149. In Vitro-Synthesized Sendai Virus RNA Fragments Induced Antitumor Immunity and Cancer Cell-Selective Apoptosis

Abstract

Three different clinical trials to treat malignant melanoma, castration-resistant prostate cancer and malignant mesothelioma using inactivated Sendai virus (hemagglutinating virus of Japan; HVJ) particles called HVJ-envelope (HVJ-E) are on-going because of the multiple-anti-cancer activities of HVJ-E. One the activities is the activation of anticancer immunity through inactivation of Treg (regulatory T cell) and promotion of NK cells activation and generation of CTL against cancers by producing interferon (IFN)-β, CXCL10, RANTES and IL-6. Another is the cancer selective apoptosis by induction of proapoptotic genes such as TRAIL and Noxa in various human cancer cells but not in normal cells. Most of these anti-cancer activities are conducted by RNA genome fragments of HVJ-E through RIG-I/MAVS (retinoic-acid inducible gene-I, mitochondrial antiviral signaling protein) signal pathway. Among various Sendai virus strains, Sendai virus Cantell strain showed the highest production of IFN-β in dendritic cells (DCs) and the strongest DC maturaion and we found that DI (defective interfering) particles of Cantell strain resulted in those immunostimulatory activities. Then, we investigated RNA molecules in Cantell HVJ DI particles which have no replication activities. The Cantell stain HVJ DI RNA has complementary termini (approximately 100nt) and exhibits the highest binding affinity to RIG-I. DI RNA genome (544 base) was isolated and transferred to human prostate cancer cell PC3 by lipofection. The DI RNA induced higher expression level of those apoptosis-related proteins such as Noxa and TRAIL and more cancer cell death than whole-genome RNA (approximately 15 kb) of complete Cantell strain without DI particles. Furthermore, we examined whether a specific structure of the DI RNA genome stimulates the RIG-I/MAVS downstream-related cancer suppressive pathways using HVJ-derived in vitro transcribed (IVT) RNAs. IVT-B2 which is derived from Cantell HVJ DI genome has a special secondary structure with a double-stranded RNA terminus and a single-stranded RNA loop. This IVT-B2 strongly stimulated RIG-I dependent proapoptotic proteins induction in prostate cancer cells. Modified IVT-B2 RNAs which had shorter dsRNA stem lost cancer cell killing activity and proapoptotic gene expressions. On the other hand, other modified IVT-RNA which had deleted ssRNA region in loop structure did not induce cancer cell-selective killing. We also found that calf intestinal alkaline-phosphatase-treated IVT-B2 RNA lost capability of inducing RIG-I/MAVS-related downstream Noxa and TRAIL expression. Finally, in vivo electroporation of IVT-B2 RNA induced intra-tumoral apoptosis and tumor suppression in human prostate cancer cell-xenograft mouse model by both direct tumor-cell killing and NK cell activation. These findings provide a novel nucleic acid medicine for the cancer treatment.