WO2024011817A1 - c-Rel特异性核糖核酸在抑制角膜移植排斥方面的应用 - Google Patents

c-Rel特异性核糖核酸在抑制角膜移植排斥方面的应用 Download PDF

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WO2024011817A1
WO2024011817A1 PCT/CN2022/134701 CN2022134701W WO2024011817A1 WO 2024011817 A1 WO2024011817 A1 WO 2024011817A1 CN 2022134701 W CN2022134701 W CN 2022134701W WO 2024011817 A1 WO2024011817 A1 WO 2024011817A1
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rel
sed
associated virus
cells
ribonucleic acid
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PCT/CN2022/134701
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French (fr)
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王婷
阮庆国
郑谦
刘芮伶
边江
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山东第一医科大学附属眼科研究所(山东省眼科研究所、山东第一医科大学附属青岛眼科医院)
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Priority to PCT/CN2022/134701 priority Critical patent/WO2024011817A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the invention belongs to the field of biomedicine and relates to a recombinant adeno-associated virus carrying c-Rel specific ribonucleic acid and its application in inhibiting corneal transplant rejection.
  • Corneal transplantation is the only treatment for irreversible corneal blindness caused by severe infection, chemical injury, etc.
  • Post-transplant rejection is the main reason for corneal transplant failure.
  • Hundreds of thousands of corneal transplants are performed worldwide every year, and the rejection rate is as high as over 50%, especially for high-risk corneal transplants.
  • the key to this problem is that the mechanism of corneal transplant rejection immunity is not clear.
  • immunosuppressants are steroid hormones, glucocorticoids, cyclosporine A, tacrolimus, mycophenolate mofetil, rapamycin, and FTY720.
  • various anti-rejection drugs used clinically have poor prevention and treatment effects, and due to lack of specificity, the drugs have large side effects and can only be used short-term to control acute inflammation.
  • the targets of antibody and fusion protein immunosuppressants that have been successfully used to prevent and treat corneal transplant immune rejection mainly include IL-2 and CD25. , TNF- ⁇ , CTLA4-Ig, IL-10, TLR2, CCR7, etc.
  • the purpose of the present invention is to reduce rejection after corneal transplantation and provide new means and strategies for clinical prevention and treatment of corneal transplant rejection.
  • the present invention provides the application of c-Rel specific ribonucleic acid in the preparation of drugs for preventing, inhibiting and/or treating corneal transplant rejection or complications, with sequences such as SED ID NO.1, SED ID As shown in NO.2 or SED ID NO.3, SED ID NO.4:
  • Antisense strand 5’AGAAGGGTATGTTCGGTTG 3’ (SED ID NO.4).
  • the present invention provides a recombinant adeno-associated virus for inhibiting the expression of c-Rel gene and/or inhibiting the expression of inflammatory factors regulated by c-Rel, which contains c-Rel as a target.
  • Rel specific ribonucleic acid or its functional equivalent, the c-Rel specific ribonucleic acid sequence is as shown in SED ID NO.1, SED ID NO.2 or SED ID NO.3, SED ID NO.4:
  • the functional equivalents have at least 95%, 96%, 97%, 98% or 99% identity with the sequences shown in SEQ ID NO: 1-4; optionally one or more substitutions, deletions and/or insertions A nucleotide sequence shown in SEQ ID NO: 1-4.
  • the serotypes of the recombinant adeno-associated virus include AAV2, AAV4, AAV5, AAV6, AAV8 and AAV9.
  • the serotype of the recombinant adeno-associated virus is AAV6, and the packaging titer is 5 ⁇ 10E 12 to 1 ⁇ 10E 13 vg/mL, and optionally the packaging titer is 6 ⁇ 10E 12 to 7 ⁇ 10E 12vg /mL.
  • the present invention also provides a pharmaceutical composition for preventing, inhibiting and/or treating corneal transplant rejection, which is characterized in that it contains c-Rel specific ribonucleic acid or its Functional equivalents, pharmaceutically acceptable carriers, and pharmaceutically acceptable excipients may also be included;
  • sequence of the c-Rel specific ribonucleic acid targeting c-Rel is as shown in SED ID NO.1, SED ID NO.2 or SED ID NO.3, SED ID NO.4:
  • the functional equivalent has at least 95%, 96%, 97%, 98% or 99% identity with the sequence shown in SEQ ID NO: 1-4;
  • the pharmaceutically acceptable vector includes plasmid vectors and/or viral vectors.
  • the pharmaceutically acceptable excipients include, but are not limited to, water, physiological saline, glucose, immunosuppressants (steroid hormones, glucocorticoids, cyclosporine A, tacrolimus, mycophenolate mofetil, rapamycin and FTY720 etc.), small molecule compounds targeting c-Rel (pentoxifylline, etc.), peptides or siRNA drugs (IL-2R monoclonal antibody, anti-CD25 monoclonal antibody, bevacizumab, TNF- ⁇ monoclonal antibody, CD154 monoclonal antibody, CTLA4-Ig, etc.).
  • immunosuppressants steroid hormones, glucocorticoids, cyclosporine A, tacrolimus, mycophenolate mofetil, rapamycin and FTY720 etc.
  • small molecule compounds targeting c-Rel pentoxifylline, etc.
  • peptides or siRNA drugs IL-2R monoclonal antibody, anti-CD25
  • the pharmaceutically acceptable carrier is an adeno-associated virus.
  • the serotype of the adeno-associated virus is AAV6, and the virus packaging titer is 5 ⁇ 10E 12 -1 ⁇ 10E 13 vg/mL; the dosage form of the pharmaceutical composition is an injection, and the pharmaceutical composition is an injection.
  • the composition is administered to the eye via conjunctival injection.
  • the present invention provides the use of the above-mentioned recombinant adeno-associated virus and pharmaceutical composition in preparing drugs for preventing, inhibiting and treating corneal transplant rejection.
  • the present invention provides the above-mentioned recombinant adeno-associated virus and pharmaceutical composition for preparing a method for inhibiting the expression of the c-Rel gene and/or the expression of inflammatory factors regulated by c-Rel in the cells of rejection patients after corneal transplantation.
  • the recombinant adeno-associated virus and the pharmaceutical composition are used in the preparation of medicaments for preventing, inhibiting and treating corneal transplant rejection, or in the preparation of intracellular c-
  • the application of Rel gene expression and/or the expression of c-Rel-regulated inflammatory factors in medicines includes administering an effective amount of c-Rel-specific ribonucleic acid to a rejection individual after corneal transplantation to inhibit macrophages in the individual's body, The process of expressing the c-Rel gene in one or more cells in dendritic cells, lymphocytes, monocytes, and CD4+T cells and/or inhibiting the expression of inflammatory factors regulated by c-Rel.
  • the inflammatory factors regulated by c-Rel include one or more of TNF- ⁇ , IL-1 ⁇ , IL-17a, IFN- ⁇ , IL-2, and IL-6.
  • the present invention provides a method for preventing, inhibiting and/or treating rejection after corneal transplantation, characterized by: administering an effective amount of the c-Rel specific ribonucleic acid to the individual after corneal transplantation. , or the recombinant adeno-associated virus, or the pharmaceutical composition.
  • Terms of the present invention such as "99% degree of identity” mean that except for 1% of the nucleic acid sequence that is different from the claimed c-Rel specific ribonucleic acid, 99% of the nucleic acid sequence is different from the claimed c-Rel specific ribonucleic acid.
  • the ribonucleic acid is the same and has the same or similar properties as the c-Rel-specific ribonucleic acid that has 99% identity with the claimed c-Rel-specific ribonucleic acid, and can achieve the technical effects of the present invention.
  • the terms "individual” or “patient” are used interchangeably herein and refer to a vertebrate animal, preferably a mammal.
  • the mammal may be a human, non-human primate, mouse, rat, dog, cat, horse or cow, but is not limited to these examples.
  • the patient is human.
  • Such “individuals” or “patients” typically suffer from or are susceptible to a condition that can be prevented or treated by administration of the above-mentioned drugs or pharmaceutical combinations of the present invention, including but not limited to the following symptoms of corneal transplant rejection: sudden tearing, red eyes , foreign body sensation in the eye, blurred vision, obvious corneal graft edema, etc.; including but not limited to increased expression of c-Rel gene, increased expression of inflammatory factors regulated by c-Rel, such as TNF- ⁇ , IL-1 ⁇ , IL- One or more of 17a, IFN- ⁇ , IL-2, and IL-6.
