CN102703389B - 痘病毒溶瘤载体 - Google Patents
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Abstract
本发明涉及包含I4L和/或F4L基因缺损的痘病毒，涉及包含该痘病毒的组合物，以及将该组合物和痘病毒用于治疗目的、更具体地用于治疗癌症的方法和用途。
Description
本申请为2008年11月17日提交的、发明名称为“痘病毒溶瘤载体”的PCT申请PCT/EP2008/009720的分案申请，所述PCT申请进入中国国家阶段的日期为2010年5月19日，申请号为200880116834.1。
技术领域
溶瘤病毒是一类用于治疗癌症，具有肿瘤依赖性自我永续独特性质的新型治疗因子（HERMISTON.Ademandfornext-generationoncolyticadenoviruses.Currentopinioninmoleculartherapeutics.2006,8卷,4号,322-30页.）。溶瘤病毒能够在恶性细胞中选择性复制，并因此提供可能远远超过癌症常规治疗的效力和特异性的水平（FISHER.Strikingoutatdisseminatedmetastases:thesystemicdeliveryofoncolyticviruses.Currentopinioninmoleculartherapeutics.2006,8卷,4号,301-13页.）。使用这些病毒的好处是，当他们复制时，他们溶解其宿主细胞。癌细胞是多种病毒的理想寄主，因为它们的抗病毒的干扰素途径灭活或者具有突变的肿瘤抑制基因，这能使病毒复制不受阻碍地继续下去（CHERNAJOVSKY,等Fightingcancerwithoncolyticviruses.Britishmedicaljournal.2006,332卷,7534号,170-2页.）。
一些病毒天然地可以在肿瘤细胞中选择性地复制，但也可以通过修饰天然存在的病毒而获得溶瘤病毒。为了这一目的，目前使用的修饰病毒的主要策略包括：主要病毒基因中的功能性缺失；控制这些病毒基因的表达的肿瘤或组织特异性启动子；向性修饰将腺病毒重定向至癌细胞表面。在不久的将来，溶瘤腺病毒需要被优化，以充分发挥其作为重要的抗癌工具的潜力，并且从而改善恶性神经胶质瘤病人的预后（JIANG,等Oncolyticadenovirusesasantigliomaagents.Expertreviewofanticancertherapy.2006,6卷,5号,697-708页.）。
例如，腺病毒ONYX-015，被选择性的修饰从而在具有p53突变的细胞中复制并杀死细胞，其正由OnyxPharmaceuticals开发，用于各种实体瘤，包括头部和颈部、胃肠和胰腺肿瘤的潜在治疗。它是一个重组腺病毒，它在E1B座位载有功能缺失性突变，其产物是结合于p53肿瘤抑制蛋白并使其灭活的55kDa的蛋白质。因此，认为ONYX-015腺病毒应使正常细胞不受影响。p53肿瘤抑制基因突变是癌症遗传异常中最常见的类型，存在于一半以上的所有主要癌症类型中。因此，这些细胞对能很容易的复制并导致细胞死亡的病毒敏感。ONYX-015正处于用于治疗复发性头部和颈部癌症的III期临床试验，结直肠、卵巢、胰腺和口腔肿瘤的II期临床试验，以及消化疾病、食道和肝脏肿瘤的I期临床试验（COHEN,等ONYX-015.OnyxPharmaceuticals.Currentopinionininvestigationaldrugs.2001,2卷,12号,1770-5页.）。
天然的溶瘤病毒是具有复制能力的病毒，其具有选择性感染并杀死肿瘤细胞的先天能力。尽管50年前被用于使用活病毒治疗癌症的最初尝试，但对天然溶瘤病毒作为癌症疗法的关注已经滞后于支持改造的腺病毒和疱疹病毒作为癌症疗法。然而，最近已经对这些天然存在的物质的高效能和选择性重新关注了（ROBERTS,等Naturallyoncolyticviruses.Currentopinioninmoleculartherapeutics.2006,8卷,4号,314-21页.）。
在天然溶瘤病毒中，痘苗病毒（痘病毒科）具有很多用于溶瘤病毒疗法的理想病毒主链所必需的关键属性。这些包括生命周期短、和快速的细胞间播散、裂解能力强、巨大的克隆能力和明确的分子生物学。此外，尽管它们能够在人类细胞中复制，但并不考虑为天然的健康问题，且它们特别好地得到表征，在消灭天花的战争中已经被递送给数百万人。使用疫苗株或基因修饰的牛痘株的早期临床结果已经证明了抗癌效果（THORNE,等Vacciniavirusandoncolyticvirotherapyofcancer.Currentopinioninmoleculartherapeutics.2005,7卷,4号,359-65页.）。
相比之下，痘病毒粘液瘤病毒是一种新型的溶瘤候选者，其没有直接用于人类的历史，因为它对兔类（兔子）有特殊的和绝对的宿主种类向性。最近已证明粘液瘤病毒也能够选择性的感染并杀死人类肿瘤细胞，该特有的向性联系在大多数人类癌症中所见的失调的细胞内信号途径。这篇综述概述了关于粘液瘤病毒对人类癌细胞向性的现有知识，以及展示其在癌症的动物模型中感染和清除肿瘤能力的临床前期数据（STANFORD,等Myxomavirusandoncolyticvirotherapy:anewbiologicweaponinthewaragainstcancer.Expertopiniononbiologicaltherapy.2007,7卷,9号,1415-25页.）。
技术问题
注射达到抗肿瘤效果所需的高剂量的痘病毒引发了毒性的问题。不利的事件大多不严重，通常与痘苗病毒有关的不良反应是自限性的，包括发烧、头痛、疲劳、肌痛、寒战、局部皮肤反应、非特异性疹、多形性红斑、淋巴结病、和种痘位置疼痛。其他反应可能需要另外的治疗方法（例如，首要的治疗VIG，第二线治疗西多福韦）。不良反应可能需要进一步的评估或治疗，包括非故意接种、全身性牛痘（GV）、牛痘性湿疹（EV）、进行性牛痘（PV）、种痘后的中枢神经系统疾病、和胎儿牛痘（CONO,等Smallpoxvaccinationandadversereactions.Guidanceforclinicians.MMWR.Recommendationsandreports:Morbidityandmortalityweeklyreport.Recommendationsandreports/CentersforDiseaseControl.2003,52卷,RR-4号,1-28页.）。
因此，需要更安全的、具有与对于其天然相应物一样好的溶瘤活性的痘病毒。
背景技术
1994年11月15日的美国5364773（VIROGENETICS公司（TROY,纽约））描述了改造的重组痘病毒，更特别是有灭活的非必要的病毒编码遗传功能的痘苗病毒，使重组痘病毒具有减弱的毒力和增强的安全性。特别地，通过缺失编码毒力因子的开放阅读框，或通过编码毒力因子的开放阅读框的插入失活，灭活遗传功能。更特别的是，这项专利描述了痘苗病毒，其中J2R、B13R+B14R、A26L、A56R、C7L-K1L和I4L的开放阅读框已被灭活。这种病毒（NYVAC）可以被改造为用于外源核酸的载体，并用作疫苗诱导宿主动物中的免疫应答。但是，NYVAC不能在大多数哺乳动物细胞中有效地复制，且不能被用作溶瘤病毒（XIANGZHI,等VacciniavirusK1Lproteinsupportsviralreplicationinhumanandrabbitcellsthroughacell-type-specificsetofitsankyrinrepeatresiduesthataredistinctfromitsbindingsiteforACAP2.JournalofVirology.2006,353卷,1号,220-233页.）。
