Treatment of Malignant Gliomas with 2-5A-anti-hTR

描述（由申请人提供）：端粒酶是一种核糖核蛋白酶，在绝大多数恶性胶质瘤中检测到，但在正常脑组织中未检测到。在恶性胶质瘤中，有端粒酶的肿瘤比没有端粒酶的肿瘤更倾向于具有恶性表型。因此，我们的长期目标是探索一种新的端粒酶靶向治疗恶性胶质瘤。为了有效地抑制端粒酶的功能，我们采用了2-5A（2 '，5'-oligoadenylate）反义系统。2-5A是干扰素作用的一个途径的介体，通过激活RNase L，导致单链RNA切割。通过将2-5A与反义连接，RNase L特异性地且有效地降解靶向RNA。本研究利用NIH的资助，合成了人端粒酶RNA组分（human telomerase RNA component，hTR）2-5A反义寡核苷酸（2-5A-anti-hTR），并研究了其对恶性胶质瘤细胞的抗肿瘤作用。用2- 5A-抗-hTR处理4天诱导大量凋亡，然后端粒长度严重缩短。相反，缺乏端粒酶的正常细胞如星形胶质细胞和成纤维细胞对2- 5A-抗-hTR不敏感。瘤内注射2- 5A-抗-hTR治疗裸鼠皮下或脑内肿瘤是有效的。基于我们以前的结果，我们假设2- 5A-抗-hTR是一种有前途的治疗恶性胶质瘤表达端粒酶的药物。然而，以下问题仍有待回答。首先，什么样的分子通路在2- 5A-抗-hTR诱导的细胞凋亡中起关键作用？第二，我们如何增强2- 5A-抗-hTR对脑内肿瘤的作用？第三，哪些基于2- 5A-抗-hTR的方案对脑内肿瘤显示出显著的联合作用？本提案的目的是解决这些问题。具体目的是：1、研究2- 5A-抗-hTR诱导细胞凋亡的分子途径。使用微阵列分析，我们将鉴定哪些基因参与2- 5A-抗-hTR诱导的细胞死亡。我们还将确定（i）与细胞死亡相关的信号通路的参与和（ii）2- 5A-抗hTR对端粒3'突出端或端粒酶阳性星形胶质细胞（有或无致瘤性）的影响。2：确定2- 5A-抗-hTR的对流增强药物递送（CEDD）对裸鼠脑内肿瘤的作用。我们将优化CEDD，然后比较CEDD和团注2- 5A-抗-hTR对脑内肿瘤的作用。3：定义基于2- 5A-抗-hTR的组合疗法的体外和体内作用。我们将确定（i）2- 5A-抗hTR和凋亡诱导剂（顺铂、紫杉醇或BCNU）联合作用的分子途径和（ii）联合治疗对脑内肿瘤的作用。

DESCRIPTION (provided by applicant): Telomerase, a ribonucleoprotein enzyme, is detected in the vast majority of malignant gliomas, but not in normal brain tissues. In malignant gliomas, tumors with telomerase tend to have more malignant phenotype than those without telomerase. Therefore, our long-term goal is to explore a novel telomerase-targeting therapy for malignant gliomas. To inhibit telomerase function effectively, we have adopted the 2-5A (2', 5'-oligoadenylate) antisense system. 2-5A is a mediator of one pathway of interferon actions by activating RNase L, resulting in single-stranded RNA cleavage. By linking 2-5A to antisense, RNase L degrades the targeted RNA specifically and effectively. With the grant supported by NIH, we synthesized the antisense oligonucleotide against human telomerase RNA component (hTR) linked to 2-5A (2-5A-anti-hTR) and investigated its anti-tumor effect on malignant glioma cells. Treatment with 2-5A-anti-hTR for 4 days induced a massive apoptosis before a telomere length shortened critically. In contrast, normal cells such as astrocytes and fibroblasts lacking telomerase were insensitive to 2-5A-anti-hTR. Treatment of subcutaneous or intracerebral tumors in nude mice with intratumoral injections of 2-5A-anti-hTR was effective. Based on our previous results, we hypothesize that 2-5A-anti-hTR is a promising agent for the treatment of malignant gliomas expressing telomerase. However, the following questions remain to be answered. First, what molecular pathways play a key role in 2-5A-anti-hTR-induced apoptosis? Second, how can we enhance the effect of 2-5A-anti-hTR on intracerebral tumors? Third, which regimens based on 2-5A-anti-hTR show a significant combination effect on intracerebral tumors? The aim of this proposal is to address these issues. The specific aims are to: 1: Characterize the molecular pathways of 2-5A-anti-hTR-induced apoptosis. Using the microarray assay, we will identify which genes are involved in 2-5A-anti-hTR-induced cell death. We will also determine (i) the involvement of the mitochondria-associated cell death signaling pathways and (ii) the effect of 2-5A-anti-hTR on telomere 3' overhang or telomerase-positive astrocytes with or without tumorigenicity. 2: Define the effect of convection-enhanced drug delivery (CEDD) of 2-5A-anti-hTR on intracerebral tumors in nude mice. We will optimize CEDD and then compare the effect of CEDD and bolus injections of 2-5A-anti-hTR on intracerebral tumors. 3: Define the in vitro and in vivo effect of combination therapy based on 2-5A-anti-hTR. We will determine (i) the molecular pathways underlying the combination effect of 2-5A-anti-hTR and apoptosis-inducing agent (cisplatin, paclitaxel, or BCNU) and (ii) the effect of combination therapy on intracerebral tumors.