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The construction of layered DNA-RNA replicons has facilitated and expanded the use of alphavirus vectors to vaccine development, construction of packaging cell lines and long-term heterologous gene expression. In these vector systems, the alphavirus replicon is under the control of a strong RNA polymerase II promoter and replicon RNA is transcribed from DNA before transport to the cytoplasm. Efficient RNA amplification catalyzed by the viral replicase results in high levels of mRNA and the recombinant protein. Recently, we developed a temperature-regulated Sindbis replicon-based DNA expression system characterized by a linear increase of expression upon decrease of the temperature from 37 degrees C to 29 degrees C. Modifications known to affect transcription and nuclear export of RNA led to a 5-fold increase in expression in BHK cells and up to over 80-fold increase in CHO cells and BF fibroblasts in transient transfection experiments. Furthermore, reducing cell proliferation resulted in a further 2- to 3-fold higher expression. While increased expression per cell was responsible for some of the enhanced production, it was primarily the number of expressing cells that made the difference in most cell lines. Further experiments indicated that a threshold amount of replicon RNA had to reach the cytoplasm in order for replication to occur. Thus, alterations that improve transcription, nuclear export and stability of the RNA had a significant impact on protein production in the pCytTS expression system and probably in other layered DNA-based viral vectors. Furthermore the results indicate that RNA replication is differentially regulated in DNA layered RNA replicons versus viral infection.

Original publication

DOI

10.1002/bit.10496

Type

Journal article

Journal

Biotechnol Bioeng

Publication Date

05/03/2003

Volume

81

Pages

553 - 562

Keywords

Active Transport, Cell Nucleus, Alphavirus, Animals, CHO Cells, Cells, Cultured, Cloning, Molecular, Cricetinae, DNA, Complementary, DNA, Viral, Gene Expression Regulation, Genetic Vectors, Kidney, Protein Engineering, RNA, Viral, Recombinant Fusion Proteins, Replicon, Sindbis Virus, Transcription, Genetic, Transfection