2004年2月19日的WO2004/014314（KIRNDAVID（美国））描述了在其病毒基因组中包含了一个或更多突变的改变的痘苗病毒。所描述的突变是以下多肽种类中的一种或多种：1）干扰素调节多肽；2）补体控制多肽；3）TNF或趋化因子调节多肽；4）丝氨酸蛋白酶抑制剂；5）IL-lp调节多肽；6）非感染性EEV形式的多肽；和7）用作抑制传染性病毒从细胞中释放的病毒多肽（抗传染病毒形式的多肽）。此外，还公开了痘苗病毒A41L或C11R中的突变。
在这一申请中更特别描述了牛痘基因组区域，如A34R、A41L、A53R、B5R、B7R、B8R、B13R、B15R、B18R、B22R、B28R、B29R、CUR、E3L、K2L、N1L、vC12L，和vCKBP。本发明的方法涉及使用任何本文中讨论的痘病毒。本发明的发明人还公开了通过给予癌细胞或患者有效量的这一改变的痘苗病毒来治疗癌症的方法。
发明公开
本发明的发明人吃惊地发现，包含缺损I4L和/或F4L基因的痘病毒具有改善的安全性，但是保持了相同的溶瘤活性（与其天然相应物相比）。
本发明涉及一种包括缺损I4L和/或F4L基因的痘病毒，条件是所述痘病毒非NYVAC。
在整个申请中所使用的术语“一个”和“一种”是用意为表示所引用的成分或步骤中的“至少一个”、“至少第一个”、“一个或多个”或“多种”，除非上下文清楚规定了其它的意思。例如，术语“一个细胞”包括多个细胞，包含其混合物。
本文所用的术语“和/或”包括，“和”、“或”和“全部或任何其它由该词连接的成分的组合”的含义。
本文所用的术语“大约”或“约”表示在给定值或范围的20％以内，优选在10％以内，且更优选在5％以内。
本文所用的术语“包含”和“包括”意在表示产物、组成物和方法包括所提及的成分或步骤，但不排除其它的。当“基本上由…组成”被用于定义产物、组成物和方法时，表示不包括其他任何具显著性的成分和步骤。因此，基本上由所述的成分组成的组合物将不排除微量污染物和可药用的载体。“由…组成”意味着排除其它不止是微量成分的成分或步骤。
本文所用的术语“包括缺损基因的痘病毒”指的是痘病毒，其在所述缺损基因的一个或多个核酸中包含缺失、取代或添加，或这些可能的任何组合，其中所述修饰导致该病毒无法产生这样的蛋白质，即具有未修改的基因所产生的蛋白质的活性的蛋白质。在本发明的优选的实施方案中，包含基因缺损的痘病毒，是指其中整个基因序列已被删除的痘病毒。可以通过本领域的技术人员所知道的许多方法，利用重组技术进行突变。本领域内有修饰痘病毒基因组的方法。例如，MCCART，等Systemiccancertherapywithatumorselectivevacciniavirusmutantlackingthymidinekinaseandvacciniagrowthfactorgenes..Cancerres..2001,61号,8751-57页.，KIM,等.SystemicarmedoncolyticansimmunologictherapyforcancerwithJX-594,atargetedpoxvirusexpressingGM-CSF.MolecularTherapeutic.2006,14号,361-70页.，2004年2月9日的WO2004/014314（KIRNDAVID（US））和1994年11月15日的US5364773（VIROGENETICSCORPORATION（TROY,NY））所公开的方法，可以被用来生产本发明的痘病毒。在本申请的实施例中公开的方法与生产根据本发明的痘病毒特别相关。本领域内可得到各种痘病毒的基因组序列，例如，痘苗病毒（vacciniavirus）、痘苗病毒（cowpoxvirus）、金丝雀痘病毒、鼠痘病毒、粘液瘤病毒基因组可在Genbank中得到（登录号分别是NC_006998、NC_003663、NC_005309、NC_004105、NC_001132）。
本发明中所使用的术语“痘病毒”是指属于痘病毒科（Poxviridaefamily）的病毒。根据优选的实施方案，根据本发明的痘病毒属于脊椎动物痘病毒亚科（Chordopoxvirinaesubfamily），更优选地属于正痘病毒属（Orthopoxvirusgenus），更加优选地属于痘苗病毒种（Vacciniavirusspecie）。
例如，可以利用痘苗病毒株DairenI、IHD-J、L-IPV、LC16M8、LC16MO、Lister、LIVP、Tashkent,、WR65-16、Wyeth,Ankara、Copenhagen、TianTan和WR。根据特别优选的实施方案，根据本发明的痘病毒是痘苗病毒株Copenhagen。
痘病毒牛痘包含大的双链DNA基因组（187千碱基对），并且是仅知的在受感染细胞的细胞质中进行复制的DNA病毒家族成员。由于被感染的细胞必须向细胞质的复制位点递送大量的DNA前体，该病毒编码并表达DNA代谢与合成所需的许多酶的活性，包括核糖核苷酸还原酶和脱氧尿嘧啶核苷5’-三磷酸核苷酸水解酶（脱氧尿苷三磷酸酶，dUTPase）。
核糖核苷酸还原酶（EC1.2317.4.1）催化核糖核苷酸还原为脱氧核糖核苷酸，该反应表示DNA生物合成中的第一实行步骤。病毒酶在亚基结构上与哺乳动物酶相似，由两个异源命名为R1和R2的亚基组成。编码病毒核糖核苷酸还原酶亚基的（多个）基因已被测序，并固定于牛痘基因组的位置上，相隔35千碱基(SLABAUGH,等.Journalofvirology.1988,卷62,页519-27.;TENGELSEN,等.Virology.1988,编号164,页121-31.;SCHMITT,等.Journalofvirology.1988,编号62,页1889-97.)。痘苗病毒大亚基的单体(命名为R1,由I4L基因编码)为86-kDa的多肽,并包含核苷酸底物变构效应体的结合位点(SLABAUGH,等.Journalofvirology.1984,编号52,页507-14.;SLABAUGH,等.Journalofvirology.1984,编号52,页501-6.)。小亚基(命名为R2,由F4L基因编码)为包含两个37-kDa的多肽的同二聚体;各多肽包含催化所需的、铁-稳定的、基于蛋白的自由基(HOWELL,等.JournalofBiologicalChemistry.1992,编号267,页1705-11.)。I4L和F4L基因的序列及其在不同痘病毒基因组中的定位可在公共数据库中获得,例如通过登录号DQ437594,DQ437593,DQ377804,AH015635,AY313847,AY313848,NC_003391,NC_003389,NC_003310,M35027,AY243312,DQ011157,DQ011156,DQ011155,DQ011154,DQ011153,Y16780,X71982,AF438165,U60315,AF410153,AF380138,U86916,L22579,NC_006998,DQ121394和NC_008291。
本文所使用的基因命名是Copenhagen牛痘株的基因命名，除非另有注明，其也被用于其他痘病毒科的同源基因。然而，基因命名可能根据痘株而不同。作为信息，Copenhage和MVA基因的对应关系可以在ANTOINE..Virology.1998,244号,365-396页.的表中找到。
根据优选的实施方案，本发明的痘病毒还包含缺损的J2R基因。
J2R基因编码一种胸苷激酶（TK），其形成嘧啶脱氧核苷酸合成补救途径的一部分。TK催化的反应涉及将γ-磷酰基部分从ATP转移至2’脱氧-胸苷（dThd），以产生胸苷5’-单磷酸（dTMP）。痘苗病毒的TK是2型TK。2型TK相对于1型具有较小的多肽链，具有约25KDa，但形成同源四聚体。它们对反馈抑制剂dTDP或dTTP敏感，其在代谢途径的最终生成。2型TK相对于1型TK具有窄得多的底物特异性，并且只能使2’脱氧尿苷（dU）和/或dThd磷酸化（ELOMARI,等.StructureofvacciniavirusthymidinekinaseincomplexwithdTTP:insightsfordrugdesign.BMCstructuralbiology.2006,6号,22页）。
