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

Abstract

Intervertebral disc (IVD) degeneration is characterized by decreased cellularity and proteoglycan synthesis and increased inflammation, catabolism, and neural/vascular ingrowth. Regenerative methods for IVD degeneration are largely cell-therapy-based or involve viral vectors, which are associated with mutagenesis and undesired immune responses. The present study used bulk electroporation and engineered extracellular vesicles (EVs) to deliver forkhead-box F1 (FOXF1) mRNA to degenerate human nucleus pulposus (NP) cells as a minimally invasive therapeutic strategy for IVD regeneration. Bulk electroporation was used to investigate FOXF1 effects on human NP cells during a 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. FOXF1 transfection significantly altered gene expression by upregulating healthy NP markers [FOXF1, keratin 19 (KRT19)], decreasing inflammatory cytokines [interleukin (IL)-1β, -6], catabolic enzymes [metalloproteinase 13 (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 FOXF1-loaded EVs into the mouse IVD in vivo resulted in a significant upregulation of FOXF1 and Brachyury, compared to controls at 7 d 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. This strategy represents a non-addictive approach for treating IVD degeneration and associated back pain.

The Role of Extracellular Vesicles in Osteoporosis: A Scoping Review

“…Engineered EVs refer to the artificially modified EVs, with great potential in the treatment of various diseases, including tumors, spinal cord injury, inflammation, cardiovascular diseases, etc. At present, there are two methods to achieve this: one is to modify the cells, co-incubate or transfect the genes or drugs so that the genes or drugs can enter into the cells, and then collect the EVs of the cells; and another is to directly add RNAs or drugs to the EVs to make the EVs have therapeutic effects [ 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 ].…”

Editorial – Disc Biology Special Issue

“…Their study demonstrated that inhibition of TLR4 can block pro-inflammatory signalling and degenerative changes to NP cell biophysical properties. Tang et al (2021) presented the delivery of engineered vesicles (EVs) loaded with forkhead-box F1 (FOXF1) mRNA as a minimally invasive, non-viral and clinically feasible approach for re-programming adult IVD cells towards healthier phenotypes.…”

Extracellular Vesicles as an Emerging Treatment Option for Intervertebral Disc Degeneration: Therapeutic Potential, Translational Pathways, and Regulatory Considerations

“…[117] Primary mouse embryonic fibroblast-derived EVs were engineered to efficiently deliver FOXF1 mRNA in vivo as a therapeutic strategy to promote a healthy NP phenotype and enhance FOXF1 and Brachyury expression. [119] Furthermore, Moen et al found that miR-223 in NPC-EVs is associated with a reduced likelihood of persistent pain following herniation. [106] Through in vivo electrophysiological measurements in a rat model, Moen et al showed that prolonged exposure of miR-223 onto dorsal nerve roots decreases C-fiber response, indicating that this specific miR has an antinociceptive effect.…”