Gene expression loop responding to hyperglycemia and application thereof

Abstract

The present invention relates to a hyperglycemia responsive gene expression loop and application thereof, the gene loop comprises a nucleic acid sequence encoding a glucose receptor and an effector, the effector is used for responding to a signal transmitted by the glucose receptor, and the glucose receptor is a glucose receptor in a non-beta cell. According to the invention, the risk that therapeutic cells integrated with the gene loop induce autoimmune response in the body of a patient with type I diabetes mellitus is reduced to the greatest extent; the size of the whole gene loop assembly is less than 5kb, so that the requirement of in-vivo gene delivery is met; an expression loop of the synthetic gene has moderate sensitivity and is exposed to hyperglycemia concentration (gt; 10mM) environment, the transcription level of the target gene in the gene loop is obviously increased, and low glucose concentration (lt; the expression level of the target gene is relatively low when the expression level of the target protein is 5mM, the kinetics released by the target protein of the gene loop is close to that of a natural pancreas islet beta cell, and the gene loop is safe and can effectively treat diabetes and complications thereof.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a hyperglycemia-responsive gene expression circuit and applications thereof. Background Art

[0002] Diabetes mellitus (DM) is a group of metabolic diseases characterized by abnormally high blood sugar levels, or hyperglycemia. DM is generally classified into two types: type 1 diabetes (T1D), which is characterized by selective autoimmune destruction of the insulin-producing beta cells of the pancreas, and the more common type 2 diabetes (T2D), which is often due to insulin resistance, a condition in which the body is unable to respond appropriately to insulin. If left untreated, diabetes-related hyperglycemia may lead to a variety of comorbidities, including cardiovascular disease, renal failure, metabolic syndrome, or diabetic enteropathy. Treatment of T1D relies on regular delivery of exogenous insulin to lower blood sugar levels while avoiding hypoglycemia. For T2D, a number of drugs have been developed to overcome peripheral tissue insulin resistance and thereby improve glycemic control. For example, glucagon-like peptide 1 (GLP-1) or other agonists of its receptors are attractive candidates for the treatment of T2D because they can act as incretins to enhance glucose-stimulated insulin secretion (GSIS), as well as other mechanisms to improve T2D. However, conventional drug administration usually follows a standard treatment regimen based on average levels, which may put individuals with above-average disease severity at risk for side effects due to under- or overdosage. Therefore, the dosage of such drugs may require fairly frequent calibration by the patient's physician based on the therapeutic effect to maintain optimal glycemic control. Compared with standard anti-diabetic therapies, smart anti-hyperglycemic drug release devices with feedback control of the delivered dose (output) and real-time blood glucose levels (input) are more suitable for diabetes treatment.

[0003] Recent advances in synthetic biology have facilitated the design of some synthetic gene circuits that can produce and deliver insulin in situ in response to exogenous or endogenous signals, representing a promising smart insulin delivery system. Exogenous signals include biosafe small molecules (e.g., clinically used drug molecules and food additives) and traceless physical signals (light, heat, magnetism, ultrasound, etc.). Endogenous signals refer to disease biomarkers, such as blood glucose. Such user-defined synthetic genetic networks operating in mammalian cells have been tested and can effectively restore glucose homeostasis to a certain extent in a variety of diabetic mouse models. Although most systems are therapeutic protein synthesis initiated by external signals with high gene expression on / off ratios, the disadvantage is that they are all open-loop control systems that rely on blood glucose level monitoring devices to achieve precise blood glucose control. In contrast, gene circuits with high blood glucose-driven feedback control loops can maintain blood glucose homeostasis like endogenous biological systems, such as pancreatic islets, thereby showing more intelligent and effective blood glucose control. A pioneering work on a glucose-driven gene circuit with closed-loop control has been reported. Professor Martin Fussenegger's system confers glucose sensitivity to chassis cells by ectopically expressing a component of a glucose sensor, a calcium channel, that evolved in natural beta cells. This design enables the engineered cells to respond to elevated extracellular glucose concentrations in a manner similar to beta cells. However, the total size of the genes that need to be ectopically expressed is close to 10kb, far exceeding the packaging capacity of standard gene therapy vectors such as AAV, which may limit its future application. In addition, engineered cells expressing key components of beta cells may be attacked by the dysfunctional immune system of T1D patients, just like beta cells.

[0004] In summary, there is still a need to develop new hyperglycemia sensing gene circuits that are expected to have minimal potential to induce autoimmune responses and an efficient and compact structure that facilitates effective delivery via standard gene therapy vectors. Integrating this gene circuit into chassis cells will result in artificial β cells that are protected from the dysregulated immune system of T1D patients, which will bring great prospects for cell-based diabetes therapy. Summary of the invention

[0005] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. In order to design a glucose sensing system that is different from that present in natural β cells, the present application studies the glucose sensing mechanism in non-β cell types. Among them, O-GlcNAc glycosylation is a glucose sensor that is commonly present in mammalian cells and is closely related to glucose metabolism. It involves adding a single O-GlcNAc (O-linked β-N-acetylglucosamine) molecule to a serine or threonine residue of a target protein. Increased glucose levels always lead to increased OGlycNAcylation of proteins. Yes-associated protein (YAP) is a target protein for O-GlcNAc glycosylation, and glucose can be O-GlcNAc glycosylated in a dose-dependent manner, thereby promoting YAP to enter the nucleus. This process gives YAP the ability to become a glucose sensor, making it an excellent candidate for the key features of the target's hyperglycemia sensing system.

[0006] Therefore, in the first aspect of the present invention, the present invention provides a gene circuit. According to an embodiment of the present invention, the gene circuit includes one or more first expression vectors, the first expression vector carries a first nucleic acid molecule, the first nucleic acid molecule encodes a glucose receptor or a fragment thereof; and a second expression vector, the second expression vector carries a second nucleic acid molecule, the second nucleic acid molecule encodes an effector; wherein the effector is used to respond to the signal transmitted by the glucose receptor, the glucose receptor is a non-β cell glucose receptor, the first nucleic acid molecule and the second nucleic acid molecule are configured to express the glucose receptor and the effector, and the glucose receptor and the effector are in a non-fused form.

[0007] The method of constructing a hyperglycemia sensing gene circuit of the present invention is different from the previous strategy based on ectopic expression of glucose sensing proteins in natural β cells. In the present invention, the hyperglycemia sensor component is constructed by reconstructing the glucose sensing mechanism commonly present in mammalian cells, so that the hyperglycemia sensing gene circuit of the present invention does not involve the natural glucose sensing proteins present in pancreatic β cells, and only uses the glucose sensing proteins present in pancreatic non-β cells, thereby minimizing the risk of therapeutic cells integrated with the gene circuit inducing autoimmune responses in patients with type 1 diabetes. In addition, the size of the entire gene circuit component is less than 5kb, which does not exceed the packaging capacity of standard gene therapy vectors and meets the requirements of in vivo gene delivery. Finally, the synthetic gene expression circuit for hyperglycemia response has moderate sensitivity. That is, when exposed to high blood sugar (>10mM), the transcription level of the target gene is significantly increased, while when exposed to low glucose concentration (<5mM), the expression level of the target gene is low. The kinetics of the release of the target protein of this gene circuit are close to those of natural pancreatic beta cells.

[0008] According to an embodiment of the present invention, the above gene circuit may further include at least one of the following additional technical features:

[0009] According to an embodiment of the present invention, the glucose sensor comprises one or more chimeric transcription factors.

[0010] According to an embodiment of the present invention, the chimeric transcription factor includes at least one of a YAP protein, a TEF-1 protein, a TetR family transcriptional regulatory factor protein and a transcriptional activation domain.

[0011] According to an embodiment of the present invention, the number of the transcription activation domains in the chimeric transcription factor is 2 to 6, preferably 4.

[0012] According to an embodiment of the present invention, the first promoter includes any promoter used in mammalian cells.

[0013] According to an embodiment of the present invention, the glucose sensor comprises two chimeric transcription factors.

[0014] According to an embodiment of the present invention, the glucose sensor comprises two identical or different chimeric transcription factors.

[0015] According to an embodiment of the present invention, the glucose sensor comprises two different chimeric transcription factors.

[0016] According to an embodiment of the present invention, the two chimeric transcription factors are chimeric transcription factor 1 and chimeric transcription factor 2, wherein the chimeric transcription factor 1 includes the YAP protein and the transcription activation domain, or the chimeric transcription factor 1 includes the YAP protein and the TetR family transcription regulatory factor protein; and / or the chimeric transcription factor 2 includes the TetR family transcription regulatory factor protein and the TEF-1 protein, or the chimeric transcription factor 2 includes the TEF-1 protein and the transcription activation domain.

[0017] According to an embodiment of the present invention, the two chimeric transcription factors are chimeric transcription factor 1 and chimeric transcription factor 2, wherein the chimeric transcription factor 1 includes the YAP protein and the transcription activation domain, and the chimeric transcription factor 2 includes the TetR family transcription regulatory factor protein and the TEF-1 protein, wherein the C-terminus of the YAP protein is connected to the N-terminus of the transcription activation domain, or the N-terminus of the YAP protein is connected to the C-terminus of the transcription activation domain, the C-terminus of the TetR family transcription regulatory factor protein is connected to the N-terminus of the TEF-1 protein, or the N-terminus of the TetR family transcription regulatory factor protein is connected to the C-terminus of the TEF-1 protein; or

[0018] The chimeric transcription factor 1 includes the YAP protein and the TetR family transcription regulatory factor protein, and the chimeric transcription factor 2 includes the TEF-1 protein and the transcription activation domain, wherein the C-terminus of the TetR family transcription regulatory factor protein is connected to the N-terminus of the YAP protein, the N-terminus of the TetR family transcription regulatory factor protein is connected to the C-terminus of the YAP protein, the C-terminus of the TEF-1 protein is connected to the N-terminus of the transcription activation domain, and the N-terminus of the TEF-1 protein is connected to the C-terminus of the transcription activation domain.

[0019] According to an embodiment of the present invention, the chimeric transcription factor 1 includes a YAP protein and a transcription activation domain, the C-terminus of the YAP protein is connected to the N-terminus of the transcription activation domain, the chimeric transcription factor 2 includes the TetR family transcription regulatory factor protein and the TEF-1 protein, and the C-terminus of the TetR family transcription regulatory factor protein is connected to the N-terminus of the TEF-1 protein; or the chimeric transcription factor 1 includes the YAP protein and the TetR family transcription regulatory factor protein, the C-terminus of the TetR family transcription regulatory factor protein is connected to the N-terminus of the YAP protein, the chimeric transcription factor 2 includes the TEF-1 protein and the transcription activation domain, and the C-terminus of the TEF-1 protein is connected to the N-terminus of the transcription activation domain.

[0020] According to an embodiment of the present invention, the chimeric transcription factor 1 also includes a first linker, wherein: the C-terminus of the YAP protein is connected to the N-terminus of the first linker, and the C-terminus of the first linker is connected to the N-terminus of the transcription activation domain, or the N-terminus of the YAP protein is connected to the C-terminus of the first linker, and the N-terminus of the first linker is connected to the C-terminus of the transcription activation domain; or the C-terminus of the TetR family transcription regulatory factor protein is connected to the N-terminus of the first linker, and the C-terminus of the first linker is connected to the N-terminus of the YAP protein, or the N-terminus of the TetR family transcription regulatory factor protein is connected to the C-terminus of the first linker, and the N-terminus of the first linker is connected to the C-terminus of the YAP protein.

[0021] According to an embodiment of the present invention, the chimeric transcription factor 2 also includes a second linker and / or a nuclear localization signal, wherein: the C-terminus of the TetR family transcription regulatory factor protein is connected to the N-terminus of the nuclear localization signal, and the C-terminus of the nuclear localization signal is connected to the N-terminus of the TEF-1 protein, or the N-terminus of the TetR family transcription regulatory factor protein is connected to the C-terminus of the nuclear localization signal, and the N-terminus of the nuclear localization signal is connected to the C-terminus of the TEF-1 protein; or the C-terminus of the nuclear localization signal is connected to the N-terminus of the TEF-1 protein, and the C-terminus of the TEF-1 protein is connected to the N-terminus of the second linker, and the C-terminus of the second linker is connected to the N-terminus of the transcription activation domain, or the C-terminus of the nuclear localization signal is connected to the N-terminus of the transcription activation domain, and the C-terminus of the transcription activation domain is connected to the N-terminus of the second linker, and the C-terminus of the second linker is connected to the N-terminus of the TEF-1 protein.

[0022] According to some preferred embodiments of the present invention, the chimeric transcription factor 1 includes a first linker, wherein: the C-terminus of the YAP protein is connected to the N-terminus of the first linker, and the C-terminus of the first linker is connected to the N-terminus of the transcription activation domain; or the C-terminus of the TetR family transcription regulatory factor protein is connected to the N-terminus of the first linker, and the C-terminus of the first linker is connected to the N-terminus of the YAP protein.

[0023] According to some preferred embodiments of the present invention, the chimeric transcription factor 2 includes a second linker and / or a nuclear localization signal, wherein: the C-terminus of the TetR family transcription regulatory factor protein is connected to the N-terminus of the nuclear localization signal, and the C-terminus of the nuclear localization signal is connected to the N-terminus of the TEF-1 protein; or the C-terminus of the nuclear localization signal is connected to the N-terminus of the TEF-1 protein, the C-terminus of the TEF-1 protein is connected to the N-terminus of the second linker, and the C-terminus of the second linker is connected to the N-terminus of the transcription activation domain.

[0024] According to an embodiment of the present invention, the first expression vector further comprises one or more first promoters.

[0025] According to an embodiment of the present invention, the first promoter is operably linked to the first nucleic acid molecule.

[0026] According to some specific embodiments of the present invention, the first promoter includes at least one of a cytomegalovirus promoter, an EF-1α promoter, and a cytomegalovirus immediate early promoter / chicken β-actin promoter.

[0027] According to some specific embodiments of the present invention, the chimeric transcription factor 1 and the chimeric transcription factor 2 are located on the same first expression vector or on different first expression vectors.

[0028] According to some specific embodiments of the present invention, when the chimeric transcription factor 1 and the chimeric transcription factor 2 are located on the same first expression vector, the first expression vector also includes a third nucleic acid molecule, which is arranged between the chimeric transcription factor 1 and the chimeric transcription factor 2, and the third nucleic acid molecule encodes a 2A peptide.

[0029] According to an embodiment of the present invention, the YAP protein includes a full-length YAP1 protein (NCBI Reference Sequence: NP_001123617.1).

[0030] According to an embodiment of the present invention, the YAP protein has an amino acid sequence shown in SEQ ID NO: 1 or 2. According to some specific embodiments of the present invention, the YAP protein used in the present invention can be amino acids 1 to 292 of the YAP1 protein, or can be amino acids 2 to 504. Furthermore, the gene circuit expressing the full length of the YAP protein can also effectively respond to glucose levels.

[0031] The sequence of amino acids 1 to 292 of the YAP1 protein (YAPΔAD1) is as follows:

[0032] MDPGQQPPPQPAPQGQGQPPSQPPQGQGPPSGPGQPAPAATQAAPQAPPAGHQIVHVRGDSETDL

[0033] EALFNAVMNPKTANVPQTVPMRLRKLPDSFFKPPEPKSHSRQASTDAGTAGALTPQHVRAHSSPASLQ

[0034] LGAVSPGTLTPTGVVSGPAATPTAQHLRQSSFEIPDDVPLPAGWEMAKTSSGQRYFLNHIDQTTTWQDP

[0035] RKAMLSQMNVTAPTSPPVQQNMMNSASGPLPDGWEGAMTQDGEIYYINHKNKTTSWLDPRLDPRFA

[0036] MNQRISQSAPVKQPPPLAPQSPQG (SEQ ID NO: 1).

