WO2025010935A1 - 用于治疗类风湿性关节炎的药物 - Google Patents

用于治疗类风湿性关节炎的药物 Download PDF

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WO2025010935A1
WO2025010935A1 PCT/CN2023/137624 CN2023137624W WO2025010935A1 WO 2025010935 A1 WO2025010935 A1 WO 2025010935A1 CN 2023137624 W CN2023137624 W CN 2023137624W WO 2025010935 A1 WO2025010935 A1 WO 2025010935A1
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vector
drug
qprt
gene therapy
injection
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恩琼格露西·万吉鲁
於邱黎阳
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Shenzhen Institute of Advanced Technology of CAS
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    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • 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
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • 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
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1077—Pentosyltransferases (2.4.2)
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/02—Pentosyltransferases (2.4.2)
    • C12Y204/02019—Nicotinate-nucleotide diphosphorylase (carboxylating) (2.4.2.19)
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to the technical field of diagnosis and treatment of rheumatoid arthritis, and in particular to a medicine for treating rheumatoid arthritis.
  • RA Rheumatoid arthritis
  • cytokines Rheumatoid arthritis
  • Abnormal cell metabolism is one of the important reasons that promote inflammatory and immune responses in RA.
  • the therapies available for RA patients are mainly based on the use of immunosuppressants such as methotrexate or strategies that target inflammatory cytokines, block cytokines or their receptors, which target downstream events of the inflammatory process.
  • immunosuppressants such as methotrexate or strategies that target inflammatory cytokines, block cytokines or their receptors, which target downstream events of the inflammatory process.
  • cytokines and their downstream events are crucial for many physiological processes, and their targeting will undoubtedly lead to adverse side effects.
  • immunotherapies such as methotrexate also have significant hepatotoxicity and renal toxicity, which is the main reason why most patients have to stop taking the drugs.
  • immunotherapies such as methotrexate
  • renal toxicity which is the main reason why most patients have to stop taking the drugs.
  • due to the heterogeneity of the clinical and pathogenesis of RA, such as cytokine production, autoantibodies, and response to specific cytokine inhibitors only a portion of patients respond to approved small molecule inhibitors and biologics.
  • Nicotinamide adenine dinucleotide plays an important role in regulating cellular metabolism and modulating important signaling pathways including cytokine production and immune response.
  • NAD+ is considered to be an upstream mechanism for tumor necrosis factor (TNF) secretion in T cells that promote RA, and the restoration of NAD+ levels in cells prevents excessive TNF production and inflammation.
  • TNF tumor necrosis factor
  • NAD+ precursors such as nicotinic acid mononucleotide (NMN) and nicotinic acid ribonucleotide (NR) in RA intervention.
  • NNN nicotinic acid mononucleotide
  • NR nicotinic acid ribonucleotide
  • these compounds can only exert short-term effects, require long-term administration, and have limited response rates, which can lead to patient intolerance and economic burden.
  • oral administration and high therapeutic doses of these compounds can lead to systemic effects, poor ability to target specific tissues and organs, and increased risk of toxicity and side effects.
  • Given the potential therapeutic role of NAD+ in RA it is necessary to identify strategies that can produce NAD+ in a targeted, long-term and sustained manner.
  • the purpose of the present invention is to provide a drug for treating RA, aiming to solve the problem that the existing drugs for treating RA have short efficacy and large dosage.
  • the purpose of the present invention is to provide a drug for treating RA, aiming to solve the problem that the existing drugs for treating RA have short efficacy and large dosage.
  • the present invention provides a drug for treating RA, comprising a gene therapy vector, wherein the gene therapy vector comprises a nucleic acid sequence encoding quinolinate phosphoribosyltransferase (QPRT).
  • QPRT quinolinate phosphoribosyltransferase
  • the gene therapy vector includes at least one of a viral vector and a non-viral vector.
  • the viral vector includes one or more of an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, and a herpes simplex viral vector.
  • the adeno-associated virus vector includes one or more of an AAV1 vector, an AAV2 vector, an AAV5 vector, an AAV6 vector, an AAV8 vector, an AAV9 vector, and an AAVrh10 vector.
  • the non-viral vector comprises one or more of a plasmid, a cationic lipid, a lipid nanoparticle, a liposome, a polymer-based carrier, or an extracellular vesicle.
  • the drug further comprises a pharmaceutically acceptable excipient.
  • the drug is in the form of an injection.
  • the drug is administered by one or more of intravenous injection, subcutaneous injection, intramuscular injection, intraarticular injection, and intradermal injection.
  • the second aspect of the present invention provides use of a reagent for detecting QPRT in the preparation of a product for diagnosing RA.
  • the product includes one of a kit and a test paper.
  • QPRT is the rate-limiting enzyme in the de novo synthesis pathway of NAD+ and is involved in a large number of important cellular processes, including inflammatory responses, pathogen resistance, and tissue remodeling. Therefore, the above-mentioned gene therapy drugs for the treatment of RA are provided.
  • the drugs provided by the present invention can restore or increase the ability of cells to produce NAD+ by providing prolonged or sustained QPRT in vivo, regulate the process in the secretory pathway, thereby preventing the pathological secretion of cytokines, stopping the inflammatory process at the point of origin, and effectively avoiding immune responses, neovascularization, and tissue destruction in the joints.
  • the gene therapy-based drugs provided by the present invention can improve the spatiotemporal distribution of QPRT in the joints, improve the therapeutic effect, reduce side effects, and can achieve prolonged or sustained release of QPRT, with long drug efficacy, and with the prolonged expression of QPRT, the dosage of the drug can be reduced.
  • Figure 1 shows the mRNA expression results of QPRT in synovial tissue, where a is the normal group and RA group, and b is the OA group and RA group.
  • a is a strip diagram of different cytokines
  • b is a concentration result diagram of TNF- ⁇
  • c is a concentration result diagram of CTGF.
  • FIG3 shows blood vessels in endothelial cells grown in different conditioned medium, wherein a is the shCtrl group and b is the shQPRT group.
  • a is a graph showing the number of vascular nodes
  • b is a graph showing the number of vascular intersections.
  • a is a curve diagram showing the changes in clinical arthritis scores as the number of weeks after injection and immunization increases
  • b is a graph showing the area under the curve in a
  • c is a curve diagram showing the changes in paw volume as the number of weeks after injection and immunization increases
  • d is a graph showing the area under the curve in c.
  • a is a strip diagram of QPRT expression
  • b is a bar diagram of QPRT expression.
  • FIG. 7 is a graph showing the results of NAD+ concentrations in serum of different groups.
  • NAD+ is an upstream process that promotes TNF secretion in T cells of RA, and its restoration in cells prevents overproduction of TNF and inflammation.
  • direct administration of NAD+ precursors is ineffective for treating RA.
  • QPRT is the rate-limiting enzyme in the de novo synthesis pathway of NAD+ and is involved in a large number of important cellular processes, including inflammatory responses, pathogen resistance, and tissue remodeling.
