 10 nucleotides. More preferably, the stem-loop structure includes a loop containing 7 to 8 nucleotides and a stem having a length of 4 to 8 nucleotides.

[0050] In a particular embodiment of the present invention, in which a stem-loop structure is to be formed, the intron may appropriately contain sequences at the splice target site as follows: -Yn-CNG / CNG-A-Zn- However, A has a length of 0 to 3 nucleotides (preferably 1 or 2 nucleotides), / represents a cleavage site, and Yn and Zn represent sequences that are complementary in nucleotide sequences when read in opposite directions and can therefore hybridize to form the stem of a stem-loop structure. Yn and Zn are preferably 3 to 10 nucleotides long, more preferably 4 to 8 nucleotides long.

[0051] In some embodiments, the intron may appropriately include sequences at the splice target site as follows: -Zn-CNG / CNG[CG]-A-Yn-, where the constituent elements have the same meaning as above. In this case, A preferably has a length of 0 nucleotides, 1 nucleotide, or 2 nucleotides.

[0052] It will be apparent that providing appropriate complementary sequences (e.g., Yn and Zn in the above structure) to give a stem structure can be achieved by adapting the sequence of an intron to provide an appropriate region that is complementary to the corresponding sequence in the adjacent coding (i.e., exon) sequence. It may also be possible or desirable to alter the sequence of the coding region to some extent, for example, by utilizing redundancy in the genetic code to alter the nucleic acid sequence without affecting the coded amino acid sequence; typically, changes in the amino acid sequence of the expression product should be avoided.

[0053] In some embodiments of the present invention, the nucleic acid expression construct comprises an inductive promoter operably ligated to a nucleic acid sequence encoding an expression product that includes a sequence encoding a controllable intron. As mentioned above, inductive promoters are known in the art. By combining the inductive promoter with the controllable intron of the present invention, a dual level of expression can be achieved, i.e., it can be regulated at both the transcriptional and translational levels. This can allow for very precise regulation of expression, for example, to avoid any expression "leaking." This may be important, for example, during the expression of a toxic protein, or in any case where the amount of background expression must be maintained at an absolute minimum before induction of expression at a desired time.

[0054] In a preferred embodiment, the inducible promoter is an unfolding protein response (UPR) inducible promoter, i.e., a promoter that is itself induced by a UPR. In such embodiments, UPR induction works to induce expression both in terms of driving transcription and in terms of enabling the expression of a functional expression product as a result of controllable intron splicing.

[0055] In embodiments of the present invention, the UPR-inducible promoter preferably includes at least one binding site to ATF6, XBP1, bZIP60, or a homolog or other equivalent transcription factor that drives protein expression in the UPR.

[0056] Appropriately, a UPR-inducible promoter contains one or more copies of at least one of the following sequences: - TGACGTG (ATF6 consensus sequence) - TGACGTGCT (the variant above) - TGACGTG[TG] (known as the UPR site), - CCAAT-N9-CCACG (known as the ERSE1 site) (SEQ ID NO: 18), and - ATTGG-N-CCACG (known as the ERSE2 site) (SEQ ID NO: 19).

[0057] These parts are bound together by ATF6, XBP1, and bZIP60.

[0058] Appropriately, the promoter includes one or more copies of the sequence TGACGTG (optionally as part of TGACGTGCT or TGACGTG[TG]), preferably three or more copies of the sequence TGACGTG[TG] (optionally as part of TGACGTGCT or TGACGTG[TG]), and preferably five or more copies of the sequence TGACGTG[TG] (optionally as part of TGACGTGCT or TGACGTG[TG]).

[0059] An example of a UPR-inducible promoter sequence is the following sequence (SEQ ID NO: 20): TGACGTGCTTGACGTGCTTGACGTGCTTGACGTGCTTGACGTGCTTGACGTGCT This sequence includes six tandem copies of the UPR region.

[0060] Another example of a UPR-inducible promoter sequence is the following sequence (SEQ ID NO: 47): This sequence contains TGACGTGCTGATGATGCGTAGCTAGTAGTTGACGTGCTGATGATGCGTAGCTAGTAGTTGACGTGCTGATGATGCGTAGCTAGTAGTTGACGTGCTGATGATGCGTAGCTAGTAGTTGACGTGCTGATGATGCGTAGCTAGTAGTTGACGTGCT. This sequence contains six copies of the UPRE site, each spaced 20 nucleotides apart.

[0061] Suitablely, the UPR-inducible promoter includes at least one of the binding sites for ATF6, XBP1, or a homolog or other equivalent transcription factor that drives the UPR, operably ligated to a minimal promoter sequence (e.g., the CMV minimal promoter). Other suitable minimal promoters are known in the art.

[0062] The CMV minimum promoter is represented by the following sequence (sequence number 21): AGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTAGATACGCCATCCACGCTGTTTTGACCTCCATAGAAGAT It has.

[0063] Therefore, the exemplary inducible promoter includes a nucleic acid containing the sequence of sequence number 20, located upstream of and operably linked to the nucleic acid having the sequence of sequence number 21.

[0064] For example, an inducible promoter would appropriately use the following sequence (SEQ ID NO: 22): TGACGTGCTTGACGTGCTTGACGTGCTTGACGTGCTTGACGTGCTTGACGTGCTGGTACCGTCGACGATATCGGATCCAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTAGATACGCCATCCACGCTGTTTTGACCTCCATAGAAGATCGCCACC It may include.

[0065] Further details on UPR-inducible promoters that can be used with controllable introns are provided below.

[0066] A nucleic acid sequence encoding an expression product is appropriately called a transgene. A transgene typically encodes a gene expression product such as RNA or polypeptide (protein). A transgene may be a full-length cDNA or genomic DNA sequence, or any fragment, subunit, or mutant having at least some biological activity. A transgene may appropriately be a minigene, i.e., a gene sequence lacking some, most, or all of its native intron sequence. A transgene may optionally contain a normal intron sequence (i.e., in addition to controllable introns). Optionally, a transgene may be a hybrid nucleic acid sequence, i.e., constructed from homogeneous and / or heterogeneous cDNA and / or genomic DNA fragments. A “mutant” means a nucleic acid sequence containing one or more nucleotides different from the wild-type or naturally occurring sequence; i.e., a mutant nucleic acid sequence contains one or more nucleotide substitutions, deletions, and / or insertions. Nucleotide substitutions, deletions, and / or insertions may result in a gene product (i.e., a protein or nucleic acid) that differs from the wild-type amino acid / nucleic acid sequence in terms of amino acid / nucleic acid sequence. In some cases, the transgene may also contain a sequence that encodes a leader peptide or signal sequence so that its transgene product will be secreted from the cell.

[0067] Conventional (i.e., uncontrollable) introns, in addition to the controllable introns discussed above, can be utilized in nucleic acid sequences encoding expression products. The term “conventional intron” encompasses any portion of an entire intron that is large enough to be recognized and spliced ​​by the nuclear splicing apparatus. Typically, short, functional intron sequences are preferred to keep the size of the expression cassette as small as possible (which facilitates the construction and manipulation of the expression cassette). In some embodiments, introns are obtained from genes encoding proteins encoded by nucleic acid sequences encoding expression products. Conventional introns may be located at the 5' end of the expression-encoding sequence, at the 3' end of the expression-encoding sequence, or within the expression-encoding sequence. Thus, in some embodiments, the nucleic acid sequence encoding the expression product further includes conventional introns. Non-limiting examples of suitable introns include mouse microvirus (MVM) introns, beta-globin introns (betalVS-II), factor IX (FIX) intron A, monkey virus (SV40) small t introns, and beta-actin introns. Introns are effective in improving expression levels, as is well known in this field.

[0068] In some preferred embodiments of the present invention, the nucleic acid sequence encoding the expression product encodes a protein. Essentially any protein can be used, and in non-limiting examples, the protein may be an enzyme, an antibody or antibody fragment (e.g., a monoclonal antibody), a viral protein (e.g., REP-CAP, REV, VSV-G, or RD114), a therapeutic protein, or a toxic protein (e.g., caspase 3, 8, or 9).

[0069] In some preferred embodiments of the present invention, the nucleic acid sequence encoding the expression product encodes a toxic protein. In this case, “toxic protein” means a protein that is toxic to the cells on which the expression product is produced during use. For example, the toxic protein may be one of the following: caspase 3, caspase 8, caspase 9, and toxic viral proteins (such as VSV-G or AAV REP protein).

[0070] In some preferred embodiments of the present invention, the nucleic acid sequence encoding the expression product encodes a therapeutic expression product. The therapeutic expression product may be a protein, for example, a secreted protein, for example, a coagulation factor, for example, factor IX or factor VIII, a cytokine, a growth factor, an antibody or nanobody, a chemokine, a plasma factor, insulin, erythropoietin, lipoprotein lipase, or a toxic protein.

[0071] Alternatively, the therapeutic expression product may be RNA such as siRNA or miRNA. Various therapeutic siRNAs have been described in the art, and non-limiting examples include siRNAs intended to treat FTDP-17 (frontotemporal dementia), DYT1 dystonia, growth hormone deficiency, BACE1 in Alzheimer's disease, leukemia (e.g., targeting c-raf, bcl-2), melanoma (e.g., targeting ATF2, BRAF), prostate cancer (e.g., targeting P110B), and pancreatic cancer (e.g., targeting K-Ras). siRNA therapy is summarized in "Therapeutic potentials of short interfering RNAs," Appl Microbiol Biotechnol, DOI 10.1007 / s00253-017-8433-z. Similarly, various miRNA therapeutic approaches that can be implemented according to the present invention are summarized in "MicroRNA therapeutics: towards a new era for the management of cancer and other diseases," Nature Reviews Drug Discovery; 16, pp. 203-222 (2017).

[0072] Appropriately, the nucleic acid expression construct includes sequences that provide or encode one or more, preferably all, ribosome-binding sites, start codons, stop codons, and transcription termination sequences.

[0073] In a further embodiment, the present invention provides a nucleic acid comprising a sequence encoding an expression product (e.g., a gene), wherein the sequence encoding the expression product comprises a sequence encoding a controllable intron, the controllable intron being an intron comprising a cleavable sequence capable of being spliced ​​out from a transcript produced from a synthetic expression construct by an unfolding protein response (UPR) system in cells, thereby resulting in a transcript encoding a functional expression product, wherein the expression product is neither an XBP1 protein, nor a Hac1 protein, nor a bZIP60 protein, nor a homolog thereof.

[0074] Therefore, the sequence encoding the expression product is not a gene that naturally contains the controllable intron according to the present invention. In other words, the controllable intron is heterogeneous to the sequence encoding the expression product in which it is found.

[0075] Preferred features of controllable introns and sequences encoding the expression product are as described above.

[0076] Such nucleic acids can be inserted into any suitable expression construct, such as an expression vector, so as to be operably linked to a promoter and any other elements required to drive the transcription of the nucleic acid. The expression of the functional expression product will be regulated by controllable introns.

[0077] In a further embodiment, the present invention provides a vector comprising a synthetic nucleic acid expression construct, as described above.

[0078] The term “vector,” as is well known in the art and as used in this application, refers to a nucleic acid molecule, such as double-stranded DNA, that can be inserted into a nucleic acid expression construct according to the present invention. A vector is appropriately used to transport the inserted nucleic acid molecule to a suitable host cell. A vector typically contains all the necessary elements to enable the transcription of the inserted nucleic acid molecule and, preferably, the translation of the transcript into a polypeptide. A vector typically contains all the necessary elements so that, once the vector is in a host cell, it can replicate independently of or concurrently with host chromosomal DNA (several copies of the vector and the inserted nucleic acid molecule may be produced). The vectors of the present invention may be episomal vectors (i.e., not integrated into the host cell genome) or vectors integrated into the host cell genome. This definition includes both non-viral and viral vectors. Non-viral vectors include, but are not limited to, plasmid vectors (e.g., pMA-RQ, pUC vectors, bluescript vectors (pBS), and pBR322, or their derivatives lacking bacterial sequences (minicircles)), and transposon-based vectors (e.g., PiggyBac (PB) vectors or Sleeping Beauty (SB) vectors). Larger vectors, such as artificial chromosomes (bacteria (BAC), yeast (YAC), or human (HAC)), may be used to accommodate larger insertion fragments. Viral vectors are derived from viruses and include, but are not limited to, retrovirus, lentivirus, adeno-associated virus, adenovirus, herpesvirus, and hepatitis virus vectors. Typically, though not always, viral vectors are replication-deficient because the viral genes essential for replication have been removed from the viral vector, thus losing the ability to proliferate in a given cell. However, some viral vectors can also be adapted to specifically replicate in certain cells, such as cancer cells, and are typically used to induce (cancer) cell-specific (tumor) lysis. Virosomals are vectors that contain both viral and non-viral elements. These are non-limiting examples, particularly those combining liposomes with inactivated HIV or influenza viruses (Yamada et al., 2003). Another example involves viral vectors mixed with cationic lipids.

[0079] In some preferred embodiments, the vector is a viral vector, such as a retrovirus, lentivirus, adenovirus, or adeno-associated virus (AAV) vector, and more preferably an AAV vector. The AAV vector is preferably used as a self-complementary double-stranded AAV vector (scAAV) to overcome one of the rate-limiting steps in AAV transduction (i.e., conversion from single-stranded AAV to double-stranded AAV) (McCarty, 2001, 2003; Nathwani et al., 2002, 2006, 2011; Wu et al., 2008), but the use of a single-stranded AAV vector (ssAAV) is also included herein.

[0080] In some preferred embodiments, the vector is a plasmid. Such a plasmid may contain a variety of other functional nucleic acid sequences, such as one or more selection markers, one or more origins of replication, and polycloning sites.

[0081] In some preferred embodiments of the present invention, the vector is an expression vector for expression in eukaryotic cells. Examples of eukaryotic expression vectors include, but are not limited to, pW-LNEO, pSV2CAT, pOG44, pXTl, and pSG available from Stratagene; pSVK3, pBPV, pMSG, and pSVL available from Amersham Pharmacia Biotech; and pCMVDsRed2-express, pIRES2-DsRed2, pDsRed2-Mito, and pCMV-EGFP available from Clontech. Many other vectors are known and commercially available. For mammalian cells, adenovirus vectors, the pSV series, and the pCMV series are particularly well known, non-limiting examples. There are many well known yeast expression vectors, which include, non-limitingly, yeast integrated plasmids (YIp) and yeast replicated plasmids (YRp). For plants, the Agrobacterium Ti plasmid is an exemplary expression vector, and plant viruses also provide suitable expression vectors, such as tobacco mosaic virus (TMV), potato virus X, and cowpea mosaic virus.