[0037] The sequence of amino acids 2 to 504 of the YAP1 protein (YAPΔAD2) is as follows:

[0038] DPGQQPPPQPAPQGQGQPPSQPPQGQGPPSGPGQPAPAATQAAPQAPPAGHQIVHVRGDSETDLEA

[0039] LFNAVMNPKTANVPQTVPMRLRKLPDSFFKPPEPKSHSRQASTDAGTAGALTPQHVRAHSSPASLQLG

[0040] AVSPGTLTPTGVVSGPAATPTAQHLRQSSFEIPDDVPLPAGWEMAKTSSGQRYFLNHIDQTTTWQDPRK

[0041] AMLSQMNVTAPTSPPVQQNMMNSASGPLPDGWEQAMTQDGEIYYINHKNKTTSWLDPRLDPRFAMN

[0042] QRISQSAPVKQPPPLAPQSPQGGVMGGSNSNQQQQMRLQQLQMEKERLRLKQQELLRQAMRNINPST

[0043] ANSPKCQELALRSQLPTLEQDGGTQNPVSSPGMSQELRTMTTNSSDPFLNSGTYHSRDESTDSGLSMSS

[0044] YSVPRTPDDFLNSVDEMDTGDTINQSTLPSQQNRFPDYLEAIPGTNVDLGTLEGDGMNIEGEELMPSLQ

[0045] EALSSDILNDMESVLAATKLDKESFLTWL (SEQ ID NO: 2).

[0046] According to an embodiment of the present invention, the TetR family transcriptional regulatory factor protein is a full-length TetR family transcriptional regulatory factor protein (GenBank: MWC77513.1) or a fragment of amino acids 2 to 208 thereof. According to a specific embodiment of the present invention, the TetR family transcriptional regulatory factor protein can effectively operate the gene circuit as long as it includes a fragment of amino acids 2 to 208. Furthermore, a gene circuit expressing the full-length TetR family transcriptional regulatory factor protein can also effectively respond to glucose levels.

[0047] According to an embodiment of the present invention, the TetR family transcriptional regulatory factor protein has an amino acid sequence shown in SE ID NO:3.

[0048] The amino acid sequence of positions 2 to 208 of the TetR family transcriptional regulatory factor protein (GenBank: MWC77513.1) is as follows:

[0049] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRP TEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS(SEQ ID NO:3).

[0050] According to an embodiment of the present invention, the TEF-1 protein is a full-length TEF-1 protein (NCBI Reference Sequence: NP_068780.2) or a fragment of amino acids 107 to 426 thereof (TEADΔTEA). According to a specific embodiment of the present invention, the TEF-1 protein can effectively operate the gene circuit as long as it includes the fragment of amino acids 107 to 426. Furthermore, the gene circuit expressing the full-length TEF-1 protein can also effectively respond to glucose levels.

[0051] According to an embodiment of the present invention, the TEF-1 protein has the amino acid sequence shown in SEQ ID NO:4.

[0052] SKLKDQTAKDKALQHMAAMSSAQIVSATAIHNKLGLPGIPRPTFPGAPGFWPGMIQTGQPGSSQDVKPFVQQAYPIQPAVTAPIPGFEPASAPAPSVPAWQGRSIGTTKLRLVEFSAFLEQQRDPDSYNKHLFVHIGHANHSYSDPLLESVDIRQIYDKFPE KKGGLKELFGKGPQNAFFLVKFWADLNCNIQDDAGAFYGVTSQYESSENMTVTCSTKVCSFGKQVVEKVETEYARFENGRFVYRINRSPMCEYMINFIHKLKHLPEKYMMNSVLENFTILLVVTNRDTQETLLCMACVFEVSNSEHGAQHHIYRLVKD(SEQ ID NO:4).

[0053] According to an embodiment of the present invention, the transcriptional activation domain is a transcriptional activation domain derived from at least one of the following: VP64, Rta, P65, VPR and YAP1; preferably, the transcriptional activation domain includes the minimal transcriptional activation domain of herpes simplex virus protein 16 (VP16).

[0054] According to an embodiment of the present invention, the transcription activation domain has the amino acid sequence shown in SEQ ID NO:5.

[0055] PADALDDFDLDMLPADALDDFDLDMLPADALDDFDLDMLPADALDDFDLDML (SEQ ID NO: 5).

[0056] According to an embodiment of the present invention, the nuclear localization signal is derived from simian virus 40 large tumor antigen (SV40 large T antigen).

[0057] According to an embodiment of the present invention, the nuclear localization sequence has the amino acid sequence shown in SEQ ID NO:6.

[0058] PKKKRKV (SEQ ID NO:6).

[0059] According to an embodiment of the present invention, the connector is a flexible connector or a rigid connector.

[0060] According to an embodiment of the present invention, the linker has an amino acid sequence shown in any one of SEQ ID NOs: 7 to 13.

[0061] GGGGSGGGGSGGGGR (SEQ ID NO:7).

[0062] GGGGGGGGGGG (SEQ ID NO: 8).

[0063] LEASPSNGSGT (SEQ ID NO:9).

[0064] ASPSNPGASNGS (SEQ ID NO: 10).

[0065] ASAEAAKEAAKAGS (SEQ ID NO: 11).

[0066] ASGAEAAAKEAAAKAGGGS (SEQ ID NO: 12).

[0067] ASAEAAAKEAAAKEAAAKAGS (SEQ ID NO: 13).

[0068] According to an embodiment of the present invention, the chimeric transcription factor 1 has the amino acid sequence shown in SEQ ID NO: 14 or 15, and / or the chimeric transcription factor 2 has the amino acid sequence shown in SEQ ID NO: 16 or 17.

[0069] According to an embodiment of the present invention, the chimeric transcription factor 1 has the amino acid sequence shown in SEQ ID NO:14, and / or the chimeric transcription factor 2 has the amino acid sequence shown in SEQ ID NO:16; or the chimeric transcription factor 1 has the amino acid sequence shown in SEQ ID NO:15, and / or the chimeric transcription factor 2 has the amino acid sequence shown in SEQ ID NO:17.

[0070] The chimeric transcription factor 1 (YAPΔAD2-10gly-VP minx4 ) The amino acid sequence is as follows: DPGQQPPPQPAPQGQGQPP SQPPQGQGPPSGPGQPAPAATQAAPQAPPAGHQIVHVRGDSETDLEALFNAVMNPKTANVPQTVPMRLRKLPDSFFKPPEPKSHSRQASTDAGTAGALTPQHVRAHSSPASLQLGAVSPGTLTPTGVVSGPAATPTAQHLRQSSFEIPDDVPLPAGWEMAKTSSGQRYFLNHIDQTTTWQDPRKAMLSQMNVTAPTSPPVQQNMMNSASGPLPDGWEQAMTQDGEIYYINHKNKTTSWLDPRL DPRFAMNQRISQSAPVKQPPPLAPQSPQGGVMGGSNSNQQQQMRLQQLQMEKERLRLKQQELLRQAMRNINPSTANSPKCQELALRSQLPTLEQDGGTQNPVSSPGMSQELRTMTTNSSDP FLNSGTYHSRDESTDSGLSMSSYSVPRTPDDFLNSVDEMDTGDTINQSTLPSQQNRFPDYLEAIPGTNVDLGTLEGDGMNIEGEELMPSLQEALSSDILNDMESVLAATKLDKESFLTWLSS GGGGGGGGGG QLPADALDDFDLDMLPADALDDFDLDMLPADALDDFDLDMLPADALDDFDLDMLPG (SEQ ID NO: 14).

[0071] The amino acid sequence of the chimeric transcription factor 1 (TetR-YAPΔAD1) is shown below: MSRLDKSKVINSALELLNEVGI EGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRALMDPGQQPPPQPAPQGQGQPPSQPPQGQGPPSGPGQPAPAATQAAPQAPPAGHQIVHVRGDSETDLEALFNAVMNPKTANVPQTVPMRLRKLPDSFFKPPEPKSHSRQASTDAGTAGALTPQHVRAHSSPASLQLGAVSPGTLTPTGVVSGPAATPTAQHLRQSSFEIPDDVPLPAGWEMAKTSSGQRYFLNHIDQTTTWQDPRKAMLSQMNVTAPTSPPVQQNMMNSASGPLPDGWEQAMTQDGEIYYINHKNKTTSWLDPRLDPRFAMNQRISQSAPVKQPPPLAPQSPQGLAATKLDKESFLTWL (SEQ ID NO:15).

[0072] The amino acid sequence of the chimeric transcription factor 2 (TetR-NLS-TEADΔTEA) is as follows: MSRLDKSKVINSALELL NEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRALPKKKRKVSKLKDQTAKDKALQHMAAMSSAQIVSATAIHNKLGLPGIPRPTFPGAPGFWPGMIQTGQPGSSQDVKPFVQQAYPIQPAVTAPIPGFEPASAPAPSVPAWQGRSIGTTKLRLVEFSAFLEQQRDPDSYNKHLFVHIGHANHSYSDPLLESVDIRQIYDKFPEKKGGLKELFGKGPQNAFFLVKFWADLNCNIQDDAGAFYGVTSQYESSENMTVTCSTKVCSFGKQVVEKVETEYARFENGRFVYRINRSPMCEYMINFIHKLKHLPEKYMMNSVLENFTILLVVTNRDTQETLLCMACVFEVSNSEHGAQHHIYRLVKD (SEQ ID NO:16).

[0073] The chimeric transcription factor 2 (NLS-TEADΔTEA-10gly-VP minx4) is as follows: PKKKRKVSK LKDQTAKDKALQHMAAMSSAQIVSATAIHNKLGLPGIPRPTFPGAPGFWPGMIQTGQPGSSQDVKPFVQQAYPIQPAVTAPIPGFEPASAPAPSVPAWQGRSIGTTKLRLVEFSAFLEQQRDPDSYNKHLFVHIGHANHSYSDPLLESVDIRQIYDKFPE KKGGLKELFGKGPQNAFFLVKFWADLNCNIQDDAGAFYGVTSQYESSENMTVTCSTKVCSFGKQVVEKVETEYARFENGRFVYRINRSPMCEYMINFIHKLKHLPEKYMMNSVLENFTILLVVTNRDTQETLLCMACVFEVSNSEHGAQHHIYRLVKDFEF GGGGGGGGGG QLPADALDDFDLDMLPADALDDFDLDMLPADALDDFDLDMLPADALDDFDLDML (SEQ ID NO: 17).

[0074] According to an embodiment of the present invention, the effector comprises one or more target proteins; preferably, the target protein comprises at least one of the following: a reporter gene encoding protein, insulin and a glucagon-like peptide.

[0075] According to an embodiment of the present invention, the second expression vector further comprises a second promoter.

[0076] According to an embodiment of the present invention, the second promoter comprises a tetracycline regulatable promoter.

[0077] According to an embodiment of the present invention, the second promoter is operably linked to the second nucleic acid molecule.

[0078] According to an embodiment of the present invention, when the effector includes multiple target proteins, the second expression vector further includes one or more fourth nucleic acid molecules, the fourth nucleic acid molecules are located between the multiple target proteins, and the fourth nucleic acid molecules encode 2A peptide.

[0079] According to an embodiment of the present invention, the reporter gene encoding protein includes at least one selected from SEAP, GFP, CAT and β-gal.

[0080] According to an embodiment of the present invention, the effector comprises at least one selected from SEAP, insulin 2, and glucagon-like peptide-1.

[0081] According to an embodiment of the present invention, the effector is SEAP; or the effector is insulin 2; or the effector is glucagon-like peptide-1; or the effector is SEAP and insulin 2; or the effector is SEAP and glucagon-like peptide-1;

[0082] According to an embodiment of the present invention, the effector has an amino acid sequence shown in any one of SEQ ID NOs: 18 to 20; or the effector has an amino acid sequence shown in SEQ ID NOs: 18 and 19; or the effector has an amino acid sequence shown in SEQ ID NOs: 18 and 20.

[0083] The amino acid sequence of the SEAP protein is as follows:

[0084] MLGPCMLLLLLLLGLRLQLSLGIIPVEEENPDFWNREAAEALGAAKKLQPAQTAAKNLIIFLGDG

[0085] MGVSTVTAARILKGQKKDKLGPEIPLAMDRFPYVALSKTYNVDKHVPDSGATATAYLCGVKGNFQTIG

[0086] LSAAARFNQCNTTRGNEVISVMNRAKKAGKSVGVVTTTRVQHASPAGTYAHTVNRNWYSDADVPAS

[0087] ARQEGCQDIATQLISNMDIDVILGGGRKYMFRMGTPDPEYPDDYSQGGTRLDGKNLVQEWLAKRQGA

[0088] RYVWNRTELMQASLDPSVTHLMGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVE

[0089] GGRIDHGHHESRAYRALTETIMFDDAIERAGQLTSEEDTLSLVTADHSHVFSFGGYPLRGSSIFGLAPGK

[0090] ARDRKAYTVLLYGNGPGYVLKDGARPDVTESESGSPEYRQQSAVPLDEETHAGEDVAVFARGPQAHL

[0091] VHGVQEQTFIAHVMAFAACLEPYTACDLAPPAGTTD (SEQ ID NO: 18).

[0092] The amino acid sequence of insulin 2 (Insulin 2 / mINS2) is as follows:

[0093] MALWMRFLPLLALLFLWESHPTQAFVKQHLCGSHLVEALYLVCGERGFFYTPMSRREVEDPQVA QLELGGGPGAGDLQTLALEVAQQKRGIVDQCCTSICSLYQLENYCN (SEQ ID NO: 19).

[0094] The amino acid sequence of the glucagon-like peptide-1 (GLP-1 / GLP1) is as follows:

[0095] AEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 20).

[0096] According to an embodiment of the present invention, the first nucleic acid molecule has a nucleotide sequence shown in at least one of SEQ ID NOs: 21 to 24, and / or the second nucleic acid molecule has a nucleotide sequence shown in at least one of SEQ ID NOs: 25 to 27.

[0097] According to an embodiment of the present invention, the first nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 21 and 22, and the second nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 25; or

[0098] The first nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 23 and 24, and the second nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 25; or

[0099] The first nucleic acid molecule has the nucleotide sequence shown in SEQ ID NOs: 23 and 24, and the second nucleic acid molecule has the nucleotide sequence shown in SEQ ID NOs: 25 and 26; or

[0100] The first nucleic acid molecule has the nucleotide sequence shown in SEQ ID NOs: 23 and 24, and the second nucleic acid molecule has the nucleotide sequence shown in SEQ ID NOs: 25 and 27.

[0101] According to an embodiment of the present invention, the third nucleic acid molecule and / or the fourth nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:26.

[0102] The nucleotide sequence encoding the chimeric transcription factor 1 (YAPΔAD2-10gly-VP minx4 ) is as follows:

[0103] gatcccgggcagcagccgccgcctcaaccggccccccagggccaagggcagccgccttcgcagcccccgcaggggcagggcccgccgtccggacccgg

[0104] gcaaccggcacccgcggcgacccaggcggcgccgcaggcaccccccgccgggcatcagatcgtgcacgtccgcggggactcggagaccgacctggagg

[0105] cgctcttcaacgccgtcatgaaccccaagacggccaacgtgccccagaccgtgcccatgaggctccggaagctgcccgactccttcttcaagccgccggagcc

[0106] caaatcccactcccgacaggccagtactgatgcaggcactgcaggagccctgactccacagcatgttcgagctcattcctctccagcttctctgcagttgggagct

[0107] gtttctcctgggacactgacccccactggagtagtctctggcccagcagctacacccacagctcagcatcttcgacagtcttcttttgagatacctgatgatgtacctc

[0108] tgccagcaggttgggagatggcaaagacatcttctggtcagagatacttcttaaatcacatcgatcagacaacaacatggcaggaccccaggaaggccatgctgt

[0109] cccagatgaacgtcacagcccccaccagtccaccagtgcagcagaatatgatgaactcggcttcaggtcctcttcctgatggatgggaacaagccatgactcag

[0110] gatggagaaatttactatataaaccataagaacaagaccacctcttggctagacccaaggcttgaccctcgttttgccatgaaccagagaatcagtcagagtgctcc

[0111] agtgaaacagccaccacccctggctccccagagcccacagggaggcgtcatgggtggcagcaactccaaccagcagcaacagatgcgactgcagcaactgc

[0112] agatggagaaggagaggctgcggctgaaacagcaagaactgcttcggcaggcaatgcggaatatcaatcccagcacagcaaattctccaaaatgtcaggagtt

[0113] agccctgcgtagccagttaccaacactggagcaggatggtgggactcaaaatccagtgtcttctcccgggatgtctcaggaattgagaacaatgacgaccaatag

[0114] ctcagatcctttccttaacagtggcacctatcactctcgagatgagagtacagacagtggactaagcatgagcagctacagtgtccctcgaaccccagatgacttcc

[0115] tgaacagtgtggatgagatggatacaggtgatactatcaaccaaagcaccctgccctcacagcagaaccgtttcccagactaccttgaagccattcctgggacaa

[0116] atgtggaccttggaacactggaaggagatggaatgaacatagaaggagaggagctgatgccaagtctgcaggaagctttgagttctgacatccttaatgacatgg

[0117] agtctgttttggctgccaccaagctagataaagaaagctttcttacatggttatctagcggaggaggtggtggaggtggaggtggaggtcaattgcctgccgacgc

[0118] cctggacgacttcgacctggacatgctgcccgccgatgccctggatgattttgatctggatatgctccctgctgacgctctcgatgacttcgatctcgatatgctgcc

[0119] agccgacgcgctagacgatttcgatctggacatgttgccaggctaa(SEQ ID NO:21).

