CN1708320A - 经修饰的树突状细胞 - Google Patents
经修饰的树突状细胞 Download PDFInfo
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Abstract
本发明说明树突状细胞受慢病毒感染而损伤其作为抗原呈递细胞的能力,抗原呈递细胞可极化天然T细胞,开辟Th1途径。所述损伤通过用含有编码IL-7、IL-12和针对IL-10RNA的siRNA的载体的慢病毒感染树突状细胞而得以恢复。
Description
相关申请案的交叉引用
本专利申请主张2002年11月7日提交的标题为“Modulation of Dendritic CellFunction”的第60/424,602号美国临时专利申请的优先权。
关于联邦资助研究的声明
本发明获得美国政府的支持,美国国家卫生总局授予资助号P50 HL59412。美国政府对于本发明具有某些权利。
技术领域
本发明涉及分子生物学、基因治疗、免疫学及病毒学领域。更具体地说,本发明涉及用慢性病毒载体(lentiviral vector,LV)转导树突状细胞从而调节树突状细胞功能的组合物和方法。
背景技术
尽管诸如人体免疫缺陷病毒(HIV)的LV与动物疾病有关,但是其所具有的将外源性核酸转移到宿主细胞中的能力已开发用于治疗疾病的基因治疗实验。对于基因治疗应用,LV提供若干种优于其它载体的优点。举例而言,HIV来源的LV采用与野生型病毒相似的细胞进入及基因组整合方法,包括感染***细胞和未***细胞的能力。感染***和未***细胞的优点使LV与常规的致癌逆转录病毒(oncoretroviral)载体相比成为更通用的基因转移媒介物。有效的整合、广泛的宿主细胞嗜性和低的组织特异性使LV比诸如与腺相关的病毒载体的其它载体更加有效及适用。
已使用LV将基因转移到树突状细胞(DC)中用于免疫疗法和疫苗应用。DC,专门的抗原呈递细胞,因具有诱导有力的T细胞应答的能力而广泛用于该等应用中。然而,如下文报告,公开说明由LV转导的DC显示出活化天然T细胞的降低的能力。在LV转导之后,DC显示出改变的细胞因子应答和表面标记表达,包括IL-10的上调及T细胞协同刺激分子的下调。根据这些研究结果,经LV转导的DC将天然T细胞极化为Th1效应细胞的能力受到损害——这种影响可能限制LV转导DC在免疫治疗及疫苗应用中的用途。
发明内容
本发明公开用于克服LV诱导的DC功能损伤的方法及组合物。在本发明的研发中,调查了一系列用于克服HIV/慢病毒感染所引起的DC诱导T细胞机能障碍的免疫调节策略,其中包括应用可溶性细胞因子及免疫调节剂。通过将诸如慢病毒免疫调节病毒的免疫调节剂传递到DC,可纠正HIV(慢病毒)感染所引起的DC及T细胞机能障碍。具体地说,受损的Th1应答可通过用含有编码IL-7、IL-12或针对IL-10RNA的siRNA的载体的慢病毒感染DC而得以恢复。此项技术提供用于在患者的治疗、疫苗接种或载体应用过程中克服与DC的HIV感染相关的免疫抑制问题的特定免疫治疗方案。
因此,本发明的特点在于一种核酸,其包含慢病毒来源的第一核苷酸序列和编码IL-7、IL-12、或对IL-10特异的siRNA的第二核苷酸序列。本发明还包含一种已将纯化核酸导入到其中的树突状细胞(例如,一种经慢病毒感染的树突状细胞),该纯化核酸包含编码从由下列各物组成的群组中选出的试剂的核苷酸序列:IL-7、IL-12、及对IL-10特异的siRNA。
本发明另一方面的特点在于一种调节树突状细胞的T细胞活化能力的方法。该方法包含调节与树突状细胞相关的IL-7、IL-10、及/或IL-12的量的步骤。举例而言,此步骤可涉及增加与该细胞相关的IL-7及/或IL-12的量,及/或降低与该细胞相关的IL-10的量。调节与树突状细胞相关的细胞因子的量可通过使该细胞与可溶性细胞因子相接触,从细胞中清除可溶性细胞因子来达到,或者通过将纯化核酸导入该细胞中来达到,该纯化核酸编码该细胞因子或降低细胞因子表达的试剂(例如,siRNA或反义核酸)。
文中所用的短语“核酸”表示诸如RNA(核糖核酸)及DNA(脱氧核糖核酸)的两种或两种以上核苷酸的链。“纯化”的核酸分子是一种已实质上与核酸天然存在于其中(例如30%、40%、50%、60%、70%、80%、90%、95%、96%、97%、98%、99%、100%无污染)的细胞或有机体中的其它核酸序列分离或离析的核酸分子。该术语包括,例如,结合到载体、质粒、病毒或原核生物或真核生物的基因组中的重组核酸分子、。纯化核酸分子的实例包括cDNA、基因组核酸片段、核酸产生的聚合酶链反应(PCR)、由基因组核酸的限制酶处理所形成的核酸、重组核酸及化学合成核酸分子。
文中所用术语“载体”表示能够转运核酸及/或病毒粒子的实体,例如质粒或病毒载体。
除非另有规定,否则本文所用的所有技术术语的含义具有与本发明所属领域的技术人员所通晓的含义相同。分子生物学术语的通晓定义可在Rieger等人的Glossary ofGenetics:Classical and Molecular,第5版,Springer-Verlag:New York,1991中,及Lewin,Genes V,Oxford University Press:New York,1994中找到。病毒学术语的通晓定义可在Granoff和Webster,Encyclopedia of Virology,第2版,Academic Press:San Diego,CA,1990,及Tidona和Darai,The Springer Index of Viruses,第1版,Springer-Verlag:New York,2002中找到。微生物学的通晓定义可在Singleton和Sainsbury,Dictionary of Microbiologyand Molecular Biology,第3版,John Wiley & Sons:New York,2002中找到。
虽然与本文所描述类似或等效的方法和材料均可用于本发明的实施或测试,但是适当的方法和材料在下文描述。所有申请案、专利申请案、专利案及其它所提及的参考文献的全文以引用的方式并入本文中。若出现矛盾,则以本发明的说明书(包括定义)为准。
附图说明
图1是显示本发明中所用各种载体结构的简略图。
图2是展示对IL-10RNA特异的siRNA的简略图。
具体实施方式
本发明提供用于克服LV所诱导的DC的T细胞活化能力损伤的方法及组合物。下列优选实施例阐述这些组合物和方法的适用性。虽然如此,但是仍然可以从这些实施例的描述出发,以下文所提供的描述为基础,做出或实施本发明的其他方面。
