WO2023231931A1

WO2023231931A1 - Red light-regulated transcriptional activation device/system for mammals, and construction method therefor and use thereof in gene therapy

Info

Publication number: WO2023231931A1
Application number: PCT/CN2023/096653
Authority: WO
Inventors: 叶海峰; 乔龙亮; 牛灵雪; 王智浩
Original assignee: 华东师范大学
Priority date: 2022-05-31
Filing date: 2023-05-26
Publication date: 2023-12-07
Also published as: CN117187302A

Abstract

Disclosed in the present invention is a red light-regulated transcriptional activation device/system (referred to as an REDLIP device/system) for mammals, which comprises a red light photosensitive protein element, a transcriptional activation element, and an effector element. The REDLIP device/system has the characteristics of a simple structure, small modules, no toxicity or side effects, a rapid response, a high transcriptional activation efficiency, a high spatio-temporal specificity, strong adjustability, etc. The REDLIP device/system can accurately and rapidly activate genetic circuits under the regulation of red light, and has an important application value in various fields such as basic biology and regenerative medicine research. The device/system has small elements and modules and can be delivered by adeno-associated viruses (AAV), thereby achieving accurate, efficient and rapid gene therapy for diseases such as diabetes and obesity. The REDLIP device/system provides an accurate, efficient and controllable tool for gene therapy, and has potential value in the clinical application of gene therapy.

Description

A mammalian red light-controlled transcription activation device/system and its construction method and application in gene therapy

Technical field

The invention relates to multidisciplinary fields such as synthetic biology, optogenetics, and gene therapy. Specifically, it relates to a mammalian red light-regulated transcription activation device/system (REDLIP) and its construction method, which can induce mammalian genes efficiently, accurately, and quickly. The transcriptional expression, and the REDLIP device/system that can be delivered by adeno-associated virus AAV, can achieve precise, efficient and long-term gene therapy for the disease.

Background technique

Synthetic biology, with its bottom-up characteristics, redefines life through the modification of genetic circuits. Precise regulation of cell life activities needs to be achieved by constructing artificially regulated gene switches. Precise control systems play an important role in various fields of basic biology and translational biomedical research, such as controlled release of therapeutic drugs, specific expression of epigenetics, etc.

The regulation of gene expression is mainly achieved through some small molecule chemicals, such as PCA, RES, ABA, etc. However, these small molecule regulatory systems have limitations such as certain toxicity and poor adjustability. Light has become an ideal inducer of gene expression because of its nontoxicity, ubiquity in nature, and strong spatiotemporal specificity. Optogenetic technology is also widely used in regulating gene expression. It can control the life activities of engineered cells with a high degree of spatiotemporal specificity, providing potential application value for traceless, remote-controlled precision-regulated medicine. In the field of genetic engineering and Precision treatment is of great significance in medicine. Currently, natural light-sensitive proteins from plants, fungi, and bacteria have been developed into a wide range of optogenetic transcription devices/systems and have been widely used in the fields of genetic engineering, translational regenerative medicine, and epigenetics. However, these current light-regulated gene transcription devices/systems still have certain limitations. Some optogenetic tools (such as CRY2/CIBN, LOV, Magnet, etc.) that use blue light or ultraviolet light as inducers have certain phototoxicity, poor tissue penetration, and poor therapeutic effect on deep diseases, limiting their use. application in clinical medicine. Gene transcription switches that use far-red light or red light as inducers are not phototoxic and have stronger tissue penetration capabilities, but the module components are often larger, such as BphP1/PpsR2; the efficiency of transcription activation of these devices/systems It is low, insensitive to light, and requires long-term illumination. These shortcomings limit its application in clinical treatment of diseases. Recently, a new transcription device/system (REDMAP) regulated by red light has been developed, which has the characteristics of high induction efficiency and rapid response to red light. However, since this system requires additional addition or expression of PCB pigments during the application process, it also limits its use in treating diseases. Implement the transformation and application of gene therapy.

As an emerging disease treatment model with remarkable therapeutic effects, gene therapy is achieved by placing the target gene into a specific vector and then introducing it into the body. The vector carrying the target gene can produce the required drug protein in the cells of the body. Treatment of disease. Adeno-associated virus (AAV) has become the safest and most effective gene delivery method in gene therapy due to its many advantages such as not integrating foreign genes into the host genome, low immunogenicity, wide host range, and long expression of foreign genes in the body. carrier. However, due to the limited packaging range of AAV (<4.7kb), its clinical application has been limited. There is an urgent need to develop a gene transcription expression device/system with small modules, simple operation, high induction efficiency, fast and sensitive, to promote the widespread clinical application of AAV gene therapy.

In order to overcome the limitations of AAV vector delivery, combined with the strong tissue penetration, precise spatiotemporal specificity, and non-toxicity of red light, a red light-controlled drug was invented in mammals that is simple to operate, safe and efficient, has high induction efficiency, and is fast. The transcription activation device/system (REDLIP), which is responsive, has small modular components and does not require the addition of additional pigments, has potential clinical application value for basic biological research, regenerative medicine applications, and precise gene therapy.

Contents of the invention

In view of the above limitations, the present invention proposes a mammalian red light-regulated transcription activation device/system (referred to as REDLIP device/system), which realizes the regulation of gene transcription activation of mammalian cells by red light. The device/system of the present invention has the characteristics of high induction efficiency, rapid response, strong spatiotemporal specificity, strong tissue penetration, no toxic side effects, small modular components and good adjustability. Compared with the REDMAP system, the device/system of the present invention does not require additional addition or expression of pigment molecules in mammals, is more safe, non-toxic, and simple to operate, and also reduces the complexity of the device/system module to a certain extent. Spend.

The present invention can efficiently and quickly activate the transcriptional expression of the reporter gene by irradiating 660nm red light for just a few seconds, and the expression level of the gene can be controlled by adjusting the light intensity of the power supply and changing the illumination time; at the same time, it can Controlling the illumination area can achieve spatial control of gene expression with strong spatiotemporal specificity. At the same time, in the present invention, the mammalian red light regulated transcription activation device/system has very low background leakage and high activation effect, can achieve high induction activation efficiency, and has strong practicability. In addition, the mammalian red light regulated transcription activation device/system of the present invention can turn off transcription activation under far-red light irradiation of 780 nm, achieving dual control of turning on and off gene transcription, and has Strong controllability.

In summary, the present invention can realize rapid, efficient and precise transcriptional activation control of gene circuits in mammalian cells, and has huge potential in the fields of mammalian basic biological research, genetic engineering, epigenetics and translational medicine. Value.

The present invention proposes for the first time a mammalian red light regulated transcription activation device/system. In the present invention, red light as an inducer has the characteristics of non-toxic side effects, high induction multiple, good spatiotemporal specificity of gene expression, precise controllability, etc. In addition, the mammalian red light regulated transcription activation device/system module has small components and can be used by AAV vectors. Delivering this system to deep tissue parts of the organism to regulate gene expression in deep tissues provides an efficient and controllable tool for gene therapy and has great potential for clinical application.

The term "spatial and temporal specificity" refers to the fact that when the expression of a target gene is induced by a specific factor, it is affected by the time and space of the induction factor, showing the dependence of gene expression on the time and space of the induction factor.

The invention proposes a mammalian red light regulated transcription activation device/system. The present invention further optimizes the structure of the red light photosensitive protein, the transcriptional activation element and the inducible promoter of the effector element in the device/system to achieve the optimal effect of red light induction and activation of gene transcription expression, so that the system has the ability to respond to red light. The maximized responsiveness increases the possibility of clinical application of mammalian red light-regulated transcription activation devices/systems.

Each nucleotide sequence or amino acid sequence described in the present invention can be prepared by artificial synthesis methods. Among them, in order to improve the conformational stability of the bacterial red light sensitive protein DrBphP under red light irradiation, the novel red light proteins PnBphP and FnBphP composed of N-terminal fusions of different NTE domains were proposed for the first time by the present invention.

The mammalian red light regulated transcription activation device/system proposed by the present invention includes: a red light sensing element, a transcription activating element and a response element.

In the present invention, the red light-sensitive protein is a bacterial-derived red light-sensitive protein DrBphP (Dr-REDLIP), PnBphP (Pn-REDLIP) that increases the NTE domain of the plant photosensitive protein PhyA, and increases the NTE structure of the fungal photosensitive protein FphA. FnBphP (Fn-REDLIP) of the domain, its amino acid sequence is as shown in SEQ ID NO.1-3, and the Gal4 protein containing the DNA binding domain, its amino acid sequence is as shown in SEQ ID NO.4; wherein, the Gal4 and The amino acid sequence of the connecting peptide between bacterial-derived red light photosensitive proteins is shown in SEQ ID NO.5.

In the present invention, the transcription activator element includes nanochaperone protein LDB3/LDB14 that interacts with red light photosensitive protein, the amino acid sequence of which is shown in SEQ ID NO. 6-7, and a transcription activator (the transcription activator has Recruiting RNA polymerase functions, including VP64, VP16, p65, VPR, p65-HSF1, whose amino acid sequence is shown in SEQ ID NO. 8-12, and the connecting peptide between LDB3/LDB14 and the transcription activator.

In the present invention, the N-terminal of the LDB3 nanochaperone protein is fused to express different copy numbers of the nuclear entry signal NLS, and the amino acid sequence of the NLS is shown in SEQ ID NO. 13; wherein, the nanochaperone protein and the transcription activator The connecting peptide between them has an amino acid sequence as shown in SEQ ID NO.14.

In the present invention, the response element includes an inducible promoter and a target gene.

Among them, the inducible promoter is composed of an operator and an inducible weak promoter, namely P _5×UAS -P _hCMVmin and P _5×UAS -P _TATA , and its nucleotide sequence is shown in SEQ ID NO. 15-16 , the red light photosensitive protein and its fusion protein Gal4 can combine with the inducible promoter. The inducible promoter cannot initiate the expression of downstream genes without recruiting RNA polymerase. The target gene can be any meaningful The gene sequence of the protein.

Wherein, the response element can be the gene sequence of any meaningful protein, including SEAP, EGFP and Luciferase reporter genes, whose amino acid sequences are shown in SEQ ID NO. 17-19; gene therapy drug proteins Insulin and TSLP, whose amino acid The sequence is shown in SEQ ID NO.20-21.

Wherein, the mammalian red light regulated transcription activation device/system can induce and activate the expression of target genes by red light with a wavelength of 660±10nm, and can turn off gene transcription under the irradiation of 780nm far-red light.

The mammalian red light regulated transcription activation device/system of the present invention and its mechanism of transcription activation are shown in Figure 1. The specific explanation is: red light photosensitive proteins DrBphP, PnBphPP, FnBphP and a protein with a DNA binding domain. Protein Gal4 is fused and expressed. Under the irradiation of 660nm red light, the conformation of the red light-sensitive protein changes, prompting its specifically recognized nanochaperone protein LDB3 and its fused transcription activator p65-HSF1 to bind to each other, using the red light-sensitive protein With the heterodimerization properties of Nanobody LDB3, the inducible promoter can recruit RNA polymerase through the transcription activator p65-HSF1 to initiate the downstream transcription expression of the target gene.

In the present invention, all modules of the mammalian red light-regulated transcription activation device/system are constructed on eukaryotic expression vectors and/or AAV expression vectors, so that transcription activation expression induced by red light can be realized in mammalian cells. And it has the effect of red light-induced activation of expression in any type of mammalian cells, such as HEK-293T, hMSC-TERT, HeLa, Hana3A, ATDC5 and other cell lines.

Wherein, the red light wavelength of the red light is 660±10nm, the light intensity is 0-2.5mW/cm ² , and the irradiation time is 0-24h. If there is no special instructions, the light intensity is 2mW/cm ² and the irradiation time is 10s; The irradiation method is continuous irradiation or specific irradiation using different red and black background pictures in different spaces to control Control gene transcription expression levels in mammalian cells. The red light source can be an LED light, a laser light, a physical therapy instrument, a screen of an electronic device such as a mobile phone, etc. The wavelength of the far-red light is 780nm.