[0120] The nucleotide sequence encoding the chimeric transcription factor 2 (TetR-NLS-TEADΔTEA) is as follows:

[0121] atgtcaagattagataaaagtaaagtgattaacagcgcattagagctgcttaatgaggtcggaatcgaaggtttaacaacccgtaaactcgcccagaagctaggtgt

[0122] agagcagcctacattgtattggcatgtaaaaaataagcgggctttgctcgacgccttagccattgagatgttagataggcaccatactcacttttgccctttagaagg

[0123] ggaaagctggcaagattttttacgtaataacgctaaaagttttagatgtgctttactaagtcatcgcgatggagcaaaagtacatttaggtacacggcctacagaaaa

[0124] acagtatgaaactctcgaaaatcaattagcctttttatgccaacaaggtttttcactagagaatgcattatatgcactcagcgctgtggggcattttactttaggttgcgta

[0125] ttggaagatcaagagcatcaagtcgctaaagaagaaagggaaacacctactactgatagtatgccgccattattacgacaagctatcgaattatttgatcaccaagg

[0126] tgcagagccagccttcttattcggccttgaattgatcatatgcggattagaaaaacaacttaaatgtgaaagtgggtccgcgtacagccgcgcgctccctaaaaaga

[0127] aacgaaaggtttccaagctaaaggatcagactgcaaaggataaggccctgcagcacatggcggccatgtcctcagcccagatcgtctcggccactgccattcat

[0128] aacaagctggggctgcctgggattccacgcccgaccttcccaggggcgccggggttctggccgggaatgattcaaacagggcagccaggatcctcacaagac

[0129] gtcaagccttttgtgcagcaggcctaccccatccagccagcggtcacagcccccattccagggtttgagcctgcatcggccccagctccctcagtccctgcctgg

[0130] caaggtcgctccattggcacaaccaagcttcgcctggtggaattttcagcttttctcgagcagcagcgagacccagactcgtacaacaaacacctcttcgtgcacat

[0131] tgggcatgccaaccattcttacagtgacccattgcttgaatcagtggacattcgtcagatttatgacaaatttcctgaaaagaaaggtggcttaaaggaactgtttgga

[0132] aagggccctcaaaatgccttcttcctcgtaaaattctgggctgatttaaactgcaatattcaagatgatgctggggctttttatggtgtaaccagtcagtacgagagttc

[0133] tgaaaatatgacagtcacctgttccaccaaagtttgctcctttgggaagcaagtagtagaaaaagtagagacggagtatgcaaggtttgagaatggccgatttgtat

[0134] accgaataaaccgctccccaatgtgtgaatatatgatcaacttcatccacaagctcaaacacttaccagagaaatatatgatgaacagtgttttggaaaacttcacaat

[0135] tttattggtggtaacaaacagggatacacaagaaactctactctgcatggcctgtgtgtttgaagtttcaaatagtgaacacggagcacaacatcatatttacaggctt

[0136] gtaaaggactga(SEQ ID NO:22).

[0137] The nucleotide sequence encoding the chimeric transcription factor 1 (TetR-YAPΔAD1) is as follows:

[0138] atgtcaagattagataaaagtaaagtgattaacagcgcattagagctgcttaatgaggtcggaatcgaaggtttaacaacccgtaaactcgcccagaagctaggtgt

[0139] agagcagcctacattgtattggcatgtaaaaaataagcgggctttgctcgacgccttagccattgagatgttagataggcaccatactcacttttgccctttagaagg

[0140] ggaaagctggcaagattttttacgtaataacgctaaaagttttagatgtgctttactaagtcatcgcgatggagcaaaagtacatttaggtacacggcctacagaaaa

[0141] acagtatgaaactctcgaaaatcaattagcctttttatgccaacaaggtttttcactagagaatgcattatatgcactcagcgctgtggggcattttactttaggttgcgta

[0142] ttggaagatcaagagcatcaagtcgctaaagaagaaagggaaacacctactactgatagtatgccgccattattacgacaagctatcgaattatttgatcaccaagg

[0143] tgcagagccagccttcttattcggccttgaattgatcatatgcggattagaaaaacaacttaaatgtgaaagtgggtccgcgtacagccgcgcgctcatggatcccg

[0144] ggcagcagccgccgcctcaaccggccccccagggccaagggcagccgccttcgcagcccccgcaggggcagggcccgccgtccggacccgggcaaccg

[0145] gcacccgcggcgacccaggcggcgccgcaggcaccccccgccgggcatcagatcgtgcacgtccgcggggactcggagaccgacctggaggcgctcttc

[0146] aacgccgtcatgaaccccaagacggccaacgtgccccagaccgtgcccatgaggctccggaagctgcccgactccttcttcaagccgccggagcccaaatcc

[0147] cactcccgacaggccagtactgatgcaggcactgcaggagccctgactccacagcatgttcgagctcattcctctccagcttctctgcagttgggagctgtttctcc

[0148] tgggacactgacccccactggagtagtctctggcccagcagctacacccacagctcagcatcttcgacagtcttcttttgagatacctgatgatgtacctctgccag

[0149] caggttgggagatggcaaagacatcttctggtcagagatacttcttaaatcacatcgatcagacaacaacatggcaggaccccaggaaggccatgctgtcccaga

[0150] tgaacgtcacagcccccaccagtccaccagtgcagcagaatatgatgaactcggcttcaggtcctcttcctgatggatgggaacaagccatgactcaggatggag

[0151] aaatttactatataaaccataagaacaagaccacctcttggctagacccaaggcttgaccctcgttttgccatgaaccagagaatcagtcagagtgctccagtgaaa

[0152] cagccaccacccctggctccccagagcccacagggattggctgccaccaagctagataaagaaagctttcttacatggttata(SEQ ID NO:23)。

[0153] The nucleotide sequence encoding the chimeric transcription factor 2 (NLS-TEADΔTEA-10gly-VP minx4 ) is as follows:

[0154] gccgccatggtccctaaaaagaaacgaaaggtttccaagctaaaggatcagactgcaaaggataaggccctgcagcacatggcggccatgtcctcagc

[0155] ccagatcgtctcggccactgccattcataacaagctggggctgcctgggattccacgcccgaccttcccaggggcgccggggttctggccgggaatgattcaaa

[0156] cagggcagccaggatcctcacaagacgtcaagccttttgtgcagcaggcctaccccatccagccagcggtcacagcccccattccagggtttgagcctgcatcg

[0157] gccccagctccctcagtccctgcctggcaaggtcgctccattggcacaaccaagcttcgcctggtggaattttcagcttttctcgagcagcagcgagacccagact

[0158] cgtacaacaaacacctcttcgtgcacattgggcatgccaaccattcttacagtgacccattgcttgaatcagtggacattcgtcagatttatgacaaatttcctgaaaa

[0159] gaaaggtggcttaaaggaactgtttggaaagggccctcaaaatgccttcttcctcgtaaaattctgggctgatttaaactgcaatattcaagatgatgctggggcttttt

[0160] atggtgtaaccagtcagtacgagagttctgaaaatatgacagtcacctgttccaccaaagtttgctcctttgggaagcaagtagtagaaaaagtagagacggagtat

[0161] gcaaggtttgagaatggccgatttgtataccgaataaaccgctccccaatgtgtgaatatatgatcaacttcatccacaagctcaaacacttaccagagaaatatatg

[0162] atgaacagtgttttggaaaacttcacaattttattggtggtaacaaacagggatacacaagaaactctactctgcatggcctgtgtgtttgaagtttcaaatagtgaaca

[0163] cggagcacaacatcatatttacaggcttgtaaaggacttcgaattcggaggaggtggtggaggtggaggtggaggtcaattgcctgccgacgccctggacgactt

[0164] cgacctggacatgctgcccgccgatgccctggatgattttgatctggatatgctccctgctgacgctctcgatgacttcgatctcgatatgctgccagccgacgcgc

[0165] tagacgatttcgatctggacatgttgccaggctaa(SEQ ID NO:24).

[0166] The nucleotide sequence encoding the SEAP protein is as follows:

[0167] atgctggggccctgcatgctgctgctgctgctgctgctgggcctgaggctacagctctccctgggcatcatcccagttgaggaggagaacccggacttctggaac

[0168] cgcgaggcagccgaggccctgggtgccgccaagaagctgcagcctgcacagacagccgccaagaacctcatcatcttcctgggcgatgggatgggggtgtct

[0169] acggtgacagctgccaggatcctaaaagggcagaagaaggacaaactggggcctgagatacccctggccatggaccgcttcccatatgtggctctgtccaaga

[0170] catacaatgtagacaaacatgtgccagacagtggagccacagccacggcctacctgtgcggggtcaagggcaacttccagaccattggcttgagtgcagccgc

[0171] ccgctttaaccagtgcaacacgacacgcggcaacgaggtcatctccgtgatgaatcgggccaagaaagcagggaagtcagtgggagtggtaaccaccacacg

[0172] agtgcagcacgcctcgccagccggcacctacgcccacacggtgaaccgcaactggtactcggacgccgacgtgcctgcctcggcccgccaggaggggtgcc

[0173] aggacatcgctacgcagctcatctccaacatggacattgacgtgatcctaggtggaggccgaaagtacatgtttcgcatgggaaccccagaccctgagtacccag

[0174] atgactacagccaaggtgggaccaggctggacgggaagaatctggtgcaggaatggctggcgaagcgccagggtgcccggtatgtgtggaaccgcactgag

[0175] ctcatgcaggcttccctggacccgtctgtgacccatctcatgggtctctttgagcctggagacatgaaatacgagatccaccgagactccacactggacccctccct

[0176] gatggagatgacagaggctgccctgcgcctgctgagcaggaacccccgcggcttcttcctcttcgtggagggtggtcgcatcgaccatggtcatcatgaaagca

[0177] gggcttaccgggcactgactgagacgatcatgttcgacgacgccattgagagggcgggccagctcaccagcgaggaggacacgctgagcctcgtcactgccg

[0178] accactcccacgtcttctccttcggaggctaccccctgcgagggagctccatcttcgggctggcccctggcaaggcccgggacaggaaggcctacacggtcctc

[0179] ctatacggaaacggtccaggctatgtgctcaaggacggcgcccggccggatgttaccgagagcgagagcgggagccccgagtatcggcagcagtcagcagt

[0180] gcccctggacgaagagacccacgcaggcgaggacgtggcggtgttcgcgcgcggcccgcaggcgcacctggttcacggcgtgcaggagcagaccttcata

[0181] gcgcacgtcatggccttcgccgcctgcctggagccctacaccgcctgcgacctggcgccccccgccggcaccaccgac(SEQ ID NO:25).

[0182] The nucleotide sequence encoding said insulin 2 is as follows: atggccctgtggatgcgcttcctgcccctgctggccctgctcttcctctgggagtcccacc ccacccaggcttttgtcaagcagcacctttgtggttcccacctggtggaggctctctacctggtgtgtggggagcgtggcttcttctacacacccatgtcccgccgta aacgggaggacccacaagtggcacaactggagctgggtggaggcccgggagcaggtgaccttcagaccttggcactggaggtggcccgccggaagcgtgg cattgtagatcagtgctgcaccagcatctgctccctctaccagctggagaactactgcaac(SEQ IDNO:26).

[0183] The nucleotide sequence encoding the glucagon-like peptide-1 is as follows: gctgaagggacctttaccagtgatgtaagttcttatttggaaggccaagctgccaaggaattcattgcttggctggtgaaaggccgagga (SEQ ID NO: 27).

[0184] The nucleotide sequence encoding the 2A peptide is as follows:

[0185] gctactaacttcagcctgctgaagcaggctggtgacgtcgaggagaatcctggcccc (SEQ ID NO: 28).

[0186] According to an embodiment of the present invention, the first expression vector has a nucleotide sequence shown in at least one of SEQ ID NOs: 29 to 33; and / or the second expression vector has a nucleotide sequence shown in any one of SEQ ID NOs: 34 to 36.

[0187] According to an embodiment of the present invention, the first expression vector has the nucleotide sequence shown in SEQ ID NO: 29 and 30; and / or the second expression vector has the nucleotide sequence shown in SEQ ID NO: 34; or

[0188] The first expression vector has the nucleotide sequence shown in SEQ ID NO: 31 and 32; and / or the second expression vector has the nucleotide sequence shown in SEQ ID NO: 34; or

[0189] The first expression vector has the nucleotide sequence shown in SEQ ID NO: 33; and / or the second expression vector has the nucleotide sequence shown in SEQ ID NO: 35; or

[0190] The first expression vector has the nucleotide sequence shown in SEQ ID NO:31 and 32; and / or the second expression vector has the nucleotide sequence shown in SEQ ID NO:36.

