In this study, we used small interfering RNA (siRNA) directed against vascular endothelial growth factor receptor 1 (vegfr1) mRNA to investigate the role of VEGFR1 in ocular neovascularization (NV). After evaluating many siRNAs, Sirna-027 was identified; it cleaved vegfr1 mRNA at the predicted site and reduced its levels in cultured endothelial cells and in mouse models of retinal and choroidal neovascularization (CNV). Compared to injection of an inverted control sequence, quantitative reverse transcriptase-PCR demonstrated statistically significant reductions of 57 and 40% in vegfr1 mRNA after intravitreous or periocular injection of Sirna-027, respectively. Staining showed uptake of 5-bromodeoxyuridine-labeled Sirna-027 in retinal cells that lasted between 3 and 5 days after intravitreous injection and was still present 5 days after periocular injection. In a CNV model, intravitreous or periocular injections of Sirna-027 resulted in significant reductions in the area of NV ranging from 45 to 66%. In mice with ischemic retinopathy, intravitreous injection of 1.0 mg of Sirna-027 reduced retinal NV by 32% compared to fellow eyes treated with 1.0 mg of inverted control siRNA. These data suggest that VEGFR1 plays an important role in the development of retinal and CNV and that targeting vegfr1 mRNA with siRNA has therapeutic potential. Gene Therapy (2006) 13, 225-234.

These data support the potential of developing sd-rxRNAs as a therapeutic for ocular disease.

Ribozymes are catalytic RNA molecules that can be designed to cleave specific RNA sequences. To investigate the potential use of synthetic stabilized ribozymes for the treatment of chronic hepatitis C virus (HCV) infection, we designed and synthesized hammerhead ribozymes targeting 15 conserved sites in the 5Ј untranslated region (UTR) of HCV RNA. This region forms an internal ribosome entry site that allows for efficient translation of the HCV polyprotein. The 15 synthetic ribozymes contained modified nucleotides and linkages that stabilize the molecules against nuclease degradation. All 15 ribozymes were tested for their ability to reduce expression in an HCV 5Ј UTR/ luciferase reporter system and for their ability to inhibit replication of an HCV-poliovirus (HCV-PV) chimera. Treatment with several ribozymes resulted in significant downregulation of HCV 5Ј UTR/luciferase reporter expression (range 40% to 80% inhibition, P F .05). Moreover, several ribozymes showed significant inhibition (G90%, Chronic infection with hepatitis C virus (HCV) can lead to cirrhosis, liver failure and/or hepatocellular carcinoma over a period of 10 to 20 years. 1,2 The Centers for Disease Control recently reported the number of chronically infected Americans to be approximately 4.5 million; thus, HCV infection is over four times as prevalent as human immunodeficiency virus infection. 3 Worldwide the prevalence of chronic HCV is similar to that found in the United States. 3 Thus, chronic HCV infection represents an important public health problem throughout the world.PTreatment of chronic HCV infection with interferon alfa leads to sustained viral clearance in only approximately 12% of patients. 4 Newer therapeutic regimes, such as the combination of interferon alfa and ribavirin, can lead to 38% to 43% of patients having a sustained virological response. 5,6 However, even with current combination regimes, approximately 60% of patients have no sustained virological benefit. Additionally, treatment with interferon alfa and ribavirin leads to significant toxicities. 5,6 Therefore, there still remains a great need for improved therapeutic modalities.HCV is a 9.5-kb, plus-strand, RNA virus that is a member of the human flavivirus family. 7,8 Although the sequence of the HCV-RNA genome is highly variable among clinical isolates, the 5Ј untranslated region (UTR) of the genome is highly conserved with respect to RNA sequence identity. 9,10 The conserved sequence/structure of the 5Ј UTR of HCV RNA contains an internal ribosome entry site (IRES) to mediate translation independent of a 5Ј-cap structure. 10,11 The HCV IRES does not require any viral protein for initiation of translation 12 and IRES elements also occur in cellular messenger RNAs. 13,14 Because the components of IRES-mediated translation are shared between cellular and HCV messenger RNAs, targeting the mechanism of HCV-IRES-mediated translation can be problematic. However, because the HCV-IRES sequence is highly conserved among viral genotypes, it is an excellent target for ribo...

No abstract

As a means of generating homogeneous populations of elongation complexes with the RNA polymerases encoded by phages T7 and SP6, transcription has been carried out in vitro on templates associated with the Gln-111 mutant of EcoRI endonuclease. The Gln-111 protein, as a result of a single amino acid substitution at position 111, lacks cleavage function yet shows higher than wild-type affinity for the EcoRI recognition sequence GAATTC. On a series of linear and circular templates associated with Gln-111 protein, blockage of the phage RNA polymerase elongation complex is observed. The 3' endpoint of the major blocked-length RNA species, just 3 bp upstream from the GAATTC, reveals an extremely close approach of polymerase's leading edge to essential contacts between Gln-111 protein and its binding site. In contrast to E. coli RNA polymerase, which is blocked stably and quantitatively by Gln-111 protein (Pavco, P.A. and Steege, D. A. (1990) J. Biol. Chem. 265, 9960-9969), the phage polymerases show substantial levels of readthrough transcription beyond the protein block.