Compositions and methods for treating inflammatory diseases

Abstract

Provided herein are oligonucleotides, compositions, and methods for treating inflammatory diseases mediated by the NLRP3 inflammasome. Particularly, provided herein are NLRP3 splice-switching antisense oligonucleotides for reducing functional NLRP3 expression and treating pathological inflammation and other disease and disorders mediated by the NLRP3 inflammasome.

Description

[0001] COMPOSITIONS AND METHODS FOR TREATING INFLAMMATORY DISEASES

[0002] FIELD

[0003] Provided herein are oligonucleotides and methods for treating inflammatory diseases mediated by the NLR family pyrin-containing protein 3 (NLRP3) inflammasome.

[0004] CROSS REFERENCE TO RELATED APPLICATIONS

[0005] This application claims the benefit of U.S. Provisional Application No. 63 / 638,296, filed April 24, 2024, the content of which is herein incorporated by reference in its entirety.

[0006] SEQUENCE LISTING STATEMENT

[0007] The content of the electronic sequence listing titled

[0008] UM 43162 601 SequenceListing.xml (Size: 42,505 bytes; and Date of Creation: April 21, 2025) is herein incorporated by reference in its entirety.

[0009] BACKGROUND

[0010] The innate immune system defends against infectious and non-infectious threats by activating pattern recognition receptors (PRR) that recognize specific features of such insults. Within this system, inflammasomes are crucial mediators of this response. These intracellular multiprotein complexes detect microbial motifs and danger-associated molecules and mount an inflammatory response. NLRP3 (NLR family pyrin-containing protein 3) senses a plethora of infectious and non-infectious signals that pose a threat to cells and initiates an inflammatory cascade to eliminate such insults.

[0011] Although inflammation is a natural way for the body to respond to and clear damaged cells and pathogens, endogenous cellular damage signals such as cholesterol crystals, amyloid-P (AP), and uric acid can lead to aberrant NLRP3 -mediated inflammation. Persistent NLRP3 inflammasome activation has been implicated in the pathogenesis of autoinfl ammatory, neurodegenerative, and metabolic diseases. Furthermore, gain of function mutations in NLRP3 cause cryopyrin-associated periodic syndrome (CAPS), a rare pediatric autoinflammatory disease resulting from dysregulation of the NLRP3 inflammasome and uncontrolled release of inflammatory cytokines such as IL-P and IL-18 and pyroptosis. Recent studies have reported on small molecule inhibitors that target NLRP3 and alleviate inflammation associated with CAPS and other conditions. However, clinical development has been hampered perhaps due to toxicity, off-target effects, difficulty penetrating the blood-brain barrier (BBB), and limited understanding of the central activation mechanism.

[0012] SUMMARY

[0013] Provided herein are oligonucleotides, compositions, and methods for inhibiting the NLRP3 inflammasome in a cell, decreasing NLRP3 signaling or activation in a cell, and treating inflammatory diseases mediated by the NLRP3 inflammasome in a subject in need thereof.

[0014] In some embodiments, the methods comprise contacting the cell (e.g., in vivo, in vitro, or ex vivo) with a splice-switching antisense oligonucleotide, or a composition thereof, targeting NLRP3.

[0015] In some embodiments, the methods comprise administering an effective amount of a splice-switching antisense oligonucleotide, or a composition thereof, targeting NLRP3 to the subject. In some embodiments, the disease or disorder is an inflammatory disease or disorder. In some embodiments, the disease or disorder comprises an inflammatory component. In some embodiments, the disease or disorder is cryopyrin-associated periodic syndrome (CAPS).

[0016] In some embodiments, the splice-switching antisense oligonucleotide comprises a sequence that is complementary to a splice donor site, a branch site, or a splice acceptor site within the pre-messenger RNA (pre-mRNA) coding NLRP3. In some embodiments, the spliceswitching antisense oligonucleotide comprises a sequence that is complementary to a splice donor site, a branch site, or a splice acceptor site for exon 2, 3, 5, 6, or 8 of a pre-mRNA encoding for NLRP3. In some embodiments, the splice-switching antisense oligonucleotide is complementary to an exon-intron boundary of exon 2, 3, 5, 6, or 8 of a pre-mRNA encoding for NLRP3. In some embodiments, the splice-switching antisense oligonucleotide induces alternative splicing of exon 2, 3, 5, 6, or 8 in a pre-mRNA encoding for NLRP3.

[0017] In some embodiments, the splice-switching antisense oligonucleotide induces alternative splicing of exon 2 in a pre-mRNA encoding for NLRP3. In some embodiments, the antisense oligonucleotide is complementary to 5’ splice site of exon 2.

[0018] In some embodiments, the splice-switching antisense oligonucleotide induces alternative splicing of exon 6 in a pre-mRNA encoding for NLRP3. In some embodiments, the spliceswitching antisense oligonucleotide is complementary to 3’ splice site of exon 6.

[0019] In some embodiments, the splice-switching antisense oligonucleotide induces alternative splicing of exon 8 in a pre-mRNA encoding for NLRP3. In some embodiments, the spliceswitching antisense oligonucleotide is complementary to 5’ splice site of exon 8. In some embodiments, the splice-switching antisense oli onucleotide comprises any one of SEQ ID NOs: 1-14.

[0020] In some embodiments, the splice-switching antisense oligonucleotide is modified with one or more internucleoside linkages. In some embodiments, the splice-switching antisense oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).

[0021] In some embodiments, the methods inhibit the NLRP3 inflammasome and / or decreases NLRP3 signaling or activation by generating an NLPR3 protein with decreased functionality, decreased stability when an oligomer, or higher rates of degradation.

[0022] Also provided herein are antisense oligonucleotides comprising a sequence complementary to an exon-intron boundary of exon 2, 3, 5, 6, or 8 of a pre-messenger RNA (pre- mRNA) encoding for NLRP3, and compositions comprising thereof. In some embodiments, hybridization of the antisense oligonucleotide to the pre-mRNA encoding for NLRP3 induces splice skipping.

[0023] In some embodiments, the antisense oligonucleotide induces alternative splicing of exon 2 in a pre-mRNA encoding for NLRP3. In some embodiments, the antisense oligonucleotide is complementary to 5’ splice site of exon 2. In some embodiments, the antisense oligonucleotide induces alternative splicing of exon 6 in a pre-mRNA encoding for NLRP3. In some embodiments, the antisense oligonucleotide is complementary to 3’ splice site of exon 6. In some embodiments, the antisense oligonucleotide induces alternative splicing of exon 8 in a pre- mRNA encoding for NLRP3. In some embodiments, the antisense oligonucleotide is complementary to 5’ splice site of exon 8.

[0024] In some embodiments, the antisense oligonucleotide comprises a sequence of any one of SEQ ID NOs: 1-14.

[0025] In some embodiments, the antisense oligonucleotide is modified with one or more internucleoside linkages. In some embodiments, the antisense oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).

[0026] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description.

[0027] BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIGS. 1A-1C show NLRP3 -targeted splice switching antisense oligonucleotides (ASOs) modulate alternative splicing. FIG. 1A: Schematic of NLRP3 spliced and protein isoforms, (left) Full-length (FL) NLRP3 mRNA and ASO-induced splice variants. Boxes are exons and lines are introns. ASOs are shown as indicated. Diagonal black lines indicate splicing of introns. Dashed diagonal lines represent natural alternative splicing. Bold, red diagonal lines indicate ASO- induced splicing event, (right) NLRP3 protein domains and predicted protein products translated from the induced spliced isoforms with molecular weights (MW). FIG. IB: Representative radioactive RT-PCR analysis of NLRP3 RNA from THP-1 cells treated with the indicated ASOs (40 pM), including a non-targeted ASO control (C), followed by activation with LPS and ATP. Primer sets used to detect the specific ASO-induced splicing event are shown at bottom of gel image. Products were separated by PAGE. Red arrowheads indicate predominant ASO-induced spliced isoform for each ASO. Some ASOs also induced a low level of other alternatively spliced products. FIG. 1C: Quantification of native spliced products (full-length) from NLRP3 mRNA relative to the predominant ASO-mediated isoforms. Data are presented as mean ± SEM, unpaired t-test compared to matched control, *P<0.05, **P<0.01, ****P<0.0001; n=3 across three independent experiments.

[0029] FIGS. 2A-2D show ASO-induced NLRP3 alternative splicing inhibits inflammasome activation in human THP-1 cells. FIG. 2A: Representative immunoblot of NLRP3 protein in THP-1 lysates treated with the indicated ASOs (40 pM), including a non-targeted ASO control (C), followed by activation with LPS and ATP or untreated and activated with LPS only, oc- tubulin was analyzed as a control. FIG. 2B: Quantification of NLRP3 protein normalized to a- tubulin control. LPS only control (-) is included. Data presented as mean ± SEM; one sample t- test compared to non-targeted ASO control (ASO-C) set to 1, *P<0.05, **P<0.01, ***P<0.001; n=3. ELISA analysis of IL-1 (FIG. 2C) and TNF-a (FIG. 2D) released from THP-1 cells treated with ASOs prior to activation with LPS and ATP. Cytokine level was normalized to LPS / ATP stimulated cells. Data are presented as mean ± SEM and analyzed by repeated measures one-way ANOVA followed by Dunnett’s multiple comparisons test relative to non-targeted ASO control (C) for IL-ip and relative to LPS for TNF-a; *P<0.05, **P<0.01, ***P<0.001; n=3 independent experiments.

[0030] FIG. 3A-3G show dose-dependent activity of ASOs targeting NLRP3 exon 2 and 6 splicing. Sequence alignment of ASO-A2 (FIG. 3A; SEQ ID NO: 1) and ASO-A6 (FIG. 3B; SEQ ID NO: 4) shown base-paired with their target sequence in NLRP3 (SEQ ID NOs: 43 and 44, respectively). The splice sites (ss) are indicated with the arrow. Exonic sequence is in capital letters and intronic sequence is in lowercase. FIG. 3C: Representative radioactive RT-PCR analysis of NLRP3 isoform expression in THP-1 cells transfected with increasing doses of ASO- A2 (FIG. 3C) or ASO-A6 (FIG. 3D), and 40 pM non-targeted ASO control (-). FL* denotes a naturally occurring isoform. Other potential spliced products indicated with asterisk (*). Products were amplified with primers specific to exon 1 and exon 4 in FIG. 3C and exons 4 and 7 in FIG. 3D and separated by PAGE. FIG. 3E: RT-PCR quantification of full-length NLRP3 mRNA relative to the predominant ASO-mediated isoforms. ASO potency was calculated using the half maximal inhibitory concentration (IC50) after plotting the data on a non-linear regression curve with a standard slope; ASO-A2 n=3; ASO-A6 n=l-2. FIG. 3F: ELISA analysis of IL-ip and TNF-a released from THP-1 cells transfected with increasing doses (2.5 pM-40 pM) of ASO-A2 or ASO-A6 and activated with LPS and ATP. Cytokine levels were normalized to LPS and ATP stimulated cells. Slopes were calculated by linear regression analysis; *P<0.05, ***P<0.001; n=2-3 across 3 independent experiments. FIG. 3G: ELISA analysis of IL-ip released from THP- 1 cells treated with MCC950 and activated with LPS and ATP. Data are presented as mean ± SEM and analyzed by one sample t-test compared to LPS / ATP set to 1; *P<0.05; n=2 independent experiments.