In the present invention, the mammalian red light regulated transcription activation device/system can control the expression amount of the reporter gene by adjusting the light intensity and light time. In addition, by controlling the spatial position of the red light illumination, the transcriptional expression of the reporter gene can be spatially controlled. The effect of specific activation, with good temporal and spatial specificity.

The invention also proposes a method for constructing the mammalian red light regulated transcription activation device/system, which includes the following steps:

(1) Construct a red light sensing element

A fusion protein of a red light photosensitive protein and a DNA-binding protein Gal4, as well as a connecting peptide between the two proteins, are constructed as the red light sensing element of the mammalian red light regulated transcription activation device/system.

Wherein, the red light photosensitive protein can be any version of the protein DrBphP(Dr-REDLIP)/PnBphP(Pn-REDLIP)/FnBphP(Fn-REDLIP), the amino acid sequence of which is shown in SEQ ID NO.1-3;

(2) Construct transcriptional activation elements.

Construct a fusion protein with the amino acid sequence of the nanochaperone protein as shown in SEQ ID NO.6-7, and the amino acid sequence of the transcription activator as shown in SEQ ID NO.8-12, as well as the connecting peptide between the two proteins, as described Transcriptional activation elements of the mammalian REDLIP apparatus/system.

Among them, both nanochaperones LDB3 and LDB14 can interact with red light photosensitive proteins under red light irradiation, and their amino acid sequences are shown in SEQ ID NO.6-7; ultimately, based on the induction efficiency of the reporter gene, LDB3 was preferred as the Transcription activator.

Among them, the nanomolecule chaperone LDB3 optimizes the device/system by adding nuclear entry signals (NLS) with different copy numbers. Its amino acid sequence is shown in SEQ ID NO.13. Finally, according to the induction efficiency of the reporter gene, two copies of the nuclear entry signal NLS (2×NLS) and LDB3 were fused for expression.

Among them, the transcription activators include p65, VP64, VPR, VP16, p65-HSF1, etc. These transcription factors all have the function of recruiting RNA polymerase, and their amino acid sequences are shown in SEQ ID NO. 8-14. Finally, p65-HSF1 was selected as a transcriptional activator based on the induction efficiency of the reporter gene.

Wherein, the connecting peptide between the two fusion proteins is 7 amino acids, and its amino acid sequence is as SEQ Shown as ID NO.14.

(3) Construct effect elements.

The constructed response element includes an inducible promoter and a downstream reporter gene. The inducible promoter includes an operator and an inducible weak promoter. The operator is a DNA sequence 5×UAS that can be specifically recognized by the DNA-binding protein Gal4, and the weak promoter is TATA or hCMVmin. The operator and the weak promoter together constitute the species-inducible promoter (P _5×UAS ), whose nucleotide sequence is shown in SEQ ID NO. 15-16; the reporter gene is any meaningful protein. Including insulin, TSLP, SEAP, EGFP, Luciferase, etc. Its amino acid sequence is shown in SEQ ID NO. 17-21.

Among them, the inducible promoter (P _5×UAS ) only activates the expression of downstream reporter genes under 660nm red light irradiation. Inducible promoters include but are not limited to TATA or hCMVmin. Finally, based on the induction efficiency of the reporter gene, (P _5×UAS , 5×UAS-P _TATA ) was preferentially selected as the inducible promoter.

The mammalian red light regulated transcription activation device/system of the present invention can respond quickly to red light in just a few seconds and efficiently initiate the transcriptional expression of downstream reporter genes.

The mammalian red light regulated transcription activation device/system of the present invention starts the expression of the reporter gene under the induction of 660nm red light, but cannot activate the expression of the reporter gene at other wavelengths.

The mammalian red light regulated transcription activation device/system of the present invention activates the transcriptional expression of the reporter gene under irradiation of 660nm red light, and turns off the transcription device/system under irradiation of 780nm far-red light.

The mammalian red light-regulated transcription activation device/system of the present invention exhibits a high degree of temporal and spatial specificity for red light.

The mammalian red light-regulated transcription activation device/system of the present invention can regulate gene expression in different mammalian cell lines.

The mammalian red light regulated transcription activation device/system of the present invention has small modular components, simple operation, and does not require the addition of additional pigments, and can be efficiently delivered by adeno-associated virus AAV.

The mammalian red light-regulated transcription activation device/system REDLIP _Cas according to the present invention can achieve the activation and expression of endogenous genes at the cellular level and the mouse body level by combining CRISPR-dCas9.

The invention also provides a eukaryotic expression vector, an AAV expression vector, an engineered mammalian cell, an engineered AAV virus particle and/or a system of a mammalian red light regulated transcription activation device/system; wherein, the engineered mammal The animal cells are mammalian cells transfected with the mammalian red light-regulated transcription activation device/system; the engineered AAV is composed of an AAV packaging plasmid and a mammalian red light-regulated transcription activation device. AAV vector plasmid packaging production of transcription activation device/system.

The eukaryotic and AAV expression vector may be a vector containing a gene encoding a red light photosensitive element alone, a vector containing a gene encoding a transcription activator element alone, or a vector alone containing a gene encoding a response element. Or a vector containing a gene encoding a red light photosensitive element and a vector encoding a transcription activator element, or a vector containing a gene encoding a transcription activator element and a vector encoding a response element. The construction methods of all the above eukaryotic expression vectors and AAV expression vectors are detailed in Table 1.

The present invention also proposes the construction and application methods of preparing eukaryotic expression vectors, AAV expression vectors, engineered cells or engineered AAV containing the mammalian red light regulated transcription activation device/system.

The preparation methods of the eukaryotic expression vector and AAV expression vector are detailed in Table 1;

The method for preparing engineered cells includes: PEI transfection and liposome Lip3000 transfection; the injection method of the engineered AAV virus is intramuscular injection. Among them, the engineered AAV was packaged and obtained by Shanghai Teltu Biotechnology Co., Ltd.

The invention also provides the preparation of the mammalian red light regulated transcription activation device/system or the eukaryotic expression vector, AAV expression vector, engineered cells and engineered AAV virus, engineered mammalian cells and/or the use of CRISPR - Application of dCas9 to activate endogenous gene expression (EGFP, Luciferase, Insulin, TSLP, etc., the amino acid sequence is shown in SEQ ID NO. 17-21), engineered AAV viruses and/or gene therapy kits for AAV delivery.

In the present invention, the kit includes a kit containing plasmids for regulating each component of the mammalian red light-regulated transcriptional activation device/system, an AAV virus kit containing a plasmid for regulating the mammalian red-light regulated transcriptional activation device/system, and Corresponding instructions.

The invention also proposes a method for regulating gene expression in mammalian cells by using the mammalian red light-regulated transcription activation device/system, which includes the steps:

a) Construct the mammalian red light-regulated transcription activation device/system into a host cell eukaryotic plasmid expression vector or AAV expression vector;

b) Introduce the expression vector containing the mammalian red light-regulated transcription activation device/system into mammalian cells;

c) Inducing the expression of reporter genes and/or drug proteins (such as SEAP, Luciferase, Insulin, TSLP, etc.) in mammals through red light;

d) Detect the expression of the target gene.

The present invention also proposes a device/system that utilizes the mammalian red light regulated transcription activation device/system to deliver the mammalian red light regulated transcription activation device/system to the mouse liver through hydrodynamic means. In the mouse liver tissue, The method of activating foreign genes specifically includes the following steps:

b) Deliver the above mixed solution containing the expression vector into the liver tissue of the mouse through hydrodynamic injection;

c) Inducing the expression of exogenous gene (Luciferase) in mouse liver through red light;

d) In vivo imaging to analyze the effect of exogenous gene activation in mouse liver tissue.

The present invention also proposes a method of using the mammalian red light-regulated transcription activation device/system REDLIP _Cas and using CRISPR-dCas9 to activate the expression of endogenous genes in mammalian cells. The endogenous genes include RHO2XF, ASCL1, TTN, MIAT, IL1RN. The nucleotide sequences of gRNA corresponding to different endogenous genes are shown in SEQ ID NO. 22-25.

Specifically, it includes the following steps:

a) Construct the mammalian red light-regulated transcription activation device/system, dCas9 and response element MS2-p65-HSF1 into a eukaryotic plasmid expression vector or AAV expression vector;

b) transfect the above expression vector into mammalian cells;

c) Red light induces the expression of MS2-p65-HSF1, thereby inducing the activation of endogenous genes in mammalian cells;

d) Extract RNA from cells and analyze the effect of endogenous gene activation in mammalian cells by RT-qPCR.

The present invention also proposes a method for gene therapy using a mammalian red light regulated transcription activation device/system, which method includes: a) constructing an AAV expression plasmid vector containing a mammalian red light regulated transcription activation device/system; b) ) Preparing an engineered AAV virus (expressing Luciferase, Insulin or TSLP) containing a mammalian red light-regulated transcription activation device/system, wherein the engineered AAV virus is prepared by Shanghai Teltu Biotechnology Co., Ltd.; c) through muscle Deliver the engineered AAV virus of the mammalian red light-regulated transcription activation device/system into the organism by injection; d) Express the target genes and/or therapeutic proteins, such as Insulin and TSLP, through red light activation, thereby achieving genetic control of the corresponding diseases. treat.

The present invention also proposes a method for treating type Ⅰ diabetes by utilizing the mammalian red light-regulated transcription activation device to pass/system through adeno-associated virus AAV as a carrier. The method includes:

a) Construct an AAV vector plasmid for a mammalian red light-regulated transcription activation device/system, and select Insulin as the reporter gene. That is, an AAV vector containing REDLIP device/system components and expressing Insulin is constructed, as shown in Table 1;

b) Package the AAV plasmid containing the mammalian red light-regulated transcription activation device/system into AAV viral particles;

c) Inject the packaged engineered AAV virus containing the mammalian red light-regulated transcription activation device/system into the mouse leg muscle tissue through intramuscular injection;

d) The level of insulin expression in AAV in mouse muscle tissue is activated through the induction of red light, thereby achieving precise and long-term blood sugar lowering effects.

The present invention also proposes a method of utilizing the mammalian red light-regulated transcription activation device/system and using the adeno-associated virus AAV as a carrier to treat obesity. The method includes:

a) Construct an AAV vector plasmid for a mammalian red light-regulated transcription activation device/system, and select TSLP as the reporter gene, that is, an AAV vector containing components of the REDLIP device/system and expressing TSLP is constructed, as shown in Table 1;

b) Package the AAV plasmid containing the mammalian red light-regulated transcription activation device/system into viral AAV particles;

c) Deliver the packaged engineered AAV virus containing the mammalian red light-regulated transcription activation device/system to the mouse muscle tissue through intramuscular injection;

d) By irradiating red light, AAV expresses TSLP in mouse muscle tissue to achieve weight loss.

In the present invention, activation is induced by red light at the animal level, the red light has a wavelength of 660±10nm, and the light source can be LED, laser light, or physical therapy instrument. Unless otherwise specified, the light intensity is 20mW/cm ² , the light time is 30 minutes, and the light frequency is once every three days.

The beneficial effects of the present invention are: the mammalian red light regulated transcription activation device/system of the present invention can achieve precise, efficient and rapid transcription activation effects by red light, and has a high degree of spatiotemporal specificity and strong tissue penetration. sex. In addition, the reporter gene transcription can be turned off by 780nm far-red light, which has strong adjustability of gene transcription expression. Since the mammalian red light-regulated transcription activation device/system has simple components and small modules, and does not require additional pigments, adeno-associated virus (AAV) can be used as a delivery vector, providing a powerful tool for the clinical application of gene therapy. . The engineered AAV containing a mammalian red light-regulated transcription activation device/system in the present invention successfully achieves Long-term, precise and controllable treatment of type 1 diabetes and obesity. The mammalian red light regulated transcription activation device/system proposed by the present invention can be widely used in a variety of basic biological research and translational medical research, and has great value in clinical applications.

Description of the drawings

Figure 1 is a schematic diagram of the mammalian red light-regulated transcription activation REDLIP device/system and its principle of activating transcription.

Figure 2 shows the selection results of different nanomolecule chaperones for red light photosensitive proteins in the REDLIP device/system for mammalian red light-regulated transcription activation.

Figure 3 shows the results of NLS optimization of different copy numbers of pre-LDB3 nuclear entry signals in the REDLIP device/system for mammalian red light-regulated transcription activation.

