WO2018177376A1 - 一种治疗自身免疫病及糖尿病的病毒载体及其构建方法和应用 - Google Patents

一种治疗自身免疫病及糖尿病的病毒载体及其构建方法和应用 Download PDF

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WO2018177376A1
WO2018177376A1 PCT/CN2018/081117 CN2018081117W WO2018177376A1 WO 2018177376 A1 WO2018177376 A1 WO 2018177376A1 CN 2018081117 W CN2018081117 W CN 2018081117W WO 2018177376 A1 WO2018177376 A1 WO 2018177376A1
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plasmid
viral vector
mfat
diabetes
cells
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PCT/CN2018/081117
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English (en)
French (fr)
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赵子建
毕欣耘
李晓曦
李芳红
林艳
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广州华真医药科技有限公司
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Priority to KR1020197032517A priority Critical patent/KR102383866B1/ko
Priority to AU2018241658A priority patent/AU2018241658A1/en
Priority to US16/497,828 priority patent/US20210269824A1/en
Priority to CA3056691A priority patent/CA3056691C/en
Priority to EP18774601.1A priority patent/EP3608414A4/en
Priority to JP2019554405A priority patent/JP2020512010A/ja
Publication of WO2018177376A1 publication Critical patent/WO2018177376A1/zh
Priority to ZA2019/06218A priority patent/ZA201906218B/en
Priority to AU2022200653A priority patent/AU2022200653A1/en

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Definitions

  • the invention relates to a therapeutic agent for autoimmune diseases induced by diabetes and immune imbalance, in particular to a viral vector for treating autoimmune diseases and diabetes, and a construction method and application thereof.
  • T lymphocyte-mediated cellular immunity is the central link in the body's immune response.
  • CD4 + T cells can differentiate into Th1, Th2, Treg and Th17 under the control of different transcription factors, and they are in a state of dynamic equilibrium through the cytokines secreted by them.
  • T1D type 1 diabetes
  • RA rheumatoid arthritis
  • Th17/Treg cells are closely related to various autoimmune diseases, especially type 1 diabetes (T1D), rheumatoid arthritis (RA). ), Multiple sclerosis (MS), Systematic lupus erythematosus (SLE), etc.
  • Type 1 diabetes called insulin-dependent diabetes mellitus (IDDM)
  • IDDM insulin-dependent diabetes mellitus
  • IDDM insulin-dependent diabetes mellitus
  • LADA Latent Autoimmune Diabetes in Adults
  • the whole process of type 1 diabetes is accompanied by a series of inflammations and immune disorders, mainly manifested by the attack of islet ⁇ cells by autoreactive T cells and the production of autoantibodies, causing islet ⁇ cell failure and necrosis, leading to insulin secretion.
  • absolutely insufficient, such as poor treatment will lead to serious complications, such as blindness, kidney failure, heart disease, amputation.
  • Type 2 diabetes is called noninsulin-dependent diabetes mellitus (NIDDM), accounting for the vast majority of diabetes.
  • NIDDM noninsulin-dependent diabetes mellitus
  • the combination of different degrees of insulin deficiency and tissue insulin resistance is the main pathogenesis of type 2 diabetes. Once type 2 diabetes is diagnosed, islet beta cell function is progressively reduced, and hepatic glucose production and release are increased, causing elevated blood glucose.
  • Type 1 diabetes the natural course of disease includes the following three stages: 1. Pre-hyperglycemia, which can last up to 10 years. Type 1 diabetes has been impaired in beta cell immunity, and its main marker is the emergence of autoantibodies such as GAD or ICA. Type 2 diabetes has no specific serological markers during this period, but is often accompanied by insulin resistance syndrome such as hyperinsulinemia and abdominal obesity. Islet function shows impaired glucose tolerance (IGT) and/or fasting blood glucose. Impaired fasting glucose (IFG). 2. High blood glucose period fasting and postprandial blood glucose to achieve diagnostic criteria for diabetes. 3. Chronic complications.
  • ITT impaired glucose tolerance
  • IGF Impaired fasting glucose
  • Th1 cells CD4 + effector lymphocytes have been divided into two major categories, Th1 and Th2, which are mutually restricted by their secreted cytokines and are in a state of dynamic equilibrium.
  • ⁇ -cell autoantigens When certain ⁇ -cell autoantigens are treated by macrophages or other antigen-presenting cells, they are presented to the surface of antigen-presenting cells together with MHC II molecules, causing the release of autoimmune signals, thereby activating Th1 cells and inhibiting Th2 cells.
  • the release of cytokines makes Th cells predominant in the Th1 cell phenotype.
  • Th1 cells further activate cytotoxic macrophages, cytotoxic T cells (CD8 + T cells) and natural killer cells (NK cells), and damage islet ⁇ cells. Inflammation of the islets is caused, resulting in a decrease or lack of insulin secretion, which eventually leads to type 1 diabetes.
  • type 2 diabetes is also an inflammatory disease.
  • the inflammatory mediator TNF- ⁇ activates several proteins that inhibit the insulin signaling pathway, attenuating the body's ability to respond to insulin, and in turn induces insulin resistance.
  • Danish scientists have found that in mice, macrophages invade diabetic pancreatic tissue in the early stages of the disease. These inflammatory cells then produce large amounts of pro-inflammatory proteins (cytokines) that act directly and damage insulin-producing beta cells in the pancreas, leading to type 2 diabetes, a finding published in the January 2014 issue of the Journal of Leukocyte Biology. Further studies have found that Th1 cells and CD8 T cells in acquired immunity promote insulin resistance, while Th2 cells and Treg cells can antagonize this effect. Therefore, controlling inflammation and regulating T cell function is also an important target for the control and treatment of type 2 diabetes.
  • cytokines pro-inflammatory proteins
  • the monoclonal antibody Anti-CD3 can reduce the degradation of insulin secretion function and reduce the increase of insulin demand in new-onset type 1 diabetes within 2 years. It plays a role in the early stage of type 1 diabetes, and the most benefit is younger or more residual ⁇ . Cell type of type 2 diabetes patients, but its clinical application is further evaluated due to side effects such as decreased immunity, fever, rash and anemia.
  • RA Rheumatoid arthritis
  • a recent epidemiological survey shows that the incidence of Chinese is about 0.4%, which is slightly less than the 1% of Caucasians. Based on this, there are about 4 to 5 million RA patients each year in China, and the prevalence of middle-aged and older women is high, with a male to female ratio of 1:3.
  • the immune system of RA patients mistakenly attacks the synovial membrane of the surrounding joints, causing inflammation, pain and joint damage.
  • the cause of RA is still unclear, but in recent years, studies have widely recognized T cell dysfunction, especially the activation of auxiliary CD4 + T cells, which is at the center of the pathogenesis of RA. In normal humans, pro-inflammatory and anti-inflammatory factors are in an equilibrium state.
  • Th1 and Th17 cells are activated, releasing inflammatory cytokines and aggravating the progression of inflammation. Inhibition of Th2 and Treg cells, while pro-inflammatory and anti-inflammatory balance processes may cause RA onset if they shift to Th1 and Th17 cells.
  • Cytokines such as IFN- ⁇ , TNF- ⁇ , IL-2, IL-1, and IL-17 in serum and joint synovial fluid of RA patients cause and continue to aggravate the occurrence of inflammation.
  • IL-17 synergizes with TNF- ⁇ and IL-1 in synovial inflammation, induces a series of cytokines and chemokines, and enhances synovial inflammation.
  • Th2 and Treg cells are involved in anti-inflammatory effects by secreting the cytokines IL-4, IL-10, and the like.
  • IL-4 secreted by Th2 cells can inhibit the activity of TNF- ⁇ , IL-1, IL-6 and IL-8 and exert an anti-inflammatory effect.
  • Treg cells can exert immunosuppressive effects in RA disease models by producing cytokines including IL-10, TGF- ⁇ and the like.
  • the imbalance of Th cell differentiation also plays a role in cartilage destruction [22] .
  • the cartilage is composed of cartilage tissue and the surrounding perichondrium, which is composed of chondrocytes, stroma and collagen fibers.
  • a balance is formed between bone formation and bone resorption during normal physiological processes, and inflammation is closely linked to bone erosion.
  • nuclear factor-kappaB receptor activating factor RANK
  • OPG nuclear factor-kappaB receptor activating factor
  • RANK nuclear factor-kappaB receptor activating factor
  • IL-17 can up-regulate the expression of RANKL and its receptors, thereby disrupting the balance of RANKL/OPG in synovial fluid and aggravating bone destruction.
  • IL-4 and IL-10 produced by Th2 cells inhibit the differentiation of osteoclasts.
  • Treg cells acting as Th17 antagonists can directly inhibit osteoclast formation, and Treg cells can up-regulate OPG expression through self-secreting IL-10, down-regulating RANKL Thereby inhibiting bone destruction.
  • RA RA-associated inflammatory fibroblasts
  • the first-line chemical drugs currently used in the treatment of RA are mainly nonsteroidal anti-inflammatory drugs (NSAIDs), including gold compounds, D-penicillamine, antimalarials, sulfasalazine, methotrexate, etc., mainly by inhibiting the immune system and reducing Destruction of the joints.
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • Infliximab, Etanercept, and Anakinra which are used to treat RA, mainly target TNF- ⁇ and IL-1, and antagonize the pro-inflammatory effects of TNF ⁇ and IL-1.
  • TNF- ⁇ is an important cytokine in the normal immune system, blocking it can cause serious side effects and increase the risk of infection. Therefore, for the current anti-rheumatic drugs, short-term and long-term tolerance and safety concerns have been raised, and it is necessary to further study new chemotherapeutic drugs, block autoimmunity, and rebalance the immunomodulatory drugs.
  • MS Multiple sclerosis
  • EAE allergical encephalomyelitis
  • MS After exposure to MBP, an immune response triggers an inflammatory response, producing an infiltration of inflammatory factors and chemokines, leading to destruction of the spinal cord sheath, causing symptoms such as visual impairment and muscle weakness in the patient [25] .
  • MS mainly responded to Th1 and Th17 reactions, and IL-2, IFN- ⁇ and IL-17 were significantly increased in the lesion area.
  • IL-4, IL-10 and TGF- ⁇ were increased, and Th1 type cytokine production was observed. It will fall. After returning with specific Th2 cells, EAE development can be prevented [27] .
  • Th2 cells After returning with specific Th2 cells, EAE development can be prevented [27] .
  • a similar situation was also found in MS clinical studies. Therefore, blocking the imbalance of Th cell differentiation will also play a crucial role in the treatment of MS.
  • Mitoxantrone inhibits the proliferation of T cells, B cells and macrophages, and exerts immunosuppressive effects.
  • the biggest side effect of Mitoxantrone is myocardial toxicity, which is prone to myocardial toxicity when the cumulative dose is greater than 140 mg/m 2 . It can be used for up to three years at a rate of 12 mg/m2 every three months. The mitoxantrone is too short-lived compared to the average 30-year course of MS [30] .
