Application of CRYAB in the treatment of STING-related diseases and aging

Abstract

The present application relates to the field of biomedical technology, and specifically to the use of CRYAB in the treatment of STING-related diseases and aging. The present application first discovered that CRYAB can inhibit STING signal activation in vitro and in vivo, and can be used as a STING inhibitor to treat STING activation-related diseases and aging.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to the use of CRYAB in preventing or treating STING-related diseases and / or aging. Background Art

[0002] cGAS-STING is an important innate immune signaling pathway in the body and a driving factor for aging-related chronic inflammation and a variety of age-related diseases. Inhibiting cGAS (Cyclic GMP-AMP synthase) or STING (Stimulator of interferon genes) in mice can delay aging caused by natural or DNA damage. In recent years, studies have shown that abnormal activation of the body's cGAS-STING pathway leads to chronic inflammation, and plays a key driving role in many diseases such as neuroinflammatory reactions, nephritis, and aging. Therefore, the development of specific inhibitors of the cGAS-STING signaling pathway has important theoretical significance and potential clinical application value for the treatment of STING-related inflammatory diseases and aging-related diseases, and also has potential application value in extending healthy life span. Currently, there are mainly two types of STIGN inhibitors: one is a covalent inhibitor that binds to the Cys88 or Cys91 residues in the transmembrane region of the STING protein. This type of inhibitor can block the palmitoylation of STING and thus inhibit the activation of STING (C176, C178, H151, etc.); the other is an inhibitor that targets the C-terminal ligand binding region of STING. This type of inhibitor can competitively bind to the endogenous ligand of STING (SN011, Compound 18, etc.).

[0003] Although some documents in the prior art mention that it is possible to kill senescent cells by inhibiting CRYAB (US20220241316A1), the research conclusion of this application is the opposite. This application found that administering protein CRYAB (rather than inhibition) can effectively inhibit the activation of STING signals in vivo and in vitro, and can be used as a STING inhibitor for the treatment of STING activation-related diseases and aging-related diseases.

[0004] In view of this, this application is proposed. Summary of the invention

[0005] To solve the above problems, this application discovered the relationship between CRYAB protein and cGAS-STING pathway for the first time through research. Knocking out cryab gene in human retinal pigment epithelial cell line ARPE-19 (ARPE) promotes the activation of STING in cells; exogenous supplementation of TAT-CRYAB recombinant protein or overexpression of cryab gene in cells can inhibit the activation and expression of STING; injection of TAT-CRYAB recombinant protein can prolong the survival rate of mice and inhibit tissue inflammatory response in TREX1 knockout mice with abnormal STING activation. The above series of results show that CRYAB can be used as an inhibitor of STING to treat STING activation-related diseases and aging-related diseases, and can also be used as a means to delay aging.

[0006] Based on the above research, this application specifically proposes the following technical solutions:

[0007] According to one aspect of this specification, the present application first provides various applications of CRYAB protein or gene, including:

[0008] 1) Use of CRYAB proteins or genes as STING inhibitors, or use of CRYAB proteins or genes in inhibiting STING activation and expression;

[0009] 2) Application of CRYAB proteins or genes in preventing or treating diseases related to STING activation;

[0010] 3) Application of CRYAB protein or gene in preventing or treating aging.

[0011] In some aspects, the STING activation-related diseases include: inflammatory diseases, autoimmune diseases, neurodegenerative diseases, and age-related macular degeneration.

[0012] Preferably, the inflammatory disease is inflammatory bowel disease and acute or chronic renal injury; the autoimmune disease is idiopathic STING-related vasculopathy, Aicardi-Goutières syndrome or systemic lupus erythematosus;

[0013] Preferably, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, ALS or multiple sclerosis.

[0014] In some aspects, the aging is physiological aging or pathological aging; preferably, the pathological aging is aging caused by abnormal activation of the cGAS-STING pathway.

[0015] In some embodiments, the CRYAB protein is a TAT-CRYAB protein, and the TAT amino acid sequence is YGRKKRRQRRR; preferably, the protein is expressed based on the non-endotoxin-producing plasmid Clear Coli BL21; preferably, the genotype of the plasmid is: F–ompT hsdSB(rB-mB-)gal dcm lonλ(DE3[lacI lacUV5-T7gene 1ind1 sam7 nin5])msbA148ΔgutQΔkdsDΔlpxLΔlpxMΔpagPΔlpxPΔeptA.

[0016] The present application also provides a pharmaceutical composition for preventing or treating STING-related diseases or aging, wherein the pharmaceutical composition comprises CRYAB protein or cryab gene; in particular, an effective dose of CRYAB protein or cryab gene.

[0017] In some aspects, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0018] In some embodiments, the CRYAB protein is a TAT-CRYAB protein, preferably, the TAT amino acid sequence is YGRKKRRQRRR; preferably, the protein is expressed based on the non-endotoxin-producing plasmid Clear Coli BL21; preferably, the genotype of the plasmid is: F–ompT hsdSB(rB-mB-)gal dcm lonλ(DE3[lacI lacUV5-T7gene 1ind1 sam7 nin5])msbA148ΔgutQΔkdsDΔlpxLΔlpxMΔpagPΔlpxPΔeptA.

[0019] The present application also provides a method for preventing or treating STING-related diseases or aging in vivo / in vitro, which includes the step of increasing the amount or activity of CRYAB protein in vivo / in vitro.

[0020] The present application also provides a method for regulating the STING pathway in vivo / in vitro, which comprises the step of regulating the amount or activity of CRYAB protein in vivo / in vitro.

