Lipid nanoparticle-delivered intrabodies for inhibiting necroptosis and pyroptosis

Deepagan, VG; Ma, X; Bazregari, F; Pang, J; Schaefer, J; Hildebrand, JM; Dempsey, RK; Doerflinger, M; Baldwin, CA; Schmidt, FI; Murphy, JM; Salvamoser, R; Vince, JE

Author(s): Deepagan, VG; Ma, X; Bazregari, F; Pang, J; Schaefer, J; Hildebrand, JM; Dempsey, RK; Doerflinger, M; Baldwin, CA; Schmidt, FI; Murphy, JM; Salvamoser, R; Vince, JE;
Details: Publication Year 2025-08-20,Volume 482,Issue #16,Page BCJ20253191
Journal Title: Biochemical Journal
Abstract: Intrabodies are intracellularly expressed high-affinity protein binders such as nanobodies and monobodies that offer an alternative approach to small molecules. However, the maturation of intrabody technology into new therapeutic modalities has been limited by the availability of a clinically relevant delivery system enabling sufficiently high levels of protein to be expressed in the cytosol. Here, we use lipid nanoparticle (LNP) systems based on clinically approved formulations for the efficient intracellular delivery of mRNAs encoding for intrabodies targeting mixed lineage kinase domain-like pseudokinase (MLKL) and apoptosis-associated speck-like protein containing a CARD (ASC), key mediators of the necrotic cell death modalities, necroptosis and pyroptosis, respectively. LNP delivery of intrabody mRNA resulted in robust protein expression, with an MLKL-binding intrabody preventing MLKL membrane translocation and protecting against necroptotic cell death. Similarly, LNP delivery of a bivalent intrabody targeting the inflammasome adaptor protein ASC protected against NLRP3 and AIM2 inflammasome-driven responses, including caspase-1 and IL-1beta activation and gasdermin D-driven pyroptotic killing. These findings establish that LNPs harbouring anti-necrotic intrabody mRNAs allow for sufficient intracellular expression to neutralize necrotic cell death signalling and provide a general, clinically relevant, strategy for delivering therapeutic intrabodies into cells.
Publisher: Portland Press
Keywords: *Liposomes; *Nanoparticles; *Necroptosis/immunology; *Pyroptosis/immunology; *Single-Domain Antibodies/genetics/immunology; *RNA, Messenger/genetics; HT29 Cells; Humans; *Protein Kinases/immunology; *CARD Signaling Adaptor Proteins/immunology; *Transfection/methods; Mlkl; inflammasome; lipid nanoparticle; nanobody; necroptosis; pyroptosis
Research Division(s): Inflammation; Infection and Global Health
PubMed ID: 40785595
Publisher's Version: https://doi.org/10.1042/BCJ20253191
Open Access at Publisher's Site: https://doi.org/10.1042/BCJ20253191
Terms of Use/Rights Notice: Refer to copyright notice on published article.

Creation Date: 2025-08-19 01:46:01

Last Modified: 2025-08-29 08:42:33