Figure 4 is a diagram showing the results of optimization of different transcription activators for LDB3 fusion expression in the mammalian red light-regulated transcriptional activation REDLIP device/system.

Figure 5 is a diagram showing the optimization results of an inducible weak promoter that initiates reporter gene expression in a mammalian red light-regulated transcriptional activation REDLIP device/system.

Figure 6 is a comparative diagram of the activation effects after optimizing the red light photosensitive protein domain in the mammalian red light-regulated transcriptional activation REDLIP device/system.

Figure 7 is a graph showing the dependence of the transcriptional expression of activated reporter genes on light time in the mammalian red light-regulated transcriptional activation REDLIP device/system.

Figure 8 is a graph showing the dependence of the transcriptional expression of activated reporter genes on light intensity in the REDLIP device/system regulated by red light in mammals.

Figure 9 is a graph showing the results of the reporter gene expression efficiency of the mammalian red light-regulated transcriptional activation REDLIP device/system sampled at different times after illumination.

Figure 10 shows the results of mammalian red light-regulated transcription activation REDLIP device/system activating reporter gene expression under light induction of different wavelengths.

Figure 11 shows the results of the mammalian red light-regulated transcription activation REDLIP device/system being turned on by 660nm red light and turned off by 780nm far-red light.

Figure 12 shows the results of the universality of mammalian red light-regulated transcriptional activation REDLIP device/system in different mammalian cell lines.

Figure 13 is a transcription table of mammalian red light-regulated transcriptional activation REDLIP device/system activation reporter gene Achieve reversible results.

Figure 14 is a diagram showing the results of verification of the spatial specificity of transcriptional expression of a reporter gene activated by the REDLIP device/system activated by red light in mammals.

Figure 15 is a schematic diagram of the principle of mammalian red light regulating transcription and activating the transcription expression of endogenous genes in the REDLIP _Cas device/system.

Figure 16 shows the results of mammalian red light-regulated transcription activation REDLIP _Cas device/system activating endogenous gene expression dependent on light intensity.

Figure 17 shows the results of mammalian red light-regulated transcription activation REDLIP _Cas device/system activating endogenous gene expression in dependence on light time.

Figure 18 is a diagram showing the reversible results of mammalian red light-regulated transcription activation REDLIP _Cas device/system activating endogenous gene expression.

Figure 19 shows the results of mammalian red light-regulated transcription activation REDLIP _Cas device/system activating endogenous gene expression in different mammalian cell lines.

Figure 20 shows the results of mammalian red light-regulated transcription activation REDLIP _Cas device/system activating the expression of different endogenous genes.

Figure 21 is a flow chart of a mammalian red light-regulated transcriptional activation REDLIP device/system that activates exogenous gene expression in mouse liver tissue through hydrodynamic means.

Figure 22 is a diagram showing the light intensity-dependent results of the mammalian red light-regulated transcriptional activation REDLIP device/system hydrodynamically activating the expression of exogenous genes in mouse liver tissue.

Figure 23 is a diagram showing the results of the mammalian red light-regulated transcriptional activation REDLIP device/system hydrodynamically activating the expression of exogenous genes in mouse liver tissue in a light time-dependent manner.

Figure 24 shows the results of the Pn-REDLIP and Fn-REDLIP devices/systems hydrodynamically activating exogenous gene expression in mouse liver tissue in a light time-dependent manner.

Figure 25 is a flow chart for the long-term stable expression of AAV virus engineered by the mammalian red light-regulated transcriptional activation Fn-REDLIP device/system in mouse muscle tissue.

Figure 26 shows the results of in vivo imaging of mammalian red light-regulated transcriptional activation Fn-REDLIP device/system engineered AAV virus activating Luciferase expression in mouse muscle tissue for long-term and stable expression.

Figure 27 is a graph showing the statistical results of long-term activation of Luciferase expression in mouse muscle tissue by the engineered AAV virus of the mammalian red light-regulated transcriptional activation Fn-REDLIP device/system.

Figure 28 shows the blood glucose monitoring results of the mammalian red light-regulated transcriptional activation Fn-REDLIP device/system engineered tri-viral AAV for the treatment of type 1 diabetes.

Figure 29 is a schematic diagram of the two-virus AAV engineered by red light-regulated transcriptional activation Fn-REDLIP device/system to treat type 1 diabetes in mammals.

Figure 30 shows the blood glucose monitoring results of the two-virus AAV engineered by the mammalian red light-regulated transcriptional activation Fn-REDLIP device/system to treat type Ⅰ diabetes.

Figure 31 is a diagram showing the monitoring results of Insulin content in vivo of mammalian red light-regulated transcriptional activation Fn-REDLIP device/system engineered two-virus AAV for the treatment of type 1 diabetes.

Figure 32 is a schematic diagram of the mammalian red light-regulated transcriptional activation Fn-REDLIP device/system engineered AAV virus for obesity gene therapy.

Figure 33 is a graph showing the statistical results of mouse body weight for obesity gene therapy using the engineered AAV virus of the mammalian red light-regulated transcriptional activation Fn-REDLIP device/system.

Figure 34 is a graph showing the statistical results of beige fat, white fat and brown fat weight in obese mice after gene therapy with mammalian red light-regulated transcriptional activation Fn-REDLIP device/system engineered AAV virus.

Figure 35 is a graph showing the statistical results of triglyceride content in the serum of obese mice subjected to gene therapy using mammalian red light-regulated transcriptional activation Fn-REDLIP device/system engineered AAV virus.

Figure 36 is a graph showing the statistical results of triglyceride content in the livers of obese mice treated with the engineered AAV virus of the mammalian red light-regulated transcriptional activation Fn-REDLIP device/system.

Detailed ways

The present invention will be further described in detail with reference to the following specific examples and drawings. These examples are only used to illustrate the invention and do not constitute any limitation on the scope of the invention. The process, conditions, experimental methods, etc. for implementing the present invention, except those specifically mentioned below, are common knowledge and common sense in the field. The reagents, instruments, etc. used in the following examples, as well as experimental methods without specifying specific conditions, were performed in accordance with conventional conditions or conditions recommended by commercial suppliers.

Materials and Methods

Plasmid construction and configuration of related reagents

Molecular cloning technology is used to construct all expression plasmids of the present invention, and the steps are common knowledge in the industry.

All primers used for PCR were synthesized by Shanghai Ruimian Biotechnology Co., Ltd. Embodiments of the invention The expression plasmids constructed in were all sequenced, and the sequencing was completed by Shanghai Paison Biotechnology Co., Ltd. Phanta Max Super-Fidelity DNA polymerase and homologous recombinase used in the examples of the present invention were purchased from Nanjing Novozan Biotechnology Co., Ltd. Endonuclease was purchased from New England Biolabs; T4 DNA ligase, DNA Marker DL15000, DNA Marker DL5000, and DNA Marker DL2000 were purchased from Baonida Biotechnology (Beijing) Co., Ltd. Yeast Extract, Trypton, agar powder, ampicillin (Amp), and agarose were purchased from Sangon Bioengineering (Shanghai) Co., Ltd. The nucleic acid dye GoldView was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; the plasmid mini-extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; the DNA gel recovery kit and PCR product purification kit were purchased from Kangwei Century Biotechnology Co., Ltd.; the other reagents such as absolute ethanol and NaCl mentioned in the examples are domestic analytically pure products.

The PCR system and program settings were carried out according to the instructions provided by Phanta Max Super-Fidelity DNA polymerase (Baori Medical Biotechnology (Beijing) Co., Ltd.); seamless cloning was carried out according to the homologous recombinase (Nanjing Novezan Biotechnology Co., Ltd.) The T4 enzyme ligation was carried out according to the instructions provided by T4 DNA ligase (Baori Medical Biotechnology (Beijing) Co., Ltd.); the enzyme digestion of DNA vectors or fragments was carried out according to the instructions provided by endonuclease (New England Biolabs). ; The gel recovery and purification recovery of DNA fragments were carried out according to the operating instructions of the DNA gel recovery kit and PCR product purification kit (Kangwei Century Biotechnology Co., Ltd.); the plasmid extraction steps were carried out according to the plasmid extract (Tiangen Biochemical Technology (Tiangen Biochemical Technology) Beijing) Co., Ltd.) extraction kit instructions.

Reagent configuration

Preparation of DH5α competent cells

The growth density of DH5α was determined by measuring the light absorption value (OD ₆₀₀ ) at a wavelength of 600 nm using an Eppendorf spectrophotometer. All reagents and consumables used to prepare the competent state need to be sterilized by high-pressure steam in advance. The ultra-clean workbench is sterilized by ultraviolet irradiation. The entire production process is strictly guaranteed to be sterile and ice bathed (the centrifuge is pre-cooled at 4°C in advance, and the reagents and Place the 1.5mL centrifuge tube in a 4°C refrigerator to pre-cool).

Specific steps are as follows:

1) Streak DH5α onto an anti-antibody solid LB plate and culture it overnight to achieve activation;

2) Pick a single clone and inoculate it into 5 mL liquid LB medium, and culture it overnight at 37°C and 210 rpm;

3) Inoculate 2 mL of seed solution into 200 mL of anti-resistant liquid LB, and culture at 37°C and 210 rpm until OD ₆₀₀ = 0.3-0.5;

4) Cool the bacterial solution on ice for 20 minutes;

5) Centrifuge at 4°C, 4500rpm for 7 minutes, discard the supernatant;

6) Add an equal volume of 0.1M CaCl ₂ to the bacterial solution, and pipe gently with a pipette to mix;

7) Centrifuge at 4500 rpm for 7 minutes at 4°C and discard the supernatant;

8) Add 1/2 volume of 0.1M CaCl ₂ to the bacterial solution, and pipe gently with a pipette to mix;

9) Centrifuge at 4500 rpm for 7 minutes at 4°C and discard the supernatant;

10) Add 10% glycerol mixed with 1/2 volume of the 0.1M CaCl ₂ solution of the bacterial solution, and gently pipet with a pipette to mix;

11) Centrifuge at 4500 rpm for 7 minutes at 4°C and discard the supernatant;

12) Resuspend in 10 mL of 10% glycerol in 0.1 M CaCl ₂ , aliquot 100 μL/tube, and store at -80°C ultra-low temperature;

Plasmid transformation

The method of transformation is CaCl _2- mediated chemical transformation.

Specific steps are as follows

1) Thaw the prepared DH5α competent state on ice and add appropriate plasmid DNA (all seamless cloning products (10 μL) and 100 ng of plasmid). The volume of plasmid DNA added generally does not exceed the total volume of the competent state. 1/10;

2) Let stand on ice for 30 minutes, heat shock at 42°C for 90 seconds, and let stand at 4°C for 2 minutes;

3) Add 600μL anti-anti LB and incubate at 210rpm and 37℃ for 45min;

4) Centrifuge at 6000 rpm for 2 minutes, discard the supernatant, resuspend in 200uL LB, and spread on an ampicillin-resistant LB solid culture plate;

5) Incubate overnight at 37°C.

Cell culture and transfection and configuration of related reagents

In the examples of the present invention, the following cell lines and PEI transfection are used as examples to illustrate the working conditions of the REDLIP device/system in cells, but this does not limit the scope of the present invention.

The mechanism of action of the REDLIP device/system and its transcriptional activation is shown in Figure 1. The specific explanation is as follows: the fusion expression of red light photosensitive proteins DrBphP, PnBphP, FnBphP and a protein Gal4 with a DNA binding domain, at 660nm red Under the irradiation of light, the conformation of the red light-sensitive protein changes, prompting the nano-chaperone protein LDB3 specifically recognized by it and its fused transcriptional activator p65-HSF1 to bind to each other, and the red-light light-sensitive protein is used to heterodimerize with the nanobody LDB3 Due to its characteristics, the inducible promoter can recruit RNA polymerase through the transcription activator p65-HSF1 to initiate the transcriptional expression of the downstream target gene.