  • immunosuppressive agents such as azathioprine, cyclophosphamide, methotrexate, mycophenolate mofetil and tacrolimus have short-term immunosuppressive effects, but the side effects are obvious, such as cyclophosphamide can cause hemorrhagic cystitis, low dose of methotrexate can still cause liver toxicity, mycophenolate Mofetil, tacrolimus and azathioprine have myelosuppressive effects.
  • Natalizumab neutralizes the adhesion molecule ⁇ -4integrin on the surface of lymphocytes, preventing it from binding to vascular endothelial cells and vascular cell adhesion molecule-1 (VCAM-1), inhibiting lymphocytes from penetrating the vascular endothelium and reaching the tissues. Thereby reducing autoimmune inflammation in brain tissue, and inevitably affecting the overall immune response, infecting JC virus and increasing the risk of progressive multifocal leukoencephalopathy (PML). Natalizumab also has severe liver toxicity. MS is a prolonged and refractory chronic autoimmune disease. Therefore, in addition to efficacy, the development of therapeutic drugs needs to consider the safety and convenience of long-term use. Effectively changing the autoimmune attack caused by immune disorders is undoubtedly the best solution to these problems. way.
  • VCAM-1 vascular cell adhesion molecule-1
  • SLE is a chronic autoimmune disease.
  • the cause of the disease is unknown, and the patient's immune system produces autoantibodies that attack the cells and tissues of the body, causing inflammation and tissue damage.
  • Common symptoms include arthritis, fever, chest pain, hair loss, mouth ulcers, swollen lymph glands, fatigue, and facial rashes.
  • the pathogenesis of SLE is not clear, and studies suggest that T cell abnormalities play an important role in the pathogenesis. Under the interaction of genetic factors, environmental factors, estrogen levels and other factors, autoreactive Th cells in SLE are activated by autoantigen and clonal expansion, and produce cytokines. In MRLP lpr lupus mice, the secretion of IFN- ⁇ increased, and the proportion of Th1/Th2 cells increased.
  • Th1/Th2 cells in peripheral blood and kidney were significantly elevated, and It is positively correlated with the degree of renal damage, suggesting that Th1 cells also play an important role in the pathogenesis of SLE.
  • the number of Treg cells in SLE patients decreased, function decreased, B cells hyperproliferated, produced a large number of autoantibodies, and combined with the corresponding autoantigens in the body to form corresponding immune complexes, deposited in skin, joints, small blood vessels, small kidneys Ball and other parts.
  • complement causing acute and chronic inflammation and tissue necrosis (such as lupus nephritis), or antibodies directly interact with tissue cell antigens, causing cell destruction, resulting in multiple system damage.
  • non-steroidal anti-inflammatory drugs include gastrointestinal discomfort, heartburn, diarrhea, and body water.
  • Corticosteroids can quickly suppress inflammation. Because of their high potency, the lowest dose is usually used to achieve maximum efficacy.
  • Short-term side effects of corticosteroids include edema, increased appetite, and weight gain.
  • Long-term side effects include osteopenia (skeletal loosening), high blood pressure, high cholesterol, high blood sugar (diabetes), arterial damage, infection, and cataracts.
  • Polyunsaturated fatty acids are linear fatty acids having 18-22 carbon atoms and containing 2-3 hydrogen bonds, usually divided into two types, omega-3 and omega-6, the most distal double bond from the carboxyl group.
  • Both omega-6 and omega-3 PUFAs are synthesized from saturated fatty acid precursors under different desaturases. Vertebrates and mammals lack specific desaturases and therefore cannot synthesize omega-6 and omega in vivo. -3, only intake of these unsaturated fatty acids from the diet.
  • Omega-3 PUFAs are extremely important for maintaining normal physiological functions, and they are key components of the structure of cellular lipid membranes. Numerous studies have shown that maintaining a proportional balance of omega-6/omega-3 PUFAs plays an important role in health. Two recent large-scale clinical studies have shown that long-term intake of omega-3 PUFAs is inversely proportional to the incidence of type 1 diabetes: a case-control study based on 2,000 Norwegian children shows that it is taken from the first year after birth.
  • Cod liver oil rich in omega-3 PUFAs can reduce the risk of type 1 diabetes in children; a follow-up survey by the American Adolescent Autoimmune Diabetes Research Program (DAISY) shows that children with a genetic risk of type 1 diabetes have a dietary intake of ⁇ -3 PUFAs can reduce the risk of autoimmune inflammation in islets.
  • free fatty acids such as palmitic acid inhibit insulin secretion in vitro, and omega-3 PUFAs can reverse this phenomenon.
  • Glucose-stimulated insulin secretion can be enhanced by dietary supplementation with omega-3 PUFAs.
  • omega-3 PUFAs have the protective effect of inhibiting inflammation, preventing and alleviating autoimmunity, and have protective effects on the pathogenesis and pathological development of RA, MS and SLE.
  • clinical attempts to treat diabetes and control body weight using fish oil capsules containing omega-3 PUFAs have mostly failed.
  • the reason is that the content of omega-6 PUFAs in the food intake is too high, relying solely on the supplement of fish oil capsules. It is difficult to balance the ratio between the two. Therefore, the present invention proposes a viral vector in which the omega-3 fatty acid desaturase encoded by the fat-1 gene promotes the conversion of omega-6 into omega-3 in vivo. , balance the ratio between the two.
  • the fat-1 gene derived from C. elegans whose protein product is an omega-3 unsaturated fatty acid desaturase, is desaturated with omega-6 PUFAs as a substrate to generate corresponding ⁇ - 3 PUFAs, while significantly increasing the content of endogenous omega-3 PUFAs in animals, reducing the content of omega-6 PUFAs and changing the proportion of omega-6/ ⁇ -3 PUFAs.
  • omega-6 PUFAs In order to make fat-1 better expressed in mammals, we have mammalianized the cDNA encoding fat-1, hence the name mfat-1.
  • Mfat-1 transgenic mouse islet ⁇ cells can significantly resist cytokine-induced anti-apoptotic effects, and omega-3 PUFAs can promote insulin secretion.
  • the first technical problem to be solved by the present invention is to provide a viral vector which is safe, non-toxic, and has therapeutic activity and can meet clinical needs.
  • a second technical problem to be solved by the present invention is to provide a method for constructing the above viral vector.
  • a third technical problem to be finally solved by the present invention is to provide a use of the above viral vector for the preparation of a medicament for treating diabetes or an autoimmune related disease.
  • the present invention provides a viral vector which is a lentiviral expression plasmid in which the mfat-1 gene is cloned or an adeno-associated virus expression plasmid in which the mfat-1 gene is cloned, the mfat- The 1 gene is shown as SEQ ID No.: 1.
  • the viral vector, the lentiviral expression plasmid is pLJM1-CMV-hPGK-EGFP plasmid, pLJM1-CMV-hPGK-mkate2 plasmid, pLenti-CMV-MCS-GFP-SV-puro plasmid, FUGW, pLenti-puro , pLenti-MP2 or pLenti plasmid;
  • the adeno-associated virus expression plasmid is pEMBL-AAV-D(+)-CMV-eGFP-SV40 plasmid, AAV GFP plasmid, AAV1 plasmid, AAV2 plasmid, rAAV2 plasmid, AAV5 plasmid, AAV8 Plasmid or pAV-FH AAV plasmid.
  • the present invention provides a method for constructing the viral vector, which is obtained by cloning the mfat-1 gene into a lentivirus expression plasmid or an adeno-associated virus expression plasmid.
  • the method for constructing the viral vector comprises the following steps:
  • mfat-1-F 5'-TATTAAGCTAGCATGGTCGCCCACAGCA-3';
  • mfat-1-R 5'-CAACCGGAATTCTCATCACTTGGCCT-3';
  • the PCR amplification product obtained in the step (1) is subjected to electrophoresis, and then the gel is recovered and purified, and the obtained PCR amplification product is digested with restriction endonucleases NheI and EcoR, and used;
  • the lentiviral expression plasmid or the adeno-associated virus expression plasmid is digested with restriction endonucleases NheI and EcoR, and used;
  • the DNA fragment obtained in the step (2) is ligated to the empty shuttle plasmid after digestion, and is obtained.
  • the method for constructing the viral vector, the PCR amplification reaction system in the step (1) comprises:
  • the template DNA is 100 ng.
  • the method for constructing the viral vector, the PCR amplification reaction procedure in the step (1) is: denaturation at 98 ° C for 5 min; 98 ° C for 10 s, 60 ° C for 15 s, 72 ° C for 2 min, a total of 30 cycles; Extend at 72 ° C for 10 min.
  • the present invention provides a recombinant viral vector constructed by the viral vector.
  • the present invention provides a virus particle which is a virus particle obtained by transforming the viral vector into a cell.
  • the viral particles are obtained by packaging the viral vector by lipofection into 293FT cell packaging.
  • the viral particles are administered by intravenous injection.
  • the present invention provides the use of the viral vector or the recombinant viral vector or the viral particle in the preparation of a medicament for treating diabetes or treating an autoimmune related disease.
  • the autoimmune-related disease is an autoimmune disease caused by imbalance of Th cell differentiation and imbalance of secreted cytokines.
  • the diabetes is autoimmune type 1 diabetes.
  • the autoimmune disease is type 1 diabetes (T1D), multiple sclerosis (MS), rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE).
  • T1D type 1 diabetes
  • MS multiple sclerosis
  • RA rheumatoid arthritis
  • SLE systemic lupus erythematosus
  • the invention provides a medicament comprising the viral vector or the recombinant viral vector or the viral particle, and a pharmaceutically acceptable carrier of the viral vector, recombinant viral vector or viral particle described above.
  • the medicament comprises preparing the viral vector or the recombinant viral vector or the viral particles into a clinically acceptable dosage form according to a conventional process by adding a conventional excipient.
  • the lentiviral expression plasmids were pLJM1-CMV-hPGK-EGFP plasmid (purchased from Addgene (Plasmid #19319)) and pLJM1-CMV-hPGK-mkate2 plasmid (purchased from addgene ) .
  • adeno-associated virus expression plasmid was pEMBL-AAV-D(+)-CMV-eGFP-SV40 plasmid (purchased from addgene).
  • the present invention adopts a gene therapy method, and uses the virus particle of the present invention to bring the mfat-1 gene into a self-expression non-obese diabetic model mouse (NOD mouse), compared with the control group, mfat-1 Lentiviral particles can significantly reduce the blood glucose level of NOD mice, and increase the serum insulin levels, which has a significant therapeutic effect.
  • Possible mechanisms for mfat-1 gene therapy for diabetes and autoimmunity include: mfat-1 converts to omega-3 PUFAs by using high-orange omega-6 PUFAs as substrates, balancing the ratios such that omega-6/ The ratio of omega-3 tends to be 1:1.
