Motomichi Kosuga

Efficient Fas-ligand gene expression in rodent liver after intravenous injection of a recombinant adenovirus by the use of a Cre-mediated switching system

An adenovirus vector AxCALNFasL was constructed in order to transduce a gene for rat Fas-ligand, requiring co-expression of Cre recombinase for its expression. In the cosmid cassette, pAxCALNFasL, a stuffer DNA fragment flanked with two loxP sequences was placed between the promoter and Fas-ligand cDNA to prevent its expression in transfected 293 cells. COS-7 cells infected with AxCALNFasL alone did not induce apoptosis in cocultivated Jurkat cells, but the cells treated with AxCALNFasL and AxCANCre (an adenovirus expressing Cre recombinase with the CAG promoter) did. BALB/c mice injected with 109 plaque-forming units of AxCALNFasL and with different doses of AxCANCre, developed lethal acute liver failure. The number of the apoptotic hepatocytes increased dramatically with increased doses of injected AxCANCre, indicating that the level of transgene expression in the rodent liver appeared to be adjustable. Based on these observations, we conclude that vectors expressing a gene to produce cytotoxic substances can be constructed by the use of a Cre-mediated switching system. Our system also demonstrated that efficient expression of the toxic gene in the rodent liver was achievable by co-infection of adenovirus vectors carrying the target gene and Cre recombinase.

Overexpression of Bcl-2 protects human hepatoma cells from Fas-antibody-mediated apoptosis.

BACKGROUND/AIMS Fas is a cell surface antigen, that triggers apoptosis upon specific ligand or antibody binding. The proto-oncogene bcl-2 prevents apoptosis induced by various treatments. The aim of our study was to evaluate whether Bcl-2 protects hepatoma cells from Fas-mediated apoptosis. METHODS Two human cell lines, HCC-T and HepG2 were used. Expression of Fas antigen and Bcl-2 was detected by flow cytometry and Western blotting. Cell viability and apoptotic change were examined after anti-Fas- and antisense oligodeoxynucleotide treatments. Apoptotic cells were detected by nick-end labelling and the TUNEL method. To test if Bcl-2 expression can protect HepG2 cells from Fas-mediated apoptosis, the cells were transduced using retroviral vector, LZBC, designed to coexpress E. coli beta-galactosidase and human Bcl-2. To further confirm the protective effect of Bcl-2 expression against Fas-mediated apoptosis in HepG2, Bcl-2 expressing plasmid vector was produced and a cell line stably expressing Bcl-2 was cloned. RESULTS Western blot analysis showed constitutive Bcl-2 expression in HCC-T cells, but not in HepG2 cells. HCC-T was resistant to apoptosis after treatment with an agonist anti-Fas antibody (1 microg/ml for 3 days), whereas 33% of the HepG2 cells were killed by this treatment. Inhibition of Bcl-2 expression by transfection of antisense oligodeoxynucleotides caused spontaneous apoptosis in HCC-T, but not in HepG2 cells, suggesting that Bcl-2 is essential for survival of HCC-T cells, whereas other proteins may substitute for it in HepG2 cells. Following LZBC infection, 10% HepG2 cells were beta-galactosidase-positive by X-gal staining and Bcl-2-positive. In cells surviving after anti-Fas treatment, the proportion of beta-galactosidase-positive cells increased to 50% and the beta-galactosidase activity increased 6-fold, indicating that Bcl-2 expression protected the cells from Fas-mediated apoptosis. In the cloned HepG2 cells stably expressing Bcl-2, the extent of Fas-mediated apoptosis was inversely related to the level of Bcl-2 expression. CONCLUSION Bcl-2 confers protection to human hepatoma cells against Fas-mediated apoptosis, and is essential for survival of some, but not all, hepatoma cells.

Long-term normalization in the central nervous system, ocular manifestations, and skeletal deformities by a single systemic adenovirus injection into neonatal mice with mucopolysaccharidosis VII.

Systemic injection of an adenovirus vector into adult mice resulted in pathological improvements in multiple visceral organs of mice with mucopolysaccharidosis VII; however, no therapeutic efficacy was observed for mental retardation, skeletal deformities, corneal clouding, and retinal degeneration. In this study, an adenovirus vector expressing human β-glucuronidase was injected into mice with mucopolysaccharidosis VII within 24 h of birth, and therapeutic efficacy was evaluated. In the brains of the mice, more than 20% of GUSB activity was maintained for at least 20 weeks after birth, and histopathological analysis showed no obvious lysosomal storage. Furthermore, no vacuolated cells were detected in corneal stroma and retinal pigment epithelium in the eyes of the mice treated in the neonatal period, while pathological improvement was not observed in adult MPSVII mice that received similar treatments. The treated mice also lacked characteristic facial skeletal deformities, and radiographic analysis demonstrated that their facial and cranial bones were morphologically normal. These results indicate that a single systemic adenovirus injection in the neonatal period could prevent the progression of mental retardation, corneal clouding, retinal degeneration, and skeletal deformities, all of which are frequently observed clinical manifestations and difficult to treat in adulthood.

