FORSKOLIN-INDUCIBLE PROMOTERS AND HYPOXIA-INDUCIBLE PROMOTERS

Abstract

The present invention relates to forskolin-inducible and hypoxia-inducible cis-regulatory elements, promoters and vectors, and methods of their use.

Claims

1. A synthetic forskolin-inducible promoter comprising one of the following structures: TABLE-US-00018 -TGAGTCA-S.sub.20-TGAGTCA-S.sub.20-TGAGTCA-S.sub.20-TGAGTCA-S.sub.20- TGAGTCA-S.sub.20-TGAGTCA-S.sub.20-TGAGTCA-S.sub.20-TGAGTCA-S.sub.59- CMV-MP; -TGACGTGCT-S.sub.20-TGACGTGCT-S.sub.20-TGACGTGCT-S.sub.20- TGAGTCA-S.sub.20-TGAGTCA-S.sub.20-TGAGTCA-S.sub.20-TGAGTCA-S.sub.20- CTGCACGTA-S.sub.20-CTGCACGTA-S.sub.20-CTGCACGTA-S.sub.61-CMV-MP; or -TGACGTCA-S.sub.10-TGACGTCA-S.sub.10-TGACGTCA-S.sub.10-  TGACGTCA-S.sub.10-TGACGTCA-S.sub.10-TGAGTCA-S.sub.10-TGAGTCA-S.sub.10- TGAGTCA-S.sub.10-TGAGTCA-YB-TATA, wherein S.sub.x represents a spacer sequence of length X nucleotides.

2. A synthetic forskolin-inducible promoter according to claim 1 which comprises one of the following sequences: TABLE-US-00019 (SEQ ID NO: 43) -TGAGTCAGATGATGCGTAGCTAGTAGTTGAGTCAGATGATGCGTAGCTA GTAGTTGAGTCAGATGATGCGTAGCTAGTAGTTGAGTCAGATGATGCGTA GCTAGTAGTTGAGTCAGATGATGCGTAGCTAGTAGTTGAGTCAGATGATG CGTAGCTAGTAGTTGAGTCAGATGATGCGTAGCTAGTAGTTGAGTCAGTA GTCGTATGCTGATGCGCAGTTAGCGTAGCTGAGGTACCGTCGACGATATC GGATCCAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCC TAGATACGCCATCCACGCTGTTTTGACCTCCATAGAAGATCGCCACC; (SEQ ID NO: 44) -TGACGTGCTGATGATGCGTAGCTAGTAGTTGACGTGCTGATGATGCGTA GCTAGTAGTTGACGTGCTGATGATGCGTAGCTAGTAGTTGAGTCAGATGA TGCGTAGCTAGTAGTTGAGTCAGATGATGCGTAGCTAGTAGTTGAGTCAG ATGATGCGTAGCTAGTAGTTGAGTCAGATGATGCGTAGCTAGTAGTCTGC ACGTAGATGATGCGTAGCTAGTAGTCTGCACGTAGATGATGCGTAGCTAG TAGTCTGCACGTAGATGATGCGTAGCTAGTAGTGCAGTTAGCGTAGCTGA GGTACCGTCGACGATATCGGATCCAGGTCTATATAAGCAGAGCTCGTTTA GTGAACCGTCAGATCGCCTAGATACGCCATCCACGCTGTTTTGACCTCCA TAGAAGATCGCCACC;  and (SEQ ID NO: 45) -TGACGTCACGATTACCATTGACGTCACGATTACCATTGACGTCACGATT ACCATTGACGTCACGATTACCATTGACGTCAGCGATTAAGATGACTCAGC GATTAAGATGACTCAGCGATTAAGATGACTCAGCGATTAAGATGACTCAG CGATTAATCCATATGCTCTAGAGGGTATATAATGGGGGCCACTAGTCTAC TACCAGAAAGCTTGGTACCGAGCTCGGATCCAGCCACC.

3. An expression cassette comprising a synthetic forskolin-inducible promoter according to any one of claim 1 or 2 operably linked to a transgene.

4. The expression cassette according to claim 3 wherein the transgene encodes a therapeutic protein or polypeptide.

5. A bioprocessing vector comprising a synthetic forskolin-inducible promoter according to any one of claims 1-2 or an expression cassette according to any one of claims 3-4.

6. A cell comprising a synthetic forskolin-inducible promoter according to any one of claims 1-2, an expression cassette according to any one of claims 3-4 or a bioprocessing vector according to claim 5.

7. The cell according to claim 6, wherein the cell is a human cell, optionally a HEK-293 cell, or a murine cell, optionally a NS0 cell or a CHO cell such as a CHO-K1SV cell or CHO-K1SV GS knock out.

8. A population of cells according to any one of claim 6 or 7.

9. A cell culture comprising a population of cells according to claim 8 and medium sufficient to support growth of the cells.

10. A reactor vessel comprising a cell culture according to claim 9.

11. Use of bioprocessing vector according to claim 5, or a cell according to claim 6 or 7 in a bioprocessing method for the manufacture of a product of interest, optionally a therapeutic product.

12. The method for producing an expression product, the method comprising: a) providing a population of cells comprising an expression cassette comprising a synthetic forskolin-inducible promoter according to any one of claims 1-2 operably linked to a transgene; b) culturing said population of cells; c) treating said population of cells so as to induce expression of the transgene present in the expression cassette and thereby produce an expression product; and d) recovering the expression product from said population of cells.

13. The method of claim 12 wherein step c) comprises administering an inducer to the cells.

14. The method of claim 13 wherein the inducer is an agent that activates adenylyl cyclase.

15. The method of claim 14 wherein the inducer is forskolin or NKH 477.

16. A synthetic hypoxia-inducible promoter comprising one of the following structures: TABLE-US-00020 -CTGCACGTA-S.sub.20-CTGCACGTA-S.sub.20-CTGCACGTA-S.sub.20- CTGCACGTA-S.sub.20-CTGCACGTA-S.sub.20-CTGCACGTA-S.sub.59- CMV-MP; -ACCTTGAGTACGTGCGTCTCTGCACGTATG-S.sub.9- ACCTTGAGTACGTGCGTCTCTGCACGTATG-S.sub.9- ACCTTGAGTACGTGCGTCTCTGCACGTATG-S.sub.9- ACCTTGAGTACGTGCGTCTCTGCACGTATG-S.sub.17-YB-TATA;  or -ACCTTGAGTACGTGCGTCTCTGCACGTATG-S.sub.9- ACCTTGAGTACGTGCGTCTCTGCACGTATG-S.sub.9- ACCTTGAGTACGTGCGTCTCTGCACGTATG-S.sub.9- ACCTTGAGTACGTGCGTCTCTGCACGTATG-S.sub.17-CMV-MP, wherein Sx represents a spacer sequence of length X nucleotides.

17. The synthetic hypoxia-inducible promoter according to claim 16 comprising one of the following sequences: TABLE-US-00021 (SEQ ID NO: 82) -CTGCACGTAGATGATGCGTAGCTAGTAGTCTGCACGTAGATGATGCGTA GCTAGTAGTCTGCACGTAGATGATGCGTAGCTAGTAGTCTGCACGTAGAT GATGCGTAGCTAGTAGTCTGCACGTAGATGATGCGTAGCTAGTAGTCTGC ACGTAGTAGTCGTATGCTGATGCGCAGTTAGCGTAGCTGAGGTACCGTCG ACGATATCGGATCCAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTC AGATCGCCTAGATACGCCATCCACGCTGTTTTGACCTCCATAGAAGATCG CCACC; (SEQ ID NO: 84) -ACCTTGAGTACGTGCGTCTCTGCACGTATGGCGATTAAGACCTTGAGTA CGTGCGTCTCTGCACGTATGGCGATTAAGACCTTGAGTACGTGCGTCTCT GCACGTATGGCGATTAAGACCTTGAGTACGTGCGTCTCTGCACGTATGGC GATTAATCCATATGCTCTAGAGGGTATATAATGGGGGCCA; (SEQ ID NO: 85) -ACCTTGAGTACGTGCGTCTCTGCACGTATGGCGATTAAGACCTTGAGTA CGTGCGTCTCTGCACGTATGGCGATTAAGACCTTGAGTACGTGCGTCTCT GCACGTATGGCGATTAAGACCTTGAGTACGTGCGTCTCTGCACGTATGGC GATTAATCCATATGCAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGT CAGATCGCCTAGATACGCCATCCACGCTGTTTTGACCTCCATAGAAGATC GCCACC; (SEQ ID NO: 86) -GATCTTTGTATTTAATTAAGACCTTGAGTACGTGCGTCTCTGCACGTAT GGCGATTAAGACCTTGAGTACGTGCGTCTCTGCACGTATGGCGATTAAGA CCTTGAGTACGTGCGTCTCTGCACGTATGGCGATTAAGACCTTGAGTACG TGCGTCTCTGCACGTATGGCGATTAATCCATATGCTCTAGAGGGTATATA ATGGGGGCCACTAGTCTACTACCAGAAAGCTTGGTACCGAGCTCGGATCC AGCCACC;  or (SEQ ID NO: 87) GATCTTTGTATTTAATTAAGACCTTGAGTACGTGCGTCTCTGCACGTATG GCGATTAAGACCTTGAGTACGTGCGTCTCTGCACGTATGGCGATTAAGAC CTTGAGTACGTGCGTCTCTGCACGTATGGCGATTAAGACCTTGAGTACGT GCGTCTCTGCACGTATGGCGATTAATCCATATGCAGGTCTATATAAGCAG AGCTCGTTTAGTGAACCGTCAGATCGCCTAGATACGCCATCCACGCTGTT TTGACCTCCATAGAAGATCGCCACC. 

18. An expression cassette comprising a synthetic hypoxia-inducible promoter according to any one of claim 16 or 17 operably linked to a transgene.

19. The expression cassette according to claim 18 wherein the transgene encodes a therapeutic protein or polypeptide.

20. A bioprocessing vector comprising a synthetic hypoxia-inducible promoter according to any one of claims 16-17.

21. A cell comprising a synthetic hypoxia-inducible promoter according to any one of claims 16-17, an expression cassette according to claims 18-19 or a bioprocessing vector according to claim 20.

22. The cell according to claim 21, wherein the cell is a human cell, optionally a HEK-293 cell, or a murine cell, optionally NS0 cell or a CHO cell such as a CHO-K1SV cell or CHO-K1SV GS knock out.

23. A population of cells according to claim 21 or 22.

24. A cell culture comprising a population of cells according to claim 23 and medium sufficient to support growth of the cells.

25. A reactor vessel comprising a cell culture according to claim 24.

26. Use of the bioprocessing vector according to claim 20, or a cell according to any one of claim 21 or 22 in a bioprocessing method for the manufacture of a product of interest, optionally a therapeutic product.

27. A method for producing an expression product, the method comprising the steps of: (a) providing a population of eukaryotic cells, optionally animal cells, optionally mammalian cells, comprising an expression cassette according to claim 18 or 19 or bioprocessing vector according to claim 20; (b) culturing said population of cells; and (c) treating said population of cells so as to induce hypoxia in the cells, such that expression from the transgene linked to the hypoxia-inducible promoter is induced and the expression product is produced; and (d) recovering the expression product.

28. The method of claim 27 wherein step (b) comprises maintaining said population of cells under suitable conditions for proliferation of the cells.

29. The method of any one of claim 27 or 28 comprising introducing into the cell an expression cassette according to claim 18 or 19 or bioprocessing vector according to claim 20.

30. The method of any one of claims 27-29 wherein step (c) comprises treating the cells by reducing the amount of oxygen supplied to the cell, e.g. such that the oxygen tension in the cells is 5% or less.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0310] FIG. 1 is an illustration of the mechanism of action of forskolin and other adenylyl cyclase activators.

[0311] FIG. 2 shows the luciferase expression from the promoters RTV-017 and RTV-019 in Huh7 (liver) without induction and after induction with 20 pM forskolin. RTV-017 and RTV-019 promoters show low background activity without induction and high activity following induction. The activity of the CMV-IE promoter does not change.

[0312] FIG. 3 shows luciferase expression from the promoter RTV-019 in C2C12 (muscle) cells without induction and after induction with 20 pM forskolin. RTV-019 promoter shows low background activity without induction and high activity following induction. CMV-IE expression was not analysed under induced conditions, and thus no result is shown in the graph.

[0313] FIG. 4 shows a graph illustrating that when the promoters RTV-017, RTV-019 and FORCYB1 are expressed from an AAV vector (therefore containing ITRs) they still maintain low background activity and high levels of expression and induction in Huh7 cells. Luciferase activity was measured without induction and after induction with 20 pM forskolin or 7.2 pM NKH477. Similarly to FIGS. 2 to 4, RTV-017, FORCYB1 and RTV-019 show low background activity without induction and high activity following induction. CMV-IE shows weaker expression in AAV vector than the tested promoters.

[0314] FIG. 5 shows the activity of the promoters after transient transfection into the suspension cell line HEK293-F. The cells were induced (at time 0 h) with 20 pM forskolin and luciferase expression was measured at 0 h, 3 h, 5 h and 24 h. All constructs showed increase in activity (to a varying degree) while the activity of CMV-IE remained constant.

[0315] FIG. 6 shows the activity of the promoters after transient transfection into the suspension cell line CHO-K1SV with and without induction by 20 pM forskolin. Luciferase expression was measured 24 h after induction. All constructs showed increase in activity (to a varying degree) following induction.

[0316] FIG. 7 shows the response of the promoter RTV-019 to increasing concentrations of forskolin and NKH477 in the stably transfected cell line CHO-K1SV.

[0317] FIG. 8 shows the SEAP expression from the promoters in the stably transfected cell line CHO-K1SV with and without induction by 20 pM forskolin and 7.2 pM NKH477.

[0318] FIG. 9 shows the same data as in FIG. 8 but the activity is expressed compared to CMV-IE.

[0319] FIG. 10 shows brightfield microscopy pictures of C2C12 cells at passage 11. A) shows the cells prior to transformation (day 2). B) shows the C2C12 cells 24 hours after transfection (day 3). C) shows the differentiated C2C12 cells after 5.5 days into differentiation medium (day 7.5). Scale bar is 50 μm

[0320] FIG. 11A shows a schematic diagram of hypoxia-inducible gene expression. Transcription factor HIF1A (HIF1a) is degraded under normal oxygen conditions, but under hypoxic conditions, it is stabilised, dimerises with HIF1B (HIF1β) to form HIF1 and is translocated to the nucleus. In the nucleus, the HIF1 complex can bind to the hypoxia response element and initiate expression of the gene of interest.

[0321] FIG. 11B shows a schematic diagram of the structural organisation of HIF1α and HIF1β. Both HIF1α and HIF1β have a bHLH domain for DNA binding. HIF1β has a Per-ARNT-Sim (PAS) domain for central heterodimerisation and HIF1α's C terminal domain (TAD N/TAD C) recruits transcriptional coregulatory proteins. When HIF1α and HIF1β dimerise, they translocate to the nucleus and turn on expression of hypoxia-regulated genes after binding to a hypoxia-responsive element.

[0322] FIG. 12 shows a schematic diagram of promoters RTV-015, RTV-016, HV3C, HYB and Synp-HYP-001. RTV-015 promoter comprises of five HRE1 and a synthetic minimal promoter MP1. These elements are spaced apart with spacers (not shown). RTV-016 promoter comprises of 6 HRE2 and CMV minimal promoter. These elements are spaced apart with spacers (not shown). HV3C promoter comprises of 4 HRE3 and CMV minimal promoter. These elements are spaced apart with spacers (not shown). HYBT promoter comprises of 4 HRE3 and a YB-TATA minimal promoter. These elements are spaced apart with spacers (not shown). Synp-HYP-001 comprises of four HRE2 and a CMV minimal promoter. The HRE2 elements are not spaced apart with spacers but there is a spacer between the last HRE2 element and the CMV minimal promoter (not shown).

[0323] FIG. 13 shows a time course of luciferase expression from the RTV-015, HYBT, RTV-016, SYNP-HYP-011, CMV-IE and HV3C constructs in transiently transduced HEK293-F cells under hypoxia. Cells were placed in hypoxia at 0 hours and then luciferase activity monitored. Luciferase expression from the CMV minimal promoter, which was used as a control, does not change but the rest of the constructs show increase in luciferase activity with time.

