Type II collagen and glycosaminoglycan expression induction in primary human chondrocyte by TGF-ß1.

BACKGROUND: A localized non-surgical delivery of allogeneic human chondrocytes (hChonJ) with irradiated genetically modified chondrocytes (hChonJb#7) expressing transforming growth factor-ß1 (TGF-ß1) showed efficacy in regenerating cartilage tissue in our pre-clinical studies and human Phase I and II clinical trials. These previous observations led us to investigate the molecular mechanisms of the cartilage regeneration. METHODS: Genetically modified TGF-ß1preprotein was evaluated by monitoring cell proliferation inhibition activity. The effect of modified TGF-ß1 on chondrocytes was evaluated based on the type II collagen mRNA levels and the amount of glycosaminoclycan (GAG) formed around chondrocytes, which are indicative markers of redifferentiated chondrocytes. Among the cartilage matrix components produced by hChonJb#7 cells, type II collagen and proteoglycan, in addition to TGF-ß1, were also tested to see if they could induce hChonJ redifferentiation. The ability of chondrocytes to attach to artificially induced defects in rabbit cartilage was tested using fluorescent markers. RESULTS: Throughout these experiments, the TGF-ß1 produced from hChonJb#7 was shown to be equally as active as the recombinant human TGF-ß1. Type II collagen and GAG production were induced in hChonJ cells by TGF-ß1 secreted from the irradiated hChonJb#7 cells when the cells were co-cultured in micro-masses. Both hChonJ and hChonJb#7 cells could attach efficiently to the defect area in the rabbit cartilage. CONCLUSIONS: This study suggests that the mixture (TG-C) of allogeneic human chondrocytes (hChonJ) and irradiated genetically modified human chondrocytes expressing TGF-ß1 (hChonJb#7) attach to the damaged cartilage area to produce type II collagen-GAG matrices by providing a continuous supply of active TGF-ß1.

Sub-acute toxicity study in female ICR mice following repetitive intramuscular injection of cervical cancer vaccines.

OBJECTIVES: The sub-acute toxic effects following repetitive intramuscular injection of two cervical cancer vaccines newly developed against human papillomaviruse (HPV)16/58/18 and HPV16 were investigated in female ICR (CrljOri: CD1) mice, and the no-observedadverse- effect-level (NOAEL) of the cervical cancer vaccines was estimated. METHODS: Female ICR mice (n=15 in each group) were exposed to a 1:1 mixture of two cervical cancer vaccines by repetitive intramuscular injection (once a week, 5 times) for 5 weeks. Mortality, body weight, organ weight, hematological/biochemical parameters, and histopathological effects were examined at different concentrations (0, 1×10(8), 5×10(8), and 2.5×10(9) copies/animal) of the cervical cancer vaccines. RESULTS: The cervical cancer vaccines did not show toxic responses for body weight, absolute/ relative organ weight, hematological/biochemical parameters, or histopathological parameters. CONCLUSIONS: Female ICR mice exposed to vaccines for cervical cancer did not show any toxic response. We suggest that a NOAEL of the vaccine following repetitive intramuscular injection for 5 weeks is >2.5×10(9) copies/animal.

Initial phase I safety of retrovirally transduced human chondrocytes expressing transforming growth factor-beta-1 in degenerative arthritis patients.