本领域内已知J2R区域缺损的痘病毒和获得它们的方法。例如MCCART,等Systemiccancertherapywithatumor-selectivevacciniavirusmutantlackingthymidinekinaseandvacciniagrowthfactorgenes.cancerresearch.2001,61卷,24号,8751-7页.、PUHLMANN,等Vacciniaasavectorfortumor-directedgenetherapy:biodistributionofathymidinekinase-deletedmutant.Cancergenetherapy.2000,7卷,1号,66-73页.、GNANT,等Systemicadministrationofarecombinantvacciniavirusexpressingthecytosinedeaminasegeneandsubsequenttreatmentwith5-fluorocytosineleadstotumor-specificgeneexpressionandprolongationofsurvivalinmice.CancerResearch.1999,59卷,14号,3396-403页.的教导可用于产生J2R区域缺失的痘病毒。
根据优选的实施方案，根据本发明的痘病毒还包含缺损的F2L基因。
脱氧尿嘧啶核苷5’-三磷酸核苷酸水解酶（dUTPase,EC3.6.1.23）在Mg离子（2+）存在下催化dUTP水解为dUMP和焦磷酸盐。在将dUTP从dNTP池中除去并产生dUMP时，dUTPase参与维持DNA复制的保真度并参与为通过胸苷酸合酶生产TMP提供前体。牛痘脱氧尿苷三磷酸酶（dUTPase）是由F2L基因编码的15kDa的蛋白质（MCGEOGH..NucleicAcidsResearch.1990,18号,4105-10页.；BROYLES..Virology.1993,195号,863-5页.）。痘苗病毒F2L基因的序列可以通过登录号M25392从GenBank中得到，不同痘病毒类基因组中F2L基因的序列和位点也可以从GenBank中获得，例如，通过登录号NC_006998、DQ121394、NC_001611、AY689436、AY689437、NC_008291、DQ437594、DQ437593、AY313847、AY313848、NC_006966、NC_005309、NC_003391、NC_003389、NC_001132、NC_003310、NC_002188、M35027、AY243312、AF170726、DQ011157、DQ011156、DQ011155、DQ011154、DQ011153、X94355、Y16780、AY318871、U94848、AF198100和M34368。
根据优选的实施方案，根据本发明的痘病毒还包含目的核酸。
在优选的实施方案中，目的核酸包含至少一种目的序列，其编码作为治疗性分子的基因产物（即治疗的基因）。“治疗性分子”是这样的分子，其在适当地施用于患者，尤其是患疾病或病症的患者，或者应受保护以避免该疾病或病症者，具有药理学的或保护性的活性。该药理学或保护性活性预期与对所述疾病或所述病症的病程或症状产生有利的影响相关。当技术人员按本发明选择编码治疗性分子的基因时，他通常将他的选择与先前获得的结果联系起来，并可以合理地预期得到这样的药理学特性，而无需实施本发明所主张以外的过多实验。根据本发明，目的序列与其被转入其中的靶细胞可以是同源的或者异源的。有利地，所述目的序列编码全部或部分的多肽，尤其是给出治疗或预防特性的治疗性或预防性多肽。多肽被理解为多核苷酸的任何翻译产物，不论其大小、糖基化与否，且包括肽和蛋白质。作为一个首要的实例，治疗性多肽包括那些可以在动物或人类有机体中补偿缺损或缺陷蛋白质的多肽，或者那些通过毒性作用从机体中限制或除去有害细胞的多肽。它们也可以是赋予免疫性的多肽，其作为内源性抗原激起体液或细胞反应，或两者兼有。
由治疗性基因编码的多肽的实例包括，编码细胞因子（α、β或γ干扰素，白细胞介素，特别是IL-2、IL-6、IL-10或IL-12，肿瘤坏死因子（TNF），集落刺激因子GM-CSF、C-CSF、M-CSF....）、免疫刺激多肽（B7.1、B7.2等）、凝结因子（FVIII、FIX...）、生长因子（转化生长因子TGF、成纤维细胞生长因子FGF等）、酶（脲酶、肾素、凝血酶、金属蛋白酶、一氧化氮合酶NOS、SOD，过氧化氢酶...）、酶抑制剂（α1-抗胰蛋白酶、抗凝血酶III、病毒蛋白酶抑制剂、纤溶酶原激活物抑制剂PAI-1）、CFTR（囊性纤维化跨膜传导调节因子）蛋白质、胰岛素、肌营养不良蛋白、I或II类MHC抗原、可以调节/调控细胞基因表达的多肽、能够抑制细菌、寄生虫或病毒感染或其发展的多肽（抗原多肽、抗原表位、通过竞争抑制天然蛋白质作用的转显性（transdominant）变体....）、凋亡诱导剂或抑制剂（Bax、Bcl2、BclX...）、细胞生长抑制剂（p21、p16、Rb...）、载脂蛋白（ApoAI、ApoAIV、ApoE...）,血管生成抑制剂（血管生成抑制素、内皮他丁（endostatin）...）、血管生成多肽（血管内皮细胞生长因子VEGF家族、FGF家族、CCN家族，包括CTGF、Cyr61和Nov）、氧自由基scaveyer、具有抗肿瘤作用的多肽、抗体、毒素，免疫毒素和标记（β-半乳糖苷酶、荧光素酶....）的基因或本领域内认为可以被用于临床症状的治疗或预防的任何其它目的基因。
适当的抗肿瘤基因包括但不限于，那些编码肿瘤抑制基因（如Rb、p53、DCC、NF-1、肾母细胞瘤、NM23、BRUSH-1、p16、p21、p56、p73以及它们各自的突变体）、自杀基因产物、抗体、抑制细胞分裂或转导信号的多肽的基因。
根据特别优选的实施方案，本发明的痘病毒还包含自杀基因。
自杀基因是指能将药物前体转化成细胞毒性化合物的蛋白质的编码基因。
自杀基因包括但不限于，编码具有胞嘧啶脱氨酶活性、胸苷激酶活性、尿嘧啶磷酸核糖转移酶活性、嘌呤核苷磷酸化酶活性和/或胸苷激酶活性的蛋白质的基因。
下表中公开了包含一个核碱基（nucleobase）部分的自杀基因和相应的药物前体的例子：
表1
根据本发明优选的实施方案，自杀基因编码具有至少一种CDase活性的蛋白质。CDase参与嘧啶代谢途径，通过该途径，外源胞嘧啶利用水解脱氨作用被转化为尿嘧啶。虽然已经在原核生物和低等真核生物中证明了CDase活性（JUND,等.JournalofBacteriology.1970,102号,607-15.页;BECK,等.JournalofBacteriology.1972,110号,219-28.页;HOEPRICH,等.JournalofInfectiousDiseases.1974,130号,112-18.页;ESDERS,等.J.biol.chem..1985,260号,3915-22.页），但它们不存在于哺乳动物中（KOECHLIN,等.Biochemicalpharmacology.1966,15号,435-46.页;POLAK,等.Chemotherapy.1976,22号,137-53.页）。
CDase也使胞嘧啶类似物，即5-氟胞嘧啶（5-FC）脱去氨基，从而形成5-氟尿嘧啶（5-FU），5-FU是一种化合物，当其被转化成5-氟UMP（5-FUMP）时，有高细胞毒性。无论是因为突变而使编码酶的基因失活还是因为其天然地缺少这种酶（就像哺乳动物细胞一样），缺少CDase活性的细胞对5-FC有抗性（JUND,等.JournalofBacteriology.1970,102号,607-15页.;KILLSTRUP,等.JournalofBacteriology.1989,171号,2124-2127页.）。相比之下，被转化了编码CDase活性的序列的哺乳动物细胞变得对5-FC敏感（（HUBER,等.CancerResearch.1993,53号,4619-4626页.;MULLEN,等.ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica.1992,89号,33-37页.；WO93/01281（USHEALTH））。此外，邻近的，未经转化的细胞也变得对5-FC敏感（HUBER,等.ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica.1994,91号,8302-6页.）。这种被称为旁观者效应的现象，是由于表达CDase活性的细胞分泌5-FU，然后其通过穿越质膜的直接扩散使邻近的细胞中毒。5-FU的这一被动扩散性质与tk/GCV参照系统相比，表现出优势，后者中旁观者效应需要与表达tk的细胞接触（MESNIL,等.ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica.1996,93号,1831-35页.）。因此可以很容易地理解CDase在基因治疗中，尤其是抗癌基因治疗中所提供的所有优势。
