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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The P/C mRNA of Sendai virus (SeV) encodes a nested set of accessory proteins, C, C, Y1, and Y2, referred to collectively as C proteins, using the ؉1 frame relative to the open reading frame of phospho (P) protein and initiation codons at different positions. The C proteins appear to be basically nonstructural proteins as they are found abundantly in infected cells but greatly underrepresented in the virions. We previously created a 4C(؊) SeV, which expresses none of the four C proteins, and concluded that the C proteins are categorically nonessential gene products but greatly contribute to viral full replication and infectivity (A. Kurotani et al., Genes Cells 3:111-124, 1998). Here, we further characterized the 4C(؊) virus multiplication in cultured cells. The viral protein and mRNA synthesis was enhanced with the mutant virus relative to the parental wild-type (WT) SeV. However, the viral yields were greatly reduced. In addition, the 4C(؊) virions appeared to be highly anomalous in size, shape, and sedimentation profile in a sucrose gradient and exhibited the ratios of infectivity to hemagglutination units significantly lower than those of the WT. In the WT infected cells, C proteins appeared to colocalize almost perfectly with the matrix (M) proteins, pretty well with an external envelope glycoprotein (hemagglutinin-neuraminidase [HN]), and very poorly with the internal P protein. In the absence of C proteins, there was a significant delay of the incorporation of M protein and both of the envelope proteins, HN and fusion (F) proteins, into progeny virions. These results strongly suggest that the accessory and basically nonstructural C proteins are critically required in the SeV assembly process. This role of C proteins was further found to be independent of their recently discovered function to counteract the antiviral action of interferon-␣/␤. SeV C proteins thus appear to be quite versatile.Sendai virus (SeV) is an enveloped virus with a linear, nonsegmented, negative-sense RNA genome of 15,384 nucleotides. It belongs to the genus Respirovirus of the family Paramyxoviridae. The genome encodes, in a 3Ј-to-5Ј order, the nucleocapsid (N) protein, phospho (P) protein, matrix (M) protein, fusion (F) protein, hemagglutinin-neuraminidase (HN), and large (L) protein. The genome RNA is tightly encapsidated with the N protein subunits and is further complexed to the polymerase comprising the L and P protein subunits (14). This ribonucleoprotein (RNP) complex represents the internal core structure of the virion. The viral envelope contains two glycoproteins, HN and F. The former mediates viral attachment to the surface of susceptible cells, and the latter is required for the fusion of the envelope with the cellular membrane to introduce the genomic material into the cytoplasm. The envelope lipid bilayer is derived from the host cell plasma membrane during the final step of assembly by budding. There is a layer of M proteins between the envelope and RNP (30). The M protein has been thought to play a critical role in a...

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.