Whole-body scanning PCR; a highly sensitive method to study the biodistribution of mRNAs, noncoding RNAs and therapeutic oligonucleotides.

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.

An imaging assay to analyze primary neurons for cellular neurotoxicity.

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.

Phosphorylated FTY720 promotes astrocyte migration through sphingosine-1-phosphate receptors.

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.

siRNA relieves chronic neuropathic pain.

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.

A role for oligonucleotide-based RNA-knock down technologies in functional genomics.

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.

Independent combinatorial effect of antisense oligonucleotides and RNAi-mediated specific inhibition of the recombinant rat P2X3 receptor.

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.

Functional downregulation of P2X3 receptor subunit in rat sensory neurons reveals a significant role in chronic neuropathic and inflammatory pain.

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.