CN100506284C - 多核苷酸构建体、药物组合物以及靶向的下调血管生成和抗癌的治疗方法 - Google Patents
多核苷酸构建体、药物组合物以及靶向的下调血管生成和抗癌的治疗方法 Download PDFInfo
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Abstract
本发明提供用于在受试者组织中下调血管生成的一种新的核酸构建体。该核酸构建体包括：(a)编码嵌合多肽的第一多核苷酸区域，所述的嵌合多肽包括与编程性细胞死亡信号分子的效应物结构域相融合的配体结合结构域；和(b)编码用于指导嵌合多肽在特异的组织或细胞中表达的顺式作用调节元件的第二多核苷酸区域；其中，选择所述的配体结合结构域使得其能与存在于特定组织或细胞中的配体相结合，而所说配体结合到所说配体结合的结构域则可激活编程性细胞死亡信号分子的效应物结构域。本发明还提供利用该核酸构建体治疗以过度或异常的新血管生成或细胞生长为特征的疾病的方法。
Description
发明背景和领域
本发明涉及核酸构建体、药物组合物和用于在受试者特异组织区域中下调血管生成的方法.更具体地说，本发明涉及可用于在特定的细胞亚群中激活编程性细胞死亡的核酸构建体，从而能够治疗以过度或异常新血管生成或以细胞生长为特征的疾病.
血管生成指新血管的生长，是一个主要依赖于毛细血管内皮细胞的移动、增殖和脉管形成的过程.在血管生成期间，内皮细胞从平静状态中苏醒并迅速增殖.虽然负责细胞转变为血管生成表型的机制未知，但导致新脉管形成事件的次序已有充分的文献记载[Hanahan，D.，Science 277，48-50，(1997)].血管的生长需要内皮出芽[Risau，W.，Nature 386，671-674，(1997)]或套迭[Patan，S.，等；Microvasc.Res.51，260-272，(1996)].在第一途径中将有下述事件发生：(a)脉管基础，通常是后毛细管小静脉和间质溶解；(b)内皮细胞向刺激物迁移；(c)尾随前导内皮细胞之后的内皮细胞增殖；(d)在内皮阵列/芽中形成腔(管道化)(e)通过芽的融合性吻合形成分支和回路以使血液流动；(f)外膜细胞(即，外周内皮细胞和平滑肌细胞)包被上述脉管；和(g)形成包围不成熟脉管的基底膜。还可以通过第二途径形成新脉管：即间质组织柱插入预先形成的脉管腔内.这些柱随后的生长和稳定化导致脉管腔的分割和局部血管网的重塑.
有多种血管生成因子控制血管生成过程.当然，在病变期间，原血管生成因子和抗血管生成因子之间的精密调节平衡被破坏，引起非自限的内皮和外周内皮细胞增殖.直到最近，对在眼新血管生成、关节炎、皮肤病和肿瘤疾病中出现的血管生成仍难以进行治疗性抑制.
因此，所有抗血管生成治疗的基本目标是使增殖微血管的病灶回复到正常的静止状态，并阻止其再生长[癌症：肿瘤学理论与实践，第五版Vincent T.DeVita，Jr.，Samuel Hellman，Steven A.Rosenberg编Lippincott-Raven出版，费城(1997)].
鉴于其可延缓肿瘤(例如，视网膜病、良性的和恶性的血管生成性肿瘤)级数生长，抗血管生成治疗是强有力的临床方法.
总的说来，任何一种由非受控毛细血管生长引起的疾病，如糖尿病性视网膜病、牛皮癣、关节炎、血管瘤，肿瘤生长和转移都是抗血管生长治疗的靶子.
例如，实体瘤的渐进生长超过临床上的潜伏大小(例如，几mm3)需要连续的新血管形成，即已知的肿瘤血管生成过程.肿瘤生长和转移是血管生成-依赖性的.肿瘤必须连续地刺激新微血管的生长以传递营养和氧供肿瘤自身生长.因此，抗血管生成肿瘤治疗的基础在于阻止肿瘤血管生成或者选择性地破坏肿瘤中已存在的血管(血管靶向疗法).
最近，已开发出许多抗血管生成药剂来治疗恶性疾病，其中一些药剂已在进行临床试验(参见Herbst等(2002)Semin.Oncol.29：66-77).
研究得最多的用于肿瘤抗血管生成治疗的目标是在人体中调节血管生成的主导过程，即血管内皮生长因子(VEGF)与其受体(VEGFR)之间的相互作用.调节VEGFR的原-血管生成作用的试剂包括(i)针对VEGF蛋白本身或其受体(例如，rhuMAb VEGF)的抗体；(ii)针对VEGFR酪氨酸激酶的小分子化合物(例如，ZD6474和SU5416)；(iii)靶向VEGFR的核酶.
其他新的血管生成抑制剂包括具有独特的抗肿瘤和抗血管生成特性的雌???二醇的天然代谢产物2-甲氧雌二醇(2-ME2)，以及分别作为纤维蛋白溶酶原和胶原XVIII的蛋白水解切割片段的血管生长抑素和内皮生长抑素.
然而在前临床模型中显示出，迄今为止所有临床试验的抗血管生成药剂在系统施用时都只有有限的成功率和相当大的毒性，包括血小板减少、白血球减少症和咯血.这些结果提示用目前的肿瘤抗血管生成药剂治疗高级恶性肿瘤可能会受到限制.O′Reilly等已经表明在抗血管生成治疗的开始以及抗肿瘤效果的出现之间的等侯期可导致在对治疗起反应之前的最初的肿瘤级数生长[O′Reilly S et.al.(1998)Proc Am Soc Clin oncol 17：217a].而且，最近的研究提示在不同的毛细管床之间的血管生成的规则可以不同，提示抗血管生成治疗可以基于器官/组织-特异性来优化[Arap等(1998)Science 279：377-380].
有趣的是，用针对平滑肌细胞增殖的抗血管生成方法治疗(例如，肝素、肝素-肽治疗)具有冠状动脉病病人的心肌缺血效果也并不理想[Liu等，血液循环，79：1374-1387(1989)；Goldman等，动脉粥样硬化，65：215-225(1987)；Wolinsky等，JACC，15(2)：475-481(1990)].与利用上述的药剂治疗心血管疾病有关的各种限制包括：(i)对患有心血管疾病的病人引起的系统性毒性危险达到不可接受的水平；(ii)在外科手术后妨碍血管伤口愈合；(iii)可能破坏周围的内皮和/或其他的中间平滑肌细胞.
由于这些以及与系统施用抗血管生成因子相关的固有障碍(即基于体外不稳定性和高剂量需求的制造限制；和归咎于次最佳效应的丸剂施用的峰动力学)限制了血管生成因子在治疗与新血管生成相关疾病中的有效使用.
随着调节血管生成过程的新基因的鉴定，已试图采用体基因治疗来突破这些限制.虽然投入了很大的努力致力于开发出癌症、心血管和外周血管的疾病的基因治疗方法，在有效且特异的基因递送方面仍存在很大的障碍[参见，Feldman AL.(2000)Cancer 89(6)：1181-94].通常以重组病毒载体作为载体、利用感兴趣的基因进行基因治疗的主要限制因素是将感兴趣的基因特异性地导向靶组织的能力.
