CN102612562A - 一种新的用于生成诱导性多能干(iPS)细胞或组织特异性细胞的蛋白质递送系统 - Google Patents
一种新的用于生成诱导性多能干(iPS)细胞或组织特异性细胞的蛋白质递送系统 Download PDFInfo
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Abstract
描述了一种新的用于生成诱导性多能干(iPS)细胞的蛋白质递送系统。该递送系统包括构建体，所述构建体具有识别体细胞中的受体的受体结合结构域、允许诱导物转移至胞质腔的转运结构域和诱导物与其连接并促进诱导物转移至细胞的货物承载结构域。
Description
发明技术领域
本发明涉及用于治疗目的的蛋白质递送系统；更特别地，所述蛋白质递送系统可以用于递送重编程因子(reprogramming factor)，致使体细胞分化为用于再生医学或疾病治疗的多能干(iPS)细胞或组织特异性细胞。
发明背景
在过去，已通过核移植和细胞融合的方式来生成多能干细胞(ShinyaYamanaka，Pluripotency and Nuclear Reprogramming，Philos Trans R SocLond B Biol Sci.363(1500)：2079-2087(June 27，2008))。两种方法都需要胚胎干细胞，这给研究和治疗使用都带来了伦理困境。通过最近发现的诱导性多能干(iPS)细胞克服了这个问题，所述诱导性多能干(iPS)细胞具有胚胎干(ES)细胞的同样吸引人的生物学特性(Yamanaka，A Fresh Look atiPS Cells.Cell 137：13-17(S.2009))。
通过迫使确定因子的表达，于2006年首次由小鼠成纤维细胞生成诱导性多能干细胞(Takahashi，Y.和S.Yamanaka，Induction of Pluripotent StemCells from Mouse Embryonic and Adult Fibroblast Cultures by DefinedFactors，Cell 126：663-676(2006))并且于2007年首次由人成纤维细胞生成诱导性多能干细胞(Yu Junying，等人，Induced Pluripotent Stem Cell LinesDerived from Human Somatic Cells，Science 318：1917-1920(2007)，Takahashi，K.，等人Induction of Pluripotent Stem Cells From Adult HumanFibroblasts by Defined Factors，Cell 131：861-872(2007))。在以下的研究中：一个包括Oct-3/4、Sox2、Klf4和c-Myc(Takahashi，Cell 126：663-676；Takahashi，Cell 131：861-872)；另一个包括Oct4、Sox2、Nanog和Lin28(Junying，Cell 318：1917-1920)，两组因子已用于引发将成年体细胞重编程为iPS细胞。通过抑制(knock down)p53的活性可以将这些确定因子的重编程效率提高10倍。
在2008年，Melton组证实了通过递送三种转录因子，Ngn3(也称为Neurog3)、Pdx1和Mafa的特定组合，将成年小鼠的分化的胰腺外分泌细胞重编程为与β-细胞非常相似的细胞(Zhou，Q.等人，In VivoReprogramming of Adult Pancreatic Exocrine Cells to β-cells，Nature 455：627-633(2008))。诱导的β-细胞与内源性胰岛β-细胞在形态上不可区分；它们表达对β-细胞功能必须的基因，并且可以通过重塑局部脉管系统和分泌胰岛素来改善高血糖症。该研究提示，在不逆转为多能干细胞状态的情况下，可以通过转录因子的特定组合直接将成年体细胞重编程为组织特异性细胞。
iPS细胞的潜力是巨大的。然而，iPS细胞的临床应用面临许多障碍(Yamanaka，Cell 137：13-17；Miura，K.等人，Variation in the Safety of InducedPluripotent Stem Cell Lines，Nature Biotechnology 27(8)：743-745(2009)；Carpenter，M.等人，Developing Safe Therapies From Human PluripotentStem Cells，Nature Biotechnology 27：606-613(2009))。一个主要障碍是与重编程因子的递送载体直接相关。最初，通过逆转录病毒载体或慢病毒载体将重编程因子基因引入躯体成纤维细胞(Takahashi，Cell 126：663-676；Junying，Cell 318：1917-1920；Takahashi，Cell 131：861-8723-5)。通过逆转录病毒载体或慢病毒载体重编程的方法仅有～0.05％的效率(Okita，K.等人，Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors，Science 322：949-953；Yamanaka，S.Elite and Stochastic Models for InducedPluripotent Stem Cell Generation，Nature 460：49-52(2009))。
癌基因的插入激活一直是使用逆转录病毒载体或慢病毒载体的主要关注的问题，因为这些载体随机地整合到宿主的基因组。尽管在iPS细胞中转基因大量沉默，但c-myc转基因的再活化可以导致肿瘤发生(Okita，K.，等人，Generation of Germline-Competent Induced Pluripotent Stem Cells，Nature 448：313-318(2007))。这些转基因的渗漏表达也可以抑制全iPS细胞的分化和成熟，导致畸胎瘤形成的更大风险(Yamanaka，Cell 137：13-17)。此外，在由iPS细胞再分化的细胞中，可以使转基因再活化和表达，导致iPS细胞的再编程分化状态或肿瘤形成的风险。