[0031] FIGS 4A-4H show AV / yG-targ eted ASOs induce exon skipping and reduce inflammasome signaling in mouse iBMDMs. FIG. 4A: Representative radioactive RT-PCR analysis of Nlrp3 RNA expression in iBMDMs transfected with the indicated ASOs (40 pM), including a nontargeted ASO control (C) followed by activation with LPS and ATP. Products were amplified using specific primer sets for each exon and separated by PAGE. Arrowheads indicate predominant ASO-induced spliced isoform for each ASO. Some ASOs also induced a low level of other alternatively spliced products. FIG. 4B: Quantification of full-length Nlrp3 mRNA relative to the predominant ASO-mediated isoforms. Data are presented as mean ± SEM, unpaired t-test compared to matched ASO control, **P<0.01, ***P<0.001, ****P<0.0001; n=4. FIG. 4C: Representative immunoblot of NLRP3, Pro-CASPl (p45) and cleaved CASP1 (p20) in iBMDM lysates and CASP1 (p20) in iBMDM supernatant treated with indicated ASOs (40 pM) or untreated, followed by LPS or LPS and ATP activation, a-tubulin was analyzed as a control. Non-specific bands are denoted with asterisk (*). FIG. 4D: Quantification of immunoblots of NLRP3 shown normalized to a-tubulin control. Data shown as mean ± SEM and analyzed by one sample t-test compared to non-targeted ASO control (ASO-C) set to 1, *P<0.05, **P<0.01; n=2-3. FIG. 4E: Quantification of immunoblots of cleaved CASP1 (p20) secreted from iBMDM treated with ASOs and activated with LPS and ATP or untreated and activated with LPS, relative to ASO-C. Data are presented as mean ± SEM and analyzed by one sample t-test compared to ASO-C set to 1 ; *P<0.05, ***P<0.001, ****P<0.0001; n=2-3. IL-10 (FIG. 4F), LDH (FIG. 4G) and TNF-a (FIG. 4H) released from iBMDMs untreated or treated with ASOs and activated with LPS or LPS and ATP. Cytokine levels were normalized to LPS and ATP stimulated cells. Data are presented as mean ± SEM and analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test relative to LPS for TNF-a and to ASO-C for IL-10 and LDH. n= 4-6; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0032] FIGS. 5A-5F show murine ASO-A2 modulates Nlrp3 splicing in a dose-dependent manner in iBMDM. FIG. 5A: Splicing analysis of Nlrp3 isoform expression in iBMDM transfected with increasing doses of ASO-A2 (2.5pM-20pM) (n=3), representative of one experiment. FIG. 5B: ASO-A2 (SEQ ID NO: 8) shown base-paired to its target sequence (SEQ ID NO: 45) at the Nlrp3 exon 2 (capital letters) / ! ntron 2 (lowercase) junction. The 5’ splice site (ss) of exon 2 is shown with an arrow. FIG. 5C: Illustration of ASO-A2 mechanism of action showing activation of a cryptic 5’ ss in exon 2 that shifts the open reading frame and results in a premature termination codon in exon 3. Boxes are exons and lines are introns. ASO binding site is labeled. Diagonal black line shows splicing of introns. Red diagonal line indicates ASO- induced splicing event. AUG and UGA indicate the translational initiation and termination codon, respectively. FIG. 5D: IL- 10 and TNF-a in the supernatant of iBMDMs transfected with increasing doses of ASO-A2 and activated with LPS and ATP (n=3). ASO potency was calculated using the half maximal inhibitory concentration (IC50) after plotting the data on a nonlinear regression curve with a standard slope. At least 3 independent experiments. FIG. 5E: RT-PCR analysis of correctly spliced exons (full-length) from Nlrp3 mRNA relative to the predominant ASO-mediated isoform from iBMDM cells transfected with increasing doses of ASO-A2 (2.5 pM-40 pM). ASO potency was determined using the half maximal inhibitory concentration (IC50) after fitting the data using a nonlinear regression curve with a standard slope; n=3 independent experiments. FIG. 5F: ELISA analysis of IL-10 and TNF-a secretion from iBMDM cells transfected with increasing doses of ASO-A2 (2.5 pM-40 pM) and activated with LPS and ATP. Cytokine levels were normalized to LPS and ATP stimulated cells. Individual data points are presented as mean ± SEM. The slopes of the dose response curves were calculated by linear regression analysis, **P<0.01; n=3 independent experiments.

[0033] FIGS. 6A-6E show ASO-induced Nlrp3 alternative exon 2 splicing reduces systemic inflammation in vivo. FIG. 6A: Schematic of dosing regimen of 7-8 week-old female wildtype (WT) mice. Mice were treated three times with either PBS vehicle (n=3), non-targeted control ASO 100 mg / kg (n=2), or ASO-A2 100 mg / kg (n=3) followed by LPS or treated 3 times with ASO-A2 100 mg / kg (n=4) followed by PBS. FIG. 6B: Splicing analysis of full-length Nlrp3 expression relative to partial A2 isoform in the liver using primers flanking exon 1-3 region. Partial A2 isoform further validated (middle gel) with a primer that binds across the new cryptic splice site in exon 2 and 0-actin included as loading control. ELISA analysis of IL-ip (FIG. 6C), IL-6 (FIG. 6D), and TNF-ot (FIG. 6E) in the plasma of the mice described in FIG. 6A. PBS vehicle (-) and non-targeted control ASO (C) were included as controls. Data are presented as mean ± SEM. Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test relative to PBS vehicle control; **P<0.01, ***P<0.001, ****P<0.0001.

[0034] FIG. 7A-7I show ASO-A2 prolongs survival and alleviates systemic inflammation in CAPS mice. FIG. 7A: Schematic of dosing regimen in Nlrp3D301N +LysM re+CAPS neonates treated with ASO-A2 or PBS control. Untreated wildtype, unaffected mice were used as controls. FIG. 7B: Kaplan-Meier survival curve of mice treated with ASO-A2 (n=l 1), PBS control (n=l 1) and untreated wildtype (WT; n=12). The day that 50% of the mice for each group were alive is shown (50% survival). Curve comparison for ASO-A2 (n=l 1) vs. PBS control (n=l 1) Gehan- Breslow-Wilcoxon test; ****P<0.0001. FIG. 7C: Images of representative ASO and control- treated pnrp^D301N / +Ly^i(:r(!+m'ce atp0Stnataiday 8 (P8) and P 13. FIG. 7D: Representative images ofH&E staining of skin of ASO-A2-treated ]\[irp^D30lN / +LysMCre+ (n=7)5PBS control- treated (n=4), or untreated WT (n=4) mice. Scale bars, 50 pm top row and 10 pm in the magnified inset. Black arrows denote representative neutrophil morphology. FIG. 7E: Quantification of neutrophil infiltration of skin from ASO-A2 -treated mice (n=7), PBS control- treated (n=4), and untreated WT (n=4) mice. One-way ANOVA with Tukey’s multiple comparisons test; *P<0.05, **P<0.01, ***P<0.001. ELISA analysis of IL-ip (FIG. 7F), IL-18 (FIG. 7G), IL-6 (FIG. 7H), and TNF-a (FIG. 71) levels in the serum of ASO-A2 (n=7), PBS control treated CAPS pups (n=7), and untreated WT pups (n=4) collected at Pl 2. Data are mean ± SEM, one-way ANOVA followed by Tukey’s post-hoc analysis; **P<0.01, ***P<0.001, nonsignificant (ns).

[0035] FIG. 8A-8E show modeling of NLRP3 Aex6 and Aex8 proteins. The NLRP3A6 and A8 isoforms are predicted to affect the stability of the inactive decamer cage assembly and potentially the NLRP3-NEK7 interaction in the active disk assembly. FIG. 8A: Comparison of the inactive decameric cage form of full-length NLRP3 (left, PDB id 7pzc; (21)) and Alphafold2 models of the A6 and A8 isoforms superimposed onto the full-length structure. FIG. 8B: Depiction of the two unique interaction surfaces of the decameric NLRP3 cage assembly between the concave (blue) and convex (red) faces of the LRR domains (left). The interactions mapped onto a linear diagram of the NLRP3 proteins reveals that the A6 isoform deletes a region of the LRR involved in the convex interaction (green box) and that the A8 isoform deletes most of the concave interaction region (green box). Both isoforms, but particularly the A8 isoform, are predicted to disrupt the stability of the decameric cage. In the case of the A6 isoform, the deletion could cause weakened convex interactions and alter the concave interactions by changing the spacing of the LRR repeats C-terminal to the deletion. FIG. 8C: Comparison of the active disk form of full-length NLRP3 (left, PDB id 8ej4; (42)) and Alphafold2 models of the A6 and A8 isoforms superimposed onto the full-length structure. FIG. 8D: Depiction of the two unique interaction surfaces of the active decameric NLRP3 disk assembly between adjacent NLRP3 monomers (red) and NEK7 (blue) (left). The interactions mapped onto NLRP3 protein linear diagrams suggests that both LRR deletions are unlikely to affect the decameric disk formation, as these interactions are mediated by the FIS and NACHT domains that are not affected in the A6 and A8 isoforms. In contrast, the A8 isoform eliminates many of the residues involved in the full- length LRR-NEK7 interaction (green box), suggesting a weaken interaction. FIG. 8E: Protein modeling of full-length NLRP3 and ASO-mediated partial A6 using SWISS-MODEL.

[0036] FIGS. 9A-9C show ASO-A2 NLRP3 exon 2 splicing in human monocyte-derived macrophages (hMDM) from persons with CAPS. FIG. 9A: Radioactive RT-PCR analysis of NLRP3 isoform expression in NLRP3Lj5 P +hMDM cells transfected with 40 pM of ASO-A2 or non-targeted ASO control (C) prior to activation with LPS. Other potential spliced products are denoted with asterisk (*). FL* denotes a naturally occurring full-length isoform. Products were amplified using a forward primer specific for NLRP3 exon 1 and a reverse primer in exon 4 and separated by PAGE. FIG. 9B: Quantification of correctly spliced exons (full-length) from NLRP3 mRNA relative to the predominant ASO-A2 induced isoforms. Data are presented as mean ± SEM, unpaired t-test compared to matched control, *P<0.05; N=3 patients. FIG. 9C: IL- ip secretion from hMDMs treated with ASO-A2 relative to control treated. Data are presented as mean ratio ± SEM and analyzed by one sample t-test set to 1, *P<0.05; n=4 representing results of experiments using cells from three different patients, and cells from one patient analyzed in two independent experiments.

[0037] FIGS. 10A-10E show analysis of weights and splicing in ASO-A2-treated CAPS mice. FIG. 10A: Growth curve of ASO-A2 (n=l 1) and PBS control (n=l 1) treated Nlrp3D301N / + Lvs ICrc mice and untreated wildtype (WT; n=12) mice. FIG. 10B: Representative image of ASO- A2- treated Nlrp3DMN / + LrsMCre+and WT mice at postnatal day 21 (P21). FIG. IOC: (top) Splicing analysis of full-length Nlrp3 expression relative to partial A2 isoform in the liver using primers in exon 1 and 3. (middle) Amplification of the exon 2 skipped isoform only, using a primer that base pairs across the ASO-induced cryptic 5’ splice site in exon 2. (bottom) -actin amplicon analyzed as a control of loading. Non-specific PCR products are denoted with asterisks (*). FIG. 10D: Spleen size relative to total body weight of ASO-A2 and PBS control treated Nlrp3D30IN / +LySMCre+ pUpS atp ] 2 / P 13 (n=7). Data are mean ± SEM, unpaired t-test. FIG. 10E: Weight at day 12. Nlrp3D301N+ / LysMCre+PBS, n=12; Nlrp3D301N+ / LysMCre+ASO, n=14.