[0324] FIG. 14 shows measurement of luciferase expression from the RTV-015, RTV-016, HV3C, HYBT and CMV-IE constructs in transiently transduced HEK293-T in normoxic conditions and after 24 hours in hypoxia. The luciferase expression from the CMV-IE promoter is the same in normoxia and hypoxia. RTV-015, RTV-016, HV3C, HYBT constructs show almost no luciferase activity in normoxia but are induced to a varying level after 24 hours in hypoxia with RTV-015 showing the lowest and HV3C showing the highest inducibility.

[0325] FIG. 15 shows measurement of luciferase expression from the RTV-015, RTV-016, HV3C, HYBT and CMV-IE constructs in transiently transduced CHO_GS suspension cell line in normoxic conditions and after 24 hours in hypoxia. The luciferase expression from the CMV-IE is the same in normoxia and hypoxia. Similar to the results shown in FIG. 4, RTV-015, RTV-016, HV3C, HYBT constructs show almost no luciferase activity in normoxia but are induced to a varying level after 24 hours in hypoxia with RTV-015 showing the lowest and HV3C showing the highest inducibility.

[0326] FIG. 16 shows measurement of SEAP expression from the RTV-015, RTV-016, HV3C, HYBT and CMV-IE constructs in stably integrated CHO-GSK1SV cell line in normoxia (24 hours after seeding—show as 0 h), followed by 24 h in normoxia or by 24 h in hypoxia. The SEAP expression from the CMV-IE construct is the same in normoxia and hypoxia. Similar to the results shown in FIGS. 4 and 5, RTV-015, RTV-016, HV3C, HYBT constructs show almost no SEAP activity in normoxia but are induced to a varying level after 24 hours in hypoxia. RTV-015 still shows the lowest inducibility and HV3C and HYBT, the highest.

[0327] FIG. 17 show cell numbers of the stably integrated CHO-GSK1SV with RTV-015, RTV-016, HV3C, HYBT and CMV-IE constructs. SEAP expression was normalised to the number of cells in the respective condition.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION AND EXAMPLES

[0328] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

[0329] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Ausubel, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (Harries and Higgins eds. 1984); Transcription and Translation (Hames and Higgins eds. 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); the series, Methods in Enzymology (Abelson and Simon, eds.-in-chief, Academic Press, Inc., New York), specifically, Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, “Gene Expression Technology” (Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (Miller and Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook of Experimental Immunology, Vols. I-IV (Weir and Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

[0330] To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

[0331] The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited features, elements or method steps.

[0332] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0333] The term “cis-regulatory element” or “CRE”, is a term well-known to the skilled person, and means a nucleic acid sequence such as an enhancer, promoter, insulator, or silencer, that can regulate or modulate the transcription of a neighbouring gene (i.e. in cis). CREs are found in the vicinity of the genes that they regulate. CREs typically regulate gene transcription by binding to TFs, i.e. they include TFBS. A single TF may bind to many CREs, and hence control the expression of many genes (pleiotropy). CREs are usually, but not always, located upstream of the transcription start site (TSS) of the gene that they regulate. “Enhancers” are CREs that enhance (i.e. upregulate) the transcription of genes that they are operably associated with, and can be found upstream, downstream, and even within the introns of the gene that they regulate. Multiple enhancers can act in a coordinated fashion to regulate transcription of one gene. “Silencers” in this context relates to CREs that bind TFs called repressors, which act to prevent or downregulate transcription of a gene. The term “silencer” can also refer to a region in the 3′ untranslated region of messenger RNA, that bind proteins which suppress translation of that mRNA molecule, but this usage is distinct from its use in describing a CRE. Generally, in the present invention, the CREs are forskolin inducible enhancers or hypoxia inducible enhancers. In the present context, it is preferred that the CRE is located 1500 nucleotides or less from the transcription start site (TSS), more preferably 1000 nucleotides or less from the TSS, more preferably 500 nucleotides or less from the TSS, and suitably 250, 200, 150, or 100 nucleotides or less from the TSS. CREs of the present invention are preferably comparatively short in length, preferably 50 nucleotides or less in length, for example they may be 40, 30, 20, 10 or 5 nucleotides or less in length.

[0334] The term “cis-regulatory module” or “CRM” means a functional module made up of two or more CREs; in the present invention the CREs are typically forskolin inducible enhancers or hypoxia inducible enhancers. Thus, in the present application a CRM typically comprises a plurality of forskolin inducible CREs or hypoxia inducible CREs. Typically, the multiple CREs within the CRM act together (e.g. additively or synergistically) to enhance the transcription of a gene that the CRM is operably associated with. There is conservable scope to shuffle (i.e. reorder), invert (i.e. reverse orientation), and alter spacing in CREs within a CRM. Accordingly, functional variants of CRMs of the present invention include variants of the referenced CRMs wherein CREs within them have been shuffled and/or inverted, and/or the spacing between CREs has been altered.

[0335] A “functional variant” of a cis-regulatory element, cis-regulatory module, promoter or other nucleic acid sequence in the context of the present invention is a variant of a reference sequence that retains the ability to function in the same way as the reference sequence, e.g. as a forskolin-inducible element or promoter or hypoxia-inducible element or promoter. Alternative terms for such functional variants include “biological equivalents” or “equivalents”.

[0336] A CRE can be considered “forskolin-inducible” if, when placed in a suitable promoter (as discussed in more detail herein), expression of a gene operably linked to said promoter can be induced by administration of forskolin to a eukaryotic cell (preferably a mammalian cell) containing said promoter.

[0337] It will be appreciated that the ability of a given CRE to function as a forskolin-inducible enhancer is determined principally by the ability of the sequence to be bound by CREB and/or AP1 (following induction by an activator of adenylyl cyclase and resulting increase in cellular cAMP levels) such that expression of an operably linked gene is induced. Accordingly, a functional variant of a CRE will contain suitable binding sites for CREB and/or AP1 (though other TFBS may also contribute). Suitable TFBS for CREB, AP1 and other TFs are discussed above.

[0338] The ability of CREB and/or AP1 (or any other TF) to bind to a given CRE can determined by any relevant means known in the art, including, but not limited to, electromobility shift assays (EMSA), binding assays, chromatin immunoprecipitation (ChIP), and ChIP-sequencing (ChIP-seq). In some embodiments the ability of CREB and/or AP1 to bind a given functional variant is determined by EMSA. Methods of performing EMSA are well-known in the art. Suitable approaches are described in Sambrook et al. cited above. Many relevant articles describing this procedure are available, e.g. Hellman and Fried, Nat Protoc. 2007; 2(8): 1849-1861.

[0339] The ability of any given CRE to function as a forskolin-inducible element can be readily assessed experimentally by the skilled person. The skilled person can thus easily determine whether any given CRE or promoter (e.g. a variant of the specific forskolin-inducible promoters or CREs recited above) is functional (i.e. whether it can be considered to be a functional forskolin-inducible promoter or CRE, or if it can be considered to be a functional variant a specific promoters or CREs recited herein). For example, any given putative forskolin-inducible promoter can be linked to a gene (typically a reporter gene) and its properties when induced by forskolin are assessed. Likewise, any given CRE to be assessed can be operatively linked to a minimal promoter (e.g. positioned upstream of a MP) and the ability of the cis-regulatory element to drive expression of a gene (typically a reporter gene) when induced by forskolin is measured. Suitable constructs to assess the functionality activity of a forskolin-inducible CRE or a forskolin-inducible promoter, can easily be constructed, and the examples set out below give suitable methodologies. For example, any given putative forskolin-inducible CRE can be substituted in place of the incumbent CRE in any of the promoters Synp-FORCSV-10, Synp-FORCMV-09, Synp-FMP-02, Synp-FLP-01, Synp-RTV-017, Synp-RTV-019, or Synp-FORCYB1 discussed below, and linked to a reporter gene (e.g. luciferase or SEAP) and its inducibility and strength of expression upon induction can be assessed. In terms of inducibility, the level of induction of the reporter after cells, e.g. CHO-K1SV cells, are exposed to 18 pM forskolin for 5 h is suitably at least a 3-fold increase in expression, more preferably a 5-, 10-, 15-, 20-, 30-, or 50-fold increase in expression. In terms of strength of the promoter, upon induction (e.g. after cells, e.g. CHO-K1SV cells, are exposed to 18 pM forskolin for 5 h) the expression level of the reporter is at least 50% of that provided by the CMV-IE promoter (i.e. when compared to an otherwise identical vector in the same cells under the same conditions, but in which expression of the transgene is under control of CMV-IE rather than the forskolin inducible promoter). More preferably the expression level of the transgene is at least 75%, 100%, 150%, 200%, 300%, 400%, 500%, 750% or 1000% of that provided by the CMV-IE promoter. Likewise, any putative forskolin-inducible promoter can be substituted for promoters Synp-FORCSV-10, Synp-FORCMV-09, Synp-FM P-02, Synp-FLP-01, Synp-RTV-017, Synp-RTV-019, or Synp-FORCYB1 in the constructs of Examples 1, 2, 3 or 4 and inducibility and power assessed (the same conditions and preferred levels of inducibility and strength apply).

[0340] A hypoxia-responsive element (HRE) is a type of cis-regulatory element (CRE). More particularly, it is an inducible enhancer that is induced when cells in which the enhancer is present are subject to hypoxic conditions. HREs comprise a plurality of hypoxia-inducible factor biding sites (HBS). As described elsewhere, under hypoxic conditions the HIF heterodimer is formed in the cells and binds to HBSs, driving expression of genes containing them. This is well-described in the literature, see, for example, Wenger R H, Stiehl D P, Camenish G. Integration of oxygen signalling at the consensus HRE. Sci STKE 2005; 306:re12. [PubMed: 16234508]. More than one HRE can be present in the vectors of the present invention, thus providing a hypoxia-responsive cis-regulatory module (CRM).

[0341] Hypoxia-inducible factors (HIFs) are transcription factors that respond to hypoxia, i.e. a decrease in available oxygen in the cellular environment. In general, HIFs are vital to development. In mammals, deletion of the HIF-1 genes results in perinatal death. HIF-1 is of particular relevance to the present invention given its preeminent role in the hypoxia response, and thus it is preferred that the HREs of the present invention are targets for HIF-1. However, other HIFs (e.g. HIF-2 or HIF-3) may also bind to the HRE, and thus they are also of relevance. HIF-1, is a heterodimer composed of an α-subunit (HIF-1α) and a β-subunit (HIF-1β), the latter being a constitutively-expressed aryl hydrocarbon receptor nuclear translocator (ARNT). The alpha subunits of HIF are hydroxylated at conserved proline residues by HIF prolyl-hydroxylases, allowing their recognition and ubiquitination by the VHL E3 ubiquitin ligase, which labels them for rapid degradation by the proteasome. This occurs only in normoxic conditions. In hypoxic conditions, HIF prolyl-hydroxylase is inhibited, since it utilizes oxygen as a co-substrate. HIF-1, when stabilized by hypoxic conditions, upregulates several genes to promote survival in low-oxygen conditions. HIF-2 or HIF-3 are similarly formed from α- and β-subunits, as is well-described in the literature. The regulation of HIF1α and 2α by hypoxia is similar and both bind to the same core motif.

[0342] A hypoxia-inducible factor biding site (HBS) is a nucleic acid sequence that acts as a binding site for HIF. In endogenous genes, HBS comprise a conserved core sequence ([AG]CGTG, SEQ ID NO: 6) and highly variable flanking sequence.

[0343] It will be appreciated that the ability of a given HRE to function as a hypoxia-inducible enhancer is determined principally by the ability of the sequence to be bound by HIF (e.g. HIF-1) under hypoxic conditions such that expression of an operably linked gene is induced. Accordingly, a functional variant of an HRE will contain suitable binding sites for HIF. Generally, the presence of the consensus HBS is required.

[0344] The ability of HIF to bind to a given HRE can determined by any relevant means known in the art, including, but not limited to, electromobility shift assays (EMSA), binding assays, chromatin immunoprecipitation (ChIP), and ChIP-sequencing (ChIP-seq). In some embodiments the ability of HIF to bind a given functional variant is determined by EMSA. Methods of performing EMSA are well-known in the art. Suitable approaches are described in Sambrook et al. cited above. Many relevant articles describing this procedure are available, e.g. Hellman and Fried, Nat Protoc. 2007; 2(8): 1849-1861. In a preferred method, the ability of a variant to bind HIF can be determined with pull down experiments.

[0345] For example, pull-down experiments can be carried out using biotinylated double-stranded probes with a variant HRE and a reference HRE. Using high stringency washing [6], the amount of HIF (e.g. assed in terms of the quantity of HIF-1a) from nuclear extract prepared from hypoxic cells can be compared between the variant HRE and reference HRE. Suitable methods are described in Stanbridge, et al. Rational design of minimal hypoxia-inducible enhancers Biochem Biophys Res Commun. 2008 Jun. 13; 370(4): 613-618 and Ebert BL, Bunn HF. Regulation of transcription by hypoxia requires a multiprotein complex that includes hypoxia-inducible factor 1, an adjacent transcription factor, and p300/CREB binding protein. Mol Cell Biol 1998; 18:4089-4096.

[0346] With regard to variants of any of the specific CRE or promoter sequences set out above, their functionality can be assessed by substituting the variant in place of the given CRE or promoter in the relevant construct of Example 1, 2, 3 or 4 and comparing the result for the construct comprising the variant against the results for the original construct. Preferably the functional variant maintains at least 50%, 60%, 70%, 80%, 90% or 100% of the inducibility of the parent construct (measured in terms of fold increase in expression as a result of induction, i.e. a 2-fold increase in expression of a reporter gene upon induction is considered to be 50% as inducible as a 4-fold increase). Preferably the functional variant maintains at least 50%, 60%, 70%, 80%, 90% or 100% of the expression strength of the reference construct upon induction. A functional variant also preferably results in a background expression level (i.e. absent any induction) that is no more than three times as high, preferably no more than twice as high, and preferably no more than 1.5 times as high when compared to the reference construct.

[0347] The ability of any given HRE to function as a hypoxia-inducible element can be readily assessed experimentally by the skilled person. The skilled person can thus easily determine whether any variant of the specific hypoxia-inducible promoters or HREs recited above remains functional (i.e. it is a functional hypoxia-inducible promoter or HRE, or if it can be considered to be a functional variant). For example, any given putative hypoxia-inducible promoter can be linked to a gene (typically a reporter gene) and its inducible properties when induced by hypoxia are assessed. Likewise, any given HRE to be assessed can be operatively linked to a minimal promoter (e.g. positioned upstream of a MP) and the ability of the cis-regulatory element to drive expression of a gene (typically a reporter gene) when induced by hypoxia is measured. Suitable constructs to assess activity of an HRE or a hypoxia-inducible promoter, can easily be constructed, and the examples set out below give suitable methodologies. For example, any given putative HRE can be placed in any of the promoters Synp-RTV-015, Synp-RTV-016, Synp-HYBT and Synp-HV3C, discussed below in place of the incumbent HRE, and linked to a reporter gene (e.g. luciferase or SEAP) and its inducibility and power can be assessed. For example, in terms of inducibility, the level of induction in after 5 h in cells when subjected to hypoxic conditions (e.g. moving from 20% oxygen to 5% oxygen) is suitably at least a 5-fold increase in expression, more preferably a 10-, 15-, 20-, 30-, or 50-fold increase in expression. For example, in terms of power, the level of expression in cells when subjected to hypoxic conditions (e.g. moving from 20% oxygen to 5% oxygen) is suitably at least 10% of the expression levels achieved by an otherwise identical constructs in which the CMV-IE promoter is used; more preferably at least 25%, 50%, 75%, 100%, 150% or 200% of the expression levels driven by CMV-IE.

[0348] Likewise, any putative hypoxia-inducible promoter can be substituted for promoters Synp-RTV-015, Synp-RTV-016, Synp-HYBT, Synp-HV3C in the constructs of examples 5, 6 or 7 and inducibility and power assessed (the same preferred levels of inducibility and power apply).

[0349] In one specific example, variants of HRE3 can be assessed by substituting the variant in place of HRE3 in the construct of HV3C or HYBT, and carrying out a suitable expression reporter assay, e.g. as described in Example 5, 6 or 7 and comparing the result for the construct comprising the variant to the results for the original HV3C or HYBT construct. Preferably the functional variant maintains at least 50%, 60%, 70%, 80%, 90% or 100% of the inducibility of the parent construct, and preferably the functional variant maintains at least 50%, 60%, 70%, 80%, 90% or 100% of the power of the parent construct.