克服这些限制的努力包括利用组织特异的与细胞毒素基因相偶联的启动子.例如，Jagger等公开了在内皮-特异的启动子控制下表达靶定内皮细胞的细胞毒素基因，他们利用KDR或E-选择性启动子在内皮细胞中特异地表达TNFα[Jaggar RT.等Hum Gene Ther(1997)8(18)：2239-47].Ozaki等用von-Willebrand因子(vWF)启动子将单纯疱疹病毒胸苷激酶(HSV-tk)递送到HUVBC[Hum GeneTher(1996)7(13)：1483-90].但是，这些启动子仅仅显示出微弱的活性并且不能进行高水平表达.
由Kong和Crystal提出了一种替代方法，这种方法包括抗血管生成因子在肿瘤中特异表达。然而迄今为止尽管在临床前模型中一些合成的抗血管生成药剂与毒性相关，但内源抗血管生成药剂的重组体形式的毒性还没有被证实[Kong和Crystal(1998)J.Natl.CancerInst.90：273-76].
血管生长抑素也已经被用作可能的抗血管生成药剂(Falkman等，Cell 1997 Jan 24；88(2)：277-85)，但由于肿瘤中涉及血管生成调节的因子过剩，单独的血管生长抑素治疗很难奏效.
因此广泛认为需要能在受试者特异的组织区域中有效下调血管生成，同时避免现有技术中抗血管生成方法的特征性毒副作用的新方法，该方法将非常有用.
发明简述
本发明一方面提供一种核酸构建体，其包含：(a)编码嵌合多肽的第一多核苷酸区域，所述的嵌合多肽包括与编程性细胞死亡信号分子的效应物结构域相融合的配体结合结构域；和(b)编码用于指导嵌合多肽在特异的组织或细胞中表达的顺式作用调节元件的第二多核苷酸区域；其中，选择所述的配体结合结构域使其能与存在于特异组织或细胞中的配体相结合，而配体结合到该配体结合结构域则可激活编程性细胞死亡信号分子的效应物结构域.
根据下述本发明的优选实施方案中的进一步描述，本发明还提供用上述核酸构建体转化的哺乳动物细胞.
根据本发明的另一方面，提供在受试者组织中下调血管生成的方法，该方法包括向受试者施用经设计并装配用于在血管生成细胞亚群中产生编程性细胞死亡的核酸构建体，所述的核酸构建体包括：(a)编码嵌合多肽的第一多核苷酸区域，所述的嵌合多肽包括与编程性细胞死亡信号分子的效应物结构域相融合的配体结合结构域；和(b)编码用于指导嵌合多肽在血管生成细胞亚群中表达的顺式作用调节元件的第二多核苷酸区域；其中，选择所述的配体结合结构域使其能与存在于或提供到血管生成细胞亚群的配体相结合，而配体结合到该配体结合结构域则可激活编程性细胞死亡信号分子的效应物结构城，由此下调该组织中的血管生成.
根据在本发明下述的优选实施方案中的进一步描述，该方法进一步包括以适合于将所述配体提供于血管生成细胞亚群的方式施用所述配体于受试者.
本发明的又一方面提供在受试者组织中下调血管生成的方法，该方法包含：(a)向受试者施用经设计并装配用于在血管生成细胞亚群中产生编程性细胞死亡的核酸构建体，该核酸构造包括：(i)编码嵌合多肽的第一多核苷酸区域，所述的嵌合多肽包括与编程性细胞死亡信号分子的效应物结构域相融合的配体结合结构域；其中，选择所述的效应物结构域使其能在配体结合到配体结合结构域后被激活；和(ii)编码用于指导嵌合多肽在血管生成细胞亚群中表达的顺式作用调节元件的第二多核苷酸区域；和(b)向受试者施用所述配体，由此下调该组织中的血管生成.
根据本发明的下述优选实施方案中的进一步描述，向受试者施用所述的配体由下述方法实现，所述方法选自：(i)全身性体内施用；(ii)间接地体内施用到从受试者机体中取出的细胞中，随后再将所述细胞引入到受试者体内；和(iii)在体内局部施用.
根据本发明下述优选实施方案中的进一步描述，所述的顺式作用调节元件是内皮细胞特异的或外周内皮细胞特异的启动子，所述启动子选自：PPE-1启动子、PPE-1-3x启动子、TIE-1启动子、TIE-2启动子、Endoglin启动子、von Willerband启动子、KDR/flk-1启动子、FLT-1启动子、Egr-1启动子、ICAM-1启动子、VCAM-1启动子、PECAM-1启动子、CArG盒元件和主动脉羧肽酶样蛋白(ACLP)启动子.
根据本发明下述优选实施方案的进一步描述，所述的配体结合结构域是细胞表面受体的配体结合结构域.
根据本发明下述优选实施方案的进一步描述，所述的细胞表面受体选自：受体酪氨酸激酶、受体丝氨酸激酶、受体苏氨酸激酶、细胞粘着分子和磷酸酶受体.
根据本发明下述优选实施方案的进一步描述，所述的编程性细胞死亡信号分子选自：Fas和TNFR.
本发明的再一方面提供用于在受试者组织中下调血管生成的药物组合物，该药物组合物包含作为活性成分的、经设计并装配用于在血管生成细胞亚群中产生编程性细胞死亡的核酸构建体和药学上可接受的载体，所述的核酸构建体包括：(a)编码嵌合多肽的第一多核苷酸区域，所述的嵌合多肽包括与编程性细胞死亡信号分子的效应物结构域相融合的配体结合结构域；和(b)编码用于指导嵌合多肽在血管生成细胞亚群中表达的顺式作用调节元件的第二多核苷酸区域；其中，选择所述的配体结合结构域使其能与存在于特异组织或细胞中的配体相结合，而配体结合到该配体结合结构域可激活编程性细胞死亡信号分子的效应物结构城.
本发明的又一方面，提供治疗与过度新血管形成相关的疾病或病症的方法，该方法包括施用治疗有效量的设计并装配用于在血管生成细胞亚群中产生编程性细胞死亡的核酸构建体，所述的核酸构建体包括：(i)编码嵌合多肽的第一多核苷酸区域，所述的嵌合多肽包括与编程性细胞死亡信号分子的效应物结构城相融合的配体结合结构域；和(ii)编码用于指导嵌合多肽在血管生成细胞亚群中表达的顺式作用调节元件的第二多核苷酸区域；和其中，选择所述的配体结合结构域使其能与存在于或提供到血管生成细胞亚群的配体相结合，而配体结合到配体结合结构域则激活编程性细胞死亡信号分子的效应物结构域，由此在所述组织中下调血管生成并治疗与过度新血管形成相关的疾病或病症.
本发明的又一方面提供治疗受试者体内肿瘤的方法，该方法包括施用治疗有效量的设计并装配用于在肿瘤细胞中产生编程性细胞死亡的核酸构建体，所述的核酸构建体包括：(i)编码嵌合多肽的第一多核苷酸区域，所述的嵌合多肽包括与编程性细胞死亡信号分子的效应物结构域相融合的配体结合结构城；和(ii)编码用于控制嵌合多肽在肿瘤细胞中表达的顺式作用调节元件的第二多核苷酸区域到，其中，选择所述的配体结合结构城使其能与存在于或提供到肿瘤细胞的配体相结合，而配体结合到该配体结合结构域则激活编程性细胞死亡信号分子的效应物结构域，由此指导该肿瘤细胞中的编程性细胞死亡.
