Interest continues to grow in the use of mRNA vaccines and therapeutics. While effective for immunization against infectious diseases, lipid nanoparticle (LNP) formulations used for other mRNA delivery applications suffer from off-target accumulation, poor immune transfection, and reactogenicity, limiting their application to immunoengineering. Development of new mRNA LNPs is severely bottlenecked by the LNP discovery process, which is historically low-throughput due to reliance on low-plex measurements. Here, we develop a high-throughput in vivo mRNA LNP screening platform based on barcoded mRNA (b-mRNA). Using this b-mRNA screening platform to simultaneously evaluate 122 LNPs, we identify novel LNP formulations capable of potent hepatic and extrahepatic transfection. We evaluate a lead LNP candidate for in situ immune modulation in a syngeneic mouse model of melanoma and demonstrate a significant reduction in tumor burden and extended survival compared to mice treated with a gold standard mRNA LNP formulation. We employ novel biochemical characterization techniques to analyze nanoparticle protein corona formation with single-particle resolution and gain insight into the influence of protein adsorption on hepatic and splenic transfection. Together, our results demonstrate the value of advanced LNP screening and characterization techniques for the development of next-generation mRNA LNPs for immunoengineering.

Regulating T cell phenotypes between activation and exhaustion remains a significant challenge for messenger RNA-based cancer immunotherapy. A potential approach to improve anti-cancer T cell activity is to co-deliver interleukin-12 (IL-12), to stimulate effector T cells, and indoleamine 2,3-dioxygenase (IDO) inhibitor, to suppress T cell exhaustion. Here we design prodrug ionizable lipid nanoparticles (pLNPs), via a library of prodrug ionizable lipids (pILs), incorporating an intracellularly cleavable IDO inhibitor within the pIL structure and encapsulating IL-12 messenger RNA. The lead pIL shows enhanced mRNA transfection over a clinically utilized ionizable lipid, as well as strong immunomodulatory effects via release of the IDO inhibitor. In a subcutaneous colon cancer mouse model, pLNP drives complete regression of primary tumours by eliciting effector T cell infiltration while reducing exhaustion, induces a memory T cell response and stimulates a systemic immune response that allows for regression of distal tumours in this study. These results highlight the promise of pLNPs for small-molecule drug and mRNA combination cancer immunotherapy.

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.

mRNA-based gene editing therapeutics offer the potential to permanently cure diseases but are hindered by suboptimal delivery platforms. Here, we devise a robust combinatorial chemistry for the plug-and-play assembly of structurally diverse biodegradable ionizable lipids from amines/thiols and dialkyl maleates. After screening 500 ionizable lipids, we obtained structure-activity relationships essential for effective in vitro mRNA delivery with the help of machine learning. Furthermore, we identified a lead ionizable lipid candidate that produced potent lipid nanoparticles for the delivery of various gene editing tools in wild-type and genetically modified mice compared to literature and industry benchmark lipid nanoparticles. Mechanistically, our lipid nanoparticles show favorable physicochemical properties, which could synergistically contribute to the superior delivery performance. This study highlights the utility of this synthetic method as well as the generality of this platform for potent in vivo gene editing.

mRNA-based cancer vaccines offer a modular and safe platform to elicit antitumor immunity, yet their efficacy is often limited by inefficient mRNA delivery and inadequate dendritic cell (DC) activation, both of which are essential for initiating robust cytotoxic T cell responses. Inadequate innate immune activation coupled with poor antigen presentation further diminishes their effectiveness, particularly in immunologically "cold" tumors. While stimulator of interferon genes (STING) agonists can enhance DC maturation and cross-presentation, their therapeutic utility is constrained by poor intracellular delivery and limited colocalization with tumor antigens. In this study, we developed a lipid nanoparticle (LNP) platform via high-throughput screening of ionizable lipids for potent mRNA delivery to DCs both in vitro and in vivo. To amplify immune activation, we coencapsulated the STING agonists c-di-AMP (AMP) and manganese (Mn2+) together with tumor antigen-encoding mRNA into the lead LNP formulation. This codelivery strategy synergistically activated type I interferon signaling, upregulated costimulatory molecules, enhanced antigen presentation, and elicited potent tumor-specific T cell responses and superior antitumor efficacy. Our results demonstrate that integrating innate immune stimulation with mRNA-LNP delivery provides a promising strategy to overcome current limitations in mRNA vaccine efficacy and to improve cancer immunotherapy outcomes.

