Makoto Asakawa

A Cytoplasmic RNA Vector Derived from Nontransmissible Sendai Virus with Efficient Gene Transfer and Expression

ABSTRACT We have recovered a virion from defective cDNA of Sendai virus (SeV) that is capable of self-replication but incapable of transmissible-virion production. This virion delivers and expresses foreign genes in infected cells, and this is the first report of a gene expression vector derived from a defective viral genome of theParamyxoviridae. First, functional ribonucleoprotein complexes (RNPs) were recovered from SeV cloned cDNA defective in the F (envelope fusion protein) gene, in the presence of plasmids expressing nucleocapsid protein and viral RNA polymerase. Then the RNPs were transfected to the cells inducibly expressing F protein. Virion-like particles thus obtained had a titer of 0.5 × 108 to 1.0 × 108 cell infectious units/ml and contained F-defective RNA genome. This defective vector amplified specifically in an F-expressing packaging cell line in a trypsin-dependent manner but did not spread to F-nonexpressing cells. This vector infected and expressed an enhanced green fluorescent protein reporter gene in various types of animal and human cells, including nondividing cells, with high efficiency. These results suggest that this vector has great potential for use in human gene therapy and vaccine delivery systems.

The RAMP2/CRLR complex is a functional adrenomedullin receptor in human endothelial and vascular smooth muscle cells

Adrenomedullin, a potently hypotensive peptide isolated from human pheochromocytoma, is known to elicit a rise in cAMP levels within mammalian endothelial and smooth muscle cells. Until now, however, little has been known about the adrenomedullin receptor. Recently, a group called receptor activity‐modifying proteins that complex with the calcitonin receptor‐like receptor, and thereby regulate its transport and ligand specificity, were identified. Here we show that mRNA for both the calcitonin receptor‐like receptor and the receptor activity‐modifying protein 2, but not the receptor activity‐modifying protein 1 or receptor activity‐modifying protein 3, are expressed in human endothelial and vascular smooth muscle cells. We also found that adrenomedullin increased cAMP levels in HeLa EBNA and 293 EBNA cells, expressing both the receptor activity‐modifying protein 2 and calcitonin receptor‐like receptor proteins. Thus, the receptor activity‐modifying protein 2/calcitonin receptor‐like receptor complex apparently serves as a functional adrenomedullin receptor in human endothelial and vascular smooth muscle cells.

Accommodation of foreign genes into the Sendai virus genome: sizes of inserted genes and viral replication

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 size‐dependent 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(−) 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.