非整合病毒载体，例如腺病毒载体随后用于递送这些重编程因子基因(Zhou，Nature 455：627-633；Stadtfeld，M.等人，Induced Pluripotent StemCells Generated Without Viral Integration，Science 322：945-949(2008))。然而，对于有效转导至缺乏腺病毒受体CAR的细胞类型，需要大量的腺病毒载体，所述大量的腺病毒载体可以引起对细胞的致细胞病变效应。此外，如果选择“非-无内容(non-gutless)”腺病毒载体作为递送载体，那么一些腺病毒基因的低水平表达可以影响转导的细胞。
也可以经直接的质粒转染来实现重编程(Okita，Science 322：949-953；Yu，J.等人，Human Induced Pluripotent Stem Cells Free of Vector andTransgene Sequences，Science 324：797-801(2009))，但其效率比逆转录病毒载体的效率低100倍多(Okita，Science 322：949-953)。腺病毒载体转导和质粒转导两者可能都不排除稳定的整合。腺病毒载体的整合频率为～10-3至10-5/细胞(Harui，A.等人，Frequency and Stability of Chromosomal Integrationof Adenovirus Vectors，J.Virology 73：6141-6146(1999))。
生产多能干细胞的方法的比较显示于表1中。
表1.用于生成多能干细胞的三种方法的优点和缺点
ES，胚胎干细胞；iPS，诱导性多能干细胞
在1998年，Frankel和Green独立地观察到了HIV-1Tat蛋白可以以不依赖受体的方式穿透细胞(Frankel，A.和C.Pabo，Cellular Uptake of the TatProtein from Human Immunodeficiency Virus，Cell 55：1189-1193(1988)；Green，M.和P.Loewenstein，Autonomous Functional Domains of ChemicallySynthesized Human Immunodeficiency Virus Tat Trans-Activator Protein，Cell55：1179-1188(1988))。包含HIV-1Tat的短的碱性富精氨酸区域(aa 48-57)的Tat蛋白转导域(PTD)在体外和体内广泛地用于递送包括肽在内的各种分子(Schwarze，S.R.等人，In Vivo Protein Transduction：Delivery of aBiologically Active Protein Into Mouse，Science 285：1569-1572(1999)；Lindsay，M.A.Peptide-Mediated Cell Delivery：Application in Protein TargetValidation，Current Opinion in Pharmacology 2：587-594(2002)；Kwon，Y.D.，等人，Cellular Manipulation of Human Embryonic Stem Cells by Tat-PdxlProtein Transduction，Molecular Therapy 12：28-32(2005)；Kim，D.，Generation of Human Pluripotent Stem Cells by Direct Delivery ofReprogramming Proteins，Cell Stem Cell 4：472-476(2009)；Wadia，J.S.等人，Transducible Tat-HA Fusogenic Peptide Enhances Escape of Tat-FusionProtein After Lipid Raft Macropinocytosis，Nature Medicine 10：310-315(2009)；Zhou，H.等人，Generation of Induced Pluripotent Stem Cells UsingRecombinant Proteins，Cell Stem Cell 4：381-384.19-24(2009))。这种通过基于蛋白质的载体递送分子的方法称为蛋白质转导。Tat-PTD通过离子相互作用结合于细胞表面导致Tat-融合蛋白通过脂质筏依赖性的大胞饮作用(lipid raft-dependent macropinocytosis)内在化(Wadia，J.S.，NatureMedicine 10：310-315)。然而，多数的Tat-融合蛋白仍然捕获于大胞饮体中，表明肽或蛋白质从大胞饮体中的脱逸是无效的过程(Wadia，J.S.，NatureMedicine 10：310-315)。最近，融合于4个重编程因子(Oct4、Sox2、Klf4和C-Myc)的C端的聚-精氨酸(11R或9R)PTD成功地将小鼠胚胎成纤维细胞(Zhou，H.等人，Generation of Induced Pluripotent Stem Cells usingRecombinant Proteins，Cell Stem Cell 4：381-384(2009))和人新生成纤维细胞(Kim，D.，Cell Stem Cell 4：472-476)重编程为iPS细胞，但效率非常低。
发明概述
