High-throughput screens identify a lipid nanoparticle that preferentially delivers mRNA to human tumors in vivo.

Lipid nanoparticles (LNPs) are a clinically relevant way to deliver therapeutic mRNA to hepatocytes in patients. However, LNP-mRNA delivery to end-stage solid tumors such as head and neck squamous cell carcinoma (HNSCC) remains more challenging. While scientists have used in vitro assays to evaluate potential nanoparticles for HNSCC delivery, high-throughput delivery assays performed directly in vivo have not been reported.

Augmented lipid-nanoparticle-mediated in vivo genome editing in the lungs and spleen by disrupting Cas9 activity in the liver.

Systemically delivered lipid nanoparticles are preferentially taken up by hepatocytes. This hinders the development of effective, non-viral means of editing genes in tissues other than the liver. Here we show that lipid-nanoparticle-mediated gene editing in the lung and spleen of adult mice can be enhanced by reducing Cas9-mediated insertions and deletions in hepatocytes via oligonucleotides disrupting the secondary structure of single-guide RNAs (sgRNAs) and also via their combination with short interfering RNA (siRNA) targeting Cas9 messenger RNA (mRNA).

Mild Innate Immune Activation Overrides Efficient Nanoparticle-Mediated RNA Delivery.

Clinical mRNA delivery remains challenging, in large part because how physiology alters delivery in vivo remains underexplored. For example, mRNA delivered by lipid nanoparticles (LNPs) is being considered to treat inflammation, but whether inflammation itself changes delivery remains understudied. Relationships between immunity, endocytosis, and mRNA translation lead to hypothesize that toll-like receptor 4 (TLR4) activation reduced LNP-mediated mRNA delivery.

Cell Subtypes Within the Liver Microenvironment Differentially Interact with Lipid Nanoparticles.

Introduction: Lipid nanoparticles (LNPs) tend to accumulate in the liver due to physiological factors. Whereas the biological mechanisms that promote LNP delivery to hepatocytes have been reported, the mechanisms that promote delivery to other cell types within the liver microenvironment are poorly understood. Single cell profiling studies have recently identified subsets of Kupffer cells and hepatic endothelial cells with distinct gene expression patterns and biological phenotypes; we hypothesized these subtypes would differentially interact with nanoparticles.

Constrained Nanoparticles Deliver siRNA and sgRNA to T Cells In Vivo without Targeting Ligands.

T cells help regulate immunity, which makes them an important target for RNA therapies. While nanoparticles carrying RNA have been directed to T cells in vivo using protein- and aptamer-based targeting ligands, systemic delivery to T cells without targeting ligands remains challenging. Given that T cells endocytose lipoprotein particles and enveloped viruses, two natural systems with structures that can be similar to lipid nanoparticles (LNPs), it is hypothesized that LNPs devoid of targeting ligands can deliver RNA to T cells in vivo.

Using Large Datasets to Understand Nanotechnology.

Advances in sequencing technologies have made studying biological processes with genomics, transcriptomics, and proteomics commonplace. As a result, this suite of increasingly integrated techniques is well positioned to study drug delivery, a process that is influenced by many biomolecules working in concert. Omics-based approaches can be used to study the vast nanomaterial chemical space as well as the biological factors that affect the safety, toxicity, and efficacy of nanotechnologies.

Testing thousands of nanoparticles using DNA barcodes.

Nanoparticles improve drug efficacy by delivering drugs to sites of disease. To effectively deliver a drug a nanoparticle must overcome physical and physiological hurdles that are not present in cell culture, yet screens are used to predict nanoparticle delivery An ideal nanoparticle discovery pipeline would enable scientists to study thousands of nanoparticles Here, we discuss technologies that enable high throughput screens, focusing on DNA barcoded nanoparticles.

Nanoparticles Containing Oxidized Cholesterol Deliver mRNA to the Liver Microenvironment at Clinically Relevant Doses.

Using mRNA to produce therapeutic proteins is a promising approach to treat genetic diseases. However, systemically delivering mRNA to cell types besides hepatocytes remains challenging. Fast identification of nanoparticle delivery (FIND) is a DNA barcode-based system designed to measure how over 100 lipid nanoparticles (LNPs) deliver mRNA that functions in the cytoplasm of target cells in a single mouse.

Barcoding chemical modifications into nucleic acids improves drug stability .

The efficacy of nucleic acid therapies can be limited by unwanted degradation. Chemical modifications are known to improve nucleic acid stability, but the (i) types, (ii) positions, and (iii) numbers of modifications all matter, making chemically optimizing nucleic acids a combinatorial problem. As a result, studies of nucleic acid stability are time consuming and expensive.

Nanoparticles That Deliver RNA to Bone Marrow Identified by in Vivo Directed Evolution.

Bone marrow endothelial cells (BMECs) regulate their microenvironment, which includes hematopoietic stem cells. This makes BMECs an important target cell type for siRNA or gene editing (e.g.

High-throughput in vivo screen of functional mRNA delivery identifies nanoparticles for endothelial cell gene editing.

Dysfunctional endothelium causes more disease than any other cell type. Systemically administered RNA delivery to nonliver tissues remains challenging, in large part because there is no high-throughput method to identify nanoparticles that deliver functional mRNA to cells in vivo. Here we report a system capable of simultaneously quantifying how >100 lipid nanoparticles (LNPs) deliver mRNA that is translated into functional protein.

Modifying a Commonly Expressed Endocytic Receptor Retargets Nanoparticles in Vivo.

Nanoparticles are often targeted to receptors expressed on specific cells, but few receptors are (i) highly expressed on one cell type and (ii) involved in endocytosis. One unexplored alternative is manipulating an endocytic gene expressed on multiple cell types; an ideal gene would inhibit delivery to cell type A more than cell type B, promoting delivery to cell type B. This would require a commonly expressed endocytic gene to alter nanoparticle delivery in a cell type-dependent manner in vivo; whether this can occur is unknown.

Analyzing 2000 in Vivo Drug Delivery Data Points Reveals Cholesterol Structure Impacts Nanoparticle Delivery.

Lipid nanoparticles (LNPs) are formulated using unmodified cholesterol. However, cholesterol is naturally esterified and oxidized in vivo, and these cholesterol variants are differentially trafficked in vivo via lipoproteins including LDL and VLDL. We hypothesized that incorporating the same cholesterol variants into LNPs-which can be structurally similar to LDL and VLDL-would alter nanoparticle targeting in vivo.

A Direct Comparison of in Vitro and in Vivo Nucleic Acid Delivery Mediated by Hundreds of Nanoparticles Reveals a Weak Correlation.

Endothelial cells and macrophages play active roles in disease and as a result are important targets for nucleic acid therapies. While thousands of chemically distinct lipid nanoparticles (LNPs) can be synthesized to deliver nucleic acids, studying more than a few LNPs in vivo is challenging. As a result, it is difficult to understand how nanoparticles target these cells in vivo.