[0021] In some aspects, the modulation is inhibition and the regulation is enhancement.

[0022] Compared with the prior art, this application has at least the following technical advantages:

[0023] The present invention discovered for the first time that CRYAB can effectively inhibit the activation and expression of STING signals in vivo and in vitro, and is expected to be used as a STING inhibitor to treat STING activation-related diseases and aging-related diseases; it is also expected to be used as a means to delay aging.

[0024] In addition, the present application constructs a recombinant protein TAT-CRYAB in one embodiment, which can promote CRYBA to enter cells; and the present application also uses an endotoxin-free plasmid to construct a recombinant TAT-CRYAB protein, which can improve protein safety and experimental reliability. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 , recombinant protein plasmid design and protein quantification results; A is the amino acid sequence of the TAT peptide and the plasmid diagram of TAT-CRYAB; B is the Coomassie brilliant blue staining results of different volumes of TAT-CRYAB protein and BSA standard.

[0027] Figure 2 , the results of knocking out CRYAB in ARPE cells promoting the expression and activation of STING; A is the detection of STING protein expression in Cryab knockout cells sgCryab1, sgCryab2 and control sg-CTRL; B and C are the detection of mRNA levels of inflammatory factors IL6 and type I interferon IFNβ in Cryab knockout cells after 6 hours of transfer of 5μg double-stranded DNA analogs poly(dT:dA) and 5μg double-stranded RNA analogs poly(I:C), respectively, and the control was lipo treatment, n=3, two-way ANOVA.

[0028] Figure 3, promoting overexpression of cryab gene to inhibit the activation and expression of STING in human ARPE cells and mouse BMDM cells; A is the protein detection result of ARPE19 cells stably expressing GFP and GFP-Cryab treated with 0.5μg normal DNA, 0.5μg DNA treated with hydrogen peroxide and 5μg cGAMP for 2 hours; BE is the result of ARPE19 cells stably expressing GFP and GFP-Cryab treated with 600μM hydrogen peroxide for 2 hours, and RNA was extracted for RT-QPCR detection of IL6, IL8, IL1β, and IFNβ, n=3 / group, two-way ANOVA; FH are heat maps of aging-related phenotypic genes and cGAS-STING-related genes sequenced by RNA sequencing of mouse BMDM cells transferred with GFP and GFP-Cryab (F); G and H are the results of GSEA enrichment analysis, showing the enrichment results of related sets of interferon response and interleukin 1 production, n=3 / group.

[0029] Figure 4 , schematic diagram of 293T cell processing and protein immunoblot detection results.

[0030] Figure 5 , the results of injecting TAT-CRYAB in the Trex1 knockout mouse model to treat inflammation and death caused by abnormal activation of STING; A is a schematic diagram of long-term injection of TAT-Cryab and ddH2O in Trex1 knockout mice; B is the survival rate of Trex1 after injection of TAT-CRYAB and ddH2O; C is the abdominal phenotype of Trex1 after injection of TAT-CRYAB and ddH2O on days 0, 14 and 35. D. Schematic diagram of short-term injection of AT-Cryab and ddH2O in Trex1 knockout mice; E is a comparison of HE staining of liver, heart, stomach and colon tissues of wild-type mice injected with ddH2O and TAT-Cryab, respectively. The white arrows indicate the location of immune cell infiltration, and the scale bar = 50μm.

[0031] Figure 6 , Cryab inhibits the results of mouse aging, among which, A is the result of whitening of abdominal hair in 12-month-old mice; B is the weight and hair color of mice at 1, 6 and 12 months of age; C is the statistics of spinal curvature in mice at 1, 6 and 12 months of age; D is the result of bone morphology and fat status of 12-month-old mice photographed by X-ray and microCT; E is the result of epidermal thickness statistics of mouse skin HE staining; F is the cell aging and cell proliferation status of mouse abdominal skin fibroblasts. DETAILED DESCRIPTION

[0032] Although the application can be implemented in many different forms, what is disclosed here is its specific illustrative embodiment that verifies the principle of the application.It should be emphasized that the application is not limited to the specific embodiments illustrated.In addition, any section heading used herein is only used for organizational purposes and is not to be interpreted as limiting the described subject matter.The following terms or definitions are provided only to help understand the application.These definitions should not be construed as having a scope less than that understood by those skilled in the art.

[0033] Unless otherwise defined below, the meaning of all technical terms and scientific terms used in the specific embodiments of the present application is intended to be the same as those generally understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present application.

[0034] The terms "comprise", "include", "have", "contain" or "involve" are inclusive or open-ended and do not exclude other unlisted elements or method steps. The term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.

[0035] When referring to a singular noun an indefinite or definite article e.g. "a" or "an", "the" or "an" is used, this includes a plural of that noun.

[0036] In addition, the terms first, second, third, (a), (b), (c), and the like in the specification and claims are used to distinguish similar elements and are not necessarily required to describe a sequential or chronological order. It should be understood that the terms so used are interchangeable under appropriate circumstances, and the embodiments described in this application can be implemented in other sequences than those described or illustrated in this application.

[0037] The term "and / or" is considered a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0038] The terms "such as" and "ie" are used merely as examples and are not intended to be limiting, and should not be construed as referring only to those items explicitly listed in the specification.

[0039] 47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more of the above values. Also included are any larger numbers or fractions therebetween.

[0040] Conversely, the term "no more than" includes every value less than the stated value. For example, "no more than 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 56, 57, 58, 59 ... 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Also included are any smaller numbers or fractions therebetween.

