Antisense Oligonucleotide Therapy Decreases IL-1β Expression and Prolongs Survival in Mutant Nlrp3 Mice

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Inflammation has been implicated in the pathogenesis of most chronic diseases affecting humans, including cancer, diabetes, neurodegenerative disease, and common diseases involving the gut, heart, kidney, and liver. A central mechanism involves chronic activation of innate immune pathways driven by nucleotide-binding and oligomerization domain-like receptors (NLRs) that serve as danger signal sensors, of which NLR family pyrin domain containing 3 (NLRP3) is the most well studied. NLRP3 has therefore become an attractive target of numerous pharmaceutical companies (1). NLRP3 forms an intracellular multiprotein complex known as the inflammasome that is comprised of the adaptor molecule apoptosis-associated speck-like protein and the effector molecule caspase-1, a cysteine protease. Activation of the inflammasome by various stimulants leads to autocleavage of caspase-1, the cleavage and activation of gasdermin D and the proinflammatory cytokine IL-1β, and ultimately the release of mature IL-1β from the cell (1).

Mutations in NLRP3 were first identified as the cause of cryopyrin-associated periodic syndrome (CAPS), a rare autoinflammatory disorder characterized by systemic inflammation, and signs and symptoms primarily involving the skin, musculoskeletal system, and/or CNS (2). CAPS is a disease continuum that includes three entities of varying severity including neonatal onset multisystem inflammatory disease (NOMID), Muckle–Wells syndrome (MWS), and familial cold autoinflammatory syndrome (FCAS) (2–4), which are all characterized by urticaria-like lesions and fever. However, each of the three entities has unique features that define prognosis and dosing of approved therapies. Heterozygous mutations in the NLRP3 gene are gain of function leading to hyperactivation of the inflammasome and increased release of IL-1β, and biologic therapies inhibiting the IL-1 pathway have been shown to improve clinical symptoms in CAPS patients (5–7). However, there remains an unmet clinical need for patients who do not respond adequately to current IL-1–targeted treatments. Small molecules targeting NLRP3 are currently in development, but a recent study suggests that at least one of these drugs may be less effective in CAPS (8), encouraging a search for new approaches including oligonucleotide-based therapies.

Antisense oligonucleotides (ASOs) are single-stranded chemically synthesized oligonucleotides composed of 12–30 nt (9–12). Binding of ASOs to the targeted mRNA or pre-mRNA in the nucleus or the cytoplasm follows in a sequence-specific manner through Watson–Crick base pairing interactions (11). After binding the target sequence, ASOs lead to RNase H–mediated RNA degradation, premature termination of transcription, or block of translation, which results in suppression of protein expression (9, 11, 13). Systemic administration of ASO leads to a broad biodistribution achieving ASO delivery to many organs and tissues. The CNS is excluded because unformulated or unconjugated ASOs cannot cross the blood–brain barrier. The organs with the highest accumulation of ASOs are the liver and kidney followed by bone marrow (10). In recent years several ASOs have been approved for therapy in a wide range of diseases, including familial hypercholesterolemia and spinal muscular atrophy (11, 12). In this study, a novel an ASO targeting Nlrp3 was designed and studied in in vitro, ex vivo, and in vivo murine models to demonstrate preclinical efficacy in CAPS.

For the efficacy screening of antisense ASOs, two murine cell lines, 4T1 and RAW 264.7, were purchased from American Type Culture Collection (LGC standards) and cultured under recommended conditions.

C57BL/6 wild-type mice were purchased from Charles River. Nlrp3 knock-in mouse strains with an aspartate 301 to asparagine (D301N) (NOMID), an alanine 350 to valine (A350V) (MWS), or a leucine 351 to proline (L351P) (FCAS) substitution were generated as previously described (available at The Jackson Laboratory: B6N.129-Nlrp3^D301NneoR/J, strain no. 017971; B6.129-Nlrp3^A350VneoR/J, strain no. 017969; and B6N.129-Nlrp3^L351PneoR/J, strain no. 017970, respectively) (14, 15). The D301N, A350V, and L351P point mutations lead to a conformational change causing a ligand-independent constitutive activation of the mutant NLRP3 inflammasome (16). Owing to the presence of an intronic floxed neomycin resistance cassette, the expression of the mutation does not occur unless the Nlrp3 knock-in mice (Nlrp3^D301NneoR, Nlrp3^A350VneoR, Nlrp3^L351PneoR) are first bred with mice expressing Cre recombinase. For in vivo studies, Nlrp3 knock-in mice were bred to those expressing Cre recombinase under the control of a lysozyme (CreL; The Jackson Laboratory, strain no. 004781, B6.129P2-Lyz2^tm1(cre)Ifo/J). For ex vivo studies, Nlrp3 knock-in mouse strains were bred to mice expressing B6.Cg-Tg(CAG-cre/Esr1*)5Amc/J (tamoxifen-inducible Cre, The Jackson Laboratory, strain no. 004682). Littermates lacking the Cre recombinase served as control mice. All murine lines were bred >10 generations to a C57BL/6 background. Experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of California, San Diego.

