R-Region cDNA Inserts in Retroviral Vectors Are Compatible with Virus Replication and High-Level Protein Synthesis from the Insert

Abstract

Protein expression from retroviral vectors is often highest when the expressed cDNA is driven by the retroviral promoter. However, the typical retroviral vector design places the cDNA downstream of the retroviral packaging signal and far from the retroviral promoter. In an attempt to improve protein production levels from cDNAs expressed in retroviral vectors, we inserted the MyoD or the purine nucleoside phosphorylase (PNP) cDNAs into the R regions of both retroviral LTRs, close to the retroviral promoter and just upstream of the polyadenylation signal present in each long terminal repeat (LTR). These R-region double-copy vectors could be produced in unrearranged form, although the titer was about seven-fold lower than that of typical vectors. R-region positioning of the MyoD cDNA resulted in five-fold higher MyoD expression compared to MyoD expression in a typical vector, whereas PNP expression was not improved. Thus, R-region double-copy vectors provide an alternative vector design that can improve protein expression from some cDNAs.

Overview summary

The level of protein production from retroviral vectors is an important determinant of their utility, particularly for gene therapy applications. Adam et al. explore an alternative “double-copy” vector design in which the cDNA to be expressed is inserted into the R region of both retroviral long terminal repeats (LTRs). For one cDNA, expression was increased five-fold in comparison to expression from a typical vector, whereas expression was reduced somewhat for another cDNA. Unfortunately, vector titer was reduced by about seven-fold as well. These results show that R-region inserts are compatible with retrovirus replication and provide an alternative design for high-level protein production. Alternative designs are important, given that vector performance is somewhat unpredictable for a given insert and construction of several vectors is sometimes required to achieve required levels of expression.