Generation of Protein-Phosphorodiamidate Morpholino Oligomer Conjugates for Efficient Cellular Delivery via Anthrax Protective Antigen.

Phosphorodiamidate morpholino oligomers (PMOs) offer great promise as therapeutic agents for translation blocking or splice modulation due to their high stability and affinity for target sequences. However, in spite of their neutral charge as compared to natural oligonucleotides or phosphorothioate analogs, they still show little permeability for cellular membranes, highlighting the need for effective cytosolic delivery strategies. In addition, the implementation of strategies for efficient cellular targeting is highly desirable to minimize side effects and maximize the drug dose at its site of action. Anthrax toxin is a three-protein toxin of which the pore-forming protein anthrax protective antigen (PA) can be redirected to a receptor of choice and lethal factor (LF), one of the two substrate proteins, can be coupled to various cargoes for efficient cytosolic cargo delivery. In this protocol, we describe the steps to produce the proteins and protein conjugates required for cytosolic delivery of PMOs through the cation-selective pore generated by anthrax protective antigen. The method relies on the introduction of a unique cysteine at the C-terminal end of a truncated LF (aa 1-254), high-yield expression of the (truncated) toxin proteins in E. coli, functionalization of a PMO with a maleimide group and coupling of the maleimide-functionalized PMO to the unique cysteine on LF by maleimide-thiol conjugation chemistry. Through co-administration of PA with LF-PMO conjugates, an efficient cytosolic delivery of PMOs can be obtained.