Cationic cholesterol-dependent LNP delivery to lung stem cells, the liver, and heart.

Adding a cationic helper lipid to a lipid nanoparticle (LNP) can increase lung delivery and decrease liver delivery. However, it remains unclear whether charge-dependent tropism is universal or, alternatively, whether it depends on the component that is charged. Here, we report evidence that cationic cholesterol-dependent tropism can differ from cationic helper lipid-dependent tropism. By testing how 196 LNPs delivered mRNA to 22 cell types, we found that charged cholesterols led to a different lung:liver delivery ratio than charged helper lipids. We also found that combining cationic cholesterol with a cationic helper lipid led to mRNA delivery in the heart as well as several lung cell types, including stem cell-like populations. These data highlight the utility of exploring charge-dependent LNP tropism.

Substituting Poly(ethylene glycol) Lipids with Poly(2-ethyl-2-oxazoline) Lipids Improves Lipid Nanoparticle Repeat Dosing.

Poly(ethylene glycol) (PEG)-lipids are used in Food-and-Drug-Administration-approved lipid nanoparticle (LNP)-RNA drugs, which are safe and effective. However, it is reported that PEG-lipids may also contribute to accelerated blood clearance and rare cases of hypersensitivity; this highlights the utility of exploring PEG-lipid alternatives. Here, it is shown that LNPs containing poly(2-ethyl-2-oxazoline) (PEOZ)-lipids can deliver messenger RNA (mRNA) to multiple cell types in mice inside and outside the liver. In addition, it is reported that LNPs formulated with PEOZ-lipids show reduced clearance from the bloodstream and lower levels of antistealth lipid immunoglobulin Ms than LNPs formulated with PEG-lipids. These data justify further exploration of PEOZ-lipids as alternatives to PEG-lipids in LNP-RNA formulations.

Nanoparticle stereochemistry-dependent endocytic processing improves in vivo mRNA delivery.

Stereochemistry can alter small-molecule pharmacokinetics, safety and efficacy. However, it is unclear whether the stereochemistry of a single compound within a multicomponent colloid such as a lipid nanoparticle (LNP) can influence its activity in vivo. Here we report that LNPs containing stereopure 20α-hydroxycholesterol (20α) delivered mRNA to liver cells up to 3-fold more potently than LNPs containing a mixture of both 20α- and 20ß-hydroxycholesterols (20mix). This effect was not driven by LNP physiochemical traits. Instead, in vivo single-cell RNA sequencing and imaging revealed that 20mix LNPs were sorted into phagocytic pathways more than 20α LNPs, resulting in key differences between LNP biodistribution and subsequent LNP functional delivery. These data are consistent with the fact that nanoparticle biodistribution is necessary, but not sufficient, for mRNA delivery, and that stereochemistry-dependent interactions between LNPs and target cells can improve mRNA delivery.

The Transcriptional Response to Lung-Targeting Lipid Nanoparticles in Vivo.

Lipid nanoparticles (LNPs) have delivered RNA to hepatocytes in patients, underscoring the potential impact of nonliver delivery. Scientists can shift LNP tropism to the lung by adding cationic helper lipids; however, the biological response to these LNPs remains understudied. To evaluate the hypothesis that charged LNPs lead to differential cellular responses, we quantified how 137 LNPs delivered mRNA to 19 cell types in vivo. Consistent with previous studies, we observed helper lipid-dependent tropism. After identifying and individually characterizing three LNPs that targeted different tissues, we studied the in vivo transcriptomic response to these using single-cell RNA sequencing. Out of 835 potential pathways, 27 were upregulated in the lung, and of these 27, 19 were related to either RNA or protein metabolism. These data suggest that endogenous cellular RNA and protein machinery affects mRNA delivery to the lung in vivo.

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.

The Extent to Which Lipid Nanoparticles Require Apolipoprotein E and Low-Density Lipoprotein Receptor for Delivery Changes with Ionizable Lipid Structure.

Lipid nanoparticles (LNPs) have delivered therapeutic RNA to hepatocytes in humans. Adsorption of apolipoprotein E (ApoE) onto these clinical LNP-mRNA drugs has been shown to facilitate hepatocyte entry via the low-density lipoprotein receptor (LDLR). Since ApoE-LDLR trafficking is conserved in mice, non-human primates, and humans, characterizing this mechanism eased clinical transition. Recently, LNPs have delivered mRNA to non-hepatocytes in mice and non-human primates, suggesting they can target new cell types via ApoE- and LDLR-independent pathways. To test this hypothesis, we quantified how 60 LNPs delivered mRNA with cell type resolution in wild-type mice and three knockout mouse strains related to lipid trafficking: ApoE-/-, LDLR-/-, and PCSK9-/-. These data suggest that the hydrophobic tail length of diketopiperazine-based lipids can be changed to drive ApoE- and LDLR-independent delivery in vivo. More broadly, the results support the hypothesis that endogenous LNP trafficking can be tuned by modifying lipid chemistry.

Piperazine-derived lipid nanoparticles deliver mRNA to immune cells in vivo.

