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

我们之前已经描述了胸苷激酶(TK)缺陷的I型单纯疱疹病毒(HSV-1)突变体d12。在CALP中，平滑肌特异性钙蛋白启动子驱动RS1基因的表达，该基因编码病毒基因的基本反式激活因子(ICP4)(Yamamura等人)。癌症研究61；3969-3977,2001)。为了提高临床前和临床试验的有效性和安全性，我们开发了一种新的有条件复制的溶瘤单纯疱疹病毒1型(d12.CALPΔRR)，其中平滑肌特异的钙蛋白转录调控序列利用内部核糖体进入位点(IRES2)通过双顺反子表达来驱动rs1基因和增强型绿色荧光蛋白(EGFP)的cDNA.工程HSV-1是携带完整TK基因的ICP4缺失突变体D120的衍生物，U_L39基因插入突变编码核糖核苷酸还原酶(ICP6)的大亚基，ICP6是病毒复制的关键酶。D12.CALPΔRR还含有大肠杆菌lacZ…更多的基因在固有的U_L39基因启动子的控制下跟踪病毒的复制。我们检测了d12.CALPΔRR在低感染复数(0.0 1~0.1个空斑形成单位/细胞)下对原代培养的有或不表达Calponin的人平滑肌肉瘤细胞的细胞病变效应。D12.CALPΔRR优先杀伤表达钙蛋白的肿瘤细胞。在体内实验中，15只荷人子宫肌肉瘤(平均肿瘤体积44 mm^3)的动物(BALB/c裸小鼠)随机分为3组，分别于移植后第21、27、34天分别以1×10~(-7)空斑形成单位(D12)、CALPΔRR/100 mm~(-3)肿瘤体积或单纯培养基瘤内治疗3次。至第39天，病毒治疗组肿瘤生长明显受到抑制(肿瘤体积平均为1281±153，n=8比342±32 mm^3，n=7)。用5×10~(-7)pfu/d的CALP-Δ-RR治疗，每4~5天静脉注射5次，至第42天肿瘤生长得到稳定而显著的抑制(平均肿瘤体积，1708±199vs.521±111 mm^3 n=5)。我们已经在小鼠身上测试了d12.CALPΔRR的安全性。BALB/c nu/nu小鼠(n=23~26)静脉接种2×10~(-7)pfu的d12，CALPΔRR存活2个月以上，血生化指标[肝、肾及代谢(糖、脂)功能]无明显异常。单次静脉注射2×10~(-7)pfu后，15min门静脉血中活性病毒显著恢复(4~6只小鼠平均1.3×10~(-5)pfu/m l)，1小时后显著下降(673 pfu/m l)，24 h及以后消失。静脉注射CALPΔRR后24 h的组织化学分析显示，LacZ和ICP4在肝实质和肺、脾细胞中几乎没有阳性表达，没有单个细胞同时表达LacZ和ICP4，表明病毒没有复制。事实上，从注射后24小时采集的脑、肺、肝和脾组织中提取的提取物显示没有活性病毒。注射后72 h，所有脏器均未见LacZ和ICP4染色。光镜下未见全部坏死和炎症反应。相反，所有接受2×10~(-7)PFU直接瘤内注射或尾静脉注射的平滑肌肉瘤移植瘤均有LacZ和ICP4的强表达。半定量聚合酶链式反应分析显示，静脉注射2×10~(-7)PFU不会导致病毒DNA(LacZ基因和糖蛋白E基因)在三叉神经节中持续不超过1周。最后，通过ICP4蛋白表达评估，阿昔洛韦(30 mg/kg/天)连续7天显著抑制移植瘤中的病毒复制(ICP4阳性细胞；对照组与阿昔洛韦治疗组，300±30vs.53±27/mm^2，n=5，p&lt；0.0005)。我们认为，在小鼠肿瘤研究中有效的剂量或以上剂量的这种新型抗平滑肌肉瘤药物d12.CALPΔRR可以安全地静脉注射和直接注射肿瘤。D12.CALPΔRR可作为全身血管注射治疗肉瘤转移至远处器官的一种具有挑战性的治疗方法，值得进一步研究。较少

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