重组AAV2/hFⅨ病毒制备及其基因治疗血友病B的实验研究

目的　制备携带人凝血因子Ⅸ (hFⅨ )基因的重组AAV2病毒 (rAAV2 /hFⅨ ) ,并对用rAAV2 /hFⅨ肌肉注射治疗血友病B模型小鼠的疗效进行评价。方法　通过“一株载体细胞 /一株辅助病毒”的双因素包装策略制备出rAAV2 /hFⅨ ,体外转导BHK 2 1、C2C12细胞后 ,检测细胞培养上清中hFⅨ的表达量 ;肌肉直接注射血友病B模型小鼠后 ,检测其血浆中hFⅨ的抗原水平和凝血活性等指标。结果　转导 2 4h后在细胞上清中即可检测到hFⅨ ,连续检测 12 0h都有表达 ,BHK 2 1、C2C12细胞 2 4h最高表达量分别达到 (5 1.0± 6 .5 )ng/ 10 5细胞和 (6 8.0± 7.2 )ng/ 10 5细胞。rAAV2 /hFⅨ经肌肉直接注射后 ,高、中、低三个剂量组均能检测到小鼠体内高效表达hFⅨ ,在给药后第 3周达到高峰 ,小鼠血浆中hFⅨ的表达量与对照组比较差异有显著性 (P <0 .0 1) ,之后缓慢下降 ,到第 10周仍可检测到低水平hFⅨ表达 ;取第 3周小鼠血浆样品检测凝血功能 ,高、中、低剂量组FⅨ活性均得到明显改善 ,小鼠的割尾实验出血时间明显缩短 ,5min失血量也相应显著减少 ,其中高剂量组hFⅨ最高表达量达到 (387.0± 12 .5 )ng/ml血浆 ,FⅨ活性达到正常水平的 (30 .0± 5 .5 ) % ;给药后第 10周 ,除在注射点外 ,其它主要脏器均未检测到AAV载体DNA。结论     

高压尾静脉注射和位点特异性整合介导的人凝血因子Ⅸ基因治疗血友病B小鼠

目的 通过高压尾静脉注射将携带attB和人凝血因子Ⅸ(hFⅨ)的质粒与表达phiC31的质粒共同导入血友病B小鼠肝脏细胞,检测目的 质粒能否整合入小鼠基因组并持续表达.方法 ①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.②用高压尾静脉注射法将该载体与表达phiC31的质粒CMV-int共注入血友病B小鼠,attB-hFⅨ-pIRES2-EGFP单独注射为对照.③ELISA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mpsL1(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.高压尾静脉注入血友病B小鼠24 h后,hFⅨ血清水平达到最高值为(1533±239)ng/ml,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 d内降到本底水平.巢式PCR的结果证实,attB-hFⅨ-pIRES2-EGFP整合到小鼠肝脏基因组的mpsL1位点.结论 phiC31可以将34 bp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.     

含hFⅨ微小基因的杂合型逆转录病毒载体在小鼠成肌细胞中高效表达的研究

目的探讨不同载体结构在成肌细胞中表达hFⅨ蛋白的效果,优化载体结构,为血友病B基因治疗提供实验依据。方法制备带有两拷贝肌酸激酶增强子(Me2)和内含子m     

人源载体pHrnF9介导的凝血因子Ⅸ基因在肠上皮sw480细胞中的表达

本研究探索肠上皮细胞和人源载体pHrnFⅨ用于血友病B基因治疗的可能性。用含人凝血因子Ⅸ（human coagulation factorⅨ，hFⅨ）基因的人源载体质粒pHrnF9转染肠上皮细胞sw480，用RT-PCR检测mRNA的转录，荧光显微镜观察转染效率，ELISA和一期法检测蛋白表达及凝血活性。结果表明：转染后的细胞中有hFⅨmRNA的转录；荧光显微镜观察到48小时转染效率最高；ELISA法测得转染后24小时细胞上清中的hFⅨ蛋白量为（11．34±0．23）ng／（10^6cells·24h），第48小时hFⅨ蛋白量最高，达（29．34±1．00）ng／（10^6cells·24h），转染后72小时降为（12．45±0．15）ng）／（10^6cells·24h）。一期法结果显示转染pHrnF9的sw480细胞分泌的FⅨ有凝血活性，48小时达峰值（6．07±0、17）％／10^6cells，第72小时降至（1．81±0．06）％／10^6cells。结论：肠上皮细胞sw480转染pHrnF9质粒后可表达有凝血活性的hFⅨ，肠上皮细胞有望成为血友病B基因治疗的靶细胞。     