生物学方法
以下描述涉及常规分子生物技术的方法。该等技术在所属领域中广泛已知,并且在诸如Molecular Cloning:A Laboratory Manual,第3版,1-3卷,Sambrook等人编辑,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y,2001;及Current Protocolsin Molecular Biology,Ausubel等人编辑,Greene Publishing and Wiley-Interscience,NewYork,1992(周期更新)的方法学论文中有详细描述。例如,Beaucage和Carruthers,Tetra.Letts.22:1859-1862,1981,及Matteucci等人,J.Am.Chem.Soc.103:3185,1981中讨论了核酸的化学合成方法。举例而言,可以在商用自动化核苷酸合成器上执行核酸的化学合成。例如在Current Protocols in Immunology,Coligan等人编辑,John Wiley &Sons,New York,1991;及Methods of Immunological Analysis,Masseyeff等人编辑,JohnWiley & Sons,New York,1992中描述了免疫学方法。基因转移和基因治疗的常规方法也可适用于本发明。例如参见Gene Therapy:Principles and Applications,T.Blackenstein等人编辑,Springer Verlag,1999;Gene Therapy Protocols(Methods in Molecular Medicine),P.D.Robbins编辑,Humana Press,1997;及Retro-vectors for Human Gene Therapy,C.P.Hodgson等人,Springer Verlag,1996。
核酸/LV
本发明提供一种核酸,该核酸包含慢病毒来源的第一核苷酸序列和编码能够调节DC功能(例如,克服LV所诱导的T细胞活化损伤)的试剂的第二核苷酸序列。本发明的核酸优选采用LV形式。已知许多不同类型的LV,包括那些基于天然慢病毒的慢病毒(如HIV-1、HIV-2)、猴免疫缺陷病毒(SIV)、猫免疫缺陷病毒(FIV)、牛免疫缺陷病毒(BIV)及其它病毒。参见美国专利案第6,207,455号。尽管本发明使用基于HIV-1的载体来描述,但也可以通过配合本文所描述的信息而使用其它慢病毒来源的其它载体。基于HIV-1的载体因提供对于基因治疗应用的许多优点而成为本发明的优选。
本发明的LV可以为(例如)假型,用于克服受限制的宿主细胞嗜性。举例而言,可使用水疱性口炎病毒G(VSV-G)病毒包膜来假型化LV。为提高安全性,也可以使用一种自我失活(self-inactivating,SIN)的LV。举例而言,可通过钝化3′U3启动子并删除除5′整合附着位点(其对于整合至宿主染色体中具有重要性)之外的所有3′U3序列来制成SIN型LV。用于设计本发明载体的尤其优选的结构为图1中所示的pTYF。
编码能够调节DC功能的试剂的第二核苷酸序列可以是一种编码如IL-7或IL-12(本文中显示两者用于克服LV所诱导的DC损伤)的细胞因子的核苷酸序列。使用含有编码IL-12、IL-12+GM-CSF及IL-7的LV的慢病毒调节DC功能(例如由慢病毒感染DC来纠正受损的Th1应答)。优选的LV包含pTYF-IL-12双顺反子载体、pTYF-IL12-GM-CSF三顺反子载体及pTYF-IL-7。本发明的优选慢病毒包括LV pTYF-IL-12双顺反子载体、pTYF-IL12-GM-CSF三顺反子载体及pTYF-IL-7,并且以VSV-G进行假型化用于拓宽其宿主细胞嗜性范围(参见Chang和Gay,Current Gene Therapy 1,237-251,2001;Chang和He,Curr Opin Mol Ther 3(5),468-75,2001)。
用于本文所描述实验的病毒载体(及相应的病毒)是基于MLV和基于SIN慢病毒(HIV-1)的载体。图1显示LV pTYF-CD80、pTYF-CD86、pTYF-Flt3-L、pTYF-IL-7、pTYF-CD40L、pTYF-IL-12、和pTYF-IL-12/GMCSF的结构。用于克隆SIN LV的启动质粒为pTYF,它是一种以中心多嘌呤管(central polypurine tract,cPPT)为特点的SIN载体。cPPT序列内含物已显示可将病毒载体活性提高大约3倍。SIN LV还含有***3′截断的长末端重复序列(LTR)之后的3′牛生长激素多聚腺苷化信号(bGHpA)。SINLV编码许多种细胞因子,其中包括IL-12,IL-12+GM-CSF及IL-7,以及诸如CD80或CD86的免疫调节分子(Liang和Sha,Curr.Opin.Immunol.14:384-390,2002;及Carreno和Collins,Annu.Rev.Immunol 20:29-53,2002),及Flt3-L。病毒载体中所含的人细胞因子cDNA序列通过RT-PCR从人外周血液淋巴细胞(CD80、CD86、GM-CSF、IL-12及IL-7),或从人肿瘤细胞(对于Flt3配体的TE671细胞)扩增。IL-12基因具有两种成分,IL-12A和IL-12B。对于在调节DC功能中的使用,IL-12两种成分的cDNA同时克隆到一双顺反子载体中,其中在这两个cDNA之间具有内部核糖体进入位点(IRES)。对于pTYF-IL-12-GMCSF载体,两个不同的IRES元素位于IL-12B/IL-12A与IL-12A/GM-CSF cDNA之间,形成三顺反子表达载体。病毒载体中的基因可受任何适宜启动子(例如,诸如人延伸因子1α,EF1α的强启动子)的支配。对于pTYF载体的结构,参见Zaiss等人,J.Virology 76:7209-7219;及Chang等人,Gene Therapy 6:715-728。可以如Zaiss等人,J.Virol.76:7209-7219,2002中所述构造MLV载体(及相应的病毒)。
