ABSTRACT
Characterizing the in vivo biodistribution pattern and relative expression levels of oligonucleotide-based molecules such as mRNA, miRNA, siRNA, and anti-miRNAs in animal models, could be a helpful first-step in the successful development of therapeutic oligonucleotides. Here we describe a simple procedure called "Whole-Body Scanning PCR" (WBS-PCR), which combines the power of PCR with that of imaging. WBS-PCR relies on 384 well-defined extractions across a mouse whole-body section followed by a single dilution step which renders the lysates compatible with various qPCR-based assays. The in vivo biodistribution maps are generated by deconvoluting the qPCR data and converting it into a TissueView compatible image file which can be overlaid with an image of the whole-body section used for extractions. WBS-PCR is a flexible platform that can be adapted to other detection systems and thereby further expand the use of this technology.
Subject(s)
Gene Expression , Molecular Imaging/methods , Oligonucleotides/genetics , Oligonucleotides/metabolism , Polymerase Chain Reaction/methods , Whole Body Imaging/methods , Animals , Cryoultramicrotomy/methods , Mice , MicroRNAs/genetics , Models, Animal , Oligonucleotides/administration & dosage , RNA, Messenger/genetics , RNA, Ribosomal, 18S/genetics , RNA, Small Interfering/genetics , Rats , Real-Time Polymerase Chain Reaction/methods , Rodentia , Tissue DistributionABSTRACT
Absorption, distribution, metabolism, and excretion properties of a small interfering RNA (siRNA) formulated in a lipid nanoparticle (LNP) vehicle were determined in male CD-1 mice following a single intravenous administration of LNP-formulated [(3)H]-SSB siRNA, at a target dose of 2.5 mg/kg. Tissue distribution of the [(3)H]-SSB siRNA was determined using quantitative whole-body autoradiography, and the biostability was determined by both liquid chromatography mass spectrometry (LC-MS) with radiodetection and reverse-transcriptase polymerase chain reaction techniques. Furthermore, the pharmacokinetics and distribution of the cationic lipid (one of the main excipients of the LNP vehicle) were investigated by LC-MS and matrix-assisted laser desorption ionization mass spectrometry imaging techniques, respectively. Following i.v. administration of [(3)H]-SSB siRNA in the LNP vehicle, the concentration of parent guide strand could be determined up to 168 hours p.d. (post dose), which was ascribed to the use of the vehicle. This was significantly longer than what was observed after i.v. administration of the unformulated [(3)H]-SSB siRNA, where no intact parent guide strand could be observed 5 minutes post dosing. The disposition of the siRNA was determined by the pharmacokinetics of the formulated LNP vehicle itself. In this study, the radioactivity was widely distributed throughout the body, and the total radioactivity concentration was determined in selected tissues. The highest concentrations of radioactivity were found in the spleen, liver, esophagus, stomach, adrenal, and seminal vesicle wall. In conclusion, the LNP vehicle was found to drive the kinetics and biodistribution of the SSB siRNA. The renal clearance was significantly reduced and its exposure in plasma significantly increased compared with the unformulated [(3)H]-SSB siRNA.
Subject(s)
Drug Carriers/metabolism , Lipids/pharmacokinetics , Nanoparticles/metabolism , RNA, Small Interfering/metabolism , Animals , Autoradiography , Chromatography, High Pressure Liquid , Drug Carriers/chemistry , Drug Carriers/pharmacokinetics , Drug Stability , Injections, Intravenous , Lipids/blood , Lipids/chemistry , Male , Mice , Mice, Inbred Strains , Nanoparticles/chemistry , RNA, Small Interfering/blood , RNA, Small Interfering/chemistry , RNA, Small Interfering/pharmacokinetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tissue Distribution , Tritium , Whole-Body CountingABSTRACT
Efficient tissue-specific delivery is a crucial factor in the successful development of therapeutic oligonucleotides. Screening for novel delivery methods with unique tissue-homing properties requires a rapid, sensitive, flexible and unbiased technique able to visualize the in vivo biodistribution of these oligonucleotides. Here, we present whole body scanning PCR, a platform that relies on the local extraction of tissues from a mouse whole body section followed by the conversion of target-specific qPCR signals into an image. This platform was designed to be compatible with a novel RT-qPCR assay for the detection of siRNAs and with an assay suitable for the detection of heavily chemically modified oligonucleotides, which we termed Chemical-Ligation qPCR (CL-qPCR). In addition to this, the platform can also be used to investigate the global expression of endogenous mRNAs and non-coding RNAs. Incorporation of other detection systems, such as aptamers, could even further expand the use of this technology.
