Non-viral reprogramming of human nucleus pulposus cells with FOXF1 via extracellular vesicle delivery: an in-vitro and in-vivo study

Intervertebral disc (IVD) degeneration is characterized by decreased cellularity, decreased proteoglycan synthesis and increased inflammation, catabolism, and neural/vascular ingrowth. Current regenerative methods for IVD degeneration are largely cell therapy based or involve viral vectors which are associated with mutagenesis and undesired immune responses. This novel study uses bulk electroporation and engineered extracellular vesicles (EVs) to deliver the transcription factor FOXF1 mRNA to degenerate human nucleus pulposus (NP) cells as a minimally invasive therapeutic strategy for regeneration of the IVD. Bulk-electroporation was used to investigate the effects of FOXF1 on human NP cells with 4-week culture in 3D agarose constructs. Engineered EV delivery of FOXF1 into human IVD cells in monolayer was determined with subsequent in-vivo validation in a pilot mouse IVD puncture model. Transfection of FOXF1 significantly altered gene expression via upregulating healthy NP markers (FOXF1, KRT19), decreasing inflammatory cytokines (IL-1β, IL6), catabolic enzymes (MMP13) and nerve growth factor (NGF) with significant increases in glycosaminoglycan accumulation in human NP cells. Engineered EVs loaded with FOXF1 demonstrated successful encapsulation of FOXF1 cargo and effective uptake by human NP cells cultured in monolayer. Injection of EVs loaded with FOXF1 into the mouse IVD in-vivo resulted in significant upregulation of FOXF1 and Brachyury compared to controls at 7-days post-injection, with no evidence of cytotoxicity. This is the first study to demonstrate non-viral delivery of FOXF1 and reprogramming of human NP cells in-vitro and mouse IVD cells in-vivo. We highlight this strategy as a non-addictive approach for treating IVD degeneration and associated back pain.