CN115969791A - 抑制Rapsyn基因表达的脂质体复合物及其应用 - Google Patents
抑制Rapsyn基因表达的脂质体复合物及其应用 Download PDFInfo
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Abstract
本发明涉及基因治疗药物技术领域,具体涉及一种能靶向白血病细胞荷载抑制Rapsyn基因表达的核酸脂质体复合物和其在制备预防和治疗白血病药物中应用。本发明提供的脂质体复合物具有靶头DSPE‑PEG2000‑scFv,能选择性靶向CD79b+白血病细胞,有效递送siRapsyn序列进入白血病细胞,不被核酸酶降解,使荷载的siRNA进入细胞质,抑制Rapsyn基因表达,具有更高的生物利用率和安全性,能有效抑制白血病细胞的增殖和肿瘤形成、延长白血病动物模型的生存期,达到治疗白血病的目的。本发明为治疗白血病药物提供了新的选择。
Description
技术领域
本发明属于基因治疗药物技术领域,具体涉及一种抑制Rapsyn基因表达的脂质体复合物及其应用。
背景技术
白血病是一类造血干细胞恶性克隆性疾病。克隆性白血病细胞因为增殖失控、分化障碍、凋亡受阻等机制在骨髓和其他造血组织中大量增殖累积,并浸润其他非造血组织和器官,同时抑制正常造血功能。临床上常将白血病分为淋巴细胞白血病、髓细胞白血病、混合细胞白血病等。随着医学的进步,每种类型的白血病都有了更加精准的治疗策略。目前主要有下列几类治疗方法:化学治疗﹑放射治疗﹑靶向治疗、免疫治疗、干细胞移植等。
虽然移植可以获得较好的生存效果,但是移植物抗宿主病等并发症可能严重影响患者的生活质量。因此,选择性免疫治疗和各种分子靶向治疗是将来治愈白血病的希望。嵌合抗原受体(CAR)-T细胞疗法是癌症免疫细胞疗法中一个进步的新支柱。它在B细胞白血病或淋巴瘤患者中产生了显著的临床反应。不幸的是,CAR-T细胞疗法的主要障碍是严重威胁生命的毒性,例如细胞因子释放综合征和有限的抗肿瘤功效(JOGALEKAR M P andRAJENDRAN R L.Front Immunol,2022,13:925985.)。因此,发现参与白血病发病机制的其他关键因素,以此为基础设计靶向治疗策略至关重要。
前期研究发现Rapsyn在白血病外周血中特异性高表达,该过表达能够促进白血病细胞的恶性增殖,并且与白血病病人短的预后生存时间有关。这些研究成果为Rapsyn可作为白血病治疗的潜在靶点提供了有力的理论依据。
然而Rapsyn在神经肌肉组织中广泛表达,与神经递质的传递有着重要作用,如果缺失会造成重症肌无力等严重疾病(LI L and CAO Y.Neuron,2016,92(5):1007-19.),用药安全性存在很大问题。
发明内容
本发明针对现有治疗技术不足,提供了一种能靶向白血病细胞荷载抑制Rapsyn基因表达核酸的脂质体复合物。本发明提供的脂质体复合物具有靶头DSPE-PEG2000-scFv,能选择性靶向CD79b+白血病细胞,有效递送抑制Rapsyn基因表达核酸进入白血病细胞,不被核酸酶降解,使荷载的siRNA进入细胞质,抑制Rapsyn基因表达。
本发明具体技术方案如下:
一种抑制Rapsyn基因表达的脂质体复合物,所述脂质体复合物具有靶头DSPE-PEG2000-scFv,脂质材料为赖氨酸谷氨酸双油醇酯OA2-Glu-Lys;所述脂质体复合物荷载有抑制Rapsyn基因表达的核酸。
Rapsyn基因核苷酸序列如SEQ ID No:49所示。
所述anti-CD79b-scFv(简称scFv)(氨基酸序列如SEQ ID No:48,USA20200207852)。本发明对anti-CD79b-scFv的氨基酸进行翻译,并对密码子优化使其适用于大肠杆菌的表达。构建了带有助溶蛋白标签MBP和scFv基因序列的pET28a质粒,提高了scFv的可溶性表达,通过异源表达纯化得到具有高活性的anti-CD79b-scFv。