Buchschacher等人,Blood 95:2499-2504,2000;Chang等人,Gene Therapy 6:715-728,1999;Emery等人,PNAS 97:9150-9155,2000;Naldini等人,Science 272:263-267,1996;Paillard等人,9:767-768,1998;Sharma等人,PNAS 93:11842-11847,1996;Reiser等人,PNAS 93:15266-15271,1996;及Chinnasamy等人,Blood 96:1309-1316,2000中讨论了重组LV及病毒粒子的结构。Miyoshi等人,J.Virol.72:8150-8157,1998;Zufferey等人,J.Virol.72:9873-9880,1998;Iwakuma等人,Virology 261:120-132,1999;Mangeot等人,J.Virol.74:8307-8315,2000;及Schnell等人,Hum.Gene Ther.11:439-447,2000中描述了SIN载体的设计。
树突状细胞
本发明提供一种已在其中导入纯化核酸的DC,该纯化核酸具有编码免疫调节剂(诸如IL-7、IL-12或对IL-10特异的siRNA)的核苷酸序列。可使用的DC包括哺乳动物DC,诸如那些来自小鼠、大鼠、豚鼠、非人灵长类动物(例如,黑猩猩和其它猿类及猴类)、牛、绵羊、猪、山羊、马、狗、猫、及人的DC。DC可以是哺乳动物患者内(活体内)的DC或离体培养物中的DC(例如离体培养以用于体外传送给患者的DC)。根据本发明的DC含有一种核酸,一种具有编码免疫调节剂(诸如IL-7、IL-12或对IL-10特异的siRNA)的核苷酸序列的纯化核酸。在优选的DC中,表达该核酸,产生多肽或RNA。
DC可从任何适宜的来源获得,包括皮肤、脾、骨髓或其它淋巴器官、***或血液。优选地,从血液或骨髓获得DC用于本发明。通常,在经外源性粒细胞-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素-4进行刺激后从骨髓及外周血液单核细胞(PBMC)产生DC。在Inaba等人,J.Exp.Med.176:1693-1702,1992;及Bai等人,Int.J.Oncol.20:247-253,2002中描述从骨髓细胞获得DC并培养DC的方法。从造血干细胞培养DC的方法(Mollah等人,J.Invest.Dermatol.120:256-265,2003)和从单核细胞培养DC的方法(Nouri-Shirazi和Guinet,Transplantation 74:1035-1044,2002)在所属领域中是已知的。在Pullarkat等人的J.Immunol.Methods 267:173-183,2002中描述了一个用于产生DC的大规模单核细胞富集程序的实例。可以在荧光活化细胞拣选(fluorescence-activated cell sorting,FACS)分析中使用DC特异性标记物从异质细胞样品中分离DC(Thomas和Lipsky J.Immunol.153:4016-4028,1994;Canque等人,Blood 88:4215-4228,1996;Wang等人,Blood 95:2337-2345,2000)。未成熟的DC具有下列特征:协同刺激分子、CD80/86、CD40的低水平表达;诱导T细胞活化的低劣能力;不能产生IL-12p70;及诱导调节性T细胞或无反应性T细胞的潜力。相比而言,成熟的DC可产生IL-12p70并表达高水平的II类MHC抗原、CD80/86及CD40、IL-12p70产物。含有DC以及经分离DC的细胞群可使用任何适宜的允许DC生长和增殖的离体培养方法进行培养。
调节DC功能
本发明还提供调节DC功能的方法。DC刺激天然T辅助细胞使其分化为产生IFN-γ的Th1细胞或产生IL-4的Th2效应细胞,它们调节不同的免疫应答。扭曲的Th应答是由LV对DC的转导和慢病毒的感染所产生的。具体地说,经慢病毒转导和慢病毒感染的DC诱导天然Th细胞朝着受损Th1应答和提高产生IL-4的Th2的应答的方向分化。本发明的组合物及方法可用于通过向DC提供细胞因子(例如免疫基因)而增加DC的免疫活化能力(例如恢复Th1应答)。适宜的细胞因子的实例包括IL-12及IL-7。其它可以提高Th1应答的细胞因子也可用于本发明。
为调节DC功能(例如,恢复Th1应答),使DC细胞与一种含有纯化核酸的LV接触,该纯化核酸包含一个慢病毒来源的核苷酸序列和至少一个非慢病毒来源的转基因。转基因可以是任何提高Th1应答的细胞因子,包括IL-12及IL-7。
在一个调节DC功能的实例中,用含有编码IL-12、IL-12加GM-CSF和IL-7的载体的慢病毒感染DC。在此实例中,未成熟的DC用Mock(293T上清液)、TYF-PLAP、TYF-IL-12、TYF-IL12-GM-CSF、或TYF-IL-7感染。在用LPS(80ng/ml)加TNF-α(20u/ml)使细胞成熟持续24小时后,收获该DC并用CD4+T细胞以1∶20的DC/T比率进行共培养。共培养5天后,在IL-2(25u/ml)存在下使T细胞另外扩增7天。接着在布雷菲德菌素A(Brefeldin A)存在下用离子霉素(ionomycin)和PMA再刺激6小时后通过细胞内IFN-γ及IL-4染色测量Th1、Th2及Th0群体。编码诸如IL-12、IL-12+GM-CSF、及IL-7的免疫调节分子的LV可有效地纠正由慢病毒感染DC所引起的受损Th1应答。
调节患者的免疫应答
用于增强及降低患者免疫应答的组合物和方法可用在多种治疗许多不同疾病的基于DC的免疫治疗策略中。成熟的DC是主要的抗原呈递细胞群,它有效地调节用于启动T细胞应答(例如,细胞毒素性T淋巴细胞的诱导)的到达有组织的淋巴组织的抗原转运。DC的正常功能是将抗原呈递给T细胞,接着T细胞特异性地识别并最终消除抗原来源。DC作为对于癌症和传染性疾病的治疗性及预防性疫苗使用。设计该等疫苗用于引发强有力的细胞免疫应答。Lundqvist和Pisa,Med.Oncol.19:197-211,2002;Herrera和Perez-Oteyza,Rev.Clin.Esp.202:552-554,2002;及Onaitis等人,Surg.Oncol.Clin.N.Am.11:645-660,2002中综述了DC生物学、向DC的基因转移及DC免疫治疗。
对细胞毒素及1型辅助(Th1)细胞应答的诱导对于针对慢性传染病或癌症的疫苗是非常需要的(P.Moingeon,J.Biotechnol.98:189-198,2002)。表达上调Th1细胞及其作用的白细胞介素的经修饰DC可用于增强机体对病原体的抵抗力(J.W.Hadden,Int.J.Immunopharmacol.16:703-710,1994)。对于HIV感染的治疗,例如,可将DC定位在活体外和活体内,启动并增强HIV特异性免疫(Piguet和Blauvelt,J.Invest.Dermatol.119:365-369,2002)。
