Biomedical subjects
J Jeffrey Goater
Publications and source records attributed to J Jeffrey Goater.
Safety and efficacy of ultraviolet-a light-activated gene transduction for gene therapy of articular cartilage defects.
BACKGROUND: Gene therapies for articular cartilage defects are limited by the absence of an in vivo delivery system that can mediate site-specific transduction restricted to within the margins of the defect during routine arthroscopy. We have proposed the use of ultraviolet light to stimulate gene expression following infection by recombinant adeno-associated virus (rAAV). However, research has demonstrated that short-wavelength ultraviolet light (ultraviolet C), while effective, is neither safe nor practical for this purpose. We evaluated the safety and efficacy of long-wavelength ultraviolet light (ultraviolet A) from a laser to induce light-activated gene transduction in articular chondrocytes in vitro and in vivo. METHODS: The effects of ultraviolet A from a 325-nm helium-cadmium laser, delivered through a fiberoptic cable, on cytotoxicity, mutagenesis, intracellular reactive oxygen species, and light-activated gene transduction of human articular chondrocytes were evaluated in dose-response experiments of primary cultures. Cytotoxicity was determined by trypan blue exclusion. The presence of pyrimidine dimers in purified genomic DNA was determined by enzyme-linked immunosorbent assays. Intracellular reactive oxygen species levels were determined by flow cytometry at one hour and twenty-four hours. In vitro light-activated gene transduction with rAAV vectors expressing the green fluorescent protein (eGFP) or beta-galactosidase (LacZ) was determined by fluorescence microscopy and bioluminescence assays, respectively. In vivo light-activated gene transduction was quantified by stereotactic immunohistochemistry for beta-galactosidase in rabbit articular cartilage defects in the patellar groove that had been irradiated with +/-6000 J/m2 of ultraviolet A one week after direct injection of 10(7) transducing units of rAAV-eGFP. RESULTS: Ultraviolet A failed to induce significant cytotoxicity at all fluencies below 6000 J/m2. Dose-dependent cytotoxicity was observed at greater fluencies. In contrast to ultraviolet C, which induced significant (p < 0.05) pyrimidine dimer formation at all fluencies in a dose-dependent manner, ultraviolet A failed to induce DNA modifications. Conversely, ultraviolet C proved to be a poor inducer of intracellular reactive oxygen species, while ultraviolet A immediately induced high levels of intracellular reactive oxygen species, which were completely resolved twenty-four hours later. Ultraviolet A demonstrated significant light-activated gene transduction effects in vitro, which were dose-dependent (p < 0.05). In vivo, ultraviolet A mediated a tenfold increase in transduction in which 40.8% of the superficial chondrocytes adjacent to the defect stained positive for green fluorescent protein compared with 5.2% in the knees treated with no ultraviolet A (p < 0.006). CONCLUSIONS: These results provide what we believe is the first formal demonstration of an agent that can induce rAAV transduction in the complete absence of cytotoxicity and DNA modification. They also suggest that the mechanism by which long-wavelength ultraviolet light mediates site-specific gene expression is by means of the induction of intracellular reactive oxygen species. Finally, laser-derived ultraviolet A can be readily transferred through a fiberoptic cable to mediate light-activated gene transduction in vivo.
Remodeling of cortical bone allografts mediated by adherent rAAV-RANKL and VEGF gene therapy.
Structural allograft healing is limited because of a lack of vascularization and remodeling. To study this we developed a mouse model that recapitulates the clinical aspects of live autograft and processed allograft healing. Gene expression analyses showed that there is a substantial decrease in the genes encoding RANKL and VEGF during allograft healing. Loss-of-function studies showed that both factors are required for autograft healing. To determine whether addition of these signals could stimulate allograft vascularization and remodeling, we developed a new approach in which rAAV can be freeze-dried onto the cortical surface without losing infectivity. We show that combination rAAV-RANKL- and rAAV-VEGF-coated allografts show marked remodeling and vascularization, which leads to a new bone collar around the graft. In conclusion, we find that RANKL and VEGF are necessary and sufficient for efficient autograft remodeling and can be transferred using rAAV to revitalize structural allografts.
