CN116687856A - 用于基因治疗的靶向药物递送的靶向纳米载体 - Google Patents
用于基因治疗的靶向药物递送的靶向纳米载体 Download PDFInfo
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Abstract
本发明涉及靶向纳米载体以及优先或主动地将用于基因转移或基因组编辑的工具或者用于基因沉默或基因表达的转录后调控的工具靶向并且递送至一系列哺乳动物细胞种类的方法。细胞特异性靶向通过使用特征在于适合的靶向锚的纳米载体来实现,这些靶向锚具有靶向部分,该靶向部分可以是碳水化合物、抗体或抗体片段、非抗体蛋白衍生物、适体、脂蛋白或其片段、肽聚糖、脂多糖或其片段、或者CpG DNA。此类靶向锚可以包括或可以不包括聚合间隔物,如聚乙二醇。纳米药物应使得能够经由不同应用途径治疗性地解决一系列哺乳动物疾病实体。这些适应症包括恶性疾病、自身免疫性疾病、遗传性障碍、代谢障碍、或传染性疾病。
Description
本申请是申请日为2016年4月29日,申请号为201680033133.6,发明名称为“用于基因治疗的靶向药物递送的靶向纳米载体”的分案申请。
技术领域
本发明涉及医学领域,并且具体地涉及通过纳米载体将基因治疗剂靶向递送至受一系列疾病实体影响的哺乳动物细胞,这些疾病实体可以通过这种治疗性干预得以治疗或治愈。
背景技术
生物活性分子靶向递送至哺乳动物细胞(如癌细胞)仍然面临许多障碍的挑战,特别是在体内。这些障碍包括(a)递送载体的组成、功能特征及稳定性,(b)包装具有治疗显著性浓度的生物活性分子,(c)在体内靶向希望的患病细胞,(d)克服不同细胞内屏障并成功向胞内靶标递送治疗浓度的生物活性分子,(e)避开宿主免疫系统的一系列成分,例如抗体、补体因子和巨噬细胞,这些成分可能会在递送载体到达其靶标之前将其破坏,(f)穿过血管壁的内皮屏障,特别是在肿瘤块的位置,(g)迁移通过若干层细胞,从而到达靶标(例如,已知实体瘤是包含肿瘤细胞和正常细胞二者的组织化结构;因此,递送载体必须穿过若干层正常细胞以接近恶性细胞),(h)迁移通过填充细胞之间的空间的胞外基质(ECM),胞外基质由糖蛋白、硫酸化糖胺聚糖、透明质酸、蛋白多糖和胶原组成,并且因此阻碍递送系统的运输,和(i)解决细胞微环境中的高间质性高血压(即血管外升高的液体静压力),这可能限制生物活性分子接近其作用部位。
已经提出了许多不同的递送载体用于核酸和药物二者的递送,包括病毒载体、非病毒非生命载体以及非病毒的生命载体。非病毒非生命载体已适用于核酸和药物二者的递送。其他两种类型的载体已经主要适用于核酸的递送。相似地,所有这些载体都有优点以及缺点。
非病毒非生命载体(运载体)的实例有阳离子聚合物(多聚复合物)、阳离子脂质(脂质体、脂质复合物)和合成纳米粒子(纳米复合物)。它们比病毒载体更通用,并且提供若干明显的优点,因为它们的分子组成可以被控制,制造与分析此类载体相当简单,它们可以适应于一系列转基因的大小,并且它们的免疫原性更低。然而,非病毒非生命载体的基因递送效率显著低于病毒载体。需要至少106个质粒拷贝来转染单个细胞,其中大约102-104个拷贝实际上到达细胞核以进行转基因表达(Feigner等人,1989;James等人,2000;Tachibana等人,2002)。这种低效率是由于非病毒非生命载体无法克服在给药部位与靶细胞的细胞核中的定位之间所遇到的大量挑战,包括(a)DNA及其递送载体在细胞外间隙的物理和化学稳定性,(b)通过胞吞作用的细胞摄取,(c)在运输到溶酶体和胞质运输之前从内体区室逃逸,以及(d)用于转录的质粒的核定位。
纳米技术的发展带来了许多科学领域的革命性的前景,其中是通过引入了在纳米尺度上操作不同材料的可能性,从而赋予在诸如基于细胞的治疗或诊断领域具有不同应用的各种结构。纳米载体代表纳米技术研究中产生的副产物之一。大量纳米粒子已被开发出来,包括碳纳米管、聚合物载体、树枝状聚合物、金属粒子(例如金纳米壳)和基于脂质的粒子(例如脂质体)(Peer D等人,Nat Nanotech[自然纳米技术]2007;2:751-60)。在所有提及的粒子中,脂质体是目前用于药物递送的最先进的系统之一,已有若干种批准用于临床使用的配制品(Wang AZ等人,Annu Rev Med[医学年评]2012;63:185-98)。
脂质体为囊泡状组织化结构,即磷脂膜双分子层包围着内部水性核心(Allen,1998)。这些系统的成功是由于其具有生物相容的且可生物降解的组成,该组成进一步使得能够将亲水性试剂封装于内部水性核心中、将疏水性药物封装于膜双分子层中或将两亲剂封装于两个区室中,从而保护它们免于降解(Allen TM,Drugs[药物]1998;56:747-56;WangAZ等人,Annu Rev Med[医学年评]2012;63:185-98)。另外,脂质体改变了封装药物的药物代谢动力学和体内分布特征(Allen TM,Drugs[药物]1998,56:747-56)。
在调理素作用之后,“第一代”脂质体配制品呈现出快速的血液清除率以及在单核巨噬细胞系统(MPS)内的累积(Immordino ML等人,Int J Nanomedicine[国际纳米医学杂志]2006;1:297-315;Wang AZ等人,Annu Rev Med[医学年评]2012;63:185-98)。通过调整脂质组成,并且尤其是通过将聚-(乙二醇))——“PEG”合并到脂质体的表面上,长循环脂质体作为“第二代”随后出现。PEG在粒子周围构建“亲水云”,从而使通过MPS的调理素作用过程和清除率最小化,提供称为脂质体的技术(Immordino ML等人,Int JNanomedicine[国际纳米医学杂志]2006;1:297-315)。对于其他粒子也应用相同的策略,以增加其血液循环时间。然而,被动靶向并不能解决特定药物递送的问题。为了规避这种限制,实际上技术人员可以进一步使用内化的靶向部分改变纳米粒子表面(Peer D.等人,NatNanotech[自然纳米技术]2007;2:751-60;Wang AZ等人,Annu Rev Med[医学年评]2012;63:185-98)。已经使用了若干种靶向部分,包括抗体、Fab’和F(ab’)2片段、适体、小肽或纳米抗体,其中,它们在细胞表面识别(过度)表达的受体并促进纳米粒子的活性内化(Peer D等人,Nat Nanotech[自然纳米技术]2007;2:751-60)。例如,Gao等人使用RGD肽来促进用于肿瘤荧光成像的量子点的靶向递送(Gao J等人,Bioconj Chem[生物共轭化学]2010;21:604-9)。尽管增加了在靶细胞(体外)中的药物积累,但胞内递送并不一定保证生物利用度的提高,因为(具体功能活性的)药物必须从内体区室逃逸。
癌症是靶向治疗的主要实例。排名仅次于心血管疾病,癌症是全球第二大死因。除了其他治疗选择,恶性肿瘤也通过化学疗法进行治疗。然而,这些侵蚀性试剂也影响快速增殖的健康旁邻细胞。因此,化学治疗药的毒性与多药耐药性发展需要安全有效的靶向治疗。Perez-Herrero和Fernandez-Medarde最近回顾说,这些靶向策略使得能够特异性地将化学治疗剂递送到肿瘤,从而避开正常的健康组织,减少全身性毒性,保护药物免于降解,并增加其半衰期、溶解度连同减少肾清除。迄今为止,市场上已经有两种聚合物-蛋白质共轭物、五种脂质体配制品和一种高分子纳米粒子。然而,由于这些新的靶向治疗具有许多优点,大量临床试验正在进行中(Pérez-Herrero E和Fernández-Medarde A.Eur JPharm Biopharm[欧洲制药学与生物制药学杂志]2015年3月23日)。“经典”癌症治疗的改进只能对患者生存期产生适度的影响。
到目前为止,核酸的细胞靶向递送仍是一个挑战。这主要是由于核酸低效递送至细胞和细胞核中,事实上,这是靶向药物递送的主题。Grunwald和Ulbert最近讨论了通过物理手段克服该问题的技术,例如体内电穿孔和与佐剂的共同给药(Grunwald T和UlbertS.,Clin Exp Vaccine Res[临床试验疫苗研究]2015)。其他用于改进的努力在于升级质粒DNA的生产与纯化方法(Xenopoulos A和Pattnaik P.,Expert Rev Vaccines[疫苗专家评论]2014;13:1537-51)。还开发了可以执行于质粒DNA上以产生持久的、高水平的基因表达的不同策略(Adijanto J和Naash MI.,Eur J Pharm Biopharm[欧洲制药学与生物制药学杂志]2015年1月12日)。
如上面已经提及的,当以游离形式给药时,核酸不是非常有效的。因此,它们用于治疗疾病的成功施用需要采用允许治疗性核酸安全有效地进入细胞的纳米载体(Silva AC等人,Curr Drug Metab[最新药物代谢]2015年4月1日)。这一需求最近由Aaldering及其同事概括表述为:“有效利用治疗性寡核苷酸(例如siRNA、反义寡核苷酸、antimiR等)的主要障碍是将其特异性递送至靶组织。治疗性寡核苷酸的不良细胞摄取阻碍了体外和体内的基因靶向有效性。”(Aaldering LJ等人,RNA Biol[RNA生物学]2015;12:412-25)。
发明内容
由于问题继续阻碍治疗的成功,因此迫切需要有靶向递送策略,该策略将选择性地递送生物活性剂至靶细胞和靶器官,抑或保护正常组织免受给药的治疗剂的损害。此类策略应通过增加治疗剂的治疗指标,同时最小化治疗相关毒性的风险来提高治疗效果。
