靶向肝脏基因组位点特异性整合治疗血友病B的初步研究
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摘要
第一部分靶向肝脏基因组位点特异性整合治疗血友病B的初步研究
研究背景和目的：血友病B是一种由于凝血因子Ⅸ缺乏导致的严重出血性疾病,为X染色体连锁隐性遗传。主要临床表现是不可预测的、反复自发性软组织及大关节出血。临床上乙型血友病的严重程度与循环中FIX (coagulation factor IX)的水平相关。目前的替代疗法已经在很大程度上降低了出血发生的频率以及继发的关节疾病,并大大的提高了血友病病人的生存期。但是血浆浓缩FIX可能引起传染性疾病,而纯化FIX所需费用巨大。血友病B是很好的基因治疗研究模型。一般来说,hFIX (human FIX)基因可以通过病毒或非病毒载体进行输送。近年来,非病毒载体的研究越来越受到重视。噬菌体整合酶phiC31可以在哺乳动物细胞内发挥作用,被应用与基因工程包括基因治疗的研究。在哺乳动物细胞,这种酶介导带有attB位点的质粒整合入宿主基因组中的假attP位点。Aneja及其同事发现外来质粒与整合酶编码质粒共注射才能实现外源基因在肺脏持续表达,而单独注射外来质粒则不能。本研究的主要目的是通过高压尾静脉注射把携带attB和hFIX的质粒与表达phiC31的质粒共同导入肝脏细胞,检测目的质粒能否整合入小鼠基因组并持续表达。
方法：首先构建表达hFIX并携带attB核心序列的真核表达载体attB-hFIX-pIRES2-EGFP并在体外验证该载体能否表达目的基因。然后用高压尾静脉注射的方法将该载体与表达phiC31的质粒CMV-int共注射血友病B小鼠,attB-hFIX-pIRES2-EGFP单独注射为对照。用ELISA的方法检测hFIX在其体内的表达；用出血时间评价血友病小鼠出血症状是否改善；用巢式PCR检测attB-hFIX-pIRES2-EGFP是否整合到基因组的整合热点mpsLl(mouse pseudo-site from liver 1)位点。
结果：经鉴定,attB-hFIX-pIRES2-EGFP载体构建成功,并在体外表达hFIX。高压尾静脉注射血友病B小鼠后24小时,hFIX血清水平达到最高值,为正常水平的30%,此时小鼠的出血症状明显改善。但是不论是否与CMV-int共注射,此后hFIX水平迅速下降,在注射后10天内降到本底水平。巢式PCR的结果证实,attB-hFIX-pIRES2-EGFP整合到了小鼠肝脏基因组的mpsL1位点。结论：phiC31可以将34bp的attB最短序列整合到小鼠基因组的整合热点mpsL1;由CMV启动hFIX的能够瞬间高表达并有效改善血友病小鼠的出血症状；但是外来DNA进入细胞后,无论是否整合到基因组均被迅速沉默,说明肺脏和肝脏对整合入基因组的CMV启动子表达调控的机制不尽相同,因此对用于基因治疗的裸DNA进行改进使其适合在靶器官表达是十分必要的。
第二部分一种新的用于血友病B基因治疗的腺病毒-整合酶嵌合系统的构建及其体外表达鉴定
研究背景和目的：目前用于血友病基因治疗的载体主要分为两大类：病毒载体和非病毒载体。这两类载体各有优缺点。病毒载体基因传递的效率高,但容易产生免疫反应和插入突变；非病毒载体制备简单,安全性好,但是基因转导的效率低。因此我们试图构建一种即可高效感染靶细胞又能实现安全定点整合入宿主基因组的腺病毒嵌合载体。
方法：通过一系列DNA操作构建腺病毒-整合酶嵌合系统。该系统包含两个腺病毒载体：一个携带转基因表达框,表达框内有hFIX,红色荧光蛋白编码序列以及attB (phiC31识别位点),表达框两侧各有一个loxp (Cre识别位点)。另一个腺病毒载体携带Cre和phiC31基因。同时构建只表达Cre和只表达phiC31的载体作为对照载体。在体外分别用脂质体转染293A细胞,用荧光显微镜观察荧光蛋白的表达,用RT-PCR鉴定各个基因的表达。
结果：经PCR,酶切及测序方法鉴定,该腺病毒-整合酶嵌合系统构建成功。体外分别用脂质体转染293A细胞后,可见绿色荧光蛋白和红色荧光蛋白表达。经RT-PCR鉴定,该系统能够成功表达各种目的蛋白。
结论：经初步鉴定,我们的腺病毒-整合酶嵌合系统构建成功,为进一步研究其治疗作用打下良好基础。
第三部分Th1 (CXCL10)和Th2 (CCL2)趋化因子在原发性免疫性血小板减少症中的表达及其临床意义的初步研究
研究背景：原发性免疫性血小板减少症(primary immune thrombocytopenia primary,ITP)是一种以血小板破坏加速为主的器官特异性自身免疫性疾病,具体发病机制未完全清楚。Th1/Th2极化失衡在ITP的发病机制中起了非常重要的作用。Th1趋化因子CXCL10和Th2趋化因子CCL2在其他自身免疫性疾病的发病机制中发挥一定的作用,但在ITP中是否发挥作用仍然未知。
目的：明确CXCL10, CCL2以及它们的受体CXCR3, CCR4和CCR2在ITP中的表达情况,对这些因子在ITP发病机制中的作用进行初步研究。
方法：用ELISA方法检测49份ITP病人以及24份正常人的血浆标本中CXCL10和CCL2的浓度。用实时定量PCR的方法检测趋化因子及其受体在24例正常人和28例活动期ITP患者外周血单个核细胞以及9例ITP患者的脾细胞中的表达。用实时定量PCR的方法检测10例ITP患者和10例正常人CD4+细胞中CCR4和CXCR3的表达。
结果：活动期ITP患者血浆样品中CXCL10的水平与正常人相比显著升高(p=0.007),缓解ITP患者降至正常水平。CCL2的水平在活动组,缓解组和正常组没有统计学差异。与正常对照相比,活动期患者外周血单个核细胞的CXCL10 mRNA水平显著升高(p=0.031),但CCR2的mRNA水平显著降低(p=0.005)。ITP患者外周血小板计数与CXCL10的水平及CXCL10/CCL2比值呈负相关。结论：活动期ITP患者的血浆CXCL10水平和CXCL10/CCL2比值与正常人相比显著升高,CXCL10可能在ITP的发病机制中发挥一定的作用。
