107. Sustained Stable Gene Expression Induced by a Cytoplasmic RNA-Virus Vector Based on Mutant Sendai Virus Strain cl.151

Abstract

Top of pageAbstract Sendai virus (SeV)|[mdash]|mouse para-influenza virus type 1|[mdash]|is a prototype virus of the Paramyxoviridae with a non-segmented negative strand RNA genome. SeV can infect a wide variety of mammalian cells in vitro and in vivo, and directs high-level gene expression from cytoplasmic viral genome RNA. As it is not pathogenic to humans, it promises to be a unique potential viral vector for human gene therapy while avoiding genotoxicity. However, current SeV vectors induce gene expression only transiently, partly because they are based on the SeV Z strain with lytic replication. To overcome this transient nature of gene expression, we have developed a vector based on the mutant SeV strain, which is capable of persistent infection. The temperature-sensitive mutant cl.151 strain, originally isolated from an SeV carrier culture (Yoshida et al., Virology 92, 139|[ndash]|154, 1979), has a defect in the late step of viral assembly (Kondo, et al., J. Biol. Chem., 268, 21924|[ndash]|21930, 1993). This mutant form readily establishes persistent infection in various cultured cells at the nonpermissive temperature (38 |[deg]|C), and expresses the viral antigen stably. We cloned the cDNA covering the entire RNA genome of this strain (15,384 nt). It has 50 nucleotide substitutions and 35 amino acid substitutions compared with the genome of the parental Nagoya strain. Using this cDNA as a starting material, we prepared recombinant SeVs carrying marker genes (for green fluorescent protein, GFP, and luciferase, Luc) by standard reverse genetics. These marker genes, flanked by transcription initiation and termination signals, were inserted immediately upstream of the NP (nucleocapsid protein) gene. The resulting cl.151-based recombinant SeV forms (r151-Luc and r151-GFP) showed temperature-dependent replication and infected various cultured cells persistently. Thus, this cloned cDNA contains all the information required for these characteristics of the cl.151 strain. These recombinant SeVs expressed the marker genes strongly as the Z strain-based vectors, and showed sustained gene expression stably for more than one month. These results strongly suggest the potential utility of this cl.151-based SeV vector as a valuable and non-toxic tool for human gene therapy. Characteristics of marker gene expression induced by cl.151-based SeV vectors are now under examination in animal tissue cells in vivo.