因此,本发明的目的是提供一种用于将活性试剂特异性递送至特定细胞种类以治疗哺乳动物中的若干种疾病的方法。
权利要求1的主题已经解决了该目的。优选实施例定义于附属权利要求中。
因此,本发明涉及通过使用纳米载体优先将药物递送至哺乳动物受试者的细胞种类的方法,该纳米载体包含特异性针对于给定细胞种类的靶向锚以治疗性地解决哺乳动物疾病实体,其中该药物是一种用于基因组编辑、基因组沉默或基因表达转录后调控的基因治疗工具,所述工具选自质粒、人工改造的限制性内切酶、特异性编码大范围核酸酶的质粒或者用于转录基因调控或转录后基因调控的工具。
通常,本发明涉及靶向纳米载体——也称为纳米药物——以及优先或主动地靶向一系列哺乳动物细胞种类并递送用于基因转移或基因组编辑的工具(即,质粒或限制性内切酶——例如锌指核酸酶、CRISPR/Cas系统或TALEN)或者用于基因沉默或基因表达的转录后调控的工具(即,微RNA、siRNA、mRNA、反义寡核苷酸或正义寡核苷酸)。细胞特异性靶向通过使用具有适合靶向锚的纳米载体来实现,该靶向锚具有靶向部分,其可以是碳水化合物、抗体或抗体片段、非抗体蛋白衍生物、适体、脂蛋白或其片段、肽聚糖、脂多糖或其片段或者CpG DNA。此类靶向锚可以包括或可以不包括聚合间隔物,如聚乙二醇。纳米药物应使得能够经由不同应用途径治疗性地解决一系列哺乳动物疾病实体。这些适应症包括恶性疾病、自身免疫性疾病、遗传性障碍、代谢障碍、或传染性疾病。
在优选的实施例中,本发明涉及封装mRNA或DNA用于在肝细胞中分别进行翻译或表达的肝细胞特异性纳米载体。
附图说明
图1:根据纯化步骤,Q-PCR显示出高度可接受的DNA封装率(每μL拷贝数)。
图2:通过针对HIV-1 5’tat剪接受体位点的正义寡核苷酸减少HIV-1感染细胞。通过免疫细胞化学鉴定表达HIV-1核心抗原p17和包膜抗原gp41/gp160二者的细胞,每个条件评估500个细胞。高度感染的细胞(黑色条)在胞质溶胶和细胞膜中均被染色,而弱染色的细胞通常仅在膜中是抗原阳性的,并且在一些病例中,胞质溶胶中也呈现暗淡的染色。总感染细胞的百分比显示为阴影条。高度感染细胞数目的减少与上清液中p24水平的降低强相关。载有正义条件的纳米载体将活跃表达HIV-1的细胞的比例降低了71%。相比之下,反义条件降低了6%,因此甚至低于对照条件,对照条件下纳米载体使用PBS填充,仅降低了18%。大型细胞聚集体——典型地如HIV-1诱导的细胞融合——可在除了正义条件以外的所有条件下被观察到。因此,封装针对5’tat剪接受体位点的正义寡核苷酸的纳米载体抑制84%的病毒蛋白的产生,并将活跃表达病毒的细胞的比例降低了71%。因此,正义条件再次导致抑制HIV-1传播,而反义条件则不会。
具体实施方式
本发明的一些实施例是指将活性剂或药物优先递送至哺乳动物受试者的细胞的方法,该哺乳动物受试者包括表达表面受体的人,这些表面受体使得靶向纳米载体能够实现由受体介导的摄取,以便向所述细胞递送其治疗性负载。细胞的分类和类型指定如下。
术语“优先”是指将靶向纳米载体递送至哺乳动物受试者的细胞(该哺乳动物受试者包括表达靶向结构的人),并且与相同的活性剂相比:(i)以其游离可溶形式存在,或(ii)当与具有不包含靶向配体的外表面的比较性纳米载体相关时,与靶向纳米载体相关的基因治疗活性剂能更有效地被所述细胞吸收。
“方法”涉及向哺乳动物受试者(包括人)注射根据本发明的靶向纳米载体,其包含基因治疗活性剂,连同受恶性疾病、自身免疫性疾病、遗传性障碍、代谢障碍、或传染性疾病影响的靶细胞种类。在靶细胞种类是干细胞的特殊情况下,基因治疗活性剂将不会解决病变细胞,而是解决用于再生治疗目的的细胞。靶细胞种类进一步定义如下。
术语“活性剂”或“药物”意指用于基因组编辑、基因组沉默抑或基因表达的转录后调控的基因治疗工具。在优选的实施例中,所述工具选自质粒、人工改造的限制性内切酶、特异性编码大范围核酸酶的质粒、或者用于转录基因调控或转录后基因调控的工具。在优选的实施例中,人工改造的限制性内切酶属于大范围核酸酶类族,例如为:锌指核酸酶(ZFN)、CRISPR/Cas系统或类转录激活因子效应物核酸酶(TALEN)。在优选的实施例中,用于转录基因调控或转录后基因调控的工具是siRNA、微RNA、mRNA、反义寡核苷酸、或正义寡核苷酸。在另一个优选的实施例中,质粒在整合入宿主细胞基因组时抑或在染色体外时表达基因,因此引发肽的或蛋白的基因产物的翻译。在优选的实施例中,人工改造的限制性内切酶或者用于转录基因调控或转录后基因调控的工具可以与包括内体逃逸肽的细胞穿透肽(CPP)共同封装。根据定义,允许CPP穿过细胞膜。可供选择的包括经典CPP(Tat48-60、穿膜肽(Antp43-58)、运输蛋白、TP10、寡聚精氨酸(R8)、MAP、MPG、MPGα)和新型CPP(hCT9-32-br、SAP、S413-PV、mPrPp、bPrPp、M918、EB1和不同CPP5。
在优选的实施例中,本发明涉及用于在肝细胞中翻译的靶向肝细胞配制品。在另一个优选的实施例中,本发明涉及用于肝细胞表达的靶向肝细胞配制品。
在优选的实施例中,细胞种类选自下组,该组由以下各项组成:干细胞、骨髓抗原呈递细胞、非骨髓抗原呈递细胞、T淋巴细胞、B淋巴细胞、先天性免疫系统的自然杀伤(NK)细胞、神经胶质细胞、肝细胞、肺泡细胞、肌细胞、神经元、角质形成细胞、成纤维细胞、软骨细胞、外分泌腺细胞、内分泌腺细胞、内皮细胞、上皮细胞、或者由健康的干细胞或体细胞经恶性退行性变产生的恶性细胞。
在一个优选的实施例中,纳米载体是基于脂质的纳米载体,优选脂质体、脂质复合物、或微团。
在另一个优选的实施例中,纳米载体不基于脂质,优选该非脂质基纳米载体是合成高分子纳米粒子、树枝状聚合物、碳纳米管、或胶体金纳米粒子。特别优选的是,合成高分子纳米粒子是聚(D,L-乳酸-乙醇酸共聚物)纳米粒子。还特别优选的是,胶体金纳米粒子是金纳米壳或金纳米笼。
在本发明的另一个优选实施例中,根据其阳离子电荷来选择纳米载体,其中可以选择强的或弱的阳离子纳米载体,优选强阳离子纳米载体。后者优选由例如1,2-二油酰基-3-三甲基铵丙烷(DOTAP)和胆固醇组成,更优选由例如53mol%DOTAP和39mol%胆固醇组成,剩余的8mol%代表靶向配体;后者优选包含更低摩尔百分比的DOTAP和胆固醇,加上中性膜磷脂。
在优选的实施例中,靶向锚包括脂质锚部分和靶向部分,该靶向部分可以是碳水化合物残基、抗体或抗体片段、非抗体蛋白质、适体、脂蛋白或其片段、肽聚糖、脂多糖或其片段、或者CpG DNA。优选地,该碳水化合物残基基于单价的或多价的、直链的或支链的岩藻糖或半乳糖或N-乙酰半乳糖胺(GalNAc),或者基于天然多糖壳聚糖。
在优选的实施例中,间隔物部分插入于脂质锚部分和靶向部分之间,特别地,其中,该间隔物部分是3,4,5-三羟基-6-(羟甲基)四氢-2H-吡喃-2-基)硫脲基)丁基)氨基甲酸酯、N-羟基琥珀酰亚胺(NHS)、1,2-二硬脂酰基-sn-甘油基-3-磷酸乙醇胺-N-[聚(乙二醇)-2000]-N-羟基琥珀酰亚胺(DSPE-PEG-NHS)、ε-氨基己酸、聚醛、聚烯烃、聚环氧乙烷、聚ω-链烯酸酯或聚甲基丙烯酸烷基酯。
在进一步优选的实施例中,靶向锚是Gal-C4-Chol或GalNAc。
在优选的实施例中,靶向锚直接连接至纳米载体,但其也可以经由间隔物连接至纳米载体。这种间隔物部分优选另外配备聚乙二醇部分或羟乙基淀粉部分。
根据本发明,靶向锚经由靶向部分特异性结合在细胞种类的表面上表达的受体分子。受体分子的实例是
-C型凝集素或非C型凝集素;
-如果靶向部分是脂蛋白、肽聚糖、脂多糖或CpG DNA,则为Toll样受体(TLR),其中TLR优选为TLR-1、TLR-2、TLR-4、TLR-6或TLR-9;
-细胞种类的表面上的标记,
-细胞种类的表面上的疾病相关标记;或
-CD20、CD30、CD33、CD52、CEA、EGFR/ErbB1、HER2/ErbB2、c-MET/HGFR、IGFR1、EphA3、TRAIL-R1、TRAIL-R2、RANKL、VEGFR、αVβ3、α5β1。
在优选的实施例中,哺乳动物疾病实体是恶性疾病、自身免疫性疾病、遗传性障碍、代谢障碍、或传染性疾病。该传染性疾病可以是由选自病毒、细菌、真菌、或原生动物的传染性病原体引起的。
在优选的实施例中,纳米载体的递送模式是经由静脉、皮下、皮内、腹膜内、胃肠道外、或肺内途径,经由肝动脉进行输注的途径、甲状腺内途径、用于肺部递送的鼻内途径、用于鼻对脑递送的鼻内途径、鞘内途径、眼后段施用、玻璃体内或局部途径、或者在重新接种到患者体内之前通过离体直接细胞处理的途径。
根据本发明的优选实施例,纳米载体可以配备有任何上述的靶向锚,具有或没有间隔物,以便产生最通用的纳米载体平台。就此而言,如果需要,可同步采用具有相同靶向部分但装载有不同基因治疗工具的任何两种至三种纳米载体,以实现患者的最佳可能治疗结果。
用于基因转移或基因组编辑的工具