OEIS complex with del(3)(q12.2q13.2)

Most cases of omphalocele-exstrophy-imperforate anusspinal defects (OEIS) complex (cloacal exstrophy) occur sporadically, but there have been several reports of recurrence in siblings [Smith et al., 1992], including monozygotic twins [Koffler et al., 1978; Redman et al., 1981; McLaughlin et al., 1984], suggesting a genetic contribution to the pathogenesis of this condition. In your journal, Thauvin-Robinet et al. [2004] recently reported a case of cloacal exstrophy with a de novo unbalanced translocationbetween the longarmof chromosome 9 and the long arm of chromosomeY, resulting in a 9q34.1-qter deletion [Thauvin-Robinet et al., 2004]. They argued that their case represented the first report of cloacal exstrophy accompanied by an unbalanced chromosomal defect and that their observation supported the notion that genetic defect(s) may be responsible for the pathogenesis of cloacal exstrophy. Here, we report a patientwith cloacal exstrophywho exhibited a de novo deletion at another chromosomal location, 3q12.2-3q13.2.

Enzyme replacement therapy attenuates disease progression in two Japanese siblings with mucopolysaccharidosis type VI

Mucopolysaccharidosis type VI (MPS VI) is a progressive, multisystem autosomal recessive lysosomal disorder resulting from deficient N-acetylgalactosamine-4-sulphatase (ASB) and the consequent accumulation of glycosaminoglycan (GAG). Preclinical and clinical studies had demonstrated clinical benefits of early initiation of systemic therapies in patients with MPS. In this case report, two siblings with MPS VI started enzyme replacement therapy (ERT) with weekly infusions of recombinant human ASB (Galsulfase) at 1mg/kg. Sibling 1 started ERT 5.6 years of age and Sibling 2 was 6 weeks old. The disease status in these two siblings prior to and for no less than 36 months of ERT was followed up and compared. The treatment was well tolerated by both siblings. During 36 months of ERT, symptoms typical of MPS VI including short stature, progressive dysmorphic facial features, hepatosplenomegaly, hearing impairment, corneal clouding, and dysostosis multiplex were largely absent in the younger sibling. Her cardiac functions and joint mobility were well preserved. On the other hand, her affected brother had typical MPS VI phenotypic features described above before commencing ERT at the equivalent age, of 3 years. There was significant improvement in the shoulder range of motion and hearing loss after 36 months of treatment and cardiac function was largely preserved. His skeletal deformity and short stature remained unchanged. The results showed that early ERT initiated at newborn is safe and effective in preventing or slowing down disease progression of MPS VI including bone deformities. These observations indicate that early diagnosis and treatment of MPS VI before development of an irreversible disease is critical for optimal clinical outcome.

Phenotype correction in murine mucopolysaccharidosis type VII by transplantation of human amniotic epithelial cells after adenovirus-mediated gene transfer.

Cell therapy with human amniotic epithelial (HAE) cells was developed as an alternative method for enzyme replacement therapy in congenital lysosomal storage disorders, but only limited therapeutic efficacy has been reported. A major drawback is insufficient production and secretion of lysosomal enzymes from HAE cells. In this study, we infected HAE cells with an E1-deleted adenoviral vector expressing human β-glucuronidase (GUSB), and generated cells overexpressing GUSB by a hundred times as much as endogenous GUSB in untreated HAE cells. GUSB secreted from the gene-transferred HAE cells were efficiently transported to murine fibroblasts with endocytosis mediated by mannose-6-phosphate receptors. The cells were administered into the spleen of the mice with the lysosomal storage disease mucopolysaccharidosis type VII (B6/MPSVII). Approximately 10–15% of the normal GUSB activity was detected in both liver and spleen 7 days after the cell administration. Histopathological examination showed that lysosomal enlargement in tissue macrophages in the liver and the spleen had disappeared by day 14. These results suggest that transplantation of the HAE cells transduced with adenoviral vectors can be employed for the treatment of congenital lysosomal storage disorders.

Newborn screening for Pompe disease in Japan.

Pompe disease is caused by a deficiency of acid alpha-glucosidase (GAA) that results in glycogen accumulation, primarily in muscle. Newborn screening (NBS) for Pompe disease has been initiated in Taiwan and is reportedly successful. However, the comparatively high frequency of pseudodeficiency allele makes NBS for Pompe disease complicated in Taiwan. To investigate the feasibility of NBS for Pompe disease in Japan, we obtained dried blood spots (DBSs) from 496 healthy Japanese controls, 29 Japanese patients with Pompe disease, and five obligate carriers, and assayed GAA activity under the following conditions: (1) total GAA measured at pH 3.8, (2) GAA measured at pH 3.8 in the presence of acarbose, and (3) neutral glucosidase activity (NAG) measured at pH 7.0 without acarbose. The % inhibition and NAG/GAA ratio were calculated. For screening, samples with GAA<8% of the normal mean, % inhibition>60%, and NAG/GAA ratio>30 were considered to be positive. Two false positive cases (0.3%) were found, one was a healthy homozygote of pseudodeficiency allele (c.1726G>A). The low false-positive rate suggests that NBS for Pompe disease is feasible in Japan.

