Mechanisms of In Vivo Nucleic Acid Delivery in Mouse Models

In vivo nucleic acid delivery in mouse models is a foundational technique in preclinical biomedical research, enabling genetic modulation for functional studies, disease modeling, and therapeutic development. Delivering nucleic acids such as plasmid DNA, siRNA, miRNA, or mRNA into living tissues requires navigation through complex biological barriers while preserving molecular integrity and promoting intracellular activity. After systemic or local administration, nucleic acids are exposed to degradation by extracellular nucleases and rapid clearance mechanisms, including renal filtration and uptake by phagocytic cells in the liver and spleen. Their large molecular size and negative charge prevent passive membrane diffusion, necessitating the use of advanced delivery vehicles to ensure cellular internalization. Lipid nanoparticles (LNPs) are a leading solution, incorporating ionizable lipids that become positively charged in acidic endosomes to trigger membrane fusion and cytoplasmic release. These formulations are often PEGylated to enhance circulation time and stability and may include targeting ligands like GalNAc or RGD peptides to direct tissue-specific uptake via receptor-mediated endocytosis. Polymeric carriers such as PEI, PBAEs, and dendrimers form electrostatic complexes with nucleic acids and promote endosomal escape, though toxicity must be carefully managed. The route of administration—whether intravenous, intratumoral, intranasal, intraperitoneal, or subcutaneous—determines tissue exposure and biodistribution, with formulation characteristics like particle size and surface charge influencing systemic behavior. Successful transfection depends not only on delivery efficiency but also on the biological context of the host organism. Mouse strain selection plays a critical role: immunocompromised models like NOD/SCID and NSG reduce immune-mediated clearance and enable higher transgene persistence, while immunocompetent strains may require immune-modulating strategies to prevent cytokine activation and transgene silencing. Once internalized by cells through mechanisms such as clathrin-mediated endocytosis or macropinocytosis, nucleic acids must escape the endosome to reach their site of action—the cytoplasm for RNA and the nucleus for DNA. Efficient transgene expression or gene silencing depends on this crucial step. These complex processes are tightly interrelated and require precise formulation and dosing strategies to achieve therapeutic or experimental objectives. Optimizing in vivo delivery platforms in mouse models continues to be a central challenge in gene therapy research, with downstream applications in oncology, immunology, infectious disease, and genetic disorders.