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.

In situ combinatorial synthesis of degradable branched lipidoids for systemic delivery of mRNA therapeutics and gene editors.

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.

High-throughput barcoding of nanoparticles identifies cationic, degradable lipid-like materials for mRNA delivery to the lungs in female preclinical models.

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.

Small-molecule-mediated control of the anti-tumour activity and off-tumour toxicity of a supramolecular bispecific T cell engager.

The broader clinical use of bispecific T cell engagers for inducing anti-tumour toxicity is hindered by their on-target off-tumour toxicity and the associated neurotoxicity and cytokine-release syndrome. Here we show that the off-tumour toxicity of a supramolecular bispecific T cell engager binding to the T cell co-receptor CD3 and to the human epidermal growth factor receptor 2 on breast tumour cells can be halted by disengaging the T cells from the tumour cells via the infusion of the small-molecule drug amantadine, which disassembles the supramolecular aggregate. In mice bearing human epidermal growth factor receptor 2-expressing tumours and with a human immune system, high intravenous doses of such a 'switchable T cell nanoengager' elicited strong tumour-specific adaptive immune responses that prevented tumour relapse, while the infusion of amantadine restricted off-tumour toxicity, cytokine-release syndrome and neurotoxicity. Supramolecular chemistry may be further leveraged to control the anti-tumour activity and off-tumour toxicity of bispecific antibodies.

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.

TGF-ßR2 signaling coordinates pulmonary vascular repair after viral injury in mice and human tissue.

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-ß signaling through TGF-ßR2 (transforming growth factor-ß 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-ßR2 were attributable to leucine-rich α-2-glycoprotein 1, a proangiogenic factor that counterbalances canonical angiostatic TGF-ß signaling. Further, we developed a lipid nanoparticle that targets the pulmonary endothelium, Lung-LNP (LuLNP). Delivery of Vegfa mRNA, a critical TGF-ßR2 downstream effector, by LuLNPs improved the impaired regeneration phenotype of EC Tgfbr2 deficiency during influenza injury. These studies defined a role for TGF-ßR2 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 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.

Upregulation of exosome secretion from tumor-associated macrophages plays a key role in the suppression of anti-tumor immunity.

Macrophages play a pivotal role in tumor immunity. We report that reprogramming of macrophages to tumor-associated macrophages (TAMs) promotes the secretion of exosomes. Mechanistically, increased exosome secretion is driven by MADD, which is phosphorylated by Akt upon TAM induction and activates Rab27a. TAM exosomes carry high levels of programmed death-ligand 1 (PD-L1) and potently suppress the proliferation and function of CD8+ T cells. Analysis of patient melanoma tissues indicates that TAM exosomes contribute significantly to CD8+ T cell suppression. Single-cell RNA sequencing analysis showed that exosome-related genes are highly expressed in macrophages in melanoma; TAM-specific RAB27A expression inversely correlates with CD8+ T cell infiltration. In a murine melanoma model, lipid nanoparticle delivery of small interfering RNAs (siRNAs) targeting macrophage RAB27A led to better T cell activation and sensitized tumors to anti-programmed cell death protein 1 (PD-1) treatment. Our study demonstrates tumors use TAM exosomes to combat CD8 T cells and suggests targeting TAM exosomes as a potential strategy to improve immunotherapies.

In situ PEGylation of CAR T cells alleviates cytokine release syndrome and neurotoxicity.

Chimeric antigen receptor T (CAR T) cell immunotherapy is successful at treating many cancers. However, it often induces life-threatening cytokine release syndrome (CRS) and neurotoxicity. Here, we show that in situ conjugation of polyethylene glycol (PEG) to the surface of CAR T cells ('PEGylation') creates a polymeric spacer that blocks cell-to-cell interactions between CAR T cells, tumour cells and monocytes. Such blockage hinders intensive tumour lysing and monocyte activation by CAR T cells and, consequently, decreases the secretion of toxic cytokines and alleviates CRS-related symptoms. Over time, the slow expansion of CAR T cells decreases PEG surface density and restores CAR T cell-tumour-cell interactions to induce potent tumour killing. This occurs before the restoration of CAR T cell-monocyte interactions, opening a therapeutic window for tumour killing by CAR T cells before monocyte overactivation. Lethal neurotoxicity is also lower when compared with treatment with the therapeutic antibody tocilizumab, demonstrating that in situ PEGylation of CAR T cells provides a materials-based strategy for safer cellular immunotherapy.

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.

Adjuvant lipidoid-substituted lipid nanoparticles augment the immunogenicity of SARS-CoV-2 mRNA vaccines.

