CN116656740A - 一种腺相关病毒基因治疗载体及其在制备治疗苯丙酮尿症的药物中的应用 - Google Patents
一种腺相关病毒基因治疗载体及其在制备治疗苯丙酮尿症的药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种腺相关病毒基因治疗载体及其在制备治疗苯丙酮尿症(PKU)的药物中的应用,所述腺相关病毒基因治疗载体包含增强子、启动子、内含子、和人PAH编码基因,所述启动子为人α1抗胰蛋白酶(alpha‑1‑antitrypsin,α1AT)启动子。本发明提供的基因治疗载体仅在肝脏中特异性表达PAH,显著提高PKU小鼠肝脏中PAH的表达水平,显著降低PKU小鼠中PHE的异常积累,并长期保持在正常小鼠水平,PKU治疗小鼠毛色也得到显著改善。结果提示本研究开发的基因治疗载体通过提高肝脏中PAH的水平,恢复其血液中PHE,从而达到PKU疾病的有效治疗。
Description
技术领域
本发明涉及生物医药技术领域,更具体地,涉及一种腺相关病毒基因治疗载体及其在制备治疗苯丙酮尿症的药物中的应用。
背景技术
苯丙酮尿症(Phenylketonuria,PKU)是临床常见重大隐性遗传氨基酸代谢病。未经治疗的PKU患儿,临床症状以智力障碍、精神和神经症状如癫痫、运动障碍、湿疹样皮肤疾病、色素脱失和鼠味尿等为特征。随着儿童成长,发育问题、异常行为和精神症状通常会变得更加明显,如果不及时治疗会导致严重且不可逆转的精神残疾,PKU为其患者及其家属带来了严重的痛苦。目前,PKU的临床治疗手段主要以低苯丙氨酸饮食疗法为主,但是长期严格的低苯丙氨酸饮食,易导致患儿营养不良,且随年龄增长,患者依从性变差,遗留不同程度的智力残疾。因此,现有饮食疗法不仅严重影响患儿生存质量,治疗效果也不理想,亟需研发新的有效方法从根本上治疗PKU。
基因治疗是利用病毒或非病毒载体把外源性遗传物质导入到特定细胞,补充缺陷的基因,从而预防和治疗疾病。基因治疗可以通过体内和体外两种策略实现,无需重复干预即可达到相对持久和稳定的治疗效果。体内基因疗法通过病毒或非病毒载体将药物或遗传物质直接递送到患者的靶器官或组织,进而纠正或补充有缺陷的基因实现疾病治疗或预防。
腺相关病毒(adeno-associated virus,AAV)因为具有相对低的致病性、高效的靶细胞感染率、独立于宿主基因组的复制性和低免疫原性等特点成为最有前景的体内基因治疗载体。本发明的发明人利用多年研究经验,开发出特定重组腺相关病毒基因治疗载体,应用静脉注射将载体递送体内,通过在肝脏中特异性表达苯丙氨酸羟化酶(PhenylalanineHydroxylase,PAH),解决由于基因突变导致的PAH功能缺陷,恢复PAH催化丙氨酸转化为酪氨酸的功能,从而让PAH活性减弱或丧失的PKU患者得到个性化基因治疗。
发明内容
本发明的目的在于提供一种腺相关病毒基因治疗载体及其在制备治疗苯丙酮尿症的药物中的应用。
在本发明的第一方面,提供了一种腺相关病毒基因治疗载体,其包含增强子、启动子、内含子和人PAH编码基因;所述启动子为人α1抗胰蛋白酶(alpha-1-antitrypsin,α1AT)启动子,其核苷酸序列如SEQ ID NO:2。
在一个优选例中,所述的增强子为肝脏特异的肝调控区HCR(hepatic controlregion,HCR)增强子。
在一个优选例中,所述的增强子包含3个相同重复的HCR,增强子核苷酸序列如SEQID NO:1所示。
在一个优选例中,所述的内含子为人β-珠蛋白和免疫球蛋白重链基因内含子,其核苷酸序列如SEQ ID NO:3所示。
