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Y Kanegae

Publications and source records attributed to Y Kanegae.

At least 37 records · Page 2Linked to original sources
PubMed1998

Persistent and secondary adenovirus-mediated hepatic gene expression using adenovirus vector containing CTLA4IgG.

Adenovirus vectors can transfer recombinant genes efficiently into a wide variety of cells in vivo, but have serious limitations: gene expression is transient and secondary gene transfer is inefficient or impossible because of cellular and humoral immune responses against adenovirus-transduced cells. To solve these limitations, we have constructed an adenovirus vector, Adex1CACTLA4IgG, that expresses CTLA4IgG molecules. After in vivo administration of Adex1CACTLA4IgG (9.0 x 10(9) PFU), the peak level of serum CTLA4IgG was 29.8 mg/ml on day 4. The serum CTLA4IgG concentration gradually fell but was still 5.7 mg/ml on day 90. However, the serum concentration of CTLA4IgG was elevated after a second administration of Adex1CACTLA4IgG. The production of antibody against adenovirus was completely prevented after treatment with Adex1CACTLA4IgG. In addition, coadministration of Adex1CALacZ with Adex1CACTLA4IgG induced persistent hepatic expression of beta-Gal molecules, while administration of Adex1CALacZ alone induced transient expression of beta-Gal molecules. More importantly, on day 160 a secondary challenge with Adex1CALacZ was possible in mice treated with Adex1CALacZ plus Adex1CACTLA4IgG. Thus, we have demonstrated that (1) gene expression of a recombinant adenovirus, Adex1CACTLA4IgG, is persistent in liver and secondary administration of this adenovirus is possible, (2) coadministration of Adex1CACTLA4IgG virus with another adenovirus, AdexCALacZ, prolongs AdexCALacZ-mediated gene expression, and (3) Adex1CACTLA4IgG is useful for secondary challenge with Adex1CALacZ.

Abatacept↗
PubMed1998

Efficient conditional transgene expression in hepatitis C virus cDNA transgenic mice mediated by the Cre/loxP system.

Conditional gene expression has greatly facilitated the examination of the functions of particular gene products. Using the Cre/loxP system, we developed efficient conditional transgene activation of hepatitis C virus (HCV) cDNA (nucleotides 294-3435) in transgenic mice. Efficient recombination was observed in transgenic mouse liver upon intravenous administration of adenovirus that expresses Cre DNA recombinase. After transgene activation, most hepatocytes were stained with anti-core polyclonal antibody, and 21-, 37-, and 64-kDa proteins were detected by Western blot analysis in liver lysates using anti-core, E1, and E2 monoclonal antibodies, respectively. Serum core protein was detected in transgenic mice 7 days after transgene activation with concurrent increases in serum alanine aminotransferase levels. Subsequently, an anti-core antibody response was detected 14 days after infection. Furthermore, a CD4 and CD8 positive cell depletion assay normalized both the serum alanine aminotransferase increases and pathological changes in the liver. These results suggest that HCV proteins are not directly cytopathic and that the host immune response plays a pivotal role in HCV infection. Thus, this HCV cDNA transgenic mouse provides a powerful tool with which to investigate the immune responses and pathogenesis of HCV infection.

Adenoviridae↗
PubMed1998

Role of Rel/NF-kappaB transcription factors during the outgrowth of the vertebrate limb.

The development of the vertebrate limb serves as an amenable system for studying signaling pathways that lead to tissue patterning and proliferation. Limbs originate as a consequence of a differential growth of cells from the lateral plate mesoderm at specific axial levels. At the tip of the limb primordia the progress zone, a proliferating group of mesenchymal cells, induces the overlying ectoderm to differentiate into a specialized structure termed the apical ectodermal ridge. Subsequent limb outgrowth requires reciprocal signalling between the ridge and the progress zone. The Rel/NF-kappaB family of transcription factors is induced in response to several signals that lead to cell growth, differentiation, inflammatory responses, apoptosis and neoplastic transformation. In unstimulated cells, NF-kappaB is associated in the cytoplasm with an inhibitory protein, I-kappaB. In response to an external signal, I-kappaB is phosphorylated, ubiquitinated and degraded, releasing NF-kappaB to enter the nucleus and activate transcription. Here we show that Rel/NF-kappaB genes are expressed in the progress zone of the developing chick limb bud. When the activity of Rel/NF-kappaB proteins is blocked by infection with viral vectors that produce transdominant-negative I-kappaBalpha proteins, limb outgrowth is arrested. Our results indicate that Rel/NF-kappaB transcription factors play a role in vertebrate limb development.