Light-activated gene transduction enhances adeno-associated virus vector-mediated gene expression in human articular chondrocytes.
OBJECTIVE: To evaluate the effects of ultraviolet (UV) light as an adjuvant for recombinant adeno-associated virus (rAAV) transduction in human articular chondrocytes. METHODS: Primary articular chondrocytes and immortalized chondrocytes (tsT/AC62) were exposed to various doses of UV light (0-1,000 J/m(2)) and infected at various multiplicities of infection (MOIs) with rAAV containing the enhanced green fluorescent protein (EGFP) gene. Cells were analyzed for viability and EGFP expression by fluorescence-activated cell sorting on days 2, 4, and 8 following infection. To evaluate the transduction efficiency in intact articular cartilage, full-thickness explants were exposed to UV light (0-200 J/m(2)), infected with rAAV-eGFP, and analyzed for transduction via immunohistochemistry. RESULTS: Toxicity from UV exposure was observed at doses > or =500 J/m(2) and > or =200 J/m(2) in primary and immortalized chondrocyte cultures, respectively. Transduction efficiency was dependent on the UV dose, MOI, and time. In the cell line, the adjuvant effect of UV on the percentage of cells transduced was modest, but 100 J/m(2) increased the mean fluorescence intensity (MFI) of the transduced cells 4-fold. In contrast, UV treatment had a profound effect on the transduction efficiency of primary chondrocytes, which reached approximately 100% after exposure to 100 J/m(2) of UV light and 10(3) MOIs for 8 days. Under the same conditions, 200 J/m(2) of UV light enhanced the MFI 7-fold. In cartilage explants, there was no difference in the number of transduced chondrocytes at the edge of the explants in the superficial, intermediate, or basal zones; however, 200 J/m(2) of UV light increased the transduction efficiency 2-fold at a low MOI. In the center of the explants, the superficial chondrocytes were efficiently transduced; those in the intermediate and basal zones could not be efficiently transduced under any condition. In the superficial chondrocytes, a low MOI and 200 J/m(2) of UV light increased the transduction efficiency 3-fold (to 100%). CONCLUSION: UV light at doses of up to 200 J/m(2) (which do not significantly affect cell viability) significantly enhances the transduction efficiency and expression of the transduced gene in cultures of rAAV-infected primary chondrocytes and in chondrocytes in the superficial zone of intact articular cartilage. These findings support the concept that UV-activated gene transduction could be used as an adjuvant for in vivo rAAV articular cartilage gene therapy with low viral titers to prevent and/or treat arthritis.
Adeno-associated virus-mediated osteoprotegerin gene transfer protects against particulate polyethylene-induced osteolysis in a murine model.
OBJECTIVE: Osteoprotegerin (OPG), a natural negative regulator of osteoclastogenesis and bone resorption, may be a potential therapeutic agent for treatment of osteolysis-associated prosthetic joint loosening. Using an in vivo adeno-associated virus (AAV)-mediated gene transfer technique, this study was designed to evaluate the protective effects of OPG transgene against orthopedic wear debris-induced bone loss in a murine model of osteolysis. METHODS: Bone tissue was implanted into established pouches on BALB/c mice, followed by the introduction of ultra-high-molecular-weight polyethylene (UHMWPE) particles to provoke inflammation and osteolysis. The viruses encoding human OPG gene (rAAV-hOPG) or beta-galactosidase marker gene (rAAV-LacZ) were injected into the air pouches, and the tissue was harvested 7 days after viral infection for histologic and molecular analyses. RESULTS: Successful transgene expression was confirmed by the detection of OPG by enzyme-linked immunosorbent assay and positive X-Gal staining of pouch tissue (LacZ). Real-time polymerase chain reaction indicated significant diminishment of messenger RNA expression of osteoclast markers in OPG-transduced pouches compared with rAAV-LacZ-transduced pouches. The transduction and expression of OPG also markedly decreased the gene copies of the biologic receptor activator of nuclear factor kappaB. The expression of OPG in the bone-implanted pouch reduced bone calcium release by a mean of 39% compared with the calcium release in the other 2 groups. Computerized image analysis revealed that expression of OPG significantly protected against bone collagen loss. CONCLUSION: OPG gene transfer mediated by rAAV effectively protects against particulate polyethylene-induced bone resorption in this experimental model. Data suggest that gene transfer using rAAV-OPG may be a feasible and effective therapeutic candidate to treat or prevent wear debris-associated osteolysis and aseptic loosening.