反转录病毒载体G1NaCⅨ的构建及其在离体细胞中的表达

目的构建高效、安全、可用于基因治疗的反转录病毒载体G1NaCⅨ。方法用限制性内切酶Bg1Ⅱ和HindⅢ将CMV－FⅨcDNA从质粒pCMVⅨ－10上切下，将该片段与BamHⅠ和HindⅢ双酶切后的G1Na连接，连接后的载体经鉴定连接正确后即为新载体G1NacⅨ，将G1NaCⅨ通过包装细胞PA317导入小鼠成纤维细胞NIH3T3、小鼠成肌细胞C2C12和人纤维肉瘤细胞HT1080中，选择培养两周后分别测定G1NaCⅨ在上述细胞中的Ⅸ因子表达量。结果（1）经酶切电泳鉴定，G1NaCⅨ连接正确；（2）G1NaCⅨ在N1H3T3中的Ⅸ因子表达量为60ng/106细胞·天-1，在C2C12中的Ⅸ因子表达量为1580ng/106细胞·天-1，在HT1080中的Ⅸ因子表达量为3600ng／106细胞·天-1，其中80％～90％的Ⅸ因子具有凝血活性。结论构建成功的反转录病毒载体G1NaCⅨ在C2C12和HT1080细胞中均能高效表达。     

逆转录病毒载体转染的人凝血因子Ⅸ基因在人脐带组织源间充质干细胞中的表达

为了研究逆转录病毒（pLEGFP—N1）转染的人凝血因子Ⅸ（hFⅨ）基因在人脐带间充质干细胞中的表达,应用DNA重组技术将hFⅨ cDNA构建入pLEGFP—N1载体,转导入包装细胞系Pheonix细胞,应用病毒上清感染人脐带组织源间充质干细胞（hUCT—MSCs）,经G418筛选10天后获得全部的转染阳性细胞,从蛋白质水平和其功能活性上检测hFⅨ的表达。结果显示：配养上清液中可检测到hFⅨ的表达,每24小时分泌量达2．68±0．36μg／10^6细胞。Westernblot检测表明,转导hFⅨ的hUCT—MSCs能分泌预期分子大小的hFⅨ入上清。功能性凝集测定实验表明了转导F／X的hUCT—MSCs 2天培养上清中hFⅨ的活性为100％-130％。结论：pLBGVe—N1-hFⅨ能有效地转导hUCT—MSCs,并在其子代细胞中表达具有凝血活性的hFⅨ,这为hUCT—MSCs成为血友病B基因治疗的细胞载体研究奠定了基础。     

人源载体pHrnF9介导的凝血因子Ⅸ基因在肠上皮sw480细胞中的表达

本研究探索肠上皮细胞和人源载体pHrnFⅨ用于血友病B基因治疗的可能性。用含人凝血因子Ⅸ（human coagulation factorⅨ，hFⅨ）基因的人源载体质粒pHrnF9转染肠上皮细胞sw480，用RT-PCR检测mRNA的转录，荧光显微镜观察转染效率，ELISA和一期法检测蛋白表达及凝血活性。结果表明：转染后的细胞中有hFⅨmRNA的转录；荧光显微镜观察到48小时转染效率最高；ELISA法测得转染后24小时细胞上清中的hFⅨ蛋白量为（11．34&#177;0．23）ng／（10^6cells&#183;24h），第48小时hFⅨ蛋白量最高，达（29．34&#177;1．00）ng／（10^6cells&#183;24h），转染后72小时降为（12．45&#177;0．15）ng）／（10^6cells&#183;24h）。一期法结果显示转染pHrnF9的sw480细胞分泌的FⅨ有凝血活性，48小时达峰值（6．07&#177;0、17）％／10^6cells，第72小时降至（1．81&#177;0．06）％／10^6cells。结论：肠上皮细胞sw480转染pHrnF9质粒后可表达有凝血活性的hFⅨ，肠上皮细胞有望成为血友病B基因治疗的靶细胞。     

