Recent advances in lipid nanoparticles for delivery of nucleic acid, mRNA, and gene editing-based therapeutics

Abstract

Lipid nanoparticles (LNPs) are becoming popular as a means of delivering therapeutics, including those based on nucleic acids and mRNA. The mRNA-based coronavirus disease 2019 vaccines are perfect examples to highlight the role played by drug delivery systems in advancing human health. The fundamentals of LNPs for the delivery of nucleic acid- and mRNA-based therapeutics, are well established. Thus, future research on LNPs will focus on addressing the following: expanding the scope of drug delivery to different constituents of the human body, expanding the number of diseases that can be targeted, and studying the change in the pharmacokinetics of LNPs under physiological and pathological conditions. This review article provides an overview of recent advances aimed at expanding the application of LNPs, focusing on the pharmacokinetics and advantages of LNPs. In addition, analytical techniques, library construction and screening, rational design, active targeting, and applicability to gene editing therapy have also been discussed.

Keywords: Base editing; DNA barcode; Gene editing; Ionizable lipid; Lipid nanoparticle; Nucleic acid; Positron emission tomography; Recombination; Targeting; mRNA.

Fig. 1

The concepts, basic components, and fundamental technologies of lipid nanoparticles for delivery of nucleic acid and mRNA-based therapeutics. Nucleic acids and mRNAs are in the lipid cores of LNPs; the outer lipid membranes of LNPs are not perfectly aligned bilayers like liposomes, but are close to micelles in structure. Ionizable lipids contribute to efficient endosomal escape and release of nucleic acids and mRNAs from LNPs after uptake by cells, in addition to the encapsulation of nucleic acids and mRNAs into LNPs. The LNPs modified with 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG) can detach PEG at a moderate rate in the blood circulation, resulting in efficient intracellular delivery of nucleic acids and mRNAs. The uptake of LNPs in the hepatocytes, after the intravenous administration, is caused via the low-density lipoprotein receptor (LDLR) because of the interaction between LNPs and apolipoprotein E (apoE) in the blood.

Fig. 2

Analysis of the intratissue distribution of LNPs using the Cre/loxP recombinase system. The transgenic mice that have reporter genes downstream of the loxP-flanked stop cassette, blocks reporter gene expression in the absence of cyclization recombinase (Cre) in the cells, but in the presence of Cre, the stop cassette is removed, and the gene is expressed. Treatment of these mice with LNPs encapsulating mRNA-encoding Cre results in stable expression of fluorescent protein in the delivered cells selectively.

Fig. 3

High-throughput screening of LNPs suitable for targeting each organ/tissue using DNA barcoding. LNPs with different compositions encapsulating nucleic acids with individual DNA or RNA sequences assigned to each as barcodes are prepared and made into libraries. By administering the mixture of these barcoded LNPs to experimental animals and performing deep sequencing for DNA or RNA barcode in each tissue, the tissue distribution characteristics of LNPs with each lipid composition can be quantified at once.

Fig. 4

The mechanisms of three types of CRISPR gene editing: non-homologous end joining (NHEJ), homology-directed repair (HDR), and base editing. The Cas9 nuclease makes a double-strand break at specific locations in the genome targeted by single-guide RNAs (sgRNAs). Mutations are introduced during subsequent repair process, causing frameshifts and premature termination codons. During the repair process, HDR occurs if there is a donor DNA, or NHEJ due to a random insertion/deletion if there is no donor DNA. In base editing, base editors, which are fusion proteins of a dead Cas9 and cytidine or adenine deaminase, are used to replace C•G base pair to T•A base pair or vice versa without cleaving the DNA strand.