CN101389273B - 基因或药物递送*** - Google Patents
基因或药物递送*** Download PDFInfo
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- CN101389273B CN101389273B CN200580026511XA CN200580026511A CN101389273B CN 101389273 B CN101389273 B CN 101389273B CN 200580026511X A CN200580026511X A CN 200580026511XA CN 200580026511 A CN200580026511 A CN 200580026511A CN 101389273 B CN101389273 B CN 101389273B
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Abstract
本发明包括用于体内递送一种或多种活性剂的组合物和方法,通过将靶器官或者组织与微泡包裹的活性剂接触并通过将微泡在靶标处暴露于超声而在所述靶标选择性释放活性剂实现所述递送,所述微泡包含电中性的脂类微泡,其装载包含一种或多种活性剂的阳离子脂质体,其中活性剂保持在微泡中受到保护直到在靶标处选择性释放。
Description
本发明的技术领域
本发明涉及用于递送活性剂的组合物和方法,更具体地,涉及使用超声和微泡组合受控地、局部递送活性剂。
发明背景
已经报导阳离子脂质体可应用于在体外和体内递送大分子到靶细胞。美国专利号4,897,355、美国专利号5,334,761和美国专利号6,034,137公开了使用阳离子脂质团聚体如脂质体、单层脂质体、多层脂质体和微团的组合物和方法,所述阳离子脂质团聚体结合带负电荷的大分子,如DNA、RNA、蛋白质和小化合物并且当与靶细胞接触时,将大分子递送到靶细胞内部或者靶细胞膜上。在基因转染中,据报导用脂质体递送的转染效率在体外很高但是在体内很低。
已经报导超声介导的微泡破坏为用于将药物、蛋白质、信号分子或基因(包括质粒载体或者病毒载体)递送到特定组织的体外或体内方法(美国专利号5,580,757):将经标记的红细胞和聚合物微球体递送到大鼠骨骼肌(Skyba,等人1998;和Price,等人1998);寡核苷酸递送到狗肾(Porter,等人1996);狗心肌(Wei,等人1997);和具有氯霉素乙酰转移酶基因的培养的HeLa、NIH/3T3和C127I细胞(Unger,等人1997)。在一个研究中,将含有处于组成型启动子控制下的β-半乳糖苷酶的重组腺病毒转基因附着到白蛋白包被的、全氟丙烷填充的微泡的表面,并通过超声介导的微泡破坏将微泡递送到大鼠心肌,导致与对照动物相比,β-半乳糖苷酶活性增加10倍(Shohet,等人2000)。
在超声定向的微泡破坏的报导中,生物活性剂用油悬浮剂捕获在微泡核心中或者通过化学、静电或者机械手段附着到微泡外壳。微泡的直径通常为约2-4微米并且为球形。它们含有包裹在外壳内的气体核心,其中气体通常是全氟化碳,但是也已经使用了空气、氮气或者六氟化硫。微泡的外壳由白蛋白、磷脂或者聚合物制造。根据电子显微镜检查,通常的微泡外壳约30-50nm厚,具有网状、可塑特征,其当暴露于正或者负压波,如超声波时发生振动。取决于所应用的超声波的振幅和频率,微泡经历空化作用,释放被微泡外壳包裹或者附着到微泡外壳的生物活性剂。
尽管已经报导了活性成分向靶位点的脂质体或者微泡递送,但是这些方法在体内还没有所希望的那么有效。对于递送生物活性DNA的情况,有几种因素限制转染效率,所以限制了它的有效性。附着到微泡的生物活性DNA可以被循环的脱氧核糖核酸酶(DNA酶)中和。当从脂质微泡释放时,DNA在靶器官内是游离的,但是可能不进入细胞膜或者核膜。而且,部分微泡外壳将保持附着到DNA分子并且从而阻止它的翻译。在递送中,其他类型的生物活性剂同样对于蛋白酶、脂肪酶、糖类切割酶和其他降解途径敏感。
发明概述
现在已经发现活性成分如药物、肽、遗传物质或者化学治疗剂可以以比迄今报导的更高的效率递送到靶标部位,如哺乳动物中的特定器官或者组织。描述了活性剂递送***,其包括在微泡和包括预先装配到脂质体的活性剂的复合体之间形成的复合体。脂质体复合体可以在所希望的时间点被破坏以允许在靶标部位释放活性成分。
本发明还包括通过使用超声定向的微泡破坏(UTMD)向靶器官或者组织体内递送生物活性剂的方法,其中电中性的脂类微泡已经装载纳球体(nanospheric)阳离子脂质体,该脂质体装载生物活性剂。
本发明包括用于体内递送一种或多种活性剂的组合物和方法,其包括步骤:将靶器官或者组织与微泡包裹的活性剂接触,其中该微泡具有电中性的脂类微泡,该脂类微泡包含预先装载的脂质体,该脂质体包含一种或多种活性剂;并通过在靶标处将微泡暴露于超声选择性释放活性剂,其中活性剂在微泡中保持受保护,直到在靶标选择性释放。活性剂可以包括一种或多种分子,例如,处于组织特异性启动子控制下的核酸区段。其他实例包括具有组织特异性基因的核酸区段,所述基因处于组织特异性启动子控制下、处于可活化的启动子控制下、处于可活化的启动子的控制下,该启动子驱动导致凋亡的基因的表达。活性剂的其他实例包括一种或多种核酸区段,其编码选自由:激素、生长因子、酶、载脂蛋白凝固因子、肿瘤抑制因子、肿瘤抗原、病毒蛋白、细菌表面蛋白,和寄生虫细胞表面蛋白组成的组的基因。
通常,微泡在药学上可接受的载体中分布。活性剂可以是可表达的基因,其选自由例如,突变或野生型的:p53、p16、p21、MMAC1、p73、zac1、C-CAM、BRCAI、Rb、Harakiri、Ad E1 B、ICE-CED3蛋白酶、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-12、IL-13、IL-14、IL-15、TNF、GMCSF、β-干扰素、γ-干扰素、VEGF、EGF、PDGF、CFTR、EGFR、VEGFR、IL-2受体、***受体、Bcl-2或Bcl-xL、ras、myc、neu、raf、erb、src、fms、jun、trk、ret、gsp、hst、abl、p53、p16、p21、MMAC1、p73、zacl、BRCAI、BRCAII、Rb、生长激素、神经生长因子、胰岛素、促肾上腺皮质激素、甲状旁腺激素、促卵泡激素、***和促甲状腺激素组成的组。这些活性剂还可以包括启动子和处于该启动子控制下的基因,该启动子选自由CMV IE、LTR、SV40 IE、HSV tk、β-肌动蛋白、胰岛素、人珠蛋白α、人珠蛋白β和人珠蛋白γ启动子组成的组。
多种超声装置以及递送和使用的方法、频率、方式、能量等等可以用于本发明。例如,超声可以以脉冲或者聚焦方式应用。超声可以以超谐波方式等应用。微泡的实例包括本领域公知的那些,在一个实例中,微泡可以是生物可降解的聚合物、生物相容的两亲性材料、具有外壳的微泡,该外壳包含生物相容的两亲性材料的外层和生物可降解的聚合物的内层,和/或由选自胶原、明胶、白蛋白或者球蛋白的两亲性材料制成的微泡。
在一组特定实例中,活性剂可以是包含处于胰岛素启动子控制下的己糖激酶的核酸载体,或者甚至是包含处于RIP启动子控制下的己糖激酶基因I的核酸载体。使用本文教导的组合物和方法递送的活性剂的另一实例包括包含hVEGF蛋白质、hVEGF mRNA或者hVEGF蛋白质和hVEGF mRNA两者的核酸载体,或者甚至包含hVEGF165蛋白质、hVEGF165mRNA或者hVEGF165蛋白质和hVEGF165 mRNA两者的核酸载体。
用于制备脂质体的脂类和它们的装载是本领域公知的并且可以包括一种或多种下面的:例如,与载体和/或质粒混合的1,2-二棕榈酰基-sn-甘油-3-磷脂酰基胆碱和1,2-二棕榈酰基-sn-甘油-3-磷脂酰基乙醇胺甘油。多种通过商业途径可获得的脂类、混合物、试剂盒等等是公知的并且可以获得。
本发明还包括治疗需要此类治疗的哺乳动物的方法,其包括向哺乳动物施用有效量的组合物并使用超声向哺乳动物释放生物活性剂,其中所述组合物具有电中性的脂质微泡,该微泡装载预先装载一种或多种生物活性剂的阳离子脂质体。哺乳动物可以是患者,可以对患者提供药学上可接受的载体中的微泡并将微泡暴露于聚焦在用于递送的部位的超声能量。
本发明的另一实施方案是用于在靶标部位超声定向的微泡破坏的药物递送组合物,其包括在微泡内部和周围预先装配的脂质体-核酸复合体。脂质体-核酸复合体可以包括阳离子脂质、阴离子脂质或者其混合物和组合。装载的微泡通常在药学上可接受的载体中处置,如以液体或者干燥形式。微泡可以重悬浮在药学上可接受的载体,例如盐水中。当以干燥形式和作为例如试剂盒的部分提供时,干燥粉剂可以与一个或多个用完扔掉的一次或多次使用的容器和递送***,例如,注射器和/或针头一起提供,并且可以还包括使用说明。通常,试剂盒组分将预先消毒。
预先装载的微泡可以用于治疗需要这种治疗的哺乳动物的方法中,该方法包括提供有效量的组合物,该组合物具有电中性的脂质微泡,该微泡装载了用生物活性剂预先装载的纳米球阳离子脂质体,并在靶标部位使用超声定向的微泡破坏来破坏微泡。
活性成分的实例包括例如,原子或者小药物、蛋白质、肽、核酸、脂类、脂肪酸、糖类、糖、多糖、维生素、矿物质和其组合和混合物。核酸的实例可以包括核糖核酸、脱氧核糖核酸,它们为正义或者反义方向、线性或环状、作为载体的部分,该载体具有例如组成型和/或组织特异的启动子、增强子、沉默子、同源重组位点等等。可以包括的肽为例如,T细胞活化抗原、激素、递质等等。蛋白质可以是前体蛋白质、抗原、抗体、融合蛋白、结构蛋白、报道分子、可检测的标记、酶(例如,蛋白酶、核酸酶、激酶、磷酸酶、代谢酶)、趋化因子、淋巴因子、干扰素、白介素、激动剂、拮抗剂、受体、traps和其混合物和组合。脂类可以是递质、膜组分、能量来源、激动剂、拮抗剂、趋化因子等等。还可以使用本发明递送营养补剂,例如,营养有效量的DNA、蛋白质、脂类、糖前体、维生素、矿物质等等。
本发明的另一实施方案是用于超声定向微泡破坏的递送组合物,该组合物包括电中性的脂质微泡,该微泡装载了用生物活性剂装载的纳米球阳离子脂质体。
附图简述
为了更完全地理解本发明的特点和优点,现在参考本发明的详细描述和附图,并且附图中:
图1包括四个小图,上部小图是来自对照大鼠(左)和UTMD处理的大鼠(右)的显微镜切片(100X)。原位PCR杂交用于染色LacZ质粒DNA,其在所处理的胰腺各处可见。可以清楚地看到胰岛(箭头)。底部小图:来自对照大鼠(左)和使用RIP-LacZ用UTMD处理的大鼠的切片(400X)。原位PCR杂交用于染色LacZ mRNA,其定位于胰岛中心。
图2包括在高倍共焦显微镜(400X)下看到的胰腺的冷冻切片的六个小图,显示了用在RIP启动子下的DsRed质粒通过UTMD处理的胰岛。顶部小图:来自相同胰岛的图像,使用不同的滤光设置以鉴定相对于β细胞的DsRed蛋白质。左上小图:胰岛中存在DsRed。中上小图:使用绿色滤光器,针对胰岛素的荧光抗体鉴定了胰岛中心的β细胞。右上小图:共焦图像证实DsRed表达与β细胞的共定位。底部小图:来自相同胰岛的相邻切片的图像,使用不同的滤光器设置以鉴定相对于α细胞的DsRed蛋白质。左下小图:胰岛中存在DsRed。中下小图:使用绿色滤光器,针对胰高血糖素的荧光抗体鉴定了沿着胰岛周围的α细胞。右下小图:共焦图像证实DsRed表达不共定位于α细胞。
图3显示了用CMV-luc(断面线条形)、RIP-荧光素酶(白色条形)或者RIP-荧光素酶加上UTMD后20%葡萄糖饲养4天处理(黑色条形)的大鼠中完整胰腺荧光素酶活性。葡萄糖饲养导致与仅RIP相比,RIP-荧光素酶表达上调4倍。注意到荧光素酶表达的显著的胰腺特异性。在肝脏和脾脏中仅注意到沿着超声路径的轻微活性。左肾也在超声束的路径上,显示出比胰腺小得多的活性,但是具有RIP-荧光素酶的可调节的表达。右肾在超声路径的外面,没有显示出荧光素酶表达。每组有3只大鼠。通过ANOVA分析,器官之间荧光素酶活性的差异是统计学显著的(F=74.86,p<0.0001)。质粒(CMV对RIP对RIP与葡萄糖饲养)中的差异也是统计学显著的(F=42.36,p<0.0001)。
图4显示了RIP-荧光素酶表达的时间过程。荧光素酶活性在第4天时从其峰值下降并且到第28天时可以忽略不计。通过ANOVA分析,荧光素酶活性在时间上的下降也是统计学显著的(F=236.4,p<0.0001)。
图5包括顶部小图:蛋白质印迹,显示了用UTMD处理后,在正常对照和DsRed处理的对照中,分离的大鼠胰腺中证明己糖激酶-1活性。左下:通过UTMD用己糖激酶处理、通过UTMD用DsRed对照处理和假手术对照处理的大鼠中的血清胰岛素水平。通过反复测量ANOVA,组差异在p=0.0033时是显著的,由事后检定(post-hoc)Scheffe检验表明在第5和10天时的显著差异。右下小图显示了通过UTMD用己糖激酶I处理、通过UTMD用DsRed对照处理和通过假手术对照处理的大鼠中的血清葡萄糖水平。通过反复测量ANOVA,组差异在p=0.0005时是显著的,由事后检定Scheffe检验表明在第5和10天时的显著差异。数据显示为平均值±一标准差,n=每组6(UTMD己糖激酶),3(UTMD对照)和3(正常对照)只大鼠。