Animals↗
PubMed1998

Enhanced and specific gene expression via tissue-specific production of Cre recombinase using adenovirus vector.

A tissue-specific promoter is potentially valuable for the study of specific gene function and for gene therapy, as it permits a linked cytotoxic or any other gene to be expressed specifically in target cells. The expression levels of such promoters are generally low, and we have therefore developed a novel and general method to enhance the expression level of a tissue-specific promoter while maintaining specificity. We constructed a "regulator" recombinant adenovirus (rAd) producing the site-specific recombinase Cre under the control of the hepatocarcinoma-specific alpha-fetoprotein (AFP) promoter. The rAd was infected to AFP-producing cells together with a "target" rAd containing a Cre-activating potent expression unit. In in vitro experiments, the double infection method gave about 50-fold higher expression than the single rAd infection directly driven by the AFP promoter, while maintaining strict specificity to AFP-producing cells. The enhanced and specific expression was also observed in in vivo tumor models. This method may contribute not only to the establishment of specific gene therapies but also to basic study for elucidating cell-type specific gene functions.

Adenoviridae↗
PubMed1998

A new system for stringent, high-titer vesicular stomatitis virus G protein-pseudotyped retrovirus vector induction by introduction of Cre recombinase into stable prepackaging cell lines.

We report here on stable prepackaging cell lines which can be converted into packaging cell lines for high-titer vesicular stomatitis virus G protein (VSV-G)-pseudotyped retrovirus vectors by the introduction of Cre recombinase-expressing adenovirus. The generated prepackaging cell lines constitutively express the gag-pol genes and contain an inducible transcriptional unit for the VSV-G gene. From this unit, the introduced Cre recombinase excised both a neomycin resistance (Neo(r)) gene and a poly(A) signal flanked by a tandem pair of loxP sequences and induced transcription of the VSV-G gene from the same promoter as had been used for Neo(r) expression. By inserting an mRNA-destabilizing signal into the 3' untranslated region of the Neo(r) gene to reduce the amount of Neo(r) transcript, we were able efficiently to select the clones capable of inducing VSV-G at high levels. Without the introduction of Cre recombinase, these cell lines produce neither VSV-G nor any detectable infectious virus at all, even after the transduction of a murine leukemia virus-based retrovirus vector encoding beta-galactosidase. They reproducibly produced high-titer virus stocks of VSV-G-pseudotyped retrovirus (1.0 x 10(6) infectious units/ml) from 3 days after the introduction of Cre recombinase. We also present evidence that VSV-G-producing cells are still fully susceptible to transduction by VSV-G pseudotypes. However, in this vector-producing system, which regulates VSV-G pseudotype production in an all-or-none manner, the integration of vector DNA into packaging cell lines would be minimized. We further show that heparin significantly inhibits retransduction of VSV-G pseudotypes in the culture fluids of packaging cell lines, leading to a two- to fourfold increase in the yield of the pseudotypes after induction. This vector-producing system was very stable and should be advantageous in human gene therapy.

Animals↗
PubMed1997

Rapid colorectal adenoma formation initiated by conditional targeting of the Apc gene.

