CN1117145C - 赋予选择性增殖活性的基因 - Google Patents
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Abstract
通过向细胞中导入编码含有以下成分的融合蛋白的基因并以配体刺激细胞而使选择性扩增细胞成为可能,融合蛋白的成分包括:(a)配体结合域,(b)当该配体结合至(a)域时发生结合的区域和(c)当结合时赋予细胞增殖活性的的结合区域。
Description
技术领域
本发明涉及遗传工程领域,特别是基因治疗领域。
背景领域
到目前为止已设计出多种方法以治疗由先天或获得性的遗传缺陷所导致的疾病,即基因疾病。在基因治疗中,有一种这样的方法,即将缺陷基因本身用正常基因替代或补充以从根本上治疗基因疾病。将正常基因准确地导入靶细胞并准确地表达该导入的基因对基因治疗的成功是重要的。将正常基因导入靶细胞的常用载体有病毒载体如逆转录病毒载体、腺病毒载体和腺伴随病毒载体和非病毒载体如脂质体。然而,所有这些载体均具有一些缺点,如基因对靶细胞的低导入效率。此外,由于其它缺陷,如导入基因的低表达效率,故它们常不足以用于治疗。在腺苷脱氨酶(ADA)缺陷中,预期导入了正常ADA基因的细胞将由于体内选择而获得存活优势或生长优势并逐渐变得占优势。在这样的实例中,尽管基因导入效率低,但获得渐进的治疗效应也许是可能的。然而,导入用于不可在体内选择的治疗基因常是必需的。因此,就有必要建立能选择性扩增含有导入基因细胞的系统。
虽然G-CSF被传统地认为是选择性扩增嗜中性细胞的细胞因子(造血因子),但最近已报导用G-CSF给药不仅增加了体内的嗜中性的细胞,而且增加了体内的造血干细胞/前体细胞库(Rinsho Ketsueki(临床血液(ClinicalBlood)),35,1080(1994))。已报导G-CSF产生这种功能的机制是由于G-CSF刺激而激活G-CSF受体时发生的G-CSF受体的二聚化(生长因子研究论文集(Proc.Growth Factor Res.),3(2),131-141(1991))。已报导G-CSF受体具有增殖诱导区和分化诱导区(细胞(Cell),74,1079-1087(1993))。此外,与G-CSF受体一样,已知雌激素受体经二聚化而激活(生物化学杂志(J.Biol.Chem.),264,2397-2400(1989)),且已有报道说雌激素受体和C-Ab1酪氨酸激酶的融合蛋白在细胞中的表达导致了C-Ab1酪氨酸激活(EMBO杂志,12,2809-2819(1993))。
发明内容
本发明通过选择性在体内或体外扩增其中导入有治疗基因的造血干细胞而寻求方法以克服基因导入效率低的困难。本发明的目的是提供用于靶向造血干细胞的基因治疗基本技术。
在目前的基因治疗领域,有许多有关基因对靶细胞的导入效率和导入基因表达效率的问题有待克服。因此,很明显,建立一个用于仅选择性地扩增含有导入基因的靶细胞的系统将产生重要的突破。特别地,如果建立这样一个用于造血干细胞的系统,那么它将在基因治疗领域产生显著的促进,造血干细胞是许多血细胞如红血细胞或白血细胞的来源并据信是用于基因治疗的最优选的靶细胞。
G-CSF,传统上认为是选择性增殖嗜中性细胞的细胞因子(造血因子),也可增殖造血干细胞。当被激活时,G-CSF受体本身二聚化。考虑到这些因素,本发明者考虑到经遗传工程操作的受体的二聚化而用于扩增造血干细胞的系统。同样,根据雌激素刺激时雌激素受体本身二聚化的事实,本发明者考虑了构建G-CSF受体基因和雌激素受体基因之间的嵌合基因、以及将该嵌合基因导入细胞并通过雌激素外部刺激该细胞而迫使该嵌合基因产物中G-CSF受体部分二聚化。
