How has the inclusion of unconventional lipids improved LNP-mediated mRNA delivery?

Safe delivery of mRNA to the brain will revolutionize the treatment of brain tumors. While lipid nanoparticles (LNPs) are clinically most advanced non-viral delivery vehicles for therapeutic mRNA, LNP-mediated mRNA delivery to the brain remains challenging. We hypothesized that rationally designed LNPs based on extracellular vesicle mimicry would enable efficient delivery of RNA therapeutics to brain cells without undue toxicity. We engineered LNPs consisting of four components similar to the formulation used in the mRNA COVID-19 vaccines (Moderna and Pfizer-BioNTech): ionizable lipid, cholesterol, helper lipid and polyethylene glycol (PEG)-lipid. We screened ten classes of helper lipids based on lipids enriched in extracellular vesicles to engineer biomimetic LNPs and tested their GFP mRNA delivery efficacy in SIM-A9 mouse microglia cell line. Several unique LNP formulations with potent delivery efficacy (>90% cells transfected) and stable GFP expression kinetics (5 days) were identified. LNP formulations with high transfection efficacy were then tested in vivo for luciferase mRNA delivery via intrathecal injection in C57BL/6 mice. Luciferase expression in vivo confirmed widespread mRNA delivery in the brain. We then tested Cre recombinase mRNA delivery in Ai9 mouse to identify LNP-targeted cells via flow cytometry and histology. Flow cytometry and expansion microscopy confirmed Cre recombinase mRNA delivery to a variety of brain cells, including microglia (75-90%), neurons (31-40%), neural stem cells (39-62%), oligodendrocytes (70-90%), and astrocytes (44-76%). LNPs were further evaluated for Cas9 mRNA and CD81 sgRNA delivery in C57BL/6 mouse brains to assess brain-targeted gene editing. Sanger sequencing showed that CRISPR-Cas9 editing was successful in ~40% of cells in the mouse brain. In summary, we engineered extracellular vesicle-based LNP library that can deliver RNA therapeutics to a variety of brain cells in vivo. With further development, this technology could potentially enable genetic and epigenetic therapies targeting drivers of brain tumors.

BackgroundThe coronavirus disease 2019 (COVID-19) pandemic has facilitated the clinical implementation of messenger RNA (mRNA) vaccines. The successful development of lipid nanoparticles (LNPs)-based mRNA vaccines, such as Spikevax (Moderna) and...

Targeted mRNA Therapy for Ornithine Transcarbamylase Deficiency.

A lipid nanoparticle platform for mRNA delivery through repurposing of cationic amphiphilic drugs.

Designed synthesis of silica based nanocarriers for mRNA delivery

Impact of tail unsaturation in ionizable lipids on mRNA delivery efficiency and immunogenicity of lipid nanoparticles.

Bacterial outer membrane vesicles (OMVs) possess intrinsic immunostimulatory properties by carrying native antigens and pathogen-associated molecular patterns (PAMPs). Lipid nanoparticles (LNPs) have become the leading platform for messenger ribonucleic acid (mRNA) delivery, underpinning the clinical success of mRNA vaccines. Here, we developed a hybrid nanoparticle formulation, OMV-coated LNPs (OMV-LNPs), that combines the high delivery efficiency of LNPs with the immunogenicity of the OMVs to enhance gene delivery. Using Escherichia coli-derived OMVs and dengue virus (DENV) E80 protein as a model antigen, we generated OMV-LNPs encapsulating DENV mRNA (OMV-LNPmRNA). These nanoparticles demonstrated improved uptake by lymph-node-resident immune cells and enhanced cytosolic mRNA delivery, resulting in robust dendritic cell maturation and proinflammatory cytokine production in vitro. In AG129 mice, the OMV-LNPmRNA elicited significantly higher titers of DENV-neutralizing antibodies compared to conventional LNPs. Collectively, these results establish OMV-LNPs as a versatile and potent platform for effective mRNA vaccine delivery.

