Gene therapy specific for lung cancer using cell-type specific promoter

使用细胞类型特异性启动子进行肺癌特异性基因治疗

• 批准号: 09670612
• 负责人: HAYASHI Seiji
• 金额: $ 1.6万
• 依托单位: OSAKA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670612/
• 关键词: Lung cancer; HSV-tk; CEA promoter; Cre; loxP system; myc oncogene; ganciclovir (GCV); nonproliferating adenoviral vector; 非増殖型アデノウイルスベクター

(1) Gene therapy specific for CEA-producing lung cancerRejection of CEA-producing tumors was not observed by intra-tumoral injection with a recombinant adenoviral vector (Ad.) expressing the HSV-tk gene under the CEA promoter (Ad.CEA-TK) followed by ganciclovir (GCV) treatment in vivo.It is conceivable that the low activity of the CPA promoter results in insufficient expression of the HSV-tk gene in cancer cells as well as in insignificant antitumor effects.To obtain enhanced expression of the HSV-tk gene exclusively in tumor cells and subsequent significant in vivo antitumor effects, we applied the Cre/loxP system to HSV-tk/GCV therapy for CEA-producing cancer.We constructed an Ad producing Cre recombinase driven by the CEA promoter (Ad.CEA-Cre) and another Ad designed for inducible expression of the HSV-tk gene by Cre (Ad.CAG-loxP-TK).Co-infection by these Ads rendered a human CEA-producing cancer cell line 8.4-fold more sensitive to GCV compared with infection by Ad.CEA-TK alone.On … More the other hand, co-infection with these Ads did not significantly change GCV sensitivity of CEA-non-producing cells.Intra-tumoral injection of Ad.CEA-Cre combined with Ad.CAG-loxP-TK followed by GCV treatment completely eradicated CEA-producing tumors established in the subcutis of 6 out of 7 athymic mice, whereas injection of Ad.CEA-TK alone with GCV administration at most retarded the growth of inoculated tumors.These results suggest distinct advantages of the Cre/loxP system applied in the conventional cell type-specific gene therapy against cancer.We are now evaluating the efficacy and side effects of this system in the peritonitis carcinomatosa model in mice, mimicking pleuritis for lung cancer in future clinical applications to human beings.(2) Gene therapy specific for Myc-overexpressing small cell lung cancer (Myc-SCLC)We previousely reported that an Ad. containing the HSV-tk gene ligated with the Myc-binding motif (Ad.Myc-TK) showed a cell-type specific expression of the HSV-tk gene in Myc-SCLC cell lines.We analyzed the efficacy of the intraperitoneal injection of Ad.Myc-TK followed by GCV treatment on Myc-SCLC inoculated in the peritoneal cavity of athymic mice.This treatment successfully reduced the weight of tumors to one fourteenth of that of untreated mice and completely eradicated tumors in 2 of 8 mice. Less

(1)针对CEA产生肺癌的特异性基因治疗在CEA启动子（Ad.CEA- tk）下，用表达HSV-tk基因的重组腺病毒载体（Ad.）在体内注射更昔洛韦（GCV）后，未观察到对CEA产生肿瘤的排斥反应。可以想象，CPA启动子的低活性导致HSV-tk基因在癌细胞中表达不足，抗肿瘤作用不显著。为了获得HSV-tk基因在肿瘤细胞中特异性表达的增强和随后显著的体内抗肿瘤效果，我们将Cre/loxP系统应用于HSV-tk/GCV治疗cea -产生癌。我们构建了一个由CEA启动子驱动的产生Cre重组酶的Ad （Ad.CEA-Cre）和一个由Cre诱导表达HSV-tk基因的Ad （Ad. cag - loxp -tk）。与Ad感染相比，这些Ad联合感染使人产生cea的癌细胞株对GCV的敏感性提高8.4倍。CEA-TK孤单。另一方面，与这些Ads共同感染没有显著改变cea -非产生细胞的GCV敏感性。肿瘤内注射Ad。CEA-Cre联合Ad。CAG-loxP-TK在GCV治疗后完全根除了7只胸腺发育小鼠中6只皮下产生的cea肿瘤，而注射Ad。CEA-TK单独加GCV最多能延缓接种肿瘤的生长。这些结果表明Cre/loxP系统在传统的细胞类型特异性基因治疗癌症方面具有明显的优势。我们目前正在对该系统在小鼠腹膜炎癌性模型中的疗效和副作用进行评估，以模拟肺癌胸膜炎在未来对人类的临床应用。(2)针对myc过表达小细胞肺癌（Myc-SCLC）的特异性基因治疗。含有与myc结合基序连接的HSV-tk基因（Ad.Myc-TK）在Myc-SCLC细胞系中显示HSV-tk基因的细胞型特异性表达。我们分析了腹腔注射Ad的疗效。接种于胸腺小鼠腹腔的Myc-SCLC接种Myc-TK后再用GCV治疗。这种治疗成功地将肿瘤的重量减少到未治疗小鼠的1 / 14，并且在8只小鼠中有2只完全根除了肿瘤。少

项目成果

Osaki T et al.: "Gene therapy for carcinoembryonic antigen-producing human lung cancer cells by cell type-specific expression of herpes simplex virus thymidine kinase gene." Cancer Res Oct. 15 ; 54(20). 5258-61 (1994)

Osaki T 等人：“通过单纯疱疹病毒胸苷激酶基因的细胞类型特异性表达，对产生癌胚抗原的人肺癌细胞进行基因治疗。”

Ueno K et al.: "Cloning and tissue expression of cDNAs from chromosome 5q21-22 which is frequently deleted in advanced lung cancer." Hum Genet. 102(1). 63-68 (1998)

Ueno K 等人：“染色体 5q21-22 cDNA 的克隆和组织表达，该染色体在晚期肺癌中经常被删除。”

Kumagai T et al.: "Eradication of Myc-overexpressing small cell lung cancer cells transfected with herpes simplex virus thymidine kinase gene containing Myc-Max response elements." Cancer Res Jan. 15 ; 56(2). 354-358 (1996)

Kumagai T 等人：“用含有 Myc-Max 反应元件的单纯疱疹病毒胸苷激酶基因转染的 Myc 过表达小细胞肺癌细胞的根除。”

Tachibana I et al.: "A 100-kDa protein tyrosine phosphorylation is concurrent with beta 1 integrin-mediated morphological differentiation in neuroblastoma and small cell lung cancer cells." Exp Cell Res Sep. 15 ; 227(2). 230-239 (1996)

Tachibana I 等人：“在神经母细胞瘤和小细胞肺癌细胞中，100 kDa 蛋白酪氨酸磷酸化与 β1 整合素介导的形态分化同时发生。”

Kumagai T et al.: "Eradication of My c-overexpressing small cell lung cancer cells transfected with herpes simplex virus thymidine kinase gene containing Myc-Max response elements." Canser Res. 15 ; 56(2). 354-358 (1996)

Kumagai T 等人：“用含有 Myc-Max 反应元件的单纯疱疹病毒胸苷激酶基因转染的 My c 过表达小细胞肺癌细胞的根除。”