在一个优选例中,所述人PAH编码基因的核苷酸序列如SEQ ID NO:4所示。
在一个优选例中,所述腺相关病毒基因治疗载体还包括转录后调控序列WPRE,其核苷酸序列如SEQ ID NO:6所示。
在一个优选例中,所述的腺相关病毒基因治疗载体为AAV1、AAV2、AAV2-retro、AAV2-QuadYF、AAV2.7m8、AAV3、AAV4、AAV5、AAV6、AAV6.2、AAV7、AAV8、AAV9、AAVrh10、AAV-PHP.eB、AAV-PHP.S、AAV-PAN、AAV-LUNG、AAV-DJ、AAV-DJ/8型腺相关病毒载体。
在一个优选例中,所述的腺相关病毒基因治疗载体为自身互补型AAV8型腺相关病毒载体。
在本发明的另一方面,提供了一种用于治疗苯丙酮尿症的药物,其含有上述任一项所述的腺相关病毒基因治疗载体。
在本发明的另一方面,提供了一种上述的腺相关病毒载体在制备治疗苯丙酮尿症的药物中的应用。
进一步的,将所述的重组腺相关病毒基因治疗载体通过静脉注射,特异性增加肝脏中酶活功能正常的PAH蛋白表达。
本发明的其它方面由于本文的公开内容,对本领域的技术人员而言是显而易见的。
与现有低苯丙氨酸饮食疗法相比,本发明提供了一种重组腺相关病毒基因载体,为开发出新型的肝脏特异性的PKU基因治疗药物奠定基础,从而有望克服长期低苯丙氨酸饮食导致患儿营养不良,生存质量差,遗留不同程度的智力残疾,治疗效果不理想等缺点。
附图说明
图1重组腺相关病毒基因治疗载体质粒图谱。包含HCR增强子、α1AT启动子、内含子、人PAH cDNA、EGFP、WPRE、BGH pA聚腺苷酸化信号和末端重复序列(ITR)。
图2细胞水平验证重组腺相关病毒基因治疗载体的特异性以及mRNA和蛋白表达效率。
图3动物水平检测重组腺相关病毒基因治疗载体的组织特异性以及mRNA和蛋白表达效率。
图4重组腺相关病毒基因治疗载体治疗效果评估。
图5重组腺相关病毒基因治疗载体安全性评估。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
实施例1pUC-3xHCR-α1AT-Intron-hPAH-T2A-EGFP-WPRE质粒的构建
以人类基因组DNA为模板,通过PCR方法扩增分别获得具有增强子功能的肝脏特异的肝调控区HCR、肝细胞特异性启动子α1AT,以mRNA逆转录产物为模板扩增获得人类PAH(hPAH)的cDNA。通过分子克隆方法构建获得携带有3个同源重复序列的3xHCR增强子组合(SEQ ID NO:1),依次将肝脏特异性启动子(α1AT,SEQ ID NO:2),可以增强转入基因长期表达的人β-珠蛋白和免疫球蛋白重链基因内含子序列(Intron,SEQ ID NO:3),人类PAH(hPAH,SEQ ID NO:4),绿色荧光标签蛋白(EGFP,SEQ ID NO:5),转录后调控序列(WPRE,SEQID NO:6)和转录终止信号序列(BGH PA,SEQ ID NO:8)克隆至载体表达框,表达框5'端和3'端分别包含反向末端重复序列:5'ITR(SEQ ID NO:9),3'(SEQ ID NO:10)。
pUC-3xHCR-α1AT-Intron-hPAH-T2A-EGFP-WPRE质粒的特征图谱如图1所示,各元件的起止位点如下:
5'ITR,起始:1,终止141;
3xHCR增强子,起始:148,终止609;
α1AT启动子,起始:616,终止852;
内含子,起始:853,终止985;
hPAH,起始:992,终止2347;
EGFP,起始:2411,终止3130;
