Lentiviral vector design using alternative RNA export elements

Taekeun Oh,Ali Bajwa,Guangfu Jia,Frank Park

doi:10.1186/1742-4690-4-38

Abstract

BackgroundLentiviral vectors have been designed with complex RNA export sequences in both the integrating and packaging plasmids in order to co-ordinate efficient vector production. Recent studies have attempted to replace the existing complex rev/RRE system with a more simplistic RNA export system from simple retroviruses to make these vectors in a rev-independent manner.ResultsTowards this end, lentiviral transfer plasmids were modified with various cis-acting DNA elements that co-ordinate RNA export during viral production to determine their ability to affect the efficiency of vector titer and transduction in different immortalized cell lines in vitro. It was found that multiple copies of the constitutive transport element (CTE) originating from different simian retroviruses, including simian retrovirus type 1 (SRV-1) and type-2 (SRV-2) and Mason-Pfizer (MPV) could be used to eliminate the requirement for the rev responsive element (RRE) in the transfer and packaging plasmids with titers >106 T.U./mL (n = 4–8 preparations). The addition of multiple copies of the murine intracisternal type A particle, the woodchuck post-regulatory element (WPRE), or single and dual copies of the simian CTE had minimal effect on viral titer. Immortalized cell lines from different species were found to be readily transduced by VSV-G pseudotyped lentiviral vectors containing the multiple copies of the CTE similar to the findings in HeLa cells, although the simian-derived CTE were found to have a lower infectivity into murine cell lines compared to the other species.ConclusionThese studies demonstrated that the rev-responsive element (RRE) could be replaced with other constitutive transport elements to produce equivalent titers using lentivectors containing the RRE sequence in vitro, but that concatemerization of the CTE or the close proximity of RNA export sequences was needed to enhance vector production.

Highlights

Lentiviral vectors have been designed with complex RNA export sequences in both the integrating and packaging plasmids in order to co-ordinate efficient vector production
The insertion of the rev-responsive element (RRE) into the transfer plasmid 5' to the PGK promoter [pHRSVR(+)cPGKnlsLacZR()W(-)] increased the viral titer to 50.4 +/- 3.5 × 103 T.U./ mL (n = 5), and there appeared to be a co-operative effect on viral titer when the RRE was included into the lentivector transfer plasmid 5' to the PGK promoter with the woodchuck post-regulatory element (WPRE) near the 3' self-inactivating long-terminal repeat (SIN long terminal repeat (LTR); 109 +/- 14 × 103 T.U./mL; n = 4)
The incorporation of the RRE into our lentivector transfer plasmid was position-dependent, since we found that the placement of the RRE near the 3' end of the lentivector resulted in a significant enhancement of viral titer to 1,080 +/- 84 × 103 T.U./mL, which was ~20-fold higher than the RRE sequence in the 5' region of the vector

Summary

Introduction

Lentiviral vectors have been designed with complex RNA export sequences in both the integrating and packaging plasmids in order to co-ordinate efficient vector production. Recent studies have attempted to replace the existing complex rev/RRE system with a more simplistic RNA export system from simple retroviruses to make these vectors in a rev-independent manner. One of the main regulatory genes, rev, has remained in many of the advanced lentivectors, and this protein is known to bind to its cis-acting DNA element, rev-responsive element (RRE). This rev/ RRE system is important to the efficient transport of unspliced viral RNA genomes from the nucleus into the cytoplasm to properly assemble the lentivector particles [8]. Earlier work by Wodrich et al [13] demonstrated that gag polyprotein (Pr55) expression can be significantly elevated if multiple copies of the MasonPfizer CTE were incorporated into the gag-pol expression plasmid compared to a single copy of the CTE, which may circumvent the requirement of the rev/RRE post-transcriptional control systems in vector production

Objectives

Methods

Results

Discussion

Conclusion