Development of sarcoma-targeting agents utilizing viral engineering

为了开发一种新的治疗肉瘤的策略，本研究的目的是开发一种新的方法来纯化具有同源病毒基因组DNA的溶瘤病毒种子库。从单纯疱疹病毒1型溶瘤病毒、针对表达钙蛋白肉瘤的ΔRR和针对肿瘤缺氧微环境的D12ODDΔRR中分离纯化病毒基因组DNA。将含有克隆性病毒基因组的BACm id转化大肠杆菌，进行大量制备和测序。克隆入BACmid，在大肠杆菌中制备，未检测到硫胺酸激酶基因序列的突变，即使是单核苷酸也未检测到突变。D12CALPfΔRR和D12ODDΔRR病毒用有限稀释法纯化。我们证实，纯化的d12ODDΔRR病毒破坏了肉瘤间皮瘤的干细胞(启动肿瘤)部分，也有效地破坏了从对化疗和分子靶向治疗耐药的肿瘤建立的培养的平滑肌肉瘤细胞。

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.