CN117551697A - 一种靶向rb1的重组腺相关病毒载体及其构建方法和应用 - Google Patents
一种靶向rb1的重组腺相关病毒载体及其构建方法和应用 Download PDFInfo
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Abstract
本申请涉及基因载体构建的技术领域,具体公开了一种靶向RB1的重组腺相关病毒载体及其构建方法和应用。该载体的表达框架包括:AAV2的Cap基因、AAV2的Rep基因、RB1基因。本申请提供的靶向RB1的重组腺相关病毒载体能够高效、精准靶向视网膜母细胞,为进一步推动RB的靶向治疗研究提供了基础和方向。
Description
技术领域
本申请涉及基因载体构建的技术领域,更具体地说,涉及一种靶向RB1的重组腺相关病毒载体及其构建方法和应用。
背景技术
视网膜母细胞瘤(RB)是一种起源于胚胎视网膜细胞的眼内恶性肿瘤,遗传方式为常染色体显性遗传,常见于儿童期,多在4岁以前发病,临床表现为早期眼底出现灰白色肿块,肿块在眼内生长时外眼正常,故很难发现。RB1基因突变是RB最重要的遗传学致病驱动畸变,RB1基因突变导致的功能缺失也是RB发生的最主要原因。因此,针对RB1基因开展基因治疗是攻克RB的根本途径。
然而,目前已发现的RB特异性RB1基因突变超过3000种,RB1基因突变的类型和突变的位点多变,无热点突变,故难以通过修复特定位点进行治疗。且RB1基因片段较大,缺乏安全有效地递送RB1基因的载体系统,导致RB1基因治疗难以实施。上述问题严重阻碍了RB的靶向治疗研究。
发明内容
本申请提供一种靶向RB1的重组腺相关病毒载体及其构建方法和应用。为了进一步推动RB的靶向治疗研究,本申请成功构建了高效、精准靶向视网膜母细胞的重组腺相关病毒(rAAV2-RB1),可高效回补重建RB1基因的功能。在此基础上,本申请以RB的PDX动物模型开展干预试验,验证本申请提供的重组腺相关病毒对RB的有效性和安全性,以期为靶向RB1基因治疗RB建立可靠方案。
第一方面,本申请提供一种靶向RB1的重组腺相关病毒载体,采用如下的技术方案:
一种靶向RB1的重组腺相关病毒载体,所述载体的表达框架包括:AAV2的Cap基因、AAV2的Rep基因、RB1基因。
可选地,所述RB1基因的核苷酸序列包含如SEQ ID NO 1-SEQ ID NO 3所示的核苷酸序列。
在一个具体的实施方式中,所述RB1基因的核苷酸序列包含如SEQ ID NO 1所示的核苷酸序列。
在一个具体的实施方式中,所述RB1基因的核苷酸序列包含如SEQ ID NO 2所示的核苷酸序列。
在一个具体的实施方式中,所述RB1基因的核苷酸序列包含如SEQ ID NO 3所示的核苷酸序列。
可选地,所述AAV2的Cap基因的核苷酸序列包含如SEQ ID NO 4-SEQ ID NO 6所示的核苷酸序列。
在一个具体的实施方式中,所述AAV2的Cap基因的核苷酸序列包含如SEQ ID NO 4所示的核苷酸序列。
在一个具体的实施方式中,所述AAV2的Cap基因的核苷酸序列包含如SEQ ID NO 5所示的核苷酸序列。
在一个具体的实施方式中,所述AAV2的Cap基因的核苷酸序列包含如SEQ ID NO 6所示的核苷酸序列。
可选地,所述AAV2的Rep基因的核苷酸序列包含如SEQ ID NO 7-SEQ ID NO 9所示的核苷酸序列。
在一个具体的实施方式中,所述AAV2的Rep基因的核苷酸序列包含如SEQ ID NO 7所示的核苷酸序列。
在一个具体的实施方式中,所述AAV2的Rep基因的核苷酸序列包含如SEQ ID NO 8所示的核苷酸序列。