本发明是在不使用基因表达载体的情况下，递送重编程因子至成年体细胞从而生成iPS细胞或组织特异性细胞的新的基于蛋白质的系统。所述系统包含具有受体结合结构域、转运结构域、货物承载结构域(cargobearing domain)和诱导物的构建体。所述受体结合结构域使构建体指向体细胞。所述转运结构域促进货物承载结构域和诱导物运输至细胞。所述货物承载结构域将诱导物递送至细胞。在本发明的一个实施方案中，所述构建体利用外毒素结构域进行构建体的结合、转运和货物功能。此类外毒素中的一些包括艰难梭菌(C.difficile)TcdA和TcdB毒素、肉毒梭菌(C.botulinum)BoNT A-G和C2毒素。为了容纳各种大小的诱导物并且使诱导物指向具有特异性结合受体的体细胞，可以使构建体的各个结构域交换。
可以使用构建体来递送多能诱导物至体细胞并且使所述细胞变为iPS细胞。本发明的另一个实施方案还提供了生成iPS细胞的方法，在所述方法中，将体细胞暴露于承载一种或多种诱导物的构建体中。所述构建体也可以与其他构建体一起使用从而生成iPS细胞，所述其他构建体例如慢病毒、小分子蛋白递送系统或小分子。
附图简述
结合附图中显示的本发明实施方案的以下描述来更详细说明本发明的以上和其他特征、方面和优点，其中：
图1A是艰难梭菌(C.difficile)结构的图示。
图1B是使用艰难梭菌(C.difficile)结构的各种可能构建体的图示。
图2A-2D是含有Oct4和TcdB/TcdA的构建体的各个结构域的图示。破折号表示结构域、标记和间隔区之间的转换(transition)。
图3A-3D是含有Sox2和TcdB/TcdA的构建体的各个结构域的图示。破折号表示结构域、标记和间隔区之间的转换。
图4A-4E是含有Oct4/Sox2及C2I和C2II的构建体的各个结构域的图示。加下划线的序列是使毒素的活性位点失活的突变。破折号表示结构域、标记和间隔区之间的转换。
图5A-5F是含有Oct4/Sox2和BoNT及其他RBD的构建体的各个结构域的图示。加下划线的序列是使毒素的活性位点失活的突变。破折号表示结构域、标记和间隔区之间的转换。
图6是时程蛋白质印迹分析(time-course Western blot analysis)，该蛋白质印迹分析显示了在诱导后2-3小时巨大芽孢杆菌(B.megaterium)的重组TcdB蛋白表达。
图7是显示了暴露于TcdB和无毒TcdB的小鼠结直肠肿瘤CT26细胞的图片。
图8A是TcdB和使用SNAP作为货物的TcdB的测试构建体的图示。
图8B是显示了纯化的SNAP-TcdB构建体的SDS-PAGE凝胶。
图8C显示了使用抗-SNAP-标记抗体的纯化样品的蛋白质印迹分析。
图8D是显示了SNAP-TcdB构建体与野生型TcdB毒性相同的图表。
详述
通过参考以下的描述，可以更好地理解以上概述的本发明，以下的描述应结合所附的权利要求书和附图进行阅读，其中同样的参考编号用于同样的部分。以下陈述的使一个人能够建立和使用本发明的实施的这种实施方案的描述无意于限制本发明，而是作为本发明的具体实例。本领域技术人员应认识到，他们可以容易地使用公开的概念和具体实施方案作为修改或设计用于实现本发明相同目的其他方法和系统的基础。本领域技术人员还应认识到，这种等同构建体和细胞系以其最宽的形式不脱离本发明的精神和范围。
如本文所用，术语“构建体”是指具有包括诱导物序列和货物递送序列的氨基酸序列的重组多肽。该构建体可以具有其他元件。例如，构建体可以包括受体结合结构域(RBD)、转运结构域(TD)、货物承载结构域(CBD)和切割序列(CS)。如以下更详细地描述的，RBD使得构建体识别并结合于承载受体的细胞。TD使得可以包括诱导物的CBD运输至胞质。CBD可以是毒素的灭活的有机活性结构域。更具体地，应理解，可以通过本领域中已知的方法使得CBD对靶细胞无毒性。在一些实施方案中，CBD可以是直接与TD连接的诱导物。CS可以是固有的半胱氨酸蛋白酶结构域(CPD)或另一种蛋白酶序列，所述蛋白酶序列允许携带例如多能诱导物的CBD从细胞内的构建体中释放。
“诱导物”是，除了其他性质以外，当引入体细胞时可以帮助体细胞转化为iPS细胞的转录因子。已经鉴定了以下的诱导物：Oct3/4、Sox2、Klf4、c-Myc、Nanog、lin28、hTERT(人端粒酶)和SV40大T-抗原。其他诱导物可以包括MafA、Pdx-1、Ngn3、SV-40T-ag、DPPA4、DPPA5、ZIC3、BCL-2、h-RAS、TPT1、SALL2、NAC1、DAX1、TERT、ZNF206、FOXD3、REX1、UTF1、p53siRNA。除了以上描述的因子之外，诱导物也可以包括p53抑制剂，例如靶向p53、siRNA、反义RNA/DNA和核酶的抗体和抗体片段。
本领域普通技术人员理解，本文描述的序列的“基本上相同的”同源物(homolog)构成了本发明的示例性实施方案。如果两个氨基酸序列(i)仅具有不显著影响所生成的多肽的折叠活性的保守氨基酸取代物；(ii)存在于两个对齐序列的最短长度中的氨基酸残基之间的缺口或者它们中的插入物、它们的缺失和取代的数量为不多于10％，优选不多于5％的该对齐序列的最短长度中的氨基酸残基的数量；或(ii)在两个序列之间不多于30％，优选不多于20％，更优选不多于15％或不多于10％的氨基酸残基改变，则该两个氨基酸序列是“基本上相同的”。如Houston等人在第US2003/0161809A1号美国申请公开中所描述的其他方法也可以用于确定两个序列是否基本上相同。
梭菌属外毒素装配有用于其N端酶结构域的有效细胞摄取和胞质递送的所有机制并且可以用于递送重编程蛋白质到成年体细胞。特别地，梭菌属外毒素显示模块化多结构域，该模块化多结构域使得毒素能够有效地将其N端酶结构域和与该N端连接的任何物质转运至胞质中。例如，肉毒梭菌(Clostridium botulinum)D型神经毒素(BoNT-D)和艰难梭菌(Clostridium difficile)B型毒素(TcdB)都表现了将附在毒素N端的货物递送至胞质的能力(Bade，S.等人，Botulinum Neurotoxin Type D EnablesCytosolic Delivery of Enzymatically Active Cargo Proteins to Neurons ViaUnfolded Translocation Intermediates，J.Neurochemistry 91：1461-147225-26(2004)；Pfeifer，G等人，Cellular Uptake of Clostridium difficile Toxin B，Translocation of the N-terminal Catalytic Domain Into the Cytosol ofEukaryotic Cells，J.of Boil.Chem.278：44535-44541(2003))。