ASOs targeting the mouse Nlrp3 pre-mRNA were designed using an in-house bioinformatics pipeline to ensure selectivity and avoid undesired off-target effects. Locked nucleic acid (LNA)–modified gapmers were ordered from Axolabs (Kulmbach, Germany) and dissolved in H₂O (stock concentration, 1 mM). Sequences of ASO and the control oligonucleotide used are shown in Table I.

Six thousand 4T1 or RAW 264.7 cells were seeded in 96-well flat-bottom plates in 100 µl of DMEM, supplemented with antibiotic/antimycotic, sodium pyruvate (1 mM), and FCS (10%). LPS (Escherichia coli O111:B4) was added at a final concentration of 1 µg/ml and oligonucleotides at 10 µM. Cells were cultured at 37°C for 3 d, then lysed for 30 min at 55°C with 50 µl of lysis mixture containing proteinase K (1:100) to 100 µl of medium per well. Cell lysates were directly used for a QuantiGene RNA singleplex assay or stored at −20°C.

Macrophages were differentiated from bone marrow cells isolated by flushing femur and tibia with PBS containing 10% FCS followed by erythrocyte lysis with RBC lysis buffer. Bone marrow cells (1.5 × 10⁶/ml) were seeded in a six-well cell culture plate in RPMI 1640 medium supplemented with antibiotic/antimycotic, sodium pyruvate (1 mM), FCS (10%), and mouse M-CSF (50 ng/ml) or GM-CSF (20 ng/ml) and cultured at 37°C. After 3 d, half of the cell culture supernatant was replaced by fresh RPMI 1640 medium, supplemented as above. Oligonucleotides were added to a final concentration of 2 or 10 µM and cells were cultured for another 4 d. Then, the cell culture supernatant of wild-type (WT) and Nlrp3 mutant bone marrow–derived macrophages (BMDMs) was replaced by serum-free RPMI 1640 medium containing LPS (E. coli O111:B4, 200 ng/ml). After 4 h, ATP (5 mM) was added to the WT BMDM cultures and the cells were incubated for 30 min at 37°C. Cell culture supernatants were collected for Western blot analysis, and cells were mechanically detached from the culture flask. Ten percent of the detached cells were resuspended in 100 µl of RPMI 1640 plus 50 µl of branched DNA (bDNA) lysis buffer containing proteinase K (1:100) and lysed at 55°C for 30 min. Cell lysates were used for a QuantiGene RNA singleplex assay or stored at −20°C. Ninety percent of cells were lysed for 20 min at 4°C in 150 µl of radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors and stored at −20°C for Western blot analysis.

Secreted IL-1β and IL-18 were measured using the DuoSet ELISA kit (R&D Systems, catalog nos. DY401-05 and DY7625-05, respectively) per the manufacturer’s instructions. Lactate dehydrogenase was measured using a CyQUANT lactate dehydrogenase cytotoxicity assay (Invitrogen, C20301) per the manufacturer’s instructions.

Nlrp3 mutant NOMID-CreL mice were injected s.c. with 20 mg/kg Nlrp3-specific ASO or control oligonucleotide starting at 2 d after birth. To get maximal in vivo exposure and knockdown in multiple different tissues, ASOs were administered every other day until a predetermined endpoint, or mouse death (17, 18).

Mice were anesthetized by Institutional Animal Care and Use Committee–approved protocols. Peripheral whole-mouse blood was collected in untreated collection tubes for serum and cell isolation. Liver and kidney tissue were harvested and snap-frozen or placed in 0.5 ml of RNAlater solution (Life Technologies, Carlsbad, CA) and frozen at −80°C.