[0041] The terms "plurality", "at least two", "two or more", "at least a second", etc., should be understood to include, but are not limited to, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 , 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more. Also includes any larger number or fraction therebetween.

[0042] The terms "approximately" and "substantially" represent the accuracy range that can be understood by those skilled in the art to still ensure the technical effect of the characteristic in question. The term usually represents ±10%, preferably ±5%, of the indicated value.

[0043] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer within the recited range and, where appropriate, fractions thereof (e.g., tenths and hundredths of integers).

[0044] Various aspects of the present disclosure are further described in detail:

[0045] 1. Therapeutic Applications

[0046] As described in the examples of this application, this application confirms the relationship between CRYAB protein and cGAS-STING pathway. Knocking out cryab gene in human retinal pigment epithelial cell line ARPE-19 (ARPE) promotes the activation of intracellular STING; exogenous supplementation of TAT-CRYAB recombinant protein or overexpression of cryab gene in cells can inhibit the activation of STING; injection of TAT-CRYAB recombinant protein can prolong the survival rate of mice and inhibit tissue inflammatory response in TREX1 knockout mice with abnormal STING activation. The above results show that CRYAB can be used as a STING inhibitor, thereby preventing or treating diseases related to STING activation, or preventing or treating aging.

[0047] Therefore, this application includes at least the following uses:

[0048] 1) Use of CRYAB proteins or genes as STING inhibitors, or use of CRYAB proteins or genes in inhibiting STING activation and expression;

[0049] 2) Use of CRYAB proteins or genes in preventing or treating diseases associated with STING activation, or in preparing drugs for preventing or treating diseases associated with STING activation;

[0050] 3) Use of CRYAB protein or gene in preventing or treating aging, or in preparing drugs for preventing or treating aging.

[0051] As used herein, the term "CRYAB protein" refers to alpha-crystallin B, and CRYAB gene refers to the corresponding gene encoding CRYAB protein.

[0052] The present application does not limit the source or form of the protein. It can be understood that both endogenous proteins and recombinantly expressed proteins can achieve the core application of the present application. Exemplarily, in a specific embodiment of the present application, in order to facilitate CRYBA to enter cells more easily, the present application prepares a recombinant TAT-CRYAB protein; preferably, the amino acid sequence of the TAT is YGRKKRRQRRR. In order to improve the safety of the protein, the present application expresses the protein based on the non-endotoxin plasmid Clear ColiBL21; preferably, the genotype of the plasmid is: F–ompT hsdSB(rB-mB-)gal dcm lonλ(DE3[lacI lacUV5-T7 gene 1ind1 sam7 nin5])msbA148ΔgutQΔkdsDΔlpxLΔlpxMΔpagPΔlpxPΔeptA.

[0053] As used herein, "STING-related diseases" or "STING activation-related diseases" have the same meaning, and refer to diseases caused by dysregulated activation of cGAS-STING signals. These diseases are already disclosed in the prior art, and exemplary include: inflammatory diseases, such as inflammatory bowel disease (IBD) or acute and chronic kidney injury; autoimmune diseases, such as spontaneous STING-associated vasculopathy (SAVI, STING-Associated Vasculopathy with Onset in Infancy), Aicardi-Goutières Syndrome (AGS), systemic lupus erythematosus (SLE); neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic Lateral Sclerosis (ALS), Multiple Sclerosis (MS), Age-related macular degeneration (AMD), etc.

[0054] As described herein, the term "aging" includes physiological aging and pathological aging. Physiological aging refers to the physiological degeneration process that occurs after maturity, and pathological aging is the senile changes caused by various external factors (including various diseases). Herein, pathological aging is particularly referred to, which is the aging of the body caused by abnormal activation of the cGAS-STING pathway. Therefore, in some embodiments of the present application, it specifically relates to the use of CRYAB in preventing or treating aging caused by abnormal activation of the cGAS-STING pathway.

[0055] 2. Pharmaceutical Composition

[0056] The present application discloses a pharmaceutical composition, which contains CRYAB protein or gene, and the pharmaceutical composition can prevent or treat STING-related diseases or aging. It can be understood that after the subject or experimenter is administered an effective dose of the pharmaceutical composition containing CRYAB protein or gene of the present application, the STING pathway can be inhibited by increasing the amount of CRYAB protein or the activity of the protein, thereby achieving the purpose of regulating the corresponding disease or aging of the pathway. Therefore, the pharmaceutical composition of the present application can be in various forms, for example, the pharmaceutical composition can be a drug at the protein level or a drug at the gene level.

[0057] In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and / or excipient according to the requirements of drug preparation.

[0058] As used herein, the term "pharmaceutically acceptable" means that the carrier, diluent, excipient and / or salt thereof is chemically and / or physically compatible with the other ingredients of the formulation and physiologically compatible with the recipient.

[0059] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with a subject and an active agent, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

[0060] As used herein, an "effective dose," "effective amount," or "therapeutically effective dose" is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of disease or promotes regression of disease, as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of impairment or disability due to disease affliction. The ability of a therapeutic agent to promote regression of disease can be assessed using a variety of methods known to skilled practitioners, such as in human subjects during clinical trials, in animal model systems used to predict efficacy in humans, or by measuring the activity of the agent in an in vitro assay.