In humans, lipid nanoparticles (LNPs) have safely delivered therapeutic RNA to hepatocytes after systemic administration and to antigen-presenting cells after intramuscular injection. However, systemic RNA delivery to non-hepatocytes remains challenging, especially without targeting ligands such as antibodies, peptides, or aptamers. Here we report that piperazine-containing ionizable lipids (Pi-Lipids) preferentially deliver mRNA to immune cells in vivo without targeting ligands. After synthesizing and characterizing Pi-Lipids, we use high-throughput DNA barcoding to quantify how 65 chemically distinct LNPs functionally delivered mRNA (i.e., mRNA translated into functional, gene-editing protein) in 14 cell types directly in vivo. By analyzing the relationships between lipid structure and cellular targeting, we identify lipid traits that increase delivery in vivo. In addition, we characterize Pi-A10, an LNP that preferentially delivers mRNA to the liver and splenic immune cells at the clinically relevant dose of 0.3 mg/kg. These data demonstrate that high-throughput in vivo studies can identify nanoparticles with natural non-hepatocyte tropism and support the hypothesis that lipids with bioactive small-molecule motifs can deliver mRNA in vivo.

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.

Universal Barcoding Predicts In Vivo ApoE-Independent Lipid Nanoparticle Delivery.

To predict whether preclinical lipid nanoparticle (LNP) delivery will translate in humans, it is necessary to understand whether the mechanism used by LNPs to enter cells is conserved across species. In mice, non-human primates, and humans, LNPs deliver RNA to hepatocytes by adsorbing apolipoprotein E (ApoE), which binds low-density lipoprotein receptor (LDLR). A growing number of LNPs can deliver RNA to nonhepatocytes, suggesting that ApoE- and LDLR-independent interactions could affect LNP tropism. To evaluate this hypothesis, we developed a universal DNA barcoding system that quantifies how chemically distinct LNPs deliver small interfering RNA in any mouse model, including genetic knockouts. We quantified how 98 different LNPs targeted 11 cell types in wildtype, LDLR-/-, very low-density lipoprotein receptor, and ApoE-/- mice, studying how these genes, which traffic endogenous lipids, affected LNP delivery. These data identified a novel, stereopure LNP that targets Kupffer cells, endothelial cells, and hepatocytes in an ApoE-independent manner. These results suggest that non-ApoE interactions can affect the tropism of LNP-RNA drugs.

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). In SpCas9 mice with acute lung inflammation, the systemic delivery of an oligonucleotide inhibiting an sgRNA targeting the intercellular adhesion molecule 2 (ICAM-2), followed by the delivery of the sgRNA, reduced the fraction of ICAM-2 indels in hepatocytes and increased that in lung endothelial cells. In wild-type mice, the lipid-nanoparticle-mediated delivery of an inhibitory oligonucleotide, followed by the delivery of Cas9-degrading siRNA and then by Cas9 mRNA and sgRNA, reduced the fraction of ICAM-2 indels in hepatocytes but not in splenic endothelial cells. Inhibitory oligonucleotides and siRNAs could be used to modulate the cell-type specificity of Cas9 therapies.

Species-dependent in vivo mRNA delivery and cellular responses to nanoparticles.

Nanoparticles are tested in mice and non-human primates before being selected for clinical trials. Yet the extent to which mRNA delivery, as well as the cellular response to mRNA drug delivery vehicles, is conserved across species in vivo is unknown. Using a species-independent DNA barcoding system, we have compared how 89 lipid nanoparticles deliver mRNA in mice with humanized livers, primatized livers and four controls: mice with 'murinized' livers as well as wild-type BL/6, Balb/C and NZB/BlNJ mice. We assessed whether functional delivery results in murine, non-human primate and human hepatocytes can be used to predict delivery in the other species in vivo. By analysing in vivo hepatocytes by RNA sequencing, we identified species-dependent responses to lipid nanoparticles, including mRNA translation and endocytosis. These data support an evidence-based approach to making small-animal preclinical nanoparticle studies more predictive, thereby accelerating the development of RNA therapies.

Optimization of lipid nanoparticles for the delivery of nebulized therapeutic mRNA to the lungs.

Lipid nanoparticles (LNPs) for the efficient delivery of drugs need to be designed for the particular administration route and type of drug. Here we report the design of LNPs for the efficient delivery of therapeutic RNAs to the lung via nebulization. We optimized the composition, molar ratios and structure of LNPs made of lipids, neutral or cationic helper lipids and poly(ethylene glycol) (PEG) by evaluating the performance of LNPs belonging to six clusters occupying extremes in chemical space, and then pooling the lead clusters and expanding their diversity. We found that a low (high) molar ratio of PEG improves the performance of LNPs with neutral (cationic) helper lipids, an identified and optimal LNP for low-dose messenger RNA delivery. Nebulized delivery of an mRNA encoding a broadly neutralizing antibody targeting haemagglutinin via the optimized LNP protected mice from a lethal challenge of the H1N1 subtype of influenza A virus, and delivered mRNA more efficiently than LNPs previously optimized for systemic delivery. A cluster approach to LNP design may facilitate the optimization of LNPs for other administration routes and therapeutics.