分别编码这两种生物的CDase的酿酒酵母（Saccharomycescerevisiae，S.cerevisiae）FCY1、白色念珠菌（CandidaAlbicans）FCA1和大肠杆菌codA基因是已知的且它们的序列已被公布（分别是SEQIDNO:4、SEQIDNO:5、SEQIDNO:6）。
在这方面，根据本发明更优选的实施方案，编码具有CDase活性的蛋白质的基因是FCY1、FCA1或CodA或其类似物。这些基因的类似物是指这样的基因，其具有的核酸序列与亲本基因的核酸序列，至少具有程度大于70％的同一性，最好大于80％，优选地大于90％，和最优选地大于95％。
专利WO2005/007857公开了一种基因，其编码具有改善的CDase活性的蛋白质。这种多肽来源于天然CDase加上一氨基酸序列。根据本发明的另一优选的实施方案，该具有CDase活性的蛋白质是WO2005/007857公开的多肽，更优选的是序列号SEQIDNO:2中描述的FCU1-8多肽及其类似物。
在原核生物和低等的真核细胞中，尿嘧啶在尿嘧啶磷酸核糖转移酶（UPRTase）的作用下被转化成UMP。这种酶将5-FU转化为5-FUMP。根据本发明的另一优选的实施方案，自杀基因编码具有UPRTase活性的蛋白质。
正在谈论的UPRTase可以是任何来源的，特别是原核的、真菌的或酵母菌来源的。以举例的方式，在本发明的情况下可以使用的核酸序列编码源自大肠杆菌（ANDERSEN,等CharacterizationoftheuppgeneencodinguracilphosphoribosyltransferaseofEscherichiacoliK12.EuropeanJournalofBiochemistry.1992,204号,51-56页.），源自乳酸乳球菌（Lactococcuslactis）（MARTINUSSEN,等Cloningandcharacterizationofupp,ageneencodinguracilphosphoribosyltransferasefromLactococcuslactis.JournalofBacteriology.1994,176卷,21号,6457-63页.），源自牛分枝杆菌（Mycobacteriumbovis）（KIM,等CompletesequenceoftheUPPgeneencodinguracilphosphoribosyltransferasefromMycobacteriumbovisBCG.Biochemistryandmolecularbiologyinternational.1997,41卷,6号,1117-24页.）和源自枯草芽孢杆菌（Bacillussubtilis）（MARTINUSSEN,等TwogenesencodinguracilphosphoribosyltransferasearepresentinBacillussubtilis.JournalofBacteriology.1995,177卷,1号,271-4页.）的UPRTase。不过，最特别优选地使用酵母UPRTase且尤其是其序列在KERN，等TheFUR1geneofSaccharomycescerevisiae:cloning,structureandexpressionofwild-typeandmutantalleles.Gene.1990,88卷,2号,149-57页.中公开的酿酒酵母（S.cerevisiae）FUR1基因所编码的UPRTase，其在此通过引用作为参考。作为指导，该基因序列和相应的UPRTase序列可以在文献和专门数据库（SWISSPROT、EMBL、Genbank、Medline等）中找到。
专利申请EP0998568A描述了FUR1基因，其在编码部分5’端缺少105个核苷酸，使得由其开始的UPRTase的合成，其在天然的蛋白质中开始的35个残基已在N端的位置缺失，且开始于在36位置的蛋氨酸。该突变基因的表达产物，命名为FUR1Δ105，其能够补充酿酒酵母的fur1突变。此外，截短的突变体表现出比天然酶高的UPRTase活性。因此，根据本发明的特别有利的实施方案，自杀基因编码天然UPRTase的缺失突变。该缺失优选地位于原UPRTaseN端的区域。其可以是完全的（影响所述N端的区域的所有残基）或部分的（影响一级结构中一个或多个连续或非连续残基）。一般而言，多肽由N端、中心和C端部分组成，每一部分约占分子的三分之一。例如，由于酿酒酵母UPRTase有251个氨基酸，其N端部分由前83个残基组成，开始于位于天然形式第一个位点的所谓的起始蛋氨酸。至于大肠杆菌UPRTase，其N端部分涵盖位点1至69。
优选的具有UPRTase活性的蛋白质包含，基本上如EP0998568A的序列号SEQIDNO:1中所描述的氨基酸序列，开始于位于位点1的Met残基并结束于位于位点216的Val残基。术语“基本上”指的是与所述的EP0998568A的SEQIDNO:1序列的同一程度大于70％，最好大于80％，优选大于90％，和最优选大于95％。但更优选的是，其包含EP0998568A的序列号SEQIDNO:1中所描述的氨基酸序列。如上所述，它可能包括额外的突变。可以特别提及位于位点2的丝氨酸残基（在天然UPRTase中的位点37）被丙氨酸残基取代。
根据本发明另一项优选的实施方案，自杀基因编码具有至少一种CDase和一种UPRTase活性的蛋白质。专利申请WO96/16183和EP0998568A描述了一种融合蛋白的用途，其编码的酶包含具有CDase和UPRTase活性的两个结构域，和展示了由表达质粒携带的融合基因codA::upp或FCY1::FUR1或FCY1::FUR1Δ105（即FCU1）的转移提高了被转化的B16细胞对5-FC的敏感性。根据本发明更优选的实施方案，自杀基因编码的多肽包含的氨基酸序列基本上如序列号SEQIDNO:3（coda::upp）、SEQIDNO:1（FCU1）或FCY1::FUR1中所述。术语“基本上”是指与所述序列的同一程度大于70％，最好大于80％，优选大于90％，和最优选大于95％。但更优选的是，其包含如序列号SEQIDNO:3（coda::upp）、SEQIDNO:1（FCU1）或FCY1::FUR1中所述的氨基酸序列。如上所述，它可包含额外的突变。
根据目前使用的常用技术，可以容易地通过克隆、通过PCR、或通过化学合成获得核酸序列。它们可以是天然基因或源自其通过突变、缺失、取代和/或添加一个或多个核苷酸的基因。此外，它们的序列被广泛地描述于本领域的技术人员能够查阅的文献中。
本领域的技术人员能够从公开的数据克隆CDase或UPRTase序列并能够进行可能的突变体，能够根据现有技术方法或者基于申请EP0998568A中所示的方案在非细胞或者细胞体系中检测突变体形式的酶活性，并能够，尤其是同相地融合具有CDase和UPRTase活性的多肽，从而融合全部或部分相应基因。
根据更优选的实施方案，本发明的痘病毒还包括核酸序列，其包含编码透性酶（permease）的基因。
透性酶是指跨膜的蛋白质，其参与包含一个核碱基（nucleobase）部分的药物，或其前体通过细胞膜的转移。
透性酶包括但不限于嘌呤透性酶、胞嘧啶透性酶和核苷转运蛋白。