[0061] As used herein, an "individual" or "subject" is a mammal. Mammals include primates (e.g., humans and non-human primates such as monkeys) and rodents (e.g., mice and rats). In certain embodiments, an individual or subject is a human. A "subject" can be a "patient" - a patient is a human subject in need of treatment, can be an individual with a CCDC112-related cancer such as colorectal cancer, a subject at risk of developing a CCDC112-related cancer such as colorectal cancer.

[0062] As used herein, the term "in vitro cell" refers to any cell cultured ex vivo. In particular, the in vitro cell may include a T cell.

[0063] 3. Control methods

[0064] As can be seen from the core technical content of this article, in the case of discovering the relationship between CRYAB and STING pathway, this application at least includes the following methods:

[0065] A method for regulating the STING pathway in vivo / in vitro, which can be achieved by regulating the amount or activity of CRYAB protein in vivo / in vitro.

[0066] As used herein, the term "regulation" generally includes the meaning of up-regulation or down-regulation in two different directions. In some cases, it can be understood as inhibition or enhancement, in some cases, it can be understood as reduction or increase, in some cases, it can be understood as reduction or increase, etc. The specific explanation is not limited and is understood and interpreted according to the actual application context. Therefore, the "regulation" of the STING pathway in this application can be understood as, for example, inhibition or enhancement of the STING pathway, especially in some cases.

[0067] Similarly, the term "regulate" also includes the meaning of up-regulating or down-regulating in two different directions. In some cases, it can be understood as inhibition or enhancement, in some cases it can be understood as reduction or improvement, in some cases it can be understood as reduction or increase, etc. The specific explanation is not limited and is understood and interpreted according to the actual application context. Therefore, the "regulation" of the amount or activity of CRYAB protein in this application can be understood as increasing or decreasing the amount or activity of CRYAB protein.

[0068] In some embodiments of the present application, the above-mentioned "regulation" and "regulation" refer in particular to "inhibition" and "increase", that is, a method of inhibiting the STING pathway in vivo / in vitro, which can be achieved by increasing the amount of CRYAB protein or protein activity in vivo / in vitro. It can be understood that increasing the amount of CRYAB protein or protein activity can include various conventional means in the art, and any means that can achieve an increase in protein amount or protein activity falls within the scope of protection of the present application. For example, in some embodiments, the means can be achieved by directly administering CRYAB protein exogenously, or by increasing the amount of CRYAB protein by genetic expression, etc., which is not limited here.

[0069] In some embodiments, the method of regulating the STING pathway described in the present application refers in particular to in vitro regulation, such as by treating isolated cells in vitro, which does not fall within the scope of disease treatment. The method can have a variety of application scenarios, such as screening or identifying candidate compounds or evaluating the activity of candidate compounds, by evaluating the effects of candidate compounds on the CRYAB protein and the STING pathway in vitro.

[0070] 4. Treatment Methods

[0071] As can be seen from the core technical content of this article, in the case where the present application discovered that CRYAB protein can inhibit the STING activation pathway, the art can conclude that by, for example, administering CRYAB protein, the purpose of inhibiting STING corresponding diseases or aging can be achieved. Therefore, the present application includes at least the following treatment methods:

[0072] 1) A method for preventing or treating a STING-related disease, which is achieved by increasing the amount or activity of CRYAB protein in vivo or in vitro in a subject;

[0073] 2) A method for preventing or treating aging, which is achieved by increasing the amount or activity of CRYAB protein in vivo or in vitro in a subject;

[0074] As used herein, the term "treatment" refers to any type of intervention or process performed on a subject, or the administration of an active agent to the subject, to achieve the purpose of reversing, alleviating, improving, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of symptoms, complications or conditions or biochemical indicators associated with the disease. In some embodiments, "treatment" includes partial remission. In another embodiment, "treatment" includes complete remission.

[0075] Increasing the amount of CRYAB protein or the activity of the protein may include various conventional means in the art, and any means capable of increasing the amount of protein or the activity of the protein belongs to the present application. For example, in some embodiments, the means may be achieved by directly administering the CRYAB protein exogenously, that is, by preparing a pharmaceutical composition comprising the CRYAB protein, or by increasing the amount of the CRYAB protein by gene expression, etc., such as by gene therapy, and the various means or forms are not limited here.

[0076] The present application is further described by the accompanying drawings and the following examples. The accompanying drawings and examples are only intended to illustrate specific embodiments of the present application and should not be construed as limiting the scope of the present application in any way.

[0077] Experimental examples, basic experimental steps of this application

[0078] 1. Isolation and culture of mouse bone marrow-derived macrophages (BMDM)

[0079] After euthanasia of 6-8 week old mice, remove the tibia from the posterior thigh of the mouse, taking care to avoid damaging the integrity of the bone to avoid contamination; soak in 70% alcohol for 30 seconds and place in pre-cooled sterile PBS for use; cut off both ends of the tibia with sterilized scissors in the clean bench; use a 1ml sterile syringe to draw 1ml of pre-cooled sterile PBS, insert the needle into one end of the tibia, slowly push the syringe, and use water pressure to elute the bone marrow; repeat twice, after collecting two tibias of a mouse; centrifuge at 500g and 25℃ for 3 minutes, remove the supernatant, and resuspend the cells with 24ml of RPMI-1640 complete medium containing 1ng / ml mouse GM-CSF; mix the resuspension evenly and add it to 2 six-well plates; culture for 72 hours and replace with fresh RPMI-1640 complete medium without GM-CSF; perform subsequent processing and analysis.

[0080] 2. RNA Extraction

[0081] In this study, the Trizol method was used to extract RNA.