Lipid nanoparticle (LNP)-formulated messenger RNA (mRNA) vaccineare a promising platform to prevent infectious diseases as demonstrated by the recent success of SARS-CoV-2 mRNA vaccines. To avoid immune recognition and uncontrolled inflammation, nucleoside-modified mRNA is used. However, such modification largely abrogates the innate immune responses that are critical to orchestrating robust adaptive immunity. Here we develop an LNP component-an adjuvant lipidoid-that can enhance the adjuvanticity of mRNA-LNP vaccines. Our results show that partial substitution of ionizable lipidoid with adjuvant lipidoid not only enhanced mRNA delivery, but also endowed LNPs with Toll-like receptor 7/8-agonistic activity, which significantly increased the innate immunity of the SARS-CoV-2 mRNA-LNP vaccine with good tolerability in mice. Our optimized vaccine elicits potent neutralizing antibodies against multiple SARS-CoV-2 pseudovirus variants, strong Th1-biased cellular immunity, and robust B cell and long-lived plasma cell responses. Importantly, this adjuvant lipidoid substitution strategy works successfully in a clinically relevant mRNA-LNP vaccine, demonstrating its translational potential.

Doxorubicin-conjugated siRNA lipid nanoparticles for combination cancer therapy.

Evasion of apoptosis is a hallmark of cancer, attributed in part to overexpression of the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2). In a variety of cancer types, including lymphoma, Bcl-2 is overexpressed. Therapeutic targeting of Bcl-2 has demonstrated efficacy in the clinic and is the subject of extensive clinical testing in combination with chemotherapy. Therefore, the development of co-delivery systems for Bcl-2 targeting agents, such as small interfering RNA (siRNA), and chemotherapeutics, such as doxorubicin (DOX), holds promise for enabling combination cancer therapies. Lipid nanoparticles (LNPs) are a clinically advanced nucleic acid delivery system with a compact structure suitable for siRNA encapsulation and delivery. Inspired by ongoing clinical trials of albumin-hitchhiking doxorubicin prodrugs, here we developed a DOX-siRNA co-delivery strategy via conjugation of doxorubicin to the surface of siRNA-loaded LNPs. Our optimized LNPs enabled potent knockdown of Bcl-2 and efficient delivery of DOX into the nucleus of Burkitts' lymphoma (Raji) cells, leading to effective inhibition of tumor growth in a mouse model of lymphoma. Based on these results, our LNPs may provide a platform for the co-delivery of various nucleic acids and DOX for the development of new combination cancer therapies.

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing.

Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and Pfizer/BioNTech. These vaccines have demonstrated the efficacy of mRNA-LNP therapeutics and opened the door for future clinical applications. In mRNA-LNP systems, the LNPs serve as delivery platforms that protect the mRNA cargo from degradation by nucleases and mediate their intracellular delivery. The LNPs are typically composed of four components: an ionizable lipid, a phospholipid, cholesterol, and a lipid-anchored polyethylene glycol (PEG) conjugate (lipid-PEG). Here, LNPs encapsulating mRNA encoding firefly luciferase are formulated by microfluidic mixing of the organic phase containing LNP lipid components and the aqueous phase containing mRNA. These mRNA-LNPs are then tested in vitro to evaluate their transfection efficiency in HepG2 cells using a bioluminescent plate-based assay. Additionally, mRNA-LNPs are evaluated in vivo in C57BL/6 mice following an intravenous injection via the lateral tail vein. Whole-body bioluminescence imaging is performed by using an in vivo imaging system. Representative results are shown for the mRNA-LNP characteristics, their transfection efficiency in HepG2 cells, and the total luminescent flux in C57BL/6 mice.

Platelet-Mimicking Nanosponges for Functional Reversal of Antiplatelet Agents.