图6通过免疫印迹显示了在来自大鼠心肌的组织匀浆中hVEGF165蛋白质的存在。在第10天时,在用UTMD-hVEGF165处理的所有三只大鼠中都看到与hVEGF165一致的明显带,但是在对照大鼠(仅UTMD,仅hVEGF165质粒,或者盐水)中仅有微弱带。还显示了阳性对照带(+C)。
图7显示了来自大鼠心肌的组织匀浆中人VEGF165 mRNA(顶部小图)和大鼠VEGF165 mRNA(底部小图)的存在的PT-PCR结果。在用UTMD处理的3只大鼠中在第5天(#1-3)和第10天(#7-9),和用UTMD处理的1只大鼠(#14)中在第30天(#13-15)中看到hVEGF165 mRNA带,但是在任何对照大鼠(#4-6,10-12,16-18)中都没有看到hVEGF165 mRNA带。为了显示的目的,每个时间期限仅显示了来自3个对照组的每一组的仅1只大鼠。在所有实验大鼠中都看到大鼠VEGF165 mRNA靶定的带(底部小图)。
图8a-8d是UTMD处理后10天心肌的组织学切片。8a是低倍(100x)苏木精伊红染色,显示了心肌的超细胞区。8b是用抗-VEGF抗体染色的超细胞区的低倍(100x)图像,证实在超细胞区中存在VEGF;8c是用BS-凝集素染色的超细胞区的高倍图像(400X)。红色箭头描绘了毛细管内皮细胞中明显的细胞核,与血管发生相一致。还有紊乱的肌细胞结构,与轻微炎症相一致;8d是用平滑肌α-肌动蛋白染色的超细胞区的高倍(400X)图像。红色箭头指向覆盖新血管的周细胞。黄色箭头指向微动脉平滑肌细胞上突出的细胞核。条线表示100μm。
图9是组织学和曲线图的合成图,显示了处理后大鼠心肌毛细血管密度的改变。顶部小图显示了在200X下用BS-凝集素染色的代表性切片。与对照心肌(左边小图)比较,用UTMD-VEGF-处理的心肌的毛细血管密度增加(右边小图)。底部小图显示了UTMD处理后毛细血管密度(凝集素+血管<10μm)随时间的平均值。毛细血管密度的平均值在所有三个时间点在所有对照中非常稳定。然而,在UTMD-VEGF-处理的大鼠中,毛细血管密度在第5天和10天时显著增加。误差线代表一个标准差。
图10是组织学和曲线图的合成图,显示了处理后大鼠心肌微动脉密度的改变。顶部小图显示了在100X下用平滑肌α-肌动蛋白染色的切片。与对照(左边)比较,微动脉密度增加(右边)。底部小图显示了UTMD处理后微动脉密度(平滑肌α-肌动蛋白+血管>30μm)随时间的平均值。微动脉密度的平均值在所有三个时间点在对照中没有显著不同。然而,在UTMD-VEGF-处理的大鼠中,微动脉密度在第5、10和30天时显著增加。误差线代表一个标准差。
发明详述
尽管下面详细讨论了做出和使用本发明的多种实施方案,但是将理解本发明提供了许多可应用的发明性概念,其可以在多种特定背景下被具体化。本文讨论的特定实施方案仅仅阐明做出和使用本发明的特定方法并且不限制本发明的范围。
为了方便理解本发明,下面定义许多术语。本文定义的术语具有本发明相关领域技术人员通常理解的含义。术语如“一个”和“这个”不意在仅仅指单数实体,而是包括特定实例可以用于阐明的一类实体。本文的术语用于描述本发明的特定实施方案,但是它们的用法不限定本发明,本发明只受到权利要求的限定。
在本说明书全文中,使用下面的缩写:TF,转录因子;ORF,开放阅读框;kb,千碱基(对);UTR,非翻译区;kD,千道尔顿;PCR,聚合酶链式反应;RT,逆转录酶。
术语“基因”用于指功能蛋白质、多肽或者肽编码单位。如本领域技术人员将理解的,该功能术语包括基因组序列、cDNA序列、其片段和/或组合,以及基因产物,包括通过人工改变的基因产物。纯化的基因、核酸、蛋白质等等用于指这样的实体,它们被鉴定并且与它通常结合的至少一种污染性核酸或者蛋白质分离。
本文所用的术语“载体”用于指核酸分子,其将DNA区段从一个细胞转移到另一个细胞。载体还可以定义为经设计用以增殖基因序列的载体,或者表达载体,其包括可操作地连接基因序列的启动子,或者经设计以导致导入这种启动子的载体。载体可以以独立于宿主细胞染色体的状态存在,或者可以整合到宿主细胞染色体中。
本文所用的术语“启动子”指RNA聚合酶结合的DNA链上的识别位点。启动子通常是帽位点或者转录启动开始位点上游5’侧翼DNA中约100到200个碱基对(bp)的DNA片段。启动子与RNA聚合酶形成起始复合体以启动和驱动转录活性。复合体可以通过称作“增强子”的激活序列或者称作“沉默子”的抑制序列修饰。通常,特定调节序列或者元件内嵌在DNA的蛋白质编码区邻近处或者内部。位置与该基因相邻的元件称作顺式作用元件。这些信号被其他可扩散的反式生物分子识别以加强转录活性。这些生物分子被称作反式作用因子。反式作用因子和顺式作用元件的存在促进基因的定时和发育表达模式。通常认为顺式作用元件是调节转录的元件并且发现位于启动子区和其他上游DNA侧翼序列内部。
如本文所用的术语“前导序列”指在结构基因的5’末端的DNA序列,其与基因一起转录。前导序列通常导致具有N-末端肽延伸(有时称作前序列(pro-sequence))的蛋白质。对于最终分泌到细胞外基质或者膜的蛋白质,该信号序列(主要是疏水的)指导该蛋白质进入内质网,该蛋白质从内质网排出到合适的目的地。
如本文所用的术语“内含子”指在基因的中部发生的DNA的部分,其不编码基因产物中的氨基酸。内含子的前体RNA被切除并且因此不转录成mRNA,也不翻译成蛋白质。
术语“表达盒”指本发明的序列,其含有将表达的核酸序列。表达盒在概念上与盒式磁带相似。每个表达盒将具有其自己的序列。从而,通过互换表达盒,载体将表达不同的序列。因为在5’和3’末端的限制性位点,表达盒可以容易地***、除去或者用另一表达盒替换。
本文所用的术语“3’非翻译区”或者“3’UTR”指在结构基因的3’末端的序列,其通常随着基因转录。该3’UTR区通常含有多聚A序列。尽管3’UTR从DNA转录,但是它在翻译成蛋白质前被切除。在本发明中,它优选具有肌原性特异的3’UTR。这使得在肌原性组织中具有特异稳定性。本文所用的术语“非编码区”或者“NCR”指与结构基因的3’UTR区连续的区域。NCR区含有转录终止信号。本文所用的术语“限制性位点”指限制性内切酶的序列特异切割位点。
本文所用的术语“载体”指某种工具,DNA片段通过它可以导入宿主生物或者宿主组织。有多种类型的载体,包括质粒、噬菌体和粘粒。
本文所用的术语“有效量”指活性剂的量,例如,UTMD递送到靶组织或者细胞,例如,胰腺的β细胞、肌原性组织或者培养物、血管生成细胞等以产生足够水平的多肽的基因或者启动子与基因的组合的量。本领域技术人员认识到该实际水平将取决于MVS的使用。该水平在处理、疫苗产生或者接种中将不同。
“质粒”通过小写字母p表示,该字母的前面和/或后面跟着大写字母和/或数字。本文的起始质粒可通过商业途径获得,可以不受限制地公开获得,或者可以按照公布的方法从此类可获得的质粒构建。此外,其他等同质粒是本领域已知的并且将是普通技术人员显而易见的。
术语“转基因”在本文用于描述可以人工***哺乳动物基因组,例如,活动物的哺乳动物细胞的遗传物质。术语“转基因动物”在本文用于描述非人动物,通常为哺乳动物,其具有非内源(即异源)核酸序列,该序列作为细胞的部分中的染色体外元件存在或者稳定整合到它的种系DNA(即,在多数或者所有它的细胞的基因组序列中)中。根据本领域公知的方法,通过例如,宿主动物的胚胎或者胚胎干细胞的遗传操作向此类转基因动物的种系中导入异源核酸。
本文所用的术语“敲除(knock-out)”包括例如,条件敲除,其中靶基因的改变可以如下激活:将动物暴露于促进靶基因改变的物质,导入促进靶基因位点的重组的酶(例如,Cre-lox***中的Cre),或者其他的用于指导靶基因改变的方法。
本文所用的术语“敲入(knock-in)”指宿主细胞基因组中导致靶基因的改变的表达(例如,增加或者减少的表达)的改变,例如,通过导入靶基因的额外拷贝,或者通过可操作地***调节序列,该调节序列提供靶基因的内源拷贝的增强的表达。敲入转基因包括靶基因的杂合敲入或者靶基因的纯合敲入并且包括额外的敲入。
一方面,本发明是通过使用超声定向的微泡破坏(UTMD),使用装载含有生物活性剂的纳米球阳离子脂质体,向靶器官或者组织体内递送生物活性剂的方法。示例性微泡包含但不限于,适于体内超声定向的微泡破坏的电中性的脂类、聚合物、金属,或者丙烯酸壳。在一个实施方案中,将生物活性剂首先包裹在纳米颗粒大小(10-60nm)的小阳离子脂质体内或者附着到该脂质体上(下文中,“装载”或者“包括”生物活性剂的纳米球阳离子脂质体指包裹在脂质体(如阳离子脂质体)内或者附着到脂质体上的任何生物活性剂),然后将该脂质体附着到电中性的脂类包被或者白蛋白包被的微泡上,该微泡填充了适于超声微泡破坏技术的气体,例如,全氟丙烷。脂质体可以附着到微泡壳的外表面,掺入微泡壳中和/或包裹在微泡壳的内部。在本发明中,一种或多种生物活性剂可以通过超声定向微泡破坏,使用装载含有生物活性剂的纳米球阳离子脂质体的电中性的脂类微泡同时地或者先后地递送。在另一方面,本发明是治疗需要此类治疗的哺乳动物的方法,其包括通过超声定向微泡破坏施用有效量的组合物,该组合物包含装载含有生物活性剂的纳米球阳离子脂质体的电中性的脂类微泡。
适于本发明的生物活性剂的实例包括药物制剂和药物、生物活性的合成有机分子、蛋白质、肽、多肽、维生素、类固醇、聚阴离子剂、遗传物质和诊断试剂。生物活性维生素、类固醇、蛋白质、肽和多肽可以是天然来源或者合成的。示例性聚阴离子剂包括但不限于硫酸化多糖、带负电荷的血清白蛋白和奶蛋白质、合成的硫酸化聚合物、聚合的阴离子表面活性剂和多磷酸盐。合适的诊断试剂包括但不限于与患者的磁共振成像、超声或者计算机化断层显象结合使用的染料和造影剂。
合适的遗传物质包括核酸、核苷、核苷酸和多核苷酸,它们可以是分离的基因组的、合成的或者重组物质;单链或者双链的;和正义或者反义方向,有或者没有对碱基、糖类残基或者磷酸二酯键的修饰。遗传物质的示例性来源包括但不限于脱氧核糖核酸(DNA)、核糖核酸(RNA)、互补DNA(cDNA)、信使RNA(mRNA)、核糖体RNA(rRNA)、短干扰RNA(siRNA)、核酶和RNA和DNA的混合的双链体和三链体。
遗传物质是在表达载体上携带的基因,所示表达载体包括但不限于辅助病毒、质粒、噬菌粒,粘粒和酵母人工染色体。适于本发明的遗传物质能够编码治疗性、调节和/或诊断蛋白质的至少一部分。此外,遗传物质可以优选编码一种以上类型的蛋白质。例如,生物活性剂可以包含质粒DNA,该质粒DNA包含编码治疗蛋白质的遗传物质和用于监视质粒DNA的递送的选择标记或者诊断标记,例如,pDsRed-人胰岛素启动子。此类蛋白质包括但不限于组织相容性抗原、细胞粘附分子、生长因子、凝血因子、激素、胰岛素、细胞因子、趋化因子、抗体、抗体片段、细胞受体、细胞内酶、转录因子、能够除去患病或者恶性细胞的毒性肽。可以通过该技术递送的其他遗传物质包括腺病毒、腺伴随病毒、逆转录病毒、慢病毒、RNA、siRNA或者选择性打开或者关闭特定基因的化学品,如聚酰胺或者肽片段。对野生型蛋白质的修饰得到野生型变体的激动剂或者拮抗剂,所述修饰落入本发明的范围。遗传物质还可以包含组织特异性启动子或者表达控制序列,如转录启动子、增强子、转录终止子、操纵基因或者其他控制序列。
本发明使用的活性剂的实例包括预先装载到脂质体并且与微泡结合的一种或多种下面的治疗剂,包括,但不限于,激素产品,如加压素和催产素和它们的衍生物、胰高血糖素和甲状腺剂,如含碘产品和抗甲状腺剂;心血管产品,如螯合剂和汞利尿剂和强心苷;呼吸产品如黄嘌呤衍生物(茶碱和氨茶碱);抗感染剂,如氨基糖苷类、抗真菌剂(例如,两性霉素)、青霉素和头孢菌素抗生素、抗病毒剂(例如,齐多夫定、利巴韦林、金刚烷胺、阿糖腺苷和阿昔洛维)、驱虫药、抗疟药和抗结核病药;生物制品如抗体(例如,抗毒素和抗蛇毒素)、疫苗抗原(例如,细菌疫苗、病毒疫苗、类毒素);抗肿瘤药(例如,亚硝脲、氮芥、抗代谢物(氟尿嘧啶、激素、***和***激动剂和/或拮抗剂);有丝***抑制剂(例如,依托泊苷和/或长春花生物碱)、放射性药物(例如,放射性碘和磷产品);和干扰素、羟基脲、甲基苄肼、达卡巴嗪、曼托坦、天冬酰胺酶和环孢菌素,包括它们的混合物和组合。
其他合适的治疗剂包括,但不限于:血栓溶解剂,如尿激酶;促凝剂,如凝血酶;抗肿瘤药,如铂化合物(例如,顺螺铂、顺铂和卡铂)、氨甲喋呤、阿霉素、紫杉醇、丝裂霉素、柄型菌素、博来霉素、阿糖胞苷、arabinosyl adsnine、巯基聚赖氨酸、长春新碱、白消安、苯丁酸氮芥、苯丙氨酸氮芥(例如,PAM、L-PAM或者苯丙氨酸氮芥)、
