CN116870195A - 一种灭活性端粒酶、具有其的腺病毒和人造mRNA及应用 - Google Patents
一种灭活性端粒酶、具有其的腺病毒和人造mRNA及应用 Download PDFInfo
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Abstract
本发明公开了一种灭活性端粒酶,该灭活性端粒酶为双突变端粒酶,双突变点分别是Y707F和D868A。本发明还公开了具有双突变点的腺病毒‑灭活性端粒酶和人造mRNA‑灭活性端粒酶,及其在治疗与非分裂性细胞端粒短缩相关的疾病中的应用。本发明能够抑制非分裂细胞内端粒酶TERT的活性成为灭活性端粒酶CI‑TERT,并同时限制端粒酶离开细胞核。以此配合能保持端粒酶在细胞核内保护端粒尾端不缩短并且防止端粒酶造成端粒延长造成细胞癌变,在降低癌变风险的情况下,使端粒酶保持在细胞核内保护端粒尾端,抑制因端粒短缩所造成疾病的恶化。
Description
技术领域
本发明涉及基因工程和细胞学技术领域。具体来说,涉及一种灭活性端粒酶及应用。
背景技术
端粒(Telomere)是在人体真核细胞染色体末端的多重复非转录DNA(Deoxyribonucleic Acid脱氧核糖核酸)和蛋白的复合体。在研究中表明,端粒与衰老(ageing),细胞分裂控制,及疾病有一定的关联。端粒酶(Telomerase),在细胞中负责端粒的延长的一种酶,是基本的核蛋白逆转录酶,可将端粒DNA加至真核细胞染色体末端,把DNA复制损失的端粒填补起来,使端粒修复延长,可以让端粒不会因细胞分裂而有所损耗。
近几十年来,基因治疗已经逐渐从科学家的梦想逐步转化成了现实。基因治疗的载体一直是整个基因治疗的关键。目前,AAV(Adenovirus Associated Virus,腺病毒)由于其能够通过转染与人类染色体整合,是一种被广泛研究的基因治疗载体。但是,腺病毒AAV在人体中对细胞的特异性辨别较低,且该病毒诱发免疫风暴的风险。研究中表明,端粒缩短与多种疾病及衰老有密切关系,端粒延长能减缓疾病或有一定的治疗效果。目前实验室中采用向实验体内直接打入腺病毒-端粒酶AAV-TERT、使有活性的端粒酶通过过表达来达到延长端粒的目的。但这种非特异性设计无法杜绝带有突变的细胞在得到具有活性端粒酶转化成癌细胞的可能。因此,如何通过合适的基因及其载体,在降低癌变风险的情况下,使端粒酶保持在细胞核内保护端粒尾端,抑制因端粒短缩所造成疾病的恶化,是本领域技术人员需要研究的方向。
发明内容
为克服现有技术的缺陷和不足,本发明旨在提供一种灭活性端粒酶、具有其的腺病毒-灭活性端粒酶和人造mRNA-灭活性端粒酶及应用。
为了实现上述目的,根据本发明的第一个方面,提供了一种灭活性端粒酶,该灭活性端粒酶为双突变端粒酶,所述的双突变点分别是Y707F和D868A。
根据本发明的第二个方面,提供了一种腺病毒-灭活性端粒酶,所述的腺病毒-灭活性端粒酶含有双突变端粒酶,所述的双突变点分别是Y707F和D868A。
根据本发明的第三个方面,提供了一种人造mRNA-灭活性端粒酶,所述的人造mRNA-灭活性端粒酶含有双突变端粒酶,所述的双突变点分别是Y707F和D868A。
根据本发明的第四个方面,提供了上述腺病毒-灭活性端粒酶或上述的人造mRNA-灭活性端粒酶在治疗与非分裂性细胞端粒短缩相关的疾病中的应用。
进一步地,非分裂性细胞包括心肌细胞、骨骼肌细胞和神经细胞。
进一步地,与非分裂性细胞端粒短缩相关的疾病优选为包括与心肌细胞端粒短缩相关的疾病。
进一步地,与心肌细胞端粒短缩相关的疾病包括扩张型心肌病DCM、肥厚型心肌病HCM、核纤层蛋白病和罕见性端粒短缩型心脏病。