在一个具体的实施方式中,所述AAV2的Rep基因的核苷酸序列包含如SEQ ID NO 9所示的核苷酸序列。
可选地,所述RB1基因、所述AAV2的Cap基因、所述AAV2的Rep基因的顺序为Cap-Rep-RB1、Rep-RB1-Cap、Rep-Cap-RB1、Cap-RB1-Rep、RB1-Cap-Rep、RB1-Rep-Cap。
在一个具体的实施方式中,所述RB1基因、所述AAV2的Cap基因、所述AAV2的Rep基因的顺序为Cap-Rep-RB1。
在一个具体的实施方式中,所述RB1基因、所述AAV2的Cap基因、所述AAV2的Rep基因的顺序为Rep-RB1-Cap。
在一个具体的实施方式中,所述RB1基因、所述AAV2的Cap基因、所述AAV2的Rep基因的顺序为Rep-Cap-RB1。
在一个具体的实施方式中,所述RB1基因、所述AAV2的Cap基因、所述AAV2的Rep基因的顺序为Cap-RB1-Rep。
在一个具体的实施方式中,所述RB1基因、所述AAV2的Cap基因、所述AAV2的Rep基因的顺序为RB1-Cap-Rep。
在一个具体的实施方式中,所述RB1基因、所述AAV2的Cap基因、所述AAV2的Rep基因的顺序为RB1-Rep-Cap。
第二方面,本申请提供一种靶向RB1的重组腺相关病毒载体的构建方法,其特征在于,所述构建方法具体包括以下步骤:
利用酶切载体使载体线性化,同时设计引物从模板中调取目的片段、扩增并胶回收目的片段,将胶回收的目的片段与线性化的载体连接重组并转化DH5α感受态细胞。通过PCR鉴定阳性克隆,摇菌抽提质粒后测序,最终构建含有目的片段的载体质粒。
第三方面,本申请提供一种靶向RB1的重组腺相关病毒载体在制备用于基因治疗视网膜母细胞瘤的组合物中的应用。
为了进一步推动RB的靶向治疗研究,本申请成功构建了高效、精准靶向视网膜母细胞的重组腺相关病毒(rAAV2-RB1),可回补重建RB1基因的功能。在此基础上,本申请以RB的PDX动物模型开展干预试验,验证本申请提供的载体对RB的有效性和安全性,以期为靶向RB1基因治疗RB建立可靠方案。
综上所述,本申请具有以下有益效果:
1.本申请提供的靶向RB1的重组腺相关病毒(rAAV2-RB1),能够为RB的治疗提供新的方向,提高RB治疗的有效性,降低眼球摘除率。
2.经过试验分析可知,在RB的PDX动物模型中注射rAAV2-RB1可显著抑制视网膜母细胞瘤的生长,并在小鼠体内具有良好的生物安全性。
附图说明
图1为本申请提供的rAAV2-RB1载体构建及包装示意图。
图2为实施例1的rAAV2-RB1靶向视网膜细胞表达的验证结果(A-小鼠眼内RB1RNA表达水平;B-小鼠眼内RB1蛋白表达水平;C-小鼠视网膜免疫荧光染色结果)。
图3为RB PDX模型的建立结果。
图4为实施例1的rAAV2-RB1在RB PDX动物模型队列上的疗效评价(A-整体实验设计图;B-经治疗后RB PDX模型鼠眼部肿瘤外观图;C-治疗后RB肿瘤大体图;D-治疗后RB肿瘤重量图;E-治疗过程中小鼠眼部肿瘤体积增殖曲线)。
图5为实施例1的rAAV2-RB1在RB PDX动物模型队列上的安全性评价(A-治疗后RBPDX模型鼠重要器官的HE染色结果和Flag蛋白的免疫组化染色结果;B-治疗后RB PDX模型鼠眼球组织学形态;C-治疗后RB PDX模型鼠视网膜电图的检测结果)。
具体实施方式
