Tetsuji Hosono

A simplified system for constructing recombinant adenoviral vectors containing heterologous peptides in the HI loop of their fiber knob

The use of recombinant adenovirus (Ad) vectors containing genetically modified capsid proteins is an attractive strategy for achieving targeted gene transfer. The HI loop of the fiber knob is a promising candidate location for the incorporation of foreign ligands for achieving this goal. However, the method of constructing an Ad vector containing a foreign ligand in the HI loop of the fiber knob has proved difficult. In this study, we developed a simple system to construct fiber-modified vectors. To do this, a vector plasmid containing a complete E1/E3-deleted Ad type 5 genome and a unique Csp45I and/or ClaI site between positions 32679 and 32680 of the Ad genome (residues threonine-546 and proline-547 of the fiber protein) was constructed. Oligonucleotides corresponding to the Arg-Gly-Asp (RGD) or Asn-Gly-Arg (NGR)-containing peptide motif (as a model) and containing a Csp45I and/or ClaI recognition site, were ligated into the Csp45I and/or ClaI-digested plasmid. The foreign transgene expression cassette was inserted into the E1 deletion site of the vector plasmid and the fiber-mutant Ad vector was produced by transfection of the PacI-digested plasmid into 293 cells. The virus containing the RGD or NGR peptide on the fiber knob was able to infect human glioma cells, which do not express coxsackievirus and adenovirus receptor (CAR), one of the Ad virus receptors, about 100–1000 times more efficient than the virus containing wild-type fiber. This suggested that the mutant virus mediated CAR-independent cell entry pathway. The simplicity of this method allows not only for easy construction of fiber-mutant Ad vectors, but also for screening of the peptides that target the vector to the desired cells and tissues.

CAR- or αv integrin-binding ablated adenovirus vectors, but not fiber-modified vectors containing RGD peptide, do not change the systemic gene transfer properties in mice

Targeted gene delivery to the tissue of interest by recombinant adenovirus (Ad) vectors is limited by the relatively broad expression of the primary receptor, the coxsackievirus and adenovirus receptor (CAR), and the secondary receptor, αv integrin. This problem could be overcome by mutating the fiber and penton base, which bind with CAR and αv integrin, respectively. In this study, we constructed CAR-binding ablated Ad vectors and αv integrin-binding ablated Ad vectors by mutation in the FG loop of fiber knob and in the RGD motif of penton base, respectively, and compared the gene transfer properties of their vectors into various types of cultured cells and mice with conventional Ad vectors. We also generated Ad vectors containing RGD peptide in the HI loop of the fiber knob. CAR-binding ablated Ad vectors mediated about 1% of gene transfer activity into CAR-positive cultured cells, compared with conventional Ad vectors, while αv integrin-binding ablated Ad vectors maintained at least 76% of gene transfer activity into cultured CAR-positive cells. Inclusion of the RGD peptide into the HI loop of the fiber knob of CAR-binding ablated Ad vectors restored gene transfer activity in vitro. On the other hand, systemically administered CAR-binding ablated Ad vectors, as well as αv integrin-binding ablated Ad vectors mediated similar levels of gene transfer into mouse liver with the conventional Ad vectors. These results suggest that continued interaction of either the fiber with CAR or the penton base with αv integrin offers an effective route of virus entry into mouse liver in vivo. Inhibition of the interaction of both the fiber with CAR and the penton base with αv integrin is likely to be crucial to the development of targeted Ad vectors.

Efficient gene transfer by fiber-mutant adenoviral vectors containing RGD peptide

One of the hurdles to adenovirus (Ad)-mediated gene transfer is that Ad vectors mediate inefficient gene transfer into cells lacking in the primary receptors, Coxsackievirus and adenovirus receptor (CAR). We previously developed a fiber-mutant Ad vector containing the Arg-Gly-Asp (RGD)-containing peptide motif on the HI loop of the fiber knob, and showed that the mutant vector had enhanced gene transfer activity to human glioma cells, which showed little CAR expression, compared to the vector containing wild type fiber. In this study, the feasibility of the Ad vector containing RGD peptide on the fiber knob was examined in a wide variety of cell types: CAR-positive or -negative human tumor cells, mouse cells, and leukemia cells. The mutant vector infected the cells, which lacked CAR expression but showed alpha(v) integrin expression, about 10-1000 times more efficiently than the vector containing wild type fiber via an RGD-integrin (alpha(v)beta3 and alpha(v)beta5)-dependent, CAR-independent cell entry pathway. The results of this study indicate that Ad vector containing RGD peptide on the fiber knob could be of great utility for gene therapy and gene transfer experiments.