Cells were lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA) containing HALT protease inhibitor cocktail (Thermo Fisher Scientific, 1:100). Then, 1000 µl of cell culture supernatant was used for concentrating the proteins with two 10-kDa Amicon Ultra-0.5 centrifugal filter units (final volume of 50 µl), after which 21µl of cell supernatant or lysate was mixed with Bolt LDS (lithium dodecyl sulfate) sample buffer (4×) and Bolt reducing agent (10×) and loaded on a Bolt 4–12% Bis-Tris Plus gel, prior to transfer to a nitrocellulose membrane. Blots were washed with iBind flex FD (fluorescence detection) solution and incubated with primary Abs specific to IL-1β (0.25 µg/ml, 1AF-401, R&D Systems) or NLRP3 (1 µg/ml, AG-20B-0014, AdipoGen Life Sciences) and secondary Ab for 2.5 h or overnight using the iBind flex western system. After incubation with secondary HRP Ab, bands were visualized with Pierce ECL substrate (Thermo Fisher Scientific). Western blot bands were quantified using the Invitrogen iBright analysis software (Thermo Fisher Scientific). Equal protein loading was verified with a β-actin Ab (anti-mouse β-actin at 0.1 µg/ml, VMA00048, Bio-Rad).

For protein extraction from livers, tissue was homogenized in RIPA buffer (Cell Signaling Technology, Danvers, MA) together with cocktails of protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, 1:100). For immunoblot analysis, 20 µg of protein lysate was resolved on Any kDa] Criterion TGX precast gels (Bio-Tad, Hercules, CA), transferred to a nitrocellulose membrane, and blocked with Intercept blocking buffer (LI-COR Biosciences, Lincoln, NE) for 1 h before incubation with primary Abs specific to IL-1β (Abcam, Cambridge, U.K., 1:500) overnight at 4°C. Membranes were incubated with IRDye secondary Ab (LI-COR Biosciences, Lincoln, NE), and protein bands were visualized via a LI-COR imaging system (LI-COR Biosciences, Lincoln, NE). Expression intensity was quantified by Image Studio (LI-COR Biosciences, Lincoln, NE). Equivalent protein loading was verified with an anti–β-actin Ab (MilliporeSigma, Burlington, MA, 1:6000).

Target expression on mRNA level was determined using a bDNA assay (QuantiGene singleplex assay kit 96-well plate format and QuantiGene sample processing kit for cultured cells, Thermo Fisher Scientific). The following probe sets were used: mouse Nlrp3 (SA-27762) and mouse Hprt1 (SA-15463). All reagents were purchased from Affymetrix/Thermo Fisher Scientific.

Mouse skin and brain samples were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, mounted on slides, deparaffinized, and rehydrated before staining with H&E or anti-myeloperoxidase (MPO) (Thermo Scientific, Waltham, MA). For MPO staining, specimens were deparaffinized and hydrated in ethanol, and the Ags were retrieved by boiling in citrate buffer (pH 6.0) for 30 min at 95°C. Following overnight incubation with anti-MPO (1:100), HRP-conjugated second Ab was applied. Slides were counterstained with hematoxylin. Images were taken using the NanoZoomer 2.0HT slide scanning system (Hamamatsu Photonics, Hamamatsu, Japan).

RNA was isolated from liver and kidney tissue using the RNeasy tissue mini kit (Qiagen, Valencia, CA), and cDNA was synthesized from 1 μg of total RNA using qScript cDNA SuperMix (Quantabio, Beverly, MA) per the manufacturer’s instructions. Real-time PCR was performed using TaqMan fast advanced master mix (Thermo Fisher Scientific, Vilnius, Lithuania). The reaction mix contained cDNA, TaqMan fast advanced master mix, and respective primers (Nlrp3 Mm04210225_m1, GAPDH Mm99999915_g1, Thermo Fisher Scientific). QuantStudio Design software (Thermo Fisher Scientific, Waltham, MA) was used for analyses.

RNA was isolated from peripheral blood using TRIzol (Life Technologies), and cDNA was synthesized using TaqMan reverse transcription reagents (Applied Biosystems). Subsequent quantitative PCR (qPCR) was performed using the Nlrp3 primers listed above and iQ SYBR Green supermix (Bio-Rad) with a Bio-Rad CFX96 real-time system. qPCR reaction parameters were as per the Bio-Rad instructions. Data were analyzed with CFX Manager v3.0 software.