Familial adenomatous polyposis coli (FAP) is a disease characterized by the development of multiple colorectal adenomas, and affected individuals carry germline mutations in the APC gene. With the use of a conditional gene targeting system, a mouse model of FAP was created that circumvents the embryonic lethality of Apc deficiency and directs Apc inactivation specifically to the colorectal epithelium. loxP sites were inserted into the introns around Apc exon 14, and the resultant mutant allele (Apc580S) was introduced into the mouse germline. Mice homozygous for Apc580S were normal; however, upon infection of the colorectal region with an adenovirus encoding the Cre recombinase, the mice developed adenomas within 4 weeks. The adenomas showed deletion of Apc exon 14, indicating that the loss of Apc function was caused by Cre-loxP-mediated recombination.

Adenomatous Polyposis Coli↗
PubMed1997

Complete rescue of lethal albino c14CoS mice by null mutation of 4-hydroxyphenylpyruvate dioxygenase and induction of apoptosis of hepatocytes in these mice by in vivo retrieval of the tyrosine catabolic pathway.

Hereditary tyrosinemia 1 (HT1) is characterized by progressive liver damage, from infancy, and by a high risk for hepatocellular carcinoma. HT1 is due to mutations in the fumarylacetoacetate hydrolase gene Fah, encoding the last enzyme in the tyrosine catabolic pathway. Lethal albino deletion c14CoS mice and mice with target-disrupted Fah are models for HT1, but they die in the perinatal period, albeit with a different phenotype from that seen in HT1 in humans. We first asked whether homozygous null mutation of the 4-hydroxyphenylpyruvate dioxygenase gene Hpd could rescue the homozygous c14CoS mice (c14CoS/c14CoS or Fah-/-). The double mutant Fah-/- Hpd-/- mice appeared normal, at least until age 18 months, and there was no evidence of liver disease, findings that facilitated examination of the effect of Fah-/- on mature and unmodified hepatocytes in vivo. The hepatocytes of Fah-/- undergo rapid apoptosis, and acute death follows. Essentially the same phenomena were observed when Fah-/- Hpd-/- mice were administered homogentisate intraperitoneally. These changes in liver pathology in Fah-/- Hpd-/- mice after the administration of homogentisate were associated with massive urinary excretion of succinylacetone. These results suggest that accumulation of fumarylacetoacetate, maleylacetoacetate, or succinylacetone seems to trigger the endogenous process of apoptosis in hepatocytes that lack fumarylacetoacetate hydrolase activity. This apoptosis may be related to the development of hepatocellular carcinomas seen in HT1 patients and pharmaceutically treated fumarylacetoacetate hydrolase-deficient mice.

4-Hydroxyphenylpyruvate Dioxygenase↗
PubMed1997

Direct involvement of the ubiquitin-conjugating enzyme Ubc9/Hus5 in the degradation of IkappaBalpha.

The NF-kappaB/Rel proteins are sequestered in the cytoplasm in association with IkappaBalpha. In response to external signals, IkappaBalpha is phosphorylated, multi-ubiquitinated, and degraded by proteasomes, thereby releasing NF-kappaB/Rel proteins to migrate to the nucleus. We have cloned a mouse ubiquitin-conjugating enzyme (mE2), which associates with IkappaBalpha. mE2 is homologous to the yeast Ubc9/Hus5 ubiquitin-conjugating enzyme. A transdominant-negative mutant of mE2 had no effect on phosphorylation of IkappaBalpha, but delayed its degradation. Correspondingly, tumor necrosis factor-alpha-inducible NF-kappaB activity was diminished. We propose that mE2 is directly involved in the ubiquitin conjugation of IkappaBalpha, a pivotal step in its degradation pathway.

Amino Acid Sequence↗
PubMed1997

Interleukin-2 gene transduction into freshly isolated lung adenocarcinoma cells with adenoviral vectors.

