[show abstract][hide abstract] ABSTRACT: RNA interference (RNAi) can be used to inhibit viral replication in mammalian cells and therefore could be a powerful new antiviral therapy. Small interfering RNA (siRNA) may be effective for RNAi, but there are some technical problems that must be solved in each case, for example, predicting the effective siRNA target site and targeting heterogeneous sequences in a virus population. We show here that diced siRNA generated from long double-stranded RNA (dsRNA) is highly effective for inducing RNAi in HuH-7 cells harboring hepatitis C virus (HCV) replicons and can overcome variations in the HCV genotype. However, in mammalian cells, long dsRNA induced an interferon response and caused cell death. Here we describe an improvement of this method, U6 promoter-driven expression of long hairpin-RNA with multiple point mutations in the sense strand. This can efficiently silence HCV RNA replication and HCV protein expression without triggering the interferon response or cell death normally caused by dsRNA. In conclusion, intracellular-diced dsRNA efficiently induces RNAi, and, despite the high rate of mutation in HCV, it should be a feasible therapeutic strategy for silencing HCV RNA.
[show abstract][hide abstract] ABSTRACT: Hepatobiliary and pancreatic carcinomas are hypovascular tumors that can proliferate under hypoxic conditions. Recent reports have demonstrated that hypoxia-inducible factor 1 alpha (HIF1alpha) plays an important role in the survival of these cancers. Given these findings, the inhibition of the HIF1alpha pathway might prove to be a powerful tool in the treatment of these cancers. To inhibit HIF1alpha expression, we used small interference RNA (siRNA) expression vectors in this study. The transient transfection of siRNA expression vectors significantly reduced both HIF1alpha mRNA levels (13% of control) and protein levels (41% of control) and significantly inhibited the growth of cancer cell lines (P<0.05). VEGF, Glut1, and aldorase A expressions were also significantly reduced by transfection with these vectors (P<0.05), and we found that these vectors induced apoptosis but not cell cycle arrest. In a subcutaneous tumor model using nude mice, transfected MIA PaCa-2 cells, stably expressing siRNAs, barely formed tumors compared to control (P<0.05). This study thus demonstrates the usefulness of siRNA expression vector in targeting HIF1alpha and points to a potential clinical role in the treatment of pancreatic and hepatobiliary carcinomas.
Cancer Gene Therapy 03/2006; 13(2):131-40. · 2.95 Impact Factor
[show abstract][hide abstract] ABSTRACT: Ribozymes are catalytic RNAs. Representative ribozymes that exist in nature include hammerhead, hairpin, hepatitis delta virus
(HDV) and Neurospora VS ribozymes; group I and II introns; the RNA subunit of RNase P; and ribosomal RNA (Birikh et al. 1997; Doudna 1998; Zhou
and Taira 1998; Walter and Burke 1998; Carola and Eckstein 1999; Gesteland et al. 1999; Lilley 1999; Scott 1999; Warashina
et al. 1999a, 2000a). These ribozymes catalyze a variety of reactions, which include site-specific cleavage and the ligation
and polymerization of nucleotides (Cech and Bass 1986; Alberts et al. 1994; Severin et al. 1997; Famulok 1999; Gesteland et
al. 1999; Lee et al. 2000; Li and Breaker 1999; Marshall and Ellington 1999; Roberts and Ja 1999; Soukup and Breaker 1999;
Wilson and Szostak 1999). RNA catalysis is of particular current interest because of the potential utility of ribozymes, which
can cleave specific sequences of interest, as therapeutic agents and as specific regulators of gene expression (Cech 1988;
Yu et al. 1995; Bertrand et al. 1997; Birikh et al. 1997; Kawasaki et al. 1998; Lan et al. 1998; Plehn-Dujowich and Altman
1998; Kuwabara et al. 1998a,b, 1999; Koseki et al. 1999; Warashina et al. 1999b; Tanabe et al. 2000a). Both hammerhead and
hairpin ribozymes have considerable potential in a clinical setting because of their small size and great specificity. The
ability of these ribozymes to cleave target RNA molecules at specific sites makes them useful as inhibitors of viral replication
and malignancy (Scanlon 1998; Rossi and Couture 1999; Krupp and Gaur 2000). Large numbers of artificial ribozymes with a great
variety of catalytic activities have also been created by in vitro selection procedures (Famulok 1999; Roberts and Ja 1999).