Engineering of a wireless single capacitive discharge conductivity-clamped gene electrotransfer probe for naked nucleic acid therapeutics

Pulsed electric field gene electrotransfer addresses the need for safe, directed “vector-free” delivery of DNA/mRNA therapeutics in deep tissue targets. We report the development of a disposable needle-type “electro-lens” probe for precision delivery of DNA/mRNA to tissues via translocation of nucleic acids into cells using focused single-pulse electric fields. We developed and manufactured a novel ganged linear bionic electrode array enhanced by incorporating integrated microfluidics to deliver naked DNA expression cassettes in a low-conductivity carrier (“conductivity-clamping,” CC). This system was packaged in a disposable wireless probe for single capacitive discharge conductivity-clamped gene electrotransfer (SCD-CC-GET), powered by an integrated capacitor with automated discharge mechanism. The electrode array was achieved by laser-etching parylene-insulated concentric stainless steel needles, with the lensing effect of the electric field tuned by varying the separation between elongated conductive elements. Our novel SCD-CC-GET probe is fully wireless and standalone, featuring integrated fluidics with minimal dead space, and an automated discharge mechanism. The probe was validated in vitro through HEK293 cell transfection of green fluorescent protein reporter plasmid DNA, where CC significantly expanded the transfection zone by up to 3×. In vivo proof of concept for deep tissue gene delivery by SCD-CC-GET was achieved in the mouse gastrocnemius muscle with long-term expression of mCherry reporter DNA. Our technology for GET is compact, low-cost, and fully wireless, with only a single point of insertion, and produces maximal electric fields at the target zone, allowing for more efficient gene therapies with minimal charge transfer, in a fine needle profile.