[0082] In some preferred embodiments, the vector is a gene therapy vector. Various gene therapy vectors are known in the art, including AAV vectors, adenovirus vectors, retrovirus vectors, and lentivirus vectors. When the vector is a gene therapy vector, the nucleic acid sequence encoding the expression product appropriately encodes a therapeutic protein. The therapeutic protein may be a secreted protein. Non-limiting examples of secreted proteins, particularly secreted therapeutic proteins, include coagulation factors such as factor VIII or factor IX, insulin, erythropoietin, lipoprotein lipase, antibodies or nanobodies, growth factors, cytokines, chemokines, plasma factors, and toxic proteins.

[0083] The nucleic acid expression constructs and vectors of the present invention may be formulated in a pharmaceutical composition with pharmaceutically acceptable excipients, i.e., one or more pharmaceutically acceptable carriers and / or additives, such as buffers, carriers, excipients, stabilizers, etc. The pharmaceutical composition may be provided in the form of a kit. As used herein, the term “pharmaceutically acceptable” means consistent with the art, compatible with other components of the pharmaceutical composition, and not harmful to its recipient.

[0084] Therefore, a further aspect of the present invention provides a pharmaceutical composition comprising a nucleic acid expression construct or vector as described herein.

[0085] In a further embodiment of the present invention, the use of nucleic acid expression constructs and vectors according to various aspects of the present invention for the production of pharmaceutical compositions is provided.

[0086] A further aspect of the present invention provides cells comprising a synthetic nucleic acid expression construct or vector according to the present invention.

[0087] Preferably, the cells are eukaryotic cells. Eukaryotic cells may appropriately be fungal cells (e.g., yeast cells), animal (metazoan) cells (e.g., mammalian cells), or plant cells.

[0088] In some embodiments of the present invention, the cells may be prokaryotic cells; although prokaryotic cells do not have UPR, they may nevertheless be useful in other steps in the preparation or handling of synthetic nucleic acid expression constructs.

[0089] In some preferred embodiments of the present invention, the cells are ex vivo, for example, in a cell culture. In other embodiments of the present invention, the cells may be part of a tissue or a multicellular organism.

[0090] In some preferred embodiments, the expression product is toxic to cells in which the construct or vector is present. In one such embodiment, the cells are cells for cell therapy (e.g., therapeutic immune cells such as therapeutic T cells) containing the synthetic nucleic acid expression construct or vector according to the present invention, and the expression product is toxic to the cells. In such embodiments, induction of UPR may be suitable for inducing cell death, i.e., as a kill switch. Suitable toxic expression products include caspases, e.g., caspase 3, caspase 8, or caspase 9.

[0091] The synthetic nucleic acid expression construct may be inserted into the cell's genome or may reside within an episomal vector.

[0092] In a further embodiment, the present invention is a) A step of supplying a population of eukaryotic cells containing a synthetic nucleic acid expression construct according to the present invention; b) A step of processing the cell population to induce an unfolding protein response, thereby inducing splicing of excisable sequences from controllable introns; c) Incubating the cell population under appropriate conditions for the production of expression products; and d) The step of isolating the expression product from the cell population. The present invention provides a method for producing an expression product, including [the specified element].

[0093] The method is, appropriately, a cell culture method. Therefore, the cells can be supplied under appropriate cell culture conditions for the cell type to be used. Appropriate cell culture conditions are well known to those skilled in the art.

[0094] Synthetic nucleic acid expression constructs may reside within the genome or in episomes.

[0095] It will be apparent that the present invention makes it possible to delay the production of an expression product (or the active form of an expression product) to a desired point in time during the cell culture process. This allows, for example, a cell population to grow to a point in time when it reaches a desired number or concentration of cells, or reaches a desired growth phase.

[0096] For example, in the case of toxic proteins, the production of functional (i.e., toxic) expression products can be avoided until the cell culture system reaches the desired stage. Once the toxic protein is expressed, the cells will, of course, be adversely affected or killed.

[0097] The method appropriately includes incubating the cell population under conditions suitable for cell proliferation, prior to step b) processing the cell population to induce an unfolding protein response (UPR).

[0098] Typically, step b) involves applying stress to the cells, such that the stress is suitable for inducing UPR. There are various stresses that can be used to induce UPR, and these are widely described in the literature.

[0099] In some preferred embodiments, the step of inducing UPR appropriately includes administering a chemical substance capable of inducing UPR in the cells (i.e., a UPR-inducing chemical).

[0100] Those skilled in the art can easily evaluate the ability of any specific stress (e.g., a chemical) to induce UPR. For example, in the method of Example 1, stress can be appropriately applied instead of DTT (e.g., by administering a candidate chemical). The ability of an active agent to induce UPR is identified by the effect that the application of stress (e.g., a chemical) has on the expression of a functional expression product, i.e., EGFP in the case of Example 1. Of course, the method of Example 1 can be modified as needed, for example, using different cell types or different constructs. In particular, Example 4 demonstrates how various candidate chemicals can be tested for their ability to induce UPR. Various other methods for evaluating the ability of stress (e.g., a chemical) to induce UPR will be obvious to those skilled in the art.

[0101] Many chemicals are known in the art that can induce ER stress and thereby induce UPR. UPR-inducing chemicals that can induce the IRE1 pathway are suitable for use in the present invention because they will result in controllable intron splicing.

[0102] Examples of UPR-inducing chemicals that can be used to induce UPR include: • Dithiothreitol (DTT) - This substance reduces the disulfide crosslinking of proteins. The denatured proteins accumulated in the ER. • Tunicamycin - This substance inhibits N-linked glycosylation. • Brefeldin A - This is commonly used as an inducer of unfolding protein responses. • Thapsigardin - This active substance is used in the sarcoplasmic reticulum / endoplasmic reticulum Ca 2+ -Inhibition of ATPase (SERCA) leads to ER Ca 2+ It will cause depletion. • 2-deoxyglucose ·A23187 (CAS number 52665-69-7) • Bortezomib (Velcade) • Quercetin Substances that disrupt the lipid balance in cells in such a way that UPR is induced, such as saturated fatty acids (e.g., palmitic acid) - that is, substances that induce lipid-induced ER stress response / UPR.

[0103] Such UPR-inducing chemicals can be administered at appropriate concentrations, which can be readily determined by those skilled in the art through routine experiments and reference to the literature. Appropriate concentrations for the administration of various active ingredients are as follows: DTT 2 mM; tunicamycin 2.5-5 μg / ml; brefelzin A 0.5 μg / ml; thapsigarzine 0.1-1 μM; 2-deoxyglucose 4 mM; A23187 (CAS number 52665-69-7) 0.5 μM; bortezomib (Velcade) 5-30 nM; and palmitic acid (or other fatty acids) 100 μM. These concentrations refer to the concentration of the active ingredient in the culture medium to which the cells are exposed.

[0104] Another UPR-inducing chemical used in this invention is forskolin. Forskolin (Coleonol) is a labdane-type diterpene produced by the Indian coleus plant (Plectranthus barbatus). Other names forskolin include pashanabhedi, Indian coleus, makandi, HL-362, and NKH477.

[0105] Dithiothreitol (DTT), tunicamycin, and thapsigardin are widely used in the literature to induce UPR and therefore represent preferred UPR-inducing chemicals in some embodiments of the present invention. Forskolin is another preferred UPR-inducing chemical, but is not limited thereto, given its safety profile for in vivo use.

[0106] In certain preferred embodiments of the present invention, a UPR-inducing chemical that can disrupt the lipid balance in the cells is administered to induce UPR. The role of lipids and lipid metabolism in inducing UPR has been widely reported in the literature, and the phenomenon is named "lipid-induced ER stress response / UPR". For example, Iwao and Shidoj, PLOS ONE | DOI:10.1371 / journal.pone.0132761, July 17, 2015; Robblee et al., "Saturated Fatty Acids Engage an IRE1a-Dependent Pathway to Activate the NLRP3 Inflammasome in Myeloid Cells," Cell Reports 14, pp. 2611-2623, March 22, 2016; Ariyama et al., "Decrease in Membrane Phospholipid Unsaturation Induces Unfolded Protein Response," THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 285, No. 29, pp. 22027-22035, July 16, 2010; Basseri and Austin, "Endoplasmic Reticulum Stress and Lipid Metabolism: Mechanisms and Therapeutic Potential," Biochemistry Research International, Vol. 2012, Article ID 841362, p. 13, doi:10.1155 / 2012 / 841362; Kitai et al., "Membrane lipid saturation activates IRE1a without inducing clustering," Genes to Cells (2013) 18, pp. 798-809.

[0107] Therefore, appropriately, UPR-inducing chemicals can alter the lipid balance of cells so that UPR is induced. There are various agents that can achieve this. For example, it has been shown that disruption of the lipid balance in cells, such as an increase in lipid saturation levels (and thus a decrease in unsaturated lipid levels), results in the induction of UPR. Therefore, appropriately, UPR-inducing chemicals can alter the lipid balance of cells so that lipid saturation levels increase.

[0108] Preferably, UPR-inducing chemicals can alter the ratio of saturated fatty acids to unsaturated fatty acids in the cell membrane so that the proportion of saturated fatty acids increases. This can be achieved in several ways, for example, by introducing saturated fatty acids into the cell or by inhibiting the activity of enzymes that convert saturated fatty acids to unsaturated fatty acids.

[0109] Therefore, in one particularly preferred embodiment, the UPR-derived chemical comprises saturated fatty acids, preferably medium-chain or long-chain saturated fatty acids. In certain embodiments of the present invention, the fatty acid has an aliphatic chain length of 6 to 26 carbon atoms, more preferably 9 to 22 carbon atoms, even more preferably 12 to 20 carbon atoms, and even more preferably 14 to 20 carbon atoms.

[0110] Preferably, the UPR-derived chemical comprises at least one fatty acid selected from the following list: caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylenic acid, lauric acid, tridecyl acid, myristic acid, pentadecyl acid, palmitic acid, margaric acid, stearic acid, nonadecyl acid, arachidonic acid, henicosyl acid, behenic acid, tricosylic acid, lignoceric acid, pentacosyl acid, and cerotic acid. More preferably, the UPR-derived chemical comprises at least one fatty acid selected from the following list: pelargonic acid, capric acid, undecylenic acid, lauric acid, tridecyl acid, myristic acid, pentadecyl acid, palmitic acid, margaric acid, stearic acid, nonadecyl acid, arachidonic acid, henicosyl acid, and behenic acid. More preferably, the UPR-derived chemical comprises at least one fatty acid selected from the following list: lauric acid, tridecyl acid, myristic acid, pentadecyl acid, palmitic acid, margaric acid, stearic acid, nonadecyl acid, and arachidonic acid.

[0111] In certain preferred embodiments, the UPR-inducing chemical comprises palmitic acid or stearic acid.

[0112] It should be noted that when fatty acids are mentioned, they may be provided in any suitable form, such as salts (palmitate, stearate, etc.).

[0113] In another preferred embodiment, the UPR-inducing chemical comprises an active agent capable of downregulating stearoyl-CoA desaturase enzyme activity in cells. Stearoyl-CoA desaturase is a commensal ER enzyme that introduces double bonds in saturated fatty acids. For example, the UPR-inducing chemical appropriately includes an inhibitor of stearoyl-CoA desaturase. MF-43 is an example of a suitable inhibitor of stearoyl-CoA desaturase. Alternatively, the UPR-inducing chemical may downregulate stearoyl-CoA desaturase expression, for example, through knockdown of stearoyl-CoA desaturase expression (e.g., by RNA interference) or knockout of the stearoyl-CoA desaturase gene. Other genes involved in fatty acid desaturation may also be targeted.

[0114] Other exemplary UPR-derived chemicals include geranylgeranic acid (GGA), 2,3-dihydroGGA, 9-cisretinoic acid, and all-transretinoic acid (Chieko Iwao and Yoshihiro Shidoj, PLOS ONE DOI:10.1371 / journal.pone.0132761, accessed July 17, 2015).

[0115] Several combinations of UPR-inducing chemicals can be used in several embodiments of the present invention. For example, saturated fatty acids (e.g., palmitic acid) can be used in combination with an active agent (e.g., MF-43) that can downregulate stearoyl-CoA desaturase enzyme activity.

[0116] In another embodiment of the present invention, the step of inducing UPR in step b) appropriately includes expressing an inducer protein in a cell population to induce a UPR response in the cell population. Such a protein is called an "inducer protein" because, when expressed in a cell population, it acts to induce a UPR response, typically by generating ER stress. The inducer protein is typically a protein different from the one encoded by the synthetic nucleic acid expression construct according to the first aspect of the present invention. The inducer protein is appropriately a heterologous protein, but in some embodiments, it may be an allologous protein to be overexpressed. Importantly, the expression of the inducer protein in cells induces UPR, which in turn results in controllable intron splicing. There are many ways in which the expression of an inducer protein can be achieved in a cell population. In a suitable embodiment, the cell population is transfected with an expression vector adapted to express an inducer protein in the cells. In one embodiment, cells can be infected with a virus that produces the expression of a viral protein resulting in ER stress and the induction of UPR. Recombinant viral vectors encoding viral or nonviral proteins can also be used, and an example of AAV being expressed in cells to induce UPR is described below; this forms one preferred embodiment of the present invention. Alternatively, essentially any other type of expression vector (e.g., plasmid) can be introduced into cells to express heterologous proteins. Appropriate methods for transfecting cells with appropriate expression vectors are well known in the art. The properties of the induced protein are typically not of particular concern, but it is generally preferable that the protein is non-toxic; rather, what is important is the ER stress that occurs in cells when heterologous protein production takes place.

[0117] In some embodiments, step b) includes transfecting the cell population with an expression vector capable of expressing an inducement protein, preferably a heterologous protein, in the cells. Alternatively, step b) may include inducing the expression of the inducement protein from an expression vector already introduced into the cells, for example, prior to step a).

