Vector for inducing and regulating expression of target gene by gene switch system and application of vector

Abstract

The invention discloses a vector for inducing and regulating expression of a target gene through a gene switch system and application of the vector. According to the gene switch system, the purpose of expressing the foreign protein on the adeno-associated virus vector with small foreign gene loading capacity can be achieved, some specific split structures have the unexpected advantage of extremely low leakage amount, and some specific split structures have the advantage of improving the expression quantity and the like.

Description

Technical Field

[0001] This invention relates to the field of gene and cell therapy, and more specifically to a vector for inducing and regulating the expression of a target gene using a gene switch system and its application. Background Technology

[0002] Gene switches are inducible gene regulatory systems used to control the expression of transgenes. Precise regulation of transgene expression is crucial for various applications, such as gene therapy, large-scale intracellular protein production, cell-based high-throughput screening experiments, functional genomics, and trait regulation in transgenic plants and animals. Several gene switches based on tetracycline inhibitors, immunosuppressive molecules, and steroid hormone receptors (including progesterone and ecdysone receptors) have been developed (Fussenegger, M. (2001) Biotechnol. Prog. 17, 1-51.).

[0003] Both CN101868241A (2008) and CN110462040A (2018) describe gene switch systems based on ecdysone receptors. The vector described in CN101868241A is a plasmid vector, viral vector, or modified cell, and all functional elements of this switch system are constructed on a single vector structure, which is protected by patent. However, the actual sequence length is too large to be widely applied to viral vectors with small exogenous gene loading, such as AAV and LV.

[0004] The vector described in patent CN110462040A (2018) is based on patent CN101868241A (2008), which involves the splitting and combination of functional elements and corresponding screening to obtain a feasible solution for CAR-T therapy. The vector is any one of lentiviral vector, retroviral vector or non-viral vector, wherein the vector is a non-viral vector and the non-viral vector is Sleeping Beauty transposon.

[0005] However, to date, there is a lack of gene switch constructs that induce and regulate the expression of target genes based on adeno-associated virus (AAV).

[0006] Therefore, there is an urgent need in this field to develop new and efficient gene switch constructs for inducing and regulating the expression of target genes based on adeno-associated virus (AAV). Summary of the Invention

[0007] The purpose of this invention is to provide a highly efficient gene switch construct for inducing and regulating the expression of target genes based on adeno-associated virus (AAV) and its application.

[0008] In a first aspect of the invention, a set of constructs for inducing and regulating the expression of a target gene based on adeno-associated virus (AAV) is provided, the set of constructs comprising a core construct selected from the group consisting of a first construct, a second construct, and an optional third construct;

[0009] Furthermore, the construct group contains a first expression cassette for expressing a first gene switch polypeptide, a second expression cassette for expressing a second gene switch polypeptide, and a third expression cassette for expressing a foreign gene product;

[0010] The first gene switch polypeptide includes a DNA-binding domain fused to a first nuclear receptor ligand-binding domain, and the second gene switch polypeptide includes a trans-activation domain fused to a second nuclear receptor ligand-binding domain. The first gene switch polypeptide and the second gene switch polypeptide are connected by a linker to form a gene switch. The promoter in the third expression cassette is a promoter regulated by the gene switch.

[0011] Furthermore, when the constructs are introduced together into mammalian cells, an adeno-associated virus (AAV)-based gene switch can be formed, and the third expression cassette is expressed under the regulation of the gene switch.

[0012] Wherein, when the third construct does not exist, one of the core constructs, the first construct and the second construct, contains one or two of the first, second and third expression boxes, while the other core construct contains the remaining expression boxes;

[0013] Alternatively, when it is a third construct, each of the first, second, and third core constructs contains a first expression box, a second expression box, and a third expression box, respectively.

[0014] In another preferred embodiment, the first expression box, the second expression box, and the third expression box are not simultaneously located on any one of the core constructs.

[0015] In another preferred embodiment, the first expression box, the second expression box, and the third expression box are each independently located in at least one core structure, and the first expression box, the second expression box, and the third expression box are not simultaneously located on any one of the core structures.

[0016] In another preferred embodiment, the first expression box, the second expression box, and the third expression box are each independently located in at least one core structure, and the first expression box, the second expression box, and the third expression box are not simultaneously located on any one of the core structures.

[0017] In another preferred embodiment, with the participation of a ligand, a second gene switch polypeptide (such as LTF) forms a stable high-affinity interaction with a first gene switch polypeptide (such as CAP) at the inducible promoter, inducing the receptor protein to recruit transcription factors and drive the expression of the target gene encoding the target protein.

[0018] In another preferred embodiment, the second gene switch polypeptide is LTF.

[0019] In another preferred embodiment, the first gene switch polypeptide is CAP.

[0020] In another preferred embodiment, the target gene encoding the exogenous gene product is at least one nucleotide sequence encoding the product of the gene of interest (GOI).

[0021] In another preferred embodiment, the gene product includes: polypeptides, RNA molecules (such as shRNA, siRNA).

[0022] In another preferred embodiment, the gene product is selected from the group consisting of: lipoprotein lipase, apolipoprotein, cytokines, interleukins, interferons, fluorescent proteins, chloramphenicol acetyltransferase, β-galactosidase, β-glucuronidase, Renilla luciferase, firefly luciferase, alkaline phosphatase, or combinations thereof.

