Rational design of lipid nanoparticles: overcoming physiological barriers for selective intracellular mRNA delivery.

This review introduces the typical delivery process of messenger RNA (mRNA) nanomedicines and concludes that the delivery involves a at least four-step SCER cascade and that high efficiency at every step is critical to guarantee high overall therapeutic outcomes. This SCER cascade process includes selective organ-targeting delivery, cellular uptake, endosomal escape, and cytosolic mRNA release. Lipid nanoparticles (LNPs) have emerged as a state-of-the-art vehicle for in vivo mRNA delivery. The review emphasizes the importance of LNPs in achieving selective, efficient, and safe mRNA delivery. The discussion then extends to the technical and clinical considerations of LNPs, detailing the roles of individual components in the SCER cascade process, especially ionizable lipids and helper phospholipids. The review aims to provide an updated overview of LNP-based mRNA delivery, outlining recent innovations and addressing challenges while exploring future developments for clinical translation over the next decade.

Investigating the Cytosolic Delivery of Proteins by Lipid Nanoparticles Using the Chloroalkane Penetration Assay.

Protein therapeutics are an expanding area for research and drug development, and lipid nanoparticles (LNPs) are the most prominent nonviral vehicles for protein delivery. The most common methods for assessing protein delivery by LNPs include assays that measure the total amount of protein taken up by cells and assays that measure the phenotypic changes associated with protein delivery. However, assays for total cellular uptake include large amounts of protein that are trapped in endosomes or are otherwise nonfunctional. Assays for functional delivery are important, but the readouts are indirect and amplified, limiting the quantitative interpretation. Here, we apply an assay for cytosolic delivery, the chloroalkane penetration assay (CAPA), to measure the cytosolic delivery of a (-30) green fluorescent protein (GFP) fused to Cre recombinase (Cre(-30)GFP) fusion protein by LNPs. We compare these data to the data from total cellular uptake and functional delivery assays to provide a richer analysis of uptake and endosomal escape for LNP-mediated protein delivery. We also use CAPA for a screen of a small library of lipidoids, identifying those with a promising ability to deliver Cre(-30)GFP to the cytosol of mammalian cells. With careful controls and optimized conditions, we expect that CAPA will be a useful tool for investigating the rate, efficiency, and mechanisms of LNP-mediated delivery of therapeutic proteins.

Harnessing non-Watson-Crick's base pairing to enhance CRISPR effectors cleavage activities and enable gene editing in mammalian cells.

Genomic DNA of the cyanophage S-2L virus is composed of 2-aminoadenine (Z), thymine (T), guanine (G), and cytosine (C), forming the genetic alphabet ZTGC, which violates Watson-Crick base pairing rules. The Z-base has an extra amino group on the two position that allows the formation of a third hydrogen bond with thymine in DNA strands. Here, we explored and expanded applications of this non-Watson-Crick base pairing in protein expression and gene editing. Both ZTGC-DNA (Z-DNA) and ZUGC-RNA (Z-RNA) produced in vitro show detectable compatibility and can be decoded in mammalian cells, including Homo sapiens cells. Z-crRNA can guide CRISPR-effectors SpCas9 and LbCas12a to cleave specific DNA through non-Watson-Crick base pairing and boost cleavage activities compared to A-crRNA. Z-crRNA can also allow for efficient gene and base editing in human cells. Together, our results help pave the way for potential strategies for optimizing DNA or RNA payloads for gene editing therapeutics and give insights to understanding the natural Z-DNA genome.

Monovalent SARS-COV-2 mRNA vaccine using optimal UTRs and LNPs is highly immunogenic and broadly protective against Omicron variants.

The emergence of highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that are resistant to the current COVID-19 vaccines highlights the need for continued development of broadly protective vaccines for the future. Here, we developed two messenger RNA (mRNA)-lipid nanoparticle (LNP) vaccines, TU88mCSA and ALCmCSA, using the ancestral SARS-CoV-2 spike sequence, optimized 5' and 3' untranslated regions (UTRs), and LNP combinations. Our data showed that these nanocomplexes effectively activate CD4+ and CD8+ T cell responses and humoral immune response and provide complete protection against WA1/2020, Omicron BA.1 and BQ.1 infection in hamsters. Critically, in Omicron BQ.1 challenge hamster models, TU88mCSA and ALCmCSA not only induced robust control of virus load in the lungs but also enhanced protective efficacy in the upper respiratory airways. Antigen-specific immune analysis in mice revealed that the observed cross-protection is associated with superior UTRs [Carboxylesterase 1d (Ces1d)/adaptor protein-3ß (AP3B1)] and LNP formulations that elicit robust lung tissue-resident memory T cells. Strong protective effects of TU88mCSA or ALCmCSA against both WA1/2020 and VOCs suggest that this mRNA-LNP combination can be a broadly protective vaccine platform in which mRNA cargo uses the ancestral antigen sequence regardless of the antigenic drift. This approach could be rapidly adapted for clinical use and timely deployment of vaccines against emerging and reemerging VOCs.