Nonviral overexpression of Scleraxis or Mohawk drives reprogramming of degenerate human annulus fibrosus cells from a diseased to a healthy phenotype.

Abstract

Intervertebral disc (IVD) degeneration is a major contributor to low back pain (LBP), yet there are no clinical therapies targeting the underlying pathology. The annulus fibrosus (AF) plays a critical role in maintaining IVD structure/function and undergoes degenerative changes such as matrix catabolism and inflammation. Thus, therapies targeting the AF are crucial to fully restore IVD function. Previously, we have shown nonviral delivery of transcription factors to push diseased nucleus pulposus cells to a healthy phenotype. As a next step in a proof-of-concept study, we report the use of Scleraxis (SCX) and Mohawk (MKX), which are critical for the development, maintenance, and regeneration of the AF and may have therapeutic potential to induce a healthy, pro-anabolic phenotype in diseased AF cells. MKX and SCX plasmids were delivered via electroporation into diseased human AF cells from autopsy specimens and patients undergoing surgery for LBP. Transfected cells were cultured over 14 days and assessed for cell morphology, viability, density, gene expression of key phenotypic, inflammatory, matrix, pain markers, and collagen accumulation. AF cells demonstrated a fibroblastic phenotype posttreatment. Moreover, transfection of SCX and MKX resulted in significant upregulation of the respective genes, as well as SOX9. Transfected autopsy cells demonstrated upregulation of core extracellular matrix markers; however, this was observed to a lesser effect in surgical cells. Matrix-degrading enzymes and inflammatory cytokines were downregulated, suggesting a push toward a pro-anabolic, anti-inflammatory phenotype. Similarly, pain markers were downregulated over time in autopsy cells. At the protein level, collagen content was increased in both MKX and SCX transfected cells compared to controls. This exploratory study demonstrates the potential of MKX or SCX to drive reprogramming in mild to moderately degenerate AF cells from autopsy and severely degenerate AF cells from surgical patients toward a healthy phenotype and may be a potential nonviral gene therapy for LBP.