Triggering inducible mRNA cyclization

Abstract

The present invention relates to a gene regulation strategy capable of programmable control of eukaryotic translation initiation, and the use of the gene regulation strategy in a variety of biomedical purposes including, but not limited to, therapeutic transgene delivery, intracellular sensing, molecular diagnosis, and gene and cell based therapy. The invention also relates to methods of detecting and eliminating cancer cells carrying fusion proteins.

Description

Field of the Invention

[0001] The present invention relates to a gene regulatory system capable of programmable control of eukaryotic translation initiation, and a method of using the system (e.g., for detecting and eliminating cancer cells containing fusion proteins). The present invention also relates to uses of the gene regulatory system, such as for various biomedical purposes, including but not limited to therapeutic transgene delivery, intracellular sensing, biocomputing, molecular diagnostics, and gene- and cell-based therapies. Background of the Invention

[0003] A core focus of synthetic biology is the engineering of biocomputational gene circuits, which enable self-sufficient therapeutic activities driven by temporal and environmentally specific regulation of mammalian cellular activity. These circuits typically consist of interconnected trigger-inducible gene switches and intracellular sensors, in which target gene expression is engineered to depend on a variety of user-defined exogenous signals and / or specific intracellular states. Specifically, gene switches are often used to experimentally study specific cellular events with high spatiotemporal precision, to monitor key biological process activities in industrial production processes, or to remotely control the expression of therapeutic transgenes in gene- and cell-based therapies. Similarly, genetically encoded sensors enable cells to detect and respond to key biological states that may be difficult to access with traditional diagnostic tools. To this end, most gene switches and sensors developed to date operate at the transcriptional level, have relatively low response rates, and are inherently limited to detecting signals transduced into the cell nucleus. A variety of fluorescent sensors have been developed based on the reconstruction of protein-protein interaction dependencies using split fluorescent proteins, but fluorescence-based sensors acting at the protein level are limited to visualization-based applications. Furthermore, when using protein-level sensors, programming more customized sensing and response activities, such as initiating therapeutic activities, will require complex redesign of the protein structure.

[0004] In summary, regulatory systems operating at the translational level of gene expression would be advantageous in many respects by directly linking the real-time detection of various intracellular target compounds with the production of user-defined proteins of interest, but the engineering of inducible translational regulatory systems with good therapeutic efficacy in vivo remains challenging. Summary of the Invention

[0005] Precise regulation of (transgenic) gene activity is crucial for achieving optimal efficacy and safety of gene- and cell-based therapies. In recent years, a number of trigger-inducible gene regulation systems have been developed for mammalian cells, enabling the expression of target genes to be controlled by various user-defined exogenous signals and / or intracellular states (Slomovic et al., 2015). For example, optogenetics can provide traceless, non-invasive, long-distance communication between electronic devices and biological systems, allowing portable electronic devices (such as smartphones) to regulate cellular activity (Shao et al., 2017). Furthermore, human cells can be programmed to sense extracellular disease markers such as blood glucose (Xie et al., 2016) or pH (D. et al., 2014), or internal signals such as redox state (Weber et al., 2006) or miRNA signatures (Xie et al., 2011), in response to tailored therapeutic actions, thus enabling the development of disease-specific control devices that provide automated diagnosis and treatment.

[0006] Most gene regulatory systems reported to date operate at the transcriptional level and therefore have relatively slow sensing and response kinetics ( et al., 2012). In contrast, regulatory systems operating at the level of protein translation are more rapid-acting and have attracted increasing interest in recent years (Dykstra et al., 2022). Translation-based systems are also suitable for sensing a wider variety of intracellular signals, as transcription-based sensors are inherently limited to detecting signals transduced into the nucleus (Nakanishi et al., 2022). However, limited information on the “convergent” molecular mechanisms of translation initiation has hindered the engineering of inducible translational regulation in mammalian cells (Jackson et al., 2010). As a result, most translational regulatory devices developed to date are repressive rather than inducible in nature (Saito et al., 2010; Wroblewska et al., 2015), where ligand responsiveness is primarily achieved by preventing the interruption and / or termination of mRNA activity (Cafferty et al., 2021; Cella et al., 2018). Inducible protein translation, that is, allowing a triggering signal to directly activate the translation of a target mRNA, remains largely elusive.

[0007] In eukaryotic cells, protein translation begins when the pre-initiation complex, consisting of the 40S ribosome and initiation factors (eIFs), is recruited to the untranslated region (UTR) of the guanine-rich 5'-cap of a mature mRNA molecule that has already been exported to the cytoplasm (Jackson et al., 2010; Mitchell and Parker, 2015). The coordinated activities of the cap-binding protein eIF4E, the RNA helicase eIF4A, the central scaffolding protein eIF4G, and the helicase enhancers eIF4B and eIF4H then trigger RNA unwinding, ribosome attachment, and codon scanning (Jackson et al., 2010). It is well known that the 3'-polyadenine tail (poly(A)) enhances the stability of mRNA in living cells, but the underlying mechanism remains a subject of debate (Jackson et al., 2010; Passmore and Coller, 2021). However, many studies support the “closed-loop” model, in which poly(A)-binding protein (PABP) is considered to be another typical eIF that can simultaneously bind poly(A) and eIF4G to form a circular mRNA conformation that is conducive to mRNA scanning, ribosome recycling, and protein translation (Gray et al., 2000; Jackson et al., 2010).

[0008] To engineer the trigger-inducible translation machinery, conventional strategies rely on integrating RNA-binding protein (RBP)-specific aptamers into the UTR of target gene mRNAs to recruit RBP-containing regulatory proteins designed to control mRNA stability or eIF4E recruitment (S. et al., 2014; Nakanishi et al., 2022). However, these approaches have shown only limited efficacy in vivo, which limits their clinical relevance.

[0009] In this work, we demonstrate that genetically encoded removal of the poly(A) signal from target mRNAs is crucial for overcoming the poor fold induction that hampered early attempts to achieve effective translational control in mammalian cells. This, in turn, paves the way for novel design approaches for the systematic engineering of therapeutically useful gene switches and intracellular sensors. For example, we show that custom-designed gene switches triggered by the FDA-approved drug grazoprevir can effectively control insulin expression and restore glucose homeostasis in diabetic mice, while remaining compatible with various DNA- and RNA-centric gene therapy delivery strategies currently in clinical use. Furthermore, we demonstrate that the unique advantage of the STIF architecture lies in the ability to custom-develop genetically encoded sensors for quantitative detection of various subcellular (mis)localized proteins, such as the BCR-ABL fusion protein of chronic myeloid leukemia (CML). Consistent with this, we describe multiple designs of intracellular protein sensors that have the potential to replace or significantly enhance state-of-the-art cell state classifier circuits, thereby generating the next generation of "therapeutic biocomputers" for future precision medicine. By illustrating issues of complexity and specificity that are likely to become relevant in a clinical setting, we conclusively demonstrate self-sufficient elimination of cancer cells in mice mediated by such protein-responsive gene therapy treatments.

[0010] Therefore, in particular aspects, the present invention relates to the following embodiments:

[0011] 1. A nucleic acid construct comprising an mRNA, the translation of which (ie, a protein synthesis event) is regulated in a trigger-inducible manner.

[0012] 2. The nucleic acid construct according to embodiment 1, wherein initiation of protein translation occurs by trigger-induced circularization of said mRNA.

[0013] 3. Nucleic acid construct according to embodiments 1-2, wherein mRNA circularization occurs by ectopic overexpression of one or several eIF4F interacting moieties that can bind to both the 3'-UTR and the 5'-UTR of said mRNA.

[0014] 4. The mRNA circularization strategy according to embodiments 2-3, wherein the eIF4F interacting part (also called eIFBP) consists of a single protein that binds to eIF4F and a specific site on the mRNA.

[0015] 5. The mRNA circularization strategy according to embodiment 4, wherein the single eIF4F interacting moiety is PABP and mutants or derivatives or fragments thereof.

[0016] 6. The mRNA circularization strategy according to embodiment 4, wherein the single eIF4F interacting moiety is NSP3 and mutants or derivatives or fragments thereof.

[0017] 7. The mRNA circularization strategy according to embodiment 4, wherein the single eIF4F interacting moiety is VPg and mutants or derivatives thereof or fragments thereof.

[0018] 8. The mRNA circularization strategy according to embodiments 2-3, wherein the single eIF4F interacting moiety is a chimeric fusion between any of the proteins of embodiments 5-7 and an RNA binding protein (RBP).

[0019] 9. The mRNA circularization strategy according to embodiment 8, wherein the RNA binding protein is L7Ae and mutants or derivatives or fragments thereof.

[0020] 10. The mRNA circularization strategy according to embodiment 8, wherein the RNA binding protein is MCP and its mutants or derivatives or fragments thereof.

[0021] 11. The mRNA circularization strategy according to embodiment 8, wherein the RNA binding protein is λ-N and mutants or derivatives or fragments thereof.

[0022] 12. The mRNA cyclization strategy according to embodiments 2-3, wherein the eIF4F interaction portion consists of two recombinant fusion proteins A (comprising the general formula XY) and B (comprising the general formula Y'-Z).

[0023] 13. The mRNA circularization strategy according to embodiment 12, wherein X of protein A is any protein of embodiments 5-7, and wherein Z of protein B is any protein of embodiments 9-11; X is genetically fused to protein Y, and Z is genetically fused to protein Y', to allow proteins A and B to associate through a specific Y:Y'-mediated interaction.

[0024] 14. The mRNA circularization strategy according to embodiment 13, wherein protein Y is DocS and protein Y' is Coh2, or wherein protein Y is Coh2 and protein Y' is DocS.

[0025] 15. The mRNA circularization strategy according to embodiment 13, wherein protein Y is FKBP and protein Y' is FRB, or wherein protein Y is FRB and protein Y' is FKBP.

[0026] 16. The mRNA circularization strategy according to embodiment 13, wherein protein Y is ABI and protein Y' is PYL1, or wherein protein Y is PYL1 and protein Y' is ABI.

[0027] 17. The mRNA circularization strategy according to embodiment 13, wherein protein Y is GAI and protein Y' is GID, or wherein protein Y is GID and protein Y' is GAI.

[0028] 18. The mRNA circularization strategy according to embodiment 13, wherein protein Y is NS3a and protein Y' is GNCR or wherein protein Y is GNCR and protein Y' is NS3a.

[0029] 19. The mRNA circularization strategy according to embodiment 13, wherein protein Y is NS3a and protein Y' is DNCR, or wherein protein Y is DNCR and protein Y' is NS3a.

[0030] 20. The mRNA circularization strategy according to embodiment 13, wherein protein Y is NS3a and protein Y' is ANR, or wherein protein Y is ANR and protein Y' is NS3a.

[0031] 21. The mRNA circularization strategy according to embodiment 13, wherein protein Y is ERK2 and protein Y' is pE59, or wherein protein Y is pE59 and protein Y' is ERK2.

[0032] 22. The mRNA circularization strategy according to embodiment 12, wherein X of protein A is any one of the proteins of embodiments 9-11, and wherein Z of protein B is any one of the proteins of embodiments 5-7; X is genetically fused to protein Y, and Z is genetically fused to protein Y', to allow proteins A and B to associate through a specific Y:Y'-mediated interaction.

[0033] 23. The mRNA circularization strategy according to embodiment 22, wherein protein Y is DocS and protein Y' is Coh2, or wherein protein Y is Coh2 and protein Y' is DocS.

[0034] 24. The mRNA circularization strategy according to embodiment 22, wherein protein Y is FKBP and protein Y' is FRB, or wherein protein Y is FRB and protein Y' is FKBP.

[0035] 25. The mRNA circularization strategy according to embodiment 22, wherein protein Y is ABI and protein Y' is PYL1, or wherein protein Y is PYL1 and protein Y' is ABI.

[0036] 26. The mRNA circularization strategy according to embodiment 22, wherein protein Y is GAI and protein Y' is GID, or wherein protein Y is GID and protein Y' is GAI.

[0037] 27. The mRNA circularization strategy according to embodiment 22, wherein protein Y is NS3a and protein Y' is GNCR or wherein protein Y is GNCR and protein Y' is NS3a.

[0038] 28. The mRNA circularization strategy according to embodiment 22, wherein protein Y is NS3a and protein Y' is DNCR, or wherein protein Y is DNCR and protein Y' is NS3a.

[0039] 29. The mRNA circularization strategy according to embodiment 22, wherein protein Y is NS3a and protein Y' is ANR, or wherein protein Y is ANR and protein Y' is NS3a.

[0040] 30. The mRNA circularization strategy according to embodiment 22, wherein protein Y is ERK2 and protein Y' is pE59, or wherein protein Y is pE59 and protein Y' is ERK2.

[0041] 31. The mRNA circularization strategy according to embodiment 12, wherein X of protein A is any protein of embodiments 5-7, and wherein Z of protein B is any protein of embodiments 9-11; X is genetically fused to protein Y, and Z is genetically fused to protein Y', to allow proteins A and B to associate with target protein Y" through specific Y:Y':Y"-mediated interactions.

[0042] 32. The mRNA circularization strategy according to embodiment 31, wherein protein Y" is a fusion gene product, an RNA binding protein, or any other intracellular or secreted protein containing one or more domains.

[0043] 33. The mRNA circularization strategy according to embodiment 31, wherein protein Y and protein Y' are two different scFvs.

[0044] 34. The mRNA circularization strategy according to embodiment 31, wherein protein Y and protein Y' are two different Nanobodies.

[0045] 35. The mRNA circularization strategy according to embodiment 31, wherein protein Y is a Nanobody and protein Y' is a scFv, or wherein protein Y is a scFv and protein Y' is a Nanobody.

[0046] 36. An mRNA circularization strategy according to embodiment 31, wherein proteins Y and Y' are any natural or synthetic proteins that bind to protein Y" with high affinity; or wherein protein Y is any natural or synthetic protein that binds to protein Y" with high affinity, and protein Y' is a scFv or a nanobody; or wherein protein Y is a scFv or a nanobody, and protein Y' is any natural or synthetic protein that binds to protein Y" with high affinity.

[0047] 37. The mRNA circularization strategy according to embodiment 12, wherein X of protein A is any protein of embodiments 9-11, and wherein Z of protein B is any protein of embodiments 5-7; X is genetically fused to protein Y, and Z is genetically fused to protein Y', to allow proteins A and B to associate with a specific target protein Y" through specific Y:Y':Y"-mediated interactions.

[0048] 38. The mRNA circularization strategy according to embodiment 37, wherein protein Y″ is a fusion gene product, an RNA binding protein, or any other intracellular or secreted protein containing one or more domains.

[0049] 39. The mRNA circularization strategy according to embodiment 37, wherein protein Y and protein Y' are two different scFvs.

[0050] 40. The mRNA circularization strategy according to embodiment 37, wherein protein Y and protein Y' are two different Nanobodies.

[0051] 41. The mRNA circularization strategy according to embodiment 37, wherein protein Y is a Nanobody and protein Y' is a scFv, or wherein protein Y is a scFv and protein Y' is a Nanobody.

[0052] 42. An mRNA circularization strategy according to embodiment 37, wherein proteins Y and Y' are any natural or synthetic proteins that bind to protein Y" with high affinity; or wherein protein Y is any natural or synthetic protein that binds to protein Y" with high affinity and protein Y' is a scFv or a nanobody, or wherein protein Y is a scFv or a nanobody and protein Y' is any natural or synthetic protein that binds to protein Y" with high affinity.

[0053] 43. The mRNA circularization strategy according to embodiments 3-28, wherein the nucleic acid sequence in the 3'-UTR or 5'-UTR of the mRNA bound by any of the eIF4F interacting moieties comprises a poly-A signal.

[0054] 44. The mRNA circularization strategy according to embodiment 3-28, wherein the nucleic acid sequence in the 3'-UTR or 5'-UTR of the mRNA bound by any of the eIF4F interacting moieties contains an RNA aptamer.

[0055] 45. The mRNA circularization strategy according to embodiment 44, wherein the RNA aptamer comprises one or more tandem copies and combinations of any of the sequences C / D-box, MS2-box, boxB or other aptamers placed in the 3'-UTR or 5'-UTR of the mRNA.

[0056] 46. The mRNA circularization strategy according to embodiment 44, wherein the RNA aptamer is placed in the 3'-UTR or 5'-UTR of the mRNA, but the native poly-A signal of the mRNA is cleaved and removed by nuclease.

[0057] 47. The mRNA circularization strategy according to embodiment 46, wherein the nuclease responsible for mRNA cleavage belongs to the RNase or CRISPR protein family.

[0058] 48. The mRNA circularization strategy according to embodiment 46, wherein cleavage of the natural poly-A signal occurs by RNA interference.

[0059] 49. The mRNA circularization strategy according to embodiment 48, wherein cleavage of the natural poly-A signal occurs by placing the siRNA binding site or multiple copies thereof in the 3'-UTR.

[0060] 50. The mRNA circularization strategy according to embodiment 48, wherein cleavage of the natural poly-A signal occurs by placing the shRNA binding site or multiple copies thereof in the 3'-UTR.

[0061] 51. The mRNA circularization strategy according to embodiment 48, wherein cleavage of the natural poly-A signal occurs by placing a miRNA binding site or multiple copies thereof in the 3'-UTR.

[0062] 52. The mRNA circularization strategy according to embodiment 44, wherein the RNA aptamer is placed in the 3'-UTR or 5'-UTR of the mRNA, but the native poly-A signal of the mRNA is cleaved and removed by a ribozyme.

[0063] 53. The mRNA circularization strategy according to embodiment 52, wherein the ribozyme responsible for mRNA cleavage is achieved by placing a self-cleaving ribozyme or multiple copies thereof or fragments thereof in the 3'-UTR.

[0064] 54. The mRNA circularization strategy according to embodiment 53, wherein the self-cleaving ribozyme is a hammerhead ribozyme (HHR).

[0065] 55. The mRNA circularization strategy according to embodiment 44, wherein the RNA aptamer is placed in the 3'-UTR or 5'-UTR of the mRNA, but the natural 5'-cap of the mRNA is cleaved and removed by a nuclease.

[0066] 56. The mRNA circularization strategy according to embodiment 55, wherein the nuclease responsible for mRNA cleavage belongs to the RNase or CRISPR protein family.

[0067] 57. The mRNA circularization strategy according to embodiment 56, wherein cleavage of the natural 5'-cap occurs by RNA interference.

[0068] 58. The mRNA circularization strategy according to embodiment 57, wherein cleavage of the natural 5'-cap signal occurs by placing the siRNA binding site or multiple copies thereof in the 5'-UTR.

[0069] 59. The mRNA circularization strategy according to embodiment 57, wherein cleavage of the natural 5'-cap signal occurs by placing an shRNA binding site or multiple copies thereof in the 5'-UTR.

[0070] 60. The mRNA circularization strategy according to embodiment 57, wherein cleavage of the natural 5'-cap signal occurs by placing a miRNA binding site or multiple copies thereof in the 5'-UTR.

[0071] 61. The mRNA circularization strategy according to embodiment 44, wherein the RNA aptamer is placed in the 3'-UTR or 5'-UTR of the mRNA, but the natural 5'-cap of the mRNA is cleaved and removed by a ribozyme.

[0072] 62. The mRNA circularization strategy according to embodiment 61, wherein the ribozyme responsible for mRNA cleavage is achieved by placing a self-cleaving ribozyme or multiple copies thereof or fragments thereof in the 5'-UTR.

[0073] 63. The mRNA circularization strategy according to embodiment 62, wherein the self-cleaving ribozyme is a hammerhead ribozyme (HHR).

[0074] 64. The nucleic acid construct according to embodiments 1-63, wherein the coding region of the mRNA flanked by 5'-UTR and 3'-UTR encodes one or more proteins or peptides.

[0075] 65. The nucleic acid construct according to embodiment 64, wherein the coding region of the mRNA flanked by 5'-UTR and 3'-UTR starts with the nucleotide sequence AUG and ends with the nucleotide sequence UAG, UAA or UGA.

[0076] 66. The nucleic acid construct according to embodiments 1-65, wherein protein translation (mRNA circularization following binding of the mRNA to the eIF4F interacting moiety) occurs outside of living cells.

[0077] 67. The nucleic acid construct according to embodiments 1-65, wherein protein translation (mRNA circularization following binding of the mRNA to the eIF4F interacting moiety) occurs within a living cell.

[0078] 68. The nucleic acid construct according to embodiment 67, wherein the living cells are of mammalian origin.

[0079] 69. The nucleic acid construct according to embodiment 68, wherein the living cells are of human origin.

[0080] 70. The nucleic acid construct according to embodiment 69, wherein the living cell is part of a living tissue of an organism.

[0081] 71. The nucleic acid construct according to embodiments 1-70, wherein the mRNA is directly delivered into living cells in the form of RNA, and the protein is directly delivered into living cells in the form of protein.

[0082] 72. The nucleic acid construct according to embodiments 1-70, wherein the mRNA is delivered into living cells via any form of vector based on encoding DNA, and the protein is delivered into living cells via any form of vector based on encoding DNA or RNA.

[0083] 73. A genetically modified living cell according to embodiments 67-72 for use as a medicament or as part of a medicament.

[0084] 74. Genetically modified living cells according to embodiments 67-72 for use in medical diagnostics and / or real-time monitoring of cellular processes.

[0085] 75. The gene regulation system according to embodiments 1-66, which is used for point-of-care testing. DETAILED DESCRIPTION

[0086] (I) Definition

[0087] To interpret this specification, the following definitions will apply, and where appropriate, terms used in the singular may also include the plural, and vice versa. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0088] Unless otherwise specified, the terms "a" and "an" as used herein mean "one," "at least one," or "one or more." Unless otherwise required by context, as used herein, singular terms shall include pluralities and plural terms shall include the singular.

[0089] As used herein, the terms "comprising" or "including" mean including the recited elements, integers, or steps but not excluding any other elements, integers, or steps. Unless otherwise indicated, when the terms "comprising" or "including" are used, the terms also encompass combinations of the elements, integers, or steps mentioned herein. In some embodiments, the terms may also mean "consisting of the recited elements, integers, or steps."

[0090] The term "about" used in combination with a numerical value is intended to encompass a value within the range of a lower limit of 5% less than the stated numerical value to an upper limit of 5% greater than the stated numerical value.

[0091] As used herein, the term "and / or" refers to any option or two or more options.

[0092] Additionally, the words "herein," "above," and "below," and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of this application.

[0093] The term "nucleic acid construct" refers to a specific type of biopolymer composed of nucleotide monomers. A "nucleotide" is defined as a chemical structure containing a 5-carbon sugar, a phosphate group, and a nitrogenous base. The two main classes of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0094] The term "translation" refers to the event of protein synthesis from an mRNA molecule carried out by ribosomes.

[0095] The term "messenger RNA (mRNA)" refers to a single-stranded RNA molecule corresponding to the genetic sequence of a gene, which is read by ribosomes during the translation process.

[0096] In the present invention, the term "eIF4F interacting moiety" refers to any protein or fragment thereof that can interact with any member of the eukaryotic eIF4F complex (also referred to as eIFBP (eIF4F binding protein) in the present examples). In specific embodiments, possible eIF4F interacting moieties include, but are not limited to, PABP, NSP3, VPg, and any member of eIF4F.

[0097] The term "RNA-binding protein (RBP)" describes a protein that can bind with sufficient affinity to a specific sequence or structure within an RNA molecule. In some embodiments, the RBP can be found in any natural (e.g., as listed in (Gerstberger et al., 2014)) or synthetic context (e.g., through directed evolution, (Fukunaga and Yokobayashi, 2021)). In specific embodiments, possible RBPs include, but are not limited to, L7Ae, MCP, or λ-N.

[0098] The term "ectopic overexpression" describes an event whereby a particular gene is forced to be expressed in a cell type in which the gene is not normally expressed at a desired level.

[0099] In the present invention, the term "mRNA circularization" relates to a specific state of translation initiation in which the 5'-cap and 3'-tail of an mRNA molecule are brought into close proximity by one or several proteins that bind simultaneously to both the 5'-UTR and 3'-UTR of the mRNA.

[0100] The term "eIF4F" refers to a heterotrimeric protein complex comprising eIF4A, eIF4B, eIF4E, and eIF4G, which binds to the 5' cap of messenger RNA (mRNA) to promote translation initiation in eukaryotes.

[0101] The term "PABP" refers to a poly(A) binding protein (NCBI-ID: XP_004402403.1) that can bind to the poly(A) signal and eIF4G (Passmore and Coller, 2021). In the present invention, PABP is considered to be an eIF4F interacting part and / or RNA binding protein.

[0102] The term "NSP3" refers to rotavirus nonstructural protein 3 (Groft and Burley, 2002; Piron et al., 1999). In some embodiments, NSP3 is derived from bovine rotavirus strain RF (NSP3; UniProtKB / Swiss-Prot: Q86504.1) or human rotavirus strain WA (hNSP3; UniProtKB / Swiss-Prot: Q82054.1). In the present invention, NSP3 is considered to be an eIF4F interacting portion and / or an RNA binding protein.

[0103] The term "VPg" refers to the VPg protein from Calicivirus (Royall and Locker, 2016). In the present invention, VPg is considered to be an eIF4F interacting part and / or RNA binding protein.

[0104] The term "fusion protein" refers to a type of hybrid protein produced by joining two or more genes that originally encoded separate proteins.

[0105] The term "fusion gene product" refers to a type of fusion protein that is naturally formed by gene mutation and / or chromosomal translocation, resulting in a new coding sequence containing portions of coding sequences from two different genes.

[0106] The term "chimeric fusion" refers to a type of synthetic fusion protein that has been engineered to retain key functions or physicochemical patterns of each individual protein that is not related in nature.

[0107] The term "L7Ae" refers to an archaeal ribosomal protein (Saito et al., 2010). In the present invention, L7Ae is considered to be an RNA binding protein.

[0108] The term "MCP" refers to the bacteriophage MS2 coat protein (GenBank: ASW25882.1). In the present invention, MCP is considered to be an RNA binding protein.

[0109] The term "λ-N" refers to the N-peptide derived from bacteriophage λ (Schoenberg et al., 2004). In the present invention, λ-N is considered to be an RNA binding protein.

[0110] The term "specific protein interaction" refers to an estimated dissociation constant below 1 μM (K D A conventional measure of the binding affinity between two different proteins (e.g., between proteins Y and Y', between proteins Y and Y", or between proteins Y' and Y").

[0111] The term "high binding affinity" refers to the ability to assess the affinity of a bond between multiple moieties, such as between two proteins (dissociation constant K D <1 μM) or between proteins and small molecules (inhibitor constant K i <1 μM).

[0112] The term "Coh2" refers to Clostridium thermocellum cohesin (Wu et al., 2020). The term "DocS" refers to Clostridium thermocellum dockerin (Wu et al., 2020). In specific embodiments, Coh2 and DocS can be Y, Y', or Y" to form a specific protein interaction.

[0113] The term "FRB" refers to the FKBP-rapamycin binding domain of the mammalian target of rapamycin (mTOR) kinase (Scheller et al., 2018). The term "FKBP" refers to FK506 binding protein (Scheller et al., 2018). In specific embodiments, FRB and FKBP can be Y, Y', or Y" to form a specific protein interaction.

[0114] The term "ABI" refers to abscisic acid-responsive PYL1 binding protein (Gao et al., 2016). The term "PYL1" refers to pyrabactin resistance (PYR)-like protein (Gao et al., 2016). In specific embodiments, ABI and PYL1 can be Y, Y', or Y" to form a specific protein interaction.

[0115] The term "GID1" refers to gibberellin-insensitive dwarf disease 1 (Gao et al., 2016). The term "GAI" refers to gibberellin-insensitive (Gao et al., 2016). In specific embodiments, GID1 and GAI can be Y, Y', or Y" to form a specific protein interaction.

[0116] The term "GNCR" refers to the grazoprevir / NS3a complex reader (Foight et al., 2019). The term "DNCR" refers to the danoprevir / NS3a complex reader (Foight et al., 2019). The term "ANR" refers to the apo NS3a reader (Cunningham-Bryant et al., 2019). The term "NS3A" refers to the hepatitis C virus protease or a mutant or fragment thereof, such as NS3a(H1) (WO2020117778A2). In specific embodiments, NS3a and GNCR can be Y, Y', or Y" to form a specific protein interaction. In other embodiments, NS3a and DNCR can be Y, Y', or Y" to form a specific protein interaction. In other embodiments, both NS3a and ANR can be Y, Y', or Y" to form a specific protein interaction.

[0117] The term "pE59" refers to a DARPin that targets phosphorylated ERK2 (Kummer et al., 2012). The term "ERK2" refers to extracellular regulated protein kinase 2 (NCBI-ID: NM_138957). In specific embodiments, pE59 and ERK2 can be Y, Y', or Y" to form a specific protein interaction.

[0118] The term "protein domain" refers to any functional and / or structural unit of a protein polypeptide chain that is self-stable and folds independently of the rest of the chain.

[0119] The term "secreted protein" refers to any protein that, after translation, is secreted outside the cell in which it is produced.

[0120] The term "intracellular protein" refers to any protein that, after translation, is present within the cell in which it is produced.

[0121] The term "single-chain variable fragment (scFv)" refers to a specific type of fusion protein between the variable regions of an immunoglobulin heavy and light chain connected by a short linker peptide.

[0122] The term "nanobody" (also called single-domain antibody) refers to antibody fragments consisting of a single monomeric variable antibody domain.

[0123] The term "poly(A) signal" or "pA" refers to a stretch of an RNA molecule (usually located in the 3'-UTR of an mRNA) composed primarily of adenine bases.

[0124] The term "aptamer" refers to a single-stranded RNA or DNA sequence that forms a secondary structure that undergoes a substantial conformational change upon binding to a specific ligand (small molecule, ion, or protein) with high affinity. In specific embodiments, possible aptamers include, but are not limited to, MS2-box, C / D-box, or boxB.

[0125] The term "MS2-box" refers to an MCP-specific aptamer. In a specific embodiment, the RNA sequence of the MS2-box is 5'-UGAGGAUCACCCA-3'.

[0126] The term "C / D-box" refers to an L7Ae-specific aptamer. In a specific embodiment, the RNA sequence of the C / D-box is 5'-GGGCGUGAUCCGAAAGGUGACCC-3'.

[0127] The term "boxB" refers to a λ-N specific aptamer. In a specific embodiment, the RNA sequence of boxB is 5'-GGGCCCUGAAGAAGGGCCC-3'.

[0128] The term "UTR" refers to the untranslated region of an mRNA molecule that does not contain protein-encoding nucleotide sequences.

[0129] The term "nuclease" refers to an enzyme that is capable of cleaving phosphodiester bonds between nucleotides in a nucleic acid.

[0130] The term "clustered regularly interspaced short palindromic repeats (CRISPR)" refers to a family of DNA sequences found in prokaryotic genomes that encode proteins capable of destroying foreign DNA during infection.

[0131] The term "ribonuclease (RNase)" refers to a class of nucleases that catalyze the degradation of RNA into smaller components.

[0132] The term "RNA interference (RNAi)" refers to a biological process in which gene expression of an mRNA is inhibited (knocked down) by small regulatory RNA (srRNA) molecules that bind to any site on the target mRNA.

[0133] The terms "siRNA," "shRNA," and "miRNA" refer to different types of srRNA molecules involved in RNA interference.

[0134] The term "ribozyme" refers to an RNA molecule with enzymatic function.

[0135] The term "hammerhead ribozyme (HHR)" refers to a ribozyme motif that catalyzes reversible cleavage and ligation reactions at a specific site within the (same) RNA molecule. In some embodiments, HHR-like self-cleaving ribozymes can be found in (Peng et al., 2021; Roberts et al., 2023; Zhong et al., 2020).

[0136] The term "5'-cap" refers to specifically altered nucleotides (eg, addition of multiple guanine nucleotides) at the 5' end of some primary transcripts, such as pre-messenger RNA.

[0137] The term "peptide" refers to a chain of interconnected amino acids that forms the basic structural unit of a protein.

[0138] The term "virulence factor" refers to molecules representative of pathogenic microorganisms and viruses that can cause disease when infecting a eukaryotic host (eg, a human).

[0139] The term "disease metabolite" refers to representative molecules in the body fluids (eg, blood, sweat, or urine) of a eukaryotic host (eg, a human) that reflect a critical health state.

[0140] The term "disease signature" refers to any molecule, such as a protein, that can represent a specific disease or abnormal cellular condition, for example, a virulence factor, such as some virus-specific antigens such as NS3, or an oncoprotein, such as a cancer-specific fusion gene product such as BCR-ABL, any other representative cytoplasmic biomarker of chronic myeloid leukemia (CML), or any other disease metabolite.

[0141] The term "environmental pollutant" refers to a molecule representative of a particular biotope (eg, water, air, or soil) that poses a specific risk to the environment and / or human health.

[0142] The term "trigger-inducible gene regulation" refers to any system that allows the expression of a specific gene of interest to be turned on only upon exposure to a specific user-defined signal (ie, a trigger).

[0143] The term "triggered repressive gene regulation" refers to any system that allows the expression of a specific gene of interest to be repressed upon exposure to a specific user-defined signal (ie, a trigger).

[0144] The term "poly(A) surrogate" refers to a synthetic protein-binding motif engineered into the 3'-UTR that can replace the native poly(A) signal or operate in parallel with the native poly(A) signal to bind proteins containing an eIF4F interacting portion. In some embodiments, potential poly(A) surrogates comprise protein-specific aptamers, such as MS2-box, C / D-box, or boxB.

[0145] The term "5'-cap surrogate" refers to a synthetic protein-binding motif engineered into the 5'-UTR that can replace the native 5'-cap or operate in parallel with the native 5'-cap to bind proteins containing an eIF4F interacting portion. In some embodiments, potential 5'-cap surrogates comprise protein-specific aptamers, such as MS2-box, C / D-box, or boxB.

[0146] The terms "tumor" and "cancer" are used interchangeably herein to encompass both solid and liquid tumors.

[0147] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is characterized by unregulated cell growth.

[0148] The term "tumor" refers to the growth and proliferation of all neoplastic cells, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein.

[0149] As used herein, "oncoprotein" refers to an antigenic determinant displayed in target cells, wherein the target cells are cells in a tumor, such as cancer cells and tumor stromal cells.

[0150] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, etc., which is administered together with an active substance.

[0151] The term "pharmaceutical composition" refers to a composition that is in such form as to permit the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition is administered.

[0152] The term "drug combination" refers to a non-fixed combination product or a fixed combination product, including but not limited to a medicine box and a pharmaceutical composition. The term "non-fixed combination" refers to an active ingredient (e.g., (i) a system of the present invention, and (ii) other therapeutic agents) being administered to a patient simultaneously, without specific time limits, or at identical or different time intervals, as separate entities, wherein these two or more activating agents are administered to provide effective levels of prevention or treatment in the patient. In some embodiments, the system of the present invention for drug combination is administered at a level not exceeding that when used alone. The term "fixed combination" refers to two or more activating agents being administered to a patient simultaneously in the form of a single entity. The dosage and / or time interval of two or more activating agents can be selected so that, in the treatment of a disease or condition, each component combination produces a greater effect than any one component used alone. Each component can take its own formulation form, which can be identical or different.

[0153] The term "combination therapy" refers to the application of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery) to treat diseases described herein. This application includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, this application includes co-applying each active ingredient to multiple or separate containers (e.g., tablets, capsules, powders, and liquids). The powder and / or liquid can be reconstituted or diluted to the desired dose before application. In addition, the application also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the treatment plan will provide the beneficial effects of the drug combination in treating the diseases described herein or the condition.

[0154] "Individual" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0155] As used herein, "treat" (or its various grammatical variations) means to slow, interrupt, prevent, alleviate, stop, reduce or reverse the progression or severity of an existing symptom, disorder, condition or disease.

[0156] As used herein, "prevention" (or its various grammatical variations) includes inhibiting the onset or progression of a disease or condition or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventive regimen. Generally, in the context of cancer, the term "prevention" refers to administering a drug prior to the onset of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0157] The term "effective amount" refers to the amount or dosage of an antibody or fragment or conjugate or composition or combination of the invention that will produce the desired effect in a patient in need of such treatment or prevention after administration to the patient in single or multiple doses.

[0158] A "therapeutically effective amount" is an amount effective to achieve the desired result at the desired dosage and for the desired period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or conjugate or composition or combination outweigh the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 20%, more preferably at least about 40%, or even more preferably at least 50%, 60%, or 70%, compared to an untreated subject.

[0159] A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result at the desired dosage and for the desired period of time. Typically, a prophylactic amount will be less than a therapeutically effective amount because a prophylactic dose is used before or at an early stage of disease in a subject.

[0160] As used herein, the term "vector" refers to a nucleic acid molecule capable of delivering and / or propagating another nucleic acid to which it is connected. The term includes vectors as self-replicating nucleic acid structures and episomal vectors (episomal vectors) delivered to the nucleus of the host cell into which it is introduced. Some vectors can instruct the expression of nucleic acids to which they are effectively connected. This type of vector is referred to as an "expression vector" in this article.

[0161] "Subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as from fresh, frozen and / or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid; cells from a subject at any time during pregnancy or development. Tissue samples may contain compounds that do not naturally mix with tissue, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0162] (II.) Gene Regulation System

[0163] In a first aspect, the present invention relates to a gene regulation strategy comprising a nucleic acid construct comprising an mRNA whose translation is regulated in a trigger-inducible manner. Preferably, initiation of protein translation occurs via trigger-inducible cyclization of the mRNA. Preferably, mRNA cyclization is achieved by ectopically overexpressing one or more eIF4F interacting moieties that bind to both the 3'-UTR and the 5'-UTR of the mRNA.