[0023] In another preferred embodiment, the gene product includes a therapeutic gene product or a reporter protein for evaluating vector transformation and expression.

[0024] In another preferred embodiment, the fluorescent protein includes: green fluorescent protein GFP and red fluorescent protein RFP.

[0025] In another preferred embodiment, the gene switch is nonconstitutive.

[0026] In another preferred embodiment, the functional elements of the gene switch (i.e., a first expression cassette for expressing a first gene switch polypeptide and a second expression cassette for expressing a second gene switch polypeptide) are distributed on different constructs (such as gene vectors).

[0027] In another preferred embodiment, the first gene-switching polypeptide and the second gene-switching polypeptide in different constructs (such as gene vectors) individually function as partial functional elements of a switch, and the expression of the target gene is induced only when all partial functional elements of the switches function in the object or cell and in the presence of a ligand (such as when administered to the cell or object).

[0028] In another preferred embodiment, the gene switch is a therapeutic switch.

[0029] In another preferred embodiment, the gene switch is a gene switch based on the ecdysone receptor (EcR).

[0030] In another preferred embodiment, the ligand binds to the EcR ligand-binding domain.

[0031] In another preferred embodiment, the ligand is a diacylhydrazine.

[0032] In another preferred embodiment, the ligand is RG-115819, RG-115932, or RG-115830.

[0033] In another preferred embodiment, the ligand is an amide ketone or an oxadiazolline.

[0034] In another preferred embodiment, the promoter regulated by the gene switch (the promoter that is operatively linked to the target gene in the third expression cassette) is FRP [6xGalRE: minimal promoter].

[0035] In another preferred embodiment, the first expression box, the second expression box, and the third expression box are each independently driven by a constitutive or inducible promoter.

[0036] In another preferred embodiment, the promoter encoding the polynucleotide sequence of at least one transcription factor is a constitutive promoter.

[0037] In another preferred embodiment, the promoter encoding the polynucleotide sequence of at least one transcription factor is an inducible promoter.

[0038] In another preferred embodiment, the promoter encoding the polynucleotide sequence of at least one co-activating protein is a constitutive promoter.

[0039] In another preferred embodiment, the promoter encoding the polynucleotide sequence of at least one co-activating protein is an inducible promoter.

[0040] In another preferred embodiment, the promoter encoding at least one transcription factor polynucleotide sequence is different from the promoter encoding at least one co-activating protein.

[0041] In another preferred embodiment, the promoter encoding at least one transcription factor and the promoter encoding at least one co-activating protein are the same.

[0042] In another preferred embodiment, the polynucleotide sequence of the encoded coactivating protein encodes a protein containing a heterodimeric chaperone box transactivation domain.

[0043] In another preferred embodiment, the encoded transcription factor's polynucleotide sequence encodes a protein comprising a DNA-binding domain and a ligand-binding domain.

[0044] In another preferred embodiment, the core constructs are selected from the group consisting of:

[0045] VG-X237(ITR-FRP-EGFP-WPRE-BGHpA-ITR) (SEQ ID NO: 1),

[0046] VG-X242(ITR-Ubc-CAP-TL-LTF-WPRE-BGHpA-ITR)(SEQ ID NO:2),

[0047] VG-X243(ITR-Ubc-CAP-P2A-LTF-WPRE-BGHpA-ITR)(SEQ ID NO:3),

[0048] VG-X244(ITR-Ubc-LTF-SV40pA-Ubc-CAP-WPRE-BGHpA-ITR) (SEQ ID NO: 4),

[0049] VG-X246(ITR-Ubc-CAP-SV40pA-FRP-EGFP-WPRE-BGHpA-ITR) (SEQ ID NO: 5),

[0050] VG-X247(ITR-Ubc-LTF-WPRE-BGHpA-ITR)(SEQ ID NO:6),

[0051] VG-X248 (ITR-Ubc-LTF-SV40pA-FRP-EGFP-WPRE-BGHpA-ITR) (SEQ ID NO:7), VG-X249 (ITR-Ubc-CAP-WPRE-BGHpA-ITR) (SEQ ID NO:8)

[0052] VG-142(ITR-Ubc-EGFP-WPRE-BGHpA-ITR).

[0053] In another preferred embodiment, the core constructs are selected from the group consisting of:

[0054] VG-X237(ITR-FRP-EGFP-WPRE-BGHpA-ITR) (SEQ ID NO: 1),

[0055] VG-X242(ITR-Ubc-CAP-TL-LTF-WPRE-BGHpA-ITR)(SEQ ID NO:2),

[0056] VG-X243(ITR-Ubc-CAP-P2A-LTF-WPRE-BGHpA-ITR)(SEQ ID NO:3),

[0057] VG-X246 (ITR-Ubc-CAP-SV40pA-FRP-EGFP-WPRE-BGHpA-ITR) (SEQ ID NO:5), VG-X247 (ITR-Ubc-LTF-WPRE-BGHpA-ITR) (SEQ ID NO:6).

[0058] In another preferred embodiment, the group of constructs includes a combination of VG-X246 and VG-X247.

