PACKAGING OF INTRON-CONTAINING GENES INTO RETROVIRUS VECTORS BY ALPHAVIRUS VECTORS

Efficient and controllable expression of a transgene usually requires the presence of intron sequences and much efforts have been made to pr oduce retrovirus vectors that can transduce and integrate genes with i ntrons. However, this has proven difficult because the viral RNA is sp liced when it is synthesized in the nucleus of a producer cell. We des cribe a novel approach to avoid this problem. In our system the retrov iral RNA is synthesized in the cytoplasm of the cell, not in the nucle us, in a reaction driven by the Semliki Forest virus (SFV) expression system. The approach was tested with a recombinant Moloney murine leuk emia virus genome containing the chloramphenicol acetyltransferase (CA T) gene in associated with an intron. This was inserted into a SFV tra nscription in vitro. BHK-21 cells were then transfected with this vect or RNA together with two additional SFV vectors that encode the Molone y murine leukemia virus packaging proteins. Retrovirus vectors contain ing intron-CAT sequences were produced at titers up to 1.3 x 10(6) inf ectious particles per ml during a 5-hr incubation period. The vectors faithfully transduced the intron-containing CAT gene into N1H 3T3 cell s, where the intron-CAT RNA was subjected to efficient splicing and us ed for high level enzyme expression. Thus, the results show that intro n containing genes can be efficiently packaged into retrovirus vectors by the SFV expression system.