[0118] The expression of the inducible protein may be regulated by constitutive or non-constitutive promoters. An example of a non-constitutive promoter is an inducible promoter (in this case, the inducible promoter is not the UPR-inducible promoter).

[0119] Other methods for inducing an unfolding protein response include exposing cells to hypoxia or carbohydrate (e.g., glucose) deficiency.

[0120] As mentioned above, given the ubiquity of IRE1-mediated intron slicing across eukaryotes, the method can be carried out in any type of eukaryotic cell. Therefore, the method can be carried out in, for example, fungal cells (e.g., yeast cells), animal (metazoan) cells (e.g., mammalian cells), and plant cells.

[0121] In a particular preferred embodiment, the eukaryotic cell population is an animal (metazoan) cell population. Appropriately, the animal cells may be invertebrate or vertebrate-derived cells.

[0122] In some preferred embodiments, the eukaryotic cell population is a mammalian cell population. A variety of mammalian cells can be used, including, but are not limited to, Chinese hamster ovary (CHO) cells, human fetal kidney (HEK) cells (e.g., HEK-293), human fetal retinal cells, human amniotic cells, and mouse myeloma lymphoblastoid cells. In such embodiments, it may be preferable that the controllable intron contains the sequence CNG / CNG[CG], i.e., a mammalian splice target consensus sequence, at one, the other, or both (preferably both) ends of the intron. Appropriately, for example, the controllable intron may contain the sequence: CNG / CNGCACUCAGACUACGUGCACCUCNG / CNGC (Sequence ID 6), CAG / CAGCACUCAGACUACGUGCACCUCUG / CUGC (Sequence ID 7), or [ka] It has (Sequence ID 17).

[0123] In other embodiments, the eukaryotic cell population is an insect cell population. Suitable insect cells used in this method include baculovirus-infected and uninfected cells, such as those derived from the following insect species: armyworm (Spodoptera frugiperda) (e.g., Sf9 or Sf21), nettle moth (Trichoplusia ni) (e.g., Hi-5), and fruit fly (Drosophila melanogaster) (e.g., Schneider 2 cells and Schneider 3 cells).

[0124] In other embodiments of the present invention, the eukaryotic cell population is appropriately a population of eukaryotic cells, preferably yeast cells. Suitable fungal cells used in this method include, but are not limited to, Saccharomyces cerevisiae, Pichia pastoris, Aspergillus species, Trichoderma species, and Myceliophthora thermophila.

[0125] In another embodiment of the present invention, the eukaryotic cell population is appropriately a population of plant cells or plant protoplasts.

[0126] In another embodiment of the present invention, the eukaryotic cell population is appropriately a population of protozoan cells, for example, Leishmania tarentolae.

[0127] Step d), that is, the step of isolating the expression product from the cell population, can be carried out using conventional techniques well known in the art. Such techniques, of course, will vary depending on the properties of the expression product.

[0128] The method may appropriately include a step of introducing the nucleic acid expression construct into cells. Many well-known methods exist for transfection into eukaryotic cells, and those skilled in the art can easily select a suitable method for any cell type. The nucleic acid expression construct can, of course, be provided in any suitable vector.

[0129] In a further embodiment, the present invention provides nucleic acid expression constructs, vectors, cells, or pharmaceutical compositions according to various aspects of the present invention, which are used in methods of treatment or therapy.

[0130] As used herein, the terms “to treat” or “treatment” refer to both therapeutic treatments and preventive or protective measures. Beneficial or desirable clinical outcomes include, but are not limited to, prevention of undesirable clinical conditions or disabilities, whether detectable or undetectable; reduction of the incidence of disability; reduction of symptoms associated with disability; reduction of the degree of disability; stabilization (i.e., non-worsening) of disability; delay or slowing of the progression of disability; improvement or mitigation of the disability; remission (whether partial or whole); or a combination thereof. “Treatment” may also mean an extension of survival compared to the expected survival without treatment.

[0131] As used herein, the terms “therapeutic treatment” or “treatment” mean treatment aimed at restoring the body or its components from an undesirable physiological change or disorder to a desirable state, for example, a less severe or uncomfortable state (e.g., improvement or relief), or to a normal, healthy state (e.g., to restore the health, physical integrity, and physical satisfaction of the subject), or to maintain it in the aforementioned undesirable physiological change or disorder (e.g., to stabilize or prevent deterioration), or to prevent or slow progression to a more severe or worse state compared to the aforementioned undesirable physiological change or disorder.

[0132] As used herein, the terms “prevention,” “preventive action,” or “suppressive action” include preventing the onset of a disease or disorder, including reducing the severity of the disease or disorder or its associated symptoms before suffering from the disease or disorder. Such pre-suffering prevention or reduction refers to the administration of the nucleic acid expression constructs, vectors, or pharmaceutical compositions described herein to patients who are not suffering from apparent symptoms of the disease or disorder at the time of administration. “Preventing” also includes, for example, preventing recurrence or relapse of the disease or disorder after a period of improvement. In embodiments, the nucleic acid expression constructs, vectors, or pharmaceutical compositions described herein may be for gene therapy.

[0133] The present invention also provides the use of nucleic acid expression constructs, vectors, or pharmaceutical compositions described herein for the manufacture of pharmaceuticals for gene therapy.

[0134] A method for gene therapy in a subject requiring such gene therapy, comprising the following steps, is also disclosed herein: - A gene therapy vector comprising a pharmaceutical composition containing a nucleic acid expression construct according to the present invention is introduced into a target, thereby delivering the nucleic acid expression construct to the target cells of the target, wherein the nucleic acid expression construct comprises a sequence encoding a therapeutic expression product; and - A step of expressing a therapeutically effective amount of a functional therapeutic expression product in target cells.

[0135] It will be clear that the expression of therapeutically effective amounts of functional therapeutic expression products occurs only in cells where UPR is active. In many cases, UPR is active in cells under stress, for example, cancerous or infected with pathogens (e.g., viruses). Therefore, the fact that the expression of functional therapeutic products (which may be toxic proteins or other cytotoxic agents) occurs only in cells where UPR is active is an advantage of the present invention. This may be useful in mitigating or avoiding undesirable off-target expression of functional therapeutic expression products.

[0136] Therefore, in various embodiments of the present invention directed toward treatment, the condition to be treated is preferably cancer or an infectious disease (e.g., a viral infection).

[0137] Alternatively, UPR can be induced in cells using appropriate UPR inducers. Various UPR inducers are discussed above, and appropriate UPR inducers include pharmaceutically acceptable agents that induce UPR (e.g., bortezomib, forskolin) and agents that disrupt the lipid balance in cells to induce UPR (e.g., saturated fatty acids discussed above). UPR inducers can be delivered directly to the target site (e.g., by injection) or administered systemically.

[0138] The therapeutic expression product may be a polypeptide / protein, such as a secreted protein or peptide, such as a coagulation factor like factor VIII or factor IX, insulin, erythropoietin, lipoprotein lipase, antibody or nanobody, growth factor, cytokine, chemokine, plasma factor, or toxic protein. Alternatively, the therapeutic expression product may be RNA such as siRNA or miRNA.

[0139] In some preferred embodiments, therapeutic expression products may be toxic to cells into which the gene therapy vector has been introduced. In such embodiments, induction of UPR can be used to induce toxicity in cells, e.g., cell death. Thus, induction of UPR and the resulting expression of therapeutic expression products can be used to induce a kill switch in the cells. Suitable toxic expression products, such as proteins, are well known in the art and include, for example, caspase 3, caspase 8, and caspase 9.

[0140] Suitable gene therapy vectors used in this aspect of the present invention are discussed above.

[0141] Gene therapy protocols are widely described in the art. These include, but are not limited to, intramuscular injection of appropriate vectors, hydrodynamic gene delivery in various tissues including muscle, interstitial injection, intravenous infusion in the airway, application to the endothelium and liver parenchyma, and intravenous or intra-arterial administration. Various devices have been developed to enhance the efficacy of DNA to target cells. A simple approach is to physically bring a DNA-containing catheter or implantable material into contact with the target cells. Another approach is to utilize needleless jet injectors that fire a column of liquid directly into the target cells under high pressure. These delivery paradigms can also be used to deliver vectors. Another approach to targeted gene delivery is the use of molecular conjugates consisting of proteins or synthetic ligands to which nucleic acid binders or DNA binders are attached for specific targeting of nucleic acids to cells (Cristiano et al., 1993).

[0142] The terms “subject” and “patient” are used interchangeably herein and refer to animals, preferably vertebrates, more preferably mammals, specifically including human patients and non-human mammals. “Mammalian” subjects include, but are not limited to, humans. A preferred patient or subject is a human subject.

[0143] As used herein, “therapeutic dose” or “therapeutic effective dose” refers to the amount of expression product effective in treating a disease or disorder in a subject, i.e., effective in obtaining the desired local or systemic effect. Thus, the term refers to the amount of expression product that elicits a biological or pharmacokinetic response in a tissue, system, animal, or human, as sought by researchers, veterinarians, physicians, or other clinicians. Such amounts typically depend on the gene product and the severity of the disease, but can be determined by those skilled in the art, perhaps through routine experimentation.

[0144] The expression level of an expression product (e.g., a protein) can be measured by various conventional means, such as antibody-based assays, e.g., Western blotting or ELISA assays, to assess whether therapeutic expression of the expression product has been achieved. The expression of an expression product can also be measured in bioassays that detect the enzymatic or biological activity of the gene product.

[0145] In a further embodiment, the present invention provides a synthetic UPR-inducible promoter comprising a synthetic UPR-responsive cis-regulatory element. Preferably, the UPR-responsive cis-regulatory element comprises at least one binding site to ATF6, XBP1, or bZIP60, or a homolog or other equivalent transcription factor that drives gene expression as part of the UPR.

[0146] Such promoters can be used to selectively drive the expression of desired expression products in eukaryotic cells by inducing UPRs. While the use of such promoters in combination with the controllable introns discussed above may be advantageous for precise regulation of gene expression, in other situations, UPR-inducible promoters can be used without controllable introns.

[0147] A UPR-responsive cis-regulatory element is a sequence containing a functional transcription factor binding site (TFBS) for one or more transcription factors that drive gene expression as part of the UPR. These include, but are not limited to, ATF6, XBP1, and bZIP60, as discussed above. A UPR-responsive cis-regulatory element containing a TFBS for ATF6 is of particular interest in this invention. Preferably, the UPR-responsive cis-regulatory element is UPR-specific, i.e., it enhances expression only during the UPR. For example, it is generally preferable that it does not contain any TFBS for transcription factors not involved in the UPR.

[0148] A synthetic UPR-inducible promoter typically comprises at least one synthetic UPR-responsive cis-regulatory element operably ligated to a minimal promoter or proximal promoter. When the cis-regulatory element is operably ligated to the proximal promoter, the proximal promoter should itself be a UPR-inducible promoter that does not drive the transcription of the operably ligated gene in eukaryotic cells when UPR is not induced. A minimal promoter typically cannot drive expression without the presence of an additional regulatory element. Examples of suitable minimal promoters used in the present invention include, but are not limited to, the CMV-minimal (SEQ ID NO: 21) promoter and the MinTk minimal promoter. Other suitable minimal promoters are known in the art.

[0149] Appropriately, a UPR-inducible promoter includes a UPR-responsive cis-regulatory element containing one or more copies of at least one of the following transcription factor target sequences: - TGACGTG (ATF6 transcription factor binding site consensus sequence) - TGACGTGCT (the variant above), - TGACGTG[TG] (known as the UPR site), - CCAAT-N9-CCACG (known as the ERSE1 site) (SEQ ID NO: 18), and - ATTGG-N-CCACG (known as the ERSE2 site) (SEQ ID NO: 19).

[0150] Appropriately, the synthetic UPR-responsive cis-regulatory element comprises two or more copies, preferably three or more, and appropriately five or more, of at least one of the transcription factor target sequences listed above. Alternatively, or additionally, the synthetic UPR-responsive cis-regulatory element appropriately comprises one or more copies of at least two of the transcription factor target sequences listed above.

[0151] Transcription factor target sequences may be directly adjacent to each other (tandem repeats), or they may be spaced apart by, for example, spacer sequences or other functional sequences (e.g., other transcription factor target sequences). Typically, if present, spacer sequences are 5 to 50 nucleotides long, but may be longer or shorter in some cases. For example, spacer sequences may be appropriately 2 to 50 nucleotides long, appropriately 4 to 30 nucleotides long, or appropriately 5 to 20 nucleotides long. Spacer sequences may preferably be multiples of 5 nucleotides in length, as this provides an integer number of half-turns of the DNA double helix (one complete turn corresponds to approximately 10 nucleotides in chromatin). Spacer sequence lengths that are multiples of 10 nucleotides may be more preferable, as this also provides an integer number of complete turns of the DNA double helix. The spacer sequence can be essentially any sequence, provided that it does not prevent the UPR-responsive cis-regulatory element from functioning as required (e.g., it contains a silencer sequence or prevents the binding of the desired transcription factor). The spacer sequences between each transcription factor target sequence may be identical or they may be different.

[0152] In a preferred embodiment, the UPR-responsive cis-regulatory element includes one or more copies of the transcription factor target sequence TGACGTG (i.e., the ATF6 consensus sequence), preferably three or more copies of the transcription factor target sequence TGACGTG, preferably five or more copies of the transcription factor target sequence TGACGTG, for example, six or more copies of the transcription factor target sequence TGACGTG. As mentioned above, these transcription factor target sequences may be tandem repeats or spaced apart from one another. Generally, it is preferable that at least two, preferably all, of the transcription factor target sequences present in the UPR-responsive cis-regulatory element are spaced apart from one another, for example, by spacer sequences as discussed above.

[0153] Appropriately, the UPR-responsive cis-regulatory element includes one or more copies of the transcription factor target sequence TGACGTGCT, preferably three or more copies of TGACGTGCT, preferably five or more copies of TGACGTGCT, for example, six or more copies of TGACGTGCT. As mentioned above, these may be tandem repeats or spaced apart from one another. Generally, it is preferable that at least two, preferably all, of the transcription factor target sequences present in the UPR-responsive cis-regulatory element are spaced apart from one another, for example, by spacer sequences as discussed above. The transcription factor target sequence TGACGTGCT has been found to be particularly effective when used in multiple copy numbers in the UPR-responsive cis-regulatory element, whether as tandem repeats or including spacer sequences.