[0059] In another preferred embodiment, the first construct has a VG-X246 structure or a Formula I structure;

[0060] ITR1-P1-CAP-pA1-FRP-GOI-WPRE-pA2-ITR2(I)

[0061] In the formula,

[0062] ITR1 is the 5' terminal ITR element of AAV, preferably an ITR element of AAV2, AAV3, AAV4, or AAV5;

[0063] P1 is the first promoter, preferably the Ubc promoter;

[0064] CAP is the coding sequence for the first gene switch polypeptide CAP;

[0065] pA1 is the first polyA element, preferably the polyA element of SV40;

[0066] FRP is 6xGalRE: minimal promoter;

[0067] GOI is the target gene (GOI);

[0068] WPRE is an absent or transcriptional enhancing element;

[0069] pA2 is a second polyA element, preferably a polyA element of BGH;

[0070] ITR2 is the 3' terminal ITR element of AAV, preferably an ITR element of AAV2, AAV3, AAV4, or AAV5;

[0071] Each "-" indicates a sequence without or without nucleotide linkage.

[0072] In another preferred embodiment, pA1 and pA2 may be the same or different.

[0073] In another preferred embodiment, the above-mentioned components are of the VG-X246 structure ( Figure 18 The corresponding elements (with corresponding nucleotide sequences) in ).

[0074] In another preferred embodiment, the second construct has a VG-X247 structure or a Formula II structure;

[0075] ITR-P1-LTF-WPRE-pA2-ITR(II)

[0076] In the formula,

[0077] ITR1 is the 5' terminal ITR element of AAV, preferably an ITR element of AAV2, AAV3, AAV4, or AAV5;

[0078] P1 is the first promoter, preferably the Ubc promoter;

[0079] LTF is the coding sequence of the second gene switch polypeptide LTF;

[0080] WPRE is an absent or transcriptional enhancing element;

[0081] pA2 is a second polyA element, preferably a polyA element of BGH;

[0082] ITR2 is the 3' terminal ITR element of AAV, preferably an ITR element of AAV2, AAV3, AAV4, or AAV5;

[0083] Each "-" indicates a sequence without or without nucleotide linkage.

[0084] In another preferred embodiment, the above-mentioned components are of the VG-X247 structure ( Figure 17 The corresponding elements (with corresponding nucleotide sequences) in ).

[0085] In another preferred embodiment, the building block group is a combination of VG-X249, VG-X247 and VG-X237.

[0086] In another preferred embodiment, the building block group is a combination of VG-X243 and VG-X237.

[0087] In a second aspect of the invention, a kit is provided, the kit comprising: a container and a set of constructs for inducing and regulating the expression of a target gene as described in the first aspect of the invention, located within the container.

[0088] In a third aspect of the invention, a method for inducing and regulating the expression of a target gene based on adeno-associated virus (AAV) is provided, comprising the steps of:

[0089] (a) Providing a set of constructs for inducing and regulating the expression of target genes based on adeno-associated virus (AAV) as described in the first aspect of the present invention;

[0090] (b) Introduce the first, second, and optional third constructs into the same cell;

[0091] (c) In the presence of a ligand, the first gene switch polypeptide and the second gene switch polypeptide are linked by a linker to form a gene switch, and the gene switch regulates the expression of the target gene by the promoter in the third expression cassette.

[0092] In a fourth aspect of the present invention, the use of the construct set for inducing and regulating the expression of a target gene based on adeno-associated virus (AAV) as described in the first aspect of the present invention and the kit as described in the second aspect of the present invention are provided for inducing and regulating the expression of a target gene.

[0093] In another preferred embodiment, the target gene is a therapeutic gene and / or a reporter gene.

[0094] In another preferred embodiment, the use is for non-therapeutic and non-diagnostic purposes.

[0095] In a fifth aspect of the invention, the use of the construct set based on adeno-associated virus (AAV) for inducing and regulating the expression of target genes as described in the first aspect of the invention and the kit described in the second aspect of the invention is provided for the preparation of drugs for the prevention and / or treatment of diseases or symptoms.

[0096] In another preferred embodiment, the disease or condition is selected from the group consisting of: hyperproliferative diseases or conditions, cardiovascular diseases or conditions, neurological diseases or conditions, autoimmune diseases or conditions, bone diseases or conditions, gastrointestinal diseases or conditions, blood diseases or conditions, metabolic diseases or conditions, inflammatory diseases or conditions, and infectious diseases or conditions.

[0097] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0098] Figure 1 This diagram illustrates the gene switch system of the present invention inducing and regulating the expression of the target gene.

[0099] Figure 2This diagram illustrates the structure of a vector for inducing and regulating the expression of a target gene in one embodiment of the present invention. CAP and LTF are located in the first plasmid, and a TSP-2 element lies between CAP and LTF; while GOI (gene of interest) is located in the second plasmid. Arrows in the plasmid structure indicate the direction of transcription, while arrows pointing towards the cell indicate introduction into the cell or application to the cell. Figure 3-10 (The arrows in the image have the same meaning.)

[0100] Figure 3 The diagram shows a schematic of the structure of a vector for inducing and regulating the expression of a target gene in one embodiment of the present invention, wherein CAP and LTF are located in the first plasmid, and a P2A element is located between CAP and LTF; while GOI (gene of interest) is located in the second plasmid.