[0038] FIGS. 11 A-l ID show murine ASO-A6 modulates Nlrp3 splicing in a dose response manner in iBMDM. FIG. 11A: Sequence alignment of ASO-A6 (SEQ ID NO: 46) to the 5’ splice site (ss) of exon 6 (SEQ ID NO: 47) located across exon 6-intron 6 junction. FIG. 1 IB: Splicing analysis of Nlrp3 isoform expression in iBMDM transfected with increasing doses of ASO-A6 (2.5pM-20pM) (n=3), representative of one experiment. Non-specific bands denoted by *. FIG. 11C: Visual illustration of ASO-A6 mechanism of action showing full skipping of exon 6 from final transcript. FIG. 11D: IL-ip and TNF-a in the supernatant of iBMDMs transfected with increasing doses of ASO-A6 and activated with LPS and ATP (n=3). ASO potency was calculated using the half maximal inhibitory concentration (IC50) after plotting the data on a nonlinear regression curve with a standard slope. At least 3 independent experiments.

[0039] FIGS. 12A and 12B show ASO-modulated Nlrp3 isoform expression in liver. Schematic of ASO dosing in WT pups collected at P16 for splicing analysis. ASO-A2 (FIG. 12A) and ASO- A6 (FIG. 12B) modulated Nlrp3 isoform expression in the liver compared to untreated controls. Untreated, n=2; ASO-A2, n=3; ASO-A6, n=2. FL, full length; P, postnatal; S.Q., subcutaneous. Data are presented as mean ± SEM, unpaired t-test; *P<0.05.

[0040] FIGS. 13A-13C show pro-inflammatory cytokine expression in the liver following LPS injection. FIG. 13A: RT-PCR of full-length Nlrp3 and P-actin included as loading control. FIG. 13B: Quantification of total Nlrp3 RNA normalized to P-actin using ImageJ. ASO-A2 PBS, n=3; PBS LPS, n=3; ASO-C LPS, n=2; ASO-A2 LPS, n=3. Data are presented as mean ± SEM and analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test relative to PBS; *P<0.05, **P<0.01. FIG. 13C: Immunoblotting of IL-ip expression in the liver following LPS injection. Untreated, n=2; PBS LPS, n=2; ASO-C LPS, n=2; ASO-A2 LPS, n=3. At least two independent experiments. FIGS. 14A-14C show neonatal ASO ICV injection modulates Nlrp3 exon 2 skipping in the hippocampus. FIG. 14A: Splicing analysis o Nlrp3 partial A2 expression in the hippocampus two weeks after ASO ICV injection in neonatal mice (Pl) ipsilateral and contralateral to the site of injection compared to an untreated control. Untreated P14, n=l; ASO-A2 P14, n=2. FIG. 14B: Splicing analysis of Nlrp3 partial A2 expression in the hippocampus 14 days, 30 days, and 60 days post ASO ICV injection compared to untreated controls. Full-length Nlrp3 mRNA expression quantified relative to ASO-mediated partial A2 isoform by radioactive RT-PCR. Untreated P14, n=2; ASO-A2 P14, n=2; ASO-A2 P30, n=2; ASO-A2 P60, n=2. FIG. 14C: Immunoblotting of NLRP3 expression in hippocampus 14 days, 30 days, and 60 days post ICV ASO injection compared to untreated and ASO-C-treated mice. Untreated Pl 4, n=2; ASO-C P14, n=2, ASO-A2 P14, n=2; Untreated P30, n=2; ASO-A2 P30, n=2; untreated P60, n=l. ASO- A2 P60, n=2. Quantification of NLRP3 relative to a-tubulin included as a loading control using ImageJ.

[0041] DETAILED DESCRIPTION

[0042] Provided herein are splice-switching antisense oligonucleotides (ASOs) and methods for treating inflammatory diseases mediated by the NLRP3 inflammasome. Splice-switching ASOs provide a specific and effective therapeutic strategy for suppressing NLRP3 -mediated inflammatory signaling by modulating NLRP3 expression at the RNA level through alternative splicing. Through in vitro experiments using human THP-1 cells and mouse iBMDMs, the ability of these ASOs to induce exon skipping, resulting in the generation of truncated NLRP3 protein isoforms lacking functional domains essential for inflammasome assembly and activation, was demonstrated. Human NLRP3 exon 2 targeted ASO induced skipping of the natural AUG codon in exon 2 and decreased full-length NLRP3 protein expression. This approach prevented NLRP3 protein expression altogether in a non-permeant manner rather than attempting to inhibit its activation, potentially offering a superior therapeutic strategy. Partial exon 6 skipping resulted in deletion of a small segment of the LRR domain (774-794 aa) and impaired inflammasome function in THP-1 cells. ASO-mediated partial exon 8 skipping shifted the reading frame and resulted in a premature termination codon in exon 9, making the transcript susceptible to nonsense-mediated mRNA decay (NMD) or protein degradation.

[0043] ASO therapy also prolonged survival and decreased systemic inflammation in a mouse model of cryopyrin-associated periodic syndrome (CAPS) expressing a missense mutation in Nlrp3 (D301N). ASO-A2-treated mice exhibited decreased IL-ip, IL-18, and IL-6 levels in the serum compared to PBS controls, suggesting ASO-mediated NLRP3 inhibition against constitutive inflammasome activation in vivo. Notably, improved skin lesions and reduction in neutrophil infiltration in the skin reveal the potential efficacy of ASO-based approaches in addressing other inflammatory skin diseases. Overall, ASO-mediated reduction in functional NLRP3 inflammasome signaling can therapeutically reduce disease-severity in CAPS mouse model. Currently, biological IL-ip treatments for CAPS must be injected daily or every several weeks and have limited efficacy in most severe cases. ASO may offer CAPS patients more specific, long-lasting treatment options that would improve their quality of life.

[0044] Definitions

[0045] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. However, two or more copies are also contemplated. The singular forms “a,” “and,” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0046] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0047] As used herein, the term “one or more” refers to, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or a greater number, if desired for a particular use.

[0048] The term “and / or” as used in a phrase such as “A and / or B” herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0049] As used herein, a “nucleic acid” or a “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and / or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793- 800 (Worth Pub. 1982)). The present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. The term “nucleic acid” or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and / or non- nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or double-stranded, and represent the sense or antisense strand. The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence,” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0050] The term “complementary” refers to specific binding between polynucleotides based on the sequences of the polynucleotides. Portions of polynucleotides are complementary to each other if they follow conventional base-pairing rules, e.g., A pairs with T (or U) and G pairs with C, although small regions (e.g., fewer than about 3 bases) of mismatch, insertion, or deleted sequence may be present. As used herein, an ASO and a pre-mRNA are complementary if they bind to each other in a hybridization assay under physiological conditions, with a Tm substantially greater than 45° C., preferably at least 50° C., and typically 60° C.-800C. or higher. Such hybridization preferably corresponds to stringent hybridization conditions. At a given ionic strength and pH, the Tm is the temperature at which 50% of a target sequence hybridizes to a complementary polynucleotide. Again, such hybridization may occur with “near” or “substantial” complementarity of the antisense oligomer to the target sequence, as well as with exact complementarity. Thus, a sequence is complementary if they bind to each other in a hybridization assay under stringent conditions, even in the presence of less than 100% complementarity due to the presence of mismatches, insertions, or deletions of bases.

[0051] As used herein, the term “antisense oligonucleotide (ASO)” refers to an oligonucleotide that is capable of interacting with and / or hybridizing to a pre-mRNA or an mRNA having a sufficiently complementary nucleotide sequence thereby modifying gene expression. Tn one embodiment, the ASOs have at least one antisense portion which hybridizes to the pre-mRNA. The antisense portion may be of any length which allows hybridization to the pre-mRNA, e.g., 10 to 50 nucleobases in length, or any range therewithin.

[0052] As used herein, the terms “pre-messenger RNA,” “precursor mRNA,” or “pre-mRNA” refer to an immature single strand of messenger ribonucleic acid (mRNA) that contains one or more intervening sequence(s) (introns). Pre-mRNA is transcribed by an RNA polymerase from a DNA template in the cell nucleus and is comprised of alternating sequences of introns and coding regions (exons). Once a pre-mRNA has been completely processed by the splicing out of introns and joining of exons, it is referred to as “messenger RNA” or “mRNA,” which is an RNA that is comprised exclusively of exons. Eukaryotic pre-mRNAs exist only transiently before being fully processed into mRNA. When a pre-mRNA has been properly processed to an mRNA sequence, it is exported out of the nucleus and eventually translated into a protein by ribosomes in the cytoplasm.

[0053] As used herein, the term “splice skipping” refers to the modification of pre-mRNA splicing by the targeting of splice donor and / or acceptor and branch sites within a pre-mRNA with one or more antisense oligonucleotide(s) (ASOs). By blocking access of a spliceosome to one or more splice donor, acceptor, or branch site, an ASO can prevent a splicing reaction thereby causing, for example, the exclusion or truncation of one or more exons from an mRNA. Splice skipping may include the masking of key sequences involved in the splicing of targeted exons by using antisense oligonucleotides (ASO) that hybridize to splice donor / acceptor, branchpoint sequences and / or by overlapping exon / intron boundaries within a pre-mRNA.

[0054] An “effective amount” refers to an amount sufficient to elicit a desired biological response (e.g., treating a condition). As will be appreciated by those skilled in the art, the effective amount may vary depending on such factors as the desired biological endpoint, the pharmacokinetics, the condition being treated, the mode of administration, and the age and health of the subject. An effective amount encompasses therapeutic and prophylactic treatment. For example, a “therapeutically effective amount” is an amount sufficient to provide a therapeutic benefit in the treatment of a condition, or to delay or minimize one or more symptoms associated with the condition. In some embodiments, a therapeutically effective amount is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to minimize one or more symptoms associated with the condition. A therapeutically effective amount means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the condition, or enhances the therapeutic efficacy of another therapeutic agent.

[0055] A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes as described herein. Likewise, subject may include either adults or juveniles (e.g., children). Moreover, subject may mean any living organism, preferably a mammal (e.g., human or non-human) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment, the mammal is a human.

[0056] A “vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, e.g., an “insert,” may be attached or incorporated so as to bring about the replication of the attached segment in a cell.

[0057] As used herein, “treat,” “treating,” and the like means a slowing, stopping, or reversing of progression of a disease or disorder when provided a peptide or composition described herein to an appropriate subject. The term also includes a reversing of the progression of such a disease or disorder to a point of eliminating or greatly reducing the disease. As such, “treating” means an application or administration of the peptides or compositions described herein to a subject, where the subject has a disease or a symptom of a disease, where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease or symptoms of the disease.

[0058] As used herein, the terms “providing,” “administering,” “introducing,” are used interchangeably herein and refer to the placement into a subject by a method or route which results in at least partial localization to a desired site. Administration can be by any appropriate route which results in delivery to a desired location in the subject.

[0059] The term “contacting” as used herein refers to bring or put in contact, to be in or come into contact. The term “contact” as used herein refers to a state or condition of touching or of immediate or local proximity. Contacting may occur by any means known to the skilled artisan.