更进一步地,罕见性端粒短缩型心脏病包括杜兴氏肌肉萎缩症DMD。
通过上述技术方案,本发明提供了突变点分别是Y707F和D868A的双突变灭活性端粒酶(CI-TERT,Catalytically Inactive TERT),其中,突变点Y707F变异使得端粒酶保留在细胞核内,限制离开会使端粒酶长期咬合在端粒尾端形成保护结构;突变点D868A变异使得端粒酶失去活性,端粒酶失去活性,会防止端粒不受控制的延长以及诱发癌症的可能性。为了实现双突变灭活性端粒酶的功能,申请人发现了腺病毒和人造mRNA可以做为双突变灭活性端粒酶的载体,通过向受体注射腺病毒-灭活性端粒酶和/或人造mRNA-灭活性端粒酶,双突变灭活性端粒酶可以在受体内产生,也就是说,本发明的技术方案能在受体细胞内产生双突变灭活性端粒酶,并通过过表达灭活性端粒酶CI-TERT,使双突变灭活性端粒酶在双突变点的相互配合下发挥相应的功能,不仅降低细胞癌变风险,还可以使端粒酶保持在细胞核内保护端粒尾端,抑制因端粒短缩所造成疾病的恶化。
相对于现有技术,本发明取得的有益效果是:
1.防止端粒缩短,预防和治疗与端粒缩短尤其是非分裂细胞端粒短缩相关的多种疾病及衰老;
2.能够使与端粒缩短尤其是非分裂细胞端粒短缩相关的多种疾病及衰老细胞的端粒延长,对减缓的疾病有一定的治疗效果;
3.抑制因端粒缩短所造成的细胞死亡;
4.保护端粒末端,防止因端粒过分生长所导致的细胞癌变的风险。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。在附图中:
图1示出了本发明的工作原理流程图;
图2示出了在健康心肌细胞中(心肌肌肉小节染色cTnT)通过QFISH定位端粒(端粒重复序列染色TelC)测量长度示意图;
图3示出了在疾病心肌细胞中(心肌肌肉小节染色cTnT)通过QFISH定位端粒(端粒重复序列染色TelC)测量长度示意图;
图4示出了在疾病心肌细胞中(心肌肌肉小节染色cTnT)端粒尾端(端粒重复序列染色TelC)及核酸受损(核酸受损信号染色53BP1)共定位免疫荧光染色;
图5示出了在疾病心肌细胞中端粒尾端(端粒重复序列染色TelC)及端粒酶(端粒酶TERT)共定位免疫荧光染色;
图6示出了端粒酶活性检测(TRAP Assay)图;
图7示出了核酸蛋白共定位(ChIP-qPCR)实验的检测图;
图8示出了在人源心肌细胞中对比人造mRNA-端粒酶(mRNA-TERT)及人造mRNA-灭活性端粒酶(mRNA-CI-TERT)的端粒长度的示意图。
具体实施方式
下面通过具体实施方式及实施例并结合附图对本发明作进一步的详细说明。需要说明的是,在不冲突的情况下,本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。
根据本发明的一典型实施例,提供了一种灭活性端粒酶,该灭活性端粒酶为双突变端粒酶,双突变点分别是Y707F和D868A。图1示出了本发明的工作原理流程图。
通过上述技术方案,突变点Y707F变异使得端粒酶保留在细胞核内,限制端粒酶离开,会使端粒酶长期咬合在端粒尾端形成保护结构;突变点D868A变异使得端粒酶失去活性,端粒酶失去活性,会防止端粒不受控制的延长以及诱发癌症的可能性。本发明提供的突变点是Y707F和D868A的双突变灭活性端粒酶CI-TERT,不仅能够抑制端粒酶TERT的活性成为灭活性端粒酶CI-TERT,并同时限制端粒酶离开细胞核。以双突变点Y707F和D868A的相互配合,保持端粒酶在细胞核内保护端粒尾端,使端粒酶长期咬合在端粒尾端形成保护结构,并且防止因端粒酶脱位造成的端粒不受控制的延长以及导致的细胞癌变,因此,本发明提供的双突变灭活性端粒酶CI-TERT能够在降低癌变风险的情况下,使端粒酶保持在细胞核内保护端粒尾端,抑制因端粒短缩所造成疾病的恶化。
