Youngsuk Yi

A novel mitochondrial septin-like protein, ARTS, mediates apoptosis dependent on its P-loop motif.

Here we describe a protein product of the human septin H5/PNUTL2/CDCrel2b gene, which we call ARTS (for apoptosis-related protein in the TGF-β signalling pathway). ARTS is expressed in many cells and acts to enhance cell death induced by TGF-β or, to a lesser extent, by other apoptotic agents. Unlike related septin gene products, ARTS is localized to mitochondria and translocates to the nucleus when apoptosis occurs. Mutation of the P-loop of ARTS abrogates its competence to activate caspase 3 and to induce apoptosis. Taken together, these observations expand the functional attributes of septins previously described as having roles in cytokinesis and cellular morphogenesis.

A novel ets-related transcription factor, ERT/ESX/ESE-1, regulates expression of the transforming growth factor-beta type II receptor.

A 2.5-kilobase cDNA clone that encodes a 371-amino acid novel transcription factor was isolated from a human placenta cDNA library using a yeast one-hybrid system. The novelets-related transcription factor (ERT) showed a homology with the ETS DNA-binding domain. Using constructs of the transforming growth factor-β (TGF-β) type II receptor (RII) promoter linked to the luciferase gene, we have demonstrated that ERT activates transcription of the TGF-β RII gene through the 5′-TTTCCTGTTTCC-3′ response element spanning nucleotides +13 to +24 and multiple additional ETS binding sites between −1816 and −82 of the TGF-β RII promoter. A specific interaction between ERT and the ETS binding sites was also demonstrated using an electrophoretic mobility shift assay. Deletion mapping of ERT protein suggests that the transactivation domain resides in the amino terminus while the DNA-binding domain is localized to the carboxyl-terminal region. Our results suggest that ERT might be a major transcription factor involved in the transcriptional regulation of the TGF-β RII gene.

Retroviral Gene Therapy: Safety Issues and Possible Solutions

The recent incidents of leukemia development in X-SCID patients after a successful treatment of the disease with retroviral gene therapy raised concerns regarding the safety of the use of retroviral vectors in clinical gene therapy. In this review, we have tried to re-evaluate the safety issues related to the use of retroviral vectors in human clinical trials and to suggest possible appropriate solutions to the issues. As revealed by the X-SCID incident, oncogenesis caused by retroviral insertional activation of host genes is one of the most prominent risks. An ultimate solution to this problem will be in re-engineering retroviral vectors so that the retroviral insertion takes place only at the desired specific sites of the host cell chromosome. This is, however, a technically demanding tasks, and it will take years to develop retroviral vectors with targeted insertion capability. In the mean time, the use of chromatin insulators can reduce chances for retrovirus-mediated oncogenesis by inhibiting non-specific activation of nearby cellular proto-oncogenes. Co-transduction of a suicidal gene under the control of an inducible promoter could also be one of the important safety features, since destruction of transduced cells can be triggered if abnormal growth is observed. Additionally, conditional expression of the transgene only in appropriate target cells via the combination of targeted transduction, cell type-specific expression, and targeted local administration will increase the overall safety of the retroviral systems. Finally, splitting of the viral genome, use of self-inactivating (SIN) retroviral vectors, or complete removal of the coding sequences for gag, pol, and env genes is desirable to virtually eliminate the possibility of generation of replication competent retroviruses (RCR).

Current Advances in Retroviral Gene Therapy

There have been major changes since the incidents of leukemia development in X-SCID patients after the treatments using retroviral gene therapy. Due to the risk of oncogenesis caused by retroviral insertional activation of host genes, most of the efforts focused on the lentiviral therapies. However, a relative clonal dominance was detected in a patient with β-thalassemia Major, two years after the subject received genetically modified hematopoietic stem cells using lentiviral vectors. This disappointing result of the recent clinical trial using lentiviral vector tells us that the current and most advanced vector systems does not have enough safety. In this review, various safety features that have been tried for the retroviral gene therapy are introduced and the possible new ways of improvements are discussed. Additional feature of chromatin insulators, co-transduction of a suicidal gene under the control of an inducible promoter, conditional expression of the transgene only in appropriate target cells, targeted transduction, cell type-specific expression, targeted local administration, splitting of the viral genome, and site specific insertion of retroviral vector are discussed here.