We evaluated the efficiency of gene transduction and of gene expression by adenoviral vectors in human lung adenocarcinoma cells. Freshly isolated cancer cells were collected from pleural effusions in adenocarcinoma patients by centrifugation with a Percoll gradient. Adenoviral vectors resulted in effective gene transduction into human lung cancer cell lines and into freshly isolated lung adenocarcinoma cells. In an experiment using the beta-galactosidase (LacZ) gene, the Adex1CA vector with a regulatory sequence of chicken beta-actin as promoter and an enhancer derived from cytomegalovirus produced a higher transduction ratio and greater expression levels than adenoviral vectors with other promoter systems. Transduction with Adex1CA vectors containing the human interleukin-2 (IL-2) gene (Adex1CAhIL-2) resulted in enhanced secretion of IL-2 from gene-modified lung cancer cells. Treatment with normal human serum inhibited gene transduction by Adex1CAhIL-2 but did not inhibit gene expression after transduction by Adex1CAhIL-2. The secretion of IL-2 from the gene-modified cells, which were irradiated at 100 Gy before transduction, continued for 8 days. In a mouse model, the intrapleural injection of IL-2 gene-modified 3LL cells transduced by Adex1CAhIL-2 could cure the pre-existing lung tumours with malignant pleural effusions to induce tumor-specific immunity. But these therapies did not show any therapeutic benefit on the pre-existing tumor in subcutaneous region. These data suggest a potentially useful but limited clinical role of Adex1CAhIL-2 in gene therapy for lung cancer patients.

Adenoviridae↗
PubMed1997

In vivo correction with recombinant adenovirus of 4-hydroxyphenylpyruvic acid dioxygenase deficiencies in strain III mice.

Tyrosinemia type 3, caused by a genetic deficiency of 4-hydroxyphenylpyruvic acid dioxygenase (HPD) in tyrosine catabolism, is characterized by convulsion, ataxia, and mental retardation. The III mouse is a model of tyrosinemia type 3. HPD activity and protein are defective in the liver and its blood tyrosine levels are elevated, the range being between 1,100 and 1,656 microM. We constructed a recombinant adenoviral vector bearing the human HPD cDNA (AdexCAGhHPD), which is expressed under the control of a potent CAG promoter. III mice were injected with 1.0 x 10(8) to 1.0 x 10(9) pfu of AdexCAGhHPD through the tail vein. When 3.0 x 10(8) - 1.0 x 10(9) pfu were injected, blood tyrosine levels decreased within 3 hr, reached a normal range (under 300 microM), and remained at a low level for 2-6 weeks. Hepatic HPD activities also increased as early as 3 hr after the injection of 5.0 x 10(8) pfu, reached the levels comparable to the control mice in 3-7 days, and then decreased, and correlated well to blood tyrosine. Hepatic HPD expression was confirmed by Northern blot and immunoblot analyses. Histology revealed no difference (gross or microscopic) between the liver injected with AdexCAGhHPD and the control. No significant changes in blood tyrosine levels were noted after the second injection of 5.0 x 10(8) pfu of AdexCAGhHPD. Thus, the intravenous administration of the adenoviral vector bearing a foreign gene seems suitable for transient, early gene transfer into the liver.

4-Hydroxyphenylpyruvate Dioxygenase↗
PubMed1997

Retrograde and anterograde labeling of cerebellar afferent projection by the injection of recombinant adenoviral vectors into the mouse cerebellar cortex.

Adenoviral vectors have recently been recognized as highly efficient systems for gene delivery into various tissues. We show that a reporter gene introduced into nerve terminals via an adenovirus can be used to label cell bodies retrogradely and then label the axons and nerve terminals of the infected neurons anterogradely in vivo. We injected a replication-defective recombinant adenovirus carrying the E. coli beta-galactosidase gene (lacZ) into the cerebellar cortex of the adult mouse. The first evidence of retrograde labeling was obtained at 2 days after the infection when neurons in the pontine nuclei and the reticulotegmental nucleus of the pons weakly expressed beta-galactosidase, and at 3 days post-infection when neurons in all precerebellar nuclei, known to project to the cerebellar cortex, were strongly stained with X-gal in a Golgi-like manner. Anterograde transport of lacZ gene products was recognized at 3 days post-infection; beta-galactosidase-positive axons arose from somata or dendrites of retrogradely labeled neurons, passed through the middle or inferior cerebellar peduncles, and entered the cerebellum. Anterogradely labeled mossy terminals were recognized on the injection side at 8 days post-infection, and on the contralateral side at 14 days post-infection. Beta-galactosidase expression persisted for up to two months, with a decrease in the total number of labeled cells over time. We could not find any signs of anterograde or retrograde transsynaptic labeling in the nuclei synaptically linked to the cerebellar cortex at any time point after injection up to 58 days post-infection.