  • omega-3 PUFAs transformed from omega-6 PUFAs promote the differentiation of Th cells into Th2 and antagonize the effect of Th1 cell activation by omega-6 PUFAs, reducing IFN- ⁇ , IL-6, TNF-
  • the secretion levels of pro-inflammatory factors such as ⁇ and IL-17 alleviate the Th1 cell-mediated inflammatory response; inhibit the infiltration of CD8 + CTL cells and macrophages in islets, alleviate the occurrence of insulitis and peri-pancreatitis; Unsaturated fatty acid dehydrogenase, therefore, it is necessary to introduce mfat-1 into the body using a lentiviral vector or an adeno-associated viral vector.
  • the above viral vector is administered by intravenous injection.
  • the viral vector of the present invention can be used for preparing a biological drug for treating type 1 diabetes and related autoimmune diseases, for example, a virus particle transfected into a cell with a viral vector is safe.
  • Figure 1 is a 1% agarose gel electrophoresis pattern of the pLJM1-mfat-1-EGFP plasmid NheI and EcoRI;
  • Figure 2 is a diagram showing the differentiation of human peripheral blood CD4 T cells after 24 hours of treatment of peripheral blood mononuclear cells in normal humans (Fig. 2A-E) and type 1 diabetic patients (Fig. 2F-J) in Example 2 of the present invention. a graph of the results of changes in cytokines;
  • FIG. 3 is a graph showing changes in peripheral blood CD4 T cell transcription factors after 24 hours of peripheral blood mononuclear cells in a patient with type 1 diabetes mellitus in vitro according to Example 2 of the present invention
  • Fig. 4 is a graph showing the results of changes in the incidence of diabetes in NOD mice after administration of lentivirus for 9 weeks in Example 3 of the present invention.
  • Figure 5 is a graph showing the results of blood glucose changes in NOD mice after administration of lentivirus in Example 3 of the present invention.
  • Figure 6 is a graph showing the results of insulin secretion of NOD mice after administration of lentivirus for 9 weeks in Example 3 of the present invention.
  • Figure 7 is a graph showing the results of changes in the development of islet inflammatory infiltration in NOD mice after 9 weeks of administration of lentivirus in Example 3 of the present invention.
  • Figure 8 is a graph showing the results of differentiation of NOD murine CD4 cells after treatment with lentivirus for 9 weeks in the Example of the present invention.
  • DH5 ⁇ E. coli supplied by TAKARA
  • LB medium supplied by Sigma
  • 293FT cell line supplied by ATCC
  • growth medium DMEM supplied by GIBCO
  • FBS fetal bovine serum supplied by GIBCO Provided
  • OPTI-MEM supplied by GIBCO
  • Lipofectamine 2000 transfection reagent supplied by Invitrogen-Thermo Fisher Scientific
  • NOD mice supplied by The Jackson Laboratory.
  • the centrifuge manufacturer is Thermo Company of the United States, model FRESCO17 high-speed refrigerated centrifuge; the electrophoresis manufacturer is BIO-RAD, the model is PowerPac TM and Mini-Sub cell GT; the gel imager manufacturer is BIO-RAD of the United States.
  • the company, model is ChemiDoc TM XRS+System.
  • Example 1 Construction of a viral vector cloned with the mfat-1 lentiviral expression plasmid.
  • the mfat-1 (as shown in SEQ ID No.: 1) gene (provided by Kingsray) was first synthesized, followed by subcloning to the pLJM1-CMV-hPGK-EGFP plasmid by NheI and EcoRI cleavage sites (addgene, Plasmid #19319), pLJM1-CMV-hPGK-EGFP-mfat-1 (PLJM1-mfat-1) expression plasmid was obtained.
  • the specific construction process is as follows:
  • the primers were as follows:
  • Upstream primer mfat-1-F 5'-TATTAAGCTAGCATGGTCGCCCACAGCA-3';
  • Downstream primer mfat-1-R 5'-CAACCGGAATTCTCATCACTTGGCCT-3';
  • the PCR amplification system is shown in Table 1.
  • the PCR amplification procedure was as follows: denaturation at 98 ° C for 5 min; 98 ° C for 10 s, 60 ° C for 15 s, and 72 ° C for 2 min for a total of 30 cycles; and finally 72 ° C for 10 min.
  • Digestion reaction conditions 37 ° C water bath for 2 h.
  • the above-mentioned digested product was subjected to electrophoresis on a 1% agarose gel to separate a target fragment, and a purified plasmid or a DNA fragment of interest was obtained using an Agarose Gel DNA Extraction Kit.
  • the DNA fragment of interest was ligated to the pLJM1 vector.
  • This embodiment also provides a virus particle, which is prepared by transforming the virus vector prepared above into a cell, and the specific steps are as follows:
  • the competent bacteria used for transformation is DH5 ⁇ E. coli, prepared by CaCl 2 method.
  • the preparation method and subsequent transformation operations refer to the second edition of Molecular Cloning, as follows:
  • Ii was transferred to ice-cold 50 ml polypropylene tube and placed in an ice bath for 10 min;
  • the C positive clones were screened by enzyme digestion and sequenced, and identified by 1% agarose gel electrophoresis. The results are shown in Figure 1, indicating that the viral vector PLJM1-mfat-1 was successfully constructed.
  • the packaging cell of lentivirus is 293FT cell line, and the growth medium is DMEM (containing 10% FBS). Adherent cells proliferate in culture to form monolayer cells.
  • step 5 Mix the plasmid reagent (PLJM1-mfat-1, psPAX2 and pMD2.G three plasmid mixture) incubated in step 3) with the diluted Lipofectamine 2000 transfection reagent in step 4), mix gently, at room temperature. The cells were incubated for 15 minutes under (20 ° C - 30 ° C) to form a transfection complex of the plasmid reagent with Lipofectamine 2000 transfection reagent.
  • step 6) Take 200 ⁇ l of step 5) Obtain the reagent (Step 5) and finally obtain the 293FT cell culture medium containing the PLJM1-mfat-1, psPAX2 and pMD2.G and Lipofectamine2000 transfection reagent transfection complex). Medium, gently shake and incubate at 37 ° C in a 5% CO 2 cell incubator.
  • virus particles as described.
  • the virus particles can be stored at 4 ° C for a short period of time ( ⁇ 3d), and stored at -80 ° C for a long time, and can be repacked to avoid repeated freezing and thawing.
  • the virus titer was determined by the TCID50 method.
  • the amplification, purification and titer of the control naked virus were determined as above.
  • the test results are shown in Fig. 2 and Fig. 3.
  • the CON in Fig. 2 is the peripheral blood lymphocytes of the normal control group without any treatment of DHA, EPA and AA in the blank control group
  • Fig. 3 The CON in the blank control group is the peripheral blood lymphocytes of patients who have not been treated with any of DHA, EPA, and AA.
  • the A, B, C, D, and E diagrams in Figure 2 are the normal human peripherals, respectively.
  • the percentage of Th1, Th2, Th1/Th2, Th17 and Treg cells in blood CD4 T cells, F, G, H, I and J are Th1, Th2 and Th1 in peripheral blood CD4 T cells of patients, respectively.
  • the percentage of /Th2, Th17, and Treg cells, and the A, B, C, and D plots in Figure 3 are the percentages of the transcription factors T-bet, GATA3, ROR ⁇ T, and Foxp3 in peripheral blood CD4 T cells, respectively.
  • the percentage of Th1 and Th17 cells in peripheral blood CD4 T cells of patients with type 1 diabetes treated with DHA, EPA and AA decreased, and the percentage of Th2 and Treg cells increased, and the ratio of Th1/Th2 was rebalanced.
  • Excessive omega-6 PUFAs in the body are substrates, which are converted into omega-3 PUFAs, and the ratio of the two is balanced so that the ratio of omega-6/omega-3 tends to 1:1, which is inhibited by Th1/Th2 and T17.
  • Autoimmune diseases caused by imbalance in /Treg ratio such as type 1 diabetes, multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.
  • Example 3 Effect of Lenti-mfat-1 lentiviral particles in the treatment of type 1 diabetes.
  • NOD mice A few NOD mice, weighing 25 g, were collected at a fixed time every day to detect random blood glucose in NOD mice. Mice with a value of more than 11.1 mmol/L for two consecutive weeks were selected for the experiment. The blood glucose was randomly taken for more than 11.1 mmol for two consecutive weeks. /L NOD mice were divided into two groups, the control group (Lenti-Con) and the lentiviral treatment group (Lenti-mfat-1), 5 in each group;
  • Lenti-mfat-1 lentiviral particles were injected into the tail vein according to the weight of the mouse with a titer of 10 8 TU/mL according to the weight of the mice at 10 9 TU/Kg, and the control group was given no mfat-1 under the same conditions.
  • the gene pLJM1-CMV-hPGK-EGFP plasmid was used to detect changes in random blood glucose in mice.
  • mice After treatment with Lenti-mfat-1 lentiviral particles in the tail of the treatment group, the mice were fed with normal drinking water compared with the control group. After 2 weeks of treatment, the body weight of the treatment group was higher than that of the control group, but there was no significant statistical significance. significance.
  • the level of polyunsaturated fatty acid in the peripheral blood of the mouse is detected.
  • the specific steps are as follows: after extracting fatty acids from the peripheral blood of the two groups of mice by conventional organic chemistry, The above extracted sample (extracted fatty acid) was dissolved in heptane and dropped into a sample bottle to be loaded for detection. The sample was injected through an autosampler and tested by the Agilent 7890A with a single injection run time of approximately one hour.
  • the peak of interest was determined by comparing the peak time, peak shape, and peak area percentage with the standard; the sum of all the area percentages of omega-3 and omega-6 polyunsaturated fatty acids was calculated, and the ratio of ⁇ -6/ ⁇ -3 was calculated.
  • the proportion of EPA administered to Lenti-mfat-1 lentiviral particles was found to increase, indicating successful administration of mfat-1 lentiviral particles (Table 5).
  • Each omega-3 and omega-6 polyunsaturated fatty acid is expressed as a relative percentage, wherein the peak area of all fatty acids detected by gas chromatography is 100%, and the content of the target fatty acid is divided by the peak area by the total peak area and Calculated.
  • Each group of data was repeated three times, using oneway-ANOVA statistics, compared with the control group *P ⁇ 0.05, NP: not detected.
  • mice with random blood glucose below 11.1 mmol/L for two consecutive weeks were considered to have returned to normal blood glucose.
  • Blood samples were taken from the eye of the Lenti-mfat-1 treatment group and the control group NOD mice using the Acce-Chek blood glucose meter. The results are shown in Figure 4-5.
  • the treatment group was smaller than the control group.
  • Rat random blood glucose has been significantly down-regulated since the second week.
  • blood glucose levels have decreased and remained at normal levels, reaching 6.3 mmol/L. It is indicated that Lenti-mfat-1 lentiviral particles have significant therapeutic effects on NOD mice.
  • the spleen lymphocytes (number of 1 ⁇ 10 5 ) in the NOD mice of the treatment group and the control group were taken into different labeled tubes, and labeled fluorescent monoclonal antibody CD3 (0.1 ⁇ g) was added to each labeled tube, and CD8 (0.1) was added.
