Development of targeted gene therapy for incurable sarcoma using a novel replication-selective and oncolytic viral vector

使用新型复制选择性和溶瘤病毒载体开发针对无法治愈的肉瘤的靶向基因疗法

基本信息

批准号：
15390468
负责人：
TAKAHASHI Katsuhito
金额：
$ 9.41万
依托单位：
Osaka Medical Center for Cancer & Cardiovascular Diseases
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

We have previously described a thymidine kinase (TK)-defective type I herpes simplex virus (HSV-1) mutant d12. CALP in which smooth muscle-specific calponin promoter drives expression of the RS1 gene encoding an essential trans-activating factor (ICP4) for viral genes (Yamamura et al. Cancer Res. 61 ; 3969-3977, 2001). In order to improve efficacy and safety for pre-clinical and clinical testing, we have developed a new conditionally replicating oncolytic HSV-1 (d12.CALPΔRR) in which smooth muscle-specific calponin transcriptional regulatory sequence drives the RS1 gene and the enhanced green fluorescent protein (EGFP) cDNA via bicistronic expression using the internal ribosomal entry site (IRES2). The engineered HSV-1 is a derivative of ICP4-null mutant d120 carrying the intact TK gene and an insertional mutation in the U_L39 gene encoding a large subunit of ribonucleotide reductase (ICP6), an essential enzyme for viral replication. d12.CALPΔRR also contains the Escherichia coli lacZ … More gene under the control of the intrinsic U_L39 gene promoter to trace viral replication. We examined the cytopathic effects of d12.CALPΔRR at low multiplicity of infection (0.01〜0.1 plaque-forming unit/cell) on primary cultures from surgically removed human leiomyosarcoma cells with or without calponin expression. d12.CALPΔRR preferentially killed calponin- expressing tumor cells. For in vivo studies, 15 animals (BALB/c nude mice) harboring human uterine leiomyosarcoma (mean tumor volume 44 mm^3) were randomly divided and treated three times intraneoplastically with either 1 x 10^7 plaque-forming units (PFU) of d12.CALPΔRR/100 mm^3 of tumor volume or medium alone on days 21, 27 and 34 after xenograft transplantation. The viral treatment group showed significant inhibition of tumor growth by day 39 (tumor volume ; mean±S.E., 1281±153, n=8 vs. 342±32 mm^3, n=7). Treatment with 5 x 10^7 PFU of d12.CALPΔRR intravenously injected five times in every 4-5 days resulted in stable and significant inhibition of tumor growth by day 42 (tumor volume ; mean±S.E., 1708±199 vs. 521±111 mm^3 n=5). We have tested the safety of d12.CALPΔRR in mice. BALB/c nu/nu mice (n=23〜26) inoculated intravenously with 2 x 10^7 PFU of d12.CALPΔRR survived for over 2 months with no apparent abnormalities in blood chemical values [liver, kidney and metabolic (glucose and lipid) functions]. After single intravenous injection of 2 x 10^7 PFU, analysis of serum samples showed significant recovery of active viruses in the portal vein blood at 15 min (mean value of 4-6 mice ; 1.3 x 10^5 PFU/ml), which significantly decreased over the first hour (673 PFU/ml) and was absent from 24 h and beyond. Histochemical analysis at 24 h post d12.CALPΔRR intravenous injection demonstrated scant positive expression of LacZ and ICP4 in liver parenchyma and cells in lung and spleen with no single cell co-expressing both LacZ and ICP4, indicating the absence of viral replication. Indeed, extracts prepared from brain, lung, liver and spleen tissues harvested at 24 h post injection demonstrated absence of active viruses. Also, no LacZ and ICP4 staining were observed in all organs examined at 72 h post injection. There was no overall necrosis and inflammation at light microscopic level in these specimens. Conversely, strong LacZ and ICP4 expression was accumulated in all leiomyosarcoma xenografts that received 2 x 10^7 PFU via direct intratumoral injection or intravenous injection from tail vein. Furthermore, semi-quantitative PCR analysis revealed that 2 x 10^7 PFU intravenous injection did not result in persistence of the viral DNA (genes for LacZ and Glycoprotein E) for no more than 1 week in the trigeminal nerve ganglia. Finally, intraperitoneal administration of aciclovir (30 mg/kg/day) for 7 days significantly inhibits viral replication in the leiomyosarcoma xenografts as assessed by ICP4 protein expression (ICP4-positive cells ; control vs. aciclovir treatment, 300±30 vs. 53±27/mm^2, n=5, p<0.0005). We conclude that this novel anti-leiomyosarcoma agent d12.CALPΔRR at or above doses that were efficacious in mouse tumor studies can be delivered safely both intravenous and direct tumor injection. d12.CALPΔRR should be investigated further as a challenging therapy for metastasis of sarcoma tumors to remote organs via systemic vascular injection. Less

我们先前已经描述了themidine激酶（TK） - 缺陷I型单纯疱疹病毒（HSV-1）突变体D12。 Yamamura等人。）。在您的内在u_l39基因启动子的控制下，我们检查了d12.calpΔrr的细胞病变作用，在低多重性感染（0.01〜0.1 〜0.1 plaque-0.1 plaque-0.1）带有或与D12的细胞。 d12.calpΔrr/100 mm ^3的斑块形成单位（PFU）在第34天后第21天单独使用肿瘤体积或单独的培养基。平均±153，n = 8 vs. 342±32 mm^3，n = 7）。 1708±199 vs。d12.小鼠的calpΔrr。血半疗法[肝，肾脏和代谢（葡萄糖和脂质）功能]在单次静脉注射2 x 10^7 PFU后，对样品的分析显示，在门静脉血液中显示出明显的核心（平均值15分钟） 6只小鼠； 1.3 x 10^5 PFU/mL），在第一个小时（673 PFU/mL）中显着降低，并且在24小时的24 h组织化学分析中不存在24小时的ravenous注入肺和脾脏中没有单一共表达LACZ和ICP4的细胞，这确实是病毒复制的。在注射后72小时内观察到的所有器官都没有整体坏死和炎症在tecime ns中，在所有平滑肌肉瘤中都可以通过2 x 10^7 pfu，在tecime ns的光中炎症。静脉注射的静脉注射不会导致病毒DNA（lacz和糖蛋白E的基因）在三叉神经神经节中不超过1周。通过ICP4蛋白质表达（ICP4阳性细胞）评估的平滑肌肉瘤Xenograghs中的离子±30 vs. 53±27/mm^2，n = 5，p <0.0005）。 D12.在小鼠肿瘤研究中，可以对静脉注射和直接的肿瘤注射进行粉碎