[0164] In an embodiment, the gene regulation strategy involves a gene regulation system that can regulate the expression of target gene mRNA, the system comprising

[0165] (i) a synthetic translation initiation factor (STIF) and an mRNA construct comprising an mRNA encoding a target protein; or

[0166] (ii) a nucleic acid encoding STIF and a nucleic acid encoding an mRNA construct comprising an mRNA encoding a target protein.

[0167] In some other embodiments, the system further comprises a poly(A) removal module.

[0168] In some embodiments, the nucleic acid is DNA or RNA.

[0169] In one embodiment, STIF comprises one or more eIF4F interacting moieties (or "eIFBPs") that can bind to the 3'-UTR or 5'-UTR of the mRNA. In an embodiment, STIF comprises or consists of at least one eIFBP (eIF4F binding protein) and at least one RBP (RNA binding protein). In some embodiments, the eIFBP and RBP can be in one protein or in separate proteins. In one embodiment, the eIF4F interacting moiety further comprises any other protein domain, which in some embodiments is inserted between the RBP and eIFBP domains, such as the calmodulin-like motif 2CaM-M13. In some embodiments, STIF further comprises a tag, such as a FLAG tag. In some embodiments, STIF is a fusion protein comprising the aforementioned moieties / proteins.

[0170] In specific embodiments, the eIFBP is selected from PABP, NSP3, VPg, and any member of eIF4F, such as eIF4A, eIF4B, eIF4E, or eIF4G.

[0171] In one embodiment, the eIFBP is PABP, a mutant or derivative thereof, or a fragment thereof having PABP function. In other embodiments, the PABP is human PABP. In other embodiments, the PABP comprises or consists of the amino acid sequence of SEQ ID NO: 108, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 108.

[0172] In another embodiment, the eIFBP is NSP3, a mutant or derivative thereof, or a fragment thereof having NSP3 function. In other embodiments, NSP3 is derived from a bovine or human rotavirus strain. In other embodiments, NSP3 comprises or consists of the amino acid sequence of SEQ ID NO: 91 or 106, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 91 or 106.

[0173] In another embodiment, the eIFBP is VPg, a mutant or derivative thereof, or a fragment thereof having VPg function. In other embodiments, the VPg is a Calicivirus VPg. In other embodiments, the VPg comprises or consists of the amino acid sequence of SEQ ID NO: 120, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 120.

[0174] In another embodiment, the eIFBP is eIF4G, a mutant or derivative thereof, or a fragment thereof having the function of eIF4G. In other embodiments, eIF4G is human eIF4G. In other embodiments, eIF4G comprises or consists of the amino acid sequence of SEQ ID NO:84, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:84.

[0175] In further embodiments, the eIFBP is eIF4E, a mutant or derivative thereof, or a fragment thereof having eIF4E function. In other embodiments, the eIF4E is human eIF4E. In other embodiments, the eIF4E is a variant having a K119A substitution. In other embodiments, the eIF4E comprises or consists of the amino acid sequence of SEQ ID NO: 83 or 314, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 83 or 314, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 83 and having a K119A substitution.

[0176] In specific embodiments, the RBP is selected from L7Ae or MCP or λ-N.

[0177] In one embodiment, the RBP is L7Ae, a mutant or derivative thereof, or a fragment thereof having L7Ae functions. In other embodiments, L7Ae is the arch ribosomal protein L7Ae. In other embodiments, L7Ae comprises or consists of the amino acid sequence of SEQ ID NO: 92, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0178] In another embodiment, the RBP is MCP and its mutants or derivatives or fragments thereof having MCP function. In other embodiments, the MCP is a phage-derived MCP. In other embodiments, the MCP comprises the amino acid sequence of SEQ ID NO: 98 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 98, or consists of said amino acid sequence. In other embodiments, the MCP is an MCP variant having a V29I substitution compared to MCP. In other embodiments, the MCP having V29I comprises the amino acid sequence of SEQ ID NO: 200 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 200, or consists of said amino acid sequence.

[0179] In another embodiment, the RBP is λ-N, a mutant or derivative thereof, or a fragment thereof having the function of λ-N. In other embodiments, λ-N is phage-derived λ-N. In other embodiments, λ-N comprises or consists of the amino acid sequence of SEQ ID NO: 100, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 100.

[0180] In a specific embodiment, STIF comprises or consists of a fusion protein comprising an eIFBP and an RBP, and optionally additional protein domains such as 2CaM-M13, or a tag such as FLAG. In one embodiment, the configuration of the fusion protein from N-terminus to C-terminus is eIFBP-RBP or RBP-eIFBP, and optionally some other protein domains are inserted or a tag is present at the N-terminus or C-terminus.

[0181] In some embodiments, STIF comprises or consists of the fusion protein L7Ae-NSP3. In some embodiments, L7Ae-NSP3 comprises or consists of the amino acid sequence of SEQ ID NO:43, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, STIF comprises or consists of the fusion protein 3xFLAG-L7Ae-NSP3. In some embodiments, L7Ae-NSP3 comprises or consists of the amino acid sequence of SEQ ID NO:49, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, STIF comprises or consists of the fusion protein L7Ae-hNSP3. In some embodiments, L7Ae-hNSP3 comprises, or consists of, the amino acid sequence of SEQ ID NO:45, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:45.

[0182] In embodiments, STIF comprises or consists of the fusion protein L7Ae-eIF4E. In some embodiments, L7Ae-eIF4E comprises or consists of the amino acid sequence of SEQ ID NO:44, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0183] In some embodiments, STIF comprises or consists of the fusion protein MCP-NSP3. In some embodiments, MCP-NSP3 comprises or consists of the amino acid sequence of SEQ ID NO:59, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, STIF comprises or consists of the fusion protein 3xFLAG-MCP-NSP3. In some embodiments, MCP-NSP3 comprises or consists of the amino acid sequence of SEQ ID NO:260, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, STIF comprises or consists of the fusion protein MCP-hNSP3. In some embodiments, MCP-hNSP3 comprises or consists of the amino acid sequence of SEQ ID NO:60, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:60.

[0184] In some embodiments, STIF comprises or consists of the fusion protein PABP-L7Ae. In some embodiments, PABP-L7Ae comprises or consists of the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, STIF comprises or consists of the fusion protein PABP-L7Ae-3xFLAG. In some embodiments, PABP-L7Ae-3xFLAG comprises or consists of the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0185] In embodiments, STIF comprises or consists of the fusion protein eIF4G-2CaM-M13-L7Ae. In some embodiments, eIF4G-2CaM-M13-L7Ae comprises or consists of the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0186] In embodiments, STIF comprises or consists of the fusion protein PABP-MCP. In some embodiments, PABP-MCP comprises or consists of the amino acid sequence of SEQ ID NO: 206, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0187] In embodiments, STIF comprises or consists of the fusion protein MCP-eIF4E. In some embodiments, MCP-eIF4E comprises or consists of the amino acid sequence of SEQ ID NO: 244, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0188] In embodiments, STIF comprises or consists of the fusion protein eIF4G-MCP. In some embodiments, eIF4G-MCP comprises or consists of the amino acid sequence of SEQ ID NO: 253, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 253.

[0189] In embodiments, STIF comprises or consists of the fusion protein MCP-VPg. In some embodiments, MCP-VPg comprises or consists of the amino acid sequence of SEQ ID NO: 275 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 275. In some embodiments, STIF comprises the fusion protein MCP V29I -VPg or consisting thereof. In some embodiments, MCP V29I-VPg comprises or consists of the amino acid sequence of SEQ ID NO: 276, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 276.

[0190] In embodiments, STIF comprises or consists of the fusion protein L7Ae-VPg. In some embodiments, L7Ae-VPg comprises or consists of the amino acid sequence of SEQ ID NO: 281, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 281.

[0191] In a specific embodiment, STIF comprises or consists of two recombinant fusion proteins A and B,

[0192] wherein protein A may be RBP-Y or Y-RBP and protein B may be Y'-eIFBP or eIFBP-Y',

[0193] Protein A may be eIFBP-Y or Y-eIFBP and protein B may be Y'-RBP or RBP-Y'.

[0194] In a preferred embodiment, STIF comprises or consists of two recombinant fusion proteins A and B,

[0195] wherein protein A may be RBP-Y and protein B may be Y'-eIFBP, or

[0196] wherein protein A may be RBP-Y and protein B may be eIFBP-Y', or

[0197] wherein protein A may be γ-RBP and protein B may be γ'-eIFBP, or

[0198] wherein protein A may be γ-RBP and protein B may be eIFBP-γ′, or

[0199] wherein protein A may be eIFBP-γ and protein B may be γ'-RBP, or

[0200] wherein protein A may be eIFBP-γ and protein B may be RBP-Y', or

[0201] wherein protein A may be γ-eIFBP and protein B may be γ'-RBP, or

[0202] wherein protein A may be Y-eIFBP and protein B may be RBP-Y', or

[0203] wherein protein A may be RBP-Y' and protein B may be Y-eIFBP, or

[0204] wherein protein A may be RBP-Y' and protein B may be eIFBP-Y, or

[0205] wherein protein A may be γ'-RBP and protein B may be γ-eIFBP, or

[0206] wherein protein A may be Y'-RBP and protein B may be eIFBP-Y, or

[0207] wherein protein A may be eIFBP-γ' and protein B may be γ-RBP, or

[0208] wherein protein A may be eIFBP-Y' and protein B may be RBP-Y, or

[0209] wherein protein A may be γ'-eIFBP and protein B may be γ-RBP, or

[0210] Wherein protein A may be Y'-eIFBP and protein B may be RBP-Y.

[0211] In one embodiment, Y and Y' may be constitutively bound to each other, or bound via a trigger or signal, or via an additional protein Y".

[0212] In some embodiments, protein A or B may comprise multiple tandem repeats of Y or Y', such as 1-5 repeats, eg, 1, 2, 3, 4, or 5 repeats.

[0213] In some embodiments, Y and Y' are constitutively bound to each other.

[0214] In some specific embodiments, protein Y is dockerin and protein Y' is cohesin; or protein Y is cohesin and protein Y' is dockerin. In some embodiments, the DocS is DocS derived from Clostridium thermocellum. In other embodiments, DocS comprises or consists of the amino acid sequence of SEQ ID NO:80, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:80. In some embodiments, the Coh2 is Coh2 derived from Clostridium thermocellum. In other embodiments, Coh2 comprises or consists of the amino acid sequence of SEQ ID NO:77, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:77.

[0215] In some embodiments, Protein A or Protein B is selected from the following fusion proteins:

[0216]

[0217]

[0218] In some specific embodiments, protein Y is hepatitis C virus protease NS3a or a mutant or fragment thereof and protein Y' is apo NS3a reader ANR; or protein Y is ANR and protein Y' is NS3a. In some embodiments, NS3a is the catalytically active NS3a-variant NS3a(H1). In other embodiments, NS3a(H1) comprises or consists of the amino acid sequence of SEQ ID NO:105, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:105. In some embodiments, the ANR comprises or consists of the amino acid sequence of SEQ ID NO:73 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:73.

[0219] In some embodiments, Protein A or Protein B is selected from the following fusion proteins:

[0220]

[0221]

[0222] In some specific embodiments, protein Y is Bcl-XL and protein Y' is LD1 or LD3; or protein Y is LD1 or LD3 and protein Y' is Bcl-XL. In some embodiments, Bcl-XL comprises or consists of the amino acid sequence of SEQ ID NO:74, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, LD1 comprises or consists of the amino acid sequence of SEQ ID NO:94, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, LD3 comprises or consists of the amino acid sequence of SEQ ID NO:95, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:95.

[0223] In some embodiments, Protein A or Protein B is selected from the following fusion proteins:

[0224]

[0225] In some specific embodiments, protein Y is EGFP and protein Y' is LaG16; or protein Y is LaG16 and protein Y' is EGFP. In some embodiments, EGFP comprises or consists of the amino acid sequence of SEQ ID NO: 82, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, LaG16 comprises or consists of the amino acid sequence of SEQ ID NO: 93, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0226] In some embodiments, Protein A or Protein B is selected from the following fusion proteins:

[0227]

[0228] In some specific embodiments, protein Y is CCmut3 and protein Y' is BCR; or protein Y is BCR and protein Y' is CCmut3. In some embodiments, CCmut3 comprises or consists of the amino acid sequence of SEQ ID NO: 222, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, BCR comprises or consists of the amino acid sequence of SEQ ID NO: 227, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0229] In some embodiments, Protein A or Protein B is selected from the following fusion proteins:

[0230]

[0231] In some specific embodiments, protein Y is ABI (iDab) and protein Y' is ABL1; or protein Y is ABL1 and protein Y' is ABI (iDab). In some embodiments, ABI (iDab) comprises or consists of the amino acid sequence of SEQ ID NO: 223, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 223. In some embodiments, ABL1 is human ABL1. In other embodiments, ABL1 comprises or consists of the amino acid sequence of SEQ ID NO: 227, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 227.

[0232] In some embodiments, Protein A or Protein B is selected from the following fusion proteins:

[0233]

[0234] In some embodiments, protein Y is an antibody or antigen-binding fragment (e.g., a nanobody, monobody, affibody, DARPin, or scFv) that specifically binds to an antigen, and protein Y' is an antigen; or protein Y is an antigen, and protein Y' is an antibody or antigen-binding fragment (e.g., a nanobody, monobody, affibody, DARPin, or scFv). In some embodiments, the antigen is NS3 or a fragment thereof (e.g., the N-terminus of NS3), such as nNS3 derived from hepatitis C virus (HCV). In other embodiments, nNS3 comprises or consists of the amino acid sequence of SEQ ID NO: 303, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 303.

[0235] In some embodiments, the antigen-binding fragment is a scFv that specifically binds to NS3 or a fragment thereof (e.g., the N-terminus of NS3), for example, the scFv comprises or consists of the amino acid sequence of SEQ ID NO: 112 or 113, or an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 112 or 113.

[0236] In some embodiments, Protein A or Protein B is selected from the following fusion proteins:

[0237]

[0238] In some embodiments, Y and Y' conditionally bind to each other in a trigger-inducible or trigger-inhibitory manner.

[0239] In some specific embodiments, protein Y is ABI and protein Y' is PYL1; or protein Y is PYL1 and protein Y' is ABI, and the binding between ABI and PYL1 is triggered by abscisic acid. In some embodiments, ABI comprises or consists of the amino acid sequence of SEQ ID NO: 71, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 71. In some embodiments, PYL1 comprises or consists of the amino acid sequence of SEQ ID NO: 111, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 111.

[0240] In some embodiments, Protein A or Protein B is selected from the following fusion proteins:

[0241]

[0242]

[0243] In some embodiments, protein Y is a DrBPhP-specific affibody and protein Y' is DrBPhP; or protein Y is DrBPhP and protein Y' is a DrBPhP-specific affibody, and the binding between the DrBPhP-specific affibody and DrBPhP is triggered by light. In some embodiments, the affibody is Aff6 V18FΔN , and comprises, or consists of, the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 72. In some embodiments, the DrBPhP comprises, or consists of, the amino acid sequence of SEQ ID NO: 81, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 81.

[0244] In some embodiments, Protein A or Protein B is selected from the following fusion proteins:

[0245]

[0246] In some specific embodiments, protein Y is hepatitis C virus protease NS3a or a mutant or fragment thereof and protein Y' is apo NS3a reader ANR; or protein Y is ANR and protein Y' is NS3a, and the binding between NS3a and ANR is inhibited by grazoprevir. In some embodiments, NS3a comprises or consists of the amino acid sequence of SEQ ID NO: 105, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 105. In some embodiments, ANR comprises or consists of the amino acid sequence of SEQ ID NO: 73, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 73.

[0247] In some embodiments, Protein A or Protein B is selected from the following fusion proteins:

[0248]

[0249]

[0250] In some specific embodiments, protein Y is CIB1 and protein Y' is Cry2; or protein Y is Cry2 and protein Y' is CIB1, and the binding between CIB1 and Cry2 is triggered by light. In some embodiments, CIB1 comprises or consists of the amino acid sequence of SEQ ID NO:76, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, Cry2 comprises or consists of the amino acid sequence of SEQ ID NO:78, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0251] In some embodiments, Protein A or Protein B is selected from the following fusion proteins:

[0252]

[0253] In some specific embodiments, protein Y is DNCR and protein Y' is hepatitis C virus protease NS3a or a mutant or fragment thereof; or protein Y is NS3a and protein Y' is DNCR, and the binding between DNCR and NS3a is triggered by danoprevir. In some embodiments, DNCR comprises or consists of the amino acid sequence of SEQ ID NO: 79, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 79. In some embodiments, NS3a comprises or consists of the amino acid sequence of SEQ ID NO: 104, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 104. In some embodiments, NS3a is a catalytically active NS3a-variant NS3a(H1). In some embodiments, NS3a(H1) NS3a comprises or consists of the amino acid sequence of SEQ ID NO: 105, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 105.

[0254] In some embodiments, Protein A or Protein B is selected from the following fusion proteins:

[0255]

[0256]

[0257] In some specific embodiments, protein Y is FKBP and protein Y' is FRB; or protein Y is FRB and protein Y' is FKBP, and the binding between FKBP and FRB is triggered by rapamycin. In some embodiments, FKBP comprises or consists of the amino acid sequence of SEQ ID NO: 86, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 86. In some embodiments, FRB comprises or consists of the amino acid sequence of SEQ ID NO: 87, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 87.

[0258] In some embodiments, Protein A or Protein B is selected from the following fusion proteins:

[0259]

[0260] In some specific embodiments, protein Y is GAI and protein Y' is GID1; or protein Y is GID1 and protein Y' is GAI, and the binding between GAI and GID1 is triggered by gibberellic acid. In some embodiments, GAI comprises the amino acid sequence of SEQ ID NO: 88, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 88, or consists of said amino acid sequence. In some embodiments, GID1 comprises the amino acid sequence of SEQ ID NO: 89, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 89, or consists of said amino acid sequence.

[0261] In some embodiments, Protein A or Protein B is selected from the following fusion proteins:

[0262]

[0263] In some specific embodiments, protein Y is GNCR and protein Y' is hepatitis C virus protease NS3a or a mutant or fragment thereof; or protein Y is NS3a and protein Y' is GNCR, and the binding between GNCR and NS3a is triggered by grazoprevir. In some embodiments, GNCR comprises or consists of the amino acid sequence of SEQ ID NO:90, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:90. In some embodiments, NS3a comprises or consists of the amino acid sequence of SEQ ID NO:104, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:104. In some embodiments, NS3a is a catalytically active NS3a-variant NS3a(H1). In other embodiments, NS3a(H1) comprises or consists of the amino acid sequence of SEQ ID NO: 105, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0264] In some embodiments, Protein A or Protein B is selected from the following fusion proteins:

[0265]

[0266]

[0267] In some embodiments, protein Y is an mCherry-specific Nanobody and protein Y' is mCherry; or protein Y is mCherry and protein Y' is an mCherry-specific Nanobody, and the binding between the Nanobody and mCherry is triggered by light. In some embodiments, the Nanobody is LaM8 AK47, and comprises, or consists of, the amino acid sequence of SEQ ID NO: 220, or an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 220. In some embodiments, mCherry comprises, or consists of, the amino acid sequence of SEQ ID NO: 97, or an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 97.

[0268] In some embodiments, Protein A or Protein B is selected from the following fusion proteins:

[0269]

[0270] In some embodiments, the binding between Y and Y' is regulated by intracellular signaling dynamics, for example, by an agent that can activate intracellular signaling.

[0271] In some specific embodiments, protein Y is ERK2 and protein Y' is pE59; or protein Y is pE59 and protein Y' is ERK2, and the binding between ERK2 and pE59 is triggered by activated MAPK signaling (e.g., epidermal growth factor EGF). In some embodiments, ERK2 comprises or consists of the amino acid sequence of SEQ ID NO: 85, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 85. In some embodiments, pE59 comprises or consists of the amino acid sequence of SEQ ID NO: 110, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 110.

[0272] In some embodiments, Protein A or Protein B is selected from the following fusion proteins:

[0273]

[0274] In some embodiments, Y and Y' can interact with each other through another protein Y". That is, Y and Y' can associate with each other only when Y" is present, because the target protein Y" triggers the Y:Y':Y"-interaction.

[0275] In some embodiments, Y" can be any protein or agent as long as it can be bound by two different proteins, preferably at different domains or different epitopes of Y".

[0276] In some embodiments, protein Y" is a fusion gene product, an oncoprotein, a virulence factor, an RNA binding protein, or any other intracellular or secreted protein containing one or more domains.

[0277] In some embodiments, protein Y and protein Y' are two different scFvs that specifically bind to protein Y", e.g., at different domains or different epitopes of Y". In some embodiments, Y" may be an antigen that can be specifically bound by two antibodies or antigen-binding fragments at different domains or different epitopes.

[0278] In some embodiments, protein Y" is selected from a disease-specific cell signature, such as an oncoprotein, such as a fusion gene product or protein complex specifically expressed in tumor cells or tumor tissues. For example, Y" can be a fusion protein BCR-ABL, or a virulence factor such as HCV or an HCV-specific protein (such as NS3 protein).

[0279] In some embodiments, protein Y and protein Y' are two different Nanobodies, which specifically bind to protein Y", e.g., at different domains or at different epitopes of Y". In some embodiments, protein Y and protein Y' are two different Affibodies, which specifically bind to protein Y", e.g., at different domains or at different epitopes of Y". In some embodiments, protein Y and protein Y' are two different monobodies, which specifically bind to protein Y", e.g., at different domains or at different epitopes of Y". In some embodiments, protein Y and protein Y' are two different DARPins, which specifically bind to protein Y", e.g., at different domains or at different epitopes of Y".

[0280] In some embodiments, protein Y is any natural or synthetic protein that binds to protein Y" with high affinity, and protein Y' is a scFv, DARPin, monobody, affibody or nanobody selected from antigen-binding fragments that bind to protein Y"; or protein Y is a scFv, DARPin, monobody, affibody or nanobody selected from antigen-binding fragments, and protein Y' is any natural or synthetic protein that binds to protein Y" with high affinity; or proteins Y and Y' are both any natural or synthetic proteins that bind to protein Y" with high affinity.

[0281] In some embodiments, protein Y is CCmut3 and protein Y' is ABI (iDab), or protein Y' is CCmut3 and protein Y" is ABI (iDab), and Y" is a protein bound by CCmut3 and ABI (iDab), such as BCR-ABL. In some embodiments, CCmut3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 222, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 222. In some embodiments, ABI (iDab) comprises, or consists of the amino acid sequence of SEQ ID NO: 223, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 223. In other embodiments, the BCR-ABL comprises or consists of the amino acid sequence of SEQ ID NO:301, or an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:301.

[0282] In some embodiments, Protein A or Protein B is selected from the following fusion proteins:

[0283]

[0284] In some embodiments, protein Y is an antibody or antigen-binding fragment (e.g., scFv, DARPin, monobody, affibody, or nanobody) that specifically binds to antigen Y", or protein Y' is another antibody or antigen-binding fragment (e.g., scFv, DARPin, monobody, affibody, or nanobody) that specifically binds to the same antigen Y". In specific embodiments, antigen Y" is NS3 or a mutant or fragment thereof (e.g., the N-terminus of NS3; nNS3). In some embodiments, nNS3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 303, or an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 303.

[0285] In some embodiments, the antigen-binding fragment Y or Y' is a scFv or fragment thereof that specifically binds to NS3 (e.g., the N-terminus of NS3; nNS3), e.g., the scFv comprises or consists of the amino acid sequence of SEQ ID NO: 112 (e.g., scFv162) or SEQ ID NO: 113 (e.g., scFv35), or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 112 or 113. In some embodiments, Y is scFv162 and Y' is scFv35. In some embodiments, Y' is scFv162 and Y is scFv35.

[0286] In some embodiments, Protein A or Protein B is selected from the following fusion proteins:

[0287]

[0288] In an embodiment, the mRNA construct comprises (i) a coding region and (ii) an RNA segment that specifically binds to STIF.

[0289] In some embodiments, the RNA segment specifically bound by STIF belongs to the 5'-UTR and / or 3'-UTR of the mRNA. In some embodiments, the RNA segment specifically bound by STIF comprises a 5'-UTR and a 3'-UTR.

[0290] In some embodiments, the coding region of the mRNA is flanked by 5'-UTR and 3'-UTR. In some embodiments, the coding region encodes one or more target proteins or peptides, for example, the mRNA can be any nucleic acid segment encoding any polypeptide of interest. In some embodiments, the coding region is any RNA sequence that starts with nucleotide AUG and ends with the nucleotide sequence UAG, UAA or UGA). For example, the target mRNA can be an mRNA encoding a target protein, and preferably, the target protein can be selected from a therapeutic protein, for example, a hormone based on protein, for example, insulin, a pro-apoptotic protein such as BAX, a fluorescent protein such as EGFP or mCherry, or any other secretory or intracellular protein that can be detected by its expression, for example, a reporter protein such as SEAP or luciferase.

[0291] In some embodiments, the RNA segment specifically bound by STIF is a poly-A signal or a poly-A surrogate, more preferably, a poly-A surrogate.

[0292] In some embodiments, the poly(A) surrogate can be any segment containing or consisting of one or more n aptamer repeats that bind to a specific RBP. In some embodiments, the poly(A) surrogate is placed in the 3'-UTR or 5'-UTR of the mRNA.

[0293] In some embodiments, the aptamer is selected from C / D-box, MS2-box, or boxB.

[0294] In an embodiment, the poly(A) surrogate contains tandem repeats of the L7Ae-specific C / D-box aptamer, e.g., (C / D-box) n , or MCP-specific MS2-box aptamer (MS2-box) n or λ-N specific aptamer (box B) n , where n can be any number between 1 and 1000, such as 5-30, such as 8, 12, 16 or 24. The choice of aptamer depends on the RBP, for example, if the RBP is L7Ae, the aptamer is typically an L7Ae-specific C / D-box aptamer, and if the RBP is MCP, the aptamer is typically an MCP-specific MS2-box aptamer.

[0295] In some embodiments, the C / D-box comprises or consists of the nucleic acid sequence of SEQ ID NO: 123. In some embodiments, the MS2-box comprises or consists of the nucleic acid sequence of SEQ ID NO: 125 or 315. In some embodiments, box B comprises or consists of the nucleic acid sequence of SEQ ID NO: 121.

[0296] In other embodiments, when the poly(A) surrogate is placed in the 3'-UTR or 5'-UTR of the mRNA, the system further comprises a construct expressing an RNase or a CRISPR family of proteins.

[0297] In other embodiments, when the poly(A) surrogate is placed in the 3'-UTR or 5'-UTR of the mRNA, the mRNA construct further comprises an RNA cleavage site for pre-programmed poly(A) removal (e.g., a self-cleaving ribozyme signal such as HHR), which is located between the aptamer and the poly(A) and is placed in the 3'-UTR.

[0298] In one embodiment, the cleavage is performed by RNA interference, and the RNA cleavage site is an siRNA binding site or multiple copies thereof, an shRNA binding site or multiple copies thereof, or a miRNA binding site or multiple copies thereof. In a specific embodiment, when the RNA cleavage site is an siRNA binding site, a construct expressing the siRNA should be included in the system of the present invention. In a specific embodiment, when the RNA cleavage site is an shRNA binding site, a construct expressing the shRNA should be included in the system of the present invention. In a specific embodiment, when the RNA cleavage site is a miRNA binding site, a construct expressing the miRNA should be included in the system of the present invention.

[0299] For example, the RNA cleavage site may comprise one or more (BS(shRNA-216)) n Repeat, wherein n can be any number between 1 and 100, preferably n can be any number between 1 and 4. In some embodiments, the RNA cleavage site is BS (shRNA-216). In some embodiments, BS (shRNA-216) comprises SEQ ID NO: 122 or consists thereof. In some embodiments, when the RNA cleavage site is BS (shRNA-216), the system further comprises a construct that expresses shRNA-216 to cleave polyA. In some embodiments, shRNA-216 comprises SEQ ID NO: 126 or consists thereof.

[0300] In other embodiments, the cleavage is performed by a ribozyme and the cleavage site is a ribozyme. In some embodiments, the ribozyme is a self-cleaving ribozyme or multiple copies thereof or fragments thereof. In some embodiments, the self-cleaving ribozyme is a hammerhead ribozyme (HHR). n , wherein n can be any number between 1 and 100, preferably n can be any number between 1 and 4. In some embodiments, the RNA cleavage site is HHR. In some embodiments, HHR comprises or consists of SEQ ID NO: 124. In some embodiments, when the RNA cleavage site is HHR, the system does not need to include an additional construct that cleaves HHR because it triggers spontaneous self-excision of the poly(A) signal.

[0301] In some embodiments, the construct comprises, from 5'-end to 3'-end, a 5'-UTR, a coding region, a poly(A) surrogate, a cleavage site, and other elements of the 3'UTR.

[0302] In some embodiments, the construct comprises, from 5'-end to 3'-end, a combination of a 5'-UTR, a coding region, and the following poly(A) surrogate cleavage sites in the 3'UTR:

[0303] combination poly(A) replacements Cleavage site 1 <![CDATA[(C / D-box)8]]> <![CDATA[(BS(shRNA-216))2]]> 2 <![CDATA[(C / D-box) 16 ]]> <![CDATA[(BS(shRNA-216))2]]> 3 <![CDATA[(C / D-box) 12 ]]> <![CDATA[(BS(shRNA-216))2]]> 4 <![CDATA[(C / D-box) 24 ]]> <![CDATA[(BS(shRNA-216))2]]> 5 <![CDATA[(C / D-box) 24 ]]> <![CDATA[(BS(shRNA-216))2]]> 6 <![CDATA[(C / D-box) 24 ]]> HHR 7 <![CDATA[(MS2-box) 24 ]]> HHR 8 <![CDATA[(MS2-box) 16 ]]> HHR 9 <![CDATA[(MS2-box) 24 ]]> HHR 10 <![CDATA[(MS2-box) 24 ]]> HHR 11 none <![CDATA[(HHR)2]]> 12 none <![CDATA[(HHR)4]]> 13 <![CDATA[(MS2-box) 24 ]]> HHR 14 none HHR 15 <![CDATA[(MS2-box) 24 ]]> HHR 16 <![CDATA[(MS2-box) 24 ]]> HHR 17 <![CDATA[(MS2-box) 16 ]]> HHR 18 <![CDATA[(C / D-box) 24 ]]> none 19 <![CDATA[(C / D-box)4]]> none 20 <![CDATA[(MS2-box) 24 ]]> HHR 21 <![CDATA[(MS2-box) 12 ]]> HHR 22 <![CDATA[(MS2-box)8]]> HHR 23 <![CDATA[(MS2-box)8]]> <![CDATA[(BS(shRNA-216))2]]>

[0304] When the poly(A) surrogate is (MS2-box)n, the STIF comprises MCP; when the poly(A) surrogate is (C / D-box)n, the STIF comprises L7Ae.

[0305] In some embodiments, the mRNA construct comprises the RNA sequence shown in any one of SEQ ID NOs: 131 to 149, or any RNA sequence in Table 2, or comprises or consists of an RNA sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 131 to 149, or any RNA sequence in Table 2.

[0306] In some embodiments, the mRNA construct may further comprise a 5'-cap or a 5'-cap surrogate. For example, the 5'-cap may be replaced by a 5'-cap surrogate by cleaving the 5'-cap. In some embodiments, a poly(A) surrogate may also be used as a 5'-cap surrogate. In some embodiments, the 5'-cap may be cleaved in the same manner as poly(A). In some embodiments, the cleavage site of poly(A) may also be used as a cleavage site for the 5'-cap.

[0307] In some embodiments, the construct comprises, from the 5'-end to the 3'-end, a 5'-UTR, a cleavage site, a 5'-cap surrogate, a coding region, and a 3'UTR. In some embodiments, the construct comprises, from the 5'-end to the 3'-end, a 5'-UTR and the following combinations of cleavage sites, 5'-cap surrogate, coding regions, and poly(A) surrogate in the 3'UTR:

[0308] In some embodiments, the construct comprises the following elements from N-terminus to C-terminus:

[0309]

[0310] Among them, BS (shRNA-216) n or (HHR) n In the formula, n can be any number between 1 and 100, preferably n can be any number between 1 and 4;

[0311] In (C / D-box) n or (MS2-box) n In , n can be any number between 1 and 1000, such as 5-30, such as 8, 12, 16 or 24;

[0312] When the poly(A) surrogate is (MS2-box) n When the poly(A) replacement is (C / D-box) n When STIF contains L7Ae.

[0313] In a specific embodiment, the gene regulation system comprises a construct expressing a synthetic translation initiation factor (STIF) (STIF construct) and an mRNA construct comprising an mRNA encoding a target protein, wherein the STIF and the expression construct each comprise the following elements:

[0314]

[0315] wherein the mRNA construct can be genetically encoded by a DNA-based expression vector or delivered as in vitro transcribed RNA, and / or wherein

[0316] STIF can be genetically encoded by a DNA-based expression vector, delivered as in vitro transcribed RNA, or delivered directly as a purified protein, and / or wherein

[0317] Y and Y' are two different proteins that bind to each other in a constitutive, trigger-inducible or protein Y"-dependent manner as described herein.

[0318] In one aspect, the present invention relates to a gene regulatory system triggered by grazoprevir.

[0319] In an embodiment, the grazoprevir-triggered system comprises:

[0320] (i) a synthetic translation initiation factor (STIF) and an mRNA construct comprising an mRNA encoding a target protein; or

[0321] (ii) a nucleic acid encoding STIF and a nucleic acid encoding an mRNA construct comprising an mRNA encoding a target protein;

[0322] wherein the STIF comprises or consists of two recombinant fusion proteins A and B,

[0323] wherein protein A may be RBP-Y or Y-RBP, and protein B may be Y'-eIFBP or eIFBP-Y', or wherein protein A may be eIFBP-Y or Y-eIFBP, and protein B may be Y'-RBP or RBP-Y'

[0324] wherein Y is NS3a and Y' is GNCR.

[0325] In one embodiment, the RBP is L7Ae or MCP.

[0326] In some embodiments, protein A or B may comprise multiple tandem repeats of GNCR or NS3A, such as 1-5 repeats, eg, 1, 2, 3, 4, or 5 repeats.

[0327] In one embodiment, Protein A has L7Ae-(NS3a) n configuration and protein B has (GNCR) n -NSP3 conformation, or protein A with L7Ae-(GNCR) n configuration and protein B has (NS3a) n -NSP3 configuration, wherein n is an integer from 1 to 10, such as 1, 2 or 3. In other embodiments, the mRNA construct comprises a coding region encoding a target protein and an RNA segment that specifically binds to STIF, wherein the RNA segment that specifically binds to STIF comprises tandem repeats of an L7Ae-specific C / D-box aptamer, such as (C / D-box) n , which is optionally placed in the 3'UTR or 5'UTR of the mRNA construct. Preferably, the mRNA construct comprises, from 5' to 3' end, the 5'UTR, the coding region, the (C / D-box) n (wherein n=1-30, for example, 24), (BS(shRNA-216)) n (where n=1, 2 or 3, e.g. 2) or (HHR) n (wherein n=1, 2, 3 or 4, eg, 1) and a 3'UTR.

[0328] In one embodiment, Protein A has MCP-(NS3a) n configuration and protein B has (GNCR) n -NSP3 conformation, or protein A with MCP-(GNCR) n configuration and protein B has (NS3a) n -NSP3 configuration, wherein n is an integer from 1 to 10, such as 1, 2 or 3. In other embodiments, the mRNA construct comprises a coding region encoding a target protein and an RNA segment that specifically binds to STIF, wherein the RNA segment that specifically binds to STIF comprises tandem repeats of an MCP-specific MS2-box aptamer, such as (MS2-box) n , which is optionally placed in the 3'UTR or 5'UTR of the mRNA construct. Preferably, the mRNA construct comprises, from 5' to 3' end, the 5'UTR, the coding region, the (MS2-box) n (wherein n=1-30, for example, 24), (BS(shRNA-216)) n(where n=1, 2 or 3, e.g. 2) or (HHR) n (wherein n=1, 2, 3 or 4, eg, 1) and a 3'UTR.

[0329] In other embodiments, the system further comprises a module for expressing shRNA-216.

[0330] In one embodiment, the target protein is selected from therapeutic proteins such as protein-based hormones, for example insulin, fluorescent proteins such as EGFP or mCherry, or any other secreted or intracellular protein whose expression can be detected, for example reporter proteins such as SEAP or luciferase.

[0331] (III) Methods for Producing Gene Regulation Systems

[0332] In one embodiment, the present invention relates to a nucleic acid encoding a STIF protein.

[0333] In further embodiments, the present invention relates to nucleic acids encoding mRNA constructs.

[0334] It is well known to those skilled in the art that, due to codon degeneracy, each amino acid sequence can be encoded by multiple nucleic acid sequences. Nucleic acid sequences encoding molecules of the present invention can be generated by methods well known in the art, for example, by de novo solid DNA synthesis or by PCR amplification.

[0335] In other embodiments, the present invention relates to a vector comprising the nucleic acid. In one embodiment, the vector is an expression vector such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, mucoids, phages, or yeast artificial chromosomes (YACs). In one embodiment, the expression vector is an episomal vector, for example, derived from pcDNA3.1(+) (Invitrogen, CA; Catalog No. V79020). In other embodiments, the expression vector is an AAV vector or a lentivirus.

[0336] AAV can be any AAV vector known in the art ((Li and Samulski, 2020)), such as AAV1, AAV2, AAV2.7m8, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10.

[0337] In some embodiments, the AAV vector comprises any one or more or all of the following elements:

[0338] (i) an ITR, e.g., an ITR comprising the nucleic acid sequence of SEQ ID NO: 212 or SEQ ID NO: 316, or comprising a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 212 or 316, or consisting of said amino acid sequence;

[0339] (ii) a nucleic acid sequence flanked by ITRs and encoding the STIF protein or said mRNA construct.