因此,通过开发出以雌激素外部刺激激活嵌合基因产物G-CSF受体部分从而选择性地扩增其中导入有基因的造血干细胞的新系统,从而将该系统应用于基因治疗领域,这样便完成了本发明。
本发明涉及包括配体结合域、当配体结合至配体结合域时发生结合的区域和结合时赋予细胞增殖活性的区域的融合蛋白、包括编码该融合蛋白基因的载体、含有该载体的细胞、及通过将此细胞暴露至类固醇激素而选择性在体内或体外扩增该细胞的方法。此外,当载体含有外源基因时,本发明涉及用于选择性扩增其中导入有外源基因的细胞的方法。
更具体地,本发明涉及:(1)融合蛋白包括(a)配体结合域,(b)当配体结合至(a)域时发生结合的区域,和(c)包括当结合时赋予细胞增殖活性的细胞因子受体或其部分的结合区域;(2)(1)的融合蛋白,其中“包括当结合时赋予细胞增殖活性的细胞因子受体或其部分的区域”从G-CSF受体衍生而来;(3)(1)的融合蛋白,其中的“配体结合域”衍生自类固醇激素受体;(4)(3)的融合蛋白,其中的类固醇激素受体为雌激素受体;(5)包括编码(1)的融合蛋白的基因的载体;(6)带有(5)的载体的细胞;(7)用于选择性扩增(6)的细胞的方法,包括将(6)的细胞暴露于能够作用于(1)的融合蛋白“配体结合域”的配体;(8)包括所需外源基因和编码包括以下成分融合蛋白基因的载体(a)配体结合域,(b)当配体结合至(a)区域时发生结合的区域,和(c)当结合时赋予细胞增殖活性的区域;(9)(8)的载体,其中“当结合时赋予细胞增殖活性的区域”从细胞因子受体衍生而来;(10)(9)的载体,其中细胞因子受体为G-CSF受体;(11)(8)的载体,其中“配体结合域”从类固醇激素受体衍生而来;(12)(11)的载体,其中类固醇激素受体为雌激素受体;(13)(8)的载体,其中“编码融合蛋白的基因”和“外源基因”位于相同的分子上;(14)(8)的载体,其中“编码融合蛋白的基因”和“外源基因”位于各自的分子上;(15)带有上述(8)到(14)中任一项的载体的细胞;(16)用于选择性扩增(15)的细胞的方法,包括将(15)的细胞暴露于能够作用于由(8)的载体中含有的基因所编码的融合蛋白的“抗体结合域”的配体;和(17)包括(a)(5)或(8)的载体和(b)能够作用于由该载体中含有的基因所编码的融合蛋白的“配体结合域”的配体的试剂盒。
只要其作用于特异性蛋白从而导致该蛋白的结合,在本发明中可使用任何配体,但类固醇激素是优选的。类固醇激素的实例包括雌激素、雄激素、孕酮、糖皮质激素和盐皮质激素。它们与其相应的受体蛋白结合使用。只要其结合时赋予细胞增殖活性,在本发明中可使用任何的细胞因子。细胞因子受体实例有包括G-CSF的细胞因子受体族和包括c-kit和flk2/flt3的酪氨酸激酶受体族。
作为本发明融合蛋白“赋予细胞增殖活性的结构域”,使用传递细胞内增殖信号的分子(例如,细胞因子受体)的完整分子是可能的。仅使用赋予细胞增殖活性分子中的结构域也是可能的,后者的方法在增殖细胞时是优越的,因为该区域只增殖其中导入有编码融合蛋白基因的细胞而不导致该细胞的分化。此外,用于本发明中的载体不仅包括含有融合蛋白编码基因的单个载体分子和含有融合蛋白编码基因和外源基因的单个载体分子,而且包括含有蛋白编码基因载体与含有外源基因载体结合的多个载体分子的载体系统,如双载体系统(binary vector system)。多个载体分子的载体系统通常通过共转化而导入细胞中。
当编码融合蛋白的基因和外源基因插入到相同的载体中时,它们可形成含有内部核糖体进入位点(IRES)(日本公开的PCT申请No.Hei 6-509713)的双顺反子(dicistronic)形式。例如,使用下面的载体是可能的,即具有从5′至3′含有启动子、外源基因、IRES和融合蛋白编码基因的结构的载体或具有从5′至3′含有启动子、融合蛋白编码基因、IRES和外源基因的结构的载体。前一种类型一般用于使表达融合蛋白基因的大多数细胞表达外源基因。