[0060] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0061] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0062] Antisense Oligonucleotides

[0063] The disclosure provides antisense oligonucleotides (ASOs) which result in modulated splicing of NLRP3, also referred to herein as splice-switching antisense oligonucleotides. ASOs are short, synthetic, modified nucleic acids that base-pair to a target pre-RNA and modulate its expression in a manner that depends on the specific ASO design. Splice-switching ASOs basepair to pre-mRNA and alter splicing by blocking the activity of splicing regulatory sequences. In some embodiments, the splice-switching ASOs target exons which result in NLRP3 dysfunction or degradation. In some embodiments, the splice-switching ASOs target exons which result in an NLRP3 protein with decreased stability (e g., stability in the oligomeric form). In some embodiments, the splice-switching ASOs induce alternative splicing of exons 2, 3, 5, 6, 7, or 8.

[0064] The ASOs may comprise a sequence that is sufficiently complementary to any splice donor site, branch site, or splice acceptor site within the pre-mRNA coding NLRP3. Donor sites (5' end of the intron or 3’ splice site of exon), branch sites (near the 31end of the intron) and acceptor sites (31end of the intron or 5’ splice site of exon) facilitate splicing of the pre-mRNA into mature mRNA. In some embodiments, the splice-switching ASOs comprise a sequence complementary to the donor site or 3’ splice site of exon, the branch site, or acceptor site or 5’ splice site of exon for any of exon 2, 3, 5, 6, or 8 of the pre-mRNA coding NLRP3. When targeting a splice site, for example, the ASO may include a sequence having its 5' end complementary to 1 to about 25 base pairs downstream of a normal splice acceptor junction in a pre-mRNA. In some embodiment, the ASO may include a sequence which encompasses the splice site or spans a splice acceptor or donor site when hybridized to the pre-mRNA. In some embodiments, the splice-switching ASOs comprise a sequence sufficiently complementary to an exon-intron boundary of pre-mRNA encoding for NLRP3 (e.g., an exonintron boundary of exon 2, 3, 5, 6, or 8 of a pre-mRNA encoding for NLRP3).

[0065] The ASO need not exhibit complete complementarity to the target region of NLRP3 pre- mRNA, e.g., the target splice donor site, branch site, splice acceptor site, or exon-intron boundary, provided that there is sufficient complementarity to result in specific hybridization / binding. Thus, the entire sequence, or only a portion, of the ASO may be complementary to the target region of the NLRP3 pre-mRNA. For example, in an oligonucleotide having 20 to 30 bases, about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 may be complementary to the target region. The ASOs may comprise at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% sequence complementarity to the target region of the NLRP3 pre-mRNA. The complementary region may be formed of contiguous bases, but may alternatively be formed of non-contiguous sequences that when placed together, e.g., from opposite ends of the oligonucleotide, constitute sequence that spans the target sequence.

[0066] In select embodiments, the ASO induces alternative splicing of exon 2 in a pre-mRNA encoding for NLRP3. In some embodiments, the ASO blocks splicing at the 5’ splice site of exon 2. For example, the ASO may be complementary and specifically bind to the 5’ splice site of exon 2 and / or adjacent sequence to block splicing at the 5’ splice site. The ASO may be complementary to the region comprising the exon 2-intron 2 boundary.

[0067] In select embodiments, the ASO induces alternative splicing of exon 6 in a pre-mRNA encoding for NLRP3. In some embodiments, the ASO blocks splicing at the 3’ splice site of exon 6. For example, the ASO may be complementary and specifically bind to the 3’ splice site of exon 6 and / or adjacent sequence to block splicing at the 3’ splice site. The ASO may be complementary to the region comprising the exon 6-intron 5 boundary. In some embodiments, the ASO blocks splicing at the 5’ splice site of exon 6. For example, the ASO may be complementary and specifically bind to the 5’ splice site of exon 6 and / or adjacent sequence to block splicing at the 5’ splice site. The ASO may be complementary to the region comprising the exon 6-intron 6 boundary. In select embodiments, the ASO induces alternative splicing of exon 8 in a pre-mRNA encoding for NLRP3. In some embodiments, the ASO blocks splicing at the 5’ splice site of exon 8. For example, the ASO may be complementary and specifically bind to the 5’ splice site of exon 8 and / or adjacent sequence to block splicing at the 5’ splice site. The ASO may be complementary to the region comprising the exon 8-intron 8 boundary.

[0068] In some embodiments, the splice-switching antisense oligonucleotides (ASOs) comprise any one of SEQ ID NOs: 1-14. The ASOs may comprise a sequence of SEQ ID NOs: 1-14, wherein one or more of the thymidines are substituted with uridines. The oligonucleotides may comprise about 15 to 30 nucleotides. In select embodiments, the oligonucleotides comprise about 18 to about 25 nucleotides. In exemplary embodiments, the oligonucleotides comprise 25 nucleotides.

[0069] The ASOs may comprise one or more additional nucleotides in addition to SEQ ID NOs: 1-14. For example. The ASOs may comprise one or more additional nucleotides at the 5’ or 3’ ends of any of SEQ ID NOs: 1-14. Additionally or alternatively, the ASOs may comprise one or more nucleotide substitutions as compared to any of SEQ ID NOs: 1-14, in so far as the ASOs retain sufficient complementarity to bind to the target region of NLRP3, e.g., an intron / exon boundary of NLRP3. In some embodiments, the oligonucleotides may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions as compared to SEQ ID NOs: 1-14.

[0070] The ASOs may be modified using one or more moieties to decrease nuclease degradation, increase stability, or increase effectiveness. The ASO may include one or more modified nucleosides having a modified sugar moiety (e.g., 2’-O-methyl, 2’-fluoro, morpholino) and / or a modified nucleobase (e.g., hypoxanthine, 7-deaza-G, 2 / 6-diamino-purine, 4-thio-uracil, difluorotoluene). For example, the ASOs may be modified using one or more modified internucleoside linkages, an intercalating group, reactive groups, groups that modify the charge and / or hydrophilicity to increase delivery to cells, saccharides, and the like.

[0071] In some embodiments, the ASOs comprise one or more modified internucleoside linkages. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphates, which contain a phosphodiester bond, phosphotriesters, methylphosphonates, phosphoramidates, phosphorothioates, phosphorodithioates, and boranophosphonate. Modified internucleoside linkages, compared to naturally occurring phosphate linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. In select embodiments, the ASO is a phosphorodiamidate morpholino oligonucleotide (PMO). The terms “morpholino oligomer” or “PMO” (phosphorami date or phosphorodiamidate morpholino oligomer) refer to an oligonucleotide analog composed of morpholino subunit structures, where the structures are linked together by phosphorus-containing linkages, one to three atoms long, preferably two atoms long, and preferably uncharged or cationic, joining the morpholino nitrogen of one subunit to a 5' exocyclic carbon of an adjacent subunit, and each morpholino ring bears a purine or pyrimidine or an equivalent base-pairing moiety effective to bind, by base specific hydrogen bonding, to a base in a polynucleotide. Variations can be made to this linkage as long as they do not interfere with binding or activity. For example, the oxygen attached to phosphorus may be substituted with sulfur (thiophosphorodiamidate). The 5' oxygen may be substituted with amino or lower alkyl substituted amino. The pendant nitrogen attached to phosphorus may be unsubstituted, monosubstituted, or disubstituted with (optionally substituted) lower alkyl. In some embodiments, the morpholino binds to an RNA target which blocks translation of the RNA target into a protein. In other embodiments, the morpholino prevents aggregation of the RNA target with itself or with other cellular RNAs, proteins, or riboproteins, such as, but not limited to, RNAs, proteins, and riboproteins associated with the cellular mRNA splicing apparatus.

[0072] A “phosphoramidate” group comprises phosphorus having three attached oxygen atoms and one attached nitrogen atom, while a “phosphorodiamidate” group comprises phosphorus having two attached oxygen atoms and two attached nitrogen atoms. In some embodiments, in the uncharged or the modified intersubunit linkages one nitrogen is always pendant to the backbone chain, whereas the second nitrogen, in a phosphorodiamidate linkage, is the ring nitrogen in a morpholino ring structure.

[0073] The PMO may be modified. For example, the PMO may comprise phosphorodiamidate morpholino oligomers comprising any number of (l-piperazino)phosphinylideneoxy, (l-(4-(co- guanidino-alkanoyl))-piperazino)phosphinylideneoxy linkages that have been described previously as PMO+ (see e.g., U.S. Pat. No. 7,943,762). The PMO may comprise at least one (B) linkage or at least one of the disclosed terminal modifications, referred to as PMO-X, (see e.g., U.S. Pat. Nos. 8,779,128 and 8,779,128).

[0074] The ASOs may be attached to a cell-penetrating peptide (CPP), e.g., to enhance delivery. The ASO-CPP may comprise multiple ASOs attached to a single CPP. The ASOs may be attached to the CPP with a cleavable linker. The ASOs described herein encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to a subject is capable of providing (directly or indirectly) the biologically active form thereof.

[0075] The disclosure also provides nucleic acids encoding the ASOs, vectors containing these segments, and cells containing the vectors. The vectors may be used to propagate the segment in an appropriate cell and / or to allow expression from the segment (e.g., an expression vector). The person of ordinary skill in the art would be aware of the various vectors available for propagation and expression of a nucleic acid sequence.

[0076] Viral and non-viral based gene transfer methods can be used to introduce the ASOs or nucleic acids encoding the ASOs herein into cells, tissues, or a subject. Such methods can be used to administer nucleic acids to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, cosmids, RNA (e.g., a transcript of a vector described herein), a nucleic acid, and a nucleic acid complexed with a delivery vehicle. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Viral vectors include, for example, retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors.

[0077] In certain embodiments, plasmids that are non-replicative, or plasmids that can be cured by high temperature may be used, such that the nucleic acid may be removed from the cells under certain conditions.

[0078] In certain embodiments, vectors of the present disclosure can drive the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, Nature (1987) 329:840, incorporated herein by reference) and pMT2PC (Kaufman, et al., EMBO J. (1987) 6:187, incorporated herein by reference). When used in mammalian cells, the expression vector's control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd eds., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference.

[0079] Vectors of the present disclosure can comprise any of a number of promoters known to the art, wherein the promoter is constitutive, regulatable or inducible, cell type specific, tissue- specific, or species specific. In addition to the sequence sufficient to direct transcription, a promoter sequence of the invention can also include sequences of other regulatory elements that are involved in modulating transcription (e.g., enhancers, Kozak sequences and introns). Many promoter / regulatory sequences useful for driving constitutive expression of a gene are available in the art and include, but are not limited to, for example, CMV (cytomegalovirus promoter), EFla (human elongation factor 1 alpha promoter), SV40 (simian vacuolating virus 40 promoter), PGK (mammalian phosphoglycerate kinase promoter), Ubc (human ubiquitin C promoter), human beta-actin promoter, rodent beta-actin promoter, CBh (chicken beta-actin promoter), CAG (hybrid promoter contains CMV enhancer, chicken beta actin promoter, and rabbit betaglobin splice acceptor), TRE (Tetracycline response element promoter), Hl (human polymerase III RNA promoter), U6 (human U6 small nuclear promoter), and the like. Additional promoters that can be used for expression of the components of the present system, include, without limitation, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Maloney murine leukemia virus (MMLV) LTR, myeoloproliferative sarcoma virus (MPSV) LTR, spleen focus- forming virus (SFFV) LTR, the simian virus 40 (SV40) early promoter, herpes simplex tk virus promoter, elongation factor 1- alpha (EF 1 -a) promoter with or without the EF 1 -a intron. Additional promoters include any constitutively active promoter. Alternatively, any regulatable promoter may be used, such that its expression can be modulated within a cell.