  • the term "effective amount” means conferring a therapeutic or inhibitory effect (e.g., controlling, alleviating, ameliorating, alleviating or slowing progression); or preventing (e.g., delaying onset or reducing the risk of progression) to an "individual" or "patient”
  • the above-mentioned medicines and pharmaceutical compositions of diseases, diseases or conditions or their symptoms will depend, for example, on the route of delivery, the activity of the particular active substance or formulation employed, the severity of corneal transplant rejection, the individual's weight and general health, and the judgment of the prescribing physician.
  • Doses may be administered once a week, or once every two days, or once a day, or even several times a day. Dosage units may be administered over a short period of time (eg, weeks to months) or over a longer period of time (months to years).
  • Targeting c-Rel to prevent and treat corneal transplant rejection has the following obvious advantages: 1) c-Rel is mainly expressed on activated lymphocytes and monocytes, and has fewer side effects than targeting the entire NF- ⁇ B family; 2) Targeting c-Rel can inhibit the expression of a series of downstream inflammatory factors. Therefore, compared with antibody drugs that only target a single inflammatory factor, targeting c-Rel can more effectively cut off the connection between innate immunity and adaptive immunity. malignant inflammatory circuit; 3) c-Rel gene knockout mice develop normally, indicating that using c-Rel as a target is safer.
  • Figure 1 Schematic diagram of recombinant plasmid construction.
  • FIG. 1 In Example 3, the results of western-blot detection of c-Rel expression in the mouse cornea of the syngeneic group and the heterogeneous group. The expression of c-Rel in the heterogeneous group is higher than that of the homologous group; B: In Example 3, The rejection score curves of mice in the syngeneic group and the heterologous group after corneal transplantation, the rejection rate of the heterologous group was significantly higher than that of the syngeneic group; C: In Example 3, the corneal graft survival curves of the syngeneic group and the heterologous group, the survival rate of the heterologous group The rate was significantly lower than that of the homologous group.
  • FIG. 1 A: Example 5, rejection score curves of wild group (WT) and knockout group (c-Rel KO) mice; B: corneal graft survival curve.
  • Figure 9 A: In Example 9, the physical images of the corneas of mice in the experimental group and the control group; B: In Example 9, the rejection score curves of the mice in the experimental group and the control group; C: In Example 9, the experimental group and Corneal graft survival curve in the control group.
  • c-Rel systemic knockout mice c-Rel KO
  • WT wild-type mice
  • compositions, step, method, article, or device that includes listed elements need not be limited to those elements, but may include other elements not expressly listed or inherent to such composition, step, method, article, or device. elements.
  • the conditions should be carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.
  • mice BALB/c and C57BL/6J mice were purchased from Spefford;
  • Adeno-associated virus and recombinant adeno-associated virus AAV6-shRel were purchased from Shandong Weizhen Biotechnology Co., Ltd.;
  • Bone marrow-derived macrophages (BMDM: Bone-marrow derived macrophage) and bone marrow-derived dendritic cells (BMDC: Bone-marrow derived dendritic cells) were extracted from the femur and tibia of C57BL/6J mice, and primary T lymphocytes were extracted from mice After the spleen cells were extracted, the primary T lymphocyte negative sorting kit of STEMCELL company was used to isolate the macrophage cell line (Raw264.7).
  • the human leukemia lymphocyte cell line (Jurkat) was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.
  • Human whole blood mononuclear cell isolation solution was purchased from Tianjin Haoyang Biological Products Technology Co., Ltd., product number: LDS1075; human peripheral blood CD4 + T cell sorting kit was purchased from STEMCELL Technologies, product number: 17952.
  • Wild-type and c-Rel knockout mice were purchased from Shanghai Southern Model Biology Company.
  • transplant rejection symptoms include but are not limited to: sudden tearing, red eyes, foreign body sensation in the eye, blurred vision, obvious corneal graft edema, etc.) and normal people Fresh peripheral blood was obtained from Qingdao Eye Hospital.
  • Adeno-associated virus AAV6-shRel refers to the recombinant adeno-associated virus disclosed in the present invention that carries specific ribonucleic acid (c-Rel-specific shRNA) targeting c-Rel and serotype 6.
  • Reagents include:
  • BCA protein concentration determination kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) includes BCA reagent A, BCA reagent B, and protein standard (5 mg/ml BSA).
  • the electrophoresis can be stopped only after the sample reaches the bottom of the electrophoresis tank.
  • the target protein is fully hybridized
  • Exposure Prepare the luminescent liquid in advance and place it in a light-proof EP tube. Use a sample gun to apply evenly to the molecular weight position of the target protein, and repeat the application evenly. Place it inside the exposure machine for exposure. The resulting bands were analyzed using Image lab software.
  • c-Rel-specific shRNA corresponding to mouse c-Rel (NCBI, NM-009044) has the sequence:
  • c-Rel-specific shRNA The specific ribonucleic acid (c-Rel-specific shRNA) corresponding to c-Rel (NCBI, NM-002908) in humans has the sequence:
  • Packaging of AAV6 adeno-associated virus The above recombinant plasmid carrying the target gene is used as a backbone vector and the helper plasmids AD Helper Vector and AAV-rep/cap Vector are co-transfected into packaging cells HEK293T. A large number of recombinant viruses will be produced in the cells 72 hours after transfection. Then collect the culture supernatant and cell pellet respectively, use PEG8000 to precipitate the virus in the culture supernatant, lyse the cell pellet to collect the virus, and combine the viruses obtained from the cell pellet and supernatant.
  • Viruses were purified using the iodixanol method. Iodixanol density gradient centrifugation can effectively separate viruses, empty capsid viruses and other cellular impurities, and enrich infectious viruses from 10% to more than 90%.
  • the titer range of adeno-associated virus AAV6-shRel is generally 5 ⁇ 10E 12 -1 ⁇ 10E 13 vg/mL.
  • the final packaged adeno-associated virus AAV6-shRel was purchased from Shandong Weizhen Biotechnology Co., Ltd., and the AAV6-shRel virus titer was 6.15 ⁇ 10E 12 vg/mL.
  • Adeno-associated virus AAV6-NC without conjugated c-Rel-specific shRNA was used as a control.
  • Penetrating corneal transplantation models were constructed using BALB/c mice and C57BL/6J mice.
  • Allo (Allo) group BALB/c mice are used as recipients and C57BL/6J are used as donors;
  • Syngeneic (Syn) group BALB/c mice serve as both donor and recipient.
  • Knockout group c-Rel knockout mice (C57BL/6J) were used as recipients, and BALB/c mice were used as donors.
  • Wild group use wild mice (C57BL/6J) as recipients and BALB/c mice as donors
  • mice BALB/c mice were used as recipients, C57BL/6J mice were used as donors, and were treated with subconjunctival injection of AAV6-shRel.
  • Control group BALB/c mice as recipients, C57BL/6J mice as donors, treated with subconjunctival injection of AAV6-NC.
  • Graft suturing add a drop of 0.9% normal saline to moisten the implant bed, spread the graft flat on the implant bed, pay attention to protect the endothelium of the graft and the lens of the recipient mouse, and use 11-0 nylon thread to suture 8 stitches intermittently , try to make the needle spacing even, leaving about 0.5mm for the knot.
  • mice that have established syngeneic and heterologous mouse corneal transplant models the rejection status of the recipient mice will be observed with a slit lamp every 3 days starting from the 7th day after surgery and photographed and recorded, and scores will be observed until 30 days after surgery.
  • Reference Qin Q, Luo D, Shi Y, et al. CD25 siRNA induces Treg/Th1 cytokine expression in rat corneal transplantation models. Exp Eye Res. 2016; 151:134-141.doi:10.1016/j.exer., according to The corneal graft opacity and edema degree and neovascularization grade score were used to determine the rejection situation and draw a scoring curve.