[0101] Figure 4 The diagram shows a schematic of the structure of a vector for inducing and regulating the expression of a target gene in one embodiment of the present invention, wherein CAP and LTF are located in the first plasmid, and a TL element is located between CAP and LTF; while GOI (gene of interest) is located in the second plasmid.

[0102] Figure 5 The diagram shows a schematic of the structure of a vector for inducing and regulating the expression of a target gene in one embodiment of the present invention, wherein CAP and LTF are located in the first plasmid, and a TSP-2 element is located between CAP and LTF, and the transcription directions of the two are opposite; while GOI (gene of interest) is located in the second plasmid.

[0103] Figure 6 The diagram shows a schematic of the structure of a vector for inducing and regulating the expression of a target gene in one embodiment of the present invention, wherein the CAP and GOI sequences are located in the first plasmid, and an FRP element is located between the CAP and GOI; while the LTF is located in the second plasmid.

[0104] Figure 7 The diagram shows a schematic of the structure of a vector for inducing and regulating the expression of a target gene in one embodiment of the present invention, wherein the LTF and GOI sequences are located in the first plasmid, and an FRP element is located between the LTF and GOI; while the CAP is located in the second plasmid.

[0105] Figure 8 The diagram shows a schematic of the structure of a vector for inducing and regulating the expression of a target gene in one embodiment of the present invention, wherein the CAP and GOI sequences are located in the first plasmid, and the FRP element is located between the CAP and GOI, and the transcription directions of the two are opposite; while the LTF is located in the second plasmid.

[0106] Figure 9The diagram shows a schematic of the structure of a vector for inducing and regulating the expression of a target gene in one embodiment of the present invention, wherein the LTF and GOI sequences are located in the first plasmid, and the FRP element is located between the LTF and GOI, and the transcription directions of the two are opposite; while CAP is located in the second plasmid.

[0107] Figure 10 The diagram shows a schematic of the structure of a vector for inducing and regulating the expression of a target gene in one embodiment of the present invention, wherein the CAP sequence is located in the first plasmid, the LTF sequence is located in the second plasmid, and the GOI sequence is located in the third plasmid.

[0108] Figure 11 The results of Example 2, showing the induction and regulation of target gene expression by a subset of gene switches with and without the addition of a ligand, are presented.

[0109] Figure 12 The results of another part of the gene switch inducing regulation of target gene expression in Example 2 with and without the addition of ligand are shown.

[0110] Figure 13 The results of another part of the gene switch inducing regulation of target gene expression in Example 2 with and without the addition of ligand are shown.

[0111] Figure 14 This shows the results of partial gene switching induction and regulation of target gene expression in Example 2 with and without the addition of a ligand. Figures 12-14 In the diagram, columns A, B, C, and D represent the detection results for 4x bright field, 4x fluorescence, 10x bright field, and 10x fluorescence, respectively.

[0112] Figure 15 The structure and sequence (SEQ ID No:1) of the construct VG-X237 are shown, with the main components highlighted in different colors.

[0113] Figure 16 The structure and sequence (SEQ ID No:2) of the construct VG-X242 are shown, with the main components highlighted in different colors.

[0114] Figure 17 The structure and sequence (SEQ ID No:3) of the construct VG-X243 are shown, with the main components highlighted in different colors.

[0115] Figure 18 The structure and sequence (SEQ ID No:5) of the construct VG-X246 are shown, with the main components highlighted in different colors.

[0116] Figure 19The structure and sequence (SEQ ID No:6) of the construct VG-X247 are shown, with the main components highlighted in different colors. Detailed Implementation

[0117] Through extensive and in-depth research, and through numerous screenings and optimizations, the inventors have developed, for the first time, a novel and unique gene switch system based on AAV for inducing and regulating the expression of target genes, as well as expression vectors (such as plasmids and AAV vectors) based on this gene switch system. Specifically, the inventors loaded the gene switch system onto two or more plasmids or adeno-associated virus vectors, thus achieving for the first time the application of the gene switch system on adeno-associated virus vectors with small exogenous gene loadings. On the one hand, this invention is suitable for expressing larger target proteins or target genes; on the other hand, the split gene switch system, CAP or LTF, can be used as needed or in stages, enabling more stable on-demand regulation. This invention was completed based on these findings.

[0118] Experiments show that the gene switch system of the present invention can not only achieve the expression of foreign proteins on adeno-associated virus vectors with small foreign gene loading, but also some specific splitting structures of the present invention have the advantages of unexpectedly low leakage and increased expression levels.

[0119] the term

[0120] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below.

[0121] As used herein, the terms "first plasmid" and "core plasmid" are used interchangeably and refer to plasmids containing the target gene (e.g., gene switch functional elements).

[0122] As used in this article, the "second plasmid" and "third plasmid" can be co-transfected with the "first plasmid" and simultaneously introduced into suitable host cells, where they are expressed and packaged into AAV viral particles.