Analyses were performed with GraphPad Prism version 8.4.2 (GraphPad Software, La Jolla, CA). Two groups were analyzed by the Student t test. ANOVA followed by Tukey’s multiple comparison was used to analyze three or more groups. The significance level was set at p < 0.05 for all comparisons. Unless otherwise stated, data are expressed as mean ± SEM or as a percentage for categorical variables.

To evaluate the efficacy of Nlrp3-specific ASOs, different ASOs targeting mouse Nlrp3 pre-mRNA were designed (Table I). More than 150 exonic and intronic targeting ASOs were screened in two murine cell lines, 4T1 and RAW 264.7. The ASO candidates that achieved a reduction of >80% of Nlrp3 mRNA expression were selected for further in vitro tolerability testing. Finally, the IC₅₀ was analyzed for the leading ASO candidates, and the top ASO candidate was selected. Treatment of the murine cell lines 4T1 and RAW 264.7 with the selected Nlrp3-specific ASO candidate at a concentration of 10 µM demonstrated a significant downregulation of Nlrp3 mRNA expression by >80%, an effect not observed with the control oligonucleotide (Fig. 1A; 4T1, *p < 0.05; RAW 264.7, **p < 0.01). A concentration–response curve of the selected Nlrp3-specific ASO candidate in RAW 264.7 cells revealed an IC₅₀ of 883 nM and a residual Nlrp3 expression of <20% at an ASO concentration of 10 µM (Fig. 1B), suggesting effective targeting. Other Nlrp3-specific ASO candidates that were not selected for subsequent functional characterization showed lower efficacy or potency of Nlrp3 downregulation in RAW 264.7 cells (Supplemental Fig. 1).

We next used WT BMDMs to assess the effects of the selected ASO candidate on NLRP3 inflammasome activation. Cells were stimulated with Nlrp3-specific ASO or nontargeting control oligonucleotide for 4 d prior to stimulation with LPS and ATP. Protein expression of NLRP3 and mature IL-1β was significantly decreased in the supernatant of cultures treated with Nlrp3-specific ASO compared with control oligonucleotide (negative control [Neg-Ctrl]) (Fig. 1C, 1D) (Nlrp3, **p < 0.01; mature IL-1β, ***p < 0.001). In cell lysates, protein expression of NLRP3 was also significantly downregulated in ASO-treated BMDMs compared with Neg-Ctrl (Fig. 1C, 1E) (NLRP3, ***p < 0.001). Consistently, there was increased presence of pro–IL-1β protein (Fig. 1C). In addition, mRNA expression of Nlrp3 was reduced in ASO-treated BMDMs (Fig. 1F) (Nlrp3, ***p < 0.001). Taken together, these data demonstrate that Nlrp3-specific ASO reduces Nlrp3 expression and downstream inflammasome activation in WT murine macrophages in vitro and ex vivo.

To determine whether ASO treatment could block intrinsic activation of the NLRP3 inflammasome, we used Nlrp3 knock-in mice. Three Nlrp3 knock-in mouse strains representing the different clinical entities of CAPS (NOMID, MWS, and FCAS) were generated as previously described (15) and bred to mice expressing inducible Cre recombinase ESR1 (CreERT), leading to mutant Nlrp3 expression in cultured cells after administration of tamoxifen (15). BMDMs derived from WT mice showed a concentration-dependent downregulation of IL-1β release after treatment with the Nlrp3-specific ASO, whereas treatment with the control oligonucleotide did not affect IL-1β release in BMDMs (Fig. 2) (2 µM, ***p < 0.001; 10 µM, ***p < 0.001). Nlrp3 mutant BMDMs from NOMID, MWS, and FCAS mice also revealed a significant, concentration-dependent reduction of IL-1β after Nlrp3-specific ASO treatment (Fig. 2) (NOMID: 2 µM, **p < 0.01; 10 µM, ***p < 0.001; MWS: 2 µM, **p < 0.01; 10 µM, ***p < 0.001; FCAS: 2 µM, *p < 0.05; 10 µM, ***p < 0.001). These inhibitory effects of the Nlrp3-specific ASO occurred with or without the second signal provided by ATP (Supplemental Fig. 2A). Nlrp3-specific ASO treatment of Nlrp3 mutant BMDMs did not show a reduction in IL-18 release or cell death compared with Neg-Ctrl ASO-treated BMDMs (Supplemental Fig. 2B, 2C). These results demonstrate that the designed Nlrp3-specific ASO is able to target mutant Nlrp3 genes and similarly and sufficiently diminish NLRP3 activation in both WT and mutant mice.

