Lipid nanoparticles (LNPs) have emerged across the pharmaceutical industry as promising vehicles to deliver a variety of therapeutics. Currently in the spotlight as vital components of the COVID-19 mRNA vaccines, LNPs play a key role in effectively protecting and transporting mRNA to cells. Liposomes, an early version of LNPs, are a versatile nanomedicine delivery platform. A number of liposomal drugs have been approved and applied to medical practice. Subsequent generations of lipid nanocarriers, such as solid lipid nanoparticles, nanostructured lipid carriers, and cationic lipid-nucleic acid complexes, exhibit more complex architectures and enhanced physical stabilities. With their ability to encapsulate and deliver therapeutics to specific locations within the body and to release their contents at a desired time, LNPs provide a valuable platform for treatment of a variety of diseases. Here, we present a landscape of LNP-related scientific publications, including patents and journal articles, based on analysis of the CAS Content Collection, the largest human-curated collection of published scientific knowledge. Rising trends are identified, such as nanostructured lipid carriers and solid lipid nanoparticles becoming the preferred platforms for numerous formulations. Recent advancements in LNP formulations as drug delivery platforms, such as antitumor and nucleic acid therapeutics and vaccine delivery systems, are discussed. Challenges and growth opportunities are also evaluated in other areas, such as medical imaging, cosmetics, nutrition, and agrochemicals. This report is intended to serve as a useful resource for those interested in LNP nanotechnologies, their applications, and the global research effort for their development.

Lipid Nanoparticles-From Liposomes to mRNA Vaccine Delivery, a Landscape of Research Diversity and Advancement.

This report examines various vaccine platforms including inactivated vaccines, protein-based vaccines, viral vector vaccines, and nucleic acid (DNA or mRNA) vaccines, and their ways of producing immunogens in cells.

A Comprehensive Review of the Global Efforts on COVID-19 Vaccine Development

Previous work has shown that lipid nanoparticles (LNP) composed of an ionizable cationic lipid, a poly(ethylene glycol) (PEG) lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and small interfering RNA (siRNA) can be efficiently manufactured employing microfluidic mixing techniques. Cryo-transmission electron microscopy (cryo-TEM) and molecular simulation studies indicate that these LNP systems exhibit a nanostructured core with periodic aqueous compartments containing siRNA. Here we examine first how the lipid composition influences the structural properties of LNP–siRNA systems produced by microfluidic mixing and, second, whether the microfluidic mixing technique can be extended to macromolecules larger than siRNA. It is shown that LNP–siRNA systems can exhibit progressively more bilayer structure as the proportion of bilayer DSPC lipid is increased, suggesting that the core of LNP–siRNA systems can exhibit a continuum of nanostructures depending on the proportions and structural preferences of component lipids. Second, it is shown that the microfluidic mixing technique can also be extended to encapsulation of much larger negatively charged polymers such mRNA (1.7 kb) or plasmid DNA (6 kb). Finally, as a demonstration of the generality of the microfluidic mixing encapsulation process, it is also demonstrated that negatively charged gold nanoparticles (5 nm diameter) can also be efficiently encapsulated in LNP containing cationic lipids. Interestingly, the nanostructure of these gold-containing LNP reveals a “currant bun” morphology as visualized by cryo-TEM. This structure is fully consistent with LNP–siRNA structure predicted by molecular modeling.

Microfluidic Mixing: A General Method for Encapsulating Macromolecules in Lipid Nanoparticle Systems B

From influenza to COVID-19: Lipid nanoparticle mRNA vaccines at the frontiers of infectious diseases.

Evolution of drug delivery systems: From 1950 to 2020 and beyond

The increasing number of approved nucleic acid therapeutics demonstrates the potential to treat diseases by targeting their genetic blueprints in vivo. Conventional treatments generally induce therapeutic effects that are transient because they target proteins rather than underlying causes. In contrast, nucleic acid therapeutics can achieve long-lasting or even curative effects via gene inhibition, addition, replacement or editing. Their clinical translation, however, depends on delivery technologies that improve stability, facilitate internalization and increase target affinity. Here, we review four platform technologies that have enabled the clinical translation of nucleic acid therapeutics: antisense oligonucleotides, ligand-modified small interfering RNA conjugates, lipid nanoparticles and adeno-associated virus vectors. For each platform, we discuss the current state-of-the-art clinical approaches, explain the rationale behind its development, highlight technological aspects that facilitated clinical translation and provide an example of a clinically relevant genetic drug. In addition, we discuss how these technologies enable the development of cutting-edge genetic drugs, such as tissue-specific nucleic acid bioconjugates, messenger RNA and gene-editing therapeutics.

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The current landscape of nucleic acid therapeutics.