根据本发明的一典型实施例,提供了一种腺病毒-灭活性端粒酶,腺病毒-灭活性端粒酶AAV-CI-TERT含有双突变端粒酶,双突变点分别是Y707F和D868A。本实施例的腺病毒-灭活性端粒酶AAV-CI-TERT通过以下步骤制备得到:
S1,使用细菌培养上述的具有突变点分别为Y707F和D868A的双突变灭活性端粒酶CI-TERT质粒;
S2,将所述的双突变灭活性端粒酶CI-TERT质粒通过脂质转染胺lipofectamin转入腺病毒-293T或AAV-293T细胞培养;
S3,培养7天后,离心提取带有双突变的灭活性端粒酶CI-TERT质粒的腺病毒或AAV病毒,
由此得到具有突变点分别是Y707F和D868A的双突变腺病毒-灭活性端粒酶CI-TERT或AAV病毒灭活性端粒酶CI-TERT。
将提取的带有双突变的灭活性端粒酶CI-TERT质粒的腺病毒或带有双突变的灭活性端粒酶CI-TERT质粒的AAV病毒腺病毒加入培养基(细胞培养)中培养24-48小时后换培养液或注射入受体,观察受体中是否自身产生腺病毒-灭活性端粒酶或称AAV-CI-TERT。
上述各步骤中,可通过基因检测证明带有双突变的灭活性端粒酶CI-TERT质粒已转化成功。在注射或注入受体后,也可通过在动物受体或生物样本上进行免疫荧光染色证明已达到预期效果。
具体来说,上述免疫荧光染色包括如下步骤:
1、样本染色:在样本周围用截流笔画在样本周围,加入10微克/毫升(ug/mL)端粒肽核酸PNA抗体进行TelC端粒重复序列染色,将玻片放入染色盒中以防光线照射。将样本盖上盖玻片放入84摄氏度烤箱中7分钟。拆下盖玻片用PNA洗液洗涤两次,每次15分钟,洗后再用PBST清洗两次,每次5分钟。清洗之后再在室温用10%小牛血清加上磷酸缓冲盐溶液所制封闭液1小时后再在室温敷染稀释一千倍的心肌小节(cTnT)一抗两小时。用封闭液再次清洗样本3次,每次5分钟,然后再敷染稀释1:1000的抗小鼠488alexa488二抗一小时。使用肽核酸PNA洗液及PBST各清洗2次,每次15分钟,在无分子纯水中加入总浓度为1微克/毫升ug/mL的细胞核染色剂DAPI染色5分钟。用磷酸缓冲盐溶液清洗3次,每次5分钟,在样本上加上封片剂封片后在四度保存至上机拍摄。
2、端粒长度测量:使用美国霍金斯大学Alan Meeker教授实验室所开发的ImageJ插件,Telometer(端粒尾端长度测量软件),选取染色样本照片中的细胞核通过TelC对比细胞核中DAPI的荧光强度来测量端粒长度。
通过上述技术方案,将带有灭活性端粒酶CI-TERT质粒的腺病毒或带有灭活性端粒酶CI-TERT质粒的AAV病毒腺病毒注射入受体,在受体内产生双突变灭活性端粒酶CI-TERT,双突变点相互配合发挥作用,其中,突变点Y707F使得端粒酶保留在细胞核内,限制端粒酶离开,使端粒酶长期咬合在端粒尾端形成保护结构;突变点D868A变异使得端粒酶失去活性,端粒酶失去活性,会防止端粒不受控制的生长以及防止恶性肿瘤的产生。本发明的技术方案以腺病毒为载体,将双突变灭活性端粒酶成功注射入受体,使受体内产生双突变灭活性端粒酶CI-TERT,在降低癌变风险的情况下,使端粒酶保持在细胞核内保护端粒尾端,抑制因端粒短缩所造成疾病的恶化。
根据本发明的一典型实施例,提供了一种人造mRNA-灭活性端粒酶,该人造mRNA-灭活性端粒酶含有双突变端粒酶,所述的双突变点分别是Y707F和D868A。人造mRNA-沉默型端粒酶DNA核酸序列的制取:
人造mRNA或称mmRNA,人造mRNA为人造信使RNA,本实施例使用的人造mRNA-灭活性端粒酶或称mmRNA-CI-TERT为Y707F突变(保留在细胞核内)及D868A突变(无活性)双重变异的人造mRNA-灭活性端粒酶或称mmRNA-CI-TERT。将突变点分别是Y707F和D868A的双突变人造mRNA-灭活性端粒酶或称mmRNA-CI-TERT注射于受体,观察受体中是否自身产生人造mRNA-灭活性端粒酶或称mmRNA-CI-TERT。
上述各步骤中,可通过基因检测证明带有双突变的灭活性端粒酶CI-TERT质粒已转化成功。在注射或注入受体后,也可通过在动物受体或生物样本上进行免疫荧光染色证明已达到预期效果。
具体来说,上述免疫荧光染色包括如下步骤:
1、样本染色:在样本周围用截流笔画在样本周围,加入10微克/毫升(ug/mL)端粒肽核酸PNA抗体进行TelC端粒重复序列染色,将玻片放入染色盒中以防光线照射。将样本盖上盖玻片放入84摄氏度烤箱中7分钟。拆下盖玻片用PNA洗液洗涤两次,每次15分钟,洗后再用PBST清洗两次,每次5分钟。清洗之后再在室温用10%小牛血清加上磷酸缓冲盐溶液所制封闭液1小时后再在室温敷染稀释一千倍的心肌小节(cTnT)一抗两小时。用封闭液再次清洗样本3次,每次5分钟,然后再敷染稀释1:1000的抗小鼠488alexa488二抗一小时。使用肽核酸PNA洗液及PBST各清洗2次,每次15分钟,在无分子纯水中加入总浓度为1微克/毫升ug/mL的细胞核染色剂DAPI染色5分钟。用磷酸缓冲盐溶液清洗3次,每次5分钟,在样本上加上封片剂封片后在四度保存至上机拍摄。
2、端粒长度测量:使用美国霍金斯大学Alan Meeker教授实验室所开发的ImageJ插件,Telometer(端粒尾端长度测量软件),选取染色样本照片中的细胞核通过TelC对比细胞核中DAPI的荧光强度来测量端粒长度。
通过上述技术方案,将具有突变点分别是Y707F和D868A的双突变灭活性端粒酶CI-TERT质粒的人造mRNA和/或mmRNA注射入受体,使受体内产生双突变灭活性端粒酶CI-TERT,双突变点相互配合发挥作用,其中,突变点Y707F使得端粒酶保留在细胞核内,限制端粒酶离开,使端粒不会缩短;突变点D868A变异使得端粒酶失去活性,端粒酶失去活性,会防止端粒不受控制的延长以及诱发癌症的可能性。本发明的技术方案以人造mRNA或称mmRNA为载体,将双突变灭活性端粒酶成功注射入受体,使受体内产生双突变灭活性端粒酶CI-TERT,在降低癌变风险的情况下,使端粒酶保持在细胞核内保护端粒尾端,抑制因端粒短缩所造成疾病的恶化。
根据本发明的一典型实施例,提供了上述实施例中腺病毒-灭活性端粒酶或上述实施例中人造mRNA-灭活性端粒酶在治疗非分裂性细胞端粒短缩相关的疾病中的应用。
值得注意的是,非分裂性细胞端粒短缩能够造成衰老、细胞分裂控制、及其他疾病。通过本发明的技术方案,通过腺病毒-灭活性端粒酶AAV-CI-TERT或人造mRNA-灭活性端粒酶mmRNA-CI-TERT,在受体内产生双突变灭活性端粒酶,突变点Y707F使得端粒酶保留在受体细胞核内,限制端粒酶离开,使端粒酶长期咬合在端粒尾端形成保护结构;突变点D868A变异使得端粒酶失去活性,端粒酶失去活性,会防止端粒不受控制的延长以及诱发癌症的可能性。因此,本发明的技术方案能够成功在受体内产生双突变灭活性端粒酶CI-TERT,有效控制端粒酶的表达和受体细胞的种类,在降低癌变风险的情况下,使具有活性的端粒酶通过过表达使端粒酶保持在细胞核内保护端粒尾端,防止端粒缩短,抑制因非分裂性细胞端粒短缩所造成的细胞死亡和疾病的恶化,从而能够预防和治疗与端粒缩短相关的多种疾病及衰老,能够使与端粒缩短相关的多种疾病及衰老细胞的端粒延长,对减缓的疾病达到治疗效果。
