CN116515773B - 一种重组腺相关病毒及在制备妊娠期内耳靶向基因治疗药物中的应用 - Google Patents
一种重组腺相关病毒及在制备妊娠期内耳靶向基因治疗药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种重组腺相关病毒及在制备妊娠期内耳靶向基因治疗药物中的应用；所述重组腺相关病毒载体结合Otogelin和/或Prestin基因启动子可提高腺相关病毒基因靶向性，另一个方面为后期遗传性耳聋部分突变基因治疗提供更加有效的治疗窗口期。此外，也为临床上胎儿期的基因治疗提供了可能。
Description
技术领域
本发明属于内耳基因治疗技术领域，具体涉及一种重组腺相关病毒及在制备妊娠期内耳靶向基因治疗药物中的应用。
背景技术
基因治疗在遗传性疾病临床治疗领域已取得重大进展。遗传性耳聋的主要致病原因为基因的异常突变，随着耳聋分子生物学研究逐步深入，多个耳聋基因的发现为基因治疗提供了精准靶点。在动物实验中，已有遗传性耳聋小鼠模型通过基因治疗手段恢复听力的案例，相关载体的内耳注射时间一般为小鼠出生后0-2天，然而，一些基因的突变甚至在出生前就可能造成永久性损害，如GJB2和SLC26A4这两个遗传性耳聋中最为常见的突变基因，其缺失或突变在出生时即造成严重的听力损失。研究表明小鼠在出生后10-14天时听功能开始出现，而人类的胎儿在孕期24-28周时即有听力反应。此外，出生后免疫系统已发育成熟，相关载体的导入易引起机体免疫反应而使得转导效率降低或副作用增多。因此，妊娠期的内耳基因治疗技术亟需解决。一方面，在孕期免疫系统幼稚阶段进行病毒载体转导或基因编码更容易被早期胚胎靶细胞接受，增大基因治疗相关载体转染效率；另一方面更早干预以期实现更好的治疗效果。
内耳由膜迷路和骨迷路组成，其中膜迷路上特定区域高度分化形成的螺旋器为听觉感受器，由感觉毛细胞和支持细胞组成。耳蜗感觉细胞包括内毛细胞(inner haircells)和外毛细胞(outer hair cells)，支持细胞包括内柱细胞(inner pillar cells)、外柱细胞(outer pillar cells)、Deiter细胞、内指细胞、内缘细胞、Hensen细胞等。传统的病毒启动子如CMV、CAG等在胚胎期内耳大量表达，缺少靶向性，限制了胚胎期病毒的应用。
因此，有必要结合新的启动子提供一种靶向性强的腺病毒，应用于妊娠期的内耳基因治疗提高相关载体转染效率，为基因治疗手段恢复听力提供新的方向。
发明内容
本发明的目的在于，提供一种靶向性强的腺病毒，应用于妊娠期的内耳基因治疗提高相关载体转染效率，为基因治疗手段恢复听力提供新的方向。
鉴于此，本发明的方案如下：
一种重组腺相关病毒，包含AAV衣壳和重组核酸分子；所述重组核酸分子包含Otogelin和/或Prestin基因启动子的核苷酸序列。
进一步地，所述Otogelin基因的启动子核苷酸序列如SEQ ID NO：1所示，所述Prestin基因的启动子核苷酸序列如SEQ ID NO：2所示。
进一步地，所述AAV衣壳来源于AAV2.7m8衣壳蛋白。
进一步地，所述重组核酸分子包含选自AAV2型的AAV反向末端重复序列。
本发明另外一个目的在于提供以上所述重组腺相关病毒的构建方法，将包含基因启动子的目标序列导入AAV质粒得到，所述AAV载体质粒含有调控元件(WPRE)。
进一步地，所述AAV质粒上表达有荧光蛋白。