术语“质粒”意指可以独立于其染色体DNA进行复制的小细胞DNA分子,并且该复制过程发生在细菌、古细菌和真核生物体中。人造质粒被用作分子克隆中的载体,在克隆过程中它们能够在宿主生物体内复制重组DNA。质粒的大小范围在大约1kbp至1,000kbp,并且可以用作根据本发明的活性剂。除了其多种天然功能以外,质粒可用于基因治疗,(i)在人或动物宿主细胞内表达用于接种疫苗目的的蛋白质,或(ii)通过将功能基因转入人或动物细胞中,以表达在给定疾病中缺乏的功能蛋白。为此,治疗性基因可能必须插入人类基因组内的预先选择的染色体靶位点或者在染色体外进行转录。如果采用质粒载体来引入功能基因,则可以优选使用锌指核酸酶以允许该同源重组。根据本发明,这甚至可以通过编码锌指核酸酶自身的质粒来实现。目前为止,质粒编码的DNA疫苗在小型啮齿类动物模型中显示出最有效的免疫原性,但仍然需要提高在人类和更大动物中的有效性。这主要是由于DNA质粒低效递送至细胞和细胞核中,事实上,这是根据本发明方法的靶向药物递送的主题。然而,普遍地无效递送质粒——例如对于一般未封装的核酸——进入细胞明显是根据本发明的靶向药物递送的主题。
细胞可以通过“分子剪刀”(限制性内切酶)保护自己免受来自病毒、真菌和细菌的遗传学攻击,该“分子剪刀”以靶向方式将来自外来生物体的遗传信息在其破坏宿主细胞之前切割成片段。此类“限制性内切酶”或“限制性内切核酸酶”在被称为限制位点的特定识别核苷酸序列处或附近,切割特定序列上的DNA链。这些限制位点在外部基因组中通常是四至六个碱基对的长度。通过贯穿DNA双螺旋的糖-磷酸主链的两个切口对DNA进行切割(Roberts RJ.Crit Rev Biochem[生物化学重要评论]1976;4:123-64;Kessler C和MantaV.Gene[基因]1990;92:1-248)。根据本发明,不同种类的限制性内切酶可以通过要求保护的方法引入细胞种类,并用作用于基因治疗目的的工具。
术语“大范围核酸酶”意指以大识别位点(12至40bp的双链DNA序列)为特征的内切脱氧核糖核酸酶,该大识别位点在任何给定的基因组中通常只出现一次。大范围核酸酶因此被认为是最特异的天然存在的限制性内切酶。大范围核酸酶的高度特异性赋予其(与其他天然存在的限制性内切酶相比)高精确度以及远远更低的毒性。大范围核酸酶的实例是来自LAGLIDADG家族的归巢内切核酸酶。ZFN、CRISP/Cas和TALEN也被共同归于新出现的大范围核酸酶药物类别中。
锌指核酸酶(ZFN)是人工改造的限制性内切酶,其被设计用于靶向基因组内的特定DNA序列。锌指是通过≥1个锌离子进行稳定的小蛋白结构基序。锌指包括多种多样的不同蛋白质结构(Klug A和Rhodes D.Cold Spring Harb Symp Quant Biol[冷泉港定量生物学专题论文集]1987;52:473-82。)。绝大多数锌指蛋白结合DNA、RNA、蛋白质、或其他分子;它们的结构变化主要改变其结合特异性。ZNF是通过融合锌指DNA结合结构域与DNA切割结构域而产生的。这使得ZNF能够靶向独特的基因组基因座并引发内源性DNA修复机制。因此,ZNF可以精确改变更高等生物体的基因组(Jabalameli HR等人,Gene[基因]2015;558:1-5),以用于治疗目的。
用于基因治疗的另一个工具来源于保护原核生物抵御外源遗传材料的分子系统:CRISPR(成簇规律间隔短回文重复序列)是包含短重复的碱基序列的DNA基因座,每个重复之后是来自之前暴露于病毒的间隔物DNA的短区段(Marraffini LA和Sontheimer EJ,NatRev Genet[遗传学自然评论]2010;11:181-90)。它们被发现于大约40%的经测序的细菌基因组中(CRISPR数据库:http://crispr.u-psud.fr/crispr/CRISPRdatabase.php),经常与编码CRISPR相关蛋白的cas基因相关,并且作为CRISPR/Cas系统,形成原核系统以获得用于抵御外源遗传元件(例如质粒和噬菌体)的获得性免疫(Barrangou R等人,Science[科学]2007;315:1709-12;Mali P等人,Nature Methods[自然方法]2013:10:957-63)。CRISPR间隔物以类似于真核RNA干扰的方式识别和切割这种外源基因材料(Marraffini LA和Sontheimer EJ,Nat Rev Genet[遗传学自然评论]2010;11:181-90)。CRISPR/Cas系统已被用于基因编辑和基因调控(Mali P等人,Nature Methods[自然方法]2013:10:957-63)。通过细胞递送Cas9和适当的指导RNA,可以在任何希望的位置切割靶标生物体的基因组(Esvelt KM等人,elife 2014:e03401)。CRISPR/Cas系统分为三种主要类型:I、II、和III,连同十二种亚型(Ain QU,Chung JY,Kim YH.J Control Release[控制释放杂志]2015;205:120-127)。
可替代地,类转录激活因子效应物核酸酶(TALEN)是通过将类转录激活因子效应物(TALE)的DNA结合结构域融合于DNA切割结构域而产生的人工改造的限制性内切酶,该DNA切割结构域可以特异性结合至任何所希望的DNA序列以进行基因组编辑(BochJ.Nature Biotech[自然生物技术]2011;29:135-6),并因此进行遗传干预(Ain QU等人,Control Release[控制释放]2015;205:120-127)。TALEN已被实验性地用于纠正导致疾病发生的遗传错误(Carlson DF等人,Mol Ther Nucleic Acids 2012[分子治疗—核酸];1:e3)。用于基因沉默或基因表达的转录后调控的工具
基因沉默下调某个基因的表达。根据本发明,用于转录基因沉默或基因表达的转录后调控的工具包括微RNA(miRNA)、反义寡核苷酸、小干扰RNA(siRNA)、和信使RNA(mRNA)。为了清楚起见,基因沉默与基因敲低(基因表达减少70%)同义,而基因敲除——如通过大范围核酸酶——完全消除来自给定基因组的各基因(基因表达减少100%)。siRNA在RNA干扰中起中心作用,即经由转录后基因沉默进行基因调控。为此,siRNA与某些蛋白成分一起形成RNA诱导沉默复合物(RISC)。如果siRNA前导链与互补mRNA接合,则该mRNA被降解抑或其翻译成蛋白的过程被阻断(Siomi H和Siomi MC.Nature[自然]2009;457:396-404)。可以采用该功能以治疗性地下调疾病相关基因的表达。
miRNA是进化上保守的小的非编码RNA,其可以调控基因表达。越来越多的证据支持miRNA在许多人类疾病中的作用,这些疾病包括癌症和自身免疫障碍。miRNA的功能可以被反义寡核苷酸有效且特异性地抑制,从而支持其作为用于开发新颖疗法的靶标的潜力。因此,miRNA是用于基因沉默的工具,其被越来越多地用于产生防治癌症和疾病(如传染性疾病和神经退行性障碍)的治疗方法。
反义寡核苷酸是在正义方向上与所选基因或基因表达序列互补的DNA或RNA的合成单链。反义DNA结合至编码蛋白的或非编码的互补RNA;在结合的情况下,DNA/RNA杂合体被RNA酶H所降解。反义RNA通过结合互补信使RNA(mRNA)来防止蛋白质翻译。可以采用反义寡核苷酸进行遗传性障碍的反义治疗。在这种情况下,互补的反义寡核苷酸结合至由各自基因产生的mRNA,从而在基因翻译成其疾病相关蛋白前,将其在转录后关闭。可替代地,反义寡核苷酸可以结合至前mRNA上的剪接位点。已经研究了反义寡核苷酸作为潜在药物用于疾病癌症(例如肺癌、结肠癌、胰腺癌、恶性胶质瘤、和恶性黑色素瘤)、糖尿病、肌萎缩性侧索硬化、迪谢内肌营养不良以及例如哮喘、关节炎和结肠袋炎的具有炎性成分的疾病(Goodchild J.Methol Biol[分子生物学方法]2011;764:1-15)。2014年,美国食品与药品管理局(FDA)批准了两种反义药物,即用于治疗巨细胞病毒性视网膜炎的Fomivirsen和针对纯合家族性高胆固醇血症的Mipomersen/>还令人感兴趣的是反义药物用于治疗若干种神经退行性障碍的用途。
尽管如此,正义寡核苷酸也已经显示出发挥治疗活性,例如抵御人类免疫缺陷病毒病毒(HIV),其中病毒繁殖受到针对于病毒的5’-tat剪接受体位点的正义20-mer寡脱氧核苷酸抑制(Sullivan SM等人,Antisense Res Dev[反义的研究与开发]1992;2:187-97)。
作为给定基因的转录物,mRNA的功能早已为人所知,即被进一步翻译成肽或蛋白质产物。如果在遗传性障碍中,一个基因功能障碍抑或完全缺失,则将相应的mRNA引入受影响的细胞可以弥补这种缺陷。因此,将这种mRNA递送到相应细胞可以被认为是基因表达的转录后调控。
基因治疗剂的靶向递送
纳米载体
如上所述,当以游离形式给药时,核酸并不是非常有效。因此,它们成功用于治疗疾病需要采用能够使得治疗性核酸安全有效地到达其靶标的纳米载体。因此,本发明人建立了另一种靶向药物递送的模型,即通过将其封装于纳米载体内发送至目的细胞。将用于基因治疗应用的此类靶向纳米载体在本申请中具体说明。
在优选的实施例中,本发明涉及封装mRNA或DNA用于在肝细胞中分别进行翻译或表达的肝细胞特异性纳米载体。
将非病毒纳米粒子——或纳米药物——细分为基于脂质/有机的和非基于脂质/无机的纳米载体。表面改性使它们适合于药物的靶向递送及其控制释放进入细胞或组织及器官中。基于脂质的纳米载体包括但不限于脂质体、脂质复合物、和微团。非基于脂质的纳米粒子包括但不限于合成高分子纳米粒子[例如聚(D,L-乳酸-乙醇酸共聚物)纳米粒子]、树状聚合物、碳纳米管、以及胶体金纳米粒子(即金纳米壳和金纳米笼)。
