CN112795595A - 一种遗传性转甲状腺素蛋白淀粉样变性疾病的基因治疗系统 - Google Patents
一种遗传性转甲状腺素蛋白淀粉样变性疾病的基因治疗系统 Download PDFInfo
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Abstract
本发明提供了一种遗传性转甲状腺素蛋白淀粉样变性疾病的基因治疗系统，利用CRISPR/Cas9系统治疗hATTR，选择了可适用于该疾病人转甲状腺素蛋白(transthyretin,TTR)基因治疗的Nme2Cas9应用于该系统，同时优化设计了高效gRNA，实现对功能基因的敲除，显示出强大的抑制突变TTR表达的能力，表现出该系统在治疗遗传性转甲状腺素蛋白淀粉样变性疾病方面的应用前景。
Description
技术领域
本发明属于生物医药技术领域，具体地，涉及一种遗传性转甲状腺素蛋白淀 粉样变性疾病的基因治疗系统。
背景技术
遗传性转甲状腺素蛋白淀粉样变性疾病(hereditary transthyretinamyloidosis, hATTR)是一类罕见的因转甲状腺素蛋白(transthyretin,TTR)基因突变导致的 进展性常染色体显性遗传性疾病。正常情况下，体内绝大部分TTR蛋白由肝细 胞合成并以稳定的四聚体形式转运甲状腺素和维生素A。迄今为止，约有120多 个发生在第2、3、4外显子中的突变被报道与hATTR相关，其中c.148G>A，即 蛋白水平的p.V30M点突变是临床上最常见的致病突变类型。TTR基因发生突变 后生成突变的TTR蛋白单体，含突变单体的TTR蛋白四聚体其结构很不稳定， 容易解聚为TTR单体，解聚后的单体会进一步发生错误折叠。错误折叠的单体 会聚集形成淀粉样纤维蛋白，沉积在细胞间隙导致组织器官的功能异常，进而引 起疾病的发生。
肝脏是生成TTR的最主要器官，临床实践证实肝脏移植能够显著减少突变 TTR的生成，进而降低外周血中TTR的浓度，明显改善患者预后。迄今为止， 肝脏移植依然是唯一可以根治hATTR的疗法。然而，肝脏移植存在健康供体稀 缺、移植手术创伤大以及术后需要长期服用免疫抑制剂等明显局限性。
专利US10060921B2公开了基因治疗药物Patisiran可以有效缓解hATTR病 情，Patisiran是siRNA类药物，通过无差异地靶向降解肝细胞内野生型和突变型 TTR mRNA降低TTR蛋白表达起作用。此外，Inotersen在临床试验也被证实可 以有效缓解hATTR病情，且两种药物分别在2018年8月和10月被美国FDA批 准上市用于治疗hATTR。Inotersen是反义寡核苷酸分子和蛋白质的复合物，同 样通过靶向结合、降解野生型和突变型TTR mRNA以降低TTR蛋白水平。然而， 因为Patisiran和Inotersen都是通过降解TTR mRNA起作用的，所以均不能从根 本上治愈该疾病，患者需要终身接受药物治疗，但这两种药物价格昂贵，hATTR患者面临着严重的经济负担和生命威胁。
CRISPR/Cas9系统的核心是一个RNA-蛋白质复合物，由能与基因组靶DNA 序列互补结合的gRNA和具有DNA切割活性的Cas9核酸酶两部分组成。在这 一系统中，gRNA引导Cas9特异性识别目标DNA序列，Cas9内切酶结合并切 割双链DNA，切割后DNA双链断裂，细胞通过同源重组和非同源末端连接两种 机制对其进行修复，后者会在DNA靶位点处产生DNA片段的插入或缺失导致 移码突变，从而造成靶基因功能丧失，实现对功能基因的敲除。利用该修复机制 对致病基因进行高效率的敲除，有望达到治疗功能获得性遗传病的目的。
但是CRISPR/Cas9系统的应用会因应用对象、靶基因的不同而有较多的复 杂影响，目前尚未有利用CRISPR/Cas9系统针对TTR基因突变导致的进展性常 染色体显性遗传性疾病的报道。
发明内容