[0191] Table 1 CAG promoter carried by plasmid 15-YAPΔAD2-10gly-VP minx4 The nucleotide sequence is as follows:

[0192] gacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggct

[0193] gaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggactatttacggtaa

[0194] actgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgacctt

[0195] atgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatgggtcgaggtgagccccacgttctgcttcactctccccatctcccccccctcccca

[0196] cccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggg

[0197] gcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaa

[0198] gcgaagcgcgcggcgggcgggagtcgctgcgttgccttcgccccgtgccccgctccgcgccgcctcgcgccgcccgccccggctctgactgaccgcgttact

[0199] cccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagcgcttggtttaatgacggctcgtttcttttctgtggctgcgtgaaagccttaaagggc

[0200] tccgggagggccctttgtgcgggggggagcggctcggggggtgcgtgcgtgtgtgtgtgcgtggggagcgccgcgtgcggcccgcgctgcccggcggctg

[0201] tgagcgctgcgggcgcggcgcggggctttgtgcgctccgcgtgtgcgcgaggggagcgcggccgggggcggtgccccgcggtgcgggggggctgcgag

[0202] gggaacaaaggctgcgtgcggggtgtgtgcgtgggggggtgagcagggggtgtgggcgcggcggtcgggctgtaacccccccctgcacccccctccccga

[0203] gttgctgagcacggcccggcttcgggtgcggggctccgtgcggggcgtggcgcggggctcgccgtgccgggcggggggtggcggcaggtgggggtgccg

[0204] ggcggggcggggccgcctcgggccggggagggctcgggggaggggcgcggcggccccggagcgccggcggctgtcgaggcgcggcgagccgcagc

[0205] cattgccttttatggtaatcgtgcgagagggcgcagggacttcctttgtcccaaatctggcggagccgaaatctgggaggcgccgccgcaccccctctagcgggc

[0206] gcgggcgaagcggtgcggcgccggcaggaaggaaatgggcggggagggccttcgtgcgtcgccgcgccgccgtccccttctccatctccagcctcggggct

[0207] gccgcagggggacggctgccttcgggggggacggggcagggcggggttcggcttctggcgtgtgaccggcggctctagagcctctgctaaccatgttcatgc

[0208] cttcttctttttcctacagctcctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaattcgccaccatggatcccgggcagcagccgccgcctcaa

[0209] ccggccccccagggccaagggcagccgccttcgcagcccccgcaggggcagggcccgccgtccggacccgggcaaccggcacccgcggcgacccagg

[0210] cggcgccgcaggcaccccccgccgggcatcagatcgtgcacgtccgcggggactcggagaccgacctggaggcgctcttcaacgccgtcatgaaccccaag

[0211] acggccaacgtgccccagaccgtgcccatgaggctccggaagctgcccgactccttcttcaagccgccggagcccaaatcccactcccgacaggccagtactg

[0212] atgcaggcactgcaggagccctgactccacagcatgttcgagctcattcctctccagcttctctgcagttgggagctgtttctcctgggacactgacccccactgga

[0213] gtagtctctggcccagcagctacacccacagctcagcatcttcgacagtcttcttttgagatacctgatgatgtacctctgccagcaggttgggagatggcaaagac

[0214] atcttctggtcagagatacttcttaaatcacatcgatcagacaacaacatggcaggaccccaggaaggccatgctgtcccagatgaacgtcacagcccccaccag

[0215] tccaccagtgcagcagaatatgatgaactcggcttcaggtcctcttcctgatggatgggaacaagccatgactcaggatggagaaatttactatataaaccataaga

[0216] acaagaccacctcttggctagacccaaggcttgaccctcgttttgccatgaaccagagaatcagtcagagtgctccagtgaaacagccaccacccctggctcccc

[0217] agagcccacagggaggcgtcatgggtggcagcaactccaaccagcagcaacagatgcgactgcagcaactgcagatggagaaggagaggctgcggctgaa

[0218] acagcaagaactgcttcggcaggcaatgcggaatatcaatcccagcacagcaaattctccaaaatgtcaggagttagccctgcgtagccagttaccaacactgga

[0219] gcaggatggtgggactcaaaatccagtgtcttctcccgggatgtctcaggaattgagaacaatgacgaccaatagctcagatcctttccttaacagtggcacctatc

[0220] actctcgagatgagagtacagacagtggactaagcatgagcagctacagtgtccctcgaaccccagatgacttcctgaacagtgtggatgagatggatacaggtg

[0221] atactatcaaccaaagcaccctgccctcacagcagaaccgtttcccagactaccttgaagccattcctgggacaaatgtggaccttggaacactggaaggagatg

[0222] gaatgaacatagaaggagaggagctgatgccaagtctgcaggaagctttgagttctgacatccttaatgacatggagtctgttttggctgccaccaagctagataaa

[0223] gaaagctttcttacatggttaTctagcggaggaggtggtggaggtggaggtggaggtcaattgcctaccgacgccctggacgacttcgacctggacatgctgcc

[0224] cgccgatgccctggatgattttgatctggatatgctccctgctgacgctctcgatgacttcgatctcgatatgctgccagccgacgcgctagacgatttcgatctgga

[0225] catgttgccaggctaa(SEQ ID NO:29).

[0226] The nucleotide sequence of CMV promoter - TetR - NLS - TEADΔTEA carried by plasmid 12 in Table 1 is as follows:

[0227] gacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggct

[0228] gaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaa

[0229] actgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgacctt

[0230] atgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacgg

[0231] ggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatg

[0232] ggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaaga

[0233] caccgggaccgatccagcctccgcggctagaccaccatgtcaagattagataaaagtaaagtgattaacagcgcattagagctgcttaatgaggtcggaatcgaa

[0234] ggtttaacaacccgtaaactcgcccagaagctaggtgtagagcagcctacattgtattggcatgtaaaaaataagcgggctttgctcgacgccttagccattgagat

[0235] gttagataggcaccatactcacttttgccctttagaaggggaaagctggcaagattttttacgtaataacgctaaaagttttagatgtgctttactaagtcatcgcgatg

[0236] gagcaaaagtacatttaggtacacggcctacagaaaaacagtatgaaactctcgaaaatcaattagcctttttatgccaacaaggtttttcactagagaatgcattata

[0237] tgcactcagcgctgtggggcattttactttaggttgcgtattggaagatcaagagcatcaagtcgctaaagaagaaagggaaacacctactactgatagtatgccgc

[0238] cattattacgacaagctatcgaattatttgatcaccaaggtgcagagccagccttcttattcggccttgaattgatcatatgcggattagaaaaacaacttaaatgtgaa

[0239] agtgggtccgcgtacagcCgcgcgcTCcctaaaaagaaacgaaaggtttccaagctaaaggatcagactgcaaaggataaggccctgcagcacatggcgg

[0240] ccatgtcctcagcccagatcgtctcggccactgccattcataacaagctggggctgcctgggattccacgcccgaccttcccaggggcgccggggttctggccg

[0241] ggaatgattcaaacagggcagccaggatcctcacaagacgtcaagccttttgtgcagcaggcctaccccatccagccagcggtcacagcccccattccagggtt

[0242] tgagcctgcatcggccccagctccctcagtccctgcctggcaaggtcgctccattggcacaaccaagcttcgcctggtggaattttcagcttttctcgagcagcagc

[0243] gagacccagactcgtacaacaaacacctcttcgtgcacattgggcatgccaaccattcttacagtgacccattgcttgaatcagtggacattcgtcagatttatgaca

[0244] aatttcctgaaaagaaaggtggcttaaaggaactgtttggaaagggccctcaaaatgccttcttcctcgtaaaattctgggctgatttaaactgcaatattcaagatgat

[0245] gctggggctttttatggtgtaaccagtcagtacgagagttctgaaaatatgacagtcacctgttccaccaaagtttgctcctttgggaagcaagtagtagaaaaagta

[0246] gagacggagtatgcaaggtttgagaatggccgatttgtataccgaataaaccgctccccaatgtgtgaatatatgatcaacttcatccacaagctcaaacacttacca

[0247] gagaaatatatgatgaacagtgttttggaaaacttcacaattttattggtggtaacaaacagggatacacaagaaactctactctgcatggcctgtgtgtttgaagtttc

[0248] aaatagtgaacacggagcacaacatcatatttacaggcttgtaaaggactga(SEQ ID NO:30).

[0249] The nucleotide sequence of CMV promoter - TetR - YAPΔAD1 carried by plasmid 20 in Table 1 is as follows:

[0250] gacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggct

[0251] gaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaa

[0252] actgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgacctt

[0253] atgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacgg

[0254] ggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatg

[0255] ggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaaga

[0256] caccgggaccgatccagcctccgcggctagaccaccatgtcaagattagataaaagtaaagtgattaacagcgcattagagctgcttaatgaggtcggaatcgaa

[0257] ggtttaacaacccgtaaactcgcccagaagctaggtgtagagcagcctacattgtattggcatgtaaaaaataagcgggctttgctcgacgccttagccattgagat

[0258] gttagataggcaccatactcacttttgccctttagaaggggaaagctggcaagattttttacgtaataacgctaaaagttttagatgtgctttactaagtcatcgcgatg

[0259] gagcaaaagtacatttaggtacacggcctacagaaaaacagtatgaaactctcgaaaatcaattagcctttttatgccaacaaggtttttcactagagaatgcattata

[0260] tgcactcagcgctgtggggcattttactttaggttgcgtattggaagatcaagagcatcaagtcgctaaagaagaaagggaaacacctactactgatagtatgccgc

[0261] cattattacgacaagctatcgaattatttgatcaccaaggtgcagagccagccttcttattcggccttgaattgatcatatgcggattagaaaaacaacttaaatgtgaa

[0262] agtgggtccgcgtacagccgcgcgctcatggatcccgggcagcagccgccgcctcaaccggccccccagggccaagggcagccgccttcgcagcccccgc

[0263] aggggcagggcccgccgtccggacccgggcaaccggcacccgcggcgacccaggcggcgccgcaggcaccccccgccgggcatcagatcgtgcacgtc

[0264] cgcggggactcggagaccgacctggaggcgctcttcaacgccgtcatgaaccccaagacggccaacgtgccccagaccgtgcccatgaggctccggaagct

[0265] gcccgactccttcttcaagccgccggagcccaaatcccactcccgacaggccagtactgatgcaggcactgcaggagccctgactccacagcatgttcgagctc

[0266] attcctctccagcttctctgcagttgggagctgtttctcctgggacactgacccccactggagtagtctctggcccagcagctacacccacagctcagcatcttcgac

[0267] agtcttcttttgagatacctgatgatgtacctctgccagcaggttgggagatggcaaagacatcttctggtcagagatacttcttaaatcacatcgatcagacaacaac

[0268] atggcaggaccccaggaaggccatgctgtcccagatgaacgtcacagcccccaccagtccaccagtgcagcagaatatgatgaactcggcttcaggtcctcttc

[0269] ctgatggatgggaacaagccatgactcaggatggagaaatttactatataaaccataagaacaagaccacctcttggctagacccaaggcttgaccctcgttttgcc

[0270] atgaaccagagaatcagtcagagtgctccagtgaaacagccaccacccctggctccccagagcccacagggattggctgccaccaagctagataaagaaagct

[0271] ttcttacatggttatag(SEQ ID NO:31).

[0272] The nucleotide sequence of CAG promoter - NLS - TEADΔTEA - 10gly - VP carried by plasmid 22 in Table 1 is as follows: minx4 is as follows:

[0273] gaccatagccaattcaatatggcgtatatggactcatgccaattcaatatggtggatctggacctgtgccaattcaatatggcgtatatggactcgtgccaattcaatat

[0274] ggtggatctggaccccagccaattcaatatggcggacttggcaccatgccaattcaatatggcggacttggcactgtgccaactggggaggggtctacttggcac

[0275] ggtgccaagtttgaggaggggtcttggccctgtgccaagtccgccatattgaattggcatggtgccaataatggcggccatattggctatatgccaggatcaatata

[0276] taggcaatatccaatatggccctatgccaatatggctattggccaggttcaatactatgtattggccctatgccatatagtattccatatatgggttttcctattgacgtag

[0277] atagcccctcccaatgggcggtcccatataccatatatggggcttcctaataccgcccatagccactcccccattgacgtcaatggtctctatatatggtctttcctatt

[0278] gacgtcatatgggcggtcctattgacgtatatggcgcctcccccattgacgtcaattacggtaaatggcccgcctggctcaatgcccattgacgtcaataggaccac

[0279] ccaccattgacgtcaatgggatggctcattgcccattcatatccgttctcacgccccctattgacgtcaatgacggtaaatggcccacttggcagtacatcaatatcta

[0280] ttaatagtaacttggcaagtacattactattggaaggacgccagggtacattggcagtactcccattgacgtcaatggcggtaaatggcccgcgatggctgccaagt

[0281] acatccccattgacgtcaatggggaggggcaatgacgcaaatgggcgttccattgacgtaaatgggcggtaggcgtgcctaatgggaggtctatataagcaatg

[0282] ctcgtttagggaaccgccattctgcctggggacgtcggagcaagcttgatttaggtgacactatagaatacaagctacttgttctttttgcaggatcccatcgatgccg

[0283] ccatggtccctaaaaagaaacgaaaggtttccaagctaaaggatcagactgcaaaggataaggccctgcagcacatggcggccatgtcctcagcccagatcgtc

[0284] tcggccactgccattcataacaagctggggctgcctgggattccacgcccgaccttcccaggggcgccggggttctggccgggaatgattcaaacagggcagc

[0285] caggatcctcacaagacgtcaagccttttgtgcagcaggcctaccccatccagccagcggtcacagcccccattccagggtttgagcctgcatcggccccagctc

[0286] cctcagtccctgcctggcaaggtcgctccattggcacaaccaagcttcgcctggtggaattttcagcttttctcgagcagcagcgagacccagactcgtacaacaa

[0287] acacctcttcgtgcacattgggcatgccaaccattcttacagtgacccattgcttgaatcagtggacattcgtcagatttatgacaaatttcctgaaaagaaaggtggc

[0288] ttaaaggaactgtttggaaagggccctcaaaatgccttcttcctcgtaaaattctgggctgatttaaactgcaatattcaagatgatgctggggctttttatggtgtaacc

[0289] agtcagtacgagagttctgaaaatatgacagtcacctgttccaccaaagtttgctcctttgggaagcaagtagtagaaaaagtagagacggagtatgcaaggtttga

[0290] gaatggccgatttgtataccgaataaaccgctccccaatgtgtgaatatatgatcaacttcatccacaagctcaaacacttaccagagaaatatatgatgaacagtgtt

[0291] ttggaaaacttcacaattttattggtggtaacaaacagggatacacaagaaactctactctgcatggcctgtgtgtttgaagtttcaaatagtgaacacggagcacaa

[0292] catcatatttacaggcttgtaaaggacttcgaattcggaggaggtggtggaggtggaggtggaggtcaattgcctaccgacgccctggacgacttcgacctggac

[0293] atgctgcccgccgatgccctggatgattttgatctggatatgctccctgctgacgctctcgatgacttcgatctcgatatgctgccagccgacgcgctagacgatttc

[0294] gatctggacatgttgccaggctaa(SEQ ID NO:32)。

[0295] The nucleotide sequence of CMV promoter - TetR - YAPΔAD1 - P2A - NLS - TEADΔTEA - 10gly - VP carried by plasmid 26 in Table 1 is as follows: minx4

[0296] gacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggct

[0297] gaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaa

[0298] actgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgacctt

[0299] atgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacgg

[0300] ggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatg

[0301] ggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaaga

[0302] ​caccgggaccgatccagcctccgcggctagaccaccatgtcaagattagataaaagtaaagtgattaacagcgcattagagctgcttaatgaggtcggaatcgaa

[0303] ggtttaacaacccgtaaactcgcccagaagctaggtgtagagcagcctacattgtattggcatgtaaaaaataagcgggctttgctcgacgccttagccattgagat

[0304] gttagataggcaccatactcacttttgccctttagaaggggaaagctggcaagattttttacgtaataacgctaaaagttttagatgtgctttactaagtcatcgcgatg

[0305] gagcaaaagtacatttaggtacacggcctacagaaaaacagtatgaaactctcgaaaatcaattagcctttttatgccaacaaggtttttcactagagaatgcattata

[0306] tgcactcagcgctgtggggcattttactttaggttgcgtattggaagatcaagagcatcaagtcgctaaagaagaaagggaaacacctactactgatagtatgccgc

[0307] cattattacgacaagctatcgaattatttgatcaccaaggtgcagagccagccttcttattcggccttgaattgatcatatgcggattagaaaaacaacttaaatgtgaa

[0308] agtgggtccgcgtacagccgcgcgctcatggatcccgggcagcagccgccgcctcaaccggccccccagggccaagggcagccgccttcgcagcccccgc

[0309] aggggcagggcccgccgtccggacccgggcaaccggcacccgcggcgacccaggcggcgccgcaggcaccccccgccgggcatcagatcgtgcacgtc

[0310] cgcggggactcggagaccgacctggaggcgctcttcaacgccgtcatgaaccccaagacggccaacgtgccccagaccgtgcccatgaggctccggaagct

[0311] gcccgactccttcttcaagccgccggagcccaaatcccactcccgacaggccagtactgatgcaggcactgcaggagccctgactccacagcatgttcgagctc

[0312] attcctctccagcttctctgcagttgggagctgtttctcctgggacactgacccccactggagtagtctctggcccagcagctacacccacagctcagcatcttcgac

[0313] agtcttcttttgagatacctgatgatgtacctctgccagcaggttgggagatggcaaagacatcttctggtcagagatacttcttaaatcacatcgatcagacaacaac

[0314] atggcaggaccccaggaaggccatgctgtcccagatgaacgtcacagcccccaccagtccaccagtgcagcagaatatgatgaactcggcttcaggtcctcttc

