LONG-TERM GENE-EXPRESSION FROM AUTONOMOUSLY REPLICATING VECTORS IN MAMMALIAN-CELLS

We tested the longevity of gene expression provided by autonomously re plicating vectors. The vectors contain segments of human genomic DNA t hat provide efficient replication initiation and sequences derived fro m Epstein-Barr virus that provide nuclear retention. In order to monit or gene expression, the vectors also carry an expression cassette cont aining the chloramphenicol acetyl transferase gene. Replicating and co ntrol vectors were transfected into rapidly dividing human 293 cells, and gene expression and DNA retention were monitored. Gene expression decreased rapidly from a control vector lacking replication and retent ion functions, reaching near background levels by to days. Vectors hav ing both replication and retention showed greatly prolonged gene expre ssion, with Vector DNA still detectable after 2 months. Autonomously r eplicating vectors also prolonged gene expression in rodent cells, in human lung epithelial cells, and in slowly dividing cell cultures. The se results demonstrate the utility of this autonomous replication syst em for long-term retention and expression of introduced genes in mamma lian cells. Such vectors could be useful for gene therapy, in combinat ion with any of several methods for introduction of DNA.