[0080] Moreover, inducible and tissue specific expression of a nucleic acid can be accomplished by placing the nucleic acid encoding such a molecule under the control of an inducible or tissue specific promoter / regulatory sequence. Examples of tissue specific or inducible promoter / regulatory sequences which are useful for this purpose include, but are not limited to, the rhodopsin promoter, the MMTV LTR inducible promoter, the SV40 late enhancer / promoter, synapsin 1 promoter, ET hepatocyte promoter, GS glutamine synthase promoter and many others. Various ubiquitous as well as tissue-specific promoters and tumor-specific are commercially available, for example from InvivoGen. In addition, promoters which are well known in the art can be induced in response to inducing agents such as metals, glucocorticoids, tetracycline, hormones, and the like, are also contemplated for use with the invention. Thus, it will be appreciated that the present disclosure includes the use of any promoter / regulatory sequence known in the art that is capable of driving expression of the desired protein or RNA operably linked thereto. The vectors of the present disclosure may direct expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Such regulatory elements include promoters that may be tissue specific or cell specific. The term “tissue specific” as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., seeds) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue. The term “cell type specific” as applied to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue. The term “cell type specific” when applied to a promoter also means a promoter capable of promoting selective expression of a nucleotide sequence of interest in a region within a single tissue. Cell type specificity of a promoter may be assessed using methods well known in the art, e.g., immunohistochemical staining.

[0081] Additionally, the vector may contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in host cells; enhancer / promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; 5 ’-and 3 ’-untranslated regions for mRNA stability and translation efficiency from highly- expressed genes like a-globin or P-globin; SV40 polyoma origins of replication and ColEl for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA; a “suicide switch” or “suicide gene” which when triggered causes cells carrying the vector to die (e.g., HSV thymidine kinase, an inducible caspase such as iCasp9), and reporter gene for assessing expression of the chimeric receptor. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art. Selectable markers also include chloramphenicol resistance, tetracycline resistance, spectinomycin resistance, streptomycin resistance, erythromycin resistance, rifampicin resistance, bleomycin resistance, thermally adapted kanamycin resistance, gentamycin resistance, hygromycin resistance, trimethoprim resistance, dihydrofolate reductase (DHFR), GPT; the URA3, HIS4, LEU2, and TRP1 genes of S. cerevisiae.

[0082] Vectors according to the present disclosure can be transformed, transfected, or otherwise introduced into a wide variety of host cells. Transfection refers to the taking up of a vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, lipofectamine, calcium phosphate co-precipitation, electroporation, DEAE-dextran treatment, microinjection, viral infection, and other methods known in the art. Transduction refers to entry of a virus into the cell and expression (e.g., transcription and / or translation) of sequences delivered by the viral vector genome. In the case of a recombinant vector, “transduction” generally refers to entry of the recombinant viral vector into the cell and expression of a nucleic acid of interest delivered by the vector genome.

[0083] Any of the vectors comprising a nucleic acid sequence that encodes the ASOs disclosed herein is also within the scope of the present disclosure. Such a vector may be delivered into host cells by a suitable method. Methods of delivering vectors to cells are well known in the art and may include DNA or RNA electroporation, transfection reagents such as liposomes or nanoparticles to delivery DNA or RNA; delivery of DNA, RNA, or protein by mechanical deformation (see, e.g., Sharei et al. Proc. Natl. Acad. Sci. USA (2013) 110(6): 2082-2087, incorporated herein by reference); or viral transduction. In some embodiments, the vectors are delivered to host cells by viral transduction. Nucleic acids can be delivered as part of a larger construct, such as a plasmid or viral vector, or directly, e.g., by electroporation, lipid vesicles, viral transporters, microinjection, and biolistics (high-speed particle bombardment). Similarly, the vector can be delivered by any method appropriate for introducing nucleic acids into a cell.

[0084] Additionally, delivery vehicles such as nanoparticle- and lipid-based mRNA or protein delivery systems can be used. Further examples of delivery vehicles include lentiviral vectors, ribonucleoprotein (RNP) complexes, lipid-based delivery system, gene gun, hydrodynamic, electroporation or nucleofection microinjection, and biolistics. Various gene delivery methods are discussed in detail by Nayerossadat et al. (Adv Biomed Res. 2012; 1 : 27) and Ibraheem et al. (Int J Pharm. 2014 Jan 1 ;459( 1 -2):70-83), incorporated herein by reference.

[0085] Also disclosed herein are compositions comprising one or more of the disclosed ASOs or nucleic acids encoding one or more of the disclosed ASOs.

[0086] The compositions may further comprise excipients or pharmaceutically acceptable carriers. The choice of excipients or pharmaceutically acceptable carriers will depend on factors including, but not limited to, the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. The term “pharmaceutically acceptable carrier,” as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material, surfactant, cyclodextrins or formulation auxiliary of any type.

[0087] Excipients and carriers may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. Some examples of materials which can serve as excipients and / or carriers are sugars including, but not limited to, lactose, glucose and sucrose; starches including, but not limited to, com starch and potato starch; cellulose and its derivatives including, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients including, but not limited to, cocoa butter and suppository waxes; oils including, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; including propylene glycol; esters including, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents including, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants including, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants. The compositions of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Techniques and formulations may be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).

[0088] The administration route and the form of the composition will dictate the type of carrier to be used. The composition may be in a variety of forms, suitable, for example, for systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral injections) or topical administration (e.g., dermal, pulmonary, nasal, aural, ocular, liposome delivery systems, or iontophoresis).

[0089] Any of the above compositions disclosed herein may further comprise at least one additional therapeutic agent.

[0090] Methods

[0091] The disclosure also provides methods for inhibiting the NLRP3 inflammasome, methods for decreasing NLRP3 signaling or activation, and methods for modulating the expression of NLRP3 (e.g., decrease mRNA or protein levels) in a cell. The methods comprise contacting a cell with one or more of the disclosed ASOs, nucleic acid(s) encoding one or more of the disclosed ASOs, or a composition comprising thereof. In some embodiments, the methods and / or the disclosed ASOs inhibit the NLRP3 inflammasome, decrease NLRP3 signaling or activation, and decrease the expression of NLRP3 (mRNA or protein) by at least about 10%, by at least about 20%, by at least about 25%, by at least about 30%, by at least about 40%, by at least about 50%, by at least about 60%, by at least about 70%, by at least about 75%, by at least about 80%, by at least about 85%, by at least about 90%, by at least about 95%, by at least about 98%, by at least about 99%, or by 100%.

[0092] The cell may be any type of cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell (e.g., a human cell).

[0093] In some cases, the cell is in culture or in vitro (e.g., immortalized cell line). Cells may be from established cell lines or they may be primary cells, where “primary cells,” “primary cell lines,” and “primary cultures” are used interchangeably herein to refer to cells and cells cultures that have been derived from a subject and allowed to grow in vitro for a limited number of passages of the culture. For example, primary cultures are cultures that may have been passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enough times go through the crisis stage. Typically, the primary cell lines are maintained for fewer than 10 passages in culture. In some embodiments, the cell is ex vivo (e.g., fresh isolate - early passage).

[0094] In some embodiments, the cell is in vivo. In some embodiments, the contacting comprises administering to a subject. In some embodiments, the subject has a disease or disorder mediated by NLRP3 or the NLRP3 inflammasome.

[0095] Thus, the disclosure also provides methods for treating disease or disorders mediated by NLRP3 or the NLRP3 inflammasome. The methods comprise administering an effective amount of one or more of the disclosed ASOs, nucleic acid(s) encoding one or more of the disclosed ASOs, or a composition comprising thereof, to a subject in need thereof. In some embodiments, the subject is a human.

[0096] In some embodiments, the disease or disorder is an inflammatory disease or disorder. In some embodiments, the disease or disorder is an inflammatory disease or disorder. Inflammatory diseases are characterized by activation of the immune system in a tissue or an organ to abnormal levels that may lead to abnormal function and / or disease in the tissue or organ. In some embodiments, the disease or disorder is an autoinflammatory disease or disorder. Autoinflammatory diseases are caused by an abnormal activation of the innate immune system, without the production of autoantibodies, leading to chronic inflammation. Exemplary inflammatory and autoinflammatory diseases and disorders include, but are not limited to, arthritis, rheumatoid arthritis, asthma, inflammatory bowel disease (Crohn's disease or ulcerative colitis), chronic obstructive pulmonary disease (COPD), allergic rhinitis, vasculitis (polyarteritis nodosa, temporal arteritis, Wegener's granulomatosis, Takayasu's arteritis, or Behcet’s syndrome), inflammatory neuropathy, psoriasis, systemic lupus erythematosus (SLE), chronic thyroiditis, Hashimoto's thyroiditis, Addison's disease, polymyalgia rheumatica, Sjogren's syndrome, Churg-Strauss syndrome NLRP12-associated disease, mevalonate kinase deficiency, PFAPA (periodic fever syndrome, aphthous stomatitis, pharyngitis, and cervical adenitis) TRAPS (tumor necrosis factor (TNF) receptor-associated periodic fever syndrome), gout, chronic recurrent multifocal osteomyelitis (CRMO), Schnitzler syndrome, DIRA, interleukin-36-receptor antagonist deficiency (DITRA), Sweet syndrome, neutrophilic panniculitis, Mevalonate kinase deficiency or Muckle-Wells syndrome, NLRP12- associated disease, Blau syndrome, familial hemophagocytic lymphohistiocytosis (HLH), Adultonset Still’s Disease (AOSD), Hyper-IgD Syndrome (HIDS), Pyrin-Associated Autoinflammatory Syndrome (PAS A), and rheumatic fever. In some embodiments, the disease or disorder comprises an inflammatory component, for example chronic diseases such as atherosclerosis, type 2 diabetes, neurodegenerative disorders, e.g., Alzheimer’s disease and Parkinson’s disease. In some embodiments, the disease or disorder is an inflammatory skin disease or disorder.

[0097] In select embodiments, the disease or disorder is cryopyrin-associated periodic syndrome (CAPS). The CAPS spectrum includes mild, moderate, and severe phenotypes. The mild phenotype is called familial cold autoinflammatory syndrome (FCAS), the moderate phenotype is also known as Muckle-Wells syndrome (MWS), and the neonatal-onset multisystem inflammatory disease (NOMID) / chronic infantile neurologic cutaneous articular syndrome (CINCA) describes the severe phenotype. The disclosed methods may be suitable for use in treating any of the mild, moderate, and severe phenotypes pf CAPGS. The CAPS phenotypes display unspecific and unique clinical signs. Dermatologic, musculoskeletal, ocular, otologic, and neurologic disease symptoms combined with chronic systemic inflammation are characteristic.

[0098] The disclosed methods may decrease any of the dermatologic, musculoskeletal, ocular, otologic, and neurologic symptoms separate from or in addition to inflammation. The disclosed methods may decrease the dermatologic, musculoskeletal, ocular, otologic, and neurologic symptoms during inflammatory flares, decrease the duration of inflammatory flares, decrease the number of occurrences of inflammatory flares, or decrease the severity of the inflammatory flares. The disclosed methods may also lessen the potential of flares or decrease the inflammatory response due to common triggers, including cold, stress, infections, or trauma and lack of sleep. In some embodiments, the methods decrease inflammatory responses, decrease neutrophilic urticaria, fever / subfebrile temperature, fatigue, influenza-like muscle pains, hearing loss, conjunctivitis or other ocular symptoms, myalgia, arthralgia, or other musculoskeletal symptoms, headaches, or other dermatologic, musculoskeletal, ocular, otologic, and neurologic symptoms.