本发明要解决的技术问题是现有技术中尚未有根治遗传性转甲状腺素蛋白 淀粉样变性疾病的药物和方法，旨在提供一种遗传性转甲状腺素蛋白淀粉样变性 疾病的基因治疗系统，该系统首次利用CRISPR/Cas9系统治疗hATTR，并创新 性地选择了适用于该疾病人转甲状腺素蛋白(transthyretin,TTR)基因治疗的 Nme2Cas9应用于该系统，同时优化设计了高效gRNA。该基因治疗系统中，gRNA 序列会引导Nme2Cas9特异性识别目标DNA序列，Nme2Cas9内切酶结合并切 割双链DNA，切割后DNA双链断裂，细胞通过同源重组和非同源末端连接两种 机制对其进行修复，后者会在DNA靶位点处产生DNA片段的插入或缺失导致 移码突变，从而造成靶基因功能丧失，实现对功能基因的敲除，显示出强大的抑 制突变TTR表达的能力，具有从根本上治愈遗传性转甲状腺素蛋白淀粉样变性 疾病的应用前景。
因此，本发明的目的是提供一种遗传性转甲状腺素蛋白淀粉样变性疾病的基 因治疗系统。
本发明的另一目的是提供一种遗传性转甲状腺素蛋白淀粉样变性疾病的基 因治疗系统的构建方法。
本发明上述目的通过以下技术方案实现：
本发明提供一种遗传性转甲状腺素蛋白淀粉样变性疾病的基因治疗系统，具 体是一种CRISPR/Cas9系统，所述CRISPR/Cas9系统的递送载体可以为AAV、 脂质体纳米颗粒或阳离子聚合物载体等。
优选地，所述Cas9为Nme2Cas9。
优选地，所述基因治疗系统还包括能靶向切割TTR基因的gRNA。
更优选地，所述基因治疗系统还包括能靶向切割TTR基因第一外显子的 gRNA。
进一步优选地，所述gRNA序列为5’-gcagaggaggagcagacgatgagaagcc-3’或 5’-ggccagcctcagacacaaataccagtcc-3’。
该基因治疗系统采用了AAV介导的CRISPR/Cas9系统，并创新性地选择了 适用于该疾病人转甲状腺素蛋白(transthyretin,TTR)基因治疗的Nme2Cas9应 用于该系统。该基因治疗系统中，gRNA序列会引导Nme2Cas9特异性识别目标 DNA序列，Nme2Cas9内切酶结合并切割双链DNA，切割后DNA双链断裂， 细胞通过同源重组和非同源末端连接两种机制对其进行修复，后者会在DNA靶 位点处产生DNA片段的插入或缺失导致移码突变，从而造成靶基因功能丧失， 实现对功能基因的敲除，显示出强大的抑制突变TTR表达的能力。
优选地，AAV用包装质粒pAAV2/8进行包装获得AAV8。
AAV没有自我复制能力，能感染不分裂细胞，具有很高的安全性和有效性， 而不同血清型的AAV在体内对不同的组织器官有相对特异的亲嗜性，AAV8亲 嗜人和小鼠的肝脏并表现出一定的肝脏靶向特异性，能作为该基因治疗系统理想 的载体。
同时，本发明还提供一种遗传性转甲状腺素蛋白淀粉样变性疾病的基因治疗 系统的构建方法，步骤如下：
S1.将gRNA片段连至Cas9_AAV载体，构建得到Cas9_AAV-gRNA的质粒：
S2.将目的质粒Cas9_AAV-gRNA和辅助质粒以及包装质粒共转染至 HEK293T细胞中生产得到同时表达Cas9和gRNA的基因治疗系统。
其中，优选地，步骤S1中使用SapI核酸内切酶切开Cas9_AAV质粒，再用 T4连接酶将gRNA片段连至Cas9_AAV载体构建Cas9_AAV-gRNA的质粒。
优选地，步骤S2中所用辅助质粒为pHelper，包装质粒为pAAV2/8。
优选地，步骤S2中，共转染至HEK293T细胞中48～120小时后，经分离、 纯化得到同时表达Cas9和gRNA的AAV。
具体地，当所用Cas9为Nme2Cas9时，本发明所述遗传性转甲状腺素蛋白 淀粉样变性疾病的基因治疗系统的构建方法如下：
S1.将gRNA片段连至Nme2Cas9_AAV载体，构建得到 Nme2Cas9_AAV-gRNA的质粒：
S2.将目的质粒Nme2Cas9_AAV-gRNA和辅助质粒pHelper以及包装质粒 pAAV2/8共转染至HEK293T细胞中生产得到同时表达Nme2Cas9和gRNA的基 因治疗系统。
更具体地，作为一种可选择的方案，步骤S2的方法是：用聚乙烯亚胺将 Nme2Cas9_AAV-gRNA、辅助质粒pHelper以及包装质粒pAAV2/8共转染至 HEK293T细胞中生产AAV8，96小时以后收集细胞并用超声破碎细胞分离得到 AAV8粗制品；AAV8粗制品经碘克沙醇密度梯度离心以及超滤浓缩得到纯化浓 缩的AAV8，即为所述基因治疗系统：AAV8介导的CRISPR/Nme2Cas9-gRNA 系统。