[0340] In embodiments, the lentivirus comprises a 5'-LTR, e.g., comprising the nucleic acid sequence of SEQ ID NO: 215, or comprising or consisting of a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 215.

[0341] In some embodiments, the vector comprises a promoter, such as a CMV promoter, a U6 promoter, a phosphoglycerate kinase gene promoter, an elongation factor 1α promoter, or a mammalian CREB1-specific promoter. In some embodiments, the promoter comprises a nucleic acid sequence of any one of SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 210, SEQ ID NO: 214, SEQ ID NO: 233-240, or comprises a nucleic acid having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or consists thereof.

[0342] The vector may also contain an IRES for expressing two or more STIF proteins and / or mRNA constructs.

[0343] In embodiments, the mRNA constructs of the present invention can be directly synthesized. For example, an expression construct comprising a gene encoding an mRNA encoding a target protein can be directly synthesized. There are several methods in the art for directly synthesizing mRNA constructs for this system, such as those listed in "(Parr et al., 2020; Yu et al., 2020)".

[0344] In other embodiments, STIF can be produced by culturing a host cell containing a nucleic acid encoding STIF under conditions suitable for expression of the STIF protein. When STIF comprises two proteins, the nucleic acid encoding each protein can be located in the same vector or in different vectors. In other embodiments, the nucleic acid encoding each STIF protein of the present invention can be introduced into the same or different host cells for expression.

[0345] In one embodiment, the host cell is a eukaryotic cell. In another embodiment, the host cell is selected from yeast cells, mammalian cells such as CHO cells (such as CHO-S, such as ExpiCHO-S) or 293 cells (such as 293F or HEK293 cells) or other cells suitable for preparing STIF. In one embodiment, the host cell is prokaryotic, such as bacteria, for example, Escherichia coli.

[0346] (IV) Pharmaceutical compositions and pharmaceutical preparations

[0347] In some embodiments, the present invention provides compositions comprising the systems described herein, preferably, the compositions are pharmaceutical compositions. In one embodiment, the compositions further comprise pharmaceutically acceptable adjuvants. In one embodiment, the compositions, such as pharmaceutical compositions, comprise a combination of the systems of the present invention and one or more other therapeutic agents or agents that can induce expression of the systems.

[0348] The present invention also includes compositions comprising the system. In some embodiments, the system comprises STIF and an mRNA construct. In some other embodiments, the system comprises a nucleic acid encoding STIF and a nucleic acid encoding an mRNA construct. In some other embodiments, the composition comprises a STIF fusion protein and an mRNA construct. In some other embodiments, the composition comprises DNA encoding STIF and DNA encoding the mRNA construct. In some embodiments, the DNA is located in a vector (such as an expression vector, such as an AAV vector such as pAAV2 / 8) or pcDNA (such as pcDNA3.1). In some embodiments, the RNA or DNA can be formulated in a protein particle (such as a virus or virus-like particle), a polymer particle (such as PEI or polymer vesicles) or a lipid particle (such as a liposome or lipid nanoparticle (LNP)). In some other embodiments, the composition comprises RNA encoding STIF and RNA encoding the mRNA construct. In specific embodiments, the RNA is mRNA. In some embodiments, the RNA is in vitro transcribed mRNA produced from a vector containing DNA encoding the system, or is directly synthesized according to the techniques described in "(Parr et al., 2020; Yu et al., 2020)".

[0349] These compositions may further comprise suitable pharmaceutically acceptable supplements, such as pharmaceutically acceptable carriers and pharmaceutically acceptable excipients, including buffers known in the art.

[0350] As used herein, "pharmaceutically acceptable carrier" includes any physiologically compatible solvents, dispersion media, isotonicity agents, and absorption retardants suitable for use in the compositions.

[0351] See also "Handbook of Pharmaceutical Excipients", 8th edition, RC Rowe, PJ Eskey and S C Owen, Pharmaceutical Press, London, Chicago, for pharmaceutically acceptable excipients used.

[0352] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), powders or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0353] Pharmaceutical formulations comprising the systems described herein can be prepared by mixing the components of the systems of the present invention having the desired degree of purity with one or more optional pharmaceutically acceptable supplementary materials, preferably in the form of a lyophilized formulation or an aqueous solution.

[0354] The pharmaceutical compositions or formulations of the present invention may also contain more than one active ingredient, such as an ingredient necessary for triggering the system, or an ingredient required for the specific indication to be treated, preferably those with complementary activities that do not adversely affect each other. The active ingredients are suitably combined in effective amounts for the intended use.

[0355] (V) Pharmaceutical Compositions and Kits

[0356] In some embodiments, the present invention also provides a pharmaceutical combination or pharmaceutical combination product comprising a system of the present invention and one or more other pharmaceutical agents.

[0357] In one embodiment, the other agent is an agent that can be used to induce, trigger, or inhibit STIF-specific translation. In specific embodiments, the other agent can be protein γ, abscisic acid, grazoprevir, danoprevir, MAPK, rapamycin, or gibberellic acid. In other embodiments, the other agent is another therapeutic agent.

[0358] Another object of the present invention is to provide a kit comprising the pharmaceutical combination of the present invention, preferably in the form of pharmaceutical dosage units.Thus, the dosage units can be provided according to the administration schedule or interval.

[0359] In one embodiment, the kit of the present invention comprises:

[0360] - one or more containers that contain each component of the system, e.g., different components may be contained together in one container, or in separate containers;

[0361] - Another container contains another pharmaceutical agent or a pharmaceutical composition containing another pharmaceutical agent.

[0362] (VI) Use and method

[0363] In one aspect, the present invention relates to a cell-based expression system for expressing one or more mRNA constructs, the translation of which is regulated in a trigger-induced manner in living cells, the expression system comprising:

[0364] delivering one or several mRNA constructs and STIF directly into living cells in the form of RNA and protein, and expressing the mRNA constructs in said living cells; or

[0365] Nucleic acids encoding the one or several mRNA constructs or expression vectors comprising the nucleic acids or nucleic acids encoding STIF or expression vectors comprising the nucleic acids are delivered into living cells, for example by transfection with expression vectors comprising the nucleic acids, and the target protein is expressed.

[0366] In a preferred embodiment, the living cell is of mammalian origin. In a more preferred embodiment, the living cell is of human origin. In another preferred embodiment, the living cell is part of a living tissue of an organism.

[0367] In other aspects, the present invention relates to the use or implementation of the gene regulation system of the present invention for mRNA in a cell-free system. Possible applications of the implementation include but are not limited to point-of-care testing involving in vitro sensing of disease metabolites, virulence factors or environmental pollutants.

[0368] (VI-I) for treatment

[0369] In another aspect, the present invention relates to the gene regulation system of the present invention for use as a medicament, eg for use in gene therapy.

[0370] In some embodiments, therapy using the systems of the present invention can achieve long-term therapeutic efficacy in vivo.

[0371] In one embodiment, the present invention relates to gene therapy, which comprises administering the gene regulatory system or pharmaceutical composition or pharmaceutical combination or kit of the present invention to a subject in need thereof.

[0372] In another embodiment, the present invention provides a method for preventing or treating a disease, comprising administering the gene regulatory system or pharmaceutical composition or pharmaceutical combination or kit of the present invention to a subject in need thereof.

[0373] In another embodiment, the present invention relates to the use of a gene regulatory system or a pharmaceutical composition or a pharmaceutical combination or a kit in the preparation of a medicament, for example, a gene therapy method for diagnosing, treating or preventing a disease.

[0374] In some specific embodiments, the disease is selected from cancer or immune disease or metabolic disease or infectious disease. In one embodiment, the cancer is a solid tumor or a blood tumor. In one embodiment, the immune disease is an autoimmune disease. In one embodiment, the metabolic disease is diabetes. In one embodiment, the infectious disease is a viral (e.g., HCV) infection.

[0375] The subject can be a mammal, such as a primate, preferably a higher primate, such as a human (e.g., an individual suffering from a disease described herein or at risk of suffering from a disease described herein). In one embodiment, the subject suffers from a disease described herein (e.g., cancer) or is at risk of suffering from a disease described herein.

[0376] In some embodiments, the systems or pharmaceutical compositions or pharmaceutical combinations or kits of the present invention can delay, attenuate, or cure the onset of a disease and / or symptoms associated with the disease.

[0377] In some embodiments, the systems or pharmaceutical compositions or drug combinations or kits of the present invention may also be used in combination with one or more other therapies, such as treatment modalities and / or other therapeutic agents, for the uses described herein, such as for the diagnosis and / or prevention and / or treatment of the relevant diseases or conditions mentioned herein.

[0378] Such combination therapy encompasses combined administration (e.g., two or more therapeutic agents are included in the same or separate formulations) and separate administration. In this case, the administration of the system of the present invention can be carried out before, simultaneously with, and / or after administration of the other therapeutic agents and / or treatment modalities.

[0379] The administration route of the system is based on known gene therapy methods and depends on the type of system. For example, for AAV treatment, injection can be used to introduce the system into the subject. The trigger can be administered based on known methods, such as oral, intravenous, intraperitoneal, intracerebral (parenchymal), intraventricular, intramuscular, ophthalmic, intraarterial, intraportal or intralesional routes; by continuous release system or by implantable device. In some embodiments, the system or trigger can be administered by bolus or by continuous infusion or by implant device.

[0380] In one embodiment, the target protein is insulin, such as human insulin, and the trigger is grazoprevir.The disease treated by the system is diabetes, such as type I diabetes.

[0381] In another aspect, the system of the present invention can be used as a translation-based protein sensor and as a next-generation therapeutic gene circuit that provides programmable, broadly controllable, and self-sufficient gene therapy for the treatment of many human diseases.

[0382] Although fusion proteins (e.g., native BCR-ABL) are the primary case where a single intracellular target protein can unambiguously identify a pathological cell state, some diseases often lack such unique biomarkers. In these cases, the "true" disease-specific cellular signature must be resolved by combining the detection of various secondary checkpoint signals.

[0383] Therefore, the system of the present invention can be used in situations where such multiplexed cell state detection is required.

[0384] In specific embodiments, the system can treat tumors or cancers, especially those involving complexity and specificity issues relevant in a clinical setting. Specifically, the system can be flexibly interconnected with other genetically encoded sensors to ultimately achieve any desired customized combination of tissue specificity and target specificity in vivo. The system of the present invention is not limited to highly specific fusion gene sensors designed systematically and empirically for treating hitherto refractory cancers, but can be used to detect any intracellular target signal for which a suitable set of protein binder moieties (e.g., nanobodies) can be found.

[0385] In one embodiment, the present invention provides a method for treating tumors, such as specifically killing tumor cells or tumor tissue.

[0386] Specifically, the system includes

[0387] (i) a synthetic translation initiation factor (STIF) and an mRNA construct comprising an mRNA encoding a target protein; or

[0388] (ii) a nucleic acid encoding STIF and a nucleic acid encoding an mRNA construct comprising an mRNA encoding a target protein;

[0389] The STIF comprises or consists of two recombinant fusion proteins A and B, wherein protein A can be RBP-Y or Y-RBP, and protein B can be Y'-eIFBP or eIFBP-Y', or wherein protein A can be eIFBP-Y or Y-eIFBP, and protein B can be Y'-RBP or RBP-Y', wherein Y and Y' can interact with each other through another protein Y". That is, Y and Y' can only associate with each other when Y" is present, because the target protein Y" triggers the Y:Y':Y"-interaction.

[0390] In one embodiment, the RBP is L7Ae or MCP.

[0391] In one embodiment, Y or Y' is any protein or fragment that can bind to Y".

[0392] In some embodiments, Y" can be any protein or agent, as long as it can be bound by two different proteins, preferably at different domains or different epitopes of Y". In some specific embodiments, protein Y" is selected from disease-specific cellular features, such as oncoproteins, such as fusion gene products or protein complexes specifically expressed in tumor cells or tumor tissues. For example, Y" can be a fusion protein BCR-ABL, or a virulence factor such as HCV or an HCV-specific protein (such as NS3 protein).

[0393] In some embodiments, protein A or B may comprise multiple tandem repeats of Y or Y', such as 1-5 repeats, such as 1, 2, 3, 4 or 5 repeats. In some embodiments, Y and Y' are two different Nanobodies, which specifically bind to protein Y", e.g., at different domains or at different epitopes of Y". In some embodiments, protein Y and protein Y' are two different Affibodies, which specifically bind to protein Y", e.g., at different domains or at different epitopes of Y". In some embodiments, protein Y and protein Y' are two different monobodies, which specifically bind to protein Y", e.g., at different domains or at different epitopes of Y". In some embodiments, protein Y and protein Y' are two different DARPins, which specifically bind to protein Y", e.g., at different domains or at different epitopes of Y".

[0394] In some embodiments, Y" is selected from disease-specific cellular features, such as fusion gene products, oncoproteins, virulence factors, RNA-binding proteins, or any other intracellular or secreted proteins containing one or more domains, and the target protein is a protein that can kill tumor cells (e.g., a pro-apoptotic protein, such as Bax protein).

[0395] (VI-II) for complex biological computation in mammalian cells or in vivo

[0396] In one aspect of the invention, the system of the invention can be used for biocomputing in vitro or in vivo.

[0397] In one embodiment, a genetic tristate buffer can be generated by regulated expression of a trigger-inducible gene switch programmed with "buffering" (BUF) and "noting" (NOT) signal processing logic in mammalian cells or in vivo.

[0398] In one embodiment, the present invention provides a transcription-translation-based gene circuit in which the control input B operating at the upstream layer (e.g., transcription) monitors the expression of a synthetic translation initiation factor (STIF), which regulates the expression of a target protein from the downstream layer (e.g., translation). The three-state buffer can be engineered to contain up to four types of gene switches: B activates STIF expression (IF1), B terminates STIF expression (IF0), A activates target protein expression (BUF), and A terminates target protein expression (NOT). For example, STIF uses the rotavirus-derived nonstructural protein 3 domain (NSP3) to bind to the pre-initiation complex eIF4F, resulting in the translation of MCP-specific mRNA after inducible binding of NSP3 and MCP-containing factors.

[0399] In one embodiment, the BUF-switch and NOT-switch can be a grazoprevir-inducible system comprising STIF comprising the triplet NS3a / GNCR / ANR controlled by grazoprevir.

[0400] In one embodiment, in order to initiate translation, a circularized mRNA conformation must be established in which the 5'- and 3'-ends are brought into close proximity by a synthetic translation initiation factor (STIF) comprising an NSP3 domain and a synthetic tether (MCP-NS3a(H1)) consisting of an RNA-binding MCP domain and a STIF-binding NS3a(H1) domain. Since grazoprevir simultaneously triggers the dissociation of the ANR from NS3a (H1) and the binding of the GNCR to NS3a(H1), STIF can be engineered to contain both the GNCR and NSP3, enabling grazoprevir-inducible translation (BUF switch). Similarly, fusion of the ANR to NSP3 results in grazoprevir-repressible translation (NOT switch).

[0401] In some embodiments of the three-state circuit, the BUF and NOT switches are managed by "active high" (IF1) or "active low" (IF0) control signals that produce "normal" or "inverted" outputs, respectively. The biological implementations of the "active high" and "active low" switches should be orthogonal to each other so that they can operate without interference in mammalian cells. For example, a vanillic acid-inducible gene switch based on a PKA / CREB1-responsive promoter activated by cAMP signaling regulated by the olfactory receptor MOR9-1 (Saxena et al., 2016) may be a potential IF1 switch, while an IF0 switch can be accomplished by the VanR-dependent mammalian transactivator (VanR-VP64), which regulates gene expression from a cognate VanO-containing promoter (Gitzinger et al., 2012).

[0402] In other embodiments, the present invention provides two additional sets of grazoprevir-responsive gene switches (e.g., Figure 25 (as shown in A).

[0403] (i) For the GEMS-based system, grazoprevir-inducible gene expression from a synthetic STAT3-specific promoter (BUF2) occurs upon dimerization of a synthetic cell membrane receptor containing an intracellular domain derived from IL6RB and an extracellular GNCR domain and NS3a (H1) domain. Exchange of the GNCR domain with ANR results in grazoprevir-repressible gene expression (NOT2).

[0404] (ii) For StaPLd-based systems, grazoprevir inhibits autolysis of the synthetic mammalian transactivator PcaV-StaPLd-VP64 or the transsilencer PcaV-StaPLd-KRAB, resulting in trigger-inducible activation (BUF3) or repression (NOT3) of gene expression from synthetic PcaV-specific promoters.

[0405] In one embodiment, the present invention provides four types of tri-state buffers based on a combination of grazoprevir-controlled (BUF and NOT) and vanillic acid-controlled gene switches (active high and active low): an active high buffer (IF1 controls BUF: BUFIF1), an active high inverting buffer (IF1 controls NOT: NOTIF1), an active low buffer (IF0 controls BUF: BUFIF0), and an active low inverting buffer (IF0 controls NOT: NOTIF0). In a biological setting, BUFIF1 exhibits logical similarity to a traditional AND gate, while NOTIF0 is logically similar to a traditional NOR gate. Similarly, NOTIF1 and BUFIF0 exhibit typical gene expression signatures of two variants of the NIMPLY (AND NOT) gate, such as Figure 27 As shown in B.

[0406] In one embodiment, the present invention provides a half-adder and half-subtractor gene circuit. The half-adder returns a digital sum S (representing 2) by binary addition of two inputs A and B. 0 bit) and carry Y (represents 2 1 Similarly, the half subtractor performs binary subtraction of B from A using two different output signals in order to borrow W (representing -1×2 1 digits) and the difference D (representing 2 0 In one embodiment, a half adder is generated by adding grazoprevir and vanillic acid regulated BUF1IF0, NOT1IF1 and BUF2IF1 tri-state buffers, e.g., Figure 30 As shown in A. In another embodiment, a half subtractor (e.g., as shown in FIG. 1 ) is assembled by three tri-state buffers BUF1IF0, NOT1IF1, and NOT2IF1. Figure 30 B).

[0407] In other embodiments, the present invention may provide different biological computing modules, for example, as listed in the following table:

[0408]

[0409]

[0410]

[0411]

[0412] "Related component (SEQ ID NO)" means that the component comprises or consists of a SEQ ID NO, or comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a SEQ ID NO. The different components of each module are shown in bold letters.

[0413] In each module, the target protein (the mRNA construct contains its mRNA encoding the target protein), such as SEAP or NanoLuc, EGFP or NanoLuc-P2A-mCherry, can be replaced by other target proteins, such as those shown in the present invention.

[0414] (VI-III) for diagnosis

[0415] In other aspects, the present invention relates to the gene control system of the present invention, which is used as a diagnostic kit, for example, for in vitro medical diagnosis or real-time monitoring of cellular processes in vivo. In one embodiment, the present invention relates to real-time monitoring of medical diagnosis or cellular processes, including diagnosing or monitoring with the gene control system. In another embodiment, the present invention relates to the use of the gene control system in the preparation of a diagnostic kit, for example, for medical diagnosis or real-time monitoring of cellular processes. In a specific embodiment, the detection can be performed in vitro or in vivo.

[0416] In some embodiments, the system can be used to detect disease metabolites, virulence factors, or environmental pollutants in vitro or in vivo.

[0417] The STIF-based translational control strategies and systems of the present invention can also be repurposed to engineer intracellular protein sensors. For example, when each split-STIF component is fused to a different member of a protein heterotrimeric system, STIF-dependent gene expression from poly(A)-deficient mRNA will be strictly dependent on the presence of the remaining members of the intact protein complex, such as Figure 34 shown.

[0418] In one embodiment, STIF comprises or consists of two recombinant fusion proteins A and B, wherein protein A can be RBP-Y or Y-RBP, and protein B can be Y'-eIFBP or eIFBP-Y', or wherein protein A can be eIFBP-Y or Y-eIFBP, and protein B can be Y'-RBP or RBP-Y', wherein Y and Y' can interact with each other through another protein Y". That is, Y and Y' can associate with each other only when Y" is present, because the target protein Y" triggers the Y:Y':Y"-interaction.

[0419] Specifically, Y and Y' in the system can be fused to the N-terminus or C-terminus of the RBP or eIFBP domain of the STIF regulatory factor, thereby allowing Y" to initiate translation initiation after triggering the circularization conformation of the mRNA containing the RBP-specific poly(A) surrogate, e.g. Figure 34 As shown in . To detect the specific target Y", a pair of proteins Y and Y', each binding to a different epitope of Y", can be fused to the N-terminus or C-terminus of the RBP or eIFBP domain of the STIF regulatory factor, such that the formation of the circularized mRNA conformation and translation initiation are completely dependent on the presence of Y" in the cell. Alternatively, the RBP-specific 5'-cap and / or poly(A) surrogate can be replaced by an adaptor motif that directly binds to Y" to form a circularized mRNA conformation in combination with Y or Y' fused to eIFBPs.

[0420] In some embodiments, Y" can be any disease characteristic, such as a disease metabolite, a virulence factor, or any other substance that requires detection, such as an environmental pollutant.

[0421] In one embodiment, the STIF-based system of the present invention can be used as a sensor for real-time detection and manipulation of cells carrying gene fusions in vivo.

[0422] In one embodiment, the invention provides methods for detecting any pathogen, such as hepatitis C virus (HCV), in a cell or in a cell-free environment or in a subject using the system of the invention (eg, by detecting virulence factors specific to the virus).

[0423] In one embodiment, Y or Y' is any protein or fragment that can bind to Y".

[0424] In some embodiments, Y" can be any protein or agent as long as it can be bound by two different proteins, preferably at different domains or different epitopes of Y". In some specific embodiments, protein Y" is selected from disease-specific cellular features, such as oncoproteins, such as fusion gene products or protein complexes specifically expressed in tumor cells or tumor tissues; virulence factors, such as HCV or HCV-specific proteins (such as NS3 protein); or any other substances that need to be detected, such as environmental pollutants.

[0425] Specifically, the system includes

[0426] (i) a synthetic translation initiation factor (STIF) and an mRNA construct comprising an mRNA encoding a target protein; or

[0427] (ii) a nucleic acid encoding STIF and a nucleic acid encoding an mRNA construct comprising an mRNA encoding a target protein;

[0428] The STIF comprises or consists of two recombinant fusion proteins A and B, wherein protein A can be RBP-Y or Y-RBP, and protein B can be Y'-eIFBP or eIFBP-Y', or wherein protein A can be eIFBP-Y or Y-eIFBP, and protein B can be Y'-RBP or RBP-Y', wherein Y and Y' can interact with each other through another protein Y". That is, Y and Y' can only associate with each other when Y" is present because the target protein Y" triggers a Y:Y':Y" interaction, wherein Y" is selected from a disease-specific cellular feature, such as a fusion gene product, an oncoprotein, a virulence factor, an RNA-binding protein or any other substance to be detected, such as an environmental pollutant or any other intracellular or secreted protein containing one or more domains, and wherein the target protein contains at least a reporter protein, such as a fluorescent protein, such as EGFP or mCherry or any secreted or intracellular protein whose expression can be detected, such as SEAP or luciferase.

[0429] In one embodiment, the RBP is L7Ae or MCP.

[0430] In some embodiments, protein A or B may comprise multiple tandem repeats of Y or Y', such as 1-5 repeats, such as 1, 2, 3, 4 or 5 repeats. In some embodiments, Y and Y' are two different Nanobodies, which specifically bind to protein Y", e.g., at different domains or at different epitopes of Y". In some embodiments, protein Y and protein Y' are two different Affibodies, which specifically bind to protein Y", e.g., at different domains or at different epitopes of Y". In some embodiments, protein Y and protein Y' are two different monobodies, which specifically bind to protein Y", e.g., at different domains or at different epitopes of Y". In some embodiments, protein Y and protein Y' are two different DARPins, which specifically bind to protein Y", e.g., at different domains or at different epitopes of Y".

[0431] In one embodiment, Y or Y' is an antibody or antigen-binding fragment (eg, scFv) that specifically binds NS3 as protein Y", eg, scF35 or scFv162.

[0432] In specific embodiments, protein A comprises or consists of the amino acid sequence of SEQ ID NO: 147, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 47. In specific embodiments, protein B comprises or consists of the amino acid sequence of SEQ ID NO: 70, 261 or 262, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 70, 261 or 262.

[0433] In another embodiment, the present invention provides a method for diagnosing chronic myeloid leukemia (CML) by the system of the present invention (especially in the early stage), comprising detecting BCR-ABL, which is a representative cytosolic biomarker of chronic myeloid leukemia (CML).

[0434] Specifically, the system includes

[0435] (i) a synthetic translation initiation factor (STIF) and an mRNA construct comprising an mRNA encoding a target protein; or

[0436] (ii) a nucleic acid encoding STIF and a nucleic acid encoding an mRNA construct comprising an mRNA encoding a target protein;

[0437] The STIF comprises or consists of two recombinant fusion proteins A and B, wherein protein A can be RBP-Y or Y-RBP, and protein B can be Y'-eIFBP or eIFBP-Y', or wherein protein A can be eIFBP-Y or Y-eIFBP, and protein B can be Y'-RBP or RBP-Y', wherein Y and Y' can interact with each other through another protein Y". That is, Y and Y' can only associate with each other when Y" is present because the target protein Y" triggers a Y:Y':Y"-interaction, wherein Y" is selected from disease-specific cellular features, such as fusion gene products, oncoproteins, virulence factors, RNA-binding proteins or any other substance to be detected, such as environmental pollutants or any other intracellular or secreted protein containing one or more domains, wherein the target protein contains at least a reporter protein, such as a fluorescent protein, such as EGFP or mCherry or any secreted or intracellular protein whose expression can be detected, such as SEAP or luciferase.

[0438] In one embodiment, the RBP is L7Ae or MCP.

[0439] In some embodiments, protein A or B may comprise multiple tandem repeats of Y or Y', such as 1-5 repeats, such as 1, 2, 3, 4 or 5 repeats. In some embodiments, Y and Y' are two different Nanobodies, which specifically bind to protein Y", e.g., at different domains or at different epitopes of Y". In some embodiments, protein Y and protein Y' are two different Affibodies, which specifically bind to protein Y", e.g., at different domains or at different epitopes of Y". In some embodiments, protein Y and protein Y' are two different monobodies, which specifically bind to protein Y", e.g., at different domains or at different epitopes of Y". In some embodiments, protein Y and protein Y' are two different DARPins, which specifically bind to protein Y", e.g., at different domains or at different epitopes of Y".

[0440] In one embodiment, Y or Y' is a protein that specifically binds to BCR or ABL, respectively. Thus, proteins A and B can specifically bind to the fusion gene product BCR-ABL as protein Y". For example, a protein that specifically binds to ABL is an ABL-specific intracellular antibody, such as ABl (iDab), which comprises the amino acids of SEQ ID NO: 223, or comprises, or consists of, an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 223. For example, a protein that specifically binds to BCR is a BCR-specific coiled-coil domain, such as CCmut3, which comprises the amino acids of SEQ ID NO: 222, or comprises, or consists of, an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 222.

[0441] In specific embodiments, protein A comprises, or consists of, the amino acid sequence of SEQ ID NO: 181 or 202, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 181 or 202. In specific embodiments, protein B comprises, or consists of, the amino acid sequence of SEQ ID NO: 201 or 258, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 201 or 258.

[0442] In one embodiment, the present invention relates to a kit comprising the system of the present invention, which can be used for diagnosis or detection of proteins, for example, proteins that bind to Y and Y' of STIF.

[0443] In certain embodiments, the system can be applied to non-biological materials such as paper discs for in vitro detection or diagnosis.

[0444] In other embodiments, the invention relates to methods of treating a disease of the invention, comprising first diagnosing the disease in vivo (e.g., by a system of the invention), and then treating the disease by a system of the invention, such as those described herein.

[0445] The present invention is further illustrated by the following examples. However, it should be understood that these examples are described by way of illustration and not limitation, and that various modifications may be made by those skilled in the art.

[0446] All embodiments of any aspect of the invention may be used in combination unless the context clearly indicates otherwise. BRIEF DESCRIPTION OF THE DRAWINGS

[0447] Figure 1 A workflow for synthetic translation initiation factor (STIF)-dependent gene expression control. Translation of a reporter mRNA with a genetically modified 3'-UTR containing a RBP-specific adaptor region and a cleavage site for preprogrammed poly(A) removal mediated by either shRNA or hammerhead ribozyme (HHR) is dependent on the presence of STIF, which is composed of different RBPs fused to different eIF4F binding proteins (eIFBPs). Because STIF is designed to mimic the action of natural poly(A) binding proteins (PABPs), simultaneously binding the target mRNA and a member of the eIF4F complex (i.e., eIF4G, eIF4E, eIF4A, eIF4B, etc.) to form a "closed-loop" mRNA configuration and activate translation, poly(A) removal is important to avoid putative crosstalk with endogenous PABP-driven processes.

[0448] Figure 2Design and validation of different STIF constructs consisting of RNA-binding proteins (RBPs) fused to different eIF4F binding moieties (eIFBPs). (A) Synthetic translation initiation factor (STIF)-mediated translation of L7Ae-specific SEAP mRNA with a poly(A) cleavable by shRNA-216. HEK-293 cells were co-transfected with a SEAP expression vector (pSL31) containing eight tandem repeats of the L7Ae-specific C / D-box, an shRNA-216 expression vector (pSL4), and expression vectors for different STIF variants (PABP-L7Ae, pLZ16; eIF4G-L7Ae, pWH127; L7Ae-eIF4E, pDJ55; L7Ae-NSP3, pLZ27; L7Ae-VPg, pLZ248) or an expression vector for the L7Ae-Coh2 protein, which is incapable of translation initiation (pSL44, negative control). SEAP levels in culture supernatants were quantified 48 hours after transfection. Data are shown as mean ± SD, n = 4 independent experiments. (B) STIF-mediated translation of MCP-specific SEAP mRNA carrying a poly(A)-cleavable shRNA-216. HEK-293 cells were co-transfected with a SEAP expression vector (pSL1331) containing eight tandem repeats of the MCP-specific MS2 box, an shRNA-216 expression vector (pSL4), and expression vectors for different STIF variants (PABP-MCP, pSL1315; eIF4G-MCP, pSL154; MCP-eIF4E, pSL1316; MCP-NSP3, pSL95; MCP-VPg, pLYL47), or an expression vector for the MCP-Coh2 protein, which is incapable of translation initiation (pSL674, negative control). SEAP levels in culture supernatants were quantified 48 hours after transfection. Data are mean ± SD, n = 4 independent experiments. (C) Correlation between the size of the C / D-box-based aptamer domain and STIF-mediated SEAP expression. SEAP expression vectors containing different tandem repeats of the L7Ae-specific C / D-box aptamer ((C / D-box)8, pSL31; (C / D-box) 12 , pSL81;(C / D-box) 16 , pSL80; (C / D-box) 24HEK-293 cells were co-transfected with an expression vector for either (a) shRNA-216 (pSL4) or a vector expressing either the PABP-L7Ae protein (ON; pLZ16) or the Coh2-L7Ae protein that cannot bind to eIF4F (OFF; pSL44). SEAP expression in the culture supernatant was recorded 48 hours after transfection. Data presented are mean ± SD, n = 4 independent experiments. (D) Correlation between MS2-box-based aptamer domain size and STIF-mediated SEAP expression. SEAP expression vectors containing different tandem repeats of the MCP-specific MS2-box aptamer ((MS2-box)8, pSL515; (MS2-box) 12 , pSL1284;(MS2-box) 16 , pSL516; (MS2-box) 24 HEK-293 cells were transfected with a constitutive expression vector for either MCP-NSP3 (ON; pSL95) or MCP-Coh2 protein that is unable to bind eIF4F (OFF; pSL674). SEAP expression was recorded in the culture supernatant 48 hours after transfection. Data presented are mean ± SD, n = 4 independent experiments.

[0449] Figure 3Characterization of the stability and nonspecific binding of STIF-specific target mRNAs. (A) Quantification of PABP binding to poly(A)-containing RNA by RIP-qPCR. HEK-293 cells were transfected with a constitutive expression vector for 3xFLAG-tagged PABP-L7Ae (pSL763), reflecting the RNA binding capacity of endogenous PABP. 24 hours later, 10 μg of in vitro transcribed EGFP mRNA with (+) or without (-) a poly(A) tail was added. 3 hours after transfection, RNA was extracted and immunoprecipitated using an anti-Flag affinity gel. Data show the results of qRT-PCR analysis as the ratio (%) of EGFP mRNA in samples before (input) and after immunoprecipitation (IP) (±SD, n = 3). (B) Effect of engineered L7Ae-specific poly(A) replacements on mRNA stability. In vitro-transcribed SEAP mRNA containing poly(A) (from pSL1091), no poly(A) (from pSL517), or a poly(A) surrogate consisting of 8 (from pSL31) or 24 (from pSL355) tandem repeats of the C / D-box in the 3'-UTR was transfected into HEK-293 cells expressing Coh2-L7Ae (24 hours after transfection with pSL44) or not (24 hours after transfection with pcDNA3.1(+)). SEAP mRNA levels were analyzed by qRT-PCR 4 (*arbitrarily set to 100%), 8, 12, and 24 hours after mRNA transfection. Data presented are mean ± SD, n = 4 independent experiments. (C) Effect of engineered MCP-specific poly(A) surrogate on mRNA stability. (Left) In vitro-transcribed SEAP mRNA containing poly(A) (from pSL1091), no poly(A) (from pSL517), or a poly(A) replacement consisting of 8 (from pSL515) or 24 (from pSL468) tandem repeats of the MS2-box in the 3'-UTR was transfected into HEK-293 cells expressing MCP-Coh2 (24 hours after transfection with pSL674) or not expressing MCP-Coh2 (24 hours after transfection with pcDNA3.1(+)). SEAP mRNA levels were analyzed by RT-PCR 4 (*arbitrarily set to 100%), 8, 12, and 24 hours after mRNA transfection. Data presented are mean ± SD, n = 4 independent experiments. (Right) HEK-293 cells were co-transfected with a SEAP expression vector containing 24 MS2-box repeats in the 3′-UTR (reporter; pSL468) and a MCP-Coh2 protein incapable of translation initiation (pSL674).SEAP levels in culture supernatants and relative mRNA expression levels in cells were quantified 48 hours after transfection. SEAP transcript levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression by setting the undetermined value to the maximum Ct of 40 cycles. Data provided are mean ± SD, n = 3. (D) mRNA stabilization by ABP / aptamer binding. HEK-293 cells were co-transfected with a SEAP expression vector (reporter; pSL88) along with shRNA-216 (pSL4) and a constitutive expression vector for the Coh2-L7Ae protein (pSL44) that is incapable of translation initiation. SEAP levels in culture supernatants (left) and mRNA expression levels in cells (right) were quantified 48 hours after transfection. SEAP transcript levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression by setting the undetermined value to the maximum Ct of 40 cycles. Data provided are mean ± SD, n = 3.

[0450] Figure 4 . Pre-programmed poly (A) cleavage cis and trans removal strategy. (A) Verification of mRNA knockdown efficiency of shRNA-216. shRNA-216 repressive SEAP-mRNA expression vector (pSL31) and different amounts of P hU6 HEK-293 cells were co-transfected with a shRNA-216 expression vector (pSL4) driven by HHR. SEAP expression in culture supernatants was recorded 48 hours after transfection. Data presented are mean ± SD, n = 4 independent experiments. (B) Quantification of HHR-mediated mRNA autocleavage. HEK-293 cells were co-transfected with SEAP expression vectors containing zero (pSL517), one (pSLM97), two (pSL767), or four (pSL768) tandem repeats of the HHR. SEAP levels were then recorded in culture supernatants 48 hours after transfection. Data presented are mean ± SD, n = 4 independent experiments.

[0451] Figure 5Translational control of SEAP, firefly luciferase, and EGFP mRNAs using a self-cleavable poly(A) motif. (A) STIF-mediated translation of L7Ae-specific SEAP mRNA containing an HHR-cleavable poly(A) motif. HEK-293 cells were co-transfected with a SEAP expression vector (pSL355) containing 24 tandem repeats of the L7Ae-specific C / D-box, along with expression vectors for different STIF variants (PABP-L7Ae, pLZ16; eIF4G-L7Ae, pWH127; L7Ae-eIF4E, pDJ55; L7Ae-NSP3, pLZ27; L7Ae-VPg, pLZ248), or a vector expressing the L7Ae-Coh2 protein (pSL44, a negative control) that is incapable of translation initiation. SEAP levels in culture supernatants were quantified 48 hours after transfection. Data are shown as mean ± SD, n = 4 independent experiments. (B) STIF-mediated translation of MCP-specific SEAP mRNA carrying an HHR-cleavable poly(A) motif. HEK-293 cells were co-transfected with a SEAP expression vector (pSL468) containing 24 tandem repeats of the MCP-specific MS2 box, along with expression vectors for different STIF variants (PABP-MCP, pSL1315; eIF4G-MCP, pSL154; MCP-eIF4E, pSL1316; MCP-NSP3, pSL95; MCP-VPg, pLYL47), or a vector expressing the MCP-Coh2 protein, which is incapable of translation initiation (pSL674, negative control). SEAP levels in culture supernatants were quantified 48 hours after transfection. Data are shown as mean ± SD, n = 4 independent experiments. (C, D) STIF-mediated regulation of gene expression at the protein level. HEK-293 cells were co-transfected with an expression vector (pSL781) for FLAG-tagged firefly luciferase (FLuc) containing an MCP-specific poly(A) replacement in the 3′-UTR, along with a constitutive expression vector for MCP-Coh2 (pSL674) or MCP-NSP3 (pSL95). Luciferase activity (C) and absolute FLuc protein levels (D) were measured by Western blotting 48 hours after transfection. Data in (C) are shown as mean ± SD, n = 4 independent experiments. (E, F) STIF-mediated translational regulation of EGFP mRNA. HEK-293 cells were co-transfected with an expression vector (pSL1308) for EGFP mRNA containing 24 tandem repeats of the MS2 box in the 3′-UTR, along with a constitutive expression vector for MCP-Coh2 (pSL674) or MCP-NSP3 (pSL95). 48 hours after transfection, EGFP expression was recorded by (E) fluorescence microscopy (scale bar: 10 μm) or (F) flow cytometry.Data are mean ± SD, n = 3.