  • the present invention provides a drug for treating RA, including a gene therapy vector, which comprises a nucleic acid sequence encoding QPRT.
  • the mRNA sequence encoding QPRT is available at NM_133686.1 of the National Center for Biotechnology Information (NCBI), and its sequence is shown in SEQ ID NO: 1.
  • the gene therapy-based drug provided by the present invention can restore or increase the ability of cells to produce NAD+ by providing prolonged or sustained QPRT in vivo, regulate the process in the secretory pathway, thereby preventing the pathological secretion of cytokines, stopping the inflammatory process at the point of origin, and effectively avoiding immune response, neovascularization and tissue destruction in the joints.
  • the gene therapy-based drug provided by the present invention can improve the spatiotemporal distribution of QPRT in the joints, improve the therapeutic effect, reduce side effects, and can achieve prolonged or sustained release of QPRT, with long drug efficacy, and with the prolonged expression of QPRT, the dosage of the drug can be reduced.
  • the gene therapy method of delivering QPRT to the joints of the present invention can provide patients with better treatment effects, because the gene therapy in the invention can provide extended or sustained QPRT in vivo without the need for repeated injections, reducing the risk of increased inflammation and other adverse reactions. Therefore, delivering QPRT to the joints of patients through gene therapy can provide patients with a safer, more cost-effective and more convenient treatment option.
  • the gene therapy vector includes at least one of a viral vector and a non-viral vector, but is not limited thereto.
  • the viral vector includes one or more of adeno-associated virus (AAV) vector, adenovirus (Ad) vector, lentivirus (LV) vector, and herpes simplex virus (HSV) vector, but is not limited thereto.
  • AAV adeno-associated virus
  • Ad adenovirus
  • LV lentivirus
  • HSV herpes simplex virus
  • the AAV vector includes one or more of an AAV1 vector, an AAV2 vector, an AAV5 vector, an AAV6 vector, an AAV8 vector, an AAV9 vector, and an AAVrh10 vector.
  • AVV can be divided into AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, and AAVrh10 according to different serotypes.
  • the gene therapy vector when the gene therapy vector is selected from AAV2, the gene therapy vector comprising the nucleic acid sequence encoding QPRT is recorded as AAV2-QPRT, and the drug for treating RA includes AAV2-QPRT.
  • the non-viral vector includes one or more of a plasmid, a cationic lipid, a lipid nanoparticle (LNP), a liposome, a polymer-based carrier, and an extracellular vesicle, but is not limited thereto.
  • the drug further comprises a pharmaceutically acceptable excipient, but is not limited thereto.
  • the pharmaceutically acceptable excipient includes, but is not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances (such as phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, cyclodextrins, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block copolymers, polyethylene glycol and lanolin.
  • the drug is in the form of an injection, which can be in the form of a lyophilized powder, a solution, or a suspension.
  • the drug is administered by one or more of intravenous (IV) injection, subcutaneous (SC) injection, intramuscular (IM) injection, intraarticular (IA) injection, and intradermal (ID) injection.
  • IV intravenous
  • SC subcutaneous
  • IM intramuscular
  • IA intraarticular
  • ID intradermal
  • the embodiment of the present invention also provides the use of a reagent for detecting QPRT in the preparation of a product for diagnosing rheumatoid arthritis.
  • the present invention confirms that the reduction of QPRT promotes the production and secretion of cytokines, angiogenesis and inflammation. Therefore, it can be proved that QPRT is a potential RA biomarker and can be used as a target for RA intervention. Therefore, the reagent for detecting QPRT can be used to diagnose RA.
  • the product includes one of a kit and a test paper, but is not limited thereto.
  • Gene expression datasets on RA were retrieved from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/). The study types were limited to array and human species expression profiling. The following keywords were used to search for datasets: “rheumatoid arthritis”, “RA”, “Homo sapiens”, “Microarray”, and “high-throughput sequencing”, i.e., “rheumatoid arthritis”, “RA”, “Homo sapiens”, “microarray”, and “high-throughput sequencing”, respectively.
  • GEO Gene Expression Omnibus
  • mRNA profiles of RA patients and controls were detected using high-throughput arrays or next-generation sequencing; (2) the datasets contained at least 3 samples each for RA and healthy controls; and (3) total RNA was extracted from human knee synovial tissue.
  • GEO dataset GSE1919
  • the groups were set as RA group, and the normal group and osteoarthritis (OA) group were used as controls, without other treatments. Then, the expression of QPRT in different groups was analyzed, i.e., RA group versus normal group and RA group versus OA group.
  • the logarithm of QPRT mRNA expression was calculated by calculating the logarithm of FKPM (fragments per kilobase) of QPRT in each group. The results are shown in Figure 1a and b. It can be seen that the mRNA expression of QPRT in the synovium of the RA group was reduced compared with the normal group and the OA group. That is, QPRT is inhibited in the RA synovium, and this inhibition is related to the inflammatory process of RA.
  • the patients in this example met the American College of Rheumatology/European League against Rheumatism classification criteria for RA.
  • Synovial tissues from 6 patients with active RA who underwent knee replacement surgery were washed three times with sterile phosphate-buffered solution ( PBS ) containing 10% streptomycin and penicillin, and then washed three times with PBS containing 1% streptomycin and penicillin.
  • PBS sterile phosphate-buffered solution
  • the washed synovial tissue was transferred to a sterile culture dish and cut into small pieces (1-2 mm in size) using a sterile scalpel. These synovial tissue pieces were then evenly distributed on the bottom of a 25 cm 2 (T25) culture flask and the flask was placed upright in an incubator to allow it to adhere. 5 mL of fibroblast culture medium containing 10% fetal bovine serum (FBS) was added to the culture flask and then incubated in an incubator at 37°C and 5% CO 2. After 5-7 days, FLSs were observed to migrate from the synovial tissue and adhere to the surface of the culture flask. The culture medium was discarded and the attached FLSs were washed with sterile PBS to remove non-adherent cells. The FLSs obtained from the tissue were considered to be passage 0 (P0).
  • P0 passage 0
  • FLSs were stored in T25 flasks and cultured at 5% CO 2 and 37°C, with the culture medium replaced every 2 days. When the cells reached >85% confluence, the culture medium was removed, the adherent cells were washed with PBS, and then added to a trypsin solution (0.25% trypsin/1 mM ethylenediaminetetraacetic acid (EDTA)) for digestion. Next, fibroblast culture medium containing 10% FBS was added to the digested tissue to stop the enzyme activity and terminate the digestion. The resulting cell suspension was centrifuged at 1200 rpm for 5 min. The cells were then divided into two parts and inoculated into new culture flasks, which was considered to be passage 1 (P1). After confluence, this process was repeated, and the cell passage n+1 was increased in each cycle.
  • the FLSs used in the following examples were from passages 3-6, which is a homogeneous cell population.
