administration of mRNA formulated in LNPs would be associated with dose-limiting inflammatory responses. inflammatory responses. To overcome this limitation, we investigated the concept of incorporating aliphatic ester prodrugs of anti-inflammatory steroids within LNPs, i.e., functionalized LNPs to suppress the inflammatory response. We show that the effectiveness of this approach depends on the alkyl chain length of the ester prodrug, which determines its retention at the site of administration. An unexpected additional benefit to this approach is the prolongation observed in the duration of protein expression. Our results demonstrate that subcutaneous administration of mRNA formulated in functionalized LNPs is a viable approach to achieving systemic levels of therapeutic proteins, which has the added benefits of being amenable to self-administration when Moxidectin chronic treatment is Moxidectin required. translation of the message to the corresponding protein that undergoes post-transcriptional modifications and folding prior to secretion.1,2 Substantial investment has been made in the last decades to modify the structural elements of the mRNA (including modifications of the nucleotides and cap structure) to enable increased protein expression and reduced immunogenicity. However, there are still some fundamental challenges with the use of mRNA as therapeutics, including stability, duration of action, pharmacokinetic/pharmacodynamic (PK/PD), and effective delivery to the target cell type or tissue. In general, the technology has progressed significantly since the first Moxidectin nonclinical studies in the 1990s and has to date been explored for vaccines, protein replacement therapies, and in regenerative medicine applications.1, 2, 3, 4, 5, 6, 7, 8, 9, 10 One of the major obstacles facing the successful development of mRNA-based therapies is the identification of a safe and effective delivery system that can offer protection of the mRNA from endonucleases and exonucleases and effectively deliver the mRNA into the cells in a manner that is acceptable to patients. Broadly speaking, RNA delivery can be mediated by viral and non-viral vectors.2,11,12 Lipid nanoparticles (LNPs), initially developed for delivery of small interfering RNA (siRNA), have also been investigated for delivery of mRNA and have shown promise as Rabbit Polyclonal to OR10H2 a nonviral delivery system.13, 14, 15, 16 LNPs are multi-component systems that typically consist of an ionizable amino lipid, a phospholipid, cholesterol, and a polyethylene glycol (PEG)-lipid, with all of the components contributing to efficient delivery of the nucleic acid drug cargo and stability of the particle.17 The cationic lipid electrostatically condenses the negatively charged RNA into nanoparticles and the use of ionizable lipids that are positively charged at acidic pH is thought to enhance endosomal escape. The most explored formulations for delivery of siRNA both clinically and non-clinically are predominantly based on cationic lipids such as DLin-MC3-DMA (MC3).11,18 Recently, the therapeutic siRNA Onpattro (patisiran) in MC3 LNPs was awarded breakthrough approval by the US Food and Drug Administration for the treatment of the polyneuropathy of hereditary transthyretin-mediated (hATTR) amyloidosis in adults.19 Building on the experience of LNP delivery of siRNA, several groups have demonstrated effective and tolerable delivery of mRNA using LNPs for transient expression of vaccine antigens as well as of secreted proteins following intravenous (i.v.) or intramuscular (i.m.) administration.4, 5, 6, 7,20, 21, 22 Consequently, a number of mRNA constructs have recently progressed into clinical trials. 23 While great progress has been made in achieving efficient and tolerable LNPs for delivery of mRNA for i.v. or i.m. administration, the challenge remains with regard to subcutaneous (s.c.) self-administration of therapeutic mRNA required for chronic/sub-chronic treatment of diseases. Following s.c. administration, LNPs and their mRNA cargo are expected to be largely retained at the site of injection, resulting in high local concentrations. Since LNPs are known to be pro-inflammatory, largely attributed to the ionizable lipid present in the LNPs, 14 then it would not be unexpected that s.c. administration of mRNA formulated in LNPs would be associated with dose-limiting inflammatory responses. Previous work has shown that co-administration of dexamethasone with LNP reduces the immune-inflammatory response following i.v. administration,24 and recently Chen et?al.25 reported on reduced immune stimulation following systemic administration by incorporating lipophilic dexamethasone prodrugs within LNP-containing nucleic acids. Herein, we report the concept of incorporating hydrophobic prodrugs of anti-inflammatory compounds (AICs) into mRNA-loaded LNPs, i.e., functionalized LNPs to minimize the inflammatory response and maintain protein expression.26 We report, for the first time, the use of functionalized LNPs that enable tolerable s.c. administration of mRNA encoding for a model secreted protein (human fibroblast growth factor 21 [hFGF21]) to demonstrate the utility of this approach for systemic protein replacement therapies. Results Systemic protein exposure and tolerability following i.v. and s.c. administration of mRNA.