[0315] ctgatggatgggaacaagccatgactcaggatggagaaatttactatataaaccataagaacaagaccacctcttggctagacccaaggcttgaccctcgttttgcc

[0316] atgaaccagagaatcagtcagagtgctccagtgaaacagccaccacccctggctccccagagcccacagggattggctgccaccaagctagataaagaaagct

[0317] ttcttacatggttaggcagtggagctactaacttcagcctgctgaagcaggctggtgacgtcgaggagaatcctggccccgccaccatggtccctaaaaagaaac

[0318] gaaaggtttccaagctaaaggatcagactgcaaaggataaggccctgcagcacatggcggccatgtcctcagcccagatcgtctcggccactgccattcataac

[0319] aagctggggctgcctgggattccacgcccgaccttcccaggggcgccggggttctggccgggaatgattcaaacagggcagccaggatcctcacaagacgtc

[0320] aagccttttgtgcagcaggcctaccccatccagccagcggtcacagcccccattccagggtttgagcctgcatcggccccagctccctcagtccctgcctggcaa

[0321] ggtcgctccattggcacaaccaagcttcgcctggtggaattttcagcttttctcgagcagcagcgagacccagactcgtacaacaaacacctcttcgtgcacattgg

[0322] gcatgccaaccattcttacagtgacccattgcttgaatcagtggacattcgtcagatttatgacaaatttcctgaaaagaaaggtggcttaaaggaactgtttggaaag

[0323] ggccctcaaaatgccttcttcctcgtaaaattctgggctgatttaaactgcaatattcaagatgatgctggggctttttatggtgtaaccagtcagtacgagagttctga

[0324] aaatatgacagtcacctgttccaccaaagtttgctcctttgggaagcaagtagtagaaaaagtagagacggagtatgcaaggtttgagaatggccgatttgtatacc

[0325] gaataaaccgctccccaatgtgtgaatatatgatcaacttcatccacaagctcaaacacttaccagagaaatatatgatgaacagtgttttggaaaacttcacaatttta

[0326] ttggtggtaacaaacagggatacacaagaaactctactctgcatggcctgtgtgtttgaagtttcaaatagtgaacacggagcacaacatcatatttacaggcttgta

[0327] aaggacttcgaattcggaggaggtggtggaggtggaggtggaggtcaattgcctaccgacgccctggacgacttcgacctggacatgctgcccgccgatgccc

[0328] tggatgattttgatctggatatgctccctgctgacgctctcgatgacttcgatctcgatatgctgccagccgacgcgctagacgatttcgatctggacatgttgccagg

[0329] cgggtccggcgctactaacttcagcctgctgaagcaggctggtgacgtcgaggagaatcctggtcccatgaccgagtacaagcccacggtgcgcctcgccacc

[0330] cgcgacgacgtccccagggccgtacgcaccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgggt

[0331] caccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccgg

[0332] agagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctg

[0333] gcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctgggcagcgccgtcgtgctccccg

[0334] gagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccg

[0335] acgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgcctga(SEQ IDNO:33).

[0336] The nucleotide sequence of tetO-SEAP carried by plasmid 16 in Table 1 is as follows:

[0337] tccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaag

[0338] tgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccct

[0339] atcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagctcggtacccgggtcgaggtaggtaggcgtgtacggtg

[0340] ggaggcctatataagcagagctcgtttagtgaaccgtcagatcgccgaattccgcattgcagagatattgtatttaagtgcctagctcgatacaataaacgccatttg

[0341] accattcaccacattggtgtgcacctcaagctaattcctgcctcgccactgtcctgctgccctccagacatgctggggccctgcatgctgctgctgctgctgctgctg

[0342] ggcctgaggctacagctctccctgggcatcatcccagttgaggaggagaacccggacttctggaaccgcgaggcagccgaggccctgggtgccgccaagaa

[0343] gctgcagcctgcacagacagccgccaagaacctcatcatcttcctgggcgatgggatgggggtgtctacggtgacagctgccaggatcctaaaagggcagaag

[0344] aaggacaaactggggcctgagatacccctggccatggaccgcttcccatatgtggctctgtccaagacatacaatgtagacaaacatgtgccagacagtggagc

[0345] cacagccacggcctacctgtgcggggtcaagggcaacttccagaccattggcttgagtgcagccgcccgctttaaccagtgcaacacgacacgcggcaacga

[0346] ggtcatctccgtgatgaatcgggccaagaaagcagggaagtcagtgggagtggtaaccaccacacgagtgcagcacgcctcgccagccggcacctacgcccc

[0347] acacggtgaaccgcaactggtactcggacgccgacgtgcctgcctcggcccgccaggaggggtgccaggacatcgctacgcagctcatctccaacatggaca

[0348] ttgacgtgatcctaggtggaggccgaaagtacatgtttcgcatgggaaccccagaccctgagtacccagatgactacagccaaggtgggaccaggctggacgg

[0349] gaagaatctggtgcaggaatggctggcgaagcgccagggtgcccggtatgtgtggaaccgcactgagctcatgcaggcttccctggacccgtctgtgacccatc

[0350] tcatgggtctctttgagcctggagacatgaaatacgagatccaccgagactccacactggaccctccctgatggagatgacagaggctgccctgcgcctgctga

[0351] gcaggaaccccgcggcttcttcctcttcgtggagggtggtcgcatcgaccatggtcatcatgaaagcagggcttaccgggcactgactgagacgatcatgttcg

[0352] acgacgccattgagagggcgggccagctcaccagcgaggaggacacgctgagcctcgtcactgccgaccactcccacgtcttctccttcggaggctaccccct

[0353] gcgagggagctccatcttcgggctggcccctggcaaggcccgggacaggaaggcctacacggtcctcctatacggaaacggtccaggctatgtgctcaagga

[0354] cggcgcccggccggatgttaccgagagcgagagcgggagccccgagtatcggcagcagtcagcagtgcccctggacgaagagacccacgcaggcgagga

[0355] cgtggcggtgttcgcgcgcggcccgcaggcgcacctggttcacggcgtgcaggagcagaccttcatagcgcacgtcatggccttcgccgcctgcctggagcc

[0356] ctacaccgcctgcgacctggcgccccccgccggcaccaccgacgccgcgcacccgggttaa(SEQ IDNO:34).

[0357] The nucleotide sequence of tetO-SEAP-P2A-mINS2 carried by plasmid 28 in Table 1 is as follows:

[0358] gagtttactccctatcagtgatagagaacgtatgtcgagtttactccctatcagtgatagagaacgatgtcgagtttactccctatcagtgatagagaacgtatgtcga

[0359] gtttactccctatcagtgatagagaacgtatgtcgagtttactccctatcagtgatagagaacgtatgtcgagtttatccctatcagtgatagagaacgtatgtcgagttt

[0360] actccctatcagtgatagagaacgtatgtcgattaggcgtgtacggtgggcgcctataaaagcagagctcgtttagtgaaccgtcagatcgcctggagccaattcc

[0361] aactcttttgccttatacctactttccgtaccacttcctaccctcgaaaggcctctgaggccatgctggggccctgcatgctgctgctgctgctgctgctgggcctgag

[0362] gctacagctctccctgggcatcatcccagttgaggaggagaacccggacttctggaaccgcgaggcagccgaggccctgggtgccgccaagaagctgcagcc

[0363] tgcacagacagccgccaagaacctcatcatcttcctgggcgatgggatgggggtgtctacggtgacagctgccaggatcctaaaagggcagaagaaggacaa

[0364] actggggcctgagatacccctggccatggaccgcttcccatatgtggctctgtccaagacatacaatgtagacaaacatgtgccagacagtggagccacagcca

[0365] cggcctacctgtgcggggtcaagggcaacttccagaccattggcttgagtgcagccgcccgctttaaccagtgcaacacgacacgcggcaacgaggtcatctcc

[0366] gtgatgaatcgggccaagaaagcagggaagtcagtgggagtggtaaccaccacacgagtgcagcacgcctcgccagccggcacctacgcccacacggtgaa

[0367] ccgcaactggtactcggacgccgacgtgcctgcctcggcccgccaggaggggtgccaggacatcgctacgcagctcatctccaacatggacattgacgtgatc

[0368] ctaggtggaggccgaaagtacatgtttcgcatgggaaccccagaccctgagtacccagatgactacagccaaggtgggaccaggctggacgggaagaatctg

[0369] gtgcaggaatggctggcgaagcgccagggtgcccggtatgtgtggaaccgcactgagctcatgcaggcttccctggacccgtctgtgacccatctcatgggtct

[0370] ctttgagcctggagacatgaaatacgagatccaccgagactccacactggacccctccctgatggagatgacagaggctgccctgcgcctgctgagcaggaac

[0371] ccccgcggcttcttcctcttcgtggagggtggtcgcatcgaccatggtcatcatgaaagcagggcttaccgggcactgactgagacgatcatgttcgacgacgcc

[0372] attgagagggcgggccagctcaccagcgaggaggacacgctgagcctcgtcactgccgaccactcccacgtcttctccttcggaggctaccccctgcgaggga

[0373] gctccatcttcgggctggcccctggcaaggcccgggacaggaaggcctacacggtcctcctatacggaaacggtccaggctatgtgctcaaggacggcgccc

[0374] ggccggatgttaccgagagcgagagcgggagccccgagtatcggcagcagtcagcagtgcccctggacgaagagacccacgcaggcgaggacgtggcgg

[0375] tgttcgcgcgcggcccgcaggcgcacctggttcacggcgtgcaggagcagaccttcatagcgcacgtcatggccttcgccgcctgcctggagccctacaccgc

[0376] ctgcgacctggcgccccccgccggcaccaccgacggcagtggagctactaacttcagcctgctgaagcaggctggtgacgtcgaggagaatcctggccccgc

[0377] caccatggtcatggccctgtggatgcgcttcctgcccctgctggccctgctcttcctctgggagtcccaccccacccaggcttttgtcaagcagcacctttgtggttc

[0378] ccacctggtggaggctctctacctggtgtgtggggagcgtggcttcttctacacacccatgtcccgccgtgaagtggaggacccacaagtggcacaactggagct

[0379] gggtggaggcccgggagcaggtgaccttcagaccttggcactggaggtggcccagcagaagcgtggcattgtagatcagtgctgcaccagcatctgctccctc

[0380] taccagctggagaactactgcaactag(SEQ ID NO:35).

[0381] The nucleotide sequence of tetO-SEAP-P2A-GLP-1 carried by plasmid 29 in Table 1 is as follows:

[0382] gagtttactccctatcagtgatagagaacgtatgtcgagtttactccctatcagtgatagagaacgatgtcgagtttactccctatcagtgatagagaacgtatgtcga

[0383] gtttactccctatcagtgatagagaacgtatgtcgagtttactccctatcagtgatagagaacgtatgtcgagtttatccctatcagtgatagagaacgtatgtcgagttt

[0384] actccctatcagtgatagagaacgtatgtcgattaggcgtgtacggtgggcgcctataaaagcagagctcgtttagtgaaccgtcagatcgcctggagccaattcc

[0385] aactcttttgccttatacctactttccgtaccacttcctaccctcgaaaggcctctgatgctggggccctgcatgctgctgctgctgctgctgctgggcctgaggctac

[0386] agctctccctgggcatcatcccagttgaggaggagaacccggacttctggaaccgcgaggcagccgaggccctgggtgccgccaagaagctgcagcctgcac

[0387] agacagccgccaagaacctcatcatcttcctgggcgatgggatgggggtgtctacggtgacagctgccaggatcctaaaagggcagaagaaggacaaactgg

[0388] ggcctgagatacccctggccatggaccgcttcccatatgtggctctgtccaagacatacaatgtagacaaacatgtgccagacagtggagccacagccacggcc

[0389] tacctgtgcggggtcaagggcaacttccagaccattggcttgagtgcagccgcccgctttaaccagtgcaacacgacacgcggcaacgaggtcatctccgtgat

[0390] gaatcgggccaagaaagcagggaagtcagtgggagtggtaaccaccacacgagtgcagcacgcctcgccagccggcacctacgcccacacggtgaaccgc

[0391] aactggtactcggacgccgacgtgcctgcctcggcccgccaggaggggtgccaggacatcgctacgcagctcatctccaacatggacattgacgtgatcctag

[0392] gtggaggccgaaagtacatgtttcgcatgggaaccccagaccctgagtacccagatgactacagccaaggtgggaccaggctggacgggaagaatctggtgc

[0393] aggaatggctggcgaagcgccagggtgcccggtatgtgtggaaccgcactgagctcatgcaggcttccctggacccgtctgtgacccatctcatgggtctctttg

[0394] agcctggagacatgaaatacgagatccaccgagactccacactggacccctccctgatggagatgacagaggctgccctgcgcctgctgagcaggaaccccc

[0395] gcggcttcttcctcttcgtggagggtggtcgcatcgaccatggtcatcatgaaagcagggcttaccgggcactgactgagacgatcatgttcgacgacgccattga

[0396] gagggcgggccagctcaccagcgaggaggacacgctgagcctcgtcactgccgaccactcccacgtcttctccttcggaggctaccccctgcgagggagctc

[0397] catcttcgggctggcccctggcaaggcccgggacaggaaggcctacacggtcctcctatacggaaacggtccaggctatgtgctcaaggacggcgcccggcc

[0398] ggatgttaccgagagcgagagcgggagccccgagtatcggcagcagtcagcagtgcccctggacgaagagacccacgcaggcgaggacgtggcggtgttc

[0399] gcgcgcggcccgcaggcgcacctggttcacggcgtgcaggagcagaccttcatagcgcacgtcatggccttcgccgcctgcctggagccctacaccgcctgc

[0400] gacctggcgccccccgccggcaccaccgacggcagtggagctactaacttcagcctgctgaagcaggctggtgacgtcgaggagaatcctggccccgccac

[0401] catggtcatggccctgtggatgcgcttcctgcccctgctggccctgctcttcctctgggagtcccaccccacccaggctcgaggtcgtcgacatgctgaagggac

[0402] ctttaccagtgatgtaagttcttatttggaaggccaagctgccaaggaattcattgcttggctggtgaaaggccgaggataa(SEQ ID NO:36)。

[0403] In a second aspect of the present invention, the present invention provides a recombinant cell, characterized in that the recombinant cell carries the gene circuit described in the first aspect. The recombinant cell is obtained by transfecting or transforming the gene circuit. According to an embodiment of the present invention, the recombinant cell can efficiently express the target protein under appropriate conditions and has a good effect of preventing and / or treating diabetes and its complications.

[0404] It should be noted that the recombinant cells of the present invention are not particularly limited and can be any eukaryotic cells, including fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cells such as armyworms, plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc. In some embodiments, the recombinant cells of the present invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells or COS cells, and do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0405] It should be noted that the "suitable conditions" described in the specification of this application refer to conditions suitable for the expression of the target protein described in this application. It is easy for those skilled in the art to understand that conditions suitable for the expression of the target protein include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell states, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the target protein according to the specific environment of the laboratory.

[0406] In a third aspect of the present invention, the present invention provides a hydrogel microcapsule. According to an embodiment of the present invention, the hydrogel microcapsule comprises the gene circuit described in the first aspect or the recombinant cell described in the second aspect. The hydrogel microcapsule according to an embodiment of the present invention can safely and effectively treat diabetes and its complications.

[0407] In the fourth aspect of the present invention, the present invention proposes the use of the gene circuit described in the first aspect, the recombinant cell described in the second aspect, or the hydrogel microcapsule described in the third aspect in the preparation of a drug, the drug is used to prevent and / or treat diabetes and its complications. As mentioned above, the gene circuit has a moderate response sensitivity to high glucose concentrations. In a high blood glucose concentration (>10mM) environment, the transcription level of the target gene in the gene circuit is significantly increased, while when exposed to low glucose concentrations (<5mM), the expression level of the target gene is low. The kinetics of the release of the target protein of the gene circuit is close to that of natural pancreatic beta cells, which is safe and effective. Furthermore, the recombinant cells and hydrogel microcapsules containing the gene circuit also have the above functions and can respond to high glucose environments, thereby effectively treating and / or preventing diabetes and its complications.