所述靶头DSPE-PEG2000-scFv为DSPE-PEG2000-Mal胶束与scFv混合制得,优选DSPE-PEG2000-Mal胶束与scFv摩尔比为1:1-50:1。
赖氨酸谷氨酸双油醇酯OA2-Glu-Lys是中国专利文献CN111087317A公开的不饱和阳离子脂质衍生物:
OA2-Glu-Lys具有良好的生物相容性和可降解性;其正电性头基可以可稳定复合DNA,提高其在递送过程中的稳定性;且其尾链引入不同的不饱和键,可以通过酸性条件下的膜融合作用提高DNA在胞质中的释放;最终提高DNA的基因转染效率。
本发明所述的脂质体复合物荷载的抑制Rapsyn基因表达的核酸包括靶向Rapsyn的反义寡核苷酸、siRNA、miRNA、shRNA、核酸适配体、转录激活RNA中的一种或几种。优选siRNA。
本发明一个具体的示例,所述siRNA靶序列核苷酸序列如SEQ ID No:1~14所示。所述的siRNA序列中经过GC含量和脱靶率评估,以及在细胞内基因和蛋白水平敲低的验证,基因沉默效率达到80%以上。优选siRNA为
siRNA1:Sense(5’-3’)CAUGAAGCCUGGCUUUGUA(SEQ ID No:15)。
Antisense(3’-5’)UACAAAGCCAGGCUUCAUG(SEQ ID No:16)。
siRNA2:Sense(5’-3’)CGAGAAGCUGUGCGAGUUU(SEQ ID No:17)。
Antisense(3’-5’)AAACUCGCACAGCUUCUCG(SEQ ID No:18)。
siRNA3:Sense(5’-3’)GCGCUAUGCCCACAACAAU(SEQ ID No:19)。
Antisense(3’-5’)AUUGUUGUGGGCAUAGCGC(SEQ ID No:20)。
本发明一个优选的方案,scFv在脂质体复合物的摩尔百分比不高于0.06%。
本发明一个优选的方案,所述脂质材料和核苷酸的氮磷比不低于3:1。
本发明另一目的在于提供本发明所述抑制Rapsyn基因表达的脂质体复合物在制备预防或治疗Rapsyn基因异常表达引起的疾病药物中的用途。所述疾病为白血病,包括淋巴细胞型血液类肿瘤和骨髓细胞型血液类肿瘤,所述淋巴细胞型血液类肿瘤为急性淋巴细胞型血病、急性原始淋巴细胞型白血病、B细胞型白血病、T细胞型白血病、何杰金氏淋巴瘤、非何杰金氏淋巴瘤、毛状细胞淋巴瘤或Burkett氏淋巴瘤;所述骨髓细胞型血液类肿瘤为急性骨髓细胞型白血病、慢性骨髓细胞型白血病、骨髓发育不全症或早幼粒细胞型白血病。
本发明另一目的在于提供一种脂质体制剂,包含本发明所述抑制Rapsyn基因表达的脂质体复合物及药学上可接受的基因药物递送载体。所述载体包括反转录病毒载体和腺病毒载体,非病毒法包括脂质体、显微注射法、磷酸钙沉淀法等。
本发明所述的脂质体复合物可通过不同途径对人体给药,具体的给药途径包括口服、非胃肠道给药、口腔吸入、透过皮肤给药、直肠给药、鼻腔给药、舌下给药、面颊给药、阴道给药及通过植入性容器给药;非胃肠道给药又包括在皮下、静脉内、肌肉内、关节内、滑膜腔内、胸骨内、鞘内、肝内、损伤部位内和颅内注射或浸渗。
本发明所述的脂质体复合物在用于人体进行肿瘤以及相关疾病治疗时,所用的剂量受多种因素的影响,这些因素包括年龄、体重、健康状况、性别、种族、饮食习惯、给药时间、排尿频率及是否使用其它药物等等。
本发明优点:
(1)目前,临床上尚没有靶向性强的沉默Rapsyn表达的抑制剂。本发明为了提高药物的安全性,在GEO数据库进行了分析,结果发现CD79b在白血病细胞表面特异性高表达,进而选择了CD79b作为本发明药物设计中的靶标。针对CD79b设计表达纯化了anti-CD79b-scFv,将scFv掺入荷载有抑制Rapsyn基因表达的核酸的脂质体中,实现选择性靶向结合白血病细胞的目的。
(2)本发明提供的脂质体复合物在体外细胞中能高效转染,并能成功递送siRNA到细胞质中。
