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Abstract Nucleic acid delivery with mRNA lipid nanoparticles are being developed for targeting a wide array of tissues and cell types. However, targeted delivery to the bone microenvironment remains a significant challenge in the field, due in part to low local blood flow and poor interactions between drug carriers and bone material. Here we report bone‐targeting ionizable lipids incorporating a piperazine backbone and bisphosphate moieties, which bind tightly with hydroxyapatite ([Ca₅(PO₄)₃OH]), a key component of mineralized tissues. These lipids demonstrate biocompatibility and low toxicity in both vitro and in vivo studies. LNP formulated with these lipids facilitated efficient cellular transfection and improved binding to hydroxyapatite in vitro, and targeted delivery to the bone microenvironment in vivo following systemic administration. Overall, our findings demonstrate the critical role of the piperazine backbone in a novel ionizable lipid, which incorporates a bisphosphonate group to enable efficient bone‐targeted delivery, highlighting the potential of rational design of ionizable lipids for next‐generation bone‐targeting delivery systems. 

Abstract Lipid nanoparticles (LNPs) have emerged as the preeminent nonviral drug delivery vehicles for nucleic acid therapeutics, as exemplified by their usage in the mRNA COVID‐19 vaccines. As a safe and highly modular delivery platform, LNPs are attractive for a wide range of applications. In addition to vaccines, LNPs are being utilized as platforms for other immunoengineering efforts, especially as cancer immunotherapies by modulating immune cells and their functionality via nucleic acid delivery. In this review, we focus on the methods and applications of LNP‐based immunotherapy in five cell types: T cells, NK cells, macrophages, stem cells, and dendritic cells. Each of these cell types has wide‐reaching applications in immunotherapy but comes with unique challenges and delivery barriers. By combining knowledge of immunology and nanotechnology, LNPs can be developed for improved immune cell targeting and transfection, ultimately working toward novel clinical therapeutics. 

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 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 Recently, targeted degradation has emerged as a powerful therapeutic modality. Relying on “event-driven” pharmacology, proteolysis targeting chimeras (PROTACs) can degrade targets and are superior to conventional inhibitors against undruggable proteins. Unfortunately, PROTAC discovery is limited by warhead scarcity and laborious optimization campaigns. To address these shortcomings, analogous protein-based heterobifunctional degraders, known as bioPROTACs, have been developed. Compared to small-molecule PROTACs, bioPROTACs have higher success rates and are subject to fewer design constraints. However, the membrane impermeability of proteins severely restricts bioPROTAC deployment as a generalized therapeutic modality. Here, we present an engineered bioPROTAC template able to complex with cationic and ionizable lipids via electrostatic interactions for cytosolic delivery. When delivered by biocompatible lipid nanoparticles, these modified bioPROTACs can rapidly degrade intracellular proteins, exhibiting near-complete elimination (up to 95% clearance) of targets within hours of treatment. Our bioPROTAC format can degrade proteins localized to various subcellular compartments including the mitochondria, nucleus, cytosol, and membrane. Moreover, substrate specificity can be easily reprogrammed, allowing modular design and targeting of clinically-relevant proteins such as Ras, Jnk, and Erk. In summary, this work introduces an inexpensive, flexible, and scalable platform for efficient intracellular degradation of proteins that may elude chemical inhibition. 

Abstract RNA‐based therapeutics have gained traction for the prevention and treatment of a variety of diseases. However, their fragility and immunogenicity necessitate a drug carrier. Lipid nanoparticles (LNPs) have emerged as the predominant delivery vehicle for RNA therapeutics. An important component of LNPs is the ionizable lipid (IL), which is protonated in the acidic environment of the endosome, prompting cargo release into the cytosol. Currently, there is growing evidence that the structure of IL lipid tails significantly impacts the efficacy of LNP‐mediated mRNA translation. Here, we optimized IL tail length for LNP‐mediated delivery of three different mRNA cargos. Using C12‐200, a gold standard IL, as a model, we designed a library of ILs with varying tail lengths and evaluated their potency in vivo. We demonstrated that small changes in lipophilicity can drastically increase or decrease mRNA translation. We identified that LNPs formulated with firefly luciferase mRNA (1929 base pairs) and C10‐200, an IL with shorter tail lengths than C12‐200, enhance liver transfection by over 10‐fold. Furthermore, different IL tail lengths were found to be ideal for transfection of LNPs encapsulating mRNA cargos of varying sizes. LNPs formulated with erythropoietin (EPO), responsible for stimulating red blood cell production, mRNA (858 base pairs), and the C13‐200 IL led to EPO translation at levels similar to the C12‐200 LNP. The LNPs formulated with Cas9 mRNA (4521 base pairs) and the C9‐200 IL induced over three times the quantity of indels compared with the C12‐200 LNP. Our findings suggest that shorter IL tails may lead to higher transfection of LNPs encapsulating larger mRNAs, and that longer IL tails may be more efficacious for delivering smaller mRNA cargos. We envision that the results of this project can be utilized as future design criteria for the next generation of LNP delivery systems for RNA therapeutics. 

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. 

Abstract The programmed cell death protein 1 (PD‐1) signaling pathway is a major source of dampened T cell activity in the tumor microenvironment. While clinical approaches to inhibiting the PD‐1 pathway using antibody blockade have been broadly successful, these approaches lead to widespread PD‐1 suppression, increasing the risk of autoimmune reactions. This study reports the development of an ionizable lipid nanoparticle (LNP) platform for simultaneous therapeutic gene expression and RNA interference (RNAi)‐mediated transient gene knockdown in T cells. In developing this platform, interesting interactions are observed between the two RNA cargoes when co‐encapsulated, leading to improved expression and knockdown characteristics compared to delivering either cargo alone. This messenger RNA (mRNA)/small interfering RNA (siRNA) co‐delivery platform is adopted to deliver chimeric antigen receptor (CAR) mRNA and siRNA targeting PD‐1 to primary human T cells ex vivo and strong CAR expression and PD‐1 knockdown are observed without apparent changes to overall T cell activation state. This delivery platform shows great promise for transient immune gene modulation for a number of immunoengineering applications, including the development of improved cancer immunotherapies. 

Abstract During healthy pregnancy, the placenta develops to allow for exchange of nutrients and oxygen between the mother and the fetus. However, placental dysregulation can lead to several pregnancy disorders, such as preeclampsia and fetal growth restriction. Recently, lipid nanoparticle (LNP)‐mediated delivery of messenger RNA (mRNA) has been explored as a promising approach to treat these disorders. Here, iterative libraries of LNPs with varied excipient molar ratios are screened in vitro for enhanced mRNA delivery to placental cells with minimal cytotoxicity when compared to an LNP formulation with a standard excipient molar ratio. LNP C5, the top formulation identified by these screens, demonstrates a fourfold increase in mRNA delivery in vitro compared to the standard formulation. Intravenous administration of LNP C5 to pregnant mice achieves improved in vivo placental mRNA delivery compared to the standard formulation and mediates mRNA delivery to placental trophoblasts, endothelial cells, and immune cells. These results identify LNP C5 as a promising optimized LNP formulation for placental mRNA delivery and further validates the design of experiments strategy for LNP excipient optimization to enhance mRNA delivery to cell types and organs of interest.