Adenoviral delivery of LIM mineralization protein-1 induces new-bone formation in vitro and in vivo.

BACKGROUND: The LIM mineralization protein-1 (LMP-1) gene encodes for an intracellular protein that induces the expression of several bone growth factors. The purpose of the present study was to determine the feasibility and the optimal dose of adenoviral delivery of the LMP-1 cDNA to promote spinal fusion. METHODS: A replication-deficient human recombinant adenovirus was constructed with the LMP-1 cDNA driven by a cytomegalovirus promoter. In phase 1, an in vitro dose-response experiment was performed to determine the optimal adenovirus-LMP-1 (AdLMP-1) concentration and infection time. In phase 2, nine rabbits had a single-level posterolateral arthrodesis of the lumbar spine with implantation of a carrier matrix loaded with bone-marrow-derived buffy-coat cells that had been infected for ten minutes with adenovirus containing the cDNA for LMP-1 (AdLMP-1) or beta-galactosidase (AdBgal). In phase 3, posterolateral arthrodesis of the spine was performed with implantation of cells infected with AdLMP-1 (ten rabbits) or cells infected with an empty adenovirus that did not contain LMP-1 cDNA (ten rabbits) and the results were compared. In this phase, peripheral-blood-derived buffy-coat cells were used instead of bone-marrow-derived cells and a collagen-ceramic-composite sponge was used as the carrier. RESULTS: In phase 1, the in vitro dose-response experiment showed that a multiplicity of infection of 0.25 plaque-forming units per cell was the most efficient dose. In phase 2, the implants that had received cells infected with AdLMP-1 induced a solid, continuous spinal fusion mass at five weeks. In contrast, the implants that had received cells infected with AdBgal or a lower dose of AdLMP-1 induced little or no bone formation. In phase 3, a solid spinal fusion was observed at four weeks in all ten rabbits that had received cells infected with AdLMP-1 and in none of the ten rabbits that had received cells infected with the empty adenovirus. Biomechanical and histological testing of the AdLMP-1-treated specimens revealed findings that were consistent with a high-quality spinal fusion. CONCLUSIONS: Adenoviral delivery of LMP-1 cDNA promotes spinal fusion in immune-competent rabbits.

Gene therapy for spine fusion.

Spine fusion is a commonly performed yet often unsuccessful surgical procedure. As many as 40% of patients undergoing spine fusion may have a nonunion or failure to form a continuous bone bridge. This clinical challenge has focused much of the attention of osteoinductive bone growth factors toward spine applications. Clinical pilot and pivotal trials will show the feasibility of recombinant and purified bone growth factors to promote spine fusion in humans. Despite this, strategies of gene therapy for spine fusion and other bone healing applications are being pursued. This article reviews the state of the art of local gene therapy and highlights specific issues that must be addressed when pursuing a gene therapy program. Perhaps the most critical step in gene therapy for bone formation is choosing an appropriate osteoinductive gene. Such choices may be limited by differences in efficacy of the chosen gene and availability because of proprietary constraints. The choice of delivery vector is crucial and depends on the potency of the gene and the specific application intended. Establishing the effective dose, transduction time, and gene transfer method are important decisions. The choice of carrier material to form the scaffold for the new bone formation is paramount to successful bone formation. Finally, a strategy for in vitro and in vivo testing must be developed to maximize the chances of success in human trials.

Lumbar spine fusion by local gene therapy with a cDNA encoding a novel osteoinductive protein (LMP-1).

STUDY DESIGN: A posterior arthrodesis animal model using local expression of a newly discovered osteoinductive protein delivered in bone marrow cells. OBJECTIVE: To introduce the concept of local gene therapy and determine its feasibility for achieving lumbar spine fusion using a gene for a novel osteoinductive protein: LIM Mineralization Protein-1 (LMP-1). SUMMARY OF BACKGROUND DATA: Extensive work is currently underway to improve the healing success and morbidity associated with the gold standard bone-grafting material of autogenous iliac crest. As a result, alternative osteoinductive proteins and new delivery methods warrant investigation. The authors' laboratory recently identified a novel gene that had osteoinductive capacity in vitro and is therefore a candidate for a new in vivo osteoinductive agent. METHODS: Single-level posterior lumbar and thoracic arthrodesis was attempted in 14 athymic rats. The graft material, which consisted of a devitalized bone matrix (no osteoinductive activity) soaked with 0.75 to 1.5 million bone marrow cells, was inserted with the dorsal spine exposed. In each rat, one site received marrow cells transfected with the cDNA encoding a novel osteoinductive protein. At the other site for a control, the marrow cells were transfected with the reverse copy of the cDNA that did not express any protein. Transfection of marrow cells for 2 hours was accomplished using the mammalian expression vector pCMV2. Rats were killed after 4 weeks, and the spines were evaluated by manual palpation, radiographs, and nondecalcified histology. RESULTS: In the pivotal experiment, successful spine fusion was obtained in 9/9 (100%) of the sites receiving marrow cells transfected with the active LMP-1 cDNA and in 0/9 (0%) of the sites receiving marrow cells transfected with the reverse (inactive) LMP-1 cDNA. Radiographs and histology confirmed the manual palpation results, demonstrating controlled new bone formation in the carrier and marrow transfected with the active LMP-1 cDNA and essentially no bone induction in the sites treated with marrow cells that did not express the protein. CONCLUSIONS: These data confirm that local delivery of the novel LMP-1 cDNA using bone marrow cells is feasible in vivo. Furthermore, these results demonstrate that posterior thoracic or lumbar spine fusion can be achieved in rats by local delivery of the LMP-1 cDNA.

Cloning and tissue expression of the rat RAB 3C GTP-binding protein.

A cDNA clone which encodes the low molecular weight GTP-binding protein rab 3C was isolated from a rat PC12 pheochromocytoma library. The 1.0 kb clone contains the entire coding region (681 bp), as well as 5' (89 bp) and 3' (230 bp) untranslated sequences. The predicted amino acid sequence of the rat rab 3C clone is 98% identical to the bovine rab 3C sequence and 85% identical to the rat rab 3A sequence. Northern blot analyses using probes containing coding and noncoding sequences of the rat rab 3C clone hybridized to a 9.5 kb transcript in brain, adrenal gland, and pituitary RNA pools.

Isolation of cDNA clones and tissue expression of rat ral A and ral B GTP-binding proteins.

cDNA clones encoding the low molecular weight GTP-binding proteins ral A (951 bp) and ral B (2073 bp), including the entire coding region (618 bp), were isolated from a rat PC12 pheochromocytoma library. Northern analyses demonstrated that both ral A and ral B are widely expressed in rat tissues. Two ral A transcripts of 1.1 and 2.9 kb were observed in most tissues in varying proportions. The 1.1 kb ral A band of testes was further shown to be composed of two closely migrating species. In contrast to these findings, a single ral B transcript of 2.3 kb was detected in most tissues. Steady-state levels of ral A transcripts appear greater than ral B. Quantitatively, the testes exhibited the highest ral A and ral B mRNA levels, with lower levels observed in the brain, adrenal and pituitary glands, kidney and ovary. Ral mRNA levels were lowest in muscle tissue, particularly skeletal muscle.