(3)本发明开发的抑制Rapsyn基因表达的脂质体复合物,能特异性识别白血病细胞,抑制Rapsyn基因在白血病细胞中的表达,抑制肿瘤细胞的生长,有效地延长白血病细胞异源移植小鼠模型的生存期,为治疗白血病的药物提供了一种新的选择。
附图说明
下面结合附图和实施例对本发明作进一步说明。
图1为实施案例1中siRNA有效序列的筛选结果其中:(A)为qRT-PCR检测三种siRNA序列对K562细胞Rapsyn基因mRNA水平的沉默作用;(B)为蛋白免疫印迹检测三种siRNA序列对K562细胞Rapsyn基因表达的抑制作用。
图2为实施案例2中OA2阳离子脂质体的制备和表征,其中:(A)为按不同N/P荷载siRNA后的的琼脂糖凝胶电泳图;(B)为本发明的阳离子脂质体/siRNA二元复合物在不同氮磷比下的粒径和电位。
图3为实施案例3中本发明的siRNA-OA2脂质体复合物在体外对四种白血病细胞的作用。
图4为白血病细胞表面分子靶标的选择与验证结果。
图5为实施案例5中anti-CD79b-scFv的表达和纯化以及其活性检测结果。其中,(A)10%SDS-PAGE检测scFv表达纯化的情况;(B)ELISA实验检测scFv不同浓度下与四种白血病细胞结合能力。
图6为实施案例6中scFv-siRNA脂质体复合物的制备与表征,其中:(A)流式细胞术检测不同比例scFv制备的靶向siRNA脂质体复合物递送Fam-siRNA至K562细胞的效率;(B)为本发明的scFv-OA2脂质体的透射电子显微镜观察图(比例尺:50μm);(C)为本发明的scFv-OA2脂质体在氮磷比为5的情况下荷载siRNA后的的琼脂糖凝胶电泳图。
图7为实施案例7中本发明的scFv-siRNA脂质体复合物在K562细胞系皮下瘤模型中对癌细胞增殖和肿瘤形成的抑制作用,分为生理盐水组、siNC-scFv-OA2组、siRNA-OA2组和siRNA-scFv-OA2,其中,(A)为小鼠体重;(B)为瘤图片;(D)为瘤体积;(C)为小鼠瘤重。
图8为实施案例8中本发明的siRNA-scFv-OA2脂质体复合物治疗K562细胞异源移植小鼠模型的生存期结果。
具体实施方式
以下通过实施例说明本发明的具体步骤,但不受实施例限制。
在本发明中所使用的术语,除非另有说明,一般具有本领域普通技术人员通常理解的含义。下面结合具体实施例并参照数据进一步详细描述本发明。应理解,这些实施例只是为了举例说明本发明,而非以任何方式限制本发明的范围。在以下实施例中,未详细描述的各种过程和方法是本领域中公知的常规方法。下面通过实施例具体说明本发明的内容:
实施例1:siRNA的筛选
针对Rapsyn基因的同源区域,用软件设计了14种针对Rapsyn基因的siRNA靶序列(SEQ ID No:1~14),并相应地设计了siRNA序列(SEQ ID No:15~42),从中根据GC含量和脱靶率评估,优选siRNA-3,再结合qRT-PCR和Western Blot实验进行细胞内验证,筛选出最优序列。14种靶序列如下:
取对数生长的K562细胞、MEG-01细胞和KU812细胞,调整细胞密度为2.5×105个/ml的细胞悬液,接种于6孔板,每孔2ml,每孔分别加入2μg的siRNA1~3,置于37℃,5%CO2恒温培养箱内培养48h。48h后收取细胞,PBS洗三遍,提取细胞总RNA或总蛋白。设置空白对照组、siNC组和siRNA1-3组。
引物序列如下:
检测结果如图1所示,qRT-PCR和蛋白免疫印迹结果显示siRNA3序列不论是在mRNA转录水平,还是蛋白的翻译水平都能有效沉默Rapsyn的表达。所以后续研究选择siRNA3作为siRNA有效序列。
实施例2:阳离子脂质体的制备与表征
空白OA2阳离子脂质体制备方法:称取适量阳离子脂质OA2(赖氨酸谷氨酸双油醇酯OA2-Glu-Lys参照中国专利文献CN111087317A公开的方法制备),用含有3ml氯仿和2ml甲醇的混合溶剂溶解,40℃减压旋蒸除去溶剂,真空干燥过夜。加入适量去离子水,在37℃水合30min,将脂质体悬液依次过0.8μm,0.45μm,0.22μm微孔滤膜各11次,即得到空白OA2阳离子脂质体溶液,置于4℃保存备用。使用DLS测量空白阳离子脂质体的粒径、电位和多分散系数(Polydispersity Index,PDI),结果如表1所示。