根据本发明的优选的实施方案，透性酶是酿酒酵母的嘌呤或胞嘧啶透性酶。酿酒酵母的核碱基转运蛋白由嘌呤-胞嘧啶透性酶（称为FCY2）和尿嘧啶透性酶（称为FUR4）组成。嘌呤-胞嘧啶透性酶FCY2介导质子和腺嘌呤、鸟嘌呤、次黄嘌呤和胞嘧啶穿越酵母质膜的同向转运（Grenson1969,Jund和Lacroute1970,Polak和Grenson1973,Chevallier等1975,Hopkins等1988）。FCY2蛋白质也介导5氟胞嘧啶的转运，5氟胞嘧啶是胞嘧啶的类似物（Grenson1969,Jund和Lacroute1970）。FCY2基因编码533个氨基酸（58kDa）的蛋白质，最初预计有10-12个跨膜区（Weber等1990），现在倾向认为有9个（Ferreira等1999）。FCY2对嘌呤核碱基和胞嘧啶表现出相似的亲和力（Brethes等1992）。尿嘧啶是由尿嘧啶透性酶FUR4介导而摄取到酿酒酵母中（Jund和Lacroute1970,Jund等1977）。FUR4是尿嘧啶-质子共转运蛋白（Hopkins等1988），预测是633个氨基酸（71.7kDa）的蛋白质，具有10个跨膜结构域和长的细胞质亲水N-和C-端尾巴（Jund等1988,Garnier等1996）。FUR4蛋白质还可以介导5氟尿嘧啶的转运，5氟尿嘧啶是尿嘧啶的类似物（Jund和Lacroute1970）。
swissprot数据库中特别提供FCY2和Fur4的氨基酸序列（登录号分别为P17064和P05316）。优选地，透性酶具有选自于，如在专利申请WO2006/048768中所公开的氨基酸序列SEQIDN0:1和SEQIDN0:2的氨基酸序列。
在这方面，根据本发明的优选的实施方案，透性酶选自于FCY2和Fur4和其类似物。Fur4和FCY2的类似物是指多肽，该多肽具有的氨基酸序列与本文上述亲本蛋白质的氨基酸序列的同一程度，至少大于70％，最好大于80％，优选大于90％，优选大于95％，并且该多肽保留了转运含有一个核碱基部分的药物穿过细胞膜的能力。
本领域的技术人员能够选择，将与包含一个核碱基部分的药物或药物前体关联的透性酶。例如，FCY2和Fur4优选地与5氟胞嘧啶（5-FC）关联。
根据更优选的实施方案，本发明的痘病毒还可包含表达目的核酸所必需的元件。
根据更优选的实施方案，本发明的痘病毒可还包含表达包括编码透性酶的基因的核酸序列所必需的元件。
这些表达目的核酸和/或包含编码透性酶的基因的核酸序列所必需的元件包括，所述DNA转录成mRNA以及（如果需要的话）mRNA翻译成多肽所必需的元件。文献中广泛地描述了适合在各种脊椎动物体系中使用的转录启动子。举例来说，适合的启动子包括病毒启动子，诸如RSV、MPSV、SV40、CMV或7.5k，牛痘启动子，诱导型启动子，等。优选的启动子是由痘病毒分离，例如痘苗病毒的7.5K、H5R、TK、p28、p11或K1L。另外，可以使用合成启动子，例如那些在CHAKRABARTI.Biotechniques.1997,23号,1094-97页.，HAMMOND,等.JournalofVirologicalMethods.1997,66号,135-38页.和KUMAR.Virology.1990,179号,151-8页.中所描述的启动子，以及早期和晚期痘病毒启动子之间的嵌合启动子。
目的核酸序列和包含编码透性酶基因的核酸序列还可以包括额外的功能元件，如内含子序列、定向序列、转运序列、分泌信号、核定位信号、IRES、多聚A转录终止序列、三联前导序列、参与复制或整合的序列。所述序列已在文献中报道并可很容易地由本领域的技术人员获得。
本发明还涉及制备根据本发明的痘病毒的方法，在其中：
（i）将根据本发明的痘病毒引入到细胞中;
（ii）将所述细胞培养在适合于能使所述痘病毒产生的条件下和;
（iii）从细胞培养物中回收所述痘病毒。
当然可以从培养上清液回收痘病毒，同时也可以从细胞中回收它。普遍采用的方法之一在于通过反复冻融循环的方法裂解细胞，以从裂解上清中收集病毒颗粒。然后可以使用本领域中的技术（色谱法、超速离心法、特别是通过氯化铯密度梯度，等）扩增和纯化病毒颗粒。
本发明还涉及一种组合物，其包含根据本发明的痘病毒，与可药用的赋形剂组合。
根据本发明的组合物更具体地被用于，通过基因治疗方法预防或治疗疾病，和更具体地针对增殖性疾病（癌症、肿瘤、再狭窄，等），或针对与破骨细胞活性增强相关的疾病（如类风湿关节炎、骨质疏松症）。
可按照惯例，以局部、肠胃外或消化途径给药为目的，制成根据本发明的组合物。特别是，治疗有效量的本发明的重组载体或痘病毒与可药用的赋形剂组合。可以设想大量的给药途径。可被提及的实例是胃内的、皮下的、心内的、肌肉内、静脉内的、腹腔内的、肿瘤内的、鼻内的、肺内的和气管内的途径。对于后三个实施方案，通过气雾剂或通过滴注法进行给药更有利。可以以单剂量进行给药，或者在特定的时间间隔后，按照一次或多次重复的剂量进行给药。适合的给药途径和剂量取决于各种因素，例如个体、将被治疗的疾病或将被转化的目的基因。可以按104至1014pfu（空斑形成单位），最好105至1013pfu，优选地106至1012pfu，更优选地106至107之间剂量的形式配制基于本发明的病毒颗粒的制剂。
该组合物还可以包含可药用的稀释剂、佐剂或赋形剂，以及增溶、稳定和防腐剂。在可注射给药的情况下，优选制剂是水溶液，非水溶液或等渗溶液。它可以以单一剂量或多剂量、液体或干燥（粉末、冻干物，等）的形式存在，该干燥形式可在使用的时候用适当的稀释剂重构。
本发明还涉及，将根据本发明的痘病毒或组合物用于制备药物的用途，该药物被用于通过基因疗法治疗人体或动物体。该药物可直接体内施用（例如，通过静脉注射、进入可进入的肿瘤、通过气雾剂方法进入肺部，使用适当的导管进入血管系统，等）。优选的用途在于，治疗或预防有害细胞增殖导致的癌症、肿瘤和疾病。可提及的可以想到的用途是乳腺癌、子宫癌（特别是乳头瘤病毒引起的）、前列腺癌、肺癌、膀胱癌、肝癌、结肠癌、胰腺癌、胃癌、食道癌、喉癌、中枢神经系统癌（如胶质母细胞瘤）和血癌（淋巴瘤、白血病，等）。另一个优选的用途在于治疗或预防类风湿性关节炎、骨质疏松症和与增加破骨细胞活性相关的其他疾病。其也可以用在心血管疾病方面，例如，为了抑制或延缓血管壁平滑肌细胞增殖（再狭窄）。最后，在传染病的情况下，可以设想该药物被应用于艾滋病。
当本发明的痘病毒、组合物或方法被用于治疗癌症时，优选的施用途径是全身途径，因为根据本发明的痘病毒能够特异地靶向肿瘤细胞。
本发明还涉及治疗疾病的方法，其特征是，向需要这种治疗的宿主生物体或细胞施用根据本发明的痘病毒、组合物。
根据有利的实施方案，该治疗应用或治疗方法还包含另外的步骤，其中向宿主生物体或细胞施用可药用数量的前药，最好是胞嘧啶类似物，特别是5-FC。举例而言，可以使用剂量为50到500mg/kg/天，优选剂量是200mg/kg/天，或者100mg/kg/天。在本发明的情况下，依照标准方法施用前药（例如，口服，系统施用）。
优选地，在施用根据本发明的治疗剂之后进行给药，优选地在施用治疗剂至少3天，更优选地至少4天，甚至更优选地至少5天之后。据本发明甚至更优选的实施方案，在施用治疗剂7天之后进行施用前药。优选的是口服途径。可以施用单剂量的前药，或者是在能使宿主生物体或细胞内产生毒性代谢产物的足够长时间段内重复施用的剂量。
此外，根据本发明的组合物或方法可以与一种或多种使5-FU的细胞毒作用增强的物质组合。可特别提到的药物是，抑制嘧啶从头生物合成途径中酶的药物（例如下文所述），药物如甲酰四氢叶酸（Waxman等,1982,Eur.J.CancerClin.Oncol.18,685-692），其在存在5-FU（5-FdUMP）的代谢产物的情况下，增加胸苷酸合酶的抑制作用，导致dTMP池减少，dTMP是复制所必需的，最后是药物如甲氨蝶呤（Cadman等,1979,Science250,1135-1137），其通过抑制二氢叶酸还原酶和提高PRPP（磷酸核糖焦磷酸）池，使得增加5-FU整合入细胞RNA。
