| Popis: |
RNAi combined with oncolytic viral vector therapy may provide a new approach to control malignant disease since allele specific shRNA to a retrogene that modulates stemness will inhibit growth of human CRC in vivo. Our original delivery vector was nonreplicating Lentiviral vectors (LV) but now oncolytic adenovirus is the vector. We have created a conditionally replicating chimeric oncolytic adenovirus (CRAd) expressing a shRNA to inhibit growth of established tumors in preclinical models. shRNA is directed to NANOGP8, a retrogene that differs from parental NANOG primarily in a single nucleotide variant: c.759G>C, p.Gln253His. Oncolytic adenoviruses were generated with a 5/3 fiber with either a control (Ad5/3-wtE1a-shNEG (Ad5/3shNEG)) or an anti-NANOGP8 shRNA (Ad5/3-wtE1a-shNp8 (Ad5/3shNp8)). LV shNEG or LV shNp8 were added 24 hr after creation of 3-D organoid cultures of human CRC cells (Clone A, CX-1 or LS174T) in serum-free medium. LV transduced 45 – 90% of cells in culture and shNp8 inhibited organoids by ~50% compared to either shNEG or untreated CRC organoids. In addition, LV injected into 7 -9 mm diameter CX-1 tumors in NOD/SCID mice reduced tumor growth in vivo by 40% for 11 days, but then tumors grew at an accelerated rate. NMF cluster analysis reveals that LVshNp8 affects a gene expression profile that is distinct from that of either LVshNEG or untreated controls. In contrast, Ad5/3shNp8 inhibited organoid growth by more than 70% and controlled growth of CX-1 and LS174T in vivo. Infection of cells in vitro was over 90%. Thus, shNp8 alters gene expression and replicating oncolytic virus with shNp8 has a more persistent inhibitory effect than that achieved with non-replicating LV-delivered shRNA. |