根据本发明下述的优选实施方案的进一步描述，该方法进一步包括以适合于将所述配体提供于所述肿瘤细胞的方式将所述配体施用于受试者。
根据本发明下述优选实施方案的进一步描述，所述的顺式作用调节元件选自：胃泌素-释放肽(GRP)启动子、hTERT启动子、己糖激酶II型启动子和L-网素启动子.
本发明通过提供一种以特异的且定向的方式有效地下调血管生成和肿瘤细胞增殖的新方法成功地克服了现有的现有技术的缺陷.
附图的简要说明
参照下述附图仅以举例的方式对本发明进行描述.现在详细地参照特定附图，需要强调的是下述具体显示的仅仅是以举例的方式达到对本发明的优选实施方案进行说明性讨论的目的，所提供的是被认为是最有用和最易于理解本发明的原理和概念的内容.在这方面，没有更详细地提供超出基本了解本发明所必需的结构细节，显而易见，结合附图的描述可使本领域的普通技术人员了解到在实践中如何体现本发明的几种形式。
在附图中：
图1a-b是克隆到pcDNA3质粒(a)或腺病毒载体(b)中的、从来自TNFR1胞外区域和Fas的跨膜和胞内区域构建的Fas嵌合基因的简图.
图2a-b说明原编程性细胞死亡基因、Fas嵌合体和TNFR1的编程性细胞死亡活性.图2a-说明用pcDNA-3-TNFR1(下)或者对照空载体(上)和编码GFP的表达质粒转染的牛主动脉内皮细胞(BAEC).图2b-说明用pcDNA-3-Fas-c(下)或者对照空载体(上)和编码GFP的表达质粒转染的293细胞.利用荧光显微镜观察转染细胞，从形态上确定编程性细胞死亡活性.
图3a-f是经原编程性细胞死亡基因转染的BAEC细胞的电子显微图像.转染后24小时，用2.5％的戊二醛固定BAEC细胞并对其进行加工.显示的是在编程性细胞死亡过程连续阶段中的细胞.
图4是指示原编程性细胞死亡基因在转染的BAEC和293细胞中定量编程性细胞死亡活性的直方图.
图5a表示AdPPE-Fas-c的PCR分析.1-2道是用包含PPB-1启动子和Fas-基因的引物所得的PCR产物.3-4道是用Fas-c引物所得的PCR产物.5-6道是在无模板DNA的情况下所得的PCR产物。
图5b是AdPPE-Fas-c转染的BAEC细胞的蛋白质斑迹法分析.蛋白样品经SDS-PAGE解析，转移到硝酸纤维素膜上，用针对TNFR1胞外部分的多克隆抗体检测.1-2道是经pcDNA3-Fas-cBAEC转染的细胞(阳性对照)。3-4道是经指示MOI的AdPPE-Fas-c病毒转染的BAEC细胞.5道是非转染细胞.6-7道是经指示MOI的AdPPE-1uc转染的BAEC细胞.
图6a-d是说明Fas-嵌合体过表达对内皮细胞编程性细胞死亡的作用的显微照片.用下述物质转染BAEC细胞：Ad-PPE-1-3x-Fas-嵌合体(图6a)；Ad-PPE-1-3x-荧光素酶(图6b)；Ad-PPE-1-3x-Fas-嵌合体和Ad-PPE1-3x-GFP(图6c)；Ad-PPE-1-3x-荧光素酶和Ad-PPE-1-3x-GFP；各在MOI1000下(图6d).显微照片是在转染后72小时拍摄的，经x10放大.
图7是说明Ad-PPE-1--Fas-嵌合体对内皮细胞编程性细胞死亡特异性作用的直方图.对经Ad-PPE-1-3x-Fas-嵌合体或对照(荧光素酶)病毒感染后72小时的内皮的(BAEC，HUVEC)和非内皮的(正常皮肤成纤维细胞-NSF)细胞用结晶紫着色，定量测定细胞活力.
图8显示施用TNFα对Fas-嵌合体介导的编程性细胞死亡的剂量反应效果.用Ad-PPE-1-3x-Fas-c感染BAEC.感染后48小时，向生长培养基中(以指示的剂量)添加TNF.24小时后通过结晶紫试验确定活力.
图9a-e是说明由TNFα配体和Fas-c受体的协同作用介导的内皮细胞-特异的编程性细胞死亡的显微照片.所指示的细胞是经Ad-PPE-1-3x-Fas-c感染后48小时在存在或不存在TNFα(10ng/ml)的情况下孵化的；结晶紫着色在感染后72小时进行.
图10a是说明依赖于TNFα的Ad-CMV-Fas-c对内皮细胞的编程性细胞死亡影响的剂量反应曲线.用指示MOI的Ad-CMV-Fas-嵌合体感染的BAEC细胞的活力在与TNFα孵化后确定.
图10b-d表示TNFα配体和Ad-CMV Fas-嵌合体对非内皮细胞NSF的编程性细胞死亡的影响.图10b是经对照病毒感染的NSF.图10c是经Ad-CMV-Fas-嵌合体感染的NSF.图10d是经Ad-CMV-Fas-嵌合体感染并与TNF(10ng/ml)孵化的NSF.
图11a-c说明Ad-PPE-1-3x-Fas-c的体内抗肿瘤作用.当肿癌可触摸到时，用Ad-PPE-1-3x-Fas-c、Ad-CMV-Fas-嵌合体、对照病毒或盐水静脉内注射接种B16黑素瘤的小鼠。图11a表示在治疗期间测量的肿瘤面积.图11b是治疗结束时的肿瘤重量.图11c是表示Ad-PPE-1-3x-Fas-c处理过的小鼠和对照小鼠中的肿瘤状态图.
优选实施方案的描述
本发明是可用于治疗以过度或异常的新血管形成或细胞生长为特征的疾病的核酸构建体和方法.特别地，本发明可用于特异靶定并杀死涉及血管生成的细胞，从而下调血管生成并进行抗肿瘤治疗.
参照附图并结合相关描述可更好地理解本发明的原理和操作.
在详细地解释至少一个实施方案之前，应当理解本发明并非限于下述描述和附图说明中所示的组分结构和排列的细节中.本发明可以有其他的实施方案或以多种不同的形式实施.同样应当理解的是，此处所用的措辞和术语是为了对本发明进行描述，不应将其理解为对本发明的限制.
抗血管生成治疗的最基本目标之一是使增殖中的微血管病灶???回归到其正常的静止状态下，并阻止其再生长.
迄今为止，大多由于在治疗个体中导致形成血小板减少、白血球减少症和/或咯血的毒性副作用，使用全身施用抗血管生成药剂的抗血管生成治疗方法仅取得了极其有限的成功.现有技术方法中的上述的毒性副作用是所使用的抗血管生成药剂非特异性表述、将健康组织暴露于这些药剂或所使用的抗血管生成药剂过剩从而使其无效力的结果。
当将本发明还原到实践中时，本发明人揭示出将原-编程性细胞死亡药剂的组织-特异性表达与特异性活化结合起来可选择性地使涉及血管生成的细胞编程性死亡，而不使非靶组织或细胞暴露于这些药剂，从而避免了现有技术中的治疗方法所产生的有毒性副作用和过剩现象。
因此，本发明的一个方面提供在受试者的组织中下调血管生成的方法。此处所用的术语“下调血管生成”指减缓或者阻止导致新血管形成的血管生成过程.