  • the pGPU6/Hygro plasmid cloned with the shRNA sequence targeting QPRT (shQPRT) and the pGPU6/Hygro plasmid cloned with the control shRNA sequence (shCtrl) were purchased from Genepharma and were respectively referred to as shQPRT plasmid and shCtrl plasmid.
  • the packaging plasmids psPAX2 (addgene #394976) and pMD2.G (addgene #12259) were from the Genepharma SuperSilencingTM shRNA Plasmid Expression Vector Set (C01001).
  • control shRNA sequence (shCtrl) is shown in SEQ ID NO: 2, specifically: GTTCTCCGAACGTGTCACGT.
  • shRNA sequences include shQPRT#1: GCCATATTTACCCAACTCAAC (as shown in SEQ ID NO: 3), shQPRT#2: GATGGTGAAGGATAACCATGT (as shown in SEQ ID NO: 4), and shQPRT#3: GGCTGACTTCACTCTGAAGGT (as shown in SEQ ID NO: 5).
  • HEK 293T cells were seeded into 6-well cell culture plates at a density of 4 ⁇ 10 5 cells/well, and 2 mL of DMEM medium was added to each well. After 24 hours, the cells were washed with sterile PBS, and 2 mL of Opti-MEM medium was added to the cells.
  • the transfection mixture was added to the above HEK 293T cells and incubated at 37°C in a constant temperature incubator with 5% CO 2 for 6 hours. The medium was then replaced with high-glucose DMEM medium containing 10% FBS and cultured for another 72 hours. The supernatant containing the lentivirus was collected by collecting the cell culture medium and filtering through a 0.45 ⁇ m syringe.
  • the supernatant containing the lentivirus was taken, and polystyrene was added thereto at a polystyrene concentration of 8 ⁇ g/mL to obtain a lentivirus supernatant containing polystyrene.
  • Human RAFLSs were seeded into 6-well cell culture plates at a density of 1 ⁇ 10 5 cells/well. After incubation for 24 hours, the cells were washed with sterile PBS, and 2 mL of lentiviral supernatant containing polystyrene was added to the cells and incubated at 37°C in a constant temperature incubator with 5% CO 2 for 24 hours. Then, new high-glucose DMEM medium containing 10% FBS was added and incubated for another 24 hours. After 48 hours, shQPRT-transfected human RA FLSs were obtained.
  • shQPRT plasmid was replaced with the shCtrl plasmid in the preparation step of the lentivirus, and finally shCtrl-transfected human RA FLSs were obtained.
  • shQPRT and shCtrl transfected human RA FLSs were starved overnight in 1% FBS conditioned medium. The next day, the medium was removed, and fresh high-glucose DMEM medium containing 10% FBS was added to the cells, and cultured in an incubator at 37°C and 5% CO 2 for 24 hours. After 24 hours, the medium was collected, centrifuged and stored at -20°C to obtain QPRT-silenced shQPRT human RAFLSs conditioned medium and shCtrl human RA FLSs conditioned medium, respectively.
  • a node is defined as a point where three or more tubes intersect, and an intersection is defined as a point where two tubes intersect.
  • the number of nodes and intersections can be used as a quantitative indicator of the complexity and branching of the tube network. Therefore, it is shown that QPRT silencing or inhibition can increase the formation of blood vessels.
  • AAV2 ITR-QPRT which contains the target gene encoding QPRT
  • pHelper carrying adenovirus-derived genes
  • pAAV-RC carrying AAV replication and the corresponding capsid gene that determines the AAV serotype
  • pCMV-SPORT6-QPRT plasmid provides the target gene encoding QPRT; rAAV-CMV-WPRE-pA plasmid; AAV2/2 (Cat.
  • PT-0545 contains key elements, and the shuttle plasmid (i.e. AAV2 ITR-QPRT, i.e. rAAV-CMV-Qprt (m) -WPRE-pA) is prepared from these two plasmids.
  • AAV2 ITR-QPRT i.e. rAAV-CMV-Qprt (m) -WPRE-pA
  • the pAAV-RC (pAAVRepCap, carrying AAV replication and the corresponding capsid genes that determine AAV serotype) plasmid and the pHelper (carrying genes from an adenoviral source) plasmid are used together with a shuttle plasmid (AAV2 ITR-QPRT, which contains the gene of interest encoding QPRT) to produce infectious AAV2 particles.
  • ITR inverted terminal repeat.
  • transfection reagent dilution solution Dropwise into the plasmid dilution solution, mix gently while adding, and place at room temperature for 20 minutes to allow the plasmid and transfection reagent to fully combine to form a stable transfection complex.
  • step 2) Freeze the cell lysate in step 2) in liquid nitrogen for 3 min, place in a 37°C water bath, and freeze and thaw 3 times.
  • step 2) Add 250U of benzonase nuclease, mix well and incubate at 37°C for 1h.
  • step 5) Add NaCl to a final concentration of 150mM, mix well and incubate at 37°C for 30min.
  • step 6) Pour the supernatant obtained after centrifugation in step 6) into the supernatant collected after centrifugation in step 2), add PEG8000 and NaCl to final concentrations of 8% and 0.5M respectively, and incubate at 4°C overnight. 8) Centrifuge at 12000rpm and 4°C for 1.5h, discard the supernatant, and resuspend the precipitate with 10mL PBS.
  • the titer of AAV2 is determined by detecting the genome copy number of the AAV2 vector in the genome through quantitative PCR to determine the number of AAV2 viral particles.
  • CIA and RA have similarities in preclinical, pathological, and immunological aspects.
  • mice Seventeen 8-week-old female DBA mice were injected intradermally at the base of their tails with 100 ⁇ L of an emulsion of chicken type II collagen and Freund's incomplete adjuvant (1:1) for immunization on day 0 to establish CIA model mice, which were then treated on day 14 after immunization.
  • mice Seventeen CIA model mice were divided into two groups. The first group was the CIA group (7 mice, not treated, injected with empty AAV2), and the second group was the AAV-QPRT group (10 mice, treated, injected with AAV2-QPRT).
  • mice Since the size of the mouse hind paw is almost twice that of the front paw, on the 14th day after immunization, the AAV-QPRT group mice were treated by percutaneous injection of 1.5 ⁇ 10 9 viral loads/paw (with PBS as the solvent, injection of 2.5 ⁇ L) into the front paws and intradermal injection of 3.3 ⁇ 10 9 viral loads/paw (with PBS as the solvent, injection of 5 ⁇ L) into the hind paws.
  • each mouse in the CIA group and the AAV-QPRT group was scored using the above-mentioned scoring criteria for the severity of arthritis, and data was recorded for each mouse, and a clinical arthritis score curve was drawn (see Figure 5a); the area under the arthritis score curve (AUC) in Figure 5a was calculated using Prism software, and the results are shown in Figure 5b. It can be seen that the score of the AAV-QPRT group was significantly lower than that of the CIA group, confirming the anti-arthritis effect of AAV2-QPRT, and the overexpression of QPRT significantly inhibited the severity of arthritis in CIA mice, and AAV2-QPRT can effectively treat RA.
