CN117343153A - 一种用于治疗亨廷顿疾病的慢病毒样颗粒 - Google Patents
一种用于治疗亨廷顿疾病的慢病毒样颗粒 Download PDFInfo
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Abstract
本发明公开了一种用于治疗亨廷顿疾病的慢病毒样颗粒;所述慢病毒样颗粒VLP‑HD由VLP载体包裹Cas9蛋白和靶向HTT基因的gRNA组成的RNP复合体组成。感染细胞后,VLP‑HD释放出Cas9:gRNA RNP,可高效地靶向敲除突变的HTT基因,从源头阻止带有PolyQ的突变HTT蛋白的产生,从而达到对因治疗HD的效果。VLP‑HD可以是单靶点VLP,递送一种CRISPR/Cas9使mHTT基因发生移码突变的方式敲除突变基因,也可以是双靶点VLP,递送两种CRISPR/Cas9删除mHTT基因中诱导疾病发生的CAG核苷酸重复片段,杜绝PolyQ的产生。
Description
技术领域
本发明属于基因治疗领域,涉及一种用于治疗亨廷顿疾病的慢病毒样颗粒,尤其涉及一种适用于治疗亨廷顿舞蹈病的经改造的慢病毒样颗粒VLP。
背景技术
亨廷顿舞蹈病(Huntington's disease,HD)是一种有精神障碍、认知障碍的有舞蹈样不自主运动特征的遗传性神经***疾病。亨廷顿为常染色体显性遗传病,患者的4号常染色体上的HTT基因外显子1上CAG核苷酸重复增加诱导的疾病。HTT基因产物HTT蛋白在人类神经元中广泛表达,具有多种功能。健康人中,HTT基因中平均有17-20个CAG重复。当CAG重复次数达到40次以上时,形成多聚谷氨酰胺(PolyQ),导致形成的亨廷顿蛋白聚集。聚集蛋白进入细胞核引起基因转录失调,聚集蛋白在细胞质破坏蛋白质稳态,包括突触功能障碍,线粒体毒性和轴突转运率降低,导致亨廷顿疾病发生。HD患者的一般在中年出现认知和运动障碍,随着年龄的增长增加。
全世界HD患病率约为2.7/10万,在西方国家中,HD的患病率为每10万人10.6-13.7人。目前临床上的治疗方式为对症治疗,而没有对因治疗的方法。随着基因治疗技术的发展,基因突变引起的遗传性疾病有了对因治疗的可能。近期,一项应用AAV载体递送microRNA(rAAV5-miHTT)敲低突变HTT(mHTT)基因的疗法AMT-130被提出应用于亨廷顿疾病治疗,II期临床试验(NCT05243017)正在进行中,在欧洲早期亨廷顿成年患者中考察AMT-130的安全性和有效性。这种治疗方法治疗策略是应用AAV载体在神经元内长期表达靶向mHTT基因的microRNA,通过抑制mHTT mRNA翻译敲低突变蛋白。然而这种方法需要外源基因长期定植细胞内,外源基因稳定性效果未知,且有外源基因整合到宿主细胞基因组的风险。
发明内容
本发明的目的在于提供解决现有技术中治疗亨廷顿舞蹈病安全性与有效性问题,提供一种用于治疗亨廷顿疾病的慢病毒样颗粒。具体的,这种治疗方法是基因编辑治疗方法;是由VLP递送CRISPR RNP靶向敲除HTT基因实现的。
具体而言,本发明的目的是通过以下技术方案来实现的:
<第一方面>
本发明涉及一种慢病毒样颗粒,所述慢病毒样颗粒由VLP载体与Cas9蛋白和靶向HTT基因的gRNA组成的RNP复合体组成。该慢病毒样颗粒VLP构成是:改造的慢病毒外壳,包裹了Cas9蛋白和具有靶向性的gRNA组成的RNP复合体。改造的慢病毒外壳是将RNA结合蛋白与慢病毒GagPol长链蛋白融合后包装成的慢病毒外壳。