Catenated networks exclusively composed of intertwining rings were first envisioned as "Olympic gels" by Pierre-Gilles de Gennes four decades ago but have not been successfully prepared in artificial materials yet due to the challenge in synthesis. Herein, we present a bio-inspired, evaporation-assisted strategy to address this issue. In our design, the evaporation of liquid catalysts that induce ring-chain equilibrium of polymer systems drives macrocycles to encounter and assists their catenation through reversible cyclization. Complete removal of liquid catalysts stabilizes the resulting ring-interlocked networks by deactivating the catenation-decatenation process. The strategy is demonstrated by the system of hydroxyethyl lipoate (LpHE) and dimethyl formamide (DMF). The results indicate that DMF evaporation leads to the formation of global LpHE polycatenane networks. This evaporation-assisted strategy displays versatility in several tested cyclic monomers, highlighting its potential as a general approach to fabricate ring-interlocked elastomers and topologically intricate materials.

The immunogenicity of lipid nanoparticles (LNPs) used for the delivery of nucleoside-modified messenger RNA limits the levels and durability of expression of the encoded protein. Here, by leveraging the Mannich reaction for ionizable lipid synthesis, and via the in vitro and in vivo screening of six combinatorial libraries of synthesized lipids, we report the identification of an antioxidant ionizable lipid, C-a16, exhibiting reduced immunogenicity. When incorporated into LNPs for mRNA delivery, C-a16 mitigated the generation of intracellular reactive oxygen species, thereby extending the duration of protein expression. In mice, and compared with commercial LNPs, LNPs incorporating C-a16 and co-delivering Cas9 mRNA and guide RNA for the editing of the transthyretin gene led to 2.8-fold higher editing efficiency; LNPs with C-a16 delivering fibroblast growth factor 21 mRNA increased the expression of the protein 3.6-fold; and when delivering mRNA encoding a tumour neoantigen or the spike protein of SARS-CoV-2, LNPs with C-a16 induced stronger antigen-specific immune responses. Our findings support the further testing of C-a16 as a promising ionizable lipid for mRNA delivery in therapeutic applications. The combinatorial synthesis of ionizable lipids allows for the identification of an antioxidant lipid exhibiting low immunogenicity for lipid nanoparticle-mediated mRNA delivery in vivo.

Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by clonal heterogeneity, immune evasion, and therapeutic resistance. Messenger RNA (mRNA) therapeutics offer programmable strategies to express therapeutic proteins and gene editors, but their efficacy is limited by poor extrahepatic delivery. To overcome these barriers, we developed a lipid nanoparticle (LNP) platform for targeted mRNA delivery to MM cells in vivo . Through combinatorial screening, we identified C16-O1, a piperazine-based ionizable lipid that efficiently transfects both CD138+ and therapy-resistant CD138-MM subclones. For tumor selectivity, LNPs were functionalized with an antibody fragment against CD38, a clinically validated MM antigen. Anti-CD38 LNPs reached the tumor-site and significantly reduced hepatic accumulation in murine xenografts. As an in vitro proof-of-concept, delivery of Cas9 mRNA and an IRF4-targeting guide RNA induced gene knockout, cell-cycle arrest, and lenalidomide sensitization. Together, these findings establish a robust framework for targeted mRNA delivery in MM and other hematologic malignancies. ### Competing Interest Statement C.G.F.-E. and M.J.M. are inventors on a U.S. Provisional Patent Application (No. 63/674,182) related to the technology described in this work. The other authors declare no competing interests. National Institutes of Health, https://ror.org/01cwqze88, F99CA284294, DP2TR002776, R01CA241661, R37CA244911, R01DK123049 American Cancer Society, https://ror.org/02e463172, 129784-IRG-16-188-38-IRG