  • the volume of the paw of each mouse in the CIA group and the AAV-QPRT group was measured at week 0, 1, 2, 3, 4, 5, 6, and 7 after immunization, and the results are shown in Figure 5c, where Sham and QPRT were the control groups for CIA and AAV-QPRT, respectively.
  • the area under the curve (AUC) of the mouse paw volume in Figure 5c was calculated using Prism software, and the results are shown in Figure 5d, which showed that the volume of the mouse paw in the AAV-QPRT group was significantly lower than that in the CIA group, confirming the anti-arthritis effect of AAV2-QPRT.
  • the overexpression of QPRT significantly inhibited the severity of arthritis in CIA mice, and AAV2-QPRT can effectively treat RA.
  • mice All mice were euthanized six weeks after AAV2-QPRT injection, and their paws were subjected to immunohistochemistry and histology.
  • the mouse paw tissue was homogenized and then lysed with RIPA buffer containing protease inhibitors and phosphatase inhibitors. 40 ⁇ g of the sample was loaded onto a 10% polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was then blocked with TBS/Tween-20 containing 5% skim milk for 1 h and then overnight at 4°C. The membrane was incubated with the secondary antibody for 1 h, and the bands were observed using ECL. The signals from the bands were quantified by ImageJ software (National Institutes of Health, USA). The results are shown in Figure 6, and it can be seen that the protein expression of QPRT increased after the mouse paw was injected with AAV2-QPRT for treatment. The two AAV-QPRT groups in a represent different mice.
  • mice Serum samples of mice were obtained and tested for NAD+ concentration. The results are shown in FIG7 . It can be seen that compared with the untreated CIA group, the NAD+ concentration in the whole blood increased after the mice were injected with AAV2-QPRT into their paws for treatment.
  • AAV2-QPRT-mediated high expression of QPRT can effectively upregulate tissue QPRT expression, reduce cell angiogenesis, increase receptor NAD+ levels, and reduce the paw thickness, volume and clinical scores of CIA model mice.
  • AAV2 delivery of QPRT provides tissue and conduction specificity, improves the spatiotemporal distribution of QPRT in the joints, thereby improving its therapeutic effect, while reducing side effects, and can achieve prolonged or sustained release of QPRT, with long-lasting drug effects. With the prolonged expression of QPRT, the dosage of the drug can be reduced.
  • immunosuppressive drugs may be detrimental to people in the asymptomatic preclinical stage of RA, and the drugs of the present invention provide an alternative method to combat immune responses.

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Abstract

提供一种用于治疗类风湿性关节炎的药物，所述药物包括基因治疗载体，所述基因治疗载体包含编码喹啉酸磷酸核糖基转移酶（QPRT）的核酸序列。提供的药物通过在体内提供延长或持续的QPRT，可以恢复或增加细胞产生NAD+的能力，调节分泌途径中的过程，从而阻止细胞因子的病理性分泌，在起源点停止炎症过程，有效避免关节中的免疫反应、新生血管形成和组织破坏。提供的基于基因治疗的药物能够改善时空分布，提高治疗效果，减少副作用，能够实现QPRT的延长或持续释放，药效长，且随着QPRT的延长表达，可以减少药物的使用剂量。

Description

用于治疗类风湿性关节炎的药物 技术领域
