Non-Viral reprogramming of degenerate Intervertebral Disc Cells induces trans-differentiation to a Healthy Phenotype with enhanced extracellular matrix accumulation
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Date
2019-03
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Abstract
Intervertebral disc (IVD) degeneration is a major contributor to chronic low back pain and is characterized by decreases in cellularity and proteoglycan synthesis, upregulation of matrix degradation, and increases in pro-inflammatory factors with neurovascular invasion. Current treatments fail to target the underlying pathology or promote tissue repair and approaches such as viral transfection raise safety concerns due to mutagenesis and unwarranted immune responses. To avoid such concerns, nonviral transfection is a viable method of gene delivery into the host cell while bypassing the caveats of viral delivery. Brachyury is expressed in the developing notochord and is associated with an immature healthy nucleus pulposus (NP). We hypothesize that Brachyury can reprogram degenerate NP cells to a healthy pro-anabolic phenotype with increased proteoglycan synthesis and decreased expression of catabolic, inflammatory and neurovascular markers. NP cells obtained from human autopsy (non-painful) and surgical tissues (painful) were transfected with plasmids encoding for Brachyury or an empty vector control via bulk electroporation. Post transfection, cells were seeded in 3D agarose constructs cultured over 4 weeks and analyzed for viability, gene expression, and proteoglycan. Results demonstrated successful transfection of both non-painful and painful NP cells. We observed long term Brachyury expression, significant increased expression of NP phenotypic markers FOXF1, KRT19, and chondrogenic marker SOX9 with decreases in inflammatory cytokines IL1-β/IL6, NGF, and MMPs and significant increase in proteoglycan synthesis. These results highlight nonviral transfection with developmental transcription factors, such as Brachyury, as a promising method to reprogram degenerate disc cells towards a healthy NP phenotype.
Description
Engineering: 3rd Place (The Ohio State University Edward F. Hayes Graduate Research Forum)
Keywords
intervertebral disc, Low Back Pain, Gene Therapy, Cell Reprogramming