此外,在本发明中,在其中导入载体的细胞包括造血干细胞、淋巴细胞和除这些血细胞之外的细胞。特别地,可自主扩增的造血干细胞在本发明中是优选的。虽然对导入本发明的细胞的外源基因没有特别限制,但一般使用与缺陷基因相应的正常基因用于基因治疗领域。
附图简述
图1(A)显示G-CSF受体和雌激素受体之间的嵌合分子(GCRER)。(B)显示G-CSF受体与雌激素受体之间嵌合分子的突变体,G-CSF受体的第5到第195个氨基酸缺失(GCRΔ(5-195)/ER)。(C)显示G-CSF受体和雌激素受体之间嵌合分子的突变体,G-CSF受体中的第5到第195个氨基酸以及第725到756个氨基酸缺失(GCRΔ(5-195,725-756)/ER)。
图2显示其中掺入了G-CSF受体和雌激素受体嵌合基因的反转录病毒载体“pMX”。
图3显示随着时间的推移,以“pCMX-GCRER”转化的Ba/F3细胞的增殖。
图4显示随着时间的推移,以“pCMX-GCRER”转化的Ba/F3细胞的增殖;细胞以不同浓度的雌二醇刺激。
图5显示随着时间的推移,以“pCMX-GCRΔ(5-195)/ER”转化的Ba/F3细胞的增殖。
图6显示质粒“pCMX-GCRER-IRES-CD24”。
图7显示质粒“pCMX-GCRΔ(5-195)/ER-IRES-CD24”。
图8显示质粒“pCMX-GCRΔ(5-195,725-756)/ER-IRES-CD24”。
图9显示通过流式细胞术检测的CD24在其中导入有“pCMX-GCRΔ(5-195)/ER-IRES-CD24”的Ba/F3细胞中的表达。上图板显示其中导入有“pCMX-GCRΔ(5-195)/ER-IRES-CD24”的Ba/F3细胞的结果;下图板显示作为对照的其中导入有“pCMX-GCRΔ(5-195)/ER”的Ba/F3细胞的结果。(注:此数据也含有来自用于检测死细胞的碘化丙锭的信号。)
图10为显示从其中导入有“vMXGCRER”的骨髓细胞衍生的粒细胞-巨噬细胞系集落的显微照片。
图11为显示从其中导入有“vMXGCRΔ(5-195)/ER”的骨髓细胞衍生的成红血细胞集落的显微照片。
图12为显示从其中导入有“vMXGCRER”的骨髓细胞分化的赖特-吉姆萨-染色的巨噬细胞的显微照片。
图13为显示从其中导入有“vMXGCRΔ(5-195)/ER的骨髓细胞分化的赖特-吉姆萨-染色的成红血细胞的显微照片。
本发明的最佳实施方式实施例1构建嵌合的G-CSF受体/雌激素受体基因(选择性扩增基因)
为了制备包括完整G-CSF受体和雌激素受体配体(雌激素)结合域的嵌合蛋白(以后简称为“GCRER”),构建具有编码各自蛋白cDNA的融合基因(图1(A))。其次,为了制备对G-CSF不具有反应性的嵌合蛋白,构建G-CSF受体胞外区从第5个残基(Glu)到第195个残基(Leu)缺失的“GCRER”融合基因的突变体(以后简称为“GCRΔ(5-195)/ER”)(图1(B))。此外,通过缺失含有该突变体G-CSF受体分化诱导域(725-756)的部分而构建突变体(以后简称为“CGRΔ(5-195,725-756)/ER”)(图1(C))。实施例2其中导入有选择性扩增基因、嵌合G-CSF受体/雌激素受体基因的Ba/F3细胞的分离。