  • the obtained suspensions were respectively IFN- ⁇ fluorescent monoclonal antibody (0.1 ⁇ g), IL- 17 fluorescent monoclonal antibody (0.1 ⁇ g), IL-4 fluorescent monoclonal antibody (0.1 ⁇ g) and Foxp3 fluorescent monoclonal antibody (0.1 ⁇ g) were labeled, and incubated at 4 ° C for 1 hour in the dark (using Foxp3 fluorescent monoclonal antibody) The labeling needs to be incubated for 8 hours); then washed three times with 0.5% saponin, centrifuged at 300g for 5min at 4°C, the supernatant was removed, fixed with mass concentration of 1% paraformaldehyde, and after 24 hours, flow cytometry was used respectively.
  • the instrument Accuri-C6 is tested to analyze the proportion of Th1, Th2, Th17 and Treg cells (the above steps Antibodies and instruments used were from BD Bioscience). The results are shown in Figure 8. After treatment with Lenti-mfat-1 lentiviral particles, the percentage of Th1 and Th17 cells in NO4 mouse CD4 T cells decreased, the percentage of Th2 and Treg cells increased, and the ratio of Th1/Th2 and T17/ were rebalanced. The Treg ratio significantly improved the inflammatory environment caused by the imbalance of Th1/Th2 and T17/Treg ratios.
  • Example 4 mfat-1 transgenic mice were completely resistant to inducers to induce their onset of MS.
  • Model establishment 3 types of Wide type mice (purchased from the Institute of Model Animals, Nanjing University), (6-8 weeks, two females and one male); maft-1 transgenic mice (provided by the Institute of Model Animals, Nanjing University, The fat-1 gene of the maft-1 transgenic mouse was expressed by pST181 prokaryotic expression vector, and the muscle creatine kinase (MCK) enhancer was designed upstream of the fat-1 gene promoter CMV- ⁇ -actin to improve fat-1. The gene was mammalianized in mammals, so it was named mfat-1 transgenic mouse) 3 (6 weeks, two females and one male).
  • MCK muscle creatine kinase
  • mice were immunized with MOG, and MOG35-55 (purchased from Sigma) was immunized to the animals by subcutaneous injection at 300 ⁇ g/body weight (kg). A second immunization was performed at the same dose six days after the first immunization. Animals survive for 24 days.
  • Model observation index behavioral observation of clinical neuropathy: record the animal behavior change (Kerlero score) every day from the 12th day after modeling; 1 point, tail weakness; 2 points, tail ⁇ ; 2.5 points Unilateral hind limbs with mild weakness; 3 points, unilateral hind limbs significantly weak; 4 points, unilateral hind limb paralysis; 4.5 points, unilateral hind limb paralysis with contralateral hind limb weakness or ipsilateral forelimb mild weakness; 5 points, bilateral Hind limb paralysis; 6 points, bilateral hind limb paralysis accompanied by unilateral forelimb paralysis.

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Abstract

提供了一种治疗自身免疫病及糖尿病的病毒载体及其构建方法和应用，该病毒载体为克隆有mfat-1基因的慢病毒表达质粒或腺相关病毒表达质粒，所述的mfat-1基因如SEQ ID No.:1所示。

Description

一种治疗自身免疫病及糖尿病的病毒载体及其构建方法和应用 技术领域
本发明涉及糖尿病和免疫失衡诱发的自身免疫疾病治疗药物，具体涉及一种治疗自身免疫病及糖尿病的病毒载体及其构建方法和应用。
背景技术
T淋巴细胞介导的细胞免疫是人体免疫应答的中心环节。其中，CD4 +T细胞分化失衡导致的免疫紊乱和炎症与多种自身免疫疾病及神经退行性疾病的相关性获得科学家持续关注，并且部分机理得以阐明。CD4 +T细胞在不同转录因子调控下可分化为Th1、Th2、Treg和Th17，并通过其所分泌的细胞因子相互制约，处于动态平衡状态。研究表明，细胞亚群Th1/Th2细胞及Th17/Treg细胞之间的失衡与多种自身免疫疾病密切相关，尤其是1型糖尿病(Type1 diabetes,T1D)，类风湿性关节炎(Rheumatoid arthritis，RA)，多发性硬化症(Multiple sclerosis，MS)，系统性红斑狼疮(Systematic lupus erythematosus，SLE)等。
1型糖尿病称为胰岛素依赖型糖尿病(insulin-dependent diabetes mellitus,IDDM)，为一种常见的器官特异性自身免疫性疾病，是严重危及儿童和青少年健康的慢性疾病之一。据美国糖尿病协会的评估,大约5％的糖尿病人为1型糖尿病，且多数于儿童或青年时期发病。在儿童及青少年患者中，T1DM所占比例约为80-90％。此外，尚有一类缓慢发病的成人隐匿性自身免疫糖尿病(Latent Autoimmune Diabetes in Adults，LADA)，在病因上亦属于自身免疫性T1DM，但由于患者起病年龄及临床表现均与2型糖尿病类似，易被误诊。我国1型糖尿病患者的绝对数已超过100万。1型糖尿病发病的整个过程中伴随一系列炎症和免疫紊乱的发生，主要表现为自身反应性T细胞对胰岛β细胞的攻击以及自身抗体的产生引起胰岛β细胞功能衰竭、坏死，导致胰岛素分泌的绝对不足，如治疗不善将发生严重的并发症，如失明、肾衰竭、心脏病、截肢等。
2型糖尿病称为非胰岛素依赖型(noninsulin-dependent diabetes mellitus,NIDDM)，占糖尿病中的绝大多数。不同程度的胰岛素缺乏和组织的胰岛素抵抗联合作用是引起2型糖尿病的主要发病机制。一旦2型糖尿病被确诊，胰岛β细胞功能进行性降低，肝脏葡萄糖生成和释放增多，引起血糖升高。
无论是1型还是2型糖尿病，自然病程都包括以下三个阶段：1.高血糖前期，此期 可长达10年左右。1型糖尿病已存在β细胞免疫性破坏，其主要标志是出现GAD或ICA等自身抗体。2型糖尿病在此期间无特定的血清学标志，但常伴有高胰岛素血症及腹型肥胖等胰岛素抵抗综合征，胰岛功能显示糖耐量减低(impaired glucose tolerance，IGT)及(或)空腹血糖受损(Impaired fasting glucose,IFG)。2.高血糖期空腹及餐后血糖达到糖尿病的诊断标准。3.慢性并发症期。
近年来，免疫调节和炎症与糖尿病的相关性获得科学家持续关注，并且部分机理得以阐明。对胰岛抗原的自身免疫应答主导了1型糖尿病的发生和发展。研究表明，Th1细胞过度极化是导致1型糖尿病的重要因素之一。长期以来，CD4 +效应性淋巴细胞一直被划分为Th1和Th2两大类，二者通过其所分泌的细胞因子相互制约，处于动态平衡状态。当一定的β细胞自身抗原被巨噬细胞或其他抗原递呈细胞处理后，与MHC II分子一起递呈到抗原递呈细胞表面，引起自身免疫信号的释放，从而激活Th1细胞，抑制Th2细胞及其细胞因子释放，使Th细胞以Th1细胞表型占优势，Th1细胞进一步激活细胞毒性巨噬细胞，细胞毒性T细胞(CD8 +T细胞)和自然杀伤细胞(NK细胞)，损伤胰岛β细胞，引发胰岛炎症，致使胰岛素分泌减少或缺乏，最终引发1型糖尿病。