[0452] Figure 6 . Characterization of endogenous RNA binding and eIF4F association of overexpressed STIF. (A) STIF associates with the endogenous eIF4F complex. HEK-293 cells were transfected with expression vectors for 3xFLAG-tagged MCP (-, pSL1084) or MCP-NSP3 (+, pSL1083), and one lysate fraction was immunoprecipitated 48 hours after transfection. Target proteins were detected in lysate fractions before (input) and after (Flag-IP) immunoprecipitation using anti-FLAG, anti-eIF4G, and anti-eIF4E antibodies. The numbers on the left axis of the Western blot represent the molecular weight (MW) of the target protein. (B) Endogenous RNA binding capacity of PABP- and NSP3-fusion proteins was quantified by RIP-qPCR. 48 hours after transfection of HEK-293 cells with expression constructs for 3xFLAG-tagged L7Ae-NSP3 (pSL762) or PABP-L7Ae (pSL763), RNA was extracted and immunoprecipitated using an anti-Flag affinity gel. Data show qRT-PCR analysis of the ratio (%) of GAPDH RNA in samples before (input) and after immunoprecipitation (IP), as a measure of the amount of RNA bound to constructs containing PABP and NSP3 (±SD, n=3). Bars represent mean and SD, and solid circles indicate individual results. ***, p<0.001.

[0453] Figure 7 Translational regulation through protein-protein interaction (PPI)-mediated STIF remodeling. The bipartite STIF system, with the NSP3 and RBP domains split into two independent proteins, allows for the flexible incorporation of different PPI systems to regulate conditional STIF assembly and translation of RBP-specific mRNAs.

[0454] Figure 8Translational regulation by spontaneous assembly of bipartite STIFs. (A) Translational regulation by constitutive STIF remodeling. HEK-293 cells were co-transfected with a plasmid encoding SEAP mRNA containing an L7Ae-specific poly(A) replacement (pSL355) and constitutive expression vectors for different combinations of L7Ae- and NSP3-fusion proteins (L7Ae-NS3a(H1) & ANR4-NSP3: pSL703 / pSL549; L7Ae-NS3a & ANR4-NSP3: pYF5 / pSL549; L7Ae-LD1 & Bcl-XL-NSP3: pSL667 / pSL615; L7Ae-LD3 & Bcl-XL-NSP3: pSL661 / pSL615; L7Ae-Coh2 & DocS-NSP3: pSL65 / pSL66). Transfection with pcDNA3.1(+) instead of the NSP3-fusion protein served as a negative control. SEAP levels in culture supernatants were quantified 48 hours after transfection. Data are shown as mean ± SD, n = 4 independent experiments. (B) Compatibility of STIF-mediated translational regulation with the split complementation approach. (Left) HEK-293 cells were cotransfected with a SEAP expression vector (pSL468) containing 24 tandem repeats of the MCP-specific aptamer, along with various combinations of constitutive expression vectors for MCP-NSP3 (pSL674) and (DocS)3-NSP3 (pSL86). (Right) HEK-293 cells were cotransfected with a SEAP expression vector (pMX116) containing a bacteriophage N-peptide (λ-N) repressor HHR upstream of a poly(A) residue, along with constitutive expression vectors for λN-Coh2 (pSL334) and DocS-mCherry (pSL1099). For the (-) condition, pcDNA3.1(+) was used instead of the expression vector for transfection. SEAP levels in the culture supernatant were recorded 48 hours after transfection. Data are shown as mean ± SD, n = 4 independent experiments.

[0455] Figure 9Translational regulation through constitutive Coh2 / DocS-mediated STIF remodeling. (A) Site-specific translational activation by eIFBP overexpression. HEK-293 cells were co-transfected with expression vectors for SEAP-mRNA containing 24 tandem repeats of the C / D-box (left panel, pSL355) or tandem repeats of the MS2-box (right panel, pSL468) in the 3′-UTR, constitutive expression vectors for L7Ae (left) or MCP (right) fused to Coh2 (L7Ae-Coh2, pSL83; MCP-Coh2, pSL674) or EGFP (L7Ae-EGFP, pSL1078; MCP-EGFP, pSL435), and constitutive expression vectors for various chimeric eIFPBP fusions containing the Coh2-specific DocS (PABP-DocS, pSL47; DocS-eIF4G, pSL87; DocS-eIF4E, pLZ312; DocS-NSP3, pSL66; DocS-VPg, pLZ311). cDNA3.1(+) was transfected instead of the vector expressing DocS or MCP as a negative control. SEAP levels in culture supernatants were quantified 48 hours after transfection. Data are shown as mean ± SD, n = 4 independent experiments. (B, C) Native 5′-cap-independent translation through site-specific recruitment of eIFBPs to the (B) 5′-UTR or (C) intergenic regions of target gene mRNAs. (B) HEK-293 cells were transfected with an expression vector (pQZ8) containing four tandem repeats of the C / D-box in the 5′-UTR, a constitutive L7Ae-(Coh2)3 expression vector (pSL83), and constitutive expression vectors for various DocS-based fusion constructs (PABP-DocS, pSL47; DocS-eIF4G, pSL87; DocS-eIF4E, pLZ312; DocS-NSP3, pSL66; DocS-VPg, pLZ311). pcDNA3.1(+) was transfected instead of the DocS-expressing vector as a negative control. SEAP levels in culture supernatants were quantified 48 hours after transfection. Data are shown as mean ± SD, n = 4 independent experiments. (C) HEK-293 cells were transfected with a reporter mRNA (pPW21) containing 24 tandem repeats of the C / D-box positioned downstream of SEAP and upstream of the NanoLuc coding region; a constitutive L7Ae-(Coh2)3 expression vector (pSL83); and constitutive expression vectors for various DocS-based fusion constructs (PABP-DocS, pSL47; DocS-eIF4G, pSL87; DocS-eIF4E, pLZ312; DocS-NSP3, pSL66; and DocS-VPg, pLZ311).pcDNA3.1(+) was used as a negative control instead of the DocS-expressing vector. SEAP (left) and NanoLuc (right) levels in the culture supernatant were quantified 48 hours after transfection. Data are shown as mean ± SD, n = 4 independent experiments.

[0456] Figure 10 Engineering a trigger-inducible gene switch using bipartite STIF. (A) Reconstructing translational regulation of L7Ae-specific mRNAs by trigger-inducible STIF. For danoprevir-inducible SEAP translation, HEK-293 cells were co-transfected with pSL355 and constitutive expression vectors for L7Ae-(NS3a)3 (pLZ76) and (DNCR)3-NSP3 (pLZ72). For abscisic acid-inducible SEAP translation, HEK-293 cells were co-transfected with pSL355 and constitutive expression vectors for L7Ae-(ABI)3 (pPW3) and (PYL1)3-NSP3 (pPW4). For gibberellic acid-inducible SEAP translation, HEK-293 cells were co-transfected with pSL355 and constitutive expression vectors for GAI-L7Ae (pPW14) and NSP3-GID1 (pPW17). For grazoprevir-inducible SEAP translation, HEK-293 cells were co-transfected with pSL355 and constitutive expression vectors for L7Ae-(NS3a)3 (pLZ76) and (GNCR)3-NSP3 (pLZ74). For rapamycin-inducible SEAP translation, HEK-293 cells were co-transfected with pSL355 and constitutive expression vectors for FKBP-L7Ae (pMX331) and FRB-NSP3 (pLZ42). For blue light-inducible SEAP translation, HEK-293 cells were co-transfected with pSL355 and constitutive expression vectors for L7Ae-CIB1 (pLZ55) and Cry2-NSP3 (pLZ68). The cells were stained with cytokines 48 h after addition of the corresponding inducers (danoprevir, 1 μM; abscisic acid, 100 μM; gibberellic acid, 100 μM; grazoprevir, 0.5 μM; rapamycin, 0.01 μM) or after exposure to blue light (450 nm; ON, 5 mW / cm 2 , 30s; OFF, 30s) or red light (660nm, constant 1W / m 2) 24 hours after inoculation, SEAP levels in the culture supernatant were recorded. Data are shown as mean ± SD, n = 3. (B) Regulation of MCP-specific mRNA by triggering inducible STIF remodeling. For danoprevir-inducible SEAP translation, HEK-293 cells were co-transfected with a SEAP expression vector (pSL468) containing a poly(A) replacement with 24 tandem repeats of the MCP-specific MS2 box, along with constitutive expression vectors for MCP-NS3a (pSL497) and DNCR-NSP3 (pLZ72). For abscisic acid-inducible SEAP translation, HEK-293 cells were co-transfected with pSL468 and constitutive expression vectors for ABI-MCP (pPW22) and (PYL1)3-NSP3 (pPW4). For gibberellic acid-inducible SEAP translation, HEK-293 cells were co-transfected with pSL468 and constitutive expression vectors for MCP-GID (pPW23) and GAI-NSP3 (pPW2). For grazoprevir-inducible SEAP translation, HEK-293 cells were co-transfected with pSL468 and constitutive expression vectors for MCP-NS3a (pSL497) and GNCR-NSP3 (pYF3). For rapamycin-inducible SEAP translation, HEK-293 cells were co-transfected with a SEAP expression vector (pSL516) containing 16 MCP-specific MS2-box tandem repeats of poly(A) substitutions, as well as constitutive expression vectors for MCP-FRB (pSL1097) and FKBP-NSP3 (pSL1098). For blue light-inducible SEAP translation, HEK-293 cells were co-transfected with pSL516 and constitutive expression vectors for MCP-CIB1 (pSL1096) and Cry2-NSP3 (pSL71). For red light-inducible SEAP translation, pSL516 and MCP-(Aff6 V18FΔN HEK-293 cells were co-transfected with constitutive expression vectors of )4 (pSL917) and DrBPhP-NSP3 (pSL901). The cells were activated 48 h after addition of the corresponding inducers (danoprevir, 0.5 μM; abscisic acid, 100 μM; gibberellic acid, 100 μM; grazoprevir, 0.5 μM; rapamycin analog, 0.1 μM) or after exposure to blue light (450 nm; ON, 5 mW / cm 2 , 30s; OFF, 30s) or 48 hours after exposure to red light (660nm, constant 1W / m 2) 24 hours after transfection, the level of SEAP in the culture supernatant was recorded. Data are shown as mean ± SD, n = 4 independent experiments. (C) Light-induced translational regulation by blue-light-activated mCherry nanobody LaM8-AK47. HEK-293 cells were transfected with a SEAP-mRNA expression vector (pSL516) containing 16 tandem repeats of the MS2-box in the 3′-UTR, as well as constitutive expression vectors for MCP-LaM8_AK47 (pSL875) and mCherry-NSP3 (pSL876). Six hours after transfection, the cells were irradiated with blue light (450 nm, 2 mW / cm 2 ) cells were irradiated for 48 hours, and the SEAP level in the culture supernatant was recorded. Data are mean ± SD, n = 4.

[0457] Figure 11 Genetically encoded signaling-specific sensors reconstituted with phosphorylation-dependent STIF. (A) L7Ae-based STIF. HEK-293 cells were co-transfected with a dual reporter vector (pSL274) containing a constitutive FLuc expression unit and a NanoLuc mRNA expression unit containing an L7Ae-specific poly(A) replacement, an shRNA-216 expression vector (pSL4), and various combinations of constitutive expression vectors for L7Ae-(pE59)2 (pSL169) and (ERK2)2-NSP3 (pSL189). Luciferase levels in culture supernatants were quantified 48 hours after the addition of 100 ng / mL recombinant human EGF. For the (-) condition, pcDNA3.1(+) was used in place of the expression vector. Data presented are mean ± SD of relative luciferase activity (NanoLuc / FLuc), n = 3 individual experiments. (B) MCP-based STIF. HEK-293 cells were co-transfected with various combinations of a constitutive FLuc expression vector (pYW99), a NanoLuc-mRNA expression vector containing an MCP-specific poly(A) replacement (pSL683), and constitutive expression vectors for MCP-(pE59)2 (pSL637) and (ERK2)2-NSP3 (pSL189), and then cultured in cell culture medium containing 2% FBS (v / v). Luciferase levels in culture supernatants were quantified 48 hours after the addition of 0 or 100 ng / mL recombinant human EGF. For the (-) condition, pcDNA3.1 (+) was used in place of the expression vector. Data presented are mean ± SD of relative luciferase activity (NanoLuc / FLuc), n = 3 individual experiments.

[0458] Figure 12STIF-mediated translational regulation by engineering 5′-cap surrogates. An RNA cleavage site can also be placed between the guanine-rich 5′-cap and the RBP-specific aptamer region to allow for preprogrammed cap removal, rendering the aptamer region a 5′-cap surrogate for recruitment of the various STIF constructs described herein.

[0459] Figure 13 Regulating therapeutic transgene expression using the FDA-approved drug grazoprevir. In vivo delivery of the genetic components of the GNCR:NS3a-dependent STIF assembly and STIF-specific target mRNA is compatible with various routes of administration for non-integrating gene therapies (e.g., DNA-encoded vectors or formulated mRNA therapeutics), allowing oral uptake of grazoprevir to trigger in situ production of a variety of therapeutic proteins of interest (e.g., insulin).

[0460] Figure 14Optimization of an L7Ae-based grazoprevir-inducible gene switch. (A) Translational regulation by grazoprevir-dependent STIF association. HEK-293 cells were transfected with plasmids encoding SEAP mRNA containing an L7Ae-specific poly(A) surrogate (generated by transfection with pSL88 & pSL4) and various grazoprevir-regulated L7Ae- and NSP3-fusion proteins (L7Ae-GNCR & NS3a-NSP3: pYF6 and pYF1; L7Ae-NS3a & GNCR-NSP3: pYF5 and pYF3). SEAP levels in culture supernatants were recorded 48 hours after the addition of 0.1 μM grazoprevir in DMSO. Data are shown as mean ± SD, n = 4 independent experiments. (B) Translational regulation by various grazoprevir-dependent NSP3-fusion proteins. HEK-293 cells were co-transfected with plasmids encoding SEAP mRNA containing an L7Ae-specific poly(A) surrogate (pSL88 & pSL4), L7Ae-NS3a (pYF5), and various NSP3 fusion proteins containing one (pYF3), two (pLZ73), or three N-terminal GNCR repeats (pLZ74). SEAP levels in culture supernatants were recorded 48 hours after the addition of 0.1 μM grazoprevir in DMSO (vehicle control). Data are shown as mean ± SD, n = 4 independent experiments. (C) Translational regulation by various grazoprevir-dependent L7Ae fusion proteins. HEK-293 cells were co-transfected with plasmids encoding SEAP mRNA (pSL88 & pSL4) containing an L7Ae-specific poly(A) replacement, GNCR-NSP3 (pYF3), and different L7Ae-fusion proteins consisting of one (pYF5), two (pLZ75), or three C-terminal NS3A repeats (pLZ76). SEAP levels in culture supernatants were recorded 48 hours after the addition of 0.1 μM grazoprevir in DMSO (vehicle control). Data are shown as mean ± SD, n = 4 independent experiments.

[0461] Figure 15Optimized L7Ae- and MCP-based grazoprevir-inducible gene switches in mammalian cells. (A) Optimized grazoprevir-inducible regulation of SEAP translation. For the L7Ae-based system, HEK-293 cells were co-transfected with plasmids encoding SEAP mRNA containing a C / D-box-based poly(A) replacement (pSL88 & pSL4), (GNCR)3-NSP3 (pLZ74), and L7Ae-(NS3a)3 (pLZ76). For the MCP-based system, HEK-293 cells were co-transfected with plasmids encoding SEAP mRNA containing an MS2-box-based poly(A) replacement (pSL468), (GNCR)3-NSP3 (pSL1032), and MCP-(NS3a)3 (pSL1042). SEAP levels in culture supernatants were recorded 48 hours after addition of 0.1 μM grazoprevir in DMSO (vehicle control). Data are shown as mean ± SD, n = 4 independent experiments. (B, C) Dose-dependent grazoprevir-induced SEAP expression. (B) HEK-293 cells were co-transfected with plasmids (pSL88 & pSL4) encoding SEAP mRNA containing an L7Ae-specific poly(A) surrogate, along with constitutive expression vectors for (GNCR)3-NSP3 (pLZ74) and L7Ae-(NS3a)3 (pLZ76). SEAP levels in culture supernatants were recorded 24 hours after addition of various concentrations of grazoprevir. Data are shown as mean ± SD, n = 4 independent experiments. (C) HEK-293 cells were co-transfected with plasmids encoding (GNCR)3-NSP3 (pSL1032), MCP-(NS3a)3 (pSL1042), and SEAP mRNA containing an MCP-specific poly(A) replacement (pSL468). SEAP levels in the culture supernatant were recorded 24 hours after the addition of various concentrations of grazoprevir. Data are shown as mean ± SD, n = 4 independent experiments.

[0462] Figure 16Comparative analysis of gene switches based on STIF-based translation and state-of-the-art transcription-centric designs. (A) Kinetics of grazoprevir-inducible SEAP expression. For grazoprevir-inducible translation, HEK-293 cells were co-transfected with plasmids encoding MCP-(NS3a)3 (pSL503) and (GNCR)3-NSP3 (pLZ74), as well as SEAP mRNA containing an MCP-specific poly(A) replacement (pSL468). For grazoprevir-inducible transcription, HEK-293 cells were co-transfected with a TetR-specific SEAP expression vector (pLZ79) and a constitutive expression vector for TetR-NS3a (pLZ88) and (GNCR)3-VP64 (pLZ85). Twenty-four hours after transfection, 0 or 0.5 μM grazoprevir was added to the culture supernatant, and SEAP levels were followed for an additional 24 hours. Data are shown as fold change ± SD calculated by dividing SEAP levels in samples treated with grazoprevir by SEAP levels in samples not treated with grazoprevir (n = 3 separate experiments). (B) Endpoint performance of grazoprevir-inducible translation and transcriptional gene switches. For grazoprevir-inducible translation, HEK-293 cells were co-transfected with a vector encoding SEAP-mRNA containing an MCP-specific poly(A) surrogate and constitutive expression vectors for MCP-(NS3a)3 (pSL503) and (GNCR)3-NSP3 (pLZ74) and then cultured in cell culture medium containing 0 or 0.5 μM grazoprevir. For grazoprevir-inducible transcription, HEK-293 cells were co-transfected with a TetR-specific SEAP expression vector (pLZ79) and constitutive expression vectors for TetR-NS3a (pLZ88) and (GNCR)3-VP64 (pLZ85). SEAP levels in culture supernatants were recorded 48 hours after addition of grazoprevir.Data are shown as fold change ± SD calculated by dividing SEAP levels in grazoprevir-treated samples by SEAP levels in non-grazoprevir-treated samples (n = 4 separate experiments).

[0463] Figure 17. Association of grazoprevir-inducible STIF with the endogenous eIF4F complex. HEK-293 cells were co-transfected with expression vectors for 3xHA-tagged (GNCR)3-NSP3 (pSL476) and FLAG-tagged MCP-NS3a (left, pSL1093) or L7Ae-(NS3a)3 (right, pSL475) and treated with (+) or without (-) grazoprevir before immunoprecipitation. Anti-FLAG, anti-HA, anti-eIF4G, and anti-eIF4E antibodies were used to detect the target protein in each lysate fraction before (input) and after (Flag-IP) immunoprecipitation. The numbers on the right axis of the Western blot represent the molecular weight (MW) of the target protein.

[0464] Figure 18 Characterization of the grazoprevir-inducible gene switch in terms of long-term performance and compatibility with RNA-only delivery. (A) Grazoprevir-inducible SEAP translation via mRNA delivery. The mRNA encoding MCP-(NS3a)3 (from pSL1085), (GNCR)3-NSP3 (from pYW361), and SEAP-(MS2-box) was used. 24 HEK-293 cells were (co)transfected with in vitro transcribed mRNA (from pSL468). SEAP levels in culture supernatants were quantified 48 hours after addition of grazoprevir. Data are shown as mean ± SD, n = 4 independent experiments. (B) Reversibility of grazoprevir-induced protein secretion in mammalian cells. HEK-293 cells were cultured LSCCS1 (Stable expression of MCP-(NS3a)3, (GNCR)3-NSP3, and NanoLuc-mRNA containing 24 tandem repeats of the MS2-box) for 7 days while continuously switching the level of grazoprevir in the culture medium between 0 and 500 nM. NanoLuc levels were measured every 12 hours. The cell density was readjusted to 1×10 every 2-3 days. 5 cells / ml.

[0465] Figure 19Design and optimization of a grazoprevir-inducible gene switch for in vivo therapeutic transgene expression. (A) Production of grazoprevir-inducible SEAP in mice. For grazoprevir-inducible SEAP production mediated by poly(A) surrogates generated by cis-acting mRNA cleavage, the corresponding encoding plasmid DNA consisting of pLZ76 / pLZ74 / pSL355 was hydrodynamically injected into the tail vein of C57BL / 6 mice. For grazoprevir-inducible SEAP production mediated by poly(A) surrogates generated by trans-acting mRNA cleavage, the corresponding encoding plasmid DNA consisting of pLZ76 / pLZ74 / pSL4 / pSL88 was hydrodynamically injected into the tail vein of C57BL / 6 mice. Six hours later, mice received the first intraperitoneal injection of grazoprevir (1 mg / kg in PBS) three times daily. SEAP levels in the mouse bloodstream were measured 24 hours after the first grazoprevir injection. Data are presented as mean ± SEM (n = 5 mice per group). (B) Dose-dependent SEAP production induced by oral administration of grazoprevir in mice. hCMV -L7Ae-(NS3a)3-pA), pLZ74(P hCMV -(GNCR)3-NSP3-pA) and pSL355 (P hCMV -SEAP-(C / D-box) 24 -HHR-pA) was hydrodynamically injected into the tail vein of C57BL / 6 mice. Six hours after injection, mice received the first oral administration of different doses of grazoprevir three times a day. SEAP levels in the bloodstream of mice were measured 24 hours after the first administration of grazoprevir. Data are expressed as mean ± SEM; n = 5 mice / group. (C) Optimized grazoprevir-inducible SEAP production in mice. Plasmid DNA composed of pSL468 / pLZ74 / pSL548 was hydrodynamically injected into the tail vein of C57BL / 6 mice. Six hours later, mice received the first intraperitoneal injection of grazoprevir (3 mg / kg, dissolved in PBS) three times a day. SEAP levels in the bloodstream of mice were measured 24 hours after the first injection of grazoprevir. Data are expressed as mean ± SEM of n = 5 mice per group. (D) In vitro expression of grazoprevir-inducible insulin. 200 ng of pSL1042 (P hCMV -MCP-(NS3a)3-pA), 200ng pSL1032(P hCMV -(GNCR)3-NSP3-pA) and different amounts of pSL1003 (P hCMVHEK-293 cells were co-transfected with a 5-well plate (NanoLuc-P2A-mINS-(MS2-box)16-HHR-pA). NanoLuc and mINS levels in the culture supernatant were recorded 48 hours after the addition of 0.5 μM grazoprevir. Data are mean ± SD, n = 3 independent experiments. The therapeutic efficacy window, represented by physiological blood insulin levels > 0.4 μg / L, is marked with a blue shaded box. ns, not significant; *, p < 0.1; ***, p < 0.001.

[0466] Figure 20 .Therapeutic efficacy and long-term performance of the grazoprevir-inducible gene switch in mice. (AC) Treatment of type 1 diabetes (T1D) by oral grazoprevir-inducible insulin expression. Plasmids encoding MCP-(NS3a)3, (GNCR)3-NSP3 and insulin-mRNA containing an MCP-specific poly(A) surrogate (pSL548 / pLZ74pSL685) were hydrodynamically injected into the tail vein of T1D mice. Six hours after injection, mice were first fed with 3 mg / kg grazoprevir three times a day. 20 hours after the first administration of grazoprevir, (A) blood insulin levels and (B) fasting blood glucose were measured in mice. An intraperitoneal glucose tolerance test (GTT) was performed 24 hours after the first administration of grazoprevir (4 hours after blood insulin quantification) (C). Data are expressed as mean ± SEM; n = 6 mice / group. (D) Long-term control of grazoprevir-inducible SEAP production in mice. C57BL / 6 mice received intravenous injections of 7×10 11 AAV2 / 8 particles were used to constitutively express (GNCR)3-NSP3, MCP-(NS3a)3, and SEAP mRNA containing an MCP-specific poly(A) surrogate. SEAP production in the bloodstream was then monitored over 10 weeks. Mice were first dosed with 3 mg / kg grazoprevir three times daily 24 hours prior to each measurement. Data are presented as mean ± SEM; n = 4 mice / group.

[0467] Figure 21.Molecular structure and design principles of a genetic tri-state buffer. (A) Principle of a tri-state buffer. In electronics and biology, a tri-state buffer comprises an upstream switch (regulated by a control input B) that directly controls a downstream switch (regulated by a data input A) in its ON / OFF state. Thus, control input B allows data input A to determine the overall activity Y of the circuit unless B is inactivated or "unplugged" by the upstream switch. In this inactive state (NOT B), the overall activity of the circuit will fall into a third "high impedance" state Z, which no longer depends on the state of A (0 or 1 value). (B) Example of a genetic tri-state buffer. In mammalian cells, a genetic tri-state buffer can be generated by regulated expression of a trigger-inducible genetic switch, which is programmed with "buffer" (BUF) and "invert" (NOT) signal processing logic. In this embodiment illustrating a transcription-translation based genetic circuit, control input B operating at the upstream layer (e.g., transcription) monitors the expression of a synthetic translation initiation factor (STIF), which regulates target protein expression from the downstream layer (e.g., translation). The tristate buffer can be engineered to contain up to four types of gene switches: B activates STIF expression (IF1), B terminates STIF expression (IF0), A activates target protein expression (BUF), and A terminates target protein expression (NOT). STIF binds to the preinitiation complex eIF4F using the rotavirus-derived nonstructural protein 3 domain (NSP3), leading to the translation of MCP-specific mRNAs following the inducible association of NSP3 with MCP-containing factors.

[0468] Figure 22Design and validation of BUF and NOT switches controlled by grazoprevir. (A) Comparative analysis of different MCP-NS3a variants for grazoprevir-inducible SEAP translation. HEK-293 cells were co-transfected with plasmids encoding MCP-specific SEAP mRNA (pSL468), (GNCR)3-NSP3 (pLZ74), and fusion proteins of MCP with NS3a (pSL497) or NS3a(H1) (pSL546). SEAP levels in culture supernatants were recorded 48 hours after addition of 0.5 μM grazoprevir in DMSO (vehicle control). Data are mean ± SD, n = 4. (B) Comparative analysis of different MCP-NS3a variants for grazoprevir-repressible SEAP translation. HEK-293 cells were co-transfected with plasmids encoding MCP-specific SEAP mRNA (pSL468), (ANR)4-NSP3 (pSL549), and fusion proteins of MCP with NS3a (pSL497) or NS3a(H1) (pSL546). SEAP levels in culture supernatants were recorded 48 hours after the addition of 0.5 μM grazoprevir dissolved in DMSO (vehicle control). Data are mean ± SD, n = 4.

[0469] Figure 23Optimizing grazoprevir-regulated BUF- and NOT-switches. (A) Grazoprevir-dependent translation in mammalian cells. To initiate translation, a circularized mRNA conformation must be established in which the 5′- and 3′-ends are brought into close proximity by a synthetic translation initiation factor (STIF) comprising an NSP3 domain and a synthetic tether (MCP-NS3a(H1)) consisting of an RNA-binding MCP domain and a STIF-binding NS3a(H1) domain. Since grazoprevir simultaneously triggers the dissociation of the ANR from NS3a (H1) and the binding of the GNCR to NS3a(H1), STIF was engineered to contain both the GNCR and NSP3, enabling grazoprevir-inducible translation (BUF switch). Similarly, fusion of the ANR to NSP3 results in grazoprevir-repressible translation (NOT switch). (B) BUF switch by grazoprevir-inducible translation. HEK-293 cells were co-transfected with plasmids encoding MCP-specific SEAP mRNA (pSL468), (GNCR)3-NSP3 (pLZ74), and different MCP fusion proteins consisting of one (pSL546), two (pSL547), or three C-terminal NS3a(H1) repeats (pSL548). SEAP levels in culture supernatants were recorded 48 h after addition of 0.5 μM grazoprevir in DMSO (vehicle control). (C) NOT inverters repressed by grazoprevir. HEK-293 cells were co-transfected with plasmids encoding MCP-specific SEAP mRNA (pSL468), (ANR)4-NSP3 (pSL549), and different MCP fusion proteins consisting of one (pSL546), two (pSL547), or three C-terminal NS3a(H1) repeats (pSL548). SEAP levels in culture supernatants were recorded 48 h after addition of 0.5 μM grazoprevir in DMSO (vehicle control).

[0470] Figure 24Design and validation of vanillic acid-controlled IF0 and IF1 switches. (A) Upstream gene switches in a biological three-state circuit. In the three-state circuit, the BUF and NOT switches are controlled by active-high (IF1) or active-low (IF0) control signals, respectively, that produce "normal" or "inverted" outputs. The biological implementations of the active-high and active-low switches should be orthogonal to each other to enable non-interference operation in mammalian cells. An example of a set of mutually orthogonal gene switches that respond to the same control input is the vanillic acid-inducible MOR9-1 system, which triggers cAMP-dependent transcription in combination with the vanillic acid-repressible mammalian transactivator VanR-VP64, driving gene expression from an inducible VanR-specific promoter. (B) Expression of vanillic acid (VA)-inducible SEAP in mammalian cells. HEK-293 cells were transfected with a constitutive MOR9-1 expression vector (pLYL76) and a cAMP-responsive SEAP expression vector (pCK53). SEAP levels in culture supernatants were recorded 48 hours after incubation in medium containing 0 or 400 μM vanillic acid (dissolved in DMSO). Data presented are mean ± SD, n = 4 independent experiments. (C) Expression of vanillic acid (VA)-repressible SEAP in mammalian cells. HEK-293 cells were transfected with a constitutive VanR-VP64 expression vector (pSL175) and a vanillic acid-inducible SEAP expression vector (pSL173). SEAP levels in culture supernatants were recorded 48 hours after incubation in medium containing 0 or 400 μM vanillic acid (dissolved in DMSO). Data presented are mean ± SD, n = 4 independent experiments. (D) Parallel operation of vanillic acid-dependent high-level active (IF1) and low-level active (IF0) gene switches in mammalian cells. HEK-293 cells were transfected with a constitutive MOR9-1 expression vector (pLYL76), a cAMP-responsive SEAP expression vector (pCK53), a constitutive VanR-VP64 expression vector (pSL175), and a VanR-specific NanoLuc expression vector (pLZ345). After 48 hours of culture in cell culture medium containing varying concentrations of vanillic acid, SEAP levels in the culture supernatant were recorded. Data presented are mean ± SD, n = 4 independent experiments. Numbers represent the fold change calculated by dividing the SEAP levels in samples containing grazoprevir by the SEAP levels in samples without grazoprevir.

[0471] Figure 25 Engineering of different sets of mutually orthogonal grazoprevir-regulated BUF / NOT switches. (A) Independent sets of BUF and NOT switches controlled by grazoprevir. Figure 23The principles of the STIF-based system (BUF1 and NOT1) are described in A. To establish a GEMS-based system, grazoprevir-inducible gene expression from a synthetic STAT3-specific promoter (BUF2) occurs upon dimerization of a synthetic cell membrane receptor containing an intracellular domain derived from IL6RB and an extracellular GNCR domain and NS3a (H1) domain. Exchange of the GNCR domain by ANR results in grazoprevir-repressible gene expression (NOT2). For the StaPLd-based system, grazoprevir inhibits the autolysis of the synthetic mammalian transactivator PcaV-StaPLd-VP64 or the trans-silencing factor PcaV-StaPLd-KRAB, resulting in trigger-induced activation (BUF3) or inhibition (NOT3) of gene expression from the synthetic PcaV-specific promoter. (B) Design and validation of grazoprevir-inducible and grazoprevir-repressible GEMS variants (BUF2 and NOT2). The GEMS receptor comprises an extracellular ligand-binding domain, a transmembrane domain GEMS TM (yellow) and the intracellular domains of receptor scaffolds derived from IL6RB-, FGFR-, or VEGFR- (grey). GEMS receptors containing NS3a (H1) and GNCR as extracellular domains (GEMS NS3a(H1) &GEMS GNCR The co-expression of GEMS receptors (GEMS) containing NS3a (H1) and ANR as extracellular domains can explain the inducible expression of target genes by grazoprevir. NS3a(H1) &GEMS ANR ) may explain the grazoprevir-repressed target gene expression. (Left) For grazoprevir-inducible target gene expression by GEMS variants containing the IL6RB-derived intracellular domain, STAT3-specific SEAP expression vector (pLZ284) and the corresponding GEMS NS3a(H1) and GEMS GNCR HEK-293 cells were co-transfected with the constitutive expression vectors (pSL890 / pSL889) of the constructs. For grazoprevir-inducible target gene expression by GEMS variants containing the intracellular domain of FGFR1, a TetR-specific SEAP expression vector (pMF111) and a TetR-Elk1 vector for TetR-Elk1 and the corresponding GEMS were used. NS3a(H1) and GEMS GNCR For grazoprevir-inducible target gene expression by GEMS variants containing a VEGFR-derived intracellular domain, a calcium-inducible SEAP expression vector (pMX57) and a constitutive expression vector for the corresponding GEMS were used. NS3a(H1) and GEMS GNCR(Right) For grazoprevir-repressible target gene expression by GEMS variants containing the IL6RB-derived intracellular domain, STAT3-specific SEAP expression vectors (pLZ284) and the corresponding GEMS were used. NS3a(H1) and GEMS ANR For grazoprevir-repressible target gene expression by GEMS variants containing the intracellular domain of FGFR1, a TetR-specific SEAP expression vector (pMF111) and a TetR-Elk1 vector for TetR-Elk1 and the corresponding GEMS were used. NS3a(H1) and GEMS ANR For grazoprevir-induced repressive target gene expression by GEMS variants containing VEGFR-derived intracellular domains, calcium-inducible SEAP expression vectors (pMX57) and the corresponding GEMS were used. NS3a(H1) and GEMS ANR Cells were co-transfected with the constitutive expression vectors (pSL894 / pLZ271) of the constructs. SEAP levels in culture supernatants were recorded 48 hours after the addition of 10 μM grazoprevir dissolved in DMSO (vehicle control). Data are mean ± SD, n = 3. (C) Design and validation of StaPLd-based grazoprevir-inducible transactivators and transsilencers (BUF3 and NOT3). (Left) For grazoprevir-repressible gene expression, HEK-293 cells were co-transfected with the constitutive PcaV-StaPLd-KRAB expression vector (pLZ417) and the PcaV-repressible SEAP expression vector (pLZ419). (Right) For grazoprevir-inducible gene expression, HEK-293 cells were co-transfected with the constitutive PcaV-StaPLd-VP64 expression vector (pLZ418) and the PcaV-specific SEAP expression vector (pSL172). SEAP levels in culture supernatants were quantified 48 hours after addition of 10 μM grazoprevir. Data are shown as mean ± SD, n = 3 independent experiments.

[0472] Figure 26 Orthogonality requirements for gene switches in three-state based gene circuits. (A) Selection criteria for downstream modules. Parallel BUFs monitoring different output signals n. n / NOT nEach individual set of switches must be orthogonal to each other for non-interfering biocomputation. (B, C) Parallel operation of grazoprevir-dependent BUF / NOT switch sets. (B) GEMS-based BUF switch (BUF2; GNCR-GEMS) that drives NanoLuc expression. IL6RB &NS3a(H1)-GEMS IL6RB &P STAT3 -NanoLuc; pSL889 / pSL890 / pLZ368) and a grazoprevir-regulated STIF-based NOT switch (NOT1; (ANR)8-NSP3&MCP-(NS3a(H1))3&P hCMV -SEAP-(MS2-box) 24 -HHR-pA; pSL582 / pSL548 / pSL468) were co-administered with a grazoprevir-regulated GEMS-based NOT switch (NOT2; ANR-GEMS) driving NanoLuc expression. IL6RB &NS3a(H1)-GEMS IL6RB &P STAT3 -NanoLuc; pLZ268 / pSL890 / pLZ368) with a grazoprevir-regulated STIF-based BUF switch (BUF1; (GNCR)3-NSP3&MCP-(NS3a(H1))3&P hCMV -SEAP-(MS2-box) 24 -HHR-pA; pSL1032 / pSL548 / pSL468) were co-administered to HEK-293 cells. After 48 h of culture in cell culture medium containing 0 or 10 μM grazoprevir, SEAP and NanoLuc levels were recorded in the culture supernatant. Data presented are mean ± SD, n = 3 independent experiments. (C) A grazoprevir-regulated StaPLd-based BUF switch (BUF3; PcaV-StaPLd-VP64; pLZ418 & pSL172) driving NanoLuc expression was co-administered with a grazoprevir-regulated STIF-based NOT switch (NOT1; ANR-NSP3 & NS3a(H1)-MCP & P hCMV -NanoLuc-(MS2-box) 24-HHR-pA; pQZ111&pSL548&pSL582), while a grazoprevir-regulated StaPLd-based NOT switch driving SEAP expression (NOT3; PcaV-StaPLd-KRAB; pLZ417&pLZ419) was co-administered with a grazoprevir-regulated STIF-based BUF switch driving NanoLuc expression (BUF1; GNCR-NSP3&NS3a(H1)-MCP&P hCMV -NanoLuc-(MS2-box) 24 -HHR-pA; pSL1032 & pSL548 & pQZ111) were co-administered to HEK-293 cells. After 48 hours of culture in cell culture medium containing 0 or 10 μM grazoprevir, the levels of SEAP and NanoLuc in the culture supernatant were recorded. Data presented are mean ± SD, n = 3 independent experiments. (D) Orthogonality of vanillic acid (VA)-inducible cAMP-dependent SEAP expression and grazoprevir-inducible STAT3-dependent NanoLuc expression in mammalian cells. A vanillic acid-inducible gene switch (IF1; pCK53) controlling SEAP expression and a grazoprevir-regulated GEMS-based BUF switch (BUF2; pSL889 / pSL890 / pLZ368) driving NanoLuc expression were co-administered to HEK-MOR9 (C0) cells and cultured in cell culture medium containing vanillic acid (VA, 400 μM) and / or grazoprevir (Gra, 10 μM). 48 hours after transfection, SEAP levels in culture supernatants were recorded. Data presented are mean ± SD, n = 3 independent experiments.