[0408] According to an embodiment of the present invention, the above-mentioned use may also include at least one of the following additional technical features:

[0409] According to an embodiment of the present invention, the diabetes includes type I diabetes and type II diabetes.

[0410] According to an embodiment of the present invention, the complication includes at least one of renal capsule damage, renal interstitial damage, retinal damage and pancreatic islet damage.

[0411] In the fifth aspect of the present invention, the present invention proposes a drug. According to an embodiment of the present invention, the drug includes the gene circuit described in the first aspect, the recombinant cell described in the second aspect, or the hydrogel microcapsule described in the third aspect. As mentioned above, the gene circuit has a moderate response sensitivity to high glucose concentrations. In a high blood glucose concentration (>10mM) environment, the transcription level of the target gene in the gene circuit is significantly increased, and when exposed to low glucose concentrations (<5mM), the expression level of the target gene is low. The kinetics of the release of the target protein of the gene circuit are close to those of natural pancreatic beta cells, which is safe and effective. Furthermore, the recombinant cells and hydrogel microcapsules containing the gene circuit also have the above functions. Therefore, the drug according to the embodiment of the present invention can also respond to high glucose environments, thereby effectively treating and / or preventing diabetes and its complications.

[0412] According to an embodiment of the present invention, the medicine provided by the present invention may further include a pharmaceutically acceptable carrier.

[0413] The term "pharmaceutically acceptable" indicates that the composition can be administered to a subject without producing adverse physiological reactions that prevent the administration of the composition. For example, "pharmaceutically acceptable carrier" refers to a carrier useful in preparing generally safe, non-toxic, and desirable drugs. Preferably, examples of these carriers or diluents include, but are not limited to: water, saline, Ringer's solution, glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, polyalkylene glycols such as polypropylene glycol, triglycerides, 5% human serum albumin, liposomes and non-aqueous vehicles, such as fixed oils, can also be used.

[0414] The medicaments of the present invention may also be administered in combination with each other or with one or more other therapeutic compounds, for example, with other therapeutic agents for the treatment of diabetes and its complications. Thus, the medicaments may also contain other therapeutic agents.

[0415] Typically, the drug is administered in an effective amount, i.e., an amount sufficient to achieve the desired therapeutic and / or preventive effect, for example, an amount that causes the prevention or alleviation of symptoms associated with the disease being treated, such as diabetes and / or diabetic complications. The effective amount of the drug administered to the subject will depend on the type and severity of the disease, as well as on the characteristics of the individual, such as general health, age, sex, weight, and tolerance to the drug; it will also depend on the severity and type of the disease, and a person skilled in the art will be able to determine an appropriate dose based on these factors, among others. The route of administration may be oral, nasal, intradermal, subcutaneous, intramuscular, or intravenous or intraperitoneal.

[0416] In the sixth aspect of the present invention, the present invention proposes the use of the gene circuit described in the first aspect, the recombinant cell described in the second aspect, the hydrogel microcapsule described in the third aspect, or the drug described in the fifth aspect in the treatment and / or prevention of diabetes and its complications. As mentioned above, the gene circuit has a moderate response sensitivity to high glucose concentrations. In a high blood glucose concentration (>10mM) environment, the transcription level of the target gene in the gene circuit is significantly increased, while when exposed to low glucose concentrations (<5mM), the expression level of the target gene is low. The kinetics of the release of the target protein of the gene circuit is close to that of natural pancreatic beta cells, which is safe and effective. Furthermore, the recombinant cells, hydrogel microcapsules and drugs containing the gene circuit also have the above functions and can effectively treat and / or prevent diabetes and its complications.

[0417] According to an embodiment of the present invention, the above-mentioned use may also include at least one of the following additional technical features:

[0418] According to an embodiment of the present invention, the diabetes includes type I diabetes and type II diabetes.

[0419] According to an embodiment of the present invention, the complication includes at least one of renal capsule damage, renal interstitial damage, retinal damage and pancreatic islet damage.

[0420] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0421] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0422] Figure 1 A schematic diagram of a synthetic gene expression circuit for hyperglycemia response containing version 1 chimeric transcription factors according to an embodiment of the present invention is shown;

[0423] Figure 2A schematic diagram of the principle of a synthetic gene expression circuit for hyperglycemia response containing version 2 chimeric transcription factors according to an embodiment of the present invention is shown;

[0424] Figure 3 The experimental results of different glucose concentrations regulating SEAP expression in a synthetic gene circuit for hyperglycemia response containing version 1 (Figure A) or version 2 (Figure B) chimeric transcription factors according to an embodiment of the present invention are shown;

[0425] Figure 4 The diagram shows the experimental results of expressing SEAP in different mammalian cells using a synthetic gene expression circuit for hyperglycemia response containing version 1 (Figure A) or version 2 (Figure B) chimeric transcription factors according to an embodiment of the present invention;

[0426] Figure 5 A graph showing the experimental results of the working efficiency of the synthetic gene expression circuit for hyperglycemia response containing version 1 or version 2 chimeric transcription factors according to an embodiment of the present invention;

[0427] Figure 6 The figure shows the experimental results of orthogonality detection of different chimeric transcription factors according to the embodiment of the present invention;

[0428] Figure 7 A schematic diagram showing the qPCR primer binding sites of the version 2 chimeric transcription factor of an embodiment of the present invention;

[0429] Figure 8 The graph shows the experimental results of integrating the synthetic gene circuit assembly containing version 2 chimeric transcription factor of the embodiment of the present invention into the genomes of multiple engineered cells, wherein HEK293T in the abscissa is the negative control group, and the rest are the numbers of the engineered cells;

[0430] Fig. 9 The experimental results of different concentrations of glucose inducing multiple engineered cells containing version 2 chimeric transcription factors to secrete the reporter gene SEAP (Figure A) and secrete mINS2 (Figure B) according to the embodiment of the present invention are shown, wherein the abscissa is the number of the engineered cells;

[0431] Fig.10 Shows enlarged views of the hydrogel microcapsule implant according to an embodiment of the present invention at different scales;

[0432] Fig.11 The fasting blood glucose values ​​of the type I diabetes model mice on days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 21 after the hydrogel microcapsules containing the synthetic gene expression circuit of the hyperglycemia response according to the embodiment of the present invention were transplanted into the mice;

[0433] Fig.12The graphs show the results of glucose tolerance experiments on the 8th day (Figure A) and the 14th day (Figure B) of the hydrogel microcapsules containing the synthetic gene expression circuit of the hyperglycemia response according to the embodiment of the present invention transplanted into the type I diabetes model mice;

[0434] Fig.13 The fasting blood glucose values ​​of the hydrogel microcapsules containing the synthetic gene expression circuit of the hyperglycemia response of the present invention were transplanted into the type II diabetes model mice on the 0th to 2nd day (Figure A) and the glucose tolerance test results on the 2nd day (Figure B);

[0435] Fig.14 The results of the H&E staining experiment of the pancreatic islets of each group of mice on the 21st day after the hydrogel microcapsules containing the synthetic gene expression circuit of the hyperglycemia response according to the embodiment of the present invention were transplanted into the type I diabetes model mice;

[0436] Fig.15 The results of the pancreatic islet immunofluorescence staining experiment of each group of mice on the 21st day after the hydrogel microcapsules containing the synthetic gene expression circuit of the hyperglycemia response according to the embodiment of the present invention were transplanted into the type I diabetes model mice are shown;

[0437] Fig.16 A graph showing the results of a histological staining experiment of the renal capsule of each group of mice on the 21st day after the hydrogel microcapsules containing a synthetic gene expression circuit responsive to hyperglycemia according to an embodiment of the present invention were transplanted into type I diabetes model mice;

[0438] Fig.17 The graph shows the results of the renal interstitial histological staining experiment of each group of mice on the 21st day after the hydrogel microcapsules containing the synthetic gene expression circuit of the hyperglycemia response of the embodiment of the present invention were transplanted into the body of type I diabetes model mice;

[0439] Fig.18 The results of immunofluorescence staining experiments of retinal ganglion cells in each group of mice on the 21st day after the hydrogel microcapsules containing the synthetic gene expression circuit of hyperglycemia response according to the embodiment of the present invention were transplanted into type I diabetes model mice are shown;

[0440] Fig.19 The figure shows the experimental results of immunofluorescence staining of retinal blood vessels of each group of mice on the 21st day after the hydrogel microcapsules containing the synthetic gene expression circuit responsive to hyperglycemia according to the embodiment of the present invention were transplanted into type I diabetes model mice. DETAILED DESCRIPTION

[0441] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0442] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0443] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0444] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0445] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0446] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0447] In this article, the term "complication" refers to a new disease or symptom that occurs during the development or treatment of an existing disease. It is not part of the primary disease itself, but is directly or indirectly related to the primary disease. For example, the complications of diabetes described herein include a variety of complications caused by long-term elevated blood sugar levels, including: islet damage, hypoglycemia, ketoacidosis, lactic acidosis, hyperosmolar hyperglycemic state, cardiovascular disease (e.g., atherosclerosis), cerebrovascular disease (e.g., stroke), kidney disease (e.g., renal capsule damage, renal interstitial damage), retinal damage, neuropathy, diabetic foot, sexual dysfunction, depression, dementia, hepatobiliary disease, skin disease, etc.

[0448] In this article, the term "pharmaceutically acceptable" indicates that the drug can be administered to a subject without producing adverse physiological reactions that prevent the administration of the composition. For example, "pharmaceutically acceptable carrier" refers to a carrier useful in preparing a generally safe, non-toxic, and desirable pharmaceutical composition. Preferably, examples of these carriers or diluents include, but are not limited to: water, saline, Ringer's solution, glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, polyalkylene glycols such as polypropylene glycol, triglycerides, 5% human serum albumin, liposomes and non-aqueous vehicles, such as fixed oils, can also be used.

[0449] For ease of understanding, the technical solution of the present invention is summarized as follows: The synthetic gene expression circuit for hyperglycemia response proposed by the present invention includes a glucose receptor that can sense blood glucose concentration and an effector that responds to the signal transmitted by the glucose receptor.

[0450] According to a preferred embodiment of the present invention, the glucose sensor comprises a promoter for expressing the glucose sensor and two chimeric transcription factors. The chimeric transcription factors have two versions: version 1 is TetR-NLS-TEADΔTEA and YAPΔAD2-10gly-VP minx4 Version 2 is TetR-YAPΔAD1 and NLS-TEADΔTEA-10gly-VP minx4 Version 2 is an optimized version based on version 1: (1) it exhibits stronger target gene expression under high-concentration glucose treatment; (2) it has better orthogonality and less interference with the endogenous YAP pathway.

[0451] The promoter can be any promoter that can work in mammalian cell lines, such as cytomegalovirus (CMV) promoter, EF-1α promoter, cytomegalovirus immediate early enhancer / chicken β-actin (CAG) promoter, etc.

[0452] Chimeric transcription factor version 1:

[0453] TetR-NLS-TEADΔTEA is a fusion of amino acids 2 to 208 of the TetR family transcriptional regulator protein (GenBank: MWC77513.1), the nuclear localization signal (NLS, PKKKRKV, SEQ ID NO: 5) derived from the simian virus 40 large tumor antigen (SV40 large T antigen), and amino acids 107 to 426 of the transcriptional enhancer factor TEF-1 protein (NCBI Reference Sequence: NP_068780.2).

[0454] YAP-10gly-VP minx4It is composed of amino acids 2-504 of the transcriptional coactivator YAP1 subtype 1 protein (NCBI Reference Sequence: NP_001123617.1), a fusion protein linker 10Gly (GGGGGGGGGG), and the minimal transcriptional activation domain (VP) from herpes simplex virus protein 16 (VP16). min ) four tandem copies (VP minx4 ) fusion.

[0455] Chimeric transcription factor version 2:

[0456] TetR-YAPΔAD1 is a fusion of amino acids 2 to 208 of the TetR family transcriptional regulator protein (GenBank: MWC77513.1) and amino acids 1 to 292 of the transcriptional coactivator YAP1 subtype 1 protein (NCBI Reference Sequence: NP_001123617.1). Different fusion protein linker sequences can be added between the two amino acid sequences.

[0457] NLS-TEADΔTEA-10gly-VP minx4 It is composed of the nuclear localization signal (NLS, PKKKRKV) derived from the simian virus 40 large tumor antigen (SV40 large T antigen), the 107-426 amino acids of the transcription enhancer factor TEF-1 protein (NCBI Reference Sequence: NP_068780.2), and the minimal transcription activation domain (VP16) from the herpes simplex virus protein. min ) four tandem copies (VP minx4 ) fusion. Among them, different fusion protein linker sequences can be added between the 107-426 amino acids of the transcription enhancer factor TEF-1 protein (NCBI Reference Sequence: NP_068780.2) and the four tandem copies of the minimal transcription activation domain from herpes simplex virus protein 16 (VP16). The four tandem copies of the minimal transcription activation domain from herpes simplex virus protein 16 (VP16) can be replaced by other transcription activation domains, such as a two-by-two combination of the three transcription activation domains of VP64, Rta, and P65, VPR, and the transcription activation domain (amino acids 293-489) from the transcription coactivator YAP1 subtype 1 protein (NCBI Reference Sequence: NP_001123617.1).

[0458] The effector includes a promoter sequence (tetracycline-regulatable promoter PhCMV*-1) and a nucleic acid sequence of a protein to be transcribed. The protein to be transcribed can be any meaningful protein, such as a protein as a reporter gene and / or a nucleic acid sequence of a drug protein or small peptide for treating a disease. The protein encoded by the reporter gene includes secretory alkaline phosphatase (SEAP), enhanced green fluorescent protein (EGFP), and luciferase; the drug protein or small peptide for treating a disease includes the nucleic acid sequence of insulin and glucagon-like peptide-1. The protein to be transcribed can be expressed simultaneously on the same expression vector through the 2A sequence, such as SEAP-2A-Insulin, SEAP-2A-GLP1.

[0459] like Figure 1 and 2 Working principle: Under high blood sugar conditions, YAPΔAD2-10gly-VP minx4 Or TetR-YAPΔAD1 will be translocated to the nucleus due to O-GlcNAc glycosylation. minx4 or TetR-YAPΔAD1 with TetR-NLS-TEADΔTEA or NLS-TEADΔTEA-10gly-VP localized in the nucleus minx4 Such a combination further promotes the DNA binding function of TetR domain and the transcription activation function of VP minx4 dimerization, thereby triggering the expression of TetR or VP minx4 Tetracycline-dependent regulation of transcription of downstream genes controlled by the promoter PhCMV*-1. Under low-glucose or normal-glucose conditions, protein O-GlcNAcylation was reduced compared with YAPΔAD2-10gly-VP minx4 or the threshold required for nuclear localization of TetR-YAPΔAD1, YAPΔAD2-10gly-VP minx4 Or if TetR-YAPΔAD1 cannot enter the cell, the gene expression and transcription cannot be turned on. The components of the synthetic gene expression circuit for hyperglycemia response are constructed in a eukaryotic expression vector by genetic engineering technology, thereby regulating the transcription and expression of the target gene. Transfection / transduction of the eukaryotic expression vector into a host cell can achieve the expression of the target gene induced by hyperglycemia in the host cell. The host cell can be any type of mammalian cell, such as Hela, HEK293T, Cos1, CHO-K1, etc.

[0460] It should be noted that, in the embodiments of the present invention, "YAPΔAD2-10gly-VP minx4 ” and “YAP-VPminx4 ” is synonymous with “TetR-NLS-TEADΔTEA”, “TetR-TEADΔTEA” is synonymous with “TetR-TEADΔTEA”, “TetR-YAPΔAD1” is synonymous with “TetR-YAPΔAD”, and “NLS-TEADΔTEA-10gly-VP minx4 ” and “TEADΔTEA-VP minx4 " is synonymous with ", that is, the NLS and the connector may or may not be shown in the name of the specific gene circuit of the embodiment of the present invention, and its specific structure is shown in Table 1 and the nucleotide sequence.

