Branched-Tail Lipid Nanoparticles for Intravenous mRNA Delivery to Lung Immune, Endothelial, and Alveolar Cells in Mice.

Lipid nanoparticles (LNPs) are proven safe and effective delivery systems on a global scale. However, their efficacy has been limited primarily to liver and immune cell targets. To extend the applicability of mRNA drugs, 580 ionizable lipidoids are synthesized and tested for delivery to extrahepatocellular targets. Of these, over 40 enabled protein expression in mice, with the majority transfecting the liver. Beyond the liver, several LNPs containing new, branched-tail ionizable lipidoids potently delivered mRNA to the lungs, with cell-level specificity depending on helper lipid chemistry. Incorporation of the neutral helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) at 16 mol% enabled highly specific delivery to natural killer and dendritic cells within the lung. Although inclusion of the cationic lipid 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (DOTAP) improved lung tropism, it decreased cell specificity, resulting in equal transfection of endothelial and lymphoid cells. DOTAP formulations are also less favorable than DOPE formulations because they elevated liver enzyme and cytokine levels. Together, these data identify a new branched-tailed LNP with a unique ability to selectively transfect lung immune cell populations without the use of toxicity-prone cationic helper lipids. This novel vehicle may unlock RNA therapies for lung diseases associated with immune cell dysregulation, including cancer, viral infections, and autoimmune disorders.

Lipid nanoparticle chemistry determines how nucleoside base modifications alter mRNA delivery.

Therapeutic mRNA has the potential to revolutionize the treatment of myriad diseases and, in 2020, facilitated the most rapid vaccine development in history. Among the substantial advances in mRNA technology made in recent years, the incorporation of base modifications into therapeutic mRNA sequences can reduce immunogenicity and increase translation. However, experiments from our lab and others have shown that the incorporation of base modifications does not always yield superior protein expression. We hypothesized that the variable benefit of base modifications may relate to lipid nanoparticle chemistry, formulation, and accumulation within specific organs. To test this theory, we compared IV-injected lipid nanoparticles formulated with reporter mRNA incorporating five base modifications (ψ, m1ψ, m5U, m5C/ψ, and m5C/s2U) and four ionizable lipids (C12-200, cKK-E12, ZA3-Ep10, and 200Oi10) with tropism for different organs. In general, the m1ψ base modification best enhanced translation, producing up to 15-fold improvements in total protein expression compared to unmodified mRNA. Expression improved most dramatically in the spleen (up to 50-fold) and was attributed to enhanced protein expression in monocytic lineage splenocytes. The extent to which these effects were observed varied with delivery vehicle and correlated with differences in innate immunogenicity. Through comparison of firefly luciferase and erythropoietin mRNA constructs, we also found that mRNA modification-induced enhancements in protein expression are limited outside of the spleen, irrespective of delivery vehicle. These results highlight the complexity of mRNA-loaded lipid nanoparticle drug design and show that the effectiveness of mRNA base modifications depend on the delivery vehicle, the target cells, and the site of endogenous protein expression.

A Potent Branched-Tail Lipid Nanoparticle Enables Multiplexed mRNA Delivery and Gene Editing In Vivo.

The clinical translation of messengerRNA (mRNA) drugs has been slowed by a shortage of delivery vehicles that potently and safely shuttle mRNA into target cells. Here, we describe the properties of a particularly potent branched-tail lipid nanoparticle that delivers mRNA to >80% of three major liver cell types. We characterize mRNA delivery spatially, temporally, and as a function of injection type. Following intravenous delivery, our lipid nanoparticle induced greater protein expression than two benchmark lipids, C12-200 and DLin-MC3-DMA, at an mRNA dose of 0.5 mg/kg. Lipid nanoparticles were sufficiently potent to codeliver three distinct mRNAs (firefly luciferase, mCherry, and erythropoietin) and, separately, Cas9 mRNA and single guide RNA (sgRNA) for proof-of-concept nonviral gene editing in mice. Furthermore, our branched-tail lipid nanoparticle was neither immunogenic nor toxic to the liver. Together, these results demonstrate the unique potential of this lipid material to improve the management of diseases rooted in liver dysfunction.

Branched-Tail Lipid Nanoparticles Potently Deliver mRNA In Vivo due to Enhanced Ionization at Endosomal pH.

The potential of mRNA therapeutics will be realized only once safe and effective delivery systems are established. Unfortunately, delivery vehicle development is stymied by an inadequate understanding of how the molecular properties of a vehicle confer efficacy. Here, a small library of lipidoid materials is used to elucidate structure-function relationships and identify a previously unappreciated parameter-lipid nanoparticle surface ionization-that correlates with mRNA delivery efficacy. The two most potent materials of the library, 306O10 and 306Oi10 , induce substantial luciferase expression in mice following a single 0.75 mg kg-1 mRNA dose. These lipidoids, which have ten-carbon tails and identical molecular weights, vary only in that the 306O10 tail is straight and the 306Oi10 tail has a one-carbon branch. Remarkably, this small difference in structure conferred a tenfold improvement in 306Oi10 efficacy. The enhanced potency of this branched-tail lipidoid is attributed to its strong surface ionization at the late endosomal pH of 5.0. A secondary lipidoid library confirms that Oi10 materials ionize more strongly and deliver mRNA more potently than lipidoids containing linear tails. Together, these data highlight the exquisite control that lipid chemistry exerts on the mRNA delivery process and show that branched-tail lipids facilitate protein expression in animals.

Lipid Nanoparticle Formulations for Enhanced Co-delivery of siRNA and mRNA.

Although mRNA and siRNA have significant therapeutic potential, their simultaneous delivery has not been previously explored. To facilitate the treatment of diseases associated with aberrant gene upregulation and downregulation, we sought to co-formulate siRNA and mRNA in a single lipidoid nanoparticle (LNP) formulation. We accommodated the distinct molecular characteristics of mRNA and siRNA in a formulation consisting of an ionizable and biodegradable amine-containing lipidoid, cholesterol, DSPC, DOPE, and PEG-lipid. Surprisingly, the co-formulation of siRNA and mRNA in the same LNP enhanced the efficacy of both drugs in vitro and in vivo. Compared to LNPs encapsulating siRNA only, co-formulated LNPs improved Factor VII gene silencing in mice from 44 to 87% at an siRNA dose of 0.03 mg/kg. Co-formulation also improved mRNA delivery, as a 0.5 mg/kg dose of mRNA co-formulated with siRNA induced three times the luciferase protein expression compared to when siRNA was not included. As not all gene therapy applications require both RNA drugs, we sought to extend the benefit of co-formulated LNPs to formulations encapsulating only a single type of RNA. We accomplished this by substituting the "helper" RNA with a negatively charged polymer, polystyrenesulfonate (PSS). LNPs containing PSS mediated the same level of protein silencing or expression as standard LNPs using 2-3-fold less RNA. For example, LNPs formulated with and without PSS induced 50% Factor VII silencing at siRNA doses of 0.01 and 0.03 mg/kg, respectively. Together, these studies demonstrate potent co-delivery of siRNA and mRNA and show that inclusion of a negatively charged "helper polymer" enhances the efficacy of LNP delivery systems.