Antisense oligonucleotides (ASOs) are a promising therapeutic modality whose efficacy and safety depend strongly on their absorption, distribution, metabolism, and excretion (ADME) characteristics. Their pharmacokinetic behavior is influenced by physicochemical properties such as ionization state, pKa, hydrophobicity, molecular size, and backbone chemistry, as well as the selected route of administration. After dosing, ASO distribution is further shaped by free drug concentration, tissue perfusion, tissue binding, local pH, and cell membrane permeability.
ASO chemistry has a major impact on plasma protein binding and tissue distribution. First- and second-generation phosphorothioate (PS)-modified ASOs are negatively charged and bind extensively to plasma proteins, especially albumin, often exceeding 85%. For example, Mipomersen shows plasma protein binding of about 95% in humans. In contrast, phosphorodiamidate morpholino oligomer (PMO)-modified ASOs are uncharged and show much lower protein binding, such as 6%–17% for Eteplirsen. As a result, PS-ASOs generally display more sustained tissue exposure and slower urinary excretion, while PMOs are cleared more rapidly.
Systemic delivery of ASOs remains challenging, particularly by the oral route, because their large molecular size and hydrophilicity limit gastrointestinal absorption and membrane permeability. Intravenous and subcutaneous administration are therefore more commonly used to achieve reliable systemic exposure. IV dosing allows rapid distribution to highly vascularized organs such as the liver, kidneys, and spleen, while distribution to muscle, heart, and lungs may be slower. SC administration can provide complete or near-complete bioavailability in some preclinical models, with delayed peak concentrations due to gradual absorption from the injection site.
Because ASOs do not readily cross cell membranes by passive diffusion, they mainly enter cells through phagocytosis or receptor-mediated endocytosis. After binding to cell surface proteins, ASOs are internalized through receptors such as EGFRs, GPCRs, and scavenger receptors. Stabilin-1 and stabilin-2 contribute to hepatic uptake of PS-ASOs, while scavenger receptor A1 is involved in PMO uptake in muscle. Once inside the cell, ASOs must escape from endosomes to reach RNA targets in the cytoplasm or nucleus. Endosomal trafficking proteins such as Rab5C, EEA1, Rab7A, and lysosomal lipids are involved in this process, although the intracellular transport mechanisms of PMOs are less well understood than those of PS-ASOs.
To improve ASO delivery, researchers are developing strategies such as conjugation to targeting ligands, fatty acids, peptides, and cell-penetrating peptides. GalNAc-conjugated ASOs have improved liver targeting through hepatocyte uptake via the asialoglycoprotein receptor, while CPPs can enhance PMO uptake, especially in muscle tissue. Local administration also offers targeted delivery for sites that are difficult to reach systemically, including intravitreal injection for ocular diseases and intrathecal injection for central nervous system disorders, as demonstrated by Nusinersen. Overall, ASO absorption and distribution are governed by chemistry, route of administration, tissue accessibility, and intracellular trafficking, making pharmacokinetic understanding essential for developing safer and more effective ASO therapies.