在本发明的优选实施例中,纳米粒子药物载体的系统性能可以通过使用聚乙二醇(PEG;MW<20,000g/mol)涂覆其表面来改变。PEG是环氧乙烷[H-(O-CH2-CH2)n-OH]的寡聚物或聚合物。取决于其分子量,该聚醚化合物也称为聚环氧乙烷(PEO;MW>20,000g/mol)或聚氧化乙烯(POE;任意分子量)。在几何上,PEG是(i)分支PEG(具有从中央核心基团发出的3-10个PEG链),(ii)星形PEG(从中央核心基团发出10-100个PEG链),和(iii)梳形PEG(具有连接至聚合物主链的多个PEG链)。大多数PEG作为分子量的混合物而来,并且因此是多分散的。它们的尺寸分布通过重均分子量(Mw)和数均分子量(Mn)(均可通过质谱进行测量)进行统计学表征。其比例被称为多分散性指数(Mw/Mn)。非离子型淀粉衍生物羟乙基淀粉(HES)是仍另一种的类似于PEG的分子类型,可以用作纳米载体的“屏蔽剂”。因此,它不是只具有作为是其最常见用途的容量扩充剂的医疗价值。除了单分子之外,纳米载体可以通过共价偶联进行PEG化,从而延长它们在血流中的循环,并且改变其药物代谢动力学和体内分布连同减少其通过血源性蛋白的调理素作用,该调理素作用将导致随后的单核吞噬细胞的非特异性摄取。
靶向部分、细胞受体、靶细胞、和适应症
根据本发明,连接至纳米载体的靶向部分的实例是肽、碳水化合物或者更确切地说是碳水化合物残基、抗体及其片段、其他(糖)蛋白、以及适体。更具体而言,该碳水化合物残基是基于单价的或多价的、直链的或支链的岩藻糖或半乳糖或N-乙酰半乳糖胺(GalNAc),或者基于天然多糖壳聚糖。甚至更具体而言,靶向锚可以是嵌入于纳米载体或与其共价连接的结构,其中细胞特异性靶向部分是碳水化合物、抗体或抗体片段、非抗体蛋白质衍生物、适体、脂蛋白或其片段、肽聚糖、脂多糖或其片段、或者CpG DNA(其中CpG序列基序代表“胞嘧啶-磷酸盐-鸟嘌呤”)。其中,基于碳水化合物的锚可以是岩藻糖(即D,L-岩藻糖、D-岩藻糖或L-岩藻糖)或甘露糖(即D,L-甘露糖、D-甘露糖或L-甘露糖)的单价或多价衍生物。此类糖为C型凝集素受体(CLR)所识别,并且因此可用于定位骨髓抗原呈递细胞以治疗性地解决病毒、细菌、真菌和寄生虫感染,或用于涉及CLR的许多其他治疗选项,其中这些受体使得能够摄取活性剂、用于疫苗或用于触发独特的信号传导途径,这些途径诱导特定细胞因子的表达,这些细胞因子决定T细胞极化的命运——并且因此决定广泛的抗原特异性免疫应答。适合作为安装于纳米载体上的靶向部分的其他碳水化合物是肝细胞特异性碳水化合物,例如半乳糖(即D,L-半乳糖、D-半乳糖或L-半乳糖)或N-乙酰半乳糖胺(GalNAc),其使得能够选择性地定位用于不同治疗应用的肝细胞。接下来,适体可以用作靶向部分。适体是短的(i)寡核苷酸(DNA或RNA或XNA)或(ii)与特定分子靶标结合的肽。迄今为止,适体已被特别用作用于靶向递送治疗性抗癌剂的活性靶向部分。此外,根据本发明,某些细菌产物可以由于其病理生理学上的结合特性而有资格作为靶向部分。包括脂蛋白(或其片段)、肽聚糖、脂多糖(或其片段)、连同CpG DNA,这些细菌产物经由其Toll样受体(TLR)而被某些细胞识别。由于它们被TLR-1(结合细菌脂蛋白和肽聚糖)、TLR-2(结合细菌肽聚糖)、TLR-4(结合细菌脂多糖)、TLR-6(结合细菌脂蛋白)、和TLR-9(识别CpG DNA)特异性识别,因此它们可用于靶向正好表达这些TLR变体的细胞或细胞家族。不可避免地,靶向部分或者(更确切地说)靶向配体的实例必须保持不完整,因为实际上能够结合至感兴趣的给定表面结构上的任何类型的分子——无论是天然存在的生物分子、修饰的生物分子或是人工分子——都可以用作纳米载体的靶向部分或者(更确切地说)靶向配体。
在本发明的优选实施例中,使用肝细胞特异性靶向配体,其使得能够选择性地定位用于不同治疗应用的肝细胞。例如,将允许选择性定位肝细胞的肝细胞特异性碳水化合物,例如半乳糖(即D,L-半乳糖、D-半乳糖或L-半乳糖)或N-乙酰半乳糖胺(GalNAc)用作安装于纳米载体上的靶向部分。此外,根据本发明,使用靶向肝细胞的糖脂。糖脂是基于Gieseler Rk等人,2005年3月21日,WO/2005/092288早期所描述的整体分子结构,例如胆甾烯-5-基氧基-N-(4-((1-亚氨基-c-b-D-硫代[CHO*]乙基)氨基)丁基)甲酰胺(*其中“CHO”代表前述单糖中的任一种)。然而,其中提及的碳水化合物被D,L-半乳糖、D-半乳糖、L-半乳糖、或GalNAc,或者其组合或排列所取代。
靶向部分可以共价地直接连接至纳米载体。可替代地,靶向部分可经由间隔物并且特别是具有适合分子大小的聚乙二醇(PEG)间隔物或羟乙基淀粉(HES)附接至纳米载体,以有益地改变靶向药物递送系统的特性(也参见上文)。可以与或可以不与PEG结合的其他类型的连接子或间隔物是3,4,5-三羟基-6-(羟甲基)四氢-2H-吡喃-2-基)硫脲基)丁基)氨基甲酸酯、N-羟基琥珀酰亚胺(NHS)、1,2-二硬脂酰基-sn-甘油基-3-磷酸乙醇胺-N-[聚(乙二醇)-2000]-N-羟基琥珀酰亚胺(DSPE-PEG-NHS)、ε-氨基己酸(又称6-氨基己酸)、聚醛、聚烯烃、聚环氧乙烷、聚ω-链烯酸酯、或聚甲基丙烯酸烷基酯。
靶向部分特异性结合至相应靶细胞或细胞种类的表面上表达的受体分子。这些靶细胞或细胞种类包括(i)干细胞,有多潜能干细胞(包括诱导的多潜能干细胞)、多能干细胞、脐带血干细胞、造血干细胞、间充质干细胞/基质细胞、神经干细胞、肿瘤干细胞、和位于解剖生态位的罕见干细胞群;(ii)免疫细胞,有骨髓抗原呈递细胞(单核细胞、未成熟或成熟的树突状细胞,朗格汉斯细胞和巨噬细胞)、非骨髓抗原呈递细胞(浆细胞样树突状细胞)、和未澄清个体发生的抗原呈递细胞(滤泡树突状细胞)、T淋巴细胞(胸腺细胞、表达αβ-T细胞受体(TCR)或γδ-TCR的T细胞、调控性T(Treg)细胞、T辅助细胞、细胞毒性T细胞、自然杀伤T(NKT)细胞、和T记忆细胞)、B淋巴细胞[祖(前-原)B细胞、原B细胞、前B细胞、未成熟B细胞、成熟B细胞、B浆细胞、和B记忆细胞)、和先天性免疫系统的自然杀伤(NK)细胞;(iii)肝细胞,有肝细胞,如原代肝细胞,例如原代人类肝细胞;肝星状细胞(又称为伊藤细胞),如人类LX-2肝星状细胞;肝癌细胞,如人类HepG2肝癌细胞;和肝窦内皮细胞;(iv)肺泡细胞,有I型和II型肺泡细胞;(v)肌细胞;(vi)神经元和神经胶质细胞,有星形胶质细胞、少突胶质细胞、和小胶质细胞;(vii)角质化细胞;(viii)成纤维细胞;(ix)软骨细胞;(x)外分泌腺细胞,有杯状细胞、帕内特细胞、布伦纳细胞、甲状旁腺主细胞、胰腺泡心细胞、立方形乳腺细胞、和前列腺腺细胞(xi)内分泌腺细胞,有甲状腺上皮细胞,滤泡旁细胞,肾上腺皮质细胞,胰腺α、β、γ和δ细胞,卵巢内分泌细胞和、睾丸内分泌细胞;(xii)内皮细胞,有在规则的血管的内表面排列的此类细胞的变体,形成血脑屏障的脑毛细血管内皮的变体、以及淋巴管的内表面的变体;和(xiii)上皮细胞,有其鳞状、柱状、立方形、和假复层的变体。细胞受体可以是健康靶细胞的表面上的常规标记。然而,当靶向部分能够定位患病细胞上被改变的受体时将引起人们的极大兴趣,这些患病细胞是由上述任何健康干细胞或体细胞的恶性退行性变、感染或其他病理性改变所造成,其中它们与其对应的健康细胞相比表现出显著增加的表达或被从头表达。
在本发明的一个优选实施例中,肝细胞(有肝细胞,如原代肝细胞,例如原代人类肝细胞;肝星状细胞(又称为伊藤细胞),如人类LX-2肝星状细胞;肝癌细胞,如人类HepG2肝癌细胞;和肝窦内皮细胞)被用作靶细胞或细胞种类。更优选地,将原代人类肝细胞用作靶细胞或细胞种类。
这种疾病相关标志物表达于患病细胞的表面,这些患病细胞位于(i)恶性疾病——包括但不限于肺癌与支气管癌、结肠癌与直肠癌、膀胱癌、黑素瘤、非霍奇金淋巴瘤、肾癌与肾盂癌、胰腺癌、肝癌(包括肝细胞癌(HCC)或胆管癌(CCC))、甲状腺癌(包括甲状腺非髓样癌与甲状腺髓样癌)、乳腺癌、前列腺癌和卵巢癌;(ii)自身免疫性疾病——包括但不限于干燥综合征、系统性红斑狼疮、自身免疫性甲状腺疾病(包括格雷夫斯氏病连同格雷夫斯氏眼眶病和桥本氏甲状腺炎)、硬皮病、幼年型特发性关节炎、类风湿性关节炎、多发性硬化症、银屑病、韦格纳氏肉芽肿病、强直性脊柱炎以及与肝相关的自身免疫性疾病(包括自身免疫性肝炎(AIH)、原发性硬化性胆管炎(PSC)和原发性胆汁性肝硬化(PBC));(iii)遗传性障碍——包括但不限于安格曼综合征、卡纳万病(又称为卡纳万-范博盖尔特-贝特朗病)、脑白质病变)、进行性腓骨肌萎缩症、囊性纤维病(又称为黏液黏稠病)、迪谢内肌营养不良、血色病,例如1型血色病(又称为HFE相关遗传性血色病)、血友病A、血友病B、神经纤维瘤、苯丙酮尿症、多囊肾病、镰状细胞病、和泰-萨克斯病;(iv)代谢障碍——包括但不限于2型糖尿病(DM1),酒精性脂肪肝病与非酒精性脂肪肝病(NAFLD),酒精性脂肪性肝炎与非酒精性脂肪性肝炎(NASH),甲、乙、丙、丁以及戊型肝炎,I型糖原贮积症(GSD I;又称为冯奇尔克氏症),粘多糖贮积症、和脂贮积症(又名为脂沉积症);和(v)传染性疾病,由以下各项所致:病毒(例如HIV-1、HIV-2、HBV、HCV、HDV、HCMV、EBV,流感病毒、冠状病毒、登革热病毒、埃博拉病毒、和马尔堡病毒之一)、专性或兼性细菌(结核分枝杆菌、麻风分枝杆菌、嗜肺军团菌、土拉热弗朗西丝菌、鼠疫耶尔森菌、贝氏柯克斯体、立克次体属物种、立氏立克次体、沙门氏菌属物种、大肠杆菌、单核细胞增生李斯特菌、奈瑟氏菌属物种、布鲁氏菌属物种和志贺菌属物种)、真菌(例如耶氏肺孢子菌和荚膜组织胞浆菌)、或原生动物(例如疟原虫属物种、鼠弓形体、小球隐孢子虫、利什曼原虫属物种、和克氏锥虫)。
