FXYD2 antisense oligonucleotide provides an efficient approach for long-lasting relief of chronic peripheral pain
Alexandre Derre, Noelian Soler, Valentine Billoux, Sebastien Benizri, Brune Vialet, Cyril Rivat, Philippe Barthélémy, Patrick Carroll, Alexandre Pattyn, Stephanie Venteo
Alexandre Derre, Noelian Soler, Valentine Billoux, Sebastien Benizri, Brune Vialet, Cyril Rivat, Philippe Barthélémy, Patrick Carroll, Alexandre Pattyn, Stephanie Venteo
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Research Article Neuroscience

FXYD2 antisense oligonucleotide provides an efficient approach for long-lasting relief of chronic peripheral pain

Abstract

Chronic pain, whether of inflammatory or neuropathic origin, affects about 18% of the population of developed countries, and most current treatments are only moderately effective and/or cause serious side effects. Therefore, the development of novel therapeutic approaches still represents a major challenge. The Na,K-ATPase modulator FXYD2 is critically required for the maintenance of neuropathic pain in rodents. Here, we set up a therapeutic protocol based on the use of chemically modified antisense oligonucleotides (ASOs) to inhibit FXYD2 expression and treat chronic pain. We identified an ASO targeting a 20-nucleotide stretch in the FXYD2 mRNA that is evolutionarily conserved between rats and humans and is a potent inhibitor of FXYD2 expression. We used this sequence to synthesize lipid-modified forms of ASO (FXYD2-LASO) to facilitate their entry into dorsal root ganglia neurons. We established that intrathecal or intravenous injections of FXYD2-LASO in rat models of neuropathic or inflammatory pain led to a virtually complete alleviation of their pain symptoms, without causing obvious side effects. Remarkably, by using 2′-O-2-methoxyethyl chemical stabilization of the ASO (FXYD2-LASO-Gapmer), we could significantly prolong the therapeutic action of a single treatment up to 10 days. This study establishes FXYD2-LASO-Gapmer administration as a promising and efficient therapeutic strategy for long-lasting relief of chronic pain conditions in human patients.

Authors

Alexandre Derre, Noelian Soler, Valentine Billoux, Sebastien Benizri, Brune Vialet, Cyril Rivat, Philippe Barthélémy, Patrick Carroll, Alexandre Pattyn, Stephanie Venteo

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Figure 1

Identification of ASOs directed against human FXYD2 mRNA.

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Identification of ASOs directed against human FXYD2 mRNA. (A) Alignment ...
(A) Alignment of 2 variants (a and b) of the mouse (M), rat (R), and human (H) FXYD2 gene coding sequences. The 20-mer and 100% conserved rat-human sequence, used to generate FXYD2-ASO that served as a model to test its efficacy in vivo using rat models, is outlined in orange. The sequence targeted by custom-made ON-TARGETplus siRNA in our previous study is shown in green. Interspecies homology is indicated by an asterisk. (B) mRNA sites containing multiloops or hairpin loops are identified in yellow, positive motifs in blue, and negative motifs in red in the common sequence encoding the 2 variants (a and b) of the human FXYD2 mRNA. The 100% conserved rat-human sequence of FXYD2-LASO that we used for in vivo tests in the rat model is underlined. (C) The secondary structure of human FXYD2 mRNA as predicted by the RNAfold program. Numbers represent the bases of the mRNA in the RefSeq annotation NM_001680. The region outlined in pink illustrates the part of the mRNA identified as susceptible to antisense inhibition in HEK293M cells. (D) Quantification of FXYD2 protein levels by Western blot of extracts of HEK293M cells after transfection by a series of ASOs directed against human FXYD2 mRNA. The levels of FXYD2 protein were normalized to actin protein. Data are represented as means ± SEM, n = 3 replicates. One-way ANOVA and post hoc Bonferroni’s test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.