[0473] Figure 27Computational (Bio)Logical Similarities of Genetically Engineered Three-State Buffers. (A) Logical characteristics of three-state buffers. The combination of grazoprevir-controlled genetic switches (BUF and NOT) with vanillic acid-controlled genetic switches (active-high and active-low) yields four different types of three-state buffers: an active-high buffer (IF1-regulated BUF: BUFIF1), an active-high inverting buffer (IF1-regulated NOT: NOTIF1), an active-low buffer (IF0-regulated BUF: BUFIF0), and an active-low inverting buffer (IF0-regulated NOT: NOTIF0). (B) Three-state buffers controlled by grazoprevir and vanillic acid in mammalian cells. In a biological context, the Hi-Z (electrically disconnected) and 0 (electrically switched off) states of a three-state buffer are considered functionally similar from the perspective of inactive gene expression (OFF). Thus, BUFIF1 shows a logical similarity to a Boolean AND gate, and NOTIF0 shows a logical similarity to a NOR gate, while NOTIF1 and BUFIF0 are logically similar to traditional NIMPLY (AND NOT) gates. For BUFIF1, cells were co-transfected with a plasmid encoding MCP-specific EGFP mRNA (pQZ112), a constitutive expression vector (pSL548) for MCP-(NS3a(H1))3 (pSL548), MOR9-1 (pLYL76), and VanR-VP64 (pSL175), and a cAMP-responsive (GNCR) 3-NSP3 expression vector (pLYL62). For NOTIF0, cells were co-transfected with pQZ112, pSL548, pSL175, and a vanillic acid-responsive (ANR) 4-NSP3 expression vector (pLYL85). For NOTIF1, cells were co-transfected with pQZ112, pSL548, pLYL76, and a cAMP-responsive (ANR) 4-NSP3 expression vector (pLYL67). For BUFIF0, cells were co-transfected with pQZ112, pSL548, pSL175, and a vanillic acid-responsive (ANR) 3-NSP3 expression vector (pLYL63). Six hours after transfection, vanillic acid (VA, 400 μM) and grazoprevir (Gra, 0.5 μM) were added. 36 hours after transfection, fluorescence images showing EGFP signals were acquired (scale bar: 100 μm), and flow cytometry analysis was performed using 10,000 cells per group ( Figure 28). Data show fluorescence units as mean ± SD, n = 3 individual experiments. (C) Tri-state buffer formed by the IF0 / IF1 switch regulated by vanillic acid and the BUF2 / NOT2 switch regulated by grazoprevir. For BUF2IF1, STAT3-specific SEAP expression vector (pLZ284) and the GEMS GNCR (pLZ285) and GEMS NS3a(H1) HEK-293 cells stably expressing MOR9-1 (HEK-MOR9(C0)) were co-transfected with a cAMP-responsive expression vector (pLZ286). For NOT2IF0, pLZ284, a constitutive VanR-VP64 expression vector (pSL175), and a vector for GEMS were used. NS3a(H1) (pLZ412) and GEMS ANR HEK-293 cells were co-transfected with a vanillic acid responsive expression vector (pLZ413). For NOT2IF1, pLZ284 and the vanillic acid responsive expression vector for GEMS were used. ANR (pLZ310) and GEMS NS3a(H1) HEK-MOR9 (C0) cells were co-transfected with a cAMP-responsive expression vector (pLZ286). For BUF2IF0, pLZ284, a constitutive VanR-VP64 expression vector (pSL175), and a cAMP-responsive expression vector for GEMS were used. NS3a(H1) (pLZ412) and GEMS GNCR HEK-293 cells were co-transfected with a vanillic acid-responsive expression vector (pLZ411). Following culture in medium containing vanillic acid (VA, 400 μM) and / or grazoprevir (Gra, 10 μM), SEAP levels in the culture supernatant were recorded 48 hours post-transfection. Data presented are mean ± SD, n = 3 independent experiments.

[0474] Figure 28 .and Figure 27 B. Correlated flow cytometry data.

[0475] Figure 29Modular assembly of Boolean logic circuits using tri-state buffers and BUF / NOT switches. For tri-state-based circuits exhibiting OR logic, cells were transfected with a constitutive expression vector for (GNCR)3-NSP3 (pLZ74; module 6) and a cAMP-responsive (ANR)8-NSP3 expression vector (pSL580; module 2). For tri-state-based circuits exhibiting XOR logic, cells were transfected with a vanillic acid-responsive (GNCR)3-NSP3 expression vector (pLYL63; module 3) and a cAMP-responsive (ANR)4-NSP3 expression vector (pLYL67; module 2). For tri-state-based circuits exhibiting NAND logic, cells were transfected with a constitutive expression vector for (ANR)8-NSP3 (pSL582; module 5) and a vanillic acid-responsive (GNCR)3-NSP3 expression vector (pLYL87; module 3). For tri-state circuits displaying XNOR logic, cells were transfected with a cAMP-responsive (GNCR)3-NSP3 expression vector (pLYL62; module 1) and a vanillic acid-responsive (ANR)8-NSP3 expression vector (pLYL95; module 4). For tri-state circuits displaying GraIMPLY VA logic, cells were transfected with a constitutive expression vector of (ANR)8-NSP3 (pSL582; module 5) and a cAMP-responsive (GNCR)3-NSP3 expression vector (pLYL62; module 1). For tri-state circuits displaying VA IMPLY Gra logic, cells were transfected with a constitutive expression vector of (GNCR)3-NSP3 (pLZ74; module 6) and a vanillic acid-responsive (ANR)8-NSP3 expression vector (pLYL95; module 4). For circuits involving modules 1 and 2, cells were further co-transfected with a constitutive expression vector of MOR9-1 (pLYL76). For the circuits involving modules 3 and 4, cells were further co-transfected with a constitutive expression vector (pSL175) of VanR-VP64. For all circuits, cells were co-transfected with a reporter plasmid (pQZ112) encoding MCP-specific EGFP mRNA and a constitutive expression vector (pSL548) of MCP-(NS3a(H1))3 to form the computational core unit. Six hours after transfection, different combinations of vanillic acid (V, 400 μM) and grazoprevir (G, 0.5 μM) were added. 36 hours after transfection, fluorescent images showing EGFP signals were acquired (scale bar: 100 μm). Representative data from three independent experiments are shown.

[0476] Figure 30 .Design and verification of half adder and half subtractor gene circuits in mammalian cells. (A) Half adder. Half adder is represented by CARRY (C: 2 1 bits) and SUM(S: represents 2 0The sum of two Boolean integers (e.g., vanillic acid and grazoprevir) is calculated using a dual output consisting of a Boolean number of bits. For biological implementation, HEK-MOR9 (C0) cells were co-transfected with plasmids encoding the computational core unit driving EGFP expression [MCP-(NS3a(H1))3 constitutive expression vector and MCP-specific EGFP mRNA (pSL548 / pQZ112)], module 3 [VanR-VP64 constitutive expression vector and vanillic acid responsive (GNCR)3-NSP3 expression vector (pSL175 / pLYL63)], module 2 [MOR9-1 constitutive expression vector and cAMP responsive (ANR)4-NSP3 expression vector (pLYL76 / pLYL67)], and module 7 [GEMS GNCR and GEMS NS3a(H1) The cells were cultured for 48 h in a medium containing different combinations of vanillic acid (V, 400 μM) and grazoprevir (G, 10 μM), and EGFP- and mCherry-expressing cells were analyzed by flow cytometry. Figure 31 A). Data are shown as mean ± SD of fluorescence units and are representative of 3 independent experiments. (B) Half subtractor. Half subtractor is constructed by returning the value of BORROW (B). O : means -1*2 1 bit) and DIFFERENCE(D: indicates 2 0 To achieve biological function, HEK-MOR9 (C0) cells were co-transfected with plasmids encoding module 8 [GEMS ANR and GEMS NS3a(H1) The cAMP-responsive expression vector and the STAT3-specific mCherry expression vector (pLZ310 / pLZ286 / pLZ287) were used as the genetic components of the 3rd module (pSL175 / pLYL63), the 2nd module (pLYL76 / pLYL67), and the computational core unit (pSL548 / pQZ112) driving EGFP expression. After culturing for 48 hours in medium containing different combinations of vanillic acid (V, 400 μM) and grazoprevir (G, 10 μM), flow cytometric analysis of EGFP- and mCherry signals was performed ( Figure 31 B). Data show fluorescence units as mean ± SD and are representative of three independent experiments.

[0477] Figure 31 .and Figure 30Related flow cytometry data. (A) Figure 30 Flow cytometry data related to A. (B) Figure 30 B. Correlated flow cytometry data.

[0478] Figure 32 : Grazoprevir and vanillic acid-responsive half-adders using BUF3. HEK-293 cells were co-transfected with plasmids encoding EGFP-mRNA with an MCP-specific poly(A) replacement (pQZ112), constitutive expression vectors for MCP-(NS3a(H1))3 (pSL548), VanR-VP64 (pSL175), and MOR9-1 (pLYL76), a cAMP-responsive (ANR)4-NSP3 (pLYL67) and NLS-PcaV-StaPLd-VP64 expression vector (pSL754), a vanillic acid-responsive (GNCR)3-NSP3 expression vector (pLYL63), and a PcaV-specific grazoprevir-inducible mCherry expression vector (pSL648). Six hours after transfection, various combinations of vanillic acid (V, 400 μM) and grazoprevir (G, 0.5 μM) were added. Fluorescence images showing EGFP signals were acquired 36 hours after transfection (scale bar: 100 μm). Representative data from three independent experiments are shown.

[0479] Figure 33Vanillic acid and grazoprevir-responsive logic gates in vivo. (A) Vanillic acid AND NOT grazoprevir gate. 420 μg of plasmid DNA (pSL683 / pSL548 / pLYL76 / pLYL67, ratio 2:6:16:3 (w / w / w / w)) was hydrodynamically injected into the tail vein of WT C57BL / 6 mice. Six hours after injection, different combinations of vanillic acid (500 mg / kg / day) and grazoprevir (9 mg / kg / day) were administered via intraperitoneal injection three times daily. NanoLuc levels in the mouse bloodstream were measured 24 hours after the first stimulation. Data are presented as mean ± SEM; n = 3 mice. (B) Vanillic acid (VA)-repressed SEAP expression (IF0). 25 μg of plasmid DNA (pSL175 / pSL173, ratio 3:2 (w / w)) was hydrodynamically injected into the tail vein of WT C57BL / 6 mice. Six hours after injection, vanillic acid (500 mg / kg / day) dissolved in PBS was administered by intraperitoneal injection three times daily. SEAP levels in the mouse bloodstream were measured 24 hours after the first vanillic acid injection. (C) Grazoprevir-repressible SEAP production (NOT1). 450 μg of plasmid DNA (pSL468 (50 μg) / pSL549 (200 μg) / pSL548 (200 μg) in a ratio of 1:4:4 (w / w / w)) was hydrodynamically injected into the tail vein of WT C57BL / 6 mice. Six hours after injection, grazoprevir (3 mg / kg) dissolved in PBS was administered by intraperitoneal injection three times daily. SEAP levels in the mouse bloodstream were measured 24 hours after the first grazoprevir injection. Data are expressed as mean ± SEM; n = 5 mice. (D) Vanillic acid (VA)-inducible SEAP expression (IF1). 430 μg of plasmid DNA (pLYL76 / pCK53, 40:3 (w / w)) was hydrodynamically injected into the tail vein of WT C57BL / 6 mice. Six hours after injection, vanillic acid (500 mg / kg / day) dissolved in PBS was administered via intraperitoneal injection three times daily. SEAP levels were measured in the mouse bloodstream 24 hours after the first vanillic acid injection. Data are presented as mean ± SEM; n = 5 mice.

[0480] Figure 34A general design strategy for STIF-based "molecular clamps" for sensing a variety of intracellular targets of interest. To sense a specific target Y", a pair of proteins Y and Y', each binding to a different epitope of Y", can be fused to the N-terminus or C-terminus of the RBP or eIFBP domain of a STIF regulatory factor, such that the formation of a circularized mRNA conformation and translation initiation are completely dependent on the presence of Y" within the cell. Alternatively, the RBP-specific 5'-cap and / or poly(A) surrogate can be replaced by an adaptor motif that directly binds to Y" to form a circularized mRNA conformation in combination with Y or Y' fused to eIFBP.

[0481] Figure 35Initial experiments to develop a STIF-based protein sensor (Part 1). (A, B) Optimization of NSP3 fusion constructs. (A) HEK-293 cells were co-transfected with plasmids encoding SEAP mRNA (pSL468) containing an MCP-specific poly(A) replacement, MCP-Coh2 (pSL674), and an NSP3 fusion protein consisting of one (pSL66), two (pSL85), or three N-terminal DocS repeats (pSL86). SEAP expression was recorded in culture supernatants 48 hours after transfection. Data are mean ± SD, n = 4. (B) HEK-293 cells were co-transfected with plasmids encoding SEAP mRNA containing an MCP-specific poly(A) replacement (pSL468), MCP-DocS (pSL1311), and an NSP3 fusion protein consisting of one (pSL241), two (pSL242), or three N-terminal Coh2 repeats (pSL243). SEAP expression in culture supernatants was recorded 48 hours after transfection. Data are mean ± SD, n = 4. (C) Translational regulation by different C / D-box-specific DocS tethers. HEK-293 cells were co-transfected with plasmids encoding SEAP mRNA containing an L7Ae-specific poly(A) replacement (pSL88 & pSL4), DocS-NSP3 (pSL66 or pcDNA3.1(+) as a negative control), and various L7Ae fusion proteins with one (pSL65), two (pSL82), or three C-terminal Coh2 repeats (pSL83). SEAP expression was recorded in culture supernatants 48 hours after transfection. Data are mean ± SD, n = 4. (D) Translational regulation by different MS2-box-specific DocS tethers. HEK-293 cells were cotransfected with plasmids encoding SEAP mRNA containing an MCP-specific poly(A) surrogate (pSL468), (DocS)3-NSP3 (pSL86), and various MCP fusion proteins consisting of one (pSL674), two (pSL1079), or three N-terminal Coh2 repeats (pSL1080). SEAP expression was recorded in culture supernatants 48 hours after transfection. Data are mean ± SD, n = 4.

[0482] Figure 36Preliminary experiments for developing STIF-based protein sensors (Part 2). (A) Translational regulation of C / D-box-containing mRNAs by different Coh2-specific eIFBPs. HEK-293 cells were co-transfected with plasmids encoding SEAP-mRNA containing an L7Ae-specific poly(A) replacement (pSL88 & pSL4), L7Ae-(Coh2)3 (pSL83), and an NSP3 fusion protein consisting of one (pSL66), two (pSL85), or three N-terminal DocS repeats (pSL86). SEAP expression was recorded in culture supernatants 48 hours after transfection. Data are mean ± SD, n = 4. (B) Translational regulation of MS2-box-containing mRNAs by different Coh2-specific eIFBPs. HEK-293 cells were cotransfected with plasmids encoding SEAP mRNA (pSL468) containing an MCP-specific poly(A) surrogate, MCP-(Coh2)3 (pSL1080), and an NSP3 fusion protein consisting of one (pSL66), two (pSL85), or three N-terminal DocS repeats (pSL86). SEAP expression in culture supernatants was recorded 48 hours after transfection. Data are mean ± SD, n = 4. (C, D) DocS-dependent NSP3-mediated activation of STIF-specific mRNA. (C) For the MCP-based system, HEK-293 cells were cotransfected with plasmids encoding SEAP mRNA (pSL468) containing an MCP-specific poly(A) surrogate, MCP-(Coh2)3 (pSL1080), and varying amounts of pSL86. (D) For the L7Ae-based system, HEK-293 cells were co-transfected with plasmids encoding SEAP mRNA containing an L7Ae-specific poly(A) surrogate (pSL88 & pSL4), L7Ae-(Coh2)3 (pSL83), and varying amounts of the (DocS)3-NSP3 expression vector (pSL86). SEAP expression in culture supernatants was recorded 48 hours after transfection. Data are mean ± SD, n = 4.

[0483] Figure 37Engineering of STIF-based protein sensors for in vitro and in vivo diagnostics. (A) Genetically encoded protein sensors using protein association-dependent STIF remodeling. HEK-293 cells were co-transfected with pSL468 and constitutive expression vectors for MCP-(Coh2)3 (pSL1080), (Coh2)3-NSP3 (pSL243), and (DocS)3 (pSL244 at varying amounts). SEAP expression in culture supernatants was recorded 48 hours after transfection. Data are shown as mean ± SD, n = 4 independent experiments. Bars represent mean ± SD, and solid circles indicate individual results. (B) Construction of a hepatitis C virus (HCV)-specific protein sensor in mammalian cells. HEK-293 cells were co-transfected with plasmids encoding SEAP-mRNA containing an L7Ae-specific poly(A) replacement (pSL88 & pSL4), L7Ae-scFv35 (pSL136), (scFv162)3-NSP3 (pSL139), and varying amounts of a synthetic trimeric target protein (nNS3)3 (pSL107). SEAP expression in the culture supernatant was recorded 48 hours after transfection. Data are mean ± SD, n = 3. (C) Implementation of synthetic gene circuits in a cell-free system. Figure 34 The STIF-based protein sensor designed according to the illustrated principle can also be applied to non-biological materials, such as paper discs. In this case, the genetic components of the STIF-based sensor (in the form of plasmid DNA, mRNA, or purified protein) can be freeze-dried onto the non-biological material along with cell lysates to enable long-term storage. Detection of a target compound (e.g., a virulence factor of bacteria or virus) in moist air triggers STIF remodeling, translation initiation, and production of a reporter protein in the paper disc, which is ultimately visible to the naked eye.

[0484] Figure 38Genetically encoded sensors for intracellular target proteins with distinct subcellular localizations. (A) Engineering of transcription- and translation-based sensors for detecting differentially localized intracellular proteins. The synthetic target protein EGFP-NS3a(H1) was targeted to different intracellular compartments by fusion with different localization signals (1. NLS (nuclear localization signal); 2. NES (nuclear export signal); 3. CAAX (prenylation motif); 4. transmembrane localization signal; 5. secretion signal peptide), allowing detection with co-expressed genetic sensors engineered based on LaG16 (EGFP nanobody) and ANR (a peptide motif that binds NS3a(H1)). For transcription-based sensing, LaG16 was fused to TetR and ANR to VP64 to allow EGFP-NS3a(H1)-dependent activation of a TetR-specific promoter. For translation-based induction, LaG16 was fused to MCP and ANR to NSP3 to allow EGFP-NS3a(H1)-dependent STIF remodeling and translation of MCP-specific mRNA. (B) Dose-dependent detection of differential translocation of EGFP-NS3a(H1). Plasmids were co-transfected with different amounts of constitutive expression vectors for different target proteins (0, native EGFP-NS3a(H1), pSL775; 1, nuclear NLS-EGFP-NS3a(H1), pSL797; 2, cytosolic NES-EGFP-NS3a(H1), pSL824; 3, prenylated EGFP-NS3a(H1)-CAAX, pSL799; 4, membrane-localized TM-EGFP-NS3a(H1), pSL798; 5, secreted SP-EGFP-NS3a(H1), pSL796). HEK-293 cells were transfected with plasmids encoding either a translation-based EGFP-NS3a(H1) sensor (pSL776 / pSL582 / pSL468: for expression of MCP-LaG16, (ANR)8-NSP3, and SEAP mRNA with an MCP-specific poly(A) replacement) or a transcription-based EGFP-NS3a(H1) sensor (pSL834 / pSL836 / pMF111: for expression of TetR-LaG16, (ANR)8-VP64, and a TetR-specific promoter controlling SEAP transcription). 48 hours after transfection, fluorescent images of cellular EGFP signals were acquired (scale bar: 10 μm) and SEAP levels in culture supernatants were analyzed. Data are presented as ±SD fold change in SEAP activity relative to basal SEAP levels in which EGFP-NS3a(H1) expression was undetectable (0 ng / bar).

[0485] Figure 39Engineering of a STIF-based protein sensor for the artificial fusion protein EGFP-NS3a(H1). (A) Translational regulation by different EGFP-specific MCP fusion proteins. HEK-293 cells were co-transfected with a plasmid encoding SEAP mRNA containing an MCP-specific poly(A) replacement (pSL468), a constitutive EGFP-NSP3 expression vector (pSL942), and expression vectors for different MCP-LaG16 variants containing one (pSL776) or two (pSL777) tandem LaG16 repeats. pcDNA3.1(+) was transfected instead of pSL942 as a negative control. 48 hours after transfection, culture supernatants were analyzed for SEAP expression. Data presented are mean ± SD, n = 4. (B) Translational regulation by different NS3a(H1)-specific NSP3 fusion proteins. HEK-293 cells were co-transfected with a plasmid encoding SEAP mRNA containing an MCP-specific poly(A) replacement (pSL468), a constitutive MCP-(NS3a(H1))3 expression vector (pSL548), and expression vectors for different ANR-NSP3 variants containing different tandem ANR repeats (ANR-NSP3, pSL704; (ANR)2-NSP3, pSL549; (ANR)6-NSP3, pSL581; (ANR)8-NSP3, pSL582). SEAP expression was analyzed in culture supernatants 48 hours after transfection. Data presented are mean ± SD, n = 4. (C) Construction of the EGFP-NS3a(H1) sensor in mammalian cells. HEK-293 cells were co-transfected with a plasmid encoding a reporter SEAP mRNA containing an MCP-specific poly(A) surrogate (pSL468), a constitutive EGFP-NS3a(H1) expression vector (pSL775), and various combinations of MCP-LaG16 (pSL776) and (ANR)8-NSP3 expression vectors (pSL582). Transfection with pcDNA3.1 (+) instead of pSL776 and / or pSL582 served as a negative control (-). Culture supernatants were analyzed for SEAP expression 48 hours after transfection. Data presented are mean ± SD, n = 4.

[0486] Figure 40Engineering of STIF-based sensors for cancer-associated fusion genes. (A) Engineering of a STIF-based sensor for the BCR-ABL fusion protein. In chronic myeloid leukemia (CML), a chromosomal translocation results in the fusion of the BCR gene at 22q11 with the gene encoding the ABL1 tyrosine kinase at 9q34, forming a hybrid oncoprotein, BCR-ABL, with enhanced kinase activity. Bipartite STIF can only be reconstituted and activate STIF-dependent translation in cells containing the fusion protein construct, in which each split component of the bipartite STIF is engineered to contain an intracellular antibody that specifically binds to the native form of each individual BCR or ABL1 protein. (B) Selectivity of the EGFP-NS3a(H1) fusion protein sensor. The EGFP-NS3a(H1) sensor (pSL468 / pSL776 / pSL582; Figure 39 (C) HEK-293 cells were co-transfected with expression vectors for EGFP (pWS164), NS3a(H1) (pSL818), or EGFP-NS3a(H1) (pSL775). SEAP expression was analyzed in culture supernatants 48 hours after transfection. Data presented are mean ± SD, n = 3 independent experiments. (C) Selectivity of BCR-ABL fusion protein sensors. HEK-293 cells were co-transfected with SEAP-producing BCR-ABL sensors (constitutively expressing MCP-ABI(iDab) (pSL860), CCmut3-NSP3 (pSL863), and MCP-specific SEAP mRNA (pSL468)) and expression vectors for BCR (pSL1045), ABL (pSL1046), or BCR-ABL (pSL1014). SEAP expression was analyzed in culture supernatants 48 hours after transfection. Data provided are mean ± SD, n = 4 independent experiments. (D) BCR-ABL-mediated association of MCP-ABI (iDab) and CCmut3-NSP3. HEK-293 cells were co-transfected with 3xFLAG-tagged MCP-ABI (iDab) (pSL1101), 3xHA-tagged CCmut3-NSP3, and expression vectors for BCR-ABL (+, pSL1014) or pcDNA3.1 (+) (- negative control) and immunoprecipitated 48 hours later. Anti-FLAG and anti-HA antibodies were used to detect the target protein in each lysate fraction before (input) and after (Flag-IP) immunoprecipitation. The numbers on the right axis of the Western blot represent the molecular weight (MW) of the target protein.

[0487] Figure 41Target specificity of the genetically encoded EGFP-NS3a(H1) protein sensor. A plasmid encoding mCherry-mRNA containing a MCP-specific poly(A) replacement (pSL683, P hCMV -NanoLuc-P2A-mCherry-(MS2-box) 24 HEK-293 cells were co-transfected with constitutive expression vectors for (-HHR-pA) and MCP-LaG16 (pSL776), (ANR)8-NSP3 (pSL582), and EGFP-NS3a(H1) (pSL775). pcDNA3.1(+) was transfected instead of pSL775 as a negative control. At 48 hours post-transfection, (A) fluorescence images showing colocalized EGFP / mCherry signals were acquired (scale bar: 50 μm), followed by (B) flow cytometric analysis (10,000 cells per group). Representative data from three independent experiments are shown.

[0488] Figure 42 Therapeutic efficacy was validated using a xenograft mouse model. Cell lines with distinct molecular signatures (hypothesized to be distinguishable by cytoplasmic target proteins) were subcutaneously implanted into the lower back of mice, and tumors were allowed to grow over 17 days. On day 7, a gene circuit engineered to selectively activate apoptosis was injected into the tumors every two days. The effects on tumor size were continuously monitored throughout the experiment.

[0489] Figure 43 Engineering of target-specific protein sensors to drive self-sufficient cancer gene therapy. (A) Validation of therapeutic efficacy using a xenograft mouse model. Plasmid DNA encoding a STIF-based protein sensor was injected into Figure 42 In the mouse tumors described above, the sensor was used to trigger the expression of the pro-apoptotic Bax protein by EGFP-NS3a(H1). In healthy cells, Bax expression remains OFF due to the lack of the EGFP-NS3a(H1) target protein. Overexpression of EGFP-NS3a(H1) in malignant cells triggers self-sufficient STIF assembly, Bax translation, and apoptosis. (BD) EGFP-NS3a(H1) specifically activates apoptosis in mice. Subcutaneous B16-F10 EGFP-NS3a(H1) Mice bearing tumors derived from the WT mice received local injections of pcDNA3.1(+) (negative control, n=5 mice / group) or pSL831 (P hCMV -mBax-(MS2-box) 24 -HHR-pA), pSL776 (P hCMV -MCP-LaG16-pA) and pSL582 (P hCMV-(ANR)8-NSP3-pA) plasmid DNA mixture (treatment group, n = 5 mice / group). (B) Calculated by V = (length * width) 2 / 2 to evaluate daily changes in tumor size. (C) Tumors were harvested on the final experimental day for image analysis and (D) Bax protein levels were measured by Western blotting. (EG) In EGFP-NS3a(H1)-deficient tissues, P hCMV The EGFP-NS3a(H1) sensor driven by the NS3a(H1) gene does not activate apoptosis. Mice bearing subcutaneous B16-F10 tumors received local injections of pcDNA3.1(+) (negative control, n = 5 mice / group) or a mixture of plasmid DNA containing pSL831 / pSL776 / pSL582 (treated group, n = 5 mice / group). (E) Calculation of V = (length * width) 2 Daily changes in tumor size were assessed using a 2-hour RT-PCR assay. (F) Tumors were harvested on the final experimental day for image analysis and (G) Bax protein levels were measured by Western blotting. The numbers on the left axis of the Western blots represent the molecular weight (MW) of the target protein.

[0490] Figure 44 A therapeutic biocomputer coupling tissue-specific detection and STIF-based protein sensing. To precisely distinguish complex cellular features within heterogeneous tissues (e.g., tumors), STIF-based protein sensors with programmable target specificity can be coupled to tissue-specific promoters (TSPs) to drive the production of STIF-specific mRNA for expression of suicide genes (e.g., Bax). Upon delivery of such gene circuits into living tissues in vivo, self-sufficient apoptosis is triggered only in cells that meet the criteria of tissue specificity (TSPs trigger transcription of poly(A)-deficient STIF-dependent Bax mRNA) and target specificity (Bax mRNA is translated only after detection of STIF-mediated intracellular protein markers).

[0491] Figure 45 Characterization of tissue and target specificity in mammalian cells and mice. (A) AFP levels were quantified using TSP-driven reporter gene expression (left panel) and qRT-PCR (right panel). MusAFPN2A, Hepa1-6, and B16-F10 cells were co-transfected with a NanoLuc expression vector (pSLM54) driven by luciferase and a constitutive FLuc expression vector (pYW99). Luciferase levels were quantified 48 hours after transfection. The data presented are the mean ± SD of relative luciferase activity (NanoLuc / FLuc), n = 3 separate experiments. (Right panel) The relative expression levels of AFP in N2A, Hepa1-6, and B16-F10 cells were analyzed by qRT-PCR using the mouse AFP-specific primers listed in Table S1 and normalized to the gene encoding mouse ribosomal protein (Rplp0). The data presented are the mean ± SD, n = 3. The bar graphs represent the mean and SD, and the solid circles show the individual results. (B) Tissue-specific induction of EGFP-NS3a (H1) in vitro. MusAFP Native (WT) or stably EGFP-NS3a(H1) transgenic N2A and Hepa1-6 cells were co-transfected with constitutive expression vectors for NanoLuc-P2A-mCherry mRNA driven by (pSL813) or containing an MCP-specific poly(A) surrogate (pSL683), as well as constitutive expression vectors for MCP-LaG16 (pSL776), (ANR)8-NSP3 (pSL582), and FLuc (pYW99). Luciferase levels were quantified 48 hours after transfection. Data presented are mean ± SD of relative luciferase activity (NanoLuc / FLuc), n = 3 separate experiments. (C) Validation of tissue-specific EGFP-NS3a(H1) induction in mice. hCMV driven by (pSL468 / pSL582 / pSL776) and P MusAFP Plasmids for the EGFP-NS3a(H1) sensor driven by (pSL857 / pSL582 / pSL776) and an EGFP-NS3a(H1) expression vector (pSL775) were hydrodynamically injected into the tail vein of C57BL / 6 mice, and SEAP levels in the bloodstream were measured 24 hours later. Mice receiving pcDNA3.1(+) instead of pSL775 served as negative controls, lacking hepatic EGFP-NS3a(H1) expression (WT). Data are presented as mean ± SEM; n = 5 mice / group.

[0492] Figure 46 . Demonstrating self-sufficient elimination of malignant cells in mice. (AD)P MusAFP - and EGFP-NS3a(H1)-specifically activate apoptosis in mice carrying subcutaneous Hepa1-6 EGFP-NS3a(H1)Mice bearing tumors derived from pcDNA3.1(+) (negative control, n=5 mice / group) or a mixture of plasmid DNA containing pSL886, pSL776, and pSL582 (treated group, n=5 mice / group) were injected locally. (A) V = (length * width) was calculated. 2 Daily changes in tumor size were assessed using a 2-hour RT-PCR assay. (B) Tumors were weighed and harvested on the final experimental day for (C) image analysis and (D) measurement of Bax protein levels by Western blotting. The numbers on the left axis of the Western blots represent the molecular weight (MW) of the target protein.

[0493] Example

[0494] Materials and methods

[0495] Vector Design. References and molecular structures of all expression vectors are provided in Table S2. Some expression vectors were constructed by in-fusion cloning using the Seamless Cloning Kit (Beyotime Biotechnology, Shanghai, China; Catalog No. D7010M). PCR amplification reactions were performed using KOD One PCR Master Mix (Toyobo, Osaka, Japan; Catalog No. KMM-201). Ligation reactions were performed using T4 DNA ligase (New England Biolabs, Beverly, MA; Catalog No. M0202L). Restriction endonucleases were purchased from New England Biolabs.

[0496] Chemicals and Recombinant Proteins. Abscisic acid (Cat. No. A8060; 100 mM stock in DMSO) and dimethyl sulfoxide (DMSO; Cat. No. D8371) were purchased from Solarbio Life Sciences (Beijing, China). Danoprevir (Cat. No. HY-10238; 10 mM stock in DMSO) and gibberellic acid (Cat. No. HY-N1964; 100 mM stock in DMSO) were purchased from MedChemExpress (Monmouth Junction, NJ). Animal-free recombinant human EGF (Cat. No. AF-100-15; 1000x stock in ddHO) was purchased from PeproTech EC (London, United Kingdom). Grazoprevir (Cat. No. S3728; 10 mM stock in DMSO (for in vitro experiments) or 100 g / L stock in DMSO (for in vivo experiments)) was purchased from Selleck Chemicals (Houston, TX). Rapamycin (Cat. No. MC0181; 10 mM stock solution in DMSO) was purchased from ImmunoWay Biotechnology (Plano, TX). Rapamycin analog (Rapalog; Cat. No. 535057; 100 μM stock solution in EtOH) was purchased from Takara Bio (Kusatsu, Japan). 4-Nitrophenyl phosphate disodium salt hexahydrate (pNPP; Cat. No. 333338-18-4) was purchased from Aladdin Biochemical Technology (Shanghai, China). Phenylmethylsulfonyl fluoride 100 mM solution (PMSF; Cat. No. ST506-2) and poly-D-lysine 5 mg / ml solution (Cat. No. C0312) were purchased from Beyotime Biotechnology (Shanghai, China). Diethanolamine (DEA; Cat. No. D807525) and anhydrous ethanol (EtOH; Cat. No. E809056) were purchased from Macklin (Shanghai, China). Polyethylenimine MAX (PEI; Product No. 24765; 1 mg / ml stock solution in ddH2O) was purchased from Polysciences (Eppelheim, Germany). Vanillic acid (Product No. R017640; 165 mM stock solution in DMSO) and isopropanol (Product No. R018247) were purchased from Rhawn Chemicals (Shanghai, China). Anhydrous D-glucose (dextrose; Product No. A610219), glycerol (Product No. A501745; 10% w / w stock solution in ddH2O), L-homoarginine hydrochloride (Product No. A602842), magnesium chloride hexahydrate (MgCl2; Product No. A610328), and Tween-20 (Product No. A100777) were purchased from Sangon Biotech (Shanghai, China).4% PFA solution (Cat. No. R20497) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Streptozotocin (STZ; Cat. No. s0130) was purchased from Sigma-Aldrich (MilliporeSigma; Burlington, MA). Anhydrous calcium chloride (CaCl2; Cat. No. 10005861), chloroform (Cat. No. 10006818), sodium chloride (NaCl; Cat. No. 10019318; 5 M stock solution in ddH2O), potassium chloride (KCl; Cat. No. 10016308), sodium acetate (Cat. No. 10018818; 3 M stock solution in ddH2O), and trisodium citrate dihydrate (Cat. No. 10019418) were purchased from Sinopharm Chemical Reagent (Shanghai, China). Puromycin dihydrochloride (Cat. No. A1113803) and blasticidin S HCl (Cat. No. R21001) were purchased from Thermo Fisher Scientific (Waltham, MA). Triton X-100 (Cat. No. X11206) was purchased from Xinyu Biological Technology (Shanghai, China). Mouse RNase inhibitor (Cat. No. R301) was purchased from Vazyme Biotech (Nanjing, China). Homemade stock solutions of 1 M Tris-HCl (pH 7.5) and 0.5 M EDTA were provided by the Westlake University Core Facility.

[0497] Cell culture and transfection. Cell lines derived from human embryonic kidney cells (HEK-293T, ATCC: CRL-3216), murine hepatoma cells (Hepa1-6, ATCC: CRL-1830), and murine neuroblastoma cells (N2A, ATCC: CRL-131) were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Waltham, MA; Catalog No. 12100046) supplemented with 10% (v / v) fetal bovine serum (Gibco FBS, Australia; Thermo Fisher Scientific, Waltham, MA; Catalog No. 10099141, Lot No. 2177370) and 1% (v / v) penicillin / streptomycin solution (PenStrep; Beyotime Biotechnology, Shanghai, China; Catalog No. ST488). Mouse melanoma cells (B16-F10, ATCC: CRL-6475) were cultured in RPMI 1640 medium (Sartorius AG, All cells were cultured at 37°C in a humidified atmosphere with 5% CO2. For subculturing, pre-confluent cells were detached by incubating in 0.05% trypsin-EDTA (Sangon Biotech, Shanghai, China; Cat. No. A610629-0050; Lot No. F319BA0030) at 37°C for 3 minutes. Cells were collected in 10 ml of cell culture medium, centrifuged at 1000 rpm for 2 minutes, and then centrifuged at 1.5 × 10 5 Cells were resuspended in fresh culture medium at a density of 10 cells / mL and seeded into new tissue culture plates. Cell number and viability were quantified using an Invitrogen Countess II AMQAX 1000 cell counter (Thermo Fisher Scientific; Cat. No. AMQAX1000).