[0154] In some embodiments of the present invention, the UPR-responsive systolic element is arranged The sequence includes TGACGTG-S-TGACGTG-S-TGACGTG-S-TGACGTG-S-TGACGTG-S-TGACGTG (Sequence ID 54), where S represents an optional spacer sequence as defined above. Preferably, the spacer sequence as defined above is located between at least two, preferably all, transcription factor target sequences (TGACGTG).

[0155] In some embodiments of the present invention, the UPR-responsive systolic element is arranged Includes TGACGTGCTTGACGTGCTTGACGTGCTTGACGTGCTTGACGTGCTTGACGTGCT (Sequence ID 20).

[0156] In another embodiment of the present invention, the UPR-responsive sys-controllable element is arranged The sequence includes TGACGTGCT-S-TGACGTGCT-S-TGACGTGCT-S-TGACGTGCT-S-TGACGTGCT-S-TGACGTGCT (Sequence ID 55), where S represents an optional spacer sequence as defined above. Preferably, the spacer sequence as defined above is located between at least two, preferably all, transcription factor target sequences.

[0157] In another embodiment of the present invention, the UPR-responsive sys-controllable element is arranged The sequence includes TGACGTGCTGATGATGCGTAGCTAGTAGTTGACGTGCTGATGATGCGTAGCTAGTAGTTGACGTGCTGATGATGCGTAGCTAGTAGTTGACGTGCTGATGATGCGTAGCTAGTAGTTGACGTGCTGATGATGCGTAGCTAGTAGTTGACGTGCT (SEQ ID NO: 56), or a sequence that is at least 50% identical thereto, more preferably at least 70% identical thereto, more preferably at least 80% identical thereto, and more preferably at least 85%, 90%, 95%, 98%, or 99% identical thereto. It is very preferable that the sequence variation exists only in sequences that are not transcription factor target sequences, i.e., sequences containing the sequence TGACGTGCT. It is generally preferable that the sequence variation exists only in spacer sequences (i.e., sequences containing the sequence GATGATGCGTAGCTAGTAGT (SEQ ID NO: 61)).

[0158] In some embodiments of the present invention, the UPR-inducible promoter is the following sequence The sequence includes TGACGTGCTTGACGTGCTTGACGTGCTTGACGTGCTTGACGTGCTTGACGTGCTGGTACCGTCGACGATATCGGATCCAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTAGATACGCCATCCACGCTGTTTTGACCTCCATAGAAGATCGCCACC (SEQ ID NO: 22), or a sequence that is at least 70% identical thereto, more preferably at least 80% identical thereto, more preferably at least 90% identical thereto, and more preferably at least 95%, 96%, 97%, 98%, or 99% identical thereto. This UPR-inducible promoter includes the UPR-responsive cis-regulatory element of SEQ ID NO: 20 operably linked to the CMV-MP minimal promoter. It is very preferable that the sequence variation exists only in sequences that are neither transcription factor target sequences, i.e., sequences having TGACGTGCT, nor CMV-MP sequences.

[0159] In another embodiment of the present invention, the UPR-inducible promoter is the following sequence: The sequence includes TGACGTGCTGATGATGCGTAGCTAGTAGTTGACGTGCTGATGATGCGTAGCTAGTAGTTGACGTGCTGATGATGCGTAGCTAGTAGTTGACGTGCTGATGATGCGTAGCTAGTAGTTGACGTGCTGATGATGCGTAGCTAGTAGTTGACGTGCTGATGATGCGTAGCTAGTAGTGCAGTTAGCGTAGCTGAGGTACCGTCGACGATATCGGATCCAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGAT (SEQ ID NO: 57), or a sequence that is at least 50% identical thereto, more preferably at least 70% identical thereto, more preferably at least 80% identical thereto, and more preferably at least 85%, 90%, 95%, 98%, or 99% identical thereto. This UPR-inducible promoter includes the UPR-responsive cis-controllable element of SEQ ID NO: 56 operably linked to the CMV-MP minimal promoter. It is highly preferable that sequence variations exist only in sequences that are neither transcription factor target sequences (i.e., sequences containing TGACGTGCT) nor CMV-MP sequences. It is generally preferable that sequence variations exist only in spacer sequences (i.e., sequences containing GATGATGCGTAGCTAGTAGT (SEQ ID NO: 61)).

[0160] In another embodiment of the present invention, the UPR-inducible promoter is the following sequence: The sequence includes TGACGTGCTGATGATGCGTAGCTAGTAGTTGACGTGCTGATGATGCGTAGCTAGTAGTTGACGTGCTGATGATGCGTAGCTAGTAGTTGACGTGCTGATGATGCGTAGCTAGTAGTTGACGTGCTGATGATGCGTAGCTAGTAGTTGACGTGCTGATGATGCGTAGCTAGTAGTGCAGTTAGCGTAGCTGAGAGGTACCGTCGACGATATCGGATCCTTCGCATATTAAGGTGACGCGTGTGGCCTCGAACACCGAG (SEQ ID NO: 58), or a sequence that is at least 50% identical thereto, more preferably at least 70% identical thereto, more preferably at least 80% identical thereto, and more preferably at least 85%, 90%, 95%, 98%, or 99% identical thereto. This UPR-inducible promoter includes the UPR-responsive cis-controllable element of SEQ ID NO: 56 operably linked to the MinTK minimal promoter. It is highly preferable that sequence variations exist only in sequences that are neither transcription factor target sequences (i.e., sequences containing TGACGTGCT) nor MinTK sequences. It is generally preferable that sequence variations exist only in spacer sequences (i.e., sequences containing GATGATGCGTAGCTAGTAGT (sequence number 61)).

[0161] A UPR-inducible promoter, preferably in eukaryotic cells, does not drive transcription of an operablely linked gene in the absence of UPR. In eukaryotic cells, the UPR-inducible promoter drives transcription of an operablely linked gene when UPR is present in the cell. The ability of a UPR-inducible promoter to selectively drive transcription upon UPR introduction can be readily assessed by those skilled in the art using various approaches, which can be tailored to specific expression systems in which the construct is intended to be used. One preferred example is the methodology described in the following examples, for example, Example 8. For example, any candidate UPR-inducible promoter to be evaluated can be substituted in the construct described in Example 8 for the exemplary ATF6-containing UPR-inducible promoter used in Example 8, and the ability of the candidate UPR-inducible promoter to selectively drive transcription upon UPR introduction can be measured by evaluating the level of luciferase expression before and after UPR induction by 2 mM DTT, as is done in Example 8. A UPR-inducible promoter can successfully induce gene expression by significantly increasing the transcription of a gene (in the case of Example 8, the luciferase gene) operably linked by UPR induction. Preferably, the UPR-inducible promoter gives at least a 5-fold increase in gene (e.g., luciferase) expression 24 hours after UPR induction at 2 mM DTT, more preferably at least a 10-fold increase, more preferably at least a 100-fold increase, and even more preferably at least a 1000-fold increase. Before UPR induction, the expression level of the gene (e.g., luciferase) is preferably minimal, preferably negligible. Minimal expression can be defined as being equal to or less than the expression level of a negative control construct (i.e., a construct in which the expression of the luciferase-coding sequence is driven by CMV-MP alone), such as the one used in Example 8, preferably less than 50%, preferably less than 20%, more preferably less than 10%, even more preferably less than 5%, and even more preferably less than 1% of the expression level of the negative control construct. The expression level is, for example, essentially undetectable using the methodology of Example 8.

[0162] The present invention also provides an expression construct or vector comprising a synthetic UPR-inducible promoter, as described above, operably linked to a nucleic acid sequence encoding an expression product. The expression construct or vector may be any expression construct or vector as discussed above in other aspects of the present invention. The expression product may be any expression product (e.g., encoding a protein) as discussed above in other aspects of the present invention.

[0163] In preferred embodiments, the expression product is not a reporter protein; that is, it does not encode a protein commonly used as an indicator of expression level. Many reporter genes are known in the art, including, in particular, fluorescent, luminescent, and pigment-producing proteins. Therefore, in some preferred embodiments, the expression product is neither a fluorescent nor a luminescent protein; for example, it is not luciferase. As indicated above, preferred expression products include therapeutic proteins and toxic proteins.

[0164] In a further embodiment, the present invention provides a method for producing an expression product, which includes: a) A step of supplying a population of eukaryotic cells comprising a synthetic nucleic acid expression construct comprising a UPR-inducible promoter operably linked to a nucleic acid sequence encoding the expression product according to the present invention; b) Processing the cell population to induce an unfolding protein response, thereby inducing transcription from the UPR-inducible promoter; c) A step of incubating the cell population under conditions optimal for the production of the expression product; and d) A step of isolating the expression product from the cell population.

[0165] Further, optional, and preferred features of the method for producing the expression product are discussed above in other aspects of the present invention, which are applicable to the present embodiment with modifications where appropriate. The expression product is preferably a therapeutic protein or a toxic protein. It is preferable that the expression product is not a reporter protein.

[0166] Accordingly, further aspects of the present invention provide a pharmaceutical composition comprising a nucleic acid expression construct or vector comprising a UPR-inducible promoter operably linked to a nucleic acid sequence encoding an expression product according to the present invention. Further, optional, and preferred features of the pharmaceutical composition are discussed above in relation to other aspects of the present invention, which are applied to the present embodiment with modifications where appropriate.

[0167] In a further embodiment of the present invention, the use of nucleic acid expression constructs and vectors comprising a nucleic acid sequence encoding an expression product according to the present invention and a UPR-inducible promoter operably linked thereto is provided for the production of pharmaceutical compositions.

[0168] Further embodiments of the present invention provide cells comprising a synthetic nucleic acid expression construct or vector containing a UPR-inducible promoter according to the present invention. Further, optional, and preferred features of such cells are discussed above in relation to other embodiments of the present invention, which are applied to the present embodiment with modifications where appropriate.

[0169] In further embodiments, the present invention provides nucleic acid expression constructs, vectors, cells, or pharmaceutical compositions comprising a UPR-inducible promoter according to the present invention, for use in methods of treatment or therapy. Further, optional, and preferred features of such methods are discussed above in other embodiments of the present invention, which are applied to the present embodiments with modifications where appropriate.

[0170] To facilitate understanding of this invention, several terms are defined below. Terms as defined herein have meanings that are generally understood by those skilled in the art. Technical terms used herein are used to describe specific embodiments of the invention, but their use does not define the scope of the invention, except as set forth in the claims.

[0171] The background discussion of the invention in this specification is included for the purpose of illustrating the subject matter of the invention. This should not be construed as an acknowledgment that any of the materials referred to were published, known, or part of the common general knowledge in any country as of the priority date of any of the claims herein.

[0172] Throughout this disclosure, various publications, patents, and published patent specifications are referenced by identifying citation. All documents referenced herein are incorporated herein by reference in whole, and in particular, the teachings or portions of such documents specifically referenced herein are incorporated by reference.

[0173] The implementation of this invention will, unless otherwise indicated, utilize the usual techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the capabilities of those skilled in the art. Such techniques are well described in the literature. For example, *Current Protocols in Molecular Biology* (Ausubel, 2000, Wiley and Son Inc., Library of Congress, USA); *Molecular Cloning: A Laboratory Manual*, 3rd edition (Sambrook et al., 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); *Oligonucleotide Synthesis* (edited by MJ Gait, 1984); U.S. Patent No. 4,683,195; *Nucleic Acid Hybridization* (edited by Harrys and Higgins, 1984); *Transcription and Translation* (edited by Hames and Higgins, 1984); *Culture of Animal Cells* (Freshney, Alan R. Liss, Inc., 1987); *Immobilized Cells and Enzymes* (IRL Press, 1986); *Perbal, A Practical Guide to Molecular Cloning* (1984); Series, *Methods in Enzymology* (edited by Abelson and Simon, Academic Press, Inc.)See, specifically, Volumes 154 and 155 (edited by Wu et al.), and Volume 185, "Gene Expression Technology" (edited by Goeddel); Gene Transfer Vectors For Mammalian Cells (edited by Miller and Calos, 1987, Cold Spring Harbor Laboratory); Immunochemical Methods in Cell and Molecular Biology (edited by Mayer and Walker, Academic Press, London, 1987); Handbook of Experimental Immunology, Volumes I-IV (edited by Weir and Blackwell, 1986); and Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986).

[0174] Terms such as "a," "an," and "the" are not intended to refer only to singular entities, but rather to include general classes for which specific examples may be used for illustrative purposes.

[0175] As used herein, the terms “comprising,” “comprises,” and “comprised of” are synonymous with “including,” “includes,” or “containing,” and are inclusive or open-ended, and do not exclude additional, unlisted features, elements, or process steps.

[0176] The enumeration of numerical ranges by endpoints includes all numbers and fractions contained within each range, as well as the enumerated endpoints.

[0177] As used herein, the term “nucleic acid” typically refers to an oligomer or polymer of any length (preferably a linear polymer) composed essentially of nucleotides. A nucleotide unit generally includes a heterocyclic base, a sugar group, and at least one, e.g., one, two, or three phosphate groups, including a modified or substituted phosphate group. Heterocyclic bases may include, among others, purine and pyrimidine bases, e.g., adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U), which are widely present in naturally occurring nucleic acids, other naturally occurring bases (e.g., xanthine, inosine, hypoxanthine), and chemically or biochemically modified (e.g., methylated), unnatural, or derivatized bases. The sugar groups may include, in particular, pentose (pentofuranose) groups, for example, preferably ribose and / or 2-deoxyribose, which are common in naturally occurring nucleic acids, or arabinose, 2-deoxyarabinose, threose, or hexose sugar groups, in addition to modified or substituted sugar groups. Nucleic acids as intended herein may include naturally occurring nucleotides, modified nucleotides, or mixtures thereof. Modified nucleotides may include modified heterocyclic bases, modified sugar moieties, modified phosphate groups, or combinations thereof. Modification of phosphate groups or sugars may be introduced to improve stability, resistance to enzymatic degradation, or several other useful properties. The term “nucleic acid” more preferably encompasses DNA, RNA, and DNA-RNA hybrid molecules, specifically including hnRNA, premRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemosynthesized) DNA, RNA, or DNA-RNA hybrids. Nucleic acids can be natural, for example, existing in nature or being isolated from nature; or they can be non-natural, for example, recombinant, i.e., produced by recombinant DNA technology, and / or partially or whole, chemically or biochemically synthesized. “Nucleic acids” can be double-stranded, partially double-stranded, or single-stranded. If single-stranded, nucleic acids can be a sense strand or an antisense strand. In addition, nucleic acids can be cyclic or linear.