I.艰难梭菌TcdA和TcdB多能性诱导物iPS细胞构建体
本发明的一个实施方案由艰难梭菌A型毒素(TcdA)和B型毒素(TcdB)构建体组成，所述构建体具有如图1所示的三个功能域：(a)RBD结合用于胞吞作用的靶细胞；(b)TD促进毒素的酶结构域的胞质递送；和(c)CBD，其是GT酶结构域的无活性部分，携带诱导物。图1A显示了艰难梭菌毒素的一级结构，其具有N端葡糖基转移酶结构域(GT)、固有的半胱氨酸蛋白酶结构域(CPD)、包括疏水区(HR)的中心转运结构域(TD)以及C端受体结合结构域(RBD)。CBD位于GT结构域内，用于改造重组蛋白。
如图1B所示，通过GT的截短(truncation)使得TcdB突变体的各种不同的构建体无毒性。野生型TcdB、无毒的TcdB(aTcdB)，以及重组融合蛋白aTcdB-M1、aTcdB-M2和aTcdB-M3的蛋白质结构。aTcdB-M1允许诱导物附在aTcdB的N端。aTcdB-M2具有与截短的葡糖基转移酶(GT)结构域连接的货物，以及aTcdB-M3具有替代整个GT结构域的货物。
作为初始步骤，TcdA的RBD和TcdB RBD结合于细胞表面碳水化合物，包括Gal-αl、3-Gal-βl、4-GalNAc。这些细胞表面碳水化合物见于大多数体细胞。因此，TcdARBD将结合大多数体细胞。在RBD结合其受体之后，通过受体介导的胞吞作用使CBD内在化。TD中的疏水区(HR)使毒素的相应部分将其自身插入到内体膜从而产生孔成为可能，通过该孔CBD可以转运至胞质中。然后转运的CBD可以与构建体的其余部分分离并通过自我蛋白酶解释放至胞质中，所述自我蛋白酶解由位于与TD的N端部分中自我切割位点相邻的固有的半胱氨酸蛋白酶结构域(CPD)催化，如图1B所示。如本文所述，结果，包括TcdA和TcdB的RBD和TD构建体可以用于将多能性诱导物引入到体细胞中。
CBD是携带诱导物的无活性的GT结构域。可以采用本领域普通技术人员已知的各种方法使该GT结构域失活。可以将GT结构域截短。GT结构域的活性也可以由其序列中的氨基酸取代破坏。在本发明的一个实施方案中，通过以下缺失D285A和D287A使TcdA的GT失活，并通过以下缺失D286A和D288A使TcdB的GT失活。在本发明的一些实施方案中，可以利用GT结构域的各种长度，其中使担负活性的结构域部分缺失。GT结构域的失活使得TcdA或TcdB无毒性并且适于在构建体中使用从而生成iPS细胞。无活性的GT结构域变为CBD，其上可以连接诱导物。图1B显示了根据本发明一种实施方案的构建体的各种可能的结构。
图2A-2D示出了根据本发明的几种实施方案的各种构建体，所述构建体利用TcdB与Oct 4和Sox 2的各个结构域。在图2A中，Oct 4-aTcdB构建体的序列(SEQ ID No 1)设有以下结构域：链霉亲和素结合肽(SBP)(aa1-55)；Oct4序列(aa57-416)；CBD(aa420-961)；CPD(aa962-1185)；TD(aa1186-2269)，其包括疏水区(aa1374-1546)；RBD(aa2270-2784)；和His标记(aa2785-2790)。在图2B中，Oct4-aTcdB(dGT)构建体的序列(SEQ ID No2)设有以下结构域：链霉亲和素结合肽(SBP)(aa1-55)；Oct4序列(aa57-416)；CBD(aa419-488)；CPD(aa489-712)；TD(aa713-1796)，其包括疏水区(aa901-1073)；RBD(aa1797-2311)；和His标记(aa2312-2317)。据了解，SBP和His标记仅仅是构建体的标记，其可以与其他标记互换或者在不影响构建体引起Oct4(诱导物)被转运至胞质腔中的能力的情况下完全去除。
在图2C中，Oct4-aTcdA构建体的序列(SEQ ID No 3)设有以下结构域：链霉亲和素结合肽(SBP)(aa1-55)；Oct4序列(aa58-416)；CBD(aa420-959)；CPD(aa960-1187)；TD(aa1188-2267)，其包括疏水区(aa900-1376)；RBD(aa2268-3128)；和His标记(aa3128-3134)。在图2D中，Oct4-aTcdA(dGT)构建体的序列(SEQ ID No 4)设有以下结构域：链霉亲和素结合肽(SBP)(aa1-55)；Oct4序列(aa58-416)；CBD(aa419-486)；CPD(aa487-714)；TD(aa715-1794)，其包括疏水区(aa903-1075)；RBD(aa1795-2655)；和His标记(aa2656-2661)。
类似地，图3A-3D示出了利用TcdA、TcdB和Sox2的构建体。在图3A中，Sox2-aTcdB构建体的序列(SEQ ID No 5)设有以下结构域：链霉亲和素结合肽(SBP)(aa1-55)；Sox2序列(aa58-373)；CBD(aa377-918)；CPD(aa919-1142)；TD(aa1143-2226)，其包括疏水区(aa1311-1503)；RBD(aa2268-3128)；和His标记(aa2227-2741)。在图3B中，Sox2-aTcdB(dGT)构建体的序列(SEQ ID No 6)设有以下结构域：链霉亲和素结合肽(SBP)(aa1-55)；Sox2序列(aa58-373)；CBD(aa376-445)；CPD(aa446-669)；TD(aa670-1753)，其包括疏水区(aa858-1030)；RBD(aa1754-2268)；和His标记(aa2269-2274)。