[0350] Levels of sequence identity between a functional variant and the reference sequence can also be an indicator or retained functionality. High levels of sequence identity in the TFBSs or HBSs and spacing between the TFBSs or HBSs is of generally higher importance than sequence identity in the spacer sequences (where there is little or no requirement for any conservation of sequence).

[0351] As used herein, the term “promoter” refers to a region of DNA that generally is located upstream of a nucleic acid sequence to be transcribed that is needed for transcription to occur, i.e. which initiates transcription. Promoters permit the proper activation or repression of transcription of a coding sequence under their control. A promoter typically contains specific sequences that are recognized and bound by plurality of TFs. TFs bind to the promoter sequences and result in the recruitment of RNA polymerase, an enzyme that synthesizes RNA from the coding region of the gene. A great many promoters are known in the art. The inducible promoters of the present invention typically drive almost none or low expression prior to being induced, and upon induction they drive a significantly higher level of expression (e.g. a 5, 10, 20, 50, 100, 150, 500, 700, or even 1000-fold increase in expression after induction).

[0352] The promoters of the present invention are synthetic promoters. The term “synthetic promoter” as used herein relates to a promoter that does not occur in nature. In the present context it typically comprises a synthetic CRE and/or CRM of the present invention operably linked to a minimal (or core) promoter. The CREs and/or CRMs of the present invention serve to provide forskolin inducible transcription of a gene operably linked to the promoter. Parts of the synthetic promoter may be naturally occurring (e.g. the minimal promoter or one or more CREs in the promoter), but the synthetic promoter as a complete entity is not naturally occurring.

[0353] As used herein, “minimal promoter” (also known as the “core promoter”) refers to a short DNA segment which is inactive or largely inactive by itself, but can mediate transcription when combined with other transcription regulatory elements. Minimum promoter sequence can be derived from various different sources, including prokaryotic and eukaryotic genes or can be synthetic. Examples of minimal promoters are discussed above, and include the synthetic MP1 promoter, cytomegalovirus (CMV) immediate early gene minimum promoter (CMV-MP) and the YB-TATA. A minimal promoter typically comprises the transcription start site (TSS) and elements directly upstream, a binding site for RNA polymerase II, and general transcription factor binding sites (often a TATA box).

[0354] As used herein, “proximal promoter” relates to the minimal promoter plus the proximal sequence upstream of the gene that tends to contain primary regulatory elements. It often extends approximately 250 base pairs upstream of the TSS, and includes specific TFBS. In the present case, the proximal promoter is suitably a naturally occurring proximal promoter that can be combined with one or more CREs or CRMs of the present invention. However, the proximal promoter can be synthetic.

[0355] The term “nucleic acid” as used herein typically refers to an oligomer or polymer (preferably a linear polymer) of any length composed essentially of nucleotides. A nucleotide unit commonly includes a heterocyclic base, a sugar group, and at least one, e.g. one, two, or three, phosphate groups, including modified or substituted phosphate groups. Heterocyclic bases may include inter alia purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) which are widespread in naturally-occurring nucleic acids, other naturally-occurring bases (e.g., xanthine, inosine, hypoxanthine) as well as chemically or biochemically modified (e.g., methylated), non-natural or derivatised bases. Sugar groups may include inter alia pentose (pentofuranose) groups such as preferably ribose and/or 2-deoxyribose common in naturally-occurring nucleic acids, or arabinose, 2-deoxyarabinose, threose or hexose sugar groups, as well as modified or substituted sugar groups. Nucleic acids as intended herein may include naturally occurring nucleotides, modified nucleotides or mixtures thereof. A modified nucleotide may include a modified heterocyclic base, a modified sugar moiety, a modified phosphate group or a combination thereof. Modifications of phosphate groups or sugars may be introduced to improve stability, resistance to enzymatic degradation, or some other useful property. The term “nucleic acid” further preferably encompasses DNA, RNA and DNA RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemically synthesised) DNA, RNA or DNA RNA hybrids. A nucleic acid can be naturally occurring, e.g., present in or isolated from nature; or can be non-naturally occurring, e.g., recombinant, i.e., produced by recombinant DNA technology, and/or partly or entirely, chemically or biochemically synthesised. A “nucleic acid” can be double-stranded, partly double stranded, or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.

[0356] The terms “identity” and “identical” and the like refer to the sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, such as between two DNA molecules. Sequence alignments and determination of sequence identity can be done, e.g., using the Basic Local Alignment Search Tool (BLAST) originally described by Altschul et al. 1990 (J Mol Biol 215: 403-10), such as the “Blast 2 sequences” algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174: 247-250).

[0357] Methods for aligning sequences for comparison are well-known in the art. Various programs and alignment algorithms are described in, for example: Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85:2444; Higgins and Sharp (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65; Pearson et al. (1994) Methods Mol. Biol. 24:307-31; Tatiana et al. (1999) FEMS Microbiol. Lett. 174:247-50. A detailed consideration of sequence alignment methods and homology calculations can be found in, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-10.

[0358] The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST™; Altschul et al. (1990)) is available from several sources, including the National Center for Biotechnology Information (Bethesda, Md.), and on the internet, for use in connection with several sequence analysis programs. A description of how to determine sequence identity using this program is available on the internet under the “help” section for BLAST™. For comparisons of nucleic acid sequences, the “Blast 2 sequences” function of the BLAST™ (Blastn) program may be employed using the default parameters. Nucleic acid sequences with even greater similarity to the reference sequences will show increasing percentage identity when assessed by this method. Typically, the percentage sequence identity is calculated over the entire length of the sequence.

[0359] For example, a global optimal alignment is suitably found by the Needleman-Wunsch algorithm with the following scoring parameters: Match score: +2, Mismatch score: −3; Gap penalties: gap open 5, gap extension 2. The percentage identity of the resulting optimal global alignment is suitably calculated by the ratio of the number of aligned bases to the total length of the alignment, where the alignment length includes both matches and mismatches, multiplied by 100.

[0360] “Synthetic” in the present application means a nucleic acid molecule that does not occur in nature. Synthetic nucleic acid expression constructs of the present invention are produced artificially, typically by recombinant technologies. Such synthetic nucleic acids may contain naturally occurring sequences (e.g. promoter, enhancer, intron, and other such regulatory sequences), but these are present in a non-naturally occurring context. For example, a synthetic gene (or portion of a gene) typically contains one or more nucleic acid sequences that are not contiguous in nature (chimeric sequences), and/or may encompass substitutions, insertions, and deletions and combinations thereof.

[0361] “Transfection” in the present application refers broadly to any process of deliberately introducing nucleic acids into cells, and covers introduction of viral and non-viral vectors, and includes transformation, transduction and like terms and processes. Examples include, but are not limited to: transfection with viral vectors; transformation with plasmid vectors; electroporation (Fromm et al. (1986) Nature 319:791-3); lipofection (Feigner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7); microinjection (Mueller et al. (1978) Cell 15:579-85); Agrobacterium-mediated transfer (Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80:4803-7); direct DNA uptake; whiskers-mediated transformation; and microprojectile bombardment (Klein et al. (1987) Nature 327:70).

[0362] The term “vector” is well known in the art, and as used herein refers to a nucleic acid molecule, e.g. double-stranded DNA, which may have inserted into it a nucleic acid sequence according to the present invention. A vector is suitably used to transport an inserted nucleic acid molecule into a suitable host cell. A vector typically contains all of the necessary elements that permit transcribing the insert nucleic acid molecule, and, preferably, translating the transcript into a polypeptide. A vector typically contains all of the necessary elements such that, once the vector is in a host cell, the vector can replicate independently of, or coincidental with, the host chromosomal DNA; several copies of the vector and its inserted nucleic acid molecule may be generated. Vectors of the present invention can be episomal vectors (i.e., that do not integrate into the genome of a host cell), or can be vectors that integrate 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 derivatives thereof that are devoid of bacterial sequences (minicircles)) transposons-based vectors (e.g. PiggyBac (PB) vectors or Sleeping Beauty (SB) vectors), etc. Larger vectors such as artificial chromosomes (bacteria (BAC), yeast (YAC), or human (HAC)) may be used to accommodate larger inserts. Viral vectors are derived from viruses and include but are not limited to retroviral, lentiviral, adeno-associated viral, adenoviral, herpes viral, hepatitis viral vectors or the like. Typically, but not necessarily, viral vectors are replication-deficient as they have lost the ability to propagate in a given cell since viral genes essential for replication have been eliminated from the viral vector. However, some viral vectors can also be adapted to replicate specifically in a given cell, such as e.g. a cancer cell, and are typically used to trigger the (cancer) cell-specific (onco)lysis. Virosomes are a non-limiting example of a vector that comprises both viral and non-viral elements, in particular they combine liposomes with an inactivated HIV or influenza virus (Yamada et al., 2003). Another example encompasses viral vectors mixed with cationic lipids.

[0363] The term “operably linked”, “operably connected” or equivalent expressions as used herein refer to the arrangement of various nucleic acid elements relative to each such that the elements are functionally connected and are able to interact with each other in the manner intended. Such elements may include, without limitation, a promoter, an enhancer and/or a regulatory element, a polyadenylation sequence, one or more introns and/or exons, and a coding sequence of a gene of interest to be expressed. The nucleic acid sequence elements, when properly oriented or operably linked, act together to modulate the activity of one another, and ultimately may affect the level of expression of an expression product. By modulate is meant increasing, decreasing, or maintaining the level of activity of a particular element. The position of each element relative to other elements may be expressed in terms of the 5′ terminus and the 3′ terminus of each element, and the distance between any particular elements may be referenced by the number of intervening nucleotides, or base pairs, between the elements. As understood by the skilled person, operably linked implies functional activity, and is not necessarily related to a natural positional link. Indeed, when used in nucleic acid expression cassettes, cis-regulatory elements will typically be located immediately upstream of the promoter (although this is generally the case, it should definitely not be interpreted as a limitation or exclusion of positions within the nucleic acid expression cassette), but this needs not be the case in vivo, e.g., a regulatory element sequence naturally occurring downstream of a gene whose transcription it affects is able to function in the same way when located upstream of the promoter. Hence, according to a specific embodiment, the regulatory or enhancing effect of the regulatory element is position-independent.

[0364] A “spacer sequence” or “spacer” as used herein is a nucleic acid sequence that separates two functional nucleic acid sequences (e.g. TFBS, CREs, CRMs, minimal promoters, etc.). It can have essentially any sequence, provided it does not prevent the functional nucleic acid sequence (e.g. cis-regulatory element) from functioning as desired (e.g. this could happen if it includes a silencer sequence, prevents binding of the desired transcription factor, or suchlike). Typically, it is non-functional, as in it is present only to space adjacent functional nucleic acid sequences from one another.

[0365] “Cell culture”, as used herein, refers to a proliferating mass of cells that may be in either an undifferentiated or differentiated state.

[0366] “Consensus sequence”—the meaning of consensus sequence is well-known in the art. In the present application, the following notation is used for the consensus sequences, unless the context dictates otherwise. Considering the following exemplary DNA sequence:

A[CT]N{A}YR

[0367] A means that an A is always found in that position; [CT] stands for either C or T in that position; N stands for any base in that position; and {A} means any base except A is found in that position. Y represents any pyrimidine, and R indicates any purine.

[0368] “Complementary” or “complementarity”, as used herein, refers to the Watson-Crick base-pairing of two nucleic acid sequences. For example, for the sequence 5′-AGT-3′ binds to the complementary sequence 3′-TCA-5′. Complementarity between two nucleic acid sequences may be “partial”, in which only some of the bases bind to their complement, or it may be complete as when every base in the sequence binds to its complementary base. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

[0369] As used herein, the phrase “transgene” refers to an exogenous nucleic acid sequence. In one example, a transgene is a gene sequence, a gene encoding an industrially or pharmaceutically useful compound, or a gene encoding a desirable trait. In yet another example, the transgene is an antisense nucleic acid sequence, wherein expression of the antisense nucleic acid sequence inhibits expression of a target nucleic acid sequence.

[0370] The terms “subject” and “patient” are used interchangeably herein and refer to animals, preferably vertebrates, more preferably mammals, and specifically include human patients and non-human mammals. “Mammalian” subjects include, but are not limited to, humans. Preferred patients or subjects are human subjects.

[0371] A “therapeutic amount” or “therapeutically effective amount” as used herein refers to the amount of expression product effective to treat a disease or disorder in a subject, i.e., to obtain a desired local or systemic effect. The term thus refers to the quantity of an expression product that elicits the biological or medicinal response in a tissue, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. Such amount will typically depend on the gene product and the severity of the disease, but can be decided by the skilled person, possibly through routine experimentation.

[0372] As used herein, the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures. Beneficial or desired clinical results include, but are not limited to, prevention of an undesired clinical state or disorder, reducing the incidence of a disorder, alleviation of symptoms associated with a disorder, diminishment of extent of a disorder, stabilized (i.e., not worsening) state of a disorder, delay or slowing of progression of a disorder, amelioration or palliation of the state of a disorder, remission (whether partial or total), whether detectable or undetectable, or combinations thereof. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0373] As used herein, the terms “therapeutic treatment” or “therapy” and the like, refer to treatments wherein the object is to bring a subject's body or an element thereof from an undesired physiological change or disorder to a desired state, such as a less severe or unpleasant state (e.g., amelioration or palliation), or back to its normal, healthy state (e.g., restoring the health, the physical integrity and the physical well-being of a subject), to keep it at said undesired physiological change or disorder (e.g., stabilization, or not worsening), or to prevent or slow down progression to a more severe or worse state compared to said undesired physiological change or disorder.

[0374] As used herein the terms “prevention”, “preventive treatment” or “prophylactic treatment” and the like encompass preventing the onset of a disease or disorder, including reducing the severity of a disease or disorder or symptoms associated therewith prior to affliction with said disease or disorder. Such prevention or reduction prior to affliction refers to administration of the nucleic acid expression constructs, vectors, or pharmaceutical compositions described herein to a patient that is not at the time of administration afflicted with clear symptoms of the disease or disorder. “Preventing” also encompasses preventing the recurrence or relapse-prevention of a disease or disorder for instance after a period of improvement. In embodiments, the nucleic acid expression constructs, vectors, or pharmaceutical compositions described herein may be for use in gene therapy.

[0375] “Hypoxia”, “hypoxic” or related terms is a condition of low oxygen tension, typically in the range 1-5% O.sub.2. Under such conditions eukaryotic cells respond through induction of various cellular responses, many of which are meditated by HIF. In a clinical context, hypoxic conditions is often found in the central region of tumours or other tissues due to poor vascularisation or disruption of blood supply. A CRE according to the eighteenth aspect of the present invention, a hypoxia-inducible promoter according to the nineteenth aspect of this invention or a gene therapy vector according to the twentieth aspect of this invention may be particularly useful in gene therapy where the tissue where therapy is required is hypoxic. This is often the case in cancer the central region of tumours and in the lymph nodes. “Normoxia” or “normoxic” is used to describe oxygen tensions between 10-20%, and “hyperoxia” for those above 20%. In the regions between 5 and 10% 02 cells may begin to show some moderate effects of hypoxia. In the present context, hypoxia can conveniently be induced by exposing cells to an oxygen tension of 5% or less.

Introduction

[0376] The ATP derivative cyclic adenosine monophosphate (cAMP, cyclic AMP, or 3′,5′-cyclic adenosine monophosphate) is a second messenger important in many biological processes. Its main function is in intracellular signal transduction in many different organisms, conveying the cAMP-dependent pathway. This pathway has been well-studied, and it is reviewed in Yan, et al., MOLECULAR MEDICINE REPORTS 13: 3715-3723, 2016.

[0377] Activation of the adenylyl cyclase (also commonly known as adenyl cyclase and adenylate cyclase, abbreviated AC) drives a cascade that, via protein kinase A, leads to activation of the transcription factor CREB which binds specific TFBS (cAMPRE: TGACGTCA) to modulate gene expression.

