A ligand-mediated lipid reprogramming approach that repurposes chemically defined, liver-tropic, ionizable lipids (lipidoids) for mRNA delivery beyond the liver, and achieves outperformed efficacy and therapeutic potential in lung-specific genome editing relative to SORT-constructed 2-t6b LNP system.
Abstract
Systemic delivery of messenger RNA (mRNA) to target tissues and cells using lipid nanoparticles (LNPs) holds transformative potential for gene therapy. However, most clinically validated LNP exhibit strong liver tropism, and redirecting their organ specificity without redesigning entirely new chemistries remains challenging. Here we present a ligand-mediated lipid reprogramming approach that repurposes chemically defined, liver-tropic, ionizable lipids (lipidoids) for mRNA delivery beyond the liver. From a library of 90 degradable lipidoids, we identified 2-t6b as a potent liver-targeting platform. By site-specific displaying of small molecule ligands onto 2-t6b headgroup, we engineered a series of reconfigured lipidoids that achieve lung-specific targeting while retaining the parent delivery scaffold. Ligand7-2-t6b-lipid-functionalized LNP achieved over 200-fold higher mRNA translation in the lungs compared to the parent liver-tropic LNP. Proteomics and molecular docking analysis revealed enhanced binding of the modified lipid to vitronectin, a serum glycoprotein that improves integrin binding and thus promotes cellular uptake and translation efficiency. Ligand-mediated 2-t6b/ligand7 LNPs achieved outperformed efficacy and therapeutic potential in lung-specific genome editing relative to SORT-constructed 2-t6b LNP system. Our modular reprogramming strategy provides a generalizable framework to upgrade existing liver-biased LNPs into lung-selective mRNA carriers, advancing next-generation tissue-specific mRNA therapies for gene editing, protein replacement therapy, and regenerative medicine.
This work presents a metabolically targeted, bioorthogonal-activated delivery strategy to address the selectivity and efficiency limitations of current mRNA medicines, providing a promising platform for precision oncology.
Mingzhe Zhang, Chunhong Wang, Xiaohan Xu et al.· Journal of the American Chem...· 0 citations
Ionizable lipid is a key component of lipid nanoparticle (LNP) for mRNA delivery. However, the discovery of new lipid scaffolds remains constrained by the limited availability of efficient synthetic platforms capable of generating structurally diverse libraries. Here, we report a photochemistry-based ionizable lipid library (PILL) platform that enables the rapid discovery of ionizable lipids for mRNA delivery. Using a visible-light-driven multicomponent reaction under 427 nm irradiation, 275 α-branched amine-containing ionizable lipids were generated in a one-pot process from amines, aldehydes, and boronic acids under mild conditions. This modular photochemical strategy enabled the rapid construction of a structurally diverse lipid library that was directly integrated with purification-free in vitro screening and subsequent in vivo validation. A18B8C14 was identified as a lead ionizable lipid candidate for mRNA delivery, and the optimized A18B8C14-LNP formulation exhibited substantially higher in vivo gene expression than the benchmark MC3-based LNP while maintaining a favorable safety profile. In addition, organ-specific delivery could be achieved by adjusting the A18B8C14-LNP formulation according to the selective organ targeting (SORT) strategy. Together, these results establish photochemistry as a practical platform for ionizable lipid discovery and provide a versatile framework for the development of next-generation LNP systems for nucleic acid therapeutics.
Simin Chun, Hyorim Nam, Donghyun Lee et al.· Journal of the American Chem...· 0 citations
Intramuscular mRNA lipid nanoparticles (LNPs) often exhibit undesirable liver accumulation, compromising safety and efficacy. While optimization efforts focus on ionizable and helper lipids, the role of trace PEG-lipids remains underexplored. To address this, we constructed a 45-member PEG-lipid library via Ugi/Passerini reactions, keeping the PEG chain constant at 2 kDa while systematically diversifying the hydrophobic tail and linker structures. High-throughput screening identified Pr-182-BNT (P1B LNP) as a lead candidate. P1B LNPs enable highly efficient and selective mRNA delivery to skeletal muscle while drastically minimizing hepatic off-targeting. This precise tropism originates from enhanced muscle cell uptake and optimized membrane interactions, driven by the lipid's unique branched, asymmetric tail. In Ai9 reporter mice, P1B LNPs drive potent muscle-specific gene editing and reduce off-target recombination. As an RSV mRNA vaccine, they elicit robust antigen-specific IgG titers and expands polyfunctional CD8+IFN-γ+ T cells, indicating a Th1-skewed response. By re-engineering only the trace PEG-lipid, this work overcomes a key targeting limitation of classical LNPs and establishes a translatable platform for safer, more effective mRNA vaccines and therapeutics.