The 10cm cell culture dish and cell culture plate used for cell culture were purchased from Thermo Fisher Scientific Company (Labserv); the Eulbecco's modified Eagle's medium (DMEM) and fetal bovine serum used were purchased from Gibico Company in the United States; penicillin and streptomycin solutions were purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; the PEI used for transfection was purchased from Polysciences, and the liposome lip3000 was purchased from Thermo Fisher Scientific (Labserv); the cell number was counted using Countess II automated cell counter; the remaining consumables were ordinary domestic consumables.

Cell culture: The cells involved in the present invention include human embryonic kidney cells (HEK-293T, ATCC: CRL-3216), which stably integrates a copy of the E1 gene (Thermo Fisher, R70507), and telomeric human mesenchymal cells (hMSC). -TERT) and HEK-293T-derived Hana3A cells, mouse chondrocytes ATDC5, all cells were cultured in Eulbecco's modified Eagle's medium (DMEM), and 10% (v/v) fetal calf serum and 1 % (v/v) penicillin and streptomycin solution; cells were cultured in an incubator containing 5% carbon dioxide at 37°C.

Transfection: There are two transfection methods for all cell lines. The first transfection uses PEI, and the second transfection uses liposome Lip3000.

PEI configuration and transfection method

PEI configuration

Fully dissolve PEI in endotoxin-free sterile water, adjust the pH to 7.0, and give a final concentration of 1 μg/μL. Sterilize the PEI through a 0.22 μm filter membrane and store it frozen in a -20°C refrigerator.

Specific steps are as follows

1) Inoculate an appropriate amount of cells in a 24-well plate/10cm cell culture dish 12-16 hours in advance, 24-well plate (6×10 ⁴ )/10cm cell culture dish (6×10 ⁵ );

2) Mix the DNA that needs to be transfected into the cells with DMEM complete medium, 50 μL/1mL in each well of the 24-well plate/10cm cell culture dish;

3) Add a corresponding volume of PEI into the mixture of DMEM and DNA, where PEI:DNA=3:1 (HeLa cells, PEI:DNA=5:1). Vortex for about 10 seconds, and use a handheld centrifuge to remove the liquid on the tube wall. , let stand at room temperature for 15 minutes;

4) Add the transfection mixture evenly into the cell culture medium and place it in a 37°C CO ₂ incubator for culture;

5) 6 hours after transfection, replace with an appropriate volume of fresh DMEM (containing fetal bovine serum and penicillin/streptomycin) medium;

Liposome Lip3000 was administered according to the instructions provided by the manufacturer.

Reporter gene detection methods and related reagent configurations:

Secreted alkaline phosphatase (SEAP)

The homoarginine, magnesium chloride, diethanolamine, and hydrochloric acid solutions used to configure the detection reporter gene reaction buffer were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.; the chromogenic substrate (p-nitrophenol phosphate) was purchased from Shanghai Jingchun Biochemical Technology Co., Ltd. (Aladdin).

Configuration of related reagents:

Adjust pH (HCL) to 9.8 and store in the dark at 4°C.

Aliquot into 2mL EP tubes and store at -20°C.

Specific steps are as follows

1) Take 200 μL of cell culture supernatant and place it in a 96-well plate;

2) Cover with plastic wrap (to prevent liquid evaporation) and place at 65°C for 30 minutes (in order to remove endogenous alkaline phosphatase, the exogenously expressed alkaline phosphatase SEAP has high temperature resistance);

3) Take 80 μL of the heated cell culture medium (or dilute it with PBS according to the experiment) supernatant in a new 96-well plate, and quickly mix it with 120 μL of reaction solution (2xbuffer:pNPP=5:1);

4) The microplate reader continuously monitors the absorption value of the reaction product at a wavelength of 405nm, and the detection time is 10 minutes;

Enzyme activity calculation

The enzyme activity of alkaline phosphatase (SEAP) is defined as: when the pH is 9.8 at 37°C, it reacts with the substrate disodium p-nitrophenyl phosphate (pNPP-Na ₂ ) to generate 1 mol/L p-nitrophenol base within 1 minute. Sex phosphatase is defined as 1 activity unit (1U). P-nitrophenol (pNPP-Na ₂ ) itself has a bright yellow color. At a wavelength of 405nm, different concentrations of p-nitrophenol (reaction product) correspond to different absorbance values. The calculation method is: the slope of the curve made from the OD values measured at different time points during the reaction between the sample and the substrate *256.8, which is the enzyme activity in U/L.

Detection of Luciferase in mice

Luciferase is a luciferase that is widely distributed in bioluminescent organisms, including bacteria, fungi, fish, insects, etc. The substrate is fluorescein (Luciferin). When Luciferase and its substrate are mixed, a rapidly attenuating yellow-green flash will be produced. This light signal can be detected with a fluorescence detector (Luminometer). The total amount of luminescence is proportional to the luciferase activity of the sample, and therefore provides an indirect estimate of the transcription of the reporter gene luciferase.

Specific steps are as follows

1) Mice were injected intraperitoneally with luciferin substrate solution (150mg/kg);

2) Place the mouse in a small animal anesthesia machine (isoflurane + oxygen);

3) Wait for 5-10 minutes and use an in vivo imager to detect the fluorescence signal;

Cell/tissue RNA extraction

All RNA extraction materials were pre-treated with RNase enzyme. Trizol was purchased from Baori Doctor Biotechnology. Technology (Beijing) Co., Ltd.; DEPC-treated ddH ₂ O was purchased from Sangon Bioengineering (Shanghai) Co., Ltd., and the isopropyl alcohol, chloroform, absolute ethanol, etc. involved are all domestic analytically pure products.

Specific steps are as follows

1) Add 200 μL of Trizol to each well of the 24-well plate. If tissue RNA is extracted, add 400 μL of Trizol. There are two replicates of the cell experiment. Mix the duplicate wells (400 μL in total) and let stand at room temperature for 5 minutes;

2) Add 80 μL (1/5 volume) of chloroform, mix by inverting until it appears milky white, and let stand at room temperature for 5 minutes;

3) Centrifuge at 12000rpm 4℃ for 15min;

4) At this time, the mixture is divided into three layers, the lower red layer is organic matter, the middle white layer is DNA, and the upper transparent layer is RNA. Carefully draw an appropriate amount of the transparent layer liquid into a new 1.5mL centrifuge tube (be careful not to suck in the middle DNA) and underlying organic matter);

5) Add an equal volume of isopropyl alcohol and let it stand at room temperature for 10 minutes (it can also be placed at 4°C or -20°C to improve the efficiency of RNA precipitation);

6) Centrifuge at 12000rpm and 4℃ for 10min, discard the supernatant;

7) Add 1 mL of 75% ethanol (DEPC-treated H ₂ O configuration) and gently suspend the precipitate at the bottom;

8) Centrifuge at 12000rpm and 4℃ for 10min, discard the supernatant;

9) After drying, dissolve the precipitate with an appropriate amount of DEPC-treated H ₂ O, and immediately invert or store at -80°C.

RT-qPCR data analysis

After extracting the RNA from the cells, follow the instructions provided by HiScript II Q Select RT SuperMix for qPCR (Vazyme, China; Cat.no.R232-01) to reverse the RNA into cDNA. The cycle threshold of the target gene was determined by Real-Time PCR Instrument (QuantStudio 3, Thermo Fisher Scientific Inc., Waltham, MA, USA) using Taq Pro Universal SYBR qPCR Master Mix (Vazyme, China; Cat. no. Q712-02). Amplification conditions were: pre-denaturation at 95°C for 10 min, followed by 40 cycles (denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s), and finally extension at 72°C for 10 min. The primers used are shown in Table 2.

Data analysis was calculated using 1/2 ^△△Ct . All samples used the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal reference. The results were expressed as relative RNA levels using dark conditions as a reference.

Hydrodynamic tail vein injection of plasmid

The main principle of the hydrodynamic method is to rapidly inject plasmid DNA solution into the mouse tail vein at high pressure. In mice, the impact of blood circulation under high pressure will cause instantaneous damage to the mouse liver, allowing the imported DNA fragments to enter the liver cells.

The plasmid DNA injected into the mouse liver was diluted with Ringer’s solution. The volume injected into each mouse was calculated based on the mouse’s body weight:

Configuration of related reagents:

Example 1 Selection of different nano-chaperones for red light photosensitive proteins in REDLIP device/system for mammalian red light-regulated transcriptional activation.

This example uses SEAP as a detection reporter gene to verify the optimal nanochaperone protein in the mammalian REDLIP device/system, but does not limit the scope of the present invention. Specific steps are as follows:

The first step is plasmid construction. The plasmid construction in this example is detailed in Table 1;

The second step is to connect the board. HEK-293T cells were seeded in 24-well plates, with two plates in total: dark group and light group;

The third step is transfection. 16-18h after inoculating the cells, PDL6 (P _5×UAS -SEAP-pA, P _5×UAS , 5×UAS-P _hCMVmin ), pQL217 (P _hCMV- Gal4-DrBphP-pA) and different nanochaperones were The plasmid vector pQL207 (P _hCMV -LDB3-VP64-pA)/pQL208 (P _hCMV -LDB14-VP64-pA) was transfected at a ratio of 1:2:2 (w/w/w) (see method materials for specific steps ). Wrap in tin foil to block light and culture in dark conditions;

The fourth step is to change the fluid. 6 hours after transfection, use a pipette tip to aspirate the cell culture medium under green light, and replace it with 500 μL of fresh DMEM (containing fetal bovine serum and penicillin/streptomycin) medium;

The fifth step is lighting. The light group was placed under LED with a light intensity of 660nm and a light intensity of 2mW/cm ² for 24 hours, and the dark group was cultured under dark conditions;

The sixth step is to detect the reporter gene (see Materials and Methods for specific steps).

The results show that in the mammalian REDLIP device/system, when LDB3 is the nanochaperone protein, the reporter gene SEAP activates the report with the highest efficiency. The experimental data are detailed in Figure 2. All data are presented as n = 3 independent replicate experiments.

Example 2 Optimization of the copy number of the transcription activator element LDB3 nuclear import signal NLS in the REDLIP device/system for mammalian red light-regulated transcription activation.

This example uses SEAP as a detection reporter gene to verify the optimal nuclear entry signal NLS copy number of the transcriptional activation element LDB3 of the mammalian REDLIP device/system, but does not limit the scope of the present invention. Specific steps are as follows:

The third step is transfection. 16-18h after the cells were inoculated, pDL6 (P _5×UAS -SEAP-pA, P _5×UAS , 5×UAS-P _hCMVmin ), pQL217 (P _hCMV- Gal4-DrBphP-pA) and pA with LDB3 plasmid vectors pQL232 (P _hCMV -3NLS-LDB3-VP64-pA), pQL250 (P _hCMV -2NLS-LDB3-VP64-pA), pQL243 (P _hCMV -1NLS-LDB3-VP64-) with different copy numbers of nuclear localization signals NLS pA), pQL207 (P _hCMV -LDB3-VP64-pA), transfected with PEI transfection reagent at a ratio of 1:2:2 (w/w/w) (see method materials for specific steps);

The fifth step is to illuminate (the specific steps are the same as Embodiment 1 of the present invention);

The sixth step is to detect the reporter gene (see Materials and Methods for specific steps);

The results show that in the mammalian REDLIP device/system, the N-terminal fusion of LDB3 expressing two copies of the nuclear entry signal NLS (P _hCMV -2NLS-LDB3-VP64-pA) has the highest efficiency in activating reporter gene expression. The experimental data are detailed in Figure 3. All data are presented in the form of n=3 independent replication experiments.

Example 3 Selection of different transcriptional activators in transcription and post-elements in REDLIP device/system for regulated transcriptional activation by mammalian red light

This example uses SEAP as a detection reporter gene to verify the impact of different activators in the mammalian REDLIP device/system on system activity, but does not limit the scope of the present invention. Specific steps are as follows:

The third step is transfection. 16-18h after inoculating the cells, pDL6 (P _5×UAS -SEAP-pA, P _5×UAS , 5×UAS-P _hCMVmin ), pQL217 (P _hCMV- Gal4-DrBphP-pA) and plasmid vector pQL251 (P _hCMV -2NLS-LDB3-VP16-pA)/pQL250 (P _hCMV -2NLS-LDB3-p65-pA)/pQL252(P _hCMV -2NLS-LDB3-VPR-pA)/pNX12(P _hCMV -2NLS-LDB3-p65-HSF1-pA), 1:2:2(w/ w/w) ratio and use PEI transfection reagent for transfection (see method materials for specific steps);

The sixth step is to detect the reporter gene (see method materials for specific steps).