  • c-Rel KO mice For wild group (WT) and knockout group (c-Rel KO) mice, from 3 days after surgery, scores were scored based on corneal graft edema, transparency, and neovascular growth (see Example 3), and total rejection was calculated. index(RI). When the RI ⁇ 5 points, the graft is considered to be rejected, and the survival curve of the corneal graft is drawn. The results are shown in Figure 4. c-Rel gene knockout mice had a lower corneal graft rejection rate, later rejection, and longer survival time (P ⁇ 0.05).
  • AAV6-shRel is used to infect a variety of immune cells, including bone marrow-derived macrophages (BMDM: Bone-marrow derived macrophage), macrophage cell lines (Raw264.7), and bone marrow-derived dendritic cells (BMDC: Bone-marrow derived dendritic cell), human leukemia lymphocyte cell line (Jurkat), primary T lymphocytes. Except for primary T lymphocytes, AAV6-shRel can successfully infect and enter the cells.
  • BMDM Bone-marrow derived macrophage
  • BMDC bone marrow-derived dendritic cells
  • Jurkat human leukemia lymphocyte cell line
  • primary T lymphocytes except for primary T lymphocytes, AAV6-shRel can successfully infect and enter the cells.
  • BMDM Bone marrow derived macrophage
  • BMDC bone marrow derived dendritic cell
  • original Seed T lymphocytes in 96-well plates respectively adjust the cell density to 0.4m/mL in the seed plate, keep the number of cells in the experimental wells and control wells consistent, and divide the cells into the control and experimental groups after 3-6 hours until the cells are completely attached.
  • BMDM Bone-marrow derived macrophage
  • BMDC bone marrow-derived dendritic cell
  • primary Generation of T lymphocytes was seeded in experimental wells and control wells at a density of 2m/mL. The number of cells in the two groups of wells remained the same.
  • Dissolve PTXF into a solution of 500 ⁇ g/mL in sterile PBS buffer and add it to the experimental group wells in equal volumes. Add sterile PBS buffer to the control wells and pretreat for 30 minutes.
  • PBMC mononuclear cells
  • RNA centrifuge and divide into three layers (upper: RNA, middle: DNA, bottom: DNA) carefully absorb the upper liquid phase, add 1ul glycogen (Thermo: R0551), and an equal volume of isopropyl alcohol, and mix well.
  • Genomic DNA removal reaction According to the formula in the table, prepare the reaction mixture on ice, and when dispensing it into the master tube, it is recommended to prepare the Master Mix according to the calculated number of reactions + 2, and finally dispense it into each reaction tube. , to ensure the consistency of the reaction system, and finally add the RNA sample.
  • Reaction solution preparation should be strictly carried out on ice. And in order to ensure the consistency of the reaction system, it is recommended that when configuring the total system Master Mix, prepare the amount of the reaction number + 2 first, and then dispense 10 ⁇ l into the tube. Perform reverse transcription reaction immediately after centrifugation and mixing.
  • STEMCELL company's human peripheral blood CD4 + T cell sorting kit (stemcell: 17952) was used to evaluate the effectiveness of adeno-associated virus AAV6-shRel.
  • the specific steps are as follows: (1) Isolate peripheral blood PBMC;
  • AAV6-NC and AAV6-shRel were injected subconjunctivally into the experimental group and the control group respectively.
  • the AAV6-shRel virus packaging titer was 6.15 ⁇ 10E 12 vg/mL. After surgery Conjunctival injections were given every 3 days until the end of the observation period.
  • the rejection status of the recipient mice was observed every 3 days with a slit lamp and photographed. The scores were observed until 30 days after the operation, and the rejection score curve and corneal graft survival curve were drawn (see Example 3). As shown in Figure 9, the corneal grafts in the AAV-shRel injection group were more translucent. After AAV6-shRel treatment, the rejection rate of mice in the experimental group was significantly reduced, and the graft rejection occurred later.
  • mice during the rejection peak period 14-21 days after shearing were homogenized (see Example 3).
  • the secretion of inflammatory factors in the corneal tissues of the experimental group and the control group was detected by ELISA, and the protein level was detected by western blot technology.
  • the secretion of inflammatory factors in mice in the AAV6-shRel treatment group was significantly reduced (P ⁇ 0.05); the expression of c-Rel was down-regulated in the mouse corneas.
  • Routine toxicology of AAV6 eyes BALB/c mice were divided into 2 groups. Group A mice were injected with AAV6-NC virus, and group B mice were injected with AAV6-shRel virus. On days 1, 3, 7 and 14 after virus treatment Conduct routine eye examinations every day, including measurement of tear secretion and corneal sensitivity. Tear secretion test: Gently open the mouse eyelids, use ophthalmic microscopic tweezers to take out the phenol red cotton thread, gently put it into the middle and outer 1/3 of the mouse's lower eyelid, use an electronic timer for 15 seconds, gently take out the phenol red cotton thread, and use The length of the tear-infiltrated part is measured using the scale on the back of the package.
  • Corneal sensitivity test When the mouse is in a quiet state, keep the mouse's eyes open naturally, adjust the length of the stylus from 6cm-1cm, shorten it by 5mm each time, and swipe the central part of the mouse cornea until a reflex blink occurs. At this time, the length of the stylus is calculated as the corneal sensitivity value. The average value of the three measurements is taken and recorded. 5.5mm-6mm is considered normal.
  • melanoma cells B16 purchased from Wuhan Pronosai Life Technology Co., Ltd.
  • BALB/C mice were divided into three groups, and 10 8 B16 cells were inoculated into each group after skin preparation.
  • 8uL PBS, AAV6-NC, and AAV6-shRel were injected subconjunctivally, and PBS, AAV6-NC, and AAV6-shRel were injected every 3 days.
  • the results are shown in Figure 12A.
  • ARPE-19, A549, Jurkat, B16, MDA-MB-231, and EL4 cell lines were used to detect cell proliferation.
  • the results are shown in Figure 12B. Configure 0.4 m/mL ARPE-19, A549, Jurkat, B16, MAD-MB-231, and EL4 cells into cell suspensions and seed them in 96-well plates respectively. Add 6 ⁇ L of AAV6-NC and AAV6-shRel to each cell. After 48 h of culture, cells were collected and washed with PBS.

