168. Non-viral transfection of human intervertebral disc cells with developmental factors induces reprogramming to a healthy anti-catabolic/inflammatory phenotype with enhanced extracellular matrix accumulation

BACKGROUND CONTEXT Intervertebral disc (IVD) degeneration is a significant contributor to low back pain (LBP) and is characterized by a decrease in proteoglycan synthesis, increased catabolism, secretion of inflammatory factors and neurovascular invasion. Although viral gene therapies have been proposed for LBP treatment associated with IVD degeneration, there are concerns due to potential mutagenesis and unwarranted immune responses. To overcome these limitations, nonviral electroporation approaches have been developed. Transcription factor, Brachyury (T) is expressed in the developing notochord and associated with a healthy immature nucleus pulposus (NP) and thus it is a promising factor to reprogram degenerate IVD cells to halt degeneration and promote repair PURPOSE To examine effects of Ttransfection on human NP cells ex-vivo. We hypothesize effective delivery of T can reprogram degenerate IVD cells into healthy cells with increased proteoglycan and decreased inflammatory, catabolic and pain factors. STUDY DESIGN/SETTING 1) Cells isolated from patient and cadaveric human NP tissue. 2) Bulk electroporated with T3) 3D cultured over 4 weeks in agarose gels. PATIENT SAMPLE N=5 samples from patients undergoing microdiscectomy (painful, 19-70yo) and N=5 cadaveric samples(nonpainful, 19-58yo). IRB: 2015H0385 OUTCOME MEASURES Gene expression (RT-qPCR) for healthy NP phenotypic markers, inflammatory/pain markers, matrix genes, and matrix cleaving enzymes, proteoglycan protein (GAG) (dimethylmethylene blue assay), cell viability (calcein/ethidium staining). METHODS Cells were enzymatically isolated from NP tissue and expanded. Subsequently, nonpainful (nPT) andpainful (PT) NP cells were electroporated with a plasmids encoding for T, or an empty vector as a SHAM control. The transfected cells were then expanded in disc cell media and seeded in 3D agarose constructs. Outcome measures were examined at day 0, week 2 and week 4. Statistical Mann-Whitney Tests at α=0.05 were used. RESULTS Cell viability remained high for all groups. Gene expression: healthy marker T was overexpressed and maintained for 4 weeks while KRT19 was significantly increased in nPT and PT cells compared to SHAM controls. Inflammatory cytokines IL-1β and IL6 decreased at 2 weeks for nPT cells and over time for PT cells. Pain marker NGF expression showed significant decrease at week 2 for PT cells and over time in nPT cells. Matrix gene ACAN was increased at 2 weeks in nPT and PT cells while MMP13 was significantly decreased in nPT cells at all time points and at week 4 for PT cells. GAG content was significantly increased in PT groups at 2 weeks compared to SHAM and observed to a lesser extent at week 4. CONCLUSIONS This study demonstrates: (i) sustained cell viability post electroporation of IVD cells, (ii) high expression of T maintained over 4 weeks, and (iii) potential of T to reprogram degenerate human NP cells to a healthy phenotype (up regulation of T and KRT19) with down regulation of inflammatory (IL-1B, 1L6), pain (NGF), and catabolic (MMP12) markers on the gene level with increase matrix synthesis at the protein level (GAG). This is the first study to demonstrate successful reprogramming of diseased human NP cells into a healthy phenotype using nonviral transfection and is a potential therapy to LBP. Future work will use novel electroporation technique, tissue nanotransfection, which has shown to reprogram and rescue diseased tissue. This allows for high efficiency delivery of factors into patient cells while bypassing caveats of viral methods to stimulate IVD repair FDA DEVICE/DRUG STATUS This abstract does not discuss or include any applicable devices or drugs.