新颖纳米药物具有显著改进癌症治疗的巨大潜力。纳米结构体(如脂质体)被广泛用于临床,而在若干国家,聚合物微团正处于临床试验的高级阶段。在过去几年中,受体介导的肿瘤靶向受到了主要关注,因为它改进了不同药物的药物代谢动力学,并且保护了患者免受由于目前大多数癌症治疗剂的非选择性本质而导致的全身性毒性与不良反应。允许摄取载有药物的靶向纳米载体的特异性受体包括但不限于肿瘤相关抗原,其被分类为(i)造血分化抗原(CD20、CD30、CD33和CD52);(ii)细胞表面分化抗原(不同糖蛋白和碳水化合物);(iii)生长因子受体(CEA、EGFR/ErbB1、HER2/ErbB2、c-MET/HGFR、IGFR1、EphA3、TRAIL-R1、TRAIL-R2、RANKL;(iv)血管靶点(VEGFR、αVβ3、α5β1)。取决于安全考虑,当用作纳米药物的靶向部分时,这些抗体中的一些连同癌症相关受体的一些天然配体可能具有相当大的另外益处。
存在一系列具有高发病率的严重肝病以及对于更好治疗方法的巨大的医疗需求。显著地,这些疾病包括非酒精性脂肪肝病(NAFLD)、非酒精性脂肪性肝炎(NASH)和肝细胞癌(HCC)。就本发明而言,所有这些障碍具有共同的特征,其在于它们提供了用于基因治疗性干预的不同靶标;因此它们可以使用携带基因有效负载的肝细胞特异性药物递送系统进行治疗性解决。NAFLD在全球西方国家与西化国家内已经演变为严重的公共健康问题。事实上,到2020年,NASH——NAFLD的最严重形式——预计将成为美国肝移植的主要原因。NAFLD和NASH的发病机制,特别是造成肝损伤和纤维化的机制,正处于密切研究的焦点。同样,HCC是世界范围的主要健康问题,并且也是癌症相关死亡的第三大常见原因。尽管事实上HCC治疗决策复杂并且取决于肿瘤分期,但Bertino及其同事详细阐述了肿瘤信号通路的分子靶向抑制剂和针对肿瘤相关抗原的免疫治疗样疫苗可能是未来最有效治疗选项的重要组成部分(Bertino G等人,Biomed Res Int[国际生物医学研究]2015;2015:731469)。这两者都可以通过由本发明的方法优先递送的靶向的适合核酸来实现。例如,Rajeev等人展示了siRNA缀合至三天线型N-乙酰半乳糖胺(GalNAc)连同单价GalNAc单元——经由通过脱唾液酸糖蛋白受体(ASGPR)介导的摄取——诱导肝脏中稳健的由RNAi介导的基因沉默。(RajeevKG等人,Chembiochem[化学生物化学]2015;16:903-8)。可以通过本发明的方法显著提高基因治疗剂的引入的效率,即通过使用以多天线型GalNAc的靶向部分为特征的纳米载体。特别是对于全世界最常见的癌症之一HCC,通过肝动脉输注进行载药纳米载体的滴注可能至少在一定百分比的患者中是选择的途径。
因此,由于优选将原代人类肝细胞用作靶细胞或细胞种类,根据本发明优选靶向以下患病细胞:以下项中的细胞:(i)肝脏恶性疾病或者更确切地说,肝癌,例如肝细胞癌(HCC)和胆管癌(CCC);(ii)肝相关的自身免疫性疾病,例如自身免疫性肝炎(AIH)、原发性硬化性胆管炎(PSC)和原发性胆汁性肝硬化(PBC);(iii)肝相关的遗传性障碍,例如血色素沉着症,如1型血色素沉着症(又称为HFE相关遗传性血色素沉着症);和(iv)肝相关的代谢障碍,例如酒精性与非酒精性脂肪肝病(NAFLD)、酒精性和非酒精性脂肪性肝炎(NASH)、以及甲、乙、丙、丁、以及戊型肝炎;更优选HCC、NAFLD和NASH中的细胞。
为了使封装的活性物质能够定位中枢神经系统中的细胞,所选择的靶向药物递送系统必须能有效地穿过血脑屏障(BBB)。BBB充当防止血源性物质(包括旨在用于治疗应用的那些物质)自由进入的物理屏障。可以通过应用靶向药物递送工具来实现治疗剂非侵入式递送入大脑,该靶向药物递送工具能够经由受体介导的胞吞转运作用穿过BBB从而运送活性剂。在这个过程中,该纳米载体-药物系统被跨细胞地运输过脑内皮,从血液到达大脑界面。就此而言,本发明人已经在本申请中展示,配备有C型凝集素特异性靶向受体的载体在一定程度上也穿过了BBB。另一种选项是将壳聚糖作为靶向分子。这种由随机分布的β-(1-4)-连接的D-葡糖胺和N-乙酰-D-葡糖胺组成的生物相容的天然线性多糖已被用于靶向药物递送以治疗神经退行性障碍。壳聚糖及其可生物降解的产物对于神经细胞和BBB具有生物活性,有益于治疗阿尔茨海默病。处于纳米复合物的形式,壳聚糖已被用于递送抗阿尔茨海默病的药物和siRNA至大脑(Sarvaiya J和Agrawal YK.Int J Biol Macromol[生物大分子国际杂志]2015;72:454-65)。
就本发明而言,该人类疾病实体是可通过靶向基因组编辑、基因组沉默或基因表达的转录后调控(在所选择核酸的变体的靶向递送时)而得以治疗或治愈的疾病实体。干细胞是一种特殊的情况,其中这些细胞本身并不发病,但可以用于再生或修复药物方面的基于细胞的疗法。
纳米载体封装的或与纳米载体相关的活性剂,其递送模式可以是静脉的、皮下的、皮内的、腹膜内的、胃肠道外的、肺内的(例如经由气溶胶)、通过经由肝动脉进行输注的、甲状腺内的、用于肺部递送的鼻内的、用于鼻对脑递送的鼻内的、鞘内的、眼后段的、通过玻璃体内施用的,或者在干细胞的情况下通过离体直接细胞处理,随后将此类处理的自体细胞重新接种入患者体内。
最后,为了实现对患者可能的最好治疗结果,采取以下任一策略会是有益的:(i)将具有相同靶向部分而装载不同基因治疗工具的两种或三种纳米载体进行组合,(ii)将具有不同靶向部分而装载相同基因治疗工具的两种或三种纳米载体进行组合,或(iii)将具有不同靶向部分且装载不同基因治疗工具的两种或三种纳米载体进行组合。在后一种情况下,(i)相同的靶细胞被不同的细胞受体经由不同的靶向部分所识别,这样使得将不同的基因治疗工具引入所述细胞以产生互补的治疗响应,或者(ii)不同的靶细胞被明显不同的细胞受体经由不同的靶向部分所识别,这样使得将不同的基因治疗工具引入不同的细胞或细胞分化阶段(例如在恶性疾病的转移性进展中),或引入受相同疾病的不同阶段所影响的不同细胞,以产生优化的治疗响应。
组合靶向系统与活性剂的互补性排列的选项是(基于证据的)个性化用药的迫切之选,其中该方法可以由药物基因组学和/或时间药理学方面的考虑进行指导(SharmaS.Curr Drug Targets[当代药物靶标]2014;15:915-30)。
纳米载体的制备在本领域技术人员的知识范围内。例如,根据之前公布的基本方案(Gieseler Rk等人,2005年3月21日,WO/2005/092288;Gieseler Rk等人,Scand JImmunol[斯堪的纳维亚免疫学杂志]2004;59:415-24.PMID15140050)配制纳米载体。然而,方案可以进行修改,修改处在于按岩藻糖衍生的锚(用于经由表达于细胞表面的C型凝集素受体来定位细胞)的5%至10%的表面密度的范围,或按半乳糖衍生的锚(用于经由表达于细胞表面的脱唾液酸糖蛋白受体来定位细胞)的5%-10%的表面密度的范围来采用靶向锚的密度。此外,脂质体组合物可以使用以下各项进行修饰:磷脂酰胆碱(PC)、1,2-二油酰基-sn-甘油基-3-磷酸胆碱(DOPC)、1,2-二油酰基-sn-甘油基-3-磷酸乙醇胺(DOPE)、1,2-二油酰基-3-三甲基铵-丙烷(DOTAP)、胆甾醇基半琥珀酸酯(CHEMS)、大豆磷脂酰胆碱(SPC-3)、和胆固醇——每个纯度>99%——使用不同的摩尔比以达成产生纳米载体的方案,这些纳米载体使得封装的基因治疗性有效负载能在靶细胞内进行核内体释放。
实例
在HIV-1的实例中,本文描述的基因-治疗方法是通过以下方法抑制病毒的增殖:(i)经由质粒编码的TALEN活性进行的基因组编辑,或(ii)经由通过正义或反义寡核苷酸阻断选择的病毒基因。HIV-1的感染可以用作实例,因为病毒基因组作为所谓的HIV-1前病毒将整合入宿主基因组。可以将相同的方法、或其变体转移至或者改编至其他适应症。
A.采用质粒DNA的研究
实例1:靶向的基于脂质的纳米载体
根据之前公布的基本方案(Gieseler Rk等人,2005年3月21日,WO/2005/092288;Gieseler Rk等人,Scand J Immunol[斯堪的纳维亚免疫学杂志]2004;59:415-24.PMID15140050)配制纳米载体。然而,对方案进行了修改,修改处在于靶向锚的密度增加至岩藻糖衍生的锚(用于经由表达于细胞表面的C型凝集素受体来定位细胞)的8%的表面密度,或半乳糖衍生的锚(用于经由表达于细胞表面的脱唾液酸糖蛋白受体来定位细胞)的8%的表面密度。此外,脂质体组合物可以使用以下各项进行修饰:磷脂酰胆碱(PC)、1,2-二油酰基-sn-甘油基-3-磷酸胆碱(DOPC)、1,2-二油酰基-sn-甘油基-3-磷酸乙醇胺(DOPE)、1,2-二油酰基-3-三甲基铵-丙烷(DOTAP)、胆甾醇基半琥珀酸酯(CHEMS)、大豆磷脂酰胆碱(SPC-3)、和胆固醇——每个纯度>99%——使用不同的摩尔比以达成产生纳米载体的方案,这些纳米载体使得封装的基因治疗性有效负载能在靶细胞内进行核内体释放。
实例2:将质粒DNA封装于纳米载体
