Clinical studies of gene therapy for cystic fibrosis (CF) suggest that the key problem is the efficiency of gene transfer to the airway epithelium. The availability of relevant vector receptors, the transient contact time between vector and epithelium, and the barrier function of airway mucus contribute significantly to this problem. We have recently developed recombinant Sendai virus (SeV) as a new gene transfer agent. Here we show that SeV produces efficient transfection throughout the respiratory tract of both mice and ferrets in vivo, as well as in freshly obtained human nasal epithelial cells in vitro. Gene transfer efficiency was several log orders greater than with cationic liposomes or adenovirus. Even very brief contact time was sufficient to produce this effect, and levels of expression were not significantly reduced by airway mucus. Our investigations suggest that SeV may provide a useful new vector for airway gene transfer.

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Unsolved issues in clinical gene therapy for cardiovascular diseases include gene transfer efficiency and the requirement of a longer exposure time. We developed a novel mononegavirus vector, recombinant Sendai virus (SeV), and tested whether it can overcome the present hurdles. SeV showed dose‐dependent and persistent gene expression in either proliferating or arrested cells, suggesting stability of RNA genome of the vector. An outstanding feature of the SeV‐mediated gene transfer was that even a brief exposure provided nearly peak gene expression in both culture cells and human veins ex vivo, as well as rabbit carotid arteries in vivo. Gene transfer to human great saphenous veins showed high efficacy in luminal and vasa vasoral endothelial cells and in adventitial fibroblasts via both intraluminal delivery and simple floating; however, only scattered cells were transfected in both neointima and media, regardless of the infusion pressure. Veins with a dissected neointima showed a clear transfection to medial cells, suggesting that the barrier in neointima reduces SeV‐mediated gene transfer to tunica media, similar to the case with adenoviruses. Although the fibromuscular neointima is a common obstacle, these findings suggest that SeV may overcome other limitations of current vectors. SeV may be an important new vector in treating subjects with vascular disorders.

Sendai virus (SeV) is an enveloped virus with a negative sense genome RNA of about 15.3 kb. We previously established a system to recover an infectious virus entirely from SeV cDNA and illustrated the feasibility of using SeV as a novel expression vector. Here, we have attempted to insert a series of foreign genes into SeV of different lengths to learn how far SeV can accommodate extra genes and how the length of inserted genes affects viral replication in cells cultured in vitro and in the natural host, mice. We show that a gene up to 3.2 kb can be inserted and efficiently expressed and that the replication speed as well as the final virus titers in cell culture are proportionally reduced as the inserted gene length increases. In vivo, such a sizedependent effect was not very clear but a remarkably attenuated replication and pathogenicity were generally seen. Our data further confirmed reinforcement of foreign gene expression in vitro from the V(3 3) version of SeV in which the accessory V gene had been knocked out. Based on these results, we discuss the utility of SeV vector in terms of both efficiency and safety.z 1999 Federation of European Biochemical Societies.

The nucleotide sequence of the Clostridium thermocellum F1 xynC gene, which encodes the xylanase XynC, consists of 1,857 bp and encodes a protein of 619 amino acids with a molecular weight of 69,517. XynC contains a typical N-terminal signal peptide of 32 amino acid residues, followed by a 165-amino-acid sequence which is homologous to the thermostabilizing domain. Downstream of this domain was a family 10 catalytic domain of glycosyl hydrolase. The C terminus separated from the catalytic domain by a short linker sequence contains a dockerin domain responsible for cellulosome assembly. The N-terminal amino acid sequence of XynC-II, the enzyme purified from a recombinant Escherichia coli strain, was in agreement with that deduced from the nucleotide sequence although XynC-II suffered from proteolytic truncation by a host protease(s) at the C-terminal region. Immunological and N-terminal amino acid sequence analyses disclosed that the full-length XynC is one of the major components of the C. thermocellum cellulosome. XynC-II was highly active toward xylan and slightly active toward p-nitrophenyl-␤-D-xylopyranoside, p-nitrophenyl-␤-D-cellobioside, p-nitrophenyl-␤-D-glucopyranoside, and carboxymethyl cellulose. The K m and V max values for xylan were 3.9 mg/ml and 611 mol/min/mg of protein, respectively. This enzyme was optimally active at 80°C and was stable up to 70°C at neutral pHs and over the pH range of 4 to 11 at 25°C.

We scrutinized the applicability and efficacy of Sendai virus (SeV) vectors expressing either LacZ or human insulin-like growth factor-I (hIGF-I) in gene transfer into skeletal muscle. Seven days after the intramuscular injection of LacZ/SeV Xgal labeled myofibers were demonstrated in rat anterior tibialis muscle with/without bupivacaine treatment and the transgene expression persisted up to 1 month after injection. Recombinant hIGF-I was detected as a major protein species in culture supernatants of a neonatal rat myoblast cell line L6 and thus induced the cells to undergo myogenetic

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal disease of unknown etiology; however, apoptosis of lung alveolar epithelial cells plays a role in disease progression. This intractable disease is associated with increased abundance of Staphylococcus and Streptococcus in the lungs, yet their roles in disease pathogenesis remain elusive. Here, we report that Staphylococcus nepalensis releases corisin, a peptide conserved in diverse staphylococci, to induce apoptosis of lung epithelial cells. The disease in mice exhibits acute exacerbation after intrapulmonary instillation of corisin or after lung infection with corisin-harboring S. nepalensis compared to untreated mice or mice infected with bacteria lacking corisin. Correspondingly, the lung corisin levels are significantly increased in human IPF patients with acute exacerbation compared to patients without disease exacerbation. Our results suggest that bacteria shedding corisin are involved in acute exacerbation of IPF, yielding insights to the molecular basis for the elevation of staphylococci in pulmonary fibrosis.

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