作为本发明的一个实施方案,表达VLP载体成分的基因序列分别位于①表达膜蛋白的质粒、②表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒、③表达慢病毒GagPol长链蛋白的质粒、④辅助质粒pRSV-Rev、⑤表达形成RNP所需Cas9蛋白的质粒和⑥表达含RNA结合蛋白所识别的RNA茎环结构的gRNA质粒上。
作为本发明的一个实施方案,表达形成RNP所需Cas9蛋白的质粒和表达含RNA结合蛋白所识别的RNA茎环结构的gRNA质粒可以为同一个质粒。即,表达VLP载体成分的基因序列分别位于①表达膜蛋白的质粒、②表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒、③表达慢病毒GagPol长链蛋白的质粒、④辅助质粒pRSV-Rev、⑤表达形成RNP所需Cas9蛋白和含RNA结合蛋白所识别的RNA茎环结构的gRNA质粒上。
作为本发明的一个实施方案,VLP载体成分中膜蛋白包括表达VSV-G、CD4识别蛋白、CD8识别蛋白、RD114、狒狒内源逆转录病毒膜蛋白或经过改造的有细胞感染特异性的膜蛋白。
作为本发明的一个实施方案,VSV-G的氨基酸序列为SEQ ID NO.7。
作为本发明的一个实施方案,经过改造的有细胞感染特异性的膜蛋白,即NVR-G蛋白,具有神经元感染特异性,NVR-G的氨基酸序列为SEQ ID NO.8。
作为本发明的一个实施方案,NVR-G的基因序列为SEQ ID NO.29。
作为本发明的一个实施方案,含RNA结合蛋白的慢病毒GagPol长链蛋白的氨基酸序列为SEQ ID NO.9。
作为本发明的一个实施方案,含RNA结合蛋白所识别的RNA茎环结构的gRNA骨架序列为SEQ ID NO.10。
作为本发明的一个实施方案,靶向HTT基因的gRNA上所携带的向导序列的靶向位点可以是HTT基因上的任意位点。携带1种Cas9:gRNA复合体的VLP靶向突变HTT(mHTT)基因产生Indel(***/缺失),发生移码突变,敲除突变HTT(mHTT)基因;携带2种Cas9:gRNA复合体的VLP靶向HTT基因分别在CAG重复序列的两端,切断CAG重复序列,防止产生引起聚集的蛋白。
作为本发明的一个实施方案,靶向HTT基因的gRNA序列为:
gRNA1:5’-GGCCTTCATCAGCTTTTCCA-3’、
gRNA4:5’-GAAGGACTTGAGGGACTCGA-3’、
gRNA7:5’-GACCCTGGAAAAGCTGATGA-3’、
gRNA8:5’-GGAGACCGCCATGGCGACCC-3’、
gRNA9:5’-CAGCTTTTCCAGGGTCGCCA-3’、
gRNA10:5’-CTTTTCCAGGGTCGCCATGG-3’
或gRNA11:5’-AGGCCTTCATCAGCTTTTCC-3’。
作为本发明的一个实施方案,靶向HTT基因的gRNA序列分别选择以下序列中的至少两种:
gRNA1:5’-GGCCTTCATCAGCTTTTCCA-3’、
gRNA2:5’-TGAGGAAGCTGAGGAGGCGG-3’、
gRNA3:5’-GGCGGCGGCTGAGGAAGCTG-3’、
gRNA4:5’-GAAGGACTTGAGGGACTCGA-3’、
gRNA5:5’-TTCATTGCCCCGGTGCTGAG-3’、
gRNA6:5’-GGCTGAGGAAGCTGAGGAGG-3’、
gRNA7:5’-GACCCTGGAAAAGCTGATGA-3’、
gRNA8:5’-GGAGACCGCCATGGCGACCC-3’、
gRNA9:5’-CAGCTTTTCCAGGGTCGCCA-3’、
gRNA10:5’-CTTTTCCAGGGTCGCCATGG-3’、