本发明涉及类风湿性关节炎诊断与治疗技术领域，尤其涉及用于治疗类风湿性关节炎的药物。
 背景技术
类风湿性关节炎（RA）是一种慢性自身免疫性疾病，其特征是过度产生细胞因子，从而导致炎症和血管生成，最终导致自身免疫和关节组织破坏。且细胞代谢异常是促进RA炎症和免疫反应的重要原因之一。目前，可用于RA患者的疗法主要是基于利用甲氨蝶呤等免疫抑制剂或靶向炎性细胞因子、阻断细胞因子或其受体的策略，这些策略针对的是炎症过程的下游事件，然而细胞因子及其下游事件对许多生理过程至关重要，它们的靶向无疑会导致不良的副作用。此外，在临床上，甲氨蝶呤等免疫制剂还存在明显的肝肾毒性，这是大部分患者不得已停药的主要原因。此外，由于RA的临床和发病机制的异质性，如细胞因子的产生、自身抗体和对特定细胞因子抑制剂的反应等，只有一部分患者对批准的小分子抑制剂和生物制剂有反应。
因此，针对上游生化和代谢事件的治疗方法在起点干预病理过程是一种非常有前景的策略，有望解决现有针对炎症过程的下游事件的策略存在的上述问题。烟酰胺腺嘌呤二核苷酸（NAD+）在调节细胞代谢和调节包括细胞因子产生和免疫反应在内的重要信号通路方面发挥着重要作用。NAD+被认为是促进RA的T细胞中肿瘤坏死因子（TNF）分泌的上游机制，NAD+水平在细胞中的恢复阻止了TNF的过量产生和炎症。因此，前期科研团队已经研究了几种NAD+前体，如烟酸单核苷酸（NMN）和烟酸核糖核苷酸（NR）在RA干预中的应用。然而，这些化合物只能发挥短期作用，需要长期给药，且响应率有限，这会导致患者的不耐受和经济负担。此外，这些化合物的口服给药和高治疗剂量会导致全身效应，靶向特定组织和器官的能力不强，有增加毒性和副作用的风险。鉴于NAD+在RA中的潜在治疗作用，有必要确定能够靶向、长期和持续产生NAD+的策略。
因此，现有技术还有待于改进和发展。
 技术问题
鉴于上述现有技术的不足，本发明的目的在于提供用于治疗RA的药物，旨在解决现有用于治疗RA的药物药效短、用量大的问题。
 技术解决方案
鉴于上述现有技术的不足，本发明的目的在于提供用于治疗RA的药物，旨在解决现有用于治疗RA的药物药效短、用量大的问题。
本发明的技术方案如下：
本发明的第一方面，提供一种用于治疗RA的药物，其中，包括基因治疗载体，所述基因治疗载体包含编码喹啉酸磷酸核糖基转移酶（QPRT）的核酸序列。
可选地，所述基因治疗载体包括病毒载体、非病毒载体中的至少一种。
可选地，所述病毒载体包括腺相关病毒载体、腺病毒载体、慢病毒载体、单纯疱疹病毒载体中的一种或多种。
可选地，所述腺相关病毒载体包括AAV1载体、AAV2载体、AAV5载体、AAV6载体、AAV8载体、AAV9载体、AAVrh10载体中的一种或多种。
可选地，所述非病毒性载体包括质粒、阳离子脂质、脂质纳米颗粒、脂质体、聚合物基载体、细胞外囊泡中的一种或多种。
可选地，所述药物还包括药学上可接受的辅料。
可选地，所述药物的剂型为注射剂。
可选地，所述药物的给药方式为静脉内注射、皮下注射、肌肉注射、关节内注射、皮内注射中的一种或多种。
本发明的第二方面，提供检测QPRT的试剂在制备用于诊断RA的产品中的应用。
可选地，所述产品包括试剂盒、试纸中的一种。
 有益效果
有益效果：发明人发现QPRT是NAD+从头合成途径中的限速酶，参与了大量重要的细胞过程，包括炎症反应、病原体耐药性和组织重塑。因此，提供了上述用于治疗RA的基因治疗药物，本发明提供的药物通过在体内提供延长或持续的QPRT，可以恢复或增加细胞产生NAD+的能力，调节分泌途径中的过程，从而阻止细胞因子的病理性分泌，在起源点停止炎症过程，有效避免关节中的免疫反应、新生血管形成和组织破坏。本发明提供的基于基因治疗的药物能够改善QPRT在关节中的时空分布，提高治疗效果，减少副作用，能够实现QPRT的延长或持续释放，药效长，且随着QPRT的延长表达，可以减少药物的使用剂量。
 附图说明
图1为滑膜组织中QPRT的mRNA表达结果，其中a为正常组和RA组，b为OA组和RA组。
图2中a为不同细胞因子的条带图，b为TNF-α的浓度结果图，c为CTGF的浓度结果图。
图3为不同条件培养基上生长的内皮细胞中的血管图，其中a为shCtrl组，b为shQPRT组。
图4中a为血管节点数量图，b为血管交叉点数量图。
图5中a为临床关节炎评分随着注射免疫周数的增加的变化曲线图，b为a中曲线下方区域的面积图，c为爪子的体积随着注射免疫周数的增加的变化曲线图，d为c中曲线下方区域的面积图。
图6中a为QPRT表达条带图，b为QPRT表达柱状图。
图7为不同组别的血清中NAD+浓度的结果图。
附图中，*表示与对照组/假手术组相比p<0.05，**表示与对照组/假手术组相比p＜0.01，***表示与对照组/假手术组相比p＜0.001； ####表示与CIA组相比p＜0.0001；ns表示不显著。
 本发明的实施方式
本发明提供一种用于治疗RA的药物，为使本发明的目的、技术方案及效果更加清楚、明确，以下对本发明进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
除非另有定义，本文所使用的所有的技术术语和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施方式的目的，不是旨在于限制本发明。
NAD+是促进RA的T细胞中TNF分泌的上游过程，其在细胞中的恢复阻止了TNF的过量产生和炎症。然而，NAD+前体的直接给药对治疗RA无效。发明人发现QPRT是NAD+从头合成途径中的限速酶，参与了大量重要的细胞过程，包括炎症反应、病原体耐药性和组织重塑。基于此，本发明提供给一种用于治疗RA的药物，其中包括基因治疗载体，所述基因治疗载体包含编码QPRT的核酸序列。编码QPRT的mRNA序列可在美国国家生物技术信息中心（NCBI）NM_133686.1获得，其序列如SEQ ID NO：1所示。
本发明提供的基于基因治疗的药物通过在体内提供延长或持续的QPRT，可以恢复或增加细胞产生NAD+的能力，调节分泌途径中的过程，从而阻止细胞因子的病理性分泌，在起源点停止炎症过程，有效避免关节中的免疫反应、新生血管形成和组织破坏。本发明提供的基于基因治疗的药物能够改善QPRT在关节中的时空分布，提高治疗效果，减少副作用，能够实现QPRT的延长或持续释放，药效长，且随着QPRT的延长表达，可以减少药物的使用剂量。
虽然细胞因子或其受体的直接靶向是目前RA治疗的常用方法，但本发明将QPRT递送到关节中的基因治疗方法可以为患者提供更好的治疗效果，因为发明中的基因治疗可以在体内提供延长或持续的QPRT，而不需要重复注射，降低增加炎症的风险及其他不良反应。因此，通过基因治疗将QPRT输送到患者的关节中，可以为患者提供更安全、更具成本效益和更方便的治疗选择。
在一些实施方式中，所述基因治疗载体包括病毒载体、非病毒载体中的至少一种，但不限于此。
在一些实施方式中，所述病毒载体包括腺相关病毒（AAV）载体、腺病毒（Ad）载体、慢病毒（LV）载体、单纯疱疹病毒（HSV）载体中的一种或多种，但不限于此。这些病毒载体具有如下优势：（1）可通过基因修饰患者的细胞进行体外基因治疗或直接将基因材料直接传递到特定的细胞或组织进行体内基因治疗，从而实现持续长期的疗效；（2）具有细胞和组织特异性靶向作用；（3）减少全身性并发症的风险。