[0178] As used herein, “nucleic acid expression construct” refers to a nucleic acid molecule comprising one or more transcriptional regulatory elements (e.g., non-limitingly, promoters, enhancers, and / or regulatory elements, polyadenylated sequences, and introns) that direct expression in one or more desired cell types, tissues, or organs. The nucleic acid expression constructs of the present invention are synthetic nucleic acid molecules.

[0179] In this application, "synthetic" means nucleic acid molecules that do not exist in nature. The synthetic nucleic acid expression constructs of the present invention are artificially produced, typically by recombinant technology. Such synthetic nucleic acids may contain naturally occurring sequences (e.g., promoters, enhancers, introns, and other such regulatory sequences), but these may be present in circumstances not present in nature. For example, a synthetic gene (or part of a gene) typically contains one or more nucleic acid sequences that are not adjacent in nature (chimeric sequences), and / or may include substitutions, insertions, deletions, and combinations thereof.

[0180] As used herein, the terms “operably linked,” “operably connected,” or equivalent expressions refer to the arrangement of various nucleic acid elements relative to each other such that the elements are functionally connected and can interact with each other in an intended manner. Such elements include, but are not limited to, promoters, enhancers, and / or regulatory elements, polyadenylated sequences, one or more introns and / or exons, and coding sequences of genes of interest to be expressed. When properly oriented or operably linked, nucleic acid sequence elements may act together to modulate each other’s activity and ultimately affect the level of expression of the expression product. Modulation means increasing, decreasing, or maintaining the level of activity of a particular element. The position of each element relative to other elements may be expressed with respect to the 5' and 3' ends of each element, and the distance between any particular elements may be referred to by the number of intervening nucleotides or base pairs between the elements. As will be understood by those skilled in the art, operably linked means functional activity and does not necessarily relate to linking in native positions. In fact, when used in nucleic acid expression cassettes, cis-regulatory elements are typically located immediately upstream of the promoter (although this is generally true, it should not be strictly interpreted as a limitation or exclusion of location within the nucleic acid expression cassette), but this is not necessarily true in vivo. For example, a regulatory element sequence that is naturally located downstream of a gene whose transcription is affected can function similarly when located upstream of its promoter. Thus, according to certain embodiments, the regulatory or enhancing effect of a regulatory element is location-independent.

[0181] "Consensus sequence" - The meaning of consensus sequence is well known in the art. In this application, unless the context indicates otherwise, the following notation is used for consensus sequences. The following exemplary DNA sequence: A[CT]N{A}YR Considering this, A means that A is always found at that position; [CT] represents either C or T at that position; N represents any base at that position; and {A} means that any base other than A is found at that position. Y represents any pyrimidine, and R represents any purine.

[0182] Unless otherwise indicated by the context, “controllable intron” in this application means a nucleic acid sequence present in an RNA molecule (typically a transcript) containing a resectable sequence adjacent to a target site for a ribonuclease, generally IRE1 or its homolog or ortholog, wherein the resectable sequence can be excised from the RNA molecule by the action of the ribonuclease as a result of an unfolding protein response. In some cases in the art, the term intron is used to refer only to sequences that are excised from an RNA molecule (i.e., cut out by splicing), but this is not very appropriate in the case of controllable introns of the present invention.

[0183] The terms "identity" and "identical" refer to the sequence similarity between two polymer molecules, for example, two nucleic acid molecules, for example, two DNA molecules. Sequence alignment and sequence identity determination can be performed using, for example, the Basic Local Alignment Search Tool (BLAST), first described by Altschul et al. in 1990 (J Mol Biol 215: pp. 403-401), or the "Blast 2 sequence" algorithm described by, for example, Tatusova and Madden in 1999 (FEMS Microbiol Lett 174: pp. 247-250).

[0184] Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are cited, for example, in Smith and Waterman (1981) Adv. Appl. Math. 2: p. 482; Needleman and Wunsch (1970) J. Mol. Biol. 48: p. 443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: p. 2444; Higgins and Sharp (1988) Gene 73: pp. 237-244; Higgins and Sharp (1989) CABIOS 5: pp. 151-153; Corpet et al. (1988) Nucleic Acids Res. 16: pp. 10881-90; Huang et al. (1992) Comp. Appl. Biosci. 8: pp. 155-155; Pearson et al. (1994) Methods Mol. Biol. 24: pp. 307-31. This is described in Tatiana et al. (1999) FEMS Microbiol. Lett. 174: pp. 247-245. A detailed examination of sequence alignment methods and homology calculations can be found, for example, in Altschul et al. (1990) J. Mol. Biol. 215: pp. 403-410.

[0185] The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST®; Altschul et al. (1990)) is available online from several sources, including the National Center for Biotechnology Information (Bethesda, MD), and is used in conjunction with several sequence analysis programs. Instructions on how to determine sequence identity using this program are available online in the "Help" section for BLAST®. For nucleic acid sequence comparison, the "Blast 2 Sequence" function of the BLAST® (Blastn) program can be used with default parameters. Nucleic acid sequences with higher similarity to a reference sequence will show an increase in percentage identity when evaluated using this method. Typically, percentage sequence identity is calculated over the entire length of the sequence.

[0186] For example, the global optimal alignment can be appropriately found using the Needleman-Wunsch algorithm with the following scoring parameters: match score: +2, mismatch score: -3; gap penalty: gap open 5, gap stretch 2. The percentage identity of the resulting optimal global alignment is appropriately calculated by multiplying the number of matched bases by 100 and the ratio of the number of matched bases to the total length of that alignment, where the length of the alignment includes both the number of matches and mismatches.

[0187] As used herein, "cell culture" refers to a proliferative mass of cells that may be in either an undifferentiated or differentiated state.

[0188] Where used herein, “cis-regulating element” or “CRE” is a term known to those skilled in the art; it relates to a region of non-coding DNA that regulates the transcription (i.e., in cis) of an adjacent gene. CREs typically regulate gene transcription by binding to transcription factors. CREs can be, for example, enhancers, promoters, insulators, or silencers. In this case, a UPR-inducible CRE is typically an enhancer element that binds to a transcription factor, acting to induce transcription as part of a UPR. In this context, if the CRE is provided as part of a gene encoding a promoter and an expression product, the UPR-inducible CRE is preferably located within 1500 nucleotides from the transcription start site (TSS), more preferably within 1000 nucleotides from the TSS, more preferably within 500 nucleotides from the TSS, and appropriately within 250 nucleotides, 200 nucleotides, 150 nucleotides, or 100 nucleotides from the TSS.

[0189] As used herein, “complementary” or “complementarity” refers to Watson-Crick base pairing between two nucleic acid sequences. For example, the sequence 5'-AGT-3' pairs with its complementary sequence 3'-TCA-5'. Complementarity between two nucleic acid sequences can be “partial,” where only some of the bases pair with their complements, or it can be complete, such as when every base in the sequence pairs with its complementary base. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

[0190] As used herein, “ordinary introns” refers to introns normally present in premRNA, typically spliceosome introns that do not encode information about protein synthesis and are removed from the mRNA molecule before mRNA translation. The term is used to distinguish such introns from the non-standard regulatory introns of the present invention.

[0191] In this application, "transfection" broadly refers to any process of systematically introducing nucleic acids into cells, encompassing the introduction of viruses and non-viral vectors, and including terms such as transformation and transduction. Examples include, but are not limited to, transfection with viral vectors; transformation with plasmid vectors; electroporation (Fromm et al. (1986) Nature 319:791-73); lipofection (Feigner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-77); microinjection (Mueller et al. (1978) Cell 15:579-85); Agrobacterium-mediated transduction (Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80:4803-77); direct DNA uptake; whiskers-mediated transformation; and particulate gun (Klein et al. (1987) Nature 327:70).

[0192] As used herein, the phrase “transgene” refers to an exogenous nucleic acid sequence. In one example, the transgene is a gene sequence, a gene encoding an industrially or pharmaceutically useful compound, or a gene encoding a desired trait. In yet another example, the transgene is an antisense nucleic acid sequence, the expression of which inhibits the expression of a target nucleic acid sequence.

[0193] As used herein, the phrase “promoter” generally refers to a region of DNA located upstream of a transcribed nucleic acid sequence that is required for transcription to occur. Promoters enable the appropriate activation or suppression of transcription of a sequence under their regulation. Promoters typically contain a specific sequence that is recognized and bound by a transcription factor, such as an enhancer sequence. The transcription factor binds to the promoter DNA sequence, resulting in the recruitment of RNA polymerase (an enzyme that synthesizes RNA from the coding region of a gene). A great many promoters are known in the art.

[0194] As used herein, “minimal promoter” refers to a short DNA segment that is inactive or nearly inactive on its own but can mediate strong transcription when combined with other transcriptional regulatory elements. Minimal promoter sequences can originate from a variety of different sources, including prokaryotic and eukaryotic genes. Examples of minimal promoters include the dopamine beta-hydroxylase gene minimal promoter, the cytomegalovirus (CMV) initial gene minimal promoter (CMV-MP), and the herpesthymidine kinase minimal promoter (MinTK).

[0195] An "RNA transcript" or "transcript" refers to the product resulting from the RNA polymerase-catalyzed transcription of a DNA sequence. When an RNA transcript is typically a complete, complementary copy of a DNA sequence, it is called a primary transcript; or, if it is an RNA sequence derived from the post-transcriptional processing of a primary transcript, it is called mature RNA.

[0196] "Messenger RNA" or "(mRNA)" refers to the processed form of transcript RNA that does not contain introns and can be translated into protein by cells.

[0197] Embodiments of the present invention are described herein by reference to the accompanying drawings, as non-limiting examples. [Brief explanation of the drawing]

[0198] [Figure 1] This figure shows the ratio of GFP fluorescence from SYNP-XBP-01 transfected cells before and after induction by 2 mM DTT. [Figure 2a] This figure shows the ratio of GFP fluorescence to the positive control before and after induction. [Figure 2b] This figure shows fluorescence microscopy observations of SYNP-XBP-01 with and without 2mM DTT. [Figure 3]This figure shows GFP expression from both plasmids after induction by 2 mM DTT. The numbers represent the ratio to CMV-IE. [Figure 4] This figure shows the raw expression data from SYNP-ATF6-01 before and after the addition of 2 mM DTT. [Figure 5] This figure shows the raw expression data from SYNP-ATF6-02 before and after the addition of 2 mM DTT. [Figure 6] This figure shows fluorescence microscopy observations of HEK293F cells transfected with SYNP-ATF6-01 or SYNP-ATF6-02 before and after treatment with 2 mM DTT. [Figure 7] This figure shows EGFP fluorescence from HEK293F cells before and after induction with wtAAV. ZsGreen is a recombinant AAV that produces a GFP variant and is used as a control, while pre-ZsGreen is untransfected (i.e., blank). [Figure 8] This figure shows EGFP expression from HEK293F cells before and after wtAAV production. [Figure 9] This figure shows EGFP expression measured as a ratio to CMV-IE. The construct used was SYNP-ATF6-02 (containing introns and an ATF6 inducible promoter). HEK293F cells were transfected using Maxcyte reagent. 24 hours after transfection, the inducer was added, and GFP expression was tracked for the next 24 hours. Inducer concentrations: DTT 2 mM, palmitate 0.5 mM, MF-43 1 μM. [Figure 10] This figure shows the plasmid map of pMA-RQ. [Figure 11] This graph shows the expression of SEAP after DTT induction in CHO cells and HEK293 cells, compared to SEAP expression under the regulation of the CMV-IE promoter (the results shown are in HEK293 cells) (the X-axis indicates time after induction). [Figure 12]Graphs showing luciferase expression after induction with DTT or palmitate in CHO cells (A) and HEK293 cells (B) (X-axis represents time). For comparison, luciferase expression under regulation with the CMV-IE promoter and the CMV minimal promoter (CMV-MP) is shown. [Figure 13] This figure shows the plasmid map of SYNP-CAS9_INT. [Figure 14] Figure 14A shows a graph of HEK293 cell viability after induction of caspase 9 (CASP9) with DTT or forskolin, compared to negative controls and caspase 9 (CASP9) under CMV-IE promoter regulation (X-axis indicates time after induction). Figure 14B shows micrographs of the fluorescent marker Spark fused with CASP9 in cells where the expression construct did not contain controllable introns (left-hand panel) and in cells where the expression construct contained controllable introns (right-hand panel). [Figure 15] This graph shows the survival rate of HEK293 cells after caspase-9 induction with DTT, compared to a negative control (HEK293 cells + DTT) and CASP9 under CMV-IE promoter regulation. [Figure 16] Figures 16A to 16D show the estimated secondary structures formed when introns are used in the following cases: A. SEAP (CAG / CAGACGGGCAACTTTACACGACGCTG / CAG) B. EGFP (CAG / CTGGAGCACTCAGACTACGTGCACCTCTG / CTG) C. CASP9 (CAG / CAGACGGGCAACTTTACACGACGCTG / CTG) D. Luciferase (CCG / CAGACGGGCAACTTTACACGACGCTG / CAG) [Figure 17] The graphs show luciferase expression after DDT induction when a UPR-responsive cis-regulatory element containing a 5×ATF6 binding site was combined with two different minimal promoters (X-axis units represent time after induction). The controls were the CMV-IE promoter and the CMV-minimal promoter without any additional regulatory elements. [Modes for carrying out the invention] [Examples]

[0199] Use of XBP-1 introns as a regulatory mechanism for protein expression A 26 bp atypical (controllable) intron is present in the mRNA of XBP-1, which encodes a nonsense protein, before splicing. Once endoplasmic reticulum (ER) stress is sensed by the IRE1 protein, the atypical intron is removed via a specific splicing site, and the mRNA is then religated to form an mRNA transcript that can be translated to produce the XBP-1 protein. ER stress can be induced in many ways, for example, by the addition of chemicals or by the expression of heterologous proteins.

