Substituting racemic ionizable lipids with stereopure ionizable lipids can increase mRNA delivery.

Lipid nanoparticles (LNPs) have delivered siRNA and mRNA drugs in humans, underscoring the potential impact of improving the therapeutic window of next-generation LNPs. To increase the LNP therapeutic window, we applied lessons from small-molecule chemistry to ionizable lipid design. Specifically, given that stereochemistry often influences small-molecule safety and pharmacokinetics, we hypothesized that the stereochemistry of lipids within an LNP would influence mRNA delivery. We tested this hypothesis in vivo using 128 novel LNPs that included stereopure derivatives of C12-200, an ionizable lipid that when formulated into LNPs delivers RNA in mice and non-human primates but is not used clinically due to its poor tolerability. We found that a novel C12-200-S LNP delivered up to 2.8-fold and 6.1-fold more mRNA in vivo than its racemic and C12-200-R controls, respectively. To identify the potential causes leading to increased delivery, we quantified LNP biophysical traits and concluded that these did not change with stereochemistry. Instead, we found that stereopure LNPs were better tolerated than racemic LNPs in vivo. These data suggest that LNP-mediated mRNA delivery can be improved by designing LNPs to include stereopure ionizable lipids.

Evaluation of Longitudinal Lymphatic Function Changes upon Injury in the Mouse Tail with Photodynamic Therapy.

PURPOSE: The lymphatic system is an essential but often understudied component of the circulatory system in comparison with its cardiovascular counterpart. Such disparity could often be explained by the difficulty in imaging lymphatics and the specialized microsurgical skills that are often required for lymphatic injury models. Recently, it has been shown that verteporfin, a photosensitive drug used for photodynamic therapy (PDT) to ablate the blood vessels, provides a similar effect on lymphatic vessels. Here, we seek to administer verteporfin and perform a modified form of PDT on collecting lymphatics in the mouse tail, a commonly used location for the study of lymphatic disorders, and examine lymphatic remodeling, contractility, and transport in response to the procedure. METHODS: Mice collecting lymphatics in the tail were injured by PDT through an intradermal injection of verteporfin in the distal tip of the tail followed by light activation on the proximal portion of the tail downstream of the injection site. Lymphatic function was evaluated using a near-infrared (NIR) imaging system weekly for up to 28 days after injury. RESULTS: PDT resulted in a loss in lymphatic function contractile frequency that persisted for up to 7 days after injury. Packet transport and packet amplitude, measurements reflective of the strength of contraction, were significantly reduced 14 days after injury. The lymphatics showed a delayed increase in lymphatic leakage at 7 days that persisted until the study endpoint on day 28. CONCLUSION: This technique provides an easy-to-use method for injuring lymphatics to understand their remodeling response to injury by PDT as well as potentially for screening therapeutics that seek to normalize lymphatic permeability or contractile function after injury.

Nanoparticle single-cell multiomic readouts reveal that cell heterogeneity influences lipid nanoparticle-mediated messenger RNA delivery.

Cells that were previously described as homogeneous are composed of subsets with distinct transcriptional states. However, it remains unclear whether this cell heterogeneity influences the efficiency with which lipid nanoparticles (LNPs) deliver messenger RNA therapies in vivo. To test the hypothesis that cell heterogeneity influences LNP-mediated mRNA delivery, we report here a new multiomic nanoparticle delivery system called single-cell nanoparticle targeting-sequencing (SENT-seq). SENT-seq quantifies how dozens of LNPs deliver DNA barcodes and mRNA into cells, the subsequent protein production and the transcriptome, with single-cell resolution. Using SENT-seq, we have identified cell subtypes that exhibit particularly high or low LNP uptake as well as genes associated with those subtypes. The data suggest that cell subsets have distinct responses to LNPs that may affect mRNA therapies.