同样的，2型糖尿病多数患者伴有慢性炎症并显示胰岛素抵抗现象，因此2型糖尿病也是一种炎症性疾病。例如，炎症介质TNF-α可以活化数种具有抑制胰岛素信号传导通路的蛋白质表达，减弱了机体对胰岛素的反应能力，继而诱发了胰岛素抵抗性。丹麦科学家们发现，在小鼠中，巨噬细胞在疾病的早期阶段侵入糖尿病胰腺组织。然后，这些炎性细胞产生大量促炎蛋白(细胞因子)，直接作用并损伤胰腺中产生胰岛素的β细胞，导致2型糖尿病，这一发现发表在2014年1月的Journal of Leukocyte Biology杂志上。进一步研究发现，获得性免疫中的Th1细胞和CD8 T细胞促进胰岛素抵抗发生，而Th2细胞和Treg细胞能够拮抗这种作用。因此，控制炎症和调控T细胞功能也是控制和治疗2型糖尿病的重要靶点。
临床上胰岛素等传统药物对2型糖尿病患者胰岛β细胞功能逐年衰退的趋势不能有效阻止或逆转，对1型糖尿病的预防和治疗同样缺乏有效手段。临床研究通过注射胰岛素和抗排异反应的药物，防止T细胞和/或炎症因子攻击胰岛β细胞，但目前的研究结果并不支持这一方案的有效性，因为免疫抑制剂通常会带来许多全身性副作用，包括诸如增加机体发生肿瘤和感染的风险，免疫抑制剂本身还会导致胰岛素抵抗和β细胞功能减低。此外，通过胰岛移植治疗1型糖尿病的前景也并不乐观。截至2011年，全球50 多家医疗机构实施了逾1000例胰岛细胞移植术，然而由于胰岛移植长期效果欠佳，胰腺供体的匮乏和需要长期使用免疫抑制剂等原因，在过去的数年中实施的临床胰岛移植的数量有所减少。单克隆抗体Anti-CD3可定向减轻胰岛素分泌功能的退化，并降低新发1型糖尿病2年内对胰岛素需求的增加，在1型糖尿病早期发挥作用，受益最多的是较年轻或有较多残存β细胞功能的2型糖尿病患者，但由于存在免疫力下降、发热、皮疹和贫血等副反应，其临床应用也在进一步评价中。因此，正确了解糖尿病炎症及自身免疫相关发病机制，探索可避免使用免疫抑制剂的新方法，寻找新的抵抗炎症和自身免疫攻击的胞内因子能够更加有效治疗及管理疾病，对于患者的身体和生活意义重大。
类风湿性关节炎(Rheumatoid arthritis，RA)是一种慢性自身免疫性疾病。最近的流行病学调查表明，中国人的发病率约为0.4％，较白种人的1％稍少。以此估之，我国每年约有400-500万RA患者，且中老年女性患病率高，男女比例为1：3。RA患者免疫系统错误地攻击周围关节滑膜，引起炎症，疼痛和关节损伤。引起RA的病因尚不清楚，但是近年研究广泛认同T细胞功能紊乱，尤其是辅助性CD4 +T细胞异常活化，在RA的发病中处于中心环节。在正常人体内，促炎因子与抗炎因子处于一个平衡状态，而对于RA患者，当某个因素启动免疫反应时，Th1和Th17细胞被激活，释放炎性细胞因子引起并加重炎症进展，同时抑制Th2和Treg细胞，而促炎和抗炎平衡过程如果向Th1和Th17细胞偏移则可能造成了RA发病。RA患者血清、关节滑液中IFN-γ、TNF-α、IL-2、IL-1、IL-17等细胞因子造成并持续加重炎症的发生。IL-17在滑膜炎症中与TNF-α和IL-1协同作用，诱导产生一系列细胞因子和趋化因子，增强了滑膜炎症。与Th1和Th17细胞相反，Th2和Treg细胞通过分泌细胞因子IL-4、IL-10等参与抗炎效应。Th2细胞分泌的IL-4能抑制TNF-α、IL-1、IL-6、IL-8的活性，发挥抗炎作用。Treg细胞可通过产生细胞因子包括IL-10、TGF-β等在RA疾病模型中起免疫抑制作用。Th细胞的分化失衡在软骨破坏中也起到作用 [22]。软骨由软骨组织及其周围的软骨膜构成，软骨组织由软骨细胞、基质及胶原纤维组成。在正常的生理过程中骨形成和骨吸收之间形成一种平衡，炎症与骨侵蚀是密切联系在一起的。近年来研究发现核因子κB受体活化因子(RANK)结合其受体RANKL，介导破骨细胞的分化成熟，而OPG能够与RANK结合，阻滞RANKL与与RANK结合进而抑制破骨细胞的分化、活化，并且诱导成熟破骨细胞的凋亡。IL-17能够上调RANKL及其受体的表达，从而破坏滑液中RANKL/OPG的平衡，加重骨破坏。Th2细胞产生的IL-4、IL-10抑制破骨细胞的分化，作为Th17拮抗效 应的Treg细胞可直接抑制破骨细胞形成，而且Treg细胞可通过自身分泌的IL-10上调OPG表达，下调RANKL从而抑制骨破坏。
目前尚没有治愈RA的方法，通常使用药物和物理方法只治疗RA患者的症状，减少炎症和止痛，延缓RA进展。目前临床用于治疗RA的一线化学药物主要为非类固醇抗炎药(Nonsteroidal Anti-inflammatory Drug,NSAID)，包括gold compounds、D-penicillamine、antimalarials、sulfasalazine、methotrexate等，主要通过抑制免疫系统，减少对于关节的破坏。治疗RA的生物药Infliximab、Etanercept、Anakinra主要以TNF-α和IL-1为靶点，拮抗TNFα和IL-1的促炎作用。这些基因工程生物药物能够降低急性发炎反应，减轻患者的肿胀与疼痛，但有30％-40％患者对TNF-α拮抗剂无疗效反应，并且有些病人会对这种嵌合抗体产生抗体，使其逐渐失去功效。此外，TNF-α在正常免疫系统中是重要的细胞因子，阻断它会产生严重的副作用，增加感染风险。因此，对于目前的抗风湿药物，已提出短期与长期耐受性及安全性疑虑，有必要进一步研究新的治标的，阻断自身免疫，重新平衡免疫调节的新型药物。
多发性硬化症(Multiple sclerosis,MS)是最常见的一种中枢神经脱髓鞘疾病。本病急性活动期中枢神经白质有多发性炎性脱髓鞘斑，陈旧病变则由于胶质纤维增生而形成钙化斑，以多发病灶、缓解、复发病程为特点，好发于视神经、脊髓和脑干，多发病于青、中年，女性较男性多见。在人的MS病理研究中，发现人MS与小鼠实验性变态反应性脑脊髓炎(experimental allergical encephalomyelitis，EAE)相似，其发生机制是针对MBP特异的Th细胞穿过血脑屏障进入中枢神经系统后，接触MBP发生免疫应答，激发炎症反应，产生炎性因子和趋化因子浸润，导致脊髓鞘破坏，造成患者出现视觉障碍、肌肉无力等症状 [25]。MS主要以Th1、Th17反应为主，病灶区IL-2和IFN-γ和IL-17产生明显增加；在恢复期，IL-4、IL-10和TGF-β产生增多，Th1型细胞因子产生随之下降。用特异性Th2细胞回输后，可阻止EAE发展 [27]。在MS临床研究中，也发现了类似的情况。因此，阻断Th细胞分化失衡也将对治疗MS起着至关重要的作用。
MS尚无根治办法，治疗皆着重于抑制免疫引起的炎症反应。目前FDA共核准六种治疗MS的药品，分为免疫调节剂(Interferon-β1a,皮下注射和肌肉注射两种剂型Interferon-β1b皮下注射,Glatiramer acetate)，免疫抑制剂(mitoxantrone)和单克隆抗体(natalizumab)。Interferon-β的优点是安全性高，但仅可降低30％复发次数，并且价格昂贵，不适合广泛推广。Mitoxantrone是一种抗癌药，主要用来治疗白血病、乳腺癌 淋巴癌和肝癌等。Mitoxantrone能够抑制T细胞和B细胞及巨噬细胞的增殖，发挥免疫抑制作用。Mitoxantrone最大的副作用是心肌毒性，当累计计量大于140mg/m 2时，容易发生心肌毒性。以每三个月使用12mg/m2计算，最多只能使用三年，相对于MS平均30年的病程，mitoxantrone使用期限过于短暂 [30]。除了mitoxantrone，其他的免疫抑制剂如azathioprine，cyclophosphamide，methotrexate，mycophenolate mofetil和tacrolimus都有短期免疫抑制疗效，但是副作用明显，如cyclophosphamide会引起出血性膀胱炎，methotrexate低剂量使用仍会造成肝毒性，mycophenolate mofetil，tacrolimus及azathioprine具有骨髓抑制作用。Natalizumab中和淋巴细胞表面的黏附分子α-4integrin，阻止其与血管内皮细胞及基质细胞上的配体(vascular cell adhesion molecule-1,VCAM-1)结合，抑制淋巴细胞穿出血管内皮到达组织，从而降低了脑组织中的自身免疫炎症，同时也不可避免的影响整体免疫反应，感染JC病毒并发生进行性多部脑白质病变(Progressive multifocal leukoencephalopathy，PML)的风险升高。Natalizumab还具有严重的肝毒性。MS是一个迁延反复难治性慢性自身免疫病，因此治疗药物的研发除了疗效，还需要考虑长期使用的安全性和方便性，有效改变免疫紊乱造成的自身免疫攻击无疑是解决这些问题的最佳途径。
SLE是一种慢性的自体免疫性疾病。发病原因不明，患者免疫系统产生自身抗体攻击自身细胞和组织，导致发炎和组织损害。常见症状包含关节炎、发烧、胸痛、脱发、口腔溃疡、淋巴腺肿大、疲倦，及脸部疹等等。SLE的发病机理不明确，研究认为，T细胞异常在其病理发生发展过程中起到重要作用。在遗传因素、环境因素、雌激素水平等各种因素相互作用下，SLE中自身反应性Th细胞被自身抗原活化后出现克隆性扩增,并产生细胞因子。在MRLP lpr狼疮鼠中IFN-γ分泌增加，Th1/Th2细胞比例升高；Ⅳ型即弥漫增殖性狼疮性肾炎(DPLN)的病人，外周血和肾脏中Th1/Th2细胞均明显升高,且与肾脏损害程度呈正相关，这表明Th1细胞也在SLE的发病中发挥重要作用。同时，SLE患者中Treg细胞数量下降，功能降低、B细胞过度增生，产生大量的自身抗体，并与体内相应的自身抗原结合形成相应的免疫复合物，沉积在皮肤、关节、小血管、肾小球等部位。在补体的参与下，引起急慢性炎症及组织坏死(如狼疮肾炎)，或抗体直接与组织细胞抗原作用，引起细胞破坏，从而导致机体的多系统损害。
迄今仍无根治SLE的方法，目前使用的治疗药物包含非类固醇抗炎药物(NSAID)、皮质类固醇、免疫抑制剂、羟氯喹，以及氨甲蝶呤。非类固醇抗炎药物的常见副作用包 括肠胃不适、胃灼热、腹泻以及身体积水，皮质类固醇可快速抑制炎症，由于其药力很强，一般使用最低剂量来达到最大的疗效。皮质类固醇的短期副作用包括浮肿、食欲大增以及体重增加，长期副作用包括骨质减弱(骨骼疏松)、高血压、高胆固醇、高血糖(糖尿病)、动脉损坏、感染及白内障。免疫抑制剂普遍副作用包括恶心、呕吐、脱发、膀胱出问题、不容易受孕、增加患癌症以及受感染的机率。上述系统性红斑狼疮疗法的患者在停止用药后，疾病有可能会复发，但是用药疗程越长，副作用的风险就越大。因此，改善SLE中Th细胞分化失衡从而缓解和逆转自身免疫攻击是彻底治愈SLE的研究方向。
多不饱和脂肪酸(PUFAs)是指具有18-22个碳原子，含有2-3个氢键的直链脂肪酸，通常分为ω-3和ω-6两种，距羧基最远端的双键在倒数第3个碳原子上的称为ω-3PUFAs，在第六个碳原子上的，则称为ω-6 PUFAs。ω-6和ω-3 PUFAs都是由饱和脂肪酸前体在不同的去饱和酶作用下合成而来，脊椎类和哺乳动物恰好缺乏特异的去饱和酶，因此不能在体内合成ω-6和ω-3，只能从膳食中摄入这些不饱和脂肪酸。
