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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 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. 

Abstract With six therapies approved by the Food and Drug Association, chimeric antigen receptor (CAR) T cells have reshaped cancer immunotherapy. However, these therapies rely on ex vivo viral transduction to induce permanent CAR expression in T cells, which contributes to high production costs and long‐term side effects. Thus, this work aims to develop an in vivo CAR T cell engineering platform to streamline production while using mRNA to induce transient, tunable CAR expression. Specifically, an ionizable lipid nanoparticle (LNP) is utilized as these platforms have demonstrated clinical success in nucleic acid delivery. Though LNPs often accumulate in the liver, the LNP platform used here achieves extrahepatic transfection with enhanced delivery to the spleen, and it is further modified via antibody conjugation (Ab‐LNPs) to target pan‐T cell markers. The in vivo evaluation of these Ab‐LNPs confirms that targeting is necessary for potent T cell transfection. When using these Ab‐LNPs for the delivery of CAR mRNA, antibody and dose‐dependent CAR expression and cytokine release are observed along with B cell depletion of up to 90%. In all, this work conjugates antibodies to LNPs with extrahepatic tropism, evaluates pan‐T cell markers, and develops Ab‐LNPs capable of generating functional CAR T cells in vivo.