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.

Long-term replication of Epstein-Barr virus-derived episomal vectors in the rodent cells.

Plasmids containing the origin of replication, oriP, of the Epstein‐Barr virus (EBV) and EBV nuclear antigen‐1 genes replicate extrachromosomally in primate cells. However, these plasmids have been believed not to replicate in rodent cells. We demonstrate here that these plasmids can replicate in some types of rodent cells over a long period. This result should offer not only the new insight into the mechanisms of species‐specific replication of EBV, but also the possibility that an EBV‐based vector can be used for gene transfer experiments in non‐primate cells and an animal experiment regarding human gene therapy.

An isoform of Nurr1 functions as a negative inhibitor of the NGFI-B family signaling.

NGFI-B, Nurr1 and NOR-1 constitute a distinct subfamily within the nuclear receptor superfamily. To clarify the transcriptional regulation by the NGFI-B family, we searched for other components that can bind to the NBRE response element, a known target sequence for these transcription factors. By low stringency hybridization using the DNA binding domain of NOR-1 as a probe, a C-terminal truncated Nurr1 isoform, named Nurr2, was isolated from a mouse MC3T3-E1 cell cDNA library. Nurr2 had a novel cryptic exon located upstream in the Nurr1 promoter region, and was generated by alternative splicing at exons 1, 2 and 6. The C-terminal region was encoded by frame-shifted exon 6, and so Nurr2 lacked the C-terminal sequences corresponding to the putative ligand binding domain or dimerization domain. Quantitative reverse transcriptase-PCR experiments confirmed the presence of the Nurr2 isoform in mouse, rat and human. It was, like Nurr1, highly expressed in the pituitary and the cerebral cortex. Nurr2 and Nurr1 were also concomitantly induced by forskolin in NIH3T3 cells. Functional analysis using a reporter gene, containing NBRE response elements, indicated that while the isoform was inactive by itself, it could inhibit transactivation by the members of the NGFI-B family. These results indicate that the C-terminal truncated isoform, Nurr2, may act as a negative regulator of the NGFI-B family signaling.

Structure and distribution of rat menin mRNA.

Menin is a protein product of a tumor suppressor gene MEN1, mutations of which are responsible for multiple endocrine neoplasia type 1, an autosomal dominant familial cancer syndrome. We isolated rat menin cDNA clones from a fetal rat brain cDNA library. We also determined the nucleotide sequence of the protein coding region of mouse menin cDNA, which was partly registered in the expressed sequence tag (EST) database. Deduced amino acid sequences of rat and mouse menin are highly homologous to human menin. All of the previously reported disease-associated missense mutations and single amino acid deletions were observed at the residues that are conserved among these three species. Rat MEN1 transcripts were detected not only in the endocrine tissues but also in the tissues of the nervous, digestive, reproductive and immune systems. The MEN1 transcripts were abundantly expressed in the developing rat brain on day 14-18 of gestation. Immunoblotting and immunocytochemical analysis of the COS-7 cells transfected with a rat menin-expression vector revealed that the translated product has a molecular mass of approximately 70 kDa, and is localized mainly in the nucleus. These findings are consistent with those reported on human menin.

Involvement of protein kinase C in the interleukin 1α-induced gene expression of matrix metalloproteinases and tissue inhibitor-1 of metalloproteinases (TIMP-1) in human uterine cervical fibroblasts