质粒形式的DNA(p-DNA)封装编码绿色荧光蛋白(GFP)的基因作为报道基因或者封装编码TALEN的序列。作为先前方案的基础,封装DNA在最开始时进行(Pupo E等人J ContrRelease[控制释放杂志]2005;104:379-96;Bailey AL和Sullivan SM.Biochim BiophysActa[生物化学与生物物理学学报]2000;1468:239-52)。然而,对方案进行了修改,修改处在于对产生小单层脂质体(SUV)的多层脂质体(MLV)的配制参数,与高压匀浆参数(挤出:50nm或100nm)进行的修改。此外,作为提高封装率的方法,引入速度混合以便最终留出可接受的有效负载封装率。通过透析从其中溶解有纳米载体的磷酸盐缓冲液中去除未封装的DNA有效负载。然而,省略透析步骤可能是未来的选项,因为易溶的核酸通常在循环中快速降解,并且因此预计不会导致任何不利影响。省去透析将减少时间和成本需求。
实例3:分析学。
本发明人通过光子相关光谱法(PCS)确定了大小(平均值和分布),通过高效薄层色谱法(HPTLC)确定了总脂质含量,并通过定量聚合酶链式反应(q-PCR)确定了有效负载封装效率。
实例4:负载DNA的纳米载体的特征。
共配制了65种特征在于岩藻糖化的靶向锚与不同膜组成的纳米载体变体。通过这些批次,我们评估了一般生产过程在脂质体成分和建立用于确定核酸封装效率的功能性方法学方法方面的大量变量。最初的批次是专门为优化不同的过程相关步骤而生产的。在所测试的不同膜组成中,最终方案采用摩尔比的变体,包括一种变体,其被指定为45.95%DOPC、45.95%DOPE和8%岩藻糖化锚定材料,负载有26μg的pEGFP mRNA或者50μg抑或100μg的TALEN编码的p-DNA。通过定量聚合酶链式反应(q-PCR),我们获得了20%-43%的DNA封装效率(即,封装入纳米载体中的DNA占最初采用的DNA量)与0.00117-0.00214的DNA负载能力(即,每单位质量脂质中的DNA的质量)。表1显示了选择代表性规格,包括配制的纳米载体的其平均尺寸(以纳米计)。图1显示了DNA封装率。
表1.封装DNA的纳米载体的规格。HIV-1p24产生的抑制。
测量细胞上清液(n=每种条件下3个)的HIV-1p24。所示为处理后第5天的测量值。在第1天和第3天,情况仅显示细微差异,如果有的话,分别为6ng/mL或20mg/mL。正义条件导致抑制HIV-1的增殖,而反义条件不会导致。SD:标准差。细胞培养条件下的浓度:纳米载体脂质:100μM封装的DNA:140nM。
关于基因组编辑的总结注释:通过启动靶向细胞的基因疗法(例如在此测试的那些)使得能够从宿主细胞基因组中切除HIV-1原病毒,可能为实现HIV疾病的治愈提供重要的步骤。对于其中可以引入基因组编辑酶的其他适应症的类似应用也是如此。
B.采用正义和反义寡核苷酸的研究
实例5:材料
试剂及生产商是(i)磷脂(阿凡提极性脂质公司(Avanti Polar Lipids),伯明翰市,阿拉巴马州,美国);(ii)胆固醇和N-琥珀酰亚胺基-S-乙酰基硫代乙酸酯(SATA)(卡比化学公司(Calbiochem),圣地亚哥市,加州,美国);(iii)植物凝集素(PHA)(PHA-P,Difco实验室,底特律市,密歇根州,美国);(iv)小鼠抗HIV-1p17、小鼠抗HIV-1gp-p41和兔抗小鼠IgG(杜邦公司/新英格兰核公司,威尔明顿市,特拉华州,美国);碱性磷酸酶/抗碱性磷酸酶(达科公司(DAKO),卡平特里亚市,加州,美国);钙黄绿素(西格玛-奥德里奇公司(Sigma-Aldrich),圣路易斯市,密苏里州,美国);(V)脱乙酰的6-羧基荧光素(分子探针公司(Molecular Probes),尤金市,俄勒冈州,美国);p24抗原捕获ELISA(雅培实验室(AbbottLaboratories),艾伯特公园市,伊利诺伊州,美国);免疫细胞化学染色剂Vector red(载体实验室(Vector Laboratories),伯林盖姆市,加州,美国);和封片剂Crystal Mount(Biomedia公司,福斯特城,加州,美国)。合成寡核苷酸,即正义序列(5’-ATTTTCAGAATTGGGTGTCG-3’)和互补的反义序列获得自时代制药公司(EpochPharmaceuticals)(雷德蒙特(Redmont),华盛顿州,美国)。通过20%聚丙烯酰胺凝胶电泳(PAGE)在7M尿素下进行分析,得到指示纯度>95%的单一条带。正义和反义的原料和批次随后由供应商以编码形式进行测序,并因此被证实代表了HIV-1 5’tat剪接受体位点的正确的正义与反义序列。
实例6:富含细胞因子的上清液的生成
血沉棕黄层是从健康献血者的全血制备而来,并且购买自美国红十字会(洛杉矶市,加州,美国)。通过在淋巴细胞分离液Lymphoprep(1.077g/mL)上进行密度梯度离心而从HIV-1阴性供血者中分离外周血白细胞(PBL)。将T细胞分离、辐照(2,000rad)并悬浮于80%RPMI 1610/20% Earle’s盐,加上10%胎牛血清(培养基80/20/FCS),并以2×106/mL接种于培养皿中。用植物凝集素(PHA)刺激细胞。48h后,将T细胞沉淀,收集上清液(T-SUP)并在-20℃下等分冷冻直至使用。将T-SUP以25%(体积/体积)添加至培养基(见下文)。实例7:未感染的PBL池。
如上所述,PBL是来自HIV-1阴性供血者的分离的血沉棕黄层。由于HIV-1的增殖在当使用来自冻存物的PBL而不是新鲜分离的PBL时的更早时间点处达到峰值,将来自供体的汇合的PBLs在80/20/FCS加10%二甲基亚砜(DMSO)的培养基中各自冷冻于液氮中(UlrichPP等人,J Med Virol[医学病毒学杂志]1988)。使用来自这些池的细胞长达四周。
实例8:HIV-1的增殖(体外)。
从八个HIV-1阳性患者(如通过HIV-1p24和针对HIV-1gag的PCR进行验证)的经EDAT处理的血液样品中分离PBL。将样品在静脉穿刺后60min内进行处理或者在处理前储存于4℃下长达3h。这些细胞通过密度梯度离心(见上文)获得。使用25cm2组织培养瓶,将来自HIV阳性患者的PBL以2×107的可见PBL(来自冷冻池,见上文)在2×107下培养于80/20/FCS加25%T-SUP(每瓶5mL)中。每隔5天,更换1mL培养基并添加5×106个未感染的汇总的供体PBL以提高病毒增殖。同样每隔5天,监测上清液的HIV-1p24核心抗原,持续总共30天的时段。
实例9:通过载有寡核苷酸的纳米载体抑制HIV-1
使用24孔组织培养孔,每孔接受2×106个未感染的门PBL加2×105个患者的细胞(处于80/20/FCS加25%T-SUP中,每孔1mL),再添加1%白蛋白、100IU/mL的青霉素、100μg/mL的链霉素和0.05mg/mL的庆大霉素。将细胞在37℃孵育3h,然后添加装载有5’tat正义、5’tat反义或只有PBS的纳米载体。在随后5天的孵育期间,不替换培养基并且不添加另外的供体细胞。在第0天连同开始后的第1天、第3天和第5天,分析三份培养物的HIV-1p24(上清液;通过酶联免疫吸附测定[ELISA])、细胞内HIV-1p17和gp41(参见下文的免疫细胞化学),以及通过台盼蓝排除法的生存力。发现纳米载体(载有正义或反义寡核苷酸或无有效负载)三种变体中的每一种的靶细胞的最大饱和摄取出现于1h后(未示出)。在添加病毒接种后1h,将负载寡核苷酸或无负载的纳米载体添加于培养物中。结果示于表2和图2中。
表2:抑制HIV-1p24的产生
测量细胞上清液(n=每种条件下3个)的HIV-1p24。所示为处理后第5天的测量值。在第1天和第3天,情况仅显示细微差异,如果有的话,分别为6ng/mL或20mg/mL。正义条件导致抑制HIV-1的增殖,而反义条件不会导致。SD:标准差。细胞培养条件的浓度:纳米载体脂质:100μM;封装的DNA:140nM。
实例10:HIV-1p17和gp41的免疫细胞化学
将细胞标本转移到载玻片上,风干,固定在丙酮中(5min,4℃),并储存于-70℃下直至评估。然后使载玻片达到室温(RT),并将细胞在Tris缓冲盐水(TBS)中再水合5min,并与正常兔血清在TBS(NRS TBS)中孵育30min以减少非特异性结合。以1:1的比率添加针对HIV-1核心抗原p17与包膜抗原gp41(与gp160进行交叉反应)的小鼠单克隆抗体,并在RT下孵育30min。载玻片用TBS洗涤3次,并且然后在RT下与兔抗小鼠IgG一起孵育30min,并且然后用TBS洗涤3次,并通过碱性磷酸酶/抗碱性磷酸酶(APAAP)(1:60,在NRS/TBS中)的三个循环进行处理。此后,将细胞制剂浸入Vector red持续20min,用自来水冲洗,用苏木精复染,并装上封片剂Crystal Mount。
关于基因表达的转录后调控的总结注释:选择HIV-1 5’tat受体位点是因为在HIV-1研究的早期,从该位点表达的病毒反式激活蛋白已显示通过作用于病毒长末端重复序列来刺激HIV-1的大量繁殖(Arya SK等人,Science[科学]1985;229:69-73;Green M和Loewenstein PM.Cell[细胞]1988;55:1179-88;Frankel AD和Pabo CO.Cell[细胞]1988;55:1189-93)。从那以后,人们已经清楚,持续存在于感染个体(接受终身抗逆转录病毒疗法)的记忆T细胞基因组中的复制能力休眠的HIV-1原病毒是根除病毒的主要障碍。需要多种分子机制来抑制病毒反式激活因子Tat并破坏导致建立潜伏病毒库的调节性Tat反馈回路(Mbonye U和Karn J.Virology[病毒学杂志]2014;454-455:328-39)。因此,通过靶向细胞的基因-治疗性启动(例如在此测试的那种)使HIV-1 5’tat受体位点失活,可能为实现HIV疾病的治愈提供重要的步骤。用于其他适应症的类似应用也是如此,其中正义或反义寡核苷酸方法适用并且似乎是可行的。