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Abstract

本发明属于生物医药领域，涉及一种载有c-Rel特异性核糖核酸的重组腺相关病毒及其在抑制角膜移植排斥方面的应用。本发明提供的重组腺相关病毒用于在角膜移植排斥个体的细胞内抑制c-Rel基因的表达和/或抑制c-Rel调控的炎症因子的表达，有力切断先天性免疫与适应性免疫间的恶性炎症回路，并且副作用小、安全性较高。

Description

c-Rel特异性核糖核酸在抑制角膜移植排斥方面的应用 技术领域
本发明属于生物医药领域，涉及一种载有c-Rel特异性核糖核酸的重组腺相关病毒及其在抑制角膜移植排斥方面的应用。
背景技术
角膜移植是治疗严重感染，化学伤等所致不可逆性角膜盲的唯一治疗手段，移植后排斥是角膜移植失败的主要原因。全球每年角膜移植数量数十万例，其排斥率高达50％以上，尤其是高危角膜移植。造成这一难题的关键在于角膜移植排斥免疫发生机制不明确。
临床应用免疫抑制剂是预防和治疗角膜移植免疫排斥最常见的方法，它的优点主要是易于操作和患者医从性较高。目前主要使用的免疫抑制剂是类固醇激素、糖皮质激素、环孢素A、他克莫司、霉酚酸酯、雷帕霉素和FTY720等。但临床上使用的各类抗排斥药物防治疗效不佳,并且由于缺乏特异性，药物毒副作用较大，只能短期使用来控制急性炎症。
近年来抗体和融合蛋白免疫抑制剂因靶点明确和作用效果明显而受到了广泛的关注，已成功用于角膜移植免疫排斥防治的抗体和融合蛋白免疫抑制剂靶点主要包括IL-2、CD25、TNF-α、CTLA4-Ig、IL-10，TLR2，CCR7等。
发明内容
本发明的目的是减少角膜移植后的排斥反应，为临床预防和治疗角膜移植排斥提供新的手段和策略。
在第一方面，本发明提供了c-Rel特异性核糖核酸在用于制备预防、抑制和/或治疗角膜移植排斥反应或并发症的药物的应用，其序列如SED ID NO.1、SED ID NO.2或SED ID NO.3、SED ID NO.4所示：
正义链5’CAACCGGACATACCCGTCT 3’(SED ID NO.1)
反义链5’AGACGGGTATGTCCGGTTG 3’(SED ID NO.2)；
或
正义链5’CAACCGAACATACCCTTCT 3’(SED ID NO.3)
反义链5’AGAAGGGTATGTTCGGTTG 3’(SED ID NO.4)。
在第二方面，本发明提供了一种用于抑制c-Rel基因的表达和/或抑制c-Rel调控的炎症因子的表达重组腺相关病毒，其包含以c-Rel为靶点的c-Rel特异性核糖核酸或其 功能等价物，所述c-Rel特异性核糖核酸序列如SED ID NO.1、SED ID NO.2或SED ID NO.3、SED ID NO.4所示：
正义链5’CAACCGGACATACCCGTCT 3’(SED ID NO.1)
反义链5’AGACGGGTATGTCCGGTTG 3’(SED ID NO.2)；
或
正义链5’CAACCGAACATACCCTTCT 3’(SED ID NO.3)
反义链5’AGAAGGGTATGTTCGGTTG 3’(SED ID NO.4)；
所述功能等价物与SEQ ID NO：1-4所示的序列具有至少95％、96％、97％、98％或99％的同一性；可选地为取代、缺失和/或插入一个或多个SEQ ID NO：1-4所示的核苷酸序列。
所述重组腺相关病毒的血清型包括AAV2、AAV4、AAV 5、AAV6、AAV8及AAV9。
在一个实施方案中，所述重组腺相关病毒的血清型为AAV6，包装滴度为5×10E 12～1×10E 13vg/mL，可选地包装滴度为6×10E 12～7×10E 12vg/mL。
在第三方面，本发明还提供了用于预防、抑制和/或治疗角膜移植排斥的药用组合物，其特征在于，包含以c-Rel为靶点的c-Rel特异性核糖核酸或其功能等价物、药用可接受载体，还可以进一步包括药用可接受辅料；
所述以c-Rel为靶点的c-Rel特异性核糖核酸，其序列如SED ID NO.1、SED ID NO.2或SED ID NO.3、SED ID NO.4所示：
正义链5’CAACCGGACATACCCGTCT 3’(SED ID NO.1)
反义链5’AGACGGGTATGTCCGGTTG 3’(SED ID NO.2)；
或
正义链5’CAACCGAACATACCCTTCT 3’(SED ID NO.3)
反义链5’AGAAGGGTATGTTCGGTTG 3’(SED ID NO.4)；
所述功能等价物与SEQ ID NO：1-4所示的序列具有至少95％、96％、97％、98％或99％的同一性；
所述药用可接受载体包括质粒载体和/或病毒载体。
所述药用可接受辅料包括但不限于水、生理盐水、葡萄糖、免疫抑制剂(类固醇激素、糖皮质激素、环孢素A、他克莫司、霉酚酸酯、雷帕霉素和FTY720等)、靶向c-Rel的小分子化合物(己酮可可碱等)、多肽或siRNA药物(IL-2R单克隆抗体、抗CD25单克隆抗体、贝伐单抗、TNF-α单克隆抗体、CD154单克隆抗体、CTLA4-Ig等)。
作为本发明一个优选的实施方案，所述药用可接受载体为腺相关病毒。
在一个实施方案中，所述腺相关病毒的血清型为AAV6,病毒包装滴度为5×10E 12-1×10E 13vg/mL；所述药用组合物的剂型为注射液，所述药用组合物通过结膜注射施用于眼部。
在第四方面，本发明提供了上述重组腺相关病毒、药用组合物在用于制备预防、抑制和治疗角膜移植排斥的药物中的应用。
在第五方面，本发明提供了上述重组腺相关病毒、药用组合物在用于制备抑制角膜移植后排斥患者细胞内c-Rel基因的表达和/或c-Rel调控的炎症因子的表达的药物中的应用。
在一个实施方案中，所述重组腺相关病毒、药用组合物在用于制备预防、抑制和治疗角膜移植排斥的药物中的应用，或在用于制备抑制角膜移植后排斥患者细胞内c-Rel基因的表达和/或c-Rel调控的炎症因子的表达的药物中的应用，包括施予角膜移植后排斥个体有效量的c-Rel特异性核糖核酸从而抑制所述个体体内巨噬细胞、树突状细胞、淋巴细胞、单核细胞、CD4+T细胞中一种或多种细胞内c-Rel基因的表达和/或抑制c-Rel调控的炎症因子的表达的过程。
在一个实施方案中，所述c-Rel调控的炎症因子包括TNF-α、IL-1β、IL-17a、IFN-γ、IL-2、IL-6中的一个或多个。
在第六方面，本发明提供了一种用于预防、抑制和/或治疗角膜移植后排斥反应的方法，其特征在于：施予角膜移植后个体有效量的所述c-Rel特异性核糖核酸、或所述重组腺相关病毒、或所述药用组合物。
本发明说明书和所附权利要求中所用的单数形式“一个”，“一种”和“所述”包括复数指示物，除非上下文另有明确规定。
本发明的术语例如“99％同一性程度”是指除了1％的核酸序列与所要求保护的c-Rel特异性核糖核酸不同外，99％的核酸序列与所要求保护的c-Rel特异性核糖核酸相同，并且与所要求保护的c-Rel特异性核糖核酸具有99％同一性程度的c-Rel特异性核糖核酸具有相同或相似的性质，均能实现本发明的技术效果。
在本发明中，术语“个体”或“患者”在此可互换地使用并且是指一种脊椎动物，优选一种哺乳动物。哺乳动物可以是人、非人灵长类动物、小鼠、大鼠、狗、猫、马或牛，但不限于这些实例。优选地，所述患者是人。这样的“个体”或“患者”典型地遭受或易于患有一种可以通过给予本发明的上述药物、药用组合来预防或治疗的病症，包括但不限于如下角膜移植排斥症状：突然流泪、眼红、眼内异物感、视物模糊、角膜植片水肿明显等；包括但不限于c-Rel基因的表达增高、c-Rel调控的炎症因子表达增 高，例如TNF-α、IL-1β、IL-17a、IFN-γ、IL-2、IL-6中的一个或多个。
本发明中，术语“有效量”是指向“个体”或“患者”赋予治疗或抑制作用(例如，控制、缓解、改善、缓和或减缓进展)；或预防(例如，延迟发作或降低发展风险)疾病、病症或病状或其症状的上述药物、药用组合物。对此用途有效的量将取决于例如递送的途径、所采用的具体的活性物质或制剂的活性、角膜移植排斥的严重性、个体体重和总体健康状况、以及处方医师的判断。剂量的给予可以每周一次，或两天或每天一次，或甚至每天几次。可以在短期(例如数周至数月)或更长时间段(数月至数年)给予剂量单元。