血友病A基因治疗的初步实验研究

目的：制备血友病A的治疗基因，从in vivo途径观察人凝血因子Ⅷ（hFⅧ）在动物体内的表达。方法：将B区缺失（760－1639位氨基酸）的hFⅧcDNA克隆至真核细胞表达载体pRC/RSV，与传染试剂DOTAP－Cholesterol liposome混合，制备治疗基因pRC/RSV-hFⅧBD-DOTAP-Cholesterol。肌肉注射给BALB／c小鼠后，在第2，10，20，30，40，50天取小鼠的血液以及心、肝、脾、肺、肾、骨髂肌等组织。检测血浆中的hFⅧ抗原和抗体，用PCR和RT－PCR方法检测hFⅧBDcDNA在小鼠各组织基因组中的分布及其转录情况，免疫组织化学染色检测各组织中hFⅧ的表达。结果：在注射结束后的第48小时，小鼠血浆和组织中即有hFⅧ的表达，第10天血浆中的hFⅧ抗原水平最高。达17＝55ng/ml，以后逐渐下降，血浆中的hFⅧ抗体在注射结束后的第10天出现，以后缓慢升高，第50天时达37．06U/ml。PCR、RT－PCR检测和免疫组织化学染色显示小鼠各组中均有hFⅧBDcDNA的存在、转录和表达，但随着时间推移逐渐减少。脾、肺、肾脏中hFⅧBDcDNA的转录和表达时间均长于心脏、肝脏和骨骼肌。结论：由pRC/RSV-hFⅧBD与DOTAP－Cholesterol liposome结合而制备的治疗基因pRC/RSV-hFⅧBD-DOTAP-Cholesterol能够通过in vivo途径在动物体内很好地表达hFⅧ，这为临床基因治疗血友病A提供了实验依据。     

基因治疗骨质疏松、骨转移癌的可行性研究

目的：研究人骨保护素（OPG）转染小鼠胚胎成肌细胞C2C12后细胞形态及细胞周期的变化,方法：采用Wesern blot方法证实OPG在真核细胞中表达,对稳定转染OPG基因的C2C12细胞的形态,细胞周期等进行检测。结果：成功克隆了人OPG编码区基因并构建了直核表达载体pcDNA3-OPG。结论：人OPG编码区基因稳定转染的小鼠胚胎成肌细胞生长状态良好,DNA合成旺盛,无诱导细胞凋亡现象,为采用OPG基因治疗骨质疏松,骨转移癌等的可行性提供了依据。     

基因治疗骨质疏松、骨转移癌的可行性研究

目的研究人骨保护素(OPG)转染小鼠胚胎成肌细胞C2C12后细胞形态及细胞周期的变化。方法采用Westernblot方法证实OPG在真核细胞中表达。对稳定转染OPG基因的C2C12细胞的形态、细胞周期等进行检测。结果成功克隆了人OPG编码区基因并构建了真核表达载体pcDNA3－OPG。结论人OPG编码区基因稳定转染的小鼠胚胎成肌细胞生长状态良好,DNA合成旺盛,无诱导细胞凋亡现象,为采用OPG基因治疗骨质疏松、骨转移癌等的可行性提供了依据。     

Major role of local immune responses in antibody formation to factor IX in AAV gene transfer

The risk of an immune response to the coagulation factor IX (F.IX) transgene product is a concern in gene therapy for the X-linked bleeding disorder hemophilia B. In order to investigate the mechanism of F.IX-specific lymphocyte activation in the context of adeno-associated viral (AAV) gene transfer to skeletal muscle, we injected AAV-2 vector expressing human F.IX (hF.IX) into outbred immune-competent mice. Systemic hF.IX levels were transiently detected in the circulation, but diminished concomitant with activation of CD4+ T and B cells. ELISPOT assays documented robust responses to hF.IX in the draining lymph nodes of injected muscle by day 14. Formation of inhibitory antibodies to hF.IX was observed over a wide range of vector doses, with increased doses causing stronger immune responses. A prolonged inflammatory reaction in muscle started at 1.5-2 months, but ultimately failed to eliminate transgene expression. By 1.5 months, hF.IX antigen re-emerged in circulation in approximately 70% of animals injected with high vector dose. Hepatic gene transfer elicited only infrequent and weaker immune responses, with higher vector doses causing a reduction in T-cell responses to hF.IX. In summary, the data document substantial influence of target tissue, local antigen presentation, and antigen levels on lymphocyte responses to F.IX.     

Determination of specific CD4 and CD8 T cell epitopes after AAV2- and AAV8-hF.IX gene therapy.

The application of AAV2 or AAV8 vectors for delivery of human coagulation factor IX (hF.IX) is a promising gene therapy for hemophilia B. One major limitation of this therapy is the development of antibodies and a cytotoxic T lymphocyte (CTL) response against both the vector capsid and the transgene. We determined the class I and class II MHC peptide epitopes for AAV2, AAV8, and hF.IX after administration of AAV-2-hF.IX or AAV8-hF.IX in H2(b) (C57BL/6), H2(d) (BALB/c), or H2(k) (C3H) strains of mice. The results indicate that the AAV2 capsid peptide AA(373-381), the AAV8 capsid peptide AA(50-58), and the hF.IX transgene peptide AA(311-319) can elicit a CTL response as indicated by an IFN-gamma ELISPOT assay and an in vivo CTL assay. Furthermore, a strong H2(k) MHC II-restricted Th1 response can be elicited in C3H mice by the AAV8 capsid peptide AA(126-140) and the hF.IX peptide AA(108-122), whereas a strong Th2 response can be elicited by the AAV2 peptide AA(475-489). These results show that specific CTL responses are generated to both AAV capsid epitopes and hF.IX epitopes after injection of AAV-hF.IX, and MHC class II epitopes derived from AAV-hF.IX promote development of either Th1 or Th2 cells.     