优选的,非分裂性细胞包括心肌细胞、骨骼肌细胞和神经细胞。非分裂细胞包括,但不限于,心肌细胞、骨骼肌细胞和神经细胞等。心肌细胞、骨骼肌细胞和神经细胞等均会因发生端粒短缩或阶段性短缩而产生异常及疾病,双突变端粒酶在非分裂细胞内,能有效控制端粒酶的表达和受体细胞的种类,达到相应的功能。
优选的,与非分裂性细胞端粒短缩相关的疾病优选为包括与心肌细胞端粒短缩相关的疾病。
优选的,与心肌细胞端粒短缩相关的疾病包括扩张型心肌病DCM、肥厚型心肌病HCM、核纤层蛋白病和罕见性端粒短缩型心脏病。
更优选的,罕见性端粒短缩型心脏病包括杜兴氏肌肉萎缩症DMD。
研究表明,心肌细胞端粒短缩是造成罕见性心脏病如杜兴氏肌肉萎缩症DMD疾病及死亡的原因。与心肌细胞端粒短缩相关的疾病还包括核纤层蛋白病(Laminopathy)、Titin疾病(Titinopathy)中的多数扩张性心肌病DCM(Dilated Cardiomyopathy)以及肥厚性心肌病HCM(Hypertrophic Cardiomyopathy)。申请人发现,与心肌细胞端粒短缩相关的疾病表现为阶段性的端粒减短。端粒短缩所造成的疾病也不限于遗传性心肌病,研究发现端粒短缩与一系列疾病都有关联。通过本发明的技术方案,以腺病毒或人造mRNA为载体,将突变点分别是Y707F和D868A的双突变灭活性端粒酶注射入受体心肌细胞内,在受体心肌细胞内成功产生具有Y707F和D868A的双突变灭活性端粒酶。突变点Y707F变异使得端粒酶保留在细胞核内,限制离开会使端粒不会缩短,突变点D868A变异使得端粒酶失去活性,端粒酶失去活性,防止端粒不受控制的生长。对于心肌细胞异常的阶梯型端粒短缩的异常,本发明的双突变灭活性端粒酶不仅能改善阶段性端粒酶短缩造成的病情持续恶化,同时在足够早的时间的介入治疗,还可防止出现端粒的短缩发生。申请人经过大量实验还发现,心衰造成的端粒短缩会造成核酸损伤,导致线粒体功能及数量下降进而影响心肌收缩功能。通过灭活性端粒酶CI-TERT保护端粒尾端能抑制疾病的恶化,且防止核酸损伤、线粒体功能及数量下降,以及心肌功能下降。
下面将结合实施例进一步说明本发明的有益效果。
实施例:
根据本发明一典型实施例,使用腺病毒-灭活性端粒酶AAV-CI-TERT治疗心脏疾病。
通过免疫荧光染色定位测量心肌病患者的心脏切片中的端粒尾端长度发现心肌病患者会出现异常端粒短缩的现象,此现象会逐渐造成细胞死亡导致病情恶化引起心衰。值得注意的是,在患者其他生物样本中未检测到端粒短缩。实验发现在人源干细胞所分化的心肌细胞中同样可以观察到这一现象,通过免疫荧光染色可以定位测量人源干细胞所分化的心肌细胞核中的端粒长度,如图2,图3所示,证明人源干细胞所分化的心肌细胞可以作为心脏疾病中异常端粒短缩的实验模型。在人源干细胞层面,健康与疾病细胞无端粒长度差别。相比健康人源干细胞所分化出来的心肌细胞,疾病人源干细胞所分化的心肌细胞呈现着端粒短缩。这表示异常端粒短缩是心肌细胞在非分裂状态下所造成的,正常情况下端粒短缩是在细胞分裂时造成的且端粒过短的细胞会在分裂中被清除。申请人通过反复实验,发现该异常端粒短缩是由于疾病发展过程中的活性氧(ROS)激发端粒酶的保护线粒体的功能,导致端粒酶离开细胞核内使得端粒尾端失去保护而造成的。申请人进一步发现,此异常端粒短缩不是急性短缩,而是阶梯式的逐渐短缩。这表示在疾病早期进行干预或进行治疗可有效的抑制或阻止端粒短缩。
