High-Throughput In Vivo Screening Identifies Differential Influences on mRNA Lipid Nanoparticle Immune Cell Delivery by Administration Route.

Immune modulation through the intracellular delivery of nucleoside-modified mRNA to immune cells is an attractive approach for in vivo immunoengineering, with applications in infectious disease, cancer immunotherapy, and beyond. Lipid nanoparticles (LNPs) have come to the fore as a promising nucleic acid delivery platform, but LNP design criteria remain poorly defined, making the rate-limiting step for LNP discovery the screening process. In this study, we employed high-throughput in vivo LNP screening based on molecular barcoding to investigate the influence of LNP composition on immune tropism with applications in vaccines and systemic immunotherapies. Screening a large LNP library under both intramuscular (i.m.) and intravenous (i.v.) injection, we observed differential influences on LNP uptake by immune populations across the two administration routes, gleaning insight into LNP design criteria for in vivo immunoengineering. In validation studies, the lead LNP formulation for i.m. administration demonstrated substantial mRNA translation in the spleen and draining lymph nodes with a more favorable biodistribution profile than LNPs formulated with the clinical standard ionizable lipid DLin-MC3-DMA (MC3). The lead LNP formulations for i.v. administration displayed potent immune transfection in the spleen and peripheral blood, with one lead LNP demonstrating substantial transfection of splenic dendritic cells and another inducing substantial transfection of circulating monocytes. Altogether, the immunotropic LNPs identified by high-throughput in vivo screening demonstrated significant promise for both locally- and systemically-delivered mRNA and confirmed the value of the LNP design criteria gleaned from our screening process, which could potentially inform future endeavors in mRNA vaccine and immunotherapy applications.

EGFR-targeted ionizable lipid nanoparticles enhance in vivo mRNA delivery to the placenta.

The full potential of ionizable lipid nanoparticles (LNPs) as an in vivo nucleic acid delivery platform has not yet been realized given that LNPs primarily accumulate in the liver following systemic administration, limiting their success to liver-centric conditions. The engineering of LNPs with antibody targeting moieties can enable extrahepatic tropism by facilitating site-specific LNP tethering and driving preferential LNP uptake into receptor-expressing cell types via receptor-mediated endocytosis. Obstetric conditions stemming from placental dysfunction, such as preeclampsia, are characterized by overexpression of cellular receptors, including the epidermal growth factor receptor (EGFR), making targeted LNP platforms an exciting potential treatment strategy for placental dysfunction during pregnancy. Herein, an EGFR antibody-conjugated LNP (aEGFR-LNP) platform was developed by engineering LNPs with increasing densities of antibody functionalization. aEGFR-LNPs were screened in vitro in immortalized placental trophoblasts and in vivo in non-pregnant and pregnant mice and compared to non-targeted formulations for extrahepatic, antibody-targeted mRNA LNP delivery to the placenta. Our top performing LNP with an intermediate density of antibody functionalization (1:5 aEGFR-LNP) mediated a âˆ¼twofold increase in mRNA delivery in murine placentas and a âˆ¼twofold increase in LNP uptake in EGFR-expressing trophoblasts compared to non-targeted counterparts. These results demonstrate the potential of antibody-conjugated LNPs for achieving extrahepatic tropism, and the ability of aEGFR-LNPs in promoting mRNA delivery to EGFR-expressing cell types in the placenta.

Antigen Presenting Cell Mimetic Lipid Nanoparticles for Rapid mRNA CAR T Cell Cancer Immunotherapy.

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.

Emerging strategies for nanomedicine in autoimmunity.

Autoimmune disorders have risen to be among the most prevalent chronic diseases across the globe, affecting approximately 5-7% of the population. As autoimmune diseases steadily rise in prevalence, so do the number of potential therapeutic strategies to combat them. In recent years, fundamental research investigating autoimmune pathologies has led to the emergence of several cellular targets that provide new therapeutic opportunities. However, key challenges persist in terms of accessing and specifically combating the dysregulated, self-reactive cells while avoiding systemic immune suppression and other off-target effects. Fortunately, the continued advancement of nanomedicines may provide strategies to address these challenges and bring innovative autoimmunity therapies to the clinic. Through precise engineering and rational design, nanomedicines can possess a variety of physicochemical properties, surface modifications, and cargoes, allowing for specific targeting of therapeutics to pathological cell and organ types. These advances in nanomedicine have been demonstrated in cancer therapies and have the broad potential to advance applications in autoimmunity therapies as well. In this review, we focus on leveraging the power of nanomedicine for prevalent autoimmune disorders throughout the body. We expand on three key areas for the development of autoimmunity therapies - avoiding systemic immunosuppression, balancing interactions with the immune system, and elevating current platforms for delivering complex cargoes - and emphasize how nanomedicine-based strategies can overcome these barriers and enable the development of next-generation, clinically relevant autoimmunity therapies.