在图3C中，Sox2-aTcdA构建体的序列(SEQ ID No 7)设有以下结构域：链霉亲和素结合肽(SBP)(aa1-55)；Sox2序列(aa58-373)；CBD(aa377-916)；CPD(aa917-1144)；TD(aa1145-2224)，其包括疏水区(aa1333-1505)；RBD(aa2225-3085)；和His标记(aa3086-3091)。在图3D中，Sox2-aTcdA(dGT)构建体的序列(SEQ ID No 8)设有以下结构域：链霉亲和素结合肽(SBP)(aa1-55)；Sox2序列(aa58-373)；CBD(aa376-443)；CPD(aa444-671)；TD(aa672-1751)，其包括疏水区(aa860-1032)；RBD(aa1752-2612)；和His标记(aa2613-2618)。
II.肉毒梭菌(C.botulinum)神经毒素多能性诱导物iPS细胞构建体
肉毒梭菌神经毒素(BoNT)是可以用于多能性诱导物递送的梭菌属外毒素的另一个候选者。BoNT构建体由识别特异性神经元受体的RBD组成。BoNT毒素的重链(Hc)作为构建体的TD。在本发明的一个实施方案中构建体的CBD可以是肉毒梭菌神经毒素轻链或者具有其非棕榈酰化的防止轻链锚定于膜的截短轻链。所述轻链选自BoNT A-G型从而提供通过构建体递送的诱导物的药理效应的不同寿命。
III.梭菌的C2毒素构建体
梭菌的C2毒素具有二元毒素结构，其中C2II链负责细胞受体结合、胞吞、孔形成和C2I酶促活性的ADP-核糖基化链的转运。根据本发明的一个实施方案的构建体可以通过多能性诱导物与C2II链的融合来创建。C2I链的部分省略使得构建体无毒并且安全的用作产生iPS细胞的启动子。可供选择地，C2I结构域可以通过活性位点的修饰或缺失来失活。而且，如下图中所述，可以改变一些残基。
图4A-4E显示了根据本发明的一个实施方案可以制备的构建体的一些代表性实例。在图4A中，基本的C2II构建体出现了SBP和His标记(SEQID No.9)。图4B显示了具有Oct4和失活的C2I结构域的构建体(SEQ ID No.10)。C2I结构域可以通过构建体的第aa600、602、804、805和807的点突变失活。SBP和组氨酸标记是可选的。图4C显示了具有Oct4和截短的C2I的构建体(SEQ ID No.11)，其中截短使其失活。图4D显示了具有Sox2和失活的C2I序列的构建体(SEQ ID No.12)。图4E显示了具有Sox2的截短的C2I(SEQ ID No 13)。
IV.梭菌属毒素结构域交换
各种梭菌属毒素结合不同的细胞类型。梭菌属毒素的RBD可以互换/交换，证实了多种不同的构建体能够递送诱导物至体细胞。通过C端RBD的结构域交换，一个人可以创建不同的用于细胞类型或组织特异性递送的构建体。在一个实施方案中，TcdA或TcdB的RBD可以用选自靶向神经元细胞的BoNT的RBD来替代。在另一个实施方案中，TcdA或TcdB的RBD可以用结合于细胞类型或组织特异性细胞表面受体并引发受体介导的内在化的配体来替代。总之，用于不同用途的完全变化的基于蛋白质的递送载体可以通过N端结构域交换和C端结构域交换的组合来生成。
超过递送载体本身，BoNT的C端RBD可以用于替代TcdA或TcdB的RBD和生成特异性靶向神经元细胞的蛋白质递送载体。类似地，TcdA或TcdB的RBD可以用于替代BoNT的RBD，导致由BoNT构建体引起的诱导物的非细胞特异性靶向。此外，BoNT轻链可以用于替代TcdB或TcdA的N端从而增强货物蛋白的需要的生物功能，所述货物蛋白与BoNT轻链的N端融合。轻链选自BoNT A-G型，优选肉毒梭菌神经毒素A型的轻链。因此，如本文所述，通过使用梭菌属外毒素之间的结构域交换，可以合成各种多能性诱导物递送载体。
图5A-5D显示了根据本发明的一个实施方案可以制备的构建体的一些代表性实例。在图5A中，Oct4与BoNT/C1连接，作为TD和TcdB RBD(SEQ ID No.14)。在图4A的构建体中，BoNT/C1的轻链的活性可以由所示的修饰破坏。图4B显示了具有BoNT/C1TD和TcdB RBD的Sox2(SEQID No.15)。图5C显示了具有作为诱导物的Oct4、作为TD的C2I和作为RBD的转铁蛋白的构建体。图5D与图5C类似，但使用Sox2代替Oct4(SEQID No.16)。图5E提供了根据本发明的一个实施方案的构建体的进一步实例，其中诱导物是Oct4，CBD和TD由BoNT/C1提供，以及RBD对应于ILGF(SEQ ID No.17)。图5F提供了根据本发明的一个实施方案的构建体的进一步实例，其中诱导物是Sox2，CBD和TD由BoNT/C1提供，以及RBD对应于ILGF(SEQ ID No.18)。
本领域普通技术人员将认识到，构建体可以通过标准方法制备。例如，构建体可以通过标准方法，用大肠杆菌(E.coli)或藻类系统表达。Humphreys，David P.等人，High-Level Periplasmic Expression inEscherichia coli Using a Eukaryotic Signal Peptide：Importance of CodonUsage at the 59End of the Coding Sequence，Protein Expression andPurification 20，252-264(2000)；Griswold，Karl E.等人，Effects of CodonUsage Versus Putative 50-mRNA Structure on the Expression of Fusariumsolani cutinase in the Escherichia coli Cytoplasm，Protein Expression andPurification 27：134-142(2003)；Mayfield，Stephen P等人，Chlamydomonasreinhardtii Chloroplasts as Protein Factories，Current Opinion inBiotechnology，18：126-133(2007)。