[0378] Furthermore, AP1 is a TF complex (dimer) composed of variations of Fos and Jun proteins of which there are many forms. These proteins have a complex regulation pathway involving many protein kinases. The activation of AP1 sites (consensus sequence TGA[GC]TCA) by forskolin and other activators of adenylyl cyclase has been documented and is believed to be related to elevated cAMP levels ability to stabilise the protein c-Fos and upregulate its transcription. Therefore, AP1 site induced gene expression is an indirect effect of activation of the adenylyl cyclase. See, for example, Hess et al. Journal of Cell Science 117, 5965-5973 and Sharma and Richards, J. Biol. Chem. 2000, 275:33718-33728.

[0379] The present invention uses cAMPRE and the AP1 TFBS to generate novel synthetic CREs and promoters that are inducible by forskolin and other activators of adenylyl cyclase.

[0380] cAMPRE is the prototypical target sequence for CREB (Craig, et al., 2001).

[0381] AP1 is a consensus sequence of AP1 transcription factor binding sequences and AP1(1), AP1(2) are variants of the consensus sequence (Hess, et al., 2004) (Sharma & Richards, 2000).

[0382] Additionally, other TFBS were used in generating novel synthetic CREs and promoters that are inducible by activators of adenylyl cyclase.

[0383] HRE1 is a consensus sequence of hypoxia responsive elements from (Schödel, et al., 2011).

[0384] ATF6 is the consensus binding sequence for activated transcription factor 6 (ATF6) in cis-regulatory element Unfolded Protein Response Element (Samali, et al., 2010).

[0385] All promoters were placed upstream of the luciferase gene (Examples 1-3) or SEAP gene (Example 4).

[0386] To compare across experiments, the strength of the inducible promoters was compared to CMV-IE promoter, which was driving the same gene as the other constructs.

[0387] Luciferase readouts were normalised to β-galactosidase to produce normalised relative luminometer units (RLUs). β-galactosidase containing pcDNA6 plasmid was used as internal control for transfection efficiency (Thermofisher, V22020). β-galactosidase activity was measured as per manufacturer's instructions (Mammalian βGalactosidase Assay Kit, 75707/75710, Thermo Scientific) using 25 μl of lysate. 25 μl of lysate was transferred into a microplate well and mixed with 25 μl of β-galactosidase Assay Reagent, equilibrated to room temperature. The mixture was incubated at 37° C. for 30 min and absorbance measured at 405 nm.

[0388] The synthetic promoters were synthesised by GeneArt.

Example 1

[0389] The forskolin-inducible promoters were initially used to drive expression of luciferase in the Huh7 human liver cell line and the C2C12 human muscle cell line. The promoter constructs were used to drive expression of luciferase in the PM-RQ plasmid. The tested promoters were synthesised directly upstream of the ATG of PM-RQ plasmid.

Huh7 Transfection

Materials

[0390] Huh7 cells which are a human liver cell line [0391] DPBS: without CaCl.sub.2, without MgCl.sub.2 (Gibco, 14190-094) [0392] DMEM (Sigma, D6546) [0393] FBS (Sigma, F9665) [0394] Pen-Strep (Sigma, P4333) [0395] Promega Fugene-HD (E2311) [0396] Forskolin (Sigma, F6886) [0397] NKH477 (Sigma, N3290) [0398] LARII (Dual Luciferase Reporter 1000 assay system, Promega, E1980) [0399] 48 well plates flat bottom (353230, Corning) [0400] Trypsin-EDTA (25200-056, Gibco) [0401] T25 & T75 flask (353108, 353136, Corning) [0402] 15 ml Falcon tubes (430791, Corning) [0403] Stripettes 5 ml, 10 ml and 25 ml (4101,4051,4251, Corning)

Day 1

[0404] Cells were seeded onto a 48 well plate at a density of 25,000 cells/300 μl

Day 2

[0405] On the day of transfection, DNA to be transfected was diluted to a 100 ng/μl stock solution. DNA to be transfected was the RTV-017, RTV-019 or CMV-IE promoter driving luciferase in the PM-RQ plasmid. [0406] Per 48 well transfection, 45 ng of DNA was mixed with 4.1 μl of Optimem medium. [0407] 0.5 μl of Fusion HD was mixed with 4 μl of Optimem medium. [0408] These 2 solutions were mixed and incubated at room temperature for 15 minutes. [0409] The final solution was then added to the well drop wise. [0410] 3 hrs after transfection the inducer was added to the appropriate wells at the indicated concentration. Inducer was forskolin at 20 pM.

Day 3

[0411] 24 hrs after induction, the luciferase activity was measured as described below.

C2C12 Transfection

Materials:

[0412] C2C12 cell line which is a muscle cell line (91031101, Sigma, lot number 15D022) [0413] DPBS without CaCl.sub.2), without MgCl.sub.2 (14190-094, Gibco) [0414] Horse Serum (16050-122, Gibco) [0415] Viafect (E4981, Promega) [0416] DMEM (high glucose, D6546, Sigma) [0417] DMEM (high glucose, no sodium pyruvate, 11960-044, Gibco) [0418] Penicillin-streptomycin Solution (15140-122, Gibco) [0419] FBS (Gibco10500-064, Lot number 08Q2771K) [0420] GlutaMax (35050-038, Gibco) [0421] 48 well plates flat bottom (353230, Corning) [0422] Trypsin-EDTA (25200-056, Gibco) [0423] T25 & T75 flask (353108, 353136, Corning) [0424] 15 ml Falcon tubes (430791, Corning) [0425] Stripettes 5 ml, 10 ml and 25 ml (4101,4051,4251, Corning)

Media Preparation:

[0426] Media was prewarmed at 37° C. before use [0427] C2C12 Complete Medium was used to grow the cells before differentiation. To prepare 50 ml of complete medium, 44 ml DMEM (high glucose, D6546, Sigma), 0.5 ml GlutaMAX, 0.5 ml Penicillin-streptomycin solution and 5 ml FBS was added. [0428] C2C12 Transfection Medium was used for transfection of the C2C12 cells. To prepare 50 ml of transfection medium, 49.5 ml DMEM (high glucose, D6546, Sigma) and 0.5 ml GlutaMAX was added. [0429] C2C12 Differentiation Medium was used to differentiate the C2C12 cells. To prepare 50 mil of differentiation medium, 48.5 ml DMEM (high glucose, no sodium pyruvate, 11960-044, Gibco), 1 ml Horse Serum (Heat Inactivated) and 0.5 GlutaMAX was added.

Transfection:

[0430] C2C12 cells were maintained at 37° C., 5% CO.sub.2. During transfection, the C2C12 cells were checked daily using an inverted microscope. C2C12 cell line was only used up to passage 12 as beyond this passage, the cells progressively lose their differentiation ability.
Day 1—Plating C2C12 cells for transformation [0431] C2C12 Complete Medium, trypsin EDTA, and PBS were prewarmed at 37° C. [0432] C2C12 cells grown in complete medium were aspirated, washed with 5 ml DPBS, aspirate and 1.0 ml Trypsin/EDTA solution (for a T75 flask) was added [0433] The cells were left at 37° C. for 3 to 5 mins, until the cells detach. Detachment was monitored under the microscope. [0434] 4 ml C2C12 Complete Medium was added to inactivate Trypsin and the cells were transferred into a 15 ml tube. [0435] The cells were centrifuged for 3 mins at 250 RCF at room temperature. [0436] The supernatant was aspirated [0437] The cells were resuspended by gentle pipetting up and down in 6 ml C2C12 Complete Medium [0438] The cells were counted in a haemocytometer by using trypan blue (Thermofisher, 15250061) to determine the how many of the cells were alive. Trypan blue stains dead cells in blue and this allows for them not to be counted. The cells were resuspended into trypan blue in ration of 1:10, e.g. 10 μl cells and 90 μl trypan blue and counted using a haemocytometer. [0439] The cells were seeded in complete medium at a density dependent on the passage number. For low passage numbers (passage 4-6), the cells were seeded at a density of 45000 cells/well. For medium passage number (passage 7-9), the cells were seeded at a density of 40000 cells/well. For high passage number (passage 10-12), the cells were seeded at a density of 38000 cells/well. Different cell densities were used at different passage numbers as the cells differentiate from myoblasts to myotubes if seeded at the wrong density. The abovementioned densities are experimentally determined for efficient differentiation and transfection.

Day 2—Transfection

[0440] 24 hours after seeding, the cells were transfected [0441] C2C12 transfection medium was prewarmed at 37° C. [0442] The DNA stock to be transfected was diluted to 100 ng/μl in deionized water (MilliQ grade). Following dilution, the concentration of the diluted DNA sample was checked by measuring absorbance at 260 nm to ensure id did not deviate more than 10% from the desired 100 ng/μl. DNA to be transfected was the RTV-019 or CMV-IE promoter driving luciferase in the PM-RQ plasmid. [0443] The media of the C2C12 cells which were seeded in the 48 well plate the previous day was changed to C2C12 Transfection Medium (300 μl of medium/well). [0444] DNA/transfection reagent complexes were prepared following the manufacturer's instructions (Viafect—E4981, Promega). For a 48 well plate, 30 μl of the following mixture was prepared per well: 26.1 μl C2C12 plain medium (DMEM high glucose, D6546, Sigma—no additives), 0.3 pg DNA (3 μl of a DNA dilution adjusted to 100 ng/μl) and 0.9 μl Viafect. The DNA was initially added to the DMEM plain medium and incubated for 5 minutes. The Viafect was then added and the mixture mixed. The mixture was incubated for 20 minutes at room temperature to allow DNA and the transfection reagent to complex. [0445] 30 μl of transfection complexes was added per well and the plate was gently swirled so that the reaction mix is evenly spread in the well and the cells were incubated at 37° C., 5% CO.sub.2.
Day 3—Switch to differentiation media [0446] 24 hours after transfection, the media of the C2C12 cells was changed to differentiation media

Day 6.5—Induction

[0447] 20 pM Forskolin was added to the appropriate wells and the cells were incubated for 24 hrs before reading.
Day 7.5—Luciferase assay [0448] 5.5 days after transfection, the luciferase assay was performed as described below

Measurement of Luciferase Activity

[0449] Luciferase activity was measured using LARII (Dual Luciferase Reporter 1000 assay system, Promega, E1980) [0450] 24 hours after induction, the media was removed from the cells [0451] The cells were washed once in 300 μl of DPBS. [0452] Cells were lysed using 100 μl of passive lysis buffer and incubated with rocking for 15 minutes. [0453] The cell debris was pelleted by centrifugation of the plate at max speed in a benchtop centrifuge for 1 min [0454] For luciferase, 10 μl sample was transferred into white 96-well plate and luminescence measured by injection of 50 μl of LARII substrate

Results

[0455] RTV-017 and RTV-019 were transiently transfected into the human liver cell line Huh7 (FIG. 2). RTV-019 was transiently transfected into the human muscle cell line C2C12 (FIG. 3). The activity of the promoters was assessed using luciferase activity. The luciferase activity was measured with and without the presence of 20 pM forskolin.

[0456] Both the RTV-017 and RTV-019 promoters in Huh7 and the RTV-019 promoter in C2C12 have low activity before the addition of forskolin. Upon addition of forskolin, the promoters have a much higher activity. In liver cells the promoters are induced 13 and 24-fold respectively whereas in muscle cells the promoter is only activated 6-fold.

Example 2

[0457] The forskolin-inducible promoters RTV-017, RTV-019 and FORCYB1 were then used to drive expression of luciferase in a pAAV vector in the Huh7 human liver cell line. The constructs were cloned into pAAV by Gibson assembly. This experiment was performed to investigate the effect of the Inverted Terminal Repeats (ITRs) on the activity of the promoters. This was done as we have observed interference from the AAV ITRs in other projects.

[0458] The cells were seeded, transfected and induced as described above. Luciferase expression was measured as described above. In this experiment, the cells were induced by using 20 pM forskolin or 7.2 pM of the water soluble derivative NKH477. Similarly, to the activity pattern observed in FIGS. 2 and 3, the promoters appeared to show low background activity before induction and the high level of luciferase activity upon induction. The induction for RTV-017 was 23 and 44-fold for with NKH477 and forskolin respectively. The induction for RTV-019 was 86 and 98-fold with NKH477 and forskolin respectively. The induction for FORCYB1 was 30 and 65-fold with NKH477 and forskolin respectively. This indicates that all promoters have good inducibility both with forskolin and the water soluble derivative NKH477. Moreover, the ITRs do not appear to substantially affect the promoter activity. This indicates that the promoters might be useful in gene therapy as transfection of Huh7 with pAAV plasmid is a good indicator of the promoter's performance in AAV viral particles.

Example 3

[0459] The forskolin-inducible promoters RTV-017, RTV-019, FORCSV-10, FORCYB-001, FOR-CMV-009, FMP-02 and FLP-01 were then used to drive expression of luciferase in a PM-RQ vector in the suspension cell line HEK293-F. The forskolin-inducible promoters RTV-017, RTV-019, FORCSV-10, FORCYB-001, FOR-CMV-009, FMP-02 and FLP-01 were also used to drive expression of luciferase in a PM-RQ vector in the suspension cell line CHO-K1SV. The tested promoters were synthesised directly upstream of the ATG of PM-RQ plasmid and the suspension cell lines were transiently transfected with the PM-RQ plasmid.

Transfection of HEK293-F Cells

[0460] 40 ml of cells were grown in a 250 ml vented Erlenmeyer flask (Sigma-Aldrich CLS431144) at 37° C., 20% O.sub.2, 8% CO.sub.2 with agitation at 100 rpm. Cells were seeded as described in the manufacturer's instructions (300,000 cells/ml). HEK293-F were obtained from Thermofisher, R79007.

[0461] One day before transfection, the cells were counted using a haemocytometer and split to 500,000 cells/ml.

[0462] On the day of transfection, the cells are seeded to 1,000,000 cells/ml in 500 μl of appropriate medium (Freestyle 293 expression medium, 12338002) in a 24 well plate. 0.625 pg of DNA per well was then added to 10 μl of OptiMem medium (Thermofisher; 11058021) and incubated for 5 minutes at room temperature.

[0463] Concurrently, 0.625 μl of Max reagent (Thermofisher, 16447100) was made up to 10 μl by addition of OptiMem and incubated for 5 minutes at room temperature. After this incubation, both mixes were added to the same tube and incubated at room temperature for 25-30 minutes. The DNA/Max reagent mix (20 μl/well) was then added directly to the cells and the cells incubated at 37 C, 8% CO.sub.2 with agitation at 100 rpm.

[0464] 24 hours after transfection, the promoters were induced by addition of 20 pM forskolin and luciferase activity was measured 0, 3, 5 and 24 hours after induction. Luciferase activity was measured as previously described.

Transient Transfection of CHO-K1SV Cells

[0465] 40 ml of cells were grown in a 250 ml vented Erlenmeyer flask (Sigma-Aldrich CLS431144) at 37° C., 20% O.sub.2, 8% CO.sub.2 with agitation at 100 rpm. Cells were seeded at 300,000 cells/ml.

[0466] One day before transfection, the cells were counted using a haemocytometer and split to 500,000 cells/ml.

[0467] On the day of transfection, the cells are seeded to 1,000,000 cells/ml in 500 μl of appropriate medium (Thermofisher, CD-CHO 10743029) in a 24 well plate. 0.625 pg of DNA per well was then added to 10 μl of OptiMem medium (Thermofisher; 11058021) and incubated for 5 minutes at room temperature.

[0468] Concurrently, 0.625 μl of Freestyle Max reagent (Thermofisher, 16447100) was made up to 10 μl by addition of OptiMem and incubated for 5 minutes at room temperature. After this incubation, both mixes were added to the same tube and incubated at room temperature for 25-30 minutes. The DNA/Max reagent mix (20 μl/well) was then added directly to the cells and the cells incubated at 37 C, 8% CO.sub.2 with agitation at 100 rpm.

[0469] The promoters were induced by addition of 20 pM forskolin and luciferase activity was measured after 24 hours. Luciferase activity was measured as described before.

Results

[0470] The forskolin-inducible promoters were used to drive expression of luciferase in the suspension cell line HEK293-F (FIG. 5) and CHO-K1SV (FIG. 6).

[0471] In the time course after induction, the promoters show a low background with a rapid increase in activity with maximal activity seen after 5 hrs. This activity is maintained until 24 hrs. Fold induction for the promoters varies from 50 to 100-fold, with FMP-02 being the weakest and RTV-019 being the strongest. The dynamic range of the promoters is also very wide with a 10-fold range at maximal activity. These results show that the promoters may be promising in bioprocessing applications due to their tight control and wide dynamic range (the ratio of the strongest promoter strength to the weakest promoter strength).