WPRE,起始:3141,终止3729;
BGH PA,起始:3765,终止3972;
3'ITR,起始:3980,终止4120;
绿色荧光标签蛋白在本实施例中仅用于验证治疗载体的靶向性,对于实际临床或治疗的腺相关病毒基因治疗载体而言,是不包含该序列的。临床或实际治疗用的基因治疗载体的全长序列如SEQ ID NO:7所示。
实施例2不同细胞系验证载体特异性和表达效率
1、实验方法
选择人肝脏细胞系Huh7和人胚肾细胞系HEK293T分别作为测试与对照细胞系。具体如下:Huh7和HEK293T复苏后,按适当比例接种至6孔板中,待细胞密度达到70%左右进行转染,5μg质粒与250μL无血清培养基混匀,5μL转染试剂(Lipofectamine)与250μL无血清培养基混匀,分别孵育5分钟后混合,进一步室温孵育20分钟,逐滴加入细胞转染孔,按十字方向轻摇混匀,二氧化碳培养箱中37℃培养24小时。隔天更换新鲜培养基,转染48小时后,收取细胞。
细胞总RNA提取及qRT-PCR:细胞的总RNA由TRIzol试剂按照生产商的规程提取,浓度由Nanodrop分光光度计2000(Thermo Fisher Scientific)测定。用0.5μg总RNA进行反转录,使用SYBR Green(Vazyme)荧光定量PCR进行检测,qPCR反应体系:0.5μL PAH-F/GAPDH-F(10μM)、0.5μL PAH-R/GAPDH-R(10μM)、10μL SYBR Green、7μL ddH2O和2μL cDNA。重复三次,采用ΔΔCt法对基因相对表达量进行分析。qPCR引物序列如下:
hPAH-F(SEQ ID NO:11):TCCTGTGTACCGTGCAAGAC;
hPAH-R(SEQ ID NO:12):GTGCAGTGGAAGACTCGGAA;
GAPDH-F(SEQ ID NO:13):GGAGCGAGATCCCTCCAAAAT;
GAPDH-R(SEQ ID NO:14):GGCTGTTGTCATACTTCTCATGG;
细胞总蛋白提取及Western Blot:加入RIPA Lysis裂解液,冰上裂解30分钟,收集细胞裂解液,4℃下以12000rpm离心30分钟,收集上清,BCA定量后,蛋白样品经12%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE),然后转移到硝酸纤维素膜进行转膜。用5%脱脂奶粉4℃封闭处理1小时,然后加入人PAH一抗,4℃孵育过夜。然后用HRP标记的二抗室温孵育2小时,最后加入ECL后进行曝光并拍照。用分子成像仪成像系统对蛋白质条带进行显示,并用Image J软件测量条带强度。
统计分析:数据以平均值±SEM表示,并使用GraphPad Prism软件进行分析。采用未配对t检验确定两组之间的差异;采用单因素方差分析确定多组间的差异。P<0.05为差异有统计学意义。
2、实验结果
qPCR结果表明,HEK293T实验组和HEK293T对照组(未转染质粒)培养48小时后均未检测到hPAH的表达(图2中的A),说明HEK293T本身不表达hPAH,并且构建的质粒在HEK293T中也不表达hPAH;质粒转染Huh7细胞48小时后,hPAH的表达量显著高于未转染质粒的Huh7对照细胞(图2中的A)。同时,进行了Western blot检测(图2中的B),统计分析结果与qPCR结果一致。以上结果表明,所构建的载体仅在肝脏细胞系Huh7中特异性表达。
实施例3pAAV-3xHCR-α1AT-Intron-hPAH-T2A-EGFP-WPRE病毒载体生产。