有益效果
以c-Rel为靶点防治角膜移植排斥具有以下明显优点：1)c-Rel主要表达于活化的淋巴细胞和单核细胞，与靶向整个NF-κB家族相比，副作用小；2)靶向c-Rel能抑制下游一系列炎症因子的表达，因而与仅以单一炎性因子为靶点的抗体药物相比，以c-Rel为靶点能更有力切断先天性免疫与适应性免疫间的恶性炎症回路；3)c-Rel基因敲除小鼠发育正常，说明以c-Rel为靶点安全性较高。
附图说明
图1、重组质粒构建示意图
图2、A:实施例3中，同系组和异系组小鼠角膜western-blot检测c-Rel表达量的结果，异系组c-Rel表达高于同系组；B:实施例3中，同系组和异系组小鼠角膜移植后排斥情况评分曲线，异系组排斥率显著高于同系组；C：实施例3中，同系组和异系组角膜植片生存曲线，异系组生存率显著低于同系组。
图3、实施例4中，受c-Rel调控的炎症因子表达ELISA检测结果。
图4、A:实施例5中，野生组(WT)和敲除组(c-Rel KO)小鼠排斥情况评分曲线；B：角膜植片生存曲线。
图5、实施例7中，PBMC细胞c-Rel表达RT-PCR及western blot检测结果。
图6、实施例7中，受c-Rel调控的炎症因子表达ELISA检测结果。
图7、实施例8中CD4 +T细胞c-Rel表达RT-PCR及western blot检测结果。
图8、实施例8中，CD4 +T细胞受c-Rel调控的炎症因子表达ELISA检测结果。
图9、A：实施例9中，实验组及对照组小鼠角膜实物图；B:实施例9中，实验组及对照组小鼠排斥情况评分曲线；C：实施例9中，实验组及对照组角膜植片生存曲线。
图10、实施例9中，受c-Rel调控的炎症因子ELISA检测结果和western-blot检测结果。
图11、实施例10中，小鼠泪液分泌量及角膜敏感度。
图12、实施例10中，小鼠荷瘤体积曲线和细胞凋亡率。
具体实施方式
为了研究腺相关病毒AAV靶向c-Rel在防治角膜移植排斥中的应用，我们首先利用同系及异系角膜移植排斥小鼠角膜中c-Rel及其调控的下游炎症因子在蛋白水平的表达量。同时对处于角膜移植术后排斥期及非排斥期的患者血液中c-Rel在蛋白水平的表达量进行测定，结果证明角膜移植排斥过程中c-Rel表达升高，受c-Rel调控的炎症因子分泌增加。
然后使用c-Rel全身敲除小鼠(c-Rel KO)和野生型(WT)小鼠，说明c-Rel促进角膜移植排斥。
在体内实验中，我们通过建立角膜移植排斥动物模型，分别对实验组及对照组小鼠结膜下注射AAV6-NC及AAV6-shRel，发现在AAV6-shRel治疗后，实验组小鼠排斥率明显降低，植片发生排斥更晚。剪取术后14-21天排斥高峰期小鼠角膜进行匀浆，通过ELISA检测实验组及对照组小鼠角膜组织中炎症因子分泌情况，发现，AAV6-shRel治疗组小鼠炎症因子分泌显著降低。另外，我们通过检测发现AAV6-shRel治疗后小鼠c-Rel表达在蛋白水平出现下调。
需要说明的是，在不冲突的情况下，本发明中的实施例及实施例中的特征可以相互组合。下面进一步由解释本发明的以下实施例进行示例性说明，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。除非另有说明，本文所使用的技术和科学术语为本发明所属领域的普通技术人员通常所理解的含义。虽然本发明实施过程中可使用类似于或等价于本文公开的那些的任何方法、条件、物质或材料，但本文描述了优选的方法、条件物质或材料。在本发明中，术语“包括”与“包含”同义。本文中所用的术语“包含”、“包括”、“具有”、“含有”或其任何其它变形，意在覆盖非排它性的包括。例如，包含所列要素的组合物、步骤、方法、制品或装置不必仅限于那些要素，而是可以包括未明确列出的其它要素或此种组合物、步骤、方法、制品或装置所固有的要素。
实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实验材料
BALB/c、C57BL/6J小鼠购买于斯贝福公司；
腺相关病毒、重组腺相关病毒AAV6-shRel购买于山东维真生物科技有限公司；
骨髓来源巨噬细胞(BMDM：Bone-marrow derived macrophage)、骨髓来源树突状细胞(BMDC：Bone-marrow derived dendritic cell)由C57BL/6J小鼠股骨及胫骨提取，原代T淋巴细胞由小鼠脾脏细胞提取后使用STEMCELL公司原代T淋巴细胞阴性分选试剂盒分离巨噬细胞细胞系(Raw264.7)，人白血病淋巴细胞细胞系(Jurkat)购于中科院上海细胞库。
人全血单个核细胞分离液购自于天津市灏洋生物制品科技有限责任公司，货号：LDS1075；人外周血CD4 +T细胞分选试剂盒购自于STEMCELL Technologies公司，货号:17952。
野生型和c-Rel敲除小鼠购买于上海南方模生物公司。
角膜移植后发生排斥患者(选择年龄大于18岁，角膜移植术后，移植排斥症状包括但不限于：突然流泪、眼红、眼内异物感、视物模糊、角膜植片水肿明显等)及正常人新鲜外周血来源于青岛眼科医院。
腺相关病毒AAV6-shRel是指本发明公开的携带以c-Rel为靶点的特异性核糖核酸(c-Rel-specific shRNA)、血清型6型的重组腺相关病毒。
实验方法
小鼠角膜匀浆制备方法：
(1)使用颈椎脱臼法处死小鼠，用15cm弯头镊小心摘取整个眼球，注意不宜用力过大损伤眼球，将眼球置于装有PBS缓冲液的1.5mL离心管中，置于冰上待用。
(2)在体式显微镜下操作，利用6cm培养皿放少量PBS缓冲液使眼球漂浮于表面，使用有齿镊固定眼球，角巩膜剪沿角膜缘外1mm的巩膜剪开，用齿镊去除晶状体、虹膜和睫状体组织。
(3)将2.0mm EP管中放入4-5颗3mm研磨珠，加入蛋白酶抑制剂的蛋白裂解液70μL及剪好的角膜组织，并用封口膜紧密封口后使用匀浆仪充分匀浆，每30s一次，后冰上静置30s，重复5次。
(4)取出研磨珠，上机离心，参数设为4℃，12000g，5min，吸取上清存于-80℃备用。
Elisa检测
试剂包括：
BCA蛋白浓度测定试剂盒(购于上海碧云天生物技术有限公司)包括BCA试剂A、BCA试剂B、蛋白标准品(5mg/ml BSA)。
1.制备蛋白标准品：取完全溶解好的蛋白标准品10μl及90μl PBS混匀，所得蛋白标准品浓度0.5mg/ml，稀释后的标准品保存于-20℃冰箱。
2.制备工作液：按照50体积BCA试剂A与1体积试剂B工作液充分混匀。
3.蛋白浓度测定：
将0.5mg/ml的蛋白标准品按照0、1、2、4、8、12、16、20μl加入96孔平底板中，用PBS加入上述孔中，保持终体积为20μl，则蛋白标品浓度可以计为0、0.025、0.05、0.1、0.2、0.3、0.4、0.5mg/ml。
4.加入4μl样本至96孔板中，同样需要使用PBS将每孔补足至体积20μl。
5.每孔加入200μl BCA工作液，37℃孵育20分钟
6.使用酶标仪于562nm读数，根据OD值绘制标准曲线，并得出公式。将样本OD值代入公式，得出所测浓度。
Western-blot检测
1.配制10％SDS-PAGE凝胶
(1)使用双蒸水清洗灌胶板，直至清亮透明，无残留水渍、胶渍，使用吹风机正反吹干。
(2)将灌胶玻璃板对齐夹于塑胶板上，保证玻璃板与灌胶架连接完好，以防止漏液。
(3)配制下层胶：将3.3ml双蒸水，4.0ml 30％的丙烯酰胺溶液，2.5ml 1.5M tris(PH＝8.8)，0.1ml 10％过硫酸铵溶液及0.1ml 10％SDS，0.004ml TMEMD充分混合，使用1ml加样枪迅速灌入两板之间的缝隙，至玻璃板2/3高度。并加入双蒸水，使液面压平。室温静置15min，待胶层与水层之间出现明显界限，则胶凝固完好。倒置制胶架，倾倒水封。折叠滤纸，尽可能吸干多余液体。