Bile acid-containing lipid nanoparticles enhance extrahepatic mRNA delivery.

Lipid nanoparticles (LNPs) have emerged as a viable, clinically-validated platform for the delivery of mRNA therapeutics. LNPs have been utilized as mRNA delivery systems for applications including vaccines, gene therapy, and cancer immunotherapy. However, LNPs, which are typically composed of ionizable lipids, cholesterol, helper lipids, and lipid-anchored polyethylene glycol, often traffic to the liver which limits the therapeutic potential of the platform. Several approaches have been proposed to resolve this tropism such as post-synthesis surface modification or the addition of synthetic cationic lipids. Methods: Here, we present a strategy for achieving extrahepatic delivery of mRNA involving the incorporation of bile acids, a naturally-occurring class of cholesterol analogs, during LNP synthesis. We synthesized a series of bile acid-containing C14-4 LNPs by replacing cholesterol with bile acids (cholic acid, chenodeoxycholic acid, deoxycholic acid, or lithocholic acid) at various ratios. Results: Bile acid-containing LNPs (BA-LNPs) were able to reduce delivery to liver cells in vitro and improve delivery in a variety of other cell types, including T cells, B cells, and epithelial cells. Our subsequent in vivo screening of selected LNP candidates injected intraperitoneally or intravenously identified a highly spleen tropic BA-LNP: CA-100, a four-component LNP containing cholic acid and no cholesterol. These screens also identified BA-LNP candidates demonstrating promise for other mRNA therapeutic applications such as for gastrointestinal or immune cell delivery. We further found that the substitution of cholic acid for cholesterol in an LNP formulation utilizing a different ionizable lipid, C12-200, also shifted mRNA delivery from the liver to the spleen, suggesting that this cholic acid replacement strategy may be generalizable. Conclusion: These results demonstrate the potential of a four-component BA-LNP formulation, CA-100, for extrahepatic mRNA delivery that could potentially be utilized for a range of therapeutic and vaccine applications.

In Vivo mRNA CAR T Cell Engineering via Targeted Ionizable Lipid Nanoparticles with Extrahepatic Tropism.

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.

mRNA Lipid Nanoparticles for Ex Vivo Engineering of Immunosuppressive T Cells for Autoimmunity Therapies.

Cell-based therapies for autoimmune diseases have gained significant traction, with several approaches centered around the regulatory T (Treg) cell─a well-known immunosuppressive cell characterized by its expression of the transcription factor Foxp3. Unfortunately, due to low numbers of Treg cells available in circulation, harvesting and culturing Treg cells remains a challenge. It has been reported that engineering Foxp3 expression in CD4+ T cells can result in a Treg-like phenotype; however, current methods result in the inefficient engineering of these cells. Here, we develop an ionizable lipid nanoparticle (LNP) platform to effectively deliver Foxp3 mRNA to CD4+ T cells. We successfully engineer CD4+ T cells into Foxp3-T (FP3T) cells that transiently exhibit an immunosuppressive phenotype and functionally suppress the proliferation of effector T cells. These results demonstrate the promise of an LNP platform for engineering immunosuppressive T cells with potential applications in autoimmunity therapies.

Orthogonal Design of Experiments for Engineering of Lipid Nanoparticles for mRNA Delivery to the Placenta.

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.

Throughput-scalable manufacturing of SARS-CoV-2 mRNA lipid nanoparticle vaccines.

Lipid nanoparticles (LNPs) are a potent delivery technology that have made it possible for the recent clinical breakthroughs in mRNA therapeutics and vaccines. A key challenge to the broader implementation of mRNA therapeutics and vaccines is the development of technology to produce precisely defined LNP formulations, with throughput that can scale from discovery to commercial manufacturing and meet the stringent manufacturing standards of the pharmaceutical industry. To address these challenges, we have developed a microfluidic chip that incorporates 1×, 10×, or 256× LNP-generating units that achieve scalable production rates of up to 17 L/h of precisely defined LNPs. Using these chips, we demonstrate that LNP physical properties and potency in vivo are unchanged as throughput is scaled. Our chips are fabricated out of silicon and glass substrates, which have excellent solvent compatibility, compatibility with pharmaceutical manufacturing, and can be fully reset and reused. SARS-CoV-2 mRNA-LNP vaccines formulated by our chips triggered potent antibody responses in a preclinical study. These results demonstrate the feasibility of directly translating microfluidic-generated LNPs to the scale necessary for commercial production.

Ionizable Lipid Nanoparticles for In Vivo mRNA Delivery to the Placenta during Pregnancy.