在一些实施方式中，所述AAV载体包括AAV1载体、AAV2载体、AAV5载体、AAV6载体、AAV8载体、AAV9载体、AAVrh10载体中的一种或多种。AVV根据血清型的不同可分为AAV1、AAV2、AAV5、AAV6、AAV8、AAV9和AAVrh10。
下文中，当基因治疗载体选自AAV2时，则包含编码QPRT的核酸序列的基因治疗载体记作AAV2-QPRT，用于治疗RA的药物包括AAV2-QPRT。
在一些实施方式中，非病毒性载体包括质粒、阳离子脂质、脂质纳米颗粒（LNP）、脂质体、聚合物基载体、细胞外囊泡中的一种或多种，但不限于此。
在一些实施方式中，所述药物还包括药学上可接受的辅料，但不限于此。所述药学上可接受的辅料包括但不限于：离子交换剂，氧化铝，硬脂酸铝，卵磷脂，血清蛋白例如人血清白蛋白，缓冲物质（如磷酸盐类），甘氨酸，山梨酸，山梨酸钾，饱和植物脂肪酸的偏甘油酯混合物，水，盐类或电解质类（例如硫酸鱼精蛋白、磷酸氢二钠、磷酸氢钾、氯化钠、锌盐），胶体二氧化硅，三硅酸镁，聚乙烯吡咯烷酮，基于纤维素的物质，聚乙二醇，环糊精类，羧甲基纤维素钠，聚丙烯酸酯类，蜡类，聚乙烯-聚氧丙烯-嵌段共聚物，聚乙二醇和羊毛脂。
在一些实施方式中，所述药物的剂型为注射剂。其形式可以是冻干粉、溶液、悬浮液。
在一些实施方式中，所述药物的给药方式为静脉内（IV）注射、皮下（SC）注射、肌肉（IM）注射、关节内（IA）注射、皮内（ID）注射中的一种或多种。
本发明实施例还提供了检测QPRT的试剂在制备用于诊断类风湿性关节炎的产品中的应用。本发明中证实了QPRT的减少促进了细胞因子的产生和分泌，促进了血管生成和炎症。因此，可以证明QPRT是一种潜在的RA生物标志物，可作为RA干预的靶点。因此，用于检测QPRT的试剂，可用于诊断RA。
在一些实施方式中，所述产品包括试剂盒、试纸中的一种，但不限于此。
下面通过具体的实施例进行详细说明。
以下实施例中所有实验均按照相关指南和规定进行。所有实验方案均经深圳先进技术研究院伦理委员会批准。将 DBA/1（H-2q）雌性小鼠品系以5-6只小鼠/笼安置在标准实验室笼中，可随意获得食物和水。
实施例1
RA滑膜、正常滑膜、骨关节炎（OA）滑膜中QPRT的表达
从Gene Expression Omnibus (GEO)数据库（https://www.ncbi.nlm.nih.gov/）中检索关于RA的基因表达数据集。研究类型仅限于阵列和人类物种的表达谱分析。使用以下关键词搜索数据集：“rheumatoid arthritis”、“RA”、“Homo sapiens”、“Microarray”和“high-throughput sequencing”即分别为“类风湿关节炎”、“RA”、“智人”、“微阵列”和“高通量测序”。采用以下标准：（1）使用高通量阵列或下一代测序检测RA患者和对照组的mRNA谱；（2）数据集中RA和健康对照组分别包含不少于3个样本；（3）从人类膝关节滑膜组织中提取总RNA。最终选定GEO数据集（GSE1919），下载了相应的基因表达谱和临床注释信息进行进一步分析。组别设置为RA组，并以正常组、骨关节炎（OA）组作为对照组，不包含其他治疗。然后分析不同组别中QPRT的表达情况，即RA 组与正常组和RA组与 OA组。通过计算各组中QPRT的FKPM（每千个碱基的片段数）的对数值来计算QPRT的mRNA表达对数值。结果如图1中a和b所示，可知，与正常（Normal）组和OA组相比，RA组滑膜中QPRT的mRNA表达减少。即QPRT在RA滑膜中受到抑制，这种抑制与RA的炎症过程有关。
实施例2人RA 成纤维样滑膜细胞（FLSs）的获取
本实施例中的患者符合美国风湿病学会/欧洲风湿病联盟的RA分类标准。
取6例接受了膝关节置换手术的活动期RA患者的滑膜组织，用含有10％链霉素和青霉素的无菌磷酸缓冲溶液 （PBS ）洗涤3次，然后用含有1％链霉素和青霉素的PBS洗涤3次。
将洗涤后的滑膜组织转移到无菌培养皿中，并使用无菌手术刀将其切成小块（大小为1-2mm），然后将这些滑膜组织小块均匀分布在25cm 2（T25）的培养瓶底部，并将瓶子竖立放置在孵箱中，使其附着。将5mL含有10％胎牛血清（FBS）的成纤维细胞培养基加入到培养瓶中，然后在37℃、5％CO 2的培养箱中孵育。5-7天后，观察到FLSs从滑膜组织中迁移并附着在培养瓶的表面上。将培养基倒掉，然后用无菌PBS清洗附着的FLSs，以去除非粘附细胞，从组织中获取的FLSs视为第0代（P0）。
FLSs保存在T25瓶中，以5％CO 2、37℃的条件培养，培养基每2天更换一次。当细胞达到>85％的融合度时，去除培养基，用PBS清洗粘附的细胞，然后加入到胰蛋白酶溶液中（0.25％胰蛋白酶/ 1 mM乙二胺四乙酸（EDTA））进行消化。接着，向消化好的组织中加入含有10％FBS的成纤维细胞培养基以停止酶活性终止消化。将得到的细胞悬液以1200rpm的速度离心5min。然后将细胞分成两部分，分别接种到新的培养瓶中，这视为第1代（P1）。汇合后，重复此过程，并且每个循环增加细胞传代n+1。以下实施例中使用的FLSs来自第3-6代，这是一种均质的细胞群。
实施例3 shRNA的转染
准备质粒：
克隆有靶向QPRT的shRNA序列(shQPRT)的pGPU6/Hygro质粒，克隆有对照shRNA 序列(shCtrl)的 pGPU6/Hygro质粒均购自Genepharma公司，分别记作shQPRT质粒和shCtrl质粒。包装质粒psPAX2(addgene #394976)和pMD2.G(addgene #12259)来自于吉玛基因SuperSilencing™ shRNA质粒表达载体套装（C01001）。
其中，对照 shRNA 序列(shCtrl)如SEQ ID NO：2所示，具体为：GTTCTCCGAACGTGTCACGT。
shRNA 序列(shQPRT)包括shQPRT#1: GCCATATTTACCCAACTCAAC（如SEQ ID NO：3所示）、shQPRT#2: GATGGTGAAGGATAACCATGT（如SEQ ID NO：4所示）、shQPRT#3: GGCTGACTTCACTCTGAAGGT（如SEQ ID NO：5所示）。
慢病毒的制备：
将HEK 293T细胞以4×10 5个细胞/孔的密度接种到6孔细胞培养板中，并向每个孔中加入2mL DMEM培养基。24小时后，用无菌PBS洗涤细胞，并向细胞中加入2mLOpti-MEM培养基。
将2μg shQPRT质粒和包装质粒稀释在100μL Opti-MEM培养基中（管A），并将18μLPEI转染试剂稀释在另外的100μL Opti-MEM培养基中（管B）。5分钟后，将管B中的物质加入到管A中并轻轻混合。将混合物在室温下孵育20分钟，得到转染混合物。
将转染混合物加入到上述HEK 293T细胞中，在5％CO 2的恒温培养箱中37℃孵育6小时。然后将培养基更换为含有10％ FBS的高糖DMEM培养基，再培养72小时。通过收集细胞培养基并通过0.45微米注射器过滤，收集包含慢病毒的上清液。