[0082] 3. RNA reverse transcription and real-time fluorescence quantitative PCR

[0083] RNA reverse transcription: DNA enzyme was reacted at 42°C for 2 minutes to remove genomic DNA; reverse transcriptase was reacted at 55°C for 15 minutes and 85°C for 5 seconds to reverse transcribe RNA into cDNA; real-time fluorescence quantitative PCR was performed using the Vazyme qPCR kit.

[0084] 4. RNA-seq sequencing analysis

[0085] RNA was extracted with Trizol and the data was obtained by library construction and sequencing. The sequencing data was analyzed for quality control using FastQ software. The adapters and low-quality fragments were removed using Trim Galore. The base sequences were aligned to genes using HISAT2 v2.2.1. The matrix file of gene expression was obtained using featureCounts v2.0.1. Differentially expressed genes DEGs (fold change ≥ 2 and FDR ≤ 0.05) were analyzed using DESeq2. Functional enrichment analysis of genes was performed using the R package "Clusterprofiler". Heat maps were drawn using the R packages "ggplot2" and "complexheatmap".

[0086] 5. Protein extraction and detection

[0087] RIPA lysis buffer was added with protease inhibitors and phosphatase inhibitors to lyse proteins; tissues or cells added with RIPA were ultrasonically disrupted, tissues (40% power, 5s on, 5s off, 10 minutes), cells (40% power, 3s on, 3s off, 2 minutes); after ultrasonication, centrifuged at 13000g for 15 minutes at 4°C; the supernatant was transferred to a new EP tube and the volume was measured; the protein concentration was detected by BCA method; 5x loading buffer was added to make the buffer concentration become 1x; 95°C metal bath for 10 minutes; ice bath cooling for 3 minutes; instant centrifugation, and placed in a -80°C refrigerator for later use. Protein immunoblotting detection.

[0088] 6. Construction of CRYAB knockout ARPE cell line

[0089] sgRNA synthesis: Single-stranded primers were synthesized by Qingke Biotechnology Co., Ltd. The sgRNA annealing reaction system is as shown in Table 1:

[0090] Table 1. sgRNA annealing reaction system

[0091]

[0092]

[0093] 1) Annealing: After the reaction system is prepared, heat the tube in a PCR instrument at 95°C for 5 minutes, then cool it to room temperature in 100°C water.

[0094] 2) Enzyme digestion: The plasmid plx459 containing 1 μg of Cas9 protein was digested overnight to obtain sticky ends for sgRNA connection;

[0095] 3) Ligation: The plasmid after enzyme digestion is cut and recovered, and connected to the sticky end of sgRNA under the action of DNA ligase to construct a circular plasmid containing sgRNA.

[0096] 4) Identification and amplification: Use Stbl3 competent cells for plasmid transformation, pick single colonies for sequencing, amplify the bacterial solution of single colonies successfully connected with sgRNA, and purify and extract the plasmid using a kit;

[0097] 5) Transfection: sgRNA was transferred into ARPE19 cells for 12 hours.

[0098] 6) Screening: Use a culture medium containing 1.25ug / ml for screening, and replace the culture medium with normal culture medium when the cells appear as single cells in the dish.

[0099] 7) Selection of monoclonal clones: Observe the monoclonal clones synthesized by single cells under a microscope, mark them with a marker pen, dry the culture medium around the monoclonal clones to form a circle to prevent a small amount of liquid from flowing out, add 5-10ul of trypsin to digest the monoclonal clones in the circle, and use a pipette tip to draw the monoclonal clones into a 48-well plate containing normal culture medium for 3 minutes. Select 5 monoclonal clones from each group.

[0100] 8) Amplification: Amplify monoclonal cells in normal culture medium, digest half of them and extract DNA for sequencing analysis after they grow to full size; further amplify monoclonal cells with disordered sequences targeted by sgRNA. Finally, verify by WB. Seed preservation: Amplify and freeze the Cryab knockout cells that have been sequenced and verified by WB and store them in liquid nitrogen for future use.

[0101] 7. Paraffin embedding and H&E staining.

[0102] (1) Paraffin embedding of skin tissue

[0103] ① Sampling: After euthanizing mice, shave off the hair on the skin surface; cut off 1 cm2 of skin with scissors, remove the fat tissue, and stick the skin flat on filter paper, and fix it in 10% formaldehyde for 24 hours.

[0104] ② Dehydration: Remove formaldehyde with 70%, 80%, 90%, 95%, and 100% alcohol gradient dehydration for 0.5 hours each;

[0105] ③ Transparent: 100% alcohol to a mixed solution of alcohol: xylene = 1:1 for 10 minutes, xylene for 10 minutes twice;

[0106] ④ Wax immersion: low melting point wax for 40 minutes, replace with new low melting point wax for 40 minutes, high melting point wax for 1 hour;

[0107] ⑤ Embedding: The cross section of the skin is perpendicular to the bottom of the embedding box, cooled on a cold table, and placed at room temperature for later use;

[0108] ⑥ Sectioning: The embedded tissue is fixed on the slicer, first trimmed with 30μm thickness, and then sliced ​​with 8μm after being flattened. Collect the slices, flatten them in cold water, attach them to the slide in 42℃ hot water, mark them and put them in the slide box for later use;

[0109] (2) H&E staining

[0110] ① Baking: Select the paraffin slices with appropriate positions and complete slices under the microscope and put them in a 65℃ oven for preliminary baking.

