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

描述(申请人提供)：端粒酶，一种核糖核蛋白酶，在绝大多数恶性胶质瘤中检测到，但在正常脑组织中检测不到。在恶性胶质瘤中，有端粒酶的肿瘤往往比没有端粒酶的肿瘤有更多的恶性表型。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的方法。为了有效地抑制端粒酶功能，我们采用了2-5A(2‘，5’-寡腺苷)反义系统。2-5A是干扰素作用途径之一，通过激活核糖核酸酶L，导致单链核糖核酸断裂。核糖核酸酶L通过将2-5A连接到反义核糖核酸上，特异性和有效地降解靶向核糖核酸。在美国国立卫生研究院的资助下，我们合成了与2-5A相连的抗人端粒酶RNA组分的反义寡核苷酸(2-5A-anti-HTR)，并研究了其对恶性胶质瘤细胞的抗肿瘤作用。2-5A-反义hTR作用4天后，端粒长度明显缩短，诱导细胞大量凋亡。相反，缺乏端粒酶的星形胶质细胞和成纤维细胞等正常细胞对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。根据我们以前的研究结果，我们推测2-5A-反义htr是治疗表达端粒酶的恶性胶质瘤的一种有前途的药物。然而，以下问题仍有待回答。首先，在2-5A-anti-HTR诱导的细胞凋亡中起关键作用的分子途径是什么？第二，如何提高2-5A-抗HTR对脑内肿瘤的疗效？第三，以2-5A-抗HTR为基础的哪些方案对脑内肿瘤有显著的联合作用？这项提议的目的是解决这些问题。其具体目的是：1：研究2-5A-反义HTR诱导细胞凋亡的分子途径。利用基因芯片技术，我们将确定哪些基因参与了2-5A-反义HTR诱导的细胞死亡。我们还将确定(I)线粒体相关的细胞死亡信号通路的参与以及(Ii)2-5A-anti-HTR对端粒3‘悬垂或端粒酶阳性星形胶质细胞的影响，无论是否具有致瘤性。2：研究2-5A-反义HTR对流增强给药对裸鼠脑内肿瘤的治疗作用。我们将优化CEDD，然后比较CEDD和2-5A-抗HTR团注治疗脑肿瘤的效果。3.明确以2-5A-抗-HTR为基础的联合治疗的体内外效应。我们将确定(I)2-5A-抗htr与凋亡诱导剂(顺铂、紫杉醇或BCNU)联合作用的分子途径，以及(Ii)联合治疗对脑内肿瘤的影响。

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.