Human Papillomavirus Gene Expression

描述(由申请人提供)： 一种安全的溶瘤病毒，适合作为治疗剂，必须以癌细胞为目标进行破坏，但不能在正常组织中繁殖。我们最近首次报道了使用器官类型的原代人类角质形成细胞的“筏”培养，形成完全复层和分化的上皮细胞，以测试靶向表达人乳头瘤病毒(HPV)癌基因E6和E7的组织的条件复制能力腺病毒(CRAD)CB016的性质(Balague等人，2001年)。嗜粘液性HPV在没有有效治疗或疫苗的情况下，会导致一系列高增殖性无生殖器和口腔病变，包括尖锐湿疣、乳头状瘤、异型增生和宫颈、阴茎、肛门和扁桃体上皮癌。HPV E7蛋白在灭活宿主肿瘤抑制蛋白pRb及相关的p107和p130方面与腺病毒E1a蛋白有相当大的功能同源性。这种功能同源性构成了开发和研究Ad5 CB016的概念基础。该病毒缺失了E1a保守区CR1和CR2。它在表达HPV-18癌基因的RAFT培养物中复制并具有细胞溶解作用，但在对照RAFT培养物中不能。然而，在仔细进行的时间进程实验中，我们进一步证明，在正常的RAFT培养中，CB016的生产性感染被显著延迟，但并未被消除。我们的长期目标是在这些初步发现的基础上，设计出新的更安全的CRAD，在正常的RAFT培养中具有最小的细胞病变影响，但在模拟良性乳头状瘤、异型增生和癌症的HPV癌基因表达的RAFT培养中具有溶瘤作用。重点将放在19 kDa Ad E4-E6/7蛋白的缺失上，该蛋白通过与E2F/DP1转录因子家族形成转录活性复合体来补充E1a突变，以及E1a蛋白的其他结构域，它也激活其他早期的腺病毒启动子。我们还打算研究加入AdE1B缺失作为正常组织的额外安全特征。

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.