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.

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.

The Promising Therapeutic Potential of Oligonucleotides for Pulmonary Fibrotic Diseases.

INTRODUCTION: Fibrotic lung diseases represent a large subset of diseases with an unmet clinical need. Oligonucleotide therapies (ONT) are a promising therapeutic approach for the treatment of pulmonary disease as they can inhibit pathways that are otherwise difficult to target. Additionally, targeting the lung specifically with ONT is advantageous because it reduces the possibilities of systemic side effects and tolerability concerns. AREAS COVERED: This review presents the chemical basis of designing various ONTs currently known to treat fibrotic lung diseases. Further, the authors have also discussed the delivery vehicle, routes of administration, physiological barriers of the lung, and toxicity concerns with ONTs. EXPERT OPINION: ONTs provide a promising therapeutic approach for the treatment of fibrotic diseases of the lung, particularly because ONTs directly delivered to the lung show little systemic side effects compared to current therapeutic strategies. Dry powder aerosolized inhalers may be a good strategy for getting ONTs into the lung in humans. However, as of now, no dry powder ONTs have been approved for use in the clinical setting, and this challenge must be overcome for future therapies. Various delivery methods that can aid in direct targeting may also improve the use of ONTs for lung fibrotic diseases.

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.

Phosphine-catalyzed divergent domino processes between γ-substituted allenoates and carbonyl-activated alkenes.

Highly enantioselective and chemodivergent domino reactions between γ-substituted allenoates and activated alkenes have been developed. In the presence of NUSIOC-Phos, triketone enone substrates smoothly reacted with γ-substituted allenoates to form bicyclic furofurans in good yields with high stereoselectivities. Alternatively, the reaction between diester-activated enone substrates and γ-substituted allenoates formed chiral conjugated 1,3-dienes in good yields with excellent enantioselectivities. Notably, by employing substrates with subtle structural difference, under virtually identical reaction conditions, we were able to access two types of chiral products, which are of biological relevance and synthetic importance.

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.

Highly Enantioselective [3 + 2] Annulation of 3-Butynoates with ß-Trifluoromethyl Enones Promoted by an Amine-Phosphine Binary Catalytic System.

We report a highly enantioselective [3 + 2] annulation between 3-butynoates and ß-trifluoromethyl enones, furnishing trifluoromethylated cyclopentenes with three contiguous stereogenic centers in good yields, high diastereoselectivities, and excellent enantioselectivities. A unique catalytic system consisting of a simple amine and a chiral phosphine was devised, and the synergistic play of Lewis basic amine and phosphine was crucial for alkyne isomerization and subsequent cyclization. The protocol disclosed herein allows facile activation of 3-butynoates in phosphine-mediated asymmetric transformations.

Phosphine-Catalyzed Asymmetric Organic Reactions.

Asymmetric phosphine catalysis showcasing remarkable progress over the past two decades has emerged as a key synthetic platform for the creation of molecular frameworks encountered in medicinal chemistry and materials science. Different types of novel chiral phosphine catalysts have been developed and employed in cornucopias of organic transformations, such as annulation, addition, Morita-Baylis-Hillman, and Rauhut-Currier reactions, among others. This review summarizes all of the literature examples from late 1990s to the end of 2017, alongside their mechanistic insights whenever possible, with a very aim to trigger more intensive research in the future to render asymmetric phosphine catalysis one of the most common and reliable tools to organic chemists.

Phosphine-Catalyzed Formal Oxa-[4 + 2] Annulation Employing Nitroethylene and Enones: Enantioselective Synthesis of Dihydropyrans.

The first phosphine-catalyzed enantioselective formal oxa-[4 + 2] reaction between nitroethylene and α-cyano-α,ß-unsaturated ketones has been developed. In the presence of a dipeptide-based phosphine catalyst and achiral Brønsted acid additives, highly functionalized 3,4-dihydro-2 H-pyrans were obtained with excellent enantio- and diastereoselectivities.

Chiral phosphine-mediated intramolecular [3 + 2] annulation: enhanced enantioselectivity by achiral Brønsted acid.

Enantioselective intramolecular [3 + 2] annulation of chalcones bearing an allene moiety has been successfully developed. The reaction was effectively promoted by amino acid-derived phosphines, in combination with achiral Brønsted acids. Dihydrocoumarin architectures were constructed in high yields and with excellent enantiomeric excesses. Theoretical studies via DFT calculations revealed that the hydrogen bonding network induced by achiral Brønsted acids/chiral phosphines could more efficiently distinguish between two enantioselective pathways, thus leading to enhanced enantioselectivity.

Catalyst-controlled regioselectivity in phosphine catalysis: the synthesis of spirocyclic benzofuranones via regiodivergent [3 + 2] annulations of aurones and an allenoate.

Catalyst-controlled regiodivergent [3 + 2] annulations of aurones and allenoates have been developed. When a dipeptide phosphine catalyst with an l-d- configuration was employed, α-selective [3 + 2] annulation products could be obtained with good regioselectivities and enantioselectivities. With the employment of l-l- dipeptide phosphines, γ-selective annulation products could be selectively obtained with excellent enantioselectivities. By simply tuning the catalyst configurations, a wide range of α-selective or γ-selective spirocyclic benzofuranones with either aryl or alkyl substitutions could be readily prepared. DFT calculations suggest that the conformation of the dipeptide phosphines influences the hydrogen bonding interactions or the distortion energy, resulting in delicate energy differentiation in the transition states, and accounting for the observed regioselectivity.

Enantioselective Phosphine-Catalyzed Formal [4+4] Annulation of α,ß-Unsaturated Imines and Allene Ketones: Construction of Eight-Membered Rings.

The first highly enantioselective phosphine-catalyzed formal [4+4] annulation has been developed. In the presence of amino-acid-derived phosphines, the unprecedented [4+4] annulations between benzofuran/indole-derived α,ß-unsaturated imines and allene ketones proceeded smoothly, thus affording azocines, bearing either a benzofuran or an indole moiety, in excellent yields and with nearly perfect enantioselectivities (≥98 % ee in most cases). This work marks the first efficient asymmetric construction of optically enriched eight-membered rings by phosphine catalysis.

Highly enantioselective synthesis of dihydrocoumarin-fused dihydropyrans via the phosphine-catalyzed [4 + 2] annulation of allenones with 3-aroylcoumarins.

Phosphine-catalyzed [4 + 2] annulation between 3-aroylcoumarins and allenones has been developed. In the presence of a dipeptide phosphine catalyst 7, dihydrocoumarin-fused dihydropyrans were prepared in high yields and with excellent enantioselectivities.

Enantioselective [4 + 2]-Annulation of Oxadienes and Allenones Catalyzed by an Amino Acid Derived Phosphine: Synthesis of Functionalized Dihydropyrans.

An enantioselective [4 + 2]-annulation process between cyano-activated oxadienes and allenones is developed. An l-valine-derived phosphine was efficient in catalyzing the reaction, and a wide range of highly functionalized dihydropyrans were prepared in high yields and with excellent enantioselectivities.

Enantioselective [3 + 2] annulation of α-substituted allenoates with ß,γ-unsaturated N-sulfonylimines catalyzed by a bifunctional dipeptide phosphine.

The first enantioselective [3 + 2] annulation of α-substituted allenoates with ß,γ-unsaturated N-sulfonylimines is described. In the presence of a dipeptide phosphine catalyst, a wide range of highly functionalized cyclopentenes bearing an all-carbon quaternary center were obtained in moderate to good yields and with good to excellent enantioselectivities.