Osteogenic protein-1 gene: gene gun transfer to intervertebral disc cells

Abstract

Takuji Matsumoto, MD, PhD, Howard S. An, MD, Koichi Masuda, MD, Eugene J. Thonar, PhD, Gunnar B. J. Andersson, MD, PhD, Chicago, IL, USAIntroduction: Therapeutic approaches to restore intervertebral disc (IVD) damage in degenerative diseases are limited. Gene therapy is one of the most attractive tools for inducing the expression of growth factors. A nonviral gene delivery system is an alternative that avoids the risks of insertional mutagenesis of retroviruses, immunogenicity of adenoviruses and acquiring replication competence. However, direct introduction of genes into the IVD has been considered to be difficult, in part because of the presence in the tissue of an extracellular matrix composed of a dense collagen framework and proteoglycans (PGs). We have recently reported the potential of a nonviral gene gun-mediated gene transfer method for efficient transfection of the IVD using a reporter gene.Purpose: The purpose of this study is to 1) to compare the efficiency of transfection of different annulus fibrosus (AF) and nucleus pulposus (NP) cells using a gene–gun transfection system and, 2) to induce, by transfection of the human osteogenic protein-1 (OP-1) gene, specific metabolic changes in IVD cells (NP and AF cells).Materials and methods: AF and NP tissues were isolated from tails of 14 to 18-month bovine steer. Cells were isolated by sequential digestion with 0.2% pronase and 0.025% collagenase. Cells were seeded at a density of 50,000 cells/well in a 12-well plate and cultured for 2 days before transfection. After the transfection, cells were cultured in DMEM/F12 medium containing 10% FBS. pCMV-b-galactosidase (Clontech) served as a reporter gene, and transgene expression was assessed using the In Situ b-galactosidase staining kit (Stratagene). Human OP-1 expression vector, pW24, was a generous gift from Dr. John C. Lee. At the time of gene transfer, a pulse of high-pressure helium gas (125 psi) was released from a helium tank through the Gold-Coat 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 tissue, and a single bombardment was carried out. After 3 days, the transfection efficiency of a Lac reporter gene construct (pCMV-b-galactosidase) in the primary monolayer cultures of normal bovine NP and AF cells was assessed using an In Situ b-galactosidase staining kit. The DNA content and the total PG content were measured in the cell layer to assess metabolic activity. PG synthesis was also measured using [35S]-sulfate labeling, followed by rapid filtration, and was compared between the OP-1–transfected (pW24) and the control (vacant vector) groups. Statistical analyses were performed by one-way analysis of variance with Fisher's PLSD test as a post hoc test.Results: The gene transfer of b-galactosidase was performed to probe the efficiency of transfection in two different cell sources. Analysis of X-gal staining demonstrated an efficiency of 14.2% in normal NP cells and 8.2% in AF cells. The DNA content and rate of PG synthesis in the three cell types did not differ significantly when the pCMV-b-gal–transfected and nontreated groups were compared. This suggested that the gene gun procedure does not have a significant adverse effect on cell metabolism. To study whether gene transfection can alter the metabolism of IVD cells, the human OP-1 gene was transfected using a pW24 vector. On day 3 after transfection, there were no significant differences in the DNA content. A small increase in the PG accumulation was observed in the OP-1-groups. However, there were no significant differences in the PG content of any cell group. On the other hand, in the OP-1–transfected group, the rate of PG synthesis was significantly higher in all cell types (AF [124%]; p<.05 and NP [144%] cells [p<.01]). NP cells were more responsive than AF cells to the transfection of the OP-1 gene.Discussion: The results of this study reveal, for the first time, that transfection of the OP-1 gene by a gene gun system to both AF and NP cells in vitro can alter the metabolism of these cells. Both the efficiency and the metabolic studies provide evidence that the NP cells may be the best target for transfection. Although it remains to be proven that OP-1 production was enhanced after gene transfer, this study suggests that gene therapy with the OP-1 expression vector can be a useful method for inducing the regeneration of IVD and articular cartilage tissues. Additional studies are now ongoing to determine if transfection of the OP-1 gene into the IVD and articular cartilage tissues can be achieved and if it can influence the metabolism of those tissues.