依照本发明该方面所提供的方法是通过向受试者施用经设计并装配用来在血管生成细胞亚群中产生编程性细胞死亡的核酸构建体来实现的。此处所用的术语＂血管生成细胞＂指任何参与或有助于血管生成过程的细胞.因此，血管生成细胞包括但是不限于内皮细胞、平滑肌细胞.
为了指导编程性细胞死亡药剂在血管生成细胞亚群中的特异性表达，本发明的核酸构建体包括编码嵌合多肽的第一多核苷酸区城，所述的嵌合多肽含有与编程性细胞死亡信号分子效应物结构域相融合的配体结合结构域，所述的配体结合结构域可以是，例如受体酪氨酸激酶的细胞表面受体结构城、受体丝氨酸激酶、受体苏氨酸激酶、细胞粘着分子或与编程性细胞死亡信号分子，例如Fas、TNFR和TRAIL的效应物结构城融合的磷酸酶受体.
所述的嵌合多肽可以包括任何与编程性细胞死亡信号结构域融合的配体结合结构域，只要配体结合结构域得到活化即可，即，通过配体结合由编程性细胞死亡信号分子的效应物结构城触发编程性细胞死亡信号.
对配体结合结构域和与之融合的编程性细胞死亡信号结构城的选择受靶向于编程性细胞死亡的血管生成细胞类型的影响.例如：当靶定特异的内皮细胞亚群时(例如，增殖内皮细胞或表现肿瘤表现型的内皮细胞)，所述的嵌合多肽包括可与天然存在于上述内皮细胞环境中，优选不存在于其他非靶组织的内皮细胞内的配体结合的配体结合结构域(例如TNF，VEGF).上述配体可由内皮细胞分泌(自分泌)、由临近的肿瘤细胞分泌(旁泌性)，或特异地靶定这些内皮细胞.
下述实施例部分中的实施例2提供了合适的嵌合多肽实例.优选地，所述嵌合多肽是下述实施例部分中实施例2-4中所描述的Fas-c嵌合体.
利用所述的嵌合多肽是特别有利的，因为如其后的实施例部分所示，其能在特异的血管生成细胞亚群中有效和有力地活化编程性细胞死亡，同时避免在其他非靶向编程性细胞死亡的细胞亚群中活化.
为进一步增强编程性细胞死亡的细胞专一性，本发明的核酸构建体进一步包括编码能指导该嵌合多肽在血管生成细胞亚群中表达的顺式作用调节元件(例如，启动子和/或增强子)的第二多核苷酸区域.
可用于本发明的核酸构建体的合适的启动子/增强子的实例包括内皮细胞-特异的启动子：前内皮素原1、PPE-1启动子(Harats D，J Clin Invest.1995 Mar；95(3)：1335-44)、PPE-1-3x启动子[PCT/IL01/01059；Varda-Bloom N，Gene Ther 2001 Jun；8(11)：819-27]，TIE-1(S79347，S79346)和TIE-2(U53603)启动子[SatoTN，Proc Natl Acad Sci U S A 1993 Oct 15；90(20)：9355-8]，Endoglin启动子[Y11653；Rius C，Blood 1998 Dec 15；92(12)：4677-90]，von Willerbrand因子[AF152417；Collins CJ Proc NatlAcad Sci U S A 1987 Jul；84(13)：4393-7]，KDR/flk-1启动子[X89777，X89776；Ronicke V，Circ Res 1996 Aug；79(2)：277-85]，FLT-1启动子[D64016 AJ224863；Morishita K，：J BiolChem 1995 Nov 17；270(46)：27948-53]，Egr-1启动子[AJ245926；Sukhatme VP，Oncogene Res 1987 Sep-Oct；1(4)：343-55]，E-selectin启动子[Y12462；Collins T J Biol Chem 1991 Feb 5；266(4)：2466-73]，内皮细胞粘着分子启动子：ICAM-1[X84737；Horley KJ EMBO J 1989 Oct；8(10)：2889-96]，VCAM-1[M92431；lademarco MF，J Biol Chem 1992 Aug 15；267(23)：16323-9]，PECAM-1[AJ313330 X96849；CD31，Newman PJ，Science 1990 Mar9；247(4947)：1219-22]，血管平滑肌特异元件CArG box X53154主动脉羧肽酶样蛋白(ACLP)启动子[AF332596；Layne MD，Circ Res.2002；90：728-736]，优选表达Gene-1的主动脉[Yen-Hsu ChenJ.Biol.Chem.，Vol.276，Issue 50，47658-47663，December14，2001].
优选地，用于本发明的核酸构建体的启动子是在正在增殖的血管生成细胞或特殊表现型的血管生成细胞(例如、肿瘤)中具有功能的.特别适合用于本发明的启动子是PPE-1-3x启动子，在下述实施例部分进行进一步描述.特别适合用于本发明的载体构建体是广泛使用的腺病毒载体及其衍生物。
本发明的核酸构建体可以进一步包括附加的多核苷酸序列比如，编码选择标记或报告多肽的序列，在细菌中编码复制起点的序列，能从单个mRNA翻译出几种蛋白的序列(IRES)，用于将启动子-嵌合多肽编码区域整合到基因组中的序列，和/或通常包括在哺乳动物表述载体中的序列，例如pcDNA3、pcDNA31(+/-)，pZeoSV2(+/-)，pSecTag2，pDisplay，pEF/myc/cyto，pCMV/myc/cyto，pCR3.1，其可购自Invitrogen，pCI，其可购自Promega，pBK-RSV和pBK-CMV其可购自Stratagene，pTRES其可购自Clontech以及它们的衍生物.
优选地，本发明的核酸构建体是通过例如全身的施用途径或经口、直肠、经粘膜(尤其是经鼻的)、肠或肠胃外给药途径施用于受试者.全身施用包括肌肉内、皮下和髓内注射以及鞘内、直接心室内、静脉内、腹腔内、鼻内、眼球内注射或肿瘤内的方式.
优选地，所述的受试者是哺乳动物，更优选人、最优选患有以过度的或异常的新血管生成为特征的疾病，如以肿瘤生长、增殖性糖尿病性视网膜病、关节炎等为特征的疾病的人.
本发明的核酸构建体可以直接或作为药物组合物的一部分(活性成分)施用于受试者.
现有技术中教导有多种递送策略可用于向多种细胞类型递送裸的或载体提供的多核苷酸(参见，例如Luft(1998)J Mol Med 76(2)：75-6；Kronenwett等(1998)Blood 91(3)：852-62；Rajur等(1997)Bioconjug Chem 8(6)：935-40；Lavigne等(1997)BiochemBiophys Res Commun 237(3)：566-71 and Aoki等(1997)BiochemBiophys Res Commun 231(3)：540-5).
此处所用的“药物组合物”指由此处所述的一种或多种活性成分或药剂与其他化学成分，如生理上可接受的载体或赋形剂组成的制剂.药物组合物的目的在于便于将化合物施用于有机体.
此处所用的短语“生理上可接受的载体”和“药学上可接受的载体”可以互换使用，指不对有机体造成明显刺激、不破坏所施用的核酸构建体的生物活性和特性的载体和稀释剂.上述词组也包括佐剂.
在此术语“赋形剂”指添加到药物组合物中更加有助于活性成分施用的惰???性物质.例如，但非限定性的赋形剂包括碳酸钙、磷酸钙、各种糖和淀粉、纤维素衍生物、明胶、植物油和聚乙二醇.
