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Abstract The ionizable lipidoid is a key component of lipid nanoparticles (LNPs). Degradable lipidoids containing extended alkyl branches have received tremendous attention, yet their optimization and investigation are underappreciated. Here, we devise an in situ construction method for the combinatorial synthesis of degradable branched (DB) lipidoids. We find that appending branch tails to inefficacious lipidoids via degradable linkers boosts mRNA delivery efficiency up to three orders of magnitude. Combinatorial screening and systematic investigation of two libraries of DB-lipidoids reveal important structural criteria that govern their in vivo potency. The lead DB-LNP demonstrates robust delivery of mRNA therapeutics and gene editors into the liver. In a diet-induced obese mouse model, we show that repeated administration of DB-LNP encapsulating mRNA encoding human fibroblast growth factor 21 alleviates obesity and fatty liver. Together, we offer a construction strategy for high-throughput and cost-efficient synthesis of DB-lipidoids. This study provides insights into branched lipidoids for efficient mRNA delivery. 

Abstract Lipid nanoparticles for delivering mRNA therapeutics hold immense promise for the treatment of a wide range of lung-associated diseases. However, the lack of effective methodologies capable of identifying the pulmonary delivery profile of chemically distinct lipid libraries poses a significant obstacle to the advancement of mRNA therapeutics. Here we report the implementation of a barcoded high-throughput screening system as a means to identify the lung-targeting efficacy of cationic, degradable lipid-like materials. We combinatorially synthesize 180 cationic, degradable lipids which are initially screened in vitro. We then use barcoding technology to quantify how the selected 96 distinct lipid nanoparticles deliver DNA barcodes in vivo. The top-performing nanoparticle formulation delivering Cas9-based genetic editors exhibits therapeutic potential for antiangiogenic cancer therapy within a lung tumor model in female mice. These data demonstrate that employing high-throughput barcoding technology as a screening tool for identifying nanoparticles with lung tropism holds potential for the development of next-generation extrahepatic delivery platforms. 

Abstract Lipid nanoparticle-mediated RNA delivery holds great potential to treat various liver diseases. However, targeted delivery of RNA therapeutics to activated liver-resident fibroblasts for liver fibrosis treatment remains challenging. Here, we develop a combinatorial library of anisamide ligand-tethered lipidoids (AA-lipidoids) using a one-pot, two-step modular synthetic method and adopt a two-round screening strategy to identify AA-lipidoids with both high potency and selectivity to deliver RNA payloads to activated fibroblasts. The lead AA-lipidoid AA-T3A-C12 mediates greater RNA delivery and transfection of activated fibroblasts than its analog without anisamide and the FDA-approved MC3 ionizable lipid. In a preclinical model of liver fibrosis, AA-T3A-C12 enables ~65% silencing of heat shock protein 47, a therapeutic target primarily expressed by activated fibroblasts, which is 2-fold more potent than MC3, leading to significantly reduced collagen deposition and liver fibrosis. These results demonstrate the potential of AA-lipidoids for targeted RNA delivery to activated fibroblasts. Furthermore, these synthetic methods and screening strategies open a new avenue to develop and discover potent lipidoids with targeting properties, which can potentially enable RNA delivery to a range of cell and tissue types that are challenging to access using traditional lipid nanoparticle formulations. 

Abstract Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable clinical success in the treatment of hematological malignancies. However, producing these bespoke cancer‐killing cells is a complicated ex vivo process involving leukapheresis, artificial T cell activation, and CAR construct introduction. The activation step requires the engagement of CD3/TCR and CD28 and is vital for T cell transfection and differentiation. Though antigen‐presenting cells (APCs) facilitate activation in vivo, ex vivo activation relies on antibodies against CD3 and CD28 conjugated to magnetic beads. While effective, this artificial activation adds to the complexity of CAR T cell production as the beads must be removed prior to clinical implementation. To overcome this challenge, this work develops activating lipid nanoparticles (aLNPs) that mimic APCs to combine the activation of magnetic beads and the transfection capabilities of LNPs. It is shown that aLNPs enable one‐step activation and transfection of primary human T cells with the resulting mRNA CAR T cells reducing tumor burden in a murine xenograft model, validating aLNPs as a promising platform for the rapid production of mRNA CAR T cells.