[0099] In some embodiments, the disease or disorder is neurodegenerative disease or disorder. A neurodegenerative disease refers to a central nervous system disease characterized by progressive, normally gradual, loss of functional neural tissue. Non-limiting examples of neurodegenerative diseases include frontotemporal dementia (FTD), Parkinson’s disease (PD), Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), Friedreich's ataxia, Multiple sclerosis, Niemann-Pick disease, Huntington's disease, transmissible spongiform encephalopathy, Charcot- Marie-Tooth disease, dementia with Lewy bodies (DLB), corticobasal degeneration, progressive supranuclear palsy, Bell’s palsy, neuronal ceroid lipofuscinoses, and hereditary spastic paraparesis.

[0100] In some embodiments, the disease or disorder is a metabolic disease or disorder. Metabolic diseases and disorders are characterized by a negative alteration in the processing and distribution of macronutrients, such as proteins, fats, and carbohydrates. Exemplary metabolic diseases and disorders include, but are not limited to, obesity, insulin-resistance, diabetes, metabolic syndrome, alcoholic steatohepatitis, and NAFLD.

[0101] A beneficial effect of the disclosed methods can easily be assessed by one of ordinary skill in the art and / or by a trained clinician who is treating the subject. Preferably, there is a positive or beneficial difference in the severity or occurrence of at least one clinical or biological score, value, or measure used to evaluate patients who have been treated with the methods of the present invention as compared to those that have not. It is not required that all effects of the disease or disorder be entirely prevented or reversed. As such, a therapeutic benefit is not necessarily a complete treatment, but rather, can encompass a result which includes reducing or preventing the symptoms, reducing or preventing the occurrence of such symptoms (either quantitatively or qualitatively), reducing the severity of such symptoms or physiological effects thereof, and / or enhancing the recovery after experiencing symptoms.

[0102] The compounds and compositions disclosed herein may be administered to a subject by a variety of methods. In any of the uses or methods described herein, administration may be by various routes known to those skilled in the art, including without limitation oral, inhalation, intravenous, intramuscular, topical, subcutaneous, systemic, and / or intraperitoneal administration to a subject in need thereof. In some embodiments, the ASOs or compositions thereof are administered by intravenous, subcutaneous, intravitreal, and / or intrathecal injection. When intravenous administration is used, and it can be continuous intravenous infusion over a period of a few minutes to an hour or more.

[0103] The specific dose level may depend upon a variety of factors including the activity of the ASO, the age, body weight, general health, and diet of the subject, time of administration, route of administration, and the nature and / or symptoms of the disease, and will be ultimately at the discretion of the attendant physician or clinician. It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the symptoms to be treated and the route of administration. Further, the dose, and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.

[0104] A wide range of second therapies may be used in conjunction with the compounds of the present disclosure. The second therapy may be administration of an additional therapeutic agent or may be a second therapy not connected to administration of another agent. Such second therapies include, but are not limited to, hearing aids, physiotherapy, orthopedic devices.

[0105] The second therapy may be administered at the same time as the initial therapy, either in the same composition or in a separate composition administered at substantially the same time as the first composition. In some embodiments, the second therapy may precede or follow the treatment of the first therapy by time intervals ranging from hours to months.

[0106] In some embodiments, a therapeutically effective amount of a compound disclosed herein, or compositions thereof, is administered alone or in combination with a therapeutically effective amount of at least one additional therapeutic agent. In some embodiments, effective combination therapy is achieved with a single composition or pharmacological formulation that includes both agents, or with two distinct compositions or formulations, administered at the same time or separated by a time interval, wherein one composition includes a compound of this invention, and the other includes the at least one additional therapeutic agent.

[0107] In some embodiments, the at least one additional therapeutic agent comprises an immune modulator, anti-IL-1 treatments (e.g., anakinra, canakinumab, and rilonacept), a steroid, nonsteroidal anti-inflammatory drugs for pain and fever, an analgesic, an antimicrobial agent, an immunotherapy, or a combination thereof.

[0108] EXAMPLES

[0109] MATERIALS AND METHODS

[0110] Antisense oligonucleotides Splice switching antisense oligonucleotides having a phosphorodiamidate morpholino (PMO) backbone were purchased from Gene-Tools, LLC (Table 1). A non-targeting PMO was used as a negative control (Gene Tools, standard control oligo). ASOs (18-25 mers) with a phosphorothioate (PS) backbone and 2’ methoxyethyl (MOE) sugar moiety were purchased from IDT. Non-targeted MOE was used as a negative control. BLAST analysis showed no off-target hybridization. Lyophilized ASOs were formulated in fdtered deionized H2O, 0.9% saline, or Dulbecco's phosphate-buffered saline (DPBS). ASO- induced NLRP3 isoforms were confirmed by Sanger sequencing. Analysis of potential off-target base-pairing of human and mouse ASO-A2 (gggenome.dbcls.jp), found no other matches in the human or mouse genomes that had fewer than four (human) or three (mouse) nucleotide mismatches and no more than 13 (human) or 11 (mouse) contiguous nucleotides. ASO AX denotes the targeted exon.

[0111] Cell culture and transfections THP-1 cells were obtained from ATCC (TIB -202) and cultured in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS) and 0.05 mM 2-Mercaptoethanol (BME) at 37 °C and 5% carbon dioxide (CO2). THP-1 cells were seeded at SxlO3cells / mL in a 24 well plate, differentiated with 50 ng / mL phorbol 12-myristate 13-acetate (PMA) for 24 hours, and allowed to rest for 18 hours before transfection. Immortalized bone-marrow derived macrophages (iBMDMs) (a gift from Dr. Venkat Magupalli and Dr. Hao Wu) were cultivated in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% FBS at 37 °C and 5% CO2. iBMDM were plated at 40-50% confluency in a 6 well plate and incubated for 18 hours. ASOs with a PMO chemistry were transfected into THP-1 and iBMDM at a final concentration of 40 pM using 9 pM and 6 pM Endo-Porter, respectively, for 48 hours according to manufacturer instructions (Gene Tools, LLC). Human monocyte-derived macrophages (hMDM) from CAPS patients harboring a L353P mutation in NLRP3 were separated by Percoll-gradient (Cytiva) centrifugation and subsequently differentiated with 20 ng / mL macrophage colony-stimulating factor (M-CSF) for 7 days. On day 6, ASO-A2 or ASO-C (40 pM final concentration) for 24 hours using Endo-Porter (Gene Tools LLC). Cells were then stimulated with 200 ng / mL LPS (Escherichia coli 0111 :B4, InvivoGen) for ~16 hours and supernatants were collected for IL-10 ELISA analysis. The lysate was collected for splicing analysis.

[0112] Inflammasome activation assay PMA-differentiated THP-1 cells and iBMDMs were transfected with ASOs as described above and incubated for 48 hours. Cells were then primed with 100 ng / mL LPS (Escherichia coli O55:B5, Sigma) in the appropriate supplemented media for 3 hours, after which time. Media was subsequently removed and cells were activated with 5 mM ATP (Invivogen) in OptiMem (ThermoFisher) for 1 hour. Cells in OptiMem (Thermo Fisher Scientific) were treated with 100 nM MCC950 (Invivogen) for 30 minutes prior to activation with 5 mM ATP for 1 hour. Media was collected for analysis of released signaling molecules and lysate was collected for RNA and protein analysis.

[0113] RNA isolation and analysis Total RNA was extracted from cells or mouse tissue using TRIzol according to the manufacturer's instructions (Invitrogen). RNA (Ipg) was reverse transcribed using GoScript reverse transcriptase (Promega) and oligo-dT primer. RT-PCR and radiolabeled RT-PCR of cDNA was performed using GoTaq Green (Promega) with or without alpha32P-deoxycytidine triphosphate (dCTP), and primers for NLRP3 (Table 1). PCR products were resolved on either a 2% agarose gel stained with ethidium bromide or a 6% non-denaturing polyacrylamide gel and quantified using Image J or a Typhoon FLA 7000 phosphorimager (GE Healthcare). The percent of the NLRP3 mRNA including the targeted exon was calculated relative to the abundance of the predominant ASO-induced isoform after the signal was corrected for the amplicon size or for the number of cytosine nucleotides for agarose and polyacrylamide gels, respectively.. Predominant ASO-induced isoforms were isolated from agarose gel, column purified (Cytiva), and sanger sequenced with primers for NLRP3 (Table 1).

[0114] Real-time qPCR Real-time qPCR was performed with PrimeTime Gene Expression Master Mix (IDT) and pre-designed NLRP3 and 0-actin probes (IDT; Table 1). Real-time PCR was performed on an Applied Biosystems (ABI) ViiA 7 Real-Time PCR System with the following thermal-cycling protocol: stage l-50°C for 2 min, 95°C for 3 min; stage 2 - 40 cycles of 95°C for 15 s, 60°C for 1 min. The relative abundance was calculated as (2 -(AACt))_

[0115] Inmmnoblot analysis Cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris-Cl pH 7.6, 1% NP-40, 0.5% Na-DOC, 0.1% SDS) containing protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Lysates were cleared by centrifugation at 10,000 x g for 20 min at 4 °C and protein concentration was determined by Bradford or Pierce BCA protein assay (1863028, 23225, Thermo Fisher Scientific). Supernatant was centrifuged using 10 kDa Ami con Ultra Centrifugal Filters (Millipore). Lysates and supernatants were prepared with 4x Bolt lithium dodecyl sulfate (LDS) and lOx dithiothreitol (DTT) and boiled in 95 °C for 5 minutes. Cell lysates were separated by 4-15% SDS-polyacrylamide electrophoresis (PAGE) tris-glycine precast gels (Bio-Rad) and supernatants were separated by 12% SDS-PAGE tris-glycine gels and both were transferred to a 0.45 [im immobilon-FL polyvinylidene difluoride (PVDF) blotting membrane (Millipore) at 75V for 1 hour. Membranes were blocked with 5% nonfat dry milk (NFDM) in Tris-buffered saline (TBS) supplemented with 0.1% Tween 20 (TBST) for 1 hr and probed with primary antibodies overnight at 4 °C. The following antibodies were used: rabbit NLRP3 (1 : 1000, D4D8T-15101, Cell Signaling), mouse caspase 1 (1 : 1000, Casper 1- AG-20B- 0042, Adipogen), goat IL-ip (1:800, AF-401-SP, R&D), rabbit a-tubulin (1 : 1000, 11224-1-AP, Proteintech). Rabbit a-tubulin (Proteintech) was incubated for 1 hr at room temperature. Horseradish peroxidase (HRP)-conjugated rabbit, mouse, and goat secondary antibodies (Invitrogen, 1 : 5000-1 :20, 000) were used for 1 hour at room temperature. Immunoblots were developed using classico (Immobilon) or SuperSignal West Femto (Thermo Scientific) substrate and exposed to film for imaging in the dark room. Western blot bands were quantified using ImageJ software.

