Teruhide Yamaguchi

Importance of Neonatal FcR in Regulating the Serum Half-Life of Therapeutic Proteins Containing the Fc Domain of Human IgG1: A Comparative Study of the Affinity of Monoclonal Antibodies and Fc-Fusion Proteins to Human Neonatal FcR

The neonatal FcR (FcRn) binds to the Fc domain of IgG at acidic pH in the endosome and protects IgG from degradation, thereby contributing to the long serum half-life of IgG. To date, more than 20 mAb products and 5 Fc-fusion protein products have received marketing authorization approval in the United States, the European Union, or Japan. Many of these therapeutic proteins have the Fc domain of human IgG1; however, the serum half-lives differ in each protein. To elucidate the role of FcRn in the pharmacokinetics of Fc domain-containing therapeutic proteins, we evaluated the affinity of the clinically used human, humanized, chimeric, or mouse mAbs and Fc-fusion proteins to recombinant human FcRn by surface plasmon resonance analysis. The affinities of these therapeutic proteins to FcRn were found to be closely correlated with the serum half-lives reported from clinical studies, suggesting the important role of FcRn in regulating their serum half-lives. The relatively short serum half-life of Fc-fusion proteins was thought to arise from the low affinity to FcRn. The existence of some mAbs having high affinity to FcRn and a short serum half-life, however, suggested the involvement of other critical factor(s) in determining the serum half-life of such Abs. We further investigated the reason for the relatively low affinity of Fc-fusion proteins to FcRn and suggested the possibility that the receptor domain of Fc-fusion protein influences the structural environment of the FcRn binding region but not of the FcγRI binding region of the Fc domain.

Development of Defective and Persistent Sendai Virus Vector A UNIQUE GENE DELIVERY/EXPRESSION SYSTEM IDEAL FOR CELL REPROGRAMMING

The ectopic expression of transcription factors can reprogram differentiated tissue cells into induced pluripotent stem cells. However, this is a slow and inefficient process, depending on the simultaneous delivery of multiple genes encoding essential reprogramming factors and on their sustained expression in target cells. Moreover, once cell reprogramming is accomplished, these exogenous reprogramming factors should be replaced with their endogenous counterparts for establishing autoregulated pluripotency. Complete and designed removal of the exogenous genes from the reprogrammed cells would be an ideal option for satisfying this latter requisite as well as for minimizing the risk of malignant cell transformation. However, no single gene delivery/expression system has ever been equipped with these contradictory characteristics. Here we report the development of a novel replication-defective and persistent Sendai virus (SeVdp) vector based on a noncytopathic variant virus, which fulfills all of these requirements for cell reprogramming. The SeVdp vector could accommodate up to four exogenous genes, deliver them efficiently into various mammalian cells (including primary tissue cells and human hematopoietic stem cells) and express them stably in the cytoplasm at a prefixed balance. Furthermore, interfering with viral transcription/replication using siRNA could erase the genomic RNA of SeVdp vector from the target cells quickly and thoroughly. A SeVdp vector installed with Oct4/Sox2/Klf4/c-Myc could reprogram mouse primary fibroblasts quite efficiently; ∼1% of the cells were reprogrammed to Nanog-positive induced pluripotent stem cells without chromosomal gene integration. Thus, this SeVdp vector has potential as a tool for advanced cell reprogramming and for stem cell research.

Characterization of in vitro and in vivo gene transfer properties of adenovirus serotype 35 vector

We have recently developed a replication-defective, recombinant adenovirus (Ad) vector composed of the whole Ad serotype 35 (Ad35), a member of subgroup B. We describe herein the in vitro and in vivo gene transfer properties of Ad35 vector in comparison with Ad serotype 5 (Ad5) and the Ad5F35 vector, which is a fiber-substituted Ad5 vector containing Ad35 fiber proteins. In vitro, Ad35 vector efficiently transduced not only human CAR-positive cells but also CAR-negative cells. Following intravenous administration into mice, both Ad5 and Ad35 vectors were rapidly cleared from the bloodstream with a half-life of approximately 3 min. Ad5 vector-mediated transgene expression predominantly occurred in liver parenchymal cells, although the Ad5 vector was delivered to both liver parenchymal and nonparenchymal cells. In contrast, Ad35 vector was efficiently taken up by liver nonparenchymal cells and mediated transduction efficiency in the liver on a level 4 log orders lower than the Ad5 vector. These findings demonstrate that Ad35 vector is an attractive vehicle for gene transfer into human cells, while the biodistribution profile of Ad35 vector in mice is much different from that of the Ad5 vector.

