Masamichi Kamihira

High-Level Expression of Single-Chain Fv-Fc Fusion Protein in Serum and Egg White of Genetically Manipulated Chickens by Using a Retroviral Vector

ABSTRACT We report here the generation of transgenic chickens using a retroviral vector for the production of recombinant proteins. It was found that the transgene expression was suppressed when a Moloney murine leukemia virus-based retroviral vector was injected into chicken embryos at the blastodermal stage. When a concentrated viral solution was injected into the heart of developing embryos after 50 to 60 h of incubation, transgene expression was observed throughout the embryo, including the gonads. For practical production, a retroviral vector encoding an expression cassette of antiprion single-chain Fv fused with the Fc region of human immunoglobulin G1 (scFv-Fc) was injected into chicken embryos. The birds that hatched stably produced scFv-Fc in their serum and eggs at high levels (∼5.6 mg/ml). We obtained transgenic progeny from a transgenic chicken generated with this procedure. The transgene was stably integrated into the chromosomes of transgenic progeny. The transgenic progeny also expressed scFv-Fc in the serum and eggs.

Genetically engineered angiogenic cell sheets using magnetic force-based gene delivery and tissue fabrication techniques

A major limitation in tissue engineering is the insufficient formation of blood vessels in implanted tissues, resulting in reduced cell density and graft size. We report here the fabrication of angiogenic cell sheets using a combination of two magnetic force-based techniques which use magnetite cationic liposomes (MCLs), magnetofection and magnetic cell accumulation. A retroviral vector encoding an expression cassette of vascular endothelial growth factor (VEGF) was labeled with MCLs, to magnetically attract the particles onto a monolayer of mouse myoblast C2C12 cells, for gene delivery. MCL-mediated infection increased transduction efficiency by 6.7-fold compared with the conventional method. During the fabrication of the tissue constructs, MCL-labeled cells were accumulated in the presence of a magnetic field to promote the spontaneous formation of a multilayered cell sheet. VEGF gene-engineered C2C12 (C2C12/VEGF) cell sheets, constructed using both magnetic force-based techniques, were subcutaneously transplanted into nude mice. Histological analyses revealed that on day 14 the C2C12/VEGF cell sheet grafts had produced thick tissues, with a high-cell density, and promoted vascularization. This suggests that the method described here represents a powerful strategy in tissue engineering.

Development of hybrid viral vectors for gene therapy

Adenoviral, retroviral/lentiviral, adeno-associated viral, and herpesviral vectors are the major viral vectors used in gene therapy. Compared with non-viral methods, viruses are highly-evolved, natural delivery agents for genetic materials. Despite their remarkable transduction efficiency, both clinical trials and laboratory experiments have suggested that viral vectors have inherent shortcomings for gene therapy, including limited loading capacity, immunogenicity, genotoxicity, and failure to support long-term adequate transgenic expression. One of the key issues in viral gene therapy is the state of the delivered genetic material in transduced cells. To address genotoxicity and improve the therapeutic transgene expression profile, construction of hybrid vectors have recently been developed. By adding new abilities or replacing certain undesirable elements, novel hybrid viral vectors are expected to outperform their conventional counterparts with improved safety and enhanced therapeutic efficacy. This review provides a comprehensive summary of current achievements in hybrid viral vector development and their impact on the field of gene therapy.

Preparation of artificial skeletal muscle tissues by a magnetic force-based tissue engineering technique

Artificial muscle tissues composed of mouse myoblast C2C12 cells were prepared using a magnetic force-based tissue engineering technique. C2C12 cells labeled with magnetite nanoparticles were seeded into the wells of 24-well ultralow-attachment culture plates. When a magnet was positioned underneath each plate, the cells accumulated evenly on the culture surface and formed multilayered cell sheets. Since the shapes of artificial tissue constructs can be controlled by magnetic force, cellular string-like assemblies were formed by using a linear magnetic field concentrator with a magnet. However, the resulting cellular sheets and strings shrank considerably and did not retain their shapes during additional culture periods for myogenic differentiation. On the other hand, when a silicone plug was positioned at the center of the well during the fabrication of a cell sheet, the cell sheet shrank drastically and formed a ring-like assembly around the plug. A histological examination revealed that the cells in the cellular ring were highly oriented in the direction of the circumference by the tension generated within the structure. Individual cellular rings were hooked around two pins separated by 10 mm, and successfully cultured for 6 d without breakage. After a 6-d culture in differentiation medium, the C2C12 cells differentiated to form myogenin-positive multinucleated myotubes. Highly dense and oriented skeletal muscle tissues were obtained using this technique, suggesting that this procedure may represent a novel strategy for muscle tissue engineering.

