Plataforma de nanoparơculas lipídicas ionizáveis para entrega de DNA in vivo

Abstract

Gene therapies and vaccines using nucleic acids have emerged as a promising approach in the treatment and prevention of various diseases, including viral diseases and cancer. However, the use of this tool requires the development of technologies that provide safe and effective delivery of the nucleic acid. In this sense, the use of ionizable lipid nanoparticles (LNPs) has proven to be an attractive solution for the clinic, since they are already being employed in approved mRNA vaccines for COVID-19, presenting a great potential to revolutionize the prevention and treatment of several diseases in the future. Therefore, the present work aims to develop and implement an LNPs-based platform for safe and effective DNA delivery through in vivo screening. To this end, a library of 29 LNPs encapsulating DNA barcoding (b-DNA) as an identification tool was developed, containing an ionizable lipid, an auxiliary lipid, cholesterol, and a PEG-coupled lipid. Among the four components, the molar composition of the auxiliary lipids (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)) and cholesterol were varied to obtain different formulations. The LNPs were formulated using a microfluidic device, combined into a pool, and injected into C57BL/6 mice intramuscularly or intravenously. To assess biodistribution and select the most promising LNPs for a given organ, high-throughput deep sequencing of b-DNA was used to identify and quantify DNA deliveries. The LNPs with the highest DNA delivery efficiency in each organ were selected and subjected to in vitro testing to assess their transfection efficiency in four cell types of interest: macrophages (RAW 264.7), fibroblasts (L929), liver cells (HepG2), and endothelial cells (bEnd.3). The results revealed that varying the composition of LNPs alters their structure and physicochemical properties such as size and zeta potential. As a consequence, we observed different tropism and biodistribution for each of the LNPs in different organs. LNP B4 demonstrated higher GFP expression in vitro, which validates its potential use in vaccine studies. Furthermore, in vivo intravenous testing showed that LNPs B4 and B10 are suitable for gene therapies that target organs such as the heart, liver, spleen, and lung.