结合图1,本申请提供的靶向RB1的重组腺相关病毒(rAAV2-RB1)以AAV2载体为基础,AAV2载体负责编码目的基因以及两个末端反向重复序列,辅助质粒包含AAV包装所需的编码病毒衣壳蛋白的Cap基因和参与病毒复制的Rep基因,以及腺病毒Helper质粒。三种质粒共同转染293T细胞后,AAV病毒开始复制和包装。获得的病毒颗粒经过超速离心纯化,并对病毒滴度进行测定。
本申请提供的靶向RB1的重组腺相关病毒载体所述载体的表达框架包括:AAV2的Cap基因、AAV2的Rep基因、RB1基因。
其中,RB1基因的核苷酸序列包含如SEQ ID NO 1-SEQ ID NO 3所示的核苷酸序列;AAV2的Cap基因的核苷酸序列包含如SEQ ID NO 4-SEQ ID NO 6所示的核苷酸序列;AAV2的Rep基因的核苷酸序列包含如SEQ ID NO 7-SEQ ID NO 9所示的核苷酸序列。
经过筛选,RB1基因的核苷酸序列包含如SEQ ID NO 1所示的核苷酸序列;AAV2的Cap基因的核苷酸序列包含如SEQ ID NO 4所示的核苷酸序列;AAV2的Rep基因的核苷酸序列包含如SEQ ID NO 7所示的核苷酸序列时,RB1 mRNA的相对表达量和RB1蛋白的相对表达量均较高。
为使本申请的目的、技术方案和优点更加清楚,下面将结合附图对本申请实施例中的技术方案进行清楚、完整地描述。基于本申请的实施例,本领域技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例,都属于本申请保护的范围。
以下结合实施例、附图、对比例以及验证试验结果对本申请作进一步详细说明。
实施例
实施例1
本实施例提供了一种靶向RB1的重组腺相关病毒(rAAV2-RB1)。
该rAAV2-RB1中,RB1基因包括如SEQ ID NO 1所述的核苷酸序列,Cap基因包括如SEQ ID NO 4所述的核苷酸序列,Rep基因包括如SEQ ID NO 7所述的核苷酸序列。三个基因的顺序依次为Cap-Rep-RB1,具体如表1所示。
上述rAAV2-RB1的构建方法具体如下:利用酶切载体(购自山东维真生物科技有限公司,AAV血清型为AAV2)使载体线性化,同时设计引物从模板中调取目的片段、扩增并胶回收目的片段,将胶回收的目的片段与线性化的载体连接重组并转化DH5α感受态细胞。通过PCR鉴定阳性克隆,摇菌抽提质粒后测序,最终构建含有目的片段的载体质粒。
该rAAV2-RB1的DNA全长序列委托第三方公司合成。
实施例2
本实施例提供了一种靶向RB1的重组腺相关病毒载体。本实施例与实施例1的不同之处在于:该rAAV2-RB1中,RB1基因包括如SEQ ID NO 2所述的核苷酸序列,具体如表1所示。
实施例3
本实施例提供了一种靶向RB1的重组腺相关病毒载体。本实施例与实施例1的不同之处在于:该rAAV2-RB1中,RB1基因包括如SEQ ID NO 3所述的核苷酸序列,具体如表1所示。
实施例4
本实施例提供了一种靶向RB1的重组腺相关病毒载体。本实施例与实施例1的不同之处在于:该rAAV2-RB1中,Cap基因包括如SEQ ID NO 5所述的核苷酸序列,具体如表1所示。
实施例5
本实施例提供了一种靶向RB1的重组腺相关病毒载体。本实施例与实施例1的不同之处在于:该rAAV2-RB1中,Cap基因包括如SEQ ID NO 6所述的核苷酸序列,具体如表1所示。
实施例6
本实施例提供了一种靶向RB1的重组腺相关病毒载体。本实施例与实施例1的不同之处在于:该rAAV2-RB1中,Rep基因包括如SEQ ID NO 8所述的核苷酸序列,具体如表1所示。
实施例7
本实施例提供了一种靶向RB1的重组腺相关病毒载体。本实施例与实施例1的不同之处在于:该rAAV2-RB1中,Rep基因包括如SEQ ID NO 9所述的核苷酸序列,具体如表1所示。