[0200] The inventors recognize that this process can be adapted to regulate protein expression at the mRNA / translation level by including controllable introns containing appropriate splice sites in the sequence of the gene of interest. Under the regulation of an appropriate promoter, mRNA is generated but cannot be processed to form a functional expression product of the gene of interest. However, once a UPR is induced, for example, by the administration of DTT or the expression of another heterologous protein, the mRNA can be processed, the controllable introns can be excised by splicing, and the mRNA can be translated into a functional protein.

[0201] This mechanism allows the production of proteins (e.g., toxic proteins) to be adjusted to suit manufacturing processes where induction is required before protein production. For example, protein translation can be delayed until a desired stage of fermentation is reached. This method can also be used for non-protein expression products such as functional RNA (e.g., siRNA or miRNA).

[0202] Splice sites and introns Consensus splice recognition site sequence for IRE1: CNG / CNG

[0203] This consensus sequence is conserved across eukaryotes. The consensus sequence CNG / CNG[CG], preferably CNG / CNGC, is typically found in mammalian cells, and the CNG / CNG consensus sequence is considered usable.

[0204] WT mammalian XBP1 splice recognition site sequence: 5' site: CCG / CAGC 3' site: CUG / CAGC

[0205] Mammalian-derived WT intron sequences: The intron sequence to be excised (SEQ ID NO: 23): cagcacucagacuacgugcaccucug

[0206] WT mammalian intron containing the sequence to be excised adjacent to the splice recognition site: CCG / cagcacucagacuacgugcaccucug / CAGC (SEQ ID NO: 24)

[0207] Construction of EGFP sequences containing controllable introns The region selected for intron insertion was determined by its sequence similarity to the splice recognition site. Only a single silent mutation from CTCG to CTGG was required at the 3' recognition site to enable intron insertion.

[0208] EGFP gene sequence (SEQ ID NO: 25) containing an underlined region for intron insertion. [ka]

[0209] EGFP protein sequence (SEQ ID NO: 26) MVSKGEELFT GVVPILVELD GDVNGHKFSV SGEGEGDATY GKLTLKFICT TGKLPVPWPT LVTTLTYGVQ CFSRYPDHMK QHDFFKSAMP EGYVQERTIF FKDDGNYKTR AEVKFEGDTL VNRIELKGID FKEDGNILGH KLEYNYNSHN VYIMADKQKN GIKVNFKIRH NIEDGSVQLA DHYQQNTPIG DGPVLLPDNH YLSTQSALSK DPNEKRDHMV LLEFVTAAGI TLGMDELYK

[0210] [[ID=I0]]The sequences used in this study are shown below. GFP is underlined, and introns and splice sites are shown in bold. In this example, an additional 3 bp sequence was added to the 5' end of the intron, generating an intron that was predicted to produce slightly suboptimal splicing. The intention was to maintain the background expression level, i.e., when the UPR has not yet been introduced, at a minimum. Floating adapted cells are typically in a slightly more stressed state than adherent cells, and thus this minor modification was expected to be effective in avoiding background expression. Of course, the wild-type intron can be used and may be preferred in some cases. The intron sequence used in this example is as follows (the additional 3 bp sequence is underlined): [Chemical formula] (SEQ ID NO: 27)

[0211] EGFP sequence with a controllable XBP-1 intron, CMV minimal promoter (CMV-MP), and SV40 polyA tail (the XBP-1 intron sequence is shown in bold, and the sequence encoding EGFP and its intron is underlined) (this construct is named SYNP-XBP-01) (SEQ ID NO: 28): [Chemical formula]

[0212] As shown above, EGFP with the controllable XBP-1 intron inserted therein can be seen to encode a truncated protein (SEQ ID NO: 29, 51, 52, 53 - SEQ ID NOs: 51, 52, 53 refer to fragmentary sequences after the first stop codon and are considered not to be translated):

Chemical formula

[0213] The DNA constructs used in this example: - CMV-MP-GFP: CMV-MP-regulated expression of EGFP - SYNP-XBP-01: CMV-MP-regulated expression of EGFP with the XBP1 intron

[0214] These constructs are identical except for the presence of the intron in the EGFP coding sequence, as shown above.

[0215] All of these constructs were synthesized by Geneart and provided in plasmid pMA-RQ (see Figure 10 for the plasmid map). Plasmid pMA-RQ containing these constructs was directly transfected into relevant cells.

[0216] The cell lines used in this example: - Freestyle HEK293F, Invitrogen catalog number: R790-07 - Freestyle CHO-S, Invitrogen catalog number: R80007

[0217] Proliferation of HEK293F and CHO-S cells - The cells were maintained according to the manufacturer's instructions. - Cells were transfected using MAX reagent (Invitrogen catalog number: 16447100).

[0218] The standard protocol for transfection of these cells was modified for 24-well plates as follows: - 40 ml of cells were grown in a 250 ml vented Erlenmeyer flask (Sigma-Aldrich CLS431144) at 37°C and 8% CO2, with stirring at 100 rpm. The cells were seeded as described in the manufacturer's instructions. - One day before transfection, the cells were counted using a hemocytometer and divided into 500,000 cells / ml. - On the day of transfection, seed the cells in 500 μl of appropriate medium at a rate of 1 million cells / ml in a 24-well plate. - Subsequently, 0.625 μg of DNA / well was added to 10 μl of OptiMem medium (Thermofisher; 11058021) and incubated at room temperature for 5 minutes. - Simultaneously, 0.625 μl of Max reagent was increased to 10 μl by adding OptiMem and incubated at room temperature for 5 minutes. - After this incubation, both mixtures were added to the same tube and incubated at room temperature for 25-30 minutes. - Subsequently, the DNA / Max reagent mixture (20 μl / well) was added directly to the cells, and the cells were incubated as previously described. - Subsequently, the cells were measured for GFP fluorescence 24 hours later. - Intron splicing from GFP constructs was measured by adding 2 mM DTT and monitoring GFP fluorescence after 1 hour. DTT is a strong reducing agent that induces ER stress by inhibiting the formation of disulfide bonds in the ER.

[0219] Measurement of GFP fluorescence: - The cell lysis buffer was prepared by diluting it 1 / 5 with sterile water from the luciferase assay system (Promega; E1500). - The cells were pelleted at 900 x g for 5 minutes and resuspended in 100 μl / 1 x 10 6 cells of the cell lysis buffer from the luciferase kit. - Then, this was incubated at room temperature for 10 - 15 minutes to cause lysis. - The cell nuclei and debris were collected at 900 x g for 5 minutes. - The supernatant was collected into a 96 - well black plate at a maximum of 100 μl / sample. - The supernatant was diluted 1 / 2 with PBS (this is to dilute the mercaptoethanol from the lysis buffer which affects the GFP signal; the GFP signal decreases in the presence of reducing agents). - The samples were incubated at room temperature for 5 minutes. - GFP fluorescence was measured using a plate reader, and the excitation and emission were set at 485 nm and 520 nm, respectively. - GFP fluorescence was also visualized directly under a fluorescence microscope.

[0220] Results SYNP - XBP - 01 experiment HEK293F experiment: - The 24 - well transfection was set up as described above. Each condition was performed in duplicates / triplicates. - 24 hours after transfection, the cells were treated either with 2 mM DTT or mock - treated with the same amount of water. - 1 hour after the treatment of the cells, GFP fluorescence was measured as described above. - The ratio of GFP fluorescence to the control plasmid was calculated before and after induction. Figure 1 shows the cumulative results of three independent experiments.

[0221] Figure 1 shows the ratio of GFP fluorescence from SYNP-XBP-01 transfected cells before and after induction by 2 mM DTT. The results clearly show that induction of ER stress induced GFP production 4–10 times more than positive controls, and the resulting activity was 2.5–7 times higher. In addition, the background was considerably lower than that of the control (dotted line), clearly indicating that EGFP expression is regulated by a controllable intron. This suggests that when placed within heterologous genes (i.e., genes other than XBP1), the intron is functional and promotes induced gene expression and higher expression levels.

[0222] CHO-S experiment: Experiments using CHO-S cells were conducted using the same method as described for HEK293F cells. The experimental results can be seen in Figures 2a and 2b.

[0223] Figure 2 shows, in panel a), the ratio of GFP fluorescence to the positive control before and after induction. Panel b) shows fluorescence microscopy observations of SYNP-XBP-01 with and without 2 mM DTT. These data indicate that in CHO-S cells, there is no background before induction, and therefore GFP fluorescence is induced more than 1000-fold with the addition of 2 mM DTT. Regulation in CHO-S cells may be even more precise than that observed in HEK293F cells. [Examples]

[0224] Use of ATF6 response elements to enhance gene expression and provide sophisticated gene regulation in the presence of controllable XBP1 introns. ATF6 is a transcription factor activated by ER stress. Once activated, this transcription factor binds to ERSE or UPRE, activating the transcription of genes that are important components of protein homeostasis (see Yoshida et al., Cell, Vol. 107, pp. 881-891, December 28, 2001).

[0225] In this study, the inventors investigated the binding of the ATF6 binding site (mammalian UPRE) and the enhancement of gene expression in the presence and absence of the XBP1 intron. This UPRE has a TGACGTG consensus sequence and is bound by XBP-1 in addition to ATF6, thereby creating a potentially strong feedback loop of ER stress-based gene expression. Intron addition also allows for research into whether regulation at the transcriptional and translational levels provides better inducibility, respectively, on its own.

[0226] For this study, we prepared the following two constructs: 1) A 6×ATF6 element (having the sequence TGACGTGCT) upstream of the CMV minimal promoter (CMV-MP) and EGFP; this construct was named SYNP-ATF6-01 (see Sequence ID 30 below). 2) EGFP having a 6×ATF6 element upstream of CMV-MP and an XBP1 intron insertion; this construct was named SYNP-ATF6-02 (see sequence number 31 below).

[0227] The intron was inserted into EGFP as described in Example 1.

[0228] HEK293F cells were used in this study. All growth conditions, transfection, and analysis were carried out as described in Example 1.

[0229] Plasmids used in this study: - pMA-RQ containing CMV-IE-GFP construct - pMA-RQ containing CMV-MP-GFP construct - pMA-RQ containing the SYNP-ATF6-01 construct - pMA-RQ containing SYNP-ATF6-02 construct

[0230] The sequence of SYNP-ATF6-01, the 6×ATF6 portion is underlined (sequence number 30): [ka]

[0231] The sequence of SYNP-ATF6-02, the 6×ATF6 region is underlined, and the excised intron sequence is shown in lowercase and bold (Sequence ID 31): [ka]

[0232] result The 24-well transfection was set up as described above. Each condition was performed in 2x / 3x series. 24 hours after transfection, the cells were treated either with 2 mM DTT or with an equal volume of water to induce UPR. GFP fluorescence was measured 1, 3, 5, and 24 hours after cell treatment, as previously discussed.

[0233] The ratio of GFP fluorescence to the control plasmid was calculated before and after induction. Figure 3 shows the cumulative results from three independent experiments.

[0234] Figure 3 shows GFP expression from both plasmids after induction by 2 mM DTT. The numbers are ratios to CMV-IE. This demonstrates that the 6×ATF6 element is highly induceable by ER stress and can enhance gene expression to three times the level of CMV-IE. Furthermore, the addition of the XBP1 intron to EGFP increases expression to four times the level of CMV-IE. Analysis of the background raw data revealed that SYNP-ATF6-01 has background activity equivalent to CMV-MP, and that the addition of its intron to EGFP reduces the background level of GFP fluorescence to virtually zero (Figures 4 and 5). This was confirmed by fluorescence microscopy observation (Figure 6). This demonstrates that the dual regulatory approach to protein expression provided herein—namely, transcription through a controllable promoter and translation through a controllable intron—can provide tightly controlled regulation and high expression levels. [Examples]

[0235] Inducible expression of EGFP from SYNP-ATF6-01 and SYNP-ATF6-02 during wtAAV production The objective of this example was to determine whether the ER stress response can be activated by the production of heterologous proteins as an alternative to (or in addition to) chemical ER stress, as previously used. Experiments were conducted in which GFP expression from SYNP-ATF6-01 and SYNP-ATF6-02 was measured during AAV production.

[0236] HEK293F cells were used in this study. All growth conditions, transfection, and analysis were carried out as described in Example 1.

[0237] In this experiment, transfection was performed in 24-well plates as previously described. The ATF6 plasmid (SYNP-ATF6-01 and SYNP-ATF6-02 constructs in the pMA-RQ vector) was transfected into HEK293F cells (using MAX reagent as described above), and GFP was measured after 24 hours. After GFP measurement, the same cells were transfected with the plasmids for wtAAV production, pGRG25AAV2 (provided by Adrien Savvy) in a 1:1 ratio and the pHelper plasmid from Takara / Clonetech. Subsequently, EGFP fluorescence was measured at 1 hour, 3 hours, 5 hours, and 24 hours (Figures 7 and 8 - not all data are shown). Both plasmids are required for wtAAV production, with pGRG25AAV2 providing the wtAAV2 genome and the pHelper plasmid providing the E2, VA, and E4 helper functions required for viral replication.

[0238] The wtAAV2 viral genome is well known in this field (see Srivastava et al., "Nucleotide sequence and organization of the adeno-associated virus 2 genome," J Virol. February 1983; 45(2): pp. 555-564), and the expression system for AAV is also well known in this field. In this case, the wtAAV2 viral genome was inserted into plasmid pGRG25 (McKenzie and Craig, "Fast, easy and efficient: site-specific insertion of transgenes into Enterobacterial chromosomes using Tn7 without need for selection of the insertion event"; BMC Microbiology 2006, 6: p. 39). The pHelper plasmid is available from Takara / Clonetech (AAVpro catalog # 6234). Suitable AAV2 expression systems for inducing AAV expression are widely available commercially, for example, the "AAVpro Helper Free System (AAV2)" from Takara (Clonetech) - see http: / / www.clontech.com / US / Products / Viral_Transduction / AAV_Vector_Systems / Helper_Free_Expression_System.

[0239] The plasmid pAAV-CMV-ZsGreen (catalog # 6231 from Takara / Clonetech's AAV vector system) was used as a control to confirm that AAV expression was achieved in cells. pAAV-CMV-ZsGreen and the pHelper plasmid were transfected in a 1:1 ratio into separate HeK293F cell populations. This confirmed successful AAV expression. Zs Green is an eGFP variant, and GFP measurements were performed on these cells as previously described.

