Accelerating ionizable lipid discovery for mRNA delivery using machine learning and combinatorial chemistry.

To unlock the full promise of messenger (mRNA) therapies, expanding the toolkit of lipid nanoparticles is paramount. However, a pivotal component of lipid nanoparticle development that remains a bottleneck is identifying new ionizable lipids. Here we describe an accelerated approach to discovering effective ionizable lipids for mRNA delivery that combines machine learning with advanced combinatorial chemistry tools. Starting from a simple four-component reaction platform, we create a chemically diverse library of 584 ionizable lipids. We screen the mRNA transfection potencies of lipid nanoparticles containing those lipids and use the data as a foundational dataset for training various machine learning models. We choose the best-performing model to probe an expansive virtual library of 40,000 lipids, synthesizing and experimentally evaluating the top 16 lipids flagged. We identify lipid 119-23, which outperforms established benchmark lipids in transfecting muscle and immune cells in several tissues. This approach facilitates the creation and evaluation of versatile ionizable lipid libraries, advancing the formulation of lipid nanoparticles for precise mRNA delivery.

Enhancing the immunogenicity of lipid-nanoparticle mRNA vaccines by adjuvanting the ionizable lipid and the mRNA.

To elicit optimal immune responses, messenger RNA vaccines require intracellular delivery of the mRNA and the careful use of adjuvants. Here we report a multiply adjuvanted mRNA vaccine consisting of lipid nanoparticles encapsulating an mRNA-encoded antigen, optimized for efficient mRNA delivery and for the enhanced activation of innate and adaptive responses. We optimized the vaccine by screening a library of 480 biodegradable ionizable lipids with headgroups adjuvanted with cyclic amines and by adjuvanting the mRNA-encoded antigen by fusing it with a natural adjuvant derived from the C3 complement protein. In mice, intramuscular or intranasal administration of nanoparticles with the lead ionizable lipid and with mRNA encoding for the fusion protein (either the spike protein or the receptor-binding domain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)) increased the titres of antibodies against SARS-CoV-2 tenfold with respect to the vaccine encoding for the unadjuvanted antigen. Multiply adjuvanted mRNA vaccines may improve the efficacy, safety and ease of administration of mRNA-based immunization.

Delivering the next generation of cancer immunotherapies with RNA.

Decades of oncologic clinical use have demonstrated that cancer immunotherapy provides unprecedented therapeutic benefits. Tragically, only a minority of patients respond to existing immunotherapies. RNA lipid nanoparticles have recently emerged as modular tools for immune stimulation. Here, we discuss advancements in RNA-based cancer immunotherapies and opportunities for improvement.

Anti-inflammatory nanoparticles significantly improve muscle function in a murine model of advanced muscular dystrophy.

Chronic inflammation contributes to the pathogenesis of all muscular dystrophies. Inflammatory T cells damage muscle, while regulatory T cells (Tregs) promote regeneration. We hypothesized that providing anti-inflammatory cytokines in dystrophic muscle would promote proregenerative immune phenotypes and improve function. Primary T cells from dystrophic (mdx) mice responded appropriately to inflammatory or suppressive cytokines. Subsequently, interleukin-4 (IL-4)- or IL-10-conjugated gold nanoparticles (PA4, PA10) were injected into chronically injured, aged, mdx muscle. PA4 and PA10 increased T cell recruitment, with PA4 doubling CD4+/CD8- T cells versus controls. Further, 50% of CD4+/CD8- T cells were immunosuppressive Tregs following PA4, versus 20% in controls. Concomitant with Treg recruitment, muscles exhibited increased fiber area and fourfold increases in contraction force and velocity versus controls. The ability of PA4 to shift immune responses, and improve dystrophic muscle function, suggests that immunomodulatory treatment may benefit many genetically diverse muscular dystrophies, all of which share inflammatory pathology.

Adsorption and desorption of DNA-functionalized beads in glass microfluidic channels.

Integrated microfluidic devices for the purification, amplification, and detection of nucleic acids are a prevalent area of research due to their potential for miniaturization, assay integration, and increased efficiency over benchtop assays. These devices frequently contain micrometer-sized magnetic beads with a large surface area for the capture and manipulation of biological molecules such as DNA and RNA. Although magnetic beads are a standard tool for many biological assays, beads functionalized with biological molecules can adhere to microchannel walls and prevent further manipulation of the beads within the channel. Here, we analyze the effects of solution composition, microchannel hydrophobicity, and bead surface hydrophobicity on DNA-functionalized bead adhesion in a borosilicate glass microfluidic device. Bead adhesion is primarily a result of adsorption of the bead-linked DNA molecule to the microchannel wall; >81% of beads are consistently removed when not functionalized with DNA. Hydrophobicities of both the microchannel walls and the microbead surface are the primary determinants of bead adhesion, rather than electrostatic interactions and ion bridging. Surprisingly, DNA-functionalized bead adhesion in a standard RNA amplification solution was virtually eliminated by using hydrophobic microbeads with hydrophobic microchannel walls; under such conditions, 96.6 ± 1.6% of the beads were removed in one 43 nl/s, 10-min wash. The efficiency of a downstream RNA amplification reaction using DNA-functionalized beads did not appear to be affected by the hydrophobicity of the microbead surface. These findings can be applied to assays that require the efficient use of magnetic beads in DNA-based microfluidic assays.