将实施例1中制备的三种选择性扩增基因导入质粒“pCMX”(癌症研究(Cancer Res.)56:4164(1996))。通过电穿孔将每种得到的10μg质粒与带有杀稻瘟素抗性基因的1μg SacI线性化的“pSV2bsr”(Kaken Pharmaceuticals)一齐导入IL-3依赖性细胞系的Ba/F3细胞中。电穿孔后,以5×105个细胞每孔将细胞分散于24孔板中,并培养于含有10μg/ml杀稻瘟素的培养基中。在导入有“pCMX-GCRER”的17孔杀稻瘟素抗性细胞中观察到有11孔中发生了增殖,在导入有“pCMX-GCRΔ(5-195)/ER”的29孔中观察到有3孔中发生增殖,且在导入“pCMX-GCRΔ(5-195,725-756)/ER”的52孔中有52孔中发生增殖。使这些杀稻瘟素抗性细胞在具有IL-3的单个孔中增殖后,以10-7M的雌二醇代替IL-3培养细胞。非IL-3依赖性且雌激素依赖性细胞在导入有“pCMX-GCRER”的11孔中观察到有7孔中发生增殖,在导入有“pCMX-GCRΔ(5-195)/ER”的3孔中有3孔发生增殖,且在导入有“pCMX-GCRΔ(5-195,725-756)/ER”的16孔中有13孔发生增殖。当用其中插入有“GCRER”的反转录病毒载体pMX”(实验血液学(Exp.Hematol)24:324(1996))(以后简称为“PMX-GCRER”)代替“pCMX-GCRER”进行类似的实验时(图2),在每孔含有一个细胞的24孔中观察到2孔中有非IL-3依赖性和雌激素依赖性细胞的增殖。同样,当以1nM G-CSF代替雌二醇加入到其中导入有“pCMX-GCRER”的细胞中时,显示出G-CSF依赖性增殖的孔与显示雌二醇依赖性增殖的孔相同。此外,当用不含质粒的Ba/F3细胞作对照时,既没有观察到G-CSF依赖性的增殖又没有观察到雌二醇依赖性的增殖。用抗GCSF受体抗体或抗雌激素受体抗体的Western印迹证实了在此细胞中产生了所需融合蛋白。实施例3通过雌二醇对细胞增殖的分析
在实施例2中通过限制性稀释而获得的克隆中,挑选出对雌二醇表现良好反应的免隆并用于下面的实验中(XTT分析)。
检测其中导入有“pCMX-GCRER”的Ba/F3细胞。有通过G-CSF或雌二醇刺激而发生增殖的非IL-3依赖性细胞(图3)。此外,当在10-14和10-17M之间改变雌二醇浓度进行相同的实验时,观察到在10-9至10-7M范围内的细胞发生增殖(图4)。该结果表明雌二醇在10-9和10-7M的浓度之间传递细胞增殖信号。
然后检测其中导入有“pCMX-GCRΔ(5-195)/ER”的Ba/F3细胞。结果表明由G-CSF刺激的细胞增殖被阻止并仅仅由雌二醇的刺激导致了细胞的增殖(图5)。
相似地,对于其中导入有“pCMX-GRΔ(5-195,725-756)/ER”的Ba/F3细胞,细胞增殖由雌激素刺激所致,但没有观察到对G-CSF的反应。实施例4 IRES-CD24表达质粒的构建
“pCMX-GCRER”以HindIII和EcoRI消化,并回收载体片段(“片段1”)。同样,回收从“pCMX-GCRER”和“pCMX-GCRΔ(5-195)/ER”的HindIII片段(“片段2”,1672bp)和KpnI片段(“片段3”,1099bp),以及EcoRI片段(“片段4”,1888bp)和KpnI片段(“片段5”,1792bp)。将PBCEC(来自EMCN,Mignta,M.,美国自然学院院报(Prol.Natl.Acad.Sci)92:12075(1995)连接至IRES和CD24的pBluescript II KS)以ApoI消化并回收含有IRES-CD24的片段(“片段6”,950bp)。通过连接“片段1”、“片段2”、“片段4”和“片段6”而构建“pCMX-GCRER-IRES-CD24”(图6)。通过连接“片段1”、“片段3”、“片段4”、和“片段6”而构建“pCMX-GCRΔ(5-195)/ER-IRES-CD24”(图7)。通过连接“片段1”、“片段3”、“片段5”和“片段6”而构建“pCMX-GCRΔ(5-195,725-756)/ER-IRES-CD24”(图8)。实施例5 CD24的细胞内表达