Persistent expression of hF.IX After tolerance induction by in utero or neonatal administration of AAV-1-F.IX in hemophilia B mice.

The major complication associated with protein replacement therapy currently used in the treatment of hemophilia B (HB) is the development of antibodies to the infused human Factor IX (hF.IX). We hypothesized that vector-mediated expression of hF.IX, either at a prenatal stage or early in life may lead to tolerance to hF.IX and long-term transgene expression. Fetal, neonatal, and adult F.IX-deficient mice were injected with AAV-1-hF.IX, and the hF.IX levels as well as antibodies to hF.IX in the circulation were assayed. In utero injection followed by postnatal re-administration of adeno-associated virus 1 (AAV-1) vector achieved persistent expression of hF.IX in all animals, with no cellular or humoral immune response to F.IX. Similar results were seen after initial injection in neonatal mice followed by re-administration, whereas all mice injected at the adult stage developed antibodies to hF.IX. In contrast, after administration of AAV-2-hF.IX in the neonatal period, antibodies to hF.IX were formed in all the injected animals. We conclude that in utero or neonatal-stage injection of AAV-1-hF.IX can lead to long-term expression and absence of immune response. The differences in immune response between the AAV-1 and AAV-2 groups suggests that tolerance may be related to differences in bio-distribution, timing of expression, and/or the initial levels of hF.IX expression. This supports the concept of a narrow "window of opportunity" for tolerance induction.     

腺相关病毒包装条件优化及重组AAV8/hFⅧ基因治疗血友病A小鼠的实验研究

目的评价携带人凝血因子FⅧ(hFⅧ)的重组腺相关病毒(AAV)血清型8(AAV8/hFⅧ)病毒治疗血友病A(HA)小鼠效果。方法应用pAAV-CB-EGFP,pH22(AAV2血清型)和pfΔ6(腺病毒辅助质粒)摸索在HEK-293细胞中AAV的包装条件,pH22、pfΔ6及pAAV-CB-EGFP质粒按照1∶1∶1的比例进行转染,免疫荧光显微镜观察四个条件(包括10 cm培养皿转10μg质粒,20 cm培养皿分别转20、30和40μg质粒)的病毒包装效果。应用最佳包装条件在HEK-293细胞中包装AAV2-EGFP,通过冻融法获得AAV2粗提液,感染HEK-293细胞和16095细胞,应用荧光显微镜观察包装效果。应用最佳转染条件将pAAV-TTR-hFⅧ、pH28、pfΔ6按1∶1∶1的比例转染HEK-293细胞获得AAV8/hFⅧ,两轮的氯化铯梯度离心法纯化病毒,以8×10^12 vg/kg剂量经尾静脉注射HA小鼠进行基因治疗,活化部分凝血活酶时间(APTT)测定分析FⅧ的活性。结果20 cm培养皿转染20μg质粒的条件能够在24、48和72 h都达到最佳的转染效果,且该条件包装的AAV2粗提液在16095细胞中具有较高的感染率。HA小鼠体内AAV8/hFⅧ能够在注射后12周仍有维持在治疗水平的FⅧ活性。结论利用优化包装条件制备的AAV8/hFⅧ能够在HA小鼠体内有效维持治疗水平的FⅧ活性达12周。     

Impact of the Underlying Mutation and the Route of Vector Administration on Immune Responses to Factor IX in Gene Therapy for Hemophilia B

Immune responses to factor IX (F.IX), a major concern in gene therapy for hemophilia, were analyzed for adeno-associated viral (AAV-2) gene transfer to skeletal muscle and liver as a function of the F9 underlying mutation. Vectors identical to those recently used in clinical trials were administered to four lines of hemophilia B mice on a defined genetic background [C3H/HeJ with deletion of endogenous F9 and transgenic for a range of nonfunctional human F.IX (hF.IX) variants]. The strength of the immune response to AAV-encoded F.IX inversely correlated with the degree of conservation of endogenous coding information and levels of endogenous antigen. Null mutation animals developed T- and B-cell responses in both protocols. However, inhibitor titers were considerably higher upon muscle gene transfer (or protein therapy). Transduced muscles of Null mice had strong infiltrates with CD8⁺ cells, which were much more limited in the liver and not seen for the other mutations. Sustained expression was achieved with liver transduction in mice with crm⁻ nonsense and missense mutations, although they still formed antibodies upon muscle gene transfer. Therefore, endogenous expression prevented T-cell responses more effectively than antibody formation, and immune responses varied substantially depending on the protocol and the underlying mutation.     

