The efficacy of lipid-encapsulated, chemically modified short interfering RNA (siRNA) targeted to hepatitis B virus (HBV) was examined in an in vivo mouse model of HBV replication. Stabilized siRNA targeted to the HBV RNA was incorporated into a specialized liposome to form a stable nucleic-acid-lipid particle (SNALP) and administered by intravenous injection into mice carrying replicating HBV. The improved efficacy of siRNA-SNALP compared to unformulated siRNA correlates with a longer half-life in plasma and liver. Three daily intravenous injections of 3 mg/kg/day reduced serum HBV DNA >1.0 log(10). The reduction in HBV DNA was specific, dose-dependent and lasted for up to 7 d after dosing. Furthermore, reductions were seen in serum HBV DNA for up to 6 weeks with weekly dosing. The advances demonstrated here, including persistence of in vivo activity, use of lower doses and reduced dosing frequency are important steps in making siRNA a clinically viable therapeutic approach.

Whereas stereochemical purity in drugs has become the standard for small molecules, stereoisomeric mixtures containing as many as a half million components persist in antisense oligonucleotide (ASO) therapeutics because it has been feasible neither to separate the individual stereoisomers, nor to synthesize stereochemically pure ASOs. Here we report the development of a scalable synthetic process that yields therapeutic ASOs having high stereochemical and chemical purity. Using this method, we synthesized rationally designed stereopure components of mipomersen, a drug comprising 524,288 stereoisomers. We demonstrate that phosphorothioate (PS) stereochemistry substantially affects the pharmacologic properties of ASOs. We report that Sp-configured PS linkages are stabilized relative to Rp, providing stereochemical protection from pharmacologic inactivation of the drug. Further, we elucidated a triplet stereochemical code in the stereopure ASOs, 3'-SpSpRp, that promotes target RNA cleavage by RNase H1 in vitro and provides a more durable response in mice than stereorandom ASOs.

To develop synthetic short interfering RNA (siRNA) molecules as therapeutic agents for systemic administration in vivo, chemical modifications were introduced into siRNAs targeted to conserved sites in hepatitis B virus (HBV) RNA. These modifications conferred significantly prolonged stability in human serum compared with unmodified siRNAs. Cell culture studies revealed a high degree of gene silencing after treatment with the chemically modified siRNAs. To assess activity of the stabilized siRNAs in vivo initially, an HBV vector-based model was used in which the siRNA and the HBV vector were codelivered via high-volume tail vein injection. More than a 3 log 10 decrease in levels of serum HBV DNA and hepatitis B surface antigen, as well as liver HBV RNA, were observed in the siRNA-treated groups compared with the control siRNAtreated and saline groups. Furthermore, the observed decrease in serum HBV DNA was 1.5 log 10 more with stabilized siRNA compared with unmodified siRNA, indicating the value of chemical modification in therapeutic applications of siRNA. In subsequent experiments, standard systemic intravenous dosing of stabilized siRNA 72 hours after injection of the HBV vector resulted a 0.9 log 10 reduction of serum HBV DNA levels after 2 days of dosing. In conclusion, these experiments establish the strong impact that siRNAs can have on the extent of HBV infection and underscore the importance of stabilization of siRNA against nuclease degradation. R NA interference (RNAi) is a recently discovered cellular mechanism that detects and destroys double-stranded RNA 1 and seems to play a role in the cell's antiviral defense system. 2 Short interfering RNA (siRNA) molecules are approximately 21-nucleotide, double-stranded RNA intermediates of the RNAi mechanism that guide a unique RNAi protein complex termed RNA-induced silencing complex to target RNA, leading to its subsequent degradation. Although in the natural RNAi pathway, siRNAs are derived from long double strand RNA that is processed by the nuclease Dicer into discrete 21-mers, 3,4 introduction of synthetic siRNAs into the cell also leads to RNAi-mediated silencing of target gene expression. 5 The use of synthetic siRNAs that use the endogenous cellular mechanism to downregulate the expression of disease-related or viral genes may lead to the development of a new therapeutic approach. In this report, we present evidence that synthetic siRNAs inhibit the replication of the hepatitis B virus (HBV) in cell culture and in a mouse model of HBV replication.Although siRNA has become an effective research tool to downregulate gene expression in cell culture, the inherent instability of RNA limits its potential use for in vivo gene silencing. The development of siRNAs as therapeutic agents will likely require improvements in the stability of siRNAs and the efficiency and specificity of tissuetargeted delivery in vivo. Chemical modifications made to synthetic siRNAs for the purpose of stabilization not only must provide resistance to nuclease degradation, but als...

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.

Screening of random oligonucleotide libraries with SELEX [systematic evolution of ligands by exponential enrichment; Tuerk, C., & Gold, L. (1990) Science 249, 505-510] has emerged as a powerful method for identifying high-affinity nucleic acid ligands for a wide range of molecular targets. Nuclease sensitivity of unmodified RNA and DNA, however, imposes considerable restrictions on their use as therapeutics or diagnostics. Modified RNA in which pyrimidine 2'-hydroxy groups have been substituted with 2'-amino groups (2'-aminopyrimidine RNA) is known to be substantially more resistant to serum nucleases. We report here on the use of SELEX to identify high-affinity 2'-aminopyrimidine RNA ligands to a potent angiogenic factor, basic fibroblast growth factor (bFGF). High-affinity ligands with the same consensus primary structure have been isolated from two independent libraries of approximately 6 x 10(14) molecules containing 30 or 50 randomized positions. Compared to unmodified RNA with the same sequence, 2'-aminopyrimidine ligands are at least 1000-fold more stable in 90% human serum. The sequence information required for high-affinity binding to bFGF is contained within 24-26 nucleotides. The minimal ligand m21A (5'-GGUGUGUGGAAGACAGCGGGUGGUUC-3'; G = guanosine, A = adenosine, C = 2'-amino-2'-deoxycytidine, U = 2'-amino-2'-deoxyuridine, and C = 2'-amino-2'-deoxycytidine or deoxycytidine) binds to bFGF with an apparent dissociation constant (Kd) of 3.5 +/- 0.3) x 10(-10) M at 37 degrees C in phosphate-buffered saline (pH 7.4). Disassociation of m21A from bFGF is adequately described with a first-order rate constant of (1.96 +/- 0.08) x 10(-3) s-1 (t1/2 = 5.9 min). The calculated value for the association rate constant (kon = k(off)/Kd) was 5.6 x 10(6) M-1 s-1. Highly specific binding of m21A to bFGF was observed: binding to denatured bFGF, five proteins from the FGF family (acidic FGF, FGF-4, FGF-5, FGF-6, and FGF-7), and four other heparin binding proteins is substantially weaker under the same conditions with KdbFGF/Kdprotein values ranging from (4.1 +/- 1.4) x 10(-2) to > 10(-6). Heparin but not chondroitin sulfate competed for binding of m21A to bFGF. In cell culture, m21A inhibited [125I]bFGF binding to both low-affinity sites (ED50 approximately 1 nM) and high-affinity sites (ED50 approximately 3 nM) on CHO cells expressing transfected FGF receptor-1.(ABSTRACT TRUNCATED AT 400 WORDS)