Therapeutic RNA Delivery for COVID and Other Diseases.

RNA can alter the expression of endogenous genes and can be used to express therapeutic proteins. As a result, RNA-based therapies have recently mitigated disease in patients. Yet most potential RNA therapies cannot currently be developed, in large part because delivering therapeutic quantities of RNA drugs to diseased cells remains difficult. Here, recent studies focused on the biological hurdles that make in vivo drug delivery challenging are described. Then RNA drugs that have overcome these challenges in humans, focusing on siRNA to treat liver disease and mRNA to vaccinate against COVID, are discussed. Finally, research centered on improving drug delivery to new tissues is highlighted, including the development of high-throughput in vivo nanoparticle DNA barcoding assays capable of testing over 100 distinct nanoparticles in a single animal.

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

Once inside the cytoplasm of a cell, mRNA can be used to treat disease by upregulating the expression of any gene. Lipid nanoparticles (LNPs) can deliver mRNA to hepatocytes in humans, yet systemic non-hepatocyte delivery at clinical doses remains difficult. We noted that LNPs have historically been formulated with phospholipids containing unconstrained alkyl tails. Based on evidence that constrained adamantyl groups have unique properties that can improve small molecule drug delivery, we hypothesized that a phospholipid containing an adamantyl group would facilitate mRNA delivery in vivo. We quantified how 109 LNPs containing "constrained phospholipids" delivered mRNA to 16 cell types in mice, then using a DNA barcoding-based analytical pipeline, related phospholipid structure to in vivo delivery. By analyzing delivery mediated by constrained phospholipids, we identified a novel LNP that delivers mRNA to immune cells at 0.5 mg/kg. Unlike many previous LNPs, these (a) did not preferentially target hepatocytes and (b) delivered mRNA to immune cells without targeting ligands. These data suggest constrained phospholipids may be useful LNP components.

A flow platform for degradation-free CuAAC bioconjugation.

The Cu-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is a cornerstone method for the ligation of biomolecules. However, undesired Cu-mediated oxidation and Cu-contamination in bioconjugates limits biomedical utility. Here, we report a generic CuAAC flow platform for the rapid, robust, and broad-spectrum formation of discrete triazole bioconjugates. This process leverages an engineering problem to chemical advantage: solvent-mediated Cu pipe erosion generates ppm levels of Cu in situ under laminar flow conditions. This is sufficient to catalyze the CuAAC reaction of small molecule alkynes and azides, fluorophores, marketed drug molecules, peptides, DNA, and therapeutic oligonucleotides. This flow approach, not replicated in batch, operates at ambient temperature and pressure, requires short residence times, avoids oxidation of sensitive functional groups, and produces products with very low ppm Cu contamination.

Modular, Step-Efficient Palladium-Catalyzed Cross-Coupling Strategy To Access C6-Heteroaryl 2-Aminopurine Ribonucleosides.

Two Pd-catalyzed methods to access 6-heteroaryl 2-aminopurine ribonucleosides from 6-chloroguanosine are described. First, Pd-132-catalyzed Suzuki-Miyaura cross-coupling using a series of boron substrates and 6-chloroguanosine forms 6-heteroaryl-2-aminopurines in a single step. The versatility of 6-chloroguanosine is further demonstrated using a modified Sonogashira coupling employing potassium iodide as an additive. Finally, the utility of the 6-alkynyl-2-aminopurine ribonucleoside as a dipolarophile in [3 + 2] cycloadditions is presented, affording triazoles and isoxazoles when reacted with azide and isonitrile 1,3-dipoles, respectively.

Strategy for Conditional Orthogonal Sequential CuAAC Reactions Using a Protected Aromatic Ynamine.

A method for conditional control of orthogonal sequential Cu-catalyzed azide-alkyne cycloaddition (CuAAC) reactions is reported. The inherent reactivity of an aromatic ynamine is controlled by a silyl protecting group that allows the selective CuAAC reaction of less reactive alkynes. Alternatively, the same protected ynamine undergoes selective CuAAC reaction via silyl deprotection in situ to give the ynamine click products. This allows complete orthogonal control of dialkyne systems and provides a unifying strategy for chemoselective CuAAC ligations in multialkyne/azide systems.

Chemoselective Sequential Click Ligations Directed by Enhanced Reactivity of an Aromatic Ynamine.

Aromatic ynamines or N-alkynylheteroarenes are highly reactive alkyne components in Cu-catalyzed Huisgen [3 + 2] cycloaddition ("click") reactions. This enhanced reactivity enables the chemoselective formation of 1,4-triazoles using the representative aromatic ynamine N-ethynylbenzimidazole in the presence of a competing aliphatic alkyne substrate. The unique chemoselectivity profile of N-ethynylbenzimidazole is further demonstrated by the sequential click ligation of a series of highly functionalized azides using a heterobifunctional diyne, dispelling the need for alkyne protecting groups.