Induction of immune tolerance to coagulation factor IX antigen by in vivo hepatic gene transfer

Gene replacement therapy is an attractive approach for treatment of genetic disease, but may be complicated by the risk of a neutralizing immune response to the therapeutic gene product. There are examples of humoral and cellular immune responses against the transgene product as well as absence of such responses, depending on vector design and the underlying mutation in the dysfunctional gene. It has been unclear, however, whether transgene expression can induce tolerance to the therapeutic antigen. Here, we demonstrate induction of immune tolerance to a secreted human coagulation factor IX (hF.IX) antigen by adeno-associated viral gene transfer to the liver. Tolerized mice showed absence of anti-hF.IX and substantially reduced in vitro T cell responses after immunization with hF.IX in adjuvant. Tolerance induction was antigen specific, affected a broad range of Th cell subsets, and was favored by higher levels of transgene expression as determined by promoter strength, vector dose, and mouse strain. Hepatocyte-derived hF.IX expression induced regulatory CD4(+) T cells that can suppress anti-hF.IX formation after adoptive transfer. With a strain-dependent rate of success, tolerance to murine F.IX was induced in mice with a large F.IX gene deletion, supporting the relevance of these data for treatment of hemophilia B and other genetic diseases.     

Intravenous administration of an E1/E3-deleted adenoviral vector induces tolerance to factor IX in C57BL/6 mice

Inbred immunocompetent C57BL/6 mice have been a favored strain to study transgene expression of human blood coagulation factor IX (hF.IX) from viral vectors because systemic expression of the secreted protein is not limited by antibody responses following intravenous (i.v.) injection of vector. For example, i.v. injection of an adenoviral (Ad) vector results in sustained expression of hF.IX in normal or hemophilic C57BL/6 mice, while anti-hF.IX antibodies rapidly emerge in other strains (Gene Therapy 4: 473; Blood 91: 784). To investigate these observations further, we injected naive C57BL/6 mice and C57BL/6 mice with pre-existing anti-hF.IX with Ad-hF.IX vector via peripheral vein. All mice expressed hF.IX antigen without detectable anti-hF.IX, even when challenged with hF.IX in different immunogenic settings at later time points. Moreover, in mice with pre-existing immunity, anti-hF.IX titers diminished to undetectable levels after i.v. administration of Ad-hF.IX. Lymphocytes from mice that had received Ad-hF.IX i.v. failed to proliferate when stimulated with hF.IX in vitro after the animals had been repeatedly challenged with hF.IX protein formulated in complete Freund's adjuvant. Thus, absence of anti-hF.IX in C57BL/6 mice after i.v. injection of Ad vector is not due to ignorance to the foreign transgene product. Similar experiments in other strains showed that immune tolerance to hF.IX does not correlate with the strain haplotype or expression of IL-10 cytokine. Given the well-documented immunogenicity of the first-generation adenoviral vector, data from C57BL/6 mice may therefore grossly underestimate immunological consequences in certain gene therapy protocols.     

Persistent delivery of factor IX in mice: gene therapy for hemophilia using implantable microcapsules.

Severe hemophilia B is a life-threatening, life long condition caused by absence of or defective coagulation factor IX. Gene therapy could provide an alternative treatment to repeated injection of plasma-derived concentrate or recombinant factor IX. We have previously described the use of implantable microcapsules containing recombinant myoblasts to deliver human factor IX in mice. This study reports the generation of improved myoblast-specific expression vectors. Mouse myoblast clones transfected with the various vectors secreted factor IX in vitro, at rates between 70 and 1000 ng/10(6) cells/day. The recombinant myoblast clones were then encapsulated and implanted into mice. Immunocompetent mice implanted with encapsulated myoblasts had up to 65 ng of factor IX per milliliter in their plasma for up to 14 days, after which antibodies to human factor IX became detectable, and this coincided with decreased factor IX in mouse plasma. In immunodeficient mice, however, factor IX delivery was maintained at a constant level for at least 6 weeks (end of experiment). Interestingly, the highest-secreting myoblast clone in vitro did not deliver the highest level of hFIX in vivo. This discrepancy observed between performance in vitro and in vivo may have important implications for the development of gene therapy protocols based on recombinant cells.