gRNA11:5’-AGGCCTTCATCAGCTTTTCC-3’、
gRNA12:5’-GAGTCGGCCCGAGGCCTCCG-3’、
gRNA13:5’-GGCGGCTGAGGAAGCTGAGG-3’、
gRNA14:5’-AGCGGGCCCAAACTCACGGT-3’、
gRNA15:5’-AGCAGCGGCTGTGCCTGCGG-3’、
gRNA16:5’-GGAAGCTGAGGAGGCGGCGG-3’、
gRNA17:5’-GCCGGGACAGGGAGCTGCAG-3’、
gRNA18:5’-TGAGGAGGCGGCGGCGGCGG-3’。
作为本发明的一个实施方案,靶向HTT基因的gRNA质粒为gRNA1、gRNA4、gRNA5、gRNA7-12中的至少一种与gRNA2、gRNA3、gRNA6、gRNA13-18中至少一种的质粒组合。
<第二方面>
本发明涉及一种前述慢病毒样颗粒的制备方法,所述方法包括如下步骤:将含有慢病毒载体基因组序列的质粒转染入病毒生产细胞,(收取上清液浓缩、纯化)制备而得;所述慢病毒载体基因组序列分别位于①表达膜蛋白的质粒、②表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒、③表达慢病毒GagPol长链蛋白的质粒、④辅助质粒pRSV-Rev,以及⑤表达形成RNP所需Cas9蛋白的质粒和⑥表达含RNA结合蛋白所识别的RNA茎环结构的HTTgRNA的质粒上,或以及⑤表达形成RNP所需Cas9蛋白和含RNA结合蛋白所识别的RNA茎环结构的HTT gRNA质粒上。
所述质粒的骨架部分(除所表达的核心序列外的部分)是不限定的。
<第三方面>
本发明涉及一种慢病毒样颗粒在制备治疗亨廷顿疾病的药物中的用途。
与现有技术相比,本发明具有如下有益效果:
1)本发明描述的VLP-HD是类慢病毒体包裹Cas9蛋白与gRNA形成的RNP,感染细胞后,VLP-HD可高效地靶向敲除突变的HTT基因,从源头阻止带有PolyQ的突变HTT蛋白的产生,从基因层面除去HD疾病的病因,从而达到对因治疗HD的效果。
2)本发明描述的VLP-HD可以是单靶点VLP,递送一种CRISPR/Cas9使mHTT基因发生移码突变的方式敲除基因,也可以是双靶点VLP,递送两种CRISPR/Cas9切断mHTT基因中诱导疾病发生的CAG核苷酸重复片段,杜绝PolyQ的产生。
3)因VLP-HD递送的是Cas9蛋白与gRNA形成的RNP结构,CRISPR/Cas9在细胞内行驶完基因切割的功能后被细胞内蛋白酶和核酸酶降解,因此VLP-HD的基因编辑具有瞬时性,瞬时存在的CRISPR/Cas9会大大降低脱靶的风险。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述,本发明的其它特征、目的和优点将会变得更明显:
图1为表达单gRNA的质粒图谱;
图2为VLP-HD的基因编辑效率;
图3为双靶点VLP-HD的基因编辑效果;
图4为双靶点VLP-HD敲除HTT蛋白示意图;
图5为VLP-HD的结构示意图;
图6为神经细胞特异性包膜蛋白在慢病毒感染不同来源细胞的效率;
图7为神经细胞特异性VLP-HD的基因编辑效果;
图8为VLP-HD-NVR-G改善亨廷顿小鼠运动协调和平衡能力数据。
具体实施方式
下面结合实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明,但不以任何形式限制本发明。应当指出的是,对本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干调整和改进。这些都属于本发明的保护范围。