根据本发明，抑制嘧啶从头生物合成途径的酶的药物，优选地选自于PALA（N-（膦酰乙酰基）-L-天冬氨酸；Moore等,1982,Biochem.Pharmacol.31,3317-3321）、来氟米特、A771726（来氟米特活性代谢产物，Davis等，1996,Biochem.35,1270-1273）和布喹那（Brequinar）（Chen等，1992,CancerRes.52,3251-3257）。
根据本发明的组合物或方法，可以与一种或多种在抗癌治疗中有效的物质组合。在抗癌治疗中有效的制药物质中，能提到的可以与根据本发明的组合物联合或组合使用的是，烷化剂例如，如丝裂霉素C、环磷酰胺、白消安、异环磷酰胺、isosfamide、美法仑、六甲三聚氰胺、塞替派、苯丁酸氮芥、或氮烯唑胺；抗代谢药物，例如吉西他滨、卡培他滨、5-氟尿嘧啶、阿糖胞苷、2-氟脱氧胞苷、甲氨蝶呤、idatrexate、拓优得或三甲曲沙；拓扑异构酶Ⅱ抑制剂，例如，阿霉素、表柔比星、依托泊苷、替尼泊苷或米托蒽醌；拓扑异构酶I抑制剂，如依立替康（CPT-11）、7-乙基-10-羟基喜树碱（SN-38）或托泊替康；抗有丝分裂的药物，例如，帕尼特西、多西他赛、长春碱、长春新碱或长春瑞滨；和铂衍生物，如顺铂、奥沙利铂、顺螺铂或carboplatinum。
根据本发明的组合物或方法也可以与放射疗法组合使用。
根据本发明的组合物或方法也可以与一种或多种这样的物质组合使用，包含但不限于免疫调节剂，例如，如α，β和γ干扰素、白细胞介素（特别是IL-2、IL-6、IL-10或IL-12）或肿瘤坏死因子；影响细胞表面受体调控的试剂，例如，如表皮生长因子受体抑制剂（特别是西妥昔单抗，Panitumumab、zalutumumab、nimotuzumab、matuzumab、吉非替尼、埃罗替尼或拉帕替尼）或人表皮生长因子受体-2抑制剂（特别是曲妥珠单抗）；以及影响血管生成的试剂，如：血管内皮生长因子抑制剂（特别是贝伐单抗或雷珠单抗）。
附图中图表简述
图1.对感染痘苗病毒的人结直肠肿瘤细胞（LoVo）5-FC的体外敏感性。以0.0001的MOI感染了指示病毒的LoVo细胞，（模拟（●）VVTK-/FCU1（■）或VVTK-I4L和/或F4L-/FCU1（△））接触不同浓度的5-FC。感染五天后测定细胞存活。以在存在或不存在药物下的细胞存活率百分比表达结果。3个单独测定的平均值±标准差（无由于病毒复制导致的细胞死亡）来表示数值。
图2.对感染痘苗病毒的人结直肠肿瘤细胞（LoVo）5-FC的体外敏感性。以0.0001的MOI感染了指示病毒的LoVo细胞，（模拟（●）VVTK-/FCU1（■）或VVTK-F4L-/FCU1（））接触不同浓度的5-FC。感染五天后测定细胞存活。以在存在或不存在药物下的细胞存活率百分比表达结果。3个单独测定的平均值±标准差（无由于病毒复制导致的细胞死亡）来表示数值。
图3.以0.0001的MOI感染了指示病毒的LoVo中，感染后第五天VVTK-/FCU1和VVTK-I4L-/FCU1的体外复制效率。以3个单独测定的平均值±标准差来表示数值。
图4.以0.0001的MOI感染了指示病毒的LoVo中，感染后第五天VVTK-/FCU1和VVTK-F4L-/FCU1的体外复制效率。以3个单独测定的平均值±标准差来表示数值。
图5.瑞士裸鼠静脉注射病毒后，皮下LoVo的平均肿瘤体积±SEM。接种肿瘤七天后（可触知的肿瘤），用107pfu的缓冲液+盐水（◇）、缓冲液+5-FC（◆）、VVTK-I4L-/FCU1+盐水（△）或VVTK-I4L-/FCU1（▲）处理小鼠。注射病毒第7天后的三周中，通过口服强饲用盐水或100mg/kg/j的5-FC处理动物，每天两次。每周测定肿瘤体积两次。
图6.瑞士裸鼠静脉注射病毒后，皮下LoVo的平均肿瘤体积±SEM。接种肿瘤七天后（可触知的肿瘤），用107pfu的缓冲液+盐水（◇）、缓冲液+5-FC（◆）、VVTK-F4L-/FCU1+盐水（□）或VVTK-F4L-/FCU1（■）处理小鼠。注射病毒第7天后的三周中，通过口服强饲用盐水或100mg/kg/j的5-FC处理动物，每天两次。每周测定肿瘤体积两次。
图7.静脉注射病毒后，瑞士裸鼠内皮下LoVo肿瘤的平均体积±SEM。接种肿瘤11天后（可触知的肿瘤），用缓冲液+水（□）、或缓冲液+5-FC（◆）、或单次注射107pfu的VVTK-I4L-/FCU1+水（○）、或单次注射107pfu的VVTK-I4L-/FCU1+5-FC（●）（5-FC在病毒注射7天后的三周中施用）；或两次注射(第11天和第33天)107pfuVVTK-I4L-/FCU1+H2O(□)；或两次注射(第11天和第33天)107pfuVVTK-I4L-/FCU1+5-FC(5-FC从第18天至第32天和从第40天至第54天施用)(■)。注射病毒7天之后的3个星期，每天以100mg/kg通过口服强饲5-FC两次来治疗这些动物。每周测定肿瘤体积两次。
图8.静脉注射病毒后，瑞士裸鼠内皮下U87-MG（胶质母细胞瘤肿瘤细胞）肿瘤的平均体积±SEM。接种肿瘤11天后（可触知的肿瘤），用缓冲液+水（□）、或缓冲液+5-FC（◆）、或107pfu的VVTK-I4L-/FCU1+水（○）、或107pfu的VVTK-I4L-/FCU1+5-FC（●）。注射病毒7天之后的3个星期，每天以100mg/kg通过口服强饲5-FC两次来治疗这些动物。每周测定肿瘤体积两次。
图9.分裂细胞对比融合细胞中病毒产量比率。用100pfu的（□）VVTK-/FCU1或（ロ）VVTK-I4L-/FCU1感染PANC1（人类胰腺肿瘤）、H1299（人类肺肿瘤）或U118MG（人神经神经胶质瘤）细胞。感染48小时后，测定病毒滴度。值是分裂细胞对融合细胞中病毒产量的比率。
图10.分裂细胞对比融合细胞中病毒产量比率。用100pfu的（□）VVTK-/FCU1或（ロ）VVTK-F4L-/FCU1感染PANC1（人类胰腺肿瘤）、H1299（人类肺肿瘤）或U118MG（人神经神经胶质瘤）细胞。感染48小时后，测定病毒滴度。值是分裂细胞对融合细胞中病毒产量的比率。
图11.用1x106pfu的VVTK-/FCU1（□）或VVTK-I4L-/FCU1（ロ）静脉注射携带人类皮下肿瘤的瑞士裸鼠后第6天及第21天，器官或肿瘤中病毒滴度（pfu/mg组织）。
图12.用1x106pfu的VVTK-/FCU1（□）或VVTK-F4L-/FCU1（ロ）静脉注射携带人类皮下肿瘤的瑞士裸鼠后第6天及第21天，器官或肿瘤中病毒滴度（pfu/mg组织）。
图13.通过静脉注射1x108pfu的VVTK-/FCU1（■）或VVTK-I4L-/FCU1（□）处理后，瑞士裸鼠的生存率。
图14.通过静脉注射1x107pfu（A）或者1x108pfu（B）的VVTK-/FCU1（■）或VVTK-I4L-/FCU1（□）处理后，免疫活性B6D2小鼠生存率。
图15.向瑞士裸鼠静脉注射1x106pfu的VVTK-/FCU1或VVTK-I4L-/FCU1后，在感染后第13天和第34天，尾巴上的平均痘疱数。
图16.向瑞士裸鼠静脉注射1x106pfu的VVTK-/FCU1或VVTK-F4L-/FCU1后，在感染后第13天和第34天，尾巴上的平均痘疱数。
图17.向瑞士裸鼠静脉注射1x107pfu的VVTK-/FCU1或VVTK-I4L-/FCU1后，在感染后第15天和第31天，尾巴上的平均痘疱数。
图18.向瑞士裸鼠静脉注射1x107pfu的VVTK-/FCU1或VVTK-F4L-/FCU1后，在感染后第15天和第31天，尾巴上的平均痘疱数。
本发明的实施方式
实施例
质粒载体的构建
