In April 2024, Cell published a landmark review article titled “From Periphery to Center Stage: 50 Years of Advancements in Innate Immunity” to mark its 50th anniversary. The article systematically highlighted that innate immunity is not merely a defensive barrier, but a central system regulating adaptive immunity and maintaining homeostasis.[1] 

Therapeutic strategies targeting innate immune pathways have opened up new treatment avenues for infections, inflammatory disorders, and cancers. Notably, the review emphasized that NLRP3 inhibitors, with their potential for “one target, multiple disease control,” may emerge as the next-generation broad-spectrum anti-inflammatory drugs, offering revolutionary treatment options for chronic inflammation driven by myeloid cells.

Figure 1. Major Signaling Pathways of the Innate Immune System. [1]

The Inflammasome: A Key Signaling Hub in Innate Immunity

The innate immune system is the body’s first line of defense against pathogens, recognizing signals such as pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) via pattern recognition receptors (PRRs). This enables the rapid and broad recognition of disease-related cellular damage, necrosis, and dysfunction, thereby initiating protective immune responses.[2]

Among these mechanisms, the inflammasome—a cytosolic multiprotein complex formed with the participation of various PRRs—acts as a key regulator of innate immunity. It activates key pro-inflammatory cytokines such as interleukin-1β (IL-1β) and interleukin-18 (IL-18), and induces pyroptosis, a form of inflammatory programmed cell death mediated by cleavage of Gasdermin family proteins.[3]

Among the different types of inflammasomes, NLRP3 is the most extensively studied. This is due to its ability to recognize a wide range of danger signals—including bacterial toxins and metabolic disturbances—and its direct association or involvement with major diseases such as Alzheimer’s disease, gout, and diabetes.[4]

Figure 2. Classification, Assembly, and Activation of Inflammasomes. [4]

The NLRP3 Inflammasome: A Key Player in Inflammation

The NOD-like receptor family pyrin domain-containing 3 (NLRP3) protein is a key component of the inflammasome that plays a critical role in its activation. The NLRP3 protein region recognizes PAMPs or DAMPs, subsequently activating caspase-1 to promote the maturation and secretion of pro-inflammatory cytokines, thereby regulating the inflammatory response.[5]

Upon detecting intracellular stress or damage, inactive monomeric NLRP3 undergoes oligomerization to form an active conformation, assembling into a highly ordered inflammasome complex. This complex activates IL-1β, IL-18, and Gasdermin D (GSDMD), triggering downstream inflammatory responses and pyroptosis.[5–6]

Notably, gain-of-function (GOF) mutations in the NLRP3 gene can result in excessive production of IL-1β and IL-18, leading to autoinflammatory disorders such as cryopyrin-associated periodic syndromes (CAPS).[7]

Figure 3. Multiple Activation Mechanisms of the NLRP3 Inflammasome.[8]

NLRP3 Inhibitors: Next-Generation Anti-Inflammatory Therapeutics

Since NLRP3 lies upstream of key inflammatory cytokines such as IL-1β and IL-18, targeting and inhibiting its activation offers a “root cause” approach to disrupt the self-perpetuating cycle of chronic inflammation—providing a crucial strategy for treating inflammation-related diseases.[5]

By inhibiting this single molecule, NLRP3 inhibitors may deliver disproportionately broad therapeutic effects across a wide range of inflammatory conditions. This has sparked comparisons to antibiotics in infectious disease treatment—“one drug for many diseases”—especially in disorders driven by myeloid cell-mediated inflammation.[1]

Currently, there are over 100 drug development programs focused on NLRP3 inhibition, with clinical trials underway in areas such as neurology, autoimmunity, inflammation, and metabolism. Several candidates have already reached Phase III clinical trials.[8–9]

Of particular interest, NLRP3 has been shown to play a significant role in central nervous system (CNS) disorders such as Alzheimer’s and Parkinson’s disease, solidifying its potential as a high-value therapeutic target.

Figure 4. NLRP3 Inflammasome Pathway Inhibitors with Different Targeting Mechanisms and Their Potential Indications. [10]

Humanized Models: a Breakthrough for NLRP3-Targeted Drug Development

Transplanting human pathogenic mutations into homologous mouse genes is a widely used strategy for constructing disease evaluation models. However, early studies revealed that introducing disease-causing human NLRP3 mutations into the mouse genome often resulted in milder phenotypes compared to those observed in human disease manifestations.[11–12]

To overcome this limitation and achieve higher translational relevance, multiple research efforts have focused on replacing the endogenous mouse Nlrp3 gene with the human NLRP3 gene, including disease-associated variants. For example, a humanized mouse model (in which the mouse Nlrp3 gene was replaced) with the human NLRP3 gene carrying the D305N mutation exhibited acute sensitivity to endotoxins and developed progressive arthritis.[13]