Regeneration of hyaline cartilage by cell-mediated gene therapy using transforming growth factor β1-producing fibroblasts

Transforming growth factor beta (TGF-beta) has been considered as a candidate for gene therapy of orthopedic diseases. The possible application of cell-mediated TGF-beta gene therapy as a new treatment regimen for degenerative arthritis was investigated. In this study, fibroblasts expressing active TGF-beta 1 were injected into the knee joints of rabbits with artificially made cartilage defects to evaluate the feasibility of this therapy for orthopedic diseases. Two to 3 weeks after the injection there was evidence of cartilage regeneration, and at 4 to 6 weeks the cartilage defect was completely filled with newly grown hyaline cartilage. Histological analyses of the regenerated cartilage suggested that it was well integrated with the adjacent normal cartilage at the sides of the defect and that the newly formed tissue was indeed hyaline cartilage. Our findings suggest that cell-mediated TGF-beta 1 gene therapy may be a novel treatment for orthopedic diseases in which hyaline cartilage damage has occurred.

Over-expression of ERT(ESX/ESE-1/ELF3), an ets -related transcription factor, induces endogenous TGF-β type II receptor expression and restores the TGF-β signaling pathway in Hs578t human breast cancer cells

The epithelium-specific transcription factor, ERT/ESX/ESE-1/ELF3, binds to the TGF-β RII promoter in a sequence specific manner and regulates its expression. In this study, we investigated whether ERT could regulate endogenous TGF-β RII expression in Hs578t breast cancer cells. Analyses of the Hs578t parental cell line revealed low RII mRNA expression and resistance to the growth inhibitory effects of TGF-β. Infection of this cell line with a retroviral construct expressing ERT induced higher levels of endogenous RII mRNA expression and protein expression relative to cells infected with chloramphenicol acetyltransferase (CATneo) as a control. Relative to control cells, the ERTneo-expressing Hs578t cells show approximately a 50% reduction in cell growth in the presence of exogenous TGF-β1, as well as a fourfold higher induction of activation in transient transfection assays using the 3TP-luciferase reporter construct. When transplanted into athymic mice, ERT-expressing Hs578t cells showed decreased and delayed tumorigenicity compared with control cells. This data strongly suggests that ERT plays an important role as a transcriptional activator of TGF-β RII expression, and that deregulated ERT expression may play a critical role in rendering Hs578t human breast cancer cells insensitive to TGF-βs growth inhibitory effects.

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.

Development of TGF-β resistance during malignant progression

Transforming growth factor-β (TGF-β) is the prototypical multifunctional cytokine, participating in the regulation of vital cellular activities activities such as proliferation and differentiation as well as a number of basic physiological functions. The effects of TGF-β are critically dependent on the expression and distribution of a family of TGF-β receptors, the TGF-β types I, II, and III. It is now know that a wide variety of human pathology can be caused by aberrant expression and function of these receptors. The coding sequence of the type II receptor (RII) appears to render it uniquely, susceptible to DNA, replication errors in the course of normal cell division. By virtue of its key role in the regulation of cell proliferation, TGF-β RII should be considered as a tumor suppressor gene. High levels of mutation in the TGF-β RII gene have been observed in a wide range of primarily epithelial malignancies, including colon and gastric cancer. It appears likely that mutation of the TGF-β RII gene may be a very critical step in the pathway of carcinogenesis.

Type II collagen and glycosaminoglycan expression induction in primary human chondrocyte by TGF-β1

BackgroundA localized non-surgical delivery of allogeneic human chondrocytes (hChonJ) with irradiated genetically modified chondrocytes (hChonJb#7) expressing transforming growth factor-β1 (TGF-β1) 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.MethodsGenetically modified TGF-β1preprotein was evaluated by monitoring cell proliferation inhibition activity. The effect of modified TGF-β1 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-β1, 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.ResultsThroughout these experiments, the TGF-β1 produced from hChonJb#7 was shown to be equally as active as the recombinant human TGF-β1. Type II collagen and GAG production were induced in hChonJ cells by TGF-β1 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.ConclusionsThis study suggests that the mixture (TG-C) of allogeneic human chondrocytes (hChonJ) and irradiated genetically modified human chondrocytes expressing TGF-β1 (hChonJb#7) attach to the damaged cartilage area to produce type II collagen-GAG matrices by providing a continuous supply of active TGF-β1.

820. Development of Efficient Self-Inactive Retroviral Vectors

Retroviral vectors have been widely used in gene therapy due to their simple genomic structure and high transduction efficiency. We previously reported a construction of Moloney murine sarcoma virus (MoMSV) and Moloney murine leukemia virus (MoMLV) hybrid-based retroviral vectors with significantly improved efficiency of transgene expression after stable incorporation into the host genome. In these vectors, contain no coding sequence for the gag, pol, or env gene that can be used for homologous recombination with sequences introduced in the packaging system for a recombination competent retroviruses (RCR) generation.