本发明再一个目的在于提供以上所述重组腺相关病毒，或以上所述构建方法在制备妊娠期内耳靶向基因治疗药物中的应用。
本发明还有一个目的在于提供一种药物组合物，包含以上所述重组腺相关病毒和一种或多种药学或生理学上可接受的载体、赋形剂和/或稀释剂。
进一步地，所述药物组合物被配制用于胚胎内耳注射给药。优选地，所述给药时间为E12.5-E13.5。
与现有技术相比，本发明有益效果包括但不限于：
本发明提供的重组腺相关病毒结合Otogelin和/或Prestin基因启动子可提高腺相关病毒基因靶向性，另一个方面为后期遗传性耳聋部分突变基因治疗提供更加有效的治疗窗口期。此外，也为临床上胎儿期的基因治疗提供了可能。
附图说明
为了更清楚地说明本发明实施例中的技术方案，下面将对实施例描述中所需要使用的附图作简单的介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明Otogelin(Otog)基因启动子驱动的腺相关病毒基因图谱。
图2为本发明Prestin基因启动子驱动的腺相关病毒基因图谱。
图3为本发明提供的rAAV-CAG-P2a-EGFP-WPREs载体图谱。
图4为本发明实施例1中P2与胚胎期注射病毒感染效率对比结果。
图5为本发明实施例2所述AAV启动子改进载体妊娠期注射靶向感染支持细胞与外毛细胞免疫荧光染色结果。
图6为本发明实施例2所述AAV启动子改进载体妊娠期注射后支持细胞与外毛细胞免疫靶向感染率统计结果。
具体实施方式
为了使本发明的目的、技术方案和有益技术效果更加清晰明白，接下来结合实施例，对本发明的技术方案做进一步地详细描述，应当理解的是，本说明书中描述的具体实施方式仅仅是为了解释本发明，并不是为了限定本发明。
Otogelin(otog)启动子是内耳所有无细胞膜的组成部分，其表达最早在胚胎第10天(E10)在听囊中检测到，在E18时可在缝隙连接(gap junction)上皮网络的细胞中检测到。申请人前期通过Otog-CreER敲除小鼠研究发现，Otog在内耳的非感觉上皮细胞中特异性表达，通过cre-loxp技术可实现支持细胞靶向基因敲除。Prestin基因特异性编码外毛细胞的运动蛋白，可实现外毛细胞靶向的基因敲入或敲除。此外，Otog及Prestin的表达出现在细胞分化之前，因此可通过构建由Otog及Prestin启动子驱动的腺相关病毒，实现胚胎期内耳支持细胞及外毛细胞靶向性感染，最终实现不同类型细胞缺陷造成的内耳疾病的靶向治疗。
在一个实施例中，提供Otog、Prestin启动子驱动的腺相关病毒的构建方法，具体如下：
(1)动物准备：选择C57背景的适龄雌鼠与雄鼠进行交配，次日中午检测到阴道栓者指定为妊娠的第0.5天(E0.5)胚胎，选取怀孕E12.5的雌鼠进行手术。
(2)腺相关病毒构建
1.Otog启动子驱动的腺相关病毒基因信息，如图1所示：rAAV-otog-2a-EGFP-WPREs(种属：小鼠)，预测小鼠源启动子，长度1200bp；在小鼠Otog基因转录激活起始区域为原点，在-1100bp-+100bp的区间，Otog启动子序列如SEQ ID NO：1所示。
2.Prestin启动子驱动的腺相关病毒基因信息，如图2所示：rAAV-prestin-EGFP-WPRE-hGh polyA(种属：小鼠)，启动子区域位于prestin基因翻译起始位点的上游552bp。prestin启动子序列如SEQ ID NO：2所示。