(4)配制上层胶，具体成分如下：将3.4ml双蒸水，0.38ml 30％的丙烯酰胺溶液，0.63ml 1.0M tris(PH＝8.8)，0.05ml 10％过硫酸铵溶液及0.05ml 10％SDS，0.005ml TMEMD。
(5)将上述液体充分混匀，使用1ml加样枪快速加入玻璃板中，尽量避免气泡，随后缓慢、垂直地插入加样梳。
(6)室温静置30min后可置于4℃保存或直接上样。
2.加样并电泳
(1)将配制好的PAGE胶转移至电泳槽中，固定牢固后加入Tris甘氨酸电泳液。
(2)取下加样梳，每孔按照计算数值上样
(3)按照正负极连接好电泳槽电泳。当样本于上层胶泳道电泳时，设定电压为80V左右，直至样本电泳至上层胶与下层胶交汇处，增大电压为120V左右。
待到样本电泳至电泳槽底部，方可停止电泳。
3.半干转转膜
(1)提前配置半干转转膜液：将转膜液、甲醇、双蒸水按照1:1:3的比例混匀，预冷备用。
(2)准备好5.5cm×8.0cm的PVDF膜，提前浸入盛有15ml甲醇的孵育盒中充分激活。
(3)准备PVDF膜大小的滤纸
(4)电泳结束后，小心撬开胶板，取出PAGE凝胶。
(5)将海绵、滤纸、PVDF膜、PAGE凝胶、滤纸、海绵浸入转膜液，并一层一层铺放整齐于半干转槽中，放置过程中需用滚轮滚平，防止气泡产生。盖好转膜夹夹紧上述“三明治”结构，转膜时间为9min。
(6)转膜后，可见Ladder Marker转移至PVDF膜上，确定转模成功，使用镊子将其小心转移至孵育盒里，TBST洗涤2次，5min每次。
4.目的蛋白充分杂交
(1)洗涤结束后，将PVDF膜置于无蛋白快速封闭液当中，室温封闭1小时。
(2)一抗：使用抗体稀释液稀释目的蛋白c-Rel及内参β-actin抗体(按照说明书建议稀释浓度配制)。4℃孵育过夜。
(3)次日，取出孵育盒，于常温复温约20min。回收一抗，保存于-20℃。
(4)使用TBST洗膜，3-4次，每次10min。
(5)加入相应二抗孵育1h，弃二抗，使用TBST漂洗3次，每次15min。
5.曝光：提前配置发光液，置于避光EP管中。使用加样枪均匀涂于目的蛋白分子量位置，反复涂匀。置于曝光机器内部曝光。所得条带使用Image lab软件分析。
实施例1 腺相关病毒AAV6-shRel的构建
(1)c-Rel特异性的核苷酸序列
对应鼠c-Rel(NCBI，NM-009044)的特异性核糖核酸(c-Rel-specific shRNA)，其序列为：
正义链5’CAACCGGACATACCCGTCT 3’(SED ID NO.1)
反义链5’AGACGGGTATGTCCGGTTG 3’(SED ID NO.2)
对应人体内c-Rel(NCBI，NM-002908)的特异性核糖核酸(c-Rel-specific shRNA)，其序列为：
正义链5’CAACCGAACATACCCTTCT 3’(SED ID NO.3)
反义链5’AGAAGGGTATGTTCGGTTG 3’(SED ID NO.4)
将上述特异性核糖核酸序列连接于酶切位点BAMHI和HindIII中间，构建重组质粒(图1)，连接产物转化为大肠杆菌DH5α感受态细胞，涂布于相应抗性的LB平板上进行筛选。然后挑取单菌落培养后提取质粒测序验证。
AAV6腺相关病毒的包装：将上述携带目的基因的重组质粒作为骨架载体与辅助质粒AD Helper Vector和AAV-rep/cap Vector共转染包装细胞HEK293T，转染72h后细胞内会产生大量重组病毒。然后分别收取培养基上清及细胞沉淀，用PEG8000沉淀培养基上清中的病毒，裂解细胞沉淀收毒，合并从细胞沉淀和上清中得到的病毒。
使用碘克沙醇法对病毒进行纯化。碘克沙醇密度梯度离心，可以有效将病毒与空衣壳病毒与其他细胞杂质分开，将有感染力的病毒由10％富集到90％以上。腺相关病毒AAV6-shRel的滴度范围一般为5×10E 12-1×10E 13vg/mL。最终包装的腺相关病毒AAV6-shRel购自山东维真生物科技有限公司，AAV6-shRel病毒滴度为6.15×10E 12vg/mL。
未连接c-Rel-specific shRNA的腺相关病毒AAV6-NC作为对照。
实施例2 构建小鼠穿透性角膜移植(PKP)模型
以BALB/c小鼠、C57BL/6J小鼠构建穿透性角膜移植模型。
分组：
异系(Allo)组：以BALB/c小鼠为受体，以C57BL/6J为供体；
同系(Syn)组：以BALB/c小鼠同时作为供体和受体
敲除组：以c-Rel基因敲除小鼠(C57BL/6J)作为受体，BALB/c小鼠作为供体
野生组：以野生小鼠(C57BL/6J)作为受体，BALB/c小鼠作为供体
实验组：BALB/c小鼠为受体，C57BL/6J小鼠作为供体，使用AAV6-shRel结膜下注射治疗。
对照组：BALB/c小鼠为受体，C57BL/6J小鼠作为供体，使用AAV6-NC结膜下注射治疗。
构建步骤：
(1)根据供受体小鼠体重及大小取0.25±0.05mL麻药，腹腔注射麻醉小鼠，同时 滴加托吡卡胺滴眼液扩散瞳孔。
(2)小鼠麻醉后，剪去供、受体胡须及睫毛，暴露角膜，使用纱布将小鼠头部垫高，调整眼球至水平位，滴加盐酸利多卡因滴眼液表面麻醉，待瞳孔充分散大，以用0.12mm平镊轻轻扩张穹隆结膜，使麻醉充分接触整眼球。使用直径2.0mm的环钻钻切供体小鼠角膜，动作轻柔，避免伤及晶状体。使用前房穿刺刀沿环钻印记，做一侧切口，房水流出，前房内压力下降，角膜塌陷，将透明质酸钠于穿刺口处注入前房，后用维纳斯剪沿钻切痕迹剪下供体小鼠角膜，即植片，取下后浸泡于0.9％的生理盐水中防止植片干涸。
(3)发现受体小鼠瞳孔充分散大，以相同方式钻切剪取受体小鼠角膜。
(4)植片缝合：滴加一滴0.9％生理盐水滋润植床，将植片展开铺平于植床上，注意保护植片内皮及受体小鼠晶状体，用11-0尼龙线间断缝合8针，尽量做到针距均匀，线结留约0.5mm，充分对合后，使用2mL注射器连32G无菌针头注入适量无菌空气以形成前房，使用10-0尼龙线缝合眼睑中央间断缝合1针，注意防止挤塌前房，涂适量左氧氟沙星眼膏于眼表，防止感染。
(5)术后第3天拆除眼睑缝线，手术7天后于全麻下拆除受体角膜缝线。
实施例3 c-Rel的表达及排斥观察
检测异系(Allo)组、同系(Syn)组小鼠角膜中c-Rel的表达及排斥情况。
在14-21天排斥高峰期，取同系组及异系组小鼠角膜匀浆提取蛋白，使用western-blot检测，异系穿透性角膜移植小鼠c-Rel的表达量显著升高(P＜0.05)(图2A)。
对已经建立同系及异系小鼠角膜移植模型的小鼠，术后第7天起，每3天裂隙灯观察受体小鼠排斥情况并拍照记录，评分观察至术后30天。参考文献Qin Q,Luo D,Shi Y,et al.CD25 siRNA induces Treg/Th1 cytokine expression in rat corneal transplantation models.Exp Eye Res.2016；151:134-141.doi:10.1016/j.exer.，根据角膜植片混浊水肿程度及新生血管分级评分，判定排斥情况，绘制评分曲线，异系移植小鼠排斥率显著高于同系小鼠(P＜0.001)(图2B)；同时作角膜植片生存曲线，异系小鼠植片生存率显著低于同系小鼠(P＜0.001)(图2C)。
实施例4 受c-Rel调控的炎症因子
在14-21天排斥高峰，取同系组及异系组小鼠角膜匀浆(参见实施例3)，通过ELISA检测c-Rel靶点的炎症因子分泌情况，结果见图3，角膜移植后发生异系小鼠角膜组织中TNF-α、IL-1β、IL-17a、IFN-γ表达量明显高于同系移植小鼠(P＜0.05)。
实施例5 c-Rel敲除实验
对野生组(WT)和敲除组(c-Rel KO)小鼠，在术后3天起，根据角膜植片水肿、透明度、新生血管生长情况进行评分(参见实施例3)，计算总排斥指数(RI)。当RI≥5分计为该植片发生排斥，绘制角膜植片生存曲线。结果如图4所示，c-Rel基因敲除小鼠角膜植片排斥率更低，排斥发生更晚，存活时间更长(P＜0.05)。
实施例6 免疫细胞感染测试