[0116] ELISA and LDH assay Supernatant, serum, plasma, and tissue were analyzed for IL-ip (human DY200, mouse DY401), IL-6 (mouse DY406), IL-18 (mouse DY7625), and TNF-a NF- a (human DY210, mouse DY410) using ELISA kits (R&D) according to manufacturer’s instructions. Optical density (450 nm - 540 nm) values are normalized to LPS and ATP stimulated cells and utilized to extrapolate the concentration from a standard curve in vivo. LDH release was measured with a CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega) and the percent of LDH was calculated relative to LPS and ATP stimulated cells. Mice All protocols met ethical standards for animal experimentation and were approved by the Institutional Animal Care and Use Committee of Rosalind Franklin University of Medicine and Science (RFUMS) and University of California in San Diego. Animals were maintained at 23 °C with a 12 h light / dark cycle with food and water available ad libitum. Wildtype C57BL / 6 mice were purchased from The Jackson Laboratory and bred at RFUMS. Mice bearing an aspartate 301 to asparagine (D301N) substitution were generated as previously described (The Jackson Laboratory: ! 6 A29-Nlrp3D301NneoR / }, strain no. 017971). The D301N point mutation results in a conformational change that leads to a ligand-independent constitutive activation of the mutant NLRP3 inflammasome. Due to the presence of an intronic- floxed neomycin resistance cassette, Nlrp3 gene expression is abolished. Expression of the mutant allele is only achieved when the Nlrp3 knock-in mice are bred with mice expressing Cre recombinase. Nlrp3 knock-in mice were bred with mice expressing Cre recombinase under the control of lysozyme promoter (CreL; The Jackson Laboratory, strain no. 004781, B6.129P2- l.\ z2lm Kcrejlfo ]), resulting in constitutive activation of mutant Nlrp3 in the myeloid lineage.

[0117] LPS challenge C57BL / 6 female mice (7-8 weeks old) were intraperitoneally (I P.) injected with ASO-A2 (100 mg / kg), ASO-C (100 mg / kg), or PBS on day 1,3, and 6. On day 7, mice were I.P. injected with 20 mg / kg ultrapure LPS (TLR-4, Invivogen) or PBS. Three hours later, the treated mice were euthanized, blood was collected in EDTA-coated tubes through cardiac puncture, and tissues were snap frozen in liquid nitrogen and stored in -80 °C until further analysis. For all but one mouse, blood was diluted 1 : 1 in PBS and added to Ficoll and centrifuged at 400 x g for 30 minutes. For one mouse, blood was collected in EDTA tube and centrifuged 2000 x g for 10 minutes. Plasma was collected and stored in -80 °C until analysis.

[0118] Sex as a biological variable Females were examined in the LPS challenge because they exhibited a greater immune response compared to male mice in pilot studies. However, the findings could apply to males and females because NLRP3 is expressed in both sexes. In the CAPS mouse model experiment, both male and female mice were used.

[0119] CAPS mouse model b / l / p3i' ''i"'''( rimicewere injected with ASO-A2 (100 mg / kg) or PBS by subcutaneous injection on Pl, P3 and then every 3 days until P24. Mice were monitored daily for growth and survival. A group of mice were euthanized at P12 and tissue and serum were collected. Tissue was snap frozen in liquid nitrogen, and stored in -80 °C until further analysis. Treated mice were monitored for growth and survival. Histological staining analysis Mouse skin lesions from the nape were collected following euthanization. Hair around the lesion was removed using hair removal cream (Nair) with a gauze-tipped applicator. After 24 hours of fixation in 10% Buffered Formalin, the paraffin-embedded skin lesions were stained with hematoxylin and eosin (H&E). The slides were then reviewed by a blinded pathologist to identify the dermis and neutrophil infiltration. Scans of all sections were taken using an Olympus VS200 Slide Scanner (UCSD Neuroscience Microscopy Core) at 40X magnification. The number of neutrophils in a single section from each mouse were quantified using a cell classifier on Qupath v0.5.1.

[0120] Structure modeling Models of the human NLRP3 A6 and A8 isoforms were initially generated from the predicted protein sequences by Alphafold2. The folds of the individual domains of the resulting models matched that of the experimental NLRP3 structures; however, the resulting conformation did not precisely match that of the known active or inactive conformations, which are related by a rigid body motion between the FISNA-NBD-HD 1 N- terminal subdomains of the NACHT domain and the WH-HD2-LRR domains. To build models of the A6 and A8 isoforms in the known active and inactive conformations, the Alphafold2- predicted A6 and A8 leucine-rich repeats were superimposed onto the LRR repeats of the full- length active conformation (PDB id 8ej4) and the full-length inactive conformation (PDB id 7pzc) (25). This analysis was performed by first calculating the affine transformation matrices to superimpose residues 651-683 of the Alphafold2 models onto residues 651-683 for each of the NLRP3 monomers in the inactive and active assemblies; this residue range includes NLRP3 regions involved in the WH-HD2-LRR rigid body motion but avoids residues deleted in the A6 and A8 isoforms. The affine transformations were then applied to the entire Alphafold2 modeled A6 and A8 LRR domains (residues 651-1034) and the transformed LRR domains were merged with residues 1-650 of the experimental structures.

[0121] Statistical analysis All data were analyzed GraphPad prism (version 9.5.1) and presented as the mean ±SEM (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). For comparison of two groups, a two-tailed Student’s t-test, with a P<0.05 considered significant, was utilized. For more than 2 groups, one way ANOVA followed by post-hoc analysis, as detailed in figure legends, was utilized. ASO potency was calculated using the half maximal inhibitory concentration (IC50) after plotting the data on a nonlinear regression curve with a standard slope. Survival curve comparison was analyzed by Gehan-Breslow-Wilcoxon test. The specific statistical test used for each experiment is detailed in the figure legends. EXAMPLE 1

[0122] Splice-switching ASOs induce NLRP3 exon skipping and suppress inflammasome activation in human THP-1 cells.

[0123] To identify the most effective approach to reducing NLRP3 activity using spliceswitching ASOs, exon 2, exon 3, and exons 5-9 were targeted (FIG. 1A). Phosphorodiamidate morpholinos (PMO) ASOs that base-pair at the 3’ or 5’ splice site regions (ss) of exon 2, 3, 5, 6, 7, 8, and 9 were designed. PMOs were transfected into differentiated, macrophage-like THP-1 cells. ASO-induced skipping of exon 2 was predicted to remove the canonical start codon and ASO-induced skipping of exons 3, 5, 6, 7, 8, and 9 was predicted to yield mRNAs with intact openreading frames that encode protein isoforms with potentially altered functions. The effect of inducing the different NLRP3 isoforms and decreasing full-length NLRP3 using ASOs was measured by activating the inflammasome complex and measuring cytokine release. A nontargeted ASO (ASO-C) as a control for non-specific effects.

[0124] Cells were primed with lipopolysaccharide (LPS) and subsequently activated with ATP to initiate the NLRP3 inflammatory response. RNA and protein were collected from the cells and splicing was analyzed by reverse transcription PCR (RT-PCR) and sequencing of the resulting amplicons. Treatment with ASO-A2, targeting exon 2, resulted in splicing from an alternative 5’ss in the upstream untranslated region of exon 2 to the 3’ss of exon 3, which splices out the natural start codon of the transcript. ASO-A3, A5, A7, and A9 induced skipping of their respective exons. ASO-A6, targeting the 3’ss of the exon, activated an in-frame, cryptic 3’ss. ASO-A8 activated an out-of-frame 5’ss, resulting in partial exon 8 skipping and creation of a stop codon in exon 9 (FIGS. 1A and IB). Some ASOs also induced a very low level of additional splicing events. Overall, treatment of cells with ASOs targeting exons 2, 3, 5, 6, 7, and 8, caused a significant decrease in full-length NLRP3 mRNA compared to ASO-C (FIG. 1C).

[0125] NLRP3 protein from ASO-treated cells was analyzed by immunoblot analysis. The predicted change in molecular weight of the protein isoforms was relatively small (8-14 kDa reduction) relative to the size of the full-length protein (-120 kDa), resulting in potential difficulty in visualizing migration differences on the blot (FIG. 1A). ASO-induced splicing of exon 2 resulted in the appearance of a lower band representing the protein isoform encoded by the induced mRNA (FIG. 2A). Modulation of NLRP3 exons 2, 5, 6, and 8 splicing also resulted in a significant decrease in NLRP3 protein abundance (FIGS. 2A and 2B). To evaluate whether ASO-mediated exon skipping and consequent modulation of NLRP3 protein expression reduced the cellular inflammatory response, IL-ip release into the media was quantified following activation. Treatment of cells with ASO-A2, ASO-A6, and ASO-A8 significantly reduced IL-ip secretion from THP-1 cells in response to LPS and ATP (FIG. 2C). There were no changes in the secreted levels of tumor necrosis factor-a (TNF-a, TNF), a proinflammatory cytokine produced by activated macrophages independently of the NLRP3 pathway (FIG. 2D).

[0126] Further testing of the most active ASOs, ASO-A2 and ASO-A6, identified their half- maximal inhibitory concentration (IC50) as 1.68 pM and 3.62 pM, respectively (FIGS. 3A-3E). Treatment of cells with these ASOs resulted in a dose-dependent decrease in IL-ip secretion following activation with LPS and ATP (FIG. 3F). The level of inhibition of the inflammatory response was similar to that observed in cells treated with MCC950, a potent and specific small molecule inhibitor of NLRP3 (FIG. 3G). TNF-a levels were not decreased, suggesting that the ASOs were acting in an on-target manner specific to reducing NLRP3 activity without exerting an immunogenic effect (FIG. 3F).

[0127] To evaluate ASO-A2 and NLRP3 A2 in an in vitro model directly relevant to clinical, pathological NLRP3 activation, human monocyte-derived macrophages (hMDMs) from individuals with CAPS harboring an NLRP3 p.L353P mutation were treated with the ASO. ASO-A2 modulated NLRP3 exon 2 skipping in hMDMs, which correlates with a decrease in IL- ip release, confirming ASO activity in human primary cells and demonstrating its potential efficacy in blocking pathological NLRP3 activity (FIG. 9).

[0128] The mechanism ofNLRP3 inactivation varies for the different ASOs. ASO-A2 induces splicing out of the native translational initiation codon in exon 2 and thereby resulted in loss of NLRP3 translation. Protein modeling of NLRP3 A6 and A8 isoforms suggested that these deletions disrupt LRR-LRR interactions in the oligomerized form. Recent cryogenic electron microscopy studies shows human NLRP3 forming an oligomer consisting of 10 subunits, creating a double cage-like structure (FIG. 8). Neighboring LRR domains from the two discs interact with each other front and back. Protein modeling of NLRP3 isoforms indicated that the A6 isoform lacks a region of the LRR involved in the convex interaction and that the A8 isoform deletes most of the regions making up the concave interactions. Both isoforms, but particularly the A8 isoform, may disrupt the stability of the decameric cage. The A6 isoform could both weaken the convex interactions as well as alter the concave interactions by changing the spacing of the LRR repeats C-terminal to the deletion. Importantly, a single ASO-induced NLRP3 isoform monomer could potentially destabilize an oligomerized structure formed with full-length NLRP3, thereby exerting a dominant negative effect that could amplify the effect of the ASO treatment. The LRR deletion in NLRP3 A6 is unlikely to affect the formation of the decameric disk, as these interactions are mediated by the FIS and NACHT domains that are not predicted to be affected in the A6 and A8 isoform structure. In contrast, the A8 isoform lacks many of the amino acids involved in the full-length LRR-NEK7 interaction (FIG. 8). Mouse ASO-A6 and A8 induce skipping of the entire targeted exon, which results in attenuation of inflammasome activation. The may be due to deletions of segments of the LRR domain. Mouse NLRP3 subunits also form a dodecamer with interacting LRR domains to support complex stability. ASO- mediated deletions of LRR segments may destabilize the 12-subunit structure, resulting in less activity.

