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Noninvasive aerosol inhalation is an established method of drug delivery to the lung, and remains a desirable route for nucleic-acid-based therapeutics. In vitro transcribed (IVT) mRNA has broad therapeutic applicability as it permits temporal and dose-dependent control of encoded protein expression. Inhaled delivery of IVT-mRNA has not yet been demonstrated and requires development of safe and effective materials. To meet this need, hyperbranched poly(beta amino esters) (hPBAEs) are synthesized to enable nanoformulation of stable and concentrated polyplexes suitable for inhalation. This strategy achieves uniform distribution of luciferase mRNA throughout all five lobes of the lung and produces 101.2 ng g -1 of luciferase protein 24 h after inhalation of hPBAE polyplexes. Importantly, delivery is localized to the lung, and no luminescence is observed in other tissues. Furthermore, using an Ai14 reporter mouse model it is identified that 24.6% of the total lung epithelial cell population is transfected after a single dose. Repeat dosing of inhaled hPBAE-mRNA generates consistent protein production in the lung, without local or systemic toxicity. The results indicate that nebulized delivery of IVT-mRNA facilitated by hPBAE vectors may provide a clinically relevant delivery system to lung epithelium., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

RNAs are a promising class of therapeutics given their ability to regulate protein concentrations at the cellular level. Developing safe and effective strategies to deliver RNAs remains important for realizing their full clinical potential. Here, we develop lipid nanoparticle formulations that can deliver short interfering RNAs (for gene silencing) or messenger RNAs (for gene upregulation). Specifically, we study how the tail length, tail geometry, and linker spacing in diketopiperazine lipid materials influences LNP potency with siRNAs and mRNAs. Eight lipid materials are synthesized, and 16 total formulations are screened for activity in vitro; the lead material is evaluated with mRNA for in vivo use and demonstrates luciferase protein expression in the spleen. In undertaking this approach, not only do we develop synthetic routes to delivery materials, but we also reveal structural criteria that could be useful for developing next-generation delivery materials for RNA therapeutics., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

B lymphocytes regulate several aspects of immunity including antibody production, cytokine secretion, and T-cell activation; moreover, B cell misregulation is implicated in autoimmune disorders and cancers such as multiple sclerosis and non-Hodgkin's lymphomas. The delivery of messenger RNA (mRNA) into B cells can be used to modulate and study these biological functions by means of inducing functional protein expression in a dose-dependent and time-controlled manner. However, current in vivo mRNA delivery systems fail to transfect B lymphocytes and instead primarily target hepatocytes and dendritic cells. Here, the design, synthesis, and biological evaluation of a lipid nanoparticle (LNP) system that can encapsulate mRNA, navigate to the spleen, transfect B lymphocytes, and induce more than 60 pg of protein expression per million B cells within the spleen is described. Importantly, this LNP induces more than 85% of total protein production in the spleen, despite LNPs being observed transiently in the liver and other organs. These results demonstrate that LNP composition alone can be used to modulate the site of protein induction in vivo, highlighting the critical importance of designing and synthesizing new nanomaterials for nucleic acid delivery., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Therapeutic nucleic acids hold great promise for the treatment of disease but require vectors for safe and effective delivery. Synthetic nanoparticle vectors composed of poly(β-amino esters) (PBAEs) and nucleic acids have previously demonstrated potential utility for local delivery applications. To expand this potential utility to include systemic delivery of mRNA, hybrid polymer-lipid nanoformulations for systemic delivery to the lungs were developed. Through coformulation of PBAEs with lipid-polyethylene glycol (PEG), mRNA formulations were developed with increased serum stability and increased in vitro potency. The formulations were capable of functional delivery of mRNA to the lungs after intravenous administration in mice. To our knowledge, this is the first report of the systemic administration of mRNA for delivery to the lungs using degradable polymer-lipid nanoparticles., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Thousands of human diseases could be treated by selectively controlling the expression of specific proteins in vivo. A new series of alkenyl amino alcohol (AAA) ionizable lipid nanoparticles (LNPs) capable of delivering human mRNA with unprecedented levels of in vivo efficacy is demonstrated. This study highlights the importance of utilizing synthesis tools in tandem with biological inspiration to understand and improve nucleic acid delivery in vivo., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)