3.载体信息：载体名称rAAV-CAG-P2a-EGFP-WPREs，如图3所示；克隆位点：MluI/AgeI。
以上载体及启动子由武汉枢密脑科学技术有限公司提供合成。
4.病毒载体构建：通过PCR扩增或者限制性核酸内切酶酶切的方法获得目的基因，再通过同源重组或者T4 DNA连接的方法组装，将外源片段构建入目的载体，转化E.coliStbl3感受态细胞，用菌落PCR对转化子进行筛选，阳性克隆送测序，经序列比对确定阳性克隆。将C端Flag标记的NLS-mNeonGreen克隆到含有目的启动子和木鱼肝炎病毒转录后调控元件(WPRE)盒的AAV质粒中，该盒由AAV2倒置末端重复区侧接。AAV血清型的载体是在HEK293T细胞中与rep-cap融合质粒和辅助质粒共同转染产生的。AAV通过碘克沙醇密度梯度超速离心法进行纯化。转染后每48小时收集两次培养基。用氯仿处理细胞裂解液，收集上清液。合并培养基和上清液，用10％的PEG 8000和1.0M的NaCl沉淀来浓缩。离心后，将颗粒重新悬浮在含有苯宗酶的PBS缓冲液中。将15％、25％、40％和60％的碘克沙醇溶液仔细分层，然后将产生的病毒悬浮液覆盖。然后在10℃下以350,000g离心90分钟。超速离心后，收集含AAV的40％馏分。交换缓冲液以去除碘克沙醇，并浓缩纯化的病毒。使用针对WPRE区域的引物，通过SYBR(罗氏)分析确定AAVs的含基因组滴度。WPRE的qPCR引物如下：
正向，5′-GTCAGGCAACGT GGCGTG-3′(SEQ ID NO：3)；
反向，5′-GGCGATGAGTTCCGCCGTGGC-3′(SEQ ID NO：4)。
用菌落PCR对转化子进行筛选，阳性克隆送测序，经序列比对确定阳性克隆。
我们前期实验锁定了AAV2.7m8病毒载体AAV2.7m8是通过体内定进化的方法产生通过将一个包含10个氨基酸的插入AAV2外壳蛋自序列的第588位，增强了AAV2与其主要受体--硫酸肝素蛋白多糖的结合，进而提高了该载体对细胞的感染效率。在乳鼠出生后2天(P2)时圆窗注射可实现内耳毛细胞与支持细胞的高效感染。
以小鼠为对象进行手术及给药方式如下：
1.麻醉：腹腔注射戊巴比妥钠(65-70毫克/公斤，Sigma公司，P3761)对小鼠进行麻醉，用非甾体抗炎药Meloxicam(5毫克/公斤，勃林格殷格翰公司)控制手术后疼痛。通过进行有害刺激试验来评估麻醉的完整性：捏爪子；捏尾巴；以及对触摸脸颊和振动区的眨眼反应。在角膜上涂抹无菌眼药膏。
2.备皮：用脱毛剂除去腹部从耻骨上区到肋骨区被毛
3.消毒后切开：雌鼠麻醉后使其处于仰卧位，覆盖无菌孔巾，按照消毒原则用络合碘棉球擦拭腹部进行消毒，沿腹中线切开皮肤1.5厘米，依次切开肌层，暴露子宫，将子宫连同胚胎一同摘出，立即用预热的(37℃)无菌生理盐水溶液冲洗腹部。
5.药物准备：定制玻璃毛细管微量注射器，使其外径为外径12-16μm，针尖斜面20°(将装好的微量注射吸管连接到微量泵(WPI,Sarasota,FL,USA)的吸管支架上。AAV2.7m8-CAG-eGFP和Fast Green FCF染料(Sigma-Aldrich,St.Louis,MO,USA)进行混合使溶液染成蓝色，而不引起明显的稀释。将病毒/Fast Green染料混合物回吸至玻璃毛细管微量注射器待用。