人RAFLSs转染：
取上述含慢病毒的上清液，将聚苯乙烯添加到其中，聚苯乙烯浓度为8μg/mL，得到含有聚苯乙烯的慢病毒上清液。
将人RAFLSs以1×10 5个细胞/孔的密度接种到6孔细胞培养板中，孵育24小时后，用无菌PBS洗涤细胞，并将2mL含有聚苯乙烯的慢病毒上清液添加到细胞中，在5％CO 2的恒温培养箱中37℃孵育24小时。然后加入新的含有10％ FBS的高糖DMEM培养基，再孵育24小时，48小时后，得到shQPRT转染的人RA FLSs。
采用类似的方法，只是在慢病毒的制备步骤中将采用的shQPRT质粒替换为shCtrl质粒，最终得到shCtrl转染的人RA FLSs。
采用蛋白质印迹法及酶联免疫吸附法分别对shQPRT转染的人RA FLSs和shCtrl转染的人RA FLSs的细胞因子进行检测，结果如图2所示，可见QPRT沉默的shQPRT转染的人RA FLSs中的各种细胞因子（细胞内的VEGF、分泌的VEGF、IL-6、TNF-α、CTGF）的产生和分泌增加。即QPRT沉默或抑制促进细胞因子的产生和分泌。
实施例4血管生成测定分析
条件培养基的制备：shQPRT和shCtrl转染的人RA FLSs分别在1%FBS条件培养基中饥饿过夜。第二天，取出培养基，将新鲜含有10％ FBS的高糖DMEM培养基加入细胞中，并在37℃、5%CO 2的培养箱中培养24小时。24小时后，收集培养基，离心并储存在-20℃中，分别得到QPRT沉默的shQPRT人RAFLSs条件培养基和shCtrl人RA FLSs条件培养基。
体外管形成测定：将人脐静脉内皮细胞系EA.hy926在含有10%FBS的高糖DMEM培养基中培养直至汇合。将15孔载玻片（ibidi）用50µL生长因子还原基质胶（Cornings）包被，并在37℃下固化30分钟。通过对细胞单层进行胰蛋白酶解，并将细胞以1×10 4个细胞/mL的比例重悬在QPRT沉默的shQPRT人RA FLSs条件培养基和shCtrl人RA FLSs条件培养基中，制备得到内皮细胞悬浮液。
向每个孔中加入50µL内皮细胞悬浮液，培养细胞24小时，并在×10放大倍数下成像。然后使用image J血管生成分析仪对图像进行分析，结果如图3和4所示，由图3和4可知，相比shCtrl人RA FLSs条件培养基，QPRT沉默的shQPRT人RA FLSs条件培养基上生长的内皮细胞的血管形成增加，且血管网络复杂性和分支增加。图4中a为血管节点数量图， b为血管交叉点数量图。节点定义为三条或三条以上管道相交的点，交叉点定义为两条管道相交的点。节点和交叉点的数量可以作为管道网络复杂性和分支的量化指标。由此，说明QPRT沉默或抑制可增加血管的形成。
实施例5 含有编码QPRT的核酸序列的AAV2（AAV2-QPRT）的制备
    1、准备质粒
准备穿梭质粒（AAV2 ITR-QPRT，其含有编码QPRT的目的基因）、pHelper(携带腺病毒来源的基因)质粒和pAAV-RC(携带AAV复制和决定AAV血清型的相应衣壳基因)质粒。这三个质粒或来自于武汉枢密脑科学技术有限公司或由武汉枢密脑科学技术有限公司合成，使用前进行抽提。其中，pCMV-SPORT6-QPRT质粒提供编码QPRT的目的基因；rAAV-CMV-WPRE-pA质粒；AAV2/2 (货号PT-0545，武汉枢密脑科学技术有限公司)含有关键元件，通过这两个质粒制备穿梭质粒（即AAV2 ITR-QPRT，也即rAAV-CMV-Qprt（m）-WPRE-pA）。
pAAV-RC（pAAVRepCap，携带AAV复制和决定AAV血清型的相应衣壳基因）质粒和pHelper（携带来自腺病毒源的基因）质粒与穿梭质粒（AAV2 ITR-QPRT，其含有编码QPRT的目的基因）一起用于产生感染性AAV2颗粒。ITR: 反向终端重复。
    2、人胚肾细胞（AAV-293细胞）培养
    2.1 AAV-293细胞复苏
  1) 预热培养基。
  2) 从液氮罐中取出AAV-293细胞冻存管，迅速放入盛有37℃水的水浴中，并不时摇动，尽快解冻。用70%酒精擦拭消毒后，移至超净工作台。
  3) 吸出AAV-293细胞悬液加入到3mL培养基中，再加入5mL培养基稀释，轻柔吹散。
  4) 1000rpm，3min离心收集AAV-293细胞。
  5) 适量培养基重悬AAV-293细胞。
    2.2 AAV-293细胞传代(10cm培养皿为例)
    1) 弃去旧培养液，加入10mL灭菌PBS，轻轻晃动，洗涤细胞生长面，然后弃去PBS。
  2) 加入1mL胰酶消化液，进行消化，直到细胞变圆开始脱落（一般消化时间为1~ 2 min）。
  3) 加入2mL完全培养基终止消化，并将皿底细胞吹打成单细胞。
  4) 按需要离心重悬计数接种，或混匀细胞直接分到新的培养瓶中，继续培养。
    2.3 AAV-293细胞冻存
  1) 取培养2～3天生长旺盛的AAV-293细胞，根据传代步骤将AAV-293细胞消化并吹打成单细胞。
  2) 1000rpm，3min离心收集AAV-293细胞。
  3) 加入适量PBS重悬AAV-293细胞，计数。
  4) 1000rpm，3min离心收集AAV-293细胞。
  5) 用冻存培养基再悬浮AAV-293细胞至 2×10 6~5×10 6个/mL，然后分装细胞悬液到2mL冷冻管，1 mL/管。
  6) 将冷冻管置于程序降温盒，然后将盒子放入-80℃冷冻过夜，第二天转移冻存细胞的冷冻管到液氮中长期保存。
    3、细胞转染
  1) 接种AAV-293细胞于10cm培养皿，48h后，汇合度达到80%，用于转染。
  2) 转染前1h取细胞培养板，去除原有细胞培养基，加入10mL的Opti-MEM培养基。
  3) 制备转染试剂和质粒的复合物。
a、将待转染的病毒载体质粒32μg(pHelper：pAAV-RC：穿梭质粒=1：1：1)溶于Opti-MEM培养基，总体积为500μL，轻轻混匀，静置5min，得到质粒稀释液；
b、将转染试剂溶于Opti-MEM培养基，总体积为500μL，轻轻混匀，静置5min，得到转染试剂稀释液；
c、将转染试剂稀释液滴加到质粒稀释液中，边加边轻轻混匀后在室温放置20min，使质粒和转染试剂充分结合形成稳定的转染复合体。
  4) 将配好的转染复合体加入到上述细胞培养皿中。
  5) 6h后吸去培养基，PBS洗一次，加入10mL新鲜完全培养基培养。
    4、病毒收获及纯化
    1) 转染60h后，使用细胞刮将细胞刮下，并收集到离心管中。     2) 1500g、4℃离心5min后，分别收集上清和细胞细胞沉淀，并将细胞沉淀重悬在9mL的裂解缓冲液（10 mL  PBS + 0.001% pluronic F68 + 200mM NaCl，pluronic F68为非离子表面活性剂）中，收集到500mL瓶中，得到细胞裂解液。