Lipid nanoparticles (LNPs) are the preeminent non-viral drug delivery vehicle for mRNA-based therapies. Immense effort has been placed on optimizing the ionizable lipid (IL) structure, which contains an amine core conjugated to lipid tails, as small molecular adjustments can result in substantial changes in the overall efficacy of the resulting LNPs. However, despite some advancements, a major barrier for LNP delivery is endosomal escape. Here, we develop a platform for synthesizing a class of branched ILs that improve endosomal escape. These compounds incorporate terminally branched groups that increase hepatic mRNA and ribonucleoprotein complex delivery and gene editing efficiency as well as T cell transfection compared to non-branched lipids. Through an array of complementary experiments, we determine that our lipid architecture induces greater endosomal penetration and disruption. This work provides a scheme to generate a class of ILs for both mRNA and protein delivery.

Lipid nanoparticles (LNPs) have emerged as pivotal vehicles for messenger RNA (mRNA) delivery to hepatocytes upon systemic administration and to antigen-presenting cells following intramuscular injection. However, achieving systemic mRNA delivery to non-hepatocytes remains challenging without the incorporation of targeting ligands such as antibodies, peptides, or small molecules. Inspired by comb-like polymeric architecture, here we utilized a multiarm-assisted design to construct a library of 270 dendron-like degradable ionizable lipids by altering the structures of amine heads and multiarmed tails for optimal mRNA delivery. Following in vitro high-throughput screening, a series of top-dendron-like LNPs with high transfection efficacy were identified. These dendron-like ionizable lipids facilitated greater mRNA delivery to the spleen in vivo compared to ionizable lipid analogs lacking dendron-like structure. Proteomic analysis of corona-LNP pellets showed enhancement of key protein clusters, suggesting potential endogenous targeting to the spleen. A lead dendron-like LNP formulation, 18-2-9b2, was further used to encapsulate Cre mRNA and demonstrated excellent genome modification in splenic macrophages, outperforming a spleen-tropic MC3/18PA LNP in the Ai14 mice model. Moreover, 18-2-9b2 LNP encapsulating therapeutic BTB domain and CNC homologue 1 (BACH1) mRNA exhibited proficient BACH1 expression and subsequent Spic downregulation in splenic red pulp macrophages (RPM) in a Spic-GFP transgene model upon intravenous administration. These results underscore the potential of dendron-like LNPs to facilitatem RNA delivery to splenic macrophages, potentially opening avenues for a range of mRNA-LNP therapeutic applications, including regenerative medicine, protein replacement, and gene editing therapies.

Targeted control of gene expression in mineralized tissue would enable the use of nucleic acids to modulate the local microenvironment at diseased sites, ultimately promoting bone regeneration. Piperazine-linked bisphosphonate ionizable lipids provide a facile approach to targeting the transfection of mineralized tissue with lipid nanoparticles (LNPs). Here, we develop a Mineralized Tissue-Targeting Expression System (MiTEX) using bisphosphonate LNPs to locally target mineralized tissues by adsorption to mineral surfaces and bone graft materials. MiTEX demonstrated a significant increase in the adsorption of RNA onto hydroxyapatite substrates, which retained the ability to transfect bone mesenchymal cells via the adsorbed layer of mRNA LNPs. Bone graft scaffolds functionalized by adsorbed Cre mRNA-LNP were implanted to genetically label newly formed bone tissues in vivo. The surface affinity and adsorption of bisphosphonate lipids provided a local reservoir in mineralized tissues, sustaining the in vivo delivery of MiTEX. Furthermore, the targeted delivery of RNA therapeutics was demonstrated using STAT3 siRNA to modulate gene expression and proinflammatory cytokine release in ex vivo periodontal tissues. The design of this new RNA-functionalized delivery platform will promote the development of precision nucleic acid therapeutics for local anti-inflammatory treatments and bone regeneration at mineralized tissue interfaces. ### Competing Interest Statement J.L., Q.C., M.J.M., K.V., I.C.Y., and L.X. have submitted a pending patent application describing this MiTEX technology. Center for Innovation & Precision Dentistry (CiPD) and Penn Health-Tech (PHT), University of Pennsylvania Center for Innovation & Precision Dentistry (CiPD) and Schoenleber Fund, University of Pennsylvania Institute for Regenerative Medicine (IRM), University of Pennsylvania

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. Utilizing an ionizable lipid nanoparticle (LNP) with extrahepatic tropism, this work demonstrates in vivo T cell transfection for CAR T cell engineering. Antibody targeting against the CD3, CD5, and CD7 pan-T cell markers is explored, and following a single intravenous injection, the CD3-LNPs generate transient CAR+ T cells capable of up to 90% B cell depletion.image

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.