实施例8
本实施例提供了一种靶向RB1的重组腺相关病毒载体。本实施例与实施例1的不同之处在于:三个基因的顺序依次为Cap-RB1-Rep,具体如表1所示。
实施例9
本实施例提供了一种靶向RB1的重组腺相关病毒载体。本实施例与实施例1的不同之处在于:三个基因的顺序依次为RB1-Cap-Rep,具体如表1所示。
实施例10
本实施例提供了一种靶向RB1的重组腺相关病毒载体。本实施例与实施例1的不同之处在于:三个基因的顺序依次为RB1-Rep-Cap,具体如表1所示。
实施例11
本实施例提供了一种靶向RB1的重组腺相关病毒载体。本实施例与实施例1的不同之处在于:三个基因的顺序依次为Rep-RB1-Cap,具体如表1所示。
实施例12
本实施例提供了一种靶向RB1的重组腺相关病毒载体。本实施例与实施例1的不同之处在于:三个基因的顺序依次为Rep-Cap-RB1,具体如表1所示。
对比例
对比例1
对比例1提供了一种靶向RB1的重组腺相关病毒载体。本对比例与实施例1的不同之处在于:该rAAV2-RB1中,RB1基因为RB1外显子序列,NCBI中Accession为NM_000321.3。
对比例2
对比例2提供了一种靶向RB1的病毒载体。本对比例与实施例1的不同之处在于:病毒载体为慢病毒(上海吉凯基因医学科技股份有限公司),RB1基因如SEQ ID NO 1所述的核苷酸序列。
验证试验
一、rAAV2-RB1靶向表达验证
对上述实施例制得的rAAV2-RB1进行靶向表达验证。
(1)验证方法
将构建获得的rAAV2-RB1通过微量注射器注射入裸鼠(先天性胸腺缺陷的突变小鼠)的玻璃体腔内(位于眼球后4/5的空腔,在晶状体后方、视网膜前方,里面充满无色透明的胶质体,即玻璃体),后用红霉素软膏覆盖伤口。
3周后,提取小鼠视网膜RNA和蛋白,并分离小鼠眼球制成病理切片,组织切片置于湿盒中,将稀释的RB1抗体(Mouse anti-RB1购于CST,美国)滴加至组织切片上,完全覆盖组织切片样本,室温孵育1h;PBS漂洗3遍后,将荧光二抗(购于Sigma-Aldrich,美国)滴加至切片上,覆盖样本,盖上湿盒,室温孵育1h;PBS漂洗3遍;取盖玻片,在中央滴加1滴Gelvatol(购于MERCK公司,美国,含DABCO的聚乙烯醇封片剂,防褪色),反转载玻片压于盖玻片上,室温避光放置20-30min,待其凝固后在荧光显微镜(购于OLYMPUS,日本)下观察并记录结果。
(2)验证结果
验证结果具体如表2所示。同时,图2为实施例1的rAAV2-RB1靶向视网膜母细胞表达的验证结果,其中,A图为小鼠眼内RB1 RNA表达水平;B图为小鼠眼内RB1蛋白表达水平;C图为小鼠视网膜母细胞免疫荧光染色结果。
由图2所知,向裸鼠玻璃体腔内注射实施例1的rAAV2-RB1 3周后,A图说明小鼠眼内RB1 RNA水平上调,同时B图说明小鼠眼内RB1蛋白和Flag蛋白(标签蛋白)的表达水平上调,且不影响GAPDH的表达。根据C图的结果可知,小鼠视网膜层细胞可稳定表达RB1蛋白。
表2实施例的靶向验证结果
由表2可知,实施例1中RB1 mRNA的相对表达量和RB1蛋白的相对表达量均较高,故本申请选择RB1基因用如SEQ ID NO 1所述的核苷酸序列,Cap基因用如SEQ ID NO 4所述的核苷酸序列,Rep基因用如SEQ ID NO 7所述的核苷酸序列。
二、RB PDX模型的建立