使用AAV6-shRel感染多种免疫细胞，包括骨髓来源巨噬细胞(BMDM：Bone-marrow derived macrophage)、巨噬细胞细胞系(Raw264.7)，骨髓来源树突状细胞(BMDC：Bone-marrow derived dendritic cell)、人白血病淋巴细胞细胞系(Jurkat)、原代T淋巴细胞。除原代T淋巴细胞外，AAV6-shRel均可成功感染进入细胞内部。
mRNA水平的检测：将骨髓来源巨噬细胞(BMDM：Bone-marrow derived macrophage)、巨噬细胞细胞系(Raw264.7)，骨髓来源树突状细胞(BMDC：Bone-marrow derived dendritic cell)、原代T淋巴细胞分别种板96孔板：将细胞密度调整为0.4m/mL种板，实验孔及对照孔细胞数目保持一致，3-6h后至细胞完全贴壁后分别于对照组及实验组分别加入5μL AAV6-NC对照病毒、AAV6-shRel病毒，包装滴度为5×10E12～1×10E13，感染48h后正倒置荧光显微镜下观察感染细胞比例，然后加入脂多糖LPS刺激36-48h后收取细胞上清，冻于-80℃备用。Jurkat：将细胞密度调整为0.4m/mL种板，实验孔及对照孔细胞量保持一致，加入5μL病毒，感染48h后正倒置荧光显微镜下观察感染细胞比例，然后加入PMA(PKC激活剂)-Ionomycin(离子霉素)浓度分别为50ng/mL及1μM刺激24-36h后收取细胞上清，冻于-80℃备用。提取各细胞mRNA后备用进行实时荧光定量实验RT-PCR。
蛋白水平的测定：将骨髓来源巨噬细胞(BMDM：Bone-marrow derived macrophage)、巨噬细胞细胞系(Raw264.7)，骨髓来源树突状细胞(BMDC：Bone-marrow derived dendritic cell)、原代T淋巴细胞使用2m/mL的密度将细胞种于实验孔及对照孔，两组孔中细胞量保持一致，无菌PBS缓冲液将PTXF溶解为500μg/mL的溶液加入实验组孔，等体积无菌PBS缓冲液加入对照孔，预处理30min，两组同时加入400ng/mL的脂多糖LPS刺激细胞，过夜，去上清，使用70μL加入蛋白酶抑制剂的RIPA裂解细胞，放置于冰上充分裂解，待30min以后，收取蛋白，观察蛋白状态，确定是否需要超声，4℃，12000g，5min离心，取上清。冻于-80℃备用。Jurkat：使用2m/mL的密度将细胞种于实验孔及对照孔，两组孔中细胞量保持一致，加入15μL病毒(包装滴度为5×10E12～1×10E13)，感染48h后正倒置荧光显微镜下观察感染细胞比例，加入PMA(PKC激活剂)-Ionomycin(离子霉素)浓度分别为50ng/mL及1 μM刺激过夜后，离心收细胞，去上清，使用70μL加入蛋白酶抑制剂的RIPA裂解细胞，放置于冰上充分裂解，待30min以后，收取蛋白，观察蛋白状态，确定是否需要超声，4℃，12000g，5min离心，取上清。冻于-80℃备用。提取蛋白后进行蛋白印迹实验western blot。
实施例7 人血液细胞检测
收集角膜移植后发生排斥患者及正常人新鲜外周血，利用人全血单个核细胞分离液，密度梯度离心法分离单个核细胞(PBMC)，具体步骤：取一支15ml离心管，加入3ml分离液，用吸管小心吸取血液样本加于分离液之液面上，22℃，500g，刹车升1降0，离心30min，离心后，此时离心管中由上至下分为四层。第一层为血浆层。第二层为环状乳白色单个核细胞层。第三层为透明分离液层。第四层为红细胞层，用吸管小心吸取第二层环状乳白色单个核细胞层到另一15ml离心管中，向所得离心管中加入10ml清洗液，混匀细胞。250g，离心10min，弃上清，用吸管以5ml清洗液重悬所得细胞，250g，离心10min。利用Trizol(Invitrogen:15596018)直接裂解PBMC细胞提取总RNA，并反转成cDNA(Takara：RR047A)，方法如下：
RNA提取：
①裂解细胞分离的PBMC用1ml trizol裂解吹打均匀；②氯仿抽提：加入200ul氯仿，剧烈手动摇晃15s,静置3min，4℃，12000g，15min；③沉淀RNA：离心后分三层(上：RNA，中：DNA，下：DNA)小心吸取上层液相，并加入1ul糖原(Thermo:R0551)，等体积异丙醇，混匀。室温静置10min，4℃，12000g，10min，弃上清；④洗涤溶解：加1ml75％乙醇(DEPC水配)，4℃，12000g，5min，弃上清，风干离心管，加入65℃预热的DEPC水30ul，置于冰上充分溶解；取1ul溶解好的RNA测浓度。(OD≈1.9)
反转成cDNA：
去除基因组DNA反应：按下表配方，在冰上配制反应混合液，且在配总管分装时，建议按计算的反应数+2的量进行配制Master Mix，最后再分装到每个反应管中，保证反应体系的一致性，最后加入RNA样品。
20μl反转录反应体系中，
Figure PCTCN2022134701-appb-000001
Green qPCR法：
Figure PCTCN2022134701-appb-000002
Figure PCTCN2022134701-appb-000003
反转录反应：
反应液配制应该严格在冰上进行。并且为了保证反应体系的一致性，建议配置总体系Master Mix时先按反应数+2的量配制，之后再应管中分装10μl。瞬时离心混匀后立即进行反转录反应。
Figure PCTCN2022134701-appb-000004
然后通过RT-PCR在mRNA水平以及利用RIPA裂解PBMC，抽提细胞总蛋白，并用western blot在蛋白水平分别检测。结果如图5、图6所示，角膜移植排斥患者(Reject)血浆PBMC中c-Rel的表达在mRNA水平及蛋白水平较正常人(Normal)均有升高；角膜移植排斥组患者PBMC中c-Rel所调控下游靶点炎症因子的表达高于正常对照组，提示c-Rel在角膜移植排斥中可能起重要作用。
实施例8 有效性实验
分离正常人的新鲜外周血PBMC后，利用STEMCELL公司人外周血CD4 +T细胞分选试剂盒(stemcell:17952)，评估腺相关病毒AAV6-shRel的有效性。具体步骤如下：(1)分离外周血PBMC；
(2)用分选细胞缓冲液(PBS+2％FBS+1mM EDTA)将细胞重悬成5×10 7细胞/mL，以每毫升细胞中加入50μL的比例，加入相应体积的大鼠血清，轻弹管壁混匀。
(3)将细胞悬液移入5mL聚苯乙烯圆底管中，以每毫升细胞中加入50μL的比例，加入相应体积的Isolation Cocktail，轻弹管壁混匀，室温孵育5min。
(4)取Streptavidin RapidSpheresTM充分涡旋30s，以每毫升细胞中加入50μL的比例加入试管中，室温孵育2.5min。
(5)加分选缓冲液至2.5mL，用1000μL移液枪轻柔吹吸2-3次混匀细胞，将试 管移至磁力架上，室温孵育5min。
(6)用3mL巴氏管小心地将细胞悬液吸出，移入新的圆底管中。
(7)离心，室温，200×g，10min，弃上清，轻柔刮弹离心管使细胞沉淀分散。
(8)用500μL分选缓冲液重悬细胞。弃去上清，重复步骤2遍。用完全培养基重悬细胞至实验需要的浓度。培养于RPIM164+10％胎牛血清+1％双抗+50UL/ml IL-2中，并且按照IBA Life science公司(货号：6-8900-050)步骤加anti-CD3/anti-CD28混合物刺激PBMC 48h，然后使用AAV6-NC和AAV6-shRel病毒以MOI＝5×10 5进行处理刺激后的CD4+T细胞24h，腺相关病毒AAV6-NC及AAV6-shRel成功感染CD4+T细胞。结果如图7、图8所示，AAV6-shRel处理组的c-Rel表达量在mRNA水平(24h)及蛋白水平(48h)均降低，同时c-Rel靶点炎症因子表达也降低。
实施例9 动物实验
按照实施例2完成建模后，于建模当天分别为实验组及对照组结膜下注射8uL AAV6-NC及AAV6-shRel，AAV6-shRel病毒包装滴度为6.15×10E 12vg/mL,术后每3天结膜注射一次，直至观察期结束。