Ionizable lipid nanoparticles (LNPs) are the most clinically advanced nonviral platform for mRNA delivery. While they have been explored for applications including vaccines and gene editing, LNPs have not been investigated for placental insufficiency during pregnancy. Placental insufficiency is caused by inadequate blood flow in the placenta, which results in increased maternal blood pressure and restricted fetal growth. Therefore, improving vasodilation in the placenta can benefit both maternal and fetal health. Here, we engineered ionizable LNPs for mRNA delivery to the placenta with applications in mediating placental vasodilation. We designed a library of ionizable lipids to formulate LNPs for mRNA delivery to placental cells and identified a lead LNP that enables in vivo mRNA delivery to trophoblasts, endothelial cells, and immune cells in the placenta. Delivery of this top LNP formulation encapsulated with VEGF-A mRNA engendered placental vasodilation, demonstrating the potential of mRNA LNPs for protein replacement therapy during pregnancy to treat placental disorders.

Clathrin senses membrane curvature.

The ability of proteins to assemble at sites of high membrane curvature is essential to diverse membrane remodeling processes, including clathrin-mediated endocytosis. Multiple adaptor proteins within the clathrin pathway have been shown to sense regions of high membrane curvature, leading to local recruitment of the clathrin coat. Because clathrin triskelia do not bind to the membrane directly, it has remained unclear whether the clathrin coat plays an active role in sensing membrane curvature or is passively recruited by adaptor proteins. Using a synthetic tag to assemble clathrin directly on membrane surfaces, here we show that clathrin is a strong sensor of membrane curvature, comparable with previously studied adaptor proteins. Interestingly, this sensitivity arises from clathrin assembly rather than from the properties of unassembled triskelia, suggesting that triskelia have preferred angles of interaction, as predicted by earlier structural data. Furthermore, when clathrin is recruited by adaptors, its curvature sensitivity is amplified by 2- to 10-fold, such that the resulting protein complex is up to 100 times more likely to assemble on a highly curved surface compared with a flatter one. This exquisite sensitivity points to a synergistic relationship between the coat and its adaptor proteins, which enables clathrin to pinpoint sites of high membrane curvature, an essential step in ensuring robust membrane traffic. More broadly, these findings suggest that protein networks, rather than individual protein domains, are likely the most potent drivers of membrane curvature sensing.

Structured and intrinsically disordered domains within Amphiphysin1 work together to sense and drive membrane curvature.

Cellular membranes undergo remodeling during many cellular processes including endocytosis, cytoskeletal protrusion, and organelle biogenesis. During these events, specialized proteins sense and amplify fluctuations in membrane curvature to create stably curved architectures. Amphiphysin1 is a multi-domain protein containing an N-terminal crescent-shaped BAR (Bin/Amphiphysin/Rvs) domain and a C-terminal domain that is largely disordered. When studied in isolation, the BAR domain of Amphiphysin1 senses membrane curvature and generates membrane tubules. However, the disordered domain has been largely overlooked in these studies. Interestingly, our recent work has demonstrated that the disordered domain is capable of substantially amplifying the membrane remodeling ability of the BAR domain. However, the physical mechanisms responsible for these effects are presently unclear. Here we elucidated the functional role of the disordered domain by gradually truncating it, creating a family of mutant proteins, each of which contained the BAR domain and a fraction of the disordered domain. Using quantitative fluorescence and electron microscopy, our results indicate that the disordered domain contributes to membrane remodeling by making it more difficult for the protein to bind to and assemble on flat membrane surfaces. Specifically, we found that the disordered domain began to significantly impact membrane remodeling when its projected area exceeded that of the BAR domain. Once this threshold was crossed, steric interactions with the membrane surface and with neighboring disordered domains gave rise to increased curvature sensing and membrane vesiculation, respectively. These findings provide insight into the synergy between structured and disordered domains, each of which play important biophysical roles in membrane remodeling.

Molecular Mechanisms of Membrane Curvature Sensing by a Disordered Protein.

The ability of proteins to sense membrane curvature is essential for the initiation and assembly of curved membrane structures. Established mechanisms of curvature sensing rely on proteins with specific structural features. In contrast, it has recently been discovered that intrinsically disordered proteins, which lack a defined three-dimensional fold, can also be potent sensors of membrane curvature. How can an unstructured protein sense the structure of the membrane surface? Many disordered proteins that associate with membranes have two key physical features: a high degree of conformational entropy and a high net negative charge. Binding of such proteins to membrane surfaces results simultaneously in a decrease in conformational entropy and an increase in electrostatic repulsion by anionic lipids. Here we show that each of these effects gives rise to a distinct mechanism of curvature sensing. Specifically, as the curvature of the membrane increases, the steric hindrance between the disordered protein and membrane is reduced, leading to an increase in chain entropy. At the same time, increasing membrane curvature increases the average separation between anionic amino acids and lipids, creating an electrostatic preference for curved membranes. Using quantitative imaging of membrane vesicles, our results demonstrate that long disordered amino acid chains with low net charge sense curvature predominately through the entropic mechanism. In contrast, shorter, more highly charged amino acid chains rely largely on the electrostatic mechanism. These findings provide a roadmap for predicting and testing the curvature sensitivity of the large and diverse set of disordered proteins that function at cellular membranes.