表1为病人心肌样本端粒长度测量。
表1证明在心肌病病人中,非分裂细胞(心肌细胞)有异常端粒短缩现象。
表2为病人平滑肌切片端粒长度测量,证明心肌病患者的异常端粒短缩只存于心肌细胞。
表3为人源干细胞及人源干细胞所分化的心肌细胞端粒长度测量。证明异常端粒短缩是在非分裂的疾病心肌细胞中发生。
表4为端粒长度追踪实验,证明此异常端粒短缩非急性短缩而为阶梯性逐渐短缩。
通过进一步对比健康的人源干细胞所分化的心肌细胞以及疾病患者的人源干细胞所分化的心肌细胞发现,疾病细胞中表现出了更高的在核内端粒尾端核酸受损信号53BP1,如图4所示,线粒体数量明显下降,且细胞收缩功能下降,即收缩力下降。
心肌细胞核酸受损信号检测 | 53BP1(核酸受损信号)阳性反应比例 |
a.健康人源干细胞分化心肌细胞 | 12.08 |
b.疾病人源干细胞分化心肌细胞 | 47.75 |
a.对比b. | P<0.0001 |
表5为测量心肌细胞的核酸受损信号,证明在疾病中心肌细胞中核酸受损信号更多。
表6为测量心肌细胞的线粒体数量,证明在疾病细胞中线粒体数量明显的下降。
表7为测量心肌细胞的收缩力,证明疾病细胞功能受影响而降低。单位为nano-newton,简称nN。
为了抑制端粒短缩且避免端粒尾端被过度延长,本发明的实施例制备了带有两个点突变的人类端粒酶序列,称为灭活性端粒酶或称CI-TERT。灭活性端粒酶或CI-TERT的两个突变点一个是Y707F,该突变点是将端粒酶保持在细胞核内,另一个是D868A,该突变点使端粒酶失去活性。通过腺病毒(AAV)或人造mRNA(mmRNA)注射灭活性端粒酶至受体,也就是说,注射腺病毒-灭活性端粒酶或人造mRNA-灭活性端粒酶至受体。如图5所示,受体能有效的表达此灭活性端粒酶且特异于核内的端粒尾端。值得注意的是,该受体可以是人源干细胞所分化的心肌细胞模型及小鼠模型,也可以是其他任何分裂性细胞。如图6和图7所示,通过端粒酶活性实验(TRAP Assay)(图6)以及染色质免疫共沉淀(ChIP)qPCR(ChIP-qPCR)(图7)证明,本发明技术方案中的双突变的灭活性端粒酶无活性,且能准确的特异在脱保护的端粒尾端上.
表8为qPCR测量CI-TERT表达,证明受体可过表达CI-TERT。
如图8所示,进一步证明本发明实施例中腺病毒-灭活性端粒酶或人造-mRNA端粒酶能有效的保护端粒尾端避免短缩,且抑制因异常端粒短缩所造成的一系列细胞反应,例如,但不限于,核酸受损,线粒体数量下降,收缩力下降等。
表9为测量注入灭活性端粒酶CI-TERT后的端粒尾端长度,证明灭活性端粒酶CI-TERT有效保护端粒尾端但不延长端粒尾端。
表10为在灭活性端粒酶CI-TERT过表达下测量核酸受损信号,证明过表达灭活性端粒酶CI-TERT能在细胞中降低核酸受损信号。
表11为在灭活性端粒酶CI-TERT过表达下测量线粒体数量,证明过表达灭活性端粒酶CI-TERT能有效减少线粒体死亡。
表12为测量过表达灭活性端粒酶CI-TERT后心肌细胞收缩力,证明过表达灭活性端粒酶CI-TERT可有效保护心肌细胞功能。
上述实施例的实验表明,本发明提供的技术方案中的腺病毒-灭活性端粒酶能有效的通过保护端粒尾端避免异常的端粒短缩及短缩造成的核酸受损,保护线粒体功能从而保护细胞功能,避免细胞死亡以及疾病的恶化导致的心衰。
