Targeting novel human transient receptor potential ankyrin 1 splice variation with splice-switching antisense oligonucleotides

Abstract

ABSTRACT: Activation of transient receptor potential ankyrin 1 (TRPA1) channels by both environmental irritants and endogenous inflammatory mediators leads to excitation of the nerve endings, resulting in acute sensation of pain, itch, or chronic neurogenic inflammation. As such, TRPA1 channels are actively pursued as therapeutic targets for various pathological nociception and pain disorders. We uncovered that exon 27 of human TRPA1 (hTRPA1) could be alternatively spliced into hTRPA1_27A and hTRPA1_27B splice variants. The resulting channel variants displayed reduced expression, weakened affinity to interact with WT, and suffered from complete loss of function because of disruption of the C-terminal coiled-coil domain. Using a human minigene construct, we revealed that binding of splicing factor serine/arginine-rich splicing factor 1 (SRSF1) to the exonic splicing enhancer was critical for the inclusion of intact exon 27. Knockdown of SRSF1, mutation within exonic splicing enhancer, or masking SRSF1 binding with antisense oligonucleotides promoted alternative splicing within exon 27. Finally, antisense oligonucleotides-induced alternative splicing produced transcript and protein variants that could be functionally determined as diminished endogenous TRPA1 activity in human Schwann cell-line SNF96.2 and hiPSCs-derived sensory neurons. The outcome of the work could potentially offer a novel therapeutic strategy for treating pain by targeting alternative splicing of hTRPA1.