The results showed that among the mammalian REDLIP devices/systems, the p65-HSF1 transcription activator was selected to activate reporter gene expression with the highest efficiency. The experimental data are detailed in Figure 4. All data are presented in the form of n=3 independent replication experiments.

Example 4 is the optimization of the inducible promoter in the response element in the REDLIP device/system for mammalian red light-regulated transcription activation.

This example uses SEAP as the detection reporter gene to verify the impact of different inducible promoters in the mammalian REDLIP device/system on the activation efficiency of the reporter gene, but does not limit the scope of the present invention. Specific steps are as follows:

The third step is transfection. 16-24h after inoculating the cells, pNX12 (P _hCMV -2NLS-LDB3-p65-HSF1-pA), pQL217 (P _hCMV -Gal4-DrBphP-pA) and plasmid vector PDL6 (P hCMV -Gal4-DrBphP-pA) with different inducible promoters were added to each group. P _5×UAS -SEAP-pA; P _5×UAS ,5×UAS-P _hCMVmin )/pYZ430 (P _5×UAS -SEAP-pA; P _5×UAS ,5×UAS-P _TATA ), 1:2 :2 (w/w/w) ratio and PEI transfection reagent for transfection (see method materials for specific steps);

The results show that in the mammalian red light-regulated transcription activation REDLIP device/system, 5×UAS-P _TATA is selected as the inducible promoter, which has the highest efficiency in activating reporter gene expression. The experimental data are detailed in Figure 5. All data are presented in the form of n=3 independent replication experiments.

Example 5 is the optimization of the red light photosensitive protein structure in the REDLIP device/system for mammalian red light-regulated transcription activation.

This example uses SEAP as the detection reporter gene to verify the impact of different structures of red light photosensitive eggs in the mammalian REDLIP device/system on the expression efficiency of the reporter gene, but does not limit the scope of the present invention. Specific steps are as follows:

The third step is transfection. 16-24h after inoculating the cells, pYZ430 (P _5×UAS -SEAP-pA; P _5×UAS , 5×UAS-P _TATA ), pNX12 (P _hCMV -2NLS-LDB3-p65-HSF1-pA) was added to each group. ), and different versions of the red light-sensitive protein plasmid vector pQL217(P _hCMV -Gal4-DrBphP-pA)/pQL325(P _hCMV -Gal4-PnBphP-pA)/pQL326(P _hCMV -Gal4-FnBphP-pA), with 1 :2:2 (w/w/w) ratio was used for transfection with PEI transfection reagent (see method materials for specific steps);

The sixth step is to detect the reporter gene (see method materials for specific steps);

The results show that in mammalian REDLIP devices/systems, compared with DrBphP (Dr-REDLIP), PnBphP (Pn-REDLIP) has better activation efficiency, and FnBphP (Fn-REDLIP) has better activation expression. The structural characteristics and experimental data of different red light-sensitive proteins are detailed in Figure 6 of the instruction manual. All data are presented in the form of n=3 independent replication experiments.

Example 6 is a study on the correlation between the expression of reporter genes and illumination time in the REDLIP device/system for regulating transcriptional activation by red light in mammals.

This example uses SEAP as the detection reporter gene to verify that the expression of the mammalian REDLIP device/system activated reporter gene is dependent on the illumination time, but does not limit the scope of the present invention. specific Proceed as follows:

The second step is to connect the board. HEK-293T cells were seeded in 24-well plates. There are 11 pieces in total, one of which is the dark group;

The third step is transfection. 16-24h after the cells were inoculated, pNX12 (P _hCMV -2NLS-LDB3-p65-HSF1-pA), pYZ430 (P _5×UAS -SEAP-pA; P _5×UAS , 5×UAS-P _TATA ) were added to each group. ) and pQL217(P _hCMV -Gal4-DrBphP-pA)(Dr-REDLIP)/pQL325(P _hCMV -Gal4-PnBphP-pA)(Pn-REDLIP)/pQL326(P _hCMV -Gal4-FnBphP) containing different versions of red light protein -pA)(Fn-REDLIP) plasmid vector, transfected with PEI transfection reagent in a ratio of 2:1:2 (w/w/w) (see method materials for specific steps);

The fifth step is lighting. Number 11 24-well plates (respectively 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11). Plate No. 1 is placed in dark conditions for culture, and plates No. 2-11 are cultured in dark conditions. 660nm, LED with a light intensity of 2mW/ ^cm2 , illuminate for different times (1s, 5s, 10s, 6min, 1h, 3h, 6h, 12h, 18h, 24h). After light treatment, place it in dark conditions for culture;

The results show that the mammalian REDLIP device/system activates the expression of reporter genes in a light time-dependent manner. The experimental data are detailed in Figure 7. All data are presented in the form of n=3 independent replication experiments.

Example 7 is a study on the correlation between the expression of reporter genes and light intensity in the REDLIP device/system for regulating transcriptional activation by red light in mammals.

This example uses SEAP as a detection reporter gene to verify that mammalian REDLIP device/system activation of reporter gene expression is dependent on light intensity, but does not limit the scope of the present invention. Specific steps are as follows:

The second step is to connect the board. Inoculate HEK-293T cells in 24-well plates, a total of 8 cells;

The third step is transfection. 16-24h after inoculating the cells, pYZ430 (P _5×UAS -SEAP-pA; P _5×UAS , 5×UAS-P _TATA ), pNX12 (P _hCMV -2NLS-LDB3-p65-HSF1-pA) was added to each group. ) and does not contain The same version of red light sensitive protein pQL217(P _hCMV -Gal4-DrBphP-pA)(Dr-REDLIP)/pQL325(P _hCMV -Gal4-PnBphP-pA)(Pn-REDLIP)/pQL326(P _hCMV -Gal4-FnBphP-pA ) (Fn-REDLIP) plasmid vector, transfected with PEI transfection reagent in a ratio of 1:2:2 (w/w/w) (see method materials for specific steps);

The fifth step is lighting. Number 8 24-well plates (respectively 1, 2, 3, 4, 5, 6, 7, 8). Plate No. 1 is placed in dark conditions for culture, and plates No. 2 to No. 8 use 660nm LEDs with different LEDs. Light intensity (0.05, 0.1, 0.25, 0.5, 0.75, 1, 2mW/cm ² ) was used for 10 seconds. After light treatment, it was placed in dark conditions for cultivation;

The results show that the expression of reporter genes activated by the mammalian REDLIP device/system is dependent on light intensity. The experimental data are detailed in Figure 8. All data are presented in the form of n=3 independent replication experiments.

Example 8 is a study on the optimal detection time of reporter gene expression efficiency using the REDLIP device/system for mammalian red light-regulated transcription activation.

This embodiment uses SEAP as a detection reporter gene to verify the optimal detection time of the mammalian REDLIP device/system, but does not limit the scope of the present invention. Specific steps are as follows:

The second step is to connect the board. HEK-293T cells were seeded in 24-well plates. Divided into light group and dark group;

The third step is transfection. 16-24h after the cells were inoculated, pYZ430 (P _5×UAS -SEAP-pA; _P5×UAS , 5×UAS-P _TATA ), pNX12 (P _hCMV -2NLS-LDB3-p65-HSF1-pA) was added to each group. , and plasmid vectors containing different versions of the red light-sensitive protein pQL325(P _hCMV -Gal4-PnBphP-pA)(Fn-REDLIP)/pQL326(P _hCMV -Gal4-FnBphP-pA)(Fn-REDLIP) at 1:2 :2 (w/w/w) ratio of PEI transfection reagent for transfection (see method materials for specific steps);

The fifth step is lighting. The illumination group was exposed to LED light at 660nm and 2mW/cm ² for 10 seconds, and then cultured in the dark. Taken at 0, 2, 4, 6, 12, 24, and 48 hours after illumination. Samples were taken, and the removed cell supernatants were stored at -20°C for unified testing;

The results show that the optimal detection time for mammalian REDLIP device/system reporter gene expression is 24 hours after illumination. The experimental data are detailed in Figure 9. All data are presented in the form of n=3 independent replication experiments.

Example 9 is a study on the spectral specificity of the REDLIP device/system for regulating transcriptional activation by red light in mammals.

This example uses SEAP as a detection reporter gene to verify the activation and shutdown gene expression characteristics of the mammalian REDLIP device/system, but does not limit the scope of the present invention. Specific steps are as follows:

The second step is to connect the board. Inoculate HEK-293T cells in 24-well plates, a total of 5 cells;

The third step is transfection. 16-24h after inoculating the cells, pYZ430 (P _5×UAS -SEAP-pA; P _5×UAS , 5×UAS-P _TATA ), pNX12 (P _hCMV -2NLS-LDB3-p65-HSF1-pA) was added to each group. ), and plasmid vectors containing different versions of the red light-sensitive protein pQL325(P _hCMV -Gal4-PnBphP-pA)(Pn-REDLIP)/pQL326(P _hCMV -Gal4-FnBphP-pA)(Fn-REDLIP), with 1: Use PEI transfection reagent in a ratio of 2:2 (w/w/w) for transfection (see method materials for specific steps);

The fifth step is lighting. Number the six 24-well plates (respectively 1, 2, 3, 4, 5). Place plate 1 under the 365nm LED, plate 2 under the 465nm LED, and plate 3 under the 530nm LED. Under the LED, plate No. 4 was placed under the 660nm LED, and plate No. 5 was placed under the 780nm LED. The illumination time was 10s. After the illumination treatment, it was placed in dark conditions for culture.

The results show that the mammalian REDLIP device/system only turns on the transcription of the reporter gene under 660nm red light irradiation, with good spectral specificity. The experimental data are detailed in Figure 10. All data are presented in the form of n=3 independent replicate experiments.

Example 10 is a study of the red light-regulated transcriptional activation REDLIP device/system in mammals to turn on/off the transcriptional expression of reporter genes.

This example uses SEAP as the detection reporter gene to verify that the mammalian REDLIP device/system is good. switching characteristics, but does not limit the scope of the present invention. Specific steps are as follows:

The first step is plasmid construction. The plasmid construction in this example is detailed in Table 1.

The second step is to connect the board. HEK-293T cells were seeded in 24-well plates. 3 pieces in total;

The third step is transfection. 16-24h after inoculating cells, pYZ430 (P _5×UAS -SEAP-pA; P _5×UAS ,5×UAS-P _TATA ), pNX12 (P _hCMV -2NLS-LDB3-p65-HSF1-pA), and Plasmid vectors of different versions of the red light-sensitive protein pQL325 (P _hCMV -Gal4-PnBphP-pA) (Pn-REDLIP)/pQL326 (P _hCMV -Gal4-FnBphP-pA) (Fn-REDLIP), in a 1:2:2 ( w/w/w) ratio of PEI transfection reagent for transfection (see method materials for specific steps);

The fifth step is lighting. The first group was placed under a 660nm LED with a light intensity of 2mW/cm ² for 10 seconds. After the second group was illuminated with a 660nm LED for 10 seconds, it was immediately illuminated with a 780nm LED with a light intensity of 2mW/cm ² for 2 minutes. The dark group was kept under Culture in dark conditions;

The results show that the mammalian REDLIP device/system can turn off the transcription of the reporter gene under 780nm far-red light irradiation after turning on the transcription of the reporter gene under 660nm red light irradiation. The system has sensitive switching characteristics. The experimental data are detailed in Figure 11. All data are presented in the form of n=3 independent replication experiments.

Example 11 is a study on the effect of mammalian red light-regulated transcriptional activation REDLIP device/system on activating reporter gene expression in different mammalian cell lines.