药物的配制和施用技术可参见＂Remington′s PharmaceuticalSciences，＂Mack Publishing Co.，Easton，PA，最新版，引入此处作为参考.
适当的给药途径可以包括例如，经口头、直肠、经粘膜、尤其经鼻的、肠或肠胃外的递送、包括肌肉内、皮下和髓内的注射，以及鞘内的、直接心室内、静脉内、腹腔内、鼻内或眼球内注射.
可替代地，也可以将上述的药物组合物以局部的而非全身的方式施用，例如将所述的药物组合物直接注射到病人的组织区域内.在本发明的上下文中，直接施用于肿瘤组织是局部施用的相关实施例.
本发明的药物组合物可以利用本领域已知的方法制造例如通过传统的混合、溶解、造粒、裹覆糖衣、研磨、乳化、封装、包埋或冻干方法.
根据本发明所使用的药物组合物可通过传统的方式用一种或多种生理上可接受的包含赋形剂和助剂的载体制备，其有助于将活性成分加工成药学上可用的制剂中.适当的剂型取决于给药途径.
对于注射，药物组合物的活性成分可在水溶液中配制，优选地在生理上可接受的缓冲液，比如Hank溶液、林格氏溶液或生理盐水缓冲液.对于经粘膜给药，在剂型中应用适合于透过屏障的渗透剂.这种渗透剂通常是本领域中已知的.
对于经口给药，所述的药物组合物可通过把活性化合物与本领域已知的药学上可接受的载体混合配制.所述的载体可使药物组合物被配制成片剂、丸剂、糖衣丸、胶囊、液体、凝胶、糖浆、浆液、悬液等等由病人口服.口服使用的药学制剂可利用固体赋形剂制造，任选地将所得的混合物磨碎，将加工颗粒混合物，需要时在其后加入适当的助剂形成片剂或糖衣丸核心.适当的赋形剂具体说来是指填料比如糖、包括乳糖、蔗糖、甘露醇，或山梨糖醇；纤维素制剂例如玉米淀粉、小麦淀粉、大米淀粉、马铃薯淀粉、明胶、黄蓍树胶、甲基纤维素、羟丙基甲基纤维素、羧甲基纤维素钠；和/或生理上可接受的聚合物，如聚乙烯吡咯烷酮(PVP).需要时可以添加崩解剂，如交联的聚乙烯基吡咯烷酮、琼脂、或藻酸或其盐，如藻酸钠.
糖衣丸核心用适当的外壳包被.为此目的可使用浓缩的糖液和适当的有机溶剂或溶剂混合物，所述的糖液可以任选地包含阿拉伯树胶、滑石、聚乙烯基吡咯烷酮、聚羧乙烯凝胶、聚乙二醇、二氧化钛、漆溶液.可向片剂或糖衣包被添加着色剂或色素以识别或表征不同的活性化合物剂量的组合.
可用于口服的药物组合物包括由明胶制成的推合胶囊，以及由明胶和成形剂，如甘油或山梨糖醇制成的软、密闭胶囊.所述的推合胶囊可以包含活性成分及与之混合的填料例如乳糖、粘合剂例如淀粉、润滑剂例如滑石或硬脂酸镁和任选地稳定剂.在软胶囊中所述活性成分可溶解或悬浮在合适的液体，如脂肪油、液体石蜡，或液体聚乙二醇中.另外，还可以添加稳定剂.所有的口服剂型都应当制成适合于所选择的给药途径的剂量.
对于面颊给药，所述的组合物可通过传统的方式制成片剂或锭剂.
对于经鼻吸入给药，根据本发明所用的活性成分可方便地通过由加压包装或喷雾器借助于适当的推进剂例如二氯二氟甲烷、三氯氟甲烷、二氯四氟乙烷或二氧化碳以气溶胶喷射剂的形式递送。对于加压气雾剂，剂量单位可通过阀门测定来输送计定的量.药剂师所用的胶囊和药筒可以被配制成包含化合物的粉末混合物和适当的粉末基材，例如乳糖或淀粉.
此处所述的药物组合物可以被配制成用于肠胃外给药，例如弹丸注射或连续的灌注.用于注射的剂型可制成单位剂量形式，例如在安瓿中，或者任选地添加防腐剂制成多剂量包装.所述的组合物可以是悬浮液、溶液或在油性或水性载体中的乳浊液，可以包含例如悬浮、稳定和/或分散剂的制剂.
用于肠胃外给药的药物组合物包括在水中可溶解的活性制剂的水溶液.此外，活性成分的悬液可制成合适的油或水基的注射悬浮液.合适的亲脂性的溶剂或载体包括脂肪油，例如芝麻油、或合成脂肪酸酯，如油酸乙酯、三酸甘油酯或脂质体.水性的注射悬液可以包含能增加悬液粘性的物质，如羧甲基纤维素钠盐、山梨糖醇或右旋糖酐.任选地，所述悬液可同时包含适当的稳定剂或能增加活性成分溶解性的试剂以制成高浓度溶液制剂.可替代地，所述的活性成分可以制成粉剂形式，在使用前与适当的载体，例如无菌无热原的水基溶液进行配制.
也可以利用传统的栓剂基材例如可可脂或其他的甘油酯将本发明的药物组合物配制成直肠用组合物，例如栓剂或保留灌肠剂.
适用于本发明上下文中所述的药物组合物包括可实现本发明目的的有效剂量的活性成分的组合物.更具体地说，治疗有效量是指可有效地阻止、减轻或改善失调(例如渐进性的骨质损失)症状或延长被治疗的受试者存活期的活性成分(例如反义寡核苷酸)的数量.
确定治疗有效量是本领域的技术人员能力范围之内的事情，尤其是参考了此处所详细公开的内容之后。
对于任何用于本发明的方法的制剂，所述的治疗有效量可在治疗开始时通过体外和细胞培养试验估计出来。例如可通过动物模型，例如骨硬化病的鼠Src缺陷模型配制剂量(Boyce等(1992)J.Clin.Invest.90，1622-1627；Lowe等(1993)Proc.Natl.Acad.Sci.USA 90，4485-4489；Soriano等(1991)Cell 64，693-702)，以达到所需的浓度或滴度.上述信息可用于更准确地确定对人体有用的剂量.
此处所述的活性成分的毒性和治疗效果可通过标准的药学方法在体外、细胞培养物或实验动物中确定.从这些体外、细胞培养试验和动物实验中获得的数据可用于配制用于人体的剂量范围.所述的剂量可根据所用的剂型和给药途径变化.精确的配方、给药途径和剂量可以由单个的内科医师根据病人的情况确定.(参见，例如Fingl，等，1975，在＂治疗学的药理学基础＂，第一章第1页).
剂量和间隔可被单独调整到活性成分足以妨碍肿瘤发展的水平(最低有效浓度，MEC).MEC因制剂的不同而不同，但可根据体外数据进行估计.实现MEC所必需的剂量取决于个体特性和给药途径.可以利用检测试验确定血浆浓度。
根据被治疗病症的严重性和反应性，给药剂量可以是单一或复合给药剂量，疗程可持续几天到几周或实现疾病状态的减轻.
所施用组合物的数量当然还将取决于接受治疗的受试者痛苦的严重程度，给药方式，开出处方的内科医师的判断等等.