实施例1:VLP-CRISPR RNP的构建(单靶点和双靶点)
在本实施例中,构建包含CRISPR RNP的VLP方法是:将表达膜蛋白的质粒(pMD.2G)、表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒(pMS2M-PH-GagPol-D64V,质粒序列如SEQ ID NO.1所示)、表达野生型慢病毒GagPol长链蛋白的质粒(pMDlg/PRRE-D64V,质粒序列如SEQ ID NO.2所示)、辅助质粒(pRSV-REV),表达Cas9蛋白的质粒(pCMV-2NLS-Cas9,质粒序列如SEQ ID NO.3所示)和表达含有MS2 stemloop的gRNA的质粒(pU6-Osp.gRNAMS2in,质粒序列如SEQ ID NO.4所示)共转染进293T细胞,收取上清液浓缩、纯化所得。用慢病毒滴度ELISA试剂盒测VLP p24浓度以定量。
构建靶向双位点的VLP-CRISPR RNP,在上述生产质粒的基础上增加一个靶向第二个位点的pU6-Osp.gRNAMS2in,即共2个表达gRNA的质粒;或将2个gRNA的表达盒放到同一个质粒上,即将2个gRNA序列的表达盒(U6-Osp.gRNAMS2in)顺式或者反式串联后***质粒pU6-Osp.gRNAMS2in中,替代原来的单个拷贝表达盒。
实施例2、设计gRNA序列并预测基因编辑效率
将HTT基因输入CRISPRon(v1.0)网站工具,设计gRNA并预测基因编辑效率,在所得的结果中筛选gRNA。筛选HTT基因外显子1上CAG重复区域5’-方向的gRNA序列,如gRNA1、gRNA4、gRNA 7-11,这些gRNA可用于构建单靶点的VLP-HD,亦可以与筛选出的CAG重复区域3’-方向的gRNA(如gRNA2、gRNA3、gRNA6和gRNA13-18)联合组成双靶点VLP-HD。筛选5’-UTR区域的gRNA序列(如gRNA5和gRNA12),与gRNA2、gRNA3、gRNA6或gRNA13-18联合组成双靶点VLP-HD。各gRNA的预测基因编辑效率见下表:
不同gRNA位点的基因编辑效率不同,在筛选的gRNA序列中除了gRNA9-11,软件预测效率都>50%。
实施例3:单靶点VLP-HD的HTT基因编辑效率和验证HTT gRNA的效率
1.生产VLP-HD。VLP-HD携带靶向HTT基因的Cas9:gRNA RNP。将质粒pMD.2G、pMS2M-PH-GagPol-D64V、pMDlg/PRRE-D64V、pRSV-REV、pCMV-2NLS-Cas9和pU6-Osp.gRNAMS2in-HTT共转染进293T细胞,收取上清液浓缩、纯化得到VLP颗粒。用慢病毒滴度ELISA试剂盒测定VLP p24浓度。
构建质粒pU6-Osp.gRNAMS2in-HTT。选取几种靶向HTT基因的gRNA序列,分别为gRNA1:5’-GGCCTTCATCAGCTTTTCCA-3’(PAM:GGG);gRNA2:5’-TGAGGAAGCTGAGGAGGCGG-3’(PAM:CGG);gRNA3:5’-GGCGGCGGCTGAGGAAGCTG-3’(PAM:AGG);gRNA4:5’-GAAGGACTTGAGGGACTCGA-3’(PAM:AGG);gRNA5:
5’-TTCATTGCCCCGGTGCTGAG-3’(PAM:CGG)gRNA12:5’-GAGTCGGCCCGAGGCCTCCG-3’(PAM:GGG);gRNA15:5’-AGCAGCGGCTGTGCCTGCGG-3’(PAM:CGG)。
