ABSTRACT
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.
Subject(s)
Oligonucleotides/chemistry , Polymerase Chain Reaction/methods , RNA, Messenger/genetics , RNA, Untranslated/chemistry , Whole Body Imaging/methods , Animals , HCT116 Cells , Humans , Image Processing, Computer-Assisted , Male , Mice , Oligonucleotides/pharmacokinetics , Oligonucleotides/therapeutic use , Organ Specificity , RNA, Messenger/chemistry , RNA, Small Interfering/chemistry , RNA, Small Interfering/genetics , RNA, Untranslated/genetics , Reproducibility of Results , Sensitivity and Specificity , Tissue DistributionABSTRACT
The development of high-content screening technologies including automated immunostaining, automated image acquisition and automated image analysis have enabled higher throughput of cellular imaging-based assays. Here we used high-content imaging to thoroughly characterize the cultures of primary rat cerebellar granule neurons (CGNs). We describe procedures to isolate and cultivate the CGNs in 96-well and 384-well format, as well as a procedure to freeze and thaw the CGNs. These methods allow the use of CGNs in 96-well format analyzing 2500 samples per experiment using freshly isolated cells. Down-scaling to 384-well format and freezing and thawing of the CGNs allow even higher throughput. A cellular assay with rat CGN cultures was established to study the neurotoxicity of compounds in order to filter out toxic compounds at an early phase of drug development. The imaging-based toxicity assay was able to reveal adverse effects of compounds on primary neurons which were not detected in neuroblastoma or other cell lines tested.
Subject(s)
Microscopy, Confocal/methods , Neurons/cytology , Neurons/drug effects , Neurotoxins/pharmacology , Adenosine Triphosphate/metabolism , Animals , Animals, Newborn , Antigens/metabolism , Astrocytes/drug effects , Astrocytes/metabolism , Bungarotoxins/toxicity , Cell Count/methods , Cells, Cultured , Cerebellum/cytology , Dimethyl Sulfoxide/pharmacology , Dose-Response Relationship, Drug , Humans , Inhibitory Concentration 50 , Nerve Net/drug effects , Neurites/drug effects , Neuroblastoma/pathology , Neurons/metabolism , O Antigens/metabolism , Oligodendroglia/drug effects , Oligodendroglia/metabolism , Proteoglycans/metabolism , Rats , Time Factors , Tubulin/metabolismABSTRACT
Sphingosine-1-phosphate (S1P) receptors are widely expressed in the central nervous system where they are thought to regulate glia cell function. The phosphorylated version of fingolimod/FTY720 (FTY720P) is active on a broad spectrum of S1P receptors and the parent compound is currently in phase III clinical trials for the treatment of multiple sclerosis. Here, we aimed to identify which cell type(s) and S1P receptor(s) of the central nervous system are targeted by FTY720P. Using calcium imaging in mixed cultures from embryonic rat cortex we show that astrocytes are the major cell type responsive to FTY720P in this assay. In enriched astrocyte cultures, we detect expression of S1P1 and S1P3 receptors and demonstrate that FTY720P activates Gi protein-mediated signaling cascades. We also show that FTY720P as well as the S1P1-selective agonist SEW2871 stimulate astrocyte migration. The data indicate that FTY720P exerts its effects on astrocytes predominantly via the activation of S1P1 receptors, whereas S1P signals through both S1P1 and S1P3 receptors. We suggest that this distinct pharmacological profile of FTY720P, compared with S1P, could play a role in the therapeutic effects of FTY720 in multiple sclerosis.
Subject(s)
Astrocytes/drug effects , Cell Movement/drug effects , Immunosuppressive Agents/pharmacology , Propylene Glycols/pharmacology , Receptors, Lysosphingolipid/physiology , Sphingosine/analogs & derivatives , 2',3'-Cyclic-Nucleotide Phosphodiesterases/metabolism , Animals , Astrocytes/physiology , Calcium Signaling/drug effects , Cell Movement/physiology , Cell Proliferation/drug effects , Cells, Cultured , Cerebral Cortex/cytology , Cyclic AMP/metabolism , Dose-Response Relationship, Drug , Embryo, Mammalian , Fingolimod Hydrochloride , Glial Fibrillary Acidic Protein/metabolism , Glutamic Acid/pharmacology , Hippocampus/cytology , Hippocampus/drug effects , Inositol Phosphates/metabolism , Organ Culture Techniques , Oxadiazoles/pharmacology , Rats , Receptors, Lysosphingolipid/agonists , Receptors, Lysosphingolipid/antagonists & inhibitors , Sphingosine/pharmacology , Thiophenes/pharmacology , beta-Alanine/analogs & derivatives , beta-Alanine/pharmacologyABSTRACT
Double stranded, short interfering RNAs (siRNA) of 21-22 nt length initiate a sequence-specific, post-trancriptional gene silencing in animals and plants known as RNA interference (RNAi). Here we show that RNAi can block a pathophysiological pain response and provide relief from neuropathic pain in a rat disease model by down regulating an endogenous, neuronally expressed gene. Rats, intrathecally infused with a 21 nt siRNA perfectly complementary to the pain-related cation-channel P2X3, showed diminished pain responses compared to missense (MS) siRNA-treated and untreated controls in models of both agonist-evoked pain and chronic neuropathic pain. This form of delivery caused no adverse effects in any of the animals receiving P2X3 siRNA, MS siRNA or vehicle. Molecular analysis of tissues revealed that P2X3 mRNA expressed in dorsal root ganglia, and P2X3 protein translocated into the dorsal horn of the spinal cord, were significantly diminished. These observations open a path toward use of siRNA as a genetic tool for drug target validation in the mammalian central nervous system, as well as for proof of concept studies and as therapeutic agents in man.