巯嘌呤、曼托坦、盐酸甲基苄肼、更生霉素(放线菌素D)、盐酸柔红霉素、盐酸阿霉素、丝裂霉素、普卡霉素(光辉霉素)、氨鲁米特、雌氮芥磷酸钠、氟他米特、醋酸亮丙瑞林、醋酸甲地孕酮、枸橼酸他莫昔芬、睾内酯、曲洛司坦、胺苯吖啶(m-AMSA)、天冬酰胺酶(L-天冬酰胺酶)、Erwinaasparaginase、依托泊苷(VP-16)、干扰素α-2a、干扰素α-2b、替尼泊苷(VM-26)、硫酸长春碱(VLB)、硫酸长春新碱、博来霉素、硫酸博来霉素、氨甲喋呤、阿霉素,和***糖(arabinosyl);血液制品,如肠胃外铁、氯高铁血红素;生物应答修饰剂,如胞壁酰二肽、胞壁酰三肽、微生物细胞壁组分、淋巴因子(例如,细菌内毒素,如脂多糖、巨噬细胞活化因子)、细菌(如分枝杆菌、棒杆菌)的亚基、合成二肽N-乙酰基-胞壁酰-L-丙氨酰-D-异谷酰胺;抗真菌剂,如酮康唑、利霉菌素、灰黄霉素、氟胞嘧啶(5-fc)、咪康唑、两性霉素B、蓖麻毒蛋白,和β-内酰胺抗生素(例如,青霉素、氨苄青霉素、磺酰胺菌素);激素,如生长激素、PDGF、EGF、CSF、GM-CSF、促黑素细胞激素、***、倍氯美松双丙酸酯、倍他米松、醋酸倍他米松和倍他米松磷酸酯钠、vetamethasone disodiumphosphate、vetamethasone sodium phosphate、醋酸可的松、***、醋酸***、***磷酸钠、flunsolide、皮质醇、氢化可的松、氢化可的松环戊丙酸酯、氢可松磷酯钠、氢化可的松琥珀酸钠酯、甲基氢化***、醋酸甲基氢化***、甲泼尼龙、醋酸对氟米松、***龙、氢化***、强的松龙磷酸钠、prednisolone rebutate、***、氢羟强的松龙、羟氢化***缩丙酮、去炎松、己酸丙炎松和醋酸氟氢可的松;维生素,如维生素C、E、A、K、ascyanocobalamin、neinoic acid、类视黄醇和衍生物,如棕榈酸维生素A,和α-生育酚;肽(例如,T细胞表位,如MAGE、GAGE、DAGE等等);蛋白质,如锰超氧化物歧化酶、醇脱氢酶、一氧化氮合酶;酶,如碱性磷酸酶;抗过敏剂,如amelexanox;抗凝剂,如苯丙香豆素和肝素;循环药物,如***;代谢增强剂,如谷胱甘肽;抗结核药,如对氨基水杨酸、异烟肼、硫酸卷曲霉素、环丝氨酸、盐酸乙胺丁醇、乙硫异烟胺、吡嗪酰胺、利福平,和硫酸链霉素;抗病毒剂,如阿昔洛维、金刚烷胺、叠氮胸苷(AZT或者叠氮胸苷)、利巴韦林和一水合阿糖胞苷(阿糖腺苷,ara-A);抗心绞痛药,如地尔硫卓、硝苯地平、维拉帕米、赤鲜醇四硝酸酯、硝酸异山梨酯、***油(三***酯)和硝酸戊四醇酯;抗凝剂,如苯丙香豆素、肝素;抗生素,如氨苯砜、氯霉素、新霉素、头孢克洛、头孢羟氨苄、头孢菌素IV、泛捷复、红霉素、克林霉素、林可霉素、阿莫西林、氨苄西林、巴氨西林、羧苄青霉素、双氯青霉素、环青霉素、picloxacillin、缩酮氯苄青霉素、甲氧苯青霉素、乙氧萘胺青霉素、苯唑西林、青霉素G、青霉素V、替卡西林、利福平和四环素;抗炎剂,如二氟苯水杨酸、布洛芬、吲哚美辛、甲氯芬那酸、甲芬那酸、甲氧萘丙酸、羟基保泰松、苯丁唑酮、吡罗昔康、舒林酸、托美汀、阿司匹林和水杨酸;抗原生动物药,如氯喹、羟氯喹、甲硝唑、奎宁和锑酸葡胺;抗风湿剂,如青霉胺;***,如止痛药;阿片制剂,如可待因、***、***、***和***;强心苷,如去乙酰毛花甙、洋地黄毒苷、地高辛、洋地黄甙和洋地黄;神经肌肉阻滞剂,如卡肌宁、弛肌碘、己芴溴铵、碘甲筒箭毒、潘库溴铵、氯化琥珀胆碱(氯化琥珀胆碱)、氯化筒箭毒碱和维库罗宁;镇静药(***),如异戊巴比妥、异戊巴比妥钠、阿普比妥、布塔巴比妥钠、水合氯醛、乙氯维诺、炔己蚁胺、盐酸氟西泮、格鲁米特、盐酸左美丙嗪、甲乙哌酮、盐酸咪达唑、副醛、戊巴比妥、戊巴比妥钠、***钠、司可巴比妥钠、他布酮、羟基安定和***仑;局部***,如盐酸布比卡因、盐酸氯普鲁卡因、盐酸依替卡因、盐酸利多卡因、盐酸甲哌卡因、盐酸普鲁卡因和盐酸丁卡因;全身***,如达哌啶醇、依托咪酯、枸橼酸芬太尼与达哌啶醇、盐酸***、甲己炔巴比妥钠和硫喷妥钠;和放射性颗粒或者离子,如锶、碘化铼和钇,和它们的组合和混合物。
前药可以在附着到微泡之前预先装载到脂质体中。前药是本领域公知的并且可以包括无活性药物前体,其经代谢而形成活性药物。技术人员将认识到如Sinkula,et al.,J.Pharm.Sci.1975 64,181-210中描述的合适的前药(和如果必要,它们的盐形式),将所述文献的相关部分引入本文作为参考。例如,前药可以包括活性药物的无活性形式,其中化学基团存在于前药上,该化学基团使得药物无活性和/或赋予药物的可溶性或者一些其他性质。在该形式中,前药通常是无活性的,但是一旦通过热、空化作用、压力和/或通过周围环境中的酶或者其他方式从前药切割了所述化学基团,就产生了活性药物。此类前药在本领域中充分描述,并且包括通过键结合到化学基团的多种药物,所述键为诸如酯到短、中等或者长链脂肪族碳酸酯、有机磷酸酯的半酯、焦磷酸酯、硫酸酯、酰胺、氨基酸、偶氮键、氨基甲酸酯、磷酰胺、葡糖苷酸酯、N-乙酰葡糖胺和β-葡糖苷。药物与亲本分子和可逆修饰或者键的实例如下:铃兰毒甙与缩酮、乙内酰脲和烷基酯、氯丙炔碘与甘氨酸或者丙氨酸酯、对乙酰氨基酚与咖啡因络合物、阿司匹林与THAM盐、阿司匹林与乙酰胺基苯基酯、纳洛酮与硫酸酯、15-甲基***素F2与甲酯、普鲁卡因与聚乙二醇、红霉素与烷基酯、克林霉素与烷基酯或者磷酸酯、四环素与内铵盐、7-酰基氨基先锋霉素类与环取代的酰氧基苄基酯、诺龙与苯基丙酸酯、硅酸酯、***与烯醇醚乙缩醛、甲基***龙与乙酸酯、睾酮与正乙酰基氨基葡糖苷葡萄糖苷酸(三甲基甲硅烷基)醚、氢化可的松或者***龙或者***与21-磷酸酯。前药还可以设计为可逆的药物衍生物并用作修饰剂来增强药物向位点特异组织的转运。具有用于影响向位点特异组织的转运或者用于增强的治疗效果的可逆修饰或键的载体分子的实例包括异氰酸酯与卤代烷基硝基脲、睾酮与丙酸酯、氨甲喋呤(3-5’-二氯甲氨蝶呤-e)与二氨基酯、阿糖胞苷与5’-酰化产物、氮芥(2,2’-二氯-N-甲基二乙基胺)、氮芥与氨基甲基四环素、氮芥与胆固醇或者***或者脱氢表雄酮酯和氮芥与偶氮苯。
技术人员将认识到可以选择可以在任一给定药物中修饰的特定化学基团以影响药物向微泡的壳或者内部的分配。所选的将化学基团连接到药物的键可以经选择以在微泡释放后具有所希望的代谢速率,例如,对于血清酯酶存在下的酯键,所述代谢为水解。此外,可以选择具体化学基团来影响用于微泡中的药物的生物分布,例如,N,N-二(2-氯代乙基)-phosphorodiamidicacid与环状磷酰胺用于卵巢腺癌。此外,用于微泡内的前药可以设计成含有可逆衍生物,其用作活性持续时间的修饰剂以提供、延长或者积蓄作用效果。
例如,烟酸可以用葡聚糖和羧甲基葡聚糖酯修饰,链霉素用藻酸盐修饰,双氢链霉素用双羟萘酸盐修饰,阿糖胞苷(ara-C)用5’-adamantoats酯修饰,ara-腺苷(ara-A)用5-palmirate和5’-苯甲酸酯修饰,两性霉素B用甲酯修饰,睾酮用17-β-烷基酯修饰,***用甲酸酯修饰,***素用2-(4-咪唑基)乙胺盐修饰,多巴胺用氨基酸酰胺修饰,氯霉素用单-和二(三甲基甲硅烷基)醚修饰,环氯胍用双羟萘酸盐修饰。在该形式中,长效药物的贮库或者贮存库可以在体内从带有前药的微泡释放。可以选择或者修饰治疗剂的特定化学结构以实现所希望的溶解性,从而治疗剂装载到脂质体后附着或装载到微泡内部、外部或者周围。类似地,其他治疗剂可以用在结构上为芳族或者甾醇的疏水基团配制以掺入微泡的表面。
适宜用于本发明的阳离子脂质体包含一种或多种单阳离子或者聚阳离子脂质,任选地与一种或多种中性或者辅助脂质组合。适于本发明的阳离子脂质可以通过商业途径得到或者可以通过本领域已知的方法制备。适于形成阳离子脂质体的阳离子脂质是本领域公知的并且包括但不限于任何磷脂相关的物质,如卵磷脂、磷脂酰乙醇胺、溶血卵磷脂、溶血磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰肌醇、鞘磷脂、脑磷脂、心磷脂、磷脂酸、脑苷脂、磷酸十六烷酯、二油酰磷脂酰胆碱(DOPC)、二棕榈酰磷脂酰胆碱(DPPC)、二油酰磷脂酰甘油(DOPG)、二棕榈酰磷脂酰甘油(DPPG)、二棕榈酰磷脂酰乙醇胺5-carboxyspermylamide(DPPES)、二油酰磷脂酰乙醇胺(DOPE)、棕榈酰油酰磷脂酰胆碱(POPC)、棕榈酰油酰磷脂乙醇胺(POPE)和二油酰磷脂酰-乙醇胺4-(N-马来酰亚胺甲基)环己烷-l-羧酸(DOPE-mal)。额外的非含磷脂类包括但不限于硬脂酰胺、十二烷基胺、十六烷基胺、棕榈酸乙酰酯、蓖麻油酸甘油酯、硬脂酸十六酯、十四烷酸异丙酯、两性丙烯酸脂聚合物、三乙醇胺-硫酸月桂酯、烷基-芳基硫酸聚氧乙烯化脂肪酸酰胺、二(十八烷基)二甲基溴化铵和类固醇,如胆固醇、麦角固醇、麦角固醇B1、B2和B3、雄甾酮、胆酸、去氧胆酸、鹅去氧胆酸、石胆酸、N-[1-(2,3-二油酰氧基)丙基]-N,N,N-三甲基氯化铵(DOTMA)、1,2-二(油酰氧基)-3-3-(三甲基氨)丙烷(DOTAP)、和5-carboxyspermylglycine二(十八烷基)酰胺(DOGS)。优选的脂质体制剂包含(3∶1,w/w)的聚阳离子脂质2,3-二油酰氧基-N-[2-(sperminecarboxaido)乙基]-N,N-二甲基-1-丙烷三氟乙酸铵(DOSPA)和中性脂质二油酰基磷脂酰乙醇胺(DOPE)at(3∶1,w/w),和它们的混合物和组合。
在本发明的方法中,阳离子脂质体装载生物活性剂。在一个实施方案中,溶解在一种或多种有机溶剂中的一种或多种脂类的阳离子脂类制剂首先干燥或冻干以除去有机溶剂,得到脂类膜。仅仅在使用前,将脂类膜与适于本发明的悬浮在合适的水性介质中的生物活性剂混合以从干燥的脂类膜形成脂质体。例如,水、水性缓冲溶液、或者组织培养基可以用于脂类膜的再水化。合适的缓冲液是磷酸缓冲盐水,即0.9%NaCl中的pH 7.4的10mM磷酸钾。再另一实施方案中,将干燥的脂类膜用合适的水性介质再水化以在加入活性剂前形成脂质体。当生物活性剂包含遗传物质时,该方法是优选的。生物活性剂向阳离子脂质体的掺入通常在约0到30℃范围的温度,例如,在室温下进行约5、10-20分钟。
在本发明的方法中,具有附着的生物活性剂的阳离子脂质体通常装载到电中性的微泡上。在优选实施方案中,这通过如下步骤完成:向具有附着的生物活性剂的阳离子脂质体加入适于制备微泡壳的脂类组合物,充分混合,然后加入合适的气体用于由微泡壳包裹,然后剧烈振动约5到60秒,优选约20秒。在优选实施方案中,在加入具有附着的生物活性剂的阳离子脂质体之前,脂类组合物保持在约0到30℃。
为了形成微泡壳,预期已知用于超声定向的微泡破坏的天然或者合成来源的任何生物相容的脂类为本发明的部分。示例性脂类可以见国际申请号WO 2000/45856并且包括但不限于脂肪酸、磷脂、糖脂、糖鞘脂类、鞘脂、脂肪族醇、脂肪族蜡、萜类、倍半萜和类固醇。优选的脂类是磷酸胆碱、磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰甘油和磷脂酰肌醇。更优选的脂类是1,2-棕榈酰-sn-甘油-3-磷酸胆碱或者1,2-棕榈酰-sn-甘油-磷脂酰乙醇胺。最优选的是L-1,2-棕榈酰-sn-甘油-3-磷酸胆碱和L-1,2-棕榈酰-sn-甘油-磷脂酰乙醇胺。
适于本发明的气体通常是惰性的和生物相容的,包括但不限于空气;二氧化碳;氮气;氧气;氟;惰性气体如氦、氖、氩和氙;基于硫的气体;氟化气体;和其混合物。气体可以是全氟丙烷,例如,八氟丙烷。
如本领域技术人员公知的,靶定配体可以附着到微泡以赋予额外的组织特异性。此类配体可以包括单克隆抗体、肽、多肽、蛋白质、糖蛋白、激素或激素类似物、单糖、多糖、类固醇或者类固醇类似物、维生素、细胞因子,或者核苷酸。
本发明的递送方法包括装载含有一种或多种生物活性剂的纳米球阳离子脂质体的电中性的微泡,所述方法提供了超声定向微泡递送***的所有优点以及脂质体递送***的所有优点。超声定向微泡递送***允许向特定器官或者组织递送药物/基因生物活性剂,而使得其他器官或者组织最小地暴露于生物活性剂。在递送期间,一种或多种生物活性剂保持在保护性阳离子脂质体内,所述脂质体保护生物活性剂免于蛋白酶、核酸酶、酯酶、糖类切割酶、自由基的破坏或者其他化学改变。该方法增加了生物活性剂的递送和其对靶组织的生物利用率。例如,在装载含有质粒DNA的纳米球阳离子脂质体的电中性的微泡的递送中,靶位点处基因表达的水平增加到高于使用相同质粒DNA的微泡递送或者脂质体递送得到的可能的表达水平。
在一方面,本发明是治疗需要此类治疗的哺乳动物的方法,其包括通过超声定向的微泡破坏施用有效量的组合物,该组合物包含装载含有生物活性剂的纳米球阳离子脂质体的电中性的脂类微泡。可以通过本领域已知的任何方法施用包含装载含有生物活性剂的纳米球阳离子脂质体的电中性的脂类微泡的组合物和这些微泡的超声定向的微泡破坏以释放所述生物活性剂。重复施用微泡是可能的,尤其用于延长治疗效果的持续时间。例如,已经表明通过超声定向的微泡破坏重复转染心肌细胞可以将心脏中荧光素酶活性的峰值持续时间从4天延长到12天(Bekeredjian et al,2003)。这可能允许将基因或者药物递送的持续时间根据特定生物学或者医学需要调整。
本发明的组合物和使用方法在下面提供的实施例中进一步详细阐明,但是不认为这些实施例以任何方式限制本发明的范围。尽管这些实施例描述了本发明,但是理解对组合物和方法的修饰是本领域技术人员能力范围之内,并且认为此类修饰在本发明的范围之内。
实施例1:装载生物活性成分的阳离子脂质体溶液的制备
为了制备装载质粒DNA pCMV-luc的阳离子脂质体溶液,仅在使用前向50微升阳离子脂质体溶液(Lipofectamine 2000;Invitrogen,Carlsbad,California)加入含有2毫克质粒DNA的50-100微升溶液并在室温下温育10-20分钟。所得脂质体包裹质粒DNA并且直径为大概250纳米。将脂质体保存在-20℃备用。