One pharmaceutical company implemented this humanized mouse model to conduct screening and in vivo evaluation of a novel small-molecule NLRP3 inhibitor. Interestingly, the compound showed minimal inhibition of the mouse NLRP3 protein, but demonstrated nanomolar-level efficacy against the human NLRP3 protein. This discovery prompted researchers to use the NLRP3-humanized mouse model to further assess the inhibitor’s ability to suppress both wild-type NLRP3 activation and disease-associated D305N variants in vivo.[14]

 

Figure 5. Establishment of an NLRP3 Humanized Mouse Peritonitis Model for In Vivo Efficacy Testing of a Novel NLRP3 Inhibitor.[14]


In addition, NodThera, a company dedicated to developing NLRP3-targeted therapeutics, has developed multiple NLRP3-humanized mouse models and conducted comprehensive studies on the species-specific physiological effects. Their research highlights that, although the NLRP3 protein is relatively conserved between humans and mice, species-specific differences in protein expression and regulation are critical when evaluating the role of NLRP3 in autoinflammatory and autoimmune diseases.[15]

As such, NLRP3 humanized mouse models offer a more accurate prediction of therapeutic efficacy for NLRP3-targeted treatments and serve as a powerful tool for advancing drug development.

Figure 6. Responses of Different Types of NLRP3 Humanized Mice to LPS Stimulation.[15]

Cyagen’s NLRP3 Humanized Models: Powerful Research Tools Drug Discovery

To advance the in vivo study of NLRP3 mechanisms and accelerate the development of novel NLRP3-targeted therapies, Cyagen has developed a humanized NLRP3 mouse model — B6-hNLRP3 Mouse (Product No.: C001616). This model carries the entire human NLRP3 gene sequence, including the downstream 3’ UTR region, providing high translational relevance for drug discovery and disease modeling.

Below are the validation data for this model.

Functional Expression of Human NLRP3 Gene

In B6-hNLRP3 mice, the human NLRP3 gene is robustly expressed in vivo, while the endogenous mouse Nlrp3 gene is no longer expressed. The tissue-specific expression pattern of the human NLRP3 gene closely mirrors that of the native gene, ensuring physiological relevance for downstream studies.

Figure 7. Expression Analysis of Human NLRP3 and Mouse Nlrp3 Genes Across Tissues in B6-hNLRP3 and Wild-Type (WT) Mice.

Successful Expression of Human NLRP3 Protein

The B6-hNLRP3 mice successfully express the human NLRP3 protein. Protein expression levels are higher in lung tissue compared to thymus tissue, which is consistent with the mRNA expression profile observed through RT-qPCR analysis.

Figure 8. Detection of Human NLRP3 Protein Expression in Thymus and Lung Tissues of Wild-Type (WT) and B6-hNLRP3 Mice.
Note: The band observed in WT mice is due to cross-reactivity of the antibody. RT-qPCR results confirm that human NLRP3 protein is specifically expressed in B6-hNLRP3 mice.

Model Summary

The B6-hNLRP3 mouse model (Product No.: C001616) enables stable in vivo expression of both the human NLRP3 gene and protein. It is well-suited for studying a broad spectrum of NLRP3 inflammasome-related diseases, including inflammatory disorders, autoimmune diseases, neurodegenerative conditions, and metabolic diseases. Moreover, it serves as an ideal preclinical tool for evaluating NLRP3-targeted drug efficacy.

In addition, Cyagen has developed humanized mouse models for other key components of the NLRP3 inflammasome signaling pathway, including IL-1B, IL-1R1, IL-1RAP, IL-18, IL-18BP, IL-18R1 (IL-18RA), and IL-18RAP (IL-18RB).

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Cyagen’s Next-Generation Humanized Mouse Models: HUGO

Cyagen has launched the HUGO (Humanized Genomic Ortholog) Project, inviting global partners to collaborate on developing novel fully humanized models to support new drug development.

HUGO-GT™ Next-Generation Humanized Models

The Humanized Genomic Ortholog for Gene Therapy (HUGO-GT™) mouse model offers a higher degree of humanization compared to traditional models to serve as an effective evaluation platform, especially for gene therapy drugs with high requirements for gene sequence integrity, such as ASO, CRISPR, and siRNA.

Our HUGO-GTTM fully humanized genome mice are developed based on the proprietary TurboKnockout-Pro technology to achieve in situ replacement of mouse genes, encompassing a broader range of intervention targets and providing full coverage of pathogenic gene mutation sites-all without patent or ownership disputes. The fully humanized target genes within these models are consistent with the pathogenic genes carried by humans and cover the majority of drug targets, significantly enhancing screening efficiency for various types of preclinical drug experiments.