将上述gRNA序列分别加入质粒pU6-Osp.gRNAMS2in的启动子和gRNA骨架之间,构建成质粒pU6-Osp.gRNAMS2in-HTT1、pU6-Osp.gRNAMS2in-HTT2、pU6-Osp.gRNAMS2in-HTT3、pU6-Osp.gRNAMS2in-HTT4、pU6-Osp.gRNAMS2in-HTT5、pU6-Osp.gRNAMS2in-HTT12、pU6-Osp.gRNAMS2in-HTT15,携带gRNA的质粒图谱示意图见图1(图中所示gRNA位置可替换为不同gRNA),质粒可以根据核苷酸序列通过基因合成得到。使用这些质粒生产的VLP分别为VLP-HD1、VLP-HD2、VLP-HD3、VLP-HD4、VLP-HD5、VLP-HD12和VLP-HD15,其中VLP-HD1、VLP-HD4和VLP-HD5为具有突变HTT基因中polyQ敲除功能的单靶点VLP-HD;VLP-HD2、VLP-HD3、和VLP-HD15需要与其它任意一种VLP-HD共同使用才具有polyQ基因敲除功能。
2.验证VLP-HD的基因编辑效率。
种293T细胞到96孔板,2×104/孔,培养24h感染各种VLP-HD,50ng/孔。感染72h后收细胞提基因组。用引物(F:5’-agggctgtcaatcatgctgg-3’和R:5’-ggttgctgggtcactctgtc-3’)做PCR扩增目的位点周围的DNA片段,Sanger测序,TIDE(https://tide.nki.nl/)检测Indel。如图2所示,最高的基因编辑效率接近90%。与预测的gRNA序列的基因编辑效率相比:VLP-HD 1-4的基因编辑效率与预测的gRNA1-4的效率有差异而没有较大差距;VLP-HD5的基因编辑效率(89.3%)远高于预测的gRNA15的效率(68.594%);VLP-HD12和VLP-HD12(Indel(***或确实)效率分别为5.8%和8.5%)的基因编辑效率远低于预测的gRNA12和gRNA15效率(分别为60.424%和62.264%,见实施例2)。
实施例4:双靶点VLP-HD敲除HTT基因
1.生产双靶点VLP-HD。VLP-HD携带靶向两个HTT基因位点的CRISPR RNP。将质粒pMD.2G、pMS2M-PH-GagPol-D64V、pMDlg/PRRE-D64V、pRSV-REV、pCMV-2NLS-Cas9和2个表达HTT gRNA的pU6-Osp.gRNAMS2in-HTT质粒共转染进293T细胞,收取上清液浓缩、纯化得到VLP颗粒。用慢病毒滴度ELISA试剂盒测定VLP p24浓度。
2个表达HTT gRNA的质粒组合为gRNA1+gRNA2、gRNA1+gRNA3、gRNA1+gRNA6、gRNA5+gRNA2、gRNA5+gRNA3、gRNA5+gRNA6。
pU6-Osp.gRNAMS2in-HTT5表达gRNA5:5’-TTCATTGCCCCGGTGCTGAG-3’(PAM:CGG);pU6-Osp.gRNAMS2in-HTT6表达gRNA6:5’-GGCTGAGGAAGCTGAGGAGG-3’(PAM:CGG)质粒可以根据核苷酸序列通过基因合成得到。
2.验证双靶点VLP-HD的基因切割情况。