[0461] The method for regulating the expression of a target gene in a host cell using the eukaryotic expression vector containing the synthetic gene expression circuit for hyperglycemia response comprises the following steps: a) constructing the synthetic gene expression circuit for hyperglycemia response in a eukaryotic plasmid expression vector. The construction method of the eukaryotic expression vector is shown in Table 1; b) introducing the eukaryotic expression vector into a host cell through transfection. The method for introducing the plasmid into the host cell includes: PEI-mediated transfection, liposome-mediated transfection, viral infection, etc.

[0462] Host cells containing synthetic gene expression circuits for hyperglycemia response can be transplanted into mice for target gene regulation expression. Engineered cells containing synthetic gene expression circuits for hyperglycemia response and engineered cell transplants can be prepared in the following ways, and the transplants are implanted into experimental subjects, such as mice: a) preparing eukaryotic plasmid expression vectors containing synthetic gene expression circuits for hyperglycemia response; b) preparing engineered cells containing synthetic gene expression circuits for hyperglycemia response; c) preparing engineered cell transplant vectors containing synthetic gene expression circuits for hyperglycemia response; d) implanting the engineered cell transplant vectors containing synthetic gene expression circuits for hyperglycemia response into mice. The transplant vector is alginate-gelatin hydrogel, and the transplant method is peritoneal transplantation.

[0463] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will appreciate that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in this area or the product instructions are used. Where the manufacturers of reagents or instruments are not indicated, they are all conventional products that can be obtained commercially.

[0464] Example 1 Preparation of engineered cells containing synthetic gene expression circuits for hyperglycemia response

[0465] 1. Construction of components of a synthetic gene expression circuit for hyperglycemia response

[0466] The synthetic gene expression circuit for hyperglycemia response was constructed according to the detailed construction method shown in Table 1, wherein all PCR primers were synthesized by Genewise Biotechnology Co., Ltd. Plasmids were obtained according to conventional molecular cloning procedures; all plasmids were sequenced by BGI Healthcare Technology (Hong Kong) Co., Ltd. (BGI, HK).

[0467] Table 1: Construction of eukaryotic expression vectors

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479] 2. Effect of glucose concentration on the expression of target genes in synthetic gene circuits responsive to hyperglycemia

[0480] HEK293T cells were inoculated into a cell culture dish, and within 16 to 24 hours after inoculation of the cells, the plasmids expressing version 1 glucose sensor (0.1 μg plasmid 12 TetR-NLS-TEADΔTEA and 0.1 μg plasmid 15 YAPΔAD2-VP minx4 ) and plasmids expressing version 2 glucose sensors (0.1 μg plasmid 20 TetR-YAPΔAD1 and 0.1 μg plasmid 22 NLS-TEADΔTEA-10gly-VP minx4 ) were respectively mixed with 0.1 μg plasmid 16P hCMV*-1 -SEAP, PEI transfection reagent and Opti-MEM were mixed and allowed to stand at room temperature for 15 minutes before being evenly added to the 12-well cell culture plate containing the HEK293T cells inoculated above. 12 to 16 hours after transfection, DMEM cell culture medium containing 2.5, 5, 10 and 15 mM glucose (purchased from Gibco TM ,cat.no11965118) were used to treat the cells. 24 h after treatment, the cell culture supernatant of each group was taken to measure the expression of alkaline phosphatase gene (SEAP), using Phosphate-Light TM SEAP Reporter Gene Assay System (Invitrogen TM , cat.no T1017) kit was used to detect the expression of SEAP. The specific operation steps refer to the kit instructions. Figure 3 As shown, when the glucose concentration is 5 mM (within the physiological range or below), the version 1 (Figure A on the left) and version 2 (Figure B on the right) gene expression circuits have no sensitive response, but when the glucose concentration is not lower than 10 mM, the higher the glucose concentration, the higher the expression of the target gene induced by the gene expression circuit.

[0481] 3. Expression of synthetic gene expression circuits for hyperglycemia response in different mammalian cells

[0482] Hela, Cos1, CHO-K1, and HEK293T cells were inoculated into cell culture dishes. Within 16 to 24 hours after inoculation of the cells, the plasmids expressing version 1 glucose sensor (0.1 μg plasmid 12 TetR-NLS-TEADΔTEA and 0.1 μg plasmid 15 YAPΔAD2-VP minx4) and plasmids expressing version 2 glucose sensors (0.1 μg plasmid 20 TetR-YAPΔAD1 and 0.1 μg plasmid 22 NLS-TEADΔTEA-VP minx4 ) were respectively mixed with 0.1 μg plasmid 16P hCMV*-1 -SEAP, PEI transfection reagent and Opti-MEM were mixed and allowed to stand at room temperature for 15 minutes, then evenly added to the 12-well cell culture plate containing the above-mentioned inoculated cells. 12 to 16 hours after transfection, DMEM cell culture medium containing 2.5 and 15 mM glucose (purchased from Gibco TM , cat.no11965118) were used to treat the above cells for 24 h, and the cell culture supernatant of the group was discarded to detect the expression of the reporter gene SEAP. The detection method was referred to the operation of Experiment 2 of this embodiment. The experimental results are shown in Figure 4 As shown, version 1 (left panel A) and version 2 (right panel B) of the hyperglycemia-responsive synthetic gene expression circuit can be expressed in different mammalian cells (HEK293T, Hela, Cos1, CHO-K1).

[0483] 4. Effects of different chimeric transcription factors on the efficiency of synthetic gene expression circuits in response to hyperglycemia

[0484] HEK293T cells were inoculated into a cell culture dish, and within 16 to 24 hours after inoculation of the cells, the plasmids expressing version 1 glucose sensor (0.1 μg plasmid 12 TetR-NLS-TEADΔTEA and 0.1 μg plasmid 15 YAPΔAD2-VP minx4 ) and plasmids expressing version 2 glucose sensors (0.1 μg plasmid 20 TetR-YAPΔAD1 and 0.1 μg plasmid 22 NLS-TEADΔTEA-10gly-VP minx4 ) were respectively mixed with 0.1 μg plasmid 16P hCMV*-1 -SEAP, PEI transfection reagent and Opti-MEM were mixed and allowed to stand at room temperature for 15 minutes before being evenly added to the 12-well cell culture plate containing the above-mentioned inoculated cells. After transfection for 12-16 hours, DMEM cell culture medium containing 2.5, 5, 10 and 15 mM glucose (purchased from Gibco TM , cat.no 11965118) were used to treat the above cells for 24 h. The cell culture supernatant of each group was taken to detect the expression of the reporter gene SEAP. The detection method was referred to the operation of Experiment 2 in this example. The experimental results are shown in Figure 5 As shown, the synthetic gene expression circuit with version 2 chimeric transcription factor secreted more target protein under high concentration glucose treatment.

[0485] 5. Orthogonality testing of different chimeric transcription factors

[0486] This experiment is used to test whether the transcription factors involved in the gene circuit will interact with the endogenous cell pathway (YAP signaling pathway). If the chimeric transcription factor interferes with the endogenous signaling pathway, it will also activate the luciferase reporter gene system, showing a high luciferase expression level; if the chimeric transcription factor has good orthogonality (i.e., it does not interfere with the endogenous YAP signaling pathway), it will not activate or activate the expression of the luciferase gene at a low level. The specific experimental operation is as follows:

[0487] HEK293T cells were seeded into a cell culture dish. Within 16 to 24 hours after seeding the cells, 0.1 μg of plasmid 12 TetR-NLS-TEADΔTEA, 0.1 μg of plasmid 15 YAPΔAD2-VP minx4 , 0.1 μg plasmid 20 TetR-YAPΔAD1 and 0.1 μg plasmid 22 NLS-TEADΔTEA-VP minx4 ) were mixed with YAP / TAZ-responsive luciferase reporter gene system, PEI transfection reagent and Opti-MEM, and then evenly added to a 12-well cell culture plate after standing at room temperature for 15 minutes. 12-16 hours after transfection, the cell culture supernatant was collected to detect the luciferase signal intensity, wherein the kit was used Reporter Assay System (Promega, E1910) was used to detect the luciferase signal intensity. The operation steps refer to the instructions of the kit. Figure 6 As shown, the YAP-VP in version 1 chimeric transcription factor minx4 Significantly activate the transcription of the luciferase gene. The two chimeric transcription factors in the optimized version 2 do not significantly activate the transcription of the luciferase gene, that is, they have better orthogonality, which may be due to the chimeric transcription factor YAPΔAD2 in version 1.

[0488] -VP minx4 Contains the entire YAP protein, thus overexpressing YAP-VP minx4 The effect is approximately equivalent to overexpressing YAP protein, thus significantly activating the transcription of the YAP / TAZ-responsive luciferase gene. Version 2 of the YAP-related chimeric transcription factor is TetR-YAPΔAD1, where YAPΔAD1 represents a YAP protein that lacks a transcriptional activation domain. Therefore, the effect of overexpressing TetR-YAPΔAD1 is equivalent to overexpressing a YAP protein variant without a transcriptional activation function, and therefore does not significantly activate the transcription of the YAP / TAZ-responsive luciferase gene.

[0489] Example 2 Preparation of hydrogel microencapsulated transplants containing engineered cells with synthetic gene expression circuits for hyperglycemia response.

[0490] HEK293T cells were inoculated into a cell culture dish. 16 to 24 hours after inoculation, 4.5 μg of plasmid 26V2STB2, 4.5 μg of plasmid 28V2STB5 and 0.5 μg of plasmid 8pCMV (CAT) T7-SB100 in Table 1 were mixed with PEI transfection reagent and Opti-MEM respectively. After standing at room temperature for 15 minutes, they were evenly added to the cell culture dish containing the above-mentioned inoculated cells. HEK293T cells that were not transfected served as a negative control. 48 hours after transfection, puromycin and G418 were used to treat for 7 days to obtain engineered cells that were stably transfected with a synthetic gene expression circuit that responds to high blood sugar. Monocloning was performed using the limiting dilution method. qPCR primers located on the synthetic gene expression circuit component were used to determine whether the expression gene circuit was integrated into the host cell genome and to detect the relative expression level of each gene circuit. The detection step was a conventional method in the art, wherein the qPCR primer binding site was such as Figure 7 As shown, Figure 8 The experimental results showed that the engineered cells successfully integrated the synthetic gene circuit components.

[0491] The engineered cell monoclonal strain with good growth status was selected for culture, and treated with cell culture medium containing low or high concentration of glucose (glucose concentration in SEAP group: 2.5, 15 mM; glucose concentration in mINS group: 5, 40 mM). After 24 hours, the cell culture supernatant was taken to detect the expression of SEAP and insulin. The detection method of SEAP expression was the same as that in Example 1, and the detection of insulin expression was performed using the Mouse Insulin ELISA kit (Mercodia, 10-1247-01). The detailed operation was carried out according to the instructions of the kit. Fig. 9 The results showed that the engineered cells secreted higher expression levels of SEAP (Figure A) and insulin (Figure B) in response to high glucose concentrations. The engineered cells in good condition were then digested with trypsin, centrifuged, and collected to make hydrogel microcapsule transplants. The engineered cells were encapsulated in alginate (PRONOVA SLG100, Novamatrix, Norway)-gelatin (1288485, sigma) hydrogel microcapsules (radius: ~150μm; approximately 140 cells per microcapsule) using a microfluidic method. The nutrients required for cell growth and the small molecule target proteins secreted by the engineered cells can freely enter and exit the microcapsules, but immune cells and the like cannot enter the microcapsules. The production method is: a density of 8×10 7Cells at a concentration of 100 μg / mL were loaded into the aqueous phase (DMEM culture medium containing 1% alginate and 5% gelatin), and Novec-7500 (3m, St. Paul, MN) was used as the oil phase. Two injection pumps were used to control the flow rates of the two phases (4 μL / min for the aqueous phase and 15 μL / min for the oil phase), and the generated alginate-gelatin droplets were collected and placed on ice to promote the solidification of the gelatin. The droplets were then dispersed onto a 70 μm cell filter, washed with a 100 mM cold calcium chloride solution, and cross-linked. The microcapsules were rinsed several times with DMEM cell culture medium to remove residual oil, and the prepared hydrogel microcapsule transplants were observed under an inverted microscope (Nikon Eclipse Ts2R). The magnified images at different scales are shown in the following figure. Fig.10 As shown, the present embodiment successfully obtained a microcapsule transplant encapsulating the above-mentioned engineered cells.

[0492] Example 3 Effect of a synthetic gene expression circuit responsive to hyperglycemia on insulin expression in type 1 diabetes model mice

[0493] First, a type I diabetes mouse model was constructed. Twelve 10-week-old male C57BL / 6J mice were injected intraperitoneally with streptozotocin (STZ, Sigma-Aldrich, USA) for 5 consecutive days, once a day, and each injection dose was 50 mg / kg body weight. After the last injection, the random blood glucose of mice was measured continuously for three days, and the type I diabetes model of mice with a mean concentration higher than 16mM was successfully constructed for subsequent experiments. Hydrogel microcapsules containing engineered cells of synthetic gene expression circuits responsive to hyperglycemia were prepared according to the method of Example 2, and the hydrogel microcapsules (about 5x 10^6cell) were resuspended in 500μL DMEM culture medium, and then the liquid was injected into the peritoneal cavity of 6 mice (T1D-CI group) using a 23-gauge needle, and 6 mice that were not injected with hydrogel microcapsules were the control group (T1D group). The fasting blood glucose levels of type 1 diabetic mice transplanted with hydrogel microcapsules were measured by taking blood from the tail on days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 21 after transplantation, and the mice were fasted for 6 hours before blood glucose measurement. Glucose tolerance experiments were performed on days 8 and 14 after transplantation. The glucose tolerance experiment was performed according to reference STWCheng, L.Chen, SYTLi, E.Mayoux, PSSPoLeung, 2016, 11, e0147391. Fig.11 The results showed that compared with the T1D group mice, the blood glucose concentration of type 1 diabetic mice transplanted with hydrogel microcapsules (T1D-CI group) was significantly reduced, and the insulin they expressed had a good blood sugar-lowering effect. Fig.12 The results showed that the glucose tolerance of diabetic mice receiving microcapsule transplants was significantly improved on days 8 and 14 after transplantation compared with type 1 diabetic mice without hydrogel microcapsules.

[0494] Example 4 Effect of a synthetic gene expression circuit responsive to hyperglycemia on GLP-1 expression in type II diabetic mice

[0495] Sixteen commercial 10-week-old gene-deficient mice C57BL / KSJ db / db and eight 10-week-old C57BL / KSJ mice were purchased from the Animal Center of the Chinese University of Hong Kong. HEK293T cells were inoculated into cell culture dishes, and within 16 to 24 hours after inoculation, the plasmids expressing version 2 glucose sensors in Table 1 (0.1 μg plasmid 20 TetR-YAPΔAD1 and 0.1 μg plasmid 22 NLS-TEADΔTEA-10gly-VP minx4 ) was mixed with 0.1 μg plasmid 29V2STB6, PEI transfection reagent and Opti-MEM, and then evenly added to the cell culture dish containing the above-mentioned inoculated cells after standing at room temperature for 15 minutes. According to the method of Example 2, hydrogel microcapsules containing engineered cells of synthetic gene expression circuits that respond to high blood sugar were prepared, and the hydrogel microcapsules were intraperitoneally transplanted into the abdominal cavity of gene-deficient mice C57BL / KSJ db / db (T2D-CI group). The transplantation method was referenced to Example 3. The C57BL / KSJ db / db mice that were not implanted with hydrogel microcapsules were the T2D group, and the WT group mice were normal C57BL / KSJ mice that did not suffer from type II diabetes. The number of mice in each group was 8. On the 0th, 1st, and 2nd day after transplantation, after fasting for 6 hours, blood was taken from the tail to determine the fasting blood glucose value of each group of mice, and the T2D and T2D-CI group mice were subjected to glucose tolerance testing on the second day after transplantation. The detection method was referenced to Example 3. Fig.13 The results showed that compared with the T2D group mice, the blood glucose concentration of the T2D-CI group mice was significantly lower on the second day. The GLP-1 expressed by them had a good blood glucose-lowering effect, and the glucose tolerance of the mice was improved.