Example 4

[0472] The forskolin-inducible promoters were then tested in a stably transfected CHO-GS-KSV1 cell line.

Generating CHO-GS-KSV1 Stable Cell Lines

Materials

[0473] CD-CHO media (Life technologies, CAT #10743029) [0474] Corning 125 mL Polycarbonate Erlenmeyer Flask with Vent Cap (CAT #734-1885). [0475] Gene Pulser® Electroporation Cuvettes, 0.4 cm gap (BioRad, CAT #165-2088) [0476] Gene Pulser Xcell Total System (BioRad, CAT, #1652660) [0477] GS-vector DNA (40 pg in 100 μL TE buffer) linearised with Sca1 [0478] Suspension cultures of CHOK1SV GS-KO host cells.

[0479] Cell suspensions of 6×10.sup.5 cells per mL were incubated on an orbital shaker set at 8% CO.sub.2, 20% O.sub.2, 37° C., 85% relative humidity and 140 rpm overnight. 2.86×10.sup.7 cells were centrifuged at 200 g for 3 minutes. Media was then aspirated and cell pellet resuspended in 2 mL fresh CD-CHO media to obtain a concentration of 1.43×10.sup.7 cells per mL. 700 μL of cell suspension was added to each of two electroporation cuvettes each containing 40 g of linearised DNA in 100 μL of sterile TE buffer (Thermofisher, 12090015). Each cuvette was electroporated, delivering a single pulse of 300V, 900 pF with resistance to infinity. Immediately after delivery of pulse electroplated cells were transferred to Erlenmeyer 125 mL flask containing 20 mL of CD-CHO media pre-warmed to 37° C. Electroporated cells from two cuvettes are combined into a single 125 mL flask to generate one pool of cells. Cells are cultured on an orbital shaker set at 8% CO.sub.2, 20% O.sub.2, 37° C., 85% relative humidity and 140 rpm. Cells are transferred to fresh CD-CHO media 24 hours after transfection and cell cultures monitored and given fresh CD-CHO media every 2-4 days. Usually after about 10-14 days cell numbers will be high enough to start passaging.

[0480] The transfected DNA was the pXC-17.4 expression vector (Lonza Biologics plc) where one of the promoter constructs (or a control promoter (CMV-IE) have been cloned upstream of the secreted alkaline phosphatase (SEAP) gene, which had been cloned into the multiple cloning site within the vector expression cassette. Promoters were closed into the pXC-17.4 vector using Gibson assembly. The pXC-17.4 expression vector is designed for making stable cell lines in the CHO-GSK1SV cell line as it contains the glutamate synthase gene which has been knocked out of the cell line. Therefore, selection of cells in glutamine drop-out medium will select for cells that have stably integrated the plasmid.

[0481] The responsiveness of the RTV-019 promoter to different concentrations of forskolin and NJH477 was tested in the stably transfected CHO-GS-KSV1 cell line. To this end, the stably transfected CHO-GS cells were seeded at 500,000 cells/ml and allowed to grow for 24 hrs. At this point the cells were induced by addition of the respective concentrations of forskolin and NKH477 and SEAP expression was measured as described below after 24 hours.

[0482] The promoter activity and inducibility of FMP-02, FORCYB-001, RTV-017, FORCSV-10, FOR-CMC-009, FLP-01 and RTV-019 were also tested in the stably transfected CHO-GS-KSV1 cell line. To this end, the stably transfected CHO-GS cells were seeded at 500,000 cells/ml and allowed to grow for 24 hrs. At this point the cells were exposed to 20 pM forskolin or 7.2 pM NKH477 for 24 hours. After this point, the SEAP activity in the medium was assessed as described below.

SEAP Assay

[0483] SEAP Reporter Gene Assay, chemiluminescent (Roche, CAT #11 779 842 001) was used to measure SEAP activity as per the manufacturer's protocol. All reagents and samples were fully pre-equilibrated at room temperature. Culture supernatant was collected from the stably transfected CHO-GS at the specific time points (0 h and 24 h). The supernatant was diluted 1:4 in dilution buffer and heat treated at 65° C. for 30 minutes. The heat-treated sample was then centrifuged for 30 s at maximum speed. 50 μl of the heat-treated sample was then added to 5 μl of inactivation buffer and incubated for 5 min at room temperature. 50 μl of substrate reagent was then added and incubated for 10 min at room temperature. The signal is then read at 477 nm and compared to a calibration curve. SEAP expression was normalized against cell number to ensure that the increase in activity was not due to increased cell numbers and was true induction.

Results

[0484] The response of the promoter RTV-019 to increasing concentrations of forskolin and NKH477 in the stably transfected CHO-GS-KSV1 cell line is shown in FIG. 7. The cell line was grown for 24 hrs then exposed to either 2, 5, 9, 18 or 32 pM of forskolin or NKH477. All concentrations induced the promoters but the optimal was 18 pM for forskolin and 8 pM for NKH477. This correlates well with the concentrations reported in the literature wherein the increase of the cAMP level by forskolin and NKH477 was measured.

[0485] The response of the promoters to 20 pM forskolin or 7.2 pM NKH477 in the stably transfected CHO-GS-KSV1 cell line is shown in FIGS. 8 and 9. FIG. 8 shows the activity of the promoters and FIG. 9 shows the maximum activity reached by each promoter compared with CMV-IE. All promoters show increased expression upon addition of either forskolin or NKH477. As before, FMP-02 is the weakest and RTV-019 is the strongest. There is up to 35-fold induction seen in this system with a range of 6-fold between the weakest and strongest expressors. In this experiment, it appears that NKH477 was a slightly more potent inducer of the promoters. NKH477 is a preferred inducer for bioprocessing because it is water soluble and it will be easier to wash it away during purification steps.

[0486] Overall these promoters show great promise for use in both bioprocessing (CHO-K1SV, HEK293-F) and gene therapy (Huh7, pAAV, C2C12). The promoters are robust in multiple cell types showing good inducibilty and strength while maintaining low background.

Hypoxia and HIF

[0487] The importance of the HIF signalling cascade is shown by knockout studies in mammals which leads to perinatal death. This is due to its role in the development of the vascular system and chondrocyte survival. In addition, HIF1 plays a central role in human metabolism as it is linked with respiration and energy generation. Furthermore, the cascade mediates the effects of hypoxia by upregulating genes important for survival in such conditions. For example, hypoxia promotes the formation of blood vessels, which is a normal response essential in development. However, in cancer, hypoxia can also lead to the vascularisation of tumours.

[0488] The main response element for the sensing and upregulation of genes involved in hypoxia stress response is the transcriptional complex HIF1. This complex is highly conserved across eukaryotes and is formed by the dimerization of 2 subunits, α and β. The β-subunit is constitutively expressed and is an aryl hydrocarbon receptor nuclear translocator (ARNT) essential for translocation of the complex to the nucleus. Both the α and β-subunits belong to the basic helix-loop-helix family of transcription factors and contain the following domains: [0489] N-terminal: bHLH domain for DNA binding [0490] Central heterodimerization domain: Per-ARNT-Sim (PAS) domain [0491] C-terminal: recruits transcriptional coregulatory proteins

HIF Mechanism of Action

[0492] Under normoxic conditions HIF1α subunits are hydroxylated at conserved proline residues. This hydroxylation by HIF prolyl-hydroxylases targets the subunits for recognition and ubiquitination by the VHL E3 ubiquitin ligase and subsequent degradation by the proteasome. However, under hypoxic conditions, oxygen limitation inhibits the HIF prolyl-hydroxylase as oxygen is an essential co-substrate for this enzyme. Once stabilised, HIF-1α subunits can heterodimerise with HIF-1β subunits and translocate to the nucleus where they can upregulate the expression of a number of genes. This is achieved by the HIF complex's binding to HIF-responsive elements (HREs) in promoters that contain the HBS sequence NCGTG (SEQ ID NO: 5) (where N is preferably either an A or G) or its reverse complement. The genes upregulated by the HIF1 complex are involved in central metabolism, such as glycolysis enzymes which allow ATP synthesis in an oxygen-independent manner, or in angiogenesis such as vascular endothelial growth factor (VEGF).

Pseudohypoxia

[0493] There are alternative ways to activate the HIF1 complex. Mutations to SDHB, one of four protein subunits forming succinate dehydrogenase, cause build-up of succinate by inhibiting electron transfer in the succinate dehydrogenase complex. This excess succinate inhibits HIF prolyl-hydroxylase, stabilizing HIF-1α.

[0494] NF-κB can also directly modulate HIF1 regulation under normoxic conditions. It is believed that NF-κB can regulate basal HIF-1α expression as increased HIF-1α levels was correlated with increased NF-κB expression.

Hypoxia Responsive Elements

[0495] Hypoxia-responsive elements tend to have a conserved HIF1 binding consensus sequence, NCGTG (SEQ ID NO: 5), where N is preferably either an A or G (Schödel, et al., 2011, Blood. 2011 Jun. 9; 117(23):e207-17.). The flanking sequence of this is notoriously variable but still contributes to the activity of the promoter.

[0496] The following exemplary HIF binding sequences (HBS) are used in the following examples: [0497] HRE1 (ACGTGC (SEQ ID NO: 8)) which is a variant of the consensus sequence ([AG]CGTG, SEQ ID NO: 6) found in HIF binding sites of hypoxia-responsive elements (Schödel, et al., 2011). [0498] HRE2 (CTGCACGTA (SEQ ID NO: 7)) was described as a superior and highly active hypoxia-inducible motif (Kaluz, et al., 2008, Biochem Biophys Res Commun. 2008 Jun. 13; 370(4):613-8). [0499] HRE3 (ACCTTGAGTACGTGCGTCTCTGCACGTATG (SEQ ID NO: 9)) was described as a strongly induced element (Ede, et al., 2016, ACS Synth. Biol., 2016, 5 (5), pp 395-404). HRE3 is a composite HBS which comprises both HRE1 and HRE2 and it was hypothesised that it may be possible to increase the strength of induction by using this element.

[0500] Synthetic promoters comprising these HBS sequences were prepared and tested as described below. [0501] Synp-HYP-001 construct (SEQ ID NO: 83) comprises 4 HRE2 elements without spacers, a spacer of 32 base pair length between the core of the last HRE2 and the TATA box of the CMV minimal promoter. This construct was designed with suboptimal spacing between the HRE2 elements and between the last HBS and the minimal promoter, and it was predicted to have relatively low inducibility and strength of expression. [0502] Synp-RTV-015 construct (SEQ ID NO: 81) comprises 5 HRE1 elements spaced apart by 40 bp spacers, followed directly by a synthetic minimal promoter TATA box (MP1). This promoter was designed to be only weakly induced by hypoxia by its suboptimal spacing of 40 bp between the HRE1 elements and a spacing of 36 bp from the core of the last HRE1 HBS to the TATA box of MP1. [0503] Synp-RTV-016 construct (SEQ ID NO: 82) comprises 6 HRE2 elements spaced apart by 20 bp spacers, with length of 65 base pairs between the core of the last HRE2 HBS and the TATA box of the CMV minimal promoter. This promoter was designed to be of medium strength, stronger than RTV-015 but weaker than HYBT and HV3C, as the spacing between the HBSs and the minimal promoter is suboptimal. [0504] Synp-HYBT construct (SEQ ID NO: 84) comprises 4 HRE3 elements spaced apart by 9 bp spacer (meaning a spacing of 21 bp between cores of adjacent HRE3 elements, and an internal spacing of 8 bp between the two core sequences of the HRE3 element), and a spacing of 29 base pairs between the core of the last HRE3 and the TATA box of the YB-TATA minimal promoter. This construct was designed to have strong activity. [0505] Synp-HV3C construct (SEQ ID NO: 85) comprises 4 HRE3 elements spaced apart by 9 bp spacer (meaning a spacing of 21 bp between cores of adjacent HRE3 elements, and an internal spacing of 8 bp between the two core sequences of the HRE3 element), and a spacing of 25 base pairs between the core of the last HRE3 and the TATA box of the CMV minimal promoter. This construct is very similar to HYBT but differs in the minimal promoter used. This construct was designed to have strong activity.

[0506] All promoters were placed upstream of the luciferase gene (Examples 1 and 2) or SEAP gene (Example 3).

[0507] To compare across experiments, the strength of the inducible promoters was compared to CMV-IE promoter, which was driving the same gene as the other constructs.

[0508] The synthetic promoters were synthesised by Geneart. The promoter constructs were used to drive expression of luciferase in the pMQ plasmid, unless otherwise stated.

Example 5

[0509] The constructs were initially used to drive luciferase expression in HEK293-F and HEK293-T in hypoxia.

Transfection of HEK293-F Cells in 24 Well Format

[0510] 40 ml of cells were grown in a 250 ml vented Erlenmeyer flask (Sigma-Aldrich CLS431144) at 37° C., 20% O.sub.2, 8% CO.sub.2 with agitation at 100 rpm. Cells were seeded as described in the manufacturer's instructions (300,000 cells/ml). HEK293-F were obtained from Thermofisher, R79007.

[0511] One day before transfection, the cells were counted using a haemocytometer and split to 500,000 cells/ml.

[0512] On the day of transfection, the cells are seeded to 1,000,000 cells/ml in 500 μl of appropriate medium (Freestyle 293 expression medium, 12338002) in a 24 well plate. 0.625 pg of DNA per well was then added to 10 μl of OptiMem medium (Thermofisher; 11058021) and incubated for 5 minutes at room temperature.

[0513] Concurrently, 0.625 μl of Max reagent (Thermofisher, 16447100) was made up to 10 μl by addition of OptiMem and incubated for 5 minutes at room temperature. After this incubation, both mixes were added to the same tube and incubated at room temperature for 25-30 minutes. The DNA/Max reagent mix (20 μl/well) was then added directly to the cells and the cells incubated at 37 C, 8% CO.sub.2 with agitation at 100 rpm. The transfected DNA was one of the vectors where the promoter constructs (RTV-015, HYBT, RTV-016, HV3C, Synp-HYP-001) or a control promoter (CMV-IE) were used to drive luciferase expression and β-galactosidase containing pcDNA6 plasmid. The β-galactosidase containing plasmid was used as internal control for transfection efficiency (Thermofisher, V22020).

[0514] After transfection the cells were incubated for 24 hrs in normoxia conditions (20% oxygen) before being switched to a gas mix of 5% oxygen, 10% carbon dioxide and 85% nitrogen (hypoxia). This was achieved by gas displacement in a sealed hypoxia chamber. Induction of the promoters was assessed by using luciferase activity after 3, 5 and 24 hrs in hypoxia. These results are shown in FIG. 13.

Transfection of HEK293-T Cells

[0515] HEK293-T are seeded at a density of 20%. Once they reached a confluence between 60 and 80%, the media is changed with DMEM (#21885-025—Thermo Scientific) supplemented with 10% FBS (Gibco, 26140). After 3 hours, the cells were transfected by a transfection mix. The transfection mix is prepared by adding DNA (2 pg per 6 well plate) and PEI 25 kDA (#23966-1—Polyscience) in a ratio of 1:3 in sterile DPBS (#14190169—ThermoFisher Scientific). After mixing, the transfection mix is incubated at room temperature for 30 minutes and then added directly to the cells. After 16 to 18 h post transfection, the media is changed to DMEM+2% FBS. The transfected DNA was one of the vectors where the promoter constructs (RTV-015, HYBT, RTV-016, HV3C) or a control promoter (CMV-IE) were used to drive luciferase expression and β-galactosidase containing pcDNA6 plasmid. The β-galactosidase containing plasmid was used as internal control for transfection efficiency (Thermofisher, V22020).

[0516] After transfection the cells were incubated for 24 hrs in normoxic conditions (20% oxygen). Induction of the promoters was assessed by using luciferase activity after 24 hrs in normoxia or hypoxia (5% oxygen, 10% carbon dioxide and 85% nitrogen). Hypoxia was achieved by gas displacement in a sealed hypoxia chamber. Results are shown in FIG. 14.

Measurement of Luciferase Activity

[0517] Luciferase activity was measured using LARII (Dual Luciferase Reporter 1000 assay system, Promega, E1980).