Preliminary study of Correction of Murine Hemophilia B by Hydrodynamic Gene Dlivery and Site-specific Genomic Integration
Background and Objective:Hemophilia B is a sex-linked hemorrhagic disease resulting from deficiency in coagulation factor IX. The main clinical manifestation of the disease is unpredictable, recurrent, spontaneous bleeding in soft tissues and/or major joints. Recurrent bleeding in large joints usually leads to crippling arthropathies in a majority of severely affected patients. The clinical severity of hemophilia B corresponds to the level of circulating FIX. Current replacement therapy has significantly reduced the frequency of bleeding episodes and subsequent joint disease, and markedly improved the life expectancy of patients with hemophilia. However, the high cost of purified factor products makes life long prophylactic infusion impractical, and plasma-derived FIX would enable the patients to be at high risk for infection from contaminant viral pathogens. Generally, the FIX gene can be delivered via either non-viral or viral mediation. Recently, researchers pay more and more attention to non-viral vector. The site-specific integrase from phiC31 bacteriophage functions in mammaliancells and is being applied for genetic engineering, including gene therapy. In mammalian cells, the enzyme mediates integration of plasmids bearing attB into pseudo attP sites in host genome. Aneja and coworkers found that foreign plasmid expression in lung could be persistent when it co-injected with the phiC31-encoding plasmid. The purpose of our current study is to determine whether the plasmid bearing attB and hFIX coding sequence could insert into mice genome and persistently express hFIX when co-injected with the integrase.
Methods:Firstly, we constructed the plasmid attB-hFIX-pIRES2-EGFP which bears attB site and hFIX coding sequence. It was proved that this plasmid could express hFIX through in vitro experiments. Then plasmid attB-hFIX-pIRES2-EGFP and CMV-int expressing integrase was co-injected by rapid infusion of a large-volume solution into the tail vein of hemophilia B mice. The mice injected attB-hFTX-pIRES2-EGFP alone served as controls. ELISA was performed to determine hFIX serum levels of hemophilia B mice. Correction of coagulation in vivo after plasmids injection was assessed by bleeding time assay. Genomic integration of foreign plasmid was determined by nested PCR.