[0498] Unless otherwise noted, transfections were performed 12 hours after seeding 50,000 mammalian cells per well of a 24-well plate. Six hours after transfection, the cell culture medium was replaced with fresh culture medium (without transfection reagent). HEK-293T cells were transfected using a PEI-based protocol with a 5:1 (w / w) PEI:DNA ratio and a transfection volume of 50 μL of native serum-free DMEM per well. N2A, Hepa1-6, and B16-F10 cells were transfected using Lipofectamine 3000 (ThermoFisher Scientific, Cat. No. L3000015) reagent according to the manufacturer's instructions. In vitro transcribed mRNA was transfected by mixing 0.75 μL of Lipofectamine 3000 (without P3000 reagent) with 500 ng of nucleic acid in a transfection volume of 50 μL of native serum-free OptiMEM medium (Thermo Fisher Scientific, Cat. No. 31985062) per well. The transfection reagent and nucleic acid were incubated at 25°C for 15 minutes before being added to the cells dropwise. For RNA transfection, the cell culture medium was replaced with medium without Lipofectamine 3000 4 hours after transfection.

[0499] Lentivirus production. 5 × 10 cells were transfected with 5 μg of pMD2.G (Addgene plasmid no. 12259), 10 μg of psPAX2 (Addgene plasmid no. 12260), and 10 μg of a transfer plasmid carrying the desired gene expression cassette. 6Recombinant, replication-defective lentiviral particles were generated by transfecting naive HEK-293T cells (cultured in 10 cm dishes). 48 hours after transfection, the culture supernatant containing the lentivirus was harvested, the medium was replaced, and the cells were cultured for an additional 48 hours. The two harvested stocks were combined and purified using a 0.45 mm filter for experimental use or stored at -80°C.

[0500] Stable cell lines were generated by co-transfecting 500 ng of pSL816 (Table S2) and 5 ng of pCMV-T7-SB100 (Addgene plasmid number 34879) into 5 × 10 4 Transgenic polyclonal HEK cells stably expressing EGFP-NS3a(H1) were constructed in natural HEK-293T, N2A, Hepa1-6 and B16-F10 cells. EGFP-NS3a(H1) 、N2A EGFP-NS3a(H1) 、Hepa1-6 EGFP-NS3a(H1) and B16-F10 EGFP-NS3a(H1) After selection with 100 μg / ml puromycin hydrochloride, the 10% surviving population with the highest EGFP expression was sorted by FACS using a MA900 multi-application cell sorter (Sony Biotechnology; San Jose, CA). 5 × 10 cells were transduced with supernatant containing lentiviral particles produced with pLZ276 (Table S2) as the transfer plasmid. 4 HEK-293T cells were used to construct monoclonal HEK-MOR9 (C0) cells stably transgenic for constitutive MOR9-1 expression. After selection with 100 μg / ml puromycin, single cell clones showing the highest MOR9-1 expression were picked and harvested.

[0501] In vitro transcription. The template DNA fragment containing the T7 promoter was isolated from the corresponding plasmid by restriction endonuclease treatment and then transcribed in the presence of 40 mM 3'-O-Me-m 7After the addition of a G(5′)ppp(5′)G RNA Cap structural analog (New England Biolabs, Beverly, MA; Cat. No. S1411L), the mRNA was transcribed using a T7 High Yield RNA Transcription Kit (Vazyme Biotech, Nanjing, China; Cat. No. TR-101). After removing the template DNA using 1 U of RNase-free DNaseI (Vazyme Biotech; Cat. No. EN401-01), the mRNA was purified by precipitation with 0.3 M sodium acetate at -20°C, followed by centrifugation at 4°C and 15,000 rpm for 30 minutes and washing with 70% EtOH. The purified mRNA was resuspended in RNA-free ddH2O and the RNA was quantified by measuring UV absorbance with a UVP crosslinker CL-3000 (Analytik Jena GmbH, Jena, Germany).

[0502] RNA extraction and cDNA synthesis. Total RNA from cells was isolated using the Trizol RNA extraction method. Briefly, 3000 cells were mixed with 1 ml of TRIzol TM Reagent (ThermoFisher Scientific, Cat. No. 15596018) was mixed and vortexed until no precipitation was observed. The cell lysate was then mixed with 200 μl of chloroform and centrifuged at 12,000 g for 15 minutes at 4 ° C. The aqueous phase was collected, mixed with 500 μl of isopropanol, and incubated at -20 ° C for 30 minutes to precipitate RNA. The RNA precipitate was collected by centrifugation at 4 ° C and 15000 g for 30 minutes. Wash with 70% EtOH, air dry and resuspended in 50 μl of RNase-free water. For cDNA synthesis, 1 μg of RNA was taken and qPCR was performed for cDNA synthesis using HiScript IIQ RT SuperMix (Vazyme Biotech; Cat. No. R223-01).

[0503] Reverse transcription-polymerase chain reaction (RT-PCR). For quantitative analysis, PCR reactions were performed on an Applied Biosystems QuantStudio 1 Real-Time PCR System (ThermoFisher Scientific) using ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech; Cat. No. Q711-02) and the primers listed in Table S1. The initial step was 95°C for 30 seconds, followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds. The relative cycle threshold (CT) was determined and normalized to the expression level of the endogenous human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or mouse ribosomal protein (Rplp0) gene.

[0504] RNA immunoprecipitation (RIP)-qPCR. 48 hours after transfection, cells were harvested with ice-cold laboratory-prepared PBS (Biomedical Experimental Technology Center, Westlake University), resuspended in RNA-free NETN300 cell lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 2 mM EDTA, 0.05% Triton X-100, 1 mM PMSF, and 50 U / ml mouse RNase inhibitor), and incubated on ice for 15 minutes. After centrifugation at 16,000 g and 4°C for 15 minutes, 4% of the supernatant was collected as the "input sample." The remaining supernatant was diluted with RNA-free NETN0 buffer (NaCl-free NETN300 cell lysis buffer) to a final NaCl concentration of 100 mM, and then immunoprecipitated by incubation with anti-Flag affinity gel (Beyotime Biotechnology; Catalog No. P2271) at 4°C for 90 minutes. The anti-Flag affinity gel was then centrifuged at 6000 g for 30 seconds and washed three times with ice-cold NETN3000 wash buffer (50 mM Tris-HCl, 300 mM NaCl, 0.05% Trion X-100, and 1 mM PMSF). RNA from the "input sample" and affinity gel was extracted for RT-PCR analysis. This method was adapted from that described elsewhere (Kim and Dekker, 2018).

[0505] Co-immunoprecipitation (co-IP). 48 hours after transfection, cells were harvested and lysed with cell lysis buffer (Beyotime Biotech; Cat. No. P0013) supplemented with 1 mM PMSF at 4°C for 30 minutes. After centrifugation at 14,000 g for 10 minutes, 100 μl of supernatant was collected as the "input sample," and the remaining supernatant was immunoprecipitated by incubation with anti-Flag affinity gel (Beyotime Biotech; Cat. No. P2271) at 4°C for 3 hours. The anti-Flag affinity gel was then centrifuged at 6000 g for 30 seconds and washed three times with IP wash buffer (20 mM Tris-HCl, 0.2 mM EDTA, 100 mM KCl, 2 mM MgCl2, 0.1% Tween 20, and 10% glycerol). The “input sample” and proteins from the affinity gel were mixed with 5X SDS PAGE loading buffer (Beyotime Biotech; Cat. No. P0015) and boiled at 98°C for 10 minutes in preparation for Western blotting. This method was adapted from that described elsewhere (DeCaprio and Kohl, 2020).

[0506] Western blotting. Samples were separated on 8%, 10%, or 12% SDS polyacrylamide gels from an SDS-PAGE preparation kit (Sangon Biotech, Shanghai, China; Catalog No. C631100) after treatment with 5X SDS PAGE loading buffer (Beyotime Biotech; Catalog No. P0015) at 98°C for 10 minutes and electroblotted onto polyvinylidene fluoride (PVDF) Western blotting membranes (Merck Millipore KGaA, Darmstadt, Germany; Catalog No. 03010040001). The membranes were incubated in BeyoECL Moon detection reagent (Beyotime Biotechnology; Cat. No. P0018FS) and probed with rabbit anti-Flag primary antibody (Sigma-Aldrich; Cat. No. F7425), rabbit anti-HA primary antibody (Cell Signaling Technology, Danvers, MA; Cat. No. 3724; Lot. 10), rabbit anti-eIF4G primary antibody (Cell Signaling Technology; Cat. No. 2498; Lot. 4), rabbit anti-eIF4E primary antibody (Cell Signaling Technology; Cat. No. 2067; Lot. 8), rabbit anti-GAPDH (6C5) primary antibody (Santa Cruz Biotechnology; Cat. No. sc-32233; Lot. L2019), or rabbit anti-Bax primary antibody (Cell Signaling Technology; Cat. No. sc-32233; Lot. L2019). The cells were stained with an Amersham Imager 600 (AI600 RGB; GE Healthcare, Uppsala, Sweden) after addition of HRP-conjugated goat anti-rabbit secondary antibody (Biyotime Biotechnology; Cat. No. A0208; Lot No. 110219200406).

[0507] Quantification of target gene expression. SEAP assay. The expression level of human placental secretory alkaline phosphatase (SEAP) in the culture supernatant was quantified based on the absorbance of p-nitrophenyl phosphate at 415 nm (Wang et al., 2015). SEAP levels in mouse serum were plotted using the SEAP chemiluminescence assay kit (Roche Diagnostics GmbH, Mannheim, Germany; Cat. No. 11779842001). NanoLuc assay. NanoLuc levels were analyzed using a luciferase assay system (Promega, Madison, WI; Catalog No. N1120). FLuc assay: Firefly luciferase levels were analyzed using a luciferase reporter gene assay kit (Yeasen Biotechnology, Shanghai, China; Catalog No. 11401ES60) after cell lysis at 4°C for 15 minutes, followed by centrifugation at 12,000 g for 5 minutes. Insulin ELISA: Modified rodent insulin (mINS) levels were quantified in culture supernatants and mouse serum using a mouse insulin ELISA kit (Mercodia, Uppsala, Sweden; Catalog No. 10-1247-01).

[0508] Flow cytometry. Cell populations were analyzed using a CytoFLEX LX flow cytometer (Beckman Coulter, Indianapolis, IN) equipped for detection of EGFP (488 nm laser, 525 / 40 emission filter) and mCherry (561 nm laser, 610 / 20 emission filter) and set to exclude dead cells and cell doublets. 10,000 cells were recorded for each data set and analyzed using FlowJo. TM Software (v10; BD Biosciences) was used for analysis. 5 The gated EGFP or mCherry expression level was determined by arbitrarily thresholding the fluorescence units and multiplying the percentage of gated cells by their median fluorescence.

[0509] Fluorescence Imaging Fluorescence microscopy was performed with a Nikon ECLIPSE Ts2-FL fluorescence microscope (Nikon Instruments, Melville, NY) equipped with a C-mount camera, an F-mount camera, a 20× objective, excitation and emission filter sets (EGFP: 488 / 509 nm; mCherry: 587 / 610 nm), and OPLENIC software (x64, version 10.1.14643.20190511).

[0510] Confocal microscopy analysis. 1 μg of plasmid DNA was transfected into 1×10 5After 24 hours in the cells, which were plated in 20 mm glass-bottomed cell culture dishes coated with 5 mg / ml poly-D-lysine (WuxiNEST Biotechnology, Wuxi, China; Cat. No. 801001), the cell culture medium was removed, the cells were washed with 1 mL of PBS, and fixed with 1 mL of 4% PFA solution. After 10 minutes, the cells were washed with PBS and stained with DAPI staining solution (Biyuntian Biotechnology; Cat. No. C1005) in the dark for 15 minutes. Finally, the cells were washed three times with PBS and imaged using an A1R HD25 confocal microscope (Nikon Instruments, Melville, NY).

[0511] AAV production. AAV2 / 8-(GNCR)3-NSP3, AAV2 / 8-MCP-(NS3a)3, and AAV2 / 8-SEAP-(MS2) were produced by PackGene Biotech (Guangzhou, China) using the transfer plasmids pSL511 (Table S2), pSL512 (Table S2), or pSL446 (Table S2), respectively. 24 -HHR-pA.

[0512] Animal experiments. Animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Westlake University (Protocol ID: 20-001-XMQ) and in accordance with the Guidelines for Animal Care of the Ministry of Science and Technology of the People's Republic of China. Hydrodynamic tail vein injection. Endotoxin-free plasmids were diluted in Ringer's solution (147 mM NaCl, 4 mM KCl, 1.13 mM CaCl2) to a final injection volume of 100 μL / g body weight and injected into the tail vein of mice >6 weeks old using a 5 mL syringe. STZT1D mouse model. Fasted 6-week-old male WT C57BL / 6 mice were injected daily with freshly diluted STZ (50 mg / kg dissolved in 200 μl ice-cold sodium citrate buffer) for five days. Chronic fasting hyperglycemia (>15 mM) developed after 3 weeks. Xenograft tumor model. 1×10 WT mice dissolved in 0.1 mL sterile PBS were injected into the tail vein of mice >6 weeks old. 6 B16-F10-or 2×10 6Hepa1-6-derived cell lines were subcutaneously injected into the right lower back of 4-week-old male WT C57BL / 6 mice. Seven days later, each animal received an intratumoral injection of 60 μL of Lipofectamine 3000 solution containing 20 μg of plasmid DNA under anesthesia on different days after cell implantation. Drug administration. Grazoprevir was administered at 100 μg / μL by intraperitoneal (ip) injection or oral gavage. Blood collection. Whole blood was collected from the submandibular vein of the mice and clotted by incubation at 4°C for 2 hours, followed by separation of serum by centrifugation at 8000 g for 8 minutes. Blood glucose measurement. Blood glucose of the mice was measured using a commercial blood glucose meter (Sinocare Plus Code blood glucose meter; detection range: 1.1-33.3 mM) purchased from a local pharmacy. Glucose tolerance test (GTT). Freshly prepared D-glucose was injected into the peritoneum of the mice at a dose of 0.75 g / kg before zero.

[0513] Data Analysis. Two-tailed unpaired Student's t-test was used to assess the statistical significance of differences between the two groups. For tumor volume studies, statistical analysis was performed using two-way analysis of variance. A P value of less than 0.05 was considered statistically significant. Statistical parameters and corresponding P values are included in the figure legends. All analyses were performed using GraphPad Prism 9 (GraphPad Software, San Diego, CA).

[0514] Example 1: Gene regulation by triggering inducible mRNA circularization in mammalian cells.

[0515] According to the "closed loop" model (Gray et al., 2000; Jackson et al., 2010), we propose that manipulating the mRNA circularization process would be an attractive access point for triggering inducible translation initiation. In the basal state (OFF), loss of the circularized conformation impairs scanning efficiency and promotes mRNA degradation by endogenous ATPases (Tang et al., 2019). Therefore, translation initiation (ON) should depend on the efficiency of establishing the closed loop conformation, a task naturally mediated by endogenous PABP binding to the poly(A) region (Jackson et al., 2010; Passmore and Coller, 2021).

[0516] To engineer similar regulatory frameworks for user-defined (trans)gene control ( Figure 1), we designed mRNA transcripts containing a control region comprising an RBP-specific aptamer (such as those listed in Table 2) in the 3′-UTR. Thus, circularization and translation of the synthetic mRNA transcripts will depend on the presence of synthetic translation initiation factors (STIFs; such as those listed in Table 1), which mimic PABP function by simultaneously binding to the aptamer-based control region and any one member of the pre-initiation complex ( Figure 1 ).

[0517] As a proof of concept, we engineered various STIF constructs by fusing different RNA binding proteins (RBPs), such as archaeal ribosomal protein L7Ae (SEQ-ID NO: 92) or phage-derived MS2 coat protein (MCP, SEQ-ID NO: 98), to different eIF4F binding proteins (eIFBPs), such as human PABP (SEQ-ID NO: 108), eIF4G (SEQ-ID NO: 84), and eIF4E (SEQ-ID NO: 83), as well as rotavirus nonstructural protein 3 (NSP3, SEQ-ID NO: 91 & 106), and calicivirus VPg (SEQ-ID NO: 120), resulting in the following specific STIF constructs:

[0518] Table 1: STIF constructs designed according to the present invention.

[0519]

[0520]

[0521] To verify their function, HEK-293 cells (ATCC: CRL-3216; see the Methods section above) were co-transfected with a SEAP expression vector containing eight tandem repeats of the L7Ae-specific C / D-box (SEQ-ID NO: 131), an shRNA-216 expression vector (SEQ-ID NO: 126), and an expression vector for different STIF variants or an L7Ae-Coh2 protein incapable of translation initiation (SEQ-ID NO: 33, negative control) as described in Table 1 above. SEAP levels in culture supernatants were quantified 48 hours after transfection, as described in the Materials and Methods section for target gene expression. Figure 2 A). Similarly, HEK-293 cellsCells were co-transfected with an expression vector containing eight tandem repeats of the MCP-specific MS2-box (SEQ-ID NO: 180), an shRNA-216 expression vector (SEQ-ID NO: 126), and an expression vector for different STIF variants or an MCP-Coh2 protein that cannot initiate translation (SEQ-ID NO: 51, negative control). 48 hours after transfection, the level of SEAP in the culture supernatant was quantified ( Figure 2 B) For STIF overexpression, any method which results in the production of the protein sequences listed in Table 1 in living cells can be used.

[0522] from Figure 2 A and Figure 2 The results of B show that

[0523] (i) Removal of poly(A) by overexpression of shRNA-216 (in the presence of SEQ-ID NO: 126) is crucial for reducing basal expression levels corresponding to the expression of Coh2 (SEQ-ID NO: 77) and L7Ae (SEQ-ID NO: 92) ( Figure 2 A) or MCP (SEQ-ID NO: 98) ( Figure 2 B) Fusion situation,

[0524] (ii) fusion of MCP (SEQ-ID NO: 98) or L7Ae (SEQ-ID NO: 92) with eIF4G (SEQ-ID NO: 84) or NSP3 (SEQ-ID NOs: 91 & 106) allows for optimal performance of RBP-dependent translation (upregulation) regulation,

[0525] (iii) the RBP and eIFBP domains can be flexibly exchanged to generate functional STIF regulators (i.e., in eIF4G-MCP (SEQ-ID NO: 253), the RBP domain MCP is located at the C-terminus; in MCP-eIF4E (SEQ-ID NO: 244), the RBP domain can also be located at the N-terminus), and

[0526] (iv) STIF may also contain any other protein domain inserted between the RBP and eIFBP domains (e.g. in the case of eIF4G-2CaM-M13-L7Ae (SEQ-ID NO: 69), wherein the calmodulin-like motif 2CaM-M13 (SEQ-ID NO: 312) is inserted between eIF4G and L7Ae).

[0527] Based on these experimental findings, it is suggested that STIF variants containing any reversed conformation not listed in Table 1 (e.g., MCP-PABP instead of PABP-MCP; L7Ae-PABP instead of PABP-L7Ae; etc.), as well as changing MCP or L7Ae to any or their mutants (e.g., in the case of MCP(V29I)-VPg, SEQ-ID NO: 276) or any other RBP (e.g., N-peptide derived from bacteriophage lambda (λN, SEQ-ID NO: 100)), will also result in STIFs that show similar Figure 2 A and Figure 2 Similar results were obtained for the functional STIF construct in B.

[0528] Next, we demonstrated that the relative fold change between basal STIF-independent expression and STIF-mediated upregulation can be further fine-tuned by increasing the number of adaptor repeats located downstream of the protein-coding region ( Figure 2 C, 2D). In the experiment, different tandem repeats of L7Ae-specific C / D-box aptamers ((C / D-box)8, SEQ-ID NO: 131; (C / D-box) 12 , SEQ-ID NO: 133; (C / D-box) 16 , SEQ-ID NO: 132; (C / D-box) 24 HEK-293 cells were co-transfected with a SEAP expression vector containing (SEQ-ID NO: 134), a shRNA-216 expression vector (SEQ-ID NO: 126), and any expression vector producing PABP-L7Ae (ON; SEQ-ID NO: 65) or a Coh2-L7Ae protein that cannot bind to eIF4F (OFF; SEQ-ID NO: 8). SEAP expression was recorded in the culture supernatant 48 hours after transfection. For the MCP-based STIF system, different tandem repeats of the MCP-specific MS2-box aptamer ((MS2-box)8, SEQ-ID NO: 179; (MS2-box) 12 , SEQ-ID NO: 178; (MS2-box) 16 , SEQ-ID NO: 138; (MS2-box) 24 HEK-293 cells were transfected with a SEAP expression vector containing either MCP-NSP3 (ON; SEQ-ID NO: 59) or an MCP-Coh2 protein that is unable to bind eIF4F (OFF; SEQ-ID NO: 51). SEAP expression was recorded in the culture supernatant 48 hours after transfection.

[0529] Therefore, the aptamer region (e.g. (C / D-box) n or (MS2-box) n ) can be considered as artificial poly(A) signals or “poly(A) surrogates” that mediate strict STIF-dependent mRNA translation, in which genetically encoded poly(A) excision allows translation to “escape” from endogenous PABP-mediated processes, thereby achieving efficient (trans)gene control ( Figure 3 A).

[0530] In fact, we quantified PABP's ability to bind to poly(A)-containing RNA using RNA immunoprecipitation-qPCR (detailed experimental procedures are described in the Methods section above). To this end, HEK-293 cells were transfected with a constitutive expression vector for 3xFLAG-tagged PABP-L7Ae (SEQ ID NO: 66) to reflect the RNA binding capacity of endogenous PABP. Twenty-four hours later, 10 μg of in vitro-transcribed EGFP-mRNA with (+) or without (-) poly(A) tail (generated from pWS164, Table S2) was added as described in the "In Vitro Transcription" section of the Methods section above. Three hours after transfection, RNA was extracted and co-immunoprecipitated using Anti-Flag Affinity Gel (Beyotime Biotech; Cat. No. P2271). Results of qRT-PCR analysis showing the proportion (%) of EGFP-mRNA in samples before (input) and after immunoprecipitation (IP) (detailed experimental procedures are described in the Methods section above) confirm that mRNA containing "poly(A) surrogate" instead of natural poly(A) is no longer bound by endogenous PABP ( Figure 3 A). In addition, the size of the engineered poly(A) surrogate (reflecting the (C / D-box) located in the 3'-UTR) n or (MS2-box) n The number of tandem repeats (n) appears to have a positive effect on delaying mRNA decay in mammalian cells ( Figure 3 B, 3C). Moreover, any aptamer-specific protein that binds to this region can confer increased stability to the target mRNA ( Figure 3 C, 3D).

[0531] Next, we engineered a single-component expression vector for poly(A)-deficient mRNA to achieve stringent STIF-specific target gene expression. To this end, we used the cis-acting hammerhead ribozyme HHR motif (SEQ-ID NO: 124) ( Figure 4 B) replaced EQ-ID NO:131-134( Figure 4A), which should trigger spontaneous self-excision of the native poly(A) signal immediately before or after nuclear mRNA export. Using this strategy, different eIFBPs were bound to L7Ae( Figure 5 A) or MCP( Figure 5 B) can also activate gene expression of poly(A)-deficient mRNA, among which L7Ae-NSP3 (SEQ-ID NO:43) and MCP-NSP3 (SEQ-ID NO:59) showed the highest induction fold between basal and activated states after HHR-mediated poly(A) removal ( Figure 5 A, 5B). Basically, these experiments are Figure 2 A and Figure 2 Similar to that described in B, except that it contains 24 L7Ae-specific C / D-box tandem repeats instead of 8 ( Figure 5 A) or MCP-specific MS2-box tandem repeats ( Figure 5 B) The SEAP expression vector is now used as a reporter vector, and the shRNA-216 specific binding site (SEQ-ID NO: 122) is exchanged for the HHR element (SEQ-ID NO: 124). Figure 5 C, 5D) or enhanced green fluorescent protein (EGFP; SEQ-ID NO: 82) ( Figure 5 When the SEAP expression vector was replaced with the isogenic target mRNA of MCP-NSP3 (SEQ-ID NO: 59), we found that MCP-NSP3 (SEQ-ID NO: 59) could also regulate a variety of other reporter genes.

[0532] Thus, any mRNA having the general description, including but not limited to "5'-UTR-GOI-(MS2-box) n -BS(shRNA) n -pA-3'", "5'-UTR-GOI-(C / D-box) n -BS(shRNA) n -pA-3'", "5'-UTR-GOI-(MS2-box) n -HHR n -pA-3'" and "5'-UTR-GOI-(C / D-box) n -HHR n-pA-3'", wherein the 3'-UTR of a specific gene of interest (GOI) consists of a poly(A) surrogate capable of binding to a specific target protein (RBP: including but not limited to L7Ae (SEQ-ID NO: 92), MCP (SEQ-ID NO: 98) and λ-N (SEQ-ID NO: 100)) and an RNA cleavage site capable of preprogrammed poly(A) removal (including but not limited to BS (shRNA-216) (SEQ-ID NO: 122) and HHR (SEQ-ID NO: 124)), and is regarded as a "STIF-specific target gene mRNA" ( Figure 1 , green boxes). In this study, the following constructs were generated and validated (Table 2):

[0533] Table 2: Synthetic mRNA transcripts containing RBP-specific poly(A) surrogates enabling STIF-dependent translation and expression of different GOIs.

[0534]

[0535]

[0536] Based on the results and findings of this application, it can be known that:

[0537] (i) The gene of interest (GOI) can be exchanged for any nucleic acid fragment encoding any polypeptide of interest (i.e., any RNA sequence starting with the nucleotide AUG and ending with the nucleotide sequence UAG, UAA, or UGA);

[0538] (ii) The poly(A) surrogate can be exchanged for any fragment containing one or more n aptamer repeats that bind to specific RNA-binding proteins (RBPs). Based on our experimental results, n can be any number between 1 and 1000, and most preferably n should be 8, 16, or 24;

[0539] (iii) For genetically encoded expression in mammalian cells, the cleavage site may comprise one or more n repeats of any ribozyme or nuclease target site. Based on our experimental results, n can be any number between 1 and 100, and most preferably, n should be any number between 1 and 4.

[0540] The full sequences of all key elements are shown in the sequence listing.To generate such sequences, any molecular cloning technique or nucleic acid synthesis strategy capable of generating the corresponding plasmid DNA or synthetic mRNA may be used.

[0541] Because STIF was designed to mimic the binding of endogenous PABP to both mRNA and the eIF4F complex, we used co-immunoprecipitation and Western blotting to experimentally confirm that STIF containing NSP3 indeed associates with the eIF4F complex in a closed-loop model ( Figure 6 A), and NSP3 showed much less nonspecific binding to endogenous RNA than PABP ( Figure 6 B). Specifically, HEK-293 cells were transfected with expression vectors for 3xFLAG-tagged MCP (-, SEQ-ID NO: 264) or MCP-NSP3 (+, SEQ-ID NO: 260), and one lysate fraction was immunoprecipitated 48 hours after transfection. Target protein was detected in lysate fractions before (input) and after immunoprecipitation (Flag-IP) using anti-FLAG (Sigma-Aldrich; Cat. No. F7425), anti-eIF4G (Cell Signaling Technology; Cat. No. 2498; Lot 4), and anti-eIF4E antibodies (Cell Signaling Technology; Cat. No. 2067; Lot 8). Details of the experimental procedures and consumables involved are described in the Methods section above. Figure 6 The Western blot results in A revealed that the colocalization of FLAG-tagged proteins with endogenous eIF4G and eIF4E was dependent on NSP3, where the input sample served as a control, showing that eIF4G / 4E were fully present in the cells. Similarly, to quantify the binding ability of PABP- and NSP3-fusion proteins to endogenous RNA, we performed RNA immunoprecipitation-qPCR using a similar experimental setup as described above (for Figure 3 A). 48 hours after HEK-293 cells were transfected with expression vectors for 3xFLAG-tagged L7Ae-NSP3 (SEQ-ID NO:49) or PABP-L7Ae (SEQ-ID NO:66), RNA was extracted and immunoprecipitated using anti-Flag affinity gel (Beyotime Biotechnology; Cat. No. P2271). qRT-PCR analysis (detailed experimental procedures are described in the Methods section above) revealed the ratio (%) of endogenous GAPDH expression levels before (input) and after (IP) immunoprecipitation (IP) as a measure of the amount of RNA bound to the PABP- and NSP3-containing constructs ( Figure 6 B) These results confirm that STIF-dependent translational regulation indeed occurs via a closed-loop model of mRNA circularization, a hitherto unexplored stage of gene expression in various cell engineering strategies.

[0542] To engineer various “sensing and responding” features, such as gene switches and genetic sensors, using the STIF-based translational regulation strategy, we will Figure 1 The conventional RBP-eIFBP or eIFBP-RBP structure described in Table 1 is split into two independent proteins RBP-Y and Y'-eIFBP ( Figure 7 ). In this bipartite STIF system, the assembly of a functional STIF capable of activating RBP-specific mRNA translation will depend on the nature of the protein-protein interaction (PPI) between any proteins Y and Y'. Notably, (i) the protein parts Y and Y' are flexibly interchangeable, (ii) each domain of each split STIF component, RBP-Y and Y'-eIFBP, can be flexibly swapped to generate Y-RBP, RBP-Y', Y-eIFBP, and eIFBP-Y' structures, and (iii) multiple tandem repeats of Y and Y' can be used to fine-tune regulatory properties. Based on these principles, the following bipartite STIF system was created for proof-of-concept:

[0543] Table 3: Bipartite STIF systems containing constitutive Y:Y' protein-protein interactions.

[0544]

[0545]

[0546] The full sequences of all key elements are shown in the sequence listing. To generate such sequences, any molecular cloning technique or nucleic acid synthesis strategy capable of producing the corresponding plasmid DNA or synthetic mRNA can be used. In this study, the constructs were validated by transfecting the encoding plasmid into HEK-293 cells, whose cell culture and transfection procedures are described above in the Methods section. To test the ability of different constitutive protein dimerizations to induce spontaneous STIF assembly ( Figure 8 A) HEK-293 cells were co-transfected with a plasmid encoding SEAP mRNA containing an L7Ae-specific poly(A) surrogate (SEQ-ID NO: 136) and constitutive expression vectors for various combinations of L7Ae- and NSP3-fusion proteins (e.g., STIF regulators listed in Table 3). Transfection with pcDNA3.1(+) (Invitrogen, CA; Cat. No. V79020) instead of the NSP3-fusion protein served as a negative control. SEAP levels in culture supernatants were quantified 48 hours after transfection. The results demonstrate that the high-affinity Coh2 / DocS (Barak et al., 2005) is effective in mediating translation of L7Ae- and MCP-specific target genes ( Figure 8Consistent with this, co-transfection of HEK-293 cells with a SEAP expression vector containing 24 tandem repeats of the MCP-specific aptamer (SEQ-ID NO: 137) and different combinations of MCP-Coh2 (SEQ-ID NO: 51) and (DocS)3-NSP3 (SEQ-ID NO: 14) constitutive expression vectors also demonstrated modular and efficient translational regulation ( Figure 8 B). In addition, we were able to use MCP-Coh2 (SEQ-ID NO: 51) or L7Ae-Coh2 (SEQ-ID NO: 33) as mRNA-specific tethers to recruit various DocS-containing eIFBP constructs to different mRNA sites, such as the 3'-UTR region ( Figure 9 A), 5'-UTR region ( Figure 9 B) or intergenic regions ( Figure 9 C) In Figure 9 In A, HEK-293 cells were transfected with expression vectors for SEAP-mRNA containing 24 tandem repeats of the C / D-box (left panel: SEQ-ID NO: 136) or tandem repeats of the MS2-box (right panel: SEQ-ID NO: 137) in the 3'-UTR, constitutive expression vectors for L7Ae or MCP fused to Coh2 (yielding SEQ-ID NO: 33 or SEQ-ID NO: 51) or EGFP (yielding SEQ-ID NO: 251 or SEQ-ID NO: 245), and constitutive expression vectors for various chimeric Coh2-specific eIFPBP fusions containing DocS (PABP-DocS, SEQ-ID NO: 207; DocS-eIF4G, SEQ-ID NO: 256; DocS-eIF4E, SEQ-ID NO: 257; DocS-NSP3, SEQ-ID NO: 12; DocS-VPg, SEQ-ID NO: 254). Figure 9 In B, HEK-293 cells were transfected with an expression vector for SEAP mRNA containing four tandem repeats of the C / D-box in the 5'-UTR (SEQ-ID NO: 176), a constitutive L7Ae-(Coh2)3 expression vector (SEQ-ID NO: 35), and a constitutive expression vector for various DocS-based fusion constructs, such as PABP-DocS (PABP-DocS, SEQ-ID NO: 207; DocS-eIF4G, SEQ-ID NO: 256; DocS-eIF4E, SEQ-ID NO: 257; DocS-NSP3, SEQ-ID NO: 12; DocS-VPg, SEQ-ID NO: 254). Figure 9In C, the reporter vector was exchanged for an mRNA construct containing 24 tandem repeats of the C / D-box placed downstream of the SEAP coding region and upstream of the NanoLuc coding region (SEQ-ID NO: 175). In all cases, pcDNA3.1(+) (Invitrogen, CA; Cat. No. V79020) was transfected instead of the vector expressing DocS or MCP as a negative control, and SEAP levels in the culture supernatant were quantified 48 hours after transfection.

[0547] We then permuted the constitutive Y:Y' association pairs into the bipartite STIF framework through various trigger-inducible PPI systems, resulting in the following regulatory systems:

[0548] Table 4: Bipartite STIF system containing trigger-inducible Y:Y' protein-protein interactions.

[0549]

[0550]

[0551]

[0552] For L7Ae based systems ( Figure 10A), SEAP-mRNA (SEQ-ID NO: 136) containing an L7Ae-specific poly(A) replacement with 24 C / D-box repeats was used as a reporter construct. For danoprevir-inducible SEAP translation, HEK-293 cells were further co-transfected with L7Ae-(NS3a)3 (SEQ-ID NO: 41) and (DNCR)3-NSP3 (SEQ-ID NO: 271) constitutive expression vectors. For abscisic acid-inducible SEAP translation, HEK-293 cells were further co-transfected with L7Ae-(ABI)3 (SEQ-ID NO: 31) and (PYL1)3-NSP3 (SEQ-ID NO: 67) constitutive expression vectors. For gibberellic acid-inducible SEAP translation, HEK-293 cells were further co-transfected with GAI-L7Ae (SEQ-ID NO: 27) and NSP3-GID1 (SEQ-ID NO: 63) constitutive expression vectors. For grazoprevir-inducible SEAP translation, HEK-293 cells were further co-transfected with L7Ae-(NS3a)3 (SEQ-ID NO: 41) and (GNCR)3-NSP3 (SEQ-ID NO: 30) constitutive expression vectors. For rapamycin acid-inducible SEAP translation, HEK-293 cells were further co-transfected with FKBP-L7Ae (SEQ-ID NO: 25) and FRB-NSP3 (SEQ-ID NO: 26) constitutive expression vectors. For blue light-inducible SEAP translation, HEK-293 cells were further co-transfected with L7Ae-CIB1 (SEQ-ID NO: 32) and Cry2-NSP3 (SEQ-ID NO: 10) constitutive expression vectors. The expression of SEAP was induced 48 h after addition of the corresponding inducers (danoprevir, 1 μM; abscisic acid, 100 μM; gibberellic acid, 100 μM; grazoprevir, 0.5 μM; rapamycin, 0.01 μM) or after exposure to blue light (450 nm; ON, 5 mW / cm 2 SEAP levels in the culture supernatant were recorded 24 hours after the onset of the culture (ON, 30 s; OFF, 30 s). The commercial information of the chemical reagents used is listed in the Chemicals and Recombinant Proteins section of the Methods section above. Figure 10B), SEAP-mRNA (SEQ-ID NO: 137) was used as a reporter construct containing an MCP-specific poly(A) replacement with 16 (SEQ-ID NO: 138) or 24 (SEQ-ID NO: 137) C / D-box repeats. For danoprevir-inducible SEAP translation, HEK-293 cells were co-transfected with SEQ ID NO: 137 and constitutive expression vectors for MCP-NS3a (SEQ-ID NO: 54) and DNCR-NSP3 (SEQ-ID NO: 270). For abscisic acid-inducible SEAP translation, HEK-293 cells were co-transfected with SEQ ID NO: 137 and constitutive expression vectors for BI-MCP (SEQ-ID NO: 259) and (PYL1)3-NSP3 (SEQ-ID NO: 67). For gibberellic acid-inducible SEAP translation, HEK-293 was co-transfected with SEQ ID NO: 137 and constitutive expression vectors for MCP-GID1 (SEQ-ID NO: 242) and GAI-NSP3 (SEQ-ID NO: 289). For grazoprevir-inducible SEAP translation, HEK-293 was co-transfected with SEQ ID NO: 137 and constitutive expression vectors for MCP-NS3a (SEQ-ID NO: 54) and GNCR-NSP3 (SEQ-ID NO: 28). For rapamycin-inducible SEAP translation, HEK-293 was co-transfected with SEQ ID NO: 138 and constitutive expression vectors for MCP-FRB (SEQ-ID NO: 243) and FKBP-NSP3 (SEQ-ID NO: 252). For blue light inducible SEAP translation, HEK-293 were co-transfected with constitutive expression vectors of SEQ ID NO: 138 and MCP-CIB1 (SEQ-ID NO: 247) and Cry2-NSP3 (SEQ-ID NO: 10). For red light inducible SEAP translation, SEQ ID NO: 138 and MCP-(Aff6 V18FΔN HEK-293 cells were co-transfected with constitutive expression vectors of )4 (SEQ-ID NO: 50) and DrBPhP-NSP3 (SEQ-ID NO: 15). 48 hours after the addition of the corresponding inducers (danoprevir, 0.5 μM; abscisic acid, 100 μM; gibberellic acid, 100 μM; grazoprevir, 0.5 μM; rapamycin analog, 0.1 μM) or after exposure to blue light (450 nm; ON, 5 mW / cm2, 30 s; OFF, 30 s) or red light (660 nm, constant 1 W / m 2 48 hours after the incubation, the level of SEAP in the culture supernatant was recorded. The commercial information of the chemical reagents used is listed in the Chemicals and Recombinant Proteins section of the Methods section above.