V.实施例
TcdB构建体的制备
我们使用巨大芽孢杆菌(B.megaterium)表达系统成功证实了艰难梭菌毒素的稳健的重组表达。TcdA和TcdB基因两者均通过PCR由艰难梭菌(VPI 10463)基因组DNA扩增。这些PCR产物被克隆至巨大芽孢杆菌表达载体pHis1522(MoBiTec，Germany)中。我们还通过使对于在葡糖基转移酶结构域中结合的底物关键的氨基酸发生突变生成了无毒的TcdB基因。这些构建体用于转染巨大芽孢杆菌原生质体(MoBiTec)。此外，我们还生成了完全合成的编码TcdB的基因构建体从而促进将来的重组构建体。还从自我切割位点上游仅具有68个氨基酸的Gal4-TF/aTcdB制备了GT截短的突变体(-ΔGT68)。选择阳性克隆用于重组蛋白表达并在离子交换分馏之后通过Ni亲和层析从细菌裂解物中进行重组His标记的重组TcdB(rTcdB)的纯化。采用这些克隆，我们进行了小规模的时程表达研究并且在木糖诱导后2-3小时发现了两种重组蛋白的峰表达，如图6所示。
从一升总细菌培养物的裂解物中，我们能够得到平均5-10mg高度纯化的重组蛋白质。用小鼠肠上皮CT26细胞在功能上测试纯化的rTcdB，以检测其致细胞病变效应和细胞毒效应以及Rac1的葡萄糖基化作用。将细胞长时间暴露于可能的最高剂量的无毒TcdB(aTcdB)中，表明突变GT几乎完全没有酶活性(～5logs)并且表现出显著降低的细胞毒性(＞5logs)。图4显示了在用aTcdB处理过的那些细胞中没有观察到致细胞病变效应。
为了证明重组TcdB能够递送融合的货物至细胞，我们将少量酶(O6-烷基鸟嘌呤-DNA烷基转移酶，被New England Biolabs称为SNAP-标记)附在TcdB的氨基端。我们可以有效地表达SNAP-标记的嵌合融合为活性重组TcdB。嵌合蛋白保留的毒性大约相当于野生型TcdB，表明货物必须已经接触细胞胞质，因为其被附在TcdB的葡糖基转移酶结构域，仅在其到达胞质之后引起毒性，如图7所示。
SNAP-TcdB融合蛋白构建、纯化和生物功能性的证实
如图8A所示，N修饰的SNAP-TcdB融合的结构域结构为270kD肽，与之相比，野生型为290kD。野生型TcdB构建体具有六聚组氨酸标记。对于亲和纯化，用六聚组氨酸标记和链霉亲和素标记来标记SNAP-TcdB构建体。图8B显示了通过链霉亲和素纯化回收的全长SNAP-TcdB产物；洗脱液(E)和流过(flow through)(FT)可以与野生型(W)样品相比较，其与预期的一样未产生产物。在图8C中，使用抗-SNAP-标记抗体，对纯化的样品进行的蛋白质印迹分析显示了强的信号。最后，在图8D中，将Vero细胞暴露于不同剂量的纯化的SNAP-TcdB或野生型TcdB中1个小时并评估细胞约数的百分比。结果证实，融合伴侣(fusion partner)添加至TcdB未对其生物功能产生不利并且依然能够递送货物至细胞。
在现有的基于细胞的系统中报道基因构建体的构建和功能测试。
以上描述的功能性TcdB肽可以用于开发根据本发明的一个实施方案的构建体。通过将多能性诱导物的序列添加至TcdB构建体的N-末端来构建该构建体。然后，可以用两个系统之一来测试多能性诱导物/TcdB构建体。
可以将用于检测Gal4DNA结合域/NFkB活化基于结构域的嵌合转录因子(Gal4-TF)的存在的哺乳动物报道基因细胞系修饰为用作多能性诱导物构建体测试的报道基因。HEK293细胞系具有整合的合成报道基因构建体，该构建体由Gal4反应性启动子组成，所述Gal4反应性启动子表达双顺反子报道基因，所述双顺反子报道基因含有YFP Venus基因，接着是高斯荧光素酶基因(Gaussia luciferase gene，Gluc基因)。Venus和Gluc通过口蹄疫(foot-and-mouth disease)病毒的“自我切割”肽2A来分离，从而允许两个报道蛋白的独立表达。两个报道基因的纳入允许在显微镜下瞬时检查细胞的Venus荧光以及检测Gluc生物发光。采用基于Gal4的转录因子作为aTcdB的货物附加物，该系统可以用于测试转录因子/多能性诱导物的功能性细胞递送。HEK293Venus/Gluc报道系统的Gal4DNA结合序列启动子可以用Oct4/Sox2元件替代，重复4次。其他多能性诱导物也可以通过将HEK293Venus/Gluc报道系统的Gal4DNA结合序列启动子用它们各自的序列替代来测试。
第二个基于Oct4/Sox2的细胞系报道基因可以用于测试构建体。该第二个系统由DNA结合区组成，所述DNA结合区是Oct4和Sox2用于位点特异性启动子活化所需要的(Ambrosetti D-C等人，Modulation of theActivity of Multiple Transcriptional Activation Domains by the DNA BindingDomains Mediates the Synergistic Action of Sox2and Oct-3on the FibroblastGrowth Factor-4Enhancer，J.Biological Chem.275：23387-23397(2000))。被称为pCATSO3的启动子报道基因构建体包括6个拷贝的存在于位于基本的SV40启动子上游的FGF-4增强子中的HMG和POU基序。HMG基序结合Sox2，而邻近的POU基序结合Oct4(Chakravarthy，H.