种HeLa细胞到96孔板,2×104/孔,培养24h感染VLP-HD,100ng/孔。感染72h后收细胞提基因组。用引物(F:5’-GGACGGGTCCAAGATGGACG-3’和R:5’-caaactcacGGTCGGTGCAG-3’)做PCR扩增目的位点周围的DNA片段做琼脂糖凝胶核酸电泳。因是双靶点切割,将移除一段含有CAG核苷酸重复的片段,预期产生2条DNA片段,一条是原片段,另一条是切割后片段。gRNA1+gRNA2预期产生410bp+282bp条带;gRNA1+gRNA3预期产生410bp+273bp条带、gRNA1+gRNA6预期产生410bp+279bp条带、gRNA5+gRNA2预期产生410bp+208bp条带、gRNA5+gRNA3预期产生410bp+199bp条带、gRNA5+gRNA6预期产生410bp+205bp条带。如图3,VLP-HD感染细胞后切割DNA条带与预期相符,切割掉了带有CAG核苷酸重复的片段,证明了VLP-HD的基因编辑能力。当VLP-HD应用到mHTT基因编辑时也可以切割掉所有的CAG核苷酸片段,从而防止产生PolyQ,引起蛋白聚集。
实施例5:双靶点VLP-HD敲除HTT蛋白
如实施例3中的方法生产VLP-HD(gRNA1+gRNA2)、VLP-HD(gRNA1+gRNA3),并感染HeLa细胞,感染5天后收取细胞沉淀,用RIPA(含1% PMSF)裂解。加10%loading buffer金属浴98℃处理10min制备样品后进行Western Blot(蛋白质印迹法)。过夜敷一抗anti-Huntingtin抗体(3E10)和anti-β-actin,室温敷二抗anti-Mouse IgG后加显影液显影。图4显示了VLP-HD切割HTT基因后,HTT蛋白被敲除。
VLP-HD的结构示意图如图5所示,由图5可知,VLP-HD为类慢病毒体外壳(VLP)包装了Cas9:gRNA RNP的结构,其中RNA不限于一种,VLP中无传统慢病毒的载体RNA基因。
实施例6:NVR-G包膜蛋白具有神经细胞感染特异性
1.构建表达融合蛋白NVR-G的质粒pNVR-G,将表达NVR-G的基因(序列为SEQ IDNO.29)序列替换pMD.2G质粒中VSV-G的基因,构成质粒pNVR-G,质粒序列如SEQ ID NO.5所示。
2.生产包膜含NVR-G蛋白的表达GFP的慢病毒,方法是:将表达膜蛋白的质粒(pNVR-G)、表达野生型慢病毒GagPol长链蛋白的质粒(pMDlg/PRRE-D64V,质粒序列如SEQID NO.2所示)、辅助质粒(pRSV-REV),表达GFP蛋白的质粒(pCCL-PGK-eGFP,质粒序列如SEQID NO.6所示)共转染进293T细胞,收取上清液浓缩、纯化所得。用慢病毒滴度ELISA试剂盒测VLP p24浓度以定量。
3.生产包膜含VSV-G蛋白的表达GFP的慢病毒,方法是:将表达膜蛋白的质粒(pMD.2G)、表达野生型慢病毒GagPol长链蛋白的质粒(pMDlg/PRRE-D64V)、辅助质粒(pRSV-REV),表达GFP蛋白的质粒(pCCL-PGK-eGFP)共转染进293T细胞,收取上清液浓缩、纯化所得。用慢病毒滴度ELISA试剂盒测VLP p24浓度以定量。
4.分别接种THP-1(人单核细胞白血病细胞)、SH-SY5Y(人神经母细胞瘤细胞)、HEB(人脑星形胶质细胞)和HeLa细胞系到48孔板,4×104细胞/孔。24h后每种细胞分别感染20ng含NVR-G包膜蛋白的慢病毒和含VSV-G包膜蛋白的慢病毒。48h后使用流式分析仪(BD&LSR Fortessa)检测各组表达GFP的细胞比例。
实验结果如图6所示,含VSV-G包膜蛋白的慢病毒具有感染广谱性,可感染多种细胞且感染效率普遍较高,含NVR-G包膜蛋白的慢病毒只有感染神经系细胞SH-SY5Y时具有较高的感染效率。VLP-HD需要编辑神经元细胞的mHTT基因发挥治疗作用,NVR-G包膜蛋白包被的慢病毒颗粒可特异性感染神经细胞,因此使用NVR-G包膜蛋白生产治疗亨廷顿的慢病毒颗粒VLP-HD-NVR-G可具有一定的神经元细胞感染靶向性。