[0123] Gene switch system

[0124] The working principle of the gene switch system of the present invention is as follows: Figure 1As shown. Similar to patents CN101868241A and CN110462040A. The gene-switching polypeptide of the present invention comprises: a first gene-switching polypeptide comprising a DNA-binding domain fused to a first nuclear receptor ligand-binding domain (preferably, this domain is expressed as a co-activating protein CAP), and a second gene-switching polypeptide comprising a trans-activating domain fused to a second nuclear receptor ligand-binding domain (preferably, this domain is expressed as a ligand-dependent transcription factor LTF), wherein the first gene-switching polypeptide and the second gene-switching polypeptide are linked by a linker. With the participation of the ligand, LTF forms a stable high-affinity interaction with CAP at the inducible promoter. Through this novel conformation, the receptor protein can be induced to recruit transcription factors, driving the expression of the target gene.

[0125] Ecdysone and its receptor

[0126] Ecdysone (20E) is a typical steroid hormone that dominates the regulation of important physiological processes in insects, such as molting, metamorphosis, and reproduction. The 20E receptor EcR-USP has been identified for nearly 20 years, and there have been many important breakthroughs in the transcriptional regulation mechanism of the 20E and its receptor complex. Existing studies have shown that: (1) the 20E receptor is formed by the nuclear receptor EcR and USP; (2) the EcR-USP heterodimer acquires DNA-binding activity with the assistance of a molecular chaperone protein complex; (3) 20E activates the EcR-USP heterodimer and initiates the transcription of downstream genes by releasing co-repressor factors and recruiting co-activators; (4) the 20E-EcR-USP ligand-receptor complex induces the expression of 20E primary response genes, and the 20E secondary response genes induced by the transcription factors encoded by the 20E primary response genes amplify the 20E signal, thereby regulating the physiological processes of insect molting, metamorphosis, and reproduction.

[0127] AAV-based splitting structure and control method

[0128] This invention provides a gene switch system based on an AAV split structure, which can be used to induce and regulate the expression of target genes.

[0129] Specifically, such as Figures 2 to 10 As shown, this invention provides nine representative AAV-based splitting structures.

[0130] by Figure 2 For example, CAP and LTF are located in the first plasmid, and a TSP-2 element is located between CAP and LTF; while GOI (gene of interest) is located in the second plasmid. The arrows in the plasmid structure indicate the direction of transcription, while arrows pointing towards the cell indicate introduction into the cell or application to the cell. Figure 3-10 (The arrows in the image have the same meaning.)

[0131] Similarly, Figures 3 to 10 Eight other splitting structures are shown.

[0132] In this invention, the expression vector includes (but is not limited to): plasmids and adeno-associated virus (AAV).

[0133] This invention also provides a method for inducing and regulating the expression of a target gene based on a gene switch system with a split structure according to this invention. Typically, the method of this invention may include (but is not limited to) the following embodiments:

[0134] Implementation Method 1: (1) A sequence encoding a target gene (GOI) that operatively binds to a factor-regulated promoter is loaded into a single plasmid, mRNA, and viral vector. (2) A polynucleotide sequence encoding at least one transcription factor and at least one co-activating protein is loaded into a single plasmid, mRNA, and viral vector. (3) (1) and (2) are introduced into the object, and the ligand is administered to the object to induce the expression of the target gene.

[0135] Implementation Method 2: (1) A polynucleotide sequence encoding at least one transcription factor is loaded into a single plasmid, mRNA, and viral vector. (2) A polynucleotide sequence encoding at least one co-activating protein and a sequence encoding a target gene that operatively binds to a promoter regulated by the factor are loaded into a single plasmid, mRNA, and viral vector. (3) (1) and (2) are introduced into the object, and a ligand is administered to the object to induce the expression of the target gene.

[0136] Implementation Method 3: (1) A polynucleotide sequence encoding at least one co-activating protein is loaded into a single plasmid, mRNA, and viral vector. (2) A polynucleotide sequence encoding at least one transcription factor and a sequence encoding a target gene that operatively binds to a promoter regulated by the factor are loaded into a single plasmid, mRNA, and viral vector. (3) (1) and (2) are introduced into the object, and a ligand is administered to the object to induce the expression of the target gene.

[0137] Implementation Method 4: (1) Loading a therapeutic polypeptide or therapeutic polynucleotide sequence that operatively binds to a promoter regulated by a factor into a single plasmid, mRNA, and viral vector. (2) Loading a polynucleotide sequence encoding at least one transcription factor into a single plasmid, mRNA, and viral vector. (3) Loading a polynucleotide sequence encoding at least one co-activating protein into a single plasmid, mRNA, and viral vector. (4) Introducing (1), (2), and (3) into the subject, and administering a ligand to the subject to induce expression of the therapeutic polypeptide or therapeutic polynucleotide.

[0138] The main advantages of this invention include:

[0139] (a) The vector structure of the split vector switch system of the present invention allows for the loading of longer target gene sequences onto adeno-associated viruses, thus truly realizing its application on adeno-associated virus vectors.

[0140] (b) The split vector switch system of the present invention is more flexible in inducing switch-initiated expression. A plasmid or adeno-associated virus carrying the switch core elements CAP and LTF can be immobilized to regulate the switch system of multiple target genes.

[0141] (c) The split carrier switching system of the present invention can avoid the continuous expression of exogenous proteins expressed by the core switching element in the subject, thus making it safer.

