Modulating NLRP3 splicing with antisense oligonucleotides to control pathological inflammation

Inflammation has an essential role in healing. However, over-active inflammation disrupts normal cellular functions and can be life-threatening when not resolved. The NLRP3 inflammasome, a component of the innate immune system, is an intracellular multiprotein complex that senses stress-associated signals, and, for this reason is a promising therapeutic target for treating unresolved, pathogenic inflammation. Alternative splicing of NLRP3 RNA has been suggested as a regulatory mechanism for inflammasome activation, as some spliced isoforms encode NLRP3 proteins with compromised function. Here, we take advantage of this natural regulatory mechanism and devise a way to control pathogenic inflammation using splice-switching antisense oligonucleotides (ASOs). To identify and induce NLRP3 spliced isoforms lacking inflammatory activity, we tested a series of ASOs, each targeting a different exon, to determine the most effective strategy for down-regulating NLRP3. We identify several ASOs that modulate NLRP3 splicing, reduce NLRP3 protein, and decrease inflammasome signaling in vitro. The most effective ASO suppresses systemic inflammation in vivo in mouse models of acute inflammation and cryopyrin-associated periodic syndrome (CAPS). Our results demonstrate a systematic approach to protein engineering using splice-switching ASOs to generate isoforms with altered activity, and identify an ASO that can treat pathological inflammation in mice by reducing functional NLRP3.