This example uses SEAP as a detection reporter gene to verify the effect of the mammalian REDLIP device/system in activating gene transcription expression in any mammalian cell line, but does not limit the scope of the present invention. Specific steps are as follows:

The second step is to connect the board. HEK-293T, hMSC-TERT, Hana3A, ATDC5, and HeLa cells were inoculated into 24-well plates and divided into light groups and dark groups;

The third step is transfection. 16-24h after inoculating cells, pYZ430 (P _5×UAS -SEAP-pA; P _5×UAS ,5×UAS-P _TATA ), pNX12 (P _hCMV -2NLS-LDB3-p65-HSF1-pA), and Different versions of red light sensitive protein pQL325(P _hCMV -Gal4-PnBphP-pA)(Pn-REDLIP)/pQL326(P _hCMV -Gal4- The plasmid vector of FnBphP-pA) (Fn-REDLIP) was transfected with PEI transfection reagent/lipofectamine Lip3000 (ATDC5) in a ratio of 1:2:2 (w/w/w) (see method materials for specific steps) ;

The fifth step is lighting. The light group was exposed to LED light at 660 nm and a light intensity of 2 mW/cm ² for 10 seconds. After the light treatment, it was placed in dark conditions for culture. The dark group was always cultured under dark conditions;

The results show that the mammalian REDLIP device/system can activate the expression of reporter genes in different mammalian cell lines and is universal. The experimental data are detailed in Figure 12. All data are presented in the form of n=3 independent replication experiments.

Example 12 is a study on the reversibility of red light-regulated transcriptional activation REDLIP device in mammals

This example uses SEAP as the detection reporter gene to verify that mammalian REDLIP activates the expression of the reporter gene in mammalian cells reversibly, but does not limit the scope of the present invention. Specific steps are as follows:

The second step is to connect the board. Inoculate HEK-293T in a 24-well plate.

The fourth step is to change the fluid. 6 hours after transfection, use a pipette tip to aspirate the cell culture medium under green light, and replace it with 500 μL of fresh DMEM (containing fetal bovine serum and penicillin/streptomycin).

The fifth step is lighting. The first group was placed under an LED with a wavelength of 660 nm and a light intensity of 1.5 mW/cm ² for 2 seconds. After the illumination ended, it was immediately placed in a dark place and cultured. After 48 hours, the light was continued for 3 seconds. The second group was first placed in a dark place and cultured for 24 hours. Illuminate for 3 seconds, then place in dark place for culture.

The results show that the mammalian REDLIP device activates gene transcription expression under 660nm red light irradiation. After being irradiated with 660nm red light again, gene transcription expression can still be activated, with good adjustability and sensitivity. The experimental data are detailed in Figure 13. All data are presented in the form of n=3 independent replication experiments.

Example 13 is a study on the spatial specificity of red light-regulated transcriptional activation REDLIP device/system in mammals

This example uses EGFP as a detection reporter gene to verify that the mammalian REDLIP device/system has spatial specificity for red light induction, but does not limit the scope of protection of the present invention. Specific steps are as follows:

The second step is to connect the board. Inoculate HEK-293T cells in a 10cm cell culture dish;

The third step is transfection. 16-24h after inoculating cells, pDQ63 (P _5×UAS -EGFP-pA; P _5×UAS ,5×UAS-P _hCMVmin ), pNX12 (P _hCMV -2NLS-LDB3-p65-HSF1-pA), and pA containing Different versions of red light sensitive protein pQL217(P _CMV -Gal4-DrBphP-pA)(Dr-REDLIP)/pQL325(P _hCMV -Gal4-PnBphP-pA)(Pn-REDLIP)/pQL326(P _hCMV -Gal4-FnBphP-pA ) (Fn-REDLIP) plasmid vector, transfected with PEI transfection reagent in a ratio of 1:2:2 (w/w/w) (see method materials for specific steps);

The fourth step is to change the fluid. 6 hours after transfection, use a pipette tip to aspirate the cell culture medium under green light, and replace it with 10 mL of fresh DMEM (containing fetal bovine serum and penicillin/streptomycin) medium;

The fifth step is lighting. Place the cell culture dish on the smartphone screen. The screen will display the pre-made picture with red letters on a black background. After 10 minutes, place it in dark conditions for culture;

The sixth step is to detect the reporter gene. The expression of green fluorescent protein EGFP is imaged by a fluorescence imager;

The results show that the mammalian REDLIP device/system induces EGFP to express the same letters as the pictures on the mobile phone, indicating good spatial specificity. The experimental data are detailed in Figure 14.

Example 14 is a study on the relationship between the endogenous gene activation effect of the mammalian red light-regulated transcriptional activation REDLIP _Cas device/system and the light intensity.

This example uses RHOXF2 as the target endogenous gene for activation to verify the mammalian REDLIP _Cas device/system using CRISPR-dCas9 to activate endogenous gene expression under different light intensities, but it does not limit the scope of protection of the present invention. Specific steps are as follows:

The third step is transfection. 16-24h after the cells were inoculated, pDQ100 (P _5×UAS -MS2-p65-HSF1-pA; P _5×UAS , 5×UAS-P _hCMVmin ), pWS69 (P _hCMV -dCas9-pA) was added to each group. pWS105 (P _U6 -sgRNA1 _RHOXF2 -pA) and pWS106 (P _U6 -sgRNA2 _RHOXF2 -pA) pNX12 (P _hCMV -2NLS-LDB3-p65-HSF1-pA), as well as pQL325 (P _hCMV - Plasmid vector of Gal4-PnBphP-pA)(Pn-REDLIP)/pQL326(P _hCMV -Gal4-FnBphP-pA)(Fn-REDLIP), with 1:10:5:5:15:15(w/w/w ) were transfected using PEI transfection reagent (see method materials for specific steps).

The fifth step is lighting. Number 8 24-well plates (respectively 1, 2, 3, 4, 5, 6, 7, 8). Plate No. 1 is placed in dark conditions for culture, and plates No. 2 to No. 8 use 660nm LEDs with different LEDs. Light intensity (0.05, 0.1, 0.25, 0.5, 0.75, 1, 1.5mW/cm ² ) was used for 10 seconds. After the light treatment, it was placed in dark conditions for cultivation;

The sixth step is to detect the reporter gene. Extract RNA and detect endogenous gene activation by RT-qPCR (see method materials for specific steps);

The results show that the activation of endogenous genes by the mammalian REDLIP _Cas device/system is dependent on light intensity. The experimental data are detailed in Figure 16. All data are presented in the form of n=3 independent replication experiments.

Example 15 is a study on the relationship between the endogenous gene activation effect of the mammalian red light-regulated transcriptional activation REDLIP _Cas device/system and the illumination time.

This example uses RHOXF2 as the target endogenous gene for activation to verify the activation of endogenous gene expression by the mammalian REDLIP device/system under different illumination times, but does not limit the scope of the present invention. Specific steps are as follows:

The second step is to connect the board. Inoculate HEK-293T cells in 24-well plates, a total of 7 plates;

The third step is transfection. 16-24h after inoculating the cells, pDQ100 (P _5×UAS -MS2- p65-HSF1-pA; P5×UAS, 5×UAS-P _hCMVmin ), pWS69 (P _hCMV -dCas9-pA), pWS105 (P _U6 -sgRNA1 _RHOXF2 -pA) and pWS106 (P _U6 -sgRNA2 _RHOXF2 -pA) pNX12 (P _hCMV -2NLS-LDB3-p65-HSF1-pA), and pQL325 (P _hCMV -Gal4-FnBphP-pA) (Pn-REDLIP)/pQL326 (P _hCMV -Gal4-FnBphP-pA) containing different versions of the red light-sensitive protein ) (Fn-REDLIP) plasmid vector, transfected with PEI transfection reagent at a ratio of 1:10:5:5:15:15 (w/w/w) (see method materials for specific steps);

The fifth step is lighting. Number 7 24-well plates (respectively 1, 2, 3, 4, 5, 6, and 7). Plate No. 1 is placed in dark conditions for culture. Plates No. 2 to No. 7 use 660nm respectively, and the light intensity is 2mW/cm. ² LEDs were illuminated for different times (1s, 3s, 5s, 10s, 1h, 12h). After the illumination treatment, they were placed in dark conditions for cultivation;

The results show that the activation of endogenous genes by the mammalian REDLIP _Cas device/system is dependent on light time. The experimental data are detailed in Figure 17. All data are presented in the form of n=3 independent replication experiments.

Example 16 is a study on the adjustability of mammalian red light-regulated transcriptional activation REDLIP _Cas device/system to activate endogenous genes.

This example uses RHOXF2 as the target endogenous gene for activation to verify that the mammalian REDLIP _Cas device/system uses CRISPR-dCas9 to turn on and off endogenous gene expression under irradiation of 660nm red light and 780nm far-red light, but it is not applicable to the present invention. The scope of protection is limited. Specific steps are as follows:

The third step is transfection. 16-24h after the cells were inoculated, pDQ100 (P _5×UAS -MS2-p65-HSF1-pA; P _5×UAS , 5×UAS-P _hCMVmin ), pWS69 (P _hCMV -dCas9-pA) was added to each group. pWS105 (P _U6 -sgRNA1 _RHOXF2 -pA) and pWS106 (P _U6 -sgRNA2 _RHOXF2 -pA), pNX12 (P _hCMV -2NLS-LDB3-p65-HSF1-pA), and pQL325 (P _hCMV ) containing different versions of the red light-sensitive protein -Gal4-PnBphP-pA)(Pn-REDLIP)/pQL326(P _hCMV -Gal4-FnBphP-pA)(Fn-REDLIP) The vector was transfected with PEI transfection reagent at a ratio of 1:100:50:50:150:150 (w/w/w) (see method materials for specific steps);

The fifth step is lighting. The first group was sampled before illumination, the second group was illuminated with 660nm LED with a light intensity of 0.1mW/cm ² 660nm for 1s, and cultured in dark conditions for 24 hours before sampling, and the third group was illuminated with 0.1mW/cm ² 660nm for 1s and placed in the dark. Samples were taken after culturing in dark conditions for 48 hours. The third group was cultured in dark conditions for 48 hours and then illuminated with 660 nm light for 1 second. Samples were taken after 24 hours (72 hours);

The results show that the activation of endogenous genes by the mammalian REDLIP _Cas device/system has good tunability at 660nm red light. The experimental data are detailed in Figure 18. All data are presented in the form of n=3 independent replication experiments.

Example 17 is a study on the effect of mammalian red light-regulated transcriptional activation REDLIP _Cas device/system on activating endogenous genes in different mammalian cell lines.

This example uses RHOXF2 as the target endogenous gene to activate to verify the effect of the mammalian REDLIP device/system in activating endogenous gene expression in different mammalian cells, but does not limit the scope of the present invention. Specific steps are as follows:

The second step is to connect the board. Inoculate HeLa, hMSC-TERT, and Hana3A cells in 24-well plates;

The third step is transfection. 16-24h after the cells were inoculated, pDQ100 (P _5×UAS- MS2-p65-HSF1-pA; P5×UAS, 5×UAS-P _hCMVmin ), pWS69 (P _hCMV -dCas9-pA), pWS105 was added to each group. (P _U6 -sgRNA1 _RHOXF2 -pA), pWS106 (P _U6 -sgRNA2 _RHOXF2 -pA), pNX12 (P _hCMV -2NLS-LDB3-p65-HSF1-pA), and pQL325 (P _hCMV - Plasmid vector of Gal4-PnBphP-pA)(Pn-REDLIP)/pQL326(P _hCMV -Gal4-FnBphP-pA)(Fn-REDLIP), with 1:10:5:5:15:15(w/w/w ) were transfected using PEI transfection reagent/lipofectamine Lip3000 (see method materials for specific steps).

The fifth step is lighting. (The specific steps are the same as Embodiment 11 of the present invention);

The results show that the mammalian REDLIP _Cas device/system uses CRISPR-dCas9 to activate endogenous genes and is universal in different mammalian cell lines. The experimental data are detailed in Figure 19. All data are presented in the form of n=3 independent replication experiments.

Example 18 is a study on the effect of mammalian red light-regulated transcriptional activation REDLIP _Cas device/system on activating endogenous genes in different mammalian cell lines.