表1本发明的空白阳离子脂质体的性质(n=3)
上述实验数据表明,本发明的各类脂质体粒径在50~150nm之间,符合基因载体的粒径要求;PDI均小于0.3,表明通过薄膜挤出法制备的脂质体粒径均一;表面电位在+20~+50mV之间,表明所制备的阳离子脂质体可以与负电性的核酸药物通过静电相互作用结合并有效压缩siRNA,符合作为基因递送载体的要求。
siRNA-OA2阳离子脂质体二元复合物的制备:固定siRNA(siRapsyn3)的质量,取相应量的siRNA体积并用DEPC水或无RNA酶水稀释至50μL,按N/P为5取对应的阳离子脂质体量并稀释至50μL,并将稀释好的质粒加入至稀释的阳离子脂质体中(50μL+50μL)混合均匀后,室温静置孵育30min,得到阳离子脂质体/siRNA二元复合物。通过琼脂糖凝胶电泳实验考察各阳离子脂质体对siRNA的荷载能力(如图2A所示),并使用DLS测定各二元复合物的粒径和电位(如图2B所示)。上述实验结果表明,本发明的阳离子脂质体OA2在N/P大于3时均能稳定荷载siRNA不发生泄漏,且二元复合物的粒径在100~200nm之间,Zeta电位在+10~+30mV之间,可进一步用于细胞转染实验。
实施例3:本发明的siRNA-OA2脂质体复合物在体外对CML细胞的抑制作用
检测本发明的siRNA-OA2脂质体复合物对白血病细胞增值的作用。取对数生长的K562细胞、MEG-01细胞、KU812细胞、Jurkat细胞,每孔5×104个细胞接种于96孔板,设置空白组、OA2组和siRNA-OA2组分别处理三种细胞,分别于转染细胞24h、48h、72h、96h后用CCK8法检测细胞增殖情况。检测结果如图3,相对于空白对照组和NC组,不同时间点敲低Rapsyn后细胞活力都显著降低,由此可知敲低Rapsyn能显著抑制白血病细胞的生长增殖。
实施例4:白血病细胞CD79b靶标的选择与验证
在GEO DataSets数据库中找到有关白血病患者和正常人外周血细胞的全基因组表达矩阵。利用GEO2R在线分析得到差异基因数据集。再利用数据库(https://www.genecards.org/)逐个分析数据组中差异高表达的基因(LogFc>1),筛选出高表达且分布在细胞膜表面的基因—CD79b。
白血病细胞是否表达CD79b的验证:收集对数生长期的K562细胞、MEG-01细胞、KU812细胞、Jurkat细胞、KG-1细胞、Sup-B15细胞,提取细胞总蛋白,Western Blot检测CD79b和内参GAPDH的表达结果如图4所示,白血病细胞均表达CD79b,因此后续选择CD79b蛋白作为药物的靶标。
实施例5:anti-CD79b-scFv的表达与纯化
构建MBP-scFv-pET28a表达质粒:利用Gibson组装将MBP助溶蛋白氨基酸序列插入到pET28a,再将经优化后由生工合成的anti-CD79b-scFv的基因序列(SEQ ID No:47)插入到质粒中,并在C端加上His标签。得到MBP-scFv-pET28a表达质粒。
重组产物转化:取100μl BL21于冰上自然融化,然后加入重组连接产物,混合后冰浴30min。42℃热激90s,再冰浴2min。移至1ml LB中,在37℃振荡摇床中培养45min。45min后离心弃上清,取800μl左右涂含有Kan的LB固体培养板上。倒置于37℃恒温培养箱过夜培养。
质粒测序验证:随机挑选5个阳性单克隆菌落于5ml含Kan抗性的LB中,在37℃、220rpm振荡培养箱中培养8h。收菌液,提取质粒送生工测序,另平行保存1ml菌液于-80℃的冰箱。将编号测序无误的质粒进行后续的蛋白表达。