利用缺失胸苷激酶基因并在牛痘合成启动子p11K7.5控制下表达FCU1基因的痘苗病毒菌株Copenhagen（登录号M35027）的DNA构建I4L缺失的穿梭质粒。利用PCR扩增I4L的DNA侧翼区。I4L下游侧翼区的引物是5’-TCCCCCGGGTTAACCACTGCATGATGTACA-3’（SEQIDNO:7；下划线为SmaI位点）和5’-GCCGAGCTCGAGGTAGCCGTTTGTAATTCT-3’（SEQIDNO:8；下划线为SacI位点）。上游区域的引物为5’–GCCTGG CCATAACTCCAGGCCGTT–3’（SEQIDNO:9；下划线为MscI位点）和5’–GCCCAGCTGATCGAGCCGTAACGATTTTCA–3’（SEQIDNO:10；下划线为PvuII位点）。用限制性内切酶SmaI/SacI或MscI/PvuII消化被扩增的DNA片段并将其连接到PpolyIII质粒的相应位点。通过PCR利用引物5’-GCCGCATGCATCCTTGAACACCAATACCGA-3’（SEQIDNO:11；下划线为SphI位点）和5’–GCTCTAGAGAGGTAGCCGTTTGTAATCTG-3’（SEQIDNO:12；下划线为XbaI位点）扩增I4L下游侧翼区的重复区域并将其插入PpolyIII质粒。在生产缺失的病毒的过程中，重复区域被用于消除选择盒。该选择盒对应于在pH5R牛痘启动子控制下的GFP/GPT融合基因，被插入到PpolyIII质粒的SacI/SphI位点。所获得的质粒即该重组穿梭质粒，因为缺失I4L基因而命名为pΔI4L。
利用痘苗病毒菌株Copenhagen（登录号M35027）的DNA构建F4L缺失的穿梭质粒。利用PCR扩增F4L的DNA侧翼区。F4L下游侧翼区的引物是5’-CGCGGATCCTTTGGTACAGTCTAGTATCCA-3’（SEQIDNO:13；下划线为BamHI位点）和5’-TCCCCCGGGTTATAACAGATGCAGTATCCA-3’（SEQIDNO:14；下划线为SmaI位点）。上游区域的引物为5’–GCCCAGCTGTTCAATGGCCATCTGAAATCC–3’（SEQIDNO:15；下划线为PvuII位点）和5’–GAAGATCTAGTATCGCATCTAAAAGATGG–3’（SEQIDNO:16；下划线为BglII位点）。用限制性内切酶BamHI/SmaI或BglII/PvuII消化被扩增的DNA片段并将其连接到PpolyIII质粒的相应位点。通过PCR利用引物5’-GCCGAGCTCACCCACACGTTTTTCGAAAAA-3’（SEQIDNO:17；下划线为SacI位点）和5’–GCCGCATGCTTATAACAGATGCAGTATCAA–3’（SEQIDNO:18；下划线为SphI位点）扩增I4L下游侧翼区的重复区域并将其插入PpolyIII质粒。在生产缺失的病毒的过程中，重复区域被用于消除选择盒。该选择盒对应于在pH5R牛痘启动子控制下的GFP/GPT融合基因，被插入到PpolyIII质粒的SacI/SmaI位点。所获得的质粒即该重组穿梭质粒，因为缺失F4L基因而命名为pΔF4L
重组痘苗病毒的产生
用VVTK-FCU1（痘苗病毒，缺损J2R激酶基因，在合成启动子p11k7.5控制下表达FCU1基因）Copenhagen株按0.1的MOI感染CEF细胞并在37℃下孵育2小时，然后用重组穿梭质粒（0.2μg）的氯化钙共沉淀物转染。将该细胞在37℃孵育48小时。然后用新产生病毒的稀释物在选择培养基中感染CEF细胞，该选择培养基含有终浓度为15μg/ml的次黄嘌呤，终浓度为250μg/ml的黄嘌呤和终浓度为250μg/ml的霉酚酸。分离荧光的（GFP）和阳性的（GPT选择）斑，并在存在GPT选择培养基的CEF细胞中进行数轮筛选。利用使用缺失区域内的引物进行40个PCR循环测定VVTk-FCU1是否存在。消除亲本病毒后，用双重缺失病毒来感染无GPT选择培养基的CEF，以消除选择盒。分离非荧光斑并在CEF中筛选2轮。在CEF中扩增最终的重组VV病毒，纯化并通过空斑测定在CEF上滴定病毒储液。
细胞对5-FC的体外敏感性。
用各重组VV在0.0001的MOI下转导人类肿瘤细胞。将总数为3×105个细胞/孔铺板于含有不同浓度5-FC的2毫升培养基的6孔培养皿中。然后将细胞在37℃下培养5天，利用台盼蓝拒染法计数活细胞。在图1、2、 3和图4中描绘的结果表明，J2R基因缺损的病毒中的FCU1活性与I4L和和J2R基因缺损的病毒、或F4L基因和J2R基因缺损的病毒中的FCU1活性相等。
培养细胞的体外复制。
用100PFU的病毒（约0.0005MOI）在6孔板中感染分裂细胞或融合细胞。对于分裂细胞使用添加10％FCS的2mL培养基，对于融合细胞使用不添加补剂的培养基。感染后48小时收获细胞。将细胞保存在-20℃并超声处理以释放病毒，也通过CEF细胞的蚀斑滴定定量病毒。在所有细胞中，分裂细胞和融合细胞之间复制的比率相似。病毒VVTK-/FCU1、VVTK-I4L-/FCU1和VVTK-F4L-/FCU1在分裂细胞中的复制都比在融合细胞中的多。
作为一个测定复制病毒的特异性的间接方法，测定分裂相对于融合肿瘤细胞（人类胰腺肿瘤PANC1；人类肺肿瘤H1299；人类神经胶质瘤U118MG）中产生病毒的产量。以1x106细胞/孔铺板融合细胞并在完全培养基中培养7天，然后在感染前1天洗涤细胞并将其培养在无血清的培养基中。感染之前一天以3x105细胞/孔铺板分裂细胞。为了评估细胞分裂水平，在铺板细胞后的5小时、24小时和48小时测定滴定过的胸苷掺入核酸的量。在此期间，在融合细胞中的胸苷掺入相对稳定而在分裂细胞中发现掺入随时间而增长。然后用100pfu病毒感染这些细胞，并在感染48小时后利用对CEF的噬斑滴定，测定分裂肿瘤细胞和融合肿瘤细胞产生的病毒产量之间的比率。在图9和10中所描述的结果表明，两种病毒VVTK-/FCU1、VVTK-I4L-/FCU1和VVTK-F4L-/FCU1在分裂细胞中的复制都比在融合细胞中的多。此外，图9和10中所示结果显示，对于两种病毒VVTK-I4L-/FCU1和VVTK-F4L-/FCU1，与VVTK-/FCU1相比，在所有不同类型细胞中比率均有增加。此在所有不同类型细胞中比率的增加是由于两种病毒VVTK-I4L-/FCU1和VVTK-F4L-/FCU1在融合细胞中均有更低的复制。这些结果表明，两种病毒VVTK-I4L-/FCU1和VVTK-F4L-/FCU1与VVTK-/FCU1相比，对于分裂细胞均显示增加的特异性。
皮下肿瘤模型
雌性瑞士裸鼠是从CharlesRiverLaboratories获得的。在研究中使用的动物年龄一致（6周）且体重范围从23至26克。以5x106LoVo细胞向瑞士裸鼠侧腹内进行皮下注射（S.C.）。当肿瘤直径达到50-70mm3，将小鼠盲式随机化，并用指示载体处理进行体内实验。
病毒的生物分布。
通过在肿瘤和器官样本中的病毒滴定评估不同病毒的存在。利用尾部静脉注射将1x106PFUVV-FCU1或VVTK-I4L-/FCU1或VVTK-F4L-/FCU1静脉（i.v.）注射到荷有确立的皮下LoVo肿瘤的裸鼠中。在指示时间点处死小鼠，并采集和称重肿瘤和其它器官。在PBS中均质化肿瘤和器官，并如前所述地用CEF测定滴度。病毒滴度标准化至每毫克组织。表2、3、4 和5中描述的结果（病毒滴度范围以pfu/mg组织表示）显示，14天后本发明的病毒多数在肿瘤中。图11和12中描述的结果显示，除了VVTK-/FCU1在尾巴中的情况以外，病毒VVTK-/FCU1、VVTK-I4L-/FCU1和VVTK-F4L-/FCU1以在肿瘤中比在被分析的其他器官中的多大约1000至10000倍的病毒来靶向肿瘤。第6天在肺、脾脏、肾脏和淋巴结中检测到少量的VVTK-/FCU1（少于10pfu/mg），在皮肤、尾巴和骨髓中发现更多，第21天在皮肤和尾巴中发现。相比之下，VVTK-I4L-/FCU1和VVTK-F4L-/FCU1均具有更高的肿瘤特异性，第6天在淋巴结中和尾巴中，第21天在肿瘤中只含有少量。
表2
| 肿瘤 | 肺 | 脾脏 | 肾脏 | 淋巴结 | 心脏 | |
| VVTK-/FCU1 | （0,2-3,3）x105 | 0.1-2 | 0-2.2 | 0-1.8 | 0-61 | 0-0.3 |
| VVTK-I4L/FCU1 | 25.6-2.2x105 | 0-0.1 | n.d | 0-1 | n.d | n.d |
表3
| 卵巢 | 皮肤 | 尾巴 | 骨髓 | 脑 | 肌肉 | |
| VVTK-/FCU1 | 2.2-74 | 0.1-24 | 13.5-7.104 | 0-800 | 0-1.8 | 0-22 |
