OLIGONUCLEOTIDE TARGETING TO ALVEOLAR MACROPHAGES BY MANNOSE RECEPTOR-MEDIATED ENDOCYTOSIS

Antisense oligonucleotides (ONs) have proven useful for selective inhi bition of gene expression. However, their effective use is limited by inefficient cellular uptake and lack of cellular targeting. In this pa per, we report a drug targeting system which utilizes mannose receptor -mediated endocytosis to enhance cellular uptake of ONs in alveolar ma crophages (AMs). The system employs a molecular complex consisting of partially substituted mannosylated poly(L-lysine) (MPL), electrostatic ally linked to a 5' fluorescently labeled ON. Upon recognition by the macrophage mannose receptors, the MPL was internalized by the receptor -mediated pathway co-transporting the ON. Om results indicated that th e AMs treated with the MPL:ON complex exhibited a significant increase in ON uptake (up to 17-fold) over free ON-treated controls. Effective ON uptake was shown to require the recognition of the mannose moiety since unmodified polylysine was much less effective in promoting ON up take. Specific internalization of the ON:MPL complex by the mannose re ceptor pathway was verified by competitive inhibition using mannosylat ed albumin. Under this condition, the ON complex uptake was inhibited. The requirement of mannose receptors for complex uptake was further d emonstrated using a macrophage cell line, J774.1, which expresses a lo w level of mannose receptors. When treated with the complex, these cel ls showed no susceptibility to ON uptake, thus suggesting the targetin g ability of the carrier system to the AMs. Following cellular interna lization, the ON complex appeared largely accumulated in endocytic ves icles. Enhanced endosomal exit of the ON was achieved using a fusogeni c peptide derived from the amino terminal sequence of influenza virus hemagglutinin HA2. Cytotoxicity studies showed that at the concentrati ons effectively enhancing ON uptake, both MPL and the fusogenic peptid e caused no toxic effects to the cells, thereby suggesting their poten tial safety and utilization in vivo.