This example selects four different endogenous genes, ASCL1, TTN, IL1RN, and MIAT, as target activation genes to verify the activation effect of the mammalian REDLIP device/system on different endogenous genes, but does not limit the scope of protection of the present invention. Specific steps are as follows:

The second step is to connect the board. Inoculate HEK-293T cells in a 24-well plate;

The third step is transfection. 16-24h after the cells were inoculated, pDQ100 (P _5×UAS -MS2-p65-HSF1-pA; P _5×UAS , 5×UAS-P _hCMVmin ), pWS69 (P _hCMV -dCas9-pA) was added to each group. pSZ83(P _U6 -sgRNA1 _ASCL1 -pA) and pSZ84(P _U6 -sgRNA2 _ASCL1 -pA)/pSZ92(P _U6 -sgRNA1 _IL1RN -pA) and pSZ93(P _U6 -sgRNA2 _IL1RN -pA)/pSZ103(P _U6 -sgRNA1 _TTN -pA) and pSZ104 (P _U6 -sgRNA2 _TTN -pA)/pYZ417 (P _U6 -sgRNA1 _MIAT -pA) and pYZ418 (P _U6 -sgRNA2 _MIAT -pA), pNX12 (P _hCMV -2NLS-LDB3-p65-HSF1 -pA), and plasmid vectors containing different versions of the red light-sensitive protein pQL325 (P _hCMV -Gal4-PnBphP-pA) (Pn-REDLIP)/pQL326 (P _hCMV -Gal4-FnBphP-pA) (Fn-REDLIP), with 1 :10:5:5:15:15 (w/w/w) ratio PEI transfection reagent for transfection (see method materials for specific steps);

The results show that the mammalian REDLIP _Cas device/system can activate different endogenous genes. The experimental data are detailed in Figure 20. All data are presented in the form of n=3 independent replication experiments.

Example 19 is a study on the relationship between activation of exogenous gene expression and light intensity by mammalian Dr-REDLIP device/system in mouse liver.

This example uses Luciferase as the exogenous gene activated in the mouse liver to verify the relationship between the mammalian Dr-REDLIP device/system activating the expression of exogenous genes in mice and light intensity, but it does not limit the scope of protection of the present invention. restricted. Specific steps are as follows:

In the second step, the plasmid DNA solution was injected into the mouse liver through the tail vein. Mixed pYZ450 (P _5×UAS -Luciferase-pA; P _5×UAS ,5×UAS-P _TATA ), pQL326 (P _hCMV -3NLS-LDB3-p65-HSF1-pA), pQL217 (P _hCMV -Gal4-PnBphP- pA) (Dr-REDLIP) plasmid vector, mix the plasmid at a ratio of 1:2:2 (w/w/w) and inject the plasmid into the liver cells of mice through the tail vein using a hydrodynamic method (see method materials for specific steps);

The third step is lighting. 16 hours after the plasmid was delivered to the hydrodynamic liver, the mice were divided into 5 groups (numbered 1, 2, 3, 4, and 5) with illumination intensities of 0, 1, 5, 10, and 20 mW/cm ² respectively, and the abdomen of the mice was illuminated for a period of time It is 1h; after 8h, the expression effect of activated genes in the liver is detected;

The fourth step is to detect the reporter gene. Detect the expression of reporter gene luciferase in mouse liver (see method materials for specific steps);

The results showed that the mammalian Dr-REDLIP device/system activated the expression of exogenous genes in the liver of mice in a light intensity-dependent manner. The experimental data are detailed in Figure 22. All data are presented in the form of n=4 independent replicate experiments.

Example 20 is a study on the relationship between the mammalian red light-regulated transcriptional activation REDLIP device/system activating exogenous gene expression in mouse liver and illumination time.

This example uses Luciferase as the exogenous gene activated in the mouse liver to verify the expression of exogenous genes in the mouse liver by different red light-sensitive proteins of the mammalian REDLIP device/system, but does not limit the scope of the present invention. . Specific steps are as follows:

In the second step, the plasmid DNA solution was injected into the mouse liver through the tail vein. Mixed pYZ450 (P _5× _UAS -Luciferase-pA; P _5×UAS ,5×UAS-P _TATA ), pQL236 (P _hCMV -3NLS-LDB3-p65-HSF1- pA), and pQL217(P _hCMV -Gal4-DrBphP-pA)(Dr-REDLIP), pQL325(P _hCMV -Gal4-PnBphP-pA)(Pn-REDLIP)/pQL326(P _hCMV - Gal4-FnBphP-pA) (Fn-REDLIP) plasmid vector was mixed at a ratio of 1:2:2 (w/w/w) and injected into the liver cells of mice through the tail vein using hydrodynamics (for specific steps, see Methods Material);

The third step is lighting. 16 hours after the plasmid was delivered to the hydrodynamic liver, the mice were divided into 5 groups (numbered 1, 2, 3, 4, and 5), in which the illumination times of the Dr-REDLIP device/system were 0, 5, 30, 60, and 120 min respectively. , the illumination times of Pn-REDLIP and Fn-REDLIP devices/systems were 0, 1, 5, 30 and 60 minutes respectively; the expression effect of activated genes in the liver was detected after 8 hours;

The results show that the mammalian REDLIP device/system activates the expression of exogenous genes in mouse liver tissue in a light time-dependent manner, and FnBphP (Fn-REDLIP) activates gene expression more quickly and efficiently. The experimental data are detailed in Figures 23-24. All data are presented in the form of n=4 independent replication experiments.

Example 21 is a study on long-term stable expression of mammalian red light-regulated transcriptional activation Fn-REDLIP device/system engineered AAV adeno-associated virus in mice.

This example uses Luciferase as a reporter gene to detect long-term expression of AAV to verify the possibility of Fn-REDLIP device/system engineering AAV to achieve long-term gene therapy in vivo, but does not limit the scope of protection of the present invention. Specific steps are as follows:

The second step is to package it into an AAV virus engineered with Fn-REDLIP device/system. The AAV vector plasmid contains pQL271 (ITR-P _5×UAS -Luciferase-P2A-Insulin-pA-ITR P _5×UAS ,5×UAS-P _TATA ), pQL383 (ITR-P _EMS -3NLS-LDB3-p65-HSF1- pA), pQL382 (ITR-P _EMS -Gal4-FnBphP-pA-ITR), the company completes the packaging of the AAV virus;

The third step is intramuscular injection of engineered AAV. The amount of virus added to the above three AAVs was 2×10 ¹¹ vg per animal, mixed in a ratio of 1:1:1, and injected into the calf muscles of mice using the three-point method;

The fourth step is lighting. Lighting was performed two weeks after AAV intramuscular injection. The light group was exposed to light for 30 minutes every week, and the expression of the reporter gene was detected immediately after 8 hours. The dark group was kept in a normal feeding environment;

The fifth step is to detect the reporter gene. Detect the expression of Luciferase in mouse muscle tissue (for specific steps, see methods and materials).

The results show that AAV viruses engineered by the Fn-REDLIP device/system can be produced and expressed stably in mice for a long time. The experimental data are detailed in Figures 26-27. All data are presented in the form of n=5 independent replication experiments.

Example 22 is a study on the gene therapy effect of mammalian red light-regulated transcriptional activation Fn-REDLIP device/system engineered AAV (three viruses) on type Ⅰ diabetes

This example uses Insulin as a therapeutic protein to verify the gene therapy effect of the Fn-REDLIP device/system on type 1 diabetes using triviral AAV, but it does not limit the scope of the present invention. Specific steps are as follows:

The second step is to package it into an AAV virus engineered with Fn-REDLIP device/system. AAV vector plasmid contains pQL271 (ITR-P _5×UAS -Luciferase-P2A-Insulin-pA-ITR P _5×UAS ,5×UAS-P _TATA ), pNX250 (ITR-P _EMS -3NLS-LDB3-p65-HSF1- pA), pQL382 (ITR-P _EMS -Gal4-FnBphP-pA-ITR), the company completes the packaging of the AAV virus;

The third step is intramuscular injection of engineered AAV. The amount of virus added to the above three AAVs was 2×10 ¹¹ vg per animal, mixed at a ratio of 1:1:1, and injected into the calf muscles of mice using the three-point method;

The fourth step is lighting. Lighting was performed two weeks after AAV intramuscular injection. The light group was exposed to light for 1 hour every 5 days (30 minutes in the morning and 30 minutes in the evening on the same day, and the test was conducted the next day). The dark group was kept in a normal feeding environment;

The fifth step is to detect the reporter gene. Test the blood glucose level of mice using a portable blood glucose monitor;

The results show that the AAV virus engineered by the Fn-REDLIP device/system achieves effective gene therapy for type 1 diabetes, and the blood sugar of mice can be maintained at normal and stable values for a long time. The experimental data are detailed in Figure 28. All data are presented in the form of n=5 independent replication experiments.

Example 23 is a study on the gene therapy effect of mammalian red light-regulated transcriptional activation Fn-REDLIP device/system engineered AAV (two viruses) on type Ⅰ diabetes

This example uses Insulin as a therapeutic protein to verify the gene therapy effect of the Fn-REDLIP device/system on type 1 diabetes using two viral AAVs, but does not limit the scope of the present invention. Specific steps are as follows:

The second step is to package it into an engineered AAV virus containing Fn-REDLIP device/system. The AAV vector plasmid contains pQL388 (ITR-P _EMS -3NLS-LDB3-p65-HSF1-pA P _5×UAS -EGFP-P2A-Insulin-pA-ITR P _5×UAS ,5×UAS-P _TATA ), pQL382 (ITR -P _EMS -Gal4-FnBphP-pA-ITR), the company completes the packaging of the AAV virus;

The third step is intramuscular injection of engineered AAV. The amount of virus added to the above two AAVs was 2×10 ¹¹ vg each, mixed at a ratio of 1:1, and injected into the calf muscles of mice using the three-point method;

The fourth step is lighting. Lighting was performed two weeks after AAV intramuscular injection. The light group was exposed to light for 1 hour every day (30 minutes in the morning and 30 minutes in the evening on the same day, and the test was conducted the next day). The dark group was kept in a normal feeding environment;

The fifth step is to detect the reporter gene. A portable blood glucose monitor was used to detect the blood glucose level of mice, and the serum obtained after taking blood from the orbit was used to detect the Insulin content in the serum using an Elisa kit;

The results show that the AAV virus engineered by the Fn-REDLIP device/system achieves effective gene therapy for type 1 diabetes. The blood sugar of mice can be maintained at a normal and stable value for a long time, and the expression of insulin also reaches a level close to normal. The experimental data are detailed in Figures 30-31. All data are presented in the form of n=6 independent replication experiments.

Example 24 is the study of the mammalian red light-regulated transcriptional activation Fn-REDLIP device/system engineered AAV (two viruses) for gene therapy of obesity diseases.

This example uses the anti-obesity cytokine TSLP as a therapeutic protein to verify the effect of the v device/system on obesity disease gene therapy delivered by two viral AAVs, but does not limit the scope of the present invention. Specific steps are as follows:

The second step is to package it into an AAV virus engineered with Fn-REDLIP device/system. The AAV vector plasmid contains pQL383 (ITR-P _EMS -3NLS-LDB3-p65-HSF1-pA P _5×UAS -TSLP-pA-ITR P _5×UAS ,5×UAS-P _TATA ) and pQL382 (ITR-P _hCMV - Gal4-FnBphP-pA-ITR), the company completes the packaging of the AAV virus;

The third step is intramuscular injection of engineered AAV. The amount of virus added to the above two AAVs was 2×10 ¹¹ vg each, mixed at a ratio of 1:1, and injected into the calf muscles of mice using the three-point method, which also included muscle. Flesh-injected nonsense AAV virus group, obese HFD group and wild-type WT group;

The fourth step is lighting. Lighting was performed two weeks after intramuscular injection of AAV. The light group was exposed to light for 30 minutes every day, while the dark group was kept in a normal feeding environment;

The fifth step is to detect the reporter gene. Monitor changes in mouse body weight;

The results showed significant weight loss during gene therapy for obesity diseases with AAV viruses engineered by the Fn-REDLIP device/system. The experimental data are detailed in Figure 33. All data are presented in the form of n=6 independent replication experiments.

Example 25 is a study on the mechanism of mammalian red light-regulated transcriptional activation Fn-REDLIP device/system engineered AAV (two viruses) for gene therapy of obesity diseases.