Fabrication of complex three-dimensional tissue architectures using a magnetic force-based cell patterning technique

We describe the fabrication of three-dimensional tissue constructs using a magnetic force-based tissue engineering technique, in which cellular organization is controlled by magnetic force. Target cells were labeled with magnetite cationic liposomes (MCLs) so that the MCL-labeled cells could be manipulated by applying a magnetic field. Line patterning of human umbilical vein endothelial cells (HUVECs) labeled with MCLs was successfully created on monolayer cells or skin tissues using a magnetic concentrator device. Multilayered cell sheets were also inducible on a culture surface by accumulating MCL-labeled cells under a uniform magnetic force. Based on these results, we attempted to construct a complex multilayered myoblast C2C12 cell sheet. Here, patterned HUVECs were embedded by alternating the processes of magnetic accumulation of C2C12 cells for cell layer formation and magnetic patterning of HUVECs on the cell layers. This technique may be applicable for the fabrication of complex tissue architectures required in tissue engineering.

Induction of functional tissue-engineered skeletal muscle constructs by defined electrical stimulation

Electrical impulses are necessary for proper in vivo skeletal muscle development. To fabricate functional skeletal muscle tissues in vitro, recapitulation of the in vivo niche, including physical stimuli, is crucial. Here, we report a technique to engineer skeletal muscle tissues in vitro by electrical pulse stimulation (EPS). Electrically excitable tissue-engineered skeletal muscle constructs were stimulated with continuous electrical pulses of 0.3 V/mm amplitude, 4 ms width, and 1 Hz frequency, resulting in a 4.5-fold increase in force at day 14. In myogenic differentiation culture, the percentage of peak twitch force (%Pt) was determined as the load on the tissue constructs during the artificial exercise induced by continuous EPS. We optimized the stimulation protocol, wherein the tissues were first subjected to 24.5%Pt, which was increased to 50–60%Pt as the tissues developed. This technique may be a useful approach to fabricate tissue-engineered functional skeletal muscle constructs.

Production of human erythropoietin by chimeric chickens

The use of transgenic avian allows cost effective and safe production of pharmaceutical proteins. Here, we report the successful production of chimeric chickens expressing human erythropoietin (hEpo) using a high-titer retroviral vector. The hEpo expressed by transgenic hens accumulated abundantly in egg white and had N- and O-linked carbohydrates. While attachment of terminal sialic acid and galactose was incomplete, portions of N- and O-linked carbohydrates were present. In vitro biological activity of egg white-hEpo was comparable to that produced by recombinant CHO cells.

Production of chimeric monoclonal antibodies by genetically manipulated chickens

Genetically manipulated chickens producing chimeric monoclonal antibodies were generated by injecting retroviral vectors encoding genes for the heavy and light chains of antibodies into developing embryos. The transgene was detected in all chickens that hatched, and they stably produced the chimeric antibodies in their serum. After sexual maturation, the antibodies were also produced in eggs laid by the manipulated hens. The stable antibody production was observed both in egg white and yolk throughout the breeding period. The chimeric antibodies produced by the chickens were properly assembled and exhibited antigen-binding activities. Furthermore, we characterized the structures of the N-linked oligosaccharide chains added to the Fc-region of the recombinant antibodies produced in the serum, egg white and yolk of the chickens.

Homologous recombination-independent large gene cassette knock-in in CHO cells using TALEN and MMEJ-directed donor plasmids

Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins.

Spheroid Formation of Hepatocytes Using Synthetic Polymer

It is known that hepatocytes show the different morphology depending on the surface condition of cultural substratum. The cells form a floating cell-aggregate called spheroid on proteoglycan-coated or positively charged dishes. The liver cell functions are generally high and maintained for the long-term in the spheroid culture. Since spheroid morphology is preferable for the construction of a bioartificial liver, it is important to develop an effective method of preparing spheroids. In this regard, we examined a preparation method of functional spheroid-like cell-aggregates, in which a synthetic polymer, Eudragit was added to culture medium for inducing liver cell-aggregation. The cell-cell attachment of the aggregate was loose at the beginning of the culture, but it became tight and spheroids were formed 2-3 days after inoculation. When 0.1% Eudragit was added to the medium, the liver functions such as albumin secretion, ammonia removal and urea synthesis were enhanced compared with monolayer and conventional spheroid cultures. The spheroid formation was also performed with suspension culture in a spinner flask. Approximately 80% of the cells inoculated formed spheroids by the addition of the polymer. Moreover, the polymer showed a protective effect from cell damage by agitation. Since this procedure does not require surface for cell attachment, a large amount of spheroids can be prepared in suspension culture.