Mild Innate Immune Activation Overrides Efficient Nanoparticle‐Mediated RNA Delivery

Abstract

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 changes delivery remains understudied. Relationships between immunity, endocytosis, and mRNA translation led us to hypothesize that TLR4 activation reduced LNP-mediated mRNA delivery. We therefore quantified LNP uptake, endosomal escape, and mRNA translation with and without TLR4 activation. We used in vivo DNA barcoding to discover a novel LNP that delivers mRNA to Kupffer cells at clinical doses; unlike most LNPs, this LNP did not preferentially target hepatocytes. TLR4 activation blocked mRNA translation in all tested cell types, without reducing LNP uptake; inhibiting TLR4 or its downstream effector PKR improved delivery. The discrepant effects of TLR4 on (i) LNP uptake and (ii) translation suggests TLR4 activation can 'override' LNP targeting, even after mRNA is delivered into target cells. Given near-future clinical trials using mRNA to modulate inflammation, this highlights the need to understand inflammatory signaling in on-and off-target cells. More generally, this suggests a LNP which delivers mRNA to one inflammatory disease may not deliver mRNA to another.

Functionalized lipid-like nanoparticles for in vivo mRNA delivery and base editing

“…As mRNA molecules are vulnerable to ribonuclease enzymes that widely exist in the biological systems and highly negatively charged, which compromise their internalization through cell membranes, suitable in vivo mRNA delivery systems are urgently needed (4). To protect and deliver mRNA, extensive efforts have been devoted to construct biocompatible carriers, such as LNPs, polymeric nanoparticles, peptides/protein-mRNA complexes, and other types of biomaterials (9,22,23). Among these reported platforms, lipid-like nanoparticles (LLNs) are one of the representative materials that have been extensively used for mRNAs delivery in vivo.…”

Nanoparticles containing constrained phospholipids deliver mRNA to liver immune cells in vivo without targeting ligands

“…To test this hypothesis, we synthesized a series of novel adamantyl‐phospholipids and quantified how 109 LNPs functionally delivered mRNA (i.e., mRNA translated into protein) to 16 cell‐types in mice (i.e., in vivo). This systematic high throughput in vivo assay 18‐20 forgoes traditional cell culture experiments. Our data suggest performing drug delivery studies directly in vivo is important; specifically, we found that in vitro nanoparticle delivery can be a poor predictor of in vivo nanoparticle delivery 21 .…”

“…To isolate the effect of constrained phospholipids on LNP formation, we used a lipid‐PEG(C 14 PEG 2000 ), cholesterol (cholesterol, 20α‐hydroxycholesterol, β‐sitosterol), and ionizable lipid (cKK‐E15) components that were previously characterized (Figure 2a), and utilized four different molar ratios (Figure 2b). In order to perform a high throughput in vivo study, we utilized Fast Identification of Nanoparticle Delivery 20 (FIND), a workflow 18‐20 that quantifies mRNA delivery mediated by many LNPs in a single mouse. Briefly, we used microfluidics to formulate 120 LNPs with Cre mRNA and a unique DNA barcode; LNP‐1, with chemical structure 1, carried Cre mRNA and DNA barcode 1, whereas LNP‐N, with chemical structure N, carried Cre mRNA and DNA barcode N. We performed a quality control analysis on all 120 LNPs by quantifying the size of each LNP using dynamic light scattering.…”

“…By isolating tdTomato + cells using fluorescence activated cell sorting (FACS), and quantifying the barcodes in those cells using next-generation DNA sequencing, we identified barcodes in cells that were transfected by functional mRNA. [18][19][20] We quantified the percentage of cells in which Cre mRNA had been functionally delivered (i.e., the tdTomato + cells) from 16 cell populations ( Figure S1b) and found that constrained phospholipids delivered mRNA to Kupffer cells and endothelial cells (ECs) efficiently ( Figure 3b). We then evaluated three additional controls.…”

Treating Cystic Fibrosis with mRNA and CRISPR

“…Importantly, mRNA delivery was rescued by inhibiting PKR and TLR4 with small molecules, illustrating the value of understanding cellular pathways that block the translation of therapeutic mRNA. 90 Modified mRNAs have been used in mRNA drugs treating CF patients. Specifically, Translate Bio, an mRNA company, is currently evaluating a treatment for CF known as MRT5005.…”