6.解剖定位：用低强度的卤素灯透射子宫，以观察胚胎在其植入部位的情况。识别跳动的心脏、脑泡、肢体芽、新生的后脑第四脑室和眼睛。每2分钟用预热的生理盐水溶液灌溉子宫以保持水分。轻轻压迫子宫以调整胚胎的方向，并确定上述的解剖学标志。确定主头静脉，其前后分支在间质区的两侧，耳囊就在其中。通过对子宫的透照，不能看到耳囊本身。耳囊位于主头静脉的前后分支之间，与静脉的主干一起，形成美式足球场上的立柱或门柱的形状。耳囊位于立柱之间的中间位置。
7.耳囊注射：双手轻轻固定胚胎头部，将微量注射器穿过子宫，其轨迹与耳囊的推定位置一致。在通过子宫后，对微量注射器进行一次注射，以观察示踪剂染料，并大致确定注射器尖端的位置。进一步将注射器推进到侧头间质进行定量注射。实验组的每个耳囊注射了1.5ul病毒溶液；对照组(假手术组)的每个耳囊注射了1.5ul无菌磷酸盐缓冲盐水(PBS)。成功的耳囊定位将显示出内淋巴管背侧的锥形和前庭的扇贝壳形。松开对子宫的压力，从胚胎/子宫中一次性取出吸管，并立即用预热的生理盐水液灌溉子宫。
8.缝合：显微注射后，用7-0外科缝线缝合腹部肌肉和真皮层的伤口。术后将雌鼠放在37℃恒温毯上进行复苏。
实施例1妊娠期内耳基因治疗使得腺相关病毒AAV转导效率增高
1.方法：
1.1分组及给药
实验分为以下4组：胚胎期假手术组、胚胎期AAV2.7m8-CAG-GFP注射组、出生后P2假手术组、出生后P2AAV2.7m8-CAG-GFP注射组，每组至少4个重复。AAV2.7m8-CAG-GFP滴度为5.0×1012，每个耳蜗给药1μl，假手术组给予1μl的无菌PBS溶液。
1.2手术操作
(1)胚胎期手术组即按照本发明第三部分发明内容进行操作；
(2)出生后P2内耳圆窗注射手术操作：出生后P2的小鼠幼崽被浸泡在冰水中进行麻醉。麻醉后，做耳后切口以暴露听泡和镫骨动脉，进而暴露圆窗。在纳升显微注射系统(WPI,Sarasota,FL,USA)上用玻璃微吸管缓慢注射1μlAAV2.7m8-CAG-GFP病毒与Fastgreen混合液至圆窗。注射后静待5分钟再将玻璃微吸管收回。之后用小米粒大小的肌肉放置圆窗口防止液体外露。7-0外科缝合线缝合切口，并将小鼠放在38℃的复温垫上进行恢复。小鼠在15-20分钟内完全恢复后被送回其母亲身边。
1.3耳蜗取材免疫荧光染色：
(1)耳蜗组织固定及脱钙：小鼠术后两周进行取材观察。小鼠称重后麻醉(同上)，运用颈椎脱臼法将其处死。剪下头颅并迅速将内耳分离取出放入4％多聚甲醛中进行蜗顶打孔固定。将处理后的耳蜗置于新的4％多聚甲醛中于4℃固定过夜。第二天将固定好的耳蜗取出，置于10％ EDTA脱钙液中脱钙1天，充分脱钙后用于解剖。
(2)耳蜗解剖：将上述处理好的耳蜗样本取出放于玻璃皿(内含0.01M PBS溶液)中。在解剖显微镜下处理耳蜗，小心去除蜗壳和外侧壁，自顶回向下逐步分离基底膜，耳蜗硬铺片分离出顶中回基底膜，耳蜗软铺片分离出顶中底全段基底膜。
(3)免疫荧光染色：上述解剖好的基底膜应用100ul 10％驴血清(含0.3％Triton-X100)，室温状态下放置1小时进行封闭。其次吸出封闭液后加入100ul一抗稀释液4℃过夜(polyclonal goat anti-sox2 antibodies，sc-17320,Santa Cruz Biotechnology，抗体:0.01M PBS溶液＝1:200),