在心肌病的情况下,非分裂细胞,如心肌细胞中端粒尾端出现了异常的阶梯型短缩。阶梯性的端粒短缩不仅仅在实验中对应了临床中所观察到病情的恶化过程,也同时表示在足够早的时间的介入进行治疗可阻止病情的恶化,即阻止端粒的持续短缩。实验证明在人源干细胞所分化的心肌细胞中可以模拟异常端粒短缩的现象,并且通过灭活性端粒酶CI-TERT(Catalytically Inactive TERT,灭活性端粒酶)可以有效的缓解此现象的一系列指标。申请人经过大量实验发现,心衰造成的端粒短缩会造成核酸损伤,导致线粒体功能及数量下降进而影响心肌收缩功能。通过灭活性端粒酶CI-TERT保护端粒尾端能抑制疾病的恶化,且防止核酸损伤,线粒体功能及数量下降,以及心肌功能下降。
本发明上述实施例提供了上述腺病毒-灭活性端粒酶在治疗与非分裂性细胞端粒短缩相关的疾病中的应用。注射腺病毒-灭活性端粒酶的治疗手段,能以有效的载体,控制端粒酶的表达即受体细胞的种类,防止在心肌细胞这一非分裂细胞体系中,因端粒尾端脱保护及短缩所引起的疾病或症状。与心肌细胞端粒短缩相关的疾病包括,但不限于,扩张型心肌病DCM、肥厚型心肌病HCM、核纤层蛋白病和罕见性端粒短缩型心脏病,如杜兴氏肌肉萎缩症DMD。本实例选取的疾病样本包含了各种与心肌细胞端粒短缩相关的疾病,例如,但不限于,扩张型心肌病DCM、肥厚型心肌病HCM、核纤层蛋白病和罕见性端粒短缩型心脏病,如杜兴氏肌肉萎缩症DMD等。
当然,使用人造mRNA-灭活性端粒酶mmRNA-CI-TERT代替注射腺病毒-灭活性端粒酶能够产生同样的实验效果。人造mRNA和腺病毒做为本发明双突变灭活性端粒酶的载体,在注射到受体后,均能在受体产生双突变灭活性端粒酶,从而起到保持端粒在细胞核内和保护端粒末端不脱位,治疗和防止端粒短缩引起的疾病和细胞死亡。
综上所述,本发明的技术方案通过具双突变的腺病毒-灭活性端粒酶和人造mRNA-灭活性端粒酶,在降低癌变风险的情况下,使端粒酶保持在细胞核内保护端粒尾端,抑制因端粒短缩所造成疾病的恶化。相对于现有技术,具有防止端粒缩短,预防和治疗与端粒缩短相关的多种疾病及衰老;抑制因端粒缩短所造成的细胞死亡;使与端粒缩短相关的多种疾病及衰老细胞的端粒延长,对减缓的疾病有一定的治疗效果。
当然,本发明还可有其他实施例,在不背离本发明精神及其实质的情况下,所属技术领域的技术人员当可根据本发明作出各种相应的改变和变形,但这些相应的改变和变形都应属于本发明的权利要求的保护范围。
Claims (10)
1.一种腺相关病毒在制备治疗与非分裂性细胞端粒短缩相关的疾病的药物中的应用,所述的腺相关病毒带有双突变的灭活性端粒酶质粒,表达灭活性端粒酶,所述的双突变点分别是Y707F和D868A。
2.根据权利要求1所述的应用,其特征在于,所述的非分裂性细胞包括心肌细胞、骨骼肌细胞和神经细胞。
3.根据权利要求2所述的应用,其特征在于,所述心肌细胞端粒短缩相关的疾病为心衰。
4.根据权利要求1所述的应用,其特征在于,所述非分裂性细胞端粒短缩相关的疾病为衰老疾病。
5.根据权利要求4所述的应用,其特征在于,所述衰老疾病为神经相关的衰老疾病。
6.一种人造mRNA在制备治疗与非分裂性细胞端粒短缩相关的疾病的药物中的应用,所述的人造mRNA带有双突变的灭活性端粒酶质粒,表达灭活性端粒酶,所述的双突变点分别是Y707F和D868A。
7.根据权利要求6所述的应用,其特征在于,所述的非分裂性细胞包括心肌细胞、骨骼肌细胞和神经细胞。
8.根据权利要求7所述的应用,其特征在于,所述心肌细胞端粒短缩相关的疾病为心衰。
9.根据权利要求6所述的应用,其特征在于,所述非分裂性细胞端粒短缩相关的疾病为衰老疾病。
10.根据权利要求9所述的应用,其特征在于,所述衰老疾病为神经相关的衰老疾病。
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