[0496] Example 5 Effects of a synthetic gene expression circuit responsive to hyperglycemia on insulin expression and its complications in type 1 diabetic model mice

[0497] In this example, three groups were set up for experiments. The construction method of type I diabetic mice (T1D group) and mice injected with hydrogel microcapsule transplants containing engineered cells of synthetic gene expression circuits responsive to hyperglycemia in Example 2 (T1D-CI group) was the same as that in Example 3. The WT group was a normal male C57BL / 6J mouse of ten weeks old that did not suffer from type I diabetes. The engineered cells containing synthetic gene expression circuits responsive to hyperglycemia and the hydrogel microcapsule transplants containing the engineered cells were prepared according to the method of Reference Example 2. The hydrogel microcapsules containing engineered cells of synthetic gene expression circuits responsive to hyperglycemia were intraperitoneally transplanted into the peritoneal cavity of the above-mentioned type I diabetic mice according to the transplantation method of Reference Example 3. On the 21st day after implantation, pancreatic islet, kidney and retinal samples of each group of mice were collected for histological staining or immunofluorescence detection. The detection steps were all routine operations in the field, and the basic operation steps were as follows: (1) Histological staining: The collected samples were fixed in 4% paraformaldehyde for 24 hours. The fixed samples were embedded in paraffin and sliced ​​every 5 μm. Sections were stained with hematoxylin and eosin (H&E), Masson's trichrome, periodic acid Schiff (PAS) or Sirius red. (2) Immunofluorescence: Pancreatic paraffin sections were incubated with anti-insulin (1:500, Sigma, 2018) and anti-glucagon (1:100, Cell signaling technology, 2760), and then detected with secondary antibodies labeled with Alex Fluor 488 (1:1000, Invitrogen, A32723) and Alex Fluor 555 (1:1000, Invitrogen, A32732). Slides were counterstained with DAPI (D9542, Sigma) and images were captured using a Nikon Ti2-E inverted fluorescence microscope. Pancreatic sections were quantified for fluorescence signal intensity and insulin / glucagon-positive areas using Image J software. For retinal sagittal sections, relative GC density was calculated by normalizing the number of bra3a-positive cells to the number of dapi-positive cells.

[0498] Fig.14 The results showed that compared with the T1D group mice, the pancreatic islet damage in the T1D-CI group mice was alleviated. Fig.15 The insulin immunofluorescence signal intensity was not significantly different from that of the WT group, and the ratio of pancreatic alpha / beta cells was significantly reduced. Fig.16 and 17 The results showed that the renal capsule and renal interstitial damage were alleviated compared with the T1D group mice. Fig.18 and Fig.19 The results showed that there were no significant differences in the density of retinal ganglion cells and the morphological characteristics of retinal blood vessels compared with the WT group mice.

[0499] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", "some implementation schemes" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0500] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A gene circuit, characterized in that: include: One or more first expression vectors, the first expression vectors carrying a first nucleic acid molecule, the first nucleic acid molecule encoding a glucose sensor or a fragment thereof; and a second expression vector carrying a second nucleic acid molecule encoding an effector; Wherein, the effector is used to respond to the signal transmitted by the glucose receptor, the glucose receptor is a non-β cell glucose receptor, the first nucleic acid molecule and the second nucleic acid molecule are configured to express the glucose receptor and the effector, and the glucose receptor and the effector are in a non-fusion form.

2. The gene circuit according to claim 1, characterized in that The glucose sensor comprises one or more chimeric transcription factors; Optionally, the chimeric transcription factor comprises at least one of a YAP protein, a TEF-1 protein, a TetR family transcriptional regulator protein, and a transcriptional activation domain; Optionally, the number of the transcriptional activation domains in the chimeric transcription factor is 2 to 6, preferably 4; Optionally, the glucose sensor comprises two chimeric transcription factors; Optionally, the glucose sensor comprises two different chimeric transcription factors; Optionally, the two chimeric transcription factors are chimeric transcription factor 1 and chimeric transcription factor 2, wherein the chimeric transcription factor 1 includes the YAP protein and the transcription activation domain, or the chimeric transcription factor 1 includes the YAP protein and the TetR family transcription regulatory factor protein; and / or The chimeric transcription factor 2 includes the TetR family transcription regulatory factor protein and the TEF-1 protein, or the chimeric transcription factor 2 includes the TEF-1 protein and the transcription activation domain.

3. The gene circuit according to claim 2, characterized in that The chimeric transcription factor 1 includes the YAP protein and the transcription activation domain, and the chimeric transcription factor 2 includes the TetR family transcription regulatory factor protein and the TEF-1 protein, wherein the C-terminus of the YAP protein is connected to the N-terminus of the transcription activation domain, or the N-terminus of the YAP protein is connected to the C-terminus of the transcription activation domain, the C-terminus of the TetR family transcription regulatory factor protein is connected to the N-terminus of the TEF-1 protein, or the N-terminus of the TetR family transcription regulatory factor protein is connected to the C-terminus of the TEF-1 protein; or The chimeric transcription factor 1 includes the YAP protein and the TetR family transcription regulatory factor protein, and the chimeric transcription factor 2 includes the TEF-1 protein and the transcription activation domain, wherein the C-terminus of the TetR family transcription regulatory factor protein is connected to the N-terminus of the YAP protein, the N-terminus of the TetR family transcription regulatory factor protein is connected to the C-terminus of the YAP protein, the C-terminus of the TEF-1 protein is connected to the N-terminus of the transcription activation domain, and the N-terminus of the TEF-1 protein is connected to the C-terminus of the transcription activation domain; Optionally, the chimeric transcription factor 1 comprises the YAP protein and the transcription activation domain, the C-terminus of the YAP protein is connected to the N-terminus of the transcription activation domain, the chimeric transcription factor 2 comprises the TetR family transcription regulatory factor protein and the TEF-1 protein, and the C-terminus of the TetR family transcription regulatory factor protein is connected to the N-terminus of the TEF-1 protein; or The chimeric transcription factor 1 includes the YAP protein and the TetR family transcription regulatory factor protein, the C-terminus of the TetR family transcription regulatory factor protein is connected to the N-terminus of the YAP protein, and the chimeric transcription factor 2 includes the TEF-1 protein and the transcription activation domain, and the C-terminus of the TEF-1 protein is connected to the N-terminus of the transcription activation domain.

4. The gene circuit according to claim 3, characterized in that The chimeric transcription factor 1 comprises a first linker, wherein: The C-terminus of the YAP protein is connected to the N-terminus of the first linker, and the C-terminus of the first linker is connected to the N-terminus of the transcriptional activation domain, or the N-terminus of the YAP protein is connected to the C-terminus of the first linker, and the N-terminus of the first linker is connected to the C-terminus of the transcriptional activation domain; or The C-terminus of the TetR family transcriptional regulatory factor protein is connected to the N-terminus of the first linker, and the C-terminus of the first linker is connected to the N-terminus of the YAP protein, or the N-terminus of the TetR family transcriptional regulatory factor protein is connected to the C-terminus of the first linker, and the N-terminus of the first linker is connected to the C-terminus of the YAP protein; Optionally, the chimeric transcription factor 2 comprises a second linker and / or a nuclear localization signal, wherein: The C-terminus of the TetR family transcriptional regulatory factor protein is connected to the N-terminus of the nuclear localization signal, and the C-terminus of the nuclear localization signal is connected to the N-terminus of the TEF-1 protein, or the N-terminus of the TetR family transcriptional regulatory factor protein is connected to the C-terminus of the nuclear localization signal, and the N-terminus of the nuclear localization signal is connected to the C-terminus of the TEF-1 protein; or The C-terminus of the nuclear localization signal is connected to the N-terminus of the TEF-1 protein, the C-terminus of the TEF-1 protein is connected to the N-terminus of the second linker, the C-terminus of the second linker is connected to the N-terminus of the transcription activation domain, or the C-terminus of the nuclear localization signal is connected to the N-terminus of the transcription activation domain, the C-terminus of the transcription activation domain is connected to the N-terminus of the second linker, and the C-terminus of the second linker is connected to the N-terminus of the TEF-1 protein; Preferably, the chimeric transcription factor 1 comprises a first linker, wherein: The C-terminus of the YAP protein is connected to the N-terminus of the first linker, and the C-terminus of the first linker is connected to the N-terminus of the transcription activation domain. The C-terminus of the TetR family transcriptional regulatory factor protein is connected to the N-terminus of the first linker, and the C-terminus of the first linker is connected to the N-terminus of the YAP protein; Preferably, the chimeric transcription factor 2 comprises a second linker and / or a nuclear localization signal, wherein: The C-terminus of the TetR family transcriptional regulatory factor protein is connected to the N-terminus of the nuclear localization signal, and the C-terminus of the nuclear localization signal is connected to the N-terminus of the TEF-1 protein; or The C-terminus of the nuclear localization signal is connected to the N-terminus of the TEF-1 protein, the C-terminus of the TEF-1 protein is connected to the N-terminus of the second linker, and the C-terminus of the second linker is connected to the N-terminus of the transcription activation domain.

5. The gene circuit according to any one of claims 1 to 4, characterized in that The first expression vector further comprises one or more first promoters; Optionally, the first promoter is operably linked to the first nucleic acid molecule; Optionally, the first promoter comprises at least one selected from the group consisting of a cytomegalovirus promoter, an EF-1α promoter, and a cytomegalovirus immediate early promoter / chicken β-actin promoter; Optionally, the chimeric transcription factor 1 and the chimeric transcription factor 2 are located on the same first expression vector or on different first expression vectors; Optionally, when the chimeric transcription factor 1 and the chimeric transcription factor 2 are located on the same first expression vector, the first expression vector further comprises a third nucleic acid molecule, the third nucleic acid molecule is disposed between the chimeric transcription factor 1 and the chimeric transcription factor 2, and the third nucleic acid molecule encodes a 2A peptide; Optionally, the YAP protein comprises YAP1 protein; Optionally, the YAP protein has the amino acid sequence shown in SEQ ID NO: 1 or 2; Optionally, the TetR family transcriptional regulatory factor protein has an amino acid sequence shown in SE ID NO: 3; Optionally, the TEF-1 protein has the amino acid sequence shown in SEQ ID NO: 4; Optionally, the transcriptional activation domain is a transcriptional activation domain derived from at least one of the following: VP64, Rta, P65, VPR and YAP1; Preferably, the transcriptional activation domain comprises the minimal transcriptional activation domain of herpes simplex virus protein 16; Optionally, the transcription activation domain has the amino acid sequence shown in SEQ ID NO:5; Optionally, the nuclear localization signal is derived from simian virus 40 large tumor antigen; Optionally, the nuclear localization sequence has the amino acid sequence shown in SEQ ID NO: 6; Optionally, the linker has an amino acid sequence as shown in any one of SEQ ID NOs: 7 to 13; Optionally, the chimeric transcription factor 1 has the amino acid sequence shown in SEQ ID NO: 14 or 15, and / or the chimeric transcription factor 2 has the amino acid sequence shown in SEQ ID NO: 16 or 17; Optionally, the chimeric transcription factor 1 has the amino acid sequence shown in SEQ ID NO: 14, and / or the chimeric transcription factor 2 has the amino acid sequence shown in SEQ ID NO: 16; or The chimeric transcription factor 1 has the amino acid sequence shown in SEQ ID NO:15, and / or the chimeric transcription factor 2 has the amino acid sequence shown in SEQ ID NO:

17.

6. The gene circuit according to claim 1, characterized in that The effector includes one or more target proteins; Optionally, the target protein includes at least one of the following: a reporter gene-encoded protein, insulin, and a glucagon-like peptide; Optionally, the second expression vector further comprises a second promoter; Optionally, the second promoter comprises a tetracycline-regulatable promoter; Optionally, the second promoter is operably linked to the second nucleic acid molecule; Optionally, when the effector includes multiple target proteins, the second expression vector further includes one or more fourth nucleic acid molecules, the fourth nucleic acid molecules are located between the multiple target proteins, and the fourth nucleic acid molecules encode a 2A peptide; Optionally, the reporter gene encoding protein includes at least one selected from SEAP, GFP, CAT and β-gal; Optionally, the effector comprises at least one selected from SEAP, insulin 2, and glucagon-like peptide-1; Optionally, the effector is SEAP; or The effector is insulin 2; or The effector is glucagon-like peptide-1; or The effectors are SEAP and insulin 2; or The effectors are SEAP and glucagon-like peptide-1; Optionally, the effector has an amino acid sequence shown in any one of SEQ ID NOs: 18 to 20; or The effector has the amino acid sequence shown in SEQ ID NO: 18 and 19; or The effector has the amino acid sequence shown in SEQ ID NO: 18 and 20.

7. The gene circuit according to claim 1, characterized in that The first nucleic acid molecule has a nucleotide sequence shown in at least one of SEQ ID NOs: 21 to 24, and / or the second nucleic acid molecule has a nucleotide sequence shown in at least one of SEQ ID NOs: 25 to 27; Optionally, the first nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 21 and 22, and the second nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 25; or The first nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 23 and 24, and the second nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 25; or The first nucleic acid molecule has the nucleotide sequence shown in SEQ ID NOs: 23 and 24, and the second nucleic acid molecule has the nucleotide sequence shown in SEQ ID NOs: 25 and 26; or The first nucleic acid molecule has the nucleotide sequence shown in SEQ ID NOs: 23 and 24, and the second nucleic acid molecule has the nucleotide sequence shown in SEQ ID NOs: 25 and 27; Optionally, the third nucleic acid molecule and / or the fourth nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 28; Optionally, the first expression vector has a nucleotide sequence shown in at least one of SEQ ID NOs: 29 to 33; and / or the second expression vector has a nucleotide sequence shown in any one of SEQ ID NOs: 34 to 36; Optionally, the first expression vector has the nucleotide sequence shown in SEQ ID NO: 29 and 30; and / or the second expression vector has the nucleotide sequence shown in SEQ ID NO: 34; or The first expression vector has the nucleotide sequence shown in SEQ ID NO: 31 and 32; and / or the second expression vector has the nucleotide sequence shown in SEQ ID NO: 34; or The first expression vector has the nucleotide sequence shown in SEQ ID NO: 33; and / or the second expression vector has the nucleotide sequence shown in SEQ ID NO: 35; or The first expression vector has the nucleotide sequence shown in SEQ ID NO:31 and 32; and / or the second expression vector has the nucleotide sequence shown in SEQ ID NO:

36.

8. A recombinant cell, characterized in that The recombinant cell carries the gene circuit according to any one of claims 1 to 7.

9. A hydrogel microcapsule, characterized in that: The hydrogel microcapsule contains the gene circuit according to any one of claims 1 to 7 or the recombinant cell according to claim 8.

10. Use of the gene circuit according to any one of claims 1 to 7, the recombinant cell according to claim 8 or the hydrogel microcapsule according to claim 9 in the preparation of a drug for preventing and / or treating diabetes and its complications; Optionally, the diabetes comprises at least one of type I diabetes and type II diabetes; Optionally, the complication includes at least one of renal capsule damage, renal interstitial damage, retinal damage and pancreatic islet damage.

11. A drug, characterized in that The method comprises the gene circuit according to any one of claims 1 to 7, the recombinant cell according to claim 8, or the hydrogel microcapsule according to claim 9.

12. Use of the gene circuit according to any one of claims 1 to 7, the recombinant cell according to claim 8, the hydrogel microcapsule according to claim 9 or the drug according to claim 11 in the treatment and / or prevention of diabetes and its complications; Optionally, the diabetes comprises at least one of type I diabetes and type II diabetes; Optionally, the complication includes at least one of renal capsule damage, renal interstitial damage, retinal damage and pancreatic islet damage.

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