[0518] Media was removed from the cells at the respective time point (0, 3, 5, 24 hrs after induction). The cells were washed once in 300 μl of DPBS. Cells were lysed by adding 100 μl of passive lysis buffer to the cells and incubation with rocking for 15 minutes. The cell debris was pelleted by centrifugation of the plate at max speed in a benchtop centrifuge for 1 min. 10 μl of supernatant was pipetted into white 96-well plate and luminescence measured by addition of 50 μl of LARII substrate.

[0519] β-galactosidase activity was measured as per manufacturer's instructions (Mammalian βGalactosidase Assay Kit, 75707/75710, Thermo Scientific) using 25 μl of lysate. 25 μl of lysate was transferred into a microplate well and mixed with 25 μl of β-galactosidase Assay Reagent, equilibrated to room temperature. The mixture was incubated at 37° C. for 30 min and absorbance measured at 405 nm.

[0520] Luciferase readouts were normalised to β-galactosidase to produce normalised relative luminometer units (RLUs).

Results

[0521] The described promoters were transiently transfected into either the suspension cell line HEK293-F (FIG. 13) or the adherent HEK293-T (FIG. 14) cell line and activity of the promoters was assessed using luciferase assay.

[0522] In FIG. 13, all of the promoters in HEK293-F cells showed a rapid increase in activity upon a switch to hypoxic conditions with an increase in luciferase activity observed after 3 hrs. Maximal activity was observed after 5 hrs for all of the promoters tested with no significant increase in activity at the 24 hr timepoint. The promoter's activities correlate with their designs, with RTV-015 being the weakest, followed by SYNP-HYP-001, RTV-016, HYBT and HV3C being the strongest. The activities of these promoters give a 13-fold dynamic range of activity with fold inductions approaching 1,000-fold. In contrast, the switch to hypoxia has no effect on the activity of the CMV-IE promoter and luciferase activity does not change. SYNP-HYP-001 has not been tested in the other examples.

[0523] In FIG. 14, the promoter's expression after 24 h in hypoxia was compared to their expression after 24 h in normoxia. In HEK293-T cells the pattern of expression is very similar to HEK293-F cells with RTV-015 being the weakest promoter and HV3C being the strongest with a 9-fold dynamic range observed across the promoters. Fold induction in this cell line is less dramatic with maximal induction of 50-fold observed. Again, there is no change in the CMV-IE promoter between normoxic and hypoxic conditions.

[0524] These results seem to validate our design principals with the strength of the promoters correlating to their theoretical relative strength.

Example 6

Transient Transfection of CHO-GS Cells

[0525] 40 ml of cells were grown in a 250 ml vented Erlenmeyer flask (Sigma-Aldrich CLS431144) at 37° C., 20% O.sub.2, 8% CO.sub.2 with agitation at 100 rpm. Cells were seeded as at 300,000 cells/ml.

[0526] One day before transfection, the cells were counted using a haemocytometer and split to 500,000 cells/ml.

[0527] On the day of transfection, the cells are seeded to 1,000,000 cells/ml in 500 μl of appropriate medium (Thermofisher, CD-CHO 10743029) in a 24 well plate. 0.625 pg of DNA per well was then added to 10 μl of OptiMem medium (Thermofisher; 11058021) and incubated for 5 minutes at room temperature.

[0528] Concurrently, 0.625 μl of Freestyle Max reagent (Thermofisher, 16447100) was made up to 10 μl by addition of OptiMem and incubated for 5 minutes at room temperature. After this incubation, both mixes were added to the same tube and incubated at room temperature for 25-30 minutes. The DNA/Max reagent mix (20 μl/well) was then added directly to the cells and the cells incubated at 37 C, 8% CO.sub.2 with agitation at 100 rpm.

[0529] The transfected DNA was one of the vectors where the promoter constructs (RTV-015, HYBT, RTV-016, HV3C) or a control promoter (CMV-IE) were used to drive luciferase expression and β-galactosidase containing pcDNA6 plasmid. The β-galactosidase containing plasmid was used as internal control for transfection efficiency (Thermofisher, V22020).

[0530] After transfection the cells were incubated for 24 hrs in normoxic conditions (20% oxygen). Induction of the promoters was assessed by using luciferase activity after 24 hrs in normoxia or hypoxia (5% oxygen, 10% carbon dioxide and 85% nitrogen). Hypoxia was achieved by gas displacement in a sealed hypoxia chamber. Luciferase activity was measured as described above. Results are shown in FIG. 15.

Results

[0531] Luciferase expression form the promoters RTV-015, RTV-016, HV3C and HYBT was assessed in the transiently transfected CHO suspension line CHO-GSK1SV in order to test their functionality in an industrially relevant CHO strain.

[0532] As can be seen from FIG. 15, the promoters behave in a similar manner in these cells as they do in HEK293 cells. The relative strength of each promoter is proportional to the HEK293 cell results with RTV-015 being the weakest and HV3C being the strongest. The dynamic range of the promoters is 13-fold with maximal induction observed at >150 fold. Again, the switch to hypoxia has no effect on the activity of the CMV-IE promoter and luciferase activity from this promoter does not differ in hypoxia or normoxia.

[0533] This demonstrates the robustness of the promoters across multiple cell lines and validates our design rules.

Example 7

Generating CHO-GS-KSV1 Stable Cell Lines

Materials

[0534] CD-CHO media (Life technologies, CAT #10743029)

[0535] Corning 125 mL Polycarbonate Erlenmeyer Flask with Vent Cap (CAT #734-1885).

[0536] Gene Pulser® Electroporation Cuvettes, 0.4 cm gap (BioRad, CAT #165-2088)

[0537] Gene Pulser Xcell Total System (BioRad, CAT, #1652660)

[0538] GS-vector DNA (40 pg in 100 μL TE buffer) linearised with Sca1

[0539] Suspension cultures of CHOK1SV GS-KO host cells.

[0540] Cell suspensions of 6×10.sup.5 cells per mL are incubated on an orbital shaker set at 8% CO.sub.2, 20% O.sub.2, 37° C., 85% relative humidity and 140 rpm overnight. 2.86×10.sup.7 cells were centrifuged at 200 g for 3 minutes. Media is then aspirated and cell pellet resuspended in 2 mL fresh CD-CHO media to obtain a concentration of 1.43×10.sup.7 cells per mL. 700 μL of cell suspension was added to each of two electroporation cuvettes each containing 40 g of linearised DNA in 100 μL of sterile TE buffer (Thermofisher, 12090015). Each cuvette was electroporated, delivering a single pulse of 300V, 900 pF with resistance to infinity. Immediately after delivery of pulse electroplated cells were transferred to Erlenmeyer 125 mL flask containing 20 mL of CD-CHO media pre-warmed to 37° C. Electroporated cells from two cuvettes are combined into a single 125 mL flask to generate one pool of cells. Cells are cultured on an orbital shaker set at 8% CO.sub.2, 20% 02, 37° C., 85% relative humidity and 140 rpm. Cells are transferred to fresh CD-CHO media 24 hours after transfection and cell cultures monitored and given fresh CD-CHO media every 2-4 days. Usually after about 10-14 days cell numbers will be high enough to start passaging. Cells were selected at 14 days and then expanded. Induction was performed once doubling time had returned to approx. 24 hrs. The cells assayed in this example were at passage number 15, 17, 19 and 21.

[0541] The transfected DNA was pXC-17.4 expression vector (Lonza Biologics plc) where one of the promoter constructs (RTV-015, HYBT, RTV-016, HV3C) or a control promoter (CMV-IE) have been cloned upstream of the SEAP gene, which had been cloned into the multiple cloning site within the vector expression cassette. Promoters were closed into the pXC-17.4 vector using Gibson assembly. The pXC-17.4 expression vector is designed for making stable cell lines in the CHO-GSK1SV cell line as it contains the glutamate synthase gene which has been knocked out of the cell line. Therefore, selection of cells in glutamine drop-out medium will select for cells that have stably integrated the plasmid.

[0542] Stably transfected CHO-GS cells were seeded at 500,000 cells/ml and allowed to grow for 24 hrs, at this point the cells were counted and placed under hypoxic conditions as previously described. After 24 hrs, the cell numbers and the expression of SEAP was measured.

SEAP Assay

[0543] SEAP Reporter Gene Assay, chemiluminescent (Roche, CAT #11 779 842 001) was used to measured SEAP activity as per manufacturers protocol. All reagents and samples were fully pre-equilibrated at room temperature. Culture supernatant was collected from the stably transfected CHO-GS at the specific time points (0 h and 24 h). The supernatant was diluted 1:4 in dilution buffer and heat treated at 65° C. for 30 minutes. The heat-treated sample was then centrifuged for 30 s at maximum speed. 50 μl of the heat-treated sample was then added to 5 μl of inactivation buffer and incubated for 5 min at room temperature. 50 μl of substrate reagent was then added and incubated for 10 min at room temperature. The signal is then read at 477 nm and compared to a calibration curve. SEAP expression was normalized against cell number to ensure that the increase in activity was not due to increased cell numbers and was true induction. The result of this experiment can be seen in FIG. 16 and FIG. 17.

Results

[0544] To be a useful tool to produce proteins in a manufacturing situation the promoters must function after stable integration into the target cells. To test this the promoters were cloned upstream of the SEAP gene in the vector pXC-17.4 in CHO-GS cells and these cells were assessed for induction by hypoxia.

[0545] The activity of SEAP at 0 hrs, 24 hrs in Hypoxia and at 24 hrs of normoxia is shown in FIG. 6. This graph shows that the promoters are induced by the hypoxic conditions with relative expression levels following a similar trend to the transient transfection assays. In stable cell lines, there is a 10-fold dynamic range of the promoters with a maximal fold induction of 20. FIG. 17 shows that there is no difference in the growth of the cells in the different conditions confirming that the activity observed is due to induction of the promoters and not cell growth.

[0546] The promoter's activity in the stable cell line validates the design principles and shows that their use in biomanufacturing is feasible.