包装方法:基于临床已被证实安全、高效靶向肝脏的AAV8血清型,对本项目构建的基因治疗载体进行包装。HEK293T细胞培养至汇合度80%左右开始转染。将上述构建的重组载体、AAV8血清型Rep/Cap质粒和pHGTI辅助质粒加到预先平衡至室温的无血清培养基并充分混匀,再加入PEI转染试剂,立即混匀后室温孵育20分钟用于转染。转染三天后收集并裂解细胞,离心取上清获取病毒颗粒。
纯化方法:通过碘克沙醇密度梯度超速离心法对病毒颗粒进一步纯化和浓缩,病毒颗粒经制备型超速离心机离心萃取后位于40%和60%梯度液之间的一层透明液中。抽取病毒液转移至超滤管,并用含0.001%F68的PBS重悬病毒液,2000g离心30分钟,重复过滤3次。最后用0.001%F68重悬,-80℃保存。
滴度测定:取5μL病毒液加入39.5μL ddH2O、5μL DNase buffer和0.5μLDNase,混匀后37℃孵育30分钟,95℃孵育10分钟。向上述反应液分别加入44μL ddH2O、5μL DNasebuffer、1μL Proteinase K(10mg/mL),混匀后37℃孵育15分钟,95℃孵育10分钟。采用SYBRGreen荧光定量PCR检测病毒滴度,qPCR反应体系:0.5μL PAH-F(10μM)、0.5μL AAV-PAH-R(10μM)、10μL SYBR Green、7μL ddH2O、2μL病毒稀释液或8个倍比稀释的质粒标准品。经科学计算,病毒滴度达到体内实验所需的>1014vp/mL。
实施例4小鼠尾静脉注射病毒载体。
实验方法
基于PAH缺陷的PKU模型小鼠,设置Homo治疗组、Homo对照组和野生型WT对照组小鼠,每组3只小鼠(8周龄)。在注射腺相关病毒基因治疗载体前记录体重、毛色、眼眶取血制备干血片,质谱法测定治疗前血液苯丙氨酸水平。注射当天上午,按小鼠体重分装病毒。AAV病毒提前半小时自冰箱取出,恢复至室温,短暂离心,注意观察有无沉淀;用0.1% F68buffer稀释病毒,稀释液浓度(即注射液浓度)为1.5E+13vp/mL,治疗剂量均为1.2E+14vp/kg,对照组按8μL/g注射0.1% F68 buffer。根据实验设计,注射器先用配制病毒相同的0.1% F68 buffer先润洗一遍,以减少AAV的吸附。润洗过的注射器吸取所需液体,按编号使用小鼠固定器固定小鼠,置于尾静脉注射显像仪上,找到小鼠尾静脉,从尾部中后段开始注射(胰岛素注射针29G),注射完后棉球按压注射点1分钟止血。
实施例5基因治疗载体的组织特异性和表达效率
1、实验方法
小动物活体成像:提前用脱毛膏对小鼠腹部脱毛,活体成像仪器制冷CCD降温至-87℃左右可开始成像,将小鼠放入诱导盒中,用异氟烷麻醉3分钟,全身麻醉后快速将小鼠转移至特制的成像暗箱,按顺序摆放小鼠,腹部朝上。在成像控制系统面板中选择“Fluorescence”,曝光数秒后获取图像。
组织总RNA提取及qRT-PCR:解剖好的肝脏、心脏、脑、脾脏、肺脏、肾脏和肌肉组织在液氮中快速冷冻后研磨成粉末,加入TRIzol试剂提取总RNA。总RNA提取及qRT-PCR方法参照上述细胞总RNA提取及qRT-PCR。
引物序列如下:
hPAH-F(SEQ ID NO:11):TCCTGTGTACCGTGCAAGAC;
hPAH-R(SEQ ID NO:12):GTGCAGTGGAAGACTCGGAA;
EGFP-F(SEQ ID NO:15):ATCATGGCCGACAAGCAGAA;
EGFP-R(SEQ ID NO:16):TCTCGTTGGGGTCTTTGCTC;
