The rapid expansion of the available genomic data continues to greatly impact biomedical science and medicine. Fulfilling the clinical potential of genetic discoveries requires the development of therapeutics that can specifically modulate the expression of disease-relevant genes. RNA-based drugs, including short interfering RNAs and antisense oligonucleotides, are particularly promising examples of this newer class of biologics. For over two decades, researchers have been trying to overcome major challenges for utilizing such RNAs in a therapeutic context, including intracellular delivery, stability, and immune response activation. This research is finally beginning to bear fruit as the first RNA drugs gain FDA approval and more advance to the final phases of clinical trials. Furthermore, the recent advent of CRISPR, an RNA-guided gene-editing technology, as well as new strides in the delivery of messenger RNA transcribed in vitro, have triggered a major expansion of the RNA-therapeutics field. In this review, we discuss the challenges for clinical translation of RNA-based therapeutics, with an emphasis on recent advances in delivery technologies, and present an overview of the applications of RNA-based drugs for modulation of gene/protein expression and genome editing that are currently being investigated both in the laboratory as well as in the clinic.

Nucleic-acid-based drugs can be designed to encode for therapeutic proteins of interest and therefore have the potential to treat a broad range of diseases. In vitro transcribed (IVT) mRNA has several properties that give it promise as a therapeutic including lack of insertional mutagenesis, the ability to transfect nondividing cells, and controlled protein expression. [1] Local delivery of IVT-mRNA to the lung is promising for the treatment of respiratory disease, for example, intratracheal delivery of IVT-mRNA encoding for surfactant protein B was demonstrated to restore expression in a deficient mouse model. [2] While intra-tracheal delivery enables small doses to be administered locally and in a wellcontrolled manner, it is invasive and lung deposition is limited to the upper airways. In contrast, noninvasive inhalation of therapeutics is a clinically used route of drug delivery that can allow for deposition throughout the entire bronchiolar and alveolar epithelium. [3] Nebulized delivery has been adopted in human clinical trials for inhaled delivery of CFTR-DNA to cystic fibrosis patients. [4] Promising yet modest improvements in lung function were reported and challenges remain in optimizing nucleic acid delivery to the lung. [5] As yet, inhaled delivery of mRNA has not previously been reported and requires the development of vectors that are efficient for cytosolic mRNA delivery, can be concentrated to high delivery have found that a cationic polymer, branched polyethylenimine 25 kDa (bPEI), can facilitate effective gene delivery via nebulization; [10,11] however, concerns regarding toxicity and accumulation of this nondegradable polymer have precluded

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 potential utility to include systemic delivery of mRNA, here we develop hybrid polymer-lipid nanoformulations for systemic delivery to the lung. Through co-formulation 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 following intravenous administration in mice. To our knowledge, this is the first description of systemic administration of mRNA for delivery to the lungs using a degradable polymer-lipid nanoparticle.

mRNA therapeutics hold great potential for treating a variety of diseases through protein-replacement, immunomodulation, and gene editing. However, much like siRNA therapy the majority of progress in mRNA delivery has been confined to the liver. Previously, we demonstrated that poly(β-amino esters), a class of degradable polymers, are capable of systemic mRNA delivery to the lungs in mice when formulated into nanoparticles with poly(ethylene glycol)-lipid conjugates. Using experimental design, a statistical approach to optimization that reduces experimental burden, we demonstrate herein that these degradable polymer-lipid nanoparticles can be optimized in terms of polymer synthesis and nanoparticle formulation to achieve a multiple order-of-magnitude increase in potency. Furthermore, using genetically engineered Cre reporter mice, we demonstrate that mRNA is functionally delivered to both the lung endothelium and pulmonary immune cells, expanding the potential utility of these nanoparticles.

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.