TABLE-US-00017 Sequences Synp-RTV-015 (SEQ ID NO: 81) ACGTGCGATGATGCGTAGCTAGTAGTGATGATGCGTAGCTAGTAGTACGTGCGATGATGCGTAG CTAGTAGTGATGATGCGTAGCTAGTAGTACGTGCGATGATGCGTAGCTAGTAGTGATGATGCGT AGCTAGTAGTACGTGCGATGATGCGTAGCTAGTAGTGATGATGCGTAGCTAGTAGTACGTGCTT GGTACCATCCGGGCCGGCCGCTTAAGCGACGCCTATAAAAAATAGGTTGCATGCTAGGCCTA GCGCTGCCAGTCCATCTTCGCTAGCCTGTGCTGCGTCAGTCCAGCGCTGCGCTGCGTAACGGC CGCC  HRE1 underlined, MP1 minimal promoter in bold. Synp-RTV-016 (SEQ ID NO: 82) CTGCACGTAGATGATGCGTAGCTAGTAGTCTGCACGTAGATGATGCGTAGCTAGTAGTCTGCAC GTAGATGATGCGTAGCTAGTAGTCTGCACGTAGATGATGCGTAGCTAGTAGTCTGCACGTAGAT GATGCGTAGCTAGTAGTCTGCACGTAGTAGTCGTATGCTGATGCGCAGTTAGCGTAGCTGAGGT ACCGTCGACGATATCGGATCCAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCG CCTAGATACGCCATCCACGCTGTTTTGACCTCCATAGAAGATCGCCACC  HRE2 underlined, CMV-MP minimal promoter bold. Synp-HYBT (SEQ ID NO: 86) GATCTTTGTATTTAATTAAGACCTTGAGTACGTGCGTCTCTGCACGTATGGCGATTAAGACCTTGA GTACGTGCGTCTCTGCACGTATGGCGATTAAGACCTTGAGTACGTGCGTCTCTGCACGTATGGC GATTAAGACCTTGAGTACGTGCGTCTCTGCACGTATGGCGATTAATCCATATGCTCTAGAGGGTA TATAATGGGGGCCACTAGTCTACTACCAGAAAGCTTGGTACCGAGCTCGGATCCAGCCACC HRE3 underlined, YB-TATA minimal promoter bold Synp-HV3C (SEQ ID NO: 87) GATCTTTGTATTTAATTAAGACCTTGAGTACGTGCGTCTCTGCACGTATGGCGATTAAGACCTTGA GTACGTGCGTCTCTGCACGTATGGCGATTAAGACCTTGAGTACGTGCGTCTCTGCACGTATGGC GATTAAGACCTTGAGTACGTGCGTCTCTGCACGTATGGCGATTAATCCATATGCAGGTCTATATA AGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTAGATACGCCATCCACGCTGTTTTGACCTC CATAGAAGATCGCCACC  HRE3 underlined, CMV-MP minimal promoter bold Synp-HYP-001 (SEQ ID NO: 83) CTGCACGTACTGCACGTACTGCACGTACTGCACGTATGGGTACCGTCGACGATATCGGATCCAG GTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTAGATACGCCATCCACGCTG TTTTGACCTCCATAGAAGATCGCCACC  HRE2 underlined, minimal promoter bold pMA-RQ luciferase vector - RTV-015 (SEQ ID NO: 88) ACGTGCGATGAGCTCCCCGGGTTAATTAACATATGACTAGTGAATTCATTGATCATAATCAGCCA TACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACA TAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAAT AGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCA TCAATGTATCTTATCATGTCTGGCGGCCGCGACCTGCAGGCGCAGAACTGGTAGGTATGGAAGA TCCCTCGAGATCCATTGTGCTGGCGGTAGGCGAGCAGCGCCTGCCTGAAGCTGCGGGCATTCC CGATCAGAAATGAGCGCCAGTCGTCGTCGGCTCTCGGCACCGAATGCGTATGATTCTCCGCCAG CATGGCTTCGGCCAGTGCGTCGAGCAGCGCCCGCTTGTTCCTGAAGTGCCAGTAAAGCGCCGG CTGCTGAACCCCCAACCGTTCCGCCAGTTTGCGTGTCGTCAGACCGTCTACGCCGACCTCGTTC AACAGGTCCAGGGCGGCACGGATCACTGTATTCGGCTGCAACTTTGTCATGCTTGACACTTTATC ACTGATAAACATAATATGTCCACCAACTTATCAGTGATAAAGAATCCGCGCCAGCACAATGGATC TCGAGGTCGAGGGATCTCTAGAGGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGC GCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGC CACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGA CTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACC TACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGACCTCGGGCCGCGT TGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCA GAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTG CGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCG TGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTG GGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTG AGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAG AGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGA AGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTC TTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGC GCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAAC GAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTA AATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAAT GCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTC CCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATAC CGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCG AGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCT AGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGT GTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACAT GATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAG TTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATC CGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGC GACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAA AGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGAT CCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTT CTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAAT GTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAG CGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAA AGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCAC GAGGCCCTGATGGCTCTTTGCGGCACCCATCGTTCGTAATGTTCCGTGGCACCGAGGACAACCC TCAAGAGAAAATGTAATCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAAGGAGACA CTTTATGTTTAAGAAGGTTGGTAAATTCCTTGCGGCTTTGGCAGCCAAGCTAGATCCGGCTGTGG AATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCA TGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTAT GCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCC CTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAG GCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTA GGCTTTTGCAAAAAGCTAGCTTGGGGCCACCGCTCAGAGCACCTTCCACCATGGCCACCTCAGC AAGTTCCCACTTGAACAAAAACATCAAGCAAATGTACTTGTGCCTGCCCCAGGGTGAGAAAGTCC AAGCCATGTATATCTGGGTTGATGGTACTGGAGAAGGACTGCGCTGCAAAACCCGCACCCTGGA CTGTGAGCCCAAGTGTGTAGAAGAGTTACCTGAGTGGAATTTTGATGGCTCTAGTACCTTTCAGT CTGAGGGCTCCAACAGTGACATGTATCTCAGCCCTGTTGCCATGTTTCGGGACCCCTTCCGCAG AGATCCCAACAAGCTGGTGTTCTGTGAAGTTTTCAAGTACAACCGGAAGCCTGCAGAGACCAATT TAAGGCACTCGTGTAAACGGATAATGGACATGGTGAGCAACCAGCACCCCTGGTTTGGAATGGA ACAGGAGTATACTCTGATGGGAACAGATGGGCACCCTTTTGGTTGGCCTTCCAATGGCTTTCCTG GGCCCCAAGGTCCGTATTACTGTGGTGTGGGCGCAGACAAAGCCTATGGCAGGGATATCGTGG AGGCTCACTACCGCGCCTGCTTGTATGCTGGGGTCAAGATTACAGGAACAAATGCTGAGGTCAT GCCTGCCCAGTGGGAGTTCCAAATAGGACCCTGTGAAGGAATCCGCATGGGAGATCATCTCTGG GTGGCCCGTTTCATCTTGCATCGAGTATGTGAAGACTTTGGGGTAATAGCAACCTTTGACCCCAA GCCCATTCCTGGGAACTGGAATGGTGCAGGCTGCCATACCAACTTTAGCACCAAGGCCATGCGG GAGGAGAATGGTCTGAAGCACATCGAGGAGGCCATCGAGAAACTAAGCAAGCGGCACCGGTAC CACATTCGAGCCTACGATCCCAAGGGGGGCCTGGACAATGCCCGTCGTCTGACTGGGTTCCAC GAAACGTCCAACATCAACGACTTTTCTGCTGGTGTCGCCAATCGCAGTGCCAGCATCCGCATTC CCCGGACTGTCGGCCAGGAGAAGAAAGGTTACTTTGAAGACCGCCGCCCCTCTGCCAATTGTGA CCCCTTTGCAGTGACAGAAGCCATCGTCCGCACATGCCTTCTCAATGAGACTGGCGACGAGCCC TTCCAATACAAAAACTAATTAGACTTTGAGTGATCTTGAGCCTTTCCTAGTTCATCCCACCCCGCC CCAGAGAGATCTTTGTGAAGGAACCTTACTTCTGTGGTGTGACATAATTGGACAAACTACCTACA GAGATTTAAAGCTCTAAGGTAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAA TTGTTTGTGTATTTTAGATTCCAACCTATGGAACTGATGAATGGGAGCAGTGGTGGAATGCCTTTA ATGAGGAAAACCTGTTTTGCTCAGAAGAAATGCCATCTAGTGATGATGAGGCTACTGCTGACTCT CAACATTCTACTCCTCCAAAAAAGAAGAGAAAGGTAGAAGACCCCAAGGACTTTCCTTCAGAATT GCTAAGTTTTTTGAGTCATGCTGTGTTTAGTAATAGAACTCTTGCTTGCTTTGCTATTTACACCACA AAGGAAAAAGCTGCACTGCTATACAAGAAAATTATGGAAAAATATTCTGTAACCTTTATAAGTAGG CATAACAGTTATAATCATAACATACTGTTTTTTCTTACTCCACACAGGCATAGAGTGTCTGCTATTA ATAACTATGCTCAAAAATTGTGTACCTTTAGCTTTTTAATTTGTAAAGGGGTTAATAAGGAATATTT GATGTATAGTGCCTTGACTAGAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTT TAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACT TGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATT TTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTCT AGCTTCGTGTCAAGGACGGTGAGG  pMA-RQ luciferase vector - RTV-016 (SEQ ID NO: 89) TAATACGACTCACTATAGGGCGAATTGGCGGAAGGCCGTCAAGGCCGCATGCATAATAAAATAT CTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGCTGCACGTAGATGATGCGTAGCTAGTA GTCTGCACGTAAGATGATGCGTAGCTAGTAGTCTGCACGTAGATGATGCGTAGCTAGTAGTCTG CACGTAGATGATGCGTAGCTAGTAGTCTGCACGTAGATGATGCGTAGCTAGTAGTCTGCACGTA GTAGTCGTATGCTGATGCGCAGTTAGCGTAGCTGAGGTACCGTCGACGATATCGGATCCAGGTC TATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTAGATACGCCATCCACGCTGTTTTGA CCTCCATAGAAGATCGCCACCATGGAAGATGCCAAAAACATTAAGAAGGGCCCAGCGCCATTCT ACCCACTCGAAGACGGGACCGCCGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGG TGCCCGGCACCATCGCCTTTACCGACGCACATATCGAGGTGGACATTACCTACGCCGAGTACTT CGAGATGAGCGTTCGGCTGGCAGAAGCTATGAAGCGCTATGGGCTGAATACAAACCATCGGATC GTGGTGTGCAGCGAGAATAGCTTGCAGTTCTTCATGCCCGTGTTGGGTGCCCTGTTCATCGGTG TGGCTGTGGCCCCAGCTAACGACATCTACAACGAGCGCGAGCTGCTGAACAGCATGGGCATCA GCCAGCCCACCGTCGTATTCGTGAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAGAA GCTACCGATCATACAAAAGATCATCATCATGGATAGCAAGACCGACTACCAGGGCTTCCAAAGCA TGTACACCTTCGTGACTTCCCATTTGCCACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAG CTTCGACCGGGACAAAACCATCGCCCTGATCATGAACAGTAGTGGCAGTACCGGATTGCCCAAG GGCGTAGCCCTACCGCACCGCACCGCTTGTGTCCGATTCAGTCATGCCCGCGACCCCATCTTCG GCAACCAGATCATCCCCGACACCGCTATCCTCAGCGTGGTGCCATTTCACCACGGCTTCGGCAT GTTCACCACGCTGGGCTACTTGATCTGCGGCTTTCGGGTCGTGCTCATGTACCGCTTCGAGGAG GAGCTATTCTTGCGCAGCTTGCAAGACTATAAGATTCAATCTGCCCTGCTGGTGCCCACACTATT TAGCTTCTTCGCTAAGAGCACTCTCATCGACAAGTACGACCTAAGCAACTTGCACGAGATCGCCA GCGGCGGGGCGCCGCTCAGCAAGGAGGTAGGTGAGGCCGTGGCCAAACGCTTCCACCTACCA GGCATCCGCCAGGGCTACGGCCTGACAGAAACAACCAGCGCCATTCTGATCACCCCCGAAGGG GACGACAAGCCTGGCGCAGTAGGCAAGGTGGTGCCCTTCTTCGAGGCTAAGGTGGTGGACTTG GACACCGGTAAGACACTGGGTGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATC ATGAGCGGCTACGTTAACAACCCCGAGGCTACAAACGCTCTCATCGACAAGGACGGCTGGCTGC ACAGCGGCGACATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTGAAGA GCCTGATCAAATACAAGGGCTACCAGGTAGCCCCAGCCGAACTGGAGAGCATCCTGCTGCAACA CCCCAACATCTTCGACGCCGGGGTCGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCCG CCGCAGTCGTCGTGCTGGAACACGGTAAAACCATGACCGAGAAGGAGATCGTGGACTATGTGG CCAGCCAGGTTACAACCGCCAAGAAGCTGCGCGGTGGTGTTGTGTTCGTGGACGAGGTGCCTA AAGGACTGACCGGCAAGTTGGACGCCCGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGG GCGGCAAGATCGCCGTGTAATGAAAGCTTGGTCTCTACGAGTAATAGACGCCCAGTTGAATTCC TTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAA AAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAAC AAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTT AAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCGATAAGGATCCGTCTGGGCCTCATGGGCCT TCCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGACAACAACAATTGCATTCATTTTATGTTTCA GGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCG ATAAGGATCCGTCTGGGCCTCATGGGCCTTCCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTG TCGTGCCAGCTGCATTAACATGGTCATAGCTGTTTCCTTGCGTATTGGGCGCTCTCCGCTTCCTC GCTCACTGACTCGCTGCGCTCGGTCGTTCGGGTAAAGCCTGGGGTGCCTAATGAGCAAAAGGC CAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCC CTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAG ATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACC GGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGT ATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCC CGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCG CCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAG TTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCT GAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGT AGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCC TTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCA TGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTA AAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGC GATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGG AGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGAACCACGCTCACCGGCTCCAG ATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATC CGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTT TGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTC ATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCG GTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGT TATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGA GTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCA ATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTC GGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCA CCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCA AAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTC AATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAA AAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC  pMA-RQ luciferase vector - HYBT (SEQ ID NO: 90) CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTA ACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGT GGCCGCTACAGGGCGCTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGG TGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTT GGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGACGTAAT ACGACTCACTATAGGGCGAATTGGCGGAAGGCCGTCAAGGCCGCATGCATAATAAAATATCTTTA TTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGGACGGATCGGGAGATCTTTGTATTTAATTAAG ACCTTGAGTACGTGCGTCTCTGCACGTATGGCGATTAAGACCTTGAGTACGTGCGTCTCTGCAC GTATGGCGATTAAGACCTTGAGTACGTGCGTCTCTGCACGTATGGCGATTAAGACCTTGAGTAC GTGCGTCTCTGCACGTATGGCGATTAATCCATATGCTCTAGAGGGTATATAATGGGGGCCACTA GTCTACTACCAGAAAGCTTGGTACCGAGCTCGGATCCAGCCACCATGGAAGATGCCAAAAACAT TAAGAAGGGCCCAGCGCCATTCTACCCACTCGAAGACGGGACCGCCGGCGAGCAGCTGCACAA AGCCATGAAGCGCTACGCCCTGGTGCCCGGCACCATCGCCTTTACCGACGCACATATCGAGGT GGACATTACCTACGCCGAGTACTTCGAGATGAGCGTTCGGCTGGCAGAAGCTATGAAGCGCTAT GGGCTGAATACAAACCATCGGATCGTGGTGTGCAGCGAGAATAGCTTGCAGTTCTTCATGCCCG TGTTGGGTGCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCTAACGACATCTACAACGAGCGCGA GCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCGTATTCGTGAGCAAGAAAGGGCTGCA AAAGATCCTCAACGTGCAAAAGAAGCTACCGATCATACAAAAGATCATCATCATGGATAGCAAGA CCGACTACCAGGGCTTCCAAAGCATGTACACCTTCGTGACTTCCCATTTGCCACCCGGCTTCAAC GAGTACGACTTCGTGCCCGAGAGCTTCGACCGGGACAAAACCATCGCCCTGATCATGAACAGTA GTGGCAGTACCGGATTGCCCAAGGGCGTAGCCCTACCGCACCGCACCGCTTGTGTCCGATTCA GTCATGCCCGCGACCCCATCTTCGGCAACCAGATCATCCCCGACACCGCTATCCTCAGCGTGGT GCCATTTCACCACGGCTTCGGCATGTTCACCACGCTGGGCTACTTGATCTGCGGCTTTCGGGTC GTGCTCATGTACCGCTTCGAGGAGGAGCTATTCTTGCGCAGCTTGCAAGACTATAAGATTCAATC TGCCCTGCTGGTGCCCACACTATTTAGCTTCTTCGCTAAGAGCACTCTCATCGACAAGTACGACC TAAGCAACTTGCACGAGATCGCCAGCGGCGGGGCGCCGCTCAGCAAGGAGGTAGGTGAGGCC GTGGCCAAACGCTTCCACCTACCAGGCATCCGCCAGGGCTACGGCCTGACAGAAACAACCAGC GCCATTCTGATCACCCCCGAAGGGGACGACAAGCCTGGCGCAGTAGGCAAGGTGGTGCCCTTC TTCGAGGCTAAGGTGGTGGACTTGGACACCGGTAAGACACTGGGTGTGAACCAGCGCGGCGAG CTGTGCGTCCGTGGCCCCATGATCATGAGCGGCTACGTTAACAACCCCGAGGCTACAAACGCTC TCATCGACAAGGACGGCTGGCTGCACAGCGGCGACATCGCCTACTGGGACGAGGACGAGCACT TCTTCATCGTGGACCGGCTGAAGAGCCTGATCAAATACAAGGGCTACCAGGTAGCCCCAGCCGA ACTGGAGAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGGGTCGCCGGCCTGCCCGA CGACGATGCCGGCGAGCTGCCCGCCGCAGTCGTCGTGCTGGAACACGGTAAAACCATGACCGA GAAGGAGATCGTGGACTATGTGGCCAGCCAGGTTACAACCGCCAAGAAGCTGCGCGGTGGTGT TGTGTTCGTGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTGGACGCCCGCAAGATCCGCGA GATTCTCATTAAGGCCAAGAAGGGCGGCAAGATCGCCGTGTAATGAAAGCTTGGTCTCTACGAG TAATAGACGCCCAGTTGAATTCCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAA CCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGT AACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAG GGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCGATAAGGA TCCGTCTGGGCCTCATGGGCCTTCCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCC AGCTGCATTAACATGGTCATAGCTGTTTCCTTGCGTATTGGGCGCTCTCCGCTTCCTCGCTCACT GACTCGCTGCGCTCGGTCGTTCGGGTAAAGCCTGGGGTGCCTAATGAGCAAAAGGCCAGCAAA AGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACG AGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCA GGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATAC CTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCA GTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACC GCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACT GGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTT GAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAG CCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCG GTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTG ATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAG ATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGT ATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGAT CTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGG GCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGAACCACGCTCACCGGCTCCAGATTT ATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGC CTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGC GCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTC AGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTA GCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATG GCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTA CTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATA CGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGG GCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCA ACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAAT GCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATA TTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAAT AAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC  pMA-RQ luciferase vector - HV3C (SEQ ID NO: 91) CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTA ACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGT GGCCGCTACAGGGCGCTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGG TGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTT GGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGACGTAAT ACGACTCACTATAGGGCGAATTGGCGGAAGGCCGTCAAGGCCGCATGCATAATAAAATATCTTTA TTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGGACGGATCGGGAGATCTTTGTATTTAATTAAG ACCTTGAGTACGTGCGTCTCTGCACGTATGGCGATTAAGACCTTGAGTACGTGCGTCTCTGCAC GTATGGCGATTAAGACCTTGAGTACGTGCGTCTCTGCACGTATGGCGATTAAGACCTTGAGTAC GTGCGTCTCTGCACGTATGGCGATTAATCCATATGCAGGTCTATATAAGCAGAGCTCGTTTAGTG AACCGTCAGATCGCCTAGATACGCCATCCACGCTGTTTTGACCTCCATAGAAGATCGCCACCATG GAAGATGCCAAAAACATTAAGAAGGGCCCAGCGCCATTCTACCCACTCGAAGACGGGACCGCC GGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGGTGCCCGGCACCATCGCCTTTACC GACGCACATATCGAGGTGGACATTACCTACGCCGAGTACTTCGAGATGAGCGTTCGGCTGGCAG AAGCTATGAAGCGCTATGGGCTGAATACAAACCATCGGATCGTGGTGTGCAGCGAGAATAGCTT GCAGTTCTTCATGCCCGTGTTGGGTGCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCTAACGAC ATCTACAACGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCGTATTCGTGA GCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAGAAGCTACCGATCATACAAAAGATCATC ATCATGGATAGCAAGACCGACTACCAGGGCTTCCAAAGCATGTACACCTTCGTGACTTCCCATTT GCCACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGGGACAAAACCATCGC CCTGATCATGAACAGTAGTGGCAGTACCGGATTGCCCAAGGGCGTAGCCCTACCGCACCGCAC CGCTTGTGTCCGATTCAGTCATGCCCGCGACCCCATCTTCGGCAACCAGATCATCCCCGACACC