[0129] EXAMPLE 2

[0130] ASO-mediated modulation of Nlrp3 splicing mitigates inflammasome signaling in immortalized mouse macrophages.

[0131] To assess the in vivo effects of inducing NLRP3 spliced isoforms with ASOs, mousespecific ASOs that targeted the exon-intron junction of exon 2 (pyrin domain), 3 (linker domain), and 5-9 (LRR domains) were designed and tested in immortalized bone-marrow derived macrophages (iBMDMs) treated with LPS and ATP. ASOs were identified that induced skipping of exons 3, 5, 6, 8 and 9 and partial skipping of exon 2 (FIGS. 4A and 4B). ASOs that induced skipping of these exons resulted in a significant decrease in full-length NLRP3 protein and / or the appearance of smaller proteins appeared on NLRP3 immunoblots, likely representing the protein isoforms encoded by mRNA with the targeted exon skipped (FIGS. 4C and 4D). Treatment of cells with ASOs targeting the different exons also resulted in a significant decrease in cleaved CASP1 (p20) secretion (FIGS. 4C and 4E). Likewise, IL-ip secretion was significantly reduced following treatment of cells with all ASOs except ASO-A7 (FIG. 4F). Because inflammatory cell death occurs in iBMDMs in response to NLRP3 activation, lactate dehydrogenase (LDH), a marker of pyroptosis, was measured and a significant decrease in levels following treatment of all ASOs except ASO-A7, which did not effectively target exon 7, was found (FIG. 4G). LPS- dependent TNF-a levels in the media were unaffected by A7 / ' / G-targctcd ASO activity (FIG. 4H).

[0132] ASO-A2, one of the most active ASOs in iBMDMs, caused the use of an alternative 5’ splice site leading to an mRNA with a shifted open reading frame, creating a premature termination codon (PTC) in exon 3 (FIGS. 5A-5C). This ASO decreased full-length Nlrp3 mRNA expression (IC50 of 16 pM) and IL-ip release in a dose-dependent manner (FIGS. 5D- 5F).

[0133] EXAMPLE 3

[0134] Splice switching ASO treatment suppresses systemic inflammation in an LPS model of sepsis.

[0135] To examine the therapeutic potential of ASO-A2 in vivo, its efficacy was tested in a mouse model of acute inflammation that employs LPS, a potent pro-inflammatory endotoxin found in the outer membrane of gram-negative bacteria that activates innate immune signaling and thereby mimics sepsis, a life-threatening sequela of infection. Previous studies have shown that peripheral LPS injection induces an NLRP3 -dependent inflammatory response in mice. To assess ASO-A2 in this model, wildtype (WT) mice were treated with three doses of ASO-A2, ASO-C, or vehicle control by intraperitoneal (IP) injection on day 1, 3, and 6. On day 7, mice were injected with LPS or vehicle control and euthanized 3 hours later (FIG. 6A). Plasma from the mice treated with ASO-A2 had significantly reduced IL- 1 P and interleukin-6 (IL-6), a downstream pro-inflammatory cytokine, but not TNF-a, compared to controls (FIGS. 6C-6E). These results demonstrate the specific anti-inflammatory activity of ASO-induced Nlrp3 exon 2 skipping in vivo. In the liver, Nlrp3 and Pro-IL-ip upregulation but no cleaved IL- 1 P was observed (FIG. 13), suggesting that LPS alone may not be sufficient to activate NLRP3 in the liver. These results provide evidence for the anti-inflammatory activity of ASO-induced Nlrp3 exon 2 skipping in vivo.

[0136] EXAMPLE 4

[0137] ASO treatment prolongs survival and alleviates inflammation in a mouse model of CAPS.

[0138] To further assess the therapeutic efficacy of ASO-A2 in a more clinically relevant model, its ability to downregulate intrinsic NLRP3 hyperactivation in A / r GD301N+ LysMCre+mouse model was investigated. ^irp^D30I^ / +^y&MCr^ mice express constitutively active NLRP3 protein in the myeloid lineage. This mutation corresponds to the human D303N mutation which causes NOMID, a severe form of CAPS. 7i 3D301N+ LysMCre+mice develop severe multi-organ inflammation characterized by excessive secretion of proinflammatory cytokines, impaired growth, skin lesions, and perinatal death. Beginning at postnatal day 1 (Pl), 7 / 7?3D301N+ / LysMCre+pups were injected with ASO-A2 or vehicle control on postnatal (P) day 1, 3, and every 3 days until P24 or mouse death (FIG. 7A). (P<0.0001, Gehan-Breslow-Wilcoxon test), with 50% of mice surviving to P30 (maximum survival to P57) compared to controls (50% survival P15; maximum survival to P20) (FIG. 7B). Unaffected, WT pups all survived to the completion of the study (FIG. 7B). Body weight and spleen size were not significantly different between ASO- treated and control groups (FIG. 10). As rash is one of the earliest and most prominent manifestations of disease in people with CAPS, skin lesions in the mice were visually and histologically assessed, present mostly on the abdomen and neck, and lower neutrophil infiltration in skin of the ASO-D2-treated mice was observed compared to vehicle controls (FIGS. 7C-7E). In addition to skin inflammation, another hallmark of CAPS is end-organ systemic inflammation mediated by cytokine release in the blood, which is evident in the mice by the elevation of LL-1 and IL-18 but not IL-6 or TNF-a (FIGS. 7F-7I). ASO-A2-treated mice had levels of IL-1 P and IL- 18 similar to untreated, WT baseline expression, demonstrating that ASO-induced Nlrp3 exon 2 skipping has a systemic anti-inflammatory, NLRP3 -dependent effect in mice with a CAPS mutation associated with constitutive NLRP3 activation. ASO-induced Nlrp3 exon 2 splicing in the liver confirms ASO activity in the treated mice (FIG. 10C). These data demonstrate that ASO-mediated modulation of Nlrp3 exon 2 splicing ameliorates the clinical phenotype of CAPS in mice.

[0139] Table 1: Splice-switching Antisense Oligonucleotides and Primers

[0140]

[0141] The scope of the present invention is not limited by what has been specifically shown and described hereinabove. Those skilled in the art will recognize that there are suitable alternatives to the depicted examples of materials, configurations, constructions, and dimensions. Variations, modifications, and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and scope of the invention.

[0142] Numerous references, including patents and various publications, are cited and discussed in the description of this invention. The citation and discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety.

Claims

CLAIMSWhat is claimed is:

1. A method for inhibiting NLRP3 inflammasome and / or decreasing NLRP3 signaling or activation in a cell comprising contacting the cell with a splice-switching antisense oligonucleotide, or a composition thereof, targeting NLRP3.

2. A method for treating a disease or disorder mediated by the NLRP3 inflammasome in a subject in need thereof comprising administering an effective amount of a splice-switching antisense oligonucleotide, or a composition thereof, targeting NLRP3 to the subject.

3. The method of claim 2, wherein the disease or disorder is an inflammatory disease or disorder or comprises an inflammatory component.

4. The method of claim 2 or 3, wherein the disease or disorder is an inflammatory skin disease or disorder.

5. The method of any of claims 2-4, wherein the disease or disorder is cryopyrin-associated periodic syndrome (CAPS).

6. The method of any of claims 1-5, wherein the splice-switching antisense oligonucleotide comprises a sequence that is complementary to a splice donor site, a branch site, or a splice acceptor site within the pre-messenger RNA (pre-mRNA) coding NLRP3.

7. The method of any of claims 1-6, wherein the splice-switching antisense oligonucleotide is complementary to an exon-intron boundary of exon 2, 3, 5, 6, or 8 of a pre-mRNA encoding for NLRP3.

8. The method of any of claims 1-7, wherein the splice-switching antisense oligonucleotide induces alternative splicing of exon 2, 3, 5, 6, or 8 in a pre-mRNA encoding for NLRP3.

9. The method of any of claims 1-8, wherein the splice-switching antisense oligonucleotide induces alternative splicing of exon 2 in a pre-mRNA encoding for NLRP3.

10. The method of claim 9, wherein the antisense oligonucleotide is complementary to 5’ splice site of exon 2.

11. The method of any of claims 1-8, wherein the splice-switching antisense oligonucleotide induces alternative splicing of exon 6 in a pre-mRNA encoding for NLRP3.

12. The method of claim 11, wherein the splice-switching antisense oligonucleotide is complementary to 3’ splice site of exon 6.

13. The method of any of claims 1-8, wherein the splice-switching antisense oligonucleotide induces alternative splicing of exon 8 in a pre-mRNA encoding for NLRP3.

14. The method of claim 9, wherein the splice-switching antisense oligonucleotide is complementary to 5’ splice site of exon 8.

15. The method of any of claims 1-14, wherein the splice-switching antisense oligonucleotide comprises any one of SEQ ID NOs: 1-14.

16. The method of any of claims 1-15, wherein the splice-switching antisense oligonucleotide is modified with one or more intemucleoside linkages.

17. The method of any of claims 1-16, wherein the splice-switching antisense oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).

18. The method of any of claims 1-17, wherein the method inhibits the NLRP3 inflammasome and / or decreases NLRP3 signaling or activation by generating an NLPR3 protein with decreased functionality, decreased stability when an oligomer, or higher rates of degradation.

19. An antisense oligonucleotide comprising a sequence complementary to an exon-intron boundary of exon 2, 3, 5, 6, or 8 of a pre-messenger RNA (pre-mRNA) encoding for NLRP3, wherein hybridization of the antisense oligonucleotide to the pre-mRNA encoding for NLRP3 induces splice skipping.

20. The antisense oligonucleotide of claim 19, wherein the antisense oligonucleotide induces alternative splicing of exon 2 in a pre-mRNA encoding for NLRP3.

21. The antisense oligonucleotide of claim 20, wherein the antisense oligonucleotide is complementary to 5’ splice site of exon 2.

22. The antisense oligonucleotide of claim 19, wherein the antisense oligonucleotide induces alternative splicing of exon 6 in a pre-mRNA encoding for NLRP3.

23. The antisense oligonucleotide of claim 22, wherein the antisense oligonucleotide is complementary to 3’ splice site of exon 6.

24. The antisense oligonucleotide of claim 19, wherein the antisense oligonucleotide induces alternative splicing of exon 8 in a pre-mRNA encoding for NLRP3.

25. The antisense oligonucleotide of claim 24, wherein the antisense oligonucleotide is complementary to 5’ splice site of exon 8.

26. The antisense oligonucleotide of any of claims 19-25, wherein the antisense oligonucleotide comprises a sequence of any one of SEQ ID NOs: 1-14.

27. The antisense oligonucleotide of any of claims 19-26, wherein the antisense oligonucleotide is modified with one or more internucleoside linkages.

28. The antisense oligonucleotide of any of claims 19-27, wherein the antisense oligonucleotide is a phosphorodi ami date morpholino oligonucleotide (PMO).

29. A composition comprising an antisense oligonucleotide of any of claims 19-28 and a carrier.

30. An antisense oligonucleotide of any of claims 19-28, or composition comprising thereof, for use in inhibiting NLRP3 inflammasome and / or decreasing NLRP3 signaling.

31. An antisense oligonucleotide of any of claims 19-28, or composition comprising thereof, for use treating a disease or disorder mediated by the NLRP3 inflammasome.

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