除HIV治疗外,本发明的经修饰DC可用于癌症免疫治疗中。经操控将肿瘤抗原呈递给二级淋巴器官及静息的、天然T细胞的DC可用于产生肿瘤特异性T细胞(A.F.Ochsenbein Cancer Gene Ther.9:1043-1055,2002)。举例而言,经修饰表达骨髓瘤相关抗原的DC可适合用作多发性骨髓瘤的抗癌疗法(Buchler和Hajek,Med.Oncol.19:213-218,2002)。已显示,表达某些细胞因子或趋化因子的DC展示出实质上改良的成熟状况、活体内迁移到二级淋巴器官的能力,及活体内刺激肿瘤特异性T细胞应答并诱导肿瘤免疫的能力。因此,经修饰表达细胞因子的DC可用于诱导肿瘤免疫,并且可与经修饰表达肿瘤抗原的DC联合使用。Lemoli等人,Haematologica 87:62-66,2002;A.F.Ochsenbein,Cancer Gene Ther.9:1043-1055,2002;Zhang等人,Biother.Radiopharm.17:601-619,2002;Di Nicola等人,Cytokines Cell Mol.Ther.4:265-273,1998;D.Avigan,Blood Rev.13:51-64,1999,及Syme等人,J.Hematother.Stem Cell Res.10:601-608,2001中综述了DC在癌症免疫疗法中的治疗作用。
在一个基于DC的疫苗策略的实例中,使用编码免疫原的LV修饰DC,导致免疫原的表达并呈递给静息的、天然T细胞。可以单独使用该抗原呈递策略或联合用作混合免疫方案的一部分,以便引发广泛的免疫应答。Onaitis等人,Surg.Oncol.Clin.N.Am.11:645-660,2002中描述了涉及将DC传送给病人的不同免疫策略。
经修饰的DC也可用于调节用于治疗自身免疫性疾病(例如,关节炎、哮喘、过敏性皮炎)的T细胞(Th1和/或Th2)应答。Th1与Th2之间的平衡在许多自身免疫性疾病中具有重要性。Th1细胞活性在患有类风湿性关节炎和胰岛素依赖型糖尿病病人的关节中起主要作用,而Th2细胞支配的应答在过敏性疾病(例如***反应性疾病)、器官特异性自身免疫性疾病(1型糖尿病和甲状腺疾病)、克罗恩氏病、同种异体移植物排斥反应(例如,肾移植急性排斥反应)及一些原因不明的习惯性流产的发病机理中(AllergyAsthma Immunol.85:9-18,2000)有所涉及。当宿主免疫***探测到同种“非已”抗原时即发生同种异体移植物排斥反应。为预防或治疗同种异体移植物排斥反应,可使用经修饰的DC诱导对组织特异性抗原的耐受性(B.Arnold Transpl.Immunol.10:109-114,2002)。表达免疫抑制分子的DC也可用作用于同种异体移植物排斥反应的疗法(Lu和Thomson,Transplantation 73:S 19-22,2002)。
经修饰的DC可进一步用于诱导针对微生物病原体(例如,病毒、细菌、真菌、原生动物和蠕虫)的免疫应答。举例而言,可以对DC进行修饰,用于表达微生物病原体来源的肽抗原。由该DC所进行的抗原递呈可能刺激针对病原体的强有力的免疫应答。
实例
本发明通过下列实例进一步进行说明。提供该等实例仅用于说明而不能以任何方式理解为对本发明范畴和内容的限制。
实例1-材料和方法
单核细胞来源的树突状细胞的产生。如先前所述(Chang和Zhang,Virology 211:157-169,1995),通过在Ficoll-Hypaque(Sigma-Aldrich,USA)中的梯度密度离心分离法,从健康供体(Civitan Blood Center,Gainesville,FL,USA)的血沉棕黄层(buffy coat)中分离外周血液单核细胞(peripheral blood mononuclear cell,PBMC)。根据Thurner等人(J.Immunol.Methods 223:1-15,1999)依照下列修改,从PBMC制备DC。在实验开始的前一天,以每孔5百万个PBMC接种到在无血清AIM-V培养基中的十二孔培养板中。于37℃下培育1小时后,将未粘附的细胞轻轻洗脱并将保持粘附的单核细胞进一步在AIM-V培养基中培养直到试验开始的第1天。小心地移除培养基,不要干扰松散粘附细胞,并添加含有重组人GM-CSF(560u/ml,Research Diagnostic Inc.Flanders NJ)和IL-4(25ng/ml,R&D Systems)的新AIM-V培养基(每孔1ml),并且将细胞在37℃、5%CO2培养箱中培养。第3天,将1ml含有GM-CSF(560u/ml)及IL-4(25ng/ml)的新鲜AIM-V培养基添加到培养物中。第5天,通过轻轻的移液操作收获未粘附的细胞。洗涤之后,将DC冷冻以备后用或立即使用。
未成熟DC的慢病毒转导和DC成熟。第5天,将未成熟的DC以每孔5×105涂在含有200ul补充了GM-CSF(560u/ml)和IL-4(25ng/ml)的培养基的24孔培养板中。通过以50-100的感染复数(multiplicity of infection,MOI)将浓缩的LV添加到细胞中来进行DC的转导。将细胞在37℃下培育2小时,每30分钟轻轻摇动,然后添加1ml DC培养基,并将病毒载体与培养物一起另外培育12小时。通过将最终浓度为80ng/ml的脂多糖(LPS)和最终浓度为20u/ml的TNF-α添加到DC培养物中并持续24小时来诱导DC成熟。在37℃、5%CO2的培养箱中用含有2mM EDTA的AIM-V培养基培育成熟的DC并持续20分钟,之后将其收获。将细胞洗涤3次,用于后续实验。
