Human Papillomavirus Gene Expression

描述（由申请人提供）： 安全的溶瘤病毒，适合作为治疗剂，必须靶向癌细胞进行破坏，但不能在正常组织中繁殖。我们最近报道了第一次使用原代人角质形成细胞的器官型“筏”培养物，其形成完全分层和分化的上皮细胞，以测试靶向表达人乳头瘤病毒（HPV）致癌基因E6和E7的组织的条件复制能力腺病毒（CRAD）CB 016的性质（Balague et al.，2001年）。亲粘膜HPV引起一系列过度增殖的肛门生殖器和口腔病变，包括宫颈、阴茎、肛门和扁桃体上皮的尖锐湿疣、乳头状瘤、发育不良和癌，没有有效的治疗或疫苗。HPV E7蛋白与腺病毒E1 A蛋白在灭活宿主肿瘤抑制蛋白pRB和相关p107和p130方面具有相当大的功能同源性。这种功能同源性形成了开发和研究Ad 5 CB 016的概念基础。该病毒缺失E1 A的保守区域CR 1和CR2。它在表达HPV-18癌基因的筏培养物中复制并溶细胞，但在对照筏培养物中不复制并溶细胞。然而，在仔细进行的时间过程实验中，我们进一步证明，CB 016的生产性感染是相当延迟，但没有消除，在正常的筏文化。我们的长期目标是在这些初步发现的基础上，设计新的更安全的CRAD，其在正常筏培养物中具有最小的细胞病变效应，但在模拟良性乳头状瘤、发育不良和癌症的表达HPV癌基因的筏培养物中具有溶瘤作用。重点将放在删除19 kDa的Ad E4-E6/7蛋白，它通过与E2 F/DP 1家族的转录因子形成转录活性复合物来补充E1 A突变，以及E1 A蛋白的其他结构域，它也激活其他早期腺病毒启动子。我们还打算调查纳入广告E1 B缺失作为一个额外的安全功能正常组织。

DESCRIPTION (provided by applicant): A safe oncolytic virus, suitable as a therapeutic agent, must target cancer cells for destruction but not propagate in normal tissues. We have recently reported the first use of organotypic "raft" cultures of primary human keratinocytes, which form fully stratified and differentiated epithelia, to test the properties of a conditional replication-competent adenovirus (CRAD) CB016 targeted to tissues expressing the human papillomavirus (HPV) oncogenes E6 and E7 (Balague et al., 2001). Mucosotropic HPVs cause a spectrum of hyper-proliferative ano-genital and oral lesions, including condylomata, papillomas, dysplasias, and carcinomas of cervical, penile, anal, and tonsillar epithelia with no effective treatment or vaccine. HPV E7 protein shares considerable functional homology with the adenovirus E1A proteins, in inactivating the host tumor suppressor protein pRB and related p107 and p130. This functional homology forms the conceptual basis for the development and investigation of Ad5 CB016. This virus is deleted of conserved regions CR1 and CR2 of E1A. It replicates and is cytolytic in raft cultures that express the HPV-18 oncogenes, but not in control raft cultures. However, in carefully conducted time course experiments, we further demonstrated that productive infection of CB016 was considerably delayed, but not eliminated, in normal raft cultures. Our long-term goal is to build upon these initial findings and design new safer CRAD having minimal cytopathic effects in normal raft cultures, but oncolytic in HPV oncogene-expressing raft cultures that simulate benign papillomas, dysplasias and cancers. Emphasis will be placed on the deletion of 19 kDa Ad E4-E6/7 protein, which complements E1A mutations by forming transcriptionally active complexes with the E2F/DP1 family of transcription factors, and on additional domains of the E1A protein, which also activate other early adenovirus promoters. We also intend to investigate the incorporation of Ad E1B deletions as an additional safety feature for normal tissues.