次日样品用PBST洗三次，然后用二抗抗体(1:200稀释，Antgene)孵育2小时在室温下孵育2小时。接下来用100ul鬼笔环肽进行标记，室温放置15分钟，注意避光。吸出鬼笔环肽，加入100ulPBS溶液泡洗基底膜3次，每次5分钟。向EP管中加入50ul DAPI染料，室温放置10分钟，注意避光；吸出DAPI染料，加入100ul PBS溶液泡洗基底膜3次，每次5分钟；用巴氏滴管将基底膜吸出放置于载玻片上，在解剖显微镜下展平基底膜，调整好基底膜摆放的位置后，滴加抗荧光淬灭剂封片，放于暗盒中待用。使用激光共聚焦显微镜观察已制备好的基底膜铺片，并使用相关软件进行拍照和三维重建。根据采集到的图像进行数据统计处理。
2实验结果：如图4所示，图4A为免疫荧光染色拍照结果，图4B为数据统计处理结果。不难看出胚胎期组内耳注射AAV2.7m8-CAG-GFP转染效率明显高于出生后P2注射组。
实施例2AAV启动子改进载体妊娠期注射可分别高效靶向感染支持细胞与外毛细胞
1.方法
1.1分组：实验分为以下4组：胚胎期假手术组、胚胎期E12.5AAV2.7m8-CAG-GFP注射组、胚胎期E12.5 AAV2.7m8-OTOG-GFP注射组，胚胎期E12.5 AAV2.7m8-Prestin-GFP注射组。每组至少4个重复。AAV2.7m8-CAG-GFP、AAV2.7m8-OTOG-GFP及AAV2.7m8-Prestin-GFP滴度为5.0×1012，每个耳蜗给药1μl，假手术组给予1μl的无菌PBS溶液。
1.2手术操作、耳蜗取材及免疫荧光染色步骤同上。结果如图5-6所示，图5为免疫荧光染色拍照结果，图6为数据统计处理结果。
2结果：从图5-6不难看出，通过本实施例所述AAV启动子改进载体妊娠期注射可高效靶向感染内耳特定细胞，其中胚胎期E12.5注射AAV2.7m8-OTOG启动子可高效靶向感染支持细胞；E12.5 AAV2.7m8-Prestin可高效靶向感染外毛细胞。
虽然本发明公开披露如上，但本发明公开的保护范围并非仅限于此。本领域技术人员在不脱离本发明公开的精神和范围的前提下，可进行各种变更与修改，这些变更与修改均将落入本发明的保护范围。
Claims (9)
1.重组腺相关病毒在制备妊娠期内耳支持细胞治疗药物中的应用，所述重组腺相关病毒包含AAV衣壳和重组核酸分子；其特征在于，所述重组核酸分子包含Otogelin基因启动子的核苷酸序列，所述Otogelin基因的启动子核苷酸序列如SEQ ID NO：1所示。
2.重组腺相关病毒在制备妊娠期内耳外毛细胞治疗药物中的应用，所述重组腺相关病毒包含AAV衣壳和重组核酸分子；其特征在于，所述重组核酸分子包含Prestin基因启动子的核苷酸序列，所述Prestin基因的启动子核苷酸序列如SEQ ID NO：2所示。
3.根据权利要求1或2所述的应用，其特征在于，所述AAV衣壳来源于AAV2.7m8 衣壳蛋白。
4.根据权利要求1或2所述的应用，其特征在于，所述重组核酸分子包含选自AAV2型的AAV反向末端重复序列。
5.根据权利要求1或2所述的应用，其特征在于，重组腺相关病毒的构建过程为，将包含基因启动子的目标序列导入AAV质粒得到，所述AAV载体质粒含有调控元件WPRE。
6.根据权利要求5所述的应用，其特征在于，所述AAV质粒上表达有荧光蛋白。
7.根据权利要求1或2所述的应用，其特征在于，所述药物还包括一种或多种药学或生理学上可接受的载体、赋形剂和/或稀释剂。
8.根据权利要求1或2所述的应用，其特征在于，所述药物被配制用于胚胎内耳注射给药。
9.根据权利要求8所述的应用，其特征在于，所述药物给药时间为E12.5-E13.5。
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