抗体染色和流式细胞术。为了由流式细胞术分析细胞表面标记的表达,将DC用正常小鼠血清培育10分钟,然后用与荧光染料联合的抗人单克隆抗体再培育30分钟,该等抗体包括HLA-ABC(Tu149,小鼠IgG2a、经FITC标记,Caltag Laboratories)、HLA-DR(TU36,小鼠IgG2b,经FITC标记,Caltag Laboratories)、CD1a(HI49,小鼠IgG1k,经APC标记,Becton Dickinson)、CD80(L307.4,小鼠IgG1k,经Cychrome标记,BectonDickinson)、CD86(RMMP-2,大鼠IgG2a,经FITC标记,Caltag Laboratories)、ICAM-1(15.2,经FITC标记,Calbiochem)、DC-SIGN(eB-h209,大鼠IgG2a,k,经APC标记,eBioscience)、CD 11c(Bly-6,小鼠IgG1,经PE标记,Becton Dickinson)、CD40(5C3,小鼠IgG1,k,经Cy-chrome标记,Becton Dickinson)、CD123(小鼠IgG1,k,经PE标记,Becton Dickinson)、CD83(HB15e,小鼠IgG1,k,经R-PE标记,BectonDickinson)。在每次染色情形中也包含相应的同型对照抗体。两次洗涤之后,将细胞重新悬浮并固定在1%多聚甲醛的PBS中,然后使用FACSCalibur流式细胞仪和CELLQUEST程序(Becton Dickinson)进行分析。通过前散射和侧散射特征控制选择活细胞,并且记录阳性细胞的百分数和细胞群平均荧光强度(mean fluorescenceintensity,MFI)。
RNA的分离、标记和阵列杂交。用逆转录病毒或腺病毒载体感染之后,收获该等细胞并用Trizole试剂(Invitrogen/Life Technologies,Carlsbad,California)使其溶解。将全部RNA分离、进行标记并准备根据厂商协议(Clontech)将其杂交至Atlas Array滤膜。用15ug经标记的cDNA产物过夜杂交。杂交并洗涤后,使用磷光成像仪(phosphorimager)(Storm 486,Molecular Dynamics)扫描阵列滤膜,并使用ClontechAtlas Array图像分析软件进行定量分析。
IL-4、IL-10和IL-12的半定量及定量RT-PCR分析。如上所述用LV转导DC并使其成熟。使用Tri-reagent试剂将总RNA纯化。对于半定量RT-PCR,使用人IL-4、IL-10和IL-12的引物及人GAPDH的对照引物来执行标准的一步RT-PCR(Promega)。对于定量RT-PCR分析,通过使用Tri-reagent试剂盒分离DC的总RNA并且使用oligo-dT和AMV逆转录酶转录第一链cDNA,然后在ABI-Prism 7000PCR循环仪(AppliedBiosystems,Foster City,CA)上执行实时RT-PCR。从ABI购得IL-12p40、IL-10、GAPDH的有效PCR引物及TaqMan MGB探针(经6FAM标记)。根据厂商说明书(Stratagene和ABI)制备PCR混合物,并且循环加热器条件如下:1×95℃,10分钟,40-50个循环变性(95℃,15秒);和联合退火/延伸(60℃,1分钟)。通过比较样品与连续稀释标准品的循环阈值(threshold cycle value)执行相对定量分析。
天然CD4+T细胞的制备。根据厂商说明书通过使用CD4+T细胞分离玫瑰花结混合物(Rosette cocktail)(StemCell Technologies)进行阴性选择而从PBMC制备CD4+T细胞。简单地说,在200ml无菌Falcon离心管中,在25℃下用2.25ml CD4+T细胞富集玫瑰花结混合物将45ml血液黄层(大约5×108PBMC)培育25分钟。接着,添加45mL含有2%FBS的PBS,稀释该血液黄层。轻轻混合之后,将30ml稀血液黄层转移,并在50ml Falcon试管中的15ml Ficoll Hypaqueh上成层,并且以1,200g离心25分钟。在Ficoll界面处收获非花结细胞,并用PBS(2%FBS)洗涤两次,计数,然后以等分样品低温贮藏在液态N2中备用。经分离的CD4+T细胞的纯度一直保持在95%以上。根据厂商说明书使用MACS(Miltenyi Biotec)磁性亲和柱基于CD45RO-细胞的阴性选择纯化CD4+CD45RA天然T细胞。
Th功能的离体诱导和细胞内细胞因子染色。离体DC:T细胞共培养方法根据CaronG等人(J.Immunol,167:3682-3686,2001)进行。简单地说,在无血清AIM-V培养基中,将经纯化的天然CD4T细胞与异源的成熟DC在不同比率(20∶1至10∶1)下共培养。第5天,添加50u/ml的rhIL-2,而且使培养物扩增,并每隔一天补给含rhIL-2的AIM-V培养基一直到3周。第12天后,洗涤静息的Th细胞并用PMA(10ng/ml或0.0162uM)和离子霉素(ionomycin)(1ug/ml,Sigma-Aldrich)再刺激5小时。在培养的最后2.5个小时中添加布雷菲德菌素A(1.5ug/ml)。接着将细胞固定、通透、并用FITC所标记的抗-IFN-y和PE所标记的抗-IL-4mAb(PharMingen)染色,然后在FACSCalibur流式细胞仪(BD Biosciences)中分析。
DC介导的混合淋巴细胞反应(MLR)。将DC从每孔10,000个细胞连续稀释到每孔313个细胞,并在96孔U底培养板中用1×105异源CD4T细胞培养5天,培养物总体积200ul。根据厂商说明书(Promega),通过将20ul的CellTiter96溶液添加到每个孔中来监控T细胞的增殖,并且获得在490nm下的OD读数。
LV结构和产生。质粒结构。如先前所述(Zais等人,J.Virol.76:7209-7219,2002)构建用于此项研究的致癌逆转录病毒(MLV)和LV(HIV-1及HIV-1 SIN)。所有的HIV-1SIN载体(pTY)都具有***3′截断的长末端重复序列(LTR)之后的3′牛生长激素多聚腺苷化信号(bGHpA)。通过将NotI消化的pHEF与人化eGFP构造来源的NotI消化的eGFP片段连接而构建增强的绿色荧光蛋白质(eGFP)表达质粒,pHEFeGFP,其中人化eGFP构造可从UF Powell基因治疗中心的载体中心获得。通过将eGFP片段(XhoI-EcoRI)从pTVdl.EFeGFP***到pTYEFnlacZ中,替换核心lacZ(nlacZ)基因来制造pTYEFeGFP。通过用从pHEFeGFP分离得到的eGFP片段(XhoI-EcoRT)替换pTVdl.EFnlacZ的nlacZ片段(XhoI-EcoRI)而产生pTVdl.EFeGFP。MLV gag-pol构造是基于pcDNA3.1/Zeo(+)(Invitrogen),其中巨细胞病毒即刻早期启动子被人延伸因子1α(EF1α)启动子所替换。表达细胞因子基因或T细胞协同刺激基因的慢病毒载体是通过如上所述将编码这些基因的cDNA***到EF1α启动子之后的pTYF-EF转导载体中而构建的。