107个Ba/F3细胞以PBS洗2次且以“OPTI-MEM1”(GIBCO-BRL)洗一次后,将此细胞悬浮于0.2ml“OPTI-MEM1”中。向细胞中加入10mg每种的“pCMX-GCRER-IRES-CD24”、“pCMX-GCRΔ(5-195)/ER-IRES-CD24”和“PMX-GCRΔ(5-195,725-756)/ER-IRES-CD24”,并用“基因脉冲仪”(伯乐(Biorad))于290V,960mF进行转化。转化后,将细胞于含有10%FCS和10U/ml mIL-3(R&D SYSTEMS)的PRMI培养基中培养2天。将106个细胞以5%FCS/PBS洗,将此细胞与1mg/ml的抗-CD24抗体(Pharmingen)于室温反应30分钟。然后将细胞以5%FCS/PBS洗2次,与1∶20稀释的PE标记的抗鼠抗体(DAKO)于室温反应30分钟,然后再以5%FCS/PBS洗两次。将此细胞悬浮于1ml的5mg/ml碘化丙锭/PBS中,并用585nm的检测器(detector)通过流式细胞仪(BectonDickinson)分析CD24的表达。测定多个其中导入有“pCMX-GCRΔ(5-195)/ER-IRES-CD24”的细胞中CD24的表达。在此实验中,其中导入有“pCMX-GCRΔ(5-195)/ER”的细胞用作其中导入有“pCMX-GCRΔ(5-195)/ER-IRES-CD24”细胞的对照。结果显示于图9和表1中。注:此数据中含有来自用于检测死细胞的碘化丙锭的信号。
表1
实施例6起源(progenitor)分析
导入的质粒 | 抗CD24抗体(-)细胞 | 抗CD24抗体(+)细胞 |
pCMX-GCRΔ(5-195)/ER-IRES-CD24 | 59.77% | 40.23% |
pCMX-GCRΔ(5-195)/ER | 85.10% | 14.90% |
将生理盐水(10mg/ml)中的5-氟尿嘧啶(5FU:Wako Pure ChemicalIndustries,Ltd.)以33ml/小鼠的剂量静脉注射至4周龄的C57BL小鼠中。注射后2天,收集股骨髓,于“Lymphocyte-M”(Cederlane)上离心(1,500rpm,25℃,22分钟)以分离单核细胞。在补充以20%FCS、100U/ml IL6和100mg/ml大鼠SCF的Iscove改进Dulbecco培养基(IMDM;Gibco)中将此单核细胞培养2天。在以CH296(Takara Shuzo;Hanenberg,H.等,自然医学(NatureMed.),2:876(1996))覆盖的平板(1146:Falcon)上,将106个以IL6和SCF预处理的骨髓细胞悬浮于含有108个反转录病毒“VMXGGCREF”(在亲嗜性包装细胞系“GP+E-86”(病毒学杂志(J.Virol.)62:1120(1988)且其中掺入有“pMX-GCRER”的培养上清中获得)或反转录病毒“VMXGCRΔ(5-195)/ER”(在亲嗜性包装细胞系“GP+E-86”且其中导入有“pMX-GCRΔ(5-195)/ER的培养上清中获得)的培养上清中。在IL6和SCF的存在下培养细胞。分别在第2、24、36和38小时更换病毒上清。在第6次更换病毒上清后的24小时,以104/孔将细胞转入含有甲基纤维素的培养基中(IMDM,1.2%甲基纤维素1,500CP;Wako,20%FCS,1%去离子BSA,10mM2-巯基乙醇,10-7Mb-雌二醇)。培养10天后,于显微镜下观察集落。制备涂片样品并以赖特-吉姆萨染色鉴定细胞。