实施例2:制备含有质粒DNA的微泡配方
根据Unger等人(Unger,等人1997.“Ultrasound enhances geneexpression of liposomal transfection,”Invest Radiol 32:723-727;美国专利号6,521,211)以前描述的方法的改进方法制备在微泡外壳内掺入质粒DNA pCMV-luc的微泡配方(下文称作“配方2”)。简言之,在可密封管中,将溶解在PBS并且预热到42℃的250微升2%1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱(C16)与1毫克质粒DNA pCMV-luc混合并在40℃温育30分钟。如需要,加入PBS以实现500微升的最终总体积。然后将管装入八氟代丙烷并在牙科搅拌机(VIALMIXBriston-Myers Squibb Medical Imaging,Inc.,North Billerica,MA)中剧烈振动20秒。除去并丢弃包含未附着的DNA pCMV-luc的液体下层清液,留下脂质包被的微泡悬浮液的乳白色上清液层。然后在输注前将所得微泡悬浮液用PBS以1∶1稀释。
实施例3:制备装载含有质粒DNA的纳米球阳离子脂质体的电中性的脂质微泡
如下制备装载阳离子脂质体/DNA复合体的电中性脂质微泡(下文称作“配方1”)。向含有50微升如实施例1制备的装载的阳离子脂质体/DNA复合体的管中加入250微升2%1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱(C16)(预热到42℃)与5微升10%白蛋白溶液和50微升甘油。用移液器充分但是温和地混合混合物。如需要,加入PBS以实现500微升的最终总体积。然后将含有混合物的管装入八氟代丙烷并在牙科搅拌机(VIALMIX)中0-4℃下剧烈振动15-35秒。在该过程中,质粒DNA首先包裹在阳离子脂质体内,然后装载的阳离子脂质体附着到微泡外壳。然后在输注前将所得微泡悬浮液用PBS以1∶1稀释。
实施例4:比较装载含有质粒DNA的纳米球阳离子脂质体的电中性的脂质微泡(配方2)和含有质粒DNA的微泡配方(配方1)
将根据实施例3的方法制备的装载含有质粒DNA的纳米球阳离子脂质体的微泡(“配方1”)和根据实施例2制备的含有质粒DNA的微泡配方2(“配方2”)的物理特征进行比较。通过Coulter计数器测量微泡的泡大小和浓度。为了测量微泡的DNA装载量,将每种配方用PBS洗涤3次以除去未附着的DNA pCMV-luc。用氯仿∶苯酚∶异丙醇(25∶24∶1)从微泡萃取DNA;通过260nm波长下的光密度测量DNA浓度;通过凝胶电泳证实DNA的完整性。对于配方2微泡,使用荧光标记的质粒的共焦显微术用于证实质粒DNA掺入到微泡的磷脂外壳。对于配方1,使用荧光标记的质粒的共焦显微术用于证实质粒DNA掺入到附着到微泡的磷脂外壳的脂质体。根据表I中概述的结果,与装载到含有质粒DNA的微泡(配方2)中的DNA的量相比,装载含有质粒DNA的纳米球阳离子脂质体的微泡(配方1)的DNA的量有显著提高。
表1:微泡制剂的物理特征
配方 | 泡大小(μm) | 浓度(每ml) | DNA(pg/每个微泡) |
1 | 2.16±0.34 | 5.25±0.125x109 | 76 |
2 | 1.94±0.26 | 1.29±0.178x109 | 1.26 |
实施例5:大鼠中的体内研究:装载含有质粒DNA pCMV-luc的纳米球阳离子脂质体的电中性脂质微泡(配方1)或者含有质粒DNApCMV-luc的微泡配方(配方2)的递送
使用重为200-300g的Sprague-Dawley雄性大鼠,检查了超声介导的微泡破坏对质粒DNA的体内递送。在一个实验组中,基因递送载体是根据实施例3中的步骤制备的装载含有质粒DNA pCMV-luc的纳米球阳离子脂质体的电中性脂质微泡。在第二个实验组中,基因递送载体是根据实施例2中的步骤制备的装载质粒DNA pCMV-luc的微泡配方。对每个实验组,用每组三只大鼠进行下面的步骤。
将重为200-300克的大鼠用2-3ml 4X阿佛丁(2克2,2,2-三溴乙醇和1.24ml 2-甲基-2-丁醇溶于38.76ml H2O)腹膜内麻醉。一旦麻醉,除去大鼠的胸和颈上的所有毛。在颈的中间外侧的颈静脉上方产生5mm切口,并通过切开向颈静脉加入导管。EKG探头附着到三只爪子用于监视,向胸部涂敷1-2厘米声耦合凝胶,并将S3转换器夹在胸部声耦合凝胶上。用S12转换器进行超声心动图(Sonos 5500,PhilipsUltrasound,Andover,MA)以定位心脏和以中短轴视图记录左心室功能,心肌和腔明显可以区分。使用连接导管的输注泵将1毫升微泡悬浮液以3mL/h的恒定速率在15-20分钟内注入大鼠的颈静脉。在微泡注入期间,夹住大鼠胸部的S3转换器以超谐波模式运行(设置:传输1/3MHz;接收3.6MHz;机械指数1.6;深度3cm;在每第四次心搏引发成像;R波峰值后延迟80ms;所有部分增益为0;接收增益为50;压缩为75;和线性处理后曲线)以将微泡破坏靶向心脏。在高机械指数超声前后每第四次心跳时监测大鼠左心室。
示例性读数以中短轴视图显示了微泡输注期间引发的谐波模式下大鼠的左心室,左视图显示了高机械指数超声前的左心室,右视图显示了高机械指数超声后的左心室(数据未显示)。通过心肌浑浊化的降低表明微泡的破坏。
研究后,除去导管,缝合切口并让动物苏醒。4天后,将大鼠处死;收获心房、肝脏、肺和后肢骨骼肌作为阳性和阴性对照。通过小心解剖分离左心室,然后将其分成前后部分。所有组织都用液氮快速冷冻并在-70℃下保存直到用于测定荧光素酶活性。
实施例6:大鼠中的体内研究:装载含有质粒DNA pCMV-luc的纳米球阳离子脂质体的电中性脂质微泡(配方1)和含有质粒DNApCMV-luc的微泡配方(配方2)的荧光素酶活性比较
使用以前描述的荧光素酶测定法(Chen,2003),对如实施例5中给出的分离的每种组织:前左心室、后左心室、心房、肝脏、肺和后肢骨骼肌测定转基因的表达。将每种组织用研钵和研杵粉碎然后在荧光素酶裂解缓冲液(0.1%NP-40,0.5%脱氧胆酸和蛋白酶抑制剂,Promega Corp.,Madison,WI)中用Polytron破坏。所得匀浆以10,000g离心10分钟,向20微升澄清上清液加入100微升荧光素酶反应缓冲液(Promega)。通过发光计(TD 20/20,Turner Designs,Inc.,Sunnyvale,CA)以每分钟相对光单位(RLU)测量光发射。使用商业试剂盒(Pierce Endogen;Rockford,IL)通过Lowry方法的改进方法(Brown,1989)测定总蛋白质含量。如表II中所述,结果表明使用装载含有质粒DNA的阳离子脂质体的微泡,质粒DNA向心房、前左心室、后左心室和肺的递送增加。在肝脏和肌肉中观察到基本无递送,表明超声定向的微泡破坏技术实现了质粒DNA的器官特异性。
实施例7:制备和表征装载含有质粒DNA的纳米球阳离子脂质体的多种电中性脂质微泡
使用实施例3中给出的步骤,用2%1,2-二苯酰基-sn-甘油-磷酸胆碱(配方1-C12)、2%1,2-二棕榈酰基-sn-甘油-磷酸胆碱(配方1-C16)、或者2%1,2-二癸酰基-sn-甘油-磷酸胆碱(配方1-C20)制备装载阳离子脂质体/DNA复合体的电中性脂质微泡。
表2:装载含有质粒DNA pCMV-luc的阳离子脂质体的微泡和含有质粒DNA pCMV-luc的微泡配方的荧光素酶活性
配方1=如实施例3中制备的装载含有pCMV-luc的阳离子脂质体的微泡;配方2=如实施例2制备的含有pCMV-luc的微泡配方;LV=左心室。
如实施例4中所述测量相应微泡的物理特征并在表III中概述。所有三种配方的泡大小和每毫升浓度都是相似的。每个微泡的DNA量随着碳数目的增加而增加:C20>C16>C12。
根据实施例5中给出的步骤对大鼠施用每种微泡配方,每个实验组有2只大鼠。根据实施例6的步骤对收获的组织进行荧光素酶测定,结果在表IV中给出。用配方1-C16处理导致质粒DNA更多地递送到靶组织。
表3:微泡制剂的物理特征
配方 | 泡大小(μm) | 浓度(每ml) | DNA(pg/每个微泡) |
1-C12 | 1.75±0.24 | 2.78±0.48x109 | 0.26 |
1-C16 | 2.16±0.34 | 5.25±0.125x109 | 76 |
1-C20 | 1.92±0.42 | 1.92±0.24x109 | 128 |
表4:装载含有质粒DNA pCMV-luc的阳离子脂质体的微泡配方的荧光素酶活性
配方1-C12=装载用2%1,2-二苯酰基-sn-甘油-磷酸胆碱(C12)制备的阳离子脂质体/DNA复合体的电中性脂质微泡;配方1-C16=装载用2%1,2-二棕榈酰基-sn-甘油-磷酸胆碱(C16)制备的阳离子脂质体/DNA复合体的电中性脂质微泡;配方1-C20=装载用2%1,2-二癸酰基-sn-甘油-磷酸胆碱(C20)制备的阳离子脂质体/DNA复合体的电中性脂质微泡;LV=左心室
实施例8:制备和表征装载载有脂质体DNA pCMV-luc的阳离子脂质体的OPTISONTM
如下制备装载阳离子脂质体/质粒DNA复合体的OPTISONTM(Amersham Health,Princeton,NJ)微泡(下文称作“OptisonFormula”)。向1.5ml离心管加入1.5ml OPTISONTM悬浮液(每毫升OPTISONTM含有5.0到8.0x108人白蛋白微球体;10mg人白蛋白,USP;0.22±0.11mg/mL八氟代丙烷;0.2mg N-乙酰色氨酸;和0.12-mg辛酸溶于0.9%氯化钠水溶液)。将OPTISONTM悬浮液以1000rpm离心1分钟,除去并丢弃下清液。临使用前,将2毫克质粒DNApCMV-luc加入100微升阳离子脂质体溶液(Lipofectamine 2000;Invitrogen,Carlsbad,California)并在室温温育15分钟。向OPTISONTM上清液加入所得的阳离子脂质体/质粒DNA复合体,并使用移液管充分但是温和地混合混合物。将含有混合物的管装入八氟代丙烷气体并用牙科混合器剧烈振动20秒。所得Optison Formula具有附着到白蛋白外壳的含有DNA的脂质体。
根据实施例5中给出的步骤将Optison Formula施用于大鼠,每个实验组三只大鼠。根据实施例6的步骤对收获的组织进行荧光素酶测定,结果在表V中给出。用配方1-C16处理导致质粒DNA更多地递送到靶组织。在靶组织中得到增加的蛋白质表达,尽管表达水平没有达到用磷脂制备的微泡观察到的表达水平。
实施例9:装载含有质粒DNA pDsRed-RIP的纳米球阳离子脂质体的电中性的脂质微泡的制备和活性
根据实施例3的步骤,更换质粒DNA,制备装载含有pDsRed-RIP的阳离子脂质体的电中性的脂质微泡。
表5:装载含有质粒DNA pCMV-luc的阳离子脂质体的OPTISONTM微泡配方的荧光素酶活性
LV=左心室
使用在实施例5中概述的超声定向的微泡破坏技术的改进形式,将新的配方递送到大鼠胰腺。所过表明胰腺中胰岛的70%的转染并且该转染是β细胞(产生胰岛素的细胞)特异的。
实施例9:用超声微泡破坏技术向胰岛的有效基因递送
该实施例描述了通过超声定向的微泡破坏(UTMD)技术向成年活动物的胰岛的基因递送的新方法。该技术包括在装载充气微泡前向磷脂外壳掺入质粒。然后将复合体输注到大鼠体内并用超声破坏胰腺微循环中的复合体。通过UTMD向胰岛β细胞进行基因特异递送通过使用含有大鼠胰岛素启动子(RIP)的质粒实现,报道基因表达受到接受RIP-荧光素酶质粒的动物中葡萄糖的适当调节。为了阐明生物学功效,用UTMD递送RIP-己糖激酶I质粒。这导致胰岛中己糖激酶I蛋白质表达的明显增加、血液中胰岛素水平增加,和循环的葡萄糖水平降低。总之,本文描述的UTMD小泡和构建体允许以足够效率将基因特异递送到胰岛以调节活动物中的β细胞功能。
糖尿病的两种主要形式都涉及β细胞破坏和机能障碍。I型糖尿病折磨美国约1百万患者1,是产生胰岛素的胰岛β细胞的自身免疫破坏引起的完全胰岛素缺乏的疾病。2型糖尿病折磨1千6百万美国人1,并且当胰岛素分泌能力不再能补偿外周胰岛素抗性时,发生与该疾病有关的高血糖症。如果可能将基因或者其他分子负荷递送到胰岛以增强胰岛素分泌或者β细胞存活,那么可以开发两种形式的糖尿病的潜在新的治疗2。尽管病毒载体已经用于离体(ex vivo)将基因有效转移到胰岛3,4,但是由于难以穿越内皮屏障,体内靶向β细胞还没有成功。此外,多数病毒基因转移载体5受到肝脏毒性、免疫原性性质、炎症、和低组织特异性,以及产生大量纯的病毒的困难和费用的限制。使用裸DNA或者脂质体载体具有低转染效率和需要通过直接注射的侵入性递送的缺点。
开发了使用超声定向的微泡破坏(UTMD)向特定组织递送基因或者药物的新技术。6-11简言之,将基因掺入到阳离子脂质体然后附着或者装载到充气微泡的磷脂或者白蛋白外壳以形成递送载体-微泡复合体。然后将递送载体-微泡复合体静脉内注射到靶器官并通过超声在靶器官的微脉管***内破坏所述复合体。本文教导的组合物和方法也用于增强组织特异性(见其他实施例),如用细胞特异性配体修饰微泡12,在转基因构建中使用细胞特异性13或者病理特异性14启动子,和通过基于导管的方法15,16或者直接注射17-19物理置换靶组织中的载体。