Monogenic blood diseases are among the most common genetic disorders worldwide. These diseases result in significant pediatric and adult morbidity, and some can result in death prior to birth. Novel ex vivo hematopoietic stem cell (HSC) gene editing therapies hold tremendous promise to alter the therapeutic landscape but are not without potential limitations. In vivo gene editing therapies offer a potentially safer and more accessible treatment for these diseases but are hindered by a lack of delivery vectors targeting HSCs, which reside in the difficult-to-access bone marrow niche. Here, we propose that this biological barrier can be overcome by taking advantage of HSC residence in the easily accessible liver during fetal development. To facilitate the delivery of gene editing cargo to fetal HSCs, we developed an ionizable lipid nanoparticle (LNP) platform targeting the CD45 receptor on the surface of HSCs. After validating that targeted LNPs improved messenger ribonucleic acid (mRNA) delivery to hematopoietic lineage cells via a CD45-specific mechanism in vitro, we demonstrated that this platform mediated safe, potent, and long-term gene modulation of HSCs in vivo in multiple mouse models. We further optimized this LNP platform in vitro to encapsulate and deliver CRISPR-based nucleic acid cargos. Finally, we showed that optimized and targeted LNPs enhanced gene editing at a proof-of-concept locus in fetal HSCs after a single in utero intravenous injection. By targeting HSCs in vivo during fetal development, our Systematically optimized Targeted Editing Machinery (STEM) LNPs may provide a translatable strategy to treat monogenic blood diseases before birth.

Lipid nanoparticles (LNPs) are widely used for mRNA delivery, with cationic lipids greatly affecting biodistribution, cellular uptake, endosomal escape and transfection efficiency. However, the laborious synthesis of cationic lipids limits the discovery of efficacious candidates and slows down scale-up manufacturing. Here we develop a one-pot, tandem multi-component reaction based on the rationally designed amine-thiol-acrylate conjugation, which enables fast (1 h) and facile room-temperature synthesis of amidine-incorporated degradable (AID) lipids. Structure-activity relationship analysis of a combinatorial library of 100 chemically diverse AID-lipids leads to the identification of a tail-like amine-ring-alkyl aniline that generally affords efficacious lipids. Experimental and theoretical studies show that the embedded bulky benzene ring can enhance endosomal escape and mRNA delivery by enabling the lipid to adopt a more conical shape. The lead AID-lipid can not only mediate local delivery of mRNA vaccines and systemic delivery of mRNA therapeutics, but can also alter the tropism of liver-tropic LNPs to selectively deliver gene editors to the lung and mRNA vaccines to the spleen.

Ionizable lipids largely determine the biocompatibility of lipid nanoparticles (LNPs) and the efficacy for mRNA delivery. Rational design and combinatorial synthesis have led to the development of potent and biodegradable ionizable lipids, yet methodologies for the stepwise optimization of ionizable lipid structure are lacking. Here we show that iterative chemical derivatization and combinatorial chemistry, and in particular the amine-aldehyde-alkyne coupling reaction, can be leveraged to iteratively accelerate the structural optimization of propargylamine-based ionizable lipids (named A3-lipids) to improve their delivery activity and biodegradability. Through five cycles of such directed chemical evolution, we identified dozens of biodegradable and asymmetric A3-lipids with delivery activity comparable to or better than a benchmark ionizable lipid. We then derived structure-activity relationships for the headgroup, ester linkage and tail. Compared with standard ionizable lipids, the lead A3-lipid improved the hepatic delivery of an mRNA-based genome editor and the intramuscular delivery of an mRNA vaccine against SARS-CoV-2. Structural criteria for ionizable lipids discovered via directed chemical evolution may accelerate the development of LNPs for mRNA delivery. Directed chemical evolution can iteratively accelerate the structural optimization of ionizable lipids to improve their delivery activity and biodegradability for applications in lipid nanoparticle-mediated mRNA delivery.