GAPDH-F(SEQ ID NO:13):GGAGCGAGATCCCTCCAAAAT;
GAPDH-R(SEQ ID NO:14):GGCTGTTGTCATACTTCTCATGG。
组织总蛋白提取及Western Blot:解剖好的肝脏、心脏、脑、脾脏、肺脏、肾脏和肌肉组织在液氮中快速冷冻后研磨成粉末,加入RIPA Lysis裂解液提取总蛋白。总蛋白提取和Western Blot方法参照上述细胞总蛋白提取及Western Blot。
统计分析:数据以平均值±SEM表示,采用2^-ΔΔCt方法对相同组织中基因相对表达量进行计算,采用ΔCt方法对不同组织中基因相对表达量进行计算。使用GraphPadPrism软件进行分析,选择单因素方差分析确定多组间的差异。P<0.05为差异有统计学意义。
2、实验结果
小动物活体成像结果表明,仅在治疗组Homo小鼠的肝脏部位检测到荧光信号(图3中的A)。RT-PCR结果表明,仅在Homo(治疗)小鼠中肝脏组织中检测到EGFP的表达(图3中的B)。此外,本发明还对Homo(治疗)小鼠的心脏、脑、肺脏、脾脏、肾脏和肌肉等组织中hPAH的表达进行WB检测,结果表明,除了在Homo(治疗)小鼠的肝脏组织中检测到PAH的表达,其余所有组织都未检测到PAH的表达(图3中的C)。以上结果表明构建的基因治疗载体具有很好的肝脏靶向性。
基因治疗载体的表达效率评估基于qPCR和WB检测hPAH的mRNA和蛋白水平的表达。qPCR结果表明,野生型和Homo(未治疗)小鼠肝脏中PAH的表达情况在转录水平没有显著差异,而Homo(治疗)小鼠肝脏中hPAH的表达量显著上调(图3中的D)。此外,对不同组小鼠肝脏中PAH蛋白的表达量进行WB检测(图3中的E),统计结果表明,与野生型小鼠相比,Homo(未治疗)小鼠肝脏中PAH蛋白表达量明显下调,而Homo(治疗)小鼠肝脏中hPAH蛋白的表达量均显著高于野生型和Homo(未治疗)小鼠(图3中的F)。以上结果表达,本发明开发的基因治疗载体可以特异性靶向肝脏,并且在转录水平和蛋白翻译水平显著提高特定基因的表达。
实施例6基因治疗载体治疗效果评估
基因治疗载体的治疗效果评估主要基于血苯丙氨酸测定和小鼠毛色的恢复情况。血液中苯丙氨酸浓度是评价PKU治疗效果的最佳指标,本发明利用串联质谱技术测定小鼠血液中苯丙氨酸浓度,结果表明,Homo(治疗)小鼠治疗后第2周(鼠龄:10周)PHE浓度恢复到正常水平(<100μmol/L),并且在治疗后8周(鼠龄:18周)仍然保持在正常水平。以上结果表明,本研究开发的基因治疗载体具有起效快、治疗效果显著和持久等优势。此外,治疗后第2周观察到Homo(治疗)小鼠毛色小面积恢复,由原来的棕黄色转变为黑色,治疗后第8周时对小鼠毛色进行拍照记录,发现Homo(治疗)小鼠毛色大面积恢复成黑色(图4中的B)。
实施例7基因治疗载体安全性评估
1、实验方法
小鼠用异氟烷麻醉后,先用0.9%生理盐水进行心脏灌流,随后更换4%多聚甲醛继续灌流,立即取下肝脏置于4%多聚甲醛中,4℃充分固定后进行石蜡包埋和切片。石蜡切片经60℃烤片后2小时,用二甲苯脱蜡,将浸泡过二甲苯的组织样本先放入无水乙醇中浸泡5分钟,使脱蜡时用的二甲苯可以被洗脱出去,使水可以进入组织中;再依次置于95%、85%、70%乙醇中各浸泡5分钟以达到充分水化的效果。
细胞凋亡检测:将水化后的组织样本切片用蒸馏水洗涤2分钟,滴加20μg/mL不含DNase的Proteinase K,37℃作用25分钟,PBS洗涤3次。根据TUNEL细胞凋亡检测试剂盒说明书配制TUNEL检测液,在样品上加50μLTUNEL检测液,37℃避光孵育60分钟。PBS洗涤3次。用含DAPI的封片剂复染15分钟,在荧光倒置光学显微镜下观察及拍照。
