In vivo transfection using cyclodextrin-containing polycations

Numerous nonviral systems have been developed for the delivery of nucleic acids to cultured cells and to particular cell types in vivo. These systems vary with regard to their toxicity, immunogenicity, and ability to target particular cell surface receptors and/or cell types. A class of linear cationic polymers containing the sugar β-cyclodextrin (β-CD) has been shown to be effective at delivering a variety of nucleic acids in vivo, including plasmid DNA, DNAzymes, and short interfering RNAs (siRNAs). These polymer-nucleic acid complexes (polyplexes) can be further modified to incorporate a targeting ligand such as transferrin to induce preferential uptake of polyplexes by cells expressing high levels of the cognate receptor. This protocol describes a procedure for the use of cyclodextrin-containing polycations (CDPs) in vivo. Salt stabilization and cell targeting are critical to the success of in vivo transfection using CDPs, so adamantane-poly(ethylene glycol) (AD-PEG) conjugates (both unmodified AD-PEG and an AD-PEG-Ligand conjugate) are included in these formulations. The amount of the AD-PEG-Ligand conjugate included depends on numerous factors, including its effect on polyplex stability (influenced by ligand size and charge) and the density of the cognate receptor on target cell type(s). Some targeting ligands may have extreme sizes or net charges that could present a challenge to their incorporation into these polyplex formulations.