ω-3 PUFAs对于维持正常的生理功能极为重要，它是构成细胞脂膜结构的关键成分。大量的研究表明，维持ω-6/ω-3 PUFAs的比例平衡对于健康起到重要作用。最近发表的两项大规模临床研究结果表明，长期摄入ω-3 PUFAs与1型糖尿病的发病率成反比：一项基于2000位挪威儿童的病例对照研究表明，从出生后第一年开始服用富含ω-3 PUFAs的鱼肝油能够降低儿童1型糖尿病的发病风险；美国青少年自身免疫性糖尿病研究项目(DAISY)的一项追踪性调查表明，具有1型糖尿病遗传风险的儿童通过饮食摄入ω-3PUFAs后，能够降低胰岛发生自身免疫炎症的风险。此外，棕榈酸等游离脂肪酸在体外抑制胰岛素分泌，而ω-3 PUFAs能够逆转这种现象。通过饮食补充ω-3 PUFAs可以增强葡萄糖刺激的胰岛素分泌水平。此外，近年有多项临床研究提示，ω-3 PUFAs具有抑制炎症，预防和缓解自身免疫的作用，对RA，MS，SLE发病和病理发展具有保护性作用。但是，临床上使用含有ω-3 PUFAs的鱼油胶丸治疗糖尿病和控制体重的尝试却大多以失败告终，究其原因，摄取的食物中ω-6 PUFAs的含量过高，单单依靠补充鱼油胶丸是难以平衡二者比例。因此，本发明提出了一种病毒载体，所述病毒载体携带的fat-1基因编码的ω-3不饱和脂肪酸去饱和酶(ω-3 fatty acid desaturase)促使体内ω-6转变为ω-3，平衡二者比例。
来源于秀丽隐杆线虫(C.elegans)的fat-1基因，其蛋白产物是ω-3不饱和脂肪酸去 饱和酶，它以ω-6 PUFAs为底物进行去饱和反应，生成相应的ω-3 PUFAs，在显著提高动物体内内源性的ω-3 PUFAs含量的同时，降低ω-6 PUFAs含量，改变ω-6/ω-3 PUFAs的比例。为了使fat-1能更好的在哺乳动物中表达，我们将编码fat-1的cDNA进行了哺乳动物化(mammalianized)，因此又将其命名为mfat-1。mfat-1转基因小鼠胰岛β细胞能够显著抵抗细胞因子诱导的抗凋亡作用，并且ω-3 PUFAs能够促进胰岛素分泌。
发明内容
本发明所要解决的第一个技术问题在于提供一种病毒载体，所述病毒载体安全无毒，且具备治疗活性，能够满足临床需求。
本发明所要解决的第二个技术问题在于提供一种上述病毒载体的构建方法。
本发明最后要解决的第三个技术问题在于提供一种上述病毒载体在制备治疗糖尿病或自身免疫相关疾病药物中的应用。
为解决上述技术问题，本发明提供了一种病毒载体，所述病毒载体为克隆有mfat-1基因的慢病毒表达质粒或克隆有mfat-1基因的腺相关病毒表达质粒，所述的mfat-1基因如SEQ ID No.:1所示。
所述的病毒载体，所述的慢病毒表达质粒为pLJM1-CMV-hPGK-EGFP质粒、pLJM1-CMV-hPGK-mkate2质粒、pLenti-CMV-MCS-GFP-SV-puro质粒、FUGW、pLenti-puro、pLenti-MP2或pLenti质粒；所述的腺相关病毒表达质粒为pEMBL-AAV-D(+)-CMV-eGFP-SV40质粒、AAV GFP质粒、AAV1质粒、AAV2质粒、rAAV2质粒、AAV5质粒、AAV8质粒或pAV-FH AAV质粒。
本发明提供了一种构建所述的病毒载体的方法，将所述mfat-1基因克隆至慢病毒表达质粒或腺相关病毒表达质粒，即得。
所述的病毒载体的构建方法，包括如下步骤：
(1)以所述mfat-1基因序列为模板，设计如下引物进行PCR扩增，使获得的所述mfat-1基因序列两端带有NheI和EcoRI酶切位点，所述引物如下：
mfat-1-F：5'-TATTAAGCTAGCATGGTCGCCCACAGCA-3'；
mfat-1-R：5'-CAACCGGAATTCTCATCACTTGGCCT-3'；
(2)将步骤(1)获得的PCR扩增产物进行电泳，然后切胶回收、纯化，将所得 PCR扩增产物进行用限制性内切酶NheI和EcoR酶切，备用；取空载的所述慢病毒表达质粒或腺相关病毒表达质粒使用限制性内切酶NheI和EcoR酶切，备用；
(3)将步骤(2)获得的DNA片段连接在酶切后所述空载穿梭质粒上，即得。
所述的病毒载体的构建方法，所述步骤(1)中的PCR扩增反应体系包括：
Figure PCTCN2018081117-appb-000001
Buffer(Mg2+plus)，10μl；
dNTP Mixture，各dNTP 2.5mM，4μl；
模板DNA，20-200 ng；
上游引物mfat-1-F，10μM，1μl；
下游引物mfat-1-R，10μM，1μl；
Figure PCTCN2018081117-appb-000002
HS DNA Polymerase，2.5U/μl，0.5μl；
补灭菌超纯水至50μL。
优选的，所述模板DNA为100ng。
所述的病毒载体的构建方法，所述步骤(1)中的PCR扩增反应程序为：98℃变性5min；98℃保持10s，60℃保持15s，72℃保持2min，共30个循环；最后72℃延伸10min。
本发明提供了一种重组病毒载体，所述重组病毒载体通过所述的病毒载体构建而成。
本发明提供了一种病毒颗粒，为将所述的病毒载体转化至细胞中后得到的病毒颗粒。
优选的，所述的病毒颗粒为所述的病毒载体通过脂质体转染至293FT细胞包装扩增所得。
优选的，所述病毒颗粒的给药方式为静脉注射。
本发明提供了一种所述的病毒载体或所述的重组病毒载体或所述的病毒颗粒在制备治疗糖尿病药物或治疗自身免疫相关疾病药物中的应用。
所述的应用，所述的自身免疫相关疾病为因Th细胞分化失衡及其分泌细胞因子失衡所导致的自身免疫病。
所述的应用，所述的糖尿病为自身免疫性1型糖尿病。
所述的应用，所述的自身免疫病为1型糖尿病(T1D)、多发性硬化症(MS)、类风湿性关节炎(RA)或系统性红斑狼疮(SLE)。
本发明提供了一种药物，包括所述的病毒载体或所述的重组病毒载体或所述的病毒颗粒，以及上述的病毒载体、重组病毒载体或病毒颗粒的药物可接受的载体。
优选的，所述的药物包括将所述的病毒载体或所述的重组病毒载体或所述的病毒颗 粒按照常规工艺，添加常规辅料制备成临床上可接受的剂型。
其中，所述的慢病毒表达质粒为pLJM1-CMV-hPGK-EGFP质粒(购自于Addgene(Plasmid#19319))和pLJM1-CMV-hPGK-mkate2质粒(购自于addgene )。
其中，所述的腺相关病毒表达质粒为pEMBL-AAV-D(+)-CMV-eGFP-SV40质粒(购自于addgene)。
本发明采用基因治疗的方法，利用本发明的所述病毒颗粒将mfat-1基因带入到自发型非肥胖糖尿病模型小鼠(NOD小鼠)体内，与对照组小鼠相比，mfat-1慢病毒颗粒能够显著降低发病NOD小鼠血糖水平，同时升高其血清中胰岛素水平，具有显著的治疗作用。mfat-1基因疗法治疗糖尿病和自身免疫的可能机制包括：mfat-1以体内过高的ω-6 PUFAs为底物，将其转化为ω-3 PUFAs，平衡二者比例，使得ω-6/ω-3的比值趋于1：1。这些由ω-6 PUFAs转化而来的内源性ω-3 PUFAs促进Th细胞向Th2方向分化，并且拮抗ω-6 PUFAs的促Th1细胞活化的效应，降低IFN-γ、IL-6、TNF-α、IL-17等促炎因子分泌水平，缓解Th1细胞介导的炎症反应；抑制CD8 +CTL细胞、巨噬细胞在胰岛中浸润，缓解胰岛炎和胰周炎的发生；由于哺乳动物体内缺乏不饱和脂肪酸脱氢酶，因此需要采用慢病毒载体或腺相关病毒载体将mfat-1导入体内。
上述病毒载体的给药方式为静脉注射。
有益效果：与现有技术相比，本发明的病毒载体可以用于制备治疗1型糖尿病和相关自身免疫性疾病的生物药物，如以病毒载体转染至细胞中的病毒颗粒是一种安全无毒具备抗炎活性的新型生物药物，以慢病毒为载体，利用基因重组技术高表达多不饱和脂肪酸脱氢酶，抗自身反应性炎症活性强，能满足使用需求，制备方法简单，容易操作，具有很好的应用前景。
附图说明
图1是pLJM1-mfat-1-EGFP质粒NheI和EcoRI酶切鉴定1％琼脂糖凝胶电泳图；
图2是本发明实施例2中慢病毒颗粒体外处理正常人(图2A-E)和1型糖尿病患者(图2F-J)外周血单核细胞24小时后，人外周血CD4 T细胞分化及其细胞因子变化的结果图；
图3是本发明实施例2中体外处理1型糖尿病患者外周血单核细胞24小时后，外周血CD4 T细胞转录因子变化的结果图；
图4是本发明实施例3中给予慢病毒治疗9周后NOD鼠糖尿病发病率变化的结果 图。
图5是本发明实施例3中给予慢病毒治疗后NOD鼠血糖变化的结果图；
图6是本发明实施例3中给予慢病毒治疗9周后NOD鼠胰岛素分泌的结果图；
图7是本发明实施例3中给予慢病毒治疗9周后NOD鼠胰岛炎症浸润发展变化的结果图；
图8是本发明实施例中3给予慢病毒治疗9周后NOD鼠CD4细胞分化的结果图。
具体实施方式
根据下述实施例，可以更好地理解本发明。然而，本领域的技术人员容易理解，实施例所描述的内容仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明。下述实施例中涉及到的：DH5α大肠杆菌，由TAKARA提供；LB培养基，由Sigma提供；293FT细胞株，由ATCC提供；生长培养基为DMEM，由GIBCO提供；FBS胎牛血清，由GIBCO提供；OPTI-MEM，由GIBCO提供；Lipofectamine2000转染试剂，由Invitrogen-Thermo Fisher Scientific提供；NOD小鼠，由The Jackson Laboratory提供。离心机生产厂家为美国Thermo公司、型号为FRESCO17高速冷冻离心机；电泳仪生产厂家为美国BIO-RAD公司、型号为PowerPac TMand Mini-Sub cell GT；凝胶成像仪生产厂家为美国BIO-RAD公司、型号为ChemiDoc TMXRS+System。
实施例1：克隆有mfat-1慢病毒表达质粒的病毒载体的构建。
先合成mfat-1(如SEQ ID No.:1所示)基因(由金斯瑞公司提供)，接着通过NheI和EcoRI酶切位点亚克隆至pLJM1-CMV-hPGK-EGFP质粒(addgene，Plasmid#19319)，得到pLJM1-CMV-hPGK-EGFP-mfat-1(PLJM1-mfat-1)表达质粒。具体构建过程如下：
1)PCR扩增mfat-1区
以合成的mfat-1基因序列为模板，设计如下引物，PCR扩增mfat-1区，使其序列两端带有NheI和EcoRI酶切位点：所述引物如下：
上游引物mfat-1-F:5'-TATTAAGCTAGCATGGTCGCCCACAGCA-3'；