After demonstrating in vitro and ex vivo effects of the Nlrp3-specific ASO on WT and mutant NLRP3 function, we next investigated the efficacy of the Nlrp3-specific ASO in vivo. Nlrp3 mutant NOMID mice were generated by breeding to mice expressing CreL (NOMID-CreL). The gain-of-function mutation leads to onset of a systemic inflammatory phenotype beginning at birth. To determine whether we could prevent or reduce the signs of NLRP3-mediated inflammation, mice were injected s.c. with Nlrp3 or control oligonucleotide beginning at 2 d of life. Fifty percent of NOMID-CreL mice treated with Nlrp3-specific ASO were alive at 32 d after birth, with some mice surviving until 45 d (Fig. 3A). In contrast, 50% of NOMID-CreL mice treated with the Neg-Ctrl oligonucleotide had perished by 21 d of life, with a maximum survival of 32 d. Weight gain analyses similarly revealed a benefit for NOMID-CreL mice treated with Nlrp3-specific ASO. Mice treated with Nlrp3-specific ASO demonstrated continuous weight gain until day 20 after birth (Fig. 3B), but NOMID-CreL mice treated with Neg-Ctrl oligonucleotide reached a weight gain plateau 10 d after birth (Fig. 3B). In addition, NOMID-CreL mice treated with Nlrp3-specific ASO showed less growth retardation and less severe skin lesions compared with mice treated with the control oligonucleotide (Fig. 3C). Taken together, these data show improvement of murine CAPS signs and symptoms in vivo.

Next, we evaluated the systemic effect of s.c. Nlrp3-specific ASO therapy in NOMID-CreL mice. Organs and tissues were collected after ASO therapy at the age of 11 d. Mice treated with Nlrp3-specific ASO showed a significant increase in body weight (Fig. 4A) (body weight, ***p < 0.001). In contrast, the spleen weight/body weight ratio was significantly reduced in Nlrp3-specific ASO-treated mice compared with mice treated with Neg-Ctrl oligonucleotide (Fig. 4A) (*p < 0.05), consistent with a decrease in systemic inflammation. H&E staining demonstrated reduced infiltration of inflammatory cells in skin as well as the meninges and perivascular areas of the brain of Nlrp3-specific ASO-treated mice compared with mice treated with Neg-Ctrl oligonucleotide (Fig. 4B, Supplemental Fig. 3). As previously observed, the infiltrating cells in the skin of the control mice were primarily neutrophils, as evident by MPO staining, and were decreased in Nlrp3-specific ASO-treated mice (Fig. 4B). Thus, these data demonstrate a reduction of systemic inflammation after Nlrp3-specific ASO therapy.

We next investigated the specific effects of Nlrp3-targeted ASO in NOMID-CreL mice at the tissue level. Naked ASOs accumulate mostly in liver and kidneys after systemic administration (10). Following ASO therapy, at the age of 11 d kidneys and livers from NOMID-CreL mice were collected and analyzed. To determine the effects of Nlrp3-targeting ASO at the molecular tissue level, we assessed gene and protein expression from isolated peripheral blood, liver, and kidneys from NOMID-CreL mice. Nlrp3 mRNA was significantly downregulated in peripheral blood, liver, and kidney tissue in NOMID-CreL mice treated with Nlrp3-specific ASO compared with Neg-Ctrl oligonucleotide (Fig. 5A) (blood, *p < 0.05; liver, ***p < 0.001; kidney, **p < 0.01). In whole-liver lysates, protein expression of mature IL-1β was reduced in Nlrp3-specific ASO-treated mice compared with Neg-Ctrl (Fig. 5B) (*p < 0.05), although pro–IL-1β remained unchanged, consistent with a specific inhibitory effect on NLRP3 function (Fig. 5B). Taken together, these data demonstrate organ-specific downregulation of Nlrp3mRNA after systemic Nlrp3-specific ASO administration.