Lipid nanoparticle (LNP) remains the most advanced platform for messenger RNA (mRNA) delivery. To date, mRNA LNPs synthesis is mostly performed by mixing lipids and mRNA with microfluidics. In this study, a cost-effective microfluidic setupfor synthesizing mRNA LNPs is developed. It allows to fine-tune the LNPs characteristics without compromising LNP properties. It is compared with a commercial device (NanoAssemblr) and ethanol injection and the influence of manufacturing conditions on the performance of mRNA LNPs is investigated. LNPs prepared by ethanol injection exhibit broader size distributions and more inhomogeneous internal structure (e.g., bleb-like substructures), while other LNPs show uniform structure with dense cores. Small angel X-ray scattering (SAXS) data indicate a tighter interaction between mRNA and lipids within LNPs synthesized by custom device, compared to LNPs produced by NanoAssemblr. Interestingly, the better transfection efficiency of polysarcosine (pSar)-modified LNPs correlateswith a higher surface roughness than that of PEGylated ones. The manufacturing approach, however, shows modest influence on mRNA expression in vivo. In summary, the home-developed cost-effective microfluidic device can synthesize LNPs and represents a potent alternative to NanoAssemblr. The preparation methods show notable effect on LNPs' structure but a minor influence on mRNA delivery in vitro and in vivo.

Enhanced mRNA delivery via incorporating hydrophobic amines into lipid nanoparticles.

Lipid nanoparticles (LNPs) have been extensively used for mRNA delivery and therapeutics, yet their delivery efficacy remains suboptimal. Advancing lipid chemistries and elucidating mechanisms of action offer avenues to enhance their efficacy. Here, we design a library of over 900 aromatic ionizable lipids and systematically study the aromatic incorporation in enhancing mRNA delivery via coordinating multiple mechanisms. Mechanistic investigations reveal that introducing aromatic groups significantly enhances the binding affinity between LNPs and mRNA, improving the encapsulation efficiency. Moreover, aromatic modification promotes membrane fluidity, facilitating superior cellular uptake and endosomal escape. During endosomal membrane remodeling, LNP dissociation and mRNA release occur simultaneously, enabling efficient cytosolic mRNA delivery. Collectively, aromatic incorporation supports multiple steps in the delivery process, resulting in substantially enhanced mRNA delivery efficacy in vivo. Notably, the top-performing candidate exhibits nearly an order-of-magnitude enhancement in delivery efficacy over FDA-approved SM-102 LNPs following both intravenous and intramuscular administration. This study not only presents a robust and efficient aromatic-derived LNP platform for mRNA delivery but also provides broader insights into the rational design and mechanistic understanding of ionizable lipids through the structural exploitation of aromatic functionalities.

Lipid nanoparticles (LNPs) have emerged as a transformative platform for mRNA delivery. However, challenges such as low endosomal escape efficiency remain key barriers in the field. Herein, we systematically investigate the clinically approved COVID-19 vaccine LNPs, extracting insights into physicochemical property-biological function relationships. Then we apply rational design principles to use monoolein (MO) as a structural helper lipid to replace phospholipid in Moderna LNP formulations. We reveal that MO incorporation induces pH-dependent mesophase transitions within LNPs with a protein corona, promoting inverse hexagonal mesophase structures that facilitate enhanced mRNA release and transfection. Comparison with COVID-19 vaccine LNPs confirms that MO-based LNPs achieve superior mRNA transfection efficiency across diverse cell types, including lung macrophages, epithelial cells, and cancer cells. Furthermore, in vivo studies validate enhanced mRNA expression of MO-based LNPs compared to the original Moderna LNPs, underscoring the capacity of both targeted pulmonary delivery by intranasal administration and targeted spleen delivery by intravenous administration. This study establishes a definitive physicochemical structure-biological function property correlation in LNP-mediated mRNA delivery, with the consideration of the protein corona effect on acidification-induced LNP internal mesophase evolution. This work provides guidance for the development of next-generation LNP platforms based on molecular biomedical engineering design principles.

Lipid nanoparticles (LNPs) have emerged as a promising platform for mRNA delivery with widespread applications across various fields, including gene editing, disease treatment, and vaccine development. Improving mRNA expression and enhancing immune responses are essential for expanding their application in mRNA vaccines. In this study, we synthesized and screened a library of guanidine-based lipids (GLs) as the fifth component to enhance LNP-mediated mRNA transfection. After screening, GL9, featuring symmetrical and long alkyl chains, was found to be the optimal fifth component for incorporation into LNPs at a 5% molar ratio to enhance mRNA expression. GL9 addition broadly improved transfection across diverse cell types and LNP formulations, without significantly altering LNP physicochemical properties. Incorporating GL9 into LNP also increased spleen-targeted mRNA expression by 12-fold via intraperitoneal injection and enhanced expression by 2.7-fold after intramuscular administration in vivo. Furthermore, GL9 promoted dendritic cell maturation, highlighting its potential as an adjuvant for mRNA vaccines. Thus, GLs, which enhance both transfection and immune activation, were incorporated into five-component LNPs to establish a promising platform for mRNA vaccines and therapeutics, offering an innovative strategy to optimize LNP-mediated mRNA delivery.