Combined delivery of VEGF and IGF-1 promotes functional innervation in mice and improves muscle transplantation in rabbits.

Microvascular muscle transfer is the gold standard for reanimation following chronic facial nerve paralysis, however, despite the regenerative capacity of peripheral motor axons, poor reinnervation often results in sub-optimal function. We hypothesized that injection of alginate hydrogels releasing growth factors directly into donor tissue would promote reinnervation, muscle regeneration, and function. A murine model of sciatic nerve ligation and neurorrhaphy was first used to assess the ability of gel delivery of vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) to promote functional reinnervation. VEGF + IGF-1 gel delivery to aged mice resulted in prolonged ability to control toe movement, increased toe spreading, and improved static sciatic index score, indicative of improved sciatic nerve and neuromuscular junction function. Further, a 26% increase in muscle fiber area, and 2.8 and 3.0-fold increases in muscle contraction force and velocity, respectively, were found compared to blank alginate in the murine model. This strategy was subsequently tested in a rabbit model of craniofacial gracilis muscle transplantation. Electromyography demonstrated a 71% increase in compound muscle action potential 9 weeks after transplantation following treatment with VEGF + IGF-1 alginate, compared to blank alginate in the rabbit model. Improving functional innervation in transplanted muscle via a hydrogel source of growth factors may enhance the therapeutic outcomes of facial palsy treatments and, more broadly, muscle transplantations.

An injectable bone marrow-like scaffold enhances T cell immunity after hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for multiple disorders, but deficiency and dysregulation of T cells limit its utility. Here we report a biomaterial-based scaffold that mimics features of T cell lymphopoiesis in the bone marrow. The bone marrow cryogel (BMC) releases bone morphogenetic protein-2 to recruit stromal cells and presents the Notch ligand Delta-like ligand-4 to facilitate T cell lineage specification of mouse and human hematopoietic progenitor cells. BMCs subcutaneously injected in mice at the time of HSCT enhanced T cell progenitor seeding of the thymus, T cell neogenesis and diversification of the T cell receptor repertoire. Peripheral T cell reconstitution increased ~6-fold in mouse HSCT and ~2-fold in human xenogeneic HSCT. Furthermore, BMCs promoted donor CD4+ regulatory T cell generation and improved survival after allogeneic HSCT. In comparison to adoptive transfer of T cell progenitors, BMCs increased donor chimerism, T cell generation and antigen-specific T cell responses to vaccination. BMCs may provide an off-the-shelf approach for enhancing T cell regeneration and mitigating graft-versus-host disease in HSCT.

Functional muscle recovery with nanoparticle-directed M2 macrophage polarization in mice.

Persistence of inflammation, and associated limits in tissue regeneration, are believed to be due in part to the imbalance of M1 over M2 macrophages. Here, we hypothesized that providing a sustained source of an antiinflammatory polarizing cytokine would shift the balance of macrophages at a site of tissue damage to improve functional regeneration. Specifically, IL-4-conjugated gold nanoparticles (PA4) were injected into injured murine skeletal muscle, resulting in improved histology and an ∼40% increase in muscle force compared with mice treated with vehicle only. Macrophages were the predominant infiltrating immune cell, and treatment with PA4 resulted in an approximately twofold increase in the percentage of macrophages expressing the M2a phenotype and an approximately twofold decrease in M1 macrophages, compared with mice treated with vehicle only. Intramuscular injection of soluble IL-4 did not shift macrophage polarization or result in functional muscle improvements. Depletion of monocytes/macrophages eliminated the therapeutic effects of PA4, suggesting that improvement in muscle function was the result of M2-shifted macrophage polarization. The ability of PA4 to direct macrophage polarization in vivo may be beneficial in the treatment of many injuries and inflammatory diseases.

Matrix stiffness and tumor-associated macrophages modulate epithelial to mesenchymal transition of human adenocarcinoma cells.

The tumor microenvironment (TME) is gaining increasing attention in oncology, as it is recognized to be functionally important during tumor development and progression. Tumors are heterogeneous tissues that, in addition to tumor cells, contain tumor-associated cell types such as immune cells, fibroblasts, and endothelial cells. These other cells, together with the specific extracellular matrix (ECM), create a permissive environment for tumor growth. While the influence of tumor-infiltrating cells and mechanical properties of the ECM in tumor invasion and progression have been studied separately, their interaction within the complex TME and the epithelial -to-mesenchymal transition (EMT) is still unclear. In this work, we develop a 3D co-culture model of lung adenocarcinoma cells and macrophages in an interpenetrating network hydrogel, to investigate the influence of the macrophage phenotype and ECM stiffness in the induction of EMT. Rising ECM stiffness increases both tumor cell proliferation and invasiveness. The presence of tumor-associated macrophages and the ECM stiffness jointly contribute to an invasive phenotype, and modulate the expression of key EMT-related markers. Overall, these findings support the utility of in vitro 3D cancer models that allow one to study interactions among key components of the TME.

Switchable Release of Entrapped Nanoparticles from Alginate Hydrogels.

Natural biological processes are intricately controlled by the timing and spatial distribution of various cues. To mimic this precise level of control, the physical sizes of gold nanoparticles are utilized to sterically entrap them in hydrogel materials, where they are subsequently released only in response to ultrasound. These nanoparticles can transport bioactive factors to cells and direct cell behavior on-demand.