Adenoviridae↗
PubMed1997

Transplantation of genetically marked cardiac muscle cells.

We examined the possibility that cardiomyocytes could be genetically marked or modified before being grafted to the heart under conditions applicable to the clinical setting. We used a replication-defective recombinant adenovirus carrying the beta-galactosidase reporter gene, and delivered it to cultured murine fetal cardiac myocytes. Virtually all fetal cardiomyocytes in a primary culture expressed beta-galactosidase 24 hours after recombinant adenovirus infection. These cells were transplanted to the hearts of syngenic adult recipient mice. Expression of the beta-galactosidase gene in the grafted cells was demonstrated by staining with 5-bromo-4-chloro-3-indoyl-beta-D-galactosidase, resulting in a blue color at the histochemical level and an electron-dense deposit on transmission electron microscopic analysis. Gene expression was recognized from 7 days to 12 weeks after transplantation. Implanted cardiomyocytes aligned themselves along the layers of the host myocardium. Formation of gap junctions was demonstrated by transmission electron microscopy. Neither inflammation nor fibrous scar tissue was detectable by histologic analysis. This study demonstrates that ex vivo gene transfer to the heart by means of the adenoviral vector is possible.

Adenoviridae↗
PubMed1996

Efficient gene activation system on mammalian cell chromosomes using recombinant adenovirus producing Cre recombinase.

To develop a method for activating genes located on cell chromosomes, an on/off switching unit regulated by the site-specific recombinase Cre was constructed. The switching unit was designed to express firstly the neo gene and secondly the reporter lacZ gene by Cre-mediated excisional deletion of the neo gene. CV1 cell lines bearing the switching unit on a cell chromosome were isolated and activation of the lacZ gene was examined after infection with a Cre-producing recombinant adenovirus. In one cell line virtually 100% of the cells stably expressed the lacZ gene, whereas in another cell line lacZ-expressing cell populations reached only to about 90% and decreased after cell divisions. The Southern blot analyses showed that the latter type of cells contained a head-to-tail array of the switching units, and that consequently the lacZ-expressing units were excised from a cell chromosome and present as extrachromosomal circular DNAs. These results showed that the system offers efficient activation of genes introduced into cell chromosomes and that the organization of the reporter units are important for efficiency and duration of the activated gene expression.

Adenoviridae↗
PubMed1996

Correction of ornithine transcarbamylase deficiency in adult spf(ash) mice and in OTC-deficient human hepatocytes with recombinant adenoviruses bearing the CAG promoter.

Ornithine transcarbamylase (OTC) deficiency, the most common and severe inborn error of the urea cycle in humans, remains without adequate treatment, and mortality rates are high. Adenoviral vectors provide an efficient system for gene delivery, but there are problems, including toxicity. Efficient promoters that reduce the amount of vector required for treatment need to be developed. We constructed two recombinant adenoviral vectors, AdexCAGhOTC and AdexSR alpha hOTC, which harbor the human OTC gene under transcriptional control of CAG (a modified chicken beta-actin promoter with CMV-IE enhancer) and SR alpha (the SV40 early promoter with the R segment and part of the US segment of the HTLV-1 LTR), respectively. Each was tested in adult spf(ash) mice, an animal model of human OTC deficiency, and in primary human hepatocytes with OTC deficiency. Spf(ash) mice have a pronounced orotic aciduria as seen in humans. A complete recovery of hepatic OTC activity with minimal tissue damage was observed in these animals following the intravenous administration of AdexCAGhOTC alone. Western blot analysis confirmed hepatic OTC expression and normalization of orotic aciduria was evident for 60 days. Enzyme activities of primary human hepatocytes infected with AdexCAGhOTC were 10-40 times higher than those with AdexSR alpha hOTC. Thus, the adenoviral vector with an efficient promoter such as CAG, can be given further consideration for possible gene therapy in humans with OTC deficiency.

