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，但在正常脑组织中没有。在恶性胶质瘤中，端粒酶存在的肿瘤比端粒酶不存在的肿瘤具有更多的恶性表型。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。为了有效抑制端粒酶的功能，我们采用了2- 5a（2', 5'-寡聚腺苷酸）反义体系。2-5A是干扰素通过激活RNase L导致单链RNA切割的一个途径的中介。RNase L通过将2-5A与反义连接，特异有效地降解目标RNA。在NIH资助下，我们合成了2-5A连接的人类端粒酶RNA成分（hTR）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-anti-hTR治疗4天后，细胞大量凋亡，端粒长度急剧缩短。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-anti-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。基于我们之前的研究结果，我们假设2-5A-anti-hTR是治疗表达端粒酶的恶性胶质瘤的一种有前景的药物。然而，以下问题仍有待回答。首先，哪些分子途径在2- 5a抗htr诱导的细胞凋亡中起关键作用？二是如何增强2-5A-anti-hTR对脑肿瘤的治疗作用？第三，哪些以2-5A-anti-hTR为基础的方案对脑肿瘤的联合治疗效果显著？这项建议的目的就是要解决这些问题。具体目的是：1：表征2- 5a抗htr诱导的细胞凋亡的分子途径。使用微阵列分析，我们将确定哪些基因参与了2- 5a抗htr诱导的细胞死亡。我们还将确定(i)线粒体相关细胞死亡信号通路的参与以及（ii） 2-5A-anti-hTR对端粒3'悬空或端粒酶阳性星形细胞是否具有致瘤性的影响。2：明确2- 5a -anti- htr对流增强给药（CEDD）对裸鼠脑内肿瘤的作用。我们将对CEDD进行优化，并比较CEDD与2-5A-anti-hTR注射剂治疗颅内肿瘤的效果。3：明确基于2-5A-anti-hTR联合治疗的体内外效果。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，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.