下游引物mfat-1-R:5'-CAACCGGAATTCTCATCACTTGGCCT-3'；
PCR扩增体系见表1。
表1
Figure PCTCN2018081117-appb-000003
Figure PCTCN2018081117-appb-000004
所述PCR扩增程序如下：98℃变性5min；98℃10s，60℃15s，72℃2min，共30个循环；最后72℃延伸10min。
2)酶切
将上述步骤获得PCR扩增产物采用1.5％琼脂糖凝胶电泳回收目的DNA片段，使用Agarose Gel DNA Extraction Kit获得纯化的目的DNA片段，将上述纯化的mfat-1的DNA片段用限制性内切酶NheI和EcoR酶切，备用；用对应的限制性内切酶NheI和EcoR酶对pLJM1-EGFP空载穿梭质粒进行酶切。所述酶切反应体系如下表2：
表2
Figure PCTCN2018081117-appb-000005
酶切反应条件：37℃水浴2h。上述酶切产物用1％琼脂糖凝胶电泳分离目的片段，使用Agarose Gel DNA Extraction Kit获得纯化的质粒或目的DNA片段。
3)连接
将目的DNA片段与pLJM1载体连接。
表3
Figure PCTCN2018081117-appb-000006
反应条件：室温(16℃)连接过夜即12小时，即得所述PLJM1-mfat-1载体。
本实施例还提供了一种病毒颗粒，通过将上述制备的病毒载体转化至细胞中制备得到所述的病毒颗粒，具体步骤如下：
1 转化使用的感受态细菌为DH5α大肠杆菌，采用CaCl 2法制备，制备方法及后续的转化操作参照《分子克隆》第二版，具体如下：
A 感受态的制备：
i 将1ml饱和菌液加入100ml LB中，摇菌，37℃，280rpm，2-3h，直到反映细菌密度的光密度OD600在0.4-0.6之间；
ii 转入冰预冷的50ml聚丙烯管中，置于冰浴中10min；
iii 4℃，1000g，离心10min；
iv 弃上清，将管倒置1min，使液体流尽，加入10ml冰预冷的0.1mol/l CaCl 2重悬沉淀，置于冰浴中30min；
v 4℃，按照1000g，离心10min；
vi 弃上清，加入2ml冰预冷的0.1mol/l CaCl 2重悬沉淀，加入0.5ml 75％的灭菌甘油(用0.1mol/l CaCl 2配制)，混匀，100μl/管分装至1.5ml离心管中，-80℃冰箱中可保存半年。
vii 转化效率鉴定
B 连接产物的转化：
i 取1管感受态细胞，置于冰上融解，加入3μl连接产物，混匀，冰浴30min；
ii 42℃水浴，静置90 sec。迅速转移至冰中，冰浴1-2min；
iii 加入900μl的LB培养基，37℃水浴10min；
iv 细菌扩增，37℃，210rpm，45min；
v 涂板，将100μl上述已转化的细菌涂布于含100μg/ml氨苄青霉素的琼脂平板上；
vi 倒置平板，37℃培养16-20h。
C 阳性克隆筛选(酶切鉴定、测序鉴定)，采用1％琼脂糖凝胶电泳鉴定，结果见图1所示，说明所述病毒载体PLJM1-mfat-1构建成功。
2.包装、扩增和纯化lenti-mfat-1慢病毒颗粒。
慢病毒的包装细胞为293FT细胞株，生长培养基为DMEM(含10％FBS)。贴壁细胞经培养生长增殖形成单层细胞。
(1)病毒扩增：
1)转染前24h，用胰蛋白酶消化对数生长期的293FT细胞，以含10％FBS胎牛血清,(由GIBCO提供)的DMEM高糖培养基(由GIBCO提供)调整细胞密度为1 x 10 5细胞/ml，接种于六孔板，37℃，5％CO 2培养箱内培养。24h后待细胞密度达0.8 x 10 6细胞/ml时即可用于转染。细胞状态对于病毒包装至关重要，因此需要保证良好的细胞状态和较少的传代次数。
2)转染前1h将细胞培养基更换为新鲜完全培养基。
3)在1.5ml EP管内加入以下质粒试剂PLJM1-mfat-1载体1.5μg，psPAX2载体Addgene(Plasmid#12260)1.125μg，pMD2.G载体Addgene(Plasmid#12259)0.375μg)，与100μl OPTI-MEM混合均匀，室温(20℃-30℃)孵育5分钟。
4)在另一个1.5ml EP管内先加100μl OPTI-MEM，再加入3μl Lipofectamine2000转染试剂，轻弹混匀，室温(20℃-30℃)孵育5分钟。
5)取步骤3)中孵育后的质粒试剂(PLJM1-mfat-1，psPAX2和pMD2.G三质粒混合物)与步骤4)中稀释后的Lipofectamine2000转染试剂进行混合，轻轻混匀，在室温下(20℃-30℃)温育15分钟，以便形成所述质粒试剂与Lipofectamine2000转染试剂的转染复合物。
6)取200μl步骤5)获得试剂(步骤5)最后得到的PLJM1-mfat-1，psPAX2和pMD2.G与Lipofectamine2000转染试剂的转染复合物)滴入步骤1)中的含293FT细胞培养基中，轻轻摇匀，于37℃，5％CO 2细胞培养箱中培养。
(2)病毒纯化：
1)将上述步骤中，含293FT细胞培养基培养24h后，弃去含有转染复和物的培养基，加入2ml新鲜完全培养基继续培养。
2)分别于48h和72h收集细胞上清，将两次收集上清混合，于1250rpm离心5min去除可能的293FT细胞，再用0.45μm滤器过滤，即得Lenti-mfat-1慢病毒颗粒，即所述的病毒颗粒。所述病毒颗粒可短期(<3d)保存于4℃，长期保存于-80℃备用，分装避免反复冻融。
(2)病毒滴度测定
采用TCID50法测定病毒滴度。对照裸病毒的扩增、纯化及滴度测定同上。
实施例2 ω-3 PUFAs对1型糖尿病患者外周血CD4 T细胞的调控作用
(1)分别抽取患者和正常人外周血5ml，用pH为7.2的PBS稀释一倍后与淋巴细胞分离液Lymphoprep(Axis-Shield,Norway)混合，3500rpm离心20min后取淋巴细胞，以RPMI1640培养基(购于Gibco)培养，然后分别采用终浓度为100μM的DHA(二十二碳六烯酸)、终浓度为100μM的EPA(二十碳五烯酸)和终浓度为100μM的AA(花生四烯酸)于体外处理上述细胞24小时后，检测上述细胞中CD4 T细胞胞内细胞因子的变化。(上述DHA、EPA和AA均购于Sigma)。
(2)CD4T细胞胞内因子的标记：分别取上述步骤中的患者和正常人的淋巴细胞(数量达1×10 5个)至不同的标记管中，每个标记管中加入标记荧光单克隆抗体CD3(0.1μg)，CD8(0.1μg)，之后每管加入1ml的质量浓度为4％多聚甲醛，混匀，室温(20℃-30℃)孵育20min，然后加入1ml破膜剂0.5％saponin，室温(20℃-30℃)孵育30min，然后于4℃，按照300g离心5min，去上清，然后加入80μl的0.5％的saponin重悬，将所获得的悬液分别使用IFN-γ荧光单克隆抗体(0.1μg)、IL-17荧光单克隆抗体(0.1μg)、IL-4荧光单克隆抗体(0.1μg)和Foxp3荧光单克隆抗体(0.1μg)标记细胞，4℃，避光孵育1小时(其中采用Foxp3荧光单克隆抗体标记的需要孵育破膜8小时)；然后采用0.5％saponin洗三次，4℃，按照300g离心5min，去上清，采用质量浓度1％多聚甲醛固定，在24小时后，分别采用流式细胞仪Accuri-C6进行检测，分析Th1、Th2、Th17和Treg细胞的比例(上述步骤中所使用的抗体和仪器均来自BD Bioscience)。
(3)CD4T细胞胞内转录因子的标记：取上述步骤1)中的患者和正常人的淋巴细胞(数量达1×10 5个)至标记管中，每个标记管中加入标记荧光单克隆抗体CD3(0.1μg)，CD8(0.1μg)。之后每管加入1ml的4％多聚甲醛，混匀，室温孵育20min；加入1ml破膜剂0.5％saponin，室温孵育30min，4℃，300g离心5min，去上清；80μl的0.5％saponin重悬，将所获得的悬液分别使用T-bet荧光单克隆抗体(0.1μg)、GATA3荧光单克隆抗体 (0.1μg)、RORγT荧光单克隆抗体(0.1μg)和Foxp3荧光单克隆抗体(0.1μg)标记细胞，4℃，避光孵育1小时(Foxp3需要破膜8小时)；0.5％saponin洗三次，4℃，300g离心5min，去上清；1％多聚甲醛固定，流式细胞仪Accuri-C6进行检测，分析上述转录因子的比例(步骤中使用抗体和仪器均来自BD Bioscience)。
(4)结果说明：检测结果如图2和图3所示，其中，图2中的CON为空白对照组未经DHA、EPA和AA任一处理的正常人的外周血的淋巴细胞，图3中的CON为空白对照组未经DHA、EPA和AA任一处理的患者的外周血的淋巴细胞，图2中的A图、B图、C图、D图和E图分别为正常人的外周血CD4 T细胞中Th1、Th2、Th1/Th2、Th17和Treg细胞的百分比，F图、G图、H图、I图和J图分别为患者的的外周血CD4 T细胞中Th1、Th2、Th1/Th2、Th17和Treg细胞的百分比，图3中的A图、B图、C图、D图分别为患者的外周血CD4 T细胞中转录因子T-bet、GATA3、RORγT和Foxp3的百分比，如图2-3所示，分别采用DHA、EPA和AA处理过的1型糖尿病患者外周血CD4T细胞中Th1、Th17细胞的百分比下降，Th2、Treg细胞的百分比上升，重新平衡了Th1/Th2比例和T17/Treg比例，同时调控Th1细胞因子表达的转录因子T-bet和调控Th17细胞因子表达的转录因子RORγT被ω-3PUFAs显著抑制，而调控Th2和Treg细胞因子表达的转录因子GATA3和Foxp3在ω-3PUFAs的作用下表达升高。这些结果充分表明，ω-3PUFAs具有重新平衡Th细胞分化，显著抑制由Th1/Th2和T17/Treg比例失衡造成的炎症，进一步说明了本发明所述的病毒颗粒携带的mfat-1基因可以通过以体内过高的ω-6 PUFAs为底物，将其转化为ω-3 PUFAs，平衡二者比例，使得ω-6/ω-3的比值趋于1：1，进而抑制由Th1/Th2和T17/Treg比例失衡造成的自身免疫病，如1型糖尿病、多发性硬化症、类风湿性关节炎和系统性红斑狼疮。
实施例3：Lenti-mfat-1慢病毒颗粒治疗1型糖尿病的效果。
(1)1型糖尿病小鼠模型的构建：
取NOD小鼠若干只，体重25g，每天固定时间采血检测NOD小鼠随机血糖，数值连续两周超过11.1mmol/L的小鼠被挑选出来用于实验，随机取上述血糖连续两周超过11.1mmol/L的NOD小鼠分为两组，对照组(Lenti-Con)和慢病毒治疗组(Lenti-mfat-1)，每组5只；
(2)给药方法：
治疗组按照病毒滴度为10 8TU/mL的浓缩液按照小鼠体重按10 9TU/Kg进行尾静脉 注射Lenti-mfat-1慢病毒颗粒后，对照组相同条件下给予不含mfat-1基因的pLJM1-CMV-hPGK-EGFP质粒，检测小鼠随机血糖的变化。