[0142] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0143] Sequence information

[0144] SEQ ID NO:1( Figure 15 (The image shows detailed location information for components such as ITR and FRP.)

[0145]

[0146] SEQ ID NO:2( Figure 16 (The image shows detailed location information for components such as ITR, Ubc, and CAPP.)

[0147]

[0148] SEQ ID NO:3( Figure 17 (The image shows detailed location information for components such as ITR, Ubc, and CAP.)

[0149]

[0150] SEQ ID NO:4

[0151]

[0152] SEQ ID NO:5( Figure 18 (The image shows detailed location information for components such as ITR, Ubc, and CAP.)

[0153]

[0154] SEQ ID NO:6( Figure 19 (The image shows detailed location information for components such as ITR, Ubc, and CAP.)

[0155]

[0156] SEQ ID NO:7

[0157]

[0158] SEQ ID NO:8

[0159]

[0160] Example 1

[0161] Construction of gene switch plasmids

[0162] Based on the ecdysone receptor-based switch system described in patent CN101868241A, the following plasmids with different gene switches were synthesized and constructed:

[0163] VG-X237(ITR-FRP-EGFP-WPRE-BGHpA-ITR) (SEQ ID NO: 1),

[0164] VG-X242(ITR-Ubc-CAP-TL-LTF-WPRE-BGHpA-ITR)(SEQ ID NO:2),

[0165] VG-X243(ITR-Ubc-CAP-P2A-LTF-WPRE-BGHpA-ITR)(SEQ ID NO:3),

[0166] VG-X244(ITR-Ubc-LTF-SV40pA-Ubc-CAP-WPRE-BGHpA-ITR) (SEQ ID NO: 4),

[0167] VG-X246(ITR-Ubc-CAP-SV40pA-FRP-EGFP-WPRE-BGHpA-ITR) (SEQ ID NO: 5),

[0168] VG-X247(ITR-Ubc-LTF-WPRE-BGHpA-ITR)(SEQ ID NO:6),

[0169] VG-X248 (ITR-Ubc-LTF-SV40pA-FRP-EGFP-WPRE-BGHpA-ITR) (SEQ ID NO:7), VG-X249 (ITR-Ubc-CAP-WPRE-BGHpA-ITR) (SEQ ID NO:8),

[0170] VG-142(ITR-Ubc-EGFP-WPRE-BGHpA-ITR).

[0171] All plasmids were constructed on the pUC57 plasmid backbone vector via recombinant cloning, and plasmids VG-X237, VG-X242, VG-X243, VG-X244, VG-X246, VG-X247, VG-X248, VG-X249 and control plasmid VG-X142 were extracted.

[0172] Example 2

[0173] Gene switch induction expression efficiency activity test

[0174] HEK293 cells cultured in a shaker using a component-defined serum-free suspension medium were removed for cell sampling and counting. The required amount of cell suspension was calculated according to Table 1. The cells were diluted with DMEM high-glucose medium containing 2% bovine serum, transferred to adherent dishes, and placed in a cell culture incubator for static culture.

[0175] Table 1

[0176]

[0177] After static culture for 1.5–2 hours, AAV-packaged triple plasmid transfection was performed using a "suspended plate" method. After transfection, the ligand diacylhydrazine was added to the drug-treated group at a final concentration of 0.01 pmol / mL. The normal transfection group and the drug-treated group were incubated together at 37°C for 72 hours. Then, fluorescence images were taken under fixed exposure parameters. The grayscale of the fluorescence images at the same magnification was analyzed using ImageJ software to evaluate whether gene switching was induced and the difference in light intensity that induced expression, thereby evaluating the gene switching induction efficiency.

[0178] The results are as follows Figures 12-14 As shown. The results are as follows:

[0179] The fluorescence results at 1.72 hours showed that the plasmid transfection groups RC2-X242, RC2-X243, RC2-X244, RC2-X247, and RC2-X249, which did not carry FRP-EGFP in their gene sequences, were all normal and did not show cell fluorescence.

[0180] 2.72-hour fluorescence results showed that the plasmid transfection groups RC2-X237, RC2-X246, and RC2-X248 carrying FRP-EGFP gene sequences, as well as the control plasmid group VG-142, all exhibited normal cell fluorescence. Except for the control plasmid group VG-142, all groups showed varying degrees of primary expression of the inducible promoter FRP. The fluorescence expression intensity was RC2-X248(VG-X248ITR-Ubc-LTF-SV40pA-FRP-EGFP-WPRE-BGHpA-ITR) > RC2-X246(VG-X246 ITR-Ubc-CAP-SV40pA-FRP-EGFP-WPRE-BGHpA-ITR) > RC2-X237(VG-X247 ITR-FRP-EGFP-WPRE-BGHpA-ITR). The core switching element LTF achieved a stronger enhancer-like function than CAP.

[0181] The fluorescence results at 3.72 hours showed that the fluorescence of RC2-X248+(VG-X248ITR-Ubc-LTF-SV40pA-FRP-EGFP-WPRE-BGHpA-ITR) and RC2-X246+(VG-X246ITR-Ubc-CAP-SV40pA-FRP-EGFP-WPRE-BGHpA-ITR) in the normal group and the group with added ligand were basically the same. Due to the lack of the switching core elements CAP and LTF, respectively, no fluorescence enhancement was observed when ligand was added, and the addition of ligand failed to achieve ligand-induced secondary expression.

