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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.

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-β1)-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-β1 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.

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.

Background A 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. Methods Genetically 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. Results Throughout 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. Conclusions This 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.

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.

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.

Retroviral vectors have been widely used in gene therapy due to their simple genomic structure and high transduction efficiency. We report 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, the residual gag gene coding sequence located in the extended region of packaging signal was removed. These vectors, therefore, 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 recombinant competent retrovirus (RCR) generation. A strong splice acceptor site obtained from the exon/intron junction of either the chimpanzee EF1-alpha gene or the human CMV major immediate early gene was placed downstream of the MoMSV packaging signal (Psi), significantly improving the efficiency of transgene expression. The 5' LTR U3 sequence was replaced with an extended human CMV major immediate early gene enhancer/promoter for a strong expression of full-length messages from the viral backbone, helping to maintain high levels of viral titer. These newly developed retroviral vectors should facilitate RCR-free gene transfer with significantly improved efficacy in clinical gene therapy trials.

A series of 5-fluorocytosine derivatives, 5'-deoxy-N-alkyloxycarbonyl-5-fluorocytosine-5'-carboxylic acid 6, were synthesized and evaluated for their antitumor activity.

A lipase gene, lipK , and a lipase modulator gene, limK , of Pseudomonas sp. strain KFCC 10818 have been cloned, sequenced, and expressed in Escherichia coli . The limK gene is located immediately downstream of the lipK gene. Enzymatically active lipase was produced only in the presence of the limK gene. The effect of the lipase modulator LimK on the expression of active lipase was similar to those of the Pseudomonas subfamily I.1 and I.2 lipase-specific foldases (Lifs). The deduced amino acid sequence of LimK shares low homology (17 to 19%) with the known Pseudomonas Lifs, suggesting that Pseudomonas sp. strain KFCC 10818 is only distantly related to the subfamily I.1 and I.2 Pseudomonas species. Surprisingly, a lipase variant that does not require LimK for its correct folding was isolated in the study to investigate the functional interaction between LipK and LimK. When expressed in the absence of LimK, the P112Q variant of LipK formed an active enzyme and displayed 63% of the activity of wild-type LipK expressed in the presence of LimK. These results suggest that the Pro 112 residue of LipK is involved in a key step of lipase folding. We expect that the novel finding of this study may contribute to future research on efficient expression or refolding of industrially important lipases and on the mechanism of lipase folding.

Pestalotia heterocornis was isolated from soil collected in yew forest and was shown to produce paclitaxel in semisynthetic liquid media. The presence of paclitaxel in the fungal extract was confirmed by FAB mass spectrometry and NMR spectroscopy. The maximum yield of paclitaxel was 31 mg per liter. Optimal paclitaxel production occurred after 5-7 days in a 20-liter scale fermentation at 23 degrees C. These results indicate that P. heterocornis is an excellent candidate for consideration in fermentation technology. Copyright 1999 John Wiley & Sons, Inc.

Background aims. TissueGene-C (TG-C) represents a cell-mediated gene therapy for localized delivery of allogeneic chondrocytes expressing transforming growth factor (TGF)- β 1 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- β 1-expressing chondrocytes (hChonJb#7) in order to help fi ll in the defect and as target cells for the actions of the expressed TGF- β 1. 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 nonoperative 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 effi cacy; however, patient numbers were not suffi cient to determine statistical signifi cance. Conclusions. Overall, there were no signifi cant 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 effi cacy of TG-C.

A series of 5-fluorocytosine derivatives, 5'-deoxy-N-alkyloxycarbonyl-5-fluorocytosine-5'-carboxylic acid 6, were synthesized and evaluated for their antitumor activity.

The recent approval of gene therapy products in Europe and Asia and the upsurge of gene therapy products in clinical trials signal the rebound of this technology not only for many orphan diseases but also for non­life threatening diseases. Following the success of induced pluripotent stem (iPS) cells in research, other modified ex vivo gene therapies are also knocking on the door of the clinic. Historically, gene therapy has experienced many ups and downs and still faces many challenges. REVIEW Submit a Manuscript: http://www.wjgnet.com/esps/ Help Desk: http://www.wjgnet.com/esps/helpdesk.aspx DOI: 10.5496/wjmg.v5.i1.1 World J Med Genet 2015 February 27; 5(1): 1-13 ISSN 2220-3184 (online) © 2015 Baishideng Publishing Group Inc. All rights reserved. World Journal of Medical Genetics W J M G February 27, 2015|Volume 5|Issue 1| WJMG|www.wjgnet.com 1 clinic. This is the right time for the physicians to have knowledge of this evolving technology that already reached the bedside. Noh MJ, Copeland O, O’Mara M, Lee KH. Cell mediated gene therapy: A guide for doctors in the clinic. World J Med Genet 2015; 5(1): 1-13 Available from: URL: http://www.wjgnet. com/2220-3184/full/v5/i1/1.htm DOI: http://dx.doi.org/10.5496/ wjmg.v5.i1.1