UTMD已经用于将报道基因和VEGF介导的血管生成靶向大鼠心肌(见下面的实施例)。4-7本发明阐明了报道基因向胰岛的安全和成功的靶向,使用大鼠胰岛素启动子实现高水平的胰岛和β细胞特异性,以及通过葡萄糖喂饲调节胰岛内所递送的转基因。此外,UTMD对己糖激酶-1基因的β细胞特异递送导致增加的胰岛素分泌。这些数据表明UTMD将转基因以足够β细胞功能的水平递送到成年活动物的胰岛β细胞,从而提供将治疗剂靶向糖尿病背景中的胰岛的可能手段。
简言之,向阳离子脂质体掺入质粒DNA,该质粒DNA具有处于CMV或者RIP启动子调节下的报道基因LacZ、DsRed、或者荧光素酶或者己糖激酶-1基因,所述阳离子脂质体然后附着到在磷脂外壳内含有全氟丙烷的微泡。微泡的平均直径和浓度分别为1.9±0.2μm和5.2±0.3x109个泡/ml。吸附到微泡的质粒的量为250±10μg/ml。经由麻醉的Sprague-Dawley大鼠(250g)的右颈内静脉在20分钟内输注1毫升质粒-微泡溶液或者对照(没有质粒的微泡)。超声指向胰腺以破坏胰腺微循环内的这些微泡;不用超声的微泡输注也用作对照。
质粒DNA的原位PCR。图1(上方小图)显示了针对质粒DNA的原位PCR的结果。在整个胰腺(包括胰岛)中看到核模式的质粒DNA。在超声束内的左肾、脾脏、肝脏部分内观察到质粒的均匀核组织定位的类似模式。在超声束外的器官:右肾或者骨骼肌中不存在质粒。对于含有CMV或者RIP启动子和LacZ或DsRed标记基因的质粒都是这种情况。对照(没有质粒的微泡或者没有超声的质粒-微泡)没有显示出胰腺内存在质粒的迹象。这些图表明超声处理在胰腺及其紧邻的附近释放质粒。
mRNA的原位RT-PCR。为了赋予胰岛特异性表达,通过UTMD递送大鼠胰岛素启动子(RIP)驱动的报道子构建体。图1(底部小图)显示了针对对应于在RIP启动子控制下表达的DsRed转录物的mRNA的原位RT-PCR的代表性实例。在整个胰岛中看到DsRed mRNA,但是在胰腺实质中没有看到DsRed mRNA,表明RIP启动子指导UTMD递送的DsRed mRNA仅在内分泌胰腺中转录。在对照中没有检测到信号,所述对照包括没有质粒的微泡、LacZ质粒-微泡或者没有超声的DsRed质粒-微泡。
通过共焦显微术阐明DsRed向胰岛β细胞的特异靶向。接着检查DsRed蛋白质的表达以确定表达是否局限于胰岛内产生胰岛素的β细胞。图2图解了胰岛细胞的中部核心内DsRed蛋白质的表达,与大鼠胰岛内β细胞的已知定位一致。用红色滤光器在568nm的激发波长和590-610nm的发射波长下鉴定DsRed蛋白质(左边小图,顶部)。通过用针对胰岛素的荧光标记的抗体免疫组织化学染色,在488nm的激发波长和490-540nm的发射波长特异鉴定β细胞(中间小图,顶部)。DsRed和胰岛素信号的共定位(右边小图,顶部)证明DsRed质粒表达存在于胰岛β细胞中。DsRed信号仅仅存在于用抗-胰岛素共同染色的胰岛组织内,表明高度β细胞特异性。此外,通过胰岛素染色鉴定了不显示出DsRed表达的胰岛。来自用对照微泡(没有质粒)或者对照质粒(LacZ)输入的大鼠的切片检查没有显示出任何可检测到的DsRed信号(数据未显示)。
DsRed表达相对于产生胰高血糖素的α细胞的定位也在图2中显示(底部小图)。使用红色滤光器,在左下小图中显示了DsRed蛋白质。通过用针对胰高血糖素的荧光抗体进行免疫组织化学染色,在胰岛外周鉴定了α细胞(亮绿色信号,中间小图,底部)。共焦显微术(右边小图,底部)表明DsRed信号从不与胰高血糖素信号共定位,胰高血糖素信号保持亮绿色并且位于胰岛外周。
通过将DsRed阳性胰岛数除以胰岛(抗胰岛素阳性)总数x100来计算胰岛转染的效率。结果在表1中显示。与CMV-DsRed质粒相比,用RIP-DsRed处理的胰岛的转染效率显著更高(67±7%对20±5%,F=235.1,p<0.0001)。如上面提到的,用对照微泡(无质粒或者LacZ质粒)处理的胰岛不显示出任何可检测到的转染。
表6.以DsRed阳性胰岛数/抗胰岛素阳性胰岛数X100确定的胰岛的转染率
大鼠#-质粒 | 载玻片1 | 载玻片2 | 载玻片3 | 总计 |
1-RIP-DsRed | 16/23(69%) | 18/22(81%) | 15/21(71%) | 49/66(74%) |
2-RIP-DsRed | 19/32(59%) | 17/27(63%) | 15/22(68%) | 51/81(63%) |
3-RIP-DsRed | 8/12(67%) | 9/14(64%) | 7/10(70%) | 24/36(67%) |
4-RIP-DsRed | 17/32(53%) | 20/29(69%) | 18/28(64%) | 55/89(62%) |
5-CMV-DsRed | 3/21(14%) | 7/25(28%) | 2/12(17%) | 12/61(19%) |
6-CMV-DsRed | 9/32(28%) | 7/30(23%) | 6/31(19%) | 22/93(24%) |
7-CMV-DsRed | 4/20(20%) | 4/17(24%) | 5/19(26%) | 13/56(23%) |
8-CMV-DsRed | 6/35(17%) | 4/30(14%) | 4/28(15%) | 14/93(15%) |
9-对照 | 0/24 | 0/32 | 0/30 | 0/86 |
一起考虑,这些数据表明UTMD与其中转基因表达受到RIP的控制的质粒的偶联导致基因以高度定向的(如果不是唯一的方式)有效递送到活的大鼠中的胰岛β细胞。
定量荧光素酶基因表达。还将胰腺中定量的基因表达与超声束内(左肾、脾脏、肝脏)和超声束外(右肾、后肢骨骼肌)的其他器官相比较。UTMD后4天处死大鼠并测量每种器官中的荧光素酶活性并对蛋白质含量表示为RLU/mg蛋白质。图3显示了为三组大鼠(每组n=3只大鼠)进行的这些器官中荧光素酶活性的比较。该研究中包括三组大鼠:接受CMV-荧光素酶微泡并用正常食物和水饲养的动物;接受RIP荧光素酶微泡并用正常食物和水饲养的动物;和接受RIP荧光素酶微泡并用正常食物和补充20%葡萄糖的水饲养的动物。对动物提供这些饮食4天后处死。在接受CMV-荧光素酶的动物中,在超声束内的所有器官中检测到低水平的活性。在超声束外的骨骼肌或者右肾中没有检测到活性。通过ANOVA,器官之间胰腺荧光素酶活性的差异是统计学显著的(F=42.4,p<0.0001),因为与其他器官相比,胰腺中具有明显更高的活性。尤其重要的是,与肝脏相比,RIP-荧光素酶质粒将胰腺活性增加了100倍(298±168RLU/mg蛋白质对2.9±0.8RLU/mg蛋白质),表明该技术避免了用病毒载体看到的肝脏摄入的问题。3
与CMV-荧光素酶相比,RIP-荧光素酶质粒将胰腺荧光素酶活性增强4倍(298±168RLU/mg蛋白质对68±34RLU/mg蛋白质,p<0.0001)。与仅有RIP-荧光素酶相比,葡萄糖喂饲进一步将胰腺荧光素酶活性增强3.5倍(1084±192RLU/mg蛋白质对298±168RLU/mg蛋白质,p<0.0001),表明被UTMD递送到胰岛后,RIP荧光素酶转基因受到葡萄糖的合适的调节。令人惊奇地,与仅有RIP-荧光素酶相比,葡萄糖喂饲还导致左肾中荧光素酶表达的调节(172±102RLU/mg蛋白质对53±23RLU/mg蛋白质,p=0.0057),提示大鼠胰岛素启动子甚至当定位于肾时也应答葡萄糖。同样地,本发明可以用于在一种以上的器官中提供受控的表达。
通过TUMD的基因表达的时程。在单独的大鼠组中,使用RIP-荧光素酶质粒测量了基因表达的时程。在UTMD后4、7、14、21和28天,通过每次处死3只大鼠测量荧光素酶活性。如图4中所示,从第4天到第7天,荧光素酶活性下降一半,到第21天时几乎检测不到(F=234,p<0.0001)。
通过己糖激酶-1基因的UTMD介导的递送调节胰岛素分泌和循环的葡萄糖水平。以前的研究已经表明由于在低葡萄糖下增加的刺激物/分泌偶联,低Km己糖激酶(例如,己糖激酶1)的过表达导致胰岛素分泌的葡萄糖剂量应答左移。3,20因此,己糖激酶I基因用于确定通过UTMD向胰岛β细胞的基因递送是否以足够允许在完整动物背景中胰岛素功能的可辨别的改变的效率发生。对六只大鼠输注微泡,其含有具有处于RIP启动子控制下的己糖激酶I基因的质粒。对照包括用含有RIP-DsRed的微泡输注的大鼠(n=3)和假手术正常大鼠(n=3)。在基线和UTMD后第5和10天得到葡萄糖和胰岛素的血清测量。
如图5中所示,在RIP-DsRed或者假手术对照组中,血清胰岛素或者葡萄糖水平随时间没有显著改变。相比,在RIP-己糖激酶I处理组中,血清胰岛素到第5天时增加4倍并且到第10天时保持升高(F=11.5,p=0.0033,通过重复测量ANOVA,处理组对比对照)。与胰岛素的增加相关,在第5天,RIP-己糖激酶I-处理的大鼠中血清葡萄糖水平降低接近30%(F=19.8,p=0.0005通过重复测量ANOVA,处理组对比对照),然后直到第10天都保持低水平。通过第10天时分离的胰岛中己糖激酶I蛋白质水平的免疫印迹分析提供了通过UTMD向胰岛高度有效递送己糖激酶I基因的进一步证据。这些数据表明在相对于对照组,用RIP-己糖激酶I质粒进行UTMD的3只大鼠的胰岛中,可免疫检测到的己糖激酶I蛋白质明显增加。总之,图5的数据清楚地表明UTMD的使用将基因高效率递送到活动物的胰岛β细胞。
UTMD的安全性。胰腺的组织切片没有揭示UTMD后的任何炎症或者坏死证据。在4只大鼠中,在基线、UTMD后1小时和24小时测量血清淀粉酶和脂肪酶;值是正常的并且不随着UTMD增加。进行UTMD的大鼠体重正常增长并且没有表现出异常行为。此外,接受RIP-DsRed质粒的大鼠的循环葡萄糖或者胰岛素水平没有经历显著改变,提示维持了正常的代谢稳态。
该实施例描述了用于将基因有效递送到胰岛的新方法。显示了质粒DNA的递送并通过针对质粒和其mRNA的原位PCR和原位RT-PCR显示了质粒DNA的随后表达。此外,当UTMD与其中用RIP指导转基因表达的质粒结合应用时,胰腺中的基因表达限制在β细胞中。此外,已经阐明RIP-荧光素酶质粒在通过UTMD递送到胰岛后保持对生理信号的应答,因为葡萄糖喂饲导致报道基因活性的明显增加。尽管有通过受精胚胎的微注射实现啮齿动物的胰岛中转基因表达的实例,21-27但是这是体内基因递送到活的成年动物的胰岛的第一个实例。
确定UTMD方法递送基因的功效以表明β细胞功能的调节。为该目的选择己糖激酶I基因。胰岛β细胞通常表达己糖激酶IV(也称作葡萄糖激酶)作为它们的主要的葡萄糖磷酸化酶,该酶对葡萄糖的高S0.5(约6mM)允许其调节葡萄糖代谢速率和在生理葡萄糖浓度下控制葡萄糖刺激的胰岛素分泌。相比,己糖激酶I对葡萄糖具有低S0.5(约0.5mM)。相比,已知大鼠胰岛中己糖激酶I的腺病毒介导的表达导致糖酵解和葡萄糖刺激的胰岛素分泌中依赖葡萄糖浓度的改变的左移。20此外,已经证明转基因小鼠的β细胞中低Km酵母己糖激酶的表达导致高胰岛素症和低血糖症。基于这些观察,正如通过高胰岛素症和低血糖症的相似表型所证实的,发现本发明通过UTMD有效地将己糖激酶I递送到β细胞,如所观察到的和在图5中概述的。
该实施例还描述了DNA构建体向β细胞的安全和有效的递送,其具有几个优点:1)有效基因转移不需要病毒载体,减少了对炎性应答或者***诱变的担心;52)在这些质粒构建体中使用RIP启动子提供了胰岛内明显程度的β细胞特异性,DsRed报道基因在产生胰高血糖素的α细胞中表达很少或者无表达;3)装载质粒的微泡可以通过全身循环递送,消除了对侵入性手术(如局部递送到胰腺脉管所需要的)的需要;和4)没有证据表明由于微泡输注和胰腺中局部应用超声引起的胰腺损伤。
与该技术的这些非常积极的特征相平衡的是一个未预料的发现。发现在肾脏中,在RIP启动子控制下实现了荧光素酶转基因的显著表达,当定向到胰脏时,所述肾脏不可避免地处于超声束的路径中。此外,发现肾脏报道基因表达应答葡萄糖。已经显示增强的大鼠胰岛素启动子在小鼠的脑、胸腺和肾脏中表达人生长激素(hGH)。28已知胰岛素影响肾脏中腺苷29和血管紧张素原30的表达。使用本发明,还可以将基因和基因递送(例如,RIP增强的肾脏基因表达)靶向肾脏。
为了减小或者避免肾脏表达,聚焦的超声换能器可以用于将微泡破坏局限于预先指定的目的区域。在这些研究中,可以使用为临床超声心动术开发的换能器,其中在超声束的整个长度、厚度和宽度发生微泡破坏。备选地,可以修饰或者截短RIP启动子从而在肾脏中不存在转基因表达时保持β细胞表达。
该实施例中描述的组合物和方法可以用于治疗主要形式的糖尿病,并且还代表评估内分泌胰腺中候选疾病基因的相关性的方法。1型糖尿病涉及胰岛β细胞的自身免疫破坏。已经提出一些方法用于保护β细胞免于免疫介导的破坏,包括通过防止细胞/细胞相互作用阻断T细胞和巨噬细胞介导的破坏,或者备选地,注射可以保护免于炎性细胞因子或者活性氧种类导致的损害的基因。2然而,这些方法的测试局限于在移植之前胰岛的转基因(种系)操作或者离体工程化。该实施例中教导的方法提供了胰岛的原位基因工程化,从而可以在1型糖尿病的动物模型中在前期糖尿病期测试用于增强胰岛存活的多种策略。