实例2-结果
病毒转导后细胞应答的cDNA微阵列分析。通过在原始的人类脐静脉内皮细胞(HUVEC)中比较不同的病毒载体来分析对病毒转导的细胞应答,该等病毒载体包括HIV-1(LV)、莫洛尼氏(Moloney)鼠白血病病毒(MLV)和腺病毒(Ad)载体。通过DNA共转染制备HIV-1和MLV载体,并且如前所述载体基因组中不包含病毒基因(Chang和Gay,Current Gene Therapy,1:237-251,2001;Zaiss等人,同上)。Ad载体是基于含有大部分腺病毒基因的E1A缺失载体***(Graham和Prevec,Manipulationof adenovirus vectors,第7卷,第11章,第109-128页,1991)。将HUVEC维持在低传代(<5)并以2-3的感染复数(moi)进行转导。为了将由包装细胞和转基因引起的变化降到最低,使此项研究中所用的3个病毒载体全部携带lacZ报告基因并且在293细胞中产生。使用一组4种Clontech Human Atlas Array 1.2印迹试剂盒研究HUVEC的细胞应答,每种印迹试剂盒含有1,176个人类cDNA、9个管家对照cDNA和阴性对照品。
用模拟品(对照品293上清液)、LV、MLV和Ad载体转导HUVEC。在感染24小时后,收获全部的polyA+ RNA,通过逆转录用32P-dATP进行标记,并用于杂交到4种相同的Clontech Atlas Human Array 1.2cDNA印迹试剂盒。使用Clontech AtlasImage 1.5软件和两两对比法分析所得结果。可由任何应用软件所记录的超过2倍的变化或10,000以上的信号强度任意地测定上调或下调基因,并通过视觉比较加以确认。结果总结为6组由Clontech任意设立的基因库:细胞周期及致癌基因、信号转导、细胞凋亡及GTP酶、转录及表面信号发放、粘附-受体-趋化因子,和应激反应-白介素-干扰素。见下表1。LV似乎比MLV和Ad载体更经常增强转录和表面信号发放基因,而且有趣的是,IL-10(一种免疫抑制细胞因子)在MLV及LV转导后出现上调。
表1.HUVEC中病毒转导对基因表达的影响。由上调(↑)、下调(↓)或不变(-)表示6个任意定义的功能基因的基因表达的变化倍数。
Ad | MLV | LV | |
A:细胞周期/致癌基因 | ↑ | ↑↑ | ↑↑ |
B:信号转导 | ↓ | ↓ | ↓ |
C:细胞凋亡,GTP酶 | - | - | - |
D:转录,表面信号发放 | ↑↑ | ↑ | ↑↑↑ |
E:粘附,受体,趋化因子 | ↑↑ | ↑↑ | ↑↑ |
F:应激反应,IL,IFN | ↑ | ↑ | ↑ |
LV转导后DC表面标记表达的分析。使用不同的抗体和流式细胞术分析LV转导后DC上的表面标记表达。用包含模拟品(对照品293上清液)、空LV粒子(含有HIV-1壳体和VSV-G包膜而无病毒基因组的粒子)、LV及MLV的载体转导外周血液单核细胞(PBM)来源的未成熟DC。也检验空LV以便观察载体粒子中存在的病毒蛋白质是否会诱导DC表型的变化。在用LPS加TNF-α处理24小时后,收获DC以用于抗体染色和流式细胞术。结果总结在表2中。在经检验的表面分子中,CD1a、CD80、CD86、ICAM-1和DC-SIGN在LV转导后出现下调,但是使用空LV或MLV时不发生此种情况。当检验携带有PLAP或Cre报告基因的LV的不同制备物时,获得相同的结果。
表2.经LV或MLV转导的DC的表面标记概况
几何平均荧光强度±SD | ||||
表面标记 | 模拟品 | 空LV | LV-PLAP | MLV |
CD11c | 48.8±3.2 | 47.2±1.3 | 52.3±2.3 | 55.3±1.1 |
CD123 | 13.0±0.4 | 13.4±0.8 | 14.9±0.6 | 15.7±0.1 |
CD1a | 27.3±1.1 | 27.6±2.9 | 21.5±0.2* | 31.0±0.3 |
CD40 | 8.6±0.1 | 8.9±0.6 | 8.6±0.1 | 9.0±0.3 |
ICAM-1 | 462.6±57.5 | 376.5±30.1 | 179.5±3.4*** | 498.5±6.9 |
CD62L | 3.3±0.1 | 3.2±0.03 | 3.7±0.1 | 3.3±0.4 |
CD80(B7-1) | 9.9±0.9 | 10.6±0.7 | 9.3±0.2* | 11.3±0.4 |
CD83 | 5.8±0.3 | 5.8±0.1 | 6.4±0.01 | 6.0±0.3 |
CD86(B7-2) | 39.6±3.5 | 39.6±2.5 | 31.4±0.4* | 47.3±1.5 |
DC-SIGN | 62.7±4.5 | 55.7±0.4 | 50.6±1.5* | 68.6±4.1 |
HLA-ABC | 13.9±1.3 | 15.8±1.0 | 14.6±0.3 | 17.2±0.9 |
HLA-DR | 31.5±0.8 | 28.6±2.2 | 26.9±0.4 | 33.2±1.7 |
结果表示为流式细胞术后的几何平均荧光强度。
星号(*)表示司图登特t-检验(Student t-test)的差异显著性(*P<0.05,**P<0.01,***P<0.001)。
受LV转导损伤的DC介导的Th1免疫。使用人DC和天然T细胞执行离体DC功能检定。在含有GM-CSF和IL-4的培养物中从PBM产生DC,并用携带PLAP报告基因的LV感染PBM来源的第5天(d5)的DC。第7天分析受感染DC的PLAP活性。在此情况下,使用LV以约30-80的感染复数转导90%以上的DC。为了观察LV感染之后DC中IL-10的表达是否受到影响,用LV感染第5天的DC,并在第6天用LPS处理DC,在接下的时间里使用抗-IL-10单核克隆抗体和流式细胞术,通过细胞内细胞因子染色(ICCS)分析IL-10表达。与HUVEC的LV转导类似,LV感染之后观察到DC中IL-10出现上调。
为了进一步表征LV感染后DC的功能,从外周血液单核细胞(PBMC)纯化天然CD4+T细胞,并且在用TNF-α和LPS诱导成熟之后与PBM来源的异源DC共培养。第5天用LV或MLV感染这些DC,诱导其成熟,并且与天然CD4+T细胞共培养。允许这些T细胞扩增并在DC启动超过7天之后进行休眠。为分析Th应答,在用离子霉素和PMA共培养后的第7天和第9天激活休眠的T细胞,并如上所述使用针对IFN-γ和IL-4的抗体进行细胞内染色(ICCS)。结果证明产生IFN-γ的Th1细胞群急剧减少,从第7天的72%和第9天的75%(对照品),降到第7天的27%和第9天的22%(经LV转导的DC),而Th2群保持不变。对于MLV转导的DC观察到了类似但不太显著的效果。