实例11:开发用于在肝细胞中翻译的靶向肝细胞的mRNA配制品1.配制品开发
嗜肝性靶向配体和肝细胞特异性纳米载体
合成了一种新颖的人类肝细胞特异性靶向配体(TL)——一种靶向肝细胞的糖脂。为了验证TL可以整合到我们的靶向脂质体平台(Gieseler RK等人,2005年3月21日,WO/2005/092288;Gieseler RK等人,Scand J Immunol[斯堪的纳维亚免疫学杂志]2004;59:415-24.PMID 15140050),根据标准方案(参见之前的引用文献)配制四种具有不同TL的摩尔分数(分别为0%、4%、8%或12%)的纳米载体批次。出于可比性的原因,采用常规脂质组合物、DOTAP和胆固醇。为了追踪通过靶肝细胞的摄取,纳米载体在其双分子层中包含0.1%的荧光染料、德克萨斯红1,2-二十六烷酰基-sn-甘油基-3-磷酸乙醇胺、三乙基铵盐(德克萨斯红-DHPE)。分析纳米载体批次的平均粒度、粒度分布、脂质含量/组成、TL的存在与否、以及视觉外观(使用PCS,通过Malvern Zetasizer v7.11软件进行评估;并使用HPTLC确定纳米载体成分,通过CAMAG Wincats 4软件进行评估)。由于与迄今生产的纳米载体相比,TL的合并不显著改变纳米载体的物理特性,因此不需要改变已经建立的生产和分析过程。体外测试批次的其细胞结合功效与效率(见下文)。肝细胞特异性纳米载体还被进一步称为HEP纳米载体。
mRNA配制品:一般制剂
为了防止RNA酶污染,研究依赖于半专用或专用材料(表3),并且分别依赖于用于防止污染的适当程序或净化程序。专门保留用于配制封装mRNA的纳米载体的化学品是完全专用的。如果能对实验室设备进行可靠的净化,则其只能用作半专用设备。净化包括用RNA酶AWAY溶液处理、用焦碳酸二乙酯(DEPC)水高压灭菌、或在大约180℃下加热灭菌至少4h。
表3.用于控制RNA酶的材料。
半专用材料包括可被使用或等分而没有受到污染风险的化学品,并且也可以用于与配制封装mRNA的纳米载体不相关的目的。
2.体外功效
如表4所示,在不同类型的肝细胞中测试HEP-纳米载体(未负载)的体外靶向功效。从德国埃森市埃森大学医院的普通、内脏与移植手术部友情提供的移植的人肝组织中富集原代肝细胞,并且然后如下详述进行培养。根据以下指定的方案培养肝细胞系。然后用HEP-纳米载体或对照批次靶向细胞,并通过免疫细胞化学进行评估。
表4.用于体外分析HEP纳米载体的测试系统。
测试系统 | 注解 |
1.原代细胞 | |
原代肝细胞 | 分离自健康人肝脏 |
2.细胞系 | |
HepG2细胞 | 肝癌细胞系 |
LX-2细胞 | 肝星状细胞系 |
原代人肝细胞
通过若干次洗涤(杜尔贝科氏磷酸盐缓冲盐水,DPBS;汉克氏缓冲盐溶液,HBSS)与灌注对手术获得的人肝组织块进行处理。用组织黏合剂Histoacryl密封任何组织开口以防止任何缓冲液或培养基的泄漏。用HBSS冲洗掉残余的血液。此后,用(1)HBSS+乙二醇四乙酸(EGTA)和(2)HBSS+CaCl2与胶原酶进行一系列灌注,直至实体组织完全变成细胞分散体。使用300mL-400mL的HBSS将细胞溶液过筛。然后将细胞分配到不同50mL Falcon管中,并在RT下以300rpm(18g)旋转10min。将重悬的沉淀合并于两个50mL试管中并用HBSS补充至50mL,并且离心(400rpm;10min;室温,RT)。将沉淀合并,重新用HBSS注满,并再次旋转一次(500rpm,10min,RT)。将该最终洗涤产生的沉淀溶解于30mL杜尔贝科氏改良伊格尔培养基(DMEM)/汉姆氏F12培养基+青霉素/链霉素/庆大霉素+10%胎牛血清(FCS)中。将细胞设置为1×106/mL。可替代地,细胞可以在6孔板或24孔板中培养。将孔板上的细胞置于培养箱中1h,并且每10min轻轻搅动直至完全沉降。然后去除培养基并用新鲜培养基替换。
HepG2细胞
根据以下文献中公开的方案培养人类HepG2肝癌细胞(HepG2细胞):Gieseler RK等人J Viral Hepat[病毒性肝炎杂志]2011;18:760-7。最开始将细胞系由冻存物(-196℃)复原并在72cm2烧瓶中于37℃、95%相对湿度(RH)下增殖七天以汇合入DMEM加10%FCS加青霉素/链霉素加谷氨酰胺(HepG2培养基)。然后将细胞分离(EDTA,4℃),用不含Mg2+/Ca2+的磷酸盐缓冲盐水(PBS)洗涤两次,传代一次并在三个72cm2培养瓶中(在HepG2培养基中)增殖五天以再次汇合。此后,将细胞分离(EDTA,4℃)并再次洗涤,在Neubauer室中进行计数,并在200μL HepG2培养基中以2×105个细胞/孔各自接种于两个96孔微量滴定板(MTP)中。为了复原用于靶向的表面受体,将细胞孵育21h至22h,然后在37℃和95%RH下进行处理。
LX-2细胞
根据以下文献中公开的方案培养人LX-2肝星状细胞系(LX-2细胞):Bechmann LP等人,Hepatol Res[肝脏病学研究]2009;39:601-8。
肝细胞靶向
将培养的细胞在培养的第五和第七天之间进行靶向,这取决于如通过光学显微镜与台盼蓝染色法所评估的各细胞群体的状态。细胞在37℃和95%RH下孵育1h,连续温和搅拌下用HEP-批次或对照纳米载体(如上文所指)以1:10、1:20或1:50进行稀释。如上所述,所有纳米载体批次都用德克萨斯红-DHPE进行标记。每种条件采用一式三份,并且免疫细胞化学的结果(见下一部分)以这一式三份的算术平均值给出。
免疫细胞化学
在与HEP-纳米载体或对照一起孵育后,通过使用FITC标记的单克隆抗体(用荧光素-5-异硫氰酸酯(FITC)进行标记)的直接免疫细胞化学(ICC)对靶向的内吞活性肝细胞受体(HepR)进行免疫细胞化学。细胞用核染料4’,6-二脒基-2-苯基吲哚(DAPI)进行复染。然后经由Zeiss LSM510 META共聚焦成像系统对细胞进行观察与拍照,并对DAPI阳性细胞、FITC阳性细胞(肝细胞)和德克萨斯红-DHPE阳性细胞(成功靶向的肝细胞)进行差异化评估。对细胞进行计数,其中每个测试条件或读数点分别为100-200个细胞。
结果
无有效负载的原代人肝细胞、HepG2细胞、和LX-2细胞
将原代肝细胞与HEP-纳米载体HEP-TargoSpheres一起孵育1h。然后对细胞进行处理用于免疫细胞化学并在激光扫描显微镜下可视化。核DAPI染色显示所有细胞。这其中,当应用HEP-纳米载体(具有12%的TL)的初始分散体的1:10稀释度(即5.7mM)时,实现了表达HepR的细胞(由FITC鉴定)中肝细胞(由德克萨斯红-DHPE鉴定)的大约80.0%的最大靶向。TL的更低的膜密度(4%或8%)实现了肝细胞的约70%的靶向效率,并且可能足以在递送mRNA时实现令人满意的转导率。相比于与HEP-纳米载体(装备有TL)一起孵育的细胞,“裸”纳米载体的非特异性摄取在减去自身荧光后为约7%。与特异性靶向的细胞不同,这些染色剂只是很微弱。采用HepG2肝癌细胞的研究产生了非常类似的结果。由于原代人肝细胞是体内定位此类细胞的基准,并且由于靶向原代肝细胞的成功进行,所以HepG2细胞仅用于对照目的。用作阴性对照的LX-2肝星状细胞系完全没有被HEP纳米载体或任何对照条件定位。总之,开发的HEP-纳米载体系统可用于定位大多数健康的实质性肝细胞。
用负载有报道mRNA的阳离子HEP纳米载体靶向的HepG2细胞
在进入体内水平之前,就HEP-纳米载体的强阳离子电荷与弱阳离子电荷对其进行进一步优化。孵育后24h对来自报道mRNA的蛋白质表达进行定量。在报道mRNA的剂量为1000ng更高时,由于细胞毒性,蛋白质的表达降低。在报道mRNA等于或低于100ng时,发现更强的阳离子HEP-纳米载体的报道蛋白的表达略微更高。与非靶向(“裸”)纳米载体相比时,使用HEP-纳米载体(装备有TL)使得蛋白质表达增加了大约2倍。
实例12.开发封装用于肝细胞表达的DNA的HEP纳米载体
为了确定经由HEP-纳米载体向人肝细胞递送质粒DNA编码的报道基因(pRG)是否能够使得报道蛋白(RP)在体外表达而进行以下实验,这为此后在使用HEP-封装的pRG处理的动物中的后续研究铺平了道路。最终目的是为具有完全基因缺陷抑或基因突变(分别导致缺失或者产生缺陷的或无效的蛋白质)的患者提供治疗。
HEP-纳米载体与对照批次
根据Rodos Biotarget建立的配制流程(也参见上文)配制多个批次。将肝细胞特异性靶向配体(TL)以8mol%掺入HEP-纳米载体中。所有的质量控制测量值(如由平均粒度、粒度分布、脂质的含量与组成、TL的存在与否、以及视觉外观所确定的)都在可接受的公差范围内(也参见上文更详细的说明)。HepG2细胞和纳米载体孵育
对于HepG2细胞的复原和培养,参见上文。
将HepG2细胞与待比较的四个批次的适当稀释物(即,分别为强阳离子型或次强阳离子型的HEP-TS或非靶向纳米载体,各自负载有pRG(参见下表5))一起孵育2h。然后使用平板转子(260g,4℃)使HepG2细胞旋转沉降,去除含纳米载体的培养基,用PBS洗涤孔,再次旋转沉降细胞,并且最终给各孔补充200μL的HepG2培养基。在收集上清液以确定转录RP的存在与活性之前,将细胞分别保存15h抑或22h。