Reduction of Natural Adenovirus Tropism to Mouse Liver by Fiber-Shaft Exchange in Combination with both CAR- and αv Integrin-Binding Ablation

ABSTRACT The primary receptor, the coxsackievirus and adenovirus receptor (CAR), and the secondary receptor, αv integrins, are the tropism determinants of adenovirus (Ad) type 5. Inhibition of the interaction of both the fiber with CAR and the penton base with the αv integrin appears to be crucial to the development of targeted Ad vectors, which specifically transduce a given cell population. In this study, we developed Ad vectors with ablation of both CAR and αv integrin binding by mutating the fiber knob and the RGD motif of the penton base. We also replaced the fiber shaft domain with that derived from Ad type 35. High transduction efficiency in the mouse liver was suppressed approximately 130- to 270-fold by intravenous administration of the double-mutant Ad vectors, which mutated two domains each of the fiber knob and shaft and the RGD motif of the penton base compared with those of conventional Ad vectors (type 5). Most significantly, the triple-mutant Ad vector containing the fiber knob with ablation of CAR binding ability, the fiber shaft of Ad type 35, and the penton base with a deletion of the RGD motif mediated a >30,000-fold lower level of mouse liver transduction than the conventional Ad vectors. This triple-mutant Ad vector also mediated reduced transduction in other organs (the spleen, kidney, heart, and lung). Viral DNA analysis showed that systemically delivered triple-mutant Ad vector was primarily taken up by liver nonparenchymal cells and that most viral DNAs were easily degraded, resulting in little gene expression in the liver. These results suggest that the fiber knob, fiber shaft, and RGD motif of the penton base each plays an important role in Ad vector-mediated transduction to the mouse liver and that the triple-mutant Ad vector exhibits little tropism to any organs and appears to be a fundamental vector for targeted Ad vectors.

CD31 (PECAM-1)-bright cells derived from AC133-positive cells in human peripheral blood as endothelial-precursor cells

To clarify the process of endothelial differentiation, we isolated AC133+ cells and induced the in vitro differentiation of these cells into endothelial cells. AC133+ cells efficiently differentiated into endothelial cells when the cells were cultured on fibronectin‐coated dishes in the presence of vascular endothelial growth factor. Time‐course analysis of the alteration of endothelial markers on cultured AC133+ cells revealed that the expression of CD31 (PECAM‐1) on AC133+ cells was the earliest marker among all of the tested markers. Based on the hypothesis that CD31 is an early indicator during the endothelial differentiation, we examined the relationship between CD31 expression and the ability to differentiate into endothelial cells in cells derived from AC133+ cells. CD31‐bright cells, which were sorted from cultured AC133+ cells, differentiated more efficiently into endothelial cells than had CD31‐positive or CD31‐negative cells, suggesting that CD31‐bright cells may be precursor cells for endothelial cells. In the present study, we identified CD31+ cells derived from cultured AC133+ cells that are able to differentiate to endothelial cells as precursor cells.

Adenovirus Vector-Mediated Doxycycline-Inducible RNA Interference

RNA interference (RNAi) is a powerful tool for the knockdown of gene expression. Here, we report on the development of an adenovirus (Ad) vector-mediated doxycycline (Dox)-inducible small interfering RNA (siRNA) expression system. We used this siRNA system to control the expression of p53 and c-Myc in human cancer cells. Coinfection of Ad vectors containing the siRNA expression system under the control of the Dox-inducible H1 promoter and Ad vectors expressing a tetracycline repressor inhibited the expression levels of p53 and c-Myc in a dose-dependent manner with both Dox and viral dose. Regulated silencing of p53 and c-Myc expression was obtained. Because an Ad vector-mediated inducible RNAi system can efficiently transduce a variety of cell types in vitro and in vivo, and the degree of loss of gene expression can be modulated according to the dose of Dox, this expression system should be a useful tool for both basic research on the analysis of gene function and therapeutic applications of RNAi.

Thyroid Hormone Targets Matrix Gla Protein Gene Associated With Vascular Smooth Muscle Calcification

Thyroid hormones have marked cardiovascular effects in vivo. However, their direct effects on vascular smooth muscle cells have been unclear. Because thyroid hormones play critical roles in bone remodeling, we hypothesized that they are also associated with vascular smooth muscle calcification, one of the pathological features of vascular sclerosis. To test this hypothesis, we examined the effects of 3′,3,5-triiodo-l-thyronine (T3) on the expression of calcification-associated genes in rat aortic smooth muscle cells (RAOSMCs). Quantitative RT-PCRs revealed that a physiological concentration of T3 (15 pmol/L free T3) increased mRNA level of matrix Gla protein (MGP), which acts as a potent inhibitor of vascular calcification in vivo, by 3-fold in RAOSMCs, as well as in cultured human coronary artery smooth muscle cells. In RAOSMCs transiently transfected with a luciferase reporter gene driven by the MGP promoter, T3 significantly stimulated luciferase activity. In addition, RNA interference against thyroid hormone receptor-&agr; gene diminished the effect of T3 on MGP expression. Aortic smooth muscle tissues from methimazole-induced hypothyroid rats (400 mg/L drinking water; 4 weeks) also showed a 68% decrease in the MGP mRNA level, as well as a 33% increase in calcium content compared with that from the control euthyroid animals, whereas hyperthyroidism (0.2 mg T3/kg IP; 10 days) upregulated MGP mRNA by 4.5-fold and reduced calcium content by 11%. Our findings suggest that a physiological concentration of thyroid hormone directly facilitates MGP gene expression in smooth muscle cells via thyroid hormone nuclear receptors, leading to prevention of vascular calcification in vivo.