由编码免疫调节基因的LV修饰DC的免疫性。如图1所示将人CD80和CD86的cDNA克隆到LV中。用携带有报告基因的LV(LV-PLAP)、携带有CD80cNA的LV(LV-CD80)或携带有CD86 cDNA的LV(LV-CD86)转导DC,12个小时后用LPS和TNF-α加以处理。LV转导后36小时,使用抗CD80和抗CD86抗体通过流式细胞术分析经转导DC的CD80和CD86表达。受到LV-PLAP感染后CD80和CD86的表达均降低,对于CD80而言从41%降到35%,对于CD86而言从61%降到49%。然而,在用编码CD80和CD86的LV转导之后,CD80和CD86的表达分别出现上调,CD80从35%上调到44%,CD86从49%上调到76%。
在其它实验中,将用模拟品、LV-PLAP、LV-PLAP加LV-CD80、或LV-PLAP加LV-CD86转导的DC与天然CD4T细胞进行共培养。8天后,再次激活T细胞并如上所述使用抗IL-4和抗IFN-γ抗体通过ICCS和流式细胞术进行分析。结果表明,LV转导之后,Th1群从24%下调到13%,而且这种损伤不能由DC中CD80和CD86的上调(分别为从13%到12%及13%)加以纠正。
在其它实验中,研究是否可以通过向DC培养物补充可溶性的IL-12和/或FL来增强DC的Th1活化功能,其中将这些细胞因子单独或一起添加到病毒转导期间的DC培养物中和DC:T细胞共培养物中。第6天和第7天再次激活经共同培养的T细胞用于Th分析。由IL-4和INF-γ ICCS对T细胞进行的第6天和第7天的分析结果均证实LV转导后Th1应答受到损伤(分别从37.5%和20%降到15.6%和10%)。然而,仅补充外源性IL-12可部分地纠正受损的Th1应答(分别为,单独补充IL-12时从15.6%和10%恢复到19.1%和11.7%,IL-12+FL时恢复到18.7%和13.2%),而单独补充FL时无作用(分别从15.6%和10.0%到14.6%和8.8%)。在其它使用更高浓度可溶性IL-12的实验中,受损的Th1应答完全得到纠正。
为了设计具有增强的关键细胞因子内源性表达的DC,构建并检验编码不同细胞因子的LV,该等细胞因子包括FL、IL-7、CD40L、双顺反子L-12及三顺反子IL-12/GM-CSF(图1)。用仅携带有报告基因的LV转导DC,或用表达不同细胞因子的LV共转导DC。通过DC:T细胞共培养物检定研究LV所转导DC的Th功能,并且12天后,如上所述再激活T细胞,并由ICCS和流式细胞术进行分析。结果显示,单独的LV报告载体转导导致降低的Th1进展(从54.6%降到37.7%)。然而,用编码双顺反子IL-12、三顺反子IL-12/GM-CSF和IL-7的LV共同转导DC,可有效地增强Th1应答,分别从37.7%增到56.2%、56.2%和50.7%。编码其它免疫调节基因(如FL、GM-CSF或CD40L)的LV未显示出任何纠正作用。
由表达针对IL-10的小干扰RNA调节DC功能。构建编码针对IL-10的小干扰RNA(small interfering RNA)的LV。选择IL-10mRNA中的两个区域作为RNA干扰靶位(图2)。由人H1 pol III启动子驱动siRNA表达盒,并将其逆向克隆到LV中。LV-siRNA载体还在邻近pol III siRNA处携带有nlacZ报告基因,以用于滴定度的测定。用报告LV和针对IL-10的LV-siRNA共同转导DC,接着在经LPS处理和ICCS后如上所述分析IL-10表达。结果再次显示,单独的LV转导上调IL-10表达,而与针对IL-10的LV-siRNA进行共同转导下调IL-10的表达。接着将两种IL-10LV-siRNA构造与LV共同转导和DC:T共培养物Th1功能检定中的LV-IL-7相比较。激活共培养的天然T细胞,并且在再次活化和Th细胞因子分析之前使其休眠20天。IL-4和IFN-y ICCS的结果表明,两种IL-10LV-siRNA载体均增强Th1应答,并且#2IL-10LV-siRNA显示出与LV-IL-7相当或高于LV-IL-7的水平的Th1应答增强。另一种Th1细胞因子TNFa ICCS的分析进一步证实了所述结果。
其它实施例
应了解,当已结合本发明的详细说明对其做出描述时,前述描述的目的在于说明而不是限制本发明的范畴,本发明的范畴由附加的权利要求界定。举例而言,可以使用非慢病毒方法将克服LV诱导的DC损伤的试剂导入到靶DC中,例如使用其它病毒载体或非基于载体的方法来进行。本发明的其它方面、优点和修改均从属于以上权利要求的范畴。
序列表
<110>郎基·常
<120>经修饰的树突状细胞
<130>7655-1WO
<160>2
<170>PatentIn version 3.2
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<211>53
<212>RNA
<213>human
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<212>RNA
<213>human
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ggguuaccug gguugccaau ucaagagauu ggcaacccag guaacccuu 49
Claims (23)
1.一种核酸,其包含慢病毒来源的第一核苷酸序列和编码能够调节树突状细胞活化T细胞能力的试剂的第二核苷酸序列。
2.根据权利要求1所述的核酸,其中所述第二核苷酸序列编码IL-7。
3.根据权利要求1所述的核酸,其中所述第二核苷酸序列编码IL-12。
4.根据权利要求1所述的核酸,其中所述第二核苷酸序列编码siRNA。
5.根据权利要求4所述的核酸,其中所述siRNA对IL-10特异。
6.根据权利要求1所述的核酸,其中所述核酸包含在一慢病毒载体中。
7.根据权利要求6所述的核酸,其中所述慢病毒载体包含在一病毒粒子中。
8.一种已在其中导入纯化核酸的树突状细胞,该纯化核酸包含一种编码能够调节所述树突状细胞活化T细胞能力的试剂的核苷酸序列。
9.根据权利要求8所述的树突状细胞,其中所述细胞包含一慢病毒载体。
10.根据权利要求8所述的树突状细胞,其中所述核苷酸序列编码IL-7。
