Lung-selective mRNA delivery of synthetic lipid nanoparticles for the treatment of pulmonary lymphangioleiomyomatosis

Safe and efficacious systemic delivery of messenger RNA (mRNA) to specific organs and cells in vivo remains the major challenge in the development of mRNA-based therapeutics. Targeting of systemically administered lipid nanoparticles (LNPs) coformulated with mRNA has largely been confined to the liver and spleen. Using a library screening approach, we identified that N-series LNPs (containing an amide bond in the tail) are capable of selectively delivering mRNA to the mouse lung, in contrast to our previous discovery that O-series LNPs (containing an ester bond in the tail) that tend to deliver mRNA to the liver. We analyzed the protein corona on the liver- and lung-targeted LNPs using liquid chromatography–mass spectrometry and identified a group of unique plasma proteins specifically absorbed onto the surface that may contribute to the targetability of these LNPs. Different pulmonary cell types can also be targeted by simply tuning the headgroup structure of N-series LNPs. Importantly, we demonstrate here the success of LNP-based RNA therapy in a preclinical model of lymphangioleiomyomatosis (LAM), a destructive lung disease caused by loss-of-function mutations in the Tsc2 gene. Our lung-targeting LNP exhibited highly efficient delivery of the mouse tuberous sclerosis complex 2 (Tsc2) mRNA for the restoration of TSC2 tumor suppressor in tumor and achieved remarkable therapeutic effect in reducing tumor burden. This research establishes mRNA LNPs as a promising therapeutic intervention for the treatment of LAM.

The use of messenger RNA (mRNA) for vaccination (1, 2), protein replacement therapy (3) and cancer immunotherapy (4), and mRNA technology encoding CRISPR/Cas nuclease for genome editing (5) holds the potential to revolutionize the treatment of a wide range of currently untreatable genetic diseases. The US Food and Drug Administration (FDA) recently authorized two mRNA vaccines enabled by nonviral lipid nanoparticles (LNPs) against COVID-19 for emergency use, representing a key milestone in mRNA therapeutics. Aside from COVID-19, other mRNA vaccines against influenza viruses (6), Cytomegalovirus (7), and advanced melanoma (8) have also been developed and are now in human clinical trials. The clinical success of these transformative therapeutics is largely reliant on the development of safe, efficient, and highly selective delivery systems to target mRNA toward specific tissues and cell types (9, 10).As one of the most advanced nonviral synthetic nanoparticles, LNPs have been proven to specifically deliver small interfering RNA (siRNA) to the liver for the treatment of hereditary transthyretin amyloidosis (11). Since mRNA predominantly accumulates in the liver and spleen following systemic delivery (12–16), much of the clinical interest to date has focused on hepatic diseases. Delivery vehicles that enable specific mRNA delivery to extrahepatic tissues are urgently needed to fully realize the potential of mRNA-based therapy. Considerable effort has been made to develop organ-targeted LNPs to bypass liver accumulation by modifying the surface of LNPs with targeting moieties such as peptides, antibodies, and proteins (17–19). Recently, targeted LNPs functionalized with alpha plasmalemma vesicle–associated protein antibody were developed for lung-targeted mRNA delivery in vivo (18). More recently, a selective organ targeting (SORT) strategy was developed to engineer LNPs to tune the biodistribution of LNPs; the incorporation of an extra excipient, the SORT molecule, can enable the precise alteration of the in vivo mRNA delivery profile (20). These strategies exhibit advantages in mitigating liver accumulation and delivering mRNA to lungs or spleens. These promising developments motivate us to continue explore innovative ways to deliver mRNA to specific locations.A major roadblock in the development of targeted LNPs is difficulty predicting the in vivo targeting behavior of newly designed LNPs due to the limited understanding of the nano-bio interactions between nanoparticles (NPs) and biological components. The outer surface of NPs can be rapidly covered with a layer of serum proteins, referred to as the “protein corona,” which remodels the surface property of NPs and substantially affects the interaction of NPs with organs and cells (21). We and others have demonstrated that the lipidoid amine head structure can impact the delivery efficacy and even the in vivo targetability of mRNA-loaded LNPs (22–24). In a recent study, we showed that imidazole-based synthetic lipidoids preferentially target mRNA to the spleen (25). For the lipidoid tail chemistry, although considerable progress has been made in the understanding of lipidoid tail length, degree of unsaturation, and degree of branching on the effect of mRNA delivery potency (26–29), the influence of lipidoid tail structures on the in vivo selectivity of LNPs remains poorly understood. To address this important knowledge gap, we synthesized a library of amide bond–containing lipidoids (N-series LNPs) via Michael addition reaction between amine heads and acrylamide tails (Fig. 1A). Surprisingly, from in vivo screening, we found that the N-series LNPs almost exclusively deliver mRNA to the lung following systemic administration (Fig. 1 B and C). Intriguingly, our previous study demonstrated that the O-series lipidoids, which contain an ester bond in the tails, tend to deliver mRNA into the liver (16). To better understand why such a small change induces such striking organ specificity, we further investigated the underlying mechanisms of these delivery differences. We hypothesized that once injected into the bloodstream, the LNPs can selectively govern the adsorption of specific plasma proteins to serve as targeting ligands that direct LNPs to selected organs. Indeed, using proteomics, we identified a group of unique plasma proteins specifically absorbed on the surface of two representative LNP candidates, 306-O12B and 306-N16B, that may affect the targetability of these LNPs. More importantly, we found that different pulmonary subcellular populations can be targeted by changing the lipidoid head structure of N-series LNPs. Furthermore, we evaluated the lung-targeting LNPs for the in vivo targeted delivery of Tsc2 mRNA to TSC2-deficient cells to restore the expression of the TSC2 tumor suppressor for the treatment of pulmonary lymphangioleiomyomatosis (LAM), a rare genetic disorder caused by biallelic mutations and loss of function of TSC complex genes. This study provides proof of concept that tuning the in vivo organ-targeting behavior of LNPs can be achieved by tailoring the composition of protein corona via simple chemistry. This work provides a strategy for the rational design of highly specific organ- and cell-selective LNPs for mRNA-based therapy.Open in a separate windowFig. 1.Synthesis and in vivo screening of N-series LNPs. (A) Synthetic route and representative chemical structure of lipidoids. Representative whole-body bioluminescence images of mice (B) and in vivo mRNA delivery efficacy (C) of N-series LNPs measured by the IVIS imaging system. Mice were injected with either of the Luc mRNA–loaded N-series LNPs at a single dose of 0.5 mg/kg. Images were taken at 6 h postinjection (n = 3). Data are presented as mean ± SD; the error bar around each data point is the SEM.