Optimization of adenovirus serotype 35 vectors for efficient transduction in human hematopoietic progenitors: comparison of promoter activities.

Adenoviral gene transfer to hematopoietic stem cells (HSCs)/progenitors would provide a new approach to the treatment of hematopoietic diseases and study of the hematopoietic system. We have previously reported that an adenovirus (Ad) vector composed of whole Ad serotype 35 (Ad35), which belongs to subgroup B, shows efficient gene transfer into human bone marrow CD34+ cells. However, Ad35 vector-mediated transduction into human HSCs/progenitors has not yet been fully optimized. In the present study, we have systematically examined promoter activity in the context of Ad35 vectors in human bone marrow CD34+ cells and primitive CD34+ subsets to optimize the transduction efficiency in human hematopoietic stem/progenitor cells. In the first of the transduction experiments, the improved in vitro ligation method was applied to Ad35 vector construction to allow for simple and efficient production of an E1/E3-deleted Ad35 vector. Using this method, we constructed a series of Ad35 vectors encoding the enhanced green fluorescence protein (GFP) under the control of a variety of strong viral and cellular promoters. Of the six types of promoters tested, significantly higher transduction efficiencies were achieved with the human elongation factor 1α promoter (EF1α promoter), the human cytomegalovirus (CMV) immediate-early 1 gene enhancer/β-actin promoter with β-actin intron (CA promoter), and the CMV promoter/enhancer with the largest intron of CMV (intron A) (CMVi promoter) in the human CD34+ cells and the immature subsets (CD34+CD38low/− and CD34+AC133+ subsets). In particular, the CA promoter was found to allow for the highest transduction efficiencies in both the whole human CD34+ cells and the immature hematopoietic subsets. Furthermore, the CA promoter-mediated GFP-expressing cells differentiated into progenitor cells of all lineages. These results indicate the construction of an optimized Ad35 vector backbone for efficient transduction into HSCs/progenitors.

Stable Transfectants of Smooth Muscle Cell Line Lacking the Expression of Myosin Light Chain Kinase and Their Characterization with Respect to the Actomyosin System

We constructed a plasmid vector having a 1.4-kilobase pair insert of myosin light chain kinase (MLCK) cDNA in an antisense direction to express antisense mRNA. The construct was then transfected to SM3, a cell line from vascular smooth muscle cells, producing a few stable transfectants. The down-regulation of MLCK expression in the transfectants was confirmed by both Northern and Western blots. The control SM3 showed chemotaxic motility to platelet-derived growth factor-BB, which was supported by lamellipodia. However, the transfectants showed neither chemotaxic motility nor developed lamellipodia, indicating the essential role of MLCK in the motility. The specificity for the targeting was assessed by a few tests including the rescue experiment. Despite this importance of MLCK, platelet-derived growth factor-BB failed to induce MLC20 phosphorylation in not only the transfectants but also in SM3. The mode in which MLCK was involved in the development of membrane ruffling is discussed with special reference to the novel property of MLCK that stimulates the ATPase activity of smooth muscle myosin without phosphorylating its light chain (Ye, L.-H., Kishi, H., Nakamura, A., Okagaki, T., Tanaka, T., Oiwa, K., and Kohama, K. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6666–6671).

Herbimycin A inhibits both dephosphorylation and translocation of cofilin induced by opsonized zymosan in macrophagelike U937 cells

We previously reported that a 21‐kDa phosphoprotein may play an important role in superoxide production through dephosphorylation by neutrophillike differentiated HL‐60 cells (Suzuki et al., 1995, Biochim Biophys Acta 1266:261–267). The phosphoprotein was identified as cofilin, an actin‐binding protein, and the activation‐induced changes in its intracellular distribution have been described elsewhere (Suzuki et al., 1995, J Biol Chem 270:19551–19556). However, the physiologic roles of cofilin in phagocytes remain to be established, and the regulatory mechanisms for dephosphorylation and translocation of cofilin are unknown. In the present study, we investigated the roles of cofilin in the opsonized zymosan (OZ)‐activated macrophagelike U937 cells by using herbimycin A, an inhibitor for protein tyrosine kinase. In the individual adherent phagocytes, OZ induced many events: 1) production of superoxide, 2) phagocytosis of the insoluble particles OZ, 3) dephosphorylation of cofilin, 4) translocation of cofilin from cytosol to plasma membrane regions, 5) decrease in intracellular pH from 7.4 to aprroximately 6.8, and 6) rapid and transient increase in filamentous actin at the cell periphery. All of these events were inhibited or reduced significantly by herbimycin A. OZ increased phosphorylation of tyrosine in 110‐, 50‐, 34‐, and 29‐kDa proteins, whereas herbimycin A inhibited it. These results suggest that tyrosine kinase plays an essential role upstream of these events through phosphorylation of such proteins. Furthermore, microinjection of anti‐cofilin antibody to the differentiated U937 cells caused inhibition of the phagocytosis. These results suggest that cofilin plays critical roles in phagocytic functions through changes in cytoskeletal organization. J. Cell. Physiol. 180:345–354, 1999.