Results:The plasmid attB-hFIX-pIRES2-EGFP was successfully constructed and could express hFIX in vitro. The hemophilia B mice produced 1533±239 ng/ml hFIX 1 day after injection of the hFIX encoding plasmid and human FIX significantly corrected the bleeding diathesis of hemophilia B mice as measured by in vivo clotting assays. But, no matter co-injected with CMV-Int or not, the hFIX levels decreased to background level in 10 days after injection. Nested-PCR results indicated that the integrase phiC31 resulted in the integration of the plasmid in mouse liver chromosomes.
Conclusion:Integrase phiC31 can catalyze recombination between 34bp attB and pseudo-attP. Human FIX driven by CMV promoter can be transiently and highly expressed afer hydrodynamic injection, but rapidly silenced no matter the insertion into genome or not. So, it is necessary to optimize the plasmid used in gene therapy according the target organ.
Construction and Identification of a Novel Adeno-Integrase Hybrid System for Hemophilia B
Background and Objective:There are two kinds of vectors for hemophilia gene therapy:viral vector and non-viral vector. Viral vectors usually display high transduction efficiency of the target cell in vitro and in vivo, but generate humoral immune response and insertional mutagenesis. In contrast, non-viral vector systems are less immunogenic and easy to prepare, but delivery efficiency of foreign DNA is low. So we try to construct an adenovirus hybrid system with high transduction efficiencies and site-specific integration.
Methods:By a serious of DNA manipulation, we constructed the hybrid system, including two adenovirus vectors. One vector contains loxp flanked transgene expression cassette, in which there are hFIX and DsRed coding sequences and attB for phiC31 recognization. The other vector carries Cre and phiC31 gene. We also constructed vectors only expressing Cre or phiC31 as controls.293A cells were transfected with the adenoviral vectors by Lipofectamine 2000 and the expression of target genes was identified by fluorescence microscope and RT-PCR.
Results:After being identified by PCR, restriction enzyme digestion and sequencing, the adeno-integrase hybrid system was successfully constructed. The system can express RFP, GFP, hFIX, Cre and phiC31 in 293A cells in vitro.
Conclusion:The adeno-integrase hybrid system was successfully constructed, which lay a good foundation for further investigation of its treatment effect.
Thl (CXCL10) and Th2 (CCL2) Chemokine Expression in Patients with Primary Immune Thrombocytopenia and their Clinical Implications
Background:Immune thrombocytopenia (ITP) is an organ-specific autoimmune disorder characterized by accelerated platelet destruction. The pathophysiology of ITP remains unclear. The imbalance of Thl/Th2 polarization might play important role in the pathogenesis of ITP. Th1 chemokine CXCL10 and Th2 chemokine CCL2 have been studied in several autoimmune diseases, but the status of these chemokines in ITP is still unknown.
Objective:To determine the expression of CXCL10 and CCL2, and their receptors, CXCR3, CCR4 and CCR2 in ITP patients, and make a preliminary study of the pathogenic roles of these factors in ITP.
Methods:Plasma samples from 49 patients with ITP, and 24 normal healthy subjects were assayed for CXCL10 and CCL2 plasma concentration by enzyme linked immunosorbent assay. Real-time quantitative PCR was performed to determine the mRNA expression of these chemokine and their receptors in the PBMNC of 24 normal controls and 28 active ITP patients as well as splenocytes of 9 ITP patients. The expression of CCR4 and CXCR3 in CD4+T cells from 10 ITP patients and 10 normal controls was compared by Real-time quantitative PCR.
Results:The CXCL10 levels in the plasma samples from patients with active ITP were significantly higher than those from healthy controls (p=0.007), and decreased to normal levels in remission ITP. In contrast, CCL2 levels were similar in active patients, remission and control subjects. PBMNC of active patients expressed more CXCLIO mRNA (p=0.031) but less CCR2 mRNA (p=0.005). Lower peripheral platelet count correlated with higher CXCL10 levels and CXCL10/CCL2 ratios.
Conclusion:Our study demonstrates that plasma levels of CXCL10 and CXC10/CCL2 ratio are higher in patients with active ITP than in healthy donors, and that CXCL10 might be a pathogenic factor of this disorder.