HUGO-Ab™ Mice for Fully Human Antibody Discovery

HUGO-AbTM (HUmanized Genomic Ortholog for Antibody Development) mice represent a leap forward in antibody discovery models:  with fully humanized genes in the antibody variable regions, these mice are capable of producing fully humanized antibodies in vivo with high affinity and low immunogenicity. Our TurboKnockout® ES technology replaces the VH and VL genes in situ to offer a higher degree of humanization with more stable phenotypic and functional outcomes in progeny than traditional transgenic methods.

HUGO-Ab™ Mouse Models for Antibody Discovery

* Fully human, full sequence diversity (Heavy, kappa, Lambda)

* Robust immune response for efficient discovery

* Human-like immune profile for vaccine development

* Numerous fixed light chain models

* Single domain models for monobody discovery

* Multiple backgrounds (B6, Balb/c, SJL)

High-throughput Fully Humanized Antibody Discovery Platform

Combining Biointron’s AbDrop™ with Cyagen’s HUGO-Ab™ mice streamlines the discovery of fully human antibodies to as few as 3 months. Our innovative High-throughput Fully Humanized Antibody Discovery Platform simplifies and accelerates drug development by eliminating the need for complex genetic modifications,  reducing costs, and leading to safer, more effective antibody therapies.



References
[1]Carpenter S, O'Neill LAJ. From periphery to center stage: 50 years of advancements in innate immunity. Cell. 2024 Apr 25;187(9):2030-2051.
[2]Pradeu T, Thomma BPHJ, Girardin SE, Lemaitre B. The conceptual foundations of innate immunity: Taking stock 30 years later. Immunity. 2024 Apr 9;57(4):613-631.
[3]Rathinam VA, Fitzgerald KA. Inflammasome Complexes: Emerging Mechanisms and Effector Functions. Cell. 2016 May 5;165(4):792-800.
[4]Ke Q, Greenawalt AN, Manukonda V, Ji X, Tisch RM. The regulation of self-tolerance and the role of inflammasome molecules. Front Immunol. 2023 Apr 4;14:1154552. 
[5]Swanson KV, Deng M, Ting JP. The NLRP3 inflammasome: molecular activation and regulation to therapeutics. Nat Rev Immunol. 2019 Aug;19(8):477-489.
[6]Andreeva L, David L, Rawson S, Shen C, Pasricha T, Pelegrin P, Wu H. NLRP3 cages revealed by full-length mouse NLRP3 structure control pathway activation. Cell. 2021 Dec 22;184(26):6299-6312.e22.
[7]Booshehri LM, Hoffman HM. CAPS and NLRP3. J Clin Immunol. 2019 Apr;39(3):277-286.
[8]Ma Q. Pharmacological Inhibition of the NLRP3 Inflammasome: Structure, Molecular Activation, and Inhibitor-NLRP3 Interaction. Pharmacol Rev. 2023 May;75(3):487-520.
[9]Yao J, Sterling K, Wang Z, Zhang Y, Song W. The role of inflammasomes in human diseases and their potential as therapeutic targets. Signal Transduct Target Ther. 2024 Jan 5;9(1):10.
[10]Zhang X, Wang Z, Zheng Y, Yu Q, Zeng M, Bai L, Yang L, Guo M, Jiang X, Gan J. Inhibitors of the NLRP3 inflammasome pathway as promising therapeutic candidates for inflammatory diseases (Review). Int J Mol Med. 2023 Apr;51(4):35.
[11]Hoffman HM. Autoinflammatory disease: New mouse models and therapies. J Allergy Clin Immunol. 2020 Jan;145(1):116-118.
[12]Putnam CD, Broderick L, Hoffman HM. The discovery of NLRP3 and its function in cryopyrin-associated periodic syndromes and innate immunity. Immunol Rev. 2024 Mar;322(1):259-282.
[13]Snouwaert JN, Nguyen M, Repenning PW, Dye R, Livingston EW, Kovarova M, Moy SS, Brigman BE, Bateman TA, Ting JP, Koller BH. An NLRP3 Mutation Causes Arthropathy and Osteoporosis in Humanized Mice. Cell Rep. 2016 Dec 13;17(11):3077-3088.
[14]Wilhelmsen, K., Deshpande, A., Tronnes, S., et al. (2024, December 22). Discovery of a Potent and Selective Inhibitor of Human NLRP3 with a Novel Binding Modality and Mechanism of Action. bioRxiv. https://doi.org/10.1101/2024.12.21.629867
[15]Koller BH, Nguyen M, Snouwaert JN, Gabel CA, Ting JP. Species-specific NLRP3 regulation and its role in CNS autoinflammatory diseases. Cell Rep. 2024 Mar 26;43(3):113852.

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