Namit Chaudhary

The broad clinical application of mRNA therapeutics has been hampered by a lack of delivery vehicles that induce protein expression in extrahepatic organs and tissues. Recently, it was shown that mRNA delivery to the spleen or lungs is possible upon the addition of a charged lipid to a standard four-component lipid nanoparticle formulation. This approach, while effective, further complicates an already complex drug formulation and has the potential to slow regulatory approval and adversely impact manufacturing processes. We were thus motivated to maintain a four-component nanoparticle system while achieving shifts in tropism. To that end, we replaced the standard helper lipid in lipidoid nanoparticles, DOPE, with one of eight alternatives. These lipids included the neutral lipids, DOPC, sphingomyelin, and ceramide; the anionic lipids, phosphatidylserine (PS), phosphatidylglycerol, and phosphatidic acid; and the cationic lipids, DOTAP and ethyl phosphatidylcholine. While neutral helper lipids maintained protein expression in the liver, anionic and cationic lipids shifted protein expression to the spleen and lungs, respectively. For example, replacing DOPE with DOTAP increased positive LNP surface charge at pH 7 by 5-fold and altered the ratio of liver to lung protein expression from 36:1 to 1:56. Similarly, replacing DOPE with PS reduced positive charge by half and altered the ratio of liver to spleen protein expression from 8:1 to 1:3. Effects were consistent across ionizable lipidoid chemistries. Regarding mechanism, nanoparticles formulated with neutral and anionic helper lipids best transfected epithelial and immune cells, respectively. Further, the lung-tropic effect of DOTAP was linked to reduced immune cell infiltration of the lungs compared to neutral or anionic lipids. Together, these data show that intravenous non-hepatocellular mRNA delivery is readily achievable while maintaining a four-component formulation with modified helper lipid chemistry.

Systemic messenger RNA (mRNA) delivery to organs outside the liver, spleen, and lungs remains challenging. To overcome this issue, we hypothesized that altering nanoparticle chemistry and administration routes may enable mRNA-induced protein expression outside of the reticuloendothelial system. Here, we describe a strategy for delivering mRNA potently and specifically to the pancreas using lipid nanoparticles. Our results show that delivering lipid nanoparticles containing cationic helper lipids by intraperitoneal administration produces robust and specific protein expression in the pancreas. Most resultant protein expression occurred within insulin-producing β cells. Last, we found that pancreatic mRNA delivery was dependent on horizontal gene transfer by peritoneal macrophage exosome secretion, an underappreciated mechanism that influences the delivery of mRNA lipid nanoparticles. We anticipate that this strategy will enable gene therapies for intractable pancreatic diseases such as diabetes and cancer.

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.