In this study, we designed a third-generation gapmer Nlrp3-specific ASO and investigated its use as a potential therapy via targeting the NLRP3 inflammasome in in vitro, ex vivo, and in vivo models. The Nlrp3-specific ASO was designed as an LNA gapmer ASO. Gapmers have ribose-modified flanks that increase target affinity and a central non–ribose-modified gap that leads to the recruitment of RNase H. Subsequently, RNase H binds and cleaves the target RNA. Fully phosphorothioated backbones prevent enzymatic degradation, promoting enhanced stability for use as a therapeutic agent. In addition, no transfection reagents and conjugates are needed for cellular delivery of LNA gapmers in both in vitro and in vivo settings. Systemic administration of gapmer ASOs results in target gene suppression in numerous tissues (10, 19–21). Taken together, these features suggest that a gapmer ASO would be a feasible therapeutic for human systemic inflammatory diseases. To date, there is no approved ASO for treatment of inflammatory diseases; however, there are approved ASOs for treatment of neuromuscular and cardiometabolic diseases, as well as amyloidosis (22–24).

The results of this study demonstrate that our newly designed gapmer ASO effectively targeted Nlrp3 in vitro, ex vivo, and in vivo. Treatment of murine cell lines and BMDMs from WT mice with Nlrp3-specific ASO showed a significant downregulation of NLRP3 and IL-1β expression. Notably, NLRP3 inflammasome activation in ex vivo BMDMs from NOMID, MWS, and FCAS Nlrp3 mutant mice after Nlrp3-specific ASO treatment was also decreased. Systemic administration of the Nlrp3-specific ASO resulted in survival and weight gain benefits in NOMID Nlrp3 mutant mice, with reduction in the severity of skin rash and systemic inflammation, indicating inhibition of NLRP3 inflammasome activation in vivo. Notably, inflammation in meninges and perivascular areas of the brain was ameliorated in mice treated with Nlrp3-specific ASO. This effect is most likely due to an improved function of the blood–brain barrier caused by reduced systemic inflammation rather than a therapeutic intracerebral effect of the ASOs. Furthermore, tissue-specific downregulation of Nlrp3 was shown in the peripheral blood, liver, and kidney. These findings are consistent with previous reports describing ASO biodistribution and accumulation primarily in the liver and kidney after systemic administration (10, 25), and they may indicate clinical utility of ASOs in more common diseases of the skin, liver, and kidney.

Although direct IL-1 blockade has been instrumental in the treatment of NLRP3-mediated autoinflammatory disorders, including CAPS, increasing numbers of patients with incomplete responses have been identified, emphasizing the need for a more specific therapy (26). Patients of all three CAPS entities NOMID, MWS, and FCAS have been shown to respond to treatment blocking IL-1 signaling; however, patients with severe symptoms tend to be less responsive to IL-1β therapy (2, 5, 6, 27, 28), suggesting that targeting the inflammasome activation pathway upstream of IL-1β release may provide more complete responses. Direct targeting of NLRP3 in CAPS has been explored by using the NLRP3 inhibitor molecule MCC950. Administration of MCC950 to MWS mutant mice leads to a survival benefit (29). Similarly, in human PBMCs of MWS patients, treatment with MCC950 led to a decrease in mature IL-1β secretion (29). However, data on the effect of direct NLRP3 inhibition are limited to MWS. Our results show that ASO treatment effectively targets Nlrp3 across the spectrum of CAPS (NOMID, MWS, and FCAS), resulting in decreased IL-1β expression and release.

The improvement in murine morbidity and mortality with administration of Nlrp3-specific ASO suggested an efficient binding of the Nlrp3-specific ASO to its target sequence despite the presence of an NLRP3 point mutation. Although the overwhelming majority of patient mutations described to date (30–33) are similarly single base pair changes, it is possible that certain mutations or insertion/deletions may render this therapy ineffective for some patients. Furthermore, efficacy in patients with CNS disease may be limited due to lack of blood–brain barrier penetration. Despite these challenges, the Nlrp3-specific ASO provides an alternate therapy to reduce NLRP3 inflammasome activation and limit the subsequent downstream inflammatory cascade.

L.B. is a site principal investigator for Novartis, Inc. H.M.H. is a consultant for Novartis. H.M.H. has research collaborations with Jecure, Inc., Zomagen, Inc., and Takeda, Inc. A.E.F. is consultant for Ventyx Bio, Inc., Novo Nordisk, and Inipharm. A.E.F. has research collaborations with Takeda, Inc. The other authors have no financial conflicts of interest.