Development of sarcoma-targeting agents utilizing viral engineering

为了开发一种新的肉瘤治疗策略，本研究的目的是开发一种新的程序，用于纯化具有同质病毒基因组DNA的溶瘤病毒种子库。将单病毒基因组DNA克隆到BACmid中，并从HSV-1溶瘤病毒中纯化，d12.CALPfΔRR靶向表达calponin的肉瘤，d12 ODD ΔRR靶向肿瘤缺氧微环境。将含有BACmid的克隆病毒基因组转化E.大肠杆菌，最大准备艾德和序列艾德。通过克隆到BACmid中并在E.杆菌用有限稀释法纯化了d12.CALPfΔRR和d12 ODD ΔRR病毒。我们证实，纯化的d12 ODD ΔRR病毒破坏肉瘤性间皮瘤的干（肿瘤起始）细胞组分，并且也有效地破坏培养的平滑肌肉瘤细胞，所述平滑肌肉瘤细胞由对化疗和分子靶向治疗均耐药的肿瘤建立。

Toward the development of a novel therapeutic strategy for sarcoma, the purpose of this study is to develop a novel procedure for purification of oncolytic viral seed stock with homogeneous viral genome DNA. Single viral geneome DNA was cloned into BACmid and was purified from HSV-1 oncolytic viruses, d12.CALPfΔRR targeting calponin-expressing sarcoma and d12ODDΔRR targeting hypoxic microenvironment of tumor. The BACmid containing clonal viral genome was transformed into E. Coli, maxi-prep'ed and sequencesed. No mutation, even in the single nucleotide was detected in the sequence of Thimidine kinase gene through cloning into BACmid and preparation in E. Coli. d12.CALPfΔRR and d12ODDΔRR viruses without BACmid sequence were purified by limited dilution methods. We confirmed that the purified d12ODDΔRR virus destroyed stem (tumot-initiating) cell fraction of sarcomatous mesothelioma, and also effective for destruction of cultured leiomyosarcoma cells which were established from tumors resistant to both chemotherapy- and molecular targeted therapy.