Abstract
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.
Highlights
Phosphorodiamidate morpholino oligomers (PMOs) are uncharged DNA analogs with therapeutic potential due to their ability to bind to target sites on RNA
For the delivery of PMOs via this mechanism, only protective antigen (PA) and the PA pore-binding domain of lethal factor (LF) are needed as protein components
We describe the preparation of the components needed to mediate cytosolic delivery of PMOs by the anthrax toxin translocation mechanism (Fig. 1a)
Summary
Phosphorodiamidate morpholino oligomers (PMOs) are uncharged DNA analogs with therapeutic potential due to their ability to bind to target sites on RNA. CPP-mediated delivery has demonstrated potential, but still does not target specific cell-surface receptors, indicating the need for a novel approach. Several groups have demonstrated that anthrax toxin, a sophisticated protein-based molecular machine that has evolved to efficiently deliver toxic catalytic proteins into the cytosol, can be employed for the functional delivery of various types of cargoes, including antisense oligonucleotides (AON) [7, 8]. For the delivery of PMOs via this mechanism, only PA and the PA pore-binding domain of LF are needed as protein components. We describe the preparation of the components needed to mediate cytosolic delivery of PMOs by the anthrax toxin translocation mechanism (Fig. 1a). After coupling maleimide-functionalized PMOs with LF, uncoupled PMO is removed via dialysis, producing LF-PMO conjugates that can be delivered to the cytosol via anthrax protective antigen (Fig. 1b).
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