其中优选地，聚乙烯亚胺、Nme2Cas9_AAV-gRNA、辅助质粒、包装质粒的 使用质量比为120μg：15μg：15μg：10μg。
本发明具有以下有益效果：
本发明首次利用CRISPR/Cas9系统治疗遗传性转甲状腺素蛋白淀粉样变性 疾病，并创新性地选择了适用于该疾病TTR基因治疗的Nme2Cas9应用于该系 统。
此外，本发明还设计了两条能高效靶向切割TTR基因第一外显子的gRNA， 该gRNA序列无法通过现有技术手段直接得出，而是申请人经过诸多尝试之后确 定的可高效切割靶位点的序列。
本发明提供的一种AAV8介导的CRISPR/Nme2Cas9-gRNA系统在治疗 hATTR方面具有很好的应用前景。
附图说明
图1A为pCCL-HCR-hAAT-hTTR(c.148G>A)-P2A-GFP慢病毒载体图谱；图 1B为报告基因GFP表达结果；图1C为免疫印迹实验结果。
图2A为F0代小鼠的PCR验证结果；图2B为F0代小鼠的报告基因GFP 表达结果；图2C显示F1代小鼠的报告基因GFP表达结果；图2D为BSP甲基 化测序结果；图2E为肝脏切片图。
图3A为Nme2Cas9_AAV质粒图谱；图3B为gRNA靶向位置和序列；图 3C为T7E1实验结果。
图4A为T7E1实验结果；图4B为实时荧光定量PCR结果；图4C为免疫 印迹实验结果；图4D为免疫印迹结果统计图；图4E为肝脏切片图。
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。
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1利用慢病毒制备人源化突变TTR转基因小鼠
(1)构建慢病毒质粒：以pCCL慢病毒骨架作为基础，将肝细胞特异性启 动子ApoE-HCR-hAAT和含点突变c.148G>A的人TTR蛋白编码序列克隆至 pCCL骨架中；同时，用P2A短肽在hTTR(c.148G>A)序列后链接GFP编码序列， 以实现用GFP作为报告基因表征hTTR(V30M)表达的目的，构建出 pCCL-HCR-hAAT-hTTR(c.148G>A)-P2A-GFP慢病毒载体(见图1A)。用该质粒 包装的慢病毒感染体外培养的小鼠肝癌细胞系Hepa1-6细胞3天后，在荧光显微 镜下可以观察到报告基因GFP表达(图1B)，同时用免疫印迹实验可以检测到 hTTR(V30M)突变蛋白(图1C)，证实由该质粒包装的慢病毒可以在肝细胞正常 表达目的蛋白。
(2)构建转基因小鼠：将表达hTTR(c.148G>A)-P2A-GFP的慢病毒通过显 微注射方式递送至CD1小鼠受精卵内使其感染受精卵，再将注射后的受精卵移 植到代孕鼠子宫中以制备转基因小鼠。代孕鼠生产后，通过PCR验证获得转基 因F0代首建鼠，同时发现肝脏GFP报告基因呈嵌合表达模式(图2A、B)。进 一步繁殖获得F1代小鼠，发现大部分F1代转基因小鼠中其转基因启动子和 hTTR(c.148G>A)DNA片段因为甲基化沉默而不能表达目的基因(图2C、D)。 进一步繁殖获得遗传背景相对一致的F2代小鼠，通过分别在出生后第2天和第 9天给予25μg和50μg的5-氮胞苷皮下注射，可以成功激活慢病毒介导的转基因 表达(图2E)。
实施例2筛选得到能高效靶向切割hTTR基因第一外显子的gRNA
用于表达Nme2Cas9和gRNA的质粒Nme2Cas9_AAV(#119924，图3A)购 买自Addgene公司。为了有效敲除hTTR(c.148G>A)，本发明经过大量优化设计、 筛选，选择了4条靶向hTTR基因1号外显子的Nme2Cas9的gRNA(图3B)， 并进一步对着四条gRNA进行试验验证。
使用SapI核酸内切酶切开Nme2Cas9_AAV质粒，再用T4连接酶将gRNA 序列克隆到Nme2Cas9_AAV质粒载体中，将质粒通过瞬时转染导入体外培养的 HEK293T细胞中。转染后2天，提取基因组DNA并通过PCR扩增靶位点序列， 用PCR产物进行T7E1实验验证4条gRNA的切割效率。结果发现G3和G4两 条gRNA可以高效切割靶位点(图3C)。
两条gRNA序列分别为：
G3:5’-gcagaggaggagcagacgatgagaagcc-3’；
G4:5’-ggccagcctcagacacaaataccagtcc-3’。