实施例7:VLP-HD-NVR-G编辑HTT基因
如实施例3中的方法,使用质粒pNVR-G替换质粒pMD.2G生产VLP-HD-NVR-G(gRNA1+gRNA2)。使用293T细胞验证基因编辑效率。
种293T细胞到96孔板,2×104细胞/孔,24h后感染100ng VLP-HD-NVR-G(gRNA1+gRNA2),感染72h后收细胞,提取基因组,用引物(F:5’-GGACGGGTCCAAGATGGACG-3’和R:5’-caaactcacGGTCGGTGCAG-3’)做PCR扩增目的位点周围的DNA片段做琼脂糖凝胶核酸电泳。因是双靶点切割,将移除一段含有CAG核苷酸重复的片段,预期产生2条DNA片段,一条是原片段,另一条是切割后片段。gRNA1+gRNA2预期产生410bp+282bp条带。如图7所示,基因编辑后的样品(图7中1+2泳道)的PCR片段显示两条预期条带。
实施例8、VLP-HD-NVR-G治疗HD小鼠有效性试验
为了研究VLP-HD缓解亨廷顿疾病异常表型的潜力,使用HD疾病特异性小鼠模型进行了体内疗效和安全性研究,该模型将内源性小鼠Htt的外显子1替换为人类突变Htt的外显子1,CAG重复约190次。小鼠模型的优点之一是它具有高水平的突变亨廷顿蛋白的表达,这使得HD症状的快速发作和进展成为可能。这使得它成为研究疾病早期阶段和测试的有用模型。亨廷顿小鼠的运动和平衡能力失调,运动协调和平衡能力可以通过平衡木试验用于评估。老鼠在一根有网格的横梁上行走1米。行走中小鼠爪子有可能掉落或滑到钢丝之间,如果发生这种情况,就会被记录为一个错误。记录总步数和总时间。通过颅内注射方式注射VLP-HD-NVR-G到8周龄的亨廷顿小鼠,对照组注射PBS,4周后分析结果。数据显示,在平衡木测试中,未处理的HD小鼠(HD Model)比野生型(WT)小鼠表现出明显更多的脚滑错误,差异具有显著性(**P<0.01)。而VLP-HD-NVR-G处理后,小鼠表现与WT小鼠无显著性差异(n.s.)(图8)。说明VLP-HD可以改善HD小鼠的运动协调和平衡能力。
以上对本发明的具体实施例进行了描述。需要理解的是,本发明并不局限于上述特定实施方式,本领域技术人员可以在权利要求的范围内做出各种变形或修改,这并不影响本发明的实质内容。
Claims (10)
1.一种慢病毒样颗粒,其特征在于,所述慢病毒样颗粒由VLP载体包裹Cas9蛋白和靶向HTT基因的gRNA组成的RNP复合体组成。
2.根据权利要求1所述的慢病毒样颗粒,其特征在于,表达VLP载体成分的基因序列分别位于①表达膜蛋白的质粒、②表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒、③表达慢病毒GagPol长链蛋白的质粒、④辅助质粒pRSV-Rev、⑤表达形成RNP所需Cas9蛋白的质粒和⑥表达含RNA结合蛋白所识别的RNA茎环结构的gRNA质粒上。
3.根据权利要求1所述的慢病毒样颗粒,其特征在于,VLP载体成分中膜蛋白包括表达VSV-G、CD4识别蛋白、CD8识别蛋白、RD114、狒狒内源逆转录病毒膜蛋白或经过改造的有细胞感染特异性的膜蛋白;VSV-G的氨基酸序列为SEQ ID NO.7;经过改造的有细胞感染特异性的膜蛋白,即NVR-G蛋白,具有神经元感染特异性,NVR-G的氨基酸序列为SEQ ID NO.8。