将保存的菌液接种于含Kan的LB中,37℃,220rpm在振荡摇床中培养过夜,第二天取200ul菌液接种至200ml含Kan的LB中,继续放置在37℃、220rpm振荡摇床中培养,至吸光度近0.8,加入IPTG(1M)200ul/200ml LB,在20℃,200rpm条件下振荡培养18h。然后收菌液,20ml的平衡液混合后用120w,超3s间6s,15min的条件超声破菌。离心取上清,先用镍柱纯化,目的蛋白在300mM咪唑溶液中,然后用糊精琼脂糖二次纯化,得到目的蛋白。用10%SDS-PAGE检测表达纯化的纯度,检测结果如图5A显示,一轮纯化还有些许杂带,再二轮纯化后能得到95%以上纯度的scFv。
scFv活性检测:将β-D-多聚赖氨酸溶液100ul 0.1mg/mL提前24小时加入96孔板中,用水洗净晾干。K562细胞、MEG-01细胞、KU812细胞和Jurkat接种于96孔板,每孔1×105个细胞,置于37℃、5% CO2培养箱中培养24h。24h后弃上清,用4%多聚甲醛固定15min,然后5% BSA溶液封闭30min后,在实验孔中加入不同浓度的anti-CD79b-scFv,孵育2h。弃上清,PBS洗三遍,加入辣根过氧化物酶标记的抗his标签单克隆抗体,37℃孵育1.5h。弃上清用PBS清洗三遍。然后加入3,3',5,5'-四甲基联苯胺100ul显色,最后加入50ul 2M H2SO4停止反应。在450nm OD下测定反应孔的吸光度。检测结果如图5B所示,随着scFv浓度的提高,与细胞的结合量也随之提升。
实施例6:scFv-OA2脂质体的制备与表征
使用后插入法制备scFv-OA2脂质体,方法如下:将DSPE-PEG2000-Mal与TCEP还原后的scFv以20:1的摩尔比混合,4℃反应2h后在冰浴下对PBS透析,制得DSPE-PEG2000-scFv。分别按计算所需量将DSPE-PEG2000-scFv与预制好的空白OA2阳离子脂质体在45℃共孵30min,制备最终scFv摩尔比为0.08%、0.06%、0.01%的scFv-OA2脂质体。使用DLS测量scFv-OA2脂质体的粒径、电位和PDI,结果如表2所示。
表2为本发明的scFv-OA2脂质体的性质(n=3)
上述实验表明,掺入靶头量过大会导致脂质体电位下降,稳定性降低而出现聚集。掺入不高于0.06%的靶头后脂质体的粒径较为稳定,相比于无靶头的空白OA2阳离子脂质体粒径增加约20~30nm,均一性较好,Zeta电位在+10~+40mV之间,可进一步用于后续实验。
不同比例scFv与OA2复合的脂质体对siRNA递送的影响:进一步制备了scFv摩尔百分比为0.08%、0.06%、0.04%、0.02%、0的scFv-OA2脂质体复合物。取对数生长的K562细胞,调整细胞密度为2.5×105个/ml的细胞悬液,接种于6孔板,每孔2ml,分组加入2μg的siRNA,置于37℃、5%CO2恒温培养箱内培养6h,6h后收取细胞,PBS洗三次,过300目筛网,用BD的流式细胞仪检测FITC通道下的荧光强度,比较他们的递送siRNA的能力。
流式细胞术检测结果如图6A所示,与对照组相比,0%、0.04%、0.06%比例的scFv-OA2都能有效递送95%以上的siRNA入K562细胞,考虑能靶向更多白血病细胞,最终选择最高比例的scFv,即0.06%scFv-OA2脂质体作为递送siRNA药物的载体。
scFv-OA2脂质体的透射电子显微镜(TEM)表征:取20μL总脂质浓度为1mg/mlscFv-OA2脂质体滴加到覆有碳膜的铜网上,静置3min后用滤纸吸去脂质体溶液,随后滴加20μL 2%磷钨酸染色3min,用滤纸吸去染色剂,干燥后通过TEM拍摄脂质体形貌。scFv-OA2脂质体的形貌如图6B所示(标尺为50nm)。上述实验表明,scFv-OA2脂质体呈近球形,并且两者的粒径与DLS测定结果一致。
按照氮磷比(N/P=5)将siRNA与OA2或scFv-OA2脂质体溶液混合,用去离子水稀释至200ul混匀,室温孵育30min,即得siRNA脂质体复合物。通过琼脂糖凝胶电泳实验考察脂质体荷载siRNA的能力,如图6C所示。结果表明,所有阳离子脂质体均能在N/P=5的条件下稳定荷载siRNA,不发生泄漏。