本文教导的组合物和方法还可以用于2型糖尿病。在该疾病中,β细胞似乎遭受功能不足和细胞质量的逐渐(但不是完全)减小的双重损害。31涉及β细胞机能障碍的发展和2型糖尿病中β细胞质量的损失的机理还没有完全理解,但是已经发展了关于慢性高脂血症和β细胞中脂类过量积累(“脂毒性”)的潜在作用,32,33以及慢性暴露于葡萄糖的损害效果(“葡萄糖毒性”)34的理论。本文教导的技术使得可以将调节脂类或者葡萄糖代谢的基因递送到2型糖尿病模型的胰岛中。此外,已知为青春晚期糖尿病(Maturity Onset Diabetes of the Young(MODY))的一组疾病似乎包括涉及控制β细胞功能的转录因子或者代谢酶的一组单基因突变。35本发明允许快速测试在成年动物背景下从人遗传研究出现的β细胞候选基因的方法。最后,随着用于抑制基因表达的技术如小干扰RNA(siRNA)的出现和它们应用于胰岛,36,37含有siRNA的质粒的UTMD介导的递送可以用于控制(上调、下调)活动物中β细胞功能和存活中的特定基因。
大鼠UTMD方案。将Sprague-Dawley大鼠(250-350g)用腹膜内***(100mg/kg)和甲苯噻嗪(5mg/kg)麻醉。通过切开将聚乙烯管(PE 50,Becton Dickinson,MD)***右颈内静脉。将外腹部剃毛并放置S3探头(Sonos 5500,Philips Ultrasound,Andover,MA)以对左肾和脾脏成像,可以容易地鉴定左肾和脾脏。胰腺位于它们之间,因此调节探头以定向胰腺并夹在适当位置。使用输注泵在20分钟内以3ml/h的恒定速率输注1ml微泡溶液。在整个输注期间,使用超谐波模式(传输1.3MHz/接收3.6MHz)以1.2-1.4的机械指数和4cm的深度实现微泡破坏。用ECG引发(R波峰后以80ms)超声脉冲以每4次心搏周期递送4帧超声爆发。以前已经表明这些设置是使用UTMD进行基因递送的最佳超声参数。5在每个研究结束时,将颈静脉打结并封闭皮肤。实验后监视所有大鼠的正常行为。4天后处死大鼠并收获胰腺。
含有质粒的脂类稳定的微泡的生产。如本发明人前面描绘的制备某些脂类稳定的微泡。5,6在本发明中,将DPPC(1,2-二棕榈酰基-sn-甘油-3-磷脂酰胆碱,Sigma,St.Louis,MO)2.5mg/ml;DPPE(1,2-二棕榈酰基-sn-甘油-3-磷脂酰乙醇胺,Sigma,St.Louis,MO)0.5mg/ml和10%甘油的溶液与2mg质粒溶液以2∶1的比例混合。将该磷脂-质粒溶液的0.5ml等分试样置于1.5ml透明小瓶中;剩余的顶部空间装入全氟丙烷气体(Air Products,Inc,Allentown,PA)。每瓶在40℃温育30分钟然后通过牙科混合器(VialmixTM,Bristol-Myers SquibbMedical Imaging,N.Billerica,MA)机械振动20秒。脂类稳定的微泡呈现为漂浮在含有未附着的质粒DNA的液体层上方的乳白色悬浮体。丢弃下层清液并用PBS洗涤微泡三次以除去未附着的质粒DNA。通过颗粒计数器(Beckman Coulter Multisizer III)测量上层中微泡的平均直径和浓度。
质粒构建体。用QIAamp Blood试剂盒(Qiagen Inc,Valencia,CA)根据生产商的使用说明从大鼠外周血提取大鼠基因组DNA。通过使用在5’末端含有限制性位点的下面的PCR引物从Sprague-Dawley大鼠DNA通过PCR扩增含有大鼠胰岛素I启动子(RIP)、外显子1、内含子1(仅内含子)和外显子2(从-412到+165)的5’末端的3bp(GTC)的DNA片段(限制性位点用下划线标出):
引物1(XhoI)5’CAACTCGAGGCTGAGCTAAGAATCCAG-3’(SEQ ID NO.:1);
引物2(EcoRI)5’-GCAGAATTCCTGCTTGCTGATGGTCTA-3’(SEQ ID NO.:2)。
对应的PCR产物通过琼脂糖凝胶电泳验证并通过QIAquick GelExtraction试剂盒(QIAGEN)纯化。为了证实该序列,用dRhodamineTerminator Cycle Sequencing试剂盒(PE Applied Biosystems,FosterCity,CA)在ABI 3100基因组分析仪(Genomic Analyzer)上进行PCR产物的直接测序。将PCR扩增的片段用XhoI和EcoRI消化并连接到pDsRed-Express-1的XhoI-EcoRI位点,pDsRed-Express-1是无启动子的Discosoma sp.红色荧光蛋白质(DsRed)质粒(BD Biosciences)。在20μl的20mM Tris-HCL,0.5mMATP,2mM二硫苏糖醇和1单位的T4DNA连接酶中进行连接反应。通过标准步骤进行该质粒的克隆、分离和纯化,并再次测序以证实不存在人为突变。
如下制备表达处于RIP启动子的己糖激酶1基因的质粒:用QIAamp试剂盒(Qiagen Inc,Valencia,CA)根据生产商的使用说明从Sprague-Dawley大鼠胰腺提取总RNA。然后用RT-PCR试剂盒的SuperScript第一链合成***(Invitrogen)将mRNA逆转录成cDNA。通过使用在5’末端含有限制性位点的下面的PCR引物通过PCR扩增己糖激酶1cDNA的全长cDNA(限制性位点用下划线标出):
引物1(EcoRI)5’-AAAGAATTCATGATCGCCGCGCAACTACTGGCCTAT-3’(SEQ ID NO.:3);
引物2(Not I)5’-AAAGCGGCCGCTTAGGCGATCGAAGGGTCTCCTCT-3’(SEQ ID NO.:4)
通过测序证实产物。将DNA用EcoR1和NotI消化然后连接到pRIP3.1载体的对应的位点。通过标准步骤进行该质粒的克隆、分离和纯化,并再次测序以证明不存在人为突变。
用于检测DsRed DNA的原位PCR。DsRed引物。使用针对DsRedDNA的一对DsRed引物;它们为DsRed 125+(5’-GAGTTCATGCGCTTCAAGGTG-3’)(SEQ ID NO.:5)和DsRed 690-(5’-TTGGAGTCCACGTAGTAGTAG-3’)(SEQ ID NO.:6)。
处死后,立即通过200ml动脉内冷却的盐水从大鼠除去血液并用100ml 2%多聚甲醛和0.4%戊二醛进行灌注固定。将胰腺切割成0.5cm小块并置于20%蔗糖溶液在4℃过夜,然后置入-86℃的OTC模子。将厚为5μm的冷冻切片置于硅烷包被的切片并在4%多聚甲醛中4℃下固定15分钟,用PBS中的10mM甘氨酸淬灭5分钟,用PBS冲洗,用PBS中的0.5%Triton X-100透化10分钟,并用PBS冲洗10分钟。使用PCR DIG Prob Synthesis试剂盒(Roche Co.;Cat.NO:1636090)。在一边用一滴指甲油固定盖玻片。然后将载玻片直接置于热循环仪的区的铝“舟皿(boat)”中。将50μl PCR反应溶液(0.8单位Taq DNA聚合酶,2μl DsRed引物,3μl DIG-dNTP,5μl 10×缓冲液和40μl水)加入每个载玻片并使用Assembly Tool(Perkin Elmer)根据生产商的使用说明通过AmpliCover Disc和Clips覆盖。用Perkin-ElmerGeneAmp***1000如下进行原位PCR:最初保持在94℃(1分钟)后,PCR进行11个循环(94℃1分钟,54℃1分钟和72℃2分钟)。扩增后,将载玻片浸入2×SSC 10分钟和0.5%多聚甲醛5分钟并浸入PBS 5分钟两次。用用于DIG检测(Roche)的荧光抗体增强剂组检测掺入洋地黄毒苷的DNA片段,然后进行组织化学染色。首先,将切片用封闭溶液温育30分钟以降低抗体与胰腺组织的非特异结合。然后,将切片与50μl抗-DIG溶液(1∶25)在湿室中37℃下温育1小时。然后摇动下用PBS洗涤载玻片3次,每次5分钟。然后将载玻片用50μl抗-小鼠-IgG洋地黄毒苷抗体溶液(1∶25)在37℃温育1小时。再次摇动下用PBS洗涤载玻片3次,每次5分钟。载玻片用50μl抗-DIG-荧光溶液(1∶25)在37℃温育1小时。再次摇动下用PBS洗涤载玻片3次,每次5分钟。最后,将切片在70%EtOH、95%EtOH和100%EtOH中脱水,每次2分钟,用二甲苯透明化并盖片。
用于检测DsRed mRNA的原位RT-PCR。DsRed引物。使用针对DsRed cDNA的一对DsRed引物,它们是DsRed 125+(5’-GAGTTCATGCGCTTCAAGGTG-3’)(SEQ ID NO.:7)和DsRed 690-(5’-TTGGAGTCCACGTAGTAGTAG-3’)(SEQ ID NO.:8)。
如上述制备灌注固定的冰冻切片。在每个载玻片上用50μl混合物溶液(Invitrogen)(5μl DNase I,5μl 10×DNase缓冲液,和40μl水)进行DNA酶处理,盖片,在25℃温育过夜,然后用PBS洗涤2次,每次5分钟。
逆转录:在每个载玻片上在50μl总体积中用50μl混合物溶液(用于RT-PCR的Superscript第一链合成***,Invitrogen试剂盒#11904-018)(1μl DsRed727-引物(5’-GATGGTGATGTCCTCGTTGTG-3’)(SEQ ID NO.:9),5μl DTT溶液,2.5μl dNTP,5μl 10×缓冲液,5μl 25mM MgCl,29μl水和2.5μl SuperScript II RT)进行第一链cDNA合成。放置盖玻片并将载玻片在42℃温育2小时;用PBS洗涤2次,每次5分钟,用100%ETOH漂洗1分钟并干燥。
用于检测DsRed蛋白质、胰岛素和胰高血糖素的免疫组织化学。将5μm厚的恒冷箱切片在4%多聚甲醛中4℃下固定15分钟并用PBS中的10mM甘氨酸淬灭5分钟。将切片用PBS漂洗3分钟,并用PBS中的0.5%Triton X-100透化10分钟。切片用10%山羊血清在37℃封闭1小时并用PBS洗涤3次。加入一级抗体(Sigma Co.)(在封闭溶液中的1∶50稀释液)并在4℃温育过夜。用PBS洗涤三次,每次5分钟后,加入二级抗体(Sigma Co.,缀合FITC的抗小鼠IgG)(封闭溶液中的1∶50稀释液)并在37℃温育1小时。将切片用PBS漂洗10分钟,5次,然后封固。
荧光素酶测定法。为了定量荧光素酶转基因的表达,将胰腺、肾脏、脾脏和骨骼肌用Polytron粉碎并用荧光素酶裂解缓冲液(PromegaCo,),0.1%NP-40,和0.5%脱氧胆酸盐和蛋白酶抑制剂温育。所得匀浆以10,000g离心10分钟,并向20μl透明上清液加入100μl荧光素酶反应缓冲液(Promega)。通过发光计(TD-20/20,Turner DesignsCo.)以RLU(相对光单位)测量光发射。通过Lowry方法(BCA蛋白质测定试剂,Pierce Co.)从每个样品的等分试样测定总蛋白质含量。荧光素酶活性表达为RLU/mg蛋白质。
己糖激酶I蛋白质印迹。在处死时(UTMD基因递送后10分钟)从每只大鼠收获完整胰腺的切片并用Tris缓冲液匀浆。来自这些组织匀浆物的等量蛋白质用12%BioRad凝胶进行电泳,封闭,并用小鼠抗-己糖激酶I抗体温育。用化学发光底物(ECL,Amersham,Piscataway,NJ,USA)显示免疫反应带。
统计学分析。通过双向ANOVA比较实验组之间的荧光素酶活性的差异。用重复测量ANOVA评估时程实验的结果。用双向重复测量ANOVA评估己糖激酶1处理的大鼠和对照组之间血清胰岛素和葡萄糖的暂时改变。认为p值<0.05是统计学显著的。仅当ANOVA F值是统计学显著的时候,进行事后检定Scheffe检验。
实施例10用微泡的超声破坏将VEGF介导的血管发生靶向大鼠心肌
使用已知为超声定向的微泡破坏(UTMD)的体内定向基因递送***在大鼠心肌中促进通过人VEGF165 cDNA介导的心肌血管发生。在心肌内微泡的超声破坏期间静脉内输入携带编码hVEGF165的质粒的微泡,或者对照溶液。在UTMD后5、10和30分钟进行基因表达和血管发生的生物化学和组织学评估。UTMD处理的心肌含有hVEGF165蛋白质和mRNA。UTMD-处理的动物的心肌显示出与hVEGF165表达和内皮细胞标记相关的细胞过多病灶。UTMD处理的毛细血管密度在5天时增加18%,在10天时增加33%,在30天时返回到对照水平(p<0.0001)。类似地,微动脉密度在5天时增加22%,在10天时增加86%,在30天时增加31%(p<0.0001)。从而,通过UTMD将hVEGF165向大鼠心肌的非侵入递送导致心肌毛细血管和微动脉密度的显著增加。
已经提出通过血管生长因子和/或表达它们的基因对新血管生长刺激作为心肌局部缺血的潜在治疗。38-41尽管在理解血管发生(新毛细血管的发育)和动脉生成(含有内膜、介质和外膜的较大血管的发育)中已经取得了很大的进展,但是还没有发生基础科学向临床上有用的疗法的转化。已经指出最近血管生长疗法的临床试验的很令人失望的结果42-52可以通过多种因素解释,这些因素包括患者选择、对最佳的血管生成剂或者其组合的不完全理解、治疗的有限时程(通常是单一固定剂量),和亚最佳递送技术。后两个问题相互关联,因为当前递送血管生成剂的方法局限于直接心肌注射或者冠状动脉内注入,它们是侵入性技术,不能很好地适于重复治疗。