等人，Identification of DPPA4 and Other Genes as Putative Sox2：Oct-3/4 TargetGenes Using a Combination of in Silico Analysis and Transcription-BasedAssays，J.Cellular Physiology，216：651-662(2008)；Rizzino，A.Sox2andOct-3/4：A Versatile Pair of Master Regulators that Orchestrate theSelf-Renewal and Pluripotency of Embryonic Stem Cells by Functioning asMolecular Rheostats，System Biology and Medicine，WIREs Syst Biol Med1(2)，(2009))。HeLa细胞，pCATSO3表达非常低水平的报道基因(CAT)。该启动子报道基因构建体对于Sox2和Oct4的存在高度敏感，在基线Sox2和Oct4在HeLa细胞中不表达。当用pCATSO3以及Sox2和Oct4的表达载体转染HeLa细胞时，存在报道基因的强劲刺激(＞40倍)。Sox2和Oct4各自刺激报道基因～3倍。考虑到Sox2和Oct4的协同响应，该检测可以提供高度灵敏的测试，从而测量Sox2-aTcdB和Oct4-aTcdB融合蛋白在一起合作工作的能力。
通过aTcdB将生物活性的Oct4和Sox2递送至Oct4/Sox2报道基因细胞系的证明。
对于基于细胞的分化检测，我们将利用Smith和Rizzino实验室的发现，该发现已经显示Oct4或Sox2水平的少量增加(～2倍)引发小鼠ES细胞的分化(Niwa H，等人，Quantitative Expression of Oct-3/4DefinesDifferentiation，Dedifferentiation or Self-Renewal of ES Cells，Nat Genet24：372-376(2000)；Kopp，J.等人，Small Increases in the Levels of Sox2Trigger the Differentiation of Embryonic Stem Cells，Stem Cells 26：903-911(2008))。在升高Oct4的情况下，ES细胞在48小时内分化为表达胚外内胚层和滋养外胚层的标记物的细胞(Niwa H，Nat Genet 24：372-376)。在升高Sox2的情况下，ES细胞在48小时内分化为表达存在于外胚层、中胚层和滋养外胚层，但不存在于内胚层中的标记物的细胞(Kopp，J.Stem Cells26：903-911)。这些分化标记物通过使用实时RT-PCR进行RNA分析容易测定(Kopp，J.Stem Cells 26：903-911)。使用这种检测，Oct4-aTcdB融合蛋白和Sox2-aTcdB融合蛋白的个体功能可以通过测定它们驱使ES细胞分化为特定组的分化的细胞的能力来评价。
对于基于细胞的转录检测，我们建议测试Oct4-aTcdB融合蛋白和Sox2-aTcdB融合蛋白刺激已经稳定地转染至HeLa细胞的启动子/报道基因构建体的能力。对于如之前所述的这种类型的检测，已经构建了这种细胞报道系统并且工作良好。在一系列蛋白质浓度中，Sox2-aTcdB融合蛋白和Oct4-aTcdB融合蛋白将单独地和组合测试。对于这些研究，我们将Sox2-aTcdB融合蛋白和Oct4-aTcdB融合蛋白刺激启动子报道基因构建体的能力与通过慢病毒载体递送的Sox2和Oct4刺激pCATSO3的能力进行比较(Nowling，T.等人，Transactivation Domain of the Transcription FactorSox-2and its Associated Coactivator，J.Biol.Chem.275：3810-3818(2000))。
此外，可以采用直接的质粒转染来构建和测试与Gal4-TF细胞系紧密相关的Oct4/Sox2报道基因细胞系统。含有表达自SO3启动子的Venus/GLuc的合成报道基因构建体可以克隆至慢病毒载体，所述合成报道基因构建体含有6个拷贝的HMG和POU基序以结合Sox2/Oct4。然后，sox2和oct4基因两者均可以被合成并克隆至pcDNA哺乳动物表达质粒中。该系统可以通过共转染HEK293细胞与报道基因构建体和sox2和oct4表达质粒来验证。该报道基因的活化可以通过GLuc检测和Venus(YFP)生物发光来监测。在证实细胞内Oct4/Sox2活性之后，可以将Oct4和Sox2单独克隆至aTcdB递送质粒中，用于生产诱导物构建体。报道基因可以通过由其他因子(即Oct4)的蛋白质递送补充的一种胚胎反式激活蛋白(即Sox2)的质粒转染来进一步验证，反之亦然，从而检测报道基因对于单独递送的蛋白质的响应。最终，aTcdB诱导物构建体可以用于递送两种因子以便测定产生iPS细胞的系统的组合效应和效率。为了比较，蛋白质递送肽(例如，Tat-Oct4)可以用于证实多能性诱导物构建体的增加的效率。
通过Sox2和Oct4经aTcdB的递送将小鼠胚胎成纤维细胞去分化为iPS细胞
融合蛋白介导的重编程的效率可以与当用表达四个表征良好的重编程转录因子的慢病毒载体转染MEFs时观察到的重编程的效率进行比较(Cox，J.L.，和Rizzino，A.Induced Pluripotent Stem Cells：What Lies Beyondthe Paradigm Shift，Experimental Biology and Medicine 235：148-158(2010))。已经使用表达多顺反子转录物的慢病毒载体来重编程MEF，所述多顺反子转录物编码通过自我切割肽(来自口蹄疫病毒的2A)相互分离的Oct4、Sox2、Klf4和c-Myc。每个多能性诱导物构建体可以通过采用表达其他多能性诱导物的可诱导慢病毒载体(Tet-on)转染MEFs来检测。例如，含有Oct4和Sox2的多能性诱导物构建体可以通过采用仅表达对强力霉素响应的Klf4和c-Myc的可诱导慢病毒载体转染MEFs来检测。然后，这些细胞可以用于评估每日添加到培养基中的Sox2-aTcdB融合蛋白和Oct4-aTcdB融合蛋白和强力霉素一起促进MEFs形成的能力。