在以“vMXGCRER”或“vMXGCRΔ(5-195)/ER”感染的骨髓细胞中,观察到了由雌二醇刺激从骨髓细胞分化出的粒细胞-巨噬细胞系集落和成红血细胞系集落。图10显示由雌二醇刺激的“vMXGCRER”感染的骨髓细胞衍生的粒细胞-巨噬细胞系集落。图11显示由雌二醇刺激的“vMXGCRΔ(5-195)/ER”感染的骨髓细胞衍生的成红血细胞系集落。当将组成这些集落的细胞制成涂片并进行赖特-吉姆萨染色时,获得了分化的血细胞图像。图12显示在由“vMXGCRER”-感染的骨髓细胞衍生的粒细胞-巨噬细胞系集落的涂片样品中观察到的以赖特-吉姆萨染色的巨噬细胞图像,图13显示在由“vMXGCRΔ(5-195)/ER”-感染的骨髓细胞衍生的成红血细胞系集落的涂片样品中观察到的以赖特-吉姆萨染色的成红血细胞的图像。
工业应用性
本发明使得人们有可能在对外部刺激作出应答时选择性地扩增其中导入有外源基因的细胞,因而即使当此基因导入靶细胞的效率低时仍可能进行有效的基因治疗。此外,由于用于选择性扩增本发明的细胞的系统可应用于多种血细胞,故在基因治疗中扩大了靶细胞的范围。因此,本发明提供了重要的基本技术,尤其是在基因治疗领域。
Claims (17)
1.一种融合蛋白,包括(a)雌激素受体的配体结合域,(b)当配体结合至(a)所述结构域时发生结合的结构域,和(c)含有通过结合而赋予细胞增殖活性的G-CSF受体或其突变体的结构域。
2.一种载体,含有编码权利要求1所述融合蛋白的基因。
3.一种细胞,携带权利要求2的载体。
4.一种选择性扩增权利要求3所述细胞的方法,包括将权利要求3所述细胞暴露于配体,该配体能够作用于权利要求1所述融合蛋白的配体结合域。
5.一种载体,含有所需的外源基因和编码权利要求1所述融合蛋白的基因。
6.权利要求5的载体,其中编码融合蛋白的基因和外源基因位于相同的分子上。
7.权利要求5的载体,其中编码融合蛋白的基因和外源基因位于不同的分子上。
8.一种细胞,携带权利要求5的载体。
9.一种细胞,携带权利要求7的载体。
10.一种选择性扩增权利要求8或9所述细胞的方法,包括将权利要求8或9所述细胞暴露于配体,该配体能够作用于由权利要求5所述载体所含基因编码的融合蛋白的配体结合域。
11.一种试剂盒,含有(a)权利要求2或5的载体,和(b)配体,所述配体能够作用于由该载体所含基因编码的融合蛋白的配体结合域。
12.权利要求1的融合蛋白,其中所述G-CSF受体突变体缺乏对G-CSF的反应性。
13.权利要求12的融合蛋白,其中所述G-CSF受体突变体缺乏野生型G-CSF的细胞外结构域。
14.权利要求13的融合蛋白,其中所述G-CSF受体突变体缺失了野生型G-CSF的第5位至第195位氨基酸残基。
15.权利要求1的融合蛋白,其中(c)所述结构域包含一种G-CSF受体突变体,该突变体缺乏对G-CSF的反应性和诱导分化的能力。
16.权利要求15的融合蛋白,其中所述G-CSF突变体同时缺乏野生型G-CSF的细胞外结构域和诱导分化的结构域。
17.权利要求16的融合蛋白,其中所述G-CSF突变体缺失了野生型G-CSF的第5位至第195位氨基酸残基以及第725位至第756位氨基酸残基。
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KR19990087525A (ko) | 1999-12-27 |
WO1997032971A1 (fr) | 1997-09-12 |
EP0887404A1 (en) | 1998-12-30 |
US20020004583A1 (en) | 2002-01-10 |
CA2248878A1 (en) | 1997-09-12 |
ATE332362T1 (de) | 2006-07-15 |
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