Efficacy of ex vivo OPG gene therapy in preventing wear debris induced osteolysis.
Aseptic loosening of prosthetic implants remains a serious orthopaedic problem and the greatest limitation to total joint arthroplasty. Central to the etiology of aseptic loosening is periprosthetic osteolysis at the bone-implant interface, which is caused by wear debris-induced inflammation. This inflammation produces the critical osteoclast differentiation factor RANKL, which directly stimulates osteoclastogenesis and osteoclastic bone resorption. A dominant factor known to counteract this process is the natural RANKL receptor antagonist protein OPG. Here we explore the potential of ex vivo OPG gene therapy for aseptic loosening by evaluating the eflicacy of stably transfected fibroblast-like synoviocytes (FLS) expressing OPG in preventing wear debris-induced osteoclastogenesis, in a mouse calvaria model. Although the stably transfected fibroblasts produced small amounts of OPG (0.3 ng/ml/72 h/10(6) cells), this protein was very effective in preventing osteoclastic resorption as determined in a bone wafer assay. More importantly. implantation of 10(7) FLS-OPG, together with 30 mg of Ti wear debris, onto the calvaria of mice, completely inhibited osteoclastogenesis 3 days after surgery. Animals given FLS-LacZ control cells, which persisted for 3 days as determined by X-gal staining, together with the Ti particles, had a 6-fold increase in osteoclastogenesis compared to controls without Ti. This increased osteoclastogenesis was completely inhibited by the FLS-OPG, as osteoclast numbers in the calvaria of these animals were similar to that seen in the SHAM controls.
Structural changes in the forefoot of individuals with diabetes and a prior plantar ulcer.
BACKGROUND: Plantar ulcers produced by diabetic foot disease are devastating and costly. Better understanding of the ulcer-producing process is important to improve detection of feet that are at risk and to improve intervention. We identified and quantified soft-tissue and osseous structural changes in the forefoot of diabetic patients with a prior plantar ulcer. METHODS: Thirty-two individuals with a mean age (and standard deviation) of 57 +/- 11 years were studied; sixteen had diabetes (of a mean of 20 +/- 11 years' duration), peripheral neuropathy, and a prior plantar ulcer, and sixteen were matched controls. Computed tomography was used to evaluate forefoot structure, including the plantar soft-tissue (muscle) density, soft-tissue thickness beneath the metatarsal heads, metatarsophalangeal joint angle, metatarsal bone density, and metatarsophalangeal joint arthropathy. RESULTS: Plantar soft-tissue (muscle) density was lower in the individuals with diabetes (mean, 1 HU [Hounsfield unit]) than it was in the controls (mean, 18 HU). There was no difference in the soft-tissue thickness beneath the metatarsal heads (mean, 10 mm) between the individuals with diabetes and the controls, but the soft-tissue thickness decreased with age. The individuals with diabetes had greater extension deformity of the first, second, and third metatarsophalangeal joints and greater arthropathy of the second, third, and fourth metatarsophalangeal joints. There were no significant differences in metatarsal bone density between the groups. CONCLUSIONS: There were significant differences between the forefeet of individuals with diabetes and a previous plantar ulcer and those of controls: plantar muscle density was decreased, and metatarsophalangeal joint extension and arthropathy were increased. Interestingly, the soft-tissue thickness under the metatarsal heads in the controls was not greater than that in the diabetic patients. CLINICAL RELEVANCE: This study demonstrated structural differences between the forefeet of patients with diabetes and a previous ulcer and those of normal age-matched controls. The information can serve to guide new interventions to prevent or treat foot ulcerations in this patient population.