重要的是,不需要作为对照的游离DNA。已经证明,与纳米载体封装的DNA相反,“裸DNA”导致不转染抑或仅非常低的转染,并且如果可能,显示与未处理对照相同的蛋白质表达(例如,Birchall JC等人,Int J Pharm[国际药剂学杂志]1999;183:195-207;Perrie Y等人,Vaccine[疫苗]2001;19:3301-10和Balbino TA等人,Langmuir[朗缪尔]2012;28:11535-45)。唯一的例外见于肌内注射(例如Cohen H等人,Gene Ther[基因治疗]2000;7:1896-905)。由于这些发现在不同系统中得到反复证实,因此省略“裸DNA”对照是现有技术水平。
参比蛋白质表达
通过生色测定法来确定RP的活性。简而言之,在RP的存在下,生色底物被水解以释放生色团,用光度法在405nm处读取其颜色。颜色强度与RP活性成比例。使用FLUOstarOmega酶标仪(BMG莱伯泰科公司,奥登伯格,德国),所用平板的材料的吸光度(Cellstar,目录号655 180;葛莱娜第一生化有限公司,索林根,德国)由读取器的软件来补偿。并行运行RP的参比标准以测量测试样本。将参比标准储存于-80℃直至使用,并在进行稀释之前立即复原。
处理条件
表5中列出了负载pRG的HEP-纳米载体与对照载体。
表5.处理条件
结果
纳米载体对HepG2细胞的影响
在任何时候(即,在最初复原后,在初次汇合的第7天,在第二次汇合的第5天,孵育于微量滴定板的21h至22h后以及用HEP-纳米载体或非靶向对照批次处理后)在显微镜下将该细胞系可视化,其均保持未受损状态。因此,在观察期内没有迹象显示阳离子纳米载体的毒性作用。
强阳离子型纳米载体
强阳离子型纳米载体引起RP表达的时间与浓度依赖性增加,并且活性高于未处理背景值。在处理后15h,靶向肝细胞的pRG与非靶向pRG几乎没有差异,但在HEP-pRG处理的HepG2细胞中,清楚明确的RP活性在处理后22h变得明显。
HEP-pRG条件明显优于非靶向条件。此外,靶向制剂显示RP表达随时间的增加比非靶向对照更强。有趣的是,该斜率在最低稀释度时最为明显。HEP-纳米载体依赖性配制品被鉴定为最佳条件。
弱阳离子型纳米载体
类似于强阳离子型配制品,弱阳离子型纳米载体引起RP表达的时间依赖性增加以及活性高于未处理背景值。然而,与强阳离子型纳米载体不同的是,其浓度依赖性达到饱和。
处理后15h,靶向肝细胞的pRG与非靶向pRG之间的差异在有利于靶向治疗方面,比强阳离子型配制品的更加明显。在处理22h,测量到HEP-pFVIII处理的HepG2细胞与未靶向条件下的相比具有清楚增加的RP表达。对于次阳离子型形成物,所计算的实际RP活性显示与强阳离子条件相同的趋势。然而,活性肯定更低。这可以由饱和动力学进行解释。同样,靶向肝细胞的配制品被鉴定为最佳条件。
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Claims (10)
1.特异性靶向肝细胞的纳米载体在制备用于治疗肝的恶性疾病和代谢障碍的药物组合物中的用途,该纳米载体由以下项组成:
-人工改造的限制性内切酶、或者特异性编码大范围核酸酶的质粒作为治疗性负载,其中该人工改造的限制性内切酶是锌指核酸酶(ZFN)、CRISPR/Cas系统、或类转录激活因子效应物核酸酶(TALEN),和
-靶向锚,该靶向锚由胆甾烯-5-基氧基-N-(4-((1-亚氨基-2-β-D-硫代[CHO*]乙基)氨基)丁基)甲酰胺组成,其中CHO*代表D,L-半乳糖、D-半乳糖、L-半乳糖、和/或N-乙酰半乳糖胺,用作安装在纳米载体上的靶向肝细胞的部分,其中该靶向锚经由该靶向肝细胞的部分特异性结合在该肝细胞的表面上表达的受体分子。
2.如权利要求1所述的用途,其中该质粒在整合入宿主细胞基因组时抑或在染色体外时表达基因,因此导致肽或蛋白基因产物的翻译。
3.如权利要求1或2所述的用途,其中该纳米载体是基于脂质的纳米载体或基于非脂质的纳米载体,优选地,
其中该基于脂质的纳米载体是脂质体、脂质复合物、或微团,且优选地,
其中该基于非脂质的纳米载体是合成高分子纳米粒子、树枝状聚合物、碳纳米管、或胶体金纳米粒子。
4.如权利要求3所述的用途,其中该高分子纳米粒子是聚(D,L-乳酸-乙醇酸共聚物)纳米粒子。
5.如权利要求1-4中任一项所述的用途,其中该靶向锚包含脂质锚部分和该靶向肝细胞的部分。
6.如权利要求5所述的用途,其中,间隔物部分插入于脂质锚部分和该靶向肝细胞的部分之间,特别是其中该间隔物部分是3,4,5-三羟基-6-(羟甲基)四氢-2H-吡喃-2-基)硫脲基)丁基)氨基甲酸酯、N-羟基琥珀酰亚胺(NHS)、1,2-二硬脂酰基-sn-甘油基-3-磷酸乙醇胺-N-[聚(乙二醇)-2000]-N-羟基琥珀酰亚胺(DSPE-PEG-NHS)、ε-氨基己酸、聚醛、聚烯烃、聚环氧乙烷、聚ω-链烯酸酯或聚甲基丙烯酸烷基酯。
7.如权利要求1-6中任一项所述的用途,其中该靶向锚直接连接于该纳米载体。
8.如权利要求6-7中任一项所述的用途,其中该间隔物部分另外配备有聚乙二醇部分或羟乙基淀粉部分。
9.如权利要求1所述的用途,其中该受体分子是(a)C-型凝集素或非C型凝集素;(b)如果该靶向部分是脂蛋白、肽聚糖、脂多糖或CpG DNA,则为Toll样受体(TLR);(c)该细胞种类的表面上的标记;(d)该细胞种类的表面上的疾病相关标记;或(f)CD20、CD30、CD33、CD52、CEA、EGFR/ErbB1、HER2/ErbB2、c-MET/HGFR、IGFR1、EphA3、TRAIL-R1、TRAIL-R2、RANKL、VEGFR、αVβ3、或α5β1。
10.用于如权利要求1-9中任一项所述的用途,其中该递送模式经由静脉、皮下、皮内、腹膜内、胃肠道外、或经由肝动脉进行输注的途径。
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BR102017016440A2 (pt) | 2017-07-31 | 2019-03-19 | Universidade Federal Do Rio Grande Do Sul | Composição para terapia gênica do sistema nervoso central, processo de obtenção e uso da mesma |
EP3676376A2 (en) | 2017-08-30 | 2020-07-08 | President and Fellows of Harvard College | High efficiency base editors comprising gam |
KR20200121782A (ko) | 2017-10-16 | 2020-10-26 | 더 브로드 인스티튜트, 인코퍼레이티드 | 아데노신 염기 편집제의 용도 |
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CA3147643A1 (en) | 2019-09-23 | 2021-04-01 | Omega Therapeutics, Inc. | Compositions and methods for modulating hepatocyte nuclear factor 4-alpha (hnf4.alpha.) gene expression |
CN110951072B (zh) * | 2019-11-28 | 2021-04-13 | 中国科学院长春应用化学研究所 | 一种具有诱导细胞钙化能力的化合物、其制备方法和应用 |
WO2021182663A1 (ko) * | 2020-03-13 | 2021-09-16 | 엘바이오 주식회사 | 암 줄기세포 특이 결합 펩타이드를 함유하고 비타민 b6가 결합된 폴리올 기반 폴리디자일리톨 유전자전달체 및 암 줄기세포 표적 치료 기술 |
DE112021002672T5 (de) | 2020-05-08 | 2023-04-13 | President And Fellows Of Harvard College | Vefahren und zusammensetzungen zum gleichzeitigen editieren beider stränge einer doppelsträngigen nukleotid-zielsequenz |
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CN114632161B (zh) * | 2020-12-16 | 2024-02-06 | 中国人民解放军军事科学院军事医学研究院 | 肿瘤坏死因子-α作为一种核酸基因药物体内递送载体的应用 |
CN114452407A (zh) * | 2022-02-11 | 2022-05-10 | 中山大学附属第三医院 | 基因编辑递送系统及其制备方法和应用 |
CN114807229A (zh) * | 2022-05-27 | 2022-07-29 | 中国科学院长春应用化学研究所 | 细胞膜、纳米疫苗及其制备方法和应用 |
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