Programming Primary Schwann Cells with Circular RNA Delivered by Listeria Drones

Efficient transient genetic engineering of primary cells remains a major challenge in regenerative medicine, immunotherapy, and other cell-based therapies, as many therapeutically relevant cell types are refractory to conventional gene delivery. Primary Schwann cells (SCs) hold particular promise for peripheral nerve and spinal cord regeneration, yet nonviral gene delivery into these cells remains challenging. Here, we demonstrate transient programming of primary rat SCs using circular RNA (circRNA) delivered by attenuated, self-destructing Listeria monocytogenes (Lm) drones. These engineered bacteria invade mammalian cells, escape the phagosome, synthesize and circularize target RNA under inducible control, and undergo programmed lysis to release circRNA into the host cytosol for IRES-mediated protein synthesis. Previously validated in cancer and immune cell lines, this platform had not been evaluated in primary cells. Using a reporter nano-luciferase (NLuc)-encoding circRNA, we optimized SC bactofection across multiplicities of infection (MOIs). At MOIs of 10-20, bactofection efficiency reached 94%, with negligible SC death. Viable Lm were completely eliminated through programmed bacterial lysis and antibiotic treatment, yielding bacteria-free SCs that continued expressing the NLuc reporter for over five days and were suitable for transplantation. The platform enables straightforward replacement of NLuc with one or more therapeutic payloads. These findings establish the first demonstration of Lm drone-mediated circRNA delivery into primary cells. By enabling transient, non-integrating expression of therapeutic factors in difficult-to-transfect SCs, this approach offers a promising strategy for developing temporally controlled cell therapies for peripheral nerve and spinal cord repair.