4.根据权利要求1所述的慢病毒样颗粒,其特征在于,含RNA结合蛋白的慢病毒GagPol长链蛋白的氨基酸序列为SEQ ID NO.9;含RNA结合蛋白所识别的RNA茎环结构的gRNA骨架序列为SEQ ID NO.10。
5.根据权利要求1所述的慢病毒样颗粒,其特征在于,靶向HTT基因的gRNA上所携带的向导序列的靶向位点是HTT基因上的任意位点。
6.根据权利要求1所述的慢病毒样颗粒,其特征在于,靶向HTT基因的gRNA序列为gRNA1:5’-GGCCTTCATCAGCTTTTCCA-3’、
gRNA4:5’-GAAGGACTTGAGGGACTCGA-3’、
gRNA7:5’-GACCCTGGAAAAGCTGATGA-3’、
gRNA8:5’-GGAGACCGCCATGGCGACCC-3’、
gRNA9:5’-CAGCTTTTCCAGGGTCGCCA-3’、
gRNA10:5’-CTTTTCCAGGGTCGCCATGG-3’
或gRNA11:5’-AGGCCTTCATCAGCTTTTCC-3’。
7.根据权利要求1所述的慢病毒样颗粒,其特征在于,靶向HTT基因的gRNA序列分别选择以下序列中的至少两种:
gRNA1:5’-GGCCTTCATCAGCTTTTCCA-3’、
gRNA2:5’-TGAGGAAGCTGAGGAGGCGG-3’、
gRNA3:5’-GGCGGCGGCTGAGGAAGCTG-3’、
gRNA4:5’-GAAGGACTTGAGGGACTCGA-3’、
gRNA5:5’-TTCATTGCCCCGGTGCTGAG-3’、
gRNA6:5’-GGCTGAGGAAGCTGAGGAGG-3’、
gRNA7:5’-GACCCTGGAAAAGCTGATGA-3’、
gRNA8:5’-GGAGACCGCCATGGCGACCC-3’、
gRNA9:5’-CAGCTTTTCCAGGGTCGCCA-3’、
gRNA10:5’-CTTTTCCAGGGTCGCCATGG-3’、
gRNA11:5’-AGGCCTTCATCAGCTTTTCC-3’、
gRNA12:5’-GAGTCGGCCCGAGGCCTCCG-3’、
gRNA13:5’-GGCGGCTGAGGAAGCTGAGG-3’、
gRNA14:5’-AGCGGGCCCAAACTCACGGT-3’、
gRNA15:5’-AGCAGCGGCTGTGCCTGCGG-3’、
gRNA16:5’-GGAAGCTGAGGAGGCGGCGG-3’、
gRNA17:5’-GCCGGGACAGGGAGCTGCAG-3’、
gRNA18:5’-TGAGGAGGCGGCGGCGGCGG-3’。
8.根据权利要求7所述的慢病毒样颗粒,其特征在于,靶向HTT基因的gRNA质粒为gRNA1、gRNA4、gRNA5、gRNA7-12中的至少一种与gRNA2、gRNA3、gRNA6、gRNA13-18中至少一种的质粒组合。
9.一种根据权利要求1所述的慢病毒样颗粒的制备方法,其特征在于,所述方法包括如下步骤:表达膜蛋白的质粒、表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒、表达慢病毒GagPol长链蛋白的质粒、辅助质粒pRSV-Rev,与表达形成RNP所需Cas9蛋白的质粒和表达含RNA结合蛋白所识别的RNA茎环结构的HTT gRNA的质粒,或与表达形成RNP所需Cas9蛋白和含RNA结合蛋白所识别的RNA茎环结构的HTT gRNA质粒共转染进病毒生产细胞,收取上清液浓缩、纯化,即得。
10.一种根据权利要求1所述的慢病毒样颗粒在制备治疗亨廷顿疾病的药物中的用途。
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