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. By using FIND to quantify how 75 chemically distinct LNPs delivered mRNA to 28 cell types in vivo, it is found that an LNP formulated with oxidized cholesterol and no targeting ligand delivers Cre mRNA, which edits DNA in hepatic endothelial cells and Kupffer cells at 0.05 mg kg−1. Notably, the LNP targets liver microenvironmental cells fivefold more potently than hepatocytes. The structure of the oxidized cholesterols added to the LNP is systematically varied to show that the position of the oxidative modification may be important; cholesterols modified on the hydrocarbon tail associated with sterol ring D tend to outperform cholesterols modified on sterol ring B. These data suggest that LNPs formulated with modified cholesterols can deliver gene‐editing mRNA to the liver microenvironment at clinically relevant doses.
a second example, nanoparticles were coated with anti-CD4 antibodies, leading to 20% target gene silencing at 1 mg kg −1 doses. [6] More recently, lipid nanoparticles (LNPs) that target hepatocytes were retargeted to T cells by coating them with CD4 antibodies, leading to 50% in vivo T cell gene silencing at 6 mg kg −1 doses. [7] These papers (and others) [8] achieve T cell delivery using peptide-, protein-, or aptamer-based targeting ligands, and more generally, ligand-based targeting is used throughout nanomedicine. However, ligands can make reproducible manufacturing at human scales more challenging. [9] One alternative to active targeting is to exploit endogenous lipid trafficking; notably, the only FDA-approved RNA nanoparticle therapy [3] utilizes LNPs without ligands that are trafficked to hepatocytes via endogenous cholesterol transport. [10] Natural trafficking has not been exploited to promote nanoparticle delivery to T cells, yet these cells can interact with viruses and lipoprotein particles, which can have diameters similar to LNPs. [11,12] We therefore hypothesized that LNPs could interact with T cells without targeting ligands. To test this hypothesis, we quantified how well 168 LNPs delivered siRNA to 9 cell types in vivo. Using traditional 1-by-1 in vivo approaches, this would require fluorescence-activated cell sorting (FACS) analysis of hundreds of mice. Thus, to generate large-scale in vivo data, we developed an siGFP/DNA barcode-based screening system. This system quantifies how over 100 nanoparticles deliver siGFP to any desired combination of on-and off-target cell types in vivo. This in vivo approach contrasts with previous LNP research, which utilizes in vitro screening to select a small number of nanoparticles for in vivo evaluation. [13] The approach is supported by evidence that in vitro nanoparticle delivery can be a poor predictor of in vivo nanoparticle delivery. [14] By combining high-throughput in vivo analyses and bioinformatics, we found that a new class of materials, named conformationally constrained lipids, can form stable LNPs. We also found that these "constrained LNPs" (cLNPs) can deliver siRNA to T cells in vivo. These data demonstrate that the conformational state of lipids can alter LNP tropism and provide intriguing preliminary evidence that natural trafficking can promote T cell delivery, offering a potential alternative to active targeting. 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.To test this hypothesis, the delivery of siRNA to 9 cell types in vivo by 168 nanoparticles using ...
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.
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