Amniotic fluid stabilized lipid nanoparticles for in utero intra-amniotic mRNA delivery

Ionizable lipid nanoparticles (LNPs) are clinically relevant non-viral vectors that allow intracellular delivery of mRNA vaccines to immune cells. To fight against notorious pathogens and cancer, mRNA vaccines necessitate the addition of an adjuvant to induce strong and durable cell-mediated immune responses. Adjuvants that stimulate Toll-like receptor 7 (TLR7) induce the secretion of type I interferons and proinflammatory cytokines, vital for generating strong immune responses. However, the intracellular delivery of TLR7 adjuvants to precisely stimulate the endosomal TLR7 receptor remains a huge challenge. This issue can be addressed by exploiting ionizable LNP platforms, which can encapsulate and carry mRNA vaccines and small molecule hydrophobic adjuvants to immune cells. CL347 is a potent lipid-based adjuvant that selectively stimulates the TLR7 receptor. In this study, we developed ionizable LNPs incorporating SM102 and CL347 adjuvant as the ionizable lipid and TLR7 adjuvant, respectively. CL347-SM102 LNPs exhibited particle sizes of less than 150nm with spherical morphology and mRNA encapsulation efficiency of greater than 95%. In vivo studies showed a two-fold increase in IFN-γ producing CD4 and CD8 T cells in the lymphoid organs of the mice immunized with adjuvanted LNPs compared to the non-adjuvanted LNPs. Human PBMCs treated with adjuvanted LNPs exhibited significantly higher CD40 expression and pro-inflammatory cytokine (IL-6 and IFN-γ) secretion than non-adjuvanted LNPs. Together, these results suggest the potential of ionizable LNPs as a platform for concurrent delivery of mRNA and adjuvants for prophylactic and therapeutic vaccine applications.

Lipid nanoparticles (LNPs) containing ionizable lipids are the most clinically advanced platform for mRNA delivery, but their application beyond the liver remains challenging. Polymer-lipid hybrid nanoparticles offer a promising alternative, combining the synthetic versatility and unique properties of polymers with the biocompatibility of lipid excipients. While the significance of alkyl tail design is well-recognized for ionizable lipids, the impact of the polymer side chain composition on interactions with lipid excipients, mRNA delivery efficacy, and tissue specificity remains poorly understood. Here, we focus on a class of ionizable amino-polyesters (APEs) that exhibit features desired for potential clinical applications, including narrow molecular weight distribution and a good safety profile, and investigate the effect of polymer side chain composition on the formulation of APE lipid nanoparticles (APE-LNPs) for mRNA delivery. A library of 36 APEs was synthesized via ring-opening polymerization of chemically diverse tertiary amino-alcohols and lactone monomers with distinct alkyl side chain compositions, including variations in length and unsaturation. We show that optimal alkyl side chain length is critical for the assembly of stable mRNA nanoparticles and efficient mRNA delivery both in vitro and in vivo. Top-performing APE-LNPs display superior delivery efficacy in vitro and in extrahepatic tissues compared to benchmark LNPs, including DLin-MC3-DMA ionizable lipid. The polymer chain composition affects the tissue selectivity of APE-LNPs, with shorter side chains (4-5 carbons) effectively targeting the spleen and lungs, while longer chains (7-9 carbons) show enhanced liver delivery. We also explored the relevance of lipid excipients in APE-LNPs, demonstrating the essential role of unsaturated phospholipids in enhancing cellular uptake and mRNA delivery, and the limited relevance of cholesterol. These findings provide valuable insights into the design of polymers for use in the LNP context, which could aid the development of polymeric alternatives to ionizable lipids and expand the utility of mRNA LNP technology to nonliver tissues.