除了测定重编程的效率(重编程的细胞的百分比)之外，所生成的iPS细胞的质量可以通过检查各种良好建立的多能干细胞的性质来评估，所述良好建立的多能干细胞的性质包括内源性多能性转录因子(例如，内源性Sox2、Oct4、Nanog、UTF1)的表达、内源性Oct4和Nanog基因的去甲基化、细胞表面标记物SSEA-1的表达，以及iPS细胞分化为表达来自使用胚状体的每个胚胎胚层的标记物的细胞的能力。Rizzino，A.Transcriptional Regulation in an In Vitro Model System for MammalianEmbryogenesis，In ″Hormones and Growth Factors in Development andNeoplasia″，eds.R.B.Dickson and D.S.Salomon，John Wiley&Sons，NewYork，pp.115-129(1998))。
结合优选的实施方案描述了本发明。为了描述本发明的构思的目的，记载了具体值、相互关系、材料和步骤，本领域技术人员将理解，如最宽泛地描述的，在不脱离本发明的基本构思和操作原理的精神或范围的情况下，如具体实施方案中所显示，可以对本发明进行多种改变和/或修改。应当认识到，根据以上教导，本领域技术人员在不脱离本文教导的前提下，可以修改那些细节。现已充分阐述了为本发明基础的构思的优选实施方案和某些修改，本领域技术人员在熟悉了此种基本构思后，将显然想到各种其他实施方案以及本文显示和描述的实施方案的某些改变和修改。所有此种修改、替代物和其他实施方案在落入其所附的权利要求书或等价物的范围之内的情况下，其意欲包括所有此种修改、替代物和其他实施方案。因此，应当理解，除非本文中另外指明，否则本发明可以实现。因此，本发明实施方案在所有方面将被认为是示例性的而非限制性的。
Claims (22)
1.一种用于生成诱导性多能干(iPS)细胞的构建体，该构建体包括：
受体结合结构域、转运结构域、货物承载结构域和诱导物。
2.根据权利要求1所述的构建体，其中，所述受体结合结构域、转运结构域和货物结构域是外毒素结构域。
3.根据权利要求2所述的构建体，其中，所述外毒素是梭菌属(Clostridium)毒素。
4.根据权利要求3所述的构建体，其中，所述梭菌属毒素是艰难梭菌(C.difficile)毒素。
5.根据权利要求4所述的构建体，其中，所述艰难梭菌毒素是TcdA。
6.根据权利要求4所述的构建体，其中，所述艰难梭菌毒素是TcdB。
7.根据权利要求2所述的构建体，其中，所述外毒素是无毒的。
8.根据权利要求3所述的构建体，其中，所述梭菌属毒素是肉毒梭菌(C.botulinum)毒素。
9.根据权利要求7所述的构建体，其中，所述肉毒梭菌毒素选自BoNTA、BoNTB、BoNTC、BoNTD、BoNTE、BoNTF和BoNTG。
10.根据权利要求7所述的构建体，其中，用非特异性受体结合结构域来替代所述肉毒梭菌毒素的受体结合结构域。
11.根据权利要求3所述的构建体，其中，所述梭菌属毒素是梭菌的C2毒素。
12.根据权利要求1所述的构建体，其中，所述诱导物是Oct3/4。
13.根据权利要求12所述的构建体，其中，所述构建体具有包含序列ID No.1的序列。
14.根据权利要求12所述的构建体，其中，所述构建体具有包含序列ID No.2的序列。
15.根据权利要求12所述的构建体，其中，所述构建体与选自序列IDNo.1和序列ID No.2的序列具有基本上相同的序列。
16.根据权利要求1所述的构建体，其中，所述诱导物是Sox2。
17.根据权利要求16所述的构建体，其中，所述构建体具有包含序列ID No.3的序列。
18.根据权利要求16所述的构建体，其中，所述构建体具有包含序列ID No.4的序列。
19.根据权利要求16所述的构建体，其中，所述构建体与选自序列IDNo.1和序列ID No.2的序列具有基本上相同的序列。
20.根据权利要求1所述的构建体，其中，所述诱导物选自Klf4、c-Myc、Nanog、lin28、hTERT(人端粒酶)和SV40大T抗原。
21.根据权利要求1所述的构建体，其中，所述货物承载结构域是无活性的外毒素结构域。
22.一种用于生成诱导性多能干(iPS)细胞的构建体，该构建体包括：
无毒的梭菌属毒素和诱导物，其中所述梭菌属毒素还包含受体结合结构域和转运结构域。
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| WO2022267942A1 (zh) * | 2021-06-22 | 2022-12-29 | 呈诺再生医学科技(珠海横琴新区)有限公司 | 特定空间结构多肽及其在制备iPSC中的应用 |
Also Published As
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|---|---|
| WO2011031568A3 (en) | 2011-10-13 |
| KR20120061053A (ko) | 2012-06-12 |
| EP2470664A4 (en) | 2013-01-16 |
| CA2772400A1 (en) | 2011-03-17 |
| US9102921B2 (en) | 2015-08-11 |
| JP2013503198A (ja) | 2013-01-31 |
| US20130288374A1 (en) | 2013-10-31 |
| US8420352B2 (en) | 2013-04-16 |
| WO2011031568A2 (en) | 2011-03-17 |
| US20110053244A1 (en) | 2011-03-03 |
| EP2470664A2 (en) | 2012-07-04 |
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