    3) 将步骤2）中的细胞裂解液置于液氮中冷冻3min，37℃水浴，反复冻融3次。     4) 加入250U的benzonase核酸酶，混匀后37℃孵育1h。     5) 加入NaCl至终浓度为150mM，混匀后37℃孵育30min。     6) 2500g、室温离心10min，取上清弃去沉淀。     7) 将步骤6）离心后得到的上清倒入步骤2）离心后收集的上清中，加入PEG8000和NaCl至终浓度分别为8%、0.5M，4℃过夜。     8)12000rpm，4℃离心1.5h，弃掉上清，沉淀用10mL PBS重悬。
    9) 将重悬后的病毒原液加入到装有碘克沙醇（其中，超速离心管中碘克沙醇的浓度从上到下分别为15%、25%、40%、60%）的超速离心管中，63000rpm、18℃离心2h。     10) 用10mL注射器抽出40%的样品装入透析袋中，4℃透析，12-16h（本实施例采用14h）换透析液，每次使用2L缓冲液。
  11）收集透析后的样品，使用超滤管进行浓缩。
    5、滴度测定
目前测定AAV2的滴度通过定量PCR检测基因组中AAV2载体的基因组拷贝数来测定AAV2的病毒颗粒数。
  1) 准备标准品：用质粒标准品，计算标准品的浓度，稀释至10 8个/μL，梯度稀释，直至10 3个/μL，共6个梯度。
  2) 准备样品：碱裂解-取5μL浓缩后的病毒样品，加入NaOH至终浓度为1M，55°C水浴30min后加入HCl中和，加入纯水或1×PBS稀释10倍。
  3) 在每个反应孔中加入反应液 18μL，然后加入 2μL的模板。
    4) 上机，退火温度设为60℃，按照标准操作得到Ct值并计算AAV2-QPRT样品中的拷贝数。经过测试得出：AAV2-QPRT样品的拷贝数为2.5×10 12。
实施例6 胶原诱导性关节炎（CIA）模型及关节炎严重程度的评分标准的建立
CIA和RA在临床前、病理学和免疫学等方面具有相似性。
取17只8周龄的雌性DBA小鼠，在第0天，在其尾部底部皮内注射100µL鸡II型胶原和弗氏不完全佐剂（1:1）的乳液进行免疫诱导，建立CIA模型小鼠，注射免疫后第14天对其进行治疗。
关节炎严重程度评分由两名独立观察者评估，小鼠每只爪子的临床评分标准如下：
0=正常；1=1个指头肿胀；2=超过1个指头或腕部或踝关节肿胀；3=整个爪子肿胀；4=畸形和/或强直，最高得分为每只小鼠16分（4只爪子×4）。
实施例7  AAV2-QPRT的治疗
（1）将17只CIA模型小鼠分为两组，第一组为CIA组（7只，不做治疗处理，注入空载的AAV2），第二组为AAV-QPRT组（10只，进行治疗，注入AAV2-QPRT）。
由于小鼠后爪的大小几乎是前爪的两倍，所以注射免疫后第14天，在AAV-QPRT组小鼠的前爪经皮注射1.5×10 9个病毒载量/爪（以PBS作为溶剂，注射2.5μL），后爪经皮内注射3.3×10 9个病毒载量/爪（以PBS作为溶剂，注射5μL），进行治疗。
同时，在注射免疫后的第0周、1周、2周、3周、4周、5周、6周、7周，使用上述关节炎严重程度的评分标准对CIA组和AAV-QPRT组中的每只小鼠进行评分，为每只小鼠记录数据，并绘制临床关节炎评分曲线（见图5中a）；使用Prism软件计算图5的a中关节炎评分曲线下的区域面积（AUC），结果如图5中b所示。可见AAV-QPRT组的评分明显低于CIA组，证实了AAV2-QPRT的抗关节炎作用，QPRT的过度表达显著抑制了CIA小鼠关节炎的严重程度，AAV2-QPRT可有效治疗RA。
同时，注射免疫后的第0周、1周、2周、3周、4周、5周、6周、7周，测定CIA组和AAV-QPRT组中每只小鼠爪子的体积，结果如图5中c所示，其中Sham和QPRT分别是CIA和AAV-QPRT的对照组；另外，使用Prism软件计算图5中c小鼠爪子体积曲线下的区域面积（AUC），结果如5中d所示，结果显示AAV-QPRT组的小鼠爪子的体积明显低于CIA组，证实AAV2-QPRT的抗关节炎作用，QPRT的过度表达显著抑制了CIA小鼠关节炎的严重程度，AAV2-QPRT可有效治疗RA。
（2）所有小鼠在注射AAV2-QPRT六周后实施安乐死，并对其爪子进行免疫组织化学和组织学分析。
取小鼠爪子组织匀浆，然后用含有蛋白酶抑制剂和磷酸酶抑制剂的RIPA缓冲液裂解，将40µg样品加载到10%聚丙烯酰胺凝胶上，并转移到聚偏二氟乙烯（PVDF）膜上。然后用含有5%脱脂乳的TBS/Tween-20封闭膜1 h，然后在4℃过夜。将膜与二抗一起孵育1 h，并使用ECL观察条带。通过ImageJ软件（美国国立卫生研究院）对来自条带的信号进行量化。结果如图6所示，可见在小鼠的爪子注射AAV2-QPRT进行治疗之后，QPRT的蛋白质表达增加。其中a中的两个AAV-QPRT组表示来源于不同的小鼠。
获取小鼠血清样品，测试NAD+浓度，结果如图7所示，可见相比未经治疗的CIA组，在小鼠的爪子注射AAV2-QPRT进行治疗之后，全血中NAD+浓度升高。
以上结果表明，AAV2-QPRT介导的QPRT高表达可以有效上调组织QPRT表达量，减少细胞血管新生，提高受体NAD+水平，并降低CIA模型小鼠足掌厚度、体积和临床评分。
本发明中，QPRT的AAV2递送提供了组织和传导特异性，改善了QPRT在关节中的时空分布，从而提高了其治疗效果，同时减少了副作用，且能够实现QPRT的延长或持续释放，药效长，随着QPRT的延长表达，可以减少药物的使用剂量。目前，免疫抑制药物可能对RA无症状临床前阶段的人不利，本发明的药物提供了一种对抗免疫反应的替代方法。
应当理解的是，本发明的应用不限于上述的举例，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，所有这些改进和变换都应属于本发明所附权利要求的保护范围。

Claims (10)

  1. 一种用于治疗类风湿性关节炎的药物，其特征在于，包括基因治疗载体，所述基因治疗载体包含编码喹啉酸磷酸核糖基转移酶的核酸序列。
  2. 根据权利要求1所述的药物，其特征在于，所述基因治疗载体包括病毒载体、非病毒载体中的至少一种。
  3. 根据权利要求2所述的药物，其特征在于，所述病毒载体包括腺相关病毒载体、腺病毒载体、慢病毒载体、单纯疱疹病毒载体中的一种或多种。
  4. 根据权利要求3所述的药物，其特征在于，所述腺相关病毒载体包括AAV1载体、AAV2载体、AAV5载体、AAV6载体、AAV8载体、AAV9载体、AAVrh10载体中的一种或多种。
  5. 根据权利要求2所述的药物，其特征在于，所述非病毒性载体包括质粒、阳离子脂质、脂质纳米颗粒、脂质体、聚合物基载体、细胞外囊泡中的一种或多种。
  6. 根据权利要求1所述的药物，其特征在于，所述药物还包括药学上可接受的辅料。
  7. 根据权利要求1所述的药物，其特征在于，所述药物的剂型为注射剂。
  8. 根据权利要求7所述的药物，其特征在于，所述药物的给药方式为静脉内注射、皮下注射、肌肉注射、关节内注射、皮内注射中的一种或多种。
  9. 检测喹啉酸磷酸核糖基转移酶的试剂在制备用于诊断类风湿性关节炎的产品中的应用。
  10. 根据权利要求9所述的应用，其特征在于，所述产品包括试剂盒、试纸中的一种。
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