研究背景和目的：血友病B是一种由于凝血因子Ⅸ缺乏导致的严重出血性疾病,为X染色体连锁隐性遗传。主要临床表现是不可预测的、反复自发性软组织及大关节出血。临床上乙型血友病的严重程度与循环中FIX (coagulation factor IX)的水平相关。目前的替代疗法已经在很大程度上降低了出血发生的频率以及继发的关节疾病,并大大的提高了血友病病人的生存期。但是血浆浓缩FIX可能引起传染性疾病,而纯化FIX所需费用巨大。血友病B是很好的基因治疗研究模型。一般来说,hFIX (human FIX)基因可以通过病毒或非病毒载体进行输送。近年来,非病毒载体的研究越来越受到重视。噬菌体整合酶phiC31可以在哺乳动物细胞内发挥作用,被应用与基因工程包括基因治疗的研究。在哺乳动物细胞,这种酶介导带有attB位点的质粒整合入宿主基因组中的假attP位点。Aneja及其同事发现外来质粒与整合酶编码质粒共注射才能实现外源基因在肺脏持续表达,而单独注射外来质粒则不能。本研究的主要目的是通过高压尾静脉注射把携带attB和hFIX的质粒与表达phiC31的质粒共同导入肝脏细胞,检测目的质粒能否整合入小鼠基因组并持续表达。
方法：首先构建表达hFIX并携带attB核心序列的真核表达载体attB-hFIX-pIRES2-EGFP并在体外验证该载体能否表达目的基因。然后用高压尾静脉注射的方法将该载体与表达phiC31的质粒CMV-int共注射血友病B小鼠,attB-hFIX-pIRES2-EGFP单独注射为对照。用ELISA的方法检测hFIX在其体内的表达；用出血时间评价血友病小鼠出血症状是否改善；用巢式PCR检测attB-hFIX-pIRES2-EGFP是否整合到基因组的整合热点mpsLl(mouse pseudo-site from liver 1)位点。
结果：经鉴定,attB-hFIX-pIRES2-EGFP载体构建成功,并在体外表达hFIX。高压尾静脉注射血友病B小鼠后24小时,hFIX血清水平达到最高值,为正常水平的30%,此时小鼠的出血症状明显改善。但是不论是否与CMV-int共注射,此后hFIX水平迅速下降,在注射后10天内降到本底水平。巢式PCR的结果证实,attB-hFIX-pIRES2-EGFP整合到了小鼠肝脏基因组的mpsL1位点。结论：phiC31可以将34bp的attB最短序列整合到小鼠基因组的整合热点mpsL1;由CMV启动hFIX的能够瞬间高表达并有效改善血友病小鼠的出血症状；但是外来DNA进入细胞后,无论是否整合到基因组均被迅速沉默,说明肺脏和肝脏对整合入基因组的CMV启动子表达调控的机制不尽相同,因此对用于基因治疗的裸DNA进行改进使其适合在靶器官表达是十分必要的。
第二部分一种新的用于血友病B基因治疗的腺病毒-整合酶嵌合系统的构建及其体外表达鉴定
研究背景和目的：目前用于血友病基因治疗的载体主要分为两大类：病毒载体和非病毒载体。这两类载体各有优缺点。病毒载体基因传递的效率高,但容易产生免疫反应和插入突变；非病毒载体制备简单,安全性好,但是基因转导的效率低。因此我们试图构建一种即可高效感染靶细胞又能实现安全定点整合入宿主基因组的腺病毒嵌合载体。
方法：通过一系列DNA操作构建腺病毒-整合酶嵌合系统。该系统包含两个腺病毒载体：一个携带转基因表达框,表达框内有hFIX,红色荧光蛋白编码序列以及attB (phiC31识别位点),表达框两侧各有一个loxp (Cre识别位点)。另一个腺病毒载体携带Cre和phiC31基因。同时构建只表达Cre和只表达phiC31的载体作为对照载体。在体外分别用脂质体转染293A细胞,用荧光显微镜观察荧光蛋白的表达,用RT-PCR鉴定各个基因的表达。
结果：经PCR,酶切及测序方法鉴定,该腺病毒-整合酶嵌合系统构建成功。体外分别用脂质体转染293A细胞后,可见绿色荧光蛋白和红色荧光蛋白表达。经RT-PCR鉴定,该系统能够成功表达各种目的蛋白。
结论：经初步鉴定,我们的腺病毒-整合酶嵌合系统构建成功,为进一步研究其治疗作用打下良好基础。
第三部分Th1 (CXCL10)和Th2 (CCL2)趋化因子在原发性免疫性血小板减少症中的表达及其临床意义的初步研究
研究背景：原发性免疫性血小板减少症(primary immune thrombocytopenia primary,ITP)是一种以血小板破坏加速为主的器官特异性自身免疫性疾病,具体发病机制未完全清楚。Th1/Th2极化失衡在ITP的发病机制中起了非常重要的作用。Th1趋化因子CXCL10和Th2趋化因子CCL2在其他自身免疫性疾病的发病机制中发挥一定的作用,但在ITP中是否发挥作用仍然未知。
目的：明确CXCL10, CCL2以及它们的受体CXCR3, CCR4和CCR2在ITP中的表达情况,对这些因子在ITP发病机制中的作用进行初步研究。
方法：用ELISA方法检测49份ITP病人以及24份正常人的血浆标本中CXCL10和CCL2的浓度。用实时定量PCR的方法检测趋化因子及其受体在24例正常人和28例活动期ITP患者外周血单个核细胞以及9例ITP患者的脾细胞中的表达。用实时定量PCR的方法检测10例ITP患者和10例正常人CD4+细胞中CCR4和CXCR3的表达。
结果：活动期ITP患者血浆样品中CXCL10的水平与正常人相比显著升高(p=0.007),缓解ITP患者降至正常水平。CCL2的水平在活动组,缓解组和正常组没有统计学差异。与正常对照相比,活动期患者外周血单个核细胞的CXCL10 mRNA水平显著升高(p=0.031),但CCR2的mRNA水平显著降低(p=0.005)。ITP患者外周血小板计数与CXCL10的水平及CXCL10/CCL2比值呈负相关。结论：活动期ITP患者的血浆CXCL10水平和CXCL10/CCL2比值与正常人相比显著升高,CXCL10可能在ITP的发病机制中发挥一定的作用。