以4周龄雌性裸鼠(由上海交通大学医学院附属第九人民医院SPF动物房提供)作为试验材料。利用临床组织标本和肿瘤细胞原代解离技术,将肿瘤原代细胞(RB)悬液解离后立即注射至裸鼠视网膜下间隙或者玻璃体腔,借助眼底照相和OCT(光学相干断层扫描)等检测技术,确认PDX小鼠的成瘤情况,并经体内传代,构建RB的PDX模型。
构建结果如图3所示。
由图3可知,与对照组相比,试验组的PDX模型在眼底出现灰白色肿块,说明RB PDX模型构建成功。从图3中还可以看出,对眼球前房和视网膜的检测结果也可以看出,与对照组相比,试验组的PDX模型均表现出异常,进一步说明RB PDX模型构建成功。
三、rAAV2-RB1在RB PDX动物模型队列上的疗效评价
利用实施例1的rAAV2-RB1在RB PDX动物模型队列进行疗效评价。
使用经序列传代及组织学稳定性评估后的RB第二代PDX模型(P2)作为本部分试验的实验用鼠,待PDX P2移植瘤生长1周,可肉眼看到白瞳症后,向玻璃体腔注射实施例1的rAAV2-RB1治疗,拍照记录治疗前眼球形态。给药后,每周对小鼠眼部肿瘤形态进行测量和记录,进行相关安全性评估。待治疗3周后终止实验,颈椎脱臼处理裸鼠,解剖取出瘤体,测量瘤体大小,拍照。解剖所得瘤体留存,相关指标检测将与未进行rAAV2-RB1治疗的对照组(Empty Vector,空载体)RB PDX模型鼠进行对比。
结果如图4所示。其中,A图为整体实验设计图,B图为经治疗后RB PDX模型鼠眼部肿瘤外观图,C图为治疗后RB肿瘤大体图;D图为治疗后RB肿瘤重量图;E图为治疗过程中小鼠眼部肿瘤体积增殖曲线。
由图4可知,由B图可以看出从外观上看,治疗后RB PDX模型鼠的眼部肿瘤有所改善。由C图可知治疗后RB肿瘤体积明显降低。由D图和E图可以看出,治疗后RB PDX模型鼠的RB肿瘤重量和体积增殖情况都有所降低。说明本申请的rAAV2-RB1能够高效、精准视网膜母细胞,显著抑制视网膜母细胞瘤的增殖。
四、rAAV2-RB1在RB PDX动物模型队列上的安全性评价
利用实施例1的rAAV2-RB1在RB PDX动物模型队列进行安全性评价。
将对照组RB PDX模型鼠和实施例1的rAAV2-RB1处理后的试验组RB PDX模型鼠治疗3周实验终止后,颈椎脱臼处理裸鼠,解剖取出小鼠的心(Heart)、肝(liver)、脾(spleen)、肺(lung)、肾(Kidney)等重要器官,利用4%多聚甲醛固定后石蜡包埋,HE染色检测重要器官的组织学形态,免疫组化检测rAAV2-RB1在小鼠重要脏器中的表达情况(Flag蛋白染色),相关指标检测与对照组RB PDX模型鼠进行对比,使用Leica DM6000光学显微镜光镜40倍镜拍摄并保存图像。
另外,对治疗后RB PDX模型鼠的眼球组织学形态和视功能进行检测,并与对照组RB PDX模型鼠和注射生理盐水的RB PDX模型鼠进行对比。其中,视功能通过视网膜电图(Electroretinography,ERG)进行检测。
ERG的检测方法如下:
(1)提前一晚将小鼠放置在动物房暗房内,暗适应过夜。
(2)于次日早晨将小鼠笼子放置在暗箱里移出动物房至ERG室。
(3)从小鼠笼子中取出实验小鼠,双眼滴加1-2滴2.5%苯肾上腺素滴眼液和0.5%托吡卡胺滴眼液扩瞳,再滴加0.5%丙美卡因麻木角膜。
(4)小鼠麻醉后,双眼滴加1-2滴0.3%羟丙甲纤维素,以缓冲角膜与电极/刺激器的接触。
(5)记录小鼠在0.01cd.s/m2和10cd.s/m2强度的白光刺激下暗视力(scotopic)的反应。
(6)检测过程中,使用系统内置的加热元件使体温维持在37℃。
(7)实验完毕后,在小鼠角膜上涂上妥布霉素地塞米松眼膏,将小鼠放置在连带温水浴的加热垫上直至复苏,再原位放回动物房。