(3)结果与讨论：
1)接受Lenti-mfat-1慢病毒颗粒治疗后NOD小鼠的一般生理情况
给予治疗组小鼠尾静脉注射Lenti-mfat-1慢病毒颗粒后，与对照组相比，小鼠摄食饮水情况正常，治疗2周后，治疗组体重率高于对照组，但无显著统计学意义。
2)在给予本发明实施1的病毒颗粒治疗9周后，检测小鼠外周血多不饱和脂肪酸变化水平，具体步骤如下：采用常规有机化学方法对两组小鼠的外周血中提取脂肪酸后，将上述提取的样品(提取的脂肪酸)溶解于庚烷中滴入上样瓶中即可上样检测。所述样品通过自动进样器进样，由安捷伦7890A进行检测，单次进样运行时间约为1小时。气相色谱条件，色谱条件为：色谱柱型号：SP2380，105m*0.53mm*0.20μm(安捷伦)；运行程序：初始温度140℃，保持3min，之后以8℃/min的速率升到220℃，保持12min；进样器、检测器温度：260℃；载气为氦气，速率设置为：12psi。最终根据样品的出峰时间与标准品(购自美国Sigma公司)，对比来确定脂肪酸类型，并以各个脂肪酸的含量百分比来得出最终结果。通过与标准品对比出峰时间、峰形、峰面积百分比来确定目的峰；计算所有ω-3和ω-6多不饱和脂肪酸面积百分比的总和，并算出ω-6/ω-3比值。色谱柱型号：SP2380，105m*0.53mm*0.20μm)发现给予Lenti-mfat-1慢病毒颗粒的小鼠EPA比例增加，说明给予的携带mfat-1慢病毒颗粒成功工作(表5)。
表5给予病毒颗粒治疗后小鼠血液多不饱和脂肪酸的改变
Figure PCTCN2018081117-appb-000007
每种ω-3和ω-6多不饱和脂肪酸使用相对百分比表示，其中由气相色谱检测出的所有 脂肪酸的峰面积为百分之百，目的脂肪酸的含量则由其出峰面积除以总的峰面积和计算得出。每组数据重复三次，使用oneway-ANOVA统计，与对照组相比*P<0.05，NP:未检测出。
3)对两组的NOD小鼠随机血糖水平、胰岛素水平及胰岛炎症的监测
随机血糖连续两周低于11.1mmol/L的小鼠被认为血糖恢复正常。对Lenti-mfat-1治疗组和对照组NOD小鼠眼眶取血后采用美国Roche公司Accu-Chek血糖仪检测血糖水平，结果见图4-5，治疗组与对照组相比，治疗组NOD小鼠的随机血糖在第二周开始已有明显下调，截至第六周，血糖水平已下降并维持在正常水平，达到6.3mmol/L。说明Lenti-mfat-1慢病毒颗粒对NOD小鼠具有显著的治疗作用。在治疗9周后，对两组小鼠眼球取血2ml，静置5min后于3500rpm离心15min，取上层血清200μl，用pH为7.4的PBS稀释100倍后采用Mercodia公司试剂盒检测血清中胰岛素浓度，结果见图6所示。在治疗9周后，在体式镜下取两组小鼠胰腺后进行常规石蜡包埋、切片，进一步HE染色后观察小鼠胰岛淋巴细胞浸润情况，结果见图7所示。
4)Lenti-mfat-1慢病毒颗粒治疗后NOD小鼠CD4 T淋巴细胞分化的改变
分别取上治疗组和对照组NOD小鼠脾脏淋巴细胞(数量达1×10 5个)至不同的标记管中，每个标记管中加入标记荧光单克隆抗体CD3(0.1μg)，CD8(0.1μg)，之后每管加入1ml的质量浓度为4％多聚甲醛，混匀，室温(20℃-30℃)孵育20min，然后加入1ml破膜剂0.5％saponin，室温(20℃-30℃)孵育30min，然后于4℃，按照300g离心5min，去上清，然后加入80μl的0.5％的saponin重悬，将所获得的悬液分别使用IFN-γ荧光单克隆抗体(0.1μg)、IL-17荧光单克隆抗体(0.1μg)、IL-4荧光单克隆抗体(0.1μg)和Foxp3荧光单克隆抗体(0.1μg)标记细胞，4℃，避光孵育1小时(其中采用Foxp3荧光单克隆抗体标记的需要孵育破膜8小时)；然后采用0.5％saponin洗三次，4℃，按照300g离心5min，去上清，采用质量浓度1％多聚甲醛固定，在24小时后，分别采用流式细胞仪Accuri-C6进行检测，分析Th1、Th2、Th17和Treg细胞的比例(上述步骤中所使用的抗体和仪器均来自BD Bioscience)。结果如图8所示，Lenti-mfat-1慢病毒颗粒治疗后NOD小鼠CD4 T细胞中Th1、Th17细胞的百分比下降，Th2、Treg细胞的百分比上升，重新平衡了Th1/Th2比例和T17/Treg比例，显著改善了由Th1/Th2和T17/Treg比例失衡造成的炎症环境。
实施例4 mfat-1转基因小鼠完全抵抗诱导剂诱导其发生MS。
(1)模型建立：Wide type小鼠(购自南京大学模式动物研究所)3只，(6-8周，二雌一雄)；maft-1转基因小鼠(由南京大学模式动物研究所提供，所述maft-1转基因小鼠的fat-1基因采用pST181原核表达载体进行表达，在fat-1基因启动子CMV-β-actin上游设计了muscle creatine kinase(MCK)增强子，提高了fat-1基因在哺乳动物中的表达效率(mammalianized)，因此将其命名为mfat-1转基因小鼠)3只(6周，二雌一雄)。所有小鼠经MOG免疫，将MOG35-55(购自Sigma)按照300μg/体重(kg)背部皮下4点注射免疫动物。第一次免疫后六天以同样剂量进行第二次免疫。动物存活24天。
(2)模型观察指标：临床神经病变的行为观察：自造模后第12天起每天观察记录一次动物行为学变化(Kerlero评分法)；1分，尾巴无力；2分，尾巴瘫痪；2.5分，单侧后肢轻度无力；3分，单侧后肢显著无力；4分，单侧后肢瘫痪；4.5分，单侧后肢瘫痪伴随对侧后肢无力或者同侧前肢轻度无力；5分，双侧后肢瘫痪；6分，双侧后肢瘫痪伴随单侧前肢瘫痪。
(3)结果说明：结果见下表6所示，正常野生型wild type小鼠造模第14天开始出现尾巴无力的轻度症状，至第23天，全部表现出单侧后肢接近瘫痪的表现；在相同的诱导条件下，mfat-1转基因小鼠全程没有表现出明显发病症状。说明，mfat-1基因在体内起到了显著抵抗或治疗MS的作用。
表6mfat-1转基因小鼠与正常wildtype小鼠关于MS的发病行为评分
Figure PCTCN2018081117-appb-000008
Figure PCTCN2018081117-appb-000009
Figure PCTCN2018081117-appb-000010

Claims (15)

  1. 一种病毒载体，其特征在于，所述病毒载体为克隆有mfat-1基因的慢病毒表达质粒或克隆有mfat-1基因的腺相关病毒表达质粒，所述的mfat-1基因如SEQ ID No.:1所示。
  2. 根据权利要求1所述的病毒载体，其特征在于，所述的慢病毒表达质粒为pLJM1-CMV-hPGK-EGFP质粒、pLJM1-CMV-hPGK-mkate2质粒、pLenti-CMV-MCS-GFP-SV-puro质粒、FUGW、pLenti-puro、pLenti-MP2或pLenti质粒；所述的腺相关病毒表达质粒为pEMBL-AAV-D(+)-CMV-eGFP-SV40质粒、AAV GFP质粒、AAV1质粒、AAV2质粒、rAAV2质粒、AAV5质粒、AAV8质粒、AAV9质粒或pAV-FH AAV质粒。
  3. 一种构建权利要求1或2所述的病毒载体的方法，其特征在于，将所述mfat-1基因克隆至慢病毒表达质粒或腺相关病毒表达质粒，即得。
  4. 根据权利要求3所述的病毒载体的构建方法，其特征在于，包括如下步骤：
    (1)以所述mfat-1基因序列为模板，设计如下引物进行PCR扩增，使获得的所述mfat-1基因序列两端带有NheI和EcoRI酶切位点，所述引物如下：
    mfat-1-F：5'-TATTAAGCTAGCATGGTCGCCCACAGCA-3'；
    mfat-1-R：5'-CAACCGGAATTCTCATCACTTGGCCT-3'；
    (2)将步骤(1)获得的PCR扩增产物进行电泳，然后切胶回收、纯化，将所得PCR扩增产物进行用限制性内切酶NheI和EcoR酶切，备用；取空载的所述慢病毒表达质粒或腺相关病毒表达质粒使用限制性内切酶NheI和EcoR酶切，备用；
    (3)将步骤(2)获得的DNA片段连接在酶切后所述空载穿梭质粒上，即得。
  5. 根据权利要求3或4所述的病毒载体的构建方法，其特征在于，所述步骤(1)中的PCR扩增反应体系包括：
    5×
    Figure PCTCN2018081117-appb-100001
    Buffer(Mg2+plus)，10μl；
    dNTP Mixture，各dNTP 2.5mM，4μl；
    模板DNA，20-200ng；
    上游引物mfat-1-F，10μM，1μl；
    下游引物mfat-1-R，10μM，1μl；
    Figure PCTCN2018081117-appb-100002
    HS DNA Polymerase，2.5U/μl，0.5μl；
    补灭菌超纯水至50μL。
  6. 根据权利要求3或4所述的病毒载体的构建方法，其特征在于，所述步骤(1)中的PCR扩增反应程序为：98℃变性5min；98℃保持10s，60℃保持15s，72℃保持2min，共30个循环；最后72℃延伸10min。
  7. 一种重组病毒载体，其特征在于，所述重组病毒载体通过权利要求1或2所述的病毒载体构建而成。
  8. 一种病毒颗粒，其特征在于，为将权利要求1或2所述的病毒载体转化至细胞中后得到的病毒颗粒。
  9. 根据权利要求8所述的病毒颗粒，其特征在于，所述病毒颗粒的给药方式为静脉注射。
  10. 权利要求1或2所述的病毒载体或权利要求7所述的重组病毒载体或权利要求8所述的病毒颗粒在制备治疗糖尿病药物或治疗自身免疫相关疾病药物中的应用
  11. 根据权利要求10所述的应用，其特征在于，所述的糖尿病为自身免疫性1型糖尿病。
  12. 根据权利要求10所述的应用，其特征在于，所述自身免疫相关疾 病为因Th细胞分化失衡及其分泌细胞因子失衡所导致的自身免疫病。
  13. 根据权利要求12所述的应用，其特征在于，所述的自身免疫病为1型糖尿病、多发性硬化症、类风湿性关节炎和系统性红斑狼疮。
  14. 一种药物，其特征在于，包括权利要求1或2所述的病毒载体或权利要求7所述的重组病毒载体或权利要求8所述的病毒颗粒，以及其药物可接受的载体。
  15. 根据权利要求14所述的药物，其特征在于，包括将权利要求1或2所述的病毒载体或权利要求7所述的重组病毒载体或权利要求8所述的病毒颗粒按照常规工艺，添加常规辅料制备成临床上可接受的剂型。
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