Optimization of ionizable lipids for aerosolizable mRNA lipid nanoparticles.

Although mRNA lipid nanoparticles (LNPs) are highly effective as vaccines, their efficacy for pulmonary delivery has not yet fully been established. A major barrier to this therapeutic goal is their instability during aerosolization for local delivery. This imparts a shear force that degrades the mRNA cargo and therefore reduces cell transfection. In addition to remaining stable upon aerosolization, mRNA LNPs must also possess the aerodynamic properties to achieve deposition in clinically relevant areas of the lungs. We addressed these challenges by formulating mRNA LNPs with SM-102, the clinically approved ionizable lipid in the Spikevax COVID-19 vaccine. Our lead candidate, B-1, had the highest mRNA expression in both a physiologically relevant air-liquid interface (ALI) human lung cell model and in healthy mice lungs upon aerosolization. Further, B-1 showed selective transfection in vivo of lung epithelial cells compared to immune cells and endothelial cells. These results show that the formulation can target therapeutically relevant cells in pulmonary diseases such as cystic fibrosis. Morphological studies of B-1 revealed differences in the surface structure compared to LNPs with lower transfection efficiency. Importantly, the formulation maintained critical aerodynamic properties in simulated human airways upon next generation impaction. Finally, structure-function analysis of SM-102 revealed that small changes in the number of carbons can improve upon mRNA delivery in ALI human lung cells. Overall, our study expands the application of SM-102 and its analogs to aerosolized pulmonary delivery and identifies a potent lead candidate for future therapeutically active mRNA therapies.

Targeted transcriptional modulation with type I CRISPR-Cas systems in human cells.

Class 2 CRISPR-Cas systems, such as Cas9 and Cas12, have been widely used to target DNA sequences in eukaryotic genomes. However, class 1 CRISPR-Cas systems, which represent about 90% of all CRISPR systems in nature, remain largely unexplored for genome engineering applications. Here, we show that class 1 CRISPR-Cas systems can be expressed in mammalian cells and used for DNA targeting and transcriptional control. We repurpose type I variants of class 1 CRISPR-Cas systems from Escherichia coli and Listeria monocytogenes, which target DNA via a multi-component RNA-guided complex termed Cascade. We validate Cascade expression, complex formation and nuclear localization in human cells, and demonstrate programmable CRISPR RNA (crRNA)-mediated targeting of specific loci in the human genome. By tethering activation and repression domains to Cascade, we modulate the expression of targeted endogenous genes in human cells. This study demonstrates the use of Cascade as a CRISPR-based technology for targeted eukaryotic gene regulation, highlighting class 1 CRISPR-Cas systems for further exploration.

Genetically Encoded Cholesterol-Modified Polypeptides.

Biological systems use post-translational modifications (PTMs) to control the structure, location, and function of proteins after expression. Despite the ubiquity of PTMs in biology, their use to create genetically encoded recombinant biomaterials is limited. We have utilized a natural lipidation PTM (hedgehog-mediated cholesterol modification of proteins) to create a class of hybrid biomaterials called cholesterol-modified polypeptides (CHaMPs) that exhibit programmable self-assembly at the nanoscale. To demonstrate the biomedical utility of CHaMPs, we used this approach to append cholesterol to biologically active peptide exendin-4 that is an approved drug for the treatment of type II diabetes. The exendin-cholesterol conjugate self-assembled into micelles, and these micelles activate the glucagon-like peptide-1 receptor with a potency comparable to that of current gold standard treatments.