Reduction of c-myc expression by an antisense approach under Cre/loxP switching induces apoptosis in human liver cancer cells.

c‐Myc has been documented to be both a positive and a negative signal for the induction of apoptosis. It is well known that overexpression of the c‐myc gene induces apoptosis of normal cells, but the result of a reduction in its expression is not fully understood. We examined whether a reduction in c‐myc expression would induce apoptosis in human liver cancer cells. Specifically, antisense and sense oligodeoxynucleotides (oligos) against the human c‐myc mRNA were synthesized, mixed with a liposome reagent at various ratios, and were applied to the liver cancer‐derived cell lines, HCC‐T, HepG2, and PLC/PRF/5. To exclude effects resulting from using oligos, plasmid vectors expressing the full‐length c‐myc cDNA in both sense and antisense orientations under the control of the Cre/loxP system were generated. Monoclonal cell lines including these plasmid vectors were produced and Cre was supplied by adenovirus infection. Apoptosis was determined morphologically and c‐Myc and Bcl‐2 expression was examined by Western blotting. The antisense myc significantly inhibited the proliferation of the cells within two days, while neither the liposome reagent alone nor sense myc did so. Most of the cells were rounded up by the antisense‐treatment and nuclear fragmentation and DNA ladder formation were detected after two days in antisense c‐myc‐treated cells. Antisense c‐myc largely reduced c‐Myc and partially Bcl‐2 expression; overexpression of Bcl‐2 partially rescued from apoptosis in HCC‐T and HepG2 cells. These results suggest that the massive reduction in c‐myc mRNA induces apoptosis in liver cancer cell lines and consequent decrease in Bcl‐2 enhances the cell death. c‐Myc reduction under the Cre/loxP switching system may be a useful tool for the clarification of c‐myc‐related cellular mechanisms in differentiation and proliferation.

Strong, long-term transgene expression in rat liver using chicken β-Actin promoter associated with cytomegalovirus immediate-early enhancer (CAG promoter)

For successful gene therapy in hepatic enzyme deficiencies, it is essential to use promoters that can maintain strong transcriptional activity for the long term in the liver. Using Gunn rats, a model animal for Crigler-Najjar syndrome type I, the long-term transcriptional function of the CAG promoter (a combination of chicken β-actin promoter and cytomegalovirus immediate-early enhancer) was evaluated in the rat liver. We constructed a plasmid pCAGGHUGT, containing expression cassettes of human bilirubin UDP-glucurono-syltransferase (BUGT) and hygromycin phosphotransferase, under the control of the CAG promoter and murine phosphoglycerate kinase promoter, respectively. Conditionally immortalized Gunn rat hepatocytes (IGRH), which had been established using mutant SV40 large T antigen (TST), were transfected with pCAGGHUGT. A stably transfected clone IGRHUGT, expressing a high level of BUGT, was obtained after selection with hygromycin. At 33°C, the cells doubled in number in approximately 72 h; however, at 37°C, cell proliferation stopped, indicating that the characteristic of temperature-dependent proliferation was retained in this clone. Ten million cells were injected into the spleen of syngeneic Gunn rats five times at 10-day intervals. Serum bilirubin levels were reduced by 45–50% at 70 days after the first transplantation and remained so throughout the duration of the study (120 days). These results suggested that the CAG promoter was able to maintain strong transcriptional activity in rat liver for at least 120 days.

Effects of idursulfase enzyme replacement therapy for Mucopolysaccharidosis type II when started in early infancy: Comparison in two siblings

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder that is progressive and involves multiple organs and tissues. While enzyme replacement therapy (ERT) with idursulfase has been shown to improve many somatic features of the disease, some such as dysostosis multiplex and cardiac valve disease appear irreversible once established, and little is known about the preventative effects of ERT in pre-symptomatic patients. We report on two siblings with severe MPS II caused by an inversion mutation with recombination breakpoints located within the IDS gene and its adjacent pseudogene, IDS-2. The siblings initiated treatment with idursulfase at 3.0 years (older brother) and 4 months (younger brother) of age, and we compared their outcomes following 2 years of treatment. At the start of treatment, the older brother showed typical features of MPS II, including intellectual disability. After 34 months of ERT, his somatic disease was stable or improved, but he continued to decline cognitively. By comparison, after 32 months of ERT his younger brother remained free from most of the somatic features that had already appeared in his brother at the same age, manifesting only exudative otitis media. Skeletal X-rays revealed characteristic signs of dysostosis multiplex in the older brother at the initiation of treatment that were unchanged two years later, whereas the younger brother showed only slight findings of dysostosis multiplex throughout the treatment period. The younger brothers developmental quotient trended downward over time to just below the normal range. These findings suggest that pre-symptomatic initiation of ERT may prevent or attenuate progression of the somatic features of MPS II. Follow-up in a larger number of patients is required to confirm the additive long-term benefits of ERT in pre-symptomatic patients.