ABSTRACT
The ocular metabolism of an siRNA duplex, SIRNA-027, was examined by ion-pair reversed-phase liquid chromatography (IP-RP-LC) coupled to electrospray ionization mass spectrometry (ESI-MS). The RNA duplex was injected intraocularly into the eyes of New Zealand white rabbits. Rabbits were sacrificed at different timepoints and the vitreous and retina/choroid tissue analyzed for siRNA by IP-RP-LC-MS. The method used a hexafluoroisopropanol (HFIP)/triethylamine (TEA) ion-pairing buffer with a methanol gradient. Using electrospray ionization, the duplex was preserved in the gas phase for analysis by a triple quadrupole mass spectrometer. With this methodology metabolites from rabbit ocular vitreous humor and retina/choroid tissue were identified and a pattern of siRNA degradation was established. Results showed that the duplex was metabolized predominantly from one end. This end of the siRNA duplex was calculated to have the weakest binding energy of the two ends indicating that the ability of the siRNA to split into single strands is a factor in its degradation.
Subject(s)
Chromatography, High Pressure Liquid/methods , Eye/metabolism , RNA, Double-Stranded/analysis , RNA, Small Interfering/analysis , Spectrometry, Mass, Electrospray Ionization/methods , Animals , Female , RNA, Antisense/analysis , Rabbits , Spectrophotometry, Ultraviolet , Vitreous Body/chemistryABSTRACT
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.
Subject(s)
Drug Delivery Systems/methods , Hepatitis B virus/genetics , Hepatitis B/therapy , Hepatitis B/virology , Liposomes/chemistry , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/chemistry , Animals , Antiviral Agents/administration & dosage , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/virology , Cell Line, Tumor , Coated Materials, Biocompatible/administration & dosage , Coated Materials, Biocompatible/chemistry , Female , Gene Targeting/methods , Genetic Therapy/methods , Hepatitis B/genetics , Hepatitis B/metabolism , Hepatitis B virus/drug effects , Humans , Liposomes/pharmacokinetics , Liver/drug effects , Liver/metabolism , Male , Metabolic Clearance Rate , Mice , RNA, Small Interfering/genetics , RNA, Small Interfering/pharmacokinetics , Tissue Distribution , Treatment OutcomeABSTRACT
Liquid chromatography/mass spectrometry (LC/MS) was used as a method for analyzing the metabolites of a model short interfering RNA (siRNA) duplex. The model siRNA duplex incorporated oligonucleotide stabilizing and protecting chemistries as these have been shown to increase the half-life of oligonucleotides. Two complementary 23 nucleotide single strands were joined to form the duplex. The intact duplex was analyzed using ion-pair reversed-phase chromatography coupled to electrospray ionization mass spectrometry (ESI-MS). The method used a hexafluoroisopropanol/triethylamine ion-pairing buffer with a methanol gradient to separate single-stranded oligonucleotide components from the duplex. This buffer system with ESI also preserved the duplex in the gas phase for analysis by a triple quadrupole mass spectrometer. Using this methodology, in vitro and in vivo metabolites from urine and rabbit ocular vitreous humor were determined and a pattern of duplex siRNA degradation was established. The masses of the metabolites were determined by ESI-MS and used with the known sequence of the siRNA duplex to identify the metabolites. Over the time course of the metabolism experiments it was shown that the breakdown products of the siRNA are consistent with the nuclease protection given by chemical modifications and that the duplex structure adds additional stability compared to the single strands alone. This study demonstrates that the ability of LC/MS to analyze duplex oligonucleotides has unique benefits for the study of siRNA metabolism.
