LAUSR

OBJECTIVE To evaluate transduction efficacy and sustainability of lentiviral vector for intervertebral disc cells both in vitro and in vivo. METHODS Human nuclear pulposus and anulus fibrosus cells isolated from disc tissue of 28 patients during surgical disc procedures were cultured and subsequently transduced using recombinant lentivirus carrying a gene for enhanced green fluorescent protein at multiplicities of infection of 0, 15, 30, 60, 90, and 150. Cell viability was determined using the trypan blue exclusion test. Transduction efficiency was measured by fluorescence-activated cell sorting analysis. In vivo experiments were done by injecting lentivirus into rat intervertebral discs. Disc tissue was harvested 7, 14, 21, and 28 days after transduction, and enhanced green fluorescent protein expression was examined using an inverted fluorescent microscope. RESULTS Intervertebral disc cells transduced with different doses of lentivirus showed equally good viabilities compared with cells in the control group, as determined by cell morphology and growth curves after transduction. The transduction ratio for disc cells after transduction reached its optimum of 97% at 60 multiplicities of infection, independent of patient age, sex, surgical procedure, diagnosis, disc level, or degeneration grade. In vivo frozen sections revealed that enhanced green fluorescent protein expression peaked on the 7th day and remained detectable the 28th day after transduction. No significant systemic symptoms were observed during the in vivo experiment. CONCLUSIONS Lentivirus appears to be an efficient and stable transduction vector for intervertebral disc cells. It has potential as a gene therapy tool for treating human degenerative disc disease.