需要时，本发明的组合物可呈现为小包或配药装置，例如经FDA核准的试剂盒，其可包括含有所述活性成分的一个或多个单位的剂型.所述的包装可以，例如包含金属或塑料薄膜，例如硬质泡沫塑料包装.所述的小包或配药装置可随附给药说明书.所述的小包或配药装置还可以在包装中随附由政府机构规定的控制药品制造、使用或销售的形式的与包装有关的注意事项，所述的注意事项反映出政府机构批准了所述组合物的形式或人或兽医给药.上述的注意事项例如可标记为经美国食品和药品管理委员会批准的处方药物或批准的产品植入注意事项.也可以制备含有在适当的药学载体中制备的本发明的制剂的组合物，放置在适当的包装中，标识出治疗指定病症，如上述的详细描述.
本发明的药物组合物可以进一步包括任何附加的成分，所述的附加成分可有利于改善细胞对本发明的核酸构建体的吸收，有利于由所述核酸构建体编码的嵌合多肽在细胞中表达，或有利于所表达的嵌合多肽的活性.
例如，用基因工程化的抗体或小肽处理载体可提高EC细胞对腺病毒载体的摄入.这样的＂腺体＂治疗显示出将腺病毒构建体定向于细胞上的EGF受体的有效性(Watkins等1997，基因治疗4：1004-1012).另外Nicklin等公开了通过噬菌体展示文库分离到的一种小肽提高了载体在内皮细胞中的专一性和效率，降低了在培养基肝细胞中的表达(Nicklin等2000，血液循环102：231-237).在最近的研究中，FGF再靶定的腺病毒载体降低了tk在小鼠中的毒性(Printz等2000，人类基因治疗11：191-204).
应当理解的是尽管优选暴露于所述配体的细胞的靶向，但本发明也注重本发明的核酸构建体在未暴露于或在天然状态下不受所述配体影响的细胞中的表达.在此情况下，本发明的方法包括将所述配体施用于经转化的细胞的步骤.这种施用可通过上述的任何给药方法进行.优选地，所述的配体以细胞靶定的方式施用，例如使用与抗体偶联的靶定，从而使对编程性细胞死亡信号的激活具有高特异性.这种编程性细胞死亡活化方法在下述的实施例部分进行详细地描述.
因此，本发明提供核酸构建体、包括所述构建体的药物组合物以及利用此种构建体在以过度的或异常的血管生成为特征的组织区城中下调血管生成的方法.
由于本发明可在特异的细胞亚群中靶定表达，它还可以经修饰来治疗各种肿瘤.
因此，本发明的另一个方面提供治疗肿瘤的方法.
根据本发明该方面的方法是通过在肿瘤细胞中表达上述的嵌合多肽来实施的.
根据本发明的这一方面，所述多肽嵌合体的表达是受肿瘤特异性元件，例如但不限于胃泌素-释放肽(GRP)启动子[AF293321S3；Morimoto E Anticancer Res 2001 Jan-Feb；21(1A)：329-31]，hTERT启动子[AH007699；Gu J，Gene Ther 2002 Jan；9(1)：30-7]，己糖激酶II型启动子[AF 148512；Katabi MM，Hum Gene Ther.1999Jan 20；10(2)：155-64.]，或L-网素启动子[L05490，AH002870，MMU82611；Peng XY，Cancer Res.2001 Jun 1；61(11)：4405-13]控制.
多肽嵌合体(例如Fas-c)在肿瘤细胞中的表达激活这些细胞中的编程性细胞死亡从而导致细胞死亡，由此引起肿瘤生长减缓或阻止或者肿瘤收缩.
审查了下述实施例后，本发明的其他目的、好处和新特点对本领域的普通技术人员而言是显而易见的，下述的实施例不作为对本发明的限制.此外，下述的实施例为上述本发明的各种实施方案和下述的权利要求部分请求保护的内容提供实验支持.
实施例
参照上述的描述和下述实施例，以非限定性的方式对本发明进行描述.一般，此处所用的命名法和下述的重组DNA技术实验方法是本领域已知并通常使用的.使用标准技术进行克隆、DNA和RNA分离、扩增和提纯.通常酶促反应涉及DNA连接酶、DNA聚合酶、限制性内切酶等，按照制造商的说明进行.这些技术和各种其他技术通常是按照Sambrook等，分子克隆实验手册，冷泉港实验室冷泉港N.Y.(1989)进行.该手册以下简称＂Sambrook＂.其他的普通参考文献在此文件中提供.此处所用的方法是本领域已知的，为方便读者在此提供出来.包含在所述文献中的全部信息均引入此处作为参考.
实施例1
在内皮细胞(BAEC)和293细胞中原编程性细胞死亡基因活性的体外实验
在癌症治疗中，抗血管生成治疗靶定滋养肿瘤生长的发展中的脉管系统[Folkman J.N Engl J Med(1995)333(26)：1757-63].随着编程性细胞死亡或程序细胞死亡的研究的进展，已鉴定出了许多编码选择性和有效的细胞死亡调节剂的基因[Strasser等Annu RevBiochem(2000)69：217-45.].
本研究筛选出了几种原编程性细胞死亡基因以鉴定最适合于抗血管生成治疗的试剂.几种原编程性细胞死亡基因包括与MORT1(FADD-Fas关联的死亡结构域蛋白，GenBank登录号NM003824)，RIP(受体-相互作用-蛋白，GenBank登录号U25995)，CASH(c-FLIP，GenBank登录号AF010127)，MACH(半胱氨酸天冬氨酸蛋白酶8GenBank登录号X98172)，CPP32(半胱氨酸天冬氨酸蛋白酶3，GenBank登录号U13737)，半胱氨酸天冬氨酸蛋白酶9(U60521)和上述两＂死亡受体＂的融合体Fas-嵌合体(Fas-c)，其是由TNFR1的胞外区域和Fas[BoldinMP等J Biol Chem(1995)270(14)：7795-8，参见图1a]的跨膜和胞内区域，构成利用本领域已知的克隆技术通过PCR扩增并克隆到pcDNA3(Invitrogen，Inc.)哺乳动物表达载体中构建的.
这些原编程性细胞死亡基因构建体是随pGFP在BAEC(牛主动脉内皮细胞)和293细胞中一起共表达，其可用作非内皮细胞对照细胞.转染后24小时利用荧光显微术分析细胞.用荧光显微镜根据典型的形态学(即小而圆的形状)鉴别编程性细胞死亡的细胞(图2a-b).用电子显微技术进一步评估编程性细胞死亡表现型(图3a-f).编程性细胞死亡效果的量化表明MORT1、TNFR1和Fas-嵌合体诱导BAEC和293细胞中最高的编程性细胞死亡活性(图4a-b).在这方面鉴别编程性细胞死亡的细胞3和9很少有效，这可能是因为它们是以非活性的酶原形式存在.根据这些结果，选择Fas-嵌合体(Fas-c)基因用于制备用于抗血管生成治疗的腺病毒-载体.
实施例2
制备在经修饰的PPE-1启动子控制下的编码Fas-嵌合体的重组腺病毒
将编码全长Fas-嵌合体的cDNA亚克隆到包含经修饰的前内皮素原1启动子的质粒pPACPPE1-3x中(参见图1b).通过将该质粒与pJM17质粒共转染到人胚肾293细胞中制备腺病毒重组体.通过PCR扩增验证成功的病毒克隆(图5a)。
为了确定Fas-c在靶细胞中的表达，用指示滴度的Ad-PPE-Fas-c转导内皮BAEC细胞.转导后72小时裂解细胞，并用非还原的SDS凝胶分离细胞蛋白.用抗TNFR1抗体(Sc-7895、Santa-CruzBiotech)进行蛋白质斑迹法分析.如图5b所示，迁移到45kD处的显著带非常明显，其表达是剂量依赖性的，表明所述嵌合蛋白的正确折叠和表达.相反，在非转导内皮细胞或用对照空病毒载体转导的细胞中没有明显的带.因此，这些结果证实腺病毒-介导的Fas-c基因转移导致靶细胞中的转基因表达.