[0182] 4.72-hour fluorescence results showed that the core switching element was separated into normal group and ligand-added group (RC2-242-237, RC2-242-237+, RC2-243-237, RC2-243-237+, RC2-244-237, RC2-244-237+, RC2-246-247, RC2-246-247+, RC2-248-249, RC2-248-249+, RC2-249-247-237, RC2-249-247-237+) co-transfected with two or more plasmid vectors to package the virus. All groups showed enhanced fluorescence in the ligand-added group, indicating that the ligand-added drug induced secondary expression of the promoter FRP.

[0183] 5.72-hour fluorescence results showed that, based on a 4x microscope comparison, the fluorescence intensity of the core switching element, divided into normal and ligand-added groups (RC2-242-237+, RC2-243-237+, RC2-244-237+), co-transfected with two or more plasmid vectors to package the virus, was slightly better than that of RC2-243-237+, and the RC2-244-237+ group was slightly better than that of RC2-242-237+. This indicates that the P2A sequence-linked switching system has advantages, especially... Figure 3 The TSP-1-CAP(AD-HP)-P2A-LTF(DBD-LBD)-3UTR structure shown in the first plasmid has significantly better expression efficiency.

[0184] Fluorescence results at 6.72 hours showed that the core switching element was separated into normal and ligand-added groups co-transfected with two or more plasmid vectors to package the virus: RC2-242-237, RC2-242-237+, RC2-243-237, RC2-243-237+, RC2-244-237, RC2-244-237+, RC2-246-247, RC2-246-247+, RC2-248-249, RC2-248-249+, RC2-249-247-237, and RC2-249-247-237+. Fluorescence also showed that the RC2-246-247 normal group had unexpectedly low primary expression.

[0185] In some applications, primary expression is defined as leakage. RC2-246-247 has the lowest primary expression leakage among all split structures, with a leakage of only 0.016% as a percentage of area. Compared with other split structures, RC2-246-247 reduces leakage by approximately 20 to 240 times. For example, 72-hour detection results show that the leakage of RC2-248-249 is 20.25 times lower than that of RC2-246-247.

[0186] The above results indicate that the RC2-246-247 split structure ( Figure 7 It helps to significantly reduce background or signal background and is particularly useful in background-sensitive applications, such as those requiring no or virtually no GOI leakage.

[0187] Some of the experimental results are summarized in Table 2 below.

[0188] Table 2

[0189]

[0190] *RC2 is a plasmid containing the AAV rep and AAV cap genes (serological type 2). In each group, plasmid co-transfer includes the RC2 plasmid, the first plasmid, the second plasmid, the third plasmid (optional), and a helper plasmid as the fourth plasmid. For example, RC2-242-237-72h-4x indicates the use of the RC plasmid, with the first plasmid being VG-X242, the second plasmid being VG-X237, the third plasmid absent, and the fourth plasmid being a helper plasmid, without the addition of ligand drugs; RC2-242-237+-72h-4x(green) indicates the use of the RC plasmid, with the first plasmid being VG-X242, the second plasmid being VG-X237, the third plasmid absent, and the fourth plasmid being a helper plasmid, with the addition of ligand drugs; and so on.

[0191] discuss

[0192] In the existing technology, taking CN101868241A as an example, because the core functional element sequence size of the vector is too large, it is not suitable for viral vectors with large target gene sequences and small exogenous gene loads.

[0193] CN101868241A discloses a single vector, describing the lengths of its core elements as follows: the promoter Ubc sequence is 400 bp, the switch system functional element - co-activating protein CAP sequence is 975 bp, the linker sequence TL is 408 bp, the switch system functional element - ligand-dependent transcription factor LTF is 1467 bp, the switch system functional element - inducible promoter FRP is 189 bp, the transcription termination signal SV40pA sequence is 221 bp, and the transcription termination signal hGHpA sequence is 627 bp, totaling 4287 bp. This vector is loaded onto an adeno-associated virus (AAV) with a foreign gene capacity of only 4.7 kb. Including the 282 bp length of the flanking ITR sequences, and excluding the operative linker sequences and some meaningless stable structural sequences, the spatial size of the expressed target gene is only 131 bp. If the commonly used 251 bp post-transcriptional regulatory sequence WPRE of AAV is added, the length will exceed the limit by 120 bp. In addition, some specific promoters and target genes with larger sequence lengths will be even more unsuitable for loading into viral vectors with small foreign gene loading capacity, such as AAV and LV.

[0194] The single carrier of CN101868241A may contain continuous switching functional elements, resulting in poor security.

[0195] The single vector of CN101868241A continuously expresses the switch system functional elements - co-activating protein CAP and switch system functional elements - ligand-dependent transcription factor LTF. The body produces ligand analogs through nutrient intake or its own reactions, which can also induce the activation of secondary expression of the target gene. It is unknown whether the continuous expression of the other two exogenous proteins will produce side effects, and the safety of this switch system is not high.

