Stable alphavirus packaging cell lines for Sindbis virus- and Semliki Forest virus-derived vectors

Alphavirus vectors are being developed for possible human vaccine and gene therapy applications. We have sought to advance this field by devising DNA- based vectors and approaches for the production of recombinant vector parti cles. In this work we generated a panel of alphavirus vector packaging cell lines (PCLs), These cell lines were stably transformed with expression cas settes that constitutively produced RNA transcripts encoding the Sindbis vi rus structural proteins under the regulation of their native subgenomic RNA promoter. As such, translation of the structural proteins was highly induc ible and was detected only after synthesis of an authentic subgenomic mRNA by the vector-encoded replicase proteins. Efficient production of biologica lly active vector particles occurred after introduction of Sindbis virus ve ctors into the PCLs, In one configuration, the capsid and envelope glycopro teins were separated into distinct cassettes, resulting in vector packaging levels of 10(7) infectious units/ml, but reducing the generation of contam inating replication-competent virus below the limit of detection. Vector pa rticle seed stocks could be amplified after low multiplicity of infection o f PCLs, again without generating replication-competent virus, suggesting ut ility for production of large-scale vector preparations. Furthermore, both Sindbis virus-based and Semliki Forest virus-based vectors could be package d with similar efficiency, indicating the possibility of developing a singl e PCL for use with multiple alphavirus-derived vectors.