实施例3AAV8介导的CRISPR/Nme2Cas9-gRNA系统的应用研究
以下呈现的是AAV8介导的CRISPR/Nme2Cas9-G3在小鼠体内高效抑制 hTTR突变蛋白表达：
用实施例2中得到的表达Nme2Cas9_AAV-G3的质粒得到同时表达 Nme2Cas9和G3gRNA的AAV8。具体制备方法如下：
用120μg聚乙烯亚胺将15μg Nme2Cas9_AAV-G3、15μg辅助质粒pHelper， 以及10μg包装质粒pAAV2/8共转染至用15cm培养皿培养的HEK293T细胞中 生产AAV8，96小时以后收集细胞并用超声破碎细胞分离得到AAV8粗制品。 AAV8粗制品经碘克沙醇密度梯度离心以及超滤浓缩得到纯化浓缩的AAV8。进 而用实时荧光定量PCR对AAV8进行滴度测定。
验证AAV8介导的Nme2Cas9-G3在小鼠体内高效抑制hTTR突变蛋白表达， 具体实施步骤如下：
将2×1012GC的AAV8用生理盐水稀释至200μl，通过尾静脉注射将AAV8 递送至实施例1中获得的4周龄的表达hTTR(V30M)的F2代转基因小鼠体内。
注射后12周处死小鼠，取肝脏组织进行分析。提取肝组织基因组DNA并 通过PCR扩增靶位点序列，用PCR产物进行T7E1实验验证肝组织基因组中靶 位点被有效切割(图4A)。
提取肝组织RNA进行mRNA表达分析，通过实时荧光定量PCR实验发现 与注射表达Nme2Cas9和非靶向gRNA的对照组比较，注射AAV8(即 Nme2Cas9_AAV8-G3)组小鼠hTTR mRNA表达下降了65％(图4B)。
提取肝组织蛋白，通过免疫印迹实验发现与对照组比较，注射Nme2Cas9_AAV8-G3组小鼠用于表征hTTR(V30M)蛋白表达的报告GFP蛋白表 达下降了71％(图4C、D)。
通过组织切片观察，发现小鼠肝脏GFP阳性细胞比例显著减少(图4E)。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施 例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替 代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (10)
1.一种遗传性转甲状腺素蛋白淀粉样变性疾病的基因治疗系统，其特征在于，所述基因治疗系统为CRISPR/Cas9系统，所述CRISPR/Cas9系统的递送载体为AAV、脂质体纳米颗粒或阳离子聚合物载体。
2.根据权利要求1所述基因治疗系统，其特征在于，所述Cas9为Nme2Cas9。
3.根据权利要求1或2所述基因治疗系统，其特征在于，所述基因治疗系统还包括能靶向切割TTR基因的gRNA。
4.根据权利要求3所述基因治疗系统，其特征在于，所述基因治疗系统还包括能靶向切割TTR基因第一外显子的gRNA。
5.根据权利要求4所述基因治疗系统，其特征在于，所述gRNA序列为5’-gcagaggaggagcagacgatgagaagcc-3’或5’-ggccagcctcagacacaaataccagtcc-3’。
6.根据权利要求1所述基因治疗系统，其特征在于，AAV用包装质粒pAAV2/8进行包装。
7.权利要求1-6任一所述基因治疗系统的构建方法,其特征在于,步骤如下：
S1.将gRNA片段连至Cas9_AAV载体，构建得到Cas9_AAV-gRNA的质粒：
S2.将目的质粒Cas9_AAV-gRNA和辅助质粒以及包装质粒共转染至HEK293T细胞中生产得到同时表达Cas9和gRNA的基因治疗系统。
8.根据权利要求7所述的构建方法，其特征在于，步骤S1中使用SapI核酸内切酶切开Cas9_AAV质粒，再用T4连接酶将gRNA片段连至Cas9_AAV载体构建Cas9_AAV-gRNA的质粒。
9.根据权利要求7或8所述的构建方法，其特征在于，步骤S2中所用辅助质粒为pHelper，包装质粒为pAAV2/8。
10.根据权利要求7所述的构建方法，其特征在于，步骤S2中，共转染至HEK293T细胞中48～120小时后，经分离、纯化得到同时表达Cas9和gRNA的AAV。
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