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

我们以前已经描述了胸苷激酶（TK）缺陷型I型单纯疱疹病毒（HSV-1）突变体d12。卡尔普，其中平滑肌特异性钙调蛋白启动子驱动编码病毒基因的必需反式激活因子（ICP 4）的RS 1基因的表达（Yamamura等，Cancer Res.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条件复制溶瘤HSV-1（d12.CALPΔRR），其中平滑肌特异性钙调蛋白转录调控序列通过使用内部核糖体进入位点（IRES 2）的双顺反子表达驱动RS 1基因和增强型绿色荧光蛋白（EGFP）cDNA。工程HSV-1是ICP 4无效突变体d120的衍生物，携带完整的TK基因和编码核糖核苷酸还原酶（ICP 6）大亚基（病毒复制必需酶）的U_L39基因中的插入突变。d12.CALPΔRR还含有大肠杆菌lacZ ...更多信息 在内在U_L39基因启动子控制下的基因以追踪病毒复制。我们检测了低感染复数（0.01 ~ 0.1空斑形成单位/细胞）下d12.CALPΔRR对手术切除的人平滑肌肉瘤细胞（有或无钙调蛋白表达）原代培养物的细胞病变效应。d12.CALPΔRR优先杀伤表达钙调蛋白的肿瘤细胞。在体内研究中，将15只携带人子宫平滑肌肉瘤（平均肿瘤体积为44 mm^3）的动物（BALB/c裸鼠）随机分组，在异种移植后第21、27和34天分别给予1 × 10^7空斑形成单位（PFU）的d12.CALPΔRR/100 mm^3肿瘤体积或单独给予培养基治疗3次。病毒治疗组在第39天显示出肿瘤生长的显著抑制（肿瘤体积;平均值±S.E.，1281±153，n=8 vs. 342±32 mm^3，n=7）。每4-5天静脉内注射5 × 10^7 PFU d12.CALPΔRR治疗5次，在第42天导致稳定和显著的肿瘤生长抑制（肿瘤体积;平均值±S.E.，1708±199与521±111 mm ^3（n=5）。我们已经在小鼠中测试了d12.CALPΔRR的安全性。静脉接种2 × 10^7 PFU d12.CALPΔRR的BALB/c nu/nu小鼠（n=23 × 26）存活超过2个月，血液化学值[肝、肾和代谢（葡萄糖和脂质）功能]无明显异常。单次静脉注射2 x 10^7 PFU后，血清样本分析显示门静脉血中的活性病毒在15 min时显著回收（4-6只小鼠的平均值; 1.3 x 10^5 PFU/ml），在第1小时内显著降低（673 PFU/ml），24 h及以后不存在。CALP ΔRR静脉注射后24 h的组织化学分析显示，LacZ和ICP 4在肝实质和肺和脾细胞中的阳性表达很少，没有单个细胞同时表达LacZ和ICP 4，表明没有病毒复制。实际上，从注射后24小时收获的脑、肺、肝和脾组织制备的提取物证明不存在活性病毒。此外，在注射后72小时检查的所有器官中均未观察到LacZ和ICP 4染色。在光镜下，这些标本中没有整体坏死和炎症。相反，通过直接瘤内注射或从尾静脉静脉注射接受2 x 10^7 PFU的所有平滑肌肉瘤异种移植物中积累了强LacZ和ICP 4表达。此外，半定量PCR分析显示，2 × 10^7 PFU静脉注射不会导致病毒DNA（LacZ和糖蛋白E的基因）在三叉神经节中持续不超过1周。最后，腹腔注射阿昔洛韦（30 mg/kg/天）7天可显著抑制平滑肌肉瘤异种移植物中的病毒复制，这通过ICP 4蛋白表达来评估（ICP 4阳性细胞;对照组vs.阿昔洛韦治疗组，300±30 vs. 53±27/mm^2，n=5，p<0.0005）。我们的结论是，这种新的抗平滑肌肉瘤剂d12.CALPΔRR在小鼠肿瘤研究中有效的剂量或以上，可以安全地通过静脉和直接肿瘤注射给药。d12.CALPΔRR应进一步研究，作为通过全身血管注射治疗肉瘤肿瘤转移至远端器官的具有挑战性的治疗方法。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