[0111] paraffin;

[0112] ② Dewaxing: After baking, return the slides to room temperature and dewax them in xylene twice, 10 minutes each time;

[0113] ③ Hydration: After dewaxing, the sections were hydrated with gradient alcohol: 100% for 5 minutes, twice, 90%, 70%, 30%, 3 minutes, once each, and finally immersed in pure water for 1 minute;

[0114] ④Hematoxylin staining: Soak in hematoxylin dye for 4 minutes, wash with pure water for 1 minute, and then wash with hydrochloric acid alcohol (75% alcohol:

[0115] Concentrated hydrochloric acid = 99:1) for 6-10 seconds, buffer in running water for 5 minutes;

[0116] ⑤Eosin staining: eosin staining for 90 seconds, pure water washing for 1 minute;

[0117] ⑥ Dehydration: 95% alcohol for 30 seconds, 2 times, 100% alcohol for 2 minutes, 2 times;

[0118] ⑦ Transparency: Soak in xylene three times for 5 minutes each time;

[0119] ⑧Seal the slides: Seal the slides with neutral resin; place them in a slice box for later use.

[0120] 8. Cell culture and treatment

[0121] The cells mainly used in this application are 293T cells and ARPE19 cells, both of which are from the ATCC cell bank; bone marrow-derived macrophages (BMDM) are isolated and extracted from mouse bone marrow in this experiment. Culture medium: 293T: DMEM high glucose culture medium + 10% fetal bovine serum FBS + 1% double anti-PS; ARPE19: F12 culture medium + 10% FBS + 1% PS; BMDM: RPMI-1640 culture medium + 10% FBS + 1% PS. All injuries, drugs or peptide treatments use the corresponding culture medium without serum and antibiotics. Cell freezing solution: 20% serum + 10% DMSO + 70% culture medium, 0.22μm filter sterilization. Cell freezing: remove the culture medium and wash the remaining culture medium with sterile PBS. Then add 1ml of preheated 0.25% trypsin, and after observing that the cells are just digested under a microscope, add 2ml of serum-containing culture medium to terminate the digestion. Centrifuge at 300g for 3 minutes at 25℃ to remove the supernatant, resuspend with 1ml freezing solution, transfer to a freezing tube, place in a gradient cooling box in a -80℃ refrigerator for 6-12 hours, and finally transfer to liquid nitrogen for storage.

[0122] Cell recovery: Take out the cells frozen in liquid nitrogen, seal them with sealing film, and quickly shake them in a 37℃ water bath to melt them; add them to a 15ml centrifuge tube containing 9ml preheated complete culture medium, and gently blow 10-15 times with a 1ml pipette tip to completely mix the cells; finally, add them to a marked 10cm dish and culture them in a cell culture incubator; after the cells adhere to the wall, replace with fresh culture medium and continue to culture.

[0123] Cell passaging: remove the culture medium, absorb the residual culture medium with sterile PBS, add preheated 0.25% trypsin to digest at room temperature, and add 1 ml of complete culture medium to terminate digestion after observing the cell boundary digestion state under a microscope; centrifuge at 300g for 3 minutes at 25℃ to remove the supernatant; resuspend with the corresponding volume of complete culture medium and add the resuspended liquid to the required culture dish; place in a cell culture incubator for continued cultivation.

[0124] 9. Mouse genotype identification

[0125] The mouse tail was cut off, 50ul of lysis solution (50mM KCl+10mM Tris-HCl pH=9.0+0.4mg / mLProteinase K+0.1% Triton X-100) was added, and lysed at 60-65℃ for 24h; inactivated in a metal bath at 95℃ for 10 minutes; PCR identification: PCR was performed on Trex1 knockout mice according to Tables 2 and 3 below, and the reaction was performed according to Table 4; DNA bands were detected by electrophoresis.

[0126] Table 2. PCR system for genotyping of Trex1 mice (20 μl)

[0127]

[0128] Table 3. PCR system for genotyping of Trex1 mice (20 μl)

[0129]

[0130] Table 4. Genotyping amplification procedures for Trex1 mice

[0131]

[0132] 10. Prepare recombinant protein TAT-CRYAB.

[0133] The present application constructs a fusion plasmid TAT-CRYAB and uses an endotoxin-free engineered bacterium to prepare the recombinant protein TAT-CRYAB. Specifically, an 11-amino acid amino acid sequence of the cell-penetrating peptide TAT (YGRKKRRQRRR) is added before the Cryab sequence, and pET28+-TAT-Cryab ( Figure 1 A). The non-endotoxin-producing plasmid ClearColi BL21 (DE3) was purchased from BIO SCI; the plasmid genotype was: F–ompT hsdSB (rB-mB-) gal dcm lonλ (DE3 [lacI lacUV5-T7 gene 1ind1 sam7 nin5]) msbA148ΔgutQΔkdsDΔlpxLΔlpxMΔpagPΔlpxPΔeptA.

