0231 - Gene Gun-mediated Gene Transfer to Intervertebral Disc Cells

INTRODUCTION: Gene therapy using retroviral and, more recently, adenoviral vectors is gaining interest in orthopedics. Particle-mediated gene transfer with a gene gun is a novel non-viral alternative that carries great promise [1]. The basic principle of particle-mediated gene transfer is to use high velocity DNA-coated gold particles that are accelerated by high pressure helium gas to penetrate outer cell layers and to introduce genetic material into living cells. This method of transfection has several advantages over viralmediated methods, including increased safety, patient acceptance, simplicity of methodology, and relative ease of future approval for clinical trials. However, to date, we are not aware of any reports in the orthopedic literature on the use of this novel method of gene transfer. In this study, we report the effectiveness of gene gun-mediated transfection of rabbit annulus fibrosus (AF) and nucleus pulposus (NP) cells in vitro with the β-galactosidase reporter gene. We also have assessed the length of transgene expression of β-galactosidase as well as effects of particlemediated gene transfer on the matrix metabolism of rabbit AF cells in 3dimensional alginate cultures. MATERIALS AND METHODS: Cell Preparation: AF and NP cells were separately isolated from lumbar and lower thoracic intervertebral discs (IVD) of adolescent New Zealand white rabbits (IACUC #98-065). AF and NP cells were resuspended at 2x10 cells in 100 μl DMEM/F12 and spread evenly onto a 1 cm diameter area on a petri dish immediately prior to transfection. Reporter Gene: The ρCMVβ β-galactosidase gene (Clontech) serves as an ideal reporter gene because its gene product is very stable. Transgene expression was assessed with Lac-Z staining using the In Situ Lac-Z Staining Kit purchased from Stratagene. Preparation of Gold Particles: The gene gun and particle preparation devices were obtained from BioRad Laboratories. The vector ρCMVβ was precipitated onto gold particles (sizes 1.0 and 1.6 μm) at DNA Loading Ratios (DLR) of 2 and 4 μg DNA per mg gold particle and washed and resuspended in absolute ethanol. The suspension was loaded into a Gold-Coat tubing which was rotated to achieve an even smearing of gold particles on the inner surface of the tube. After drying with nitrogen gas, the tubing was cut into 0.5-inch segments and loaded onto the gene gun cartridge. Gene Transfer: At gene transfer, a pulse of high pressure helium gas (range 50 to 250 psi) was released from the helium tank through the GoldCoat tubing, accelerating the DNA-coated gold particles on the inside of the tubing cartridge to penetrate the target cells. The gene gun was positioned at a minimal distance from the petri dish and a single bombardment was carried out. Negative controls consisted of untransfected cells and cells which underwent gene gun bombardment with gold particles not coated with DNA. Transfection was accomplished in about 30 seconds for each sample. Immediately after the bombardment the cells were collected and placed into alginate beads as previously described [2] to maintain the phenotype of the cultured cells. Biochemical Assays: At 1, 2 and 3 weeks after gene transfer, following proteinase-K digestion, the DNA and sulfated proteoglycan (PG) contents of AF beads were analyzed using Hoechst 33258 dye and the DMMB assay, respectively. PG synthesis was also assessed using S-sulfate labeling followed by a rapid filtration assay [2]. For each time point, all analyses of beads were performed on triplicate cultures of 9 beads each. The standard deviations are shown in Fig. 2. Statistical analyses were performed by oneway ANOVA with Fisher’s PLSD test as a post hoc test. RESULTS: Particle-mediated gene transfer carried out at helium gas pressures above 150 psi resulted in significant cell dislodgement from tissue culture wells when tested initially on the AF monolayer cell cultures. Bombardment pressures ranging between 125 and 150 psi were found to be optimal for maximizing transfection efficiency. The level of gene expression was highest when using a particle size of 1.6 μm at a DLR of 2 μg of DNA per mg gold with the gene gun pressured at 125 psi. Under these conditions the transfection efficiency with the ß-galactosidase gene was approximately 10%, as measured by manual cell count after Lac-Z staining (Fig. 1). Transgene expression of β-galactosidase by both NP and AF cells in 3dimensional alginate cultures was maintained over the 3 weeks of culture after transfection. The content of DNA increased in a similar fashion in all 3 groups of AF cells after transfection, suggesting that the procedure did not cause significant cell damage (data not shown). There was no significant difference in the rate of PG synthesis by AF cells at any time point (Fig. 2, left panel). PG content in the beads increased similarly in all groups over the 21 days of culture. There were no differences among the 3 groups (Fig. 2, right panel), except at the one week time point (Particles only >Control: p < 0.05).