Ionizable lipid nanoparticles (LNPs) are widely used for delivery of CRISPR/Cas9 payloads to hepatocytes, but conventional hepatic uptake is strongly influenced by adsorption of apolipoprotein E and subsequent low-density lipoprotein receptor (LDLR)-mediated internalization. This dependence may limit specificity and reduce efficacy in LDLR-deficient settings. Here, we designed an aptamer-functionalized LNP platform to enable hepatocyte-selective genome editing through an LDLR-independent route and validated its performance using a genetically defined LDLR-knockout HepG2 model. Ionizable LNPs co-encapsulating Cas9 mRNA and an LDLR-targeting guide RNA were surface-decorated with the hepatocellular carcinoma-targeting TLS11a aptamer using thiol-maleimide chemistry. Comprehensive physicochemical analysis using cryo-electron microscopy, dynamic light scattering, pKa titration, UV and circular dichroism spectroscopy, X-ray photoelectron spectroscopy, and molecular beacon assays confirmed uniform nanoparticles of approximately 105 nm, preserved mRNA integrity, retained endosomal charge-switching behavior with a pKa of approximately 6.3 to 6.5, and maintained correctly folded surface-displayed TLS11a. TLS11a decoration increased Cas9 mRNA delivery to HepG2 cells from 39% to 79% Cy5-positive cells, while reducing uptake in receptor-low control cells, supporting aptamer-associated and cell-preferential delivery. In parallel, CRISPR/Cas9-mediated deletion of LDLR exon 2 generated a validated LDLR-deficient HepG2 line, confirmed at genomic, transcript, and protein levels. LDLR loss reduced LDL binding and uptake by approximately 85%, while transferrin uptake was preserved, indicating selective impairment of LDLR-dependent endocytosis. Cholesterol depletion activated the SCAP-SREBP-2 pathway and induced cholesterol biosynthesis genes. Together, these findings establish a modular aptamer-guided LNP system for targeted genome-editing delivery and a validated LDLR-null hepatocyte model for studying LDLR-dependent biology and disease.
Beyond their deployment as COVID-19 vaccines, lipid nanoparticles (LNPs) have emerged as versatile vehicles for therapeutic nucleic acid delivery. However, achieving efficient and cell-targeted transfection in extrahepatic tissues, particularly pancreatic β cells, remains a major challenge. Here, we develop a dual-targeting LNP engineering strategy that integrates high-throughput compositional screening with surface conjugation of β cell-specific targeting ligands to enable selective gene delivery to pancreatic β cells. Compositional optimization identified LNP formulations that achieved over a 148-fold increase in β cell transfection efficiency in vitro and more than an 8-fold increase in pancreatic selectivity in vivo compared to the Moderna LNP formulation. Surface conjugation of the ZnT8-specific monoclonal antibody (mAb43), which recognizes the zinc transporter ZnT8 highly expressed on murine β cells, further increased pancreatic transgene expression by more than 2-fold and achieved over 70% β cell transfection in murine models. To improve translational potential, we conjugated a high-affinity camelid single-domain antibody (4hD29 nanobody) targeting dipeptidyl peptidase-6 (DPP6), a biomarker enriched on human β cells, to compositionally optimized LNPs to deliver human STAT2-siRNA. These dual-targeting LNPs reduced STAT2 expression in human β cells under IFN-α stimulation to below baseline levels observed in unstimulated controls and induced > 4-fold increase in PDL1 expression. Together, this integrated LNP design for β cell-directed gene delivery establishes a versatile platform for RNA therapeutics and gene-editing applications in a pro-inflammatory type 1 diabetes context.
Di Yu, Yining Zhu, A. Roca-Rivada et al.· ACS Nano· 0 citations
Lipid nanoparticles (LNPs) are effective carriers for RNA and have become the standard of care; however, their natural affinity for the liver restricts their use elsewhere. To address this limitation, the “biological identity" of LNPs must be reprogrammed by modifying the three interconnected factors of lipid composition, physicochemical properties and protein corona modulation. Many previous studies have tended to evaluate individual regulatory factors in isolation; therefore, building on prior research, this review establishes a comprehensive conceptual framework that integrates these three dimensions to systematically elucidate the design principles for achieving extrahepatic targeting of LNPs. Targeting strategies are classified according to the mechanism of organ accumulation: (1) Endogenous corona-mediated (SORT, ENDO, POST), these approaches are independent of direct exogenous ligand–receptor interactions and rely on the recruitment of specific plasma proteins; within this category, POST is further distinguished by peptide-remodeled coronas; (2) Covalent active targeting - surface-conjugated ligands that directly bind to cell-surface receptors; and (3) Passive accumulation - regulated by vascular permeability. This review summarizes recent advances in delivery to the lungs, spleen, brain, heart, kidney, bone, and pancreas, discusses major translational bottlenecks (species differences, immunogenicity, and manufacturing), and proposes a direction for predictive design.
Yu Liu, Xiao Guo, Qin Hu et al.· Materials Today Bio· 0 citations
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