[0134] The specific steps of plasmid transformation, protein expression purification and quantification are as follows:

[0135] 1) Transformation: Add 50 ng of plasmid to 50 ul of bacterial solution and place on ice for 30 minutes, heat shock at 42°C for 60 seconds, quickly place on ice for 5 minutes, then add 500 ul of LB medium without antibiotics. Transfer to a bacterial incubator to recover for 1 hour;

[0136] 2) Plate coating: Take 100ul of liquid LB medium containing bacterial solution, evenly spread it on solid LB medium containing Kana antibiotics, and place it in a bacterial incubator for overnight culture;

[0137] 3) Pick a single colony: After 24 hours, use a sterilized pipette tip to pick up a single colony and culture it overnight in 3 ml of LB medium containing Kana antibiotics;

[0138] 4) Induce protein expression: dilute the bacterial solution at a ratio of 1:20, continue culturing for 2 hours until the OD value of the bacterial solution is about 0.6, add IPTG to induce protein expression at a final concentration of 1 mM for 5-6 hours;

[0139] 5) Centrifuge at 5000g for 30 minutes at 4°C and remove the supernatant. Keep the bacterial pellet at -80°C for later use;

[0140] 6) Purification: Purify with the Bio-Tech His-Tag Protein Purification Kit (P2229S). For every 100 ml of bacterial precipitate, add 8 ml of denaturing lysis buffer (add protease inhibitor Selleck, P8340), 250w, on 2s, off 2s ultrasonic for 30 minutes; centrifuge at 13000g, 4℃ for 30 minutes, and take the supernatant. Mix with Ni ion gel and gently shake and incubate on a 4℃ shaker for 1 hour; elute through the chromatography column, wash 4 times with 2 ml of lysis buffer each time, wash 12 times with 1 ml of washing buffer containing 20 mM imidazole each time, and elute the protein 4 times with 0.5 ml of elution buffer containing 200 mM imidazole each time. Collect the solution eluted by the eluent, which is the liquid containing protein.

[0141] 7) Desalting: Use Thermo Fisher desalting column (89891) to remove salt components in the protein solution in one step;

[0142] 8) Quantification: Protein quantification analysis was performed by Coomassie Brilliant Blue staining and BSA standard (results are shown in Figure 1 B).

[0143] Example 1: Knockout of CRYAB in human ARPE cells promotes the expression and activation of STING

[0144] In this example, a CRYAB ko ARPE cell line was constructed. Western blotting results showed that the protein level of STING increased in both CRYAB ko cell lines sg-Cryab1 and sg-Cryab2 ( Figure 2 A).

[0145] Next, this example verifies the specific effect of CRYAB on STING: CRYAB ko ARPE19 cells were treated with 5 μg of a synthetic double-stranded RNA analog poly(I:C); and 5 μg of a double-stranded DNA analog poly(dA:dT). QRT-PCR results showed that the mRNA levels of proinflammatory factors IL6 and type I interferon IFNβ in Cryab ko cells after the introduction of Poly(dA:dT) were significantly increased compared with control cells, but there was no significant difference in the levels of IL6 and IFNβ between CRYAB ko cells and wild-type cells after the introduction of Poly(I:C) ( Figure 2 B / C). These results indicate that the activation of inflammatory factors IL6 and type I interferon IFNβ induced by CRYAB ko is mainly achieved through the activation of the cGAS-STING signaling pathway.

[0146] Example 2: Promoting overexpression of cryab gene in human ARPE cells and mouse BMDM cells to inhibit activation and expression of STING

[0147] In this example, an ARPE19 cell line stably expressing GFP and GFP-Cryab was constructed. First, ARPE19 cells stably expressing GFP and GFP-CRYAB were treated with 0.5 μg normal DNA, 0.5 μg DNA treated with hydrogen peroxide, and 5 μg cGAMP, respectively, and protein detection was performed 2 hours after treatment. The results showed that under different stimulation conditions, cells overexpressing GFP-CRYAB could effectively reduce the protein expression of STING and P-TBK1 ( Figure 3 A). The cells were then treated with 600 μM hydrogen peroxide. The results showed that the RNA expression of inflammatory factors IL6, IL8, IL1β and interferon IFNβ was upregulated after hydrogen peroxide treatment, while cells overexpressing GFP-Cryab could effectively inhibit the expression of proinflammatory factors and interferon induced by hydrogen peroxide ( Figure 3 BE). This indicates that stable expression of GFP-CRYAB can inhibit the expression of STING, the secretion of inflammatory factors and the production of type I interferon in human ARPE19 cells.

[0148] In the human ARPE19 cell line stably expressing GFP-Cryab, the present application detected the expression and activation of STING. Then, GFP-CRYAB and GFP were transiently transfected in mouse BMDM cells, and RNA was extracted for bulk-RNA sequencing after 48 hours of recovery. After quality control, filtering, alignment and differential analysis, the present application compared aging-related secretory phenotype genes such as metalloproteinase family proteins Mmp8, Mmp11, Mmp12, Mmp13 and Mmp14, cGAS-STING related genes Cgas, Sting1, Ifnb, Mx1 and Irf3 through heat maps. The results showed that the expression of this type of gene in BMDMs transferred with GFP-Cryab was reduced compared with the group transferred with GFP ( Figure 3 F). GSEA enrichment analysis also showed that GFP-Cryab treatment reduced the expression of genes related to interferon-β response and interleukin-1 production ( Figure 3 G).

[0149] Example 3: Recombinant protein TAT-CRYAB inhibits exogenous STING activation in human 293T cells

[0150] Previous studies have reported that STING is not expressed in 293T cells, so this application selected 293T as a relatively pure cell environment to study the inhibitory effect of exogenous CRYAB on STING. 293T cells were first treated with 5μg TAT-Cryab or ddH2O for 24h, and then 1μg Flag empty vector and Flag-STING plasmid were transferred for 24h, and finally treated with 5μg cGAMP for 6h, and then WB analysis was performed ( Figure 4 A).

[0151] Experimental results ( Figure 4 B), TAT-Cryab has no effect on TBK1 phosphorylation in 293T cells without Flag-STING; but it can inhibit the expression of phosphorylated TBK1 in Flag-STING cells. After cGAMP was introduced into cells with Flag-STING, the phosphorylation level of TBK1 increased significantly, while TAT-Cryab inhibited the phosphorylation of TBK1 induced by cGAMP activation of STING. This result shows that TAT-Cryab can effectively enter cells and inhibit the expression and activation of STING.

