Biomedical subjects

Pierre Martin

Publications and source records attributed to Pierre Martin.

5 recordsLinked to original sources

PubMed2004

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. 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.

Animals↗

PubMed2003

mRNA fusion constructs serve in a general cell-based assay to profile oligonucleotide activity.

A cellular assay has been developed to allow measurement of the inhibitory activity of large numbers of oligonucleotides at the protein level. The assay is centred on an mRNA fusion transcript construct comprising of a full-length reporter gene with a target region of interest inserted into the 3'-untranslated region. Luciferase and fluorescent reporter genes were used in the constructs. The insert can be from multiple sources (uncharacterised ESTs, partial or full-length genes, genes from alternate species, etc.). Large numbers of oligonucleotides were screened for antisense activity against a number of such constructs bearing different reporters, in different cell lines and the inhibitory profiles obtained were compared with those observed through screening the oligonucleotides against the corresponding endogenous genes assayed at the mRNA level. A high degree of similarity in the profiles was obtained indicating that the fusion constructs are suitable surrogates for the endogenous messages for characterisation of antisense oligonucleotides (ASOs). Furthermore, the results support the hypothesis that the secondary structure of mRNAs are divided into domains, the nature of which is determined by primary nucleotide sequence. Oligonucleotides whose activity is dependent on the local structure of their target mRNAs (e.g. ASOs, short interfering RNAs) can be characterised via such fusion RNA constructs.

Animals↗

PubMed2003

Cell cycle modulation of gene targeting by a triple helix-forming oligonucleotide.

Successful gene-targeting reagents must be functional under physiological conditions and must bind chromosomal target sequences embedded in chromatin. Triple helix-forming oligonucleotides (TFOs) recognize and bind specific sequences via the major groove of duplex DNA and may have potential for gene targeting in vivo. We have constructed chemically modified, psoralen-linked TFOs that mediate site-specific mutagenesis of a chromosomal gene in living cells. Here we show that targeting efficiency is sensitive to the biology of the cell, specifically, cell cycle status. Targeted mutagenesis was variable across the cycle with the greatest activity in S phase. This was the result of differential TFO binding as measured by cross-link formation. Targeted cross-linking was low in quiescent cells but substantially enhanced in S phase cells with adducts in approximately 20-30% of target sequences. 75-80% of adducts were repaired faithfully, whereas the remaining adducts were converted into mutations (>5% mutation frequency). Clones with mutations could be recovered by direct screening of colonies chosen at random. These results demonstrate high frequency target binding and target mutagenesis by TFOs in living cells. Successful protocols for TFO-mediated manipulation of chromosomal sequences are likely to reflect a combination of appropriate oligonucleotide chemistry and manipulation of the cell biology.

Animals↗

PubMed2002

Minimum number of 2'-O-(2-aminoethyl) residues required for gene knockout activity by triple helix forming oligonucleotides.

Triple helix forming oligonucleotides (TFOs) that bind chromosomal targets in living cells may become tools for genome manipulation, including gene knockout, conversion, or recombination. However, triplex formation by DNA third strands, particularly those in the pyrimidine motif, requires nonphysiological pH and Mg(2+) concentration, and this limits their development as gene-targeting reagents. Recent advances in oligonucleotide chemistry promise to solve these problems. For this study TFOs containing 2'-O-methoxy (2'-OMe) and 2'-O-(2-aminoethyl) (2'-AE) ribose substitutions in varying proportion have been constructed. The TFOs were linked to psoralen and designed to target and mutagenize a site in the hamster HPRT gene. T(m) analyses showed that triplexes formed by these TFOs were more stable than the underlying duplex, regardless of 2'-OMe/2'-AE ratio. However, TFOs with 2'-AE residues were more stable in physiological pH than those with only 2'-OMe sugars, as a simple function of 2'-AE content. In contrast, gene knockout assays revealed a threshold requirement--TFOs with three or four 2'-AE residues were at least 10-fold more active than the TFO with two 2'-AE residues. The HPRT knockout frequencies with the most active TFOs were 300-400-fold above the background, whereas there was no activity against the APRT gene, a monitor of nonspecific mutagenesis.

Animals↗

PubMedDate not supplied

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.

Drug Industry↗