细胞炎症检测:将水化后的组织样本切片用蒸馏水洗涤2分钟,之后用移液枪吸取已经预先配制好的苏木素染色液,每个组织切片滴加100μL,充分染色10分钟。染色完毕后使用蒸馏水洗去多余的苏木素染色液,然后再使用1%的盐酸乙醇进行分化。分化完成后,再用双蒸水将组织切片冲洗干净。为了使苏木素染蓝色,使用弱碱性的促蓝液加入组织切片中,让细胞核染蓝色。反蓝结束后先用清水进行清洗,再用双蒸水将组织切片冲洗干净。向上一步驟的组织样本切片中加入伊红染液,并使组织可以充分染色3分钟,染色完毕后,再将组织切片进行梯度脱水,分别使用浓度为80%、95%以及无水乙醇进行操作。80%的乙醇脱水5秒,95%乙醇脱水2分钟,无水乙醇脱水2分钟。将脱水后的组织样本切片使用二甲苯浸泡2次,每次持续4分钟,然后将组织样本切片晾干,并使用中性树胶封片。最后在显微镜下观察并拍照
2、实验结果
通过TUNEL染色研究本发明开发的腺相关病毒基因治疗载体是否会引起细胞凋亡,与野生型和Homo(未治疗)相比,Homo(治疗)小鼠肝脏中未观察到明显增多的凋亡标志物(图5中的A)。同时,通过H&E染色研究本发明开发的腺相关病毒基因治疗载体是否会引起细胞炎症,与野生型和Homo(未治疗)相比,Homo(治疗)小鼠肝脏中未观察到炎症反应(图5中的B)。说明本发明开发的腺相关病毒基因治疗载体既不会引起细胞凋亡,也不会导致细胞炎症,安全性良好,具有极大的体内基因治疗应用前景。
Claims (10)
1.一种腺相关病毒基因治疗载体,其特征在于包含增强子、启动子、内含子和人PAH编码基因;所述启动子为人α1抗胰蛋白酶(alpha-1-antitrypsin,α1AT)启动子,其核苷酸序列如SEQ ID NO:2。
2.如权利要求1所述的腺相关病毒基因治疗载体,其特征在于所述的增强子为肝脏特异的肝调控区HCR(hepatic control region,HCR)增强子。
3.如权利要求2所述的腺相关病毒基因治疗载体,其特征在于所述的增强子包含3个相同重复的HCR,增强子核苷酸序列如SEQ ID NO:1所示。
4.如权利要求1所述的腺相关病毒基因治疗载体,其特征在于所述的内含子为人β-珠蛋白和免疫球蛋白重链基因内含子,其核苷酸序列如SEQ ID NO:3所示。
5.如权利要求1所述的腺相关病毒基因治疗载体,其特征在于所述人PAH编码基因的核苷酸序列如SEQ ID NO:4所示。
6.如权利要求1所述的腺相关病毒基因治疗载体,其特征在于所述腺相关病毒基因治疗载体还包括转录后调控序列WPRE,其核苷酸序列如SEQ ID NO:6所示。
7.如权利要求1所述的腺相关病毒基因治疗载体,其特征在于为AAV1、AAV2、AAV2-retro、AAV2-QuadYF、AAV2.7m8、AAV3、AAV4、AAV5、AAV6、AAV6.2、AAV7、AAV8、AAV9、AAVrh10、AAV-PHP.eB、AAV-PHP.S、AAV-PAN、AAV-LUNG、AAV-DJ、AAV-DJ/8型腺相关病毒载体。
8.如权利要求7所述的腺相关病毒基因治疗载体,其特征在于,为自身互补型AAV8型腺相关病毒载体。
9.一种用于治疗苯丙酮尿症的药物,其特征在于,含有如权利要求1-8任一项所述的腺相关病毒基因治疗载体。
10.如权利要求1-8任一项所述的腺相关病毒载体在制备治疗苯丙酮尿症的药物中的应用。
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