CellGenTech Inc.

Chiba, Japan

CellGenTech Inc.

Chiba, Japan
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Kuroda M.,Chiba University | Aso M.,CellGenTech Inc. | Saito Y.,Chiba University
Journal of Diabetes Investigation | Year: 2011

Because of its availability and recent advances in cell biology, adipose tissue is now considered an ideal target site for the preparation of recipient cells and for the transplantation of gene-transduced cells for supplementation of therapeutic proteins. Inherited or acquired serum protein deficiencies are the ideal targets for gene therapy. However, to develop an effective exvivo gene therapy-based protein replacement treatment, the requirements for the recipient cells are different from those for standard gene therapy that is intended to correct the function of the recipient cells themselves. To meet the requirements for such a therapeutic strategy, recent invitro and animal model studies have developed new methods for the preparation, culture, expansion and manipulation of adipose cells using advanced gene transduction methods and transplantation scaffolds. In this short review, we introduce the progress made in novel adipose tissue-based therapeutic strategies for the treatment of protein deficiencies by our group and other investigators, and describe their future applications for diabetes and other metabolic diseases. © 2011 Asian Association for the Study of Diabetes and Blackwell Publishing Asia Pty Ltd.

Aoyagi Y.,Chiba University | Aoyagi Y.,CellGenTech Inc | Kuroda M.,Chiba University | Kuroda M.,CellGenTech Inc | And 8 more authors.
Experimental and Molecular Medicine | Year: 2011

The development of clinically applicable scaffolds is important for the application of cell transplantation in various human diseases. The aims of this study are to evaluate fibrin glue in a novel protein replacement therapy using proliferative adipocytes and to develop a mouse model system to monitor the delivery of the transgene product into the blood and the fate of the transduced cells after transplantation. Proliferative adipocytes from mouse adipose tissue were transduced by a retroviral vector harboring the human lecithin-cholesterol acyltransferase (lcat) gene, and were subcutaneously transplanted into mice combined with fibrin glue. The lcat gene transduction efficiency and the subsequent secretion of the product in mouse adipocytes were enhanced using a protamine concentration of 500 μg/ml. Adipogenesis induction did not significantly affect the lcat gene-transduced cell survival after transplantation. Immunohistochemistry showed the ectopic enzyme production to persist for 28 days in the subcutaneously transplanted gene-transduced adipocytes. The increased viability of transplanted cells with fibrin glue was accompanied with the decrease in apoptotic cell death. The im-munodetectable serum LCAT levels in mice implanted with the fibrin glue were comparable with those observed in mice implanted with Matrigel, indicating that the transplanted lcat gene-transduced adipocytes survived and functioned in the transplanted spaces with fibrin glue as well as with Matrigel for 28 days. Thus, this in vivo system using fibrin is expected to serve as a good model to further improve the transplanted cell/scaffold conditions for the stable and durable cell-based replacement of defective proteins in patients with LCAT deficiency.

Aoyagi Y.,Chiba University | Kuroda M.,Chiba University | Asada S.,Chiba University | Tanaka S.,CellGenTech Inc. | And 7 more authors.
Experimental Cell Research | Year: 2012

Adipose tissue is expected to provide a source of cells for protein replacement therapies via auto-transplantation. However, the conditioning of the environment surrounding the transplanted adipocytes for their long-term survival and protein secretion properties has not been established. We have recently developed a preparation procedure for preadipocytes, ceiling culture-derived proliferative adipocytes (ccdPAs), as a therapeutic gene vehicle suitable for stable gene product secretion. We herein report the results of our evaluation of using fibrin glue as a scaffold for the transplanted ccdPAs for the expression of a transduced gene in a three-dimensional culture system. The ccdPAs secreted the functional protein translated from an exogenously transduced gene, as well as physiological adipocyte proteins, and the long viability of ccdPAs (up to 84. days) was dependent on the fibrinogen concentrations. The ccdPAs spontaneously accumulated lipid droplets, and their expression levels of the transduced exogenous gene with its product were maintained for at least 56. days. The fibrinogen concentration modified the adipogenic differentiation of ccdPAs and their exogenous gene expression levels, and the levels of exogenously transduced gene expression at the different fibrinogen concentrations were dependent on the extent of adipogenic differentiation in the gel. These results indicate that fibrin glue helps to maintain the high adipogenic potential of cultured adipocytes after passaging in a 3D culture system, and suggests that once they are successfully implanted at the transplantation site, the cells exhibit increased expression of the transduced gene with adipogenic differentiation. © 2011 Elsevier Inc.

Asada S.,Chiba University | Asada S.,CellGenTech Inc. | Kuroda M.,Chiba University | Kuroda M.,CellGenTech Inc. | And 9 more authors.
Molecular Genetics and Metabolism | Year: 2011

We report the in vitro efficacy of recombinant LCAT produced by lcat gene-transduced proliferative adipocytes (ccdPA/. lcat), which has been developed for enzyme replacement therapy. ApoA-I-specific immunodetection in combination with 1D and 2D gel electrophoreses showed that the disturbed high-density lipoprotein subpopulation profile was clearly ameliorated by the in vitro incubation with ccdPA/. lcat-derived recombinant LCAT. Thus, these results using ccdPA/. lcat strongly suggest the cell implantation could contribute the enzyme replacement for the patients with LCAT deficiency. © 2010 Elsevier Inc.

Kuroda M.,Chiba University | Kuroda M.,CellGenTech Inc | Aoyagi Y.,Chiba University | Aoyagi Y.,CellGenTech Inc | And 12 more authors.
Open Gene Therapy Journal | Year: 2011

Human proliferative adipocytes propagated via ceiling culture technique from subcutaneous fat tissue (designated as ccdPA) were herein evaluated for their potential as a recipient for retroviral vector-mediated gene transduction of a therapeutic protein delivery. Exposure to the ZsGreen-expressing vector supernatant using a cell preparation generated by a 7-day ceiling culture induced a 40-50% transduction efficiency, with less than two integrated copies of viral genome per cell on average. The lcat gene-transduced human ccdPA secreted functional LCAT protein, correlating with the integrated copy number of vector genome. The gene-transduced cells could be expanded up to nearly 10 12 cells from 1 g of fat tissue within one month after fat tissue preparation. The cells also maintained the potential to differentiate into adipocytes in vitro. The presence of human LCAT protein in serum was immunologically identified upon transplantation of lcatexpressing ccdPA into the adipose tissue of immune-deficient mice. These results indicated that human ccdPA has a novel therapeutic potential for LCAT-deficient patients. The clinical application in combination with cell transplantation shed a light on a development of a life-long protein replacement therapy for LCAT-deficient patients. © Kuroda et al.

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