Preliminary study of Correction of Murine Hemophilia B by Hydrodynamic Gene Dlivery and Site-specific Genomic Integration
Background and Objective:Hemophilia B is a sex-linked hemorrhagic disease resulting from deficiency in coagulation factor IX. The main clinical manifestation of the disease is unpredictable, recurrent, spontaneous bleeding in soft tissues and/or major joints. Recurrent bleeding in large joints usually leads to crippling arthropathies in a majority of severely affected patients. The clinical severity of hemophilia B corresponds to the level of circulating FIX. Current replacement therapy has significantly reduced the frequency of bleeding episodes and subsequent joint disease, and markedly improved the life expectancy of patients with hemophilia. However, the high cost of purified factor products makes life long prophylactic infusion impractical, and plasma-derived FIX would enable the patients to be at high risk for infection from contaminant viral pathogens. Generally, the FIX gene can be delivered via either non-viral or viral mediation. Recently, researchers pay more and more attention to non-viral vector. The site-specific integrase from phiC31 bacteriophage functions in mammaliancells and is being applied for genetic engineering, including gene therapy. In mammalian cells, the enzyme mediates integration of plasmids bearing attB into pseudo attP sites in host genome. Aneja and coworkers found that foreign plasmid expression in lung could be persistent when it co-injected with the phiC31-encoding plasmid. The purpose of our current study is to determine whether the plasmid bearing attB and hFIX coding sequence could insert into mice genome and persistently express hFIX when co-injected with the integrase.
Methods:Firstly, we constructed the plasmid attB-hFIX-pIRES2-EGFP which bears attB site and hFIX coding sequence. It was proved that this plasmid could express hFIX through in vitro experiments. Then plasmid attB-hFIX-pIRES2-EGFP and CMV-int expressing integrase was co-injected by rapid infusion of a large-volume solution into the tail vein of hemophilia B mice. The mice injected attB-hFTX-pIRES2-EGFP alone served as controls. ELISA was performed to determine hFIX serum levels of hemophilia B mice. Correction of coagulation in vivo after plasmids injection was assessed by bleeding time assay. Genomic integration of foreign plasmid was determined by nested PCR.