该实施例阐明了非侵入性方法:超声定向的微泡破坏(UTMD),其允许将基因治疗特异定向到心脏。简言之,将含有质粒DNA的阳离子脂质体附着到直径为2-4μm的充气微泡的磷脂外壳。这些微泡-脂质体复合体静脉内输注并通过低频率超声在心肌微循环内破坏。如上文显示的,UTMD可以用于将报道基因选择性递送到胰腺和肾脏。已经显示了递送到心脏的其他实例;;53-55然而,还没有报导其用于实现生物学效应。UTMD通过将人血管内皮生长因子165(hVEGF165)表达构建体非侵入性递送到大鼠心肌用于促进血管发生。
雄性Sprague-Dawley大鼠经历用含有编码hVEGF165基因的质粒的微泡、或三种不同的对照:没有微泡的hVEGF165质粒、没有质粒的微泡或者盐水进行的大鼠UTMD处理。所有大鼠都耐受UTMD程序而没有并发症并且在该程序后存活到它们的指定的在5、10或30天时的处死。在UTMD处理的或者对照大鼠之间左心室质量和部分区域缩短没有显示出显著差异(表7),提示没有发生由于UTMD引起的左心室肥大或者收缩期机能障碍。处死时,动物在活力或喂饲方面没有显示出改变,并且缺少水肿、血管瘤或者其他肿瘤的证据。
大鼠心肌中hVEGF165的存在。免疫印迹揭示处理后10天在心脏组织的匀浆中与hVEGF165一致的显著的37kDa带(图6)。在对照动物中看到可能代表内源VEGF的弱带。hVEGF165蛋白质的增加局限于UTMD定向的组织。与超声定向的区域相邻但是在该区域外的器官(如肝脏、肺和脾脏)的匀浆没有显示出hVEGF165蛋白质的相似增加。这些发现证实局限于超声化区域的外源血管发生基因的组织特异性。在任何对照动物中都没有检测到hVEGF165。
表7.处理动物对比对照动物中左心室(LV)缩短分数和质量。
与蛋白质印迹的结果一致的是,RT-PCR揭示了在第5天组和第10天组以及第30天组中一只大鼠中hVEGF165的表达(图7),但是在对照组中没有hVEGF165的表达。为了避免任何交叉污染,PCR阳性对照不用于hVEGF165。通过测序证实人VEGF165RT-PCR产物(数据未显示)。
在处理后10天,组织学揭示了UTMD处理的动物(图8)但不在对照动物中的细胞过多病灶。这些细胞过多病灶显示出用抗-VEGF抗体的染色,证明了外源血管生成基因的成功转移和表达。此外,这些病灶显示出用内皮细胞特异性标记CD-31和BS-I凝集素的染色。这些区域中内皮细胞显示出突出的细胞核和偶然的有丝***像。平滑肌α-肌动蛋白染色显示出覆盖血管的周细胞,其是血管发生的进一步证据。嗜中性粒细胞、单核细胞、浆细胞和淋巴细胞明显稀少并且没有肌细胞坏死。然而,存在成纤维细胞增殖与肌纤维结构的破坏,与轻微炎症相一致。到第30天时,这些病灶显示出炎症的消退。在对照动物中都不存在这些细胞过多病灶。
使用BS-1凝集素染色组织学评估心肌毛细血管密度(图9顶部小图)。在所有三个时间期间,三个对照组中毛细血管密度明显相似,平均为2606±150/mm2(图9,底部小图)。在UTMD-处理的大鼠中,在第5天时毛细血管密度增加18%(3079±86/mm2),第10天时增加33%(3465±283毛细血管/mm2),但是在第30天时返回到对照水平(2683±145/mm2)。通过ANOVA,这些处理组之间毛细血管密度的改变是统计学显著的(F=19.25,p<0.0001)。
使用平滑肌α-肌动蛋白(sm-α-肌动蛋白)染色评估微动脉密度(图10顶部小图)。在研究的三个时间点处,对照组之间的微动脉密度没有显著差异,平均为71±10/mm2(图10,底部小图)。在UTMD处理的大鼠中,微动脉密度在第5天时增加23%(87±3/mm2),第10天时增加86%(132±43/mm2),第30天时增加31%(93±7/mm2)。通过ANOVA,处理组之间微动脉密度的改变是统计学显著的(F=11.05,p<0.0001)。
该实施例阐明血管发生基因可以非侵入性地靶向心脏,并改变心肌微脉管***。特别地,毛细血管密度有暂时升高,微动脉密度有更持续的升高。值得注意的,这是首次证据证明转基因向心脏的非侵入递送具有治疗潜力,因为它导致导致心肌中基因表达和生物学改变。
阐明了hVEGF165质粒基因转移后在心肌中增加的毛细血管和微动脉密度。有限的质粒表达(仅在基因递送后10天在所有治疗大鼠中检测到hVEGF165蛋白质)在治疗后10天增加了毛细血管和微动脉密度。然而,到第30天时,观察到毛细血管回复到基线水平。可能这是由于质粒表达的暂时性质或者在正常而不是局部缺血心肌背景中随后的毛细血管去募集。
微动脉也从第10天时的峰值下降到处理后30天。然而,30天时的微动脉密度仍然显著高于对照,表明hVEGF165治疗后持续动脉生成。这是重要的新发现,其可能涉及UTMD后比直接注射或者冠状动脉内灌注更长的hVEGF165表达。在VEGF的条件转换的小鼠模型中,短暂暴露于VEGF导致血管的暂时生长,其在VEGF撤除后消失。56相比,10-14天的VEGF刺激产生了血管生成应答,其中成熟血管不消溶。56在该实例中,UTMD在处理后10天在大鼠心肌中产生了通过蛋白质印迹可以容易地检测的hVEGF165蛋白质。使用UTMD延长hVEGF165表达的持续时间也可以促进以前描述的平滑肌细胞-内皮细胞相互作用对新近形成的微循环的保护效应和它在血管重建中的重要作用。57-60
仅用hVEGF165质粒或者仅用微泡破坏都观察不到毛细血管或者微动脉密度的增加。并不惊奇的是静脉内VEGF不促进血管发生,这是因为循环DNA酶的影响和不存在循环质粒穿过内皮屏障的机制。然而,Song等人61阐明静脉内注射白蛋白微泡后暴露于低频超声的大鼠骨骼肌中的动脉生成,提示微泡破坏可能促进血管重建。已知超声微泡破坏导致空化、热效应、微束和自由基产生,它们是将可能与内皮细胞潜在地相互作用而导致它们的活化的因素。62-65而且,微脉管***内微泡的机械破坏产生毛细血管破裂;66,67这些破裂部位的愈合可能促进了它们的模型中动脉生成的某个方面。该研究中仅使用UTMD时血管生成效应的缺乏可以是由于与骨骼肌相比在心肌中对微泡破坏的不同应答、白蛋白和脂类微泡外壳之间的差异,或者其他未知的实验变量。
在UTMD组中注意到的轻微炎症和肌细胞的破坏可能是UTMD的VEGF介导的血管发生的结果。已知VEGF通过一些机制促进炎症,所述机制包括内皮细胞粘附标记、基质金属蛋白酶,和α-防卫素。68-70在对照组中没有这些组织学发现表明仅仅微泡的简单破坏不足以导致炎症,灌注没有微泡载体而仅仅VEGF质粒也不足以导致炎症。然而,VEGF质粒和微泡破坏的组合可能在产生炎性应答中是协同的。重要的是注意到炎性应答不导致左心室肥大或者收缩期机能障碍,证实了来自本发明人以前对心脏中微泡破坏的显著生物效应的缺乏的研究的结果。71该实施例还表明在这里使用的相似微泡浓度和超声功率下,微泡破坏不在体内诱导心脏基因表达。72最后,在使用通过UTMD递送到心脏的报道基因的以前研究中,我们通过组织学71或者基因表达72没有发现任何炎症证据。
hVEGF165的表达构建体的UTMD递送用于刺激正常心肌中的毛细血管和微动脉生长。由于对组织学评估的需要,UTMD的效应仅仅是对三个特定时间点-第5、10和30天时的血管生长。技术人员使用本文教导的组合物和方法而不用过多实验就可以确定转基因表达的时程或最大量的确立,如可以确定中间时间点的毛细血管或者微动脉应答的最大量一样。类似地,可以观察更长的时帧,例如,30天后,以确定微动脉密度是否返回到基线水平或者低氧条件是否可以维持如Hershey等人在兔后肢局部缺血模型中描述的动脉生成过程。72心脏中报导基因构建体的表达可以通过重复应用UTMD延长。72通过现存小毛细血管74的生长或者新的微动脉75的从头形成可以引起动脉生成。
有许多其他已知的血管生成因子,如成纤维细胞生长因子(FGF)、血小板衍生生长因子(PDGF)、angiopoetin-2、或者低氧可诱导的因子1-α(HIF-1α),76其可以用UTMD产生优良的血管生成或者动脉生成应答,可能没有VGEF的一些炎性后果。还应该指出血管滋养管中的血管发生可以促进或促使动脉粥样硬化,,77-81其是在该研究中没有处理的VEGF基因治疗的潜在不利效果。
UTMD可以用于将基因成功地递送到更大哺乳动物(如人、猴、狗或者猪)的心脏中。大鼠的小尺寸使得它们更适于UTMD,因为心脏足够小而完全被超声束的宽度包围和因为有较少的组织弱化或者肺干扰。
超声定向的微泡破坏(UTMD)将hVEGF165表达定向到大鼠心肌,导致毛细血管和微动脉密度的增加。该方法是非侵入性的并且允许基因表达特异靶向心脏和其他器官。还似乎对LV功能是安全的,没有有害效果。通过UTMD的心肌转染的确切分子机制仍然未确定。
动物制备和基因递送。根据NIH推荐和公共机构的动物研究委员会(institutional animal research committee)的批准进行动物研究。将雄性Sprague Dawley大鼠(200到250g,Harlan)用腹膜内***(60mg/kg)和甲苯噻嗪(5mg/kg)麻醉。从心前区和颈剔除毛发,并将聚乙烯管(PE 50,Becton Dickinson,MD)通过切断***右颈内静脉。大鼠接受四种治疗之一:装载编码处于增强的CMV的启动子下的hVEGF165基因的质粒的微泡(0.6mg DNA/kg),未附着到微泡的这些相同质粒(0.6mg DNA/kg),没有附着的质粒的单独的微泡,或者生理盐水。接受泡溶液的动物在20分钟内通过泵(Genie,KentScientific)输注与0.5ml PBS混合的0.5ml泡。1ml无泡质粒或者盐水溶液在20分钟内不加稀释以总共1ml类似地输入。
在输入期间,用通过商业途径可获得的换能器(S3,Sonos 5500,Philips Ultrasound,Bothell,WA)将超声定向到心脏。得到心脏的中间心室、短轴视图并且优化像平面后,将探针夹在合适位置。然后以超谐波模式(传输1.3MHz/接收3.6MHz)以1.6的机械指数应用超声。使用R波峰值后45-70ms延迟,通过ECG将超声的四次爆发引发到每第四次收缩末期。已经表明这些设置对于使用该仪器通过UTMD进行质粒递送是最优的。54泡破坏在所有大鼠中在视觉上是明显的。在心肌中到第四次搏动时,在视觉上看不到回波-对比信号。UTMD后,将颈静脉打结,封闭皮肤,并允许动物恢复。UTMD后使用超剂量的戊巴比妥钠(120mg/kg)在第5天(n=12)、第10天时(n=12)和第30天(n=12)时处死动物。基于发明人以前的对UTMD后报道基因表达的发现选择这些时间点。.54,55收获心脏、肺、肝、脾脏和肾用于组织学和通过蛋白质印迹评估hVEGF165蛋白质,通过RT-PCR评估mRNA。
免疫组织化学。收获的组织在methyl carnosyl中固定然后在70%乙醇中固定并用石蜡包埋。得到5μm切片、脱蜡,并通过在0.01M柠檬酸钠,pH 6.0中通过微波以900W加热20分钟进行CD31、hVEGF165和平滑肌α-肌动蛋白的抗原回收。将切片用10%山羊血清封闭并用甲醇中的0.3%H2O2淬灭内源过氧化物酶活性。根据生产商的推荐用一级单克隆抗体温育切片:1∶50稀释度的抗-CD31、1∶20稀释度的抗平滑肌α-肌动蛋白,和1∶100稀释度的抗人VEGF-165,接着是生物素化的二级抗体:CD31和平滑肌α-肌动蛋白的抗-小鼠IgG和VEGF的抗山羊IgG。用Griffonia simplicifolia凝集素I∶BS-I凝集素生物素化的抗体(Sigma-Aldrich,St Louis,MO,USA)进行凝集素染色,用10%山羊血清封闭后不进行抗原回收并如上淬灭。所有染色都用HRP-链霉抗生物素蛋白进行然后用DAB色原显影,然后用苏木精复染。
RT-PCR。用RNeasy Mini试剂盒(QIAGEN)根据生产商的使用说明从标本制备总RNA。用Sensiscript RT试剂盒(QIAGEN)在总共20μl反应液中用30ng总RNA进行cDNA合成。用GeneAmp PCRSystem 9700(PE ABI)在50μl体积中对所有样品进行PCR,所述50μl体积含有2μl cDNA,25μl HotStarTaq Master Mix(QIAGEN)和20pmol每种引物:5’GGAGGAGGGCAGAATCATCAC 3’(正义)(SEQID NO.:10);5’CGCTCTGAGCAAGGCCCACAGG 3’(反义)(SEQID NO.:11),所述PCR在下面的条件下进行:最初加热到94℃10分钟,然后48个循环的94℃20秒,56℃20秒,72℃30秒;然后72℃5分钟。然后在2%琼脂糖凝胶上分析RT-PCR产物。使用大鼠VEGF引物5’ACAGAAGGGGAGCAGAAAGCCCAT 3’(正义引物)(SEQID NO.:12);5’CGCTCTGACCAAGGCTCACAGT 3’(反义引物)(SEQ ID NO.:13)的PCR反应作为阳性对照。