11.根据权利要求8所述的树突状细胞,其中所述核苷酸序列编码IL-12。
12.根据权利要求8所述的树突状细胞,其中所述核苷酸序列编码siRNA。
13.根据权利要求12所述的树突状细胞,其中所述siRNA对IL-10特异。
14.根据权利要求9所述的树突状细胞,其中所述慢病毒载体包含编码所述试剂的核苷酸序列,该试剂从由下列各物组成的群组中选出:IL-7、IL-12和对IL-10特异的siRNA。
15.一种调节树突状细胞的T细胞活化活性的方法,该方法包含调节至少一种与所述树突状细胞相关的细胞因子的量的步骤。
16.根据权利要求15所述的方法,其中所述至少一种细胞因子从由下列各物组成的群组中选出:IL-7、IL-10及IL-12。
17.根据权利要求16所述的方法,其中增加与所述细胞相关的IL-7的量。
18.根据权利要求16所述的方法,其中增加与所述细胞相关的IL-12的量。
19.根据权利要求16所述的方法,其中降低与所述细胞相关的IL-10的量。
20.根据权利要求15所述的方法,其中所述调节至少一种与所述树突状细胞相关的细胞因子的量的步骤包含使该细胞与一种可溶性细胞因子相接触。
21.根据权利要求15所述的方法,其中所述调节至少一种与所述树突状细胞相关的细胞因子的量的步骤包含将一纯化核酸导入所述树突状细胞中,该纯化核酸包含一种编码从由下列各物组成的群组中选出的试剂的核苷酸序列:IL-7、IL-12和对IL-10特异的siRNA。
22.一种包含慢病毒来源的第一核苷酸序列和编码siRNA的第二核苷酸序列的核酸。
23.一种调节树突状细胞中基因表达的方法,该方法包含将一核酸导入该树突状细胞中的步骤,该核酸包含慢病毒来源的第一核苷酸序列和编码siRNA的第二核苷酸序列。
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CN108676815A (zh) * | 2018-05-31 | 2018-10-19 | 深圳市免疫基因治疗研究院 | 一种b型血友病慢病毒载体、慢病毒及其制备方法和应用 |
CN111647563A (zh) * | 2020-08-06 | 2020-09-11 | 北京翊博普惠生物科技发展有限公司 | 靶向Survivin全抗原的DC细胞、CTL细胞及其制备方法和应用 |
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US20090010948A1 (en) * | 2006-12-15 | 2009-01-08 | The University Of Hong Kong | Anti-tumor vaccines delivered by dendritic cells devoid of interleukin-10 |
WO2009048560A1 (en) * | 2007-10-08 | 2009-04-16 | Intrexon Corporation | Engineered dendritic cells and uses for the treatment of cancer |
RU2015144160A (ru) | 2013-03-15 | 2017-04-24 | Те Брод Инститьют, Инк. | Генная экспрессия при ответе дендритной клетки, их композиции и способы применения |
CN103834691B (zh) * | 2014-01-21 | 2016-06-29 | 宁波大学 | 靶向il-33基因rna干扰重组慢病毒载体的构建方法 |
BR112018008911A2 (pt) * | 2015-11-09 | 2018-11-27 | Immune Design Corp | composições compreendendo vetores lentivirais que expressam il-12 e métodos de uso das mesmas |
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US5965535A (en) * | 1997-09-12 | 1999-10-12 | Ludwig Institute For Cancer Research | Mage-3 peptides presented by HLA class II molecules |
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DE19956568A1 (de) * | 1999-01-30 | 2000-08-17 | Roland Kreutzer | Verfahren und Medikament zur Hemmung der Expression eines vorgegebenen Gens |
JP4637368B2 (ja) * | 1999-04-14 | 2011-02-23 | ノバルティス バクシンズ アンド ダイアグノスティックス,インコーポレーテッド | アルファウイルスに基づくベクター系を利用する免疫応答を生成するための組成物および方法 |
WO2001016342A1 (en) * | 1999-08-27 | 2001-03-08 | The Regents Of The University Of California | Use of lentiviral vectors for antigen presentation in dendritic cells |
US6627442B1 (en) * | 2000-08-31 | 2003-09-30 | Virxsys Corporation | Methods for stable transduction of cells with hiv-derived viral vectors |
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CN111647563A (zh) * | 2020-08-06 | 2020-09-11 | 北京翊博普惠生物科技发展有限公司 | 靶向Survivin全抗原的DC细胞、CTL细胞及其制备方法和应用 |
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