UNLABELLED: BACKGROUND AIMS. TissueGene-C (TG-C) represents a cell-mediated gene therapy for localized delivery of allogeneic chondrocytes expressing transforming growth factor (TGF)-ß1 directly to the damaged knee joint. Untransduced human chondrocytes (hChonJ cells) have also been incorporated into the TG-C product at a 3:1 ratio with TGF-ß1-expressing chondrocytes (hChonJb#7) in order to help fill in the defect and as target cells for the actions of the expressed TGF-ß1. METHODS: A phase I dose-escalating clinical trial was performed to evaluate the safety and biologic activity of TG-C in patients with advanced osteoarthritis of the knee joint (full thickness cartilage defect) that was refractory to existing non-operative therapies. Following a single intra-articular injection into the joint space of the damaged knee, patients were monitored for safety, and an evaluation was performed to assess the pharmacokinetics and biologic activity of TG-C. RESULTS: There were no treatment-related serious adverse events. Swelling, effusion and minor localized reactions such as warming sensation or itching were observed in a dose-dependent manner at the injection site. Knee evaluation scores seemed to indicate a dose-dependent trend toward efficacy; however, patient numbers were not sufficient to determine statistical significance. CONCLUSIONS: Overall, there were no significant safety issues related to the administration of TG-C, with only some minor injection site reactions observed. Additionally, knee scoring analyzes indicated a possibility that TG-C may contribute to improvement of arthritic symptoms. More study is warranted to evaluate further the safety and determine the potential efficacy of TG-C.

Pre-clinical studies of retrovirally transduced human chondrocytes expressing transforming growth factor-beta-1 (TG-C).

BACKGROUND AIMS: The aim was to evaluate cartilage regeneration in animal models involving induced knee joint damage. Through cell-mediated gene therapy methods, a cell mixture comprising a 3:1 ratio of genetically unmodified human chondrocytes and transforming growth factor beta-1 (TGF-beta1)-secreting human chondrocytes (TG-C), generated via retroviral transduction, resulted in successful cartilage proliferation in damaged regions. METHODS: Non-clinical toxicology assessments for efficacy, biodistribution and local/systemic toxicity of single intra-articular administration of the cell mixture in mice, rabbits and goats was conducted. RESULTS: Administration of the mixture was tolerated well in all of the species. There was evidence of cartilage proliferation in rabbits and goats. As an additional precautionary step, the efficacy of TGF-beta1 secretion in irradiated human chondrocytes was also demonstrated. CONCLUSIONS: Four studies in rabbits and goats demonstrated the safety and efficacy of TG-C following direct intra-articular administration in animal models involving induced knee joint damage. Based on these pre-clinical studies authorization has been received from the USA Food and Drug Administration (FDA) to proceed with an initial phase I clinical study of TG-C for degenerative arthritis.

Irradiated human chondrocytes expressing bone morphogenetic protein 2 promote healing of osteoporotic bone fracture in rats.

Bone morphogenetic protein 2 (BMP2) was selected as a transgene to regenerate osteoporotic bone defects after several BMPs were tested using a bone formation study in nude mice. Human chondrocytes were transduced with a BMP2-containing retroviral vector, and single clones were selected. The cells were characterized over numerous passages for growth and BMP2 expression. The single clones were irradiated and tested for viability. BMP2 expression lasted for 3 weeks before dying off completely after approximately 1 month. Irradiated and non-irradiated transduced chondrocytes successfully healed fractures in osteoporotic rats induced by ovariectomy. The osteoinducing effect of irradiated cells was better than that of their non-irradiated counterparts or a chondrocytes-only control. This study showed that delivering BMP2 from the transduced and irradiated chondrocytes could be an effective and safe method of repairing osteoporotic bone fractures.

Hyaline cartilage regeneration using mixed human chondrocytes and transforming growth factor-beta1- producing chondrocytes.