VEGF蛋白质印迹。在基因递送后的每个时间点(5、10和30天)将从组织匀浆收获的等量蛋白质通过12%SDS聚丙烯酰胺凝胶进行电泳并转移到聚偏二氟乙烯膜(Immobilon,Millipore,Billerica,MA,USA),封闭,并用抗-人-VEGF抗体温育。用化学发光底物(ECL,Amersham,Piscataway,NJ,USA)显现免疫反应带。
毛细血管和微动脉密度测量。认为通过免疫组织化学显示具有<10μm直径的BS-I凝集素阳性血管和直径>30μm的平滑肌α-肌动蛋白阳性血管分别是毛细血管和微动脉。使用光学显微术在400X放大倍数下计数毛细血管。从每个载玻片照5张显微照片并在每个显微照片上放置网格。使用随机数字发生器,从每个网格的选择5个切片用于计数,每只得到大鼠总共25个视野。毛细血管密度表达为每mm2数目。仅仅计数与血管垂直取向的切片。以相似的方式用200X放大倍数计数微动脉密度,因为微动脉血管比毛细血管数目少得多。读取毛细血管或者微动脉密度的研究人员对于治疗组和处死时间是未知的。
超声波心动描记法。用从12MHz宽带换能器(S12探头,PhilipsUltrasound,Bothell,WA)获得的数字图像得到LV质量和部分区域缩短的超声心动图测量。通过面积-长度方法如下计算LV质量:
LV质量=1.05{[5/6A1(L+t)]-[5/6A2(L)]},
其中A1=心外膜面积并且A2=在舒张末期从短轴视图得到的心内膜面积;L=在舒张末期长轴视图得到的从LV顶点到二尖瓣环的中央的左心室(LV)长度;t=从短轴腔区反过来计算的心肌厚度。
从下面的公式评估部分区域缩短:
FS=(LVEDA-LVESA)/LVEDA,
其中LVEDA=左心室舒张末期面积(cm2)并且LVESA=左心室收缩末期面积(cm2)。
数据分析。用Statview软件(SAS,Cary,NC)分析数据。结果表达为平均值±1标准差。通过ANOVA用Fisher的事后检定分析差异并认为p<0.05时是显著的。
含有质粒的脂类稳定化的微泡的生产。如本发明人以前描述的54,55制备脂类稳定化的微泡。简言之,将DPPC(1,2-二棕榈酰基-sn-甘油-3-磷脂酰胆碱,Sigma,St.Louis,MO)2.5mg/ml;DPPE(1,2-二棕榈酰基-sn-甘油-3-磷脂酰乙醇胺,Sigma,St.Louis,MO)0.5mg/ml;和10%甘油的溶液置于1.5ml透明小瓶中;剩余的顶部空间装入全氟丙烷气体(Air Products,Inc,Allentown,PA)。每瓶在室温温育30分钟然后通过牙科混合器(VialmixTM,Bristol-Myers Squibb MedicalImaging,N.Billerica,MA)机械振动20秒。脂类稳定的微泡呈现为液体层的上方漂浮的乳白色悬浮体。丢弃下层清液并用颗粒计数器(Beckman Coulter Multisizer III)测量上层中微泡的平均直径和浓度。通过用2mg质粒DNA与50μl阳离子脂质体溶液(lipofectamine2000,Invitrogen)混合并在室温温育15分钟来制备含有质粒DNA的阳离子脂质体。这形成了纳米球大小的阳离子脂质体复合体,其包裹质粒DNA。79如上通过向250μl磷脂包被的微泡加入50μl脂质体并在混合器中室温下摇动20秒,用全氟丙烷气体填充小瓶的顶部空间来制备具有阳离子脂质体-质粒复合体的微泡。
质粒构建和DNA制备。如下制备处于增强的CMV启动子下的具有内含子的表达hVEGF165基因的质粒:用QIAamp Blood试剂盒(Qiagen Inc,Valencia,CA)根据生产商的使用说明从健康志愿者血液提取总mRNA。然后用用于RT-PCR试剂盒(Invitrogen)的SuperScript第一链合成***将mRNA逆转录成cDNA。使用下面的PCR引物通过PCR扩增hVEGF165 cDNA的全长cDNA,所述引物在5’末端含有限制性位点(限制性位点加下划线):
引物1(XhoI)5’-TTCCTCGAGAATGAACTTTCTGCTGCTGTCTTG-3’(SEQ ID NO.:14);
引物2(Sma1)5’-AAACCCGGGTCACCGCCTCGG CTTGTCA-3’(SEQ ID NO.:15).
通过测序证实产物。将DNA用XhoI和SmaI消化然后连接到pCI-neo(Promega)的对应位点中。该质粒的克隆、分离和纯化通过标准步骤进行,80并再次测序以证实不存在人为突变。
将理解本文描述的具体实施方案通过阐明进行说明并且不作为本发明的限制。本发明的主要特征可以用于多种实施方案而不背离本发明的范围。本领域技术人员将认识到或者使用仅仅常规实验就可以确定本文描述的特定步骤的许多等同方案。认为此类等同方案在本发明的范围内并且本权利要求覆盖。
说明书中提到的所有出版物和专利申请指出本发明所属领域的技术人员的技术水平。所有出版物和专利申请都引入本文作为参考,就好像每个单独的出版物或者专利申请都特别并单独指出引入作为参考一样。
在权利要求中,所有过渡短语如“包含”、“包括”、“携带”、“具有”、“含有”、“包括”等等都理解为是开放的,即表示包括但不限于。仅仅过渡短语“由…组成”和“基本由…组成”分别将是封闭或半封闭的过渡短语。
本文公开和要求保护的所有组合物/或方法都可以按照本公开做出和实施而不用过多实验。尽管本发明的组合物和方法已经按照优选实施方案描绘,但是本领域技术人员将显而易见的是变通方案可以应用于本文描述的组合物和/或方法和方法的步骤中或者方法的步骤顺序中而不背离本发明的概念、精神和范围。更特别地,将显而易见的是,化学和生理学相关的一些试剂可以代替本文描述的试剂而将实现相同或相似结果。本领域技术人员显而易见的所有此类相似的替代和修饰都被认为在如通过所附权利要求定义的本发明的精神、范围和概念之内。
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Claims (33)
1.用于递送活性剂的组合物,其中所述组合物包含电中性的脂类微泡,该脂类微泡包含预装配的脂质体,该脂质体包含处于启动子控制下的核酸片段,其中所述微泡可以通过在靶目标处暴露于超声而选择性释放所述核酸片段,其中该核酸片段保持在微泡中受保护直到在靶标处选择性释放。
2.如权利要求1的组合物,其中该核酸片段包含处于组织特异性启动子控制下的组织特异性基因。
3.如权利要求1的组合物,其中该核酸片段处于可活化的启动子控制下。
4.如权利要求1的组合物,其中该核酸片段处于可活化的启动子控制下,所述启动子驱动导致细胞凋亡的基因的表达。
5.如权利要求1的组合物,其中该核酸片段编码选自由:激素、生长因子、酶、载脂蛋白凝固因子、肿瘤抑制因子、肿瘤抗原、病毒蛋白、细菌表面蛋白,和寄生虫细胞表面蛋白组成的组的基因。
6.如权利要求1的组合物,其中该微泡分布在药学上可接受的载体中。
7.如权利要求1的组合物,其中该核酸片段包含可表达基因,该基因选自由p53、p16、p21、MMAC1、p73、zac1、C-CAM、BRCAI、Rb、Harakiri、Ad E1 B、ICE-CED3蛋白酶、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-12、IL-13、IL-14、IL-15、TNF、GMCSF、β-干扰素、γ-干扰素、VEGF、EGF、PDGF、CFTR、EGFR、VEGFR、IL-2受体、***受体、Bcl-2或Bcl-xL、ras、myc、neu、raf、erb、src、fms、jun、trk、ret、gsp、hst、ab1、p53、p16、p21、MMAC1、p73、zac1、BRCAI、BRCAII、Rb、生长激素、神经生长因子、胰岛素、促肾上腺皮质激素、甲状旁腺激素、促卵泡激素、***和促甲状腺激素组成的组。
8.如权利要求1的组合物,其中该启动子选自由CMV IE、LTR、SV40IE、HSV tk、β-肌动蛋白、胰岛素、人珠蛋白α、人珠蛋白β和人珠蛋白γ启动子组成的组。
9.如权利要求1的组合物,其中该超声以脉冲和聚焦模式应用。
10.如权利要求1的组合物,其中该超声以超谐波模式应用。
11.如权利要求1的组合物,其中该微泡包含生物可降解的聚合物。
12.如权利要求1的组合物,其中该微泡包含生物相容性两亲性物质。
13.如权利要求1的组合物,其中该微泡包含具有外壳的微泡,所述外壳包含生物相容性两亲性物质的外层和生物可降解的聚合物的内层。
14.如权利要求1的组合物,其中该微泡由选自胶原、明胶、白蛋白或者球蛋白的两亲性物质制成。
15.如权利要求1的组合物,其中该核酸片段包含核酸载体,该载体包含处于胰岛素启动子控制下的己糖激酶基因。
16.如权利要求1的组合物,其中该核酸片段包含核酸载体,该载体包含处于RIP启动子控制下的己糖激酶基因I。
17.如权利要求1的组合物,其中所述的活性剂中包含核酸载体,所述的核酸载体包含hVEGF蛋白质、hVEGF mRNA或者hVEGF蛋白质和hVEGF mRNA两者。
18.如权利要求1的组合物,其中该核酸片段包含核酸载体,该载体包含hVEGF165蛋白质、hVEGF165mRNA或者hVEGF165蛋白质和hVEGF165mRNA两者。
19.如权利要求1的组合物,其中该脂质体包含与质粒混合的1,2-二棕榈酰基-sn-甘油-3-磷脂酰胆碱和1,2-二棕榈酰基-sn-甘油-3-磷脂酰乙醇胺甘油。
20.用作药物的用于递送活性剂的组合物,该组合物包含含有脂质体的电中性脂类微泡,所述脂质体含有处于启动子控制下的一种或多种核酸片段,所述核酸片段在靶标处使用超声释放。
21.如权利要求20的组合物,其中该微泡在药学上可接受的载体中。
22.用于超声定向的微泡破坏的药物递送组合物,其包含
包含于脂质体中处于启动子控制下的核酸片段以形成脂质体-核酸复合体;所述脂质体-核酸复合体包含于电中性的脂类微泡中,其中所述脂质体-核酸复合体保持在所述脂类微泡中受保护直到超声定向的微泡破坏。
23.如权利要求22的组合物,其中该脂质体-核酸复合体包含阳离子脂类、阴离子脂类或者其混合物和组合。
24.如权利要求22的组合物,其中该微泡分布在药学上可接受的载体中。
25.如权利要求22的组合物,其中该脂质体-核酸复合体包含可表达基因,该基因选自由p53、p16、p21、MMAC1、p73、zac1、C-CAM、BRCAI、Rb、Harakiri、Ad E1 B、ICE-CED3蛋白酶、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-12、IL-13、IL-14、IL-15、TNF、GMCSF、β-干扰素、γ-干扰素、VEGF、EGF、PDGF、CFTR、EGFR、VEGFR、IL-2受体、***受体、Bcl-2或Bcl-xL、ras、myc、neu、raf、erb、src、fms、jun、trk、ret、gsp、hst、ab1、p53、p16、p21、MMAC1、p73、zac1、BRCAI、BRCAII、Rb、生长激素、神经生长因子、胰岛素、促肾上腺皮质激素、甲状旁腺激素、促卵泡激素、***和促甲状腺激素组成的组。
26.如权利要求22的组合物,其中该脂质体-核酸复合体包含启动子和处于该启动子控制下的基因,该启动子选自由CMV IE、LTR、SV40 IE、HSV tk、β-肌动蛋白、胰岛素、人珠蛋白α、人珠蛋白β和人珠蛋白γ启动子组成的组。
27.如权利要求22的组合物,其中该核酸片段包含己糖激酶基因,且所述启动子是胰岛素启动子。
28.如权利要求22的组合物,其中该核酸片段包含己糖激酶基因I,且所述启动子包含RIP启动子。
29.如权利要求22的组合物,其中该脂质体-核酸复合体包含核酸载体,该载体包含hVEGF蛋白质、hVEGF mRNA或者hVEGF蛋白质和hVEGF mRNA两者。
30.如权利要求22的组合物,其中该脂质体-核酸复合体包含核酸载体,该载体包含hVEGF165蛋白质、hVEGF165mRNA或者hVEGF165蛋白质和hVEGF165mRNA两者。
31.如权利要求22的组合物,其中该脂质体-核酸复合体包含与质粒混合的1,2-二棕榈酰基-sn-甘油-3-磷脂酰胆碱和1,2-二棕榈酰基-sn-甘油-3-磷脂酰乙醇胺甘油。
32.如权利要求22的组合物,其还包含所述电中性的脂类微泡上的包衣。
33.如权利要求22的组合物,其还包含一种或多种含铁试剂。
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EP1793865A2 (en) | 2007-06-13 |
WO2007008220A3 (en) | 2007-12-13 |
WO2007008220A2 (en) | 2007-01-18 |
US20070207194A1 (en) | 2007-09-06 |
US20140134234A1 (en) | 2014-05-15 |
EP1793865A4 (en) | 2009-05-13 |
CN101389273A (zh) | 2009-03-18 |
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WO2007008220A8 (en) | 2008-02-07 |
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