Bone fractures such as pelvic fracture or sternal fracture could be a serious, life-threatening event requiring emergency medical care and lengthy rehabilitation. We have selected BMP-2 as a possible answer for the treatment of severe bone fractures. BMP-2 is known as the powerful inducers of osteogenic differentiation. Due to the short half-life of BMPs in vivo, continuous production of BMPs by gene therapy could be one of the good methods for the successful regeneration of a bone defect. We have tested ex-vivo approach for the regeneration of critical sized bone defect. We investigated whether xenogeneic BMP-2 producing cells had the ability to regenerate cortical bone at the diaphysis of a long bone in a rabbit without using a scaffold. We have constructed BMP-2 producing NIH3T3 and human foreskin fibroblasts. The expression levels of BMP2 were confirmed by ELISA. Osteogenic activities were determined by alkaline phaosphatase assay. Radiographic analysis was performed at 1, 2, 3, 4, 5 and 6 weeks after injection of the cells. Radiological union was observed beginning at 3 weeks after injection and complete thickness cortical bone was observed at 6 weeks after injection. The tibia was harvested at 6 weeks and histological examination was performed. The regenerated bone was almost identical to the normal cortical bone. Fibrous tissue was formed at the end of normal bone without the evidence of bone formation in the control.Immunohistochemical and histological staining was used to analyze the regenerated tissue. At one week after injection, inflammatory cells were observed. At four weeks after injection, cortical was similar to normal bone. At 6 weeks after injection, the structure of regenerated bone tissue was almost identical to that of the normal bone tissue.Osteocalcin expression was observed from regenerated bone osteocytes. Our results suggest that bone regeneration with BMP-2 could have induced the osteoblastic transformation of the pre-osteogenic cells which were recruited into the defect area. These results show that ex vivo gene therapy using BMP-2 producing fibroblasts could be an effective tool for the promoting of bone formation.

We have identified a transcription termination site in the 3' flanking region of the mouse nkx-1.2 gene. A downstream transcription regulatory element in the mouse nkx-1.2 gene was characterized by transferring its 3'-fragment into a chloramphenicol acetyl transferase (CAT) expression vector. Analysis of recombinant plasmids transfected into mouse NIH3T3 cells by CAT assay showed the possible region of regulation. There were two direct repeat structures containing poly(dG-dT) x poly(dC-dA) sequences (GT repeats) in this region. The precise location of transcription termination was mapped by nuclease S1 analysis of the transcripts from recombinant plasmids transfected into COSM6 cells. It was approximately 20 nucleotides upstream of the first GT repeat within the 5' sequences of the first element of the two direct repeats. Gel mobility shift assay and footprinting analysis demonstrated that nuclear DNA binding proteins bound specifically to the sequences where the termination occurred as well as the other sequences in the second element of the direct repeats. Southwestern analysis showed that 90-, 54-, 36- and 15-kDa nuclear proteins bound to the region of the termination. It is possible that one or more of those proteins are involved in blocking the elongation of the mouse nkx-1.2 gene transcript and then result in termination.

BamHI endonuclease and BamHI methylase were used to investigate their specific interaction with the common recognition sequence, GGATCC. Five derivatives of the oligonucleotide, GACGGATCCGTC, containing a variety of single-base analog substitutions within the hexameric recognition core were synthesized. Steady-state kinetics for the reaction of the endonuclease and the methylase showed that both enzymes recognize the sequences by contacting with functional groups exposed in both major and minor grooves of the site but in different ways. Removal or substitution of the 5-methyl group in thymidine blocked the endonuclease reaction completely but still allowed the methylase reaction with less efficiency. The data also showed that the methylase made a critical minor groove contact with the 2-amino group of the first G but the endonuclease did with that of the second G.

Top of pageAbstract Gene therapy approaches to a variety of diseases will require the therapeutic gene to be expressed in a regulated fashion so that the precise amount of the gene product can be delivered in the appropriate tissues at the desired time and for the desired duration. This may be particularly important when the therapeutic gene is used for controlled regeneration of tissues. We have developed the RheoSwitch|[reg]| Therapeutic System (RTS) that can be precisely controlled by an orally active synthetic small molecule ligand (Activator Drug) for regulated expression of therapeutic genes. The RTS consists of two component proteins that heterodimerize to create a functional inducible transcription factor complex. The ligand- binding component is the DEF domain of a mutant ecdysone receptor that is fused to a Gal4 DNA binding domain. The second component is the EF domain of a chimeric RXR that is fused to the VP16 activation domain. These proteins can be constitutively expressed under a ubiquitous or tissue-specific promoter, and, in the presence of a potent ligand, can induce in vivo gene expression from a responsive promoter. The RTS components have been assembled into single vector systems that are adapted for delivery by retroviral as well as other viral vector systems. The RTS has been tested for the expression of many therapeutic proteins including cytokines and growth factors. We now demonstrate the adaptation of the RTS in retroviral vectors and the potential for the repair of damaged skeletal tissue in a variety of degenerative diseases by controlled delivery of BMP2 from fibroblast cells that are transduced by retroviral vectors carrying the BMP-2 gene under the RTS. As safety is becoming paramount in the development of cell and gene therapies, having the ability to control the gene expression in-vivo adds a new level of control to product development. Moreover, regenerative processes such as osteogenesis often require differential expression of genes for varying times. This poster will present the preliminary data of the integration of BMP-2 and the RTS system, and discuss some of our future plans in the development of a regulated system for expression of growth factors in bone healing applications.