Results:The plasmid attB-hFIX-pIRES2-EGFP was successfully constructed and could express hFIX in vitro. The hemophilia B mice produced 1533±239 ng/ml hFIX 1 day after injection of the hFIX encoding plasmid and human FIX significantly corrected the bleeding diathesis of hemophilia B mice as measured by in vivo clotting assays. But, no matter co-injected with CMV-Int or not, the hFIX levels decreased to background level in 10 days after injection. Nested-PCR results indicated that the integrase phiC31 resulted in the integration of the plasmid in mouse liver chromosomes.
Conclusion:Integrase phiC31 can catalyze recombination between 34bp attB and pseudo-attP. Human FIX driven by CMV promoter can be transiently and highly expressed afer hydrodynamic injection, but rapidly silenced no matter the insertion into genome or not. So, it is necessary to optimize the plasmid used in gene therapy according the target organ.
Construction and Identification of a Novel Adeno-Integrase Hybrid System for Hemophilia B
Background and Objective:There are two kinds of vectors for hemophilia gene therapy:viral vector and non-viral vector. Viral vectors usually display high transduction efficiency of the target cell in vitro and in vivo, but generate humoral immune response and insertional mutagenesis. In contrast, non-viral vector systems are less immunogenic and easy to prepare, but delivery efficiency of foreign DNA is low. So we try to construct an adenovirus hybrid system with high transduction efficiencies and site-specific integration.
Methods:By a serious of DNA manipulation, we constructed the hybrid system, including two adenovirus vectors. One vector contains loxp flanked transgene expression cassette, in which there are hFIX and DsRed coding sequences and attB for phiC31 recognization. The other vector carries Cre and phiC31 gene. We also constructed vectors only expressing Cre or phiC31 as controls.293A cells were transfected with the adenoviral vectors by Lipofectamine 2000 and the expression of target genes was identified by fluorescence microscope and RT-PCR.
Results:After being identified by PCR, restriction enzyme digestion and sequencing, the adeno-integrase hybrid system was successfully constructed. The system can express RFP, GFP, hFIX, Cre and phiC31 in 293A cells in vitro.
Conclusion:The adeno-integrase hybrid system was successfully constructed, which lay a good foundation for further investigation of its treatment effect.
Thl (CXCL10) and Th2 (CCL2) Chemokine Expression in Patients with Primary Immune Thrombocytopenia and their Clinical Implications
Background:Immune thrombocytopenia (ITP) is an organ-specific autoimmune disorder characterized by accelerated platelet destruction. The pathophysiology of ITP remains unclear. The imbalance of Thl/Th2 polarization might play important role in the pathogenesis of ITP. Th1 chemokine CXCL10 and Th2 chemokine CCL2 have been studied in several autoimmune diseases, but the status of these chemokines in ITP is still unknown.
Objective:To determine the expression of CXCL10 and CCL2, and their receptors, CXCR3, CCR4 and CCR2 in ITP patients, and make a preliminary study of the pathogenic roles of these factors in ITP.
Methods:Plasma samples from 49 patients with ITP, and 24 normal healthy subjects were assayed for CXCL10 and CCL2 plasma concentration by enzyme linked immunosorbent assay. Real-time quantitative PCR was performed to determine the mRNA expression of these chemokine and their receptors in the PBMNC of 24 normal controls and 28 active ITP patients as well as splenocytes of 9 ITP patients. The expression of CCR4 and CXCR3 in CD4+T cells from 10 ITP patients and 10 normal controls was compared by Real-time quantitative PCR.
Results:The CXCL10 levels in the plasma samples from patients with active ITP were significantly higher than those from healthy controls (p=0.007), and decreased to normal levels in remission ITP. In contrast, CCL2 levels were similar in active patients, remission and control subjects. PBMNC of active patients expressed more CXCLIO mRNA (p=0.031) but less CCR2 mRNA (p=0.005). Lower peripheral platelet count correlated with higher CXCL10 levels and CXCL10/CCL2 ratios.
Conclusion:Our study demonstrates that plasma levels of CXCL10 and CXC10/CCL2 ratio are higher in patients with active ITP than in healthy donors, and that CXCL10 might be a pathogenic factor of this disorder.
引文
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