实施例3
Ad-PPE-Fas-c表达诱导内皮细胞中的编程性细胞死亡
确定了Ad-PPE-Fas嵌合体诱导内皮细胞编程性细胞死亡的能力.如图6a-b所示，前内皮素原控制的腺病毒-介导的内皮细胞转导导致明显的、大量的细胞死亡，用Ad-PPE-Fas-c(103MOI)感染的HUVEC和BAEC有经受编程性细胞死亡的粘附细胞的形态特征，包括膜气泡、圆而缩小以及从从培养皿脱离.相反，用对照病毒细胞以相同的MOI感染可维持正常的表型和生长速率.用100MOI转导的细胞仅表现出最低程度的细胞死亡(数据未显示).
通过在表达受PPE-1启动子控制的报道基因GFP的细胞中表达这一病毒来进一步评估Ad-PPE-Fas-c的细胞毒素性质.从图6c-d中明显可见，大部分转导细胞在转导后72小时表现出典型的编程性细胞死亡，而用对照病毒和Ad-PPE-GFP共转导的细胞表现正常.
利用结晶紫着色对Fas-c的细胞毒素效果进行定量.如图7所示，用Ad-PPE-Fas-c感染BAEC和HUVEC分别导致57％和65％的死亡率，而对照病毒不影响细胞的存活力.
用这一载体感染NSF(正常皮肤成纤维细胞)证明原编程性细胞死亡载体Ad-PPE-Fas对内皮细胞的专一性.这些表达低水平PPE-1[Varda-Bloom，N.等Gene Ther 8，819-27.(2001)]的细胞不受Ad-PPE-Fas-c感染的影响.相反，重组体载体Ad-CMV-Fas-c在这些细胞中诱导编程性细胞死亡.
实施例4
共同施用Ad-PPE-Fas-c受体和TNFα配体以选择性的方式增加原编程性细胞死亡效果
对TNFα增加表达Fas-c的细胞编程性细胞死亡效果进行了研究。将人TNFα添加到用Ad-PPE-Fas-c(MOI 100)病毒感染后48小时的内皮细胞培养物中.24小时后测定细胞活力.如图8所示TNFα(10ng/ml)诱导Ad-PPE-Fas-c感染的细胞存活力下降73％，而对照病毒(Ad-Luc)感染的细胞没有显著的死亡，而单独的TNFα也不对死亡率造成显著的影响.
为了证实TNFα的效果阐明了细胞专一性.用Ad-PPE-Fas-c或对照病毒感染NSF(正常皮成纤维细胞)、DA3(小鼠乳腺癌)、d122(Lewis肺癌)和B16黑素瘤细胞.48小时后，向培养物中添加TNFα，用结晶紫着色后评定细胞形态.如图9a-e所示，用Ad-PPE-Fas-c感染的非内皮细胞表现出正常的表型且不受TNF的影响.另一方面，腺病毒介导的Fas-c感染BAEC在添加TNF时导致细胞存活力明显的下降.图10a表示由CMV启动子驱动的Fas-c的非选择性编程性细胞死亡活性，其说明Ad-CMV-Fas-c对内皮细胞的TNF依赖性的编程性细胞死亡的影响.用TNF孵化后确定用指示MOI的Ad-CMV-Fas-嵌合体感染的BAEC细胞的存活力.
显著地，非内皮细胞特异性载体Ad-CMV-Fas-c引起内皮和非内皮细胞的TNFα依赖的编程性细胞死亡(图10b-d).
实施例5
Ad-PPE1-Fas-c引起B16黑素瘤在小鼠体内生长停滞
用B16黑素瘤小鼠模型检验PPE1-3x启动子驱动表达的Fas-c的抗肿瘤效果.将b16黑素瘤细胞(8 x 105)皮下注射到40c57b1/6小鼠侧腹区域.当肿瘤明显(～5x5毫米)时，将小鼠随机分成4组如下：(i)对照-盐水注射；(ii)对照病毒(包含受PPE启动子控制的荧光素酶的腺病毒)；(iii)包含受前内皮素原(PPE)启动子控制的Fas-TNF受体嵌合的基因的Ad PPE1-3x-Fas-c-病毒；和(iv)包含受非内皮特异的CMV启动子控制的Fas-TNF受体嵌合的基因的Ad-CMV-Fas-c-病毒.
用手持卡尺测量肿瘤大小(长和宽).如图111a所示与对照小鼠相比，用Ad-PPE1-3x-Fas-c或Ad-CMV-Fas-c处理的小鼠肿瘤尺寸小.在治疗过程结束时Ad-PPE1-3x-Fas-c处理的小鼠中的肿瘤的重量也更低(图11b).用Ad-PPE1-3x-Fas-c注射的小鼠显示出明显的肿瘤坏死(图11C).
尽管结合特异的实施方案对本发明进行了描述，显然，各种替代、修饰和变化对本领域的技术人员而言是显而易见的.因此本发明的宗旨是包括所有这些替代、修饰和变化，它们均落入本发明的精神和宽范围之内。本说明书中提及的全部的出版物、专利、专利申请和由其登录号确定的序列均全文引入本说明书作为参考，引入的程度如同各单独的出版物、专利、专利申请和由其登录号确定的序列特定地、单独地引入此处作为参考一样。另外本申请中所引证或鉴定的任何参考并不被看作是使这些参考成为相对于本发明的现有技术.