(8)采用Espion V6软件测量不同强度的白光刺激后ERG的a波和b波振幅大小。其中,a波振幅以基线为起始点,以负向波谷为终止点;b波振幅以负向波谷为起始点,终止点为正向波峰。记录并分析检测结果。
上述结果如图5所示。其中,A图为治疗后RB PDX模型鼠重要器官的HE染色结果和Flag蛋白的免疫组化染色结果;B图为治疗后RB PDX模型鼠眼球组织学形态;C图为治疗后RB PDX模型鼠视网膜电图的检测结果。
由图5可知,A图中实施例1的rAAV2-RB1处理后的试验组RB PDX模型鼠和对照组RBPDX模型鼠在小鼠的心(Heart)、肝(liver)、脾(spleen)、肺(lung)、肾(Kidney)均呈现出相同相近的组织学形态,说明rAAV2-RB1在RB PDX动物模型队列上具有较好的安全性,对小鼠重要器官的组织学形态没有影响。同时,Flag蛋白的免疫组化染色结果表明rAAV2-RB1在小鼠心(Heart)、肝(liver)、脾(spleen)、肺(lung)、肾(Kidney)等重要脏器中均无特异性表达,说明rAAV2-RB1眼部靶向性良好,没有外溢到除眼部以外的器官表达。
B图和C图中,对比注射生理盐水的RB PDX模型鼠、对照组RB PDX模型鼠以及治疗后RB PDX模型鼠的眼球组织学形态和视网膜电图,可知治疗后RB PDX模型鼠眼球组织学形态和视网膜电图未发生改变,说明本申请的rAAV2-RB1对眼球组织学形态和视功能具有较好的安全性。
最后应说明的是:以上实施例仅用以说明本申请的技术方案,而非对其限制;尽管参照前述实施例对本申请进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本申请各实施例技术方案的精神和范围。
Claims (9)
1.一种靶向RB1的重组腺相关病毒载体,其特征在于,所述载体的表达框架包括:AAV2的Cap基因、AAV2的Rep基因、RB1基因。
2.根据权利要求1所述的重组腺相关病毒载体,其特征在于,所述RB1基因的核苷酸序列包含如SEQ ID NO 1-SEQ ID NO 3所示的核苷酸序列。
3.根据权利要求2所述的重组腺相关病毒载体,其特征在于,所述RB1基因的核苷酸序列包含如SEQ ID NO 1所示的核苷酸序列。
4.根据权利要求1所述的重组腺相关病毒载体,其特征在于,所述AAV2的Cap基因的核苷酸序列包含如SEQ ID NO 4-SEQ ID NO 6所示的核苷酸序列。
5.根据权利要求4所述的重组腺相关病毒载体,其特征在于,所述AAV2的Cap基因的核苷酸序列包含如SEQ ID NO 4所示的核苷酸序列。
6.根据权利要求1所述的重组腺相关病毒载体,其特征在于,所述AAV2的Rep基因的核苷酸序列包含如SEQ ID NO 7-SEQ ID NO 9所示的核苷酸序列。
7.根据权利要求6所述的重组腺相关病毒载体,其特征在于,所述AAV2的Rep基因的核苷酸序列包含如SEQ ID NO 7所示的核苷酸序列。
8.一种权利要求1-7中任一项所述的靶向RB1的重组腺相关病毒载体的构建方法,其特征在于,所述构建方法具体包括以下步骤:
利用酶切载体使载体线性化,同时设计引物从模板中调取目的片段、扩增并胶回收目的片段,将胶回收的目的片段与线性化的载体连接重组并转化DH5α感受态细胞;通过PCR鉴定阳性克隆,摇菌抽提质粒后测序,最终构建含有目的片段的载体质粒。
9.一种权利要求1-7中任一项所述的靶向RB1的重组腺相关病毒载体在制备用于基因治疗视网膜母细胞瘤的组合物中的应用。
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