[0240] These data indicate that both plasmids can be induced to CMV-IE levels by wtAAV production. Furthermore, while ATF6-01 has background levels similar to previous experiments, ATF6-02 has zero background levels, and it can be observed that only wtAAV synthesis can induce EGFP production. This supports our finding that both ER stress elements are required for complete regulation of expression. [Examples]

[0241] Induction of UPR with various inducers Experiments were conducted to evaluate the ability of several candidate activators to induce UPR. The ability of these candidate inducers to induce UPR was evaluated using essentially the techniques described above. Additional candidates tested were 0.5 mM palmitic acid, 1 μM MF-43 (2-methyl-5-(6-(4-(2-(trifluoromethyl)phenoxy)piperidine-1-yl)pyridazin-3-yl)-1,3,4-thiadiazole) (a stearoyl-CoA desaturase inhibitor), and combinations of both (each at the same concentration).

[0242] In these experiments, transfection was performed in 24-well plates as previously described. HEK293F cells were transfected with the SYNP-ATF6-02 plasmid (i.e., SYNP-ATF6-02 in the pMA-RQ vector), and GFP was measured after 24 hours. Subsequently, UPR induction was stimulated using the aforementioned inducer, and samples were collected and measured at 0, 1, 3, 5, and 24 hours after induction. GFP expression was measured as previously described. The results are the mean of three independent experiments, and the error bars represent the standard deviation.

[0243] Summary of the experimental procedure: - 40 ml of cells were grown in a 250 ml vented Erlenmeyer flask (Sigma-Aldrich CLS431144) at 37°C and 8% CO2, with stirring at 100 rpm. The cells were seeded as described in the manufacturer's instructions. - One day before transfection, the cells were counted using a hemocytometer and divided into 500,000 cells / ml. - On the day of transfection, seed the cells in 500 μl of appropriate medium at a rate of 1 million cells / ml in a 24-well plate. - Subsequently, 0.625 μg of DNA / well was added to 10 μl of OptiMem medium (Thermofisher; 11058021) and incubated at room temperature for 5 minutes. - Simultaneously, 0.625 μl of Max reagent was increased to 10 μl by adding OptiMem and incubated at room temperature for 5 minutes. - After this incubation, both mixtures were added to the same tube and incubated at room temperature for 25-30 minutes. - Subsequently, the DNA / Max reagent mixture (20 μl / well) was added directly to the cells, and the cells were incubated as previously described. - Subsequently, the cells were measured for GFP fluorescence 24 hours later. - Intron splicing from GFP constructs was measured by adding one of the following: 2 mM DTT, 0.5 mM palmitate, 1 μM MF-43, or a combination of 0.5 mM palmitate and 1 μM MF-43, and by monitoring GFP fluorescence 1, 3, 5, and 24 hours after AAV synthesis.

[0244] AAV production in UPR induction: The ATF6 plasmid (i.e., SYNP-ATF6-02 in the pMA-RQ vector) was transfected into HEK293F cells as previously described. GFP was measured after 24 hours. After GFP measurement, the same cells were transfected with plasmids for wtAAV production, pGRG25AAV2 and pHelper plasmids in a 1:1 ratio, as described above. EGFP fluorescence was then measured at 1 hour, 3 hours, 5 hours, and 24 hours.

[0245] Measurement of GFP fluorescence - The cell lysis buffer was prepared by diluting a luciferase assay system (Promega; E1500) to one-fifth of its original volume with sterile water. - Pellet the cells at 900xg for 5 minutes, then add 100 μl / 1×10 of cell lysis buffer from the luciferase kit. 6 It was resuspended in the cells. - This was then incubated at room temperature for 10-15 minutes to induce dissolution. - Cell nuclei and debris were collected at 900xg for 5 minutes. - The supernatant was collected in a 96-well black plate at a maximum rate of 100 μl / sample. - The supernatant was diluted by half with PBS (this dilutes the mercaptoethanol derived from the lysis buffer, which affects the GFP signal; the GFP signal is reduced in the presence of a reducing agent). - The sample was incubated at room temperature for 5 minutes. - GFP fluorescence was measured using a plate reader, and the excitation and emission were set to 485 nm and 520 nm, respectively. - GFP fluorescence was also visualized directly under a fluorescence microscope.

[0246] The results of these experiments are shown in Figure 9, which shows that all of the active ingredients successfully induced UPR, as indicated by the expression of functional EGFP after the addition of each ingredient. There were varying intensities of induction from each ingredient. DTT was a very potent inducer of UPR. Both palmitate and MF-43, which affect the lipid balance in cells, individually effectively induced UPR, and when combined, the level of induction was more potent. wtAAV production was an effective inducer, but its effect was not stronger than that of the other inducers.

[0247] An interesting and potentially useful observation from this experiment is that different inducers can be used to induce UPR at different levels, thus enabling splicing of the functional expression product (in this case, EGFP) and regulation of its expression level. Alternatively, or in addition, the expression level of the functional expression product can be regulated using different dose levels of various activators.

[0248] The procedure set out in this embodiment can be used to evaluate the ability of any active substance to induce UPR. [Examples]

[0249] Use of controllable introns for the regulation of secretory alkaline phosphatase (SEAP) expression Secretory alkaline phosphatases are standard proteins used as markers in the bioprocessing industry. Secretory alkaline phosphatases are ideal markers for secretory proteins because they experience all checkpoints in the cellular protein quality control (transcription, translation, post-translational modification, and subsequent secretion).

[0250] Construction of SEAP sequences containing controllable introns The construct SEAP-ATF6-001 was synthesized by chemical synthesis at GeneART.

[0251] The UPR-inducible cis-controllable element (enhancer region) used is 6×ATF6(TGACGTGCT), with each ATF6 spaced 20bp apart and linked to the CMV-MP. This is a modification of the tandem-repeat 6×ATF6 promoter used above, and its sequence is underlined in the sequence below.

[0252] An intron was inserted between two CAG codons at position 1314 of the SEAP coding sequence. The DNA encoding the region to be excised by the inserted intron was as follows: CAGACGGGCAACTTTACACGACGCTG (Sequence No. 32) When a splice site is included, this results in the following sequence: CAG / CAGACGGGCAACTTTACACGACGCTG / CAG (Sequence number 33)

[0253] This sequence does not result in the intronic secondary structure described for the XBP1 wild-type intron in the literature (see Example 6 below for a discussion on this point).

[0254] The SEAP expression construct sequence, including the promoter and controllable introns, is as follows (excised intron sequences are shown in bold, and the 6×ATF6 enhancer region is underlined): [ka] (Sequence ID 34)

[0255] Translation of coding sequences containing introns results in the following truncated protein sequences: MLLLLLLLGLRLQLSLGIIPVEEENPDFWNREAAEALGAAKKLQPAQTAAKNLIIFLGDGMGVSTVTAARILKGQKKDKLGPEIPLAMDRFPYVALSKTYNVDKHVPDSGATATAYLCGVKG NFQTIGLSAAARFNQCNTTRGNEVISVMNRAKKAGKSVGVVTTTRVQHASPAGTYAHTVNRNWYSDADVPASARQEGCQDIATQLISNMDIDVILGGGRKYMFRMGTPDPEYPDDYSQGGTRL DGKNLVQEWLAKRQGARYVWNRTELMQASLDPSVTHLMGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVEGGRIDHGHHESRAYRALTETIMFDDAIERAGQLTSEEDTLSLVTADHSHVFSFGGYPLRGSSIFGLAPGKARDRKAYTVLLYGNGPGYVLKDGARPDVTESESGSPEYRQQTGNFTRRCSQQCPWTKRHTQARTWRCSRAARRRTWFTACRSRPS (Sequence ID 35)

[0256] Removal of introns enables the complete translation of the SEAP protein: MLLLLLLLGLRLQLSLGIIPVEEENPDFWNREAAEALGAAKKLQPAQTAAKNLIIFLGDGMGVSTVTAARILKGQKKDKLGPEIPLAMDRFPYVALSKTYNVDKHVPDSGATATAYLCGVKGNFQTIGLSA AARFNQCNTTRGNEVISVMNRAKKAGKSVGVVTTTRVQHASPAGTYAHTVNRNWYSDADVPASARQEGCQDIATQLISNMDIDVILGGGRKYMFRMGTPDPEYPDDYSQGGTRLDGKNLVQEWLAKRQGARY VWNRTELMQASLDPSVTHLMGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVEGGRIDHGHHESRAYRALTETIMFDDAIERAGQLTSEEDTLSLVTADHSHVFSFGGYPLRGSSIFGLAPGKARDRKAYTVLLYGNGPGYVLKDGARPDVTESESGSPEYRQQSAVPLDEETHAGEDVAVFARGPQAHLVHGVQEQTFIAHVMAFAACLEPYTACDLAPPAGTTDAAHPGYSRVGAAGRFEQT (Sequence ID 36)

[0257] The experiment was carried out essentially as described above, i.e., HEK or CHO-s cells were transfected with the aforementioned construct (pMA-RQ) and incubated for 24 hours. After this time, the activating compound DTT was added at a final concentration of 2 mM, and SEAP activity was then measured at 3, 5, and 24 hours post-induction.

[0258] For more details: The transfection protocol for the 24-well plate was as described in Example 1. Subsequently: - The supernatant was measured for SEAP activity after 24 hours. - Intron splicing from SEAP constructs was measured by monitoring SEAP activity 3 hours, 5 hours, and 24 hours after the addition of 2 mM DTT. - SEAP activity was measured according to the manufacturer's instructions for use (SEAP reporter gene assay kit obtained from Roche, Sigma-Aldrich, catalog No. 11 779 842 001).

[0259] As can be seen in Figure 11, there was no SEAP expression before induction. However, the addition of DTT resulted in a rapid increase in SEAP activity, and indeed, after 3 hours, the level of SEAP activity was similar to that of CMV-IE (strong constitutive promoter). This expression rate was too fast to be explained by transcription alone, suggesting that mRNA was generated from the leaky promoter and translated almost immediately by intron removal. Indeed, after 24 hours, the expression levels in both cell types were approximately twice that of CMV-IE.

[0260] These results indicate that introns are suitable for use with secreted proteins. Furthermore, considerable flexibility is observed with respect to the secondary structure formed by the intron, and with respect to the central intron sequence adjacent to the splice site target sequence (i.e., the sequence referred to as Xn in the above example), suggesting that the splice site target sequence is an essential factor for successful splicing of introns from mRNA. The controllable intron used in this experiment (CAGACGGGCAACUUUACACGACGCUG (SEQ ID NO: 37)) is quite different from the wild-type XBP1 intron sequence (CAGCACUCAGACUACGUGCACCUCUG (SEQ ID NO: 23)), yet effective splicing was still achieved. Moreover, as discussed in more detail below, the intron used in this example is not expected to form a secondary structure similar to that formed by the wild-type XBP1 intron. [Examples]

[0261] Use of controllable introns for controlling protein expression using the firefly luciferase gene Prior art has asserted that the secondary structure formed by the XBP1 intron is essential for splicing. However, all of the above experiments used intron structures that were not expected to form such secondary structures or structures similar to wild-type XBP1 introns, and nevertheless, the performance of the splicing system was very robust.

[0262] Therefore, one aspect of this experiment was to determine the effect of introns specifically designed to have the secondary structures described in the prior art. Luciferase, an intracellular protein, was selected as the reporter protein, and luciferase is also advantageous due to the ease of assaying its expression levels.

[0263] Construction of a luciferase sequence containing controllable introns The expression construct was chemically synthesized at GeneART. The enhancer region, as described above, consists of 6×ATF6 (TGACGTGCT) molecules linked to CMV-MP at 20 bp intervals.

[0264] An intron was inserted at position 1447 of the luciferase coding sequence, between the CCG and CAG codons. The DNA encoding the region to be excised by the inserted intron sequence was as follows: CAGACGGGCAACTTTACACGACGCTG (Sequence No. 32) When a splice site is included, this results in the following sequence: CCG / CAGACGGGCAACTTTACACGACGCTG / CAG (Sequence ID 38)

[0265] This controllable intron sequence, when inserted into the luciferase gene as described below, is expected to provide the same secondary structure as the XBP1 wild-type intron at its native location in the XBP1 gene (calculated using the RNAfold web server at http: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi). The putative secondary structure formed by the intron in luciferase is shown in Figure 16D. For comparison, the putative secondary structures formed by the intron when inserted into related genes, such as those used in other experiments, are also shown in Figure 16: - Figure 16B - Intron used in EGFP (i.e., CAG / CTGCAGCACTCAGACTACGTGCACCTCTG / CTG, coded by sequence number 48, as used in Examples 1-4); - Figure 16A - Intron used in SEAP (i.e., CAG / CAGACGGGCAACTTTACACGACGCTG / CAG, coded by Sequence ID No. 33, as used in Example 5); - Figure 16C - Intron as used in CASP9 (i.e., CAG / CAGACGGGCAACTTTACACGACGCTG / CTG, coded in Sequence ID No. 43, as used in Example 7), see Figure 16C.

[0266] The luciferase expression construct sequence, including the promoter and controllable introns, is as follows (intron sequences are shown in bold, and the 6×ATF6 enhancer region is underlined):

[0267] Translation of the aforementioned intron-containing coding sequence results in the following truncated protein: MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMGISQPTVVF VSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLG YLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAADGQLYTTLQTSCWNTVKP (Sequence ID 39)

[0268] Intron removal enables the complete translation of the luciferase protein. (Sequence No. 40)

[0269] The experiment was carried out essentially as described in Example 5, i.e., HEK or CHO-s cells were transfected with the aforementioned construct and incubated for 24 hours. After this time, the activating compound DTT or palmitate was added. Subsequently, luciferase activity was measured at 3 hours, 5 hours, and 24 hours after induction. Luciferase was measured as follows: - The cell lysis buffer was prepared by diluting a luciferase assay system (Promega; E1500) to one-fifth of its original volume with sterile water. - Pellet the cells at 900xg for 5 minutes, then add 100 μl / 1×10 of cell lysis buffer from the luciferase kit. 6 It was resuspended in the cells. - This was then incubated at room temperature for 10-15 minutes to induce dissolution. - Cell nuclei and debris were collected at 900xg for 5 minutes. - The supernatant was collected and 10 μl / sample was added to a 96-well white plate. - Bioluminescence was measured using a plate reader with an automated injection device, and 50 μl of substrate was injected. The substrate was prepared according to the manufacturer's instructions for use (Promega; E1500).