[0553] Translational regulation via trigger-inducible STIF systems can also be engineered in various other ways. For example, we engineered blue light-dependent LaM8 AK47 The nanobody (SEQ-ID NO: 220) was integrated into the STIF framework, demonstrating the versatility and design flexibility of translational regulation ( Figure 10 C) In this configuration, a SEAP-mRNA expression vector (SEQ-ID NO: 138) containing 16 tandem repeats of the MS2-box in the 3'-UTR and MCP-LaM8 were used. AK47 HEK-293 cells were transfected with constitutive expression vectors of mCherry-NSP3 (SEQ-ID NO: 241) and mCherry-NSP3 (SEQ-ID NO: 250). Six hours after transfection, the cells were illuminated with blue light (450 nm, 2 mW / cm 2 ) irradiated cells for 48 hours, and then SEAP levels in the culture supernatant were recorded to assess blue light-triggered gene switching. To demonstrate that it is also possible to engineer genetically encoded sensors that respond to intracellular signaling dynamics, we integrated the ERK2-specific pE59 DARPin system (SEQ-ID NO: 110) into the bipartite STIF framework. Since pE59 was developed to specifically bind phosphorylated ERK2 during MAPK signaling, the single STIF component RBP-(pE59) n (SEQ-ID NO:46,249&280) and (ERK2) n -NSP3 (SEQ-ID NO: 24 & 277) assembles and activates RBP-specific reporter gene expression only in cells with high MAPK activity ( Figure 11 ). For the L7Ae-based RBP system ( Figure 11 A), HEK-293 cells were co-transfected with different combinations of a dual reporter vector (SEQ-ID NO: 135) containing a constitutive FLuc expression unit and a NanoLuc-mRNA expression unit containing an L7Ae-specific poly(A) surrogate, an shRNA-216 expression vector (SEQ-ID NO: 126), and constitutive expression vectors for L7Ae-(pE59)2 (SEQ-ID NO: 46) and (ERK2)2-NSP3 (SEQ-ID NO: 24). Figure 11B) HEK-293 cells were co-transfected with various combinations of a constitutive FLuc expression vector (SEQ-ID NO: 305), an expression vector for NanoLuc mRNA containing an MCP-specific poly(A) surrogate (SEQ-ID NO: 139), and constitutive expression vectors for MCP-(pE59)2 (SEQ-ID NO: 249) and (ERK2)2-NSP3 (SEQ-ID NO: 24), and then cultured in medium containing 2% FBS (v / v) (Gibco FBS, Australia; Thermo Fisher Scientific, Waltham, MA; Cat. No. 10099141, Lot No. 2177370). Luciferase levels in culture supernatants were quantified 48 hours after the addition of 100 ng / mL recombinant human EGF (PeproTech EC, Cat. No. AF-100-15) as described above. For (-) conditions, pcDNA3.1(+) (Invitrogen, CA; Cat. No. V79020) was used instead of the expression vector for transfection. The results showed that the increase in reporter levels was associated with the increase in MAPK activity induced by EGF ( Figure 11 In the future, similar sensors could be easily engineered for other signaling pathways by replacing the pE59 (SEQ-ID NO: 110) and ERK2 (SEQ-ID NO: 85) domains with other signal-specific PPI systems. This would facilitate cell-based applications for monitoring signaling pathway dynamics in real time, whereas current techniques (e.g., genetically encoded fluorescent reporters and / or Western blot analysis) are limited to visualization-centric studies and endpoint measurements, respectively.

[0554] Based on these results, we believe that mRNA circularization can also be triggered by engineering 5'-cap replacements at the 5'-UTR, similar to how poly(A) replacements work in the 3'-UTR. In this case, the shRNA or HHR-based RNA cleavage site would be placed directly downstream of the guanine-rich 5'-cap and upstream of the RBP-specific adaptor region to allow preprogrammed cap removal ( Figure 12 Thus, the aptamer region will act as a 5'-cap surrogate to recruit the same STIF constructs described in Tables 1, 3, and 4 for constitutive or trigger-induced initiation of mRNA circularization and target gene translation ( Figure 12 In this case, the corresponding target gene mRNA will contain the following structure:

[0555] Table 5: Synthetic mRNA transcripts containing RBP-specific 5'-cap substitutions enabling STIF-dependent translation and expression of different genes of interest (GOIs).

[0556]

[0557]

[0558] Example 2: Grazoprevir-controlled gene expression in mammalian cells.

[0559] A trigger-inducible translation control system based on STIF-mediated mRNA circularization Figure 7 and Table 4) can realize a variety of cell-based applications. For example, based on the grazoprevir-inducible GNCR:NS3a system ( Figure 10 ), we show how to generate clinically relevant gene switches for regulating the activity of a variety of mammalian cells or for long-term therapeutic transgene delivery in vivo. Grazoprevir-inducible gene switches have two major advantages. First, grazoprevir is an FDA-approved drug for the treatment of hepatitis C and is therefore bioavailable, non-toxic, and metabolically inert, and should be suitable for regulating a variety of protein therapeutics without interfering with their efficacy in vivo. At the same time, translation-based gene switches are designed to be compatible with a variety of clinically approved gene therapy products; they can be administered to patients using AAV vectors for DNA-encoded therapies, or formulated directly into in vitro prepared mRNA drugs ( Figure 13 ).

[0560] In order to use Figure 7 By fine-tuning the design strategies and building blocks described in Table 4 for the grazoprevir-inducible gene switch, we demonstrated that switching between the L7Ae-NS3a / GNCR-NSP3 and L7Ae-GNCR / NS3a-NSP3 configurations ( Figure 14 A) and increase GNCR (SEQ-ID NO: 90) ( Figure 14 B) and NS3a tandem repeats (SEQ-ID NO: 104) ( Figure 14C) are important choices for increasing the fold change in gene expression triggered by grazoprevir. In the experiments, HEK-293 cells were transfected with plasmids encoding SEAP mRNA containing L7Ae-specific poly(A) surrogates (SEQ-ID NOs: 126 & 134) and different grazoprevir-regulated L7Ae- and NSP3-fusion proteins (SEQ-ID NOs: 36 & 62 or 39 & 28), different NSP3 fusion proteins containing one (SEQ-ID NO: 28), two (SEQ-ID NO: 29), or three N-terminal GNCR repeats (SEQ-ID NO: 30), or different L7Ae fusion proteins consisting of one (SEQ-ID NO: 39), two (SEQ-ID NO: 40), or three C-terminal NS3a repeats (SEQ-ID NO: 41). Specifically, the constructs were validated by transfecting the encoding plasmids into HEK-293 cells, whose cell culture and transfection methods are described above in the Methods section. SEAP levels in the culture supernatant were recorded 48 hours after the addition of 0.1 μM grazoprevir in DMSO. Finally, the split-STIF constructs, each containing three tandem repeats of GNCR and NS3a ((GNCR)3-NSP3, SEQ ID NO: 30), combined with L7Ae-(NS3a)3 (SEQ-ID NO: 41, specific for target mRNA containing C / D-box) or MCP-(NS3a)3 (SEQ-ID NO: 55, specific for target mRNA containing MS2-box) showed no significant difference in fold change ( Figure 15 A), dose dependence ( Figure 15 B, 15C) and activation kinetics ( Figure 16 ) showed the best regulation performance.

[0561] For the L7Ae-based system, HEK-293 cells were co-transfected with plasmids encoding SEAP-mRNA (SEQ-ID NO: 126 & 134) containing a C / D-box-based poly(A) surrogate, (GNCR)3-NSP3 (SEQ-ID NO: 30), and L7Ae-(NS3a)3 (SEQ-ID NO: 41). For the MCP-based system, HEK-293 cells were co-transfected with plasmids encoding SEAP-mRNA (SEQ-ID NO: 137) containing an MS2-box-based poly(A) surrogate, (GNCR)3-NSP3 (SEQ-ID NO: 30), and MCP-(NS3a)3 (SEQ-ID NO: 55). For fold change analysis ( Figure 15A, 16B), SEAP levels in culture supernatants were recorded 48 hours after addition of 0.1 μM grazoprevir in DMSO (vehicle control). For dose-dependent analysis ( Figure 15 B, 15C), SEAP levels in culture supernatants were recorded 48 hours after addition of different concentrations of grazoprevir dissolved in DMSO (vehicle control). For activation rate analysis ( Figure 16 A), SEAP levels in culture supernatants were recorded 24 hours after addition of different concentrations of grazoprevir. Thus, we could demonstrate that the STIF-based grazoprevir-inducible translational gene switch, by expressing TetR-NS3a (SEQ-ID NO: 299), (GNCR)3-VP64 (SEQ-ID NO: 300), and a TetR-inducible promoter (SEQ-ID NO: 168) driving SEAP transcription, is significantly faster than the corresponding state-of-the-art gene switch operating at the transcriptional level, resulting in not only higher fold induction over the entire 24-hour experimental time span ( Figure 16 A), and produced similar absolute expression intensities over longer time spans ( Figure 16 B). Importantly, we used co-immunoprecipitation and Western blotting techniques (e.g. Figure 6 A and described in the Methods section above) to confirm that endogenous (GNCR) 3-NSP3-specific eIF4G and eIF4E colocalized with RBP-(NS3a)3 (SEQ-IDNOs: 48 & 263) only in the presence of grazoprevir ( Figure 17 ), which is the mechanistic basis for triggering the induction of the circularized mRNA configuration. This experiment is also critical for identifying the gene regulatory system developed based on the present invention; if the circularized mRNA configuration triggered by grazoprevir is not formed, eIF4G (detected by anti-eIF4G antibodies such as Cell Signaling Technology's Product No. 2498, Lot 4), eIF4E (detected by anti-eIF4E antibodies such as Cell Signaling Technology's Product No. 2067, Lot 8), and HA-tagged NSP3 (detected by anti-HA antibodies such as Cell Signaling Technology's Product No. 3724, Lot 10) will not co-localize with FLAG-tagged L7Ae or MCP constructs. Therefore, demonstrating that this co-localization is strictly dependent on the presence of a user-defined trigger signal (in this case, grazoprevir) is key to demonstrating the concept of future regulatory systems using the STIF-based strategy developed in this work (Example 1).

[0562] Example 3: In vivo gene therapy regulated by grazoprevir.

[0563] In this example, we used the grazoprevir inducible translational control system (described in Example 2) to demonstrate how STIF-mediated gene switches can be developed into clinically qualified gene therapy products for in vivo applications. Figure 13 ), the translational regulatory system can be administered to patients using AAV vectors for DNA-encoded therapeutics or formulated directly into in vitro prepared mRNA drugs. Thus, we show that our grazoprevir-inducible translational gene switch does indeed work with either DNA-encoded ( Figure 15 C, 16A) and RNA-centric delivery strategies ( Figure 18 A) compatible. To demonstrate grazoprevir-inducible SEAP translation via mRNA delivery, HEK-293 cells were (co-)transfected with in vitro transcribed mRNA encoding MCP-(NS3a)3 (SEQ-ID NO: 55, from pSL1085), (GNCR)3-NSP3 (SEQ-ID NO: 30, from pYW361), and a SEAP expression vector containing an MCP-specific poly(A) surrogate (SEQ-ID NO: 137, from pSL468). In vitro transcription was performed using the T7 High Yield RNA Transcription Kit (Vazyme Biotech, Nanjing, China; Cat. No. TR-101), with detailed setup described in the Methods section above. To demonstrate grazoprevir-induced SEAP translation via DNA delivery, HEK-293 cells were transfected with plasmids encoding the corresponding constructs (e.g., (GNCR)3-NSP3, MCP-(NS3a)3, and SEAP-mRNA containing a poly(A) replacement based on the MS2-box; SEQ-ID NOs: 30, 55, and 137). SEAP levels in the culture supernatant were quantified 48 hours after the addition of grazoprevir. In direct comparison, DNA-based delivery facilitates various long-term applications in mammalian cells, such as the selection of stable cell lines, enabling reversible sensing and response kinetics over extended periods of time ( Figure 18 B) and / or integrate the genetic components into AAV-based vectors for various therapeutic purposes in vivo. To conduct reversibility studies, we first created stably transgenic HEK-293 LSCCS1Cell line for grazoprevir-triggered co-expression of (SEQ-ID NO: 101) and murine insulin (mINS; SEQ-ID NO: 99). Specifically, pSL721 (a Sleeping Beauty (SB)-specific transposon expressing (MCP-(NS3a)3, SEQ-ID NO: 55), pSL722 (a SB-specific transposon expressing (GNCR)3-NSP3, SEQ-ID NO: 30), pSL688 (a SB-specific transposon expressing NanoLuc and mINS mRNA with an MCP-specific poly(A) replacement, SEQ-ID NO: 140), and pCMV-T7-SB100 (Addgene plasmid number 34879, for constitutive expression of SB transposase) were transfected into HEK-293 cells and then treated with 1 μg / ml puromycin (ThermoFisher Scientific; Cat. No. A1113803), 10 μg / ml blasticidin (Thermo Fisher Scientific; Cat. No. R21001), and 100 μg / ml bleomycin (Thermo Fisher Scientific; Cat. No. 21 monoclonal cell lines were harvested and selected by quantifying NanoLuc expression after 24 hours of treatment with 500 nM grazoprevir. LSCCS1 Cultures were maintained for 7 days, with the level of grazoprevir in the culture medium continuously shifted between 0 and 500 nM by medium exchange and washed three times with medium without grazoprevir. NanoLuc levels were measured every 12 hours. Cell density was readjusted to 1 × 10 cells every 2–3 days. 5 For AAV production, the transfer plasmids pSL511 (expressing SEAP-mRNA with an MCP-specific poly(A) replacement, SEQ-ID NO: 137), pSL512 (expressing MCP-(NS3a)3, SEQ-ID NO: 55), and pSL446 (expressing (GNCR)3-NSP3, SEQ-ID NO: 30) were constructed as described in the methods section above to produce the corresponding AAV2 / 8-(GNCR)3-NSP3, AAV2 / 8-MCP-(NS3a)3, and AAV2 / 8-SEAP-(MS2-box) 24 -HHR-pA particles.

[0564] To evaluate grazoprevir-inducible transgene regulation in vivo, we hydrodynamically injected a vector encoding STIF and an L7Ae-specific SEAP expression vector into the tail vein of mice ( Figure 19A). Specifically, as described in the methods section above, 300 μg of plasmids containing pLZ74 (encoding (GNCR)3-NSP3, SEQ-ID NO: 30), pLZ76 (encoding L7Ae-(NS3a)3, SEQ-ID NO: 41), and pSL355 (SEQ-ID NO: 136: for HHR-mediated cis-removal of poly(A)) or pSL88 & pSL4 (SEQ-IDs NO: 126 and 134: for shRNA-216-mediated trans-removal of poly(A)) were administered to C57BL / 6 mice by hydrodynamic tail vein injection. Six hours later, mice received the first intraperitoneal injection of grazoprevir (1 mg / kg, dissolved in PBS) three times a day. SEAP levels in the bloodstream of mice were measured 24 hours after the first injection of grazoprevir. According to our experimental results ( Figure 19 A), HHR-dependent reporter (SEQ-ID NO: 136) was identified as a class of STIF-specific target mRNAs for in vivo applications ( Figure 19 A), where an oral dose of 3 mg / kg grazoprevir is sufficient to fully activate this system ( Figure 19 B, 19C). Next, using insulin as an exemplary therapeutic output, we tested the therapeutic potential of the grazoprevir-inducible gene switch ( Figure 19 D). First, the therapeutic window of insulin expression was determined by co-transfecting HEK-293 cells with 200 ng of pSL1042 (expressing MCP-(NS3a)3, SEQ-ID NO: 55), 200 ng of pSL1032 (expressing (GNCR)3-NSP3, SEQ-ID NO: 30), and varying amounts of pSL1003 (expressing NanoLuc and mINS-mRNA containing an MCP-specific poly(A) surrogate, SEQ-ID NO: 174). NanoLuc and mINS levels were recorded in the culture supernatant 48 hours after the addition of 0.5 μM grazoprevir. Thus, a gene switch configuration in which the grazoprevir-triggered ON state reached physiological insulin levels ( Figure 19The blue shaded box in D) shows that the basal OFF state decreases below this threshold (50 ng of pSL1003). In line with this, plasmids encoding MCP-(NS3a)3, (GNCR)3-NSP3, and insulin-mRNA containing an MCP-specific poly(A) surrogate (SEQ-ID NOs: 30, 55, and 140) were hydrodynamically injected into the tail vein of type 1 diabetic (T1D) mice. Six hours after injection, mice were first fed with 3 mg / kg grazoprevir three times a day, and then blood insulin levels in mice were measured 20 hours after the first administration of grazoprevir ( Figure 20 A) and fasting blood glucose ( Figure 20 B). Intraperitoneal glucose tolerance test (GTT) was also performed 24 hours after the first administration of grazoprevir (4 hours after blood insulin quantification according to the experimental procedure described in the Methods section above). In summary, our results show that oral administration of grazoprevir almost completely corrected insulin deficiency in type 1 diabetic mice treated with gene therapy ( Figure 20 A) Hyperglycemia Figure 20 B) and glucose intolerance ( Figure 20 C), the gene therapy consisted of genetic components for grazoprevir-inducible insulin translation (i.e., MCP-(NS3a)3, SEQ-ID NO: 55; (GNCR)3-NSP3, SEQ-ID NO: 30; and mINS-mRNA containing MCP-specific poly(A) surrogates, SEQ-ID NO: 140 & 174). To test the long-term control of grazoprevir-inducible SEAP production in vivo, C57BL / 6 mice were injected intravenously with 7×10 11 AAV2 / 8-(GNCR)3-NSP3, AAV2 / 8-MCP-(NS3a)3 and AAV2 / 8-SEAP-(MS2-box) 24 -HHR-pA particles, constitutively expressing GNCR)3-NSP3 (SEQ-ID NO: 30), MCP-(NS3a)3 (SEQ-ID NO: 55) and SEAP-mRNA containing an MCP-specific poly(A) surrogate (SEQ-ID NO: 137), and then monitoring SEAP production in the bloodstream for 10 weeks. 24 hours before each measurement, mice were first fed 3 mg / kg of grazoprevir three times a day. The results showed that mice injected with AAV2 / 8 particles carrying the grazoprevir-inducible gene switch maintained the expected regulatory protein secretion profile for at least 10 weeks, indicating the potential for long-term therapeutic efficacy in vivo ( Figure 20 D). Thus, we were able to engineer a grazoprevir-inducible gene switch based on the high-affinity grazoprevir:NS3a interaction (K i140 pM (Foight et al., 2019), demonstrating rapid, tightly controlled activation kinetics in vitro and compatibility with therapeutic transgene delivery in vivo. Furthermore, we show that STIF-dependent gene switches are compatible with state-of-the-art gene therapy delivery strategies.

[0565] Example 4: Glazoprevir-controlled gene expression can be used for complex biological computation in mammalian cells and mice.

[0566] Engineering genetic circuits composed of interconnected three-state buffers to achieve complex biological computations in mammalian cells has long been an elusive goal. However, the genetic implementation of three-state buffers has been hampered by the lack of robust and mutually compatible genetic switches that can be integrated into complex genetic networks. In a three-state buffer, the connectivity of a binary switch regulated by input A must be strictly controlled by an upstream switch through another signal B ( Figure 21 A). Thus, control input B allows data input A to determine the overall activity Y of the circuit, unless B is deactivated or "unplugged" by the upstream switch. In this inactive state (NOT B), the overall activity of the circuit will fall into a third "high impedance" state Z, which no longer depends on the state of A (0 or 1 value) ( Figure 21 A). To implement this tri-state buffer in mammalian cells, it is necessary to control the expression of a trigger-inducible gene switch programmed with "buffer" (BUF) and "invert" (NOT) signal processing logic. Therefore, the NS3a / GNCR / ANR triplet controlled by grazoprevir (SEQ-ID NO: 73, 90 & 104: in Tables 3 & 4; Figure 8-10 ) into the STIF-based translational regulatory framework and would be an ideal candidate for creating potential BUF and NOT switches ( Figure 21 B). The grazoprevir-inducible translational gene switches designed in Examples 2 and 3 essentially follow the BUF logic, wherein SEAP-mRNA (SEQ-ID NO: 137) and (GNCR)3-NSP3 (SEQ-ID NO: 30) containing MCP-specific poly(A) replacements are combined with MCP-NS3a (SEQ-ID NO: 54) or MCP-NS3a (H1) (SEQ-ID NO: 56) to constitute the relevant genetic components ( Figure 22A). To engineer a grazoprevir-repressible translational gene switch using the NOT logic circuit, HEK-293 cells were co-transfected with plasmids encoding a fusion protein between SEAP-mRNA (SEQ-ID NO: 137), (ANR)4-NSP3 (SEQ-ID NO: 3), and either NS3a (producing MCP-NS3a, SEQ-ID NO: 54) or NS3a(H1) (producing MCP-NS3a(H1), SEQ-ID NO: 56) containing an MCP-specific poly(A) replacement. SEAP levels in culture supernatants were scored 48 h after addition of 0.5 μM grazoprevir in DMSO (vehicle control). Figure 22 B). Therefore, STIF was engineered to contain GNCR (SEQ-ID NO:90) and NSP3 (SEQ-ID NO:106), enabling grazoprevir-inducible translation (BUF switch), while fusion of the ANR motif (SEQ-ID NO:73) to NSP3 (SEQ-ID NO:106) resulted in grazoprevir-repressible translation (NOT switch) ( Figure 23 A). In addition, by increasing the number of NS3a(H1) tandem repeats (SEQ-ID NO: 105) added to MCP (SEQ-ID NO: 98), the corresponding BUF-switch ( Figure 23 B) and NOT-switch ( Figure 23 Specifically, HEK-293 cells were co-transfected with plasmids encoding: MCP-specific SEAP mRNA (SEQ-ID NO: 137), (GNCR)3-NSP3 (SEQ-ID NO: 30), and different MCP fusion proteins consisting of one (SEQ-ID NO: 56), two (SEQ-ID NO: 57), or three C-terminal NS3a (H1) repeats (SEQ-ID NO: 58). Figure 23 B); or co-transfected with plasmids encoding: MCP-specific SEAP mRNA (SEQ-ID NO: 137), (ANR)4-NSP3 (SEQ-ID NO: 3), and different MCP fusion proteins consisting of one (SEQ-ID NO: 56), two (SEQ-ID NO: 57), or three C-terminal NS3a (H1) repeats (SEQ-ID NO: 58). Figure 23 C) SEAP levels in culture supernatants were recorded 48 h after addition of 0.5 μM grazoprevir in DMSO (vehicle control).

[0567] To form a tri-state buffer ( Figure 21A), the expression of these grazoprevir-controlled BUF and NOT switches must be regulated by an upstream gene switch, which in turn is controlled by another control input B ( Figure 21 B). By default, this upstream gene switch can generate an inverted output signal (called a "low-level active" control signal generated by the "IF0" switch) or a non-inverted output signal (called a "high-level active" control signal generated by the "IF1" switch). Therefore, a tri-state buffer can contain up to 4 types of gene switches: B activates STIF expression (IF1), B terminates STIF expression (IF0), A activates target protein expression (BUF), and A terminates target protein expression (NOT). For implementation in mammalian cells, it is necessary that the antagonistic upstream switches IF0 and IF1 are orthogonal to each other and are controlled by the same trigger signal at the same time ( Figure 24 A). For example, a vanillic acid-inducible gene switch based on a PKA / CREB1-responsive promoter activated by cAMP signaling regulated by the olfactory receptor MOR9-1 (Saxena et al., 2016) could be a potential IF1 switch, while an IF0 switch could be accomplished by regulating gene expression from a homologous VanO-containing promoter via the VanR-dependent mammalian transactivator (VanR-VP64) (Gitzinger et al., 2012). Figure 24 B), HEK-293 cells were transfected with a constitutive MOR9-1 expression vector (SEQ-ID NO: 208) and a cAMP-responsive SEAP expression vector (SEQ-ID NO: 172). Figure 24 C), HEK-293 cells were transfected with a constitutive VanR-VP64 expression vector (SEQ-ID NO: 296) and a vanillic acid-inducible SEAP expression vector (SEQ-ID NO: 171). After incubation in medium containing 0 or 400 μM vanillic acid (dissolved in DMSO), SEAP levels in the culture supernatant were recorded 48 hours after transfection. To demonstrate the parallel and orthogonal operation of IF1 and IF0 in mammalian cells, HEK-293 cells were further transfected with MOR9-1 (SEQ-ID NO: 208) and VanR-VP64 (SEQ-ID NO: 296) constitutive expression vectors, a cAMP-responsive SEAP expression vector (SEQ-ID NO: 172), and a VanR-specific NanoLuc expression vector (SEQ-ID NO: 170). After 48 hours of incubation in cell culture medium containing various concentrations of vanillic acid, SEAP levels in the culture supernatant were recorded. The results showed that the IF0 and IF1 switches triggered by vanillic acid did not crosstalk with each other when introduced into the same cells ( Figure 24D), thus meeting the eligibility requirements for upstream gene switches in three-state-based gene circuits ( Figure 24 A).

[0568] Using tri-state buffers, the same set of upstream IF1 / IF0 switches can be flexibly combined in parallel with multiple sets of downstream BUF / NOT switches, thereby achieving resource-efficient data transmission without sacrificing switching speed. Figure 23 A; named BUF1 / NOT1), we also created two other sets of grazoprevir-responsive gene switches that can be combined with (vanillic acid-regulated) IF1 / IF0 switching ( Figure 25 A). In one group (called BUF2 / NOT2), the mutually exclusive triad NS3a(H1) / GNCR / ANR (SEQ-ID NOs: 73, 90 & 105) was introduced into the framework of synthetic GEMS receptors (Scheller et al., 2018). GEMS receptors typically contain an antibody-derived extracellular ligand-binding domain, an EpoR-derived transmembrane domain (GEMS TM , SEQ-ID NO: 182) and an intracellular signaling domain that mediates the activation of different signaling pathways in human cells upon dimerization of cell surface receptors. To generate GEMS-based BUF / NOT switches regulated by grazoprevir, we replaced the antibody domain of the traditional GEMS construct with NS3a (H1) (SEQ-ID NO: 105), GNCR (SEQ-ID NO: 90) or ANR (SEQ-ID NO: 73) Figure 25 B) Each GEMS variant was subsequently tested for different intracellular signaling domains, such as IL-6RB m (triggering JAK / STAT3 signaling, SEQ-ID NO: 184), FGFR1 int (triggering MAPK signaling, SEQ-ID NO: 186) or VEGFR2 int (triggering NFAT signaling, SEQ-ID NO: 185). For grazoprevir-inducible target gene expression by GEMS variants containing an IL6RB-derived intracellular domain, a STAT3-specific SEAP expression vector (SEQ-ID NO: 169) and a STAT3-specific SEAP expression vector for the corresponding GEMS were used. NS3a(H1) and GEMS GNCRHEK-293 cells were co-transfected with the constitutive expression vectors of the constructs (SEQ-ID NOs: 191 & 192). For grazoprevir-inducible target gene expression by GEMS variants containing the intracellular domain derived from FGFR1, a TetR-specific SEAP expression vector (SEQ-ID NO: 168) and a TetR-Elk1 (SEQ-ID NO: 199) vector were used in combination with the corresponding GEMS. NS3a(H1) and GEMS GNCR Cells were co-transfected with the constitutive expression vectors of the constructs (SEQ-ID NO: 193 & 194). For grazoprevir-inducible target gene expression by GEMS variants containing a VEGFR-derived intracellular domain, a calcium-inducible SEAP expression vector (SEQ-ID NO: 167) and a vector for the corresponding GEMS were used. NS3a(H1) and GEMS GNCR Cells were co-transfected with the constitutive expression vectors of the constructs (SEQ-ID NO: 195 & 196). For grazoprevir-repressible target gene expression by GEMS variants containing an intracellular domain derived from IL6RB, a STAT3-specific SEAP expression vector (SEQ-ID NO: 169) and a vector for the corresponding GEMS were used. NS3a(H1) and GEMS ANR Cells were co-transfected with the constitutive expression vectors of the constructs (SEQ-ID NO: 187 & 192). For grazoprevir-repressible target gene expression by GEMS variants containing an intracellular domain derived from FGFR1, a TetR-specific SEAP expression vector (SEQ-ID NO: 168) and a TetR-Elk1 (SEQ-ID NO: 199) vector were used in conjunction with the corresponding GEMS. NS3a(H1) and GEMS ANR Cells were co-transfected with the constitutive expression vectors of the constructs (SEQ-ID NO: 189 & 194). For grazoprevir-repressible target gene expression by GEMS variants containing a VEGFR-derived intracellular domain, calcium-inducible SEAP expression vectors (SEQ-ID NO: 167) and the vectors for the corresponding GEMS were used. NS3a(H1) and GEMS ANR The cells were co-transfected with the constitutive expression vectors of the constructs (SEQ-ID NO: 190 & 196). 48 h after the addition of 10 μM grazoprevir dissolved in DMSO (vehicle control), the SEAP levels in the culture supernatant were recorded. Figure 25 The experimental results shown in B) were obtained by NS3a(H1)-GEMS TM -IL-6RB m (named GEMS NS3a(H1) , SEQ-ID NO: 192), GNCR-GEMSTM -IL-6RB m (named GEMS GNCR , SEQ-ID NO: 191) and a reporter gene expression vector driven by a synthetic STAT3-specific promoter (SEQ-ID NO: 164 & 169) were co-transfected to establish a grazoprevir-inducible BUF2 switch. Similarly, a new grazoprevir-repressible NOT2 switch contains (ANR) n -GEMS TM -IL-6RB m Instead of GEMSGNCR (named GEMS ANR , SEQ-IDNO: 187&188; n can be any number between 1 and 1000, most preferably 4 or 8) ( Figure 25 To construct a third set of BUF / NOT switches regulated by grazoprevir, we utilized the NS3a-containing self-cleaving degradation determinant (degron) StaPLd (SEQ-ID NO: 116). Since StaPLd triggers autolysis of each polypeptide construct in which it is located, the engineered transcription factors PcaV-StaPL-VP64 (SEQ-ID NO: 198) and PcaV-StaPL-KRAB (SEQ-ID NO: 197) will undergo spontaneous degradation unless grazoprevir is present to inhibit the self-cleavage activity of StaPLd by binding to its NS3a-domain ( Figure 25 Thus, grazoprevir enables PcaV-StaPL-VP64 to transactivate a minimal PcaV-specific promoter in a typical BUF3 manner, while NOT3 results from grazoprevir-dependent silencing of a constitutive promoter harboring a binding site for PcaV-StaPL-KRAB ( Figure 25 C). For grazoprevir-repressible gene expression (NOT3), HEK-293 cells were co-transfected with a constitutive PcaV-StaPL-KRAB expression vector (SEQ-ID NO: 197) and a PcaV-repressible SEAP expression vector (EQ-ID NO: 166). For grazoprevir-inducible gene expression (BUF3), HEK-293 cells were co-transfected with a constitutive PcaV-StaPL-VP64 expression vector (SEQ-ID NO: 198) and a PcaV-specific SEAP expression vector (EQ-ID NO: 165). SEAP levels in culture supernatants were quantified 48 hours after addition of 10 μM grazoprevir.

[0569] In order to combine different groups of BUF n / NOT nWhen integrating the switches into the same three-state based gene circuit, it is essential that there is no signal crosstalk between each individual set of gene switches when regulating different output modules in parallel. Figure 26 A). Therefore, we co-expressed the NOT1 switch that controls secreted alkaline phosphatase (SEAP, SEQ-ID NO: 114) as the first reporter gene and the BUF2 switch that controls secreted nanoluciferase (NLuc, SEQ-ID NO: 101) as the second reporter gene. We also co-expressed the NOT2 switch that controls NLuc and the BUF1 switch that controls SEAP in the same cells ( Figure 26 B). Specifically, HEK-293 cells were co-transfected with a plasmid encoding the grazoprevir-regulated BUF2 switch (GNCR-GEMS) driving NanoLuc expression. IL6RB &NS3a(H1)-GEMS IL6RB &P STAT3 -NanoLuc; SEQ-ID NO: 191, 192 & 164) and a NOT1 switch regulated by grazoprevir that drives SEAP expression ((ANR)8-NSP3 & MCP-(NS3a(H1))3 & SEAP-mRNA with MCP-specific poly(A) replacement; SEQ-ID NO: 5, 58 & 137). Similarly, HEK-293 cells were co-transfected with a plasmid encoding a NOT2 switch regulated by grazoprevir that drives NanoLuc expression (ANR-GEMS IL6RB &NS3a(H1)-GEMS IL6RB & STAT3-specific NanoLuc expression vector; SEQ-ID NO: 188, 192 & 164)) and a BUF1 switch regulated by grazoprevir to drive SEAP expression ((GNCR)3-NSP3 & MCP-(NS3a(H1))3 & SEAP-mRNA with MCP-specific poly(A) replacement; SEQ-ID NO: 30, 58 & 137). After 48 hours of culture in cell culture medium containing 0 or 10 μM grazoprevir, the levels of SEAP and NanoLuc in the culture supernatant were recorded. Similar experiments were also performed to demonstrate non-interfering operation between NOT1 & BUF3 and NOT3 & BUF1 ( Figure 26C). For example, a grazoprevir-regulated BUF3 switch (SEQ-ID NO: 198 & 165) driving SEAP expression was co-administered to HEK-293 cells with a grazoprevir-regulated NOT1 switch (SEQ-ID NO: 5, 58 & 163) driving NanoLuc expression. Furthermore, a grazoprevir-regulated BUF3 switch (SEQ-ID NO: 197 & 166) driving SEAP expression was co-administered to HEK-293 cells with a grazoprevir-regulated NOT1 switch (SEQ-ID NO: 30, 58 & 163) driving NanoLuc expression. After 48 hours of culture in cell culture medium containing 0 or 10 μM grazoprevir, the levels of SEAP and NanoLuc were recorded in the culture supernatant. The results showed that each individual switch operated in a highly autonomous manner when triggered by grazoprevir, demonstrating robust and non-interfering performance in mammalian cells.

[0570] Because IF1 and BUF2 / NOT2 utilize distinct intracellular signaling pathways in mammalian cells, we needed to test for potential signaling crosstalk between their key components before inserting a new set of grazoprevir-regulated BUF / NOT switches into the output lines of the vanillic acid-controlled upstream module. To this end, a vanillic acid-inducible gene switch (IF1; SEQ-ID NO: 172) controlling SEAP expression was co-administered with a grazoprevir-regulated GEMS-based BUF switch (SEQ-ID NO: 191, 192 & 164) driving NanoLuc expression to HEK-293 cells stably expressing MOR9-1 (HEK-MOR9(C0)). The cells were then cultured in cell culture medium containing vanillic acid (VA, 400 μM) and / or grazoprevir (Gra, 10 μM). SEAP levels were recorded in the culture supernatant 48 hours after transfection. The experiments demonstrated that these IF1 switches controlling SEAP expression could operate in parallel with the BUF2 switch driving NanoLuc expression in the same cells. This not only supports the feasibility of generating a tri-state buffer but also generally suggests a potential orthogonality between intracellular cAMP and STAT3 signaling from a cell biology perspective ( Figure 26 D).