The purpose of this study was to investigate the efficacy of cartilage regeneration when using a mixture of transforming growth factor-beta1 (TGF-beta1)-producing human chondrocytes (hChon-TGF-beta1) and primary human chondrocytes (hChon) ("mixed cells"), compared with either hChon-TGF-beta1 or hChon cells alone. Specifically, mixed cells or hChon cells were first injected intradermally into the backs of immune-deficient nude mice to test the feasibility of cartilage formation in vivo. Both the mixed cells and the hChon-TGF-beta1 cells alone induced cartilage formation in nude mice, whereas hChon cells alone did not. To further test the efficacy of the cells in generating cartilage, an artificially induced partial thickness defect of the femoral condyle of a rabbit knee joint was loaded with hChon-TGF-beta1 cells with or without mixing additional untransduced hChon cells, and hyaline cartilage regeneration was observed at 4 or 6 weeks. The efficiency of complete filling of the defect and the quality of tissue generated after implanting were evaluated on the basis of a histological grading system modified from O'Driscoll et al. (J. Bone Joint Surg. 70A, 595, 1988). Significantly, mixed cells (14.2 +/- 0.9) produced significantly better results than hChon-TGF-beta1 (9.0 +/- 1.7) or hChon (8.0 +/- 1.8) cells alone. Histological and immunohistochemical staining of the newly repaired tissues produced after treatment with either mixed cells or hChon-TGF-beta1 cells alone showed hyaline cartilage- like characteristics. These results suggest that the implantation of mixed cells may be a clinically efficient method of regenerating hyaline articular cartilage.

Continuous transforming growth factor beta1 secretion by cell-mediated gene therapy maintains chondrocyte redifferentiation.

One of the most important factors in the production of cartilage is transforming growth factor beta1 (TGF-beta1). To obtain sustained release of TGF-beta1, a cell-mediated gene therapy technique was introduced. We infected chondrocytes with a retroviral vector carrying the TGF-beta1 gene. The single clone derivative showed sustained TGF-beta1 secretion. It also showed constitutive type II collagen expression. Whereas the TGF-beta1 protein itself is unable to induce formation of cartilage in vivo, human chondrocytes engineered to express a retroviral vector encoding TGF-beta1 showed cartilage formation in vivo when cells were injected into nude mice intradermally. These data suggest that cell-mediated gene therapy using TGF-beta1 as a transgene would be a promising treatment for osteoarthritis.

Regeneration of hyaline articular cartilage with irradiated transforming growth factor beta1-producing fibroblasts.

The regeneration of hyaline articular cartilage by cell-mediated gene therapy using transforming growth factor beta(1) (TGF-beta(1))-producing fibroblasts (NIH 3T3-TGF-beta(1)) has been reported previously. In this study, we investigated whether TGF-beta(1)-producing fibroblasts irradiated with a lethal dose of radiation are still capable of inducing the regeneration of hyaline articular cartilage. NIH 3T3TGF-beta(1) fibroblasts were exposed to doses of 20, 40, or 80 Gy, using a irradiator, and then injected into artificially made partial defects on the femoral condyle of rabbit knee joints. The rabbits were killed 3 or 6 weeks postinjection and hyaline articular cartilage regeneration was evaluated by histological and immunohistochemical staining (n = 5 per each group). Irradiated NIH 3T3-TGFbeta(1) fibroblasts started to die rapidly 3 days after irradiation; moreover, the kinetics of their viability were similar regardless of the radiation intensity. TGF-beta1 expression, measured by ELISA, showed that the TGF-beta(1) protein produced from the irradiated cells peaked 5 days after irradiation and thereafter declined rapidly. Complete filling of the defect with reparative tissue occurred in all the groups, although variations were observed in terms of the nature of the repair tissue. Histological and immunohistochemical staining of the repair tissue showed that the tissue newly formed by irradiated NIH 3T3-TGF-beta(1) fibroblasts after exposure to 20 Gy had hyaline cartilage-like characteristics, as was observed in the nonirradiated controls. On the other hand, the repair tissue formed by NIH 3T3-TGF-beta(1) fibroblasts irradiated with 40 or 80 Gy showed more fibrous cartilage-like tissue. These results suggest that TGF-beta(1)-producing fibroblasts irradiated up to a certain level of lethal dose (i.e., 20 Gy) are able to induce normal-appearing articular cartilage in vivo. Therefore, irradiated heterologous cell-mediated TGF-beta(1) gene therapy may be clinically useful and an efficient method of regenerating hyaline articular cartilage.