术后第7天起，每3天裂隙灯观察受体小鼠排斥情况并拍照记录，评分观察至术后30天，绘制排斥评分曲线及角膜植片生存曲线(参见实施例3)。如图9所示，注射AAV-shRel组的角膜植片情况更透亮，在AAV6-shRel治疗后，实验组小鼠排斥率明显降低，植片发生排斥更晚。
剪取术后14-21天排斥高峰期小鼠角膜进行匀浆(参见实施例3)，通过ELISA检测实验组及对照组小鼠角膜组织中炎症因子分泌情况，通过western blot技术在蛋白水平检测，如图10所示，AAV6-shRel治疗组小鼠炎症因子分泌显著降低(P＜0.05)；小鼠角膜中c-Rel表达下调。
实施例10 毒副作用
1、常规检查
我们分别给正常BALB/c小鼠结膜下注射AAV6-NC、AAV6-shRel，第1、2、3、7和14天对三组小鼠眼睛进行眼常规检查，主要检测三组小鼠泪液分泌，角膜敏感度情况。如图11所示，差异不大，差异不具有统计学意义(P＞0.05)。
AAV6眼常规毒理：将BALB/c小鼠分为2组，A组小鼠注射AAV6-NC病毒，B组小鼠注射AAV6-shRel病毒，在病毒处理后的第1、3、7和14天对眼睛进行常规检查，包括：泪液分泌、角膜敏感的测定。泪液分泌检测：将小鼠眼睑轻轻拉开，用眼科显微镊取出酚红棉线，轻轻放入小鼠下眼睑中外1/3处，使用电子计时器计时15s， 轻取酚红棉线，使用包装背面的刻度尺测量泪液浸润部分长度，每只眼睛测量3次，取平均值。角膜敏感度检测:在小鼠处于安静状态时，保持小鼠眼睛自然张开状态，从6cm-1cm调整触针长度，每次缩短5mm，轻扫小鼠角膜中央部位，直至出现反射性瞬目反应，此时触针长度计为角膜敏感度数值，3次测量取平均值并记录，5.5mm-6mm计为正常。
2、肿瘤测试
我们使用黑色素瘤细胞B16(购于武汉普诺赛生命科技有限公司)构建小鼠皮肤表面荷瘤模型，将BALB/C小鼠分为三组，分别备皮后均接种10 8个B16细胞，后隔天分别于结膜下注射8uL PBS、AAV6-NC、AAV6-shRel，每隔3天注射一次PBS、AAV6-NC、AAV6-shRel。观察并使用游标卡尺测量三组小鼠肿瘤体积(长*宽*高),观察10天后情况发现差异无统计学意义(P＞0.05)，结果见图12A。
3、细胞凋亡测试
使用ARPE-19、A549、Jurkat、B16、MDA-MB-231、EL4细胞株(均购买于上海细胞库)检测细胞增殖情况，结果见图12B。将0.4m/mL的ARPE-19、A549、Jurkat、B16、MAD-MB-231、EL4细胞配置成细胞悬液分别种于96孔板中，每种细胞中加入6μL AAV6-NC、AAV6-shRel培养48h后收集细胞，PBS洗涤细胞。加入500μl的Bind Buffer悬浮细胞，先后加入5μl Annexin V-FITC和5μl 7-AAD混匀后室温避光孵育15min，上流式细胞仪分析检测。发现三组处理间细胞凋亡差异无统计学意义(P＞0.05)。
以上对本发明实施例进行了详细介绍，本文中应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明仅用于帮助理解本发明的方法及其核心思想。同时，本领域技术人员依据本发明的思想，基于本发明的具体实施方式及应用范围上做出的改变或变形之处，都属于本发明保护的范围。综上所述，本说明书内容不应理解为对本发明的限制。

Claims (11)

  1. c-Rel特异性核糖核酸在用于制备预防、抑制和/或治疗角膜移植排斥的药物的应用，其序列如SED ID NO.1、SED ID NO.2或SED ID NO.3、SED ID NO.4所示：
    正义链5’CAACCGGACATACCCGTCT 3’(SED ID NO.1)
    反义链5’AGACGGGTATGTCCGGTTG 3’(SED ID NO.2)；
    或
    正义链5’CAACCGAACATACCCTTCT 3’(SED ID NO.3)
    反义链5’AGAAGGGTATGTTCGGTTG 3’(SED ID NO.4)。
  2. 一种重组腺相关病毒，其特征在于，用于在角膜移植排斥个体的细胞内抑制c-Rel基因的表达和/或抑制c-Rel调控的炎症因子的表达，所述重组腺相关病毒包含以c-Rel为靶点的c-Rel特异性核糖核酸或其功能等价物，所述c-Rel特异性核糖核酸序列如SED ID NO.1、SED ID NO.2或SED ID NO.3、SED ID NO.4所示：
    正义链5’CAACCGGACATACCCGTCT 3’(SED ID NO.1)
    反义链5’AGACGGGTATGTCCGGTTG 3’(SED ID NO.2)；
    或
    正义链5’CAACCGAACATACCCTTCT 3’(SED ID NO.3)
    反义链5’AGAAGGGTATGTTCGGTTG 3’(SED ID NO.4)；
    所述功能等价物与SEQ ID NO：1-4所示的序列具有至少95％、96％、97％、98％或99％的同一性；
    所述重组腺相关病毒的血清型包括AAV2、AAV4、AAV5、AAV6、AAV8及AAV9。
  3. 如权利要求2所述的重组腺相关病毒，其特征在于，所述重组腺相关病毒的血清型为AAV6，包装滴度为5×10E 12～1×10E 13vg/mL。
  4. 如权利要求2所述的重组腺相关病毒，其特征在于，所述c-Rel调控的炎症因子包括TNF-α、IL-1β、IL-17a、IFN-γ、IL-2、IL-6中的一个或多个。
  5. 一种用于抑制角膜移植排斥的药用组合物，其特征在于，包含以c-Rel为靶点的c-Rel特异性核糖核酸或其功能等价物、药用可接受载体和/或药用可接受辅料；
    所述以c-Rel为靶点的c-Rel特异性核糖核酸，其序列如SED ID NO.1、SED ID NO.2或SED ID NO.3、SED ID NO.4所示：
    正义链5’CAACCGGACATACCCGTCT 3’(SED ID NO.1)
    反义链5’AGACGGGTATGTCCGGTTG 3’(SED ID NO.2)；
    或
    正义链5’CAACCGAACATACCCTTCT 3’(SED ID NO.3)
    反义链5’AGAAGGGTATGTTCGGTTG 3’(SED ID NO.4)；
    所述功能等价物与SEQ ID NO：1-4所示的序列具有至少95％、96％、97％、98％或99％的同一性；
    所述药用可接受载体包括质粒载体和/或病毒载体；所述病毒载体包括腺相关病毒。
  6. 如权利要求5所述的药用组合物，其特征在于，所述腺相关病毒的血清型为AAV6,病毒包装滴度为5×10E 12～1×10E 13vg/mL；可选地包装滴度为6×10E 12～7×10E 12vg/mL。
  7. 如权利要求5所述的药用组合物，其特征在于，所述药用组合物的剂型为注射液，所述药用组合物通过结膜注射施用于眼部。
  8. 如权利要求2-4所述的重组腺相关病毒、或如权利要求5-7所述的药用组合物在用于制备预防、抑制和治疗角膜移植排斥的药物中的应用。
  9. 如权利要求2-4所述的重组腺相关病毒、或如权利要求5-7所述的药用组合物在用于制备抑制角膜移植后排斥患者细胞内c-Rel基因的表达和/或c-Rel调控的炎症因子的表达的药物中的应用。
  10. 如权利要求9所述的应用，其特征在于，包括施予角膜移植后个体有效量的c-Rel特异性核糖核酸从而抑制所述个体体内巨噬细胞、树突状细胞、淋巴细胞、单核细胞、CD4 +T细胞中一种或多种细胞内c-Rel基因的表达和/或抑制c-Rel调控的炎症因子的表达的过程。
  11. 一种用于预防、抑制和/或治疗角膜移植后排斥反应的方法，其特征在于：施予角膜移植后个体有效量的如权利要求1所述的c-Rel特异性核糖核酸、或如权利要求2-4所述的重组腺相关病毒、或如权利要求5-7所述的药用组合物。
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