Actins↗
PubMed1996

Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.

An efficient method of constructing recombinant adenoviruses (Ads) has been established. The expression unit to be introduced into recombinant Ad was first inserted into the unique Swa I site of the full-length Ad genome cloned in a cassette cosmid. The cassette bearing the expression unit was then cotransfected into human embryonic kidney 293 cells together with the Ad DNA-terminal protein complex digested at several sites with Eco T22I or Ase I/EcoRI. The use of the parent Ad DNA-terminal protein complex instead of the deproteinized Ad genome DNA allowed very efficient recovery of the desired recombinant Ad, and the above restriction digestion drastically reduced regeneration of the parent virus. Several hundred virus clones were readily obtained in each experiment, and about 70% of the clones were the desired recombinant viruses. Furthermore, because the cassette contained the full-length Ad genome, any position of the genome could be easily modified to develop a new vector design. We established construction systems for two types of Ad vectors, the E1-substitution type and the E4-insertion type. This method may greatly facilitate the application of recombinant Ads and should be useful for further improvement of Ad vectors.

Adenovirus E4 Proteins↗
PubMed1996

A neural cell-type-specific expression system using recombinant adenovirus vectors.

We demonstrated neural cell-type-specific gene expression using an adenovirus vector, which is useful for delivering foreign genes into quiescent neural cells. We produced eight recombinant replication-defective adenoviruses carrying the lacZ reporter gene driven by various promoters, including those of the L7/PCP2 gene (highly restricted expression in cerebellar Purkinje cells), and the myelin basic protein (in oligodendrocytes) gene. We demonstrated in vitro and in vivo promoter-driven, neural cell-type-specific gene expression by recombinant adenoviruses. The genes were transferred into these recombinant adenoviruses and expressed with high levels of efficiency in vitro and in vivo. In primary culture, the recombinant adenoviruses AdexL7-NL-LacZ and AdexMBP-NL-LacZ appeared to be expressed in a Purkinje cell and oligodendrocyte-specific manner. Introduction of 10(8) pfu of these viruses into the adult rat cerebellum by stereotactic injection yielded neural cell-type-specific expression without apparent toxicity to the animals. Thus, adenovirus vectors are useful for cell-type-specific therapeutic uses and in studies requiring neural cell-type-specific gene expression.

Adenoviruses, Human↗
PubMed1996

Gene therapy for alpha-fetoprotein-producing human hepatoma cells by adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene.

We have developed a recombinant replication-defective adenovirus containing human alpha-fetoprotein (AFP) promoter/enhancer to direct cell type-specific expression of the herpes simplex virus thymidine kinase (HSVtk) gene to AFP-producing hepatocellular carcinoma (HCC) cells. After an in vitro infection by a recombinant adenovirus carrying the lacZ gene under the control of human AFP promoter/enhancer (AdAFPlacZ), an expression of the lacZ gene was demonstrated efficiently in AFP-producing HuH-7 and HepG2 cell lines, but not in AFP-nonproducing HLE and HLF cell lines, although lacZ gene expression was demonstrated in all these cell lines when infected with adenovirus vector carrying lacZ gene driven by the beta-actin-based promoter. Expression of the HSVtk gene by adenovirus, from AFP promoter/enhancer (AdAFPtk) induced the cells sensitive to ganciclovir (GCV) in the AFP-producing cell line efficiently, but not in AFP-nonproducing HLF hepatoma cells. An in vitro bystander effect was observed when only 10% of the cells were infected with AdAFPtk. These findings suggest that the AFP promoter/enhancer sequence can provide the tumor-specific activity for the therapeutic gene expression, and that the AdAFPtk vector induces the selective growth inhibition by GCV in the adenovirus-infected human hepatoma cells in vitro. Recombinant adenovirus transfer of the HSVtk gene under the control of tumor-specific promoter followed by GCV may have promise as a targeted in situ treatment for solid neoplasms.

Adenoviridae↗