[0152] Example 4: Injection of TAT-CRYAB in Trex1 knockout mouse model to treat inflammation and death caused by abnormal STING activation

[0153] Trex1 knockout mice are a commonly used in vivo model for monitoring STING inhibitors. TREX1 is a DNA exonuclease in the cytoplasm. In TREX1 knockout mice, due to the lack of TREX1 enzyme activity, undegraded DNA accumulates in the cytoplasm, leading to abnormal activation of STING. Mice will show chronic inflammation and autoimmune reactions, similar to human diseases such as Aicardi-Goutières Syndrome (AGS), Systemic Lupus Erythematosus (SLE), Inflammatory Bowel Disease (IBD), acute and chronic kidney injury, premature organ aging, and pathological characteristics of mouse death about 1 month after birth.

[0154] In this example, 4-week-old Trex1 knockout mice were intraperitoneally injected with TAT-CRYAB and ddH2O proteins to study whether supplementation of CRYAB protein can inhibit the death and tissue inflammation of mice caused by Trex1 knockout. First, a long-term intraperitoneal injection was performed, once a day at 15:30, 50 μg each time, for 5 consecutive days per week and 2 days of rest ( Figure 5 A), and analyze their survival rate.

[0155] The results showed that injection of TAT-CRYAB could effectively improve the survival rate of Trex1 knockout mice by 75% ( Figure 5 B). Secondly, the present application compares the phenotypes of mice at different stages, and injection of TAT-CRYAB can reduce abdominal hair loss and scab formation in Trex1 knockout mice ( Figure 5 C).

[0156] Finally, this application also conducted a short-term intraperitoneal injection to study whether TAT-CRYAB has a relieving effect on its tissue inflammation: 50 μg was injected once a day at 15:30 for 6 consecutive days, and samples were taken for HE staining on the 8th day ( Figure 5 D). The results showed that immune cell infiltration in the liver, heart, stomach, and colon of Trex1 knockout mice treated with TAT-Cryab was reduced ( Figure 5 E).

[0157] The above results indicate that TAT-Cryab can inhibit the inflammatory response caused by Trex1 KO.

[0158] Example 5: Cryab inhibits aging in mice

[0159] To further confirm the role of CRYAB in mice, the present application also observed and compared 12-month-old wild-type (WT) mice and Cryab knockout (Cryab ko) mice. The results showed that Cryab ko mice showed obvious whitening of abdominal hair, which is one of the phenotypes of aging ( Figure 6 A). The weight and hair color of mice at 1, 6, and 12 months of age were recorded. The results showed that Cryab ko mice of different ages had different degrees of weight loss compared with WT mice, and the degree of weight loss was more serious in female mice. It is worth noting that 12-month-old Cryab ko female mice showed negative weight gain, while male mice did not ( Figure 6 B), which shows that 12-month-old Cryab KO female mice have already experienced aging characterized by weight loss; in addition, both female and male mice began to develop white hair at 6 months of age, a phenomenon not observed in WT mice ( Figure 6 B). At the same time, in the statistical process of this example, it was also observed that Cryab ko mice began to develop spinal curvature at 6 months old, and the probability of spinal curvature in 12-month-old Cryab ko female mice was greater than 80% ( Figure 6 D). In order to demonstrate this phenomenon intuitively, this application selected 12-month-old WT and Cryab ko mice for X-ray and microCT imaging of their bone morphology and fat status ( Figure 6C), the results also confirmed that Cryab ko mice did show obvious spinal curvature and that both subcutaneous fat and visceral fat in Cryab ko mice were significantly lost.

[0160] The above results indicate that knocking out Cryab will induce aging phenomena in mice, manifested by weight loss, graying of hair and spinal curvature.

[0161] In this example, the epidermal thickness of the mouse skin was statistically analyzed by HE staining, and the results showed that the epidermal layer of the Cryab KO mouse skin was significantly thinner ( Figure 6 E).

[0162] To further verify the relationship between Cryab and aging, this example isolated mouse abdominal skin fibroblasts, selected the fourth generation cells for experiments, and detected cell senescence by senescence-associated-β-galactosidase (SA-β-Gal) staining, and detected cell proliferation by Edu staining. The results showed that Cryab ko cells had a higher proportion of SA-β-Gal-positive cells (WT about 20%, Cryab ko about 80%), and a lower proportion of Edu-positive cells (WT about 25%, Cryab ko about 1%) ( Figure 6 F).

[0163] The foregoing description of the specific exemplary embodiments of the present application is for the purpose of illustration and illustration. These descriptions are not intended to limit the present application to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present application and its practical application, so that those skilled in the art can realize and utilize the various exemplary embodiments of the present application and various selections and changes. The scope of the present application is intended to be limited by the claims and their equivalents.

Claims

1. Use of CRYAB protein or gene in the preparation of a drug for preventing or treating a disease associated with STING activation; the disease associated with STING activation is spontaneous STING-associated vasculopathy or Aicardi-Goutières syndrome.

2. Use of TAT-CRYAB protein in the preparation of a drug for preventing or treating a disease associated with STING activation, wherein: The amino acid sequence of TAT is YGRKKRRQRRR; the STING activation-related disease is spontaneous STING-related vasculopathy or Aicardi-Goutières syndrome.

Images