Subject(s)
Neuralgia/therapy , Purinergic P2 Receptor Antagonists , RNA, Small Interfering/therapeutic use , Animals , Chronic Disease , Hyperalgesia/therapy , Neuralgia/metabolism , RNA Interference , Rats , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2/metabolism , Receptors, Purinergic P2X3ABSTRACT
Functional genomics is inundating the pharmaceutical industry with large numbers of potential gene targets from several sources such as gene expression profiling experiments (DNA microchips, proteomics) or database mining. Oligonucleotide-based RNA-knock down technologies such as antisense or RNA interference can aid in the filtering and prioritization of target candidates in the drug discovery process.
Subject(s)
Oligoribonucleotides/chemical synthesis , Drug Industry/methods , Genomics , Oligonucleotide Array Sequence Analysis , RNA/genetics , RNA, Small Interfering/chemistry , RNA, Small Interfering/geneticsABSTRACT
Synthetic 21-bp-long short interfering RNAs (siRNA) can stimulate sequence-specific mRNA degradation in mammalian cell cultures, a process referred to as RNA interference (RNAi). In the present study, the potential of RNAi was compared to the traditional antisense approach, acting mainly via RnaseH, for targeting the recombinant rat pain-related cation-channel P2X3 expressed in CHO-K1 and a rat brain tumour-derived cell line, 33B. Downregulation of the P2X3 receptor was evaluated at the mRNA, protein, and functional levels. In this study, four siRNA duplexes induced up to 95% sequence-specific inhibition of the P2X3 mRNA, independent of the type of 2 nt 3'-overhang modification and the location of the targeted sequences. Furthermore, we detected and characterised an independent combinatorial effect of antisense oligonucleotides (ASOs) and RNAi-mediated specific inhibition of the P2X3 receptor. Enhanced downregulation was observed only when siRNA was combined with nonhomologous ASO, targeting distant regions on the common P2X3 mRNA. The two reagents resulted in more efficient downregulation of P2X3 mRNA when administered in combination rather than separately. To our knowledge, this is the first investigation at the molecular level of the potential benefits of mixed antisense and RNAi-mediated treatment for inhibiting expression of a medically relevant pain-related gene.
Subject(s)
Oligonucleotides, Antisense/metabolism , RNA, Double-Stranded/metabolism , Receptors, Purinergic P2/genetics , Animals , CHO Cells , Cricetinae , Gene Expression Regulation , Oligonucleotides, Antisense/genetics , RNA Interference , RNA, Double-Stranded/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Receptors, Purinergic P2X3 , Recombinant Proteins/genetics , Transfection/methods , Tumor Cells, CulturedABSTRACT
The excitation of nociceptive sensory neurons by ATP released in injured tissue is believed to be mediated partly by P2X3 receptors. Although an analysis of P2X3 knock-out mice has revealed some deficits in nociceptive signaling, detailed analysis of the role of these receptors is hampered by the lack of potent specific pharmacological tools. Here we have used antisense oligonucleotides (ASOs) to downregulate P2X3 receptors to examine their role in models of chronic pain in the rat. ASOs and control missense oligonucleotides (180 microg/d) were administered intrathecally to naive rats for up to 7 d via a lumbar indwelling cannula attached to an osmotic minipump. Functional downregulation of the receptors was confirmed by alphabeta-methylene ATP injection into the hindpaw, which evoked significantly less mechanical hyperalgesia as early as 2 d after treatment with ASOs relative to controls. At this time point, P2X3 protein levels were significantly downregulated in lumbar L4 and L5 dorsal root ganglia. After 7 d of ASO treatment, P2X3 protein levels were reduced in the primary afferent terminals in the lumbar dorsal horn of the spinal cord. In models of neuropathic (partial sciatic ligation) and inflammatory (complete Freund's adjuvant) pain, inhibition of the development of mechanical hyperalgesia as well as significant reversal of established hyperalgesia were observed within 2 d of ASO treatment. The time course of the reversal of hyperalgesia is consistent with downregulation of P2X3 receptor protein and function. This study demonstrates the utility of ASO approaches for validating gene targets in in vivo pain models and provides evidence for a role of P2X3 receptors in the pathophysiology of chronic pain.