实施例7:siRNA-scFv-OA2脂质体复合物的对体内肿瘤细胞的抑制作用
在裸鼠皮下接种了K562细胞的原位裸鼠模型,评估siRNA-scFv-OA2复合物的抗肿瘤作用。具体而言,将K562细胞注射到4周龄NOD-SCID雌性小鼠侧腹,当肿瘤体积达到50mm3,时,将小鼠随机分为4个治疗组:生理盐水组、空载scFv-OA2组、siRNA-OA2组和siRNA-scFv-OA2组。10ug siRNA/只或等体积的生理盐水或等量的scFv-OA2,每隔1天瘤内给药一次,直到瘤体积超过1500mm3时停止给药。期间监测小鼠肿瘤体积和重量,结果如7A,各组小鼠体重无异常变化,随着时间增长,对照组和NC组的瘤子增长速度显著提高(如图7C和7D),而OA2-siRNA组瘤子生长缓慢,scFv-OA2-siRNA组瘤体积减少,结合最后提取的瘤重数据(图7C),结果发现敲低Rapsyn能显著抑制瘤子的生长,并且带有scFv的siRNA脂质体复合物能更高效的抑制瘤子的生长。
实施例8:本发明的scFv-OA2-siRNA脂质体复合物治疗K562细胞异源移植小鼠模型的生存期检测。
构建K562细胞异源移植小鼠模型:将对数生长期K562细胞1×10^7个/200μl尾静脉注射入4周龄NCG雌鼠中,40只小鼠随机分成4组(n=10):生理盐水组、siNC-scFv-OA2组、siRNA-OA2组和siRNA-scFv-OA2组。7天后开始尾静脉分组给药,每组2.5nmol siRNA。每隔两天给药,记录每只小鼠的生存时间。结果如图8所示,与生理盐水组、siNC-scFv-OA2组和siRNA-OA2组相比,siRNA-scFv-OA2能有效延长小鼠的生存期,而非靶向的siRNA-OA2与生理盐水组和siNC-scFv-OA2组一样,无治疗效果。
Claims (10)
1.一种抑制Rapsyn基因表达的脂质体复合物,其特征在于,所述脂质体复合物具有靶头DSPE-PEG2000-scFv,脂质材料为赖氨酸谷氨酸双油醇酯OA2-Glu-Lys;所述脂质体复合物荷载有抑制Rapsyn基因表达的核酸,所述scFv为anti-CD79b-scFv。
2.根据权利要求1所述的脂质体复合物,其特征在于,所述核酸包括靶向Rapsyn的反义寡核苷酸、siRNA、miRNA、shRNA、核酸适配体、转录激活RNA中的一种或几种。
3.根据权利要求2所述的脂质体复合物,其特征在于,所述siRNA的靶序列如SEQ IDNo:1~14所示。
4.根据权利要求1所述的脂质体复合物,其特征在于,所述anti-CD79b-scFv氨基酸序列如SEQ ID No:48所示。
5.根据权利要求1所述的脂质体复合物,其特征在于,所述靶头DSPE-PEG2000-scFv为DSPE-PEG2000-Mal胶束与anti-CD79b-scFv以摩尔比1:1-50:1混合制得。
6.根据权利要求1所述的脂质体复合体,其特征在于,scFv在脂质体复合物的摩尔百分比不高于0.06%。
7.根据权利要求1所述的脂质体复合体,其特征在于,所述脂质材料和核酸的氮磷比不低于3:1。
8.权利要求1-7任一项所述抑制Rapsyn基因表达的脂质体复合物在制备预防或治疗Rapsyn基因异常表达引起的疾病药物中的用途。
9.根据权利要求8所述的用途,其特征在于,所述疾病为白血病,包括淋巴细胞型血液类肿瘤和骨髓细胞型血液类肿瘤,所述淋巴细胞型血液类肿瘤为急性淋巴细胞型血病、急性原始淋巴细胞型白血病、B细胞型白血病、T细胞型白血病、何杰金氏淋巴瘤、非何杰金氏淋巴瘤、毛状细胞淋巴瘤或Burkett氏淋巴瘤;所述骨髓细胞型血液类肿瘤为急性骨髓细胞型白血病、慢性骨髓细胞型白血病、骨髓发育不全症或早幼粒细胞型白血病。
10.一种脂质体制剂,其特征在于,包含权利要求1-7任一项所述抑制Rapsyn基因表达的脂质体复合物及药学上可接受的基因药物递送载体。
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