GCTATCCTCAGCGTGGTGCCATTTCACCACGGCTTCGGCATGTTCACCACGCTGGGCTACTTGA TCTGCGGCTTTCGGGTCGTGCTCATGTACCGCTTCGAGGAGGAGCTATTCTTGCGCAGCTTGCA AGACTATAAGATTCAATCTGCCCTGCTGGTGCCCACACTATTTAGCTTCTTCGCTAAGAGCACTC TCATCGACAAGTACGACCTAAGCAACTTGCACGAGATCGCCAGCGGCGGGGCGCCGCTCAGCA AGGAGGTAGGTGAGGCCGTGGCCAAACGCTTCCACCTACCAGGCATCCGCCAGGGCTACGGCC TGACAGAAACAACCAGCGCCATTCTGATCACCCCCGAAGGGGACGACAAGCCTGGCGCAGTAG GCAAGGTGGTGCCCTTCTTCGAGGCTAAGGTGGTGGACTTGGACACCGGTAAGACACTGGGTG TGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATCATGAGCGGCTACGTTAACAACC CCGAGGCTACAAACGCTCTCATCGACAAGGACGGCTGGCTGCACAGCGGCGACATCGCCTACT GGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTGAAGAGCCTGATCAAATACAAGGGCTA CCAGGTAGCCCCAGCCGAACTGGAGAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGG GGTCGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCCGCCGCAGTCGTCGTGCTGGAAC ACGGTAAAACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTTACAACCGCCAA GAAGCTGCGCGGTGGTGTTGTGTTCGTGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTGGA CGCCCGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGCGGCAAGATCGCCGTGTAATG AAAGCTTGGTCTCTACGAGTAATAGACGCCCAGTTGAATTCCTTCGAGCAGACATGATAAGATAC ATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGT GATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTC ATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAA TGTGGTAAAATCGATAAGGATCCGTCTGGGCCTCATGGGCCTTCCGCTCACTGCCCGCTTTCCA GTCGGGAAACCTGTCGTGCCAGCTGCATTAACATGGTCATAGCTGTTTCCTTGCGTATTGGGCG CTCTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGGTAAAGCCTGGGGTGCCTA ATGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCAT AGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCG ACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGA CCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAG CTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAA CCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAA GACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGG CGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTA TCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAA ACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGAT CTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAA GGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGT TTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAG GCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGAT AACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGAACCACGC TCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGT CCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTC GCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGT TTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTG TGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGT TATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTT CTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCT TGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTG GAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAA CCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAA AACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATA CTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTG AATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC  pMA-RQ luciferase vector - Synp-HYP-001 (SEQ ID NO: 92) AATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGCTGCACGTACTGCACGTA CTGCACGTACTGCACGTATGGGTACCGTCGACGATATCGGATCCAGGTCTATATAAGCAGAGCT CGTTTAGTGAACCGTCAGATCGCCTAGATACGCCATCCACGCTGTTTTGACCTCCATAGAAGATC GCCACCATGGAAGATGCCAAAAACATTAAGAAGGGCCCAGCGCCATTCTACCCACTCGAAGACG GGACCGCCGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGGTGCCCGGCACCATC GCCTTTACCGACGCACATATCGAGGTGGACATTACCTACGCCGAGTACTTCGAGATGAGCGTTC GGCTGGCAGAAGCTATGAAGCGCTATGGGCTGAATACAAACCATCGGATCGTGGTGTGCAGCG AGAATAGCTTGCAGTTCTTCATGCCCGTGTTGGGTGCCCTGTTCATCGGTGTGGCTGTGGCCCC AGCTAACGACATCTACAACGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGT CGTATTCGTGAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAGAAGCTACCGATCATA CAAAAGATCATCATCATGGATAGCAAGACCGACTACCAGGGCTTCCAAAGCATGTACACCTTCGT GACTTCCCATTTGCCACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGGGAC AAAACCATCGCCCTGATCATGAACAGTAGTGGCAGTACCGGATTGCCCAAGGGCGTAGCCCTAC CGCACCGCACCGCTTGTGTCCGATTCAGTCATGCCCGCGACCCCATCTTCGGCAACCAGATCAT CCCCGACACCGCTATCCTCAGCGTGGTGCCATTTCACCACGGCTTCGGCATGTTCACCACGCTG GGCTACTTGATCTGCGGCTTTCGGGTCGTGCTCATGTACCGCTTCGAGGAGGAGCTATTCTTGC GCAGCTTGCAAGACTATAAGATTCAATCTGCCCTGCTGGTGCCCACACTATTTAGCTTCTTCGCT AAGAGCACTCTCATCGACAAGTACGACCTAAGCAACTTGCACGAGATCGCCAGCGGCGGGGCG CCGCTCAGCAAGGAGGTAGGTGAGGCCGTGGCCAAACGCTTCCACCTACCAGGCATCCGCCAG GGCTACGGCCTGACAGAAACAACCAGCGCCATTCTGATCACCCCCGAAGGGGACGACAAGCCT GGCGCAGTAGGCAAGGTGGTGCCCTTCTTCGAGGCTAAGGTGGTGGACTTGGACACCGGTAAG ACACTGGGTGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATCATGAGCGGCTAC GTTAACAACCCCGAGGCTACAAACGCTCTCATCGACAAGGACGGCTGGCTGCACAGCGGCGAC ATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTGAAGAGCCTGATCAAAT ACAAGGGCTACCAGGTAGCCCCAGCCGAACTGGAGAGCATCCTGCTGCAACACCCCAACATCTT CGACGCCGGGGTCGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCCGCCGCAGTCGTCG TGCTGGAACACGGTAAAACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTTAC AACCGCCAAGAAGCTGCGCGGTGGTGTTGTGTTCGTGGACGAGGTGCCTAAAGGACTGACCGG CAAGTTGGACGCCCGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGCGGCAAGATCGC CGTGTAATGAAAGCTTGGTCTCTACGAGTAATAGACGCCCAGTTGAATTCCTTCGAGCAGACATG ATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGT GAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACA ATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAAC CTCTACAAATGTGGTAAAATCGATAAGGATCCGTCTGGGCCTCATGGGCCTTCCGCTCACTGCC CGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAACATGGTCATAGCTGTTTCCTTGCGT ATTGGGCGCTCTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGGTAAAGCCTGG GGTGCCTAATGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCG TTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGC GAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCC TGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTT TCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTG TGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAA CCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAG GTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACA GTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATC CGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGA AAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAA CTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTA AAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTA ATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGT CGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGA GAACCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGC AGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGT AAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCAC GCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCC CCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGC CGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAA GATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCG AGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCT CATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTT CGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGG TGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAA TACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATA CATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCC ACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTT TAACCAATAGGCCGAA  Synp-FORCSV-10 (SEQ ID NO: 46) CCATTGACGTCACGATTACCATTGACGTCACGATTACCATTGACGTCACGATTACCATTGACGTC ACGATTACCATTGACGTCAGCGATTAAGATGACTCAGCGATTAAGATGACTCAGCGATTAAGATG ACTCAGCGATTAAGATGACTCACTAGCCCGGGCTCGAGATCTGCGATCTGCATCTCAATTAGTC AGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCA TTCTCCGCCCCATCGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCT GAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTGGC ATTCCGGTACTGTTGGTAAAGCCACC  cAMPRE and AP1 underlined, minimal promoter bold Synp-FORCMV-09 (SEQ ID NO: 47) CCATTGACGTCACGATTACCATTGACGTCACGATTACCATTGACGTCACGATTACCATTGACGTC ACGATTACCATTGACGTCAGCGATTAAGATGACTCAGCGATTAAGATGACTCAGCGATTAAGATG ACTCAGCGATTAAGATGACTCAGCGATTAATCCATATGCAGGTCTATATAAGCAGAGCTCGTTTA GTGAACCGTCAGATCGCCTAGATACGCCATCCACGCTGTTTTGACCTCCATAGAAGATCGCCA CC  CAMPRE and AP1 underlined, minimal promoter bold Synp-FMP-02 (SEQ ID NO: 41) TGAGTCAGATGATGCGTAGCTAGTAGTTGAGTCAGATGATGCGTAGCTAGTAGTTGAGTCAGATG ATGCGTAGCTAGTAGTTGAGTCAGATGATGCGTAGCTAGTAGTTGAGTCAGATGATGCGTAGCTA GTAGTTGAGTCAGATGATGCGTAGCTAGTAGTTGAGTCAGATGATGCGTAGCTAGTAGTTGAGTC AGTAGTCGTATGCTGATGCGCAGTTAGCGTAGCTGAGGTACCGTCGACGATATCGGATCCTTCG CATATTAAGGTGACGCGTGTGGCCTCGAACACCGAGCGACCCTGCAGCGACCCGCTTAA  AP1 underlined, minimal promoter bold Synp-FLP-01 (SEQ ID NO: 42) TGAGTCAGATGATGCGTAGCTAGTAGTTGAGTCAGATGATGCGTAGCTAGTAGTTGAGTCAGATG ATGCGTAGCTAGTAGTTGAGTCAGATGATGCGTAGCTAGTAGTTGAGTCAGATGATGCGTAGCTA GTAGTTGAGTCAGATGATGCGTAGCTAGTAGTTGAGTCAGATGATGCGTAGCTAGTAGTTGAGTC AGTAGTCGTATGCTGATGCGCAGTTAGCGTAGCTGAGGTACCGTCGACGATATCGGATCCGGGC ATATAAAACAGGGGCAAGGCACAGACTCATAGCAGAGCAATCACCACCAAGCCTGGAATAAC TGCAGCCACC  AP1 underlined, minimal promoter bold Synp-RTV-017 (SEQ ID NO: 43) TGAGTCAGATGATGCGTAGCTAGTAGTTGAGTCAGATGATGCGTAGCTAGTAGTTGAGTCAGATG ATGCGTAGCTAGTAGTTGAGTCAGATGATGCGTAGCTAGTAGTTGAGTCAGATGATGCGTAGCTA GTAGTTGAGTCAGATGATGCGTAGCTAGTAGTTGAGTCAGATGATGCGTAGCTAGTAGTTGAGTC AGTAGTCGTATGCTGATGCGCAGTTAGCGTAGCTGAGGTACCGTCGACGATATCGGATCCAGGT CTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTAGATACGCCATCCACGCTGTTTT GACCTCCATAGAAGATCGCCACC  AP1 underlined, minimal promoter bold. Synp-RTV-019 (SEQ ID NO: 44) TGACGTGCTGATGATGCGTAGCTAGTAGTTGACGTGCTGATGATGCGTAGCTAGTAGTTGACGT GCTGATGATGCGTAGCTAGTAGTTGAGTCAGATGATGCGTAGCTAGTAGTTGAGTCAGATGATG CGTAGCTAGTAGTTGAGTCAGATGATGCGTAGCTAGTAGTTGAGTCAGATGATGCGTAGCTAGTA GTCTGCACGTAGATGATGCGTAGCTAGTAGTCTGCACGTAGATGATGCGTAGCTAGTAGTCTGC ACGTAGATGATGCGTAGCTAGTAGTGCAGTTAGCGTAGCTGAGGTACCGTCGACGATATCGGAT CCAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTAGATACGCCATCCACG CTGTTTTGACCTCCATAGAAGATCGCCACC  Inducible elements underlined, minimal promoter bold. Synp-FORCYB1 (SEQ ID NO: 45) CCATTGACGTCACGATTACCATTGACGTCACGATTACCATTGACGTCACGATTACCATTGACGTC ACGATTACCATTGACGTCAGCGATTAAGATGACTCAGCGATTAAGATGACTCAGCGATTAAGATG ACTCAGCGATTAAGATGACTCAGCGATTAATCCATATGCTCTAGAGGGTATATAATGGGGGCCA CTAGTCTACTACCAGAAAGCTTGGTACCGAGCTCGGATCCAGCCACC  cAMPRE underlined, minimal promoter bold. PM-RQ vector (SEQ ID NO: 48) ATGGAAGATGCCAAAAACATTAAGAAGGGCCCAGCGCCATTCTACCCACTCGAAGACGGGACCG CCGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGGTGCCCGGCACCATCGCCTTTA CCGACGCACATATCGAGGTGGACATTACCTACGCCGAGTACTTCGAGATGAGCGTTCGGCTGGC AGAAGCTATGAAGCGCTATGGGCTGAATACAAACCATCGGATCGTGGTGTGCAGCGAGAATAGC TTGCAGTTCTTCATGCCCGTGTTGGGTGCCCTGTTCATCGGTGTGGCTGTGGCCCCAGCTAACG ACATCTACAACGAGCGCGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTCGTATTCGT GAGCAAGAAAGGGCTGCAAAAGATCCTCAACGTGCAAAAGAAGCTACCGATCATACAAAAGATC ATCATCATGGATAGCAAGACCGACTACCAGGGCTTCCAAAGCATGTACACCTTCGTGACTTCCCA TTTGCCACCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGGGACAAAACCATC GCCCTGATCATGAACAGTAGTGGCAGTACCGGATTGCCCAAGGGCGTAGCCCTACCGCACCGC ACCGCTTGTGTCCGATTCAGTCATGCCCGCGACCCCATCTTCGGCAACCAGATCATCCCCGACA CCGCTATCCTCAGCGTGGTGCCATTTCACCACGGCTTCGGCATGTTCACCACGCTGGGCTACTT GATCTGCGGCTTTCGGGTCGTGCTCATGTACCGCTTCGAGGAGGAGCTATTCTTGCGCAGCTTG CAAGACTATAAGATTCAATCTGCCCTGCTGGTGCCCACACTATTTAGCTTCTTCGCTAAGAGCAC TCTCATCGACAAGTACGACCTAAGCAACTTGCACGAGATCGCCAGCGGCGGGGCGCCGCTCAG CAAGGAGGTAGGTGAGGCCGTGGCCAAACGCTTCCACCTACCAGGCATCCGCCAGGGCTACGG CCTGACAGAAACAACCAGCGCCATTCTGATCACCCCCGAAGGGGACGACAAGCCTGGCGCAGT AGGCAAGGTGGTGCCCTTCTTCGAGGCTAAGGTGGTGGACTTGGACACCGGTAAGACACTGGG TGTGAACCAGCGCGGCGAGCTGTGCGTCCGTGGCCCCATGATCATGAGCGGCTACGTTAACAA CCCCGAGGCTACAAACGCTCTCATCGACAAGGACGGCTGGCTGCACAGCGGCGACATCGCCTA CTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTGAAGAGCCTGATCAAATACAAGGG CTACCAGGTAGCCCCAGCCGAACTGGAGAGCATCCTGCTGCAACACCCCAACATCTTCGACGCC GGGGTCGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCCGCCGCAGTCGTCGTGCTGGA ACACGGTAAAACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTTACAACCGCC AAGAAGCTGCGCGGTGGTGTTGTGTTCGTGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTG GACGCCCGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGCGGCAAGATCGCCGTGTAA GTGTAATGAAAGCTTGGTCTCTACGAGTAATAGACGCCCAGTTGAATTCCTTCGAGCAGACATGA TAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTG AAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAA TTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACC TCTACAAATGTGGTAAAATCGATAAGGATCCGTAACAACAACAATTGCATTCATTTTATGTTTCAG GTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCGA TAAGGATCCGTCTGGGCCTCATGGGCCTTCCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGT CGTGCCAGCTGCATTAACATGGTCATAGCTGTTTCCTTGCGTATTGGGCGCTCTCCGCTTCCTCG CTCACTGACTCGCTGCGCTCGGTCGTTCGGGTAAAGCCTGGGGTGCCTAATGAGCAAAAGGCC AGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCC TGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGA TACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCG GATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTAT CTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCC GACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGC CACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGT TCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTG AAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTA GCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCT TTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCAT GAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAA AGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGC GATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGG AGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGAACCACGCTCACCGGCTCCAG ATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATC CGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTT TGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTC ATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCG GTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGT TATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGA GTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCA ATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTC GGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCA CCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCA AAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTC AATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAA AAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC  SEAP coding sequence (SEQ ID NO: 49) ATGCTGCTGCTGCTGCTGCTGCTGGGCCTGAGGCTACAGCTCTCCCTGGGCATCATCCCAGTTG AGGAGGAGAACCCGGACTTCTGGAACCGCGAGGCAGCCGAGGCCCTGGGTGCCGCCAAGAAG CTGCAGCCTGCACAGACAGCCGCCAAGAACCTCATCATCTTCCTGGGCGATGGGATGGGGGTG TCTACGGTGACAGCTGCCAGGATCCTAAAAGGGCAGAAGAAGGACAAACTGGGGCCTGAGATA CCC  pAAV vector: (SEQ ID NO: 50) CGATAGATCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCA CTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGC GAGCGAGCGCGCAGCTGCCTGCAGGCAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGACT GGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCG TAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATG GCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATAUGTCAAAGCA ACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGT GACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCA CGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGC TTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCC TGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAA ACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCG GCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGT TTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGA CACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGA CAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCG CGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTT AGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATA CATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGG AAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCT GTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAG TGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACG TTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCG GGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGT CACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGA GTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTT TTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCC ATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTAT TAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAA GTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAG CCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTA TCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGA GATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGAT TGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACC AAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATC TTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGC GGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAG CGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTA GCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGT CGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAA CGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTAC AGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAA GCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTT TATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGG GGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCC TTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGA GTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAG CGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTG GCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTC ACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAG CGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGAATTGCCTGCAGGCAGCTGC GCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGC CCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTC CTATC 

REFERENCES

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