[0196] The vector disclosed in CN110462040A (2018) is based on patent CN101868241A (2008), which involves the splitting and combination of functional elements and corresponding screening to obtain a feasible solution for CAR-T therapy. The vector is any one of lentiviral vector, retroviral vector or non-viral vector, wherein the vector is a non-viral vector and the non-viral vector is Sleeping Beauty transposon, which cannot be used for AAV.

[0197] In this invention, the inventors for the first time split each functional element of the switch onto two or more plasmids and adeno-associated virus vectors. The split switch system, CAP or LTF, can be entered as needed or in stages, thereby achieving more stable on-demand control, lower leakage, and / or higher expression efficiency.

[0198] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A construct for inducing and regulating the expression of a target gene based on adeno-associated virus (AAV), characterized in that, The group of structures includes core structures selected from the following group: a first structure, a second structure, and an optional third structure; Furthermore, the construct group contains a first expression cassette for expressing a first gene switch polypeptide, a second expression cassette for expressing a second gene switch polypeptide, and a third expression cassette for expressing a foreign gene product; The first gene switch polypeptide includes a DNA-binding domain fused to a first nuclear receptor ligand-binding domain, and the second gene switch polypeptide includes a trans-activation domain fused to a second nuclear receptor ligand-binding domain. The first gene switch polypeptide and the second gene switch polypeptide are connected by a linker to form a gene switch. The promoter in the third expression cassette is a promoter regulated by the gene switch. Furthermore, when the constructs are introduced together into mammalian cells, an adeno-associated virus (AAV)-based gene switch can be formed, and the third expression cassette is expressed under the regulation of the gene switch. Wherein, when the third construct does not exist, one of the core constructs, the first construct and the second construct, contains one or two of the first, second and third expression boxes, while the other core construct contains the remaining expression boxes; Alternatively, when it is a third construct, each of the first, second, and third core constructs contains a first expression box, a second expression box, and a third expression box, respectively.

2. The group of structures as claimed in claim 1, characterized in that, The first expression box, the second expression box, and the third expression box are not located on any one type of core construct at the same time.

3. The group of structures as described in claim 1, characterized in that, With the participation of ligands, the second gene switch peptide (such as LTF) forms a stable high-affinity interaction with the first gene switch peptide (such as CAP) at the inducible promoter, inducing the receptor protein to recruit transcription factors and drive the expression of the target gene encoding the target protein.

4. The group of structures as claimed in claim 1, characterized in that, The gene product is selected from the group consisting of: lipoprotein lipase, apolipoprotein, cytokines, interleukins, interferons, fluorescent proteins, chloramphenicol acetyltransferase, β-galactosidase, β-glucuronidase, Renilla luciferase, firefly luciferase, alkaline phosphatase, or combinations thereof.

5. The group of structures as claimed in claim 1, characterized in that, The gene switch is based on the ecdysone receptor (EcR).

6. The group of structures as claimed in claim 1, characterized in that, The core constructs are selected from the following group: VG-X237(ITR-FRP-EGFP-WPRE-BGHpA-ITR) (SEQ ID NO: 1), VG-X242(ITR-Ubc-CAP-TL-LTF-WPRE-BGHpA-ITR)(SEQ ID NO:2), VG-X243(ITR-Ubc-CAP-P2A-LTF-WPRE-BGHpA-ITR)(SEQ ID NO:3), VG-X244(ITR-Ubc-LTF-SV40pA-Ubc-CAP-WPRE-BGHpA-ITR) (SEQ ID NO: 4), VG-X246(ITR-Ubc-CAP-SV40pA-FRP-EGFP-WPRE-BGHpA-ITR) (SEQ ID NO: 5), VG-X247(ITR-Ubc-LTF-WPRE-BGHpA-ITR)(SEQ ID NO:6), VG-X248(ITR-Ubc-LTF-SV40pA-FRP-EGFP-WPRE-BGHpA-ITR) (SEQ ID NO:7), VG-X249(ITR-Ubc-CAP-WPRE-BGHpA-ITR)(SEQ ID NO:8), VG-142(ITR-Ubc-EGFP-WPRE-BGHpA-ITR).

7. A reagent kit, characterized in that, The kit comprises: a container and a set of constructs for inducing and regulating the expression of a target gene based on adeno-associated virus (AAV) as described in claims 1-6, located within the container.

8. A method for inducing and regulating the expression of a target gene based on adeno-associated virus (AAV), characterized in that, Including the following steps: (a) Provides the constructs for inducing and regulating the expression of target genes based on adeno-associated virus (AAV) as described in claims 1-6; (b) Introduce the first, second, and optional third constructs into the same cell; (c) In the presence of a ligand, the first gene switch polypeptide and the second gene switch polypeptide are linked by a linker to form a gene switch, and the gene switch regulates the expression of the target gene by the promoter in the third expression cassette.

9. The use of the constructs for inducing and regulating the expression of a target gene based on adeno-associated virus (AAV) as described in claims 1-6, and the kit as described in claim 7, characterized in that, Used to induce and regulate the expression of target genes.

10. The use of the constructs for inducing and regulating the expression of a target gene based on adeno-associated virus (AAV) as described in claims 1-6, and the kit as described in claim 7, characterized in that, Used to prepare medicines for the prevention and / or treatment of diseases or conditions.

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