BACKGROUND: During long-term antiplatelet agents (APAs) administration, patients with thrombotic diseases take a fairly high risk of life-threatening bleeding, especially when in need of urgent surgery. Rapid functional reversal of APAs remains an issue yet to be efficiently resolved by far due to the lack of any specific reversal agent in the clinic, which greatly restricts the use of APAs. METHODS: Flow cytometry analysis was first applied to assess the dose-dependent reversal activity of platelet-mimicking perfluorocarbon-based nanosponges (PLT-PFCs) toward ticagrelor. The tail bleeding time of mice treated with APAs followed by PLT-PFCs was recorded at different time points, along with corresponding pharmacokinetic analysis of ticagrelor and tirofiban. A hemorrhagic transformation model was established in experimental stroke mice with thrombolytic/antiplatelet therapy. Magnetic resonance imaging was subsequently applied to observe hemorrhage and thrombosis in vivo. Further evaluation of the spontaneous clot formation activity of PLT-PFCs was achieved by clot retraction assay in vitro. RESULTS: PLT-PFCs potently reversed the antiplatelet effect of APAs by competitively binding with APAs. PLT-PFCs showed high binding affinity comparable to fresh platelets in vitro with first-line APAs, ticagrelor and tirofiban, and efficiently reversed their function in both tail bleeding and postischemic-reperfusion models. Moreover, the deficiency of platelet intrinsic thrombotic activity diminished the risk of thrombogenesis. CONCLUSIONS: This study demonstrated the safety and effectiveness of platelet-mimicking nanosponges in ameliorating the bleeding risk of different APAs, which offers a promising strategy for the management of bleeding complications induced by antiplatelet therapy.

Ligand-tethered lipid nanoparticles for targeted RNA delivery to treat liver fibrosis.

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.

Solid tumor growth depends on an intricate equilibrium of malignant cell states.

Control of cell identity and number is central to tissue function, yet principles governing organization of malignant cells in tumor tissues remain poorly understood. Using mathematical modeling and candidate-based analysis, we discover primary and metastatic pancreatic ductal adenocarcinoma (PDAC) organize in a stereotypic pattern whereby PDAC cells responding to WNT signals (WNT-R) neighbor WNT-secreting cancer cells (WNT-S). Leveraging lineage-tracing, we reveal the WNT-R state is transient and gives rise to the WNT-S state that is highly stable and committed to organizing malignant tissue. We further show that a subset of WNT-S cells expressing the Notch ligand DLL1 form a functional niche for WNT-R cells. Genetic inactivation of WNT secretion or Notch pathway components, or cytoablation of the WNT-S state disrupts PDAC tissue organization, suppressing tumor growth and metastasis. This work indicates PDAC growth depends on an intricately controlled equilibrium of functionally distinct cancer cell states, uncovering a fundamental principle governing solid tumor growth and revealing new opportunities for therapeutic intervention.

Nanotechnology-based strategies against SARS-CoV-2 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has already infected more than 500 million people globally (as of May 2022), creating the coronavirus disease 2019 (COVID-19) pandemic. Nanotechnology has played a pivotal role in the fight against SARS-CoV-2 in various aspects, with the successful development of the two highly effective nanotechnology-based messenger RNA vaccines being the most profound. Despite the remarkable efficacy of mRNA vaccines against the original SARS-CoV-2 strain, hopes for quickly ending this pandemic have been dampened by the emerging SARS-CoV-2 variants, which have brought several new pandemic waves. Thus, novel strategies should be proposed to tackle the crisis presented by existing and emerging SARS-CoV-2 variants. Here, we discuss the SARS-CoV-2 variants from biological and immunological perspectives, and the rational design and development of novel and potential nanotechnology-based strategies to combat existing and possible future SARS-CoV-2 variants. The lessons learnt and design strategies developed from this battle against SARS-CoV-2 variants could also inspire innovation in the development of nanotechnology-based strategies for tackling other global infectious diseases and their future variants.

Rational Design of Bisphosphonate Lipid-like Materials for mRNA Delivery to the Bone Microenvironment.

The development of lipid nanoparticle (LNP) formulations for targeting the bone microenvironment holds significant potential for nucleic acid therapeutic applications including bone regeneration, cancer, and hematopoietic stem cell therapies. However, therapeutic delivery to bone remains a significant challenge due to several biological barriers, such as low blood flow in bone, blood-bone marrow barriers, and low affinity between drugs and bone minerals, which leads to unfavorable therapeutic dosages in the bone microenvironment. Here, we construct a series of bisphosphonate (BP) lipid-like materials possessing a high affinity for bone minerals, as a means to overcome biological barriers to deliver mRNA therapeutics efficiently to the bone microenvironment in vivo. Following in vitro screening of BP lipid-like materials formulated into LNPs, we identified a lead BP-LNP formulation, 490BP-C14, with enhanced mRNA expression and localization in the bone microenvironment of mice in vivo compared to 490-C14 LNPs in the absence of BPs. Moreover, BP-LNPs enhanced mRNA delivery and secretion of therapeutic bone morphogenetic protein-2 from the bone microenvironment upon intravenous administration. These results demonstrate the potential of BP-LNPs for delivery to the bone microenvironment, which could potentially be utilized for a range of mRNA therapeutic applications including regenerative medicine, protein replacement, and gene editing therapies.