Lipid nanoparticle (LNP) formulations of nucleic acid are leading vaccine candidates for COVID-19, and enabled the first approved RNAi therapeutic, Onpattro. LNPs are composed of ionizable cationic lipids, phosphatidylcholine, cholesterol, and polyethylene glycol (PEG)-lipids, and are produced using rapid-mixing techniques. These procedures involve dissolution of the lipid components in an organic phase and the nucleic acid in an acidic aqueous buffer (pH 4). These solutions are then combined using a continuous mixing device such as a T-mixer or microfluidic device. In this mixing step, particle formation and nucleic acid entrapment occur. Previous work from our group has shown that, in the absence of nucleic acid, the particles formed at pH 4 are vesicular in structure, a portion of these particles are converted to electron-dense structures in the presence of nucleic acid, and the proportion of electron-dense structures increases with nucleic acid content. What remained unclear from previous work was the mechanism by which vesicles form electron-dense structures. In this study, we use cryogenic transmission electron microscopy and dynamic light scattering to show that efficient siRNA entrapment occurs in the absence of ethanol (contrary to the established paradigm), and suggest that nucleic acid entrapment occurs through inversion of preformed vesicles. We also leverage this phenomenon to show that specialized mixers are not required for siRNA entrapment, and that preformed particles at pH 4 can be used for in vitro transfection.

Spontaneous, solvent-free entrapment of siRNA within lipid nanoparticles.

Gene editing mediated by CRISPR/Cas9 systems is due to become a beneficial therapeutic option for treating genetic diseases and some cancers. However, there are challenges in delivering CRISPR components which necessitate sophisticated delivery systems for safe and effective genome editing. Lipid nanoparticles (LNPs) have become an attractive nonviral delivery platform for CRISPR-mediated genome editing due to their low immunogenicity and application flexibility. In this review, we provide a background of CRISPR-mediated gene therapy, as well as LNPs and their applicable characteristics for delivering CRISPR components. We then highlight the challenges of CRISPR delivery, which have driven the significant development of new, safe, and optimized LNP formulations in the past decade. Finally, we discuss considerations for using LNPs to deliver CRISPR and future perspectives on clinical translation of LNP-CRISPR gene editing.

Lipid-Nanoparticle-Based Delivery of CRISPR/Cas9 Genome-Editing Components

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide expansion in the HTT gene, which encodes for an abnormal polyglutamine tract in the huntingtin protein (HTT). This review examines the known mechanisms of HTT gene regulation. We discuss HTT expression patterns, features of the HTT promoter, regulatory regions of the HTT promoter with functional significance, and HTT regulators located outside of the proximal promoter region. The factors that influence HTT expression in the brain and the mechanisms of HTT transcriptional regulation are currently poorly understood, despite continuing research. Expanding knowledge of HTT regulation will inform future studies investigating HTT function. Improving understanding of HTT expression and control may also uncover novel therapeutic approaches for HD through the development of methods to modulate mHTT levels.

Transcriptional Regulation of the Huntingtin Gene.

Background Huntington's disease is a late onset neurological disorder caused by a trinucleotide CAG repeat expansion mutation in the HTT gene encoding for the protein huntingtin. Despite considerable ongoing research, the wild-type function of huntingtin is not yet fully understood. Objective To improve knowledge of HTT gene regulation at the transcriptional level and inform future studies aimed at uncovering the HTT gene's normal function. Methods The HTT gene region was functionally characterized through an in silico analysis using publicly available data sets. ChIP-seq data sets and the online STRING database were used to identify putative transcription factor binding sites (TFBSs) and protein-protein interactions within the HTT promoter region. siRNA-mediated knockdown and ChIP-qPCR of STAT1, a TF identified from the in silico analysis, were used to validate the bioinformatics screen. Results 16 regions containing potential regulatory genomic markers were identified. TFBSs for 59 transcription factors (TFs) were detected in one or more of the 16 candidate regions. Using these TFs, 15 clusters of protein-protein interactions were identified using STRING. siRNA-mediated knockdown of STAT1 resulted in an increase in HTT expression, and ChIP-qPCR detected enrichment of STAT1 binding at one of the predicted regions. These assays confirmed the utility of the bioinformatic analysis. Conclusions Putative regulatory regions outside of the immediate HTT promoter region have been identified with specific protein-protein interactions. Future work will focus on in vitro and in vivo studies to examine the effect of modulating identified TFBSs and altering the levels of specific TFs of interest in regulating HTT gene expression.

Sarah B. Thomson

Papers

The current landscape of nucleic acid therapeutics.

Spontaneous, solvent-free entrapment of siRNA within lipid nanoparticles.

Lipid-Nanoparticle-Based Delivery of CRISPR/Cas9 Genome-Editing Components

Transcriptional Regulation of the Huntingtin Gene.

Computational Analysis of Transcriptional Regulation Sites at the HTT Gene Locus.