[0571] Finally, we connected the vanillic acid (VA)-regulated IF0 and IF1 switches (upstream module) and the glazoprevir (Gra)-regulated BUF and NOT switches (downstream module), resulting in four different types of tri-state buffers: BUFIF1 (where IF1 regulates the active-high buffer of BUF), NOTIF1 (where IF1 regulates the active-high inverting buffer of NOT), BUFIF0 (where IF0 regulates the active-low buffer of BUF), and NOTIF0 (where IF0 regulates the active-low inverting buffer of NOT) ( Figure 27 A). In a biological context, BUFIF1 shows logical similarity to a traditional AND gate, while NOTIF0 is logically similar to a traditional NOR gate. Similarly, NOTIF1 and BUFIF0 show typical gene expression signatures of two variants of the NIMPLY (AND NOT) gate ( Figure 27 B). For BUFIF1, HEK-293 cells were co-transfected with a plasmid encoding MCP-specific EGFP mRNA (SEQ-ID NO: 149), constitutive expression vectors for MCP-(NS3a(H1))3 (SEQ-ID NO: 58) and MOR9-1 (SEQ-ID NO: 208), and a cAMP-responsive (GNCR) 3-NSP3 expression vector (SEQ-ID NO: 162). For NOTIF0, cells were co-transfected with SEQ-ID NO: 149, constitutive expression vectors for MCP-(NS3a(H1))3 (SEQ-ID NO: 58) and VanR-VP64 (SEQ-ID NO: 296), and a vanillic acid-responsive (ANR) 4-NSP3 expression vector (SEQ-ID NO: 161). For NOTIF1, cells were co-transfected with constitutive expression vectors of SEQ-ID NO: 149, MCP-(NS3a(H1))3 (SEQ-ID NO: 58), and MOR9-1 (SEQ-ID NO: 208), as well as a cAMP-responsive (ANR) 4-NSP3 expression vector (SEQ-ID NO: 160). For BUFIFO, cells were co-transfected with constitutive expression vectors of SEQ-ID NO: 149, MCP-(NS3a(H1))3 (SEQ-ID NO: 58), and VanR-VP64 (SEQ-ID NO: 296), as well as a vanillic acid-responsive (GNCR) 3-NSP3 expression vector (SEQ-ID NO: 159). Six hours after transfection, vanillic acid (VA, 400 μM) and grazoprevir (Gra, 0.5 μM) were added. 36 hours after transfection, fluorescence images showing EGFP signals were acquired (scale bar: 100 μM), and flow cytometry analysis was performed using 10,000 cells per group ( Figure 28The same procedure can be performed using a second set of grazoprevir (Gra)-regulated BUF and NOT switches (BUF2 / NOT2) ( Figure 27 C) For BUF2IF1, a STAT3-specific SEAP expression vector (SEQ-ID NO: 169) and a GEMS GNCR (SEQ-ID NO: 158) and GEMS NS3a(H1) HEK-293 cells stably expressing MOR9-1 (HEK-MOR9(C0)) were co-transfected with a cAMP-responsive expression vector (SEQ-ID NO: 157). For NOT2IF0, SEQ-ID NO: 169, a constitutive VanR-VP64 (SEQ-ID NO: 296) expression vector and GEMS NS3a(H1) (SEQ-ID NO: 156) and GEMS ANR HEK-293 cells were co-transfected with a vanillic acid responsive expression vector (SEQ-ID NO: 155). ANR (SEQ-ID NO: 154) and GEMS NS3a(H1) HEK-MOR9 (C0) cells were co-transfected with a cAMP-responsive expression vector (SEQ-ID NO: 157). For BUF2IF0, SEQ-ID NO: 169, a constitutive VanR-VP64 (SEQ-ID NO: 296) expression vector and GEMS NS3a(H1) (SEQ-ID NO: 156) and GEMS GNCR HEK-293 cells were co-transfected with a vanillic acid-responsive expression vector (SEQ-ID NO: 313). After culture in a medium containing vanillic acid (VA, 400 μM) and / or grazoprevir (Gra, 10 μM), SEAP levels in the culture supernatant were recorded 48 hours after transfection. n / NOT n The combination of switches creates four tri-state buffers, BUFIF1, NOTIF1, BUFIF0, and NOTIF0, whose logic is analogous to the AND, NOR, and IMPLY gates in mammalian cells. This sets the stage for designing a wide variety of complex mammalian cell functions of interest, similar to the computational logic of any 2-input, 1-output Boolean logic gate or 2-input, 2-output Boolean operation. For example, the combination of NOTIF1 with another BUF switch creates a gene circuit that displays the expression profile of a conventional OR gate ( Figure 29). In the experiment, HEK-293 cells were transfected with a (GNCR)3-NSP3 constitutive expression vector (SEQ-ID NO:30) and a cAMP-responsive (ANR)8-NSP3 expression vector (SEQ-ID NO:153). Similarly, NAND gate-like logic was achieved by adding NOT to BUFIF0 (HEK-293 cells were transfected with (ANR)8-NSP3 (SEQ-ID NO:5) and VanR-VP64 (SEQ-ID NO:296) constitutive expression vectors and a vanillic acid-responsive (GNCR)3-NSP3 expression vector (SEQ-ID NO:152)), while the combination of NOT with BUFIF1 or adding BUF to NOTIF0 produced two variants of the IMPLY gate ( Figure 29 For Gra IMPLY VA logic, cells were transfected with a constitutive expression vector for (ANR)8-NSP3 (SEQ-ID NO: 5) and a cAMP-responsive (GNCR)3-NSP3 expression vector (SEQ-ID NO: 162). For VA IMPLY Gra logic, cells were co-transfected with constitutive expression vectors for (GNCR)3-NSP3 (SEQ-ID NO: 30) and VanR-VP64 (SEQ-ID NO: 296) and a vanillic acid-responsive (ANR)8-NSP3 expression vector (SEQ-ID NO: 151). XOR logic was achieved by combining BUFIFO with NOTIF1 (HEK-293 cells were transfected with a constitutive VanR-VP64 (SEQ-ID NO: 296) expression vector, a vanillic acid responsive (GNCR) 3-NSP3 expression vector (SEQ-ID NO: 159), and a cAMP responsive (ANR) 4-NSP3 expression vector (SEQ-ID NO: 160)), while XNOR was generated by superposition of NOTIF0 with BUFIF1 (HEK-293 cells were transfected with a constitutive VanR-VP64 (SEQ-ID NO: 296) expression vector, a cAMP-responsive (GNCR) 3-NSP3 expression vector (SEQ-ID NO: 162), and a vanillic acid responsive (ANR) 8-NSP3 expression vector (SEQ-ID NO: 151)) Figure 29). For all circuits, cells were co-transfected with a reporter plasmid encoding MCP-specific EGFP mRNA (SEQ-ID NO: 149) and a constitutive expression vector for MCP-(NS3a(H1))3 (SEQ-ID NO: 58), forming the computational basis for running all the different circuits. All experiments were performed in native HEK-293 cells (by co-transfection with a constitutive MOR9-1 (SEQ-ID NO: 208) expression vector or in HEK-293 cells stably expressing MOR9-1 (HEK-MOR9(C0)). The methods used to generate stable cell lines are as described above. Six hours after transfection, different combinations of vanillic acid (V, 400 μM) and grazoprevir (G, 0.5 μM) were added. 36 hours after transfection, fluorescent images showing the EGFP signal were acquired (scale bar: 100 μm).

[0572] Although Boolean logic gates follow pre-programmed algorithms 39 Converts multiple input signals into a single output signal, but calculators often produce multiple output signals. For example, adders and subtractors are shown in Figures 2 and 3 where each bit is shown to represent a different 2 n The half adder performs Boolean algebra between two or more inputs by performing binary addition of two inputs A and B. For example, a half adder returns the sum S (which represents 2 0 bit) and carry Y (represents 2 1 Similarly, the half subtractor performs binary subtraction of B from A using two different output signals in order to borrow W (representing -1×2 1 digits) and the difference D (representing 2 0 For this reason, tri-state buffers also allow modular and systematic assembly of various Boolean calculators in mammalian cells. For example, a half adder ( Figure 30 A). In the experiment, constitutive expression vectors of VanR-VP64 (SEQ-ID NO: 296), MOR9-1 (SEQ-ID NO: 208), MCP-(NS3a(H1))3 (SEQ-ID NO: 58) and MCP-specific EGFP mRNA (SEQ-ID NO: 149), vanillic acid responsive (GNCR)3-NSP3 expression vector (SEQ-ID NO: 152), (ANR)4-NSP3, GEMS GNCR and GEMS NS3a(H1)HEK-MOR9 (C0) cells were co-transfected with cAMP-responsive expression vectors (SEQ-ID NO: 157, 158 & 160) and STAT3-specific mCherry expression vector (SEQ-ID NO: 150). After 48 hours of culture in medium containing different combinations of vanillic acid (V, 400 μM) and grazoprevir (G, 10 μM), flow cytometric analysis of EGFP- and mCherry signals was performed ( Figure 31 A). Data are shown as weighted fluorescence units as mean ± SD and are representative of three independent experiments. A half-adder was also created based on BUF1IF0, NOT1IF1, and BUF3IF1 ( Figure 32 Specifically, HEK-293 cells were co-transfected with a plasmid encoding EGFP-mRNA (SEQ-ID NO: 149) containing an MCP-specific poly(A) replacement, a constitutive expression vector for MCP-(NS3a(H1))3 (SEQ-ID NO: 58), VanR-VP64 (SEQ-ID NO: 296), and MOR9-1 (SEQ-ID NO: 208), a cAMP-responsive expression vector for (ANR)4-NSP3 and NLS-PcaV-StaPLd-VP64 (SEQ-ID NO: 160 & 130), a vanillic acid-responsive (GNCR)3-NSP3 expression vector (SEQ-ID NO: 159), and a PcaV-specific mCherry expression vector (SEQ-ID NO: 141). 6 hours after transfection, different combinations of vanillic acid (V, 400 μM) and grazoprevir (G, 0.5 μM) were added. 36 hours after transfection, fluorescence images showing EGFP signals were acquired (scale bar: 100 μm), and flow cytometry analysis was performed (10,000 cells per group). In addition, a half-subtractor was assembled by three tri-state buffers BUF1IF0, NOT1IF1, and NOT2IF1 ( Figure 30 B). Specifically, (ANR)4-NSP3, GEMS ANR and GEMS NS3a(H1)HEK-MOR9 (C0) cells were co-transfected with a cAMP-responsive expression vector for STAT3 (SEQ-ID NO: 160, 154 & 157) and a STAT3-specific mCherry expression vector (SEQ-ID NO: 150), a constitutive expression vector for MCP-(NS3a(H1))3 (SEQ-ID NO: 58), VanR-VP64 (SEQ-ID NO: 296) and MOR9-1 (SEQ-ID NO: 208), a vanillic acid-responsive (GNCR)3-NSP3 expression vector (SEQ-ID NO: 159), and an EGFP-mRNA containing an MCP-specific poly(A) surrogate (SEQ-ID NO: 149). After culturing for 48 hours in medium containing different combinations of vanillic acid (V, 400 μM) and grazoprevir (G, 10 μM), flow cytometric analysis of EGFP- and mCherry signals was performed ( Figure 31 B). In vivo, only vanillic acid and not grazoline are functional ( Figure 33 A), possibly due to the transcription-based IFO switch not operating properly in mice ( Figure 33 B). However, in translation (NOT1; Figure 33 C) and cell signaling levels (IF1; Figure 33D) All other components of the operation are fully functional in mice, suggesting that currently used transcription-based gene switches may have limitations in their in vivo applications. To test the vanillic acid AND NOT grazoprevir logic (NOTIF1) in vivo, 420 μg of plasmid DNA (pSL683 / pSL548 / pLYL76 / pLYL67, ratio 2:6:16:3 (w / w / w / w)) was hydrodynamically injected into the tail vein of WT C57BL / 6 mice (generating SEQ-ID NOs: 208, 58, 160 & 139) according to the experimental details described in the Methods section above. Six hours after injection, different combinations of vanillic acid (500 mg / kg / day) and grazoprevir (9 mg / kg / day) were administered by intraperitoneal injection (3 times a day). 24 hours after the first stimulation, the NanoLuc level in the bloodstream of the mice was measured. To test the vanillic acid (VA) repressible IFO switch in vivo, 25 μg of plasmid DNA (pSL175 / pSL173, ratio 3:2 (w / w)) was hydrodynamically injected into the tail vein of WT C57BL / 6 mice. 171 & 296). Six hours after injection, vanillic acid (500 mg / kg / day) dissolved in PBS was administered by intraperitoneal injection (3 times a day). 24 hours after the first vanillic acid injection, SEAP levels in the bloodstream of mice were measured. To test the in vivo grazoprevir repressible NOT1 switch, 450 μg of plasmid DNA (pSL468 / pSL549 / pSL548, ratio 1:4:4 (w / w / w)) was hydrodynamically injected into the tail vein of WT C57BL / 6 mice (generating SEQ-ID NO: 171 & 296). NO: 3, 58 & 137). Six hours after injection, grazoprevir (3 mg / kg) dissolved in PBS was administered three times daily by intraperitoneal injection. 24 hours after the first grazoprevir injection, SEAP levels in the mouse bloodstream were measured. To test the in vivo vanillic acid (VA)-inducible IF1 switch, 430 μg of plasmid DNA (pLYL76 / pCK53, 40:3 (w / w)) was hydrodynamically injected into the tail vein of WT C57BL / 6 mice (generating SEQ-ID NO: 172 & 208). Six hours after injection, vanillic acid (500 mg / kg / day) dissolved in PBS was administered by intraperitoneal injection (three times daily). 24 hours after the first vanillic acid injection, SEAP levels in the mouse bloodstream were measured.

[0573] Example 5: Engineering of intracellular protein sensors for in vitro diagnostics in cell-based and cell-free environments.

[0574] STIF-based translational control strategies can also be repurposed to engineer intracellular protein sensors. For example, when each split STIF component is fused to a different member of the protein heterotrimeric system, STIF-dependent gene expression from poly(A)-deficient mRNA becomes strictly dependent on the presence of the remaining members of the intact protein complex ( Figure 34 To this end, we first demonstrated that it is possible to construct a bipartite STIF-based MCP-Coh2 / DocS-NSP3 ( Figure 35 A) and the MCP-DocS / Coh2-NSP3 combination ( Figure 35 B) Two to engineer the bipartite STIF construct. We then determined that Coh2 (SEQ-ID NO: 77) contains three tandem repeats ( Figure 35 C, 35D) and DocS (SEQ-ID NO: 80) ( Figure 36 The STIF assembly of (A, 36B) showed optimal efficiency in the dose-dependent translation initiation of poly(A)-deficient SEAP mRNA ( Figure 36 C, 36D). Therefore, we fused three tandem Coh2 repeats to the RBP-domain (L7Ae or MCP) and the NSP3 domain and used the STIF components MCP-(Coh2)3 (SEQ-ID NO: 273) and (Coh2)3-NSP3 (SEQ-ID NO: 9) as highly specific "molecular clamps" that detect cytosolic (DocS)3 (SEQ-ID NO: 302) in a dose-dependent manner ( Figure 37 A). For example ( Figure 35 A), HEK-293 cells were co-transfected with a plasmid encoding SEAP-mRNA (SEQ-ID NO: 137), MCP-Coh2 (SEQ-ID NO: 51), and an NSP3 fusion protein consisting of one (SEQ-ID NO: 12), two (SEQ-ID NO: 13), or three N-terminal DocS repeats (SEQ-ID NO: 14). Alternatively ( Figure 35 B), HEK-293 cells were co-transfected with plasmids encoding SEAP-mRNA (SEQ-ID NO: 137), MCP-DocS (SEQ-ID NO: 246), and NSP3 fusion proteins consisting of one (SEQ-ID NO: 268), two (SEQ-ID NO: 269), or three N-terminal Coh2 repeats (SEQ-ID NO: 9). Figure 35C), HEK-293 cells were co-transfected with plasmids encoding SEAP-mRNA containing L7Ae-specific poly(A) replacements (SEQ-ID NO: 126 & 134), DocS-NSP3 (SEQ-ID NO: 12), and L7Ae fusion proteins consisting of one (SEQ-ID NO: 33), two (SEQ-ID NO: 34), or three C-terminal Coh2 repeats (SEQ-ID NO: 35). For MCP-Coh2-based bipartite STIF ( Figure 35 D), HEK-293 cells were co-transfected with plasmids encoding SEAP-mRNA (SEQ-ID NO: 137) containing an MCP-specific poly(A) surrogate, (DocS)3-NSP3 (SEQ-ID NO: 14), and an MCP fusion protein consisting of one (SEQ-ID NO: 51), two (SEQ-ID NO: 272), or three C-terminal Coh2 repeats (SEQ-ID NO: 273). For the bipartite STIF based on DocS-NSP3 ( Figure 36 A, 36B), HEK-293 cells were co-transfected with plasmids encoding NSP3 fusion protein consisting of one (SEQ-ID NO: 12), two (SEQ-ID NO: 13), or three N-terminal DocS repeats (SEQ-ID NO: 14), SEAP-mRNA containing L7Ae-specific poly(A) replacement (SEQ-ID NO: 126 & 134) and L7Ae-(Coh2)3 (SEQ-ID NO: 35) ( Figure 36 A) or SEAP-mRNA (SEQ-ID NO: 137) and MCP-(Coh2)3 (SEQ-ID NO: 273) containing MCP-specific poly(A) substitutions ( Figure 36 B). Dose-dependent translation activation of MCP-(Coh2)3-specific mRNA ( Figure 36 C), HEK-293 cells were co-transfected with plasmids encoding SEAP-mRNA (SEQ-ID NO: 137), MCP-(Coh2)3 (SEQ-ID NO: 273), and different amounts of (DocS)3-NSP3 expression vector (SEQ-ID NO: 14) containing MCP-specific poly(A) replacement. Dose-dependent translational activation of L7Ae-(Coh2)3-specific mRNA ( Figure 36C), HEK-293 cells were co-transfected with plasmids encoding SEAP-mRNA containing L7Ae-specific poly(A) replacement (SEQ-ID NO: 126 & 134), L7Ae-(Coh2)3 (SEQ-ID NO: 35) and different amounts of (DocS)3-NSP3 expression vector (SEQ-ID NO: 14). To validate the STIF-based protein sensor ( Figure 37 A) HEK-293 cells were co-transfected with a plasmid encoding SEAP mRNA (SEQ-ID NO: 137) containing an MCP-specific poly(A) surrogate, along with constitutive expression vectors for MCP-(Coh2)3 (SEQ-ID NO: 273), (Coh2)3-NSP3 (SEQ-ID NO: 9), and (DocS)3 (SEQ-ID NO: 302, via varying amounts of pSL244). SEAP expression was recorded in culture supernatants 48 hours after transfection.

[0575] Once highly specific binder pairs for the same target are available, this molecular clamp can be used to detect a variety of proteins of interest in living cells. Therefore, as a general blueprint for engineering a STIF-based sensor for a specific target Y", a pair of proteins Y and Y' is required, each of which binds to a different epitope of Y". Y and Y' can then be fused to the N-terminus or C-terminus of the RBP or eIFBP domain of the STIF regulator, allowing Y" to initiate translation initiation after triggering the circularization conformation of an mRNA containing an RBP-specific poly(A) surrogate ( Figure 34For example, we designed a sensor for the hepatitis C virus (HCV)-specific nNS3 protein (SEQ-ID NO: 303) by fusing two different nNS3-specific scFvs to L7Ae and NSP3, generating L7Ae-scFv35 (SEQ-ID NO: 47) and (scFv162)3-NSP3 (SEQ-ID NO: 262). HEK-293 cells were then co-transfected with plasmids encoding SEAP mRNA containing an L7Ae-specific poly(A) surrogate (SEQ-ID NO: 126 & 134), L7Ae-scFv35 (SEQ-ID NO: 47), (scFv162)3-NSP3 (SEQ-ID NO: 262), and varying amounts of overexpressed nNS3 (SEQ-ID NO: 303). SEAP expression was recorded in the culture supernatant 48 hours after transfection. The results showed that co-expression of L7Ae-scFv35 and (scFv162)3-NSP3 in mammalian cells activated the translation of target mRNA containing L7Ae-specific poly(A) replacement in a strictly nNS3-dependent manner ( Figure 37 B), demonstrating the potential application of this STIF-based protein sensor for molecular diagnostics by delivering genes into living cells or by developing point-of-care testing devices based on synthetic gene circuits operating in cell-free systems (Pardee et al., 2014) ( Figure 37 C).

[0576] To demonstrate the superiority of translation-based sensors over state-of-the-art transcription-based sensors, we created a synthetic EGFP-NS3a(H1) protein (SEQ-ID NO: 17) as a model target signal for detection ( Figure 38A). This synthetic target protein, EGFP-NS3a(H1), was then targeted to different intracellular compartments by fusing with different localization signals (NLS (nuclear localization signal, SEQ-ID NO: 103), resulting in SEQ-ID NO: 21; NES (nuclear export signal, SEQ-ID NO: 102), resulting in SEQ-ID NO: 20; CAAX (prenylation motif, SEQ-ID NO: 75), resulting in SEQ-ID NO: 19; TM (transmembrane localization signal, SEQ-ID NO: 118), resulting in SEQ-ID NO: 23; SP (secretion signal peptide, SEQ-ID NO: 115), resulting in SEQ-ID NO: 22), thereby allowing detection of each differentially localized protein using a co-expressed genetic sensor consisting of LaG16 (EGFP nanobody, SEQ-ID NO: 93) and ANR (NS3a(H1) binding peptide motif, SEQ-ID NO: 105). For transcription-based sensing, LaG16 (SEQ-ID NO:93) was fused to TetR (SEQ-ID NO:117), while different repeats of ANR (SEQ-ID NO:105) were fused to VP64 (SEQ-ID NO:119), resulting in EGFP-NS3a(H1)-dependent transcriptional activation of the TetR-specific promoter. For translation-based sensing, LaG16 (SEQ-ID NO:93) was fused to MCP (SEQ-ID NO:98), while different repeats of ANR (SEQ-ID NO:105) were fused to NSP3 (SEQ-ID NO:106), resulting in EGFP-NS3a(H1)-dependent STIF remodeling and translation of MCP-specific mRNA. To establish the STIF-based sensor of EGFP-NS3a(H1), we found that one LaG16 repeat was sufficient to associate with EGFP ( Figure 39 A), while NS3a(H1) detection requires multiple tandem ANR peptide motifs fused to the N-terminus of NSP3 ( Figure 39B). For example, HEK-293 cells were co-transfected with a plasmid encoding SEAP mRNA (SEQ ID NO: 137) containing an MCP-specific poly(A) surrogate, a constitutive EGFP-NSP3 (SEQ ID NO: 18) expression vector, and expression vectors for different MCP-LaG16 variants consisting of one (SEQ ID NO: 52) or two tandem repeats of LaG16 (SEQ ID NO: 53). pcDNA3.1(+) (Invitrogen, CA; Catalog No. V79020) was used as a negative control in place of the EGFP-NSP3 expression vector. Similarly, HEK-293 cells were co-transfected with a plasmid encoding SEAP-mRNA (SEQ-ID NO: 137) containing an MCP-specific poly(A) surrogate, a constitutive MCP-(NS3a(H1))3 (SEQ-ID NO: 58) expression vector, and an expression vector for ANR-NSP3 variants containing different numbers of ANR tandem repeats (SEQ-ID NO: 2-5 & 267). 48 hours after transfection, SEAP expression in the culture supernatant was analyzed. Thus, MCP-LaG16 (SEQ-ID NO: 52), (ANR)8-NSP3 (SEQ-ID NO: 5), and a reporter mRNA containing an MCP-specific poly(A) surrogate (Table 2) were established as highly accurate genetic sensors of intracellular EGFP-NS3a(H1) ( Figure 39 C). To demonstrate the accuracy of induction, HEK-293 cells were co-transfected with a plasmid encoding a reporter SEAP mRNA containing an MCP-specific poly(A) surrogate (SEQ-ID NO: 137), a constitutive EGFP-NS3a(H1) (SEQ-ID NO: 17) expression vector, and various combinations of MCP-LaG16 (SEQ-ID N...

Claims

1. A gene regulation system capable of regulating the expression of target gene mRNA, the system comprising (i) a synthetic translation initiation factor (STIF) and an mRNA construct comprising an mRNA encoding a target protein; or (ii) A nucleic acid encoding STIF and a nucleic acid encoding an mRNA construct comprising an mRNA encoding a target protein, for example, the nucleic acid is DNA or RNA. in, The STIF comprises or consists of at least one eIFBP (eIF4F binding protein) and at least one RBP (RNA binding protein).

2. The system according to claim 1, wherein: The eIFBP is selected from PABP, NSP3, VPg and any member of eIF4F such as eIF4A, eIF4B, eIF4E or eIF4G; and / or the RBP is selected from L7Ae, MCP or λ-N.

3. The system according to claim 2, wherein PABP comprises or consists of the amino acid sequence of SEQ ID NO: 108, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:

108. NSP3 comprises or consists of the amino acid sequence of SEQ ID NO: 91 or 106, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 91 or 106. VPg comprises or consists of the amino acid sequence of SEQ ID NO: 120, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 120; eIF4G comprises, or consists of, the amino acid sequence of SEQ ID NO:84, or an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:84; eIF4E comprises, or consists of, the amino acid sequence of SEQ ID NO:83 or 314, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:83 or 314, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:83 and having a K119A substitution. L7Ae comprises, or consists of, the amino acid sequence of SEQ ID NO:92, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:92; The MCP comprises or consists of the amino acid sequence of SEQ ID NO: 98, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In other embodiments, the MCP is an MCP variant having a V29I substitution compared to the MCP; or the λ-N comprises or consists of the amino acid sequence of SEQ ID NO: 100, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

4. The system according to any one of claims 1 to 3, wherein the configuration of the fusion protein from N-terminus to C-terminus is eIFBP-RBP or RBP-eIFBP, and optionally has some other protein domains inserted (e.g., 2CaM-M13 or a tag) or has a tag at the N-terminus or C-terminus, preferably, STIF contains fusion proteins L7Ae-NSP3; 3Xflag-L7Ae-NSP3; L7Ae-hNSP3; L7Ae-eIF4E; MCP-NSP3; MCP-hNSP3; PABP-L7Ae; PABP-L7Ae-3xFLAG; eIF4G-2CaM-M13-L7Ae; PABP-MCP; MCP-eIF4E; eIF4G-MCP; 3xFLAG-MCP-NSP3; MCP-VPg; MCP V29I -VPg; or L7Ae-VPg, or consisting thereof.

5. The system according to any one of claims 1 to 3, wherein the STIF comprises or consists of two recombinant fusion proteins A and B, wherein wherein protein A may be RBP-Y and protein B may be Y'-eIFBP, or wherein protein A may be RBP-Y and protein B may be eIFBP-Y', or wherein protein A may be γ-RBP and protein B may be γ'-eIFBP, or wherein protein A may be γ-RBP and protein B may be eIFBP-γ′, or wherein protein A may be eIFBP-γ and protein B may be γ'-RBP, or wherein protein A may be eIFBP-Y and protein B may be RBP-Y', or wherein protein A may be γ-eIFBP and protein B may be γ'-RBP, or wherein protein A may be Y-eIFBP and protein B may be RBP-Y', or wherein protein A may be RBP-Y' and protein B may be Y-eIFBP, or wherein protein A may be RBP-Y' and protein B may be eIFBP-Y, or wherein protein A may be γ'-RBP and protein B may be γ-eIFBP, or wherein protein A may be Y'-RBP and protein B may be eIFBP-Y, or wherein protein A may be eIFBP-γ' and protein B may be γ-RBP, or wherein protein A may be eIFBP-Y' and protein B may be RBP-Y, or wherein protein A may be γ'-eIFBP and protein B may be γ-RBP, or wherein protein A may be Y'-eIFBP and protein B may be RBP-Y; Wherein Y and Y' may interact constitutively with each other, or through a trigger or signal or through another protein Y".

6. The system of claim 5, wherein the protein A or B comprises a plurality of tandem repeats of Y or Y', such as 1-5 repeats, such as 1, 2, 3, 4 or 5 repeats.

7. The system according to claim 5 or 6, wherein said Y and Y' constitutively interact with each other, preferably, Protein Y is DocS and protein Y' is Coh2; or protein Y is Coh2 and protein Y' is DocS; Protein Y is NS3a(H1) and protein Y' is ANR; or protein Y is ANR and protein Y' is NS3a(H1); protein Y is Bcl-XL and protein Y' is LD1 or LD3; or protein Y is LD1 or LD3 and protein Y' is Bcl-XL; Protein Y is EGFP and protein Y' is LaG16; or protein Y is LaG16 and protein Y' is EGFP; Protein Y is CCmut3 and protein Y' is BCR; or protein Y is BCR and protein Y' is CCmut3; Protein Y is ABI (iDab) and protein Y' is ABL1; or protein Y is ABL1 and protein Y' is ABI (iDab); protein Y is an antibody or antigen-binding fragment (e.g., VHH or scFv) that specifically binds to an antigen and protein Y' is the antigen; or protein Y is an antigen and protein Y' is an antibody or antigen-binding fragment (e.g., VHH or scFv), for example, the antigen is NS3 or a fragment thereof, such as the N-terminus of NS3.

8. The system according to claim 5 or 6, wherein Y and Y' interact with each other by trigger induction or trigger inhibition using an agent, such as Protein Y is ABI and protein Y' is PYL1; or protein Y is PYL1 and protein Y' is ABI, e.g., the protein-protein interaction (binding) between ABI and PYL1 can be induced by abscisic acid triggering; Protein Y is Aff6 and protein Y' is DrBPhP; or protein Y is DrBPhP and protein Y' is Aff6, for example, the protein-protein interaction (binding) between Aff6 and DrBPhP can be induced by red light triggering; Protein Y is ANR and protein Y' is NS3a(H1); or protein Y is NS3a(H1) and protein Y' is ANR, e.g., the protein-protein interaction (binding) between ANR and NS3a(H1) can be inhibited by triggering grazoprevir; Protein Y is CIB1 and protein Y' is Cry2; or protein Y is Cry2 and protein Y' is CIB1, for example, the protein-protein interaction (binding) between CIB1 and Cry2 can be induced by blue light triggering; Protein Y is DNCR and protein Y' is NS3a or NS3a(H1); or protein Y is NS3a or NS3a(H1) and protein Y' is DNCR, e.g., protein-protein interaction (binding) between DNCR and NS3a or NS3a(H1) and induction is triggered by danoprevir; Protein Y is FKBP and protein Y' is FRB; or protein Y is FRB and protein Y' is FKBP, for example, the protein-protein interaction (binding) between FKBP and FRB can be induced by rapamycin triggering; Protein Y is GAI and protein Y' is GID1; or protein Y is GID1 and protein Y' is GAI, for example, the protein-protein interaction (binding) between GAI and GID1 can be induced by gibberellic acid; Protein Y is GNCR and protein Y' is NS3a or NS3a(H1); or protein Y is NS3a or NS3a(H1) and protein Y' is GNCR, e.g., the protein-protein interaction (binding) between GNCR and NS3a or NS3a(H1) can be triggered and induced by grazoprevir; or Protein Y is LaM8AK47 and protein Y' is mCherry; or protein Y is mCherry and protein Y' is LaM8AK47, for example, protein-protein interaction (binding) between LaM8AK47 and mCherry can be induced by blue light triggering.

9. The system according to claim 5 or 6, wherein said Y and Y' are capable of specifically interacting with each other through signal transduction, wherein Protein Y is ERK2 and protein Y' is pE59; or protein Y is pE59 and protein Y' is ERK2, for example, the protein-protein interaction (binding) between ERK2 and pE59 can be induced by MAPK signaling (e.g., an agent that activates MAPK signaling, such as MAPK, EGF).

10. The system according to claim 5 or 6, wherein Y and Y' can interact with each other via another protein Y", and when Y" is present, Y and Y' can associate with the target protein through a specific Y:Y":Y'-mediated interaction, preferably, protein Y and protein Y' are two different scFvs that specifically bind to protein Y", for example, specifically bind to different domains or epitopes of protein Y".

11. The system of claim 10, wherein Y" is selected from: (i) disease-specific cellular signatures, such as oncoproteins, e.g., fusion proteins or protein complexes specifically expressed in tumor cells or tumor tissues, e.g., BCR-ARL, or infection-specific proteins, e.g., HCV or HCV-specific proteins (e.g., NS3 protein); (ii) a fusion protein of Y and Y″, or a fusion protein comprising a binding domain of Y and a binding domain of Y′, wherein Y and Y′ can constitutively interact with each other, such as an EGFP-NS3a (H1) fusion protein; (iii) intracellular or secreted proteins containing one or more domains; (iv) an antigen having at least two or more epitopes or domains.

12. The system according to any one of claims 1 to 11, wherein the mRNA construct comprises a coding region and a STIF-binding RNA segment, and the coding region is flanked by 5'-UTR and 3'-UTR.

13. The system according to claim 12, wherein the coding region is any RNA sequence starting with the nucleotide AUG and ending with the nucleotide sequence UAG, UAA or UGA, for example, the coding region encodes a target protein (preferably, the target protein can be selected from therapeutic proteins such as insulin, such as human insulin or pro-apoptotic proteins such as BAX (such as human Bax)), a protein whose expression can be detected (such as a marker protein or a reporter protein (such as SEAP), luciferase, a fluorescent protein (such as GFP or EGFP)).

14. The system according to claim 12 or 13, wherein the RNA segment bound by STIF is a poly-A signal or a poly(A) surrogate, wherein the poly(A) surrogate can be any segment containing or consisting of one or more n aptamer repeats that bind to a specific RBP and is placed in the 3'-UTR or 5'-UTR of the mRNA.

15. The system of claim 14, wherein the aptamer is selected from C / D-box, MS2-box or boxB.

16. The system of claim 15, wherein If the RBP of STIF is L7Ae, the poly(A) surrogate is a tandem repeat of the L7Ae-specific C / D-box aptamer, e.g., (C / D-box)n; or if the RBP of STIF is MCP, the poly(A) surrogate is a tandem repeat of the MCP-specific MS2-box aptamer (MS2-box)n or the λ-N-specific aptamer (boxB)n, wherein n can be any number between 1 and 1000, e.g., 5 to 30, e.g., 8, 12, 16, or 24.

17. The system of claim 16, wherein The C / D-box comprises or consists of the amino acid sequence of SEQ ID NO: 123; The MS2-box comprises or consists of the amino acid sequence of SEQ ID NO: 125 or 315; or Box B comprises or consists of the amino acid sequence of SEQ ID NO:

121.

18. The system according to claims 14 to 17, wherein the mRNA construct further comprises an RNA cleavage site capable of pre-programmed poly(A) removal, preferably, the site is located between the aptamer and the poly(A) and is placed in the 3'-UTR.

19. The system of claim 18, wherein the cleavage is performed by RNA interference and the RNA cleavage site is a siRNA binding site or multiple copies thereof, a shRNA binding site or multiple copies thereof, or a miRNA binding site or multiple copies thereof.

20. The system according to claim 19, wherein the system further comprises a construct expressing shRNA-216 to cleave the polyA, and wherein the RNA cleavage site may comprise one or more n repeats (BS(shRNA-216))n, wherein n may be any number between 1 and 100 and preferably n may be any number between 1 and 4, preferably, the BS(shRNA-216) comprises or consists of SEQ ID NO: 122, and / or the shRNA-216 comprises or consists of SEQ ID NO:

126.

21. The system of claim 18, wherein the cleavage is performed by a ribozyme and the cleavage site is a ribozyme, eg, the ribozyme is a self-cleaving ribozyme or multiple copies thereof or fragments thereof.

22. The system according to claim 21, wherein the self-cleaving ribozyme is a hammerhead ribozyme (HHR)n, wherein n can be any number between 1 and 100, preferably n can be any number between 1 and 4, for example, the HHR comprises or consists of SEQ ID NO:

124.

23. The system according to claim 12, wherein the mRNA construct comprises, from N-terminus to C-terminus, a 5'UTR, a coding region, a Poly(A) surrogate, a cleavage site and a 3'UTR, preferably, the combination of the Poly(A) surrogate and the cleavage site is selected from 24. The system according to claims 12-23, wherein the mRNA construct may further comprise a 5'-cap or a 5'-cap surrogate, preferably, the 5'-cap surrogate may be a poly(A) surrogate as defined in any one of claims 14-17.

25. The system according to claim 24, wherein the mRNA construct further comprises cleavage as defined in any one of claims 19-22.

26. The system according to claim 12, wherein the system is triggered by grazoprevir, wherein the STIF comprises or consists of two recombinant fusion proteins A and B, and wherein (i) protein A is in the configuration of L7Ae-(NS3a)n and protein B is (GNCR)n-NSP3, or protein A is in the configuration of L7Ae-(GNCR)n and protein B is (NS3a)n-NSP3, wherein n is an integer from 1 to 10; and the mRNA construct comprises, from N-terminus to C-terminus, a 5'UTR, a coding region, a (C / D-box)n (e.g., n=1-30, e.g., 24), a (BS(shRNA-216))n (e.g., n=1, 2 or 3, e.g., 2), and a 3'UTR; or (ii) Protein A is MCP-(NS3a) n configuration and protein B is (GNCR) n -NSP3, or protein A is MCP-(GNCR) n configuration and protein B is (NS3a)n-NSP3, wherein n is an integer from 1 to 10; and the mRNA construct comprises, from N-terminus to C-terminus, a 5'UTR, a coding region, (MS2-box)n (e.g., n=1-30, e.g., 24), (BS(shRNA-216))n (e.g., n=1, 2 or 3, e.g., 2) or (HHR) n (eg, n=1, 2, 3, or 4, such as 1) and a 3'UTR; And the system further comprises a construct expressing shRNA-216; Preferably, the coding region encodes insulin, such as human insulin.

27. A nucleic acid encoding STIF and / or an mRNA construct of the system according to any one of claims 1 to 26, wherein the nucleic acid is DNA or RNA.

28. A vector comprising the nucleic acid of claim 27, wherein the vector is an expression vector, such as a eukaryotic expression vector, such as pcDNA3.1 or AAV.

29. A pharmaceutical composition, kit or non-biological material comprising the system of any one of claims 1 to 26.

30. A pharmaceutical combination comprising the system of any one of claims 5 to 26 and one or more other agents, such as agents that trigger or induce or inhibit expression of the system, such as protein γ", abscisic acid, grazoprevir, danoprevir, MAPK, rapamycin, gibberellic acid; or other therapeutic agents.

31. A method for preventing or treating a disease, comprising administering the gene regulatory system according to any one of claims 1 to 26, the pharmaceutical composition according to claim 29, or the pharmaceutical combination according to claim 30 to a subject in need thereof.

32. Use of the system of any one of claims 1 to 26 for in vitro or in vivo biocomputing.

33. A method for diagnosing a disease, comprising using the system of any one of claims 10 to 26 to detect a disease characteristic of the disease, wherein the disease characteristic is Y" which can be bound by Y and Y' of STIF.

34. A method for detecting a protein in vitro, comprising using the system of any one of claims 10 to 26 to detect a protein acting as Y" bound by both Y and Y' of STIF, preferably, the system being contained in a non-biological material, such as a paper disc.

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