Claims (23)
1.一种核酸构建体，其包括：
(a)编码嵌合多肽的第一多核苷酸区域，所述的嵌合多肽包括与能够诱导内皮细胞中编程性细胞死亡的编程性细胞死亡信号分子的效应物结构域相融合的配体结合结构域；和
(b)编码选自PPE-1启动子和PPE-1-3X启动子的内皮细胞特异性顺式作用调节元件的第二多核苷酸区域；其中，选择所述的配体结合结构域使其能与存在于所说的内皮细胞中的配体相结合，而所说的配体结合到所说的配体结合结构域则激活所述编程性细胞死亡信号分子的所述效应物结构域。
2.如权利要求1所述的核酸构建体，其中，所述的配体结合结构域是细胞表面受体的配体结合结构域。
3.如权利要求2所述的核酸构建体，其中，所述的细胞表面受体选自：受体酪氨酸激酶、受体丝氨酸激酶、受体苏氨酸激酶、细胞粘着分子和磷酸酶受体。
4.如权利要求1所述的核酸构建体，其中，所述的编程性细胞死亡信号分子选自Fas、TNFR、TRAIL。
5.用如权利要求1所述的核酸构建体转化的哺乳动物细胞，条件是所述哺乳动物细胞不是原位人细胞。
6.一种核酸构建体在生产用于下调组织中血管生成的药物中用途，所述的核酸构建体包括：
(a)编码嵌合多肽的第一多核苷酸区域，所述的嵌合多肽包括与能够诱导内皮细胞中编程性细胞死亡的编程性细胞死亡信号分子的效应物结构域相融合的配体结合结构域；和
(b)编码选自PPE-1启动子和PPE-1-3X启动子的内皮细胞特异性顺式作用调节元件的第二多核苷酸区域；其中，选择所述的配体结合结构域使其能与存在于或提供到血管生成内皮细胞的配体相结合，而所述的配体结合到所说的配体结合结构域则激活所述编程性细胞死亡信号分子的所述效应物结构域。
7.如权利要求6所述的用途，其中，所述的配体结合结构域是细胞表面受体的配体结合结构域。
8.如权利要求7所述的用途，其中，所述的细胞表面受体选自：受体酪氨酸激酶、受体丝氨酸激酶、受体苏氨酸激酶、细胞粘着分子和磷酸酶受体。
9.如权利要求6所述的用途，其中，所述的编程性细胞死亡信号分子选自Fas、TNFR和TRAIL。
10.一种核酸构建体在生产用于在受试者组织中下调血管生成的药物中的用途，该核酸构建体包括：
(a)编码嵌合多肽的第一多核苷酸区域，所述的嵌合多肽包括与能够诱导内皮细胞中编程性细胞死亡的编程性细胞死亡信号分子的效应物结构域相融合的配体结合结构域；其中，选择所述的效应物结构域使其能在配体结合到所述配体结合结构域后被激活；和
(b)编码选自PPE-1启动子和PPE-1-3X启动子的内皮细胞特异性顺式作用调节元件的第二多核苷酸区域。
11.如权利要求10所述的用途，其中，所述的配体结合结构域是细胞表面受体的配体结合结构域。
12.如权利要求11所述的用途，其中，所述的细胞表面受体选自：受体酪氨酸激酶、受体丝氨酸激酶、受体苏氨酸激酶、细胞粘着分子和磷酸酶受体。
13.如权利要求10所述的用途，其中，所述的编程性细胞死亡信号分子选自Fas、TNFR和TRAIL。
14.一种在受体组织中下调血管生成的药物组合物，该药物组合物包含作为活性成分的、经设计并装配用于在血管生成细胞中产生编程性细胞死亡的核酸构建体和药学上可接受的载体，所述的核酸构建体包括：
(a)编码嵌合多肽的第一多核苷酸区域，所述的嵌合多肽包括与能够诱导内皮细胞中编程性细胞死亡的编程性细胞死亡信号分子的效应物结构域相融合的配体结合结构域；和
(b)编码选自PPE-1启动子和PPE-1-3X启动子的内皮细胞特异性顺式作用调节元件的第二多核苷酸区域；其中，选择所述的配体结合结构域使其能与存在于所述血管生成细胞中的配体相结合，而所说的配体结合到所说的配体结合结构域则可激活所述编程性细胞死亡信号分子的所述效应物结构域。
15.如权利要求14所述的药物组合物，其中，所述的配体结合结构域是细胞表面受体的配体结合结构域。
16.如权利要求15所述的药物组合物，其中，所述的细胞表面受体选自：受体酪氨酸激酶、受体丝氨酸激酶、受体苏氨酸激酶、细胞粘着分子和磷酸酶受体。
17.如权利要求14所述的药物组合物，其中，所述的编程性细胞死亡信号分子选自Fas、TNFR和TRAIL。
18.一种核酸构建体在生产用于治疗与过度新血管形成相关的疾病或病症的药物中的用途，所述的核酸构建体包括：
(a)编码嵌合多肽的第一多核苷酸区域，所述的嵌合多肽包括与能够诱导内皮细胞中编程性细胞死亡的编程性细胞死亡信号分子的效应物结构域相融合的配体结合结构域；和
(b)编码选自PPE-1启动子和PPE-1-3X启动子的内皮细胞特异性顺式作用调节元件的第二多核苷酸区域；其中，选择所述的配体结合结构域使其能与存在于或提供到血管生成内皮细胞的配体相结合，而所述配体结合到所述配体结合结构域则激活所述编程性细胞死亡信号分子的所述效应物结构域。
19.如权利要求18所述的用途，其中，所述的配体结合结构域是细胞表面受体的配体结合结构域。
20.如权利要求19所述的用途，其中，所述的细胞表面受体选自：受体酪氨酸激酶、受体丝氨酸激酶、受体苏氨酸激酶、细胞粘着分子和磷酸酶受体。
21.如权利要求18所述的用途，其中，所述的编程性细胞死亡信号分子选自Fas、TNFR和TRAIL。
22.一种核酸构建体在生产用于治疗受试者体内的肿瘤的药物中的用途，所述的核酸构建体包括：
(a)编码嵌合多肽的第一多核苷酸区域，所述的嵌合多肽包括与能够诱导内皮细胞中编程性细胞死亡的编程性细胞死亡信号分子的效应物结构域相融合的配体结合结构域；和
(b)编码选自PPE-1启动子和PPE-1-3X启动子的内皮细胞特异性顺式作用调节元件的第二多核苷酸区域，其中，选择所述的配体结合结构域使其能与存在于或提供到所述肿瘤的血管生成内皮细胞中的配体相结合，所说配体结合到所说配体结合结构域则激活所说编程性细胞死亡信号分子的所述这效应物结构域。
23.一种用于下调受试者的组织血管生成的包含核酸构建体的药物组合物，该核酸构建体包括：
(a)编码嵌合多肽的第一多核苷酸区域，所述的嵌合多肽包括与能够诱导内皮细胞中编程性细胞死亡的编程性细胞死亡信号分子的效应物结构域相融合的配体结合结构域，其中选择所述的效应物结构域使其能在配体与所述的配体结合结构域结合之后被活化；和
(b)编码选自PPE-1启动子和PPE-1-3X启动子的内皮细胞特异性顺式作用调节元件的第二多核苷酸区域。
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Also Published As
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| WO2003033514A1 (en) | 2003-04-24 |
| CA2463816A1 (en) | 2003-04-24 |
| JP2005532031A (ja) | 2005-10-27 |
| DE60237777D1 (de) | 2010-11-04 |
| EP2277887A3 (en) | 2011-02-16 |
| ATE481986T1 (de) | 2010-10-15 |
| US8415318B2 (en) | 2013-04-09 |
| EP2277887A2 (en) | 2011-01-26 |
| CN101570764A (zh) | 2009-11-04 |
| EP2223932A1 (en) | 2010-09-01 |
| US7989427B2 (en) | 2011-08-02 |
| CN1602315A (zh) | 2005-03-30 |
| EP1436313A1 (en) | 2004-07-14 |
| AU2002307793B2 (en) | 2007-01-25 |
| US20040197860A1 (en) | 2004-10-07 |
| US20110251122A1 (en) | 2011-10-13 |
| EP1436313A4 (en) | 2006-05-03 |
| CA2463816C (en) | 2014-07-08 |
| KR100866117B1 (ko) | 2008-10-31 |
| US8916378B2 (en) | 2014-12-23 |
| ZA200402756B (en) | 2006-05-31 |
| US20130272998A1 (en) | 2013-10-17 |
| US20080305088A1 (en) | 2008-12-11 |
| US7585666B2 (en) | 2009-09-08 |
| KR20040094391A (ko) | 2004-11-09 |
| NZ532348A (en) | 2006-06-30 |
| MXPA04003514A (es) | 2004-07-23 |
| EP1436313B1 (en) | 2010-09-22 |
| JP4173446B2 (ja) | 2008-10-29 |
| HK1067641A1 (en) | 2005-04-15 |
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