| VVTK-I4L/FCU1 | 0-102 | n.d | 26,3 | n.d | n.d | n.d |
表4
| 肿瘤 | 肺 | 脾脏 | 肾脏 | 淋巴结 | 卵巢 | |
| VVTK-/FCU1 | (0,2-3,3)x105 | 0.1-2 | 0-2.2 | 0-1.8 | 0-61 | 2.2-74 |
| VVTK-F4L/FCU1 | 51.8-3.8x104 | n.d | n.d | n.d | 0-2.1 | n.d |
表5
| 尾巴 | 骨髓 | 肠 | 脑 | 肌肉 | 心脏 | |
| VVTK-/FCU1 | 13.5-7.104 | 0-800 | n.d | 0-1.8 | 0-22 | 0-0.3 |
| VVTK-F4L/FCU1 | 0-7.9 | n.d | n.d | n.d | n.d | n.d |
本发明的痘病毒在皮下肿瘤模型中的抗肿瘤活性。
分别用1.107PFU剂量的指示载体一次静脉注射（通过尾巴静脉）处理荷有已确立的皮下LoVo肿瘤（50-70mm3）的裸鼠。注射病毒后第7天开始，持续3周每天两次以100mg/kg（0.5ml5-FC0.5%水溶液）口服强饲5-FC。每周用卡尺测量肿瘤大小2次。使用公式（p/6）以mm3（长x宽2）计算肿瘤体积。如图5和6所示的结果表明，不同病毒具有相似的功效（p<0.05），其溶瘤活性能够控制肿瘤生长，并具有与施用5-FC的组合活性（病毒的溶瘤活性和FCU1基因的治疗活性），施用5-FC可以进一步改进肿瘤生长的控制（p<0.01）。
用1.107PFU剂量的指示载体，也用静脉注射（通过尾巴静脉）处理荷有确立的皮下LoVo肿瘤(50-70mm3)的裸鼠，按照如下进行：接种肿瘤11天后（可触知的肿瘤），用缓冲液+水、或缓冲液+5-FC、或单次注射107pfuVVTK-I4L-/FCU1+H2O、或单次注射107pfuVVTK-I4L-/FCU1+5-FC（5-FC在病毒注射7天后的三周中施用）、或两次注射(第11天和第33天)107pfuVVTK-I4L-/FCU1+H2O、或两次注射(第11天和第33天)107pfuVVTK-I4L-/FCU1+5-FC(5-FC从第18天至第32天和从第40天至第54天施用)处理小鼠。通过口服强饲用100mg/kg的5-FC每天处理这些动物两次。用卡尺每周测定肿瘤大小2次。使用公式（p/6）（长x宽2）以mm3计算肿瘤体积。如图7所示的结果显示在一或两次注射之后，单独的病毒没有抗肿瘤活性。至第50天时，当与载体组和单独的病毒(无5-FC)相比，添加5-FC处理显示显著的肿瘤生长抑制(p<0.05)。当与载体组和两次注射单独的病毒(无5-FC)相比，如同单次注射，两次静脉注射VVTK-I4L-/FCU1+5-FC显示显著的抗肿瘤活性(p<0.05)。此外，在组合了5-FC处理的病毒的一次和两次注射之间，从第56天观察到肿瘤发展的显著差异(p<0.05)。荷有确立的皮下U87-MG(胶质母细胞瘤肿瘤细胞)的裸鼠用指示载体以1.107PFU的剂量静脉处理(尾部静脉)，按如下进行:接种肿瘤11天后（可触知的肿瘤）,小鼠用缓冲液+H2O,或缓冲液+5-FC,或107pfuVVTK-I4L-/FCU1+H2O,或107pfuVVTK-I4L-/FCU1+5-FC处理。注射病毒7天之后的3个星期，每天以100mg/kg通过口服强饲5-FC两次来治疗这些动物。用卡尺每周测定肿瘤体积两次。使用公式（π/6）（长x宽2）以mm3计算肿瘤体积。如图8所示的结果VVTK-I4L-/FCU1对U87-MG细胞的高溶癌活性，其导致强抗癌活性(p<0.0001)。
通过口服强饲，添加了5-FC的组合活性，得到相似活性(p<0.0001)。
病毒的致病性
用对瑞士裸鼠（图13）和免疫活性的B6D2小鼠（图14）进行存活研究以评估病毒的致病性。用100μL每鼠缓冲液中的1.107或1.108PFU所有VVTK-/FCU1和VVTK-I4L-/FCU1静脉注射小鼠。在整个实验过程中每天观察小鼠。瑞士裸鼠中（图13），注射1x108PFU的VVTK-/FCU1导致40％的动物在感染3天后死亡。其余的老鼠在感染后第50至第80天死亡。施用VVTK-I4L-/FCU1致病性较低，大部分动物在第65至第140天死亡（p<0.01）。两种病毒在107pfu没有观察到毒性的迹象（图14(A)）。所有小鼠在静脉注射108pfuVVTK-/FCU1后死亡（图14(B)）。用VVTK-I4L-/FCU1处理的组，与用VVTK-/FCU1感染的小鼠相比，具有显著的延长存活达70％（图14(B)）。因此，这一结果表明了，双重缺失的病毒VVTK-I4L-/FCU1的毒性降低。
痘疱尾巴损害模型
对瑞士裸鼠静脉注射1.106（图15和16）或1.107PFU（图17和18）的每种病毒。每周数一次尾巴损害。如图15（A）和图16（A）所示，在感染后第13天，用1.106PFU的VVTK-I4L-/FCU1或VVTK-F4L-/FCU1注射的小鼠，具有的痘疱/小鼠少于1，相比于注射VVTK-/FCU1的小鼠，平均各鼠8个痘疱。在注射后的第34天结果类似，VVTK-I4L-/FCU1或VVTK-F4L-/FCU1平均痘疱为4，相对比于注射VVTK-I4L-/FCU1的约为1（p<0.0001），如图15（B）和图16（B）所示。在感染后15天，用1.107PFU的VVTK-I4L-/FCU1或VVTK-F4L-/FCU1注射的小鼠，各鼠分别平均有3和2个痘疱，相对比于，注射1.107PFU的VVTK-/FCU1，各鼠平均有10个痘疱（图17（A）和图18（A））。在感染后第31天，用1.107PFU的VVTK-I4L-/FCU1或VVTK-F4L-/FCU1注射的小鼠，分别平均有1.5个痘疱/鼠和2个痘疱/鼠，相对比于，注射VVTK-/FCU1的，平均有7个痘疱/鼠（图17（B）和图18（B））。VVTK-/FCU1与VVTK-I4L-/FCU1和VVTK-F4L-/FCU1两者之间痘疱数量的差异是统计上显著的（p<0.01）。该痘疱的形成与尾巴中病毒的复制相关，也与毒性和毒力相关。静脉注射VVTK-I4L-/FCU1或VVTK-F4L-/FCU1的毒性比单一缺失的TK病毒低。
统计分析。
采用非参数Mann-WhitneyU检验和STATISTICA7.1软件（StatSoft，公司）进行统计分析。P<0.05被认为是统计上显著的。
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Claims (16)
1.溶瘤痘病毒在制备用于治疗癌症的药物中的用途，其中所述溶瘤痘病毒的I4L和/或F4L基因缺损。
2.根据权利要求1的用途，其中所述溶瘤痘病毒的J2R基因进一步缺损。
3.根据权利要求1或2的用途，其中所述溶瘤痘病毒的F2L基因进一步缺损。
4.根据权利要求1或2的用途，其中所述溶瘤痘病毒属于脊椎动物痘病毒亚科。
5.根据权利要求4的用途，其中所述溶瘤痘病毒属于痘苗病毒种。
6.根据权利要求5的用途，其中所述溶瘤痘病毒是痘苗病毒Copenhagen株。
7.根据权利要求5的用途，其中所述溶瘤痘病毒是痘苗病毒WR株。
8.根据权利要求1的用途，其中所述溶瘤痘病毒还包含编码透性酶的基因。
9.根据权利要求8的用途，其中所述透性酶是酿酒酵母的嘌呤或胞嘧啶透性酶。
10.根据权利要求9的用途，其中所述透性酶选自FCY2和Fur4。
11.根据权利要求8的用途，其中所述溶瘤痘病毒还包含表达编码透性酶的基因所必需的元件。
12.包含根据权利要求1至11中任一项定义的溶瘤痘病毒和可药用的赋形剂的组合物在制备用于治疗癌症的药物中的用途。
13.根据权利要求12的用途，其中所述组合物通过全身途径施用。
14.根据权利要求12或13的用途，其中还施用可药用数量的前药。
15.根据权利要求14的用途，其中在施用所述溶瘤痘病毒至少3天之后施用所述前药。
16.根据权利要求14的用途，其中在施用所述溶瘤痘病毒7天之后施用所述前药。
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