This example uses the same method as Example 25 to measure the weight of white fat, beige fat and brown fat in mice after undergoing Fn-REDLIP device/system engineering AAV gene therapy, and measure triglycerides in serum and liver tissue. content to verify the mechanism of Fn-REDLIP device/system engineered AAV gene therapy for obesity, but does not limit the scope of protection of the present invention. Specific steps are as follows:

The first step is to obtain mouse serum. After 6 weeks of treatment, serum was obtained from mice in each group by taking blood from the orbit;

The second step is to detect the triglyceride content in serum and liver tissue;

The third step is to separate the beige fat, white fat, and brown fat of the mouse and weigh them separately;

The results showed that after gene therapy for obesity diseases by the Fn-REDLIP device/system, all adipose tissues in the mice were reduced, and the triglyceride content in the serum and liver was significantly reduced. The experimental data are detailed in Figures 34-36. All data are presented in the form of n=8 independent replication experiments.

Table 1 Related plasmid information

Table 2 qPCR related primer information

SEQ ID NO.1: Amino acid sequence of red light photosensitive protein DrBphP

SEQ ID NO.2: Amino acid sequence of red light photosensitive protein Pn BphP

SEQ ID NO.3: Amino acid sequence of red light photosensitive protein Fn BphP

SEQ ID NO.4: Amino acid sequence of Gal4

SEQ ID NO.5: Amino acid sequence of the connecting peptide between red light photosensitive protein and Gal4

SEQ ID NO.6: Amino acid sequence of nanochaperone protein LDB3

SEQ ID NO.7: Amino acid sequence of nanochaperone protein LDB14

SEQ ID NO.8: Amino acid sequence of transcription activator VP64

SEQ ID NO.9: Amino acid sequence of transcription activator VP16

SEQ ID NO.10: Amino acid sequence of transcription activator p65

SEQ ID NO 11: Amino acid sequence of transcription activator VPR

SEQ ID NO.12: Amino acid sequence of transcription activator p65-HSF1

SEQ ID NO.13: Amino acid sequence of N-terminal nuclear import signal NLS of LDB3 nanochaperone protein

SEQ ID NO.14: Amino acid sequence of the connecting peptide between LDB3 and the transcription activator

SEQ ID NO.15: Nucleotide sequence of inducible promoter P _5×UAS (hCMVmin)

SEQ ID NO.16: Nucleotide sequence of inducible promoter P _5×UAS (TATA)

SEQ ID NO.17: Amino acid sequence of alkaline phosphatase SEAP

SEQ ID NO.18: Amino acid sequence of green fluorescent protein EGFP

SEQ ID NO.19: Amino acid sequence of reporter gene Luciferase

SEQ ID NO.20: Amino acid sequence of therapeutic protein Insulin

SEQ ID NO.21: Amino acid sequence of therapeutic protein TSLP

SEQ ID NO.22-23: Nucleotide sequence of RHOXF2 gRNA

SEQ ID NO.24-25: Nucleotide sequence of ASCL1 gRNA

SEQ ID NO.26-27: Nucleotide sequence of IL1RN gRNA

SEQ ID NO.28-29: Nucleotide sequence of TTN gRNA

SEQ ID NO.30-31: Nucleotide sequence of MIAT gRNA

SEQ ID NO.32-33: Nucleotide sequence of Ascl1 gRNA

The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that those skilled in the art can think of are included in the present invention, and are protected by the appended claims.

Claims

A mammal red light regulated transcription activation device/system, characterized by comprising a red light sensing element, a transcription activating element and a response element.
The mammalian red light regulated transcription activation device/system according to claim 1, wherein the red light sensing element includes: bacterial-derived red light photosensitive proteins DrBphP, PnBphP, FnBphP, and Gal4 containing a DNA binding domain. proteins and fusion peptides between two proteins.
The mammalian red light-regulated transcription activation device/system according to claim 1, wherein the transcription activation element includes: nanochaperone proteins LDB3, LDB14, transcription activators and LDB3/ Linking peptide between LDB14 and transcriptional activators.
The mammalian red light regulated transcription activation device/system according to claim 1, wherein the response element includes: an inducible promoter and a target gene.
The mammalian red light regulated transcription activation device/system according to claim 4, wherein the inducible promoter includes: an operator and an inducible weak promoter; the target gene can be any protein of interest. gene sequence.
The mammalian red light regulated transcription activation device/system according to claim 2, wherein the amino acid sequence of the bacterial-derived red light photosensitive protein DrBphP is shown in SEQ ID NO.1; the bacterial-derived red light-sensitive protein DrBphP The photosensitive protein PnBphP is the NTE domain of the DrBphP protein fused to the N-terminus of the Arabidopsis red photoprotein PhyA, and its amino acid sequence is shown in SEQ ID NO.2; the bacterial-derived red photosensitive protein FnBphP is the N-terminal fusion of the DrBphP protein The NTE domain of fungal red light protein FphA, its amino acid sequence is shown in SEQ ID NO.3.
The mammalian red light regulated transcription activation device/system according to claim 2, wherein the Gal4 is a protein that can bind to a specific DNA sequence, and its amino acid sequence is as shown in SEQ ID NO.4; wherein, the Gal4 The amino acid sequence of the connecting peptide between Gal4 and the bacterial-derived red light photosensitive protein is shown in SEQ ID NO. 5.
The mammalian red light-regulated transcription activation device/system according to claim 3, wherein the nanochaperones LDB3 and LDB14 can interact with the bacterial-derived red light photosensitive proteins DrBphP, PnBphP, and FnBphP, and The amino acid sequence is shown in SEQ ID NO.6-7.
The mammalian red light-regulated transcription activation device/system according to claim 3, wherein the transcription activator has the function of recruiting RNA polymerase, including VP64, VP16, p65, VPR, p65-HSF1, and the amino acid sequence As shown in SEQ ID NO.8-12.
The mammalian red light regulated transcription activation device/system according to claim 3, characterized in that, The N-terminal fusion of the LDB3 nanochaperone protein expresses nuclear entry signal NLS with different copy numbers, and the amino acid sequence of the NLS is shown in SEQ ID NO. 13; wherein, the connection between the nanochaperone protein and the transcription activator The peptide has an amino acid sequence as shown in SEQ ID NO. 14.
The mammalian red light regulated transcription activation device/system according to claim 5, wherein the inducible promoters are P 5×UAS- (P hCMVmin ) and P 5×UAS- (P TATA ), Its nucleotide sequence is shown in SEQ ID NO. 15-16; wherein, the inducible promoter cannot initiate the transcription expression of downstream genes without a transcription activator to recruit RNA polymerase.
The mammalian red light-regulated transcription activation device/system according to claim 4, wherein the response element can be the gene sequence of any meaningful protein, including SEAP, EGFP and Luciferase reporter genes, whose amino acid sequences are respectively as follows SEQ ID NO.17-19 is shown; the gene therapy drug proteins Insulin and TSLP have their amino acid sequences as shown in SEQ ID NO.20-21.
The mammalian red light regulated transcription activation device/system according to claim 1, characterized in that the device/system can induce and activate the expression of the target gene by red light with a wavelength of 660±10nm, and the expression of the target gene can be induced by irradiation of far-red light of 780nm. down to turn off gene transcription.
A method for constructing a mammalian red light-regulated transcription activation device/system, which is characterized by including the following steps:

(1) Construct a red light sensing element: The red light sensing element includes red light sensitive proteins DrBphP, PnBphP, and FnBphP containing different structural domains, whose amino acid sequences are shown in SEQ ID NO.1-3, and contains DNA binding The Gal4 domain has an amino acid sequence as SEQ ID NO.4, and a fusion protein is formed between the two through a connecting peptide. The amino acid sequence of the connecting peptide is as shown in SEQ ID NO.5;

(2) Construct a transcriptional activation element: The transcriptional activation element includes the nanochaperone protein LDB3 that interacts with the red light photosensitive protein. Its amino acid sequence is shown in SEQ ID NO. 6, in which the N-terminal fusion of LDB3 expresses two copy numbers. The nuclear entry signal is 2×NLS. The amino acid sequence of NLS is shown in SEQ ID NO.13. and p65-HSF1, a transcriptional activator that recruits RNA polymerase. Its amino acid sequence is shown in SEQ ID NO.12. A fusion protein is formed between the two through a connecting peptide. The amino acid sequence of the connecting peptide is shown in SEQ ID NO.14. ;

(3) Construct a response element: the response element is an inducible promoter P 5×UAS -P TATA , whose nucleotide sequence is shown in SEQ ID NO. 16, and the target gene, whose amino acid sequence is shown in SEQ ID NO. As shown in 17-21.
The method of claim 14, wherein in step (1), the red light photosensitive egg White DrBphP, PnBphP, and FnBphP can undergo conformational changes in response to 660nm red light; in step (2), the nanochaperone protein LDB3 and the transcription activator p65-HSF1 fusion protein can react with red light that undergoes conformational changes after being irradiated with 660nm red light. The inducible promoter described in step (3) of photosensitive protein binding to each other activates the expression of downstream target genes after p65-HSF1 recruits RNA polymerase.
The mammalian red light regulated transcription activation device/system constructed by the method of claim 14 or 15, wherein the red light photosensitive protein DrBphP is called the Dr-REDLIP device/system, and PnBphP is called the Pn-REDLIP device /system, FnBphP is called Pn-REDLIP device/system.
A eukaryotic expression vector and/or AAV expression vector and/or engineered mammalian cells and/or engineered AAV virus and/or system, characterized in that the eukaryotic expression vector and/or AAV expression vector and/or Or engineered mammalian cells and/or engineered AAV viruses and/or systems and/or kits, including the mammalian red light regulated transcription activation device/system according to any one of claims 1 to 13.
The mammalian red light regulated transcription activation device/system according to any one of claims 1 to 13 or the expression eukaryotic expression vector and/or AAV expression vector and/or engineered mammalian cells according to claim 17 and /or the use of engineered AAV viruses and/or systems in the preparation of engineered mammalian cells and/or the use of CRISPR-dCas9 to activate endogenous gene expression, engineered AAV viruses, and/or gene therapy kits for AAV delivery.
The application of claim 18, wherein the induced expression products include SEAP, EGFP, Luciferase, Insulin and TSLP, and the amino acid sequence of the expression product is as shown in SEQ ID NO. 17-21.
A method for regulating gene expression in host cells using the mammalian red light-regulated transcription activation device/system according to any one of claims 1 to 13, characterized in that the method includes the following steps:

a) Constructing the mammalian red light-regulated transcription activation device/system according to any one of claims 1 to 13 into a host cell eukaryotic plasmid expression vector and/or an AAV expression vector; b) Introducing the expression vector into the mammal In animal cells; c) By inducing the transcription and expression of the target gene in engineered mammalian cells with 660 nm red light, the mammalian red light regulated transcription activation device/system can induce the transcription expression of activated genes in mammalian cells.
A mammalian red light regulated transcription activation device/system REDLIP Cas , a method for inducing mammalian endogenous gene expression using CRISPR-dCas9, characterized in that the method includes: a) constructing a method containing any of claims 1 to 13 The mammalian red light regulated transcription activation device/system REDLIP Cas -related response element MS2-p65-HSF1 eukaryotic plasmid expression vector and/or AAV expression vector described in one item; b) Introducing the mammalian red light regulated transcription activation device/system REDLIP Cas plasmid expression vector as described in any one of claims 1 to 13 into the mammalian cells; c) Inducing and activating mammalian expression through red light irradiation The expression of the response element MS2-p65-HSF1 ultimately up-regulates the expression of endogenous genes.
A method for gene therapy using a mammalian red light regulated transcription activation device/system, characterized in that the method includes: a) constructing a mammalian red light regulated transcription activation device/system as described in any one of claims 1 to 13 An AAV expression plasmid vector of a transcription activation device/system; b) Preparing an engineered AAV virus comprising the mammalian red light regulated transcription activation device/system according to any one of claims 1 to 13, wherein the engineering AAV virus is prepared by Shanghai Teltu Biotechnology Co., Ltd.; c) Deliver the engineered AAV virus of the mammalian red light-regulated transcription activation device/system into the organism by intramuscular injection; d) Activate expression by red light Target genes and/or therapeutic proteins to achieve gene therapy for corresponding diseases.