The role of protein kinase C in the interleukin 1 (IL-1)-mediated production of pro-matrix metalloproteinases (proMMPs) and tissue inhibitor-1 of metalloproteinases (TIMP-1) in human uterine cervical fibro-blasts has been investigated. IL-1 and a protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (TPA) augmented the production of proMMP-1 (interstitial procollagenase), proMMP-3 (prostromelysin-1) and TIMP-1, but their effects were inhibited by the protein kinase C inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and staurosporine in a dose-dependent manner. The suppressive effect of H-7 and staurosporine on the IL-1-induced production of proMMPs-1 and -3 and TIMP-1 resulted from the decrease in the steady-state levels of their mRNAs. When protein kinase C was down-regulated by treating the cells with a high level of TPA, the inductive effect of IL-1 upon proMMP-3 production was reduced considerably. These results indicate that protein kinase C mediates the IL-1-induced production of proMMPs-1 and -3 and TIMP-1 at the pretranslational level in human uterine cervical fibroblasts. On the other hand, neither IL-1 nor TPA modulated the production of proMMP-2 (progelatinase A). Both IL-1 and TPA also accelerated the production of prostaglandin E2 (PGE2) by cervical fibroblasts. However, the treatment of the cells with staurosporine in the presence of IL-1 or TPA further augmented PGE2 synthesis, suggesting that the increased synthesis of PGE2 by IL-1 treatment is mediated via signalling pathways distinct from those of proMMPs-1 and -3 and TIMP-1.

Translational augmentation of pro-matrix metalloproteinase 3 (prostromelysin 1) and tissue inhibitor of metalloproteinases (TIMP)-1 mRNAs induced by epidermal growth factor in human uterine cervical fibroblasts

The mechanisms by which epidermal growth factor (EGF) enhances the production of pro‐matrix metalloproteinase 3 (proMMP‐3/prostromelysin 1) and tissue inhibitor of metalloproteinases (TIMP)‐1 were investigated using human uterine cervical fibroblasts. The treatment of the cells with EGF for 24 h resulted in about 5–6‐fold increase in the production of proMMP‐3 and TIMP‐1 compared with the untreated control cells. This increase was accompanied by an increase of proMMP‐3 and TIMP‐1 mRNAs. However, an about 3‐ and 2‐fold increase in the production of proMMP‐3 and TIMP‐1, respectively, was observed as early as 1 h after the treatment of the cells with EGF, and it was not accompanied by any apparent increase in proMMP‐3 and TIMP‐1 mRNAs. This early effect of EGF on the enhanced production of proMMP‐3 and TIMP‐1 was not inhibited by actinomycin D, even though actinomycin D inhibited the synthesis of the total RNA in both the EGF‐treated and untreated cells. These results indicate that EGF enhances the apparent production of proMMP‐3 and TIMP‐1 by two mechanisms: one by the accelerated translation of their mRNAs; and the other by the enhanced transcription of their genes. The former event takes place much earlier than the latter.

Effects of beta-tricalcium phosphate particles on primary cultured murine dendritic cells and macrophages

Beta-tricalcium phosphate (β-TCP) is widely used for bone substitution in clinical practice. Particles of calcium phosphate ceramics including β-TCP act as an inflammation mediators, which is an unfavorable characteristic for a bone substituent or a prosthetic coating material. It is thought that the stimulatory effect of β-TCP on the immune system could be utilized as an immunomodulator. Here, in vitro effects of β-TCP on primary cultured murine dendritic cells (DCs) and macrophages were investigated. β-TCP particles enhanced expression of costimulatory surface molecules, including CD86, CD80, and CD40 in DCs, CD86 in macrophages, and MHC class II and class I molecules in DCs. DEC205 and CCR7 were up-regulated in β-TCP-treated DCs. Production of cytokines and chemokines, including CCL2, CCL3, CXCL2, and M-CSF, significantly increased in DCs; CCL2, CCL3, CCL4, CCL5, CXCL2, and IL-11ra were up-regulated in macrophages. The results of the functional assays revealed that β-TCP caused a prominent reduction in antigen uptake by DCs, and that conditioned medium from DCs treated with β-TCP facilitated the migration of splenocytes in the transwell migration assay. Thus, β-TCP induced phenotypical and functional maturation/activation of DCs and macrophages; these stimulating effects may contribute to the observed in vivo effect where β-TCP induced extensive migration of immune cells. When compared to lipopolysaccharide (LPS), an authentic TLR ligand, the stimulatory effect of β-TCP on the immune systems is mild to moderate; however, it may have some advantages as a novel immunomodulator. This is the first report on the direct in vitro effects of β-TCP against bone marrow-derived DCs and macrophages.