Nanoparticles are promising for drug delivery applications, with several clinically approved products. However, attaining high nanoparticle accumulation in solid tumours remains challenging. Here we show that tumour cell-derived small extracellular vesicles (sEVs) block nanoparticle delivery to tumours, unveiling another barrier to nanoparticle-based tumour therapy. Tumour cells secrete large amounts of sEVs in the tumour microenvironment, which then bind to nanoparticles entering tumour tissue and traffic them to liver Kupffer cells for degradation. Knockdown of Rab27a, a gene that controls sEV secretion, decreases sEV levels and improves nanoparticle accumulation in tumour tissue. The therapeutic efficacy of messenger RNAs encoding tumour suppressing and proinflammatory proteins is greatly improved when co-encapsulated with Rab27a small interfering RNA in lipid nanoparticles. Together, our results demonstrate that tumour cell-derived sEVs act as a defence system against nanoparticle tumour delivery and that this system may be a potential target for improving nanoparticle-based tumour therapies. Cancer cell-derived small extracellular vesicles bind to therapeutic nanoparticles leading them from tumours to the liver for degradation. This mechanism is another barrier for the development of efficient nanoparticle-based cancer therapies.

Disruption of pulmonary vascular homeostasis is a central feature of viral pneumonia, wherein endothelial cell (EC) death and subsequent angiogenic responses are critical determinants of the outcome of severe lung injury. A more granular understanding of the fundamental mechanisms driving reconstitution of lung endothelium is necessary to facilitate therapeutic vascular repair. Here, we demonstrated that TGF-β signaling through TGF-βR2 (transforming growth factor–β receptor 2) is activated in pulmonary ECs upon influenza infection, and mice deficient in endothelial Tgfbr2 exhibited prolonged injury and diminished vascular repair. Loss of endothelial Tgfbr2 prevented autocrine Vegfa (vascular endothelial growth factor α) expression, reduced endothelial proliferation, and impaired renewal of aerocytes thought to be critical for alveolar gas exchange. Angiogenic responses through TGF-βR2 were attributable to leucine-rich α-2-glycoprotein 1, a proangiogenic factor that counterbalances canonical angiostatic TGF-β signaling. Further, we developed a lipid nanoparticle that targets the pulmonary endothelium, Lung-LNP (LuLNP). Delivery of Vegfa mRNA, a critical TGF-βR2 downstream effector, by LuLNPs improved the impaired regeneration phenotype of EC Tgfbr2 deficiency during influenza injury. These studies defined a role for TGF-βR2 in lung endothelial repair and demonstrated efficacy of an efficient and safe endothelial-targeted LNP capable of delivering therapeutic mRNA cargo for vascular repair in influenza infection.

In situ cancer vaccination refers to any approach that exploits tumour antigens available at a tumour site to induce tumour-specific adaptive immune responses. These approaches hold great promise for the treatment of many solid tumours, with numerous candidate drugs under preclinical or clinical evaluation and several products already approved. However, there are challenges in the development of effective in situ cancer vaccines. For example, inadequate release of tumour antigens from tumour cells limits antigen uptake by immune cells; insufficient antigen processing by antigen-presenting cells restricts the generation of antigen-specific T cell responses; and the suppressive immune microenvironment of the tumour leads to exhaustion and death of effector cells. Rationally designed delivery technologies such as lipid nanoparticles, hydrogels, scaffolds and polymeric nanoparticles are uniquely suited to overcome these challenges through the targeted delivery of therapeutics to tumour cells, immune cells or the extracellular matrix. Here, we discuss delivery technologies that have the potential to reduce various clinical barriers for in situ cancer vaccines. We also provide our perspective on this emerging field that lies at the interface of cancer vaccine biology and delivery technologies. In situ cancer vaccines generate tumour-specific T cell responses and hold promise for the treatment of solid tumours. This Review discusses drug delivery technologies such as lipid nanoparticles, hydrogels and scaffolds that are being developed to boost the antitumour immune effects of several types of in situ cancer vaccine.