Cancer dual-targeting of an infectivity-enchanced CRAd

描述（由申请人提供）：5型腺病毒（Ad 5）的许多特征使其成为适用于多种癌症治疗方法的最佳基因治疗/病毒治疗载体。尽管有这些优点，但总体有效性仍然受到次优腺病毒递送至癌组织的限制。克服这种限制的一种概念性方法是通过感染后递送的腺病毒的选择性复制来实现腺病毒感染性的放大效应，使得可以发生子代病毒的侧向扩散。在这种方法中，有条件复制能力的病毒在转导的肿瘤细胞中但不在正常组织中进行溶瘤性复制。从转导的肿瘤细胞产生腺病毒后代将允许感染邻近的肿瘤细胞。使用条件复制型腺病毒（CRAd）代表了实现有效的肿瘤细胞溶瘤和减轻肿瘤细胞感染限制的方法。然而，考虑到基于Ad 5的CRAd具有天然的肝向性，正常组织中的任何背景复制活性（和随后的肝毒性）提出了一个特别关键的问题，该问题需要开发新的方法来改善癌症特异性。在该提案中，我们的目标是通过引入2种不同水平的癌症靶向Ad 5 E1 A基因表达来降低非癌细胞中的背景复制活性。首先，这可以通过将CRAd复制功能所必需的E1 A基因置于转录控制下来实现。为了实现这一点，我们将使用CXCR 4基因启动子，它在头颈部鳞状细胞癌（HNSCC）中具有独特的癌症特异性激活特征。第二，我们将通过在E1 A mRNA编码序列上游设计成纤维细胞生长因子2（FGF 2）的高度结构化的5 '-非翻译区（5'-UTR）序列来引入癌症特异性蛋白质翻译控制。这将使得这种嵌合mRNA的有效翻译依赖于翻译起始因子eIF-4 E，其在大多数正常组织中以有限的量表达，并且在大多数癌细胞中过表达。因此，我们假设癌症特异性E1 A mRNA转录与癌症特异性蛋白质翻译的双重水平将显著增强E1 A基因向癌症组织的靶向表达，超过单独使用任一控制元件，从而显著改善CRAd的癌症特异性复制。成功完成所提出的研究，以开发和验证HNSCC细胞系中转基因表达的双重靶向原理，将产生用于癌症病毒治疗的全新类别的CRAd。

DESCRIPTION (provided by applicant): A number of characteristics of the Adenovirus type 5 (Ad5) make it an optimal gene therapy/virotherapy vector suitable for a wide array of cancer therapeutic approaches. Despite these advantages, overall ifficacy remains limited by sub-optimal adenovirus delivery to cancer tissues. 1 conceptual approach to overcome this limitation is to achieve an amplification effect of adenovirus infectivity via selective replication of the delivered adenovirus post-infection, such that lateral spread of progeny viruses may occur. In this approach, a conditionally replication-competent virus replicates oncolytically in transduced tumor cells but not in normal tissue. Production of adenovirus progeny from transduced tumor cells would then allow infection of the neighboring tumor cells. Using conditionally replicative adenoviruses (CRAds) represent a method to achieve efficient tumor cell oncolysis and mitigate tumor cell infection limitations. However, given that Ad5-based CRAds have a natural liver tropism, any background replication activity in normal tissues (and subsequent liver toxicity) presents a particularly critical problem that warrants development of novel approaches to improve cancer specificity. In this proposal, we aim specifically to reduce background replication activity in non-cancer cells by introducing 2 different levels of cancer targeting for the Ad5 E1A gene expression. First, this can be achieved by placing E1A gene, which is essential for CRAd replication function, under transcriptional control. To accomplish this, we will use the CXCR4 gene promoter, which has a distinct cancer-specific activation profile in squamous cell carcinomas of the head and neck (HNSCC). Second, we will introduce cancer-specific protein translational control by engineering a highly structured 5'-untranslated region (5'-UTR) sequence of the Fibroblast Growth Factor 2 (FGF2) upstream of the E1A mRNA coding sequence. This will render efficient translation of such a chimeric mRNA dependent upon the translation initiation factor elF-4E, which is expressed in limiting amounts in most normal tissues and is over expressed in most cancer cells. Thus, we hypothesize that the dual level of cancer-specific E1A mRNA transcription together with cancer-specific protein translation will significantly enhance target expression of E1A gene to cancer tissues beyond the use of either control element alone, and thereby dramatically improve cancer-specific replication of the CRAd. The successful completion of the proposed research to develop and validate the principle of dual-targeting of transgene expression in HNSCC cell lines, will result in a totally new class of CRAds for cancer virotherapy.