[0270] As can be seen in Figure 12, luciferase expression was absent before induction, but the addition of either inducer resulted in a rapid increase in activity. In fact, after 3 hours, the level of luciferase activity was 2 to 2.5 times that of CMV-IE (strong constitutive promoter). This expression rate, again in this case, is too fast to be explained by transcription alone, suggesting that some mRNA is already being generated from the promoter and translated almost immediately after intron removal following induction. Indeed, after 24 hours, the expression levels in both cell types were approximately 3 to 4 times that of CMV-IE.

[0271] These results seem to suggest to some extent that a “complete” intronic secondary structure may, in some cases, yield better induction levels or higher splicing efficiency. However, its structure is clearly not important to the overall function of the system. Furthermore, SEAP and luciferase are entirely different proteins, and a simple comparison is not possible; SEAP is secreted and therefore undergoes further modification, which is another bottleneck for expression. [Examples]

[0272] Regulation of caspase-9 gene expression using controllable introns This experiment was conducted to confirm that the regulation of expression from controllable introns is appropriately precise in regulating the expression of toxic expression products that are lethal to the expressing cells. The controllable introns were incorporated into the apoptotic protein caspase 9. Overexpression of this protein in HEK cells causes rapid death. Therefore, even small amounts of expression from the unmodified sequence of this protein result in a significant increase in dead cells in culture. This prompted the inventors to determine the precision of the regulation provided by the introns. Caspase 9 was also fused with GFP-Spark (the introns are invisible if they are present in the transcript).

[0273] Construction of a luciferase sequence containing controllable introns For expression, the intron was ligated to the CMV-IE constitutive promoter. The intron construct was cloned into the plasmid vector SYNP-CASPSp-001 using a BsaI restriction site and two complementary oligonucleotides. The plasmid was digested with BsaI (NEB R0535S), and the oligonucleotides were annealed by heating to 98°C for 5 minutes to melt the secondary structure, followed by incubation at 55°C for 20 minutes. This allowed the oligonucleotides to form double-stranded DNA. This DNA was designed to have overhangs at both the 5' and 3' ends, which would allow ligation to the digested SYNP-CASPSp-001. Subsequently, the double-stranded DNA was ligated to the plasmid and then transformed into one-shot top ten chemically competent cells (Thermofisher, C404003). The isolated DNA was sequenced to confirm the presence of the intron. The complete plasmid is called SYNP-CASP9-INT, and its plasmid map is shown in Figure 13.

[0274] The intron oligo attached to the vector has the following sequence: INTC9FP: ACGTCAGACGGGCAACTTTACGACGCTG (Sequence ID 41) INTC9RP: ACGTCAGCGTCGTGTAAAGTTGCCCGTCTG (Sequence ID 42)

[0275] In this way, a controllable intron was incorporated between the CCAG codon and the CTG codon at position 1087 of the caspase 9 coding sequence.

[0276] The DNA encoding the region to be excised from the inserted intron sequence was as follows: CAGACGGGCAACTTTACACGACGCTG (Sequence No. 32) When a splice site is included, this results in the following sequence: CAG / CAGACGGGCAACTTTACACGACGCTG / CTG (Sequence ID 43)

[0277] This sequence does not result in the "complete" intronic secondary structure described for the XBP1 wild-type intron.

[0278] The following is a SYNP-CASP9-INT vector containing the CASP9-intron construct sequence (intron sequences are shown in bold): [ka] [ka] [ka] [ka] (Sequence ID 44)

[0279] Intron-containing coding sequences result in the following truncated proteins: MDEADRRLLRRCRLRLVEELQVDQLWDALLSRELFRPHMIEDIQRAGSGSRRDQARQLIIDLETRGSQALPLFISCLEDTGQDMLASFLRTNRQAAKLSKPTLENLTPVVLRPEIRKPEVLRPETPRPVDIGSGGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWSAGDPGRHL(Sequence ID 45)

[0280] Intron removal enables the complete translation of the caspase-9 protein: MDEADRRLLRRCRLRLVEELQVDQLWDALLSRELFRPHMIEDIQRAGSGSRRDQARQLIIDLETRGSQALPLFISCLEDTGQDMLASFLRTNRQAAKLSKPTLENLTPVVLRPEIRKPEVLRPETPRPVDIGSGGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLEL AQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWSADGQLYTTLLETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSGGGGS (Sequence ID 46)

[0281] Transcription was performed as previously described. DTT was added at 2 mM, and forskolin at 10 μM. The latter was added to determine whether forskolin could induce intron removal. In particular, forskolin has potential applications in gene therapy. Induction of intron removal was measured as a function of cell death, with higher cell death rates indicating intron removal.

[0282] The experiment was carried out as described in the method, i.e., HEK or CHO-s cells were transfected with the aforementioned constructs and incubated for 24 hours. After this time, the activating compound DTT or forskolin was added, and then % cell death was measured at 3 hours, 5 hours, and 24 hours post-induction.

[0283] Intron induction was measured as a function of cell death using a Countess II cell counter (Thermofisher). Cell viability was confirmed by staining cells with trypan blue. 10 μl of cell suspension was added to a hemocytometer and analyzed for count.

[0284] As can be seen from Figure 14A, there was no cell death before induction, but the addition of any of the inducers resulted in a rapid increase in the percentage of dead cells. In fact, after 3 hours with respect to DTT, the number of dead cells was close to that of the control construct without introns. Indeed, after 5 hours of DTT treatment, the percentage of dead cells was the same as the positive control, but forskolin treatment only reached this level after 24 hours, suggesting that it is a weak splicing inducer. Cells without an inducer showed little to no cell death and showed no caspase-9 expression until the induction substance was added. This indicates that the regulation of this gene expression is very precise (in fact, seemingly absolute) and at the mRNA level, because the CMV-IE promoter is constitutive and therefore the transcript is continuously produced. Figure 14B also shows that the regulation of gene expression from the intron is very precise. This is because caspase 9 is fused with a GFP variant called SPARK, and therefore, caspase 9 expression will lead to GFP variant expression. This figure shows that there is no GFP expression until the inducer is added and cell death begins.

[0285] In further experiments, the same construct (SYNP-CASP9-INT) was used to measure the rate of cell death. This figure shows that approximately 50% of the cells died within one hour of induction, demonstrating that the response is incredibly precise and rapid. The results of this experiment are shown in Figure 15. [Examples]

[0286] UPR-inducible promoters using different minimal promoters The objective of this experiment was to test ATF6-containing UPR-responsive cis-regulatory elements (also referred to herein as enhancers) as used in the above examples, with different minimal promoters, and to further evaluate the inducibility and rigor of their regulation. To achieve this objective, the ATF6-containing UPR-responsive cis-regulatory elements were operably linked to the minimal promoters of CMV-MP and MinTK (herpesthymidine kinase minimal promoter).

[0287] The structures were synthesized by chemical synthesis at GeneART, as described above.

[0288] As described above, the enhancer sequence contains six repeats of the sequence TGACGTGCT (containing the ATF6 consensus sequence, TGACGTG), spaced apart by a 20bp spacer sequence. The enhancer sequence was concatenated with either MinTK (within construct ATF06-MP001) or CMV-MP (within construct ATF06-MP002).

[0289] HEK293-F cells were transfected as previously described. DTT was added at a concentration of 2 mM. Luciferase activity was measured as previously described. The results are shown in Figure 17 - DTT was added at time = 0, as shown on the X axis (units are time).

[0290] Both constructs showed good inducibility and negligible expression before the addition of DTT. Slightly higher expression was observed with the promoter containing CMV-MP compared to the promoter containing MinTK. However, both promoter constructs showed high expression levels after UPR induction by DTT, which were significantly higher than those provided by the constitutive CMV-IE promoter.

[0291] ATF06-MP-001 (ATF06 and MinTK promoter sequences are underlined) [ka] (Sequence ID 59)

[0292] ATF06-MP-002 (ATF06 and CMV-MP promoter sequences are underlined) [ka] [ka] (Sequence ID 60)

[0293] While the fabrication and use of various embodiments of the present invention have been discussed in detail above, it should be recognized that the present invention provides many applicable inventive concepts that can be embodied in various specific circumstances. The specific embodiments discussed herein merely illustrate specific methods for fabricating and using the present invention and do not define the scope of the invention.

Claims

1. A synthetic nucleic acid expression construct for producing an expression product in a cell, wherein the synthetic nucleic acid expression construct comprises a promoter sequence operably linked to a nucleic acid sequence encoding the expression product, the nucleic acid sequence encoding the expression product comprises a sequence encoding a controllable intron, the controllable intron being a cleavable sequence capable of being spliced ​​out from a transcript produced from the synthetic nucleic acid expression construct by an unfolding protein response (UPR) system in a cell by the IRE1 protein or its homolog or ortholog, thereby resulting in a transcript encoding a normally functioning expression product. Synthetic nucleic acid expression constructs whose expression product does not contain the XBP1 protein or an XBP1 protein lacking a DNA-binding domain.

2. The synthetic nucleic acid expression construct according to claim 1, wherein the expression product is a protein.

3. The excision of controllable introns in splicing of excisable sequences enables translation of transcripts from nucleic acid sequences encoding the expression product, thereby enabling the production of the expression product, and / or, The presence of introns in the transcript from the nucleic acid sequence encoding the expression product can result in the translation of proteins from transcripts that cannot function properly, or The excision of controllable introns in splicing of excisable sequences eliminates stop codons in the transcript, or The excision of a controllable intron during splicing of a resectable sequence results in a reading frame shift for sequences in transcripts located downstream of the controllable intron, or A synthetic nucleic acid expression construct according to claim 1 or 2, wherein the nucleotide length of the excisable sequence is not divisible by 3.

4. A synthetic nucleic acid expression construct according to any one of claims 1 to 3, wherein the controllable intron comprises the sequence CNG / CNG-Xn-CNG / CNG, where Xn represents a sequence of n bases in length, / represents a cleavage site, the sequence CNG-Xn-CNG is excised from the transcript, and n represents a nucleotide length of 10 to 500 nucleotides.

5. Controllable introns, sequence CNG / CNG-Xn-CNG / CNG A synthetic nucleic acid expression construct according to claim 4, comprising [CG], wherein / represents a cleavage site where the excisable sequence CNG-Xn-CNG is excised from the transcript by splicing, and the nucleotide at the 5' end of sequence Xn is C or G.

6. A synthetic nucleic acid expression construct according to claim 4 or 5, wherein Xn comprises the sequence CACUCAGACUACGUGCACCU (SEQ ID NO: 1) or a sequence that is at least 90% identical thereto.

7. The controllable intron contains the sequence CNG / CNGCACUCAGACUACGUGCACCUCNG / CNGC (sequence number 6) or a sequence that is at least 90% identical thereto, or A controllable intron contains or consists of the sequence CAG / CAGCACUCAGACUACGUGCACCUCUG / CUGC (Sequence ID 7) or a sequence that is at least 90% identical thereto, The controllable introns are in the following sequence: - CNG / CAGCACUCAGACUACGUGCACCUCUG / CNG(Sequence ID 8); - CNG / CAGCACUCAGACUACGUGCUCCUCUG / CNG(Sequence ID 9); - CNG / CAGCACUCAGACUACGUGCCCCUCUG / CNG(Sequence ID 10); - CNG / CAGCACUCAGACUACGUGCGCCUCUG / CNG(Sequence ID 11); and - CNG / CAGCACUCAGACUAUGUGCACCUCUG / CNG (Sequence ID 12) It includes one of the following, or consists of one of the following, The controllable introns are in the following sequence: - CAG / CAGCACUCAGACUACGUGCACCUCUG / CUGC(Sequence ID 7); - CAG / CAGCACUCAGACUACGUGCUCCUCUG / CUGC(Sequence ID 13); - CAG / CAGCACUCAGACUACGUGCCCCUCUG / CUGC(Sequence ID 14); - CAG / CAGCACUCAGACUACGUGCGCCUCUG / CUGC(Sequence ID 15); and - CAG / CAGCACUCAGACUAUGUGCACCUCUG / CUGC (Sequence ID 16) It includes one of the following, or consists of one of the following, A controllable intron contains the sequence CAG / CUGCAGCACUCAGACUACGUGCACCUCUG / CUG (SEQ ID NO: 17) or CAG / CUGCAGCACUCAGACUACGUGCACCUCUG / CUGG (SEQ ID NO: 27), where / represents a cleavage site. A synthetic nucleic acid expression construct according to any one of claims 1 to 6.

8. A synthetic nucleic acid expression construct according to any one of claims 1 to 7, comprising an inducible promoter operably linked to a nucleic acid sequence encoding an expression product.

9. A nucleic acid comprising a sequence encoding an expression product, wherein the sequence encoding the expression product comprises a sequence encoding a controllable intron, the controllable intron being an intron comprising a cleavable sequence capable of being spliced ​​out from a transcript produced from the sequence encoding the expression product by an unfolding protein response (UPR) system in cells by the IRE1 protein or its homolog or ortholog, thereby resulting in a transcript encoding an expression product that can function normally, wherein the expression product is neither the XBP1 protein, nor the XBP1 protein lacking a DNA-binding domain, nor the Hac1 protein, nor the bZIP60 protein, nor its homolog.

10. A synthetic nucleic acid expression construct according to any one of claims 1 to 8, or a vector comprising the nucleic acid according to claim 9.

11. Ex vivo cells comprising a synthetic nucleic acid expression construct according to any one of claims 1 to 8, a nucleic acid according to claim 9, or a vector according to claim 10.

12. a) A step of supplying a population of eukaryotic cells comprising a synthetic nucleic acid expression construct according to any one of claims 1 to 8; b) Processing the cell population to induce an unfolding protein response, thereby inducing splicing of excisable sequences from controllable introns; c) A step of incubating the cell population under conditions optimal for the production of the expression product; and d) The step of isolating the expression product from the cell population. An ex vivo method for producing an expression product, including [a specific expression].

13. Step b) includes applying stress to the cells, wherein the stress is suitable for inducing UPR, or Step b) includes administering a chemical substance that can induce UPR in the cells, or Step b) includes expressing an inducible protein in the eukaryotic cell population in order to induce a UPR response in the cell population, or The ex vivo method according to claim 12, wherein step b) includes exposing cells to hypoxia or carbohydrate deficiency.

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